Complementary DNA cloning, protein expression, and characterization of alpha-class GSTs from Macaca fascicularis liver.

Large species differences exist in sensitivity to aflatoxin B(1) (AFB(1))-induced liver cancer. Mice are resistant to AFB(1)-induced liver cancer because they express an alpha-class GST (mGSTA3-3) that has high activity toward the reactive intermediate aflatoxin B(1)-8,9-epoxide (AFBO). Rats constitutively express only small amounts of a GST with high AFBO activity (rGSTA5-5) and thus are sensitive to AFB(1)-induced hepatocarcinogenesis, although induction of rGSTA5-5 can confer resistance in rats. In contrast to rodents, constitutively expressed human hepatic alpha-class GSTs have little or no AFBO detoxifying activity. Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), has significant constitutive hepatic GST activity toward AFBO and most of this activity belongs to mu-class GSTs. To determine if any alpha-class GSTs in Mf liver have AFBO activity, a cDNA library from a male Mf liver was constructed and screened using the human alpha-class GstA1 cDNA as a probe. Three different cDNA clones with full-length open reading frames were identified from the Mf hepatic cDNA library. Analyses of the cDNA deduced protein sequences indicated that these three alpha-class cDNA clones were 97-98% homologous with each other, and shared 93, 95, and 95% identity with human GSTA1, and were named mfaGSTA1, mfaGSTA2, and mfaGSTA3, respectively. Bacterially expressed mfaGSTA1-1 recombinant protein had similar activities toward classic GST substrates such as DCNB, CHP, and ECA, but slightly lower CDNB conjugating activity relative to human GSTA1-1. However, similar to hGSTA1-1, mfaGSTA1-1 had no AFBO conjugating activity. In addition, similar to human GSTA1 gene, cDNA-derived amino acid sequence analyses demonstrated that all of these Mf alpha-class GSTs genes (mfaGSTA1, mfaGSTA2, and mfaGSTA3) had none of the six critical residues that were identified previously to confer high AFBO activity in mouse alpha-class GSTA3-3. Thus, in contrast to rodents but similar to humans, alpha-class GSTs from the nonhuman primate, Mf, have little conjugating activity toward AFBO.     

Effects of dietary butylated hydroxytoluene on aflatoxin B1-relevant metabolic enzymes in turkeys.

We have shown previously that the extreme sensitivity of turkeys to aflatoxin B(1) (AFB(1)) is due to a combination of efficient AFB(1) activation by cytochrome P450s (CYPs) 1A and deficient detoxification by glutathione S-transferases (GSTs). Phenolic antioxidants such as butylated hydroxytoluene (BHT) have been shown to be chemoprotective in some animal models due, in part, to modulation of AFB(1)-relevant phase I and/or phase II activities, and we wished to determine whether BHT has a similar effect in turkeys. Ten-day-old male turkeys were maintained on diets amended with 1000 or 4000 ppm of BHT for 10 days, then sampled. Hepatic microsomal CYP 1A activity as well as conversion of AFB(1) to the putative toxic metabolite, the exo-AFB(1)-8,9-epoxide (AFBO), were significantly lower compared with control. Conversely, dietary BHT significantly increased activities of several isoforms of hepatic cytosolic GST, as well quinone oxidoreductase (QOR). Western immunoblotting confirmed that dietary BHT increased expression of homologues to rodent GST isoforms Yc1, Yc2 and Ya. There was, however, no observable BHT-related increase in GST-mediated specific conjugation with microsomally-generated AFBO. In total, our data indicates that dietary BHT modulates a variety of AFB(1)-relevant phase I and phase II enzymes, while having no measurable effect towards specific AFB(1) detoxification by GST.     

Biochemical basis for the extreme sensitivity of turkeys to aflatoxin B(1)

