SHORT COMMUNICATION: Evaluation of the post-initiation effects of oltipraz on aflatoxin B1-induced preneoplastic foci in a rat model of hepatic tumorigenesis

Previous studies have demonstrated that ingestion of 5-(2-pyimmyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) during the aflatoxin B¹ (AFB₁)treatment phase completely prevented hepatic cancer. In thisstudy we evaluated the effect of feeding oltipraz during thepost-AFB₁ treatment phase. Fifty-five male F344 rats were dividedinto five groups. All rats were gavaged with 25 µg AFB₁/rat,five times a week for two successive weeks. The rats were fedthe oltipraz-supplemented diet according to three differentfeeding regimes: during the AFB₁ treatment phase (1 week priorto, during and 1 week after the last gavage with AFBj); duringthe post-treatment phase; or throughout the entire time of theexperiment. Phenobarbital-supplemented diet was fed during post-treatmentphase to one group and this was used as a positive control forthe promotion of AFB₁-induced focal growth. The burden of putative,preneoplastk, hepatic glutathione S-transferase P-positive fociwas evaluated at 13 weeks after the AFB₁ treatment phase. Asseen previously, oltipraz fed during the AFB₁ treatment phasesignificantly inhibited focal development, i.e. the volume percentof the liver occupied with foci was reduced by 87%. Oltiprazwhen fed during the post-treatment phase neither inhibited norenhanced focal development.     

R-Goitrin- and BHA-induced modulation of aflatoxin B1, binding to DNA and biliary excretion of thiol conjugates in rats

Previous studies have shown that dietary R-goitrin is a potentinducer of hepatic glutathione S-transferase (GST) and epoxidehydrolase activities but has no effect on components of themixed function oxidase system (ethoxycoumarin O-deethylase andcytochrome P-450). In the present work effects of dietary R-goitrin(200 p.p.m.) or butylated hydroxyanisole (BHA) (7500 p.p.m.)on GST activity, binding of aflatoxin B₁, (AFB]) to DNA, invivo, and biliary excretion of thiol conjugates of AFB₁ in ratswere studied. Increases of GST activities (1.9- and 2.1-fold)were accompanied by reductions in AFB₁-DNA binding (43% and85%) and increases (1.7-and 2.2-fold) in biliary excretion ofAFB₁-thiol conjugates in R-goitrin and BHA groups, respectively.Microsomal aflatoxin 8,9-epoxidase activities were not increasedin either treatment group. The role of GST induction in thecarcinogenesis of AFB₁ and the anticarcinogenic potential ofR-goitrin are discussed.     

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.     

Dose-related inhibition of aflatoxin B1 induced hepatocarcinogenesis by the phenolic antioxidants, butylated hydroxyanisole and butylated hydroxytoluene

The effect of butylated hydroxyanisole (BHA) or butylated hydroxytoluene(BHT) on the carcinogenicity in rats of aflatoxin B₁ (AFB₁)was investigated. AFB₁ was administered by gastric intubationto male F344 rats at 25µg/kg body wt three times a weeksuch that a total dose of 1.5 mg/kg (0.48mmol/kg) body wt wasgiven over a period of 20 weeks and diets containing either1000 or 6000 p.p.m. BHA or BHT were fed starting one more weekbefore carcinogen, during administration and for one week aftercessation. Animals were killed during exposure and at intervalsup to 24 weeks after cessation. Liver altered foci and neoplasmswere quantified using the exclusion of cellular iron after iron-loadingand -glutamyl transpeptidase reaction, as well as conventionalstaining for identification. Exposure to AFB₁ alone inducedsubstancial numbers of altered foci after 20 weeks and at 24weeks after cessation of exposure, the incidence of hepato-cellularneoplasms was 63%. In the groups receiving BHA or BHT togetherwith AFB₁ the numbers of altered foci were decreased at alltime points and at termination, the final incidence of livercell neoplasms and number of neoplasms peranimal were also reducedin a dose-related manner. Neoplasms in other organs were rareand were not affected by antioxidant treatment, except for apossible reduction of colon cancer. Thus, BHA and BHT inhibitedthe hepatocarcino-genesis of concurrently administered AFB₁without shifting the organotropism.     

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.     

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.     

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.     

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.     

Ethoxyquin alone induces preneoplastic changes in rat kidney whilst preventing induction of such lesions in liver by aflatoxin B1

Pretreatment of Fischer 344 rats with the antioxidant ethoxy-quin(EQ), followed by administration of aflatoxin B₁, (AFB) in thecontinuing presence of EQ was used to examine the effect ofthe antioxidant on liver and kidney. EQ (0.5% in diet) completelyprevented the formation of AFB₁-induced preneoplastic liverlesions as judged by morphological alteration, or by markerssuch as gamma glutamyl transpeptidase, glutathione S-transferaseP or J1, an unknown membrane-bound antigen. While protectionwas afforded to the liver, EQ alone caused severe damage tothe kidney. Many changes were those of chronic glomerulonephrosis,such that EQ appeared to accelerate the ageing process. In addition,many hyperplastic and putative preneoplastic tubules were visible,suggesting that EQ may be exerting a carcinogenic effect inthe kidney.     

