Hydrogen sulfide : metabolism , biological and medical role

Hydrogen sulfide (H2S) is a signaling molecule that is actively synthesized in the tissues and is involved in the regulation of vascular tone, neuromodulation, cytoprotection, inflammation and apoptosis. In recent years, new data on animal and human H2S metabolism and function under the effect of various endogenous and exogenous factors, including drugs were collected. This review is provided to introduce generalized information about the main and alternative H2S metabolism and regulation, peculiarities of transport, signaling, biological role and participation in pathogenesis. Submitted data describe H2S content and activity of H2S-synthesizing enzymes in different organs, H2S effect on blood coagulation and platelet aggregation based on our research results. The working classification of H2S metabolism modulators, which are used in biology and medicine, is proposed: 1) agents that increase H2S content in tissues (inorganic and organic H2S donors; H2S-synthesizing enzymes substrates and their derivatives, H2S-releasing drugs; agents that contain H2S-synthesizing enzymes cofactors and activators, agents that inhibit H2S utilization); 2) agents that reduce H2S content in tissues (specific and nonspecific inhibitors of H2S-synthesizing enzymes), 3) agents with uncertain impact on H2S metabolism (some medicines). It was demonstrated that vitamin-microelement and microelement complexes with H2S-synthesizing enzymes cofactors and activators represent a promising approach for H2S content correction in tissues.

Pirogov Vinnytsya National Memorial Medical University, Ukraine e-mail: nzaichko@mail.ruH ydrogen sulfide (H 2 S) is well-known as a toxic gas with obnoxious odour, which is mainly formed in the process of putrefaction of animal and plant proteins.Its role in living organisms was considered for a long time only from the viewpoint of exotoxicant and endotoxicant, meaning the inhibitor of cytochrome c oxidase, monoamine oxidase, neurotoxin, and lung irritant.The interest to H 2 S significantly increased from the beginning of the 90's of the 20 th century because of determination of its connection with regulation of animal and human physiological functions.The results of studies of Kazuho Abe та Hideo Kimura (1996) have triggered more deep research of H 2 S biological role.The studies described peculiarities of this metabolite production in rats' brain and identified its first molecular target: N-methyl-D-aspartate receptors (NMDA-receptors) [1].
H 2 S is up to date a significant member of gasotransmitter family, including nitrogen monoxide (NO) and carbon monoxide (СO), and is involved іn vascular tone regulation, neuromodulation, cytoprotection, inflammation, apoptosis and other processes [2][3][4][5][6][7].Despite of the great number of foreign articles describing the role of H 2 S in biology and medicine, the Ukrainian works on this issue are scarce.New information has been accumulated recently on metabolism and H 2 S functions in organisms under the effect of different endogenous and exogenous factors, including medicines, which we tried to generali ze in this review.
H 2 S physicochemical properties and membrane transport.H 2 S is a short-living molecule with half-life of a few minutes [2].In aqueous solutions 20-30% of H 2 S exist in a non-dissociated form at pH 7.4 and 70-80% in a form of hydrosulfide anion (H 2 S ↔ Н + + HS -; рk а 6.89), partially transforming into sulfide anion (S 2-) [3,8].H 2 S possesses a high lipophility and is solved in lipid membranes two times easier (partition coefficient -2.0 ± 0.6), than in water [8].H 2 S is characterized by high permeability coefficient of lipid membrane (P m = 3 cm•s -1 ) and easily diffuses throughout cell membranes [8].In conditions of organism at рН 7.4, the transmembrane diffusion of H 2 S proceeds slower (P m7.4 = 0.85 cm•s -1 ), that leads to local accumulation of this molecule near a cell-producent.It has been determined throughout 3D-mathematic modelling that the sphere of H 2 S biological action is determined by that distance, at which metabolite's concentration is no less than 10% of its concentration in the place of synthesis, and is spread over 200 neighbouring cells per 1 sec on the average [8].Thus, H 2 S acts as paracrine signaling molecule, but the distant effect is not excluded, because this metabolite is a part of blood plasma and may be transported by erythrocytes.It is determined that H 2 S transport by human erythrocytes proceeds in 4 stages: 1) diffusion of H 2 S throughout cell membranes or gas channels; 2) extracellular deprotonation of H 2 S into HS -; 3) arrival of HS -into erythrocyte throughout anion transporter -protein AE1 at the exchange for Cl − ; 4) intracellular HS -protonation to H 2 S [9].The membrane transport of H 2 S can also occur throughout aquaporins -water channels [10].
