DNA polymerase ζ limits chromosomal damage and promotes cell survival following aflatoxin exposure.

Routine dietary consumption of foods that contain aflatoxins is the second leading cause of environmental carcinogenesis worldwide. Aflatoxin-driven mutagenesis is initiated through metabolic activation of aflatoxin B1 (AFB1) to its epoxide form that reacts with N7 guanine in DNA. The resulting AFB1-N7-dG adduct undergoes either spontaneous depurination or imidazole-ring opening yielding formamidopyrimidine AFB1 (AFB1-Fapy-dG). Because this latter adduct is known to persist in human tissues and contributes to the high frequency G-to-T mutation signature associated with many hepatocellular carcinomas, we sought to establish the identity of the polymerase(s) involved in processing this lesion. Although our previous biochemical analyses demonstrated the ability of polymerase ζ (pol ζ) to incorporate an A opposite AFB1-Fapy-dG and extend from this mismatch, biological evidence supporting a unique role for this polymerase in cellular tolerance following aflatoxin exposure has not been established. Following challenge with AFB1, survival of mouse cells deficient in pol ζ (Rev3L-/-) was significantly reduced relative to Rev3L+/- cells or Rev3L-/- cells complemented through expression of the wild-type human REV3L. Furthermore, cell-cycle progression of Rev3L-/- mouse embryo fibroblasts was arrested in late S/G2 following AFB1 exposure. These Rev3L-/- cells showed an increase in replication-dependent formation of γ-H2AX foci, micronuclei, and chromosomal aberrations (chromatid breaks and radials) relative to Rev3L+/- cells. These data suggest that pol ζ is essential for processing AFB1-induced DNA adducts and that, in its absence, cells do not have an efficient backup polymerase or a repair/tolerance mechanism facilitating survival.

Pathways of Metabolite-Related Damage to a Synthetic p53 Gene Exon 7 Oligonucleotide Using Magnetic Enzyme Bioreactor Beads and LC-MS/MS Sequencing.

Reactive metabolites of environmental chemicals and drugs can cause site specific damage to the p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high-throughput, cell-free, 96-well plate magnetic bead-enzyme system interfaced with LC-MS/MS sequencing for bioactivating test chemicals and identifying resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using eight microsomal cytochrome (cyt) P450 enzymes from different organs coated on magnetic beads. All cyt P450s converted aflatoxin B1 to aflatoxin B1-8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites and then strand breaks. This is the first demonstration in a cell-free medium that the aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1-8,9-epoxide to G in codon 249 precedes the SN2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method of identifying metabolite-related gene damage sites may facilitate predictions of organ specific cancers for test chemicals via correlations with mutation sites.

DNA Sequence Modulates Geometrical Isomerism of the trans-8,9- Dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)- 9-hydroxy Aflatoxin B1 Adduct.

Aflatoxin B(1) (AFB(1)), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B(1)-8,9-epoxide, which alkylates DNA at N7-dG. Under basic conditions, this N7-dG adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (AFB(1)−FAPY) adduct. The AFB(1)−FAPY adduct exhibits geometrical isomerism involving the formamide moiety. NMR analyses of duplex oligodeoxynucleotides containing the 5'-XA-3', 5'-XC-3', 5'-XT-3', and 5'-XY-3' sequences (X = AFB(1)−FAPY; Y = 7-deaza-dG)demonstrate that the equilibrium between E and Z isomers is controlled by major groove hydrogen bonding interactions.Structural analysis of the adduct in the 5'-XA-3' sequence indicates the preference of the E isomer of the formamide group,attributed to formation of a hydrogen bond between the formyl oxygen and the N(6) exocyclic amino group of the 3'-neighboradenine. While the 5'-XA-3' sequence exhibits the E isomer, the 5'-XC-3' sequence exhibits a 7:3 E:Z ratio at equilibrium at 283K. The E isomer is favored by a hydrogen bond between the formyl oxygen and the N(4)-dC exocyclic amino group of the 3'-neighbor cytosine. The 5'-XT-3' and 5'-XY-3' sequences cannot form such a hydrogen bond between the formyl oxygen and the 3'-neighbor T or Y, respectively, and in these sequence contexts the Z isomer is favored. Additional equilibria between α and ß anomers and the potential to exhibit atropisomers about the C5−N(5) bond do not depend upon sequence. In each of the four DNA sequences, the AFB(1)−FAPY adduct maintains the ß deoxyribose configuration. Each of these four sequences feature the atropisomer of the AFB(1) moiety that is intercalated above the 5'-face of the damaged guanine. This enforces the Ra axialc onformation for the C5−N(5) bond.

