Hydrogen sulfide and the liver.

Nitric Oxide xxx (2014) xxx–xxx

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Nitric Oxide journal homepage: www.elsevier.com/locate/yniox

Hydrogen sulfide and the liver Sarathi Mani a,b, Wei Cao b,c,d, Lingyun Wu b,c,e, Rui Wang a,b,⇑ a

Department of Biology, Lakehead University, Thunder Bay, Canada Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, Canada c Thunder Bay Regional Research Institute, Thunder Bay, Canada d Department of Natural Medicine & Institute of Materia Medica, Fourth Military Medical University, Xi’an, China e Department of Health Sciences, Lakehead University, Thunder Bay, Canada b

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Hydrogen sulfide Liver Diabetes Lipids Cirrhosis Oxidative stress

a b s t r a c t Hydrogen sulfide (H2S) is a gasotransmitter that regulates numerous physiological and pathophysiological processes in our body. Enzymatic production of H2S is catalyzed by cystathionine c-lyase (CSE), cystathionine b-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MST). All these three enzymes present in the liver and via H2S production regulate liver functions. The liver is the hub for metabolism of glucose and lipids, and maintains the level of circulatory lipids through lipoprotein metabolism. Hepatic H2S metabolism affects glucose metabolism, insulin sensitivity, lipoprotein synthesis, mitochondrial biogenetics and biogenesis. Malfunction of hepatic H2S metabolism may be involved in many liver diseases, such as hepatic fibrosis and hepatic cirrhosis. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Hydrogen sulfide (H2S) is a gasotransmitter that is synthesized in mammalian cells, and exerts a regulatory impact on many physiological and pathophysiological processes [1]. Mammalian cells generate H2S through either non-enzymatic or enzymatic mechanisms. The non-enzymatic generation of H2S involves phosphogluconate pathway in erythrocytes and the reduction of elemental sulfur produced from the reducing equivalents of oxidized glucose during glycolysis [2,3]. Most significant to endogenous H2S level is the enzymatic H2S production [1,4–6]. Cystathionine c-lyase (CSE), cystathionine b-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MST) in concert with cysteine aminotransferase (CAT) are responsible for the synthesis of endogenous H2S (Fig. 1). These enzymes all use cysteine and/or homocysteine or their derivatives as the substrates [1,7,8]. The distribution of H2Sproducing enzymes is tissue specific. For example, CSE is the predominant H2S-producing enzyme in the cardiovascular system [9–11], the liver and kidney [12–14], the pancreas [15], vascular smooth muscle cells (VSMC) [16–18], and the respiratory system [19,20]. CSE mRNA has been detected in the brain [21]. However, CSE inhibitors, DL-propargylglycine (PPG) and b-cyano-L-alanine ⇑ Corresponding author at: Office of Vice President, (Research, Economic Development and Innovation), Lakehead University, 955 Oliver Road, Thunder Bay, Ontario P7B5E1, Canada. Fax: +1 (807) 766 7105. E-mail address: [email protected] (R. Wang).

(BCA), had no effect on brain production rate of H2S [22], while they suppressed H2S generation in the liver and kidney [23]. Unlike CSE, CBS expression is rare in the cardiovascular system. CBS is the predominant H2S-producing enzyme in the central nervous system or neurons [1], highly expressed in the hippocampus and cerebellum in the brain [22,24]. CSE, CBS, and MST proteins have been detected in the liver, and they contribute to liver production of H2S to different extents. In order to maintain proper physiological levels of H2S, our body eliminates H2S through oxidation, methylation, scavenging and expiration [1,8,11] (Fig. 1). Oxidation and methylation are the most important mechanisms for H2S elimination. Mitochondria first oxidize H2S to thiosulfate, then to sulfite or sulfate, which are excreted by the kidney in urine. Whereas the methylation of H2S takes place in the cytosol, oxidation in mitochondria is much faster than methylation [25]. In addition, methemoglobin and some other proteins scavenge H2S in the blood and tissues. Lastly, H2S is exhaled through the lung. In healthy individuals, very little H2S is eliminated through the lung as alveolar air only contains 25–50 ppb H2S [26,27]. The liver plays a key role in glucose and lipid metabolism, xenobiotic metabolism, and antioxidant defence. Hepatic H2S metabolism affects glucose metabolism, insulin sensitivity, lipoprotein synthesis, mitochondrial bioenergetics and biogenesis. Malfunction of hepatic H2S metabolism may be involved in the pathogenesis of many liver diseases, such as hepatic fibrosis and cirrhosis.

http://dx.doi.org/10.1016/j.niox.2014.02.006 1089-8603/Ó 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: S. Mani et al., Hydrogen sulfide and the liver, Nitric Oxide (2014), http://dx.doi.org/10.1016/j.niox.2014.02.006

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S. Mani et al. / Nitric Oxide xxx (2014) xxx–xxx

Fig. 1. Endogenous production of H2S in the liver and its effects on liver functions. CAT: cysteine aminotransferase; CBS: cystathionine b-synthase; CSE: cystathionine c-lyase; GSH: reduced glutathione; H2S: hydrogen sulfide; 3-MP: 3-mercaptopyruvate; MST: 3-mercaptopyruvate sulfurtransferase; sulfHb: sulfhemoglobin; VLDL: Very low density lipoprotein.

