The model Ah-receptor agonist {beta}-naphthoflavone inhibits aflatoxin B1--DNA binding in vivo in rainbow trout at dietary levels that do not induce CYP1A enzymes

ß-Naphthoflavone (BNF), a well-known Ah-receptor agonist,has been believed to inhibit aflatoxin B₁(AFB₁) carcinogenesisin rats and rainbow trout primarily through induction of thecytochrome P450 1A (CYP1A) enzyme subfamily and consequent diversionof AFB₁ to the less carcinogenic phase I metabolite aflatoxinM₁(AFM₁). This study investigates the dose responsive effectsof dietary BNF treatment on CYP1A induction, AFM₁ formation,AFB₁–8, 9-epoxide formation and AFB₁-DNA binding in thetrout model. Pre-feeding diet containing 10–200 p.p.m.BNF after AFB₁ i.p. injection provided dose-dependent inductionof CYP1A-dependent ethoxyresorufin-O-deethylase (EROD) activityand inhibition of in vivo AFB₁-DNA binding. However, most ofthe observable inhibition of DNA adduction (45% inhibition)had occurred at 10 p.p.m. BNF without detectable EROD induction;higher doses of BNF up to 200 p.p.m. induced EROD >6-foldbut provided only another 15% inhibition of DNA adduction invivo. When in vitro AFB₁-DNA binding was assessed using livermicrosomes from trout fed 10–100 p.p.m. BNF, induced microsomalEROD activity correlated moderately with reduction of in vitroAFB₁-DNA binding activity. However, BNF treatment in a low doserange (0.2–10 p.p.m.) also strongly inhibited in vivohepatic AFB₁-DNA binding (69% inhibition at 5 p.p.m. BNF inthis experiment), in a dose-dependent manner, in the completeabsence of detectable EROD induction. The microsomes from 5p.p.m. BNF-treated trout had no more EROD activity than controlmicrosomes, and no less capacity for catalyzing AFB₁-DNA bindingin vitro than control microsomes. Thus, the potent inhibitionof hepatic AFB₁-DNA binding in vivo by 5 p.p.m. BNF was a resultof neither CYP1A enzyme induction nor irreversibly reduced catalyticcapacity for AFB₁–8, 9-epoxide formation. Direct analysisof AFB₁ metabolites formed in vitro by liver microsomes fromtrout fed 10, 100 and 500 p.p.m. BNF showed that low dietaryBNF (10 p.p.m.) neither induced microsomal CYP1A-mediated AFM₁formation nor altered AFB₁–8, 9-epoxide formation comparedto the control. By comparison, 100 and 500 p.p.m. BNF pretreatmentsignificantly elevated microsome-catalyzed AFM₁ formation invitro (P0.001), and this increase was highly correlated withincreased EROD activity (r² = 0.999, P< 0.001). Upon in vitroaddition, BNF was found to be a potent inhibitor of microsome-mediatedAFB₁–8,9-exo-epoxide formation (IC₅₀=2.6±0.1µM)and AFB₁-DNA binding (inhibition constant K₁= 3.03±0.25µM).These findings indicate that CYP1A enzyme induction can contributemodestly to BNF protection against AFB₁ in this species bothin vivo and in vitro at higher BNF doses, but does not do soat lower doses. Instead, enzyme inhibition by BNF against AFB₁8,9-epoxidation appears to be the predominant protective mechanismat higher BNF doses, and the sole protective mechanism at lowdoses, in the rainbow trout. These findings demonstrate thatmechanisms of chemoprevention can change with anticarcinogendose, and caution that even potent induction of phase I or phaseII activities does not assure that pathway to be a predominantprotective mechanism in vivo.     

