Non-specific inhibition of cytochrome P450 activities by chlorophyllin in human and rat liver microsomes

Chlorophyllin, a copper/sodium salt of chlorophyll used in thetreatment of geriatric patients, inhibits the mutagenicity of2-amino-3-methylimidazo[4, 5-f)quinoline (IQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), aflatoxin B₁ and benzo[a]pyrene (B[a]P).Recent in vitro and in vivo studies have shown that a molecularcomplex is formed between IQ and chlorophyllin, suggesting thatthis complex formation might be responsible for the antigenotoxiceffect of chlorophyllin observed. Cytochrome P450 (P450) enzymesappear to be the major catalysts in the bioactivation of thesecarcinogens. We have investigated the in vitro effects of chlorophyllinon several P450 activities including ethoxyresorufin O-deethylation,benzyloxyreso-rufin O-debenzylation, coumarin 7-hydroxylation,7-ethoxycoumarin O-deethylation, B[a]P 3-hydroxylation, andchlorzoxazone 6-hydroxylation. Chlorophyllin non-specificallyinhibited all of P450 activities observed. Spectrally detectableP450 was also destroyed in microsomes and purified P450 in areconstituted system in the presence of chlorophyllin and anNADPH-generating system. These results suggest that the antigenotoxiceffect of chlorophyllin might be due to inhibition of P450 enzymesinvolving bioactivation of carcinogens in addition to molecularcomplex formation between carcinogens and chlorophyllin. Comparisonof the apparent K₁ for P450 inactivation with previously estimatedconstants for chlorophyllin-IQ complexation suggest that P450inhibition should be the dominant mechanism of inhibition.     

Procarcinogen activation by cytochrome P450 3A4 and 3A5 expressed in Escherichia coli and by human liver microsomes

Recent studies indicate that cytochrome P450 (P450) 3A4 playsimportant roles in the activation of procarcinogens such asaflatoxin B₁ and sterigmatocystin, as well as in the oxidationof a number of structurally diverse chemicals and endogenouscompounds. Since P450 3A5 has been reported to be present atsignificant levels in liver microsomes in     

Roles of cytochrome P450IIE1 in the dealkylation and denitrosation of N-nitrosodimethylamine and N-nitrosodiethylamine in rat liver microsomes

N-Nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are widely occurring nitrosamines and require enzyme-catalyzed activation for their carcinogenic actions. The low Km forms of the enzyme are generally considered to be important in the activation of environmental carcinogens. In this work we examined the role of cytochrome P450IIE1--a constitutive enzyme that is also inducible by acetone, ethanol, fasting and other factors--in catalyzing the dealkylation and denitrosation of these two carcinogens. The experimentally determined Km value of NDMA demethylase depended upon the experimental conditions and was lower when lower protein concentrations were used. Low Km values of 15-20 microM were observed for NDMA demethylase with different preparations of microsomes. In the deethylation of NDEA, a low Km of approximately 40 microM was observed for both control and acetone-induced microsomes. Immunoinhibition studies indicated that P450IIE1 was responsible for almost all the low Km NDMA demethylase activity in acetone-induced microsomes and greater than 80% in control microsomes. This enzyme was also responsible for about three-quarters of the low Km NDEA deethylase activity in acetone-induced microsomes and about half in control microsomes. The denitrosation of NDMA and NDEA was inhibited to approximately the same extents as the dealkylation reactions under different experimental conditions, suggesting the involvement of the same enzyme and perhaps a common initial intermediate in these two types of reactions. The relevance of this work and its relationship to related information in the literature are discussed.     

