Abstract
A consequence of mitochondrial (mt) electron transport and energy production is continual formation of reactive oxygen species (ROS). MtROS production is usually compensated by high activities of antioxidants, e.g., superoxide dismutases 1 and 2. However, under various physiological or pathological states, mtROS production exceeds antioxidant defenses and oxidative (Ox) modification of mt proteins and mtDNA occurs. Current techniques to detect mtOx stress include estimations of ROS, Southern blotting to measure mtDNA fragmentation, or immunostaining and HPLC to measure Ox stress biomarkers such as 8‐hydroxy‐2′‐deoxyguanosine (8OHdG). These techniques are laborious and require specialized procedures (use of radioisotopes) or equipment (HPLC). Our goal was to devise and evaluate a new, simple method of assessing mtOx stress. Total cellular or mtDNA was isolated from liver and heart from Zucker lean and obese rats. Equal amounts of DNA were fixed to a nitrocellulose membrane and an immunoblotting protocol was performed with an antibody against 8OHdG. Levels of 8OHdG in heart mtDNA were less in Zucker lean than those from obese rats, consistent with other measurements from our laboratories showing greater mtROS production in obese compared to lean rats. The proposed method is useful to measure ROS‐induced DNA damage because it is convenient, semi‐quantitative, and accurate even with small amounts of DNA.
Published Version
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