Kinetic parameters of key and alternative pyridoxal 5′-phosphate dependent reactions of H 2 S synthesis, which have been studied in vitro on the example of human recombinant enzymes CSE and CBS, are significantly distinct.Investigations of Singh (2009) have shown that 96% of H 2 S is produced in the condensation of L-cysteine with L-homocysteine with the participation of CBS with V max 18.7 U/mg and k m 3.2 mМ.Alternative reactions of H 2 S synthesis with the participation of CBS have such kinetic parameters: 1) desulfuration of L-cysteine into pyruvate with V max 0.82 U/mg protein and k m 27.3 mМ; 2) lanthionine synthesis from L-cysteine with V max 0.77 U/mg protein and k m 45,6 mМ [13].
Chiku et al. have shown (2009) that among reactions of H 2 S synthesis with the participation of CSE only cysteine desulfuration (α, β-elimination) can play the main role because its kinetic parameters are V max 0.6 U/mg protein and k m 1.7 mМ.It appeared that k m of alternative CSE-dependent reactions of H 2 S synthesis associated with lanthionine, homolanthionine and homoserine creation are significantly higher: 33; 5.9; 2.7 mМ at V max 1.2; 6.6; 1.2 U/mg, respectively [14].Kinetic parameters of H 2 S synthesis from cystine are not determined, because under conditions close to physiological ones, the reaction does not proceed.

Enzyme
Scheme of reaction Ref.  [11,12] anion into thiosulfite-anion, which is further reduced to H 2 S with the participation of ТSТ [11,12,18].The question is how H 2 S general production may provide reactions, which necessitate significantly high substrate concentrations (L-cysteine and L-homocysteine).As it is known, under organizm conditions, the pull of free sulfur-containing amino acids is significantly lower, than that of their bound and disulfide forms.The content of general homocysteine in human blood plasma does not exceed 15 µМ, that of cysteine is 300 µМ, but the content of their free (thiol) forms is about 0.13-0.17and 24-27 µМ, respectively [19].Their quantity under pathology conditions may grow significantly: under homozygous deficit of CBS the level of general homocysteine in the blood plasm increases up to 100-500 µМ and above [20], the content of free cysteine may 60 times exceed the norm under chronic kidney failure.Part of free cysteine in blood plasma of pre-hemodialysis patients was 40.9% compared Thiosulfatedithiol sulfurtransferase (EC 2.8.1.5)[22,29] Огляди to 1.6% in healthy persons [21].Obviously, the role of alternative reactions of H 2 S synthesis at ordinary concentrations of sulfur-containing amino acids in organism is insignificant, but it may increase under hyperhomocysteinemia or hypercysteinemia, which are often combined [19].

T a b l e 2. Alternative enzymatic reactions of Н 2 S creation in tissues of animals and humans
The role of pyridoxal phosphate-independent ways of H 2 S creation (from D-cysteine and thiosulfate) in animal and human organism has not been studied completely.Studies of Shibuya and Kimura (2013) prove that optimal conditions for H 2 S synthesis from D-cysteine exist in organism (рН 7.4).However, this amino acid is not formed in the cells and may arrive only from exogenous sources [12].The role of thiosulfate-anion and TST in creation of H 2 S in tissues is the least studied.We have no clear data as of today regarding the content of thiosulfate-anion in organs and tissues, thus it is difficult to estimate the importance of this enzyme.Allowing for the concentration of thiosulfate-anion in blood plasma and urine of humans (1.13 ± 0.11 mg/dl and 0.28 ± 0.02 mg/dl, respectively) [22], the above-mentioned way of H 2 S formation in tissues is possible.
Н 2 S catabolism by SQR and its direct reaction with thiols results in formation of unstable persulfides (R-S-S*-H, thiotaurine, thiocysteine) which contain active sulfane sulfur (S 0 ) [23].Н 2 S is deposited in sulfane sulfur form in different tissues (brain, heart, liver, kidney) and can be released on demand.3-MST and CAT provide Н 2 S deposition in polysulfides [25,26].The quantity of polysulfides in cells with 3-MST and CAT expression is twice as much in comparison with cells without these enzymes [26].
