Glucuronidation of a number of carboxyl-containing drugs generates reactive acyl glucuronide metabolites. Theseelectrophilic species alkylate cell proteins and may be implicated in the pathogenesis of a number of toxic syndromesseen in patients receiving the parent aglycones. Whether acyl glucuronides also attack nuclear DNA is unknown,although the acyl glucuronide formed from clofibric acid was recently found to decrease the transfection efficiency ofphage DNA and generate strand breaks in plasmid DNA in vitro. To determine if such a DNA damage occurs within acellular environment, the comet assay (i.e. single-cell gel electrophoresis) was used to detect DNA lesions in the nucleargenome of isolated mouse hepatocytes cultured with clofibric acid. Overnight exposure to 50 mM and higherconcentrations of clofibric acid produced concentration-dependent increases in the comet areas of hepatocyte nuclei,with 1 mM clofibrate producing a 3.6-fold elevation over controls. These effects closely coincided with culture mediumconcentrations of the glucuronide metabolite formed from clofibric acid, 1-O-b-clofibryl glucuronide. Consistent with arole for glucuronidation in the DNA damage observed, the glucuronidation inhibitor borneol diminished glucuronideformation from 100 mMclofibrate by 98% and returned comet areas to baseline levels. Collectively, these results suggestthat the acyl glucuronide formed from clofibric acid is capable of migrating from its site of formation within theendoplasmic reticulum to generate strand nicks in nuclear DNA. © 2002 Elsevier Science Ireland Ltd. All rights reserved.