ToxSci Advance Access originally published online on May 19, 2006
Toxicological Sciences 2006 93(1):146-155; doi:10.1093/toxsci/kfl022
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Variable In Vivo Embryoprotective Role for Ataxia-Telangiectasia–Mutated against Constitutive and Phenytoin-Enhanced Oxidative Stress in Atm Knockout Mice
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* Faculty of Pharmacy and Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 3M2
3 To whom correspondence should be addressed at Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada. Fax: +1 416 267 7797. E-mail: pg.wells{at}utoronto.ca.
Received January 6, 2006; accepted April 30, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Knockout mice lacking the ataxia-telangiectasia–mutated (Atm) protein exhibit impaired detection and repair of DNA damage and increased embryopathies from ionizing radiation in vivo, and vehicle or phenytoin in embryo culture. Here we determined if Atm-deficient mice are more susceptible in vivo to phenytoin embryopathies. Wild-type (+/+) or heterozygous (+/–) Atm knockout dams were mated with +/– males, pregnant dams were treated with phenytoin (65 mg/kg ip) or its vehicle, and resorptions and fetuses were genotyped and characterized. This strain proved resistant to phenytoin-initiated cleft palates but not to other spontaneous and phenytoin-enhanced embryopathies. With vehicle-treated +/– dams, fetal body weight was lower in homozygous Atm-null (–/–) fetuses compared to +/– and +/+ littermates (p < 0.05). Phenytoin enhanced this Atm-dependent embryopathic pattern (p < 0.05). It also enhanced DNA oxidation in –/– Atm-deficient embryos compared to its +/– Atm-deficient (p < 0.001) and +/+ Atm-normal (p < 0.001), phenytoin-exposed littermates and to its –/– vehicle controls (p < 0.01). Postpartum lethality was greater in both +/– and –/– Atm-deficient fetuses compared to +/+ littermates, independent of treatment (0.05 < p < 0.1). By maternal genotype, +/– Atm-deficient dams had fewer implantations than +/+ dams, independent of treatment, and phenytoin decreased litter size (p < 0.05). Conversely, phenytoin-exposed +/+ fetuses were more likely than –/– littermates to die in utero (p < 0.05), and in +/+ dams fetal resorptions and postpartum lethality were variably higher and enhanced by phenytoin (p < 0.05). Despite variable actions in vivo, the embryoprotective effects of Atm suggest a role for reactive oxygen species and oxidative DNA damage in some spontaneous and phenytoin-enhanced embryopathies.
Key Words: Atm; ataxia-telangiectasia; oxidative stress; reactive oxygen species; phenytoin; development; embryopathy; developmental toxicology.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ataxia-telangiectasia–mutated (ATM) protein is involved in the detection and repair of DNA damage or initiation of apoptosis (Banin et al., 1998
; Canman et al., 1998). The molecular details of this action and its role in the disease ataxia-telangiectasia (A-T) are provided in Bhuller and Wells (2006). In brief, ATM deficiency is associated with a spectrum of clinical manifestations (Meyn, 1999), including enhanced sensitivity to ionizing radiation (IR), genetic instability, and enhanced tumorigenesis (Kamsler et al., 2001; Xu and Baltimore, 1996). Irradiated Atm-deficient cells have impaired repair of DNA double-strand breaks (Rotman and Shiloh, 1999) and exhibit elevated residual chromosomal damage (Pandita and Hittelman, 1992). Adult Atm-null knockout mice accumulate oxidative DNA damage (Kamsler et al., 2001; Quick and Dugan, 2001), but Atm-null mice have not been reported to exhibit birth defects or other developmental abnormalities, suggesting that ATM may not be required for normal development.
Oxidative stress and the accompanying macromolecular damage have been implicated in several diseases (Halliwell and Gutteridge, 1999) including A-T (Kamsler et al., 2001; Quick and Dugan, 2001) but are particularly important in teratogenesis because the developing embryo has relatively little protection against both endogenous and exogenous sources of reactive oxygen species (ROS) (Nicol et al., 2000; Wells and Winn, 1996; Wells et al., 2005). Knockout mice deficient in p53 are also more susceptible to the embryopathic effects of several DNA-damaging teratogens (Moallem and Hales, 1998; Nicol et al., 1995), including the ROS-initiating anticonvulsant drug phenytoin (Laposa and Wells, 1995; Wong and Wells, 2002), suggesting the potential importance of DNA damage and repair in teratogenesis. This hypothesis is supported by in vivo studies in which Atm-deficient embryos were more susceptible to the teratogenic effects of IR (Laposa et al., 2004) and by in vitro embryo culture studies showing a similar gene dose-dependent susceptibility of Atm-deficient embryos to the ROS-initiating teratogen phenytoin (Bhuller and Wells, 2006), suggesting that Atm, like p53, may serve as a teratological suppressor gene.
