Carbon Monoxide: Exogenous Sources and Toxic Effects Bench-to-bedside Review: Carbon Monoxide -from Mitochondrial Poisoning to Therapeutic Use

Carbon monoxide (CO) is generated during incomplete combustion of carbon-containing compounds and leads to acute and chronic toxicity in animals and humans depending on the concentration and exposure time. In addition to exogenous sources, CO is also produced endogenously by the activity of heme oxygenases (HOs) and the physiological significance of HO-derived CO has only recently emerged. CO exerts vasoactive, anti-proliferative, anti-oxidant, anti-inflammatory and anti-apoptotic effects and contributes substantially to the important role of the inducible isoform HO-1 as a mediator of tissue protection and host defense. Exoge-nous application of low doses of gaseous CO might provide a powerful tool to protect organs and tissues under various stress conditions. Experimental evidence strongly suggests a beneficial effect under pathophysiological conditions such as organ trans-plantation, ischemia/reperfusion, inflammation, sepsis, or shock states. The cellular and molecular mechanisms mediating CO effects are only partially characterized. So far, only a few studies in humans are available, which, however, do not support the promising results observed in experimental studies. The protective effects of exogenous CO may strongly depend on the pathological condition, the mode, time point and duration of application, the administered concentration, and on the target tissue and cell. Differences in bioavailability of endogenous CO production and exogenous CO supplementation might also provide an explanation for the lack of protective effects observed in some experimental and clinical studies. Further randomized, controlled clinical studies are needed to clarify whether exogenous application of CO may turn into a safe and effective preventive and therapeutic strategy to treat pathophysiological conditions associated with inflammatory or oxidative stress. High concentrations of carbon monoxide (CO) are generated during incomplete combustion of carbon-containing compounds such as wood, coal, gas, oil, or tobacco. CO is a colorless and odorless gas that causes acute and chronic toxicity in humans and animals. CO mediates its toxic effects primarily by strongly binding to hemoglobin and forming carboxyhemoglobin (COHb), thereby reducing the oxygen-carrying capacity of the blood. The affinity of hemoglobin for CO is approximately 210 to 250 times that for oxygen [1]. Both decreased arterial oxygen content (impaired O 2 binding to hemoglobin) and decreased tissue oxygen pressure (PO 2 ; increased affinity of COHb for O 2) lead to tissue hypoxia [2,3]. There is a linear correlation between the inspired level of CO and arterial COHb levels [4]. Although the percentage of COHb in blood represents the best predictive marker for extrapolating the total amount …

smokers may have more severe symptoms since their COHb levels are already elevated.
Carbon monoxide appears to be the leading cause of injury and death due to poisoning worldwide [10]. Since tissue hypoxia is the underlying mechanism of CO-induced injury, increasing the inspired oxygen concentration represents the treatment for CO poisoning. In severe poisoning, hyperbaric oxygen therapy is regarded as the therapy of choice [11]. Both normobaric and hyperbaric oxygen improve oxygen delivery by increasing the amount of oxygen dissolved in plasma and by reducing the half-life of COHb. However, the results from existing randomized, controlled trials of hyperbaric versus normobaric oxygen in the treatment of acute CO poisoning provide conflicting results regarding the effectiveness of hyperbaric oxygen for the prevention of neurological symptoms [12]. An ongoing phase IV randomized clinical trial investigates important clinical outcomes (for example, 6-week cognitive sequelae) of patients with acute CO poisoning randomized to receive either one or three hyperbaric oxygen treatments [13]. The estimated study completion date is May 2009. If treatment of CO poisoning is timely, most patients are able to recover, but even with adequate treatment CO poisoning may result in permanent memory loss or brain damage. For the long-term sequelae of acute CO poisoning, only symptomatic therapy is available. Chronic exposure to CO may lead to myocardial hypertrophy [14].

