Roles of different forms of cytochrome P450 in the activation of the promutagen 6-aminochrysene to genotoxic metabolites in human liver microsomes

We reported previously that the potent mutagen 6-aminochryseneis catalyzed principally by rat liver microsomal P4501A andP4502B enzymes to reactive metabolites that induce umu geneexpression in O-acetyltransferase-over-expressing strain Salmonellatyphimurium NM2009; the proposal was made that there are differentmechanisms in the formation of reactive N-hydroxylated and diolepoxidemetabolites by P450 enzymes (Yamazaki,H. and Shimada, T., Biochem.Pharmacol., 44, 913–920, 1992). Here we further examinedthe roles of human liver P450 enzymes and the mechanism of activationof 6-aminochrysene by rat and human P450 enzymes in the Salmonellatester strains. Liver microsomes from 18 different human samplescatalyzed activation of 6-aminochrysene more efficiently inS.typhimurium NM2009 than in the original strain of S.typhimuriumTA1535/pSK1002. The rates of 6-aminochrysene activation in 18human liver samples showed good correlation to the contentsof P4502B6 as well as contents of P4503A4 and the respectivemono-oxygenase activities catalyzed by P4503A4. Among purifiedP450 enzymes examined, P4501A2 as well as P4503A4 were highlyactive in transforming 6-aminochrysene to reactive metabolites,suggesting the involvement of different human P450 enzymes inthe reaction. Four human samples that contained relatively highlevels of particular P450 enzymes in their microsomes were selectedand used for further characterization. Liver microsomes fromhuman samples HL-13 and HL-4 that contained the highest levelsof P4502B6 and P4503A4 respectively, were sensitive to the respectiveantibodies raised against monkey P4502B and human P4503A4; theactivity in sample HL-16 having the highest level of P4501A2was inhibited by anti-P4501A2 IgG.     

Roles of human liver cytochrome P4502C and 3A enzymes in the 3-hydroxylation of benzo(a)pyrene.

The major oxidation product of the classic polycyclic hydrocarbon carcinogen benzo(a)pyrene [B(a)P] is 3-hydroxy B(a)P. Numerous studies have been concerned with the measurement of B(a)P 3-hydroxylation activity in experimental animals and human tissues. Although human liver is the main site of this reaction, systematic studies had not been carried out to define the roles of individual cytochrome P-450 (P-450) enzymes involved. Purified human P4502C8 and P4503A4 showed appreciable catalytic activity; purified human P4501A2 and yeast recombinant (human) P4502C9 and P4502C10 had less activity. No B(a)P 3-hydroxylation activity was observed with purified human P4502A6, P4502D6, P45602E1, or P4502CMP. When microsomes prepared from different human liver samples were compared, B(a)P 3-hydroxylation activity was well correlated with nifedipine oxidation (a P4503A4 marker) but not markers of other P-450s, including tolbutamide hydroxylation (P4502C9 and 2C10), chlorzoxazone 6-hydroxylation (P4502E1), (S)-mephenytoin 4'-hydroxylation (P4502CMP), and coumarin 7-hydroxylation (P4502A6). In three of the liver microsomal samples with relatively high B(a)P 3-hydroxylation activity, immunoinhibition was observed with anti-P4503A greater than anti-P4502C (and no inhibition with several other antibodies). The selective chemical inhibitors gestodene and troleandomycin (P4503A enzymes) and sulfaphenazole (P4502C enzymes) reduced the B(a)P 3-hydroxylation activity of the more active microsomal preparations to rates seen in the preparations with low activity. This residual activity (and most of the activity in the low activity samples) was refractory to all of the chemical inhibitors and antibodies. The addition of 7,8-benzoflavone dramatically stimulated B(a)P 3-hydroxylation in all of the microsomal samples (and also stimulated purified P4503A4), arguing against an important role for P4501A1 or P4501A2. We conclude that roles of human P-450 enzymes for B(a)P 3-hydroxylation follow the order P4503A4 greater than or equal to P4502C8 greater than P4502C9/10 in human liver and that the other P-450s examined here do not have major roles. P4502C8 and P4502CMP (but not P4503A4) were found to activate B(a)P to products genotoxic in Salmonella typhimurium; this pathway would appear to involve products other than 3-hydroxy B(a)P and B(a)P 7,8-dihydrodiols.     