The ability of ATM to respond to other types of DNA damage, such as DNA adducts or oxidized DNA, is uncharacterized. However, IR enhances the formation of ROS, including hydroxyl radicals, which can produce an array of oxidative damage to cellular molecules (Halliwell and Gutteridge, 1999), not all of which result in DNA double-strand breaks. Furthermore, ROS-mediated oxidative damage resulting from even endogenous oxidative stress can have embryopathic effects when antioxidative pathways are compromised (Nicol et al., 2000). Since ATM is an upstream regulator of p53 (Banin et al., 1998; Canman et al., 1998), which is also a regulator in the genotoxic response pathway and a teratological suppressor gene (Bhuller and Wells, 2006, Fig. 1; Nicol et al., 1995), we hypothesized that Atm may have a significant developmental role in protecting the embryo from more subtle forms of oxidative DNA damage caused by endogenous and xenobiotic-enhanced ROS.
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Our results herein corroborate this hypothesis and provide the first in vivo evidence that Atm is expressed in the embryo during organogenesis and protects against phenytoin-enhanced oxidative DNA damage and some spontaneous and phenytoin-enhanced embryopathies, including fewer implantations, decreased fetal weight, and possible postpartum lethality. In contrast, Atm appeared to enhance the risk of fetal resorptions. Although Atm appeared to protect against postpartum lethality measured by fetal genotype, the incidence of postpartum lethality was lower in +/– than +/+ dams, suggesting that maternal genotype may modulate the teratological outcome. Overall, Atm appears to be responsive to subtle ROS effects and plays a complex role in the developmental response to constitutive and drug-enhanced oxidative stress.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
8-Oxo-2'-deoxyguanosine (8-oxo-dG) was obtained from Cayman Chemical Co (Ann Arbor, MI). Nuclease P1 and Escherichia coli alkaline phosphatase were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). Redistilled phenol was from Aldrich Chemical Co (Oakville, Ontario, Canada), chloroform:isoamyl alcohol:phenol (CIP, 24:1:25) from Life Technologies, Inc (Burlington, Ontario, Canada), and proteinase K and complete, Mini, EDTA-free protease inhibitor cocktail tablets from Roche Diagnostics (Laval, Quebec, Canada). PCR primers were purchased from ACGT Corporation (Toronto, Ontario, Canada). Taq polymerase and dNTPs were purchased from Perkin Elmer (Mississauga, Ontario, Canada). All other reagents used were of analytical or high-performance liquid chromatography (HPLC) grade.
Animals.
Heterozygous (+/–) Atm-deficient dams (129S6/SvEv Tac-Atmtm1Awb, Jackson Laboratory, Bar Harbor, ME) were mated with CD-1 males in order to generate a sustainable Atm colony. The animals were housed in plastic cages with ground corncob bedding (Beta Chip; Northeastern Products, Warrensburg, NY) and maintained in a temperature-controlled room with a 12-h light/dark cycle. Food (Laboratory Rodent Chow 5001; PMI Feeds, St Louis, MO) and tap water were provided ad libitum. Third-generation Atm +/+ or +/– females were mated with +/– males for the study, since –/– males and females generated for this study were infertile, consistent with previously published results (Barlow et al., 1996; Yamashita et al., 1996). One male was housed with three +/– or +/+ females from 5:00 P.M. to 9:00 A.M. the next day. The presence of a vaginal plug was considered as gestational day (GD) 1, and these females were separated from the colony and housed together in groups of up to four animals per cage.
Evaluation of fetal development.