Role of H2S in hepatic lipid metabolism As a key metabolic organ, the liver plays a vital role in various aspects of lipid metabolism through interacting with the intestinal tract and adipose tissue [28]. The liver synthesizes fatty acids and triglycerides from excess carbohydrates and proteins, which are either exported and stored in adipose tissue or oxidized by the liver itself to produce energy [29]. Also, the liver synthesizes large quantities of cholesterol, phospholipids and apoproteins, which are utilized for the transportation of lipids in the circulation in the form of lipoproteins to the rest of the body [30]. The excess lipids are catabolised and excreted by the liver to support whole-body lipid homeostasis. Liver dysfunction leads to deregulation of lipid metabolism such as alterations in fatty acid biosynthesis, beta-oxidation and very low density lipoprotein (VLDL) secretion, which subsequently changes serum concentrations of cholesterol and lipoproteins [31–33]. Fatty liver disease is an important causative factor of cardiovascular diseases and obesity-related disorders [34,35]. The liver is an important organ for H2S production and its clearance [1,3,8]. CSE and CBS abundantly present in the liver, involved in the endogenous production of H2S and its metabolism. CBS is important for normal liver function and its deficiency leads to diverse clinical disorders, especially fatty liver. The mouse with homozygous mutation of CBS exhibits hyperhomocysteinemia, oxidative stress, fibrosis and hepatic steatosis, the features shared by CBS deficient patients [36,37]. Moreover, CBS deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice [38]. CSE-KO mice have been developed in our laboratories [9]. These mice have a phenotype of age-dependent hypertension and hyperhomocysteinemia [9]. CSE-KO mice are indistinguishable

with wild-type (WT) mice in terms of liver morphology and function [39]. Both CBS and CSE expression levels and activities in the liver were significantly increased in WT mice fed with high fat diet [40]. Norris et al. had proposed that the liver is a key regulator of H2S levels by maintaining a high capacity for H2S clearance from circulation [41]. There is only limited information available on the impact of H2S on liver lipid metabolism. Namekata et al. reported abnormal lipid metabolism in CBS-KO mice as a consequence of hyperhomocysteinemia [42]. Serum total cholesterol in CBS-KO mice did not significantly differ from WT mice. Serum and liver triglyceride, non-esterified cholesterol and non-esterified fatty acid levels were significantly up-regulated in CBS-KO mice, whereas serum high density lipoprotein (HDL) and phospholipids in HDL were decreased. Namekata et al. further showed that impaired b-oxidation of fatty acid and decreased thiolase activity and VLDL secretion from the liver caused hepatic steatosis in CBS-KO mice [42]. Unfortunately, liver H2S level was unknown in this study and its correlation with abnormal lipid metabolism and subsequent steatosis development could not be determined [42]. Zhou et al. reported that hyperhomocysteinemia in atherogenic dietfed C57BL/6 J mice did not independently cause dyslipidemia [43]. Therefore, abnormal lipid metabolism in CBS-KO mice could not be caused solely by hyperhomocysteinemia. We recently discovered that under physiological conditions CBS proteins exist in both the cytosol and mitochondria of hepatocytes. Under stress conditions, CBS proteins are accumulated in the mitochondrion to produce more H2S in hepatocytes [6]. In addition to CBS, CSE is an important enzyme for H2S production in the liver. We recently reported the impact of altered endogenous H2S levels on plasma lipid metabolism in mice fed with high-fat atherogenic diet



[10]. CSE-KO mice on atherogenic diet showed increased plasma total cholesterol and low density lipoprotein (LDL) cholesterol levels with decreased HDL cholesterol, and early development of atherosclerosis when compared to WT mice [10]. Furthermore, we treated these atherogenic diet-fed CSE-KO mice with sodium hydrosulfide (NaHS), which improved their plasma lipid profile and decreased atherosclerotic lesions [10]. Although CSE-KO mice did not show hepatic steatosis or other pathologic features in the liver on regular chow diet feeding [9,39], abnormal lipid metabolism in the liver can be postulated based on the observed abnormal plasma lipid profile [10]. Jain et al. examined fasting blood levels of H2S and lipids in healthy human subjects, and showed a significantly positive correlation between H2S and HDL-cholesterol and negative correlation between H2S and LDL/HDL-cholesterol ratio [44]. Our previous study also recorded a negative correlation between endogenous H2S and mouse plasma LDL levels [10]. Plasma LDL levels were significantly increased in CSE-KO mice, and administration of NaHS to these animals decreased plasma LDL levels [10]. Statins have been used to treat hypercholesterolemia through the inhibition of liver 3-hydroxy-3-methyl-glutaryl-CoA (HMGCoA) reductase, the rate limiting enzyme in cholesterol biosynthesis pathway. Hypercholesterolemia has been associated with fatty liver and cardiovascular diseases. Wójcicka et al. examined differential effects of statins on endogenous H2S formation in perivascular adipose tissue and liver of male Wistar rats [45]. Pravastatin and atorvastatin treatment of Wistar rats increased liver H2S production by 52% and 71%, respectively. In addition to H2S production, pravastatin and atorvastatin also reduced the oxidation of H2S in the liver by 46% and 53%, respectively [45]. Ubiquinone is a co-factor of sulfide:quinineoxidoreductase (SQR), a mitochondrial H2S oxidation enzyme. The statin-suppressed H2S oxidation is due to the depletion of ubiquinone since the addition of exogenous synthetic ubiquinone (30 lM) to isolated mitochondria markedly increased H2S oxidation. These results suggest that cardiovascular protection of statins is executed not only by reduction in plasma LDL-cholesterol levels, but also by increased endogenous H2S level to normalize liver lipid metabolism (Fig. 1). Garlic is a known folk remedy for various health issues, including liver diseases [46]. Polysulfides contained in garlic can be converted to H2S by human erythrocytes through non-enzymatic reactions [2]. Diallyl trisulfide, a garlic derived organic polysulfide compound that acts as an H2S donor, is commercially available, and its vasoactivity has been proven [47]. Zeng et al. showed that garlic oil containing 31.1% diallyl disulfide and 29.3% diallyl trisulfide ameliorated ethanol-induced hepatic steatosis by modulating sterol regulatory element-binding transcription factor 1 (SREBP1), peroxisome proliferator activated receptor alpha (PPARa) and cytochrome P450 2E1 (CYP2E1) [48]. Garlic oil treatment of mice significantly reduced fat infiltration into liver and plasma aminotransferase activities and prevented the development of ethanolinduced fatty liver disease [48]. These findings indicate that endogenous H2S or H2S donor supplement has the potential to preserve normal lipid metabolism in physiological or pathophysiological conditions. The mechanisms by which H2S shapes lipoprotein profile and controls liver lipid metabolism, however, remain unclear.

