Human cytochrome P450 2E1 and sulfotransferase 1A1 coexpressed in Chinese hamster V79 cells enhance spontaneous mutagenesis

Genetic engineering of target cells for investigating the genotoxicity associated with specific xenobiotic‐metabolizing enzymes is useful for elucidating metabolic activation and inactivation processes. We constructed a V79‐derived cell line expressing both human cytochrome P450 (CYP) 2E1 and human sulfotransferase (SULT) 1A1. We previously reported that this cell line (V79‐hCYP2E1‐hSULT1A1) efficiently activates various important pro‐genotoxicants. Here we present data on the expression level and stability of the heterologous enzymes, measured by immunoblotting, enzyme activities, and mutagenic responses to CYP2E1‐ and SULT1A1‐dependent promutagens. Unexpectedly, these cells demonstrated greatly elevated spontaneous gene mutation frequencies (determined at the Hprt locus), and elevated frequencies of sister chromatid exchange, as compared with control V79 cells and V79‐derived lines engineered for other enzymes. Therefore, V79‐hCYP2E1‐hSULT1A1 cells require regular cleansing in aminopterin‐containing medium when used for Hprt gene mutation assays. In a 4‐week time course without such selection, V79‐hCYP2E1‐hSULT1A1 demonstrated a progressive increase in the spontaneous mutant frequency from 2.9 to 155 × 10^?6. This phenomenon was moderately, strongly, and completely prohibited in the presence of CYP2E1 inhibitor 1‐aminobenzotriazole, SULT1A1 inhibitor pentachlorophenol and both in combination, respectively. This protection indicates that the enhanced spontaneous mutagenicity involves the activity of the expressed enzymes rather than being caused by an accidental genetic alteration that might have occurred during transfection. We postulate that human CYP2E1 and SULT1A1 activate an endogenous cellular molecule or a medium component to become mutagenic. It will be challenging to identify this compound and to see whether it is involved in spontaneous mutagenesis and carcinogenesis in vivo. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Human CYP2E1-dependent mutagenicity of benzene and its hydroxylated metabolites in V79-derived cells: Suppression and enhancement by ethanol pretreatment

Benzene is a human carcinogen that requires metabolic activation. We previously observed that benzene and its hydroxylated metabolites induce micronuclei in mammalian cells expressing human CYP2E1. This study was initially aimed to study another endpoint, the induction of gene mutations by those compounds in the same cell models. A V79-derived cell line expressing human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1) pretreated with ethanol (a CYP2E1 stabilizer) was used in the hprt gene mutagenicity assay. Phenol, hydroquinone, catechol, and 1,2,4-trihydroxybenzene all induced gene mutations, while they were inactive, or only weakly positive (hydroquinone), in parental V79-Mz cells. Unexpectedly, benzene was non-mutagenic in both cell lines, but it became positive in V79-hCYP2E1-hSULT1A1 cells using regimes of short exposure/long recovery without ethanol pretreatment, for both gene mutations and micronuclei formation. In silico molecular simulation showed binding energies and positions favorable for each compound to be oxidized by human CYP2E1, benzene demonstrating the highest affinity. By tunnel analysis, ethanol binding did not limit benzene to pass tunnel S, which was specifically active for benzene. However, its end product, acetic acid, decreased the occurrence of tunnel S from 5.4 to 2.2% and extended the length of its bottleneck from 5.5 to 9.0 Å. With residual ethanol molecules still being present in CYP2E1 for a period of time after benzene exposure, the acetic acid formed could limit the entrance of benzene, thus inhibit its metabolic activation. In summary, ethanol may interfere with the activation of benzene to mutagenic metabolites, at least in cultured cells.     

Analysis of human CYP1A1 and CYP1A2 genes and their shared bidirectional promoter in eight world populations

