Expression changes in DNA repair enzymes and mitochondrial DNA damage in aging rat lens

Abstract

Purpose: 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.

Figure 1

Increased mtDNA damage compared to nDNA in aged rat lens. Long fragments of mtDNA (13.4 kb) and nDNA (12.5 kb) from lens were measured. These data were normalized by the measured levels of the short fragment of mtDNA and nDNA obtained using the same DNA sample. A: there was a significant decrease in mtDNA amplification at 16 months (p=0.03, n=8) and at 26 months (p<0.01, n=8), compared with the 2 month group. There was a significantly increase at 26 months compared to 16 months group (p<0.01, n=8). B: there was no significant change in the relative amplification of nDNA at 16 months (p=0.149, n=8) and 26 months (p=0.06, n=8), compared with 2 month group. There was not a significantly change at 26 months (p=0.606, n=8) compared to 16 months group.

Figure 2

Increased levels of deleted mtDNA in aged rat lens. Measurements of levels of the PCR products of non-deleted (A) and deleted (B) mtDNA normalized by total mtDNA were done using the EvaGreen reagent. A: there were no significant change in non-deleted mtDNA at 16 months (p=0.51, n=8) and at 26 months (p=0.93, n=8), compared with the 2 month group. There was not a significantly change at 26 months compared to 16 months group (p=0.56, n=8). However, there was a significant increase in deleted mtDNA at 16 months (p=0.002, n=8) and at 26 months (p<0.001, n=8), compared with the 2 month group in B. There was a significantly increase at 26 months (p<0.01, n=8) compared to 16 months group.

Figure 3

Increased 8-OHdG in aged rat lens. The amount of 8-OHdG in DNA was quantitatively measured by 8-OHdG ELISA. The level of 8-OHdG in DNA was significantly increased at 16 months (p<0.001, n=8) and at 26 months (p<0.001, n=8), compared with 2 month group. There was a significantly increase at 26 months (p<0.01, n=8) compared to 16 months group.

Figure 4

mRNA levels of BER enzymes are decreased in aged rat lens. Comparisons of the mRNA levels of BER enzymes: OGG1 (A), APE1 (B), and Polγ (C). GAPDH was used as internal standard for normalization. A: the expression level of OGG1 was decreased at 16 months (0.92±0.03, p<0.001, n=6) and 26 months (0.78±0.02, p<0.001, n=6), compared to the 2 month old group, and there was significantly decreased at 26 months (0.78±0.02, p<0.001, n=6) compared to 16 months group (0.92±0.03, n=6). B: it is shown that the expression level of APE1 was decreased at 26 months (0.78±0.03,p<0.001, n=6) as compared to the 16 month (0.95±0.06, n=6) and 2 month old group, and there was no significant change between 16 month (0.95±0.06, p=0.075,n=6) and 2 month old group. C: the expression level of Polγ was decreased at 26 months (0.90±0.05, p<0.001, n=6) compared to the 2 month old group, and there was a significantly decrease at 26 months (0.90±0.05, p=0.03, n=6) compared to the 16 months group (0.95±0.04, n=6).

Figure 5

Protein levels of BER enzymes are decreased in aged rat lens. There were significant decreased in OGG1, APE1, and Polγ protein levels (0.46±0.01, p=0.003, n=3; 0.68±0.02, p=0.009, n=3;0.42±0.05, p=0.006, n=3,respectively) at 26 months when comparing to the 2 month old group (0.73±0.07, n=3; 0.99±0.004, n=3 and 1.00±0.18, n=3, respectively).

Figure 6

Immunohistochemical localization of OGG1 and APE1. Sectioned equals 7 μm thickness. OGG1 and APE1 were localized by immunohistochemistry within LECs and superficial fiber cells. A: Immunohistochemical localization of OGG1 in 2 months old rat lens. B: Immunohistochemical localization of OGG1 in 26 months old rat lens. C: Immunohistochemical localization of APE1 in 2 months old lens. D: Immunohistochemical localization of APE1 in 26 months old lens.