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Octanol antagonism of ethanol teratogenesis¹

SHAO-YU CHEN^*, MICHAEL F. WILKEMEYER, KATHLEEN K. SULIK^* and MICHAEL E. CHARNESS²

^* University of North Carolina Bowles Center for Alcohol Studies, and Department of Cell Biology and Anatomy, University of North Carolina School of Medicine, Chapel Hill North Carolina 27599, USA;
Neurology Service, VA Boston Healthcare System, West Roxbury, Massachusetts 02132; USA; and Department of Neurology, Harvard Medical School, Boston, and Department of Neurology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA

²Correspondence: Department of Neurology (127), Harvard Medical School, VA Boston Healthcare System, 1400 VFW Parkway, West Roxbury, MA 02132, USA. E-mail: mcharness{at}hms.harvard.edu

SPECIFIC AIMS

Alcohol may cause birth defects in part by disrupting the developmentally critical L1 cell adhesion molecule. Because 1-octanol antagonizes ethanol inhibition of L1-mediated cell adhesion, we used mouse whole embryo culture to test whether 1-octanol also prevents ethanol teratogenicity.

PRINCIPAL FINDINGS

1. Low concentrations of 1-octanol are not toxic in whole embryo culture
To determine the toxicity of 1-octanol, gestational day 8 (GD8) whole mouse embryos were cultured for 6 h in the absence and presence of 1-octanol and transferred to control medium for an additional 20 h. Somite pairs were counted after a total of 26 h in culture. Based on somite counts, treatment with 3 µM 1-octanol did not produce a delay in embryonic development, whereas 10 µM and higher concentrations of 1-octanol caused increasing dysmorphogenesis (Fig. 1a ).

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of 1-octanol and ethanol on the morphology of cultured C57Bl/6J GD8 mouse embryos. a) Mean ± SE number of somite pairs after treatment for 6 h with the indicated concentrations of 1-octanol and after an additional 20 h of culture (n=4–6). b) Mean ± 95% confidence interval and number of somite pairs in individual embryos treated for 6 h under control conditions (n=18), in the presence of 100 mM ethanol alone (n=23) or with 100 mM ethanol combined with 3 µM 1-octanol (n=23) and after an additional 20 h of culture. *P < 0.001, ethanol plus octanol vs. ethanol alone; **P < 0.001, ethanol vs. control; P=0.024, ethanol plus octanol vs. control. c) Illustrated is a representative embryo (median number of somites) from each of the three experimental groups: control, 19 somites; ethanol (EtOH), 13 somites; ethanol plus octanol (EtOH/Oct), 17 somites.

2. 1-Octanol decreases ethanol-induced dysmorphology
Embryos cultured for 6 h with 100 mM ethanol showed markedly delayed in vitro development (13.2±0.6 somite pairs after 26 h, n=23) compared with control embryos (19.1±0.4 somite pairs, n=18, P<0.001) (Fig. 1b , c ). The toxicity of 100 mM ethanol was significantly reduced by coincubating embryos with 3 µM 1-octanol (16.9±0.6 somite pairs; n=23, P<0.001). Only 8.7% of the embryos treated with ethanol alone had 17 somite pairs; in contrast, 94.4% of the control embryos and 60.9% of the embryos treated with 1-octanol and ethanol had 17 somite pairs. Conversely, whereas 43.5% of the embryos treated with ethanol alone had 12 somite pairs, none of the control embryos and only 4.3% of the embryos treated with ethanol plus 1-octanol were this small.

3. 1-Octanol decreases ethanol-induced apoptosis
To determine whether 1-octanol antagonizes ethanol-induced apoptosis, GD8 mouse whole embryos were cultured for 6 h in the absence and presence of alcohols and then an additional 6 h in alcohol-free medium. Compared with controls, ethanol-exposed embryos showed excessive Nile blue sulfate staining in the lateral margins of the cranial neuroepithelium (Fig. 2b , d , f ), including the region of the neural crest. TUNEL staining was also increased in the ethanol exposed embryos and the distribution of TUNEL staining was similar to that obtained with Nile blue sulfate (Fig. 2c , e , g ). Coincubation of embryos with 3 µM 1-octanol greatly reduced the ethanol-induced increase in Nile blue sulfate and TUNEL staining, indicating that 1-octanol decreased ethanol-induced apoptosis.

