Species, sex and organ differences in induction of a cytochrome P-450 isozyme responsible for carcinogen activation: effects of dietary hepatocarcinogenic tryptophan pyrolysate components in mice and rats

Both sexes of BALB/cxDBA/2 F₁ mice and F344 rats were treatedfor 1 week with a diet containing 0.02% of hepatocarcinogenictryptophan pyrolysate component (Trp P-1 or Trp P-2), and changesin the carcinogen activation enzyme activity in various organswere examined comparatively using a mutation test with Salmonellatyphimurium TA98 as a tester bacterium. Hepatic enzymes fromuntreated mice and rats showed a definite catalytic activityfor mutagenic activations of Trp P-1 and Trp P-2, whereas theactivities of other organs —such as lung, kidney, smallintestine and colon—were undetectable or very low. Inboth mice and rats either the Trp P-1 or Trp P-2 feeding resultedin induction of cytochrome P-450 isozyme(s), which could mediatein the liver but not in other organs the mutagenic activationof the carcinogen itself. As to the sex difference, the inductionof the activation enzyme(s) was greater in the female animalsthan in the males. Species difference in the activity of hepaticenzymes catalyzing the Trp P-1 and Trp P-2 mutageneses was alsoobserved in animals treated with the basal diet; the activitywas higher in mice than in the sex-matched rats (Trp P-1, {smalltilde}1.5-fold; Trp P-2, {small tilde}7-fold). When diet containingTrp P-1 or Trp P-2 was fed for 1 week, the activity of the ratliver for Trp P-1 mutagenesis was of a level similar to thatof the sex-matched mice, but for Trp P-2 mutagenesis it wasless than half that in the mice. The induced hepatic enzymesin mice and rats were suggested to be 3-methylcholanthrene-induciblecytochrome P-448 isozymes as determined by mutation tests withTrp P-1, Trp P-2 and two other substrates and by immunochemicalanalyses of rat hepatic cytochrome P-450 using monoclonal antibodiesagainst rat cytochrome P-448 isozymes. These results indicatethat a form of cytochrome P-450 responsible for activation ofTrp P-1 and Trp P-2 is inducible by dietary treatment of miceor rats with these carcinogens and that the amount of the cytochromeP-450, including resident and induced forms, is related to thespecies, sex and organ differences in their carcinogenic susceptibilityto these chemicals.     

Fetal mouse susceptibility to transplacental lung and liver carcinogenesis by 3-methylcholanthrene: positive correlation with responsiveness to inducers of aromatic hydrocarbon metabolism

The role of metabolic activation of carcinogens in fetal tissueas a determinant of sensitivity in transplacental carcinogenesiswas investigated in a pharmacogenetic experiment utilizing backcrossesof C57BL/6 (Ah^bAh^b, responsive to induction of aromatic hydrocarbonmetabolism) and DBA/2 (Ah^dAh^d, non-responsive) mice. Responsive(C57BL/6 x DBA/2)F₁ and non-responsive DBA mothers, all carryingboth responsive (Ah^bAh^d) and non-responsive (Ah^dAh^d) fetuses,were given i.p. doses of the carcinogen 3-methylcholanthrene(MC) ranging from 5 to 175 mg/kg on gestation day 17. At 10months of age the metabolic phenotype of each offspring wasdetermined, and correlated with number of lung and liver tumors.Both male and female Ah^bAh^d (responsive) offspring in most dosegroups presented a consistent two- to three-fold higher incidenceof lung tumors than did non-responsive Ah^dAh^d littermates. Thedifference held for offspring of both (C57BL/6 x DBA)F₁ andDBA mothers and it was of statistical significance for one orboth sexes at most dosage levels. Hepatocellular tumors werealso significantly more frequent in responsive male Ah^bAh^d progenyof (C57BL/6 x DBA/2)F₁ mothers than in non-responsive Ah^dAh^dlittermates. Progeny of the DBA mothers exhibited significantlymore liver and lung tumors than did those of the (C57BL/6 xDBA/2)F₁ mothers receiving the same dose. These results suggestthat in this model system both maternal and fetal genotype forresponsiveness to induction of aromatic hydrocarbon metabolismare important factors modulating fetal carcinogenic risk.     