CBS is considered the major producer of H 2 S in the central nerve system; it is expressed in the hippocampus, cerebellum, cortex and brain stem [5,15,34,35].Creation and deposition of Н 2 S as polysilfude in brain are provided by CAT and 3-МST, which are expressed in the cortex, cerebellum and retina [25].CBS is localized in astrocytes, and 3-MST -in neurons [17].Contribution of CSE to production of H 2 S in brain is the least, however, this way is considered important in support of redox homeostasis of the brain [36].CSE activity in the mice brain is about 1% of its activity in the liver.CSE is present in neurons of human brains and its activity is several times higher than that in mice [36].
In blood vessels H 2 S is synthesized with participation of CSE which is mainly expressed in smooth myocytes, and with CAT/3-MST, which are expressed in endothelium.The expression of these enzymes has been found in the aorta, lung artery, mesenteric and kidney arteries [3,5,11,17].CAT/3MST and CSE are expressed in the myocardium [37][38][39], but CAT/3-MST, CSE and CBS are expressed in skeletal muscles [40].Considerably high ability of

t a b l e 4. Main h 2 S-synthesizing enzymes expression in laboratory animals and human tissues
Organ, tissue H 2 S-synthesizing enzymes Ref. [47,48] Notation.Enzyme expression in 1 rat (rattus norvegicus), 2 mouse (Mus musculus) and 3 human tissues H 2 S creation was detected in the myometrium of rats and humans, in which CBS and CSE are expressed [41,42].The expression of CSE and CBS is detected in the intestine, stomach, β-cells of pancreas [43][44][45], adipocytes [46], lungs [47,48].H 2 S synthesis regulation.CBS and CSE cells are localized in the cytozole, while 3-MST and CAT -in the mitochondria and cytozole (division of their fractions depends on tissue) [17,26,49].
Human and rat CBS is homotetramer and consists of 4 equal subunits with molecular weight of 63 kDa.Each subunit is bound to the cofactors pyridoxal 5′-phosphate, heme and S-adenosylmethionine [5].S-adenosylmethionine is an allosteric activator of CBS.That is why CBS-dependent H 2 S production may increase, if S-adenosylmethionine concentration is elevating in cells [1].CBS activity is inhibited by CO, which binds with high affinity with heme (k і 5.6 µМ), and NO -only in high supraphysiological concentrations (k і 360 µМ) [50].One more mechanism of impact on CBS activity, which proves possible participation of enzyme in epigenetic regulation and gene expression, was detected recently.C-terminal fragment of CBS contains a tandem of two domains, which undergoes SUMO-modification (SUMO -small ubiquitin-like modifier) of lysine in position 211 that causes a decrease of enzyme's activity.CBS sumoylation is inhibited by cystathionine [51].Biological importance of SUMO-modification of CBS remains unidentified so far.Hypothetically, such form of CBS translocation into nucleus with further desumoylation and renovation of ability to H 2 S synthesis can take place [51].
H 2 S formation by CSE is activated under the action of calmodulin in the presence of 1-2 mМ Са 2+ [52].However, basic concentration of Са 2+ in cells is around 100 nМ, that is why the role of Са 2+ /calmodulin in regulation of CSE-dependent H 2 S production necessitates further study [6].Under in vitro conditions the ability of recombinant human CSE to sumoylation is established, and this process is not blocked by cystathionine [51].Whether SUMOmodi fication of CSE under conditions of organism exists and what kind of role this process plays has not been determined yet.Under hypoxia when concentration of cytosolic Са 2+ increases, CSE translocation from cytosole to mitochondrium may happen, the process is accompanied by the increase of mitochondrial H 2 S production and АТР synthesis [26].
In contrast to CSE, H 2 S formation with participation of CAT/3-МSТ is dose-dependently inhibited by Са 2+ (with full blocking in the presence of 2.9 µМ Са 2+ ) and does not depend on calmodulin [17].CAT activity decreases with L-aspartate increase in the medium [49].3-МSТ-dependent H 2 S production increases with the increase of thioredoxin and dihydrolipoic acid content in cells [17].
Concentration of endogenous H 2 S. H 2 S content in blood plasma of animals (rats) and humans is around 50-80 µМ [2,3,53].H 2 S presence is more considerable in tissues, in particular, the content of this metabolite in the animal brain is around 50-160 µМ [17,2].According to other data, H 2 S content in the brain, myocardium and kidneys of rats is 2.6; 11.4 and 6.7 µg/g of tissue, respectively [54].We have to mention that measurement of H 2 S content in tissues in the majority of investigations is performed by colorimetric method (in compliance with the reaction with N,N-dimethyl-p-phenylenediamine in the presence of FeCl 3 ), which needs highly acidic medium and does not exclude H 2 S release from the tissue depot.Presumably, intracellular free H 2 S concentration is much more less, because pH is around 7-8 in the mitochondria.Free H 2 S concentration determined by the method of gas chromatography, was 0.12 µmol/kg of protein (14 nМ) in the rat brain, and concentration of acid-labile sulfur was 916 µmol/kg of protein [55].