On GDs 12 and 13, pregnant Atm +/+ and +/– females were treated ip with a teratogenic dose of phenytoin (65 mg/kg) (Sigma Chemical Co, St Louis, MO) dissolved in 0.9% NaCl containing 0.002 N NaOH or its vehicle. Pregnant CD-1 mice (Charles River Canada, St Constant, Quebec, Canada), which are known to be susceptible to phenytoin teratogenicity, including cleft palates (Wells et al., 2005), were similarly treated as positive controls. On GD 19, 1 day prior to spontaneous delivery, females were sacrificed by cervical dislocation. Following laparotomy, the uterine horns were exteriorized and the location and number of resorptions (embryonic death) and fetuses noted. The number of implantations was calculated by adding the total number of fetuses born alive or dead with the number of resorptions. For each parameter, the data were analyzed for each litter, and the mean was determined for all litters. Fetal resorptions were calculated by dividing the number of resorptions by the total number of implantations (resorptions + fetuses). In addition, resorptions were dissected free of the uterus and analyzed for embryonic Atm genotype. Resorptions with a particular genotype were compared according to both the total number of resorptions and the total number of implantations. The fetuses were removed and viable fetuses placed under a heat lamp at 30°C for 2 h to determine postpartum survival. Postpartum lethality was calculated by dividing the total number of fetuses born alive and dying within the 2 h by the total number of viable fetuses born, for a given fetal genotype. At the end of the period, all the fetuses were weighed, sexed, and examined for any other abnormalities. Prior to fixing the fetuses in Carnoy's solution (100% absolute ethanol, chloroform, and glacial acetic acid [6:3:1, vol/vol/vol]) (Sigma Chemical Co) for at least a week, tail snips were taken from the fetuses for Atm genotyping. Resorptions were dissected from the uterus, immediately snap frozen in liquid nitrogen, and stored at – 80°C for genotyping. After fixation was complete, fetuses were examined for cleft lips and palates and other external malformations.
DNA isolation.
To determine the potential role for DNA oxidation as a potential molecular target mediating phenytoin teratogenicity in Atm-deficient embryos, Atm +/– dams (Jackson Laboratory) were mated to +/– males. Pregnant females were treated on GDs 12 and 13 with a teratogenic dose of phenytoin or its vehicle. On GD 13, 6 h after treatment, pregnant dams were sacrificed under isoflurane (Bimeda-MTC, Animal Health Inc, Cambridge, Ontario, Canada) anesthesia, and the embryos and yolk sacs were removed, snap frozen in liquid nitrogen, and stored at – 80°C until analyzed. Yolk sacs were used as the source for genotyping the embryos. The cerebellum was also removed from the pregnant dams and, prior to being snap frozen, separated from the rest of the brain.
Nuclear DNA was isolated from the embryo, the maternal cerebellum, and "whole" brain (without the cerebellum) (Nicol et al., 2000). Briefly, each sample was homogenized (T8-Turrax, IKA Scientific, Wilmington, NC) in 500 µl of DNA buffer (100mM Tris-HCl [pH 8], 5mM EDTA [pH 8], 0.2% SDS, and 200mM NaCl) prior to incubation overnight with proteinase K (Sigma) at 55°C. Samples were transferred, using glass pipettes, to phase-lock gel microfuge tubes containing approximately 0.1 ml of phase extraction gel. The samples were thoroughly extracted with one volume (500 µl) of CIP (24:1:25 in Tris-HCl buffer) (GIBCO Laboratories, Toronto, Ontario, Canada), thoroughly shaken (no vortex), and separated by microcentrifugation at maximum speed, for 1 min. The supernatant was then transferred to a new tube and the DNA precipitated by adding 250 µl of 7.5M ammonium acetate and two original volumes (1 ml) of 100% precooled (– 20°C) ethanol. Samples were thoroughly shaken, and the DNA was pelleted by microcentrifugation for 1 min. The 100% ethanol was aspirated and the pellet washed with 750 µl of precooled (– 20°C) 75% ethanol to wash off excess salt. The sample was thoroughly shaken (no vortex) and the DNA pelleted again by centrifugation at maximum speed for 1 min in a microcentrifuge. The 75% ethanol was aspirated and the pellet dried with nitrogen gas for approximately 4 s per tube. The dried pellet was then dissolved in 500 µl of 0.1M phosphate buffer (PBS) (pH 7.4) and incubated at 65°C for at least 2 h to facilitate solubilization. The samples were cooled on ice for about 10 min, and prior to incubation at 37°C, 2.5 µl of ribonuclease T1 (50 units/ml) (Sigma) and 20 µl of ribonuclease A (100 µg/ml) (Sigma) were added to digest residual RNA. The samples were then transferred to phase-lock gel microfuge tubes, one volume of CIP was used to reextract the dissolved DNA, followed by microcentrifugation for 1 min, and the DNA was precipitated as described above. The isolated DNA pellet was dissolved in 500 µl of 20mM sodium acetate buffer (pH 4.8) and incubated at 65°C for 1 h. The DNA was quantified using an UV/Vis spectrophotometer (Lambda 3 System, Perkin Elmer, Ltd, Woodbridge, ON) at 260 nm with calf thymus DNA as the standard. The isolated DNA samples were digested to nucleotides by incubation at 37°C with 6.7 µl of nuclease P1 (67 µg/ml) (Sigma) for 30 min, followed by the addition of 34 µl of 1M Tris-HCl (pH 7.4). The samples were mixed using a vortex and incubated at 37°C, for 60 min, with 10.5 µl of E. coli alkaline phosphate (0.37 units/ml) (Sigma). The resulting mixture was filtered (0.22 µm) and analyzed by HPLC with electrochemical detection.
