Rumsby P C, Yardley-Jones A, Anderson D, Phillimore H E, Davies M J
Department of Molecular Biology, BIBRA International, Surrey, United Kingdom.
Teratog Carcinog Mutagen. 1996;16(1):65-74. doi: 10.1002/(SICI)1520-6866(1996)16:1<65::AID-TCM7>3.0.CO;2-E.
It is important to determine sensitive biomarkers for both exposure and susceptibility since differences in individual susceptibility to potentially hazardous chemicals may represent a major variable in the assessment of risk. Induction of cytochrome P450IA1 (CYP1A1) may be a measure of environmental exposure to aromatic hydrocarbons in cigarette smoke. This study investigated the use of reverse transcriptase-polymerase chain reaction (RT-PCR) to detect constitutive levels of CYP1A1 mRNA in the peripheral lymphocytes of a population of smokers and non-smokers as a potential marker of exposure. In addition, the presence of an Msp 1 restriction fragment length polymorphism was analyzed using a simple PCR method as a biomarker for susceptibility. DNA and RNA were isolated from the peripheral lymphocytes of 20 smokers and a matched group of non-smokers. RT-PCR was used to detect the endogenous levels of CYP1A1 mRNA with glyceraldehyde-3-phosphate dehydrogenase as a control gene. The 3'-region of CYP1A1 gene was amplified by PCR and underwent restriction digestion with Msp 1 to detect the polymorphism. The endogenous CYP1A1 expression as detected by RT-PCR was very low and variable and there was a slight but not significant increase in the smokers by comparison with non-smokers. Thirty-two of the volunteers were homozygous for the normal allele while 8 were heterozygous for the uncommon Msp 1 allele and none was homozygous for the polymorphism. The allele frequency (0.1) was found to be in Hardy-Weinberg equilibrium. Since only a slight increase was seen in endogenous CYP1A1 mRNA levels in the peripheral lymphocytes of smokers by comparison with non-smokers, the effect may have been diluted by variation in sensitivity to dose, a threshold of exposure effect, or the return of mRNA to baseline between exposures. The wide variation in mRNA levels may reflect the influence and exposure of different environmental factors. The sensitivity of PCR-based methods suggests that they may have an important role in future overall biomonitoring of exposure and susceptibility to environmental chemicals.
确定暴露和易感性的敏感生物标志物非常重要，因为个体对潜在有害化学物质易感性的差异可能是风险评估中的一个主要变量。细胞色素P450IA1（CYP1A1）的诱导可能是衡量香烟烟雾中芳香烃环境暴露的指标。本研究调查了使用逆转录聚合酶链反应（RT-PCR）检测吸烟者和非吸烟者群体外周淋巴细胞中CYP1A1 mRNA的组成水平，作为潜在的暴露标志物。此外，使用简单的PCR方法分析Msp 1限制性片段长度多态性的存在，作为易感性的生物标志物。从20名吸烟者和一组匹配的非吸烟者的外周淋巴细胞中分离DNA和RNA。以甘油醛-3-磷酸脱氢酶作为对照基因，用RT-PCR检测CYP1A1 mRNA的内源性水平。通过PCR扩增CYP1A1基因的3'区域，并用Msp 1进行限制性消化以检测多态性。RT-PCR检测到的内源性CYP1A1表达非常低且变化不定，与非吸烟者相比，吸烟者有轻微但不显著的增加。32名志愿者为正常等位基因纯合子，8名志愿者为罕见Msp 1等位基因杂合子，没有人为该多态性纯合子。发现等位基因频率（0.1）处于哈迪-温伯格平衡。由于与非吸烟者相比，吸烟者外周淋巴细胞中内源性CYP1A1 mRNA水平仅略有增加，这种影响可能因对剂量的敏感性变化、暴露效应阈值或暴露之间mRNA恢复到基线而被稀释。mRNA水平的广泛变化可能反映了不同环境因素的影响和暴露。基于PCR方法的敏感性表明，它们可能在未来环境化学物质暴露和易感性的整体生物监测中发挥重要作用。
