The Transcriptional Repressor Polycomb Group Factor 6, PCGF6, Negatively Regulates Dendritic Cell Activation and Promotes Quiescence.

Pro-inflammatory signals provided by the microenvironment are critical to activate dendritic cells (DCs), components of the innate immune system that shape both innate and adaptive immunity. However, to prevent inappropriate immune activation, mechanisms must be in place to restrain DC activation to ensure DCs are activated only once sufficient stimuli have been received. Here, we report that DC activation and immunogenicity are regulated by the transcriptional repressor Polycomb group factor 6 (PCGF6). Pcgf6 is rapidly downregulated upon stimulation, and this downregulation is necessary to permit full DC activation. Silencing PCGF6 expression enhanced both spontaneous and stimulated DC activation. We show that PCGF6 associates with the H3K4me3 demethylase JARID1c, and together, they negatively regulate H3K4me3 levels in DCs. Our results identify two key regulators, PCGF6 and JARID1c that temper DC activation and implicate active transcriptional silencing via histone demethylation as a previously unappreciated mechanism for regulating DC activation and quiescence.

Transplacental transfer and metabolism of buprenorphine in preterm human placenta.

We sought to determine whether gestational age affects the transplacental transfer and metabolism of buprenorphine (BUP). Transfer of BUP (10 ng/mL) and its [ (3)H]-isotope was determined across placentas of 30 to 34 weeks of gestation utilizing the technique of dual perfusion of placental lobule. Concentration of the drug in trophoblast tissue and in maternal and fetal circuits was determined by liquid scintillation spectrometry. Microsomes prepared from placentas of 17 to 37 weeks of gestation were divided into three groups: late second, early third, and late third trimesters. Antibodies raised against human cytochrome P450 (CYP) isoforms were utilized to identify the enzyme(s) catalyzing BUP biotransformation by preterm placental microsomes. The amount of norbuprenorphine formed was determined by liquid chromatography-mass spectrometry (LC-MS). BUP transfer across the placentas of 30 to 34 weeks of gestation was similar to those at term. CYP19 antibodies caused 60% inhibition of BUP metabolism by microsomes of late second and early third trimesters and 85% by microsomes of late third trimester. The developmental changes occurring in human placenta between 30 weeks of gestation through term do not affect the transfer of BUP across human placenta. CYP19 is the major enzyme responsible for biotransformation of BUP beginning at 17 weeks of gestation until term.

Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite.

The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.

Epigenetic regulation of the alternatively activated macrophage phenotype.

Alternatively activated (M2) macrophages play critical roles in diverse chronic diseases, including parasite infections, cancer, and allergic responses. However, little is known about the acquisition and maintenance of their phenotype. We report that M2-macrophage marker genes are epigenetically regulated by reciprocal changes in histone H3 lysine-4 (H3K4) and histone H3 lysine-27 (H3K27) methylation; and the latter methylation marks are removed by the H3K27 demethylase Jumonji domain containing 3 (Jmjd3). We found that continuous interleukin-4 (IL-4) treatment leads to decreased H3K27 methylation, at the promoter of M2 marker genes, and a concomitant increase in Jmjd3 expression. Furthermore, we demonstrate that IL-4-dependent Jmjd3 expression is mediated by STAT6, a major transcription factor of IL-4-mediated signaling. After IL-4 stimulation, activated STAT6 is increased and binds to consensus sites at the Jmjd3 promoter. Increased Jmjd3 contributes to the decrease of H3K27 dimethylation and trimethylation (H3K27me2/3) marks as well as the transcriptional activation of specific M2 marker genes. The decrease in H3K27me2/3 and increase in Jmjd3 recruitment were confirmed by in vivo studies using a Schistosoma mansoni egg-challenged mouse model, a well-studied system known to support an M2 phenotype. Collectively, these data indicate that chromatin remodeling is mechanistically important in the acquisition of the M2-macrophage phenotype.

Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells.

