The strong magnetic moment of the unpaired electron promotes both spin lattice and spin-spin relaxation of the surrounding water protons, resulting with decrease in their spin-lattice (T1) and spinspin (T2) relaxation times or enhancement in signal intensity on T1 or T2 weighted MR images. Relaxation constants of (MGD)2-Fe(II)-NO, (MGD)2-Fe(II) at 500 MHz\* and 300 MHz\*\* is shown indicating that relaxivity is not magnetic field dependent over this range.

Table 2. Relaxation constants of biosensor complexes are shown for NO detection in aqueous solutions.

Inhibition of Nitric Oxide Synthase Gene Expression:

hormone molecules in tissue.

reference Fujii, et al. 2007.

MR contrast due to trapped NO can be expressed in Eq 2, 3:

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 129

1/T1[NO-Fe(II)(MGD)2] = 1/T1(observed) – 1/T\* = r1*i*[NO-Fe(II)(MGD)2] × concentration (2)

 1/Tobs = 1/T*\**+ r1*i*[NO-Fe(II)(MGD)2]; *i* = 1, 2... (3) where T1(observed) is long longitudinal relaxation time constant and T1\* is short longitudinal relaxation time constant in absence of [NO-Fe(II)(MGD)2], r1i is longitudinal relaxivity dpends on I = 1, 2, 3 the number of carbons in branch of MGD chain, [NO-Fe(II)(MGD)2], concentration and interaction with NO specific bioactive receptor, enzyme, antibody,

Fig. 5. Imaging of preformed (MGD)2-Fe(II)-NO complex. Transverse T1-weighted MR images in the axial plane of the liver of Wistar rats. Two ml of 9.5 mM (MGD)2-Fe(II)-NO complex was injected i.p. in the lower abdomen of 250 g rats (n = 3). **a:** Control, before injection; **b:** 60 min after injection of (MGD)2-Fe(II)-NO complex. A sketch at bottom shows the interaction of No with heme as basis of MRI signal. Reproduced with permission for

In other study, NO complex was prepared from NO gas and (MGD)2-Fe(II) in glass capillaries. The strong magnetic moment of the unpaired electrons promote both spin lattice and spin-spin relaxation of the surrounding water protons and show a decrease in their spin-lattice (T1) and spin-spin (T2) relaxation constants or show enhanced signal intensity in T1 or T2 weighted MR images as shown in Table 2 and Figure 5 [Hortelano, et al. 2005]. Major points were:


In previous study, (MGD)2-Fe(II)-NO and LPS complexes were used as in vivo MR contrast agents [Fujii, et al. 2007]. First, intraperitoneal injection of 2 ml of 9.1 mM (MGD)2-Fe(II)-NO complex (150 nmol/g tissue) showed relative difference on T1-weighted MR images of rat liver site before and after injection of NO complex as shown in Figure 5.

 The images indicated the NO complex very effective ''intrinsic contrast agent,'' to enhance the contrast in several other organs.

The Second, LPS injected animals after 6 hours of (MGD)2-Fe(II) injection, showed subsequent MRI detection and reproducible visualization of NO generated in liver of animals as shown in Figure 5 and Fig. 6a (indicated by the arrow) as plots against time with the intensity of the reference water sample. Figure 6b showed that the image intensity in the liver increased with the NO generation, without any intensity change in reference sample in the presence of the specific NOS inhibitor *N*-monomethyl L-arginine (L-NMMA).

Interestingly, other face of (MGD)2-Fe(II)-NO complex is 'spin trap'. Spin traps are chemical compounds used to detect unstable free radicals such as hydroxyl (\*OH) and superoxide (O2 \*-) in biological systems. Spin trapping compounds (spin traps) selectively react with NO and trap the nitric oxide. The spin-trap stock (MGD)2-Fe(II) complex solution (mixture of deoxygenated saline MGD (100 mM) and FeSO4 (20 mM) solutions in nitrogen) makes (MGD)2-Fe(II)-NO complex by mixing (MGD)2-Fe(II) with either pure NO gas or *S*-nitroso-*N*acetyl DL-penicillamine (SNAP). The pure NO gas is prepared by passing NO gas through a KOH solution. Major concern was the coordination of NO to the Fe complex altered the solubility of N0-Fe(II)(MGD)2 < 1 mM. The in vivo half life of N0-Fe(II)(MGD)2 was 41 minutes and only 40% trapped NO was estimated by detection of Fe(II)(MGD)2 induced inhibited NOS and decrease in NO formation and perfusion in the tissue [Fichtlscherer, et al. 2000].

$$\begin{aligned} \text{2NO} + \text{Fe(III)(MGD)} & \xrightarrow{\text{H}\_2\text{O} \text{ (\*OH)}} \text{NO-Fe(II)(MGD)} + \text{NO}\_2 \\\\ \text{NO} + \text{Fe(III)(MGD)} & \xrightarrow{\text{Reducing Equivalent}} \text{NO-Fe(II)(MGD)} \text{2(40\%)} \end{aligned} \tag{1}$$

In other study, NO complex was prepared from NO gas and (MGD)2-Fe(II) in glass capillaries. The strong magnetic moment of the unpaired electrons promote both spin lattice and spin-spin relaxation of the surrounding water protons and show a decrease in their spin-lattice (T1) and spin-spin (T2) relaxation constants or show enhanced signal intensity in T1 or T2 weighted MR images as shown in Table 2 and Figure 5 [Hortelano, et al. 2005].

The relaxation constants of (MGD)2-Fe(II)-NO in aqueous media were characteristic and

T1 and T2 relaxation constants of liver were measured 1.17(1/mM \* sec) and 1.32

 T1 relaxation constant of (MGD)2-Fe(II), was reported 0.044 (1/mM \* sec) at both 20 MHz and 85 MHz magnetic fields. The T1 relaxation constant was field independent; Distinct increase in relaxivity after complexing NO with (MGD)2-Fe(II). These unique properties of complex suggested its feasibility to visualize the regional distribution in

In previous study, (MGD)2-Fe(II)-NO and LPS complexes were used as in vivo MR contrast agents [Fujii, et al. 2007]. First, intraperitoneal injection of 2 ml of 9.1 mM (MGD)2-Fe(II)-NO complex (150 nmol/g tissue) showed relative difference on T1-weighted MR images of rat

The images indicated the NO complex very effective ''intrinsic contrast agent,'' to

The Second, LPS injected animals after 6 hours of (MGD)2-Fe(II) injection, showed subsequent MRI detection and reproducible visualization of NO generated in liver of animals as shown in Figure 5 and Fig. 6a (indicated by the arrow) as plots against time with the intensity of the reference water sample. Figure 6b showed that the image intensity in the liver increased with the NO generation, without any intensity change in reference sample in

Interestingly, other face of (MGD)2-Fe(II)-NO complex is 'spin trap'. Spin traps are chemical compounds used to detect unstable free radicals such as hydroxyl (\*OH) and superoxide (O2

in biological systems. Spin trapping compounds (spin traps) selectively react with NO and trap the nitric oxide. The spin-trap stock (MGD)2-Fe(II) complex solution (mixture of deoxygenated saline MGD (100 mM) and FeSO4 (20 mM) solutions in nitrogen) makes (MGD)2-Fe(II)-NO complex by mixing (MGD)2-Fe(II) with either pure NO gas or *S*-nitroso-*N*acetyl DL-penicillamine (SNAP). The pure NO gas is prepared by passing NO gas through a KOH solution. Major concern was the coordination of NO to the Fe complex altered the solubility of N0-Fe(II)(MGD)2 < 1 mM. The in vivo half life of N0-Fe(II)(MGD)2 was 41 minutes and only 40% trapped NO was estimated by detection of Fe(II)(MGD)2 induced inhibited NOS

\*-)

(1)

the presence of the specific NOS inhibitor *N*-monomethyl L-arginine (L-NMMA).

and decrease in NO formation and perfusion in the tissue [Fichtlscherer, et al. 2000].

2NO + **Fe(III)**(MGD)2 H2O (\*OH)\_\_\_\_\_\_\_ NO-**Fe(II)**(MGD)2 + NO2-

NO + **Fe(III)**(MGD)2 \_\_\_\_Reducing Equivalent\_\_\_\_ NO-**Fe(II)(**MGD)2 (40%)

(1/mM \* sec) respectively at 500 MHz at increasing concentration of NO;

liver site before and after injection of NO complex as shown in Figure 5.

Major points were:

complex concentration dependent;

tissue *in vivo* where the NO was trapped.

enhance the contrast in several other organs.

MR contrast due to trapped NO can be expressed in Eq 2, 3:

$$\text{1/} \text{Tl} \text{[NO-Fe(II)(MGD)} \text{]} = \text{1/} \text{Tl} \text{(cherved)} - \text{1/} \text{T\*} = \text{r1} \text{[NO-Fe(II)(MGD)} \text{]} \times \text{concentration} \quad \text{(2)}$$

$$1/\Gamma\_{\rm obs} = 1/\Gamma^\* + \text{r1}\_i [\text{NO-Fe(II)(MGD)}\_2]; i = 1, 2... \tag{3}$$

where T1(observed) is long longitudinal relaxation time constant and T1\* is short longitudinal relaxation time constant in absence of [NO-Fe(II)(MGD)2], r1i is longitudinal relaxivity dpends on I = 1, 2, 3 the number of carbons in branch of MGD chain, [NO-Fe(II)(MGD)2], concentration and interaction with NO specific bioactive receptor, enzyme, antibody, hormone molecules in tissue.

Fig. 5. Imaging of preformed (MGD)2-Fe(II)-NO complex. Transverse T1-weighted MR images in the axial plane of the liver of Wistar rats. Two ml of 9.5 mM (MGD)2-Fe(II)-NO complex was injected i.p. in the lower abdomen of 250 g rats (n = 3). **a:** Control, before injection; **b:** 60 min after injection of (MGD)2-Fe(II)-NO complex. A sketch at bottom shows the interaction of No with heme as basis of MRI signal. Reproduced with permission for reference Fujii, et al. 2007.

Inhibition of Nitric Oxide Synthase Gene Expression:

(PARAVISION 3.2).

detecting solid tissues.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 131

experiments, anesthesia was administered as sodium pentobarbital (30 mg/kg, i.p.) and ketamine (20 mg/kg, intra-muscular injection) before injection of contrast agent in animals. For imaging, animals were anesthetized by intubating with 14 gauge, 2 inch intravenous catheter (Abbocath Lab, IL) on nose with 30% oxygen/70% nitrogen mixture containing 5% isoflurane/air mixture to continuous supply through nose during MRI session. Animals were kept in vertical direction to the side of MRI gantry and 30 mm diameter RF insert covering kidneys in the center of magnet. During six hours after administration of LPS to Wistar rats (150–200 g) they were injected intraparitoneally with 3 ml of (MGD)2-Fe(II) [MGD:100 mM, Fe:20 mM] at different intervals. MR images of the anesthetized rats (n = 5) were obtained after 1 hr spin trap injection with a Bruker Biospin 500 MHz scanner

Different imaging techniques are available in our lab on 500 MHz and 900 MHz scanners. In vivo NO induction was carried out according to previously reported methods with slight

 Axial T2-weighted spin echo sequence with fat suppression (TR 2000 ms, TE 100 ms, flip angle 30; EC 1/1 15.6 kHz) was used for more detailed T2-weighted information in

 Axial T1-weighted images were acquired at TR/TE/flip angle = 750 ms/ 4.18 ms/25º, FOV/matrix size/spatial resolution = 2.6 x 1.4 cm2/ 256 x 256 ×75 μm, and the inversion time (TI approximately 250 ms) set to null the normal the background tissue. Gradient echo sequence in-phase and opposed-phase(TR 180 ms, TE 2.3 ms/4.5 ms, flip angle 30 were done as a dual echo sequence to show renal lesions hypointense and parenchyma isointense with hyperintense protein rich cyst. Opposed phased T1-

 Axial T1-weighted gradient echo sequence for dynamic perfusion/ diffusion weighted imaging (TR 130 ms, TE 1.0 ms, flip angle 30) was done after 30 ml intravenous gadolinium contrast injection for acquiring pre-contrast and post-contrast images in arterial phase to distinguish perfusion in the tissues. Parameters for T1-weighted spinecho images were TR 1500 msec, TE 10 msec, 2 NEX, 1-mm slice thickness, 0.5-mm slice

The original bird-cage coil with 2.6 cm diameter Rf insert was used for MR imaging. We established in lab MR image analysis by shareware software NIH Image J, a PC version supplied by Scion Corporation. The analysis of averaged image intensities from selected regions of interest 15 x 3 x 15, and matrix , 256 x 256 served as best for measurement of signal enhancement. An external reference (saline solution in a sealed test tube) placed near the animal serves to normalize intensities from both control and test groups. Percentage enhancements can be calculated from the normalized data [35]. Functional dependence of the MR imaging signal intensity *S* on the intrinsic properties (proton spin density *N*(*H*), T1, T2) has been derived based on the assumption of a monoexponential dependence on T1 and T2 with a spin-echo sequence: *S*(TE,TR) = *N*(*H*)exp(-TE/T2) [1 -exp[-(TR/ TE)/T1]]. The term on the right reflects the T1 dependence, and the exponential expression on the left reflects the T2 effects. TE << T2 generates a T1-weighted. T1 image is obtained by means of least-squares fitting (Marquardt-Levenberg algorithm) of the mean intensity values versus

modifications [Lai, et al. 1994, Yoshimura, et al. 1996, Green, et al. 1998].

weighted gradient echo sequences were sensitive to fat.

gap, field of view 12 x 3 x 12 cm3, and matrix, 256 x 256.

Fig. 6. Imaging of NO in six phantoms and LPS-treated rats. **a:** Transverse T1-weighted MR images focussed on a selected region of the liver in LPS-doped rats. The MR images were measured at the times indicated. Six hours after LPS injection, the NO spin-trap [3 ml of (MGD)2-Fe(II), MGD: 100 mM, Fe: 20 mM] was administered i.p. **b:** Plot of MR image intensity with time. Signal intensities were averaged over the selected region indicated by the arrow in a (7\*7 mm2) for three different animals (filled symbols), normalized to intensity of a reference (water in a tube placed next to the animal, open symbols). At zero time, the spin-trap, (MGD)2- Fe(II), was added. **c:** Comparison of the signal intensity in a selected slice region of the liver in the presence and absence of the NOS inhibitor, L-NMMA. The image intensity in the liver was first normalized to the external standard noted above. The averaged intensity in the selected regions (7 x 7 mm2) was averaged (n = 3). L-NMMA (50 mg/kg in saline) was injected i.p. 3 h after LPS injection (Reproduced from reference Fujii et al. 2007).

#### **2.4 MRI measurement protocol design**

We developed a MRI protocol to investigate the regional distribution of (MGD)2-Fe(II)-NO in various organs. For whole animal imaging, protocols for relaxivity of (MGD)2-Fe(II)-NO were designed on a Bruker 500 MHz, to achieve millimolar relaxivity. For the animal

Fig. 6. Imaging of NO in six phantoms and LPS-treated rats. **a:** Transverse T1-weighted MR images focussed on a selected region of the liver in LPS-doped rats. The MR images were measured at the times indicated. Six hours after LPS injection, the NO spin-trap [3 ml of (MGD)2-Fe(II), MGD: 100 mM, Fe: 20 mM] was administered i.p. **b:** Plot of MR image intensity with time. Signal intensities were averaged over the selected region indicated by the arrow in a (7\*7 mm2) for three different animals (filled symbols), normalized to intensity of a reference (water in a tube placed next to the animal, open symbols). At zero time, the spin-trap, (MGD)2- Fe(II), was added. **c:** Comparison of the signal intensity in a selected slice region of the liver in the presence and absence of the NOS inhibitor, L-NMMA. The image intensity in the liver was first normalized to the external standard noted above. The averaged intensity in the selected regions (7 x 7 mm2) was averaged (n = 3). L-NMMA (50 mg/kg in saline) was injected i.p. 3 h

We developed a MRI protocol to investigate the regional distribution of (MGD)2-Fe(II)-NO in various organs. For whole animal imaging, protocols for relaxivity of (MGD)2-Fe(II)-NO were designed on a Bruker 500 MHz, to achieve millimolar relaxivity. For the animal

after LPS injection (Reproduced from reference Fujii et al. 2007).

**2.4 MRI measurement protocol design** 

experiments, anesthesia was administered as sodium pentobarbital (30 mg/kg, i.p.) and ketamine (20 mg/kg, intra-muscular injection) before injection of contrast agent in animals. For imaging, animals were anesthetized by intubating with 14 gauge, 2 inch intravenous catheter (Abbocath Lab, IL) on nose with 30% oxygen/70% nitrogen mixture containing 5% isoflurane/air mixture to continuous supply through nose during MRI session. Animals were kept in vertical direction to the side of MRI gantry and 30 mm diameter RF insert covering kidneys in the center of magnet. During six hours after administration of LPS to Wistar rats (150–200 g) they were injected intraparitoneally with 3 ml of (MGD)2-Fe(II) [MGD:100 mM, Fe:20 mM] at different intervals. MR images of the anesthetized rats (n = 5) were obtained after 1 hr spin trap injection with a Bruker Biospin 500 MHz scanner (PARAVISION 3.2).

Different imaging techniques are available in our lab on 500 MHz and 900 MHz scanners. In vivo NO induction was carried out according to previously reported methods with slight modifications [Lai, et al. 1994, Yoshimura, et al. 1996, Green, et al. 1998].


The original bird-cage coil with 2.6 cm diameter Rf insert was used for MR imaging. We established in lab MR image analysis by shareware software NIH Image J, a PC version supplied by Scion Corporation. The analysis of averaged image intensities from selected regions of interest 15 x 3 x 15, and matrix , 256 x 256 served as best for measurement of signal enhancement. An external reference (saline solution in a sealed test tube) placed near the animal serves to normalize intensities from both control and test groups. Percentage enhancements can be calculated from the normalized data [35]. Functional dependence of the MR imaging signal intensity *S* on the intrinsic properties (proton spin density *N*(*H*), T1, T2) has been derived based on the assumption of a monoexponential dependence on T1 and T2 with a spin-echo sequence: *S*(TE,TR) = *N*(*H*)exp(-TE/T2) [1 -exp[-(TR/ TE)/T1]]. The term on the right reflects the T1 dependence, and the exponential expression on the left reflects the T2 effects. TE << T2 generates a T1-weighted. T1 image is obtained by means of least-squares fitting (Marquardt-Levenberg algorithm) of the mean intensity values versus

Inhibition of Nitric Oxide Synthase Gene Expression:

functional MRI [Caramia, et al. 1998].

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 133

Fig. 7. NO sensitive biomarker imaging contrast agents. Example of NO-Fe(II)(DTC)2 is cited to illustrate the signal change during changes in cerebral blood flow and possibility of

Two decades ago, NO activation-induced signal changes in functional MRI (fMRI) was observed [Di Salle, et al. 1997]. Hb–NO was found to produce marked stimulator dose and concentration-dependent NO sensitive fMRI signal intensity changes (increased after aqueous NO solution, nitrite, or dithionite and nitrite added to blood while it decreased on the addition of ascorbic acid) examined by T1-, T2-, and T2 -weighted MRI in venous erythrocytes with possibility of physiological MRI evaluation associated with NO change [Di Salle, et al. 1997]. Transient formation of two paramagnetic species (met Hb and NO-Hb) enhanced the signal intensity, while ascorbate reduced the signal intensity in fMRI experiment on healthy volunteers during standard tasks. Further, infusion of NO precursor, L-arginine increased the cerebral blood volume (measured by MRI) in nonischemic spontaneously hypertensive rats [Foster, et al. 1998]. In other study, administration of an NO donor, isosorbide dinitrate (ISDN) increased both tumor blood flow and partial oxygen pressure in mice implanted with liver tumor in the thigh [Jordan, et al. 2000]. These findings indicate blood flow-independent change in MRI signal produced by different additives or stimulators. These observations open a new perspective on the monitoring of NO and the in vivo speculation of NO effects to test new drugs by using magnetic resonance techniques. Fuji et al. 1999 first time reported *in vivo* MRI of NO using Fe(MGD)2 as NO–Fe(MGD)2 spintrapping technique in a septic shock rat model [Fujii, et al. 1999]. (Fig. 6A). The reduced signal after administration of an iNOS inhibitor confirmed the MRI visibility of NO–

**NO-Fe(II)(DTC)2**

TR. T2 weighted image is obtained by TR >> T2. Mean intensity values are fitted versus the TE (Marquardt Levenberg algorithm).

T1 weitghted MR contrast due to trapped NO as NO-Fe(II)X spin trap in tissue can be expressed in Eq 4, 5:

$$1/\text{T1}[\text{NO-Fe(II)X}] = 1/\text{T1}\_{\text{(observed)}} - 1/\text{T} = \text{r1}\_{i}[\text{NO-Fe(II)X}] \times \text{concentration} \tag{4}$$

$$1/\Gamma\_{\rm obs} = 1/\Gamma^\* + \text{r1}\_i [\text{NO-Fe(II)}\lambda]; i = 1, 2... \tag{5}$$

where T1(observed) is long longitudinal relaxation time constant and T1\* is short longitudinal relaxation time constant in absence of [NO-Fe(II)X],

r1i is longitudinal relaxivity dpends on i= 1, 2, 3 the number of carbons in branch of X contrast agent, its concentration and interaction with NO specific bioactive receptor, enzyme, antibody, hormone molecules in tissue.

#### **2.5 Approaches to NO evaluation by Magnetic Resonance Imaging (MRI) techniques**

Two major developments reported in context with NO. First, a search of NO specific stable multimodal MR/EPR sensitive spin trap complex; second, calibrating imaging characteristics of spin trap complex on NMR/MRI/PEDRI techniques. NO is perfused in gaseous phase or remains in ionic phase by redox state in the tissues. Both forms of NO are captured or trapped by Fe(II)-(DTC)2 complexes and show MRI signal change due to reactive nitrosylation (reaction of intracellular NO with Fe(II)(DTC)2 to form NO-Fe(II)(DTC)2 complex) shown in Eq 6. Second-order rate constant of the reaction of NO with Fe(II)(ProDTC)2 was reported to be (1.1 0.3)×108 M-1 s-1[Caramia, et al. 1998].

$$\text{Fe(II)(DTC)}\_2 \blackheadrightarrow \text{Fe(II)(ProDTC)}\_2 + \text{NO} \twoheadrightarrow \text{NO-Fe(II)(DTC)}\_2 \tag{6}$$

Recently, attempts have been made to overcome the limitations of EPR-based imaging with low resolution and large sample size by employing new approaches of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques. Several paramagnetic metal complexes have been widely used as contrast agents in magnetic resonance imaging (MRI) due to their ability to enhance the NMR relaxation of neighboring protons [von Bohlen, et al. 2003]. Paramagnetic NO-Fe-DTC complex has been utilized to visualize NO generated in living rats with septic shock [Fichtlscherer, et al. 2000, Fujii, et al. 1999]. Paramagnetic nitrosyl iron complex showed contrast properties to enhance the signal intensity of SNP-perfused rat liver in MRI [Fichtlscherer, et al. 2000, Fujii, et al. 1999]. Thus, a paramagnetic nitrosyl iron complex may be potentially useful as a microscopic localizer MRI contrast agent specific for NO accumulation in living organisms. Other protonelectron-double-resonance-imaging (PEDRI) technique was based on the enhancement of proton NMR signal intensity in the presence of radicals through the Overhauser effect Mulsch et al. 1999,Foster, et al.1998]. Rat livers were exposed to SNP as an NO donor exhibited intense PEDRI images [Rast, et al. 2001, Jordan, et al.2004]. PEDRI imaging is a potential tool to study NO in large biological specimens. Still imaging application of MR and EPR spectroscopy using nitric oxide synthase inhibitors, oxygenation or perfusion are in infancy [von Bohlen, et al. 2003, Zhou, et al. 2009, Nagano, et al. 2002, Anbar, 2000]

TR. T2 weighted image is obtained by TR >> T2. Mean intensity values are fitted versus the

T1 weitghted MR contrast due to trapped NO as NO-Fe(II)X spin trap in tissue can be

1/T1[NO-Fe(II)X] = 1/T1(observed) – 1/T\* = r1*i*[NO-Fe(II)X] × concentration (4)

 1/Tobs = 1/T*\**+ r1*i*[NO-Fe(II)X]; *i* = 1, 2... (5) where T1(observed) is long longitudinal relaxation time constant and T1\* is short longitudinal

r1i is longitudinal relaxivity dpends on i= 1, 2, 3 the number of carbons in branch of X contrast agent, its concentration and interaction with NO specific bioactive receptor,

**2.5 Approaches to NO evaluation by Magnetic Resonance Imaging (MRI) techniques**  Two major developments reported in context with NO. First, a search of NO specific stable multimodal MR/EPR sensitive spin trap complex; second, calibrating imaging characteristics of spin trap complex on NMR/MRI/PEDRI techniques. NO is perfused in gaseous phase or remains in ionic phase by redox state in the tissues. Both forms of NO are captured or trapped by Fe(II)-(DTC)2 complexes and show MRI signal change due to reactive nitrosylation (reaction of intracellular NO with Fe(II)(DTC)2 to form NO-Fe(II)(DTC)2 complex) shown in Eq 6. Second-order rate constant of the reaction of NO with

 Fe(II)(DTC)2Fe(II)(ProDTC)2 + NO NO-Fe(II)(DTC)2 (6) Recently, attempts have been made to overcome the limitations of EPR-based imaging with low resolution and large sample size by employing new approaches of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques. Several paramagnetic metal complexes have been widely used as contrast agents in magnetic resonance imaging (MRI) due to their ability to enhance the NMR relaxation of neighboring protons [von Bohlen, et al. 2003]. Paramagnetic NO-Fe-DTC complex has been utilized to visualize NO generated in living rats with septic shock [Fichtlscherer, et al. 2000, Fujii, et al. 1999]. Paramagnetic nitrosyl iron complex showed contrast properties to enhance the signal intensity of SNP-perfused rat liver in MRI [Fichtlscherer, et al. 2000, Fujii, et al. 1999]. Thus, a paramagnetic nitrosyl iron complex may be potentially useful as a microscopic localizer MRI contrast agent specific for NO accumulation in living organisms. Other protonelectron-double-resonance-imaging (PEDRI) technique was based on the enhancement of proton NMR signal intensity in the presence of radicals through the Overhauser effect Mulsch et al. 1999,Foster, et al.1998]. Rat livers were exposed to SNP as an NO donor exhibited intense PEDRI images [Rast, et al. 2001, Jordan, et al.2004]. PEDRI imaging is a potential tool to study NO in large biological specimens. Still imaging application of MR and EPR spectroscopy using nitric oxide synthase inhibitors, oxygenation or perfusion are in

Fe(II)(ProDTC)2 was reported to be (1.1 0.3)×108 M-1 s-1[Caramia, et al. 1998].

infancy [von Bohlen, et al. 2003, Zhou, et al. 2009, Nagano, et al. 2002, Anbar, 2000]

TE (Marquardt Levenberg algorithm).

relaxation time constant in absence of [NO-Fe(II)X],

enzyme, antibody, hormone molecules in tissue.

expressed in Eq 4, 5:

#### **NO-Fe(II)(DTC)2**

Fig. 7. NO sensitive biomarker imaging contrast agents. Example of NO-Fe(II)(DTC)2 is cited to illustrate the signal change during changes in cerebral blood flow and possibility of functional MRI [Caramia, et al. 1998].

Two decades ago, NO activation-induced signal changes in functional MRI (fMRI) was observed [Di Salle, et al. 1997]. Hb–NO was found to produce marked stimulator dose and concentration-dependent NO sensitive fMRI signal intensity changes (increased after aqueous NO solution, nitrite, or dithionite and nitrite added to blood while it decreased on the addition of ascorbic acid) examined by T1-, T2-, and T2 -weighted MRI in venous erythrocytes with possibility of physiological MRI evaluation associated with NO change [Di Salle, et al. 1997]. Transient formation of two paramagnetic species (met Hb and NO-Hb) enhanced the signal intensity, while ascorbate reduced the signal intensity in fMRI experiment on healthy volunteers during standard tasks. Further, infusion of NO precursor, L-arginine increased the cerebral blood volume (measured by MRI) in nonischemic spontaneously hypertensive rats [Foster, et al. 1998]. In other study, administration of an NO donor, isosorbide dinitrate (ISDN) increased both tumor blood flow and partial oxygen pressure in mice implanted with liver tumor in the thigh [Jordan, et al. 2000]. These findings indicate blood flow-independent change in MRI signal produced by different additives or stimulators. These observations open a new perspective on the monitoring of NO and the in vivo speculation of NO effects to test new drugs by using magnetic resonance techniques. Fuji et al. 1999 first time reported *in vivo* MRI of NO using Fe(MGD)2 as NO–Fe(MGD)2 spintrapping technique in a septic shock rat model [Fujii, et al. 1999]. (Fig. 6A). The reduced signal after administration of an iNOS inhibitor confirmed the MRI visibility of NO–

Inhibition of Nitric Oxide Synthase Gene Expression:

MRI technique [Fujii, et al.1999].

**3.2 Emission enhancement mechanism** 

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 135

contrast in the vascular structure, such as the hepatic vein and inferior vena cava, which can be reduced after administration of an iNOS inhibitor.Initially, NO mapping was reported by whole body EPR imaging at poor resolution [Claudette, et al. 2005,Yoshimura, et al. 1996]. Recently, MRI strategies combined with in vivo spin-trapping map ''NO distributions'' within tissues and organs showed much higher spatial resolution using (MGD)2-Fe(II)-NO as a NMR contrast agent in vivo [Fichtlscherer, et al. 2000]. The MR images also provided follow-up of NO generation kinetics at different sites within the organ at much higher spatial resolution than with EPR [Claudette, et al. 2005,Yoshimura, et al. 1996]. NO makes stable complex such as (MGD)2-Fe(II)-NO. In vivo, hemoglobin is normally the natural NO spin-trap such that NO tends to bind with hemoglobin or oxidize the hemoglobin, followed by conversion to nitrosyl-hemoglobin or methemoglobin, both of these complexes are paramagnetic. Authors believe that during brain stimulation, NO is generated in some regions and it is quite possible that (paramagnetic) nitrosyl-hemoglobin and methemoglobin are formed in these active regions in brain. The MRI signal intensity enhancement in these regions results from changes in blood flow rich with these complexes. However, for blood flow–independent effects in MRI, the paramagnetic relaxation from spin-trapped NO might provide a new fMRI contribution in future. To our knowledge, there have been no reports successfully imaging or detecting free radicals such as NO in vivo by NMR or fMRI. Further, both short lifetime and rapid diffusion of NO in time preclude any effective relaxation enhancement mechanisms. NMR spin trapping serves to overcome these problems such as NO short lifetime and its fast diffusion in tissues to some extent. In other classic study on rats, spin relaxation constants of dinitrosyl-iron bound albumin or GSH and mononitrosyl-iron dithiocarbamate MGD complexes showed concentration dependence by

Present state of art in NO detection was developed over last 2 decades using fluorescein cheletropic compounds visualized by emission enhancement and photoinduced electron transfer mechanisms. Fluorescence properties of fluorescein derivatives are controlled by the photoinduced electron transfer (PET) process from the benzoic acid moiety to the fluorophore, the xanthene ring. PET is a widely accepted mechanism for fluorescence quenching in which electron transfer from the PET donor to the excited fluorophore diminishes the fluorescence of the fluorophore. A brief survey of these compounds and their fluorescence mechanisms will catch the attention of readers with the newer developments of NO detection in tissues. EPR (also known as electron spin resonance or ESR) explores the magnetic moment associated with the unpaired electron(s) in free radicals and paramagnetic metal ions [Mader, 1998, Swartz, 2007]. It has been applied for the detection of free radicals and paramagnetic metal ions in various biological conditions [Berliner, et al. 2004]. When an unpaired electron in a paramagnetic molecule is subjected to an external magnetic field, the energy level of the electron splits into two quantum states with different energy levels. Typically, the magnetic field is scanned with a fixed microwave frequency of approximately 9.5 GHz (usually called the X-band). Specimens less than about 100 μL are measurable in an Xband spectrometer. However, larger biological samples (e.g., tissues, organs, and live animals) cannot be measured with a conventional X-band spectrometer due to the high dielectric loss of water at such frequencies and the small size of the EPR cavity resonator.

Fe(MGD)2 complex especially in inflammation which has high levels of NO. Now many MRI visible nitrosyl-iron complexes are known for MRI mapping of NO using a conventional 1.5 T MR scanner. The principle is that after exposure to NO in tissues Fe(MGD)2 imaging agents form nitrosyl-iron complexes, which shorten the T1 and T2 relaxation time in a concentration-dependent manner [Hong, et al. 2009]. Mainly, four major techniques are reported in quest of developing new NO sensitive MRI contrast agents. First, unpaired electrons of the nitrosyl–iron complexes can enter into dynamic nuclear polarization with water protons, a technique called proton-electron-double-resonance imaging (PEDRI). Second, superparamagnetic iron oxide (SPIO) or ferumoxides dephase T2 weighted MRI signal dependent on SPIO: NO donor ratios indicating inverse relation of contrast enhancement by SPIO with increasing levels of NO in tissue. The MRI signal change was due to reduction of ferric to ferrous iron with result of decrease in paramagnetic relaxation of water protons [Kojima, et al.2001]. Third, technical development emerged as irreversible paramagnetic chemical exchange saturation transfer (PARACEST) based MRI contrast agents in imaging [Liu, et al. 2007, Zhang, et al. 2003, Wu, et al. 2008]. A PARACEST MRI agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) orthoaminoanilide (Yb-DO3A-OAA), was developed for NO detection (Fig. 6B) [9]. Fourth, NO and O2 combine by irreversible covalent bond that causes a quick disappearance of the PARACEST effect captured in the MR images. PARACEST MRI is fast data acquisition (in few minutes) vs SPIO based imaging is slow (in few hours). PARACEST effect has poor sensitivity (only detects millimolar concentrations of NO).

#### **2.6 Approach of real-time NO synthase inhibition as blood oxygenation dependent MRI signal**

Nitric oxide synthase inhibition (NOSi) in humans by blood oxygenation level-dependent (BOLD) MRI as reduced response to NOSi was reported using BOLD MRI to compare changes in R2\* to direct measures of renal medullary oxygen levels and blood flow using Nnitro-L-arginine methyl ester invasive probes (OxyLite/OxyFlo) to examine for the first time the effect of NOSi on intrarenal oxygenation in humans [Li, et al. 2009, Di Salle, et al. 1997]. Authors showed that NOSi decreased medullary pO2 and blood flow in a dose-dependent manner, and BOLD MRI showed an increase in medullary R2\* consistent with the invasive pO2 measurements.

Other alternate approach of positron emission tomography (PET) was proposed in recent study using nitric oxide imaging probe prepared from [18F] 6-(2-fluoropropyl)-4-methylpyridin-2-amine by substitution at position-6 of substituted 2-amino-4-methylpyridine with favorable properties as a PET tracer to image iNOS activation/expression with PET of animal lungs [Zhou, et al. 2009].

#### **3. Major developments in NO bioimaging probes**

#### **3.1 Magnetic resonance relaxation enhancement mechanism**

In 1999, the first in vivo MRI of NO was reported using the spintrapping technique in a septic shock rat model [Fujii, et al. 1999]. NO was trapped by Fe(MGD)2 in vivo, which can be visualized by MRI. The NO–Fe(MGD)2 complex displayed significantly enhanced

Fe(MGD)2 complex especially in inflammation which has high levels of NO. Now many MRI visible nitrosyl-iron complexes are known for MRI mapping of NO using a conventional 1.5 T MR scanner. The principle is that after exposure to NO in tissues Fe(MGD)2 imaging agents form nitrosyl-iron complexes, which shorten the T1 and T2 relaxation time in a concentration-dependent manner [Hong, et al. 2009]. Mainly, four major techniques are reported in quest of developing new NO sensitive MRI contrast agents. First, unpaired electrons of the nitrosyl–iron complexes can enter into dynamic nuclear polarization with water protons, a technique called proton-electron-double-resonance imaging (PEDRI). Second, superparamagnetic iron oxide (SPIO) or ferumoxides dephase T2 weighted MRI signal dependent on SPIO: NO donor ratios indicating inverse relation of contrast enhancement by SPIO with increasing levels of NO in tissue. The MRI signal change was due to reduction of ferric to ferrous iron with result of decrease in paramagnetic relaxation of water protons [Kojima, et al.2001]. Third, technical development emerged as irreversible paramagnetic chemical exchange saturation transfer (PARACEST) based MRI contrast agents in imaging [Liu, et al. 2007, Zhang, et al. 2003, Wu, et al. 2008]. A PARACEST MRI agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) orthoaminoanilide (Yb-DO3A-OAA), was developed for NO detection (Fig. 6B) [9]. Fourth, NO and O2 combine by irreversible covalent bond that causes a quick disappearance of the PARACEST effect captured in the MR images. PARACEST MRI is fast data acquisition (in few minutes) vs SPIO based imaging is slow (in few hours). PARACEST effect has poor

**2.6 Approach of real-time NO synthase inhibition as blood oxygenation dependent** 

Nitric oxide synthase inhibition (NOSi) in humans by blood oxygenation level-dependent (BOLD) MRI as reduced response to NOSi was reported using BOLD MRI to compare changes in R2\* to direct measures of renal medullary oxygen levels and blood flow using Nnitro-L-arginine methyl ester invasive probes (OxyLite/OxyFlo) to examine for the first time the effect of NOSi on intrarenal oxygenation in humans [Li, et al. 2009, Di Salle, et al. 1997]. Authors showed that NOSi decreased medullary pO2 and blood flow in a dose-dependent manner, and BOLD MRI showed an increase in medullary R2\* consistent with the invasive

Other alternate approach of positron emission tomography (PET) was proposed in recent study using nitric oxide imaging probe prepared from [18F] 6-(2-fluoropropyl)-4-methylpyridin-2-amine by substitution at position-6 of substituted 2-amino-4-methylpyridine with favorable properties as a PET tracer to image iNOS activation/expression with PET of

In 1999, the first in vivo MRI of NO was reported using the spintrapping technique in a septic shock rat model [Fujii, et al. 1999]. NO was trapped by Fe(MGD)2 in vivo, which can be visualized by MRI. The NO–Fe(MGD)2 complex displayed significantly enhanced

sensitivity (only detects millimolar concentrations of NO).

**3. Major developments in NO bioimaging probes** 

**3.1 Magnetic resonance relaxation enhancement mechanism** 

**MRI signal** 

pO2 measurements.

animal lungs [Zhou, et al. 2009].

contrast in the vascular structure, such as the hepatic vein and inferior vena cava, which can be reduced after administration of an iNOS inhibitor.Initially, NO mapping was reported by whole body EPR imaging at poor resolution [Claudette, et al. 2005,Yoshimura, et al. 1996]. Recently, MRI strategies combined with in vivo spin-trapping map ''NO distributions'' within tissues and organs showed much higher spatial resolution using (MGD)2-Fe(II)-NO as a NMR contrast agent in vivo [Fichtlscherer, et al. 2000]. The MR images also provided follow-up of NO generation kinetics at different sites within the organ at much higher spatial resolution than with EPR [Claudette, et al. 2005,Yoshimura, et al. 1996]. NO makes stable complex such as (MGD)2-Fe(II)-NO. In vivo, hemoglobin is normally the natural NO spin-trap such that NO tends to bind with hemoglobin or oxidize the hemoglobin, followed by conversion to nitrosyl-hemoglobin or methemoglobin, both of these complexes are paramagnetic. Authors believe that during brain stimulation, NO is generated in some regions and it is quite possible that (paramagnetic) nitrosyl-hemoglobin and methemoglobin are formed in these active regions in brain. The MRI signal intensity enhancement in these regions results from changes in blood flow rich with these complexes. However, for blood flow–independent effects in MRI, the paramagnetic relaxation from spin-trapped NO might provide a new fMRI contribution in future. To our knowledge, there have been no reports successfully imaging or detecting free radicals such as NO in vivo by NMR or fMRI. Further, both short lifetime and rapid diffusion of NO in time preclude any effective relaxation enhancement mechanisms. NMR spin trapping serves to overcome these problems such as NO short lifetime and its fast diffusion in tissues to some extent. In other classic study on rats, spin relaxation constants of dinitrosyl-iron bound albumin or GSH and mononitrosyl-iron dithiocarbamate MGD complexes showed concentration dependence by MRI technique [Fujii, et al.1999].

#### **3.2 Emission enhancement mechanism**

Present state of art in NO detection was developed over last 2 decades using fluorescein cheletropic compounds visualized by emission enhancement and photoinduced electron transfer mechanisms. Fluorescence properties of fluorescein derivatives are controlled by the photoinduced electron transfer (PET) process from the benzoic acid moiety to the fluorophore, the xanthene ring. PET is a widely accepted mechanism for fluorescence quenching in which electron transfer from the PET donor to the excited fluorophore diminishes the fluorescence of the fluorophore. A brief survey of these compounds and their fluorescence mechanisms will catch the attention of readers with the newer developments of NO detection in tissues. EPR (also known as electron spin resonance or ESR) explores the magnetic moment associated with the unpaired electron(s) in free radicals and paramagnetic metal ions [Mader, 1998, Swartz, 2007]. It has been applied for the detection of free radicals and paramagnetic metal ions in various biological conditions [Berliner, et al. 2004]. When an unpaired electron in a paramagnetic molecule is subjected to an external magnetic field, the energy level of the electron splits into two quantum states with different energy levels. Typically, the magnetic field is scanned with a fixed microwave frequency of approximately 9.5 GHz (usually called the X-band). Specimens less than about 100 μL are measurable in an Xband spectrometer. However, larger biological samples (e.g., tissues, organs, and live animals) cannot be measured with a conventional X-band spectrometer due to the high dielectric loss of water at such frequencies and the small size of the EPR cavity resonator.

Inhibition of Nitric Oxide Synthase Gene Expression:

fluorescent probes in NO bioimaging.

Fig. 8. Representative spin-trapping agents for EPR imaging of NO.

Fe(MGD)2 or Fe(DETC)2 have also been tested for EPR imaging to map the spatial distribution of NO generation in the ischemic myocardium [Kuppusamy, et al. 1996]. Subsequently, Fe(MGD)2 was used in both EPR and MR imaging in rats with sepsis shock [Fujii, et al.1999]. It was reported that Fe(MGD)2 can reduce nitrite to NO at physiological pH, which may cause some detection Inaccuracy as shown in Figure 8, although the rate

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 137

AX is almost nonfluorescent [Nagano, et al. 2002]. Thus, a small change in the size of conjugated aromatics, namely, from naphthalene to anthracene, causes a great alteration of the fluorescence properties. Authors compared the HOMO levels of the benzoic acid moieties with xanthine ring and naphthoic acid. Napthoic acid moiety is present in highly fluorescent fluorescein and NX. The HOMO levels of benzoic acid moiety were lower than that of the xanthene ring, while the HOMO level of anthracenecarboxylic acid moiety in fluorescent AX, was higher than that of the xanthene. These results were consistent with the idea that a PET process controls the fluorescence properties of fluorescein derivatives and that these properties can be predicted from the HOMO level of the benzoic acid moiety, with a threshold at around -8.9 eV. This in turn provides a basis for developing novel fluorescence probes with fluorescein-derived structure. Still much remains to justify the

EPR spectroscopy at lower frequencies, the L-band (0.4–1.6 GHz) or S-band(1.6–4 GHz), can be used for in vivo EPR imaging. EPR imaging is generally considered to be the most effective technique for noninvasive observation of the spatial distribution of free radicals [Kleschyov, et al. 2007]. Direct EPR detection of endogenous free radicals in biological samples requires that the radical of interest is present at a concentration higher than the practical detection limit (0.1–0.01 μM) and has a relatively long lifetime. Although such requirement can be met at cryogenic temperature, in vivo detection is almost impossible since most radicals do not have a sufficiently long lifetime. Therefore, spin-trapping techniques have been developed in which the spin traps can react with unstable free radicals to form a relatively stable adduct [Berliner, et al. 2004, Davie, et al. 2004]. The formation of the long lived adduct results in accumulation of steady-state free radicals which can be detected readily by EPR spectroscopy.

In principle, the fluorescence properties of fluorescein derivatives are controlled by the photoinduced electron transfer (PET) process from the benzoic acid moiety to the fluorophore, the xanthene ring. PET is a widely accepted alternate mechanism for fluorescence quenching in which electron transfer from the PET donor to the excited fluorophore diminishes the fluorescence of the fluorophore. The fluorescein structure has two parts confirmed by X-ray analysis, i.e., the benzoic acid moiety as the PET donor and the xanthene ring shown as the fluorophore in previous study [Liu, et al. 2007]. The study showed that only small alterations in absorbance were observed among fluorescein and its derivatives. The dihedral angle () between the benzoic acid moiety and the xanthene ring was almost 90o. It also suggested that there is little ground-state interaction measured by HOMO energy levels between these two parts.

The relationship of HOMO energy levels and () angle of fluorescent compounds predict the fluorescence mechanism of benzoic acid moiety in the compounds. If the HOMO energy level of the benzoic acid moiety is high enough for electron transfer to the excited xanthene ring, the value will be small. In other words, fluorescein derivatives with a high value must have benzoic moieties with low HOMO energy levels. The HOMO energy levels of 3 aminobenzoic acid, 3-benzamidobenzoic acid, 3,4-diaminobenzenecarboxylic acid (DAB-COOH), and benzotriazole-5-carboxylic acid (BT-COOH), which are the benzoic moieties of aminofluorescein, benzamidofluorescein, DAF-2, and DAF-2T, respectively were estimated by semi-empirical (PM3) calculations. DAB-COOH and aminobenzoic acid, which are the benzoic acid moieties of weakly fluorescent fluorescein derivatives, have higher HOMO levels than BT-COOH and amidobenzoic acid. These results were consistent with the PET mechanism.

Further, to confirm this mechanism 9-[2-(3-carboxy)-naphthyl]-6-hydroxy-3H-xanthen-3-one (NX) and 9-[2-(3-carboxy)anthryl]-6-hydroxy-3H-xanthen-3-one (AX) were synthesized [Nagano, et al. 2002]. It is well-known that expanding the size of aromatic conjugates makes their HOMO levels higher, similar with by introducing electron-donating substituents. Thus, naphthoic acid and anthracene-2-carboxylic acid would have higher HOMO levels than benzoic acid, and therefore, the fluorescence properties of NX and AX would differ from those of fluorescein.. The absorbance maximum (Exmax) and emission maximum (Emmax) of NX and AX were closer and not much altered among these fluorescein derivatives. However, the values were greatly altered; NX is highly fluorescent, whereas

EPR spectroscopy at lower frequencies, the L-band (0.4–1.6 GHz) or S-band(1.6–4 GHz), can be used for in vivo EPR imaging. EPR imaging is generally considered to be the most effective technique for noninvasive observation of the spatial distribution of free radicals [Kleschyov, et al. 2007]. Direct EPR detection of endogenous free radicals in biological samples requires that the radical of interest is present at a concentration higher than the practical detection limit (0.1–0.01 μM) and has a relatively long lifetime. Although such requirement can be met at cryogenic temperature, in vivo detection is almost impossible since most radicals do not have a sufficiently long lifetime. Therefore, spin-trapping techniques have been developed in which the spin traps can react with unstable free radicals to form a relatively stable adduct [Berliner, et al. 2004, Davie, et al. 2004]. The formation of the long lived adduct results in accumulation of steady-state free radicals which can be

In principle, the fluorescence properties of fluorescein derivatives are controlled by the photoinduced electron transfer (PET) process from the benzoic acid moiety to the fluorophore, the xanthene ring. PET is a widely accepted alternate mechanism for fluorescence quenching in which electron transfer from the PET donor to the excited fluorophore diminishes the fluorescence of the fluorophore. The fluorescein structure has two parts confirmed by X-ray analysis, i.e., the benzoic acid moiety as the PET donor and the xanthene ring shown as the fluorophore in previous study [Liu, et al. 2007]. The study showed that only small alterations in absorbance were observed among fluorescein and its derivatives. The dihedral angle () between the benzoic acid moiety and the xanthene ring was almost 90o. It also suggested that there is little ground-state interaction measured by

The relationship of HOMO energy levels and () angle of fluorescent compounds predict the fluorescence mechanism of benzoic acid moiety in the compounds. If the HOMO energy level of the benzoic acid moiety is high enough for electron transfer to the excited xanthene ring, the value will be small. In other words, fluorescein derivatives with a high value must have benzoic moieties with low HOMO energy levels. The HOMO energy levels of 3 aminobenzoic acid, 3-benzamidobenzoic acid, 3,4-diaminobenzenecarboxylic acid (DAB-COOH), and benzotriazole-5-carboxylic acid (BT-COOH), which are the benzoic moieties of aminofluorescein, benzamidofluorescein, DAF-2, and DAF-2T, respectively were estimated by semi-empirical (PM3) calculations. DAB-COOH and aminobenzoic acid, which are the benzoic acid moieties of weakly fluorescent fluorescein derivatives, have higher HOMO levels than BT-COOH and amidobenzoic acid. These results were consistent with the PET

Further, to confirm this mechanism 9-[2-(3-carboxy)-naphthyl]-6-hydroxy-3H-xanthen-3-one (NX) and 9-[2-(3-carboxy)anthryl]-6-hydroxy-3H-xanthen-3-one (AX) were synthesized [Nagano, et al. 2002]. It is well-known that expanding the size of aromatic conjugates makes their HOMO levels higher, similar with by introducing electron-donating substituents. Thus, naphthoic acid and anthracene-2-carboxylic acid would have higher HOMO levels than benzoic acid, and therefore, the fluorescence properties of NX and AX would differ from those of fluorescein.. The absorbance maximum (Exmax) and emission maximum (Emmax) of NX and AX were closer and not much altered among these fluorescein derivatives. However, the values were greatly altered; NX is highly fluorescent, whereas

detected readily by EPR spectroscopy.

HOMO energy levels between these two parts.

mechanism.

AX is almost nonfluorescent [Nagano, et al. 2002]. Thus, a small change in the size of conjugated aromatics, namely, from naphthalene to anthracene, causes a great alteration of the fluorescence properties. Authors compared the HOMO levels of the benzoic acid moieties with xanthine ring and naphthoic acid. Napthoic acid moiety is present in highly fluorescent fluorescein and NX. The HOMO levels of benzoic acid moiety were lower than that of the xanthene ring, while the HOMO level of anthracenecarboxylic acid moiety in fluorescent AX, was higher than that of the xanthene. These results were consistent with the idea that a PET process controls the fluorescence properties of fluorescein derivatives and that these properties can be predicted from the HOMO level of the benzoic acid moiety, with a threshold at around -8.9 eV. This in turn provides a basis for developing novel fluorescence probes with fluorescein-derived structure. Still much remains to justify the fluorescent probes in NO bioimaging.

Fig. 8. Representative spin-trapping agents for EPR imaging of NO.

Fe(MGD)2 or Fe(DETC)2 have also been tested for EPR imaging to map the spatial distribution of NO generation in the ischemic myocardium [Kuppusamy, et al. 1996]. Subsequently, Fe(MGD)2 was used in both EPR and MR imaging in rats with sepsis shock [Fujii, et al.1999]. It was reported that Fe(MGD)2 can reduce nitrite to NO at physiological pH, which may cause some detection Inaccuracy as shown in Figure 8, although the rate

Inhibition of Nitric Oxide Synthase Gene Expression:

**Fluorescence imaging of NO**:

similar to that of DAF-2T.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 139

In following section, we will summarize the progress to date on multimodality imaging of NO and NOS expression and address future research directions and obstacles for NO/NOS imaging using biosensors as outlined in Figure 9. For interested readers, basic methods of

2,7-dichlorofluorescin was introduced for NO detection. When it is oxidized by NO, a fluorescent compound (dichlorofluorescein) is formed. Besides a relatively poor detection sensitivity (~ 10 μM of NO), 2,7-dichlorofluorescein also has very low specificity for NO. Later several iron (II) complexes, fluorescently labeled cytochrome c (a hemoprotein that binds NO selectively), cobalt complexes and fluorescent NO cheletropic traps (FNOCTs) were introduced. The iron(II) complex of quinoline pendant cyclam has fluorescence emission at 460 nm, which can be quenched effectively by NO from NO releasing agents. 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) labeled with acridine and Fe(II)-(N-(dithiocarboxy) sarcosine)2 complex (Fe(DTCS)2) (Fig.5), was developed. The addition of an NO-releasing reagent causes a decrease in the

NO detection and imaging are given in Appendix in then end of this chapter.

fluorescence signal due to the irreversible binding of NO to the Fe(II).

 DAFCs are the most successful and extensively investigated agents for NO imaging. In vivo imaging of NO in whole living vertebrate with the DAF compounds was reported, where diaminofluorophore 4-amino-5-methylamino-2′-7′-difluorofluorescein diacetate (DAF-FM-DA) was used to detect NO production sites in the zebrafish Danio rerio. The specificity of the fluorescent signal was confirmed by a decrease in animals exposed to a NO scavenger or a NOS inhibitor, as well as an increase in the presence of a NO donor. Local changes in NO production in response to stressful conditions could also be imaged by this agent, suggesting that DAF-FM-DA can be used to monitor changes in NO production which may have future applications in drug screening and molecular pharmacology. Best example of NO imaging in vivo with the DAF compound in zebrafish is quite transparent. The major limitation of the DAF-2 compound is that it is not specific to NO because it can also react with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles

 Similar to DAF and DAR, rhodamine fluorophore was developed and it was termed "the DAR". To image NO in living cells, DARs should be membrane permeable. Therefore, an ethyl group was introduced into the carboxyl group of DAR-1 (4,5-diamino-N,N,N′,N′ tetra ethyl rhodamine), anticipating that the ethyl ester would be hydrolyzed by cytosolic

 New class of DAQ compounds of DAFC family called diaminocyanines (DACs) were prepared with a tricarbocyanine as the NIR fluorophore and o-phenylenediamine as the NO sensitive fluorescence modulator. It was reported that these compounds can react faster with NO than the DAFs and the reaction efficiency was at least 50 times higher than that of DAF-2. Although it was demonstrated that NO can be imaged in isolated rat

esterases after permeation through the cell membrane [Kojima et al. 2001].

kidneys, in vivo imaging of NO using these compounds has not been reported. A new class of difluoroboradiaza-s-indacene-based agents have been widely used in the detection of NO in cells or tissues [Huang, et al. 2002a]. One of these compounds, 1,3,5,7-tetramethyl-8-(4′-aminophenyl-N-(2′′-amino)-phenzyl)-difluoroboradiaza-sindacene (TMAPABODIPY), was shown to have high photostability, high sensitivity (detection limit is 5 nMof NO), and no pH dependency over a wide pH range (see

constant is quite slow (1–5 M−2 s−1) [Tsuchiya, et al. 2000]. Fe(DETC)2 has been used for imaging vascular NO production in rabbits [Kleschyov, et al. 2000], animal brain injury [Ziaja, et al. 2007], vascular disease [Khoo, et al. 2004], and even in plants [Xu, et al. 2004, Vanin, et al. 2007]. Heme proteins, such as myoglobin, cytochrome c, and catalase, have been used as NO-trapping agents in various biological specimens[Kleschyov, et al. 2000]. Although the EPR signals of heme–NO species can be observed in tissues exposed to elevated levels of NO, it is almost impossible to discriminate between the individual nitrosylated heme proteins due to overlapping of the signal. Generally, the dominant heme– NO EPR signal belongs to the major heme protein expressed in the tissue (e.g., myoglobin in the heart [Konorev, et al. 1996]). The most frequently used heme protein in EPR research is hemoglobin (Hb) [Kosaka, et al. 1994], which can interact with NO to generate several paramagnetic Hb–NO derivatives detectable by EPR spectroscopy [Picknova, et al. 2005]. Different preparations of deoxy-Hb have been used for NO trapping in isolated cells and organelles [Kozlov, et al. 1996].

#### **3.3 NO imaging and fluorescent dyes**

Several fluorescence indicators for direct detection of NO have been generated, which might be useful for in vitro or in vivo NO-imaging. These substances interact with NO to form a fluorescent complex. Several fluorescent dyes have been developed, which can be used for NO bioimaging. However, several of these promising fluorescent dyes have been found not to be very specific such as 2′ ,7′ -dichlorofluorescein (DCF) or to be cytotoxic such as 2,3 diaminonaphthalene (DAN). Of specific mention, iron (II) N-(dithiocarboxyl) sarcosine(Fe(DTCS)2; Fig. 5) trapping agents within 1 h after injection of the spin trap generated stable signal which indicated that the formation of the steady-state free radical had reached equilibrium. The same trapping agent was also tested for NO imaging in lipopolysaccharide-treated mice [Quaresima, et al. 1996]. Subsequently, EPR measurements and EPR-CT (CT denotes computed tomography) imaging with Fe(DTCS)2 were carried out in live mice [Yokoyama, et al. 1997]. The use of Fe(DTCS)2 resulted in a clear EPR-CT image showing high intensity areas in the ventral regions, while other NO-bound iron complexes such as Nmethyl-D-glucamine dithiocarbamate (Fe (MGD)2; Fig. 5) or N,Ndiethyldithiocarbamate (Fe(DETC)2) gave much lower signal-to-noise ratios. Several compounds such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Fig. 5), α-phenyl-N-tertbutylnitrone(PBN), or 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), NO cheletopic traps (NOCTs), aci anions of nitroalkanes, iron (II)–dithiocarbamate(DTC) complex and(DAFs) are fluorescein derivatives. DAFs themselves scarcely fluoresce while DAF-Ts are strong fluorescent compounds. However, the mechanisms accounting for the diminution of fluorescence in DAFs and fluorescence enhancement in DAF-Ts remain unclear.Other fluorescent Fe(DTCS)2 and Cobalt based dyes have been tested in some cell culture systems, but these substances have not been tested in in vivo or in vitro acute brain slice preparations or tissues [von Bohlen, et al. 2003, Zhou, et al. 2009]. Until now, several other fluorescent dyes have been promising candidates for the evaluation of the temporal and spatial aspects of NO generation and distribution even under microscopic observation, including diaminofluoresceins (DAFC), diaminorhodamines (DAR), diaminoanthraquinone (DAQ), fluorescent resonance energy transfer (FRET) and the "fluorescent nitric oxide cheletropic traps" as shown in Table 3 [von Bohlen, et al. 2003, Zhou, et al. 2009].

In following section, we will summarize the progress to date on multimodality imaging of NO and NOS expression and address future research directions and obstacles for NO/NOS imaging using biosensors as outlined in Figure 9. For interested readers, basic methods of NO detection and imaging are given in Appendix in then end of this chapter.

#### **Fluorescence imaging of NO**:

138 Enzyme Inhibition and Bioapplications

constant is quite slow (1–5 M−2 s−1) [Tsuchiya, et al. 2000]. Fe(DETC)2 has been used for imaging vascular NO production in rabbits [Kleschyov, et al. 2000], animal brain injury [Ziaja, et al. 2007], vascular disease [Khoo, et al. 2004], and even in plants [Xu, et al. 2004, Vanin, et al. 2007]. Heme proteins, such as myoglobin, cytochrome c, and catalase, have been used as NO-trapping agents in various biological specimens[Kleschyov, et al. 2000]. Although the EPR signals of heme–NO species can be observed in tissues exposed to elevated levels of NO, it is almost impossible to discriminate between the individual nitrosylated heme proteins due to overlapping of the signal. Generally, the dominant heme– NO EPR signal belongs to the major heme protein expressed in the tissue (e.g., myoglobin in the heart [Konorev, et al. 1996]). The most frequently used heme protein in EPR research is hemoglobin (Hb) [Kosaka, et al. 1994], which can interact with NO to generate several paramagnetic Hb–NO derivatives detectable by EPR spectroscopy [Picknova, et al. 2005]. Different preparations of deoxy-Hb have been used for NO trapping in isolated cells and

Several fluorescence indicators for direct detection of NO have been generated, which might be useful for in vitro or in vivo NO-imaging. These substances interact with NO to form a fluorescent complex. Several fluorescent dyes have been developed, which can be used for NO bioimaging. However, several of these promising fluorescent dyes have been found not

diaminonaphthalene (DAN). Of specific mention, iron (II) N-(dithiocarboxyl) sarcosine(Fe(DTCS)2; Fig. 5) trapping agents within 1 h after injection of the spin trap generated stable signal which indicated that the formation of the steady-state free radical had reached equilibrium. The same trapping agent was also tested for NO imaging in lipopolysaccharide-treated mice [Quaresima, et al. 1996]. Subsequently, EPR measurements and EPR-CT (CT denotes computed tomography) imaging with Fe(DTCS)2 were carried out in live mice [Yokoyama, et al. 1997]. The use of Fe(DTCS)2 resulted in a clear EPR-CT image showing high intensity areas in the ventral regions, while other NO-bound iron complexes such as Nmethyl-D-glucamine dithiocarbamate (Fe (MGD)2; Fig. 5) or N,Ndiethyldithiocarbamate (Fe(DETC)2) gave much lower signal-to-noise ratios. Several compounds such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Fig. 5), α-phenyl-N-tertbutylnitrone(PBN), or 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), NO cheletopic traps (NOCTs), aci anions of nitroalkanes, iron (II)–dithiocarbamate(DTC) complex and(DAFs) are fluorescein derivatives. DAFs themselves scarcely fluoresce while DAF-Ts are strong fluorescent compounds. However, the mechanisms accounting for the diminution of fluorescence in DAFs and fluorescence enhancement in DAF-Ts remain unclear.Other fluorescent Fe(DTCS)2 and Cobalt based dyes have been tested in some cell culture systems, but these substances have not been tested in in vivo or in vitro acute brain slice preparations or tissues [von Bohlen, et al. 2003, Zhou, et al. 2009]. Until now, several other fluorescent dyes have been promising candidates for the evaluation of the temporal and spatial aspects of NO generation and distribution even under microscopic observation, including diaminofluoresceins (DAFC), diaminorhodamines (DAR), diaminoanthraquinone (DAQ), fluorescent resonance energy transfer (FRET) and the "fluorescent nitric oxide cheletropic


organelles [Kozlov, et al. 1996].

to be very specific such as 2′

**3.3 NO imaging and fluorescent dyes** 

,7′

traps" as shown in Table 3 [von Bohlen, et al. 2003, Zhou, et al. 2009].

2,7-dichlorofluorescin was introduced for NO detection. When it is oxidized by NO, a fluorescent compound (dichlorofluorescein) is formed. Besides a relatively poor detection sensitivity (~ 10 μM of NO), 2,7-dichlorofluorescein also has very low specificity for NO. Later several iron (II) complexes, fluorescently labeled cytochrome c (a hemoprotein that binds NO selectively), cobalt complexes and fluorescent NO cheletropic traps (FNOCTs) were introduced. The iron(II) complex of quinoline pendant cyclam has fluorescence emission at 460 nm, which can be quenched effectively by NO from NO releasing agents. 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) labeled with acridine and Fe(II)-(N-(dithiocarboxy) sarcosine)2 complex (Fe(DTCS)2) (Fig.5), was developed. The addition of an NO-releasing reagent causes a decrease in the fluorescence signal due to the irreversible binding of NO to the Fe(II).


Inhibition of Nitric Oxide Synthase Gene Expression:

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 141

Fig. 9. Imaging NO with diamino-aromatic fluorescent compounds. (A) The general reaction

(B) Fluorescence image of live zebrafish larvae loaded with DAF-FM-DA. Blue arrow, heart; orange arrow, the cleithrum; purple arrow, the notochord; yellow arrow, the caudal fin. (C) Distribution of NO in B16F10 tumors grown in the cranial window in mouse. Left: microangiography using tetramethylrhodamine-dextran; middle: representative

that leads to NO detection by fluorescence with this class of agents.


Figure 9). Subsequently, a similar compound, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8- (3′,4′diaminophenyldifluoroboradiaza-s-indacene (TMDCDABODIPY), was applied for real-time imaging of NO in cells [Huang, et al. 2002b].

Table 3. Properties of fluorescent NO-indicators


Emission (color) ~520 nm (green) ~515 nm(green) >580 nm(red) 460 nm(blue) Excitation ~460 nm ~495 nm ~550 nm ~520 nm Reacts with ROS Generates No -- No

Neurotoxic -- No -- No

 In copper-based fluorescent compounds copper (II) is coordinated with certain fluorophores, the base-catalyzed reaction with NO leads to the reduction of the metal center and the intramolecular nitrosylation of a secondary amine. Based on this mechanism, a series of fluophore coordinated copper (II) complexes were designed for NO imaging [Lim, 2007]. Reduction of the Cu (II) core by NO to Cu(I) with nitrosation of the fluorescent ligand is accompanied by bright fluorescence emission. Any nitrogen/oxygen reactive species or ascorbic acid do not cause any significant fluorescence alteration in Cu(II)-based agents. Cu(II) complexes are quite successful in NO imaging in cell culture but they have not been tested in vivo likely due to the suboptimal emission wavelength, the potential toxicity, and the poor in vivo stability of the Cu(II) complex. Recently, several benzimidazole derivatives have been tested in vivo for NO imaging [Quyang, et al. 2008]. The fluorescent 5′-chloro-2-(2′-hydroxyphenyl)-1Hnaphtho[2,3-d]imidazol and 4-methoxy-2-(1H-naphtho [2,3-d]imidazol-2-yl)phenol can coordinate with Cu(II) to form a nonfluorescent coordination compound, which can detect NO production in inflammation with a turn-on fluorescence. NO production was imaged successfully in both activated murine macrophages and an acute severe hepatic injury (ASHI) mouse model. Although these agents represent the first successful attempt of in vivo fluorescence imaging of NO, the actual imaging was performed ex vivo on frozen tissue slices due to the suboptimal fluorescence emission wavelength as shown in Figure 10. Much further optimization of these Cu(II)-based agents will be needed in the future, in particular fluorescence emission in the NIR range and reduction of the toxicity. Imaging NO/NOS with FRET: FRET agents react with NO covalently a irreversible sensors for NO. Further, some of the organic dyes can accumulate in subcellular membranes and emit fluorescence signals in an NO-independent manner which can cause significant background signal and interfere with the detection of NO. To overcome these limitations, a NO-selective sensor with the heme domain of soluble guanylate cyclase (sGC) was developed [Barker, et al. 1999]. The heme domain of the sGC was labeled with a fluorescent dye and domain changes in the fluorescence intensity of the dye based on the sGC heme domain's characteristic binding of NO. It was found that these sensors have fast, linear, and reversible responses to NO unaffected by other radical species. However, the detection limit is only about 10 μM of

real-time imaging of NO in cells [Huang, et al. 2002b].

Table 3. Properties of fluorescent NO-indicators

NO which makes less useful.

Tested in

neurotoxic tissues

Figure 9). Subsequently, a similar compound, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8- (3′,4′diaminophenyldifluoroboradiaza-s-indacene (TMDCDABODIPY), was applied for

MGD DAF DAR DAQ

No Yes No Yes

Fig. 9. Imaging NO with diamino-aromatic fluorescent compounds. (A) The general reaction that leads to NO detection by fluorescence with this class of agents.

(B) Fluorescence image of live zebrafish larvae loaded with DAF-FM-DA. Blue arrow, heart; orange arrow, the cleithrum; purple arrow, the notochord; yellow arrow, the caudal fin. (C) Distribution of NO in B16F10 tumors grown in the cranial window in mouse. Left: microangiography using tetramethylrhodamine-dextran; middle: representative

Inhibition of Nitric Oxide Synthase Gene Expression:

imaging/detection.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 143

Subsequently, a genetically encoded fluorescent indicator for NO was developed based on FRET, which can reversibly detects NO with high sensitivity (detection limit of 0.1 nM) and visualizes the nanomolar dynamics of NO in single living cells on NO induction [Jares-Erijman, et al. 2003]. However, this approach requires genetic encoding and it generates the biologically active molecule cyclic guanosine monophosphate (cGMP), which can induce other cellular responses. The cGMP molecules can bind to both the NO-associated and the No free probe, resulting in an increase in fluorescence. Therefore, the signal generated is a reflection of the intracellular concentration of cGMP rather than that of NO per se. Recently, the use of confocal based spectral imaging with NO-sensitive FRET reporters enabled NO imaging in the vasculature of intact, isolated perfused mouse lung[St Croix, et al. 2008]. Using calibration spectra, it is possible to unambiguously separate the cross-talk between overlapping donor and acceptor emissions. Overall, FRET is a very powerful technique in cell-based experiments but with only very limited potential applications in animal studies as shown in Figure 11.

**3.4 NO bioimaging by bioluminescence and chemiluminescence imaging** 

The major application of chemiluminescence in NO imaging/ detection lies in gas phase or liquid phase measurements[Taha, et al. 2003]. The major chemiluminescence activators for NO imaging include ozone, luminol, and lucigenin. Gas phase chemiluminescence detection of NO is generally based on the reaction of NO with ozone to produce excited-state nitrogen dioxide. When it returns to the normal state, light is emitted at about 600 nm wavelength. Such gas phase detection has been widely used for measuring NO in exhaled breath to detect inflammatory diseases of the lung. Gas phase chemiluminescence detection has very high specificity, as well as extreme sensitivity, which can reach parts per billion (ppb). However, drawbacks include inconvenient instrumentation, high cost, and timeconsuming detection procedure. To overcome these problems, an optimized chemiluminescence detection system for NO was developed. For liquid phase chemiluminescence imaging of NO, currently the luminol/H2O2 detection system is the most useful [Robinson, et al. 1999]. Alkaline luminol can react with NO to generate luminescence. The presence of H2O2 as an activator can enhance the sensitivity by about 20-fold. Two approaches have been used for luminol/H2O2-based imaging of NO. In the first approach, the sample is directly added to the luminol/H2O2 mixture. This strategy can detect very low levels of NO, which is quite useful for visualizing the production of NO from cells or organs, but it lacks NO specificity since luminol can react with many free radical species. In the second strategy, a selective dialysis membrane is placed between the sample and the luminol/H2O2 mixture. This procedure can offer better specificity yet it suffers from poor sensitivity, slow response time, and undesirable NO detection limit (μM level). Besides ozone and luminol, lucigenin is can also be used for NO detection. Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide formation. In particular, lucigenin at a concentration of 5 μM could be used as an indirect probe to estimate basal vascular NO release. Generally, chemiluminescence is a very sensitive method for NO imaging/detection. However, the disadvantages such as low specificity and time-consuming procedure make chemiluminescence not very useful as an independent technique for NO

Several other imaging techniques have been investigated for NO detection. As an important component of molecular imaging, ultrasound is not inherently suitable for direct NO imaging, although it can be used for evaluating the endothelial function or vasodilation

microfluorography captured 1 h after the loading of DAF-2 in tumors; right: The animals were treated with NOS inhibitor. Color bar shows calibration of the fluorescence intensity with known concentrations of DAF- 2T. (D) Left: fluorescent image of endothelial cells with DAR-4M AM at 10 min after the stimulation. Right: an antagonist of the stimuli strongly abolished the fluorescence intensity. (E) Confocal laser fluorescence microscopy image of cells loaded with DAQ and stimulated to produce NO. Left: bright-field; middle: 488 nm excitation; right: 543 nm excitation. (F) Bright-field and fluorescence images of activated PC12 cells loaded with TMDCDABODIPY in the presence of arginine. Adapted from [Huang, et al. 2007a, 2007b].

Fig. 10. Copper-based fluorescence imaging of NO. (A) CuFL detection of NO produced by iNOS in macrophage cells after different time of incubation. Top: fluorescence images; Bottom, bright-field images. (B) Fluorescence images of excised liver slices harvested from live animals after intravenous injection MNIP-Cu. Left: normal mouse liver; center: ASHI liver; right: Kupffer cell-depleted ASHI liver. Adapted from reference [Quyang, et al. 2008].

microfluorography captured 1 h after the loading of DAF-2 in tumors; right: The animals were treated with NOS inhibitor. Color bar shows calibration of the fluorescence intensity with known concentrations of DAF- 2T. (D) Left: fluorescent image of endothelial cells with DAR-4M AM at 10 min after the stimulation. Right: an antagonist of the stimuli strongly abolished the fluorescence intensity. (E) Confocal laser fluorescence microscopy image of cells loaded with DAQ and stimulated to produce NO. Left: bright-field; middle: 488 nm excitation; right: 543 nm excitation. (F) Bright-field and fluorescence images of activated PC12 cells loaded with TMDCDABODIPY in the presence of arginine. Adapted from

Fig. 10. Copper-based fluorescence imaging of NO. (A) CuFL detection of NO produced by iNOS in macrophage cells after different time of incubation. Top: fluorescence images; Bottom, bright-field images. (B) Fluorescence images of excised liver slices harvested from live animals after intravenous injection MNIP-Cu. Left: normal mouse liver; center: ASHI liver; right: Kupffer cell-depleted ASHI liver. Adapted from reference [Quyang, et al. 2008].

[Huang, et al. 2007a, 2007b].

Subsequently, a genetically encoded fluorescent indicator for NO was developed based on FRET, which can reversibly detects NO with high sensitivity (detection limit of 0.1 nM) and visualizes the nanomolar dynamics of NO in single living cells on NO induction [Jares-Erijman, et al. 2003]. However, this approach requires genetic encoding and it generates the biologically active molecule cyclic guanosine monophosphate (cGMP), which can induce other cellular responses. The cGMP molecules can bind to both the NO-associated and the No free probe, resulting in an increase in fluorescence. Therefore, the signal generated is a reflection of the intracellular concentration of cGMP rather than that of NO per se. Recently, the use of confocal based spectral imaging with NO-sensitive FRET reporters enabled NO imaging in the vasculature of intact, isolated perfused mouse lung[St Croix, et al. 2008]. Using calibration spectra, it is possible to unambiguously separate the cross-talk between overlapping donor and acceptor emissions. Overall, FRET is a very powerful technique in cell-based experiments but with only very limited potential applications in animal studies as shown in Figure 11.

#### **3.4 NO bioimaging by bioluminescence and chemiluminescence imaging**

The major application of chemiluminescence in NO imaging/ detection lies in gas phase or liquid phase measurements[Taha, et al. 2003]. The major chemiluminescence activators for NO imaging include ozone, luminol, and lucigenin. Gas phase chemiluminescence detection of NO is generally based on the reaction of NO with ozone to produce excited-state nitrogen dioxide. When it returns to the normal state, light is emitted at about 600 nm wavelength. Such gas phase detection has been widely used for measuring NO in exhaled breath to detect inflammatory diseases of the lung. Gas phase chemiluminescence detection has very high specificity, as well as extreme sensitivity, which can reach parts per billion (ppb). However, drawbacks include inconvenient instrumentation, high cost, and timeconsuming detection procedure. To overcome these problems, an optimized chemiluminescence detection system for NO was developed. For liquid phase chemiluminescence imaging of NO, currently the luminol/H2O2 detection system is the most useful [Robinson, et al. 1999]. Alkaline luminol can react with NO to generate luminescence. The presence of H2O2 as an activator can enhance the sensitivity by about 20-fold. Two approaches have been used for luminol/H2O2-based imaging of NO. In the first approach, the sample is directly added to the luminol/H2O2 mixture. This strategy can detect very low levels of NO, which is quite useful for visualizing the production of NO from cells or organs, but it lacks NO specificity since luminol can react with many free radical species. In the second strategy, a selective dialysis membrane is placed between the sample and the luminol/H2O2 mixture. This procedure can offer better specificity yet it suffers from poor sensitivity, slow response time, and undesirable NO detection limit (μM level). Besides ozone and luminol, lucigenin is can also be used for NO detection. Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide formation. In particular, lucigenin at a concentration of 5 μM could be used as an indirect probe to estimate basal vascular NO release. Generally, chemiluminescence is a very sensitive method for NO imaging/detection. However, the disadvantages such as low specificity and time-consuming procedure make chemiluminescence not very useful as an independent technique for NO imaging/detection.

Several other imaging techniques have been investigated for NO detection. As an important component of molecular imaging, ultrasound is not inherently suitable for direct NO imaging, although it can be used for evaluating the endothelial function or vasodilation

Inhibition of Nitric Oxide Synthase Gene Expression:

developed multimodal techniques in their infancy.

significantly help in this aspect [Cai, et al.2007a, 2007b].

**3.5.1 Optical imaging of NOS expression** 

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 145

in the same subjects [Cai, et al. 2008a, 2008b]. In the clinical setting, molecular imaging can significantly aid in early lesion detection, patient stratification, and treatment monitoring, which can allow for much earlier diagnosis, earlier treatment, better prognosis, and individualized patient management. To date, the multimodal imaging modalities for visualization of NO include optical imaging (fluorescence and chemiluminescence), electron paramagnetic resonance (EPR) imaging, and MRI. Meanwhile, NOS has been mainly investigated using optical and PET imaging. In following section, we describe recently

Imaging enzyme expression and function is quite challenging. Compared to the vast number of literature reports on NO imaging, much less effort has been directed toward NOS imaging. Briefly, the major modalities for NOS imaging include optical imaging, PET, and some other techniques such as electron microscopy. Historically, the study of NOS physiology was constrained by the lack of suitable probes to detect NOS in living cells or animals. In one pioneering study, a fluorescent iNOS inhibitor, pyrimidine imidazole FITC (PIF), was developed and investigated for microscopy of iNOS in living cells [Panda, et al.2005]. It is an isoform-selective molecule with high affinity to iNOS and the binding is essentially irreversible, which allowed for iNOS imaging in many cell types as well as freshly obtained human lung epithelium. This study indicated that fluorescent probes (e.g., PIF) can be valuable for studying iNOS cell biology and understanding the pathophysiology of diseases that involve dysfunctional iNOS expression. In another report, immunofluorescence staining was performed to evaluate nNOS expression on left inferior alveolar nerve (IAN) injury in ferrets [Davies, et al. 2004]. A possible translocation of nNOS protein from the cell body to the site of nerve injury was proposed. Lastly, some ruthenium(II) and rhenium(I)-diimine wires were reported to be capable of binding to iNOS in vitro to generate NIR fluorescence (710 nm emission), which may potentially be useful as a fluorescent imaging probe for iNOS [Dunn, et al. 2005]. The inflammatory modulating effects of iNOS on laser therapy were studied using bioluminescence imaging (BLI) [Moriyama, et al. 2005]. In this study, the efficiency of different wavelengths laser therapy at different wavelengths in modulating iNOS expression was determined using transgenic animals with a luciferase gene driven by the iNOS gene. On induction of inflammation followed by laser treatment, imaging of iNOS expression was performed at various times by measuring the bioluminescence signal. When compared with the nonirradiated animals, a significant increase in the bioluminescence signal was observed after laser irradiation which demonstrated that iNOS expression can be detected by noninvasive BLI. Comparing the two strategies for NOS imaging with optical techniques, many fluorescently labeled NOS inhibitors have charges which makes them not cell membrane permeable. NOS-controlled luciferase expression appears to be a preferable choice, yet much further optimization will be needed in future studies. Generally speaking, the limited penetration of light in tissue renders optical imaging only suitable for a limited number of scenarios (e.g., superficial regions-of-interest and endoscopy-accessible tissues). Another drawback of optical imaging is that it cannot give accurate quantitative analysis of the imaging result. Combination of quantum dot optical and radionuclide-based imaging techniques (e.g., PET) may

caused by NO [Heiss, et al. 2008]. It is worth noting that ultrasound at different frequencies can stimulate NO production in different contexts such as in endothelial cells, osteoblasts, and vascular dilation. A few other techniques such as time-resolved photoion and photoelectron imaging, Hb spectral alteration imaging, laser induced fluorescence detection (LIF), and calcium amplification imaging have also been studied for NO detection. In particular, LIF imaging was able to detect the NO release in a single cell, which makes it a very powerful tool for studying the kinetics of NO release by neuronal cells during neurotransmission. PET imaging of anesthetized dogs with inhaled radiolabeled 13NO has been performed to study the in vivo kinetics of NO [McCarthy, et al. 1996]. In future, molecular imaging may provide more accurate information on NO in intact living systems. EPR-CT [Yokoyama, et al. 1997], EPR-MRI [Berliner, et al. 2004], and EPRchemiluminescence imaging [Nagano, 2002] have all been attempted and much further research/optimization will be needed before any potential clinical investigation can be in place.

#### **3.5 Multimodal bioimaging of NOS gene expression**

Initially NOS was considered a sole enzyme to produce NO. Nitric oxide synthase (NOS) is also under extensive investigation [Crowell et al. 2003]. NO is biosynthesized as three distinct mammalian NOS isoforms shown in section 1. Neuronal NOS (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) are constitutively expressed in neuronal and endothelial cells, respectively and both are referred to as cNOS. Inducible NOS (iNOS or NOS II) is expressed in cells involved in inflammation, such as macrophages and microglias when stimulated by cytokines and/or endotoxins. Although cNOS and iNOS do not have a significant difference in activity (both function through NO), cNOS expression can be stimulated by Ca2+ whereas iNOS activity is Ca2+-independent. Generally, the NO levels produced by cNOS in stimulated endothelial and neuronal cells are much lower (in nanomolar [nM] range) than those generated by iNOS in macrophages (micromolar [μM] range). Aside from NOS synthesis, recent studies have pointed out that nitrite reduction can also serve as a possible source of biologically relevant NO [Gladwin, et al. 2005]. Excessive production of NO is believed to be responsible for various pathophysiologies, while very low levels of NO are needed to maintain normal physiological conditions. Since the production of NO is closely associated with the expression of NOS, blocking NOS expression is a more convenient approach to inhibit the function of NO than intervention through NO itself.

Molecular imaging refers to the characterization of NO/NOS and measurement of biological processes at the molecular level [Massoud, et al. 2003]. It takes advantage of traditional diagnostic imaging techniques and introduces molecular probes to measure the expression of indicative molecular markers at different stages of diseases. Molecular imaging modalities include molecular magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), optical imaging, targeted ultrasound, single photon emission computed tomography (SPECT), and positron emission tomography (PET). Many hybrid systems that combine two or more of these modalities are already commercially available and certain others are under active development. Molecular imaging can give whole body readout in an intact system at the cost of less workload, inexpansive drug development trial, and provide more statistically relevant results since longitudinal studies can be performed in the same subjects [Cai, et al. 2008a, 2008b]. In the clinical setting, molecular imaging can significantly aid in early lesion detection, patient stratification, and treatment monitoring, which can allow for much earlier diagnosis, earlier treatment, better prognosis, and individualized patient management. To date, the multimodal imaging modalities for visualization of NO include optical imaging (fluorescence and chemiluminescence), electron paramagnetic resonance (EPR) imaging, and MRI. Meanwhile, NOS has been mainly investigated using optical and PET imaging. In following section, we describe recently developed multimodal techniques in their infancy.

#### **3.5.1 Optical imaging of NOS expression**

144 Enzyme Inhibition and Bioapplications

caused by NO [Heiss, et al. 2008]. It is worth noting that ultrasound at different frequencies can stimulate NO production in different contexts such as in endothelial cells, osteoblasts, and vascular dilation. A few other techniques such as time-resolved photoion and photoelectron imaging, Hb spectral alteration imaging, laser induced fluorescence detection (LIF), and calcium amplification imaging have also been studied for NO detection. In particular, LIF imaging was able to detect the NO release in a single cell, which makes it a very powerful tool for studying the kinetics of NO release by neuronal cells during neurotransmission. PET imaging of anesthetized dogs with inhaled radiolabeled 13NO has been performed to study the in vivo kinetics of NO [McCarthy, et al. 1996]. In future, molecular imaging may provide more accurate information on NO in intact living systems. EPR-CT [Yokoyama, et al. 1997], EPR-MRI [Berliner, et al. 2004], and EPRchemiluminescence imaging [Nagano, 2002] have all been attempted and much further research/optimization will be needed before any potential clinical investigation can be in

Initially NOS was considered a sole enzyme to produce NO. Nitric oxide synthase (NOS) is also under extensive investigation [Crowell et al. 2003]. NO is biosynthesized as three distinct mammalian NOS isoforms shown in section 1. Neuronal NOS (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) are constitutively expressed in neuronal and endothelial cells, respectively and both are referred to as cNOS. Inducible NOS (iNOS or NOS II) is expressed in cells involved in inflammation, such as macrophages and microglias when stimulated by cytokines and/or endotoxins. Although cNOS and iNOS do not have a significant difference in activity (both function through NO), cNOS expression can be stimulated by Ca2+ whereas iNOS activity is Ca2+-independent. Generally, the NO levels produced by cNOS in stimulated endothelial and neuronal cells are much lower (in nanomolar [nM] range) than those generated by iNOS in macrophages (micromolar [μM] range). Aside from NOS synthesis, recent studies have pointed out that nitrite reduction can also serve as a possible source of biologically relevant NO [Gladwin, et al. 2005]. Excessive production of NO is believed to be responsible for various pathophysiologies, while very low levels of NO are needed to maintain normal physiological conditions. Since the production of NO is closely associated with the expression of NOS, blocking NOS expression is a more convenient approach to inhibit the function of NO than intervention

Molecular imaging refers to the characterization of NO/NOS and measurement of biological processes at the molecular level [Massoud, et al. 2003]. It takes advantage of traditional diagnostic imaging techniques and introduces molecular probes to measure the expression of indicative molecular markers at different stages of diseases. Molecular imaging modalities include molecular magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), optical imaging, targeted ultrasound, single photon emission computed tomography (SPECT), and positron emission tomography (PET). Many hybrid systems that combine two or more of these modalities are already commercially available and certain others are under active development. Molecular imaging can give whole body readout in an intact system at the cost of less workload, inexpansive drug development trial, and provide more statistically relevant results since longitudinal studies can be performed

place.

through NO itself.

**3.5 Multimodal bioimaging of NOS gene expression** 

Imaging enzyme expression and function is quite challenging. Compared to the vast number of literature reports on NO imaging, much less effort has been directed toward NOS imaging. Briefly, the major modalities for NOS imaging include optical imaging, PET, and some other techniques such as electron microscopy. Historically, the study of NOS physiology was constrained by the lack of suitable probes to detect NOS in living cells or animals. In one pioneering study, a fluorescent iNOS inhibitor, pyrimidine imidazole FITC (PIF), was developed and investigated for microscopy of iNOS in living cells [Panda, et al.2005]. It is an isoform-selective molecule with high affinity to iNOS and the binding is essentially irreversible, which allowed for iNOS imaging in many cell types as well as freshly obtained human lung epithelium. This study indicated that fluorescent probes (e.g., PIF) can be valuable for studying iNOS cell biology and understanding the pathophysiology of diseases that involve dysfunctional iNOS expression. In another report, immunofluorescence staining was performed to evaluate nNOS expression on left inferior alveolar nerve (IAN) injury in ferrets [Davies, et al. 2004]. A possible translocation of nNOS protein from the cell body to the site of nerve injury was proposed. Lastly, some ruthenium(II) and rhenium(I)-diimine wires were reported to be capable of binding to iNOS in vitro to generate NIR fluorescence (710 nm emission), which may potentially be useful as a fluorescent imaging probe for iNOS [Dunn, et al. 2005]. The inflammatory modulating effects of iNOS on laser therapy were studied using bioluminescence imaging (BLI) [Moriyama, et al. 2005]. In this study, the efficiency of different wavelengths laser therapy at different wavelengths in modulating iNOS expression was determined using transgenic animals with a luciferase gene driven by the iNOS gene. On induction of inflammation followed by laser treatment, imaging of iNOS expression was performed at various times by measuring the bioluminescence signal. When compared with the nonirradiated animals, a significant increase in the bioluminescence signal was observed after laser irradiation which demonstrated that iNOS expression can be detected by noninvasive BLI. Comparing the two strategies for NOS imaging with optical techniques, many fluorescently labeled NOS inhibitors have charges which makes them not cell membrane permeable. NOS-controlled luciferase expression appears to be a preferable choice, yet much further optimization will be needed in future studies. Generally speaking, the limited penetration of light in tissue renders optical imaging only suitable for a limited number of scenarios (e.g., superficial regions-of-interest and endoscopy-accessible tissues). Another drawback of optical imaging is that it cannot give accurate quantitative analysis of the imaging result. Combination of quantum dot optical and radionuclide-based imaging techniques (e.g., PET) may significantly help in this aspect [Cai, et al.2007a, 2007b].

Inhibition of Nitric Oxide Synthase Gene Expression:

al. 2009]

et al. 2009]

**4. Biological applications of NO imaging** 

fluctuations during a MRI scanning of NO-related diseases.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 147

Fig. 11. (on left) Imaging NO with FRET. (A) Basic principles of FRET between CFP and YFP. CFP: cyan fluorescent protein; YFP: yellow fluorescent protein. (B) Pseudocolor images of the CFP/YFP emission ratio of the cell before (1570 s) and after addition of NOC-7, an NO-donating reagent that releases 2 mol of NO per mole. Adapted from reference [Hong et

Fig. 12. (on right) Several NOS inhibiters and an antisense oligonucleotide for iNOS mRNA have been radiolabeled for PET imaging of NOS expression.Adapted from reference [Hong,

Still development and applications of fluorescent agents in cell imaging and NO sensitive MRI contrast agents for in vivo physiological fMRI are in infancy. MRI imaging of NO based on nitrite concentration can detect physiological changes such as cerebral blood volume change in nonischemic spontaneously hypertensive rats [Caramia et al. 1998], increase in tumor blood flow and partial oxygen pressure after NO donor administration [Jordan et al. 2000], NO production alteration in humans after a 30-min exposure to a 1.5 T magnetic field [Sirmatel, et al. 2007]. Interestingly, high magnetic strength can cause NO concentration

#### **3.5.2 PET imaging of NOS expression**

Small animal PET scanners have very good spatial resolution (1 mm) and they are also becoming increasingly widely available. PET does not have a tissue penetration issue, it is highly quantitative, and it can be used for both diagnosis and treatment monitoring. To date, PET imaging of NOS expression has been focused on the clarification of nNOS function in neurological diseases [Pomper, et al. 2000, Zhang, et al. 1997]. PET imaging of nNOS was initially investigated with a 11C-labeled nNOS inhibitor, S-methyl-Lthiocitrulline (MTICU) (FIG 11, 12) [Zhang, et al. 1997]. Biodistribution of 11C-MTICU gave a brain-to-blood ratio of 1:2 at 30 min postinjection. Uptake in the cerebellum (high nNOS expression) was 20% higher than that of the cortex or the brain stem (low nNOS expression). Blockage using 1 mg/kg of MTICU was able to reduce the uptake in the cerebellum and the cortex, but not in the brain stem. Study demonstrated the potential of 11CMTICU as a potentially useful tracer in determining nNOS levels in vivo. In another report, two nNOS-selective PET tracers, AR-R 17443 [N-(4-(2-(phenylmethyl) (methyl) amino)ethyl)phenyl)-2-thiophenecarboximidamide)] and AR-R 18512 [(N(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-yl)-2 thiophenecarboximidamide)] were synthesized and evaluated in rodents and primates [Pomper et al. 2000]. Another strategy has been employed for iNOS imaging using antisense oligonucleotide with hybridization properties for iNOS mRNA using RT-PCR. The oligonucleotide was then labeled with 18F (FIG 12) [de Varies, et al. 2004]. Cellular uptake or efflux showed no selectivity for iNOS expressing cells. Thus PET imaging of NOS expression has not been very successful which is likely due to several reasons. First, the expression of NOS is in the cytoplasm. Although the radiolabeled NOS inhibitors usually have high affinity for NOS, they may not permeate through cell membrane efficiently which dramatically affects the tracer uptake. Second, the specificity of these NOS inhibitors may not be high enough for imaging applications; many of them can undergo nonspecific adsorption to other proteins such as albumin. Third, the stability of those tracers in vivo is a major concern. Uptake in the tumor or other targeted organs may not truly reflect the distribution of the intact tracer. Lastly, these NOS inhibitors are small molecules. Although replacing the 12CH3 with 11CH3 will not change the chemical structure, in many cases this cannot be achieved. Recently, a novel NOS inhibitor design strategy, termed the "anchored plasticity approach," may give rise to novel NOS inhibitors for PET imaging applications in the future [Garcin et al. 2008].

