Grantee Research Project Results
Final Report: Assessing the Effects of BPA Exposure on Early Human Development
EPA Grant Number: R834678C002Subproject: this is subproject number 002 , established and managed by the Center Director under grant R834678
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Center for Integrative Research on Childhood Leukemia and the Environment - 2015
Center Director: Metayer, Catherine
Title: Assessing the Effects of BPA Exposure on Early Human Development
Investigators: McMaster, Michael , Fisher, Susan J. , Woodruff, Tracey J.
Institution: University of California - San Francisco
EPA Project Officer: Hahn, Intaek
Project Period: May 1, 2010 through April 30, 2013
RFA: Children's Environmental Health and Disease Prevention Research Centers: Formative Centers (with NIEHS) (2009) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
To gain an understanding of the effects of BPA exposure on early human development using human embryonic stem cells (hESC) as a model system. To validate the candidate targets and pathways we identify by differentiating BPA-treated hESCs and studying their composition with regard to the progeny of the three definitive germ layers.
Summary/Accomplishments (Outputs/Outcomes):
Initial experiments showed that BPA treatment of a federally approved hESC cell line (UCSF4) resulted in modulation of nuclear receptors, transcription factors and their target genes as well as markers of differentiation. For example, the brachyury gene, encoding a factor important for establishment of the mesodermal lineage during gastrulation in early human embryos, was upregulated by BPA treatment. We then focused our analyses on the effects of BPA on expression of families of transcriptional regulators known to play key roles in hESC self-renewal and differentiation. Hox family members (e.g., HOX-C6, -C9, -B13, -C12, -C10, and DLX2) involved in early morphogenesis were particularly sensitive to regulation by BPA. These and other data support the hypothesis that BPA tips the balance from proliferation in an undifferentiated state toward differentiation of particular lineages in early embryos. This conclusion is also bolstered by experiments showing that BPA treatment inhibits proliferation and increases differentiation as assessed by morphological changes we observed in hESC colonies. During the remainder of the funding period, we completed work on both Aims. Specifically, we performed microarray-based global transcriptional profiling experiments in which hESCs were exposed to BPA in two doses (10 and 50 nM) or to estrogen or vehicle/ethanol controls. The unexpected and very interesting result was that BPA had a global effect on mRNA splicing rather that frank changes in the levels of expression of individual genes. The microarray data analysis strategy to determine exon-level changes is not included in the established methodologies and required significant methods development in our group. The current generation of Affymetrix microarrays, whole gene arrays, use random primers to initiate cDNA formation, thus capturing the entire length of transcripts from the 5’ to the 3’ end. To complement this approach, the probes on the arrays are designed to cover the entire length of each transcript. Thus, a single chip provides approximately 200,000 probes for 100,000 exons to 20,000 annotated genes and allows interrogation of quantitative differences in specific mRNA transcripts, including splice variants, at the exon level. We adapted a method using whole gene arrays and open-source analytical tools within R/Bioconductor, which allowed us to interrogate the microarray data set for the presence and extent of alternative splicing across the genome that resulted from BPA treatment. Thus, a major outcome of this work is the development of bioinformatics approaches that allow us to interrogate the splicome using standard microarray datasets.
By applying this methodology, we showed that a number of genes with diverse functions in hESCs were alternatively spliced in response to BPA treatment. Two patterns were observed. In the first, BPA and estrogen had similar effects suggesting common pathways. In the second, BPA had effects that were not observed in the estrogen-treated samples. These results suggest that in the hESC system BPA had estrogenic and nonestrogenic effects.
Interestingly, TIE1 transcripts encoding an angiopoietin receptor expressed by endothelial cells and hematopoietic precursors was alternatively spliced in response to both doses of BPA (but not estrogen). TIE1 regulates cell-adhesion molecules (VCAM-1, E-selectin and ICAM-1) and plays a key role in angiogenesis and blood vessel stability. Its role in early development is poorly understood, but alternatively spliced transcript variants encoding multiple isoforms with biological activity in vascular and inflammatory pathways have been described.
Another gene whose splicing was affected by BPA is SPG7, encoding paraplegin, part of the m-AAA protease complex responsible for ribosome assembly. Best known for mutations that cause spastic paraplegia type 7, altered splicing of this transcript has the potential to globally interfere with mRNA processing and translation thereby affecting diverse cellular processes including membrane trafficking, organelle biogenesis, protein folding and proteolysis. A splice variant of SPG7 was recently described in mice that is implicated in neurodegenerative processes. Other pathways that our data suggest may be impacted in BPA-induced splicing variation included growth factor signaling, cell motility and mitosis.
Alternative splicing is a central process for modulation of gene expression, affecting over 90 percent of human genes and greatly increasing the number of distinct protein products encoded in the genome. Constitutive and alternative splicing pathways are well-characterized, involve hundreds of genes and are known to be affected by various drugs and xenobiotics. Our data showing BPA-induced disruption of pre-mRNA splicing in hESCs suggests that gestational exposure to this ubiquitous environmental chemical may interfere with early development by mechanisms that involves changes in protein isoform expression.
To pursue this hypothesis, we have extended our analyses to include specific inquiries into splicing changes induced by BPA and other environmental chemicals we are testing. These include disruptions in splicing machinery as well as changes observed in transcripts with known roles in controlling hESC renewal in an undifferentiated state or differentiation into cells with characteristics of differentiated embryonic lineages. In addition, the microarray datasets we have generated through this work, in both control (untreated) and test conditions, have formed the foundation for incorporating the transcriptome of hESCs into studies we are now carrying out that monitor changes in the epigenome (DNA methylation and histone modifications) that accompany altered transcription and pre mRNA splicing in response to xenobiotic exposures.
Conclusions:
Evidence from our group and others increasingly supports the hypothesis that endocrine disruptors like BPA exert at least a portion of their developmental effects by altering the epigenome in early development.
Supplemental Keywords:
Risk assessment, metabolism, bioavailability, ethnic groups, endocrine disruptors, sensitive populations, decision making, measurement methods, RFA, Scientific Discipline, Health, INTERNATIONAL COOPERATION, Biochemistry, Children's Health, Environmental Policy, Biology, prenatal exposure, biological response, developmental toxicity, chemical mixtures, assessment of exposure, children's vulnerablity, children's environmental healthRelevant Websites:
UCSF Program on Reproductive Health and the Environment Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R834678 Center for Integrative Research on Childhood Leukemia and the Environment - 2015 Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R834678C001 Assessing Maternal and Fetal Exposure to Chemicals
R834678C002 Assessing the Effects of BPA Exposure on Early Human Development
R834678C003 (Pilot Study): Predictors of Maternal Exposure to BPA During Pregnancy
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.