Role of cytochrome P1 in the induction of NAD(P)H:quinone reductase in a murine hepatoma cell line and its mutants

NAD(P)H:Quinone oxidoreductase (QR) is a widely-distributedenzyme that promotes obligatory two-electron reductions of quinonesand thereby protects cells against the cytotoxicity of quinonesand their metabolic precursors. QR is induced by a wide varietyof chemoprotectors in many animal tissues as well as in theHepa 1c1c7 murine hepatoma cell line. Such inducers fall intotwo families: dual inducers (e.g. polycydic aromatics, azo dyes,ß-naphthoflavone) that elevate QR as well as cytochromeP₁-450, and selective inducers of QR (e.g. tert-butylhydroquinoneand other redoxlabile diphenols). Induction by the first familyof inducers depends on binding to the Ah (Aryl hydrocarbon)receptor and the associated expression of a functional cytochromeP₁-450 enzyme, whereas the induction by redox-labile diphenolsdoes not appear to be receptor-mediated. In order to analyzethe possible role of the cytochrome P₁-450 system in the inductionof QR, we examined this process in the Hepa 1c1c7 cells andin four mutants of this cell line that are defective in theinduction or expression of functional cytochrome P₁-450. tert-Butylhydroquinonewas an effective inducer of QR in all of the cell lines, andthis process does not, therefore, depend on a functional cytochromeP₁-450 enzyme. In contrast, azo dyes and polycyclic aromaticsinduce QR in the parent cell line but not in the various typesof cytochrome P₁-450-defective mutants. We conclude that theAh receptor and cytochrome P₁-450 function are involved in theinduction of QR by certain azo dyes and polycyclic aromatics,but not by phenolic antioxidants.     

Immunohistochemically demonstrated altered expression of cytochrome P-450 molecular forms and epoxide hydrolase in N-ethyl-N-hydroxyethylnitrosamine-induced rat kidney and liver lesions

A comparison of N-ethyl-N-hydroxyethylnistrosamine (EHEN)-inducedpreneoplastic and neoplastic lesions in the rat liver and kidneywas made with respect to the expression of different drug metabolizingenzymes. Four cytochrome P-450 species (cyt. P-450 UT₅₀, PB_3a,MC₁ and MC₂) and microsomal epoxide hydrolase (mEHb) were investigatedalong with two glutathione S-transferase species (GST-P andA forms) earlier shown to be elevated in putative preneoplasticlesions in the liver and kidney, respectively. In contrast tothe liver lesions, which showed clear decrease in all formsof cyt. P-450s and increase of mEHb, elevated levels of cyt.P-450 PB_3a and, to a lesser extent, the other P-450 forms andearly elevation to late decrease in mEHb characterized the renaltubular lesions. Thus opposite shift in enzyme phenotype wasobserved in carcinogen-induced focal lesions of the two organs.Variation in binding levels in the different nephron segmentsand zones of the liver acinus indicated physiological specializationwith regard to the enzymes investigated and suggested that thealtered phenotype of preneoplastic populations might be of adaptivesignificance.     

Induction of alkoxyresorufin O-dealkylases, epoxide hydrolase, and liver weight gain: correlation with liver tumor-promoting potential in a series of barbiturates

