BACKGROUND
carbon monoxide  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  <cit> .
furthermore, co is an endogenous product of heme degradation by heme-oxygenase , 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  <cit> .
in hepatocytes and/or liver models, co appears to act as an anti-apoptotic molecule.
by stimulating atp production, co activates p <dig> mapk signalling, preventing apoptosis in human hepatocytes  <cit> .
co rescues mice from fulminant hepatitis, presenting a marked reduction of tnf-alpha-induced apoptosis  <cit>  or via no generation  <cit> .
in primary cultures of rat hepatocytes, co limits cytotoxicity induced by glucose deprivation through suppression of erk mapk activation  <cit> .
in an endotoxic shock model, co protects hepatocytes from apoptosis by augmenting inos expression  <cit> .
it is also described that superoxide anion-induced apoptosis is inhibited by co via limiting jnk activity  <cit> .
co treatment protects hepatocytes from cell death by inducing nf-kb activation, which is dependent on ros generation, since inhibition of ros generation  reverses co-induced cytoprotection  <cit> .
among all publications showing co as anti-apoptotic molecule in hepatic model, only kim and colleagues  <cit>  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  <cit> .
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  these factors are released into the cytosol and cell death becomes an irreversible process  <cit> .
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  <cit> .
therefore, mitochondria become a crucial target to modulate cell death in several models.
based on the following facts:  co is an anti-apoptotic molecule in several hepatic models, hepatocytes and/or liver and  mitochondria are central executers of cell death process, via the mitochondrial membrane permeabilization ; we explored the direct effect of co into isolated liver mitochondria  and the involvement of ros in this process.
mmp was assessed by mitochondrial depolarization, inner membrane permeabilization and mitochondrial swelling.
RESULTS
assessment of co toxicity and establishment of optimal co concentration in isolated liver mitochondria
in order to evaluate the toxicity of carbon monoxide  on isolated liver mitochondria, swelling and depolarization assays were performed with different doses of co .
up to  <dig> μm and for  <dig> minutes, co is not able to trigger mitochondrial swelling.
however co triggers swelling at concentrations of  <dig> and  <dig> μm , indicating that at higher concentrations this gas induces mitochondrial damage.
because mitochondrial depolarization is an earlier event compared to swelling, lower concentrations of co have induced lost of ΔΨm, only at  <dig> μm there is no depolarization induction .
pre-treatment with  <dig>   <dig> or  <dig> μm of co for  <dig> minutes partially prevents mitochondrial swelling induced by calcium .
since  <dig> μm presents the highest protection and does not induce mitochondrial depolarization, this concentration is the optimal one to be used in all other assays.
co inhibits mitochondrial membrane permeabilization  in isolated liver mitochondria
co partially inhibits mitochondrial swelling  when liver isolated mitochondria were exposed to  <dig> μm of co during  <dig> minutes at room temperature prior to addition of calcium to induce mmp  and of atractyloside, a ligand of ant that prevents adp/atp translocation and induces its pore forming function   <cit> .
swelling quantification analysis was performed at different time points for calcium or atractyloside because of their different mode of action and kinetics .
loss of ΔΨm, or mitochondrial depolarization, induced by atractyloside or calcium was also prevented by prior addition of co at  <dig> μm  and quantification analysis was done  for calcium  and for atractyloside .
changes in the inner membrane permeability  were assessed by an enzymatic assay based on the accessibility of citrate synthase, which is a soluble matrix enzyme  <cit> .
the atractyloside induction of inner membrane permeabilization is partially prevented by co .
due to the nature of this enzymatic assay, the quantification of the effect has to be done by calculating the slopes of the different curves over time, which were normalised relatively to ca2+  <dig> μm .
co pre-treatment decreases the slope compared to ca2+  <dig> μm  and atra  <dig> μm .
this reduction demonstrates co delay in the induction of inner membrane permeabilization.
