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Bisphenol A induces otolith malformations during vertebrate embryogenesis.

Gibert Y, Sassi-Messai S, Fini JB, Bernard L, Zalko D, Cravedi JP, Balaguer P, Andersson-Lendahl M, Demeneix B, Laudet V - BMC Dev. Biol. (2011)

Bottom Line: Here, we investigated BPA effects during embryonic development using the zebrafish and Xenopus models.As no effects on otolith development were seen with exposure to micromolar concentrations of thyroid hormone, 17-ß-estradiol or of the estrogen receptor antagonist ICI 182,780 we conclude that the effects of BPA are independent of estrogen receptors or thyroid-hormone receptors.The data suggest that the spectrum of BPA action is wider than previously expected and argue for a systematic survey of the developmental effects of this endocrine disruptor.

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Affiliation: Institut de Génomique Fonctionnelle de Lyon; Université de Lyon; Université Lyon 1; CNRS; INRA; Ecole Normale Supérieure de Lyon; 46 allée d'Italie, 69364 Lyon Cedex 07, France. vincent.laudet@ens-lyon.fr

ABSTRACT

Background: The plastic monomer and plasticizer bisphenol A (BPA), used for manufacturing polycarbonate plastic and epoxy resins, is produced at over 2.5 million metric tons per year. Concerns have been raised that BPA acts as an endocrine disruptor on both developmental and reproductive processes and a large body of evidence suggests that BPA interferes with estrogen and thyroid hormone signaling. Here, we investigated BPA effects during embryonic development using the zebrafish and Xenopus models.

Results: We report that BPA exposure leads to severe malformations of the otic vesicle. In zebrafish and in Xenopus embryos, exposure to BPA during the first developmental day resulted in dose-dependent defects in otolith formation. Defects included aggregation, multiplication and occasionally failure to form otoliths. As no effects on otolith development were seen with exposure to micromolar concentrations of thyroid hormone, 17-ß-estradiol or of the estrogen receptor antagonist ICI 182,780 we conclude that the effects of BPA are independent of estrogen receptors or thyroid-hormone receptors. Na+/K+ ATPases are crucial for otolith formation in zebrafish. Pharmacological inhibition of the major Na+/K+ ATPase with ouabain can rescue the BPA-induced otolith phenotype.

Conclusions: The data suggest that the spectrum of BPA action is wider than previously expected and argue for a systematic survey of the developmental effects of this endocrine disruptor.

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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).
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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).

Mentions: Next, we checked whether the phenotype implicated a defect in hindbrain patterning as this can occur when specific pathways (e.g. FGF, RA) are disrupted [50,51]. The expression of egr2a, a marker of rhombomere 3 and 5 segmentation at 18 hpf was thus examined. egr2a expression is not affected (Additional file 3A,B), suggesting that hindbrain patterning in the region of the otic vesicle is unaltered by the BPA treatment. We next tested if the antero-posterior or the dorso-ventral patterning of the otic vesicle was affected by BPA treatment. Using atonal1 (atoh1a) and hmx3, two markers expressed in the anterior part of the otic vesicle at 26 hpf, we showed that the antero-posterior patterning is not affected in BPA treated embryos (Additional file 3C-F). Similarly, dorso-ventral patterning is not altered, as displayed by dlx3b labelling at 26 hpf (Additional file 3G and 3H) [52]. Recently, Petko and colleagues [53] identified otoconin 90 (oc90) as essential for normal otolith development during zebrafish embryogenesis. When translation of this gene was abolished, otoliths were smaller in size, altered in shape or absent. To test for possible links between the BPA-induced phenotype and the oc90 phenotype, we studied oc90 expression in BPA treated embryos at 24 hpf. In our hands, oc90 is expressed in the ventral portion of the otic epithelium (Figure 3A). In BPA-treated embryos a stronger signal of oc90 expression is detected (Figure 3B). A similar up -regulation of oc90 is observed in embryos treated with another bis-phenol leading to otolith malformation, BPE (Additional file 4). Therefore, we conclude that BPA acts by altering directly or indirectly the expression of genes implicated in otolith development. To better understand the relationship between oc90 and BPA action, we injected a published morpholino against the oc90 gene [53]. The most dominant phenotype observed is a single posterior otolith (Additional file 5C) with sometimes the presence of 2 or 3 small otoliths in contact with each other. This finding recalls those of Petko and colleagues [53]. However, when we treated these oc90 morphants with BPA we observed a full BPA phenotype (i.e. high numbers of otholith aggregates) in the posterior developing otolith (Figure 5D). Therefore, even in the absence of oc90, BPA exposure can lead to otolith aggregation in a similar fashion to control treated embryos (see Additional file 5B and 5D).


