METABOLIC ACTIVATION OF AROMATIC CARCINOGENS

多环芳烃（PAHs）是环境中普遍存在的一类有机污染物 污染物和一些被认为是人类癌症的原因。这 该提案有两个目标。 第一个目标是阐明 3-甲基胆蒽（3 MC）的代谢活化机制， 是一种强致癌物，也是一种广泛使用的细胞色素P450同工酶诱导剂 作为癌症和药物研究的模型化合物。第二个目标是 检查近致癌代谢物的形成， 随后在代谢中氧化为最终致癌产物 苯并[a]芘（BaP）和3 MC在酶体系中的 含有人类肝脏P450的十三种形式之一 具体形式的 人P450（1A 2、2A 3、2B 7、2C 8、2C 9、2D 6、2 E1、2F 1、3A 3、3A 4、3A 5和 4 B1）各自包含在感染有 含有人P450 cDNA的重组牛痘病毒或AHH-1 淋巴母细胞样细胞，其中编码人P450 1A 1的cDNA是 整合到疱疹样载体中。 苯并[a]芘在代谢上 沿着BaP->BaP 7，8-环氧化物->BaP 7，8-二氢二醇->BaP 7，8-二醇-9，10-环氧化物途径。 然而，代谢途径 3 MC的激活不太清楚。 我们建议在体外和 体内方法来阐明3 MC的激活途径。个人 cDNA表达的人P450将用于确定P450特异性 活化/解毒代谢途径。 3 MC通过哺乳动物药物代谢酶系统代谢， 代谢物的复杂混合物。 在合成的3 MC衍生物中， 已知2-羟基-3MC（2-OH-3 MC）和3 MC-2-酮是有效的 致癌物质。 根据我们实验室的最新发现， 提出了一种假设，即三个高度光学活性的9， 2S-羟基-3MC进一步代谢产生的10-二醇-7，8-环氧化物 （2S-OH-3 MC）、3 MC-2-酮和3-羟甲基胆蒽（3-OHMC）可以是 负责3 MC的致癌活性。 每个激活 与BaP通路的细节不同，涉及四个或五个 由细胞色素P450催化的对映选择性酶促步骤， 微粒体酶复合物中的环氧化物水解酶。 实验 包括高效液相色谱分离， 代谢产物的理化表征、DNA结合和 在小鼠皮肤上的肿瘤引发活性试验被设计用于确定 3 MC的活化/解毒途径。 在P450表达方面已确立的物种差异表明， 不太可能可靠地推断啮齿动物致癌物活化数据， 对人类 人体致癌物P450代谢产物的直接分析 因此，必须确定负责以下方面的形式： 致癌物质活化和解毒。 结果可能最终 使我们能够特异性地诱导某些P450参与解毒， 与致癌物活化相关的过程， 降低毒性。 这项研究将有助于急需的人类 基于酶的环境致癌物风险评估。

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and some are believed to be causal in human cancer. This proposal has two objectives. The first objective is to elucidate the mechanism(s) of metabolic activation of 3-methylcholanthrene (3MC), which is a potent carcinogen and a cytochrome P450 isozyme inducer widely used as a model compound in cancer and drug research. The second objective is to examine the formation of proximate carcinogenic metabolites and subsequent oxidation to ultimate carcinogenic product in the metabolism of benzo[a]pyrene (BaP) and 3MC respectively in an enzyme system containing one of thirteen forms of human liver P450s. Specific forms of human P450s (1A2, 2A3, 2B7, 2C8, 2C9, 2D6, 2E1, 2F1, 3A3, 3A4, 3A5, and 4B1) are each contained in human hepatoma Hep G2 cells infected with recombinant vaccinia virus containing a human P450 cDNA or in AHH-1 lymphoblastoid cells in which the cDNA encoding for human P450 1A1 is incorporated in a herpes-like vector. BaP is known to be metabolically activated along the BaP->BaP 7,8-epoxide->BaP 7,8-dihydrodiol->BaP 7,8-diol-9,10-epoxide pathway. However, pathway(s) of metabolic activation of 3MC is less clear. We propose to employ in vitro and in vivo methods to elucidate the activation pathways of 3MC. Individual cDNA-expressed human P450s will be used to determine P450-specific activation/detoxification metabolic pathways. 3MC is metabolized by mammalian drug metabolizing enzyme system to form a complex mixture of metabolites. Among synthetic 3MC derivatives, 2-hydroxy-3MC (2-OH-3MC) and 3MC-2-one are known to be potent carcinogens. Based on recent findings in our laboratory, we have developed a hypothesis that three highly optically active 9, 10-diol-7,8-epoxides derived from further metabolism of 2S-hydroxy- 3MC (2S-OH-3MC), 3MC-2-one, and 3-hydroxymethylcholanthrene (3-OHMC) may be responsible for the carcinogenic activities of 3MC. Each activation pathway differs in detail from that of BaP and involves four or five enantioselective enzymatic steps catalyzed by cytochrome P450s and epoxide hydrolase in the microsomal enzyme complex. Experiments including high-performance liquid chromatographic isolation and physicochemical characterization of metabolites, DNA binding, and tumor-initiating activity test on mouse skin are designed to determine activation/detoxification pathways of 3MC. The well-established species differences in P450 expression indicate that it is unlikely to reliably extrapolate rodent carcinogen activation data to humans. Direct analysis of human P450-based carcinogen metabolite formations is therefore essential to identify the form(s) responsible for carcinogen activation and detoxification. The results may eventually allow us to specifically induce certain P450s engaged in detoxification processes relative to those involved in carcinogen activation and thus reduce toxicity. This research will contribute to the much needed human enzyme-based risk assessment of environmental carcinogens.