The water framework directive re-evaluation proposes the integration of effect-based tools, increasing the need for alternative methods. Especially within aquatic toxicology, coverage of specific toxicity pathways is scarce, and most applications are based on mammalian or bacterial models, not reflecting realistic exposure scenarios. The use of transient reporter gene assays in cells from organisms of interest could be a quick and inexpensive solution. However, interference with cellular homeostasis may impact the system beyond the function of the manipulated gene and thus lead to non-specific results. We describe how varying vector geometry and different regulatory gene elements on plasmids used for transfection in zebrafish hepatocytes and embryonic fibroblasts may lead up to a tenfold difference in potency. Cells were transiently co-transfected with an Nrf2-responsive Firefly luciferase reporter plasmid and eight different Renilla luciferase normalization plasmids. Transfected cells were exposed to two different regimes (0.1-100 µM and 7.8-250 µM) of the oxidative stress-inducing compounds, sulforaphane, tertbutylhydroquinone, and metazachlor. Nrf2 activity was measured in dual-luciferase assays. In parallel, cytotoxicity was assessed for different endpoints (energy metabolism, protein amount, membrane stability, and cell proliferation) in non-transfected cells and cells co-transfected with constructs of increasing size, to be used for normalization. Transfected cells were more susceptible to cytotoxicity in a vector size-dependent manner. Conclusively, we report that vector geometries (size, backbones, gene-regulatory units), cell line (tissue origin), applied transfection methods, and signal normalization may alter the sensitivity of reporter bioassays in a synergistic manner. Further, we propose that thorough bioassay design is needed to ensure reliability and regulatory acceptance.

中文翻译：

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暂时性体外报告基因生物测定的潜力和陷阱：重组斑马鱼细胞系受到载体几何形状和细胞毒性的干扰。

水框架指令的重新评估提出了基于效果的工具的集成，从而增加了对替代方法的需求。特别是在水生毒理学中，很少有特定的毒性途径的报道，并且大多数应用是基于哺乳动物或细菌模型的，不能反映实际的暴露情况。在感兴趣的生物体的细胞中使用瞬时报告基因测定可能是一种快速而廉价的解决方案。但是，对细胞动态平衡的干扰可能会影响系统，超出操纵基因的功能，从而导致非特异性结果。我们描述了如何改变载体几何形状和用于斑马鱼肝细胞和胚胎成纤维细胞中转染的质粒上的不同调控基因元件如何导致功效提高十倍。用Nrf2反应性萤火虫荧光素酶报道质粒和八个不同的海肾荧光素酶标准化质粒瞬时共转染细胞。将转染的细胞暴露于两种不同的氧化应激诱导化合物方案（0.1-100 µM和7.8-250 µM），萝卜硫烷，叔丁基对苯二酚和间草胺。Nrf2活性在双荧光素酶测定中进行了测量。同时，对未转染的细胞和用大小逐渐增加的构建体共转染的细胞中不同终点（能量代谢，蛋白质量，膜稳定性和细胞增殖）的细胞毒性进行了评估，以用于标准化。转染的细胞以载体大小依赖性方式更容易受到细胞毒性作用。最后，我们报告了载体的几何形状（大小，骨架，基因调控单位），细胞系（组织来源），应用的转染方法和信号归一化可能以协同方式改变报告基因生物测定的灵敏度。此外，我们建议需要进行彻底的生物测定设计，以确保可靠性和法规接受性。