METHYLENE DIANILINE--A SELECTIVE BILE DUCT TOXICANT?

DAPM(4，4‘-二氨基二苯甲烷)被认为是一种选择性胆管 毒物，因为DAPM对胆管上皮造成形态损伤 肝内胆管细胞(BEC)对肝细胞无影响 并迅速损害胆道功能，特别是葡萄糖重吸收 来自胆道，但不改变肝细胞的转运 功能。人们对DAPM和其他疾病的机制知之甚少 破坏胆管的药物会破坏胆管。DAPM后3小时，BEC 显示线粒体超微结构改变，管腔丢失 高尔基体池的微绒毛和扩张。关于肝脏的研究 DAPM治疗3小时后线粒体未见改变 酶活性，线粒体通透性特征，或 组织化学染色模式。BEC线粒体的观察 是DAPM损伤的早期部位可以解释DAPM损伤的速度和严重性 DAPM造成的损害，并提供了一个独特的机会 探讨胆道毒物的作用机制。 我们的总体目标是了解DAPM是如何以及为什么导致早期的， 选择性损伤胆管细胞。此应用程序的重点是 胆汁中排出的DAPM代谢物及其对BEC的损伤能力。 我们的实验方法将使用25毫克/公斤的DAPM剂量，这会导致 BEC中中度损伤性的功能/结构改变，但 不改变胆汁流量或损伤肝细胞。代谢物的研究 麻醉大鼠胆汁排泄和胆道功能 十二指肠内注入牛磺胆酸盐以维持胆汁流动。目标1 将阐明DAPM对紧密连接渗透性的影响 细胞旁标记和细胞化学超微结构方法。目标2 将表征体内的DAPM代谢物，特别是胆汁中的代谢物 将使用分离的细胞在体外评估DAPM的细胞毒性 肝细胞与BEC。目标3将确定抑制剂是否 结合反应减少了BEC和BEC中的Il相酶 肝细胞，通过测定确定可能的DAPM的近端毒物 胆汁中DAPM排泄对大鼠损伤程度的影响 在体内调节，并评估可疑近端的相对毒性 毒物的体外细胞毒性测定。我们的新数据提供了这样的信息 DAPM对BEC的选择性作用的有力证据，特别是 在BEC线粒体上，一个新的AIM 4提出了生理和 体外培养的BEC线粒体功能的生化研究 使用DAPM。 拟议中的研究将对我们的理解做出重大贡献 BEC损伤的机制，并提供了一种新的模型系统 关于BEC在胆汁形成中的作用的问题。

DAPM (4,4'-diaminodiphenylmethane) is proposed as a selective bile duct toxicant because DAPM causes morphological injury to biliary epithelial cells (BEC) of the intrahepatic bile ducts without effects on hepatocytes and rapidly impairs biliary functions, particularly glucose reabsorption from the biliary traCt, but does not alter hepatocellular transport functions. Little is known about the mechanisms by which DAPM and other cholangiodestructive agents damage bile ducts. By 3 hr after DAPM, BEC show ultrastructural alterations in mitochondria, loss of lumenal microvilli and dilation of Golgi Cisternae. Studies of liver mitochondria 3 hr after DAPM treatment demonstrate no alterations in enzyme activities, mitochondrial permeability characteristics, or histochemical staining patterns. The observation that BEC mitochondria are an early site of DAPM injury may explain the rapidity and severity of lesions caused by DAPM, and provides a unique opportunity to investigate mechanisms of bile duct toxicants. Our overall objective is to understand how and why DAPM causes early, selective injury to bile duct cells. The focus of this application will be on DAPM metabolites excreted in bile and their capacity to damage BEC. Our experimental approach will use the 25 mg/kg dose of DAPM which causes moderately injurious functional/structural alterations in BEC but does not alter bile flow or damage hepatocytes. Studies of metabolite excretion in bile and biliary function will be done in anesthetized rats infused with taurocholate intraduodenally to maintain bile flow. AIM 1 will clarify the effects of DAPM on tight junction permeability using paracellular markers and cytochemical ultrastructural methods. AIM 2 will characterize DAPM metabolites in vivo, particularly in bile, and will use isolated cells to assess DAPM cytotoxicity in vitro in hepatocytes versus BEC. AIM 3 will determine if inhibitors of conjugation reactions decrease Phase Il enzymes in both BEC and hepatocytes, identify possible proximate toxicants of DAPM by determining extent of injury in rats whose biliary excretion of DAPM has been modulated in vivo, and assess relative toxicities of suspect proximate toxicants by in vitro cytotoxicity assays. Our new data provide such strong evidence of the selective effect of DAPM on BEC, and particularly on BEC mitochondria, that a new AIM 4 proposes physiological and biochemical studies of mitochondrial function in isolated BEC treated with DAPM. The proposed research will contribute significantly to our understanding of the mechanisms of BEC injury and provide a new model system to ask questions about the role of BEC in bile formation.