Oncogenic and tumor suppressor pathways in subchronic aflatoxicosis in rats: Association with serum and urinary aflatoxin exposure biomarkers.

In this study, the changes in oncogenic and tumor suppressor signaling pathways in liver and their association with serum and urinary biomarkers of aflatoxin exposure were evaluated in Wistar rats fed diets containing aflatoxin B1 (AFB1) for 90 days. Rats were divided into four groups (n = 15 per group) and assigned to dietary treatments containing 0 (control), 50 (AFB50), 100 (AFB100) and 200 µg AFB1 kg-1 diet (AFB200). Multiple preneoplastic foci of hepatocytes marked with glutathione-S-transferase-placental form (GST-P) were identified in AFB100 and AFB200 groups. Hepatocellular damage induced by AFB1 resulted in overexpression of cyclin D1 and ß-catenin. The liver expression of retinoblastoma (Rb) and p27Kip1 decreased in AFB100 and AFB200 groups, confirming the favorable conditions for neoplastic progression to hepatocellular carcinoma. All samples from rats fed AFB1-contaminated diets had quantifiable AFB1-lysine in serum or urinary AFM1 and AFB1-N7-guanine, with mean levels of 20.42-50.34 ng mL-1, 5.31-37.68 and 39.15-126.37 ng mg-1 creatinine, respectively. Positive correlations were found between AFB1-lysine, AFM1 or AFB1-N7-guanine and GST-P+, ß-catenin+ and cyclin D1+ hepatocytes, while Rb + cells negatively correlated with those AFB1 exposure biomarkers. The pathways evaluated are critical molecular mechanisms of AFB1-induced hepatocarcinogenesis in rats.

The role of selected cytochrome P450 enzymes on the bioactivation of aflatoxin B1 by duck liver microsomes.

A study was conducted to determine the cytochrome (CYP) P450 enzymes responsible for the bioactivation of aflatoxin B1 (AFB1) into its epoxide form (AFBO) in duck liver microsomes. Six male and six female 6-week-old Pekin ducks were used. The biochemical toxicology strategies applied included the use of selective inhibitors, prototype substrate activity for specific human P450s, correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates, and the expression of specific CYP450 enzymes using antibodies against human CYP450s. Enzymatic activity was detected for the duck orthologues CYP1A1/2, CYP2A6 and CYP3A4 but not for the CYP2D6 orthologue. Immunoreactive proteins for CYP1A1, CYP2A6 and CYP3A4 were also detected. Inhibition studies suggested that the duck turkey CYP2A6 orthologue and, to a lesser extent, the CYP1A1 orthologue are involved in the bioactivation of AFB1. Correlation studies, however, suggest that CYP3A4, CYP2A6 and CYP1A1/2 are all involved in AFBO formation. The finding that four CYP enzymes may be involved in AFB1 bioactivation in ducks could explain the high sensitivity of this species to AFB1. Further studies are needed to fully elucidate the phase I hepatic metabolism of AFB1 in ducks, the only poultry species that develops hepatic cancer from AFB1 exposure.

Aflatoxin B1 albumin adducts in plasma and aflatoxin M1 in urine are associated with plasma concentrations of vitamins A and E.