Effect of manipulation of {gamma}-glutamyl transpeptidase levels on biliary excretion of aflatoxin B1 conjugates

The reactive intermediate of aflatoxin B₁ (AFB₁) forms a glutathioneconjugate (AFB₁-GSH) and this has been shown to be a substratefor -glutamyl transpeptidase (GGT) in vitro. This study describesthe biliary excretion of AFB₁-GSH and the product of GGT activity,the cysteinylglycyl conjugate (AFB₁-Cys-Gly), following i.v.injection of AFB₁ (5 µmol kg^–1) in control malerats and in rats that had been maintained on a toxic diet containing4 p.p.m. AFB₁ for 10–12 weeks prior to the experiment.AFB₁ metabolites in the bile were analyzed by reverse phaseh.p.l.c. and GGT activity in the liver was assessed histochemicallyand by quantitative fluorimetric assay. In the control malerats (n = 6) AFB₁-GSH and AFB₁-Cys-Gly together were detectedas 4.2 ± 2.3% of the i.v. dose over the first two hoursof bile collection (AFB₁-GSH: AFB₁-Cys-Gly, 5.5:1). GGT activity(38.2 + 7.9 nmol product formed/g liver) was located in thebile duct epithelium. The group maintained on a toxic diet (n= 2) showed higher levels of AFB₁-Cys-Gly (AFB₁ GSH: AFB₁-Cys-Gly,1:1). GGT activity was elevated (5–10 x control levels)and located in numerous foci throughout the liver. The involvementof GGT in the biliary excretion of AFB₁-Cys-Gly was demonstratedby in vivo inhibition of GGT by administering AT125 to a groupof animals (n = 3) 15 min prior to the injection of AFB₁. Histochemicaland quantitative estimation of GGT confirmed total inhibitionthroughout the liver and conversion of AFB₁-GSH to AFB₁-Cys-Glywas almost completely blocked. Female Fischer 344 rats (n =3) showed slightly elevated AFB1-Cys-Gly excretion and higherGGT activity (79.3 + 26.3 nmol/min/g liver) compared to controlmale rats.     

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.     

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.     

Evidence for an indirect mechanism of aflatoxin B1 inhibition of rat liver nuclear RNA polymerase II activity in vivo

Previous studies suggested multiple sites of action of aflatox-inB₁ (AFB₁) in vivo to inhibit rat liver nuclear RNA synthesis- it impairs nucleolar DNA template function and inhibits RNAPolymerase II activity. We have previously shown that AFB₁ activatedin vitro inhibits nucleolar RNA synthesis. The question is whetherAFB₁ can inhibit RNA polymerase II under these in vitro conditions.Male Sprague-Dawley rats, 200 g, were injected i.p. with 0.6mg AFB₁ and liver nuclei were isolated 2 h later. When the totalnuclear free RNA polymerases were extracted and assayed in theabsence and presence of a-amanitin (3.2 µg/ml), we foundthat only -amanitin-sensitive (i.e., RNA polymerase II) activitywas inhibited (97%). DEAE-Sephadex chromatography confirmedthis result. When total nuclear free RNA polymerases were incubatedwith AFB₁ activated in vitro under conditions producing 70%inhibition of nucleolar RNA synthesis, no inhibition was observedfor either -amanitin-sensitive or -resistant activities. Similarresults were obtained with low and high (28 and 167 µg/ml)concentrations of AFB₁. This was further confirmed using highlypurified RNA polymerase II. We conclude that AFB₁ inhibitionof RNA polymerase II activity in vivo is not a result of directinteraction of AFB₁ to the enzyme.     

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.     

Effect of choline-deficiency and methotrexate administration on peroxisomal {beta}-oxidation, palmitoyl-CoA hydrolase activity and the glutathione content in rat liver

Hepatic metabolism of long-chain fatty acids was studied inmale rats fed a defined choline-defkient (CD) diet with andwithout choline and after methotrexate (MTX) administration.Peroxisomal ß-oxidation was increased 4-fold in theperoxisome-enriched fraction of CD-fed animals, whereas thecatalase activity was increased 1.3-fold. The urateoxidase activitywas marginally affected. The CD-fed rats also revealed elevatedcapacity for hydrolysis of palmitoyl-CoA in the cytosolic fraction(2.0-fold), whereas the microsomal palmitoyl-CoA hydrolase activitywas decreased. Notably, the increased peroxisomal ß-oxdiation,the catalase activity and palmitoyl-CoA hydrolase activities(the membrane-bounded and cytosolic) were almost fully preventedby adding choline to the CD-diet. Thus, the change in theseenzyme activities appears to be a consequence of a choline-deficiencyprovoked bythe CD diet. MTX administration of normal fed rats(ND diet) had no effects on the peroxisomal ß-oxidation,catalase activity and urate oxidase activity. MTX treatmentof the ND-fed animals, however, increased the mitochondrialpalmitoyl-CoA hydrolase activity and decreased the microsomalenzyme activity. As choline-deficiency and MTX increased thehepatic lipid level, the overall results suggest that fat accumulationis not an ‘induction signal’ for increased peroxisomalß-oxidation. The CD diet alone increased the reducedghitathione content in liver, whereas MTX did not significantlychange this level. Whether the changes of H₂O₂-generating peroxisomaloxidation of long-chain fatty acids may be an important stepin a chain of events, which eventually results in tumour formationby choline-deficiency, should be considered.     

