Xenobiotic-Metabolizing Enzymes

在哺乳动物中，存在大量的酶来代谢药物和其他外源物质。细胞色素P450是这些酶中最重要的，参与了大多数治疗药物的新陈代谢。此外，P450还催化化学致癌物的代谢活化。参与异源代谢的P450存在于细胞色素P450、细胞色素P450、细胞色素P450和细胞色素P450家族中。这些家族中的每一个都由两个或更多的子家族组成。事实上，P450在体外可以代谢激活毒素和前致癌物，这意味着它们与毒性和癌症有关。然而，目前尚不清楚P450是否对完整动物体内的化学物质的毒性和致癌性是必需的。唯一表明P450在癌症病因学中的作用的实验是细胞培养中的间接化学诱导转化分析，以及涉及ah基因的小鼠遗传实验。在一个完整的动物模型系统中，没有直接证据表明P450是致癌所必需的。为了评估P450在急性化学毒性和化学致癌过程中的潜在作用，并确定它们在哺乳动物发育和生理动态平衡中的作用，我们制造了P450基因缺失的小鼠。其他致癌物代谢酶如微粒体环氧化物水解酶(Meh)和苯醌氧化还原酶(NQO1)的表达缺失。 对CYP1B1缺失小鼠的研究表明，这种P450对于多环芳烃的致癌作用是必需的。缺乏meh的小鼠对致癌物也很敏感，这表明经典的二元醇-环氧化物致癌物活化途径是体内最重要的致癌途径。 为了制造能够更准确地预测人类药物和致癌物代谢的小鼠模型，正在使用细菌人工染色体制备P450人源化小鼠。将携带人类基因的转基因小鼠与P450基因缺失小鼠杂交，产生人源化小鼠。表达人细胞色素P1A1、细胞色素P1A2、细胞色素P2E1、细胞色素P4D6、细胞色素P3A4和细胞色素P3A7的小鼠已经建立并鉴定。最近对CYP1A2人源化小鼠的一项研究发现，人的CYP1A2比相应的小鼠P450产生更多的饮食致癌物2-amino-1-methyl-6-phenylimidazo[4，5-b]pyridine(PhIP)的N-羟基代谢物。对CYP3A4人源化小鼠的研究表明，在特定情况下，它可以控制血清雌激素水平。这些小鼠还被用来建立潜在的性别差异的CYP3A4表达(女性和男性更高)，这可以解释药物代谢和反应的性别差异。我们正在发起一项代谢组学的新倡议，以寻找和开发尿液中P450表达的小分子生物标记物。利用UPLC-MS(Qtof)对野生型和各种缺陷型小鼠的品系、年龄和性别匹配的小鼠的尿液进行分析，以寻找与特异性P450表达相关的生物标志物。这些方法有可能被开发用于人类，以检测单个人类P450形式的表达程度。这种方法也可以用来寻找癌症的生物标志物。

In mammals, a large number of enzymes exist that metabolize drugs and other xenobiotics. Cytochrome P450s are among the most important of these enzymes that are involved in metabolism of most therapeutically-used drugs. In addition, P450s catalyze the metabolic-activation of chemical carcinogens. The P450s involved in xenobiotic metabolism are found in the CYP1, CYP2 and CYP3 families. Each of these families consist of two or more subfamilies. The fact that P450s can metabolically-activate toxins and procarcinogens in vitro implies that they are involved in toxicity and cancer. However, it is not known whether P450s are required for the toxicity and carcinogenicity of chemicals in an intact animal. The only experiments suggestive of a role for P450s in cancer etiology are indirect chemically-induced transformation assays in cell culture, and genetic experiments in mice involving the Ah locus. No direct evidence is available to establish that P450s are necessary for carcinogenesis in an intact animal model system. To assess the potential contribution of P450s to acute chemical toxicity and the process of chemical carcinogenesis, and to determine their roles, if any, in mammalian development and physiological homeostasis, P450-null mice were produced. Mice lacking expression of other carcinogen metabolizing enzymes like the microsomal epoxide hydrolase (mEH) and the quinone oxidoreductase (NQO1) were also made. Studies with CYP1B1-null mice have revealed that this P450 is required for the carcinogenic efforts of polycyclic aromatic hydrocarbons. Mice lacking mEH are also sensitive to the carcinogens thus indicating that the classic diol-epoxide route of carcinogen activation is the most important or carcinogenesis in vivo. In order to produce mouse models that can more accurately be used to predict human drug and carcinogen metabolism, P450-humanized mice are being prepared using bacterial artificial chromosomes. Transgenic mice carrying the human genes are bred with P450 null-mice to produce humanized mice. Mice expressing human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7 have been produced and characterized. A recent study with CYP1A2-humanized mice found that human CYP1A2 produces more of the N-hydroxy metabolite of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) than does the corresponding mouse P450. Studies with the CYP3A4-humanized mouse revealed that it can, under certain circumstances, control the serum levels of estrogen. These mice were also used to establish a potential gender differences in CYP3A4 expression (higher in female and male) that could account for gender differences in drug metabolism and response.We are launching a new initiative in metabonomics in order to find and develop small molecule biomarkers for P450 expression in urine. The urine of strain, age and sex matched mice from wild-type and various null mice are being analyzed by use of UPLC-MS(Qtof) in order to find biomarkers that correlate with expression of specific P450s. These could potentially be developed for use in humans to examine the extent of expression of individual human P450 forms. This methodology can also be used to find biomarkers for cancer.