![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Pharmacology and Toxicology and School of Environmental Studies, Queen's University, Kingston, Ontario, Canada
Received August 11, 2005; accepted December 23, 2005
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). Furthermore, valproic acid has also proven to be effective in the treatment of bipolar disorder (Bowden, 2003), migraine prophylaxis (Freitag, 2003), and neuropathic pain (Covington, 1998). Despite its effectiveness and widespread use, valproic acid is teratogenic in both animals (Brown et al., 1980) and humans (Robert and Rosa, 1983). Of particular concern is the 1 to 2% risk of neural tube defects, most of which are spina bifida, with the use of valproic acid during the first trimester of pregnancy (Bjerkedal et al., 1982). This is 10 to 20 times the prevalence rate for neural tube defects in the general population (Frey and Hauser, 2003). However, the molecular mechanism by which valproic acid causes neural tube defects has not been elucidated.
The teratogenicity of many drugs and chemicals is believed to be initiated through the bioactivation of the parent compound to form a reactive intermediate, followed by the formation of ROS (Wells and Winn, 1996; Wells et al., 1997). The developing embryo is particularly sensitive to increases in ROS formation because enzymes in embryonic and fetal tissue that detoxify ROS generally tend to have low activities (Wells et al., 1997). If ROS levels exceed the cellular detoxifying capabilities and a state of oxidative stress ensues, detrimental consequences to the embryo can result and may ultimately lead to teratogenesis (Wells et al., 1997).
Several studies suggest indirectly that valproic acid-initiated teratogenicity may be caused by oxidative stress. For example, previous research has shown that catalase, which detoxifies hydrogen peroxide, prevented valproic acid-induced lymphocyte toxicity and that 1,10-phenanthroline, an iron chelator, also decreased valproic acid-mediated cytotoxicity in vitro (Tabatabaei and Abbott, 1999). These results suggest that the production of hydrogen peroxide and the succeeding iron-catalyzed formation of hydroxyl radicals may be the specific ROS that mediates valproic acid-induced toxicity (Tabatabaei and Abbott, 1999). In addition, valproic acid has been shown to inhibit cardiomyocyte differentiation of embryoid bodies derived from murine pluripotent embryonic stem cells through an increase in ROS (Na et al., 2003). Alternatively, the antioxidant vitamin E has been shown to decrease the frequency of valproic acid-induced neural tube defects in mice, suggesting that ROS may play a role in the failure of the neural tube to develop properly (Al Deeb et al., 2000).
One of the consequences of increased ROS production is oxidative DNA damage (Klaunig and Kamendulis, 2004). Previous research suggests that the mechanism of teratogenesis for both thalidomide and phenytoin may be mediated through an increase in oxidative DNA damage (Winn and Wells, 1995; Parman et al., 1999). Oxidized bases can be repaired by base excision repair and nucleotide excision repair; however, during the repair process, DNA double-strand breaks can be generated (Lindahl and Wood, 1999). For example, when oxidized bases located close together and on opposite strands are being repaired through base excision repair and nucleotide excision repair, the simultaneous excision of the damaged bases can result in a double-strand break (Pfeiffer et al., 2000). Alternatively, single-strand breaks that are generated during the repair of oxidative DNA damage can be converted to double-strand breaks during replication (van den Bosch et al., 2002). In addition, doublestrand breaks can be caused by free radicals themselves (van Gent et al., 2001).
DNA double-strand breaks are repaired through two mechanisms: nonhomologous end-joining, and HR (van den Bosch et al., 2002). Although HR often repairs DNA double-strand breaks with a high degree of fidelity, it has been shown that HR is not always an error-free process and can contribute to genomic instability through the production of genetic changes, including the loss of heterozygosity (LOH) and gene deletions and duplications (Bishop and Schiestl, 2003). Because of its potential to contribute to genetic changes, HR is believed to play a major role in the development of some diseases, including cancer (Bishop and Schiestl, 2003). Because proper development of the embryo requires highly regulated gene expression, HR that results in genetic changes has the potential to disrupt these tightly coordinated processes and may underlie the mechanism of valproic acidinduced neural tube defects.
