The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol

The effects of ethanol on the metabolism of nitrosamines byrat liver microsomes have been studied. Treatment of rats with10 or 15% ethanol in drinking water for 3 days causes a 4- to5-fold enhancement in microsomal N-nitrosodimethylamine demethylase(NDMAd) activity and a 40–60% increase in gross P-450content. The enhancement is mainly due to the induction of alow K_m form (K_m = 0.07 mM) of NDMAd. The treatment induces proteinspecies with molecular weights between 50 000 and 52 000, someof which are believed to be P-450 isozymes with high affinityto NDMA. In addition to NDMA, treatment with ethanol also enhancesthe metabolism of N-nitroso-N-methylethylamine, N-nitrosomethylamline,and N-nitroso-N-methylbenzylamine. When added to the incubationmixture, ethanol and its homologs inhibit the demethylationof these nitrosamines by microsomes. Ethanol is a competitiveinhibitor of the low K_m NDMAd with a K_i of 0.31 mM and is lesseffective in inhibiting the metabolism of more lipophilic nitrosamines.     

The effects of ascorbic acid deficiency and excess on the metabolism and toxicity of N-nitrosodimethylamine and N-nitrosodiethylamine in the guinea pig

The influence of ascorbate deficiency and megadosage on themetabolism of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine(NDEA) was investigated in the guinea pig. After 21 days ona scorbutogenic diet, microsomal cytochrome P-450 and cytochromeb₅ levels fell by 51 and 32%, respectively, while cytochromec reductase activity remained constant. The activities of NDMAand NDEA dealkylase I were also depressed significantly. TheV_max of NDMA demethylase I and NDEA deethylase I was significantlydepressed. Also, ascorbate deficiency significantly decreasedthe plasma clearance of both nitrosamines though the LD₅₀ ofneither were altered by ascorbate nutrition. Covalent bindingof ¹⁴C from [¹⁴C]NDMA and [¹⁴C]NDEA to DNA obtained from liverslices was significantly lower in the deficient than in thecontrol samples; megadosage appeared to have the opposite effect.     

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.     

Hepatic cytochrome P-450 isozyme(s) induced by dietary carcinogenic aromatic amines preferentially in female mice of DBA/2 and other strains

DBA/2, BALB/c or (BALB/cxDBA/2)F₁ (CDF₁) mice of both sexeswere treated for 1 week with a dietary hepatocarcinogenic tryptophanpyrolysate component (Trp P-1 or Trp P-2), and the activityof hepatic microsomal enzyme(s) for mutagenk activations ofTrp P-1 and Trp P-2 were assessed by means of a mutation testwith Salmonella typhimurium TA98. In both Ah-responsive (BALB/cand CDF₁) and Ah-nonresponsive (DBA/2) mice, the dietary treatmentwith Trp P-l or Trp P-2 resulted in a significant increase ofthe enzyme activity for mutagenic activations of Trp P-1 andTrp P-2 in females but not in males, except the case of maleBALB/c mice treated with dietary Trp P-1 Also induction of enzyme(s)in female mice was suppressed by an administration of testosterone.The induced hepatic microsomal enzyme(s) was demonstrated tobe cytochrome P-450 isozyme(s) (mol. wt of 55 000 daltons) byimmunoblots with use of an anti-rat cytochrome P-448 monoclonalantibody and by selective inhibition of the activity by additionof 7,8-benzoflavone into the mutation assay system. These findingsindicate that carcinogic aromatic amines such as Trp P-1 andTrp P-2 are able to induce hepatic cytochrome P-450 isozyme(s)not only in Ah-responsive mice (BALB/c and CDF₁) but also inAh-nonresponsive DBA/2 mice and that the cytochrome P-450 inductionis controlled by androgen(s).     

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.     

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.     

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.     

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.     

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.     

Enzymatic mechanisms in the metabolic activation of N-nitrosodialkylamines

The metabolism of several N-nitrosodialkylamines was studied using rat liver microsomes and purified cytochrome P450 isozymes in a reconstituted monooxygenase system. With purified acetone/ethanol-inducible cytochrome P450 (P450ac), high N-nitrosodimethylamine (NDMA) demethylase activity was observed. Cytochrome b5 was also involved in NDMA metabolism by decreasing the Km of NDMA demethylase. A close relationship between the demethylation and denitrosation of this substrate was observed. P450ac was also active in the metabolism of N-nitrosoethylmethylamine (NEMA), but was less active than phenobarbital-inducible cytochrome P450 (P450b) in the metabolism of N-nitrosobutylmethylamine (NBMA), especially in catalysing the debutylation reaction. Similar substrate specificity was demonstrated with liver microsomes from rats treated with other inducers. With different P450 isozymes and microsomes, a close relationship between metabolism and activation of nitrosamines to mutagens to V79 cells was demonstrated. DNA alkylation by NDMA in vitro was correlated with the rate of metabolism of these compounds, whereas DNA alkylation in vivo was more complex and was dose-dependent. The work demonstrates the importance of knowledge of the substrate specificity of cytochrome P450 isozymes in understanding the mechanisms of the metabolic activation of nitrosamines.     

