Pharmacological Reports 66 (2014) 87–92
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Original research article
The effect of lipoic acid on cyanate toxicity in the rat heart Maria Sokołowska a,*, Maciej Kostan´ski a, Elz˙bieta Lorenc-Koci b, Anna Bilska a, Małgorzata Iciek a, Lidia Włodek a a b
The Chair of Medical Biochemistry, Jagiellonian University, Collegium Medicum, Krako´w, Poland Department of Neuropsychopharmacology, Institute of Pharmacology, Polish Academy of Science, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 12 February 2013 Received in revised form 26 June 2013 Accepted 2 August 2013 Available online 1 February 2014 Keywords: Cyanate Lipoic acid Cardiovascular diseases Ur(a)emia Sulfane sulfur
A B S T R A C T
Background: Cyanate is a uremic toxin formed principally via spontaneous urea biodegradation. Its active isoform, isocyanate, is capable of reaction with proteins by –N and –S carbamoylation, which influences their structure and function. Sulfurtransferases implicated in anaerobic cysteine transformation and cyanide detoxification belong to the enzymes possessing –SH groups in their active centers. The present studies aimed to demonstrate the effect of cyanate and lipoic acid on the activity of these enzymes as well as on the level of antioxidants and prooxidants in the rat heart. Methods: Wistar rats, which received intraperitoneal injections of cyanate and lipoic acid alone and in combination were sacrificed 2.5 h after the first injection. The hearts were isolated and homogenized in phosphate buffer and next biochemical assays were performed comprising determination of the level of glutathione, malondialdehyde and sulfane sulfur and the activity of antioxidant enzymes as well as glutathione S-transferase and gamma glutamyl transferase. Results: Sulfurtransferases and glutathione S-transferase were deactivated by cyanate treatment. It was accompanied by the decreased level of glutathione and sulfane sulfur and the increased level of reactive oxygen species and malondialdehyde. In parallel, antioxidant enzymes: catalase, glutathione peroxidase and gamma glutamyl transferase were activated under such circumstances. Lipoic acid, administered in combination with cyanate prevented the decrease in the level of glutathione and reduction of a pool of sulfane sulfur-containing compounds, concomitantly preserving the activity of antioxidant enzymes. Conclusions: Since uremia, characterized by the elevated cyanate/isocyanate level, is accompanied by frequent cases of cardiovascular diseases, the addition of lipoic acid to the therapy seems promising in prophylaxis of heart diseases in uremic patients. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Isocyanate, an active isoform of cyanate, is capable of reaction with proteins by –N and –S carbamoylation, thereby influencing their structure and function. In biological systems cyanate shows the highest reactivity with sulfhydryl (SH) groups of peptides and proteins [2,42]. Since the enzymes participating in anaerobic cysteine transformation, namely cystathionase (CSE EC 4.2.1.15) and mercaptopyruvate sulfurtransferase (MST EC 2.8.1.2), as well as the sulfane sulfur transporting enzyme, thiosulfate sulfurtransferase
Abbreviations: OCN, cyanate; NCO, isocyanate; DHLA, dihydrolipoic acid; S*, sulfane sulfur; GSH, glutathione; ROS, reactive oxygen species; GPx, glutathione peroxidase; TST, rhodanese; MST, mercaptopyruvate sulfurtransferase; CSE, cystathionase; CN, cyanide; SCN, thiocyanate; gGT, g-glutamyl transferase; cLDL, carbamoylated LDL. * Corresponding author. E-mail addresses: [email protected] (M. Sokołowska), [email protected] (E. Lorenc-Koci), [email protected] (A. Bilska), [email protected] (M. Iciek), [email protected] (L. Włodek).
