Metabolic Activation Of Aflatoxin B1 Research Articles

Frequencies and spectra of aflatoxin B1-induced mutations in liver genomes of NEIL1-deficient mice as revealed by duplex sequencing.

Increased risk for the development of hepatocellular carcinoma (HCC) is driven by a number of etiological factors including hepatitis viral infection and dietary exposures to foods contaminated with aflatoxin-producing molds. Intracellular metabolic activation of aflatoxin B1 (AFB1) to a reactive epoxide generates highly mutagenic AFB1-Fapy-dG adducts. Previously, we demonstrated that repair of AFB1-Fapy-dG adducts can be initiated by the DNA glycosylase NEIL1 and that male Neil1-/- mice were significantly more susceptible to AFB1-induced HCC relative to wild-type mice. To investigate the mechanisms underlying this enhanced carcinogenesis, WT and Neil1-/- mice were challenged with a single, 4 mg/kg dose of AFB1 and frequencies and spectra of mutations were analyzed in liver DNAs 2.5 months post-injection using duplex sequencing. The analyses of DNAs from AFB1-challenged mice revealed highly elevated mutation frequencies in the nuclear genomes of both males and females, but not the mitochondrial genomes. In both WT and Neil1-/- mice, mutation spectra were highly similar to the AFB1-specific COSMIC signature SBS24. Relative to wild-type, the NEIL1 deficiency increased AFB1-induced mutagenesis with concomitant elevated HCCs in male Neil1-/- mice. Our data establish a critical role of NEIL1 in limiting AFB1-induced mutagenesis and ultimately carcinogenesis.

Following the adverse outcome pathway from micronucleus to cancer using H2B-eGFP transgenic healthy stem cells

In vitro assessment of genotoxicity as an early warning tool for carcinogenicity mainly relies on recording cytogenetic damages (micronuclei, nucleoplasmic bridges) in tumour-derived mammalian cell lines like V79 or CHO. The forecasting power of the corresponding standardised test is based on epidemiological evidence between micronuclei frequencies and cancer incidence. As an alternative to destructive staining of nuclear structures a fish stem cell line transgenic for a fusion protein of histone 2B (H2B) and enhanced green fluorescent protein (eGFP) was established. The cells are derived from koi carp brain (KCB) and distinguish from mammalian culturable cells by non-tumour-driven self-renewal. This technology enables the analysis of genotoxic- and malign downstream effects in situ in a combined approach. In proof-of concept-experiments, we used known carcinogens (4-Nitroquinoline 1-oxide, colchicine, diethylstilbestrol, ethyl methanesulfonate) and observed a significant increase in micronuclei (MNi) frequencies in a dose-dependent manner. The concentration ranges for MNi induction were comparable to human/mammalian cells (i.e. VH-16, CHL and HepG2). Cannabidiol caused the same specific cytogenetic damage pattern as observed in human cells, in particular nucleoplasmic bridges. Metabolic activation of aflatoxin B1 and cyclophosphamide could be demonstrated by pre-incubation of the test compounds using either conventional rat derived S9 mix as well as an in vitro generated biotechnological alternative product ewoS9R. The presented high throughput live H2B-eGFP imaging technology using non-transformed stem cells opens new perspectives in the field of in vitro toxicology. The technology offers experimental access to investigate the effects of carcinogens on cell cycle control, gene expression pattern and motility in the course of malign transformation. The new technology enables the definition of Adverse Outcome Pathways leading to malign cell transformation and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is suggested. A vertebrate cell model showing "healthy" stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology.

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.

Cytochrome P450 2A13 is an efficient enzyme in metabolic activation of aflatoxin G1 in human bronchial epithelial cells