taken together, co acts directly on mitochondria in order to limit their membrane permeabilization, shown by its prevention of swelling, depolarization and inner membrane permeabilization.
inner membrane permeabilization is followed by absorbance at  <dig> nm.
the slope was calculated between  <dig> to  <dig> minutes for each different condition in order to assess the effect of co in the inner membrane permeabilization after treatment with ca2+ or atractyloside.
for slope calculation, absorbance values for each different condition were normalized by the absorbance value corresponding to the control non-treated mitochondria.
the difference between control mitochondria and ca2+ -treated mitochondria slopes correspond to an increase of 100%.
ros are important molecules for co prevention of mmp in liver mitochondria
mitochondrial generated reactive oxygen species  are described as imperative signalling molecules for co biological functions  <cit> .
thus, the role of ros in co-induced protection in isolated liver mitochondria was also evaluated.
first, it was verified whether co increases ros generation in isolated liver mitochondria in a dose-response manner and this generation is limited by pre-treatment with ß-carotene .
to disclose ros role in this system, their level augmentation was prevented by treating mitochondria with ß-carotene prior to co addition and mmp induction.
indeed, co inhibition of mitochondrial swelling , depolarization  and inner membrane permeabilization  were, at least, partially prevented by the anti-oxidant ß-carotene.
thus, at mitochondrial level, ros appear to be important signalling molecules for co mode of action during inhibition of mitochondrial membrane permeabilization.
inner membrane permeabilization is followed by absorbance at  <dig> nm.
the slope was calculated between  <dig> to  <dig> minutes for each different condition in order to assess the effect of co in the inner membrane permeabilization in the presence or absence of ß-carotene and after treatment with ca2+.
for slope calculation, absorbance values for each different condition were normalized by the absorbance value corresponding to the control non-treated mitochondria.
the difference between control mitochondria and ca2+ -treated mitochondria slopes correspond to an increase of 100%.
low concentrations of co transiently prevents cytochrome c oxidase  activity
major intracellular sources of ros are the oxidative phosphorilation complexes.
moreover, accordingly to the literature, co is described to prevent cytochrome c oxidase  activity  <cit> .
thus, co direct effect on the cox activity was assessed in isolated liver mitochondria.
co at  <dig> μm transiently prevents cox activity up to  <dig> minutes, while  <dig> minutes later the effect is inverted; co slightly accelerates cox activity .
discussion
carbon monoxide has been described to be involved in protection of hepatocytes against cell death.
kim and colleagues  <cit>  have demonstrated that co decreases bcl- <dig> family proteins translocation into mitochondria, limiting cytochrome c release into the cytosol  <cit> .
despite the crucial role of mitochondria in cell death control and the potent anti-apoptotic property of co in hepatocytes, the direct effect of co in isolated liver mitochondrial membrane permeabilization has never been reported before.
herein it is shown that low doses of this gaseous molecule prevent mitochondrial membrane permeabilization.
recently, we have shown that co limits mitochondrial membrane permeabilization in non-synaptic mitochondria isolated from rat brain cortex  <cit> , which was accompanied by inhibition of cytochrome c release from mitochondria and by glutathionylation of adenine nucleotide translocase .
in this cerebral model, co protects astrocytes against cell death and ros generation appears to be important for this pathway  <cit> .
furthermore, in other systems, it is generally recognized that several co biological functions are dependent on mitochondrial ros generation and signalling  <cit> .
in the present work, ros also emerge as significant molecules involved in the signal transduction at the mitochondrial sub-cellular level.
inhibition of mitochondrial ros generation by an anti-oxidant addition  reverses co prevention of liver mitochondrial membrane permeabilization , which confirms their key role.
still one might hypothesize that ros promote post-translational modifications on mitochondrial proteins, as described for ant glutathionylation in non-synaptic mitochondria  <cit> .
the most accepted hypothesis for co-induced mitochondrial ros production is via partial inhibition of cytochrome c oxidase, accumulating electrons at complex iii level.
