Evaluation of Cysteine Metabolism in the Rat Liver and Kidney Following Intravenous Cocaine Administration and Abstinence.

Many toxic effects of cocaine are attributed to reactive oxygen species (ROS) generated during its metabolism. Recently, it has been suggested that the biological action of ROS is often confused with endogenously generated reactive sulfur species (RSS). The aim of this study was to evaluate the impact of cocaine on thiols and RSS in the rat liver and kidney in the drug self-administration (SA) paradigm and the cocaine yoked delivery model (YC) followed by drug abstinence with extinction training. The level of thiols as well as RSS formed during anaerobic metabolism of cysteine and sulfate were assayed. In addition, the activity of enzymes involved in RSS formation and glutathione metabolism were determined. In the liver, following direct cocaine administration (SA and YC), the RSS levels decreased, while in the kidneys, cocaine increased the RSS contents in both groups. These changes were maintained in these tissues during drug abstinence. The level of sulfates was changed by cocaine only in the liver. In the kidney, cocaine shifted cysteine metabolism towards an anaerobic pathway. Our study demonstrates for the first time the changes in cysteine metabolism and thiol levels in the liver and kidney of rats after cocaine self-administration and abstinence.

Is aldehyde dehydrogenase inhibited by sulfur compounds? In vitro and in vivo studies.

Aldehyde dehydrogenase (ALDH) catalyzes the critical step of ethanol metabolism, i.e. transformation of toxic acetaldehyde to acetic acid. It is a redox sensitive protein with the key Cys in its active site. Recently, it has been documented that activity of some proteins can be modified by sulfur-containing molecules called reactive sulfur species leading to the formation of hydro- persulfides. The aim of the present study was to examine whether ALDH activity can be modified in this way. Studies were performed in vitro using yeast ALDH and various reactive sulfur species, including Na2S, GSSH, K2Sx, Na2S2O3, and garlic-derived allyl sulfides. The effect of garlic-derived trisulfide on ALDH activity was also studied in vivo in the rat liver. The obtained results clearly demonstrated that ALDH could be regulated by sulfur species which inhibited its enzymatic activity. The results also suggested that not H2S but polysulfides or hydropersulfides were the oxidizing species responsible for this modification. This process was easily reversible by reducing agents. After the treatment with polysulfides or hydropersulfides the level of protein-bound sulfur increased, while the activity of the enzyme dramatically decreased. Moreover, the study demonstrated that ALDH activity was inhibited in vivo in the rat liver after garlic-derived trisulfide administration. This is the first study reporting the regulation of ALDH activity by sulfane sulfur species and the results suggest that it leads to the inhibition of the enzyme.

The effect of NaCl on the level of reduced sulfur compounds in rat liver. Implications for blood pressure increase.

BACKGROUND: It is commonly known that excessive salt intake is a risk factor of hypertension. Currently, there is an increasing interest in reduced reactive sulfur species (RSS), mainly H2S and sulfane sulfur (SS) as new gasotransmitters showing vasorelaxant properties. The aim of the present study was to determine the effect of repeated administration of low sodium chloride dose included in physiological saline on blood pressure, on the level of RSS and activity of enzymes involved in their biosynthesis in the rat. METHODS: Two separate experiments were carried out on male Wistar rats: one with intravenous injections of saline and the second one with intraperitoneal saline injections. Blood pressure was measured during the experiment. The level of RSS and other biochemical assays were conducted in the rat liver, because of an intense cysteine metabolism to RSS in this organ. RESULTS: Intravenous administration of saline induced a significant increase in systolic blood pressure while intraperitoneal injections showed only a tendency towards increasing blood pressure. The RSS (H2S and SS) level as well as the activity of the main enzyme responsible for their production in the liver of animals after iv saline injections were decreased. Animals injected with physiological saline by ip route did not reveal any statistically significant differences in SS, H2S, and activities of sulfurtransferases, although a tendency to decrease in the RSS was observed. CONCLUSIONS: The repeated iv saline injection induced a slight hypertension accompanied by disturbances in anaerobic cysteine metabolism in the rat liver.

The Role of Hemoproteins: Hemoglobin, Myoglobin and Neuroglobin in Endogenous Thiosulfate Production Processes.