The human CYP1A1_CYP1A2 locus comprises the CYP1A1 (5,988 bp) and CYP1A2 (7,759 bp) transcribed regions, oriented head‐to‐head, sharing a bidirectional promoter of 23,306 bp. The older CYP1A1 gene appears more conserved and responsible for critical life function(s), whereas the younger CYP1A2 gene might have evolved more rapidly due to environmental (dietary) pressures. A population genetics study might confirm this premise. We combined 60 CYP1A1_CYP1A2 SNPs found in the present study (eight New Guinea Highlanders, eight Samoans, four Dogrib, four Teribe, four Pehuenche, and one Caucasian) with those found in a previous study (six West Africans, four Han Chinese, six Germans, four Samoans, and four Dogrib), yielding a total of 106 SNPs in 106 chromosomes. Resequencing of Oceanians plus Amerindians in the present study yielded 21 New World SNPs (～20%), of which 17 are not previously reported in any SNP database. Various tests revealed selective pressures for both genes and both haploblocks; unfortunately, differences in rates of evolution between the two genes were undetectable. Fay & Wu's H test revealed a “hitchhiking event” centered around four SNPs in the CYP1A1 3′‐UTR; a study in silico identified different microRNA‐binding patterns in the hitchhiked region, when the mutations were present compared with the mutations absent. Hum Mutat 30:1–14, 2009. © 2009 Wiley‐Liss, Inc.     

CYP1A1、CYP2D6基因多态性对白血病发生的影响

目的 分析细胞色素P450 CYP1A1和CYP2D6的多态性基因型在湖南地区白血病患者和健康人群中的分布及其对白血病发生的影响.方法 采用PCR及PCR-RFLP技术分析多态性基因型频率.结果 CYP1A1和CYP2D6基因的野生型、杂合突变型及纯合突变型的分布频率在急性淋巴细胞性白血病、急性非淋巴细胞性白血病、慢性粒细胞性白血病患者组与健康对照组之间无显著性差异;携带一个突变等位基因型的个体患白血病的风险与相应野生型携带者比较均无显著性差异;急性非淋巴细胞性白血病患者组的CYP1A1杂合突变型与CYP2D6杂合突变型的联合基因型频率高于健康对照组.结论 单独的CYP1A1或CYP2D6基因的多态性变异与白血病易感性不相关;CYP1A1杂合突变与CYP2D6杂合突变的联合基因型增加患急性非淋巴细胞性白血病的风险.     

韩国人CYP450 1A1、CYP450 2E1和GSTM1、GSTT1、GSTP1基因座遗传多态性分析

目的 调查代谢相关的CYP4501A1、CYP4502E1和GSTM1、GSIT1、GSTP1基因座在韩国人群中的遗传多态性分布状况。方法 采用多重聚合酶链式反应、聚合酶链式反应-限制性片段长度多态性技术，分析300名韩国健康大学生的CYP1A1基因3′端限制性内切酶Msp Ⅰ位点、CYP2E1基因5′端转录调节区Pst Ⅰ位点和GSTM1、GSTT1缺失与存在、GSTP1基因第5外显子BsmA Ⅰ位点的基因型，计算基因型和基因频率。结果 CYP1A1基因型频率为ml／ml型39．7％、ml／m2型49．7％、m2／m2型10．7％，基因频率为ml 0．645、m2 0．355。CYP2E1基因型频率为cl／cl型66．7％、cl／c2型30％、c2／c2型3．3％，基因频率为C1 0．818、C2 0．182。GSTM1基因缺失型频率为53．3％。GSTT1基因缺失型频率为54．7％。GSTP1基因型频率为Ile／Ile型62％、Ile／Val型34．3％、VaL／Val型3．7％，基因频率为Ile 0．792、Val 0．208。基因分布符合Hardy-Weirtberg平衡定律。结论 韩国人CYP1A1、CYP2E1、GSTM1、GSTT1基因分布与我国人群较为相近，半数以上人缺乏GSTM1和GSTT1基因，纯合缺失型频率超过印度人的3倍。     

Genotoxicity of 7H-dibenzo[c,g]carbazole and its tissue-specific derivatives in human hepatoma HepG2 cells is related to CYP1A1/1A2 expression