Poultry are the most susceptible food animal species to the toxic effects of the mycotoxin aflatoxin B(1) (AFB(1)). Feed contaminated with even small amounts of AFB(1) results in significant adverse health effects in poultry. The purpose of this study was to explain the biochemical mechanism(s) for this extreme sensitivity. We measured microsomal activation of AFB(1) to the AFB(1)-8,9-epoxide (AFBO), the putative toxic intermediate, as well as cytosolic glutathione S-transferase (GST)-mediated detoxification of AFBO, in addition to other hepatic phase I and phase II enzyme activities, in 3-week-old male Oorlop strain turkeys. Liver microsomes prepared from these turkeys activated AFB(1) in vitro with an apparent K(m) of 109 microM and a V(max) of 1.25 nmol/mg/min. Preliminary evidence for the involvement of cytochromes P450 (CYP) 1A2 and, to a lesser extent, 3A4 for AFB(1) activation was assessed by the use of specific mammalian CYP inhibitors. The possible presence of avian orthologues of these CYPs was supported by activity toward ethoxyresorufin and nifedipine, as well as by Western immunoblotting using antibodies to human CYPs. Cytosol prepared from turkey livers exhibited GST-mediated conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB), but at a much lower rate than that observed in other species. Western immunoblotting indicated the presence of alpha and sigma class GSTs and another AFB(1)-detoxifying enzyme, AFB(1)-aldehyde reductase (AFAR). Turkey liver cytosol also had quinone oxidoreductase (QOR) activity. Importantly, cytosol exhibited no measurable GST-mediated detoxification of microsomally activated AFB(1), indicating that turkeys are deficient in the most crucial AFB(1)-detoxification pathway. In total, our data indicate that the extreme sensitivity of turkeys to AFB(1) may be attributed to a combination of efficient AFB(1) activation and deficient detoxification by phase II enzymes, such as GSTs.     

Mouse strain differences in glutathione S-transferase activity and aflatoxin B1 biotransformation

Previous studies have suggested that mice are resistant to the carcinogenic effects of aflatoxin B1 (AFB1) and that this resistance is largely the result of expression of an isoenzyme of glutathione S-transferase (GST) with high activity toward AFB1-8,9-epoxide. Significant interstrain differences in cytosolic GST activities toward a variety of substrates have been reported in mice. If such differences exist for the conjugation of AFB1-8,9-epoxide, then there may be significant mouse strain differences in susceptibility to AFB1-induced hepatocarcinogenicity. The hepatic microsomal and cytosolic biotransformation of AFB1 was studied in 8 different strains of mice fed a purified diet. GST-mediated conjugation of AFB1-8,9-epoxide with glutathione and GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide (CHP) were determined with cytosolic fractions from 8-10 pooled livers. Specific activities of cytochrome-P-450-mediated oxidation of AFB1 to aflatoxin Q1 (AFQ1), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1), as well as the reactive intermediate AFB1-8,9-epoxide, were determined with hepatic microsomal fractions from each mouse strain. No striking differences in specific activity between mouse strains were observed for any of the P-450- or GST-mediated enzymatic pathways measured, although some statistically significant differences were found. GST specific activities toward AFB1-8,9-epoxide, CDNB, DCNB, ECA and CHP ranged from 1.5-2.1, 2,830-5,370, 81-144, 38-69 and 32-73 nmol/mg protein/min, respectively. The rate of formation of AFB1-8,9-epoxide ranged from 208 to 465 pmol/mg protein/min. The specific activities of AFQ1,AFM1, and AFP1 formation by microsomes ranged from 36-70, 161-326, and 252-426 pmol/mg protein/min, respectively. Mice fed a standard rodent chow diet showed evidence of microsomal and cytosolic enzyme induction when compared to mice fed a purified diet. The lack of substantial differences in enzyme specific activities between mouse strains suggests that interstrain variations in the hepatocarcinogenic effects of AFB1 in mice should not be large.     

Biotransformation of methyl parathion by glutathione S-transferases.

The organo(thio)phosphate esters are one of the most widely used classes of insecticides. Worldwide, organophosphate insecticides (OPs) result in numerous poisonings each year. In insects, glutathione S-transferases (GSTs) play an important role in OP resistance; limited data suggest that GST-mediated O-dealkylation occurs in humans as well. To characterize the capacity of mammalian GSTs to detoxify OPs, we investigated mammalian GST biotransformation of the widely used OP, methyl parathion (MeP). Cytosolic fractions isolated from rat, mouse, and ten individual adult human livers biotransformed 300 microM MeP at rates of 2.36, 1.76, and 0.70 (mean rate) nmol desmethyl parathion/min/mg, respectively. Our study focused on human GSTs; in particular, we investigated hGSTs M1-1 and T1-1, since deletion polymorphisms occur commonly in these genes. However, we found no correlation between hGSTM1/T1 genotypes and MeP O-dealkylation activities of the ten human liver cytosolic samples. We also measured MeP O-dealkylation activities of several purified recombinant GSTs belonging to the alpha (human GSTs A1-1 and A2-2, mouse GSTA3-3, rat GSTA5-5), mu (human GSTs M1a-1a, M2-2, M3-3, M4-4), pi (human GSTP1-1, mouse GSTs P1-1, P2-2), and theta (human GSTT1-1) classes. At 1 mM glutathione and 300 microM MeP concentrations, hGSTT1-1 and hGSTA1-1 exhibited the highest O-dealkylation activities: 545.8 and 65.0 nmol/min/mg, respectively. When expression level and enzymatic activity are considered, we estimate that hGSTA1-1 is responsible for the majority of MeP O-dealkylation in human hepatic cytosol. In target organs such as brain and skeletal muscle, where hGSTT1-1 is expressed, hGSTT1-1-mediated biotransformation of MeP may be important.     