Cytochrome P450 2A13 (CYP2A13) is an extrahepatic enzyme that mainly expresses in human respiratory system, and it is reported to mediate the metabolic activation of aflatoxin B1. Due to the structural similarity, AFG1 is predicted to be metabolized by CYP2A13. However, the role of CYP2A13 in metabolic activation of AFG1 is unclear. In present study, human bronchial epithelial cells that stably express CYP2A13 (B-2A13) were used to conduct the effects of AFG1 on cytotoxicity, apoptosis, DNA damages, and their response protein expression. Low concentrations of AFG1 induced significant cytotoxicity and apoptosis, which was consistent with the increased expressions of pro-apoptotic proteins, such as C-PARP and C-caspase-3. In addition, AFG1 increased 8-OHdG and γH2AX in the nuclies and induced S phase arrest and DNA damage in B-2A13 cells, and the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and γH2AX, were activated. All the above effects were inhibited by nicotine (a substrate of CYP2A13) or 8-MOP (an inhibitor of CYP enzymes), confirming that CYP2A13 mediated the AFG1-induced cytotoxicity and DNA damages. Collectively, our findings first demonstrate that CYP2A13 might be an efficient enzyme in metabolic activation of AFG1 and helps provide a new insight into adverse effects of AFG1 in human respiratory system.

Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100μM nicotine (a substrate of CYP2A13) or 1μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system.

Metabolism Comparative Cytotoxicity Assay (MCCA) and Cytotoxic Metabolic Pathway Identification Assay (CMPIA) with cryopreserved human hepatocytes for the evaluation of metabolism-based cytotoxicity in vitro: Proof-of-concept study with aflatoxin B1

Recently, we have improved the cryopreservation procedures for human hepatocytes, leading to cells that can be cultured after thawing (“plateable” cryopreserved human hepatocytes). The ability to culture cryopreserved human hepatocytes allows application of the cells for prolonged incubations such as long-term (days) metabolism studies, enzyme induction studies, and cytotoxicity studies. We report here the application of the plateable cryopreserved human hepatocytes to evaluate the relationship between xenobiotic metabolism and toxicity. Two assays were developed: The Metabolism Comparative Cytotoxicity Assay (MCCA) and the Cytotoxic Metabolic Pathway Identification Assay (CMPIA). The MCCA was designed for the initial identification of the role of metabolism in cytotoxicity by comparing the cytotoxic potential of a toxicant in a metabolically competent (primary human hepatocytes) and a metabolically incompetent (Chinese hamster ovary (CHO)) cell type, as well as the evaluation of the role of P450 metabolism by comparing the cytotoxicity of the toxicant in question in human hepatocytes in the presence and absence of a nonspecific, irreversible P450 inhibitor, 1-aminobenzotriazole (ABT). The CMPIA was designed for the identification of the P450 isoforms involved in metabolic activation via the evaluation of the cytotoxicity of the toxicant in the presence and absence of isoform-selective P450 inhibitors. Results of a proof-of-concept study with the MCCA and CMPIA with a known hepatotoxicant, aflatoxin B1 (AFB1), are reported. AFB1 is known to require P450 metabolism for its toxicity. In the MCCA, AFB1 was found to have significantly higher cytotoxicity in human hepatocytes than CHO cells, therefore confirming its requirement for biotransformation to be toxic. ABT was found to effectively attenuate AFB1 cytotoxicity, confirming that P450 metabolism was involved in its metabolic activation. In the CMPIA, AFB1 cytotoxicity was found to be attenuated by ketoconazole and diethyldithiocarbamate, but not by furafylline, quinidine, and sulfaphenazole. Results with the isoform-selective inhibitors suggest that the isoforms inhibited by ketoconazole (mainly CYP3A4) and diethyldithiocarbamate (mainly CYP2A6, and CYP2E1), but not the isoforms inhibited by furafylline (mainly CYP1A2), sulfaphenazole (mainly CYP2C9) and quinidine (mainly CYP2D6) are involved in the metabolic activation of AFB1. This proof-of-concept study suggests that MCCA and CMPIA with cryopreserved human hepatocytes are potentially useful for the evaluation of the relationship between human xenobiotic metabolism and toxicity.

