Abstract
Valproic acid (VPA) has been shown to cause neural tube defects in humans and mice, but its mechanism of action has not been elucidated. We hypothesize that alterations in embryonic antioxidant status and Hoxa2 gene expression play an important role in VPA-induced teratogenesis. A whole embryo culture system was applied to explore the effects of VPA on total glutathione, on glutathione in its oxidized (GSSG) and reduced (GSH) forms [GSSG/GSH ratio] and on Hoxa2 expression in cultured CD-1 mouse embryos during their critical period of organogenesis. Our results show that VPA can (1) induce embryo malformations including neural tube defects, abnormal flexion, yolk sac circulation defects, somite defects, and craniofacial deformities such as fusion of the first and second arches, and (2) alter glutathione homeostasis of embryos through an increase in embryonic GSSG/GSH ratio and a decrease in total GSH content in embryos. Western blot analysis and quantitative real-time RT-PCR show that VPA can inhibit Hoxa2 expression in cultured embryos at both the protein and mRNA level, respectively. The presence of ascorbic acid in the culture media was effective in protecting embryos against oxidative stress induced by VPA and prevented VPA-induced inhibition of Hoxa2 gene expression. Hoxa2 null mutant embryos do not exhibit altered glutathione homeostasis, indicating that inhibition of Hoxa2 is downstream of VPA-induced oxidative stress. These results are first to suggest VPA may, in part, exert its teratogenicity through alteration of the embryonic antioxidant status and inhibition of Hoxa2 gene expression and that ascorbic acid can protect embryos from VPA-induced oxidative stress.
Similar content being viewed by others
References
Akin ZN, Nazarali AJ (2005) Hox genes and their candidate down stream targets in the developing central nervous system Cell. Mol Neurobiol 25:697–741
Al Deeb S, Al Moutaery K, Arshaduddin M, Tariq M (2000) Vitamin E decreases valproic acid induced neural tube defects in mice. Neurosci Lett 292:179–182
Allen RG (1991) Oxygen-reactive species and antioxidant responses during development: the metabolic paradox of cellular differentiation. Proc Soc Exp Biol Med 196:117–129
Aoki Y, Okada A, Fujiwara M (2001) In vitro effects of anti-epileptic drugs (AEDs) on HOX gene expression in human EC cell line NT2/D1. The Japanese teratology society, 41st annual meeting, Congenit. Anomal (Kyoto), Abstract # O-38, 41:249
Baker MA, Cerniglia GJ, Zaman A (1990) Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal Biochem 190:360–365
Bannai S, Tateishi N (1986) Role of membrane transport in metabolism and function of glutathione in mammals. J Membr Biol 89:1–8
Barnes GL, Mariani BD, Tuan RS (1996) Valproic acid-induced somite teratogenesis in the chick embryo: relationship with Pax-1 gene expression. Teratology 54:93–102
Brown NA, Colhoun CW (1984) Valproic acid teratogenesis, 1. In vivo/in vitro comparisons and effects on somite morphogenesis. Teratology society 24th annual meeting program teratology. 29: Abstract# 20A–21A
Bruckner A, Lee YJ, O’Shea KS, Henneberry RC (1983) Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture. Teratology 27:29–42
Ceylan S, Duru S, Ceylan S (2001) Valproic acid sodium-induced spina bifida occulta in the rat. Neurosurg Rev 24:31–34
Chang TK, Abbott FS (2006) Oxidative stress as a mechanism of valproic acid-associated hepatotoxicity. Drug Metab Rev 38:627–639
Clayton-Smith J, Donnai D (1995) Fetal valproate syndrome. J Med Genet 32:724–727
