Pyruvate dehydrogenase kinase isozyme 4 (Pdk4) mRNA expression.

<p>Real-time PCR quantification of Pdk4 gene was performed on the left ventricle (LV), right ventricle (RV), left atrium (LA) and the right atrium (RA) samples of rhesus monkey (Macaca mulatta) fetuses at birth. Fetuses were exposed maternally to a 400 µg/Kg. body weight of BPA dose (B) or 150 µL ethanol (C) during late gestation (LG, days 100±2–term). Data were analyzed by tissue and bars represent the mean ± S.E.M. of the log₂ (fold change). A two sample t-test was performed to identify significant BPA effects and a two-tailed p≤0.05 was considered statistically significant.</p

Differentially expressed genes observed using microarray on rhesus fetal heart exposed maternally to BPA or vehicle during early gestation, LG (days; 100±2–term).

<p>ND - below differential expression filter criteria.</p

Maternal Bisphenol A Exposure Impacts the Fetal Heart Transcriptome

<div><p>Conditions during fetal development influence health and disease in adulthood, especially during critical windows of organogenesis. Fetal exposure to the endocrine disrupting chemical, bisphenol A (BPA) affects the development of multiple organ systems in rodents and monkeys. However, effects of BPA exposure on cardiac development have not been assessed. With evidence that maternal BPA is transplacentally delivered to the developing fetus, it becomes imperative to examine the physiological consequences of gestational exposure during primate development. Herein, we evaluate the effects of daily, oral BPA exposure of pregnant rhesus monkeys (<i>Macaca mulatta</i>) on the fetal heart transcriptome. Pregnant monkeys were given daily oral doses (400 µg/kg body weight) of BPA during early (50–100±2 days post conception, dpc) or late (100±2 dpc – term), gestation. At the end of treatment, fetal heart tissues were collected and chamber specific transcriptome expression was assessed using genome-wide microarray. Quantitative real-time PCR was conducted on select genes and ventricular tissue glycogen content was quantified. Our results show that BPA exposure alters transcription of genes that are recognized for their role in cardiac pathophysiologies. Importantly, myosin heavy chain, cardiac isoform alpha (<i>Myh6</i>) was down-regulated in the left ventricle, and ‘A Disintegrin and Metalloprotease 12’, long isoform (<i>Adam12-l</i>) was up-regulated in both ventricles, and the right atrium of the heart in BPA exposed fetuses. BPA induced alteration of these genes supports the hypothesis that exposure to BPA during fetal development may impact cardiovascular fitness. Our results intensify concerns about the role of BPA in the genesis of human metabolic and cardiovascular diseases.</p></div

Differentially expressed genes observed using microarray on rhesus fetal heart exposed maternally to BPA or vehicle during early gestation, EG (days; 50–100±2).

<p>ND - below differential expression filter criteria.</p

Taqman MGB primer & probe system.

<p>Sequences of primers; forward (FP), reverse (RP), Taqman probe (TP) specific to gene transcripts analyzed.</p

Total number of significantly altered genes observed using microarray global transcriptome expression analysis.

<p>Whole transcriptome analysis was performed on the left ventricle (LV), right ventricle (RV), left atrium (LA) and the right atrium (RA) of rhesus monkey (Macaca mulatta) fetuses that were exposed maternally to a 400 µg/kg body weight, Bisphenol A (BPA), relative to matched control fetuses, either during, (A) early gestation, EG (days; 50–100±2) or, (B) late gestation, LG (days 100±2–term). Genes that changed by greater than ±1 log₂ fold change (1 LFC = 2 fold change) at an unadjusted p≤0.01 were considered significant and differentially expressed. Bars represent the number of upregulated (positive y-axis) or downregulated (negative y-axis) genes in each of the tissues.</p

Real-time PCR quantification of glycogenin (Gyg1) expression.

<p>Expression of Gyg1 mRNA in the left ventricle (LV), right ventricle (RV), left atrium (LA) and the right atrium (RA) of rhesus monkey (Macaca mulatta) fetuses at birth was measured. Fetuses were exposed maternally to a 400 µg/Kg. body weight of BPA dose (B) or 150 µL ethanol (C) during late gestation (LG, days 100±2–term). Data were analyzed by tissue and bars represent the mean ± S.E.M. of the log₂ (fold change). A two sample t-test was performed to identify significant BPA effects and a two-tailed p≤0.05 was considered statistically significant.</p

Altered fast myosin heavy chain expression.

<p>Real-time PCR quantification of myosin heavy chain 6, cardiac isoform alpha (Myh6) expression in the left ventricle (LV), right ventricle (RV), left atrium (LA) and the right atrium (RA) of rhesus monkey (Macaca mulatta) fetuses at birth. Fetuses were exposed maternally to a 400 µg/Kg. body weight of BPA dose (B) or 150 µL ethanol (C) during late gestation (LG, days 100±2–term). Data were analyzed by tissue and bars represent the mean ± S.E.M. of the log₂ (fold change). A two sample t-test was performed to identify significant BPA effects and a two-tailed p≤0.05 was considered statistically significant. Marginal significance between the groups was indicated with a ‘#’ symbol.</p

Expression changes in Adam12-l mRNA.

<p>Real-time PCR quantification of the long isoform of A Disintegrin And Metalloprotease (Adam12-l) expression in the left ventricle (LV), right ventricle (RV), left atrium (LA) and the right atrium (RA) of rhesus monkey (Macaca mulatta) fetuses at birth. Fetuses were exposed maternally to a 400 µg/Kg. body weight of BPA dose (B) or 150 µL ethanol (C) during late gestation (LG, days 100±2–term). Data were analyzed by tissue and bars represent the mean ± S.E.M. of the log₂ (fold change). A two sample t-test was performed to identify significant BPA effects and a two-tailed p≤0.05 was considered statistically significant. Asterisk (*) indicate significantly different groups.</p

Tissue glycogen estimation.

<p>Ventricular glycogen content was assessed using a calorimetric method on the left ventricle (LV) and the right ventricle (RV) of rhesus monkey (Macaca mulatta) fetuses at birth. Fetuses were exposed maternally to a 400 µg/Kg. body weight of BPA dose (B) or 150 µL ethanol (C) during late gestation (LG, days 100–term). Data were analyzed by tissue and bars represent the mean ± S.E.M., of the tissue glycogen content measured in µg for every milligram tissue. A two sample t-test at a two-tailed p≤0.05 was considered statistically significant.</p