Structural characterization of the major adducts obtained after reaction of an ultimate carcinogen aflatoxin B1-dichloride with calf thymus DNA in vitro

The major adduct formed on acid hydrolysis of calf thymus DNA which has been reacted with 8,9-dichloro-8,9-dihydroaflatoxin B1, a chemical model of the ultimate carcinogen 8,9-dihydro-8,9-epoxyaflatoxin B1 (AFB1-epoxide), has been characterized by proton nuclear magnetic resonance and fast atom bombardment mass spectroscopy. This adduct has been identified as an N7-substituted guanine adduct analogous to that formed on reaction of AFB1-8,9-epoxide with DNA in vivo and in vitro, namely trans-8,9-dihydro-8-(7-guanyl)-9-hydroxy AFB1. This 8,9-dichloro-8,9-dihydroaflatoxin B1 adduct in DNA, like its equivalent B1 adduct in DNA, like its equivalent AFB1-epoxide adduct, is prone to quantitative imidazole ring opening of the substituted guanine in mildly alkaline conditions and to substantial depurination under mildly acidic conditions.     

In vivo formation of aflatoxin B1-DNA adducts in parenchymal and non-parenchymal cells of rat liver.

The induction of hepatocellular carcinoma from liver parenchymal cells in laboratory animals by aflatoxin B1 (AFB1) is well documented. In contrast no tumours arising from the sinusoidal cell population have been reported after exposure to AFB1. The apparent resistance of the latter cell type was investigated at the level of DNA adduct formation in vivo in male Sprague-Dawley rats. Liver parenchymal and non-parenchymal cell populations were isolated from rats at 20 min and 1, 24 and 72 h after administration of 240 microCi (0.6 mg) [G-3H]AFB1/kg. AFB1-DNA binding was observed in both liver cell subpopulations and was 3- to 5-fold higher in parenchymal cells than in non-parenchymal cells. The major DNA adduct found in parenchymal cells at 1 h after AFB1 administration was 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-gua), whereas at later time points the persistent secondary adduct, AFB1-formamidopyrimidine, predominated. In contrast, AFB1-gua was not observed at any time in DNA from non-parenchymal cells and the secondary adducts predominated throughout. These observations are discussed with reference to the susceptibility of different liver cell types to AFB1-carcinogenesis and the possible roles of the major AFB1-DNA adduct species.     

Comparative formation and removal of aflatoxin B1-DNA adducts in cultured mammalian tracheal epithelium

Aflatoxin B1 (AFB1) DNA binding, adduct formation, and AFB1-DNA adduct repair were studied in tracheal explants from rabbit, hamster, and rat. These species vary in populations of cytochrome P-450-containing nonciliated tracheal epithelial cells. Explants were cultured in media containing 0.5 microM AFB1 for 12 h. After the 12-h treatment, the explants were cultured for time intervals up to 84 h and then analyzed for AFB1-DNA adducts. Binding of AFB1 to DNA was highest in rabbit tracheal explants (78 pmol/mg DNA), followed by the hamster (28 pmol/mg DNA), with the rat (3 pmol/mg DNA) showing minimal AFB1-DNA binding. Repair rates in the hamster and rat were constant over time with removal of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 accounting for the majority of adduct disappearance. The rabbit demonstrated biphasic repair of adducts; all adduct types [8,9-dihydro-8-(2-amino-6-formamido-4-oxo-3,4-dihydropyrimid-5- ylamino)-9- hydroxyaflatoxin B1] were rapidly removed during the first 12 h posttreatment with AFB1, followed by a slower removal phase of primarily 8,9-dihydro-8-N7-guanyl)-9-hydroxyaflatoxin B1. After 84 h, 90, 72, and 55% of the initial adducts were removed in the rabbit, hamster, and rat, respectively. Labeled thymidine studies showed that cells of the tracheal epithelium did not turn over sufficiently to bias the apparent repair rates. These results demonstrated that carcinogen activation and repair capabilities of tracheal epithelium vary among species and that these processes likely relate to the presence of smooth endoplasmic reticulum containing nonciliated tracheal epithelial cells in those species.     

