Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in human lung and liver microsomes and cytochromes P-450 expressed in hepatoma cells.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen in animals, has been linked to tobacco-related cancers in humans. The cytochrome(s) P-450 (P-450) responsible for the metabolic activation of NNK in humans has not been identified. The present work investigated the ability of human lung and liver microsomes and 12 forms of human P-450, expressed in Hep G2 (hepatoma) cells, to metabolize NNK. Of the 12 P-450 forms, P-450 1A2 had the highest activity in catalyzing the conversion of NNK to the keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone. P-450s 2A6, 2B7, 2E1, 2F1, and 3A5 also had measurable activities in the formation of keto alcohol. The apparent Km and Vmax for the formation of keto alcohol in the P-450 1A2-expressed Hep G2 cell lysate were 309 microM and 55 pmol/min/mg protein, respectively. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol, a reductive product, was the major metabolite formed, whereas the formation of keto alcohol and its aldehyde and acid derivatives (all alpha-hydroxylation products) constituted approximately 1% of the initial amount of NNK in P450-expressed Hep G2 cell lysate. A similar metabolite pattern was observed with human lung or liver microsomes. In human lung microsomes, the apparent Kms for the formation of 4-hydroxy-4-(3-pyridyl)butyric acid, 4-oxo-1-(3-pyridyl)-1-butanone, NNK-N-oxide, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were 526, 653, 531, and 573 microM, respectively; the formation of keto alcohol was not observed. For human lung microsomes, there was no sex-related difference in NNK metabolism. Carbon monoxide (90% atmosphere) significantly inhibited the metabolism of NNK in human lung and liver microsomes. 7,8-Benzoflavone, an inhibitor of P-450s 1A1 and 1A2, had no effect on NNK metabolism in human lung microsomes but decreased the formation of keto alcohol by 47% in human liver microsomes. Similarly, antibodies against human P-450s 1A2 and 2E1 decreased keto alcohol formation by 42% and 53%, respectively, in human liver microsomes but did not affect NNK metabolism in lung microsomes. Inhibitory antibodies against P-450s 2A1, 2C8, 2D1, or 3A4 had little or no effect on the metabolism of NNK in human liver or lung microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Thalidomide metabolism by the CYP2C subfamily.

PURPOSE: This research investigated the biotransformation of thalidomide by cytochrome P-450 (CYP). EXPERIMENTAL DESIGN: We used liver microsomes from humans and/or animals and the recombinant specific CYP isozymes to investigate CYP-mediated metabolism of thalidomide. RESULTS: Thalidomide was biotransformed into 5-hydroxythalidomide (5-OH) and diastereomeric 5'-hydroxythalidomide (5'-OH) by liver microsomes. The human liver microsomes with higher CYP2C19 activity formed more metabolites than those with lower CYP2C19 activity and had less activity in metabolite formations than those from rats. Recombinant human CYP2C19 and rat CYP2C6 isozymes were primarily responsible for forming these metabolites, and the male rat-specific CYP2C11 formed only 5'-OH. 5-OH was subsequently hydroxylated to 5,6-dihydroxythalidomide by CYP2C19, CYP2C9, and CYP1A1 in humans and by CYP2C11, CYP1A1, CYP2C6, and CYP2C12 in rats. Incubations with S-mephenytoin and omeprazole, substrates of CYP2C19, inhibited metabolism by human liver microsomes, supporting the involvement of CYP2C19. alpha-Naphthoflavone, an inhibitor of CYP1A, simultaneously stimulated the 5-OH formation and inhibited cis-5'-OH formation catalyzed by human liver microsomes. The contribution of the CYP2C subfamily was supported by the immunoinhibition study using human liver microsomes. When we used the microsomes from treated rats, the metabolite formations did not increase by inducers for CYP1A, CYP2B, CYP2E, CYP3A, or CYP4A, suggesting that these could not be involved in the main metabolic pathway in rats. CONCLUSIONS: We discovered that the polymorphic enzyme CYP2C19 is responsible for 5- and 5'-hydroxylation of thalidomide in humans. In rats, thalidomide was hydroxylated extensively by CYP2C6 as well as the sex-specific enzyme CYP2C11.     

Bioactivation of tegafur to 5-fluorouracil is catalyzed by cytochrome P-450 2A6 in human liver microsomes in vitro.

