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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.)

Received April 3, 2003; accepted April 28, 2003.

	Abstract

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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.

Although ROS are generated in many physiological processes, oxidative stress can occur with xenobiotic bioactivation, leading to an imbalance between ROS formation and detoxification (Gutteridge and Halliwell, 2002). Excessive production of ROS, including superoxide radical anions, hydroperoxyl radicals, hydrogen peroxide, and the highly reactive hydroxyl radical, has been implicated in many disease processes, including carcinogenesis and teratogenesis.

Damage to DNA is a critical cellular lesion involved in cell death, carcinogenesis, and teratogenesis (Nicol et al., 1995; Nickoloff, 2002). DNA damage includes arylation, oxidation, and DSBs. Oxidized and arylated DNA is repaired by base excision repair and nucleotide excision repair, and both pathways seem to be important for removing different types of oxidized DNA bases. 2'-Deoxyguanosine (2dG) and thymine can be oxidized to 8-OH-2dG and thymine glycol, respectively, which are typically repaired via base excision repair (Lindahl and Wood, 1999), although these lesions seem to be repaired by nucleotide excision repair if base excision repair capacity is saturated (Klungland et al., 1999). Other forms of oxidized DNA, such as purine cyclodeoxynucleotides, are predominantly repaired by nucleotide excision repair (Kuraoka et al., 2000). Interestingly, regions of single-stranded DNA or DNA nicks produced by these two repair mechanisms can be converted to DSBs, which are then repaired via homologous recombination (Haber, 1999). Therefore, ROS can directly induce DSBs as well as oxidize nucleotides that are subsequently converted to DSBs during replication (Brennan and Schiestl, 1998; Haber, 1999).

DSBs are repaired by homologous recombination or nonhomologous end-joining. Homologous recombination between direct repeats can occur by gene conversion with or without associated crossovers, and by a nonconservative mechanism termed single-strand annealing, with crossovers and single-strand annealing resulting in deletions (Nickoloff, 2002). For larger repeats (>1 kbp), homologous recombination typically involves gene conversion without associated crossovers (Nickoloff, 1992; Taghian and Nickoloff, 1997). Nonhomologous end-joining can be precise if broken ends are ligatable, such as those induced by nucleases, but DSBs initiated by chemical or physical agents are typically repaired by imprecise end-joining, leading to loss (and sometimes gain) of nucleotides at junctions. Although homologous recombination can result in error-free repair of DSBs, some events are associated with rearrangements that may be deleterious, such as large-scale loss of heterozygosity, deletions, duplications, and translocations that contribute to genome instability and carcinogenesis (Nickoloff, 2002).

ROS can be produced from normal metabolic processes and from the bioactivation of xenobiotics, such as phenytoin (Kim et al., 1997). Although antioxidants are essential for ROS detoxification, they cannot completely prevent ROS-initiated DNA damage, and cells have evolved many DNA repair pathways to cope with residual damage. DSBs can be initiated by ROS and repaired through homologous recombination. However, excessive homologous recombination can lead to deleterious genetic changes. We hypothesize that phenytoin-initiated ROS production increases oxidative DNA damage, which results in DSBs that in turn promote aberrant hyper-recombination. The increase in recombination may be an underlying mechanism for phenytoin-initiated toxicity. To gain support for this hypothesis, we used a previously characterized Chinese hamster ovary (CHO) cell line carrying a neo direct repeat recombination substrate. We demonstrate that treatment of cells with phenytoin increases both oxidative DNA damage and homologous recombination. These results provide the first direct evidence implicating increased homologous recombination as a molecular mechanism in the toxicity of phenytoin and, potentially, other ROS-initiating xenobiotics.

	Materials and Methods

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Cell Culture. The previously characterized CHO strain 3-6 carries a single copy of a neo direct repeat recombination substrate (Fig. 1) (Nickoloff, 1992). One copy of neo is driven by the mouse mammary tumor virus promoter but is inactivated by an insertion of a HindIII linker. The second copy of neo has wild-type coding capacity but is inactive because it lacks a promoter. Homologous recombination can produce a functional neo gene that confers resistance to G418 (Invitrogen, Burlington, ON, Canada). Cells were maintained in -minimum essential media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen) at 37°C in 5% CO₂.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Structure of the neo direct repeat recombination substrate. The upstream neo is regulated by the MMTV promoter but is inactivated by insertion of a HindIII linker in the MscI site. The downstream neo has wild-type coding capacity but is inactive because it lacks a promoter. The neo genes flank a simian virus 40 promoter-driven E. coli gpt gene. Homologous recombination by gene conversion without crossover results in loss of the HindIII site (left); crossovers, unequal sister chromatid exchange, or single-strand annealing deletes one copy of neo and SVgpt. All of these events result in loss of the HindIII site and confer G418 resistance.