The effects of a series of barbiturates, of known and varyingliver tumor-promoting ability, on several short-term endpointsincluding liver weight and liver-to-body weight ratio increasesand induction of cytochromes(s) P-450 and epoxide hydrolaseactivities were examined. Male F344 rats (3 months of age) wereadministered barbiturates in the drinking water for 12 days.At the end of the treatment period they were killed, body andliver weights were taken, microsomal p-nitroanisole O-demethylationand epoxide hydration, and liver S-9 O-dealkylation of ethoxy-,pentoxy-andbenzyloxyresonifin were measured. The latter two substrateshave been shown to be preferentially metabolized by the majorphenobarbital inducible form of cytochrome(s) P-450 (P-450_band were employed since they offered a means of differentiatingmore dearly varying levels of P-450 induction. Exposure to sodiumbarbital (SB) and sodium phenobarbital (PB) resulted in significantincreases in liver weight and liver-to-body weight ratios. Inductionof cytochrome(s) P-450 and epoxide hydrolase activities by thevarious barbiturates depended on the functional groups on C₅.When ranked in terms of decreasing induction potency, the followingorder was obtained for each enzyme activity quantitated: PB,SB, sodium pentobarbital, amobarbital, hexobarbital and theC₅ substituted parent compound (barbituric acid). Thus, thebarbiturates were found to exhibit a spectrum of induction potendes,with PB and SB, the most potent liver twnor promoters, yieldingthe greatest degree of liver weight increase and induction ofcytochrome(s) P-450 and epoxide hydrolase activities.     

Specificity of rat liver cytochrome P-450 isozymes in the mutagenic activation of benzo[a]pyrene, aromatic amines and aflatoxin B1

The ability of three purified forms of rat liver cytochromeP-450 to metabolically activate benzo[a]pyrene, trans-benzo-[a]pyrene-7,8-dihydrodiol,2-aminofluorene, afiatoxin B₁, dimethylnitrosamine, and a pyrolysisproduct of tryptophan(3-amino-l-methyl-5H-pyrido(4,3-b)indole)(Trp-P-2) to muta-genic products was examined using Salmonellatyphimurium strains TA98 and G46 in a reconstituted monooxygenasesystem. The isozymes examined were cytochrome P-450-PB (themajor phenobarbital inducible form), and the two major 3-MCinducible forms (cytochromes P-448₅₂ and P-448₅₅). CytochromesP-448₅₂ and P-448₅₅ preferentially metabolize 2-aminofluoreneand Trp-P-2 to mutagenic products. However, only cytochromeP-448₅₅ metabolizes benzo[a]pyrene and its 7,8-dihydrodiol derivativeto mutagenic products. Both cytochrome P-448₅₂ and P-448₅₅ metabolizeafiatoxin B, to mutagenic products at a much faster rate thancytochrome P-450-PB. Dimethylnitrosamine was not activated byany of the isozymes tested.     

Inhibitory effect of dietary 4-ipomeanol on DNA adduct formation by the food mutagen 2-amino-3-methylimidazo [4,5-f]quinoline (IQ)in male CDF1 mice

The food mutagen 2-amino-3-methlimidazo[4,5-f]quinoline (IQ)is carcinogenic in the CDF₁ mouse liver, lungs and stomach.IQ Is activated to its ultimate carcinogenic form by N-hydroxylation,catalyzed principally by hepatic microsomal cytochrome P450IA2,and further esterification, resulting in the formation of N-(deoxyguanosin-8-yl)-IQand other adducts. The furanoterpenold 4-ipomeanol (IPO) isa naturally occurring pneumotoxin which exerts its specifictoxicity in Clara cells of the lung after activation by microsomalcytochrome P450. Because IPO is activated in the liver by acytochrome P450IA2 enzyme, we evaluated IPO as a possible chemopreventiveagent by assessing its ability to inhibit IQ-DNA adduct formationin the CDF₁ mouse. Mice were put on an AIN-76A diet with orwithout 0.075% IPO from day 0 to 54. IQ (0.01%) was added tothe diets from day 22 to 41 and animals were killed (four animals/timepoint) on days 42, 44, 46, 48, 50 and 54. Blood (for white bloodcell isolation), liver, lungs, stomach, small intestine, cecun,colon, kidneys, spleen and heart were collected for analysisof IQ-DNA adducts by .³²P-post-labeling. During the 12 day periodafter cessation of IQ exposure (days 42–54) IQ-DNA adductformation was significantly inhibited in the liver (33.6–46.4%),lungs (29.9–58.6%), stomach (33.2–51.5%) and whiteblood cells (24.5–63.7%), but not in the other organs.Except in the colon, adduct removal from organs during days42–54 was relatively slow (36.0–81.9% of day 42levels remaining on day 54, 9.4–16.7% in the colon), butthe presence of IPO in the diet did not influence the rate ofadduct removal. Measurement of hepatic microsomal ethoxyresorufindeethylase, an activity specific for cytochrome P450IA isozymes,showed that the enzyme could be inhibited (14.1–68.1%)by IPO (0.05–10.0 mM) in vitro. It is concluded that IPOinhibits IQ-DNA adduct formation in target organs of the CDF₁mouse and that IPO may act by inhibiting N-hydroxylation ofIQ. It is therefore possible that IPO may be a candidate chemopreventiveagent against IQ-induced carcinogenesis.     

