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.     

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline induces and inhibits cytochrome P450 from the IA subfamily in chick and rat hepatocytes.

Several heterocyclic amines, found in cooked food, are powerful mutagens in the Ames Salmonella mutagenicity test system. One of these, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is one of the most mutagenic chemicals tested in this assay. In primary cultures of chick and rat hepatocytes, MeIQ, by itself, induced cytochrome P450 from the IA subfamily but was a weak inducer compared to 3-methylcholanthrene. However, in both chick and rat hepatocytes in culture, MeIQ decreased the amount of 3-methylcholanthrene-induced ethoxyresorufin deethylase activity, which is catalyzed by cytochrome P450 IA. The protein moiety of cytochrome P450 IA was decreased at MeIQ concentrations of 2.5 micrograms/ml or greater in chick hepatocytes and 25 micrograms/ml in rat hepatocytes. In hepatic microsomes from methylcholanthrene-treated chicks and rats, MeIQ was a competitive inhibitor of both ethoxyresorufin deethylase activity, a reaction catalyzed mainly by rodent cytochrome P450 IA1, and uroporphyrinogen oxidation, a reaction catalyzed by rodent P450 IA2. In cultured chick hepatocytes, MeIQ also decreased cytochrome P450-mediated oxidation of uroporphyrinogen by intact cells. The ability of MeIQ to inhibit as well as to induce cytochrome P450s of the IA subfamily may be important in assessing the mutagenic and carcinogenic effects of MeIQ in mammals.     

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.     

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.     

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.     

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.      

N,N',N'-triethylenethiophosphoramide (thio-TEPA) oxygenation by constitutive hepatic P450 enzymes and modulation of drug metabolism and clearance in vivo by P450-inducing agents.

The cancer chemotherapeutic drug N,N',N'-triethylenephosphoramide (thio-TEPA) is oxidatively desulfurated to yield the active metabolite N,N',N'-triethylenephosphoramide (TEPA) in a reaction catalyzed by the phenobarbital-inducible rat liver P450 enzyme IIB1. In the current study, the role of constitutively expressed P450 enzymes in thio-TEPA metabolism was studied using purified P450s, isolated liver microsomes, and intact rats. Metabolism of thio-TEPA (100 microM) to TEPA by uninduced adult female and male rat liver microsomes proceeded at initial rates of 0.10 and 0.28 nmol TEPA formed/min/mg microsomal protein, respectively. Although these rates are low compared to those catalyzed by phenobarbital-induced liver microsomes (3.5 nmol TEPA/min/mg), they are sufficient to contribute to the systemic metabolism of this drug. Thio-TEPA metabolism catalyzed by uninduced female liver microsomes was approximately 70% inhibitable by antibodies selectively reactive with P450 IIC6. For the uninduced male liver microsomes, which exhibit a severalfold higher rate of thio-TEPA metabolism, enzyme activity was only 15-20% inhibitable by these antibodies but was 80-85% inhibited by an anti-P450 IIC6 monoclonal antibody cross-reactive with P450 IIC11, which is expressed only in the males. Consistent with these observations, purified P450s IIC11 and IIC6 both oxidized thio-TEPA in reconstituted systems (turnover, 1.1 and 0.3 min-1 P450-1, respectively, at 100 microM substrate), while several other constitutive hepatic P450s exhibited significantly lower or undetectable activities (turnover, less than or equal to 0.15 min-1 P450-1). Metabolism of thio-TEPA by purified P450 IIC11 was associated with a time-dependent inactivation of the cytochrome analogous to that previously shown to accompany thio-TEPA metabolism catalyzed by P450 IIB1. Depletion of hepatic P450 IIC11 by cisplatin treatment of adult male rats led to a 70% reduction of TEPA formation catalyzed by the isolated liver microsomes, suggesting that cisplatin may influence thio-TEPA pharmacokinetics when these two drugs are given in combination. The extent to which hepatic P450s contribute to thio-TEPA metabolism and clearance in vivo was assessed by monitoring thio-TEPA and TEPA pharmacokinetics in rats that exhibit widely differing rates of microsomal thio-TEPA metabolism, i.e., uninduced female and male rats, and male rats treated with the P450 IIB1 inducers clofibrate and phenobarbital. In accord with the microsomal activities, conversion of thio-TEPA to TEPA was less extensive and thio-TEPA elimination slower in female than in male rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.     