Cotariu D, Evans S, Zaidman JL, Marcus O (1990) Early changes in hepatic redox homeostasis following treatment with a single dose of valproic acid. Biochem Pharmacol 40:589–593
Dawson JE, Raymond AM, Winn LM (2006) Folic acid and pantothenic acid protection against valproic acid-induced neural tube defects in CD-1 mice. Toxicol Appl Pharmacol 211:124–132
Eikel D, Hoffmann K, Zoll K, Lampen A, Nau H (2006a) S-2-pentyl-4-pentynoic hydroxamic acid and its metabolite s-2-pentyl-4-pentynoic acid in the NMRI-exencephaly-mouse model: pharmacokinetic profiles, teratogenic effects, and histone deacetylase inhibition abilities of further valproic acid hydroxamates and amides. Drug Metab Dispos 34:612–620
Eikel D, Lampen A, Nau H (2006b) Teratogenic effects mediated by inhibition of histone deacetylases: evidence from quantitative structure activity relationships of 20 valproic acid derivatives. Chem Res Toxicol 19:272–278
Eyer P, Podhradsky D (1986) Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman’s reagent. Anal Biochem 153:57–66
Faiella A, Wernig M, Consalez GG, Hostick U, Hofmann C, Hustert E, Boncinelli E, Balling R, Nadeau JH (2000) A mouse model for valproate teratogenicity: parental effects, homeotic transformations, and altered HOX expression. Hum Mol Genet 9:227–236
Finnell RH (1991) Genetic differences in susceptibility to anticonvulsant drug-induced developmental defects. Pharmacol Toxicol 69:223–227
Freeman SJ, Steele CE (1986) Post-implantation whole embryo culture and the study of teratogenesis. Food Chem Toxicol 24:619–622
Fuller LC, Cornelius SK, Murphy CW, Wiens DJ (2002) Neural crest cell motility in valproic acid. Reprod Toxicol 16:825–839
Gendron-Maguire M, Mallo M, Zhang M, Gridley T (1993) Hoxa-2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest. Cell 75:1317–1331
Graf WD, Oleinik OE, Glauser TA, Maertens P, Eder DN, Pippenger CE (1998) Altered antioxidant enzyme activities in children with a serious adverse experience related to valproic acid therapy. Neuropediatrics 29:195–201
Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207–212
Hamza AA, Amin A (2007) Apium graveolens modulates sodium valproate-induced reproductive toxicity in rats. J Exp Zool Part A Ecol Genet Physiol 307:199–206
Hao Z, Yeung J, Wolf L, Doucette R, Nazarali A (1999) Differential expression of Hoxa-2 protein along the dorsal-ventral axis of the developing and adult mouse spinal cord. Dev Dyn 216:201–217
Harris C, Stark KL, Juchau MR (1988) Glutathione status and the incidence of neural tube defects elicited by direct acting teratogens in vitro. Teratology 37:577–590
Hegde KR, Varma SD (2004) Protective effect of ascorbate against oxidative stress in the mouse lens. Biochim Biophys Acta 1670:12–18
Held KD, Biaglow JE (1994) Mechanisms for the oxygen radical-mediated toxicity of various thiol-containing compounds in cultured mammalian cells. Radiat Res 139:15–23
Hishida R, Nau H (1998) VPA-induced neural tube defects in mice. I. Altered metabolism of sulfur amino acids and glutathione. Teratog Carcinog Mutagen 18:49–61
Hurd RW, Van Rinsvelt HA, Wilder BJ, Karas B, Maenhaut W, De Reu L (1984) Selenium, zinc, and copper changes with valproic acid: possible relation to drug side effects. Neurology 34:1393–1395
Hutter DE, Till BG, Greene JJ (1997) Redox state changes in density-dependent regulation of proliferation. Exp Cell Res 232:435–438
Ingram JL, Rodier PM (1998) Valproic acid alters expression of Hoxa1 in rat embryos: a mechanism of teratogenesis? Teratology society: platform sessions abstracts. Teratology 57(4/5):191