Effect of an extract of the root of Scutellaria baicalensis and its flavonoids on aflatoxin B1 oxidizing cytochrome P450 enzymes

The inhibition of aflatoxin B1 (AFB1) metabolism by a water extract of the root of Scutellaria baicalensis and its flavonoids was examined in liver microsomes. AFB1 is known to be metabolized to aflatoxin M1 (AFM1), aflatoxin Q1 (AFQ1), and AFB1-8,9-epoxide (AFBO). The water extract potently inhibited the production of AFM1 by cytochrome P450 (CYP)1A1/2 and slightly reduced AFBO formation by CYP1A1/2, CYP2B1, CYP2C11 and CYP3A1/2 in TCDD-treated rat liver microsomes. IC50 values for AFM1 and AFBO formation were 6.8 and 122.4 microg/ml, respectively. Wogonin showed the highest inhibitory activity towards AFM1 formation among the flavonoids isolated from the extract. On the other hand, the extract had no effects on the formation of AFBO and AFQ1 in human liver microsomes, and on the activities of CYP2B1, CYP2C11 and CYP3A1/2 which were detected by hydroxylation patterns of testosterone. These results demonstrated that the extract of the root of Scutellaria baicalensis has a specific inhibitory effect on CYP1A1/2 among CYP enzymes involved in AFB1 metabolism by rat and human microsomes.     

Protective effects of coffee diterpenes against aflatoxin B1-induced genotoxicity: mechanisms in rat and human cells.

The coffee-specific diterpenes cafestol and kahweol (C + K) have been reported to be anticarcinogenic in several animal models. Proposed mechanisms involve a co-ordinated modulation of several enzymes responsible for carcinogen detoxification, thus preventing reactive agents interacting with critical target sites. To address the human relevance of the chemoprotective effects of C + K against aflatoxin B(1) (AFB1) genotoxicity observed in rat liver, and to compare the mechanisms of protection involved in both species, animal and human hepatic in vitro test systems were applied. In rat primary hepatocytes, C + K reduced the expression of cytochrome P450 CYP 2C11 and CYP 3A2, the key enzymes responsible for AFB1 activation to the genotoxic metabolite aflatoxin B1-8,9 epoxide (AFBO). In addition, these diterpenes induced significantly GST Yc2, the most efficient rat GST subunit involved in AFBO detoxification. These effects of C + K resulted in a marked dose-dependent inhibition of AFB1-DNA binding in this rat in vitro culture system. Their relevance in humans was addressed using liver epithelial cell lines (THLE) stably transfected to express AFB1 metabolising cytochrome P450s. In these cells, C + K also produced a significant inhibition of AFB1-DNA adducts formation linked with an induction of the human glutathione S-transferase GST-mu. Altogether, these results suggest that C + K may have chemoprotective activity against AFB1 genotoxicity in both rats and humans.     

Comparative expression of two alpha class glutathione S-transferases in human adult and prenatal liver tissues