Investigation of covalent aflatoxin B1-DNA adducts formed in vivo in rainbow trout (Salmo gairdneri) embryos and liver

The formation of covalent aflatoxin-DNA adducts has been studied in embryo and yearling stages of the rainbow trout (Salmo gairdneri). A linear relationship was found between the amount of aflatoxin B1 (AFB1) absorbed into 21-day-old eggs and the level of covalent modification of embryo DNA after exposure to 0.25 to 0.5 p.p.m. aqueous solutions of AFB1 for 1 h; higher concentrations resulted in absorption of a greater proportion of AFB1. The principal covalent product, identified after acid and enzymatic hydrolysis of isolated embryo DNA, was chromatographically identical to 2,3-dihydro-2-(N7-guanyl)-3-hydroxy aflatoxin B1. Additional AFB1 derivatives which were present are believed to be formed by chemical transformation of this product in DNA producing an aflatoxin-formamidopyrimidine adduct and the metabolic activation of other aflatoxin metabolites, such as aflatoxin M1 and aflatoxin P1. Although the eggs were exposed to AFB1 for a short time, covalent modification of DNA was evident over an extended period. The highest level of covalent products was present at approximately 24 h after exposure to 0.5 p.p.m. AFB1. Ninety-six hours later, this level had decreased slightly; however, the distribution of covalent adducts had changed: formamidopyrimidine adducts now represented up to 50% of the hydrolyzed aflatoxin derivatives. A similar pattern of covalent aflatoxin derivatives was found in the liver DNA of yearling trout 10 h after the administration of 0.3 mg/kg AFB1.     

Immunological and HPLC detection of aflatoxin adducts in human tissues after an acute poisoning incident in S.E. Asia

Acid hydrolysed, purified DNA, extracted from formalin fixed human tissues from persons acutely exposed to aflatoxins during a poisoning incident, was found to inhibit antibody binding in a competitive aflatoxin inhibition ELISA both before and after immunoaffinity column purification. HPLC analysis of acid hydrolysates of the DNA revealed a peak with a retention time 3 min earlier than 8,9-dihydro-2-(N7-guanyl)-9-hydroxy AFB1 synthesized either by peracid activation or direct reaction of the 8,9-oxide with DNA. The major peak seen when DNA was extracted from formalin fixed tissues from rats treated with aflatoxin B1 was identical to that seen in the formalin fixed human tissues. Adduct levels ranged from 0 to 170 x 10(6) nucleotides depending on tissue type and individual examined.     

Immunological detection of aflatoxin B1-DNA adducts formed in vivo

Two monoclonal antibodies (6A10 and 12F5) were obtained after fusion of mouse P3X63-AG.8.653 myeloma cells with spleen cells isolated from BALB/c mice immunized with imidazole ring-opened aflatoxin B1 (AFB1)-DNA and characterized by competitive enzyme-linked immunosorbent assays. Both antibodies are highly specific for imidazole ring opened AFB1-DNA and show some cross-reactivity with AFB1-DNA and no cross-reactivity with 8,9-dihydro-8-(7-guanyl)-9-hydroxy-AFB1, AFB1 conjugated with bovine serum albumin, aflatoxin M1 conjugated with bovine serum albumin, AFB1, or aflatoxin G1. Antibody 6A10 was further characterized and showed no cross-reactivity with DNA modified by several other carcinogens. It could detect adducts with 4-fold higher sensitivity in highly modified DNA (2.5 adducts/100 nucleotides) than in low modified DNA (4 adducts/10(5) nucleotides). With low modified DNA the limit of sensitivity is 5 adducts/10(7) nucleotides. Antibody 6A10 reliably detected adducts formed in vivo in rats and mice treated with AFB1. In a pilot study, AFB1 adducts were detected in liver tissues from individuals living in areas with suspected exposure to AFB1. Monitoring adduct levels in human tissue may provide information not only on carcinogen exposure but also on the relationship among infection with hepatitis B virus, dietary exposure to aflatoxin B1, and liver cancer.     