#### **3.5.3 Imaging NOS expression with other techniques**

Besides optical and PET imaging, other techniques have been explored for NOS-related research, mainly electron microscopy and immunohistochemistry [Heinrich, et al. 2005, Lajoix, et al. 2006]. Electron microscopy was applied to visualize the subcellular localization of NOS in different cell organelles using gold particles conjugated with anti-NOS antibodies, which gave exquisite distribution information of NOS in cells. Immunohistochemistry also uses anti-NOS antibodies, easier to perform than electron microscopy yet the resolution is not as high. Both approaches are invasive and not suitable for in vivo imaging; however, the experimental findings are typically quite reliable and thus they can serve as ex vivo validation for in vivo imaging studies.

Small animal PET scanners have very good spatial resolution (1 mm) and they are also becoming increasingly widely available. PET does not have a tissue penetration issue, it is highly quantitative, and it can be used for both diagnosis and treatment monitoring. To date, PET imaging of NOS expression has been focused on the clarification of nNOS function in neurological diseases [Pomper, et al. 2000, Zhang, et al. 1997]. PET imaging of nNOS was initially investigated with a 11C-labeled nNOS inhibitor, S-methyl-Lthiocitrulline (MTICU) (FIG 11, 12) [Zhang, et al. 1997]. Biodistribution of 11C-MTICU gave a brain-to-blood ratio of 1:2 at 30 min postinjection. Uptake in the cerebellum (high nNOS expression) was 20% higher than that of the cortex or the brain stem (low nNOS expression). Blockage using 1 mg/kg of MTICU was able to reduce the uptake in the cerebellum and the cortex, but not in the brain stem. Study demonstrated the potential of 11CMTICU as a potentially useful tracer in determining nNOS levels in vivo. In another report, two nNOS-selective PET tracers, AR-R 17443 [N-(4-(2-(phenylmethyl) (methyl) amino)ethyl)phenyl)-2-thiophenecarboximidamide)] and AR-R 18512 [(N(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-yl)-2 thiophenecarboximidamide)] were synthesized and evaluated in rodents and primates [Pomper et al. 2000]. Another strategy has been employed for iNOS imaging using antisense oligonucleotide with hybridization properties for iNOS mRNA using RT-PCR. The oligonucleotide was then labeled with 18F (FIG 12) [de Varies, et al. 2004]. Cellular uptake or efflux showed no selectivity for iNOS expressing cells. Thus PET imaging of NOS expression has not been very successful which is likely due to several reasons. First, the expression of NOS is in the cytoplasm. Although the radiolabeled NOS inhibitors usually have high affinity for NOS, they may not permeate through cell membrane efficiently which dramatically affects the tracer uptake. Second, the specificity of these NOS inhibitors may not be high enough for imaging applications; many of them can undergo nonspecific adsorption to other proteins such as albumin. Third, the stability of those tracers in vivo is a major concern. Uptake in the tumor or other targeted organs may not truly reflect the distribution of the intact tracer. Lastly, these NOS inhibitors are small molecules. Although replacing the 12CH3 with 11CH3 will not change the chemical structure, in many cases this cannot be achieved. Recently, a novel NOS inhibitor design strategy, termed the "anchored plasticity approach," may give rise to novel NOS inhibitors for PET imaging applications in the

**3.5.2 PET imaging of NOS expression** 

future [Garcin et al. 2008].

validation for in vivo imaging studies.

**3.5.3 Imaging NOS expression with other techniques** 

Besides optical and PET imaging, other techniques have been explored for NOS-related research, mainly electron microscopy and immunohistochemistry [Heinrich, et al. 2005, Lajoix, et al. 2006]. Electron microscopy was applied to visualize the subcellular localization of NOS in different cell organelles using gold particles conjugated with anti-NOS antibodies, which gave exquisite distribution information of NOS in cells. Immunohistochemistry also uses anti-NOS antibodies, easier to perform than electron microscopy yet the resolution is not as high. Both approaches are invasive and not suitable for in vivo imaging; however, the experimental findings are typically quite reliable and thus they can serve as ex vivo

Fig. 11. (on left) Imaging NO with FRET. (A) Basic principles of FRET between CFP and YFP. CFP: cyan fluorescent protein; YFP: yellow fluorescent protein. (B) Pseudocolor images of the CFP/YFP emission ratio of the cell before (1570 s) and after addition of NOC-7, an NO-donating reagent that releases 2 mol of NO per mole. Adapted from reference [Hong et al. 2009]

Fig. 12. (on right) Several NOS inhibiters and an antisense oligonucleotide for iNOS mRNA have been radiolabeled for PET imaging of NOS expression.Adapted from reference [Hong, et al. 2009]

#### **4. Biological applications of NO imaging**

Still development and applications of fluorescent agents in cell imaging and NO sensitive MRI contrast agents for in vivo physiological fMRI are in infancy. MRI imaging of NO based on nitrite concentration can detect physiological changes such as cerebral blood volume change in nonischemic spontaneously hypertensive rats [Caramia et al. 1998], increase in tumor blood flow and partial oxygen pressure after NO donor administration [Jordan et al. 2000], NO production alteration in humans after a 30-min exposure to a 1.5 T magnetic field [Sirmatel, et al. 2007]. Interestingly, high magnetic strength can cause NO concentration fluctuations during a MRI scanning of NO-related diseases.

Inhibition of Nitric Oxide Synthase Gene Expression:

\*NO imaging [Kojima, et al. 2001, Kojima, et al. 2001].

lungs, heart, and liver [Kuppusamy, et al. 2001].

**4.4 Calcium-imaging and NO-imaging** 

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 149

biomedical applications in molecular imaging [Liu, et al. 2007]. In other study, the clinical detection of evolving acute tubular necrosis (ATN) and differentiating it from other causes of renal failure was performed. Herein, sodium magnetic resonance imaging (23Na MRI) was applied to study the early alteration in renal sodium distribution in rat kidneys 6 h after the induction of ATN combined with inhibition of nitric oxide and prostaglandin synthesis. Hence, 23Na MRI non-invasively quantified changes in the corticomedullary sodium gradient in the ATN kidney during morphologic tubular injury [Maril, et al. 2006]. Other newer application of 19F NMR spin-trapping technique for in vivo \*NO detection was employed to elucidate the significance of \*NO availability in animal models of hypertension. In vivo \*NO-induced conversion of the hydroxylamine from fluorinated nitrosyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) was evidenced in hypertensive ISIAH and OXYS rat strains [Bobko, et al. 2005]. The NMR detected positive levels of nitrite/nitrate for *in vivo* evaluation of \*NO production and provided the basis for in vivo

The cardiovascular ischemia in tissues generates the nitric oxide (NO). It is an enzymeindependent mechanism of NO generation and more pathogenesis. So, the NO formation in mice after cardiopulmonary arrest could be imaged. Real-time measurement of NO generation was performed by detection of naturally generated NO-heme complexes in tissues using L-band electron paramagnetic resonance (EPR) spectroscopy. Measurements of NO generation were imaged on the intact animal at the levels of the head, thorax, and abdomen within 3 hours [Ueno, et al. 2002]. Very recently, nitric oxide was recognized as biomarker of vascular dysfunction in renal, clitoris and penile sex organs to detect the physiology [Ockalli, et al. 1999, Kakiailatu, 2000, Burnett, 2002, Barouch, et al. 2002, Hillebrandt, et al. 2009, Ciplak, et al. 2009, Gragasin, et al. 2004, Richards, et al. 2003]. The NO complexes were found to have maximum levels in lung, heart, and liver by threedimensional spatial mapping of the NO complex in the intact animal subjected to cardiopulmonary arrest. The images also confirmed the maximum NO formation in the

The combined real-time bioimaging of calcium and nitric oxide with continuous electrophysiological intracellular recording has gained enormous interest. For intracellular monitoring by calcium imaging in cells, Fura-2 is a calcium-sensitive fluorescent probe. Using Fura-2, intracellular free calcium ion concentration ([Ca2+]i) of single neurons was measured [Richards, et al. 2003]. Calcium-imaging with Fura-2 was used to monitor and modulate the intracellular Ca2+ evoked by NMDA and AMPA in cultured hippocampal pyramidal cells [Keelan, et al. 1999] Calcium and NO play role in excitotoxic mitochondrial depolarization in the hippocampus [Berkels, et al. 2000]. The combination of NO bioimaging with calcium imaging may distinguish the temporal rise in NO and calcium to suggest that NO is being produced as a consequence of Ca2+ enhancement and Ca2+ induced NO Synthase activity). Recently, imaging intracellular calcium imaging was achieved together with NO imaging by using two monochromators DAF-2 and Fura-2 AM as excitation light source. The DAF-2 excited at 485 nm and Fura-2 AM excited at 340 nm [Berkels, et al. 2000]. The emission maximum of fura-2 is about 510 nm different while the emission maximum of DAF-2 is about 515 nm. By using dichroic or multichroic mirrors calcium and NO levels can

The major advantages of MRI include high resolution, good soft tissue contrast, wide availability, sufficiently large field-of-view, and the ability to provide both functional and anatomical information. Nitric oxide bioimaging has emerged as technique to visualize the intracellular metabolism based on redox reactions, mitochondrial oxidation and nitric oxide synthase enzyme NOSi [Anbar, 2000, Zhao, et al. 2009]. Recently, nitric oxide was invented as active mechanism to play role in vascular physiology in lungs, kidney, heart, brain, sex organs. Endothelial cell, smooth muscle cells, oxygenation and perfusion, hypoxia, ischemia are the possible targets of nitric oxide biotransformation. These physiological changes are likely to be visualized by nitric oxide bioimaging in real time manner and reviewed in the following sections.

#### **4.1 Cardiovascular and endothelial cell injury**

DAF-FM is useful tool to visualize the temporal and spatial distribution of intracellular NO. Endogenous ATP plays a central role in HTS-induced NOS in endothelial cell injury. Endothelial cNOS, a calmodulin-dependent enzyme is critical for vascular homeostasis and makes detectable basal level of NO production at low extracellular Ca2+. cNOS expression can be stimulated by Ca2+ while iNOS activity is Ca2+-independent. Actin microfilaments in primary artery endothelial cells (PAEC) regulates L-arginine transport and this regulation can affect NO production by PAEC, DAR-4M may be useful for bioimaging of samples that have strong autofluorescence [Lai, et al. 1994, Kojima, et al. 2001]

#### **4.2 Apoptosis in cells**

Apoptosis plays a key role in many pathological circumstances, such as neurodegenerative diseases. In these processes, the involvement of nitric oxide (NO) has been well established, and the ability of NO to exert cellular damage due to its reactive oxidative properties is perhaps the primary neurotoxic mechanism. Emerging evidences indicate that apoptosis contributes to neuronal death to explain neurodegeneration [Fujii et al. 1997]. The major biochemical change in activation of the cystein protease (caspase-3) was reported to execute apoptosis in the central nervous system disorders such as stroke, spinal cord trauma, head injury and Alzheimer's disease [Green,1998, Nagata,1997 ]. Although numerous techniques have been described to evaluate apoptosis, these approaches involve invasive techniques and cannot provide detailed information about apoptosis *in vivo* [Anbar, 2000].

#### **4.3 Renal and cardiovascular imaging**

A new approach of irreversible responsive PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) for MRI contrast agents constituted a new type of agent for molecular imaging. A novel PARACEST MRI contrast agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid)-orthoaminoanilide (Yb-DO3A-oAA), was developed to image nitric oxide (NO). The agent exhibited two CEST effects at -11 ppm and +8 ppm chemical shifts, which were assigned to chemical exchange from amide and amine functional groups, respectively. This responsive PARACEST MRI contrast agent indicated the change in the presence of NO and O(2), which caused an irreversible disappearance of both PARACEST effects from MR images. This report highlighted the advantages of irreversible MRI contrast agents and demonstrated that large changes in PARACEST can be used to in vivo

The major advantages of MRI include high resolution, good soft tissue contrast, wide availability, sufficiently large field-of-view, and the ability to provide both functional and anatomical information. Nitric oxide bioimaging has emerged as technique to visualize the intracellular metabolism based on redox reactions, mitochondrial oxidation and nitric oxide synthase enzyme NOSi [Anbar, 2000, Zhao, et al. 2009]. Recently, nitric oxide was invented as active mechanism to play role in vascular physiology in lungs, kidney, heart, brain, sex organs. Endothelial cell, smooth muscle cells, oxygenation and perfusion, hypoxia, ischemia are the possible targets of nitric oxide biotransformation. These physiological changes are likely to be visualized by nitric oxide bioimaging in real time manner and reviewed in the

DAF-FM is useful tool to visualize the temporal and spatial distribution of intracellular NO. Endogenous ATP plays a central role in HTS-induced NOS in endothelial cell injury. Endothelial cNOS, a calmodulin-dependent enzyme is critical for vascular homeostasis and makes detectable basal level of NO production at low extracellular Ca2+. cNOS expression can be stimulated by Ca2+ while iNOS activity is Ca2+-independent. Actin microfilaments in primary artery endothelial cells (PAEC) regulates L-arginine transport and this regulation can affect NO production by PAEC, DAR-4M may be useful for bioimaging of samples that

Apoptosis plays a key role in many pathological circumstances, such as neurodegenerative diseases. In these processes, the involvement of nitric oxide (NO) has been well established, and the ability of NO to exert cellular damage due to its reactive oxidative properties is perhaps the primary neurotoxic mechanism. Emerging evidences indicate that apoptosis contributes to neuronal death to explain neurodegeneration [Fujii et al. 1997]. The major biochemical change in activation of the cystein protease (caspase-3) was reported to execute apoptosis in the central nervous system disorders such as stroke, spinal cord trauma, head injury and Alzheimer's disease [Green,1998, Nagata,1997 ]. Although numerous techniques have been described to evaluate apoptosis, these approaches involve invasive techniques

A new approach of irreversible responsive PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) for MRI contrast agents constituted a new type of agent for molecular imaging. A novel PARACEST MRI contrast agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid)-orthoaminoanilide (Yb-DO3A-oAA), was developed to image nitric oxide (NO). The agent exhibited two CEST effects at -11 ppm and +8 ppm chemical shifts, which were assigned to chemical exchange from amide and amine functional groups, respectively. This responsive PARACEST MRI contrast agent indicated the change in the presence of NO and O(2), which caused an irreversible disappearance of both PARACEST effects from MR images. This report highlighted the advantages of irreversible MRI contrast agents and demonstrated that large changes in PARACEST can be used to in vivo

and cannot provide detailed information about apoptosis *in vivo* [Anbar, 2000].

following sections.

**4.2 Apoptosis in cells** 

**4.3 Renal and cardiovascular imaging** 

**4.1 Cardiovascular and endothelial cell injury** 

have strong autofluorescence [Lai, et al. 1994, Kojima, et al. 2001]

biomedical applications in molecular imaging [Liu, et al. 2007]. In other study, the clinical detection of evolving acute tubular necrosis (ATN) and differentiating it from other causes of renal failure was performed. Herein, sodium magnetic resonance imaging (23Na MRI) was applied to study the early alteration in renal sodium distribution in rat kidneys 6 h after the induction of ATN combined with inhibition of nitric oxide and prostaglandin synthesis. Hence, 23Na MRI non-invasively quantified changes in the corticomedullary sodium gradient in the ATN kidney during morphologic tubular injury [Maril, et al. 2006]. Other newer application of 19F NMR spin-trapping technique for in vivo \*NO detection was employed to elucidate the significance of \*NO availability in animal models of hypertension. In vivo \*NO-induced conversion of the hydroxylamine from fluorinated nitrosyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) was evidenced in hypertensive ISIAH and OXYS rat strains [Bobko, et al. 2005]. The NMR detected positive levels of nitrite/nitrate for *in vivo* evaluation of \*NO production and provided the basis for in vivo \*NO imaging [Kojima, et al. 2001, Kojima, et al. 2001].

The cardiovascular ischemia in tissues generates the nitric oxide (NO). It is an enzymeindependent mechanism of NO generation and more pathogenesis. So, the NO formation in mice after cardiopulmonary arrest could be imaged. Real-time measurement of NO generation was performed by detection of naturally generated NO-heme complexes in tissues using L-band electron paramagnetic resonance (EPR) spectroscopy. Measurements of NO generation were imaged on the intact animal at the levels of the head, thorax, and abdomen within 3 hours [Ueno, et al. 2002]. Very recently, nitric oxide was recognized as biomarker of vascular dysfunction in renal, clitoris and penile sex organs to detect the physiology [Ockalli, et al. 1999, Kakiailatu, 2000, Burnett, 2002, Barouch, et al. 2002, Hillebrandt, et al. 2009, Ciplak, et al. 2009, Gragasin, et al. 2004, Richards, et al. 2003]. The NO complexes were found to have maximum levels in lung, heart, and liver by threedimensional spatial mapping of the NO complex in the intact animal subjected to cardiopulmonary arrest. The images also confirmed the maximum NO formation in the lungs, heart, and liver [Kuppusamy, et al. 2001].

#### **4.4 Calcium-imaging and NO-imaging**

The combined real-time bioimaging of calcium and nitric oxide with continuous electrophysiological intracellular recording has gained enormous interest. For intracellular monitoring by calcium imaging in cells, Fura-2 is a calcium-sensitive fluorescent probe. Using Fura-2, intracellular free calcium ion concentration ([Ca2+]i) of single neurons was measured [Richards, et al. 2003]. Calcium-imaging with Fura-2 was used to monitor and modulate the intracellular Ca2+ evoked by NMDA and AMPA in cultured hippocampal pyramidal cells [Keelan, et al. 1999] Calcium and NO play role in excitotoxic mitochondrial depolarization in the hippocampus [Berkels, et al. 2000]. The combination of NO bioimaging with calcium imaging may distinguish the temporal rise in NO and calcium to suggest that NO is being produced as a consequence of Ca2+ enhancement and Ca2+ induced NO Synthase activity). Recently, imaging intracellular calcium imaging was achieved together with NO imaging by using two monochromators DAF-2 and Fura-2 AM as excitation light source. The DAF-2 excited at 485 nm and Fura-2 AM excited at 340 nm [Berkels, et al. 2000]. The emission maximum of fura-2 is about 510 nm different while the emission maximum of DAF-2 is about 515 nm. By using dichroic or multichroic mirrors calcium and NO levels can

Inhibition of Nitric Oxide Synthase Gene Expression:

multichroic mirrors in synaptic plasticity.

**4.7 Alveolar cells** 

**4.8 Endothelial cells** 

production via the O2-

2002].

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 151

neuronal processes with continuous monitoring the calcium and NO by dichroic or

Human alveolar macrophages and alveolar epithelium is the site of nitric oxide synthase-2 (NOS-2) messenger ribonucleic acid (mRNA) and protein expression in alveolar wall in lung of patients with pulmonary malignancies. In the process of NO production, proinflammatory cytokines interleukin (IL)-1β, tumour necrosis factor (TNF)-, interferon (IFN)- or lipopolysaccharide (LPS) play significant role to induce the effect of human surfactant protein-A (SP-A) on IFN--mediated NOS-2 mRNA expression [Pechkovsky, et al.

Kojima et al. 2001, reported NO bioimaging in cultured bovine aortic endothelial cells. The cultured bovine aortic endothelial cells were incubated with DAF-FM DA for 1 h for dye loading. After stimulation with bradykinin, the fluorescence intensity in the cells increased and that in the cytosol increased more than that in the nucleus. NO is produced in the cytosol, where NOS exists [von Bohlan, et al. 2002]. This observation implies that little of the produced NO diffuses into the nucleus or if it does diffuse into the nucleus little of it is oxidized there. The augmentation of the fluorescence intensity was suppressed by an NOS inhibitor. In conclusion, DAF-FM is a useful tool for visualizing the temporal and spatial distribution of intracellular NO. Kimura et al.2001, examined the effects of acute glucose overload on the cellular productivity of NO in bovine aortic endothelial cells (BAEC) using DAF-2. ATP induced an increase in NO production, assessed by DAF-2, mainly due to Ca2+ entry as shown in Figure 13. In contrast, the ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload. The results indicate that glucose overload impairs NO

examined in detail the mechanism by which mechanical stress induces NOS in endothelium. Hypotonic stress (HTS) induced ATP release, which evoked Ca2+ transients in BAEC. HTS also induced NOS, assessed by DAF-2 fluorescence. The results obtained indicate that endogenous ATP plays a central role in HTS-induced NOS in BAEC. Broillet et al. 2001, reported that Ca2+, Mg2+, or incident light promoted the reaction of DAF-2 and NO. However, Nagano et al. 2002, probed that the enhancement of fluorescence intensity was caused not by increasing the reaction rate between DAF-2 and NO but by promotion of NOreleasing rate of NO donors. Endothelial NOS, a Ca2+/calmodulin-dependent enzyme, is critical for vascular homeostasis [Suzuki, et al. 2002]. To determine the signaling pathway for endogenous eNOS, Lin et al. 2000, directly measured NO production in bovine pulmonary artery endothelial cells (PAEC). Endothelial cells grown in a monolayer produce very low levels of NO which are below the detection range of traditional assays. Therefore, they employed DAF-2 DA to measure NO production in real-time. PAEC had detectable basal NO production which increased slightly after thapsigargin treatment in the presence of low extracellular Ca2+. In contrast, a large increase in NO production was detected in the presence of 4 mM extracellular Ca2+, suggesting that capacitative Ca2+ entry regulates wildtype eNOS activity. Zharikov et al. 2001, investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/NO pathway in pulmonary artery


be measured simultaneously [Pittner, et al. 2003]. Other example of monochromator complex is DAQ and DAR-4M AM. These are calcium sensitive non-neurotoxic dyes with unique fluorescent patterns of DAQ (excitation 520 nm; emission 580 nm) and DAR-4M AM (excitation 550 nm; emission 580 nm). These can also combine with the calciumsensitive dye Fura-2 as potential tools in fluorescence microscopy.

#### **4.5 Kupffer cell NO bioimaging**

A new mechanism was reported in nonparenchymal cells based on Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, was upregulated in vitro and in vivo by nitric oxide (NO) downregulation of BNIP3 gene expression as one mechanism of hepatocyte cell death and liver damage. Authors proposed that inflammatory stresses can lead to the modulation of BNIP3 [Metukuri, et al. 2009].

#### **4.6 Combining electrophysiology and NO-imaging**

The calcium-imaging with electrophysiological recording was excitement in the hippocampus imaging art because of calcium accumulation in hippocampus pyramidal cells during synaptic activation [Regehr, et al. 1989]. Now a day, calcium-imaging combined with electrophysiological stimulation and recording has emerged as present state of art of calcium-signaling and intracellular free calcium ion concentration [Ca2+]i of single neurone. Moreover, little is understood how neuronal signals stimulate NO production. Potential applications of fluorescent NO indicators in bioimaging are to monitor NO in response to pharmacological manipulation using NO-donors, NOS substrate, and NOS or NOS isoform inhibitors as enhancers of NO production in vivo and in vitro [Moore, et al. 1997, Southan, et al. 1996]*.* Other emerging techniques of bioimaging by NO inhibitor are targeting the differential cofactor requirements of the various isoforms; pharmacological agents targeting the differential substrate requirements of cell expression of various isoforms of the NOS [Salerno, et al. 2002]. However, this approach suffers due to lack a marked isoform selectivity or direct affecting the arginine transport into cells, like *N*G-methyl-L-arginine (L-NMA). Other example of NOS inhibitors such as 7-nitroindazole (7-NI), might have unexpected side-effects in neuronal tissues, since 7-NI interferes with monoamine oxidase [Castagnoli, et al. 1999, Desvignes, et al. 1999]. However, selective NOS-inhibitors together with fluorescent NO-imaging appear as useful tools for the investigation of the biological functions of the different NOS-isoforms in neuronal tissues [Chen, et al. 2001, Brown, et al. 1999]. The combined NO-imaging with in vitro electrophysiology methods visualize the NO generation and NO distribution with localization of NO induced by neurotransmitter stimulations, e.g., NO formation after neuronal stimulation with NMDA, in normal synaptic transmission, in paired-pulse facilitation, in long-term depression or LTP. Thus, in a single preparation, the spatial distribution of NO after induction of hippocampal LTP has been evaluated [Schuppe, et al. 2002]. Main unsolved issues of precise time-point of NO release and selective NOS isoform-inhibitors in neuronal activity still remain to investigate before the combined fluorescent NO-imaging and electrophysiology method becomes an acceptable bioimaging tool. In near future, the researchers will thrive to combine electrophysiology with simultaneous calcium-imaging and NO-imaging to get better answers of the role and interaction of calcium and NO in physiological and pathological neuronal processes with continuous monitoring the calcium and NO by dichroic or multichroic mirrors in synaptic plasticity.

#### **4.7 Alveolar cells**

150 Enzyme Inhibition and Bioapplications

be measured simultaneously [Pittner, et al. 2003]. Other example of monochromator complex is DAQ and DAR-4M AM. These are calcium sensitive non-neurotoxic dyes with unique fluorescent patterns of DAQ (excitation 520 nm; emission 580 nm) and DAR-4M AM (excitation 550 nm; emission 580 nm). These can also combine with the calcium-

A new mechanism was reported in nonparenchymal cells based on Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, was upregulated in vitro and in vivo by nitric oxide (NO) downregulation of BNIP3 gene expression as one mechanism of hepatocyte cell death and liver damage. Authors proposed that inflammatory stresses can lead to the modulation of BNIP3 [Metukuri, et al. 2009].

The calcium-imaging with electrophysiological recording was excitement in the hippocampus imaging art because of calcium accumulation in hippocampus pyramidal cells during synaptic activation [Regehr, et al. 1989]. Now a day, calcium-imaging combined with electrophysiological stimulation and recording has emerged as present state of art of calcium-signaling and intracellular free calcium ion concentration [Ca2+]i of single neurone. Moreover, little is understood how neuronal signals stimulate NO production. Potential applications of fluorescent NO indicators in bioimaging are to monitor NO in response to pharmacological manipulation using NO-donors, NOS substrate, and NOS or NOS isoform inhibitors as enhancers of NO production in vivo and in vitro [Moore, et al. 1997, Southan, et al. 1996]*.* Other emerging techniques of bioimaging by NO inhibitor are targeting the differential cofactor requirements of the various isoforms; pharmacological agents targeting the differential substrate requirements of cell expression of various isoforms of the NOS [Salerno, et al. 2002]. However, this approach suffers due to lack a marked isoform selectivity or direct affecting the arginine transport into cells, like *N*G-methyl-L-arginine (L-NMA). Other example of NOS inhibitors such as 7-nitroindazole (7-NI), might have unexpected side-effects in neuronal tissues, since 7-NI interferes with monoamine oxidase [Castagnoli, et al. 1999, Desvignes, et al. 1999]. However, selective NOS-inhibitors together with fluorescent NO-imaging appear as useful tools for the investigation of the biological functions of the different NOS-isoforms in neuronal tissues [Chen, et al. 2001, Brown, et al. 1999]. The combined NO-imaging with in vitro electrophysiology methods visualize the NO generation and NO distribution with localization of NO induced by neurotransmitter stimulations, e.g., NO formation after neuronal stimulation with NMDA, in normal synaptic transmission, in paired-pulse facilitation, in long-term depression or LTP. Thus, in a single preparation, the spatial distribution of NO after induction of hippocampal LTP has been evaluated [Schuppe, et al. 2002]. Main unsolved issues of precise time-point of NO release and selective NOS isoform-inhibitors in neuronal activity still remain to investigate before the combined fluorescent NO-imaging and electrophysiology method becomes an acceptable bioimaging tool. In near future, the researchers will thrive to combine electrophysiology with simultaneous calcium-imaging and NO-imaging to get better answers of the role and interaction of calcium and NO in physiological and pathological

sensitive dye Fura-2 as potential tools in fluorescence microscopy.

**4.6 Combining electrophysiology and NO-imaging** 

**4.5 Kupffer cell NO bioimaging** 

Human alveolar macrophages and alveolar epithelium is the site of nitric oxide synthase-2 (NOS-2) messenger ribonucleic acid (mRNA) and protein expression in alveolar wall in lung of patients with pulmonary malignancies. In the process of NO production, proinflammatory cytokines interleukin (IL)-1β, tumour necrosis factor (TNF)-, interferon (IFN)- or lipopolysaccharide (LPS) play significant role to induce the effect of human surfactant protein-A (SP-A) on IFN--mediated NOS-2 mRNA expression [Pechkovsky, et al. 2002].

#### **4.8 Endothelial cells**

Kojima et al. 2001, reported NO bioimaging in cultured bovine aortic endothelial cells. The cultured bovine aortic endothelial cells were incubated with DAF-FM DA for 1 h for dye loading. After stimulation with bradykinin, the fluorescence intensity in the cells increased and that in the cytosol increased more than that in the nucleus. NO is produced in the cytosol, where NOS exists [von Bohlan, et al. 2002]. This observation implies that little of the produced NO diffuses into the nucleus or if it does diffuse into the nucleus little of it is oxidized there. The augmentation of the fluorescence intensity was suppressed by an NOS inhibitor. In conclusion, DAF-FM is a useful tool for visualizing the temporal and spatial distribution of intracellular NO. Kimura et al.2001, examined the effects of acute glucose overload on the cellular productivity of NO in bovine aortic endothelial cells (BAEC) using DAF-2. ATP induced an increase in NO production, assessed by DAF-2, mainly due to Ca2+ entry as shown in Figure 13. In contrast, the ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload. The results indicate that glucose overload impairs NO production via the O2- -mediated attenuation of Ca2+ entry. In addition, Kimura et al. 2000, examined in detail the mechanism by which mechanical stress induces NOS in endothelium. Hypotonic stress (HTS) induced ATP release, which evoked Ca2+ transients in BAEC. HTS also induced NOS, assessed by DAF-2 fluorescence. The results obtained indicate that endogenous ATP plays a central role in HTS-induced NOS in BAEC. Broillet et al. 2001, reported that Ca2+, Mg2+, or incident light promoted the reaction of DAF-2 and NO. However, Nagano et al. 2002, probed that the enhancement of fluorescence intensity was caused not by increasing the reaction rate between DAF-2 and NO but by promotion of NOreleasing rate of NO donors. Endothelial NOS, a Ca2+/calmodulin-dependent enzyme, is critical for vascular homeostasis [Suzuki, et al. 2002]. To determine the signaling pathway for endogenous eNOS, Lin et al. 2000, directly measured NO production in bovine pulmonary artery endothelial cells (PAEC). Endothelial cells grown in a monolayer produce very low levels of NO which are below the detection range of traditional assays. Therefore, they employed DAF-2 DA to measure NO production in real-time. PAEC had detectable basal NO production which increased slightly after thapsigargin treatment in the presence of low extracellular Ca2+. In contrast, a large increase in NO production was detected in the presence of 4 mM extracellular Ca2+, suggesting that capacitative Ca2+ entry regulates wildtype eNOS activity. Zharikov et al. 2001, investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/NO pathway in pulmonary artery

Inhibition of Nitric Oxide Synthase Gene Expression:

**4.9 Smooth muscle cells** 

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 153

Fig. 14. Fluorescence images of a rat brain slice loaded with DAF-2 DA. The fluorescence intensity is shown in pseudocolor. (Reproduced from reference Nagano, et al. 2002)

DAF-2 DA was applied to the imaging of NO in cultured rat aortic smooth muscle cells by using a fluorescence microscope equipped with fluorescence filters for fluorescein chromophores. Confocal laser scanning microscopy indicated that fluorescence was emitted from the whole cell body, including the nucleus. This implies that DAF-2 regenerated intracellularly by esterase is distributed throughout the whole cell. There was no indication of any cell damage caused by loading the dye. The results show that the fluorescence intensity in endotoxin- and cytokine-activated cells increased owing to DAF-2 T production from DAF-2 by reaction with NO. The fluorescence did not increase in inactivated cells, which did not produce NO. The probe is very useful for bioimaging of NO in cultured rat aortic smooth muscle cells. Itoh et al. detected DAF-FM T using reversed-phase highperformance liquid chromatography with a fluorescence detector [Itoh et al. 2000]. This sensitive method enabled them to detect the spontaneous and substance P-induced NO release from isolated porcine coronary arteries, both of which were dependent entirely on the NOS activity in vascular endothelial cells. Furthermore, they obtained fluorescence images of cultured smooth muscle cells of the rat urinary bladder after loading with DAF-FM DA. In the cells pretreated with cytokines, the fluorescence intensity increased with time after DAF-FM loading. In recent studies on imaging of nitric oxide in nitrergic neuromuscular neurotransmission suggested the new insight of molecular mechanisms in

animal organs [Thatte, et al. 2009, Perrier, et al. 2009, Di Cesare Mannelli, et al. 2009].

endothelial cells (PAEC). DAF-2 DA was used for detection of NO in PAEC. They conclude that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC. Berkels et al. 2000, presented a new method to measure intracellular Ca2+ and NO simultaneously in endothelial cells. The method makes it possible to follow intracellular Ca2+ and NO distributions online and is sensitive enough to monitor changes of NO formed by the constitutive endothelial NOS. Franz et al. 2000, reported DAR-4M AM applied to imaging of NO in bovine aortic endothelial cells using aminotroponiminates[Franz et al. 2000]. DARs are more photostable than DAFs. The fluorescence in cells was observed after stimulation with bradykinin (see Figure 14), which raises the intracellular Ca2+ level and thereby activates NOS, and this increase was suppressed by addition of an NOS inhibitor. DAR-4M should be useful for bioimaging of samples that have strong autofluorescence in the case of 490 nm excitation in which the intracellular pH may fall below 6. Hiratsuka et al. 2009, reported in vivo visualization of nitric oxide and intracellular inflammatory interactions among platelets, leukocytes, and endothelium following hemorrhagic shock and reperfusion.

Fig. 13. Bright-field and fluorescence images of cultured bovine aortic endothelial cells loaded with DAR-4M AM. The upper images are the bright-field, the fluorescence, and the fluorescence ratio images at the start of measurement, respectively. The middle and the lower images are fluorescence ratio images at the indicated times after the start of measurement. The fluorescence ratio images indicate the ratio of the intensity to the initial intensity at the start. Adopted from reference Kimura, et al. 2001

Fig. 14. Fluorescence images of a rat brain slice loaded with DAF-2 DA. The fluorescence intensity is shown in pseudocolor. (Reproduced from reference Nagano, et al. 2002)

#### **4.9 Smooth muscle cells**

152 Enzyme Inhibition and Bioapplications

endothelial cells (PAEC). DAF-2 DA was used for detection of NO in PAEC. They conclude that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC. Berkels et al. 2000, presented a new method to measure intracellular Ca2+ and NO simultaneously in endothelial cells. The method makes it possible to follow intracellular Ca2+ and NO distributions online and is sensitive enough to monitor changes of NO formed by the constitutive endothelial NOS. Franz et al. 2000, reported DAR-4M AM applied to imaging of NO in bovine aortic endothelial cells using aminotroponiminates[Franz et al. 2000]. DARs are more photostable than DAFs. The fluorescence in cells was observed after stimulation with bradykinin (see Figure 14), which raises the intracellular Ca2+ level and thereby activates NOS, and this increase was suppressed by addition of an NOS inhibitor. DAR-4M should be useful for bioimaging of samples that have strong autofluorescence in the case of 490 nm excitation in which the intracellular pH may fall below 6. Hiratsuka et al. 2009, reported in vivo visualization of nitric oxide and intracellular inflammatory interactions among platelets, leukocytes, and

Fig. 13. Bright-field and fluorescence images of cultured bovine aortic endothelial cells loaded with DAR-4M AM. The upper images are the bright-field, the fluorescence, and the fluorescence ratio images at the start of measurement, respectively. The middle and the lower images are fluorescence ratio images at the indicated times after the start of

measurement. The fluorescence ratio images indicate the ratio of the intensity to the initial

intensity at the start. Adopted from reference Kimura, et al. 2001

endothelium following hemorrhagic shock and reperfusion.

DAF-2 DA was applied to the imaging of NO in cultured rat aortic smooth muscle cells by using a fluorescence microscope equipped with fluorescence filters for fluorescein chromophores. Confocal laser scanning microscopy indicated that fluorescence was emitted from the whole cell body, including the nucleus. This implies that DAF-2 regenerated intracellularly by esterase is distributed throughout the whole cell. There was no indication of any cell damage caused by loading the dye. The results show that the fluorescence intensity in endotoxin- and cytokine-activated cells increased owing to DAF-2 T production from DAF-2 by reaction with NO. The fluorescence did not increase in inactivated cells, which did not produce NO. The probe is very useful for bioimaging of NO in cultured rat aortic smooth muscle cells. Itoh et al. detected DAF-FM T using reversed-phase highperformance liquid chromatography with a fluorescence detector [Itoh et al. 2000]. This sensitive method enabled them to detect the spontaneous and substance P-induced NO release from isolated porcine coronary arteries, both of which were dependent entirely on the NOS activity in vascular endothelial cells. Furthermore, they obtained fluorescence images of cultured smooth muscle cells of the rat urinary bladder after loading with DAF-FM DA. In the cells pretreated with cytokines, the fluorescence intensity increased with time after DAF-FM loading. In recent studies on imaging of nitric oxide in nitrergic neuromuscular neurotransmission suggested the new insight of molecular mechanisms in animal organs [Thatte, et al. 2009, Perrier, et al. 2009, Di Cesare Mannelli, et al. 2009].

Inhibition of Nitric Oxide Synthase Gene Expression:

regulate ion channel activity.

**4.13 Inflammation** 

**4.12 Fertilization and developmental biology** 

and necessary and sufficient for successful fertilization.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 155

inactivation. The biological signaling mechanisms that regulate the persistence of Na+ channels are not well understood. Ahern et al. 2000, showed that in nerve terminals and ventricular myocytes, NO reduces the inactivation of Na+ current. This effect was independent of cGMP and blocked by *N*-ethylmaleimide. Thus, ROS act directly on the channel or a closely associated protein. Application of ionomycin to raise the intracellular Ca2+ concentration in myocytes activated NOS. The NO produced in response to ionomycin was detected with DAF-2 DA. They concluded that NO is a potential endogenous regulator of persistent Na+ current under physiological and pathophysiological conditions. Choi et al. 2000, reported that the NMDA receptor (NMDAR)-associated ion channel is modulated not only by exogenous NO, but also by endogenous NO. They examined inhibition of NMDAR responses by endogenous NO to determine the underlying molecular mechanism. For this purpose, HEK 293 cells transfected with nNOS were transiently transfected with wild-type NR1/NR2A or mutant NR1/NR2A (C399A) receptors. NMDA responses were monitored by digital Ca2+ imaging with fura-2. Production of endogenous NO by these HEK-nNOS cells was measured with DAF-2. It was concluded that endogenous *S*-nitrosylation may

Kuo et al. 2000, demonstrated that NO is necessary and sufficient for egg activation at fertilization. The early steps that lead to the rise in Ca2+ and egg activation at fertilization are unknown but are of great interest, particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. They showed that active NOS is present at high concentration in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the Ca2+ pulse of fertilization. They used DAF-2 DA for detection of NO. It was concluded that NOS- and NO-related bioactivity satisfy the primary criteria of an egg activator, i.e. they are present in an appropriate place, active at an appropriate time,

NO plays important roles in inflammatory processes. Lopez-Figueroa et al. 2000, examined whether changes in NOS mRNA expression lead to similar temporal and anatomical changes in NO production in an experimental model of CNS inflammation. iNOS mRNA expression was analyzed 2, 4, 6, and 24 h after intracerebroventricular (icv) injection of interleukin-1β or the vehicle. Increased expression of iNOS mRNA was observed surrounding the microinjection site and meninges. Using DAF-2 DA for the direct detection of NO production, they observed a significant increase in NO production after 4 and 6 h. They conclude that increase in iNOS mRNA following icv administration of IL-1β leads to increases in NO production. They proposed that the DAF-2 DA method can be used as a potential marker in the diagnosis of CNS inflammation. MRI of NO is very useful because NO is available at high concentrations in millimolar levels in immune-related diseases (e.g., inflammation).Terashima et al. 2010, reported in vivo detection of iNOS expression in murine carotid lesions by biuoluminescence may provide a valuable approach for monitoring vascular gene expression and inflammation in small animal models. The phasic

#### **4.10 Brain and neuronal behavior**

Reif et al. 2009, reported the influence of functional variant of neuronal nitric oxide synthase on impulsive behaviors in humans. The biological functions of NO in the neuronal system remain controversial. Using DAF-2 DA for direct detection of NO, Nagano et al. 2002, examined both acute rat brain slices and organotypic culture of brain slices to ascertain NO production sites. The fluorescence intensity sensitive to Ca++ in the CA1 region of the hippocampus was augmented, especially after stimulation with NMDA, in acute brain slices. This NO production in the CA1 region was also confirmed in cultured hippocampus. Glutamate and NO were important mediators, since antagonists of both N-methyl-D-aspartate (NMDA) and NOS inhibitors were effective in attenuating neurotoxicity. This is the first direct evidence of NO production in the CA1 region as shown in Figure 4. There were also fluorescence cells in the cerebral cortex after stimulation with NMDA. Imaging techniques using DAF-2 DA should be very useful for the clarification of neuronal NO functions. Calcium sensitive fluorescent probe can monitor intracellular calcium by FURA-2. Combined calcium and NO together can be imaged by excitation light source (DAF-2) excited at 485 nm, and Fura-2 AM excited at 340 nm using dichroic or multichroic mirrors.DAQ, DAR-4M AM are other choices. NOS-II may be involved in Alzheimer's disease as b-amyloid induces NOS-II expression in astrocytes Astrocytes which which can be potentiated by cytokines [Vodovotz et al. 1996].

DAF-FM DA was also applied to imaging of NO generated in rat hippocampus slices by exposure to an aglycemic medium [Reif et al. 2009]. NO production was observed mainly in the CA1 area and was dependent on the concentration of O2. During exposure to an anoxicaglycemic medium, NO was hardly produced while marked elevation of intracellular Ca2+ was observed. Production of NO increased sharply as soon as the perfusate was changed to the normal medium. These results suggest that NOS is activated after reperfusion rather than during ischemia. ROS and NO are important participants in signal transduction that could provide the cellular basis for activity-dependent regulation of neuronal excitability [Yermolaieva, et al. 2000]. In young rat cortical brain slices and undifferentiated PC 12 cells, paired application of depolarization/agonist stimulation and oxidation induces long-lasting potency of subsequent Ca2+ signaling that is reversed by hypoxia. This potency critically depends on NO production and involves cellular ROS utilization. Using the fluorescent dye DAF-2, NO production was measured in PC 12 cells during depolarization or histamine application [Yermolaieva, et al. 2000]. Application of 100 µM histamine induced a significant increase in NO production. The crucial role for NO in oxidative potentiation of Ca2+ signaling triggered by pairing of electrical/agonist stimulation and oxidation demonstrated here provides an insight into the cellular mechanisms involved in neuronal developmental plasticity. NO could influence its effectors by stimulating the cGMP-dependent signaling pathway and/or by acting as a weak radical. Perrier et al. 2009, reported the effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells in heart. In other report, Sergeant et al. 2009, reported the spontaneous Ca2+ waves in rabbit corpus cavernosum were modulated by nitric oxide. Role of cGMP was crucial in regulation of calcium dependent neuroactive energy changes.

#### **4.11 Ion channels**

Most voltage-gated Na+ channels are almost completely inactivated at depolarized membrane potentials, but in some cells a residual Na+ current is seen that is resistant to inactivation. The biological signaling mechanisms that regulate the persistence of Na+ channels are not well understood. Ahern et al. 2000, showed that in nerve terminals and ventricular myocytes, NO reduces the inactivation of Na+ current. This effect was independent of cGMP and blocked by *N*-ethylmaleimide. Thus, ROS act directly on the channel or a closely associated protein. Application of ionomycin to raise the intracellular Ca2+ concentration in myocytes activated NOS. The NO produced in response to ionomycin was detected with DAF-2 DA. They concluded that NO is a potential endogenous regulator of persistent Na+ current under physiological and pathophysiological conditions. Choi et al. 2000, reported that the NMDA receptor (NMDAR)-associated ion channel is modulated not only by exogenous NO, but also by endogenous NO. They examined inhibition of NMDAR responses by endogenous NO to determine the underlying molecular mechanism. For this purpose, HEK 293 cells transfected with nNOS were transiently transfected with wild-type NR1/NR2A or mutant NR1/NR2A (C399A) receptors. NMDA responses were monitored by digital Ca2+ imaging with fura-2. Production of endogenous NO by these HEK-nNOS cells was measured with DAF-2. It was concluded that endogenous *S*-nitrosylation may regulate ion channel activity.

#### **4.12 Fertilization and developmental biology**

Kuo et al. 2000, demonstrated that NO is necessary and sufficient for egg activation at fertilization. The early steps that lead to the rise in Ca2+ and egg activation at fertilization are unknown but are of great interest, particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. They showed that active NOS is present at high concentration in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the Ca2+ pulse of fertilization. They used DAF-2 DA for detection of NO. It was concluded that NOS- and NO-related bioactivity satisfy the primary criteria of an egg activator, i.e. they are present in an appropriate place, active at an appropriate time, and necessary and sufficient for successful fertilization.

#### **4.13 Inflammation**

154 Enzyme Inhibition and Bioapplications

Reif et al. 2009, reported the influence of functional variant of neuronal nitric oxide synthase on impulsive behaviors in humans. The biological functions of NO in the neuronal system remain controversial. Using DAF-2 DA for direct detection of NO, Nagano et al. 2002, examined both acute rat brain slices and organotypic culture of brain slices to ascertain NO production sites. The fluorescence intensity sensitive to Ca++ in the CA1 region of the hippocampus was augmented, especially after stimulation with NMDA, in acute brain slices. This NO production in the CA1 region was also confirmed in cultured hippocampus. Glutamate and NO were important mediators, since antagonists of both N-methyl-D-aspartate (NMDA) and NOS inhibitors were effective in attenuating neurotoxicity. This is the first direct evidence of NO production in the CA1 region as shown in Figure 4. There were also fluorescence cells in the cerebral cortex after stimulation with NMDA. Imaging techniques using DAF-2 DA should be very useful for the clarification of neuronal NO functions. Calcium sensitive fluorescent probe can monitor intracellular calcium by FURA-2. Combined calcium and NO together can be imaged by excitation light source (DAF-2) excited at 485 nm, and Fura-2 AM excited at 340 nm using dichroic or multichroic mirrors.DAQ, DAR-4M AM are other choices. NOS-II may be involved in Alzheimer's disease as b-amyloid induces NOS-II expression in astrocytes

Astrocytes which which can be potentiated by cytokines [Vodovotz et al. 1996].

crucial in regulation of calcium dependent neuroactive energy changes.

Most voltage-gated Na+ channels are almost completely inactivated at depolarized membrane potentials, but in some cells a residual Na+ current is seen that is resistant to

**4.11 Ion channels** 

DAF-FM DA was also applied to imaging of NO generated in rat hippocampus slices by exposure to an aglycemic medium [Reif et al. 2009]. NO production was observed mainly in the CA1 area and was dependent on the concentration of O2. During exposure to an anoxicaglycemic medium, NO was hardly produced while marked elevation of intracellular Ca2+ was observed. Production of NO increased sharply as soon as the perfusate was changed to the normal medium. These results suggest that NOS is activated after reperfusion rather than during ischemia. ROS and NO are important participants in signal transduction that could provide the cellular basis for activity-dependent regulation of neuronal excitability [Yermolaieva, et al. 2000]. In young rat cortical brain slices and undifferentiated PC 12 cells, paired application of depolarization/agonist stimulation and oxidation induces long-lasting potency of subsequent Ca2+ signaling that is reversed by hypoxia. This potency critically depends on NO production and involves cellular ROS utilization. Using the fluorescent dye DAF-2, NO production was measured in PC 12 cells during depolarization or histamine application [Yermolaieva, et al. 2000]. Application of 100 µM histamine induced a significant increase in NO production. The crucial role for NO in oxidative potentiation of Ca2+ signaling triggered by pairing of electrical/agonist stimulation and oxidation demonstrated here provides an insight into the cellular mechanisms involved in neuronal developmental plasticity. NO could influence its effectors by stimulating the cGMP-dependent signaling pathway and/or by acting as a weak radical. Perrier et al. 2009, reported the effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells in heart. In other report, Sergeant et al. 2009, reported the spontaneous Ca2+ waves in rabbit corpus cavernosum were modulated by nitric oxide. Role of cGMP was

**4.10 Brain and neuronal behavior** 

NO plays important roles in inflammatory processes. Lopez-Figueroa et al. 2000, examined whether changes in NOS mRNA expression lead to similar temporal and anatomical changes in NO production in an experimental model of CNS inflammation. iNOS mRNA expression was analyzed 2, 4, 6, and 24 h after intracerebroventricular (icv) injection of interleukin-1β or the vehicle. Increased expression of iNOS mRNA was observed surrounding the microinjection site and meninges. Using DAF-2 DA for the direct detection of NO production, they observed a significant increase in NO production after 4 and 6 h. They conclude that increase in iNOS mRNA following icv administration of IL-1β leads to increases in NO production. They proposed that the DAF-2 DA method can be used as a potential marker in the diagnosis of CNS inflammation. MRI of NO is very useful because NO is available at high concentrations in millimolar levels in immune-related diseases (e.g., inflammation).Terashima et al. 2010, reported in vivo detection of iNOS expression in murine carotid lesions by biuoluminescence may provide a valuable approach for monitoring vascular gene expression and inflammation in small animal models. The phasic

Inhibition of Nitric Oxide Synthase Gene Expression:

**4.16 Mitochondria** 

mitochondrial NOS.

**4.17 Retina** 

**4.18 Drosophila** 

**4.19 Plants** 

response mechanisms to hypoxia.

nitrotyrosine within cellular proteins in a dose-dependent manner.

measured the intracellular NO level in real-time using DAF-2.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 157

irradiation doses of 2-50 Gy stimulated the expression of iNOS. The activation of iNOS was accompanied by an increase in the fluorescence of DAF-2 and accumulation of 3-

NO has been implicated in the modulation of mitochondrial respiration, membrane potential, and subsequently apoptosis. Although the presence of mitochondrial NOS was reported while there is no direct evidence in vivo of the presence of NO within mitochondria. Using DAF-2 DA, Lopez-Figueroa et al. observed NO production in PC 12 and COS-1 cells by conventional and confocal fluorescence microscopy [Lopez-Figueroa, et al. 2000]. The subcellular distribution of NO production is consistent with the presence of a

In the retina, NO functions in network coupling, light adaptation, neurotransmitter receptor function, and synaptic release. Neuronal NOS is present in the retina of every vertebrate species so far investigated. However, although nNOS can be found in every retinal cell type, little is known about the production of NO in specific cells or about the diffusion of NO within the retina. Blute et al. 2000, used DAF-2 to image real-time NO production in turtle retina in response to stimulation with NMDA. In response to NMDA, NO was produced in somata in the ganglion cell and inner nuclear layers, in synaptic boutons and processes in the inner plexiform layer, in processes in the outer plexiform layer, and in photoreceptor inner segments. They concluded that NO may function at specific synapses, modulate gene expression, or coordinate events throughout the cell. Matsuo et al. 2000, reported that basal NO production is enhanced by hydraulic pressure in cultured human trabecular cell. He

Wingrove and O'Farrell 1999, reported that *Drosophila* utilizes components of the NO/cGMP signaling pathway to respond to hypoxia. Hypoxic exposure rapidly induced exploratory behavior in larvae and arrested the cell cycle. These behavioral and cellular responses were diminished by an inhibitor of NOS and by a polymorphism of cGMP-dependent protein kinase. Regions of the larvae that specialize in responding to hypoxia should express significant levels of the activities involved. They used DAF-2 DA to define possible foci of function. DAF-2 DA stained the pouch of tissue surrounding the anterior spiracles as well as neuronal-like processes within the pouch. This staining near the openings of the tracheal system is interesting because the location is consistent with a possible role in governing the opening of the spiracles to increase access to oxygen. DAF-2 DA is useful for clarification of

Leaves and callus of *Kalanchoe daigremontiana* and *Taxus brevifolia* were used to investigate NO-induced apoptosis in plant cells [Huang, et al. 2000]. NO production was visualized in cells and tissues with DAF-2 DA. The NO burst preceded a significant increase in nuclear

nature of inflammation is also associated with a decrease in NOS activity. NOS inhibitors are effective at alleviating pain and have disease modifying effects. Induction of NOS-II is seen in models of septic shock. NOS-II inhibitors (dexamethasone) prevent hypotension and decrease in PO2.

Fig. 15. Optical imaging of iNOS expression. (A) Fluorescence imaging of iNOS in living cells. Left: cells were induced for iNOS expression; Right: cells were not induced for iNOS expression. (B) Bioluminescence signal from the knee joints (circle) indicates iNOS expression following inflammation induction. Adapted from reference [Panda, et al. 2000].

#### **4.14 Adrenal zona glomerulosa**

Adrenal zona glomerulosa (ZG) cells do not contain NOS. Hanke et al. 2000, conferred endothelial NOS activity upon adrenal ZG cells through transduction with a recombinant adenovirus encoding the endothelial NOS gene (AdeNOS) to determine the effect of endogenous NO on aldosterone synthesis. AdeNOS-transduced cells exhibited DAF-2 DA fluorescence, which was blocked by pretreatment with an NOS inhibitor. In conclusion, adenovirus-mediated gene transfer of eNOS in ZG cells results in the expression of active endothelial NOS enzyme, and this endogenous NO production by ZG cells decreases aldosterone synthesis.

#### **4.15 Bone marrow stromal cells**

Gorbunov et al. 2000, reported that using the reverse transcription-polymerase chain reaction and immunofluorescence analysis of murine bone marrow stromal cells after -

irradiation doses of 2-50 Gy stimulated the expression of iNOS. The activation of iNOS was accompanied by an increase in the fluorescence of DAF-2 and accumulation of 3 nitrotyrosine within cellular proteins in a dose-dependent manner.

#### **4.16 Mitochondria**

156 Enzyme Inhibition and Bioapplications

nature of inflammation is also associated with a decrease in NOS activity. NOS inhibitors are effective at alleviating pain and have disease modifying effects. Induction of NOS-II is seen in models of septic shock. NOS-II inhibitors (dexamethasone) prevent hypotension and

Fig. 15. Optical imaging of iNOS expression. (A) Fluorescence imaging of iNOS in living cells. Left: cells were induced for iNOS expression; Right: cells were not induced for iNOS

expression following inflammation induction. Adapted from reference [Panda, et al. 2000].

Adrenal zona glomerulosa (ZG) cells do not contain NOS. Hanke et al. 2000, conferred endothelial NOS activity upon adrenal ZG cells through transduction with a recombinant adenovirus encoding the endothelial NOS gene (AdeNOS) to determine the effect of endogenous NO on aldosterone synthesis. AdeNOS-transduced cells exhibited DAF-2 DA fluorescence, which was blocked by pretreatment with an NOS inhibitor. In conclusion, adenovirus-mediated gene transfer of eNOS in ZG cells results in the expression of active endothelial NOS enzyme, and this endogenous NO production by ZG cells decreases

Gorbunov et al. 2000, reported that using the reverse transcription-polymerase chain reaction and immunofluorescence analysis of murine bone marrow stromal cells after -

expression. (B) Bioluminescence signal from the knee joints (circle) indicates iNOS

decrease in PO2.

**4.14 Adrenal zona glomerulosa** 

**4.15 Bone marrow stromal cells** 

aldosterone synthesis.

NO has been implicated in the modulation of mitochondrial respiration, membrane potential, and subsequently apoptosis. Although the presence of mitochondrial NOS was reported while there is no direct evidence in vivo of the presence of NO within mitochondria. Using DAF-2 DA, Lopez-Figueroa et al. observed NO production in PC 12 and COS-1 cells by conventional and confocal fluorescence microscopy [Lopez-Figueroa, et al. 2000]. The subcellular distribution of NO production is consistent with the presence of a mitochondrial NOS.

#### **4.17 Retina**

In the retina, NO functions in network coupling, light adaptation, neurotransmitter receptor function, and synaptic release. Neuronal NOS is present in the retina of every vertebrate species so far investigated. However, although nNOS can be found in every retinal cell type, little is known about the production of NO in specific cells or about the diffusion of NO within the retina. Blute et al. 2000, used DAF-2 to image real-time NO production in turtle retina in response to stimulation with NMDA. In response to NMDA, NO was produced in somata in the ganglion cell and inner nuclear layers, in synaptic boutons and processes in the inner plexiform layer, in processes in the outer plexiform layer, and in photoreceptor inner segments. They concluded that NO may function at specific synapses, modulate gene expression, or coordinate events throughout the cell. Matsuo et al. 2000, reported that basal NO production is enhanced by hydraulic pressure in cultured human trabecular cell. He measured the intracellular NO level in real-time using DAF-2.

#### **4.18 Drosophila**

Wingrove and O'Farrell 1999, reported that *Drosophila* utilizes components of the NO/cGMP signaling pathway to respond to hypoxia. Hypoxic exposure rapidly induced exploratory behavior in larvae and arrested the cell cycle. These behavioral and cellular responses were diminished by an inhibitor of NOS and by a polymorphism of cGMP-dependent protein kinase. Regions of the larvae that specialize in responding to hypoxia should express significant levels of the activities involved. They used DAF-2 DA to define possible foci of function. DAF-2 DA stained the pouch of tissue surrounding the anterior spiracles as well as neuronal-like processes within the pouch. This staining near the openings of the tracheal system is interesting because the location is consistent with a possible role in governing the opening of the spiracles to increase access to oxygen. DAF-2 DA is useful for clarification of response mechanisms to hypoxia.

#### **4.19 Plants**

Leaves and callus of *Kalanchoe daigremontiana* and *Taxus brevifolia* were used to investigate NO-induced apoptosis in plant cells [Huang, et al. 2000]. NO production was visualized in cells and tissues with DAF-2 DA. The NO burst preceded a significant increase in nuclear

Inhibition of Nitric Oxide Synthase Gene Expression:

NO sensitive imaging techniques as shown in Table 4.

**5.1 Real-time NO imaging** 

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 159

about the potential toxicity caused by such studies. Lastly, relatively long data acquisition time is typically needed to generate a detectable MR signal in MR imaging. Since the lifetime of NO and/or related species is usually short, the accuracy of MRI measurement of NO concentration is questionable. After these issues are resolved, it will need sequence of investigations to calibrate linearity of NO concentration and image signal intensity, image reproducibility, image component analysis, signal measurement accuracy and precision of

**Target Modality Resolution Sensitivity Penetration Clinical potential** 

cNOS PET +++ ++++ ++++ Medium-High

Nitric oxide (NO) is a small uncharged free radical abundant in nanomolar quantities that is

 When generated in vascular endothelial cells, NO plays a key role in vascular tone regulation and EPR/optical/MRI visible signals visualize the vascular physiology (induced fitting of L-arginine specific NOS active site) as prosposed by author to inhibit

In this direction, short lived NO specific amplifier-coupled fluorescent indicator was reported to visualize physiological nanomolar dynamics of NO in living cells (detection limit of 0.1 nM)[Ueno, et al. 2002, Burnett, et al. 2002]. An amplifier-coupled fluorescent indicator visualizes NO in single living cells. Its domain structures are sGCα, sGCβ, CGY, sGCα-CGY, and sGCβ-CGY. This genetically encoded high-sensitive indicator revealed that ≈1 nM of NO was enough to relax blood vessels [Ueno, et al. 2002, Burnett, et al. 2002, Barouch, et al. 2002, Hillebrand et al. 2009, Ciplak, et al. 2009, Gragasin, et al. 2004]. The nanomolar range of basal endothelial NO thus revealed appeared to be fundamental to vascular homeostasis. A fuorescent probe, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3,4 diaminophenyl)-diXuoroboradiaza-*s*-indacence (TMDCDABODIPY), produced real-time image NO of PC12 cells, Sf9 cells and human vascular endothelial cells in the presence of Larginine with inverted fuorescence microscope [Manuel, et al. 2002]. NO production in the cells was successfully captured and imaged with improved temporal and spatial resolution. The bioimaging applications of TMDCDABODIPY as a fuorescent imaging probe for NO in living cells was compared with the other fuorescent imaging probes for NO. The TMDCDABODIPY had the advantages of shorter reaction time, higher photostability and minimal background. The study has indicated that TMDCDABODIPY-based fuorescence microscopy may be a promising approach for NO-related pathological and physiological

NO Optical ++++ ++++ + Low NO EPR ++ ++++ +++ Medium NO MRI +++ + ++++ Medium NO PET ++ ++++ +++ Medium cNOS Optical ++++ +++ ++ Low

Table 4. Comparison of molecular imaging of NO and cNOS expression

involved in diverse physiological and pathophysiological mechanisms.

NOS and low NO production shown in Figure 5.

DNA fragmentation and cell death. L-NMMA significantly decreased NO production and apoptosis in both species. Pedroso et al. 2000a, 2000b concluded that NO is involved in DNA damage leading to cell death and proposed a potential role of NO as a signal molecule in these plants. Foissner et al.2000, used DAF-2 DA, in conjunction with confocal laser scanning microscopy, for in vivo real-time imaging of an elicitor-induced NO burst in tobacco. A growing body of evidence suggests that NO, an important signaling and defense molecule in mammals, plays a key role in activating disease resistance in plants, acting as a signaling molecule and possibly also as a direct antimicrobial agent. The results revealed additional similarities between plant and animal host responses to infection.

#### **5. NO/NOS bioimaging: Future prospects**

The existing techniques based on quenching spin trap or paramagnetic heavy metals pose a concern of contrast agent toxicity with limitations of event capturing to generate NO image of isolated cells by EPR or in vivo body image by MGD enhanced MRI. However, unique role of short lived NO molecule in the cell is peculiar to give insight of rapid ionic regulatory mechanisms such as calcium, sodium dependent signaling events including dietary effects, antimicrobial therapy on inflammation, angiogenesis and hypoxia reported in last three years [Manuel, et al. 2002, Palombo, et al. 2009]. To date, the main imaging modalities for visualization of NO include fluorescence and bio/chemiluminescence optical imaging, electron paramagnetic resonance (EPR) imaging, and MRI. cNOS has been mainly investigated using optical and PET imaging. Paper has presented a progress to date on multimodality imaging of NO and addressed the future research directions and obstacles of NO imaging. Other major developments are expected in design of robust imaging hardware with improved image generating capability and highly sensitive to the rapid changes of NO/NOS molecular EPR signal and MRI frequency with time. In this direction, active research efforts suggest the development and availability of less toxic ion sensitive contrast agents attached with EPR and MRI visible molecules as labels sensitive to fluorometric and magnetic resonance techniques respectively. It is attributed that cytochromes are best candidates as fluorochromic agents while lipopolysaccharides and carbamates with paramagnetic agents are potential as MRI contrast agents. More likely, fast imaging techniques such as parallel phase array, SENSE, FISP MRI imaging, xenon based PARACEST techniques may be available to capture rapid NO changes or distribution associated with metabolic short lived events such as oxygen sensitivity, sodium sensitivity in the intact tissue. In future, advances in ionic ratiomatric techniques will be available to capture the short living NO molecular distribution in cells as real-time events associated with motion. Still major issues to resolve are:


The biggest disadvantage of MRI is its low molecular sensitivity. The need for a high dose of imaging agents (typically more than 100 mM injection) undoubtedly raises major concerns about the potential toxicity caused by such studies. Lastly, relatively long data acquisition time is typically needed to generate a detectable MR signal in MR imaging. Since the lifetime of NO and/or related species is usually short, the accuracy of MRI measurement of NO concentration is questionable. After these issues are resolved, it will need sequence of investigations to calibrate linearity of NO concentration and image signal intensity, image reproducibility, image component analysis, signal measurement accuracy and precision of NO sensitive imaging techniques as shown in Table 4.


Table 4. Comparison of molecular imaging of NO and cNOS expression

#### **5.1 Real-time NO imaging**

158 Enzyme Inhibition and Bioapplications

DNA fragmentation and cell death. L-NMMA significantly decreased NO production and apoptosis in both species. Pedroso et al. 2000a, 2000b concluded that NO is involved in DNA damage leading to cell death and proposed a potential role of NO as a signal molecule in these plants. Foissner et al.2000, used DAF-2 DA, in conjunction with confocal laser scanning microscopy, for in vivo real-time imaging of an elicitor-induced NO burst in tobacco. A growing body of evidence suggests that NO, an important signaling and defense molecule in mammals, plays a key role in activating disease resistance in plants, acting as a signaling molecule and possibly also as a direct antimicrobial agent. The results revealed

The existing techniques based on quenching spin trap or paramagnetic heavy metals pose a concern of contrast agent toxicity with limitations of event capturing to generate NO image of isolated cells by EPR or in vivo body image by MGD enhanced MRI. However, unique role of short lived NO molecule in the cell is peculiar to give insight of rapid ionic regulatory mechanisms such as calcium, sodium dependent signaling events including dietary effects, antimicrobial therapy on inflammation, angiogenesis and hypoxia reported in last three years [Manuel, et al. 2002, Palombo, et al. 2009]. To date, the main imaging modalities for visualization of NO include fluorescence and bio/chemiluminescence optical imaging, electron paramagnetic resonance (EPR) imaging, and MRI. cNOS has been mainly investigated using optical and PET imaging. Paper has presented a progress to date on multimodality imaging of NO and addressed the future research directions and obstacles of NO imaging. Other major developments are expected in design of robust imaging hardware with improved image generating capability and highly sensitive to the rapid changes of NO/NOS molecular EPR signal and MRI frequency with time. In this direction, active research efforts suggest the development and availability of less toxic ion sensitive contrast agents attached with EPR and MRI visible molecules as labels sensitive to fluorometric and magnetic resonance techniques respectively. It is attributed that cytochromes are best candidates as fluorochromic agents while lipopolysaccharides and carbamates with paramagnetic agents are potential as MRI contrast agents. More likely, fast imaging techniques such as parallel phase array, SENSE, FISP MRI imaging, xenon based PARACEST techniques may be available to capture rapid NO changes or distribution associated with metabolic short lived events such as oxygen sensitivity, sodium sensitivity in the intact tissue. In future, advances in ionic ratiomatric techniques will be available to capture the short living NO molecular distribution in cells as real-time events associated

lack of NO sensitive EPR or MRI contrast agent to generate quicker and enough MRI or

 the images generated from MRI visible paramagnetic ions or EPR visible spin trap agents may represent several physical and molecular events sensitive to attached NO quenching carbamates or polysaccharides for MRI and cytochrome proteins for EPR. The biggest disadvantage of MRI is its low molecular sensitivity. The need for a high dose of imaging agents (typically more than 100 mM injection) undoubtedly raises major concerns

the EPR and MRI signals are not true representative of NO concentrations;

additional similarities between plant and animal host responses to infection.

**5. NO/NOS bioimaging: Future prospects** 

with motion. Still major issues to resolve are:

EPR signal visible as image;

Nitric oxide (NO) is a small uncharged free radical abundant in nanomolar quantities that is involved in diverse physiological and pathophysiological mechanisms.

 When generated in vascular endothelial cells, NO plays a key role in vascular tone regulation and EPR/optical/MRI visible signals visualize the vascular physiology (induced fitting of L-arginine specific NOS active site) as prosposed by author to inhibit NOS and low NO production shown in Figure 5.

In this direction, short lived NO specific amplifier-coupled fluorescent indicator was reported to visualize physiological nanomolar dynamics of NO in living cells (detection limit of 0.1 nM)[Ueno, et al. 2002, Burnett, et al. 2002]. An amplifier-coupled fluorescent indicator visualizes NO in single living cells. Its domain structures are sGCα, sGCβ, CGY, sGCα-CGY, and sGCβ-CGY. This genetically encoded high-sensitive indicator revealed that ≈1 nM of NO was enough to relax blood vessels [Ueno, et al. 2002, Burnett, et al. 2002, Barouch, et al. 2002, Hillebrand et al. 2009, Ciplak, et al. 2009, Gragasin, et al. 2004]. The nanomolar range of basal endothelial NO thus revealed appeared to be fundamental to vascular homeostasis. A fuorescent probe, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3,4 diaminophenyl)-diXuoroboradiaza-*s*-indacence (TMDCDABODIPY), produced real-time image NO of PC12 cells, Sf9 cells and human vascular endothelial cells in the presence of Larginine with inverted fuorescence microscope [Manuel, et al. 2002]. NO production in the cells was successfully captured and imaged with improved temporal and spatial resolution. The bioimaging applications of TMDCDABODIPY as a fuorescent imaging probe for NO in living cells was compared with the other fuorescent imaging probes for NO. The TMDCDABODIPY had the advantages of shorter reaction time, higher photostability and minimal background. The study has indicated that TMDCDABODIPY-based fuorescence microscopy may be a promising approach for NO-related pathological and physiological

Inhibition of Nitric Oxide Synthase Gene Expression:

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 161

NOS in living subjects with high fidelity which will greatly facilitate scientists and clinicians in the development of new drugs and patient management[Oliveira, et al. 2010, Payne, et al. 2010]. Lastly, novel NO/NOS imaging agents with optimal in vivo stability and suitable pharmacokinetics such as pteridines, imidazoles, quinolines for clinical translation will

Imaging of NOS expression mainly relies on labeling high affinity, preferably also high specificity, NOS inhibitors with optimal membrane permeability and pharmacokinetics. Of all the fluorescence imaging agents for NO, the DAFC compounds are the most widely used. The requirement of oxygen restricts their applicability in cancer-related NO imaging to a certain extent, although some have been tested for NO imaging during tumor angiogenesis [Kashiwagi, et al. 2005]. Copper-based fluorescent compounds do not require oxygen for NO imaging, yet the stability and toxicity of those compounds are questionable. FRET-based NO imaging is a clever approach, yet it is too technically challenging to be widely available and useful. With spin-trapping technology, EPR imaging of NO can be quite accurate, yet it is not applicable for clinical studies due to many reasons. MRI can have high resolution and large field-of-view for potential clinical imaging studies, yet it is usually quite slow in data acquisition and the intrinsic low sensitivity makes molecular MRI unlikely to succeed in the clinic. NOS imaging is also important in elucidating NO-related physiological and pathological processes. NOS imaging has not been well studied to date. To the best of our knowledge, no clinical studies of NO or NOS imaging have been reported. Whether NO/NOS imaging can help and improve patient management remains to be demonstrated in the future. The deregulation of iNOS in melanoma has been correlated directly with poor survival [Madunapantula, et al. 2008] and NOS expression in patients with neurological disorder has been studied [Broholm, et al. 2004]. Given the fact that NO and NOS plays diverse roles in many diseases such as inflammation, neurodegeneration, cancer, and vascular malfunctions, further understanding of the disease mechanisms and clarification of the temporal/ spatial NO/NOS expression are certainly very important in patient management. Whether imaging NO or NOS is more relevant depends primarily on the disease. eNOS and nNOS typically only produces low levels of NO; therefore, imaging NO is expected to be more difficult than imaging the NOSs themselves. On the other hand, iNOS can produce high concentrations of NO in immunological processes and/or cancer; hence imaging NO may be more desirable in these scenarios. Granted that NO imaging is more relevant since it directly affects the physiological and pathological processes, imaging of NO may not always be feasible and imaging NOS expression (as an alternative) can provide valuable insights. The notion that nitrite reduction can also serve as a possible source of biologically relevant NO tilted the balance toward NO imaging [Bryan, 2006]. Nitrite reduction to NO can occur via several routes involving enzymes, proteins, vitamins, or even simple protons [Galdwin, 2004, Lundberg, et al. 2005]. Nitrite is now under active investigations in physiology, pathophysiology, and therapeutics. The ideas that nitrite can be a "storage" form of NO and the possible importance of dietary nitrite are two vibrant research areas today. Similar to the important role of L-arginine for optimal NOS activity, nitrite may emerge as an essential nutrient. This pathway may serve as a backup system for NO generation in conditions such as hypoxia, in which the NOS/L-arginine system is compromised. We envision that imaging NO and NOS in the same system will undoubtedly give a more thorough picture in understanding the functions of NO and NOS. For potential clinical translation of NO/NOS imaging probes, PET tracers have the best chances of

benefit in patient management [Payne, et al. 2010, Bonnefous, et al. 2009].

studies [Hiratsuka, et al. 2009]. A recent review highlighted the possibilities if nitric oxide bioimaging becomes reality as clinical imaging tool [Hong, et al. 2009]. Current state-of-theart multimodality imaging may detect NO and NOS enzyme expression [Matter et al. 2004, Zhang, et al. 2011]. Optical (fluorescence, chemiluminescence, and bioluminescence), electron paramagnetic resonance (EPR), magnetic resonance (MR), and positron emission tomography (PET) noninvasive imaging techniques may reveal the biodistribution of NO or

Fig. 15. (on top) Formation and release of NO in tissues is shown. No is trapped by Fe2+(MGD)2 to make MRI/EPR visible trap NO-(MGD)2-Fe3+. (in middle row)The figure represents how different levels of Nitric oxide (1) activate cytosolic guanylate cyclase (2) and thus elevate intracellular levels of cyclic GMP (3 and 4) as secondary messenger. The cyclic GMP stimulates response (5) in different tissues and nitric oxide can be detected as biomarker of tissue pathology.(at bottom) The relation of released NO across the membrane is shown with different biological and physiological changes to generate EPR and MRI signal visible by imaging techniques. Notice the NO regulation by enzyme guanylate cyclase over intracellular responses (cell injury, inflammation etc.) sensitive to spin trap MRI/EPR image signals. A proposal of multimodal nitric oxide imaging is shown for development of multimodal spin trap applicable in imaging.

studies [Hiratsuka, et al. 2009]. A recent review highlighted the possibilities if nitric oxide bioimaging becomes reality as clinical imaging tool [Hong, et al. 2009]. Current state-of-theart multimodality imaging may detect NO and NOS enzyme expression [Matter et al. 2004, Zhang, et al. 2011]. Optical (fluorescence, chemiluminescence, and bioluminescence), electron paramagnetic resonance (EPR), magnetic resonance (MR), and positron emission tomography (PET) noninvasive imaging techniques may reveal the biodistribution of NO or

Fig. 15. (on top) Formation and release of NO in tissues is shown. No is trapped by Fe2+(MGD)2 to make MRI/EPR visible trap NO-(MGD)2-Fe3+. (in middle row)The figure represents how different levels of Nitric oxide (1) activate cytosolic guanylate cyclase (2) and thus elevate intracellular levels of cyclic GMP (3 and 4) as secondary messenger. The cyclic

GMP stimulates response (5) in different tissues and nitric oxide can be detected as

multimodal spin trap applicable in imaging.

biomarker of tissue pathology.(at bottom) The relation of released NO across the membrane is shown with different biological and physiological changes to generate EPR and MRI signal visible by imaging techniques. Notice the NO regulation by enzyme guanylate cyclase over intracellular responses (cell injury, inflammation etc.) sensitive to spin trap MRI/EPR image signals. A proposal of multimodal nitric oxide imaging is shown for development of

NOS in living subjects with high fidelity which will greatly facilitate scientists and clinicians in the development of new drugs and patient management[Oliveira, et al. 2010, Payne, et al. 2010]. Lastly, novel NO/NOS imaging agents with optimal in vivo stability and suitable pharmacokinetics such as pteridines, imidazoles, quinolines for clinical translation will benefit in patient management [Payne, et al. 2010, Bonnefous, et al. 2009].

Imaging of NOS expression mainly relies on labeling high affinity, preferably also high specificity, NOS inhibitors with optimal membrane permeability and pharmacokinetics. Of all the fluorescence imaging agents for NO, the DAFC compounds are the most widely used. The requirement of oxygen restricts their applicability in cancer-related NO imaging to a certain extent, although some have been tested for NO imaging during tumor angiogenesis [Kashiwagi, et al. 2005]. Copper-based fluorescent compounds do not require oxygen for NO imaging, yet the stability and toxicity of those compounds are questionable. FRET-based NO imaging is a clever approach, yet it is too technically challenging to be widely available and useful. With spin-trapping technology, EPR imaging of NO can be quite accurate, yet it is not applicable for clinical studies due to many reasons. MRI can have high resolution and large field-of-view for potential clinical imaging studies, yet it is usually quite slow in data acquisition and the intrinsic low sensitivity makes molecular MRI unlikely to succeed in the clinic. NOS imaging is also important in elucidating NO-related physiological and pathological processes. NOS imaging has not been well studied to date. To the best of our knowledge, no clinical studies of NO or NOS imaging have been reported. Whether NO/NOS imaging can help and improve patient management remains to be demonstrated in the future. The deregulation of iNOS in melanoma has been correlated directly with poor survival [Madunapantula, et al. 2008] and NOS expression in patients with neurological disorder has been studied [Broholm, et al. 2004]. Given the fact that NO and NOS plays diverse roles in many diseases such as inflammation, neurodegeneration, cancer, and vascular malfunctions, further understanding of the disease mechanisms and clarification of the temporal/ spatial NO/NOS expression are certainly very important in patient management. Whether imaging NO or NOS is more relevant depends primarily on the disease. eNOS and nNOS typically only produces low levels of NO; therefore, imaging NO is expected to be more difficult than imaging the NOSs themselves. On the other hand, iNOS can produce high concentrations of NO in immunological processes and/or cancer; hence imaging NO may be more desirable in these scenarios. Granted that NO imaging is more relevant since it directly affects the physiological and pathological processes, imaging of NO may not always be feasible and imaging NOS expression (as an alternative) can provide valuable insights. The notion that nitrite reduction can also serve as a possible source of biologically relevant NO tilted the balance toward NO imaging [Bryan, 2006]. Nitrite reduction to NO can occur via several routes involving enzymes, proteins, vitamins, or even simple protons [Galdwin, 2004, Lundberg, et al. 2005]. Nitrite is now under active investigations in physiology, pathophysiology, and therapeutics. The ideas that nitrite can be a "storage" form of NO and the possible importance of dietary nitrite are two vibrant research areas today. Similar to the important role of L-arginine for optimal NOS activity, nitrite may emerge as an essential nutrient. This pathway may serve as a backup system for NO generation in conditions such as hypoxia, in which the NOS/L-arginine system is compromised. We envision that imaging NO and NOS in the same system will undoubtedly give a more thorough picture in understanding the functions of NO and NOS. For potential clinical translation of NO/NOS imaging probes, PET tracers have the best chances of

Inhibition of Nitric Oxide Synthase Gene Expression:

Press Inc. Totowa, NJ. Pp 293-311.

**Detection of Nitric Oxide Radical** 

1. NO•-Specific Microelectrode

cellular lipid membrane. iron-MGD and iron-DETC.

2. ESR with NO•-Specific Spin Traps

tetramethylpyrrolidine

micromolar range.

3. ESR measurement:

functional NO•. **Materials needed:** 

(K2K2)(2sb)2(2s\*)2(2pb)4(2pb)2(2p\*)1

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 163

Spin trap contrast agents may enable the simultaneous EPR-MRI and fluorescent imaging of NO distribution in the organs and tissues in muscle vascular system, retina, adrenal gland, brain, bone marrow to monitor intracellular metabolic events such as apoptosis, inflammation, nerve activation and calcium ions etc. To our knowledge, there are no reports of successfully imaging or detecting free radicals such as NO in vivo clinical imaging. Due to short lifetime and rapid diffusion of NO in tissue, it is a challenge to devise any effective relaxation enhancement mechanism(s) for noninvasive clinical imaging. NMR spin trapping may serve to overcome these problems of NO short lifetime and diffusion to some extent.

Source: Cai H., Dikalov S, Griendling K.K., Harrison, D.G.(2007) Detection of reactive oxygen species and nitric oxide in vascular cells and tissues. Chapter 20 In: Methods in Molecular Medicine, Vascular Biology Protocols. Editor: Sreejayan N. and Ron J., Humana

It has been challenging to detect nitric oxide radical (NO•) directly from biological samples. NO• "production" is measured by NO• synthase activity using the L-arginine conversion assay or NO• metabolites nitrite and nitrate using the Griess reagent. New methods are NO•-selective electrode and ESR represent specific and quantitative assays for detection of

Nafion and o-PD coated carbon electrode can directly detect NO• in the low

Dithiocarbamate (DTC), N-methylglucamine dithiocarbamate (MGD) detect extracellular NO\*, and diethyldithiocarbamate (DETC) trap and detect the NO\* in

Cyclic hydroxylamine CPH or 1-hydroxy-3-methoxycarbonyl-2,2,5,5-

 USA) in modified Kreb's/HEPES buffer containing metal chelator, 25–50mM deferoximine, and 3 5mM DETC. This stoc k solution should be de-oxygenated by nitrogen gas continuously to maintain low background oxidation of the spin traps.

 Lysis buffer containing protease inhibitors: 50mM Tris–HCl buffer, pH 7.4, containing 0.1mM ethylenediamine tetraacetic acid (EDTA), 0.1mM ethylene

**7. Appendix 1: Protocols on NOS and NO\* radical detection** 

Electronic configuration in NO\* with 11 valence electrons:

Endothelial or vascular smooth muscle cells.

Modified Krebs/HEPES buffer (see Subheading 2.1.1.2.).

(CMH) stock solution (10 mM) (Alexis Biochemicals, San Diego, CA,

success. Each imaging modality has its advantages and disadvantages in terms of sensitivity, spatial resolution, temporal resolution, probe availability, and cost [Massoud, et al. 2003]. With the development of hybrid imaging systems such as PET/CT, SPECT/CT, and PET/MRI Catana et al. 2006; Sharma, 2002, 2008, 2011;Townsend, et al. 2002], a multimodality approach may offer synergistic advantages over any single modality alone in the future.

### **6. Conclusion**

Nitric oxide (NO) *in vivo* image visualizes the distribution of NO using the ''spin-trapping'' technique. Available NO visualizing chemical probes combined with the spin traps and fluorescent dyes are promising. Physiological mechanisms of NO production by NOS and role of NOS gene expression altered by NOS inhibitors is illustrated. **Scope:** Different multimodal contrast mechanisms are introduced using nitrosyl-iron complexes as MRI-PET signal intensity enhancers and fluorescent or bioluminescent NO visualizing cheletropic trap agents. NO synthase enzyme is the source of NO formation in presence of competitive inhibitor N-monomethyl-L-arginine in the tissues.

	- Nitric oxide bioimaging is emerging as rapid noninvasive multimodal molecular imaging techniques by fluorescent, MRI, PET, optical bioimaging for wider applications;
	- Significant progress is made to explain the mechanism of multimodal NO spin trap contrast agent enhanced MRI and fluorescent bioimaging;
	- Both MRI and fluorescent contrast agents may serve as potential MRI/fluorescent multimodal spin traps as well as contrast agents in clinical or bioimaging;
	- NO sensitive MRI of vital soft tissues is major achievement to visualize very short lived NO in real time manner.
	- Development of NOS inhibitors or NO production suppressors and in vivo NO/NOS evaluation is a booming industry in diagnostics of stroke, sepsis, neurodegeneration, inflammation and new drug discovery.

Spin trap contrast agents may enable the simultaneous EPR-MRI and fluorescent imaging of NO distribution in the organs and tissues in muscle vascular system, retina, adrenal gland, brain, bone marrow to monitor intracellular metabolic events such as apoptosis, inflammation, nerve activation and calcium ions etc. To our knowledge, there are no reports of successfully imaging or detecting free radicals such as NO in vivo clinical imaging. Due to short lifetime and rapid diffusion of NO in tissue, it is a challenge to devise any effective relaxation enhancement mechanism(s) for noninvasive clinical imaging. NMR spin trapping may serve to overcome these problems of NO short lifetime and diffusion to some extent.

### **7. Appendix 1: Protocols on NOS and NO\* radical detection**

Source: Cai H., Dikalov S, Griendling K.K., Harrison, D.G.(2007) Detection of reactive oxygen species and nitric oxide in vascular cells and tissues. Chapter 20 In: Methods in Molecular Medicine, Vascular Biology Protocols. Editor: Sreejayan N. and Ron J., Humana Press Inc. Totowa, NJ. Pp 293-311.

Electronic configuration in NO\* with 11 valence electrons: (K2K2)(2sb)2(2s\*)2(2pb)4(2pb)2(2p\*)1

#### **Detection of Nitric Oxide Radical**

It has been challenging to detect nitric oxide radical (NO•) directly from biological samples. NO• "production" is measured by NO• synthase activity using the L-arginine conversion assay or NO• metabolites nitrite and nitrate using the Griess reagent. New methods are NO•-selective electrode and ESR represent specific and quantitative assays for detection of functional NO•.

#### **Materials needed:**

162 Enzyme Inhibition and Bioapplications

success. Each imaging modality has its advantages and disadvantages in terms of sensitivity, spatial resolution, temporal resolution, probe availability, and cost [Massoud, et al. 2003]. With the development of hybrid imaging systems such as PET/CT, SPECT/CT, and PET/MRI Catana et al. 2006; Sharma, 2002, 2008, 2011;Townsend, et al. 2002], a multimodality approach may offer synergistic advantages over any single modality alone in

Nitric oxide (NO) *in vivo* image visualizes the distribution of NO using the ''spin-trapping'' technique. Available NO visualizing chemical probes combined with the spin traps and fluorescent dyes are promising. Physiological mechanisms of NO production by NOS and role of NOS gene expression altered by NOS inhibitors is illustrated. **Scope:** Different multimodal contrast mechanisms are introduced using nitrosyl-iron complexes as MRI-PET signal intensity enhancers and fluorescent or bioluminescent NO visualizing cheletropic trap agents. NO synthase enzyme is the source of NO formation in presence of competitive

 **General Significance:** NO makes stable paramagnetic with N-methyl-D-glucamine dithiocarbamate (MGD), as (MGD)2-Fe(II)-NO metal complex *in vivo* by spin trapping and may serve as effective EPR and/or MRI contrast agent than other less stable nitrogen containing radicals, such as bound nitric oxides. MRI spin-trapping induces the magnetic resonance relaxation changes of neighboring protons and visualizes spatial distributions of NO free radicals in pathologic organs and tissues but not confirmed. Other paramagnetic NO-Fe-dithiacarbamate metal complexes serve as contrast agents in EPR bioimaging. Recently, real time physiological in vivo monitoring using tissue physical properties such as fluorescence and magnetic resonance in tissues

 **Major conclusions**: 1. More applications of NO bioimaging and fluorescent probes are emerging in imaging apoptosis, vascular imaging, smooth muscle cells, neurodegeneration, calcium channel imaging and endothelial stress. 2. The status of NO production by NOS in cells gives insight of inflammation, fertilization, mitochondrial oxidation injury, bone structure and adrenal gland and retina. 3. The real time NO

 Nitric oxide bioimaging is emerging as rapid noninvasive multimodal molecular imaging techniques by fluorescent, MRI, PET, optical bioimaging for wider

Significant progress is made to explain the mechanism of multimodal NO spin trap

 Both MRI and fluorescent contrast agents may serve as potential MRI/fluorescent multimodal spin traps as well as contrast agents in clinical or bioimaging; NO sensitive MRI of vital soft tissues is major achievement to visualize very short

 Development of NOS inhibitors or NO production suppressors and in vivo NO/NOS evaluation is a booming industry in diagnostics of stroke, sepsis,

is revisted to visualize nanomolar range of nitric oxide mapping.

contrast agent enhanced MRI and fluorescent bioimaging;

neurodegeneration, inflammation and new drug discovery.

imaging application is emerging in animals and plants.

the future.

**6. Conclusion** 

**Major highlights:** 

applications;

lived NO in real time manner.

inhibitor N-monomethyl-L-arginine in the tissues.

	- Endothelial or vascular smooth muscle cells.
	- Modified Krebs/HEPES buffer (see Subheading 2.1.1.2.).
	- Cyclic hydroxylamine CPH or 1-hydroxy-3-methoxycarbonyl-2,2,5,5 tetramethylpyrrolidine
	- (CMH) stock solution (10 mM) (Alexis Biochemicals, San Diego, CA,
	- USA) in modified Kreb's/HEPES buffer containing metal chelator, 25–50mM
	- deferoximine, and 3 5mM DETC. This stoc k solution should be de-oxygenated by
	- nitrogen gas continuously to maintain low background oxidation of the spin traps.
	- Lysis buffer containing protease inhibitors: 50mM Tris–HCl buffer, pH 7.4,
	- containing 0.1mM ethylenediamine tetraacetic acid (EDTA), 0.1mM ethylene

Inhibition of Nitric Oxide Synthase Gene Expression:

mm vessel segments).

receiver gain, 900.

mm vessel segments).

**8. Acknowledgements** 

28810-5.

**9. References** 

**Iron-DETC Protocol for Intracellular Trapping of NO•** 

NO• signal using ESR at the following settings:

**Iron-MGD Protocol for Extracellular Trapping of NO•** 

1:10 (final Fe concentration: 0.5 mM).

immediately add to culture dish.

modulation amplitude, 3 G; receiver gain, 1×104; and four scans.

then make stock solutions of FeSO4·7H2O, 4 mM, and MGD, 20 mM.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 165

Culture endothelial cells on 100-mm Petri dishes or prepare vascular segments (6–12, 2-

 Aspirate media and rinse cells with warm PBS once, add 1.5 ml modified Kreb's/HEPES buffer with or without desired agonists, then mix Fe2+(DETC)2, and

 Bruker EMX: Field sweep, 160 G; microwave frequency, 9.39 GHz; microwave power, 10MW; modulation amplitude, 3 G; conversion time, 2621 ms; time constant, 328 ms;

 Miniscope 200: Biofield, 3267; field sweep, 100 G; microwave frequency, 9.78 GHz; microwave power, 40 mW; modulation amplitude, 10 G; 4096 points resolution; and

Culture endothelial cells on 100-mm Petri dishes or prepare vascular segments (6–12, 2-

Bubble freshly prepared saline (0.9% NaCl) with nitrogen gas to remove oxygen, and

Prepare stock solutions of Fe2+MGD by mixing FeSO4 and MGD at the ratio of 1:5 or

 Aspirate media and rinse cells with warm PBS once, add 1.5 ml modified Kreb's/HEPES buffer with or without desired agonists, then mix Fe2+(MGD)2, and

 Incubate in cell culture incubator for desired period for cumulative trapping of NO•. Collect 1 ml of post-incubation supernatant into a 1-ml insulin syringe and snap freeze in liquid nitrogen, then transfer sample column into a finger dewer, and capture

Fe2+MGD–NO• signal using ESR settings as described above for Fe2+(DETC)2.

manuscript. Prof Avdhesh Sharma, JNV University, corrected manuscript.

