Little is known about the way that allyl alcohol causes hepatocellular necrosis. The damage occurs after metabolic conversion of the alcohol into acrolein, a highly reactive aldehyde that produces lethal cellular injury. Previous studies in isolated hepatocytes indicated that modification of protein thiol groups is important for allyl alcohol-mediated cytotoxicity and that lipid peroxidation is not essential for lethality. The present application proposes to continue the investigation of allyl alcohol's toxic mechanism by examining specific toxic events that might result from protein thiol modification and lead eventually to cell death. The proposed work takes advantage of an intervention (post-treatment with dithiothreitol) that permits interruption of the toxic process at a stage before irreversible cell injury has been initiated. This ability to rescue hepatocytes at an early stage in the toxic process provides a useful tool for determining which events are necessary for the eventual loss of cell viability. Four biochemical mechanisms of cell injury will be explored using this model: 1) disturbance of calcium homeostasis, 2) compromise in energy statue, 3) collapse of the mitochondrial membrane potential, and 4) alteration of pyridine nucleotide ratios. Measurements will be made at various time points during the development of toxicity to see if allyl alcohol causes a rise in cytosolic Ca2+, depletion of ATP, leakage of triphenylmethylphosphonium cation from mitochondria, and changes in NAD, NADH, NADP or NADPH concentrations in the hepatocytes. The approach will be to determine the timing of these biochemical disturbances in relation to the occurrence of glutathione depletion, protein sulfhydryl loss, appearance of cell surface protrusions, and loss of viability. The primary mechanism of toxicity should occur in allyl alcohol-exposed cells, it should precede other signs of toxicity, and it should be prevented or reversed by post-treatment with dithiothreitol. The proposed research will contribute mainly to our understanding of allyl alcohol-induced cytotoxicity, but information about the sequence of events that culminates in cell death from this toxicant may also be relevant to irreversible damage from other chemicals. The long term goal of this research is to understand more about the genesis of lethal injury from toxic compounds.