Detection of 8-oxo-dG.
Oxidation of 2'-dG to 8-oxo-dG was quantified using an isocratic Series 200 HPLC system (Perkin Elmer Instruments LLC, Shelton, CT) equipped with a 5-µm Exsil 80A-ODS C-18 column (5 cm x 4.6 mm, Jones Chromatography, Ltd, Brockville, ON), an electrochemical detector (Coulochem II), a guard cell (model 5020), an analytical cell (model 5010) (Coulochem, ESA Inc, Chelmsford, MA), and an integrator (Perkin Elmer NCI 900 Interface). Samples were filtered (0.22 µm), injected into the HPLC-EC system, and eluted using a mobile phase consisting of 50mM KH2PO4 buffer (pH 5.5)–methanol (95:5, vol/vol) at a flow rate of 0.8 ml/min with a detector oxidation potential of + 0.4 V (Winn and Wells, 1995). Chromatographs were analyzed using a commercial chromatography software program (TotalChrom version 6.2.0, Perkin Elmer Instruments LLC).
Genotyping.
Resorptions, tail snips from dams and fetuses, and yolk sacs from embryos (see below) were genotyped by a PCR-based assay using DNA that was purified using a resin-based DNA extraction kit (Qiagen, Mississauga, ON). Sample preparation and reaction conditions were as described previously (Bhuller and Wells, 2006), and the PCR products were separated on a gel consisting of 1.5% (wt/vol) agarose, 89mM Tris, 89mM boric acid, and 2mM EDTA.
Immunoprecipitation/Western blotting.
The cerebellum from Atm +/+ and –/– dams was separated from the rest of the brain, and the cerebellum and whole brain (without the cerebellum) were immediately snap frozen in liquid nitrogen for subsequent determination of protein levels. Cerebellum from +/+ mice was used as a positive control because the presence of degenerative neurons has been reported in the cerebellar cortex of Atm-deficient mice (Kuljis et al., 1997), and in humans the ATM protein has been detected in cerebellar cortex but not in the cerebral cortex (Oka and Takashima, 1998). Wild-type whole brain (without the cerebellum) and Atm –/– cerebellum were used as negative controls. Wild-type embryos resulting from the mating of Atm +/+ males and females were removed from nontreated dams on GDs 12 and/or 13 and individual embryos snap frozen in liquid nitrogen and stored at – 80°C. Individual Atm +/+ embryos and cerebella were homogenized mechanically for 1 min (T8-Turrax, IKA Scientific) on ice in 300 µl of RIPA buffer (1x PBS, 1% Nonidet P-40 [Amaresco, Solon, OH], 0.5% sodium deoxycholate, and 0.1% SDS), which was supplemented immediately prior to use with a mammalian protease inhibitor cocktail (Sigma). Brain samples (without cerebellum) were also homogenized for 1 min in 1 ml of RIPA buffer. Cellular debris was removed by centrifugation at 10,000 x g for 5 min in a microcentrifuge for 10 min at 4°C. The supernatant was removed and total cellular protein quantified using a high-sensitivity adaptation of the Lowry assay (Markwell et al., 1981). A 500-µg sample of total cellular protein was used in order to immunoprecipitate the protein of interest as described previously (Bhuller and Wells, 2006). Proteins were separated electrophoretically under reducing conditions and the bands detected with a chemiluminescence kit (ECL, Amersham Pharmacia Biotech, Piscataway, NJ) (Bhuller and Wells, 2006).
Statistical analysis.
The statistical significance of differences between treatment groups in each study group was determined using a standard computerized statistical program (SigmaStat software, version 2.03; Jandel Scientific, San Rafael, CA). For each parameter, the data were analyzed for each litter and the mean was determined for all litters. Groups were compared using one-way ANOVA, and the significance of differences between specific pairs was determined by a Tukey test or Dunn's method. Binomial data were examined using the chi-square test or Fisher's exact test. A probability of p < 0.05 was chosen as the level of statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phenytoin Teratogenicity
Phenytoin causes a characteristic syndrome of fetal teratological anomalies, including cleft palate in susceptible murine models, such as the CD-1 strain (Wells et al., 2005
). This was confirmed in a preliminary validation study using CD-1 mice, in which phenytoin increased the incidence of cleft palates, fetal resorptions, and postpartum lethality and decreased fetal body weight, compared to the vehicle controls (p < 0.05) (Fig. 1). This CD-1 study served as a positive control for a comparison with the Atm strain, which has not been characterized for its in vivo susceptibility to phenytoin teratogenicity. The results from the Atm study indicated that this strain is resistant to phenytoin teratogenicity. Specifically, no cleft palates or other external anomalies were observed in any Atm wild-type or knockout fetuses exposed to phenytoin. Nevertheless, this strain exhibited Atm-dependent susceptibility to several other embryopathic effects of endogenous and phenytoin-enhanced oxidative stress.
