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TOXICOLOGY

Oxidative Stress-Induced Homologous Recombination As a Novel Mechanism for Phenytoin-Initiated Toxicity

Department of Pharmacology and Toxicology and School of Environmental Studies, Queen's University, Kingston, Ontario, Canada (L.M.W., P.M.K.); and Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico (J.A.N.)

Although the mechanism(s) of phenytoin-initiated toxicity is unknown, phenytoin can be enzymatically bioactivated to a reactive intermediate leading to increased formation of reactive oxygen species, which can damage essential macromolecules, including DNA. The oxidation of DNA can induce DNA double-strand breaks (DSBs), which may be repaired through homologous recombination. Increased levels of DSBs may induce hyper-recombination, leading to deleterious genetic changes. We hypothesize that these genetic changes mediate phenytoin-initiated toxicity. To investigate this hypothesis we used a Chinese hamster ovary cell line containing a neo direct repeat recombination substrate to determine whether phenytoin-initiated DNA oxidation increases homologous recombination. Cells were treated with 0 to 800 µM phenytoin for 5 or 24 h, and homologous recombination frequencies and recombinant product structures were determined. Phenytoin-initiated DNA oxidation was determined by measuring the formation of 8-hydroxy-2'-deoxyguanosine. We demonstrate that phenytoin increases both DNA oxidation and homologous recombination in a concentration- and time-dependent manner. All recombination products analyzed arose via gene conversion without associated crossover. Our data demonstrate that phenytoin-initiated DNA damage can induce homologous recombination, which may be a novel mechanism mediating phenytoin-initiated toxicity.

Address correspondence to: Dr. Louise M. Winn, Department of Pharmacology and Toxicology and School of Environmental Studies, Queen's University, Kingston, ON, Canada. E-mail: winnl{at}biology.queensu.ca

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