Mouse CYP2A5 Research Articles

Identification of 5-hydroxymethylfurfural in cigarette smoke extract as a new substrate metabolically activated by human cytochrome P450 2A13

Cytochrome P450 2A13 (CYP2A13) is an extrahepatic enzyme mainly expressed in the human respiratory system and is reported to mediate tobacco-specific N-nitrosamines (TSNA) metabolism in cigarette smoke. This study aimed to identify other new substrates of CYP2A13 in cigarette smoke and their corresponding respiratory toxicity. Following separation by HPLC, GC–MS/MS, NMR and cytotoxicity assays in BEAS-2B cells stably expressing CYP2A13 (B-2A13), 5-Hydroxymethylfurfural (5-HMF) was screened and identified in the 4–5 min section of cigarette smoke extract (CSE). In vitro metabolism results showed that CYP2A13 mediated the fast clearance of 5-HMF and formed the metabolite 5-HMF acid (5-HMFA). CSE 5-HMF (CSE-5-HMF) showed cytotoxicity similar to that of standard 5-HMF in B-2A13 and B-2A5 cells, which was inhibited by 8-methoxypsoralen (8-MOP), a CYP enzyme inhibitor. Mouse CYP2A5, a homologous CYP enzyme to CYP2A13, shares many substrates with CYP2A13 in cigarette smoke. Thus, CYP2A5−/− mice were generated to explore the role of CYP2A5 in 5-HMF bioactivation. Compared with CYP2A5−/− mice, WT mice showed serious histological lung and nasal olfactory mucosa damage, as well as increased inflammatory cells and elevated TNF-α and IL-6 levels in bronchoalveolar lavage fluid. Besides, nasal microsomes undertook fast 5-HMFA formation in WT mice than that in CYP2A5−/− mice, which could be inhibited by 8-MOP. This study is the first to identify 5-HMF as a new toxic substrate of human CYP2A13 in cigarette smoke, it may play a potential role in cigarette smoke-induced respiratory injuries.

Effect of miR-433-3p and miR-883b-5p on murine CYP 3A family enzymes in AML12 cells.

Here we searched for microRNAs that could interact with cytochrome P450 (CYP) enzymes in silico, and then investigated their effects on Cyp gene expressions using the cultured mouse liver cell line AML12. Among the mouse Cyp3a genes, some miRNAs were found to interact with Cyp3a11, 13, 16, and 44 by the in silico analysis using the miRWalk2.0 database. In addition to this software, which included twelve miRNA target prediction algorithms, we also applied our in-house-developed Excel VBA algorithm to obtain predictions more efficiently. Finally, two miRNAs, miR-433-3p and miR-883b-5p, were extracted as candidates that interact with Cyp3a genes. To evaluate the effects of these miRNAs on Cyp3a gene expression, we first examined whether they actually interacted with the Cyp3a 3'-untranslated region (3'-UTR) using a luciferase assay system in AML12 cells. We then evaluated whether the expression of each miRNA affected the expression of Cyp3a mRNAs and their transcribed proteins. We found that the transiently expressed miRNAs significantly reduced the reporter activity of the Cyp3a 3'-UTR site in AML12 cells. In addition, the mRNA and protein expressions of the corresponding Cyp3as were significantly decreased in the miRNA-treated AML12 cells. Using cultured cells, we clearly demonstrated that miR-433-3p and miR-883b-5p, which were identified by in silico prediction, actually bind to Cyp3a mRNAs and regulate Cyp gene expressions.