Biological and chemical studies on 8,9-dihydroxy-8,9-dihydro-aflatoxin B1 and some of its esters

A number of aflatoxin B₁ (AFB₁) derivatives have been synthesizedincluding 8-acyloxy- and 8-benzoyloxy-9-hydroxy-8,9-dihydro-AFB₁compounds, AFB₁-8,9-diol and [³H] AFB₁ labelled at the 9 position.AFB₁-hydroxyesters appear to be models of AFB₁-8,9-oxide inthat they are bacterial mutagens, stimulate unscheduled DNAsynthesis in HeLa cells and react with DNA to give trans-8,9-dihydro-8(7-guanyl)-9-hydroxy-AFB₁as the major adduct after hydrolysis. The potency of the hydroxyestersincreases with ease of release of the ester grouping at position8. Absence of the hydroxyl at position 9 gives compounds whichare readily hydrolysed in water but are not biologically active.The hydroxyesters hydrolyse in water to give AFB₁-diol, providinga convenient means of synthesis of this compound. Studies withAFB₁-diol show that it reacts with one molecule of Tris base,probably through the ring-opened furan form, with the aminogroup of the Tris. Acidification results in ring closure inan analogous manner to AFB₁-diol. AFB₁-diol binds to DNA invitro as well as to liver slice DNA. The compound is mutagenictowards S. typhimurium TA100 without metabolic activation. Theimplications of these findings are discussed in relation tothe mechanisms of AFB₁ carcinogenicity.     

DNA adducts of the carcinogen, 15, 16-dihydro-11-methylcyclo-penta(a)phenanthren-17-one, in vivo and in vitro: high pressure liquid chromatographic separation and partial characterization

The 11-methyl derivative (11-methyl ketone) is the most carcinogenicof the series of methylated derivatives based on 15, 16-dihydro-cyclopenta[a]phenanthren-17-one.The nudeoside adducts derived from [³H]-11-methyl ketone-modifiedmouse skin DNA have been separated by both Sephadex LH-20 chromatographyand reverse-phase h.p.l.c and compared to those derived fromDNA modified in vitro with the [¹⁴C]-11-methyl ketone usingrat liver microsomes. The in vivo modified DNA separated togive 6 adducts (designated I- VI) on h.p.l.c. The major in vivoadduct (80% total adducts) co-chromatographed with the majorin vitro adduct. The metabolites of the 11-methyl ketone (designateda-g) have been separated by h.p.l.c, and the adducts derivedfrom each of these individual metabolites determined by furthermetabolism in the presence of DNA. H.p.l.c. separation of theseadducts has allowed characterization of the in vivo adducts.The major adduct (V) and possibly one of the minor adducts (IV)were derived from the 3, 4-dihydro-3, 4-diol of the 11-methylketone (metabolite e). Adducts II and III were derived fromthe 16- and 15-monohydroxylated derivatives of the 11-methylketone and also from their corresponding 3, 4-diols and thereforeare likely to be the 16- and 15-hydroxy derivatives, respectively,of adduct V. Adduct VI, however, although derived from the 15-hydroxy-3,4-diol had a late retention time on h.p.l.c, suggesting eithera non-diol-epoxide adduct or a deoxyadenosine adduct. The useof [³H-G]DNA has established that the major adduct (V) and the16-hydroxy-derived adduct (II) contain deox-yguanosine. Reactionof the carcinogen with [³H-A]poly(dA-dT) gave adduct VI whichwas the only adduct peak shown to contain [³H]deoxyadenosine.     

Fluorine probes for investigating the mechanism of activation of indeno[1,2,3-cd]pyrene to a tumorigenic agent

