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.     

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.     

Roles of different cytochrome P450 enzymes in bioactivation of the potent hepatocarcinogen 3-methoxy-4-aminoazobenzene by rat and human liver microsomes

The potent hepatocarcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) has been reported to be bioactivated to mutagenic intermediates by rat liver microsomal cytochrome P450 (P450) and to be a selective inducer of rat P450IA2. In this study we have further investigated the roles of individual rat and human P450 enzymes in the bioactivation of this hepatocarcinogen in a Salmonella typhimurium TA1535/pSK1002 system where umu response is indicative of DNA damage. 3-MeO-AAB was found to be bioactivated by liver microsomal enzymes from rats and humans in this assay system. The liver microsomal activities are increased by pretreatment of rats with various P450 inducers such as phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX), acetone, ethanol, isoniazid (INH), diphenylhydantoin and valproic acid, and can be inhibited considerably by SKF-525A and metyrapone. alpha-Naphthoflavone (ANF) is also an inhibitor for the reaction catalyzed in BNF-treated rats, but stimulated the microsomal activity in DEX-treated rats. Evidence has also been obtained that specific antibodies raised against P450IIB1, P450IA1 or IA2, P450IIE1, and P450IIIA2 inhibited the activation in liver microsomes from rats pretreated with PB, BNF, INH and DEX respectively, suggesting the possible roles of several P450 enzymes in the bioactivation of 3-MeO-AAB. The results obtained with reconstituted monooxygenase systems containing various rat P450 enzymes are highly supportive of this conclusion. Human liver microsomal activation of 3-MeO-AAB was also inhibited to various extents by antibodies raised against P450IA2, P450MP, P450IIE1 and P450IIIA4. In a reconstituted system containing purified forms of human P450, P450IA2 was the most active in catalyzing 3-MeO-AAB, followed by P450IIIA4 and P450MP. ANF, a known activator of P450IIIA-catalyzed reactions, caused an increase in activation of 3-MeO-AAB in human liver microsomal and P450IIIA4- and P450MP-containing reconstituted systems. From these results it is concluded that multiple P450 enzymes in rat and human liver microsomes are involved in the bioactivation of 3-MeO-AAB, regardless of its selective induction of the rat P450IA2 gene.     

Selective expression and induction of cytochrome P450PB and P450MC during the development of hereditary hepatitis and hepatoma of LEC rats

The Long-Evans rat with a cinnamon-like coat color (LEC rat)is a mutant strain displaying hereditary hepatitis with severejaundice. The age related difference in microsomal dealkylationof pentoxyresorufin and ethoxyresorufin was examined. The enzymeactivity levels of pentoxyresorufin O-depentylase in LEC ratswere decreased to 25% of the levels in control [Long-Evans ratswith an agouti coat color (LEA rats)]. In contrast, ethoxyresorufinO-deethylase exhibited a much less marked difference betweenthe strains. In parallel with these strain differences in enzymeactivities, a decrease in phenobarbital (PB) indudble P450 isozymes,mainly P450b and P450e, was observed by Western blot analysis.The level of P450_PB in LEC rats was more markedly depressedthan in the LEA strain. On the other hand, microsomes from uninducedLEC rat liver had more 3-methylcholanthrene (MC) inducible P450_MC,mainly P450c and P450d, than microsomes from LEA rat liver andthese isozymes in the LEC were markedly induced by 3-methylcholanthrenetreatment. The great difference in cytochrome P450_PB contentof the liver microsomes between LEC and LEA rats and the maintainedconstitutive levels of hepatic cytochrome P450_MC in the LECrats suggest a possible role of these cytochrome isozymes inthe onset of spontaneous hepatitis and hepatoma.     

