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.

工作总结：原代大鼠肝细胞 (HPC) 培养物和肝细胞/非实质细胞 (HPC/ NPC) 共培养物用 Wyeth-14,643 (WY)（一种强效啮齿动物）进行处理 致癌物、复制 DNA 合成和特定 mRNA 通过逆转录酶测定转录水平 聚合酶链反应 (RT-PCR) 和 Northern 印迹。一个 在 48 时检测到 HPC 复制 DNA 合成增加 WY 处理的 HPC 和 HPC/NPC 共培养相对时间 来控制。在 HPC 中，这一增长大约是 3 倍和 6 倍 和 HPC/NPC 文化，分别是 WY 浓度依赖性。 PPARa 转录水平 依赖基因（细胞色素 P4504A1、酰基辅酶 A 氧化酶 （AOxase）和肝脂肪酸结合蛋白（L-FABP）） 转录本被确定为 PPARα 激活的指标。 这些转录物在 48 小时内均呈剂量依赖性增加。 HPC 和 HPC/NPC 培养物浓度高达 10 mM WY。我们也 研究诱导DNA复制的机制 WY合成。 WY诱导的复制DNA合成是 浓度依赖性，并且在 HPC 中显着更大 与单独培养的 HPC 相比，与 NPC 一起培养。 10-十一碳酸抑制 WY 诱导的 DNA 合成 （4504A1抑制剂）以剂量依赖性方式，完全 在 250 mM 时实现抑制。此外，WY诱导的DNA 合成受到吲哚美辛（环氧合酶抑制剂）的抑制 呈剂量依赖性，在 10 mM 时观察到完全抑制。 总之，需要有活性的 P4504A1 和 COXII WY 诱导复制 DNA 合成，并且由于 HPC 和 NPC 是最佳响应所必需的，我们假设 COXII 和 P4504A1 位于 NPC 和 HPC 中， 分别。目前正在进行检查此问题的工作 通过评估 WY 和其他过氧化物酶体的影响来验证假设 从 COXI 和 COXII 中分离出 HPC 和 NPC 上的增殖细胞 体内和体外基因敲除小鼠。