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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2004 as doi:10.1096/fj.03-0850fje. |
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Bowles Center for Alcohol Studies and Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
1 Correspondence: Bowles Center for Alcohol Studies, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7178, USA. E-mail: syc{at}med.unc.edu
SPECIFIC AIMS
Fetal alcohol spectrum disorders (FASD) comprise a well-recognized cadre of birth defects that result from maternal alcohol consumption. Based on in vitro studies that have illustrated prevention of ethanol-induced cell death and malformations by antioxidants, the purpose of this study was to examine, using an in vivo model, the anti-teratogenic potential of a potent synthetic superoxide dismutase (SOD) plus catalase mimetic, EUK-134.
PRINCIPAL FINDINGS
1. In vivo ethanol exposure resulted in major limb malformations in C57BL/6J mouse fetuses
A high incidence (67.3%) of predominantly right-sided limb reduction defects was observed in C57BL/6J mouse fetuses after gestational day 9 (GD 9) maternal ethanol treatment. No defects were observed in 37 fetuses from 5 saline-treated control litters. Among 33 of 49 viable ethanol-exposed fetuses examined that were abnormal, the forelimb was affected unilaterally (63.6% of abnormal fetuses) or bilaterally (36.4% of abnormal fetuses). Of those with unilateral defects, all except two fetuses were affected on the right side (Table 1
). As observed both by gross examination and in cleared skeletal preparations, malformations as a whole represent postaxial reduction defects involving the autopod and zeugopod. Absence or reduction in number of postaxial metacarpals and of the distal portion of the ulna resulted in ulnar deviation of the autopod. The most severely affected specimens presented with a single, apparently syndacylous, radial digit. Intermediate digit loss (apparently involving digit 4) was not uncommon, occurring in 7 of the abnormal fetuses. Of the total normal complement of forelimb digits, 24.5% were missing in the ethanol-treated group. In addition to digit loss, some affected autopods presented with the full complement of digits but abnormal soft tissue loss (splits) between digits 3 and 4 or 4 and 5. Toxicity of ethanol was also evidenced by the fact that it increased the incidence of resorption from 8.1% in saline control litters to 22.2% in the ethanol-treated group.
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2. EUK-134 significantly decreased the incidence and severity of ethanol-induced limb defects
Of 77 viable GD 18 fetuses from control dams treated with EUK-134, alone, no defects were observed, and the incidence of resorption was 4.9%. Administered concurrently with ethanol, this antioxidant significantly reduced both the percentage of fetuses that presented with limb malformations (from 67.3% to 35.9%, P<0.01) and the percentage that resorbed (from 22.2% to 7.1%, P<0.01) as a result of ethanol treatment alone. Additionally, only 9.8% of the total number of possible forelimb digits in EUK-134 cotreated fetuses were missing, which is significantly lower than that in the ethanol-treated group (24.5%; P<0.0001). Besides a significant difference in total number of missing digits, frequency distribution of missing digits in affected limb buds was different between EUK-134 cotreated fetuses and those treated with ethanol alone. More specifically, a less severe phenotype (loss of only 1 digit or interdigital splits) was far more common in the EUK-134 cotreated specimens (37.5% of affected limbs in the EUK-134 cotreated group vs. 17.8% of affected limbs in the ethanol-treated group) than in the fetuses exposed to ethanol alone, where loss of 2 or more digits was more commonly seen. (82.2% of the affected limbs in ethanol-treated group vs. 62.5% of the affected limbs in the EUK-134 cotreated group; P<0.05).
3. EUK-134 decreases ethanol-induced apoptosis in the developing apical ectodermal ridge (AER)
Examination of GD 9 embryos at 15 h after initial treatment of the dam with saline, ethanol, or ethanol in combination with EUK-134 entailed a qualitative assessment of amount and distribution of cell death in developing limb buds. Vital staining with Nile blue sulfate showed localization of excessive amounts of stain uptake in the distal rim of the limb buds of the ethanol-exposed embryos as compared to the control and co-EUK-134-treated embryos (Fig. 1
a–c). This was consistent with TUNEL assays in which the dark brown staining identified sites of DNA fragmentation (apoptosis) in histological sections through the limb buds. As compared with stage-matched controls and co-EUK-134-treated embryos, excessive apoptosis was readily evident in developing the AER of ethanol-exposed embryos (Fig. 1d-f
).
