Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus)

Whole-body autoradiography of [³H]aflatoxin B₁ (³H-AFB₁) inmarmoset monkeys (Callithrix jacchus) showed a localizationof bound labelling, in addition to the liver, in the nasal olfactoryand respiratory mucosa and the mucosa of the nasopharyngealduct, the pharynx, the larynx, the trachea and the oesophagus.In vitro microautoradiography of these tissues incubated with³H-AFB₁ showed a localization of bound radioactivity in somecells in the epithelial linings of the tissues. This bindingwas abolished when the incubations were performed in the presenceof the cytochrome P-450 inhibitor metyrapone. These resultsindicate a cytochrome P-450-dependent bioactivation of AFB₁in some structures in the epithelia of the nasal olfactory mucosaand the upper respiratory and alimentary pathways, in additionto the liver, in the marmoset monkey. Quantitation of the invitro formation of tissue-bound radioactivity from the ³H-AFB₁showed that the nasal olfactory mucosa had the highest capacityto form bound metabolites among the tissues examined. Liquidchromatography of lipid-soluble metabolites formed by the nasalolfactory mucosa and the liver showed that aflatoxin M₁ (ATM₁)was the major metabolite. AFM₁ was also the major metabolitefound in the liver in vivo. In the pigmented tissues of themarmosets there was an accumulation of nonmetabolized AFB₁.This can be related to a melanin-affinity of AFB₁. The describedtissues with a capacity to accumulate and metabolize AFB₁ maybe potential targets for the carcinogenicity of this substancein the marmoset monkey.     

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.     

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.     

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 -hepatocarcinogenesis in rats by {beta}-naphthoflavone

Effects of ß-naphthonflavoe (ßNF) on theactivity of hepatic microsomal aflatoxin B₁ (AFB₁)-4-hydroxylase- the cytochrome P-450-dependent enzyme system which catalyzesthe metabolism of AFB₁ to AFM₁ - and on AFB₁ induced in vivohepatocarcinogenesis were investigated in weanling male Fischerrats. A single i.p. injection of ßNF in doses of 20mg/kg and 150 mg/kg induced AFB₁ -4-hydroxylase 3- and 4-fold,respectively, 48 h post injection. Feeding of diet containing0.01% ßNF for a period of 9-weeks induced AFB₁ 2-fold.AFB₁, given by intubatlon in a dose of 25 µg five times/weekfor 8 weeks, produced 42 weeks later a 100% incidence of liverlesions (neoplastic foci, nodules or tumors), but feeding ßNFin diet at a con centration of 0.015% for one week prior toand during the 8 weeks of AFB treatment inhibited AFB₁ hepatocarcinogenesis by -7%. These results are in accord with the suggestionthat AFB₁ induction may be associated with the inhibition ofAFB₁ carcinogenesis, possibly occurring as a consequence ofaccelerated detoxi-fication of AFB₁ via its conversion to AFM₁     

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.     

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.     

Binding and metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene by seven purified forms of cytochrome P-450

