Acetaminophen, a safe analgesic when dosed properly but hepatotoxic at overdoses, has been reported to induce DNA strand breaks but it is unclear whether this event preceeds hepatocyte toxicity or is only obvious in case of overt cytotoxicity. Moreover, it is not known whether the formation of reactive oxygen species (ROS) is involved in the formation of the DNA strand breaks. In the present study, the dose-response curves for cytotoxicity and DNA strand breaks and the response to antioxidant protection have been compared. In primary hepatocytes from untreated male rats, cytotoxicity as measured by the MTT test and by Neutral Red accumulation was obvious at 10 mM acetaminophen but DNA strand breaks as measured by the comet assay were only found at 25-30 mM acetaminophen. Non-cytotoxic concentrations of three compounds with antioxidant activity, the glutathione precursor N-acetylcysteine (100 micro M), the plant polyphenol silibin (25 micro M) and the antioxidant vitamin alpha-tocopherol (50 micro M), were not able to inhibit acetaminophen toxicity at any acetaminophen concentration, while they completely prevented the formation of DNA strand breaks at 25-30 mM acetaminophen. The occurrence of oxidative stress in our experiments was indicated by a slight increase of malondialdehyde formation at 40 mM acetaminophen and by an adaptive increase in catalase mRNA concentration. We conclude that in acetaminophen-treated hepatocytes ROS-independent cell death and ROS-dependent DNA strand breaks occur which appear not to be causally related as judged from their dose dependency and their response to antioxidants.