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DEPLETION OF CELLULAR PROTEIN THIOLS AS AN INDICATOR OF ARYLATION IN ISOLATED TROUT HEPATOCYTES EXPOSED TO 1,4-BENZOQUINONE
Citation:
Tapper, M A., B R. Sheedy, D E. Hammermeister, AND P. K. Schmieder. DEPLETION OF CELLULAR PROTEIN THIOLS AS AN INDICATOR OF ARYLATION IN ISOLATED TROUT HEPATOCYTES EXPOSED TO 1,4-BENZOQUINONE. TOXICOLOGICAL SCIENCES 55:327-334, (2000).
Description:
A method for the measurement of protein thiols (PrSH), un-reacted as well as oxidized, i.e. dithiothreitol recoverable, was adapted for the determination of PrSH depletion in isolated rainbow trout hepatocytes exposed to an arylating agent, 1,4-benzoquinone (BQ). Toxicant analysis revealed rapid conversion of BQ to 1,4-hydroquinone (HQ) upon addition to hepatocytes. Hepatocytes exposed to 200 uM BQ + HQ showed 80% decline in glutathione (GSH) (1 h), 30% loss of PrSH (6 h), and no loss of viability (24 h). Recoverable oxidized PrSH was detected only after 24 h (200 uM BQ + HQ). Exposure to 600 uM BQ + HQ caused rapid (10 min) loss of > 90% GSH and > 60% PrSH, with eventual cell death. Half of the PrSH depletion at 6 h observed in hepatocytes exposed to 600 uM BQ + HQ was recoverable by reduction with dithiothreitol. Following the loss of GSH in hepatocytes exposed to 600 uM BQ + HQ, , cellular PrSH were susceptible to direct arylation and oxidation. Rainbow trout hepatocytes, which contained 10-fold less GSH than rat cells, had a GSH:PrSH ratio of 1:82 compared with rat ratios of 1:2 to 1:6. The methods reported are useful for further study and discrimination of reactive modes of action needed for prediction of aquatic organism susceptibility to these types of toxicants.