Cytochromes P450 in cynomolgus monkeys mutagenically activate 2-amino-3-methylimidazo(4, 5-f)quinoline (IQ) but not 2-amino-3, 8-dimethylimidazo(4, 5-f)quinoxaline(MeIQx)

THE PROMUTAGENIC AND PROCARCINOGENIC HETEROCYCLIC AMINES (HAS) FOUND IN COOKED MEATS ARE N-HYDROXYLATED BY MICRO-SOMAL CYTOCHROME P450 ENZYMES AS THE FIRST STEP IN THEIR METABOLIC ACTIVATION. IN CYNOMOLGUS MONKEYS, ONE OF THE HAS, 2-AMINO-3-METHYLIMIDAZO[4, 5-F: quinoline (IQ), has been shown to be a potent hepatocarcinogen.However, the structurally similar HA 2-amino-3, 8-dimethylimidazo[4,5-fquinoxaline (MelQx) lacks this potency to induce hepato-cellularcarcinoma in monkeys. Liver microsomes from cynomolgus monkeysshow a striking substrate specificity for the metabolic activationof IQ and MelQx, the former being a far better substrate forN-hydroxylation. Western blot analysis showed that cynomolgusmonkey hepatic microsomes constitutively express P450s immunologicallyrelated to the human CYP3A, CYP2C, and low levels of CYP1A1.For comparison, Western blot analysis of rat, human and patasmonkey microsomes was also carried out. Treatment of cynomolgusmonkeys with rifampicin induced hepatic cytochromes P450 relatedto human CYP3A4 and CYP2C9/10 without inducing CYP1A1 or CYP1A2.Immunoblot analysis also showed that chronic exposure of cynomolgusmonkeys to IQ induced hepatic microsomal cytochrome CYP1A1 andCYP1A2, similarly but lesser in magnitude to that observed with2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCCD) induction. Usingthe Ames Salmonella mutagenicity assay, we examined the effectof the inducers on the mutagenic activation (i.e. N-hydroxylation)of IQ and MelQx by cynomolgus monkey hepatic microsomes. Wealso examined the mutagenic activation of these HAs by rat,human and patas monkey liver microsomes. Microsomes from cynomolgusmonkeys treated with rifampicin showed a 3-fold increase inthe mutagenic activation of IQ but showed no increase in themutagenic activation of MelQx. Since cytochromes P4503A and/orP4502C are constitutively expressed in cynomolgus monkey hepaticmicrosomes, and upon induction with rifampicin are associatedwith an increased metabolic activation of IQ but not MelQx,it appears that CYP3A and/or CYP2C are the isoform(s) showingthe selective substrate specificity in the metabolic activationof IQ over MelQx. Treatment of monkeys with TCDD significantlyincreased the mutagenic activation of both IQ and MelQx, concomitantwith an induction of CYP1A isozymes. Thus, it appears that TCDD-inducibleCYP1A enzymes W-hydroxylate both substrates without selectivity.Together, these findings suggest that CYP3A and CYP2C are theprincipal isoforms in the cynomolgus monkey, associated withthe metabolic activation implicated in the induction of hepatocarcinogenicityby IQ. Furthermore, the poor metabolic activation of MelQx byCYP3A and CYP2C, coupled with low constitutive levels of CYP1Aisozymes, provide a metabolic explanation for the low hepatocarcinogenicpotency of MelQx in cynomolgus monkeys.     

Expression of cytochrome P450 in LEC rats during the development of hereditary hepatitis and hepatoma

The expression of 14 forms of cytochrome P450 in the liver aswell as changes in the testosterone hydroxylation activitiesof hepatic microsomes were investigated during the developmentof hepatitis in Long-Evans Cinnamon (LEC) rats. P4501A1 and-1A2 (3-methylcholanthrene-inducible forms) and P4502B1 and-2B2 (phenobarbital-inducible forms) were barely detected inthe hepatic microsomes of male and female LEC rats. In immaturemale rats, the levels of male-specific forms (P4502C11 and -2C13)were higher in LEC rats than in control Long-Evans Agouti (LEA)rats. P4502C11 appeared in female LEC rats from 4 to 16 weeksof age, reflecting that testosterone 2- and 16-hydroxylationactivities were detected at significant levels in female LECrats. In immature female rats, the level of P4502C12 (a majorfemale-specific form) was higher in LEC rats than in LEA rats.The level of P4502C13 in male LEC rats and that of P4502C12in female LEC rats decreased markedly with ageing or duringthe development of hepatitis. The level of P4503A2 (a male-predominantform) was especially high in immature male and female LEC rats,reflecting that both rats had high 2ß- and 6ß-hydroxylationactivities toward testosterone. These sex-specific forms areregulated by androgens and by pituitary growth hormone. Thus,there may be abnormalities of the hypothalamo–pituitary-gonadalaxis in LEC rats. Furthermore, P4503A2 efficiently activatesaflatoxin B1, a potent hepatocarcinogen, and the increased levelsof this form in LEC rats may be related to the onset of hepatitisor liver cancer.     

