Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage
Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage
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DOI：
10.1016/j.mito.2006.11.026
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发表时间：
2007-02-01
期刊：
影响因子：
4.4
通讯作者：
Majima, Hideyuki J.
中科院分区：
文献类型：
--
作者：
Indo, Hiroko P.;Davidson, Mercy;Majima, Hideyuki J.
线粒体损伤是线粒体相关疾病的一个众所周知的原因。线粒体相关疾病发展的主要机制被认为是线粒体电子传递链（ETC）损伤引起的细胞内氧化应激增加。然而，对于线粒体DNA （mtDNA）受损的细胞，细胞内自由基产生的明确证据尚未明确提供。本研究利用新型荧光染料2-[6-（4′-羟基）phenoxy- 3h - xanthen3 - on9 -yl]苯甲酸（HPF）检测羟基自由基（(OH)- o -），在143B细胞（亲本细胞）、143B-rho(0)细胞（mtDNA缺失细胞）、87 wt （cybrid）和4977-bp mtDNA缺失（常见缺失）细胞中检测细胞内自由基的形成，其中缺失频率分别为0%、5%、50%和bbb99 % （HeLacot、BH5、BH50和BH3.12）。采用激光共聚焦显微镜检测方法。ETC抑制剂（鱼藤酮、3-硝基丙酸、烯酰三氟丙酮、抗霉素A和氰化钠）也被检测，以确定抑制剂处理是否增加细胞内活性氧（ROS）的生成。与143B细胞相比，143B-rho(0)细胞的ROS明显增加。然而，对于87重量的杂交种，没有观察到增加。在mtdna缺失的细胞BH50和BH3.12中也观察到增加。ETC抑制剂增加了143B和14313-rho(0)细胞内的ROS。此外，在每张荧光图像中，荧光染料都出现在细胞核周围。为了明确定位，我们用染料和MitoTracker Red对细胞进行了双重染色。所产生的荧光始终位于线粒体中。此外，锰超氧化物歧化酶（MnSOD） cdna转染的细胞ROS减少。这些结果表明，在ETC被抑制和mtdna受损的细胞中，线粒体产生了更多的ROS，从而损害了ETC。(c) 2006 Elsevier B.V.和线粒体研究协会。版权所有。
Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals ((OH)-O-.), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 14313-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC. (c) 2006 Elsevier B.V. and Mitochondria Research Society. All rights reserved.