**8. Measurement of O2 consumption in murine tissues**

A novel *in vitro* system is developed to measure O2 consumption by various murine tissues over several hours (Al-Salam et al., 2011; Al Samri et al., 2011; Al Shamsi et al., 2010). Small tissue specimens excised from male Balb/c mice were immediately immersed in ice-cold Krebs-Henseleit buffer (115 mM NaCL, 25 mM NaHCO3, 1.23 mM NaH2PO4, 1.2 mM Na2SO4, 5.9 mM KCL, 1.25 mM CaCl2, 1.18 mM MgCl2 and 6 mM glucose, pH ~7.4), saturated with 95% O2:5% CO2. The samples were incubated at 37oC in the same buffer and continuously gassed with O2:CO2 (95:5). Normal tissue histology at hr 5 was confirmed by light and electron microscopy. NaCN inhibited O2 consumption, confirming the oxidation occurred in the mitochondrial respiratory chain. A representative experiment of pneumatocyte respiration is shown in Fig. 8. The rate of lung tissue respiration incubated *in vitro* for 3.9 < *t* <12.4 hr was 0.24 ± 0.03 M O2 min-1 mg-1 (mean ± SD, n = 28). The corresponding rate for the liver was 0.27 ± 0.13 (n = 11, *t* <4.7 hr), spleen 0.28 ± 0.07 (*t* <5 hr, n = 10), kidney 0.34 ± 0.12 (*t* <5 hr, n = 7) and pancreas 0.35 ± 0.09 (*t* <4 hr, n = 10), Table 2. This approach provided accurate assessment of tissue bioenergetics *in vitro* over several hours.

Phosphorescence Oxygen Analyzer as a Measuring Tool for Cellular Bioenergetics 247

**Tissues Respiration** 

Lung 0.24 ± 0.03 Liver 0.27 ± 0.13 Spleen 0.28 ± 0.07 Kidney 0.34 ± 0.12 Pancreas 0.35 ± 0.09

Values are mean ± SD. For unit conversion, 1.0 mL O2 = 1.4276 mg or 0.0446125 mmol.

**9. Biocompatibility of calcined mesoporous silica particles with murine** 

The rate of respiration (k) was set as negative of the slope of [O2] vs. time; the values of k

Fig. 9. Pneumatocyte respiration with and without 200 g/mL MCM41-cal.

(in M O2 min-1 mg-1) are shown at the bottom of the runs. Zero minute corresponds to the addition of

Normal tissue architecture and histology were confirmed by light microscopy. Intracellular accumulation of the particles in the studied tissues was evident by electron microscopy. The results show reasonable *in vitro* biocompatibility of the mesoporous silicas with murine

The *in vitro* system discussed in Section 8 is used to investigate the effects of two forms of calcined mesoporous silica particles (MCM41-cal and SBA15-cal) on cellular respiration of mouse tissues (Al Shamsi et al., 2010; Al-Salam et al., 2011; Tao et al., 2008c). O2 consumption by lung, liver, kidney, spleen and pancreatic tissues was unaffected by exposure to 200 g/mL MCM41-cal or SBA15-cal for several hours. A representative

Table 2. O2 consumption by murine tissues.

the particles. U, untreated; and T, treated.

experiment of pneumatocyte respiration is shown in Fig. 9.

**tissue bioenergetics** 

**(M O2 min-1 mg-1)** 

The reactions contained 1.5 x 106 cells in PBS plus 10 mM glucose and 68 M Ac-DEVD-AMC with and without 20 μM zVAD-fmk. The suspensions were incubated at 37oC for 2 hr without other additions (left panel) or with the addition of ~100 M aflatoxin (right panel). At the end of the incubation period, the cells were disrupted and their supernatants were separated on HPLC and monitored by fluorescence. The retention time for Ac-DEVD-AMC was ~2.4 min and for the released AMC ~8.7 min.

Fig. 7. Caspase activation by aflatoxin B1 in human peripheral blood mononuclear cells parenthesis for PBMC:PBMC

Specimens were excised from the lung of an anesthetized mouse and immediately immersed in ice-cold Krebs-Henseleit buffer saturated with 95% O2:5% CO2. The samples were incubated at 37oC in the same buffer with continuous gassing with O2:5%CO2. At indicated time periods, specimens were removed from the incubation mixture, weighed and placed in Krebs-Henseleit buffer containing 0.5% albumin and 3 μM Pd phosphor for O2 measurement. The rate of respiration was set as the negative of the slope of [O2] vs. time. The weight is shown at the top and the respiration rate (in μM O2 min-1 mg-1) at the bottom of each run (Al Samri et al., 2011).

Fig. 8. Representative experiment of O2 consumption by lung tissue.


Values are mean ± SD. For unit conversion, 1.0 mL O2 = 1.4276 mg or 0.0446125 mmol.

Table 2. O2 consumption by murine tissues.

