Sensitive quantification of carbon monoxide in vivo reveals a protective role of circulating hemoglobin in CO intoxication.

Carbon monoxide (CO) is a gaseous molecule known as the silent killer. It is widely believed that an increase in blood carboxyhemoglobin (CO-Hb) is the best biomarker to define CO intoxication, while the fact that CO accumulation in tissues is the most likely direct cause of mortality is less investigated. There is no reliable method other than gas chromatography to accurately determine CO content in tissues. Here we report the properties and usage of hemoCD1, a synthetic supramolecular compound composed of an iron(II)porphyrin and a cyclodextrin dimer, as an accessible reagent for a simple colorimetric assay to quantify CO in biological samples. The assay was validated in various organ tissues collected from rats under normal conditions and after exposure to CO. The kinetic profile of CO in blood and tissues after CO treatment suggested that CO accumulation in tissues is prevented by circulating Hb, revealing a protective role of Hb in CO intoxication. Furthermore, hemoCD1 was used in vivo as a CO removal agent, showing that it acts as an effective adjuvant to O2 ventilation to eliminate residual CO accumulated in organs, including the brain. These findings open new therapeutic perspectives to counteract the toxicity associated with CO poisoning.

The CO-releasing molecule CORM-3 protects adult cardiomyocytes against hypoxia-reoxygenation by modulating pH restoration.

Several studies have reported that CORM-3, a water-soluble carbon monoxide releasing molecule, elicits cardioprotection against myocardial infarction but the mechanism remains to be investigated. Numerous reports indicate that inhibition of pH regulators, the Na+/H+ exchanger (NHE) and Na+/HCO3- symporter (NBC), protect cardiomyocytes from hypoxia/reoxygenation injury by delaying the intracellular pH (pHi) recovery at reperfusion. Our goal was to explore whether CORM-3-mediated cytoprotection involves the modulation of pH regulation. When added at reoxygenation, CORM-3 (50 µM) reduced the mortality of cardiomyocytes exposed to 3 h of hypoxia and 2 h of reoxygenation in HCO3--buffered solution. This effect was lost when using inactive iCORM-3, which is depleted of CO and used as control, thus implicating CO as the mediator of this cardioprotection. Interestingly, the cardioprotective effect of CORM-3 was abolished by switching to a bicarbonate-free medium. This effect of CORM-3 was also inhibited by 5-hydroxydecanoate, a mitochondrial ATP-dependent K+ (mKATP) channel inhibitor (500 µM) or PD098059, a MEK1/2 inhibitor (10 µM). In additional experiments and in the absence of hypoxia-reoxygenation, intracellular pH was monitored in cardiomyocytes exposed to cariporide to block NHE activity. CORM-3 inhibited alkalinisation and this effect was blocked by PD098059 and 5-HD. In conclusion, CORM-3 protects the cardiomyocyte against hypoxia-reoxygenation injury by inhibiting a bicarbonate transporter at reoxygenation, probably the Na+/HCO3- symporter. This cardioprotective effect of CORM-3 requires the activation of mKATP channels and the activation of MEK1/2.

Nrf2 activators modulate oxidative stress responses and bioenergetic profiles of human retinal epithelial cells cultured in normal or high glucose conditions.

