Raaka S, Hassett C, Omiencinski C J
Department of Environmental Health, University of Washington, Seattle 98105-6099, USA.
Carcinogenesis. 1998 Mar;19(3):387-93. doi: 10.1093/carcin/19.3.387.
Microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of xenobiotic epoxides, including various epoxide derivatives of the procarcinogenic polyaromatic hydrocarbons. Levels of mEH enzymatic activity among different cell types and between individuals within the population vary considerably. Genetic polymorphisms within the structural region of the human mEH gene exist and appear to contribute to the population variance in functional expression. In this study, we used single strand conformational polymorphism analysis and direct DNA sequencing approaches to identify seven additional polymorphic sites within the upstream region of the mEH gene, spanning -743 to +185 bp, relative to the transcription initiation site. Allelic frequencies and linkages of the polymorphic nucleotides were determined in 51 individuals using restriction fragment length polymorphism or competitive oligonucleotide priming assays. To determine the functional significance of the individual nucleotide substitutions, DNA fragments representing the variant alleles were cloned into the heterologous pBRAMScat2 reporter vector, transfected into HepG2 cells and assessed for reporter gene expression. Results indicated that certain of these polymorphic loci might differentially regulate transcription, with the maximum contribution of any of the variants modifying levels of reporter gene activity by approximately 30%. These observations establish that genetic variation in the 5' flanking sequence of mEH gene is likely an additional contributing factor to the range of functional mEH expression existing in human populations.
微粒体环氧化物水解酶（mEH）催化外源性环氧化物的水解，包括致癌性多环芳烃的各种环氧化物衍生物。不同细胞类型之间以及人群中个体之间的mEH酶活性水平差异很大。人类mEH基因结构区域内存在基因多态性，这似乎导致了功能表达的人群差异。在本研究中，我们使用单链构象多态性分析和直接DNA测序方法，在mEH基因上游区域（相对于转录起始位点，跨度为-743至+185 bp）鉴定出另外7个多态性位点。使用限制性片段长度多态性或竞争性寡核苷酸引物分析，在51名个体中确定了多态性核苷酸的等位基因频率和连锁关系。为了确定单个核苷酸替代的功能意义，将代表变异等位基因的DNA片段克隆到异源pBRAMScat2报告载体中，转染到HepG2细胞中，并评估报告基因的表达。结果表明，这些多态性位点中的某些位点可能对转录有不同的调节作用，任何一个变异体对报告基因活性水平的最大影响约为30%。这些观察结果表明，mEH基因5'侧翼序列的遗传变异可能是导致人类群体中功能性mEH表达范围的另一个因素。