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 mediated bioactivation of methyleugenol to 1'- hydroxymethyleugenol in Fischer 344 rat and human liver microsomes

Cytochrome P450 mediated metabolism of methyleugenol to the proximate carcinogen 1'-hydroxymethyleugenol has been investigated in vitro. Kinetic studies undertaken in liver microsomes from control male Fischer 344 rats revealed that this reaction is catalyzed by high affinity (Km of 74.9 +/- 9.0 microM, Vmax of 1.42 +/- 0.17 nmol/min/nmol P450) and low affinity (apparent Km several mM) enzymic components. Studies undertaken at low substrate concentration (20 microM) with microsomes from livers of rats treated with the enzyme inducers phenobarbital, dexamethasone, isosafrole and isoniazid indicated that a number of cytochrome P450 isozymes can catalyze the high affinity component. In control rat liver microsomes, 1'- hydroxylation of methyleugenol (assayed at 20 microM substrate) was inhibited significantly (P < 0.05) by diallylsulfide (40%), p- nitrophenol (55%), tolbutamide (30%) and alpha-naphthoflavone (25%) but not by troleandomycin, furafylline, quinine or cimetidine. These results suggested that the reaction is catalyzed by CYP 2E1 and by another as yet unidentified isozyme(s) (most probably CYP 2C6), but not by CYP 3A, CYP 1A2, CYP 2D1 or CYP 2C11. Administration of methyleugenol (0-300 mg/kg/day for 5 days) to rats in vivo caused dose- dependent auto-induction of 1'-hydroxylation of methyleugenol in vitro which could be attributed to induction of various cytochrome P450 isozymes, including CYP 2B and CYP 1A2. Consequently, high dose rodent carcinogenicity studies are likely to over-estimate the risk to human health posed by methyleugenol. The rate of 1'-hydroxylation of methyleugenol in vitro in 13 human liver samples varied markedly (by 37- fold), with the highest activities being similar to the activity evident in control rat liver microsomes. This suggests that the risk posed by dietary ingestion of methyleugenol could vary markedly in the human population.      

Comparative metabolism of N-nitrosopiperidine and N-nitrosopyrrolidine by rat liver and esophageal microsomes and cytochrome P450 2A3

N-nitrosopiperidine (NPIP) is a potent esophageal carcinogen in rats whereas structurally similar N-nitrosopyrrolidine (NPYR) induces liver, but not esophageal tumors. NPIP is a possible causative agent for human esophageal cancer. Our goal is to explain mechanistically these differing carcinogenic activities in the esophagus. We hypothesize that differences in metabolic activation of these nitrosamines could be one factor accounting for their differing carcinogenicity. alpha-Hydroxylation is the key metabolic activation pathway leading to nitrosamine-induced carcinogenesis. In this study, we examined the alpha-hydroxylation rates of [3,4-(3)H]NPIP and [3,4-(3)H]NPYR by male F344 rat esophageal and liver microsomes. The major alpha-hydroxylation products of NPIP and NPYR, 2-hydroxytetrahydro-2H-pyran (2-OH-THP) and 2-hydroxytetrahydrofuran (2-OH-THF), respectively, were monitored by high performance liquid chromatography with radioflow detection. NPIP or NPYR (4 microM) was incubated with varying concentrations of esophageal microsomes and co-factors. Microsomes converted NPIP to 2-OH-THP with a 40-fold higher velocity than NPYR to 2-OH-THF. Similar results were observed in studies with NPIP and NPYR at substrate concentrations between 4 and 100 micro M. Kinetics of NPIP alpha-hydroxylation were biphasic; K(M) values were 312 +/- 50 and 1600 +/- 312 microM. Expressed cytochrome P450 2A3, found in low levels in rat esophagus, was a good catalyst of NPIP alpha-hydroxylation (K(M) = 61.6 +/- 20.5 microM), but a poor catalyst of NPYR alpha-hydroxylation (K(m) = 1198 +/- 308 micro M). Cytochrome P450 2A3 may play a role in the preferential activation of NPIP observed in rat esophagus. Liver microsomes metabolized NPYR to 2-OH-THF (V(max)/K(M) = 3.23 pmol/min/mg/ microM) as efficiently as NPIP to 2-OH-THP (V(max)/K(M) = 3.80-4.61 pmol/min/mg/ microM). We conclude that rat esophageal microsomes activate NPIP but not NPYR whereas rat liver microsomes activate NPIP and NPYR. These results are consistent with previous findings that tissue-specific activation of nitrosamines contributes to tissue-specific tumor formation.     

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.     