Structure of the Human Lung Cytochrome P450 2A13

The human lung cytochrome P450 2A13 (CYP2A13) activates the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into DNA-altering compounds that cause lung cancer. Another cytochrome P450, CYP2A6, is also present in human lung, but at much lower levels. Although these two enzymes are 93.5% identical, CYP2A13 metabolizes NNK with much lower K(m) values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35A by x-ray crystallography and compared with structures of CYP2A6. As expected, the overall CYP2A13 and CYP2A6 structures are very similar with an average root mean square deviation of 0.5A for the Calpha atoms. Like CYP2A6, the CYP2A13 active site cavity is small and highly hydrophobic with a cluster of Phe residues composing the active site roof. Active site residue Asn(297) is positioned to hydrogen bond with an adventitious ligand, identified as indole. Amino acid differences between CYP2A6 and CYP2A13 at positions 117, 300, 301, and 208 relate to different orientations of the ligand plane in the two protein structures and may underlie the significant variations observed in binding and catalysis of many CYP2A ligands. In addition, docking studies suggest that residues 365 and 366 may also contribute to differences in NNK metabolism.

Efficient activation of aflatoxin B1 by cytochrome P450 2A13, an enzyme predominantly expressed in human respiratory tract

The worldwide human exposure to aflatoxin B1 (AFB1), particularly in developing countries, remains to be a serious public health concern. Although AFB1 is best known as a hepatocarcinogen, epidemiological studies have shown a positive association between human lung cancer occurrence and inhalation exposure to AFB1. Cytochrome P450 (CYP)-catalyzed metabolic activation is required for AFB1 to exert its carcinogenicity. Previous studies have identified CYP1A2 and CYP3A4 as the major enzymes for AFB1 activation in human liver. However, the key CYP enzymes in human lung that can efficiently activate AFB1 in situ are unknown. In the present study, we demonstrate that CYP2A13, an enzyme predominantly expressed in human respiratory tract, has a significant activity in metabolizing AFB1 to its carcinogenic/toxic AFB1-8,9-epoxide and AFM1-8,9-epoxide at both low (15 microM) and high (150 microM) substrate concentrations. Under the same conditions, there was no detectable AFB1 epoxide formation by CYP2A6, which was also reported to be involved in the metabolic activation of AFB1. Consistent with the activity data, there was an approximately 800-fold difference in LC50 values of AFB1 (48-hr treatment) between Chinese hamster ovary (CHO) cells expressing CYP2A13 and CYP2A6 (50 nM versus 39 microM). We further demonstrate that amino acid residues Ala117 and His372 in CYP2A13 protein are important for AFB1 epoxidation and its related cytotoxicity. Our results suggest that CYP2A13-catalyzed metabolic activation in situ may play a critical role in human lung carcinogenesis related to inhalation exposure to AFB1.

Cytochrome P450 expression and related metabolism in human buccal mucosa.

Constituents in food and fluids, tobacco chemicals and many drugs are candidates for oral absorption and oxidative metabolism. On this basis, the expression of cytochrome P450 isozymes (CYPs) and the conversion of CYP substrates were analysed in reference to buccal mucosa. A RT-PCR based analysis of human buccal tissue from 13 individuals demonstrated consistent expression of mRNA for the CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5. CYP 2D6 was expressed in six out of the 13 specimens, whereas all samples were negative for 2A6 and 2B6. Serum-free monolayer cultures of the Siman virus 40 large T-antigen-immortalized SVpgC2a and the carcinoma SqCC/Y1 buccal keratinocyte lines expressed the same CYPs as tissue except 3A4/7 and 3A5 (SVpgC2a), and 2C, 2D6 and 3A4/7 (SqCC/Y1). Dealkylation of ethoxyresorufin and methoxyresorufin in both normal and transformed cells indicated functional 1A1 and 1A2, respectively. SVpgC2a showed similar activity as normal keratinocytes for both substrates, whereas SqCC/Y1 showed about 2-fold lower 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation activities. SVpgC2a showed detectable and many-fold higher activity than the other cell types towards chlorzoxazone, a substrate for 2E1. Absent or minute catalytic activity of 2C9, 2D6 and 3A4 in the various cell types was indicated by lack of detectable diclofenac, dextromethorphan and testosterone metabolism (<0.2-0.5 pmol/min/mg). Metabolic activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the mycotoxin aflatoxin B1 (AFB1) to covalently bound adducts was indicated by autoradiographic analysis of both monolayer and organotypic cultures of SVpgC2a. In contrast, SqCC/Y1 showed lower or absent metabolic activity for these substrates. Finally, measurements of various non-reactive AFB1 metabolites indicated rates of formation <0.1 pmol/min/mg in both normal and transformed cells. The results indicate presence of several CYPs of which some may contribute to significant xenobiotic metabolism in human buccal epithelium. Notably, metabolic activation of AFB1 was not previously implicated for oral mucosa. Further, the results show that CYP-dependent metabolism can be preserved or even activated in immortalized keratinocytes. Metabolic activity in SVpgC2a under both monolayer and organotypic culture conditions suggests that this cell line may be useful to pharmaco-toxicological and carcinogenesis studies.

