Each year, approximately 1,500 infants are born with limb reduction malformations in the United States. Although the causes of most are unknown, prenatal exposures that have been associated include ones that reduce uteroplacental vascular perfusion such as cocaine, phenytoin, vasoactive and cardioactive agents and possibly chorionic villus sampling. Because of the common feature of reduced conceptal perfusion, it has been suggested that embryonic tissue hypoxia is the salient teratogenic mechanism. Preliminary findings presented here, however, argue for a major role for toxic reactive oxygen species generated in the mitochondria of teratogenically sensitive tissues during embryonic reperfusion. Consequently, the overall goal of the proposed studies is to examine the role of reactive oxygen species in teratogenesis during late gestation. Experimental protocols employing rat embryos in vivo and in vitro appear to accurately model the morphology and timing of limb reduction malformations in humans, enabling us to study relevant teratogenic mechanisms. We propose that during reperfusion, embryos generate toxic oxygen species by a process analogous to that associated with myocardial infarction, stroke and numerous other diseases. Unlike adults, embryos are uniquely ill-equipped to perform the enzymatic detoxification of potential oxidants and tissue injury occurs at sites where reactive oxygen species are generated in the greatest quantity. We propose to investigate the following hypotheses: (l) hypoxia/reoxygenation of late gestational conceptuses results in the generation of reactive oxygen species; (2) mitochondria represent the primary source of reactive oxygen species generated during conceptal reoxygenation and (3) reactive oxygen species generated locally during hypoxia/reoxygenation induce limb reduction malformations. The proposed studies will test these hypotheses in vitro and confirm the applicability of findings in vivo.
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