A catalytic antioxidant metalloporphyrin blocks hydrogen peroxide-induced mitochondrial DNA damage

Abstract

Reactive oxygen species (ROS) have been implicated as the cause of cumulative damage to DNA, proteins and lipids that can ultimately result in cell death. A common problem when measuring oxidative DNA damage has been the introduction of modifications in the native state of the molecule by many DNA isolation methods. We circumvented this problem by employing direct PCR (DPCR) of whole cell lysates. DPCR of mouse lung fibroblasts performed better than PCRs containing template acquired by phenol/chloroform extraction or a commercially available genomic DNA isolation kit. We investigated the direct use of whole cell preparations in the polymerase chain reaction (PCR) to detect hydrogen peroxide (H(2)O(2))-mediated DNA damage. We observed a concentration-dependent decrease in amplification efficiency of a 4.3 kb mitochondrial (mt)DNA target in H(2)O(2)-treated mouse lung fibroblasts (MLFs). At low doses the efficiency of amplification returns to control levels over 24 h. We detected no change in amplification efficiency of a plasmid control containing our mtDNA target under any of the culture conditions employed in these studies. Treatment of MLFs with the catalytic antioxidant manganese(III) meso -tetrakis(4-benzoic acid)porphyrin (MnTBAP) attenuates the effects of H(2)O(2)exposure. When quantitated with an external standard the use of DPCR in tandem with a PCR amplification efficiency assay provides a powerful approach to assess oxidative mtDNA damage.

Figure 1

The 4.3 kb PCR product from the mouse mitochondrial genome. The mouse mitochondrial genome is illustrated with the tRNA coding sequences indicated according to the amino acid single letter designation. Open reading frames are shown as shaded boxes with arrows in the direction of transcription; rRNAs appear as cross-hatched boxes. The 4.3 kb amplification product is shown as a hatched box.

Figure 2

A comparison of direct PCR with two DNA purification techniques. Direct PCR amplification of whole MLFs was compared with two methods of DNA purification. Lane 1 shows a 4.3 kb mtDNA amplification product obtained by DPCR of 2 × 10³ MLFs. Template DNA in lanes 2 and 3 is from phenol/chloroform extraction and Qiagen purification of 2 × 10³ whole MLFs. The template in lane 4 is 100 ng of phenol/chloroform-extracted whole mouse lung DNA. A 5 µl aliquot of a 50 µl PCR was analyzed.

Figure 3

Example of external standard PCR and the PCR standard curve. External standard reactions were generated with each PCR assay. The amplification products were quantitated using the Pico Green dsDNA assay and generated a standard curve that was used to normalize each assay run. A linear relationship was found for amplification up to 7.5 × 10⁵ template molecules.

Figure 4

Effects of increasing concentrations of H₂O₂ on the efficiency of PCR amplification. DNA damage was assessed after a 1 h incubation in serum-free DMEM containing 0, 200, 400 and 800 µM H₂O₂ immediately (0 h) after treatment and 24 h later by amplification of a 4277 bp mtDNA. (A) 1% agarose gel of 10 µl of each 50 µl PCR reaction. Amplification efficiency of (B) the mtDNA target and (C) the pGEMT4.3 target were analyzed with a fluorescence plate reader using the Pico Green dsDNA assay. The amplification efficiencies are shown as a percentage of the control, which had no hydrogen peroxide treatment. H₂O₂ treatment produced a concentration-dependent decrease in mtDNA amplification.

Figure 5

Protection against H₂O₂-induced DNA damage by MnTBAP. Amplification efficiencies of mtDNA and nDNA targets were measured at 0 and 24 h in cells treated with increasing concentrations of MnTBAP and exposed to H₂O₂. MLF were exposed to 0, 25, 50 and 100 µM MnTBAP for 1 h at room temperature, washed, then treated with (+) or without (–) 400 µM H₂O₂ for 1 h in serum-free DMEM. DPCR was performed immediately (0 h) after H₂O₂ treatment and again 24 h later. Amplification was measured for (A) a 4277 bp mtDNA target and (B) a 4440 bp pGEMT4.3 target. Data is presented as a percentage of the untreated (–) H₂O₂, 0 µM MnTBAP control.