Pericardial edema – fluid accumulation within the pericardium – is a frequently observed malformation in zebrafish embryo-based chemical toxicity screens. We recently discovered that the severity of triphenyl phosphate (TPHP)-induced pericardial edema was dependent on the ionic strength of exposure media. TPHP is an aryl phosphate ester (APE) widely used as a plasticizer and flame retardant. APEs are characterized by having one or more aryl groups bound to a phosphate center, with TPHP containing only unsubstituted aryl groups. Therefore, the objective of this study was to begin investigating whether, similar to TPHP, pericardial edema induced by other structurally related APEs is dependent on the ionic composition of exposure media. We first mined the peer-reviewed literature to identify other APEs that 1) induced pericardial edema in zebrafish embryos within a minimum of three peer-reviewed publications, and 2) demonstrated a statistically significant induction of pericardial edema in at least 70 % of the studies evaluated. Based on this meta-analysis, we identified four other APEs that caused pericardial edema in zebrafish embryos: isopropylated triphenyl phosphate (IPTPP), cresyl diphenyl phosphate (CDP), tricresyl phosphate (TMPP), and 2-ethylhexyl diphenyl phosphate (EDHPHP). Using TPHP as a positive control and pericardial edema as a readout, we developed concentration-response curves for all four APEs based on static exposure from 24 to 72 h post-fertilization (hpf). We then conducted co-exposures with D-Mannitol (an osmotic diuretic) and exposures within reverse osmosis (RO) water determine whether the ionic composition of exposure media mitigated APE-induced pericardial edema at 72 hpf. Using pericardial edema as an endpoint, the approximate EC50s for TPHP (positive control), IPTPP, CDP, TMPP, and EDHPHP were 6.25, 3.125, 3.125, 25, and 100 µM, respectively, based on exposure from 24 to 72 hpf. Interestingly, similar to our findings with TPHP, co-exposure with D-Mannitol and exposure within ion-deficient water significantly mitigated IPTPP- CDP-, TMPP-, and EDHPHP-induced pericardial edema in zebrafish embryos, suggesting that chemically-induced pericardial edema may be 1) dependent on the ionic composition of exposure media and 2) driven by a disruption in osmoregulation across the embryonic epidermis. Therefore, similar to other assay parameters, our findings underscore the need to standardize the osmolarity of exposure media in order to minimize the potential for false positive/negative hits in zebrafish embryo-based chemical toxicity screens conducted around the world.

中文翻译：

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斑马鱼胚胎中芳基磷酸酯诱导的心包水肿受暴露培养基的离子组成的影响


心包水肿 – 心包内积液 – 是基于斑马鱼胚胎的化学毒性筛查中经常观察到的畸形。我们最近发现磷酸三苯酯 （TPHP） 诱导的心包水肿的严重程度取决于暴露介质的离子强度。TPHP 是一种芳基磷酸酯 （APE），广泛用作增塑剂和阻燃剂。APE 的特征是具有一个或多个与磷酸盐中心结合的芳基，其中 TPHP 仅包含未取代的芳基。因此，本研究的目的是开始研究与 TPHP 类似，由其他结构相关的 APE 诱导的心包水肿是否取决于暴露介质的离子组成。我们首先挖掘同行评议的文献以确定其他 APE，这些 1） 在至少三篇同行评议的出版物中诱导斑马鱼胚胎心包水肿，以及 2） 在至少 70% 的评估研究中证明心包水肿的诱导具有统计学意义。基于这项荟萃分析，我们确定了导致斑马鱼胚胎心包水肿的其他四种 APE：异丙基磷酸三苯酯 （IPTPP）、甲酚二苯磷酸酯 （CDP）、磷酸三甲苯酯 （TMPP） 和 2-乙基己基二苯基磷酸酯 （EDHPHP）。使用 TPHP 作为阳性对照和心包水肿作为读数，我们根据受精后 24 至 72 小时 （hpf） 的静态暴露开发了所有四种 APE 的浓度-反应曲线。然后，我们与 D-甘露醇（一种渗透性利尿剂）进行了共暴露，并在反渗透 （RO） 水中进行了暴露，以确定暴露介质的离子组成是否减轻了 72 hpf 时 APE 诱导的心包水肿。 以心包水肿为终点，TPHP（阳性对照）、IPTPP、CDP、TMPP 和 EDHPHP 的近似 EC50 分别为 6.25、3.125、3.125、25 和 100 μM，基于 24 至 72 hpf 的暴露。有趣的是，与我们对 TPHP 的研究结果类似，与 D-甘露醇的共同暴露和暴露在离子缺乏的水中显着减轻了斑马鱼胚胎中 IPTPP-CDP-、TMPP-和 EDHPHP 诱导的心包水肿，这表明化学诱导的心包水肿可能是 1） 取决于暴露介质的离子组成和 2） 由胚胎表皮的渗透调节破坏驱动。因此，与其他检测参数类似，我们的研究结果强调了标准化暴露介质渗透压的必要性，以最大限度地减少在世界各地进行的基于斑马鱼胚胎的化学毒性筛选中出现假阳性/阴性命中的可能性。