Indeno[1,2,3-cd]pyrene (IP) is a non-alternant polycyclic aromatic hydrocarbon that has tumor-initiating activity on mouse skin and is carcinogenic in newborn mice and in rat lungs. Previous studies have shown that 8- and 9-hydroxyIP and IP-1,2-diol are major metabolites formed in vivo in mouse skin. 8-HydroxyIP-1,2-diol and 9-hydroxyIP-1,2-diol are also observed as in vivo metabolites of IP. Although 8-hydroxyIP had marginal tumor-initiating activity on mouse skin, IP-1,2-diol and its epoxide precursor, IP-1,2-oxide, had similar tumorigenic activity as IP. In the present study fluorine probes have been employed to investigate the contribution of metabolic activation at the 1,2 and 7-10 positions of IP. At a total initiating dose of 4.0 mumol, 2-fluoroIP induced skin tumors in 76% of the treated animals with an average of 3.9 tumors/mouse. At the same dose, IP induced a 72% incidence of tumor-bearing mice with 2.1 tumors/mouse. In contrast, 8,9-difluoroIP elicited a tumorigenic response in 40% of the treated animals with 0.6 tumors/animal. Five mice from each experimental group were killed at the conclusion of the initiation phase of the bioassay and DNA was isolated from the treated areas of skin. 32P-Postlabeling analysis of the hydrolyzed DNA indicated that IP forms one major detectable DNA adduct that migrates close to the origin. This adduct is absent in mice treated with 8,9-difluoroIP. In contrast, 2-fluoroIP forms one major adduct spot with different retention behavior as compared with the adduct formed from IP. DNA from mice treated topically with IP-1,2-diol and IP-1,2-oxide was subjected to 32P-postlabeling analysis. IP-1,2-diol forms one major DNA adduct spot with mobility similar to that observed for the IP-DNA adduct. IP-1,2-oxide displayed an intense pattern of DNA adducts centered around the location of the IP-DNA adduct. No adducts were detected which had mobility similar to that formed from 2-fluoroIP. These results are consistent with IP undergoing metabolic activation at positions 7-10 either alone or in conjunction with dihydrodiol formation at the 1,2 position.     

Aflatoxin B1 formamidopyrimidine adducts are preferentially repaired by the nucleotide excision repair pathway in vivo

Aflatoxin B(1) (AFB(1)), the most potent member of the aflatoxin family of hepatocarcinogens, upon metabolic activation reacts with DNA and forms a population of covalent adducts. The most prevalent adduct, 8,9-dihydro-8-(N(7)-guanyl-)-9-hydroxyaflatoxin (AFB(1)-N(7)-dG), as well as the AFB(1) formamidopyrimidine adduct (AFB(1)-FAPY), resulting from imidazole ring opening of the major adduct, are thought to be responsible for mutations caused by AFB(1). The AFB(1)-N(7)-dG lesions are rapidly removed in Escherichia coli and mammals, whereas the AFB(1)-FAPY lesions persist in mammalian cells, which along with the higher stability of this lesion suggests that AFB(1)-FAPY might significantly contribute to the observed toxicity and carcinogenicity of AFB(1) in higher organisms. Other workers have shown in vitro evidence that AFB(1)-FAPY lesions are substrates for both nucleotide excision repair (NER) and base excision repair (BER). The present study, done in vivo, utilized a modified host cell reactivation assay and showed that AFB(1)-FAPY lesions are preferentially repaired in E.coli by NER. Comparisons of repair in wild-type, NER-deficient (uvrA), BER-deficient (mutM) and NER/BER double mutant E.coli strains transformed with plasmids enriched for AFB(1)-N(7)-dG or AFB(1)-FAPY lesions indicate that both lesions are efficiently repaired by NER-proficient cells (both wild-type and BER-deficient strains). We have found that the level of activity of the reporter gene is significantly affected by the presence of either lesion in NER-deficient strains due to the lack of repair. This effect is similar in NER-deficient and NER/BER-deficient strains indicating that BER (specifically in the strains we investigated) does not contribute significantly to the repair of these lesions in vivo. Consistent with this finding, in vitro analysis of AFB(1)-FAPY adduct excision by purified MutM and its functional analog human 8-oxoguanine DNA glycosylase using site-specifically modified oligonucleotides indicates that this lesion is a poor substrate for both proteins compared with canonical substrates for these enzymes, such as 7,8-dihydro-8-oxoguanine and methylformamidopyrimidine.     