Relationships of DNA adduct formation, K-ras activating mutations and tumorigenic activities of 6-nitrochrysene and its metabolites in the lungs of CD-1 mice

6-Nitrochrysene (6-NC), an environmental pollutant and a potentmouse lung carcinogen, is activated by two major metabolic pathwaysto yield DNA adducts derived from either trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene(1,2-DHD-6-AC) or N-hydroxy-6-aminochrysene (N-OH-6-AC). Whilethe former pathway has been shown to be the major activationpathway leading to DNA adducts in mice treated with 6-NC, thepotential contribution of the minor nitroreduction pathway totumorigenicity in this system is not clear. To evaluate theroles of these activation pathways and the resulting DNA adductsin mouse lung tumorigen-esis, we studied DNA adduct formation,the induction of tumors and tumor K-ras mutational spectra inthe lungs of male CD-1 mice treated with 6-NC and its metabolites.6-NC, 6-AC and 1,2-DHD-6-AC produced predominantly a singlechromatographically identical dG adduct, and 6-nitrosochrysene(6-NOC) gave a single major adduct that was most likely derivedfrom reaction at the C8 position of deoxyadenosine. 6-NC-, 1,2-DHD-6-AC-and 6-NOC-treated mice developed both adenomas and adenocarcin-omasin the lung, whereas only lung adenomas were observed in 6-AC-treatedanimals. K-ras mutations in adenomas resulting from 6-NC andits metaboliteswere primarily at G: C basepairs in codons 12and 13, while adenocarcinomas had K-ras mutations distributedbetween codons 12, 13 and 61, and involved both G:C and A:Tbasepairs. The K-ras mutational spectra in codons 12 and 13were similar in both adenomas and adenocarcinomas, whereas ahigher percentage of mutations at A:T incodon 61 was found inadenocarcinomas. These results support the conclusion that the1,2-DHD-6-AC-derived adduct is associated with both adenomaand adenocarcinoma formation and is the primary lesion involvedin the induction of mouse lung tumors by 6-NC. The major adductdetected after 6-NOC treatment, which is derived from N-OH-6-AC,is apparently less efficient as an inducer of mouse lung tumorsand is associated more specifically with adenocarcinoma formation.     

Cytochrome P450 forms in the rodent lung involved in the metabolic activation of food-derived heterocyclic amines

The metabolic activation of the promutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoline(IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx)and 2-amino-1-methyI-6-phenylimidazo[4,5-b]pyridine (PhIP) byrat and mouse lung microsomes was studied using Salmonella mutagenicity(strain TA98). Lungs from uninduced animals were found to activateall three compounds. A 4–6 fold higher mutagenic activitywas obtained with IQ compared to MeIQx and the mutagenic responseof PhIP was 1–2 orders of magnitude lower than that ofIQ. In order to characterize the forms of P450 in the lung responsiblefor the metabolic activation of these food mutagens Westernblots were performed with microsomes and partially purifiedP450 fractions from the lung. Western blots revealed the presenceof cytochrome P450 2A, 2B and 4A forms in untreated rats. Inthe lung CYP 1A1 was only detectable after BNF treatment ofrats. The CYP 4A isozymes, which have not previously been describedin the rat lung, were further identified after PCR amplificationfrom lung mRNA as 4A2 and 4A8. Antibody inhibition studies showedthat CYP 2A3 catalyzed a major part (70%) of the metabolic activationof IQ by uninduced rat lung microsomes. The metabolic activationof MeIQx was not influenced by this antibody. An antibody againstCYP 2B isozymes also partially inhibited the activation of IQby uninduced rat lung microsomes. However, since induction ofCYP 2B isozymes in the liver by phenobarbital treatment didnot increase the metabolic activation of the heterocyclic aminesover controls it is unlikely that the rat lung CYP 2B1 is participatingin the activation of heterocyclic amines. The inhibition ofthe IQ-dependent mutagenicity by the CYP 2B antibody is probablydue to cross-reaction with CYP 2A3. Alfa-naptho-flavone (ANF),considered to be a specific inhibitor of CYP 1A isozymes at10 µM, partly inhibited the activation of IQ (30–40%)and MeIQx (60–80%) by uninduced rat and mouse lung microsomes.Upon pretreatment of rats with BNF, lung microsomes activatedMeIQx at a rate that was 2–10-fold higher than controllung microsomes, whereas the increase in EROD activity was approximately100-fold in the same lung preparations. These results suggestthat CYP 1A1 may not be the enzyme responsible for the activationof MeIQx in the control rat despite the inhibition with ANF.It is likely that ANF can inhibit other P450 enzymes in thelung, including CYP 2A3. The involvement of CYP 2A3. The involvementof CYP 2A3 in the metabolic activation of IQ by uninduced ratlung shows that CYP forms that are not of major importance inthe liver may play a significant role in extra-hepatic activationof heterocyclic amines.     

