Mechanisms of anti-carcinogenesis: the distribution and metabolism of aflatoxin B1 in rainbow trout fed Aroclor 1254

Polychlorinated biphenyls (PCBs) have previously been shownto be inhibitors of carcinogenesis in trout. The mechanism ofthis inhibition was investigated by studying the effects ofPCBs on aflatoxin B₁ (AFB₁) distribution, metabolism and DNAadduct formation, both in vivo and in vitro. A 24 h distributionstudy of injected tritiated AFB₁ showed more radioactivity inblood, liver and bile in fish fed PCBs, but less in residualcarcass. The metabolites of AFB₁ found in vivo in blood plasmaand liver homogenates were shifted by PCB pre-treatment towardsgreater production of the polar metabolite aflatoxin M₁ (AFL-M₁)and glucuronide conjugates. The major metabolite in bile ofPCBs fish was the glucuronide of aflatoxi-col M₁ (AFL-M₁), whichwas enhanced 15-fold over controls. Levels of aflatoxicol (AFL)glucuronide, the major conjugate in controls, were unalteredby PCBs. The pattern of AFB1 metabolism in isolated bepatocytesfrom PCB-prefed fish was consistent with in vivo metabolism.AFB₁-DNA adduct formation in a 1 h assay was similar in hepatocytesfrom PCB-fed and control fish. However, the total rate of AFB₁metabolism was significantly elevated in hepatocytes from PCB-fedfish such that the degree of AFB₁-DNA adduct formed per unitAFB₁ metabolized was 42% lower than control. Similarly, adductformation in vivo during the first 24 h post-AFB₁ injectionin PCB fish was not significantly different from controls. However,over a longer 21 day period, adduct levels in PCB fish wereonly 48–69% of controls (P < 0.005, analysis of variance),once peak adduct formation was reached. Thus, initial ratesof adduct formation may be misleading in the absence of furtherinformation on rates of carcinogen metabolism in vitro and/orpharmacokinetics of peak adduct formation in vivo. These resultsindicate that PCB inhibition of AFB₁ carcinogenesis in troutinvolves dramatic initial changes in carcinogen distribution,metabolism and elimination which, over time, results in a netreduction of DNA adduct formation.     

Aflatoxin B1 metabolism and DNA binding in isolated hepatocytes from rainbow trout (Salmo gairdneri)

The metabolism of aflatoxin B₁ (AFB₁) was examined in freshlyisolated hepatocytes from rainbow trout. Intracellular DNA adductformation was linearly related to AFB₁ dose, and qualitativelysimilar to adducts formed in vivo. The rate of adduct accumulationwas constant during the first hour following completion of thepreparation, after which an increase and gradual decrease inrate routinely occurred. The relative rates of production ofthe major unbound AFB₁ metabolites aflatoxicol, aflatoxin M₁,and polar conjugates, also remained constant over the firsthour of preparation age, but subsequently changed in a mannerconsistent with the changes in DNA binding. The common solventvehicles ethanol and dimethyl sulfoxide were shown to seriouslyperturb AFB₁ metabolism and DNA binding even at levels <1%.A simple method is reported for removal of ethanol prior tointroduction of hepatocytes for incubation with AFB₁. The influenceof cell concentration was also examined. DNA binding and relativedistribution of AFB₁ metabolites showed little or no dependencein the range 1–6 x 10⁶ cell/ml, but were substantiallyaltered above 10⁷ cells/ml. Under defined conditions, studiesin isolated hepatocytes appear to reflect in vivo cell capabilitiesfor AFB₁ metabolism.     