Comprehensive analysis of the mouse cytochrome P450 family responsible for omega-3 epoxidation of eicosapentaenoic acid

Metabolites generated via oxygenation of the omega-3 double bond (omega-3 oxygenation) in eicosapentaenoic acid (EPA) have recently been identified as novel anti-inflammatory lipid mediators. Therefore, oxygenase(s) responsible for this metabolic pathway are of particular interest. We performed genome-wide screening of mouse cytochrome P450 (CYP) isoforms to explore enzymes involved in omega-3 oxygenation of EPA. As a result, 5 CYP isoforms (mouse Cyp1a2, 2c50, 4a12a, 4a12b, and 4f18) were selected and identified to confer omega-3 epoxidation of EPA to yield 17,18-epoxyeicosatetraenoic acid (17,18-EpETE). Stereoselective production of 17,18-EpETE by each CYP isoform was confirmed, and molecular modeling indicated that chiral differences stem from different EPA binding conformations in the catalytic domains of respective CYP enzymes.

Detection of dioxin-induced demethylation of mouse Cyp1a1 gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end

Environmental factors stimulate alteration of DNA methylation level. Investigation of the genome-wide DNA methylation status is important for environmental health studies. We here designed a genomic DNA amplification and labeling protocol using a methylation-sensitive restriction enzyme HinP1 I. This method can specifically amplify genomic DNA fragments possessing methyl-CpG at the end. The fragments are a relatively short size and dominantly located on CpG-islands. By using the samples prepared by this method, a dioxin-induced change in the methylation level of the mouse Cyp1a1 promoter was successfully evaluated using oligonucleotide probes covalently bound onto a glass plate. The method developed in this paper would be useful for other genome-wide analysis platforms for the large scale epigenome-wide association studies (EWAS) including human epidemiological samples.

T0901317-induced hepatic steatosis dramatically decreases CYP3A and CYP2C activities in mice

T0901317-induced hepatic steatosis dramatically decreases CYP3A and CYP2C activities in mice

Comparison of In Vitro Hepatic Scoparone 7-O-Demethylation between Humans and Experimental Animals.

Scoparone is a natural bioactive compound in Chinese herbal medicines. It has numerous pharmacological actions, including liver protective, hypolipidemic, antitumor, and anti-inflammatory effects. The primary metabolism route of scoparone is O-demethylation to scopoletin or isoscopoletin catalyzed by CYP enzymes. The aims of our study were to identify the human CYP enzymes catalyzing scoparone 7-O-demethylation to scopoletin and to compare this oxidation reaction in liver microsomes among different species. A high throughput fluorescent-based assay method was developed to determine the scoparone 7-O-demethylation to scopoletin rate. The rate was 100 - 400 nmol/(min×g protein) in mouse and rabbit liver microsomes, 10 - 20 nmol/(min×g protein) in pig microsomes, 1 - 3 nmol/(min×g protein) in human and less than 1 nmol/(min×g protein) in rat liver microsomes. Human CYP1A1 (Km13 µM and Vmax0.8 min-1), CYP1A2 (Km48 µM and Vmax0.3 min-1), and CYP2A13 (Km10 µM and Vmax22 min-1) were the most efficient catalysts of the reaction. The CYP2A6 selective inhibitor pilocarpine and an antibody against mouse CYP2A5 inhibited scoparone 7-O-demethylation to scopoletin in rabbit, mouse, and pig liver microsomes, indicating involvement of CYP2A enzymes in the reaction. Hepatic scoparone 7-O-demethylation to scopoletin differed between species both with respect to the rate of reaction and catalyzing enzymes. These species differences need to be taken into account when testing scoparone pharmacokinetics in animals and humans.

High-Fat Diet Feeding Alters Expression of Hepatic Drug-Metabolizing Enzymes in Mice.

