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JPET #52639
1
April 28, 2003
OXIDATIVE STRESS-INDUCED HOMOLOGOUS RECOMBINATION AS A NOVEL
MECHANISM FOR PHENYTOIN-INITIATED TOXICITYa
Louise M. Winn, Perry M. Kim and Jac A. Nickoloff
Department of Pharmacology and Toxicology and School of Environmental Studies, Queen’s
University, Kingston, Ontario, Canada (LMW, PMK) and Department of Molecular Genetics
and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico,
U.S.A. (JAN).
Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.
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a) Running Title: Homologous recombination and phenytoin toxicity
b) Corresponding author: Louise M. Winn, Department of Pharmacology and Toxicology
and School of Environmental Studies, Queen’s University, Kingston, Ontario, Canada.
Email: winnl@biology.queensu.ca, Telephone: (613) 533-6465, Fax: (613) 533-6412.
c) Number of text pages: 12
Number of tables: 1
Number of figures: 4
Number of references: 40
Number of words in the Abstract: 182
Number of words in the Introduction: 653
Number of words in the Discussion: 1081
d) List of abbreviations:
CHO, Chinese hamster ovary, 2dG, 2’-deoxyguanosine; DSB, double-strand break; 8-
OH-2dG, 8-hydroxy-2’-deoxyguanosine; ROS, reactive oxygen species.
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ABSTRACT
While 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 (ROS) 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
(CHO) cell line containing a neo direct repeat recombination substrate to determine whether
phenytoin-initiated DNA oxidation increases homologous recombination. Cells were treated
with 0-800 µM phenytoin for 5 or 24 hr, 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 (8-OH-2dG). 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
chemical-initiated toxicity.
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The toxicity associated with the anticonvulsant drug phenytoin, including teratogenesis,
may be due to its bioactivation to reactive intermediates (reviewed in: Winn and Wells, 1995a).
Phenytoin can be bioactivated by peroxidases such as prostaglandin H synthase to free radical
intermediates that can initiate the formation of reactive oxygen species (ROS) such as hydroxyl
radicals (Kim and Wells, 1996), which in turn may oxidize lipid, protein and DNA (Liu and
Wells, 1995; Winn and Wells, 1995b, 1999). This damage can ultimately lead to embryopathy
which can be completely abolished by the actions of either superoxide dismutase or catalase
(Winn and Wells 1995b, 1999), implicating a role for oxidative stress in phenytoin-initiated
toxicity.
While ROS are generated in many physiological processes, oxidative stress can occur
with xenobiotic bioactivation leading to an inbalance 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 appear 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 appear 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
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(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 (Haber, 1999; Brennan
and Schiestl, 1998).
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). While 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
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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.
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MATERIALS AND METHODS
Cell Culture
The previously characterized CHO strain 3-6 carries a single copy of a neo direct repeat
recombination substrate (figure 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
(Gibco Life Technologies, Burlington, ON). Cells were maintained in α-minimum essential
media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco Life
Technologies, Burlington, ON) at 37°C in 5% CO2.
Analysis of DNA Oxidation
Cells were treated as above, except after 5 or 24 hr exposure to phenytoin, genomic DNA
was immediately isolated from cells by using a DNeasy Tissue Kit (Qiagen Inc., 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.) at two separate oxidizing potentials of 500 mV and 750 mV for 8-OH-2dG and 2dG,
respectively.
Recombination Assays
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Homologous recombination frequencies were determined by seeding CHO strain 3-6
cells at a density of 1 x 106 per 10 cm (diameter) culture dish. Plating efficiency (cell survival)
was determined by seeding cells at a density of 300 cells per 10 cm dish. After 5 hr cells were
treated with phenytoin (80, 240 or 800 µM; Sigma Chemical Co., St. Louis, MO) or the vehicle
control (0.002N NaOH) for 5 or 24 hr. After drug exposure, cells were washed with PBS and
fresh medium containing G418 (500 µg/ml) was added. For plating efficiency, cells were treated
as above except G418 was omitted. Cells were grown for either 1 week to score plating
efficiency, or two 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 (Nickoloff, 1992).
Statistical Analysis
Results were analyzed using a standard, computerized statistical program (GraphPad
Prism 3.0). Groups were compared using a one factor analysis of variance (ANOVA) and a
Dunnett’s Post Test. The minimum level of significance used throughout was p<0.05.
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RESULTS
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 dose and higher
doses to yield further mechanistic information, for either 5 or 24 hr. We observed increased
levels of 8-OH-2dG with increasing phenytoin concentration and increasing exposure time
(figure 2). Exposure to 80, 240 or 800 µM phenytoin for 5 hr significantly increased 8-OH-2dG
compared to vehicle control (p<0.04). With 24 hr exposures, 240 and 800 µM phenytoin
significantly increased 8-OH-2dG vs. vehicle control (p<0.007). With a 24 hr 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.