The essential role of the functional group in alkyl isothiocyanates for inhibition of tobacco nitrosamine-induced lung tumorigenesis

The importance of the isothiocyanate group in alkyl isothiocyanatefor inhibition of tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis was examined in A/J mice. Ourprevious structure-activity relationship study of isothiocyanatesshowed that 1-dodecyl isothiocyanate [CH₃(CH₂)₁₁NCS], a simplealkyl isothiocyanate, is a potent inhibitor of NNK-induced lungtumorigenesis. It was chosenfor this study due to its structuralfeatures and potency. A single dose of 1-dodecyl isothiocyanategiven by gavage at 1 µmol/mouse 2 h prior to NNK administrationcompletely inhibited lung tumorigenesis, while removal of theisothiocyanate group or replacing it with a hydroxyl group abolishedthe inhibitory activity. These results demonstrate that theisothiocyanate functional group is critical for the inhibitoryactivity of isothiocyanates in NNK-induced lung tumorigenesis.To gain more insights into the relationship of in vivo inhibitionof tumorigenesis with the cytochrome P-450 enzyme inhibitoryactivity, the effects of these compounds on metabolism of NNKin mouse lung microsomes were studied. 1-Dodecyl isothiocyanateinhibited all three known oxidative pathways of NNK metabolism,with a stronger inhibitory activity toward NNK N-oxidation (IC₅₀430 nM) and keto alcohol formation (IC₅₀ 500 nM) than keto aldehydeformation (IC₅₀ 13 000 nM). 1-Dodecanol had a similar selectivityin inhibition of these metabolic pathways, but was less potentthan 1-dodecyl isothiocyanate. Dodecane showed little or noinhibitory activity in the same concentration range. These resultsindicate that the isothiocyanate group of 1-dodecyl isothiocyanateis important for inhibition of NNK-induced lung tumorigenesisand also for effective inhibition of cytochrome P-450 enzymesinvolved in NNK oxidation.     

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.     

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.     

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.     

Aflatoxin B1 carcinogenesis and its relation to DNA adduct formation and adduct persistence in sensitive and resistant salmonid fish