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.     

Role of cytochrome P450IIIA4 in the metabolism of the pyrrolizidine alkaloid senecionine in human liver

Studies were carried out to investigate the metabolism of senecionine by human liver microsomes and the role of human cytochrome P450IIIA4 in this process. Human liver microsomes metabolized senecionine to two major products, (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) and senecionine N-oxide. The rates of product formation (DHP and senecionine N-oxide) varied widely with the microsomal samples tested. There was a 30-fold difference in DHP formation and a 25-fold difference in N-oxidation between the poorest metabolizer and the highest metabolizer of senecionine. The conversion of senecionine to DHP and senecionine N-oxide in human liver microsomes was markedly inhibited by the mechanism-based inactivators of P450IIIA4, gestodene and triacetyloleandomycin. Anti-P450IIIA4 IgG, at a concentration of 1 mg/nmol of P450, was found to inhibit completely the formation of DHP and senecionine N-oxide in human liver microsomes (HL101) having low activity toward senecionine. At 5 mg IgG/nmol P450, anti-P450IIIA4 inhibited 90 and 84% respectively of the formation of DHP and senecionine N-oxide in liver microsomes (HL110) with the highest activity toward senecionine. The formation of DHP or senecionine N-oxide was highly correlated with the amount of P450IIIA4 measured in the microsomes using polyclonal anti-P450IIIA4 IgG. The rate of DHP production also had a strong correlation with the rate of senecionine N-oxide formation (r = 0.999) and with the rate of nifedipine oxidation (r = 0.998). Our present studies provide evidence that P450IIIA4 is the major enzyme catalyzing the bioactivation (DHP formation) and detoxication (senecionine N-oxide formation) of senecionine in human liver.     

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.     

A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiple human cytochrome P450s including the polymorphic human cytochrome P4502D6

We have developed a human B-lymphoblastoid cell line, designated 2D6/Hol, which stably expresses human cytochrome P450 CYP2D6 cDNA. This cell line exhibits bufuralol 1'-hydroxylase activity and immunologically detectable CYP2D6 protein. The specific activity of (+)-bufuralol 1'-hydroxylase in microsomes from 2D6/Hol cells was comparable to that observed in human liver microsomes. This cell line was used to examine the mutagenicity activation of three tobacco smoke-derived nitrosamines, N-nitrosonornicotine (NNN), 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal) (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2D6. Exposure of 2D6/Hol cells to NNK concentrations of 30-90 micrograms/ml induced a concentration-dependent decrease in relative survival and increase in mutant fraction at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus. In contrast, NNK was non-mutagenic and non-cytotoxic to control cells at exposure concentrations up to 150 micrograms/ml. NNK mutagenicity in 2D6/Hol cells was compared to the responses observed in isogenic cell lines expressing human CYP1A2 (1A2/Hol), human CYP2A3 (2A3/Hol) and human CYP2E1 (2E1/Hol). These three additional human cytochrome P450-expressing cell lines were also found to be sensitive to NNK-induced mutagenicity and cytotoxicity. We found no evidence for CYP2D6-mediated activation of NNN or NNA. NNN was non-cytotoxic and non-mutagenic to both control and 2D6/Hol cells. NNA was equally cytotoxic and mutagenic to control cells and 2D6/Hol cells. The activation of NNA to a mutagen may have been carried out by P450 native to the AHH-1 TK +/- cell line. The 2D6/Hol cell line, in conjunction with the control cell line and other isogenic cell lines expressing other human cytochrome P450 cDNAs provides a useful system for the examination of the role of the polymorphic CYP2D6 in human procarcinogen activation and drug metabolism.     

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.      

Metabolism of N-nitrosodialkylamines by human liver microsomes

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

Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of cytochrome P450 1B1 in lung, liver and kidney of rats exposed to diesel exhaust.