The ability of the fetus to detoxify transplacental drugs and chemicals can be a critical determinant of teratogenesis and developmental toxicity. Developmentally regulated expression of alpha class glutathione S-transferases (GSTs) is of particular interest, since these isozymes have high activity toward peroxidative byproducts of oxidative injury that are linked to teratogenesis. The present study was initiated to examine the expression and catalytic activities of alpha class GST isozymes in human prenatal liver. Northern analysis demonstrated the presence of hGSTA1 and/or A2 (hGSTA1/2) and hGSTA4 steady-state mRNAs in second trimester prenatal livers. Western blotting of prenatal liver proteins provided corroborating evidence via detection of an hGSTA1/2-reactive protein in both cytosol and mitochondria and of hGSTA4-4-reactive protein in mitochondria alone. Catalytic studies demonstrated that prenatal liver cytosolic GSTs were active toward 1-chloro-2,4-dinitrobenzene (a general GST reference substrate), delta5-androstene-3,17-dione (relatively specific for hGSTA1-1), and 4-hydroxynonenal, a highly mutagenic alpha,beta-unsaturated aldehyde produced during oxidative damage and a substrate for hGSTA4-4. Total GSH-peroxidase and GST-dependent peroxidase activities were 9- and 18-fold higher, respectively, in adult liver than in prenatal liver. Multiple tissue array analyses demonstrated considerable tissue-specific and developmental variation in GST mRNA expression. In summary, our results demonstrate the presence of two important alpha class GSTs in second trimester human prenatal tissues, and indicate that mitochondrial targeting of GST may represent an important pathway for removal of cytotoxic products in prenatal liver. Furthermore, the relatively inefficient prenatal reduction of hydroperoxides may underlie an increased susceptibility to maternally transferred pro-oxidant drugs and chemicals.     

Metabolism of aflatoxin B1 by normal human bronchial epithelial cells

Aflatoxin B, (AFB1) is a potent hepatocarcinogen in animal models and a suspected carcinogen in humans. High concentrations of AFB, have been found in respirable grain dusts, and may therefore be a risk factor for human lung cancer in certain occupations. To study the potential for AFB, activation in human lung, cytochrome P-450 (CYP)-mediated activation and glutathione S-transferase (GST)-mediated detoxification of AFB1 were examined in cultured normal human bronchial epithelial (NHBE) cells. Cells were exposed to 0. 15 microM or 1.5 microM AFB, for 48 h and media was collected for metabolite analysis by high-performance liquid chromatography (HPLC). At 0. 15 microM, AFB1 was metabolized only to the detoxified metabolite aflatoxin Q1 (AFQ1). At 1.5 microM AFB1, both aflatoxin M1 (AFM1), and AFQ1 were produced. Cells pretreated with 50 degrees M 3-methylcholanthrene (3MC), a CYP 1A inducer, for 72 h prior to 0.15 microM AFB1, produced the activated AFB1 8,9-epoxide (AFBO). Similarly, microsomes prepared from 3MC-pretreated cells formed AFBO, but microsomes from noninduced cells did not. While AFB1-DNA adducts were not detected at low AFB1 concentrations in untreated NHBE, 3MC induction caused the production of AFB1-DNA adducts at 0.015 and 0.15 microM AFB1. Western immunoblots showed that the primary CYP isoforms responsible for AFB1 activation in the liver, 1A and 3A4, to be constitutively expressed in NHBE cells. Expression of CYP 1A was significantly increased in 3MC-pretreated cells, while CYP 3A4 expression increased slightly, but not to the extent of the 1A isoforms. The principal AFBO detoxifying enzyme, glutathione S-transferase (GST), was constitutively expressed in NHBE cells, and was increased approximately twofold by 3MC pretreatment. Cytosolic fractions from neither control nor 3MC-induced NHBE had measurable AFBO conjugating activity, indicating that these cells may lack AFB1-relevant GST activity. From these data, it appears that NHBE cells activate AFB1 inefficiently, but possess CYPs reportedly responsible for metabolism of AFB1. These data support earlier findings showing modest CYP-mediated AFB1 activation in human airways, but indicate that exposure to polycyclic aromatic hydrocarbons (PAHs), such as 3MC, which induce CYP(s) that specifically activate AFB1 may increase the harmful effects of AFB1 exposures in human airways.     

Variability in aflatoxin B(1)-macromolecular binding and relationship to biotransformation enzyme expression in human prenatal and adult liver