Analysis by Fetal Genotype
Mean fetal body weight.
In untreated (vehicle controls) +/– dams, which produce all three fetal genotypes, fetal body weight was 38% lower in Atm-null (–/–) fetuses compared to +/– Atm-deficient fetuses and 43% lower compared to +/+ Atm-normal littermates (p < 0.05) (Fig. 2).
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In +/– dams, phenytoin decreased the body weight of –/– Atm-deficient fetuses by 35 and 36%, respectively, compared to their +/– Atm-deficient and +/+ Atm-normal, phenytoin-exposed littermates (p < 0.05), and by 10% compared to –/– vehicle controls (p < 0.05) (Fig. 2). Furthermore, phenytoin decreased the body weights of Atm +/+ and +/– fetuses by 19 and 13% compared to their respective vehicle controls (p < 0.05).
In contrast, with +/+ dams, which produce only +/+ and +/– fetal Atm genotypes, Atm-dependent effects on fetal body weight were not observed in either vehicle- or phenytoin-exposed fetuses, nor did phenytoin reduce fetal weight compared to vehicle controls with either fetal genotype (Fig. 2, inset).
Fetal postpartum lethality.
In +/– dams treated with either vehicle or phenytoin, postpartum lethality in both +/– and –/– fetuses was between two- and threefold greater than that in +/+ Atm-normal littermates, although this apparent enhancement was not statistically significant (0.5 < p 
0.1) (Fig. 3). There was no apparent enhancement by phenytoin in any fetal genotype.
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In +/+ dams, phenytoin appeared to enhance postpartum lethality in both fetal genotypes, with increases of 83 and 63% in +/+ and +/– fetuses, respectively, compared to their vehicle-exposed littermates of the matching genotype, but this trend was not significant (p = 0.07) (Fig. 3, inset). There was no Atm-dependent effect between the two fetal genotypes.
Fetal resorptions.
In +/– dams treated with phenytoin, there were fewer –/– Atm-deficient fetal resorptions compared to both +/– Atm-deficient and +/+ Atm-normal fetal resorptions from the same litters (p < 0.05). There were no –/– Atm-deficient resorptions following either vehicle or phenytoin exposure, and all the implantations with this genotype resulted in viable fetuses. For both vehicle- and phenytoin-treated +/– dams, there appeared to be a gene dose-dependent increase in the number of fetal resorptions from –/– to +/– to +/+, which was more apparent when exposed to phenytoin, but this was not significant (0.5 < p <1) (Fig. 4).
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In +/+ dams, phenytoin treatment appeared to increase the number of fetal resorptions for both +/+ and +/– Atm genotypes, in comparison to their respective vehicle controls, but this increase was not significant (0.05 < p < 1) (Fig. 4, inset).
Analysis by Maternal Genotype
Atm-dependent embryoprotective effects against endogenous and/or phenytoin-enhanced oxidative stress were observed for some but not all parameters. Embryoprotection was observed in +/+ Atm-normal dams for implantation in both vehicle- and phenytoin-exposed embryos and for litter size at birth in phenytoin-exposed embryos (Fig. 5), as detailed below.
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In contrast, both vehicle- and phenytoin-treated +/+ dams had a higher incidence of resorption than the respectively treated +/– dams, and phenytoin significantly enhanced resorptions over vehicle controls only in +/+ dams, as detailed below. In addition, +/+ dams treated with phenytoin had a higher incidence of postpartum lethality than phenytoin-treated +/– dams, and phenytoin enhanced postpartum lethality only in +/+ dams.
Independent of treatment, implantations were 41% lower in +/– dams than +/+ dams (p < 0.05) (Fig. 5A). Furthermore, Atm +/+ dams treated with vehicle had a total of 119 implantations, 56 of which were +/+ and 63 were +/–, a ratio of 1:1.125, similar to the theoretical ratio of 1:1, predicted from Mendelian genetics. A 1:1 ratio was also seen in phenytoin-treated +/+ dams, wherein 58 of the total implantations were +/+ and 56 were +/–. Similarly, Atm +/– dams treated with vehicle or phenytoin had implantations with genotypes in the ratio of approximately 1:2:1 for +/+, +/–, and –/– implantations, respectively (data not shown), also as predicted from Mendelian genetics.
Litter size at birth.
