**7. Aflatoxin B1 impairs human lymphocyte respiration**

Aflatoxins (most notably, aflatoxin B1) are highly carcinogenic compounds, commonly found in food contaminated by aspergillus flavus, parasiticus and penicillium species (Williams, et al., 2004; Eaton & Gallagher, 1994). These potent mycotoxins create major health problems, especially where food storage is subjected to heat and humidity. A high rate of dietary exposure is reported in Sahara Africa, China and Taiwan. For example, in eastern China (where liver cancer exceeds 1 per 10,000 population per year), an average human exposure to aflatoxins is estimated to be 2.2g/kg/day. For comparison, the exposure in the United States is about 3 orders of magnitude less (Wang et al., 1996).

Biotransformation of aflatoxin B1 is critical for its activation. The parent compound undergoes oxidation by monooxygenases, especially the hepatic cytochrome P450 3A4. The active metabolite, AFB exo-8,9-epoxide, undergoes base-catalyzed rearrangement to a dialdehyde, which rapidly reacts with guanyl N7 in DNA and lysine in proteins (Johnson et al., 1996).

Exposure to aflatoxin B1 has been associated with hepatocellular carcinoma (Montesano et al., 1997), mutagenesis (e.g., in the tumor suppressor gene p53) and immune suppression (Corrier, 1991). Most of the information on immunotoxicity of aflatoxin B1 is derived from animal studies (Stec et al., 2009; Reddy et al., 1987; Reddy et al., 1989; Jiang et al., 2008; reviewed in Williams, et al., 2004]. In healthy humans, exposure to aflatoxin B1 is associated

The reaction mixtures contained PBS, 3 M Pd phosphor, 0.5% fat-free bovine serum albumin, 50 g/mL glucose oxidase and shown concentrations of β-glucose. The values of 1/τ (mean ± SD, n = 1200

Aflatoxins (most notably, aflatoxin B1) are highly carcinogenic compounds, commonly found in food contaminated by aspergillus flavus, parasiticus and penicillium species (Williams, et al., 2004; Eaton & Gallagher, 1994). These potent mycotoxins create major health problems, especially where food storage is subjected to heat and humidity. A high rate of dietary exposure is reported in Sahara Africa, China and Taiwan. For example, in eastern China (where liver cancer exceeds 1 per 10,000 population per year), an average human exposure to aflatoxins is estimated to be 2.2g/kg/day. For comparison, the

exposure in the United States is about 3 orders of magnitude less (Wang et al., 1996).

Biotransformation of aflatoxin B1 is critical for its activation. The parent compound undergoes oxidation by monooxygenases, especially the hepatic cytochrome P450 3A4. The active metabolite, AFB exo-8,9-epoxide, undergoes base-catalyzed rearrangement to a dialdehyde, which rapidly reacts with guanyl N7 in DNA and lysine in proteins (Johnson et

Exposure to aflatoxin B1 has been associated with hepatocellular carcinoma (Montesano et al., 1997), mutagenesis (e.g., in the tumor suppressor gene p53) and immune suppression (Corrier, 1991). Most of the information on immunotoxicity of aflatoxin B1 is derived from animal studies (Stec et al., 2009; Reddy et al., 1987; Reddy et al., 1989; Jiang et al., 2008; reviewed in Williams, et al., 2004]. In healthy humans, exposure to aflatoxin B1 is associated

flashes over 2 min) as a function of [β-glucose] are shown. The lines are linear fits.

**7. Aflatoxin B1 impairs human lymphocyte respiration** 

Fig. 4. Calibration with -glucose plus glucose oxidase

al., 1996).

with lower perforin (a cytolytic protein produced by natural killer lymphocytes) expression on CD8+ T-lymphocytes (Jiang et al., 2008). A dose-related decrease in DNA synthesis in lymphocyte cultures (with and without mitogens) is found in mice exposed *in vivo* to aflatoxin B1 (Reddy et al., 1987). A decrease in DNA synthesis is also observed in normal splenic mouse lymphocytes cultured *in vitro* with >10 M aflatoxin B1; a decrease in RNA synthesis is observed at dosing >25 M and a decrease in protein synthesis at dosing >100 M (Reddy et al., 1989).