Functions of endogenous carbon monoxide production
Coburn and colleagues [15] demonstrated that CO is endogenously produced in animals and humans. The vast majority of endogenous CO is derived from the oxidative breakdown of heme by microsomal heme oxygenases (HOs). HO catalyzes the first and rate-limiting step in heme degradation, yielding equimolar amounts of CO, iron, and biliverdin-IXα (Figure 1), which is further converted to bilirubin by biliverdin reductase [16]. Two isoforms of HO have been described, namely HO-1 [17,18] and HO-2 [19,20]. Furthermore, a third isoform has been found in rats [21], which represents a processed pseudogene derived from the gene for HO-2 [22]. HO-2 is constitutively expressed in many tissues, with high activity in testes, central nervous system, liver, kidney, and intestine. A basal expression of HO-1 is found in tissues that degrade senescent red blood cells, predominantly spleen, reticuloendothelial cells of the liver and bone marrow [23]. HO-1 is the inducible isoform, and induction of HO-1 gene expression occurs in response to a wide variety of endogenous and exogenous stimuli, such as chemical or physical stimuli, xenobiotics, hyperoxia, hypoxia, ischemia/reperfusion, inflammation, surgical procedures, or anesthetics [24][25][26][27][28][29].
The critical role of HO-1 under physiological conditions was demonstrated in the first described case of human HO-1 deficiency. The boy in this case presented with severe growth retardation, persistent hemolytic anemia, and severe, persistent endothelial damage [30] and died at the age of 6 years [31]. Over the past decade the function of HO-1 has expanded from a heme-degrading enzyme to a key mediator of tissue protection and host defense, and its cytoprotective effects have been described in vivo and in vitro [24,25,28,[32][33][34][35][36][37][38][39][40][41][42].
The products of the HO pathway -CO, iron, and biliverdin/ bilirubin -have long been regarded solely as waste products. Recently, the unique biological functions of the products and their contribution to the protective effects of the HO system have attracted great interest. Thus, the HO system has different functions: besides the breakdown of heme, a prooxidant [43], it produces cytoprotective substances, and the inducibility of HO-1 renders it a powerful endogenous cytoprotective system. Bilirubin has been described as a potent endogenous antioxidant [44] with potential clinical implications [45]. Free iron exhibits oxidizing capacities, although the iron released during heme degradation stimulates the synthesis of ferritin [46], which sequesters unbound iron, thereby serving as an additional anti-oxidant [47]. The observation that CO can weakly activate soluble guanylate cyclase (sGC), thereby stimulating the production of cGMP, suggested an important role of CO as an intracellular messenger molecule, thus acting in a similar way to nitric oxide [48,49]. The functions of CO as a neural messenger have since been described [50]. Vasoactive effects of CO have been reported in the pulmonary vasculature [51] and in the liver [37,52], where CO acts to maintain portal venous vascular tone in a relaxed state [37]. In addition to the biological functions of CO under physiological conditions, the substantial contribution of CO to the protective effects of induced HO activity has recently been recognized and includes vasoactive, anti-oxidative, antiinflammatory, anti-apoptotic, and anti-proliferative properties. Thus, CO has advanced from a toxic waste product to a physiological regulator and the importance of endogenously derived CO to control homeostasis under both physiological and pathophysiological conditions is increasingly recognized in every organ system and cell type.
Although different mechanisms explaining the effects of CO have been described, the exact underlying signaling mechanisms and precise molecular targets of CO are only partially elucidated. Effects mediated by CO-induced activation of sGC/cGMP include inhibition of platelet activation and aggregation, smooth muscle relaxation, vasoactive effects, inhibition of cellular proliferation, and effects on neurotransmission [37,[49][50][51][52][53][54][55][56]. cGMP-independent mechanisms of vasoregulation have also been suggested. CO may directly activate calcium-dependent potassium channels, thus mediating dilation of blood vessels [57]. Recent evidence suggests an important role of CO as a signaling molecule in modulating mitogen-activated protein kinases (MAPKs), especially p38 MAPK in response to oxidative stress and inflammation (reviewed in [58,59]). CO-mediated activation of p38 MAPK has been shown to exert anti-inflammatory [60], anti-apoptotic, and anti-proliferative effects [61,62]. Downstream target molecules of CO-dependent p38 MAPK activation have been identified, namely heat shock protein 70 and caveolin-1 [61,62]. Zhang and colleagues [63] demonstrated that the anti-apoptotic effects of CO involve both phosphatidylinositol 3-kinase/Akt and p38 MAPK signaling pathways in endothelial cells in a model of anoxia-reoxygenation injury. In hepatocytes, CO activated nuclear factor-κB (NF-κB) through a mechanism that involves reactive oxygen species-induced Akt phosphorylation and protected against cell death [64]. Figure 2 provides a simplified overview of the described CO-mediated signal transduction pathways.