Author acknowledge the manuscript preparation, corrections, expert suggestions by Dr Soonjo Kwon, Utah State University, Logan, UT and Professor Ching J Chen at Florida State University, Tallahassee for extending expert modifications and comments in this

Ahern, G.P., Hsu, S.F., Klyachko,V.A., Jackson, M.B. (2000) Induction of persistent sodium

current by exogenous and endogenous nitric oxide. J Biol Chem, Vol 275, No 37, pp

Bubble freshly prepared saline (0.9% NaCl) with nitrogen gas to remove oxygen.

immediately add to culture dish (500µl of each solution, final volume 2.0 ml). Incubate in cell culture incubator for desired period for cumulative trapping of NO•. Aspirate buffer, gently collect cells into a 1-ml insulin syringe, snap freeze in liquid nitrogen, then transfer sample column into a finger dewer, and capture Fe2+(DETC)2–

	- NADPH (0.2 mM).
	- Xanthine (0.1 mM).

#### **Detection of Nitric Oxide Radical**

*NO•-Specific Microelectrode* 


#### **Iron-DETC for Trapping of NO•**


#### **Methods:**

#### *Detection of Nitric Oxide Radical*

#### *NO•-Specific Microelectrode*


#### **Iron-DETC Protocol for Intracellular Trapping of NO•**

164 Enzyme Inhibition and Bioapplications

Carbon fiber electrodes (100µm length and 30µm outer diameter; Word Precision

Fe2+(DETC)2 : FeSO4·7H2O, 4.45 mg/10 ml for 1.6 mmol/l stock and DETC, 7.21 mg/10

Coat bare carbon fiber electrodes (100µm length and 30µm outer diameter; Word

 Precision Instruments) with nafion and o-PD. Coat with freshly made o-PD solution at constant potential (+0.9V vs. Ag/AgCl reference electrode) for 45 min. Dip in nafion solution for 3 s and dry for 5 min at 85°C. The nafion-coating cycle should be repeated

Culture endothelial cells on 35-mm dishes or prepare fresh tissue samples in freshly

Place the electrode tip at the surface of an individual cell, endocardium, or lumen of

 Record NO•-dependent oxidation currents (voltage clamp mode, hold at 0.65 V, approximately the voltage for peak NO• oxidation) immediately after addition of agonists using an Axopatch 200B amplifier (Axon Instruments). A silver/silver chloride reference electrode is used. Use pCLAMP 7.0 program (Axon Instruments) for delivery

Calculate NO• concentrations from a standard curve obtained using dilutions of de-

bestatin, 1mM pepstatin, and 2mM leupeptin.

 o-PD solution (in 0.1M PBS with 100µM ascorbic acid). Nafion (5% in aliphatic alcohols; Sigma-Aldrich).

N-methyl-d-glucamine dithiocarbamate MGD (20 mM).

Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA).

 NADPH (0.2 mM). Xanthine (0.1 mM).

**Detection of Nitric Oxide Radical** 

Instruments, Sarasota, FL, USA).

Modified Kreb's/HEPES buffer.

**Iron-DETC for Trapping of NO•** 

ml for 3.2 mmol/l stock.

Modified Kreb's/HEPES buffer.

made modified Kreb's/HEPES buffer.

oxygenated, saturated NO\* gas solutions.

blood vessels, and then withdraw precisely 5µm.

of voltage protocols and data acquisition and analysis.

 Culture endothelial cells. Saline (0.9% NaCl).

Ferrous sulfate (4 mM).

*Detection of Nitric Oxide Radical* 

*NO•-Specific Microelectrode* 

10–15 times.

PBS.

**Methods:** 

Silver/silver chloride reference electrode.

*NO•-Specific Microelectrode* 

glycol tetraacetic acid (EGTA), 1mM phenylmethyl sulfonyl fluoride, 2mM


#### **Iron-MGD Protocol for Extracellular Trapping of NO•**


#### **8. Acknowledgements**

Author acknowledge the manuscript preparation, corrections, expert suggestions by Dr Soonjo Kwon, Utah State University, Logan, UT and Professor Ching J Chen at Florida State University, Tallahassee for extending expert modifications and comments in this manuscript. Prof Avdhesh Sharma, JNV University, corrected manuscript.

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**1. Introduction**

et al., 2010).

Transcriptional bursting, also known as transcriptional pulsing, is a fundamental property of genes from bacteria to humans (Chubb et al., 2006; Golding et al., 2005; Raj et al., 2006). Transcription of genes, the process which transforms the stable code written in DNA into the mobile RNA message can occur in "bursts" or "pulses". This phenomenon has recently come to light with the advent of new technologies, such as MS2 tagging, to detect RNA production in single cells, allowing precise measurements of RNA number, or RNA appearance at the gene. Other, more widespread techniques, such as Northern Blotting, Microarrays, RT-PCR and RNA-Seq, measure bulk RNA levels from homogeneous population extracts. These techniques lose dynamic information from individual cells, and give the impression transcription is a continuous smooth process. The reality is that transcription is irregular, with strong periods of activity, interspersed by long periods of inactivity. Averaged over millions of cells, this appears continuous. But at the individual cell level, there is considerable variability,

**in the Tryptophan Operon of** *E. coli* **and Its Effect on the System Stochastic Dynamics** 

*Centro de Investigación y de Estudios Avanzados del IPN, Unidad Monterrey,* 

**Transcriptional Bursting** 

*México* 

**6**

Emanuel Salazar-Cavazos and Moisés Santillán

*Parque de Investigación e Innovación Tecnológica, Apodaca NL* 

The bursting phenomenon, as opposed to simple probabilistic models of transcription, can account for the high variability in gene expression occurring between cells in isogenic populations (Blake et al., 2003). This variability in turn can have tremendous consequences on cell behaviour, and must be mitigated or integrated. In certain contexts, such as the survival of microbes in rapidly changing stressful environments, or several types of scattered differentiation, the variability may be essential (Losick & Desplan, 2008). Variability also impacts upon the effectiveness of clinical treatment, with resistance of bacteria to antibiotics demonstrably caused by non-genetic differences (Lewis, 2010). Variability in gene expression may also contribute to resistance of sub-populations of cancer cells to chemotherapy (Sharma

Bursting may result from the stochastic nature of biochemical events superimposed upon a 2 or more step fluctuation. In its most simple form, the gene can exist in 2 states, one where activity is negligible and one where there is a certain probability of activation (Raj & van Oudenaarden, 2008). Only in the second state does transcription readily occur. Whilst the nuclear and signalling landscapes of complex eukaryotic nuclei are likely to favour more than two simple states—for example, there are over twenty post-translational modifications

and for most genes, very little activity at any one time.


## **Transcriptional Bursting in the Tryptophan Operon of** *E. coli* **and Its Effect on the System Stochastic Dynamics**

Emanuel Salazar-Cavazos and Moisés Santillán *Centro de Investigación y de Estudios Avanzados del IPN, Unidad Monterrey, Parque de Investigación e Innovación Tecnológica, Apodaca NL México* 

#### **1. Introduction**

178 Enzyme Inhibition and Bioapplications

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Transcriptional bursting, also known as transcriptional pulsing, is a fundamental property of genes from bacteria to humans (Chubb et al., 2006; Golding et al., 2005; Raj et al., 2006). Transcription of genes, the process which transforms the stable code written in DNA into the mobile RNA message can occur in "bursts" or "pulses". This phenomenon has recently come to light with the advent of new technologies, such as MS2 tagging, to detect RNA production in single cells, allowing precise measurements of RNA number, or RNA appearance at the gene. Other, more widespread techniques, such as Northern Blotting, Microarrays, RT-PCR and RNA-Seq, measure bulk RNA levels from homogeneous population extracts. These techniques lose dynamic information from individual cells, and give the impression transcription is a continuous smooth process. The reality is that transcription is irregular, with strong periods of activity, interspersed by long periods of inactivity. Averaged over millions of cells, this appears continuous. But at the individual cell level, there is considerable variability, and for most genes, very little activity at any one time.

The bursting phenomenon, as opposed to simple probabilistic models of transcription, can account for the high variability in gene expression occurring between cells in isogenic populations (Blake et al., 2003). This variability in turn can have tremendous consequences on cell behaviour, and must be mitigated or integrated. In certain contexts, such as the survival of microbes in rapidly changing stressful environments, or several types of scattered differentiation, the variability may be essential (Losick & Desplan, 2008). Variability also impacts upon the effectiveness of clinical treatment, with resistance of bacteria to antibiotics demonstrably caused by non-genetic differences (Lewis, 2010). Variability in gene expression may also contribute to resistance of sub-populations of cancer cells to chemotherapy (Sharma et al., 2010).

Bursting may result from the stochastic nature of biochemical events superimposed upon a 2 or more step fluctuation. In its most simple form, the gene can exist in 2 states, one where activity is negligible and one where there is a certain probability of activation (Raj & van Oudenaarden, 2008). Only in the second state does transcription readily occur. Whilst the nuclear and signalling landscapes of complex eukaryotic nuclei are likely to favour more than two simple states—for example, there are over twenty post-translational modifications

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<sup>181</sup> Transcriptional Bursting

tRNA

*T*

RNA Polymerase

DNA, structural gene Trp molecules

D

Fig. 1. Schematic representation of the three regulatory mechanisms found in the tryptophan operon: A) repression, B) transcription attenuation, and C) enzyme inhibition. A glossary with the meaning of all the geometric forms in this figure is shown in panel D. See the main

**Transcription attenuation** works by promoting an early termination of mRNA transcription, see Figure 1B. The transcription starting site in the *trp* operon is separated from *trpE* by a leader region responsible for attenuation control. The transcript of this leader region consists of four segments (termed Segments 1, 2, 3, and 4) which can form three stable hairpin structures between consecutive segments. After the first two segments are transcribed they form a hairpin which stops transcription (c.f. Figure 1B-I). When a ribosome binds the nascent mRNA, it disrupts Hairpin 1:2 and transcription is re-initiated along with translation. Segment 1 contains two tryptophan codons in tandem. If there is scarcity of tryptophan, and thus of loaded tRNATrp, the ribosome stalls in the first segment. The development of Hairpin 2:3 (the antiterminator) is then facilitated, and transcription proceeds until the end (c.f. Figure 1B-II). However, if tryptophan is abundant, the ribosome rapidly finishes translation of Segments 1 and 2 and promotes the formation of a stable hairpin structure between Segments 3 and 4 (c.f. Figure 1B-III). RNA polymerase molecules recognize this

hairpin structure as a termination signal and transcription is prematurely terminated.

**Enzyme inhibition** takes place through anthranilate synthase, the first enzyme to catalyse a reaction in the catalytic pathway that leads to the synthesis of tryptophan from chorismate. This enzyme is a hetero-tetramer consisting of two TrpE and two TrpD polypeptides. Anthranilate synthase is inhibited by tryptophan through negative feedback. This feedback inhibition is achieved when the TrpE subunits in anthranilate synthase are individually bound by a tryptophan molecule, see Figure 1C. Therefore, an excess of intracellular tryptophan inactivates most of the anthranilate synthase protein avoiding the production of more

Repressor molecule Ribosome

Ribosome

Trp polypeptides Anthranilate synthase

mRNA hairpin 3:4

mRNA hairpin 2:3

I

mRNA hairpin 1:2

II

III

*trpP trpL trpE*

TrpB TrpA TrpC

Anthranilate Synthase

*R*

*P*

text for further explanation.

tryptophan.

Tryptophan Biosynthesis

of nucleosomes known, this simple two step model provides a reasonable framework for understanding the changing probabilities affecting transcription. What do the restrictive and permissive states represent? An attractive idea is that the repressed state is a closed chromatin conformation whilst the permissive state is an open one. Another hypothesis is that the fluctuations reflect transition between bound pre-initiation complexes (permissive) and dissociated ones (restrictive) (Blake et al., 2003; Ross et al., 1994). Bursts may also result from bursty signalling, cell cycle effects or movement of chromatin to and from transcription factories. Nonetheless, to the best of our knowledge, there is no generally accepted explanation for this phenomenon. Transcriptional bursting in prokaryotic cell is particularly puzzling given the simplicity of the transcription initiation process, as opposed to eukaryotic cells.

There is evidence that the cooperative interaction of distant operators through a single repressor molecule has a strong influence on the transcriptional bursting observed in the *lac* operon of *E. coli* (Choi et al., 2008). Since the repression regulatory mechanism in *E. coli*'s *trp* operon involves cooperativity between two repressor molecules bound to neighbouring operators, it is interesting to investigate whether such cooperative interaction has any effect upon the system transcriptional dynamics. The present chapter is advocated to tackling this question from a mathematical modelling perspective. We also investigate the effects of the enzymatic feedback-inhibition regulatory mechanism (also present on the *trp* operon regulatory pathway) on the system dynamic behaviour.

#### **2. Theory**

#### **2.1 The** *trp* **operon**

The amino acid tryptophan can be synthesized by bacteria like *E. coli* through a series of catalysed reactions. The catalysing enzymes in *E. coli* are made up of the polypeptides encoded by the tryptophan operon genes: *trpE*, *trpD*, *trpC*, *trpB*, and *trpA*, which are transcribed from *trpE* to *trpA*. Transcription is initiated at promoter *trpP*, which is located upstream from gene *trpE*. The *trp* operon is regulated by three different negative-feedback mechanisms: repression, transcription attenuation, and enzyme inhibition. Below these regulatory mechanisms are briefly reviewed based on (Brown et al., 1999; Grillo et al., 1999; Jeeves et al., 1999; Xie et al., 2003; Yanofsky, 2000; Yanofsky & Crawford, 1987). The reader should consult Figure 1 for a better understanding.

**Repression** in the *trp* operon is mediated by three operators (*O1*, *O2*, and *O3*) overlapping with the operon promoter, *trpP* (see Figure 1A). When an active repressor is bound to an operator it blocks the binding of a RNA polymerase to *trpP* and prevents transcription initiation. The *trp* repressor normally exists as a dimeric protein (called the *trp* aporepressor) and may or may not be complexed with tryptophan (Trp). Each portion of the *trp* aporepressor has a binding site for tryptophan.

The *trp* aporepressor cannot bind the operators tightly when not complexed with tryptophan. However, if two tryptophan molecules bind to their respective binding sites the *trp* aporepressor is converted into a functional repressor. The resulting functional repressor complex can tightly bind to the *trp* operators and so the synthesis of the tryptophan producing enzymes is prevented. This sequence constitutes the repression negative-feedback mechanism: an increase in the concentration of tryptophan induces an increase in the concentration of the functional repressor, thus preventing the synthesis of tryptophan.

2 Will-be-set-by-IN-TECH

of nucleosomes known, this simple two step model provides a reasonable framework for understanding the changing probabilities affecting transcription. What do the restrictive and permissive states represent? An attractive idea is that the repressed state is a closed chromatin conformation whilst the permissive state is an open one. Another hypothesis is that the fluctuations reflect transition between bound pre-initiation complexes (permissive) and dissociated ones (restrictive) (Blake et al., 2003; Ross et al., 1994). Bursts may also result from bursty signalling, cell cycle effects or movement of chromatin to and from transcription factories. Nonetheless, to the best of our knowledge, there is no generally accepted explanation for this phenomenon. Transcriptional bursting in prokaryotic cell is particularly puzzling given the simplicity of the transcription initiation process, as opposed

There is evidence that the cooperative interaction of distant operators through a single repressor molecule has a strong influence on the transcriptional bursting observed in the *lac* operon of *E. coli* (Choi et al., 2008). Since the repression regulatory mechanism in *E. coli*'s *trp* operon involves cooperativity between two repressor molecules bound to neighbouring operators, it is interesting to investigate whether such cooperative interaction has any effect upon the system transcriptional dynamics. The present chapter is advocated to tackling this question from a mathematical modelling perspective. We also investigate the effects of the enzymatic feedback-inhibition regulatory mechanism (also present on the *trp* operon

The amino acid tryptophan can be synthesized by bacteria like *E. coli* through a series of catalysed reactions. The catalysing enzymes in *E. coli* are made up of the polypeptides encoded by the tryptophan operon genes: *trpE*, *trpD*, *trpC*, *trpB*, and *trpA*, which are transcribed from *trpE* to *trpA*. Transcription is initiated at promoter *trpP*, which is located upstream from gene *trpE*. The *trp* operon is regulated by three different negative-feedback mechanisms: repression, transcription attenuation, and enzyme inhibition. Below these regulatory mechanisms are briefly reviewed based on (Brown et al., 1999; Grillo et al., 1999; Jeeves et al., 1999; Xie et al., 2003; Yanofsky, 2000; Yanofsky & Crawford, 1987). The reader

**Repression** in the *trp* operon is mediated by three operators (*O1*, *O2*, and *O3*) overlapping with the operon promoter, *trpP* (see Figure 1A). When an active repressor is bound to an operator it blocks the binding of a RNA polymerase to *trpP* and prevents transcription initiation. The *trp* repressor normally exists as a dimeric protein (called the *trp* aporepressor) and may or may not be complexed with tryptophan (Trp). Each portion of the *trp* aporepressor

The *trp* aporepressor cannot bind the operators tightly when not complexed with tryptophan. However, if two tryptophan molecules bind to their respective binding sites the *trp* aporepressor is converted into a functional repressor. The resulting functional repressor complex can tightly bind to the *trp* operators and so the synthesis of the tryptophan producing enzymes is prevented. This sequence constitutes the repression negative-feedback mechanism: an increase in the concentration of tryptophan induces an increase in the concentration of the functional repressor, thus preventing the synthesis of tryptophan.

regulatory pathway) on the system dynamic behaviour.

should consult Figure 1 for a better understanding.

has a binding site for tryptophan.

to eukaryotic cells.

**2. Theory**

**2.1 The** *trp* **operon**

Fig. 1. Schematic representation of the three regulatory mechanisms found in the tryptophan operon: A) repression, B) transcription attenuation, and C) enzyme inhibition. A glossary with the meaning of all the geometric forms in this figure is shown in panel D. See the main text for further explanation.

**Transcription attenuation** works by promoting an early termination of mRNA transcription, see Figure 1B. The transcription starting site in the *trp* operon is separated from *trpE* by a leader region responsible for attenuation control. The transcript of this leader region consists of four segments (termed Segments 1, 2, 3, and 4) which can form three stable hairpin structures between consecutive segments. After the first two segments are transcribed they form a hairpin which stops transcription (c.f. Figure 1B-I). When a ribosome binds the nascent mRNA, it disrupts Hairpin 1:2 and transcription is re-initiated along with translation. Segment 1 contains two tryptophan codons in tandem. If there is scarcity of tryptophan, and thus of loaded tRNATrp, the ribosome stalls in the first segment. The development of Hairpin 2:3 (the antiterminator) is then facilitated, and transcription proceeds until the end (c.f. Figure 1B-II). However, if tryptophan is abundant, the ribosome rapidly finishes translation of Segments 1 and 2 and promotes the formation of a stable hairpin structure between Segments 3 and 4 (c.f. Figure 1B-III). RNA polymerase molecules recognize this hairpin structure as a termination signal and transcription is prematurely terminated.

**Enzyme inhibition** takes place through anthranilate synthase, the first enzyme to catalyse a reaction in the catalytic pathway that leads to the synthesis of tryptophan from chorismate. This enzyme is a hetero-tetramer consisting of two TrpE and two TrpD polypeptides. Anthranilate synthase is inhibited by tryptophan through negative feedback. This feedback inhibition is achieved when the TrpE subunits in anthranilate synthase are individually bound by a tryptophan molecule, see Figure 1C. Therefore, an excess of intracellular tryptophan inactivates most of the anthranilate synthase protein avoiding the production of more tryptophan.

(2004) found that the probability that transcription is not prematurely terminated due to

<sup>183</sup> Transcriptional Bursting

in the Tryptophan Operon of *E. coli* and Its Effect on the System Stochastic Dynamics

*PA* <sup>=</sup> <sup>1</sup> <sup>+</sup> <sup>2</sup>*α<sup>T</sup>*

It follows from the considerations in the previous paragraphs that a promoter in the restrictive global state is completely incapable of being expressed, but its activity level when it is in the permissive global state is a function of the tryptophan level *T* and is given by the product of *PR* and *PA*. Therefore, if *kE* denotes the rate of enzyme synthesis by a fully active promoter, the enzyme synthesis rate when the promoter is in the permissive state at a given tryptophan

Tryptophan is synthesized by proteins which are assembled from the polypeptides coded by the *trp* genes. Conversely, tryptophan is mainly consumed in the synthesis of all kinds of

*dt* <sup>=</sup> *kTEPI*(*T*) <sup>−</sup> *<sup>ρ</sup>*(*T*) <sup>−</sup> *<sup>μ</sup>T*, where *μ* is the bacterial growth rate, *kT* is the tryptophan rate of synthesis per enzyme

*PI*(*T*) = *<sup>K</sup><sup>n</sup>*

is the probability that an enzyme molecule is not feedback inhibited by tryptophan, and

*ρ*(*T*) = *ρmax*

is the rate of tryptophan consumption associated to protein synthesis. If we assume that these processes are much faster than those associated to gene expression and protein degradation, then we can make the following quasi-steady state approximation: *dT*/*dt* = 0, and the tryptophan molecule count can be uniquely calculated in terms of the enzyme molecule count

Following previous modelling studies we assume that the enzyme degradation rate is negligible as compared with the bacterial growth rate, *μ*. On the other hand, instead of considering a cell that grows exponentially, we assume that we have a constant-volume cell

The facts previously discussed in the present section provide enough information to develop a model for the *trp* operon regulatory pathway. This model consists of four chemical reactions: promoter activation, promoter inactivation, enzyme synthesis, and enzyme degradation, whose rates are *k*−, *k*+(*T*(*E*))*RA*(*T*(*E*)), *kEPR*(*T*(*E*))*PA*(*T*(*E*)), and *μE*, respectively. Figure 2 provides a schematic representation of such a model. It is worth emphasizing that the repression regulatory feedback loop is implicitly accounted for by functions *k*+(*T*(*E*)) and *PR*(*T*(*E*)), that function *PA*(*T*(*E*)) corresponds to the attenuation feedback regulatory mechanism, and the feedback enzyme inhibition is implicit in the function *T*(*E*), obtained

*I T<sup>n</sup>* + *K<sup>n</sup> I*

> *T T* + *K<sup>ρ</sup>*

*kTEPI*(*T*) − *ρ*(*T*) − *μT* = 0. (7)

proteins in *E. coli*. Thus, the equation governing the tryptophan-level dynamics is:

*dT*

as the root of the following algebraic equation:

and that the effective enzyme degradation rate is *μ*.

after solving Eqn. (7).

(<sup>1</sup> <sup>+</sup> *<sup>α</sup>T*)<sup>2</sup> , (5)

*kEPR*(*T*)*PA*(*T*). (6)

transcriptional attenuation is:

level turns out to be:

molecule

with *α* a parameter to be estimated.

#### **3. Methods**

#### **3.1 Model development**

There are three different repressor binding sites (operators) overlapping the *trp* promoter. Hence, the promoter can be in eight different states, with each operator being either free or bound by a repressor molecule. Furthermore, when two repressor molecules are bound to the first and second operators, they do it cooperatively (Grillo et al., 1999). As discussed in Appendix A, this cooperativity allows the grouping of the promoter states into two different sets that we term the permissive and the restrictive global states. The transitions within each global state and those from the permissive global state to the restrictive global state being much faster than those from the restrictive global to the permissive global states. This fact justifies the assumption that the system "instantaneously" reaches a stationary probability distribution for the states within every global state. This supposition in turn permits the derivation of the following expressions for the transition rates from the permissive into the restrictive global states (*k*+), and vice versa (*k*−):

$$k^{+} = \frac{k\_i^{+} \mathcal{R}\_A / \mathcal{K}\_{\dot{j}} + k\_j^{+} \mathcal{R}\_A / \mathcal{K}\_{\dot{i}}}{1 + \mathcal{R}\_A / \mathcal{K}\_{\dot{i}} + \mathcal{R}\_A / \mathcal{K}\_{\dot{j}}},\tag{1}$$

$$k^{-} = \frac{k\_i^{-} + k\_j^{-}}{k\_c},\tag{2}$$

where *k*<sup>+</sup> *<sup>i</sup>* and *<sup>k</sup>*<sup>+</sup> *<sup>j</sup>* are the rates for the reactions where a repressor molecule binds to the first and second operators, respectively; *k*− *<sup>i</sup>* and *k*<sup>−</sup> *<sup>j</sup>* are the rates for the reactions in which a repressor molecule detaches from the first and second operator; *RA* is the number of active repressors; *Ki* = *k*<sup>−</sup> *<sup>i</sup>* /*k*<sup>+</sup> *<sup>i</sup>* ; and *Kj* = *k*<sup>−</sup> *<sup>j</sup>* /*k*<sup>+</sup> *j* .

On the other hand, when the promoter is in the permissive global state, the probability that it is not bound by any repressor and so it is free to be bound by a polymerase to start transcription is

$$P\_R = \frac{1}{(1 + R\_A/K\_k)(1 + R\_A/K\_i + R\_A/K\_j)}.\tag{3}$$

Repressor molecules are activated when they are bound by a couple of tryptophan molecules. The kinetics of repressor activation were analysed in (Santillán & Zeron, 2004), where the number of active repressors is demonstrated to be given by

$$R\_A = R\_T \left(\frac{T}{T + K\_T}\right)^2,\tag{4}$$

where *RT* stands for the total number of repressor molecules, while *T* is the tryptophan molecule count. Substitution of Eqn. (4) into Eqns. (1) and (3) permits the calculation of the promoter inactivation rate (*k*+) and the probability *PR* in terms of the tryptophan level (*T*).

Due to transcriptional attenuation, only a fraction of the polymerase molecules that initiate transcription reach the end of the *trp* genes and produce functional mRNA molecules, which in turn are translated to produce the proteins coded by the *trp* genes. Santillán and Zeron (2004) found that the probability that transcription is not prematurely terminated due to transcriptional attenuation is:

$$P\_A = \frac{1 + 2\alpha T}{(1 + \alpha T)^2} \tag{5}$$

with *α* a parameter to be estimated.

4 Will-be-set-by-IN-TECH

There are three different repressor binding sites (operators) overlapping the *trp* promoter. Hence, the promoter can be in eight different states, with each operator being either free or bound by a repressor molecule. Furthermore, when two repressor molecules are bound to the first and second operators, they do it cooperatively (Grillo et al., 1999). As discussed in Appendix A, this cooperativity allows the grouping of the promoter states into two different sets that we term the permissive and the restrictive global states. The transitions within each global state and those from the permissive global state to the restrictive global state being much faster than those from the restrictive global to the permissive global states. This fact justifies the assumption that the system "instantaneously" reaches a stationary probability distribution for the states within every global state. This supposition in turn permits the derivation of the following expressions for the transition rates from the permissive into the

*<sup>i</sup> RA*/*Kj* <sup>+</sup> *<sup>k</sup>*<sup>+</sup>

*<sup>i</sup>* + *k*<sup>−</sup> *j kc*

1 + *RA*/*Ki* + *RA*/*Kj*

*<sup>i</sup>* and *k*<sup>−</sup>

a repressor molecule detaches from the first and second operator; *RA* is the number of active

On the other hand, when the promoter is in the permissive global state, the probability that it is not bound by any repressor and so it is free to be bound by a polymerase to start transcription

Repressor molecules are activated when they are bound by a couple of tryptophan molecules. The kinetics of repressor activation were analysed in (Santillán & Zeron, 2004), where the

> *T T* + *KT*

where *RT* stands for the total number of repressor molecules, while *T* is the tryptophan molecule count. Substitution of Eqn. (4) into Eqns. (1) and (3) permits the calculation of the promoter inactivation rate (*k*+) and the probability *PR* in terms of the tryptophan level

Due to transcriptional attenuation, only a fraction of the polymerase molecules that initiate transcription reach the end of the *trp* genes and produce functional mRNA molecules, which in turn are translated to produce the proteins coded by the *trp* genes. Santillán and Zeron

<sup>2</sup>

(1 + *RA*/*Kk*)(1 + *RA*/*Ki* + *RA*/*Kj*)

*<sup>j</sup> RA*/*Ki*

*<sup>j</sup>* are the rates for the reactions where a repressor molecule binds to the

, (1)

. (3)

, (4)

, (2)

*<sup>j</sup>* are the rates for the reactions in which

**3. Methods**

where *k*<sup>+</sup>

is

(*T*).

*<sup>i</sup>* and *<sup>k</sup>*<sup>+</sup>

repressors; *Ki* = *k*<sup>−</sup>

**3.1 Model development**

restrictive global states (*k*+), and vice versa (*k*−):

first and second operators, respectively; *k*−

*<sup>i</sup>* ; and *Kj* = *k*<sup>−</sup>

number of active repressors is demonstrated to be given by

*<sup>i</sup>* /*k*<sup>+</sup>

*<sup>k</sup>*<sup>+</sup> <sup>=</sup> *<sup>k</sup>*<sup>+</sup>

*<sup>k</sup>*<sup>−</sup> <sup>=</sup> *<sup>k</sup>*<sup>−</sup>

*<sup>j</sup>* /*k*<sup>+</sup> *j* .

*PR* <sup>=</sup> <sup>1</sup>

*RA* = *RT*

It follows from the considerations in the previous paragraphs that a promoter in the restrictive global state is completely incapable of being expressed, but its activity level when it is in the permissive global state is a function of the tryptophan level *T* and is given by the product of *PR* and *PA*. Therefore, if *kE* denotes the rate of enzyme synthesis by a fully active promoter, the enzyme synthesis rate when the promoter is in the permissive state at a given tryptophan level turns out to be:

$$k\_E P\_\mathcal{R}(T) P\_\mathcal{A}(T). \tag{6}$$

Tryptophan is synthesized by proteins which are assembled from the polypeptides coded by the *trp* genes. Conversely, tryptophan is mainly consumed in the synthesis of all kinds of proteins in *E. coli*. Thus, the equation governing the tryptophan-level dynamics is:

$$\frac{dT}{dt} = k\_T E P\_I(T) - \rho(T) - \mu T\_{\nu}$$

where *μ* is the bacterial growth rate, *kT* is the tryptophan rate of synthesis per enzyme molecule

$$P\_I(T) = \frac{K\_I^n}{T^n + K\_I^n}$$

is the probability that an enzyme molecule is not feedback inhibited by tryptophan, and

$$
\rho(T) = \rho\_{\text{max}} \frac{T}{T + K\_{\rho}}
$$

is the rate of tryptophan consumption associated to protein synthesis. If we assume that these processes are much faster than those associated to gene expression and protein degradation, then we can make the following quasi-steady state approximation: *dT*/*dt* = 0, and the tryptophan molecule count can be uniquely calculated in terms of the enzyme molecule count as the root of the following algebraic equation:

$$k\_T E P\_I(T) - \rho(T) - \mu T = 0.\tag{7}$$

Following previous modelling studies we assume that the enzyme degradation rate is negligible as compared with the bacterial growth rate, *μ*. On the other hand, instead of considering a cell that grows exponentially, we assume that we have a constant-volume cell and that the effective enzyme degradation rate is *μ*.

The facts previously discussed in the present section provide enough information to develop a model for the *trp* operon regulatory pathway. This model consists of four chemical reactions: promoter activation, promoter inactivation, enzyme synthesis, and enzyme degradation, whose rates are *k*−, *k*+(*T*(*E*))*RA*(*T*(*E*)), *kEPR*(*T*(*E*))*PA*(*T*(*E*)), and *μE*, respectively. Figure 2 provides a schematic representation of such a model. It is worth emphasizing that the repression regulatory feedback loop is implicitly accounted for by functions *k*+(*T*(*E*)) and *PR*(*T*(*E*)), that function *PA*(*T*(*E*)) corresponds to the attenuation feedback regulatory mechanism, and the feedback enzyme inhibition is implicit in the function *T*(*E*), obtained after solving Eqn. (7).

0.5 1 1.5 2 2.5 3 Active Interval (min)

1 1.5 2 2.5 3 3.5

A

in the Tryptophan Operon of *E. coli* and Its Effect on the System Stochastic Dynamics

Activity Cumulative Sum

0 500 1000 1500 2000 2500 3000 Enzyme Count

*<sup>x</sup>* and *k*<sup>−</sup>

0 20 40 60 80 Inactive Interval (min)

B C

D E

μ = 8.2e4 CV = 0.16

0 2e4 4e4 6e4 8e4 10e4 12e4 Tryptophan Count

*<sup>x</sup>* (*x* = *I*, *j*, *k*) by a factor of 100. In this way, the promoter switching rate

*<sup>x</sup>* by the same factor, and is in agreement with the fact that

0 0.05 0.1 0.15 0.2 0.25 0.3

0 0.02 0.04 0.06 0.08 0.1 0.12

Normalized Frequency

Fig. 3. Statistical analysis of the simulation corresponding to the wild-type strain. A) Plot of the cumulative promoter activity vs. time. B) Histogram of the promoter permissive time intervals and best fit to an exponential distribution. C) Histogram of the promoter restrictive time intervals and best fit to an exponential distribution. D) Histogram of the enzyme molecule count and best fit to a gamma distribution. E) Histogram of the tryptophan

In order to understand the influence of the slow promoter gating between the global permissive and restrictive states on the operon dynamics, we increased the value of

among all its available states gets faster, without altering each state's stationary probability. We repeated the simulation described in the previous paragraph with this new parameter set

We observe by comparing Figures 3A and 4A that there are many more alternated activity and inactivity periods in the fast-switching model than in that corresponding to the wild type strain. Concomitantly, the periods are shorter in the former case. Interestingly, the accumulated promoter activity is quite similar for both models. This last result comes from

In Figures 4B and 4C we present the histograms for the activity and inactivity periods, and the corresponding fits to exponential distributions. By comparing with the wild-type period distributions we can see that the mean values of both the activity and inactivity periods decrease. However the decrease of the activity-period average is about twice as large as that

Finally, the histograms for the enzyme and tryptophan molecule counts are plotted in Figures 4D and 4E. Notice that the histogram for the enzyme count is well approximated by a gamma distributions with parameters *k* = 2079 and *θ* = 1. Therefore, by making the promoter switching rate faster we increased the frequency of bursting, but decreased in the same

the mean enzyme and tryptophan counts are quite similar in both models (see below).

Normalized Frequency

μ = 0.35 min μ = 8.88 min

0 100 200 300 400 Time (min)

<sup>185</sup> Transcriptional Bursting

0 0.1 0.2 0.3 0.4 0.5

> μ = 1988 CV = 0.19

0 0.02 0.04 0.06 0.08 0.1 0.12

Normalized Frequency

molecule count.

parameters *k*<sup>+</sup>

*<sup>x</sup>* and *k*<sup>−</sup>

our increasing parameters *k*<sup>+</sup>

of the average of the inactivity period.

and the results are condensed in Figure 4.

Normalized Frequency

Fig. 2. Schematic representation of the mathematical model here developed for tryptophan operon gene regulatory circuit.

#### **3.2 Parameter estimation**

We paid special attention to the estimation of all the model parameters from reported experimental data. The parameter values we employ in the present work and the detailed procedure to estimate them are presented in Appendix B.

#### **3.3 Numerical methods**

The time evolution of the reaction network that models the tryptophan operon regulatory pathway was simulated by means of the Gillespie algorithm, which we implemented in Python.

#### **4. Results**

We carried out stochastic simulations with the model described in the previous section. As formerly stated, we made use of Gillespie's algorithm to mimic the system dynamic evolution for 200,000 min. In the first simulation we employed the parameter values estimated in Appendix B, which correspond to a wild-type bacterial strain. The results are summarized in Figure 3. In Figure 3A the cumulative sum of the promoter activity is plotted vs. time. We can appreciate there the existence of alternated activity and inactivity periods, just like it has been observed in transcriptional bursting. To further investigate this phenomenon we calculated the histograms of the permissive and restrictive period lengths. The results are shown in Figures 3B and 3C, respectively. Observe that both histograms are well fitted by exponential distributions, in agreement with the reported experimental data on transcriptional bursting. Finally, we present in Figures 3D and 3E the histograms for the enzyme and tryptophan molecule counts, respectively.

One feature worth noticing is that the histogram for the enzyme abundance is well fitted by a gamma distribution with parameters *k* = 32.5 and *θ* = 63. This last fact is in agreement with the existence on transcriptional bursting in the *trp* operon of *E. coli*. It has been proved that in such a case, the protein count obeys a gamma distribution, with parameters *k* and *θ* respectively interpreted as the average number of transcriptional bursts occurring during an average protein lifetime and the mean number of proteins produced per burst (Shahrezaei & Swain, 2008). It is also interesting to point out that the coefficient of variation in the tryptophan molecule count is similar to that of the enzyme molecule count.

6 Will-be-set-by-IN-TECH

*kE P*R(*T* (*E* )) *PA*(*T* (*E* ))

Fig. 2. Schematic representation of the mathematical model here developed for tryptophan

We paid special attention to the estimation of all the model parameters from reported experimental data. The parameter values we employ in the present work and the detailed

The time evolution of the reaction network that models the tryptophan operon regulatory pathway was simulated by means of the Gillespie algorithm, which we implemented in

We carried out stochastic simulations with the model described in the previous section. As formerly stated, we made use of Gillespie's algorithm to mimic the system dynamic evolution for 200,000 min. In the first simulation we employed the parameter values estimated in Appendix B, which correspond to a wild-type bacterial strain. The results are summarized in Figure 3. In Figure 3A the cumulative sum of the promoter activity is plotted vs. time. We can appreciate there the existence of alternated activity and inactivity periods, just like it has been observed in transcriptional bursting. To further investigate this phenomenon we calculated the histograms of the permissive and restrictive period lengths. The results are shown in Figures 3B and 3C, respectively. Observe that both histograms are well fitted by exponential distributions, in agreement with the reported experimental data on transcriptional bursting. Finally, we present in Figures 3D and 3E the histograms for the enzyme and tryptophan

One feature worth noticing is that the histogram for the enzyme abundance is well fitted by a gamma distribution with parameters *k* = 32.5 and *θ* = 63. This last fact is in agreement with the existence on transcriptional bursting in the *trp* operon of *E. coli*. It has been proved that in such a case, the protein count obeys a gamma distribution, with parameters *k* and *θ* respectively interpreted as the average number of transcriptional bursts occurring during an average protein lifetime and the mean number of proteins produced per burst (Shahrezaei & Swain, 2008). It is also interesting to point out that the coefficient of variation in the tryptophan

molecule count is similar to that of the enzyme molecule count.

Tryptofano (*T* )

Enzymes (E)

γ*E* ∅

Active Promoter

procedure to estimate them are presented in Appendix B.

operon gene regulatory circuit.

**3.2 Parameter estimation**

**3.3 Numerical methods**

molecule counts, respectively.

Python.

**4. Results**

Inactive Promoter

*<sup>k</sup>*+(*<sup>T</sup>*(*E* ))*R*<sup>A</sup> *<sup>k</sup>* (*T* (*E* )) -

Fig. 3. Statistical analysis of the simulation corresponding to the wild-type strain. A) Plot of the cumulative promoter activity vs. time. B) Histogram of the promoter permissive time intervals and best fit to an exponential distribution. C) Histogram of the promoter restrictive time intervals and best fit to an exponential distribution. D) Histogram of the enzyme molecule count and best fit to a gamma distribution. E) Histogram of the tryptophan molecule count.

In order to understand the influence of the slow promoter gating between the global permissive and restrictive states on the operon dynamics, we increased the value of parameters *k*<sup>+</sup> *<sup>x</sup>* and *k*<sup>−</sup> *<sup>x</sup>* (*x* = *I*, *j*, *k*) by a factor of 100. In this way, the promoter switching rate among all its available states gets faster, without altering each state's stationary probability. We repeated the simulation described in the previous paragraph with this new parameter set and the results are condensed in Figure 4.

We observe by comparing Figures 3A and 4A that there are many more alternated activity and inactivity periods in the fast-switching model than in that corresponding to the wild type strain. Concomitantly, the periods are shorter in the former case. Interestingly, the accumulated promoter activity is quite similar for both models. This last result comes from our increasing parameters *k*<sup>+</sup> *<sup>x</sup>* and *k*<sup>−</sup> *<sup>x</sup>* by the same factor, and is in agreement with the fact that the mean enzyme and tryptophan counts are quite similar in both models (see below).

In Figures 4B and 4C we present the histograms for the activity and inactivity periods, and the corresponding fits to exponential distributions. By comparing with the wild-type period distributions we can see that the mean values of both the activity and inactivity periods decrease. However the decrease of the activity-period average is about twice as large as that of the average of the inactivity period.

Finally, the histograms for the enzyme and tryptophan molecule counts are plotted in Figures 4D and 4E. Notice that the histogram for the enzyme count is well approximated by a gamma distributions with parameters *k* = 2079 and *θ* = 1. Therefore, by making the promoter switching rate faster we increased the frequency of bursting, but decreased in the same

0.5 1 1.5 2 2.5 Active Interval (min)

1 1.01 1.02 1.03 1.04 1.05 1.06 1.07

A

in the Tryptophan Operon of *E. coli* and Its Effect on the System Stochastic Dynamics

Activity Cumulative Sum

10 20 30 40 50 60 70 Enzyme Count

> 0 0.2 0.4 0.6 0.8 1

Fig. 6. Plots of the normalized enzyme count and the normalized tryptophan count,

promoter-transition (red line), and the inhibition-less (green line) *E. coli* strains.

averaged over 1,000 independent simulations, vs. time for the wild-type (blue line), the fast

mutant because of the reduced enzyme count, we fitted the histogram in Figure 5D and found that the best fit is obtained with parameters *k* = 40 and *θ* = 1.2. Since the values of *k* for the inhibition-less and the wild-type strains are similar, we conclude that the burst frequency is

0 0.2 0.4 0.6 0.8 1

Normalized Enz. Count

Normalized Trp Count

0 10 20 30 40 50 Inactive Interval (min)

B C

D E

μ = 3.4e5 CV = 2.92

> 1e5 2e5 3e5 4e5 5e5 6e5 Tryptophan Count

0 0.1 0.2 0.3 0.4 Normalized Frequency

0 100 200 300 400 Time (min)

<sup>187</sup> Transcriptional Bursting

μ = 0.30 min μ = 6.37 min

0 0.05 0.1 0.15 0.2 0.25 Normalized Frequency

Fig. 5. Statistical analysis of the simulation corresponding to the inhibition-less strain. A) Plot of the cumulative promoter activity vs. time. B) Histogram of the promoter permissive time intervals and best fit to an exponential distribution. C) Histogram of the promoter restrictive time intervals and best fit to an exponential distribution. D) Histogram of the enzyme molecule count and best fit to a gamma distribution. E) Histogram of the tryptophan

> 0 50 100 150 200 Time (min)

> 0 50 100 150 200 Time (min)

0 0.1 0.2 0.3 0.4 0.5 0.6

> μ = 46 CV = 0.15

0 0.1 0.2 0.3 0.4

Normalized Frequency

molecule count.

Normalized Frequency

Fig. 4. Statistical analysis of the simulation corresponding to the strain with fast promoter-transition rates. A) Plot of the cumulative promoter activity vs. time. B) Histogram of the promoter permissive time intervals and best fit to an exponential distribution. C) Histogram of the promoter restrictive time intervals and best fit to an exponential distribution. D) Histogram of the enzyme molecule count and best fit to a gamma distribution. E) Histogram of the tryptophan molecule count.

amount the number of proteins synthesized per burst. In that way, the average enzyme count remains the same (compare with Figure 3D). However, the variation coefficient is much smaller in the fast promoter switching model than in the wild-type one. Recall that, in the gamma distribution, the mean and the standard deviation are: *μ* = *θk* and *σ* = *θ* <sup>√</sup>*k*, while the variation coefficient is given by *CV* <sup>=</sup> *<sup>σ</sup>*/*<sup>μ</sup>* <sup>=</sup> 1/√*k*.

We further simulated a bacterial mutant strain lacking the feedback inhibition regulatory mechanism. To mimic this mutation we increased the value of parameter *KI* by two orders of magnitude, up to *KI* = 500, 000 molecules. We analysed this last simulation in a similar way than the previous ones and present the results in Figure 5.

A comparison of Figures 3A and 5A reveals that the promoter level of activation is generally smaller in the inhibition-less mutant strain than in the wild-type strain, because the accumulated activity is about three times smaller in the former case. Nonetheless the length of the activity and inactivity periods seem to be similar. This last assertion is corroborated by the plots in Figures 5B and 5C, where we can see the the activity and inactivity period histograms are well fitted by exponential distributions, and that the corresponding mean values are similar to the corresponding ones in the wild-type strain.

In agreement with the fact that the promoter level of activity is smaller in the inhibition-less than in the wild-type strain, the mean protein count is smaller in the first case (compare Figures 3D and 5D). On the other hand, the coefficient of variation (CV) is similar in both cases. To understand why this happens when one would expect a larger CV in the inhibition-less 8 Will-be-set-by-IN-TECH

0 10 20 30 40 Inactive Interval (min)

B C

D E

μ = 8.5e4 CV = 0.018

> 8e4 82000 84000 86000 88000 90000 Tryptophan Count

> > <sup>√</sup>*k*, while the

0 0.1 0.2 0.3 0.4

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

Normalized Frequency

promoter-transition rates. A) Plot of the cumulative promoter activity vs. time. B) Histogram of the promoter permissive time intervals and best fit to an exponential distribution. C) Histogram of the promoter restrictive time intervals and best fit to an exponential distribution. D) Histogram of the enzyme molecule count and best fit to a gamma

amount the number of proteins synthesized per burst. In that way, the average enzyme count remains the same (compare with Figure 3D). However, the variation coefficient is much smaller in the fast promoter switching model than in the wild-type one. Recall that, in the

We further simulated a bacterial mutant strain lacking the feedback inhibition regulatory mechanism. To mimic this mutation we increased the value of parameter *KI* by two orders of magnitude, up to *KI* = 500, 000 molecules. We analysed this last simulation in a similar way

A comparison of Figures 3A and 5A reveals that the promoter level of activation is generally smaller in the inhibition-less mutant strain than in the wild-type strain, because the accumulated activity is about three times smaller in the former case. Nonetheless the length of the activity and inactivity periods seem to be similar. This last assertion is corroborated by the plots in Figures 5B and 5C, where we can see the the activity and inactivity period histograms are well fitted by exponential distributions, and that the corresponding mean

In agreement with the fact that the promoter level of activity is smaller in the inhibition-less than in the wild-type strain, the mean protein count is smaller in the first case (compare Figures 3D and 5D). On the other hand, the coefficient of variation (CV) is similar in both cases. To understand why this happens when one would expect a larger CV in the inhibition-less

Normalized Frequency

μ = 0.063 min μ = 4.37 min

0 100 200 300 400 Time (min)

0.2 0.4 0.6 0.8 1 Active Interval (min)

1 1.5 2 2.5 3 3.5 A

Activity Cumulative Sum

1900 1950 2000 2050 2100 2150 2200 2250 Enzyme Count

Fig. 4. Statistical analysis of the simulation corresponding to the strain with fast

gamma distribution, the mean and the standard deviation are: *μ* = *θk* and *σ* = *θ*

0 0.2 0.4 0.6 0.8 Normalized Frequency

> μ = 2079 CV = 0.022

distribution. E) Histogram of the tryptophan molecule count.

variation coefficient is given by *CV* <sup>=</sup> *<sup>σ</sup>*/*<sup>μ</sup>* <sup>=</sup> 1/√*k*.

than the previous ones and present the results in Figure 5.

values are similar to the corresponding ones in the wild-type strain.

0 0.05 0.1 0.15 0.2 0.25 0.3

Normalized Frequency

Fig. 5. Statistical analysis of the simulation corresponding to the inhibition-less strain. A) Plot of the cumulative promoter activity vs. time. B) Histogram of the promoter permissive time intervals and best fit to an exponential distribution. C) Histogram of the promoter restrictive time intervals and best fit to an exponential distribution. D) Histogram of the enzyme molecule count and best fit to a gamma distribution. E) Histogram of the tryptophan molecule count.

Fig. 6. Plots of the normalized enzyme count and the normalized tryptophan count, averaged over 1,000 independent simulations, vs. time for the wild-type (blue line), the fast promoter-transition (red line), and the inhibition-less (green line) *E. coli* strains.

mutant because of the reduced enzyme count, we fitted the histogram in Figure 5D and found that the best fit is obtained with parameters *k* = 40 and *θ* = 1.2. Since the values of *k* for the inhibition-less and the wild-type strains are similar, we conclude that the burst frequency is

shift. This burst allows most of the cells to reach tryptophan levels superior to two thirds

• Enzyme inhibition also has important dynamic effects. It increases the noise level in the enzyme count, but decreases the noise level in the number of tryptophan molecules. Furthermore, this regulatory mechanism also increases the system response time. Knowing that the presence of a strong negative feedback is capable of reducing the noise in a biological system (Austin et al., 2006; Becskei & Serrano, 2000; Dublanche et al., 2006), we can explain the above observations as follows: the fact that the wild-type *E. coli* strain has lower tryptophan levels than the inhibition-free strain means that the transcriptional-attenuation and the repression feedback loops are weaker in the first strain; this weakening of both negative feedback loops is responsible for the increment of the noise level in the enzyme count. For the same reasons, the presence of the enzyme-inhibition feedback loop reduces the noise level in the tryptophan count, but makes it necessary to produce much more enzymes to fulfil the required tryptophan production. This increased

<sup>189</sup> Transcriptional Bursting

in the Tryptophan Operon of *E. coli* and Its Effect on the System Stochastic Dynamics

Finally, if we assume that having a tryptophan operon with short response times and low noise levels in the tryptophan molecular count are beneficial traits for *E. coli* then we can speculate from the previously discussed facts that evolution has bestowed this system with an optimal

This research was partially supported by Consejo Nacional de Ciencia y Tecnología

Three different operators are overlap with the *trp* promoter and each of them can be bound by a repressor molecule. Therefore, the eight promoter states can be denoted as {*i*, *j*, *k*}, where *i*, *j*, *k* = 0, 1 represent the binding state of the first, second, and third repressors, respectively. A zero (one) value means that the corresponding operator is free from (bound by) a repressor

respectively represent the dissociation rate of a repressor molecule solely bound to the first,

It is known that the first and second operators are bound by repressor molecules cooperatively and the cooperativity constant *kc >* 1 has been measured. Here we assume that this cooperativity means that the rate of dissociation for a repressor molecule bound to either the

are bound by repressor molecules. Under this assumption, the eight different promoter states can be grouped into two sets that we call the permissive and the restrictive global states. The permissive global state consists of states (0, 0, 0), (1, 0, 0), (0, 1, 0), (0, 0, 1), (1, 0, 1), and (0, 1, 1), while the restrictive global state consists of (1, 1, 0), and (1, 1, 1). From the

first, second, and third operators, when the other two are free. Similarly, let *k*−

first or the second operator is respectively given by *k*−

*<sup>k</sup>* respectively denote the rates of binding of a repressor molecule to the

*<sup>i</sup>* /*kc* or *k*<sup>−</sup>

*<sup>i</sup>* , *k*<sup>−</sup>

*<sup>j</sup>* /*kc*, when both operators

*<sup>j</sup>* , and *k*<sup>−</sup> *k*

enzyme synthesis requirement lengthens the system response time.

trade-off between short response times and low tryptophan noise.

of the steady-state level.

**6. Acknowledgements**

**7. Appendix**

molecule. Let *k*<sup>+</sup> *<sup>i</sup>* , *<sup>k</sup>*<sup>+</sup>

(CONACyT, MEXICO) under Grant: 55228.

**A. Promoter dynamics modelling**

*<sup>j</sup>* , and *<sup>k</sup>*<sup>+</sup>

second, and third operators.

comparable in both cases. However, the number of proteins produced per burst is notably smaller in the inhibition-less strain due to the reduced promoter activity, as well as to the increased level of transcriptional attenuation.

We note that, in contrast with the two previous simulations, the variation coefficient of the tryptophan molecular count in the inhibition-less strain is much larger than the coefficient of variation for the enzyme count. To our understanding, this happens because, being enzyme inhibition absent, there is a highly non-linear relation between the enzyme and tryptophan molecule counts.

In order to investigate the effect of the inhibition-less mutation and of the increased promoter switching rate on the *trp* operon response time, we carried out 1,000 simulations with each bacterial strain (including the wild type), starting with the promoter in the restrictive state and zero enzyme and tryptophan molecules as initial conditions. Then, we averaged the enzyme and the tryptophan counts over all the simulations, and plotted the results in Figure 6 to compare how fast each strain approaches the steady state. We can see there that the inhibition-less strain is the one with the shortest response time, followed by the wild type and the fast promoter switching strains, respectively.

#### **5. Concluding remarks**

In this work we have introduced a stochastic mathematical model for the tryptophan operon. Our objectives were twofold: 1) to investigate whether the reported reaction rates of the interaction between repressor and the operators can give rise to transcriptional bursting; and 2) to study the dynamic effects of transcriptional bursting, if it exists, and of the feedback enzyme-inhibition regulatory mechanism.

Regarding the first objective our results indicate that, indeed, the reported reaction rates make the promoter switching between its available states slow enough so as to give rise to transcriptional bursting. As previously discussed, this assertion is supported by the agreement between our model results and a number of reported experimental facts. Interestingly, experiments on the *lac* operon also suggest that transcriptional bursting has its origin in the kinetics of the repressor-operator interaction. As a matter of fact, Choi et al. (2008) demonstrated that the cooperativity present when a single repressor molecule is bound to two distant operators is responsible of generating two different types of bursts in the expression of *lac* operon.

In our model we have assumed that the trp promoter activation rate *k*− is independent of the tryptophan concentration. This assumption is supported by direct and indirect experimental measurements of the promoter activation and deactivation rates on the *lac* and several other promoters (Choi et al., 2008; So et al., 2011). Those reports demonstrate that modulation of gene expression is mainly achieved by changing the promoter deactivation rate.

Regarding our second objective, our results allow us to put forward the following conclusions:

• Transcriptional bursting increases the noise level and decreases the system response time after a nutritional shift. To the best of our understanding, the noise level is increased because the promoter-transition events become less frequent as the promoter-repressor interactions slow down, thus enhancing the concomitant stochastic effects. On the other hand, the faster system response can be explained by a single burst of intense transcriptional activity, occurring during the first couple of minutes after the nutritional shift. This burst allows most of the cells to reach tryptophan levels superior to two thirds of the steady-state level.

• Enzyme inhibition also has important dynamic effects. It increases the noise level in the enzyme count, but decreases the noise level in the number of tryptophan molecules. Furthermore, this regulatory mechanism also increases the system response time. Knowing that the presence of a strong negative feedback is capable of reducing the noise in a biological system (Austin et al., 2006; Becskei & Serrano, 2000; Dublanche et al., 2006), we can explain the above observations as follows: the fact that the wild-type *E. coli* strain has lower tryptophan levels than the inhibition-free strain means that the transcriptional-attenuation and the repression feedback loops are weaker in the first strain; this weakening of both negative feedback loops is responsible for the increment of the noise level in the enzyme count. For the same reasons, the presence of the enzyme-inhibition feedback loop reduces the noise level in the tryptophan count, but makes it necessary to produce much more enzymes to fulfil the required tryptophan production. This increased enzyme synthesis requirement lengthens the system response time.

Finally, if we assume that having a tryptophan operon with short response times and low noise levels in the tryptophan molecular count are beneficial traits for *E. coli* then we can speculate from the previously discussed facts that evolution has bestowed this system with an optimal trade-off between short response times and low tryptophan noise.

#### **6. Acknowledgements**

This research was partially supported by Consejo Nacional de Ciencia y Tecnología (CONACyT, MEXICO) under Grant: 55228.

#### **7. Appendix**

10 Will-be-set-by-IN-TECH

comparable in both cases. However, the number of proteins produced per burst is notably smaller in the inhibition-less strain due to the reduced promoter activity, as well as to the

We note that, in contrast with the two previous simulations, the variation coefficient of the tryptophan molecular count in the inhibition-less strain is much larger than the coefficient of variation for the enzyme count. To our understanding, this happens because, being enzyme inhibition absent, there is a highly non-linear relation between the enzyme and tryptophan

In order to investigate the effect of the inhibition-less mutation and of the increased promoter switching rate on the *trp* operon response time, we carried out 1,000 simulations with each bacterial strain (including the wild type), starting with the promoter in the restrictive state and zero enzyme and tryptophan molecules as initial conditions. Then, we averaged the enzyme and the tryptophan counts over all the simulations, and plotted the results in Figure 6 to compare how fast each strain approaches the steady state. We can see there that the inhibition-less strain is the one with the shortest response time, followed by the wild type and

In this work we have introduced a stochastic mathematical model for the tryptophan operon. Our objectives were twofold: 1) to investigate whether the reported reaction rates of the interaction between repressor and the operators can give rise to transcriptional bursting; and 2) to study the dynamic effects of transcriptional bursting, if it exists, and of the feedback

Regarding the first objective our results indicate that, indeed, the reported reaction rates make the promoter switching between its available states slow enough so as to give rise to transcriptional bursting. As previously discussed, this assertion is supported by the agreement between our model results and a number of reported experimental facts. Interestingly, experiments on the *lac* operon also suggest that transcriptional bursting has its origin in the kinetics of the repressor-operator interaction. As a matter of fact, Choi et al. (2008) demonstrated that the cooperativity present when a single repressor molecule is bound to two distant operators is responsible of generating two different types of bursts in the expression

In our model we have assumed that the trp promoter activation rate *k*− is independent of the tryptophan concentration. This assumption is supported by direct and indirect experimental measurements of the promoter activation and deactivation rates on the *lac* and several other promoters (Choi et al., 2008; So et al., 2011). Those reports demonstrate that modulation of

Regarding our second objective, our results allow us to put forward the following conclusions: • Transcriptional bursting increases the noise level and decreases the system response time after a nutritional shift. To the best of our understanding, the noise level is increased because the promoter-transition events become less frequent as the promoter-repressor interactions slow down, thus enhancing the concomitant stochastic effects. On the other hand, the faster system response can be explained by a single burst of intense transcriptional activity, occurring during the first couple of minutes after the nutritional

gene expression is mainly achieved by changing the promoter deactivation rate.

increased level of transcriptional attenuation.

the fast promoter switching strains, respectively.

enzyme-inhibition regulatory mechanism.

molecule counts.

**5. Concluding remarks**

of *lac* operon.

#### **A. Promoter dynamics modelling**

Three different operators are overlap with the *trp* promoter and each of them can be bound by a repressor molecule. Therefore, the eight promoter states can be denoted as {*i*, *j*, *k*}, where *i*, *j*, *k* = 0, 1 represent the binding state of the first, second, and third repressors, respectively. A zero (one) value means that the corresponding operator is free from (bound by) a repressor molecule.

Let *k*<sup>+</sup> *<sup>i</sup>* , *<sup>k</sup>*<sup>+</sup> *<sup>j</sup>* , and *<sup>k</sup>*<sup>+</sup> *<sup>k</sup>* respectively denote the rates of binding of a repressor molecule to the first, second, and third operators, when the other two are free. Similarly, let *k*− *<sup>i</sup>* , *k*<sup>−</sup> *<sup>j</sup>* , and *k*<sup>−</sup> *k* respectively represent the dissociation rate of a repressor molecule solely bound to the first, second, and third operators.

It is known that the first and second operators are bound by repressor molecules cooperatively and the cooperativity constant *kc >* 1 has been measured. Here we assume that this cooperativity means that the rate of dissociation for a repressor molecule bound to either the first or the second operator is respectively given by *k*− *<sup>i</sup>* /*kc* or *k*<sup>−</sup> *<sup>j</sup>* /*kc*, when both operators are bound by repressor molecules. Under this assumption, the eight different promoter states can be grouped into two sets that we call the permissive and the restrictive global states. The permissive global state consists of states (0, 0, 0), (1, 0, 0), (0, 1, 0), (0, 0, 1), (1, 0, 1), and (0, 1, 1), while the restrictive global state consists of (1, 1, 0), and (1, 1, 1). From the

**B. Estimation of the model parameters**

with

acid.

with

obtain

in this bacterium is

by the following function:

In this work we consider a bacterial doubling time of 40 min, and thus

in the Tryptophan Operon of *E. coli* and Its Effect on the System Stochastic Dynamics

duplicates (every 40 min), then the average tryptophan consumption rate is

assumed that the consumption rate for this amino acid is given by

*<sup>μ</sup>* � 0.017 min<sup>−</sup>1.

<sup>191</sup> Transcriptional Bursting

From the website E. coli Statistics, the total number of proteins in *E. coli* is about 3.6 millions, while the average protein size is 360 residues. On the other hand, we have from the website B1ONUMB3RS (http://bionumbers.hms.harvard.edu/) that the abundance of tryptophan in the *E. coli* proteins is around 1.1%. The data above imply that there are of the order of 14.256 million tryptophan molecules assembles in the *E. coli* proteins at any given time. If we further consider that all the proteins in a bacterium have to be doubled before it

*ρmax* � 360, 000 molecules/min. Since this consumption rate cannot be maintained when the tryptophan level is too low, we

*K<sup>ρ</sup>* = 1, 000 molecules. This choice for *K<sup>ρ</sup>* guarantees that the tryptophan consumption rate is most of the time very close to *ρmax*, except when there are of the order of a few thousand molecules of the amino

According to the website E. coli Statistics (http://redpoll.pharmacy. ualberta.ca/CCDB/cgi-bin/STAT\_NEW.cgi), the average tryptophan molecule count

*T*<sup>∗</sup> � 80, 000 molecules. From Morse et al. (1968), the average number of anthranilate synthase enzymes in *E. coli* is

*E*<sup>∗</sup> � 2, 000 molecules. Caligiuri & Bauerle (1991) found from their experimental data that the probability that an anthranilate synthase enzyme is not feedback inhibited by tryptophan can be approximated

*PI*(*T*) = *<sup>K</sup><sup>n</sup>*

*KI* � 2, 500 molecules, and *n* � 1.2. Let *kT* be the tryptophan synthesis rate per anthranilate synthase molecule. Given, that the average tryptophan synthesis rate must equal the consumption rate for this amino acid, we can solve for *kT* from the following equation *kTE*∗*PI*(*T*∗) = *ρmax*. After doing the math we

*kT* � 12, 000 molecules/min.

*I Kn <sup>I</sup>* <sup>+</sup> *<sup>T</sup><sup>n</sup>* ,

*T T* + *K<sup>ρ</sup>* ,

*ρ*(*T*) = *ρmax*

way the were constructed, the transitions within each global state and the transitions from the permissive to the restrictive global state are much faster than the transitions from the restrictive to the permissive global state. From the above considerations we make an adiabatic approximations for all the transitions within the permissive state, and thus:

$$P(0,0,0)\frac{\mathbb{R}\_A}{\mathbb{K}\_\prime} = P(1,0,0)\_\prime$$

$$P(0,0,0)\frac{\mathbb{R}\_A}{\mathbb{K}\_\prime} = P(0,1,0)\_\prime$$

$$P(0,0,0)\frac{\mathbb{R}\_A}{\mathbb{K}\_\prime} = P(0,0,1)\_\prime$$

$$P(0,0,1)\frac{\mathbb{R}\_A}{\mathbb{K}\_\prime} = P(1,0,1)\_\prime$$

$$P(0,0,1)\frac{\mathbb{R}\_A}{\mathbb{K}\_\prime} = P(0,1,1)\_\prime$$

where *P*(*i*, *j*, *k*) stands for the probability of state *i*, *j*, *k* within the restrictive global state, *RA* denotes the number of active repressors, and *Kx* = *k*− *<sup>x</sup>* /*k*<sup>+</sup> *<sup>x</sup>* (*x* = *i*, *j*, *k*). It follow from the equations above and the constraint *P*(0, 0, 0) + *P*(1, 0, 0) + *P*(0, 1, 0) + *P*(0, 0, 1) + *P*(1, 0, 1) + *P*(0, 1, 1) = 1 that

$$\begin{split}P(0,0,0) &= \frac{1}{(1+\mathcal{R}\_A/\mathcal{K}\_k)(1+\mathcal{R}\_A/\mathcal{K}\_i+\mathcal{R}\_A/\mathcal{K}\_j)},\\P(1,0,0) &= \frac{\mathcal{R}\_A/\mathcal{K}\_i}{(1+\mathcal{R}\_A/\mathcal{K}\_k)(1+\mathcal{R}\_A/\mathcal{K}\_i+\mathcal{R}\_A/\mathcal{K}\_j)},\\P(0,1,0) &= \frac{\mathcal{R}\_A/\mathcal{K}\_j}{(1+\mathcal{R}\_A/\mathcal{K}\_k)(1+\mathcal{R}\_A/\mathcal{K}\_i+\mathcal{R}\_A/\mathcal{K}\_j)},\\P(0,0,1) &= \frac{\mathcal{R}\_A/\mathcal{K}\_k}{(1+\mathcal{R}\_A/\mathcal{K}\_k)(1+\mathcal{R}\_A/\mathcal{K}\_i+\mathcal{R}\_A/\mathcal{K}\_j)},\\P(1,0,1) &= \frac{\mathcal{R}\_A/\mathcal{K}\_k \times \mathcal{R}\_A/\mathcal{K}\_k}{(1+\mathcal{R}\_A/\mathcal{K}\_k)(1+\mathcal{R}\_A/\mathcal{K}\_i+\mathcal{R}\_A/\mathcal{K}\_j)}.\\\end{split}$$

Let *k*<sup>+</sup> (*k*−) be the transition rate from each of the states in the permissive (restrictive) global state to each if the states in the restrictive (permissive) global state. From Zeron and Santillán (2010), these rates are given by

$$k^{+} = k\_{\rm i}^{+}(P(0,1,0) + P(0,1,1)) + k\_{\rm j}^{+}(P(1,0,0) + P(1,0,1)) = \frac{k\_{\rm i}^{+} \mathcal{R}\_{A}/\mathcal{K}\_{\rm j} + k\_{\rm j}^{+} \mathcal{R}\_{A}/\mathcal{K}\_{\rm j}}{1 + \mathcal{R}\_{A}/\mathcal{K}\_{\rm i} + \mathcal{R}\_{A}/\mathcal{K}\_{\rm j}}.$$

and

$$k^{-} = \frac{k\_i^{-} + k\_j^{-}}{k\_c} \left( P(1, 1, 0) + P(1, 1, 1) \right) = \frac{k\_i^{-} + k\_j^{-}}{k\_c} \rho$$

since *P*(1, 1, 0) + *P*(1, 1, 1) = 1.

Finally, when the promoter is in the permissive global state, the probability that it is not bound by any repressor and so it is free to be bound by a polymerase to start transcription is

$$P\_{\mathcal{R}} = P(0,0,0) = \frac{1}{(1 + \mathcal{R}\_A/\mathcal{K}\_k)(1 + \mathcal{R}\_A/\mathcal{K}\_l + \mathcal{R}\_A/\mathcal{K}\_{\bar{j}})}.$$

Thus *PR* can be interpreted as the operator activity level.

#### **B. Estimation of the model parameters**

In this work we consider a bacterial doubling time of 40 min, and thus

$$\mu \simeq 0.017 \text{ min}^{-1}.$$

From the website E. coli Statistics, the total number of proteins in *E. coli* is about 3.6 millions, while the average protein size is 360 residues. On the other hand, we have from the website B1ONUMB3RS (http://bionumbers.hms.harvard.edu/) that the abundance of tryptophan in the *E. coli* proteins is around 1.1%. The data above imply that there are of the order of 14.256 million tryptophan molecules assembles in the *E. coli* proteins at any given time. If we further consider that all the proteins in a bacterium have to be doubled before it duplicates (every 40 min), then the average tryptophan consumption rate is

*ρmax* � 360, 000 molecules/min.

Since this consumption rate cannot be maintained when the tryptophan level is too low, we assumed that the consumption rate for this amino acid is given by

$$
\rho(T) = \rho\_{\text{max}} \frac{T}{T + K\_{\rho}},
$$

with

12 Will-be-set-by-IN-TECH

way the were constructed, the transitions within each global state and the transitions from the permissive to the restrictive global state are much faster than the transitions from the restrictive to the permissive global state. From the above considerations we make an adiabatic

*Ki* = *P*(1, 0, 0),

*Kj* = *P*(0, 1, 0),

*Kk* = *P*(0, 0, 1),

*Ki* = *P*(1, 0, 1),

*Kj* = *P*(0, 1, 1),

(1+*RA*/*Kk* )(1+*RA*/*Ki*+*RA*/*Kj*),

(1+*RA*/*Kk* )(1+*RA*/*Ki*+*RA*/*Kj*),

(1+*RA*/*Kk* )(1+*RA*/*Ki*+*RA*/*Kj*),

(1+*RA*/*Kk* )(1+*RA*/*Ki*+*RA*/*Kj*),

(1+*RA*/*Kk* )(1+*RA*/*Ki*+*RA*/*Kj*),

(1+*RA*/*Kk* )(1+*RA*/*Ki*+*RA*/*Kj*).

*<sup>j</sup>* (*P*(1, 0, 0) + *P*(1, 0, 1)) =

(1 + *RA*/*Kk*)(1 + *RA*/*Ki* + *RA*/*Kj*)

(*P*(1, 1, 0) + *P*(1, 1, 1)) =

Finally, when the promoter is in the permissive global state, the probability that it is not bound

by any repressor and so it is free to be bound by a polymerase to start transcription is

*PR* <sup>=</sup> *<sup>P</sup>*(0, 0, 0) = <sup>1</sup>

*k*+

*k*− *<sup>i</sup>* + *k*<sup>−</sup> *j kc*

*<sup>i</sup> RA*/*Kj* <sup>+</sup> *<sup>k</sup>*<sup>+</sup>

,

.

1 + *RA*/*Ki* + *RA*/*Kj*

*<sup>j</sup> RA*/*Ki*

,

*<sup>x</sup>* /*k*<sup>+</sup>

*<sup>x</sup>* (*x* = *i*, *j*, *k*). It follow from the

where *P*(*i*, *j*, *k*) stands for the probability of state *i*, *j*, *k* within the restrictive global state, *RA*

equations above and the constraint *P*(0, 0, 0) + *P*(1, 0, 0) + *P*(0, 1, 0) + *P*(0, 0, 1) + *P*(1, 0, 1) +

approximations for all the transitions within the permissive state, and thus:

*P*(0, 0, 0) *RA*

*P*(0, 0, 0) *RA*

*P*(0, 0, 0) *RA*

*P*(0, 0, 1) *RA*

*P*(0, 0, 1) *RA*

*P*(0, 0, 0) = <sup>1</sup>

*P*(1, 0, 0) = *RA*/*Ki*

*<sup>P</sup>*(0, 1, 0) = *RA*/*Kj*

*P*(0, 0, 1) = *RA*/*Kk*

*P*(1, 0, 1) = *RA*/*Ki*×*RA*/*Kk*

*<sup>P</sup>*(0, 1, 1) = *RA*/*Kj*×*RA*/*Kk*

Let *k*<sup>+</sup> (*k*−) be the transition rate from each of the states in the permissive (restrictive) global state to each if the states in the restrictive (permissive) global state. From Zeron and Santillán

denotes the number of active repressors, and *Kx* = *k*−

*P*(0, 1, 1) = 1 that

(2010), these rates are given by

since *P*(1, 1, 0) + *P*(1, 1, 1) = 1.

*<sup>i</sup>* (*P*(0, 1, 0) + *<sup>P</sup>*(0, 1, 1)) + *<sup>k</sup>*<sup>+</sup>

*<sup>k</sup>*<sup>−</sup> <sup>=</sup> *<sup>k</sup>*<sup>−</sup>

Thus *PR* can be interpreted as the operator activity level.

*<sup>i</sup>* + *k*<sup>−</sup> *j kc*

*k*<sup>+</sup> = *k*<sup>+</sup>

and

$$K\_{\rho} = 1,000 \text{ molecules.}$$

This choice for *K<sup>ρ</sup>* guarantees that the tryptophan consumption rate is most of the time very close to *ρmax*, except when there are of the order of a few thousand molecules of the amino acid.

According to the website E. coli Statistics (http://redpoll.pharmacy. ualberta.ca/CCDB/cgi-bin/STAT\_NEW.cgi), the average tryptophan molecule count in this bacterium is

$$T^\* \simeq 80\text{, } 000\text{ molecules.}$$

From Morse et al. (1968), the average number of anthranilate synthase enzymes in *E. coli* is

$$E^\* \simeq 2,000\text{ molecules.}$$

Caligiuri & Bauerle (1991) found from their experimental data that the probability that an anthranilate synthase enzyme is not feedback inhibited by tryptophan can be approximated by the following function:

$$P\_I(T) = \frac{K\_I^n}{K\_I^n + T^n}.$$

with

$$K\_I \simeq 2,500 \text{ molecules} \quad \text{and} \quad n \simeq 1.2.$$

Let *kT* be the tryptophan synthesis rate per anthranilate synthase molecule. Given, that the average tryptophan synthesis rate must equal the consumption rate for this amino acid, we can solve for *kT* from the following equation *kTE*∗*PI*(*T*∗) = *ρmax*. After doing the math we obtain

$$k\_T \simeq 12,000 \text{ molecules/min.}$$

**8. References**

439(7076): 608–611.

*Nature* 405(6786): 590–593.

*Nature* 422(6932): 633–637.

*typhimurium*, *J. Biol. Chem.* 266: 8328–8335.

gene, *Current Biology* 16(10): 1018–1025.

analysis, *Molecular Systems Biology* 2: 41.

*Escherichia coli*, *J Bacteriol* 167(1): 272–8.

*J Biochem* 265(3): 919–28.

*Theor. Biol.* 231: 287–298.

105(45): 17256–17261.

individual bacteria, *Cell* 123(6): 1025–1036.

repressor-operator recognition, *J. Mol. Biol.* 287: 539–554.

of *Escherichia coli*, *Proc. Natl. Acad. Sci.* 60(4): 1428–1435.

in mammalian cells, *Plos Biology* 4(10): 1707–1719.

and its consequences, *Cell* 135(2): 216–226.

Austin, D., Allen, M., McCollum, J., Dar, R., Wilgus, J., Sayler, G., Samatova, N., Cox, C.

<sup>193</sup> Transcriptional Bursting

in the Tryptophan Operon of *E. coli* and Its Effect on the System Stochastic Dynamics

Becskei, A. & Serrano, L. (2000). Engineering stability in gene networks by autoregulation,

Blake, W., Kaern, M., Cantor, C. & Collins, J. (2003). Noise in eukaryotic gene expression,

Brown, M. P., Grillo, A. O., Boyer, M. & Royer, C. A. (1999). Probing the role of water in the

Caligiuri, M. G. & Bauerle, R. (1991). Identification of amino acid residues involved

Choi, P. J., Cai, L., Frieda, K. & Xie, S. (2008). A stochastic single-molecule event triggers

Chubb, J., Trcek, T., Shenoy, S. & Singer, R. (2006). Transcriptional pulsing of a developmental

Dublanche, Y., Michalodimitrakis, K., Kuemmerer, N., Foglierini, M. & Serrano, L. (2006).

Golding, I., Paulsson, J., Zawilski, S. & Cox, E. (2005). Real-time kinetics of gene activity in

Grillo, A. O., Brown, M. P. & Royer, C. A. (1999). Probing the physical basis for *trp*

Gunsalus, R. P., Miguel, A. G. & Gunsalus, G. L. (1986). Intracellular *trp* repressor levels in

Jeeves, M., Evans, P. D., Parslow, R. A., Jaseja, M. & Hyde, E. I. (1999). Studies of the *Escherichia*

Morse, D. E., Baker, R. F. & Yanofsky, C. (1968). Translation of the tryptophan messenger RNA

Raj, A., Peskin, C. S., Tranchina, D., Vargas, D. Y. & Tyagi, S. (2006). Stochastic mRNA synthesis

Raj, A. & van Oudenaarden, A. (2008). Nature, nurture, or chance: Stochastic gene expression

Ross, I. L., Browne, C. M. & Hume, D. A. (1994). Transcription of individual genes in eukaryotic cells occurs randomly and infrequently, *Immunol Cell Biol* 72(2): 177–85. Santillán, M. & Zeron, E. S. (2004). Dynamic influence of feedback enzyme inhibition and

Shahrezaei, V. & Swain, P. S. (2008). Analytical distributions for stochastic gene expression,

Sharma, S. V., Lee, D. Y., Li, B., Quinlan, M. P., Takahashi, F., Maheswaran, S., McDermott,

transcription attenuation on the tryptophan operon response to nutritional shifts, *J.*

*Proceedings of the National Academy of Sciences of the United States of America*

U., Azizian, N., Zou, L., Fischbach, M. A., Wong, K.-K., Brandstetter, K., Wittner, B.,

Lewis, K. (2010). Persister cells, *Annual Review of Microbiology, Vol 64, 2010* 64: 357–372. Losick, R. & Desplan, C. (2008). Stochasticity and cell fate, *Science* 320(5872): 65–68.

*coli trp* repressor binding to its five operators and to variant operator sequences, *Eur*

in feedback regulation of the anthranilate synthase complex from *Salmonella*

Noise in transcription negative feedback loops: simulation and experimental

tryptophan repressor-operator complex, *Protein Sci* 8(6): 1276–85.

phenotype switching of a bacterial cell, *Science* 322(5900): 442–446.

& Simpson, M. (2006). Gene network shaping of inherent noise spectra, *Nature*

Consider the promoter model discussed in Section 3. Let *Pact* be the probability that the promoter is in the global permissive state. We have thus that, in the stationary state, *<sup>k</sup>*+*RAPact* <sup>=</sup> *<sup>k</sup>*−(<sup>1</sup> <sup>−</sup> *Pact*). Then, by solving for *Pact* in the previous equation and taking into consideration that, when the promoter is in the global permissive state, the probability that it is ready to be bound by a polymerase is *PR*. The stationary promoter activity level as a function of the tryptophan molecule count is

$$\frac{1}{1 + k^+(T)R\_A(T)/k^-}P\_R(T).$$

It is straightforward to test that the promoter activity level equals one when *T* = 0 molecules. On the other hand, Yanofsky & Horn (1994) measured that the operon expression level is maximal under conditions of tryptophan starvation, and that the repression regulatory mechanism decreases the promoter activity level by 60 times when the tryptophan level reaches its normal value. Thus, we must have that

$$\frac{1}{1 + k^+(T^\*)R\_A(T^\*)/k^-}P\_R(T^\*) = 1/60.5$$

This last result can then be used to estimate parameter *KT*—see Eqn. (4). Thus, from Eqn. (4) and given that (Gunsalus et al., 1986)

$$R\_T \simeq 400 \text{ molecules}$$

we obtain after some algebra that

$$K\_T \simeq 1.7 \times 10^6 \text{ molecules.}$$

Grillo et al. (1999) estimated the following values for the promoter-repressor interaction rates:

$$\begin{aligned} k\_i^+ &\simeq 8.1 \text{ molecules}^{-1} \text{min}^{-1}, \\ k\_i^- &\simeq 6.0 \text{ min}^{-1}, \\ k\_j^+ &\simeq 0.312 \text{ molecules}^{-1} \text{min}^{-1}, \\ k\_j^- &\simeq 0.198 \text{ min}^{-1}, \\ k\_k^+ &\simeq 0.3 \text{ molecules}^{-1} \text{min}^{-1}, \\ k\_k^- &\simeq 36.0 \text{ min}^{-1}, \\ k\_\mathbb{C} &\simeq 40.0 \end{aligned}$$

The probability that transcription is not prematurely terminated due to transcriptional attenuation is given by Eqn. (5). On the other hand, Yanofsky & Horn (1994) measured that one of every ten polymerases that have initiated transcription finish transcribing the operon genes when the tryptophan level is at its normal value. This means that *PA*(*T*) = 0.1. We obtain from this that

$$
\alpha \simeq 2.3 \times 10^{-4} \text{ molecules}^{-1}.
$$

Finally, the value of parameter *kE* is chosen so that, when *T* = *T*∗, the average enzyme molecule count is *E*∗. We found by inspection that

$$k\_E \simeq 30\text{\textquotedblleft}{0}000\text{ molecules/min.}$$

complies with this requirement.

#### **8. References**

14 Will-be-set-by-IN-TECH

Consider the promoter model discussed in Section 3. Let *Pact* be the probability that the promoter is in the global permissive state. We have thus that, in the stationary state, *<sup>k</sup>*+*RAPact* <sup>=</sup> *<sup>k</sup>*−(<sup>1</sup> <sup>−</sup> *Pact*). Then, by solving for *Pact* in the previous equation and taking into consideration that, when the promoter is in the global permissive state, the probability that it is ready to be bound by a polymerase is *PR*. The stationary promoter activity level as a

1 <sup>1</sup> <sup>+</sup> *<sup>k</sup>*+(*T*)*RA*(*T*)/*k*<sup>−</sup> *PR*(*T*). It is straightforward to test that the promoter activity level equals one when *T* = 0 molecules. On the other hand, Yanofsky & Horn (1994) measured that the operon expression level is maximal under conditions of tryptophan starvation, and that the repression regulatory mechanism decreases the promoter activity level by 60 times when the tryptophan level

<sup>1</sup> <sup>+</sup> *<sup>k</sup>*+(*T*∗)*RA*(*T*∗)/*k*<sup>−</sup> *PR*(*T*∗) = 1/60.

This last result can then be used to estimate parameter *KT*—see Eqn. (4). Thus, from Eqn. (4)

*RT* � 400 molecules,

*KT* � 1.7 <sup>×</sup> 106 molecules. Grillo et al. (1999) estimated the following values for the promoter-repressor interaction rates:

*<sup>i</sup>* � 8.1 molecules<sup>−</sup>1min−1,

*<sup>j</sup>* � 0.312 molecules<sup>−</sup>1min−1,

*<sup>k</sup>* � 0.3 molecules<sup>−</sup>1min−1,

The probability that transcription is not prematurely terminated due to transcriptional attenuation is given by Eqn. (5). On the other hand, Yanofsky & Horn (1994) measured that one of every ten polymerases that have initiated transcription finish transcribing the operon genes when the tryptophan level is at its normal value. This means that *PA*(*T*) = 0.1. We

*<sup>α</sup>* � 2.3 <sup>×</sup> <sup>10</sup>−<sup>4</sup> molecules<sup>−</sup>1.

Finally, the value of parameter *kE* is chosen so that, when *T* = *T*∗, the average enzyme

*kE* � 30, 000 molecules/min.

*<sup>i</sup>* � 6.0 min<sup>−</sup>1,

*<sup>j</sup>* � 0.198 min<sup>−</sup>1,

*<sup>k</sup>* � 36.0 min<sup>−</sup>1,

*kc* � 40.0

1

*k*+

*k*−

*k*+

*k*−

*k*+

*k*−

molecule count is *E*∗. We found by inspection that

function of the tryptophan molecule count is

reaches its normal value. Thus, we must have that

and given that (Gunsalus et al., 1986)

we obtain after some algebra that

obtain from this that

complies with this requirement.


**7** 

*1USA 2India* 

Rakesh Sharma1,2

*1Center of Nanomagnetics and Biotechnology, Florida State University, Tallahassee, Florida* 

**Mechanisms of Hepatocellular Dysfunction** 

**Nitroimidazole and Human Liver Regeneration** 

Enzymes so called (Enz'-a-ai-am) are biologically active protein molecules responsible of biochemical reactions in the bacteria, cells and organs. Enzymes regulate the rates of biochemical reactions to maintain the metabolism to keep active physiological actions in the body. High enzyme activities or high rates of reactions cause higher product formation or deposits to initiate disease. Drugs are used as enzyme competitors to normalize the reactions to correct disease. Most of diseases are cured by 'enzyme inhibition'. Enzyme inhibition can be three types: competitive, non-competitive, and uncompetitive. Enzyme inhibition also provides a kind of defense in cells by regulation of metabolism to inhibit or stimulate the biochemical processes. Most of the drugs undergo detoxification and biotransformation in liver to regenerate or formation of new hepatocytes and Kupffer cells. Major biochemical events in liver regeneration are regulated by enzymes in energy

Present chapter describes an example of liver cell enzyme battery to regulate the carbohydrate, lipid and protein metabolism in liver cells, mainly hepatocytes and Kupffer cells in the light of liver damage due to amoebic liver abscess and role of enzyme inhibition in liver regeneration by 2'-nitroimidazole. Liver damage by amoeba is manifested by elevated enzymes in cells. As a result, two major clinical manifestations of *Entamoeba histolytica* infection are amoebic colitis and amoebic liver abscesses. To cure amoebic liver abscess, liver regeneration and amoebic killing by 2'-nitroimidazole therapy is routine in clinical practice. 2'-nitroimidazole acts in liver to perform enzyme inhibition at the level of carbohydrate, lipid, and protein metabolic regulatory steps. Earlier, nitroimidazole derivatives were considered drug of choice in treatment of hepatic hypoxia (low oxygen) conditions in parasitic infections, cancer and recently nitroimidazole derivatives are emerging as hypoxia markers and radiosensitizers in

Since 1960, hepatocytes were investigated rich in major enzymes regulating the energy metabolism located in cytoplasm and mitochondria for glycolysis, TCA cycle while drug

**1. Introduction** 

metabolism, growth factors and cytokine molecules.

tumor treatment [Sharma 2001, Sharma 2011a, 2011b].

**and Regeneration: Enzyme Inhibition by** 

*2Amity Institute of Nanotechnology, Amity University, NOIDA UP,* 

Ramaswamy, S., Classon, M. & Settleman, J. (2010). A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations, *Cell* 141(1): 69–80.


## **Mechanisms of Hepatocellular Dysfunction and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration**

#### Rakesh Sharma1,2

*1Center of Nanomagnetics and Biotechnology, Florida State University, Tallahassee, Florida 2Amity Institute of Nanotechnology, Amity University, NOIDA UP, 1USA 2India* 

#### **1. Introduction**

16 Will-be-set-by-IN-TECH

194 Enzyme Inhibition and Bioapplications

So, L.-H., Ghosh, A., Zong, C., Sepulveda, L. A., Segev, R. & Golding, I. (2011).

Xie, G., Keyhani, N. O., Bonner, C. A. & Jensen, R. A. (2003). Ancient origin of the

Yanofsky, C. (2000). Transcription attenuation, once viewed as a novel regulatory strategy, *J.*

Yanofsky, C. & Crawford, I. P. (1987). The tryptophan operon, *in* F. C. Neidhart, J. L. Ingraham,

Yanofsky, C. & Horn, V. (1994). Role of regulatory features of the *trp* operon of *Escherichia coli* in mediating a response to a nutritional shift, *J. Bacteriol.* 176: 6245–6254.

drug-tolerant state in cancer cell subpopulations, *Cell* 141(1): 69–80.

43(6): 554–U84.

*Rev.* 67: 303–342.

*Bacteriol.* 182: 1–8.

DC, pp. 1453–1472.

Ramaswamy, S., Classon, M. & Settleman, J. (2010). A chromatin-mediated reversible

General properties of transcriptional time series in escherichia coli, *Nature Genetics*

tryptophan operon and the dynamics of evolutionary change, *Microbiol. Mol. Biol.*

K. B. Low, B. Magasanik & H. E. Umbarger (eds), *Escherichia coli and Salmonella thyphymurium: Cellular and Molecular Biology, Vol. 2*, Am. Soc. Microbiol., Washington,

> Enzymes so called (Enz'-a-ai-am) are biologically active protein molecules responsible of biochemical reactions in the bacteria, cells and organs. Enzymes regulate the rates of biochemical reactions to maintain the metabolism to keep active physiological actions in the body. High enzyme activities or high rates of reactions cause higher product formation or deposits to initiate disease. Drugs are used as enzyme competitors to normalize the reactions to correct disease. Most of diseases are cured by 'enzyme inhibition'. Enzyme inhibition can be three types: competitive, non-competitive, and uncompetitive. Enzyme inhibition also provides a kind of defense in cells by regulation of metabolism to inhibit or stimulate the biochemical processes. Most of the drugs undergo detoxification and biotransformation in liver to regenerate or formation of new hepatocytes and Kupffer cells. Major biochemical events in liver regeneration are regulated by enzymes in energy metabolism, growth factors and cytokine molecules.

> Present chapter describes an example of liver cell enzyme battery to regulate the carbohydrate, lipid and protein metabolism in liver cells, mainly hepatocytes and Kupffer cells in the light of liver damage due to amoebic liver abscess and role of enzyme inhibition in liver regeneration by 2'-nitroimidazole. Liver damage by amoeba is manifested by elevated enzymes in cells. As a result, two major clinical manifestations of *Entamoeba histolytica* infection are amoebic colitis and amoebic liver abscesses. To cure amoebic liver abscess, liver regeneration and amoebic killing by 2'-nitroimidazole therapy is routine in clinical practice. 2'-nitroimidazole acts in liver to perform enzyme inhibition at the level of carbohydrate, lipid, and protein metabolic regulatory steps. Earlier, nitroimidazole derivatives were considered drug of choice in treatment of hepatic hypoxia (low oxygen) conditions in parasitic infections, cancer and recently nitroimidazole derivatives are emerging as hypoxia markers and radiosensitizers in tumor treatment [Sharma 2001, Sharma 2011a, 2011b].

> Since 1960, hepatocytes were investigated rich in major enzymes regulating the energy metabolism located in cytoplasm and mitochondria for glycolysis, TCA cycle while drug

Mechanisms of Hepatocellular Dysfunction

Glucose 6 Pase Glucose 6 PDH Phoshogluconate DH Phosphofructokinase

Citrate Synthase Isocitrate DH Succinate DH Malate DH

NADH Oxidase

Acid DNAase

Peroxidase Catalase

Guanase

Ca++ ATPase 5' Nucleotidase Acid Lipase Acid Phosphatase Alkaline Phosphatase Beta glucuronidase

Cytochrome C Oxidase NADPH Cyt C Reducatase

Glutathione Reductase

Superoxide dismutase

Adinosine Deaminase Leucine aminopeptidase

high(+++), extreme(++++ or more).

Tyrosine Aminotransferase Aniline Hydroxylase Aminopyrine demethylase

Aldolase Pyruvate kinase Pyruvate DH LDH

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 197

number of lysosomal enzymes acting as destroyers actually lyse and digest the bacteria, virus and toxicants. Microsomal enzymes are active in detoxification of drugs or drug biotransformaton to interact (stimulation or inhibition of enzymes) with metabolic regulation in liver cells. Let us introduce a bit of enzymes in liver cells performing different metabolic regulatory functions in following description. Abscesses occupying large areas of the liver can be cured without drainage, and even by one single dose of nitroimidazole.[Powell et al. 1969, Lasserre et al. 1983, Akgun et al. 1999]. Today, role of ultrasound or percutaneous therapeutic aspiration guided by CT in the treatment of

Enzyme Amoeba Hepatocyte Kupffer cell

Source: Sharma R. Effect of Nitroimidazole on isolated liver cells in development of amoebic liver

Table 1. Carbohydrate metabolizing enzymes in amoeba, hepatocytes and Kupffer cells. Abundance of enzyme activities in cells are shown. Comparative enzyme database of amoeba and isolated liver cells is shown and sketched in Figures 3,5, and 6. Strengths of different enzyme activities in amoeba, liver cells are shown as weak(+), moderate(++),

abscess. Ph.D dissertation submitted to Indian Institute of Technology, Delhi, 1995.