Studies of transplacental transfer of aflatoxin B(1) (AFB(1)) suggest that the developing human fetus may be a sensitive target for AFB(1) injury. Because AFB(1) requires metabolic activation to the reactive AFB(1)-8,9-exo-epoxide (AFBO) to exert its carcinogenic effects, ontogenic and interindividual differences in AFB(1) biotransformation enzymes may underlie susceptibility to AFB(1)-induced cell injury. The present study was initiated to compare the rates of in vitro AFB(1)-DNA and AFB(1)-protein adduct formation among a panel of 10 adult and 10 second-trimester prenatal livers and to examine the relationship among AFB(1) metabolizing enzyme expression and AFB(1) binding. Mixtures of cytosolic and microsomal proteins from prenatal and adult livers catalyzed the formation of AFB(1)-DNA and AFB(1)-protein adducts at relatively similar rates, although greater individual variability in AFB(1) adduct formation was observed in adult tissues. Extensive interindividual variation among adult tissues was observed in the expression of the AFB(1) activation enzymes cytochrome P4501A2 (CYP1A2), CYP3A4/5, and lipoxygenase (LO). Prenatal CYP3A7 expression was also highly variable. LO expression was eightfold higher in prenatal liver tissues than adults, whereas the expression of the AFBO detoxification enzyme microsomal epoxide hydrolase was twofold higher in adult liver. The levels of the polymorphic glutathione S-transferase M1 (hGSTM1-1), which may potentially protect against AFBO injury, were higher in the hGSTM1-1-expressing tissues of adults in relation to prenatal livers. In general, there was not a strong relationship among AFB(1)-DNA or AFB(1)-protein adduct formation and expression levels of individual AFB(1) metabolizing enzymes. In summary, despite the presence of marked individual and ontogenic differences in the expression of AFB(1) metabolizing enzymes, human second trimester prenatal liver tissues compared to adults do not exhibit a marked sensitivity to the in vitro formation of macromolecular AFB(1) adducts.     

Protective activity of different hepatic cytosolic glutathione S-transferases against DNA-binding metabolites of aflatoxin B1

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.     

Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione S-transferase mu.

Ovarian carcinoma cells 10-fold resistant to the alkylating agent chlorambucil (CBL) were isolated after repeated exposure of the parent cells to gradually escalating concentrations of the drug. The resistant variant, A2780(100), was highly cross-resistant (9-fold) to melphalan and showed lower-level resistance to other cross-linking agents. The resistant A2780(100) cells had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The pi-class GST(s) was the major isoform(s) in both cell lines. However, the resistant A2780(100) cells had at least 11-fold higher GST mu as compared with the parental cells, in which this isoform was barely detectable. A significant induction of GST mu was observed in A2780 cells, but not in the resistant cells, 18 hr after a single exposure to 100 microM CBL. The induction of GST mu by CBL was both time- and concentration-dependent. Assays of the conjugation of CBL with GSH showed that the human mu-class GST had 3.6- and 5.2-fold higher catalytic efficiency relative to the pi- and alpha-class GSTs, respectively. This difference was reflected in the relatively higher (about 6-fold) efficiency of CBL conjugation in A2780(100) cells as compared with the parental cells. These results have demonstrated for the first time a near-linear correlation between CBL resistance and overexpression of mu-class GSTs and suggest that this overexpression maybe responsible, at least in part, for the acquired resistance of ovarian carcinoma cells to CBL, and possibly the other bifunctional alkylating agents. Consistent with this hypothesis, we found evidence for decreased formation of DNA lesions in A2780(100) compared with the drug-sensitive A2780 cells after exposure to CBL.     

Genotoxicity of 4-hydroxy-2-nonenal in human colon tumor cells is associated with cellular levels of glutathione and the modulation of glutathione S-transferase A4 expression by butyrate.

The cellular production of 4-hydroxy-2-nonenal (HNE), a product of endogenous lipid peroxidation, constitutes a genotoxic risk factor for carcinogenesis. Our previous studies have shown that human HT29 colon cells developed resistance toward HNE injury after treatment with butyrate, a diet-associated gut fermentation product. This resistance was attributed to the induction of certain glutathione S-transferases (hGSTP1-1, hGSTM2-2, and hGSTA1-1) and also for the tripeptide glutathione (GSH) synthesizing enzymes. In the present study, we have investigated in HT29 cells whether hGSTA4-4, which has a high substrate specificity for HNE, was also inducible by butyrate and, thus, could contribute to the previously observed chemoresistance. In addition, we investigated if cellular depletion of GSH by L-buthionine-S,R-sulfoximine (BSO) enhances chemosensitivity to HNE injury in HT29 cells. Incubation of HT29 cells with butyrate (2-4 mM) significantly elicited a 1.8 to 3-fold upregulation of steady state hGSTA4 mRNA over 8-24 h after treatment. Moreover, 4 mM butyrate tended to increase hGSTA4-4 protein concentrations. Incubation with 100 microM BSO decreased cellular GSH levels by 77% without significant changes in cell viability. Associated with this was a 2-fold higher level of HNE-induced DNA damage as measured by the comet assay. Collectively, the results of this study and our previous work indicate that the genotoxicity of HNE is highly dependent on cellular GSH status and those GSTs that contribute toward HNE conjugation, including hGSTA4-4. Since HNE contributes to colon carcinogenesis, the favorable modulation of the GSH/GST system by butyrate may contribute to chemoprevention and reduction of the risks.     