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CONCLUSIONS AND SIGNIFICANCE
Results of this study show that maternal antioxidant treatment is effective in diminishing major malformations caused by prenatal ethanol exposure. For the purpose of this investigation, using the limb as the model system proved advantageous. Advantages of this model system include 1) provision of an easily quantifiable endpoint with respect to incidence and severity of malformation; 2) ready application of whole mount techniques that allow a comprehensive assessment of pertinent ethanol-induced cellular pathogenesis; and 3) an existing wealth of information regarding the developmental biology of the system, information that provides important clues regarding ethanol’s teratogenic mechanisms of action.
A number of studies have demonstrated that maternal ethanol exposure can directly or indirectly induce oxidative stress. Umbilical vessel spasm and transient impairment of umbilical circulation has been reported after maternal ethanol administration in monkeys. Not only vasoconstriction and tissue ischemia, but also the subsequent reperfusion results in cellular damage that is largely the result of free radical generation. Free radical generation associated with ethanol’s teratogenesis could also result from ethanol metabolism via the microsomal ethanol oxidizing system as well as from ethanol oxidation-mediated excess NADH generation and disturbance of mitochondrial electron transport. Use of spin trapping and electron paramagnetic resonance spectroscopy has confirmed that both acute and chronic alcohol exposure in vivo and in vitro results in the generation of free radicals.
Included among the damaging effects of reactive oxygen species (ROS) are oxidation of cellular membrane polyunsaturated fatty acids and subsequent changes in membrane fluidity and permeability, protein fragmentation and cross-linking, and nucleic acid damage, each of which could ultimately lead to cell death. ROS have been proposed to be common mediators of apoptosis, and antioxidants have been shown to be able to prevent apoptosis whether initiated by ROS or not.
In addition to the direct effects on key cell molecules, ROS may act as secondary messengers and subsequently alter gene expression. It has long been recognized that integrity of the AER is required for continued proximo-distal limb outgrowth. The AER appears to function by maintaining immediately subjacent cells in a proliferative state. Its formation and maintenance depend on genetic cascades that involve signaling between the mesenchyme and limb ectoderm. Whether mediated by ROS or not, the AER cell death observed in ethanol-exposed limb buds in this study may be a direct result of altered gene expression. Defining a cascade of ethanol-induced genetic alterations remains a major challenge.
Most eukaryotic cells possess inherent antioxidant defense mechanisms that include both nonenzymatic neutralization and enzymatic detoxification of oxygen-derived radicals and other reactive species. However, early embryos might be expected to have only minimal antioxidant capabilities, since the amount of O2 they metabolize is very low. Indeed, studies by Davis et al. have provided evidence that neural crest cells cultured from chick embryos have a deficiency of SOD. Consequently, exposure to even small amounts of oxidative stress during early stages of development might be expected to be particularly deleterious.
Although SOD has been used to diminish ethanol-induced cell death and teratogenesis in both cell and whole embryo culture, prevention is not complete. In addition to the fact that teratogenic mechanisms other than free radical damage may result from ethanol treatment, only partial prevention by SOD might be due in part to the accumulation of hydrogen peroxide from the dismutation of superoxide anion by SOD in the absence of an adequate amount of catalase or peroxidase. Combined SOD and catalase activity, which ensures immediate conversion of hydrogen peroxide to oxygen at the site of its formation, renders EUK complexes potentially advantageous over other antioxidants.
In conclusion, the fact that in vivo cotreatment with SOD/catalase mimetic EUK-134 resulted in diminished AER cell death and parallel reductions in the incidence and severity of limb defects not only supports the premise of a causal link between excessive ethanol-induced cell death and subsequent malformations, but also shows that free radicals are major players with respect to ethanol’s cellular toxicity. This, coupled with the fact that synthetic or naturally occurring antioxidants such as vitamin C or E can readily be applied therapeutically in human populations, provides hope for diminishing the effects of prenatal alcohol exposure in the offspring of women who are unable or unwilling to curtail their use of alcohol.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0850fje; doi: 10.1096/fj.03-0850fje
This article has been cited by other articles:
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  P. Wentzel and U. J. Eriksson Ethanol-Induced Fetal Dysmorphogenesis in the Mouse Is Diminished by High Antioxidative Capacity of the Mother Toxicol. Sci., August 1, 2006; 92(2): 416 - 422. [Abstract] [Full Text] [PDF]
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