Benzene metabolism by human liver microsomes in relation to cytochrome P450 2E1 activity

Low levels of benzene from sources including cigarette smokeand automobile emissions are ubiquitous in the environment.Since the toxicity of benzene probably results from oxidativemetabolites, an understanding of the profile of biotransformationof low levels of benzene is critical in making a valid riskassessment. To that end, we have investigated metabolism ofa low concentration of [¹⁴C]benzene (3.4 µM) by microsomesfrom human, mouse and rat liver. The extent of phase I benzenemetabolism by microsomal preparations from 10 human liver samplesand single microsomal preparations from both mice and rats wasthen related to measured activities of cytochrome P450 (CYP)2E1. Measured CYP 2E1 activities, as determined by hydroxylationof p-nitrophenol, varied 13-fold (0.253–3.266 nmol/min/mg)for human samples. The fraction of benzene metabolized in 16min ranged from 10% to 59%. Also at 16 min, significant amountsof oxidative metabolites were formed. Phenol was the main metaboliteformed by all but two human microsomal preparations. In thosesamples, both of which had high CYP 2E1 activity, hydro-quinonewas the major metabolite formed. Both hydroquinone and catecholformation showed a direct correlation with CYP 2E1 activityover the range of activities present. A simulation model wasdeveloped based on a mechanism of competitive inhibition betweenbenzene and its oxidized metabolites, and was fit to time-coursedata for three human liver preparations. Model calculationsfor initial rates of benzene metabolism ranging from 0.344 to4.442 nmol/mg/min are directly proportional to measured CYP2E1 activities. The model predicted the dependence of benzenemetabolism on the measured CYP 2E1 activity in human liver samples,as well as in mouse and rat liver samples. These results suggestthat differences in measured hepatic CYP 2E1 activity may bea major factor contributing to both interindividual and interspeciesvariations in hepatic metabolism of benzene. Validation of thissystem in vivo should lead to more accurate assessment of therisk of benzene's toxicity following low-level exposure.     

Anticancer drugs as inhibitors of two polymorphic cytochrome P450 enzymes, debrisoquin and mephenytoin hydroxylase, in human liver microsomes

To identify potential substrates for the debrisoquin and mephenytoin hydroxylation polymorphisms, we performed in vitro inhibition studies with human liver microsomes and the respective prototype substrates in the absence and presence of several anticancer drugs. (+)-Bufuralol 1'-hydroxylation (as the prototype reaction for the debrisoquin polymorphism) was tested at 5 microM substrate concentration and in the presence of cyclophosphamide (0 to 200 microM), teniposide (0 to 100 microM), vinblastine (0 to 220 microM), etoposide (0 to 200 microM), flavone acetic acid (0 to 1000 microM), or ifosphamide (0 to 200 microM). (S)-Mephenytoin 4-hydroxylation was tested at 60 microM substrate concentration and in the presence of the same drugs as above; vincristine was also tested at 0 to 200 microM. Teniposide competitively inhibited the 4-hydroxylation of (S)-mephenytoin, with a Ki of 12 microM (Km of the reaction = 65 microM). Etoposide and flavone acetic acid were weaker inhibitors of this reaction. The only agent to inhibit bufuralol hydroxylation was vinblastine, which did so with a Ki of 90 microM (Km of the enzyme for the substrate = 12 microM). We conclude that teniposide and high concentrations of flavone acetic acid could spuriously alter mephenytoin phenotype determination in cancer patients, and that teniposide deserves further investigation as a possible substrate for the genetically regulated mephenytoin hydroxylase.     

Evidence for cytochrome P450 2A6 and 3A4 as major catalysts for N'- nitrosonornicotine alpha-hydroxylation by human liver microsomes