Expression of hepatic and ovarian antioxidant enzymes during estrous cycle in rats

The regulation of antioxidant enzymes has received increased attention in terms of protection from many diseases. Despite reports that administered estradiol derivatives can change antioxidant enzyme levels, comprehensive information is not available regarding the effects of the human menstrual cycle or the rat estrous cycle on the expression of the antioxidant enzyme system. The present study was performed to determine the expression levels of cytosolic antioxidant enzymes, including superoxide dismutase-1, catalase, glutathione peroxidase-1, glutathione reductase, peroxiredoxin (Prx)-1, Prx-2, thioredoxin-1, gamma-glutamylcysteine ligase catalytic subunit, alpha-class glutathione S-transferase (GST), pi-class GST, and mu-class GST, in the liver and ovaries of female rats during diestrus and proestrus. Our results indicate that hepatic expression of Prx-1 and Prx-2, and ovarian expression of alpha-class GST were increased significantly during the proestrus phase compared with the diestrus phase. These results suggest that the hepatic Prx family and ovarian alpha-class GST are sensitive to changes during the estrous cycle. Further studies are needed to determine the physiological significance of the regulation of the Prx family and alpha-class GST during the estrous cycle.     

Identification of aflatoxin M1-N7-guanine in liver and urine of tree shrews and rats following administration of aflatoxin B1

Epidemiological studies have shown that exposure to aflatoxin B(1) (AFB(1)) and concurrent infection with hepatitis B lead to a multiplicative risk of developing liver cancer. This chemical-viral interaction can be recapitulated in the tree shrew (Tupia belangeri chinensis). As an initial characterization of this model, the metabolism of AFB(1) in tree shrews has been examined and compared to a sensitive bioassay species, the rat. Utilizing LC/MS/MS, an unreported product, aflatoxin M(1)-N(7)-guanine (AFM(1)-N(7)-guanine), was detected in urine and hepatic DNA samples 24 h after administration of 400 microg/kg AFB(1). In hepatic DNA isolated from tree shrews, AFM(1)-N(7)-guanine was the predominant adduct, 0.74 +/- 0.14 pmol/mg DNA, as compared to 0.37 +/- 0.07 pmol/mg DNA of AFB(1)-N(7)-guanine. Conversely, in rat liver, 6.56 +/- 2.41 pmol/mg DNA of AFB(1)-N(7)-guanine and 0.42 +/- 0.13 pmol/mg DNA of AFM(1)-N(7)-guanine were detected. Rats excreted 1.00 +/- 0.21 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.29 +/- 0.10 pmol AFM(1)-N(7)-guanine/mg creatinine as compared to 0.60 +/- 0.12 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.69 +/- 0.16 pmol AFM(1)-N(7)-guanine/mg creatinine excreted by the tree shrew. Furthermore, tree shrew urine contained 40 times more of the hydroxylated metabolite, AFM(1), than was excreted by rats. In vitro experiments confirmed this difference in oxidative metabolism. Hepatic microsomes isolated from tree shrews failed to produce aflatoxin Q(1) or aflatoxin P(1) but formed a significantly greater amount of AFM(1) than rat microsomes. Bioassays indicated that the tree shrew was considerably more resistant than the rat to AFB(1) hepatocarcinogenesis, which may reflect the significant differences in metabolic profiles of the two species.     

GlutathioneS-Transferase-Catalyzed Conjugation of Bioactivated Aflatoxin B1in Rabbit Lung and Liver