Thiosulfate formation and biodegradation processes link aerobic and anaerobic metabolism of cysteine. In these reactions, sulfite formed from thiosulfate is oxidized to sulfate while hydrogen sulfide is transformed into thiosulfate. These processes occurring mostly in mitochondria are described as a canonical hydrogen sulfide oxidation pathway. In this review, we discuss the current state of knowledge on the interactions between hydrogen sulfide and hemoglobin, myoglobin and neuroglobin and postulate that thiosulfate is a metabolically important product of this processes. Hydrogen sulfide oxidation by ferric hemoglobin, myoglobin and neuroglobin has been defined as a non-canonical hydrogen sulfide oxidation pathway. Until recently, it appeared that the goal of thiosulfate production was to delay irreversible oxidation of hydrogen sulfide to sulfate excreted in urine; while thiosulfate itself was only an intermediate, transient metabolite on the hydrogen sulfide oxidation pathway. In the light of data presented in this paper, it seems that thiosulfate is a molecule that plays a prominent role in the human body. Thus, we hope that all these findings will encourage further studies on the role of hemoproteins in the formation of this undoubtedly fascinating molecule and on the mechanisms responsible for its biological activity in the human body.

Lipoic Acid as a Possible Pharmacological Source of Hydrogen Sulfide/Sulfane Sulfur.

The aim of the present study was to verify whether lipoic acid (LA) itself is a source of H2S and sulfane sulfur. It was investigated in vitro non-enzymatically and enzymatically (in the presence of rat tissue homogenate). The results indicate that both H2S and sulfane sulfur are formed from LA non-enzymatically in the presence of environmental light. These results suggest that H2S is the first product of non-enzymatic light-dependent decomposition of LA that is, probably, next oxidized to sulfane sulfur-containing compound(s). The study performed in the presence of rat liver and kidney homogenate revealed an increase of H2S level in samples containing LA and its reduced form dihydrolipoic acid (DHLA). It was accompanied by a decrease in sulfane sulfur level. It seems that, in these conditions, DHLA acts as a reducing agent that releases H2S from an endogenous pool of sulfane sulfur compounds present in tissues. Simultaneously, it means that exogenous LA cannot be a direct donor of H2S/sulfane sulfur in animal tissues. The present study is an initial approach to the question whether LA itself is a donor of H2S/sulfane sulfur.

The Effects of Different Garlic-Derived Allyl Sulfides on Anaerobic Sulfur Metabolism in the Mouse Kidney.

Diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) are major oil-soluble organosulfur compounds of garlic responsible for most of its pharmacological effects. The present study investigated the influence of repeated intraperitoneally (ip) administration of DAS, DADS and DATS on the total level of sulfane sulfur, bound sulfur (S-sulfhydration) and hydrogen sulfide (H2S) and on the activity of enzymes, which catalyze sulfane sulfur formation and transfer from a donor to an acceptor in the normal mouse kidney, i.e., γ-cystathionase (CSE) and rhodanese (TST). The activity of aldehyde dehydrogenase (ALDH), which is a redox-sensitive protein, containing an -SH group in its catalytic center, was also determined. The obtained results indicated that all tested compounds significantly increased the activity of TST. Moreover, DADS and DATS increased the total sulfane sulfur level and CSE activity in the normal mouse kidney. ALDH activity was inhibited in the kidney after DATS administration. The results indicated also that none of the studied allyl sulfides affected the level of bound sulfur or H2S. Thus, it can be concluded that garlic-derived DADS and DATS can be a source of sulfane sulfur for renal cells but they are not connected with persulfide formation.

The Effect of Chronic Mild Stress and Imipramine on the Markers of Oxidative Stress and Antioxidant System in Rat Liver.

Liver abnormalities have been reported to occur in up to 20 % of patients on a long-term therapy with the tricyclic antidepressant drug imipramine (IMI). The mechanism involved in this IMI-induced process is unknown but a contribution of oxidative stress is highly likely. Chronic mild stress (CMS) is widely used for modeling depressive-like behavior in rats. In the present study, we examined the effects of CMS and chronic IMI treatment, applied alone or in combination, on the levels of oxidative stress markers, such as reactive oxygen species (ROS), malondialdehyde (MDA), non-protein sulfhydryl groups, and sulfane sulfur as well as on activities of key antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase in the rat liver. Administration of IMI for 5 weeks to rats subjected to CMS resulted in a gradual significant reduction of anhedonia measured by sucrose intake, in a majority of animals (CMS IMI-reactive, CMS IMI-R), although about 20 % of rats did not respond to the IMI treatment (CMS IMI non-reactive, CMS IMI-NR). CMS-induced hepatic oxidative stress, estimated by increased ROS and MDA concentrations, was not prevented by the IMI administration, moreover, in CMS IMI-NR animals, the level of the marker of lipid peroxidation, i.e., MDA was increased in comparison to CMS-subjected rats and activity of antioxidant enzymes (GPx and CAT) was decreased compared to IMI-treated rats. The clinical significance of this observation remains to be established.

Cysteine Metabolism and Oxidative Processes in the Rat Liver and Kidney after Acute and Repeated Cocaine Treatment.