++++ ++++ +++ +++ ++ ++ ++ + ++++ +++ ++ +++ + + + ++ ++ ++ ++ ++ +++ ++ ++ ++ + ++ ++ ++ ++ ++ +++ ++

++ ++ + ++ ++ + ++ ++ + ++ + ++ ++ +++ ++ + + + + + + + + ++ + + + + + + + ++

+ + + + + + + +++ ++ ++ + ++ ++ ++ ++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ ++++ ++++ ++++ ++++ +++++ +++++ +++

uncomplicated amoebic liver-abscess by surgical drainage is controversial.

metabolizing and redox enzymes are located in lysosomes and microsomes. Other enzymes also participate in defense processes such as respiratory burst, HMP shunt, oxygenase pathways and inflammatory cytokines. Present chapter reviews the ongoing developments with clear and complete information on action of new nitroimidazole derivatives as 'enzyme inhibitors' in liver selective cytotoxicity, oxygen depletion, hepatocellular DNA and enzyme normalization before enzyme can be used in hypoxia monitoring and therapy [Sharma, 2011b]. The paucity of information on nitroimidazole-liver tissue interaction is poor and available data of initial sequential biochemical changes in liver cells is scanty which further leads to detectable hypoxia [Sharma 2011a, 2011b]. It is believed that initially enzyme regulated glucose and calcium hemostasis are the primary targets of hepatic hypoxia followed by enzyme regulated induced metabolic integrity loss leads to apoptosis, regulatory failure in glycolytic, TCA cycle, gluconeogenesis and Ca++ mediated cAMP related biodegradation of molecules [Sharma et al.2011c].

The present chapter focuses on hepatic enzyme inhibition by nitroimidazole at different levels of energy metabolism, respiratory burst and drug metabolizing enzymes. In following section, a molecular basis of enzyme inhibition and hepatocellular hypoxia and dysfunction criteria is described in detail. In following section, regulatory behavior of rate limiting enzymes of glycolysis, TCA cycle, phagocytosis is described with examples: glucokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NADPH cytochrome P450 reductase, phosphodieaterase, lysosomal enzymes in serum, liver biopsy samples. In next section, role of enzymes in liver abscess is described.

#### **1.1 Enzymes in liver abscess**

*Entamoeba histolytica* is a human-specific pathogen and reproducible animal models of intestinal amoebiasis have proved elusive to describe interaction between *E. histolytica* and liver cells. The most common extraintestinal manifestation of disease is amoebic liverabscess. Amoebic liver-abscesses arise from haematogenous spread (probably via the portal circulation) of amoebic trophozoites that have breached the colonic mucosa.[Thompson et al. 1985, Abuabara et al. 1982, Shandera et al. 1998, Barnes et al. 1987, Adams 1977, Lancet 2003]. Effective nitroimidazole treatment and rapid diagnosis showed the mortality rates to 1–3%. *Entamoeba histolytica* trophozoites can lyse neutrophils *in vitro*, causing them to release toxic substance killer enzymes such as superoxide dismutases, collagenases, elastases and cathepsins [Jarumilinta et al. 1964, Guerrant et al. 1981]. On these lines, author proposed a 'Hepatocellular dysfunction criteria' to make systematic observations in abscess development and step by step method of medical/surgical intervention [Sharma et al.2011]. In previous reports, patient symptoms, leucocytosis without eosinophilia, mild anaemia, raised concentrations of alkaline phosphatase, and a high rate of erythrocyte sedimentation were the most common laboratory findings.[ Thompson et al. 1985, Abuabara et al. 1982, Shandera et al. 1998, Barnes et al. 1987, Adams 1977, Lancet 2003].

Amoeba cause amoebic liver abscesses, which are circumscribed regions of dead hepatocytes, liquefied cells and cellular debris surrounded by a rim of connective tissue, some inflammatory cells and few amoebic trophozoites [Sharma et al. 2011]. The adjacent liver parenchyma is usually completely normal. At same sites in liver so called 'acini' fixed sinusoidal phagocytic cells (Kupffer cells) provide defense against any bacteria, virus or foreign drug. Kupffer cells have two intracellular structures filled with enzymes: lysosomes (Lai-zo-somes) and microsomes (Mai-kro-zomes). Lysososmal bags filled with a large

metabolizing and redox enzymes are located in lysosomes and microsomes. Other enzymes also participate in defense processes such as respiratory burst, HMP shunt, oxygenase pathways and inflammatory cytokines. Present chapter reviews the ongoing developments with clear and complete information on action of new nitroimidazole derivatives as 'enzyme inhibitors' in liver selective cytotoxicity, oxygen depletion, hepatocellular DNA and enzyme normalization before enzyme can be used in hypoxia monitoring and therapy [Sharma, 2011b]. The paucity of information on nitroimidazole-liver tissue interaction is poor and available data of initial sequential biochemical changes in liver cells is scanty which further leads to detectable hypoxia [Sharma 2011a, 2011b]. It is believed that initially enzyme regulated glucose and calcium hemostasis are the primary targets of hepatic hypoxia followed by enzyme regulated induced metabolic integrity loss leads to apoptosis, regulatory failure in glycolytic, TCA cycle, gluconeogenesis and Ca++ mediated cAMP

The present chapter focuses on hepatic enzyme inhibition by nitroimidazole at different levels of energy metabolism, respiratory burst and drug metabolizing enzymes. In following section, a molecular basis of enzyme inhibition and hepatocellular hypoxia and dysfunction criteria is described in detail. In following section, regulatory behavior of rate limiting enzymes of glycolysis, TCA cycle, phagocytosis is described with examples: glucokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NADPH cytochrome P450 reductase, phosphodieaterase, lysosomal enzymes in serum, liver biopsy samples. In next

*Entamoeba histolytica* is a human-specific pathogen and reproducible animal models of intestinal amoebiasis have proved elusive to describe interaction between *E. histolytica* and liver cells. The most common extraintestinal manifestation of disease is amoebic liverabscess. Amoebic liver-abscesses arise from haematogenous spread (probably via the portal circulation) of amoebic trophozoites that have breached the colonic mucosa.[Thompson et al. 1985, Abuabara et al. 1982, Shandera et al. 1998, Barnes et al. 1987, Adams 1977, Lancet 2003]. Effective nitroimidazole treatment and rapid diagnosis showed the mortality rates to 1–3%. *Entamoeba histolytica* trophozoites can lyse neutrophils *in vitro*, causing them to release toxic substance killer enzymes such as superoxide dismutases, collagenases, elastases and cathepsins [Jarumilinta et al. 1964, Guerrant et al. 1981]. On these lines, author proposed a 'Hepatocellular dysfunction criteria' to make systematic observations in abscess development and step by step method of medical/surgical intervention [Sharma et al.2011]. In previous reports, patient symptoms, leucocytosis without eosinophilia, mild anaemia, raised concentrations of alkaline phosphatase, and a high rate of erythrocyte sedimentation were the most common laboratory findings.[ Thompson et al. 1985, Abuabara et al. 1982,

Amoeba cause amoebic liver abscesses, which are circumscribed regions of dead hepatocytes, liquefied cells and cellular debris surrounded by a rim of connective tissue, some inflammatory cells and few amoebic trophozoites [Sharma et al. 2011]. The adjacent liver parenchyma is usually completely normal. At same sites in liver so called 'acini' fixed sinusoidal phagocytic cells (Kupffer cells) provide defense against any bacteria, virus or foreign drug. Kupffer cells have two intracellular structures filled with enzymes: lysosomes (Lai-zo-somes) and microsomes (Mai-kro-zomes). Lysososmal bags filled with a large

related biodegradation of molecules [Sharma et al.2011c].

section, role of enzymes in liver abscess is described.

Shandera et al. 1998, Barnes et al. 1987, Adams 1977, Lancet 2003].

**1.1 Enzymes in liver abscess** 

number of lysosomal enzymes acting as destroyers actually lyse and digest the bacteria, virus and toxicants. Microsomal enzymes are active in detoxification of drugs or drug biotransformaton to interact (stimulation or inhibition of enzymes) with metabolic regulation in liver cells. Let us introduce a bit of enzymes in liver cells performing different metabolic regulatory functions in following description. Abscesses occupying large areas of the liver can be cured without drainage, and even by one single dose of nitroimidazole.[Powell et al. 1969, Lasserre et al. 1983, Akgun et al. 1999]. Today, role of ultrasound or percutaneous therapeutic aspiration guided by CT in the treatment of uncomplicated amoebic liver-abscess by surgical drainage is controversial.


Source: Sharma R. Effect of Nitroimidazole on isolated liver cells in development of amoebic liver abscess. Ph.D dissertation submitted to Indian Institute of Technology, Delhi, 1995.

Table 1. Carbohydrate metabolizing enzymes in amoeba, hepatocytes and Kupffer cells. Abundance of enzyme activities in cells are shown. Comparative enzyme database of amoeba and isolated liver cells is shown and sketched in Figures 3,5, and 6. Strengths of different enzyme activities in amoeba, liver cells are shown as weak(+), moderate(++), high(+++), extreme(++++ or more).

Mechanisms of Hepatocellular Dysfunction

that requires live active trophozoites.

seen as a DNA ladder on separating gels.

**1.2 Entamoeba histolytica and programmed cell death** 

independent of Fas–Fas-ligand interactions and TNFR1.

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 199

placed in human intestinal xenografts [Ankri et al.1998]. It suggests that amoebic cysteine proteinases must exert their effects on gut inflammation and tissue damage after an initial step

Other important role of caspase enzymes was explored when *E. histolytica* trophozoites were incubated with common types of mammalian cells. Normally trophozoites kill mammalian cells in a contact dependent manner by two processes: by lytic necrosis [Berninghausen et al.1997] or undergo apoptosis[Ragland et al.1994]. Apoptosis, or programmed cell death, is an ordered system of cell death with an initiator or signaling phase followed by an effector stage that causes cell death by degrading various cellular components. The effector stage involves the activation of caspase enzymes, cysteine proteinases with specificity for aspartate residues that form a cascade, converging on caspase 3. A key biochemical marker of apoptosis is endonuclease cleavage of chromatin between histone bodies, which can be

Detection of DNA fragmentation *in situ* by the endlabeling of single- or double-stranded DNA breaks with terminal deoxyribonucleotidyltransferase (TUNEL assay) has also been used as a biomarker of apoptosis [Schulte-Hermann et al.1994]. However, *E. histolytica* can induce apoptosis *in vitro* raises doubt if apoptosis is a component of the cell death commonly seen in amoebic liver abscess. To test this fact, amoebae were inoculated into the livers of SCID mice and evidence for apoptosis was sought by gel separation of DNA from infected livers and TUNEL staining of amoebic liver abscesses [Seydel et al.1998]. DNA ladder formation was detected in samples obtained from SCID mice by 1 h after amoebic inoculation. TUNEL staining revealed significant areas of apoptosis within amoebic liver abscesses common in inflammatory cells and hepatocytes close to amoebic trophozoites [Stenley, 2001]. However, some distant liver cells also were TUNEL positive. Thus, in the murine model of amoebic liver abscess, *E. histolytica* induced cellular apoptosis appears to be a significant component of cell death as shown in Fig. 3. Interestingly, *E. histolytica*induced apoptosis was not blocked by Bcl-2 [Ragland et al.1994] and did not appear to involve either of the two of the major pathways for the induction of apoptosis: ligation of the Fas receptor and ligation of TNFα1 receptor 1 (TNFR1). In both C57Bl/6 MRL-lpr/lpr mice with no Fas receptor, and C57Bl/6.C3H-gld/gld mice with no Fas ligand, apoptosis was detected in amoebic liver abscesses by DNA ladder formation and TUNEL staining [Seydel et al. 1998]. Apoptosis was also present in hepatocytes in amoebic liver abscesses from both TNFR1 knockout mice and the heterozygote controls[Seydel et al. 1998].]. These data indicate that *E. histolytica* causes apoptosis in mouse liver by a mechanism that is

Genetic approaches and new models of amoebic liver abscesses have provided good prospects on the complex interactions between *E. histolytica* and the host liver cells [Hamann et al.1995]. Well-known *E. histolytica* enzymes lyse cells and digest extracellular matrix proteins. Enzymes have capability to induce apoptosis in hepatocytes and inflammatory cells. Enzymes can stimulate, and perhaps enhance, an NF-κB-mediated human liver inflammatory response that contributes to tissue damage. These new enzyme regulated pathways might offer an explanation for some of the clinical and pathological differences

Characterization of enzymes in amoeba has been a long time quest to explore the possibility of amebic enzyme inhibition by new drugs in drug discovery. Several antiamoebic drugs are in market as potent enzyme inhibitors since last four decades. Author proposed a comparative enzyme database of amoeba and hepatocellular cells in culture (see Table 1 and Figure 1). Of specific mention, cysteine proteinase enzymes are secreted in large quantities by the parasite during immune response and can cleave extracellular matrix proteins, which might facilitate amoebic invasion[Scholze et al. 1988, Keene 1986]. Mainly carbohydrate metabolizing, energy metabolizing enzymes and lyssomal enzymes are abundant and participate in amoebic –host liver cell interaction as shown in Figures 2,3a,3b and 3c. These proteins act as virulence factors in animal models of amoebic liver abscess [Stanley et al.1995, Ankri et al.1999]. A family of six genes encodes *E. histolytica* cysteine proteinases (*ehcp1–6* ) but ~90% of the proteinase activity is related to three proteinase enzymes, EhCP1, EhCP2 and EhCP5 [Bruchhaus, et al. 2003]. Although targeted disruption of selected *E. histolytica* genes has not yet been achieved, stable episomal expression of foreign DNA is possible in amoebae by maintaining continuous selective pressure [Hamann et al. 1995, Vines et al. 1995]. This has enabled the investigators to target specific molecules in *E. histolytica* by the episomal expression of antisense mRNA or of genes encoding dominant negative mutants [Ankri et al. 1998]. The antisense approach has been applied to *E. histolytica* cysteine proteinases and episomal expression of an antisense RNA to *ehcp5* could reduce total amoebic proteinase activity by 80–90% [Ankri et al. 1998]. To assess the role of *E. histolytica* cysteine proteinases in amoebiasis, in earlier study, human xenografts in SCID-HU-INT mice were infected with amoebic trophozoites expressing the *ehcp5* antisense RNA(proteinase-deficient amoebae) or amoebic trophozoites containing the same plasmid without the antisense insert (the control group)[Zhang et al. 2000]. Major findings were: 1. no obvious defect was apparent in the ability of cysteine-proteinase deficient amoebae to inhabit and survive within the colonic lumen; 2. post-24 h after infection, cysteine proteinase-deficient amoeba had, in contrast to the control group, failed to induce significant amounts of human IL-1α or IL-8 from infected intestine; 3. gut inflammation was also reduced in human intestine infected with cysteine-proteinase-deficient *E. histolytica* trophozoites; 4. control *E. histolytica* trophozoites damaged the intestinal permeability barrier at 24 h but there was only a minimal increase in intestinal permeability in human intestinal xenografts infected with cysteine-protease-deficient amoeba; 5. histological studies at 24 h, human xenografts infected with control amoebae showed damage to the colonic mucosa, invasion of amoebic trophozoites into submucosal tissues and neutrophil-predominant inflammation; 6. by contrast, xenografts infected with cysteine-proteinase-deficient amoebae showed less mucosal damage, almost no evidence for amoebic invasion into submucosal tissue and little inflammation. Authors speculated that cysteine-proteinase-deficient amoeba might have a defect in their ability to induce gut inflammation and to invade into the submucosal tissues [Zhang et al. 2000]. How amoebic cysteine proteinases contribute to gut inflammation and tissue damage in amoebiasis? Possibly, *Entamoeba histolytica* trophozoites expressing the *ehcp5*  antisense RNA might have reduced the phagocytic capabilities or reduced virulence in amoebiasis [Ankri et al.1998]. However, protease-deficient amoebae maintain their ability to lyse target cells, so a defect in cell killing does not underlie the reduced virulence of cysteine proteinase-deficient amoebae [Seydel et al.1997]. In addition, it is unlikely to be a direct effect of cysteine proteases on intestinal epithelial cells, because amoebic lysates rich in cysteine protease activity fail to induce high levels of cytokine production or inflammation when placed in human intestinal xenografts [Ankri et al.1998]. It suggests that amoebic cysteine proteinases must exert their effects on gut inflammation and tissue damage after an initial step that requires live active trophozoites.

#### **1.2 Entamoeba histolytica and programmed cell death**

198 Enzyme Inhibition and Bioapplications

Characterization of enzymes in amoeba has been a long time quest to explore the possibility of amebic enzyme inhibition by new drugs in drug discovery. Several antiamoebic drugs are in market as potent enzyme inhibitors since last four decades. Author proposed a comparative enzyme database of amoeba and hepatocellular cells in culture (see Table 1 and Figure 1). Of specific mention, cysteine proteinase enzymes are secreted in large quantities by the parasite during immune response and can cleave extracellular matrix proteins, which might facilitate amoebic invasion[Scholze et al. 1988, Keene 1986]. Mainly carbohydrate metabolizing, energy metabolizing enzymes and lyssomal enzymes are abundant and participate in amoebic –host liver cell interaction as shown in Figures 2,3a,3b and 3c. These proteins act as virulence factors in animal models of amoebic liver abscess [Stanley et al.1995, Ankri et al.1999]. A family of six genes encodes *E. histolytica* cysteine proteinases (*ehcp1–6* ) but ~90% of the proteinase activity is related to three proteinase enzymes, EhCP1, EhCP2 and EhCP5 [Bruchhaus, et al. 2003]. Although targeted disruption of selected *E. histolytica* genes has not yet been achieved, stable episomal expression of foreign DNA is possible in amoebae by maintaining continuous selective pressure [Hamann et al. 1995, Vines et al. 1995]. This has enabled the investigators to target specific molecules in *E. histolytica* by the episomal expression of antisense mRNA or of genes encoding dominant negative mutants [Ankri et al. 1998]. The antisense approach has been applied to *E. histolytica* cysteine proteinases and episomal expression of an antisense RNA to *ehcp5* could reduce total amoebic proteinase activity by 80–90% [Ankri et al. 1998]. To assess the role of *E. histolytica* cysteine proteinases in amoebiasis, in earlier study, human xenografts in SCID-HU-INT mice were infected with amoebic trophozoites expressing the *ehcp5* antisense RNA(proteinase-deficient amoebae) or amoebic trophozoites containing the same plasmid without the antisense insert (the control group)[Zhang et al. 2000]. Major findings were: 1. no obvious defect was apparent in the ability of cysteine-proteinase deficient amoebae to inhabit and survive within the colonic lumen; 2. post-24 h after infection, cysteine proteinase-deficient amoeba had, in contrast to the control group, failed to induce significant amounts of human IL-1α or IL-8 from infected intestine; 3. gut inflammation was also reduced in human intestine infected with cysteine-proteinase-deficient *E. histolytica* trophozoites; 4. control *E. histolytica* trophozoites damaged the intestinal permeability barrier at 24 h but there was only a minimal increase in intestinal permeability in human intestinal xenografts infected with cysteine-protease-deficient amoeba; 5. histological studies at 24 h, human xenografts infected with control amoebae showed damage to the colonic mucosa, invasion of amoebic trophozoites into submucosal tissues and neutrophil-predominant inflammation; 6. by contrast, xenografts infected with cysteine-proteinase-deficient amoebae showed less mucosal damage, almost no evidence for amoebic invasion into submucosal tissue and little inflammation. Authors speculated that cysteine-proteinase-deficient amoeba might have a defect in their ability to induce gut inflammation and to invade into the submucosal tissues [Zhang et al. 2000]. How amoebic cysteine proteinases contribute to gut inflammation and tissue damage in amoebiasis? Possibly, *Entamoeba histolytica* trophozoites expressing the *ehcp5*  antisense RNA might have reduced the phagocytic capabilities or reduced virulence in amoebiasis [Ankri et al.1998]. However, protease-deficient amoebae maintain their ability to lyse target cells, so a defect in cell killing does not underlie the reduced virulence of cysteine proteinase-deficient amoebae [Seydel et al.1997]. In addition, it is unlikely to be a direct effect of cysteine proteases on intestinal epithelial cells, because amoebic lysates rich in cysteine protease activity fail to induce high levels of cytokine production or inflammation when

Other important role of caspase enzymes was explored when *E. histolytica* trophozoites were incubated with common types of mammalian cells. Normally trophozoites kill mammalian cells in a contact dependent manner by two processes: by lytic necrosis [Berninghausen et al.1997] or undergo apoptosis[Ragland et al.1994]. Apoptosis, or programmed cell death, is an ordered system of cell death with an initiator or signaling phase followed by an effector stage that causes cell death by degrading various cellular components. The effector stage involves the activation of caspase enzymes, cysteine proteinases with specificity for aspartate residues that form a cascade, converging on caspase 3. A key biochemical marker of apoptosis is endonuclease cleavage of chromatin between histone bodies, which can be seen as a DNA ladder on separating gels.

Detection of DNA fragmentation *in situ* by the endlabeling of single- or double-stranded DNA breaks with terminal deoxyribonucleotidyltransferase (TUNEL assay) has also been used as a biomarker of apoptosis [Schulte-Hermann et al.1994]. However, *E. histolytica* can induce apoptosis *in vitro* raises doubt if apoptosis is a component of the cell death commonly seen in amoebic liver abscess. To test this fact, amoebae were inoculated into the livers of SCID mice and evidence for apoptosis was sought by gel separation of DNA from infected livers and TUNEL staining of amoebic liver abscesses [Seydel et al.1998]. DNA ladder formation was detected in samples obtained from SCID mice by 1 h after amoebic inoculation. TUNEL staining revealed significant areas of apoptosis within amoebic liver abscesses common in inflammatory cells and hepatocytes close to amoebic trophozoites [Stenley, 2001]. However, some distant liver cells also were TUNEL positive. Thus, in the murine model of amoebic liver abscess, *E. histolytica* induced cellular apoptosis appears to be a significant component of cell death as shown in Fig. 3. Interestingly, *E. histolytica*induced apoptosis was not blocked by Bcl-2 [Ragland et al.1994] and did not appear to involve either of the two of the major pathways for the induction of apoptosis: ligation of the Fas receptor and ligation of TNFα1 receptor 1 (TNFR1). In both C57Bl/6 MRL-lpr/lpr mice with no Fas receptor, and C57Bl/6.C3H-gld/gld mice with no Fas ligand, apoptosis was detected in amoebic liver abscesses by DNA ladder formation and TUNEL staining [Seydel et al. 1998]. Apoptosis was also present in hepatocytes in amoebic liver abscesses from both TNFR1 knockout mice and the heterozygote controls[Seydel et al. 1998].]. These data indicate that *E. histolytica* causes apoptosis in mouse liver by a mechanism that is independent of Fas–Fas-ligand interactions and TNFR1.

Genetic approaches and new models of amoebic liver abscesses have provided good prospects on the complex interactions between *E. histolytica* and the host liver cells [Hamann et al.1995]. Well-known *E. histolytica* enzymes lyse cells and digest extracellular matrix proteins. Enzymes have capability to induce apoptosis in hepatocytes and inflammatory cells. Enzymes can stimulate, and perhaps enhance, an NF-κB-mediated human liver inflammatory response that contributes to tissue damage. These new enzyme regulated pathways might offer an explanation for some of the clinical and pathological differences

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 201

followed by detectable necrosis in liver. Hypothesis was that liver cells undergo a series of changes - hepatocytes undergo metabolic energy loss and oxygen depletion (hypoxia) while Kupffer cells undergo phagocytosis, respiratory burst and proteolysis. Nitroimidazole induces enzyme inhibition in liver cells to normalize the elevated enzymes during metabolic energy loss and oxygen depletion (hypoxia) in liver cell (in mitochondria) and lysosomal enzyme inhibition to combat the phagocytosis. The approach is applicable possibly in design of drugs to treat oxygen starved tumor cells or infected liver lesions and abscesses. Previous reports on first initial stage of nitroimidazole induced liver cell cytotoxicity indicated the apoptosis, glutathione, DNA interaction, oxygen depletion, lactate inhibition, enhanced NADPH cytochrome reductase and superoxide dismutase enzymes [Ersoz et al.2001, Brezden et al.1997, Noss et al.1988, Sugiyama et al.1993, Berube et al. 1992, Sapora et al.1992, Moselen et al. 1995, Stratford et al.1981, Adams et al.1980, Noss et al. 1989, Mulcahy et al.1989, Widel et al. 1982, Rauth et al. 1981, Chao et al. 1982, Whitmore et al.1986]. Still nitroimidazole structure-function relationship between hypoxia, radiosensitization and cytotoxicity or liver regeneration remain less understood perhaps due to nitroimidazole low sensitivity to clinical investigations and significant enzyme alterations only at the very late necrotic stage [Cowan et al.1994, Melo et al. 2000, Moller et al. 2002, Ballinger et al. 1996, Edwards et al. 1981, Edwards et al.1982, Ballinger et al.2001, Riche et al.2001, Melo et al.2000]. However, nitroimidazole cytotoxicity was reported continuously in last three decades indicating many altered enzymes involved in nitric oxide production, cytokines, oxygen depletion (hypoxia) and immunoactive substance release from both hepatic infections and cancer tumor tissues [Su et al.1999, Rumsey et al.1994, Koch et al.2003, Hodgkiss et al.1997, Sharma 2009a, Sharma et al.2009b]. In following section, author puts an evidence of hepatic metabolic integrity loss (in mitochondria and cytoplasm) and lysosomal stimulation associated with hypoxia in liver. Nitroimidazole is still considered clearly hepatotoxic, renal and neurotoxic drug with several reports of diffused damage of parenchymal and nonparenchymal liver cells as initial stage of hepatic damage observed by electron microscopy and biochemical markers in serum with emphasis of pathophysiology [Mahy et al.2004]. Our previous observation on nitroimidazole induced effects to act against hepatic amoebiasis and amoebic abscess development indicated the possibility of nitroimidazole concentration-dependent effects upon oxygen depletion, nitric oxide production, cytokine synergy and liver cell enzyme alterations as inter-related consequences of liver regeneration or bringing back elevated enzyme levels [Chu et al.2004]. The major players were the energy metabolizing and drug metabolizing lysosomal enzymes in initial liver damage with assumption that liver cell enzyme profile defines the hypoxia sensitivity of nitroimidazole by 'Hepatocellular Hypoxia Criteria'. The novelty of hepatocellular hypoxia criteria was detail cytomorphic-biochemical information extracted from altered enzyme activities during initial cytomorphic changes in hypoxic hepatic cells simultaneously with microstructure changes by electron microscopy to confirm the role of cell organelle in hypoxia development. The criterion can be used to evaluate common liver hypoxia conditions such as hepatic tumors, hepatic infections etc. Several new nitroimidazole derivatives are emerging as potential cancer chemosensitizers, hypoxia markers and hypoxia imaging contrast agents acting as enzyme inhibitors [Gronroos et al.2004, Ziemer et al.2003, Jankovic et al. 2006, Papadopoulou et al.2003, Eschmann et

al.2005, Heimbrook et al.1988, Berube et al.1992, Sharma et al. 2009].

between amoebiasis and amoebic liver abscess. The hepatocyte inhabited apoptosis would predominate in amoebic liver abscesses. If these mechanisms prove to be important in human disease, they will provide new targets in both the host and the parasite for interventions designed to ameliorate or inhibit amoebiasis and amoebic liver abscesses. Recently, author in team established that apoptosis results with hypoxia in liver cells and evaluated nitroimidazole cytotoxicity [Kwon et al. 2009]. In following section, a criterion of hepatocellular hypoxia is described based on liver cell enzymes and cytomorphology.

Fig. 1. Entamoeba histolytica trophozoites induce apoptosis in inflammatory cells and hepatocytes in amoebic liver abscesses. Section of liver stained for apoptotic cells with the TUNEL method from an amoebic liver abscess in a C57Bl/6 MRL-lpr/lpr mouse. Amoebic trophozoites (long arrow) are adjacent to a cluster of inflammatory cells (short arrow), which show marked TUNEL staining. TUNEL staining in nuclei and cytoplasm is also evident in a band of dead hepatocytes and inflammatory cells (D). TUNEL-positive nuclei (brown staining) are also visible in hepatocytes in regions (H) flanking the dead cells. Reproduced, with permission, from Ref. Seydel et al. 1998.

#### **2. Liver enzymes and enzyme inhibition in human liver: Hepatocellular criteria**

The liver is made of parenchymal hepatocytes and nonparenchymal Kupffer cells as sole targets that exhibit their intracellular biochemical changes as hepatocellular enzyme biomarker profile. Liver cells are rich in enzymes. Enzyme inhibition in liver is characterized by two ways: 1. intact hepatocellular enzymes in hypoxia serum and liver homogenates; 2. Enzyme regulatory behavior by characterizing enzymes in presence of additives added to cultures of isolated liver cells. Additives and drugs are mainly biotransformed and detoxified in liver by a battery of drug metabolizing enzymes: lysosomal and microsomal enzymes. Drugs inhibit enzymes. In following description, readers are introduced with the importance of enzyme inhibition as research tool for two purposes: 1. drug testing and hypoxia disease monitoring (Hepatocellular Hypoxia Criteria) to understand the hypoxia and liver cell interactions with amoeba and nitroimidazole; 2. Enzyme regulatory behavior in presence of additives in isolated liver cells in culture (hepatocytes and Kupffer cells).

#### **2.1 Hepatocellular hypoxia criteria**

Author proposed a "Hepatocellular Hypoxia Criteria". 'Hepatocellular hypoxia criteria' assumes that initially liver cells loose metabolic integrity (ATP and NADPH insufficiency from glucose to cause oxygen insufficiency in mitochondria) and undergoes apoptosis

between amoebiasis and amoebic liver abscess. The hepatocyte inhabited apoptosis would predominate in amoebic liver abscesses. If these mechanisms prove to be important in human disease, they will provide new targets in both the host and the parasite for interventions designed to ameliorate or inhibit amoebiasis and amoebic liver abscesses. Recently, author in team established that apoptosis results with hypoxia in liver cells and evaluated nitroimidazole cytotoxicity [Kwon et al. 2009]. In following section, a criterion of hepatocellular hypoxia is described based on liver cell enzymes and cytomorphology.

Fig. 1. Entamoeba histolytica trophozoites induce apoptosis in inflammatory cells and hepatocytes in amoebic liver abscesses. Section of liver stained for apoptotic cells with the TUNEL method from an amoebic liver abscess in a C57Bl/6 MRL-lpr/lpr mouse. Amoebic trophozoites (long arrow) are adjacent to a cluster of inflammatory cells (short arrow), which show marked TUNEL staining. TUNEL staining in nuclei and cytoplasm is also evident in a band of dead hepatocytes and inflammatory cells (D). TUNEL-positive nuclei (brown staining) are also visible in hepatocytes in regions (H) flanking the dead cells.

**2. Liver enzymes and enzyme inhibition in human liver: Hepatocellular** 

The liver is made of parenchymal hepatocytes and nonparenchymal Kupffer cells as sole targets that exhibit their intracellular biochemical changes as hepatocellular enzyme biomarker profile. Liver cells are rich in enzymes. Enzyme inhibition in liver is characterized by two ways: 1. intact hepatocellular enzymes in hypoxia serum and liver homogenates; 2. Enzyme regulatory behavior by characterizing enzymes in presence of additives added to cultures of isolated liver cells. Additives and drugs are mainly biotransformed and detoxified in liver by a battery of drug metabolizing enzymes: lysosomal and microsomal enzymes. Drugs inhibit enzymes. In following description, readers are introduced with the importance of enzyme inhibition as research tool for two purposes: 1. drug testing and hypoxia disease monitoring (Hepatocellular Hypoxia Criteria) to understand the hypoxia and liver cell interactions with amoeba and nitroimidazole; 2. Enzyme regulatory behavior in presence of additives in isolated liver cells in culture (hepatocytes and Kupffer cells).

Author proposed a "Hepatocellular Hypoxia Criteria". 'Hepatocellular hypoxia criteria' assumes that initially liver cells loose metabolic integrity (ATP and NADPH insufficiency from glucose to cause oxygen insufficiency in mitochondria) and undergoes apoptosis

Reproduced, with permission, from Ref. Seydel et al. 1998.

**2.1 Hepatocellular hypoxia criteria** 

**criteria** 

followed by detectable necrosis in liver. Hypothesis was that liver cells undergo a series of changes - hepatocytes undergo metabolic energy loss and oxygen depletion (hypoxia) while Kupffer cells undergo phagocytosis, respiratory burst and proteolysis. Nitroimidazole induces enzyme inhibition in liver cells to normalize the elevated enzymes during metabolic energy loss and oxygen depletion (hypoxia) in liver cell (in mitochondria) and lysosomal enzyme inhibition to combat the phagocytosis. The approach is applicable possibly in design of drugs to treat oxygen starved tumor cells or infected liver lesions and abscesses.

Previous reports on first initial stage of nitroimidazole induced liver cell cytotoxicity indicated the apoptosis, glutathione, DNA interaction, oxygen depletion, lactate inhibition, enhanced NADPH cytochrome reductase and superoxide dismutase enzymes [Ersoz et al.2001, Brezden et al.1997, Noss et al.1988, Sugiyama et al.1993, Berube et al. 1992, Sapora et al.1992, Moselen et al. 1995, Stratford et al.1981, Adams et al.1980, Noss et al. 1989, Mulcahy et al.1989, Widel et al. 1982, Rauth et al. 1981, Chao et al. 1982, Whitmore et al.1986]. Still nitroimidazole structure-function relationship between hypoxia, radiosensitization and cytotoxicity or liver regeneration remain less understood perhaps due to nitroimidazole low sensitivity to clinical investigations and significant enzyme alterations only at the very late necrotic stage [Cowan et al.1994, Melo et al. 2000, Moller et al. 2002, Ballinger et al. 1996, Edwards et al. 1981, Edwards et al.1982, Ballinger et al.2001, Riche et al.2001, Melo et al.2000]. However, nitroimidazole cytotoxicity was reported continuously in last three decades indicating many altered enzymes involved in nitric oxide production, cytokines, oxygen depletion (hypoxia) and immunoactive substance release from both hepatic infections and cancer tumor tissues [Su et al.1999, Rumsey et al.1994, Koch et al.2003, Hodgkiss et al.1997, Sharma 2009a, Sharma et al.2009b]. In following section, author puts an evidence of hepatic metabolic integrity loss (in mitochondria and cytoplasm) and lysosomal stimulation associated with hypoxia in liver. Nitroimidazole is still considered clearly hepatotoxic, renal and neurotoxic drug with several reports of diffused damage of parenchymal and nonparenchymal liver cells as initial stage of hepatic damage observed by electron microscopy and biochemical markers in serum with emphasis of pathophysiology [Mahy et al.2004]. Our previous observation on nitroimidazole induced effects to act against hepatic amoebiasis and amoebic abscess development indicated the possibility of nitroimidazole concentration-dependent effects upon oxygen depletion, nitric oxide production, cytokine synergy and liver cell enzyme alterations as inter-related consequences of liver regeneration or bringing back elevated enzyme levels [Chu et al.2004]. The major players were the energy metabolizing and drug metabolizing lysosomal enzymes in initial liver damage with assumption that liver cell enzyme profile defines the hypoxia sensitivity of nitroimidazole by 'Hepatocellular Hypoxia Criteria'. The novelty of hepatocellular hypoxia criteria was detail cytomorphic-biochemical information extracted from altered enzyme activities during initial cytomorphic changes in hypoxic hepatic cells simultaneously with microstructure changes by electron microscopy to confirm the role of cell organelle in hypoxia development. The criterion can be used to evaluate common liver hypoxia conditions such as hepatic tumors, hepatic infections etc. Several new nitroimidazole derivatives are emerging as potential cancer chemosensitizers, hypoxia markers and hypoxia imaging contrast agents acting as enzyme inhibitors [Gronroos et al.2004, Ziemer et al.2003, Jankovic et al. 2006, Papadopoulou et al.2003, Eschmann et al.2005, Heimbrook et al.1988, Berube et al.1992, Sharma et al. 2009].

Mechanisms of Hepatocellular Dysfunction

autophagy & lysosomal

membrane damage

5. Hepatocytology:

irritation

4. Liver pathology changes: raised diaphragm

Cell proliferation advancing tumor

Cell debris tissue growth on

6. Liver cell recovery negative liver

Tissue shrinkage Hypoxia monitoring

Surgical intervention

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 203

Morphological changes Clinical changes Liver biochemical changes 3. Cellular organelle damage: poor drug response slow metabolic disorder: mitochondria(M) oxidative phosphorylation microsomes (MI) drug metabolizing enzymes

lysosome (L) initial phagocytosis

nuclear (N) DNA fragmentation(beads)

mitochondrial damage amebic liver scan +ve stimulation of Kupffer cells exfoliative ER hyperplasia of Kupffer cells anisonucleosis loss of metabolic control

cytosolic granulation increased molecule imbalance fatty liver appearance with increased lipid synthesis

Hepatocellular degeneration and necrosis

vascularization

ultrasound

Nitroimidazole single dose therapy schedule

scan/ultrasound

positive

recovery

Source: Sharma R. Effect of Nitroimidazole on isolated liver cells in development of amoebic liver

damage in infected hepatitis or hepatic tumors. Different clinical methods suggest

Table 2. A step by step scheme of "hepatocellular hypoxia criteria" to evaluate liver hypoxia

composite picture of hepatic hypoxia and associated biochemical and cytomorphic changes.

abscess. Ph.D dissertation submitted to Indian Institute of Technology, Delhi, 1995.

OR if unchanged or poor

cytosol (C) glucose/protein/respiratory burst Hepatocellular Oxygen insufficiency (inflammation)

increased water accumulation

surgical aspirates (altered

proteins, lipids, enzymes)

normal liver function test


abnormal ELISA,enzymes

Components of Hepatocellular Hypoxia Criteria: The criterion is described a step by step sequence of oxygen depletion in hepatocellular damage: 1. initial loss of metabolic integrity; 2. programmed regulatory failure of cell oxygen and energy metabolism; 3. liver tissue inflammation and immunity loss; 4. necrosis and active death. The purpose of metabolic integrity in hepatocytes is keeping cells intact by maintaining balance between glucose formation and glucose breakdown to maintain energy flow of NADPH and ATP molecules. Sequence: The nitroimidazole induced liver cell cytotocity leads to enhanced glucose breakdown and more demand of ATP and NADPH. With course of time, high energy demand at the cost of cell metabolic resources leads to metabolic integrity loss or energy loss and oxygen deprivation followed by possibility of step by step programmed cell death (only in tumor cells). In normal liver cells, the nitroimidazole cytotoxicity effect cause less damage and cells sustain the effect while struggling oxygen starved infected or tumor cells further loose capability to stay alive (such cells die after nitroimidazole induced infected or tumor cell killing). In normal cells, liver regeneration recovers the damage.

Hepatic hypoxia criterion has 3 major enzyme components: 1. major event of high glycolytic rate is immediate result of high glucose turnover such as glycolysis followed by secondary metabolic cycles viz. tricarboxylic acid, glycogenolysis, gluconeogenesis, pentose phosphate pathways; 2. changes in peripheral biomarkers such as cytokine immunity, altered glutathione reductase, nitric oxide production, super oxide dismutase enzymes; 3. after severe energy loss, changes occur in liver cellular morphology and tissue shrinkage [Sharma et al.2011, Kwon et al. 2009]. The sensitivity of different enzymes as cytomorphic manifestation of hypoxia in liver abscess and nitroimidazole induced liver regeneration is a simple scheme of "Hepatocellular Hypoxia Criteria" in steps is shown in Table 2. In following section, different enzymes are described.


Components of Hepatocellular Hypoxia Criteria: The criterion is described a step by step sequence of oxygen depletion in hepatocellular damage: 1. initial loss of metabolic integrity; 2. programmed regulatory failure of cell oxygen and energy metabolism; 3. liver tissue inflammation and immunity loss; 4. necrosis and active death. The purpose of metabolic integrity in hepatocytes is keeping cells intact by maintaining balance between glucose formation and glucose breakdown to maintain energy flow of NADPH and ATP molecules. Sequence: The nitroimidazole induced liver cell cytotocity leads to enhanced glucose breakdown and more demand of ATP and NADPH. With course of time, high energy demand at the cost of cell metabolic resources leads to metabolic integrity loss or energy loss and oxygen deprivation followed by possibility of step by step programmed cell death (only in tumor cells). In normal liver cells, the nitroimidazole cytotoxicity effect cause less damage and cells sustain the effect while struggling oxygen starved infected or tumor cells further loose capability to stay alive (such cells die after nitroimidazole induced infected or tumor

Hepatic hypoxia criterion has 3 major enzyme components: 1. major event of high glycolytic rate is immediate result of high glucose turnover such as glycolysis followed by secondary metabolic cycles viz. tricarboxylic acid, glycogenolysis, gluconeogenesis, pentose phosphate pathways; 2. changes in peripheral biomarkers such as cytokine immunity, altered glutathione reductase, nitric oxide production, super oxide dismutase enzymes; 3. after severe energy loss, changes occur in liver cellular morphology and tissue shrinkage [Sharma et al.2011, Kwon et al. 2009]. The sensitivity of different enzymes as cytomorphic manifestation of hypoxia in liver abscess and nitroimidazole induced liver regeneration is a simple scheme of "Hepatocellular Hypoxia Criteria" in steps is shown in Table 2. In

Morphological changes Clinical changes Liver biochemical changes

hepatic infiltration hepatomegaly liver function tests(elevated)

Loss of cellular metabolic integrity

Hepatocellular enlargement (Apoptosis)

2.Electron microscopy: hepatomegaly with altered hepatocyte enzymes of: Mitochondria(M) diffused injury low ATP/ADP; NADPH/NADP

endoplasmic reticulum(ER) gluconeogenesis peroxisome (P) glycogenolytic lysosome (L) lysosomal enzymes nuclear changes (N) oxygen flux related

cell killing). In normal cells, liver regeneration recovers the damage.

following section, different enzymes are described.


1. Physical examination:

Table 2. Continued


Source: Sharma R. Effect of Nitroimidazole on isolated liver cells in development of amoebic liver abscess. Ph.D dissertation submitted to Indian Institute of Technology, Delhi, 1995.

Table 2. A step by step scheme of "hepatocellular hypoxia criteria" to evaluate liver hypoxia damage in infected hepatitis or hepatic tumors. Different clinical methods suggest composite picture of hepatic hypoxia and associated biochemical and cytomorphic changes.

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 205

Nitroimidazole

Fig. 3a. The histogram bars show the effect of nitroimidazole on biomarker enzymes and comparison of different enzymes in hepatocytes in control vs nitroimidazole treated subjects

Fig. 3b. The histogram bars show the effect of nitroimidazole on biomarker enzymes and comparison of different enzymes in Kupffer cells in control vs nitroimidazole treated

subjects in control vs nitroimidazole treated subjects

#### **2.2 Origin of hypoxia and enzyme regulation**

Oxygen insufficiency or hypoxia begins with low NADPH and ATP supply from glucose. Glucokinase, aldolase, pyruvate kinase and lactate dehydrogenase possibly serve as initial glycolytic regulatory enzymes for energy flow to generate tricarboxylic acid cycle (TCA) precursors. TCA cycle being as main source of energy molecule synthesis and gluconeogenesis, it serves as sole source of central ATP pool in glucose homeostasis during liver cytototoxicity or regenerated liver cell [Sharma et al.2007, Sharma et al.2010]. The citrate synthase, malate dehydrogenase, isocitrate dehydrogenase and succinate dehydrogenase enzymes serve as TCA cycle regulatory enzymes. These enzymes in hepatocytes characterize the tumor hypoxia and hepatocyte response after nitroimidazole therapy [Horlen et al.2000, Tabak et al.2003, Sapora et al.1992, Moselen et al.1995, Stratford et al.1981]. At the cost of ATP from TCA cycle and oxygen from the cell, oxidative phosphorylation maintains the metabolic integrity and continuous flow of energy. In the state of low NADPH and low oxygen cell undergoes state of metabolic integrity loss and "hypoxia". NADPH dependent cytochrome redox enzymes are biomarker of oxidative phosphorylation status and oxygen insufficiency (hypoxia) in liver. Other glutathione reductase and superoxide dismutase enzymes are biomarkers of hypoxic state of liver cells [Larrey et al.2000, Michaopoulos et al.1997]. Any energy imbalance and oxygen insufficiency (hypoxia) in liver cell are indicated by these enzymes [Michaopoulos et al.1997, Ramirez-Emiliano et al.2007]. The behavior of these enzymes in isolated liver cells is described in following section on isolated liver cell enzymes.

Fig. 2. The liver scan (on left panel) is shown for assessing the position of hepatic hypoxia and associated hepatomegaly and for biopsy collection site as shown with arrow.

Oxygen insufficiency or hypoxia begins with low NADPH and ATP supply from glucose. Glucokinase, aldolase, pyruvate kinase and lactate dehydrogenase possibly serve as initial glycolytic regulatory enzymes for energy flow to generate tricarboxylic acid cycle (TCA) precursors. TCA cycle being as main source of energy molecule synthesis and gluconeogenesis, it serves as sole source of central ATP pool in glucose homeostasis during liver cytototoxicity or regenerated liver cell [Sharma et al.2007, Sharma et al.2010]. The citrate synthase, malate dehydrogenase, isocitrate dehydrogenase and succinate dehydrogenase enzymes serve as TCA cycle regulatory enzymes. These enzymes in hepatocytes characterize the tumor hypoxia and hepatocyte response after nitroimidazole therapy [Horlen et al.2000, Tabak et al.2003, Sapora et al.1992, Moselen et al.1995, Stratford et al.1981]. At the cost of ATP from TCA cycle and oxygen from the cell, oxidative phosphorylation maintains the metabolic integrity and continuous flow of energy. In the state of low NADPH and low oxygen cell undergoes state of metabolic integrity loss and "hypoxia". NADPH dependent cytochrome redox enzymes are biomarker of oxidative phosphorylation status and oxygen insufficiency (hypoxia) in liver. Other glutathione reductase and superoxide dismutase enzymes are biomarkers of hypoxic state of liver cells [Larrey et al.2000, Michaopoulos et al.1997]. Any energy imbalance and oxygen insufficiency (hypoxia) in liver cell are indicated by these enzymes [Michaopoulos et al.1997, Ramirez-Emiliano et al.2007]. The behavior of these enzymes in isolated liver cells is described in

Fig. 2. The liver scan (on left panel) is shown for assessing the position of hepatic hypoxia

and associated hepatomegaly and for biopsy collection site as shown with arrow.

**2.2 Origin of hypoxia and enzyme regulation** 

following section on isolated liver cell enzymes.

Fig. 3a. The histogram bars show the effect of nitroimidazole on biomarker enzymes and comparison of different enzymes in hepatocytes in control vs nitroimidazole treated subjects

Fig. 3b. The histogram bars show the effect of nitroimidazole on biomarker enzymes and comparison of different enzymes in Kupffer cells in control vs nitroimidazole treated subjects in control vs nitroimidazole treated subjects

Mechanisms of Hepatocellular Dysfunction

Tables 2-4.

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 207

aldolase (in 80%), pyruvate kinase (in 100%), malate dehydrogenase (in 80%), ICDH (in 80%), citrate dehydrogenase (in 70%), phosphogluconate dehydrogenase (in 80%). Kupffer cells showed higher enzyme levels of β-glucuroronidase (in 80%), leucine aminopeptidase (in 70%), acid phosphatase (in 80%) and aryl sulphatase (in 88%). In these 10 biopsy samples from subjects, the electron microscopy cytomorphology observations showed swollen bizarre mitochondria, proliferative endoplasmic reticulum, and anisonucleosis. In 2' nitronidazole clinical trial patients, after 2'-Nitroimidazole effect in liver cell damage was manifested as enzyme inhibition in liver cells as shown in Figure 3[Sharma 2008]. Nitroimidazole treated patients shows altered enzymes: glucokinase levels were more or less normal in both serum and liver biopsy (100%); phosphofructokinase levels were nonspecific in nitroimidazole treated subjects or normal (80%); elevated lactate dehydrogenase levels were reversed to normal. In nitroimidazole treated subjects the elevated enzyme levels due to amoebic abscess brought back to normal after nitroimidazole treatment due to enzyme inhibitory effect of nitroimidazole: isocitrate dehydrogenase enzyme normal levels (80 %); the citrate synthase normal levels (80%); phosphogluconate dehydrogenase normal levels (80 %). In nitroimidazole treated subjects, the succinate dehydrogenase levels were normal (60 %) in both. The enzymes are shown in Figure 3 and

**3.1 Enzyme inhibition in Kupffer cell lysosomal enzymes and Hypoxia** 

cells and serum (80%). The enzymes are shown in Figure 2 and Table 3.

In nitroimidazole treated patients showed elevated enzymes brought back to normal: βglucuronidase normal levels (50 %) in Kupffer cells and normal in serum (60 %); acid phosphatase normal levels in both; leucine aminopeptidase levels were normal in serum (80 %) and remained high in Kupffer cells (60%); Guanase levels were normal in both Kupffer

Kupffer cell hyperplasia was observed with swollen lysosomal contents as shown in Figure 4. Altered glycolytic enzymes in cytosol, TCA cycle enzymes in mitochondria, lysosomes and increased synthesis of enzymes by endoplasmic reticulum showed correlation with clear liver cell degeneration of microbodies. After nitroimidazole treatment, observations of both electron microscopy and biochemical parameters suggested the reversed hepatocellular changes towards normal recovery or liver regeneration. In following section, I describe the outcome of nitroimidazole effect in liver regeneration and enzyme inhibition as defense.

The enzyme biomarkers could be analyzed in serum and liver cells (hepatocytes and Kupffer cells) as clinically significant indicator of hepatic damage in disease or cytotoxicity of drug. We assume that initially metabolic integrity loss leads to over-secretion of liver cell enzymes including lysosomal enzymes. Soon after, the ultrastructural changes in liver cells become evident by electron microscopy suggestive of acute organelle degeneration. Ultrastructural hepatocyte cytotoxicity was associated with nitroimidazole overdosage (normal dosage is 2 × 3 gm one time in amoebic hepatitis and thrice in amoebic abscess). The regenerative changes were consistently observed after nitroimidazole therapy to reverse liver damage. Few pathology reports are available to show nitroimidazole cytotoxicity and no electron microscopy study is available to support asymptomatic inflammatory or unequivocal diffuse parenchymal injury exhibiting diffused sinusoidal and portal

Fig. 3c. The histogram bars show the effect of nitroimidazole on biomarker enzymes and comparison of different enzymes in serum biomarker enzymes.

#### **3. Enzyme inhibition in initial loss of metabolic integrity, glycolysis and ATP in hepatocytes**

Enzymes in serum and liver cells from positive control amoebic liver abscess patients exhibited more or less specific characteristic changes in enzymes [Sharma et al.2008]. First goal was to establish elevated enzyme levels in patients and to prove subsequent enzyme inhibition after nitroimidazole therapy. Author reported ten control subjects with 2' nitroimidazole therapy follow up for their carbohydrate metabolizing enzymes in serum and hepatocellular enzymes in liver biopsy tissues. Proven ten hepatic hypoxia control subjects were studied for hypoxia by enzyme assays [Sharma et al.2008]. The 2' nitroimidazole treated paired ten subjects were studied for hypoxia related enzyme inhibition using enzyme assays and hepatocellular cytomorphology by electron microscopy. Out of ten positive control subjects, nine patients showed elevated carbohydrate metabolizing and lysosomal enzyme levels in serum: Glucokinase (in 80% samples), aldolase (in 80% samples), phosphofructokinase (in 80% samples), malate dehydrogenase (in 75% samples), isocitrate dehydrogenase (ICDH) (in 60% patients) were elevated while succinate dehydrogenase and lactate dehydrogenase (LDH) levels remained unaltered. Lysosomal β-glucuronidase, alkaline phosphatase, acid phosphatase enzymes showed enhanced levels in the all patient serum samples. In ten control liver biopsies, the isolated hepatocytes and Kupffer cell preparations showed altered liver cell enzyme levels. Hepatocytes showed reduced glucokinase (in 80%), LDH (in 80%), and higher content of

Fig. 3c. The histogram bars show the effect of nitroimidazole on biomarker enzymes and

**3. Enzyme inhibition in initial loss of metabolic integrity, glycolysis and ATP** 

Enzymes in serum and liver cells from positive control amoebic liver abscess patients exhibited more or less specific characteristic changes in enzymes [Sharma et al.2008]. First goal was to establish elevated enzyme levels in patients and to prove subsequent enzyme inhibition after nitroimidazole therapy. Author reported ten control subjects with 2' nitroimidazole therapy follow up for their carbohydrate metabolizing enzymes in serum and hepatocellular enzymes in liver biopsy tissues. Proven ten hepatic hypoxia control subjects were studied for hypoxia by enzyme assays [Sharma et al.2008]. The 2' nitroimidazole treated paired ten subjects were studied for hypoxia related enzyme inhibition using enzyme assays and hepatocellular cytomorphology by electron microscopy. Out of ten positive control subjects, nine patients showed elevated carbohydrate metabolizing and lysosomal enzyme levels in serum: Glucokinase (in 80% samples), aldolase (in 80% samples), phosphofructokinase (in 80% samples), malate dehydrogenase (in 75% samples), isocitrate dehydrogenase (ICDH) (in 60% patients) were elevated while succinate dehydrogenase and lactate dehydrogenase (LDH) levels remained unaltered. Lysosomal β-glucuronidase, alkaline phosphatase, acid phosphatase enzymes showed enhanced levels in the all patient serum samples. In ten control liver biopsies, the isolated hepatocytes and Kupffer cell preparations showed altered liver cell enzyme levels. Hepatocytes showed reduced glucokinase (in 80%), LDH (in 80%), and higher content of

comparison of different enzymes in serum biomarker enzymes.

**in hepatocytes** 

aldolase (in 80%), pyruvate kinase (in 100%), malate dehydrogenase (in 80%), ICDH (in 80%), citrate dehydrogenase (in 70%), phosphogluconate dehydrogenase (in 80%). Kupffer cells showed higher enzyme levels of β-glucuroronidase (in 80%), leucine aminopeptidase (in 70%), acid phosphatase (in 80%) and aryl sulphatase (in 88%). In these 10 biopsy samples from subjects, the electron microscopy cytomorphology observations showed swollen bizarre mitochondria, proliferative endoplasmic reticulum, and anisonucleosis. In 2' nitronidazole clinical trial patients, after 2'-Nitroimidazole effect in liver cell damage was manifested as enzyme inhibition in liver cells as shown in Figure 3[Sharma 2008]. Nitroimidazole treated patients shows altered enzymes: glucokinase levels were more or less normal in both serum and liver biopsy (100%); phosphofructokinase levels were nonspecific in nitroimidazole treated subjects or normal (80%); elevated lactate dehydrogenase levels were reversed to normal. In nitroimidazole treated subjects the elevated enzyme levels due to amoebic abscess brought back to normal after nitroimidazole treatment due to enzyme inhibitory effect of nitroimidazole: isocitrate dehydrogenase enzyme normal levels (80 %); the citrate synthase normal levels (80%); phosphogluconate dehydrogenase normal levels (80 %). In nitroimidazole treated subjects, the succinate dehydrogenase levels were normal (60 %) in both. The enzymes are shown in Figure 3 and Tables 2-4.

#### **3.1 Enzyme inhibition in Kupffer cell lysosomal enzymes and Hypoxia**

In nitroimidazole treated patients showed elevated enzymes brought back to normal: βglucuronidase normal levels (50 %) in Kupffer cells and normal in serum (60 %); acid phosphatase normal levels in both; leucine aminopeptidase levels were normal in serum (80 %) and remained high in Kupffer cells (60%); Guanase levels were normal in both Kupffer cells and serum (80%). The enzymes are shown in Figure 2 and Table 3.

Kupffer cell hyperplasia was observed with swollen lysosomal contents as shown in Figure 4. Altered glycolytic enzymes in cytosol, TCA cycle enzymes in mitochondria, lysosomes and increased synthesis of enzymes by endoplasmic reticulum showed correlation with clear liver cell degeneration of microbodies. After nitroimidazole treatment, observations of both electron microscopy and biochemical parameters suggested the reversed hepatocellular changes towards normal recovery or liver regeneration. In following section, I describe the outcome of nitroimidazole effect in liver regeneration and enzyme inhibition as defense.

The enzyme biomarkers could be analyzed in serum and liver cells (hepatocytes and Kupffer cells) as clinically significant indicator of hepatic damage in disease or cytotoxicity of drug. We assume that initially metabolic integrity loss leads to over-secretion of liver cell enzymes including lysosomal enzymes. Soon after, the ultrastructural changes in liver cells become evident by electron microscopy suggestive of acute organelle degeneration. Ultrastructural hepatocyte cytotoxicity was associated with nitroimidazole overdosage (normal dosage is 2 × 3 gm one time in amoebic hepatitis and thrice in amoebic abscess). The regenerative changes were consistently observed after nitroimidazole therapy to reverse liver damage. Few pathology reports are available to show nitroimidazole cytotoxicity and no electron microscopy study is available to support asymptomatic inflammatory or unequivocal diffuse parenchymal injury exhibiting diffused sinusoidal and portal

Mechanisms of Hepatocellular Dysfunction

nitroimidazole breakdown products.

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 209

[Das et al.1999]. The cause of the ultrastructural changes after nitroimidazole cytotoxicity were supported by initial enzyme alterations reflecting loss of metabolic integrity probably induced by free radical formation from nitroimidazole [Das et al.1999]. Since ultrastructural changes in liver were completely reversible after nitroimidazole therapy within 7-9 days, it is quite reasonable that pathogenesis of diffused hepatocyte damage was due to

Fig. 5. The sketch of nitroimidazole induced intracellular enzyme inhibition changes indicating the points of metabolism and metabolic control during hypoxia and subsequent liver recovery after nitroimidazole treatment. The enzymes distribution in organelle and enzyme location in different hepatic sites explains the liver damage and enzymatic basis of hepatocellular hypoxia criteria [reproduced from Sharma R. 1990. Ph.D dissertation].

As indicated in previous section, 'Hepatocellular hypoxia criteria' is a sequence of events in hepatic cell injury at molecular level. The initial loss of hepatocellular metabolic integrity leads to hepatic injury. Glucose-energy metabolic integrity loss and nitric oxide formation with Ca++ homeostasis are main initial determinants of hypoxia [Ramirez-Emiliano et al.2007]. In following section, present chapter extends the enzyme regulatory behavior during glucose metabolizing pathways viz. glycolysis, TCA cycle and gluconeogenesis to establish the value of enzyme inhibition in liver regeneration. In isolated liver cells, metabolic alterations of energy metabolism pathways explain the events in hepatitis and

infiltration events [Tabak et al.2003, Larrey et al.2000]. Biomarker liver cell enzymes with electron microscopy put evidence of amoebic cytotoxicity in liver biopsy samples and liver regeneration after nitroimidazole treatment by 'hepatic hypoxia criteria'. Degenerative changes of hepatocytes suggested possible necrosis.

Fig. 4. Ultrastructural changes are shown in hepatocyte organelles during hypoxia: exfoliation of endoplasmic reticulum (top on left); anisonucleosis (mid and bottom on left); intercellular junction gaps (top and mid panels in center); nuclear inclusions (bottom on center); swollen and bizarre mitochondria(top on right); inclusions in peroxisome (mid on right); mitochondrial atrophy with lipid vesicles (bottom on right). Hepatocyte mitochondria became swollen, bizarre with dense matrix and showed destorted cristae, endoplasmic reticulum dilated vesicles, giant nuclei with diffuse proliferation of endoplasmic reticulum and clear anisonucleosis features. The ultrastructure of liver cells showed characteristic organelle changes in nitroimidazole treated liver.

Evidence of hepatic regeneration and nitroimidazole cytotoxicity (hypoxia induced changes) was characterized previously in terms of cytokine synergy, unusual degree of anisonucleosis, nitric oxide production and the presence of giant nucleus in hepatocytes after liver regenerative therapy [Michalopoulous et al.1997]. Endoplasmic reticulum showed a diffuse and intense proliferative activity in these liver cells with normal appearance of mitochondria as earlier reported [Ramirez-Emiliano et al.2007, Das et al.1999]. However, intramitochondrial inclusion bodies were absent while they were very prominent features

infiltration events [Tabak et al.2003, Larrey et al.2000]. Biomarker liver cell enzymes with electron microscopy put evidence of amoebic cytotoxicity in liver biopsy samples and liver regeneration after nitroimidazole treatment by 'hepatic hypoxia criteria'. Degenerative

changes of hepatocytes suggested possible necrosis.

Fig. 4. Ultrastructural changes are shown in hepatocyte organelles during hypoxia:

right); mitochondrial atrophy with lipid vesicles (bottom on right). Hepatocyte

showed characteristic organelle changes in nitroimidazole treated liver.

exfoliation of endoplasmic reticulum (top on left); anisonucleosis (mid and bottom on left); intercellular junction gaps (top and mid panels in center); nuclear inclusions (bottom on center); swollen and bizarre mitochondria(top on right); inclusions in peroxisome (mid on

mitochondria became swollen, bizarre with dense matrix and showed destorted cristae, endoplasmic reticulum dilated vesicles, giant nuclei with diffuse proliferation of

endoplasmic reticulum and clear anisonucleosis features. The ultrastructure of liver cells

Evidence of hepatic regeneration and nitroimidazole cytotoxicity (hypoxia induced changes) was characterized previously in terms of cytokine synergy, unusual degree of anisonucleosis, nitric oxide production and the presence of giant nucleus in hepatocytes after liver regenerative therapy [Michalopoulous et al.1997]. Endoplasmic reticulum showed a diffuse and intense proliferative activity in these liver cells with normal appearance of mitochondria as earlier reported [Ramirez-Emiliano et al.2007, Das et al.1999]. However, intramitochondrial inclusion bodies were absent while they were very prominent features [Das et al.1999]. The cause of the ultrastructural changes after nitroimidazole cytotoxicity were supported by initial enzyme alterations reflecting loss of metabolic integrity probably induced by free radical formation from nitroimidazole [Das et al.1999]. Since ultrastructural changes in liver were completely reversible after nitroimidazole therapy within 7-9 days, it is quite reasonable that pathogenesis of diffused hepatocyte damage was due to nitroimidazole breakdown products.

Fig. 5. The sketch of nitroimidazole induced intracellular enzyme inhibition changes indicating the points of metabolism and metabolic control during hypoxia and subsequent liver recovery after nitroimidazole treatment. The enzymes distribution in organelle and enzyme location in different hepatic sites explains the liver damage and enzymatic basis of hepatocellular hypoxia criteria [reproduced from Sharma R. 1990. Ph.D dissertation].

As indicated in previous section, 'Hepatocellular hypoxia criteria' is a sequence of events in hepatic cell injury at molecular level. The initial loss of hepatocellular metabolic integrity leads to hepatic injury. Glucose-energy metabolic integrity loss and nitric oxide formation with Ca++ homeostasis are main initial determinants of hypoxia [Ramirez-Emiliano et al.2007]. In following section, present chapter extends the enzyme regulatory behavior during glucose metabolizing pathways viz. glycolysis, TCA cycle and gluconeogenesis to establish the value of enzyme inhibition in liver regeneration. In isolated liver cells, metabolic alterations of energy metabolism pathways explain the events in hepatitis and

Mechanisms of Hepatocellular Dysfunction

analogues.

**3.2 Role of enzymes in energy metabolic integrity in hypoxia** 

**3.3 Role of lysosomal enzymes in hepatocellular damage** 

In previous section, role of secretory lysosomal enzymes was described in defense of liver cells in context of intestinal amoebiasis and amoebic liver abscess. Conceptually, lysosomal hydrolases cause foreign body damage by several reactions including redox reaction, phosphorylation reaction, glucuronidation reaction, hydroxylation reaction, dehydroxylation reaction, nucleation reaction, terminal breakdown reaction, transcarbamylation reaction. Other studies also reported molecular mechanisms of enzymes in pathogenesis [Das et al.1999], liver cell damage [Virk et al.1989, Virk et al.1988], phagocytosis [Sharma 2009], cyclooxygenase-2 expression [Sanchez-Ramirez et al.2000].

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 211

Hexokinase, a rate limiting enzyme in cytosol for glucose turnover was estimated due to high glucose conversion into glucose 6P showed high enzyme secretion from hepatocytes. Aldolase, rate controlling enzyme which splits fructose-1, 6 Diphosphate into two 3 phosphoglyceric acid and dehydroxyacetone phosphate, showed high enzyme secretion from heptocytes. Phosphofructokinase, rate controlling enzyme which phosphorylates fructose 6P into fructose 1, 6 DiP, enhanced as its more demand in hepatocellular glucose turnover which probably induced higher secretion of phosphofructokinase. Pyruvate kinase, transferring phosphoryl group from phosphoenolpyruvate to ADP with pyruvate formation, showed high enzyme secretion from hepatocytes. It may be attributed due to high concentration of glycolytic intermediates as terminal step was blocked or due to 2, 3 Di-Phosphoglycerate regulated abnormal oxygen dissociation. Malate dehydrogenase and isocitrate dehydrogenase enzymes, catalyzing malate oxidation into oxaloacetate and oxidative decarboxylation of isocitrate into α-ketoglutarate respectively both require NAD+. Both enzymes were elevated in damaged cells due to reducing equivalent low ratio of NADH/NAD+ pushing in forward direction. Succinate dehydrogenase, oxidizing succinate to fumerate using FAD, showed decreased enzyme levels due to low iron sulphur proteins resulting with lowered electron transport system in inner mitochondrial membrane i.e. supply of electrons to molecular oxygen by electron transfer from FADH2 to Fe+++ (SDH) in amoebic recruited liver cells. Citrate synthase, synthesizing citrate from oxaloacetate and acetyl CoA by aldol condensation followed by hydrolysis, showed elevated levels due to rapid turnover of oxaloacetate and acetyl CoA molecules during cytolysis. Moreover, high TCA cycle activity in hepatocyte during liver damage conditions was described earlier [Sharma 2010]. In serum, the enzymes exhibit their significance but aldolase, pyruvate kinase and LDH, MDH, ICDH observed as distinguishing the diffused injury or abscess formation [Virk et al.1989]. Phosphogluconate dehydrogenase elevated levels may be attributed due to ribulose 5P formation and transaldolase and transketolase control, for phosphogluconate pathway. Nitroimidazole induced cytotoxicity perhaps have insignificant impact on ATP supply. In following section, different enzyme regulatory behaviors of nitroimidazole induced enzyme inhibition are described during liver cell regeneration. A strategy was developed to isolate hepatocytes and Kupffer cells in monoaxenic cultures from excised liver samples. Mixing different inhibitors or additives in liver cell cultures stimulate the enzyme regulatory behavior in presence of specific growth factors, metabolite

hepatic tumors as best correlated respective enzyme dysfunctions and ultrastructural changes observed in cells in biopsy. The following description is broad explanation of different enzymes secreted from hepatocytes as a result of nitroimidazole induced cytotoxicity and oxygen depletion. From biochemical stand point, different regulatory enzymes are discussed as biochemical events of hypoxia development as shown in Figures 3a,3b,3c and 5.

Fig. 6. The sketch of relative biomarker enzyme changes (thickness of arrow) in liver hypoxia. The +ve sign denotes the relative increase in enzyme activity and –ve sign denotes the decrease in enzyme activity in liver cells in different organelles due to ameba induced hypoxia. The figure sketch also represents a relative enzyme activities in sequence of metabolic steps as 'hepatocellular hypoxia criteria'. Notice the liver regenerative or enzyme inhibitory action of nitroimidazole at different enzyme reactions is shown [Reproduced from Sharma R. 1995. Ph.D dissertation].

#### **3.2 Role of enzymes in energy metabolic integrity in hypoxia**

210 Enzyme Inhibition and Bioapplications

hepatic tumors as best correlated respective enzyme dysfunctions and ultrastructural changes observed in cells in biopsy. The following description is broad explanation of different enzymes secreted from hepatocytes as a result of nitroimidazole induced cytotoxicity and oxygen depletion. From biochemical stand point, different regulatory enzymes are discussed as biochemical events of hypoxia development as shown in Figures

Fig. 6. The sketch of relative biomarker enzyme changes (thickness of arrow) in liver hypoxia. The +ve sign denotes the relative increase in enzyme activity and –ve sign denotes the decrease in enzyme activity in liver cells in different organelles due to ameba induced hypoxia. The figure sketch also represents a relative enzyme activities in sequence of metabolic steps as 'hepatocellular hypoxia criteria'. Notice the liver regenerative or enzyme inhibitory action of nitroimidazole at different enzyme reactions is shown [Reproduced

from Sharma R. 1995. Ph.D dissertation].

3a,3b,3c and 5.

Hexokinase, a rate limiting enzyme in cytosol for glucose turnover was estimated due to high glucose conversion into glucose 6P showed high enzyme secretion from hepatocytes. Aldolase, rate controlling enzyme which splits fructose-1, 6 Diphosphate into two 3 phosphoglyceric acid and dehydroxyacetone phosphate, showed high enzyme secretion from heptocytes. Phosphofructokinase, rate controlling enzyme which phosphorylates fructose 6P into fructose 1, 6 DiP, enhanced as its more demand in hepatocellular glucose turnover which probably induced higher secretion of phosphofructokinase. Pyruvate kinase, transferring phosphoryl group from phosphoenolpyruvate to ADP with pyruvate formation, showed high enzyme secretion from hepatocytes. It may be attributed due to high concentration of glycolytic intermediates as terminal step was blocked or due to 2, 3 Di-Phosphoglycerate regulated abnormal oxygen dissociation. Malate dehydrogenase and isocitrate dehydrogenase enzymes, catalyzing malate oxidation into oxaloacetate and oxidative decarboxylation of isocitrate into α-ketoglutarate respectively both require NAD+. Both enzymes were elevated in damaged cells due to reducing equivalent low ratio of NADH/NAD+ pushing in forward direction. Succinate dehydrogenase, oxidizing succinate to fumerate using FAD, showed decreased enzyme levels due to low iron sulphur proteins resulting with lowered electron transport system in inner mitochondrial membrane i.e. supply of electrons to molecular oxygen by electron transfer from FADH2 to Fe+++ (SDH) in amoebic recruited liver cells. Citrate synthase, synthesizing citrate from oxaloacetate and acetyl CoA by aldol condensation followed by hydrolysis, showed elevated levels due to rapid turnover of oxaloacetate and acetyl CoA molecules during cytolysis. Moreover, high TCA cycle activity in hepatocyte during liver damage conditions was described earlier [Sharma 2010]. In serum, the enzymes exhibit their significance but aldolase, pyruvate kinase and LDH, MDH, ICDH observed as distinguishing the diffused injury or abscess formation [Virk et al.1989]. Phosphogluconate dehydrogenase elevated levels may be attributed due to ribulose 5P formation and transaldolase and transketolase control, for phosphogluconate pathway. Nitroimidazole induced cytotoxicity perhaps have insignificant impact on ATP supply. In following section, different enzyme regulatory behaviors of nitroimidazole induced enzyme inhibition are described during liver cell regeneration. A strategy was developed to isolate hepatocytes and Kupffer cells in monoaxenic cultures from excised liver samples. Mixing different inhibitors or additives in liver cell cultures stimulate the enzyme regulatory behavior in presence of specific growth factors, metabolite analogues.

#### **3.3 Role of lysosomal enzymes in hepatocellular damage**

In previous section, role of secretory lysosomal enzymes was described in defense of liver cells in context of intestinal amoebiasis and amoebic liver abscess. Conceptually, lysosomal hydrolases cause foreign body damage by several reactions including redox reaction, phosphorylation reaction, glucuronidation reaction, hydroxylation reaction, dehydroxylation reaction, nucleation reaction, terminal breakdown reaction, transcarbamylation reaction. Other studies also reported molecular mechanisms of enzymes in pathogenesis [Das et al.1999], liver cell damage [Virk et al.1989, Virk et al.1988], phagocytosis [Sharma 2009], cyclooxygenase-2 expression [Sanchez-Ramirez et al.2000].

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 213

The addition of triamcinolone with insulin in combination showed significant increase in glucokinase activity in all hepatocyte groups. It pointed out to the enhancement in hormone dependent glucokinase enzyme synthesis in hepatocytes and enzyme activity showed highest enzyme synthesis rate in hepatic abscess recruited hepatocytes while nitroimidazole treated hepatocytes showed lesser increase in hormone dependent glucokinase in comparison with hepatocytes as shown in Figure 7. The increase in enzyme activity or enhanced glucokinase enzyme synthesis in hepatocytes showed that glucokinase enzyme behavior was hormone dependent. Different concentration of insulin and progesterone hormones further indicated

The addition of progesterone hormone 0.1 µgm/ml in hepatocyte cultures showed maximum enhanced glucokinase enzyme activity and nitroimidazole treated hepatocytes showed less enhanced glucokinase activity but more than control hepatocyte enzyme

In control group, glucokinase enzyme activity in untreated hepatocytes did not show any chnage in their glucokinase enzyme activity in presence of nitroimidazole additive. The yield of hepatocytes and glucokinase enzyme activities from hepatic abscess liver biopsy were similar as yield of cells and glucokinase enzyme activities in untreated and nitroimidazole treated hepatocytes. So, it was clear that addition of actinomycin-D and insulin with triamicilone both enhanced the glucokinase enzyme synthesis in hepatocytes as

Fig. 7. The nitroimidazole effect on glucokinase enzyme activity in guinea pig normal and infected hepatocytes is shown in presence of different enzyme regulatory additives actinomycin D, insulin-triamcilone. Notice the enhanced effect of insulin-triamicilone on glucokinase enzyme (P value <0.0001; r2 0.9872) in nitroimidazole treated vs liver abscess

the regulatory behavior of glucokinase enzyme as hormone dependent.

added

added

progesterone

activity as shown in histogram bars (see Figure 7).

shown in Figure 7 histograms bars.

biopsy heaptocytes.

#### **4. Enzyme inhibition properties and enzyme regulatory behavior in isolated liver cells**

Isolated liver cells with characteristic enzyme properties are research tools. In our previous study, the hepatocyte yield from livers was 2.5× 108 per mg liver. The cytosol fraction showed the removal of all cell organelle from supernatant and resultant supernatant as cytosol was free of any remnant by microscopic observation by trypan blue exclusion. Isolation of human Kupffer cells was developed by a modified pronasecollagenase enzymatic isolation and purity of Kupffer cells by using biomarker enzyme assay. Earlier reports used similar practice of enzymatic digestion and enzymatic assays as indicators of cell viability and yield [Hendriks et al.1990, Neaud et al.1995, McCuskey et al.1990]. A new rapid method was used for the isolation and fractionation of both rat and human Kupffer cells without the need of liver perfusion techniques. The study used rat livers or small human liver wedge biopsies obtained peroperatively and incubated with pronase under continuous pH registration. Kupffer cells were subsequently separated from other nonparenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation [Neaud et al.1995]. Identification of Kupffer cells was achieved on the basis of ultrastructural analyses and immunophenotyping [Neaud et al.1995]. The fractionation of Kupffer cells gave good yield comparable with other studies [Hendriks et al.1990, Neaud et al.1995, McCuskey et al.1990]. For details of protocol of liver cell isolation, see the Appendix in the end of this chapter. Human liver cell enzymes in isolated liver cells (hepatocytes and Kupffer cells in cultures) showed characteristic regulatory behavior in presence of additives.

Liver cell hypoxia is represented as a state of oxygen depletion in cell. Initially liver cell gets its ATP and NADPH supply from glycolysis and TCA cycle. Consequently, electron transfer chain (oxidative phosphorylation) through series of cytochrome redox reactions converts available cell oxygen to water to produce high energy metabolites in the cell (metabolic integrity). Infected liver cells show loss of metabolic integrity and less available oxygen (oxygen starved or hypoxia). Nitroimidazole was established two decades ago as potential oxygen quenching drug in most of the infected and tumor cells. "Nitroimidazole oxygen quenching action" makes oxygen depleted or starved cells that further undergo worse to die and leaving normal cells fully functional that can be observed as potential tumor hypoxia therapy and antiparasitic treatment by nitroimidazole.

In following section, enzyme inhibition characteristics in energy metabolism regulation including glycolysis, TCA cycle regulatory enzyme behavior and drug metabolizing proteolytic lysosomal enzymes induced by additives in liver cell cultures are described.

#### **4.1 Glucokinase**

The actinomycin D showed non-significant inhibitory change in glucokinase enzyme activity as shown in Figure 7 at the top histogram bars. The nitroimidazole showed enhanced glucokinase activity and reversed the inhibitory effect of actinomycin D. The glucokinase activity in hepatic abscess hepatocytes did not show any significant effect of nitroimidazole. Overall actinomycin D alone did not show any change in glucokinase enzyme activity in hepatic abscess hepatocytes or nitroimidazole treated hepatocytes.

**4. Enzyme inhibition properties and enzyme regulatory behavior in isolated** 

Isolated liver cells with characteristic enzyme properties are research tools. In our previous study, the hepatocyte yield from livers was 2.5× 108 per mg liver. The cytosol fraction showed the removal of all cell organelle from supernatant and resultant supernatant as cytosol was free of any remnant by microscopic observation by trypan blue exclusion. Isolation of human Kupffer cells was developed by a modified pronasecollagenase enzymatic isolation and purity of Kupffer cells by using biomarker enzyme assay. Earlier reports used similar practice of enzymatic digestion and enzymatic assays as indicators of cell viability and yield [Hendriks et al.1990, Neaud et al.1995, McCuskey et al.1990]. A new rapid method was used for the isolation and fractionation of both rat and human Kupffer cells without the need of liver perfusion techniques. The study used rat livers or small human liver wedge biopsies obtained peroperatively and incubated with pronase under continuous pH registration. Kupffer cells were subsequently separated from other nonparenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation [Neaud et al.1995]. Identification of Kupffer cells was achieved on the basis of ultrastructural analyses and immunophenotyping [Neaud et al.1995]. The fractionation of Kupffer cells gave good yield comparable with other studies [Hendriks et al.1990, Neaud et al.1995, McCuskey et al.1990]. For details of protocol of liver cell isolation, see the Appendix in the end of this chapter. Human liver cell enzymes in isolated liver cells (hepatocytes and Kupffer cells in

cultures) showed characteristic regulatory behavior in presence of additives.

Liver cell hypoxia is represented as a state of oxygen depletion in cell. Initially liver cell gets its ATP and NADPH supply from glycolysis and TCA cycle. Consequently, electron transfer chain (oxidative phosphorylation) through series of cytochrome redox reactions converts available cell oxygen to water to produce high energy metabolites in the cell (metabolic integrity). Infected liver cells show loss of metabolic integrity and less available oxygen (oxygen starved or hypoxia). Nitroimidazole was established two decades ago as potential oxygen quenching drug in most of the infected and tumor cells. "Nitroimidazole oxygen quenching action" makes oxygen depleted or starved cells that further undergo worse to die and leaving normal cells fully functional that can be observed as potential tumor hypoxia therapy and antiparasitic treatment by

In following section, enzyme inhibition characteristics in energy metabolism regulation including glycolysis, TCA cycle regulatory enzyme behavior and drug metabolizing proteolytic lysosomal enzymes induced by additives in liver cell cultures are described.

The actinomycin D showed non-significant inhibitory change in glucokinase enzyme activity as shown in Figure 7 at the top histogram bars. The nitroimidazole showed enhanced glucokinase activity and reversed the inhibitory effect of actinomycin D. The glucokinase activity in hepatic abscess hepatocytes did not show any significant effect of nitroimidazole. Overall actinomycin D alone did not show any change in glucokinase enzyme activity in hepatic abscess hepatocytes or nitroimidazole treated hepatocytes.

**liver cells** 

nitroimidazole.

**4.1 Glucokinase** 

The addition of triamcinolone with insulin in combination showed significant increase in glucokinase activity in all hepatocyte groups. It pointed out to the enhancement in hormone dependent glucokinase enzyme synthesis in hepatocytes and enzyme activity showed highest enzyme synthesis rate in hepatic abscess recruited hepatocytes while nitroimidazole treated hepatocytes showed lesser increase in hormone dependent glucokinase in comparison with hepatocytes as shown in Figure 7. The increase in enzyme activity or enhanced glucokinase enzyme synthesis in hepatocytes showed that glucokinase enzyme behavior was hormone dependent. Different concentration of insulin and progesterone hormones further indicated the regulatory behavior of glucokinase enzyme as hormone dependent.

The addition of progesterone hormone 0.1 µgm/ml in hepatocyte cultures showed maximum enhanced glucokinase enzyme activity and nitroimidazole treated hepatocytes showed less enhanced glucokinase activity but more than control hepatocyte enzyme activity as shown in histogram bars (see Figure 7).

In control group, glucokinase enzyme activity in untreated hepatocytes did not show any chnage in their glucokinase enzyme activity in presence of nitroimidazole additive. The yield of hepatocytes and glucokinase enzyme activities from hepatic abscess liver biopsy were similar as yield of cells and glucokinase enzyme activities in untreated and nitroimidazole treated hepatocytes. So, it was clear that addition of actinomycin-D and insulin with triamicilone both enhanced the glucokinase enzyme synthesis in hepatocytes as shown in Figure 7 histograms bars.

Fig. 7. The nitroimidazole effect on glucokinase enzyme activity in guinea pig normal and infected hepatocytes is shown in presence of different enzyme regulatory additives actinomycin D, insulin-triamcilone. Notice the enhanced effect of insulin-triamicilone on glucokinase enzyme (P value <0.0001; r2 0.9872) in nitroimidazole treated vs liver abscess biopsy heaptocytes.

Mechanisms of Hepatocellular Dysfunction

ADP additives (2-5 mM ADP).

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 215

Fig. 8b. The pyruvate kinase enzyme activity in control, amoebic liver abscess and nitroimidazole treated hepatocytes is shown in presence of different enzyme regulatory

Fig. 8c. The pyruvate kinase enzyme activity in control, amoebic liver abscess and progesterone treated liver cells is shown in presence of different enzyme regulatory

additives (ATP, PEP+FDP,PEP inhibitors).

#### **4.2 Pyruvate kinase**

The altered behavior of pyruvate kinase regulation in isolated hepatocytes from nitroimidazole and progesterone induced cultures showed characteristic sigmoid behavior in normal group but towards linear relationship at increased pH of hepatocyte culture media. In other words, a pH increase alters the enzyme velocity in same proportion with ATP concentration changes in linear manner in liver cell medium. At high ATP concentration, enzyme synthesis in nitroimidazole stimulated cells remained significantly elevated while at pH 7.0-7.4 cells did not show sudden enzyme elevation and showed a fall in enzyme activity as observed in normal hepatocytes (see Figure 8). The higher ADP/ATP ratio kept the enzyme synthesis high at pH 7.4 in progesterone while nitroimidazole stimulated the hepatocyte cells in cultures (see Figure 8).

The intricacy was reported as ATP interacts with H+ and Mg++ to give complex distribution of many ionized and metal complex species. However, Mg++ free ADP itself has three subspecies viz. ADP--, ADPK--, ADPH— whose relative proportions vary with change in pH. Out of which ADP— happens to be true substrate of pyruvate kinase which presumably was utilized by hepatocytes stimulated by nitroimidazole. It further suggested the binding of ADP to unbound and fully bound enzyme conformations involving different groups within active site. The binding of ADP and ADP free conformation of enzyme did not involve groups that ionized within applied pH range in nitroimidazole and progesterone stimulated hepatocytes but binding of ADP to the fully bound enzyme conformation (cooperativity) involved the ionizable groups with high pK values (at pH 6.6-7.4). Moreover, phosphoenol pyruvate bound enzyme ionization has been previously reported to promote cooperative binding of ADP in diseased cell. In present study, ADP binding becomes less cooperative by stimulated hepatocytes. It is possible that enzyme may have two active conformations. So, allosteric interactions actually might have altered the mode of ADP binding in normal cells while they were non-effective in pyruvate kinase enzyme of stimulated hepatocytes. Pyruvate kinase active site conformation perhaps seems to change the ADP binding.

Fig. 8a. The pyruvate kinase enzyme activity in control, amoebic liver abscess and nitroimidazole treated hepatocytes is shown in presence of different enzyme regulatory additives (A-I).

The altered behavior of pyruvate kinase regulation in isolated hepatocytes from nitroimidazole and progesterone induced cultures showed characteristic sigmoid behavior in normal group but towards linear relationship at increased pH of hepatocyte culture media. In other words, a pH increase alters the enzyme velocity in same proportion with ATP concentration changes in linear manner in liver cell medium. At high ATP concentration, enzyme synthesis in nitroimidazole stimulated cells remained significantly elevated while at pH 7.0-7.4 cells did not show sudden enzyme elevation and showed a fall in enzyme activity as observed in normal hepatocytes (see Figure 8). The higher ADP/ATP ratio kept the enzyme synthesis high at pH 7.4 in progesterone while nitroimidazole

The intricacy was reported as ATP interacts with H+ and Mg++ to give complex distribution of many ionized and metal complex species. However, Mg++ free ADP itself has three subspecies viz. ADP--, ADPK--, ADPH— whose relative proportions vary with change in pH. Out of which ADP— happens to be true substrate of pyruvate kinase which presumably was utilized by hepatocytes stimulated by nitroimidazole. It further suggested the binding of ADP to unbound and fully bound enzyme conformations involving different groups within active site. The binding of ADP and ADP free conformation of enzyme did not involve groups that ionized within applied pH range in nitroimidazole and progesterone stimulated hepatocytes but binding of ADP to the fully bound enzyme conformation (cooperativity) involved the ionizable groups with high pK values (at pH 6.6-7.4). Moreover, phosphoenol pyruvate bound enzyme ionization has been previously reported to promote cooperative binding of ADP in diseased cell. In present study, ADP binding becomes less cooperative by stimulated hepatocytes. It is possible that enzyme may have two active conformations. So, allosteric interactions actually might have altered the mode of ADP binding in normal cells while they were non-effective in pyruvate kinase enzyme of stimulated hepatocytes.

Pyruvate kinase active site conformation perhaps seems to change the ADP binding.

Fig. 8a. The pyruvate kinase enzyme activity in control, amoebic liver abscess and nitroimidazole treated hepatocytes is shown in presence of different enzyme regulatory

stimulated the hepatocyte cells in cultures (see Figure 8).

**4.2 Pyruvate kinase** 

additives (A-I).

Fig. 8b. The pyruvate kinase enzyme activity in control, amoebic liver abscess and nitroimidazole treated hepatocytes is shown in presence of different enzyme regulatory ADP additives (2-5 mM ADP).

Fig. 8c. The pyruvate kinase enzyme activity in control, amoebic liver abscess and progesterone treated liver cells is shown in presence of different enzyme regulatory additives (ATP, PEP+FDP,PEP inhibitors).

Mechanisms of Hepatocellular Dysfunction

**4.4 Phosphodiesterase** 

did not respond or responded slow after progesterone addition.

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 217

[Pilkis et al.1979, Sanchez-Martinez et al.2000]. However, nitroimidazole added hepatocytes

Cyclic AMP dependent phosphodiesterase regulation over prostaglandin E2 and F2 synthesis was stimulated in Kupffer cells added with trophozoits and nitroimidazole. Phosphodiesterase activity in Kupffer cells was dependent upon calcium ion concentration which was regulatory factor for cyclic AMP pool and prostaglandin production. In amoebic trophozoite added cultures, cells exhibited insignificant elevated enzyme activity while nitroimidazole addition did not alter the enzyme activity as shown in Figure 10. The enzyme behavior was similar in nature for its dependence on cyclic AMP secretion, prostaglandin synthesis and calcium ion. However, tropozoites stimulated more cyclic AMP secretion and prostaglandins. It provides the probable mechanism of trophozoite stimulated macrophagal action. Earlier calmodulin dependent adenylate cyclase and phosphodiesterase regulation for cyclic AMP pool have been described [Hidi et al.2000]. Moreover, the stimulated prostaglandin synthesis was described as calmodulin dependent cyclic AMP regulated mechanism [Hidi et al.2000]. Thus it seems that amoebic trophozoite addition with hepatocytes regulates intracellular calcium ion concentration which controls prostaglandin synthesis via phospholipid and arachidonic acid precursors in hepatocytes. Thus synthesized prostaglandins regulate the cyclic AMP intracellular levels. The similar

mechanism has been described as "self-limiting" mechanism [Martina et al.2006].

Fig. 10. Cyclic AMP dependent phosphodisesterase activity and secretion of prostaglandin E2 and F2 in isolated kupffer cells from different groups. The phosphodiesterase enzyme activity in amebic liver abscess group showed highest enzyme activity after additives were added.

Fig. 9. The effect of nitroimidazole on phosphofructokinase enzyme activity of isolated hepatocytes from control, amebiasis and nitroimidazole groups. Hepatocytes from each group were sedimented and homogenized in Kreb Hanseleit buffer pH 8.0 containing 2.5 mM dithiothretiol, 0.1 mM EDTA and 50 mM NaF for 90 seconds followed by centrifugation at 27000 g for 10 minutes. One unit enzyme activity catalyzed 1 micromole fructose 6 phosphate to fructose 1,6 DiP per minute per mg enzyme protein.

#### **4.3 Phosphofructokinase**

Phosphofructokinase enzyme regulation in isolated hepatocytes cultures added with amoebic trophozoites, nitroimidazole and progesterone exhibited specific regulatory behavior. Addition of fructose 1,6 Diphosphate additive exhibited a stimulation of phosphofructokinase activity (see Figure 9). The effect of progesterone was seen as best response at 10 nM in hepatocytes of all groups while fructose 1,6 diphosphate showed maximum effect at concentration of 10 nM on the hepatocytes in presence of trophozoites added in hepatocyte cultures. However, hepatocyte cultures in other groups did not show any alteration (see Figure 9). Similarly, progesterone addition to hepatocytes in presence of trophozoites exhibited maximum fructose 1,6 diphosphate in the medium. The addition of progesterone concentration was within the physiological range (see Figure 9). Progesterone was presumed to effect enzyme activity by influencing intracellular level of various allosteric effectors of enzyme. Fructose 1,6 diphosphate acts as potent activator of enzyme activity. The phosphofructkinase enzyme activity was reported as cyclic AMP dependent [Pilkis et al.1979, Sanchez-Martinez et al.2000]. However, nitroimidazole added hepatocytes did not respond or responded slow after progesterone addition.

#### **4.4 Phosphodiesterase**

216 Enzyme Inhibition and Bioapplications

Fig. 9. The effect of nitroimidazole on phosphofructokinase enzyme activity of isolated hepatocytes from control, amebiasis and nitroimidazole groups. Hepatocytes from each group were sedimented and homogenized in Kreb Hanseleit buffer pH 8.0 containing 2.5 mM dithiothretiol, 0.1 mM EDTA and 50 mM NaF for 90 seconds followed by centrifugation

at 27000 g for 10 minutes. One unit enzyme activity catalyzed 1 micromole fructose 6

Phosphofructokinase enzyme regulation in isolated hepatocytes cultures added with amoebic trophozoites, nitroimidazole and progesterone exhibited specific regulatory behavior. Addition of fructose 1,6 Diphosphate additive exhibited a stimulation of phosphofructokinase activity (see Figure 9). The effect of progesterone was seen as best response at 10 nM in hepatocytes of all groups while fructose 1,6 diphosphate showed maximum effect at concentration of 10 nM on the hepatocytes in presence of trophozoites added in hepatocyte cultures. However, hepatocyte cultures in other groups did not show any alteration (see Figure 9). Similarly, progesterone addition to hepatocytes in presence of trophozoites exhibited maximum fructose 1,6 diphosphate in the medium. The addition of progesterone concentration was within the physiological range (see Figure 9). Progesterone was presumed to effect enzyme activity by influencing intracellular level of various allosteric effectors of enzyme. Fructose 1,6 diphosphate acts as potent activator of enzyme activity. The phosphofructkinase enzyme activity was reported as cyclic AMP dependent

phosphate to fructose 1,6 DiP per minute per mg enzyme protein.