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coneogenesis in the liver [49]. The failure of insulin to trigger above-mentioned actions in liver cells is defined as hepatic insulin resistance [50], which is a common feature of type 2 diabetes and metabolic syndrome. Yusuf et al. showed that hepatic H2S level in streptozotocin-induced diabetic rats was significantly higher than non-diabetic controls [51]. Conversely, mice treated with metformin (100 mg/kg), the mostly prescribed insulin sensitizer for type 2 diabetes, significantly increased liver H2S production rate [52]. Chang et al. has demonstrated that H2S interacted with methylglyoxal, an intermediate of glucose metabolism [16]. This interaction would affect the formation of advanced glycation end product and the development of insulin resistance [16]. Only recently the role of H2S in hepatic glucose metabolism has been explored in details in our laboratory [53]. In this study, we incubated HepG2 cells with 500 nM insulin for 24 h in the presence of high concentration of glucose (33 mM) to develop a hepatic insulin resistance cellular model. Using this model, we found that NaHS significantly impaired basal and insulin-stimulated glucose uptake and glycogen storage, and enhanced gluconeogenesis and glycogenolysis. The inhibitory effect of endogenous H2S on glycogen storage has been also demonstrated since adenovirus-mediated CSE overexpression in HepG2 cells reduced glycogen content but knockdown of CSE expression with siRNA or the application of PPG increased glycogen content. In accordance with these in vitro results, glycogen content was significantly higher in liver tissues from CSE-KO mice compared to that from WT mice. Together, these data demonstrate that H2S is a novel endogenous inhibitor for glucose consumption and storage in the liver. H2S down-regulated glucose uptake and glycogen storage are mediated by decreased activation of AMPK and glucokinase. On the other hand, H2S-increased glucose production might be due to increased activity of phosphoenolpyruvate carboxykinase (Fig. 2). At the physiological concentration range,

Role of H2S in hepatic glucose metabolism The sophisticated homeostatic control of glucose metabolism in the liver is primarily directed by insulin and glucagon. As a hormone with extensive effects on the whole body metabolism, insulin promotes glucose disposal in adipose tissue and muscles, and prevents glucose production by inhibiting glycogenolysis and glu-

Fig. 2. Molecular mechanisms for H2S-mediated hepatic glucose metabolism. H2S down-regulates glucose uptake by inactivating AMPK and glucokinase. H2S also increases glucose production by inhibiting Akt and activating phosphoenolpyruvate carboxykinase. Insulin at physiological level increases glucose uptake and downregulates glucose production through the inhibition of endogenous production of H2S by CSE. Once in the state of insulin resistance, insulin stimulates CSE expression and endogenous H2S production.