The role of cocaine in modulating the metabolism of sulfur-containing compounds in the peripheral tissues is poorly understood. In the present study we addressed the question about the effects of acute and repeated (5 days) cocaine (10 mg/kg i.p.) administration on the total cysteine (Cys) metabolism and on the oxidative processes in the rat liver and kidney. The whole pool of sulfane sulfur, its bound fraction and hydrogen sulfide (H2S) were considered as markers of anaerobic Cys metabolism while the sulfate as a measure of its aerobic metabolism. The total-, non-protein- and protein- SH group levels were assayed as indicators of the redox status of thiols. Additionally, the activities of enzymes involved in H2S formation (cystathionine γ-lyase, CSE; 3-mercaptopyruvate sulfurtransferase, 3-MST) and GSH metabolism (γ-glutamyl transpeptidase, γ-GT; glutathione S-transferase, GST) were determined. Finally, we assayed the concentrations of reactive oxygen species (ROS) and malondialdehyde (MDA) as markers of oxidative stress and lipid peroxidation, respectively. In the liver, acute cocaine treatment, did not change concentrations of the whole pool of sulfane sulfur, its bound fraction, H2S or sulfate but markedly decreased levels of non-protein SH groups (NPSH), ROS and GST activity while γ-GT was unaffected. In the kidney, acute cocaine significantly increased concentration of the whole pool of sulfane sulfur, reduced the content of its bound fraction but H2S, sulfate and NPSH levels were unchanged while ROS and activities of GST and γ-GT were reduced. Acute cocaine enhanced activity of the CSE and 3-MST in the liver and kidney, respectively. Repeatedly administered cocaine enhanced the whole pool of sulfane sulfur and reduced H2S level simultaneously increasing sulfate content both in the liver and kidney. After repeated cocaine, a significant decrease in ROS was still observed in the liver while in the kidney, despite unchanged ROS content, a marked increase in MDA level was visible. The repeated cocaine decreased 3-MST and increased γ-GT activities in both organs but reduced GST in the kidney. Our results show that cocaine administered at a relatively low dose shifts Cys metabolism towards the formation of sulfane sulfur compounds which possess antioxidant and redox regulatory properties and are a source of H2S which can support mitochondrial bioenergetics.

INACTIVATION OF ALDEHYDE DEHYDROGENASE BY NITROGLYCERIN IN THE PRESENCE AND ABSENCE OF LIPOIC ACID AND DIHYDROLIPOIC ACID. IMPLICATIONS FOR THE PROBLEM OF DIFFERENTIAL EFFECTS OF LIPOIC ACID IN VITRO AND IN VIVO.

Lipoic acid (LA-(SS), LA) and its reduced form - dihydrolipoic acid DHLA-(SH)2, DHLA) are synthesized mainly in the mammalian liver. In this study, we investigated in viti the inactivation of yeast aldehyde dehydrogenase (ALDH) by nitroglycerin (GTN) in the presence and absence of LA and DHLA. In vivo studies were performed to answer the question whether LA administered jointly with GTN for 8 days will affect the ALDH activity in the rat liver. The results indicated that in vito both LA and DHLA restored and protected ALDH activity against GTN-induced inactivation, while treatment of rats with LA and GTN in combination did not provide any protection against GTN-induced ALDH inhibition. In summary, the obtained results seem to confirm earlier reports indicating the differential effects of LA in vitio and in vivo.

S-sulfhydration as a cellular redox regulation.

For many years reactive oxygen and nitrogen species (ROS and RNS) have been recognized as key messengers in the process of thiol-based redox regulation. Relatively recently, literature reports began to mention reactive sulfur species (RSS) and their role in thiol regulation. This review is focused on biogenesis and biological properties of RSS, including: hydropersulfides, polysulfides and hydrogen sulfide (H2S). Based on the most up-to-date literature data, the paper presents biological significance of S-sulfhydration process. In this reaction, sulfane sulfur is transferred to the-SH groups forming hydropersulfides. Protein cysteine residues, called 'redox switches' are susceptible to such reversible modifications. In line with the most recent reports, it was emphasized that sulfane sulfur-containing compounds (mainly hydrogen persulfides and polysulfides) are real and better mediators of S-sulfhydration-based signalling than H2S. We also overviewed proteins participating in the formation and transport of RSS and in mitochondrial H2S oxidation. In addition, we reviewed many reports about proteins unrelated to sulfur metabolism which are modified by S-sulfhydration that influences their catalytic activity. We also addressed the problem of the regulatory function of S-sulfhydration reaction in the activation of KATP channels (vasorelaxant) and transcription factors (e.g. NFκB) as well as in the mechanism of therapeutic action of garlic-derived sulfur compounds. Some aspects of comparison between RNS and RSS are also discussed in this review.

The effects of cocaine on different redox forms of cysteine and homocysteine, and on labile, reduced sulfur in the rat plasma following active versus passive drug injections.

