Dna damage in human leukocytes after ischemia/reperfusion injury

Leukocytes have been shown to play an important role in the development of tissue injury after ischemia and reperfusion (I/R). In the present study, t he effects of tourniquet-ischemia on induction of DNA damage in peripheral leukocytes and on respiratory burst of neutrophils in humans were examined. The DNA damage was measured as increased migration of DNA using the single -cell gel-electrophoresis technique (comet assay). Intracellular production of reactive oxygen species by neutrophils was measured flow-cytometrically using dihydrorhodamine 123 as indicator. Postischemic, significantly incre ased migration of DNA was found in leukocytes of 20 patients (tourniquet-is chemia of the lower limb for 65-130 min, anterior-cruciate-ligament-reconst ruction) and in 10 experiments (1 volunteer, repeated tourniquet-ischemia o f the upper limb for 60 min, no operation). DNA effects were most pronounce d 5-30 min after tourniquet release, and then declined over a 2 h period, b ut did not return to preischemic baseline values. A similar time course sho wed the oxidative status of unstimulated granulocytes during reperfusion. S imultaneously, opposing changes were measured in formyl peptide (f-MLP)- or phorbol ester (PMA)-stimulated granulocytes, which showed a significantly declined respiratory burst reaction after tourniquet-release indicating pre activation of neutrophils by I/R. Our data suggest that I/R induces genotox ic effects in human leukocytes presumably in response to oxidative stress d uring reperfusion. (C) 2000 Elsevier Science Inc.