Carbon monoxide (CO) is usually considered a harmful and toxic molecule due to its high affinity to heme proteins. However, recent evidences show that low doses of CO can be cytoprotective, presenting several biological properties, namely, anti-apoptosis, anti-proliferation, anti-inflammation and vasodilatation [1
]. Furthermore, CO is an endogenous product of heme degradation by heme-oxygenase (HO), generating free iron and biliverdin as by-products. In fact, HO system is essential for tissue response to diverse pathological contexts, aiming at restoring and/or maintaining cellular homeostasis [2
In hepatocytes and/or liver models, CO appears to act as an anti-apoptotic molecule. By stimulating ATP production, CO activates p38 MAPK signalling, preventing apoptosis in human hepatocytes [3
]. CO rescues mice from fulminant hepatitis, presenting a marked reduction of TNF-alpha-induced apoptosis [4
] or via
NO generation [5
]. In primary cultures of rat hepatocytes, CO limits cytotoxicity induced by glucose deprivation through suppression of ERK MAPK activation [6
]. In an endotoxic shock model, CO protects hepatocytes from apoptosis by augmenting iNOS expression [7
]. It is also described that superoxide anion-induced apoptosis is inhibited by CO via
limiting JNK activity [8
]. CO treatment protects hepatocytes from cell death by inducing NF-kB activation, which is dependent on ROS generation, since inhibition of ROS generation (via anti-oxidant addition or by using respiratory deficient cells) reverses CO-induced cytoprotection [9
]. Among all publications showing CO as anti-apoptotic molecule in hepatic model, only Kim and colleagues [10
] have mentioned the involvement of mitochondria. CO protects hepatocytes from TNF-alpha/Actinomycin D-induced apoptosis by activating NF-kB, which is associated with a reduction in cytochrome c release from mitochondria [10
]. However, no data demonstrate the direct role of CO into isolated liver mitochondria.
Mitochondria play a key role in the intrinsic pathways of apoptosis. Many pro-apoptotic factors are confined in the inter-membrane space, and upon mitochondrial membrane permeabilization (MMP) these factors are released into the cytosol and cell death becomes an irreversible process [11
]. MMP marks a point of no return in the apoptotic intrinsic pathways by activating both caspase-dependent and caspase-independent mechanisms. The rupture of mitochondrial membrane also leads to the functional impairment of mitochondria, bioenergetic and redox crisis with ATP depletion and strong oxidative stress [12
]. Therefore, mitochondria become a crucial target to modulate cell death in several models.
Based on the following facts: (i) CO is an anti-apoptotic molecule in several hepatic models, hepatocytes and/or liver and (ii) mitochondria are central executers of cell death process, via the mitochondrial membrane permeabilization (MMP); we explored the direct effect of CO into isolated liver mitochondria (MMP modulation) and the involvement of ROS in this process. MMP was assessed by mitochondrial depolarization, inner membrane permeabilization and mitochondrial swelling.