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.     

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.     

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.     

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.     

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.     

Aflatoxin B1: effect on the synthesis and degradation of mitochondrial proteins in hepatocyte monolayers

The effect of aflatoxin B₁ (AFB₁), a hepatocarcinogen, on mitochondrialand general protein synthesis and degradation has been studied.AFB₁ (0.003, 0.03, 0.25 µg ml^–1) inhibited totalprotein synthesis over a 5 h period by 30, 64 and 82%, respectively,measured by incorporation of [³H]leucine. After 24 h in thepresence of AFB₁ inhibition was 23, 77 and 100%, respectively.AFB₁ inhibited total hepatocyte protein degradation in a concentrationindependent manner by 50% i.e., from 1.4% h ^–1 to 0.7%h^–1. The immediate effect of AFB₁ on mitoribosomal andtotal mitochondrial protein synthesis and mitochondrial degradationhas been assessed by two methods. Mitoribosomal synthesis ofproteins was inhibited over a 5 h period by AFB₁ in a concentrationindependent manner by 43%. Total mitochondrial protein synthesisshowed a 23 and 45% inhibition by AFB₁ (0.003 and 0.03 µgAFB₁ ml^–1) over a 4 h period and 25 and 72% inhibition,respectively, after 24 h in culture. The rate of mitochondrialprotein degradation was not altered. AFB₁ inhibits dibutyrylcyclic AMP-induced tyrosine amino transferase (TAT) activityin hepatocytes by 57% at 0.003 ug ml^–1 and 100% at 0.03µg ml^–1 over a 24 h period. Dibutyryl cyclic AMPincreases the rate of degradation of proteins in hepatocytemonolayers from 1.4% h^–1 to 2.7% h^–1 and was inhibitedat both concentrations of AFB₁ used. AFB₁ causes a rapid inhibitionof total hepatocyte protein synthesis, synthesis of proteinsin hepatocyte mitochondria and the synthesis of imported mitochondrialproteins. General hepatocyte and dibutyryl cyclic AMP-inducedprotein degradation are significantly inhibited by AFB₁ whereasthe degradation of mitochondrial proteins is unaffected.     

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.     

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.     

Hepatic and extrahepatic bioactivation and GSH conjugation of aflatoxin B1 in sheep

Whole-body autoradiography of [³H]aflatoxin B₁ ([³H]AFB₁ inlamb showed a localization of bound labelling, in addition tothe liver, in the nasal olfactory and respiratory mucosa, inthe mucosa of the nasopharynx, pharynx, oesophagus, larynx,trachea, bronchi and bronchioles and in the palpebral and bulbarconjunctiva. Microautoradiography revealed that the bound materialwas confined to specific cell types in extrahepatic tissues.Whole-body autoradiography also showed a labelling of pigmentedtissues (such as the eye melanin), which can be ascribed toa melanin affinity of AFB₁ In vivo experiments, performed withmicrosomal preparations of tissues from ewe and lamb showedthat several of the extrahepatic tissues were more efficientthan the liver in forming DNA-bound AFB₁ metabolites. The nasalolfactory mucosa was by far the most effective tissue in thisrespect. AFB, induced a high number of gene mutations in Salmonellatyphimurium TA100 when incubated with super natant preparations(9000 g) of the nasal olfactory mucosa, whereas incubationswith preparations of the liver resulted in a lower effect. Ithas been reported that AFB₁ can induce nasal tumours in sheep.When microsomal preparations of various tissues were incubatedin the presence of reduced glutathione (GSH), but without anyaddition of cytosolic glutathione-S-transferase (GST), a drasticdecrease in the AFB₁-DNA binding was seen. Analyses of the water-solublemetabolites formed in the microsomal incubations supple mentedwith GSH showed fluorescent and ninhydrin-positive metabolitesthat were not present in the absence of GSH. These results indicatethat sheep tissues have intrinsic microsomal GST or cytosolicGSTs associated with the microsomal fraction with a high capacityto catalyse the conjugation of bioactivated AFB₁ to GSH. Theresults of the present study show that several extrahepatictissues of sheep have a potent capacity to bioactivate AFB₁and also a high capacity to GSH conjugate the bioactivated AFB₁.     

Lack of influence of butylated hydroxytoluene on modification of lung microsome mediated aflatoxin B1-DNA binding: role of pulmonary glutathione S-transferase

Phenolic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are known to inhibit tumor formation due to several chemical carcinogens including aflatoxin B1 (AFB1). Metabolic activation of AFB1 by lung microsomes and possible modification by dietary BHA was reported in an earlier communication (Allameh et al. (1988) Cancer Lett., 40, 49). Here we report the effect of dietary BHA at a high dose (0.75% for 15 days) and a low dose (0.06% for 180 days) on the activation and inactivation of AFB1 by subcellular preparations of lung. BHT at high dose alone induced hepatic cytosolic glutathione (GSH) S-transferases activity while the pulmonary enzyme was unaffected by BHT feeding. This observation was substantiated when the addition of lung cytosol from control and BHT-treated rats showed similar inhibition (50%) in the microsome mediated AFB1-DNA binding. Thus BHT appears to have little influence on the pulmonary metabolism of AFB1.