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Reprod Toxicol
2019 Mar 01;84:65-74. doi: 10.1016/j.reprotox.2018.12.005.
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Bisphenol A, Bisphenol AF, di-n-butyl phthalate, and 17β-estradiol have shared and unique dose-dependent effects on early embryocleavage divisions and development in Xenopus laevis.
Arancio AL
,
Cole KD
,
Dominguez AR
,
Cohenour ER
,
Kadie J
,
Maloney WC
,
Cilliers C
,
Schuh SM
.
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Bisphenol A (BPA), Bisphenol AF (BPAF), and di-n-butyl phthalate (DBP) are widespread compounds used in the production of plastics. We used Xenopus laevis to compare their effects on early embryo cell division and development. Directly after in vitro fertilizations, embryos were exposed to BPA, BPAF, DBP, or 17β-estradiol (E2) for up to 96 h. BPA (1-50 μM) and BPAF (0.003-25 μM) caused disrupted cleavage divisions, slowed cytokinesis, and cellular dissociation within 1-6 h. Flexures of the spinal cord, shorter body axis/tail, craniofacial malformations, and significant mortality occurred with environmentally relevant doses of BPAF (LC50 = 0.013 μM). DBP (10-200 μM) showed similar effects, but with severe ventral edema. There were both shared and unique effects of all compounds, with BPAF having the greatest potency and toxicity (BPAF > BPA > estradiol > DBP). These findings underscore the pleiotropic effects of widespread toxicants on early development and highlight the need for better toxicological characterization.
Fig. 1. The progression of developmental defects with increasing doses of BPA or BPAF. Representative embryos exposed from 0.5 hrs (2 h.p.f.) to 72 hrs, to doses of 0, 10, 25, or 50 μM BPA (A) or 0, 0.03, 0.3, 3, or 25 μM BPAF or 10 μM E2 (B). Higher doses of the chemicals resulted in increased developmental defects including irregular, asymmetrical mitotic divisions and neurulation (white arrows), truncated body axis/tail, curved spinal cord, craniofacial defects, pigment reduction, and ventral blisters and edema, along with decreased survival rates (black and white arrows; right columns; scale bar = 500 μm).
Fig. 2. BPA and BPAF disrupt cleavage divisions and neurulation at very early stages of development. A) At stage 6.5 around 4 hrs of development, control embryos (left) exhibit concise and regular mitotic cell divisions and cell membranes are clearly defined. The embryos exposed to increasing doses of BPA (A) or BPAF (B) had increasing irregular cleavage divisions, cellular dissociation, delayed development, and greatly reduced numbers of cells, which were larger in size. The bottom panels show cell counts and tracked cell borders. Estradiol-treated embryos (A, right) had some cellular dissociation, but were similar to controls (scale bar = 250 μm). C) Quantification of mean cell counts per embryo at 4 hrs show the effect of the toxicants on cleavage divisions (3–5 trials; *P < 0.05; **P < 0.005). D) Abnormal neurulation at 24 hrs was observed in about 60% of BPA-treated embryos (middle) with irregular neural folds and neural grooves (45% of embryos), incomplete and open neural tubes (10%), and extra neural tubes (5%), compared to controls (left). BPAF resulted in severely abnormal and incomplete neurulation in 95% of embryos (right).
Fig. 3. Dose-dependent effects of BPA and BPAF on the number of normal surviving embryos over the span of 72 hours. The mean percentage of normally developing embryos exposed to increasing concentrations of BPA (A, a–d) or BPAF (B, e–h) compared to control embryos (black) from 0 to 72 hours. Also shown are the mean percentages of normal embryos treated with 10 μM estradiol (E2; purple) for comparison (B, e). Legends indicate the concentrations (mean + SEM; 3–7 trials; ~300–1,000 embryos/treatment; all solutions with 0.03% ethanol; *P < 0.05; **P < 0.005).
Fig. 4. Comparison of the effects of DBP, BPA, BPAF, and E2 on Xenopus tadpoles. A) Control embryos (top) developing from stage 27 to 40 compared to embryos treated with 25 µM DBP (bottom), showing malformed head and curved, shorter body axis and tail (black arrows), ventral blisters (white arrows), and overall delayed development at the corresponding times. B) The mean percent of normal embryos at various doses of DBP over the span of 96 hours (mean + SEM; 3 replicates; 300–350 embryos/treatment; *P < 0.05). C) Control embryos at 96 hrs compared to those exposed to 10 μM estradiol (D), 10 μM BPA (E), 0.03 μM BPAF (F), 0.3 μM BPAF (G), or 200 μM DBP (H) (all solutions with 0.03% ethanol). E2 caused bent tails, pigmentation reduction, long loosely coiled gut, and a ventral blister (D, arrows). BPA caused body axis defects, and shorter bent tails, head and eye defects, and occasional blisters (E, arrows). BPAF caused craniofacial defects, shorter tails, ventral blisters, edema and peritoneal effusion at the lowest doses tested (F, arrows). All embryos exposed to 200 μM DBP had severe body malformations and ventral edema (H, arrows; scale bar = 1 mm).
Supplemental Figure 1: Early cleavage division defects caused by BPA and BPAF. Control embryos (left column), compared to embryos exposed to BPA (50 M; middle column) or BPAF (3 M; right column) at 1, 2 and 6 hours of exposure. Irregular, asymmetrical mitotic division, slowed cytokinesis, and cellular dissociation were observed in the majority of BPA- and BPAF-treated embryos (arrows).
Supplemental Figure 2. : Early embryological defects caused by E2, BPA, and BPAF. A) Control tailbud stage embryos at 48 hours of development compared to tadpoles exposed to 10 M estradiol (B), BPA at 1 (C), 10 (D), and 25 M (E), and BPAF at 0.03 M (F), showing curved body axis defects, neural tube defects including two neural tubes, gut blisters, and overall extreme abnormalities (arrows; scale bar = 1 mm).