Aflatoxin B₁(AFB₁) requires bioactivation to AFB₁-8,9-epoxide for carcinogenicity, and glutathioneS-transferase (GST)-catalyzed conjugation of activated AFB₁with glutathione (GSH) is a critical determinant of susceptibility to the mycotoxin. Incubations containing [³H]AFB₁, rabbit liver microsomes, an NADPH-generating system, 1 mMGSH, and GST-containing lung or liver cytosol were performed to assess the abilities of lung and liver GSTs to conjugate AFB₁-8,9-epoxide. [³H]AFB₁–GSH was isolated by isocratic reverse-phase high-performance liquid chromatography (HPLC) and quantitated by liquid scintillation spectroscopy. Maximal [³H]AFB₁–GSH formation rates were significantly lower for lung than for liver (0.3 ± 0.1 and 1.7 ± 0.4 nmol/mg/hr, respectively). Immunoprecipitation of rabbit pulmonary cytosolic GSTs with anti-alpha or anti-mu GST antisera decreased [³H]AFB₁–GSH production by approximately 45 and 51%, respectively, indicating that alpha-class and mu-class GSTs are of similar importance in catalyzing this reaction in the lung. Because mu-class GSTs comprise only a small proportion of total lung GST content, these enzymes have high specific activity toward AFB₁-8,9-epoxide. In contrast, the pi-class GST appeared to play a negligible role. Using a rat liver microsomal system to generate both AFB₁exo- andendo-epoxide isomers, and analysis based on chiral HPLC, we found that rabbit liver cytosolic GSTs catalyzed formation of both AFB₁exo- andendo-epoxide–GSH conjugates, whereas pulmonary cytosolic GSTs catalyzed formation of only theexostereoisomer at detectable levels. Despite a preference for conjugating the more mutagenic AFB₁exo-epoxide isomer, the relatively low capacity for GST-catalyzed detoxification of bioactivated AFB₁in lung may be an important factor in the susceptibility of the lung to AFB₁toxicity.     

Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B1-8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide

As part of the studies of the biochemical basis for species differences in biotransformation of the carcinogen aflatoxin B1 (AFB1) and its modulation by phenolic antioxidants, we have investigated the role of mouse liver glutathione S-transferase (GST) isoenzymes in the conjugation of AFB1-8,9-epoxide. Isoenzymes of GST were purified to electrophoretic homogeneity from Swiss-Webster mouse liver cytosol by affinity chromatography and chromatofocusing. The isoenzyme fractions were characterized in terms of activity toward surrogate substrates and immunologic cross-reactivity with antisera to rat GSTs. The major isoenzymes were identified as SW 4-4, SW 3-3, and SW 1-1. The specific activity of SW 4-4 toward AFB1-8,9-epoxide was at least 50- and 150-fold greater than that of SW 3-3 and SW 1-1, respectively. Relatively high activity toward another epoxide carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, was observed with both SW 4-4 and SW 3-3. SW 1-1 had the highest activity toward 1-chloro-2,4-dinitrobenzene (CDNB) whereas SW 4-4 had relatively low CDNB activity. Following pretreatment with 0.75% butylated hydroxyanisole in the diet, the fraction of total GST contributed by SW 1-1 appeared to increase dramatically, whereas in control mice SW 3-3 constituted the predominant isoenzyme. The high GST activity of mouse liver cytosol toward AFB1-8,9-epoxide is apparently due to an isoenzyme that contributes little to the overall cytosolic CDNB activity.     

METABOLISM AND CYTOTOXICITY OF AFLATOXIN B 1 IN CYTOCHROME P-450-EXPRESSING HUMAN LUNG CELLS

The mycotoxin aflatoxin B 1 (AFB 1 ) is a hepatocarcinogen in many animal models and probably a human carcinogen. Besides being a dietary carcinogen, AFB 1 has been detected in dusts generated in the processing and transportation of AFB 1 -contaminated products. Inhalation of grain dusts contaminated with AFB 1 may be a risk factor in human lung cancer. Aflatoxin B 1 requires cytochrome P-450 (CYP)-mediated activation to form cytotoxic and DNA-reactive intermediates, and this activation in human liver is mediated by the CYP 1A2 and 3A4 isoforms. Which isoforms are important in AFB 1 activation in human lung is not well understood. To investigate whether these CYPs can activate AFB 1 at low, environmentally relevant concentrations in human lung cells, SV40 immortalized human bronchial epithelial cells (BEAS-2B) that were transfected with cDNA for CYPs 3A4 (B3A4) or 1A2 (B-CMV1A2) were used. B-CMV1A2 cultured in 15 n M AFB 1 produced the AFB 1 -glutathione conjugate (AFB 1 -GSH) and aflatoxin M 1 (AFM 1 ), while B3A4 cells produced only aflatoxin Q 1 (AFQ 1 ) at 0.15 M AFB 1 . Nontransfected BEAS-2B cells produced no metabolites, even at 1.5 m M AFB 1 . Microsomes prepared from B-CMV1A2 and B3A4 cells activated AFB 1 to AFB 1 8,9-epoxide (AFBO), while those from BEAS-2B cells did not produce AFBO. Cytosol from all three cell types was ineffective at glutathione S -transferase (GST)-mediated trapping of enzymatically generated AFB 1 8,9-epoxide. B-CMV1A2 cells were 100-fold more sensitive to AFB 1 compared to B3A4 cells, and were 6000-fold more sensitive than control BEAS-2B cells. Western immunoblots confirmed that only B-CMV1A2 cells expressed CYP 1A2 protein, while CYP 3A4 was only in B3A4 cells. B-CMV1A2 cells were the most sensitive to AFB 1 , followed by B3A4 cells. CYP 3A4, which has been predicted to activate AFB 1 primarily at higher AFB 1 concentrations, was also responsible for significant AFB 1 toxicity at low concentrations. These data indicate that human lung cells expressing these CYP isoforms are capable of activating AFB 1 , even at environmentally relevant concentrations.     