This study was conducted to determine whether valproic acid increases HR through an increase in ROS formation and subsequent oxidative DNA damage in the Chinese hamster ovary 3-6 (CHO 3-6) recombination reporter cell line. In addition, PEG-catalase was used to determine whether antioxidant administration could decrease valproic acid-induced HR. The results of this study show that valproic acid does increase ROS levels and HR frequency but not DNA oxidation. These data support our hypothesis that HR may be the underlying mechanism mediating valproic acid-induced teratogenesis.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-minimum essential media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all from Gibco Life Technologies) at 37°C in 5% CO2.
|
). The cells were then washed twice with phosphate-buffered saline, and fresh media containing G418 (500 µg/ml) were added to the plates. The cells were incubated for 2 weeks at 37°C and then stained with 1% crystal violet in methanol. For plating efficiency studies to assess cell death, the above protocol was used, except that 300 cells/10-cm culture dish were plated, G418 was not added, and the cells were grown for 1 week. To determine HR frequency, the number of G418-resistant colonies per live cells plated was calculated and expressed as a -fold increase from the respective control group.
For HR studies using PEG-catalase, a similar protocol was used as described above, except PEG-catalase (200 and 400 U/ml) was administered immediately after cell plating, and the cells were incubated for 24 h before being dosed with valproic acid (10 mM) for 24 h (Ceolotto et al., 2004).
Southern Hybridization. To determine the types of HR events induced by valproic acid, CHO 3-6 cells were treated with valproic acid (5 and 10 mM) or vehicle control (media) for 24 h and then treated with G418 as described above. Ten G418-resistant colonies from each treatment group consisting of 20 cells or more were identified under a light microscope, isolated, and grown to confluence in six-well plates (Corning Inc.). The DNA was then extracted using the QIAGEN DNeasy Tissue Kit (QIAGEN, Mississauga, ON, Canada). Genomic DNA was digested with ScaI and HindIII restriction endonucleases (New England BioLabs Inc., Mississauaga, ON, Canada), and Southern hybridization was conducted using 32P-labeled neomycin cDNA as the probe to determine the size of the HR products as described previously (Nickoloff, 1992). A 7.9-kb band represents a conservative HR event, whereas a 2.6-kb band represents a nonconservative HR event.
ROS Studies. CHO 3-6 cells were plated at a density of 1 x 106 cells/10-cm culture dish and were allowed to adhere for 4 h. Cells were preincubated for 30 min with 10 µM concentration of the ROS-sensitive dye 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate (CM-H2DCF) or vehicle (dimethyl sulfoxide) before dosing with valproic acid (5 or 10 mM) for 0.5, 1, 2, or 4 h. The interaction of CM-H2DCF with ROS results in intracellular dichlorofluorescein derivatives, which are highly fluorescent (Li et al., 2002). Therefore, dichlorofluorescein fluorescence was measured by flow cytometry as an indication of the relative amount of intracellular ROS. For the ROS studies using PEG-catalase, a similar protocol as described above was used except that PEG-catalase was added to the plates when the cells were initially plated for 24 h before treatment with CM-H2DCF, and the cells were exposed to valproic acid (10 mM) for 1 h.
DNA Oxidation. CHO 3-6 cells were treated as described for the recombination frequency assays, except that DNA was immediately isolated after a 1-, 4-, or 24-h exposure to valproic acid using the QIAGEN DNeasy Tissue Kit (QIAGEN). The DNA was then processed to individual nucleosides using a modified method as described previously (Huang et al., 2001). 8-OH-2'-dG and 2'-dG were separated and quantified using high-pressure liquid chromatography with electrochemical detection by using a YMCbasic 150 x 4.6 mm column (YMC, Inc., Milford, MA) under isocratic conditions consisting of a mobile phase of 5% methanol and 95% 100 mM sodium acetate buffer, pH 5.2. The separated nucleosides were detected using a CoulArray Electrochemical Detector (ESA, Inc., Chelmsford, MA).
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 twoway analysis of variance if required and/or a one-way analysis of variance. The Neumann-Keuls multiple comparison test was used for post hoc analysis. The minimum level of significance used throughout was p < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
|
|
DNA Oxidation as a Result of Valproic Acid Exposure. To determine whether the ROS generated after valproic acid exposure caused oxidative DNA damage, CHO 3-6 cells were exposed to valproic acid (10 mM) for 1, 4, or 24 h, and the ratio of 8-OH-2'-dG/2'-dG, which is a measure of DNA oxidation, was calculated. No significant differences in the ratio of 8-OH-2'-dG/2'-dG were observed in cells treated with valproic acid compared with control (Fig. 7).