The tobacco specific carcinogen N'-nitrosonornicotine (NNN), is believed to be a causative agent for esophageal cancer in smokers. NNN requires metabolic activation to exert its carcinogenic potential. Metabolism occurs through cytochrome P450 (P450) catalyzed 2'- and 5'- hydroxylation, which generates unstable metabolites that decompose to 4- hydroxy-1-(3-pyridyl)-1-butanone ('keto alcohol') and 4-hydroxy-4-(3- pyridyl)butanal, respectively. The latter cyclyzes to 5-(3-pyridyl)-2- hydroxytetrahydrofuran ('lactol'). 2'-Hydroxylation of NNN is believed to be the pathway critical for esophogeal NNN carcinogenesis in the rat. The ability of human liver microsomes and expressed human P450s to metabolize [5-(3)H]NNN to keto alcohol and lactol was determined by reverse phase HPLC with radioflow detection. At low NNN concentrations, 11 human liver microsomes metabolized NNN primarily by 5'-hydroxylation to lactol. This reaction was strongly correlated (r = 0.92) with coumarin 7-hydroxylation, suggesting that NNN 5'-hydroxylation is catalyzed mainly by P450 2A6. 2'-Hydroxylation of NNN by human liver microsomes correlated with 6beta-hydroxylation of testosterone, a P450 3A4-specific activity (r = 0.94). The relative rates of 2'- and 5'- hydroxylation by human P450s 2A6, 2E1, 2D6 and 3A4 expressed in Sf9 cells by the baculovirus-insect cell expression system, and human P450 3A4 produced by stable expression in Chinese hamster ovary cells, were determined. Human P450 2A6 metabolized 1 microM NNN exclusively by 5'- hydroxylation. The rate of lactol formation was 317 pmol/min per nmol P450. Human P450s 2E1 and 2D6 also metabolized NNN only to lactol, but at much lower rates, 0.4 and 0.8 pmol/min per nmol of P450 respectively. In contrast, the metabolism of NNN by expressed human P450 3A4 was specific for keto alcohol formation. The Km for 5'- hydroxylation by baculovirus-expressed P450 2A6 was 2.1 microM, and k(cat) was 953 pmol/min per nmol of P450. The Km for lactol formation by human liver microsomes containing high levels of P450 2A6, was 5 microM . Human liver microsomes exhibited a Km of 312 microM for keto alcohol formation. Coumarin, 8-methoxypsoralen (P450 2A6 inhibitors), and anti-2A6 monoclonal antibody were strong inhibitors of NNN-derived lactol formation in human liver microsomes. Troleandomycin, an inhibitor of P450 3A4, effectively inhibited the metabolism of NNN to keto alcohol by human liver microsomes. These results are consistent with P450 2A6 mediated 5'-hydroxylation and P450 3A4 mediated 2'- hydroxylation of NNN in human liver microsomes.      

Androgen-dependent renal microsomal cytochrome P-450 responsible for N-hydroxylation and mutagenic activation of 3-methoxy-4-aminoazobenzene in the BALB/c mouse

A murine renal microsomal enzyme responsible for the mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) was characterized by its catalytic activity for the mutagenic and metabolic conversion of 3-MeO-AAB. Incubation of 3-MeO-AAB with a renal or hepatic microsome fraction from male BALB/c mice in the presence of NADPH and NADH yielded N-hydroxy and 4'-hydroxy metabolites of 3-MeO-AAB as determined by two-dimensional thin layer chromatography, and the enzyme responsible for the N-hydroxylation was named 3-MeO-AAB N-hydroxylase. A mutagenicity test using Salmonella typhimurium TA98 bacteria as a tester strain has revealed that N-hydroxy-3-MeO-AAB is a potent direct mutagen but that 4'-hydroxy-3-MeO-AAB is not mutagenic. Although 3-MeO-AAB N-hydroxylase activity in liver microsomes showed no sex difference, the enzyme activity in the kidney was detected from male mice but not from females. However, administration of testosterone to female mice induced the enzyme in the kidney. Castration of male mice depressed the activity of 3-MeO-AAB N-hydroxylase in renal microsomes but it little affected the hepatic activity, and on administration of testosterone to the castrated mice the depressed renal microsomal activity recovered to a normal level. The activity of 3-MeO-AAB hydroxylase and the amount of cytochrome P-450 in renal microsomes showed a close correlation. Both renal and hepatic microsomes required NADPH as a main cofactor to mutagenize 3-MeO-AAB and to yield N-hydroxy-3-MeO-AAB from 3-MeO-AAB, and the enzyme activity was strongly inhibited by 7,8-benzoflavone. When the activities of renal and hepatic 3-MeO-AAB N-hydroxylase were compared on the basis of the amount of cytochrome P-450, the renal type enzyme showed about 8 times greater activity than hepatic type enzyme. These results indicate that the kidney contains an androgen-dependent microsomal 3-MeO-AAB hydroxylase which is different from an isozyme present in the liver and which is a new type of cytochrome P-450 isozyme.     