Comparative effect of dietary butylated hydroxyanisole and {beta}-naphthoflavone on aflatoxin B1 metabolism, DNA adduct formation, and carcinogenesis in rainbow trout

Butylated hydroxyanisole (BHA) and ß-naphthoflavone(BNF), both chemicals with anti-carcinogeneic properties insome experimental animals, were compared for effects on afiatoxinB1 (AFB1) metabolism, hepatic DNA adduct formation and carcinogenesisin the rainbow trout. Dietary BHA had no effect on the hepatictumor incidence when fed at 0.03 or 0.3% 4 weeks prior to andduring a 4 week dietary exposure of 10 p.p.b. AFB1. BNF, whenfed at 0.005 or 0.05% under similar conditions, significantlyreduced tumor response, which confirms previous results in trout(Nixon et al.9 Carcinogenesis, 5, 615–619, 1984). BHAfed at either 0.03 or 0.3% for 8 weeks had no post-initiationeffect on the 52 week hepatic tumor incidence of trout exposedto a 0.5 p.p.m. AFB1 solution as embryos. A similar post-initiationexposure to 0.05% BNF significantly enhanced AFB1 tumor response.The influence of dietary BHA and BNF on AFB1 metabolism andDNA adduct formation and persistence in trout were examined.A 3 week pre-treatment with 0.3% dietary BHA had no effect onin vivo hepatic nuclear AFB1-DNA adduct formation at 0.5, 1,2 and 7 days after AFB1 i.p. injection. By contrast 0.05% dietaryBNF reduced hepatic AFB1-DNA adducts to 33–60% of controllevels at 0.5, 1, 2 and 4 days after AFB1 exposure. This wasaccompanied by significantly lower blood and liver levels ofAFB1 during the first 24 h after i.p. injection. Livers of BNFtrout also contained 4-fold more of the less carcinogenic metabolite,aflatoxin M1, and 50% less aflatoxicol (AFL), a metabolite withsimilar carcinogenicity as AFB1. Bile AFL-glucuronide levelswere significantly decreased in BNF-fed trout, but total bileglucuronides were significantly increased due to a 15-fold increasein aflatoxicol-M1 glucuronide. Freshly isolated hepatocytesfrom BHA-fed fish, when incubated with AFB1 for 1 h, showedno difference in levels of AFB1-DNA adducts or ratios of AFB1metabolites when compared to hepatocytes isolated from fishfed a control diet only. By contrast, dietary BNF has been previouslyshown to greatly enhance AFM1 production, reduce AFL production,and significantly reduce AFB1-DNA adduct formation in isolatedtrout hepatocytes (Bailey et al., Natl. Cancer Inst. Monograph,65, 379–385, 1984). These results indicate that dietaryBHA up to 0.3% does not alter AFB1 metabolism or DNA adductionin trout, nor does it inhibit or promote AFB1 hepatocarcinogenesisin this species. This is in contrast to anti-oxidant enhancementof AFB1-glutathione conjugation, reduction of AFB1-DNA binding,and consequent reduction of tumor response in rats. The nullresults in trout thus support enhanced glutathione conjugationas the major mechanism for BHA inhibition of AFB1 cardnogenesisin mammalian models. By contrast, BNF dietary pre-treatmentappears to inhibit AFB1 carcinogenicity in trout by enchancingglucuronide formation and elimination of the carcinogen, leadingto reduced DNA adduct formation in target tissue.     

Bioactivation of aflatoxin B1 in the bovine olfactory mucosa: DNA binding, mutagenicity and induction of sister chromatid exchanges.

Nasal olfactory tumours occur in cattle in relatively high frequencies in several developing countries. Since affected animals sometimes show signs of severe aflatoxicosis, a role of aflatoxin B1 (AFB1) in tumorigenesis can be proposed. The results of the present study show that microsomal preparations of the bovine olfactory mucosa have a much higher ability than liver microsomes to induce covalent binding of AFB1 to calf thymus DNA and to microsomal proteins. The major DNA adduct formed was 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1. Incubations of microsomal preparations of the bovine nasal olfactory mucosa with glutathione (GSH) and cytosolic fractions of the nasal mucosa resulted in decreased AFB1 DNA binding. A more pronounced decrease was observed when cytosolic fractions of mouse liver were added to the incubations. Mouse liver is known to contain a glutathione-S-transferase with a high ability to scavenge the reactive AFB1-epoxide via conjugation to GSH. Our results indicate that AFB1-GSH conjugation occurs less efficiently in the bovine nasal olfactory mucosa than in the mouse liver. Supernatant preparations (9000 g) of the bovine nasal olfactory mucosa incubated with AFB1 were shown to have the capacity to induce a strong genotoxic response both as regards induction of gene mutations in Salmonella typhimurium TA100 and the induction of sister chromatid exchanges in Chinese hamster ovary cells. Preparations of the bovine liver (9000 g) has a much lower ability to induce these effects. The results of the present study show that the bovine nasal olfactory mucosa has a high AFB1-bioactivating capacity, which can be related to the potent DNA damaging and mutagenic effects observed. It is considered that our results support the assumption that AFB1 plays a role in the aetiology of nasal tumours in cattle.     