Inhibition of 2-aminofluorene mutagenesis in bacteria by inducers of cytochrome P-450d

Certain chemical inducers of rat liver microsomal cytochromeP-450d are tightly bound to the cytochrome. We investigatedthe ability of two inducers of cytochrome P-450d, Aroclor 1254and isosafrole, to inhibit the microsomal activation of 2-aminofluoreneto a mutagen as measured in Salmonella typhimurium. Butanoltreatment of microsomes from isosafrole-treated rats removedan inhibitory metabolite of isosafrole and increased 2-aminofluorenemutagenesis by {small tilde}2-fold over controls. Butanol treatmentof microsomes from Aroclor 1254-treated rats failed to eitherremove any of the Aroclor 1254 associated with microsomal cytochromeP-450 or affect 2-aminofluorene-induced mutagenesis. However,addition of Aroclor 1254 to butanol-treated microsomes fromisosafrole-treated rats almost completely inhibited 2-aminofluorenemutagenesis. Aroclor 1254 completely inhibited the cytochromeP-450d-dependent estradiol 2-hydroxylase activity of butanoi-treatedmicrosomes from isosafrole-treated rats. Thus, we suspect thatcertain congeners from Aroclor 1254, a widely used mixture forinduction of cytochrome P-450 activities, could inhibit cytochromeP-450d and partially mask its ability to metabolize some chemicalsto mutagens.     

Metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone as measured by DNA alkylation in vitro and its inhibition by isothiocyanates

The bioactivation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by microsomes from target organs was studied with an in vitro microsome-mediated DNA alkylation system. Mouse lung, rat lung, and rat nasal microsomes catalyzed a time- and protein-dependent DNA methylation by [methyl-3H]NNK with activities of 4.11, 0.95, and 137.4 pmol/mg DNA/mg protein/h, respectively. The DNA methylation of NNK catalyzed by all three microsomal systems was inhibited by cytochrome P-450 inhibitors, such as carbon monoxide and metyrapone, but not by the cyclooxygenase inhibitor, aspirin, or by prolonged preincubation in the absence of NADPH. The possible involvement of specific P450 isozymes was assessed by specific inhibitory antibodies. An anti-P450IIB1&2 antibody significantly inhibited the DNA methylation by 45 and 32% in mouse lung and rat lung, respectively, whereas anti-P450IA1 and anti-P450IIE1 antibodies failed to show significant inhibition. All antibodies showed no inhibition in rat nasal microsomes. Glutathione inhibited the DNA methylation in a concentration-dependent manner in all three microsomal systems. Phenethyl isothiocyanate (PEITC), at doses of 0.25 and 1.00 mmol/kg body weight, was given intragastrically 2 h before sacrifice to mice and 24 h before sacrifice to rats, respectively; both mouse and rat lung microsomal activities were inhibited by about 40 and 90% by the low- and high-dose PEITC treatments, respectively. The rat nasal microsomes were only inhibited by the high-dose PEITC treatment by about 40%. PEITC, 4-phenylbutyl isothiocyanate, and 6-phenylhexyl isothiocyanate all inhibited the microsome-mediated DNA methylation of NNK in vitro, with 4-phenylbutyl isothiocyanate and 6-phenylhexyl isothiocyanate being more potent than PEITC and the mouse lung microsomes more sensitive than the rat lung and nasal microsomes. All three microsomal systems were shown to catalyze the in vitro DNA pyridyloxobutylation by [5-3H]NNK. On an equal protein basis, the rat nasal microsomes were much more active in catalyzing the DNA pyridyloxobutylation.     

Characterization of cytochrome P450-dependent dimethylhydrazine metabolism in human colon microsomes

Polyclonal antibodies to components of the rat liver cytochrome P450 system were used to examine the composition and function of the microsomal cytochrome P450-dependent monooxygenase system of human colonic mucosal cells. Anticytochrome P450 reductase antibody gave a strong band of immunocross-reactivity in human colon microsomes at the same molecular weight level as purified cytochrome P450 reductase from rat liver, as well as hepatic microsomes isolated from untreated or phenobarbital-treated rats. These results demonstrate the presence of cytochrome P450 reductase in human colon cells. Similarly, cytochromes P450 IIB1 and IIA1 also appear to be present in Western blots of human colon microsomes. These antibodies, as well as antibodies to reductase and cytochrome b5, inhibit dimethylhydrazine metabolism in human colon microsomes to varying degrees. These data argue for a functional P450-dependent drug metabolism system in colon capable of activating/metabolizing the colon-specific model carcinogen, 1,2-dimethylhydrazine.     