According to the results of our own studies (Tab le 5), general H 2 S content and specific activity of H 2 S-synsethizing enzymes in rats' tissues are on the highest level in the liver and kidneys, slightly lower in myocardium, brain and aorta.Determination of H 2 S content and production in tissues was performed as it has been described [22,[56][57][58].
H 2 S biological role and molecular targets.H 2 S in organism plays a role of signaling molecule, gasotransmitter; no specific receptors have been found for it.Different ion channels, receptors, enzymes and proteins, regulating numerous biochemical and physiological processes, play a role of H 2 S molecular targets (Table 6).
A key mechanism of H 2 S-signaling is S-sulfhydration of proteins, post-translation modification with convertation of -SH groups into -SSH, which significantly increases reactivity of cysteine residues and increases functional activity of molecular targets as well [6,17,59].H 2 S also reduces disulfide bonds of cystine residues with -SH groups releasing [23].Redox-modification of proteins with participation of H 2 S may be of independent regulatory importance as well as serve as a preparatory stage for S-sulfhydration and other kinds of post-translational S-modification (S-nitrosylation, S-homocysteinylation, S-glutathionylation).For example, activation of NMDA-receptors with participation of H 2 S proceeds in two stages: 1) NMDA-receptors become active under reduction of their disulfide form into thiol form, 2) transition of thiol form into persulfide form (S-sulfhydration) with a further, more considerable increase of NMDA-receptors activity [17].Mustafa et al. (2009) obtained direct proofs that S-sulfhydration raises the activity of АТР-sensitive potassium channels (-SSH modification of Kir6,1 subunit), glyceraldehyde-3-phosphate dehydrogenase (-SSH modification Сys150) and increases actin abili ty to polymerization [59].S-sulfhydration increases neuroprotective activity of ubiquitin E3 ligase [15] H 2 S together with NO participates in S-nitrosylation of proteins and low-molecular thiols [28,27].S-nitrosylation, in contrast to S-sulfhydration, decreases cystein's ability to reaction and leads Огляди to further lost of activity of molecular targets.[6,17,59].For example, glyceraldehyde-3-phosphate dehydrogenase is inactivated during S-nitrozylation Сys150 [59].H 2 S, in its turn, may express NО, activating guanilate cyclase and its own signaling ways, from nitrosothiols [27,28].

T a b l e 5. Н 2 S content and activity of h 2 S-synthesizing enzymes in rats' organs (M ± m, n = 10)
H 2 S also acts through interaction with prosthetic groups of metalloproteins, reactive oxygen species (ROS), low-molecular electrophilic derivatives.H 2 S in high concentrations blocks the activity of cytochrome c oxidase, reducing by electrons heme aa3, cuB and cytochrome с [23].H 2 S may reduce Fe 3+ into Fe 2+ of methemoglobin with its conversion into a form able to bind and transport oxygen [23].Under H 2 S interaction with ROS, generation of thiyl radical and thiosulfate anion which is transformed into oxygen-containing sulfur derivatives (sulfites and sulfates) is going on.It is not excluded that this way also mediates H 2 S signaling, because SO 2 participates in regulation of vascular tonus and contraction of the myocardium [60].It is proved that Na 2 SO 3 /NaHSO 3 causes dose-dependent relaxation of isolated rings of rats' aorta in vitro [61].As a nucleophile, H 2 S easily interacts with electrophile lipid derivatives, including 4-hydroxynonenal, which is a strong modulator of oxidative stress, cell proliferation, apoptosis [62].
