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.     

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.     

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.     

DNA binding and adduct formation of aflatoxin B1 in cultured human and animal tracheobronchial and bladder tissues

DNA binding and adduct formation of aflatoxin B₁ (AFB₁) wasstudied in cultured bladder and tracheobronchial explants fromhuman, monkey, dog, hamster and rat. Explants were exposed to[³H]AFB₁ (1 µM final concentration) in PFMR-4 medium (pH7.4) without serum for 24 h, after which epithetial cell DNAwas isolated by hydroxylapatite chromatography. Binding (µmolAFB₁/mol deoxyribo-nucledetide, mean ± SD) was higherin tracheobronchial tissues (human, 2.2 ± 2.4; rat, 5.7± 2.4; dog, 10.6 ± 6.6; hamster 134.6 ±44.6) than in bladder tissues (human, 1.5 ± 2.3; monkey,2.5 ± 1.1; rat, 3.8 ± 1.1; dog, 5.2 ± 2.3;hamster, 26.2 ± 13.3). These binding levels were notcorrelated with the relative susceptibilities of these speciesto AFB₁ hepatocarcinogenesis, in that the hamster and the dogare insensitive, but exhibited the highest binding, while thesusceptible species, the rat and the monkey, had lower binding.After acid hydrolysis of the isolated DNA, the [³H]AFB₁-DNAadducts were separated by h.p.l.c. In all cases, almost allof the [³H]AFB1-DNA represented addition of AFB₁ to the N⁷ atomof guanine, the major adduct (40–79% of the total) being8, 9-dihydro-8-(N⁵-formyl-2', 5', 6' -triamino-4' -oxo-N⁵-pyrimidyl)-9-hydroxyAFB₁,with minor amounts (7–28%) of 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyAFB₁.In some cases small amounts (0–8%) of unknown, polar adductscould be detected. It is concluded that, qualitatively, AFB₁-DNAadduct formation by human and animal bladder and tracheobronchilalexplants is similar to that found in other in vitro and in vivoextrahepatic and hepatic systems, but that in vitro bindingdata of AFB₁ to extrahepatic animal tissues can probably notbe used to predict the susceptibility of the human to AFB₁-relatedcardnogenesis in these tissues.     

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.     

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 mutagenesis in Salmonella typhimurium and DNA damage in cultured rat and human tracheobronchial tissues by ellagic acid

Ellagic acid (EA), a plant phenol found in various fruits andnuts, was examined for its ability to inhibit aflatoxin B₁ (AFB₁)mutagenesis in strain TA 100 of Salmonella typhimurium. In thepresence of rat liver S-9 microsomal preparation, EA (1.5 µg/plate)inhibited the number of mutations induced by AFB, (0.5 µg/plate)by 50%. EA at a dose of 1000 µg/plate inhibited the mutationfrequency by >90%. EA was also tested for its ability toinhibit the DNA binding and adduct formation of AFB₁ in culturedexplants of rat trachea and human tracheobronchus. Explantswere incubated in medium containing EA at concentrations of10, 50 and 100 µM for 16 h foUowed by the addition of1 µM [₃H]AFB₁ and EA for 24 h. DNA was isolated by phenolextraction and hydroxylapatite chromatography. EA caused a dose-dependentinhibition in the covalent binding of AFB₁ to the DNA of boththe rat trachea (9—57% inhibition) and human tracheobronchus(24—79% inhibition). After acid hydrolysis of the isolatedDNA, the AFB₁—DNA adducts were separated by h.p.l.c. Intissues from both species, the major AFB₁—DNA adductswere AFB₁-N⁷-Gua [8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyAFB₁] andAFB₁-N⁷-FaPyr (major) [8,9-dihydro-8- (2, 6-diamino-4-oxo-3,4-dihydro-pyrimid-5-ylformamido)-9-hydroxyAFB₁], and the formation of these adductswas reduced by 28—76% in the presence of EA. These dataindicate that EA has the potential to act as a naturally occurringinhibitor of AFB₁-related respiratory damage in rats and inhumans.     