Mouse endometrium stromal cells express a polycyclic aromatic hydrocarbon-inducible cytochrome P450 that closely resembles the novel P450 in mouse embryo fibroblasts (P450EF)

Stromal cells from mouse endometrium, E041 cells, at passages21, 23 and 26 were metabolically active towards 7,12-dimethylbenz[a]anthracene(DMBA). The total DMBA-metabolizing activity of E041 cells waspreferentially increased by benz[a]anthracene (BA) relativeto 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment (7-foldby BA and 4-fold by TCDD), but the relative proportions of DMBAmetabolites formed remained unchanged. Profiles of DMBA metabolitesgenerated from E041 cell microsomes were very different fromthat of mouse P4501A1 but exhibited similarities to that ofP450EF, the polycyclic aromatic hydrocarbon (PAH)-induciblecytochrome P450 in the mouse embryo fibroblast cell line C3H10T1/2(10T1/2). Notably, both induced and uninduced E041 cell microsomeswere very effective in the formation of the proximate carcinogenDMBA-3,4-dihydrodiol (15–20% of total) and DMBA-10,11-dihydrodiol(13–18% of total) but ineffective in forming the 7-hydroxymethylderivative of DMBA (<1% of total). Antibodies to P450EF completelyinhibited the DMBA-metabolizing activities of both induced anduninduced E041 cell microsomes, with an effectiveness similarto that in microsomes prepared from identically treated 10T1/2cells. Anti-P4501A1 had no inhibitory effect on DMBA metabolismby either induced or uninduced E041 cell microsomes. Total DMBA-metabolizingactivities in BA- and TCDD-induced E041 cells were consistently2-fold lower compared to those in similarly treated 10T1/2 cells.In addition, both induced and uninduced E041 cell microsomesformed a lower proportion of DMBA dihydrodiols relative to phenolsin comparison to identically treated 10T1/2 cell microsomes(0.5 versus 1). Addition of exogenous epoxide hydrolase to E041cell microsomes resulted in a product distribution indistinguishablefrom that in 10T1/2 cells. Immunoblots demonstrated 5-fold lowerlevels of epoxide hydrolase in E041 cell microsomes comparedto 10T1/2 cell microsomes. Anti-P450EF immunoblotted a 55 kdprotein in E041 cell microsomes that was induced 14-fold byBA and 6-fold by TCDD, thus paralleling the increases in therespective DMBA metabolism. It is therefore concluded that followingPAH exposure endometrial stromal cells express the novel PAH-induciblemouse embryo fibroblast P450 and fail to express P4501A1.     

Genotoxicity of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): formation of 2-hydroxamino-PhIP, a directly acting genotixic metabolite

Hepatocytes isdated from Aroclor 1254 (PCB) pretreated ratsmetabolized 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine(PhIP) to a reactive metabolite that induced DNA damage measuredby alkaline elution or as increased unscheduled DNA synthesis.PhIP induced mutations in Salmonella typhimuniun TA98 and DNAstrand breaks and sister chromatid exchange(s) in Chinese hamsterV79 cells co-incubated with PCR-hepatocytes. No, or only minorgenotoxic, effects were observed when hepatocytes from non-inducedrats were used. The bacterial mutagenicity could be inhibitedby -naphthoflavone, indicating a role of P-450 in the activationof PhIP. At least eight different metabolites could be separatedon HPLC after PhIP had been incubated with PCB-hepatocytes.All of the directly acting mutagenicity towards S.typhimuriumTA98 co-eluted with one of the metabolites. The identity ofthis metabolite was concluded to be 2-hydroamino-PhIP basedon the following evidence: (i) it reduced ferric ion to ferrousion as hydroxylamines do, (ii) it had an identical UV spectrumand chromatographic properties as a species formed upon redudionof 2-nitro-PhIP by NADPH P-450 reductase. This product displayeda major peak at m/z 241 during thermospray mass spectrometryin the positive-ion mode as would be expected from 2-hydroxamino-PhIP.2-Hydroxamino-PhIP was directly genotoxic both to TA98 and V79cells. The genotoxic activity of the medium after removing thehepatocytes remained stable for several hours. Compared to 2-amino-3,4-dimethylimidazo-[4,5-f]quinoline(MeIQ), PhIP caused a much larger increase in DNA damage inV79 cells (with hepatocyte activation), whereas MeIQ was morepotent with respect to DNA damage induced in hepatocytes andbacteria.     

Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes.

An acetyltransferase-overexpressing strain of Salmonella typhimurium (NM2009) has been used to investigate roles of human liver microsomal cytochrome P450 (P450) enzymes in the activation of carcinogenic nitrosamine derivatives, including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines, to genotoxic products. Studies employing correlation of activities with several P450-dependent monooxygenase reactions in different human liver samples, inhibition of microsomal activities by antibodies raised against human P450 enzymes and by specific P450 inhibitors, and reconstitution of activities with purified P450 enzymes suggest that the tobacco-smoke-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N-nitrosonornicotine (NNN) as well as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are oxidized to genotoxic products by different P450 enzymes, particularly P450 2E1 and 2A6. The activation of NDMA and NNN by liver microsomes was suggested to be catalyzed more actively by P450 2E1 than by other P450 enzymes because the activities were well correlated with NDMA N-demethylation and aniline p-hydroxylation in different human samples, and purified P450 2E1 had the highest activities in reconstituted monooxygenase systems. The relatively high contribution of P450 2A6 to the activation of NDEA and NNK was supported by the correlation seen with coumarin 7-hydroxylation in human liver microsomes, and antibodies raised against P450 2A6 inhibited both activities by approximately 50%. P450 3A4, 2D6 and 2C enzymes appear not to be extensively involved in the activation of these nitrosamines as judged by several criteria examined. Thus, this work indicates that several P450 enzymes, particularly P450 2E1 and 2A6, catalyze metabolic activation of nitrosamine derivatives including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines in human liver microsomes.     