(TST EC 2.8.1.1) possess –SH groups in their active centers [33], isocyanate can potentially influence the activity of these enzymes. Consequently, it can influence cysteine transformation to hydrogen sulfide (H2S) and sulfane sulfur compounds, i.e. polysulfides (R-SSn*-S-R), thiosulfate (S2O32), persulfides (R-S-S*H) and others, which contain a labile highly reactive sulfur atom (S*) in 0 or 1 oxidation state, covalently bound to another sulfur atom. Sulfane sulfur compounds are formed by biodegradation of mixed disulfide of homocysteine and cysteine and b-elimination of L-cysteine, and both processes are catalyzed by CSE. On the other hand, H2S is formed by decomposition of cysteine (b- and a,b-elimination) in the presence of CSE, desulfuration of 3-mercaptopyruvate in the presence of MST and during reactions of persulfides with an excess of thiols [8,15,32]. S*-containing compounds play an important role in cyanide (CN) to thiocyanate (SCN) detoxification catalyzed by TST and MST, as well as by CSE [22,32]. Results of our previous studies demonstrated peroxidative character of cyanate remaining in balance with isocyanate, its ability to lower glutathione and sulfane sulfur levels and to inhibit sulfurtransferase activities in the rat liver. On the other hand, these
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.08.009
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studies demonstrated a stimulating effect of lipoic acid on the glutathione (GSH) production and activity of sulfurtransferases, and its antioxidant properties confirmed by a number of reports [5,6,28,29]. Since cyanate/isocyanate are the spontaneous biodegradation products of urea [4,10], the level of which is extremely high in uremic patients (higher than other substances), the results of our studies can to some extent be applicable to uremia. Uremia is characterized by retention of fluid and electrolytes, the considerable part of which affects the organism inducing negative biological changes. These are the so-called uremic toxins and urea is one of them [36,37]. Currently used defense strategies against uremic toxins entail intensification of their removal or attenuation of their toxic effects [36]. Since uremia is accompanied by frequent cases of cardiovascular diseases, we decided to perform similar investigations on the rat heart [9,36,44]. The present studies aimed to demonstrate in vivo the effect of the combined treatment with cyanate and lipoic acid both on the
anaerobic cysteine transformation, and peroxidative processes in the rat cardiomyocytes in relation to cyanate and lipoic acid alonetreated animals. Lipoic acid, which can react with cyanate due to the presence of two –SH groups in its molecule [5] seems to be an agent which can diminish cyanate toxicity and potentially decrease the risk of heart diseases in uremic patients. Materials and methods Animals The experiments were carried out on male Wistar rats weighing approximately 250 g. The animals were kept under standard laboratory conditions and were fed a standard diet. All procedures were approved by the Ethics Committee for Animal Research in Krako´w. Animals were assigned to 4 groups, containing 6–7 animals each. Groups were treated as follows:
Group 1
Saline
saline
saline
0
30
60
Saline
cyanate
saline
0
30
60
lipoic acid
saline
lipoic acid
0
30
60
lipoic acid
cyanate
lipoic acid
0
30
60
sacrifice
150 min
Group 2
sacrifice
150 min
Group 3
sacrifice
150 min
Group 4
sacrifice
150 min
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Table 1 The influence of cyanate, lipoate and the combined treatment with cyanate and lipoate on the sulfurtransferases activities and sulfane sulfur level in the rat heart. Enzymes activities and sulfane sulfur level
TST MST CSE S*
Groups of animals Saline
Lipoate
Cyanate
Lipoate + cyanate
0.899 0.115 9.223 0.595 0.014 0.002 2.551 0.201
0.875 0.16 8.643 0.889 0.015 0.002 3.032 0.096a
0.623 0.074a 6.946 1.111a 0.011 0.002a 2.194 0.044b
0.835 0.063c 8.333 0.941d 0.013 0.001d 2.772 0.263c
TST – rhodanese, MST – 3-mercaptopyruvate sulfotransferase, CST – cystathionase [U/mg of protein] (mmol/(min mg of protein)), and S* – sulfane sulfur [nmol/mg of protein]. a Post hoc LSD test, differences statistically significant with respect to the control (saline) group p < 0.001. b p < 0.01. c Differences are statistically significant with respect to the cyanate group (p < 0.001). d p < 0.05.