Co-expression of human CYP1A1 and a human analog of cytochrome P450-EF in response to 2,3,7,8-tetrachloro-dibenzo-pdioxin in the human mammary carcinoma-derived MCF-7 cells

Cultured human mammary carcinoma (MCF-7) cells exhibited constitutivecytochrome P450-dependent metabolism of 7,12-dimethylbenz[a]anthracene(DMBA) (45–75 pmol/mg microsomal protein). Exposure ofthe cells to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), whichis known to induce CYP1A1, not only resulted in a 30-fold increasein the total microsomal metabolism of DMBA but produced substantialdifferences in the distribution of DMBA metabolites formed.This suggested that different cytochrome P450 (P450) forms predominatedin untreated and induced cells. Comparative studies with TCDD-inducedhuman hepatoblastoma (HepG2) and skin cell carcinoma (SCC-13)cells and also recombinantly expressed human CYP1A1, confirmedthat the DMBA metabolite profile in TCDD-induced MCF-7 cellswas that of human CYP1A1. This distribution, however, differedsubstantially from the regioselectivity of rat CYP1A1 and mouseCypla-1. Rabbit antibodies to rat CYP1A1 completely inhibitedthe DMBA-metabolizing activity of TCDD-induced MCF-7 cells buthad no inhibitory effect on constitutive DMBA metabolism whichwas, however, completely inhibited by chicken antibodies tothe novel P450 in mouse embryo fibroblasts (P450-EF). Anti-P450-EFinhibited only 10% of the DMBA-metabolizing activity in theTCDD-induced MCF-7 cell microsomes. Microsomes from untreatedMCF-7 cells expressed a 52 kDa protein that was immunodetectableby rabbit anti-P450-EF and failed to express immunodetectablelevels of human CYP1A1. DMBA metabolism, therefore, s from P450-EFin uninduced microsomes to CYP1A1 in TCDD-induced microsomes.The mobility of the P450-EF-like protein in MCF-7 cells washigher than that of P450-EF from C3H/10T1/2CL8 (10T1/2) cells(55 kDa). The 52 kDa protein from MCF-7 cells was induced     

Relative contribution of various forms of cytochrome P450 to the metabolism of benzo[a]pyrene by human liver microsomes

A variety of cytochromes P450 have been implicated in the hepaticmetabolism of benzo[a]pyrene (BP), including forms that areconstitutively expressed and those that are highly inducible.In the present study the metabolism of BP to organic solvent-solublederivatives by eight forms of cytochrome P450 isolated fromrat liver and by a series of 11 human liver microsomal sampleswas investigated. The relative contribution of specific P450forms to the human hepatic metabolism was evaluated. A 4-foldvariation in formation of total organic solvent-soluble BP metaboliteswas observed, as well as differences in the regio- and stereo-selectivityof this metabolism between the three individuals studied. Thelevels of expression of cytochromes P450 from five gene sub-families,as determined by Western blot analysis, did not show any correlationwith the rate of BP metabolism to organic solvent-soluble derivativesin these livers. No reduction in metabolism was observed inthree livers in which either the debrisoqulne P450 (P450IID1)was not expressed or bufuralol 1-hydroxylase activity was low.Of six different antibodies to forms of rat liver P450 tested,only those to P450s MC_la (P450IA2), MC_lb (P450IA1) and UT (P450IIA1)consistently inhibited BP metabolism. This inhibition was generallylimited and rarely exceeded 30%. An antibody to cytochrome P450PB (P450 did, however, inhibit the formation of metabolitesat the 4,5- and 9,10-positions of BP by microsomal fractionsof livers from one individual who had been receiving the drugphenytoin. These data indicate that several forms of P450 inhuman liver are involved in the metabolism of BP and that bothconstitutively expressed as well as inducible forms are importantin its disposition in man.     