the generated anion superoxide is rapidly converted into hydrogen peroxide  <cit> .
according to our results, and using low concentrations of co , cox inhibition occurs only up to  <dig> minutes after co treatment .
one might speculate that this transient inhibition assures sufficient ros generation to signal protective pathways, although not enough to induce damage.
on the other hand, after  <dig> minutes cox activity is enhanced by co treatment .
interestingly it is in accordance to our previous data showing an increase on atp/adp translocase activity of ant  <cit>  or a mitochondrial hyperpolarization by low concentrations of co  <cit> .
in summary, low doses of co appear to accelerate mitochondrial oxidative phosphorylation and oxygen consumption.
another hypothesis to be considered is whether transient inhibition of cox activity also decreases calcium uptake protecting mitochondria against mmp.
further studies are needed to elucidate the mechanisms implicated in ros signalling, in particular how co modifies and/or accelerates mitochondrial oxidative phosphorylation and oxygen consumption.
CONCLUSIONS
thus, for the first time, it was demonstrated that co inhibits mmp in isolated liver mitochondria, by preventing mitochondrial swelling, mitochondrial depolarization and the opening of a non-specific pore through inner membrane.
additionally, small amounts of ros generation are essential for signalling mmp inhibition by co. in conclusion, it can be hypothesized that part of the co's anti-apoptotic property in hepatocytes and/or liver is due to its capacity to limit mitochondrial membrane permeabilization, preventing the release of pro-apoptotic factors into the cytosol.

 carbon monoxide 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. furthermore, co is an endogenous product of heme degradation by heme-oxygenase, 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. in hepatocytes and/or liver models, co appears to act as an anti-apoptotic molecule. by stimulating atp production, co activates p mapk signalling, preventing apoptosis in human hepatocytes. co rescues mice from fulminant hepatitis, presenting a marked reduction of tnf-alpha-induced apoptosis or via no generation. in primary cultures of rat hepatocytes, co limits cytotoxicity induced by glucose deprivation through suppression of erk mapk activation. in an endotoxic shock model, co protects hepatocytes from apoptosis by augmenting inos expression. it is also described that superoxide anion-induced apoptosis is inhibited by co via limiting jnk activity. co treatment protects hepatocytes from cell death by inducing nf-kb activation, which is dependent on ros generation, since inhibition of ros generation reverses co-induced cytoprotection. among all publications showing co as anti-apoptotic molecule in hepatic model, only kim and colleagues 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. 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 these factors are released into the cytosol and cell death becomes an irreversible process. 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. therefore, mitochondria become a crucial target to modulate cell death in several models. based on the following facts: co is an anti-apoptotic molecule in several hepatic models, hepatocytes and/or liver and mitochondria are central executers of cell death process, via the mitochondrial membrane permeabilization; we explored the direct effect of co into isolated liver mitochondria and the involvement of ros in this process. mmp was assessed by mitochondrial depolarization, inner membrane permeabilization and mitochondrial swelling.  assessment of co toxicity and establishment of optimal co concentration in isolated liver mitochondria in order to evaluate the toxicity of carbon monoxide on isolated liver mitochondria, swelling and depolarization assays were performed with different doses of co. up to μm and for minutes, co is not able to trigger mitochondrial swelling. however co triggers swelling at concentrations of and μm, indicating that at higher concentrations this gas induces mitochondrial damage. because mitochondrial depolarization is an earlier event compared to swelling, lower concentrations of co have induced lost of ΔΨm, only at μm there is no depolarization induction. pre-treatment with or μm of co for minutes partially prevents mitochondrial swelling induced by calcium. since μm presents the highest protection and does not induce mitochondrial depolarization, this concentration is the optimal one to be used in all other assays. co inhibits mitochondrial membrane permeabilization in isolated liver mitochondria co partially inhibits mitochondrial swelling when liver isolated mitochondria were exposed to μm of co during minutes