Tegafur is a prodrug of 5-fluorouracil (5-FU) consisting of a new class of oral chemotherapeutic agents, tegafur/uracil and S-1, which are classified as dihydropyrimidine dehydrogenase inhibitory fluoropyrimidines. It is bioactivated to 5-FU via 5'-hydroxylation mediated by cytochrome P-450 (CYP). However, which isoform(s) of CYP is responsible for the bioactivation process of tegafur remains unclear. The purpose of the present study was to identify the human CYP isoform(s) involved in the metabolic activation of tegafur using human liver microsomes and cDNA-expressed human CYPs. The formation of 5-FU from tegafur in human liver microsomes showed biphase kinetics with Km and Vmax values for the high-affinity component of 0.43 +/- 0.05 mM and 4.02 +/- 1.70 nmol/mg/min (mean +/- SD, n = 4), respectively. In the correlation study using a panel of 10 human liver microsomes, the formation of 5-FU from tegafur showed a significant correlation (r = 0.98; P < 0.001) with coumarin 7-hydroxylation, a marker activity of CYP2A6. In addition, a specific substrate of CYP2A6 and anti-CYP2A6 antibody inhibited the formation of 5-FU by 90% in human liver microsomes. Moreover, cDNA-expressed CYP2A6 showed the highest activity for the formation of 5-FU among 10 cDNA-expressed CYPs, with a Km value similar to that found for the high-affinity component in human liver microsomes. These findings clearly suggest that CYP2A6 is a principal enzyme responsible for the bioactivation process of tegafur in human liver microsomes. However, to what extent the bioactivation of tegafur by CYP2A6 accounts for the formation of 5-FU in vivo remains unclear, because the formation of 5-FU from tegafur is also catalyzed by the soluble fraction of a 100,000 x g supernatant and also derived from spontaneous degradation of tegafur.     

Metabolic conversion of methoxymorpholinyl doxorubicin: from a DNA strand breaker to a DNA cross-linker.

Methoxymorpholinyl doxorubicin (MMDX) is a novel anti-cancer anthracycline that differs from doxorubicin in its mechanisms of action, pattern of resistance and metabolism. Whereas doxorubicin is primarily an inhibitor of topoisomerase II, MMDX inhibits both topoisomerases I and II, resulting in predominantly single-strand DNA cleavage and, to a lesser extent, double-strand DNA breakage. MMDX is equally cytotoxic in vitro against the doxorubicin-sensitive and -resistant uterine sarcoma cell lines, MES-SA and Dx5. Using fluorescent laser cytometry, MMDX was retained intracellularly to a similar extent in MES-SA and Dx5; the intracellular retention of MMDX was 7.5-fold higher than that of doxorubicin in Dx5. The cytotoxicity of MMDX on an ovarian carcinoma cell line, ES-2, was potentiated 50-fold by preincubating the drug with human liver microsomes and NADPH. This cytotoxic potentiation was associated with the appearance of DNA interstrand cross-links. The in vitro potentiation of MMDX was inhibited by cyclosporin A, which is a substrate for human cytochrome P450 IIIA.     

A new form of cytochrome P-450 responsible for mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline in human livers.

Antibodies to P-450IA2 strongly inhibited the mutagenic activation of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate but not aflatoxin B1 in human liver microsomes. The anti-rat P-450IA2 antibodies were capable of recognizing two proteins which show different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human liver microsomes. A new form of cytochrome P-450 (designated P-450-HM4) cross-reactive with anti-rat P-450IA2 antibodies showing that the smaller molecular weight was purified from human liver microsomes by means of the fast-performance liquid chromatography system. The molecular weight of P-450-HM4 was estimated to be 49,000, which was apparently different from that of P-450PA (human P-450IA2). The antibodies to P-450-HM4 did not cross-react with P-450PA (human P-450IA2) but inhibited to various extents the mutagenic activation of IQ in microsomes from human livers. In addition, P-450-HM4 showed significant mutagen-producing activity from IQ in a reconstituted system. Together with these and other results reported previously, it is concluded that at least two forms of cytochrome P-450 [P-450-HM4 and P-450PA (human P-450IA2)] are involved in the mutagenic activation of IQ in human liver.     