In +/– dams treated with phenytoin, the litter size at birth was reduced compared to its vehicle-treated control (p < 0.05) (Fig. 5B). There was no Atm-dependent effect with either treatment, and phenytoin did not affect litter size in +/+ dams. Phenytoin-treated +/+ dams on average gave birth to approximately six fetuses, which was similar to the number born from +/– dams treated with vehicle.
Fetal resorptions.
Independent of treatment, Atm +/+ dams exhibited higher incidence of fetal resorptions compared to treated +/– dams (p < 0.05) (Fig. 5C). In +/+ dams, phenytoin enhanced the incidence of resorptions compared to vehicle controls (p < 0.05), and a similar but nonsignificant trend was observed in +/– dams (p = 0.08).
Resorptions were dissected from the uterus and subjected to PCR analysis to determine the embryonic genotype. Despite the potential for DNA degradation during disintegration, 100% of a total of 113 resorptions were genotyped. Overall, +/– dams had no resorption with a –/– genotype, compared to a substantial collective incidence with +/– (45%) and +/+ (55%) genotypes in +/+ and +/– dams, independent of treatment conditions (p < 0.05) (data not shown).
Postpartum lethality.
Atm +/+ dams treated with phenytoin showed an increase in postpartum lethality compared to vehicle controls (p < 0.05), whereas no phenytoin effect was observed in +/– dams (Fig. 5D). An Atm-dependent effect on postpartum lethality was observed in phenytoin-treated animals, with +/+ dams having a higher incidence than +/– dams (p < 0.05). No Atm-dependent effect on postpartum lethality was observed between the vehicle-treated control groups, which exhibited an incidence similar to that observed in phenytoin-treated +/– dams.
Biochemical Analysis of Embryos
Embryoprotective role for Atm against phenytoin-enhanced DNA oxidation.
In +/– dams treated with phenytoin, DNA oxidation was enhanced 1.7- and 2.1-fold in –/– Atm-deficient embryos compared to its +/– Atm-deficient (p < 0.001) and +/+ Atm-normal (p < 0.001), phenytoin-exposed littermates, respectively, and 1.8-fold compared to its –/– vehicle controls (p < 0.01) (Fig. 6). DNA oxidation was enhanced 1.3-fold in both +/– Atm-deficient and +/+ Atm-normal, phenytoin-exposed embryos compared to their vehicle controls; however, this was not statistically significant. A trend for a gene dose-dependent pattern was seen in both vehicle- and phenytoin-treated +/+ Atm-normal and +/– and –/– deficient embryos.
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Embryonic Atm protein expression during organogenesis.
Immunochemical detection was used to determine whether and to what degree Atm was expressed in +/+ Atm-normal embryos during organogenesis. Substantial Atm expression was detected in GDs 12 and 13 embryos compared to positive and negative controls from adult animals (Fig. 7). As a positive control, Atm was expressed in adult Atm +/+ cerebellum, while in negative controls, Atm was not expressed in adult Atm +/+ whole brain (without cerebellum), nor in adult Atm-deficient –/– cerebellum.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results show for the first time that Atm is expressed in the developing mouse embryo in vivo during organogenesis. The protein level was evaluated to determine whether wild-type embryos express Atm during this period when the embryo is at risk for various spontaneous embryopathies, including in utero death. This is also the critical gestational period for susceptibility to phenytoin teratogenicity. The substantial levels of embryonic Atm protein observed herein are consistent both with a previously published study that found an increase in Atm mRNA, albeit not protein activity, in rat embryos (Vinson and Hales, 2003
) and with the Bhuller and Wells (2006) study showing a substantial level of Atm protein found in murine embryos in culture.
Atm was found to exert a protective effect against some, albeit not all, spontaneous and phenytoin-enhanced embryopathies. Atm-deficient mice were more susceptible to phenytoin-enhanced embryonic oxidative DNA damage and to fewer implantations, decreased litter size, decreased fetal body weight, and possibly increased neonatal postpartum lethality either arising spontaneously and/or initiated by phenytoin. Several lines of evidence suggest that the protective mechanism may involve Atm-dependent repair of oxidative DNA damage resulting from constitutive or phenytoin-enhanced formation of ROS. This evidence includes (1) the known role of ATM in DNA repair, (2) the substantial levels of embryonic Atm protein observed herein, (3) the enhanced embryonic oxidative DNA damage reported in published studies of several ROS-initiating teratogens including phenytoin (Wells et al., 2005), and (4) the enhanced oxidative DNA damage in phenytoin-exposed, –/– Atm-deficient embryos in vivo, observed herein.
