Perturbation of retinoic acid (RA)-mediated limb development suggests a role for diminished RA signaling in the teratogenesis of ethanol§

Authors

  • Corey S. Johnson,

    1. The Department of Cell and Developmental Biology, and Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill, North Carolina
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  • Robert M. Zucker,

    1. Reproductive Toxicology Branch, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina
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  • Edward Sidney Hunter III,

    Corresponding author
    1. Reproductive Toxicology Branch, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina
    • Reproductive Toxicology Branch, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina
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  • Kathleen K. Sulik

    1. The Department of Cell and Developmental Biology, and Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill, North Carolina
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  • This article is a US Government work and, as such, is in the public domain in the United States of America.

  • This work has been presented, in part, at the 62nd annual meeting of the Society for Developmental Biology, July 30–August 3, 2003 in Boston, MA and at the 44th annual meeting of the Teratology Society, June 26–July 1, 2004 in Vancouver, BC.

  • §

    Disclaimer: Although the research described in this article has been supported in part by the United States Environmental Protection Agency, it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Abstract

BACKGROUND:

A proposed mechanism for ethanol teratogenicity entails ethanol-mediated reductions in retinoic acid (RA). This premise was investigated utilizing a mouse model, with limb reduction defects as the teratogenic end point.

METHODS:

Ethanol, Disulfiram, or BMS-189453 was administered to C57BL/6J mice on the 9th day of pregnancy. Forelimb morphology was assessed on gestation day 18 using Alcian blue and Alizarin red staining. Nile blue sulfate or LysoTracker Red (LTR) vital staining identified cell death in the limb bud. The ability of RA to prevent ethanol-induced cell death was assessed by coadministration followed by laser scanning confocal microscopic examination of LTR-staining. In situ hybridization and qPCR were used to examine gene expression in treated limb buds.

RESULTS:

Ethanol, Disulfiram, and BMS-189453 resulted in postaxial ectrodactyly, intermediate ectrodactyly, and other digital defects. Excessive Nile blue sulfate staining was evident in the presumptive AER following each of the three exposures. Ethanol-induced LTR staining was prevented by RA supplementation. Both in situ hybridization and qPCR illustrated decreases in Shh and Tbx5 in ethanol-exposed embryos as compared to control.

CONCLUSIONS:

Contrary to studies of prolonged RA deficiency, acute exposure to functional antagonists of RA results in limb defects that are morphologically similar to those caused by ethanol. The rescue of ethanol-induced cell death by RA and similar changes in Shh transcription further suggest that RA contributes to ethanol-induced limb dysmorphology. Moreover, the repression of key mediators of limb development soon after ethanol exposure adds to the existing knowledge of the pathogenic effects of ethanol. Birth Defects Research (Part A), 2007. © 2007 Wiley-Liss, Inc.

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