The metabolism of aflatoxin B1 by hepatocytes isolated from rats following the in vivo administration of some xenobiotics

Isolated rat hepatocytes, an intact cellular system capableof performing phase I and phase II metabolism, have been usedto investigate metabolism of aflatoxin B₁ These cells were foundto metabolise [¹⁴C] aflatoxin B₁ to aflatoxins M₁ and Q₁, andto radiolabelled polar material, presumably conjugates, as analysedby h.p.l.c., t.l.c. and radioactive determination. In vivo administrationof the mixed function oxidase inducers, phenobarbitone and 3-methylcholanthrene,resulted in enhanced hepatocyte phase I (microsomal) metabolismof aflatoxin B₁ In contrast to metabolism of AFB₁ by in vitrosubcellular systems increased production of polar material (conjugatedmetabolites) derived from [¹⁴C] aflatoxin B₁ was also detectedin hepatocytes isolated from these pretreated animals. Formationof aflatoxin Q₁ by isolated hepatocytes appeared to be mediatedby cytochrome P450-linked enzymes whereas cytochrome P448-linkedenzymes were apparently involved in aflatoxin M₁ production.Chronic feeding of aflatoxin B₁ to rats enhanced hepatocyteproduction of conjugated material only and did not elevate cellularcytochrome P450 levels, thus suggesting that aflatoxin B₁ isnot an inducer of its own primary metabolism.     

Metabolism and DNA binding of aflatoxicol and aflatoxin B1 in vivo and in isolated hepatocytes from rainbow trout (Salmo gairdneri)

The purpose of this study was to compare the metabolism andDNA binding of aflatoxicol (AFL) with that of aflatoxin B₁ (AFB₁)in vivo and in isolated hepatocytes from Mt Shasta strain rainbowtrout (Salmo gairdneri). Maximum total binding of [³H]AFL toliver DNA from trout exposed by intraperitoneal injection was38–47% of that of [³H]AFB₁ over a 1–7 day period.The average AFL/AFB₁ DNA binding ratio in 1-h incubations withisolated hepatocytes was 0.67±0.36 (n=13). In freshlyisolated hepatocytes, substantial interconversion between AFB₁and AFL via reductase and dehydrogenase enzymes was observed.Total in vivo excretion of conjugates in bile over 4 days wasgreater for [³H]AFL substrate than for [³H]AFB₁. To determineif AFL binding was due to direct activation or to prior metabolismto AFB₁ followed by activation, AFL with a tritium atom on thecarbon containing the cyclopentenol function ([1-³H]AFL, wassynthesized and incubated with hepatocytes. Binding of [1-³H]AFLwas 3% that of [³H]AFB₁ and represents only direct binding ofthe intact cyclopentenol epoxide molecule before transformationto AFB₁ and consequent loss of ³H. H.p.l.c. analysis of DNAhydrolyzed after incubation with [1-³H]AFL resulted primarilyin production of non-radioactive 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxinB₁ (AFB₁-N⁷-guanine). A radioactive peak estimated to be 1%as abundant as the AFB₁-N⁷-guanine was also observed. The overallbinding of generally labeled [³H]AFL to trout liver DNA in vivoand in freshly prepared hepatocytes correlates well with availabletumor incidence and mutagenicity data. Conclusions from thesefindings are that direct interaction of AFL-8,9-epoxide withDNA is of relatively minor quantitative importance in rainbowtrout hepatocytes and that the major adduct results from conversionof AFL to AFB₁ prior to epoxide formation.     

Comparative metabolism and DNA binding of aflatoxin B1, aflatoxin M1, aflatoxicol and aflatoxicol-M1 in hepatocytes from rainbow trout (Salmo gairdneri)

DNA binding and metabolism patterns of ³H-labeled aflatoxinB₁ (AFB₁) and its phase I metabolites, aflatoxicol (AFL), aflatoxinM₁ (AFM₁) and aflatoxlcol-M₁ (AFL-M₁), were compared in freshlyprepared rainbow trout (Salmo gairdneri) hepatocytes. Aflatoxinswere incubated with hepatocytes for periods up to 1 h, cellularDNA was isolated and specific activities determined by scintillationcounting and Burton analysis. Data for (pmol bound aflatoxin/µgDNA)/(µmol dose) versus time fit a linear function (P< 0.002)passing nearly through the origin for each aflatoxin.DNA binding at 1 h relative to AFB was: AFL, 0.53 ? 0.07; AFM0.81 ? 0.20 AFL-M₁ 0.83 ? 0.24. Statistical analysis indicatedthat binding of AFL, AFM₁ and AFL-M₁ were significantly lessthan that of AFB HPLC analysis of the cellular supernatantsindicated that the major metabolites were AFL, AFB₁ AFL-M₁ andAFM₁ from AFB₁ AFL, AFM and AFL-M₁ substrates, respectively.Small quantities of hydroxylated metabolites and glucuronidesalso were detected in some of the incubations. The time-coursedata suggested that initial formation of major metabolites wasrapid and that, by 20–30 min, net changes in metabolitelevels decreased or approached zero. Because the four compoundspossessa 8,9-double bond, DNA binding could be due to activationof the parent substrates as well as of their phase I metaholites.Based on current mutagenicity data and limited carcinogenicitystudies, AFM₁, and AFL-M₁ have binding levels which are higherthan expectedcompared to AFB₁ and AFL.     

