Toxicology 291 (2012) 32–42 Contents lists available at SciVerse ScienceDirect Toxicology j ourna l ho me pag e: www.elsevier.com/locate/toxicol Multiple point action mechanism of valproic acid-teratogenicity alleviated by folic acid, vitamin C, And N-acetylcysteine in chicken embryo model Chiu-Lan Hsieh a , Hui-Er Wang b , Wan-Jane Tsai a , Chiung-Chi Peng c,d,e,∗ , Robert Y. Peng e,f a Graduate Institute of Biotechnology, Changhua University of Education, Jin-De Campus, 1 Jin-De Rd., Changhua 50007, Taiwan b Department of Food Science and Applied Technology, Hungkuang University, 34 Chung-Chie Rd., Shalu County, Taichung Hsien 43302, Taiwan c Graduate Institute of Rehabilitation Science, College of Health Care, China Medical University, 91 Hsueh-Shih Rd., Taichung 40202, Taiwan d Department of Physical Therapy, College of Health Care, China Medical University, 91 Hsueh-Shih Rd., Taichung 40202, Taiwan e Graduate Institute of Clinical Medicine, Taipei Medical University, 250 Wu-Xin St., Taipei 110, Taiwan f Research Institute of Biotechnology, Hungkuang University, 34 Chung-Chie Rd., Shalu County, Taichung Hsien 43302, Taiwan a r t i c l e i n f o Article history: Received 17 September 2011 Received in revised form 19 October 2011 Accepted 20 October 2011 Available online 25 October 2011 Keywords: Valproic acid Folic acid Ascorbic acid N-acetylcysteine Histone deacetylase a b s t r a c t The teratogenicity of antiepilepsy drug valproic acid (VPA) mostly is found in genetic and somatic levels, causing teratogenesis involving neurotubular defects (NTDs), anencephaly, lumbosacral meningomyelo- cele, and leg dysfunction due to spina bifida aperta. A diversity of nutraceutics have been tried to alleviate the risk of VPA-teratogenicity. The effect was varying. In order to promote the preventive prescription, to find out its action mechanism can be rather crucial. We used chicken embryo model to try the effect of folic acid (FA), ascorbic acid (AA), and N-acetyl cysteine (NAC). VPA at 30 mM showed the higher malformation rate (66.7%) with the least mortality (22.2%). Pathological findings indicated that the cer- vical muscle was more susceptible to VPA injury than the ankle muscle. VPA downregulated levels of superoxide dismutase (SOD), glutathione (GSH), histone deacetylase (HDAC) and folate, and upregulated H 2 O 2 and homocysteine. FA, AA, and NAC significantly upregulated SOD, but only AA alone activated GSH. AA and NAC downregulated H 2 O 2 , while FA was totally ineffective. All three nutraceutics compara- bly rescued HDAC with simultaneously suppressed homocysteine accumulation and folate re-elevation, although less effectively by NAC. Based on these data, we conclude VPA possesses “Multiple Point Action Mechanism”. In addition to affecting the cited transcription and translation levels, we hypothesize that VPA competitively antagonize the glutamic acid to couple with pteroic acid in biosynthesis of dihydro- folic acid (DHFA). H 2 O 2 directly destroyed the NADPH reducing system at dihydrofolate reductase (DHFR) and methylene tetrahydrofolate reductase (MTHFR) levels, while completely restored by AA, an implica- tion in preservation of intact apoenzymes. In addition, the GSH–GSSG system is sandwiched between the reducing systems NADPH/NADP and DHA–AA, its net balance is highly dependent on in situ in vivo Redox state, hence folic acid transformation is varying. To rescue the VPA-induced teratogenicity, simultaneous multiple prescriptions are suggested. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Comparing to the mouse model (Faiella et al., 2000), the chicken egg and developing embryo are useful models for the study of ter- atogenicity (Whitsel et al., 2002; Rosenquist et al., 2010; Lie et al., 2010) and embryotoxic potency (Jelinek and Peterka, 1985). The teratogenicity of VPA mostly is found in genetic and somatic levels (Whitsel et al., 2002; Eikel et al., 2006), mostly involving neurotubu- lar defects (NTDs), anencephaly, lumbosacral meningomyelocele (Lindhout et al., 1992; Koch et al., 1996), and spina bifida aperta ∗ Corresponding author at: 4F-3, No.133, Song-Pin Rd. Taipei, Taiwan. Tel.: +886 2 27585767; fax: +886 2 27585767; mobile: +886 953 002 072. E-mail address: misspeng@ms2.hinet.net (C.-C. Peng). (Mominoki et al., 2006). The cited mechanism of VPA to elicit terato- genicity involve (i) promoting folic acid deficiency (Johannessen, 2000; Chango, 2009) and acting as a disrupter of methylene tetrahy- drofolate reductase (MTHFR) (Karabiber et al., 2003; Roy et al., 2008); (ii) inducing oxidative stress (Tabatabaei and Abbott, 1999); (iii) leading to the - and -oxidation (Lheureux et al., 2005); (iv) inhibiting histone deacetylase (Menegola et al., 2006; Hrzenjak et al., 2006); (v) antiangiogenesis (Rosenberg, 2007); and (vi) DNA damages (Schulpis et al., 2006). In addition, reduced folate lev- els may result in hyperhomocysteinemia. Homocysteine has been considered a mediator of the teratogenic potential of VPA (Verrotti et al., 2000; Karabiber et al., 2003). Reduced folate can compromise DNA (Fig. S1, van Gelder et al., 2010). Mild MTHFR deficiency and reduced maternal erythrocyte folate concentration is a particularly strong risk factor for NTD (Amorim et al., 2007). 0300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2011.10.015