Medical conditions accompanying obesity often require drug therapy, but whether and how obesity alters the expression of drug-metabolizing enzymes and thus drug pharmacokinetics is poorly defined. Previous studies have shown that high-fat diet (HFD) feeding and subsequent obesity in mice lead to altered expression of transcriptional regulators for cytochrome P450 CYP2D6, including hepatocyte nuclear factor 4α (HNF4α, a transcriptional activator of CYP2D6) and small heterodimer partner (SHP, a transcriptional repressor of CYP2D6). The objective of this study was to examine whether diet-induced obesity alters CYP2D6 expression by modulating HNF4α and SHP expression. Male CYP2D6-humanized transgenic (Tg-CYP2D6) mice were fed with HFD or matching control diet for 18 weeks. Hepatic mRNA expression of CYP2D6 decreased to a small extent in the HFD group (by 31%), but the differences in CYP2D6 protein and activity levels in hepatic S9 fractions were found insignificant between the groups. Although hepatic SHP expression did not differ between the groups, HNF4α mRNA and protein levels decreased by ∼30% in the HFD group. Among major mouse endogenous cytochrome P450 genes, Cyp1a2 and Cyp2c37 showed significant decreases in the HFD group, whereas Cyp2e1 expression did not differ between groups. Cyp2b10 and Cyp3a11 expression was higher in the HFD group, with corresponding 2.9-fold increases in hepatic CYP3A activities in HFD-fed mice. Together, these results suggest that obesity has minimal effects on CYP2D6-mediated drug metabolism, although it modulates the expression of mouse endogenous P450s in a gene-specific manner.

Genomic Landscape of Experimental Bladder Cancer in Rodents and Its Application to Human Bladder Cancer: Gene Amplification and Potential Overexpression of Cyp2a5/CYP2A6 Are Associated with the Invasive Phenotype.

Non-muscle invasive (superficial) bladder cancer is a low-grade malignancy with good prognosis, while muscle invasive (invasive) bladder cancer is a high-grade malignancy with poor prognosis. N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induces superficial bladder cancers with papillary morphology in rats and invasive bladder cancers with infiltrating phenotype in mice. In this study, we analyzed genomic landscapes of rodent BBN-induced bladder cancers using array-based comparative genomic hybridization (array CGH). While no significant copy number alterations were detected in superficial bladder tumors in rats, copy number gains in chromosomal regions 2D-E1, 7qA3, 9F2, and 11C-D were detected in invasive bladder tumors in mice. Amplification of representative genes located on 2D-E1 and 7qA3 chromosomal regions was confirmed by quantitative PCR. Cyp2a22 and Cyp2a5 genes but not Cyp2g1, Cyp2a12, and Rab4b genes on mouse chromosome 7qA3 were amplified in invasive bladder cancers. Although the human ortholog gene of Cyp2a22 has not been confirmed, the mouse Cyp2a5 gene is the ortholog of the human CYP2A6 gene located in chromosomal region 19q13.2, and CYP2A6 was identified by database search as one of the closest human homolog to mouse Cyp2a22. Considering a possibility that this region may be related to mouse 7qA3, we analyzed CYP2A6 copy number and expression in human bladder cancer using cell lines and resected tumor specimens. Although only one of eight cell lines showed more than one copy increase of the CYP2A6 gene, CYP2A6 amplification was detected in six out of 18 primary bladder tumors where it was associated with the invasive phenotype. Immunohistochemical analyses of 118 primary bladder tumors revealed that CYP2A6 protein expression was also higher in invasive tumors, especially in those of the scattered type. Together, these findings indicate that the amplification and overexpression of the CYP2A6 gene are characteristic of human bladder cancers with increased malignancy and that CYP2A6 can be a candidate prognostic biomarker in this type of cancer.

Celery extract inhibits mouse CYP2A5 and human CYP2A6 activities via different mechanisms.

Human cytochrome P450 (CYP) 2A6 participates in the metabolism of nicotine and precarcinogens, thus the deliberate inhibition of CYP2A6 may reduce cigarette consumption and therefore reduce the risk of developing the types of cancer associated with smoking. The inhibitory effects and mechanisms of celery (Apium graveolens) extract on mouse CYP2A5 and human CYP2A6 activity remain unclear. These effects were investigated in mouse and human liver microsomes using coumarin 7-hydroxylation in a probe reaction. Celery extract reduced CYP2A5 and CYP2A6 activities in vitro in a dose-dependent manner. In vivo experiments also showed that celery extract markedly decreased CYP2A5 activity. The inhibition of celery extract on CYP2A5 was time- and nicotinamide adenine dinucleotide phosphate (NADPH)-independent, and was markedly reduced by ultracentrifugation. Additionally, the inhibition of celery extract on CYP2A6 was time and NADPH-dependent. Levels of inhibition were characterized by a Ki, the measure of the tightness of bonds between the enzyme and its inhibitor, of 266.4 µg/ml for CYP2A5, and a Ki of 1,018 µg/ml and Kinact of 0.3/min for CYP2A6. Kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor. The coumarin derivative 5-methoxypsoralen present in celery extract did not solely to the inhibition of CYP2A5/6 activity. In conclusion, celery extract inhibited the levels of mouse CYP2A5 and human CYP2A6 activity via different mechanisms: Mixed competitive inhibition for CYP2A5 and mechanism-based inhibition for CYP2A6.