Seven different forms of cytochrome P-450 have been purifiedfrom rat liver microsomes. The major 3-methylcholanthrene (MC)inducible cytochrome P-450 (form c) exhibits the greatest activitytoward both benzo[a]pyrene (BP) (58 min^-1) and 7,12-dimethylbenz[a]anthracene(DMBA) (29 min^-1) and forms substantially high spin, high affinitycomplexes (K_d = 10 nM) with both hydrocarbons. Cytochrome P-450d,a minor MC-inducidble form, has far lower activity for metabolismof both polycyclic aromatic hydrocarbons (PAH), yet also formshigh affinity complexs (K_d {small tilde} 100 nM) with both PAH,retaining the full high spin state of the free cytochrome. Althoughtwo phenobarbital (PB)-induced forms (P-450's b and e) differby only 13 amino acids, they exhibit significant differencesin metabolism of PAH and in complex formation. Whereas P-450bis only active in metabolism of DMBA (9.8 min^-1 versus 1.9 min^-1for BP), P-450e has low activity for both substrates (3.3 and1.2 min^-l). Nevertheless, P-450e forms a high affinity complex(K_d {small tilde} 100 nM) with both PAH that enhances the proportionof the high spin state (from 30% to 70%). Failure to displacen-octylamine (NOA) suggests binding that is removed from theheme. P-450b remains low spin in the presence of PAH and NOAis again not displaced. In addition, the two forms can be distinguishedby their regioselectivities for both PAH. P-450' a, h, and pregnenolone-l6-carbonitrile(PCN) exhibit little activity toward BP or DMBA, but P-450 PCNdoes form a low spin complex with BP (not DMBA). Regioselectivityin metabolism of DMBA by PB-induced microsomes does not agreewith that of the major constituent forms. Only the minor, lessactive purified forms (e and a) mediate substantial 12-hydroxylationand 3,4-epoxidation of DMBA. Thus, additional factors in microsomalreactlons must contribute to these differences.     

Specificity of hepatic cytochrome P-450 isoenzymes from PCB-treated rats and participation of cytochrome b5 in the activation of aflatoxin B1

Employing six forms of cytochrome P-450s fractionated from thehepatic microsomes of PCB-treated rats, the activation of aflatoxinB₁ (AFB₁) was examined in the reconstituted cytochrome P-450system. AFB₁ was specifically activated into DNA-binding formby cytochrome P-450 I-a, which is one of P-450 type cytochromesand possesses an absorption peak at 450.0 nm in its carbon monoxidedifference spectrum. This activation was enhanced by cytochromeb₅ and the maximal enhancement (1.6-fold of the control) wasobserved with the molar ratio of 0.25 cytochrome b₅:1.0 cytochromeP-450.     

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.     

Formation of tissue-bound N'-nitrosonornicotine metabolites by the target tissues of Sprague-Dawley and Fisher rats

The distribution and metabolism of N' -(¹⁴C)nitrosonor-nicotine((¹⁴C)NNU) was studied in Sprague-Dawley and Fisher rats, inwhich NNN induces tumours preferentially of the nasal cavityand the oesophagus. After i.v. injections of (¹⁴C)NNN, a localizationof tissue-bound metabolites was found in the mucosa of the nasalcavity and oesophagus. These tissues were also found to havea capacity to form tissue-bound metabolites from NNN in vitro.These data suggest that in situ metabolic activation of NNNplays an important role in the pathogenesis of the NNN-inducedtumours of the nasal cavity and the oesophagus in the rat.     

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.     

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 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.     

Immunochemical study on the contributions of two molecular species of cytochrome P-450 in mutagenesis by selected aminoazo dyes

The relative contributions of two species of cytochrome P-450,the major cytochrome P-450 components of liver microsomes ofphenobarbital-treated rats (PB-P-450) and of 3-methylcholanthrene-treatedrats (MC-P-448), in the mutagenic activation of 3'-methyl-N,N-dimethyl-4-aminoazobenzene(3'-Me-DAB) and its eight metabolites were studied in the Salmonellaassay system using specific antibodies and inhibitors. The antibodyagainst MC-P-448 considerably inhibited the mutagenicities of3'-Me-DAB, 3'-CH₂OH-DAB, their three N-demethylated compounds,3'-CHO-DAB, and 3'-Me-4'-OH-DAB in strain TA98, whereas theantibody against PB-P-450 inhibited their mutagenicities <29%.In contrast, the antibody against MC-P-448 caused no or slight(29%) inhibition of the mutagenicities of 3'-hydoxymethyl-N-methyl-4-aminoazobenzene(3'-CH₂OH-MAB) and 3'-COOH-DAB. However, the mutagenicitiesof both compounds were considerably inhibited by 7,8-benzo-flavone,like those of other seven aminoazo dyes. These results demonstratethat rat liver cytochrome P-450, especially MC-P-448, is involvedin mutagenic activation of the aminoazo dyes. Participationof a second form of cytochrome P-448 in mutagenic activationof 3'-CH₂OH-MAB and 3'-COOH-DAB is discussed.     