Cytochrome P-450 3A4 (nifedipine oxidase) is responsible for the C-oxidative metabolism of 1-nitropyrene in human liver microsomal samples.

The nitrated polycyclic aromatic hydrocarbon 1-nitropyrene is a ubiquitous environmental pollutant. The role of cytochromes P-450 in the human metabolism of [3H]-1-nitropyrene was investigated using human liver microsomes. The range of microsomal metabolism from 16 individual liver specimens was 0.13 to 0.99 nmol/min/mg protein. Using 3 microsomal samples exhibiting different maximal velocities, the Km of 1-nitropyrene metabolism was 3.3 +/- 0.5 microM, indicating that perhaps a single or similar cytochromes P-450 was involved in the metabolism of 1-nitropyrene in these samples. The P-450 3A inhibitor triacetyloleandomycin inhibited 86 +/- 8% of the microsomal metabolism of 1-nitropyrene. Further evidence for the role of P-450 3A in human microsomal metabolism of 1-nitropyrene was gained using inhibitory anti-P-450 3A antibodies. Using 3 separate microsomal samples, antibody conditions that inhibited approximately 90% of the metabolism of the P-450 3A4-specific substrate nifedipine inhibited approximately 60-70% of the metabolism of 1-nitropyrene. Human liver microsomes demonstrated a preference for 1-nitropyren-3-ol formation over 1-nitropyren-6-ol or 1-nitropyren-8-ol, which is in contrast to that noted in rodents where the 6-ol and 8-ol are preferentially formed over the 3-ol, yet in agreement with earlier studies on the metabolism of 1-nitropyrene using Vaccinia-expressed human cytochromes P-450. These results indicate that the human hepatic metabolism of 1-nitropyrene is carried out by at least two or more P-450s including those in the P-450 3A subfamily. These studies also suggest that the metabolism of this compound by humans may differ from that in rodents in both the cytochromes that are involved and the specific metabolites that are formed.     

In vivo antitumor activity and host toxicity of methoxymorpholinyl doxorubicin: role of cytochrome P450 3A

Methoxymorpholinyl doxorubicin (MMDX; PNU 152243) is a promising doxorubicin derivative currently undergoing clinical evaluation. Previous in vitro studies suggested that the compound undergoes hepatic biotransformation by cytochrome P450 (CYP) 3A into a more cytotoxic metabolite(s). The present study examined the role of CYP3A-mediated metabolism in the in vivo antitumor activity and host toxicity of MMDX in the mouse model and investigated the potential for increasing the therapeutic effectiveness of the drug by inducing its hepatic CYP-catalyzed activation. We found that MMDX cytotoxicity for cultured M5076 tumor cells was potentiated 22-fold by preincubating the drug with NADPH-supplemented liver microsomes from untreated C57BL/6 female mice. A greater (50-fold) potentiation of MMDX cytotoxicity was observed after its preincubation with liver microsomes isolated from animals pretreated with the prototypical CYP3A inducer pregnenolone-16alpha-carbonitrile. In contrast, in vivo administration of the selective CYP3A inhibitor troleandomycin (TAO) reduced both potentiation of MMDX cytotoxicity and the rate of CYP3A-catalyzed N-demethylation of erythromycin by isolated liver microsomes (55.5 and 49% reduction, respectively). In vivo antitumor activity experiments revealed that TAO completely suppressed the ability of 90 microg/kg MMDX i.v., a dose close to the LD10, to delay growth of s.c. M5076 tumors in C57BL/6 mice and to prolong survival of DBA/2 mice with disseminated L1210 leukemia. Moreover, TAO administration markedly inhibited the therapeutic efficacy of 90 microg/kg MMDX i.v. in mice bearing experimental M5076 liver metastases; a complete loss of MMDX activity was observed in liver metastases-bearing animals receiving 40 microg/kg MMDX i.v. plus TAO. However, pregnenolone-16alpha-carbonitrile pretreatment failed to enhance MMDX activity in mice bearing either s.c. M5076 tumors or experimental M5076 liver metastases. Additional experiments carried out in healthy C57BL/6 mice showed that TAO markedly inhibited MMDX-induced myelosuppression and protected the animals against lethal doses of MMDX. Taken together, these findings demonstrate that an active metabolite(s) of MMDX synthesized via CYP3A contributes significantly to its in vivo antitumor activity and host toxicity.     