Activation of the food-derived mutagen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by rabbit and human liver microsomes and purified forms of cytochrome P-450

The specificity of rabbit cytochrome P-450 involved in the mutagenicactivation of 2-amino-1-methyl-6-phenylimid-azo[4, 5-b]pyridine(PhIP) was assessed using control and induced rabbit liver andlung microsomes, and six purified forms of cytochrome P-450.The number of revertants produced/2.5µg PhIP by controlrabbit liver was 260 ± 196/10 µg of microsomalprotein (mean ± SD; n = 3), and this increased to 1265± 248 when 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD)-inducedliver microsomes were used as the activation source in the Amestest. Microsomes form phenobarbital-, rifampicin-and acetone-pretreatedrabbits showed no increase in activity over controls. Controllung microsomes did not activate PhIP to a mutagen, whereasTCDD-induced lung microsomes produced 1443 ± 136 (mean± SD; n=4) Ames/Salmonella revertants/100 µg protein.In reconstitution experiments cytochrome P450 forms 4 and 6were found to be efficient activators of PhIP to a mutagen.Form 6 was 3.1-fold more active than form 4 and produced 4577revertants/10 pmol with a 20-min preincubation step in the Amestest. Cytochrome form 5 produced 17 revertants/10 pmol and forms2, 3b and 3c were not active in metabolizing PhIP to a mutagen.A highly significant statistical correlation existed betweenthe capacity of control and induced liver microsomes to activatePhIP to a mutagen and their cytochrome P-450 form 4 (r = 0.97,r² = 0.94) and form 6 (r = 0.95, r² = 0.90) content. These datastrongly support the involvement of polycyclic hydrocarbon-inducibleforms of cytochrome P450 in the activation of PhIP in the rabbit.Anti-rabbit forms 4 and 6 IgGs recognized proteins in sevenhuman liver microsomes of comparable mol. wt to rabbit cytochromeP-450 forms 4 and 6. However, no correlation existed betweenthe content of these proteins and the capacity of human livermicrosomes to activate PhIP.     

Metabolism and DNA binding of the environmental pollutant 6-nitrochrysene in primary culture of human breast cells and in cultured MCF-10A,MCF-7 and MDA-MB-435s cell lines

The environmental pollutant 6-nitrochrysene (6-NC) is a potent mammary carcinogen in the rat. To determine if the results obtained in 6-NC-treated rodents can be applicable to humans, we examined its metabolic activation in primary cultures of human breast cells prepared from tissues obtained from reduction mammoplasty from 3 women and in a cultured, immortalized human mammary epithelial cell line (MCF-10A), as well as estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-435s) human breast cancer cell lines. Metabolites identified following 24 hr incubations of [(3)H]6-NC (2.5, 5.0 and 10 microM) with human breast cells were derived from ring-oxidation (trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene [1,2-DHD-6-NC]) and nitro-reduction (6-aminochrysene [6-AC]); chrysene-5,6-quinone (C-5,6-Q) was also detected. Levels of metabolites (pmol/mg protein) varied greatly depending on the concentration of 6-NC and the individual breast tissue used; 1,2-DHD-6-NC, ranged from not detected to 15.6 +/- 1.0; 6-AC, from 11.5 +/- 4.0 to 155 +/- 10.2; C-5,6-Q, from 18.3 +/- 10.8 to 196.7 +/- 15.4. Qualitatively similar metabolic profiles were obtained upon incubation of [(3)H]6-NC with MCF-10A, MCF-7 and MDA-MB-435s. We also detected 1,2-dihydroxy-6-aminochrysene (1,2-DH-6-AC; ranged from not detected to 50.4 +/- 9.8). Some of the metabolites identified in our study are known to be proximate carcinogenic forms of 6-NC in rodents. MCF-7 was the most efficient cell line in catalyzing 6-NC to genotoxic metabolites, and we demonstrated that the major DNA adduct is chromatographically identical to that found in the mammary gland of rats treated by gavage with 6-NC and that obtained from the incubation of [(3)H]1,2-DHD-6-NC with MCF7 cells or from nitro-reduction of 1,2-DHD-6-NC in the presence of 2'-deoxyguanosine 5'-monophosphate in vitro. This is the first report to demonstrate the ability of human breast cells, MCF-10A, and breast cancer cell lines to activate 6-NC to metabolites that can damage DNA.     