BACKGROUND: Although aflatoxin exposure has been associated with micronutrient deficiency in animals, there are few investigations on the effects of aflatoxin exposure on micronutrient metabolism in humans. OBJECTIVE: To examine the relationship between aflatoxin B1 (AFB1) albumin adducts (AF-ALB) in plasma and the aflatoxin M1 (AFM1) metabolite in urine and plasma concentrations of retinol (vitamin A) and alpha-tocopherol (vitamin E) in Ghanaians. METHODS: A cross-sectional study of 147 adult participants was conducted. Blood and urine samples were tested for aflatoxin and vitamins A and E levels. RESULTS: Multivariable analysis showed that participants with high AF-ALB (>or=0.80 pmol/mg albumin) had increased odds of having vitamin A deficiency compared to those with lower AF-ALB [Odds Ratio (OR)=2.61; CI=1.03-6.58; p=0.04]. Participants with high AF-ALB also showed increased odds of having vitamin E deficiency but this was not statistically significant (OR=2.4; CI=0.96-6.05; p=0.06). Conversely, those with higher AFM1 values had a statistically nonsignificant reduced odds of having vitamin A deficiency (OR=0.31; CI=0.09-1.02; p=0.05) and a statistically significant reduced odds of having vitamin E deficiency (OR=0.31; CI=0.10-0.97; p=0.04). Participants with high AF-ALB or high AFM1 (>or=437.95 pg/dL creatinine) were almost 6 times more likely to be hepatitis B virus surface antigen (HBsAg)-positive (OR=5.88; CI=1.71-20.14; p=0.005) and (OR=5.84; CI=1.15-29.54; p=0.03) respectively. CONCLUSIONS: These data indicate that aflatoxin may modify plasma micronutrient status. Thus, preventing aflatoxin exposure may reduce vitamin A and E deficiencies.

Dealing with aflatoxin B1 dihydrodiol acute effects: Impact of aflatoxin B1-aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effects.

Aflatoxin B1 aldehyde reductase (AFAR) enzyme activity has been associated to a higher resistance to the aflatoxin B1 (AFB1) toxicity in ethoxyquin-fed rats. However, no studies about AFAR activity and its relationship with tolerance to AFB1 have been conducted in poultry. To determine the role of AFAR in poultry tolerance, the hepatic in vitro enzymatic activity of AFAR was investigated in liver cytosol from four commercial poultry species (chicken, quail, turkey and duck). Specifically, the kinetic parameters Vmax, Km and intrinsic clearance (CLint) were determined for AFB1 dialdehyde reductase (AFB1-monoalcohol production) and AFB1 monoalcohol reductase (AFB1-dialcohol production). In all cases, AFB1 monoalcohol reductase activity saturated at the highest aflatoxin B1 dialdehyde concentration tested (66.4 µM), whereas AFB1 dialdehyde reductase did not. Both activities were highly and significantly correlated and therefore are most likely catalyzed by the same AFAR enzyme. However, it appears that production of the AFB1 monoalcohol is favored over the AFB1 dialcohol. The production of alcohols from aflatoxin dialdehyde showed the highest enzymatic efficiency (highest CLint value) in chickens, a species resistant to AFB1; however, it was also high in the turkey, a species with intermediate sensitivity; further, CLint values were lowest in another tolerant species (quail) and in the most sensitive poultry species (the duck). These results suggest that AFAR activity is related to resistance to the acute toxic effects of AFB1 only in chickens and ducks. Genetic selection of ducks for high AFAR activity could be a means to control aflatoxin sensitivity in this poultry species.

Inherent stereospecificity in the reaction of aflatoxin B(1) 8,9-epoxide with deoxyguanosine and efficiency of DNA catalysis.

Kinetic analysis of guanine alkylation by aflatoxin B(1) exo-8,9-epoxide, the reactive form of the hepatocarcinogen aflatoxin B(1), shows the reaction to be >2000 times more efficient in DNA than in aqueous solution, that is, with free 2'-deoxyguanosine. Thermodynamic analysis reveals AFB(1) exo-8,9-epoxide intercalation as the predominant source of the observed DNA catalytic effect. However, the known exo > endo epoxide stereospecificity of the DNA alkylation is observed even with free deoxyguanosine (ratio >20:1 determined by LC-MS and NMR measurements), as predicted by theoretical calculations [ Bren , U. , et al. ( 2007 ) Chem. Res. Toxciol. 20 , 1134 - 1140 ].

