Aquatic Toxicology 80 (2006) 396–404 Vulnerable windows for developmental ethanol toxicity in the Japanese medaka fish (Oryzias latipes) Sharon L. Oxendine a,b,1 , John Cowden b , David E. Hinton c , Stephanie Padilla b,∗ a Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States b Cellular and Molecular Toxicology Branch, Neurotoxicology Division (B105-06), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, United States c Division of Environmental Sciences and Policy, Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC 27708, United States Received 28 July 2006; received in revised form 17 October 2006; accepted 19 October 2006 Abstract Ethanol (EtOH) is a well-known developmental toxicant that produces a range of abnormal phenotypes in mammalian systems including craniofacial abnormalities, cognitive deficits and growth retardation. While the toxic potential of developmental EtOH exposure is well characterized clinically, the effect of timing on the extent of toxicity remains unknown. Fish models such as the Japanese medaka, Oryzias latipes, provide a convenient system for investigating the effects of developmental EtOH exposure in vivo. In this study, medaka embryo toxicity tests were used to assess temporal variations in developmental EtOH toxicity. Fertilized eggs were collected and incubated during early, middle or late egg development (e.g., 0–3, 3–6 or 6–9 days post-fertilization) with various sub-lethal concentrations of EtOH [0.1% (17.2 mM), 0.5% (86.0 mM) or 1% (172 mM)]. Uptake of EtOH by the embryo was 60–68% of the solution concentration across all windows. Time to hatch, head width, total body length and whole embryo caspase activity were used to assess toxicity. Hatching delays were noted only at the highest concentration of EtOH. Head width was affected at all ethanol levels, regardless of the window of exposure. EtOH-induced decreases in body length, however, appeared to be most pronounced when exposure occurred either during the first or last window. The effect on caspase-3/7 activity also depended on the window of exposure, with increases in caspase noted in embryos treated on days 1 or 2 (first window) and decreases seen in embryos treated on day 6 (second window) or day 8 (third window). In general, these data suggest that critical periods for heightened sensitivity to developmental EtOH exposure may vary according to the specific endpoint used to assess toxicity. © 2006 Elsevier B.V. All rights reserved. Keywords: Ethanol; FAS; Developmental toxicity; Embryo; Vulnerable windows; Window of sensitivity; Fish 1. Introduction Women who consume large amounts of ethanol during pregnancy often give birth to children exhibiting phenotypic abnormalities collectively referred to as the Fetal Alcohol Syndrome (FAS). These anomalies include growth deficiency, cognitive impairment and distinctive craniofacial features (reviewed in Coles, 1993). Ethanol is a well-known developmen- ∗ Corresponding author. Tel.: +1 919 541 3956; fax: +1 919 541 3335. E-mail addresses: Oxendine.Sharon@epa.gov (S.L. Oxendine), Cowden.john@epa.gov (J. Cowden), DHinton@Duke.edu (D.E. Hinton), Padilla.stephanie@epa.gov (S. Padilla). 1 Present address: National Center for Environmental Economics, U.S. Envi- ronmental Protection Agency (1809 T), 1200 Pennsylvania Avenue, NW, Washington, DC 20460, United States. tal toxicant in humans and laboratory animals. The mechanism for the toxic actions of ethanol on the developing organism is, however, unknown (for a recent review, see Goodlett et al., 2005). Many hypotheses exist, including interaction with neu- rotrophins (Kentroti, 1997), cell-adhesion molecules (Bearer, 2001), or specific receptors (e.g., Costa and Guizzetti, 2002); increased apoptosis (Olney et al., 2002a,b); or increased oxida- tive stress (Cohen-Kerem and Koren, 2003). Not only are the mechanisms as yet unknown (or multifunctional), but the issue of sensitive periods remains unresolved. That is, it is unclear if certain stage(s) of development are more sensitive to ethanol exposure. Very few studies have systematically examined differences in the vulnerability of the entire central nervous system at distinct periods throughout brain development. There are pri- mate studies, however, that compared short periods of ethanol 0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.10.007