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.     

Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450

The cis isomer of N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen for the Syrian golden hamster, is metabolized by hamster liver microsomes to yield N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product. Rabbit liver microsomes catalyze the metabolism of cis-NNDM to HPOP at a rate slower than that observed with hamster microsomes, but significantly faster than that obtained with rat microsomes. Pretreatment of rabbits with phenobarbital results in a 6-fold increase in the cis-NNDM hydroxylase activity of the rabbit microsomes to levels equal to that observed with the hamster; pretreatment of rabbits with other xenobiotics had no effect on the hydroxylation of cis-NNDM. The role of rabbit liver microsomal cytochrome P-450 in the metabolism of the cis isomer of NNDM was studied in the reconstituted system consisting of NADPH:cytochrome P-450 reductase, phospholipid, and cytochrome P-450. Cytochrome P-450LM2, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The Vmax for the formation of HPOP was 1.78 nmol/min/nmol cytochrome P-450LM2, and the apparent Km was 360 microM. Cytochrome P-450LM3a also catalyzed the metabolism of NNDM to HPOP at a significant rate (0.25 nmol/min/nmol cytochrome P-450). Of the four other isozymes of cytochrome P-450 (forms 3b, 3c, 4, and 6) tested in the reconstituted system, only forms 3b and 3c exhibited measurable activities (approximately 0.04 nmol of HPOP formed/min/nmol cytochrome P-450). The addition of antibodies to isozyme 2 to microsomes from phenobarbital-treated rabbits resulted in approximately 95% inhibition of the metabolism of NNDM, while the addition of antibodies to LM3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM2 and anti-LM3a antibodies was 50 and 64%, respectively. The addition of antibodies to isozyme 3a to microsomes isolated from ethanol-treated rabbits caused approximately 90% inhibition of the metabolism of NNDM. These data conclusively demonstrate that several forms of cytochrome P-450 can catalyze the metabolism of cis-NNDM and that isozymes 2 and 3a play important roles in the rabbit hepatic metabolism of NNDM to HPOP, the proximate carcinogenic metabolite.     

Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes.

PURPOSE: Nemorubicin (3'-deamino-3'-[2'(S)-methoxy-4'-morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3'-deamino-3',4'-anhydro-[2'(S)-methoxy-3'(R)-oxy-4'-morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPH-supplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682. EXPERIMENTAL DESIGN: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively. RESULTS: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts. CONCLUSIONS: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.     

Association of DNA cross-linking with potentiation of the morpholino derivative of doxorubicin by human liver microsomes

The morpholino analog of doxorubicin (DOX), 3'-deamino-3'-(4"-morpholinyl)-doxorubicin (MRA), is 0.5- to 10-fold more potent than DOX in vitro but 100- to 200-fold more potent in vivo, which indicated that biotransformation in vivo may generate a highly potent metabolite(s). A likely mechanism for such biotransformation is hepatic mixed-function oxidation. At a concentration of 5 microM, MRA was incubated for 30 minutes at 37 degrees C with 1 mg of human liver microsomes/mL and 0.45 mM of NADPH. The cytotoxicity of the microsome- and NADPH-treated MRA was 44-fold higher than that of the untreated MRA in the human ovarian carcinoma cell line ES-2. This potentiation did not occur for MRA treated with boiled microsomes and NADPH, active microsomes in the absence of NADPH, or Tris buffer plus NADPH. No potentiation was observed with DOX or the highly potent cyanomorpholino derivative of DOX, MRA-CN, under any of the above conditions. After 2 hours of exposure of the ES-2 cells to microsome- and NADPH-treated MRA, dose-dependent DNA cross-links were observed with 5 nM or more of MRA, whereas only DNA strand breaks were detected in cells exposed to 500 nM of untreated MRA or MRA incubated under other conditions. These data indicate that MRA is biotransformed by the hepatic mixed-function oxidases to a potent DNA-alkylating metabolite(s), which may be important in the determination of the pharmacologic and toxicologic profile of MRA. The active metabolite(s) of MRA may be analogous to MRA-CN, which cross-links DNA without requiring bioactivation.     