Inhibition of aflatoxin B1 carcinogenesis in rainbow trout by flavone and indole compounds

Several compounds such as flavonoids, selenium, anti-oxidantsand retinoids reportedly reduce the induction of cancer in experimentalanimals, and some have been suggested to function by affectingthe mixed-function oxidase (MFO) system. The following compounds:50 and 500 p.p.m. ß-naphthoflavone (BNF), 1000 p.p.m.flavone, 1000 p.p.m. of a tangeretin-nobilitin mixture, 1000p.p.m. ß-ionone, 1000 p.p.m. indole-3-carbinol (13C)and 2000 p.p.m. quercetin were examined for protection againstaflatoxin B, (AFB₁) hepatocardnogenesis, induction of the MFOsystem and metabolism of AFB₁ in rainbow trout. These compoundswere fed to fingerting rainbow trout for 8 weeks. At that timethe activity of several MFO enzymes and cytochrome P450 contentwere measured and the trout were exposed for 2 weeks to 20 p.p.b.AFB₁ in the same diets. After feeding the test diets withoutAFB₁ for another 6 weeks and basal diet for another 52 weeks,the tumor incidence was determined. The effect of BNF and I3Con in vivo binding of AFB₁ to DNA was also measured in separategroups of trout. BNF induced the trout MFO system in a dose-dependentmanner, tangeretin-nobilitin was less effective and I3C didnot induce. BNF showed significant alterations in the metabolismof AFB₁ to aflatoxicol and aflatoxin M₁ using cell fractionsfrom pretreated fish. None of the other compounds, includingI3C showed such an effect. Despite the apparent lack of in vitroeffect of I3C, both BNF and I3C reduced AFB₁- DNA binding invivo. I3C and BNF provided marked protection against AFB inducedhepatocardnogenesis, while the other compounds were less effective.The 58 weeks tumor incidences were 4% for 1000 p.p.m. DC, 6%for 500 p.p.m. BNF and 18% for 50 p.p.m. BNF, compared to 38%for the AFB₁-positive control. These data demonstrate that grossinduction of the MFO system was not necessarily required foralterations in DNA adduct formation in vivo or protection againstAFB₁ carcinogenesis. Both BNF and I3C provided marked protectionbut only BNF induced the MFO system.     

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity

DNA binding levels were determined and compared in culturedhepatocytes from male and female rats as well as other animalspecies following exposure to aflatoxin B₁ (AFB₁) or 2-acetylaminofluorene(2-AAF). When human, rat (both male and female) and mouse hepatocytesin primary culture were exposed to 2.0 ? 10^–7 M [³H]AFB₁(sp. act. 2.63 µCi/nmol) for 24 h, male rat hepatocyteshad the highest degree of [³H]AFB₁-DNA binding (203 pmol/mgDNA) and human hepatocytes contained the next highest bindinglevel (42 pmol/mg DNA). Hepatocytes from female rats contained38 pmol/mg DNA while cultured mouse hepatocytes contained only1.4 pmol/mg DNA. When the same dose of [³H]AFB₁ was administeredto the cultured male rat hepatocytes at 24 h, 48 h, 72 h and1 week after seeding, and incubated for 24 h, the DNA bindinglevels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallelexperiments to the cultured male rat hepatocytes above, theAFB₁-DNA binding levels in the cultured female hepatocytes were42, 41, 37 and 34 pmol/mg DNA respectively. Human, male andfemale rat hepatocytes in primary culture were exposed to 5.2? 10^–5 M 2-acetyl-amino [9–¹⁴C]fluorene (sp. act.0.0094 µCi/nmol) for 24 h. It was determined that malerat hepatocytes contained the highest amount of radioactivelylabeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), femalerat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytescontained 0.29 nmol/mg DNA. Results from our in vitro hepatocyteculture system correlate well with in vivo animal studies dealingwith species and sex differences in DNA binding and carcinogenicsusceptability. This indicates that hepatocytes in vitro maintainmany of the biological properties necessary for carcinogen responsesimilar to liver cells in vivo. In addition, comparison of genotoxiceffect in cultured hepatocytes from animals as well as humansmay be useful in evaluating carcinogenic potential of xenobioticsin human liver.     

Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/lacl transgenic rats and mice following exposure to aflatoxin B1

In vivo mutations were studied in lambda/lacI (Big Blueb.®)transgenic C57BL/6 mice and F344 rats following exposure toeither AFB₁ (aflatoxin B₁) or DMSO vehicle. Fourteen days afterexposure, livers were removed for DNA extraction and subsequentmutational analysis of the lacI gene. Mice injected with a singlei.p. dose of AFB₁ at 2.5 mg/kg did not show a significant increasein liver mutant frequency relative to vehicle-treated controls.DNA sequence analysis of lacI mutations collected from the AFB₁-treatedmice showed a pattern of mutation similar to that of the previouslyobserved spontaneous mouse liver mutational spectrum. In contrast,rats subjected to one-tenth the mouse AFB₁ dosage respondedwith an approximate 20-fold induction in liver mutant frequencyover background. Sequencing of lacI mutations also revealedspectral differences between vehicle- and AFB₁-treated rats.A large increase in G: C     

Comparative activation of aflatoxin B1 to mutagens by isolated hepatocytes from rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch)

Isolated hepatocytes from rainbow trout readily activated aflatoxinB₁ (AFB₁) to mutagens detectable by S. typhimurium TA 98. Characterizationstudies demonstrated that activation efficiency was essentiallyLinear with respect to hepatocyte concentration (5 ? 10⁵–2?10⁷ cell/ml) and AFB₁ dose (0–10 µg/ml). This systemwas employed to assess possible differences in AFB₁ activationin hepatocytes from rainbow trout and coho salmon, two specieswhich have been shown in in vivo studies to differ widely insensitivity to AFB₁ carcinogenesis. Activation efficiency wasapproximately three times greater in trout hepatocytes comparedwith salmon hepatocytes. This difference was more marked whenS20 Liver fractions from the two species were used. Analysisof unbound [³H]AFB₁ metabolites performed on supernatants ofhepatocyte incubations revealed that under tbe normal conditionsof assay, addition of bacteria does not perturb the variouspathways of AFB₁ metabolism within hepatocytes. These resultssupport other studies which suggest that the greater sensitivityof trout to AFB₁ carcinogenicity resides largely in increasedinitial DNA damage, compared with coho salmon.     

The activation of aflatoxin B1 in liver slices and in bacterial mutagenicity assays using livers from different species including man

The activation of aflatoxin B₁ (AFB₁) has been compared in twoin vitro systems: (1) binding to DNA in liver slices incubatedwith [¹⁴C] or [³H]AFB₁; (2) standard bacterial mutation systemsusing 9000 x g supernatant (S-9) fractions from uninduced liversfor activation. Several factors which modify aflatoxin carcinogenesiswere investigated, namely species, sex, phenobarbitone pretreatmentand aflatoxin G₁ (AFG₁) compared with AFB₁. The results fromDNA binding in liver slices showed the following trends: rat> hamster > mouse, control > phenobarbitone-pretreatedrat and AFB₁ > AFG₁ which correlated directly with trendsin carcinoenicity. An exception to this trend was the similarlevel of binding found in male and female rat livers, the latterbeing less susceptible to AFB₁ carcinogenesis. This result suggeststhat sex differences in AFB₁ carcinogenicity may be due to differencesin repair of lesions or during the promotion phase of carcinogenesis.The levels of binding of [³H]AFB₁ to DNA slices from fresh humanliver biopsies showed considerable variation between the sixsamples. Values ranged from 0.7–8.5 ng AFB₁/mg DNA, whichare in between values observed in the hamster and mouse. Mutagenicitydata did not correlate with carcinogenicity in relation to speciesdifferences (hamster > rat > mouse) nor phenobarbitonepretreatment. Supplementation of the top agar mixture with glutathioneand/or pre-incubation of S-9, AFB₁ and cofactors did not improvethis correlation. Nevertheless, it is expected that differencesbetween these two systems are due to limitations of the metabolizingsystem in mutagenicity tests, rather than either DNA bindingor bacterial mutation being the more valid end point.     

