Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland.
School of Architecture, Civil and Environmental Engineering, EPFL Lausanne, 1015 Lausanne, Switzerland.
Chem Res Toxicol. 2020 Nov 16;33(11):2863-2871. doi: 10.1021/acs.chemrestox.0c00315. Epub 2020 Oct 14.
In view of the steadily increasing number of chemical compounds used in various products and applications, high-throughput toxicity screening techniques can help meeting the needs of 21st century risk assessment. Zebrafish (), especially its early life stages, are increasingly used in such screening efforts. In contrast, cell lines derived from this model organism have received less attention so far. A conceivable reason is the limited knowledge about their overall capacity to biotransform chemicals and the spectrum of expressed biotransformation pathways. One important biotransformation route is the mercapturic acid pathway, which protects organisms from harmful electrophilic compounds. The fully functional pathway involves a succession of several enzymatic reactions. To investigate the mercapturic acid pathway performance in the zebrafish embryonic cell line, PAC2, we analyzed the biotransformation products of the reactions comprising this pathway in the cells exposed to a nontoxic concentration of the reference substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, we used targeted proteomics to measure the expression of cytosolic glutathione S-transferases (GSTs), the enzyme family catalyzing the first reaction in this pathway. Our results reveal that the PAC2 cell line expresses a fully functional mercapturic acid pathway. All but one of the intermediate CDNB biotransformation products were identified. The presence of the active mercapturic acid pathway in this cell line was further supported by the expression of a large palette of GST enzyme classes. Although the enzymes of the class alpha, one of the dominant GST classes in the zebrafish embryo, were not detected, this did not seem to affect the capacity of the PAC2 cells to biotransform CDNB. Our data provide an important contribution toward using zebrafish cell lines, specifically PAC2, for animal-free high- throughput screening in toxicology and chemical hazard assessment.
鉴于各种产品和应用中使用的化学化合物数量不断增加，高通量毒性筛选技术可以帮助满足 21 世纪风险评估的需求。斑马鱼（），尤其是其早期生命阶段，越来越多地用于此类筛选工作。相比之下，到目前为止，源自该模式生物的细胞系受到的关注较少。一个可以想象的原因是，人们对其整体化学转化能力和表达的生物转化途径的范围了解有限。一个重要的生物转化途径是硫醚氨酸途径，它可以保护生物体免受有害的亲电化合物的侵害。该功能齐全的途径涉及一系列酶促反应。为了研究斑马鱼胚胎细胞系 PAC2 中的硫醚氨酸途径的性能，我们分析了暴露于无毒浓度参考底物 1-氯-2,4-二硝基苯（CDNB）的细胞中包含该途径的反应的生物转化产物。此外，我们使用靶向蛋白质组学来测量催化该途径中第一个反应的胞质谷胱甘肽 S-转移酶（GSTs）的表达。我们的研究结果表明，PAC2 细胞系表达了一个功能齐全的硫醚氨酸途径。除了一个之外，所有的中间 CDNB 生物转化产物都被鉴定出来。该细胞系中存在活性硫醚氨酸途径，这进一步得到了大量 GST 酶类表达的支持。尽管未检测到在斑马鱼胚胎中占主导地位的 GST 酶类之一的 alpha 类酶，但这似乎并没有影响 PAC2 细胞对 CDNB 的生物转化能力。我们的数据为使用斑马鱼细胞系，特别是 PAC2，进行无动物高通量筛选在毒理学和化学危害评估方面提供了重要贡献。