Analysis of DNA Oxidation. Cells were treated as described above, except after 5- or 24-h exposure to phenytoin, genomic DNA was immediately isolated from cells by using a DNeasy tissue kit (QIAGEN, Valencia, CA) and processed to nucleosides according to Ravanat et al. (1998). 8-OH-2dG and 2dG were separated using a Sulpelco LC18-DB 5-µm column (250 x 4.6 mm) under isocratic conditions, which consisted of a mobile phase of 5% methanol and 95% 100 mM sodium acetate buffer, pH 5.2. The separated nucleosides were then detected using a CoulArray electrochemical detector (ESA, Inc., Chelmsford, MA) at two separate oxidizing potentials of 500 and 750 mV for 8-OH-2dG and 2dG, respectively.

Recombination Assays. Homologous recombination frequencies were determined by seeding CHO strain 3-6 cells at a density of 1 x 10⁶/10-cm-diameter culture dish. Plating efficiency (cell survival) was determined by seeding cells at a density of 300 cells/10-cm dish. After 5 h, cells were treated with phenytoin (80, 240, or 800 µM; Sigma-Aldrich, St. Louis, MO) or the vehicle control (0.002 N NaOH) for 5 or 24 h. After drug exposure, cells were washed with phosphate-buffered saline and fresh medium containing G418 (500 µg/ml) was added. For plating efficiency, cells were treated as described above except G418 was omitted. Cells were grown for either 1 week to score plating efficiency, or 2 weeks to score homologous recombination, and the resulting colonies were stained with 1% crystal violet in methanol and counted. Homologous recombination frequencies were calculated as the number of G418-resistant colonies per viable cell plated in G418 medium (Nickoloff, 1992). Recombinant product structures were determined by Southern hybridization using a 1.4-kbp neo fragment as probe as described previously (Nickoloff, 1992).

Statistical Analysis. Results were analyzed using a standard, computerized statistical program (GraphPad Prism 3.0; GraphPad Software Inc., San Diego, CA). Groups were compared using a one-factor analysis of variance and a Dunnett's post test. The minimum level of significance used throughout was p < 0.05.

	Results

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Phenytoin-Induced DNA Oxidation. Phenytoin is known to create ROS and its cytotoxic and teratogenic effects can be blocked by superoxide dismutase, catalase, or antioxidants (Wells and Winn, 1997). To examine DNA oxidation by phenytoin, we measured the levels of 8-OH-2dG in CHO strain 3-6 treated with various concentrations of phenytoin, including a therapeutically relevant concentration and higher concentrations to yield further mechanistic information, for either 5 or 24 h. We observed increased levels of 8-OH-2dG with increasing phenytoin concentration and increasing exposure time (Fig. 2). Exposure to 80, 240, or 800 µM phenytoin for 5 h significantly increased 8-OH-2dG compared with vehicle control (p 0.04). With 24-h exposures, 240 and 800 µM phenytoin significantly increased 8-OH-2dG versus vehicle control (p 0.007). With a 24-h exposure to 80 µM phenytoin, 8-OH-2dG increased 5-fold but this was not significantly different than the control level due to high variability in this experiment.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Phenytoin-initiated DNA oxidation. Levels of DNA oxidation in CHO strain 3-6 cells treated with phenytoin (80, 240, and 800 µM) for 5 or 24 h. Values represent averages for five determinations (+ S.D.). Significant differences from the vehicle-treated group (0.002 N NaOH) are indicated by the asterisk (p < 0.05) and by the daggers (p < 0.001).

Phenytoin is Cytotoxic at High Concentrations. Despite the high level of DNA damage in cells treated with 80 to 240 µM phenytoin (Fig. 2), these concentrations had no effect on cell survival with either 5- or 24-h treatments (Fig. 3). At 800 µM, cell survival was reduced to 50% of untreated cells with a 5-h exposure and to 30% with a 24-h exposure. It is important to note that although both plating efficiency and homologous recombination frequency were scored at later times than DNA oxidation, both of the former assays measure early effects of DNA damage. The data shown in Figs. 2 and 3 indicate that the cytotoxic effects of phenytoin are not directly proportional to the level of DNA damage, suggesting that the DNA repair pathway(s) that processes these lesions become saturated at phenytoin concentrations above 240 µM.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Phenytoin-initiated cell death. Percentage of cell survival of CHO strain 3-6 after 5- or 24-h exposure to various doses of phenytoin as indicated. Cell survival was determined by calculating the number of colonies formed after 1 week divided by the total number of cells plated. Values represent averages (+ S.D.) for four determinations. Asterisks indicate differences from the vehicle-treated group (0.002 N NaOH) (p < 0.05).

