METABOLIC ACTIVATION OF AROMATIC CARCINOGENS

多环芳烃 (PAH) 是普遍存在的环境污染物 污染物，其中一些被认为是导致人类癌症的原因。这个 提案有两个目标。 第一个目标是阐明 3-甲基胆蒽 (3MC) 的代谢激活机制，其中 是一种强效致癌物，也是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 检查近似致癌代谢物的形成和 随后在代谢中氧化成最终致癌产物 酶系统中分别含有苯并[a]芘 (BaP) 和 3MC 含有十三种形式的人类肝脏 P450 之一。 具体形式 人 P450（1A2、2A3、2B7、2C8、2C9、2D6、2E1、2F1、3A3、3A4、3A5 和 4B1) 均包含在感染了 含有人 P450 cDNA 或 AHH-1 的重组痘苗病毒 类淋巴母细胞，其中编码人 P450 1A1 的 cDNA 为 并入疱疹样载体中。 BaP 已知具有代谢性 沿 BaP->BaP 7,8-环氧化物->BaP 7,8-二氢二醇->BaP 激活 7,8-二醇-9,10-环氧化物途径。 然而，代谢途径 3MC 的激活不太清楚。 我们建议采用体外和体内 体内方法阐明 3MC 的激活途径。个人 cDNA 表达的人 P450 将用于确定 P450 特异性 激活/解毒代谢途径。 3MC经哺乳动物药物代谢酶系统代谢形成 代谢物的复杂混合物。 在合成的3MC衍生物中， 已知 2-羟基-3MC (2-OH-3MC) 和 3MC-2-one 有效 致癌物质。 根据我们实验室的最新发现，我们 提出了一个假设，三个高度光学活性的 9， 10-二醇-7,8-环氧化物源自2S-羟基-3MC的进一步代谢 (2S-OH-3MC)、3MC-2-酮和3-羟甲基胆蒽(3-OHMC)可以是 3MC 的致癌活性负责。 每次激活 该途径与 BaP 的途径在细节上有所不同，涉及四到五个 由细胞色素 P450 催化的对映选择性酶促步骤和 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和 代谢物的理化特征、DNA 结合和 对小鼠皮肤进行肿瘤引发活性测试旨在确定 3MC 的激活/解毒途径。 P450 表达的明确物种差异表明 不太可能可靠地推断啮齿动物致癌物激活数据 对人类。 直接分析人类 P450 致癌代谢物 因此，确定负责的形式至关重要 致癌物质的激活和解毒。 结果可能最终 允许我们专门诱导某些参与解毒的 P450 与涉及致癌物激活的过程相关的过程，因此 减少毒性。 这项研究将为急需的人类做出贡献 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.