title: Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage.
abstract: 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), 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 143B-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.
authors.affiliation: Department of Oncology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
authors.forename: Hiroko P
authors.initials: HP
authors.lastname: Indo
authors.forename: Mercy
authors.initials: M
authors.lastname: Davidson
authors.forename: Hsiu-Chuan
authors.initials: HC
authors.lastname: Yen
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Suenaga
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tomita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nishii
authors.forename: Masahiro
authors.initials: M
authors.lastname: Higuchi
authors.forename: Yasutoshi
authors.initials: Y
authors.lastname: Koga
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ozawa
authors.forename: Hideyuki J
authors.initials: HJ
authors.lastname: Majima
mesh_terms: D000968:Antimycin A / Q000494:pharmacology; D045744:Cell Line, Tumor; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006822:Hybrid Cells; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009574:Nitro Compounds / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D012966:Sodium Cyanide / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000235:genetics; D013804:Thenoyltrifluoroacetone / Q000494:pharmacology; D014162:Transfection
chemical_list: C520960:2-(6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl)benzoic acid; D004272:DNA, Mitochondrial; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009574:Nitro Compounds; D011422:Propionates; D017382:Reactive Oxygen Species; D012402:Rotenone; D013804:Thenoyltrifluoroacetone; D000968:Antimycin A; D013482:Superoxide Dismutase; D012966:Sodium Cyanide; C015392:3-nitropropionic acid