Therapeutic applications of carbon monoxide
The observation that induction of HO-1 gene expression under pathological conditions plays an important role in organ preservation strongly suggests that CO might be substantially involved in mediating these effects. This is supported by the observation in models of HO-1 deficiency or after blockade of HO activity that the protective effects of induction of HO-1 are mimicked by low amounts of exogenous CO [54,59,65]. However, pre-induction of the HO-1 system by exogenous stimuli to induce local CO release or exogenous application of CO to potentiate the endogenous protective effects may be challenging. To increase the availability of CO, different approaches have been developed, including induction of HO-1 gene expression with pharmacological and genetic strategies, inhalation of low doses of CO, and application of CO-releasing molecules. Figure 3 briefly summarizes the protective effects and the potential therapeutic applications of CO in a variety of disorders and diseases of different organ systems.

Induction of HO-1 gene expression
Strategies to induce HO-1 as a protective mechanism against a subsequent stress event include pharmacological approaches such as volatile anesthetics [40] or heme derivatives [32,33], and genetic approaches [39] as well as the use of other inducers as described above. Long-term overexpression of HO-1 by targeted gene transfer has become a powerful tool to investigate the specific role of the HO-1 enzyme [66]. The amount of CO released by the induced activity of HO-1 is unknown. In addition, induction of HO-1 increases the concentration of all products of the pathway, and the contribution of CO to the observed protective effects is difficult to evaluate.

Exogenous application of carbon monoxide
Inhalation of CO represents a novel therapeutic approach and exerts both local effects on the lungs and systemic effects. The challenge remains to reach safe and effective concentrations in target tissues without producing deleterious effects caused by CO-mediated tissue hypoxia. The tolerance to CO exposure has been investigated in rodents and conflicting results have been obtained: while continuous application of 500 ppm CO for 2 years had no deleterious effects [67], 200 ppm for 20 h per day over 14 days induced myocardial hypertrophy [14].
The CO-releasing properties of transition metal carbonyls were first described by Herrman [68]. Motterlini and his group have developed CO-releasing molecules (CO-RMs) as a new strategy to deliver defined amounts of CO for therapeutic applications [6,69] without significantly affecting COHb levels [70]. In particular, the synthesis of a water-soluble compound might be promising. So far, only experimental data are available. The use of CO-RMs to characterize CO-mediated cytoprotection has been reviewed by Foresti and colleagues [6].