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.     

Comparative mutagenicity of aflatoxins using a Salmonella/trout hepatic enzyme activation system

A modification of the Ames assay using rainbow trout (Salmogairdneri) liver postmitochondrial fraction (PMF) was developedto investigate the relative mutagenic potential of a seriesof aflatoxins (AFs). Preliminary experiments revealed that the20 000 x g (S20) liver fraction contained a higher metabolicactivity than either the S9 or S30 fractions, and that 5 mgof S20 protein/plate gave the highest mutagenic response. A9–24 h preincubation period at 25°C was also required.The results from comparative mutagenicity experiments showedthe following relative potencies: AFB₁ > AFL > AFG₁ >AFM₁ > AFB₂ > AFP₁ > AFQ₁. The relative potencies observedwith this in vitro system qualitatively correlated with thein vivo carcinogenic activity seen in trout, indicating thatthis assay is of value in predicting the carcinogenic potentialof mycotoxins in this species.     

Different inhibition of DNA synthesis by transforming growth factor {beta} and phenobarbital on GST-P-positive and GST-P-negative hepatocytes

Hepatocyte resistance against inhibition of DNA synthesis bytransforming growth factor ß₁ (TGF-ß₁) wasstudied in vitro. Hepatocytes were isolated from rats that hadreceived diethylnitrosamine (DEN) for 6 weeks. The effect ofTGF-ß₁ and phenobarbital (PB) on DNA synthesis indifferent cell populations was studied using bromodeoxyuridine(BrdU) incorporation and placental glutathione S-transferase(GST-P) as markers. It was found that GST-P-positive cells wereresistant to the growth inhibitory effect of TGF-ß₁and PB, whereas GST-P-negative cells were inhibited. It is concludedthat resistance to TGF-ß₁-dependent growth controlmay develop early during DEN-induced hepatocarcinogenesis.     

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.     

Enhancement of glutathione S-transferase placental-form positive liver cell foci development by microcystin-LR in aflatoxin B1-initiated rats

The objective of this study was to elucidate whether microcystin-LR(MC-LR), a hepatotoxic blue-green algal toxin in drinking water,is carcinogenic or possesses the ability to modulate aflatoxinB₁ (AFB₁)-induced hepatocarcinogenicity. In a medium-term liverbioassay, male Fischer 344 rats were given a single i.p. injectionof diethylnitrosamine (DEN, 200 mg/kg) followed by an i.p. injectionof MC-LR for 6 weeks after 2 weeks of DEN treatment. To studythe synergism between AFB₁ and MC-LR, DEN-treated rats weregiven an i.p. injection of AFB₁ (0.5 mg/kg) dissolved in dimethylsulfoxide (DMSO) followed by MC-LR at 2 weeks after the treatment.In a separate experiment, the rats were first given AFB₁ (0.5mg/kg) and 2 weeks later an i.p. injection of 1 or 10 µg/kgof MC-LR twice a week for 6 weeks. Most rats were subjectedto a two-thirds partial hepatectomy (PH) at week 3 and werekilled under anesthesia at week 8. Liver sections were analyzedfor glutathione S-transferase placental form (GST-P) expression,and subjected to histopathological examination for phenotypicalteration of hepatocellular foci. In rats that did not receiveDEN, MC-LR did not cause a significant increase in the numbersof GST-P-positive foci, whereas AFB₁ induced a slight increasein GST-P-positive foci development. In rats given DEN, MC-LRenhanced the expression of GST-P-positive foci, as did AFB₁but no synergism was observed. Histopathological analysis revealedthat the area of eosinophilic foci, a biomarker for preneoplasticliver lesion, markedly increased because of MC-LR. In rats givenAFB₁ as an initiator, treatment with MC-LR resulted in a synergisticincrease in the development of GST-P-positive foci. These resultssuggest that the hepatocarcinogenicities of MC-LR and AFB₁ canbe predicted in experimental animals with a medium-term bioassay.Furthermore, tumor promoting activity of MC-LR was demonstratedin rats treated with AFB₁.     