Group 1 received 3 intraperitoneal injections of physiological saline (0.9% NaCl) at 30 min intervals. Group 2 was injected with 0.9% NaCl, cyanate - KCNO (200 mg/ kg b.w.) 30 min later and saline after the next 30 min. The dose of cyanate was determined according to the data presented in the article by Tor-Agbidye et al. [34]. Group 3 was given lipoic acid (50 mg/kg b.w.), 0.9% NaCl 30 min later and lipoic acid (50 mg/kg b.w.) after the next 30 min. Group 4 was administered lipoic acid (50 mg/kg b.w.), KCNO (200 mg/kg b.w.) 30 min later and again lipoic acid (50 mg/kg b.w.) after the next 30 min.
9. Activity of glutathione peroxidase (GPx) was assayed by the method of Flohe and Gunzler [11]. 10. Glutathione S-transferase (GST) activity assay was determined according to the method of Habig et al. [13]. 11. Gamma-glutamyl transferase (gGT) activity was assayed by the method of Orłowski and Meister [25]. 12. Protein content was determined according to Lowry’s method [18].
Animals were sacrificed 2.5 h after the first injection, the heart tissues were isolated and stored at 80 8C until further experiments were performed.
The results are presented as the mean plus standard deviation (SD) of the mean for each group. Statistically significant differences between groups were calculated using a two-way ANOVA, followed (if significant) by an LSD-test.
Statistical analysis
Biological material Results The heart tissues were weighed and homogenized in 4 ml of the ice-cold phosphate buffer, pH 7.4 per 1 g of the tissue. Chemicals Potassium cyanide (KCN), dithiothreitol, p-phenylenodiamine, N-ethylmaleimide (NEM), b-nicotinamide adenine dinucleotide reduced form (NADH), 5,50 -dithio-bis-2-nitrobenzoic acid (DTNB), mercaptopyruvic acid sodium salt, L-homoserine, pyridoxal 50 phosphate monohydrate, 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate, lactic dehydrogenase (LDH), potassium cyanate (KNCO), and a-lipoic acid sodium salt were provided by Sigma Chemical Co. (St. Louis, MO, USA). Formaldehyde, ferric chloride (FeCl3), thiosulfate and all the other reagents were obtained from the Polish Chemical Reagent Company (P.O.Ch, Gliwice, Poland).
Cyanate decreased activity of the enzymes participating in the formation and transport of sulfane sulfur: rhodanese (TST), cystathionase (CSE) and mercaptopyruvate sulfurtransferase (MST), which was accompanied by the decrease in sulfane sulfur (S*) level in the rat heart (Table 1). Lipoic acid alone did not affect sulfurtransferase activities. However, when administered in combination with cyanate, it elevated sulfane sulfur level and sulfurtransferase activities (TST, CST and MST) in relation to the cyanate-treated group (Table 1). Cyanate statistically significantly lowered NPSH level; while lipoic acid given alone significantly increased NPSH level in relation to the control. The combined cyanate plus lipoic acid treatment significantly enhanced NPSH level vs. cyanate alone and slightly elevated it vs. control group (Fig. 1). Cyanate treatment was accompanied by a significant increase in ROS and MDA level. In opposite, lipoate alone significantly lowered
Methods 1. Sulfane sulfur (S*) level was assayed by a cold cyanolysis method [43]. 2. Activity of rhodanese (TST) was determined using the method of So¨rbo [30]. 3. MST was assayed with the method of Valentine and Frankenfeld [35]. 4. Cystathionase (CSE) activity was determined by the method of Matsuo and Greenberg [20]. 5. Nonprotein sulfhydryl groups (NPSH) were assayed according to the method of Ellman [26]. 6. Reactive oxygen species (ROS) level was assayed by the method of Bondy and Guo [7]. 7. Determination of lipid peroxidation (MDA) was based on the method of Ohkawa et al. [24]. 8. Catalase activity was determined by the method of Aebi [1].
Fig. 1. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the NPSH level (expressed in nmole/mg of protein) in the rat hearts. Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of differences in post hoc LSD test, DDD ***p < 0.001, *p < 0.05 vs. control (C); p < 0.001 vs. cyanate-treated group (CY).
