Depletion of cellular protein thiols as an indicator of arylation in isolated trout hepatocytes exposed to 1,4-benzoquinone.
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.
Tapper
M A
MA
U.S. Environmental Protection Agency, National Health and Environmental Effects Laboratory, Mid-Continent Ecology Division, Duluth, Minnesota 55804, USA.
Sheedy
B R
BR
Hammermeister
D E
DE
Schmieder
P K
PK
D000478:Alkylation; D000818:Animals; D016227:Benzoquinones / Q000378:metabolism / Q000633:toxicity*; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004229:Dithiothreitol; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D016227:Benzoquinones; D006873:Hydroquinones; D013438:Sulfhydryl Compounds; C004532:quinone; D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide; C031927:hydroquinone