Effects of bisphenol A and triclocarban on brain-specific expression of aromatase in early zebrafish embryos Eunah Chunga,1,2, Maria C. Gencoa,2,3, Laura Megrelisa,4, and Joan V. Rudermana,b,5 aDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; and bMarine Biological Laboratory, Woods Hole, MA 02543 Contributed by Joan V. Ruderman, September 15, 2011 (sent for review July 26, 2011) Estrogen regulates numerous developmental and physiological pesticides (7–9), surfactant derivatives (10, 11), and the plastics processes. Most effects are mediated by estrogen receptors (ERs), monomer bisphenol A (BPA) (12–14). Many xenoestrogens can which function as ligand-regulated transcription factors. Estrogen directly regulate the transcriptional activities of ERs (7, 8, 15– also regulates the activity of GPR30, a membrane-associated G 17), but their effects are not always identical to those of natural protein-coupled receptor. Many different types of environmental estrogen (18). Xenoestrogens can also affect physiological and contaminants can activate ERs; some can bind GPR30 as well. There developmental programs through membrane-associated subsets is growing concern that exposure to some of these compounds, of ERs and GPR30 (14, 19). termed xenoestrogens, is interfering with the behavior and re- Most mammals contain a single aromatase gene (CYP19), the productive potential of numerous wildlife species, as well as expression of which is controlled by multiple tissue-specific pro- affecting human health. Here, we investigated how two common, moters that respond to estrogen or other physiological regulators environmentally pervasive chemicals affect the in vivo expression (1). In contrast, many fish, including zebrafish, contain a pair of of a known estrogen target gene in the brain of developing zebra- aromatase genes: cyp19a/cyp19a1 and cyp19b/cyp19a2 (20), re- fish embryos, aromatase AroB, which converts androgens to estro- ferred to here as AroA and AroB, respectively. The AroA gene, gens. We confirm that, like estrogen, the well-studied xenoestrogen which is expressed most strongly in the ovary, lacks recognizable bisphenol A (BPA, a plastics monomer), induces strong brain-specific estrogen response elements and is not induced by estrogen. The overexpression of aromatase. Experiments using ER- and GPR30- AroB gene, which is expressed most strongly in the developing and selective modulators argue that this induction is largely through adult brains of both sexes, contains estrogen response elements nuclear ERs. BPA induces dramatic overexpression of AroB RNA in and is estrogen-responsive. Sawyer et al. (21) identified the period the same subregions of the developing brain as estrogen. The anti- around 72 to 96 h postfertilization (hpf) as a time when the AroB bacterial triclocarban (TCC) by itself stimulates AroB expression only gene is especially sensitive to further induction by exogenous es- slightly, but TCC strongly enhances the overexpression of AroB that trogen. Thus, as noted in that study and others, the AroB gene has is induced by exogenous estrogen. Thus, both BPA and TCC have the the potential to be useful in screening chemicals and environ- potential to elevate levels of aromatase and, thereby, levels of en- mental samples for estrogen-like activities and their effects on the dogenous estrogens in the developing brain. In contrast to estro- CNS. Here, we have investigated the effects of two pervasive gen, BPA-induced AroB overexpression was suppressed by TCC. synthetic chemicals, BPA and triclocarban (TCC), on expression These results indicate that exposures to combinations of certain of AroB in developing zebrafish embryos. hormonally active pollutants can have outcomes that are not easily BPA was among several orally active, nonsteroidal estrogenic predicted from their individual effects. compounds synthesized in the 1930s, but its activity was too low for clinical applications. In the 1950s, BPA found new use in the endocrine disruptors | environmental estrogens production of polycarbonate plastics and epoxy resins, which are now used in a wide variety of consumer products, including many strogen plays important roles in many developmental and food-packaging materials. Currently, BPA is one of the highest Ephysiological processes (1). Best understood are its re- production volume chemicals worldwide (13, 22). BPA’s estro- productive functions in females, where circulating estrogen genic properties were, in a way, accidentally rediscovered in the produced by the ovaries regulates many of the events involved in early 1990s as an activity that leached from polycarbonate lab- sexual maturation and fertility. Estrogen also affects the de- oratory flasks in quantities sufficient to stimulate ER-dependent velopment and