Modulation of microsome-mediated aflatoxin B1 binding to exogenous and endogenous DNA by cytosolic glutathione S-transferases in rat and hamster livers

Microsome mediated aflatoxin B₁ (AFB₁) binding to exogenousand endogenous DNA and its modulation by cytosolic glutathione(GSH) S-transferases have been examined in rat and hamster livers.Kinetic studies over a wide range of cytosol concentrationsindicate that cytosol from the hamster is several-fold moreeffective than that from the rat in inhibiting AFB₁ bindingto exogenous calf thymus DNA mediated by microsomes from eitherspecies. Low concentrations of GSH (0.1–0.2 mM) are requiredfor 50% inhibition of AFB₁—DNA binding by cytosol. Withexogenous DNA, combined microsome-cytosol fractions from thehamster give more AFB₁—DNA binding than those from therat. However, with nuclei as a source of endogenous DNA, AFB₁—DNAbinding is less with combined microsome-cytosol fractions fromthe hamster than those from the rat. Cytosolic inhibition ofAFB₁—DNA binding is almost completely reversed in thepresence of 1 mM levels of either trichloropropene oxide orstyrene oxide. Quantitation of AFB₁—DNA binding and AFB₁-GSH conjugation indicate an inverse relationship between thesetwo processes. Cytosol from the rat has less capacity than thatfrom the hamster to form an AFB₁—GSH conjugate. HepaticGSH levels are about equal (6–7 mM) in both species. I.p.administration of [¹⁴C]AFB₁ 2 h before sacrifice gives moreAFB₁ binding to hepatic nuclear DNA in rats than in hamsters.However, depletion of hepatic GSH levels by 80% by i.p. administrationof diethylmaleate (600 mg/kg) increases AFB₁—DNA binding2- to 3-fold in both species. The role of cytosolic GSH S-transferasesin modulating hepatic AFB₁—DNA binding in rats and hamstersis discussed.     

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene(N-OAc-AAF) have previously been shown to induce dose-dependentDNA strand breaks in primary hepatocytes from mice and rats.In an attempt to determine the relationship between the extentof DNA strand breaks and the formation of specific DNA-carcinogenbound adducts in murine liver, the capability of N-OH-AAF andN-OAc-AAF to induce both DNA single strand breaks and adductformation in in vivo and in primary hepatocytes was measured.N-OH-AAF induced a low level of DNA damage in F344 rats (10mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment.The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene(Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene(Gua-C8-AF) 34% versus 67% and Mguanin-N²-yl)-2-acetylaminofluorene(Gua-N²-AAF) 11% versus 10%, respectively, for rat and mouseliver. An additional unknown adduct (12%) was detected in mouseliver. Dose dependent DNA binding and formation of individualDNA adducts were observed in rat and mouse primary hepatocytesfollowing 1 h exposure to [ring-³H]-N-OH-AAF (0.1-20 µM)and [ring-³-N-OAc-AAF (5–20 /M). The patterns of DNA adductsin mouse and rat primary hepatocytes exposed to N-OH-AAF andN-OAc-AF were similar to those obtained in liver following invivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon(10^–4M) completely inhibited DNA damage induced by N-OH-AAFin mouse and partially in rat hepatocytes while DNA damage causedby N-OAc-AAF was only partially inhibited by paraoxon (10^–4M) in both species. Parallel experiments showed that paraoxon,at low concentration (10^– M), did not alter either thelevel of DNA binding or the pattern of adduct formation in rathepatocytes treated with N-OH-AAF (20 µM). However, at10^–4 M paraoxon partially blocked DNA binding (60%) andthe formation of Gua-C8-AAF (95%) and Gua-N²-AAF (80%) whileGua-C8-AF was increased twofold. In mouse hepatocytes paraoxonpretreatment (10^–4M) inhibited the formation of Gua-C8-AFby 70% following exposure to N-OH-AAF (20 µM). Gua-C8-AAFand Gua-N²-AAF were also inhibited but only at 10^–4M paraoxon.Paraoxon (10^–6 and 10^–4 M) pretreatment induceddosedependent partial inhibition of the covalent binding ofN-OAc-AAF to rat DNA and the formation of all guanine adducts.In the mouse, paraoxon (10^–6 and 10^–4 M) inhibitedthe formation of Gua-C8-AF while it increased Gua-C8-AAF. Theseresults indicate that a positive correlation exists betweenthe extent of DNA strand breaks and the formation of eitherGua-C8-AAF or Gua-C8-AF.     

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.     