Activation of amino-alpha-carboline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and a copper phthalocyanine cellulose extract of cigarette smoke condensate by cytochrome P-450 enzymes in rat and human liver microsomes

The ability of cigarette smoke condensate to induce a genotoxic response has been measured in liver microsomal and reconstituted monooxygenase systems containing rat and human cytochrome P-450 (P-450) enzymes, as determined by umu gene expression in Salmonella typhimurium TA1535/pSK1002. The reactivities of amino-alpha-carboline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), two compounds known to be present at considerable levels in cigarette smoke condensate, were also determined and compared with regard to genotoxicity. Amino-alpha-carboline and PhIP are activated principally by P-450 1A2 enzymes in human and rat liver microsomes: (a) activation of both compounds was catalyzed efficiently by liver microsomes prepared from rats treated with 5,6-benzoflavone, isosafrole, or the commercial polychlorinated biphenyl mixture Aroclor 1254, and the activities could be considerably inhibited by antibodies raised against P-450 1A1 or 1A2; (b) the rates of activation of these compounds were correlated with the amount of human P-450 1A2 and of phenacetin O-deethylation activity in different human liver microsomal preparations, and these activities were inhibited by anti-P-450 1A2; (c) reconstituted enzyme systems containing P-450 1A enzymes isolated from rats and humans showed the highest rates of activation of amino-alpha-carboline and PhIP. In rat liver microsomes PhIP may also be activated by P-450 3A enzymes; activity was induced in rats treated with pregnenolone 16 alpha-carbonitrile and was inhibited by anti-human P-450 3A4. However, in humans the contribution of P-450 3A enzymes could be excluded as judged by the very low effects of anti-P-450 3A4 on the microsomal activities and poor correlation with P-450 3A4-catalyzed activities in various liver samples. Cigarette smoke condensate strongly inhibited the activation of several potent procarcinogens by human liver microsomes, particularly the reactions catalyzed by P-450 1A2, but was not so inhibitory of the activation reactions catalyzed by P-450 3A4 and of P-450 2D6-catalyzed bufuralol 1'-hydroxylation. Genotoxic components of the cigarette smoke condensate were extracted by using copper phthalocyanine cellulose (blue cotton). Genotoxicity of this extract was observed only after activation by P-450, and the inhibition of P-450 1A2 activities by these extracts was slight.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of human cytochrome P450 3A4 in metabolism of medroxyprogesterone acetate.

Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced or recurrent breast cancer and endometrial cancer. The drug is extensively metabolized in the intestinal mucosa and in the liver. Cytochrome P450s (CYPs) involved in the metabolism of MPA were identified by using human liver microsomes and recombinant human CYPs. In this study, the overall metabolism of MPA was determined as the disappearance of the parent drug from an incubation mixture. The disappearance of MPA in human liver microsomes varied 2.6-fold among the 18 samples studied. The disappearance of MPA in the same panel of 18 human liver microsomes was significantly correlated with triazolam alpha-hydroxylase activity, a marker activity of CYP3A (r = 0.764; P < 0.001). Ketoconazole, an inhibitor of CYP3A4, potently inhibited the disappearance of MPA in 18 human liver microsomes. Anti-CYP3A antibody also inhibited 86% of the disappearance of MPA in human liver microsomes. Although sulfaphenazole (an inhibitor of CYP2C9) and S-mephenytoin (an inhibitor of CYP2C19) partially inhibited the disappearance of MPA, no effect of the anti-CYP2C antibody was observed. The disappearance of MPA did not correlate with either the activity metabolized via CYP2C9 (diclofenac 4'-hydroxylase activity) or the activity metabolized via CYP2C19 (S-mephenytoin 4'-hydroxylase activity). Among the 12 recombinant human CYPs (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) studied, only CYP3A4 showed metabolic activity of MPA. These results suggest that CYP3A4 is mainly involved in the overall metabolism of MPA in human liver microsomes.     

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.      

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.     

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.     

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     

Characterization of covalent DNA binding of morpholino and cyanomorpholino derivatives of doxorubicin.