Biological effect of H 2 S may be significantly distinct depending on tissue localization of molecular target.H 2 S impact on ion channels, for example, is going on in such directions [7,15,26,63,64]: 1) activation of АТР-K + -channels of smooth myocytes of blood vessels, that is associated with vasodilation, lowering of arterial pressure, cardioprotection under ischemia/reperfusion, inhibition of insulin secretion by the β-cells, anti-inflammatory, antinociceptive and anti-apoptosis effects; 2) inhibition of Са 2+ -channels of L-type in cardiomyocytes (with Са 2+ level decrease) and their activation in neurons (with an increase of Са 2+ level); inhibition of large K + (Са 2+ )-channels in carotide sinuses and their activation in pituitary cells; 3) activation of Са 2+ -channels of Т-type and TRPV 1 channels, that is associated with H 2 S-induced hyperalgesia and pronociceptive effect; activation of TRPA 1 -channels with transportation of Са 2+ into astrocytes; 4) H 2 Sinduced contraction of out-vascular smooth muscles and increase Сl -secretion in gastrointestinal tract are connected with activation of TRPV 1 and TRPА 1 channels; 5) Сl -channels activation and prevention from neurons oxytosis.
Year after year more and more mechanisms of H 2 S-signaling are identified, where kinases, transcription factors, growth factors and other regulator molecules are involved.H 2 S action may be mediated through: 1) modulation of adenylate cyclase, phosphodiesterase activity and сАМР content in cells; H 2 S causes inhibition of adenylate cyclase and decrease of сАМР content in neurons of morphindependent mice [65]; phosphodiesterase activity increase in juxtaglomerulal cells of rats' kidneys with renovascular hypertension [66]; сАМР level and activity of protein kinase A increase but сАМРdependent phosphodiesterase activity decrease in isolated mitochondria of rats' hepatocytes [67]; сАМР synthesis increase in culture of neurons and glial cells [68].
4) modulation of NO and CO synthesis and their signaling pathways; H 2 S up-regulates eNOS expression and increases endothelial NO production [23]; decreases іNOS production and nitrosative stress [75]; up-regulates heme oxygenase-1 expression and CO production in cardiomyocytes and other cells [75,76].
5) impact on aging and circadian rythm genes (SIrt1, klotho) expression, through which formation of age-associated changes in tissues is mediated [77].
Workers of Blood Circulation Physiology Department of the O. O. Bohomolets Institute of Physio logy of NAS of Ukraine (Kyiv) under leadership of V. F. Sahach, Corresponding Member of NAS of Ukraine, doctor of medical science, assessed that H 2 S is involved into opening of mitochondrial permeability transition pore, which is a key player in development of apoptosis and necrosis, and this effect is realized through K + -АТР-channels modulation [102][103][104].
Direction of H 2 S action depends on its content in tissues.At low (physiological) concentrations H 2 S shows its vasodilating, cytoprotector, antioxidant, anti-inflammatory and anti-apoptotic effects.It also increases sensibility of NMDA-receptors of neurons to glutamate, stimulates Са 2+ transportation to astrocytes and increases synaptic activity.H 2 S activates cystine-glutamate antiporters, stimulates transportation of cysteine into mitochondria, and increases activity of γ-glutamylcysteine synthetase (glutamatecysteine ligase, EC 6.3.2.2) and glutathione synthesis in neurons and other cells [4,17,23,26,68].H 2 S in supraphysiological and toxic concentration induces mithohondrial dysfunction, blocks tissue respiration and oxidative phosphorylation, increases vascular tonus, causes hyperalgesia, promotes inflammation and apoptosis, inhibits synaptic transmission.H 2 S enables biogen amins effects (γ-aminobutiric acid, glutamate, serotonin, dophamine, epinephrine and norepinephrine) and acetylcholine by up-regulation of their receptors exression and inhibition of monoamine oxidase and acetylcholine esterase activity [17,68].Low H 2 S concentrations increase basal tension, smooth muscle contractions and motility of the gastric antrum, but high H 2 S concentrations, on the contrary, decrease all the above-mentioned functions [78].H 2 S in concentration of 0.1-1 µM increased electron transport and ATP content, and being in a concentration of 3-30 µM it inhibited cytochrome c oxidase and oxidative phosphorylation in mitochondria of isolated hepatocytes [79].
Different pathological conditions are associated with disorder of H 2 S content in tissues.A decrease of basic H 2 S content in blood plasma is noticed in patients with arterial hypertension, ischaemic heart disease, deep venous thrombosis, Alzheimer's disease, hyperhomocysteinemia [3,19,53].An increase of H 2 S content is observed in patients with Down's syndrome, with decompensated liver cirrhosis, sepsis, ischaemic stroke, chronic obstructive pulmonary diseases [19,3].

H 2 S metabolism modulators in biology and medicine.