Studies on the detoxication of microsomally-activated aflatoxin B1 by glutathione and glutathione transferases in vitro

Aflatoxin B₁ (AFB₁)-8,9-oxide, the proposed ultimate carcinogenis conjugated enzymically with glutathione (GSH) to give 8-S-glutathionyl)-9-hydroxy-8,9-dihydroAFB₁ (AFB₁-SG). The GSH conjugate isolated from rat bile wasshown, on the basis of ¹ H n. m. r. to be identical to AFB₁-SG. Of the seven soluble rat liver GSH transferases 1-1, 1-2,2-2, 3-3, 3-4, 4-4 and 5-5 (see reference 1 for the new systemof nomenclature), only the first three were active with microsomallygenerated AFB₁ -88, 9-oxide, their rates of conjugation being1.1, 0.61, and 0.64 nmol/min/mg enzyme, respectively. AFB₁ -SGis a thioacetal, but it was not formed from the incubation ofthe hemiacetal, AFB₁ -8,9-dihydrodiiol, with GSH or GSH plusGSH transferase 1-1 plus 1-2. The covalent binding of in vitromicrosomally activated AFB₁ to DNA and the formation of AFB₁-SGwere linearly related to AFB₁concentration in range of 0.2–2µg/ml.DNA binding was decreased by 38% by the competing formationof AFB₁-SG throughout this range of concentrations. These resultsare in accord with the observation of Scott Appleton et al.(Cances Res., 42, 3629-3662) that, in the rat in vivo, thereis no evident threshold for the binding of AGB₁ to DNA. Thesefindings are also consistent with further observation, reportedin this paper that GSH and GSH transferases have no effectnon the mutagenicity of microsomally activated AFB₁ to Salmonellatyphimurium TA 100.     

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.     

Production of monoclonal antibodies to guanine imidazole ring-opened aflatoxin B1DNA, the persistent DNA adduct in vivo

Monoclonal antibodies were produced following immunisation ofmice with guanine imidazole ring-opened aflatoxin B₁ DNA (iroAFB₁ DNA), coupled electrostatically to methylated keyhole limpethaemocyanin. Three monoclonal hybridoma lines producing antibodiesspecific for iro AFB₁ DNA were grown as ascites tumours andsuitable dilutions of the ascitic fluid (1:8000–1:50 000)used in a competitive enzyme linked immunosorbent assay (ELISA)to measure reactivity of the antibodies to a variety of aflatoxinand nucleic acid-related compounds. These antibodies recogniseAFB₁ bound to DNA at levels 10⁴–10⁵ times lower concentrationthan unmodified calf thymus DNA or 8,9-dihydro-8,9-dihydroxy-aflatoxinB₁; and show 2–5 times the affinity to iro AFB₁ DNA comparedto AFB₁ DNA. The concentration of AFB₁ in iro AFB₁ DNA producing50% inhibition in a competitive ELISA was 1.8 x 10^–7 molar.Using the most sensitive hybridoma line, levels of 1 adductin 300 000 nudeotides would be detectable, which is the levelof binding found in the rat and hamster in vivo. These monoclonalantibodies should therefore prove useful in detecting theselesions in animal and human tissue samples exposed to aflatoxins.     

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.     

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.     

