PMID- 19834037
OWN - NLM
STAT- MEDLINE
DCOM- 20100217
LR  - 20121115
IS  - 1552-5783 (Electronic)
IS  - 0146-0404 (Linking)
VI  - 51
IP  - 2
DP  - 2010 Feb
TI  - Mitochondrial DNA damage induced by 7-ketocholesterol in human retinal pigment 
      epithelial cells in vitro.
PG  - 1164-70
LID - 10.1167/iovs.09-3443 [doi]
AB  - PURPOSE: To assess oxysterol-induced mitochondrial DNA (mtDNA) damage and 
      mitochondrial dysfunction in cultured human retinal pigment epithelial cells 
      (ARPE- 19). METHODS: ARPE-19 cultures were exposed for 6 and 24 hours to 40 
      microg/mL 7-ketocholesterol (7kCh), and total DNA was extracted. Long-extension 
      polymerase chain reaction was performed to amplify the full-length mtDNA genome. 
      The products were separated by electrophoresis on a 0.8% agarose gel stained with 
      ethidium bromide. Superoxide and reactive oxygen/nitrogen species (ROS/RNS; 
      hydrogen peroxide, peroxynitrite anions, and peroxyl radicals) were measured with 
      an amine-reactive green-dye assay and 2',7'-dicholorodihydrofluorescein diacetate 
      (H(2)DCFDA) dye assay, respectively. The changes in mitochondrial membrane 
      potential (DeltaPsim) were measured with a cationic (green) dye assay. Western 
      blot analysis was used to assess porins, a structural protein of the 
      mitochondrial membranes. RESULTS: The 7kCh-treated cultures showed significant 
      increase in ROS/RNS production (P < 0.001) compared with untreated controls, but 
      the superoxide levels were unchanged. The 7kCh-treated ARPE-19 cultures had 
      diminished levels of the full-length 16.2-kb mtDNA band, a 2.2-fold decrease of 
      the DeltaPsim compared with control cultures (P < 0.001), and decreased levels of 
      porins. CONCLUSIONS: 7kCh causes significant damage to the full-length intact 
      mtDNA and mitochondrial dysfunction in ARPE-19 cells. These observations suggest 
      that the mitochondria and its DNA may be targets for oxysterol-induced oxidative 
      stress and may play a role in the pathogenesis of retinal diseases.
FAU - Gramajo, Ana L
AU  - Gramajo AL
AD  - Department of Ophthalmology, University of California, Irvine, California, USA.
FAU - Zacharias, Leandro C
AU  - Zacharias LC
FAU - Neekhra, Aneesh
AU  - Neekhra A
FAU - Luthra, Saurabh
AU  - Luthra S
FAU - Atilano, Shari R
AU  - Atilano SR
FAU - Chwa, Marilyn
AU  - Chwa M
FAU - Brown, Donald J
AU  - Brown DJ
FAU - Kuppermann, Baruch D
AU  - Kuppermann BD
FAU - Kenney, M Cristina
AU  - Kenney MC
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20091015
PL  - United States
TA  - Invest Ophthalmol Vis Sci
JT  - Investigative ophthalmology & visual science
JID - 7703701
RN  - 0 (DNA, Mitochondrial)
RN  - 0 (Ketocholesterols)
RN  - 0 (Reactive Oxygen Species)
RN  - 11062-77-4 (Superoxides)
RN  - 9007-49-2 (DNA)
RN  - O7676FE78M (7-ketocholesterol)
SB  - IM
MH  - Blotting, Western
MH  - Cells, Cultured
MH  - DNA/isolation & purification
MH  - *DNA Damage
MH  - DNA, Mitochondrial/*metabolism
MH  - Electrophoresis, Agar Gel
MH  - Humans
MH  - Ketocholesterols/*pharmacology
MH  - Membrane Potential, Mitochondrial/drug effects
MH  - Polymerase Chain Reaction
MH  - Reactive Oxygen Species/metabolism
MH  - Retinal Pigment Epithelium/*drug effects/metabolism
MH  - Superoxides/metabolism
EDAT- 2009/10/17 06:00
MHDA- 2010/02/18 06:00
CRDT- 2009/10/17 06:00
PHST- 2009/10/17 06:00 [entrez]
PHST- 2009/10/17 06:00 [pubmed]
PHST- 2010/02/18 06:00 [medline]
AID - iovs.09-3443 [pii]
AID - 10.1167/iovs.09-3443 [doi]
PST - ppublish
SO  - Invest Ophthalmol Vis Sci. 2010 Feb;51(2):1164-70. doi: 10.1167/iovs.09-3443. 
      Epub 2009 Oct 15.