EPR determination of low molecular weight iron content applied to whole rat hepatocytes

Electron paramagnetic resonance (EPR) has been described as suitable for the evaluation of low molecular weight (LMW) iron in liver homogenates after chelation by desferrioxamine. LMW iron is a highly toxic iron species incriminated in free radical production. The first aim of this study was to evaluate the conditions of EPR application for LMW iron content determination in whole rat hepatocytes. For this purpose, LMW iron was simultaneously quantified by EPR and by atomic absorption spectrometry. EPR determination of LMW iron needed a preincubation of hepatocyte cultures with the iron chelator for at least one hr. Deferiprone as LMW iron chelator was revealed to be more suited than desferrioxamine. Secondly, we showed the applicability of this method for evaluating the prooxidant status during an oxidative stress. As an example, oxidative stress induced by ethanol in hepatocytes was studied during inflammatory circumstances, well-known to lead to nitric oxide production. In hepatocyte cultures supplemented with ethanol, an elevation of LMW iron content was observed in cells. But when nitric oxide donors or a supplementation constituted of lipopolysaccharide and gamma-interferon, able to induce nitric oxide synthase, were added, LMW iron content decreased. Thus EPR determination of LMW iron content in whole hepatocytes could give some insight about the mechanism of induction or inhibition of an oxidative stress.