Multipotential naive CD4(+) T cells differentiate into distinct lineages including T helper 1 (Th1), Th2, Th17, and inducible T regulatory (iTreg) cells. The remarkable diversity of CD4(+) T cells begs the question whether the observed changes reflect terminal differentiation with heritable epigenetic modifications or plasticity in T cell responses. We generated genome-wide histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) trimethylation maps in naive, Th1, Th2, Th17, iTreg, and natural Treg (nTreg) cells. We found that although modifications of signature-cytokine genes (Ifng, Il4, and Il17) partially conform to the expectation of lineage commitment, genes encoding transcription factors like Tbx21 exhibit a broad spectrum of epigenetic states, consistent with our demonstration of T-bet and interferon-gamma induction in nTreg cells. Our data suggest an epigenetic mechanism underlying the specificity and plasticity of effector and regulatory T cells and also provide a framework for understanding complexity of CD4(+) T helper cell differentiation.

Thymocyte proliferation induced by pre-T cell receptor signaling is maintained through polycomb gene product Bmi-1-mediated Cdkn2a repression.

Thymocytes undergo massive proliferation before T cell receptor (TCR) gene rearrangement, ensuring the diversification of the TCR repertoire. Because activated cells are more susceptible to damage, cell-death restraint as well as promotion of cell-cycle progression is considered important for adequate cell growth. Although the molecular mechanism of pre-TCR-induced proliferation has been examined, the mechanisms of protection against cell death during the proliferation phase remain unknown. Here we show that the survival of activated pre-T cells induced by pre-TCR signaling required the Polycomb group (PcG) gene product Bmi-1-mediated repression of Cdkn2A, and that p19Arf expression resulted in thymocyte cell death and inhibited the transition from CD4(-)CD8(-) (DN) to CD4(+)CD8(+) (DP) stage upstream of the transcriptional factor p53 pathway. The expression of Cdkn2A (the gene encoding p19Arf) in immature thymocytes was directly regulated by PcG complex containing Bmi-1 and M33 through the maintenance of local trimethylated histone H3K27. Our results indicate that this epigenetic regulation critically contributes to the survival of the activated pre-T cells, thereby supporting their proliferation during the DN-DP transition.

Interactions of two major metabolites of prasugrel, a thienopyridine antiplatelet agent, with the cytochromes P450.

The biotransformation of prasugrel to R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid) involves rapid deesterification to R-95913 (2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone) followed by cytochrome P450 (P450)-mediated formation of R-138727, the metabolite responsible for platelet aggregation. For identification of the P450s responsible for the formation of the active metabolite, the current studies were conducted with R-95913 as the substrate. Incubations required supplementation with reduced glutathione. Hyperbolic kinetics (K(m) 21-30 microM), consistent with a single enzyme predominating, were observed after incubations with human liver microsomes. Correlation analyses revealed a strong relationship between R-138727 formation and CYP3A-mediated midazolam 1'-hydroxylation (r(2) = 0.98; p < 0.001) in a bank of characterized human liver microsomal samples. The human lymphoblast-expressed enzymes capable of forming R-138727, in rank order of rates, were CYP3A4>CYP2B6>CYP2C19 approximately CYP2C9>CYP2D6. A monoclonal antibody to CYP2B6 and the CYP3A inhibitor ketoconazole substantially inhibited R-138727 formation, whereas inhibitors of CYP2C9 (sulfaphenazole) and CYP2C19 (omeprazole) did not. Scaling of in vitro intrinsic clearance values from expressed enzymes to the whole liver using a relative abundance approach indicated that either CYP3A4 alone or CYP3A4 and CYP2B6 are the major contributors to R-138727 formation. R-95913 and R-138727 were also examined for their ability to inhibit metabolism mediated by five P450s. R-138727 did not inhibit the P450s tested. In vitro, R-95913 inhibited CYP2C9, CYP2C19, CYP2D6, and CYP3A, with K(i) values ranging from 7.2 microM to 82 microM, but did not inhibit CYP1A2. These K(i) values exceed circulating concentrations in humans by 3.8- to 43-fold. Therefore, neither R-95913 nor R-138727 is expected to substantially inhibit the P450-mediated metabolism of coadministered drugs.

Cytochrome P450 3A and 2B6 in the developing kidney: implications for ifosfamide nephrotoxicity.