Metabolism of the potent carcinogen 3-methylcholanthrylene by rat liver microsomes

The products formed in the metabolism of 3-methylcholanthrylene (3MCE), either in the presence or in the absence of an epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide (TCPO), with an NADPH-regenerating system and liver microsomes from 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats were separated by reversed-phase and normal-phase HPLC. The metabolites were characterized by UV-visible absorption spectral analysis, and by comparing their retention times on reversed-phase and normal-phase HPLC with authentic 3MC derivatives whenever available. In addition to 3MC trans-1,2-diol, 3MC-1-one, and 3MC-2-one reported earlier by other investigators, 3-hydroxymethylcholanthrylene (3-OHMCE), 3-OHMCE trans-11,12-dihydrodiol, 3MCE trans-11,12-dihydrodiol, 3MCE trans-9, 10-dihydrodiol. 9- and 10-hydroxy-3MCE. 3MC-2-one trans-9,10-dihydrodiol, and a chemically unstable 3MCE 1,2-epoxide were identified as metabolites of 3MCE. 3MC cis-1,2-diol, a previously reported metabolite of 3MCE, was not detectable. In the presence of TCPO, metabolites that have been identified include 3-OHMCE, 3-OHMCE 11,12-epoxide. 3MCE 11,12-epoxide, 3MC-2-one, 3MC-1-one, 9-hydroxy-3MCE, 10-hydroxy-3MCE, and an unstable metabolic intermediate 3MCE 1,2-epoxide. The results suggest that 3MCE 1,2-epoxide, 3MCE 9,10-diol-7,8-epoxide, and 3MC-2-one 9,10-diol-7,8-epoxide may be involved in the metabolic activation of 3MCE to carcinogenic form.     

Metabolic deactivation of furylfuramide by cytothrome P450 in human and liver microsomes

Metabolic deactivation of furylfuramide by human and rat livermicrosomal cytochrome P450 enzymes has been investigated ina system measuring induction of umu gene expression responsein Salmonella typhimurium TA1535/pSK1002. Both human and ratliver microsomes catalyzed the metabolism of furylfuramide toinactive form(s) that are incapable of inducing umu gene expressionin the tester strain. The reaction required an NADPH-generatingsystem and molecular oxygen and was inhibited by carbon monoxide,suggesting that a cytochrome P450-linked monooxygenase systemis prerequisite for the deactivation reaction. With liver microsomesfrom variously pretreated rats, 3-methylcholanthrene was foundto be a powerful inducer for the furylfuramide-metabolizingactivity, and antibodies raised against rat P450IA1(BNF-B, c)and P450IA2(ISF-G, d) inhibited the microsomal activity. Humanliver microsomal furylfuramide-metabolizing activity was alsoinhibited significantly by anti-P450IA2 lgG but weakly by anti-P450IA2IgG. In liver microsomes prepared from seven different humansamples, the activities of deactivation of furylfuramide werefound to correlate with the amounts of immunoreactive proteinrelated to rat P450IA2 and with the monooxygenase activitiesof metabolic activation of 2-amino-3,4-dimethyl-imidazo [4,5-]quinoline(MeIQ) and of ethoxyresorufin O-deethylation. These resultssuggest that P450IA1 and P450IA2 in rats, and P45OPA (IA2, thephenacetin O-deethylase and ortholog of rat P450IA2) in humansare the major enzymes involved in the deactivation of furylfuramidein liver microsomes. The metabolic studies involving HPLC analysisof products followed by spectrophotometric examination havealso suggested that furylfuramide can be degraded very rapidlythrough the aerobic metabolism by liver microsomes.     

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.     