Compared to CD-1 mice and other strains susceptible to the truly teratogenic effects of phenytoin, evidenced by structural birth defects, the Atm strain proved to be resistant in vivo to the cleft palates characteristic of phenytoin teratogenicity. Nevertheless, the protective pattern observed for other embryopathies is consistent with the complementary in vitro study of Bhuller and Wells (2006) of this Atm knockout strain in embryo culture, which avoids the confounding effects of maternal processes. This embryo culture study found for all parameters that Atm protected against the embryopathic effects of both endogenous and phenytoin-enhanced oxidative stress in a gene dose-dependent fashion, with heterozygotes exhibiting a clear intermediate risk for most embryopathies (Bhuller and Wells, 2006). In the in vivo study herein, an apparent intermediary susceptibility for +/– fetuses was not statistically significant, possibly in part because this strain was relatively resistant to phenytoin embryopathies. This strain resistance appears at least in part to involve reduced susceptibility to oxidative stress, since fetal oxidative DNA damage was only enhanced by phenytoin in –/– Atm fetuses. Although not statistically significant, Atm heterozygotes appeared to exhibit intermediary risk for postpartum lethality and fetal resorptions, but confirmation of this apparent trend would require studies with a higher dose of phenytoin and/or a larger number of animals. The risk for heterozygotes is of potential clinical importance, since this genotype is relatively common (1–2%) in humans (Smilenow et al., 2001). The protective effect of Atm against phenytoin embryopathies in the study herein is also consistent with its observed in vivo role as a teratological suppressor gene in protecting against the teratogenic and other embryopathic effects of low-dose IR during organogenesis (Laposa et al., 2004), against earlier gestational IR exposure during gastrulation (Heyer et al., 2000), and against postnatal exposure of the CNS to high IR doses (Chong et al., 2000; Herzog et al., 1998). The substantial "runting" or decreased fetal body weight of Atm-deficient fetuses compared to Atm-normal littermates exposed to IR during organogenesis (Laposa et al., 2004) is remarkably similar to that observed for –/– Atm fetuses in this in vivo study of endogenous and phenytoin-enhanced ROS. Taken together, the modulatory role of Atm in reducing embryonic oxidative damage herein and similarly reducing spontaneous embryopathies and those caused by the ROS-initiating teratogen phenytoin and even low-dose IR suggest a broad developmental role for Atm in protecting the embryo from the embryopathic effects of endogenous and environmental sources of oxidative stress, which may be mediated by ROS-dependent signal transduction and/or oxidative DNA damage.
However, unlike in embryo culture where consistent protection was afforded by Atm against all embryopathic effects of endogenous and phenytoin-enhanced ROS, the effects of Atm in this in vivo study were more complex, possibly modulated in part by the maternal genotype, and likely complicated by the resistance of the background strain to the teratogenic effects of phenytoin. In +/– dams, genotyping of the fetal resorptions showed that phenytoin-exposed +/+ embryos were more likely than their –/– littermates to die in utero, and a similar albeit nonsignificant pattern was observed for the vehicle-exposed embryos, suggesting that embryonic Atm can enhance some embryopathies. Interestingly, this pattern of Atm-enhanced in utero embryonic death was opposite to that observed with IR (Laposa et al., 2004), possibly due in part to the earlier GD 9.5 exposure for IR. Similarly, when analyzed by maternal genotype, some embryopathies were enhanced in vivo by Atm, as evidenced by increased fetal resorptions independent of treatment in +/+ Atm-normal dams, and increased fetal postpartum lethality in phenytoin-treated +/+ dams. Resorptions may involve a mechanism different from that for those embryopathies conversely reduced by Atm, and the resorption increase observed by maternal analysis is consistent with the lack of any resorptions having an Atm-null genotype. The mechanism for the increased incidence of resorptions in +/+ dams exposed only to vehicle may involve Atm-dependent apoptosis, which constitutes the major Atm-dependent alternative to interruption of the cell cycle and enhanced DNA repair. However, in the case of increased phenytoin-enhanced resorptions and postpartum lethality in +/+ dams, our laboratory, in other studies, has found no evidence for phenytoin-initiated embryonic apoptosis with in utero exposure (Laposa and Wells, 1995), suggesting that other mechanisms may be involved.
Atm –/– males and females are known to be infertile (Barlow et al., 1996; Yamashita et al., 1996), and the study herein provides the first evidence for reduced fertility in +/– females. Independent of treatment, Atm +/– females had 41% lower implantations than +/+ dams (p < 0.05), suggesting a potentially important developmental role for ATM in fertility, early development, and/or implantation. The decreased implantations in +/– dams in all treatment groups was likely due to endogenous ROS, since phenytoin was given after the developmental event was complete. Since the ratio of fetal genotypes was similar to that predicted by Mendelian genetics (1:2:1), it is possible that maternal Atm is the critical determinant for successful early development and implantation. Mitogenic events in the egg postulated to make Atm –/– mice infertile (Barlow et al., 1996; Yamashita et al., 1996) may also contribute to the fewer number of implantations seen in +/– dams.