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insulin inhibited CSE expression and decreased phosphorylation of Akt in HepG2 cells. However, in the insulin resistance state after exposing cells to high levels of insulin (500 nM) and glucose (33 mM), CSE expression in hepatocytes was increased [53]. This bi-phasic regulation of CSE expression by insulin is further elucidated in Fig. 2. Based on these observations, one would predict that the inhibition of hepatic H2S biosynthesis or CSE activity may be an organ-specific approach to attenuate the progress of diabetes and its complications. The inhibition of hepatic and pancreatic H2S biosynthesis would increase insulin sensitivity and insulin release from these organs [15], respectively, during the induction phase of diabetes. Supplementation/donation of H2S, on the other hand, may be a therapeutic avenue to protect against the development of cardiovascular complications of diabetes such as diabetic vasculopathy, nephropathy, and cardiomyopathy [54]. Role of H2S in hepatic oxidative stress Mammalian cells produce reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion (O2 ), singlet oxygen molecules and hydroxyl radicals when needed for regulating innate immune defense, apoptosis, anti-inflammation, cell growth and cell–cell interaction. In this sense, ROS serve as signaling molecules [55,56]. High concentration of ROS, however, creates oxidative stress [57–59]. An imbalance between antioxidants and ROS would cause oxidative damage to lipids, proteins, and DNA [59]. H2S is increasingly being recognized as an important antioxidant. It has been shown that H2S prevented oxidative modification of LDL through a mechanism involving the reduction of lipid hydroperoxide content [60,61], and inhibited hyperglycemia-induced intrarenal renin-angiotensin system activation via attenuation of ROS generation [62]. Endogenous H2S deficiency leads to decreased glutathione peroxidase and glutathione reductase, resulting in reduced glutathione (GSH) biosynthesis from glutathione disulfide [10]. Osborne et al. also reported that H2S-releasing derivative of aspirin (ACS14) stimulates GSH production to overcome glutamate mediated oxidative stress [63]. H2S maintains intracellular GSH levels by decreasing the activities of GSH catabolizing enzymes [1]. Oxidative stress affects liver functions. Elevated production of ROS and decreased antioxidants are implicated in hepatic insulin resistance, fatty liver, and liver fibrosis [64–66]. The roles of H2S in the regulation of oxidative status have been reported in several cell types including hepatocytes [59]. NaHS treatment of hepatocytes significantly elevated GSH levels; and decreased ROS levels, cyclooxygenase (COX) expression, CYP2E1 activity and lipid peroxidation [67–69]. H2S stimulated nuclear translocation and binding of NFE2p45-related factor 2 (Nrf2) with the antioxidant gene promoters, glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and GSH reductase [70]. In mouse embryonic fibroblast (MEF) from CSE-KO mice, oxidative stress level was higher than that of MEF from WT mice, which was rescued with NaHS treatment [70]. Nrf2 is also involved in the production H2S via the regulation of CBS and CSE expressions [71]. Treatment of WT MEFs with NaHS increased mRNA levels of CBS by 2.2-fold and that of CSE by 3.2-fold, whereas this increase in CBS and CSE expression was not apparent in Nrf2-deficient MEFs [71]. These studies showed that the antioxidant effect of H2S is related to the dissociation of Kelch-like ECH-associated protein-1 (Keap1) from Nrf2, which results in Nrf2 translocation and increased expression of cytoprotective genes in association with oxidative stress. All three H2S producing enzymes (CBS, CSE, and MST) are involved in the regulation of hepatic oxidative stress [24,37,39,72,73]. Robert et al. studied the expression of various oxidative stress genes in the liver of CBS-KO mice using atlas mouse stress cDNA expression membranes [24]. CBS deficiency increased the expression of heme oxygenase-1 and CYP7B1, and

decreased the expression and activity of paraoxonase/arylesterase 1 (PON1) [24]. PON1, a protein component of HDL, destroys biologically active lipids in oxidized LDL to prevent oxidized LDL buildup. CBS-KO mice exhibited 30% of increased protein oxidation and lipid peroxidation. Their liver injury was increased about 50 folds due to mitochondrial damage in association with hepatic stellate cells (HSC) activation [37]. Whether exogenous H2S supplementation would reverse this increased oxidative stress and liver injury in CBS-KO mice was not further explored. Recently, we reported that CSE-KO mice fed with high fat diet exhibited hyperhomocysteinemia with reduced plasma total thiol levels, GSH, superoxide dismutase (SOD) activity and increased plasma malondialdehyde levels [10]. Reduced liver GSH levels in CSE-KO mice, even in the absence of dietary manipulation, further reveals the importance of CSE/H2S pathway [10]. NaHS supplement to CSE-KO mice significantly increased plasma GSH, total thiol and SOD activity, and decreased plasma malondialdehyde levels [10]. It would be interesting to further investigate whether the supplementation of H2S also rescues the liver from increased oxidative stress in CSE-KO mice. Lipid peroxidation is the most important causative factor for hepatic damage. Lipid peroxidation produces several cytotoxic degradation products such as malondialdehyde and 4-hydroxynonenal [24,37,74–76]. CBS-KO mice had higher malondialdehyde and 4-hydroxynonenal levels when compared to WT controls, which leads to increased carbonyl formation in the liver [37]. Increased carbonyl formation is an indicator of oxidative stress. Single dose of acetaminophen (650 mg/kg, i.p.) to male Swiss mice induced hepatotoxicity. NaHS supplementation to acetaminophentreated mice significantly decreased serum alanine aminotransferase (ALT) and hepatic malondialdehyde with a concurrent increase in hepatic GSH content [77]. We also previously reported that NaHS treatment significantly decreased plasma lipid peroxidation in CSE-KO mice [10]. Hence, H2S could be hepatoprotective against oxidative stress-induced liver damage. Raw garlic homogenates or garlic oil corrected the elevated malondialdehyde level and decreased GSH level in diabetic rat liver [78] or ethanol-induced mouse fatty liver [48]. In contrast to the reported antioxidant role of H2S, treatment of rat primary hepatocytes with 500 lM of NaHS increased ROS formation through the depletion of GSH and the inhibition of cytochrome c oxidase in electron transport chain [79]. These results suggest that H2S is protective against hepatic oxidative stress at low concentrations but may become detrimental at high concentrations. Oxidative stress itself also lowered endogenous production of H2S. Modis et al. investigated the effect of H2O2-induced oxidative stress on the activity of MST to produce H2S from 3-mercaptopyruvate (3-MP) supplementation. They used mouse recombinant MST enzyme and the mitochondrial preparations isolated from cultured murine hepatoma cells. H2O2 treatment concentration-dependently decreased the activity of recombinant mouse MST and mitochondrial H2S production from 3-MP [73]. The authors hypothesized that this inhibitory effect of H2O2 on the H2S synthesizing activity of MST may result from the oxidative modification of MST [73]. Hyperhomocysteinemia, the blood homocysteine level being higher than 15 lM, is a well-known risk factor for cardiovascular diseases, hepatic fibrosis, and neuropsychiatric illness [36]. Hyperhomocysteinemia induced hepatic stellate cell proliferation and subsequent progression of hepatic fibrosis through activation of NAD(P)H oxidases and upregulation of p22phox expression and p47phox phosphorylation [80]. CSE-KO mice have a phenotype of hyperhomocysteinemia [9,11]. Challenging these mice with galactosamine/lipopolysaccharide induced acute liver failure and significantly decreased plasma and liver homocysteine levels. WT mice receiving the same challenge did not develop acute liver failure. The lack of CSE/H2S signaling in this animal model seems



