Discriminating redox cycling and arylation pathways of reactive chemical toxicity in trout hepatocytes.

The toxicity of four quinones, 2,3-dimethoxy-1,4-naphthoquinone (DMONQ), 2-methyl-1,4-naphthoquinone (MNQ), 1,4-naphthoquinone (NQ), and 1,4-benzoquinone (BQ), which redox cycle or arlyate in mammalian cells, was determined in isolated trout (Oncorhynchus mykiss) hepatocytes. More than 70% of cells died in 3 h when exposed to BQ or NQ; 50% died in 7 h when exposed to MNQ, with no mortality compared to controls after 7 h DMONQ exposure. A suite of biochemical parameters was assessed for ability to discriminate these reactivity pathways in fish. Rapid depletion of glutathione (GSH) with appearance of glutathione disulfide (GSSG) and increased dichlorofluoroscein fluorescence were used as indicators of redox cycling, noted with DMONQ, MNQ, and NQ. Depletion of GSH with no GSSG accumulation, and loss of free protein thiol (PrSH) groups (nonreducible) indicated direct arylation by BQ. All toxicants rapidly oxidized NADH, with changes in NADPH noted later (BQ, NQ, MNQ) or not at all (DMONQ). Biochemical measures including cellular energy status, cytotoxicity, and measures of reactive oxygen species, along with the key parameters of GSH and PrSH redox status, allowed differentiation of responses associated with lethality. Chemicals that arylate were more potent than redox cyclers. Toxic pathway discrimination is needed to group chemicals for potency predictions and identification of structural parameters associated with distinct types of reactive toxicity, a necessary step for development of mechanistically based quantitative structure-activity relationships (QSARs) to predict chemical toxic potential. The commonality of reactivity mechanisms between rodents and fish was also demonstrated, a step essential for species extrapolations.