Inactivation of 1,3-, 1,6-, and 1,8-dinitropyrene by cytochrome P-450 enzymes in human and rat liver microsomes

NADPH-fortified human liver microsomes were examined with regard to ability to detoxicate several chemicals that do not require enzymatic oxidation to elicit a genotoxic response in a Salmonella typhimurium TA1535/pSK1002 system where umu response is used as an indicator of DNA damage. Microsomes did not affect the response seen with daunomycin, mitomycin C, 2,4,7-trinitro-9-fluorene, 1-nitropyrene, doxorubicin, 1-methyl-3-nitro-1-nitrosoguanidine, 2-nitrofluorene, or 1-ethyl-3-nitro-1-nitrosoguanidine (cited in order of decreasing umu response per mol). Human and rat liver microsomes did inactivate 1,3-, 1,6-, and 1,8-dinitropyrene; with human liver microsomes the activity of 1,3-dinitropyrene was most strongly inhibited, while with rat liver microsomes the genotoxicities of all three dinitropyrenes were inhibited to a similar extent. NADPH-cytochrome P-450 reductase was demonstrated to inactivate 1,6- and 1,8-dinitropyrene but not 1,3-dinitropyrene. Both rat cytochrome P-450 beta NF-B (P-450 IA1) and P-450ISF-G (P-450 IA2) inactivated 1,3-dinitropyrene, with the former being more effective. Correlation studies done with liver microsomes prepared from variously treated rats and immunoinhibition studies suggest that cytochrome P-450 beta NF-B and P-450ISF-G are both involved in the detoxication of all three of the dinitropyrenes in rat liver microsomes. In a series of assays done with various human liver microsomal preparations, the inactivation of the three dinitropyrenes was not correlated to each other at all. Correlation analysis and inhibition studies with 7,8-benzoflavone and antibodies indicate that human cytochrome P-450 enzymes in the IA family are most effective in detoxicating this compound; the contribution of cytochrome P-450PA (P-450 IA2, the phenacetin O-deethylase) is deemed more important, but a role for the small amount of cytochrome P1-450 (P-450 IA1) in the liver cannot be ruled out. In contrast to the case of 1,3-dinitropyrene, the inactivation of 1,6-dinitropyrene is well correlated with levels of cytochrome P-450NF (P-450 IIIA4, nifedipine oxidase) and its catalytic activities. The inactivation of 1,8-dinitropyrene was not correlated with any of the above parameters and only correlated with the conversion of benzo(a)pyrene to its 3-hydroxy and 4,5-dihydrodiol products, for which the principal enzymes involved in human liver are unknown. Thus, distinct human cytochrome P-450 enzymes are involved in the detoxication of different dinitropyrene congeners, and the situation appears to contrast with that in rat liver.     

Contributions of human liver cytochrome P450 enzymes to the N-oxidation of 4,4'-methylene-bis(2-chloroaniline).

4,4'-Methylene-bis(2-chloroaniline) (MOCA) can produce tumors in rodents and dogs and an increased incidence of bladder tumors has been reported in exposed workers. It is therefore of interest to identify the human cytochrome P450 (P450) enzymes involved in MOCA N-oxidation, the primary reaction involved in the formation of an electrophilic product. Human liver microsomes were fractionated and MOCA N-oxidation activity was monitored through the procedure. The most active enzyme fraction corresponded to P450 3A4, as determined by immunochemical assays and N-terminal amino acid sequence analysis. Yeast recombinant P450 3A4 also had MOCA N-oxidation activity. Purified human liver P450 2A6 showed catalytic activity; however, anti-P450 2A6 inhibited less than 20% of the microsomal activity while anti-P450 3A4 inhibited up to 75%. Levels of marker activities of both P450 3A4 (nifedipine oxidation) and P450 2A6 (coumarin 7-hydroxylation) were measured in a set of human liver microsomes and both were correlated with MOCA N-oxidation rates. Gestodene and troleandomycin inhibited up to half of the microsomal MOCA N-hydroxylation activity but 7,8-benzoflavone showed only slight inhibition. Anti-P450 3A4 inhibited (up to 80% of) the microsomal transformation of MOCA to a product genotoxic as judged by bacterial SOS response. The work indicates that P450 3A4 makes a major contribution to human liver microsomal MOCA N-oxidation, and P450 2A6 has a minor role. P450 1A2, which catalyzes the hydroxylation of many arylamines, does not contribute to a great extent.     