Repeated administration of agents (e.g., cancer chemotherapy) that can cause drug-induced nephrotoxicity may lead to acute or chronic renal damage. This will adversely affect the health and well-being of children, especially when the developing kidney is exposed to toxic agents that may lead to acute glomerular, tubular or combined toxicity. We have previously shown that the cancer chemotherapeutic ifosfamide (IF) causes serious renal damage substantially more in younger children (less than 3 years of age) than among older children. The mechanism of the age-related IF-induced renal damage is not known. Our major hypothesis is that renal CYP P450 expression and activity are responsible for IF metabolism to the nephrotoxic chloroacetaldehyde. Presently, the ontogeny of these catalytic enzymes in the kidney is sparsely known. The presence of CYP3A4, 3A5 and 2B6 was investigated in human fetal, pediatric and adult kidney as was the metabolism of IF (both R-IF and S-IF enantiomers) by renal microsomes to 2-dechloroethylifosfamide (2-DCEIF) and 3-dechloroethylifosfamide (3-DCEIF). Our analysis shows that CYP 3A4 and 3A5 are present as early as 8 weeks of gestation. IF is metabolized in the kidney to its two enantiomers. This metabolism can be inhibited with CYP 3A4/5 and 2B6 specific monoclonal inhibitory antibodies, whereby the CYP3A4/5 inhibitory antibody decreased the production of R-3-DCEIF by 51%, while the inhibitory CYP2B6 antibody decreased the production of S-2-DCEIF and S-3-DCEIF by 44 and 43%, respectively, in patient samples. Total renal CYP content is approximately six-fold lower than in the liver.

Rat and human liver cytochrome P-450 isoform metabolism of ecteinascidin 743 does not predict gender-dependent toxicity in humans.

Ecteinascidin 743 (ET743, NSC648766) is a marine natural product with potent in vivo activity in human xenograft models. Hepatotoxicity was the most prominent toxicity in preclinical studies and was greater in female rats than in male rats. To assess the potential implications for human toxicities, the in vitro metabolism of ET743 was characterized using rat and human preparations. NADPH-dependent ET743 metabolism was greater with male rat liver microsomal preparations than with preparations from female rats and was induced by pretreatment of rats with phenobarbital and dexamethasone but not by pretreatment with 3-methylcholanthrene. Rat and human microsomal metabolism of ET743 was reduced in the presence of chemical CYP3A inhibitors or antirat CYP3A2 antiserum and to a much lesser extent by CYP2E, CYP2C, and CYP2A inhibitors. In human liver panel studies, ET743 disappearance was highly correlated with CYP3A activities and to a lesser extent with CYP2C activities. ET743 was metabolized by a number of cDNA-expressed rat P-450 isoforms, including male-predominant CYP2A2 and CYP3A2. ET743 was metabolized by cDNA-expressed human CYP3A4 and to a much lesser extent by CYP2C9, CYP2D6, and CYP2E1 preparations. Three oxidative metabolites were detected in cDNA-expressed isoform incubations, including the N-demethylated metabolite ET729 and two additional products characterized by laser capture-mass spectrometry analyses. The plasma pharmacokinetics and biliary excretion of ET743 were characterized in rats. There were no gender-dependent differences in half-life or total body clearance values. Although very modest, the biliary excretion of ET743 in male rats (0.48%) was greater than in female rats (0.28%). In contrast, the biliary excretion of the cytotoxic N-demethylated metabolite ET729 was 5-fold greater in the female rat (1.05% of dose) than in the male rat (0.19% of dose). Biliary excretion of ET729 may contribute to the hepatic toxicity in rats. These data are consistent with a major role for CYP3A isoforms in ET743 rat and human metabolism. Although there are conflicting data in the literature, expression of CYP3A isoforms in human tissues and elimination of CYP3A substrates have not been shown to vary substantially by gender. There are no indications that the other CYP isoforms implicated in ET743 metabolism are expressed differently in males and females. Thus, although it is not possible to rule out gender differences in ET743 human toxicities, our data do not predict major gender-dependent differences in the toxicity of ET743 based on metabolism.

