Gibert Y , Sassi-Messai S , Fini JB , Bernard L , Zalko D , Cravedi JP , Balaguer P , Andersson-Lendahl M , Demeneix B , Laudet V .

	Figure 1. BPA treatment induces otolith abnormalities. A: BPA induced otolith malformation is dose dependent. Embryos were treated with different concentrations of BPA from 5 hpf onwards. Black bars represent embryos showing no otolith abnormalities, red bars represent treated embryos showing otolith abnormalities. (*: P < 0.01, Fisher test after Bonferroni correction). B: Anterior otolith (ao, upper left) and posterior otolith (po, upper middle) of control embryo and ao (lower left) and po (lower middle) of embryos treated with 70 μM BPA from 5 to 50 hpf showing otolith aggregation. In some rare cases a single otolith (upper right) or extra otolith (eo lower right) are observed. C: Upper panel: control embryo at 50 hpf (Upper right: close up of the otic vesicle). Lower panel: overall shape of BPA treated embryos form 6-50 hpf resembles control embryos, but display otolith aggregates (arrow). Lower right: close up of the otic vesicle in a BPA treated embryo form 24-50 hpf showing normal otoliths. D: After 75 hours of exposure, the concentration of BPA measured in embryos is 0.7 μg equivalent per mg fresh weight and 0.22 μg of BPF equivalent per mg fresh weight; see also Figure 2 C-E. E: BPA induces malformation of the otic vesicle in Xenopus embryos. Xenopus embryos were exposed to BPA (5 or 10 μM) from stage NF 18 to stage NF 40 (48 h) and examined at stage NF 45. The upper panel shows the morphology of the otoliths (or otoconia [73]) in control and BPA treated embryos. Note that 5 and 10 μM of BPA induce a progressive reduction in the size of the otoconia. The lower panel shows the semi-circular canals with a reduction in the distance between the two semi circular canals (arrowheads) induced by BPA. Similarly, the morphology of the developing semi circular canals is flattened under BPA treatment.
	Figure 2. Bisphenol effects are time and compound specific. A: Live pictures of the developing otic vesicle from 18 to 50 hpf in zebrafish embryo. B: BPA affects otolith in a restricted time window. Diagram showing the effect of 70 μM BPA treatments with different starting points or length of exposure. Upper panel: BPA pulse treatments (red bars) were performed from 10 hpf. At different stage of development (18, 24, 30 and 38 hpf) embryos were washed and developed in BPA free medium (gray bars). Otic vesicles were scored at 50 hpf. Lower panel: BPA acts prior 22 hpf to induce otolith defects. Treatments started prior or at 18 hpf lead to 100% of otolith defects. Treatment started from 20 hpf onwards lead to 85% of embryos with otolith defect. Treatment started at 22 hpf lead to only 2% of affected embryos. Treatments started later did not lead to any otolith defect. Red bars represent time when embryos were exposed to BPA. Gray bars represent time when embryos were not exposed to this compound. C: Various bisphenols affect otolith development and/or pigmentation. Embryos were treated from 5 to 72 hpf and malformed otolith or pigmentation defects scored (+++ indicates maximum defect, i.e. malformed otolith or total lack of pigment). Treatment of embryos with either BPA (70 μM) or BPE (70 μM) resulted in malformed otoliths. BPE also affected pigmentation, as did BPF (50 μM). BPC (70 μM) was without clear effects in these assays. D: Pictures of the effect of BPC, BPE and BPF on otolith development. All embryos were exposed to 70 μM of bisphenol. Note that only BPE gives an otolith phenotype similar to what in observed in BPA treated embryos with otolith aggregates marked by a black arrow. E: Chemical structures of BPA, BPC, BPE and BPF.