promoting acute liver failure [81]. Whether the expression of CSE or CBS enzymes and their activities in the liver can be affected by galactosamine/lipopolysaccharide stimulation is unknown. Galactosamine decreased NO production in primary hepatocytes and caused NO deficiency-mediated hepatocytes toxicity [82]. Similar to galactosamine/lipopolysaccharide, carbon tetrachloride is also hepatotoxic. Carbon tetrachloride decreased endogenous production of H2S from rat livers in a dose- and time-dependent manner [69,83]. Role of H2S in hepatic mitochondrial bioenergetics The mitochondrion plays a critical role in energy production, ROS generation, b-oxidation of fatty acid and regulation of calcium homeostasis [84–87]. At toxic concentrations H2S inhibits cytochrome c oxidase (a key component of mitochondrial respiratory chain complex IV), decreases ATP synthesis, and increases ischemia/reperfusion injury [67]. Exogenous administration of H2S significantly ameliorated myocardial and hepatic ischemia/ reperfusion injury due to the preservation of mitochondrial function [67,88]. These observations confirm that, similar to NO, H2S at physiological levels exerts protective effects on mitochondrial function, oxidative phosphorylation, and cellular bioenergetics [73,89–91]. Traditionally, it was believed that H2S is produced in the cytosol and consumed through oxidation in the mitochondrion [92]. Permeable nature of H2S to lipid bilayer makes this hypothesis conceivable. We recently reported that CSE proteins in the cytosol of vascular smooth muscle cells were translocated into mitochondria upon hypoxia or calcium stress, aided by Tom20 [33]. This translocation facilitates metabolism of cysteine

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and H2S production in the mitochondrion, maintaining ATP production under hypoxia or other stress conditions (Fig. 3). On the other hand, CBS proteins, but not CSE proteins, present in liver mitochondria under normal resting conditions [6]. Under stress condition, CBS proteins are accumulated in mitochondria due to their decreased degradation [6,89] (Fig. 3). CBS protein levels in liver mitochondria are constitutively regulated by Lon-protease in a heme-based and oxygen-sensitive fashion [6]. Ischemia/ hypoxia significantly increased mitochondrial CBS protein levels, which subsequently leads to increased mitochondrial H2S production to prevent Ca2+ induced cytochrome c release from mitochondria and mitochondrial swelling [6]. Hypoxia contributes to hepatic injury through increased production of ROS [93,94]. Our study revealed that mitochondrial H2S produced by accumulated CBS ameliorated ROS generation and increased ATP production during hypoxia [6]. Treatment of cultured murine hepatoma cell line Hepa1c1c7 with 10–100 nM 3-MP, the substrate of MST, stimulated mitochondrial H2S production and enhanced mitochondrial electron transport and cellular bioenergetics [95]. The stimulating effect of 3-MP on mitochondrial bioenergetics was abolished by silencing MST with gene specific siRNA (Fig. 3) [72]. These results confirmed that in addition to CBS and CSE, MST also plays a physiological role in the maintenance of mitochondrial electron transport and cellular bioenergetics. Role of H2S in hepatic fibrosis and cirrhosis The principle forms of chronic liver diseases are chronic viral hepatitis, and alcoholic and non-alcoholic fatty liver disease

Fig. 3. Role of H2S in mitochondrial bioenergetics. CAT: cysteine aminotransferase; CBS: cystathionine b-synthase; CSE: cystathionine c-lyase; e : electron; GSH: reduced glutathione; H2O2: Hydrogen peroxide; HSP70: Heat shock protein 70; MST: 3-mercaptopyruvate sulfurtransferase; O2 : Super oxide anion; ROS: Reactive oxygen species.