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Fig. 2. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the level of ROS (expressed in nmole of 2,7-dichlorofluorescein/mg of protein) in the rat hearts. Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of DD p < 0.01 vs. CY group. differences in post hoc LSD test, ***p < 0.001 vs. control (C);
Fig. 5. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the enzymatic activities of glutathione peroxidase (GPx) in the rat hearts. Activity of glutathione peroxidase was expressed in U/mg (mmol of GSH, oxidized in the presence of enzyme during 1 min/mg protein). Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of differences in post hoc LSD test, **p < 0.01, *p < 0.05 vs. control (C) group.
ROS and MDA level. After lipoic acid administration in combination with cyanate, free radical and MDA concentrations dropped to the control level (Figs. 2 and 3). Catalase and GPx activities were raised after cyanate treatment in relation to the control group (Figs. 4 and 5). Lipoic acid alone significantly lowered catalase activity, but did not affect GPx activity. After lipoic acid administered in combination with cyanate, catalase and GPx activities were observed to be unchanged in relation to the cyanate group (Figs. 4 and 5). gGT activity was elevated after cyanate and lowered after lipoic acid treatment in comparison with the control group. Lipoic acid administered in combination with cyanate did not affect gGT activity (Fig. 6). Fig. 6. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the enzymatic activities of gglutamyl transferase (g-GT) expressed in U/mg (mmol of p-nitroaniline formed during 1 min/mg protein) in the rat hearts. Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of differences in post hoc LSD test, ***p < 0.001, *p < 0.05, vs. control (C).
Glutathione transferase activity (GST) declined after cyanate treatment (Fig. 7) while lipoic acid treatment did not change it vs. control group. On the contrary, GST activity was lowered vs. control, but unchanged vs. cyanate group after the combined treatment with cyanate and lipoic acid (Fig. 7). Discussion
Fig. 3. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the level of MDA (expressed in nmole of thiobarbituric acid/mg of protein) in the rat hearts. Data are presented as mean SD, n = 18 for each group. Symbols indicates significance of differences in post DDD hoc LSD test, ***p < 0.001, **p < 0.01, vs. control (C); p < 0.001 vs. CY group.
The present in vivo studies evidenced the cyanate-induced decrease in sulfane sulfur (S*) level in the rat heart which was
Fig. 4. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the enzymatic activities of catalase in the rat hearts. Activity of catalase was expressed in U/mg (mmol of H2O2 degraded under the catalytic influence of enzyme/mg protein/min). Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of differences in post hoc LSD test, *p < 0.05 vs. control (C).
Fig. 7. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the enzymatic activities of glutathione transferase (GST) in the rat hearts. Activity of glutathione transferase was expressed in U/mg (mmol of 2,4-dinitrofenyl-S-glutathione formed during 1 min/mg protein). Data are presented as mean SD, n = 7 for each group. Symbols indicates significance of differences in post hoc LSD test, **p < 0.01, *p < 0.05 vs. control (C) group.