View larger version (68K): [in this window] [in a new window]   Figure 2. Effect of 1-octanol on ethanol-induced cell death. Twelve hours after the initiation of culture, control embryos have 8–10 somite pairs and have begun to close their neural tube (a). The midbrain/hindbrain junction is indicated by the curved arrows; arrowheads indicate the edge of the anterior neural folds; the asterisk is in the position of the motor nucleus of the trigeminal nerve. Comparably staged control (b, c), ethanol-exposed (d, e), and ethanol+octanol-exposed (f, g) embryos vitally stained with Nile blue sulfate (b, d, f) or using whole-mount TUNEL (c, e, g) illustrate 1-octanol-mediated amelioration of apoptosis. d) Inset illustrates the pattern of ethanol-induced cell death as viewed from the lateral aspect of the embryo’s head and neck. The rest of the images are dorsal views. Shown are representative results from 10–13 (Nile blue) and 5–6 (TUNEL) independent experiments.

4. L1 is expressed in GD8 mouse embryos
If 1-octanol antagonizes the early embryotoxicity of ethanol through effects on L1, then L1 expression should be detectable in GD8–9 whole mouse embryos. Anti L1 monoclonal antibody 74–5H7 (Covance; Richmond, CA) stained virtually all cell populations present in histological sections through the head and neck of GD8 embryos. The heaviest stained cells were localized to regions populated by neural crest cells, a cell type that is particularly sensitive to ethanol. Expression of predominantly the RSLE- negative isoform of L1 was confirmed by the polymerase chain reaction (PCR) and sequencing of PCR products.

CONCLUSIONS

The brain abnormalities of children with alcohol-related birth defects, including fetal alcohol syndrome, can resemble those of children with L1 gene mutations. For this reason, we have speculated that inhibition of L1-mediated cell–cell adhesion contributes to the teratogenic effects of ethanol. The major finding of these experiments is that 3 µM 1-octanol, a long-chain alcohol, significantly antagonizes the teratogenic effects of 100 mM ethanol, a short-chain alcohol, in mouse whole embryo culture. The same concentration of 1-octanol half-maximally antagonizes ethanol inhibition of L1-mediated cell–cell adhesion. The structural similarity of these alcohols raises the possibility that 1-octanol decreases ethanol teratogenesis by blocking its interaction with L1.

Animal studies suggest that multiple actions of alcohol account for its teratogenicity, including induction of apoptosis. Our current study shows that 1-octanol greatly reduces ethanol-induced apoptosis, although the underlying mechanism is unclear. 1-Octanol could be an antagonist for multiple effects of ethanol on cell signaling; however, 1-octanol is a more potent agonist than ethanol in many of these signaling pathways. At this time, the only action for which 1-octanol is a demonstrated antagonist is ethanol inhibition of L1-mediated cell–cell adhesion.

How might ethanol inhibition of cell adhesion lead to apoptosis? Disruption of cell–cell or cell–substrate adhesion promotes apoptosis through a reactive oxygen-dependent process known as anoikis. If ethanol induces apoptosis by disrupting L1-mediated cell adhesion, then 1-octanol might prevent ethanol teratogenesis by blocking ethanol’s interaction with L1 (Fig. 3 ). L1 was detected in control mouse embryos as early as GD8, an early stage of vulnerability to ethanol teratogenesis. The paradoxical inhibition of ethanol teratogenicity by a longer chain 1-alcohol suggests a strategy for developing compounds that prevent alcohol-related birth defects.

View larger version (41K): [in this window] [in a new window]   Figure 3. Hypothetical mechanism whereby 1-octanol antagonizes ethanol-induced apoptosis. A) L1-mediated cell–cell adhesion inhibits apoptosis. B) Ethanol inhibits L1-mediated cell–cell adhesion. Loss of cell contact promotes apoptosis. C) 1-Octanol antagonizes ethanol inhibition of L1-mediated cell–cell adhesion. Cell contact inhibits apoptosis. Proteins that interact with L1 might also be targets of ethanol. Ethanol could also inhibit L1-mediated cell–substrate adhesion.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0862fje ; to cite this article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0862fje

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