ENZYMATIC BASIS OF AOM AND MAM ACTIVATION

AOM 和 MAM 激活的酶学基础

• 批准号: 6458789
• 负责人: OCK S SOHN
• 金额: $ 28.57万
• 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/6458789
• 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（由申请人提供）：甲基偶氮氧基甲醇（MAM）， 甲基偶氮氧乙酸甲酯（MAMAc），MAM的稳定形式，以及偶氮氧甲烷 (AOM)MAM的化学和代谢前体， 啮齿动物体内的致癌物质AOM通过羟基化活化为MAM，其产生 自发或通过酶催化反应的DNA烷基化物质。两 AOM和MAMAc，尤其是AOM，已被广泛用于啮齿动物 旨在确定结肠癌化学预防剂的研究。使 为了合理利用从这些研究中获得的数据， 存在足够的信息基础上，在体内激活这些 致癌物质。由于目前缺乏这类信息，往往导致明显的 矛盾和无法最佳解释数据外推 认识到这些不足，我们的目标是扩大我们的 了解负责AOM代谢活化的酶系统 和MAM。我们以前已经表明，MAM和AOM都可以通过代谢作用 在体外被CYP 2 E1激活。最近，使用cyp 2 e1-null和-wild类型 小鼠，我们明确表明，CYP 2 E1也参与激活 的AOM和MAM的DNA反应性物种在体内。然而，同样的研究 清楚地表明，其他酶，也许是其他的酶家族成员， 也参与其中。这些研究还表明，CYP 2 E1 水平，如在零和野生型小鼠，可以深刻地影响 AOM和MAMAc的活化和致瘤性-相对于野生型 小鼠，AOM的致瘤性降低，MAM的致瘤性增加， 空型小鼠。化学预防剂的影响， 诱导或抑制CYP 2 E1和AOM和MAM激活其它酶， 预计会相似。作为特定目标1，我们建议鉴定CYP同工酶 CYP 2 E1除外，其在14 C-MAM的体外代谢中具有活性， 14 C-AOM，使用表达特异性人β-内酰胺酶同工酶的昆虫细胞微粒体， 通过独特的HPLC分析系统分析代谢物。作为逻辑 因此，在具体目标2中，我们建议使用特定的酶修饰剂， 获得证据表明，在具体目标1中鉴定的同工酶，事实上， 参与体内AOM和MAM的激活，如DNA中所反映的 烷基化作为具体目标3，我们测试了在 本发明的目的是利用野生型和 null-CYP 2 E1小鼠及其对结肠异常隐窝的影响 阵具体目标4下的工作将检查其他酶， 包括醇脱氢酶和前列腺素合酶， 参与致癌物质的体外和体内代谢。