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associated with a drop in sulfurtransferase activities (Table 1). The decrease in the level of S*-containing compounds observed in our experiments can be explained by a partial inhibition of CSE activity, which also suggests the inhibition of cystathionine biodegradation to cysteine [32]. On the other hand, the drop of cysteine, a rate-limiting amino acid in glutathione synthesis, leads to the decrease in the level of GSH which constitutes about 95% of the pool of non-protein sulfhydryl (NPSH) groups. Further, the drop in GSH level, can be aggravated by a direct reaction of cyanate with –SH groups [31,42]. Theoretically, a harmful effect of cyanate on GSH level could be abolished by gGT activation (Fig. 6), since this enzyme participates in GSH decomposition, promoting de novo synthesis of this compound [21]. However, the concomitant increase in ROS and MDA (Figs. 2 and 3) levels indicates that cyanate enhances pro-oxidant processes in the heart cells. gGT, which catalyzes GSH biodegradation to cysteinylglycine (Cysgly), can be implicated in this process since Cys-gly generates free radicals in the presence of ferric ions. Such action of gGT was confirmed by the presence of this enzyme in atherosclerotic foci and by its participation in low-density lipoprotein cholesterol oxidation. The elevated gGT activity has recently been also accepted as an independent risk factor for death from heart diseases [19]. Besides, the cyanate-induced activation of catalase and GPx (Figs. 4 and 5) can indicate mobilization of cardiomyocytes in oxidative environment. Although a majority of researchers have demonstrated a drop in antioxidant enzyme activities during oxidative stress, a similar compensatory effect, i.e. the increase in GPx activity, was seen in the rabbit heart during daunorubicin-induced oxidative stress [38]. In contrast, the cyanateinduced decrease in GST activity (Fig. 7), which participates in the removal of xenobiotics in the form of glutathione S-conjugates was probably caused by GSH (a GST substrate) depletion. The cyanate-induced decline in S* level and in TST and MST activities may also result in lowering of the ability to detoxify the highly toxic cyanide (CN) to the less toxic thiocyanate (SCN). Since CN inhibits respiratory chain in the mitochondria, impairing ATP production, its removal can be also significant for normal heart function [14,16,23]. CSE and to a lesser extent MST are the major H2S producing enzymes in the cardiovascular system [8,15,32]. Thus, the blockade of these enzymes by cyanate can lead to the limitation of H2S-mediated vasorelaxation and signal transduction. Cyanate may also contribute to pathogenesis of atherosclerosis. Low-density lipoproteins both oxidized (oxLDL) and carbamoylated (cLDL) are taken in by macrophages which transform into foam cells and later give rise to atherosclerotic plaques. Since cLDL promotes also in vitro smooth muscle cell proliferation and apoptosis of endothelial cells, it can be expected that they participate both in the initiation (endothelial cell damage) and progression of atherosclerosis [3,27,40]. Many papers have described ROS scavenging properties of both lipoic acid and dihydrolipoic acid (DHLA), also useful in the prevention of cardiovascular diseases [5,12,28], which was confirmed by our studies, as well. The decrease in ROS and MDA level (Figs. 2 and 3) after the combined treatment with cyanate and lipoic acid, together with the increase in GSH level (Fig. 1) and restoration of normal catalase activity (Fig. 4) indicates that lipoic acid has a normalizing effect on the antioxidant status in cardiomyocytes. The increased CSE activity which elevates the level of cysteine, a GSH precursor, may contribute to that effect. The parallel increase in TST and MST activities (Table 1) probably promotes recovery of cyanide detoxifying abilities in cardiomyocytes and by increasing of S* level, further increases their antioxidant power. It should be emphasized that the S*-containing compounds are stronger antioxidants than thiol compounds, including GSH. The ability of lipoic acid to restitute the activity of TST and other sulfurtransferases possessing S* binding domain