246 Bioenergetics

The reactions contained 1.5 x 106 cells in PBS plus 10 mM glucose and 68 M Ac-DEVD-AMC with and without 20 μM zVAD-fmk. The suspensions were incubated at 37oC for 2 hr without other additions (left panel) or with the addition of ~100 M aflatoxin (right panel). At the end of the incubation period,

fluorescence. The retention time for Ac-DEVD-AMC was ~2.4 min and for the released AMC ~8.7 min. Fig. 7. Caspase activation by aflatoxin B1 in human peripheral blood mononuclear cells

Specimens were excised from the lung of an anesthetized mouse and immediately immersed in ice-cold Krebs-Henseleit buffer saturated with 95% O2:5% CO2. The samples were incubated at 37oC in the same buffer with continuous gassing with O2:5%CO2. At indicated time periods, specimens were removed from the incubation mixture, weighed and placed in Krebs-Henseleit buffer containing 0.5% albumin and 3 μM Pd phosphor for O2 measurement. The rate of respiration was set as the negative of the slope of [O2] vs. time. The weight is shown at the top and the respiration rate (in μM O2 min-1 mg-1) at the

the cells were disrupted and their supernatants were separated on HPLC and monitored by

parenthesis for PBMC:PBMC

bottom of each run (Al Samri et al., 2011).

Fig. 8. Representative experiment of O2 consumption by lung tissue.

## **9. Biocompatibility of calcined mesoporous silica particles with murine tissue bioenergetics**

The *in vitro* system discussed in Section 8 is used to investigate the effects of two forms of calcined mesoporous silica particles (MCM41-cal and SBA15-cal) on cellular respiration of mouse tissues (Al Shamsi et al., 2010; Al-Salam et al., 2011; Tao et al., 2008c). O2 consumption by lung, liver, kidney, spleen and pancreatic tissues was unaffected by exposure to 200 g/mL MCM41-cal or SBA15-cal for several hours. A representative experiment of pneumatocyte respiration is shown in Fig. 9.

The rate of respiration (k) was set as negative of the slope of [O2] vs. time; the values of k (in M O2 min-1 mg-1) are shown at the bottom of the runs. Zero minute corresponds to the addition of the particles. U, untreated; and T, treated.

Fig. 9. Pneumatocyte respiration with and without 200 g/mL MCM41-cal.

Normal tissue architecture and histology were confirmed by light microscopy. Intracellular accumulation of the particles in the studied tissues was evident by electron microscopy. The results show reasonable *in vitro* biocompatibility of the mesoporous silicas with murine

Phosphorescence Oxygen Analyzer as a Measuring Tool for Cellular Bioenergetics 249

Minutes zero correspond to collecting the liver tissue specimens at 3 hr post injections. Two runs were done for each condition. Rates of respiration (*kc*, in μM O2 min-1 mg-1) are shown at the bottom of the

Fig. 10. Representative experiment for liver tissue respiration in C57BL/6 mice 3 hr post

Thus, Con A treatment produced a concurrent impairment of hepatocyte respiration. The lower rate of respiration at 12 hr post treatment (Table 3) concurred with large areas of necrosis and the enhanced rate of respiration at ~3 hr post treatment (Fig. 10) concurred with inflammatory infiltrates limited to the perivascular space without any notable necrosis. The latter finding suggests a role for inflammatory mediators, such as TNF- and IL-2 (both known to peak 3 hr post Con A treatment) in modulating hepatocyte energy metabolism (Louis et al., 1997; Gottlieb et al., 2000). The mechanism for the presumed inflammationinduced increase in hepatocyte oxygen consumption could be uncoupling oxidative phosphorylation *vs.* up-regulating the energy metabolism. Nevertheless, for both assumptions, there is a large demand for energy supply to prevent fulminant liver necrosis. In an *in vitro* experiment, liver tissue respiration was measured with and without IL-2 (added directly to the O2 measuring vial). The rate of respiration without IL-2 was 0.21 M O2 min-1 mg-1 and with IL-2 0.087 M O2 min-1 mg-1 (~60% inhibition). Thus, similar to TNF-

The above *in vitro* system was also used to measure human spermatozoa respiration. O2 concentrations in solutions containing glucose and human spermatozoa declined linearly with time. Sodium cyanide also inhibited sperm oxygen consumption, confirming the oxidations occurred in the respiratory chain. The rate of respiration (mean + SD, n = 10) was 1.0 + 0.3 M O2 min-1 per 108 sperm. Immediate decline in the rate of sperm respiration was noted when toxic agents [e.g., 4-hydroperoxycyclophosphamide (4OOH-CP), 9 tetrahydrocannabinol (9-THC) or 8-tetrahydrocannabinol (8-THC)] were added to washed sperm or neat semen. The inhibition was concentration-dependent and irreversible

injection of PBS (left panel) or 12 mg/kg Con A (right panel)

, IL-2 also inhibits cellular respiration *in vitro* (Gottlieb et al., 2000).

**11. Spermatozoa respiration** 

(Badawy et al., 2009a-b).

runs.

tissue bioenergetics. Therefore, the measurements of respiration can be used to explore biocompatibility and viability of tissues and cells as a result of various treatments.
