Abstract
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.
Footnotes
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These studies were supported by the National Cancer Institute of the National Institutes of Health (CA77693 to J.A.N.) and by the Queen's University Research Advisory Committee (to L.M.W. and P.M.K.). A preliminary report of this research was presented at the 41st Annual Meeting of the Society of Toxicology (U.S.A.) [Toxicol Sci66 (Suppl 1):235].
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DOI: 10.1124/jpet.103.052639.
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ABBREVIATIONS: ROS, reactive oxygen species; DSB, double-strand break; 2dG, 2′-deoxyguanosine; 8-OH-2dG, 8-hydroxy-2′-deoxyguanosine; kbp, kilobase pair(s); CHO, Chinese hamster ovary.
- Received April 3, 2003.
- Accepted April 28, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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