Ketoconazole Stereoisomers Differentially Induce Cytochrome P450 1A1 Between Human Hepatoma HepG2 and Mouse Hepatoma Hepa1c1c7 Cells

Ketoconazole (KTZ) has 2 chiral centers with the therapeutically active form being a racemic mixture of 2 cis-enantiomers, namely, (2R,4S)-(+)-KTZ and (2S,4R)-(−)-KTZ. The aims of the present study were to examine the effects of (+)-KTZ, (−)-KTZ, and (±)-KTZ on aryl hydrocarbon receptor activation and subsequently CYP1A1 induction in both human HepG2 and murine Hepa1c1c7 hepatoma cells, and to further test their inhibitory effect using recombinant human and mouse CYP1A1 enzymes. Our results demonstrated that (+)-KTZ induced human CYP1A1 more than (−)-KTZ, whereas on the other hand (−)-KTZ induced murine Cyp1a1 more than (+)-KTZ at the mRNA, and activity levels. Human CYP1A1 showed higher affinity to 7ER compared with murine Cyp1a1 (Km values 13.29 nM for human vs. 168.1 nM for murine). The intrinsic clearance values for human and murine CYP1A1 were 194.1 and 87.6 μL/pmol P450/min, respectively, whereas, Vmax values were 2.58 and 14.73 pmol/pmol P450/min, respectively. (+)-KTZ and (−)-KTZ directly inhibited CYP1A1 activity by noncompetitive mechanism. The affinity of (−)-KTZ to interact with human CYP1A1 and murine Cyp1a1 was significantly different from (+)-KTZ, as the Ki values for human CYP1A1 and murine Cyp1a1 were 199.4 and 413.7 nM, respectively, for (+)-KTZ, and 269.3 and 230.8 nM, respectively, for (−)-KTZ.

Thalidomide-induced limb abnormalities in a humanized CYP3A mouse model.

Thalidomide is a teratogen in humans but not in rodents. It causes multiple birth defects including malformations of limbs, ears, and other organs. However, the species-specific mechanism of thalidomide teratogenicity is not completely understood. Reproduction of the human teratogenicity of thalidomide in rodents has previously failed because of the lack of a model reflecting human drug metabolism. In addition, because the maternal metabolic effect cannot be eliminated, the migration of unchanged thalidomide to embryos is suppressed, and the metabolic activation is insufficient to develop teratogenicity. Previously, we generated transchromosomic mice containing a human cytochrome P450 (CYP) 3A cluster in which the endogenous mouse Cyp3a genes were deleted. Here, we determined whether human CYP3A or mouse Cyp3a enzyme expression was related to the species difference in a whole embryo culture system using humanized CYP3A mouse embryos. Thalidomide-treated embryos with the human CYP3A gene cluster showed limb abnormalities, and human CYP3A was expressed in the placenta, suggesting that human CYP3A in the placenta may contribute to the teratogenicity of thalidomide. These data suggest that the humanized CYP3A mouse is a useful model to predict embryonic toxicity in humans.

Gene Silencing and Haploinsufficiency of Csk Increase Blood Pressure.

