At What Stage (Tier), Endocrine Disrupting Effects Of Chemicals Are Evaluated In Whole Animal?

doi: 10.1016/j.cbpc.2017.09.007. Epub 2017 Sep 21.

Assaying uptake of endocrine disruptor compounds in zebrafish embryos and larvae

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• PMID: 28943455
• PMCID: PMC5862746
• DOI: 10.1016/j.cbpc.2017.09.007

Gratuitous PMC article

Assaying uptake of endocrine disruptor compounds in zebrafish embryos and larvae

J Paige Souder  et al. Comp Biochem Physiol C Toxicol Pharmacol. 2018 Jun .

Free PMC article

Abstract

To written report the furnishings of ecology endocrine disruptor compounds (EDCs) on aquatic animals, embryos and larvae are typically incubated in water containing defined concentrations of EDCs. Still, the amount of EDC uptake into the animal is oft hard to determine. Using radiolabeled estradiol ([³H]E2), we previously developed a rapid, straightforward analysis to measure estradiol uptake from water into zebrafish embryos and larvae. Here, we extend this approach to measure the uptake of two additional EDCs, bisphenol A (BPA) and ethinyl estradiol (EE2). As with E2, the uptake of each compound by individual larvae was low (<6%), and increased with increasing concentration, duration, and developmental stage. Nosotros establish that E2 and EE2 had similar uptake under equivalent exposure conditions, while BPA had comparatively lower uptake. One awarding of this assay is to exam factors that influence EDC uptake or efflux. It has been suggested that persistent organic pollutants (POPs) inhibit ABC transporters that may normally efflux EDCs and their metabolites, inducing toxicity in aquatic organisms. We measured [³H]E2 levels in zebrafish in the presence or absenteeism of the POP PDBE-100, and cyclosporine A, a known inhibitor of ABC transporters. Neither chemical significantly affected [³H]E2 levels in zebrafish, suggesting that zebrafish maintain estradiol efflux in the presence of PDBE-100, independently of cyclosporine A-responsive transporters. These uptake results will be a valuable reference for EDC exposure studies in developing zebrafish, and provide a rapid analysis to screen for chemicals that influence estrogen-like EDC levels in vivo.

Keywords: Bisphenol A; Endocrine disruptors; Estrogens; Zebrafish.

Copyright © 2017 Elsevier Inc. All rights reserved.

Figures

Figure 1. EE2 uptake is greater than BPA at multiple exposure concentrations and durations

(A–F) Embryos at 48 hours postal service fertilization were exposed to iii unlike concentrations of [³H]EE2 (A–C) or [³H]BPA (D–F) for one hour (A,D) or 24 hours (B,E). Radioactive decay was measured using a scintillation counter and used to summate pmol per embryo (black circles) and pct uptake per embryo (white circles). For each compound, pmol uptake increased in a dose-dependent manner (teal or royal lines connecting black circles), whereas percent [³H]E2 uptake remained constant (grey lines connecting white circles). Each circle represents the hateful uptake from a unmarried experiment (n=three) assaying ten embryos per experiment. Unlike embryos were assayed at each dose. Lines connect the mean values of the iii experiments for each group. Horizontal dotted line represents the limit of detection (0.01 pmol). (C) Both pmol and percent [ⁱⁱⁱH]EE2 uptake increased approximately half dozen-fold following 24-hour exposure compared to 1-hour exposure. Horizontal lines represent the mean fold change in pmol (teal) or percentage uptake (grey). (F) Both pmol and percent [³H]BPA uptake increased approximately 13-fold following 24-hour exposure compared to 1-60 minutes exposure. Horizontal lines correspond the mean fold change in pmol (purple) or percent uptake (grey).

Effigy two. EE2 and BPA uptake depend on developmental stage in zebrafish

(A, B) Embryos and larvae were exposed to five nM [³H]EE2 (A,B) or 10 nM [^threeH]BPA (C,D) for one hour (A,C) or 24 hours (B,D) starting at five different developmental stages betwixt 6 and 96 hours mail service fertilization (hpf). Radioactivity was measured using a scintillation counter and used to calculate pmol per embryo (black circles) and percent uptake per embryo (white circles). Each circle represents the mean uptake from a single experiment (north=iii–five) assaying 10 embryos per experiment. Different embryos were assayed at each treatment catamenia. Lines connect the mean values of the iii–5 experiments for each grouping. Horizontal dotted line represents the limit of detection (0.01 pmol). Brackets denote percent uptake comparisons that are non statistically significant (ns), all other comparisons are significant (i-way ANOVA, p < 0.05).