We have shown previously that diesel exhaust particle (DEP) extracts (DEPE) and 1-nitropyrene were genotoxically activated by human cytochrome P450 1B1 in SOS/umu assay. In this study, the in vivo induction of P450 family 1 enzymes in rats by exposure to diesel exhaust was investigated with regard to mRNA levels, P450 enzyme content, drug oxidation activities in the microsomes and umu gene expression of typical P450 substrates and DEPE itself catalyzed by the microsomes. Male Fischer 344 rats (4 weeks old) were exposed to 0.3 and 3.0 mg/m(3) DEP for 12 h per day for 4 weeks; the former dose corresponded to the typical daily airborne particle concentration. The levels of mRNA of rat P450 1B1 and P450 1A1 in the lung and liver were significantly increased 1.1-1.4-fold by exposure to 0.3 mg/m(3) DEP. Diesel exhaust particle extracts induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of a functional P450 system and were further activated by human recombinant P450 1B1. Using an O-acetyltransferase overexpressing Salmonella strain, genotoxic activation of P450 1B1 marker chemicals (1-nitropyrene, 1-aminopyrene and DEPE) by lung, liver and kidney microsomes was increased 1.7-4.2-, 1.4-1.5- and 1.0-1.3-fold, respectively, by exposure to 0.3 mg/m(3) DEP. Activation of 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1; marker for P450 1A1) by lung microsomes and the P450 1A2 content in liver microsomes were slightly increased by exposure to 3.0 mg/m(3) DEP. This is the first report to suggest that typical daily contaminant levels (0.3 mg particle/m(3)) of diesel exhaust can induce P450 1B1 in rats and that the induced P450 1B1 may catalyze the genotoxic activation of DEP.     

Metabolism of azoxymethane, methylazoxymethanol and N-nitrosodimethylamine by cytochrome P450IIE1

The metabolism of azoxymethane (AOM), methylazoxymethanol (MAM) and N-nitrosodimethylamine (NDMA) by liver microsomes from acetone-induced rats as well as by a reconstituted system containing purified cytochrome P450IIE1 was examined. The products consisted of MAM from AOM; methanol and formic acid from MAM; and methylamine, formaldehyde, methanol, methylphosphate and formic acid from NDMA. Compared to liver microsomes from untreated rats, the metabolic activity of acetone-induced microsomes was approximately 4 times higher for all three carcinogens. Using the reconstituted system, the enzyme activities (nmol substrate metabolized/nmol P450/min) for AOM, MAM and NDMA were 2.88 +/- 1.14, 2.87 +/- 0.59 and 9.47 +/- 2.24 respectively. Incubations carried out in the presence of a monoclonal antibody to cytochrome P450IIE1 resulted in a 85-90% inhibition of all three reactions in this system. These results provide conclusive evidence that AOM, MAM and NDMA are metabolized by the same form of rat liver cytochrome P450. In addition, the stoichiometry of NDMA products formed in these reactions indicates that denitrosation, a presumed detoxication process, and alpha-hydroxylation, an activation reaction, are also catalyzed by the same cytochrome P450 isozyme.     

Effects of advanced leukemia on hepatic drug-metabolizing activity in the mouse

Summary Mice that had received 10⁶ P388 leukemia cells IV 8 days previously exhibited a decrease in the components of the hepatic microsomal mixed function oxidase, with a 58% decrease in cytochrome P-450, and up to a 60% decrease in hepatic microsomal metabolism of biphenyl. Liver weight was increased by 49% due to infiltration of the liver with leukemic cells. Changes in liver drug-metabolizing activity and liver weight were not seen 6 days after administration of P388 leukemia. There was a small increase in serum liver enzyme but no increase in total serum bilirubin in tumor-bearing mice. In vivo total-body plasma clearance of cyclophosphamide, a drug metabolized by hepatic cytochrome P-450, was decreased to 53 ml/min/kg in mice that had received P388 cells 8 days earlier, as against 97.2 ml/min/kg in control mice. Cytochrome P-450-independent metabolism of [¹⁴C]5-fluorouracil, measured by means of [¹⁴C]CO₂in the breath over 3 h, was decreased to 21% of the dose administered by 8 days after tumor cell administration, compared with 31% of the dose in control mice. P388 leukemia cells growing in the ascitic form in the intraperitoneal cavity of mice did not release an inhibitor of 5-fluorouracil metabolism into the ascitic fluid. Total-body plasma clearance of indocyanine green was decreased to 11 ml/min/kg by 8 days after P388 cell administration, compared with 36 ml/min/kg in control mice. The decrease in indocyanine green clearance might reflect a decrease in hepatic blood flow in the tumor-bearing mice. A possible explanation for the decrease in hepatic drug metabolism caused by P388 leukemia is that the hepatocytes are deprived of oxygen and nutrients by the tumor in the liver, coupled with or caused by a physical obstruction of hepatic blood flow.     