The following basic approaches are used to study H 2 S role in vivo and in vitro: 1) introduction of inorganic and organic H 2 S donors; 2) introduction of specific inhibitors of H 2 S-synthesizing enzymes; 3) modification of efficiency and toxicity of pharmacological remedies with the help of H 2 S; 4) modification of H 2 S metabolism with the help of pharmacological drugs.Examples of some H 2 S metabolism modulators are presented in Table 7.
H 2 S under conditions of organism generally works in the form of HS-, that is why NaHS or Na 2 S are used as its donors.Information on doses, ways and duration of administration to animals of inorganic H 2 S donors varies [80].NaHS/Na 2 S, according to most studies, are injected parenterally (intraperitoneally, intravenously) in doses from 100 µg/kg to 3 mg/kg of the animal body weight, and duration of experiment may vary from several hours to 2-3 weeks [80].In long-term studies (8-10 weeks) NaHS is added to drinking water in concentrations of about 30 µmol/l; animals drink it ad libitum [81].We should note that LD 50 for NaHS under parenteral injection is 14.6 ± 1.0 mg/kg [82], in compliance with other data it is 60.2 mg/kg, and conventionally therapeutic doses of NaHS are about 1/20 LD 50 [83].NaHS/Na 2 S in such concentration range decrease the ischaemic-reperfusion damages under conditions of myocardial infarction, kidney ischemia, intestinal ischemia, show antioxidative, cerebroprotective, antiinflammatory features [7,17,34,68,80,81].
Vasodilatory, cytoprotective, antioxidative, anti-inflammatory effects of NaHS/Na 2 S in studies in vitro are realized in broader range concentrations from 1 to 1000 µМ [80].
We have shown that H 2 S donors demonstrate antiaggregation and anticoagulant effect: NaHS in concentration of 1 mМ inhibited aggregation of human platelets, induced by ADP and collagen, and decreased amidolytic activity of thrombin (ІС 50 = 65.3 ± 3.76 µМ) in vitro; the injection of Na 2 S•9H 2 O (Sigma, USA) to rats in the amount of 3.36 mg/kg once a day during 7 days caused the increase of prothrombin time and activated partial thromboplastic time and decrease of Ха factor activi ty [19].According to data of Nishikawa et al. (2013), NaHS inhibits thrombocyte aggregation, induced by ADP and collagen in plasma enriched by thrombocytes in concentrations of 0.1-0.3mМ, and in a suspension of washed up thrombocytes of rabbits in concentrations of 1-3 mМ, respectively [84].and causes mitochondria swelling [102][103][104].Gradual swelling of rats' heart's mitochondria is observed in potassium-free medium in the presence of NaHS in concentrations of 10 -12 -10 -8 М [102].Age distinctions regarding H 2 S impact over pore creation in the heart mitochondria have been observed: NaHS protector effect in adult rats is realized in a more broad range of concentrations (10 -6 , 10 -5 and 5•10 -5 М) than in old rats (10 -5 М) [102].NaHS in the range of concentrations 10 -9 -10 -6 М causes dose-depending decrease of oxygen absorption by isolated mitochondria of adult rats' heart in the presence of succinate and ADP at concentration of 10 9 and 10 -8 М and increases connection between oxidation and phosphorylation [103].Preincubation of isolated mitochondria with inhibitor of K + -АТР-channels 5-hydroxydecanoate significantly decreased the ability of NaHS (10 -4 М) to prevent mitochondrial swelling under ischemia/ reperfusion of myocardium [104].
Inhalation of H 2 S aimed at cytoprotection is used in some studies, and concentrations of 50-400 ррm which cause mitochondrial dysfunction, are sublethal.H 2 S in concentration up to 10 ррm does not create disorder in activity of mitochondrial enzymes [26].The perfusion of lungs by gas mixture with H 2 S in concentration of 50 and 100 µmol/l decreased oxidative disorders at pulmonal ischemia/ reperfusion in mice [80].
