Oxidative stress-induced homologous recombination as a novel mechanism for phenytoin-initiated toxicity.
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 microM 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.
Winn
Louise M
LM
Department of Pharmacology and Toxicology and School of Environmental Studies, Queen's University, Kingston, ON, Canada. winnl@biology.queensu.ca
Kim
Perry M
PM
Nickoloff
Jac A
JA
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D064420:Drug-Related Side Effects and Adverse Reactions; D005260:Female; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000502:physiology*; D010672:Phenytoin / Q000494:pharmacology*; D011995:Recombination, Genetic / Q000187:drug effects* / Q000502:physiology*
D010672:Phenytoin; D004247:DNA