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Covington, 1998; Bowden, 2003; Freitag, 2003). However, in utero exposure to valproic acid during the first trimester of pregnancy is associated with a 1 to 2% risk of neural tube defects (Bjerkedal et al., 1982). Given the teratogenic potential of valproic acid and the expanding use of this drug clinically, elucidating the mechanism of valproic acid-induced neural tube defects is critical. In the present study, we investigated the novel idea that HR, a DNA double-strand break repair mechanism, may mediate valproic acid-induced teratogenesis by analyzing HR frequency and the types of HR events that occur after valproic acid exposure. We also investigated the role of valproic acid-mediated ROS production in initiating HR. Our results show that valproic acid exposure generates ROS and increases HR frequency in CHO 3-6 cells, suggesting a new and novel mechanism by which valproic acid may mediate neural tube defects.
Both carcinogenic and teratogenic agents have been shown to induce HR. For example, the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin and metabolites of benzene metabolism, which are carcinogens, and the teratogen phenytoin have been shown to induce HR in the CHO 3-6 cell line (Winn, 2003; Winn et al., 2003; Chan et al., 2004). In the present study, we demonstrated that exposure to 5 and 10 mM valproic acid for 24 h significantly increased HR frequency in CHO 3-6 cells. Because DNA damage induces HR, our results suggest that valproic acid is capable of being genotoxic. Previous studies that examined the genotoxicity of valproic acid by analyzing sister chromatid exchanges in peripheral lymphocytes from patients treated with valproic acid have shown contradictory results. Sister chromatid exchanges occur via the HR repair pathway and are used as an indicator of chromosomal stability after exposure to a potentially mutagenic agent. A significant increase in sister chromatid exchanges was observed in peripheral lymphocytes from epileptic children treated with valproic acid (Hu et al., 1990). However, another study demonstrated that valproic acid treatment did not induce sister chromatid exchanges in peripheral lymphocytes in adult male patients (Schaumann et al., 1989). Further evidence indicates that valproic acid may contribute to genomic instability, because a growing body of evidence demonstrates that valproic acid alters chromatin structure because it can function as a histone deacetylase inhibitor, leading to the acetylation of histone tails (Marchion et al., 2005). This change in the chromatin structure relaxes the conformation of DNA, making it more susceptible to DNA damage, including double-strand breaks (Coyle et al., 2005). Valproic acid's histone deacetylase activity has been attributed to certain pathologies, including valproic acidinduced teratogenesis and valproic acid-associated acute leukemia, and is the basis behind the current testing of the effectiveness of valproic acid in cancer therapy (Phiel et al., 2001; Camphausen et al., 2005; Coyle et al., 2005).
Although HR is a DNA repair process, it is not error-free and can contribute to genetic instability through the formation of gene deletions, duplications, translocations, and LOH (Bishop and Schiestl, 2003). Because HR can ultimately lead to genomic instability, considerable evidence associating HR with carcinogenesis has been documented (Bishop and Schiestl, 2003). Through Southern blot analysis, we show that valproic acid induces HR between direct repeats through gene conversion without associated crossovers. A gene conversion event can lead to a genetic mutation if a mutated allele is copied via HR to repair the double-strand break (Bishop and Schiestl, 2003). Therefore, the significance of gene conversion is that it can result in an LOH. This type of event is important in the initiation of some cancers, because tumor suppressor genes may be deleted by this mechanism, or mutated alleles can be used as a template during HR (Bishop and Schiestl, 2003). We propose that similar to the progression of carcinogenesis, teratogenesis can occur in this manner, because genes critical to the developing embryo may be altered through an LOH. We postulate that the fidelity of double-strand break repair via HR is especially important in the embryo, in which both the rapid replication and differentiation of cells are necessarily linked to the tight regulation of numerous developmental genes. Increases in HR and lack of HR fidelity would increase the chances of critical genes either being turned off (because of the loss of a gene) or turned on (multiplications) at inappropriate times during embryonic development.
