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Toxicological Sciences 55, 327-334 (2000)
Copyright © 2000 by the Society of Toxicology

Depletion of Cellular Protein Thiols as an Indicator of Arylation in Isolated Trout Hepatocytes Exposed to 1,4-Benzoquinone

M. A. Tapper, B. R. Sheedy, D. E. Hammermeister and P. K. Schmieder1

U.S. Environmental Protection Agency, National Health and Environmental Effects Laboratory, Mid-Continent Ecology Division, Duluth, Minnesota 55804

A method to measure protein thiols (PrSH), reduced and oxidized, was adapted to determine PrSH depletion in isolated rainbow trout hepatocytes exposed to 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 µM 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 µM BQ+HQ). Exposure to 600 µM 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 µM BQ+HQ was recoverable by reduction with dithiothreitol. Following the loss of GSH in hepatocytes exposed to 600 µM 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.

Key Words: glutathione; 1,4-hydroquinone; redox cycling; Oncorhynchus mykiss; monobromobimane.


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P. K. Schmieder, M. A. Tapper, R. C. Kolanczyk, D. E. Hammermeister, B. R. Sheedy, and J. S. Denny
Discriminating Redox Cycling and Arylation Pathways of Reactive Chemical Toxicity in Trout Hepatocytes
Toxicol. Sci., March 1, 2003; 72(1): 66 - 76.
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