COMPARISON OF METABOLISM AND EXPOSURE BIOMARKERS IN ANIMAL AND HUMAN TISSUES

动物和人体组织中代谢和暴露生物标志物的比较

• 批准号: 6106668
• 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/6106668
• 关键词: acyl coA; biomarker; carcinogen testing; chemical carcinogen; chemical carcinogenesis; cytochrome P450; drug metabolism; environment related neoplasm /cancer; environmental toxicology; enzyme activity; herbicides; human tissue; laboratory mouse; laboratory rat; liver neoplasms; mixed tissue /cell culture; mutagen testing; mutagens; oxidoreductase; peroxisome

Summary of Work: Primary rat hepatocyte (HPC) cultures and Hepatocyte/nonparenchymal cell (HPC/ NPC) cocultures were treated with Wyeth-14,643 (WY), a potent rodent carcinogen, and replicative DNA synthesis and specific mRNA transcript levels were determined by reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting. An increase in HPC replicative DNA synthesis was detected at 48 hours in both WY- treated HPC and HPC/NPC cocultures relative to controls. This increase was approximately 3 and 6 fold in HPC and HPC/NPC cultures, respectively, and was WY concentration-dependent. The levels of PPARa-transcriptionally dependent genes (cytochrome P4504A1, acyl-CoA oxidase (AOxase), and liver-fatty acid binding protein (L-FABP)) transcripts were determined as indicators of PPARa activation. These transcripts increased dose- dependently at 48 hours in both HPC and HPC/NPC cultures up to 10 mM WY. We also investigated the mechanisms of induction of replicative DNA synthesis by WY. WY-induced replicative DNA synthesis was concentration-dependent and was significantly greater in HPC incubated with NPC compared to HPC cultured separately. WY-induced DNA synthesis was inhibited by 10-undecynoic acid (4504A1 inhibitor) in a dose-dependent manner, with complete inhibition achieved at 250 mM. Further, WY-induced DNA synthesis was inhibited by indomethacin (cyclooxygenase inhibitor) in a dose- dependent with complete inhibition observed at 10 mM. In conclusion, an active P4504A1 and COXII are required for the induction of replicative DNA synthesis by WY, and since both HPC and NPC are required for an optimal response, we hypothesized that COXII and P4504A1 are localized in NPC and HPC, respectively. Work is currently in progress to examine this hypothesis by assessing the effects of WY and other peroxisome proliferators on HPC and NPC isolated from COXI and COXII knockout mice both in vivo and in vitro.

工作摘要：初级大鼠肝细胞（HPC） 培养和肝细胞/非核细胞（HPC/ NPC） 通过有效的啮齿动物Wyeth-14,643（WY）处理共培养 致癌和复制性DNA合成和特定mRNA 通过反向转录酶确定转录水平 聚合酶链反应（RT-PCR）和北印迹。一个 在48处检测到HPC复制性DNA合成的增加 WY处理过的HPC和HPC/NPC共培养相关的小时 进行控件。 HPC的增长约为3和6倍 和HPC/NPC培养物，并且是WY 浓度依赖性。 PPARA转录的水平 依赖基因（细胞色素P4504a1，酰基-COA氧化酶 （AOXASE）和肝脂肪酸结合蛋白（L-FABP）） 确定转录本作为PPARA激活的指标。 这些成绩单在48小时时在48小时内增加了剂量 HPC和HPC/NPC培养物高达10毫米WY。我们也是 研究了复制DNA诱导的机制 WY的合成。 WY诱导的复制DNA合成为 浓度依赖性，HPC明显更大 与分别培养的HPC相比，与NPC孵育。 WY诱导的DNA合成被10-毫秒酸抑制 （4504a1抑制剂）以剂量依赖性方式，完整 在250毫米处实现的抑制作用。此外，WY诱导的DNA 合成被吲哚美辛（环氧合酶抑制剂）抑制 在剂量依赖性中，在10 mm处观察到完全抑制。 总之，需要一个主动的P4504A1和COXII WY诱导复制DNA合成，并且由于HPC都是 最佳响应需要NPC，我们假设 该Coxii和P4504A1位于NPC和HPC中， 分别。目前正在进行研究以检查此事 假设通过评估WY和其他过氧化物酶体的影响 从Coxi和Coxii分离的HPC和NPC上的扩散剂 敲除体内和体外的小鼠。