MECHANISMS OF TOXICITY IN SINUSOIDAL ENDOTHELIAL CELLS

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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).