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(NAFLD) [96–98]. All chronic liver diseases can lead to liver fibrosis, a scarring process that reflects liver’s response to injury [99]. NAFLD has been considered being a key cause of hepatic fibrosis. Like skin, of which the wound healing is constituted of the deposition of collagen and other matrix constituents, the liver also repairs its injury through the deposition of new collagen. Eventually, this process can disrupt the architectural organization of hepatic functional units and blood flow through the liver, which consequently leads to liver cirrhosis. Liver cirrhosis may cause several complications, including portal hypertension, liver failure, and hepatic carcinoma. Once liver cirrhosis has developed, the risk of liver cancer will be higher [100]. H2S participates in the regulation of hepatic physiological and pathophysiological conditions and its deficiency can lead to liver cirrhosis [101,102]. Several studies reported that carbon tetrachloride treatment in rats decreased hepatic CSE expression and endogenous H2S production, leading to liver cirrhosis and portal hypertension [69,83]. The effect of carbon tetrachloride on CSE expression is time dependent. Liver CSE expression and H2S production as well as serum H2S levels declined 4 weeks after carbon tetrachloride treatment and dropped even more after 8 and 12 weeks [69]. Significantly elevated ALT, aspartate aminotransferase (AST), and hyaluronic acid levels, and reduced level of albumin in the blood of carbon tetrachloride-treated 12-week rats were reversed by NaHS supplementation, but worsened by PPG treatment [69]. The protective effect of NaHS is related to its anti-oxidation, anti-inflammation, cytoprotection, and anti-fibrosis actions [69]. It should be noticed, however, that the effects of NaHS or other H2S donors may not truly represent that of authentic H2S or endogenous H2S. The release of H2S from its donors can vary and the delivered concentration may not be at the level as expected. It should also be cautious to interpret the effect of PPG and other inhibitors for H2S-generating enzymes since these inhibitors may have non-specific effects [1]. Majority of NAFLD patients developed cardiovascular diseases, type 2 diabetes, obesity, insulin resistance, dyslipidemia, or hypertension [103,104]. Feeding mice for 5 weeks with high-fat diet induced fatty liver with significantly increased expression of hepatic CSE and CBS mRNA and proteins [40]. Hepatic H2S production in these treated animals also increased. Increased CBS/CSE expressions and endogenous H2S production may be a compensative response to pathological stimuli. Robert et al. reported that CBS deficiency in mice on normal chow diet was associated with spontaneous fibrosis and steatosis, concomitant with an enhanced expression of metalloprotease-1, procollagen type-1, TGF-b1 and proinflammatory cytokines [37]. Another study on CBS-KO mouse liver showed that hepatocytes were enlarged and multinucleated with micro-vesicular lipid droplets, but no signs of obvious fibrosis was observed [36]. This observation was made on CBS-KO mice of 3–8 weeks old. In contrast, Robert et al. found liver steatosis in 8– 32-week CBS-KO mice [37]. The development of liver fibrosis is a chronic process. Although young animals did not show liver fibrosis with low levels of endogenous liver H2S, the liver damage might already occurred. The lack of endogenous H2S would speed up the fibrosis, but it only shows up at the adult or old ages. The liver contains two major cell types, namely parenchymal and non-parenchymal cells. Parenchymal cells are commonly referred to hepatocytes, constituting about 80% of liver volume. Non-parenchymal cells take up about 6.5% of liver volume. Hepatic stellate cells (HSC), categorized as non-parenchymal cells, are located between sinusoids and hepatocytes of the liver, and involved in the formation of scar tissue and liver fibrosis in response to liver damage [105]. Exogenous H2S inhibited carbon tetrachloride-induced liver fibrosis in rats through the reduction of TGF-b1protein expression level and extracellular matrix sedimentation in liver tissue [106]. Fan et al. investigated the in vitro and in vivo effects

of H2S on activated hepatic stellate cells (HSC-T6) and carbon tetrachloride-induced hepatic fibrosis in rats. NaHS treatment at 500 lM suppressed ferric nitrilotriacetate-induced HSC-T6 cell proliferation. This suppression was achieved by G1 phase arrest and apoptosis, and by reduced intra cellular ROS production [107]. Decreased phospho-p38 and increased phospho-Akt expressions were the underlying mechanism for the effect of NaHS on the nitrilotriacetate-induced HSC-T6 cell proliferation and prevention of hepatic fibrosis [107]. Portal hypertension is a common and dreadful complication of chronic liver disease. Carbon tetrachloride-induced rat liver cirrhosis resulted in a significantly elevated portal pressure [69]. Administration of NaHS (10 lmole/kg, i.p.) significantly reduced portal hypertension in these cirrhotic rats, whereas PPG (30 mg/kg, i.p.) increased portal hypertension [69]. One key mechanism for H2S-induced vasorelaxation is the opening of KATP channels [17]. Moreau et al. showed that blockade of KATP channels by glibenclamide reduced portal pressure in portal hypertensive rats [108]. Whether endogenously produced H2S controls the functional status of KATP channels in portal circulation has not been reported. CSE mRNA and protein expression levels were significantly decreased in cirrhotic liver when compared to normal rat livers [101]. L-cysteine, a substrate of CSE, significantly decreased vasoconstriction in normal rat livers but failed to do so in the livers of cirrhotic rats [101]. In vitro studies on HSC demonstrated that spontaneous activation of HSC down-regulated CSE expression and H2S production, leading to abrogation of relaxation induced by L-cysteine [101]. These results confirm that CSE-derived H2S is involved the regulation of portal venous pressure and its deficiency might contribute to the development of portal hypertension.

