ENZYMATIC BASIS OF AOM AND MAM ACTIVATION

AOM 和 MAM 激活的酶学基础

• 批准号: 6718214
• 负责人: OCK S SOHN
• 金额: $ 28.66万
• 依托单位: INSTITUTE FOR CANCER PREVENTION
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-16 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6718214
• 关键词: DNA damage; alkylation; azo compounds; chemical carcinogen; chemical carcinogenesis; cytochrome P450; enzyme activity; enzyme deficiency; enzyme induction /repression; enzyme mechanism; gene targeting; genetically modified animals; isozymes; laboratory mouse

DESCRIPTION (provided by applicant): Methylazoxymethanol (MAM), methylazoxymethyl acetate (MAMAc), the stable form of MAM, and azoxymethane (AOM), the chemical and metabolic precursor of MAM, are potent colon carcinogens in rodents. AOM is activated by hydroxylation to MAM, which yields a DNA alkylating species spontaneously or by enzyme-catalyzed reactions. Both AOM and MAMAc, but most especially AOM, have been used extensively in rodent studies seeking to identify colon cancer chemopreventive agents. To make rational use of the data obtained from such studies, it is necessary that there exist an adequate information base on the in vivo activation of these carcinogens. The current absence of such information has often led to apparent paradoxes and the inability to optimally interpret the data for extrapolation to man. Recognizing these deficiencies, our goal here is to expand our knowledge of the enzyme systems responsible for the metabolic activation of AOM and MAM. We have shown previously that both MAM and AOM can be metabolically activated by CYP2E1 in vitro. Very recently, using cyp2e1-null and -wild type mice, we showed unambiguously that CYP2E1 also participates in the activation of AOM and MAM to DNA-reactive species in vivo. However, the same studies clearly showed that other enzymes, perhaps other members of the CYP family, were also involved. These studies also demonstrated that differences in CYP2E1 levels, as in the null- and wild-type mice, can profoundly influence the activation and tumorigenicity of AOM and MAMAc - relative to the wild-type mice, the tumorigenicity of AOM is decreased, and that of MAM is increased, in the null-type mice. The influences of chemopreventive agents which may either induce or inhibit CYP2E1 and other enzymes of AOM and MAM activation is expected to be similar. As Specific Aim 1, we propose to identify CYP isozymes other than CYP2E1, which are active in the in vitro metabolism of 14C-MAM and 14C-AOM using insect cell microsomes expressing specific human CYP isozymes, with analysis of metabolites by a unique HPLC analytical system. As a logical consequence, in Specific Aim 2 we propose to use specific enzyme modifiers to obtain evidence that the isozymes identified in Specific Aim 1 are, in fact, involved in the activation of AOM and MAM in vivo, as reflected in DNA alkylation. As Specific Aim 3, we test the relevance of the results obtained in the previous Aims by using specific modifiers of CYP isozymes in wild- and null-CYP2E1 mice and determining their effects on colonic aberrant crypt formation. The work under Specific Aim 4 will examine whether other enzymes, including alcohol dehydrogenase and prostaglandin synthase are additionally involved in the in vitro and in vivo metabolism of the carcinogens.

描述(申请人提供)：甲氧基甲醇(MAM)， 甲氧基乙酸甲酯(MAMAC)，MAM的稳定形式，以及偶氮甲烷 (AOM)是MAM的化学和代谢前体，是一种有效的结肠 啮齿动物体内的致癌物。AOM通过羟基化生成MAM而被激活，生成 脱氧核糖核酸一种自发或通过酶催化反应的DNA烷基化物种。两者都有 AOM和MAMAC，尤其是AOM，在啮齿动物中得到了广泛的应用 寻求确定结肠癌化学预防药物的研究。使 合理利用从这类研究中获得的数据，有必要 存在足够的关于这些基因在体内激活的信息基础 致癌物质。目前此类信息的缺乏往往导致明显的 悖论和无法以最佳方式解释数据进行外推 对人类来说。认识到这些不足，我们的目标是扩大我们的 AOM代谢激活的酶系统知识 和MAM。我们以前已经证明，MAM和AOM都可以代谢 在体外被细胞色素P450-2E_1激活。最近，使用了CYP2E1-空和野生型 小鼠，我们明确地表明，CYP2E1也参与了激活 AOM和MAM对体内DNA活性物种的影响。然而，同样的研究 清楚地表明，其他酶，也许是CYP家族的其他成员， 也牵涉其中。这些研究还表明，细胞色素P4502E1中的差异 水平，就像在空白和野生型小鼠中一样，可以深刻地影响 AOM和MAMAC的激活和致瘤性--相对于野生型 AOM对小鼠的致瘤性降低，MAM的致瘤性增强。 零型老鼠。化学预防药物的影响可能是 诱导或抑制CYP2E1等AOM和MAM酶的激活 预计会是相似的。作为特定的目标1，我们建议鉴定细胞色素P450同工酶 在~(14)C-MAM的体外代谢中起作用的不是细胞色素P_2E_1，而是 14C-AOM使用表达特定人类CYP同工酶的昆虫细胞微粒体， 用独特的高效液相分析系统分析代谢物。作为一个合乎逻辑的 因此，在特定目标2中，我们建议使用特定的酶修饰剂来 获得在特定目标1中确定的同工酶实际上是 参与体内AOM和MAM的激活，反映在DNA中 烷基化反应。作为具体目标3，我们检验了在以下方面所获得的结果的相关性 以前的目的是通过使用野生和野生的CYP同工酶的特定修饰物 Null-CYP2E1小鼠及其对结肠畸形隐窝的影响 队形。在特定目标4下的工作将检查是否有其他酶， 包括酒精脱氢酶和前列腺素合成酶 参与致癌物的体外和体内代谢。