	Figure 3. Expression of markers of inner ear development in zebrafish embryos are affected by BPA treatment. (A,B)oc90 a gene require for otolith formation in zebrafish is up-regulated under BPA treatment at 24 hpf (n=30). In situ for control and BPA treated embryos were performed in the same tube with the tip of the tail removed for the control embryos. (C,D)aldh1a3 is detected in the anterior cristae (ac), the cranial epithelial projection (cp), the endolymphatic duct (ed), the posterior cristae (pc) and the anterior macula (am) in wild type zebrafish embryo at 50 hpf (M), its expression is reduced in ac, cp and am and is absent in ed and pc in BPA treated embryos from 6 hpf onward (D). (E,F) Expression of ugdh at 50 hpf in control otic vesicle (E) is detected in the ac, cp, ed and pc. In BPA treated embryos from 6 hpf onwards (D), ugdh expression is severely reduced and remains solely detected in the ac and the cp. (G,H) Expression of bmp4 in control embryos at at 50 hpf (G) in the anterior (ac) lateral (lc) in the posterior cristae (pc) and the endolymphatic duct (ed) remains unaffected after BPA treatment (H). (I,J) Confocal microscopy of acetulated tubulin antibody staining showing the presence of the ciliated macula (white arrow) in control embryos (I) and in BPA treated embryos (white arrow in J).
	Figure 4. BPA effect on otolith development is estrogen receptor-independent. Morphology of the inner ear of zebrafish embryos at 48 hpf following exposure to BPA with or without ER antagonists (ICI 182 780) or agonists (17-β estradiol, E2). Treatment with either 1 μM ICI (A) or 1 μM 17-β estradiol (B) from 5 to 48 hpf does not induce any malformation of the developing semi-circular canals nor otoliths. Moreover, the BPA-induced otolith phenotype is not rescued when embryos are co-treated with BPA 5 μM +ICI 1 μM (A) or BPA 5 μM +βE2 1 μM (B). Interestingly, co-treatment with BPA 5 μM +ICI 1 μM lead to in increased ratio of affected otolith than a treatment with BPA 5 μM alone (A). On the other hand co-treatment with BPA 5 μM +βE2 1 μM gives a similar ratio than a treatment with BPA 5 μM alone (B).
	Figure 5. BPA effect on otolith development is thyroid hormones independent. (A) Control otic vesicle at 50 hpf with two otoliths. (B) Treatment with 1 μM of T3, from 5 hpf onwards lead to a normal otolith development. (C) 70 μM BPA treated embryo from 5 hpf onwards lead to otolith aggregates. (D) Half dose of BPA, 35 μM, does not affect otolith development. (E) Co-treatment of half dose of BPA, 35 μM, supplemented with 1 μM of T3 does not affect otolith development. (F) Co-treatment of full dose of BPA, 70 μM supplemented with 1 μM of T3 lead to a similar otolith phenotype than BPA 70 μM alone (compare C with F).
	Figure 6. Chemical inhibition of Na/K ATPase rescues the BPA induced phenotype. (A) 50 hpf control embryo with two normal otolith. (B) Treatment with 1.8 mM ouabain from 6 hpf onwards leads to one misformed otolith. (C) Embryo treated with 70 μM BPA from 5 hpf onwards leads to otolith aggregates. (D) Co treatment with 70 μM BPA and 1.8 mM oubain from 6 hpf onwards leads to normal otolith. (E) Co treatment with 70 μM BPA and 0.9 mM oubain from 6 hpf onwards leads to a mild BPA like phenotype. In some cases one almost normal otolith is found (arrow). (F) Co treatment with 50 μM BPA and 1.8 mM oubain from 6 hpf onwards leads to two normal otoliths. (G)α1a1 morphants display a complete absence of otolith. (H) 70 μM BPA of α1a1 morphants did not rescue otolith formation and result in a complete absence of otolith. (I)α1a1 is expressed in the lower membrane of the otic vesicle at 24 hpf. (J) A similar expression of α1a1 is detected in BPA treated embryos.