The phosphorescence oxygen analyzer is used to monitor the effects of aflatoxin B1 on human lymphocyte mitochondrial oxygen consumption. These experiments investigate whether aflatoxin B1 impairs respiration of the lymphoid tissue, an organ that is typically targeted by this potent mycotoxin. Aflatoxin B1 (1.0 mg = 3.2 micromol) was freshly dissolved in 1.0 mL dry methanol and immediately added to cell suspensions with vigorous mixing. Alternatively, aflatoxin B1 powder was directly added to the cell suspension with vigorous mixing. The concentrations were determined by the absorbance at 350 nm (10 L aflatoxin B1 stock solution or cell-free supernatant in 1.0 mL dry methanol), using an extinction coefficient of 21,500 M-1 cm-1 (Nesheim et al., 1999); the aflatoxin B1 excitation wavelength is 366 nm and the emission wavelength 455 nm. The reactions were carried out in glass vials and protected from light.

PBMC (0.6 x 107 cells/mL) were suspended in 6.0 mL PBS, 10 mM glucose, 3 M Pd phosphor and 0.5% fat-free bovine serum albumin. The mixture was divided into 2 equal aliquots. Methanol (25 L per mL, Fig. 5, left panel) or aflatoxin B1 (25 M, Fig. 5, right panel) was then added and the incubation continued at 37oC (open to air with gentle stirring). At *t* = 10 and 110 min, 1.0 mL of each mixture was simultaneously placed in the instruments for O2 measurement. The rate of respiration (*k*, in M O2 min-1) for *t* = 10 to 78 min for the methanol-treated cells was 2.0 and for the aflatoxin B1-treated cells 2.1. The values of *k* for *t* = 110 to 174 were 2.2 and 1.2, respectively (corresponding to 45% inhibition of lymphocyte respiration).

PBMC were incubated at 37oC with 25 μL per mL methanol (left panel) or 25 μM aflatoxin B1 (right panel). Minute zero corresponds to the addition of aflatoxin B1. At t = 10 and t = 110 min, 1.0 mL of each mixture was simultaneously placed in the instruments for O2 measurement. Rates of respiration (k) were calculated from the best-fit linear curves. Additions of 5.0 mM NaCN and 50 g/mL glucose oxidase are shown.

Fig. 5. Effect of aflatoxin B1 on human PBMC respiration.

Phosphorescence Oxygen Analyzer as a Measuring Tool for Cellular Bioenergetics 245

releases the fluorogenic AFC; the latter can be separated on HPLC and detected by

Caspase-3 activity in lymphocytes exposed to aflatoxin B1 is shown in Fig. 7. The purpose of these experiments is to confirm caspases are activated within the time period required for inhibition of respiration. The mixtures (final volume, 0.5 mL) contained 1.5 x 106 cells in PBS, 10 mM glucose and 68 M Ac-DEVD-AMC (N-acetyl-asp-glu-val-asp-7-amino-4-methyl coumarin, a caspase-3 substrate) with and without 20 M zVAD-fmk (benzyloxycarbonylval-ala-DL-asp-fluoromethylketone, a pan-caspase inhibitor) (Slee et al., 1996). The suspensions were incubated at 37oC for 2 hr without other additions (Fig. 7, left panel) or with the addition of ~100 M aflatoxin B1 (Fig. 7, 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 results show AMC moieties (the cleavage product of Ac-DEVD-AMC) appear in the cells about 2 hr after the addition of aflatoxin B1. This 2-hr period is the same as that observed for aflatoxin B1-induced inhibition of respiration (see Fig. 5). Thus, the results suggest aflatoxin B1 impairs human lymphocyte mitochondrial

The above findings also demonstrate the lymphocyte preparation contain monooxygenases that activate aflatoxin B1. These results are consistent with previous reports (Stec et al., 2009; Rossano et al., 1999; Savel et al., 1970; Wang et al., 1999). In one study, the addition of aflatoxins B1 at concentrations up to 32 M had a minimum effect on phytohemagglutininp-stimulated human lymphocyte proliferation (Meky et al., 2001). However, an earlier study on human lymphocytes by Savel et al. (1970) showed a reduced phytohemagglutinin-pstimulated lymphocyte proliferation with 16 M aflatoxin B1. More recently, aflatoxin B1 was shown to inhibit *in vitro* concanavalin A-induced proliferation of pig blood lymphocytes; in 72-hr cultures, the concentration of aflatoxin B1 producing 50% inhibition (IC50) was 60 nM (Stec et al., 2009). In other studies, aflatoxin G1 induced *in vitro* apoptosis in human lymphocytes (Wang et al., 1999; Sun et al., 2002). In summary, the data presented show human lymphocytes exposed *in vitro* to aflatoxin B1 exhibit impairments of cellular respiration, which could result from caspase activation. The results substantiate the potent

