Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
Toxicol Sci. 2017 Aug 1;158(2):465-474. doi: 10.1093/toxsci/kfx107.
Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies.
斑马鱼是一种强大的模型系统，可用于评估在胚胎发育过程中暴露于有毒化学物质对分子和细胞的影响。为了研究环境内分泌干扰物的影响，通常将胚胎和幼鱼暴露于水中超生理浓度的这些化合物中，但它们在斑马鱼中的生物利用度在很大程度上是未知的。一种假设是，水中的超生理浓度雌激素是达到体内生理水平所必需的；然而，这尚未得到直接测试。为了验证这一假设，我们开发了一种使用放射性标记的雌二醇([3H]E2)的测定法，以测量从水中在 0 到 5 天受精后(postfertilization，dpf)发育中的斑马鱼在多个浓度和暴露时间的摄取。我们发现[3H]E2 的摄取随浓度、持续时间和发育阶段的增加而增加。从处理溶液的总体积中摄取的百分比随暴露持续时间和发育阶段的增加而增加，但随浓度的增加而保持不变。我们还发现，在 3 dpf 之前一直包围着胚胎的无细胞卵壳(chorion)不会显著影响[3H]E2 的摄取。最后，我们发现，在 1 dpf 时，E2 在多个暴露时间优先被卵黄吸收，而在 2 dpf 时，E2 优先被胚胎体吸收。我们的结果支持这样一种假设，即暴露于斑马鱼胚胎和幼鱼的超生理浓度雌激素是体内达到生理相关剂量所必需的。这里报告的同位素测定法将为确定其他化合物的摄取量用于致畸性、毒理学和药物发现研究提供基础。