**4.3 Phosphofructokinase** 

Cyclic AMP dependent phosphodiesterase regulation over prostaglandin E2 and F2 synthesis was stimulated in Kupffer cells added with trophozoits and nitroimidazole. Phosphodiesterase activity in Kupffer cells was dependent upon calcium ion concentration which was regulatory factor for cyclic AMP pool and prostaglandin production. In amoebic trophozoite added cultures, cells exhibited insignificant elevated enzyme activity while nitroimidazole addition did not alter the enzyme activity as shown in Figure 10. The enzyme behavior was similar in nature for its dependence on cyclic AMP secretion, prostaglandin synthesis and calcium ion. However, tropozoites stimulated more cyclic AMP secretion and prostaglandins. It provides the probable mechanism of trophozoite stimulated macrophagal action. Earlier calmodulin dependent adenylate cyclase and phosphodiesterase regulation for cyclic AMP pool have been described [Hidi et al.2000]. Moreover, the stimulated prostaglandin synthesis was described as calmodulin dependent cyclic AMP regulated mechanism [Hidi et al.2000]. Thus it seems that amoebic trophozoite addition with hepatocytes regulates intracellular calcium ion concentration which controls prostaglandin synthesis via phospholipid and arachidonic acid precursors in hepatocytes. Thus synthesized prostaglandins regulate the cyclic AMP intracellular levels. The similar mechanism has been described as "self-limiting" mechanism [Martina et al.2006].

Fig. 10. Cyclic AMP dependent phosphodisesterase activity and secretion of prostaglandin E2 and F2 in isolated kupffer cells from different groups. The phosphodiesterase enzyme activity in amebic liver abscess group showed highest enzyme activity after additives were added.

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 219

Fig. 12. The enzyme activity was estimated in Kupffer cells cultures added with sheep erythrocytes, trophozoites, nitroimidazole. The Kupffer cells were maintained for 48 hours or more to determine superoxide anion production in presence of 1.0 mM and 1.0 mM sodium cyanide additive added in culture medium. In brief, 3 x 105 Kupffer cells were suspended in Kreb's Henseleit buffer containing 5 mM glucose, 3 mM calcium chloride pH 7.4 in presence of 50 M ferricytochrome in total 1.0 ml reaction mixture and cells were incubated for 15 minutes in presence of different sodium cyanide additives. The reaction was started by addition of zymosan 500 g per ml to the medium after 10 minutes at 37 C supernatant was taken, centrifuged at 8000 x g in cold for two minutes and assayed for

The O2 superoxide anion radical production as result of respiratory burst activity showed proportionate elevated enzyme activity in isolated Kupffer cells from cultures added with trophozoites and nitroimidazole additives. The active role of reactive oxygen species and superoxide dismutase activity in cultured Kupffer cell damage during hypoxia is shown in Figure 12. Previous study supported these observations on cellular damage and cytotoxicity from reactive oxygen species from superoxide anion acting as a scavenger [Sharma et al.2010]. Another possibility of Kupffer cell toxicity by additives may be associated with cytokines, specifically TNF-α which was associated with the cytotoxicity [Sharma et al.2007]. Previous study also reported a decreased hepatocyte cell viability associated with altered cAMP phosphodiesterase and HMP shunt activity as a result of hypoxia [Carre et al.2010]. Authors suggested that the oxygen insufficiency is possibly a consequence of energy depletion and increased oxygen reactive species [Keeling et al.1982]. Other reports support this view of vasodilatory sensitive ATP depletion in Kupffer cells [Ana et al.2011]. The reactive oxygen species production also seems as another cause of hypoxia as suggested by

cytochrome C reductase enzyme activity. Ref. Sharma, R. et al. 2010.

#### **4.5 Enzyme inhibition in respiratory burst of liver cells**

An initial evidence of liver cell survival at different oxygen pressures is shown in Figure 11 to highlight respiratory burst of enzymes. The inhibited enzymes related with cell respiration are presumably considered responsible of induced respiratory burst (hypoxia) in liver cells. Respiratory burst process including enzyme alterations (superoxide dismutase, glutathione reductase, NADPH oxidase enzyme activity) was suggested as initial Kupffer cell stimulation or respiratory burst by additives and followed by energy and oxygen depletion as possible cause of hypoxia and cell viability loss as important findings in following section.

Fig. 11. Effect of oxygen supply (shown in different pressures) to liver cells. Notice the low oxygen supply depletes the oxygen content in cells while high oxygen supply keeps cells in in good survival. For long survival, reoxygenation of cells is needed(see green vertical line).

#### **4.6 Superoxide dismutase enzyme activity in respiratory burst of Kupffer cells**

The superoxide dismutase enzyme activity in isolated cells was comparable and showed minimal difference in both control and additive triggered Kupffer cells. Addition of sodium cyanide concentration showed small effect on elevated superoxide dismutase activities in both control and nitroimidazole triggered Kupffer cells. However, Kupffer cells showed elevated respiratory burst activity significantly high after adding 10 nM sodium cyanide in the cell culture medium as shown in Figure 12. However, the response of Kupffer cells with sodium cyanide addition was more in trophozoites added cell cultures at 10 mM sodium cyanide concentration. Zymosan stimulation on superoxide production was insignificantly high in the Kupffer cells in both groups. Earlier stimulation of zymosan induced superoxide anion production in Kupffer cells by sodium cyanide was reported to show the effect of cyanides on cytosolic superoxide dismutase enzyme activity [D'Alessandro et al.2011, Shahhosseini et al.2006]. Moreover, zymosan stimulated superoxide production was reported in Kupffer cells as reaction of cytochrome C reduction [Duan et al.2004].

An initial evidence of liver cell survival at different oxygen pressures is shown in Figure 11 to highlight respiratory burst of enzymes. The inhibited enzymes related with cell respiration are presumably considered responsible of induced respiratory burst (hypoxia) in liver cells. Respiratory burst process including enzyme alterations (superoxide dismutase, glutathione reductase, NADPH oxidase enzyme activity) was suggested as initial Kupffer cell stimulation or respiratory burst by additives and followed by energy and oxygen depletion as possible cause of hypoxia and cell viability loss as important findings in

Fig. 11. Effect of oxygen supply (shown in different pressures) to liver cells. Notice the low oxygen supply depletes the oxygen content in cells while high oxygen supply keeps cells in in good survival. For long survival, reoxygenation of cells is needed(see green vertical line).

The superoxide dismutase enzyme activity in isolated cells was comparable and showed minimal difference in both control and additive triggered Kupffer cells. Addition of sodium cyanide concentration showed small effect on elevated superoxide dismutase activities in both control and nitroimidazole triggered Kupffer cells. However, Kupffer cells showed elevated respiratory burst activity significantly high after adding 10 nM sodium cyanide in the cell culture medium as shown in Figure 12. However, the response of Kupffer cells with sodium cyanide addition was more in trophozoites added cell cultures at 10 mM sodium cyanide concentration. Zymosan stimulation on superoxide production was insignificantly high in the Kupffer cells in both groups. Earlier stimulation of zymosan induced superoxide anion production in Kupffer cells by sodium cyanide was reported to show the effect of cyanides on cytosolic superoxide dismutase enzyme activity [D'Alessandro et al.2011, Shahhosseini et al.2006]. Moreover, zymosan stimulated superoxide production was

**4.6 Superoxide dismutase enzyme activity in respiratory burst of Kupffer cells** 

reported in Kupffer cells as reaction of cytochrome C reduction [Duan et al.2004].

**4.5 Enzyme inhibition in respiratory burst of liver cells** 

following section.

Fig. 12. The enzyme activity was estimated in Kupffer cells cultures added with sheep erythrocytes, trophozoites, nitroimidazole. The Kupffer cells were maintained for 48 hours or more to determine superoxide anion production in presence of 1.0 mM and 1.0 mM sodium cyanide additive added in culture medium. In brief, 3 x 105 Kupffer cells were suspended in Kreb's Henseleit buffer containing 5 mM glucose, 3 mM calcium chloride pH 7.4 in presence of 50 M ferricytochrome in total 1.0 ml reaction mixture and cells were incubated for 15 minutes in presence of different sodium cyanide additives. The reaction was started by addition of zymosan 500 g per ml to the medium after 10 minutes at 37 C supernatant was taken, centrifuged at 8000 x g in cold for two minutes and assayed for cytochrome C reductase enzyme activity. Ref. Sharma, R. et al. 2010.

The O2 superoxide anion radical production as result of respiratory burst activity showed proportionate elevated enzyme activity in isolated Kupffer cells from cultures added with trophozoites and nitroimidazole additives. The active role of reactive oxygen species and superoxide dismutase activity in cultured Kupffer cell damage during hypoxia is shown in Figure 12. Previous study supported these observations on cellular damage and cytotoxicity from reactive oxygen species from superoxide anion acting as a scavenger [Sharma et al.2010]. Another possibility of Kupffer cell toxicity by additives may be associated with cytokines, specifically TNF-α which was associated with the cytotoxicity [Sharma et al.2007]. Previous study also reported a decreased hepatocyte cell viability associated with altered cAMP phosphodiesterase and HMP shunt activity as a result of hypoxia [Carre et al.2010]. Authors suggested that the oxygen insufficiency is possibly a consequence of energy depletion and increased oxygen reactive species [Keeling et al.1982]. Other reports support this view of vasodilatory sensitive ATP depletion in Kupffer cells [Ana et al.2011]. The reactive oxygen species production also seems as another cause of hypoxia as suggested by

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 221

lesser inhibition activity of hypoxic GSH enzyme initially during 24-48 hours. **Inhibition of NADH oxidase in respiratory burst:** The respiratory burst activity was described as increased activity in liver macrophages during hepatic cell damage showing increased oxygen consumption, stimulation of HMP shunt and elevated superoxide production with activated membrane NADPH oxidase enzyme as one of the mode of phagocytosis [Dusting et al. 2005]. Moreover, our experiments suggested the specific roles of additives on respiratory burst biomarkers: 1. The increased sodium cyanide concentration showed stimulation of superoxide dismutase activity; 2. The live erythrocytes and trophozoites showed HMP shunt stimulation while inert latex particles showed no effect on HMP shunt activity suggesting the association of additive nature as determinant of the HMP shunt activity or trio-enzyme inhibition; 3. The glutathione plays a critical role in surviving Kupffer cells during hypoxia and indicates the stimulated reactive oxygen species in respiratory burst activity [Bhatnagar et al.1981]. All these respiratory burst biomarker activities showed dependence on the nature of additives in corroboration with earlier reports [Kumar et al.2002; Forman et al. 2002; Ding et al. 1988]. In present chapter, both HMP shunt activity and respiratory burst activity (superoxide dismutase, glutathione reductase, NADPH oxidase enzyme activity) are outlines for suggested initial Kupffer cell stimulation by additives and later followed by energy and oxygen depletion as possible cause of hypoxia and cell viability

activity was highly elevated in all groups cultures incubated with sheep erythrocytes and amoebic trophozoits while cells with latex particles did not show any activation. Perhaps latex particles being biologically inert could not change the cellular sinusoidal function activity. Sheep erythrocytes and trophozoits perhaps stimulated the glucose metabolism via hexose monophosphate pathway but latex particles were phagocytosed without altered shunt activity. It suggested that HMP shunt may be a triggering event in Kupffer cell activation dependent not upon intracelllular fate of ingested material or particles [Knook et al. 1980]. The trophozoits added Kupffer cells showed direct interaction with increased glucose-6-phosphate dehydrogenase and 6 phosphogluconate dehydrogenase enzyme activities as described as a result of enhanced hexose monophosphate shunt activity. Earlier zymosan stimulated and iodoacetate additives inhibited Kupffer cell glucose 6 phosphate dehydrogenase and 6 phosphogluconate dehydrogenase activities have been reported [Heuff et al. 1994]. **Inhibition of Glutathione Reductase enzyme activity in respiratory burst:** The glutathione reductase GSH specific enzyme activity was measured in hypoxic hepatocytes in presence of different GSH enzyme inhibitors such as DEM, antimycin, BSO as additive effectors. To demonstrate the dependence of enzyme activity on exposure time of inhibitors, the hypoxic hepatocytes were incubated in presence of effectors and enzyme activity was measured in medium. At 0 hour, the enzyme specific activity was same in hepatocytes in all groups. After 24 hours, the activity in hepatocytes was inhibited maximum by actinomycin while DEM and BSO inhibited the enzyme at similar extent and hypoxic hepatocytes exhibited no difference in enzyme activities (P value > 0.001) (see Figure 14). After 48 hours, the GSH enzyme activity was inhibited maximum by addition of BSO and minimum by antimycin in cell cultures. However, the behavior pattern of enzyme inhibition by DEM and BSO was similar. After 72 hours, the inhibition pattern of GSH enzyme activitity by inhibitors was same as after 48 hours. After 96 hours, all inhibitors showed maximal inhibition and hypoxic hepatocytes showed same specific activities in all groups. Antimycin inhibitor exhibited

the nature of superoxide dismutase as additive cyanide sensitive enzyme. We observed a good reason of association between superoxide anion scavenger, glutathione reductase and NADPH oxidase as cause of cell viability loss leading further to Kupffer cell damage. In this direction, ample evidence supports the possibility of activation of HMP shunt due to depleted ATP and NADH in the Kupffer cells [Grahm, 2000; Spolarics et al. 1991].

#### **4.7 HMP shunt pathway in respiratory burst**

Hexose monophosphate shunt activation during respiratory burst in isolated Kupffer cells exposed with amoebic trophozoites and nitroimidazole showed enhanced enzyme activities as shown in Figures 13A-13C. HMP shunt activation is measured in three ways: 1. HMP shunt activity in presence of additives such as latex, sheep erythrocytes, trophozoites; 2. HMP shunt activity as radiolabeled glucose conversion to carbon dioxide; 3. HMP shunt activity as enzyme inhibition.


the nature of superoxide dismutase as additive cyanide sensitive enzyme. We observed a good reason of association between superoxide anion scavenger, glutathione reductase and NADPH oxidase as cause of cell viability loss leading further to Kupffer cell damage. In this direction, ample evidence supports the possibility of activation of HMP shunt due to

Hexose monophosphate shunt activation during respiratory burst in isolated Kupffer cells exposed with amoebic trophozoites and nitroimidazole showed enhanced enzyme activities as shown in Figures 13A-13C. HMP shunt activation is measured in three ways: 1. HMP shunt activity in presence of additives such as latex, sheep erythrocytes, trophozoites; 2. HMP shunt activity as radiolabeled glucose conversion to carbon dioxide; 3. HMP shunt

a. The HMP shunt activity was maximum and it elevated in Kupffer cells after incubation with sheep erythrocytes and trophozoites while Kupffer cells exposed with inert latex particles did not show any activation or change in HMP shunt activity. Sheep erythrocytes and trophozoites both stimulated glucose metabolism via hexose monophosphate shunt activity. Perhaps latex particles being biologically inert nature could not change the cellular activity at all. So, latex particles were phagocytosed without altered HMP shunt activity. It suggested that HMP shunt activity may be a triggering event in Kupffer cell activation dependent not solely upon intracellular fate of ingested material or particles. However, other additives such as zymosan, erythrocytes and trophozoites also showed same response. Previous study reported similar response of zymosan stimulated and iodoacetate inhibited Kupffer cell glucose-6-phosphate dehydrogenase and

b. The HMP shunt activity as [14C] glucose conversion to [14C]-CO2 liberated product indicated the active inflammation elicited by macrophage response of Kupffer cells [Schorlemmer et al. 1999]. The HMP shunt activity in control and triggered Kupffer cells was measured by [14C]CO2 release in dpm from labeled [14C] glucose per minute per mg protein in presence of different effectors known as quenching the hexose monophosphates in glycolysis. The HMP shunt activity was similar with no difference (P value > 0.001) in control compared with triggered Kuffer cells with effector and inert latex particles did not alter the HMP shunt activity. The HMP shunt activity was significantly enhanced in triggered Kupffer cells in presence of trophozoites (P value < 0.001) but the HMP shunt enhancement was lesser in presence of erythrocytes. It indicated the dependance of HMP shunt activity on nature of effector either inert or surface active. The latex was inert, erythrocytes were nonvirulent and trophozoits were virulent as a result HMP shunt activity was enhanced in the order of latex <

c. The trophozoites further activated the Kupffer cell glucose-6-phosphate and phosphogluconate dehydrogenase enzymes in association of HMP shunt activity. In following section, enzyme inhibition in liver cells is reported in control, hypoxic cells in different groups. Hexose monophosphate shunt activation was enhanced during respiratory burst in isolated Kupffer cells in amoebic trophozoits and nitroimidazole added cultures. In the cultures added with trophozoits and nitroimidazole both, Kupffer cells exhibited significantly elevated HMP shunt activation. The HMP shunt

phosphogluconate dehydrogenase enzymes.[Sharma et al.2010]

erythrocytes < trophozoits added in the medium of Kupffer cells.

depleted ATP and NADH in the Kupffer cells [Grahm, 2000; Spolarics et al. 1991].

**4.7 HMP shunt pathway in respiratory burst** 

activity as enzyme inhibition.

activity was highly elevated in all groups cultures incubated with sheep erythrocytes and amoebic trophozoits while cells with latex particles did not show any activation. Perhaps latex particles being biologically inert could not change the cellular sinusoidal function activity. Sheep erythrocytes and trophozoits perhaps stimulated the glucose metabolism via hexose monophosphate pathway but latex particles were phagocytosed without altered shunt activity. It suggested that HMP shunt may be a triggering event in Kupffer cell activation dependent not upon intracelllular fate of ingested material or particles [Knook et al. 1980]. The trophozoits added Kupffer cells showed direct interaction with increased glucose-6-phosphate dehydrogenase and 6 phosphogluconate dehydrogenase enzyme activities as described as a result of enhanced hexose monophosphate shunt activity. Earlier zymosan stimulated and iodoacetate additives inhibited Kupffer cell glucose 6 phosphate dehydrogenase and 6 phosphogluconate dehydrogenase activities have been reported [Heuff et al. 1994].


Mechanisms of Hepatocellular Dysfunction

1

**4.8 Cytochrome oxidase** 

0

10

20

GSH activity (IU)

30

mM DEM

M antimycin A

hepatocytes in culture. Ref. Sharma, R. et al. 2010.

C at 550 nm as described by Emmelot et al.1964.

**4.9 Inhibition of NADPH cytochrome P450 reductase** 

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 223

1 mM BSO

Fig. 14. Effect of added stimulators on Glutathione reductase enzyme activity in hypoxic

Cytochrome C oxidase was determined based upon oxidation of ferrocytochrome C by decrease in absorbance at 550 nm on spectrophotometer. In 3 ml reaction mixture containing 66 mM potassium phosphate buffer pH 7.4. 20µM reduced cytochrome C and 20µl mitochondrial protein reaction was followed at 30°C for two minutes. Reading was recorded after two minutes for absorbance of completely oxidised cytochrome C by adding 0.05 M potassium ferricyanide. The activity of cytochrome C oxidase was expressed in n moles of cytochrome C oxidase/mg protein/min which is equal to concentration of cytochrome C x K divided by concentration of protein. For calculation of cytochrome C concentration a millimolar extinction coefficient of 19.1 was employed for reduced vs oxidized cytochrome

*Cytochrome P 450:* Cytochrome P 450 was measured by spectrophotometer in absence or presence of carbon monoxide and determined by the method [Guengerich et al.2009]. In brief, 0.1 ml microsomal fraction in two cuvettes test and standard which mere added 1,5 mg sodium dithionite and carbon monoxide passed in only test sample cuvette for 25 seconds and same repeated again and ΔA recorded at 450 nm for cytochrome P 450 content

in n moles per mg protein calculated by extinction coefficient 91 mM/cm path length.

0 hour 24 hours 48 hours 72 hours 96 hours

loss as important finding. However, association of Kupffer cell stimulation and hypoxia is not conclusive and needs further investigation.

Fig. 13. Hexose monophosphate shunt activity is shown in isolated Kupffer cells from ameba infected and nitroimidazole treated cells in presence of erythrocytes, trophozoits and latex additives (see panel on top). In 3.0 ml medium, 1 micro Curie 14C glucose was added and simultaneously particles added with Kupffer cells in ratio 5:1(particle:cell) for erythrocytes, 2:1 for amebic trophozoits and 40:1 for latex. After one hour, 2 ml of cell free medium soaked by filter paper wetted with 100 µL of 10% KOH in counter well which further was added with 200 µL 1.0 N H2SO4 and after 15 minutes filter paper strip elute in tritosol were measured for scientillation counts. The trophozoits showed maximum activity while inert latex beads did not show any change in activity(see panel at bottom).Ref. Sharma, R. et al. 2010.

Fig. 14. Effect of added stimulators on Glutathione reductase enzyme activity in hypoxic hepatocytes in culture. Ref. Sharma, R. et al. 2010.

#### **4.8 Cytochrome oxidase**

222 Enzyme Inhibition and Bioapplications

Fig. 13. Hexose monophosphate shunt activity is shown in isolated Kupffer cells from ameba infected and nitroimidazole treated cells in presence of erythrocytes, trophozoits and latex additives (see panel on top). In 3.0 ml medium, 1 micro Curie 14C glucose was added and simultaneously particles added with Kupffer cells in ratio 5:1(particle:cell) for erythrocytes, 2:1 for amebic trophozoits and 40:1 for latex. After one hour, 2 ml of cell free medium soaked by filter paper wetted with 100 µL of 10% KOH in counter well which further was added with 200 µL 1.0 N H2SO4 and after 15 minutes filter paper strip elute in tritosol were measured for scientillation counts. The trophozoits showed maximum activity while inert latex beads did

not show any change in activity(see panel at bottom).Ref. Sharma, R. et al. 2010.

is not conclusive and needs further investigation.

loss as important finding. However, association of Kupffer cell stimulation and hypoxia

Cytochrome C oxidase was determined based upon oxidation of ferrocytochrome C by decrease in absorbance at 550 nm on spectrophotometer. In 3 ml reaction mixture containing 66 mM potassium phosphate buffer pH 7.4. 20µM reduced cytochrome C and 20µl mitochondrial protein reaction was followed at 30°C for two minutes. Reading was recorded after two minutes for absorbance of completely oxidised cytochrome C by adding 0.05 M potassium ferricyanide. The activity of cytochrome C oxidase was expressed in n moles of cytochrome C oxidase/mg protein/min which is equal to concentration of cytochrome C x K divided by concentration of protein. For calculation of cytochrome C concentration a millimolar extinction coefficient of 19.1 was employed for reduced vs oxidized cytochrome C at 550 nm as described by Emmelot et al.1964.

#### **4.9 Inhibition of NADPH cytochrome P450 reductase**

*Cytochrome P 450:* Cytochrome P 450 was measured by spectrophotometer in absence or presence of carbon monoxide and determined by the method [Guengerich et al.2009]. In brief, 0.1 ml microsomal fraction in two cuvettes test and standard which mere added 1,5 mg sodium dithionite and carbon monoxide passed in only test sample cuvette for 25 seconds and same repeated again and ΔA recorded at 450 nm for cytochrome P 450 content in n moles per mg protein calculated by extinction coefficient 91 mM/cm path length.

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 225

Fig. 15. Adenylate cyclase enzyme activity is shown in control, nitroimidazole, progesterone

*Nitroimidazole stimulation of adenylate cyclase activity:* Nitroimidazole is recently emerged as anti-tumor and radiosensitizer compound. It is used in tumor imaging, therapy and antiamoebic applications. Nitroimidazole has phenolic rings and phenolic side chains as shown in Figure 6. So, it is obvious that the side chains interfere with the dephosphorylation of ATP by phosphodiesterase enzyme. It appears to result with inhibition of hepatocyte adenylate cyclase catalytic subunit function in the presence of nitroimidazole. The cytotoxicity of nitroimidazole is poorly studied and reported. However, its anti-amoebic effect has been proven to decrease hepatic and intestinal infection rate. The nitroimidazole is widely reported as single dose in treatment of amoebic liver abscess. Moreover, nitroimidazole induced cytotoxic effect on human hepatocytes are still not known. DNA strand breakage,

and amoebic liver abscess recruited liver cells in presence of GITP/GTP stimulators,

protein\* additives. Ref. Sharma, R. et al. 2010.

#### **4.10 Inhibition of adenylate cyclase**

*Adenylate cyclase regulatory properties*: Adenylate cyclase enzyme in isolated hepatocytes has been reported as tool of regulatory studies of hormones, enzymes in the presence of Gs bound receptors [Small et al. 2000]. Membrane adenylate cyclase activity is defined due to its multi-pass transmembrane protein made of two cytosolic segments as shown in Figure 15. These segments play active roles in association of Gsα-GTP. The GTP induced Gs<sup>α</sup> binding with adenylate cyclase can be a key mechanism of adenylate cyclase stimulation or inhibition during progesterone bound α-adrenergic receptor [Sunahara et al. 1997]. The possible mechanism of adenylate cyclase activation or inhibition by progesterone is shown in Figure 15. The membrane adenylate cyclase activity can be an indicator of membrane viability. However, the enzyme activity depends on the type and nature of effecter. The behavior of liver cells in culture in the presence of effectors may indicate the regulatory effect of stimulators *in vivo*. However, in cultures, hepatocytes and Kupffer cells may likely experience different physiological environment different from *in vivo*. In controlled liver cell culture conditions of medium, the liver cells showed adenylate cyclase stimulation in a specific manner by effectors such as progesterone, nitroimidazole, GTP, GITP, and prostaglandins [Aoshiba et al. 1997]. The receptors for prostaglandins interact with Gs<sup>α</sup> subunits. Gsα-GTP complex binds with α-adrenalgic receptor made of seven subunits as shown in Figure 5. Binding of receptor with Gsα-GTP protein inhibits adenylate cyclase activity and cAMP formation. The present study highlights the behavior of liver cells and the characteristic features of their cyclic AMP dependent adenylate cyclase regulatory system in culture.

*Progesterone modulation of adenylate cyclase activity*: Progesterone modulates the activity of adenylate cyclase and adenylate cyclase response is hormone sensitive [Gilman et al. 1984]. The progesterone stimulates the receptor activating the adenylate cyclase by stimulatory Gsα-GTP complex resulting with formation of cAMP. Hepatocytes with progesterone added in medium exhibited the enhanced enzyme activity. The addition of GTP stimulator did not change the adenylate cyclase activity. In contrast, GITP stimulated cells exhibited an apparent adenylate cyclase activation. Unlike the adenylate cyclase enzyme activity in the presence of different effectors in stimulated hepatocytes, the rates of cyclic AMP production was delayed in control hepatocytes. However, progesterone addition to hepatocytes did not exhibit significant delay in GITP stimulated adenylate cyclase activity. Thus reason can be speculated that progesterone binds with stimulatory receptors on hepatocytes which lead to increase in intracellular cyclic AMP accumulation through adenylate cyclase activation via guinine nucleotide regulated mechanism [Cohen-Tannoudji et al. 1991]. The Kupffer cells also showed similar guanine regulatory enzyme properties.

The progesterone response of adenylate cyclase activity is a two phase process [Ko, et al.1999; Martin et al. 1987]. First, progesterone receptor and catalytic subunit (C complex) of adenylate cyclase occupy the place on membrane followed by Gsα protein interaction with enzyme to make G protein C complex [Ko et al. 1999]. However, the addition of GITP stimulated the adenylate cyclase to a greater extent in hepatocytes more than pre-exposed hepatocytes to progesterone. Recently, G protein-enzyme complex formation by GTP/GITP stimulation has been reviewed [Niu et al. 2003].

*Adenylate cyclase regulatory properties*: Adenylate cyclase enzyme in isolated hepatocytes has been reported as tool of regulatory studies of hormones, enzymes in the presence of Gs bound receptors [Small et al. 2000]. Membrane adenylate cyclase activity is defined due to its multi-pass transmembrane protein made of two cytosolic segments as shown in Figure 15. These segments play active roles in association of Gsα-GTP. The GTP induced Gs<sup>α</sup> binding with adenylate cyclase can be a key mechanism of adenylate cyclase stimulation or inhibition during progesterone bound α-adrenergic receptor [Sunahara et al. 1997]. The possible mechanism of adenylate cyclase activation or inhibition by progesterone is shown in Figure 15. The membrane adenylate cyclase activity can be an indicator of membrane viability. However, the enzyme activity depends on the type and nature of effecter. The behavior of liver cells in culture in the presence of effectors may indicate the regulatory effect of stimulators *in vivo*. However, in cultures, hepatocytes and Kupffer cells may likely experience different physiological environment different from *in vivo*. In controlled liver cell culture conditions of medium, the liver cells showed adenylate cyclase stimulation in a specific manner by effectors such as progesterone, nitroimidazole, GTP, GITP, and prostaglandins [Aoshiba et al. 1997]. The receptors for prostaglandins interact with Gs<sup>α</sup> subunits. Gsα-GTP complex binds with α-adrenalgic receptor made of seven subunits as shown in Figure 5. Binding of receptor with Gsα-GTP protein inhibits adenylate cyclase activity and cAMP formation. The present study highlights the behavior of liver cells and the characteristic features of their cyclic AMP dependent adenylate cyclase regulatory

*Progesterone modulation of adenylate cyclase activity*: Progesterone modulates the activity of adenylate cyclase and adenylate cyclase response is hormone sensitive [Gilman et al. 1984]. The progesterone stimulates the receptor activating the adenylate cyclase by stimulatory Gsα-GTP complex resulting with formation of cAMP. Hepatocytes with progesterone added in medium exhibited the enhanced enzyme activity. The addition of GTP stimulator did not change the adenylate cyclase activity. In contrast, GITP stimulated cells exhibited an apparent adenylate cyclase activation. Unlike the adenylate cyclase enzyme activity in the presence of different effectors in stimulated hepatocytes, the rates of cyclic AMP production was delayed in control hepatocytes. However, progesterone addition to hepatocytes did not exhibit significant delay in GITP stimulated adenylate cyclase activity. Thus reason can be speculated that progesterone binds with stimulatory receptors on hepatocytes which lead to increase in intracellular cyclic AMP accumulation through adenylate cyclase activation via guinine nucleotide regulated mechanism [Cohen-Tannoudji et al. 1991]. The Kupffer cells also showed similar guanine regulatory enzyme

The progesterone response of adenylate cyclase activity is a two phase process [Ko, et al.1999; Martin et al. 1987]. First, progesterone receptor and catalytic subunit (C complex) of adenylate cyclase occupy the place on membrane followed by Gsα protein interaction with enzyme to make G protein C complex [Ko et al. 1999]. However, the addition of GITP stimulated the adenylate cyclase to a greater extent in hepatocytes more than pre-exposed hepatocytes to progesterone. Recently, G protein-enzyme complex formation by GTP/GITP

**4.10 Inhibition of adenylate cyclase** 

system in culture.

properties.

stimulation has been reviewed [Niu et al. 2003].

Fig. 15. Adenylate cyclase enzyme activity is shown in control, nitroimidazole, progesterone and amoebic liver abscess recruited liver cells in presence of GITP/GTP stimulators, protein\* additives. Ref. Sharma, R. et al. 2010.

*Nitroimidazole stimulation of adenylate cyclase activity:* Nitroimidazole is recently emerged as anti-tumor and radiosensitizer compound. It is used in tumor imaging, therapy and antiamoebic applications. Nitroimidazole has phenolic rings and phenolic side chains as shown in Figure 6. So, it is obvious that the side chains interfere with the dephosphorylation of ATP by phosphodiesterase enzyme. It appears to result with inhibition of hepatocyte adenylate cyclase catalytic subunit function in the presence of nitroimidazole. The cytotoxicity of nitroimidazole is poorly studied and reported. However, its anti-amoebic effect has been proven to decrease hepatic and intestinal infection rate. The nitroimidazole is widely reported as single dose in treatment of amoebic liver abscess. Moreover, nitroimidazole induced cytotoxic effect on human hepatocytes are still not known. DNA strand breakage,

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 227

Fig. 16. Lysosomal and microsomal enzymes in isolated nonparenchymal liver cells are shown in control, nitroimidazole, progesterone, amoebic liver abscess recruited livers. Note inhibitory action of nitroimidazole on liver cells to bring normal activities in comparison to

amoebic liver abcess recruited cells. Ref. Sharma, R. et al. 2009.

energy regulatory enzymes, phagocytosis, and immune response have been reported as nitroimidazole induced cytotoxic effects in animals [Zheng et al. 1997; Whitaker et al. 1992; Singh et al. 1991]. The role of nitroimidzole is understood to restore energy regulatory process involving adenylate cyclase, phosphodiesterase, cAMP formation in hepatocytes and reported as hormone and GTP specific in action. The nitroimidazole derivatives inhibited anterior pituitary cell function apparently by their direct effect on the catalytic subunit of the adenylate cyclase holoenzyme [Stalla et al. 1989]. The nonparenchymal Kuffer cells play a significant role of phagocytosis sensitive to prostaglandins. Recently, nitroimidazole derivatives have been identified as anticancer potential agents. Still the role of imidazoles as anticancer agent is not established [Anderson et al. 2006]. The indicated study the association of imidazole ring structure with the cyclic AMP dependent adenylate cyclase catalytic subunit regulatory mechanism in liver cells.

#### **5. Inhibition of drug metabolizing enzymes in isolated liver cells**

Drug metabolizing enzymes are abundant in microsomes and lysosomes. Most of these enzymes are elevated or secreted at the sites of inflammation, disease conditions and in presence of foreign bodies. An elevated enzyme in cells and secretion from lysosomes or microscomes action is in defense by 'suicide action' of microbody against disease or foreign bodies. Drugs inhibit these enzymes or bring back any disease or inflammation induced effect on enzyme activity in liver cell. Characteristic inhibition of enzyme activities in different groups by nitroimidazole are shown in Figure 15. Recently, we reported the enzyme inhibition by nitroimidazole in detail [Sharma 2011a, Sharma 2011b]. In general, lysosomal enzymes are peptidases, act at acidic pH, and participate as terminal scavengers of harmful anions, radicals and peptides. For details, read section 2.

Nitroimidazole inhibits the liver enzymes by making adduct for glyoxyl-glutamine metabolism in liver cells as author described in detail [Sharma, et al. 2011].

#### **6. Phagocytosis**

Although the clearance and distribution of ligand molecules in circulation represent the function of hepatic sinusoidal cells, these mechanisms reveal a network that is more intricate than would at first seem, since several receptors are common to not only one type of cell, but also to two or three types of cells in the liver. In the case of latex particles in which their uptake by a particular cell type seems to be determined by their size, sinusoidal endothelial cells are able to internalize particles up to 0.23 microns under physiologic conditions, in vivo, and larger particles are taken up by Kupffer cells. However, when the phagocytic function of Kupffer cells were impaired by frog virus or alcohol, the endothelial cells were reported to take up particles larger than 1 micron in diameter after the injection of an excess amount of latex particles. Endothelial cells would thus constitute a second line of defense in the liver in that they remove foreign materials from the blood when Kupffer cell phagocytic function is totally disturbed [Schoremmer et al. 1990]. This potential role may not, however, be fully expressed under physiologic conditions when Kupffer cells are active in clearing foreign substance from the circulation. The functions of liver sinusoidal cells are varied and complex and these cells can be regarded as "a sinusoidal cell unit." This cellular interaction must be taken into account for any quantitative analysis [Kampas et al. 1999].

energy regulatory enzymes, phagocytosis, and immune response have been reported as nitroimidazole induced cytotoxic effects in animals [Zheng et al. 1997; Whitaker et al. 1992; Singh et al. 1991]. The role of nitroimidzole is understood to restore energy regulatory process involving adenylate cyclase, phosphodiesterase, cAMP formation in hepatocytes and reported as hormone and GTP specific in action. The nitroimidazole derivatives inhibited anterior pituitary cell function apparently by their direct effect on the catalytic subunit of the adenylate cyclase holoenzyme [Stalla et al. 1989]. The nonparenchymal Kuffer cells play a significant role of phagocytosis sensitive to prostaglandins. Recently, nitroimidazole derivatives have been identified as anticancer potential agents. Still the role of imidazoles as anticancer agent is not established [Anderson et al. 2006]. The indicated study the association of imidazole ring structure with the cyclic AMP dependent adenylate

Drug metabolizing enzymes are abundant in microsomes and lysosomes. Most of these enzymes are elevated or secreted at the sites of inflammation, disease conditions and in presence of foreign bodies. An elevated enzyme in cells and secretion from lysosomes or microscomes action is in defense by 'suicide action' of microbody against disease or foreign bodies. Drugs inhibit these enzymes or bring back any disease or inflammation induced effect on enzyme activity in liver cell. Characteristic inhibition of enzyme activities in different groups by nitroimidazole are shown in Figure 15. Recently, we reported the enzyme inhibition by nitroimidazole in detail [Sharma 2011a, Sharma 2011b]. In general, lysosomal enzymes are peptidases, act at acidic pH, and participate as terminal scavengers

Nitroimidazole inhibits the liver enzymes by making adduct for glyoxyl-glutamine

Although the clearance and distribution of ligand molecules in circulation represent the function of hepatic sinusoidal cells, these mechanisms reveal a network that is more intricate than would at first seem, since several receptors are common to not only one type of cell, but also to two or three types of cells in the liver. In the case of latex particles in which their uptake by a particular cell type seems to be determined by their size, sinusoidal endothelial cells are able to internalize particles up to 0.23 microns under physiologic conditions, in vivo, and larger particles are taken up by Kupffer cells. However, when the phagocytic function of Kupffer cells were impaired by frog virus or alcohol, the endothelial cells were reported to take up particles larger than 1 micron in diameter after the injection of an excess amount of latex particles. Endothelial cells would thus constitute a second line of defense in the liver in that they remove foreign materials from the blood when Kupffer cell phagocytic function is totally disturbed [Schoremmer et al. 1990]. This potential role may not, however, be fully expressed under physiologic conditions when Kupffer cells are active in clearing foreign substance from the circulation. The functions of liver sinusoidal cells are varied and complex and these cells can be regarded as "a sinusoidal cell unit." This cellular interaction

cyclase catalytic subunit regulatory mechanism in liver cells.

**5. Inhibition of drug metabolizing enzymes in isolated liver cells** 

of harmful anions, radicals and peptides. For details, read section 2.

**6. Phagocytosis** 

metabolism in liver cells as author described in detail [Sharma, et al. 2011].

must be taken into account for any quantitative analysis [Kampas et al. 1999].

Fig. 16. Lysosomal and microsomal enzymes in isolated nonparenchymal liver cells are shown in control, nitroimidazole, progesterone, amoebic liver abscess recruited livers. Note inhibitory action of nitroimidazole on liver cells to bring normal activities in comparison to amoebic liver abcess recruited cells. Ref. Sharma, R. et al. 2009.

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 229

Fig. 17. Phagocytic activity of Kupffer cells is shown as β glucuronidase enzyme activity of cells after adding zymosan, erythrocytes, entamoeba histolytica trophozoits, latex beads additives, and nitroimidazole as drug in cultures of Kupffer cells from liver biopsy samples. The enzyme activity was measured after 1, 2, 3, 4 days of incubation. Each point value

represents the average of 3 readings of observations. Ref Sharma,R. et al. 2009.

Fig. 18. Phagocytic activity of Kupffer cells is shown as lactate dehydrogenase enzyme activity of cells after adding zymosan, erythrocytes, entamoeba histolytica trophozoits, latex beads additives, and nitroimidazole as drug in cultures of Kupffer cells from liver biopsy samples. The enzyme activity was measured after 1, 2, 3, 4 days of incubation. Each point value represents the average of 3 readings of observations. Ref Sharma,R. et al. 2009.


 **Enzyme inhibition in phagocytosis**: In our experiments, liver cell cultures were mixed with zymosan (7:1 particle cell ratio); erythrocytes (5:1 particle cell ratio); amebic trophozoits (2:1 particle ratio), latex (20:1 particle cell ratio) and nitroimidazole was added 1 µM/106 cells. Kupffer cell phagocytic activation in isolated Kupffer cells from human livers (added with amoebic trophozoits) was elevated 2-3 fold over normal Kupffer cells. Kupffer cells incubated with zymosan and trophozoits exhibited maximum phagocytic activity while with latex and sheep erythrocytes these exhibited minimum and insignificantly increased activity respectively. Increased phagocytic activity was time dependent which was exhibited by -glucuronidase, lactate dehydrogenase and plasminogen activator activity. Phagocytosis of zymosan and sheep erythrocytes triggered the immediate release of glucuronidase, stimulated synthesis of cellular lactate dehydrogenase and induced delayed production with secretion of plasminogen activator. Inhibition of these enzymes as nitroimidazole response is shown in Figures 16-18, in detail. Major findings were: 1. effect of nitroimidazole and latex were minimum. Similar observations were reported in earlier study [Kamps et al. 1999, Bhatnagar et al. 1981, Kuttner et al. 1982]; 2. nitroimidazole was biotransformed into water soluble products and cleared by drug metabolizing enzyme system of Kupffer cells; 3. latex particles being inert confirmed the independence of Kupffer cell activation from intracellular fate of ingested material; 4. amoebic trophozoits with their toxins or specific membrane surface signals stimulate Kupffer cells for phagocytosis resulting early release of lysosomal hydrolases than the release by other particles [Virk et al 1989, Virk et al. 1988]. However, increased lysosomal enzymes in Kupffer cells incubated with amoebic trophozoits were reported as a consequence of phagocytic activation [Froh et al. 2003]. Recently, these phagocytic activation changes due to nitroimidazole have been reported as associated with prostaglandin synthesis and their release

 The glucuronidase activity showed the enhanced enzyme activity trend in all additives but different enzyme enhancement for different additives. In control cells, the activity enhanced during 24-36 hours in incubation. The erythrocytes showed most vulnerability to Kupffer cells in initial 24 hours. Zymosan beads showed slow response to phagocytosis while latex remained inert to Kupffer cells. The nitroimidazole showed normal enzyme pattern. It was interesting that maximum rate of phagocytosis was

 Lactate dehydrogenase enzyme activity showed the enhanced enzyme activity trend in all additives except latex additive but different enzyme enhancement for different additives. In control cells, the activity enhanced during 12-36 hours in incubation. The erythrocytes showed most vulnerability to Kupffer cells in initial 24 hours. Zymosan beads showed maximum response to phagocytosis while latex remained inert to Kupffer cells. The nitroimidazole showed inhibitory enzyme activity in Kupffer cells but after 24 hours the response was stimulatory to enzyme activity (delayed enhanced enzyme stimulation as a result phagocytosis). It was interesting that maximum rate of phagocytosis was observed during 48 hours. However, the leaked out LDH activity represented the probable phagocytic activity of Kupffer cells relationship with time. Nitroimidazole addition in incubation medium perhaps showed the minimizing amoebic virulence [Sharma 2010]. Earlier lysosomal enzyme release with increased

observed during 24 and 36 hours except erythrocytes.

LDH cellular levels has been reported [Koppele et al. 1991].

[Spolarics et al. 1997].

Fig. 17. Phagocytic activity of Kupffer cells is shown as β glucuronidase enzyme activity of cells after adding zymosan, erythrocytes, entamoeba histolytica trophozoits, latex beads additives, and nitroimidazole as drug in cultures of Kupffer cells from liver biopsy samples. The enzyme activity was measured after 1, 2, 3, 4 days of incubation. Each point value represents the average of 3 readings of observations. Ref Sharma,R. et al. 2009.

Fig. 18. Phagocytic activity of Kupffer cells is shown as lactate dehydrogenase enzyme activity of cells after adding zymosan, erythrocytes, entamoeba histolytica trophozoits, latex beads additives, and nitroimidazole as drug in cultures of Kupffer cells from liver biopsy samples. The enzyme activity was measured after 1, 2, 3, 4 days of incubation. Each point value represents the average of 3 readings of observations. Ref Sharma,R. et al. 2009.

Mechanisms of Hepatocellular Dysfunction

liver abscess in following section.

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 231

 Increased RNA synthesis also increases ribonucleotide pool, rise in RNA polymerase activity, increase in polyamine synthesis and s-RNA methylation. The temporal relationship between kinetic changes in the DNA channel and the RNA channel seem to indicate that RNA channel is primer of DNA channel. In other words, cyclic AMP level may initiate both DNA and RNA synthesis beginning at the nucleotide level under hormonal regulation. Best example of elevated RNA sysnthesis in liver regeneration is elevated orotate pyrophosphorylase/decarboxylase to keep CTP/UTP pool in presence of UTP/CDP coenzymes for glycogenesis and lipogenesis respectively. UMP stimulated orotate-tRNA labeling around 18s-28s RNA complex step is preceded by an increase in tRNA methylation by tRNA Methylase enzyme. For its action, short chain of monocistronic mRNA on 18s component acts as primer for synthesis of constitutive RNA and export protein synthesis. Nobel Prize was awarded in 2009 on resolving the intricacies of ribosomal subunit interplay and consequent role of DNA/RNA Polymerase enzyme reactions in protein synthesis during drug induced regeneration [Schmeing et al.2009]. However, other enzymes RNase activity, cataionic polyamine play role in stabilization of ribosomal RNA (labeled methionine to spermidine) in protein synthesis. Increased polymine synthesis enhances ornithine decarboxylase to increase amino acid pool in presence of cAMP induced prostaglandins mediated growth hormone. Moreover, growth hormone stimulates thymidine kinase in liver. Author describes DNA synthesis in isolated Kupffer cells during liver regeneration after nitroimidazole treatment in amoebic

 Kupffer cells isolated from human amoebic liver abscess liver-biopsies and progesterone treated Kupffer cell in cultures were induced for DNA synthesis by adding nitroimidazole. This offered a simple model to study the mechanism of cell DNA induction and interdependence between two cell populations. Earlier such interactions between cells were reported regulated by thymidine kinase (thymidine pathway) and ribonucleotide reductase (thymidylate pathway)[Bresnick et al.1970]. Conceptually, immediately after disease in regeneration, liver cell gets confronted a challenge of increased protein synthesis for cell division involving metabolic enzymes leading to DNA synthesis (binding of ribonucleotides with deoxyribonucleotides). Two pathways play role in this process by Thymidine pathway and thymidylate pathway. Thymidylate pathway for DNA synthesis needs dTTP supply, thymidine kinase, TDP kinase to keep TMP pool. Basically, a repressor molecule (thymidine) inhibits transcription of thymidine kinase by allosteric regulation. Thymidine kinase is a TTP insensitive dimeric form (one catalytic site and other allosteric site) means Km for enzyme decreases with elevated ATP concentration (ATP positive effector and TTP negative effector). dimeric form and shows sigmoid behavior of thymidine kinase [Barroso et al. 2005]. Activation of thymidine kinase also depends on cyclic AMP activated protein kinase. On other side, ribonucleotise reductase reduces CDP in presence of dCMP deaminase to make dUMP (a substrate for thymidylate synthesis) by thymidylate sysnthetase in presence of N5,N10 hydroxymethyl-FH4. dCMP is good enzyme inhibitor of thymidylate sysnthetase and activator of folate reductase to keep net high TMP pool. Ribonucleotide synthesis is regulated by

polymerization proceeds from 5'-phosphate end to the growing 3'-OH end.

chromosomal DNA and deoxyribonucleotides. Priming DNase action on native and denatured DNA initiates DNA synthesis. Earlier study reported that arginase enzyme acts strong DNA synthase inhibitor perhaps due to lack of neosynthesized histone (arginine-rich histone) by reducing priming state of chromosomal DNA. DNA sysnthesis by means of additive or replicative DNA polymerase needs 3'-OH roup and

 The plasminogen activator induced lysis of Kupffer cells showed the enhanced plasminogen activity only after 36 hours. Initially the activity trend in all additives was same and immune to any additive added. In control cells, the plasminogen activity remained unaltered while kupffer cells showed immunity to zymosan and latex additives throughout the incubation period. However Kupffer cells showed sharp enhancement of plasminogen activity after 36 hours during 36-48 hours in incubation. The trophozoits showed maximum effect on plasminogen activator in Kupffer cells after 36 hours. Plasminogen activator induction has been reported dependent on de novo enzyme synthesis in stimulated macrophage by endotoxins and asbestos [Nagaoka et al. 2003].

Fig. 19. Phagocytic activity of Kupffer cells is shown as plasminogen activator activity of cells after adding zymosan, erythrocytes, entamoeba histolytica trophozoits, latex beads additives, and nitroimidazole as drug in cultures of Kupffer cells from liver biopsy samples. The enzyme activity was measured after 1, 2, 3, 4 days of incubation. Each point value represents the average of 3 readings of observations. Each point on graph represents the activity as extent of cell lysis in percent increase over basal value of 100. Ref Sharma et al. 2009.

#### **7. DNA synthesis in Kupffer cells**

Stimulated DNA synthesis is considered as defense mode. From physiological point of view, it is true that drastic change in enzymatic pattern in liver regeneration permits DNA synthesis. Major enzyme players are DNA polymerase and DNase (allosteric complex enzyme) in mitosis. We present five sequence steps of events: deoxyribonucleotides, ribonucleotides, DNA, RNA and proteins (see Figure 20) [Bresnick et al.1970].

 DNase exposes 3'-OH end groups thus increases capacity of chromosomal DNA in vivo. DNA polymerase plays a passive role in DNA synthesis in presence of high

 The plasminogen activator induced lysis of Kupffer cells showed the enhanced plasminogen activity only after 36 hours. Initially the activity trend in all additives was same and immune to any additive added. In control cells, the plasminogen activity remained unaltered while kupffer cells showed immunity to zymosan and latex additives throughout the incubation period. However Kupffer cells showed sharp enhancement of plasminogen activity after 36 hours during 36-48 hours in incubation. The trophozoits showed maximum effect on plasminogen activator in Kupffer cells after 36 hours. Plasminogen activator induction has been reported dependent on de novo enzyme synthesis in stimulated macrophage by endotoxins and asbestos [Nagaoka et al. 2003].

Fig. 19. Phagocytic activity of Kupffer cells is shown as plasminogen activator activity of cells after adding zymosan, erythrocytes, entamoeba histolytica trophozoits, latex beads additives, and nitroimidazole as drug in cultures of Kupffer cells from liver biopsy samples. The enzyme activity was measured after 1, 2, 3, 4 days of incubation. Each point value represents the average of 3 readings of observations. Each point on graph represents the activity as extent of

Stimulated DNA synthesis is considered as defense mode. From physiological point of view, it is true that drastic change in enzymatic pattern in liver regeneration permits DNA synthesis. Major enzyme players are DNA polymerase and DNase (allosteric complex enzyme) in mitosis. We present five sequence steps of events: deoxyribonucleotides,

 DNase exposes 3'-OH end groups thus increases capacity of chromosomal DNA in vivo. DNA polymerase plays a passive role in DNA synthesis in presence of high

cell lysis in percent increase over basal value of 100. Ref Sharma et al. 2009.

ribonucleotides, DNA, RNA and proteins (see Figure 20) [Bresnick et al.1970].

**7. DNA synthesis in Kupffer cells** 

chromosomal DNA and deoxyribonucleotides. Priming DNase action on native and denatured DNA initiates DNA synthesis. Earlier study reported that arginase enzyme acts strong DNA synthase inhibitor perhaps due to lack of neosynthesized histone (arginine-rich histone) by reducing priming state of chromosomal DNA. DNA sysnthesis by means of additive or replicative DNA polymerase needs 3'-OH roup and polymerization proceeds from 5'-phosphate end to the growing 3'-OH end.


Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 233

Isolated lysosomal preparations from L[1-14C] leucine (25 Ci per 100 gm wt) intraperitonially injected human Kupffer cell cultures exhibited percent degradation at pH 3.0, 4.0,5.0, 6.0,7.0, 8.0 and 9.0 at temperatures 10 °C, 20 °C, 30 °C, 40 °C, 50 °C and 60 °C, in presence of amoebic trophozoits after 15 minutes(short lived) and 15 hours(long-lived) addition as shown in Figures 21. Major players in this liver regeneration process are ribosomes (sitting on endoplasmic reticulum), GTP (ribotide pool) and coenzymes for ribotide polymerase enzymes (a balance in thymidine kinase and thymidylate synthetase). Other enzyme inhibition is LDH-M type gets converted to LDH-H type or lactate pyruvate (accelerated glycolysis). In simultaneous other set of experiment, Kupffer cell lysosomes in presence of leupeptin, pepstatin inhibitors and nitroimidazole in 100 g, 100 g and 50 mM per ml concentration respectively showed aryl sulphatase enzyme activity changes (see Figures 21,22) in the presence of 0.3 M sucrose at 37 °C for 60 minutes at pH 5.5 in incubation mixture of Kupffer cell lysosomes and additives. Proteolysis rate and degradation of short and long lived proteins in isolated lysosomes from the Kupffer cell

**8. Inhibition of liver lysosomal enzymes in protein metaboilism** 

Fig. 21. Figure shows the effect of of radiolabeled [14C]-Leucine in Kupffer cultures represented by aryl sulphatase enzyme activity in normal and ALA recruited cells at different pH and temperatures. Notice the low enzyme activity in normal cells while

maximum leucine degradation was at pH 6 and 40 °C.

ribonucleotide reductase. Increase in CTP build up dCTP, purine deoxyribonucleotides and TTP pools[Cheung et al.1970].

 Several types of growth factors have been reported as regulatory substances participating in liver regeneration process. Stimulation of DNA synthesis in Kupffer cells by amoeba trophozoites is shown here as response induced by colony stimulating factor. Since measured increase of radio label uptake by increasing amoeba trophozoite concentration in medium reflected an increase of cell number entering into S phase rather than higher rate of [3H] TdR incorporation seen in all the cells, it was possible that different Kupffer cells normal or pretreated with amoeba (from these different liver cell populations) may vary in susceptibility to colony stimulating factor (CSF). High concentrations of amoeba caused a reduction of thymidine uptake and remarkably reduction was measured on whole cover slip scintillation counts. It reflected lower incorporation of [3H] TdR into each DNA synthesizing cells. This phenomenon may be due to reduced capacity of Kupffer cells uptake as a result of DNA synthesis but it requires further inhibitor study or presence of CSF. Thus higher concentration of amoebae trophozoites caused a reduced Kupffer cell uptake capacity during CSF production.

Fig. 20. DNA synthesis in liver cells in culture medium is shown in presence of macrophage growth factor measured as cpm/flask and cpm/cover slip during 4-10 days. Notice the nitroimidazole induced inhibition of DNA synthesis in amebic treated cells.Source: Sharma, R.(1990) Ph.D dissertation.

#### **8. Inhibition of liver lysosomal enzymes in protein metaboilism**

232 Enzyme Inhibition and Bioapplications

 Several types of growth factors have been reported as regulatory substances participating in liver regeneration process. Stimulation of DNA synthesis in Kupffer cells by amoeba trophozoites is shown here as response induced by colony stimulating factor. Since measured increase of radio label uptake by increasing amoeba trophozoite concentration in medium reflected an increase of cell number entering into S phase rather than higher rate of [3H] TdR incorporation seen in all the cells, it was possible that different Kupffer cells normal or pretreated with amoeba (from these different liver cell populations) may vary in susceptibility to colony stimulating factor (CSF). High concentrations of amoeba caused a reduction of thymidine uptake and remarkably reduction was measured on whole cover slip scintillation counts. It reflected lower incorporation of [3H] TdR into each DNA synthesizing cells. This phenomenon may be due to reduced capacity of Kupffer cells uptake as a result of DNA synthesis but it requires further inhibitor study or presence of CSF. Thus higher concentration of amoebae trophozoites caused a reduced

Fig. 20. DNA synthesis in liver cells in culture medium is shown in presence of macrophage growth factor measured as cpm/flask and cpm/cover slip during 4-10 days. Notice the nitroimidazole induced inhibition of DNA synthesis in amebic treated cells.Source: Sharma,

and TTP pools[Cheung et al.1970].

R.(1990) Ph.D dissertation.

Kupffer cell uptake capacity during CSF production.

ribonucleotide reductase. Increase in CTP build up dCTP, purine deoxyribonucleotides

Isolated lysosomal preparations from L[1-14C] leucine (25 Ci per 100 gm wt) intraperitonially injected human Kupffer cell cultures exhibited percent degradation at pH 3.0, 4.0,5.0, 6.0,7.0, 8.0 and 9.0 at temperatures 10 °C, 20 °C, 30 °C, 40 °C, 50 °C and 60 °C, in presence of amoebic trophozoits after 15 minutes(short lived) and 15 hours(long-lived) addition as shown in Figures 21. Major players in this liver regeneration process are ribosomes (sitting on endoplasmic reticulum), GTP (ribotide pool) and coenzymes for ribotide polymerase enzymes (a balance in thymidine kinase and thymidylate synthetase). Other enzyme inhibition is LDH-M type gets converted to LDH-H type or lactate pyruvate (accelerated glycolysis). In simultaneous other set of experiment, Kupffer cell lysosomes in presence of leupeptin, pepstatin inhibitors and nitroimidazole in 100 g, 100 g and 50 mM per ml concentration respectively showed aryl sulphatase enzyme activity changes (see Figures 21,22) in the presence of 0.3 M sucrose at 37 °C for 60 minutes at pH 5.5 in incubation mixture of Kupffer cell lysosomes and additives. Proteolysis rate and degradation of short and long lived proteins in isolated lysosomes from the Kupffer cell

Fig. 21. Figure shows the effect of of radiolabeled [14C]-Leucine in Kupffer cultures represented by aryl sulphatase enzyme activity in normal and ALA recruited cells at different pH and temperatures. Notice the low enzyme activity in normal cells while maximum leucine degradation was at pH 6 and 40 °C.

Mechanisms of Hepatocellular Dysfunction

pH 5.0 at temperature range 30 C-40 C as shown in Figure 23.

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 235

Infected Kupffer cell lysosomes exhibited more pronounced elevated lysosomal degradation at

Fig. 23. Figure shows a comparison of % proteolysis inhibition and % protein degradation in presence of nitroimidazole, pepstatin and leupeptin. Proteolysis is represented in terms of arylsulphatase enzyme activity. Notice the size of peptide length makes difference shown by \* for long duration and short duration of reaction in each additive. Control cells showed slow % protein degradation and low enzyme concentration. Source. Sharma R 1990,Ph.D dissertation.

During metabolic integrity loss and resultant hypoxia state, other factors in liver cell signal the alarm to produce nitric oxide, release of cytokines (IFN-α with IL-1β or TNF-α). These changes also trigger the sequence of Kupffer cell stimulation and lysosomal action simultaneously. As a result, lysosomal enzymes, growth factor, plasminogen stimulating factor consistently participate or stimulate the Kupffer cell suicidal phagocytic action (hepatic necrosis) by nitroimidazole analogs and previously reviewed [Michalopoulos, 1990; Thurman, 2000]. In drug induced hepatitis, liver lysosomal enzymes have been reported elevated as stimulation of hepatocellular defense apart from initiating respiratory burst and chemotaxis in liver cells

**9. Liver regeneration and role of lysosomal enzymes** 

Fig. 22. Figure shows the effect of radiolabeled [14C]-Leucine in Kupffer cultures represented by aryl sulphatase enzyme activity in normal and ALA recruited cells. Notice the threshold of leucine turnover at 40 IU in terms of enzyme activity. Control cells showed slow % protein degradation and low enzyme concentration. Source. Sharma 1990. Ph.D dissertation.

cultures added with trophozoits, nitroimidazole, exhibited increased protein degradation. Lysosome as proteolytic component was shown to contribute for protein degradation as earlier various reports indicated protein size dependence [Neff et al.1990]. Proteolytic rate in isolated lysosomes increased several fold with decreased intralysosomal pH. However, leakage of hydrolytic enzymes (elevated orotate phosphorylase/decarboxylase) may cause degradation of extra lysosomal orotate substrates. Thus incubation time should be short. Degradation of short and long-lived proteins in isolated lysosomes demonstrates that after 15 minutes of [14C] leucine labeling, these lysosomes were active in degradation of short time labeled proteins. Since transit time for lysosomal enzymes from endoplasmic reticulum to lysosomes was around 2-3 hours [Zhu et al. 1994]. So after, 15 minutes some other proteins may be thought to enter lysosomes. The degradation rate in isolated lysosomes was sometimes faster for short time labeling because of short lived proteins as cytosolic or secretory in nature. On the other hand, membrane proteins had half lives in range of few days. So these long labeled proteins were degraded at different rates within lysosomes. Moreover short lived proteins were reported more susceptible to digestion by lysosomal proteases. The effect of proteolytic inhibitors on protein breakdown in isolated lysosomes for distinguishing lysosomal from extra lysosomal protein degradation was dependent upon cell type, culture medium, labeling schedule and inhibitors used. Inhibitors of lysosomal enzymes impede their supposed target of action viz. the lysosomes. But complete inhibition of proteolysis could not be possible due to lysosomal and nonlysosomal proteolysis. Pepstatin inhibited only selective aspartic proteinase cathepsin D, leupeptin inhibited thiol proteinase. Similarly nitroimidazole either intralysosomal pH and restored proteinases. Moreover, cathepsin D inhibition by pepstatin, cathepsin B, H and L inhibition by leupeptin are reported commonly [Katunuma, 2011]. Thus lysosomes were active in degradation of both short and long lived proteins during basal state.

Fig. 22. Figure shows the effect of radiolabeled [14C]-Leucine in Kupffer cultures represented by aryl sulphatase enzyme activity in normal and ALA recruited cells. Notice the threshold of leucine turnover at 40 IU in terms of enzyme activity. Control cells showed slow % protein degradation and low enzyme concentration. Source. Sharma 1990. Ph.D dissertation.

cultures added with trophozoits, nitroimidazole, exhibited increased protein degradation. Lysosome as proteolytic component was shown to contribute for protein degradation as earlier various reports indicated protein size dependence [Neff et al.1990]. Proteolytic rate in isolated lysosomes increased several fold with decreased intralysosomal pH. However, leakage of hydrolytic enzymes (elevated orotate phosphorylase/decarboxylase) may cause degradation of extra lysosomal orotate substrates. Thus incubation time should be short. Degradation of short and long-lived proteins in isolated lysosomes demonstrates that after 15 minutes of [14C] leucine labeling, these lysosomes were active in degradation of short time labeled proteins. Since transit time for lysosomal enzymes from endoplasmic reticulum to lysosomes was around 2-3 hours [Zhu et al. 1994]. So after, 15 minutes some other proteins may be thought to enter lysosomes. The degradation rate in isolated lysosomes was sometimes faster for short time labeling because of short lived proteins as cytosolic or secretory in nature. On the other hand, membrane proteins had half lives in range of few days. So these long labeled proteins were degraded at different rates within lysosomes. Moreover short lived proteins were reported more susceptible to digestion by lysosomal proteases. The effect of proteolytic inhibitors on protein breakdown in isolated lysosomes for distinguishing lysosomal from extra lysosomal protein degradation was dependent upon cell type, culture medium, labeling schedule and inhibitors used. Inhibitors of lysosomal enzymes impede their supposed target of action viz. the lysosomes. But complete inhibition of proteolysis could not be possible due to lysosomal and nonlysosomal proteolysis. Pepstatin inhibited only selective aspartic proteinase cathepsin D, leupeptin inhibited thiol proteinase. Similarly nitroimidazole either intralysosomal pH and restored proteinases. Moreover, cathepsin D inhibition by pepstatin, cathepsin B, H and L inhibition by leupeptin are reported commonly [Katunuma, 2011]. Thus lysosomes were active in degradation of both short and long lived proteins during basal state. Infected Kupffer cell lysosomes exhibited more pronounced elevated lysosomal degradation at pH 5.0 at temperature range 30 C-40 C as shown in Figure 23.

Fig. 23. Figure shows a comparison of % proteolysis inhibition and % protein degradation in presence of nitroimidazole, pepstatin and leupeptin. Proteolysis is represented in terms of arylsulphatase enzyme activity. Notice the size of peptide length makes difference shown by \* for long duration and short duration of reaction in each additive. Control cells showed slow % protein degradation and low enzyme concentration. Source. Sharma R 1990,Ph.D dissertation.

#### **9. Liver regeneration and role of lysosomal enzymes**

During metabolic integrity loss and resultant hypoxia state, other factors in liver cell signal the alarm to produce nitric oxide, release of cytokines (IFN-α with IL-1β or TNF-α). These changes also trigger the sequence of Kupffer cell stimulation and lysosomal action simultaneously. As a result, lysosomal enzymes, growth factor, plasminogen stimulating factor consistently participate or stimulate the Kupffer cell suicidal phagocytic action (hepatic necrosis) by nitroimidazole analogs and previously reviewed [Michalopoulos, 1990; Thurman, 2000]. In drug induced hepatitis, liver lysosomal enzymes have been reported elevated as stimulation of hepatocellular defense apart from initiating respiratory burst and chemotaxis in liver cells

Mechanisms of Hepatocellular Dysfunction

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 237

homeostasis during the entire regenerative process [Michalopoulos et al. 2007]. Recently, role of tissue engineering concept showed several advantages in liver transplantation. However, several factors are significant in regeneration: matrices design, cell mass up to whole-organ equivalents, optimized microarchitecture, celladhesion peptides, growth factors and extracellular matrix molecules to the polymeric scaffold, hepatic enzymes in microenvironment [Mooney et al. 1992]. The use of enzyme characterization is very helpful in hepatocyte culture to test biocompatibility required for the retention of tissue-specific gene expression [Kim et al. 2000; Voytik-Harbin et al. 1997]. Liver cell transplantation into polymeric matrices (cell-matrix-constructs) is emerging as routine in hospitals to meet absolute requirement of the transplanted hepatocytes for hepatotrophic factors that liver constantly receives through its portal circulation [Starzl et al. 1973]. Apart from cellular therapies, two experimental approaches are worth mentioning for successful clinical translation. The first "cell sheet" technology developed by Sasagawa et al.,2007. Ott et al. reported perfusion

decellularization to generate whole organ scaffolds [Ott et al. 2008].

 In previous report, investigators developed a similar perfusion decellularization method for the liver [Baptista et al.2009] for organ bioengineering. These bioscaffolds preserve their tissue microarchitecture and an intact vascular network that can be readily used as a route for recellularization (intact enzymes of regulatory metabolic reactions) by perfusion of culture medium containing different liver cell populations. Significant enzyme inhibition assays are used as biomarkers of liver cell viability. In following example, a bioengineered liver (the regenerated hepatic tissue) is shown clearly visible by the naked eye after 7 days in the bioreactor (Fig. 24A). Regenerated tissue was highly cellular (Fig. 24B) and displays biliary duct structures positive for cytokeratin 19 (Fig. 24C), as well as clusters of hepatocytes expressing cytochrome P450 3A and albumin (Fig. 24D, E, respectively). Expression of endothelial cell nitric oxide synthase (eNOS) enzyme in seeded human endothelial umbilical vein cells (hUVECs) were also observed coating and spreading from vascular structures (Fig. 24F). In other approach, Uygun et al. 2010 decellularized rat livers and repopulated them with rat primary hepatocytes, showing promising hepatic function and the ability of heterotopically transplanting these bioengineered livers into animals for up to eight hours [Uygun et al. 2010]. Bioengineered livers displayed some functions of a native human liver (albumin and urea secretion, CYP450 enzyme expression, etc) and also exhibited an endothelial vascular network that prevented platelet adhesion and aggregation, critical for blood vessel patency after transplantation. Hence, this liver regeneration technology with proper active enzyme functions has the potential to translate in the future into the bioengineering of human size

in regenerating rat liver and various tumors. Apart from enzyme inhibition, many growth factors and cytokines such as most notably hepatocyte growth factor, epidermal growth factor, transforming growth factor-α, interleukin-6, tumor necrosis factor-α, insulin, and norepinephrine, appear to play important roles in this enzyme inhibition and liver response. Many reviews attempted to integrate in last three decades and looked toward clues to the nature of triggering mechanisms in liver and cellular response. [Michalopoulos et al. 1997]. The major events are signaling cascades involving growth factors, cytokines, matrix remodeling, and several feedbacks of stimulation and inhibition of growth related signals. Liver manages to restore any lost mass and adjust its size to that of the organism, while at the same time providing full support for body

[Vician et al.2009]. Our study showed the associated electron microscopic observations that Kupffer cells accumulate around the site of hypoxia and exhibited hyperplasia condition showing degenerated nucleus, enlarged lysosomal vesicles [Sharma, 2009]. The enlarged lysosomal vesicles were further correlated by elevated lysosomal enzyme levels in serum. In liver biopsies, these lysosomal enzymes were significantly enhanced. Acid phosphatase, leucine aminopeptidase enzymes catalyzing phosphorylation and amino-peptidization, seem to be secreted more and suggestive of active protein degradation. β-glucuronidase and aryl sulphatase high enzyme secretion was suggestive of continuous breakdown of aryl substituted and β-glucuronidation reactions during cytolysis and proteolysis [Knook et al. 1981]. No previous data is available on liver cell enzymes estimated in liver cells isolated from liver biopsy of nitroimidazole treated livers in hypoxia. Our observations of enzyme inhibition in hypoxic liver cells are very valuable to design a nitroimidazole based radiosensitizer or drug for parasites, myocardial ischemia, tuberculosis, neurotoxicosis etc. Liver cell enzymes may extend the better biochemical explanation of complex hypoxia state at molecular level. However, several scattered studies on serum choline esterase, alkaline phosphatase, glucose-6P-dehydrogenase, ornithine carbamoyl transferase, cyclooxygenase-2, lactate dehydrogenase enzymes have been reported significant in hepatic hypoxia or hepatic damage evaluation with addition of new members of enzymes [Carlson et al. 1976; Koppele et al. 1991]. Enzyme inhibition in liver cells is significant information to design new liver cell metabolic drug stimulators in detoxification, evaluation of hypoxia imaging probes, and cultivation of type of monoaxenic liver cell cultures (hepatocytes and Kupffer cells) with specific enzyme profile.

There appear two main reasons of enzyme level hepatic cell recovery by nitroimidazole. First, altered enzyme changes were recovered by nitroimidazole therapy as drug inhibits the enzymes participating in energy status in hepatocytes and reduces Kupffer cell macrophagal activity with reduced hepatic enzyme secretion to bring back enzyme levels to normal. Second, regenerating hepatocytes may also regulate the normal cell recovery process and initialize the signaling Kupffer cells to store enough lysosomal enzymes.

#### **10. Role of enzyme inhibition in liver transplantation and tissue engineering**

Liver regeneration after the enzyme inhibition or loss of hepatic tissue is a fundamental parameter of liver response to injury. Enzyme inhibition phenomenon is recognized as specific external stimuli involving sequential changes in gene expression, growth factor production, and morphologic structure. Author sums up an account on different enzymes in following description:

 Angiotensin Converting enzyme inhibition by lisinopril degrades bradykinin and subsequently induces liver regeneration in rats. During this enzyme inhibition process (liver regeneration) induces 3.4 S DNA polymerase activity in both cytoplasmic fraction and nuclear fractions as constant while 6 S to 8 S DNA polymerase activities in the cytoplasmic fraction increased 6- to 7-fold [Ramalho et al.2001; Chang et al.1972]. The activity of histidine decarboxylase was markedly increased in regenerating liver tissues put. Ornithine decarboxylase is an enzyme catalyze in polyamine synthesis. Inhibition of ornithine decarboxylase, histidine decarboxylase, and other amino acid decarboxylases, was reported in regenerating rat liver and several rat tumors.[Russell, et al.1969]. Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity

[Vician et al.2009]. Our study showed the associated electron microscopic observations that Kupffer cells accumulate around the site of hypoxia and exhibited hyperplasia condition showing degenerated nucleus, enlarged lysosomal vesicles [Sharma, 2009]. The enlarged lysosomal vesicles were further correlated by elevated lysosomal enzyme levels in serum. In liver biopsies, these lysosomal enzymes were significantly enhanced. Acid phosphatase, leucine aminopeptidase enzymes catalyzing phosphorylation and amino-peptidization, seem to be secreted more and suggestive of active protein degradation. β-glucuronidase and aryl sulphatase high enzyme secretion was suggestive of continuous breakdown of aryl substituted and β-glucuronidation reactions during cytolysis and proteolysis [Knook et al. 1981]. No previous data is available on liver cell enzymes estimated in liver cells isolated from liver biopsy of nitroimidazole treated livers in hypoxia. Our observations of enzyme inhibition in hypoxic liver cells are very valuable to design a nitroimidazole based radiosensitizer or drug for parasites, myocardial ischemia, tuberculosis, neurotoxicosis etc. Liver cell enzymes may extend the better biochemical explanation of complex hypoxia state at molecular level. However, several scattered studies on serum choline esterase, alkaline phosphatase, glucose-6P-dehydrogenase, ornithine carbamoyl transferase, cyclooxygenase-2, lactate dehydrogenase enzymes have been reported significant in hepatic hypoxia or hepatic damage evaluation with addition of new members of enzymes [Carlson et al. 1976; Koppele et al. 1991]. Enzyme inhibition in liver cells is significant information to design new liver cell metabolic drug stimulators in detoxification, evaluation of hypoxia imaging probes, and cultivation of type of monoaxenic liver cell cultures (hepatocytes and Kupffer cells) with specific enzyme profile.

There appear two main reasons of enzyme level hepatic cell recovery by nitroimidazole. First, altered enzyme changes were recovered by nitroimidazole therapy as drug inhibits the enzymes participating in energy status in hepatocytes and reduces Kupffer cell macrophagal activity with reduced hepatic enzyme secretion to bring back enzyme levels to normal. Second, regenerating hepatocytes may also regulate the normal cell recovery process and

**10. Role of enzyme inhibition in liver transplantation and tissue engineering**  Liver regeneration after the enzyme inhibition or loss of hepatic tissue is a fundamental parameter of liver response to injury. Enzyme inhibition phenomenon is recognized as specific external stimuli involving sequential changes in gene expression, growth factor production, and morphologic structure. Author sums up an account on different enzymes in

 Angiotensin Converting enzyme inhibition by lisinopril degrades bradykinin and subsequently induces liver regeneration in rats. During this enzyme inhibition process (liver regeneration) induces 3.4 S DNA polymerase activity in both cytoplasmic fraction and nuclear fractions as constant while 6 S to 8 S DNA polymerase activities in the cytoplasmic fraction increased 6- to 7-fold [Ramalho et al.2001; Chang et al.1972]. The activity of histidine decarboxylase was markedly increased in regenerating liver tissues put. Ornithine decarboxylase is an enzyme catalyze in polyamine synthesis. Inhibition of ornithine decarboxylase, histidine decarboxylase, and other amino acid decarboxylases, was reported in regenerating rat liver and several rat tumors.[Russell, et al.1969]. Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity

initialize the signaling Kupffer cells to store enough lysosomal enzymes.

following description:

in regenerating rat liver and various tumors. Apart from enzyme inhibition, many growth factors and cytokines such as most notably hepatocyte growth factor, epidermal growth factor, transforming growth factor-α, interleukin-6, tumor necrosis factor-α, insulin, and norepinephrine, appear to play important roles in this enzyme inhibition and liver response. Many reviews attempted to integrate in last three decades and looked toward clues to the nature of triggering mechanisms in liver and cellular response. [Michalopoulos et al. 1997]. The major events are signaling cascades involving growth factors, cytokines, matrix remodeling, and several feedbacks of stimulation and inhibition of growth related signals. Liver manages to restore any lost mass and adjust its size to that of the organism, while at the same time providing full support for body homeostasis during the entire regenerative process [Michalopoulos et al. 2007].


Mechanisms of Hepatocellular Dysfunction

**12. Current developments and future prospects** 

cells with high degree of accurate enzyme estimation.

**13. Conclusion** 

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 239

Nitroimidazoles are rediscovered antiamoebic drugs having great potential of radiosensitizers in diagnosis and treatment of tumors, tuberculosis, myocardial infarction, and hypoxia. Nitroimidazole cytotoxicity was recently capitalized in hypoxic tumor detection and tumor killing. Nitroimidazoles act as smart enzyme inhibitors on one hand and play as enzyme modulators. Liver enzyme inhibition is unique for developing drugs in drug discovery at molecular level. The major challenge is to explore enzyme inhibitor that selectively and specifically catalyzes enzyme inhibition in only abnormal or diseased liver cells leaving safe normal cells in acute stage of liver damage such as hepatic coma, threatening hepatitis, hepatic cancer. The success of enzyme inhibition mainly depends on intracellular physiological conditions such as pH, temperature, concentration of active enzyme or inhibitor, coenzyme, co-factor, enzyme substrate structural-functional conformation state, type of biochemical reaction such as coupled, cascade or individual enzyme reaction and finally regulatory enzyme behavior to control metabolism. Currently, art is developing in liver transplantation using cultured cells to replace diseased or non-functional liver cells from diseased liver. Tissue engineering has opened the new opportunity of using conditioned monoaxenic and monoclonal liver cells in both experimental and pre-clinical or clinical therapeutics in both industries and academics. Recently, gene therapy in liver was a great success and it will be a boom in medical science of detoxification or drug metabolism if and when clinically accepted. Battery of enzymes in hepatocytes and rate limiting enzyme regulatory behavior in presence of additives or inhibitors will certainly enhance better understanding of disease development and new drug pharmaceutical action in drug discovery. Still today, liver cells remain as major research tool to discover drugs in therapeutics and search of transplantable disease resistant liver cells because of liver as sole organ responsible of bile formation, detoxification and drug metabolism. Cirrhosis is a serious health hazard that solely requires functional liver. In future, it remains to see if molecular biology will assist in silencing genes ON or OFF responsible of enzyme stimulation or inhibition in liver as method of disease therapy and cure. The major discoveries will be discovery and better understanding of new growth factors, colony stimulating factors affecting enzyme reactions in hepatocytes and Kupffer cells as major engineered cancer resistant or cirrhosis resistant liver cells. In future, chip technology or silica or polymer coated enzyme estimation techniques may be more reliable in small sample collections or in liver

The 2'nitroimidazole is both antiaparasitic drug and radiosensitizer hypoxia marker in tumor therapy. It shows hepatocellular cytotoxicity. The 'hepatocellular hypoxia criterion' distinguishes the nitroimidazole induced hepatocellular oxygen depletion and associated organelle changes by enzyme levels in serum, biopsy samples and cell organelles by electron microscopy. Initially, glucose regulation leads to metabolic integrity loss. Later, it may be oxygen insufficiency and slow cell death. The 2'-nitroimidazole is drug of choice in hepatic infections and its derivatives are emerging choice of tumor treatment. Its action may be evaluated rapidly by enzyme biochemical estimations without time consuming drug monitoring and therapeutic assay techniques. Entamoeba histolytica, an intestinal

livers to offer readily available liver for drug discovery applications and for liver transplantation, overcoming organ shortage.

Fig. 24. Human bioengineered livers are highly cellular and display some of the functions observed in native hepatic tissue. (A) Macroscopic appearance of a seeded liver bioscaffold 7 days after seeding with primary human fetal liver and endothelial cells. (B) H&E showing broad recellularization of the bioscaffold with the formation of biliary ductal structures. (C) Immunofluorescence staining for cytokeratin 19 (red) showing a biliary duct formed within the bioscaffold with a visible lumen. (D) Immunofluorescence staining of cytochrome P450 3A (orange) and (E) albumin (purple) showing groups of hepatocytic lineage cells expressing these more mature hepatic markers. (F) Immunofluorescence staining of eNOS (purple) showing hUVECs coating and spreading from vascular structures (arrows). All nuclei stained with YO-PRO1 (green) or DAPI (blue). Reproduced with permission from reference Baptista et al. 2010.

#### **11. Challenges, limitations in enzyme inhibition**

There are two major challenges. First challenge was to get enough biopsy to estimate several enzymes. Second challenge was to choose significant enzymes as representative of hepatocellular criteria. The main limitation was that the 'hepatocellular hypoxia criteria' was used in small number of human subjects. The biopsy samples for electron microscopy observations needs more thoroughly controlled experiments. Other limitation was that the enzyme estimations in serum and biopsy samples may not be perfect representative samples and it needs to establish the measurable and actual enzyme activities in cells. The most crucial issue is that hypoxia is a combination of sequence of several metabolic and subphysiological reactions in cells. Moreover, other inflammatory cytokines, apoptosis, nitric oxide production and phagocytosis are associated changes during hypoxia. It further needs tracer technique to track the details of hypoxia in cell.

#### **12. Current developments and future prospects**

238 Enzyme Inhibition and Bioapplications

Fig. 24. Human bioengineered livers are highly cellular and display some of the functions observed in native hepatic tissue. (A) Macroscopic appearance of a seeded liver bioscaffold 7 days after seeding with primary human fetal liver and endothelial cells. (B) H&E showing broad recellularization of the bioscaffold with the formation of biliary ductal structures. (C) Immunofluorescence staining for cytokeratin 19 (red) showing a biliary duct formed within the bioscaffold with a visible lumen. (D) Immunofluorescence staining of cytochrome P450

expressing these more mature hepatic markers. (F) Immunofluorescence staining of eNOS (purple) showing hUVECs coating and spreading from vascular structures (arrows). All nuclei stained with YO-PRO1 (green) or DAPI (blue). Reproduced with permission from

There are two major challenges. First challenge was to get enough biopsy to estimate several enzymes. Second challenge was to choose significant enzymes as representative of hepatocellular criteria. The main limitation was that the 'hepatocellular hypoxia criteria' was used in small number of human subjects. The biopsy samples for electron microscopy observations needs more thoroughly controlled experiments. Other limitation was that the enzyme estimations in serum and biopsy samples may not be perfect representative samples and it needs to establish the measurable and actual enzyme activities in cells. The most crucial issue is that hypoxia is a combination of sequence of several metabolic and subphysiological reactions in cells. Moreover, other inflammatory cytokines, apoptosis, nitric oxide production and phagocytosis are associated changes during hypoxia. It further

3A (orange) and (E) albumin (purple) showing groups of hepatocytic lineage cells

reference Baptista et al. 2010.

**11. Challenges, limitations in enzyme inhibition** 

needs tracer technique to track the details of hypoxia in cell.

transplantation, overcoming organ shortage.

livers to offer readily available liver for drug discovery applications and for liver

Nitroimidazoles are rediscovered antiamoebic drugs having great potential of radiosensitizers in diagnosis and treatment of tumors, tuberculosis, myocardial infarction, and hypoxia. Nitroimidazole cytotoxicity was recently capitalized in hypoxic tumor detection and tumor killing. Nitroimidazoles act as smart enzyme inhibitors on one hand and play as enzyme modulators. Liver enzyme inhibition is unique for developing drugs in drug discovery at molecular level. The major challenge is to explore enzyme inhibitor that selectively and specifically catalyzes enzyme inhibition in only abnormal or diseased liver cells leaving safe normal cells in acute stage of liver damage such as hepatic coma, threatening hepatitis, hepatic cancer. The success of enzyme inhibition mainly depends on intracellular physiological conditions such as pH, temperature, concentration of active enzyme or inhibitor, coenzyme, co-factor, enzyme substrate structural-functional conformation state, type of biochemical reaction such as coupled, cascade or individual enzyme reaction and finally regulatory enzyme behavior to control metabolism. Currently, art is developing in liver transplantation using cultured cells to replace diseased or non-functional liver cells from diseased liver. Tissue engineering has opened the new opportunity of using conditioned monoaxenic and monoclonal liver cells in both experimental and pre-clinical or clinical therapeutics in both industries and academics. Recently, gene therapy in liver was a great success and it will be a boom in medical science of detoxification or drug metabolism if and when clinically accepted. Battery of enzymes in hepatocytes and rate limiting enzyme regulatory behavior in presence of additives or inhibitors will certainly enhance better understanding of disease development and new drug pharmaceutical action in drug discovery. Still today, liver cells remain as major research tool to discover drugs in therapeutics and search of transplantable disease resistant liver cells because of liver as sole organ responsible of bile formation, detoxification and drug metabolism. Cirrhosis is a serious health hazard that solely requires functional liver. In future, it remains to see if molecular biology will assist in silencing genes ON or OFF responsible of enzyme stimulation or inhibition in liver as method of disease therapy and cure. The major discoveries will be discovery and better understanding of new growth factors, colony stimulating factors affecting enzyme reactions in hepatocytes and Kupffer cells as major engineered cancer resistant or cirrhosis resistant liver cells. In future, chip technology or silica or polymer coated enzyme estimation techniques may be more reliable in small sample collections or in liver cells with high degree of accurate enzyme estimation.

#### **13. Conclusion**

The 2'nitroimidazole is both antiaparasitic drug and radiosensitizer hypoxia marker in tumor therapy. It shows hepatocellular cytotoxicity. The 'hepatocellular hypoxia criterion' distinguishes the nitroimidazole induced hepatocellular oxygen depletion and associated organelle changes by enzyme levels in serum, biopsy samples and cell organelles by electron microscopy. Initially, glucose regulation leads to metabolic integrity loss. Later, it may be oxygen insufficiency and slow cell death. The 2'-nitroimidazole is drug of choice in hepatic infections and its derivatives are emerging choice of tumor treatment. Its action may be evaluated rapidly by enzyme biochemical estimations without time consuming drug monitoring and therapeutic assay techniques. Entamoeba histolytica, an intestinal

Mechanisms of Hepatocellular Dysfunction

osmium tetra oxide staining [46].

**Reagents Used** 

al. 1994; Kirn et al.1982].

spectrophotometer Cecil Inc. England [Sharma, 2009].

**Art of liver cell isolation and enzyme characterization** 

enzyme was purchased from Boerhinger Meinheim GMbH.

needle [45].

and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration 241

The ELISA was done by method of Prakash et al.[44]. Liver biopsy was taken by Manghini

Biochemical assays: In biopsy sonipreps and serum samples the hepatocellular enzymes were estimated as described elsewhere [Sharma, 2009]. All substrates of enzymes were obtained from Sigma Chemical Company, St Louis, USA. The estimations were done on UV

Electron microscopy: The biopsy specimens at stored in glutaraldehyde at -20°C. For fixing samples were fixed in dental wax and emersed in pool of 0.1 M phosphate buffer containing 0.1 M Ca++. The samples were cut 1 mm cube by Gillette blade. The cubes were fixed for 2 hours in 0.2 M phosphate buffer in 4 changes at 30 minutes interval in capped vials. The vials were washed for 2 hours in 0.2 M phosphate buffer 4 times at 30 minutes interval in capped vials. The vials were post fixed in buffered 1% osmium tetroxide for 1-2 hours at 4°C, dehydrated in cold 10% ethanol for about 5 minutes followed by dehydrations in cold 50%, 70%, 80%, 95% ethanol for about 5 minutes each and tissues were kept at room temperature. Further tissues were dehydrated at 100% ethanol for 15 minutes. Electron Microscope PA model was used for screening hepatic cell damage by epoxy resin blocks and

The procedures of liver biopsy excision and preparation of liver cells were followed as per

The collagenase enzyme was purchased from Sigma-Aldrich, St Louis, MO and pronase

Isolation and preparation of Kupffer cells: The liver biopsy samples (6-10 grams) were digested with collagenase enzyme (Sigma-Aldrich, St Louis, MO) 15 mg/ml for 10 minutes and bottom pallet was used for hepatocyte experiments. Other remained supernatant part of digested liver was further digested with 2 mg/ml pronase enzyme (Boerhinger Meinheim GMbH) in 0.2 % Kreb's Hansleit Buffer pH 7.4 containing 5 mm glucose and 3 mM calcium chloride after separation of hepatocytes. The said suspension was incubated for 45 minutes at 37C stirring 250 revolutions per minute. After incubation, the cell suspension was filtered through nylon sieve 79 × 79 microns and filtrate was centrifuged and washed twice with KHB containing 5 mM glucose and 3 mM calcium chloride pH 7.4. After centrifugation and final washing the filtrate was centrifuged at 350 × g in ice cold centrifuge. The settled pallet at bottom was washed with TC 199 medium. The number of Kupffer cells was counted on

Kupffer cell fractionation: The isolated Kupffer cells were divided in two parts. One part was used for Kupffer cellular enzymes and other part was used for in vitro experiments. For enzyme estimations Kupffer cells from freshly harvested preparations were sonicated at zero degree temperature in said KHB buffer in soniprep. Later these were fractionated for different cellular fractions as described following by methods [Alabraba et al.2007; Heuff et

ethical committee of institute as described in following section [Sharma, 2009].

Naubourgh counting chamber and visibility was determined by trypan blue.

protozoan parasite can proliferate, lyse and destroy human liver cells. Within the last decade, new animal models of E. histolytica infection showed amoebic trophozoites cause intestinal disease and liver abscess. Recent studies have expanded our understanding of a large number of enzyme inhibition reactions in hepatic regeneration after liver abscess is treated by nitroimidazole as remarkable drug to kill parasites. Still interactions between E. histolytica and human intestine; and between E. histolytica and hepatocytes or Kupffer cells are not fully known. In present chapter, a scheme of enzyme changes in liver cells during development of amoebic liver abscess and enzyme inhibition is explored in detail as rehabilitative action of nitroimidazole to regenerate liver cells. Characteristics of enzyme regulatory behavior highlight the metabolic integrity loss, energy depletion, oxidative phosphorylation, fatty deposition, phagocytosis and slow cell death as main culprits in development of liver abscess and hypoxia. Nitroimidazole inhibits several rate limiting enzymes to control oxygen (hypoxia) levels and keeps balance of NADPH, ATP, fatty acids, amino acids and proteins. In hepatocytes, enzyme inhibition and regulatory behavior of apoptosis (caspases, cysteine protease); carbohydrate metabolizing (glucokinase, pyruvate kinase, phosphofructokinase); energy metabolism (NADPH cytochrome P450 Reductase, Phosphodiesterase); DNA synthesis (Thymidylate Synthetase) describe the process of liver regeneration. In Kupffer cells, enzyme inhibition of drug metabolizing enzymes (oxidoreductases, proteases, esterases, phosphatases) predicts the liver regeneration and recovery from liver abscess. In last decade, several studies reported the use of enzyme inhibition mechanisms in enhancing liver regeneration and recovery as tool of tissue engineering and liver transplantation. In conclusion, enzyme inhibition mechanisms in liver damage and regeneration explain the better information of liver transplantation to maintain complex liver cell environment in balance. Enzyme inhibition mechanisms throw a light on characterization of enzyme regulation in engineered liver cells, amoebic liver abscess development and desired design of targeted nitroimidazole analogues to treat liver abscess.

#### **14. Appendix 1: A protocol of hepatocellular dysfunction**

Patients: In 10 control subjects, stool, ELISA, serum and liver biopsies were collected1. Only proved 10 subjects free from any hepatic disease were examined for enzyme estimations in isolated liver cells from biopsy specimens. Other ten subjects were treated with nitroimidazole (Tiniba and Zil from Hindustan Lever Ltd, Bombay) therapy 2 x 5 gm one-time dose thrice at 2 days interval. At the end of dose, liver biopsy and serum collection was done.

 All the 10 control subjects had no clinical findings of any hepatic disease and showed stool -ve, ELISA -ve. All 10 showed normal liver and no abscess confirmed by ultrasound and liver scan. The subjects were selected of 35 ± 15 years (mean age ± sd), average monthly income 1750 ± 100 INR, no hepatomegaly, no diarrhea, fever and pain. The subjects after nitroimidazole treatment showed induced hepatomegaly insignificant less than 2 cm by liver scan. The subjects with amoebiasis and amoebic liver abscess are not included and not shown here.