Inorganic H 2 S donors quickly increase its content in blood plasma and tissues, but they are also quickly eliminated from the organism in a form of sulfides, thiosulfates, sulfites, sulfates.More slow increase of H 2 S content in tissues provide H 2 Ssynthesing enzymes substrates such as L-cysteine, D-cysteine; L-cysteine derivatives such as N-acetylcysteine, S-propalgyl-L-cysteine, S-propyl-Lcysteine, S-allyl-L-cysteine; polysulfides (diallyl disulfide, diallyl trisulfide) and artificial organic donor of H 2 S -Lawesson's reagent [68,85,105].Sodium thiosulfate may also play the role of inorganic substrate for endogenous H 2 S production.For example, the injection of L-cysteine (15 and 100 mg/kg) stimulated epithelialization of gastric ulcer in rats [86].Dcysteine protects neurons of cerebellum from oxidative stress and decreases damage of kidneys under condition of ischeamia-reperfusion more effectively than L-cysteine [12].S-propalgyl-L-cysteine, S-propyl-L-cysteine, S-allyl-L-cysteine showed cardioprotective action under acute myocardial infarction in rats, increased H 2 S content and increased activity of superoxide dismutase (Mn-SOD) in cardiomyocytes [105].Administration of sodium thiosulfate (3 mg/ ml with drinking water for 6 weeks) normalized cardial H 2 S production in rats with chronic heart failure [87].
To reach a decrease of H 2 S endogenous production the H 2 S-synthesizing enzymes have been used: inhibitors of CSE -DL-propargylglycine, β-cyanoalanine or inhibitors of CBS -hydroxylamine, aminooxyacetate.As a rule, inhibitors of H 2 S-synthesizing enzymes demonstrate vasoconstriction, cytotoxic, pro-oxidant and pro-inflammatory action [7,15,17,34,68,80,81].For example, the injection of DL-propargylglycine caused a decrease in activity of mitochondrial electron transport chain Complex І, decrease of glutathione content and development of oxidative stress in the brain of mice [36]; increased ischaemia-reperfusion damages and increased size of myocardial infarction, worsened functional condition of kidneys and increased content of creatinine in blood serum of rats [80].Seven-day injection of propargylglycine (50 mg/kg) caused activation of blood coagulation and increased ADP-induced aggregation of thrombocytes in rats [88].With excessive H 2 S in tissues, the inhibitors of H 2 S-synthesizing enzymes may show cytoprotector action.Propargylglycine injection (8-10 mg/kg) decreased features of acute turbular necrosis, prevented the increase of serum creatinine and decreased H 2 S creation in rats' kidneys under gentamicyne administration [89].
Taking into consideration H 2 S physiological effects, the attempts have been made to modify effectiveness and toxicity of medicines with the help of its donors.Thus there appeared H 2 S-releasing drugs, and H 2 S-derivatives of nonsteroidal anti-inflammatory drugs (NSAIDs) became their first example.It has been proved that one of the mechanisms of gastroduodenal toxicity of NSAIDs is a decrease of H 2 S endogenous production [90].H 2 S-derivatives, S-diclofenac, S-naproxen, S-aspirin possess a higher anti-inflammatory action and lower gastrotoxicity than their prototypes [86,91].(H 2 S+NO)-derivatives of aspirin and H 2 S-derivatives of other pharmacological means -L-DOPA (ACS 83-86 ), sildenafil (ACS6) and mesalamin (ATB-429) were made later [68].
Pharmaceutical means may influence H 2 S content in animals' organs in different ways.For example, paracetamol decreased H 2 S concentration in brain, but increased its concentration in the heart, liver and kidneys of mice [92].Amplodipin caused a decrease of H 2 S level in the brain and liver of mice in a dose of 3 and 10 mg/kg, but caused its increase in the heart and kidneys in a dose of 3 mg/kg and its decrease in a dose of 10 mg/kg [56].It has been demonstrated as well that aspirin decreased H 2 S level in the brain and increased its level in the liver of mice [93].Carvedilol (in a dose of 10 mg/kg) caused an increase of H 2 S content in the heart, kidneys and brain of mice [94].The same effect was caused by digoxin [54], atorvastatin [92], ramipril [95], and metformin [96].
Medicines' influence on H 2 S content in tissues, and their interaction with H 2 S donors and inhibitors of H 2 S-synthesising enzymes are not known well.It is proved that atorvastatin increases H 2 S content in perivascular adipose tissue at the expence of inhibition of its mitochondria utilization, but does not influence the CSE activity.Atovastatin disrupts endogenous production of cofactor of sulfide quinone oxidoreductase -ubiquinone that significantly decreases its content in the blood plasma and tissues of animals [97].Other drugs may be non-specific activators or inhibitors of H 2 S-synthesizing enzymes.We have shown that a single injection of cysplatine (7 mg/kg) causes a significant decrease of CSE, CBS and CAT activity in the rat kidneys, that is associated with a decrease of H 2 S level and increase of homocysteine level in the blood plasma.The injection of DL-propargylglycine (50 mg/kg) potentiated nephrotoxic effect of cysplatine, while NaHS injection (3 mg/kg), on the contrary, decreased cysplatineinduced nephropathia, increased glutathione content and glutamate-cysteine ligase in the rat kidneys [98].