Activation of trans-l,2-dihydro-l,2-dihydroxy-6-aminochrysene to genotoxic metabolites by rat and human cytochromes P450

In order to address the hypothesis that 6-aminochrysene (6-AC)is converted to genotoxic products by cytochrome P450 enzymesvia two activation pathways (N-hydroxylation and epoxidation),the activation of 6-AC and trans-l,2-dihydro-l,2-dihydroxy-6-aminochrysene(6-AC-diol) to genotoxic metabolites was examined in rat andhuman liver microsomal cytochrome P450 enzymes using Salmonellatyphimurium TA1535/pSK1002 and TA1535/pSK1002/pNM12 (NM2009)as tester strains. The latter bacteria, an O-acetyl-transferase-overexpressingstrain, was highly sensitive to metabolites derived from activationof 6-AC, but not those from 6-AC-diol, using liver microsomesfrom phenobarbital-treated rats or a reconstituted monooxygenasesystem containing P4502B1 or -2B2, thus suggesting the rolesof P450 and acetyltransferase systems in the activation process.6-AC-diol, on the other hand, was activated very efficientlyby liver microsomes prepared from ß-naphthoflavone-treatedrats or a reconstituted system containing P4501A1 or -1A2; theactivation reaction is considered to proceed through diol-epoxideformation. The contribution of rat P4501A enzymes towards activationof 6-AC-diol was confirmed by the inhibitory effects on theactivation process of -naphthoflavone, a specific inhibitorof P4501 A-related activities, and antibodies raised againstpurified P4501A1 and -1A2. In humans, P4501A2 was found to bethe major enzyme involved in the activation of 6-AC-diol togenotoxic metabolites while the parent compound 6-AC was activatedmainly by P4503A4. Experiments using recombinant P450 proteinsexpressed in human lymphoblastoid cells lines showed that humanP4501A1 could also activate 6-AC-diol to reactive metabolitesat almost the same rate measured with P4501A2. In addition,P4502B6 was found to efficiently catalyze the activation of6-AC to genotoxic metabolites, and P4503A4 was active in theactivation of 6-AC-diol as well as 6-AC. Addition of purifiedrat epoxide hydrolase to the incubation mixture containing purifiedrat P4501A1 or microsomes expressing human P4501A1 caused inhibitionof activation of 6-AC-diol. These results suggest the existenceof different enzymatic activation pathways for 6-AC and 6-AC-diol.The former carcinogen may be N-hydroxylated principally by P4502Benzymes in rats and P4503A4 and -2B6 in humans and activationto its ultimate metabolites may proceed through esterificationof the N-hydroxy metabolites by an N-acetyltransferase. The6-AC-diol is metabolized to its ultimate diolepoxide productby P4501A enzymes in rat and human liver microsomes. P4503A4(humans) and P4503A2 (rats) may also contribute to some extentin the activation of 6-AC-diol, albeit at lower rates than thoseof P4501A enzymes.     

Identification of the cytochrome P450 isozymes involved in the metabolism of N-nitrosodipropyl-, N-nitrosodibutyl- and N-nitroso-n-butyl-n-propylamine

The metabolism of N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine(NDBA) and N-nitroso-n-butyl-n-propylamine (NBPA) was investigatedin vitro using liver microsomes and purified isoforms of cytochromeP450 in a reconstituted system. Liver microsomes were preparedfrom rats pretreated with phenobarbital (PB), pyridine (PYR),ß-naphthoflavone (BNF), butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), clofibrate (CLO) or from untreatedrats. The purified cytochrome P450s used in the reconstitutedsystem were rat 1A1 and 2B1and rabbit 2E1. The rates of metabolismand the product profiles for NDPA, NDBA and NBPA changed significantlydepending on the pretreatment of the rats or the identity ofthe purified cytochrome P450 isoforms. Induction by PB dramaticallyincreased cleavage of NDPA, NDBA and NBPA at C-N bonds, leadingto substantial increases in formation of the respective aldehydesand the overall metabolic rates. Microsomes from PYR-pretreatedrats exhibited increased activities for formation of formaldehydeand propionaldehyde from NDPA and NBPA. Microsomes from BHT-pretreatedrats showed a slight increase in activity for N-dealkylationof NDBA and NBPA. Treatment with BHA decreased the overall metabolismof NDBA, but slightly increased N-dealkylation of NBPA. Microsomalmetabolism of NDPA, NDBA and NBPA was decreased by pretreatmentwith BNF and CLO. Results from studies using the reconstitutedsystem with purified cytochrome P450 isoforms demonstrated thatcytochrome P450 2B1 specifically catalyzed     

