Vanden Heuvel J P, Clark G C, Thompson C L, McCoy Z, Miller C R, Lucier G W, Bell D A
School of Pharmacology and Toxicology, Purdue University, West Lafayette, IN 47907.
Carcinogenesis. 1993 Oct;14(10):2003-6. doi: 10.1093/carcin/14.10.2003.
Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.
准确的人体风险评估需要灵敏的方法来评估剂量反应关系，尤其是在低水平暴露之后。我们开发了一种逆转录聚合酶链反应（RT-PCR）方法来定量人血淋巴细胞中细胞色素P450-1A1（CYP1A1）的mRNA水平。许多多环芳烃（PAH），如苯并[a]芘，以及氯代PAH，如多氯二苯并二恶英、二苯并呋喃和联苯，通过激活内源性蛋白——芳烃受体（Ah受体）来诱导CYP1A1的表达。我们使用一种包括合成内标的定量竞争性RT-PCR方法，测定了静息以及有丝分裂原刺激的人血淋巴细胞中CYP1A1 mRNA的拷贝数。在有丝分裂原刺激的人血淋巴细胞中，对于这个低表达基因的测量，测定变异约为10%，并且mRNA水平与乙氧异吩唑酮-O-脱乙基酶（EROD）活性密切相关。用10 nM四氯二苯并二恶英培养人淋巴细胞后，mRNA的表达诱导了20倍。在未刺激、未诱导的淋巴细胞中，CYP1A1水平极低（1000个mRNA拷贝/10(4)个细胞），无法通过EROD活性进行测量。关于化学暴露人群中CYP1A1 mRNA表达的研究正在进行中。