Jones BG, Rose FA, Tudball N (1994) Lipid peroxidation and homocysteine induced toxicity. Atherosclerosis 105:165–170
Jurima-Romet M, Abbott FS, Tang W, Huang HS, Whitehouse LW (1996) Cytotoxicity of unsaturated metabolites of valproic acid and protection by vitamins C and E in glutathione-depleted rat hepatocytes. Toxicology 112:69–85
Kultima K, Nyström A-M, Scholz B, Gustafson A-L, Dencker L, Stigson M (2004) Valproic acid teratogenicity: a toxicogenomics approach. Environ Health Perspect 112:1225–1235
Maconochie M, Krishnamurthy R, Nonchev S, Meier P, Manzanares M, Mitchell PJ, Krumlauf R (1999) Regulation of Hoxa2 in cranial neural crest cells involves members of the AP-2 family. Development 126:1483–1494
Mahmood R, Mason IJ, Morriss-Kay GM (1996) Expression of Fgf-3 in relation to hindbrain segmentation, otic pit position and pharyngeal arch morphology in normal and retinoic acid-exposed mouse embryos. Anat Embryol 194:13–22
Menegola E, Broccia ML, Di Renzo F, Prati M, Giavini E (2000) In vitro teratogenic potential of two antifungal triazoles: triadimefon and triadimenol. In Vitro Cell Dev Biol Anim 36:88–95
Menegola E, Di Renzo F, Broccia ML, Prudenziati M, Minucci S, Massa V, Giavini E (2005) Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity. Birth Defects Res B Dev Reprod Toxicol 74:392–398
Michoulas A, Tong V, Teng XW, Chang TK, Abbott FS, Farrell K (2006) Oxidative stress in children receiving valproic acid. J Pediatr 149:692–696
Miranda AF, Wiley MJ, Wells PG (1994) Evidence for embryonic peroxidase-catalyzed bioactivation and glutathione-dependent cytoprotection in phenytoin teratogenicity: modulation by eicosatetraynoic acid and buthionine sulfoximine in murine embryo culture. Toxicol Appl Pharmacol 124:230–241
Na L, Wartenberg M, Nau H, Hescheler J, Sauer H (2003) Anticonvulsant valproic acid inhibits cardiomyocyte differentiation of embryonic stem cells by increasing intracellular levels of reactive oxygen species. Birth Defects Res A Clin Mol Teratol 67:174–180
New DA (1978) Whole embryo culture and the study of mammalian embryos during organogenesis. Biol Rev Camb Philos Soc 53:81–122
Nosel PG, Klein NW (1992) Methionine decreases the embryotoxicity of sodium valproate in the rat: in vivo and in vitro observations. Teratology 46:499–507
Okada A, Kushima K, Aoki Y, Bialer M, Fujiwara M (2005) Identification of early-responsive genes correlated to valproic acid-induced neural tube defects in mice. Birth Defect Res A Clin Mol Teratol 73:229–238
Ornoy A (2006) Neuroteratogens in man: an overview with special emphasis on the teratogenicity of antiepileptic drugs in pregnancy. Reprod Toxicol 22:214–226
Ozolinš TRS, Hales BF (1997) Oxidative stress regulates the expression and activity of transcription factor activator protein-1 in rat conceptus. J Pharmacol Exp Ther 280:1085–1093
Padmanabhan R, Ahmed I (1996) Sodium valproate augments spontaneous neural tube defects and axial skeletal malformations into mouse fetuses. Reprod Toxicol 10:345–363
Pippenger CE, Meng XZ, Rothner AD, Cruse RP, Erenberg G, Solano R (1991) Free radical scavenging enzyme activity profiles in risk assessment of idiosyncratic drug reactions. In: Levy RH, Penry JK (eds) Idiosyncratic reactions to valproate: clinical risk patterns and mechanisms of toxicity. Raven Press, New York, pp 75–88
Polifka JE, Friedman JM (2002) Medical genetics: 1. Clinical teratology in the age of genomics. CMAJ 167:265–273
Prince V, Lumsden A (1994) Hoxa2 expression in normal and transposed rhombomeres: independent regulation in the neural tube and neural crest. Development 120:911–923