Cytochrome P4502B6 and 2C9 do not metabolize midazolam: kinetic analysis and inhibition study with monoclonal antibodies.

We determined the contribution of cytochrome P450 (CYP) isoforms to the metabolism of midazolam by kinetic analysis of human liver microsomes and CYP isoforms and by examining the effect of chemical inhibitors and monoclonal antibodies against CYP isoforms in vitro. Midazolam was metabolized to 1'-hydroxymidazolam (1'-OH MDZ) by human liver microsomes with a Michaelis-Menten constant (Km) of 4.1 (1.0) (mean (SD)) micromol litre(-1) and a maximum rate of metabolism (Vmax) of 5.5 (1.1) nmol min(-1) mg protein(-1) (n = 6). Of the nine representative human liver CYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, three (CYP2B6, 3A4 and 3A5) showed midazolam 1'-hydroxylation activity, with Kms of 40.7, 1.7 and 3.0 micromol litre(-1), respectively, and Vmax values of 12.0, 3.3 and 13.2 nmol min(-1) nmol P450(-1), respectively (n = 4). Midazolam 1'-hydroxylation activity of human liver microsomes correlated significantly with testosterone 6beta-hydroxylation activity, a marker of CYP3A activity (r2 = 0.77, P = 0.0001), but not with S-mephenytoin N-demethylation activity, a marker of CYP2B6 activity (r2 < 0.01, P = 0.84) (n = 11). Troleandomycin and orphenadrine, chemical inhibitors of CYP isoforms, inhibited the formation of 1'-OH MDZ by human liver microsomes. Monoclonal antibody against CYP3A4 inhibited the formation of 1'-OH MDZ by 79%, whereas monoclonal antibody against CYP2B6 had no effect on midazolam 1'-hydroxylation by human liver microsomes (n = 5). These results indicate that only CYP3A4, but not CYP2B6 or CYP2C, is involved in the metabolism of midazolam in vitro.

Immunolocalisation of cytochrome P-450 3A enzymes in human breast carcinoma: relationship with tumour differentiation and steroid receptors.

Cytochrome P-450 3A enzymes belong to the most abundant subfamily of the cytochrome P-450 system. They are predominantly found in the liver where they metabolize numerous drugs and endogenous substances such as oestrogens. However, they are also expressed by normal and tumoural extrahepatic tissues. Accordingly, immunolocalization was assessed in malignant breast tumours (n=32) and normal counterparts, by using a monoclonal antibody that recognizes all human CYP3A proteins. We investigated a potential relation between expression of CYP3A protein expression, the degree of tumour differentiation assessed by the histological grade and the proliferation index assessed by Ki-67 immunostaining. Immunodetection of CYP3A was observed in 27 of the 32 tumours analyzed (84%). A focal staining was also observed in the adjacent normal breast tissue in 33% of the samples, but expression was always fainter than in tumours. A significant negative association was found between CYP3A and the proliferation index, but there was no relation with receptor status or tumour differentiation. While CYP3A protein expression can be found in normal breast tissues, these data highlight higher and more frequent CYP3A in malignant breast cells. Such expression in malignant breast cells appears inversely related to the proliferation index whereas no relation is found with tumour differentiation.

Differential induction of rat hepatic cytochromes P450 3A1, 3A2, 2B1, 2B2, and 2E1 in response to pyridine treatment.

