Increased 8-hydroxyguanine formation and endonuclease activity for its repair in ischemic-reperfused hearts of rats

A type of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua) formation, and the activity for its subsequent repair, 8-OH-Gua endonuclease activity, wer e examined in an ischemia-reperfusion model of isolated rat hearts. The lev el of 8-OH-Gua in myocardial DNA was measured by a high performance liquid chromatography (HPLC) equipped with an electrochemical detector, and the 8- OH Gua endonuclease activity was analysed by the endonuclease nicking assay using a synthetic double-stranded oligonucleotide containing an 8-OH-Gua r esidue as a substrate. The Langendorff-perfused rat hearts were subjected t o 30 or 60 min of global ischemia, followed by reperfusion with an oxygenat ed or a nitrogenated Krebs-Henseleit solution. The 8-OH-Gua content in the DNA of the ischemic hearts reperfused with an oxygenated solution was three to four times higher than that of the control hearts. The levels of 8-OH-G ua did not increase either in the ischemic hearts reperfused with a nitroge nated solution or in the ischemic-reperfused hearts treated with SOD, manni tol or allopurinol. When the myocardial extract was incubated with the 8-OH -Gua-containing oligonucleotide substrate, a specific cleavage at the site of an 8-OH-Gua residue was detected. The endonuclease activity responsible for this cleavage increased two-fold in the ischemic-reperfused hearts, com pared to the control. This study demonstrates that the formation of 8-OH-Gu a in DNA as well as the level of its repair process, 8-OH-Gua endonuclease activity, increase in the ischemic-reperfused rat hearts in response to oxi dative stress due to higher levels of oxygen free radicals. (C) 2000 Academ ic Press.