<sup>1</sup>The patients were studied for ongoing other research study on amoebic hepatitis vs amoebic liver abscess by Hepatocellular Dysfunction Criteria at Liver Unit, AIIMS. The results of hepatic damage are shown in Figure 3.

The ELISA was done by method of Prakash et al.[44]. Liver biopsy was taken by Manghini needle [45].

Biochemical assays: In biopsy sonipreps and serum samples the hepatocellular enzymes were estimated as described elsewhere [Sharma, 2009]. All substrates of enzymes were obtained from Sigma Chemical Company, St Louis, USA. The estimations were done on UV spectrophotometer Cecil Inc. England [Sharma, 2009].

Electron microscopy: The biopsy specimens at stored in glutaraldehyde at -20°C. For fixing samples were fixed in dental wax and emersed in pool of 0.1 M phosphate buffer containing 0.1 M Ca++. The samples were cut 1 mm cube by Gillette blade. The cubes were fixed for 2 hours in 0.2 M phosphate buffer in 4 changes at 30 minutes interval in capped vials. The vials were washed for 2 hours in 0.2 M phosphate buffer 4 times at 30 minutes interval in capped vials. The vials were post fixed in buffered 1% osmium tetroxide for 1-2 hours at 4°C, dehydrated in cold 10% ethanol for about 5 minutes followed by dehydrations in cold 50%, 70%, 80%, 95% ethanol for about 5 minutes each and tissues were kept at room temperature. Further tissues were dehydrated at 100% ethanol for 15 minutes. Electron Microscope PA model was used for screening hepatic cell damage by epoxy resin blocks and osmium tetra oxide staining [46].

#### **Art of liver cell isolation and enzyme characterization**

The procedures of liver biopsy excision and preparation of liver cells were followed as per ethical committee of institute as described in following section [Sharma, 2009].

#### **Reagents Used**

240 Enzyme Inhibition and Bioapplications

protozoan parasite can proliferate, lyse and destroy human liver cells. Within the last decade, new animal models of E. histolytica infection showed amoebic trophozoites cause intestinal disease and liver abscess. Recent studies have expanded our understanding of a large number of enzyme inhibition reactions in hepatic regeneration after liver abscess is treated by nitroimidazole as remarkable drug to kill parasites. Still interactions between E. histolytica and human intestine; and between E. histolytica and hepatocytes or Kupffer cells are not fully known. In present chapter, a scheme of enzyme changes in liver cells during development of amoebic liver abscess and enzyme inhibition is explored in detail as rehabilitative action of nitroimidazole to regenerate liver cells. Characteristics of enzyme regulatory behavior highlight the metabolic integrity loss, energy depletion, oxidative phosphorylation, fatty deposition, phagocytosis and slow cell death as main culprits in development of liver abscess and hypoxia. Nitroimidazole inhibits several rate limiting enzymes to control oxygen (hypoxia) levels and keeps balance of NADPH, ATP, fatty acids, amino acids and proteins. In hepatocytes, enzyme inhibition and regulatory behavior of apoptosis (caspases, cysteine protease); carbohydrate metabolizing (glucokinase, pyruvate kinase, phosphofructokinase); energy metabolism (NADPH cytochrome P450 Reductase, Phosphodiesterase); DNA synthesis (Thymidylate Synthetase) describe the process of liver regeneration. In Kupffer cells, enzyme inhibition of drug metabolizing enzymes (oxidoreductases, proteases, esterases, phosphatases) predicts the liver regeneration and recovery from liver abscess. In last decade, several studies reported the use of enzyme inhibition mechanisms in enhancing liver regeneration and recovery as tool of tissue engineering and liver transplantation. In conclusion, enzyme inhibition mechanisms in liver damage and regeneration explain the better information of liver transplantation to maintain complex liver cell environment in balance. Enzyme inhibition mechanisms throw a light on characterization of enzyme regulation in engineered liver cells, amoebic liver abscess development and desired

design of targeted nitroimidazole analogues to treat liver abscess.

**14. Appendix 1: A protocol of hepatocellular dysfunction** 

not included and not shown here.

shown in Figure 3.

days interval. At the end of dose, liver biopsy and serum collection was done.

Patients: In 10 control subjects, stool, ELISA, serum and liver biopsies were collected1. Only proved 10 subjects free from any hepatic disease were examined for enzyme estimations in isolated liver cells from biopsy specimens. Other ten subjects were treated with nitroimidazole (Tiniba and Zil from Hindustan Lever Ltd, Bombay) therapy 2 x 5 gm one-time dose thrice at 2

 All the 10 control subjects had no clinical findings of any hepatic disease and showed stool -ve, ELISA -ve. All 10 showed normal liver and no abscess confirmed by ultrasound and liver scan. The subjects were selected of 35 ± 15 years (mean age ± sd), average monthly income 1750 ± 100 INR, no hepatomegaly, no diarrhea, fever and pain. The subjects after nitroimidazole treatment showed induced hepatomegaly insignificant less than 2 cm by liver scan. The subjects with amoebiasis and amoebic liver abscess are

1The patients were studied for ongoing other research study on amoebic hepatitis vs amoebic liver abscess by Hepatocellular Dysfunction Criteria at Liver Unit, AIIMS. The results of hepatic damage are The collagenase enzyme was purchased from Sigma-Aldrich, St Louis, MO and pronase enzyme was purchased from Boerhinger Meinheim GMbH.

Isolation and preparation of Kupffer cells: The liver biopsy samples (6-10 grams) were digested with collagenase enzyme (Sigma-Aldrich, St Louis, MO) 15 mg/ml for 10 minutes and bottom pallet was used for hepatocyte experiments. Other remained supernatant part of digested liver was further digested with 2 mg/ml pronase enzyme (Boerhinger Meinheim GMbH) in 0.2 % Kreb's Hansleit Buffer pH 7.4 containing 5 mm glucose and 3 mM calcium chloride after separation of hepatocytes. The said suspension was incubated for 45 minutes at 37C stirring 250 revolutions per minute. After incubation, the cell suspension was filtered through nylon sieve 79 × 79 microns and filtrate was centrifuged and washed twice with KHB containing 5 mM glucose and 3 mM calcium chloride pH 7.4. After centrifugation and final washing the filtrate was centrifuged at 350 × g in ice cold centrifuge. The settled pallet at bottom was washed with TC 199 medium. The number of Kupffer cells was counted on Naubourgh counting chamber and visibility was determined by trypan blue.

Kupffer cell fractionation: The isolated Kupffer cells were divided in two parts. One part was used for Kupffer cellular enzymes and other part was used for in vitro experiments. For enzyme estimations Kupffer cells from freshly harvested preparations were sonicated at zero degree temperature in said KHB buffer in soniprep. Later these were fractionated for different cellular fractions as described following by methods [Alabraba et al.2007; Heuff et al. 1994; Kirn et al.1982].

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#### **15. Acknowledgements**

The author acknowledges the assistance provided by Professor Rakesh K Tandon to provide assistant research officer job during this work with grant assistance from ICMR, New Delhi and Dr V.S.Singh for guidance and valuable Council of Scientific and Industrial Research fellowship and research grant from ICMR, New Delhi to do Ph.D.

#### **16. References**


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Kupffer cells in cultures: The Kupffer cell cultures were maintained for 48 hours after isolation from pronase perfused liver biopsy samples [Brouwer et al. 1988]. The in vitro cultured cells were screened for trypan blue exclusion [Froh et al.2003]. The Kupffer cells were used for their in vitro phagocytic action upon foreign particles which were sheep erythrocytes, latex beads and zymosan suspended in 10 mM phosphate buffer saline pH 7.4 at 4°C. After harvesting Kupffer cells were added in phosphate buffered saline pH 7.4 containing 4.6 x 106 adherent cells and three ml PBS-TC 199 medium in culture flasks containing Kupffer cells in mediukm with particle cell ratio 1:5 for erythrocytes, 1:40 for latex beads and 1:4 for trophozoits. The ratio for zymosan and cell was 20:1. They were kept up to 48 hours or more in carbogen atmosphere (95 % O2 and 5% CO2) in desiccator with

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**8** 

*Brazil* 

**Reversible Inhibition of Tyrosine Protein** 

Daniela Cosentino-Gomes and José Roberto Meyer-Fernandes

Protein phosphorylation/dephosphorylation is considered a widespread mechanism of protein regulation. The covalent addition of a phosphate group to tyrosine residues in cellular proteins may occur as an appropriate response to a series of morphological and biochemical processes, with particular importance in complex functions such as growth, proliferation, differentiation, adhesion and motility. Protein tyrosine phosphorylation is regulated in the cell by the opposing activities of two classes of enzymes: protein tyrosine kinases, which phosphorylate specific tyrosine residues in protein using ATP as the phosphate source, and protein tyrosine phosphatases, which hydrolyze the phosphotyrosines yielding the restored amino acid residue and inorganic phosphate as products (Kolmodin & Åqvist, 2001; Mayer, 2008). The number of active protein phosphatases with the ability to dephosphorylate phosphotyrosine are very similar to the number of active tyrosine phosphatases. Both types of enzymes also display comparable tissue distribution patterns (Alonso et al., 2004). Abnormalities in tyrosine phosphorylation play a role in the pathogenesis of numerous inherited and acquired human diseases from

Phosphorylation, acetylation, ubiquitinylation and glycosylation are among the most wellknown post-translational modifications. The concept of protein oxidation as a posttranslational modification has only gained acceptance more recently. Protein oxidation occurs as an outcome of a chemical attack by reactive oxygen species (ROS) or reactive nitrogen species (RNS) on susceptible amino acids such as tyrosine, tryptophan, histidine, lysine, methionine, and cysteine (Spickett et al., 2006). Indeed, it is important to note that signal transduction must occur in a coordinated manner in response to a previous stimuli. The key elements of a signaling response is reversibility and specificity (Tonks, 2005). In this way, an oxidative-dependent chain reaction may be short and employ low concentration of

In this chapter, we will discuss the following: the structural mechanism of action of ROS in the enzymatic activity of tyrosine phosphatases and how it interacts with their target molecules; the reversible regulation of this enzyme by oxidants and antioxidants; and the

**1. Introduction** 

cancer to immune deficiencies (Alonso et al., 2004)

oxidants to avoid irreversible damage to cellular components.

major consequences of this tightly controlled mechanism on cell signaling.

**Phosphatases by Redox Reactions** 

*Universidade Federal do Rio de Janeiro (UFRJ),* 

*Instituto de Bioquímica Médica (IbqM)* 


## **Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions**

Daniela Cosentino-Gomes and José Roberto Meyer-Fernandes *Universidade Federal do Rio de Janeiro (UFRJ), Instituto de Bioquímica Médica (IbqM) Brazil* 

#### **1. Introduction**

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determined by pulsed-field gel electrophoresis. Int J Radiat Biol Vol. 61, No 1, pp

Entamoeba histolytica cysteine proteinases with interleukin-1� converting enzyme (ICE) activity cause intestinal inflammation and tissue damage in amoebiasis. Mol. Protein phosphorylation/dephosphorylation is considered a widespread mechanism of protein regulation. The covalent addition of a phosphate group to tyrosine residues in cellular proteins may occur as an appropriate response to a series of morphological and biochemical processes, with particular importance in complex functions such as growth, proliferation, differentiation, adhesion and motility. Protein tyrosine phosphorylation is regulated in the cell by the opposing activities of two classes of enzymes: protein tyrosine kinases, which phosphorylate specific tyrosine residues in protein using ATP as the phosphate source, and protein tyrosine phosphatases, which hydrolyze the phosphotyrosines yielding the restored amino acid residue and inorganic phosphate as products (Kolmodin & Åqvist, 2001; Mayer, 2008). The number of active protein phosphatases with the ability to dephosphorylate phosphotyrosine are very similar to the number of active tyrosine phosphatases. Both types of enzymes also display comparable tissue distribution patterns (Alonso et al., 2004). Abnormalities in tyrosine phosphorylation play a role in the pathogenesis of numerous inherited and acquired human diseases from cancer to immune deficiencies (Alonso et al., 2004)

Phosphorylation, acetylation, ubiquitinylation and glycosylation are among the most wellknown post-translational modifications. The concept of protein oxidation as a posttranslational modification has only gained acceptance more recently. Protein oxidation occurs as an outcome of a chemical attack by reactive oxygen species (ROS) or reactive nitrogen species (RNS) on susceptible amino acids such as tyrosine, tryptophan, histidine, lysine, methionine, and cysteine (Spickett et al., 2006). Indeed, it is important to note that signal transduction must occur in a coordinated manner in response to a previous stimuli. The key elements of a signaling response is reversibility and specificity (Tonks, 2005). In this way, an oxidative-dependent chain reaction may be short and employ low concentration of oxidants to avoid irreversible damage to cellular components.

In this chapter, we will discuss the following: the structural mechanism of action of ROS in the enzymatic activity of tyrosine phosphatases and how it interacts with their target molecules; the reversible regulation of this enzyme by oxidants and antioxidants; and the major consequences of this tightly controlled mechanism on cell signaling.

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 255

The intracellular protein tyrosine phosphatases contain a single catalytic domain and various amino or carboxyl terminal extensions. They may contain SH2 (Src homology domain) domains that have targeting or regulatory functions, PEST (Pro-Glu-Ser-Thr domain) domains for proteolytic control, phosphorylation sites for protein/protein interactions, and enzymatic activity regulation. Cytosolic tyrosine phosphatases are characterized by regulatory sequences that flank the catalytic domain and control activity either directly, by interactions at the active site that modulate activity or by controlling substrate specificity. This class of enzymes is also activated through its own tyrosine phosphorylation, being essentially inactive under normal basal conditions. Among the members of this group are the prototype PTP1B, the most studied tyrosine phosphatase, the low molecular weight-PTP (LMW-PTP), and the Shp2 phosphatase (Alonso et al., 2004;

Tyrosine phosphorylation is common to all cytosolic enzymes, including those that do not contain an SH2 domain, such as PTP1B or LMW-PTP. Both phosphatases have been reported to be activated through autophosphorylation. At the same time, autodephosphorylation has been reported for several tyrosine phosphatases, including Shp2, RPTPα, Shp1, and LMW-PTP (den Hertog et al., 1994; Giannoni et al., 2005; Meng et al., 2002; Zhao et al., 1994). PTP1B action encompass numerous substrates, including different PTK receptors, such as the EGFR (epidermal growth factor receptor), PDGFR (platelet-derived growth factor receptor), CSF-1 (colony-stimulating factor 1), IR (insulin receptor) and IGF-1 (insulin-like growth factor-1) receptors, and cytoplasmic PTKs members, such as c-Src and JAK2 (Janus protein tyrosine kinase 2). The non-transmembrane Shp1 and Shp2 (protein tyrosine phosphatase Src homology 1/2 (SH2) containing domain) perform opposing tasks in signaling pathways. Shp-1, predominantly expressed in hematopoietic cells, is a negative regulator in the signaling pathways mediated by the chemokine and cytokine receptors and by other receptor tyrosine kinases. Unlike other tyrosine phosphatases that negatively regulate signaling, Shp2 phosphatases can positively regulate cell signaling through the dephosphorylation of substrates that are negatively regulated by tyrosine phosphorylation and can participate in the propagation of the ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3 kinase)/Akt (protein kinase B) pathways in which multiple receptor PTKs play a role (C. Y.

The dual-specific protein phosphatases (DSPs) can dephosphorylate a variety of substrates, including phosphotyrosine-containing proteins, phosphothreonine, phosphoserine residues and phospholipids, and is the most diverse group in terms of substrate specificity because of this feature. The dual-specific protein phosphatases are much more diverse than tyrosinespecific phosphatases and can be divided into several subgroups. Eleven of the 61 dualspecific PTPs encoded by the human genome are specific for the mitogen-activated protein kinases (MAPK); the other ones can be represented by the well-known cell cycle regulators Cdc25 phosphatases and the tumor suppressor phosphatase (PTEN), which is also able to dephosphorylate lipid substrates (Alonso et al., 2004; Chiarugi & Buricchi, 2007). The dualspecific tyrosine protein phosphatases also include the VH1-like enzymes, which are related to the prototypic VH1 DSP, a 20-kDa protein that is a virulence factor of the vaccinia virus

**2.1.2 The intracellular protein tyrosine phosphatases** 

Chiarugi & Buricchi, 2007).

Chen et al., 2009; Tiganis & Bennett, 2007).

(Tonks, 2005).

**2.2 The dual-specific tyrosine protein phosphatases** 

#### **2. Protein tyrosine phosphatases family**

The protein tyrosine phosphatase (PTP) superfamily is encoded by approximately 100 genes in the human genome. They all possess an overall structure with a core catalytic domain composed of four parallel β-strands surrounded on both sides by α-helices. The active site sequence Cys(X)5Arg defines the PTP family, and this sequence is referred to as the "PTP signature motif". Moreover, this conserved motif creates a very similar structural motif, termed the PTP loop, which connects a central β -strand to an α -helix at the center of the catalytic site. The diversity within the families is conferred by the regulatory domains (Salmeen & Barford, 2005; Tiganis & Bennett, 2007). All protein tyrosine phosphatases are characterized by: a) their sensitivity to vanadate; b) an ability to hydrolyze the artificial substrate *p*-nitrophenyl phosphate; c) an insensitivity to okadaic acid; and d) a lack of metal ion dependence for catalysis (Chiarugi & Buricchi, 2007; Denu & Dixon, 1998). PTPs have the capacity to function both positively and negatively in the regulation of signal transduction, being able to stimulate or inhibit protein functions through their specific dephosphorylation activity (Tonks, 2006).

The protein tyrosine phosphatase superfamily can be grouped into two subfamilies, the tyrosine-specific phosphatases that comprise 38 different enzymes and the dual-specific phosphatases with 61 components in the human genome. The common classification of the protein tyrosine phosphatase family is depicted in table 1. Despite different subcellular localizations and substrate specificities, the protein tyrosine phosphatase classes employ a similar chemical mechanism for phosphate hydrolysis involving a transient cysteinylphosphate intermediate (Chiarugi et al., 2005; Östman et al., 2011).

#### **2.1 Protein tyrosine-specific phosphatases**

The subfamily of tyrosine-specific phosphatases is constituted by the well-known classical protein tyrosine phosphatase (PTP), which acts strictly on phosphotyrosine-containing proteins. The active site of this group is deeper than the catalytic site of dual-specific protein phosphatase, which makes these enzymes much more selective for p-Tyr-containing substrates than the dual-specific protein phosphatase. The classical PTPs can also be divided into transmembrane, receptor-like enzymes (RPTPs) and the cytosolic, nonreceptor PTPs (NRPTPs) (Chiarugi & Buricchi, 2007).

#### **2.1.1 The receptor-like protein tyrosine phosphatases**

The receptor-like PTPs such as RPTPα and CD45 are cell-surface receptors and generally have an extracellular ligand-binding domain, a single transmembrane region, and one or two cytoplasmic catalytic domains. Receptor-like PTPs are mainly negatively regulated through dimerization, a known regulatory mechanism for many signal transduction molecules, such as receptor proteins (Chiarugi & Buricchi, 2007). The number of PTP catalytic domains encoded by the human genome is greater than the number of PTP genes because many of the receptor-type proteins have tandem catalytic domains (Alonso et al., 2004). Receptor-like tyrosine phosphatases have the potential to regulate signaling through ligand-controlled protein tyrosine dephosphorylation. Many of these tyrosine phosphatases display features of cell-adhesion molecules in their extracellular segment and can be implicated in processes that involve cell-cell and cell-matrix contact (Tonks, 2006).

#### **2.1.2 The intracellular protein tyrosine phosphatases**

254 Enzyme Inhibition and Bioapplications

The protein tyrosine phosphatase (PTP) superfamily is encoded by approximately 100 genes in the human genome. They all possess an overall structure with a core catalytic domain composed of four parallel β-strands surrounded on both sides by α-helices. The active site sequence Cys(X)5Arg defines the PTP family, and this sequence is referred to as the "PTP signature motif". Moreover, this conserved motif creates a very similar structural motif, termed the PTP loop, which connects a central β -strand to an α -helix at the center of the catalytic site. The diversity within the families is conferred by the regulatory domains (Salmeen & Barford, 2005; Tiganis & Bennett, 2007). All protein tyrosine phosphatases are characterized by: a) their sensitivity to vanadate; b) an ability to hydrolyze the artificial substrate *p*-nitrophenyl phosphate; c) an insensitivity to okadaic acid; and d) a lack of metal ion dependence for catalysis (Chiarugi & Buricchi, 2007; Denu & Dixon, 1998). PTPs have the capacity to function both positively and negatively in the regulation of signal transduction, being able to stimulate or inhibit protein functions through their specific

The protein tyrosine phosphatase superfamily can be grouped into two subfamilies, the tyrosine-specific phosphatases that comprise 38 different enzymes and the dual-specific phosphatases with 61 components in the human genome. The common classification of the protein tyrosine phosphatase family is depicted in table 1. Despite different subcellular localizations and substrate specificities, the protein tyrosine phosphatase classes employ a similar chemical mechanism for phosphate hydrolysis involving a transient cysteinyl-

The subfamily of tyrosine-specific phosphatases is constituted by the well-known classical protein tyrosine phosphatase (PTP), which acts strictly on phosphotyrosine-containing proteins. The active site of this group is deeper than the catalytic site of dual-specific protein phosphatase, which makes these enzymes much more selective for p-Tyr-containing substrates than the dual-specific protein phosphatase. The classical PTPs can also be divided into transmembrane, receptor-like enzymes (RPTPs) and the cytosolic, nonreceptor PTPs

The receptor-like PTPs such as RPTPα and CD45 are cell-surface receptors and generally have an extracellular ligand-binding domain, a single transmembrane region, and one or two cytoplasmic catalytic domains. Receptor-like PTPs are mainly negatively regulated through dimerization, a known regulatory mechanism for many signal transduction molecules, such as receptor proteins (Chiarugi & Buricchi, 2007). The number of PTP catalytic domains encoded by the human genome is greater than the number of PTP genes because many of the receptor-type proteins have tandem catalytic domains (Alonso et al., 2004). Receptor-like tyrosine phosphatases have the potential to regulate signaling through ligand-controlled protein tyrosine dephosphorylation. Many of these tyrosine phosphatases display features of cell-adhesion molecules in their extracellular segment and can be

implicated in processes that involve cell-cell and cell-matrix contact (Tonks, 2006).

phosphate intermediate (Chiarugi et al., 2005; Östman et al., 2011).

**2. Protein tyrosine phosphatases family** 

dephosphorylation activity (Tonks, 2006).

**2.1 Protein tyrosine-specific phosphatases** 

(NRPTPs) (Chiarugi & Buricchi, 2007).

**2.1.1 The receptor-like protein tyrosine phosphatases** 

The intracellular protein tyrosine phosphatases contain a single catalytic domain and various amino or carboxyl terminal extensions. They may contain SH2 (Src homology domain) domains that have targeting or regulatory functions, PEST (Pro-Glu-Ser-Thr domain) domains for proteolytic control, phosphorylation sites for protein/protein interactions, and enzymatic activity regulation. Cytosolic tyrosine phosphatases are characterized by regulatory sequences that flank the catalytic domain and control activity either directly, by interactions at the active site that modulate activity or by controlling substrate specificity. This class of enzymes is also activated through its own tyrosine phosphorylation, being essentially inactive under normal basal conditions. Among the members of this group are the prototype PTP1B, the most studied tyrosine phosphatase, the low molecular weight-PTP (LMW-PTP), and the Shp2 phosphatase (Alonso et al., 2004; Chiarugi & Buricchi, 2007).

Tyrosine phosphorylation is common to all cytosolic enzymes, including those that do not contain an SH2 domain, such as PTP1B or LMW-PTP. Both phosphatases have been reported to be activated through autophosphorylation. At the same time, autodephosphorylation has been reported for several tyrosine phosphatases, including Shp2, RPTPα, Shp1, and LMW-PTP (den Hertog et al., 1994; Giannoni et al., 2005; Meng et al., 2002; Zhao et al., 1994). PTP1B action encompass numerous substrates, including different PTK receptors, such as the EGFR (epidermal growth factor receptor), PDGFR (platelet-derived growth factor receptor), CSF-1 (colony-stimulating factor 1), IR (insulin receptor) and IGF-1 (insulin-like growth factor-1) receptors, and cytoplasmic PTKs members, such as c-Src and JAK2 (Janus protein tyrosine kinase 2). The non-transmembrane Shp1 and Shp2 (protein tyrosine phosphatase Src homology 1/2 (SH2) containing domain) perform opposing tasks in signaling pathways. Shp-1, predominantly expressed in hematopoietic cells, is a negative regulator in the signaling pathways mediated by the chemokine and cytokine receptors and by other receptor tyrosine kinases. Unlike other tyrosine phosphatases that negatively regulate signaling, Shp2 phosphatases can positively regulate cell signaling through the dephosphorylation of substrates that are negatively regulated by tyrosine phosphorylation and can participate in the propagation of the ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3 kinase)/Akt (protein kinase B) pathways in which multiple receptor PTKs play a role (C. Y. Chen et al., 2009; Tiganis & Bennett, 2007).

#### **2.2 The dual-specific tyrosine protein phosphatases**

The dual-specific protein phosphatases (DSPs) can dephosphorylate a variety of substrates, including phosphotyrosine-containing proteins, phosphothreonine, phosphoserine residues and phospholipids, and is the most diverse group in terms of substrate specificity because of this feature. The dual-specific protein phosphatases are much more diverse than tyrosinespecific phosphatases and can be divided into several subgroups. Eleven of the 61 dualspecific PTPs encoded by the human genome are specific for the mitogen-activated protein kinases (MAPK); the other ones can be represented by the well-known cell cycle regulators Cdc25 phosphatases and the tumor suppressor phosphatase (PTEN), which is also able to dephosphorylate lipid substrates (Alonso et al., 2004; Chiarugi & Buricchi, 2007). The dualspecific tyrosine protein phosphatases also include the VH1-like enzymes, which are related to the prototypic VH1 DSP, a 20-kDa protein that is a virulence factor of the vaccinia virus (Tonks, 2005).

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 257

Protein tyrosine phosphatases are a family of enzymes with high structural multiplicity. Cysteine-based phosphatases share the common function of hydrolyzing phosphorester bonds in proteins and/or lipids via a conserved cysteine-based mechanism. The most significant feature of the protein tyrosine phosphatase superfamily is the presence of a signature motif Cys(X)5Arg, where X is any amino acid. This well-conserved motif forms the phosphate-binding loop in the active site, known as the P-loop or PTP-loop, and is responsible for structural features required for phosphate recognition and phosphorester hydrolysis. The structural arrangement ensures that the catalytic cysteine functions as the nucleophile in catalysis, which specifically binds negatively charged substrates, and the arginine, which is involved in phosphate binding, remains in close proximity to form a cradle to hold the phosphate group of the substrate in the correct place for nucleophilic attack (Chiarugi & Buricchi, 2007; Salmeen & Barford, 2005; Tabernero et al., 2008). Figure 1

Fig. 1. (A) Crystal structure of PTP1B complexed with phosphotyrosine (PDB entry 1PTV) (Jia et al., 1995). A substrate positioned in the active site is represented by phosphotyrosine (ball and stick). The cysteine (C215) represented in the active site is the nucleophile that attacks the substrate phosphorous atom, forming the cysteinyl-phosphate intermediate. The

Specific protein tyrosine phosphatases utilize a two-step reaction for phosphate monoester hydrolysis (Fig. 2). The first step is initiated by a nucleophilic attack by the active-site cysteine (C215) on the phosphorus atom of the bound substrate that leads to the formation of a cysteinyl-phosphate intermediate. Besides substrate binding, the arginine is also involved in the stabilization of this intermediate. During the formation of the transition-state intermediate, the only aspartic residue in the WPD (Trp-Pro-Asp) loop acts as a general acid

arginine (R221) is involved in the intermediate stabilization and substrate binding (Tabernero et al., 2008). (B) Binding of phosphotyrosine to the catalytic site. Here the

nucleophile cysteine 215 is mutated to serine 215 (Jia et al., 1995).

**3. Structural characteristics of the cysteine-based protein tyrosine** 

shows the crystal structure of PTP1B complexed with phosphotyrosine.

**phosphatase catalytic domain** 

The MAPK phosphatases are characterized by dual phosphothreonine and phosphotyrosine specificity and the presence of a CH2 region and other MAPK-targeting motifs. They can dephosphorylate tyrosine and threonine residues within the activation loop of MAPKs. The different specificities towards the various MAPKs mean that MAPK phosphatase plays a critical negative regulation on the MAPK-mediated signaling process (Tiganis & Bennett, 2007). Cdc25 phosphatases are involved in the dephosphorylation of cyclin-dependent kinases at their inhibitory, dually phosphorylated N-terminal Thr-Tyr-motifs. The reaction is required for activation of these kinases to drive progression of cell cycle (Alonso et al., 2004). Another subgroup of dual-specific protein phosphatases, the atypical dual-specific protein phosphatases, includes a number of poorly characterized enzymes that lack specific MAPKtargeting motifs and tend to be much smaller enzymes. This is the case for PIR (phosphatase interacting with RNA/RNP), which dephosphorylates mRNA. Cdc14 is involved in the inactivation of cyclin-dependent kinases and in exit from mitosis, while the slingshots and PRLs (phosphatase of regenerating liver) are poorly understood. PTEN dephosphorylates phosphatidylinositol-3,4,5-trisphosphate at the plasma membrane, while the myotubularins primarily dephosphorylate phosphatidylinositol-3-phosphate on internal surface of cell membranes (Alonso et al., 2004).

Regarding substrate specificity, dual-specific protein phosphatases may present a preference for either tyrosine or serine/threonine residues. For cyclin-dependent kinase (CDK) associated phosphatase (KAP), the phosphatase preferentially dephosphorylates the threonine residue in the activation loop of CDKs, while for the VH1-related dual-specific phosphatases, tyrosine is the preferred residue in MAPKs (Tonks, 2006).


Table 1. Classification of the protein tyrosine phosphatase family and some of its principal members and ligand-inducing oxidation, according to Tonks in 2006. (PDGF, plateletderived growth factor; EGF, epidermal growth factor; TNFα, tumor-necrosis factor-α; TCPTP, T-cell protein tyrosine phosphatase; MKP, mitogen-activated protein kinase phosphatase; LMW-PTP, low molecular weight protein tyrosine phosphatase).

The MAPK phosphatases are characterized by dual phosphothreonine and phosphotyrosine specificity and the presence of a CH2 region and other MAPK-targeting motifs. They can dephosphorylate tyrosine and threonine residues within the activation loop of MAPKs. The different specificities towards the various MAPKs mean that MAPK phosphatase plays a critical negative regulation on the MAPK-mediated signaling process (Tiganis & Bennett, 2007). Cdc25 phosphatases are involved in the dephosphorylation of cyclin-dependent kinases at their inhibitory, dually phosphorylated N-terminal Thr-Tyr-motifs. The reaction is required for activation of these kinases to drive progression of cell cycle (Alonso et al., 2004). Another subgroup of dual-specific protein phosphatases, the atypical dual-specific protein phosphatases, includes a number of poorly characterized enzymes that lack specific MAPKtargeting motifs and tend to be much smaller enzymes. This is the case for PIR (phosphatase interacting with RNA/RNP), which dephosphorylates mRNA. Cdc14 is involved in the inactivation of cyclin-dependent kinases and in exit from mitosis, while the slingshots and PRLs (phosphatase of regenerating liver) are poorly understood. PTEN dephosphorylates phosphatidylinositol-3,4,5-trisphosphate at the plasma membrane, while the myotubularins primarily dephosphorylate phosphatidylinositol-3-phosphate on internal surface of cell

Regarding substrate specificity, dual-specific protein phosphatases may present a preference for either tyrosine or serine/threonine residues. For cyclin-dependent kinase (CDK) associated phosphatase (KAP), the phosphatase preferentially dephosphorylates the threonine residue in the activation loop of CDKs, while for the VH1-related dual-specific

Table 1. Classification of the protein tyrosine phosphatase family and some of its principal members and ligand-inducing oxidation, according to Tonks in 2006. (PDGF, plateletderived growth factor; EGF, epidermal growth factor; TNFα, tumor-necrosis factor-α; TCPTP, T-cell protein tyrosine phosphatase; MKP, mitogen-activated protein kinase phosphatase; LMW-PTP, low molecular weight protein tyrosine phosphatase).

phosphatases, tyrosine is the preferred residue in MAPKs (Tonks, 2006).

membranes (Alonso et al., 2004).

#### **3. Structural characteristics of the cysteine-based protein tyrosine phosphatase catalytic domain**

Protein tyrosine phosphatases are a family of enzymes with high structural multiplicity. Cysteine-based phosphatases share the common function of hydrolyzing phosphorester bonds in proteins and/or lipids via a conserved cysteine-based mechanism. The most significant feature of the protein tyrosine phosphatase superfamily is the presence of a signature motif Cys(X)5Arg, where X is any amino acid. This well-conserved motif forms the phosphate-binding loop in the active site, known as the P-loop or PTP-loop, and is responsible for structural features required for phosphate recognition and phosphorester hydrolysis. The structural arrangement ensures that the catalytic cysteine functions as the nucleophile in catalysis, which specifically binds negatively charged substrates, and the arginine, which is involved in phosphate binding, remains in close proximity to form a cradle to hold the phosphate group of the substrate in the correct place for nucleophilic attack (Chiarugi & Buricchi, 2007; Salmeen & Barford, 2005; Tabernero et al., 2008). Figure 1 shows the crystal structure of PTP1B complexed with phosphotyrosine.

Fig. 1. (A) Crystal structure of PTP1B complexed with phosphotyrosine (PDB entry 1PTV) (Jia et al., 1995). A substrate positioned in the active site is represented by phosphotyrosine (ball and stick). The cysteine (C215) represented in the active site is the nucleophile that attacks the substrate phosphorous atom, forming the cysteinyl-phosphate intermediate. The arginine (R221) is involved in the intermediate stabilization and substrate binding (Tabernero et al., 2008). (B) Binding of phosphotyrosine to the catalytic site. Here the nucleophile cysteine 215 is mutated to serine 215 (Jia et al., 1995).

Specific protein tyrosine phosphatases utilize a two-step reaction for phosphate monoester hydrolysis (Fig. 2). The first step is initiated by a nucleophilic attack by the active-site cysteine (C215) on the phosphorus atom of the bound substrate that leads to the formation of a cysteinyl-phosphate intermediate. Besides substrate binding, the arginine is also involved in the stabilization of this intermediate. During the formation of the transition-state intermediate, the only aspartic residue in the WPD (Trp-Pro-Asp) loop acts as a general acid

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 259

hydroxyl group of the serine or threonine residue in the signature motif is important for stabilizing the thiolate form of the active site cysteine, which results in the characteristically lower pKa. The P-loop is found at the N-terminus of an α-helix and should also contribute to the thiolate stabilization, as well as, the presence of neighbouring acid (Asp or Glu) and basic (Arg, His or Lys) amino acids near the catalytic cysteine (Hess et al., 2005; Kolmodin

The structure of cytoplasmatic tyrosine phosphatases is highly conserved with only minor differences in the main structural core. The catalytic domain is formed by about 280 amino acids, which is responsible for the specific folding of the enzyme and other differing characteristics. The catalytic domain often exhibits relatively broad substrate specificities *in vitro*; however, under *in vivo* conditions, full-length tyrosine phosphatases have more stringent specificities. This is in part due to the presence of additional regulatory domains that direct their subcellular localization and interactions with specific substrates (Tabernero

Reactive oxygen species (ROS) are a group of molecules produced in cells when oxygen is metabolized and are more reactive than molecular oxygen (Groeger et al., 2009). In aerobic organisms, ROS can originate from different sources, including mitochondrial electron transport chain, xanthine oxidase, myeloperoxidase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox enzymes), and lipoxygenase (Groeger et al., 2009; Östman et al., 2011; Salmeen & Barford, 2005). The last two are responsible for the production of ROS in response to hormones, growth factors and cytokines (Bae et al., 1997; Salmeen & Barford, 2005; Sundaresan et al., 1995). Among these oxygen metabolites are superoxide anions (O2•-), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) (Chiarugi & Buricchi, 2007). These species are not all equally reactive regarding to prospective targets. Many of them have very short half-lives, leading to little relevance in terms of signaling. For example, the hydroxyl radical is the most unstable radical, reflecting its limited ability to transmit signals across any significant distance. At the same time, superoxide and hydrogen peroxide can be considered more stable species and because of these, may be the most

After being considered important components of the biological process by McCord and Fridovich in 1969, free radicals emerged in the subsequent years as a dangerous byproduct of cellular metabolism by bringing deleterious consequences to DNA, lipids and proteins. The first research on reactive oxygen species has focused primarily on their chemical ability to react with and cause toxic and mutagenic effects on cellular components. There are exceptions, such as phagocytes, that deliberately generate reactive oxygen species as a main defense mechanism (Lambeth, 2002). Almost ten years later in 1977, Mittal and Murad showed the first evidence of the potential for free radicals to act in cellular signaling in metabolism. Now, it is largely accepted that living organisms have not only adapted to cope with oxidizing species but also have developed mechanisms for the advantageous use of free radicals (Dröge, 2002). ROS can act as second messengers that are required for the downstream signaling events (Cross & Templeton, 2006) However, mechanisms by which

**4. Sources of oxidizing species in biological systems** 

favorable ROS to operate as a signaling molecule (K. Chen et al., 2009).

and Åqvist, 2001).

et al., 2008).

**4.1 Reactive oxygen species** 

by donating its proton to the phenolic oxygen of the tyrosyl-leaving group, thus cleaving the phosphate of tyrosine to form the cysteine-phosphate intermediate. This first substitution reaction allows the phosphate group to be covalently attached to the nucleophile via a thioester linkage. In the second step of catalysis, the same aspartic residue functions as a general base by accepting a proton from water during hydrolysis of the cysteinyl phosphate intermediate, thus restoring the free enzyme to its normal basal Cys-SH conformation (Kolmodin & Åqvist 2001; Tabernero et al., 2008; Tiganis & Bennett, 2007). Figure 2 illustrates the general two-step catalysis of protein tyrosine phosphatases.

Most of the cysteine residues within proteins present a normal pKa of 8.3, and this characteristic ensures that this amino acid has a relatively good nucleophile attribute. Because of the unique environment of the tyrosine phosphatase active site, the catalytic cysteine presents an unusually low pKa value. Unlike the free cysteines, which are shown to be protonated at physiological pH, the catalytic cysteines of the tyrosine-specific PTPs are more acidic and are therefore proposed to be deprotonated in the free enzymes at a physiological pH (or even at the pH optimum of 5-6), existing as a thiolate anion (Cys-S−). This property enables them to act as nucleophiles in the first step of catalysis but also makes them highly susceptible to oxidations by different reactive oxygen species and, to a lesser degree, with reactive nitrogen species. Exposure of thiolate anions to reactive nitrogen species causes S-nitrosothiol formation, whereas treatment with peroxynitrite yields Snitrothiol formation (Brandes et al., 2009; Chiarugi et al., 2005; Salmeen & Barford, 2005).

Fig. 2. Schematic mechanism of two-step catalysis of protein tyrosine phosphatases. The first step is initiated by a nucleophilic attack of the active site cysteine, which is in its thiolate anion conformation (Cys-S−), on the phosphorus atom of the bound substrate (1). The dephosphorylated protein tyrosine is released from the active site of the phosphatase while a cysteinyl-phosphate intermediate is formed. In the second step of catalysis, the aspartic residue in the P-loop accepts a proton from water during hydrolysis of the cysteinyl phosphate intermediate, thus restoring the free enzyme to its basal Cys-S− conformation (2).

After oxidation, the modified cysteine can no longer function as a phosphate acceptor in the first step of catalysis, which abrogates the catalytic function of tyrosine phosphatase (Chiarugi et al., 2005; Salmeen & Barford, 2005). A network of hydrogen bonds stabilizes the negative charges in the active site. Among other interactions in the PTPs, it is found that the hydroxyl group of the serine or threonine residue in the signature motif is important for stabilizing the thiolate form of the active site cysteine, which results in the characteristically lower pKa. The P-loop is found at the N-terminus of an α-helix and should also contribute to the thiolate stabilization, as well as, the presence of neighbouring acid (Asp or Glu) and basic (Arg, His or Lys) amino acids near the catalytic cysteine (Hess et al., 2005; Kolmodin and Åqvist, 2001).

The structure of cytoplasmatic tyrosine phosphatases is highly conserved with only minor differences in the main structural core. The catalytic domain is formed by about 280 amino acids, which is responsible for the specific folding of the enzyme and other differing characteristics. The catalytic domain often exhibits relatively broad substrate specificities *in vitro*; however, under *in vivo* conditions, full-length tyrosine phosphatases have more stringent specificities. This is in part due to the presence of additional regulatory domains that direct their subcellular localization and interactions with specific substrates (Tabernero et al., 2008).

### **4. Sources of oxidizing species in biological systems**

#### **4.1 Reactive oxygen species**

258 Enzyme Inhibition and Bioapplications

by donating its proton to the phenolic oxygen of the tyrosyl-leaving group, thus cleaving the phosphate of tyrosine to form the cysteine-phosphate intermediate. This first substitution reaction allows the phosphate group to be covalently attached to the nucleophile via a thioester linkage. In the second step of catalysis, the same aspartic residue functions as a general base by accepting a proton from water during hydrolysis of the cysteinyl phosphate intermediate, thus restoring the free enzyme to its normal basal Cys-SH conformation (Kolmodin & Åqvist 2001; Tabernero et al., 2008; Tiganis & Bennett, 2007). Figure 2

Most of the cysteine residues within proteins present a normal pKa of 8.3, and this characteristic ensures that this amino acid has a relatively good nucleophile attribute. Because of the unique environment of the tyrosine phosphatase active site, the catalytic cysteine presents an unusually low pKa value. Unlike the free cysteines, which are shown to be protonated at physiological pH, the catalytic cysteines of the tyrosine-specific PTPs are more acidic and are therefore proposed to be deprotonated in the free enzymes at a physiological pH (or even at the pH optimum of 5-6), existing as a thiolate anion (Cys-S−). This property enables them to act as nucleophiles in the first step of catalysis but also makes them highly susceptible to oxidations by different reactive oxygen species and, to a lesser degree, with reactive nitrogen species. Exposure of thiolate anions to reactive nitrogen species causes S-nitrosothiol formation, whereas treatment with peroxynitrite yields Snitrothiol formation (Brandes et al., 2009; Chiarugi et al., 2005; Salmeen & Barford, 2005).

Fig. 2. Schematic mechanism of two-step catalysis of protein tyrosine phosphatases. The first step is initiated by a nucleophilic attack of the active site cysteine, which is in its thiolate anion conformation (Cys-S−), on the phosphorus atom of the bound substrate (1). The dephosphorylated protein tyrosine is released from the active site of the phosphatase while a cysteinyl-phosphate intermediate is formed. In the second step of catalysis, the aspartic residue in the P-loop accepts a proton from water during hydrolysis of the cysteinyl

phosphate intermediate, thus restoring the free enzyme to its basal Cys-S− conformation (2).

After oxidation, the modified cysteine can no longer function as a phosphate acceptor in the first step of catalysis, which abrogates the catalytic function of tyrosine phosphatase (Chiarugi et al., 2005; Salmeen & Barford, 2005). A network of hydrogen bonds stabilizes the negative charges in the active site. Among other interactions in the PTPs, it is found that the

illustrates the general two-step catalysis of protein tyrosine phosphatases.

Reactive oxygen species (ROS) are a group of molecules produced in cells when oxygen is metabolized and are more reactive than molecular oxygen (Groeger et al., 2009). In aerobic organisms, ROS can originate from different sources, including mitochondrial electron transport chain, xanthine oxidase, myeloperoxidase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox enzymes), and lipoxygenase (Groeger et al., 2009; Östman et al., 2011; Salmeen & Barford, 2005). The last two are responsible for the production of ROS in response to hormones, growth factors and cytokines (Bae et al., 1997; Salmeen & Barford, 2005; Sundaresan et al., 1995). Among these oxygen metabolites are superoxide anions (O2•-), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) (Chiarugi & Buricchi, 2007). These species are not all equally reactive regarding to prospective targets. Many of them have very short half-lives, leading to little relevance in terms of signaling. For example, the hydroxyl radical is the most unstable radical, reflecting its limited ability to transmit signals across any significant distance. At the same time, superoxide and hydrogen peroxide can be considered more stable species and because of these, may be the most favorable ROS to operate as a signaling molecule (K. Chen et al., 2009).

After being considered important components of the biological process by McCord and Fridovich in 1969, free radicals emerged in the subsequent years as a dangerous byproduct of cellular metabolism by bringing deleterious consequences to DNA, lipids and proteins. The first research on reactive oxygen species has focused primarily on their chemical ability to react with and cause toxic and mutagenic effects on cellular components. There are exceptions, such as phagocytes, that deliberately generate reactive oxygen species as a main defense mechanism (Lambeth, 2002). Almost ten years later in 1977, Mittal and Murad showed the first evidence of the potential for free radicals to act in cellular signaling in metabolism. Now, it is largely accepted that living organisms have not only adapted to cope with oxidizing species but also have developed mechanisms for the advantageous use of free radicals (Dröge, 2002). ROS can act as second messengers that are required for the downstream signaling events (Cross & Templeton, 2006) However, mechanisms by which

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 261

three groups of nonphagocytic Nox members are summarized in table 2. The production of reactive species by NADPH oxidases occurs during inflammatory conditions in cells of the immune system or by external stimuli, such as growth factors in most mammalian cells, and

Lipoxygenase is a mixed function oxidase involved in the synthesis of leukotrienes from arachidonic acids. Which are released from membrane phospholipids via the activity of cytosolic phospholipase A2, and are also responsible for the production of superoxide

Superoxide is not highly reactive and cannot cross cellular membranes; its diffusion is dependent of anion channels in the membranes. In most cases, the potential effects of superoxides are restricted to a single intracellular compartment that can be directed by some organelles like mitochondria, endosomes, and phagosomes (K. Chen et al., 2009). However, superoxide can readily be converted to hydrogen peroxide by superoxide dismutase, which

Hydrogen peroxide is a two-electron oxidant that acts as an electrophile and can react with protein thiol moieties to produce a variety of sulfur oxidation states, as will be discussed later. Most of the times, this reactivity affords reversible posttranslational modification of proteins that would be important for cellular signaling. Moreover, the relatively longer halflife of hydrogen peroxide compared with other ROS in biologic systems affords better activity as second messengers (K. Chen et al., 2009). Both, superoxide and hydrogen peroxide are involved in the inhibitory oxidation of the catalytic sites of the protein tyrosine

The reactive nitrogen species (•NO) is produced in higher organisms by the oxidation of one of the terminal guanidino-nitrogen atoms of L-arginine. This process is catalyzed by the enzyme nitric oxide synthase (NOS). Nitric oxide (NO) can be generated by the reduction of NOx species that are derived from exogenous or endogenous sources (Hess et al., 2005). Depending on the microenvironment, NO can be converted to various other reactive nitrogen species (RNS), such as nitrosonium cation (NO+), nitroxyl anion (NO−) or peroxynitrite (ONOO−)(Dröge, 2002). Separate genes code for different NOS isoforms, for example, neuronal NOS (nNOS or NOS1) and for endothelial NOS (eNOS or NOS3), which are named after the tissues in which they were discovered, as well as, for inducible/Ca2+-independent NOS (iNOS or NOS2). (Hess et al., 2005). Nitric oxide is produced in large amounts, ranging in nanomoles concentration, for several hours. In contrast, the constitutive NOS found in some tissues remains active for relatively short periods of time (Do et al., 1996). The attachment of the NO moiety to a nucleophilic group or a transition metal is called nitrosyl. Ingeneral, the NO moiety can be provided by NO itself, nitrite, other NOx species (higher NO oxides), metal-NO complexes. Nitric oxide has high reactivity for thiol groups that are present in proteins, reduced glutathione, or cysteine residues. The chemical addition of NO moiety to a reactive cysteine thiol group (nitrosation) is called S-nitrosylation (SNO). S-Nitrosylation-induced conformation can lead to the formation of S-nitrosoproteins, S-nitrosoglutathione, or Snitrosocysteine. These nitrosylated molecules can also serve as source of NO moieties. (Do et al., 1996; Janssen-Heininger et al., 2008). Accumulation of protein SNOs is the only mechanism

their relevance is related to intracellular signaling (Dorgë, 2002; Lambeth, 2002).

(Chiarugi & Buricchi, 2007; Kim et al., 2008).

phosphatases.

**4.2 Reactive nitrogen species** 

finally penetrates membranes (Cross & Templeton, 2006).

proteins are able to sense ROS and translate signaling into an effective response are not totally clear.

Superoxide anions are derived by the univalent reduction of triplet-state molecular oxygen (3O2). This process is mediated by enzymes, such as xanthine oxidase, NADPH oxidases (Nox) and dual oxidases (Duox) (also known as Nox enzymes), or nonenzymatically by redox reactive compounds, such as the semi-ubiquinone of mitochondria. Within mitochondria, superoxide is continually generated as a result of electron leakage from the electron transport chain mainly at complex I (NADH/ubiquinone oxidoreductase) and complex III (ubiquinol/cytochrome c oxido-reductase) (Cross & Templeton, 2006; Harrison, 2002).


Table 2. Nox members in nonphagocytic cells.

Xanthine oxidase catalyzes the oxidation of xanthine to uric acid and O2•-. Further O2•- can be dismuted into hydrogen peroxide and molecular oxygen. The enzyme is expressed mainly in the liver and epithelia (Harrison, 2002).

Regarding the Nox family, seven mammalian members are known. The seven members are Nox1-5 and dual oxidases (Duoxs) 1 and 2. Nox proteins are integral membrane proteins with six transmembrane domains, which together are thought to form a channel to allow the successive transfer of electrons. They operate by transferring electrons from NADPH (converting it to NADP−) to FAD, then to heme and finally to oxygen to make O2 •-. Duox1 and 2 both have a peroxidase-like domain in their N-terminal region, which means that these two members of the family produce hydrogen peroxide and not O2 •- (Groeger et al., 2009). The NADPH oxidase complexes were firstly thought to be specific to macrophages, but now these enzymes are recognized and expressed almost universally in nonphagocytic cell types, and are also able to produce ROS via a similar mechanism (Chiarugi & Buricchi, 2007; Cross & Templeton, 2006). The wide-ranging distribution of Nox members, suggests that ROS production may have a broad implications for the different cellular phenotypes (K. Chen et al., 2009). Besides the NADPH oxidase from phagocytic cells (Nox 2), Nox family can be divided into three groups that present distinct regulatory patterns from those of Nox 2. The three groups of nonphagocytic Nox members are summarized in table 2. The production of reactive species by NADPH oxidases occurs during inflammatory conditions in cells of the immune system or by external stimuli, such as growth factors in most mammalian cells, and their relevance is related to intracellular signaling (Dorgë, 2002; Lambeth, 2002).

Lipoxygenase is a mixed function oxidase involved in the synthesis of leukotrienes from arachidonic acids. Which are released from membrane phospholipids via the activity of cytosolic phospholipase A2, and are also responsible for the production of superoxide (Chiarugi & Buricchi, 2007; Kim et al., 2008).

Superoxide is not highly reactive and cannot cross cellular membranes; its diffusion is dependent of anion channels in the membranes. In most cases, the potential effects of superoxides are restricted to a single intracellular compartment that can be directed by some organelles like mitochondria, endosomes, and phagosomes (K. Chen et al., 2009). However, superoxide can readily be converted to hydrogen peroxide by superoxide dismutase, which finally penetrates membranes (Cross & Templeton, 2006).

Hydrogen peroxide is a two-electron oxidant that acts as an electrophile and can react with protein thiol moieties to produce a variety of sulfur oxidation states, as will be discussed later. Most of the times, this reactivity affords reversible posttranslational modification of proteins that would be important for cellular signaling. Moreover, the relatively longer halflife of hydrogen peroxide compared with other ROS in biologic systems affords better activity as second messengers (K. Chen et al., 2009). Both, superoxide and hydrogen peroxide are involved in the inhibitory oxidation of the catalytic sites of the protein tyrosine phosphatases.

#### **4.2 Reactive nitrogen species**

260 Enzyme Inhibition and Bioapplications

proteins are able to sense ROS and translate signaling into an effective response are not

Superoxide anions are derived by the univalent reduction of triplet-state molecular oxygen (3O2). This process is mediated by enzymes, such as xanthine oxidase, NADPH oxidases (Nox) and dual oxidases (Duox) (also known as Nox enzymes), or nonenzymatically by redox reactive compounds, such as the semi-ubiquinone of mitochondria. Within mitochondria, superoxide is continually generated as a result of electron leakage from the electron transport chain mainly at complex I (NADH/ubiquinone oxidoreductase) and complex III (ubiquinol/cytochrome c oxido-reductase) (Cross & Templeton, 2006; Harrison,

**Nox Members Expressing cells Main characteristics**

Duox 1 Thyroid, lung Presents a calmodulin-like

Xanthine oxidase catalyzes the oxidation of xanthine to uric acid and O2•-. Further O2•- can be dismuted into hydrogen peroxide and molecular oxygen. The enzyme is expressed

Regarding the Nox family, seven mammalian members are known. The seven members are Nox1-5 and dual oxidases (Duoxs) 1 and 2. Nox proteins are integral membrane proteins with six transmembrane domains, which together are thought to form a channel to allow the successive transfer of electrons. They operate by transferring electrons from NADPH (converting it to NADP−) to FAD, then to heme and finally to oxygen to make O2•-. Duox1 and 2 both have a peroxidase-like domain in their N-terminal region, which means that these two members of the family produce hydrogen peroxide and not O2•- (Groeger et al., 2009). The NADPH oxidase complexes were firstly thought to be specific to macrophages, but now these enzymes are recognized and expressed almost universally in nonphagocytic cell types, and are also able to produce ROS via a similar mechanism (Chiarugi & Buricchi, 2007; Cross & Templeton, 2006). The wide-ranging distribution of Nox members, suggests that ROS production may have a broad implications for the different cellular phenotypes (K. Chen et al., 2009). Besides the NADPH oxidase from phagocytic cells (Nox 2), Nox family can be divided into three groups that present distinct regulatory patterns from those of Nox 2. The

Duox 2 Thyroid, colon peroxidase domain.

Similar to phagocytic Nox 2 because of their structure and enzymatic activity.

Presents a calmodulin-like domain and can be activated by calcium.

domain, and an amino-terminal

Nox 1 Colon, vascular smooth muscle, prostate

Nox 3 Fetal liver, fetal lung, fetal spleen, fetal

Nox 4 Kidney, osteoclasts, pancreas, placenta,

Nox 5 Spleen, sperm, lymphnode, ovary,

Table 2. Nox members in nonphagocytic cells.

mainly in the liver and epithelia (Harrison, 2002).

kidney

skeletal muscle, ovary, astrocytes

placenta, pancreas

totally clear.

2002).

The reactive nitrogen species (•NO) is produced in higher organisms by the oxidation of one of the terminal guanidino-nitrogen atoms of L-arginine. This process is catalyzed by the enzyme nitric oxide synthase (NOS). Nitric oxide (NO) can be generated by the reduction of NOx species that are derived from exogenous or endogenous sources (Hess et al., 2005). Depending on the microenvironment, NO can be converted to various other reactive nitrogen species (RNS), such as nitrosonium cation (NO+), nitroxyl anion (NO−) or peroxynitrite (ONOO−)(Dröge, 2002). Separate genes code for different NOS isoforms, for example, neuronal NOS (nNOS or NOS1) and for endothelial NOS (eNOS or NOS3), which are named after the tissues in which they were discovered, as well as, for inducible/Ca2+-independent NOS (iNOS or NOS2). (Hess et al., 2005). Nitric oxide is produced in large amounts, ranging in nanomoles concentration, for several hours. In contrast, the constitutive NOS found in some tissues remains active for relatively short periods of time (Do et al., 1996). The attachment of the NO moiety to a nucleophilic group or a transition metal is called nitrosyl. Ingeneral, the NO moiety can be provided by NO itself, nitrite, other NOx species (higher NO oxides), metal-NO complexes. Nitric oxide has high reactivity for thiol groups that are present in proteins, reduced glutathione, or cysteine residues. The chemical addition of NO moiety to a reactive cysteine thiol group (nitrosation) is called S-nitrosylation (SNO). S-Nitrosylation-induced conformation can lead to the formation of S-nitrosoproteins, S-nitrosoglutathione, or Snitrosocysteine. These nitrosylated molecules can also serve as source of NO moieties. (Do et al., 1996; Janssen-Heininger et al., 2008). Accumulation of protein SNOs is the only mechanism

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 263

through a supportive role to the peroxiredoxins. Besides their function as ROS scavengers, these enzymes can actually protect proteins from oxidations or regenerate oxidized proteins (K. Chen et al., 2009; Chiarugi & Buricchi, 2007). The overall catalysis mechanism of these

2 2 22 2

2 2 2 2 Catalase 2H O 2H O + O

As mentioned above nitrosative stress results from increased or deregulated production of reactive nitrogen species (Hess et al., 2005). The formation of S-nitrosoglutathione is regulated by the coordinately action of the enzyme S-nitrosoglutathione reductase (GSNO reductase), also known as glutathione-dependent formaldehyde dehydrogenase. This enzyme converts GSNO to GSNHOH, ultimately forming ammonia and oxidized glutathione. GSNO reductase is responsible for the regulation of the steady-state levels of Snitrosylated proteins, that are in equilibrium with GSH. Denitrosylation can also be achieved by specific metal ions, such as Cu+, the low-molecular-weight antioxidant

SOD O O 2H H O +O (1)

2+ 3+ • - H O + Fe Fe + OH + OH 2 2 (2)

GPx or Prx (3)

•• +

ascorbate, or thioredoxins enzymes (Janssen-Heininger et al., 2008).

**5. Redox inhibition of cysteine-based protein tyrosine phosphatases** 

Reversible oxidation of regulatory proteins by hydrogen peroxide or NO/SNO has been implicated in broad cellular signaling mechanisms. Tyrosine phosphatases can be differentially regulated by phosphorylation, intra- and intermolecular interactions, alternative splicing, transcription, and translation, but recently, it has become apparent that this family of enzymes may also be regulated by reversible oxidation. In general, the redox regulation of proteins occurs primarily when cysteine residues within the protein or bound to transition metals react with ROS, such as H2O2 or superoxide, or with reactive nitrogen species, such as nitric oxide, *S*-nitrosothiols, or peroxynitrite. Reactions of cysteines with mild oxidizing or nitrosylating reagents, such as low concentrations of H2O2 or •NO, can lead to reversible modulations of protein activity. Oxidation or nitrosylation of the catalytic cysteine of protein tyrosine phosphatases renders the residue unable to act as a nucleophile, and the enzyme loses its phosphatase activity (Chiarugi et al., 2005). Protein phosphatases are among the earliest characterized targets of inhibition by thiol group modification. Thiol group modifications are thought to play a central role in cellular signaling process through

The first evidence of protein regulation by the redox modulation of a cysteine was described in 1967 by Pontremoli and colleagues, which showed that disulfide formation with cysteamine stimulated fructose-1,6-bisphosphatase activity. The redox regulation of tyrosine phosphatases depends on different oxidative stress conditions and is tissue specific

antioxidant enzymes is summarized below:

**4.3.2. Antioxidants of Nitrosylated Molecules** 

ROS modifications (Cross & Templeton, 2006).

(Chiarugi et al., 2005).

of NO toxicity that is able to induce nitrosative stress. Nitrosative stress that results from increased or deregulated production of reactive nitrogen species is countered by several cellular mechanisms triggered by nitrosative modifications of proteins that mobilize adaptive and protective responses (Hess et al., 2005).

#### **4.3 Antioxidant mechanisms**

#### **4.3.1 Antioxidants of reactive oxygen species**

The transitory fluctuations in ROS provide important regulatory functions, but when present in high amounts, they can induce severe damage to cellular components. To avoid further deleterious outcomes, cells have evolved with the ability to handle increases in ROS production. These include various nonenzymatic and low-molecular-weight antioxidants, for example, reduced glutathione, vitamins A, C, and E, flavonoids, and enzymatic scavengers (Chiarugi & Buricchi, 2007). Reduced glutathione is a tripeptide synthesized in cells by the sequential addition of cysteine to glutamate followed by the addition of glycine, giving rise to the molecular γ-L-glutamyl-L-cysteinyl-glycine. The sulfhydryl group (-SH) of the cysteine can be involved in reduction reaction, removing peroxides, or it can be associated in conjugation reactions with enzymes such as glutathione peroxidases (GPx) and the isoforme 6 of peroxiredoxins (Psdx 6). These enzymes catalyze the reduction of hydrogen peroxide by GSH into H2O and oxidized glutathione (GSSG). In mammalian cells, GSH is present in millimolar concentrations and the conversion of GSH to its oxidized form GSSG by enzymatic activities serves as a redox buffer system within cells. The GSSG can then be reduced back originating two GSH molecules by the action of glutathione reductase enzymes, at the expense of NADH or NADPH (Cross & Templeton, 2006; Forman et al., 2009). Glutathione also is important for maintaining ascorbate (vitamin c), itself a potent intracellular antioxidant, in a reduced state (K. Chen et al., 2009).

In cells, superoxide can be converted nonenzymatically to hydrogen peroxide and singlet oxygen (1O2), or through the enzymatic action of superoxide dismutases (SOD), superoxide can be then converted into H2O2, which is a less reactive specie. In mammals, three isoforms of superoxide dismutase are recognized: the cytoplasmatic SOD, which is a copper/zincdependent dismutase (SOD1); the mitochondrial manganese SOD (SOD 2); and the extracellular Cu/Zn-dependent SOD (SOD3).

In the presence of reduced transition metals, such as ferrous or cuprous ions, hydrogen peroxide can be converted into a highly reactive hydroxyl radical (HO−). This radical is a less stable radical species and acts in a nonselective, oxidative manner on organic molecules. There are several families of enzymes, *e.g.,* catalases, glutathione peroxidases (GPx), and peroxiredoxins (Prx), that break down hydrogen peroxide directly (Dröge, 2002; Salmeen & Barford, 2005). Catalases are localized entirely in peroxisomes and efficiently converts hydrogen peroxide to water and molecular oxygen, while glutathione peroxidases are largely restricted to the cytosol and mitochondria. Glutathione peroxidases reduce peroxides by transferring electrons from GSH with the generation of oxidized glutathione. Peroxiredoxins exhibit a higher affinity toward hydrogen peroxide and are also abundant in the cytosol. Peroxiredoxins remove hydrogen peroxide to yield water, while form an intermolecular disulfide bond, which then can be reduced by thioredoxins (Trx). Consequently, thioredoxins are not directly involved in the removal of ROS, but indirectly through a supportive role to the peroxiredoxins. Besides their function as ROS scavengers, these enzymes can actually protect proteins from oxidations or regenerate oxidized proteins (K. Chen et al., 2009; Chiarugi & Buricchi, 2007). The overall catalysis mechanism of these antioxidant enzymes is summarized below:

$$\text{O}\_2\text{"}^- + \text{O}\_2\text{"}^- + 2\text{H}^+ \xrightarrow{\text{SOD}} \text{ H}\_2\text{O}\_2 + \text{O}\_2 \tag{1}$$

$$\text{CH}\_2\text{O}\_2 + \text{Fe}^{2+} \rightarrow \text{Fe}^{3+} + \text{"OH} + \text{OH}"\tag{2}$$

$$2\text{H}\_2\text{O}\_2 \xrightarrow[\text{GPx or Prx}]{\text{Catalase}} 2\text{H}\_2\text{O} + \text{O}\_2\tag{3}$$

#### **4.3.2. Antioxidants of Nitrosylated Molecules**

262 Enzyme Inhibition and Bioapplications

of NO toxicity that is able to induce nitrosative stress. Nitrosative stress that results from increased or deregulated production of reactive nitrogen species is countered by several cellular mechanisms triggered by nitrosative modifications of proteins that mobilize adaptive

The transitory fluctuations in ROS provide important regulatory functions, but when present in high amounts, they can induce severe damage to cellular components. To avoid further deleterious outcomes, cells have evolved with the ability to handle increases in ROS production. These include various nonenzymatic and low-molecular-weight antioxidants, for example, reduced glutathione, vitamins A, C, and E, flavonoids, and enzymatic scavengers (Chiarugi & Buricchi, 2007). Reduced glutathione is a tripeptide synthesized in cells by the sequential addition of cysteine to glutamate followed by the addition of glycine, giving rise to the molecular γ-L-glutamyl-L-cysteinyl-glycine. The sulfhydryl group (-SH) of the cysteine can be involved in reduction reaction, removing peroxides, or it can be associated in conjugation reactions with enzymes such as glutathione peroxidases (GPx) and the isoforme 6 of peroxiredoxins (Psdx 6). These enzymes catalyze the reduction of hydrogen peroxide by GSH into H2O and oxidized glutathione (GSSG). In mammalian cells, GSH is present in millimolar concentrations and the conversion of GSH to its oxidized form GSSG by enzymatic activities serves as a redox buffer system within cells. The GSSG can then be reduced back originating two GSH molecules by the action of glutathione reductase enzymes, at the expense of NADH or NADPH (Cross & Templeton, 2006; Forman et al., 2009). Glutathione also is important for maintaining ascorbate (vitamin c), itself a potent

In cells, superoxide can be converted nonenzymatically to hydrogen peroxide and singlet oxygen (1O2), or through the enzymatic action of superoxide dismutases (SOD), superoxide can be then converted into H2O2, which is a less reactive specie. In mammals, three isoforms of superoxide dismutase are recognized: the cytoplasmatic SOD, which is a copper/zincdependent dismutase (SOD1); the mitochondrial manganese SOD (SOD 2); and the

In the presence of reduced transition metals, such as ferrous or cuprous ions, hydrogen peroxide can be converted into a highly reactive hydroxyl radical (HO−). This radical is a less stable radical species and acts in a nonselective, oxidative manner on organic molecules. There are several families of enzymes, *e.g.,* catalases, glutathione peroxidases (GPx), and peroxiredoxins (Prx), that break down hydrogen peroxide directly (Dröge, 2002; Salmeen & Barford, 2005). Catalases are localized entirely in peroxisomes and efficiently converts hydrogen peroxide to water and molecular oxygen, while glutathione peroxidases are largely restricted to the cytosol and mitochondria. Glutathione peroxidases reduce peroxides by transferring electrons from GSH with the generation of oxidized glutathione. Peroxiredoxins exhibit a higher affinity toward hydrogen peroxide and are also abundant in the cytosol. Peroxiredoxins remove hydrogen peroxide to yield water, while form an intermolecular disulfide bond, which then can be reduced by thioredoxins (Trx). Consequently, thioredoxins are not directly involved in the removal of ROS, but indirectly

and protective responses (Hess et al., 2005).

**4.3.1 Antioxidants of reactive oxygen species** 

intracellular antioxidant, in a reduced state (K. Chen et al., 2009).

extracellular Cu/Zn-dependent SOD (SOD3).

**4.3 Antioxidant mechanisms** 

As mentioned above nitrosative stress results from increased or deregulated production of reactive nitrogen species (Hess et al., 2005). The formation of S-nitrosoglutathione is regulated by the coordinately action of the enzyme S-nitrosoglutathione reductase (GSNO reductase), also known as glutathione-dependent formaldehyde dehydrogenase. This enzyme converts GSNO to GSNHOH, ultimately forming ammonia and oxidized glutathione. GSNO reductase is responsible for the regulation of the steady-state levels of Snitrosylated proteins, that are in equilibrium with GSH. Denitrosylation can also be achieved by specific metal ions, such as Cu+, the low-molecular-weight antioxidant ascorbate, or thioredoxins enzymes (Janssen-Heininger et al., 2008).

#### **5. Redox inhibition of cysteine-based protein tyrosine phosphatases**

Reversible oxidation of regulatory proteins by hydrogen peroxide or NO/SNO has been implicated in broad cellular signaling mechanisms. Tyrosine phosphatases can be differentially regulated by phosphorylation, intra- and intermolecular interactions, alternative splicing, transcription, and translation, but recently, it has become apparent that this family of enzymes may also be regulated by reversible oxidation. In general, the redox regulation of proteins occurs primarily when cysteine residues within the protein or bound to transition metals react with ROS, such as H2O2 or superoxide, or with reactive nitrogen species, such as nitric oxide, *S*-nitrosothiols, or peroxynitrite. Reactions of cysteines with mild oxidizing or nitrosylating reagents, such as low concentrations of H2O2 or •NO, can lead to reversible modulations of protein activity. Oxidation or nitrosylation of the catalytic cysteine of protein tyrosine phosphatases renders the residue unable to act as a nucleophile, and the enzyme loses its phosphatase activity (Chiarugi et al., 2005). Protein phosphatases are among the earliest characterized targets of inhibition by thiol group modification. Thiol group modifications are thought to play a central role in cellular signaling process through ROS modifications (Cross & Templeton, 2006).

The first evidence of protein regulation by the redox modulation of a cysteine was described in 1967 by Pontremoli and colleagues, which showed that disulfide formation with cysteamine stimulated fructose-1,6-bisphosphatase activity. The redox regulation of tyrosine phosphatases depends on different oxidative stress conditions and is tissue specific (Chiarugi et al., 2005).

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 265

Different cysteine residues can take part in the disulfide bridge, such as what may occur with the catalytic cysteine of LMW-PTP or PTEN with another cysteine residue or between the classical protein tyrosine phosphatases such as PTP1B and a cysteine from GSH (Barrett et al., 1999; Chiarugi et al., 2001; den Hertog et al., 2005; Kwon et al., 2004; Lee et al., 1998; Salmeen & Barford, 2005). The tripeptide GSH can also induce alterations of proteins function by the formation of mixed disulfide bonds, a reversible process referred to as glutathionylation. The last conformation can result in a temporarily inhibition of PTP activity that protects enzyme from irreversible oxidation. Glutathionylation can be easily reversed by the action of glutaredoxins or thioredoxins, restoring normal enzyme function

It is assumed that both disulfide bonds or sulfenyl-amide conformations protect the reactive cysteine from a higher-order, irreversible oxidation or expose the oxidized cysteine to cellular reducing agents, which facilitates the reactivation of the oxidized cysteine, because sulfenyl-amide rings or disulfide bonds represent reversibly oxidized states of cysteine and can be readily reduced (Chiarugi et al., 2005; Salmeen & Barford, 2005; Tonks, 2005; Tabernero et al., 2008). Moreover, these two conformational changes may represent a signal that the given protein tyrosine phosphatase is in a temporarily inactive state (Tabernero et al., 2008), which may be important to stabilize associations within protein complexes, to modify structures, to create, destroy or modulate functional sites, or to regulate enzymatic

Dual-specific phosphatases, such as Cdc25, PTEN, and LMW-PTP, and classical protein tyrosine phosphatases are also a target of ROS oxidation. Nevertheless, these enzymes contain a second cysteine residue within the active site. Following oxidation of the cysteine in the signature motif, a disulfide bond is formed with the vicinal cysteine, which protects the enzyme from higher oxidation and irreversible inactivation. Intramolecular disulfide bond formation involving the catalytic cysteine of classical PTPs has not been observed (den

Different phosphatases present distinct sensitivities to oxidation. Regulation of the oxidized states of the cysteine may vary with the structural distances between the cysteines involved in the formation of disulfide bridge and the availability of thiolate ions. This may represent a degree of specificity for protein tyrosine phosphatase redox regulation and lead to a

Inactivation of the protein tyrosine phosphatases by hydrogen peroxide could be reversed by thiol-reducing agents such as dithiothreitol (DTT), β-mercaptoethanol, reduced glutathione, cysteine or by the action of some enzymes in which their catalytic cycle involves either a mono- or dithiol mechanism, such as glutaredoxins, thioredoxins, and protein disulfide-isomerase that have a CXXC motif in the active site. Thioredoxins are able to reduce sulfenic acids and disulfide bonds, while glutaredoxins are involved in the reversibility of mixed disulfides with GSH and S-nitrosothiols (Chiarugi & Buricchi, 2007). The oxidative modification of protein tyrosine phosphatases can be stable over prolonged periods (Salmeen and Barford, 2005). In some cases, the sulfhydryl group is open to further irreversible oxidation if no cysteine derivatives or compounds containing thiol groups are close enough to facilitate the formation of a disulfide bridge or a sulfenyl-amide ring. The addition of one or two oxygen molecules results in the formation of sulfinic acid (Cys-SO2)

or transcriptional activities of proteins (Poole & Nelson, 2008).

Hertog et al., 2005; Lee et al., 2002; Tonks, 2005).

different extent of oxidation (Chiarugi & Buricchi, 2007).

(Cross & Templeton, 2006).

Fig. 3. Sources of reactive oxygen species. Superoxide (O2•-) can be produced from different sources as within the mitochondrial electron transport chain, the activity of xanthine oxidases, Nox members like NADPH oxidase or Dual oxidase, and lipoxygenases. Later, superoxide may be dismuted to hydrogen peroxide (H2O2) through the action of superoxide dismutase (SOD). H2O2 can then act as second messenger in cell signaling.

For tyrosine phosphatases, the typical element of redox regulation occurs within the catalytic site of the enzyme. Two specific amino acid residues are essential to the catalysis of tyrosine phosphatases, Cys215 and Arg221. Oxidation of the catalytic cysteine of the protein tyrosine phosphatases occurs *in vivo* in response to intracellular ROS production, which can occur in response to a variety of stimuli, such as platelet-derivative growth factor (PDGF), epidermal growth factor (EGF), insulin, several other cytokines, and integrins (Rhee et al., 2003; Tonks, 2005). Cysteine residues are also affected by extracellularly added ROS or by an imbalance of the redox potential within the cell. Therefore, hydrogen peroxide, Snitrosylation, or other radical species may oxidize cysteine residues in proteins to form cysteine sulfenic acid (Cys-SOH), which can be further stabilized by the formation of interor intramolecular disulfide (S-S) or sulfenyl-amide bonds. The last conformation is characterized by a five-membered ring that is formed by the covalent linkage of an atom of the catalytic cysteine to an amide nitrogen in a neighboring residue (Chiarugi & Buricchi, 2007; Denu & Tanner, 1998; Janssen-Heininger et al., 2008).

NADPH oxidase

> O2 •−

> > H2O2

Cell signaling

Fig. 3. Sources of reactive oxygen species. Superoxide (O2•-) can be produced from different sources as within the mitochondrial electron transport chain, the activity of xanthine oxidases, Nox members like NADPH oxidase or Dual oxidase, and lipoxygenases. Later, superoxide may be dismuted to hydrogen peroxide (H2O2) through the action of superoxide

For tyrosine phosphatases, the typical element of redox regulation occurs within the catalytic site of the enzyme. Two specific amino acid residues are essential to the catalysis of tyrosine phosphatases, Cys215 and Arg221. Oxidation of the catalytic cysteine of the protein tyrosine phosphatases occurs *in vivo* in response to intracellular ROS production, which can occur in response to a variety of stimuli, such as platelet-derivative growth factor (PDGF), epidermal growth factor (EGF), insulin, several other cytokines, and integrins (Rhee et al., 2003; Tonks, 2005). Cysteine residues are also affected by extracellularly added ROS or by an imbalance of the redox potential within the cell. Therefore, hydrogen peroxide, Snitrosylation, or other radical species may oxidize cysteine residues in proteins to form cysteine sulfenic acid (Cys-SOH), which can be further stabilized by the formation of interor intramolecular disulfide (S-S) or sulfenyl-amide bonds. The last conformation is characterized by a five-membered ring that is formed by the covalent linkage of an atom of the catalytic cysteine to an amide nitrogen in a neighboring residue (Chiarugi & Buricchi,

dismutase (SOD). H2O2 can then act as second messenger in cell signaling.

2007; Denu & Tanner, 1998; Janssen-Heininger et al., 2008).

**SOD**

oxidase Lipoxygenases

Dual

Xanthine oxidase

Different cysteine residues can take part in the disulfide bridge, such as what may occur with the catalytic cysteine of LMW-PTP or PTEN with another cysteine residue or between the classical protein tyrosine phosphatases such as PTP1B and a cysteine from GSH (Barrett et al., 1999; Chiarugi et al., 2001; den Hertog et al., 2005; Kwon et al., 2004; Lee et al., 1998; Salmeen & Barford, 2005). The tripeptide GSH can also induce alterations of proteins function by the formation of mixed disulfide bonds, a reversible process referred to as glutathionylation. The last conformation can result in a temporarily inhibition of PTP activity that protects enzyme from irreversible oxidation. Glutathionylation can be easily reversed by the action of glutaredoxins or thioredoxins, restoring normal enzyme function (Cross & Templeton, 2006).

It is assumed that both disulfide bonds or sulfenyl-amide conformations protect the reactive cysteine from a higher-order, irreversible oxidation or expose the oxidized cysteine to cellular reducing agents, which facilitates the reactivation of the oxidized cysteine, because sulfenyl-amide rings or disulfide bonds represent reversibly oxidized states of cysteine and can be readily reduced (Chiarugi et al., 2005; Salmeen & Barford, 2005; Tonks, 2005; Tabernero et al., 2008). Moreover, these two conformational changes may represent a signal that the given protein tyrosine phosphatase is in a temporarily inactive state (Tabernero et al., 2008), which may be important to stabilize associations within protein complexes, to modify structures, to create, destroy or modulate functional sites, or to regulate enzymatic or transcriptional activities of proteins (Poole & Nelson, 2008).

Dual-specific phosphatases, such as Cdc25, PTEN, and LMW-PTP, and classical protein tyrosine phosphatases are also a target of ROS oxidation. Nevertheless, these enzymes contain a second cysteine residue within the active site. Following oxidation of the cysteine in the signature motif, a disulfide bond is formed with the vicinal cysteine, which protects the enzyme from higher oxidation and irreversible inactivation. Intramolecular disulfide bond formation involving the catalytic cysteine of classical PTPs has not been observed (den Hertog et al., 2005; Lee et al., 2002; Tonks, 2005).

Different phosphatases present distinct sensitivities to oxidation. Regulation of the oxidized states of the cysteine may vary with the structural distances between the cysteines involved in the formation of disulfide bridge and the availability of thiolate ions. This may represent a degree of specificity for protein tyrosine phosphatase redox regulation and lead to a different extent of oxidation (Chiarugi & Buricchi, 2007).

Inactivation of the protein tyrosine phosphatases by hydrogen peroxide could be reversed by thiol-reducing agents such as dithiothreitol (DTT), β-mercaptoethanol, reduced glutathione, cysteine or by the action of some enzymes in which their catalytic cycle involves either a mono- or dithiol mechanism, such as glutaredoxins, thioredoxins, and protein disulfide-isomerase that have a CXXC motif in the active site. Thioredoxins are able to reduce sulfenic acids and disulfide bonds, while glutaredoxins are involved in the reversibility of mixed disulfides with GSH and S-nitrosothiols (Chiarugi & Buricchi, 2007).

The oxidative modification of protein tyrosine phosphatases can be stable over prolonged periods (Salmeen and Barford, 2005). In some cases, the sulfhydryl group is open to further irreversible oxidation if no cysteine derivatives or compounds containing thiol groups are close enough to facilitate the formation of a disulfide bridge or a sulfenyl-amide ring. The addition of one or two oxygen molecules results in the formation of sulfinic acid (Cys-SO2)

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 267

Cellular signaling processes are controlled by the coordinated activity of protein phosphatases and kinases. Protein tyrosine kinases (PTKs) are usually divided into two families, the transmembrane receptor family and the nonreceptor family (Blume-Jensen & Hunter, 2001). Receptor tyrosine kinases (RTKs) are cell-surface, transmembrane receptors carrying a multidomain extracellular segment that binds polypeptide ligands, a single-pass transmembrane helix, and a cytoplasmic segment containing a tyrosine kinase domain together with several regulatory sequences located both N- and C-terminal to the kinase domain. The receptor tyrosine kinase family includes epidermal growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and the insulin receptor kinase (IRK),

The kinase activities of protein tyrosine kinases are closely regulated through autoregulatory mechanisms and by the action of tyrosine phosphatases. In general, most tyrosine kinases are maintained in a low activity state through a variety of these autoregulatory mechanisms and by avoiding the most favorable configuration of the kinase active site. Activation of receptor tyrosine kinases is achieved through a ligand binding to the extracellular domain, which stabilizes a dimeric receptor arrangement and facilitates *trans*-phosphorylation in the cytoplasmic domain. Autophosphorylation of one or two conserved tyrosines within the juxtamembrane domain of these receptors is required for

The classic receptor-mediated signaling involves a ligand engagement, followed by the production of a diffusible second messenger that interacts with a target to affect the signal. This arrangement supports both signal transduction over space and signal amplification, because most second messengers are produced via an enzymatic process. Typically, second messengers are small, diffusible molecules that rapidly activate effector proteins such as protein kinases, phosphatases or ion channels. These molecules can act through binding or

It is now widely accepted that hydrogen peroxide may function as a second messenger within cells by being able to induce tyrosine phosphorylation of cellular proteins, which is strongly potentiated by a combination treatment with vanadate. The mechanism was believed to be attributed to the inhibition of tyrosine phosphatase, the activation of tyrosine kinases, or both (Chiarugi & Buricchi, 2007). Furthermore, the addition of exogenous hydrogen peroxide to cells can mimic the effects of growth factors or hormones and lead to hyperphosphorylation of receptor tyrosine kinases. Mitogen-activated protein kinases have also been shown to be activated by extracellular hydrogen peroxide through a specific

Little is known about how hydrogen peroxide is actually distributed to the cytoplasm. Most of the time, Nox family members are responsible for producing hydrogen peroxide near the receptor at the plasma membrane; in addition, hydrogen peroxide, which is produced in

**6. Cellular signaling through oxidative inhibition of the protein tyrosine** 

**phosphatases** 

**6.1 Receptor tyrosine kinases** 

among others (Chiarugi & Buricchi, 2007).

kinase cascade (Torres, 2003).

complete kinase activation (Chiarugi & Buricchi, 2007).

**6.2 Hydrogen peroxide as a second messenger in cellular signaling** 

chemical modification, thus promoting signal transduction (K. Chen et al., 2009).

or sulfonic acid (Cys-SO3), respectively. These oxidative modifications are irreversible, and the phosphatase will be unable to become active again, even in a reducing environment (Groeger et al., 2009). However, it has been shown recently that sulfinic acids can be reduced by the action of a specific enzyme called sulfiredoxin, which has only been described in mammals and yeast so far (Biteau et al., 2003; Woo et al., 2003).

The redox balance of the cells can affect the ability of ROS to oxidize a given protein. At normal cellular conditions, the cellular redox potential may vary from –260 mV to –150 mV. In addition, protein oxidation will depend on the distance between the site of ROS production and the target protein and on the concentration of scavenger enzymes available. The length of time that the protein will be inactivated will also depend on the proximity to enzymes such as glutaredoxin and thioredoxin, which catalyze thiol reduction (Salmeen & Barford, 2005). Regarding protein tyrosine phosphatases, the differences in pKa values of catalytic cysteines, the availability of thiolate ions and the structural distance between the two cysteines involved in the disulfide bridge may also strongly determine the ability of the phosphatases to be rapidly regulated by changes in the intracellular redox conditions, thereby representing a degree of specificity for tyrosine phosphatase regulation (Chiarugi et al., 2005).

Fig. 4. The redox modulation of a protein tyrosine phosphatase thiolate anion. Because of its low pKa value, the cysteine residue in the active site of the tyrosine phosphatase is present in its thiolate anion form. This conformation facilitates the inhibition of the enzyme by oxidation, which would become inactivated as the sulfenic acid form. Furthermore, sulfenic acid is converted to a more stable conformation, such as a disulfide bridge or sulfenyl-amide ring. Both conformations are stable and reversible through the action of reductant agents. However, if oxidation persists for a long period, stronger and higher oxidation could lead to the formation of sulfinic and sulfonic acids, which are irreversible conformations. In some cases, sulfinic acid can be reversed enzymatically through the action of sulfiredoxin (Sphix) enzyme.

#### **6. Cellular signaling through oxidative inhibition of the protein tyrosine phosphatases**

#### **6.1 Receptor tyrosine kinases**

266 Enzyme Inhibition and Bioapplications

or sulfonic acid (Cys-SO3), respectively. These oxidative modifications are irreversible, and the phosphatase will be unable to become active again, even in a reducing environment (Groeger et al., 2009). However, it has been shown recently that sulfinic acids can be reduced by the action of a specific enzyme called sulfiredoxin, which has only been described in

The redox balance of the cells can affect the ability of ROS to oxidize a given protein. At normal cellular conditions, the cellular redox potential may vary from –260 mV to –150 mV. In addition, protein oxidation will depend on the distance between the site of ROS production and the target protein and on the concentration of scavenger enzymes available. The length of time that the protein will be inactivated will also depend on the proximity to enzymes such as glutaredoxin and thioredoxin, which catalyze thiol reduction (Salmeen & Barford, 2005). Regarding protein tyrosine phosphatases, the differences in pKa values of catalytic cysteines, the availability of thiolate ions and the structural distance between the two cysteines involved in the disulfide bridge may also strongly determine the ability of the phosphatases to be rapidly regulated by changes in the intracellular redox conditions, thereby representing a

> **PTP-S-O2H Sulfinic acid**

**ROS ROS ROS**

Sphix

Fig. 4. The redox modulation of a protein tyrosine phosphatase thiolate anion. Because of its low pKa value, the cysteine residue in the active site of the tyrosine phosphatase is present in its thiolate anion form. This conformation facilitates the inhibition of the enzyme by oxidation, which would become inactivated as the sulfenic acid form. Furthermore, sulfenic acid is converted to a more stable conformation, such as a disulfide bridge or sulfenyl-amide ring. Both conformations are stable and reversible through the action of reductant agents. However,

**PTP-S-S-Protein Disulfide with protein**

if oxidation persists for a long period, stronger and higher oxidation could lead to the formation of sulfinic and sulfonic acids, which are irreversible conformations. In some cases, sulfinic acid can be reversed enzymatically through the action of sulfiredoxin (Sphix) enzyme.

**Protein-SH H2O**

**PTP-S-N Sulfenylamide**

**Reversible**

**PTP-S-O3H Sulfonic acid**

**Irreversible**

mammals and yeast so far (Biteau et al., 2003; Woo et al., 2003).

**PTP-S-Thiolate anion**

Reduction

Reduction

Reduction

degree of specificity for tyrosine phosphatase regulation (Chiarugi et al., 2005).

**PTP-S-OH Sulfenic acid**

**H2O Amino acid-NH**

Cellular signaling processes are controlled by the coordinated activity of protein phosphatases and kinases. Protein tyrosine kinases (PTKs) are usually divided into two families, the transmembrane receptor family and the nonreceptor family (Blume-Jensen & Hunter, 2001). Receptor tyrosine kinases (RTKs) are cell-surface, transmembrane receptors carrying a multidomain extracellular segment that binds polypeptide ligands, a single-pass transmembrane helix, and a cytoplasmic segment containing a tyrosine kinase domain together with several regulatory sequences located both N- and C-terminal to the kinase domain. The receptor tyrosine kinase family includes epidermal growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and the insulin receptor kinase (IRK), among others (Chiarugi & Buricchi, 2007).

The kinase activities of protein tyrosine kinases are closely regulated through autoregulatory mechanisms and by the action of tyrosine phosphatases. In general, most tyrosine kinases are maintained in a low activity state through a variety of these autoregulatory mechanisms and by avoiding the most favorable configuration of the kinase active site. Activation of receptor tyrosine kinases is achieved through a ligand binding to the extracellular domain, which stabilizes a dimeric receptor arrangement and facilitates *trans*-phosphorylation in the cytoplasmic domain. Autophosphorylation of one or two conserved tyrosines within the juxtamembrane domain of these receptors is required for complete kinase activation (Chiarugi & Buricchi, 2007).

#### **6.2 Hydrogen peroxide as a second messenger in cellular signaling**

The classic receptor-mediated signaling involves a ligand engagement, followed by the production of a diffusible second messenger that interacts with a target to affect the signal. This arrangement supports both signal transduction over space and signal amplification, because most second messengers are produced via an enzymatic process. Typically, second messengers are small, diffusible molecules that rapidly activate effector proteins such as protein kinases, phosphatases or ion channels. These molecules can act through binding or chemical modification, thus promoting signal transduction (K. Chen et al., 2009).

It is now widely accepted that hydrogen peroxide may function as a second messenger within cells by being able to induce tyrosine phosphorylation of cellular proteins, which is strongly potentiated by a combination treatment with vanadate. The mechanism was believed to be attributed to the inhibition of tyrosine phosphatase, the activation of tyrosine kinases, or both (Chiarugi & Buricchi, 2007). Furthermore, the addition of exogenous hydrogen peroxide to cells can mimic the effects of growth factors or hormones and lead to hyperphosphorylation of receptor tyrosine kinases. Mitogen-activated protein kinases have also been shown to be activated by extracellular hydrogen peroxide through a specific kinase cascade (Torres, 2003).

Little is known about how hydrogen peroxide is actually distributed to the cytoplasm. Most of the time, Nox family members are responsible for producing hydrogen peroxide near the receptor at the plasma membrane; in addition, hydrogen peroxide, which is produced in

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 269

dephosphorylate receptor tyrosine kinases becomes temporarily compromised because of the oxidation of the catalytic site of the tyrosine phosphatases. These events are involved in the enhancement of the signaling response through an overall increase in tyrosine phosphorylation. Therefore, complete activation of the tyrosine kinase signaling cascades requires both an inhibition of protein tyrosine phosphatases by oxidation and an activation of receptor tyrosine kinases by phosphorylation (Salmeen & Barford, 2005). Redox signaling through the reversible oxidation of tyrosine protein phosphatases must occur through a reaction that is chemically reversible under physiological conditions to ensure the continuity

Fig. 5. After ligand engagement, the receptor tyrosine kinase (RTK) needs the first phase

Besides their regulatory functions, the regulatory domains in tyrosine phosphatases are also involved in the sensitivity to oxidation, which probably contributes to the intrinsic sensitivity differences between various protein tyrosine phosphatases to oxidative inhibition

in which the tyrosine phosphorylation level must be high to guarantee signal propagation. This high level is granted by a transient inhibition of protein tyrosine phosphatase (PTP) due to oxidation promoted by hydrogen peroxide and by the disrupted association with the receptor substrates. Hydrogen peroxide is produced by NADPH oxidase that also became activated by ligand stimulation of the receptor tyrosine

kinase. The concomitant tyrosine phosphorylation of oxidized protein tyrosine phosphatase, which is performed in response to receptor tyrosine kinase activation, is ineffective on the abolished enzymatic activity and preparative for the second phase. In the signaling termination phase, the tyrosine phosphatase recovers its enzymatic activity due to a re-reduction of the catalytic thiol group and immediately becomes hyperactive due to the previous tyrosine phosphorylation, thus promoting the rapid and efficient termination of the signal. The tyrosine phosphatase recurrence after oxidation is followed by a dephosphorylation of the activated receptors, which consequently terminates the

of cellular mechanisms.

signal (Chiarugi et al., 2005).

response to stimulation by a ligand, is released outside of the cell to then be diffused locally into the cell. Also, NADPH oxidase and lipoxygenase, which can be localized within the endoplasmic reticulum and the nuclear membrane, are able to release hydrogen peroxide into the luminal spaces of the specific organelles (Chiarugi & Buricchi, 2007; Tonks, 2005).