An unusually high production of hepatic aflatoxin B1-dihydrodiol, the possible explanation for the high susceptibility of ducks to aflatoxin B1.

A study was conducted to determine the enzymatic kinetic parameters Vmax, KM, and intrinsic clearance (CLint) for the hepatic in vitro production of aflatoxin B1-dihydrodiol (AFB1-dhd) from aflatoxin B1 (AFB1) in four commercial poultry species, ranging in sensitivity to AFB1 from highest (ducks) to lowest (chickens). Significant but small differences were seen for Vmax, while large significant differences were observed for KM. However, the largest inter-species differences were observed for the CLint parameter, with ducks being extraordinarily efficient in converting AFB1 into AFB1-dhd. Since AFB1-dhd is considered the metabolite responsible for the acute toxic effects of AFB1, the high hepatic production of AFB1-dhd from AFB1 in ducks is the possible biochemical explanation for the extraordinary high sensitivity of this poultry species to the adverse effects of AFB1.

Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods.

Aflatoxins are a class of carcinogenic mycotoxins produced by Aspergillus fungi and are known to contaminate a large portion of the world's food supply. Aflatoxin B1 (AFB1) is the most potent of these compounds and has been well-characterized to lead to the development of hepatocellular carcinoma (HCC) in humans and animals. This review focuses on the metabolism of AFB1, including epoxidation and DNA adduction, as it concerns the initiation of cancer and the underlying mechanisms. The link between AFB1 consumption and HCC is also discussed including synergistic interactions with the hepatitis B virus. Toxic effects of AFB1, including growth suppression, malnutrition, and immunomodulation, are also covered. This review also describes recent reports of AFB1 occurrence in global food supplies and exposures in occupational settings. Furthermore, a summary of recent detoxification methods is included to indicate the present state of the field in developing aflatoxin control methods. This information shows that AFB1 occurs frequently in food supplies at high concentrations, particularly in maize. Regarding detoxification methods, chemical control methods were the fastest methods that still retained high detoxification efficacy. The information presented here highlights the need to implement new and/or existing detoxification methods to reduce the global burden of AFB1 toxicity.

Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat.

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B(1) (AFB(1))-induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB(1) to alter repair of AFB(1)-DNA damage was determined. Plasmid DNA containing AFB(1)-N(7)-guanine or AFB(1)-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and approximately 6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB(1)-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB(1) i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB(1)-N(7)-guanine damage was approximately 3.5-fold higher in liver of AFB(1)-treated mice relative to control. This is the first study to show that in vivo treatment with AFB(1) can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB(1), and selective induction of repair in liver contribute to the susceptibility of mice to AFB(1)-induced lung tumorigenesis relative to hepatocarcinogenesis.

Editor's Highlight: Pregnancy Alters Aflatoxin B1 Metabolism and Increases DNA Damage in Mouse Liver.

Pregnancy is a complex physiological state, in which the metabolism of endogenous as well as exogenous agents is ostensibly altered. One exogenous agent of concern is the hepatocarcinogen aflatoxin B1 (AFB1), a foodborne fungal toxin, that requires phase I metabolic oxidation for conversion to its toxic and carcinogenic form, the AFB1-8,9-exo-epoxide. The epoxide interacts with cellular targets causing toxicity and cell death; these targets include the covalent modification of DNA leading to mutations that can initiate malignant transformation. The main detoxification pathway of the AFB1-epoxide involves phase II metabolic enzymes including the glutathione-S-transferase (GST) family. Pregnancy can modulate both phase I and II metabolism and alter the biological potency of AFB1. The present work investigated the impact of pregnancy on AFB1 exposure in mice. A single IP dose of 6 mg/kg AFB1 was administered to pregnant C57BL/6 J mice at gestation day 14 and matched non-pregnant controls. Pregnant mice accumulated 2-fold higher AFB1-N7-guanine DNA adducts in the liver when compared with nonpregnant controls 6 h post-exposure. Enhanced DNA adduct formation in pregnant animals paralleled elevated hepatic protein expression of mouse CYP1A2 and mouse homologs of human CYP3A4, phase I enzymes capable of bioactivating AFB1. Although phase II enzymes GSTA1/2 showed decreased protein expression, GSTA3, the primary enzymatic protection against the AFB1-epoxide, was unaffected at the protein level. Taken together, our results reveal that pregnancy may constitute a critical window of susceptibility for maternal health, and provide insight into the biochemical factors that could explain the underlying risks.

