Research ReportAltered gene expression in neural crest cells exposed to ethanol in vitro
Introduction
Ethanol exposure causes facial and cardiac malformations in the offspring, both in humans (Jones et al., 1973, Lemoine et al., 1968) and animals (Chernoff, 1977, Henderson and Schenker, 1977, Kronick, 1976, Tze and Lee, 1975). Neural crest cells from the cranial region (cNCC) of the neural tube are crucial for the development of the facial skeleton (Imai et al., 1996, Sant'Anna and Tosello, 2006) and heart (Kirby et al., 1983, Poelmann et al., 1998), whereas the neural crest cells of the trunk (tNCC) are primarily involved in the genesis of the peripheral nervous system. Indeed, both the mandibular arch and the heart primordium of the embryo need an influx of cNCC for normal development, and, furthermore correct closure of the neural tube requires normally developed NCC (Moase and Trasler, 1992). This suggests that ethanol-induced facial and cardiac malformations may be the result of an impaired NCC development (Amini et al., 1996).
Previous work has suggested that ethanol-induced oxidative stress is involved in the etiology of congenital anomalies. Administration of vitamin E and beta-carotene to ethanol-exposed rat hippocampal cells in vitro improved viability and morphology (Mitchell et al., 1999), and vitamin E, as well as mitochondrially targeted vitamin E, enhanced survival of ethanol-exposed neuronal cells in vitro (Heaton et al., 2004, Siler-Marsiglio et al., 2005). Addition of SOD to ethanol-exposed embryos in vitro blocked dysmorphogenesis (Kotch et al., 1995) and SOD, catalase and vitamin E addition rectified NCC development after ethanol exposure in vitro (Chen and Sulik, 1996). Likewise, administration of folic acid to pregnant and lactating rats diminished ethanol-induced decrease in GSH levels and attenuated increased TBARS and carbonyls in the offspring (Cano et al., 2001). Supplementation of a SOD/catalase mimetic, EUK-134, diminished ethanol-induced limb malformations in mice (Chen et al., 2004). Over-expression of the antioxidant enzymes catalase and peroxiredoxin 5 protected Xenopus embryos against alcohol-induced ocular and gastrointestinal defects (Peng et al., 2004) and, likewise, over- and under expression of superoxide dismutase (SOD) partly protected, and failed to protect, respectively, mouse embryos subjected to ethanol in utero (Wentzel and Eriksson, 2006). Administration of N-acetylcysteine (NAC) diminished maldevelopment of rat embryos exposed to ethanol in vitro (Wentzel and Eriksson, 2008) and the use of black ginseng diminished ethanol-induced mouse embryo maldevelopment (Lee et al., 2009). Taken together, these data suggest that ethanol exposure may cause embryonic oxidative stress (Chen et al., 2002, Chen et al., 1997, Devi et al., 1994), subsequent apoptosis (Ramachandran et al., 2001, Ramachandran et al., 2003) and, later, developmental damage (Cartwright et al., 1998, Sulik et al., 1988).
In the present study we elected to evaluate the effect of ethanol exposure on the development of one cell type, the NCC. We isolated cNCC and tNCC from inbred rats of a strain with ethanol-sensitive embryogenesis and subsequently exposed the NCC to a teratogenic or non-teratogenic environment followed by the recording of key genetic responses. We used exposure to 88 mM ethanol as a teratogen, and addition of the GSH-sparing antioxidant N-acetylcysteine (NAC) as a putative anti-teratogenic agent. The genes chosen for study are involved in apoptosis, oxidative defense, cellular metabolism, NCC development or inflammation, and several of them have been estimated in whole embryos exposed to ethanol in vivo and in vitro (Wentzel and Eriksson, 2008). The observed differences in gene expression between the cNCC and tNCC exposed to the teratogenic milieu may reflect etiological differences in teratological susceptibility of these two NCC populations.
Section snippets
Results
Evaluation of the slides stained with either DLX or AP 2-α antibodies revealed that approximately 75% of the cells were of NCC origin, with no difference in the cranial or trunk cell cultures (Fig. 1B–E).
Discussion
One important finding in the present study was the ethanol-induced pro-apoptotic influence in both cNCC and tNCC, reflected as increased Bax/Bcl-2 ratio. This finding is in line with several previous studies describing enhanced apoptosis in embryonic neural tissue exposed to ethanol (Dunty et al., 2001, Green et al., 2005, Lee et al., 2005, Miller, 1996, Olney et al., 2002, Ramachandran et al., 2001), in particular has enhanced cell death been found in cNCC of chicken embryos exposed to ethanol
Animals
The Uppsala Regional Ethical Committee on Animal Experiments approved the research protocol including all experimental procedures involving animals. Inbred Sprague–Dawley rats were fed a commercial pelleted diet (R36, Lactamin, Kimstad, Sweden) and had free access to both food and tap water during the whole experiment. Female rats were mated with males during the night, conception was verified the following morning by the presence of sperm in the vaginal smear, and this day was denoted
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
The Swedish Labor Market Insurance Company, and The Swedish Research Council Grant No. 54X-21117 supported this study.
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