Pyridine (PY) effects on rat hepatic cytochromes P450 (CYP) 3A1 and 3A2 expression were examined at the levels of metabolic activity, protein, and mRNA and were compared with those of CYP2B1/2 and CYP2E1. CYP3A metabolic activity as well as CYP3A protein and mRNA levels increased following treatment of rats with PY. CYP3A1 and CYP3A2 were differentially affected by PY treatment in terms of induction levels, dose dependence, and stability of mRNA. CYP3A1 mRNA levels maximally increased ~42-fold after PY treatment, whereas CYP3A2 mRNA level increased ~4-fold. Moreover, CYP3A1 mRNA levels decreased more rapidly than those of CYP3A2 as determined following inhibition of transcription with actinomycin D or cordycepin. Treatment of rats with PY resulted in a dose-dependent increase in CYP3A1, CYP3A2, and CYP2B1/2B2 protein levels. In contrast to the effects of PY treatment on CYP3A1 and 2B, CYP2E1 protein levels increased in the absence of a concomitant increase in CYP2E1 mRNA levels. Treatment of rats with PY at 200 mg/kg/day for 3 days increased both protein and mRNA levels of CYP3A2, whereas treatment with higher than 200 mg/kg/day for 3 days increased CYP3A2 protein levels without an increase in CYP3A2 mRNA levels. These data demonstrated that PY regulates the various CYPs examined in this study at different levels of expression and that PY regulates CYP3A1 expression through transcriptional activation and CYP3A2 expression through transcriptional and post-transcriptional activation at a low- and high-dose PY treatment, respectively.

Indications for the involvement of a CYP3A-like iso-enzyme in the metabolism of chlorobornane (Toxaphene) congeners in seals from inhibition studies with liver microsomes.

The different isoforms of the cytochrome P450 (CYP) system can metabolise a suite of classes of lipophilic, anthropogenic compounds. The bioaccumulative potential as well as the toxicity of xenobiotics may be significantly altered in the process. To compare the metabolic ability of different wildlife species, it is important to identify the different iso-enzymes of CYP, which are responsible for the metabolism of different classes of compounds. This can be achieved with in vitro incubation assays. In the present study, preparations of hepatic microsomes of a harbour seal (Phoca vitulina) and a grey seal (Halichoerus grypus) demonstrated that the chlorobornane (CHB) congeners CHB-32 and -62 were metabolised enzymatically to their hydroxylated derivatives. These derivatives were partially characterised by their NCI mass-spectra. Inhibition studies were carried out to identify the specific CYP isoform(s) responsible for the metabolism of CHB-32 and -62. Ketoconazole has been shown to inhibit CYP3A enzymes in human and rat studies. In this study, ketoconazole caused concentration-dependent inhibition of metabolism of CHB-32 and -62, reaching 80% at the 1.0 microM treatment level. Ellipticine (1.0 microM), which has been shown to inhibit CYP1A1/2, also inhibited CHB-32 and -62 metabolism in the microsomes of grey seal, but to a much lower degree of less than 10 and 24%, respectively. In the same experiment the metabolism of 4,4'-dichlorobiphenyl was already inhibited 70% by ellipticine treatment at the same concentration. This non-ortho substituted PCB congener can easily attain a planar molecular configuration, and therefore served as a model CYP1A substrate. Inhibition of chlorobornane metabolism was not observed after the addition of goat anti-rat CYP2B antibodies or Aldrin, which is a model CYP2B substrate in rat. Cautious interpretation is advised for results obtained with so-called selective competitive inhibitors. Regardless, these studies indicated for the first time the possible involvement a CYP3A isoform in the mediation of chlorobornane metabolism in seals. The immunochemical cross-reactivity of mouse, rabbit or sheep anti-rat antibodies in the hepatic microsomes of harbour seal confirmed the presence of CYP1A1/2, CYP1A1, CYP2B1/2, CYP3A and CYP4A isoenzymes. Enantioselective metabolism by the microsomes of harbour seal was observed for both CHB-32 and -62. Stereochemical preferences of biotransformation enzymes can have an influence on the environmental distribution of both enantiomers of optically active compounds.

Coxsackievirus B3 infection induces anti-flavoprotein antibodies in mice.