Predominant 4-hydroxylation of estradiol by constitutive cytochrome P450s in the female ACI rat liver

The ACI rat is extremely sensitive to estrogens as mammary carcinogens, whereas the Sprague-Dawley strain is relatively resistant. Comparison of the disposition and effects of estrogens in these two strains should provide insights into the mechanisms of estrogen carcinogenicity. We have begun this investigation by comparing the metabolism of [(3)H]17beta-estradiol (E2) by liver microsomes prepared from female rats from each strain. Both strains produce estrone (E1) as the major product at E2 concentrations >1 microM, with smaller amounts of 2-hydroxy-E2 formed. As the E2 concentration is decreased, however, aromatic hydroxylation becomes a more dominant pathway for both strains. At starting E2 concentrations as low as 3 nM, Sprague-Dawley liver microsomes produced comparable yields of 2-hydroxy-E2 and E1. In contrast, ACI liver microsomes yielded a profound shift to aromatic hydroxylation as the dominant pathway as E2 concentrations dropped below 1 microM, and this shift reflected the production of 4-hydroxy-E2 as the predominant product. The apparent K(m) for 4-hydroxylation of E2 is <0.8 microM, as opposed to approximately 4 microM for 2-hydroxylation, suggesting that different cytochrome P450s (CYPs) are responsible. Western immunoblotting of the liver microsomal preparations from ACI and Sprague-Dawley rats for CYPs known to catalyze 2- and 4-hydroxylation of E2 revealed that both strains contained comparable amounts of CYP 2B1/2 and 3A1/2, but no detectable amounts of CYP 1B1, the proposed E2 4-hydroxylase. Although this enzyme is not a constitutive CYP in Sprague-Dawley rat liver, its presence in ACI liver could provide a ready explanation for the predominance of 4-hydroxy-E2 as a product. The identity of the estradiol 4-hydroxylase in ACI rat liver and the role of this unique reaction in the heightened sensitivity to E2 carcinogenicity remain to be elucidated.     

Cytochrome P450 metabolic dealkylation of nine N-nitrosodialkylamines by human liver microsomes

The metabolic dealkylation of nine nitrosodialkylamines, includingfive symmetrical (nitrosodimethylamine, nitroso-diethylamine,nitrosodipropylamine, nitrosodibutylamine and nitrosodiamylamine)and four asymmetrical nitro-sodialkylamines (nitrosomethylethylamine,nitrosomethyl-propylamine, nitrosomethylbutylamine and nitrosomethyl-amylamine),was investigated in 14 samples of human liver microsomes. Allthese nitrosodialkylamines were dealkylated to aldehydes thatwere separated by reversed phase HPLC and UV detected as dinitrophenylhydrazones.As the length of the alkyl chain increased from methyl to pentyl,dealkylation of symmetrical nitrosodialkylamines became lessefficiently catalyzed by cytochrome P450. Conversely, oxidationof the methyl moiety of asymmetrical nitrosomethylalkylaminesincreased with the size of the alkyl moiety, while dealkylationof the longer alkyl group decreased. N-Dealkylase activitieswere significantly correlated with P450 activities measuredin human liver micro-somes. These catalytic activities involveCYP2A6 (coumarin 7-hydroxylation), CYP2C (mephenytoin 4-hydroxylationand tolbutamide hydroxylation), CYP2D6 (dextromethor-phan O-demethylation),CYP2E1 (chlorzoxazone and p-nitrophenol hydroxylation) and CYP3A4(nifedipine oxidation). By using 10 heterologously expressedP450s, it was shown that nitrosodimethylamine was mainly demethylatedby CYP2E1. However, such enzyme specificity was lost with increasingsize of the alkyl group. Therefore, the chain length of thealkyl group of nitrosodialkylamines determined the P450 involvedin its oxidation. All these results emphasize that the catalyticsite of P450 2E1 has a geometric configuration such that onlysmall molecules like nitrosodimethylamine fit favorably withinthe putative active site of the enzyme. Furthermore, there isgood evidence that P450s other than P450 2E1, such as P450 2A6,2C8/2C9/2C19 and 3A4, are involved in the metabolism of nitrosodialkylaminesbearing bulky alkyl chains.     

