EPR DETERMINATION OF LOW-MOLECULAR-WEIGHT IRON CONTENT APPLIED TO WHOLE RAT HEPATOCYTES

Electron paramagnetic resonance (EPR) has been described as suitable f or the evaluation of low molecular weight (LMW) iron in liver homogena tes after chelation by desferrioxamine. LMW iron is a highly toxic iro n species incriminated in free radical production. The first aim of th is study was to evaluate the conditions of EPR application for LMW iro n content determination in whole rat hepatocytes. For this purpose, LM W iron was simultaneously quantified by EPR and by atomic absorption s pectrometry. EPR determination of LMW iron needed a preincubation of h epatocyte cultures with the iron chelator for at least one hr. Deferip rone as LMW iron chelator was revealed to be more suited than desferri oxamine. Secondly, we showed the applicability of this method for eval uating the prooxidant status during an oxidative stress. As an example , oxidative stress induced by ethanol in hepatocytes was studied durin g inflammatory circumstances, well-known to lead to nitric oxide produ ction. In hepatocyte cultures supplemented with ethanol, an elevation of LMW iron content was observed in cells. But when nitric oxide donor s or a supplementation constituted of lipopolysaccharide and gamma-int erferon, able to induce nitric oxide synthase, were added, LMW iron co ntent decreased. Thus EPR determination of LMW iron content in whole h epatocytes could give some insight about the mechanism of induction or inhibition of an oxidative stress.