Rainbow trout (Salmo gairdneri)and coho salmon (Oncorhyn-chuskisutch) were exposed to aflatoxin B1(AFB1) either by passiveembryo uptake or by dietary treatment after hatching and feedingonset. Trout exposed as embryos to an aqueous solution of 0.5p.p.m. AFB1 for 15 min showed a 62% tumor incidence 12 monthslater, whereas coho salmon exposed to a similar solution for30 min showed only a 9% incidence. The difference between salmonand trout response was even greater by dietary AFB1 treatment.Trout exposed for 4 weeks to 20 p.p.b. dietary AFB1 had a 62%tumor response 12 months later, whereas salmon exposed to 40p.p.b. dietary AFB1 for 4 weeks failed to develop tumors. A5% tumor incidence was observed in salmon 12 months after 3weeks exposure to 5000 p.p.b. dietary AFB1, a lethal dose fortrout. In addition to a lower tumor incidence when comparedto trout, the neoplastic response of salmon to AFB1 is to producebenign hepatic adenomas in contrast to the malignant hepatocellularcarcinomas seen in trout. AFB1 metabolism, DNA adduct formation,adduct persistence in vivo and in vitro and cytochrome P-450isozyme composition were compared in livers of trout and salmonto understand the role of metabolism and initiation in thisspecies difference. AFB1-DNA binding was 7–56 times greaterin trout than salmon liver at various times after AFB1 injection,20 times greater in embryos or in freshly isolated trout hepatocytepreparations after a 1 h incubation with aflatoxin Bl, and 18times greater in trout liver after a three week dietary (80p.p.b.) exposure. The major AFB1-DNA adduct was 8, 9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxinB1 in both species. Persistence of AFB1-DNA adducts in vivoin liver was high compared to mamalian systems, implying thatactive enzymatic removal of bulky DNA adducts is low in bothspecies and probably not a factor in their differential responseto aflatoxin. Species differences in other phase I and phaseII metabolism pathways and in AFB1 elimination were, overall,much less striking than those previously observed for troutfed inhibitors of aflatoxin carcinogenesis. Rates of bileeliminationof AFB1 detoxication products, and total excretion of aflatoxinsinto water after AFB1 exposure, were not significantly differentbetween trout and salmon. Since detoxication differences werenot observed, the species difference in AFB1-DNA binding appearsto reflect less efficient cytochrome P-450 metabolism of aflatoxinto the reactive 8, 9-epoxide in salmon, compared to trout. Insupport of this hypothesis, trout liver microsomes displayeda K_m (7.5 µM)for AFB1-DNA adduction in vitro that was7-fold lower than salmon (52 µM). Furthermore, immunoquantitationof various P-450 isozymes suggest that salmon liver microsomeshave much lower amounts of an isozyme immunochemically relatedto trout P-450 LM₂ which has previously been shown to be themajor isozyme catalyzing AFB1 8, 9-epoxidation. Other, post-initiationdifferences were not ruled out by these studies and may contributeto the differential response of rainbow trout and coho salmonto AFB1 hepatocarcino-genesis.     

Metabolism of aflatoxin B1 in the bovine olfactory mucosa

Carcinomas of the ethmoidal region of the nose are observedrelatively frequently in cattle in several countries in tropicaland subtropical latitudes. Viruses have been implicated as causativeagents, but it has been observed that affected animals sometimessuffer from aflatoxicosis, and a role of aflatoxin B₁ (AFB₁)in the aetiology has also been proposed. We have examined whetherthe bovine nasal olfactory mucosa has a capacity to metabolizeAFB₁. The contents of cytochrome P–450 and cytochromeb₅, and the NADPH cytochrome c reductase activity in the nasalolfactory mucosa have also been determined. Comparative experimentshave been performed with the liver. Incubations with ³H-labelledAFB₁ showed that the nasal olfactory mucosa has a much highercapacity than the liver to form lipid-soluble, water-solubleand tissue-bound AFB₁-metabolites. High-resolution microautoradiographyshowed a strong localization of tissue-bound metabolites inthe sustentacular cells in the apical portion of the olfactorysurface epithelium and in Bowman's glands in the olfactory laminapropria mucosae. Especially in the sustentacular cells the labellingwas preferentially located in the nuclei of the cells. Liquidchromatography of chloroform extracts of the nasal olfactorymucosa and the liver incubated with ³H-AFB₁ showed formationof several metabolites. The dominating peak in both tissueswas aflatoxin M₁ (AFM₁). However, the amount of AFM₁ was higherin the nasal olfactory mucosa than in the liver, and the amountsand proportions of several other metabolites also differed markedlybetween the two tissues. The level of cytochrome P-450 in thenasal olfactory mucosa was found to be about one quarter ofthat in the liver, but the NADPH cytochrome c reductase activitywas much higher in the nasal olfactory mucosa than in the liver.In addition, the cytochrome b₅: cytochrome P-450 ratio was higherin the nasal olfactory mucosa than in the liver. The highermetabolism of AFB₁ in the nasal olfactory mucosa than in theliver may be related to differences in the cytochrome P-450isoenzyme profile. In addition, the microsomal electron transportto cytochrome P-450 may be facilitated by the high reductase:cytochrome P–450 ratio and the high cytochrome b₅: cytochromeP–450 ratio in the nasal olfactory mucosa. It is consideredthat the results of the present study strengthen the hypothesisthat exposure of AFB₁-contaminated feed may be an importantaetiological factor in the development of nasal tumours in cattle.     

