Studies on Hg(II)-induced H 2O 2 formation and oxidative stress in vivo and in vitro in rat kidney mitochondria

Abstract

Studies were undertaken to investigate the principal actions underlying mercury-induced oxidative stress in the kidney. Mitochondria from kidneys of rats treated with HgCl 2 (1.5 mg/kg i.p.) demonstrated a 2-fold increase in hydrogen peroxide (H 2O 2) formation for up to 6 hr following Hg(II) treatment using succinate as the electron transport chain substrate. No increase in H 2O 2 formation was observed when NAD-linked substrates (malate/glutamate) were used, suggesting that Hg(II) affects H 2O 2 formation principally at the ubiquinone-cytochrome b region of the mitochondrial respiratory chain in vivo. Together with increased H 2O 2 formation, mitochondrial glutathione (GSH) content was depleted by more than 50% following Hg(II) treatment, whereas formation of thiobarbiturate reactive substances (TBARS), indicative of mitochondrial lipid peroxidation, was increased by 68%. Studies in vivo revealed a significant concentration-related depolarization of the inner mitochondrial membrane following the addition of Hg(II) to mitochondria isolated from kidneys of untreated rats. This effect was accompanied by significantly increased H 2O 2 formation, GSH depletion and TBARS formation linked to both NADH dehydrogenase (rotenone-inhibited) and ubiquinone-cytochrome b (antimycin-inhibited) regions of the electron transport chain. Oxidation of pyridine nucleotides (NAD[P]H) was also observed in mitochondria incubated with Hg(II) in vitro. In further studies in vitro, the potential role of Ca 2+ in Hg(II)-induced mitochondrial oxidative stress was investigated. Ca 2+ alone (30–400 nmol/mg protein) produced no increase in H 2O 2 and only a slight increase in TBARS formation when incubated with kidney mitochondria isolated from untreated rats. However, Ca 2+ significantly increased H 2O 2 and TBARS formation elicited by Hg(II) at the ubiquinone-cytochrome b region of the mitochondrial electron transport chain, whereas TBARS formation was decreased significantly when the Ca 2+ uptake inhibitors, ruthenium red or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), were included with Hg(II) in the reaction mixtures. These findings support the view that Hg(II) causes depolarization of the mitochondrial inner membrane with consequent increased H 2O 2 formation. These events, coupled with Hg(II)-mediated GSH depletion and pyridine nucleotide oxidation, create an oxidant stress condition characterized by increased susceptibility of mitochondrial membranes to iron- dependent lipid peroxidation (TBARS formation). Since increased H 2O 2 formation, GSH depletion and lipid peroxidation were also observed in vivo following Hg(II) treatment, these events may underlie oxidative tissue damage caused by mercury compounds. Moreover, Hg(II)-induced alterations in mitochondrial Ca 2+ homeostasis may excerbate Hg(II)-induced oxidative stress in kidney cells.