Phenytoin is Cytotoxic at High Concentrations
Despite the high level of DNA damage in cells treated with 80-240 µM phenytoin (figure
2), these concentrations had no effect on cell survival with either 5 or 24 hr treatments (figure 3).
At 800 µM, cell survival was reduced to ∼50% of untreated cells with a 5 hr exposure, and to
∼30% with a 24 hr exposure. It is important to note that while both plating efficiency and
homologous recombination frequency were scored at later time periods than DNA oxidation,
both of the former assays measure early effects of DNA damage. The data shown in figures 2
and 3 indicate that the cytotoxic effects of phenytoin are not directly proportional to the level of
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DNA damage, suggesting that the DNA repair pathway(s) that process these lesions become
saturated at phenytoin concentrations above 240 µM.
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 hr to
80-800 µM phenytoin (figure 4). With 5 hr exposures, 240 and 800 µM phenytoin significantly
enhanced recombination by at least 2-fold (p<0.05), and with 24 hr 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 (figure 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,
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1992) and for DSB-induced recombination in CHO cells carrying a related neo recombination
substrate (Taghian and Nickoloff, 1997).
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DISCUSSION
Phenytoin can be bioactivated by peroxidases to free radical intermediates, initiating the
formation of ROS that can oxidize lipid, protein and DNA (reviewed in: 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 phenylbutylnitrone, can protect against
toxicity and teratogenesis induced by phenytoin and other xenobiotics including benzo[a]pyrene
and thalidomide (Parman et al., 1999; Winn and Wells, 1995b, 1999; reviewed in: 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, while 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
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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 appears 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
(reviewed in: Bishop and Schiestl, 2000), and bacteria (Quinto and Radman, 1987). Many of
these toxicants can covalently bind and/or oxidize DNA, or form interstrand crosslinks.
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 (Deng and Nickoloff, 1994;
Bhattacharyya et al. 1990). Thus, at least for UV, recombination is enhanced by damage, not by
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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 E. 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
following 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 (reviewed in: 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
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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 to 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 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.
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Evidence for reactive oxygen species-mediated DNA oxidation in the molecular mechanism of
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FOOTNOTES
a) 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, 2002.
b) Send reprint requests to: Louise M. Winn, Department of Pharmacology and Toxicology
and School of Environmental Studies, Queen’s University, Kingston, Ontario, Canada.
Email: winnl@biology.queensu.ca, Telephone: (613) 533-6465, Fax: (613) 533-6412.
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Table 1. Phenytoin-initiated homologous recombination products.
Dose Gene conversion products Deletion products
80 µM (n=10) 10 0
240 µM (n=4) 4 0
800 µM (n=12) 12 0
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FIGURE LEGENDS
Figure 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 an SV40-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.
Figure 2. Phenytoin-initiated DNA oxidation. Levels of DNA oxidation in CHO strain 3-6
cells treated with phenytoin (80, 240, 800 µM) for 5 or 24 hr. Values represent averages for 5
determinations (+SD). Significant differences from the vehicle-treated group (0.002N NaOH) are
indicated by the asterisk (p<0.05) and by the daggers (p<0.001).
Figure 3. Phenytoin-initiated cell death. Percent cell survival of CHO strain 3-6 after 5 or 24
hr exposure to various doses of phenytoin as indicated. Cell survival was determined by
calculating the number of colonies formed after one week divided by the total number of cells
plated. Values represent averages (+SD) for 4 determinations. Asterisks indicate differences
from the vehicle-treated group (0.002N NaOH) (p<0.05).
Figure 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 hr. Homologous recombination frequency was determined by counting the number of
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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 (0.002N NaOH) are indicated by the asterisk (p<0.05)
and by the daggers (p<0.001).
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ScaI ScaI
7.9 kbp Sca I fragment 2.6 kbp Sca I fragment
ScaI
ScaI ScaI
HindIIISVgpt
SVgpt
neo
neo
Gene conversionwithout crossover Deletion
MMTVneo
MMTVneo+ MMTVneo+
ScaI
FIGURE 1
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0 80 240 8000
5
10
155 hr Incubation24 hr Incubation
FIGURE 2
*
Phenytoin Concentrations (µM)
*
*
*
†
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0
25
50
75
100
*
*
FIGURE 3
0 80 240 800 0 80 240 800
5 hr 24 hr
Phenytoin Concentration (µM)
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0.0
2.5
5.0
7.5
10.0
5 hr 24 hr
(8)(8)
(8)
(5)
(20)
(18) (19)
(10)
*
0 80 240 800 0 80 240 800
Phenytoin Concentration (µΜ)
FIGURE 4
†
†† †
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