Preclinical experimental studies
In most experimental models, acute rather than chronic inhalation of CO is applied (10 to 1,000 ppm for 1 to 24 h). Depending on the concentration, different exposure times are required to reach COHb equilibrium [71]. CO inhalation has been shown to be protective in experimental inflammatory and non-inflammatory disease models (reviewed in [6,25,[72][73][74][75]). The majority of studies investigating the effects of low amounts of inhaled CO concentrate on disease models in the lungs. In addition to local effects in the lungs, inhaled CO is also able to affect systemic organ dysfunction.
Lung The protective effects of inhaled CO have been investigated in models of acute lung injury, acute respiratory distress syndrome (ARDS), ischemia/reperfusion, asthma, and remote lung injury. The first in vivo evidence to suggest a therapeutic potential of low dose gaseous CO was provided by Otterbein and colleagues [76]. Rats exposed to low concentrations of CO exhibited a significant attenuation of hyperoxia-induced lung injury and increased survival. CO exposure exerted anti-inflammatory and anti-apoptotic effects. The molecular mechanisms of the observed inhibition of proinflammatory cytokines involve the MKK3/p38 MAPK pathway [77]. In contrast, low levels of CO were not protective in a similar rat model of hyperoxic acute lung injury [4]. Inhalation of CO attenuated the development of hypoxia-induced pulmonary artery hypertension in rats, presumably through activation of Ca 2+ -activated K + channels [78] and was also able to reverse established pulmonary hypertension [79]. Inhalation of CO for 6 h after intratracheal injection of acidic solution in mice reduced early neutrophil recruitment without affecting chemokine levels in bronchoalveolar fluid [80]. The pathomechanisms of allergen-induced asthma include inflammation and bronchoconstriction. In ovalbumin-induced asthma, CO treatment of mice for 2 h before aerosol challenge led to a specific reduction of the pro-inflammatory cytokine IL-5 while other pro-inflammatory or anti-inflammatory cytokines were unaffected [81]. In the same model of inflammation, Ameredes and colleagues [82] showed a CO-induced, cGMP-dependent reduction of airway hyper-responsiveness. Carbon monoxide signal transduction pathways. CO, carbon monoxide; HSF, heat shock factor; HSP, heat shock protein; MAPK, mitogenactivated protein kinase; NFκB, nuclear factor-κB; NO, nitric oxide; sGC, soluble guanylate cyclase.