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.     

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.     

High-affinity nitrosamine dealkylase system in rat liver microsomes and its induction by fasting

In order to elucidate the enzymic basis of nitrosamine metabolism, the in vitro metabolism of nitrosamines by rat liver microsomes and the effects of fasting on the microsomal enzymes have been studied. Fasting for 1 to 3 days causes a 2- to 3-fold enhancement of the reduced nicotinamide adenine dinucleotide phosphate-dependent nitrosodimethylamine demethylase (NDMAD) activity. The cytochrome P-450 content and the activities of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase and benzphetamine demethylase, however, are only modestly increased. Gel electrophoretic analysis reveals the induction of a 50,000-dalton protein band during fasting. The induction of this protein band as well as the enhancement of NDMAD activity are inhibited by CoCl2 and inhibitors of protein and RNA biosynthesis. The involvement of cytochrome P-450 in the NDMAD is supported by the fact that microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase is required for the demethylase activity. Kinetic analysis indicates that a low-Km form of NDMAD (apparent Km, 0.07 mM) is markedly induced by fasting. With microsomes of control rats, there are at least three apparent Km values (0.07, 0.38, and 38.6 mM) for NDMAD; but with microsomes of fasting rats, the low-Km (0.07 mM) form is predominant. These results suggest that rat liver microsomes contain a cytochrome P-450 isozyme which has high affinity for nitrosodimethylamine, and this isozyme is induced by fasting. In addition to nitrosodimethylamine, the oxidative demethylation of N-nitroso-N-methylethylamine, N-nitroso-N-methylbutylamine, N-nitroso-N-methylaniline, and N-nitroso-N-methylbenzylamine is also enhanced by fasting. The extent of enhancement and substrate dependency of these reactions, however, is different from that of NDMAD.     

Metabolism of N-nitrosodialkylamines by human liver microsomes

The metabolism of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine, N-nitrosobenzylmethylamine, and N-nitrosobutylmethylamine was investigated in incubations with human liver microsomes. All of the 16 microsomal samples studied were able to oxidize NDMA to both formaldehyde and nitrite at NDMA concentrations as low as 0.2 mM; the rates of product formation of the samples ranged from 0.18 to 2.99 nmol formaldehyde/min/mg microsomal protein (median, 0.53 nmol). At a concentration of 0.2 mM NDMA, the rates of denitrosation (nitrite formation) were 5 to 10% (median, 6.3%) those of demethylation (formaldehyde formation); the ratio of denitrosation to demethylation increased with increases in NDMA concentration, in a similar manner to rat liver microsomes. Immunoblot analysis with antibodies prepared against rat P-450ac (an acetone-inducible form of cytochrome P-450) indicated that the P-450ac [P-450j (isoniazid-inducible form)] orthologue in human liver microsomes had a slightly higher molecular weight than rat P-450ac and the amounts of P-450ac orthologue in human liver microsomes were highly correlated with NDMA demethylase activities (r = 0.971; P less than 0.001). Analysis of four selected microsomal samples showed that human liver microsomes exhibited at least three apparent Km and corresponding Vmax values for NDMA demethylase. This result, suggesting the metabolism of NDMA by different P-450 enzymes, is similar to that obtained with rat liver microsomes, even though most of the human samples had lower activities than did the rat liver microsomes. The high affinity Km values of the four human samples ranged from 27 to 48 microM (median, 35 microM), which were similar to or slightly lower than those observed in rat liver microsomes, indicating that human liver microsomes are as efficient as rat liver microsomes in the metabolism of NDMA. The human liver microsomes also catalyzed the dealkylation and denitrosation of other nitrosamines examined. The rates of product formation and the ratios of denitrosation to dealkylation varied with the structures and concentrations of the substrates as well as with the microsomal samples tested. The results indicate that human liver microsomes are capable of metabolizing N-nitrosodialkylamines via the pathways that have been established with rat liver microsomes.     

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

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

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 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.     

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.