Metabolism of aflatoxin B1 by rabbit and rat nasal mucosa microsomes and purified cytochrome P450, including isoforms 2A10 and 2A11

The nasal mucosa of some mammalian species are susceptible tothe toxicity of aflatoxin B1 (AFB1), a potent hepato-carcinogen,but little is known about the nasal enzymes involved in themetabolic activation of AFB1 or the metabolites produced. Inthe present study, the metabolism of AFB1 was studied with nasalmicrosomes from rats and rabbits and with several purified isozymesof rabbit P450 in a reconstituted enzyme system. The rates ofAFB1-N7-guanine DNA adduct formation with rabbit and rat nasalmicrosomes are over 3- and 10-fold higher, respectively, thanwith liver microsomes from the same species. On the other hand,the rates of formation of AFM1 (9a-hydroxy-AFB1) and AFQ1 (3-hydroxy-AFB1)products known to be less toxic, are lower with nasal than withliver microsomes. Of particular interest, nasal microsomes producehigh levels of six unidentified polar metabolites that are notformed by microsomes from liver or several other tissues. Thesesame products are also generated by P450 NMa purified from rabbitnasal microsomes in a reconstituted system, but not by fiveother isozymes of cytochrome P450 (1A2, 2B4, 2E1, 2G1, 3A6)that are known to be present in nasal microsomes. AFB1-DNA adductsare formed by P450 NMa at a rate 3-fold higher than that bynasal microsomes. The DNA adducts are formed at much slowerrates by P450s 2G1, 2B4, and 1A2, and adducts are not formedat measurable rates by P450s 2E1 and 3A6. Moreover, AFB1—     

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION: Protection by green tea, black tea, and indole-3-carbinol against 2-amino-3-methylimidazo[4,5-f]quinoline-induced DNA adducts and colonic aberrant crypts in the F344 rat

Male F344 rats were exposed for 8 weeks to extracts of greentea (2% w/v) or black tea (1% w/v), or to 0.1% dietary indole-3-carbinol(I3C). In weeks 3 and 4 of the study, rats were given 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) every other day by oral gavage (50 mg/kgbody wt) in order to induce aberrant crypt foci (ACF) in thecolon. Compared with controls given IQ alone, all three inhibitorsreduced the number of total aberrant crypts per colon, and greentea and I3C inhibited significantly the mean number of ACF (P< 0.05). Rats pre-treated with green tea, black tea, or I3Cand given a single p.o. injection of 50 mg IQ/kg body wt 24–48h before sacrifice had reduced levels of IQ-DNA adducts in theliver, and excreted lower amounts of IQ and other promutagensin the urine and feces. Inhibitors also reduced the excretionof IQ-sulfamate in the urine, but increased the relative amountsof IQ-5-O-sulfate and IQ-5-O-glucuronide. Western blotting togetherwith assays for 7-ethoxyresorufin O-deethylase and methoxyresorufinO-demethylase established that I3C preferentially induced cytochromeP4501A1 over 1A2, consistent with the altered profile of urinarymetabolites. However, both teas caused slight induction of cytochromeP4501A2 versus 1A1, which would be predicted to enhance theactivation of IQ. Thus, green tea and black tea are likely toprotect against IQ-DNA adducts and ACF by mechanisms other thaninduction of cytochromes P450, such as inhibition of enzymeswhich activate IQ or the scavenging of reactive intermediates.     

Metabolic activation of the potent carcinogen dibenzo[a, l]pyrene by human recombinant cytochromes P450, lung and liver microsomes

The metabolic activation of dibenzo[a, l]pyrene (DB[a, l]P),recently considered the most potent carcinogen among all polycyclicaromatic hydrocarbons, to the 11, 12-dihydrodiol, a precursorof the ultimate carcinogens, the 11, 12-diol-13, 14-epoxides,was investigated using eleven human recombinant cytochrome P450s,as well as human lung and liver microsomes. Of all human P450s,1A1 was the most active in the metabolism of DB[a, l]P (310pmol/min, nmol P450) and had 5–23-fold higher catalyticactivity than other P450s examined. The order of activity inthe formation of the 11, 12-dihydrodiol was as follows: 1A1(116 pmol/min, nmol P450) > 2C9 (29) > 1A2 (22) > 2B6(18) > 3A4 (16) > others (     