The aim of the present studies was to evaluate cocaine-induced changes in the concentrations of different redox forms of cysteine (Cys) and homocysteine (Hcy), and products of anaerobic Cys metabolism, i.e., labile, reduced sulfur (LS) in the rat plasma. The above-mentioned parameters were determined after i.p. acute and subchronic cocaine treatment as well as following i.v. cocaine self-administration using the yoked procedure. Additionally, Cys, Hcy, and LS levels were measured during the 10-day extinction training in rats that underwent i.v. cocaine administration. Acute i.p. cocaine treatment increased the total and protein-bound Hcy contents, decreased LS, and did not change the concentrations of Cys fractions in the rat plasma. In turn, subchronic i.p. cocaine administration significantly increased free Hcy and lowered the total and protein-bound Cys concentrations while LS level was unchanged. Cocaine self-administration enhanced the total and protein-bound Hcy levels, decreased LS content, and did not affect the Cys fractions. On the other hand, yoked cocaine infusions did not alter the concentration of Hcy fractions while decreased the total and protein-bound Cys and LS content. This extinction training resulted in the lack of changes in the examined parameters in rats with a history of cocaine self-administration while in the yoked cocaine group an increase in the plasma free Cys fraction and LS was seen. Our results demonstrate for the first time that cocaine does evoke significant changes in homeostasis of thiol amino acids Cys and Hcy, and in some products of anaerobic Cys metabolism, which are dependent on the way of cocaine administration.

The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells.

The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H(2)O(2)) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H(2)O(2) formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis.

Effects of different garlic-derived allyl sulfides on peroxidative processes and anaerobic sulfur metabolism in mouse liver.

Biological activity of garlic has been attributed to organosulfur compounds, most of all to oil-soluble allyl sulfides, such as diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS). This study evaluated the effectiveness of garlic-derived allyl sulfides in influencing peroxidative processes, levels of thiols and sulfane sulfur and its metabolic enzymes in normal mouse liver cells. Various allyl sulfides (DAS, DADS and DATS) dissolved in corn oil were given intraperitoneally to mice for 10 days. After sacrificing the mice, biochemical assays were performed in liver homogenates and in plasma in order to establish liver function. All allyl sulfides under study had a beneficial effect in the mouse liver since they decreased reactive oxygen species and malondialdehyde levels and increased glutathione S-transferase activity and non-protein sulfhydryl group level. Moreover, DADS and DATS elevated total sulfane sulfur pool and activity of sulfane sulfur biosynthetic enzymes. The increase in sulfane sulfur level entailed augmentation of its antioxidant and regulatory capacities. Garlic-derived allyl sulfides exhibited antioxidant action in the liver and elevated anaerobic cysteine metabolism leading to the formation of sulfane sulfur-containing compounds. Thus, DADS and DATS showed beneficial action in the liver, which can be used for protection of normal liver cells during chemotherapy or for alleviation of liver damage.

Thiol levels, protein carbonylation and anaerobic sulfur metabolism in erythrocytes of peritoneal dialysis and predialysis patients.

AIM: The goal of the present study was to investigate the changes in sulfur metabolism in erythrocytes of end-stage renal failure patients. METHODS: The following substances were determined in erythrocytes of chronic kidney disease patients before dialysis, patients treated with continuous ambulatory peritoneal dialysis, and in a group of healthy volunteers: (i) sulfane sulfur level and activity of the enzymes involved in its metabolism and in cyanide detoxification; (ii) concentration of total and non-protein sulfhydryl groups -SH; and (iii) protein carbonylation rate. RESULTS: Erythrocytes of chronic kidney disease patients in predialysis period contained lower levels of sulfane sulfur, non-protein thiols, total thiols and 3-mercaptopyruvate sulfotransferase. On the other hand, in erythrocytes of end-stage renal failure patients treated with continuous ambulatory peritoneal dialysis, sulfane sulfur, non-protein thiols, total thiols and 3-mercaptopyruvate sulfotransferase activity remained at the level observed in healthy controls. These changes indicate a disturbed thiol balance and anaerobic cysteine metabolism in non-dialysis patients, whereas continuous ambulatory peritoneal dialysis patients did not show these disorders. γ-Cystathionase activity was equally elevated in predialysis period and in peritoneal dialysis patients, which means that chronic kidney disease pathology is accompanied by an increased expression of this enzymatic activity in erythrocytes. Erythrocytic rhodanese activity was unchanged and stayed at the control level in both groups. Protein carbonylation rate was equally enhanced in both patient groups, which indicated acceleration of oxidative processes and inability of continuous ambulatory peritoneal dialysis to correct these changes in erythrocytes. CONCLUSION: The CAPD as a replacement therapy helps to preserve thiol levels and anaerobic sulfur metabolism in erythrocytes.