Involvement of microsomal cytochrome P450 and cytosolic thymidine phosphorylase in 5-fluorouracil formation from tegafur in human liver.

Recently, we have reported that tegafur, an anticancer agent, is biotransformed into active drug 5-fluorouracil (5-FU) by cytochromes P450 1A2, 2A6, and 2C8 in human liver microsomes (T. Komatsu et al., Drug Metab. Dispos, 28: 1457-1463, 2000). Because the conversion of tegafur into 5-FU has also been reported to be catalyzed by cytosolic thymidine phosphorylase (dThdPase), the involvement of human liver microsomes and cytosol and individual differences in 5-FU formation from tegafur were investigated. In 14 human samples, the mean rates of 5-FU formation in liver microsomes were 5-fold and 2-fold higher than those in liver cytosol at substrate concentrations of 100 microM and 1 mM tegafur, respectively. In the presence of 5-chloro-2,4-dihydroxypyridine, a dihydropyrimidine dehydrogenase inhibitor, the rates of 5-FU formation by the combination of liver microsomes and cytosol showed 5- and 3-fold interindividual differences at 100 microM and 1 mM tegafur, respectively. Kinetic analysis of human liver cytosolic 5-FU formation indicated an apparent higher Km value (16 +/- 4 mM) than that of liver microsomes (1.8 +/- 0.3 mM) with similar Vmax values. Human liver cytosolic 5-FU formation was confirmed to be catalyzed by dThdPase with correlation and chemical inhibition studies. These results suggested that 5-FU formation from tegafur in human liver was mainly catalyzed by microsomal P450 at low concentrations of tegafur, but the contribution of cytosolic 5-FU formation by dThdPase would be important at high concentrations.     

Benzene metabolism by ethanol-, acetone-, and benzene-inducible cytochrome P-450 (IIE1) in rat and rabbit liver microsomes

Ethanol is known to exert a synergistic effect on the toxicity of benzene. In the present investigation it was found that benzene was metabolized at a rate 20-65-fold higher in liver microsomes from ethanol- or acetone-treated rats than in microsomes from control animals. One high affinity site [Km = 19 +/- 5 (SD) microM] and one low affinity site [Km = 0.3 +/- 0.1 mM] for benzene metabolism were present in microsomes of acetone-treated rats, and similar sites were seen in microsomes from control or ethanol-treated rats. Treatment of the animals with either ethanol or acetone mainly influenced the Vmax values for benzene metabolism. Also benzene treatment of rats caused an increased rate of microsomal benzene metabolism. The hepatic microsomal NADPH-dependent metabolism of benzene was inhibited by compounds known to interact with the ethanol-inducible form of P-450 such as imidazole, ethanol, aniline, and acetone but was unaffected by addition of metyrapone. Anti-IgG against ethanol-inducible cytochrome P-450 from rat (P-450j) or rabbit liver (P-450 LMeb) inhibited the microsomal benzene metabolism effectively in rat or rabbit liver microsomes, respectively, whereas preimmune IgG was without effect. The level of rat ethanol-inducible P-450 (P-450j) was induced to an extent similar to that for the microsomal benzene metabolism, by either benzene, acetone, or ethanol. The data indicate that benzene is metabolized mainly by the ethanol-inducible P-450 form in liver microsomes and that the induction of this isozyme by ethanol can provide an explanation for the synergistic action of ethanol on benzene toxicity.