Associate Professor, Arcadia University, PA
Our group is interested in the effects of environmental toxicants on development using the model system Drosophila melanogaster. Bisphenol-A (BPA) is produced in high quantities world-wide and is ubiquitously found in commercially available products. Human exposure to BPA through dietary and other means has been well documented. BPA is a known endocrine disruptor and exposure during development has been associated with a number of reproductive as well as other defects in a wide range of vertebrate and invertebrate model systems. We have recently reported that administration of BPA during Drosophila development promotes increased larval growth and an earlier onset of pupariation. Growth and maturation during development in Drosophila is determined, in part, by activation of several endocrine pathways. The effects of BPA on larval growth in our model system are consistent with previously reported phenotypes resulting from increased activity of the Insulin/Insulin-like growth factor (IIS)/ Target of Rapamycin (TOR) signaling network. In addition, BPA may directly affect ecdysteroid signaling through interaction with ecdysone receptor (EcR), as a weak interaction has been reported in cultured cells. Finally, recent reports suggest that BPA binds with high specificity to human estrogen-related receptor gamma (ERRg) in receptor binding assays, although BPA binding to the Drosophila ortholog, dERR, has not been established to date. We have initiated RNA-seq experiments in order to gain insight into the complex mechanisms underlying BPA toxicity in Drosophila. This study will focus on the effects of BPA (0.1 mg/L) after 48 hours of exposure (72 hours after egg laying, AEL), and the findings will serve as a basis for comparison for future RNA-seq studies carried out at different BPA concentrations and developmental time points.
In our recent study, we have found that the effects of BPA on growth are observed 24 hours following treatment (48 hours after egg laying, AEL), with the highest growth rate observed between 72 and 96 hours AEL. In order to gain insight into the putative mode(s) of BPA toxicity, we performed an RNA-sequence analysis at the 72 hour time point to identify gene expression patterns associated with activation of specific endocrine pathways, as well as to reveal other potential effects (i.e. effects on metabolism). Since the IIS/TOR endocrine pathways are highly conserved from flies to humans, the findings of this study will be of significant interest to the scientific community at large. Conversely, since specific endocrine pathways (i.e. estrogen signaling) have not been reported in flies, the generation of an RNA-seq data set from flies allows for the opportunity to analyze putative activation of endocrine pathways in the absence of estrogen signaling. Experiments were carried out in the fly strain Oregon-R-modENCODE (25211), obtained from the Bloomington Stock Center, Bloomington, IN, and used by several of the modENCODE contributors. This stock was chosen for our original study as expression datasets are currently available for larval developmental time points through the modENCODE consortium and can be used as a basis for comparison to newly generated datasets. In order to correlate changes in gene expression with phenotype, a growth assay was performed prior to processing larvae for RNA-seq analysis. At 72 hours AEL larvae were collected and imaged using a Leica FireCam V 3.2.0 (Leica Microsystems, Inc., Buffalo Grove IL, USA). The area of each individual larva was determined using ImageJ software and average larval area was determined. We used 15 larvae for each biological sample, and RNA was extracted using TRIzol reagent, followed by DNase I treatment and column purification.
RNA-seq dataset to be provided at a later time.
Materials are under development.
BPA exposure affects larval growth.
Mean larval area was determined by imaging groups of treated and control larvae and tracing individual larvae using ImageJ tools. Mean values are shown for 72 hours after egg laying (AEL) (top panel). Examples of larvae whose area (actual) approximated the calculated means for each treatment group at 72 hours AEL are shown (bottom panel).