Phenytoin Induces Homologous Recombination. Many DNA-damaging agents stimulate homologous recombination, including UV, ionizing radiation, and genotoxic chemicals. To determine whether increased levels of oxidative DNA damage in phenytoin-treated cells correlate with increased recombination, we determined the frequency of G418-resistant recombinants of CHO strain 3-6 cells exposed for 5 or 24 h to 80 to 800 µM phenytoin (Fig. 4). With 5-h exposures, 240 and 800 µM phenytoin significantly enhanced recombination by at least 2-fold (p < 0.05), and with 24-h exposures, recombination was significantly enhanced at all three doses by 2- to 4-fold (p < 0.001). The neo direct repeats in CHO strain 3-6 allow detection of gene conversion without associated crossover, which preserves the gross structure of the recombination substrate, and deletion events that result in loss of one copy of neo and the intervening sequences (Fig. 1). To determine the types of homologous recombination events induced by phenytoin, genomic DNA from G418-resistant colonies was isolated and digested with ScaI and HindIII and analyzed via Southern blot, using a 1.4-kbp neo fragment as a probe (Nickoloff, 1992). Gene conversion products result in a single 7.9-kbp band and deletion products result in a 2.6-kbp band; in either case, the HindIII site is lost. We isolated genomic DNA from a total of 26 G418-resistant colonies from cells treated with either 80, 240, or 800 µM phenytoin. All 26 recombinants displayed a single 7.9-kbp band (Table 1), indicating that all arose by gene conversion without associated crossovers. Conversion without crossover also was predominant for spontaneous events in CHO strain 3-6 (Nickoloff, 1992) and for DSB-induced recombination in CHO cells carrying a related neo recombination substrate (Taghian and Nickoloff, 1997).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Phenytoin-initiated homologous recombination. Frequency of homologous recombination in CHO strain 3-6 cells treated with various doses of phenytoin as indicated for 5 or 24 h. Homologous recombination frequency was determined by counting the number of G418-resistant colonies versus the total number of surviving cells plated. Numbers in parentheses indicate the number of independent determinations per treatment. Significant differences from the vehicle-treated group (0.002 N NaOH) are indicated by the asterisk (p < 0.05) and by the daggers (p < 0.001).

	Discussion

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Phenytoin can be bioactivated by peroxidases to free radical intermediates, initiating the formation of ROS that can oxidize lipid, protein, and DNA (for review, see Wells and Winn, 1997). Superoxide dismutase, catalase, antioxidants such as caffeic acid, vitamin E, N-acetyl-cysteine (a glutathione precursor), and the free radical trapping agent phenyl-butylnitrone can protect against toxicity and teratogenesis induced by phenytoin and other xenobiotics, including benzo[a]pyrene and thalidomide (Winn and Wells, 1995b, 1999; Parman et al., 1999; for review, see Wells and Winn, 1997).

Because DNA is the sole repository of genetic information, it is a critical molecular target of ROS-initiated toxicity. If not repaired, oxidative modifications to DNA have been shown to disrupt transcription, translation, and replication, and to give rise to mutations and ultimately cell death (Ames et al., 1993). In bacterial studies, mutagenicity initiated by phenytoin is dependent upon P450-catalyzed enzymatic bioactivation, requiring preincubation with a metabolic activating system (S9 liver fraction) (Sezzano et al., 1982). However, a subsequent study gave conflicting results as phenytoin was not mutagenic in all bacterial strains tested (Leonard et al., 1984). Conflicting results have also been reported for phenytoin-induced sister chromatid exchange in patients. Hadebank et al. (1982) found a significant increase in sister chromatid exchange in phenytoin monotherapy patients, whereas Hunke and Carpenter (1978) found no difference. More recently Kaul and Goyle (1999) demonstrated that phenytoin monotherapy increases sister chromatid exchange in epilepsy patients that was independent of the disease itself. Phenytoin induces micronucleus formation in rat skin fibroblasts (Kim et al., 1997) and studies in vivo, in vitro, and in cultured embryos, have shown that phenytoin increases DNA oxidation, producing 8-OH-2dG (this study; Liu and Wells, 1995; Winn and Wells, 1995b). Thus, substantial evidence indicates that phenytoin is a strong genotoxin.