Participation of cytochrome P-450 in reductive metabolism of 1-nitropyrene by rat liver microsomes

Reductive metabolism of carcinogenic 1-nitropyrene by rat liver microsomes and reconstituted cytochrome P-450 systems was investigated. Under the nitrogen atmosphere, 1-aminopyrene was the only detected metabolite of 1-nitropyrene. The reductase activity in liver 105,000 X g supernatant fraction was ascribed to DT-diaphorase, aldehyde oxidase, and other unknown enzyme(s) from the results of cofactor requirements and inhibition experiments. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2,4-dichloro-6-phenylphenoxyethylamine, and n-octylamine. Flavin mononucleotide markedly enhanced the activity, and 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride also enhanced it, but slightly. The microsomal activity was induced by the pretreatment of rats with 3-methylcholanthrene, sodium phenobarbital, or polychlorinated biphenyl, and the increments of the activity correlated well with those of the specific contents of cytochrome P-450 in microsomes. The reductase activity could be reconstituted by NADPH-cytochrome P-450 reductase and forms of cytochrome P-450 purified from liver microsomes of polychlorinated biphenyl-induced rats. Among four forms of cytochrome P-450 examined, an isozyme P-448-IId which showed high activity in hydroxylation of benzo(a)pyrene catalyzed most efficiently the reduction of 1-nitropyrene. The results of this study indicate the central role of cytochrome P-450 in the reductive metabolism of 1-nitropyrene in liver microsomes.     

Differential metabolism of gefitinib and erlotinib by human cytochrome P450 enzymes.

PURPOSE: To examine the enzyme kinetics of gefitinib and erlotinib metabolism by individual cytochrome P450 (CYP) enzymes, and to compare their effects on CYP3A activity, with the aim to better understand mechanisms underlying pharmacokinetic variability and clinical effects. EXPERIMENTAL DESIGN: Enzyme kinetics were examined by incubating gefitinib or erlotinib (1.5-50 micromol/L) with recombinant human CYP3A4, CYP3A5, CYP2D6, CYP1A1, CYP1A2, and CYP1B1 (10-160 pmol/mL). Their effects on CYP3A activity were examined by comparing midazolam metabolism in the presence and absence of gefitinib or erlotinib in human liver and intestinal microsomes. Parent compounds and metabolites were monitored by high-performance liquid chromatography with a photodiode detector or tandem mass spectrometer. RESULTS: Both drugs were metabolized primarily by CYP3A4, CYP3A5, and CYP1A1, with respective maximum clearance (Cl(max)) values for metabolism of 0.41, 0.39, and 0.57 mL/min/nmol for gefitinib and 0.24, 0.21, 0.31 mL/min/nmol for erlotinib. CYP2D6 was involved in gefitinib metabolism (Cl(max), 0.63 mL/min/nmol) to a large extent, whereas CYP1A2 was considerably involved in erlotinib metabolism (Cl(max), 0.15 mL/min/nmol). Both drugs stimulated CYP3A-mediated midazolam disappearance and 1-hydroxymidazolam formation in liver and intestinal microsomes. CONCLUSIONS: Gefitinib is more susceptible to CYP3A-mediated metabolism than erlotinib, which may contribute to the higher apparent oral clearance observed for gefitinib. Metabolism by hepatic and extrahepatic CYP1A may represent a determinant of pharmacokinetic variability and response for both drugs. The differential metabolizing enzyme profiles suggest that there may be differences in drug-drug interaction potential and that stimulation of CYP3A4 may likely play a role in drug interactions for erlotinib and gefitinib.     

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.     

Carcinogenicity of 3''-hydroxymethyl-N,N-dimethyl-4-aminoazobenzene in rat liver

Male Sprague-Dawley rats were fed a cube diet containing 2.51mmol/kg 3' -hydroxymethyl-N, N-dimethyl-4-aminoazobenzene (3'-CH₂OH-DAB) for a period of 1 or 3 months. (This is the molarequivalent of 0.06% 3' -Me-DAB in the diet). The oral administrationof 3' -CH₂OH-DAB for 3 months resulted in a high incidence ofliver tumors at 6 months and the 1 month feeding also causedthe development of liver tumors. Histologically, the tumorswere cholangiocellular carcinomas and hepatocellular carcinomas.The development of tumors in other sites was not seen. Consequently,3' -CH₂OH-DAB, a recently identified metabolite of 3' -Me-DAB,is a potent hepatocarcinogen.