Until recently, it was thought that hydrogen peroxide would cross biological membranes freely; however, many membranes were shown to be almost impermeable to hydrogen peroxide as well as to water (Antunes & Cadenas, 2000; Makino et al., 2004; Seaver & Imlay, 2001). Thus, for transport, hydrogen peroxide must be carried by an appropriate transporter that has not yet been well characterized (Chiarugi & Buricchi, 2007).

#### **6.3 Nitric oxide as a second messenger in cellular signaling**

Nitric oxide is a gas, which readily diffuses across membranes and does not act at conventional membrane associated receptors (Do et al., 1996). Like hydrogen peroxide, signal transduction through induced-nitric-oxide modifications relies on the system of Cysbased posttranslational modifications. Accordingly, S-nitrosylation of proteins plays an essential role in downstream cascades.

Nitric oxide exerts a ubiquitous influence on cellular signaling in large part by means of Snitrosylation/denitrosylation of protein cysteine residues. SNO, or higher oxides is considered a post-translational protein modification that is precisely regulated, conferring specificity to NO-derived effects. These NO-dependent modifications influence protein activity, protein-protein interactions, and protein location. S-nitrosylation thus serves as the prototypical redox-based signal (Janssen-Heininger et al., 2008).

S-Nitrosylation has been implicated in transmitting signals downstream of all classes of receptors, including G-protein-coupled receptor (GPCR), receptor tyrosine kinase, tumor necrosis factor, Toll-receptors, and glutaminergic receptors, acting locally within subcellular signaling domains as well conveying signals from the cell surface to intracellular compartments, including the mitochondria and the nucleus (Janssen-Heininger et al., 2008).

Regarding cellular signaling through S-nitrosylation, specific features of this kind of modification can be considered useful tools in signaling transduction According to Janssen-Heininger et al. (2008). These features include:


#### **6.4 The role of protein tyrosine phosphatases in cellular signaling**

Physiological stimuli, such as growth factors or the engagement of antigen receptors, can trigger localized and controlled production of ROS. Protein tyrosine phosphatases, because of their high susceptibility to inactivation by oxidation at physiological pH, are targets of ligand-induced ROS generation. Thus, the ability of tyrosine phosphatases to dephosphorylate receptor tyrosine kinases becomes temporarily compromised because of the oxidation of the catalytic site of the tyrosine phosphatases. These events are involved in the enhancement of the signaling response through an overall increase in tyrosine phosphorylation. Therefore, complete activation of the tyrosine kinase signaling cascades requires both an inhibition of protein tyrosine phosphatases by oxidation and an activation of receptor tyrosine kinases by phosphorylation (Salmeen & Barford, 2005). Redox signaling through the reversible oxidation of tyrosine protein phosphatases must occur through a reaction that is chemically reversible under physiological conditions to ensure the continuity of cellular mechanisms.

268 Enzyme Inhibition and Bioapplications

response to stimulation by a ligand, is released outside of the cell to then be diffused locally into the cell. Also, NADPH oxidase and lipoxygenase, which can be localized within the endoplasmic reticulum and the nuclear membrane, are able to release hydrogen peroxide into the luminal spaces of the specific organelles (Chiarugi & Buricchi, 2007; Tonks, 2005). Until recently, it was thought that hydrogen peroxide would cross biological membranes freely; however, many membranes were shown to be almost impermeable to hydrogen peroxide as well as to water (Antunes & Cadenas, 2000; Makino et al., 2004; Seaver & Imlay, 2001). Thus, for transport, hydrogen peroxide must be carried by an appropriate transporter

Nitric oxide is a gas, which readily diffuses across membranes and does not act at conventional membrane associated receptors (Do et al., 1996). Like hydrogen peroxide, signal transduction through induced-nitric-oxide modifications relies on the system of Cysbased posttranslational modifications. Accordingly, S-nitrosylation of proteins plays an

Nitric oxide exerts a ubiquitous influence on cellular signaling in large part by means of Snitrosylation/denitrosylation of protein cysteine residues. SNO, or higher oxides is considered a post-translational protein modification that is precisely regulated, conferring specificity to NO-derived effects. These NO-dependent modifications influence protein activity, protein-protein interactions, and protein location. S-nitrosylation thus serves as the

S-Nitrosylation has been implicated in transmitting signals downstream of all classes of receptors, including G-protein-coupled receptor (GPCR), receptor tyrosine kinase, tumor necrosis factor, Toll-receptors, and glutaminergic receptors, acting locally within subcellular signaling domains as well conveying signals from the cell surface to intracellular compartments, including the mitochondria and the nucleus (Janssen-Heininger et al., 2008). Regarding cellular signaling through S-nitrosylation, specific features of this kind of modification can be considered useful tools in signaling transduction According to Janssen-

5. Enzymatic control of S-nitrosylation through the action of S-nitrosoglutathione

Physiological stimuli, such as growth factors or the engagement of antigen receptors, can trigger localized and controlled production of ROS. Protein tyrosine phosphatases, because of their high susceptibility to inactivation by oxidation at physiological pH, are targets of ligand-induced ROS generation. Thus, the ability of tyrosine phosphatases to

1. Temporal regulation of response through a rapid and controlled stimulation; 2. The existence of motifs within proteins that provides S-nitrosylation specificity;

that has not yet been well characterized (Chiarugi & Buricchi, 2007).

**6.3 Nitric oxide as a second messenger in cellular signaling** 

prototypical redox-based signal (Janssen-Heininger et al., 2008).

essential role in downstream cascades.

Heininger et al. (2008). These features include:

4. Reversibility of protein S-nitrosylation;

reductase.

3. Colocalization of target proteins with a source of NO;

**6.4 The role of protein tyrosine phosphatases in cellular signaling** 

Fig. 5. After ligand engagement, the receptor tyrosine kinase (RTK) needs the first phase in which the tyrosine phosphorylation level must be high to guarantee signal propagation. This high level is granted by a transient inhibition of protein tyrosine phosphatase (PTP) due to oxidation promoted by hydrogen peroxide and by the disrupted association with the receptor substrates. Hydrogen peroxide is produced by NADPH oxidase that also became activated by ligand stimulation of the receptor tyrosine kinase. The concomitant tyrosine phosphorylation of oxidized protein tyrosine phosphatase, which is performed in response to receptor tyrosine kinase activation, is ineffective on the abolished enzymatic activity and preparative for the second phase. In the signaling termination phase, the tyrosine phosphatase recovers its enzymatic activity due to a re-reduction of the catalytic thiol group and immediately becomes hyperactive due to the previous tyrosine phosphorylation, thus promoting the rapid and efficient termination of the signal. The tyrosine phosphatase recurrence after oxidation is followed by a dephosphorylation of the activated receptors, which consequently terminates the signal (Chiarugi et al., 2005).

Besides their regulatory functions, the regulatory domains in tyrosine phosphatases are also involved in the sensitivity to oxidation, which probably contributes to the intrinsic sensitivity differences between various protein tyrosine phosphatases to oxidative inhibition

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 271

physiologically significant rates with a hydroperoxide, such as hydrogen peroxide, unless the reaction is catalyzed. However, thiolates (S−) react faster with hydroperoxides depending on their local environment (Forman et al., 2004). The reaction rate of hydrogen peroxide with protein tyrosine phosphatase is about 105 times slower than with peroxiredoxin. The question appears on how hydrogen peroxide can react specifically with the active site of PTPs before its metabolization by enzymatic scavengers. Two

1. Hydrogen peroxide is generated by NADPH oxidase close to PTP so that the

2. PTPs could be compartmentalized to the sites of ROS generation and far from scavenger

Besides, specificity of ROS to a given PTP may be influenced by contributions from the surrounding amino acids near the catalytic cysteine. The prevalence of basic amino acids near the active site of the enzyme can contribute to the extension formation of thiol ionization giving rise to thiolate anions within active cysteine. Chemical reactivity secondary to the local amino acids environments is likely to contribute to the modification of cysteine residues (Cross & Templeton, 2006). This is the case of disulfide bonds or

Receptor signaling in which receptor activation induces both the activation of downstream kinases and the oxidative inactivation of inhibitory phosphatases are components required for efficient signaling. The regulated and localized production of ROS can inactivate a subpopulation of phosphatase molecules near the site of ROS generation. The specificity of ROS for tyrosine phosphatase exists through different intrinsic sensitivities to oxidation and a tight control over the production of ROS. This production can be achieved by the controlled action of Nox enzymes, including their requirement for assembly into a multiprotein complex, the phosphorylation of regulatory subunits, the binding of inositol phospholipids and the small GTPase, Rac. In addition, regulation of Duox and NADPH oxidases by Ca2+ adds further control of ROS generation in cells (Tonks, 2006; Poole et al., 2004). As an example, Nox4 can regulate the oxidation of PTP1B in response to insulin and localizes with PTP1B on the intracellular membranes. Ecto-phosphatases, which are protein phosphatases that present a catalytic site facing the extracellular medium, are also able to respond to the endogenous production of hydrogen peroxide from mitochondria. The addition of a proton ionophore, which was able to reduce mitochondrial hydrogen peroxide production, was also shown to induce ectophosphatase activity due to the reduction of hydrogen peroxide outside of the cell

The mechanism by which ROS/RNS exerts a regulatory function in cysteine-based protein tyrosine phosphatases is well known, as well as the role of antioxidants in reverse enzyme oxidation. The reversible inhibition of tyrosine phosphatases by ROS has been intensely investigated in some diseases such as cancer and diabetes, but the complete pathway in which these reactive species are involved is not elucidated yet. Some questions still

concentration could be sufficient to cause enzyme modification; or

enzymes (Chiarugi et al., 2005; Forman et al., 2004).

possible mechanisms can arise:

sulfenyl-amide conformations.

(Cosentino-Gomes et al., 2009).

**7. Conclusion** 

(Groeger et al., 2009; Östman et al., 2011). The redox regulation of tyrosine phosphatases may depend on different oxidative stress conditions and the extent of oxidation, as it is tissue and differentiation dependent (Chiarugi et al., 2005). As mentioned above, the transient negative regulation of tyrosine phosphatases due to oxidants is dependent on ligand stimulation of the receptor tyrosine kinases.

Hydrogen peroxide is recognized as one of the main ROS capable of acting as a second messenger because of it is relative stability. Cytokines, growth factors and integrins activate the multicomponent NADPH oxidase enzymes as part of the receptor-mediated signaling in cells. Both superoxide and, hence, hydrogen peroxide are generated, with the latter serving a particularly significant role as a second messenger in signaling, which mediates the regulation of survival pathways in cells (Poole & Nelson, 2008; Groeger et al., 2009).

The tyrosine phosphorylation level of a given protein is the result of the collective action of kinases and phosphatases. Signal transduction by ROS through reversible tyrosine phosphatase inhibition represents a widespread and conserved mechanism that is triggered by the ligand stimulation of receptor tyrosine kinases. Conformational changes lead to an increase in ROS production, which causes a transient negative regulation of protein tyrosine phosphatases that are associated with receptor tyrosine phosphatases, and at the same time decreases receptor tyrosine phosphatase dephosphorylation that leads to a high stimulation of its activity and signal propagation. The same PTP that suffered oxidation can be highly phosphorylated by the protein tyrosine kinase that is now activated. The oxidation of the active site of the tyrosine phosphatase protects the enzyme from autodephosphorylation. When the tyrosine phosphatase returns to the reduced form, its highly phosphorylated state influences a hyperactivation of the enzyme's activity, which is essential to terminating the signaling cascade in a fast way (Chiarugi & Buricchi, 2007). This mechanism represents a strategy adopted by cells to promote receptor tyrosine kinase signaling by avoiding its prompt inactivation by tyrosine phosphatases. After the redox potential has been restabilized, tyrosine phosphatase recovers its activity and the signaling triggered by the receptor tyrosine kinases is terminated (Chiarugi et al., 2005; Chiarugi & Buricchi, 2007). Thus, tyrosine kinase activation by oxidation is considered an indirect effect of tyrosine phosphatase inhibition. The overall mechanism is represented in figure 5.

The distinct but complementary function of kinases and phosphatases clarifies the role of both enzymes in controlling cellular signaling. Therefore, it is possible to affirm that kinases are implicated in controlling the amplitude of a signaling response whereas phosphatases are thought to have an important role in controlling the rate and duration of the response (Tonks, 2006).

A key element of a signaling response is reversibility. Regulated reversible phosphorylation of proteins and other cellular molecules plays a ubiquitous role in the control of cellular behavior. For oxidation to illustrate the mechanism for reversible regulation of protein tyrosine phosphatase function, it is essential that the active site cysteine residue not be oxidized to sulfinic or sulfonic acids, which is usually irreversible (Poole et al., 2004; Tonks, 2005). As mentioned elsewhere, thiol groups (SH) do not react at physiologically significant rates with a hydroperoxide, such as hydrogen peroxide, unless the reaction is catalyzed. However, thiolates (S−) react faster with hydroperoxides depending on their local environment (Forman et al., 2004). The reaction rate of hydrogen peroxide with protein tyrosine phosphatase is about 105 times slower than with peroxiredoxin. The question appears on how hydrogen peroxide can react specifically with the active site of PTPs before its metabolization by enzymatic scavengers. Two possible mechanisms can arise:


Besides, specificity of ROS to a given PTP may be influenced by contributions from the surrounding amino acids near the catalytic cysteine. The prevalence of basic amino acids near the active site of the enzyme can contribute to the extension formation of thiol ionization giving rise to thiolate anions within active cysteine. Chemical reactivity secondary to the local amino acids environments is likely to contribute to the modification of cysteine residues (Cross & Templeton, 2006). This is the case of disulfide bonds or sulfenyl-amide conformations.

Receptor signaling in which receptor activation induces both the activation of downstream kinases and the oxidative inactivation of inhibitory phosphatases are components required for efficient signaling. The regulated and localized production of ROS can inactivate a subpopulation of phosphatase molecules near the site of ROS generation. The specificity of ROS for tyrosine phosphatase exists through different intrinsic sensitivities to oxidation and a tight control over the production of ROS. This production can be achieved by the controlled action of Nox enzymes, including their requirement for assembly into a multiprotein complex, the phosphorylation of regulatory subunits, the binding of inositol phospholipids and the small GTPase, Rac. In addition, regulation of Duox and NADPH oxidases by Ca2+ adds further control of ROS generation in cells (Tonks, 2006; Poole et al., 2004). As an example, Nox4 can regulate the oxidation of PTP1B in response to insulin and localizes with PTP1B on the intracellular membranes. Ecto-phosphatases, which are protein phosphatases that present a catalytic site facing the extracellular medium, are also able to respond to the endogenous production of hydrogen peroxide from mitochondria. The addition of a proton ionophore, which was able to reduce mitochondrial hydrogen peroxide production, was also shown to induce ectophosphatase activity due to the reduction of hydrogen peroxide outside of the cell (Cosentino-Gomes et al., 2009).

### **7. Conclusion**

270 Enzyme Inhibition and Bioapplications

(Groeger et al., 2009; Östman et al., 2011). The redox regulation of tyrosine phosphatases may depend on different oxidative stress conditions and the extent of oxidation, as it is tissue and differentiation dependent (Chiarugi et al., 2005). As mentioned above, the transient negative regulation of tyrosine phosphatases due to oxidants is dependent on

Hydrogen peroxide is recognized as one of the main ROS capable of acting as a second messenger because of it is relative stability. Cytokines, growth factors and integrins activate the multicomponent NADPH oxidase enzymes as part of the receptor-mediated signaling in cells. Both superoxide and, hence, hydrogen peroxide are generated, with the latter serving a particularly significant role as a second messenger in signaling, which mediates the regulation of survival pathways in cells (Poole & Nelson, 2008; Groeger et

The tyrosine phosphorylation level of a given protein is the result of the collective action of kinases and phosphatases. Signal transduction by ROS through reversible tyrosine phosphatase inhibition represents a widespread and conserved mechanism that is triggered by the ligand stimulation of receptor tyrosine kinases. Conformational changes lead to an increase in ROS production, which causes a transient negative regulation of protein tyrosine phosphatases that are associated with receptor tyrosine phosphatases, and at the same time decreases receptor tyrosine phosphatase dephosphorylation that leads to a high stimulation of its activity and signal propagation. The same PTP that suffered oxidation can be highly phosphorylated by the protein tyrosine kinase that is now activated. The oxidation of the active site of the tyrosine phosphatase protects the enzyme from autodephosphorylation. When the tyrosine phosphatase returns to the reduced form, its highly phosphorylated state influences a hyperactivation of the enzyme's activity, which is essential to terminating the signaling cascade in a fast way (Chiarugi & Buricchi, 2007). This mechanism represents a strategy adopted by cells to promote receptor tyrosine kinase signaling by avoiding its prompt inactivation by tyrosine phosphatases. After the redox potential has been restabilized, tyrosine phosphatase recovers its activity and the signaling triggered by the receptor tyrosine kinases is terminated (Chiarugi et al., 2005; Chiarugi & Buricchi, 2007). Thus, tyrosine kinase activation by oxidation is considered an indirect effect of tyrosine phosphatase

The distinct but complementary function of kinases and phosphatases clarifies the role of both enzymes in controlling cellular signaling. Therefore, it is possible to affirm that kinases are implicated in controlling the amplitude of a signaling response whereas phosphatases are thought to have an important role in controlling the rate and duration of the response

A key element of a signaling response is reversibility. Regulated reversible phosphorylation of proteins and other cellular molecules plays a ubiquitous role in the control of cellular behavior. For oxidation to illustrate the mechanism for reversible regulation of protein tyrosine phosphatase function, it is essential that the active site cysteine residue not be oxidized to sulfinic or sulfonic acids, which is usually irreversible (Poole et al., 2004; Tonks, 2005). As mentioned elsewhere, thiol groups (SH) do not react at

ligand stimulation of the receptor tyrosine kinases.

inhibition. The overall mechanism is represented in figure 5.

al., 2009).

(Tonks, 2006).

The mechanism by which ROS/RNS exerts a regulatory function in cysteine-based protein tyrosine phosphatases is well known, as well as the role of antioxidants in reverse enzyme oxidation. The reversible inhibition of tyrosine phosphatases by ROS has been intensely investigated in some diseases such as cancer and diabetes, but the complete pathway in which these reactive species are involved is not elucidated yet. Some questions still

Reversible Inhibition of Tyrosine Protein Phosphatases by Redox Reactions 273

Chiarugi, P., Taddei, M.L. & Ramponi, G. (2005). Oxidation and tyrosine phosphorylation:

Cosentino-Gomes, D., Russo-Abrahão, T., Fonseca-de-Souza, A.L., Ferreira, C.R., Galina, A.

Cross, J.V. & Templeton, D.J. (2006). Regulation of signal transduction through protein

den Hertog, J., Groen, A. & van der Wijk, T. (2005). Redox regulation of protein-tyrosine

den Hertog, J., Tracy, S. & Hunter, T. (1994). Phosphorylation of receptor protein-tyrosine

Denu, J.M. & Dixon, J.E. (1998). Protein tyrosine phosphatases: mechanisms of catalysis and

Denu, J.M. & Tanner, K.G. (1998). Specific and reversible inactivation of protein tyrosine

Do, K.Q., Benz, B., Grima, G., Gutteck-Amsler, U., Kluge, I. & Salt, T.E. (1996). Nitric oxide

Forman, H.J., Zhanq, H. & Rinna, A. (2009). Glutathione: overview of its protective roles,

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#### **8. References**


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**9** 

*Chile* 

**Feasible Novozym 435-Catalyzed Process to** 

**Frying Oil: Role of Lipase Inhibition** 

*Scientific and Technological Bioresources Nucleous, La Frontera University* 

Laura Azócar, Gustavo Ciudad, Robinson Muñoz, David Jeison, Claudio Toro and Rodrigo Navia

**Fatty Acid Methyl Ester Production from Waste** 

Fatty acid methyl ester (FAME) or biodiesel is a biofuel conventionally produced from edible oil and methanol, using an alkaline catalyst, through a transesterification reaction. As FAME is mostly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. In addition, between 70 and 80% of the total FAME production costs correspond to the vegetable oils. Therefore, the use of waste lipids such as waste frying oils (WFO), waste fats and soapstock has been proposed as low-cost alternative to feedstock. Non-edible oils such as jatropha, pongamia and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstock for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole cell catalysts, avoiding soap production since the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered and the

Lipase-catalyzed processes have been widely investigated for FAME production from alternatives raw material. Although interesting results have been reached up to date, the enzymatic catalysis has not become competitive compared to the conventional chemical process. The main reasons explaining this issue are the long reaction time (until 48 h), the loss of enzymatic activity due to methanol use in the reaction and the high operational costs because the lipases cannot be reused. The present chapter described an investigation to a get a feasible lipase-catalyzed process to FAME production from WFO, avoiding lipase

purification of FAME is simplified using biological catalysts.

**1. Introduction** 

inhibition.

Zhao, Z., Larocque, R., Ho, W.T., Fischer, E.H. & Shen, S.H. (1994). Purification and characterization of PTP2C, a widely distributed protein tyrosine phosphatase containing two SH2 domains. *The Journal of Biological Chemistry*, Vol. 269, No 12, pp. 8780-8785, ISSN 1083-351X

## **Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl Ester Production from Waste Frying Oil: Role of Lipase Inhibition**

Laura Azócar, Gustavo Ciudad, Robinson Muñoz, David Jeison, Claudio Toro and Rodrigo Navia *Scientific and Technological Bioresources Nucleous, La Frontera University Chile* 

#### **1. Introduction**

276 Enzyme Inhibition and Bioapplications

Zhao, Z., Larocque, R., Ho, W.T., Fischer, E.H. & Shen, S.H. (1994). Purification and

8780-8785, ISSN 1083-351X

characterization of PTP2C, a widely distributed protein tyrosine phosphatase containing two SH2 domains. *The Journal of Biological Chemistry*, Vol. 269, No 12, pp.

> Fatty acid methyl ester (FAME) or biodiesel is a biofuel conventionally produced from edible oil and methanol, using an alkaline catalyst, through a transesterification reaction. As FAME is mostly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. In addition, between 70 and 80% of the total FAME production costs correspond to the vegetable oils. Therefore, the use of waste lipids such as waste frying oils (WFO), waste fats and soapstock has been proposed as low-cost alternative to feedstock. Non-edible oils such as jatropha, pongamia and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstock for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole cell catalysts, avoiding soap production since the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered and the purification of FAME is simplified using biological catalysts.

> Lipase-catalyzed processes have been widely investigated for FAME production from alternatives raw material. Although interesting results have been reached up to date, the enzymatic catalysis has not become competitive compared to the conventional chemical process. The main reasons explaining this issue are the long reaction time (until 48 h), the loss of enzymatic activity due to methanol use in the reaction and the high operational costs because the lipases cannot be reused. The present chapter described an investigation to a get a feasible lipase-catalyzed process to FAME production from WFO, avoiding lipase inhibition.

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

Table 1. Variable and levels used in the response surface methodology

between terms and their effects on FAME production yield.

04) and flash point (ASTM Standard D 56-02a).

Independent variables Symbols

yield may be written as follows (Eq. 1):

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 279

WFO in feedstock [wt %] X1 0 25 50 75 100 Methanol to oil ratio [mol/mol] X2 1.50 2.25 3.00 3.75 4.50 Temperature [°C] X3 35 40 45 50 55 Novozym 435 [wt %] X4 3 6 9 12 15

The experimental data obtained were fitted to a second-order polynomial equation. This equation describes the relationship between the predicted response variable (FAME production yield) and the independent variables (Table 1). The polynomial model for FAME

Where β (0 = intercept, i = linear, ii = quadratic and ij = interaction) and Xi, Xj (i = 1, 4; j = 1, 4; i ≠ j represent the coded independent variables) are the model coefficients. With the fitted quadratic polynomial equation, contour plots were developed to analyze the interaction

To identify and quantify the fatty acids in the feedstock a Clarus 600 chromatograph coupled to a Clarus 500T mass spectrometer of Perkin Elmer (GC-MS) was utilized. An Elite-5ms capillary column with a length of 30 m, thickness of 0.1 µm and internal diameter of 0.25 mm was used. The vials were prepared by adding 3 µg of sample to 100 µL methyl heptadecanoate as an internal standard (initial concentration of 1300 mg/L). The following temperature program was used: 50°C for 1 min and then increasing temperature at a rate of 1.1°C/min up to 187°C. Both the injector and detector temperatures were 250°C and He was used as the carrier gas. Before injection into the GC-MS equipment, the WFO and rapeseed oil were methylated according to Araújo (1995). Specific gravity was measured at 20°C using a manual densimeter. Kinematic viscosity was measured at 40°C using a capillary viscosimeter. The acid value was determined by titration with KOH using phenolphthalein as an indicator. The peroxide value was determined by titration with Na2S2O3, and the iodine value was determined by the Wijs method (Araújo 1995). The following parameters were measured according to ASTM Standard Methods: water and sediments (ASTM Standard D 1976-97), pour point (ASTM Standard D 97-04), sulfur (ASTM Standard D 7039-

To establish the degree of oil conversion, monoacylglycerols (MG), diacylglycerols (DG), triacylglycerides (TG) and FFA in the oil feedstock were quantified using an HP 6890 series gas chromatography system (GC-MS) with an adaptation of the EN-14214 (The)methodology. TG was determined by mass balance. A 50-m long BPX-5 column with a thickness of 0.5 µm and an internal diameter of 0.32 mm was used. The vials were prepared by combining 10 mg of sample with 0.8 µL of 1,2,3-butanetriol and 10 µL of 1,2,3 tricaprinoylglycerol dissolved in pyridine as internal standards. N-methyl-N-

44 4 2 0 i i ii i ij i j i=1 i=1 i<j=1

FAME yield=<sup>β</sup> <sup>+</sup> <sup>β</sup> X + <sup>β</sup> X + <sup>β</sup> X X (1)

Levels -2 -1 0 1 2

#### **2. Lipase catalyzed process to FAME production from waste frying oil: Improving the yield**

In spite of that some investigations have been carried using WFO instead of edible oils in FAME production using lipases, there is not clear the effect in the process of replace the raw material. Using *Rhizopus oryzae* as the biocatalyst, FFA from a synthetic WFO were esterified to produce FAME with an improved reaction yield (Li et al., 2007). In addition, using *Thermomyces lanuginosus* lipases immobilized on a microporous polymer, 97% FAME content from edible sunflower oil was reached, while only 90.2% FAME content was obtained from WFO (Dizge et al., 2009). Watanabe et al. (2001) tested the immobilized the *Candida antartica* lipase immobilized on acrylic resin (Novozym 435) and obtained a 5.5% reduction in FAME conversion yield when using WFO compared to edible oil as the feedstock. They concluded that the oxidized fatty acid compounds in WFO may be responsible for this decrease.

As the effects of using WFO instead of edible oil in lipase-catalyzed processes are not clear, the aim of this study was to elucidate the effect of WFO incorporation in feedstock mixed with rapeseed oil on FAME production yield using Novozym 435 as the catalyst by means of the response surface methodology (RSM). In addition, specific WFO and rapeseed oil chemical characteristics were investigated to identify the components that were responsible for these results. Finally, a preliminary study to establish the optimal time for methanol addition during the reaction was proposed.

Both filtered WFO collected from restaurants and crude rapeseed oil from a local factory from Southern Chile were used as the feedstocks. Novozym 435 from Sigma-Aldrich was used as the catalyst. Methyl heptadecanoate, 1,2,3-butanetriol and 1,2,3-tricaprinoylglycerol were used as internal standards and were chromatographically pure.

RSM was used to analyze and optimize the interaction effects of four variables on FAME production yield (Table 1): the WFO content in the feedstock mixture (% wt), the final methanol-to-oil ratio (mol/mol), temperature (°C) and Novozym 435 dosage (% wt based on oil weight). A central composite matrix with 5 levels was used and 30 runs were carried out in a random order. Each run was performed in triplicate.

All reactions were incubated in flasks containing 1 mL of oil at 200 rpm. The volume of the flasks was selected to maintain perfect agitation of the samples when using both the highest dosage of catalyst and the lowest methanol-to-oil molar ratio. Under these conditions, different combinations of feedstock mixture, dosage of catalyst, methanol-to-oil molar ratio and temperature were used. Methanol was added in two steps to avoid lipase inhibition (Shimada et al., 2002). In the first step, one-third of the total molar ratio was added according to Table 1, while in the second step, the remaining two-thirds of the total molar ratio was added to generate the final methanol-to-oil molar ratio.

To establish the reaction and second methanol addition times, a preliminary study was performed. This experiment was carried out for 48 h using the RSM central point, with 50% (wt) WFO in a mixed feedstock, a methanol/oil molar ratio of 3:1 and 9% (wt) Novozym 435 at 45°C and stirring 200 rpm.

Samples were immediately stored at 4°C to stop the reaction. The upper layer was analyzed by gas chromatography for FAME quantification.

In spite of that some investigations have been carried using WFO instead of edible oils in FAME production using lipases, there is not clear the effect in the process of replace the raw material. Using *Rhizopus oryzae* as the biocatalyst, FFA from a synthetic WFO were esterified to produce FAME with an improved reaction yield (Li et al., 2007). In addition, using *Thermomyces lanuginosus* lipases immobilized on a microporous polymer, 97% FAME content from edible sunflower oil was reached, while only 90.2% FAME content was obtained from WFO (Dizge et al., 2009). Watanabe et al. (2001) tested the immobilized the *Candida antartica* lipase immobilized on acrylic resin (Novozym 435) and obtained a 5.5% reduction in FAME conversion yield when using WFO compared to edible oil as the feedstock. They concluded that the oxidized fatty acid compounds in WFO may be

As the effects of using WFO instead of edible oil in lipase-catalyzed processes are not clear, the aim of this study was to elucidate the effect of WFO incorporation in feedstock mixed with rapeseed oil on FAME production yield using Novozym 435 as the catalyst by means of the response surface methodology (RSM). In addition, specific WFO and rapeseed oil chemical characteristics were investigated to identify the components that were responsible for these results. Finally, a preliminary study to establish the optimal time for methanol

Both filtered WFO collected from restaurants and crude rapeseed oil from a local factory from Southern Chile were used as the feedstocks. Novozym 435 from Sigma-Aldrich was used as the catalyst. Methyl heptadecanoate, 1,2,3-butanetriol and 1,2,3-tricaprinoylglycerol

RSM was used to analyze and optimize the interaction effects of four variables on FAME production yield (Table 1): the WFO content in the feedstock mixture (% wt), the final methanol-to-oil ratio (mol/mol), temperature (°C) and Novozym 435 dosage (% wt based on oil weight). A central composite matrix with 5 levels was used and 30 runs were carried out

All reactions were incubated in flasks containing 1 mL of oil at 200 rpm. The volume of the flasks was selected to maintain perfect agitation of the samples when using both the highest dosage of catalyst and the lowest methanol-to-oil molar ratio. Under these conditions, different combinations of feedstock mixture, dosage of catalyst, methanol-to-oil molar ratio and temperature were used. Methanol was added in two steps to avoid lipase inhibition (Shimada et al., 2002). In the first step, one-third of the total molar ratio was added according to Table 1, while in the second step, the remaining two-thirds of the total molar

To establish the reaction and second methanol addition times, a preliminary study was performed. This experiment was carried out for 48 h using the RSM central point, with 50% (wt) WFO in a mixed feedstock, a methanol/oil molar ratio of 3:1 and 9% (wt) Novozym 435

Samples were immediately stored at 4°C to stop the reaction. The upper layer was analyzed

were used as internal standards and were chromatographically pure.

in a random order. Each run was performed in triplicate.

ratio was added to generate the final methanol-to-oil molar ratio.

**2. Lipase catalyzed process to FAME production from waste frying oil:** 

**Improving the yield** 

responsible for this decrease.

at 45°C and stirring 200 rpm.

by gas chromatography for FAME quantification.

addition during the reaction was proposed.


Table 1. Variable and levels used in the response surface methodology

The experimental data obtained were fitted to a second-order polynomial equation. This equation describes the relationship between the predicted response variable (FAME production yield) and the independent variables (Table 1). The polynomial model for FAME yield may be written as follows (Eq. 1):

$$\text{FAME yield} = \beta\_0 + \sum\_{i=1}^{4} \beta\_i \mathbf{X}\_i + \sum\_{i=1}^{4} \beta\_{\text{ii}} \mathbf{X}\_i^2 + \sum\_{i \preceq j=1}^{4} \sum\_{j=1}^{4} \beta\_{\overline{j}} \mathbf{X}\_i \mathbf{X}\_j \tag{1}$$

Where β (0 = intercept, i = linear, ii = quadratic and ij = interaction) and Xi, Xj (i = 1, 4; j = 1, 4; i ≠ j represent the coded independent variables) are the model coefficients. With the fitted quadratic polynomial equation, contour plots were developed to analyze the interaction between terms and their effects on FAME production yield.

To identify and quantify the fatty acids in the feedstock a Clarus 600 chromatograph coupled to a Clarus 500T mass spectrometer of Perkin Elmer (GC-MS) was utilized. An Elite-5ms capillary column with a length of 30 m, thickness of 0.1 µm and internal diameter of 0.25 mm was used. The vials were prepared by adding 3 µg of sample to 100 µL methyl heptadecanoate as an internal standard (initial concentration of 1300 mg/L). The following temperature program was used: 50°C for 1 min and then increasing temperature at a rate of 1.1°C/min up to 187°C. Both the injector and detector temperatures were 250°C and He was used as the carrier gas. Before injection into the GC-MS equipment, the WFO and rapeseed oil were methylated according to Araújo (1995). Specific gravity was measured at 20°C using a manual densimeter. Kinematic viscosity was measured at 40°C using a capillary viscosimeter. The acid value was determined by titration with KOH using phenolphthalein as an indicator. The peroxide value was determined by titration with Na2S2O3, and the iodine value was determined by the Wijs method (Araújo 1995). The following parameters were measured according to ASTM Standard Methods: water and sediments (ASTM Standard D 1976-97), pour point (ASTM Standard D 97-04), sulfur (ASTM Standard D 7039- 04) and flash point (ASTM Standard D 56-02a).

To establish the degree of oil conversion, monoacylglycerols (MG), diacylglycerols (DG), triacylglycerides (TG) and FFA in the oil feedstock were quantified using an HP 6890 series gas chromatography system (GC-MS) with an adaptation of the EN-14214 (The)methodology. TG was determined by mass balance. A 50-m long BPX-5 column with a thickness of 0.5 µm and an internal diameter of 0.32 mm was used. The vials were prepared by combining 10 mg of sample with 0.8 µL of 1,2,3-butanetriol and 10 µL of 1,2,3 tricaprinoylglycerol dissolved in pyridine as internal standards. N-methyl-N-

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

diminishing the reaction productivity.

12 hours.

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 281

This two-step technique of methanol addition was shown to diminish the reaction time by obviously preventing enzyme inhibition. Li et al. (2009) showed that in spite of the high yield reached using stepwise methanol addition, both the reaction time and rate of FAME conversion were slower. In experiments using a three-step methanol addition and 4% (wt) Novozym 435 as the catalyst, high FAME production yields were reached but only after 50 hours of reaction (Du et al., 2004; Watanabe et al., 2001). Similarly, in another recent study, an reaction time of 50 hours was also used (Ognjanovic et al., 2009). Usually, the second methanol addition starts after one-third (33.3%) of the FAME content has been generated and the initially added methanol has been completely consumed (Du et al., 2004; Shimada et al., 2002). This methodology produces a stationary phase of FAME production yield,

As Novozym 435 was shown to be a robust and stable catalyst compared to other lipases in the presence of short chain alcohols (Hernandez-Martin et al., 2008), all of the experiments in this study were performed using this commercially available immobilized enzyme. When approximately 28.6% of the FAME content was reached after 8 hours of reaction, the second aliquot of methanol was added. This stepwise addition of methanol increased the reaction productivity by avoiding the stationary production phase and led to a plateau in FAME production at 12 hours. Similar reductions in the reaction time by shortening the stationary phase of FAME production have already been reported (Li et al., 2009). Therefore, in the RSM experiments, the first amount of methanol (one-third of the molar ratio shown in Table 1) was added at the start of the reaction, while the second aliquot of methanol (two-thirds of the molar ratio shown in Table 1) was added after 8 hours, and the total reaction time was

*Optimization of methanolysis conditions.* Four variables previously shown to have significant effects on FAME production yield were investigated. The methanol-to-oil ratio, temperature and Novozym 435 dosage were established based on previous studies in the literature (Du et al., 2004; Fukuda et al., 2001; Kose et al., 2002). According to previous experiments (data not shown), the effect of the WFO content in the feedstock mixture was also tested in the lipasecatalyzed process. The experimental central composite design matrix is presented in Table 1. To understand and optimize the relationship between the tested variables, the obtained experimental data were analyzed by second-order polynomial equations by means of the RSM. The analysis of variance (ANOVA) of the quadratic polynomial model showed low pvalues (0.125) and both high determination coefficients (R2=95.3%) and high adjustment of the determination coefficients (Adj. R2=94.5%). The low p-value obtained indicates that the model accurately represented the relationship between response and the variables. The R2 value obtained indicates that the variation in FAME production yield correlated with 95.3% of the independent variables and the obtained Adj. R2 value indicates a 94.5% correlation between the independent variables. The lack of fit refers to the fact that a simple linear regression model may not adequately fit the experimental data. The obtained p-value not indicate a significant

lack of fit and, therefore, gave no reason to evaluate a more complex model.

The p-values obtained from the regression analysis results showed that all of the coefficients of the linear, interaction and quadratic terms had a significant effect on FAME production yield. All of the linear and quadratic terms, as well as the interaction terms X1X3, X2X4 and X3X4, were significant at the 1% level. The interaction terms X1X2, X1X4 and X2X3 were

trimethylsilyltrifluoroacetamide (MSTFA) was added to the vials, which were then shaken and incubated for 15 min to transform the sample into more volatile siliade components. Subsequently, the preparation was dissolved in 0.8 mL of n-heptane. The following temperature program was used: 15ºC for 1 min and three consecutive ramps of 15ºC/min to 180ºC, 7ºC/min to 230ºC and 10ºC/min to 320ºC and 320ºC for 15 min. The detector temperature was 380ºC and a split rate 10 was used for injection of 1 µL of sample.

*Methanol addition.* Methanol solubility is less than 1.5:1 methanol/oil (mol/mol), but a molar ratio of 3:1 methanol/oil is necessary to complete the transesterification reaction for FAME production (Shimada et al., 2002). However, very high initial concentrations of methanol could lead to the inhibition of the lipase used in this study due to its low solubility in oil (Du et al., 2004; Shimada et al., 2002). Therefore, the experiment was designed so that the methanol was added in two steps (Fig. 1).

Fig. 1. FAME content and productivity during the reaction time. Operational conditions: 50 wt% WFO in the mixed feedstock, methanol to oil molar ratio of 3:1, 9 wt % Novozym 435 and 45°C, at 200 rpm. Arrows indicate methanol addition.

The experiment was started with an initial concentration of one-third of the necessary moles of methanol. When about one-third of the oil was converted to FAME after 8 hours, the remaining two-thirds of the methanol were added (Fig. 1). The addition of the higher concentration of methanol in the second step was determined based on Shimada et al. (2002), who established that methanol is more soluble in FAME than in TG.

trimethylsilyltrifluoroacetamide (MSTFA) was added to the vials, which were then shaken and incubated for 15 min to transform the sample into more volatile siliade components. Subsequently, the preparation was dissolved in 0.8 mL of n-heptane. The following temperature program was used: 15ºC for 1 min and three consecutive ramps of 15ºC/min to 180ºC, 7ºC/min to 230ºC and 10ºC/min to 320ºC and 320ºC for 15 min. The detector

*Methanol addition.* Methanol solubility is less than 1.5:1 methanol/oil (mol/mol), but a molar ratio of 3:1 methanol/oil is necessary to complete the transesterification reaction for FAME production (Shimada et al., 2002). However, very high initial concentrations of methanol could lead to the inhibition of the lipase used in this study due to its low solubility in oil (Du et al., 2004; Shimada et al., 2002). Therefore, the experiment was designed so that the

time [h]

Fig. 1. FAME content and productivity during the reaction time. Operational conditions: 50 wt% WFO in the mixed feedstock, methanol to oil molar ratio of 3:1, 9 wt % Novozym 435

The experiment was started with an initial concentration of one-third of the necessary moles of methanol. When about one-third of the oil was converted to FAME after 8 hours, the remaining two-thirds of the methanol were added (Fig. 1). The addition of the higher concentration of methanol in the second step was determined based on Shimada et al.

0 10 20 30 40 50 60

time [h] 0 10 20 30 40 50

temperature was 380ºC and a split rate 10 was used for injection of 1 µL of sample.

methanol was added in two steps (Fig. 1).

productivity

and 45°C, at 200 rpm. Arrows indicate methanol addition.

[g FAME/g catalyst/h]

0.0

(2002), who established that methanol is more soluble in FAME than in TG.

0.2

0.4

0.6

0.8

1.0

FAME content [%]

0

20

40

60

80

100

This two-step technique of methanol addition was shown to diminish the reaction time by obviously preventing enzyme inhibition. Li et al. (2009) showed that in spite of the high yield reached using stepwise methanol addition, both the reaction time and rate of FAME conversion were slower. In experiments using a three-step methanol addition and 4% (wt) Novozym 435 as the catalyst, high FAME production yields were reached but only after 50 hours of reaction (Du et al., 2004; Watanabe et al., 2001). Similarly, in another recent study, an reaction time of 50 hours was also used (Ognjanovic et al., 2009). Usually, the second methanol addition starts after one-third (33.3%) of the FAME content has been generated and the initially added methanol has been completely consumed (Du et al., 2004; Shimada et al., 2002). This methodology produces a stationary phase of FAME production yield, diminishing the reaction productivity.

As Novozym 435 was shown to be a robust and stable catalyst compared to other lipases in the presence of short chain alcohols (Hernandez-Martin et al., 2008), all of the experiments in this study were performed using this commercially available immobilized enzyme. When approximately 28.6% of the FAME content was reached after 8 hours of reaction, the second aliquot of methanol was added. This stepwise addition of methanol increased the reaction productivity by avoiding the stationary production phase and led to a plateau in FAME production at 12 hours. Similar reductions in the reaction time by shortening the stationary phase of FAME production have already been reported (Li et al., 2009). Therefore, in the RSM experiments, the first amount of methanol (one-third of the molar ratio shown in Table 1) was added at the start of the reaction, while the second aliquot of methanol (two-thirds of the molar ratio shown in Table 1) was added after 8 hours, and the total reaction time was 12 hours.

*Optimization of methanolysis conditions.* Four variables previously shown to have significant effects on FAME production yield were investigated. The methanol-to-oil ratio, temperature and Novozym 435 dosage were established based on previous studies in the literature (Du et al., 2004; Fukuda et al., 2001; Kose et al., 2002). According to previous experiments (data not shown), the effect of the WFO content in the feedstock mixture was also tested in the lipasecatalyzed process. The experimental central composite design matrix is presented in Table 1.

To understand and optimize the relationship between the tested variables, the obtained experimental data were analyzed by second-order polynomial equations by means of the RSM. The analysis of variance (ANOVA) of the quadratic polynomial model showed low pvalues (0.125) and both high determination coefficients (R2=95.3%) and high adjustment of the determination coefficients (Adj. R2=94.5%). The low p-value obtained indicates that the model accurately represented the relationship between response and the variables. The R2 value obtained indicates that the variation in FAME production yield correlated with 95.3% of the independent variables and the obtained Adj. R2 value indicates a 94.5% correlation between the independent variables. The lack of fit refers to the fact that a simple linear regression model may not adequately fit the experimental data. The obtained p-value not indicate a significant lack of fit and, therefore, gave no reason to evaluate a more complex model.

The p-values obtained from the regression analysis results showed that all of the coefficients of the linear, interaction and quadratic terms had a significant effect on FAME production yield. All of the linear and quadratic terms, as well as the interaction terms X1X3, X2X4 and X3X4, were significant at the 1% level. The interaction terms X1X2, X1X4 and X2X3 were

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

100

80

60

40

20

0

method for FAME production on an industrial scale.

20.0

**WFO in mixed feedstock [%]**

methanol to oil ratio 3:1 mol/mol.

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 283

40.0

60.0

Fig. 2. Contour plot of FAME production yield predicted from the model at 45ºC and molar

Recently, Issariyakul et al. (2008) investigated the use of mixed WFO and rapeseed oil using an alkaline catalyst. However, lower ester production yields were observed when WFO was incorporated in the reaction mixtures. These results agreed with Fukuda et al. (2001), who established that the FFA in WFO can be completely converted to FAME using lipases as catalysts, whereas soap is produced when an alkaline catalyst is used, which diminishes the production yield. Although the cost of lipases is significantly higher than that of alkaline catalysts, their use may partially solve the main drawbacks of the conventional biodiesel production process: the use of pure vegetable oils (which account for around 80% of the cost of the total process) and the competition with food products (Gui et al., 2008). We have shown that the incorporation of WFO in the feedstock to partially replace rapeseed oil in processes catalyzed by Novozym 435 may diminish the production costs while simultaneously increasing the production yield, which makes it a potential alternative

FAME production yield was more sensitive to both the methanol-to-oil ratio and Novozym 435 dosage, compared to temperature (Fig. 3). FAME production yield was enhanced when the temperature increased to 45-50ºC; however, the opposite tendency was observed for temperatures higher than 50ºC. These results agree with the results obtained by Kose et al. (2002), who established that at about 50ºC, FAME production yield decreases as a result of enzyme deactivation at high temperatures (Fig. 3A). An enhancement in FAME production yield was observed when both the methanol-to-oil ratio and Novozym 435 dosage increased (Fig. 3B). When using a high immobilized biocatalyst load, large amounts of alcohol were

**Novozym 435 [wt%]**

5.0 7.5 10.0 12.5 15.0

80.0

significant at the 5% level. Therefore, the final response model equation in terms of the variable factors can be written as follows (Eq. 2):

$$\begin{aligned} FAME\_{yield} &= -896.52 + 1.90 \cdot X\_1 + 91.40 \cdot X\_2 + 28.06 \cdot X\_3 + 20.00 \cdot X\_4 - 0.01 \cdot X\_1^2 \\ &- 14.46 \cdot X\_2^2 - 0.24 \cdot X\_3^2 - 0.85 \cdot X\_4^2 - 0.12 \cdot X\_1 \cdot X\_2 - 0.02 \cdot X\_1 \cdot X\_3 \\ &- 0.03 \cdot X\_1 \cdot X\_4 - 0.59 \cdot X\_2 \cdot X\_3 + 4.13 \cdot X\_2 \cdot X\_4 - 0.26 \cdot X\_3 \cdot X\_4 \end{aligned} \tag{2}$$

The main factors that affect the FAME production yield were the linear terms X1, X2, X3 and X4, the quadratic term X22 and the interaction term X2X4. The linear terms with positive coefficients indicate an increase in FAME production yield. As X1 is a positive term, it is possible to establish that WFO incorporation could increase FAME production yield.

To validate the model obtained, an experimental point was compared to the model prediction (Eq. 2), and an error of less than 8% was obtained. In addition, the optimal methanolysis conditions were obtained through the regression model (Eq. 2) according to the limit criterion of the maximum response of FAME production yield. The obtained optimal conditions were 100% (wt) WFO, a methanol-to-oil ratio of 3.8:1 (mol/mol), 15% (wt) Novozym 435 and 44.5°C, which generated 100% FAME production yield. These predicted conditions show that WFO incorporation increases FAME production yield.

Moreover, using this model, it was also possible to predict several optimal conditions to reach the highest FAME production yields, while simultaneously reducing production costs and enzyme use. For instance, 96% FAME production yield could be reached using 75% (wt) WFO in the mixed feedstock, with a methanol-to-oil ratio of 3.75:1 (mol/mol), 12% (wt) Novozym 435 and 40°C.

To illustrate that several optimal combinations are able to produce the highest FAME production yields, contour plots were generated. Figure 2 and 3 show the effect of variable interaction on FAME production yield in contour plots predicted by the model. The contour plots were generated to show the effect of two variables on the response, while the other two independent variables were held constant.

Figure 2 shows the interaction between two independent variables (WFO in the mixed feedstock and Novozym 435 dosage) and their effects on the response variable FAME production yield, while the other two variables were held at zero. At these conditions, an increase in Novozym 435 dosage led to an enhancement in FAME production yield. This change is additionally increased when WFO is incorporated at up to 80% in the mixed feedstock. WFO seems to be a more available substrate compared to rapeseed oil for Novozym 435 catalysis under the conditions investigated.

FAME production yield was more sensitive to Novozym 435 dosage, since high doses were necessary to obtain high FAME production yields. In this sense, in order to decrease the production costs for future economically sound industrial applications, further experiments should be conducted to investigate the effect of WFO incorporation using another inexpensive biologic catalyst instead of Novozym 435. In addition, because Novozym 435 has shown high residual activity in successive applications (Hernandez-Martin et al., 2008), further experiments with a subsequent recovery protocol and reuse of the immobilized catalyst should be conducted under the optimal conditions established in this work.

significant at the 5% level. Therefore, the final response model equation in terms of the

*FAMEyield X X X XX*

 

The main factors that affect the FAME production yield were the linear terms X1, X2, X3 and X4, the quadratic term X22 and the interaction term X2X4. The linear terms with positive coefficients indicate an increase in FAME production yield. As X1 is a positive term, it is

To validate the model obtained, an experimental point was compared to the model prediction (Eq. 2), and an error of less than 8% was obtained. In addition, the optimal methanolysis conditions were obtained through the regression model (Eq. 2) according to the limit criterion of the maximum response of FAME production yield. The obtained optimal conditions were 100% (wt) WFO, a methanol-to-oil ratio of 3.8:1 (mol/mol), 15% (wt) Novozym 435 and 44.5°C, which generated 100% FAME production yield. These predicted conditions show that WFO incorporation increases FAME production yield.

Moreover, using this model, it was also possible to predict several optimal conditions to reach the highest FAME production yields, while simultaneously reducing production costs and enzyme use. For instance, 96% FAME production yield could be reached using 75% (wt) WFO in the mixed feedstock, with a methanol-to-oil ratio of 3.75:1 (mol/mol), 12% (wt)

To illustrate that several optimal combinations are able to produce the highest FAME production yields, contour plots were generated. Figure 2 and 3 show the effect of variable interaction on FAME production yield in contour plots predicted by the model. The contour plots were generated to show the effect of two variables on the response, while the other

Figure 2 shows the interaction between two independent variables (WFO in the mixed feedstock and Novozym 435 dosage) and their effects on the response variable FAME production yield, while the other two variables were held at zero. At these conditions, an increase in Novozym 435 dosage led to an enhancement in FAME production yield. This change is additionally increased when WFO is incorporated at up to 80% in the mixed feedstock. WFO seems to be a more available substrate compared to rapeseed oil for

FAME production yield was more sensitive to Novozym 435 dosage, since high doses were necessary to obtain high FAME production yields. In this sense, in order to decrease the production costs for future economically sound industrial applications, further experiments should be conducted to investigate the effect of WFO incorporation using another inexpensive biologic catalyst instead of Novozym 435. In addition, because Novozym 435 has shown high residual activity in successive applications (Hernandez-Martin et al., 2008), further experiments with a subsequent recovery protocol and reuse of the immobilized

catalyst should be conducted under the optimal conditions established in this work.

14.46 0.24 0.85 0.12 0.02 0.03 0.59 4.13 0.26

possible to establish that WFO incorporation could increase FAME production yield.

2 3 4 12 13 14 23 24 34

896.52 1.90 91.40 28.06 20.00 0.01

*X X X XX XX XX XX XX XX*

222

1 2 3 41

2

(2)

variable factors can be written as follows (Eq. 2):

Novozym 435 and 40°C.

two independent variables were held constant.

Novozym 435 catalysis under the conditions investigated.

Fig. 2. Contour plot of FAME production yield predicted from the model at 45ºC and molar methanol to oil ratio 3:1 mol/mol.

Recently, Issariyakul et al. (2008) investigated the use of mixed WFO and rapeseed oil using an alkaline catalyst. However, lower ester production yields were observed when WFO was incorporated in the reaction mixtures. These results agreed with Fukuda et al. (2001), who established that the FFA in WFO can be completely converted to FAME using lipases as catalysts, whereas soap is produced when an alkaline catalyst is used, which diminishes the production yield. Although the cost of lipases is significantly higher than that of alkaline catalysts, their use may partially solve the main drawbacks of the conventional biodiesel production process: the use of pure vegetable oils (which account for around 80% of the cost of the total process) and the competition with food products (Gui et al., 2008). We have shown that the incorporation of WFO in the feedstock to partially replace rapeseed oil in processes catalyzed by Novozym 435 may diminish the production costs while simultaneously increasing the production yield, which makes it a potential alternative method for FAME production on an industrial scale.

FAME production yield was more sensitive to both the methanol-to-oil ratio and Novozym 435 dosage, compared to temperature (Fig. 3). FAME production yield was enhanced when the temperature increased to 45-50ºC; however, the opposite tendency was observed for temperatures higher than 50ºC. These results agree with the results obtained by Kose et al. (2002), who established that at about 50ºC, FAME production yield decreases as a result of enzyme deactivation at high temperatures (Fig. 3A). An enhancement in FAME production yield was observed when both the methanol-to-oil ratio and Novozym 435 dosage increased (Fig. 3B). When using a high immobilized biocatalyst load, large amounts of alcohol were

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

Novozym 435, through direct FFA esterification (Li et al., 2007).

Physicochemical properties

Fatty acid composition [wt%]

Acylglycerols and FFA composition [wt%]

analysis).

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 285

temperatures. The kinematic viscosity of WFO was also measured and was slightly higher compared to rapeseed oil, probably as a result of polymerization reactions. As WFO is refined oil, the sulfur content was lower in WFO compared to rapeseed oil, which is an unrefined oil. In addition, a higher FFA content in WFO compared to rapeseed oil was observed, which may enhance FAME production yield using a biological catalyst, such as

 Specific gravity [kg/m3 at 20°C] 925 927 Kinematic viscosity at 40°C [cst] 37 34 Acid value [mg KOH/g] 4.5 0.8 Iodine value [g I2/100 g oil] 111 105 Peroxide value [meq/kg] 15.2 4.1 Water and sediments [%v/v] 1.3 < 0.01 S [mg/L] 2.0 7.0 Pour point [°C] -6.7 -23.3 Flash point [°C] >100 >100

 Palmitic [C16:0] 12.2 4.2 Stearic [C18:0] 4.1 1.3 Oleic [C18:1] 28.5 66.6 Linoleic [C18:2] 49.5 18.7 Linolenic [C18:3] 3.5 7.7 Arachidic [C20:0] 0.2 0.4 Eicosenic [C20:1] 0.1 1.0 Eicosapentaenoic [C20:5] 0.2 0.1 Erucic [C22:1] 0.0 0.1 Others 1.7 0.0

 Monoacylglycerol 0.1 0.0 Diacylglycerol 3.3 0.5 Triacylglycerol 93.5 98.7 Free fatty acid 3.1 0.8

Table 2. Properties and composition of oil feedstocks (samples were filtered before to the

Feedstock

WFO Rapeseed oil

needed to provide sufficient liquid to maintain a uniform suspension of the biocatalyst (Hernandez-Martin et al., 2008). In addition, as Novozym 435 is a robust biocatalyst in the presence of short chain alcohols, the excess alcohol kept the glycerol in solution, which prevented deactivation of Novozym 435 by glycerol blockage of the entrance to catalyst pores (Hernandez-Martin et al., 2008). Therefore, the levels of Novozym 435 used and the stepwise methanol addition seem to avoid the possible diffusion limitations, which resulted in high production yields.

Fig. 3. Contour plots of FAME production yield predicted from the model for 50% (wt) WFO at (A) a methanol-to-oil ratio of 3:1 (mol/mol) and (B) 45ºC.

*Properties of feedstock that affect methanolysis optimization*. The feedstock characteristics were investigated to determine the components responsible for the results obtained in the RSM, particularly the increase in the FAME yield when WFO was incorporated in the process (Table 2).

Differences were found in most of the physical properties of the oil feedstock measured (Table 2). The food frying process produced an increase in both acid and peroxide values from WFO due to the hydrolysis and oxidation reactions, respectively (Araújo 1995). The high peroxide value of the WFO could positively affect the FAME characteristics by increasing the oxygen content, which enhances its burning efficiency (Lin et al., 2007). However, a higher oxygen content in oils may also promote a higher nitrogen oxide concentrations during the burning process (Lin et al., 2007).

The analyzed WFO was originally an edible soybean oil and sunflower oil mixture, which is characterized by a typical iodine value of about 130 g I2/100 g oil. As the iodine value is diminished by polymerization reactions during the frying process, a similar iodine value compared to rapeseed oil was found for WFO (Table 2). A lower iodine value increases the pour point and may also improve the oxidative stability of the oil (Canakci 2007). However, a high pour point can negatively affect FAME performance in an engine, when used at low

needed to provide sufficient liquid to maintain a uniform suspension of the biocatalyst (Hernandez-Martin et al., 2008). In addition, as Novozym 435 is a robust biocatalyst in the presence of short chain alcohols, the excess alcohol kept the glycerol in solution, which prevented deactivation of Novozym 435 by glycerol blockage of the entrance to catalyst pores (Hernandez-Martin et al., 2008). Therefore, the levels of Novozym 435 used and the stepwise methanol addition seem to avoid the possible diffusion limitations, which resulted

80.0

40.0

60.0

5.0 7.5 10.0 12.5 15.0

**Novozym 435 [wt%]**

A B Fig. 3. Contour plots of FAME production yield predicted from the model for 50% (wt) WFO

**Methanol to oil molar ratio [mol/mol]**

4.5

4.0

3.5

3.0

2.5

2.0

1.5

20.0

*Properties of feedstock that affect methanolysis optimization*. The feedstock characteristics were investigated to determine the components responsible for the results obtained in the RSM, particularly the increase in the FAME yield when WFO was incorporated in the process

Differences were found in most of the physical properties of the oil feedstock measured (Table 2). The food frying process produced an increase in both acid and peroxide values from WFO due to the hydrolysis and oxidation reactions, respectively (Araújo 1995). The high peroxide value of the WFO could positively affect the FAME characteristics by increasing the oxygen content, which enhances its burning efficiency (Lin et al., 2007). However, a higher oxygen content in oils may also promote a higher nitrogen oxide

The analyzed WFO was originally an edible soybean oil and sunflower oil mixture, which is characterized by a typical iodine value of about 130 g I2/100 g oil. As the iodine value is diminished by polymerization reactions during the frying process, a similar iodine value compared to rapeseed oil was found for WFO (Table 2). A lower iodine value increases the pour point and may also improve the oxidative stability of the oil (Canakci 2007). However, a high pour point can negatively affect FAME performance in an engine, when used at low

at (A) a methanol-to-oil ratio of 3:1 (mol/mol) and (B) 45ºC.

60.0

5.0 7.5 10.0 12.5 15.0

**Novozym 435 [wt%]**

80.0 80.0

concentrations during the burning process (Lin et al., 2007).

in high production yields.

20.0

40.0

(Table 2).

**Temperature [°C]**

55

50

45

40

35

temperatures. The kinematic viscosity of WFO was also measured and was slightly higher compared to rapeseed oil, probably as a result of polymerization reactions. As WFO is refined oil, the sulfur content was lower in WFO compared to rapeseed oil, which is an unrefined oil. In addition, a higher FFA content in WFO compared to rapeseed oil was observed, which may enhance FAME production yield using a biological catalyst, such as Novozym 435, through direct FFA esterification (Li et al., 2007).


Table 2. Properties and composition of oil feedstocks (samples were filtered before to the analysis).

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

performance (Wang et al., 2006).

catalyzing was proposed (Tamalampudi et al., 2008).

been recently proposed (Royon et al., 2007; Yu et al., 2010).

WFO, jatropha, microalgae and crude palm oil (Azócar et al., 2010b).

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 287

producing FAME with a high acid value. Therefore, to accomplish with international biofuel standards, additional treatment are needed (Fukuda et al., 2008). In addition, this problem could be enhanced by using alternative feedstock characterized by high acid value, such

To solve these drawbacks the addition of water adsorbents to the lipase-catalyzed reaction has been recently investigated (Li et al., 2009; Wang et al., 2006). Some reported absorbents are blue silica gel, maceo-pored silica gel, fine-pored silica gel, and molecular sieves of 3 Å, 4 Å and 5 Å (Li et al., 2009; Wang et al., 2006). Among the adsorbents examined, blue silica gel has been reported as the best one, increasing FAME yield when immobilized lipase from *Penicillium expansum* was used as catalyst (Li et al., 2009). However, high dosage of silica gel could provoke low biodiesel yield. In fact, as the pore size of silica gel is much larger than the methanol molecule size, silica adsorbs methanol negatively affecting the reaction

The possible advantages of using water adsorbents in lipase-catalyzed processes to improve FAME production yield may be in contradiction with reports showing that lipases activation may need water presence. In fact, lipases activation involves the active site restructuration, which requires the presence of an oil-water interface (Yu et al., 2010). As a result, small dosages of water in the reaction have been proposed to achieve an effective process (Yu et al., 2010). Novozym 435 has been shown to contain enough water in its support medium to preserve its catalytic activity in an anhydrous medium (Ognjanovic et al., 2009; Tamalampudi et al., 2008). In a response surface methodology study, Organovic et al. (2009) correlated Novozym 435 concentration with water concentration, achieving the highest biodiesel yield of 92% using the medium with the lowest water content level (0%) and the highest enzyme concentration (5% based on oil weight). In another study, Novozym 435 showed low activity in the presence of water and an anhydrous medium for efficient

Novozym 435 has been widely reported as an effective biocatalyst for promoting high FAME production yields (Azócar et al., 2010b). Optimal conditions to reach 100% FAME yield using WFO as feedstock were determined to be the following: Methanol-to-oil molar ratio 3.8:1 (wt), 15% (wt) Novozym 435 and incubation at 44.5°C for 12 h with agitation at 200 rpm (Azócar et al., 2010a). Novozym 435 activity loss and regeneration have been also investigated. It has been established that the immobilization material (acrylic resin) could adsorb polar components such as methanol and glycerol, provoking enzyme inactivation. Therefore, Novozym 435 reutilization by means of washing processes using acetone, soybean oil, *tert*-butanol, isopropanol and 2-butanol has been investigated (Chen et al., 2003; Samukawa et al., 2000). In addition, the incorporation of a co-solvent in the reaction has

According to previous research, Novozym 435 has been proved to be an effective catalyst in FAME production and has been shown to preserve its catalytic activity in an anhydrous medium. However, Novozym 435 has not been investigated yet regarding catalyzedprocesses when adsorbent materials are added to the reaction in order to obtain high quality biodiesel with low acid value. In this sense, it is necessary to evaluate the behavior of the enzymatic reaction in anhydrous medium. In addition, it is necessary to determine the effect

The FFA esterification reaction produces 1 mol of water per 1 mol of FAME produced. In this work, water and sediments were quantified and low levels were found in both rapeseed oil and WFO, with a slightly higher water content for WFO (Table 2). However, as WFO has a high FFA content, incorporation of WFO in the mixed feedstock may increase the water content through FFA esterification reactions. Although lipases need an optimal amount of water to maintain their activity in organic media, a recent work established that Novozym 435 itself appears to contain sufficient water to preserve its catalytic activity without requiring the presence of an oil/water interface (Ognjanovic et al., 2009). It has also been shown that Novozym 435 needs a nearly anhydrous reaction medium to be effective (Salis et al., 2005). According to these previous studies, when WFO was incorporated in a mixed feedstock, the reaction yield diminished as a result of the high water content. Therefore, an improvement in FAME production yield when WFO is incorporated into a feedstock should not be produced due to the presence of water in the transesterification reaction.

Differences between the fatty acid compositions of WFO and rapeseed oil were also detected (Table 2). In order to analyze the role of fatty acids in FAME production, the input and output fatty acid compositions were compared. However, no significant differences between input and output fatty acid composition were detected. (data not shown). Products from oil conversion, i.e., MG, DG, TG and FFA, in the used feedstock were compared using GC-MS. MG, DG and FFA account for about 6.5% of the WFO composition, compared to only 1.3% in the case of rapeseed oil (Table 2). Turkan & Kalay (2006) established that in Novozym 435-catalyzed transesterification, the first step (i.e., conversion of TG to DG) is the ratedetermining step, since TG is converted to FAME without significant accumulation of MG and DG. This fact indicates that MG, DG and FFA can be more easily converted to FAME compared to TG. Therefore, WFO is a more available substrate in the process investigated, compared to rapeseed oil, and incorporation of WFO in feedstock increased the FAME production yield.

Finally, is possible to establish that according to the RSM, the optimized predicted combination of conditions to obtain 100% FAME production yield was the use of 100% (wt) WFO, a methanol-to-oil ratio of 3.8:1 (mol/mol), 15% (wt) Novozym 435 and 44.5°C at 200 rpm. Methanol addition in two steps was previously optimized. An earlier second addition of methanol after 8 hours of reaction time was an effective technique to decrease the total reaction time to 12 hours, while preventing enzyme inhibition. The model obtained from the RSM predicted that several optimal conditions were able to reach the highest FAME production yield. According to this model, the addition of WFO increased the FAME production yield, and this effect was mainly attributed to the higher contents of MG, DG and FFA of WFO compared to rapeseed oil, which are more available substrates for enzymatic catalysis. Therefore, a partial replacement of rapeseed oil by WFO in processes catalyzed by Novozym 435 may diminish the cost of biodiesel production by using a less expensive feedstock that simultaneously increases the production yield.

#### **3. Enzymatic biodiesel production in an anhydrous medium with lipase reutilization**

A major problem in lipase-catalyzed processes to FAME production is the high acidity of the final product, mainly caused by water presence. In fact, water favors the hydrolysis reaction

The FFA esterification reaction produces 1 mol of water per 1 mol of FAME produced. In this work, water and sediments were quantified and low levels were found in both rapeseed oil and WFO, with a slightly higher water content for WFO (Table 2). However, as WFO has a high FFA content, incorporation of WFO in the mixed feedstock may increase the water content through FFA esterification reactions. Although lipases need an optimal amount of water to maintain their activity in organic media, a recent work established that Novozym 435 itself appears to contain sufficient water to preserve its catalytic activity without requiring the presence of an oil/water interface (Ognjanovic et al., 2009). It has also been shown that Novozym 435 needs a nearly anhydrous reaction medium to be effective (Salis et al., 2005). According to these previous studies, when WFO was incorporated in a mixed feedstock, the reaction yield diminished as a result of the high water content. Therefore, an improvement in FAME production yield when WFO is incorporated into a feedstock should

not be produced due to the presence of water in the transesterification reaction.

production yield.

**reutilization** 

Differences between the fatty acid compositions of WFO and rapeseed oil were also detected (Table 2). In order to analyze the role of fatty acids in FAME production, the input and output fatty acid compositions were compared. However, no significant differences between input and output fatty acid composition were detected. (data not shown). Products from oil conversion, i.e., MG, DG, TG and FFA, in the used feedstock were compared using GC-MS. MG, DG and FFA account for about 6.5% of the WFO composition, compared to only 1.3% in the case of rapeseed oil (Table 2). Turkan & Kalay (2006) established that in Novozym 435-catalyzed transesterification, the first step (i.e., conversion of TG to DG) is the ratedetermining step, since TG is converted to FAME without significant accumulation of MG and DG. This fact indicates that MG, DG and FFA can be more easily converted to FAME compared to TG. Therefore, WFO is a more available substrate in the process investigated, compared to rapeseed oil, and incorporation of WFO in feedstock increased the FAME

Finally, is possible to establish that according to the RSM, the optimized predicted combination of conditions to obtain 100% FAME production yield was the use of 100% (wt) WFO, a methanol-to-oil ratio of 3.8:1 (mol/mol), 15% (wt) Novozym 435 and 44.5°C at 200 rpm. Methanol addition in two steps was previously optimized. An earlier second addition of methanol after 8 hours of reaction time was an effective technique to decrease the total reaction time to 12 hours, while preventing enzyme inhibition. The model obtained from the RSM predicted that several optimal conditions were able to reach the highest FAME production yield. According to this model, the addition of WFO increased the FAME production yield, and this effect was mainly attributed to the higher contents of MG, DG and FFA of WFO compared to rapeseed oil, which are more available substrates for enzymatic catalysis. Therefore, a partial replacement of rapeseed oil by WFO in processes catalyzed by Novozym 435 may diminish the cost of biodiesel production by using a less

expensive feedstock that simultaneously increases the production yield.

**3. Enzymatic biodiesel production in an anhydrous medium with lipase** 

A major problem in lipase-catalyzed processes to FAME production is the high acidity of the final product, mainly caused by water presence. In fact, water favors the hydrolysis reaction producing FAME with a high acid value. Therefore, to accomplish with international biofuel standards, additional treatment are needed (Fukuda et al., 2008). In addition, this problem could be enhanced by using alternative feedstock characterized by high acid value, such WFO, jatropha, microalgae and crude palm oil (Azócar et al., 2010b).

To solve these drawbacks the addition of water adsorbents to the lipase-catalyzed reaction has been recently investigated (Li et al., 2009; Wang et al., 2006). Some reported absorbents are blue silica gel, maceo-pored silica gel, fine-pored silica gel, and molecular sieves of 3 Å, 4 Å and 5 Å (Li et al., 2009; Wang et al., 2006). Among the adsorbents examined, blue silica gel has been reported as the best one, increasing FAME yield when immobilized lipase from *Penicillium expansum* was used as catalyst (Li et al., 2009). However, high dosage of silica gel could provoke low biodiesel yield. In fact, as the pore size of silica gel is much larger than the methanol molecule size, silica adsorbs methanol negatively affecting the reaction performance (Wang et al., 2006).

The possible advantages of using water adsorbents in lipase-catalyzed processes to improve FAME production yield may be in contradiction with reports showing that lipases activation may need water presence. In fact, lipases activation involves the active site restructuration, which requires the presence of an oil-water interface (Yu et al., 2010). As a result, small dosages of water in the reaction have been proposed to achieve an effective process (Yu et al., 2010). Novozym 435 has been shown to contain enough water in its support medium to preserve its catalytic activity in an anhydrous medium (Ognjanovic et al., 2009; Tamalampudi et al., 2008). In a response surface methodology study, Organovic et al. (2009) correlated Novozym 435 concentration with water concentration, achieving the highest biodiesel yield of 92% using the medium with the lowest water content level (0%) and the highest enzyme concentration (5% based on oil weight). In another study, Novozym 435 showed low activity in the presence of water and an anhydrous medium for efficient catalyzing was proposed (Tamalampudi et al., 2008).

Novozym 435 has been widely reported as an effective biocatalyst for promoting high FAME production yields (Azócar et al., 2010b). Optimal conditions to reach 100% FAME yield using WFO as feedstock were determined to be the following: Methanol-to-oil molar ratio 3.8:1 (wt), 15% (wt) Novozym 435 and incubation at 44.5°C for 12 h with agitation at 200 rpm (Azócar et al., 2010a). Novozym 435 activity loss and regeneration have been also investigated. It has been established that the immobilization material (acrylic resin) could adsorb polar components such as methanol and glycerol, provoking enzyme inactivation. Therefore, Novozym 435 reutilization by means of washing processes using acetone, soybean oil, *tert*-butanol, isopropanol and 2-butanol has been investigated (Chen et al., 2003; Samukawa et al., 2000). In addition, the incorporation of a co-solvent in the reaction has been recently proposed (Royon et al., 2007; Yu et al., 2010).

According to previous research, Novozym 435 has been proved to be an effective catalyst in FAME production and has been shown to preserve its catalytic activity in an anhydrous medium. However, Novozym 435 has not been investigated yet regarding catalyzedprocesses when adsorbent materials are added to the reaction in order to obtain high quality biodiesel with low acid value. In this sense, it is necessary to evaluate the behavior of the enzymatic reaction in anhydrous medium. In addition, it is necessary to determine the effect

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

the FAME yield.

the recovery treatment.

were repeated 4 times.

eliminate residual *tert*-butanol in the enzymes.

reaction time. Methanol was added in two steps as described previously.

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 289

operational conditions were the following: Methanol-to-oil ratio of 3.8:1 (mol/mol); 15% Novozym 435 (% wt based on oil weight); 44.5 ºC; stirring at 200 rpm; and 12 hours of

For each reaction, 8 mL of WFO (7.4 g) with an acid value of 5.61 mg KOH/g oil were used. To maintain an anhydrous medium, 0.95 g of 3 Å molecular sieves were added to each reaction flask, which were calculated according to Eq. (3). Each reaction was carried out in triplicate. Samples of 70 µL were taken consecutively at 3, 6, 9 and 12 hours of reaction time. The samples were centrifuged and the upper layer was extracted to analyze

For enzyme recovery, the molecular sieves were separated with a strainer after the transesterification reaction. A paper filter over a vacuum system was placed underneath the strainer containing the molecular sieves to retain the enzymes. The product of the reaction was passed through the two filters: The molecular sieves captured in the strainer were eliminated, whereas the enzymes captured in the paper filter were placed in a new flask for

Three treatments to reutilize the enzymes were performed according to the reference studies (Chen et al., 2003; Samukawa et al., 2000; Yu et al., 2010) and a previous experiments (data not shown). A control experiment reusing the enzymes without treatment was also carried out. The treatments were the following: (A) Acetone washing: The enzymes were washed by adding a small dosage of acetone to the flask containing the enzymes. The flask was shaken and the liquid residue was eliminated. This was repeated successively until the liquid was clear. The total volume used in this process was about 10 mL of acetone per gram of enzyme. Subsequently, a wash with 10 mL of WFO per gram of enzyme was carried out to eliminate residual acetone in the enzymes. (B) Waste frying oil washing: The enzymes were washed in a sufficient quantity of WFO to maintain the enzymes submerged in the flask. The flask was shaken and the liquid residue was eliminated. The washing was repeated 3 times. (C) *tert-*Butanol washing: The washing was carried out by adding a specific dosage of *tert*-butanol to the flask containing the enzymes. Then, the mixture was shaken and the residual liquid was eliminated. This sequence was repeated consecutively until the liquid was clear. The total volume used in this process was about 10 mL of *tert*-butanol per gram of enzyme. After this, a wash with about 10 mL of WFO per gram of enzyme was carried out to

After each washing step, a known dosage of WFO was added to the flasks containing the enzymes, which were incubated in oil overnight at ambient temperature. After about 10 hours, both methanol and 3 Å molecular sieves were added to carry out a new FAME production reaction in the same incubation medium. The control was carried out by transferring the enzymes directly to a new reaction for FAME production. Cycles that consisted of both a reaction for FAME production and a treatment for enzyme recovery

As an alternative to enzyme reuse for FAME production using an anhydrous medium, the addition of *tert*-butanol as a co-solvent in the reaction was investigated. To carry out the experiments, consecutive cycles of enzymatic FAME production using the same enzymes (reutilized) were carried out in an anhydrous medium and in a *tert*-butanol system. Each

of water absence in the enzyme activity during consecutive reactions and to evaluate alternatives for enzyme recovery during successive reactions.

The aim of this work was to test an anhydrous medium in Novozym 435 catalyzed-process to produce FAME with a low acid value. In addition, the behavior of Novozym 435 in the anhydrous medium was discussed and enzyme recovery alternatives were proposed.

Filtered WFO collected from restaurants and crude rapeseed oil from a local factory in Southern Chile were used as feedstock. The WFO was characterized by an acid value of 5.61 mg KOH/g oil, 2.8% FFA, 1.3% (v/v) water content, 0.1% (wt) MG, 3.3% (wt) DG and 925 Kg/m3 of specific gravity. The rapeseed oil was characterized by an acid value of 0.8 mg KOH/g oil, 1.6% FFA and 927 Kg/m3 of specific gravity. Novozym 435 donated by Novo Industries (Denmark) was used as catalyst. 3 Å molecular sieves (Sigma-Aldrich) were used to generate the anhydrous medium. Chromatographically pure methyl heptadecanoate, 1,2,3-butanetriol and 1,2,3-tricaprinoylglycerol were used as internal standards.

FAME production using Novozym 435 in an anhydrous medium was studied by adding 3 Å molecular sieves to the reaction. All reactions were carried out in flasks containing 1 mL of WFO (0.925 g) and 15% Novozym 435 (% wt based on oil weight) as the biocatalyst. A methanol-to-oil molar ratio of 4:1 was used. Methanol was added in two steps in order to avoid lipase inhibition (according to previous experiments in section 2). The flasks were incubated in a shaker at 35 °C and stirred at 200 rpm. Each flask was managed as a destructive sample at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours of reaction time and each condition was carried out in triplicate. Controls were carried out under the same conditions but without molecular sieves.

To calculate the dosage of molecular sieves, first the maximal theoretical water content in the reaction was estimated. This value was established by taking the initial water content in the WFO and the amount of water produced during the reaction by esterification of FFA contained in the WFO. The water produced by esterification of FFA was estimated by using the mass balance, considering that 1 mol of esterified FFA produces 1 mol of water. With an FFA content of 2.8% and water content of 1.3% in the WFO described above, a total water content of 4.1% was used to estimate the molecular sieve dosage in the reaction, as shown in Eq. 3.

$$\text{Molecular sives} = \frac{\text{Voil} \cdot \text{\textbullet } \rho \text{ oil} \cdot \text{\textbullet} \, H\_2O}{\text{WAC}} \approx \frac{1 \cdot \text{\textbullet } 0.925 \cdot \text{\textbullet } 4.1}{20} = 0.19 \text{ g} \tag{3}$$

Where Voil (mL) is the volume of oil added, ρoil (g/mL) is the density of the oil, H2O is the total water content during the reaction (water content in the oil more water produced by esterification of FFA) (% v/v) and WAC (%) is the water absorption capacity of the sieves. At the end of the reaction period, the samples were stored at 4 °C to stop the reaction. The samples were centrifuged and the upper layer was extracted for the analysis of FAME yield and acid value.

Different treatments to recover enzyme activity for further reutilization were investigated. Prior to the enzyme recovery treatments, reactions of FAME production were carried out in flasks which were incubated in a shaker under the same operating conditions. The

of water absence in the enzyme activity during consecutive reactions and to evaluate

The aim of this work was to test an anhydrous medium in Novozym 435 catalyzed-process to produce FAME with a low acid value. In addition, the behavior of Novozym 435 in the anhydrous medium was discussed and enzyme recovery alternatives were proposed.

Filtered WFO collected from restaurants and crude rapeseed oil from a local factory in Southern Chile were used as feedstock. The WFO was characterized by an acid value of 5.61 mg KOH/g oil, 2.8% FFA, 1.3% (v/v) water content, 0.1% (wt) MG, 3.3% (wt) DG and 925 Kg/m3 of specific gravity. The rapeseed oil was characterized by an acid value of 0.8 mg KOH/g oil, 1.6% FFA and 927 Kg/m3 of specific gravity. Novozym 435 donated by Novo Industries (Denmark) was used as catalyst. 3 Å molecular sieves (Sigma-Aldrich) were used to generate the anhydrous medium. Chromatographically pure methyl heptadecanoate,

FAME production using Novozym 435 in an anhydrous medium was studied by adding 3 Å molecular sieves to the reaction. All reactions were carried out in flasks containing 1 mL of WFO (0.925 g) and 15% Novozym 435 (% wt based on oil weight) as the biocatalyst. A methanol-to-oil molar ratio of 4:1 was used. Methanol was added in two steps in order to avoid lipase inhibition (according to previous experiments in section 2). The flasks were incubated in a shaker at 35 °C and stirred at 200 rpm. Each flask was managed as a destructive sample at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours of reaction time and each condition was carried out in triplicate. Controls were carried out under the same conditions

To calculate the dosage of molecular sieves, first the maximal theoretical water content in the reaction was estimated. This value was established by taking the initial water content in the WFO and the amount of water produced during the reaction by esterification of FFA contained in the WFO. The water produced by esterification of FFA was estimated by using the mass balance, considering that 1 mol of esterified FFA produces 1 mol of water. With an FFA content of 2.8% and water content of 1.3% in the WFO described above, a total water content of 4.1% was used to estimate the molecular sieve dosage in the reaction, as shown in

*V oil oil H O Molecular sieves <sup>g</sup> WAC*

Where Voil (mL) is the volume of oil added, ρoil (g/mL) is the density of the oil, H2O is the total water content during the reaction (water content in the oil more water produced by esterification of FFA) (% v/v) and WAC (%) is the water absorption capacity of the sieves. At the end of the reaction period, the samples were stored at 4 °C to stop the reaction. The samples were centrifuged and the upper layer was extracted for the analysis of FAME yield

Different treatments to recover enzyme activity for further reutilization were investigated. Prior to the enzyme recovery treatments, reactions of FAME production were carried out in flasks which were incubated in a shaker under the same operating conditions. The

<sup>2</sup> 1 0.925 4.1 0.19 20

(3)

1,2,3-butanetriol and 1,2,3-tricaprinoylglycerol were used as internal standards.

alternatives for enzyme recovery during successive reactions.

but without molecular sieves.

Eq. 3.

and acid value.

operational conditions were the following: Methanol-to-oil ratio of 3.8:1 (mol/mol); 15% Novozym 435 (% wt based on oil weight); 44.5 ºC; stirring at 200 rpm; and 12 hours of reaction time. Methanol was added in two steps as described previously.

For each reaction, 8 mL of WFO (7.4 g) with an acid value of 5.61 mg KOH/g oil were used. To maintain an anhydrous medium, 0.95 g of 3 Å molecular sieves were added to each reaction flask, which were calculated according to Eq. (3). Each reaction was carried out in triplicate. Samples of 70 µL were taken consecutively at 3, 6, 9 and 12 hours of reaction time. The samples were centrifuged and the upper layer was extracted to analyze the FAME yield.

For enzyme recovery, the molecular sieves were separated with a strainer after the transesterification reaction. A paper filter over a vacuum system was placed underneath the strainer containing the molecular sieves to retain the enzymes. The product of the reaction was passed through the two filters: The molecular sieves captured in the strainer were eliminated, whereas the enzymes captured in the paper filter were placed in a new flask for the recovery treatment.

Three treatments to reutilize the enzymes were performed according to the reference studies (Chen et al., 2003; Samukawa et al., 2000; Yu et al., 2010) and a previous experiments (data not shown). A control experiment reusing the enzymes without treatment was also carried out. The treatments were the following: (A) Acetone washing: The enzymes were washed by adding a small dosage of acetone to the flask containing the enzymes. The flask was shaken and the liquid residue was eliminated. This was repeated successively until the liquid was clear. The total volume used in this process was about 10 mL of acetone per gram of enzyme. Subsequently, a wash with 10 mL of WFO per gram of enzyme was carried out to eliminate residual acetone in the enzymes. (B) Waste frying oil washing: The enzymes were washed in a sufficient quantity of WFO to maintain the enzymes submerged in the flask. The flask was shaken and the liquid residue was eliminated. The washing was repeated 3 times. (C) *tert-*Butanol washing: The washing was carried out by adding a specific dosage of *tert*-butanol to the flask containing the enzymes. Then, the mixture was shaken and the residual liquid was eliminated. This sequence was repeated consecutively until the liquid was clear. The total volume used in this process was about 10 mL of *tert*-butanol per gram of enzyme. After this, a wash with about 10 mL of WFO per gram of enzyme was carried out to eliminate residual *tert*-butanol in the enzymes.

After each washing step, a known dosage of WFO was added to the flasks containing the enzymes, which were incubated in oil overnight at ambient temperature. After about 10 hours, both methanol and 3 Å molecular sieves were added to carry out a new FAME production reaction in the same incubation medium. The control was carried out by transferring the enzymes directly to a new reaction for FAME production. Cycles that consisted of both a reaction for FAME production and a treatment for enzyme recovery were repeated 4 times.

As an alternative to enzyme reuse for FAME production using an anhydrous medium, the addition of *tert*-butanol as a co-solvent in the reaction was investigated. To carry out the experiments, consecutive cycles of enzymatic FAME production using the same enzymes (reutilized) were carried out in an anhydrous medium and in a *tert*-butanol system. Each

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

Control

Anhydrous medium

performance.

0

FAME content [%]

0

20

40

60

80

100

1

2

3

4

5

6

7

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 291

et al. (2008) who suggest that Novozym 435 transesterification activity is inhibited by the presence of added water and that it needs a nearly anhydrous medium for an efficient

> [h] 0 2 4 6 8 10 1 2

> > **B**

**A** 

Fig. 4 Acid values (A) and FAME yield (B) during the reaction in an anhydrous medium with a methanol-to-oil ratio of 4:1 (mol/mol), 15% (wt) Novozym 435 (based on the weight

time [h] 0 2 4 6 8 10 12

of oil), 35 ºC, 200 rpm and 12 hours of reaction time.

Control

Anhydrous medium

experiment was carried out in flasks by adding 5 mL of WFO (4.6 g), 15% of Novozym 435 (% wt based on oil weight) and a methanol-to-oil ratio of 3.8:1 (mol/mol). Methanol was added to the flask in two steps, similar to the previous experiment. In addition, both the selected dosage of *tert*-butanol and an amount of 3 Å molecular sieves (estimated according to Eq. 1) were added to the flasks. The *tert-*butanol dosage was established according to previous experiments at 0.75% (V/V) (data not show). Under these conditions, the flasks were incubated at 44.5 °C and 200 rpm during 12 hours of reaction time. All the experiments were carried out in triplicate and samples were taken throughout the reaction time. The upper layer of the centrifuged samples was analyzed to determine the FAME yield, according to the analytical procedures.

The acid value and FFA content in both the feedstock and throughout the reaction time were determined by titration with a KOH solution of known concentration and using phenolphthalein as indicator. Water and sediments were measured according to ASTM Standard Method D 1976-97(American). FAME, MG and DG in feedstock and reaction products were quantified according to methodology previously described in section 2.

*Biocatalysis in an anhydrous medium.* In order to reduce the acid value of the produced FAME, the alternative of carrying out FAME production reaction in an anhydrous medium was studied. Adsorbent materials such as blue silica gel and molecular sieves of different sizes have been shown to be effective in water removal during biodiesel production (Li et al., 2009). However, in this study, blue silica gel was discarded due to results from previous experiments showing possible methanol adsorption, negatively interfering with the reaction (data not shown). The adsorption of methanol could also occur when molecular sieves with large pore size are used. Thus, 3 Å molecular sieves were chosen to produce an anhydrous medium through water absorption.

The acid value and FAME yield results obtained using an anhydrous medium and a control run are shown in Fig. 4. The acid value obtained from the reaction using the anhydrous medium was maintained in about 1 mg KOH/g oil throughout the entire reaction time, whereas the control showed a value higher than 3 mg KOH/g oil by the end of the reaction (Fig. 4A). The values obtained using the anhydrous medium are close to those established by the biodiesel norm. As FFA present in WFO were esterified producing water and FAME at the start to the reaction (Fig. 4A), water was removed from the reaction by adsorption onto the molecular sieves, avoiding hydrolysis reactions and further FFA production. Therefore, the biocatalysis in an anhydrous medium produces a higher quality product compared to a typical standard reaction using Novozym 435.

FAME yield was also analyzed in an anhydrous medium (Fig. 4B). The results obtained show that water removal during FAME production generated a significant increase in FAME yield using Novozym 435 as the catalyst. These results are in agreement with those obtained by Li et al. (2007) who established that using 3 Å molecular sieves as adsorbent to remove excessive water could significantly increase the FAME yield when 100% FFA is used as raw material for biodiesel production. In this study, WFO containing only 2.8% FFA and 1.3% water was employed as the raw material, but FAME yield increased drastically. In addition, the control run only using molecular sieves indicates that this material did not catalyze the reaction (data not shown). Therefore, the increment in FAME yield is only related to the anhydrous medium. This corroborates the results obtained by Tamalampudi

experiment was carried out in flasks by adding 5 mL of WFO (4.6 g), 15% of Novozym 435 (% wt based on oil weight) and a methanol-to-oil ratio of 3.8:1 (mol/mol). Methanol was added to the flask in two steps, similar to the previous experiment. In addition, both the selected dosage of *tert*-butanol and an amount of 3 Å molecular sieves (estimated according to Eq. 1) were added to the flasks. The *tert-*butanol dosage was established according to previous experiments at 0.75% (V/V) (data not show). Under these conditions, the flasks were incubated at 44.5 °C and 200 rpm during 12 hours of reaction time. All the experiments were carried out in triplicate and samples were taken throughout the reaction time. The upper layer of the centrifuged samples was analyzed to determine the FAME yield,

The acid value and FFA content in both the feedstock and throughout the reaction time were determined by titration with a KOH solution of known concentration and using phenolphthalein as indicator. Water and sediments were measured according to ASTM Standard Method D 1976-97(American). FAME, MG and DG in feedstock and reaction products were quantified according to methodology previously described in section 2.

*Biocatalysis in an anhydrous medium.* In order to reduce the acid value of the produced FAME, the alternative of carrying out FAME production reaction in an anhydrous medium was studied. Adsorbent materials such as blue silica gel and molecular sieves of different sizes have been shown to be effective in water removal during biodiesel production (Li et al., 2009). However, in this study, blue silica gel was discarded due to results from previous experiments showing possible methanol adsorption, negatively interfering with the reaction (data not shown). The adsorption of methanol could also occur when molecular sieves with large pore size are used. Thus, 3 Å molecular sieves were chosen to produce an anhydrous

The acid value and FAME yield results obtained using an anhydrous medium and a control run are shown in Fig. 4. The acid value obtained from the reaction using the anhydrous medium was maintained in about 1 mg KOH/g oil throughout the entire reaction time, whereas the control showed a value higher than 3 mg KOH/g oil by the end of the reaction (Fig. 4A). The values obtained using the anhydrous medium are close to those established by the biodiesel norm. As FFA present in WFO were esterified producing water and FAME at the start to the reaction (Fig. 4A), water was removed from the reaction by adsorption onto the molecular sieves, avoiding hydrolysis reactions and further FFA production. Therefore, the biocatalysis in an anhydrous medium produces a higher quality product

FAME yield was also analyzed in an anhydrous medium (Fig. 4B). The results obtained show that water removal during FAME production generated a significant increase in FAME yield using Novozym 435 as the catalyst. These results are in agreement with those obtained by Li et al. (2007) who established that using 3 Å molecular sieves as adsorbent to remove excessive water could significantly increase the FAME yield when 100% FFA is used as raw material for biodiesel production. In this study, WFO containing only 2.8% FFA and 1.3% water was employed as the raw material, but FAME yield increased drastically. In addition, the control run only using molecular sieves indicates that this material did not catalyze the reaction (data not shown). Therefore, the increment in FAME yield is only related to the anhydrous medium. This corroborates the results obtained by Tamalampudi

according to the analytical procedures.

medium through water absorption.

compared to a typical standard reaction using Novozym 435.

et al. (2008) who suggest that Novozym 435 transesterification activity is inhibited by the presence of added water and that it needs a nearly anhydrous medium for an efficient performance.

Fig. 4 Acid values (A) and FAME yield (B) during the reaction in an anhydrous medium with a methanol-to-oil ratio of 4:1 (mol/mol), 15% (wt) Novozym 435 (based on the weight of oil), 35 ºC, 200 rpm and 12 hours of reaction time.

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

Samukawa et al. (2000).

