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1. 1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined. 2. CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation. 3. Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower k value than other CYP2A13.1 variants. 4. CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene. 5. Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2 A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.
Naphthalene, phenanthrene, biphenyl, and their derivatives having different ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding spectra more readily than with P450 2A6. Among the various substituted derivatives examined, 2-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl propargyl ether had larger ΔAmax/Ks values in inducing Type I binding spectra with P450 2A13 than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or 4-hydroxybiphenyl, respectively, at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra.
1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and its derivatives in the active site of P450 2A13, with lower interaction energies (U values) than observed for P450 2A6 and that several amino acid residues (including Ala-301, Asn-297 and Ala-117) play important roles in directing the orientation of these PAHs in the P450 2A13 active site. In addition, Phe-231 and Gly-329 were found to interact with pyrene to orient this compound in the active site of P450 1B1. 5. These results suggest that P450 2A13 is one of the important enzymes that oxidizes these PAH compounds and may determine how these chemicals are detoxicated and bioactivated in humans.
In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼ 44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
PURPOSE - The purpose of this study was to investigate the role(s) of cytochrome P450 epoxygenases (CYPs) and their products, the epoxyeicosatrienoic acids (EETs), in hypoxia-induced VEGF production and pathologic retinal angiogenesis.
METHODS - Human retinal astrocytes, Müller cells, and retinal microvascular endothelial cells (HRMEC) were exposed to hypoxia, and relative CYP2C expression was measured by RT-PCR. Astrocyte and Müller cell VEGF production was measured by ELISA after exposure to hypoxia and treatment with the general CYP inhibitor, SKF-525a. Human retinal microvascular endothelial cells were treated with the CYP product, 11,12-epoxyeicosatrienoic acid [EET], or SKF-525a in the presence or absence of VEGF. Proliferation of HRMEC and tube formation were assayed. Oxygen-induced retinopathy (OIR) was induced in newborn rats. Retinal CYP2C11 and CYP2C23 expression were measured by RT-PCR. The OIR rats received SKF-525a by intravitreal injection and preretinal neovascularization (NV) was quantified. Retinal VEGF protein levels were measured by ELISA.
RESULTS - Human retinal astrocytes were the only cells to exhibit significant induction of CYP2C8 and CYP2C9 mRNA expression by hypoxia. Astrocytes, but not Müller cells, exhibited reduced hypoxia-induced VEGF production when treated with SKF-525a. 11,12-EET induced HRMEC proliferation and tube formation, and SKF-525a inhibited VEGF-induced proliferation. Oxygen-induced retinopathy induced expression of CYP2C23, but had no effect on CYP2C11. SKF-525a inhibited retinal NV and reduced retinal VEGF levels in OIR rats.
CONCLUSIONS - The CYP-derived 11,12-EET may exhibit a proangiogenic biological function in the retina following stimulation by hypoxia in astrocytes. Inhibition of CYP may provide a rational therapy against retinal NV, because it can reduce VEGF production and VEGF-induced angiogenic responses in endothelial cells.
Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
The influence of genetic variation on warfarin dose requirement is limited for paediatric patients. We performed a retrospective, cross-sectional study to examine the effect of variant CYP2C9 and VKORC1 genotypes on warfarin dose in 100 children. Those with VKORC1 genotype AA required 48% of the dose of homozygous wild-type (GG, P < 0·0001). Patients with any variant CYP2C9 allele required 71% of the dose for wild-type (P = 0·001). The effect of variant VKORC1 alleles tended to vary with age, suggesting developmental ontogeny may influence warfarin sensitivity. Age, CYP2C9 genotype, VKORC1 genotype and age:VKORC1 interaction accounted for 53% of warfarin dose variability.
© 2014 John Wiley & Sons Ltd.
We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41-48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.
Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated by H. Yamazaki et al. ( ( 2013 ) Chem. Res. Toxicol. 26 , 486 - 489 ) using the model substrate midazolam in various in vitro and in vivo models. Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pretreatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting the activation of upstream transcription factors involved in detoxication, e.g., the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of coregulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 coregulator-derived nuclear receptor-interaction motifs and coregulator and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5-hydroxythalidomide displayed significant modulation of coregulator interaction with PXR and CAR ligand-binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation.
In the US alone, around 60,000 lives/year are lost due to colon cancer. Diet and environment have been implicated in the development of sporadic colon tumors. The objective of this study was to determine how dietary fat potentiates the development of colon tumors through altered B(a)P biotransformation, using the Adenomatous polyposis coli with Multiple intestinal neoplasia mouse model. Benzo(a)pyrene was administered to mice through tricaprylin, and unsaturated (USF; peanut oil) and saturated (SF; coconut oil) fats at doses of 50 and 100 μg/kg via oral gavage over a 60-day period. Blood, colon, and liver were collected at the end of exposure period. The expression of B(a)P biotransformation enzymes [cytochrome P450 (CYP)1A1, CYP1B1 and glutathione-S-transferase] in liver and colon were assayed at the level of protein, mRNA and activities. Plasma and tissue samples were analyzed by reverse phase high-performance liquid chromatography for B(a)P metabolites. Additionally, DNA isolated from colon and liver tissues was analyzed for B(a)P-induced DNA adducts by the (32)P-postlabeling method using a thin-layer chromatography system. Benzo(a)pyrene exposure through dietary fat altered its metabolic fate in a dose-dependent manner, with 100 μg/kg dose group registering an elevated expression of B(a)P biotransformation enzymes, and greater concentration of B(a)P metabolites, compared to the 50 μg/kg dose group (P<.05). This effect was more pronounced for SF group compared to USF group (P<.05). These findings establish that SF causes sustained induction of B(a)P biotransformation enzymes and extensive metabolism of this toxicant. As a consequence, B(a)P metabolites were generated to a greater extent in colon and liver, whose concentrations also registered a dose-dependent increase. These metabolites were found to bind with DNA and form B(a)P-DNA adducts, which may have contributed to colon tumors in a subchronic exposure regimen.
OBJECTIVES - An efavirenz-based antiretroviral therapy (ART) regimen is preferred for children more than 3 years of age with tuberculosis. However, rifampin, a key component of antituberculosis therapy, induces CYP2B6. An increased dose of efavirenz is recommended in adults weighing more than 50 kg who require rifampin, but there is scant information in children being treated for tuberculosis.
DESIGN - Plasma efavirenz concentrations were compared in 40 children during concomitant treatment for tuberculosis and HIV-1, after stopping rifampicin, and in a control group of children without tuberculosis. Associations with antituberculosis treatment, metabolizer genotype (based on CYP2B6 516G→T, 983T→C, and 15582C→T), weight, and time after dose were evaluated.
RESULTS - Compared to children with extensive metabolizer genotypes, efavirenz concentrations were increased 1.42-fold (95% confidence interval, CI 0.94–2.15) and 2.85-fold (95% CI 1.80–4.52) in children with intermediate and slow metabolizer genotypes, respectively. Concomitant antituberculosis treatment increased efavirenz concentrations 1.49-fold (95% CI 1.10–2.01) in children with slow metabolizer genotypes, but did not affect efavirenz concentrations in extensive or intermediate metabolizer genotypes. After adjustment for dose/kg, each kilogram of weight was associated with a 2.8% (95% CI 0.9–4.7) decrease in efavirenz concentrations. Despite higher milligram per kilogram doses, a higher proportion of children in the lowest weight band (10–13.9 kg) had efavirenz concentrations less than 1.0 mg/l than larger children.
CONCLUSION - Antituberculosis treatment was not associated with reduced efavirenz concentrations in children, which does not support increased efavirenz doses. Children with slow metabolizer genotype have increased efavirenz concentrations during antituberculosis treatment, likely due to isoniazid inhibiting enzymes involved in accessory metabolic pathways for efavirenz.