Rijli FM, Mark M, Lakkaraju S, Dierich A, Dollé P, Chambon P (1993) A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene. Cell 75:1333–1349
Samaei A, Nobahar M, Vafaei AA (2009) An experimental design for finding of minimum dosage of carbamazepine and valproate in preventing of seizure attacks. Pak J Pharm Sci 22:180–183
Santagati F, Minoux M, Ren S-Y, Rijli FM (2005) Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis. Development 132:4927–4936
Seçkin S, Basaran-Küçükgergin C, Uysal M (1999) Effect of acute and chronic administration of sodium valproate on lipid peroxidation and antioxidant system in rat liver. Pharmacol Toxicol 85:294–298
Sen CK, Packer L (1996) Antioxidant and redox regulation of gene transcription. FASEB J 10:709–720
Stodgell CJ, Ingram JL, O’Bara M, Tisdale BK, Nau H, Rodier PM (2006) Induction of the homeotic gene Hoxa1 through valproic acid’s teratogenic mechanism of action. Neurotoxicol Teratol 28:617–624
Tabatabaei AR, Abbott FS (1999) LC/MS analysis of hydroxylation products of salicylate as an indicator of in vivo oxidative stress. Free Radic Biol Med 26:1054–1058
Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27:502–522
Tong V, Teng XW, Chang TK, Abbott FS (2005a) Valproic acid I: time course of lipid peroxidation biomarkers, liver toxicity, and valproic acid metabolite levels in rats. Toxicol Sci 86:427–435
Tong V, Teng XW, Chang TK, Abbott FS (2005b) Valproic acid II: effects on oxidative stress, mitochondrial membrane potential, and cytotoxicity in glutathione-depleted rat hepatocytes. Toxicol Sci 86:436–443
Trainor PA, Krumlauf R (2001) Hox genes, neural crest cells and branchial arch patterning. Curr Opin Cell Biol 13:698–705
Van Maele-Fabry G, Delhaise F, Picard JJ (1990) Morphogenesis and quantification of the development of post-implantation mouse embryos. Toxicol In Vitro 4:149–156
Verrotti A, Scardapane A, Franzoni E, Manco R, Chiarelli F (2008) Increased oxidative stress in epileptic children treated with valproic acid. Epilepsy Res 78:171–177
Wang X, Zhang W, Nazarali AJ (2007) Toxic effects of valproic acid on mouse palatal development in vitro. Proc. SECOTOX conference, Skiathos, Greece, vol 1, pp 139–144
Wegner C, Nau H (1991) Diurnal variation of folate concentrations in mouse embryo and plasma: the protective effect of folinic acid on valproic-acid-induced teratogenicity is time dependent. Reprod Toxicol 5:465–471
Wilder BJ, Hurd RW (1991) Free radical scavenger deficiency and valproic acid. In: Levy RH, Penry JK (eds) Idiosyncratic reactions to valproate: clinical risk patterns and mechanisms of toxicity. Raven Press, New York, pp 89–95
Williams JA, Mann FM, Brown NA (1997) Gene expression domains as markers in developmental toxicity studies using mammalian embryo culture. Int J Dev Biol 41:359–364
Yüksel A, Cengiz M, Seven M, Ulutin T (2001) Changes in the antioxidant system in epileptic children receiving antiepileptic drugs: two-year prospective studies. J Child Neurol 16:603–606
Acknowledgments
This study was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC). Dr. Benzhong Zhang is recipient of a scholarship from the China Scholarship Council.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhang, B., Wang, X. & Nazarali, A.J. Ascorbic Acid Reverses Valproic Acid-Induced Inhibition of Hoxa2 and Maintains Glutathione Homeostasis in Mouse Embryos in Culture. Cell Mol Neurobiol 30, 137–148 (2010). https://doi.org/10.1007/s10571-009-9438-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-009-9438-7