STUDIES ON HG(II)-INDUCED H2O2 FORMATION AND OXIDATIVE STRESS INVIVO AND INVITRO IN RAT-KIDNEY MITOCHONDRIA

Studies were undertaken to investigate the principal actions underlyin g mercury-induced oxidative stress in the kidney. Mitochondria from ki dneys of rats treated with HgCl2 (1.5 mg/kg i.p.) demonstrated a 2-fol d increase in hydrogen peroxide (H2O2) formation for up to 6 hr follow ing Hg(II) treatment using succinate as the electron transport chain s ubstrate. No increase in H2O2 formation was observed when NAD-linked s ubstrates (malate/glutamate) were used, suggesting that Hg(II) affects H2O2 formation principally at the ubiquinone-cytochrome b region of t he mitochondrial respiratory chain in vivo. Together with increased H2 O2 formation, mitochondrial glutathione (GSH) content was depleted by more than 50% following Hg(II) treatment, whereas formation of thiobar biturate reactive substances (TBARS), indicative of mitochondrial lipi d peroxidation, was increased by 68%. Studies in vivo revealed a signi ficant concentration-related depolarization of the inner mitochondrial membrane following the addition of Hg(II) to mitochondria isolated fr om kidneys of untreated rats. This effect was accompanied by significa ntly increased H2O2 formation, GSH depletion and TBARS formation linke d to both NADH dehydrogenase (rotenone-inhibited) and ubiquinone-cytoc hrome b (antimycin-inhibited) regions of the electron transport chain. Oxidation of pyridine nucleotides (NAD[P]H) was also observed in mito chondria incubated with Hg(II) in vitro. In further studies in vitro, the potential role of Ca2+ in Hg(II)-induced mitochondrial oxidative s tress was investigated. Ca2+ alone (30-400 nmol/mg protein) produced n o increase in H2O2 and only a slight increase in TBARS formation when incubated with kidney mitochondria isolated from untreated rats. Howev er, Ca2+ significantly increased H2O2 and TBARS formation elicited by Hg(II) at the ubiquinone-cytochrome b region of the mitochondrial elec tron transport chain, whereas TBARS formation was decreased significan tly when the Ca2+ uptake inhibitors, ruthenium red or [ethylenebis(oxy ethylenenitrilo)]tetraacetic acid (EGTA), were included with Hg(II) in the reaction mixtures. These findings support the view that Hg(II) ca uses depolarization of the mitochondrial inner membrane with consequen t increased H2O2 formation. These events, coupled with Hg(II)-mediated GSH depletion and pyridine nucleotide oxidation, create an oxidant st ress condition characterized by increased susceptibility of mitochondr ial membranes to iron-dependent lipid peroxidation (TBARS formation). Since increased H2O2 formation, GSH depletion and lipid peroxidation w ere also observed in vivo following Hg(II) treatment, these events may underlie oxidative tissue damage caused by mercury compounds. Moreove r, Hg(II)-induced alterations in mitochondrial Ca2+ homeostasis may ex cerbate Hg(II)-induced oxidative stress in kidney cells.