Effigy three. BPA uptake is less than E2 and EE2 uptake

(A,B) EE2 and BPA uptake compared to E2 uptake in 48 hours mail service fertilization (hpf) embryos exposed to 5 nM (A) or 10 nM (B) of each chemical compound for 1 or 24 hours. (C,D) EE2 and BPA pct uptake compared to E2 in half dozen–96 hpf zebrafish exposed to 5 nM [ⁱⁱⁱH]EE2 or [^threeH]E2 or 10 nM [³H]BPA for one hr (C) or 24 hours (D). Fold-change compared to E2 was calculated past dividing the boilerplate pmol or percent uptake of [ⁱⁱⁱH]EE2 or [³H]BPA (n=3 experiments, 10 embryos per experiment) by the pmol or percent uptake of [^threeH]E2 at each exposure status. Horizontal lines represent the mean fold change in pmol (teal/royal) or percent uptake (grayness). Horizontal grayness line at y=1 represents no change in uptake compared to E2. [^threeH]E2 data from Souder and Gorelick, 2017.

Effigy 4. E2 uptake is not affected by ABC transporter inhibition

(A) Embryos were exposed at 96 hpf to vehicle or 0.v µM rhodamine B (RhB) with or without 5 µM cyclosporin A (CsA) and not-radioactive 5 nM E2 for 24 hours in the dark. Embryos were imaged following treatment and mean integrated density of fluorescence for whole embryos was measured (mean fluorescence). Each circumvolve on the graph represents the mean fluorescence from a single experiment (n=two–3 experiments, 10 embryos per experiment), horizontal blue line represents the hateful. Horizontal dotted line at y=666 represents mean fluorescence of vehicle-treated embryos (due north=10 embryos). Representative fluorescence images for each group are shown to the right of the graph. CsA treatment reduced RhB efflux, indicated past significantly increased RhB fluorescence. E2 exposure had no meaning effect on CsA-dependent RhB efflux (oneway ANOVA, ns=not significant, ***p<0.001, ****p<0.0001). Fluorescence of each grouping was significantly increased compared to vehicle (p<0.05). (B) Embryos at 96 hpf were exposed to five nM [³H]E2 with or without v µM CsA for 24 hours. Radioactivity was measured using a scintillation counter and used to summate pmol per embryo (blackness circles) and pct uptake per embryo (white circles). Horizontal blueish lines represent mean pmol uptake, gray lines correspond mean percentage uptake, due north= v–vi experiments, 10 embryos per experiment. CsA failed to increase uptake of [³H]E2. Instead, percent and pmol uptake were slightly decreased with the improver of CsA (unpaired Pupil's t-examination, **p<0.01 percentage uptake, p<0.05 pmol uptake, CsA + E2 vs vehicle + E2). (C) Embryos at 96 hpf were exposed to 10 nM [^threeH]BPA with or without 5 µM CsA for 24 hours. Radioactivity was measured using a scintillation counter and used to summate pmol per embryo (blackness circles) and percent uptake per embryo (white circles). Horizontal regal lines represent hateful pmol uptake, gray lines stand for mean percent uptake, north=2–three experiments, 10 embryos per experiment. Percentage [³H]BPA uptake was non significantly changed with the addition of CsA (unpaired Student's t-test, ns not pregnant, p≥0.05). (D) Embryos at 72 hpf were exposed to 5 nM [³H]E2 together with vehicle or with PBDE-100 (20 – 200 nM). Radioactivity was measured using a scintillation counter and used to calculate pmol per embryo (blackness circles) and percentage uptake per embryo (white circles). Each circle represents a single embryo, northward=ten embryos per treatment. Horizontal blue lines represent mean pmol uptake, gray lines represent mean percent uptake. No significant alter was found between groups (i-mode ANOVA, p≥0.05). B-D, Horizontal dotted line represents the limit of detection (0.01 pmol).

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