ObjectiveRecent genome-wide association studies have identified 33 human genetic loci that influence blood pressure. The 15q24 locus is one such locus that has been confirmed in Asians and Europeans. There are 21 genes in the locus within a 1-Mb boundary, but a functional link of these genes to blood pressure has not been reported. We aimed to identify a causative gene for blood pressure change in the 15q24 locus.Methods and ResultsCSK and ULK3 were selected as candidate genes based on eQTL analysis studies that showed the association between gene transcript levels and the lead SNP (rs1378942). Injection of siRNAs for mouse homologs Csk, Ulk3, and Cyp1a2 (negative control) showed reduced target gene mRNA levels in vivo. However, Csk siRNA only increased blood pressure while Ulk3 and Cyp1a2 siRNA did not change it. Further, blood pressure in Csk+/- heterozygotes was higher than in wild-type, consistent with what we observed in Csk siRNA-injected mice. We confirmed that haploinsufficiency of Csk increased the active form of Src in Csk+/- mice aorta. We also showed that inhibition of Src by PP2, a Src inhibitor decreased high blood pressure in Csk+/- mice and the active Src in Csk+/- mice aorta and in Csk knock-down vascular smooth muscle cells, suggesting blood pressure regulation by Csk through Src.ConclusionsOur study demonstrates that Csk is a causative gene in the 15q24 locus and regulates blood pressure through Src, and these findings provide a novel therapeutic target for the treatment of hypertension.

Inhibitory effects and oxidation of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin via human CYP2A6 and its mouse and pig orthologous enzymes

1. Information about the metabolism of compounds is essential in drug discovery and development, risk assessment of chemicals and further development of predictive methods.2. In vitro and in silico methods were applied to evaluate the metabolic and inhibitory properties of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin with human CYP2A6, mouse CYP2A5 and pig CYP2A19.3. 6-Methylcoumarin was oxidized to fluorescent 7-hydroxy-6-methylcoumarin by CYP2A6 (Km: 0.64–0.91 µM; Vmax: 0.81–0.89 min−1) and by CYP2A5 and CYP2A19. The reaction was almost completely inhibited at 10 µM 7-methylcoumarin in liver microsomes of human and mouse, but in pig only 40% inhibition was obtained with the anti-CYP2A5 antibody or with methoxsalen and pilocarpine. 7-Methylcoumarin was a mechanism-based inhibitor for CYP2A6, but not for the mouse and pig enzymes. 7-Formylcoumarin was a mechanism-based inhibitor for CYP2As of all species.4. Docking and molecular dynamics simulations of 6-methylcoumarin and 7-methylcoumarin in the active sites of CYP2A6 and CYP2A5 demonstrated a favorable orientation of the 7-position of 6-methylcoumarin towards the heme moiety. Several orientations of 7-methylcoumarin were possible in CYP2A6 and CYP2A5.5. These results indicate that the active site of CYP2A6 has unique interaction properties for ligands and differs in this respect from CYP2A5 and CYP2A19.

Relative Contributions of CYP1A2 and CYP2E1 to the Bioactivation and Clearance of 4-Aminobiphenyl in Adult Mice.

4-Aminobiphenyl (ABP), a prototypical aromatic amine carcinogen in rodents and humans, requires bioactivation to manifest its toxic effects. A traditional model of ABP bioactivation, based on in vitro enzyme kinetic evidence, had postulated initial N-hydroxylation by the cytochrome P450 isoform CYP1A2. This is followed by phase 2 O-conjugation and hydrolysis to form a reactive nitrenium ion that covalently binds to DNA and produces tumor-initiating mutations. However, Cyp1a2(-/-) mice still possess significant liver ABP N-hydroxylation activity, DNA damage, and incidence of ABP-induced liver tumors, and in vivo induction of CYP1A2 paradoxically reduces levels of ABP-induced DNA damage. Competing ABP detoxification pathways can include N-acetylation by arylamine N-acetyltransferase 1 (NAT1) and/or NAT2; however, wild-type and Nat1/2(-/-) mice have similar in vivo ABP clearance rates. Together, these studies suggest the existence of novel ABP bioactivating and clearance/detoxification enzymes. In the present study, we detected similar reductions in Vmax for ABP N-hydroxylation by liver microsomes from Cyp1a2(-/-) and Cyp2e1(-/-) mice when compared with wild-type mice. In addition, recombinant mouse CYP1A2 and CYP2E1 were both able to N-hydroxylate ABP in mouse hepatoma cells. However, the in vivo clearance of ABP was significantly reduced in Cyp1a2(-/-) but not in Cyp2e1(-/-) mice. Our results support a significant role for CYP2E1 as a novel ABP N-oxidizing enzyme in adult mice, and suggest a more important contribution of CYP1A2 to the in vivo plasma clearance and thus detoxification of ABP.

