Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

中文翻译：

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Oatp1d1 膜转运蛋白的功能性敲除影响双氯芬酸在斑马鱼胚胎中的毒性


有机阴离子转运多肽 （OATP） 促进细胞摄取大量化合物。斑马鱼 Oatp1d1 与人 OATP 直系同源物的功能能力相匹配，特别是在激素和药物转运方面。它在肝脏和胚胎发育的后期高度表达，表明它在斑马鱼生理学和发育中起着关键作用。先前体外分析的数据显示，斑马鱼 Oatp1d1 对药物和外源性物质具有高亲和力，为进一步体内研究其防御和发育功能提供了基础。使用 CRISPR-Cas9 技术，我们生成了 Oatp1d1 斑马鱼突变体，与野生型 （WT） 相比，该突变体在胚胎和成人组织中的 Oatp1d1 表达显著降低。使用定制抗体确认 Oatp1d1 的存在。为了评估其生态毒理学相关性，突变体和 WT 胚胎暴露于浓度不断增加的双氯芬酸，一种以其广泛和频繁的使用、环境伪持久性和生态影响而闻名的非甾体抗炎药。WT 胚胎在较高的双氯芬酸浓度下表现出发育迟缓和畸形，例如脊柱弯曲、心脏水肿和血液淤积，而 Oatp1d1 突变胚胎表现出显着的弹性，具有较轻的发育缺陷和延迟的毒性作用。这些观察结果表明，Oatp1d1 的缺失阻碍了双氯芬酸有效进入肝细胞，从而减缓了其生物转化为可能毒性更强的代谢物。此外，其他摄取转运蛋白的转录表达变化揭示了一个高度可能且复杂的补偿机制网络。 因此，本研究的结果指出了 Oatp1d1 介导的双氯芬酸转运的重要性，正如首次使用 Oatp1 缺陷斑马鱼系在体内证明的那样。最后，我们的数据表明，需要考虑具有重叠底物偏好的其他转运蛋白的补偿作用，以便可靠地理解膜转运蛋白的生理和/或防御作用。