Characterization of a microsomal fraction from rat small intestine for metabolic activation of some promutagens

Rat small intestine possesses cytochrome P-450 mixed functionoxidase activity and is thus potentially capable of activatingprocarcinogens during absorption. The low spontaneous tumourincidence at this site may be due partially to the detoxificationactivity of the intestinal mucosa. To study the contributionof the intestine to the metabolism of foreign chemicals, microsomeshave been obtained from rat small intestine by a procedure facilitatingrecovery of preparations with consistently high enzyme activitiesand abilities to activate some selected promutagens in the Salmonellamutagenicity assay. Intestinal microsomes from animals withand without pretreatment with inducers have been used for investigationsof biochemical properties and ability to activate several mutagensin the Salmonella plate incorporation assay. The effects ofmicrosomal protein concentration and inhibitors were also studied.The results are compared with data obtained using liver microsomesfrom the same animals. Despite the induction of lower numbersof revertants, intestinal microsomes were at least as efficientas liver preparations for the activation of all the promutagensused when the data were corrected for cytochrome P-450 contents.Differences in dose-response curves for certain mutagens usingliver and intestinal microsomes are discussed in relation tovariation in metabolism of promutagens. Activation was linearlydependent on microsomal protein concentration, for both liverand small intestinal microsomes. The intestine was generallyless susceptible to the effects of cytochrome P-450 and P-448inducers, although sensitivity to orally administered phenobarbitonewas increased by extending treatment times. SKF525A and ß-naphthoflavoneinhibited microsomes from both sources, equal inhibition beingobserved for each type following incorporation of the inhibitor,although they differed in their ability to activate 2-acetylaminofluorenein the presence of the deacylase inhibitor, NaF. The data arediscussed in relation to the possible role of the small intestinein metabolic activation in vivo and the utility of microsomestherefrom for the in vitro detection of putative dietary carcinogens.     

Differential rates of metabolic activation and detoxication of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by different cytochrome P450 enzymes

Rat liver microsomes metabolized the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to the genotoxic metabolite 2-hydroxamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-hydroxamino-PhIP) and to the detoxified product 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP). A 25-fold higher rate of metabolism was measured in microsomes from polychlorinated-biphenyl-treated rats (94 nmol/mg proteins/30 min) in comparison with those from untreated rats. Other effective inducers of PhIP metabolism were beta-naphthoflavone and isosafrole (ISF), whereas phenobarbital was ineffective. About twice as much 2-hydroxamino-PhIP as 4'-hydroxy-PhIP was formed in microsomes irrespective of the inducer the rats had been treated with. The metabolism was dependent on NADPH and was abolished by the cytochrome P450 inhibitor alpha-naphthoflavone. In a reconstituted enzyme system purified rat cytochrome P450 IA2 (P450ISF-G) had the highest N-hydroxylation rate (30 nmol/nmol P450/30 min) closely followed by the rat cytochrome P450 IA1 (P450BNF-B). Less activity was seen with rat P450 IIC11 (P450UT-A) and rabbit P450 IA2 (P450 LM4). Rat P450 IIE1 (P450j), P450 IIB1 (P450PB-B) and rabbit P450 IIB4 (P450 LM-2) and P450 IIE1 (P450 LM3a) were essentially inactive. Rat P450 IA1 (P450BNF-B) produced five times more 4'-hydroxy-PhIP (32 +/- 2 nmol/nmol P450/30 min) than did P450 IA2 (P450ISF-G). Hence, the measured ratio of activation to detoxication for rat P450 IA2 (P450ISF-G) enzyme was 7-fold higher than that of the other active P450 enzymes.     

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.     