MP36-04 GENE AMPLIFICATION AND OVEREXPRESSION OF CYP2A6 IN EARLY STAGE OF INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-04 GENE AMPLIFICATION AND OVEREXPRESSION OF CYP2A6 IN EARLY STAGE OF INVASIVE BLADDER CANCER Kazuhiro Kanemoto, Katsuhiro Fukuta, Noriyasu Kawai, Keiichi Tozawa, Masako Ochiai, Koji Okamoto, Hiromi Sakamoto, Teruhiko Yoshida, Yae Kanai, Masaru Katoh, Hitoshi Nakagama, and Kenjiro Kohri Kazuhiro KanemotoKazuhiro Kanemoto More articles by this author , Katsuhiro FukutaKatsuhiro Fukuta More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Keiichi TozawaKeiichi Tozawa More articles by this author , Masako OchiaiMasako Ochiai More articles by this author , Koji OkamotoKoji Okamoto More articles by this author , Hiromi SakamotoHiromi Sakamoto More articles by this author , Teruhiko YoshidaTeruhiko Yoshida More articles by this author , Yae KanaiYae Kanai More articles by this author , Masaru KatohMasaru Katoh More articles by this author , Hitoshi NakagamaHitoshi Nakagama More articles by this author , and Kenjiro KohriKenjiro Kohri More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.732AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In bladder cancers, invasive recurrence after transurethral resection (TUR) based curative treatment of superficial cancer is serious problem in the clinic, and biomarkers predicting invasive recurrence are necessary for appropriate clinical care of bladder cancer patients. Genomic alterations at the chromosomal level are rare events in superficial papillary cancers, but are frequently observed in invasive cancers. By analyzing the genomic landscapes of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced rodent bladder cancers, we observed genomic copy number aberrations (CNAs) and upregulation of the Cyp2a5 gene in early invasive cancers in mice. In order to identify genetic markers linked to early invasive carcinogenesis in humans, focusing on the genomic instability of invasive cancers, we searched CNAs in human invasive cancers and demonstrated that the distinction of invasiveness leads to different expression patterns regarding the immunohistochemical staining intensity of CYP2A6 (the human ortholog of mouse Cyp2a5). METHODS Immunohistochemical staining was performed on TUR tissues (59 invasive and 45 superficial papillary cancers). In addition, statistical differences in immunostaining were analyzed in 240 invasive lesions, 53 superficial papillary cancers, and 27 carcinoma in situ (CIS). Furthermore, we investigated the correlations between CNAs on genomic quantitative PCR and differential expression on immunostaining in TUR tissues of 9 invasive and 9 superficial papillary cancers. RESULTS In superficial papillary cancers, dilute cells were homogeneously immunostained. In contrast, cells with a high expression of CYP2A6 in invasive lesions were increased in aggressive cancers, represented by a scattered lesion pattern that differed from aggregated lesions (P < 0.01). Moreover, CIS had the highest expression score. The average CNA was +1.2 in invasive cancers versus +0.2 in superficial papillary cancers (P = 0.0085). Only invasive cancers showed a positive correlation between copy number gains and increased expression of CYP2A6 (r = 0.59, P = 0.048). CONCLUSIONS Application of genomic landscapes of BBN-induced bladder cancer in rodents to human bladder cancer indicate that gene amplification and overexpression of CYP2A6 are characteristic features in human bladder cancers with a more malignant phenotype. CYP2A6 may be clinically useful as a diagnostic biomarker for the prognosis, therapeutic optimization, and early detection of invasive bladder cancer patients. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e429 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kazuhiro Kanemoto More articles by this author Katsuhiro Fukuta More articles by this author Noriyasu Kawai More articles by this author Keiichi Tozawa More articles by this author Masako Ochiai More articles by this author Koji Okamoto More articles by this author Hiromi Sakamoto More articles by this author Teruhiko Yoshida More articles by this author Yae Kanai More articles by this author Masaru Katoh More articles by this author Hitoshi Nakagama More articles by this author Kenjiro Kohri More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of methoxsalen, a CYP2A5/6 inhibitor, on nicotine dependence behaviors in mice