Roles of H2S in hepatoprotection and hepatotoxicity Similar to the other two gasotransmitters NO and CO, H2S induces often a biphasic concentration-dependent effect, cytoprotective vs. cytotoxic [109]. Ischemia–reperfusion is common in major surgery and liver organ transplantation [110]. H2S protects against total hepatic I/R injury and dysfunction [67]. In one rat study, hepatic ischemia was induced by occlusion of the portal vein and hepatic artery with a vascular clamp, and the reperfusion initiated 30 min later by removing the clamp [111]. Intraperitoneal injection of NaHS (14 lmole/kg) 30 min before I/R significantly attenuated the severity of liver injury [111]. Serum levels of H2S and liver H2S production activity were significantly increased 1, 3, and 6 h after 30 min of I/R injury, suggesting a self-protective response of the liver to I/R. During I/R, the release of pro-inflammatory cytokines and the increased apoptosis in the liver cause damage to the liver. H2S would protect the liver from I/R damage by the virtue of its anti-inflammatory and anti-apoptosis actions. Jha et al. observed that intravenous administration of IK1001, a H2S donor, 5 min before reperfusion protected murine liver against 60 min of I/R injury [67]. Serum ALT and AST levels were decreased and the protein levels of HSP90 and Bcl-2 increased. Furthermore, H2S improved balance between liver reduced GSH and oxidized glutathione (GSSG), attenuated formation of liver lipid hydroperoxides, and increased expression of liver thioredoxin-1. The latter is a small redox protein that transfers electrons between species for redox signalling [67]. Hepatotoxicity of H2S is tightly related to H2S concentrations. Previous clinical studies have suggested that H2S can induce hepatic damage as the incidence of cholecystitis, cholangitis, and cholelithiasisis is higher than normal in oil refinery workers. These workers are often exposed to H2S as a by-product of fuel desulfurization process [112]. One case report showed that a sewer worker exposed to deadly concentrations of H2S had abnormal liver



function test results with elevated ALT and AST levels of 56 and 83 IU/L, respectively [113]. Data from animal studies also demonstrated different degrees of hepatic damage after sub-chronic or acute exposure to H2S, such as enlarged livers in mice exposed to 63 ppm H2S for 16 h and severe liver hyperemia in monkeys exposed to 500 ppm for 22 min [112,114]. Intoxication of mice with NaHS (60 lg/g, i.p.) elevated the sulfide concentration in liver by 18% [115]. An in vitro experiment further showed that incubation of hepatocytes with 0.5 mM NaHS for 2 h led to rapid hepatocyte necrosis [79]. Numerous studies have described the beneficial effects of H2S in multiple models of hepatic diseases such as liver cirrhosis and fibrosis [69,101], portal hypertension [69], NAFLD [40], hepatic ischemia and reperfusion injury [67,111], and acetaminophen-induced liver failure [77]. However, some recent studies indicate that H2S exerts detrimental effects on the liver in sepsis and endotoxemia models [116,117]. Li et al. reported that the plasma level of H2S was increased both in humans with septic shock and in mice administrated with E. coli lipopolysaccharide [116]. Further studies demonstrated that NaHS (14 lmole/kg, i.p.) administration resulted in increased liver myeloperoxidase (MPO, a marker of tissue neutrophil infiltration) activity in mice, and raised plasma TNF-a concentration. In contrast, PPG (50 mg/kg, i.p.) exhibited marked anti-inflammatory activity as evidenced by reduced liver MPO activity and ameliorated liver tissue damage. These findings suggest that H2S exhibits pro-inflammatory activity in endotoxic shock. Perturbed hepatic sinusoidal perfusion is a critical factor in the development of focal tissue hypoxia leading to liver injury during sepsis [118]. Using an isolated perfused normal rat liver system, it was found that portal infusion of H2S donor, Na2S, increased portal pressure in vivo [117]. During Na2S infusion, decreased sinusoidal diameter was found using intra vital microscopy. Endothelin-1-stimulated sinusoidal constriction produces heterogeneous perfusion at the level of individual sinusoids and involved in the development of sepsis [119]. In endotoxin treated rats, treatment with CSE inhibitor PPG significantly attenuated sinusoidal sensitization to endothelin-1. It has been known that H2S hyperpolarizes vascular smooth muscle cells (VSMC) via activation of KATP channels, which leads to vasorelaxation [120]. Within the liver, VSMCs are present around the portal terminal venules and hepatic arterioles. The hepatic sinusoids lack VSMCs and are primarily regulated by HSCs. Therefore, the role of H2S in the hepatic sinusoid is likely different from that in most resistance vessels, including the portal venules. H2S-induced hepatotoxicity involves at least two mechanisms. Firstly, H2S inhibits mitochondrial electron transport and increases cellular NADH/NAD+, placing the cell under reductive stress (excessive levels of reducing equivalents) [112,121,122]. The site of inhibition is similar to that of hydrogen cyanide (HCN) and involves cytochrome c oxidase (CCO) [123]. In this context, H2S was classified as a cellular asphyxiant, together with CO, HCN, and azide [122]. CCO activity is inhibited by the formation of a covalent bond between H2S and the Fe atom coordinated by heme A [124]. The binding of H2S to CCO causes accelerated degradation of CCO, reduces the amount of fully assembled and functionally active CCO, and inhibits aerobic metabolism [122,125]. H2S is a more potent inhibitor of CCO, approximately four times more cytotoxic than cyanide in hepatocytes. Secondly, production of reactive sulfur species leads to depletion of hepatocyte GSH and activation of oxygen to form ROS [79]. Truong et al. found that exposure of freshly isolated rat hepatocytes to high concentrations of NaHS (0.5 mM) led to GSH depletion and rapid necrosis [79]. In aqueous solution, H2S is a weak acid with two acid dissociation constants. The first dissociation yields HS–, and the second yields S2–, with pKa values for each of these dissociations of 7.04 and 11.96, respectively. Hence, at physiological pH 69% of H2S exists as HS . In the