Previous research suggests that genetic instability can result from oxidative stress (Limoli et al., 2003). To determine whether valproic acid induces a state of oxidative stress in the CHO 3-6 cell line, ROS levels were measured after exposure to valproic acid using the ROS-sensitive dye CM-H2DCF. Our results show that valproic acid does, in fact, increase the generation of ROS in the CHO 3-6 cell line. These results are consistent with a previous study which showed that valproic acid causes an increase in ROS formation in embryoid bodies derived from murine pluripotent embryonic stem cells (Na et al., 2003). In the present study, PEG-catalase was administered to determine whether antioxidants could protect against valproic acid-induced ROS formation. Catalase protects cells from hydrogen peroxide by the enzymatic conversion of hydrogen peroxide to water and molecular oxygen (Mates et al., 1999). Although hydrogen peroxide is a mild oxidant itself, it can be converted to the highly toxic and short-lived hydroxyl radical via the Fenton reaction (Mates et al., 1999). Hydroxyl radicals have been implicated in the formation of DNA damage, including the formation of 8-OH-2'-dG (Klaunig and Kamendulis, 2004). Our results demonstrate that preincubation with PEG-catalase before the administration of valproic acid significantly decreased the production of ROS, although ROS levels did not decrease to baseline levels. In a previous study using in vitro human lymphocyte preparations, catalase significantly protected against valproic acid-induced cytotoxicity; however, similar to findings in the present study, cell death levels were not completely attenuated to baseline levels (Tabatabaei and Abbott, 1999). Given that increased DCF fluorescence can result because of exposure to various ROS and reactive nitrogen species, DCF is considered a general marker for these species (Li et al., 2002). This suggests that valproic acid exposure not only leads to increased hydrogen peroxide production but to other ROS as well. Currently, the source or the mechanism of valproic acid-mediated ROS production is not known. Therefore, it is possible that ROS, such as the superoxide radical, are generated upon exposure to valproic acid, which catalase does not detoxify.
The formation of ROS can lead to DNA damage, including base and sugar modifications, DNA cross-links, and single- and double-strand breaks (Klaunig and Kamendulis, 2004). One of the major base modifications caused by ROS is the formation of 8-OH-2'-dG, which results from the hydroxylation of the C-8 position of guanine (Barnes and Lindahl, 2004). 8-OH-2'-dG is commonly used as a biomarker of oxidative DNA damage, and increased levels of 8-OH-2'-dG have been associated with both cancer promotion and teratogenesis (Winn and Wells, 1995; Barnes and Lindahl, 2004). Our results show that valproic acid exposure did not cause an increase in the formation of 8-OH-2'-dG. Likewise, in a previous study, ROS levels were significantly increased in chromosomally unstable cells compared with chromosomally stable cells; however, an increase in 8-OH-2'-dG formation was not apparent in those cells that possessed elevated ROS levels (Limoli et al., 2003). In addition, it has been demonstrated that administration of 4-ene valproic acid, a potentially hepatotoxic and teratogenic metabolite of valproic acid, in rats resulted in a depletion of the mitochondrial glutathione pool (Tang et al., 1995). Therefore, if valproic acid-induced ROS production is due to the depletion of antioxidant defense mechanisms in the mitochondria, the excess ROS may not be in a close enough proximity to the nucleus to cause oxidative DNA damage. It is also possible that some of the oxidative damage could have been repaired before the direct measurement of 8-OH-2'-dG.
To determine whether the increased production of ROS after exposure to valproic acid was associated with the increase in HR, HR studies were conducted using PEG-catalase. Our results show that pretreatment with the antioxidative enzyme PEG-catalase completely blocked the observed increase in HR initiated by exposure to valproic acid in CHO 3-6 cells. These results support our hypothesis that valproic acid causes oxidative stress, which then increases HR. The protective effects of PEG-catalase observed in this study are consistent with other in vitro studies showing a protective effect of catalase against ROS production and ROS-initiated HR (Shen et al., 1996; Winn, 2003).
In summary, our results show that valproic acid increases HR frequency after dosing for 24 h in the CHO 3-6 cell line. Mechanistically, we have demonstrated that valproic acidinduced HR occurs through a conservative mechanism without the loss of DNA. Although we have shown that valproic acid does induce a state of oxidative stress in the CHO 3-6 cell line, the precise contribution of valproic acid-mediated ROS formation to the induction of HR requires further analysis. The results of this study suggest that valproic acidinduced neural tube defects could possibly result from aberrant HR, leading to an LOH in genes critical to proper neural tube development.
|
Footnotes |
|---|
This work was presented previously at the following conferences: Defoort E, Kim PM, and Winn LM (2005) Valproic acid (VPA) increases reactive oxygen species formation and homologous recombination frequency: a novel mechanism for VPA-induced teratogenesis, 8th Annual Meeting for Basic and Clinical Trainees, Faculty of Health Sciences, Queen's University, Kingston; Defoort E, Kim PM, and Winn LM (2005) Valproic acid increases conservative homologous recombination frequency: implications for a mechanism of valproic acid-induced neural tube defects, 44th Annual Meeting Society of Toxicology, New Orleans, Louisiana; Defoort E, Kim PM, and Winn LM (2004) Valproic acid increases reactive oxygen species formation and homologous recombination frequency: a novel mechanism for valproic acid-induced neural tube defects, 37th Annual Symposium of The Society of Toxicology of Canada, Montreal, Quebec; and Defoort E, Kim PM, and Winn LM (2004) The role of homologous recombination in valproic acid-induced neural tube defects, 7th Annual Meeting for Basic and Clinical Trainees, Faculty of Health Sciences, Queen's University, Kingston.