Enteroviruses, the most common cause of acute myocarditis, are also supposed aetiological agents of dilated cardiomyopathy. Autoantibodies (anti-M7; Klein & Berg, Clin Exp Immunol 1990; 58:283-92) directed against flavoproteins with covalently bound flavin (alphaFp-Ab; Otto et al., Clin Exp Immunol 1998; 111:541-2) are detected in up to 30% of sera of patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM). Mice inoculated with a myocarditic variant of coxsackievirus B3 (CVB3) were employed to study the occurrence of serum alphaFp-Ab following viral infection. The presence of alphaFp-Ab was analysed by Western blotting with the flavoprotein antigens 6-hydroxy-D-nicotine oxidase (6HDNO) and sarcosine oxidase (SaO). Of 10 sera from CVB3-infected mice, five showed a strong reaction with both antigens. The sera were reactive also to the mitochondrial covalently flavinylated proteins dimethylglycine dehydrogenase and sarcosine dehydrogenase. Sera of non-infected mice did not react with these antigens. A 6HDNO mutant protein with non-covalently bound FAD no longer reacted on Western blots with sera of CVB3-infected mice. Preincubation with FAD abolished or reduced the reaction of the sera with the 6HDNO antigen. At 2 weeks p.i. the alphaFp-Ab were of the IgM and IgG isotypes, at 7 and 9 weeks p.i. of the IgG isotype. The sera of CVB3-infected mice reproduced closely the antigenic specificity of the anti-M7 sera of patients, lending further support to the role of coxsackieviruses in the pathogenesis of IDCM.

CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants.

The in vitro biotransformation of bupropion to hydroxybupropion was studied in human liver microsomes and microsomes containing heterologously expressed human cytochromes P450 (CYP). The mean (+/-S.E.) K(m) in four human liver microsomes was 89 (+/-14) microM. In microsomes containing cDNA-expressed CYPs, hydroxybupropion formation was mediated only by CYP2B6 at 50 microM bupropion (K(m) 85 microM). A CYP2B6 inhibitory antibody produced more than 95% inhibition of bupropion hydroxylation in four human livers. Bupropion hydroxylation activity at 250 microM was highly correlated with S-mephenytoin N-demethylation activity (yielding nirvanol), another CYP2B6-mediated reaction, in a panel of 32 human livers (r = 0.94). The CYP2B6 content of 12 human livers highly correlated with bupropion hydroxylation activity (r = 0.96). Thus bupropion hydroxylation is mediated almost exclusively by CYP2B6 and can serve as an index reaction reflecting activity of this isoform. IC(50) values for inhibition of a CYP2D6 index reaction (dextromethorphan O-demethylation) by bupropion and hydroxybupropion were 58 and 74 microM, respectively. This suggests a low inhibitory potency versus CYP2D6, the clinical importance of which is not established. Since bupropion is frequently coadministered with other antidepressants, IC(50) values (microM) for inhibition of bupropion hydroxylation were determined as follows: paroxetine (1.6), fluvoxamine (6.1), sertraline (3.2), desmethylsertraline (19.9), fluoxetine (59.5), norfluoxetine (4.2), and nefazodone (25.4). Bupropion hydroxylation was only weakly inhibited by venlafaxine, O-desmethylvenlafaxine, citalopram, and desmethylcitalopram. The inhibition of bupropion hydroxylation in vitro by a number of newer antidepressants suggests the potential for clinical drug interactions.

Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity.

The purpose of this study was to establish bupropion (BUP) hydroxylation as a selective in vitro marker of cytochrome P450 (CYP) 2B6 catalytic activity. Among a panel of 16 human liver microsomes (HLMs), BUP hydroxylase activity varied 80-fold when assayed at 500 microM substrate and significantly correlated with CYP2B6 blotting density (r(2) = 0.99) and S-mephenytoin N-demethylase activity (r(2) = 0.98). Kinetic analysis of BUP hydroxylation was performed in a subset of seven HLMs representative of the 80-fold range in activity. Sigmoidal kinetics suggestive of allosteric activation was observed in five HLMs exhibiting low or high activity; the mean apparent K(m) for BUP hydroxylation in these HLMs (130 microM) was similar to the K(m) for cDNA-expressed CYP2B6 (156 microM). Nonsaturable, biphasic kinetics was observed in two HLMs exhibiting low activity. Among a panel of cDNA-expressed P450 isoforms, CYP2B6 and CYP2E1 demonstrated the highest rates of BUP hydroxylation at 12 mM BUP (7.0 and 2.4 pmol/min/pmol of P450, respectively). The relative contributions of CYP2B6 and CYP2E1 to BUP hydroxylation were estimated by using immunoinhibitory monoclonal antibodies (MAB) to these enzymes. MAB-2B6 produced 88% maximum inhibition of BUP hydroxylation when assayed at 12 mM BUP in a high activity HLM, whereas MAB-2E1 produced 81% maximum inhibition in a low activity HLM. However, negligible inhibition by MAB-2E1 was observed when low and high activity HLMs were assayed at 500 microM BUP. These results demonstrate selectivity of BUP hydroxylation for CYP2B6 at 500 microM BUP, thereby validating its use as a diagnostic in vitro marker of CYP2B6 catalytic activity.