Characterization of oxidative and reductive metabolism in vitro of nitrofluoranthenes by rat liver enzymes

Nitrofluoranthenes (NFs) are mutagenic and carcinogenic environmental pollutants found in incomplete combustion products and urban air particulate. We have studied both oxidative and reductive metabolism in vitro of different NF isomers mediated by subcellular rat liver fractions. Under aerobic conditions only ring hydroxylation of NFs by rat liver microsomes occurred and the isomeric position of the nitro group affected both the amount and the type of phenolic metabolites formed. Liver microsomes from 3-methylcholanthrene-induced rats were most effective in giving ring hydroxylated 7- and 8-nitrofluoranthene, whereas liver microsomes from phenobarbital-pretreated rats were the most active in metabolizing 1- and 3-nitrofluoranthene. Under anaerobic conditions, only reduction of NFs mediated by both cytosolic and microsomal rat liver enzymes occurred. Cofactor requirements and inhibition experiments indicated that the reductase activity in rat liver cytosolic fractions could be ascribed to DT-diaphorase, aldehyde oxidase and/or other unknown enzymes. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride and n-octylamine, and slightly by cytochrome c; flavin mononucleotide greatly enhanced this activity. 3-Nitrofluoranthene microsomal nitroreductase activity was increased by phenobarbital rat pretreatment and this increment correlated well with the content of cytochrome P450. These results indicate a participation of cytochrome P450 in the reductive metabolism of NFs by rat liver microsomes.     

Cytochrome P450IA expression in adult and fetal human liver.

A monoclonal antibody has been produced that recognizes the cytochrome P450 form, cytochrome P450IA1, but not cytochrome P450IA2 in rats and recognizes a single protein band of similar mol. wt on immunoblots of human liver microsomes. Immunohistochemical studies have been carried out with this antibody to investigate the localization and distribution of cytochrome(s) P450 of the P450IA family in human liver. Cytochrome P450IA was identified in both adult and fetal liver and in each case it was localized predominantly to hepatocytes. In adult liver there was a heterogeneous distribution of cytochrome P450IA immunoreactivity with cytochrome P450IA mainly present in zone 3 hepatocytes of the liver acinus. Within fetal liver there was a uniform distribution of cytochrome P450IA immunoreactivity with no apparent zonal distribution. Bile duct epithelium did not show definite immunostaining for cytochrome P450IA in either adult or fetal liver.     

Induction of cytochrome P450IA1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone

It is known that consumption of cruciferous vegetables protects against the chemical induction of cancer in many organs. It has been suggested that this protection is mediated through an effect on the cytochrome P450 monooxygenase system. This system is responsible for the activation of a number of chemical carcinogens to their ultimate forms. In the present study, the effect of indole-3-carbinol (I3C) and 5,6-benzoflavone (5,6BF) on the expression of cytochrome P450IA1 in rat colon and liver has been investigated. Cytochrome P450IA1 mRNA was induced in colon following a single oral administration of I3C or 5,6BF. A biphasic induction profile was obtained with maxima at 4 and 16 h post-administration. Both inducers caused an approximately 2-fold increase in P450IA1 mRNA at 4 h and a 10-fold increase at 16 h. In contrast, both cytochrome P450IA1 and IA2 mRNAs was increased over the control between 4 and 24 h. The total amount of P450IA mRNAs in liver at 4 and 16 h was increased about 2- and 4-fold respectively by I3C; 5,6BF induced the P450IA mRNAs 4- and 5-fold respectively. The expression of cytochrome P450IA1 and IA2 is induced by I3C and several flavones present in cruciferous vegetables. This suggests that one of the protective effects of cruciferous vegetables in the reduction of chemically induced cancer may be regulation of cytochrome P450s involved in the metabolism of the chemical carcinogens.     

Pseudoenzymatic reduction of N-hydroxy-2-acetylaminofluorene to 2-acetylaminofluorene mediated by cytochrome P450

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) was reduced to 2-acetylaminofluorene by rat liver microsomes in the presence of both NAD(P)H and FAD under anaerobic conditions. The microsomal reduction proceeds as if it were an enzymatic reaction. However, when the microsomes were boiled, the activity was not abolished, but was enhanced. The activity was also observed with cytochrome P450 2B1 alone, without NADPH-cytochrome P450 reductase, in the presence of these cofactors. Hematin also exhibited a significant reducing activity in the presence of both a reduced pyridine nucleotide and FAD. The activities of microsomes, cytochrome P450 2B1 and hematin were also observed upon the addition of photochemically reduced FAD instead of both NAD(P)H and FAD. The microsomal reduction of N-OH-AAF appears to be a non-enzymatic reaction by the reduced flavin, catalyzed by the heme group of cytochrome P450.