Photoactivated aflatoxins are mutagens to Salmonella typhimurium and bind covalently to DNA in vitro

The mutagenesis of Salmonella typhimurium TA100 and covalentbinding in vitro of photoactivated aflatoxin B₁ (AFB₁), aflatoxinB₂ (AFB₂), aflatoxin G₁ (AFG₁) and aflatoxin G₂ (AFG₂) wereinvestigated. Covalent binding levels of 1140, 225, 330 and8 pmol aflatoxin per µmol nucleotide phosphate were obtainedfor AFB₁, AFG₁, AFB₂ and AFG₂, respectively, at 50 µMmycotoxin after 2 h of irradiation. Mutant frequencies to histidineprototrophy were 97, 19, 49 and 0 x 10^–6 for AFB₁, AFG₁,AFB₂ and AFG₂ respectively, after 2 h irradiation at 100 µMmycotoxin in the surviving fraction of the mutagenized cultures.Toxicity to Salmonella was 0.59, 0.03, 0.31 and 0 lethal hitsunder the conditions specified for mutagenesis for AFB₁, AFG₁,AFB₂ and AFG₂, respectively.     

Short Communication: Interaction of aflatoxin B2 with rat liver DNA and histones in vivo

The interaction of aflatoxin B₂ (AFB₂) in vivo with rat livernuclear macromolecules was examined in an attempt to correlatethis binding with biological potency. The incorporation of [³H]AFB₂residues into rat liver his tones and DNA was determined 2,24 and 48 h following administration of a single i.p. dose of1 mg (³H)AFB₂/kg body weight. At each time point, hist one H1and the total histone fraction contained 5–30-fold more[³H]AFB₂ moieties than did DNA on a weight basis. Analyticalreversed-phase h.p.l.c. of the acid hydrolysis products resultingfrom AFB² binding to DNA revealed that 85% of the radioactivityco-chromatographed with the major aflatoxin B₁-DNA adduct, 2,3-dinydro-2-(N⁷-guanyl)-3-hydroxyaftatoxinB₁. These studies revealed an apparent correlation between AFB₂derived binding to DNA in vivo in rats and its potency as atoxin and carcinogen in this species.     

Evidence for membrane-mediated chromosomal damage by aflatoxin B1 in human lymphocytes

The hepatocarcinogen aflatoxin B₁ (AFB₁) was found to be a potentclastogen for phytohemagglutinin stimulated human lymphocytes.It also induced sister chromatid exchanges. These types of chromosomaldamage were induced at very low levels of covalent AFB₁-DNAadducts suggesting that AFB₁ operates in part by indirect actionbecause of its membrane-active character. The membrane-activecharacter of AFB₁ is documented by the following results: (i)AFB₁ stimulated the excretion of hydroxy- and/or hydroperoxy-arachidonicacid (AA) and free AA into the culture medium; (ii) the phospholipaseA₂ inhibitor p-bromophenacylbromide was anticlastogenic; (iii)the inhibitors of the oxidative metabolism of AA indomethacin,flufenamic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiareticacid and BN 1015 were anticlastogenic. These results are compatiblewith the induction of DNA damage by indirect action or the formationof covalent adducts via metabolic activation by cooxygenation.The observation that CuZn superoxide dismutase was antilastogenicindicates the intermediacy of superoxide in DNA damage formationand supports the former mechanism.     