Figure 3
Protective effects and potential therapeutic applications of carbon monoxide. ALI, acute lung injury; ARDS, acute respiratory distress syndrome; CO, carbon monoxide; I/R, ischemia/reperfusion.
Mechanical ventilation may cause profound lung injury and inflammatory responses. Dolinay and colleagues [87] described a CO-mediated suppression of tumor necrosis factor (TNF)-alpha release and neutrophil recruitment and postulated an involvement of the p38 MAPK pathway. A study in knock-out mice suggests a key role of Egr-1 as a pro-inflammatory regulator in ventilator-induced lung injury. Moreover, peroxysome proliferator-activated receptor-gamma, an antiinflammatory nuclear regulator, seems to be involved in the protective effects of CO [88].
In addition to attenuating local lung injury, CO also protects against remote lung injury. After ischemia and reperfusion of the lower extremities, CO significantly reduced ischemia/ reperfusion-induced acute lung injury [89]. Pretreatment with inhaled CO reduced pulmonary inflammatory response and provided anti-apoptotic effects in a model of cardiopulmonary bypass in pigs [90].
Liver Effects of CO on the liver have been investigated in models of inflammation-and ischemia/reperfusion-induced hepatocellular injury as well as in burn injury. TNF-alphainduced hepatocyte cell death in mice was prevented by CO inhalation. CO-induced activation of NF-κB and inducible nitric oxide synthase and nitric oxide-induced HO-1 expression were required for the protective effects [91]. In addition, CO-stimulated liver ATP generation through the activation of sGC was a prerequisite for CO to protect against TNF-alpha-induced apoptosis [92]. In models of liver ischemia and reperfusion, HO-1 induction plays an important role in maintaining hepatocellular integrity [38] and induction of HO-1 before (low flow) ischemia can attenuate the subsequent hepatic injury [32,40]. A role for CO in preventing hypoxia-induced decreases in hepatocyte ATP levels was postulated in a mouse model of hemorrhagic shock and resuscitation [93]. In cold ischemia reperfusion associated with liver transplantation, CO inhalation suppressed the inflammatory response. Downregulation of MEK/ERK1/2 seems to play a role in mediating the protective effects while the NF-κB signaling pathway does not seem to be affected [94]. CO-RM-liberated CO attenuates liver injury in burn mice by mechanisms involving downregulation of pro-inflammatory mediators and suppression of the pro-adhesive phenotype of endothelial cells [95,96].
Intestine The protective effects of CO in the intestine have been investigated in a variety of animal models of postoperative ileus and cold ischemia/reperfusion injury associated with transplantation. The development of postoperative ileus may occur after mild manipulation of the small bowel during surgery, which initiates an inflammatory response within the intestinal muscularis [97] that is characterized by the release of pro-inflammatory mediators, increased expression of adhesion molecules on the vascular endothelium, and recruitment of leukocytes from the systemic circulation [98,99]. Inhalation of CO significantly attenuated the surgically induced molecular inflammatory response and the associated decline in gastrointestinal contractility that is characteristic of postoperative ileus [100,101]. Similar effects could be observed after intraperitoneal injection of CO-saturated Ringer`s lactate solution, possibly in a sGC-dependent manner [102].
Nakao and colleagues [103] provide a large body of evidence that inhaled CO is also protective by improving posttransplant motility and attenuating the inflammatory cytokine response in the syngeneic rat transplant model. In addition, CO is anti-apoptotic and significantly improves animal survival [104]. Similar protective results can be achieved after storage of grafts in University of Wisconsin solution saturated with CO [105].
Vascular diseases Short-term administration of CO has been shown to be protective against vascular injury. CO rescued the pro-thrombotic phenotype of Hmox1 deficiency during oxidative stress [106]. Intravenous injection of CO-saturated saline produced vasodilatation and improved microvascular hemodynamics in a hamster skinfold window chamber preparation, possibly via increased cardiac output and local cGMP content [107]. Otterbein and colleagues [55] described a beneficial effect of inhaled CO in preventing arteriosclerotic lesions that occur following aorta transplantation.
Heart Experimental models of heart transplantation or cardiopulmonary bypass have been used to investigate CO effects on accompanying organ injury. CO reduced ischemia/ reperfusion injury and cardiac rejection of mouse to rat cardiac transplants via anti-apoptotic, anti-inflammatory and vasodilatory mechanisms, and suppression of platelet aggregation and fibrinolysis [65]. Treatment of the donor (CO inhalation) and graft (CO-saturated storage solution) but not the recipient protected against ischemia/reperfusion injury via anti-apoptotic mechanisms [108]. In contrast, low-dose CO inhalation of the recipient after transplantation effectively ameliorated heart allograft rejection via downregulation of pro-inflammatory mediators [109].
In a clinically relevant model of cardiopulmonary bypass surgery in pigs, treatment with CO improved cardiac energetics, prevented edema formation and apoptosis, and facilitated recovery [110]. In a rat model of ischemia/ reperfusion injury induced by occlusion of the left anterior descending coronary artery, pre-exposure to CO significantly reduced infarct size and migration of macrophages into infarct areas. In addition, TNF-alpha expression was reduced.
The protective effects were mediated by CO-induced activation of p38 MAPK, protein kinase B (Akt), endothelial nitric oxide synthase, and cGMP in the myocardium [111].
Kidney Most of the studies of CO effects in kidneys concentrate on models of cold ischemia/reperfusion injury in transplantation. Ischemia/reperfusion injury of kidney grafts is one of the major deleterious factors affecting successful renal transplantation. Renal ischemia/reperfusion injury causes delayed graft function and plays a significant role in the development of chronic allograft nephropathy [112,113]. Exposure to low concentrations of CO prevented fibroinflammatory changes associated with chronic allograft nephropathy and preserved long-term renal allograft function [114]. Storage of kidneys with cold preservation solutions containing CO-RMs also improved their function upon reperfusion [115]. Hypoxia-inducible factor-1-mediated upregulation of vascular endothelial growth factor seems to contribute to the protective mechanisms [116]. Nakao and colleagues [117] provide evidence that prevention of cytochrome P450 degradation, maintenance of normal intracellular heme levels and a reduction of lipid peroxidation participate in the protective effects of CO-RMs during storage of kidney grafts.
Systemic inflammation As a model of systemic inflammation, lipopolysaccharide (LPS)-induced inflammatory response and organ injury has widely been used to study protective COmediated effects. In rodents and pigs injected with LPS, inhalation of CO leading to 14.08 ± 1.34% COHb significantly reduced LPS-induced cytokine response [118,119] and improved long-term survival [120]. Further mechanisms of CO-mediated protection against LPS-induced multiple injury in rats have been described and include anti-oxidative, anti-inflammatory and anti-apoptotic effects, and up-regulation of HO-1 expression [121]. In contrast, in a randomized, controlled study in pigs, CO exposure did not alter LPSinduced levels of pro-and anti-inflammatory cytokines [122]. The lack of protective effects observed in this study might possibly be explained by the low level of COHb measured (5% compared to 14%) [118].