IN VITRO INHIBITION OF CARCINOGEN-INDUCED MUTAGENICITY BY CASSIA OCCIDENTALIS AND EMBLICA OFFICINALIS

Aqueous extracts of Cassia occidentalis Linn. (Leguminoceae) and Emblica officinalis Gaertn. (Euphorbiaceae) were screened for effectiveness in inhibiting mutagenicity of aflatoxin B1 (AFB1) and benzo[a]-pyrene (B[a]P) in the Ames test. Antimutagenicity was evaluated using Salmonella typhimurium strains TA 98 and TA 100. In the assay, metabolic activation of AFB1 (0.5 μg/plate) and B[a]P (1 μg/plate) was mediated by rat liver S9 preparation. Although both plants inhibited mutagenicity, E. officinalis had more inhibitory effect than C. occidentalis. Their action is possibly mediated through interactions with micro somal activating enzymes. Their inhibitory action on chromosomal aberrations together with present results suggest that these plants have potent antimutagenic and anticarcinogenic activities against mutagens requiring metabolic activation.

Metabolic activation of aflatoxin B1 to aflatoxin B1-8,9-epoxide in woodchucks undergoing chronic active hepatitis.

Chronic hepatitis B virus infection as well as consumption of food contaminated with the mycotoxin aflatoxin B1 are considered to be 2 major risk factors for the development of primary liver cancer in humans. Furthermore, epidemiological surveys indicate that hepatitis B virus and aflatoxin B1 might act synergistically to induce primary liver cancer. In the present study, we have tested the hypothesis that the metabolic activation of aflatoxin B1 to aflatoxin B1-8,9-epoxide, the ultimate mutagenic and carcinogenic mycotoxin metabolite, is enhanced in an experimental model of chronic hepatitis using woodchucks, chronically infected with the woodchuck hepatitis virus. Woodchuck liver microsomes were incubated with radiolabeled aflatoxin B1, the resulting aflatoxin B1-8,9-epoxide was trapped as a glutathione conjugate and its formation rate was determined by a reversed-phase HPLC analysis. In woodchuck hepatitis virus-positive woodchucks, activation of aflatoxin B1 to aflatoxin B1-8,9-epoxide was reduced when compared to woodchuck hepatitis virus-free animals, and the extent of the reduction was dependent on the severity of the hepatitis. Hence, at least in woodchucks, a chronic hepadnaviral infection does not lead to an enhanced activation of aflatoxin B1.

Food Restriction Reduces Aflatoxin B1 (AFB1)-DNA Adduct Formation, AFB1-Glutathione Conjugation, and DNA Damage in AFB1-Treated Male F344 Rats and B6C3F1 Mice ,

The objective of this study was to examine effects of food restriction (FR) on the metabolic activation of aflatoxin B1 (AFB1) in rats and mice, which are AFB1-sensitive and -resistant rodent species, respectively. Forty percent FR [60% of ad libitum (AL) food consumption] reduced the metabolic activation of AFB1 in both rats and mice, causing formation of hepatic AFB1-DNA adducts to be 43% and 31% lower, respectively. The AFB1-DNA adduct 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) was predominantly formed in rat liver DNA; the formation of the ring-open analogue of AFB1-N7-Gua, AFB1-formamidopyrimidine (AFB1-FAP), was predominantly found in mouse liver DNA. In contrast to the in vivo results, the in vitro AFB1-DNA adduct formation mediated by the microsomes of liver, kidney or lung from FR-mice was greater than the formation of AFB1-DNA adducts mediated by the tissue microsomes from the AL-mice. Food restriction induced hepatic glutathione S-transferase (GST) activity, as measured by the formation of AFB1-glutathione conjugates (AFB1-SG), in both rats and mice; AFB1-SG was also formed in mouse kidney. Food restriction–induced GST activity assayed in an in vitro system, using [3H]AFB1-8,9-epoxide and glutathione (GSH) as substrates, was also found when mouse kidney and lung cytosolic fractions were used. Food restriction inhibited the AFB1-induced DNA double strand breaks in mouse kidney. The reduction of levels of AFB1-DNA adduct formation in mouse kidney was comparable to the degree of AFB1-induced DNA strand breakages. The results of this study indicate that the metabolic activation of AFB1 can be modulated by FR through the alteration of the formation of AFB1-DNA adducts and AFB1-SG conjugation. However, species and tissue specificities exist regarding the metabolic activation of AFB1.