Characterization of atrazine biotransformation by human and murine glutathione S-transferases.

Atrazine is one of the most widely used herbicides in the United States and has been detected, occasionally, at low levels in drinking water sources. The biotransformation of atrazine in humans has not been fully characterized. Rodent studies suggest Phase I-dominated biotransformation with minor Phase II-mediated biotransformation by glutathione S-transferase(s) (GST). In human urine, mercapturates of atrazine are significant metabolites, yet the specific GST form(s) responsible for glutathione (GSH) conjugation have not been identified. Using recombinant alpha, mu, pi and theta class human GSTs, we demonstrated that only hGSTP1-1 displays significant activity toward atrazine (7.1 nmol/min/mg protein). We also confirmed that mouse GST Pi (pi) protein is responsible for the GSH-dependent biotransformation of atrazine in mouse liver; recombinant mGSTP1-1 had a specific activity of 7.3-nmol/min/mg protein. Furthermore, cytosolic fractions from mouse and human liver conjugated atrazine with glutathione at rates of 282.3 and 3.0 pmol/min/mg, respectively. Docking studies of the atrazine-GST conjugate in the hGSTP1-1 substrate-binding site were used to elucidate a basis for the dramatic difference in activity between mouse GSTP1-1 and GSTP2-2 (7.14 versus 0.02 nmol/min/mg protein, respectively). The inactivity of mGSTP2-2 appears to be attributable to an indirect structural disruption of the G-site by Pro12. Possible effects of the hGSTP1 polymorphisms were investigated. No significant differences in catalytic-specific activity were noted among purified proteins corresponding to the four hGSTP1 variants: hGSTP1(*)A (most common form), hGSTP1(*)B (Ile105Val), hGSTP1(*)C (Ile105Val, Ala114Val), and hGSTP1(*)D (Ala114Val). Overall, this work supports a physiological role for GSTs in atrazine biotransformation and indicates a novel diagnostic substrate for human and mouse GSTP1-1 proteins.     

Dominant contribution of P450 3A4 to the hepatic carcinogenic activation of aflatoxin B1

The hepatic carcinogen aflatoxin B1 (AFB1) is metabolized in the liver by at least four different P450s, all of which exhibit large interindividual differences in the expression levels. These differences could affect the individual risk of hepatocellular carcinoma (HCC). We investigated the metabolism of AFB1 in a panel of 13 human liver microsomal preparations using a hepatic abundance model, which takes into account the specific kinetic parameters and the expression levels of these P450s. We found a 12-fold variability in the production rate of the carcinogenic metabolite AFB1-8,9-epoxide (AFBO) and a 22-fold variability in the production of the detoxification product AFQ1. The ratio between the AFBO and the AFQ1 production rates varied between 1:19 and 1:1.7. P450 3A4 contributed a majority of AFBO and AFQ1, and its expression level was the most important determinant of the AFB1 disposition toward these primary metabolites. P450 3A5, which exclusively produced AFBO, was the second-most important enzyme activating AFB1 to AFBO, followed by P450 3A7 and P450 1A2. The relative contribution of AFBO by P450 3A5 strongly depended on the concomitant expression of P450 3A4, and it was as high as 15% in a P450 3A5 high expressor with the lowest P450 3A4 expression of all livers. The P450 1A2-specific AFB1 detoxification product AFM1 was not detected. In conclusion, the variable expression of P450s has a major effect on the carcinogenic activation of AFB1, which may affect the individual predisposition to HCC. P450 3A4 expression is the most important determinant of AFB1 activation to AFBO. The contribution of P450 1A2 to AFB1 metabolism appears to be negligible and may have been overestimated. Targeted chemoprevention of AFB1-associated HCC should consider P450 3A4 inhibitors and avoidance of P450 3A4 inducers.