Fluorescence spectral evidence that benzo[a]pyrene is activated by metabolism in mouse skin to a diol-epoxide and a phenol-epoxide

Hydrolysates of DNA that had been isolated from mouse skin treated with 3H-labelled benzo[a]pyrene were subjected to chromatography on Sephadex LH20. Two major products were eluted in the region expected for deoxyribonucleoside-hydrocarbon adducts and these were purified further by h.p.l.c. The fluorescence emission and excitation spectra of one of the adducts were identical to that of the adduct obtained from DNA that was treated with BP-7,8-diol 9,10-oxide (r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a] pyrene). The fluorescence emission and excitation spectra of the other adducts were identical to the published spectra of 9-OHBP-4,5-diol (4,5-dihydro-4,5,9-trihydroxy-benzo[a]pyrene) and of the deoxyribonucleoside-hydrocarbon adduct obtained from DNA that had been incubated with 9-OHBP (9-hydroxybenzo[a] pyrene) in the presence of a rat-liver microsomal system. The metabolic activation of benzo[a]pyrene in mouse skin, a target tissue for carcinogenesis by this hydrocarbon, thus appears to involve the formation of adducts derived from both BP-7,8-diol 9,10-oxide and 9-OHBP 4,5-oxide (9-hydroxybenzo-[a]pyrene 4,5-oxide), although quantitatively, the adduct derived from 9-OHBP 4,5-oxide is a minor product.     

Metabolism of aflatoxin B1 in the upper airways of the rabbit: role of the nonciliated tracheal epithelial cell

Short-term tracheal explant cultures from the rabbit were used to study the metabolism of the carcinogen aflatoxin B1 (AFB1) and to determine the cell types that are susceptible to damage by AFB1 and their relative contents of monooxygenase enzymes. Tracheas were cultured in serum-free medium for 0.5-24 h with 0.7 microM [3H]AFB1, and metabolism was measured by determining the level of binding of the carcinogen to DNA and by the release of metabolites into the medium. The binding of aflatoxin B1 was time dependent and appeared to peak at 12 h in culture. In addition, the metabolites aflatoxicol, aflatoxin M1, and aflatoxin Q1 were produced by the explants. Ultrastructural evaluation of cultured tracheas showed degenerative changes exclusively in nonciliated secretory cells after 4 h in culture. Extensive nonciliated secretory cell necrosis was evident by 12 h. Ciliated cells did not show degenerative changes until 12 h and appeared much more viable after 24-h exposure to AFB1 relative to the nonciliated cells. Tracheal sections stained to demonstrate rabbit lung cytochrome P-450, Forms 2 and 5, and cytochrome P-450 reduced nicotinamide adenine dinucleotide phosphate reductase by an immunoperoxidase technique showed intense staining selectively within nonciliated cells. In total, the data revealed that: (a) rabbit tracheal explants are able to metabolize aflatoxin B1; (b) the nonciliated secretory cell population in this tissue is the target cell for cytotoxicity of this carcinogen; and (c) as is the case in the more distal airways, the nonciliated epithelial cells appear to have a high content of components of the pulmonary cytochrome P-450 monooxygenase system, which may be an important factor in the susceptibility of these cells and this region of the airways to suspected airborne carcinogens.     