Effect of dietary restriction on partial hepatectomy-induced liver regeneration of aged F344 rats.

Fourteen weeks-old male F344 rats maintained on a reduced caloric diet (60% of ad libitum (AL) food consumption) for 6 weeks or for 14 months did not affect the hepatic cell proliferation in terms of % S phase population, determined by evaluation of DNA synthesis in hepatocytes isolated from either young (5 months) or aged (18 months) rats. However, hepatic basal cellular DNA synthesis estimated by [3H]thymidine incorporation was reduced through acute dietary restriction (DR) in young rats, but increased in aged animals after 14 months restriction. Partial hepatectomy (PH) on aged rats stimulated hepatocyte regeneration and restored some aging-associated biochemical functions, such as drug metabolizing enzyme-dependent xenobiotic metabolic activation which was determined by measuring the formation of carcinogen-DNA adducts. Forty-eight hours after partial hepatectomy, the % of S phase population and the basal nuclear DNA synthesis of hepatocytes isolated from the partial hepatectomized DR-rats were 4- and 2.8-fold, respectively, greater than those of hepatocytes from AL-animals. DR reduced aflatoxin B1 (AFB1) metabolizing enzyme activity and decreased the AFB1-DNA adduct formation in young rats treated with AFB1. In aged AL-rats, the formation of AFB1-DNA adducts diminished to the same level as that of DR-groups and probably was due to the faster decline of drug metabolizing enzymes in aging AL-rats. However, 48 h after PH, the metabolic activation of AFB1 was restored in AL- and DR-groups which resulted in the increase of AFB1-DNA binding by 4.2 and 1.9-fold, respectively. During the liver regeneration of old PH-rats, DR inhibited the AFB1-DNA adduct formation after the PH-rats received a single dose of AFB1. DR increased benzo[a]pyrene (BaP) metabolic activation in both young and aged rats. Aging also decreased BaP-DNA adduct formation in both DR and AL-rats. The increase of BaP-DNA adduct formation in PH-groups was attributed to the restoration of BaP-metabolizing enzyme activity during liver regeneration. The PH-stimulated BaP-DNA adduct formation in AL- and DR-rats was 3.4- and 2.0-fold greater than control aged rats. Our results indicated that the stimulation of PH-induced liver regeneration by DR in aged animals may be attributed to the retardation of aging by DR and the retention of more active biochemical and enzymological functions in old DR-animals.

Metabolism of aflatoxin B1 by rabbit and rat nasal mucosa microsomes and purified cytochrome P450, including isoforms 2A10 and 2A11.