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may be attributed to the fact that it can be an acceptor of S* from their active centers [39]. Cystathionase and mercaptopyruvate sulfurtransferase activation by the combined cyanate and lipoic acid treatment can elevate the level of H2S, which shows antioxidant properties and acts as a vasodilator, and this can contribute to the improvement of heart function. It was corroborated, for instance, by the alleviation of ventricular dysfunction and inhibition of myocardial apoptosis in the developing heart failure model after the administrations of NaHS, a H2S precursor [41]. In addition, lipoate ability to chelate Fe2+ cations can lead to the diminution of pro-oxidant action of cysteinylglycine. Further, DHLA can suppress LDL oxidation and can repair oxidative protein damage, thereby contributing to reconstruction of the damaged vascular endothelium [5]. Since lipoate is a target of carbamoylation, its administration can decrease LDL carbamoylation thus lowering the risk of atherosclerosis progression. Conclusions Cyanate showed pro-oxidant action in cardiomyocytes which was manifested by the elevation of ROS and MDA level and gGT activity in parallel with the decrease in the main cellular antioxidant GSH and the pool of sulfane sulfur (S*)-containing compounds. It was accompanied by the decreased activity of sulfurtransferases and the increased activities of antioxidant enzymes, i.e. catalase and GPx which can suggest adaptation of cardiomyocytes under oxidative conditions. Lipoic acid administered in combination with cyanate lowered ROS and MDA to the control level thus restoring physiological GSH and S* concentrations and sulfurtransferase activities, which could result in the increase in H2S level. It suggests a possibility to recover the capability of cyanide detoxification and vasorelaxation in the rat heart after lipoic acid treatment. In addition, the ability of lipoic acid to maintain H2S homeostasis can be decisive for the heart function since it was proven that the treatment with the H2S precursor, NaHS could protect cardiomyocytes of patients with heart problems from apoptosis. Conflict of interest The authors declare that there are no conflicts of interest. Funding No financial support for the conduct of the research and preparation of the article. References [1] Aebi H. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysis, vol. 77. Weinheim: Verlag: Chemic Academic Press, Inc.; 1981. p. 325. [2] Arlandson M, Decker T, Roongta VA, Bonilla L, Mayo KH, MacPherson JC, et al. Eosinophil peroxidase oxidation of thiocyanate. J Biol Chem 2001;276:215–24. [3] Asci G, Basci A, Shah SV, Basnakian A, Toz H, Ozkahya M, et al. Carbamylated low-density lipoproteins induced proliferation and increases adhesion molecule expression of human coronary artery smooth muscle cells. Nephrology 2008;13:480–6. [4] Beddie C, Webster CE, Hall MB. Urea decomposition facilitated by a urease model complex: a theoretical investigation. Dalton Trans 2005;21:3542–51. [5] Biewenga G, Haenen G, Bast A. The pharmacology of the oxidant lipoic acid. Gen Pharmacol 1997;29:315–31. [6] Bilska A, Dudek M, Iciek M, Kwiecien´ I, Sokołowska-Jez˙ewicz M, Filipek B, et al. Biological actions of lipoic acid associated with sulfane sulfur metabolism. Pharmacol Rep 2008;60:225–32. [7] Bondy SC, Guo SX. Effect of ethanol treatment on indices of cumulative oxidative stress. Eur J Pharmacol 1994;270:349–55. [8] Chen X, Jhee KH, Kruger WD. Production of the neuromodulator H2S by cystathionine b-synthase via the condensation of cysteine and homocysteine. J Biol Chem 2004;279:52082–86.
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
The effect of lipoic acid on cyanate toxicity in the rat heart Maria Sokołowska a,*, Maciej Kostan´ski a, Elz˙bieta Lorenc-Koci b, Anna Bilska a, Małgorzata Iciek a, Lidia Włodek a a b
The Chair of Medical Biochemistry, Jagiellonian University, Collegium Medicum, Krako´w, Poland Department of Neuropsychopharmacology, Institute of Pharmacology, Polish Academy of Science, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 12 February 2013 Received in revised form 26 June 2013 Accepted 2 August 2013 Available online 1 February 2014 Keywords: Cyanate Lipoic acid Cardiovascular diseases Ur(a)emia Sulfane sulfur
A B S T R A C T