Formation of DNA adducts in mouse skin treated with metabolites of chrysene

Analysis by 32P-postlabelling of DNA isolated from mouse skin that had been treated in vivo with the polycyclic hydrocarbon chrysene revealed the presence of 7 adducts. All 7 adducts were also present in DNA from mice treated with trans-1,2-dihydro-1,2-dihydroxychrysene (chrysene-1,2-diol), and one of them, adduct 2, was formed from the triol derivative 9-hydroxy-trans-1,2- dihydro-1,2-dihydroxychrysene (9-hydroxychrysene-1,2-diol) and from 3-hydroxychrysene. Adducts were not detected in DNA from mice treated with trans-3,4-dihydro-3,4-dihydroxychrysene (chrysine-3,4-diol) or with 1-, 2-, 4-, 5- or 6-hydroxychrysene. In vitro modification of DNA by the anti-isomer of the bay-region diol-epoxide yielded adducts 3-7, while the corresponding triol-epoxide yielded adducts 2. It is concluded that chrysene activation in mouse skin proceeds principally via the bay-region diol-epoxide and to a lesser extent via the related bay-region triol-epoxide.     

Modulation of aflatoxin metabolism, aflatoxin-N7-guanine formation, and hepatic tumorigenesis in rats fed ethoxyquin: role of induction of glutathione S-transferases

The effects of dietary administration of ethoxyquin (EQ) on aflatoxin B1 (AFB1) metabolism, DNA adduct formation and removal, and hepatic tumorigenesis were examined in male Fischer rats. Rats were fed a semipurified diet containing 0.4% EQ for 1 wk, gavaged with 250 micrograms of AFB1 per kg 5 times a wk during the next 2 wk, and, finally, restored to the control diet 1 wk after cessation of dosing. At 4 mo, focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transpeptidase. Treatment with EQ reduced by greater than 95% both area and volume of liver occupied by gamma-glutamyl transpeptidase-positive foci. Utilizing the same multiple dosing protocol, patterns of covalent modifications of DNA by AFB1 were determined. EQ produced a dramatic reduction in the binding of AFB1 to hepatic DNA: 18-fold initially and 3-fold at the end of the dosing period. Although binding was detectable at 3 and 4 mo postdosing, no effect of EQ was observed, suggesting that these persistent adducts are not of primary relevance to AFB1 carcinogenesis. Analysis of nucleic acid bases by high-performance liquid chromatography revealed no qualitative differences in adduct species between treatment groups. The inhibitory effect of EQ on AFB1 binding to DNA and tumorigenesis appears related to induction of detoxication enzymes. Rats fed 0.4% EQ for 7 days showed a 5-fold increase in hepatic cytosolic glutathione S-transferase (GST)-specific activities. Multiple molecular forms of GST were induced, and concomitant elevations in messenger RNA levels coding for the synthesis of GST subunits were observed. Correspondingly, biliary elimination of AFB1-glutathione conjugate was increased 4.5-fold in animals on the EQ diet during the first 2 h following p.o. administration of 250 micrograms of AFB1 per kg. Thus, induction by EQ of enzymes important to AFB1 detoxication, such as GST, can lead to enhanced carcinogen elimination, as well as reductions of AFB1-DNA adduct formation and subsequent expression of preneoplastic lesions, and, ultimately, neoplasia.     

Aflatoxin-DNA adduct formation in chronically dosed rats fed a choline-deficient diet

Nutritional modulation of male Fischer rats by a choline-deficient/methionine-low diet dramatically increases hepatocarcinogenesis and reduces time to first tumors induced by aflatoxin B1 (AFB1). The effect of this diet on hepatic aflatoxin-DNA adduct burden in male Fischer rats dosed with a carcinogenic regimen of AFB1 was examined in this study. After 3 weeks of ingestion of a choline-deficient/methionine-low diet or control semi-purified diet, rats were administered a carcinogenic regimen of 25 micrograms [3H]AFB1 for 5 days a week over 2 weeks. Six choline-deficient and four control diet rats were killed 2 h after each dose, and liver DNA isolated. In addition, hepatic DNA was isolated from animals 1, 2, 3, and 11 days after the last [3H]AFB1 administration. At all time points HPLC analysis of aflatoxin-DNA adducts was performed to confirm radiometric determinations of DNA binding levels. No significant quantitative differences in AFB1-DNA adduct formation between the dietary groups were observed following the first exposure to [3H]AFB1; however, total aflatoxin-DNA adduct levels in the choline-deficient animals were significantly increased during the multiple dose schedule. When total aflatoxin-DNA adduct levels were integrated over the 10 day dose period, a 41% increase in adduct burden was determined for the choline-deficient animals. While this increase in DNA damage is consistent with the hypothesis that DNA damage is related to tumor outcome, the biochemical basis for this effect still needs to be elucidated.     