ABBREVIATIONS: ROS, reactive oxygen species; 8-OH-2'-dG, 8-hydroxy-2'-deoxyguanosine; 2'-dG 2'-deoxyguanosine; CHO, Chinese hamster ovary; DCF, dichlorodihydrofluorescein; CM-H2DCF, 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate; HR, homologous recombination; LOH, loss of heterozygosity; PEG, polyethylene glycol; kb, kilobase(s).
Address correspondence to: Dr. Louise M. Winn, Department of Pharmacology and Toxicology and School of Environmental Studies, Queen's University, Kingston, Ontario, Canada. E-mail: winnl{at}biology.queensu.ca
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Barnes DE and Lindahl T (2004) Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annu Rev Genet 38: 445-476.[CrossRef][Medline]
Bishop AJR and Schiestl RH (2003) Role of homologous recombination in carcinogenesis. Exp Mol Pathol 74: 94-105.[CrossRef][Medline]
Bjerkedal T, Czeizel A, Goujard J, Kallen B, and Mastroiacova P (1982) Valproic acid and spina bifida. Lancet 2: 1096.[Medline]
Bowden CL (2003) Valproate. Bipolar Disord 5: 189-202.[Medline]
Brown NA Kao J and Fabro S (1980) Teratogenic potential of valproic acid. Lancet 1: 660-661.[Medline]
Camphausen K, Cerna D, Scott T, Sproull M, Burgan WE, Cerra MA, Fine H, and Tofilon PJ (2005) Enhancement of in vitro and in vivo tumor cell radiosensitivity by valproic acid. Int J Cancer 114: 380-386.[CrossRef][Medline]
Chan CY, Kim PM, and Winn LM (2004) TCDD-induced homologous recombination: the role of the Ah receptor versus oxidative DNA damage. Mutat Res 563: 71-79.[Medline]
Chapman A, Keane PE, Meldrum BS, Simiand J, and Vernieres JC (1982) Mechanism of anticonvulsant action of valproate. Prog Neurobiol 19: 315-359.[CrossRef][Medline]
Ceolotto G, Bevilacqua M, Papparella I, Baritono E, Franco L, Corvaja C, Mazzoni M, Semplicini A, and Avogara A (2004) Insulin generates free radicals by an NAD(P)H, phosphatidylinositol 3'-kinase-dependent mechanism in human skin fibroblasts ex vivo. Diabetes 53: 1344-1351.
Covington EC (1998) Anticonvulsants for neuropathic pain and detoxification. Cleve Clin J Med 65 (Suppl 1): SI21-SI29, discussion SI45-SI47.[Medline]
Coyle TE, Bair AK, Stein C, Vajpayee N, Mehdi S, and Wright J (2005) Acute leukemia associated with valproic acid treatment: a novel mechanism for leukemogenesis? Am J Hematol 78: 256-260.[Medline]
Freitag FG (2003) Divalproex in the treatment of migraine. Psychopharmacol Bull 37: 98-115.[Medline]
Frey L and Hauser WA (2003) Epidemiology of neural tube defects. Epilepsia 44: 4-13.[Medline]
Hu LJ, Lu XF, Lu BQ, and Huang YQ (1990) The effect of valproic acid on sister chromatid exchange aberration frequency in epileptic children. Chin Med J 103: 415-417.[Medline]
Huang X, Powell J, Mooney LA, Li C, and Frenkel K (2001) Importance of complete DNA digestion in minimizing variability of 8-oxo-DG analyses. Free Radic Biol Med 31: 1341-1351.[CrossRef][Medline]
Klaunig JE and Kamendulis LM (2004) The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol 44: 239-267.[CrossRef][Medline]
Limoli CL, Giedzinski E, Morgan WF, Swarts SG, Jones GD, and Hyun W (2003) Persistent oxidative stress in chromosomally unstable cells. Cancer Res 63: 3107-3111.