Biotransformation of aflatoxin B1 in human lung

In addition to being a potent hepatocarcinogen, aflatoxin B₁(AFB₁) is a pulmonary carcinogen in experimental animals, andepidemiological studies have shown an association between AFB₁exposure and lung cancer in humans. This study investigatedAFB₁ bioactivation and detoxification in human lung tissue obtainedfrom patients under-going clinically indicated lobectomy. [³H]AFB₁was bioactivated to a DNA binding metabolite by human wholelung cytosols in a time-, protein concentration-, and AFB₁ concentration-dependentmanner. Cytosolic activation of [³H]AFB₁ correlated with lipoxygenase(LOX) activity and was inhibited by the LOX inhibitor nordihydroguaiareticacid (NDGA; 100 µM), indicating that LOXs were largelyresponsible for the observed cytosolic activation of AFB₁. Inwhole lung microsomes, low levels of indomethacin inhibitableprostaglandin H synthase (PHS)-mediated [³H]AFB₁-DNA bindingand cytochrome P-450 (P450)-mediated [³H]AFB₁-DNA binding wereobserved. Cytosolic glutathione S-transferase (GST)-catalyzeddetoxification of AFB₁–8,9-epoxide, produced by rabbitliver microsomes, was minimal at 1 and 10 µM [³H]AFB₁.With 100 µM [³H]AFB₁, [³H]AFB₁–8, 9-epoxide conjugationwith reduced glutathione was 0.34 ± 0.26 pmol/mg/h (n= 10). In intact, isolated human lung cells, [³H]AFB₁ bindingto cellular DNA was higher in cell fractions enriched in macrophagesthan in either type II cell-enriched fractions or fractionscontaining unseparated cell types. Indomethacin produced a 63–100%decrease in [³H]AFB₁-DNA binding in macrophages from five ofseven patients, while NDGA inhibited [³H]AFB₁ -DNA adduct formationby 19, 40 and 56% in macrophages from three of seven patients.In alveolar type O cells, NDGA decreased [³H]AFB₁-DNA bindingby 30–100% in cells from three patients and indomethacinhad little effect. SKF525A, an isozyme non-selective P450 inhibitor,enhanced [³H]AFB₁ binding to cellular DNA in unseparated cells,macrophages, and type II cells, suggesting that P450-mediatedbioactivation of AFB₁ is not a major pathway by which AFB₁–8,9-epoxideis formed in human lung cells. Overall, these studies suggestthat P450 has a minor role in the bioactivation of AFB₁ in humanlung. Rather, LOXs and PHS appear to be important bioactivationenzymes. Co-oxidative bioactivation of AFB₁, in combinationwith the low conjugating activity displayed by human lung cytosolicGSTs, likely contributes to human pulmonary susceptibility toAFB₁.     

The relationship between N-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes

The metabolism of N-nitrosodimethylamine (NDMA) and its methylationof DNA were simultaneously determined in hepatocytes isolatedfrom untreated and saline- and pyrazole-treated male Sprague-Dawleyrats. Metabolism of NDMA was directly measured by monitoringits disappearance via gas chromatography coupled with a sensitiveand specific detector for N-nitrosamines. DNA methylation wasdetermined in the same cells employed in the metabolism studiesusing a monoclonal antibody-based competitive ELISA procedurespecific for O⁶-methyldeoxyguanosine (6-Me-dG). The apparentK_m and V_max, for NDMA metabolism are 61 µM and 56 pmol/min/10⁶cells respectively for hepatocytes isolated from untreated rats.It was found that the addition of pyrazole to the in vitro hepatocyteincubations caused a dose-dependent inhibition of both metabolismand DNA methylation. However, when DNA methylation is expressedas a function of NDMA metabolized, there is no significant differencebetween hepatocyte incubations without or with pyrazole, withan average value of 79 nmol 6-Me-dG/mol dG/nmol NDMA metabolized.Based on the pyrazole inhibition studies, cyto-chrome P450IIE1is responsible for at least 60% of the DNA methylation in rathepatocytes. In pyrazole-pretreated rats there was an inconsistentincrease in NDMA metabolism, but when metabolism was elevatedso was DNA methylation. In contrast, microsomes isolated frompyra zole-pretreated rats consistently showed elevated metabolismof NDMA. Based on the simultaneous determination of adduct levelsand metabolism, there is 1 6-Me-dG adduct formed/133 000 NDMAmolecules metabolized in the uninduced hepatocytes.     

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.     

The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures

Primary cultures of rat hepatocytes were exposed to [ring-³H]-N-hydroxy-2-acetylaminofluorene(N-OH-AAF) for 4 h, and the RNA and DNA nucleoside adducts wereisolated and identified by h.p.l.c. The DNA adducts were shownto be N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF),N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxy-guanosin-8-yl)-2-acetylaminofluorene(dG-N2-AAF), while the RNA adducts were N-(guanosin-8-yl)-2-acetyl-aminofluorene,and N-(guanosin-8-yl)-2-aminofluorene. The removal of theseadducts was measured up to 38 h following the cessation of exposureof the hepatocytes to N-OH-AAF. The dG-C8-AAF adduct was removedwith a half-life of approximately 10 h, while dG-N²-AAF anddG-C8-AF remained constant for 14 h, followed by a slow rateof removal. The dG-C8-AAF adduct initially composed about 60%of the total DNA adducts of primary hepatocytes in contrastto the 20% found in liver in vivo. The formation of the 3 DNAadducts and the different rates of repair indicate that primarycultured rat hepatocytes may be a valuable system to study initiationof liver carcinogenesis by N-OH-AAF.