Clinical studies
While a large body of experimental evidence suggests the potential of low amounts of inhaled CO to protect the lungs and systemic organs and tissues against oxidative and inflammatory insults, only a few studies on therapeutic applications of CO inhalation in humans have been published.
In a randomized, double-blinded, placebo-controlled, two-way cross-over trial experimental endotoxemia was induced in healthy volunteers by injection of 2 ng/kg LPS. The potential anti-inflammatory effects of CO inhalation were investigated by inhalation of 500 ppm CO (leading to an increase in COHb from 1.2% to 7%) versus synthetic air as a placebo for 1 h. CO inhalation had no effect on the inflammatory response as measured by systemic cytokine production (TNF-alpha, IL-6, IL-8, IL-1α and IL-1β) [123]. In this study, no adverse side effects of CO inhalation were observed.
This study is in contrast to the above described results obtained in most experimental models of endotoxemia. Possible explanations for this discrepancy could be that blood from different species has different affinities for CO, different COHb half-lives, different hemoglobin CO saturation points (different COHb levels at the same CO concentration), or different basic physiologies, such as heart rate.
COPD is characterized by an inflammatory and oxidative stress response. Furthermore, COPD is accompanied by increased COHb levels that correlate with exhaled CO [124]. However, the endogenous CO release might not be sufficient to protect against the development and progression of COPD. In a randomized, placebo-controlled, cross-over study 20 ex-smoking patients with stable COPD were examined to assess safety, feasibility, and potential anti-inflammatory effects of CO inhalation. Inhalation of 100 to 125 ppm CO for 2 h per day on 4 consecutive days led to a maximal individual COHb level of 4.5%. In two patients, exacerbations of COPD occurred during or after the CO inhalation period; otherwise the treatment was well tolerated. The primary study endpoint was sputum neutrophil counts. Although there was a trend towards reduction in sputum eosinophils and improvement of bronchial responsiveness, no significant therapeutic effects were observed [125]. The results of this pilot study are interesting, since they provide some evidence for a potential therapeutic use of inhaled CO. However, whether CO inhalation increases the risk of COPD exacerbations needs to be determined.
One clinical study investigating the effects of low amounts of inhaled CO is currently in progress [126]. A single blinded, randomized, placebo controlled phase I study in healthy subjects investigates the potential of inhaled carbon monoxide in preventing lung inflammatory responses following local endotoxin instillation. The study is ongoing, but currently not recruiting participants.

Conclusion
CO has long been regarded solely as a toxic environmental or endogenous waste product. In addition to cytoprotective properties of endogenous CO, recent evidence strongly suggests protective effects of low concentrations of exogenous CO under pathophysiological conditions such as organ transplantation, ischemia/reperfusion, inflammation, sepsis, or shock states. Studies in humans are scarce and so far do not support the promising results observed in pre-clinical experimental studies. A potential beneficial effect of exogenous CO may highly depend on the pathological condition, the mode, time point and duration of application, the administered concentration, and on the target tissue. Further randomized, controlled clinical trials are needed to clarify whether exogenous application of CO, either by inhalation or intravenous application of CO-RMs, may become a safe and effective preventive and therapeutic tool to treat pathophysiological conditions associated with inflammatory or oxidative stress.

Competing interests
The authors declare that they have no competing interests This article is part of a review series on Gaseous mediators, edited by Peter Radermacher.
Other articles in the series can be found online at http://ccforum.com/series/gaseous_mediators