maintenance of numerous organ systems in both gene transcription (12). Human exposures appear to come sexes, including bone, the cardiovascular system, and the central mainly from BPA that has leached from food containers lined nervous system. In the brain, for example, estrogen affects with BPA-based resins (23). BPA is also a significant contami- neuronal cell migration and survival, axonal outgrowth, synaptic nant of wastewater and biosolids from sewage treatment plants, plasticity, cognitive and social development, and memory (1, 2). with potential for affecting wildlife at environmentally relevant Nuclear and membrane-associated estrogen receptors (ERs) are concentrations (24–26). Experimental work with mammals shows expressed in many regions of the brain, as is GPR30, a mem- that fetal, neonatal, and adolescent exposures can affect de- brane-associated G protein-coupled estrogen receptor (2–6). Aromatase, the enzyme that synthesizes estrogen, is also expressed in the brains of both sexes, thus generating estrogen Author contributions: E.C., M.C.G., L.M., and J.V.R. designed research, performed re- locally. Among the many different signals that can regulate search, analyzed data, and wrote the paper. aromatase gene expression is estrogen itself, providing oppor- The authors declare no conflict of interest. tunities for both positive and negative feedback regulation (1). Over the past several decades, a steadily increasing number of Freely available online through the PNAS open access option. 1 ’ synthetic chemicals used in agriculture, manufacturing, and Present address: Cincinnati Children s Hospital Medical Center, Cincinnati, OH 45229. consumer products have been identified as xenoestrogens (also 2E.C. and M.C.G. contributed equally to this work. called environmental estrogens), compounds that can mimic or 3Present address: Brandeis University, Waltham, MA 02454. interfere with the actions of endogenous estrogens and thus act 4Present address: Université Paris Descartes, 75104 Paris, France. as endocrine disruptors. Well-studied examples include many 5To whom correspondence should be addressed. E-mail: [email protected]. 17732–17737 | PNAS | October 25, 2011 | vol. 108 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1115187108 Downloaded by guest on September 30, 2021 veloping organ systems in ways that have adverse consequences A in later life. For example, transient early exposures to BPA result 100 in alterations in the morphology of developing mammary glands 80 60 and increase the incidence of mutagen-induced breast cancer in Aro A 40 adults (13, 27, 28). In the brain, early exposures to BPA can lead 20 to permanent changes in brain morphology, disruptions in be- 0 havior that include increased levels of aggression and anxiety, 100 and alterations in learning, memory, exploration, and emotional 80 fi 60 responsiveness (13, 29). In humans, the rst prospective epide- Aro B miological study of prenatal BPA exposure and childhood be- 40 Expression (fold change) havior suggests that it may be associated with increases in 20 0 hyperactivity and aggression in a subset of 2-y-olds (30). 0n1 10 100 1000 M E2 TCC is an antimicrobial agent that is widely used in soaps and other personal care products, with some absorption occurring through the skin (31). TCC is one of the most frequently B Aro B detected contaminants of wastewaters and biosolids derived 100 from sewage treatment plants (32). Several studies have shown 80 that TCC can bioaccumulate, raising concerns about potential 60 — 40 effects on soil microorganisms, wildlife and via application of 20 fold change biosolids to agricultural fields—humans (26). TCC by itself 0 exhibits little or no agonist activity, but it increases hormone- 150 10 100 1105 stimulated transcription of androgen receptor (AR) and ER reporter constructs in mammalian tissue culture cells by up to nM E2 M BPA 30% (33). In vivo, TCC strongly amplifies effects of testosterone Fig. 1. Estrogen and BPA induce strong overexpression of AroB in zebrafish on male sex organs in castrated male rats (34, 35). In snails, TCC embryos. Groups of 30 zebrafish embryos were treated from 72 to 96 hpf with can stimulate an increase in embryo production, a response that the indicated concentrations of 17-β-estradiol (E2), BPA, or DMSO. Levels of has been used previously in that system to assay for xenoes- AroA and AroB RNAs relative to β-actin RNA were determined by qRT-PCR. (A) SCIENCES ’ trogens (36). To date, no studies of TCC s effects on transcrip- AroA induction (Upper); AroB induction (Lower). (B)AroBinduction. ENVIRONMENTAL tion of known estrogen target genes have been reported. Here, we confirm that BPA, like estrogen, induces strong overexpression of AroB in early zebrafish embryos. Experiments