ABSTRACT

Prenatal smoke exposure (PSE) is a risk factor for nicotine dependence. One susceptibility gene for nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher activity of the CYP2A6 enzyme is associated with nicotine dependence, but no research has addressed the PSE effects on the CYP2A6 gene or its mouse homologue Cyp2a5. We hypothesized that PSE affects Cyp2a5 promoter methylation, Cyp2a5 mRNA levels, and nicotine metabolism in offspring. We used a smoke-exposed pregnant mouse model. RNA, DNA, and microsomal protein were isolated from liver tissue of foetal, neonatal, and adult offspring. Enzyme activity, Cyp2a5 mRNA levels, and Cyp2a5 methylation status of six CpG sites within the promoter region were analysed via HPLC, RT-PCR, and bisulphite pyrosequencing. Our data show that PSE induced higher cotinine levels in livers of male neonatal and adult offspring compared to controls. PSE-induced cotinine levels in neonates correlated with Cyp2a5 mRNA expression and promoter methylation at CpG-7 and CpG+45. PSE increased methylation in almost all CpG sites in foetal offspring, and this effect persisted at CpG-74 in male neonatal and adult offspring. Our results indicate that male offspring of mothers which were exposed to cigarette smoke during pregnancy have a higher hepatic nicotine metabolism, which could be regulated by DNA methylation. Given the detected persistence into adulthood, extrapolation to the human situation suggests that sons born from smoking mothers could be more susceptible to nicotine dependence later in life.

摘要

产前烟雾暴露（PSE）是尼古丁依赖的危险因素。尼古丁依赖的一个易感基因是细胞色素 P450 (CYP) 2A6，一种负责将尼古丁转化为可替宁并在肝脏中清除尼古丁的酶。CYP2A6 酶的较高活性与尼古丁依赖相关，但没有研究解决 PSE 对CYP2A6基因或其小鼠同源基因Cyp2a5的影响。我们假设 PSE 影响子代Cyp2a5启动子甲基化、Cyp2a5 mRNA 水平和尼古丁代谢。我们使用暴露于烟雾的怀孕小鼠模型。从胎儿、新生儿和成年后代的肝组织中分离出 RNA、DNA 和微粒体蛋白。通过 HPLC、RT-PCR 和亚硫酸氢盐焦磷酸测序分析启动子区域内六个 CpG 位点的酶活性、Cyp2a5 mRNA 水平和Cyp2a5甲基化状态。我们的数据显示，与对照组相比，PSE 诱导男性新生儿和成年后代肝脏中的可替宁水平更高。PSE 诱导的新生儿可替宁水平与Cyp2a5 mRNA 表达和 CpG-7 和 CpG+45 启动子甲基化相关。PSE 增加了胎儿后代中几乎所有 CpG 位点的甲基化，并且这种效应在男性新生儿和成年后代中的 CpG-74 处持续存在。我们的研究结果表明，怀孕期间接触香烟烟雾的母亲所生的男性后代具有较高的肝脏尼古丁代谢，这可以通过 DNA 甲基化来调节。鉴于检测到的尼古丁持续到成年期的情况，对人类情况的推断表明，吸烟母亲所生的儿子在以后的生活中可能更容易受到尼古丁依赖。