Cell Damage Following Carbon Monoxide Releasing Molecule Exposure: Implications for Therapeutic Applications

Abstract

The cytoprotective properties of carbon monoxide (CO) gas and CO-releasing molecules (CORMs) are well established. Despite promising pre-clinical results, little attention has been paid to the toxicological profile of CORMs. The effects of CORM-2 and its CO-depleted molecule (iCORM-2) (20-400 lM) were compared in primary rat cardiomyocytes and two cell lines [human embryonic kidney (HeK) and Madine-Darby canine kidney Cells (MDCK)]. Cells were assessed for cell viability, apoptosis, necro-sis, cytology, mitochondrial energetics, oxidative stress and cell cycle arrest markers. In separate experiments, the anti-apoptotic effects of CORM-2 and i-CORM-2 treatment were compared against CO gas treatment in HeK and MDCK lines. H 2 O 2-induced cellular damage, measured by lactate dehydrogenase (LDH) release from primary cardiomyocytes, was reduced by 20 lM CORM-2; LDH activity, however, was directly inhibited by 400 lM CORM-2. Both CORM-2/iCORM-2 and CO gas decreased cisplatin-induced caspase-3 activity in MDCK and HeK cells suggesting an anti-apoptotic effect. Conversely, both CORM-2 and iCORM-2 induced significant cellular toxicity in the form of decreased cell viability, abnormal cell cytology, increased apoptosis and necrosis, cell cycle arrest and reduced mitochondrial enzyme activity. Comparison of these markers after CO gas administration to MDCK cells found significantly less cellular toxicity than in 100 lM CORM-2/iCORM-2-treated cells. CO gas did not have an adverse effect on mitochondrial energetics and integrity. Release of CO by low concentrations of intact CORM-2 molecules provides cyto-protective effects. These results show, however, that the ruthenium-based CORM by-product, iCORM-2, is cytotoxic and suggest that the accumulation of iCORM-2 would seriously limit any clinical application of the ruthenium-based CORMs. Carbon monoxide (CO), iron and biliverdin are products of the catabolism of haem by the enzyme haem oxygenase (HO) [1]. Both CO administration and HO-1 induction have been demonstrated to afford cytological and organ protection in a number of models [2,3]. CO provides cytoprotection via a number of different mechanisms that include anti-inflammatory [4], vasodilatory [5,6], anti-coagulative [7], anti-apoptotic [7,8] and anti-proliferative/fibrotic pathways [3]. The anti-apoptotic properties of CO have been demonstrated in both in vivo and in vitro models to involve modulation of intrinsic pathways, extrinsic pathways and also up-regulation of protective anti-apoptotic Bcl-X L /Bax interaction [6-9]. CO exerts a variety of effects on mitochondrial function dependent on its concentration, duration of exposure and cell type studied. Both endogenous and exogenous CO have been demonstrated to diminish cellular respiration via inhibition of complex IV [10,11]. However, inhibition of complex IV by CO may account in part for some of its cytoprotective effects, by creating a 'metabolic hypoxia' [12] and activating anti-inflammatory p38 MAPK pathways via ROS release [13]. Exposure of rats to CO (50 ppm;...

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