Bisphenol A (BPA) is a widely used industrial compound found in polycarbonate plastics, epoxy resin, and various polymer materials, leading to its ubiquitous presence in the environment. The toxicity of BPA to aquatic organisms has been well documented following in vivo exposure scenarios, with known cytotoxic and endocrine-disrupting effects. As such, BPA was used in this study as a well-characterized chemical to implement more ethical and resource-efficient scientific practices in toxicity testing through new approach methods (NAMs). Due to the frequent use of Daphnia spp. as a model organism in toxicology, we developed an in vitro cell culture system from Daphnia magna embryos, with optimized medium to support cell longevity. The cultures were maintained for up to two months, demonstrating their stability and suitability for cytotoxicity studies. Using this novel system, lethal concentration 50 (LC50) values were determined at the 24 and 48 h time points following BPA exposure. Subsequently, oxidative stress, endocrine disruption, and DNA damage were assessed through gene expression, activity assays, and a comet assay in BPA-exposed cells. LC50 values of 52 µM and 20 µM BPA were calculated after 24 and 48 h exposures, respectively. BPA cells exposed to 20 and 52 µM had significantly increased GSH, GPx, and GST activity levels. mRNA expression analysis revealed significant upregulations in the expression of hsp70, hsp90, gst, gpx, vtg1, and cyp4, with downregulations of sod, cat, and ecr following BPA exposure. Furthermore, comet assays showed a significantly higher level of DNA damage induced by BPA compared to controls, with greater comet and tail lengths. This study established a novel in vitro Daphnia model, using BPA as a case study for determining toxic effects, further highlighting the importance and applicability of utilizing alternative methods in ecotoxicological research through reducing animal use.

中文翻译：

---

建立大型溞细胞培养物作为（生态）毒理学测定的体外模型：使用双酚 A 作为代表性细胞毒性和内分泌干扰化学物质的案例研究


双酚 A （BPA） 是一种广泛使用的工业化合物，存在于聚碳酸酯塑料、环氧树脂和各种聚合物材料中，因此在环境中无处不在。在体内暴露情景之后，BPA 对水生生物的毒性已得到充分证明，具有已知的细胞毒性和内分泌干扰作用。因此，BPA 在本研究中被用作一种特征明确的化学品，通过新方法方法 （NAM） 在毒性测试中实施更合乎道德和资源效率的科学实践。由于在毒理学中经常使用水蚤属作为模式生物，我们从大型水蚤胚胎中开发了一种体外细胞培养系统，并使用优化的培养基来支持细胞寿命。培养物维持长达两个月，证明其稳定性和适用于细胞毒性研究。使用这种新型系统，在 BPA 暴露后 24 小时和 48 小时时间点测定致死浓度 50 （LC50） 值。随后，通过在 BPA 暴露的细胞中通过基因表达、活性测定和彗星测定来评估氧化应激、内分泌破坏和 DNA 损伤。分别在暴露 24 和 48 小时后计算 52 μM 和 20 μM BPA 的 LC50 值。暴露于 20 μM 和 52 μM 的 BPA 细胞的 GSH 、 GPx 和 GST 活性水平显著增加。mRNA 表达分析显示 hsp70 、 hsp90 、 gst 、 gpx 、 vtg1 和 cyp4 的表达显著上调，BPA 暴露后 sOD 、 CAT 和 ECR 的表达下调。此外，彗星测定显示，与对照组相比，BPA 诱导的 DNA 损伤水平显著更高，彗星和尾部长度更大。 本研究建立了一种新的体外水蚤模型，以 BPA 作为确定毒性作用的案例研究，进一步强调了通过减少动物使用在生态毒理学研究中利用替代方法的重要性和适用性。