Aflatoxin B1 hydroxylation by the pregnenolone-16{alpha}-carbonitrile-inducible form of rat liver microsomal cytochrome P-450

The effects of treating rats with various pregnenolone-16-carbonitrile(PCN)-type inducers of cytochrome P-450p on the liver microsomalmetabolism of aflatoxin B₁ (AFB₁) were investigated. Treatmentof male rats with PCN resulted in a 6-fold increase in the 9-hydroxylationof AFB₁ to aflatoxin Q₁ (AFQ₁). Treatment of female rats withPCN resulted in a 16-fold increase in the formation of AFQ₁.The age-dependent decline in constitutive cytochrome P-450plevels in female but not male rats resulted in a sex differencein the formation of AFQ₁ in liver microsomes from untreatedrats (male: female 3: 1). The formation of AFQ₁ was stimulatedup to 5.4-fold when liver microsomes from triacetyloleandomycin(TAO)-treated rats were treated with potassium ferricyanide,which dissociates the complex between cytochrome P-450p andTAO. Treatment of male rats with the cytochrome P-450p inducer,dexamethasone, increased ( 7-fold) the 9-hydroxylation of AFB₁to AFQ₁ by liver microsomes, and also enhanced ( 2-fold) themicrosomal activation of AFB₁ to metabolites that were mutagenicto Salmonella typhimurium TA98 and TA100. These results indicatethat the 9-hydroxylation of AFB₁ to AFQ₁ is catalyzed by ratliver microsomal cytochrome P-450p.     

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.     

Metabolism of the cis and trans isomers of N-nitroso-2,6-dimethylmorpholine and their deuterated analogs by liver microsomes of rat and hamster

Liver microsomes from male Syrian golden hamsters and SpragueDawley rats metabolize the cis and trans isomers of N-nitroso-2,6-dimethylmorpholine(NNDM) to N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (HPOP)as the major product detectable by h.p.l.c. The rates of totalmetabolism are similar for both the cis and trans isomers; butthe cis isomer of NNDM yields >70% of the total product asHPOP while the trans isomer yields HPOP only as a minor product(20–30%) in both hamster and rat. The inability to identifyother products could be attributed to -hydroxylation which leadsto fragmentation of NNDM and loss of tritium label to water.In order to investigate the possibility of the participationof an -hydroxylation reaction, the metabolism of NNDM fullydeuterated at either the 3 and 5 (-d₄) or the 2 and 6 (ß-d₂)positions was examined and compared to the metabolism of theundeuterated compound (d0). Although the rates of metabolismof all of the cis and trans derivatives of NNDM were similar(^Vmax = 2.13 nmol/min/mg hamster microsomal protein) as determinedfrom measurements of substrate disappearance, the yields ofHPOP were different. Maximum HPOP yields were observed withcis -(d₄) NNDM (93.9% of the total), followed by cis d₀ NNDM(72.3%), trans -(d₄) NNDM (60.1%), trans d₀ NNDM (30.2%), cisß-(d₂) NNDM (19.5%) and trans ß-(d₂) NNDM(8.5%). These results suggest that a-hydroxylation is an alternativeto ß-hydroxylation. Since the carcinogenic potencyof the various deuterium derivatives of NNDM for the Syriangolden hamster parallels their ability to yield HPOP, ß-hydroxylationis closely related to pancreatic carcinogenesis in the hamster.Rat liver microsomal fractions showed the same patterns of HPOPformation to total metabolite yields as hamster liver microsomeswith both the cis and trans isomers. However, rates of NNDMmetabolism and HPOP formation were 7 times faster with hamsterthan with rat liver microsomes. Such a difference may be relatedto the failure of the cis isomer to induce pancreatic cancerin rats.     

