Endothelial Cell DNA Integrity following Anoxia/Reoxygenation Injury

Abstract

Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases including stroke, heart attack and hemorrhagic shock. This study investigated the extent of DNA damage in an in vitro system of hypoxia/reoxygenation that may be useful for screening for drugs to reduce this form of injury. The usual method of subjecting cells to reduced oxygen (O2) by purging with nitrogen is a slow process and does not reproduce the rapid local reduction in O2 that occurs in vivo during ischemia. Glucose oxidase (GOX), however, rapidly oxidizes extracellular glucose producing a very low O2 level (< 0.4 μM) within 5 min with only minor reductions in glucose levels. We previously showed 10 min of anoxia followed by reoxygenation of human umbilical vein endothelial cells (HUVEC) damaged various biomolecules, such as lipids, proteins, and DNA. In the present study, human umbilical vein endothelial cells (HUVEC) were subjected to GOX‐induced hypoxia for 10 min and returned to medium and normoxia for up to 6 hrs. The most commonly used biomarker of DNA oxidation, in epidemiological as well as experimental studies, is 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo), however this methods requires large amounts of DNA. Here we used a high‐throughput assessment of DNA damage utilizing a high‐sensitivity long‐run real‐time PCR technique for DNA‐damage quantification in both the mitochondrial and nuclear genome. Following hypoxia cells were provided normoxia (3% oxygen) for up to 6 hours, fixed in 50% isopropanol and DNA extracted with DNAzol (MRC, Cincinnati, OH). The isolated DNA was subjected to real‐time polymerase chain reaction (RT‐PCR) with primers designed to amplify large and small regions of mitochondrial and nuclear DNA where the extent of template amplification is inversely proportional to the lesion frequency within a given DNA sequence. Detected lesions per 10kb DNA were determined. Compared to hydrogen peroxide induced DNA damage, the test conditions produced extensive damage to both mitochondrial and nuclear DNA that could readily be detected by 4 and 6 hours. These results were also correlated with HPLC analysis of 8‐oxoguanine that indicated that this biomarker to oxidative stress was enhanced about 50%. This in vitro system of hypoxia/reoxygenation can be used to further understand responses of cells to I/RI and identify drugs and potential therapeutic windows in vitro that may be translated to ameliorate ischemia/reperfusion injury in vivo.Support or Funding InformationFunded by US Army Medical Research Materiel Command