Some studies on the relationship between the cytotoxicity of aflatoxin B1 to rat hepatocytes and metabolism of the toxin

Aflatoxin B₁ (AFB₁) caused marked, rapid (1 h) inhibition ofRNA synthesis and subsequent cytotoxic response in isolatedand primary cultured hepatocytes, from control rats, which areknown to metabolise AFB₁. A rat liver-derived cell line, BL8L,was much less susceptible to these effects of AFB₁ These cellshave no detectable AFB₁ metabolising capacity, but a less potent,anti-mitotic action of AFB₁ was observed in the BL8L cell line.Thus AFB₁ would seem to require metabolism to exert its acutecytotoxic action which is found at very low AFB₁ concentrations,although a direct antimitolic effect, independent of metabolism,is seen in dividing cells. Phenobarbitone and 3-methylcholanthrenein vivo pretreatments, known inducers of AFB₁ metabolism, resultedin reduced AFB₁ inhibition of RNA synthesis and cytotoxicityin hepatocytes, but only at lower concentrations of AFB₁ used,whereas cells from AFB₁ fed rats were much less susceptibleto AFB toxicity at all concentrations used. This resistanceto cytotoxicity of AFB₁ would appear to involve detoxificationmechanisms, primarily the formation of polar conjugates of AFB₁metabolites, particularly glutathione conjugates. These cellculture systems are useful for studying association betweenmetabolism and cytotoxicity of AFB₁ and other xenobiotics.     

Inhibition of aflatoxin B1 binding to hepatic DNA by dehydroepiandrosterone in vivo

Dehydroepiandrosterone (DHEA) a naturally occurring steroid,has been reported to inhibit the binding of N-dimethylnitrosamineand 7,12-dimethylbenz[a]anthracene to DNA in vivo and to increaseglutathione transferase activity. In this study, we have investigatedif DHEA could protect hepatic DNA from damage by the potenthepatocarcinogen aflatoxin B₁ (AFB₁). Young male Fischer 344(2-month-old) rats were fed a diet containing 0.8% DHEA for14 days. Control rats were pair-fed the same diet without DHEA.The rats were then administered a single i.p. dose of [³H]AFB₁in dimethylsulfoxide (0.6 mg/kg body weight; 200 mCi/mmol) andkilled after 3 h. Liver weight, mitochondrial, microsomal andcytosolic protein, cytochrome P450 content and glutathione transferaseactivity increased significantly (P < 0.001) in DHEA-fedrats; however, the hepatic DNA content was not altered. DHEAfeeding increased the total amount of AFB₁ bound to hepaticprotein but decreased the extent of DNA binding. In in vitroexperiments, there was less total binding to DNA and proteinby AFB₁ when using microsomes from DHEA-fed rats. These resultssuggest that DHEA inhibits the binding of AFB₁ to DNA by modifyingthe biotransformation of the carcinogen.     

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.     

Effect of diet and route of administration on the DNA binding of aflatoxin B1 in the rat

The effects of dietary Brussels sprouts and indole-3-carbinol(I3C) on xenobiotic-metabolizing enzyme activities and hepaticaflatoxin B₁ (AFB₁)-DNA binding were detennined in rats. Animalswere dosed intraperitoneally (i.p.) or intragastrically (i.g.)with [³H]AFB₁ and killed 2 (i.p.) or 3 (i.g.) h later. Brusselssprouts caused a significant (P < 0.01) 50–60% decreasein hepatic AFB₁ binding, and increased hepatic and intestinalglutathione S-transferase (GST) activities. Hepatic mono-oxygenase(AHH and ECD) activities were not altered in sprouts-fed rats,but >2-fold increases in intestinal AHH and ECD activitieswere found. Although I3C increased intestinal AHH and ECD activitiessimilarly to Brussels sprouts, I3C did not significantly decreaseAFB₁ binding, nor did it increase hepatic or intestinal GSTactivity. Route of administration did not alter the percentageinhibition of binding in comparison to control rats in eithertreatment group, suggesting that the small intestine may notplay a significant role in the metabolism of AFB₁. In a secondexperiment, rats were dosed either i.p. or i.g. with [³H]AEB₁and killed 2, 6, 12, 24 or 48 h later. Hepatic AFB₁-DNA bindingand tissue radioactivity levels were determined. Brussels sproutsonce again significantly (P<0.001) decreased hepatic AFB₁-DNAbinding. Route of administration of the carcinogen did not affectDNA binding over time in sprouts-fed animals, confirming ourprevious results.     