Bisphenol A induces otolith malformations during vertebrate embryogenesis.

Gibert Y, Sassi-Messai S, Fini JB, Bernard L, Zalko D, Cravedi JP, Balaguer P, Andersson-Lendahl M, Demeneix B, Laudet V - BMC Dev. Biol. (2011)

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).
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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).
Mentions: Next, we checked whether the phenotype implicated a defect in hindbrain patterning as this can occur when specific pathways (e.g. FGF, RA) are disrupted [50,51]. The expression of egr2a, a marker of rhombomere 3 and 5 segmentation at 18 hpf was thus examined. egr2a expression is not affected (Additional file 3A,B), suggesting that hindbrain patterning in the region of the otic vesicle is unaltered by the BPA treatment. We next tested if the antero-posterior or the dorso-ventral patterning of the otic vesicle was affected by BPA treatment. Using atonal1 (atoh1a) and hmx3, two markers expressed in the anterior part of the otic vesicle at 26 hpf, we showed that the antero-posterior patterning is not affected in BPA treated embryos (Additional file 3C-F). Similarly, dorso-ventral patterning is not altered, as displayed by dlx3b labelling at 26 hpf (Additional file 3G and 3H) [52]. Recently, Petko and colleagues [53] identified otoconin 90 (oc90) as essential for normal otolith development during zebrafish embryogenesis. When translation of this gene was abolished, otoliths were smaller in size, altered in shape or absent. To test for possible links between the BPA-induced phenotype and the oc90 phenotype, we studied oc90 expression in BPA treated embryos at 24 hpf. In our hands, oc90 is expressed in the ventral portion of the otic epithelium (Figure 3A). In BPA-treated embryos a stronger signal of oc90 expression is detected (Figure 3B). A similar up -regulation of oc90 is observed in embryos treated with another bis-phenol leading to otolith malformation, BPE (Additional file 4). Therefore, we conclude that BPA acts by altering directly or indirectly the expression of genes implicated in otolith development. To better understand the relationship between oc90 and BPA action, we injected a published morpholino against the oc90 gene [53]. The most dominant phenotype observed is a single posterior otolith (Additional file 5C) with sometimes the presence of 2 or 3 small otoliths in contact with each other. This finding recalls those of Petko and colleagues [53]. However, when we treated these oc90 morphants with BPA we observed a full BPA phenotype (i.e. high numbers of otholith aggregates) in the posterior developing otolith (Figure 5D). Therefore, even in the absence of oc90, BPA exposure can lead to otolith aggregation in a similar fashion to control treated embryos (see Additional file 5B and 5D).

Bottom Line: Here, we investigated BPA effects during embryonic development using the zebrafish and Xenopus models.As no effects on otolith development were seen with exposure to micromolar concentrations of thyroid hormone, 17-ß-estradiol or of the estrogen receptor antagonist ICI 182,780 we conclude that the effects of BPA are independent of estrogen receptors or thyroid-hormone receptors.The data suggest that the spectrum of BPA action is wider than previously expected and argue for a systematic survey of the developmental effects of this endocrine disruptor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon; Université de Lyon; Université Lyon 1; CNRS; INRA; Ecole Normale Supérieure de Lyon; 46 allée d'Italie, 69364 Lyon Cedex 07, France. vincent.laudet@ens-lyon.fr

ABSTRACT

Background: The plastic monomer and plasticizer bisphenol A (BPA), used for manufacturing polycarbonate plastic and epoxy resins, is produced at over 2.5 million metric tons per year. Concerns have been raised that BPA acts as an endocrine disruptor on both developmental and reproductive processes and a large body of evidence suggests that BPA interferes with estrogen and thyroid hormone signaling. Here, we investigated BPA effects during embryonic development using the zebrafish and Xenopus models.

Results: We report that BPA exposure leads to severe malformations of the otic vesicle. In zebrafish and in Xenopus embryos, exposure to BPA during the first developmental day resulted in dose-dependent defects in otolith formation. Defects included aggregation, multiplication and occasionally failure to form otoliths. As no effects on otolith development were seen with exposure to micromolar concentrations of thyroid hormone, 17-ß-estradiol or of the estrogen receptor antagonist ICI 182,780 we conclude that the effects of BPA are independent of estrogen receptors or thyroid-hormone receptors. Na+/K+ ATPases are crucial for otolith formation in zebrafish. Pharmacological inhibition of the major Na+/K+ ATPase with ouabain can rescue the BPA-induced otolith phenotype.

Conclusions: The data suggest that the spectrum of BPA action is wider than previously expected and argue for a systematic survey of the developmental effects of this endocrine disruptor.

Show MeSH
Related in: MedlinePlus