Li H, Junk P, Huwiler A, Burkhardt C, Wallerath T, Pfeilschifter J, and Forster-mann U (2002) Dual effect of ceramide on human endothelial cells: induction of oxidative stress and transcriptional upregulation of endothelial nitric oxide synthase. Circulation 106: 2250-2256.
Lindahl T and Wood RD (1999) Quality control by DNA repair. Science (Wash DC) 286: 1897-1905.
Marchion DC, Bicaku E, Daud AI, Sullivan DM, and Munster PN (2005) Valproic acid alters chromatin structure by regulation of chromatin modulation proteins. Cancer Res 65: 3815-3822.
Mates JM, Perez-Gomez C, and Nunez De Castro I (1999) Antioxidant enzymes and human diseases. Clin Biochem 32: 595-603.[CrossRef][Medline]
Na L, Wartenberg M, Nau H, Hescheler J, and Sauer H (2003) Anticonvulsant valproic acid inhibits cardiomyocyte differentiation of embryonic stem cells by increasing intracellular levels of reactive oxygen species. Birth Defects Res 67: 174-180.
Nickoloff JA (1992) Transcription enhances intrachromosomal homologous recombination in mammalian cells. Mol Cell Biol 12: 5311-5318.
Parman T, Wiley MJ, and Wells PG (1999) Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nat Med 5: 582-585.[CrossRef][Medline]
Pfeiffer P, Goedecke W, and Obe G (2000) Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis 15: 289-302.
Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, and Klein PS (2001) Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer and teratogen. J Biol Chem 276: 36734-36741.
Robert E and Rosa F (1983) Valproate and birth defects. Lancet 2: 1142.[Medline]
Schaumann BA, Winge VB, and Garry VF (1989) Sister chromatid exchanges in adult epilepsy patients on valproate monotherapy. Epilepsy Res 3: 182-184.[CrossRef][Medline]
Shen Y, Shen H-M, Shi C-Y, and Ong C-N (1996) Benzene metabolites enhance reactive oxygen species in HL60 human leukemia cells. Hum Exp Toxicol 15: 422-427.[Medline]
Tabatabaei AR and Abbott FS (1999) Assessing the mechanism of metabolismdependant valproic acid-induced in vitro cytotoxicity. Chem Res Toxicol 12: 323-330.[Medline]
Tang W, Borel AG, Fujimiya T, and Abbott FS (1995) Fluorinated analogues as mechanistic probes in valproic acid hepatotoxicity: hepatic microvesicular steatosis and glutathione status. Chem Res Toxicol 8: 671-682.[CrossRef][Medline]
Thisted E and Ebbesen F (1993) Malformations, withdrawal manifestations and hypoglycemia after exposure to valproate in utero. Arch Dis Child 69: 288-291.[Abstract]
van den Bosch M, Lohman PHM, and Pastink A (2002) DNA double-strand break repair by homologous recombination. Biol Chem 383: 873-892.[CrossRef][Medline]
van Gent DC, Hoeijmakers HJ, and Kanaar R (2001) Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet 2: 196-206.[CrossRef][Medline]
Wells PG, Kim PM, Laposa RR, Nicol CJ, Parman T, and Winn LM (1997) Oxidative damage in chemical teratogenesis. Mutat Res 396: 65-78.[Medline]
Wells PG and Winn LM (1996) Biochemical toxicology of chemical teratogenesis. Crit Rev Biochem Mol Biol 31: 1-40.[Medline]
Winn LM (2003) Homologous recombination initiated by benzene metabolites: a potential role of oxidative stress. Toxicol Sci 72: 143-149.
Winn LM, Kim PM, and Nickoloff JA (2003) Oxidative stress-induced homologous recombination as a novel mechanism for phenytoin-initiated toxicity. J Pharmacol Exp Ther 306: 523-527.
Winn LM and Wells PG (1995) Phenytoin-initiated DNA oxidation in murine embryo culture and embryo protection by the antioxidative enzymes superoxide dismutase and catalase: evidence for reactive oxygen species-mediated DNA oxidation in the molecular mechanism of phenytoin teratogenicity. Mol Pharmacol 48: 112-120.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  Y. Kawai and I. J. Arinze Valproic Acid-Induced Gene Expression through Production of Reactive Oxygen Species. Cancer Res., July 1, 2006; 66(13): 6563 - 6569. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, significant difference from the 10 mM valproic acid treatment group (**, p < 0.01, 