Chloroperbenzoic acid induced DNA damage and peracid activation of alfatoxin B1

The activated form of aflatoxin B₁ (AFB₁) in vitro and in vitrois believed to be the 2,3-oxide, which cannot be isolated, presumablydue to its reactivity. In addition to in vitro activation bycrude metabolic enzymes, two chemical procedures are available,one involving oxidation with the mild organic oxidant chloroperbenzoicacid and another involving the synthesis of AFB₁,-2,3-dichloride,an electronic analog of AFB₁,-2,3-oxide. Here we show that chloroperbenzoicacid by itself can modify DNA, primarily at non-basepaired adenineand guanine residues, as revealed by ‘replication block’analysis.     

Comparative kinetic studies on aflatoxin B1- DNA binding and aflatoxin B1-glutathione conjugation with rat and hamster livers in vitro

Inhibition of microsome mediated aflatoxin B₁ (AFB₁) bindingto exogenous or endogenous DNA by cytosolic glutathione (GSH)S-transferases is well established from our earlier studies.Correlation between inhibition of AFB₁-DNA binding and AFB₁-GSHconjugation in vitro using rat and hamster liver subcellularfractions is elucidated in this report. Even though hamsterliver microsomes catalyzed AFB₁ binding to exogenous DNA threetimes as much as the rat, hamster cytosol inhibited AFB₁-DNAbinding catalyzed by either microsomes severalfold more thanthe rat cytosol. AFB₁ - DNA binding is found to be inverselyrelated to AFB₁-GSH conjugation at all AFB₁ concentrations (2–100µM)studied. Presence of either styrene oxide or 3,3,3-trichloropropeneoxide at 1 mM level diminished AFB₁-GSH formation in vitro confirmingsome competition by these epoxides with AFB₁-epoxide for cytosolicGSH S-transferases. In a reconstituted system with endogenousDNA, the ratio of AFB₁-GSH to AFB₁-DNA binding was found tobe 10–15 times higher with the hamster in comparison withthe rat indicating enhanced inactivation of the ultimate carcinogenicmetabolite in the hamster. These results are discussed in relationto AFB₁-DNA binding and AFB₁ hepatocardnogenicity in resistantand sensitive species.     

Absence of p53 mutation at codon 249 in duck hepatocellular carcinomas from the high incidence area of Qidong (China)

Dietary aflatoxin and hepatitis B virus infection may play arole in generating the p53 tumor suppressor gene codon 249 hotspotmutation found in human hepatocellular carcinomas (HCCs) fromQidong (China) and southern Africa. No data are available onthe HCC site-specific mutation of the p53 gene in hepadnavirus-infectedanimals exposed to AFB₁. We have searched for the presence ofp53 gene codon 249 mutations in both duck hepatitis B virus(DHBV) positive and negative HCCs of domestic ducks from Qidong,where the human p53 hotspot is so prevalent, as well as in duckHCCs experimentally induced by AFB₁. Direct sequencing of DNAamplification products encompassing p53 codon 249 did not revealany mutations in 11 HCCs from Qidongducks, regardless of thestatus of DHBV infection. In addition no mutation was detectedin four HCCs from AFB₁-treated ducks. This contrasts with thehuman data; however, in humans, the mutation and the preferentialbinding of AFB₁ to codon 249 occurs at the third nucleotideG, while in duck, the codon 249 lacks this G residue. The DNAsequence of adjacent codons is also different in the two specieseven though the amino acid sequence is identical. This may explainthe low frequency of mutation we have observed. In addition,species differences in metabolism and DNA repair could influencethe occurrence of codon 249 mutations.