Depletion of cellular protein thiols as an indicator of arylation in isolated trout hepatocytes exposed to 1,4-benzoquinone

• Published in: Toxicological sciences 2000-06, Vol.55 (2), p.327-334
• Main Authors: TAPPER, M. A, SHEEDY, B. R, HAMMERMEISTER, D. E, SCHMIEDER, P. K
• Format: Article
• Language: English
• Subjects: Agnatha. Pisces; Alkylation; Animal, plant and microbial ecology; Animals; Applied ecology; arylation; Benzoquinones - metabolism; Benzoquinones - toxicity; Biological and medical sciences; Cell Separation; Cell Survival - drug effects; Cells, Cultured; Dithiothreitol; Ecotoxicology, biological effects of pollution; Effects of pollution and side effects of pesticides on vertebrates; Female; Fundamental and applied biological sciences. Psychology; Glutathione - metabolism; Glutathione Disulfide - metabolism; Hydroquinones - metabolism; Liver - cytology; Liver - drug effects; Liver - metabolism; Male; Oncorhynchus mykiss; Oncorhynchus mykiss - metabolism; Oxidation-Reduction; protein thiols; quinone; Sulfhydryl Compounds - metabolism
• ISSN: 1096-6080; 1096-0929; 1096-0929
• DOI: 10.1093/toxsci/55.2.327

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 microM 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 microM BQ+HQ). Exposure to 600 microM 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 microM BQ+HQ was recoverable by reduction with dithiothreitol. Following the loss of GSH in hepatocytes exposed to 600 microM 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.