Probiotic supplementation reduces a biomarker for increased risk of liver cancer in young men from Southern China.

BACKGROUND: In vitro and in vivo studies suggest that selected strains of probiotic bacteria can form tight complexes with aflatoxin B(1) and other carcinogens. OBJECTIVE: The aim of the present study was to determine whether administration of probiotic bacteria could block the intestinal absorption of aflatoxin B(1) and thereby lead to reduced urinary excretion of aflatoxin B(1)-N(7)-guanine (AFB-N(7)-guanine), a marker for a biologically effective dose of aflatoxin exposure. Elevated urinary excretion of this aflatoxin-DNA adduct is associated with an increased risk of liver cancer. DESIGN: Ninety healthy young men from Guangzhou, China, were randomly assigned to 2 groups; one group received a mixture of Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii subsp. shermanii strains 2 times/d for 5 wk, and the other group received a placebo preparation. The subjects provided 4 urine samples: at baseline, at 3 and 5 wk after starting the supplementation, and at the end of the 5-wk postintervention period. RESULTS: The percentage of samples with negative AFB-N(7)-guanine values tended to be higher in the probiotic group than in the placebo group during the 5-wk intervention period (odds ratio: 2.63, P = 0.052), and a statistically significant decrease in the concentration of urinary AFB-N(7)-guanine was observed in the probiotic group. The reduction was 36% at week 3 and 55% at week 5. The geometric means for the probiotic and placebo groups were 0.24 and 0.49 ng AFB-N(7)-guanine/mL, respectively, during the intervention period (P = 0.005). CONCLUSION: A probiotic supplement reduces the biologically effective dose of aflatoxin exposure and may thereby offer an effective dietary approach to decrease the risk of liver cancer.

Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots.

The major mutational hot spots in human cancers occur at CpG sequences in the p53 gene. It is generally presumed that the majority of mutations at these sites result from the endogenous deamination of methylated cytosine. Using a UvrABC incision method, we have found that cytosine methylation greatly enhances guanine alkylation at all CpG sites in the p53 gene by a variety of carcinogens, including benzo(a)pyrene diol epoxide, benzo(g)chrysene diol epoxide, aflatoxin B1 8,9-epoxide, and N-acetoxy-2-acetylaminofluorene. These findings suggest that mutational hot spots at methylated CpG sequences in the p53 gene may be a consequence of preferential carcinogen binding at these sites.

Molecular dosimetry in rat urine of aflatoxin-N7-guanine and other aflatoxin metabolites by multiple monoclonal antibody affinity chromatography and immunoaffinity/high performance liquid chromatography.

The development of molecular dosimetry methods will simplify the identification of people at high risk for cancer. A combined monoclonal antibody immunoaffinity chromatography/high performance liquid chromatography method has been devised to isolate and quantify aflatoxin-DNA adducts and other metabolites in rat urine samples. We report the production of 11 different monoclonal antibodies recognizing aflatoxin B1, aflatoxin Q1, aflatoxin G1, aflatoxicol, and aflatoxin M1 and the application of these antibodies to a multiple monoclonal antibody affinity chromatography technique. Using the multiple monoclonal antibody affinity column with rat urines obtained from dosed animals, between 90 and 95% of total aflatoxin metabolites can be bound to the column and isolated. Analytical immunoaffinity chromatography/high performance liquid chromatography analysis of these isolated aflatoxins reveals that more than 55% of the aflatoxins in rat urine are aflatoxin-dihydrodiol, aflatoxin-N7-guanine, aflatoxin Q1, aflatoxin M1, aflatoxin P1, and aflatoxin B1, accounting for 1.5, 9.6, 1.8, 34.5, 8.0, and 1.0% of the total aflatoxins, respectively. Further, a perchloric acid digestion of the aflatoxin-N7-guanine peak was used to confirm its identity by its conversion to guanine. The measurement of aflatoxin-N7-guanine excretion in rat urine was examined to assess its utility as a marker of DNA adduct formation in the liver, and a dose-dependent excretion in urine was found with a correlation coefficient of 0.99. A comparison of the dose-dependent residual levels of aflatoxin binding to liver DNA with the amount of aflatoxin-N7-guanine excreted in urine showed a correlation coefficient of 0.98. Besides the nucleic acid adduct excretion data, aflatoxin M1 and aflatoxin P1 were evaluated as molecular dosimeters in the urine. Aflatoxin M1 was found to be an excellent marker, whereas no linear relationship between dose and aflatoxin P1 excretion in urine was found.