Metabolism of nicotine to inactive cotinine by hepatic enzyme CYP2A6 is the principal pathway by which active nicotine is removed from circulation. We therefore hypothesized that inhibition of mouse CYP2A5, the ortolog of human CYP2A6, by methoxsalen (8-methoxypsoralen) alter dependence-related behaviors of nicotine in the mouse. Conditioned place preference (CPP) test was used to assess the appetitive reward-like properties and precipitated nicotine withdrawal to assess physical (somatic and hyperalgesia) and affective (anxiety-related behaviors) measures. The nicotine plasma levels were also measured with or without methoxsalen pretreatment. Methoxsalen (15 and 30 mg/kg, intraperitoneally) pretreatment enhanced nicotine-induced preference in mice (p < 0.05). However, there was a lack of enhancement of nicotine in the CPP test after the highest dose of the CYP-2A5 inhibitor. Similarly to the CPP results, repeated administration of methoxsalen increased the intensity of mecamylamine-precipitated withdrawal signs. The potentiation of nicotine preference and withdrawal intensity by methoxsalen was accompanied by significant increase in nicotine plasma levels in mice (p < 0.05). Finally, methoxsalen enhanced the ability of a very low dose of nicotine (0.05 mg/kg) to reverse withdrawal signs in mice undergoing spontaneous withdrawal after chronic nicotine infusion (p < 0.05). In conclusion, inhibition of nicotine metabolism by methoxsalen alters the behavioral effects of nicotine in the mouse. Combining CYP2A6 inhibitors with low dose nicotine replacement therapies may have a beneficial role in smoking cessation because it will decrease the drug elimination rate and maintain plasma and brain nicotine levels.

Cytochrome P450 mRNA Expression in the Rodent Brain: Species-, Sex-, and Region-Dependent Differences

Cytochrome P450 (P450) enzymes play a critical role in the activation and detoxication of many neurotoxic chemicals. Although research has largely focused on P450-mediated metabolism in the liver, emerging evidence suggests that brain P450s influence neurotoxicity by modulating local metabolite levels. As a first step toward better understanding the relative role of brain P450s in determining neurotoxic outcome, we characterized mRNA expression of specific P450 isoforms in the rodent brain. Adult mice (male and female) and rats (male) were treated with vehicle, phenobarbital, or dexamethasone. Transcripts for CYP2B, CYP3A, CYP1A2, and the orphan CYP4X1 and CYP2S1 were quantified in the liver, hippocampus, cortex, and cerebellum by quantitative (real-time) polymerase chain reaction. These P450s were all detected in the liver with the exception of CYP4X1, which was detected in rat but not mouse liver. P450 expression profiles in the brain varied regionally. With the exception of the hippocampus, there were no sex differences in regional brain P450 expression profiles in mice; however, there were marked species differences. In the liver, phenobarbital induced CYP2B expression in both species. Dexamethasone induced hepatic CYP2B and CYP3A in mice but not rats. In contrast, brain P450s did not respond to these classic hepatic P450 inducers. Our findings demonstrate that P450 mRNA expression in the brain varies by region, regional brain P450 profiles vary between species, and their induction varies from that of hepatic P450s. These novel data will be useful for designing mechanistic studies to examine the relative role of P450-mediated brain metabolism in neurotoxicity.