The metabolism of indole alkaloid tumor promoter, (-)-indolactam V, which has the fundamental structure of teleocidins, by rat liver microsomes

Metabolic activation and/or deactivation of indole alkaloidtumor promoter, (–)-indolactam V (ILV), was examined usingrat liver microsomes. Reaction of ILV with the microsomes supplementedwith NADPH and MgCl₂ gave three major metabolites, which wereidentified as (–)-N¹³-des-methylindolactam V and two diastereomersof (–)-2-oxy-indolactam V at C-3. The tumor-promotingactivities of these metabolites were evaluated by inductionof Epstein-Barr virus early antigen and inhibition of specificbinding of [³H]-12-O-tetradecanoylphorbol-13-acetate to a mouseepidermal partculate fraction, and proved to be conspicuouslylower than that of ILV. These results demonstrate that the metabolismof ILV results in detoxification, and that it itself is thetumor-promoting entity. Studies on the enzymes concerned withthis metabolism suggested the involvement of cytochrome P-450-containingmixed-function oxidases. Similar deactivation seems to be possibleby skin, where the mixed-function oxidases are known to exist.     

Gamma interferon induction depresses murine hepatic promutagen/procarcinogen activation

In vivo induction of gamma interferon (IFN-) by sensitizationof mice with Mycobacterium bovis strain BCG and subsequent challengewith tuberculin depressed the ability of liver homogenates fromtreated animals to metabolically activate promutagens. The AmesSalmonella typhimurium revertant assay was used for analysesof metabolic conversion of promutagens by liver homogenates.Relative to the mutant frequencies determined with control liverbomogenates, induction of IFN- depressed the abilities of homogeoatesfrom treated animals to activate N-acetylaminofluorene (AAF),aflatoxin B₁ (AFB₁), and benzo[a]pyrene (BP) by 55%, 44% and95%, respectively. Within 18–24 h of Aroclor 1254 treatment,liver P-450 content had increased 43%, and the relative mutantyields per unit protein for all three promutagens had approximatelydoubled. In vivo induction of IFN- suppressed the Aroclor 1254-dependeotincreases in mutagenesis by AAF (63%), AFB₁ (90%), and BP (reducedto a level 23% below non-Araclor 1254 treatment). In all cases,the levels of depression of promutagen activation qualitativelycorrelated with cytochrome P-450 content and the induction ofIFN-.     

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.     

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.     

A monoclonal antibody against cytochrome P-450 enhances mutagen activation of N-nitrosodimethylamine by mouse liver S9: studies on the mode of action

The effect of the monoclonal antibody MAb 2-66-3, directed againstthe major rat liver phenobarbital (PB)-induced cytochrome P-450(P-450), on the S9-mediated mutagenicity of N-nitrosodimethylamine(DMN) in Salmonella typhimurium strain TA1530 was studied usingliver S9 from PB-treated mice. This MAb enhanced {small tilde}2-fold S9-mediated mutagenicity of DMN but inhibited both itsN-demethylation and N-denitrosation by 50%. Thus MAb-mediatedenhancement of DMN mutagenesis does not result from alteredactivationl inactivation pathways, both known to involve P-450isozymes. DMSO, a hydroxyl radical (HO⁺) scavenger and desferrioxamine,an inhibitor of HO⁺-dependent reactions, quenched the MAb-mediatedenhancement of DMN mutagenesis, implicating the HO⁺-dependentactivation of DMN to mutagenic species. As a mechanism, we proposethat the binding of this MAb to P-450 isozyme implicated inDMN metabolism decreases the functional coupling between thereductase and the P-450 complex, leading to an increased electronflow from the reductase towards molecular oxygen to form reducedoxygen species (HO⁺) at the expense of the monooxygenase functions.