Expression changes in DNA repair enzymes and mitochondrial DNA damage in aging rat lens.
OBJECTIVE
To determine if there is increased mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage with age in the lenses of rats. We also explored the immunolocalization of 8-oxoguanine DNA glycosylase 1 (OGG1) and AP endonuclease 1 (APE1) in the lens and studied three of the predominant base excision repair (BER) enzymes: OGG1, APE1, and DNA polymerase gamma (Polgamma).


METHODS
The methods used by this study include the selection of twenty-six male Wistar rats in each group (2 months old and 26 months old) and fourteen male Wistar rats in the 16 months old group. The total DNA of lenses were isolated and the DNA genome was amplified by a long extension-polymerase chain reaction (LX-PCR). We examined mtDNA and nDNA damage with a quantitative polymerase chain reaction (QPCR) assay that was combined with EvaGreen. We also studied the gene expression of mRNA and protein in these key BER enzymes with real time-polymerase chain reaction (RT-PCR) and western blot analysis.


RESULTS
There was an increase in oxidative DNA damage, which exists primarily in the mtDNA. The amount of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) in DNA was significantly increased with age. Our experiments demonstrated that the gene expression of mRNA and protein in these key BER enzymes decreased with age. OGG1 and APE1 were localized by immunohistochemistry within lens epithelial cells (LECs) and superficial fiber cells.


CONCLUSIONS
The gene expression of mRNA and protein in these key BER enzymes decreased with age, which caused a decrease in the repairing capability of the mtDNA and the accumulation of mtDNA damage. The increased mtDNA damage and decreased expression of BER enzymes may cause a "vicious cycle" of oxidative stress that contributes to the accumulation of mtDNA mutations and age-related cataract pathogenesis.
Zhang
Yi
Y
Eye hospital, The First Affiliated Hospital, Harbin Medical University, Harbin, China.
Zhang
Lu
L
Zhang
Lan
L
Bai
Jie
J
Ge
Hongyan
H
Liu
Ping
P
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000375:Aging / Q000235:genetics / Q000473:pathology*; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic; D007150:Immunohistochemistry; D007908:Lens, Crystalline / Q000201:enzymology* / Q000473:pathology*; D008297:Male; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion
D004272:DNA, Mitochondrial; D012333:RNA, Messenger; D000080242:8-Hydroxy-2'-Deoxyguanosine; D045643:DNA Repair Enzymes; D003849:Deoxyguanosine
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