Phenytoin metabolism by human cytochrome P450: involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation.

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes.

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.

NADH-dependent reduction of sulphamethoxazole hydroxylamine in dog and human liver microsomes.

1. Reduction of hydroxylamine drug metabolites by NADH-dependent hydroxylamine reductase (NDHR) has been suggested to be involved in the pathogenesis of idiosyncratic sulphonamide toxicity in humans. The dog represents a naturally occurring clinical model for sulphonamide toxicity in humans. he purpose of these studies, therefore, was to characterize the presence of hepatic NADH-dependent hydroxylamine reductase activity in the dog and to compare this activity with that found in humans. 2. NDHR activity was characterized by the presence of two enzymes in both dog and human liver microsomes, with comparable estimates of Km (Km1 = 75 microM, Km2 = 404 microM in dog; Km1 = 69 microM, Km2 = 503 microM in human). Estimates of maximal velocity were significantly, but not dramatically, higher for dog NDHR (Vmax1 = 2.09 nmole mg(-1) min(-1) Vmax2 = 4.58 nmole mg(-1) min(-1) compared with human NDHR (Vmax1 = 0.42 nmole mg(-1) min(-1), Vmax2 = 1.56 nmole mg(-1) min(-1)). NDHR in dog, as in humans, preferred NADH to NADPH, was more active at pH 6.3 than at 7.4 and was not inhibited by carbon monoxide, azide, anaerobic conditions, the CYP substrate inhibitors tolbutamide, dextromethorphan, or erythromycin, or antibodies directed against CYP2C, CYP2D or CYP3A. 3. It is concluded that two forms of NDHR are present in dog and humans with similar biochemical characteristics. Although NDHR activity has been attributed to a CYP2D isoform in pig, there is no evidence for involvement of CYP450 in the reduction of sulphamethoxazole hydroxylamine in either dogs or humans.

Ethynylestradiol-mediated induction of hepatic CYP3A9 in female rats: implication for cyclosporine metabolism.

Repeated treatment of female rats with the synthetic estrogen ethynylestradiol (EE(2)) increases the formation of the cyclosporine A (CyA) metabolites AM1c and AM9 by 3-fold, whereas the formation of AM1 and AM4N is not significantly enhanced. The formation of all four CyA metabolites was inhibited by greater than 80% by the CYP3A-selective substrate midazolam or polyclonal anti-rat CYP3A IgGs in liver microsomes of untreated and EE(2)-induced rats. In contrast, anti-rat CYP2C6 IgGs had little effect, indicating the involvement of a CYP3A but not 2C6 in this EE(2)-stimulated CyA metabolism. Semiquantitative reverse-transcriptase polymerase chain reaction was used to determine the mRNA content for four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in livers of control and EE(2)-treated female rats. EE(2) selectively induced CYP3A9 by 3.3-fold whereas the expression of CYP3A18 and CYP3A23 was slightly decreased; neither CYP3A2 mRNA nor CYP3A1 mRNA was detectable in these EE(2)-treated livers. To determine whether rat liver microsomal CYP3A9 was indeed responsible for the EE(2)-stimulated CyA metabolism, a recombinant CYP3A9 was heterologously expressed in Escherichia coli. When functionally reconstituted, this enzyme was active in metabolizing CyA preferentially to its AM9 and AM1c metabolites as compared with CYP3A4. These findings thus support the notion that the increased CyA-metabolizing capacity of EE(2)-treated female rat liver microsomes is due to the induction of the CYP3A9 enzyme.