Essential Role of the Cytochrome P450 Enzyme CYP2A5 in Olfactory Mucosal Toxicity of Naphthalene

Naphthalene (NA), a ubiquitous environmental pollutant that can cause pulmonary and nasal toxicity in laboratory animals, requires cytochrome P450 (P450)-mediated metabolic activation to cause toxicity. Our recent study using a Cyp2f2-null mouse showed that CYP2F2 plays an essential role in NA-induced lung toxicity, but not in NA-induced nasal toxicity. The aim of this study was to determine whether mouse CYP2A5, abundantly expressed in nasal olfactory mucosa (OM) and the liver, but less in the lung, plays a major role in the bioactivation and toxicity of NA in the OM. We found, by comparing Cyp2a5-null and wild-type (WT) mice, that the loss of CYP2A5 expression led to substantial decreases in rates of NA metabolic activation by OM microsomes. The loss of CYP2A5 did not cause changes in systemic clearance of NA (at 200 mg/kg, i.p.). However, the Cyp2a5-null mice were much more resistant than were WT mice to NA-induced nasal toxicity (although not lung toxicity), when examined at 24 hours after NA dosing (at 200 mg/kg, i.p.), or to NA-induced depletion of total nonprotein sulfhydryl in the OM (although not in the lung), examined at 2 hours after dosing. Thus, mouse CYP2A5 plays an essential role in the bioactivation and toxicity of NA in the OM, but not in the lung. Our findings further illustrate the tissue-specific nature of the role of individual P450 enzymes in xenobiotic toxicity, and provide the basis for a more reliable assessment of the potential risks of NA nasal toxicity in humans.

Gender-dependent activity of CYP3A is indirectly modified by GR in the noradrenergic system

BackgroundThe noradrenergic system is involved in the regulation of cytochrome P450 activity in the liver.We investigated the effect of selective ablation of the glucocorticoid receptor in the noradrenergic systemon the activity of theCYP3Aisoforminmouse liver. MethodsThe activity of CYP3A was studied by measuring the rate of testosterone 6β-hydroxylation in liver microsomes. ResultsIn mutant mice, the activity of CYP3A was reduced to 68% of the control in females, but remained unchanged in males. Chronic restraint stress increased CYP3A activity in mutant mice only. ConclusionsThe total basal activity of mouse CYP3A may be indirectly modulated by the glucocorticoid receptor in the noradrenergic system during a pubertal period.

Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver

Here, we studied the effects of cytochrome P450 (CYP)3A deficiency on the mRNA expression of genes encoding regulators of hepatic cholesterol levels using Cyp3a-knockout (Cyp3a(-/-)) mice. The mRNA expression levels of genes encoding enzymes involved in cholesterol biosynthesis in the livers of Cyp3a(-/-) mice were higher than those of wild-type (WT) mice. Nuclear levels of sterol regulatory element-binding protein-2 (SREBP-2), which enhances cholesterol biosynthesis, were also higher in the livers of Cyp3a(-/-) mice. Binding of SREBP-2 to the Hmgcs1 gene promoter was more abundant in the livers of Cyp3a(-/-) mice. These results suggest that deficiency of CYP3A enzymes enhances transcription of genes encoding enzymes involved in cholesterol biosynthesis via activation of SREBP-2. On the other hand, hepatic cholesterol levels in Cyp3a(-/-) mice were 20% lower than those in WT mice. The mRNA expression levels of genes encoding enzymes involved in bile acid synthesis, plasma levels of 7α-hydroxy-4-cholesten-3-one and hepatic levels of total bile acid were significantly higher in Cyp3a(-/-) mice than in WT mice. These findings suggest that reduction of hepatic total cholesterol in Cyp3a(-/-) mice would be the consequence of enhanced bile acid synthesis. Therefore, CYP3A enzymes appear to play roles in the synthesis of cholesterol and bile acid in vivo.