at room temperature prior to addition of calcium to induce mmp and of atractyloside, a ligand of ant that prevents adp/atp translocation and induces its pore forming function. swelling quantification analysis was performed at different time points for calcium or atractyloside because of their different mode of action and kinetics. loss of ΔΨm, or mitochondrial depolarization, induced by atractyloside or calcium was also prevented by prior addition of co at μm and quantification analysis was done for calcium and for atractyloside. changes in the inner membrane permeability were assessed by an enzymatic assay based on the accessibility of citrate synthase, which is a soluble matrix enzyme. the atractyloside induction of inner membrane permeabilization is partially prevented by co. due to the nature of this enzymatic assay, the quantification of the effect has to be done by calculating the slopes of the different curves over time, which were normalised relatively to ca2+ μm. co pre-treatment decreases the slope compared to ca2+ μm and atra μm. this reduction demonstrates co delay in the induction of inner membrane permeabilization. taken together, co acts directly on mitochondria in order to limit their membrane permeabilization, shown by its prevention of swelling, depolarization and inner membrane permeabilization. inner membrane permeabilization is followed by absorbance at nm. the slope was calculated between to minutes for each different condition in order to assess the effect of co in the inner membrane permeabilization after treatment with ca2+ or atractyloside. for slope calculation, absorbance values for each different condition were normalized by the absorbance value corresponding to the control non-treated mitochondria. the difference between control mitochondria and ca2+ -treated mitochondria slopes correspond to an increase of 100%. ros are important molecules for co prevention of mmp in liver mitochondria mitochondrial generated reactive oxygen species are described as imperative signalling molecules for co biological functions. thus, the role of ros in co-induced protection in isolated liver mitochondria was also evaluated. first, it was verified whether co increases ros generation in isolated liver mitochondria in a dose-response manner and this generation is limited by pre-treatment with ß-carotene. to disclose ros role in this system, their level augmentation was prevented by treating mitochondria with ß-carotene prior to co addition and mmp induction. indeed, co inhibition of mitochondrial swelling, depolarization and inner membrane permeabilization were, at least, partially prevented by the anti-oxidant ß-carotene. thus, at mitochondrial level, ros appear to be important signalling molecules for co mode of action during inhibition of mitochondrial membrane permeabilization. inner membrane permeabilization is followed by absorbance at nm. the slope was calculated between to minutes for each different condition in order to assess the effect of co in the inner membrane permeabilization in the presence or absence of ß-carotene and after treatment with ca2+. for slope calculation, absorbance values for each different condition were normalized by the absorbance value corresponding to the control non-treated mitochondria. the difference between control mitochondria and ca2+ -treated mitochondria slopes correspond to an increase of 100%. low concentrations of co transiently prevents cytochrome c oxidase activity major intracellular sources of ros are the oxidative phosphorilation complexes. moreover, accordingly to the literature, co is described to prevent cytochrome c oxidase activity. thus, co direct effect on the cox activity was assessed in isolated liver mitochondria. co at μm transiently prevents cox activity up to minutes, while minutes later the effect is inverted; co slightly accelerates cox activity. discussion carbon monoxide has been described to be involved in protection of hepatocytes against cell death. kim and colleagues have demonstrated that co decreases bcl- family proteins translocation into mitochondria, limiting cytochrome c release into the cytosol. despite the crucial role of mitochondria in cell death control and the potent anti-apoptotic property of co in hepatocytes, the direct effect of co in isolated liver mitochondrial membrane permeabilization has never been reported before. herein it is shown that low doses of this gaseous molecule prevent mitochondrial membrane permeabilization. recently, we have shown that co limits mitochondrial membrane permeabilization in non-synaptic mitochondria isolated from rat brain cortex, which was accompanied by inhibition of cytochrome c release from mitochondria and by glutathionylation of adenine nucleotide translocase. in