Abel, Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system. 1999, Pubmed

Abel, Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system. 1999, Pubmed
Avanesov, Mutations that affect the survival of selected amacrine cell subpopulations define a new class of genetic defects in the vertebrate retina. 2005, Pubmed
Baba, Bisphenol A disrupts Notch signaling by inhibiting gamma-secretase activity and causes eye dysplasia of Xenopus laevis. 2009, Pubmed , Xenbase
Bardet, Characterization of oestrogen receptors in zebrafish (Danio rerio). 2002, Pubmed
Basheer, Endocrine disrupting alkylphenols and bisphenol-A in coastal waters and supermarket seafood from Singapore. 2004, Pubmed
Belfroid, Occurrence of bisphenol A in surface water and uptake in fish: evaluation of field measurements. 2002, Pubmed
Bertrand, Unexpected novel relational links uncovered by extensive developmental profiling of nuclear receptor expression. 2007, Pubmed
Bever, Three-dimensional morphology of inner ear development in Xenopus laevis. 2003, Pubmed , Xenbase
Björnström, Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. 2005, Pubmed
Blasiole, Separate Na,K-ATPase genes are required for otolith formation and semicircular canal development in zebrafish. 2006, Pubmed
Blasiole, Differential expression of Na,K-ATPase alpha and beta subunit genes in the developing zebrafish inner ear. 2003, Pubmed
Busch-Nentwich, The deafness gene dfna5 is crucial for ugdh expression and HA production in the developing ear in zebrafish. 2004, Pubmed
Cabaton, Biotransformation of bisphenol F by human and rat liver subcellular fractions. 2008, Pubmed
Calafat, Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population. 2005, Pubmed
Chapin, NTP-CERHR expert panel report on the reproductive and developmental toxicity of bisphenol A. 2008, Pubmed
Chen, Acute toxicity, mutagenicity, and estrogenicity of bisphenol-A and other bisphenols. 2002, Pubmed
Colantonio, The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear. 2009, Pubmed
Crain, An ecological assessment of bisphenol-A: evidence from comparative biology. 2007, Pubmed
Duan, Individual and joint toxic effects of pentachlorophenol and bisphenol A on the development of zebrafish (Danio rerio) embryo. 2008, Pubmed
Escriva, Neofunctionalization in vertebrates: the example of retinoic acid receptors. 2006, Pubmed , Xenbase
Fini, An in vivo multiwell-based fluorescent screen for monitoring vertebrate thyroid hormone disruption. 2007, Pubmed , Xenbase
Focazio, A national reconnaissance for pharmaceuticals and other organic wastewater contaminants in the United States--II) untreated drinking water sources. 2008, Pubmed
Haddon, Early ear development in the embryo of the zebrafish, Danio rerio. 1996, Pubmed
Hammond, Hedgehog signalling is required for correct anteroposterior patterning of the zebrafish otic vesicle. 2003, Pubmed
Hans, Changes in retinoic acid signaling alter otic patterning. 2007, Pubmed
Hawkins, Large scale gene expression profiles of regenerating inner ear sensory epithelia. 2007, Pubmed
Heimeier, The xenoestrogen bisphenol A inhibits postembryonic vertebrate development by antagonizing gene regulation by thyroid hormone. 2009, Pubmed , Xenbase
Hiroi, Differential interactions of bisphenol A and 17beta-estradiol with estrogen receptor alpha (ERalpha) and ERbeta. 1999, Pubmed , Xenbase
Hughes, Mixing model systems: using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development. 2006, Pubmed
Ikezuki, Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. 2002, Pubmed
Imaoka, Bisphenol A causes malformation of the head region in embryos of Xenopus laevis and decreases the expression of the ESR-1 gene mediated by Notch signaling. 2007, Pubmed , Xenbase
Iwamuro, Effects of bisphenol A on thyroid hormone-dependent up-regulation of thyroid hormone receptor alpha and beta and down-regulation of retinoid X receptor gamma in Xenopus tail culture. 2006, Pubmed , Xenbase
Kausch, Biomarkers for exposure to estrogenic compounds: gene expression analysis in zebrafish (Danio rerio). 2008, Pubmed
Kimmel, Stages of embryonic development of the zebrafish. 1995, Pubmed , Xenbase
Kolpin, Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000: a national reconnaissance. 2002, Pubmed
Kuch, Determination of endocrine-disrupting phenolic compounds and estrogens in surface and drinking water by HRGC-(NCI)-MS in the picogram per liter range. 2001, Pubmed