Aflatoxin B1-induced DNA damage and its repair

Aflatoxin B(1) (AFB(1))-N(7)-guanine is the predominant adduct formed upon the reaction of AFB(1)-8,9-exo-epoxide with guanine residues in DNA. AFB(1)-N(7)-guanine can convert to the ring-opened formamidopyrimidine, or the adducted strand can undergo depurination. AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine are thought to be predominantly repaired by nucleotide excision repair in bacteria, yeast and mammals. Although AFB(1)-formamidopyrimidine is removed less efficiently than AFB(1)-N(7)-guanine in mammals, both lesions are repaired with equal efficiencies in bacteria, reflecting differences in damage recognition between bacterial and mammalian repair systems. Furthermore, DNA repair activity and modulation of repair by AFB(1) seem to be major determinants of susceptibility to AFB(1)-induced carcinogenesis.     

Comparative biotransformation of aflatoxin B1 in mammalian airway epithelium

Aflatoxin B1 (AFB1) appears to be a risk factor for upper respiratory tumors in individuals occupationally exposed to AFB1-contaminated grain dusts. To study the potential effects of this mycotoxin in the upper airways, the metabolism of AFB1 was investigated in tracheal cultures and purified tracheal microsomes from rabbit, hamster and rat. These species differ in the proportion of P450-containing non-ciliated epithelial (NC) cells in the upper airway (17, 41, 0% respectively). Cultures from the rabbit produced the highest level of the AFB1 metabolites AFB1-dihydrodiol (AFB1-diol), GSH-AFB1, AFM1, AFB2a and the highest tracheal microsomal pentoxyresorufin-O-dealkylase (PROD) activity (an indicator of that P450 activity which activates AFB1) and greater cytosolic GSH-transferase activity compared to hamster and rat. Tracheal microsomal epoxide hydrolase activity, AFB1-diol production, cytochrome P450 content, P450 reductase and ethoxyresorufin-O-dealkylase (EROD) activity (an indicator of AFB1 detoxification) were highest in the hamster. Although the overall metabolic activity in rat tracheal epithelium was low, PROD-related activity appeared to predominate. Conjugation with GSH was the major detoxification pathway in rabbit and rat upper airways, although levels of AFB1-GSH and activities of glutathione transferase were significantly lower in the rat than in the rabbit and hamster. Hydrolysis of the putative AFB1-2,3-epoxide via epoxide hydrolase appeared to be the major AFB1 detoxification pathway in hamster tracheal epithelium as indicted by corresponding high tracheal microsomal AFB1-diol production and EH activity compared to rabbit and rat. Glucuronide and sulfate conjugates of AFB1 and its metabolites were formed in tracheal explant cultures from these three species, although amounts formed were minor. These results indicate that rabbit upper airway epithelium contains metabolic activity primarily involved in AFB1 activation, whereas AFB1 detoxification pathways predominante in hamster. Furthermore, the characteristics of carcinogen metabolism are not predictable based solely on airway morphology.     

Characterisation of the imidazole ring-opened forms of trans-8,9-dihydro-8,9-dihydro-8-(7-guanyl)9-hydroxy aflatoxin B1

Hydroxyl ion attack on aflatoxin B₁ (AFB₁)-adducted DNA followedby acid hydrolysis releases two AFB₁ derivatives, 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-ylformamido) 9-hydroxy AFB₁ (major compound) and 8, 9-dihydro-8-(2-ammino-6-formamido-4-oxo-3,4dihydropyrimid-5-ylamino)9-hydroxy AFB₁. The minor compound converts into the majorisomer in aqueous dimethylsulphoxide.