this cerebral model, co protects astrocytes against cell death and ros generation appears to be important for this pathway. furthermore, in other systems, it is generally recognized that several co biological functions are dependent on mitochondrial ros generation and signalling. in the present work, ros also emerge as significant molecules involved in the signal transduction at the mitochondrial sub-cellular level. inhibition of mitochondrial ros generation by an anti-oxidant addition reverses co prevention of liver mitochondrial membrane permeabilization, which confirms their key role. still one might hypothesize that ros promote post-translational modifications on mitochondrial proteins, as described for ant glutathionylation in non-synaptic mitochondria. the most accepted hypothesis for co-induced mitochondrial ros production is via partial inhibition of cytochrome c oxidase, accumulating electrons at complex iii level. the generated anion superoxide is rapidly converted into hydrogen peroxide. according to our results, and using low concentrations of co, cox inhibition occurs only up to minutes after co treatment. one might speculate that this transient inhibition assures sufficient ros generation to signal protective pathways, although not enough to induce damage. on the other hand, after minutes cox activity is enhanced by co treatment. interestingly it is in accordance to our previous data showing an increase on atp/adp translocase activity of ant or a mitochondrial hyperpolarization by low concentrations of co. in summary, low doses of co appear to accelerate mitochondrial oxidative phosphorylation and oxygen consumption. another hypothesis to be considered is whether transient inhibition of cox activity also decreases calcium uptake protecting mitochondria against mmp. further studies are needed to elucidate the mechanisms implicated in ros signalling, in particular how co modifies and/or accelerates mitochondrial oxidative phosphorylation and oxygen consumption.  thus, for the first time, it was demonstrated that co inhibits mmp in isolated liver mitochondria, by preventing mitochondrial swelling, mitochondrial depolarization and the opening of a non-specific pore through inner membrane. additionally, small amounts of ros generation are essential for signalling mmp inhibition by co. in conclusion, it can be hypothesized that part of the co's anti-apoptotic property in hepatocytes and/or liver is due to its capacity to limit mitochondrial membrane permeabilization, preventing the release of pro-apoptotic factors into the cytosol. 
interestingly, cytochrome c oxidase transiently responds to low concentrations of co by decreasing its activity in the first min, later on there is an increase of cytochrome c oxidase activity, which were detected up to min. mitochondria are key organelles in cell death control via their membrane permeabilization and the release of pro-apoptotic factors. direct and indirect approaches were used to evaluate mmp inhibition by co: mitochondrial swelling, mitochondrial depolarization and inner membrane permeabilization. herein, we show that co prevents mitochondrial membrane permeabilization in liver isolated mitochondria. co directly prevents mitochondrial membrane permeabilization, which might be implicated in the hepatic apoptosis inhibition by this gaseoustransmitter. additionally, co increases mitochondrial reactive oxygen species generation, and their scavenging, by ß-carotene addition, decreases co protection, which reveals the key role of ros. low concentrations of carbon monoxide protect hepatocytes against apoptosis and confers cytoprotection in several models of liver.
 low concentrations of carbon monoxide protect hepatocytes against apoptosis and confers cytoprotection in several models of liver. mitochondria are key organelles in cell death control via their membrane permeabilization and the release of pro-apoptotic factors.  herein, we show that co prevents mitochondrial membrane permeabilization in liver isolated mitochondria. direct and indirect approaches were used to evaluate mmp inhibition by co: mitochondrial swelling, mitochondrial depolarization and inner membrane permeabilization. additionally, co increases mitochondrial reactive oxygen species generation, and their scavenging, by ß-carotene addition, decreases co protection, which reveals the key role of ros. interestingly, cytochrome c oxidase transiently responds to low concentrations of co by decreasing its activity in the first min, later on there is an increase of cytochrome c oxidase activity, which were detected up to min.  co directly prevents mitochondrial membrane permeabilization, which might be implicated in the hepatic apoptosis inhibition by this gaseoustransmitter. 