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.     

Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodiinethylamine

Capsalcin (trans-8-methyl-N-vanillyl-6-nonenamide) is a majorpungent and irritating ingredient of hot chill peppers, whichare frequently consumed as spices. This dietary phytochemicalhas been found to interact with microsomal xenobiotic metabolizingenzymes in rodents. Capsalcin and its saturated analog dihydrocapsaicin(trans 8-methyl-N-vanlllyl-6-nonanamide) have been proposedto inactivate cytochrome P-450 IIE1 by irreversibly bindingto the active sites of the enzyme. Besides cytochrome P 450IIE1, other isoforms of the P-450 superfamily were also reportedto be inhibited by capsaicin. The inhibition by capsaicin ofmicrosomal monooxygenases involved in carcinogen activationimplies its chemopreventive potential. As part of a programto investigate chemoprotective properties of capsalcin we initiallydetennined the effect of capsalcin on vinyl carbamate (VC)-and N-nitrosodlmethyl amine (NDMA)-induced mutagenesis in Salmonellatyphi murium TA 100. Capsaicin (0.42 mM) attenuated the bacterialmutagenicity of VC and NDMA by 50% and 42% respectively. Diallylsulfide, a thloether found in garlic with selective P-450 IIE1inhibitory activity, also lessened the mutagenicity of the abovecarcinogens in a concentration-dependent manner. The suppressionof VC- and NDMA Induced mutagenesis by capsaicin and diallylsulfide correlated with their inhibition of P-450 IIE1-mediatedp-nltrophenol hydroxylation and NDMA N-demethylation. Pretreatmentof female ICR mice with a topical dose of capsalcin loweredthe average number of VC-induced skin tumors by 62% at 22 weeksafter promotion. A similar degree of protection was attainedwith oral administration of diallyl sulfide before carcinogentreatment. The results of this study suggest that capsaicinand diallyl sulfide suppress VC- and NDMA-induced mutagenesisor tumorigenesis In part through inhibition of the cytochromeP-450 IIE1 isoform responsible for activation of these carcinogens.     

Sex-dependent induction of hepatic enzymes for mutagenic activation of a tryptophan pyrolysate component, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole, by feeding in mice

