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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.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: To determine if the timing of BPA action corresponds to that of inner ear organogenesis in zebrafish, two series of experiments were done. First, BPA pulse treatments of different lengths were started at 10 hpf and continued until 18, 24, 30 or 38 hpf, a time frame covering the main steps of otic vesicle development that are shown on Figure 2A[47]. The morphology of the otoliths was examined at 50 hpf. In all cases, exposure to BPA (70 μM) during these critical periods induced otolith malformation with 100% of the treated embryos displaying otolith aggregates (Figure 2B, Upper red bars, n = 30). Otolith aggregates were never observed in control embryos (n = 30). In a second series of experiments, treatment started from 6 hpf onwards or at 2 h intervals thereafter till 30 hpf (ie earliest treatment starting at 6 hpf and latest treatment starting at 30 hpf). Again, the otolith morphology was examined at 50 hpf. When BPA exposure began prior to or at 18 hpf, 100% of the embryos displayed malformed otoliths (i.e. otolith aggregates). When treatment began at 20 hpf the proportion of affected embryos decreased to 85% (n = 40, Figure 2B, lower panel). However, almost no effects (only 2% of the embryos affected, n = 40) were seen when treatments began at 22 hpf (Figure 2B, lower panel). Treatments starting after 22 hpf did not induce any otolith defect (Figure 2B, lower panel and data not shown). Of note is the fact that phenotype severity (i.e; number of aggregates) is dependent on time of treatment onset. The earlier the BPA exposure (i.e. the closer to mid-blastula transition, 6 hpf), the stronger the phenotype (See Additional file 2). According to Riley and colleagues [48] and Colantonio et al., [49], otic vesicle formation occurs at 18-18.5 hpf. Thus, our results suggest that BPA acts prior to actual otolith formation per se, potentially acting on determination of cells that will give rise to the otoliths.


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)

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.
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Related In: Results  -  Collection

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Show All Figures
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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.
Mentions: To determine if the timing of BPA action corresponds to that of inner ear organogenesis in zebrafish, two series of experiments were done. First, BPA pulse treatments of different lengths were started at 10 hpf and continued until 18, 24, 30 or 38 hpf, a time frame covering the main steps of otic vesicle development that are shown on Figure 2A[47]. The morphology of the otoliths was examined at 50 hpf. In all cases, exposure to BPA (70 μM) during these critical periods induced otolith malformation with 100% of the treated embryos displaying otolith aggregates (Figure 2B, Upper red bars, n = 30). Otolith aggregates were never observed in control embryos (n = 30). In a second series of experiments, treatment started from 6 hpf onwards or at 2 h intervals thereafter till 30 hpf (ie earliest treatment starting at 6 hpf and latest treatment starting at 30 hpf). Again, the otolith morphology was examined at 50 hpf. When BPA exposure began prior to or at 18 hpf, 100% of the embryos displayed malformed otoliths (i.e. otolith aggregates). When treatment began at 20 hpf the proportion of affected embryos decreased to 85% (n = 40, Figure 2B, lower panel). However, almost no effects (only 2% of the embryos affected, n = 40) were seen when treatments began at 22 hpf (Figure 2B, lower panel). Treatments starting after 22 hpf did not induce any otolith defect (Figure 2B, lower panel and data not shown). Of note is the fact that phenotype severity (i.e; number of aggregates) is dependent on time of treatment onset. The earlier the BPA exposure (i.e. the closer to mid-blastula transition, 6 hpf), the stronger the phenotype (See Additional file 2). According to Riley and colleagues [48] and Colantonio et al., [49], otic vesicle formation occurs at 18-18.5 hpf. Thus, our results suggest that BPA acts prior to actual otolith formation per se, potentially acting on determination of cells that will give rise to the otoliths.

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