Cytochrome P450 species involved in the metabolism of quinoline

Quinoline is a hepatocarcinogen in rats and mice and a well-knownmutagen in bacteria after incubation with rat liver microsomes.The specific cytochrome P450 enzymes involved in quinoline metabolismin human and rat liver microsomes were determined using cDNA-expressedcytochrome P450s, correlations with specific cytochrome P450-linkedmonooxygenase activities in human liver microsomes and inhibitionby specific inhibitors and antibodies. CYP2A6 is the principalcytochrome P450 involved in the formation of quinoline-1-oxidein human liver micro-somes (correlation coefficient r = 0.95),but is formed in only minute quantities in rat liver microsomes.CYP2E1 is the principal cytochrome P450 involved in the formationof 3-hydroxyquinoline (r = 0.93) in human liver microsomes andis involved in the formation in rat liver microsomes. A highcorrelation coefficient (r = 0.91) between CYP2A6 activity andquinoline-5,6-diol formation in human liver microsomes was observed,but this most likely reflects the involvement of CYP2A6 in theformation of quinoline-5,6-epoxide, from which the quinoline-5,6-diolis formed, as conversion of quinoline-5,6-epoxide to quinoline-5,6-diolon incubation of the epoxide with CYP2A6 could not be demonstrated.A cDNA-expressed human microsomal epoxide hydrolase, however,efficiently converted the epoxide to the diol and the microsomalepoxide inhibitor cyclohexene oxide inhibited quinoline-5,6-diolformation in rat liver microsomes. A preliminary kinetic analysisof quinoline metabolism in human liver microsomes was carriedout and Eadie-Hofstee plots indicate that the formation of quinoline-5,6-diolis monophasic, while that of quinoline-1-oxide and 3-hydroxyquinolineis biphasic.     

Activation of 2-amino-6-methyldipyrido[1, 2-a: 3', 2'-d]imidazole, a mutagenic pyrolysis product of glutamic acid, to bind to microsomal protein by NADPH-dependent and -independent enzyme systems

The conversion of 2-amino-6-methyldipyrido[l, 2-a: 3',2'-d]-imidazole(Glu-P-1), a highly mutagenic principle in a pyro-lysate ofglutamic acid, to protein-bound metabolites in vitro was examinedwith microsomes from various tissues of female F344 rats. Additionof NADPH to the incubation mixture containing microsomes and[¹⁴C]Glu-P-1 increased the binding of its metabolites to microsomalproteins linearly with time for up to 30 min, while on additionof arachidonic acid the binding increased linearly only forthe first 2-4 min of incubation and then levelled off. However,due to the initial rapid binding, addition of arachidonic addresulted in 6-fold greater binding of metabolites to small intestinalmicrosomes than addition of NADPH on incubation for 4 min, andwith microsomes from liver and colon, arachidonic acid was foundto be a better cofactor than NADPH for activation of Glu-P-1.Indomethadn significantly inhibited the increase in bindingby arachidonic acid. Additions of linoleic and linolenic addsalso increased the binding, but addition of oteic acid had noinfluence. With hepatic microsomes from 3-methykholanthrene-treatedrats, binding within 4 min after addition of arachidonic addwas greater than that after addition of NADPH and the reverseon further incubation. These findings suggest that prostaglandinsynthetase may serve as an alternative enzyme to cytochromeP-450 monooxygenases for conversion of Glu-P-1 to active intermediatesin all the rat tissues investigated.     

Metabolic activation of a protein pyrolysate promutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by rat liver microsomes and purified cytochrome P-450

The enzymatic activation of a promutagenic pyrolysate, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx), was studied using the Ames mutagenesis test system.The enzyme catalyzing the mutagenic activation of MeIQx is mainlylocalized in the microsomal fraction. A large number of revertantswas observed in the presence of hepatic microsomes obtainedfrom 3-methylcholanthrene (3-MC)- or polychlorinated biphenyl(PCB)-treated rats but only a minimal number with the hepaticmicrosomes from untreated or phenobarbital (PB)-treated rats.In addition, the microsomal activation was reduced efficientlyby known inhibitors of cytochrome P-450- mediated reactionssuch as 7,8-benzoflavone, ellipticine and flavone. Among fiveforms of purified rat cytochrome P-450, the highest sp. act.(no. of revertants induced/nmol cytochrome P-450) for the activationof MeIQx was observed with a high-spin form of cytochrome P-450,P-448-H, followed by the low-spin form, P-448-L, and to a lesserextent by PB-inducible forms, P-450b and P-450e. P-450-male,which is a main constitutive form of cytochrome P-450 In malerat livers, showed considerable catalysis for the mutagenicactivation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) andMeIQx. These results Indicate that the metabolic activationof MeIQx is catalyzed mainly by two forms of cytochrome P-450,P-448-H and P-488-L, in the livers of PCB- or 3-MC-treated rats,but also that P-450-male may play an important role in the activationin livers of Intact male rats.     

Metabolic activation of heterocyclic aromatic amines catalyzed by human arylamine N-acetyltransferase isozymes (NAT1 and NAT2) expressed in Salmonella typhimurium