Oltipraz-mediated changes in aflatoxin B(1) biotransformation in rat liver: implications for human chemointervention.

Oltipraz (OPZ) is currently being considered for human use to protect against aflatoxin B1 (AFB)-induced hepatocarcinogenesis based on its proven protective effect in rats. The effectiveness of this treatment presumes that orthologous cytochrome P450 and glutathione S-transferase (GST) isozymes metabolize AFB in humans as they do in rats. In this study, alterations in the expression of multiple forms of cytochrome P450 and GST were evaluated after treatment with OPZ, as well as other known P450 inducers, including 3-methylcholanthrene, pregnenolone-16alpha-carbonitrile, and ciprofibrate. Evidence is presented that the male-specific rat CYP 3A2, an orthologue of human CYP 3A4, may be primarily responsible for AFB activation in rat liver at both high and low AFB substrate concentrations. The CYP 1A2 enzyme does not appear to play a role in AFB activation in rat liver at any substrate concentration, whereas the major human P450 enzyme capable of activating AFB at a low substrate concentration has been identified as CYP 1A2. Surprisingly, we found that the CYP 1A2 steady-state mRNA level and the CYP 1A2-associated methoxyresorufin-O-demethylase activity were induced approximately 3- and 2-fold, respectively, by OPZ in rat liver. However, because CYP 1A2 does not appear to participate in AFB activation, induction of CYP 1A2 may be insignificant for AFB-induced hepatocarcinogenesis in rat models. In the rat, a heterodimeric alpha class GST enzyme containing the Yc2 subunit is the only polypeptide characterized to date in this species with high catalytic activity for the conjugation of activated AFB with glutathione. The GST Yc2 steady-state mRNA level was induced 5-fold by OPZ treatment. This induction was mirrored by significant increases in both the corresponding protein level and AFB-8,9-epoxide-conjugating enzyme activity, which may contribute significantly to protection against AFB-induced carcinogenesis in the rat. Investigations from this and other laboratories have not revealed any evidence for a Yc2-like GST isozyme with high AFB-8,9-epoxide-conjugating activity in human liver. We have also been unable to demonstrate that the two major human alpha class GST isozymes, A1-1 and A2-2, purified from bacteria expressing the corresponding cDNAs, exhibit any significant AFB-8,9-epoxide-conjugating activity. Our results suggest that humans may not be protected to the same extent as rats against AFB-induced hepatocarcinogenesis by treatment with OPZ and that further investigations are needed to establish the usefulness of OPZ for protection against human exposure to AFB.

Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1.

Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.

Assessment of aflatoxin exposure using serum and urinary biomarkers in São Paulo, Brazil: A pilot study.

