MECHANISMS OF TOXICITY IN SINUSOIDAL ENDOTHELIAL CELLS

肝静脉闭塞症(Hvod)是一种经常致命的并发症。 在骨髓移植前使用的化疗药物， 尤其是在活体无关的骨髓移植中。长期的 目的：探讨毒素诱导的高密度脂蛋白血症的发病机制。 以开发治疗方案。我的初步工作表明，毒素 可能对肝窦内皮细胞有选择性毒性 (SEC)而不是肝细胞，谷胱甘肽(GSH)可能参与其中 在抵御所研究的毒素方面。具体目标：工作 假设Secs是导致HVOD的毒素的目标，并且 严重的GSH耗竭是毒性的常见机制。具体的 目标是：(1)确定在肝脏中的靶细胞的药物 引起HVOD并检查为什么毒性针对肝脏；(2) 检查GSH动态平衡并以秒为单位确定潜在的前体；(3) 表征谷胱甘肽动态平衡的破坏与 细胞死亡；以及(4)建立体内模型，以在体外证实 调查结果并为今后的调查提供线索。方法：(1)体外培养 毒性研究将在Secs、肝细胞和非肝细胞中进行 使用硫唑嘌呤、白消安、达卡巴津的内皮细胞和上皮细胞 和野百合碱；这些类型的细胞代谢各自的能力 将对毒素进行检测。(2)在SECs和非肝内皮细胞中，GSH 合成速率将在无细胞提取物中确定，外排将 在培养细胞中被测量；含硫氨基酸和 完整的GSH补充GSH将在SECS和非肝脏中进行比较 内皮细胞。(3)GSH对毒素的保护作用将得到确认 在秒内，谷胱甘肽耗竭的时间进程将与 毒性；谷胱甘肽耗竭的原因(合成障碍、氧化应激或 接合作用)将被研究；将寻找毒素- 诱导的细胞CSH水平变化，线粒体功能障碍和 毒性，以及这种相关性是否持续存在于 保护剂(抗氧化剂、自由基清除剂和Ru 红色)。(4)建立多次给药的小鼠体内模型 毒素。在体内模型中，控制GSH水平的效果将 通过提供合成SEC GSH的前体和通过 使用抗氧化剂和自由基清除剂(去铁胺)。

Hepatic veno-occlusive disease (HVOD) is a frequently fatal complication of chemotherapeutic agents used prior to bone marrow transplantation, especially in living-unrelated bone marrow transplantation. The long-term objective is to explore the mechanisms involved in toxin-induced HVOD and to develop therapeutic options. My preliminary work suggests that toxins that induce HVOD may be selectively toxic to sinusoidal endothelial cells (SECs) rather than hepatocytes and that glutathione (GSH) may be involved in protecting against the toxins studied. Specific Aims: The working hypothesis is that SECs are the target of toxins that cause HVOD and that profound GSH depletion is the common mechanism of toxicity. The specific aims are: (1) to determine the target cell in the liver for agents that cause HVOD and examine why toxicity is directed toward the liver; (2) to examine GSH homeostasis and determine potential precursors in SECs; (3) to characterize the relationship between disruption of GSH homeostasis and cell death; and (4) to develop an in vivo model to confirm in vitro findings and provide leads for future investigation. Methods: (1) In vitro toxicity studies will be done in SECs, hepatocytes and non-hepatic endothelial and epithelial cells using azathioprine, busulfan, dacarbazine and monocrotaline; the ability of these cell types to metabolize each toxin will be examined. (2) In SECs and non-hepatic endothelial cells, GSH synthetic rates will be determined in cell free extracts and efflux will be measured in cultured cells; the ability of sulfur amino acids and intact GSH to replete GSH will be compared in SECs vs non-hepatic endothelial cells. (3) GSH protection against the toxins will be confirmed in SECs; time-course of GSH depletion will be correlated with onset of toxicity; cause of GSH depletion (impaired synthesis, oxidant stress or conjugation) will be examined; correlation will be sought between toxin- induced change in cellular CSH levels, mitochondrial dysfunction and toxicity and whether the correlation persists in the presence of protective agents (antioxidants, free radical scavengers and ruthenium red). (4) An in vivo model will be developed in mice using multiple doses of toxin. In the in vivo model the effect of manipulating GSH levels will be examined by providing precursors for the synthesis of SEC GSH and by administering anti-oxidants and free radical scavengers (deferoxamine).