ROS induces DNA damage, including base damage and apurinic/apyrimidinic sites that are thought to be repaired primarily by the base excision repair pathway, although nucleotide excision repair seems to provide a back-up function (Huang et al., 1994; Klungland et al., 1999). A number of studies indicated that various toxicants, including carcinogens, can induce DNA damage and homologous recombination in mammalian cells (Wang et al., 1988), yeast (for review, see Bishop and Schiestl, 2000), and bacteria (Quinto and Radman, 1987). Many of these toxicants can covalently bind and/or oxidize DNA, or form interstrand cross-links. Furthermore, in yeast cells DNA recombination stimulated by the known human leukemogen benzene is diminished by the free radical scavenger N-acetyl cysteine (Brennan and Schiestl, 1998), consistent with the idea that ROS-induced DNA damage is recombinogenic. Here, we provide the first evidence that phenytoin induces homologous recombination.

There are two distinct mechanisms by which oxidative DNA damage could induce homologous recombination. Base excision and nucleotide excision repair produce single-strand strand breaks or gaps that can be converted to recombinogenic DSBs when encountered by the replication machinery. Alternatively, unrepaired DNA damage may block replication, and the damage can be bypassed by a recombinational mechanism in which the undamaged sister chromatid is used as a template. Thus, in the first mechanism, recombination is dependent on repair activity, whereas the second operates in the absence of repair. UV-induced DNA damage strongly induces recombination, and in mammalian cells, recombination is enhanced to a greater extent when nucleotide excision repair is reduced or absent (Bhattacharyya et al., 1990; Deng and Nickoloff, 1994). Thus, at least for UV, recombination is enhanced by damage, not by repair. In yeast, spontaneous recombination is enhanced in mutants with defects in base and nucleotide excision repair pathways (Swanson et al., 1999), indicating that unrepaired base damage is processed by one or more recombinational pathways. In contrast, oxidative DNA damage induced by nitric oxide induces recombination in Escherichia coli by a mechanism that is promoted by base excision repair enzymes (Spek et al., 2002). Thus, for oxidative DNA damage, recombination may be stimulated by base excision repair-dependent and -independent mechanisms. Further studies are required to distinguish these possibilities.

Various DNA alterations may be directly dependent on DSBs that result from oxidative DNA damage (Bishop and Schiestl, 2000). DSBs induced by oxidative DNA damage are formed through the loss of at least one nucleotide in each DNA strand at the break site; therefore, in the absence of some form of nucleolytic process these ends are virtually unligatable (Pastwa et al., 2001). DSB repair by nonhomologous end-joining or homologous recombination can result in limited or large-scale loss of heterozygosity, deletions, insertions, and translocations (Nickoloff, 2002). Inappropriate DSB repair can lead to oncogenic activation of transcription factors after chromosomal rearrangements produced by aberrant immunoglobulin V(D)J site-specific recombination (Cleary, 1991). It is also well known that loss of heterozygosity and gene deletions are associated with increased cancer risk (Gonzalez et al., 1999) and that chromosomal rearrangements such as translocations are prevalent in hematologic cancers and sarcomas (Elliott and Jasin, 2002). Many studies in yeast have shown that genotoxins, including those that produce oxidative damage, stimulate deletions between direct repeats (for review, see Bishop and Schiestl, 2000). It is important to note that the recombination substrate used in these yeast studies was designed to detect deletions via crossovers, single-strand annealing, or unequal sister chromatid exchange, but does not detect conservative gene conversion. We show here that phenytoin induces high levels of oxidative DNA damage and recombination between direct repeats, primarily involving gene conversion without associated crossovers. Although we did not detect deletions in our limited analysis of recombinant products, it seems likely that high-level oxidative DNA damage producing genome-wide increases in recombination will result in deleterious rearrangements.

Although we did not evaluate phenytoin-initiated homologous recombination in embryos, the teratogenic effects of phenytoin may reflect its mutagenic and/or recombinogenic effects. Interestingly, cells with defective p53 display hyper-recombination phenotypes (Bertrand et al., 1997; Mekeel et al., 1997), and p53-deficient mice are more susceptible to the teratogenicity of phenytoin (Laposa et al., 1996) and benzo-[a]pyrene (Nicol et al., 1995). Thus, the enhanced teratogenic effects of phenytoin in p53-defective mice may be due to additive or synergistic increases in recombination induced by oxidative damage in a p53-defective background. Our results are consistent with the hypothesis that phenytoin-initiated ROS formation increases oxidative DNA damage that can stimulate homologous recombination, and thus we propose that ROS-initiated homologous recombination may be an important mechanism mediating the teratogenicity of phenytoin. Both large-scale chromosomal rearrangements and more subtle genetic alterations in the embryo are known to cause physical and mental defects. In light of the linkage between replication and recombination (Haber, 1999), we propose that phenytoin-induced homologous recombination may be particularly important in embryonic tissues that undergo rapid replication and differentiation.

	Footnotes

 
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 Sci 66 (Suppl 1):235].

DOI: 10.1124/jpet.103.052639.

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.

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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