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.     

Effect of varying the onset of exposure to DDT on its modulation of AFB1-induced hepatocarcinogenesis in the rat

We have previously reported that p, p'-dichlorodiphenyl-trichloroethane(DDT) inhibited the hepatocarcinogenicity of aflatoxin B₁ (AFB₁)if given to rats 1 week prior to AFB₁ but could enhance thecarcinogenesis when given 1 week after the completion of AFB₁treatment. However, simultaneous administration of DDT withAFB₁ resulted in a slight reduction in the incidence of livertumours. In the present experiment in which the dose of AFB₁was reduced to about half of that used previously, we observedthat DDT markedly inhibited the hepatocarcinogenesis if givento animals starting at the same time with AFB₁. On the otherhand, giving DDT to animals starting in the middle of AFB₁ treatmentresulted in a significant enhancement of hepatocarcinogenesis.DDT exhibited a maximal tumour promoting effect when given either1 or 3 weeks after completion of AFB₁ treatment. It enhancedthe number of animals bearing liver carcinomas as well as thenumber of carcinomas per animal. Determination of gammaglutamyltranspeptidasein the serum revealed that the activity increased only in animalsbearing big and/or a number of carcinomas in the livers especiallyin those promoted by DDT. These results therefore demonstratedthat DDT will act as an inhibitor of AFB₁-induced hepatocarcinogenesisif it is given to animals starting either prior to or at thesame time as carcinogen. On the other hand, it will act as atumour promoter if given to animals starting either in the middleof or after the completion of AFB₁ treatment.     

Activation of K-ras in aflatoxin B1-induced lung tumors from AC3F1 (A/JxC3H/HeJ) mice

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. Since point mutations atcodons 12, 13 and 61 of the K-ras protooncogene are often implicatedin chemically induced mouse lung tumors and in human lung adeno-carcinomas,we undertook an investigation of the role of K-ras activationin AFB₁-induced pulmonary carcino-genesis. Female AC3F1 (A/JxC3H/HeJ)mice were treated with AFB₁ (150 mg/kg i.p., divided into 24doses over 8 weeks), and 6–14 months after the completionof dosing mice were killed and pulmonary adenomas and carcinomasremoved. Of the 76 AFB₁-induced lung tumors analyzed by singlestrand conformation polymorphism (SSCP) and direct sequencing,75 possessed K-ras codon 12 mutations (46 GTT, 14 GAT, 13 TGTand 2 TTT; normal, GGT) and one had a GGC     

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     

Identification of an aflatoxin G1-serum albumin adduct and its relevance to the measurement of human exposure to aflatoxins

A major aflatoxin G₁ (AFG₁)-albumin adduct has been identifiedand characterized in rats following exposure to AFG₁. The productisolated from a Pronase digest of in vivomodified albumin wasidentical by chromatographic retention time to the syntheticproduct obtained by the acylase-catalysed deacetylation productof N-acetyl-L-lysine with 8,9-dihydro- 8,9-dibromo-AFG₁. Thein vitro product, AFG₁-lysine, was characterized by UV, fluorescence,¹H- and ¹³C-NMR spectroscopy and fast atom bombardment MS. Acompetitive enzyme-linked immunoassay for this adduct was establishedusing polyclonal antibodies to AFB, and this was used togetherwith an HPLC-fluorescence technique to quantitate the in vivoformation of AFG₁–albumin adducts in comparison to AFB₁.A linear dose–response relationship was observed in ratsfollowing single exposures to 0.1– 3 mg AFG₁/kg body wt.The levels of AFG₁-albumin adducts were determined to be 5.7-and 2.8-fold lower than with equivalent doses of AFB₁ as determinedby immunoassay and HPLC fluorescence respectively. The lowerbinding of AFG₁ and the lower levels in the human food supplycompared to AFB, suggest that the newly identified adduct couldbe added as an internal standard for methods using the measurementof aflatoxin-albumin adducts to quantitate human exposure toaflatoxin.