BACKGROUND: The doxorubicin analogues cyanomorpholino doxorubicin (MRA-CN) and morpholino doxorubicin (MRA) were synthesized in an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy. MRA-CN forms interstrand DNA cross-links without requiring microsomal metabolic activation in the presence of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) to form a product that alkylates DNA, but MRA requires metabolic activation. Alkylation produces DNA cross-links, which are associated with potentiation of the cytotoxicity of some drugs. PURPOSE: Our purpose was to study the DNA binding of MRA-CN and MRA with and without metabolic activation in order to better understand the mechanisms for cross-linking DNA. METHODS: We used [3H]MRA and [3H]MRA-CN, with the 3H labeled at C-2 and C-6 of the morpholino ring. MRA (10 nM) was incubated with human liver microsomes with or without NADPH to measure DNA binding. In addition, a filter elution assay was used to determine the nature and extent of drug binding to DNA in the human ovarian carcinoma cell line ES-2. We studied the appearance of interstrand cross-links versus total DNA adducts in pBR322 plasmid DNA incubated with 100 nM MRA-CN in cell-free medium and then subjected to denaturation and agarose gel electrophoresis. RESULTS: Regardless of the extracellular concentration of the drug (1-100 nM), 85% of intracellular MRA-CN was covalently bound to DNA, and the total amount of drug bound to DNA was proportional to extracellular drug concentration. No covalent binding of MRA to DNA was found in cells exposed to 10 nM MRA alone for 2 hours. In contrast, 10% of the intracellular drug was bound to DNA if the cells were exposed to MRA preincubated with human liver microsomes and NADPH. The percentage of plasmids containing at least one interstrand cross-link rose from 35% at 15 minutes to 92% at 2 hours. We estimate that eight molecules of MRA-CN were adducted per molecule of pBR322 DNA (or one drug adduct per 545 base pairs), with a minimum of 12% of the adducts forming interstrand cross-links. CONCLUSIONS: These results suggest that the carbons at positions 2 and 6 of the morpholino ring of both MRA-CN and the activated metabolite of MRA are retained in the drug-DNA adduct. They also indicate that the formation of interstrand DNA cross-links by MRA-CN is preceded by formation of drug adducts to a single strand of DNA.     

16Alpha-hydroxylation of estrone by human cytochrome P4503A4/5

The cytochrome P450 (P450) enzymes that catalyse metabolism of the estrogen, estrone (E1), to the putative carcinogen 16alpha-hydroxy E1 (16alpha-OHE1) in humans were determined. The potential of the most abundant circulating form of estrogen, estrone 3-sulfate (E1S), to be the substrate was also investigated. Human liver microsomal sulfatases convert E1S to E1, an essential prerequisite for formation of 16alpha- OHE1 from added E1S in this system. E1 metabolism to 16alpha-OHE1 in a panel of 15 human liver microsomal preparations correlated with total P450 concentrations (r2 = 0.63) and with activities associated with P450 forms CYP3A4 and 3A5 (r2 = 0.72). E1 16alpha-hydroxylase activity in human liver microsomes was inhibited by 75% by monoclonal anti human CYP3A4/5 antibodies at 4 mg antibody/nmol total P450, and by troleandomycin, a specific CYP3A4/5 inhibitor. Rates of E1 metabolism to 16alpha-OHE1 were 1.6-fold higher when E1 was generated in situ from E1S than when E1 was added. Microsomal preparations of cDNA expressed CYP3A4 or 3A5, with NADPH-P450-reductase co-expressed, both metabolized E1 to 16alpha-OHE1, and added cytochrome b5 increased the rates 5.1- and 7.5-fold, respectively. In these systems rates of E1 metabolism to 16alpha-OHE1 were 2.8-fold higher when E1 was generated in situ from E1S than when E1 was added. Kinetic values for E1 metabolism to 16alpha- OHE1 by human liver microsomes and for the expressed CYP3A4 system were Km 154 and 172 microM, respectively, and Vmax 238 pmol/min/nmol total P450 and 1050 pmol/min/nmol CYP3A4, respectively. Thus, formation of the putative carcinogen 16alpha-OHE1 is catalysed by CYP3A4 and 3A5 and stimulated by cytochrome b5. E1S is not a substrate but formation of E1 from E1S in situ stimulates formation of 16alpha-OHE1, possibly because E1S is more water soluble and in situ generation of E1 provides for facilitated exposure of E1 to the P450 substrate binding sites. Blocking of the pathway of E1 to 16alpha-OHE1 could provide a therapeutic approach for diminishing the risk of estrogen dependent breast cancer.