Although it has been implied that ATM-mediated protection against IR is elicited primarily by the recognition and repair of DNA double-strand breaks (Barlow et al., 2000; Khanna, 2000), ATM can interact with multiple pathways to maintain DNA integrity, as well as serve as a "sensor" of oxidative stress (Bhuller and Wells, 2006, Fig. 1). The ability of ATM to respond to more subtle forms of developmental oxidative stress, such as oxidative DNA damage reflected by 8-oxo-dG formation, has not been well characterized. Several in vivo and in vitro studies have found that DNA may be a molecular target mediating phenytoin teratogenesis (Wells et al., 2005), and p53-deficient mice lacking DNA repair are more susceptible to ROS-initiating teratogens like phenytoin (Laposa and Wells, 1995; Wong and Wells, 2002), cyclophosphamide (Moallem and Hales, 1998), and benzo[a]pyrene (Nicol et al., 1995). Since ATM is an upstream regulator of p53, which is also a regulator in the genotoxic response pathway (Banin et al., 1998; Canman et al., 1998) and a teratological suppressor gene (Nicol et al., 1995), the protective effects of Atm may involve the repair of oxidative DNA damage in the embryo. This would be consistent with the increase in DNA oxidation observed herein in phenytoin-exposed –/– Atm fetuses and with the results of preliminary studies using oxoguanine glycosylase 1 (ogg1) and Cockayne Syndrome B (csb) knockout mice, which cannot repair the 8-oxo-dG lesion, and exhibited enhanced fetal oxidative DNA damage and postnatal neurodevelopmental deficits following in utero exposure to the ROS-initiating drug methamphetamine (Jeng et al., 2005; Wong et al., 2004, 2005). Alternatively, ATM may have a broader role as a sensor of oxidative stress and ROS-mediated signal transduction, since (1) other DNA repair mechanisms are preserved in A-T patients, (2) there is little evidence of elevated levels of DNA double-strand breaks (in the absence of IR) in A-T children, and (3) it has been difficult to explain how loss of the known function of ATM leads to degeneration of a specific population of postmitotic neurons (Quick and Dugan, 2001). Since ROS-mediated signal transduction involving Ras (Winn and Wells, 2002) and NF-kB (Kennedy et al., 2004) has also been implicated in the mechanism of phenytoin teratogenicity, Atm-dependent responses to altered signal transduction may contribute to the mechanism of its developmental effects. Atm has also been reported to be colocalized with the peroxisomal matrix protein catalase and may be important in regulating catalase activity (Kamsler et al., 2001), which we have found to be protective against ROS-initiating teratogens such as phenytoin (Winn and Wells, 1995, 1999). The in vivo study herein provides the first evidence that Atm responds to effects of ROS more subtly than DNA double-strand breaks, namely, phenytoin-initiated DNA oxidation, although a contribution from Atm as a ROS sensor cannot be excluded.
In conclusion, this in vivo study of constitutive and phenytoin-enhanced oxidative DNA damage and embryopathies in combination with the complementary study in embryo culture of Bhuller and Wells (2006) indicates that Atm responds to embryopathic effects more subtly than IR-initiated double-strand breaks, possibly including ROS-mediated signal transduction and oxidative DNA damage. Unlike the consistently protective role of Atm in embryo culture, shown in Bhuller and Wells (2006), Atm in vivo exhibits a variable modulatory role, protecting the embryo from some embryopathies, while adversely affecting other developmental outcomes. The increased risk of –/– Atm fetuses to phenytoin-enhanced oxidative DNA damage and some embryopathies is consistent with a role for oxidative DNA damage in the mechanism of phenytoin teratogenesis and for DNA repair in the mechanism of protection by ATM.
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1 Present address: Therapeutic Products Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada.
2 Present address: Covance Laboratories Inc, Vienna, VA 22182-1699, USA.
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ACKNOWLEDGMENTS |
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The authors are grateful to Crystal Lee for her assistance in the analysis of oxidative DNA damage. Preliminary reports of this research were presented at the 2001 Annual Meeting of the Society of Toxicology [Supplement: The Toxicologist, 66(Suppl. 1), 28 (Abstract 114)] and at the 2002 Annual Meeting of the Society of Toxicology, Canada. This research was supported by a grant from the Canadian Institutes of Health Research.
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