The aim of this study was to evaluate the human exposure of individuals from Pirassununga, Brazil, to dietary aflatoxins B1 (AFB1) and M1 (AFM1) by determination of serum AFB1-lysine and urinary aflatoxin biomarkers (AFM1 and AFB1-N(7)-guanine). The participants were recruited among employees from a Campus of the University of São Paulo, which provided food samples from their homes, as well as serum and urine samples four times every three months, from June 2011 until March 2012. The probable daily intake (PDI) of aflatoxin was estimated by using the results from analysis of food products collected by the time of samples collection, and data from a 24-hour dietary recall questionnaire. Analyses of AFB1 and AFM1 in food samples were conducted by high-performance liquid chromatography with fluorescence detection. Biomarkers in serum and urine were determined by tandem mass spectrometry. AFB1 and AFM1 were detected in 38 samples of cereals (28%, N=136) and 31 milk products (36%, N=86), respectively. AFB1-lysine and AFB1-N(7)-guanine and were not detected in serum or urine samples, respectively. However, AFM1 was found in 74 urine samples (65%), at mean levels in the 4 sampling times ranging from 0.37±0.23 to 1.70±2.88pg/mg creatinine. The mean PDI varied among different sampling times, ranging from 0.09±0.09 to 1.35±5.98ng/kg body weight/day. A modest though significant correlation (r=0.45; p=0.03; N=23) was found for the first time in Brazil between the AFM1 concentration in urine and the PDI for total aflatoxins (AFB1+AFM1) in sampling 1 (June 2011). Urinary AFM1 was confirmed as very sensitive for monitoring the human exposure to dietary aflatoxin. Further studies using serum and urinary biomarkers are needed to estimate the aflatoxin exposure of populations in higher risk areas in Brazil.

Contribution of the glutathione S-transferases to the mechanisms of resistance to aflatoxin B1.

The harmful effects of Aflatoxin B1 (AFB1) are a consequence of it being metabolized to AFB1-8,9-epoxide, a compound that serves as an alkylating agent and mutagen. The toxicity of AFB1 towards different cells varies substantially; sensitivity can change significantly during development, can be modulated by treatment with xenobiotics and is decreased markedly in preneoplastic lesions as well as in tumors. Three types of resistance, namely intrinsic, inducible and acquired, can be identified. The potential resistance mechanisms include low capacity to form AFB1-8,9-epoxide, high detoxification activity, increase in AFB1 efflux from cells and high DNA repair capacity. Circumstantial evidence exists that amongst these mechanisms the glutathione S-transferases, through their ability to detoxify AFB1-8,9-epoxide, play a major role in determining the sensitivity of cells to AFB1.

Human variability in hepatic glutathione S-transferase-mediated conjugation of aflatoxin B1-epoxide and other substrates.

Hepatic cytosolic fractions prepared from 14 human donors were analysed for glutathione S-transferase (GST) activity towards synthetic aflatoxin B1-8,9-epoxide (AFBO). In addition, GST-AFBO activity of pooled human liver cytosols was compared with rat, hamster, and mouse liver cytosol GST-AFBO activities. Consistent with previous studies, human liver cytosolic GSTs exhibited little activity towards AFBO. Hepatic GST-AFBO activities of rat, hamster, and mouse were 48-, 56-, and 312-fold greater, respectively, than observed for human liver using synthetic AFBO, and 70-, 465-, and 3545-fold greater, respectively, than observed for human liver using microsomally-generated AFBO. Furthermore, there was a 58-fold variation in hepatic GST-AFBO activities among the 14 human samples using synthetic AFBO as a substrate. Large interindividual variations were also observed with respect to GST activities towards bromosulfophthalein (BSP, 92-fold variation) and 3,4-dichloronitrobenzene (DCNB, 36-fold variation). Lesser interindividual variations were observed with respect to human liver GST activities towards benzo(a)pyrene-4,5-oxide (BaPO, 9-fold variation), 1-chloro-2,4-dinitrobenzene (CDNB, 8.5-fold variation), cumene hydroperoxide (CHP, 5-fold variation), and p-nitrophenyl acetate (NPA, 4-fold variation). No correlation was found among GST-AFBO activities and the presence of GST mu as determined by enzyme-linked immunosorbent assay (ELISA) or GST-trans-stilbene oxide (TSO) catalytic activity. Our observations support those of previous studies indicating that human liver cytosolic GSTs are relatively ineffective at conjugating AFBO. Furthermore, our data indicate that humans exhibit large inter-individual differences with respect to hepatic cytosolic GST conjugation of AFBO and certain other GST substrates.