FAME content [%]

FAME content [%]

0

FAME content [%]

0

20

40

60

80

20

40

60

80

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 293

sufficient quantity of water to preserve its catalytic activity in an anhydrous medium (Ognjanovic et al., 2009; Tamalampudi et al., 2008). Therefore, the enzyme activity may be reduced when using an acetone washing step, because the enzyme might lose its flexibility conformation due to the lack of bound water (Yu et al., 2010). The enzyme washing step using WFO followed by overnight incubation in WFO was also studied (Fig. 5C). The activity of Novozym 435 was higher in the third cycle in comparison to the two previous alternatives. Samukawa et al. (2000) reported that Novozym 435 preincubated in methyl oleate and subsequently in soybean oil could enhance the rate of FAME production through impregnation of these compounds into the enzyme support material. In this study, it was assumed that the enzyme could contain methyl ester residues when incubated in WFO and therefore, the treatment should be similar to that carried out by

FAME content [%]

0

20

40

60

80

100

<sup>100</sup> **A B** 

Fig. 5. Comparison of reutilization treatments of Novozym 435 after the reactions to FAME production with a methanol-to-oil ratio of 3.8:1 (mol/mol), 15% (wt) Novozym 435 (based on the weight of oil), 44.5 ºC, 200 rpm and 12 hours of reaction time. A) Control, B) Acetone

0

Time [h]

20

40

60

80

100

FAME content [%]

<sup>100</sup> **C D** 

time [h] 0 10 20 30 40 50

time [h] 0 10 20 30 40 50

This treatment using WFO is advantageous compared to the acetone washing process because it is a cheaper and more environmental friendly process. In addition, the same WFO that was used for the incubation was used subsequently for the reaction of FAME

washing, C) Waste frying oil washing D) *tert*-Butanol washing.

time [h] 0 10 20 30 40 50

time [h] 0 10 20 30 40 50

A reasonable explanation for the high FAME yield obtained may be related to the work of Cabrera et al. (2009). They suggest that lipase may exist in two different structural forms; in one form, the site of the lipase is isolated from the medium whereas in the other form, the active site is exposed to the reaction medium. In a homogeneous aqueous medium the lipase is in equilibrium between these two structures. In the case of Novozym 435, it is immobilized in acrylic resin with hydrophilic properties. Therefore, the higher yield in the reaction may occur because of interactions with the hydrophobic medium, where the lipase shifts towards the open structure form, increasing its activity. These conformational changes enable lipases to be greatly altered by controlled immobilization of the support material properties. This is in accordance with the results obtained by Samukawa et al. (2000) who established that preincubation of the enzyme in oil prior to the reaction improves the yield. This is because water adsorbed during the reaction prevented oil penetration, whereas this did not occur with the preincubated enzyme.

*Treatments for enzyme reutilization in an anhydrous medium.* The main advantage of using immobilized lipases is that the enzyme can be used repeatedly in a semi-continuous process. However, this objective is not always reached under the optimized conditions as short chain acyl acceptors can produce a loss of enzyme activity in successive reactions (Ognjanovic et al., 2009). In this study, the stability of the enzyme and treatments for its reutilization in an anhydrous medium were examined. Fig. 5 shows the results obtained by different treatments to maintain lipase activity after each FAME production reaction in an anhydrous medium. Fig. 5A shows the reutilization of Novozym 435 without treatment after reaction. According to the results obtained, FAME yield decreased considerably in the second cycle. This tendency was maintained in successive cycles, with values close to 0% of FAME yield in the fourth cycle. These results are in accordance with the findings of Ognjanovic et al. (2009), who reported 0% FAME yield in the fourth cycle using Novozym 435 and methanol as the acyl acceptor in an organic medium. Thus, although the activity of the enzyme increased in the first cycle using an anhydrous medium compared to the control (Fig. 4), the loss of activity seems to be similar in both types of medium when successive cycles are carried out (Fig. 5A). Lipases have shown high synthesis activity and stability in hydrophobic solvents, but alcohol and glycerol are immiscible in those solvents (Halim et al., 2008). This situation could produce poor solubilization of polar compounds in the medium, leading to their adsorption onto the lipases hydrophilic support and therefore provoking a low transesterification rate (Halim et al., 2008). So far, an alternative process is needed to allow the reuse of the enzyme achieving a low-cost process which can feasibly be implemented at an industrial scale.

The results of the first treatment studied for reusing the enzyme are shown in Fig. 5B. Applying an acetone washing step a higher FAME yield was achieved in the second cycle compared to the control (Fig. 5B and 5A, respectively). This result is caused by the fact that acetone is a hydrophilic solvent that could remove the glycerol and the methanol adsorbed onto the hydrophilic support material of the enzyme. However, in the third cycle the FAME yield diminished drastically (Fig. 5B). Acetone is a hydrophilic solvent with a very low log P (partition coefficient in a standard octanol-water two-phase system) value of -0.24. According to Yu et al. (2010) water has higher affinity to these hydrophilic solvents rather than to the enzyme. It has been reported that Novozym 435 can contain a

A reasonable explanation for the high FAME yield obtained may be related to the work of Cabrera et al. (2009). They suggest that lipase may exist in two different structural forms; in one form, the site of the lipase is isolated from the medium whereas in the other form, the active site is exposed to the reaction medium. In a homogeneous aqueous medium the lipase is in equilibrium between these two structures. In the case of Novozym 435, it is immobilized in acrylic resin with hydrophilic properties. Therefore, the higher yield in the reaction may occur because of interactions with the hydrophobic medium, where the lipase shifts towards the open structure form, increasing its activity. These conformational changes enable lipases to be greatly altered by controlled immobilization of the support material properties. This is in accordance with the results obtained by Samukawa et al. (2000) who established that preincubation of the enzyme in oil prior to the reaction improves the yield. This is because water adsorbed during the reaction prevented oil penetration, whereas this

*Treatments for enzyme reutilization in an anhydrous medium.* The main advantage of using immobilized lipases is that the enzyme can be used repeatedly in a semi-continuous process. However, this objective is not always reached under the optimized conditions as short chain acyl acceptors can produce a loss of enzyme activity in successive reactions (Ognjanovic et al., 2009). In this study, the stability of the enzyme and treatments for its reutilization in an anhydrous medium were examined. Fig. 5 shows the results obtained by different treatments to maintain lipase activity after each FAME production reaction in an anhydrous medium. Fig. 5A shows the reutilization of Novozym 435 without treatment after reaction. According to the results obtained, FAME yield decreased considerably in the second cycle. This tendency was maintained in successive cycles, with values close to 0% of FAME yield in the fourth cycle. These results are in accordance with the findings of Ognjanovic et al. (2009), who reported 0% FAME yield in the fourth cycle using Novozym 435 and methanol as the acyl acceptor in an organic medium. Thus, although the activity of the enzyme increased in the first cycle using an anhydrous medium compared to the control (Fig. 4), the loss of activity seems to be similar in both types of medium when successive cycles are carried out (Fig. 5A). Lipases have shown high synthesis activity and stability in hydrophobic solvents, but alcohol and glycerol are immiscible in those solvents (Halim et al., 2008). This situation could produce poor solubilization of polar compounds in the medium, leading to their adsorption onto the lipases hydrophilic support and therefore provoking a low transesterification rate (Halim et al., 2008). So far, an alternative process is needed to allow the reuse of the enzyme achieving a low-cost process which can feasibly be

The results of the first treatment studied for reusing the enzyme are shown in Fig. 5B. Applying an acetone washing step a higher FAME yield was achieved in the second cycle compared to the control (Fig. 5B and 5A, respectively). This result is caused by the fact that acetone is a hydrophilic solvent that could remove the glycerol and the methanol adsorbed onto the hydrophilic support material of the enzyme. However, in the third cycle the FAME yield diminished drastically (Fig. 5B). Acetone is a hydrophilic solvent with a very low log P (partition coefficient in a standard octanol-water two-phase system) value of -0.24. According to Yu et al. (2010) water has higher affinity to these hydrophilic solvents rather than to the enzyme. It has been reported that Novozym 435 can contain a

did not occur with the preincubated enzyme.

implemented at an industrial scale.

sufficient quantity of water to preserve its catalytic activity in an anhydrous medium (Ognjanovic et al., 2009; Tamalampudi et al., 2008). Therefore, the enzyme activity may be reduced when using an acetone washing step, because the enzyme might lose its flexibility conformation due to the lack of bound water (Yu et al., 2010). The enzyme washing step using WFO followed by overnight incubation in WFO was also studied (Fig. 5C). The activity of Novozym 435 was higher in the third cycle in comparison to the two previous alternatives. Samukawa et al. (2000) reported that Novozym 435 preincubated in methyl oleate and subsequently in soybean oil could enhance the rate of FAME production through impregnation of these compounds into the enzyme support material. In this study, it was assumed that the enzyme could contain methyl ester residues when incubated in WFO and therefore, the treatment should be similar to that carried out by Samukawa et al. (2000).

Fig. 5. Comparison of reutilization treatments of Novozym 435 after the reactions to FAME production with a methanol-to-oil ratio of 3.8:1 (mol/mol), 15% (wt) Novozym 435 (based on the weight of oil), 44.5 ºC, 200 rpm and 12 hours of reaction time. A) Control, B) Acetone washing, C) Waste frying oil washing D) *tert*-Butanol washing.

This treatment using WFO is advantageous compared to the acetone washing process because it is a cheaper and more environmental friendly process. In addition, the same WFO that was used for the incubation was used subsequently for the reaction of FAME

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

reactions.

FAME content [%]

0

20

40

60

80

100

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 295

catalyst. Therefore, the positive effect of *tert*-butanol in the reaction could be first related to the increment in the oil-methanol miscibility, preventing direct contact between enzyme and alcohol at the beginning of the reaction and improving the reaction yield. Secondly, due to *tert*-butanol's hydrophilic properties, it is able to dissolve both methanol and glycerol during the reaction, preventing enzyme inhibition throughout the reaction. Thirdly, *tert*butanol's hydrophobic properties maintained the high lipase activity through the successive

Control

Anhydrous medium with *tert*-butanol

time [h] 0 20 40 60 80 100 120 140 160 180 200

Fig. 6. Time course of FAME yield during 17 batch reaction cycles with a methanol-to-oil ratio of 3.8:1 (mol/mol), 15% (wt) Novozym 435 (based on the weight of oil), 44.5 ºC, 0.75% v/v of *tert*-butanol, 0.9 g of molecular sieves, 200 rpm and 12 hours of reaction time.

According to Yu et al. (2010), different properties such as viscosity, dielectric constant, solubility parameters and log P should be considered when choosing a co-solvent to improve enzyme performance in biodiesel production. Another aspect which should be considered is the feasibility of recovering the co-solvent through distillation, which is related to the boiling point. In this sense, *tert*-butanol has advantages compared to others cosolvents reported, such as amyl alcohol, which has very similar properties compared to the *tert*-butanol but a higher boiling point (102 °C) close to the boiling point of the water. As the boiling point of *tert*-butanol is 82 °C and the distillation of methanol occurs at 65 °C, the recovery of *tert-*butanol should not increase the amount of energy spent that much in comparison to the advantages of using this co-solvent. Therefore, employing *tert*-butanol as a co-solvent could allow the use of an anhydrous medium as an industrial alternative for

Symbols: in *tert*- butanol system (closed circles), control (open circles).

FAME production using Novozym 435 as biocatalyst.

production, and therefore less equipment is needed to carry out this process. In another study, Chen and Wu (2003) achieved a FAME yield five times higher when the enzyme was incubated overnight in soybean oil. However, when they used the same oil in incubation experiments to recover the enzyme after the reaction, the activity began to decay after the fourth reutilization. Although the advantages of using WFO in enzyme recovery, FAME yield also declined in this study, as reported by Chen and Wu (2003). The reason may be the low solubility of alcohol and glycerol in the oil (hydrophobic wash), which impedes the efficient washing of the enzyme by oil.

As the washing process with hydrophobic and hydrophilic solvents did not allow an efficient recovery of the enzyme activity by more than three successive reactions in an anhydrous medium, a moderate polar solvent was also investigated. *Tert*-butanol, a tertiary alcohol with a log P value of 0.35 has been shown to improve enzyme activity more than linear alcohols. This fact could be attributed to the differences in miscibility with triglycerides, as compared to alcohols with the same carbon numbers, branched ones have better miscibility with triglycerides compared to linear isomers (Yu et al., 2010). Therefore, a washing treatment of the enzymes with *tert*-butanol, followed by incubation in WFO, was conducted. The results showed that this enzyme pretreatment achieved the best results, maintaining a higher activity over the time compared to the previous experiments (Fig. 5D). It is likely that *tert*-butanol recovered the enzyme activity because it has the advantages of both hydrophilic and hydrophobic solvents but none of the drawbacks. Therefore, *tert*-butanol should promote the removal of both glycerol and methanol from the lipase support material because of its hydrophilic properties, while its hydrophobic properties should help maintain a high level of lipase activity. This is in accordance to Chen and Wu (2003) who found that *tert*-butanol can be even used to regenerate a deactivated, immobilized enzyme such as Novozym 435. Therefore, *tert*butanol is a promising alternative for enzyme recovery after reaction in an anhydrous medium, and is also highly stable and less reactive than other butanol isomers. Based on these results, a new experiment was carried out which included *tert*-butanol as a cosolvent in the reaction in an anhydrous medium.

*Successive reactions to FAME production in an anhydrous medium using tert-butanol as a cosolvent.* A previous study found that *tert*-butanol is inert in the methanolysis system, whereas it is also a potential co-solvent that could maintain enzymatic activity (Li et al., 2006). According to this, the performance of *tert*-butanol as a co-solvent was studied in an anhydrous medium to determine its ability to maintain Novozym 435 activity in successive reactions. In Fig. 6 is shown the successive reactions for FAME production that were carried out in an anhydrous medium under previously optimized operational conditions, using a previously selected dosage of *tert*-butanol (0.75% v/v, data not show). A control without *tert*-butanol as co-solvent was also carried out, showing that FAME yield was drastically reduced in the second cycle (< 20% FAME yield). The best results were obtained in the system using the co-solvent, where FAME yield was maintained over 50% after 17 cycles (Fig. 6). According to similar findings from other studies, the enzyme is inhibited throughout the reaction (Royon et al., 2007). The inhibitory effect at the beginning of the reaction is due to the presence of methanol which has poor miscibility in oil. Subsequently, once methanol concentration decreases, inhibition is caused by a glycerol layer coating the

production, and therefore less equipment is needed to carry out this process. In another study, Chen and Wu (2003) achieved a FAME yield five times higher when the enzyme was incubated overnight in soybean oil. However, when they used the same oil in incubation experiments to recover the enzyme after the reaction, the activity began to decay after the fourth reutilization. Although the advantages of using WFO in enzyme recovery, FAME yield also declined in this study, as reported by Chen and Wu (2003). The reason may be the low solubility of alcohol and glycerol in the oil (hydrophobic wash), which impedes the

As the washing process with hydrophobic and hydrophilic solvents did not allow an efficient recovery of the enzyme activity by more than three successive reactions in an anhydrous medium, a moderate polar solvent was also investigated. *Tert*-butanol, a tertiary alcohol with a log P value of 0.35 has been shown to improve enzyme activity more than linear alcohols. This fact could be attributed to the differences in miscibility with triglycerides, as compared to alcohols with the same carbon numbers, branched ones have better miscibility with triglycerides compared to linear isomers (Yu et al., 2010). Therefore, a washing treatment of the enzymes with *tert*-butanol, followed by incubation in WFO, was conducted. The results showed that this enzyme pretreatment achieved the best results, maintaining a higher activity over the time compared to the previous experiments (Fig. 5D). It is likely that *tert*-butanol recovered the enzyme activity because it has the advantages of both hydrophilic and hydrophobic solvents but none of the drawbacks. Therefore, *tert*-butanol should promote the removal of both glycerol and methanol from the lipase support material because of its hydrophilic properties, while its hydrophobic properties should help maintain a high level of lipase activity. This is in accordance to Chen and Wu (2003) who found that *tert*-butanol can be even used to regenerate a deactivated, immobilized enzyme such as Novozym 435. Therefore, *tert*butanol is a promising alternative for enzyme recovery after reaction in an anhydrous medium, and is also highly stable and less reactive than other butanol isomers. Based on these results, a new experiment was carried out which included *tert*-butanol as a co-

*Successive reactions to FAME production in an anhydrous medium using tert-butanol as a cosolvent.* A previous study found that *tert*-butanol is inert in the methanolysis system, whereas it is also a potential co-solvent that could maintain enzymatic activity (Li et al., 2006). According to this, the performance of *tert*-butanol as a co-solvent was studied in an anhydrous medium to determine its ability to maintain Novozym 435 activity in successive reactions. In Fig. 6 is shown the successive reactions for FAME production that were carried out in an anhydrous medium under previously optimized operational conditions, using a previously selected dosage of *tert*-butanol (0.75% v/v, data not show). A control without *tert*-butanol as co-solvent was also carried out, showing that FAME yield was drastically reduced in the second cycle (< 20% FAME yield). The best results were obtained in the system using the co-solvent, where FAME yield was maintained over 50% after 17 cycles (Fig. 6). According to similar findings from other studies, the enzyme is inhibited throughout the reaction (Royon et al., 2007). The inhibitory effect at the beginning of the reaction is due to the presence of methanol which has poor miscibility in oil. Subsequently, once methanol concentration decreases, inhibition is caused by a glycerol layer coating the

efficient washing of the enzyme by oil.

solvent in the reaction in an anhydrous medium.

catalyst. Therefore, the positive effect of *tert*-butanol in the reaction could be first related to the increment in the oil-methanol miscibility, preventing direct contact between enzyme and alcohol at the beginning of the reaction and improving the reaction yield. Secondly, due to *tert*-butanol's hydrophilic properties, it is able to dissolve both methanol and glycerol during the reaction, preventing enzyme inhibition throughout the reaction. Thirdly, *tert*butanol's hydrophobic properties maintained the high lipase activity through the successive reactions.

Fig. 6. Time course of FAME yield during 17 batch reaction cycles with a methanol-to-oil ratio of 3.8:1 (mol/mol), 15% (wt) Novozym 435 (based on the weight of oil), 44.5 ºC, 0.75% v/v of *tert*-butanol, 0.9 g of molecular sieves, 200 rpm and 12 hours of reaction time. Symbols: in *tert*- butanol system (closed circles), control (open circles).

According to Yu et al. (2010), different properties such as viscosity, dielectric constant, solubility parameters and log P should be considered when choosing a co-solvent to improve enzyme performance in biodiesel production. Another aspect which should be considered is the feasibility of recovering the co-solvent through distillation, which is related to the boiling point. In this sense, *tert*-butanol has advantages compared to others cosolvents reported, such as amyl alcohol, which has very similar properties compared to the *tert*-butanol but a higher boiling point (102 °C) close to the boiling point of the water. As the boiling point of *tert*-butanol is 82 °C and the distillation of methanol occurs at 65 °C, the recovery of *tert-*butanol should not increase the amount of energy spent that much in comparison to the advantages of using this co-solvent. Therefore, employing *tert*-butanol as a co-solvent could allow the use of an anhydrous medium as an industrial alternative for FAME production using Novozym 435 as biocatalyst.

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

GC-MS methods.

Simulate initial rate of reaction, *u* [mol L-1 min-1]

0.000

WFO concentration of 300 mol L-1.

0.001

0.002

0.003

0.004

0.005

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 297

the kinetic constant KIM was obtained using Excel solver to find the minimum objective function that compares the measured rate of the reaction with those predicted by the proposed kinetic equation. Each assay were performed in quadruplicate in Erlenmeyer flask of 25mL and incubated in an orbital shaker during 4 hr under the same operational condition: 15% (wt) of Novozym 435, 44°C, 0.75% (V/V) of *tert-*butanol, 0.5 g of molecular sieves and 200 rpm. In order to remove the *tert*-butanol, the samples were centrifuged and heated at 85°C during 30 minutes. The supernatant was used to quantify FAME yield using

Using the experimental results the kinetic parameters estimated for the model were: Vmáx= 0.018 mol/L/min, KM, metanol = 1030 mol/L, KW, WFO= 397 mol/L y KIM, metanol= 1,815 mol/L. The Fig. 7 shows a comparison between the model predictions and the experimental data of

> Experimental initial rate of reaction, *<sup>u</sup>*[mol L-1 min-1] 0.000 0.001 0.002 0.003 0.004 0.005

Fig. 7. Comparison between the experimental results and the Ping-Pong kinetic model equation with the estimated constants to different initial methanol concentration and initial

the initial rate of reaction at different methanol concentration using Novozym 435.

The results of this section showed that the use of an anhydrous medium (resulting from water extraction by using molecular sieves), FAME yield was improved by avoiding hydrolysis and esterification reactions, producing FAME mainly through transesterification of TG. The enzyme activity cannot be recovered successively neither using a hydrophilic washing step with acetone nor by hydrophobic washing with WFO. However, 17 successive cycles of FAME production using *tert*-butanol as a moderate polar co-solvent, show that Novozym 435 can be reused in anhydrous medium. These results also show that the anhydrous medium could enable the implementation of lipase-catalyzed processes on an industrial scale for biodiesel production mainly by transesterification reaction. Different raw materials could be used for this, achieving properties close to the norm and potentially avoiding post-treatments to refine the produced biodiesel.

#### **4. Enzymatic transesterification reaction kinetic in a** *tert***-butanol system for biodiesel production**

For the design of suitable reactors to FAME production using lipases, kinetic information of the rate of product formation and the effects of changes in system conditions is needed. Investigation about kinetic of FAME production using different lipases has been recently reported. The first researches were focused in the esterification of FFA, where the Ping Pong model with competitive inhibition by methanol was used to describe the reaction (Krishna et al., 2001). After, Al-Zuhair et al. (2007) proposed a kinetic model based in the transesterification of TG. The aim of this work was study the kinetic of the transesterification to FAME production catalyzed by Novozym 435. The investigation was carried out in anhydrous medium, using WFO as raw material, methanol as acyl acceptor and *tert-*butanol as co-solvent. In addition, the set-up of a semi-continuous enzymatic bioreactor was carried out. To realize the study Ping Pong model with competitive inhibition by methanol was used (Eq. 4).

$$\nu = \frac{V\_{\text{max}}}{1 + \frac{K\_{\text{IV}}}{\left[VV\right]} \left[1 + \frac{\left[M\right]}{K\_{\text{IM}}}\right] + \frac{K\_{\text{M}}}{\left[M\right]}} \tag{4}$$

Where υ (mol/L/min) is the initial reaction rate, Vmáx is the maximum rate of reaction (mol/L/min), KW and KM are the binding constants for the WFO (W) and the methanol (M) (mol/L), [W] is WFO concentration (mol/L), [M] is methanol concentration (mol/L) and KIM is the inhibition constant for the methanol (mol/L).

To determinate the sole effect of methanol in the transesterification, the experiments was run using methanol concentrations in the range of 100-3000 mol/L, equivalent at methanol to oil molar ratio 0.6-15 (mol/mol), at a constant WFO concentration of 300 mol/L. The WFO concentration was chosen to ensure operating outside WFO limitation or inhibition region. To determinate the sole effect of WFO in the transesterification , the experiments was run using WFO concentrations in the range of 200-350 mol/L, at a constant methanol concentration of 1400 mol/L. The methanol concentration was chosen to ensure operating outside alcohol limitation or inhibition region. Michaelis-menten kinetic was used to estimate the kinetic constants Vmáx, KW y KM. Subsequently, the values were optimized and

The results of this section showed that the use of an anhydrous medium (resulting from water extraction by using molecular sieves), FAME yield was improved by avoiding hydrolysis and esterification reactions, producing FAME mainly through transesterification of TG. The enzyme activity cannot be recovered successively neither using a hydrophilic washing step with acetone nor by hydrophobic washing with WFO. However, 17 successive cycles of FAME production using *tert*-butanol as a moderate polar co-solvent, show that Novozym 435 can be reused in anhydrous medium. These results also show that the anhydrous medium could enable the implementation of lipase-catalyzed processes on an industrial scale for biodiesel production mainly by transesterification reaction. Different raw materials could be used for this, achieving properties close to the norm and potentially

**4. Enzymatic transesterification reaction kinetic in a** *tert***-butanol system for** 

For the design of suitable reactors to FAME production using lipases, kinetic information of the rate of product formation and the effects of changes in system conditions is needed. Investigation about kinetic of FAME production using different lipases has been recently reported. The first researches were focused in the esterification of FFA, where the Ping Pong model with competitive inhibition by methanol was used to describe the reaction (Krishna et al., 2001). After, Al-Zuhair et al. (2007) proposed a kinetic model based in the transesterification of TG. The aim of this work was study the kinetic of the transesterification to FAME production catalyzed by Novozym 435. The investigation was carried out in anhydrous medium, using WFO as raw material, methanol as acyl acceptor and *tert-*butanol as co-solvent. In addition, the set-up of a semi-continuous enzymatic bioreactor was carried out. To realize the study Ping Pong model with competitive

max

*V K K M WK M*

Where υ (mol/L/min) is the initial reaction rate, Vmáx is the maximum rate of reaction (mol/L/min), KW and KM are the binding constants for the WFO (W) and the methanol (M) (mol/L), [W] is WFO concentration (mol/L), [M] is methanol concentration (mol/L) and KIM

To determinate the sole effect of methanol in the transesterification, the experiments was run using methanol concentrations in the range of 100-3000 mol/L, equivalent at methanol to oil molar ratio 0.6-15 (mol/mol), at a constant WFO concentration of 300 mol/L. The WFO concentration was chosen to ensure operating outside WFO limitation or inhibition region. To determinate the sole effect of WFO in the transesterification , the experiments was run using WFO concentrations in the range of 200-350 mol/L, at a constant methanol concentration of 1400 mol/L. The methanol concentration was chosen to ensure operating outside alcohol limitation or inhibition region. Michaelis-menten kinetic was used to estimate the kinetic constants Vmáx, KW y KM. Subsequently, the values were optimized and

[] [] *W M IM*

(4)

[ ] 1 1

 

avoiding post-treatments to refine the produced biodiesel.

**biodiesel production** 

inhibition by methanol was used (Eq. 4).

is the inhibition constant for the methanol (mol/L).

the kinetic constant KIM was obtained using Excel solver to find the minimum objective function that compares the measured rate of the reaction with those predicted by the proposed kinetic equation. Each assay were performed in quadruplicate in Erlenmeyer flask of 25mL and incubated in an orbital shaker during 4 hr under the same operational condition: 15% (wt) of Novozym 435, 44°C, 0.75% (V/V) of *tert-*butanol, 0.5 g of molecular sieves and 200 rpm. In order to remove the *tert*-butanol, the samples were centrifuged and heated at 85°C during 30 minutes. The supernatant was used to quantify FAME yield using GC-MS methods.

Using the experimental results the kinetic parameters estimated for the model were: Vmáx= 0.018 mol/L/min, KM, metanol = 1030 mol/L, KW, WFO= 397 mol/L y KIM, metanol= 1,815 mol/L. The Fig. 7 shows a comparison between the model predictions and the experimental data of the initial rate of reaction at different methanol concentration using Novozym 435.

Fig. 7. Comparison between the experimental results and the Ping-Pong kinetic model equation with the estimated constants to different initial methanol concentration and initial WFO concentration of 300 mol L-1.

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

reactor during all the cycles. In Fig. 9 are showed the results obtained.

produce FAME.

**5. Conclusions** 

catalyzed process.

**6. Acknowledgement** 

11110282.

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 299

The results of the kinetic study were applied in the operation of a semi-continuous reactor to enzymatic FAME production. The bioreactor was made of glass with a volume of 0.5 L. The temperature was controller by a thermostat. The agitation of the bioreactor was supply by a magnetic stirrer. In order to extract water constantly from media, a glass column filled with molecular sieves was connected to the bioreactor, where the media was recycling using a peristaltic pump. The glycerol was removed from bottom by a settler connected to the bioreactor. The operational conditions to each reaction cycle were: methanol to oil molar ratio 8/1 (mol/mol), 15 % (wt) Novozym 435, 0.75% (v/v) of *tert*-butanol, 44.5 °C, 200 rpm and 4 hours of reaction time. The enzymes were reused successively remaining into the

The bioreactor was operated several cycles of charge and discharge during 30 h, adding only raw material and changing the molecular sieves to maintain an anhydrous media. Under these conditions was possible to maintain FAME yields over 80% during 7 reaction cycles (Fig. 8). Therefore, the use of both an anhydrous media and *tert-*butanol as cosolvent are effective strategies for the implementation of a semi-continuous enzymatic process that can become economically competitive with traditional chemical catalysts to

The short reaction time of the proposed process and the reuse of the enzymes generate a feasible alternative to be implemented at industrial scale. This process has several advantages compared to the chemical process. Although the chemical process requires only 1 hour of reaction time, after the process a washing step to remove catalyst residues is necessary. This washing step is not necessary in the enzymatic process because Novozym 435 is a solid catalyst. In addition, the enzymatic process has the advantage to be flexible allowing the use of alternative and low cost raw materials. Therefore, the results obtained in this work have generated an enzymatic process that could become not only environmentally friendly but also economically competitive with the chemical-

The kinetic study and the reactor operation showed that Novozym 435 was not inhibited to high methanol concentrations and could be reused without significant loss of activity. These results shown that under the conditions investigated enzymatic FAME production could be a competitive process to be implemented to industrial scale. To reach this objective is necessary to carry on the bioreactor operation in the long time, in order to establish the lifespan of the enzymes. In addition is necessary to establish a molecular sieves recuperation protocol and to

Research described was supported by Desert Bioenergy S.A., Chilean project "Inserción de Capital Humano Avanzado en el Sector Productivo Chileno 78110106", Chilean Fondecyt project 3120171, Chilean CONICYT Project 79090009 and FONDECYT iniciación project

looking for inexpensive material such as zeolites to decrease the operational cost.

According to Fig.7 the kinetic model is suitable to predict the behavior of the reaction, indicating that the use Michaelis-Mentel kinectic could simplify the calculation of kinectic constant of complex model such as Ping Pong kinetic model. The model and empirical data showed that Novozym 435 presented a low inhibition by methanol, even at higher concentration maintaining a high initial reaction rate until a methanol to WFO molar ratio of 8/1 (mol/mol). The model predicts a moderate decrease in the initial reaction rate for higher concentration of methanol. This behavior could be associated to the incorporation of the cosolvent in the reaction due its capacity to improve the miscibility of reaction mix. This is an advantage because higher concentration of methanol favor the production formation since the transesterification is an equilibrium reaction, decreasing the operation time of the FAME production using enzymatic catalysts.

Fig. 8. FAME yield during the set-up of a semi-continuous enzymatic reactor in anhydrous medium with enzymes reutilization. Operational conditions: methanol-to-oil ratio of 8/1 (mol/mol), 15% (wt) Novozym 435 (based on the weight of oil), 44.5 ºC, 0.75% v/v of *tert*butanol, 200 rpm and 4 hours of reaction time.

The results of the kinetic study were applied in the operation of a semi-continuous reactor to enzymatic FAME production. The bioreactor was made of glass with a volume of 0.5 L. The temperature was controller by a thermostat. The agitation of the bioreactor was supply by a magnetic stirrer. In order to extract water constantly from media, a glass column filled with molecular sieves was connected to the bioreactor, where the media was recycling using a peristaltic pump. The glycerol was removed from bottom by a settler connected to the bioreactor. The operational conditions to each reaction cycle were: methanol to oil molar ratio 8/1 (mol/mol), 15 % (wt) Novozym 435, 0.75% (v/v) of *tert*-butanol, 44.5 °C, 200 rpm and 4 hours of reaction time. The enzymes were reused successively remaining into the reactor during all the cycles. In Fig. 9 are showed the results obtained.

The bioreactor was operated several cycles of charge and discharge during 30 h, adding only raw material and changing the molecular sieves to maintain an anhydrous media. Under these conditions was possible to maintain FAME yields over 80% during 7 reaction cycles (Fig. 8). Therefore, the use of both an anhydrous media and *tert-*butanol as cosolvent are effective strategies for the implementation of a semi-continuous enzymatic process that can become economically competitive with traditional chemical catalysts to produce FAME.

#### **5. Conclusions**

298 Enzyme Inhibition and Bioapplications

According to Fig.7 the kinetic model is suitable to predict the behavior of the reaction, indicating that the use Michaelis-Mentel kinectic could simplify the calculation of kinectic constant of complex model such as Ping Pong kinetic model. The model and empirical data showed that Novozym 435 presented a low inhibition by methanol, even at higher concentration maintaining a high initial reaction rate until a methanol to WFO molar ratio of 8/1 (mol/mol). The model predicts a moderate decrease in the initial reaction rate for higher concentration of methanol. This behavior could be associated to the incorporation of the cosolvent in the reaction due its capacity to improve the miscibility of reaction mix. This is an advantage because higher concentration of methanol favor the production formation since the transesterification is an equilibrium reaction, decreasing the operation time of the FAME

> Cicle [N°] 01234567

time [h] 0 10 20 30 40 50

Fig. 8. FAME yield during the set-up of a semi-continuous enzymatic reactor in anhydrous medium with enzymes reutilization. Operational conditions: methanol-to-oil ratio of 8/1 (mol/mol), 15% (wt) Novozym 435 (based on the weight of oil), 44.5 ºC, 0.75% v/v of *tert*-

production using enzymatic catalysts.

FAME content [%]

0

butanol, 200 rpm and 4 hours of reaction time.

20

40

60

80

100

The short reaction time of the proposed process and the reuse of the enzymes generate a feasible alternative to be implemented at industrial scale. This process has several advantages compared to the chemical process. Although the chemical process requires only 1 hour of reaction time, after the process a washing step to remove catalyst residues is necessary. This washing step is not necessary in the enzymatic process because Novozym 435 is a solid catalyst. In addition, the enzymatic process has the advantage to be flexible allowing the use of alternative and low cost raw materials. Therefore, the results obtained in this work have generated an enzymatic process that could become not only environmentally friendly but also economically competitive with the chemicalcatalyzed process.

The kinetic study and the reactor operation showed that Novozym 435 was not inhibited to high methanol concentrations and could be reused without significant loss of activity. These results shown that under the conditions investigated enzymatic FAME production could be a competitive process to be implemented to industrial scale. To reach this objective is necessary to carry on the bioreactor operation in the long time, in order to establish the lifespan of the enzymes. In addition is necessary to establish a molecular sieves recuperation protocol and to looking for inexpensive material such as zeolites to decrease the operational cost.

#### **6. Acknowledgement**

Research described was supported by Desert Bioenergy S.A., Chilean project "Inserción de Capital Humano Avanzado en el Sector Productivo Chileno 78110106", Chilean Fondecyt project 3120171, Chilean CONICYT Project 79090009 and FONDECYT iniciación project 11110282.

Feasible Novozym 435-Catalyzed Process to Fatty Acid Methyl

*Enzymology,* 1547, 262-267.

medium. *J Mol Catal B: Enzym*, 58-62.

*Process Biochem,* 42, 1481-1485.

*Catal B: Enzym,* 17, 133-142.

129.

648–653.

185-189.

Ester Production from Waste Frying Oil: Role of Lipase Inhibition 301

Kose, O., Tuter, M. & Aksoy, H. (2002). Immobilized *Candida antarctica* lipase-catalyzed

Krishna, S. H. & Karanth, N. G. (2001). Lipase-catalyzed synthesis of isoamyl butyrate - A

Li, L., Du, W., Liu, D., Wang, L. & Li, Z. (2006). Lipase-catalyzed transesterification of

Li, N., Zong, M. & Wu, H. (2009). Highly efficient transformation of waste oil to biodiesel by immobilized lipase from *Penicillium expansum*. *Process Biochem,* 44, 685-688. Li, W., Du, W. & Liu, D. (2007). *Rhizopus oryzae* IFO 4697 whole cell-catalyzed methanolysis

Lin, C. & Lin, H. (2007). Engine performance and emission characteristics of a three-phase emulsion of biodiesel produced by peroxidation. *Fuel Process. Tech.,* 88, 35-41. Ognjanovic, N., Bezbradica, D. & Knezevic-Jugovic, Z. (2009). Enzymatic conversion of

Royon, D., Daz, M., Ellenrieder, G. & Locatelli, S. (2007). Enzymatic production of

Salis, A., Pinna, M., Monduzzi, M. & Solinas, V. (2005). Biodiesel production from triolein and short chain alcohols through biocatalysis. *J Biotechnol,* 119, 291-299. Samukawa, T., Kaieda, M., Matsumoto, T., Ban, K., Kondo, A., Shimada, Y., Noda, H. &

Shimada, Y., Watanabe, Y., Sugihara, A. & Tominaga, Y. (2002). Enzymatic alcoholysis for

Tamalampudi, S., Talukder, M., Hama, S., Numata, T., Kondo, A. & Fukuda, H. (2008).

The British Standards Institution (2003): BS EN 14214:2003. *Automotive fuels. Fatty acid* 

Turkan, A. & Kalay, S. (2006). Monitoring lipase-catalyzed methanolysis of sunflower oil by

Wang, L., Du, W., Liu, D., Li, L. & Dai, N. (2006). Lipase-catalyzed biodiesel production

mechanisms of lipases. *J. Chromatogr. A,* 1127, 34-44.

system. *J Mol Catal B: Enzym,* 43, 29-32.

biodiesel fuel production from plant oil. *J Biosci Bioeng,* 90, 180-183.

immobilized system stability. *Bioresour Technol,* 100, 5146-5154.

alcoholysis of cotton seed oil in a solvent-free medium. *Bioresour. Technol.,* 83, 125-

kinetic study. *Biochimica Et Biophysica Acta-Protein Structure and Molecular* 

rapeseed oils for biodiesel production with a novel organic solvent as the reaction

of crude and acidified rapeseed oils for biodiesel production in *tert*-butanol system.

sunflower oil to biodiesel in a solvent-free system: Process optimization and the

biodiesel from cotton seed oil using *t*-butanol as a solvent. *Bioresour Technol,* 98,

Fukuda, H. (2000). Pretreatment of immobilized *Candida antarctica* lipase for

biodiesel fuel production and application of the reaction to oil processing. *J Mol* 

Enzymatic production of biodiesel from Jatropha oil: A comparative study of immobilized-whole cell and commercial lipases as a biocatalyst. *Biochem Eng J,* 39,

*methylesters (FAME) for diesel engines*. Requirements and test methods, BSI, London.

reversed-phase high-performance liquid chromatography: Elucidation of the

from soybean oil deodorizer distillate with absorbent present in *tert*-butanol

#### **7. References**


Al-Zuhair, S., Wei, F. & Jun, L. (2007). Proposed kinetic mechanism of the production of

American Society for Testing and Materials, ASTM (2008): *Annual Book of ASTM Standards:* 

Araújo, J. (1995). Oxidação de Lipidios, p. 1- 64. *In* Imprensa Universitária (ed.), Química de

Azócar, L., Ciudad, G., Heipieper, H., Muñoz, R. & Navia, R. (2010a). Improving fatty acid

Azócar, L., Ciudad, G., Heipieper, H. & Navia, R. (2010b). Biotechnological processes for biodiesel production using alternative oils. *Appl Microbiol Biotechnol,* 88, 621–636. Cabrera, Z., Fernandez-Lorente, G., Fernandez-Lafuente, R., Palomo, J. & Guisan, J. (2009).

Canakci, M. (2007). The potential of restaurant waste lipids as biodiesel feedstocks. *Bioresour* 

Chen, J. & Wu, W. (2003). Regeneration of immobilized *Candida antarctica* lipase for

Dizge, N., Aydiner, C., Imer, D. Y., Bayramoglu, M., Tanriseven, A. & Keskinlera, B. (2009).

Du, W., Xu, Y., Liu, D. & Zeng, J. (2004). Comparative study on lipase-catalyzed

Fukuda, H., Hama, S., Tamalampudi, S. & Noda, H. (2008). Whole-cell biocatalysts for

Fukuda, H., Kondo, A. & Noda, H. (2001). Review: Biodiesel fuel production by

Gui, M., Lee, K. & Bhatia, S. (2008). Feasibility of edible oil vs. non-edible oil vs. waste edible

Halim, S. & Kamaruddin, A. (2008). Catalytic studies of lipase on FAME production from waste cooking palm oil in a *tert*-butanol system. *Process Biochem,* 43, 1436–1439. Hernandez-Martin, E. & Otero, C. (2008). Different enzyme requirements for the synthesis

Issariyakul, T., Kulkarni, M., Meher, L., Dalai, A. & Bakhshi, N. (2008). Biodiesel

of biodiesel: Novozym (R) 435 and Lipozyme (R) TL IM. *Bioresour Technol,* 99,

production from mixtures of canola oil and used cooking oil. *Chem Eng J,* 140, 77-

*Section 5 - Petroleum Products, Lubricants, and Fossil Fuels*. ASTM International, West

methyl ester production yield in a lipase-catalyzed process using waste frying oils

Novozym 435 displays very different selectivity compared to lipase from *Candida antarctica* B adsorbed on other hydrophobic supports. *J Mol Catal B: Enzym,* 57, 171–

Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer.

transformation of soybean oil for biodiesel production with different acyl

biodiesel from palm oil using lipase. *Process Biochem,* 42, 951-960.

alimentos: Teoría e prática. Universidad Federal de Viçosa. Viçosa.

as feedstock. *J Biosci Bioeng,* 109, 609-614.

transesterification. *J Biosci Bioeng,* 95, 466-469.

acceptors. *J Mol Catal B: Enzym,* 30, 125-129.

biodiesel fuel production. *Trends Biotechnol,* 26, 668-673.

transesterification of oils. *J Biosci Bioeng,* 92, 405-416.

oil as biodiesel feedstock. *Energy,* 33, 1646-1653.

*Bioresour Technol,* 100, 1983-1991.

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176.

277-286.

85.

*Technol,* 98, 183–190.


**10** 

*Pakistan* 

**Urease Inhibition** 

*of Engineering and Technology Karachi* 

Muhammad Raza Shah1 and Zahid Hussain Soomro2 *1International Center for Chemical and Biological Sciences, H.E.J.* 

The design, synthesis, characterization and exploring the broad spectrum of potency of biologically active molecular building blocks have attracted the attention of scientific community since last couple of decades. The emergence of pathogenic resistance is a natural phenomenon and investigations toward the advances of new structurally diverse inhibitors have always been at the esteem of pharmaceutical research. This growing field has ever become an active area of research for the synthetic, biological and medicinal perspectives, looking at the molecular level. Enzyme inhibition has attracted great attention of biomedical scientist since last couple of decades. A variety of inhibitors have been discovered and used for the control of various diseases. One of the key features of enzyme is its selectivity towards certain inhibitors; the inhibitor can be a simple organic molecule or complex molecular architecture. The specificity of inhibitor depends on the size, shape and the interactive forces which result in the exact matching of inhibitor and the enzyme. These inhibitions block the activity of the enzyme under the physiological conditions. Urea and urease are the landmark molecules in the early days of synthetic organic chemistry. Urea was the first organic molecule synthesized in laboratory and urease was being first crystallized out from jack bean (Amtul et al., 2002; Hagar et al., 1925; Mobley et al., 1989; Amtul et al., 2006; Zonia et al., 1995). The significance of the urease enzyme is critically investigated due to its capacity to serve as a virulence factor of infections in the urinary and gastrointestinal tracts, maintaining equilibrium in the nitrogen cycles of nitrogenous wastes in the rumens of domestic livestock, and its importance in environmental transformations of nitrogenous compounds, including urea based fertilizers. The physiology of microbial ureases depends on their cellular location, regulation, genetic makeup and the relationships between the microbial urease and the well-characterized potent range of inhibitors. The urease (urea amido hydrolase; EC 3.5.1.5) is a nickel containing enzyme which hydrolyzes urea into ammonia and carbamic acid, the later compound produce ammonia and carbon dioxide on further decomposition. The hydrolysis of the urea yields high concentrations of ammonia which accompanying in pH elevation. Urease has been regarded as the important enzyme for the manipulation of urea and other nitrogenous ingredients in the biological, agricultural and environmental fields. Urease occurs in many plants, selected fungi, and a

**1. Introduction** 

*Research Institute of Chemistry, University of Karachi 2Institute of Materials Science and Research Dawood College* 


## **Urease Inhibition**

Muhammad Raza Shah1 and Zahid Hussain Soomro2

*1International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi 2Institute of Materials Science and Research Dawood College of Engineering and Technology Karachi Pakistan* 

#### **1. Introduction**

302 Enzyme Inhibition and Bioapplications

Watanabe, Y., Shimada, Y., Sugihara, A. & Tominaga, Y. (2001). Enzymatic conversion of

Yu, D., Tian, L., Wu, H., Wang, S., Wang, Y., Ma, D. & Fang, X. (2010). Ultrasonic irradiation

703-707.

*Biochem,* 45, 519–525.

waste edible oil to biodiesel fuel in a fixed-bed bioreactor. *J Am Oil Chem Soc,* 78,

with vibration for biodiesel production from soybean oil by Novozym 435. *Process* 

The design, synthesis, characterization and exploring the broad spectrum of potency of biologically active molecular building blocks have attracted the attention of scientific community since last couple of decades. The emergence of pathogenic resistance is a natural phenomenon and investigations toward the advances of new structurally diverse inhibitors have always been at the esteem of pharmaceutical research. This growing field has ever become an active area of research for the synthetic, biological and medicinal perspectives, looking at the molecular level. Enzyme inhibition has attracted great attention of biomedical scientist since last couple of decades. A variety of inhibitors have been discovered and used for the control of various diseases. One of the key features of enzyme is its selectivity towards certain inhibitors; the inhibitor can be a simple organic molecule or complex molecular architecture. The specificity of inhibitor depends on the size, shape and the interactive forces which result in the exact matching of inhibitor and the enzyme. These inhibitions block the activity of the enzyme under the physiological conditions. Urea and urease are the landmark molecules in the early days of synthetic organic chemistry. Urea was the first organic molecule synthesized in laboratory and urease was being first crystallized out from jack bean (Amtul et al., 2002; Hagar et al., 1925; Mobley et al., 1989; Amtul et al., 2006; Zonia et al., 1995). The significance of the urease enzyme is critically investigated due to its capacity to serve as a virulence factor of infections in the urinary and gastrointestinal tracts, maintaining equilibrium in the nitrogen cycles of nitrogenous wastes in the rumens of domestic livestock, and its importance in environmental transformations of nitrogenous compounds, including urea based fertilizers. The physiology of microbial ureases depends on their cellular location, regulation, genetic makeup and the relationships between the microbial urease and the well-characterized potent range of inhibitors. The urease (urea amido hydrolase; EC 3.5.1.5) is a nickel containing enzyme which hydrolyzes urea into ammonia and carbamic acid, the later compound produce ammonia and carbon dioxide on further decomposition. The hydrolysis of the urea yields high concentrations of ammonia which accompanying in pH elevation. Urease has been regarded as the important enzyme for the manipulation of urea and other nitrogenous ingredients in the biological, agricultural and environmental fields. Urease occurs in many plants, selected fungi, and a

Urease Inhibition 305

al., 2009). Literature is rich on the role o*f H. pylori w*hich is the main cause for the gastric infections. Urease is a prominent antigen of the *H. Pylori* which has served as a powerful immunogen for this organism. There have been various reports on large number of patients who have shown gastritis significantly elevated response in immunoglobulins G and A along with urease in the blood serum samples with relative comparison of pre-infected levels. Range of enzymatic assays is reported to measure the immune responses and these are used for the diagnostic purposes in monitoring the antibiotic therapy of *H-Pylori* for the

The inhibition mechanism is mainly based on the binding of the substrate and enzyme. The urease has a bi metallic centric structure which binds with range of molecules depending on the kinetics, dynamics, mechanism of action and the possible secondary interactions involved in this phenomenon. The mechanism of inhibition can be categorized on the basis of inhibitor, either as irreversible inhibitors or the reversible inhibitor where as the later one is further classified into two categories based on the binding with enzyme either in a competitive manner or in a non competitive manner. The process involved in case of reversible inhibitors mostly occur through non covalent interactions such as hydrogen bonding, hydrophobic interaction and orientation of inhibitor and enzyme in an organized fashion (Amtul et al., 2002). The irreversible inhibitors interact through its functional groups with amino acids in the active site, irreversibly e.g. the nerve gases and pesticides, which contain an organophosphorus that bind with serine residues in the enzyme acetylcholine esterase. The reversible inhibitors are of two types either as competitive or non-competitive. The competitive inhibitors compete with the substrate molecules for the active site while the inhibitor's action is proportional to its concentration that resembles with the substrate's structure closely. The non-competitive inhibitors are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. The non-competitor inhibitor follows the allosteric sites in the enzyme for the binding (Figure 2.1). The reversible inhibitors can be washed out of the solution of enzyme by dialysis.

Fig. 1. Model representation of the binding of the enzyme active site and inhibitor.

epidemiological studies (Amtul et al., 2002).

**2. Inhibition mechanism** 

wide variety of prokaryotes. This enzyme is having important negative implications in medicine, agriculture and environment. The urease inhibitors can play a vital role to counter effect the negative role of urease in living organisms. Urease inhibitors are effective against several serious infections caused by the secretion of urease by Helicobactor pylori which includes gastric tract syndromes, proteus related species in the urinary tract, struvite urolithiasis mainly in dogs and cats. The urease inhibitors have also received great attention from scientists in the soil sciences whereas the nutrient miss-management is associated to the excessive use of synthetic fertilizer and excess of urea products. Research on urease inhibitions yielded several vital therapeutic drugs (Amtul et al., 2002). Hagar and Magath have reported in 1925 that urease is the primary source for the biochemical formation of stone in urinary tract (Hagar et al., 1925). Urease serves as virulence factor in the pathogens responsible for kidney stones entailed in urolithiasis that contributes toward the acute pyelonephritis with other urinary tract infection which induced arthritis and gastric intestinal infections and ultimately the urease imbalance lead to peptic ulcers (Mobley et al., 1989). Urease controls the nitrogen contents in the physiological systems, while it provides a protection mechanism against predators and phytphathogenic organisms (Amtul et al., 2006; Zonia et al., 1995). The high concentrations of ammonia arising from the biochemical reactions of urease, as well as pH elevation result into important negative implications in medicine and agriculture (Zonia et al., 1995; Collins et al., 1993; Montecucco et al 2001; Zhengping et al). High concentrations of ureases cause significant environmental and economic problems by releasing abnormally large amounts of ammonia into the atmosphere during urea fertilization. It induces plant damage primarily by depriving plants of their essential nutrients and secondarily by ammonia toxicity which result in pH increase of the soil (Bremner et al., 1995). The urease inhibitors have played a vital role in the controlling the Helicobacter pylori which caused an imbalance of ammonia levels in cirrhotic patients (Zullo et al., 1998). The urease based liquid crystal-sensors are also used for the detection of heavy metals; these urease based sensors are useful in monitoring the inhibition of enzymatic activities that has been widely investigated for the detection of heavy metals (HMs) (Verma et al., 2005). In many cases, after inhibition of immobilized enzymes with HMs, chelating agents such as EDTA are usually used to regenerate the active site of the enzyme (Bracka et al., 2000). Urease-based sensors, which are inexpensive and sensitive to HMs, have been widely exploited in the scientific community (Volotovsky et al., 1997). Several analytical methods for monitoring HMs are based on the enzyme-catalyzed hydrolysis of urea into ammonia and carbon dioxide, as the consumption of urease inhibitors is vital for the production of grains over the long-term, meanwhile the coating of urea to reduce ammonia volatilization loss from urea fertilizer is gaining great attention of researchers in order to control the depletion of active ingredients in the soil. The use of fertilizer is very much common in the areas where the active ingredients do not occur in sufficient quantities. The nitrogen losses can be reduced in these situations and the use of urease inhibitor is applied to the fertilizer to maintain the optimum pH of the soil (Schwedt et aj., 1993; Saboury et al., 2010). Recently, the LCs has been doped with functional molecules that have been used to develop novel types of sensors. There are series of compounds with 4-pentyl-biphenyl-4-carboxylic acid (PBA), which contains pH-sensitive functional groups could be used in an LC-based pH sensor to monitor small amounts of H+ released from enzymatic reactions, especially in solutions with a high buffer capacity (Bi et

wide variety of prokaryotes. This enzyme is having important negative implications in medicine, agriculture and environment. The urease inhibitors can play a vital role to counter effect the negative role of urease in living organisms. Urease inhibitors are effective against several serious infections caused by the secretion of urease by Helicobactor pylori which includes gastric tract syndromes, proteus related species in the urinary tract, struvite urolithiasis mainly in dogs and cats. The urease inhibitors have also received great attention from scientists in the soil sciences whereas the nutrient miss-management is associated to the excessive use of synthetic fertilizer and excess of urea products. Research on urease inhibitions yielded several vital therapeutic drugs (Amtul et al., 2002). Hagar and Magath have reported in 1925 that urease is the primary source for the biochemical formation of stone in urinary tract (Hagar et al., 1925). Urease serves as virulence factor in the pathogens responsible for kidney stones entailed in urolithiasis that contributes toward the acute pyelonephritis with other urinary tract infection which induced arthritis and gastric intestinal infections and ultimately the urease imbalance lead to peptic ulcers (Mobley et al., 1989). Urease controls the nitrogen contents in the physiological systems, while it provides a protection mechanism against predators and phytphathogenic organisms (Amtul et al., 2006; Zonia et al., 1995). The high concentrations of ammonia arising from the biochemical reactions of urease, as well as pH elevation result into important negative implications in medicine and agriculture (Zonia et al., 1995; Collins et al., 1993; Montecucco et al 2001; Zhengping et al). High concentrations of ureases cause significant environmental and economic problems by releasing abnormally large amounts of ammonia into the atmosphere during urea fertilization. It induces plant damage primarily by depriving plants of their essential nutrients and secondarily by ammonia toxicity which result in pH increase of the soil (Bremner et al., 1995). The urease inhibitors have played a vital role in the controlling the Helicobacter pylori which caused an imbalance of ammonia levels in cirrhotic patients (Zullo et al., 1998). The urease based liquid crystal-sensors are also used for the detection of heavy metals; these urease based sensors are useful in monitoring the inhibition of enzymatic activities that has been widely investigated for the detection of heavy metals (HMs) (Verma et al., 2005). In many cases, after inhibition of immobilized enzymes with HMs, chelating agents such as EDTA are usually used to regenerate the active site of the enzyme (Bracka et al., 2000). Urease-based sensors, which are inexpensive and sensitive to HMs, have been widely exploited in the scientific community (Volotovsky et al., 1997). Several analytical methods for monitoring HMs are based on the enzyme-catalyzed hydrolysis of urea into ammonia and carbon dioxide, as the consumption of urease inhibitors is vital for the production of grains over the long-term, meanwhile the coating of urea to reduce ammonia volatilization loss from urea fertilizer is gaining great attention of researchers in order to control the depletion of active ingredients in the soil. The use of fertilizer is very much common in the areas where the active ingredients do not occur in sufficient quantities. The nitrogen losses can be reduced in these situations and the use of urease inhibitor is applied to the fertilizer to maintain the optimum pH of the soil (Schwedt et aj., 1993; Saboury et al., 2010). Recently, the LCs has been doped with functional molecules that have been used to develop novel types of sensors. There are series of compounds with 4-pentyl-biphenyl-4-carboxylic acid (PBA), which contains pH-sensitive functional groups could be used in an LC-based pH sensor to monitor small amounts of H+ released from enzymatic reactions, especially in solutions with a high buffer capacity (Bi et

al., 2009). Literature is rich on the role o*f H. pylori w*hich is the main cause for the gastric infections. Urease is a prominent antigen of the *H. Pylori* which has served as a powerful immunogen for this organism. There have been various reports on large number of patients who have shown gastritis significantly elevated response in immunoglobulins G and A along with urease in the blood serum samples with relative comparison of pre-infected levels. Range of enzymatic assays is reported to measure the immune responses and these are used for the diagnostic purposes in monitoring the antibiotic therapy of *H-Pylori* for the epidemiological studies (Amtul et al., 2002).

#### **2. Inhibition mechanism**

The inhibition mechanism is mainly based on the binding of the substrate and enzyme. The urease has a bi metallic centric structure which binds with range of molecules depending on the kinetics, dynamics, mechanism of action and the possible secondary interactions involved in this phenomenon. The mechanism of inhibition can be categorized on the basis of inhibitor, either as irreversible inhibitors or the reversible inhibitor where as the later one is further classified into two categories based on the binding with enzyme either in a competitive manner or in a non competitive manner. The process involved in case of reversible inhibitors mostly occur through non covalent interactions such as hydrogen bonding, hydrophobic interaction and orientation of inhibitor and enzyme in an organized fashion (Amtul et al., 2002). The irreversible inhibitors interact through its functional groups with amino acids in the active site, irreversibly e.g. the nerve gases and pesticides, which contain an organophosphorus that bind with serine residues in the enzyme acetylcholine esterase. The reversible inhibitors are of two types either as competitive or non-competitive. The competitive inhibitors compete with the substrate molecules for the active site while the inhibitor's action is proportional to its concentration that resembles with the substrate's structure closely. The non-competitive inhibitors are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. The non-competitor inhibitor follows the allosteric sites in the enzyme for the binding (Figure 2.1). The reversible inhibitors can be washed out of the solution of enzyme by dialysis.

Fig. 1. Model representation of the binding of the enzyme active site and inhibitor.

Urease Inhibition 307

E.I = Enzyme-Inhibitor Complex

believe that it make a bridge between the two nickle ions. Similarly it is also suggested that the E-I and E-I\* type of interactions occurs, forming an initial monodentate species that bridges the two nickel ions at the active site (Amtul et al., 2002). The non-steady-state studies have also been employed with other competitive inhibitors, the specific examples for the kinetic analysis is the interaction of phenylphosphoro diamidate with urease (*K. aerogenes*) with dissociation constant of 4.7x10 -5 s-1. The proposed studies suggests that the E-I state where an inhibitor bound in unidentate mode to one nickel ion while the EI\* species involves the bridging of the two nickel ion with inhibitor where R is the aromatic ring *via* the formation of tetrahedral intermediate rather that the hypothetical trigonal bipyramidyl intermediate. Here the

The modes of action of enzyme have been investigated on the thiol based compounds. As the presence of an auxochrome sulphar (thio) moiety in the inhibitor help in characterization *via* UV-Visible spectroscopy for a variety of enzymes *e. g* jack bean and *K aerogenes* ureases, these spectroscopic perturbation is consistent with the development of thiolate. It is well documented that the Ni (II) charge-transfer complex is kinetically controlled process. Since a competition of thiol with urea for the active sites of the enzyme in binding the nickel

A B Scheme 2. Structural hypothesis for the competitive interaction of the urease active sites

provide a relative reference to further study the action of inhibitor over the enzyme.

= Stable Enzyme-Inhibitor Complex

Ni O

HN

OH

O Ni

CH3

E.I\*

NH

OH

Ni Ni

HN

HO Ni

E.I\*

hydrolysis occurs with simple in-line displacement (Andrews et al., 1984).

NH2

S

with thiourea (A) and hydroxyurea (B).

Ni Ni

Scheme 1. Bi-dentate ligand complex of substrate and enzyme.

Ni O

H2N

E.I

**2.2 Action of inhibition** 

H2N

CH3

OH

In the competitive inhibition the inhibitor directly binds to the active site of the enzyme *via* a specific host and guest complex, while in the non-competitive mechanism the substrate binds somewhere else and not specifically on the active site in the enzyme this binding of substrate affect the structure of the enzyme and induce inhibition. Furthermore another mode of binding of inhibitor is the un-competitive mechanism, in this mechanism the inhibitor irreversibly binds with the enzyme and result an intermediate complex (E-I\*) between the enzyme and the inhibitor. This portion of enzyme is known as allosteric site (Figure 1). The binding of enzyme with the substrate occurs in an organized fashion and the substrates have specific affinity toward the enzyme. Though the active site is a specific region in the enzyme and this specificity depends on the pro-non covalent interaction available for interaction in the inhibitors. These non covalent interactions between the enzyme and inhibitor are the main feature for the chemoselectivity of the substrate and enzymes during formation of the complex.

These interactions mainly provide a surface to regenerate the enzyme for the reaction cycle as a catalyst. The unique geometrical shapes and topology of the active site is complimentary for the substrate molecule, which fit like lock and key arrangement. The enzymes specifically react with only few compounds of the similar structural features, these structural feature in the inhibitor is known as pharmacohores, while the potency of the inhibitor is managed by appending various functional groups to the main skeleton of the inhibitor by making target oriented derivative containing these features and consequently resulted in the controlled target oriented synthesis of the potent and fascinating inhibitors. As there are so many theories which explain the mechanistic perspective of the enzymatic action, only the 'lock and key' and the 'induced fit' theories are well accepted (Donald et al., 2005). The mechanistic studies of the biological systems undergoes *via* a complex phenomenon, a large number of literature reports are available involving the structurebased design and testing of novel pharmacophores model for the recognition of urease inhibitors. The intimated details of the molecular geometry of urease enzyme as well as its mechanism of urea hydrolysis have been confirmed with XRD-analysis.

#### **2.1 Kinetics of enzyme activity**

The rate of reaction provide a clue about the affinity of the inhibitor towards the enzyme, while at the same time it also effect the potency of the inhibitor. The kinetic effect of rebeprazole has been investigated on the urease inhibition, it is reported that rebeprazole act as an irreversible noncompetitive inhibitor. Mainly the associated inhibitory potency of rabeprazole is dependent on the pH of reaction mixture and *Ki* value which varies at the progressive inactivation of urease by rabeprazole, initially it proceeds according to pseudofirst-order kinetics with respect to the remaining enzymatic activity at pH 7.0 and 37 °C, with a second-order rate constant of 0.0017 M-1 s-1. This inhibitor competes with substrate to bind to the enzyme and form as an E-I (enzyme and inhibitor) complex, that slowly transformed to an E-I\* complex which is stable (Scheme 1) (Park et al., 1996).

The use of non-steady-state kinetic measurements is important to properly analyze the non competitive inhibition (Mobley et al., 1989; Rosenstein et al, 1984). A number of mechanistic studies have been carried out on various inhibitors. It is reported that hydroxamic acid which suggests that a bidentate complex form with one of the nickel ions, quite few scientists

In the competitive inhibition the inhibitor directly binds to the active site of the enzyme *via* a specific host and guest complex, while in the non-competitive mechanism the substrate binds somewhere else and not specifically on the active site in the enzyme this binding of substrate affect the structure of the enzyme and induce inhibition. Furthermore another mode of binding of inhibitor is the un-competitive mechanism, in this mechanism the inhibitor irreversibly binds with the enzyme and result an intermediate complex (E-I\*) between the enzyme and the inhibitor. This portion of enzyme is known as allosteric site (Figure 1). The binding of enzyme with the substrate occurs in an organized fashion and the substrates have specific affinity toward the enzyme. Though the active site is a specific region in the enzyme and this specificity depends on the pro-non covalent interaction available for interaction in the inhibitors. These non covalent interactions between the enzyme and inhibitor are the main feature for the chemoselectivity of the substrate and

These interactions mainly provide a surface to regenerate the enzyme for the reaction cycle as a catalyst. The unique geometrical shapes and topology of the active site is complimentary for the substrate molecule, which fit like lock and key arrangement. The enzymes specifically react with only few compounds of the similar structural features, these structural feature in the inhibitor is known as pharmacohores, while the potency of the inhibitor is managed by appending various functional groups to the main skeleton of the inhibitor by making target oriented derivative containing these features and consequently resulted in the controlled target oriented synthesis of the potent and fascinating inhibitors. As there are so many theories which explain the mechanistic perspective of the enzymatic action, only the 'lock and key' and the 'induced fit' theories are well accepted (Donald et al., 2005). The mechanistic studies of the biological systems undergoes *via* a complex phenomenon, a large number of literature reports are available involving the structurebased design and testing of novel pharmacophores model for the recognition of urease inhibitors. The intimated details of the molecular geometry of urease enzyme as well as its

The rate of reaction provide a clue about the affinity of the inhibitor towards the enzyme, while at the same time it also effect the potency of the inhibitor. The kinetic effect of rebeprazole has been investigated on the urease inhibition, it is reported that rebeprazole act as an irreversible noncompetitive inhibitor. Mainly the associated inhibitory potency of rabeprazole is dependent on the pH of reaction mixture and *Ki* value which varies at the progressive inactivation of urease by rabeprazole, initially it proceeds according to pseudofirst-order kinetics with respect to the remaining enzymatic activity at pH 7.0 and 37 °C, with a second-order rate constant of 0.0017 M-1 s-1. This inhibitor competes with substrate to bind to the enzyme and form as an E-I (enzyme and inhibitor) complex, that slowly

The use of non-steady-state kinetic measurements is important to properly analyze the non competitive inhibition (Mobley et al., 1989; Rosenstein et al, 1984). A number of mechanistic studies have been carried out on various inhibitors. It is reported that hydroxamic acid which suggests that a bidentate complex form with one of the nickel ions, quite few scientists

mechanism of urea hydrolysis have been confirmed with XRD-analysis.

transformed to an E-I\* complex which is stable (Scheme 1) (Park et al., 1996).

enzymes during formation of the complex.

**2.1 Kinetics of enzyme activity** 

Scheme 1. Bi-dentate ligand complex of substrate and enzyme.

believe that it make a bridge between the two nickle ions. Similarly it is also suggested that the E-I and E-I\* type of interactions occurs, forming an initial monodentate species that bridges the two nickel ions at the active site (Amtul et al., 2002). The non-steady-state studies have also been employed with other competitive inhibitors, the specific examples for the kinetic analysis is the interaction of phenylphosphoro diamidate with urease (*K. aerogenes*) with dissociation constant of 4.7x10 -5 s-1. The proposed studies suggests that the E-I state where an inhibitor bound in unidentate mode to one nickel ion while the EI\* species involves the bridging of the two nickel ion with inhibitor where R is the aromatic ring *via* the formation of tetrahedral intermediate rather that the hypothetical trigonal bipyramidyl intermediate. Here the hydrolysis occurs with simple in-line displacement (Andrews et al., 1984).

#### **2.2 Action of inhibition**

The modes of action of enzyme have been investigated on the thiol based compounds. As the presence of an auxochrome sulphar (thio) moiety in the inhibitor help in characterization *via* UV-Visible spectroscopy for a variety of enzymes *e. g* jack bean and *K aerogenes* ureases, these spectroscopic perturbation is consistent with the development of thiolate. It is well documented that the Ni (II) charge-transfer complex is kinetically controlled process. Since a competition of thiol with urea for the active sites of the enzyme in binding the nickel provide a relative reference to further study the action of inhibitor over the enzyme.

Scheme 2. Structural hypothesis for the competitive interaction of the urease active sites with thiourea (A) and hydroxyurea (B).

Urease Inhibition 309

benzoquinone etc. The inhibitors have broad applications in combination with urea fertilizers that has been proposed for soil urease activity control (Bundy et al, 1973; Amtul et al., 2002). There is an example of kinetic studies on quinone-induced inhibition of urease has also been investigated, one among those is polyhalogenated benzoand naphthoquinones, in which the quinones were found to be non-competitive inhibitors of jack bean and bacterial ureases of different strengths (Zaborska et al., 2002; Pearson et al., 1997). Several other potent inhibitors are being used as first line of treatment for infections causes by urease-producing bacteria which are available in current market. The most effective inhibitors with safe great potency profile for considerable control of urease-related ailments are still needed. The available data provide a basis for urease inhibition properties of thiol-compounds, different cysteine derivatives (CysDs) with cysteine-like scaffold arylidene barbiturates have been studied as urease inhibitors such as *N*-Substituted Hydroxyureas (Amtul et al., 2002; Amtul et al., 2004; Todd et al., 1989). Acetohydroxamates are the bacterial urease inhibitor and used as therapeutic potential in hyperammonaemic states (Carlini et al., 2002). The urease inhibitors have also been employed for the coating of urea in the agricultural soils to reduce the ammonia mitigating. The nitrogen loss depends on the rate of biodegradation and constant volatilization in agricultural soils of urea fertilizer, such coating can improve the bioavailability of nitrogen and consequently increase dry matter yield and nitrogen uptake. These increases of urea contents in soil result from delayed urea hydrolysis by urease inhibitors and coating materials. The value of inhibitors in nitrogen mitigating is depending on the rate of biodegradation and persistence in soils (Amtul et al., 2007). The urease inhibitors have been investigated for the treatment of bacterial infections and also for the excessive urea break down in soil, therefore studies on the potent and specific inhibitors have been an active area of research. Taking in view the great potential of urease in medicine and soil sciences, several classes of urease inhibitor have been discovered in the recent years (Ara et al., 2007; Mobley et al., 1989; Mulvaney et al, 1981; Rosenstein et al., 1984). The urease inhibitors can also be classified into two categories either as metal complexes or organic compounds. The organic compounds are further classified into the three main classes such as hydroxamic acid analogs, phosphoramide compounds and thiourea derivatives. Hydroxamic acid (Scheme 3) analogs are the most commonly known urease inhibitors and first member of its series was hydroxamic acid which was discovered in 1962. The acetohydraxamic acid with other numerous derivatives have been synthesized and studied against ureases of plants and bacterial origin (Mobley et al., 1989). The other members of the series include *n*-aliphatic hydroxamic acid, ortho or para substituted benzohydroxamic acid, *trans*-cinnamoyl

hydroxamic acid with other functional groups and substituents.

Scheme 3. General structure of hydroxamic acid.

R N

O

H

Phosphoramide compounds are another class of potent inhibitors of the urease enzyme, this class of inhibitors comprise of a large number of simple and complex compounds. Mainly

OH

The mechanism of action related to hydrolysis of urease to ammonia and carbamate has been investigated in depth, the later compound spontaneously decomposes to the carbamic acid, accompanying the deprotonation generating equilibrium as a result of increase in pH. The research on urease has contributed extensively towards development of medicinal organic chemistry (Summer et al., 1926) and crystallization (Amtul et al., 2002). The urea binds into the pocked adjacent to Ni near Wat-502. The H2-H4 mobile flap may open to allow access into the active site of enzyme, once the active functional group binds to Ni-1 *via* coordination. The urea is then placed in a manner that allows the coordination of its oxygen with the N epsilon of His 219, by forming hydrogen bond. The urea also accepts a hydrogen bond from the interaction of its amide nitrogen with side chain of His 320. These non covalent interactions are oriented to urea in an organized fashion so that the carbonyl group of the urea undergoes polarization and attacked by hydroxide (Wat-502), that coordinates other metal atom of Ni-2 and forms tetrahedral intermediate. As one proton is transferred from His-320 to the nitrogen (leaving group), meanwhile the intermediate collapses into ammonia and carbamate. The carbamate undergoes hydrolysis to form carbamic acid and second molecule of ammonia (Amtul et al., 2002). The suboptimal interactions provide a significant source of substrate binding energy; this energy is commonly found in the enzymes with mobile active site loops which undergo induced fit. The side chain of His 320 in urease is in close proximity to the side chain of Asp 221 and Arg 336, whereas the geometry is not optimal for the hydrogen bonding between the residues. His 320 also interacts with Wat-170 *via* hydrogen bonding. The Wat-170 also forms hydrogen bond with two main chain carbonyls. Wat-170 does not have the ability to donate three hydrogen bonds, so one of the H-bond at any given time is unable to form, making the interactions of water suboptimal. There are also suboptimal interactions between the nickel metallo center and solvent. The active site water molecules (Wat-500, Wat, 501, Wat-502) have inter-oxygen distance that is too short to allow for simultaneous occupancy. Since the occupancy of the active site is too small to accommodate all three water molecules, there appears a competition for occupancy at the three positions. All these interactions must contribute to the flexibility of the mobile flap by the destabilizing the closed positions. These interactions are the main driving force for the catalytic mechanism of the urease in the various biological processes. The most of the biochemical reactions occurs by decreasing the free energy difference between the unbound enzyme with substrate and enzyme-substrate complex which accomplished in two ways with enzyme binding transition state which is highly favorable or the initial state which has higher free energy, possibly the addition of suboptimal interactions. Such interaction makes possible transition state which usually is not favorable, therefore most of the urease mechanisms involve a number of suboptimal interactions to derive the hydrolysis of urea in the systems (Mobley et al., 1989; Mulvaney et al., 1981).

#### **3. Potent inhibitors**

A vast range of naturally isolated and synthetic urease inhibitors has been reported in the literature though pharmacological importance and the developed resistance in the bacterial infections have attracted the attention of the scientific community to explore new versatile urease inhibitors.

Urease inhibitors are classified broadly into two major classes as metal-organic and organic compounds, the later examples are such as acetohydroxamic acid, humic acid and 1,4-

The mechanism of action related to hydrolysis of urease to ammonia and carbamate has been investigated in depth, the later compound spontaneously decomposes to the carbamic acid, accompanying the deprotonation generating equilibrium as a result of increase in pH. The research on urease has contributed extensively towards development of medicinal organic chemistry (Summer et al., 1926) and crystallization (Amtul et al., 2002). The urea binds into the pocked adjacent to Ni near Wat-502. The H2-H4 mobile flap may open to allow access into the active site of enzyme, once the active functional group binds to Ni-1 *via* coordination. The urea is then placed in a manner that allows the coordination of its oxygen with the N epsilon of His 219, by forming hydrogen bond. The urea also accepts a hydrogen bond from the interaction of its amide nitrogen with side chain of His 320. These non covalent interactions are oriented to urea in an organized fashion so that the carbonyl group of the urea undergoes polarization and attacked by hydroxide (Wat-502), that coordinates other metal atom of Ni-2 and forms tetrahedral intermediate. As one proton is transferred from His-320 to the nitrogen (leaving group), meanwhile the intermediate collapses into ammonia and carbamate. The carbamate undergoes hydrolysis to form carbamic acid and second molecule of ammonia (Amtul et al., 2002). The suboptimal interactions provide a significant source of substrate binding energy; this energy is commonly found in the enzymes with mobile active site loops which undergo induced fit. The side chain of His 320 in urease is in close proximity to the side chain of Asp 221 and Arg 336, whereas the geometry is not optimal for the hydrogen bonding between the residues. His 320 also interacts with Wat-170 *via* hydrogen bonding. The Wat-170 also forms hydrogen bond with two main chain carbonyls. Wat-170 does not have the ability to donate three hydrogen bonds, so one of the H-bond at any given time is unable to form, making the interactions of water suboptimal. There are also suboptimal interactions between the nickel metallo center and solvent. The active site water molecules (Wat-500, Wat, 501, Wat-502) have inter-oxygen distance that is too short to allow for simultaneous occupancy. Since the occupancy of the active site is too small to accommodate all three water molecules, there appears a competition for occupancy at the three positions. All these interactions must contribute to the flexibility of the mobile flap by the destabilizing the closed positions. These interactions are the main driving force for the catalytic mechanism of the urease in the various biological processes. The most of the biochemical reactions occurs by decreasing the free energy difference between the unbound enzyme with substrate and enzyme-substrate complex which accomplished in two ways with enzyme binding transition state which is highly favorable or the initial state which has higher free energy, possibly the addition of suboptimal interactions. Such interaction makes possible transition state which usually is not favorable, therefore most of the urease mechanisms involve a number of suboptimal interactions to derive the hydrolysis of urea in

the systems (Mobley et al., 1989; Mulvaney et al., 1981).

A vast range of naturally isolated and synthetic urease inhibitors has been reported in the literature though pharmacological importance and the developed resistance in the bacterial infections have attracted the attention of the scientific community to explore new versatile

Urease inhibitors are classified broadly into two major classes as metal-organic and organic compounds, the later examples are such as acetohydroxamic acid, humic acid and 1,4-

**3. Potent inhibitors** 

urease inhibitors.

benzoquinone etc. The inhibitors have broad applications in combination with urea fertilizers that has been proposed for soil urease activity control (Bundy et al, 1973; Amtul et al., 2002). There is an example of kinetic studies on quinone-induced inhibition of urease has also been investigated, one among those is polyhalogenated benzoand naphthoquinones, in which the quinones were found to be non-competitive inhibitors of jack bean and bacterial ureases of different strengths (Zaborska et al., 2002; Pearson et al., 1997). Several other potent inhibitors are being used as first line of treatment for infections causes by urease-producing bacteria which are available in current market. The most effective inhibitors with safe great potency profile for considerable control of urease-related ailments are still needed. The available data provide a basis for urease inhibition properties of thiol-compounds, different cysteine derivatives (CysDs) with cysteine-like scaffold arylidene barbiturates have been studied as urease inhibitors such as *N*-Substituted Hydroxyureas (Amtul et al., 2002; Amtul et al., 2004; Todd et al., 1989). Acetohydroxamates are the bacterial urease inhibitor and used as therapeutic potential in hyperammonaemic states (Carlini et al., 2002). The urease inhibitors have also been employed for the coating of urea in the agricultural soils to reduce the ammonia mitigating. The nitrogen loss depends on the rate of biodegradation and constant volatilization in agricultural soils of urea fertilizer, such coating can improve the bioavailability of nitrogen and consequently increase dry matter yield and nitrogen uptake. These increases of urea contents in soil result from delayed urea hydrolysis by urease inhibitors and coating materials. The value of inhibitors in nitrogen mitigating is depending on the rate of biodegradation and persistence in soils (Amtul et al., 2007). The urease inhibitors have been investigated for the treatment of bacterial infections and also for the excessive urea break down in soil, therefore studies on the potent and specific inhibitors have been an active area of research. Taking in view the great potential of urease in medicine and soil sciences, several classes of urease inhibitor have been discovered in the recent years (Ara et al., 2007; Mobley et al., 1989; Mulvaney et al, 1981; Rosenstein et al., 1984). The urease inhibitors can also be classified into two categories either as metal complexes or organic compounds. The organic compounds are further classified into the three main classes such as hydroxamic acid analogs, phosphoramide compounds and thiourea derivatives. Hydroxamic acid (Scheme 3) analogs are the most commonly known urease inhibitors and first member of its series was hydroxamic acid which was discovered in 1962. The acetohydraxamic acid with other numerous derivatives have been synthesized and studied against ureases of plants and bacterial origin (Mobley et al., 1989). The other members of the series include *n*-aliphatic hydroxamic acid, ortho or para substituted benzohydroxamic acid, *trans*-cinnamoyl hydroxamic acid with other functional groups and substituents.

Scheme 3. General structure of hydroxamic acid.

Phosphoramide compounds are another class of potent inhibitors of the urease enzyme, this class of inhibitors comprise of a large number of simple and complex compounds. Mainly

Urease Inhibition 311

There is vast reports on the structure of urease, the *Klebsiella areogenes,* the urease structure has a trimer of alpha, beta and gamma units in a triangular arrangement with three active sites per enzyme. These subunits include a 60.3 KD apha, a 11.7 KD beta and a 11.1 KD gamma subunit (Jabr et al., 1992). The interactions between the individual subunits of all three α subunits make extensive contacts with each other to build a trimer. Each alpha subunit packs between two beta and two gamma subunits to form the side of the triangle arrangement (Jabri et al., 1995). Each beta subunit packs between the two of the adjacent alpha subunits at each corners of the triangle. The remaining gamma subunit interacts with two gamma subunits at the crystallographic three fold axis. Because of the high degree of interaction, it is not obvious that which alpha, beta and gamma chains make up the primary units. High conservation of sequences and the extensive interactions of the trimer suggest a similar trimer structure in all the urease which is further subdivided into sub-units. The gamma subunit consists of an alpha-beta domain with four helices and two anti parallel stands. Two helices (b and c) and the two strands pack together with right-handed up-down-up-down topological order. One of the remaining helices (a) is lined up peripherally and interacts at the three fold axis with the other two gamma subunits, tightly packing against itself and to other (a) helices in an orthogonal manner. The last helix (d) experience single turn. The first fifteen residues of the beta subunits form two antiparallels beta sheets with the amino-terminal residues of the alpha subunits. The core of the beta subunit forms an imperfect six-stranded antiparallel beta sheet (Mobley et al., 1989; Hausinger et al., 1993). This subunit stabilizes the trimer by interacting with domain two of its own alpha subunits and domain one of a symmetry related alpha subunit. The alpha subunit consists of two domains, an (alpha, beta) 8 barrel domain and a primarily beta domain. The (alpha, beta) 8 barrel is the only domain that contributes to the active site. Its barrel is rather elliptical with the long axis connecting strands 1 and 7. Strands 9 and 10 extend the lower portion of strands 1 and 3. The active site is located at the carboxyl terminal of the strands. A flap is formed that covers the active site by a helical excursion between strand 7 and helix 7. Mixed four-strands and eight-stranded beta sheets form the wall of U-Shaped canyon which makes up the second beta domain. The walls are connected by the long strands 5 and 6 together with strands 10 and 11 which go down on one side, and up on the other side. The structure activity relationship of the various urease inhibitors are reported in literature, the studies on *in-vitro* with *in-vivo* efficacy of the effect benzimidazoles suggested that the orientation and position of the pyridyl moiety larger and more lipophillic chromophore of the

Ni

H2O O

NHis

O

Asp

NHis

Ni

H2O

NHis

NHis

O H

O O

NLys

Scheme 4. Active center of urease.

the less complex examples includes of phosphoramidates and diamidophos phate with substituted phenylphosphoradiamidates, while a range of N-acyl phosphoric triamidates (Humayun et al., 2010).

Thio substituted compounds are also strong inhibitors of the urease enzyme. This includes cysteamine containing a cationic -amino cysteamine, which exhibit highest affinity for the urease, on the other hand the thiolates containing anionic carboxyl groups are uniformly poor inhibitors. The pH dependence studies demonstrate that the actual inhibitor is the thiolate anion (Ansari et al., 2005). A series of bezothiazepines have also been investigated bearing significant urease inhibitory activities (Kühler et al., 1998). The studies on structure activity relationship of the thio substituted benzimidazoles with *in-vitro* and *in-vivo* efficacy model, where the potency of the substrate is depend on the substituents positions in the pharmacophore (Creason et al., 1990). The inhibitory effects of *N-(n*-butyl) thiophosphoratriamides (NBPT) and its oxon analogs in the soil are also been reported (Zaborska et al., 2004). There are several other potent inhibitors readily available in the market the most common is NBPT (N-[n-butyl] thiophosphoratriamide) which is sold worldwide under the trade name of Agrotain. This fertilizer has amended the coats of urea preventing the urease enzyme from breaking down the urea for up to fourteen days of life. As this increases the probability that urea will be absorbed into the soil after a rain rather than volatilized into the atmosphere which reduce the nitrogen content in the soil. The rich content of urea in soils causes hydroxylation under the surface of soil and decreases atmospheric losses of the major ingredients of fertilizers. The use of potent inhibitors also decreases the localized zones of high pH which is common in untreated urea agricultural fields. These remedies are commonly employed to maintain the pH of the soil and controlled ammonia volatilization. The use of inhibitors increases the efficiency of urea in delivering nitrogen to soil.

A wide range of metal complexes with meager to good urease inhibitory activities are reported which includes heavy metal ions, such as Cu2+, Zn2+, Pd2+ and Cd2+ (Zaborska et al., 2001; Asato et al., 1997). There are reports on the urease inhibitory activities of organotin(IV), vanadium(IV), bismuth(III), copper(II), and cadmium(II) complexes (Khan et al., 2007; Ara et al., 2007; Hou et al., 2008; You et al., 2008; Tanaka et al., 2003; Cheng et al., 2007; You et al., 2010). A series of metal complexes of Schiff bases such as thiocyanato-coordinated manganese(III) complexes based on N-ethylethane-1,2-diamine and N-(2-hydroxyethyl)ethane-1,2-diamine have shown significant urease (Huang et al., 2011). There is library of compounds are available in various databases and the recent developments is continuously adding numerous class of of hetero-dinuclear CuII–ZnII complexes (You et al., 2011).

#### **4. Structure Activity Relations (SAR)**

The hydroxamic is the most commonly used urease inhibitor coordinating to nickel of urease through carboxylic acid and hydroxyl amine (Scheme 3). The structural studies on the urease from *Klebsiella aerogenes*, *Bacillus pasteurii,* and *Helicobacter pylori* have revealed that it contains a dinuclear Ni active site with a modified amino acid side chain-containing a carbamylated lysine residue that bridges the deeply buried metal atoms as shown in Scheme 3 (Benini et al., 1999; Jabri et al., 1995; Ha et al., 2001). The crystal structure has been resolved only for bacterial ureases; it has an active centre which contains two simple coordinated water molecules and a bridging OH group. The specificity of the enzyme is closely related to the shape of its active centre (Mobley et al., 1995).

the less complex examples includes of phosphoramidates and diamidophos phate with substituted phenylphosphoradiamidates, while a range of N-acyl phosphoric triamidates

Thio substituted compounds are also strong inhibitors of the urease enzyme. This includes cysteamine containing a cationic -amino cysteamine, which exhibit highest affinity for the urease, on the other hand the thiolates containing anionic carboxyl groups are uniformly poor inhibitors. The pH dependence studies demonstrate that the actual inhibitor is the thiolate anion (Ansari et al., 2005). A series of bezothiazepines have also been investigated bearing significant urease inhibitory activities (Kühler et al., 1998). The studies on structure activity relationship of the thio substituted benzimidazoles with *in-vitro* and *in-vivo* efficacy model, where the potency of the substrate is depend on the substituents positions in the pharmacophore (Creason et al., 1990). The inhibitory effects of *N-(n*-butyl) thiophosphoratriamides (NBPT) and its oxon analogs in the soil are also been reported (Zaborska et al., 2004). There are several other potent inhibitors readily available in the market the most common is NBPT (N-[n-butyl] thiophosphoratriamide) which is sold worldwide under the trade name of Agrotain. This fertilizer has amended the coats of urea preventing the urease enzyme from breaking down the urea for up to fourteen days of life. As this increases the probability that urea will be absorbed into the soil after a rain rather than volatilized into the atmosphere which reduce the nitrogen content in the soil. The rich content of urea in soils causes hydroxylation under the surface of soil and decreases atmospheric losses of the major ingredients of fertilizers. The use of potent inhibitors also decreases the localized zones of high pH which is common in untreated urea agricultural fields. These remedies are commonly employed to maintain the pH of the soil and controlled ammonia volatilization.

The use of inhibitors increases the efficiency of urea in delivering nitrogen to soil.

hetero-dinuclear CuII–ZnII complexes (You et al., 2011).

**4. Structure Activity Relations (SAR)** 

the shape of its active centre (Mobley et al., 1995).

A wide range of metal complexes with meager to good urease inhibitory activities are reported which includes heavy metal ions, such as Cu2+, Zn2+, Pd2+ and Cd2+ (Zaborska et al., 2001; Asato et al., 1997). There are reports on the urease inhibitory activities of organotin(IV), vanadium(IV), bismuth(III), copper(II), and cadmium(II) complexes (Khan et al., 2007; Ara et al., 2007; Hou et al., 2008; You et al., 2008; Tanaka et al., 2003; Cheng et al., 2007; You et al., 2010). A series of metal complexes of Schiff bases such as thiocyanato-coordinated manganese(III) complexes based on N-ethylethane-1,2-diamine and N-(2-hydroxyethyl)ethane-1,2-diamine have shown significant urease (Huang et al., 2011). There is library of compounds are available in various databases and the recent developments is continuously adding numerous class of of

The hydroxamic is the most commonly used urease inhibitor coordinating to nickel of urease through carboxylic acid and hydroxyl amine (Scheme 3). The structural studies on the urease from *Klebsiella aerogenes*, *Bacillus pasteurii,* and *Helicobacter pylori* have revealed that it contains a dinuclear Ni active site with a modified amino acid side chain-containing a carbamylated lysine residue that bridges the deeply buried metal atoms as shown in Scheme 3 (Benini et al., 1999; Jabri et al., 1995; Ha et al., 2001). The crystal structure has been resolved only for bacterial ureases; it has an active centre which contains two simple coordinated water molecules and a bridging OH group. The specificity of the enzyme is closely related to

(Humayun et al., 2010).

Scheme 4. Active center of urease.

There is vast reports on the structure of urease, the *Klebsiella areogenes,* the urease structure has a trimer of alpha, beta and gamma units in a triangular arrangement with three active sites per enzyme. These subunits include a 60.3 KD apha, a 11.7 KD beta and a 11.1 KD gamma subunit (Jabr et al., 1992). The interactions between the individual subunits of all three α subunits make extensive contacts with each other to build a trimer. Each alpha subunit packs between two beta and two gamma subunits to form the side of the triangle arrangement (Jabri et al., 1995). Each beta subunit packs between the two of the adjacent alpha subunits at each corners of the triangle. The remaining gamma subunit interacts with two gamma subunits at the crystallographic three fold axis. Because of the high degree of interaction, it is not obvious that which alpha, beta and gamma chains make up the primary units. High conservation of sequences and the extensive interactions of the trimer suggest a similar trimer structure in all the urease which is further subdivided into sub-units. The gamma subunit consists of an alpha-beta domain with four helices and two anti parallel stands. Two helices (b and c) and the two strands pack together with right-handed up-down-up-down topological order. One of the remaining helices (a) is lined up peripherally and interacts at the three fold axis with the other two gamma subunits, tightly packing against itself and to other (a) helices in an orthogonal manner. The last helix (d) experience single turn. The first fifteen residues of the beta subunits form two antiparallels beta sheets with the amino-terminal residues of the alpha subunits. The core of the beta subunit forms an imperfect six-stranded antiparallel beta sheet (Mobley et al., 1989; Hausinger et al., 1993). This subunit stabilizes the trimer by interacting with domain two of its own alpha subunits and domain one of a symmetry related alpha subunit. The alpha subunit consists of two domains, an (alpha, beta) 8 barrel domain and a primarily beta domain. The (alpha, beta) 8 barrel is the only domain that contributes to the active site. Its barrel is rather elliptical with the long axis connecting strands 1 and 7. Strands 9 and 10 extend the lower portion of strands 1 and 3. The active site is located at the carboxyl terminal of the strands. A flap is formed that covers the active site by a helical excursion between strand 7 and helix 7. Mixed four-strands and eight-stranded beta sheets form the wall of U-Shaped canyon which makes up the second beta domain. The walls are connected by the long strands 5 and 6 together with strands 10 and 11 which go down on one side, and up on the other side. The structure activity relationship of the various urease inhibitors are reported in literature, the studies on *in-vitro* with *in-vivo* efficacy of the effect benzimidazoles suggested that the orientation and position of the pyridyl moiety larger and more lipophillic chromophore of the

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#### **5. Conclusion**

The chapter is focused on the importance and future implications of the urease. The emphases have been laid on urease keeping in mind its role as virulence factor for the urinary tract infections and gastrointestinal infections while the inhibition of urease is also effective in the agricultural sector. Urease enzyme inhibition has become a growing area of research at the interface of the biomedical sciences. The broad range of potent inhibitors has been reported in literature from simple organic molecules to complex molecular building block with metal complexes. These molecular building blocks are expected to bring revolution in pharmaceutical, agricultural and environmental fields. Urease based sensors, adsorbent and other devices have also been discussed that are commercially used.

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## *Edited by Rakesh R. Sharma*

Enzyme Inhibition and Bioapplications is a concise book on applied methods of enzymes used in drug testing. The present volume will serve the purpose of applied drug evaluation methods in research projects, as well as relatively experienced enzyme scientists who might wish to develop their experiments further. Chapters are arranged in the order of basic concepts of enzyme inhibition and physiological basis of cytochromes followed by new concepts of applied drug therapy; reliability analysis; and new enzyme applications from mechanistic point of view.

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Enzyme Inhibition and Bioapplications

Enzyme Inhibition and

Bioapplications

*Edited by Rakesh R. Sharma*