The nasal mucosa of some mammalian species are susceptible to the toxicity of aflatoxin B1 (AFB1), a potent hepatocarcinogen, but little is known about the nasal enzymes involved in the metabolic activation of AFB1 or the metabolites produced. In the present study, the metabolism of AFB1 was studied with nasal microsomes from rats and rabbits and with several purified isozymes of rabbit P450 in a reconstituted enzyme system. The rates of AFB1-N7-guanine DNA adduct formation with rabbit and rat nasal microsomes are over 3- and 10-fold higher, respectively, than with liver microsomes from the same species. On the other hand, the rates of formation of AFM1 (9a-hydroxy-AFB1) and AFQ1 (3-hydroxy-AFB1) products known to be less toxic, are lower with nasal than with liver microsomes. Of particular interest, nasal microsomes produce high levels of six unidentified polar metabolites that are not formed by microsomes from liver or several other tissues. These same products are also generated by P450 NMa purified from rabbit nasal microsomes in a reconstituted system, but not by five other isozymes of cytochrome P450 (1A2, 2B4, 2E1, 2G1, 3A6) that are known to be present in nasal microsomes. AFB1-DNA adducts are formed by P450 NMa at a rate 3-fold higher than that by nasal microsomes. The DNA adducts are formed at much slower rates by P450s 2G1, 2B4, and 1A2, and adducts are not formed at measurable rates by P450s 2E1 and 3A6. Moreover, AFB1-DNA adduct formation is also catalyzed by cDNA-derived, heterologously expressed P450s 2A10 and 2A11, both of which are known to be present in the purified P450 NMa preparation. The Km and Vmax values of the two isozymes for DNA adduct formation are comparable to those for nasal microsomes. Furthermore, the formation of AFB1-DNA adducts by nasal microsomes is decreased by nicotine, a known inhibitor of P450 NMa. These data indicate that members of the P450 2A gene subfamily play an important role in the metabolic activation of AFB1 in rabbit and rat nasal mucosa and suggest a molecular basis for assessing the health risk associated with inhalation exposure to this procarcinogen in humans.

Effect of caloric restriction on the metabolic activation of xenobiotics.

The effect of caloric restriction (CR) on xenobiotic metabolizing enzyme activities results in alterations in the metabolic activation of chemical carcinogens, with a resultant impact on DNA-carcinogen adduct formation and DNA repair. Using aflatoxin B1 (AFB1) and benzo[a]pyrene (BP) as model carcinogens, we studied the effect of CR on the metabolic activation of these carcinogens and carcinogen-induced DNA damage and repair in terms of AFB1-DNA and BP-DNA adduct formation and removal. Male Fischer 344 rats fed calorie restricted diets (60% of the food consumption for ad libitum-fed rats) showed a reduction in the metabolic activation of AFB1 and decrease in both the in vitro and in vivo AFB1-DNA adduct formation. However, CR increased the activity of BP metabolizing enzymes resulting in an enhancement of BP-DNA adduct formation. Our results indicate that the effect of CR on metabolic activation of xenobiotics is dependent upon the selected xenobiotic metabolizing enzymes whose activities may be significantly altered by CR, and upon the nature of the chemical carcinogens which exert different structure-activity relationships during the process of chemically induced carcinogenesis.

Metabolic activation of aflatoxin B1 by human placental microsomes.

The metabolic activation of aflatoxin B1 by human placental microsomes was studied. Aflatoxin B1 showed relatively high mutagenic activity in Ames test when incubated with human placental microsomes. Addition of alpha-naphthoflavone or aminoglutethimide, known inhibitors of cytochrome P450 1A and P450 19, respectively, into the test system partially inhibited the mutagen-producing activity. It was suggested that the activation of aflatoxin B1 in human placental microsomes is mediated by at least these two forms of cytochrome P450.

Human fetal liver cytochrome P-450: capacity to form genotoxic metabolites.

Unlike most experimental animals, human fetal liver possesses forms of cytochrome P-450. Thus, the purpose of this study was to clarify the toxicological significance of these forms of cytochrome P-450 to understand possible roles of these cytochromes in producing genotoxic metabolites from promutagens. In fact, human fetal livers showed considerable capacity to activate aflatoxin B1 and IQ (2-amino-3-methylimidazo [4,5-f] quinoline). Three of four forms of cytochrome P-450, P-450HFLa-d, which we could purify from human fetal livers were capable of activating promutagens to mutagens. One of these three forms, namely P-450HFLa, catalyzed the metabolic activation of aflatoxin B1 and IQ. An expression plasmid containing HFL33 cDNA encoding P-450HFLa was constructed and the protein expressed in insect (Sf9) cells and in human cancer cells, MCF-7. Aflatoxin B1 was efficiently activated to a mutagen upon addition of the lysate of Sf9 cells to the incubation mixture for the assay. Transformants of MCF-7 cells expressing P-450IIIA7 (HFLa) showed higher sensitivity to aflatoxin B1 than the parental MCF-7 cells as detected by cytotoxicity.

Liver DNA adducts in methyl-deficient rats administered a single dose of aflatoxin B1.