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cell-free system tested, the depletion of GSH occurred at pH 6.0, not at pH 8.0 [79]. Because cellular acidosis occurred within 30 min of H2S exposure, the reactive species which caused GSH depletion may have been formed from H2S. Further evidence for the notion that H2S autoxidation products caused hepatocyte GSH depletion was derived from the experiments with metal chelators [diethylene-triaminepentaacetic acid (DETAPAC) and desferoximine]. The concurrent application of these chelators to hepatocytes at the time of NaHS addition prevented hepatocyte GSH depletion caused by NaHS (0.5 mM) [79]. Furthermore, ROS could be generated via cytochrome P450-catalyzed oxidation of H2S so that the cytotoxicity of NaHS can be partly prevented by P450 inhibitors [126]. Although cytochrome P450 isozymes, e.g. CYP 2E1 and CYP 2B6, are important sources of endogenous ROS in hepatocytes, ROS formation is not mainly determined by P450s, but rather by free iron (ferrous state) through the Fenton reaction [79,126–128]. Thus, the molecular events of H2S-induced cytotoxicity may follow this sequence: ROS generated by P450s in hepatocytes initiate H2S autoxidation to form cytotoxic OH, while H2S reduces the ferritin-ferric complex to release ferrous iron into the cytosol which can be used for the Fenton reaction. In short, H2S and GSH may contribute to the redox cycling of Fe3+/Fe2+ needed for OH formation, leading to hepatocyte damage [79,126,129]. Conclusion The liver is a major organ producing endogenous H2S under physiological conditions. Endogenously produced H2S participates in the regulation of liver glucose metabolism, lipoprotein synthesis, hepatic circulation, liver bioenergetics, and oxidative stress. Deficiency of endogenous production of H2S leads to several hepatic complications, including hepatic fibrosis and cirrhosis, but increased endogenous H2S may be involved in insulin resistance and progression of diabetes. Low level of H2S is cytoprotective and higher concentration of H2S may exert hepatotoxic effects. Targeting at H2S production and the expression of its producing enzymes may prove to be an effective strategy for modulating liver physiology and managing hepatic disorders. Conflict of interest None. Acknowledgments The study has been supported by an operating grant to RW from Canadian Institutes of Health Research and a grant-in-aid to LW from the Heart and Stroke Foundation of Canada. References [1] R. Wang, Physiological implications of hydrogen sulfide: a whiff exploration that blossomed, Physiol. Rev. 92 (2012) 791–896. [2] D.G. Searcy, S.H. Lee, Sulfur reduction by human erythrocytes, J. Exp. Zool. 282 (1998) 310–322. [3] R. Wang, Two’s company, three’s a crowd: can H2S be the third endogenous gaseous transmitter?, FASEB J 16 (2002) 1792–1798. [4] J. Furne, A. Saeed, M.D. Levitt, Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values, Am. J. Physiol. Regul. Integr. Comp. Physiol. 295 (2008). R1479-85. [5] D.J. Lefer, A new gaseous signaling molecule emerges: cardioprotective role of hydrogen sulfide, Proc. Natl. Acad. Sci. USA 104 (2007) 17907–17908. [6] H. Teng, B. Wu, K. Zhao, G. Yang, L. Wu, R. Wang, Oxygen-sensitive mitochondrial accumulation of cystathionine beta-synthase mediated by Lon protease, Proc. Natl. Acad. Sci. USA 110 (2013) 12679–12684. [7] C. Szabo, Hydrogen sulphide and its therapeutic potential, Nat. Rev. Drug Discov. 6 (2007) 917–935.


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Toxicology of hydrogen sulfide.

Hydrogen sulfide intoxication.

Electrochemical hydrogen sulfide biosensors.

Hydrogen sulfide augments survival signals in warm ischemia and reperfusion of the mouse liver.

Environmental toxicology of hydrogen sulfide.

Hydrogen sulfide in signaling pathways.

Hydrogen Sulfide Oxidation by Myoglobin.

Hydrogen Sulfide and Cellular Redox Homeostasis.

Signaling molecules: hydrogen sulfide and polysulfide.

Hydrogen sulfide and polysulfides as signaling molecules.

Hydrogen sulfide and polysulfides as biological mediators.

Is exogenous hydrogen sulfide a relevant tool to address physiological questions on hydrogen sulfide?

Hydrogen Sulfide: Biogenesis, Physiology, and Pathology.

Epithelial Electrolyte Transport Physiology and the Gasotransmitter Hydrogen Sulfide.

Neuroprotective effects of hydrogen sulfide and the underlying signaling pathways.

Endothelial dysfunction: the link between homocysteine and hydrogen sulfide.

Hydrogen Sulfide in Physiology and Diseases of the Digestive Tract.

The metallization and superconductivity of dense hydrogen sulfide.

A novel hydrogen sulfide prodrug, SG1002, promotes hydrogen sulfide and nitric oxide bioavailability in heart failure patients.

Hydrogen sulfide promotes adipogenesis in 3T3L1 cells.

Modulation of hydrogen sulfide by vascular hypoxia.

Interactions of hydrogen sulfide with myeloperoxidase.

A passive sampler for hydrogen sulfide.

Hydrogen sulfide in hemostasis: friend or foe?