The goal of this study was to investigate the genotoxicity of 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, and its methyl derivatives, 5,9-dimethylDBC (DiMeDBC), a strict hepatocarcinogen, and N-methylDBC (N-MeDBC), a specific sarcomagen in human hepatoma HepG2 cells, and to infer potential mechanisms underlying the biological activity of particular carcinogen. All dibenzocarbazoles, regardless the tissue specificity, induced significant DNA strand break levels and micronuclei in HepG2 cells; though a mitotic spindle dysfunction rather than a chromosome breakage was implicated in N-MeDBC-mediated micronucleus formation. While DBC and N-MeDBC produced stable DNA adducts followed with p53 protein phosphorylation at Ser-15, DiMeDBC failed. A significant increase in DNA strand breaks following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggested that either oxidative DNA damage or unstable DNA-adducts might underlie DiMeDBC genotoxicity in human hepatoma cells. DiMeDBC and N-MeDBC increased substantially also the amount of CYP1A1/2 expression in HepG2 cells. Pretreatment of cells with substances affecting AhR-mediated CYP1A family of enzymes expression; however, diminished DiMeDBC and N-MeDBC genotoxicity. Our data clearly demonstrated differences in the mechanisms involved in the biological activity of DiMeDBC and N-MeDBC in human hepatoma cells; the genotoxicity of these DBC derivatives is closely related to CYP1A1/2 expression.     

Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss

The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.     

Mannan‐binding lectin attenuates acetaminophen‐induced hepatotoxicity by regulating CYP2E1 expression via ROS‐dependent JNK/SP1 pathway

Mannan‐binding lectin (MBL) acts as a soluble pattern recognition molecule in the innate immune system, which is primarily produced by the liver. MBL deficiency occurs with high frequency in the population and is reported to be associated with susceptibility to several liver diseases. In the present study, we investigated the pathophysiological role of MBL in acetaminophen (APAP)‐induced hepatotoxicity. After APAP treatment, MBL‐deficient (MBL^?/?) mice had significantly higher mortality and aggravated hepatic necrosis as well as elevated serum lactate dehydrogenase and alanine aminotransferase levels compared to control mice. The enhanced hepatotoxicity in MBL^?/? mice was associated with increased concentration of APAP toxic metabolisms. Furthermore, we demonstrated here that genetic ablation of MBL resulted in excessive reactive oxygen species (ROS) production and enhanced c‐Jun N‐terminal kinase (JNK) activation, leading to up‐regulated specificity protein 1 (SP1) nuclear expression, thus promoted CYP2E1 hepatic expression and consequently exacerbated APAP‐induced liver injury in mice. Importantly, we have validated that MBL protected against APAP toxicity in human HepaRG cells in vitro with the same mechanism. Our study revealed an unexpected function of MBL in drug metabolism, thus providing new insight into the drug‐induced liver injury in patients with MBL deficiency.     

Detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells using flow cytometry

ABCC2 has a wide tissue distribution and can mediate the efflux of a number of therapeutic compounds from cells and contribute to potential treatment failure. Its diverse expression and ability to efflux a number of substrates imply a number of physiological and pharmacological roles. CYP2B6 and CYP3A4 are responsible for the metabolism of a number of therapeutic compounds. Reports on the expression of these proteins in various cells and tissues have been contradictory mainly due to differences in experimental approach and cell type studied. With the advances in commercially available antibodies we describe here a simplified technique for the detection of ABCC2, CYP2B6 and CYP3A4 in human peripheral blood mononuclear cells (PBMC) by flow cytometry. Results are expressed as mean increase in fluorescence compared to isotypically matched controls. Using these assays we confirmed the expression of these proteins in human PBMC. These methods are rapid and reproducible and have potential use for both in vitro and clinical applications.     

细胞色素P4501A1基因多态性与肺癌遗传易感性的研究

探讨细胞色素Ｐ４５０１Ａ１基因型和异亮氨酸－缬氨酸基因型与肺癌易感性的关系。方法以病例－对照的研究方法，采用ＰＣＲ－ＲＦＬＰ和ＡＳＡ技术检测５９例原发性肺癌、５９例名住院对照和７３名健康对照ＣＹＰ１Ａ１ＭｓｐⅠ和Ｉｌｅ－Ｖａｌ基因型。     

Haplotypes in the 5′‐untranslated region of the CYP1A2gene are inversely associated with lung cancer risk but do not correlate with caffeine metabolism