Kuiper, The estrogen receptor beta subtype: a novel mediator of estrogen action in neuroendocrine systems. 1998, Pubmed
Legler, Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity. 2002, Pubmed
Maffini, Endocrine disruptors and reproductive health: the case of bisphenol-A. 2006, Pubmed
Markov, The evolution of the ligand/receptor couple: a long road from comparative endocrinology to comparative genomics. 2008, Pubmed
Meltser, Estrogen receptor beta protects against acoustic trauma in mice. 2008, Pubmed
Menuet, Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions. 2002, Pubmed
Moriyama, Thyroid hormone action is disrupted by bisphenol A as an antagonist. 2002, Pubmed
Morvan Dubois, Deiodinase activity is present in Xenopus laevis during early embryogenesis. 2006, Pubmed , Xenbase
Nicolson, The genetics of hearing and balance in zebrafish. 2005, Pubmed
Pastva, Morphological effects of Bisphenol-A on the early life stages of medaka (Oryzias latipes). 2001, Pubmed
Pennie, Differential activation by xenoestrogens of ER alpha and ER beta when linked to different response elements. 1998, Pubmed
Perez, The estrogenicity of bisphenol A-related diphenylalkanes with various substituents at the central carbon and the hydroxy groups. 1998, Pubmed
Petko, Otoc1: a novel otoconin-90 ortholog required for otolith mineralization in zebrafish. 2008, Pubmed
Pittlik, Expression of zebrafish aldh1a3 (raldh3) and absence of aldh1a1 in teleosts. 2008, Pubmed
Ramakrishnan, Impact of bisphenol-A on early embryonic development and reproductive maturation. 2008, Pubmed
Richter, Estradiol and Bisphenol A stimulate androgen receptor and estrogen receptor gene expression in fetal mouse prostate mesenchyme cells. 2007, Pubmed
Riley, A critical period of ear development controlled by distinct populations of ciliated cells in the zebrafish. 1997, Pubmed
Rüsch, Thyroid hormone receptor beta-dependent expression of a potassium conductance in inner hair cells at the onset of hearing. 1998, Pubmed
Sassi-Messai, The phytoestrogen genistein affects zebrafish development through two different pathways. 2009, Pubmed
Satoh, Study on anti-androgenic effects of bisphenol a diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives using cells stably transfected with human androgen receptor, AR-EcoScreen. 2004, Pubmed
Schönfelder, In utero exposure to low doses of bisphenol A lead to long-term deleterious effects in the vagina. 2002, Pubmed
Sisneros, Steroid-dependent auditory plasticity leads to adaptive coupling of sender and receiver. 2004, Pubmed
Solomon, Genetic interactions underlying otic placode induction and formation. 2004, Pubmed
Staples, An examination of the physical properties, fate, ecotoxicity and potential environmental risks for a series of propylene glycol ethers. 2002, Pubmed
Sun, Determination of bisphenol A in human breast milk by HPLC with column-switching and fluorescence detection. 2004, Pubmed
Takeuchi, Serum bisphenol a concentrations showed gender differences, possibly linked to androgen levels. 2002, Pubmed
Thalmann, Development and maintenance of otoconia: biochemical considerations. 2001, Pubmed
Thisse, Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. 1993, Pubmed
Waring, Endocrine disrupters: a human risk? 2005, Pubmed
Wetherill, In vitro molecular mechanisms of bisphenol A action. 2007, Pubmed
Whitfield, Mutations affecting development of the zebrafish inner ear and lateral line. 1996, Pubmed
Wozniak, Xenoestrogens at picomolar to nanomolar concentrations trigger membrane estrogen receptor-alpha-mediated Ca2+ fluxes and prolactin release in GH3/B6 pituitary tumor cells. 2005, Pubmed
Zalko, Biotransformations of bisphenol A in a mammalian model: answers and new questions raised by low-dose metabolic fate studies in pregnant CD1 mice. 2003, Pubmed
Zoeller, Bisphenol-A, an environmental contaminant that acts as a thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters RC3/neurogranin expression in the developing rat brain. 2005, Pubmed
Zoeller, Environmental chemicals as thyroid hormone analogues: new studies indicate that thyroid hormone receptors are targets of industrial chemicals? 2005, Pubmed
Zsarnovszky, Ontogeny of rapid estrogen-mediated extracellular signal-regulated kinase signaling in the rat cerebellar cortex: potent nongenomic agonist and endocrine disrupting activity of the xenoestrogen bisphenol A. 2005, Pubmed