Using an 8 week Solt-Farber protocol with selection pressure (2-acetylaminofluorene/partial hepatectomy) applied during weeks 6 and 7, we have observed that a single oral administration of aflatoxin B1 (AFB1) to Fischer 344 rats on day 1 of the study, followed by a 3 week feeding regimen of either a methyl-deficient (CMD) or a basal (CMS) diet, results in a relative increase in hepatic preneoplastic lesions in CMD diet fed rats. It has previously been shown that a multiple dosing regimen with AFB1, started after 3 weeks of CMD diet, enhances tumor incidence. In the present study, the role of metabolic activation in the induction of preneoplastic lesions, and liver DNA adduct levels after the first dose of AFB1 in the tumorigenesis model have been investigated. AFB1-DNA adducts were determined at 2-168 h following a single non-necrogenic (100 micrograms/kg body wt) or necrogenic (600 micrograms/kg body wt) dose of AFB1 on day 1 or day 21 of a 3 week treatment with a complete basal or CMD diet. In all rats irrespective of dose, dietary treatment or time of AFB1 dosing, the patterns of adduct formation and repair did not change. In rats receiving AFB1 on day 1, total DNA adduct levels between the diet or dose groups were not significantly different, and quantitatively did not correlate with the observed increase in preneoplastic lesions, suggesting a contribution by additional factors in the initiation of these lesions. Administration of AFB1 on day 21, however, resulted in significantly reduced levels of total adducts at both dose levels in CMD diet fed rats compared to controls. Serum biochemistry data suggest that a prolonged exposure to CMD diet may cause pathological and/or biochemical alterations in hepatocytes with a resultant decrease in metabolic activation of AFB1, thus making it difficult to evaluate whether DNA damage is directly related to tumorigenesis.

DNA binding, adduct characterisation and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal, Kupffer and endothelial cells

In vitro studies with rat liver parenchymal, Kupffer and endothelial cells isolated from male Sprague-Dawley rats were undertaken to investigate cell-specific bioactivation of aflatoxin B1, DNA binding and adduct formation. In the mutagenicity studies, using homogenates of all three separated liver cell populations (co-incubated with NADP+ and glucose-6-phosphate as cofactors for the cytochrome P-450 monooxygenase system) parenchymal, Kupffer and endothelial cells were able to activate aflatoxin B1 to a metabolite mutagenic to Salmonella typhimurium TA 98. In the case of nonparenchymal cells (i.e. Kupffer and endothelial cells) 10-fold higher concentrations of aflatoxin B1 had to be used to obtain a similar number of revertants to that observed with parenchymal cells. Induction studies with Aroclor 1254 led to a striking decrease in the activation of aflatoxin B1 in parenchymal cells, whereas nonparenchymal cells had a slightly enhanced metabolic activation capacity for aflatoxin B1. Metabolism studies with microsomes from induced and noninduced cells using testosterone as substrate revealed comparable results: after induction with Aroclor 1254, parenchymal cells showed a 60% decrease in the formation rate of 2 alpha-hydroxytestosterone, whereas the formation rate of this metabolite remained unchanged in nonparenchymal cells; 2 alpha-hydroxytestosterone is specifically formed by cytochrome P-450 IIC11, which also catalyses the activation of aflatoxin B1 to its epoxide. When freshly isolated, intact cells were incubated with tritiated aflatoxin B1, a dose-dependent aflatoxin B1 binding to DNA in parenchymal and nonparenchymal cells was observed. HPLC analysis of DNA acid hydrolysates of all three cell types showed the major adduct to be 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1.

Activation of the hepatocarcinogen aflatoxin B1 by forms of human hepatic cytochromes P-450: Some do and some don't

Twelve forms of human hepatic cytochrome P-450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P-450's were analyzed for their abilities to activate the potent hepatocarcinogens aflatoxin B1 to metabolites having mutagenic or DNA-binding properties. Five forms, P-450's IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B1 to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P-450's catalyzed conversion of aflatoxin B1 to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P-450 forms. Seven other human P-450's, IIC8, IIC9, IID6, IIE1, IIIA5, and IVB1, did not significantly activate aflatoxin B1 as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P-450 antibodies that crossreact with individual human P-450's IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B1 activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish the metabolic activation of aflatoxin B1 in human liver involves the contribution of multiple forms of P-450.