Male and female BALB/c X DBA/2 F1 mice were treated with a diet containing 0.02% 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole (Trp P-1), a hepatocarcinogenic tryptophan pyrolysate component, and the capacities of subcellular fractions of the liver to catalyze the mutagenic activation of Trp P-1 and its analogue Trp P-2 (4-demethylated Trp P-1) were examined by the in vitro Salmonella test with strain TA 98. In mice on control diet, both 9000 X g supernatant (S-9) and microsomal fractions from female mice livers displayed only 1.1- to 1.3-fold higher capacities for the mutagenic activation of either Trp P-1 or Trp P-2 than did those from male mice livers. When mice were treated with the Trp P-1 diet for 1 week, the S-9 activity in male mice for the Trp P-1 mutagenesis did not change, but that in females was increased to 2.5-fold of the female control. Treatment of mice with the dietary Trp P-1 for 2 weeks increased the S-9 activities to 2.8-fold in males and 4.9-fold in females of the same sex controls and the increased S-9 activities were not significantly changed by additional Trp P-1 feeding for 2 weeks. Similar changes in the S-9 activity were observed for the Trp P-2 mutagenesis. The overall changes in the S-9 activities induced by feeding Trp P-1 were reflected in the isolated microsomes. However, microsomes derived from the same volume of S-9 used exhibited only about one-half (Trp P-1) or one-third (Trp P-2) of the activity of the respective complete S-9 mixtures. Addition of liver cytosolic fractions (105,000 X g supernatants) from untreated or Trp P-1-treated mice to microsomes resulted in enhanced activities. Cytosols alone did not activate the compounds to mutagens. The microsome-mediated mutagenicity of either Trp P-1 or Trp P-2 was diminished by removal of NADPH from the assay system. It was also inhibited by addition of 7,8-benzoflavone and to a lesser extent by SKF 525A. Enzyme(s) for the mutagenic activation of Trp P-1 was induced by an i.p. injection of 3-methylcholanthrene to mice and to a lesser extent by an injection of phenobarbital, but no sex differences were observed in these enzyme inductions as opposed to the Trp P-1 feeding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Susceptibility to phorbol ester skin tumor promotion in (C57BL/6xDBA/2) F1 mice is inherited as an incomplete dominant trait: evidence for multi-locus involvement

Since current evidence suggests that the tumor promotion stageis a primary determinant in susceptibility to multistage carcinogenesis,we have characterized the genetics of susceptibility to phorbolester skin tumor promotion in inbred mice. Susceptibility ofhybrids (B6D2F₁), beween DBA/2 (sensitive) and C57BL/6 (resistant)parents, initiated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and promoted with 12-O-tetradecanoylphorbol-13-acetate(TPA) was similar to DBA/2 mice at doses of 13.6 nmol per mousebut clearly less when doses of 1.7–6.8 nmol per mousewere used. In addition, no significant differences were observedbetween male andfemale B6D2F₁ mice in terms of tumor incidencealthough some differences were observed in tumor multiplicitiesbetween male and female F₁ mice at the highest TPA dose. ReciprocalF₁ mice initiated with DMBA (i.e. D2B6F₁) were also responsiveto TPA. Female D2B6F₁ mice were of lower sensitivity at lowerdoses of TPA, compared to female DBA–2, a finding similarto that observed with B6D2F₁ mice initiated with MNNG. Furtheranalyses of the susceptibility of B6D2F₂ and B6D2F₁xC57BL–6backcross mice to TPA promotion indicated that more than onedominant genetic locus must account for the differences in promotionsensitivity between DBA/2 and C57BL/6 mice. To understand furtherthe genes responsible for promotion sensitivity, histologkalevaluations were performed on DBA/2, C57BL/6 and B6D2F₁ mice.Histological examination revealed that the epidermis of DBA/2mice showed a marked hyperplasia and the presence of a muchgreater number of dark basal keratinocytes (DCs) compared withC57BL/6 mice 48 h after the last of four applications of TPA(doses 3.4 nmol). A marked dermal infiltration of polymorphonuclearleukocytes (PMNs) was observed in DBA/2 mice, whereas littleinfiltration was observed in the skin of C57BL/6 mice. The hyperplasiain the skin of B6D2F₁ mice was intermediate between DBA/2 andC57BL/6 mice at all TPA doses examined except the lowest dose(1.7 nmol), whereas the DC response, although significantlylower at doses of 6.8 nmol or below, was similar to DBA/2 miceat higher TPA doses (13.6 and 17.0 nmol). The infiltration ofPMNs in the dermis of B6D2F₁ mice was similar to or greaterthan DBA/2 mice at all doses of TPA tested. Our results suggestthat (i) susceptibility to TPA promotion is inherited as anincomplete dominant trait; (ii) neither cytoplasmic geneticdeterminants nor the X-chromosome appear to play a significantrole in susceptibility to TPA; and (iii) the degree of sustainedepidermal hyperplasia and especially the induction of DCs aftermultiple applications of TPA show an excellent correlation withinherited susceptibility to promotion. Our data are consistentwith a model where allelic differences at more than one geneticlocus contribute to the sensitivity of DBA/2 mice to phorbolester promotion.     

