CNC-Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Biocant Park, 3060-197 Cantanhede, Portugal.
IIIUC-Institute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugal.
Toxicol Sci. 2018 Jul 1;164(1):191-204. doi: 10.1093/toxsci/kfy068.
Human skin fibroblasts present technical advantages for the study of mitochondrial-induced toxicity, because those cells can be isolated from patients by lowly invasive methods and present specific cumulative cellular damage and mutations of particular conditions. Several drugs lead to organ toxicity, with some of these drugs having been already withdrawn from the market. Frequently, drug-induced toxicity is attributed to mitochondrial liabilities. One of the approaches to identify drug-induced mitochondrial toxicity is using glucose-free/galactose/glutamine/pyruvate-containing cell culture media that force cells to be more dependent on oxidative phosphorylation for energy production. However, the effects of this modified culture medium itself on the mitochondrial phenotype of human skin fibroblasts have not been explored in detail. Our objective was to assess the mitochondrial biology of human skin fibroblasts under standard or modified culture conditions so that system can be validated and used in a more reliable way to disclose mitochondrial liabilities of drug candidates or intrinsic metabolic differences in fibroblasts. Our results showed that forcing mitochondrial remodeling in human skin fibroblasts increased oxygen consumption rate, ATP levels, and mitochondria-related transcripts and proteins. Moreover, the metabolic remodeling increased cytotoxicity of mitochondrial poisons. In general, no alterations in gene expression related with differentiation status were observed in human skin fibroblasts, with exception of increased paxilin gene expression. Not only the current work highlights the importance of using human skin primary cells to study drug-induced mitochondrial toxicity, it also reinforces the use of this tool to detect specific mitochondrial defects in skin fibroblasts from patients.
人皮肤成纤维细胞在研究线粒体诱导毒性方面具有技术优势，因为这些细胞可以通过微创方法从患者中分离出来，并且具有特定的累积细胞损伤和特定条件下的突变。一些药物会导致器官毒性，其中一些药物已经从市场上撤出。通常，药物诱导的毒性归因于线粒体的缺陷。一种识别药物诱导的线粒体毒性的方法是使用不含葡萄糖/半乳糖/谷氨酰胺/丙酮酸的细胞培养基，这种培养基迫使细胞更加依赖氧化磷酸化来产生能量。然而，这种改良的培养基本身对人皮肤成纤维细胞线粒体表型的影响尚未详细研究。我们的目的是评估人皮肤成纤维细胞在标准或改良的培养条件下的线粒体生物学，以便该系统可以得到验证，并以更可靠的方式用于揭示候选药物的线粒体缺陷或成纤维细胞的固有代谢差异。我们的结果表明，在人皮肤成纤维细胞中强制进行线粒体重塑会增加耗氧率、ATP 水平以及与线粒体相关的转录本和蛋白质。此外，代谢重塑增加了线粒体毒物的细胞毒性。总的来说，在人皮肤成纤维细胞中没有观察到与分化状态相关的基因表达改变，除了 paxilin 基因表达增加。本研究不仅强调了使用人皮肤原代细胞研究药物诱导的线粒体毒性的重要性，还强调了使用该工具检测来自患者的皮肤成纤维细胞中特定的线粒体缺陷。