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

fluorescence with a great sensitivity (Tao et al., 2007).

immunosuppressive activity of aflatoxins in human.

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

function by activating caspases.

hours.

The time-course for aflatoxin B1-induced inhibition of lymphocyte respiration was investigated (Fig. 6). PBMC (1.3 x 107 cells/mL) were suspended in 3.0 mL PBS, 10 mM glucose and divided into 2 equal aliquots. Aflatoxin B1 powder was directly added to one aliquot with vigorous mixing (final concentration, ~75 M). The 2 aliquots were then incubated at 37oC for 60 min (open to air with continuous stirring). At *t* = 60 min, 5 mg albumin and 2.0 M Pd phosphor were added to each suspension. The samples were then simultaneously placed in the chambers for O2 measurement. For the untreated cells, O2 consumption was linear with time (*k* = 2.4 M O2/min, *R*2 > 0.916). For the treated cells, O2 consumption was exponential with time, *R*2 > 0.934. Changes at 5-min intervals for the treated cells showed a sharp decline in the values of *k* for *t* = 60 to 90 min, followed by a steady low rate for *t* = 95 to 175 min. In contrast, the values of *k* remained relatively stable for *t* = 60 to 140 min. The mean + SD (coefficient of variation) for the values of k for the untreated cells was 2.43 + 0.55 (Cv = 23%) and for the treated cells 1.56 + 1.80 (Cv = 87%); *p*value < 0.02 (Fig. 6, insert).

The lines are linear fit for the untreated cells (R2 > 0.916) and exponential fit for the treated cells (R2 > 0.936). Insert, changes in the values of k at 5-min intervals.

Fig. 6. Time-course of the effect of aflatoxin B1 on human lymphocyte respiration.

The exponential profile of O2 consumption in the presence of aflatoxin B1 is similar to dactinomycin (Tao et al., 2006b; Tao et al., 2008a). This pattern of bioenergetic derangements could stem from progressive mitochondrial and metabolic disturbances, ranging from uncoupling oxidative phosphorylation (which accelerates O2 consumption and rapidly depletes the metabolic fuels) to mitochondrial respiratory chain function collapse. Experimentally, these two phases are clearly distinguishable in our system (Fig. 6, insert).

Caspase activation in lymphocytes treated with aflatoxin B1 was then examined. Many of the caspases (e.g., caspase-3, -2 and -7) target the asp-glu-Val-asp (DEVD) motif and cleave at sites next to the last aspartate residue (Nicholson et al., 1997). Synthetic cell-permeable substrates, such as N-acetyl-DEVD-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC) and N-acetyl-DEVD-7-amino-4-methyl coumarin (Ac-DEVD-AMC) have been used to investigate caspase activities. For example, cleavage of Ac-DEVD-AFC by specific caspases

The time-course for aflatoxin B1-induced inhibition of lymphocyte respiration was investigated (Fig. 6). PBMC (1.3 x 107 cells/mL) were suspended in 3.0 mL PBS, 10 mM glucose and divided into 2 equal aliquots. Aflatoxin B1 powder was directly added to one aliquot with vigorous mixing (final concentration, ~75 M). The 2 aliquots were then incubated at 37oC for 60 min (open to air with continuous stirring). At *t* = 60 min, 5 mg albumin and 2.0 M Pd phosphor were added to each suspension. The samples were then simultaneously placed in the chambers for O2 measurement. For the untreated cells, O2 consumption was linear with time (*k* = 2.4 M O2/min, *R*2 > 0.916). For the treated cells, O2 consumption was exponential with time, *R*2 > 0.934. Changes at 5-min intervals for the treated cells showed a sharp decline in the values of *k* for *t* = 60 to 90 min, followed by a steady low rate for *t* = 95 to 175 min. In contrast, the values of *k* remained relatively stable for *t* = 60 to 140 min. The mean + SD (coefficient of variation) for the values of k for the untreated cells was 2.43 + 0.55 (Cv = 23%) and for the treated cells 1.56 + 1.80 (Cv = 87%); *p*-