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.     

Effect of modifying agents on the phenotypic expression of cytochrome P-450, glutathione S-transferase molecular forms, microsomal epoxide hydrolase, glucose-6-phosphate dehydrogenase and {gamma}-glutamyltranspeptidase in rat liver preneoplastic lesions

The expression of A and P forms of glutathione S-transferase(GST-A and P), two cytochrome P-450 isoenzymes (P-450 PB_3a andP-450 MC₂), microsomal epoxide hydrolase (mEHb), glucose-6-phosphatedehydrogenase (G6PD) and -glutamyltranspeptidase (-GT) was comparedin preneo-plastic liver lesions and background parenchyma ofF344 rats post-treated with butylated hydroxyanisole (BHA),ethoxyquin (EQ) or acetaminophen (AAP). These latter three compoundshave been shown to inhibit hepatocarcinogenesis after initialtreatment with N-ethyl-N-hydroxyethylnitrosamine (EHEN) anda significant decrease in the number of enzymealtered foci andnodules positive for GST-P, GST-A, G6PD and -GT and negativefor P-450 PB_3a, P-450 MC₂ was associated with their administration.Whereas in the foci case the decrease was most prominent fornon-discrete (heterogeneous) type lesions, the results of quantitationof nodules revealed a most significant alteration in the discretehomogeneously staining population. This indicates that BHA,EQ and AAP have the potential to inhibit the growth of the phenotypicallystable lesions thought most likely to be the immediate precursorsof hepatocellular carcinomas. The two anti-oxidants were associatedwith periportal increase of all enzymes investigated, whereasAAP induced GST species and mEHb in the perivenular zone. Irrespectiveof slightly elevated enzyme levels in surrounding parenchyma,mEHb antibody binding levels within lesions showed a reciprocalshift from positive to negative in rats treated with BHA, EQand AAP.     

Selective interactions of cytochromes P-450 with the hydroxymethyl derivatives of 7, 12-dimethylbenz[a]anthracene

Competition between a hydroxylated metabolite and the parentpolycyclic aromatic hydrocarbon (PAH) for metabolism at cytochromesP-450 may result in the generation of hydroxylated dihydrodiolepoxides. The effectiveness of the competition between 7-hydroxymethyI-12-methylbenz[a]-anthracene(7HOMMBA) or 12-hydroxymethyl-1–7-methyl-benz[a]anthracene(12HOMMBA) and 7, 12-dimethylbenz[a]-anthracene (DMBA) is highlydependent on the form(s) of cytochrome P-450 in the microsomes.The inhibitory effects of exogenously added 7HOMMBA or 12HOMMBAon DMBA metabolism were 30- to 50-fold greater in 3-methyl-cholanthrene(MC-induced rat liver microsomes (K_i = 0.4 µM) comparedto either uninduced or phenobarbital (PB-induced liver microsomes(K_i = 14 and 11 µM, respectively). Similarly, productinhibition of total DMBA metabolism by metabolites generatedin situ was significant only in MC-induced liver microsomes(K'_i = 2.5 µM). Metabolism of 7HOMMBA in these microsomeswas strongly restricted by an unusual substrate inhibition derivedfrom the inhibitory binding of a second molecule of 7HOMMBA.This same phenomenon was observed with reconstituted cytochromeP-450c but not with PB-induced or uninduced microsomes. Complexformation by binding of DMBA, 7HOMMBA, and 12HOMMBA to purifiedP-450c reconstituted in phospholipid micelles was determinedby optical spectroscopy and fluorescence quenching. Bindingaffinities of both the 7HOMMBA and 12HOMMBA (K_d = 95 and 110nM, respectively), were 2.5-fold higher compared to that ofDMBA (K_d = 265 nM). These results provide a first demonstrationthat hydroxylation of a PAH can lead to preferential metabolismthrough an increased affinity for cytochrome P-450.