Acetaminophen (APAP) is a commonly used analgesic agent that has been associated with acute liver and kidney damage when taken in overdoses. APAP or its nephrotoxic metabolite, para-aminophenol (PAP), may be responsible for the kidney damage. APAP-induced kidney damage has not been well investigated and the mechanisms responsible for injury have not been identified. The present proposal is designed to test the hypothesis that cell death due to APAP or PAP, regardless of the outcome, is initiated by apoptosis. Cell death is thought to occur by one of two processes, oncosis (necrosis) or apoptosis. Chemical-induced cytotoxicity has been considered to represent cell death by oncosis, but recent evidence suggests that low concentrations of chemicals may activate apoptotic pathways that allow deletion of sublethally injured cells. The current proposal will test the involvement of oncotic and apoptotic pathways in APAP and PAP-induced cytotoxicity by addressing the following specific aims: (1) examination of the time course and dose-dependence of DNA damage in vivo following administration of APAP and PAP, (2) examination of the time course and concentration-dependence of the appearance of apoptotic markers in vitro following incubation of LLC-PK1 cells with APAP or PAP, (3) examination of cytochrome c release from mitochondria in LLC-PK1 cells following incubation with APAP or PAP, and (4) determination of reactive oxygen intermediate (ROI) formation in LLC-PK1 cells following incubation with APAP or PAP. Evidence documenting apoptosis in vivo will be obtained by examining tissue collected from rats receiving nontoxic and toxic doses of APAP and PAP for DNA damage. Evidence documenting oncosis and apoptosis in vitro will be obtained by examining markers for both pathways in LLC-PK1 cells incubated with various concentrations of APAP and PAP. Mitochondria represent a target for APAP and PAP and mitochondrial damage may cause cytochrome c release from the inner mitochondrial membrane. Cytochrome c release will be investigated by incubating LLC-PK1 cells with concentrations of APAP and PAP shown to cause apoptosis. Cytosol and mitochondrial proteins will be separated by polyacrylamide gel electrophoresis and Western blotting for cytochrome c. ROI formation will be assessed using specific dyes that respond to these chemical intermediates. The results of these studies will shed important insight into mechanisms of cytotoxicity following exposure to APAP and PAP and into the relationship between apoptosis and oncosis.
|Foreman, Brooke D; Tarloff, Joan B (2008) Contribution of reactive oxygen species to para-aminophenol toxicity in LLC-PK1 cells. Toxicol Appl Pharmacol 230:144-9|
|Kendig, Derek M; Tarloff, Joan B (2007) Inactivation of lactate dehydrogenase by several chemicals: implications for in vitro toxicology studies. Toxicol In Vitro 21:125-32|