Retinal pigment epithelial cells exert an important supporting role in the eye and develop adaptive responses to oxidative stress or high glucose levels, as observed during diabetes. Endogenous antioxidant defences are mainly regulated by Nrf2, a transcription factor that is activated by naturally-derived and electrophilic compounds. Here we investigated the effect of the Nrf2 activators dimethylfumarate (DMF) and carnosol on antioxidant pathways, oxygen consumption rate and wound healing in human retinal pigment epithelial cells (ARPE-19) cultured in medium containing normal (NG, 5mM) or high (HG, 25 mM) glucose levels. We also assessed wound healing using an in vivo corneal epithelial injury model. We found that Nrf2 nuclear translocation and heme oxygenase activity increased in ARPE cells treated with 10 µM DMF or carnosol irrespective of glucose culture conditions. However, HG rendered retinal cells more sensitive to regulators of glutathione synthesis or inhibition and caused a decrease of both cellular and mitochondrial reactive oxygen species. Culture in HG also reduced ATP production and mitochondrial function as measured with the Seahorse XF analyzer and electron microscopy analysis revealed morphologically damaged mitochondria. Acute treatment with DMF or carnosol did not restore mitochondrial function in HG cells; conversely, the compounds reduced cellular maximal respiratory and reserve capacity, which were completely prevented by N-acetylcysteine thus suggesting the involvement of thiols in this effect. Interestingly, the scratch assay showed that wound closure was faster in cells cultured in HG than NG and was accelerated by carnosol. This effect was reversed by an inhibitor of heme oxygenase activity. Moreover, topical application of carnosol to the cornea of diabetic rats significantly accelerated wound healing. In summary, these data indicate that culture of retinal epithelial cells in HG does not affect the activation of the Nrf2/heme oxygenase axis but influences other crucial oxidative and mitochondrial-dependent cellular functions. The additional effect on wound closure suggests that results obtained in in vitro experimental settings need to be carefully evaluated in the context of the glucose concentrations used in cell culture.

Isothiocyanate-cysteine conjugates protect renal tissue against cisplatin-induced apoptosis via induction of heme oxygenase-1.

Heme oxygenase-1 (HO-1) is a redox sensitive inducible enzyme endowed with important antioxidant and cytoprotective activities. Here we report that two water-soluble isothiocyanate-cysteine conjugates, S-[N-benzyl(thiocarbamoyl)]-l-cysteine (BTTC) and S-[N-(3-phenylpropyl)(thiocarbamoyl)]-l-cysteine (PTTC), potently increase HO-1 protein expression and heme oxygenase activity in renal tubular epithelial cells at 5 and 10µM, while higher concentrations are themselves cytotoxic and pro-apoptotic. Inhibitors of the pro-survival pathways ERK, MAPK and PI3K almost completely abolished the increase in HO-1 induction and heme oxygenase activity, while the JNK pathway appeared to be mainly involved in the apoptosis triggered by the isothiocyanates. We also found that renal cells exposed to 50µM cisplatin (CDDP), a chemotherapeutic agent known for its nephrotoxic actions, displayed a marked increase in caspase-3 activity and the number of apoptotic cells. These effects were abolished by pre-incubation of cells with concentrations of BTCC or PTCC that maximize HO-1 induction and were reversed by the inhibitor of heme oxygenase activity tin protoporphyrin IX (SnPPIX). Moreover, in a model of CDDP-induced nephrotoxicity in vivo, pre-treatment of rats with a daily dose of BTCC or PTCC (25mg/kg, i.p.) completely abolished the increase in serum creatinine and urea levels and markedly reduced the severity of renal tissue apoptosis caused by CDDP. The renoprotective effects of BTCC and PTCC in vivo were markedly attenuated by administration of rats with SnPPIX. These findings indicate that water-soluble isothiocyanates counteract renal dysfunction and apoptosis by up-regulating the HO-1 system and could be used as a supplementary treatment to mitigate CDDP-induced nephrotoxic effects.

Mitochondrial and cellular heme-dependent proteins as targets for the bioactive function of the heme oxygenase/carbon monoxide system.

The toxic effect of high concentrations of CO gas in living organisms is coherently typified at biochemical levels by the high affinity of CO for hemoglobin and cytochromes, heme-dependent proteins that are indispensable for oxygen transport and mitochondrial respiration. However, the basal production of CO during heme degradation and the ability of heme oxygenase-1 (HO-1) to increase CO availability pose the question of how this gaseous molecule interacts with metal centers within the intracellular milieu to serve as one of the most unconventional signaling mediators. Emerging evidence indicates that the diverse and multifaceted beneficial effects exerted by "low concentrations" of CO cannot be explained solely by the activation of classic prototypic targets (i.e., guanylate cyclase/potassium channels) but entails the dynamic and concerted activation/inhibition of a group of CO-responsive proteins. As the complexity of the temporal and spatial action of CO is progressively being appreciated, this review aims to (a) highlight the current knowledge on certain metal-containing proteins that interact directly with CO; (b) analyze the latest notions on their functional role in response to CO; and finally (c) propose a rational view on the mode these CO targets may interrelate with and be regulated by the HO/CO pathway.