Background: Cyanate is a uremic toxin formed principally via spontaneous urea biodegradation. Its active isoform, isocyanate, is capable of reaction with proteins by –N and –S carbamoylation, which influences their structure and function. Sulfurtransferases implicated in anaerobic cysteine transformation and cyanide detoxification belong to the enzymes possessing –SH groups in their active centers. The present studies aimed to demonstrate the effect of cyanate and lipoic acid on the activity of these enzymes as well as on the level of antioxidants and prooxidants in the rat heart. Methods: Wistar rats, which received intraperitoneal injections of cyanate and lipoic acid alone and in combination were sacrificed 2.5 h after the first injection. The hearts were isolated and homogenized in phosphate buffer and next biochemical assays were performed comprising determination of the level of glutathione, malondialdehyde and sulfane sulfur and the activity of antioxidant enzymes as well as glutathione S-transferase and gamma glutamyl transferase. Results: Sulfurtransferases and glutathione S-transferase were deactivated by cyanate treatment. It was accompanied by the decreased level of glutathione and sulfane sulfur and the increased level of reactive oxygen species and malondialdehyde. In parallel, antioxidant enzymes: catalase, glutathione peroxidase and gamma glutamyl transferase were activated under such circumstances. Lipoic acid, administered in combination with cyanate prevented the decrease in the level of glutathione and reduction of a pool of sulfane sulfur-containing compounds, concomitantly preserving the activity of antioxidant enzymes. Conclusions: Since uremia, characterized by the elevated cyanate/isocyanate level, is accompanied by frequent cases of cardiovascular diseases, the addition of lipoic acid to the therapy seems promising in prophylaxis of heart diseases in uremic patients. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Isocyanate, an active isoform of cyanate, is capable of reaction with proteins by –N and –S carbamoylation, thereby influencing their structure and function. In biological systems cyanate shows the highest reactivity with sulfhydryl (SH) groups of peptides and proteins [2,42]. Since the enzymes participating in anaerobic cysteine transformation, namely cystathionase (CSE EC 4.2.1.15) and mercaptopyruvate sulfurtransferase (MST EC 2.8.1.2), as well as the sulfane sulfur transporting enzyme, thiosulfate sulfurtransferase
Abbreviations: OCN, cyanate; NCO, isocyanate; DHLA, dihydrolipoic acid; S*, sulfane sulfur; GSH, glutathione; ROS, reactive oxygen species; GPx, glutathione peroxidase; TST, rhodanese; MST, mercaptopyruvate sulfurtransferase; CSE, cystathionase; CN, cyanide; SCN, thiocyanate; gGT, g-glutamyl transferase; cLDL, carbamoylated LDL. * Corresponding author. E-mail addresses: [email protected] (M. Sokołowska), [email protected] (E. Lorenc-Koci), [email protected] (A. Bilska), [email protected] (M. Iciek), [email protected] (L. Włodek).
(TST EC 2.8.1.1) possess –SH groups in their active centers [33], isocyanate can potentially influence the activity of these enzymes. Consequently, it can influence cysteine transformation to hydrogen sulfide (H2S) and sulfane sulfur compounds, i.e. polysulfides (R-SSn*-S-R), thiosulfate (S2O32), persulfides (R-S-S*H) and others, which contain a labile highly reactive sulfur atom (S*) in 0 or 1 oxidation state, covalently bound to another sulfur atom. Sulfane sulfur compounds are formed by biodegradation of mixed disulfide of homocysteine and cysteine and b-elimination of L-cysteine, and both processes are catalyzed by CSE. On the other hand, H2S is formed by decomposition of cysteine (b- and a,b-elimination) in the presence of CSE, desulfuration of 3-mercaptopyruvate in the presence of MST and during reactions of persulfides with an excess of thiols [8,15,32]. S*-containing compounds play an important role in cyanide (CN) to thiocyanate (SCN) detoxification catalyzed by TST and MST, as well as by CSE [22,32]. Results of our previous studies demonstrated peroxidative character of cyanate remaining in balance with isocyanate, its ability to lower glutathione and sulfane sulfur levels and to inhibit sulfurtransferase activities in the rat liver. On the other hand, these
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.08.009
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studies demonstrated a stimulating effect of lipoic acid on the glutathione (GSH) production and activity of sulfurtransferases, and its antioxidant properties confirmed by a number of reports [5,6,28,29]. Since cyanate/isocyanate are the spontaneous biodegradation products of urea [4,10], the level of which is extremely high in uremic patients (higher than other substances), the results of our studies can to some extent be applicable to uremia. Uremia is characterized by retention of fluid and electrolytes, the considerable part of which affects the organism inducing negative biological changes. These are the so-called uremic toxins and urea is one of them [36,37]. Currently used defense strategies against uremic toxins entail intensification of their removal or attenuation of their toxic effects [36]. Since uremia is accompanied by frequent cases of cardiovascular diseases, we decided to perform similar investigations on the rat heart [9,36,44]. The present studies aimed to demonstrate in vivo the effect of the combined treatment with cyanate and lipoic acid both on the