It is not improbable that inorganic and organic donors of H 2 S may significantly impact biotransformation of medicines.NaHS peroral administration (5 mg/kg) caused a decrease of CYP2C9 activity, increase of CYP3A4 activity and had no influence on CYP2B6, CYP2D6 and CYP2C19 activity in rats [99].
Search for safe and effective H 2 S modulators is continued.In our opinion, the drugs containing activators and cofactors of H 2 S-synthesizing enzymes, vitamin-microelements complexes, are perspective.We have showed in some studies that under longterm hyperhomocystenemia in rats H 2 S content in the blood plasma and H 2 S-synthesizing enzymes activity (CSE, CBS, CAT, ТSТ) in the liver, kidneys, aorta is decreased.Administration of vitaminmicroelement complex (VMC), which contains vitamins В 6 , В 9 , В 12 and coordinating compositions of zinc (Zn 2+ ) and chrome (Cr 3+ ) with N-2,3-dimethyl-phenylanthranilic acid and ammonium vanadate (V 5+ ), effectively increased endogenous H 2 S production from cysteine, homocysteine, thiosulfate in rats' organs.The H 2 S production was also increased under the condition of combination of cysteine, homocysteine, thiosulfate in rats' organs with hyperhomocysteinemia and also under its combination with nitric oxide synthase inhibitor L-NAME [57].A complex of essential microelements (iron, copper, zinc, cobalt, manganese, chrome) and oxygencontaining salts of ultramicroelements (vanadium, molybdenum and selenium) -esmin (Esmin, PC "Kyiv Vitamin Factory") in a dose of 35 mg/kg decreased age-associated reduction of H 2 S production in the myocardium, aorta and kidneys of rats [100].The above-mentioned microelements are necessary for the broad range of biochemical processes; in particular, they are cofactors or activators of antioxidant enzymes and enzymes of sulfur-containing amino acids metabolism, involved in cardiovascular homeostasis and tissue respiration.
Thus a great body of scientific information about H 2 S role in biology and medicine (Scheme) was accumulated within the last ten years.H 2 S metabolism modulation has assumed great significance in determining mechanisms of formation of different pathological conditions, in development of new approaches to their preventive maintenance and correction, in increasing effectiveness and safety of pharmacotherapy and in development of many other directions, which amount increases.In our opinion, there is a necessity for systematization of H 2 S metabolism modulators and creation of their working classification.We propose to divide modulators of H 2 S metabolism, which are used in biology and medi cine, into groups and subgroups in respect of their impact on endogenous H 2 S in tissues and mechanism of their action (Table 8): 1) means that increase H 2 S content in tissues (with regard to action mechanism, they are divided into H 2 S donors; H 2 S-synthesising substrates of enzymes and their derivatives; H 2 S-releasing drugs; remedies containing cofactors and activators of H 2 Ssynthesizing enzymes; drugs inhibiting H 2 S utilization); 2) means that decrease H 2 S content in tissues (with regard to action mechanism, they are divided into specific and non-specific inhibitors of H 2 S-synthesizing enzymes); 3) means with indeterminate mechanism of impact on H 2 S metabolism.This group contains phar-

Т а b l e 8. Classification of H 2 S metabolism modulators, which are used in biology and medicine
Groups Representatives 1. Agents that increase H 2 S content in tissues . ZaIchko, a. V. MelNIk, M. M. YoltUkhIVSkYY, a. S. olhoVSkIY, I. V. PalaMarchUk

Т а b l e 7 .
Examples of H 2 S metabolism modulators It has been showed in works of N. A. Strutynska, O. M. Semenykhina et al (2011, 2013) that NaHS та L-cystein in physiological concentrations improve functional conditions of mitochondria in the heart, prevent Са 2+ -induced opening of mitochondrial pore and swelling of mitochondria at the time when CSE inhibitor propargylglycine increases sensitivity of mitochondria pore up to the action of Са 2+ inductor N. V. ZAICHKO, A. V. MELNIK, M. M. YOLTUKHIVSKYY et al.
2 S in tissues paracetamol (in brain), amlodipinScheme.Main directions of metabolism and biological effects of h 2 S