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Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid.

ABSTRACT: Fetal Alcohol Syndrome (FAS), a severe consequence of the Fetal Alcohol Spectrum Disorders, is associated with craniofacial defects, mental retardation, and stunted growth. Previous studies in C57BL/6J and C57BL/6N mice provide evidence that alcohol-induced pathogenesis follows early changes in gene expression within specific molecular pathways in the embryonic headfold. Whereas the former (B6J) pregnancies carry a high-risk for dysmorphogenesis following maternal exposure to 2.9 g/kg alcohol (two injections spaced 4.0 h apart on gestation day 8), the latter (B6N) pregnancies carry a low-risk for malformations. The present study used this murine model to screen amniotic fluid for biomarkers that could potentially discriminate between FAS-positive and FAS-negative pregnancies.B6J and B6N litters were treated with alcohol (exposed) or saline (control) on day 8 of gestation. Amniotic fluid aspirated on day 17 (n = 6 replicate litters per group) was subjected to trypsin digestion for analysis by matrix-assisted laser desorption-time of flight mass spectrometry with the aid of denoising algorithms, statistical testing, and classification methods.We identified several peaks in the proteomics screen that were reduced consistently and specifically in exposed B6J litters. Preliminary characterization by liquid chromatography tandem mass spectrometry and multidimensional protein identification mapped the reduced peaks to alpha fetoprotein (AFP). The predictive strength of AFP deficiency as a biomarker for FAS-positive litters was confirmed by area under the receiver operating characteristic curve.: These findings in genetically susceptible mice support clinical observations in maternal serum that implicate a decrease in AFP levels following prenatal alcohol damage.

SUBMITTER: Datta S

PROVIDER: S-EPMC2803677 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid.

Datta Susmita S Turner Delano D Singh Reetu R Ruest L Bruno LB Pierce William M WM Knudsen Thomas B TB

Birth defects research. Part A, Clinical and molecular teratology 20080401 4

<h4>Background</h4>Fetal Alcohol Syndrome (FAS), a severe consequence of the Fetal Alcohol Spectrum Disorders, is associated with craniofacial defects, mental retardation, and stunted growth. Previous studies in C57BL/6J and C57BL/6N mice provide evidence that alcohol-induced pathogenesis follows early changes in gene expression within specific molecular pathways in the embryonic headfold. Whereas the former (B6J) pregnancies carry a high-risk for dysmorphogenesis following maternal exposure to ...[more]

PMID: 18240165

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Project description:Alcohol consumption during pregnancy causes fetal alcohol spectrum disorder (FASD), which includes a range of developmental deficits. Fetal alcohol syndrome is the most severe form of FASD and can be diagnosed with pathognomonic facial features such as a smooth philtrum, short palpebral fissure, and thin upper vermilion. However, many children with developmental damage because of prenatal alcohol exposure exhibit none, or only a subset, of the above features, making diagnosis difficult. This study explored novel analyses to quantify the effect of a known dose of alcohol on specific facial measurements in substrains C57BL/B6J (B6J) and C57BL/6NHsd (B6N) mice. Mouse dams were provided alcohol (Alc) consisting of 4.8% (vol/vol) alcohol in a liquid diet for 16 days prepregnancy and chow and water diet during mating, and then the alcohol liquid diet was reinstated on gestational days 7 (E7) to gestational day 17 (E17). Treatment controls included a pair-fed (PF) group given matched volumes of an alcohol-free liquid diet made isocalorically and a group given ad lib access to lab chow and water (Chow). Maternal diet intake (Alc and PF), blood alcohol concentrations (BACs), embryo weights, and 15 morphometric facial measurements for E17 embryos were analyzed. B6N dams drank more alcohol during pregnancy and generated higher BAC than B6J dams. Both the Alc and PF treatments induced significant reductions in embryo weights relative to Chow in both substrains. Alcohol treatments produced significant changes, relative to controls, in 4 of the 15 facial measures for the B6N substrain but only in two measures for the B6J substrain. Discriminant analysis demonstrated successful classification of the alcohol-exposed versus nonalcohol-exposed B6N embryos, with a high sensitivity of 86%, specificity 80%, and overall classification (total correct 83%), whereas B6J mice yielded sensitivity of 80%, specificity 78%, and overall correct classification in 79%. In addition, B6N mice showed significantly more effects of pair feeding on these facial measures than did B6J mice, suggesting that the B6N substrain may be more vulnerable to nutritional stress during pregnancy. Overall, these data indicate that both B6N and B6J mice were vulnerable to alcohol but show differences in the severity and location of alcohol-induced dysmorphic facial features and may parallel findings from human studies comparing different ethnic groups. Furthermore, these findings suggest that discriminant analysis may be useful in predicting alcohol exposure in either mouse substrains.

Project description:Fetal alcohol spectrum disorders (FASD) describe many of the well-known neurodevelopmental deficits afflicting children exposed to alcohol in utero. The effects of alcohol on the maternal-fetal interface, especially the placenta, have been less explored. We herein hypothesized that chronic binge alcohol exposure during pregnancy significantly alters the placental protein profile in a rat FASD model.Pregnant rats were orogastrically treated daily with alcohol (4.5 g/kg, gestational day [GD] 5 to 10; 6.0 g/kg, GD 11 to 19) or 50% maltose dextrin (isocalorically matched pair-fed controls). On GD 20, placentae were collected, flash-frozen, and stored until tissues were homogenized. Protein lysates were denatured, reduced, captured on a 10-kDa spin filter, and digested. Peptides were eluted, reconstituted, and analyzed by a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer.Mass spectrometry (MS) analysis identified 2,285 placental proteins based on normalized spectral counts and 2,000 proteins by intensity-based absolute quantification. Forty-five placental proteins were significantly (p < 0.05) altered by gestational alcohol exposure by both quantification approaches. These included proteins directly related to alcohol metabolism; specific isoforms of alcohol dehydrogenase and aldehyde dehydrogenase were up-regulated in the alcohol group. Ingenuity analysis identified ethanol degradation as the most significantly altered canonical pathway in placenta, and fetal/organ development as most altered function, with increased risk for metabolic, neurological, and cardiovascular diseases. Physiological roles of the significantly altered proteins were related to early pregnancy adaptations, implantation, gestational diseases, fetal organ development, neurodevelopment, and immune functions.We conclude that the placenta is a valuable organ not only to understand FASD etiology but it may also serve as a diagnostic tool to identify novel biomarkers for detecting the outcome of fetal alcohol exposure. Placental MS analysis can offer sophisticated insights into identifying alcohol metabolism-related enzymes and regulators of fetal development.

Dataset Information

Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid.

Publications

Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid.

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