Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, Illinois, USA.
Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, Illinois, USA.
J Biol Chem. 2021 Jan-Jun;296:100206. doi: 10.1074/jbc.RA120.014885. Epub 2020 Dec 31.
Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus infection, and their use can cause mitochondrial toxicity, including mitochondrial DNA (mtDNA) depletion in several cases. The first-generation NRTIs, including 2',3'-dideoxycytidine (ddC), were originally and are still pursued as anticancer agents. NRTI-sensitive DNA polymerases localizing to mitochondria allow for the opportunity to poison proliferating cancer cell mtDNA replication as certain cancers rely heavily on mitochondrial functions. However, mtDNA replication is independent of the cell cycle creating a significant concern that toxicants such as ddC impair mtDNA maintenance in both proliferating and nonproliferating cells. To examine this possibility, we tested the utility of the HepaRG cell line to study ddC-induced toxicity in isogenic proliferating (undifferentiated) and nonproliferating (differentiated) cells. Following ddC exposures, we measured cell viability, mtDNA copy number, and mitochondrial bioenergetics utilizing trypan blue, Southern blotting, and extracellular flux analysis, respectively. After 13 days of 1 μM ddC exposure, proliferating and differentiated HepaRG harbored mtDNA levels of 0.9% and 17.9% compared with control cells, respectively. Cells exposed to 12 μM ddC contained even less mtDNA. By day 13, differentiated cell viability was maintained but declined for proliferating cells. Proliferating HepaRG bioenergetic parameters were severely impaired by day 8, with 1 and 12 μM ddC, whereas differentiated cells displayed defects of spare and maximal respiratory capacities (day 8) and proton-leak linked respiration (day 14) with 12 μM ddC. These results indicate HepaRG is a useful model to study proliferating and differentiated cell mitochondrial toxicant exposures.
核苷逆转录酶抑制剂（NRTIs）是最初用于治疗人类免疫缺陷病毒感染的药物，它们的使用会导致线粒体毒性，包括在几种情况下线粒体 DNA（mtDNA）耗竭。第一代 NRTIs，包括 2',3'-双脱氧胞苷（ddC），最初是并且仍然被作为抗癌药物来研究。定位于线粒体的 NRTI 敏感 DNA 聚合酶使增殖的癌细胞 mtDNA 复制中毒成为可能，因为某些癌症严重依赖线粒体功能。然而，mtDNA 复制不依赖于细胞周期，这使得像 ddC 这样的毒物在增殖和非增殖细胞中损害 mtDNA 维持的可能性变得非常重要。为了检验这种可能性，我们测试了 HepaRG 细胞系在研究同基因增殖（未分化）和非增殖（分化）细胞中 ddC 诱导的毒性的效用。在 ddC 暴露后，我们分别使用台盼蓝、Southern 印迹和细胞外通量分析来测量细胞活力、mtDNA 拷贝数和线粒体生物能。在 1 μM ddC 暴露 13 天后，增殖和分化的 HepaRG 细胞的 mtDNA 水平分别为对照细胞的 0.9%和 17.9%。暴露于 12 μM ddC 的细胞甚至含有更少的 mtDNA。到第 13 天，分化细胞的活力得到维持，但增殖细胞的活力下降。增殖 HepaRG 细胞的生物能参数在 1 和 12 μM ddC 存在下，在第 8 天受到严重损害，而分化细胞在第 8 天显示备用和最大呼吸能力以及质子漏相关呼吸的缺陷（第 14 天）与 12 μM ddC。这些结果表明 HepaRG 是研究增殖和分化细胞线粒体毒物暴露的有用模型。