In this study, we analyzed the influence of CYP1A2 genetic variation and enzyme activity on lung cancer risk in a high‐incidence area. A total of 95 lung cancer patients and 196 controls were genotyped for the ?3860G/A, ?3113A/G, ?2467T/delT, ?739T/G, and ?163C/A polymorphisms in the 5′‐untranslated region of the gene. In addition, a subset of 70 patients and 115 controls were phenotyped by high‐performance liquid chromatography determination of the caffeine metabolic ratio (CMR). The ?2467T/delT polymorphism and the CYP1A2*1V haplotype (‐163C>A, ?2467T>delT) were inversely associated with lung cancer risk (odds ratio [OR] = 0.47 [0.2–0.9]; P = 0.02 and OR = 0.13 [0.02‐1.0]; P = 0.04; respectively). In addition, the CYP*1A/*1V and *1F (‐163C>A)/*1D (?163C>A, ?2467T>delT) diplotypes were absent in the patients group, whereas accounting for 7.1% (P = 0.017) and 5.6% (P = 0.037) of controls, respectively. Mean CMR was significantly higher in patients than in controls (10.50 ± 17.31 vs. 6.52 ± 6.26, P = 0.01) but regression analyses did not yield significant ORs for the association with lung cancer risk. Similarly, no significant correlations were found between any genetic variant and enzyme activity. Several CYP1A2 haplotypes and diplotypes containing the ?2467delT variant were associated with lower lung cancer risk; however, they did not correlate with significant changes in CYP1A2 metabolic activity toward caffeine. Environ. Mol. Mutagen., 2013. © 2012 Wiley Periodicals, Inc.     

Toward the evaluation of function in genetic variability: characterizing human SNP frequencies and establishing BAC-transgenic mice carrying the human CYP1A1_CYP1A2 locus

Interindividual differences in human CYP1A1 and CYP1A2 expression appear to be associated with variability in risk toward various types of environmental toxicity and cancer. These two genes are oriented head-to-head on human chromosome 15; the 23.3-kb spacer region might contain distinct regulatory regions for CYP1A1 and distinct regulatory regions for CYP1A2, or the regulatory regions for the two genes might overlap one another. From 24 unrelated subjects of five major, geographically-isolated subgroups, we resequenced both genes (all exons and all introns) plus some 3' flanking sequences and the entire spacer region (39.6 kb total); 85 SNPs were found, 49 of which were not currently in the National Center for Biotechnology Information (NCBI) database. Of the 57 double-hit SNPs, we carried out SNP-typing in 94 Africans, 96 Asians, and 83 Caucasians and found striking ethnic differences in SNP frequencies and haplotype evolution; the two CYP1A1 SNPs and the one CYP1A2 SNP that are most commonly used in epidemiological studies were shown not to be representative haplotype tag SNPs across these three human subgroups. Four BAC-transgenic mouse lines, carrying the human CYP1A2 and 15,190 bp of 5' flank, expressed only negligible basal or inducible CYP1A2 mRNA. A fifth BAC-transgenic mouse line, carrying both the human CYP1A1 and CYP1A2 genes and ample amounts of 3' flanking sequences, plus all of the spacer region--in the absence of the mouse Cyp1a1 or Cyp1a2 genes--expressed the human CYP1A1 and CYP1A2 mRNA, protein and enzyme activities in liver and nonhepatic tissues very similar to that of the mouse. Comparison of this hCYP1A1_1A2 transgenic line with hCYP1A1_1A2 lines carrying other common human haplotypes will enable us to evaluate function in human CYP1A1_CYP1A2 locus variability, with regard to toxicity and cancer caused by combustion products.     

Immunohistochemical localization of cytochrome P450 2E1 in human pulmonary carcinoma and normal bronchial tissue

Cytochrome P450 2E1 (CYP2E1) is a major xenobiotic-metabolizing enzyme but data concerning its extrahepatic expression are few. CYP2E1 can metabolically activate many procarcinogens and therefore its presence in the lung might play a role in bioactivation of procarcinogens, so we studied the expression and localization of CYP2E1 in primary pulmonary carcinomas and surrounding normal bronchial tissue from 28 patients. Seromucous glands showed expression of CYP2E1 in 19 and bronchial epithelium in 18 of the 28 samples of normal bronchial tissue. Thirteen of the corresponding cases of primary pulmonary carcinoma showed staining for CYP2E1. In 11 of these 13 cases, CYP2E1 was also present in normal bronchial tissue. There was no statistically significant difference in the expression of CYP2E1 between adenocarcinomas and squamous cell carcinomas. No association was observed between the expression of CYP2E1 in tumour tissue and normal bronchial tissue. However, there was a significant correlation between the expression of CYP2E1 in seromucous glands and bronchial epithelium (r=0.61, P<0.01) of normal tissue. We conclude that CYP2E1 can be present in both normal and neoplastic bronchial tissue.