Heterocyclic aromatic amines formed during the cooking of meatand meat-derived products can be activated to reactive metaboliteswhich bind to DNA, induce mutations and cause tumors in animals.A principal route of metabolic activation is N-oxidation tohydroxylamines, and their subsequent activation by acetyltransferase-catalyzedO-acetylation. We have used mutagenicity assays to study O-acetylationof heterocyclic arylhydroxylamines by the two isozymes of humanN-acetyltransferase, NAT1 and NAT2, expressed in Salmonellatyphimurium. N-Acetylation was also examined, using an HPLCmethod. In addition, Salmonella strains with endogenous acetyltransferaseand lacking this activating activity were used. Hydroxylaminesof nine heterocyclic aromatic amines, IQ, isoIQ, MeIQ, MeIQx,NI, PhIP, Glu-P-1, Glu-P-2, and Trp-P-2 were generated in situby rat liver S9 mix. The strains expressing human NAT1 and lackingacetyltransferase activity showing little or no ability to activatethese substrates. The strains expressing human NAT2 and Salmonellaacetyltransferase supported to different extents the activationof all the compounds except PhIP and Trp-P-2. N-Acetylationof IQ, MeIQx and PhIP was slow or not detectable. In conclusion,human NAT2 but not NAT1 can O-acetylate heterocyclic hydroxylamines.NAT2 probably plays a key role in the genotoxic effects of theabove heterocyclic amines except for PhIP and Trp-P-2, whichhave NAT2-independent mutagenic activity.     

Activation of the carcinogens N-hydroxy-N-2-fluorenylbenzamide and N-hydroxy-N-2-fluorenylacetamide via deacylations and acetyl transfers by rat peritoneal serosa and liver

Intraperitoneally administered N-hydroxy-N-2-fluorenylbenzamide(N-OH-2-FBA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA)are carcinogenic for rat peritoneum. The potential of peritonealserosa to activate these compounds via deacylations and acyltransfers was compared to that of liver. N-Deacylations of N-OH-2-FBAand N-OH-2-FAA to N-2-fluorenylhydroxylamine (N-OH-2-FA) werefaster by liver than serosa and by microsomes than cytosols.N-Debenz-oylations of N-OH-2-FBA were 73- to 123-fold fasterthan N-deacetylations of N-OH-2-FAA. The esters, N-benzoyloxy-2-FBAand N-acetoxy-2-FAA, were O- and N-deacylated to N-OH-2-FA byliver, and the benzoate by serosa. Inhibition by paraoxon ofthe above deacylations implicated a serine carboxylesterase.Liver and serosa cytosols catalyzed acetyl CoA-, but not benzoylCoA-, dependent and iodoacetamide (IAA)-sensitive N-acylationof N-2-fluorenamine (2-FA), implicating an acetyltransferase.In hepatic microsomes this activity was LAA-insensitive andpartially inhibited by para-oxon. Liver cytosol, but not microsomes,used N-OH-2-FAA as an acyl donor and neither used N-OH-2-FBA.Liver and serosa catalyzed binding to DNA of N-OH-2-[ring-³H]FBAwhich was paraoxon-sensitive and increased by acetyl CoA, butnot benzoyl CoA. Binding to DNA of N-OH-2-[ring-³H]FAA catalyzedby cytosols was 22-fold greater in liver than in serosa andwas IAA-sensitive. Microsome-catalyzed binding of this compoundin both tissues was increased 2-fold by acetyl CoA. The resultssupport a two-step activation of N-OH-2-FBA in the liver consistingof esterase-catalyzed N-debenzoylation to N-OH-2-FA and an acyltrans-ferase-catalyzedO-acetylation to the putative electrophile N-acetoxy-2-FA. Inthe serosa, binding to DNA appears to be due to rapid N-debenzoylationto N-OH-2-FA, a fraction of which is O-acetylated. Whereas activationof N-OH-2-FAA by liver and serosa microsomes may also involveN-OH-2-FA and/or its O-acetate, activation by the cytosols isconsistent with N, O-acetyltransfer of N-OH-2-FAA to yield N-acetoxy-2-FA.The study provides first evidence for activation of N-OH-2-FBAby rat liver and of both compounds by peritoneum in vitro.     

Genotoxicity of the flame retardant tris(2,3-dibromopropyl)phosphate in the rat and Drosophila: effects of deuterium substitution

Formation of 2-bromoacrolein (2BA) from tris(2,3-dibromo- propyl)phosphate(Tris-BP) by cytochrome P450 (cyt. P450) activity is most likelyresponsible for the high mutagenicity of Tris-BP for bacteriain vitro when rat liver microsomes are used for metabolic activation.As yet, it has not been established whether cyt. P450 playsa role in the formation of genotoxic metabolites from Tris-BPin higher organisms in vivo. This was studied by comparing theeffects of completely deuterated Tris-BP (D15-Tris-BP) withnormal Tris-BP in various in vivo test systems for genotoxicity:deuterium substitution should decrease cyt. P450-dependent bioactivationof Tris-BP. Three test systems in Drosophila were used to measuredifferent types of genetic damage: (I) the ring-X chromosomeloss (CL) test to detect clastogenicity, (II) the sex-linkedrecessive lethal (RL) test to detect forward mutations and deletionsand (III) the white/white⁺ (w/w⁺) eye mosaic assay to detectpredominantly mitotic recombin ation. Tris-BP was positive inall test systems, while D15-Tris-BP was without effect. Therelative clastogenic efficiency of a compound, defined as theratio of CL over RL, can be used to distinguish monofunctionalagents (CL/;RL ratio