Cyt P450 Oxidation in Chemical Toxicity /Carcinogenicity

化学毒性/致癌性中的 Cyt P450 氧化

• 批准号: 7174348
• 负责人: BURHAN I GHANAYEM
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7174348
• 关键词: acrylamides; biotransformation; cancer risk; chemical carcinogen; chemical carcinogenesis; cytochrome P450; environmental exposure; environmental toxicology; enzyme activity; epoxides; flow cytometry; genetically modified animals; germ cells; laboratory mouse; liquid chromatography mass spectrometry; northern blottings; oxidation; polymerase chain reaction; site directed mutagenesis; toxin metabolism; western blottings

Exposure to environmental chemicals is considered a contributing factor to increased human susceptibility to cancer, reproductive and developmental disorders, and other diseases. The mechanism(s) of action of these chemicals is not well understood, however, most of these environmental chemicals are metabolized to epoxide intermediates via CYP2E1. We therefore hypothesized that epoxidation of these chemicals may be responsible for the induction of mutagenicity. Using acrylamide (AA) as a model chemical, studies were undertaken to investigate the metabolic and molecular basis for the induction of somatic and germ cell mutagenicity by epoxide-forming chemicals. AA is an animal carcinogen, neurotoxin, and reproductive toxin. AA is formed in baked and fried carbohydrate-rich foods as a result of processing at high temperatures. Metabolism of AA occurs via epoxidation to glycidamide (GA) or direct conjugation with glutathione. Studies were undertaken to assess the role of CYP2E1 in the epoxidation of AA to GA and the formation of DNA and hemoglobin (HGB) adducts. AA was administered to CYP2E1-null or wild-type mice at 50 mg/kg. Using LC-ES/MS/MS, AA, GA, and DNA- and HGB-adducts were measured. While the plasma levels of AA and GA were 115?14.0 and 1.7?0.31uM in CYP2E1-null mice, they were 0.84?0.80 and 33.0?6.3uM in the plasma of AA-treated wild-type mice. Administration of AA to wild-type mice caused a large increase in N7-GA-Gua and N3-GA-Ade adducts in the liver, lung, and testes. While traces of N7-GA-Gua adducts were measured in the tissues of AA-treated CYP2E1-null mice, these levels were 52-66-fold lower than in wild-type mice. Significant elevation of both AA- and GA-HGB adducts was detected in AA-treated wild-type mice. To explore the role of CYP2E1 metabolism in the germ cell mutagenicity of AA, CYP2E1-null and wild-type were treated by intraperitoneal injection with 50mg AA/kg/day for 5 consecutive days. At defined times after exposure, males were mated to untreated B6C3F1 females. Females were sacrificed in late gestation and uterine contents were examined. Dose-related increases in resorption moles (chromosomally aberrant embryos) and decreases in the numbers of pregnant females and the proportion of living fetuses were seen in females mated to AA-treated wild-type mice. No changes in any fertility parameters were seen in females mated to AA-treated CYP2E1-null mice. In a second set of experiments, we examined the role of CYP2E1 in AA-induced somatic cell damage. Female wild type and CYP2E1-null mice were administered AA (0, 25, 50 mg/kg) by intraperitoneal injection once daily for 5 consecutive days. Erythrocyte micronucleus frequencies were determined using flow cytometry and DNA damage was assessed in leukocytes, liver, and lung using the alkaline (pH>13) single cell gel electrophoresis (Comet) assay. Results were consistent with our observations in male germ cells: significant dose-related increases in micronucleated erythrocytes and DNA damage in somatic cells were induced in acrylamide-treated wild type but not in the CYP2E1-null mice. These results constitute the first unequivocal demonstration that genetic damage in somatic and germ cells of mice-treated with AA is dependent upon metabolism of the parent compound to GA by CYP2E1.

接触环境化学物质被认为是增加人类对癌症、生殖和发育障碍以及其他疾病的易感性的一个因素。这些化学物质的作用机制尚不清楚，然而，大多数这些环境化学物质通过 CYP2E1 代谢为环氧化物中间体。因此，我们假设这些化学物质的环氧化可能是诱发诱变的原因。使用丙烯酰胺（AA）作为模型化学品，进行了研究以调查环氧化物形成化学品诱导体细胞和生殖细胞致突变性的代谢和分子基础。 AA是一种动物致癌物、神经毒素和生殖毒素。 AA 是在富含碳水化合物的烘焙和油炸食品中经过高温加工而形成的。 AA 的代谢通过环氧化为缩水甘油酰胺 (GA) 或与谷胱甘肽直接结合而发生。研究评估了 CYP2E1 在 AA 环氧化为 GA 以及 DNA 和血红蛋白 (HGB) 加合物形成中的作用。以 50 mg/kg 的剂量向 CYP2E1 缺失或野生型小鼠施用 AA。使用 LC-ES/MS/MS 测量 AA、GA 以及 DNA 和 HGB 加合物。在CYP2E1缺失小鼠中，AA和GA的血浆水平分别为115×14.0和1.7×0.31uM，而在AA处理的野生型小鼠的血浆中，AA和GA的水平分别为0.84×0.80和33.0×6.3uM。给野生型小鼠施用AA导致肝脏、肺和睾丸中的N7-GA-Gua和N3-GA-Ade加合物大量增加。虽然在 AA 处理的 CYP2E1 缺失小鼠的组织中检测到 N7-GA-Gua 加合物的痕迹，但这些水平比野生型小鼠低 52-66 倍。在 AA 处理的野生型小鼠中检测到 AA- 和 GA-HGB 加合物显着升高。 为了探讨CYP2E1代谢在AA生殖细胞致突变性中的作用，CYP2E1缺失型和野生型通过腹腔注射50mg AA/kg/天连续5天进行治疗。在暴露后的规定时间，雄性与未经处理的 B6C3F1 雌性交配。在妊娠后期处死雌性并检查子宫内容物。在与 AA 处理的野生型小鼠交配的雌性小鼠中，观察到与剂量相关的吸收痣（染色体异常胚胎）增加以及怀孕雌性数量和活胎比例的减少。在与 AA 处理的 CYP2E1 缺失小鼠交配的雌性小鼠中，未发现任何生育参数发生变化。在第二组实验中，我们研究了 CYP2E1 在 AA 诱导的体细胞损伤中的作用。雌性野生型和 CYP2E1 缺失小鼠通过腹腔注射 AA（0、25、50 mg/kg），每天一次，连续 5 天。使用流式细胞术测定红细胞微核频率，并使用碱性（pH>13）单细胞凝胶电泳（彗星）测定评估白细胞、肝脏和肺中的DNA损伤。结果与我们在雄性生殖细胞中的观察结果一致：在丙烯酰胺处理的野生型小鼠中，微核红细胞和体细胞中的 DNA 损伤出现显着的剂量相关增加，但在 CYP2E1 缺失小鼠中则不然。这些结果首次明确证明，用 AA 处理的小鼠体细胞和生殖细胞的遗传损伤取决于 CYP2E1 将母体化合物代谢为 GA。