The lines are linear fit for the untreated cells (R2 > 0.916) and exponential fit for the treated cells (R2 >

The exponential profile of O2 consumption in the presence of aflatoxin B1 is similar to dactinomycin (Tao et al., 2006b; Tao et al., 2008a). This pattern of bioenergetic derangements could stem from progressive mitochondrial and metabolic disturbances, ranging from uncoupling oxidative phosphorylation (which accelerates O2 consumption and rapidly depletes the metabolic fuels) to mitochondrial respiratory chain function collapse. Experimentally, these two phases are clearly distinguishable in our system (Fig. 6, insert). Caspase activation in lymphocytes treated with aflatoxin B1 was then examined. Many of the caspases (e.g., caspase-3, -2 and -7) target the asp-glu-Val-asp (DEVD) motif and cleave at sites next to the last aspartate residue (Nicholson et al., 1997). Synthetic cell-permeable substrates, such as N-acetyl-DEVD-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC) and N-acetyl-DEVD-7-amino-4-methyl coumarin (Ac-DEVD-AMC) have been used to investigate caspase activities. For example, cleavage of Ac-DEVD-AFC by specific caspases

Fig. 6. Time-course of the effect of aflatoxin B1 on human lymphocyte respiration.

0.936). Insert, changes in the values of k at 5-min intervals.

value < 0.02 (Fig. 6, insert).

releases the fluorogenic AFC; the latter can be separated on HPLC and detected by fluorescence with a great sensitivity (Tao et al., 2007).

Caspase-3 activity in lymphocytes exposed to aflatoxin B1 is shown in Fig. 7. The purpose of these experiments is to confirm caspases are activated within the time period required for inhibition of respiration. The mixtures (final volume, 0.5 mL) contained 1.5 x 106 cells in PBS, 10 mM glucose and 68 M Ac-DEVD-AMC (N-acetyl-asp-glu-val-asp-7-amino-4-methyl coumarin, a caspase-3 substrate) with and without 20 M zVAD-fmk (benzyloxycarbonylval-ala-DL-asp-fluoromethylketone, a pan-caspase inhibitor) (Slee et al., 1996). The suspensions were incubated at 37oC for 2 hr without other additions (Fig. 7, left panel) or with the addition of ~100 M aflatoxin B1 (Fig. 7, 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 results show AMC moieties (the cleavage product of Ac-DEVD-AMC) appear in the cells about 2 hr after the addition of aflatoxin B1. This 2-hr period is the same as that observed for aflatoxin B1-induced inhibition of respiration (see Fig. 5). Thus, the results suggest aflatoxin B1 impairs human lymphocyte mitochondrial function by activating caspases.

The above findings also demonstrate the lymphocyte preparation contain monooxygenases that activate aflatoxin B1. These results are consistent with previous reports (Stec et al., 2009; Rossano et al., 1999; Savel et al., 1970; Wang et al., 1999). In one study, the addition of aflatoxins B1 at concentrations up to 32 M had a minimum effect on phytohemagglutininp-stimulated human lymphocyte proliferation (Meky et al., 2001). However, an earlier study on human lymphocytes by Savel et al. (1970) showed a reduced phytohemagglutinin-pstimulated lymphocyte proliferation with 16 M aflatoxin B1. More recently, aflatoxin B1 was shown to inhibit *in vitro* concanavalin A-induced proliferation of pig blood lymphocytes; in 72-hr cultures, the concentration of aflatoxin B1 producing 50% inhibition (IC50) was 60 nM (Stec et al., 2009). In other studies, aflatoxin G1 induced *in vitro* apoptosis in human lymphocytes (Wang et al., 1999; Sun et al., 2002). In summary, the data presented show human lymphocytes exposed *in vitro* to aflatoxin B1 exhibit impairments of cellular respiration, which could result from caspase activation. The results substantiate the potent immunosuppressive activity of aflatoxins in human.