anaerobic cysteine transformation, and peroxidative processes in the rat cardiomyocytes in relation to cyanate and lipoic acid alonetreated animals. Lipoic acid, which can react with cyanate due to the presence of two –SH groups in its molecule [5] seems to be an agent which can diminish cyanate toxicity and potentially decrease the risk of heart diseases in uremic patients. Materials and methods Animals The experiments were carried out on male Wistar rats weighing approximately 250 g. The animals were kept under standard laboratory conditions and were fed a standard diet. All procedures were approved by the Ethics Committee for Animal Research in Krako´w. Animals were assigned to 4 groups, containing 6–7 animals each. Groups were treated as follows:
Group 1
Saline
saline
saline
0
30
60
Saline
cyanate
saline
0
30
60
lipoic acid
saline
lipoic acid
0
30
60
lipoic acid
cyanate
lipoic acid
0
30
60
sacrifice
150 min
Group 2
sacrifice
150 min
Group 3
sacrifice
150 min
Group 4
sacrifice
150 min
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Table 1 The influence of cyanate, lipoate and the combined treatment with cyanate and lipoate on the sulfurtransferases activities and sulfane sulfur level in the rat heart. Enzymes activities and sulfane sulfur level
TST MST CSE S*
Groups of animals Saline
Lipoate
Cyanate
Lipoate + cyanate
0.899 0.115 9.223 0.595 0.014 0.002 2.551 0.201
0.875 0.16 8.643 0.889 0.015 0.002 3.032 0.096a
0.623 0.074a 6.946 1.111a 0.011 0.002a 2.194 0.044b
0.835 0.063c 8.333 0.941d 0.013 0.001d 2.772 0.263c
TST – rhodanese, MST – 3-mercaptopyruvate sulfotransferase, CST – cystathionase [U/mg of protein] (mmol/(min mg of protein)), and S* – sulfane sulfur [nmol/mg of protein]. a Post hoc LSD test, differences statistically significant with respect to the control (saline) group p < 0.001. b p < 0.01. c Differences are statistically significant with respect to the cyanate group (p < 0.001). d p < 0.05.
Group 1 received 3 intraperitoneal injections of physiological saline (0.9% NaCl) at 30 min intervals. Group 2 was injected with 0.9% NaCl, cyanate - KCNO (200 mg/ kg b.w.) 30 min later and saline after the next 30 min. The dose of cyanate was determined according to the data presented in the article by Tor-Agbidye et al. [34]. Group 3 was given lipoic acid (50 mg/kg b.w.), 0.9% NaCl 30 min later and lipoic acid (50 mg/kg b.w.) after the next 30 min. Group 4 was administered lipoic acid (50 mg/kg b.w.), KCNO (200 mg/kg b.w.) 30 min later and again lipoic acid (50 mg/kg b.w.) after the next 30 min.
9. Activity of glutathione peroxidase (GPx) was assayed by the method of Flohe and Gunzler [11]. 10. Glutathione S-transferase (GST) activity assay was determined according to the method of Habig et al. [13]. 11. Gamma-glutamyl transferase (gGT) activity was assayed by the method of Orłowski and Meister [25]. 12. Protein content was determined according to Lowry’s method [18].
Animals were sacrificed 2.5 h after the first injection, the heart tissues were isolated and stored at 80 8C until further experiments were performed.
The results are presented as the mean plus standard deviation (SD) of the mean for each group. Statistically significant differences between groups were calculated using a two-way ANOVA, followed (if significant) by an LSD-test.
Statistical analysis
Biological material Results The heart tissues were weighed and homogenized in 4 ml of the ice-cold phosphate buffer, pH 7.4 per 1 g of the tissue. Chemicals Potassium cyanide (KCN), dithiothreitol, p-phenylenodiamine, N-ethylmaleimide (NEM), b-nicotinamide adenine dinucleotide reduced form (NADH), 5,50 -dithio-bis-2-nitrobenzoic acid (DTNB), mercaptopyruvic acid sodium salt, L-homoserine, pyridoxal 50 phosphate monohydrate, 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate, lactic dehydrogenase (LDH), potassium cyanate (KNCO), and a-lipoic acid sodium salt were provided by Sigma Chemical Co. (St. Louis, MO, USA). Formaldehyde, ferric chloride (FeCl3), thiosulfate and all the other reagents were obtained from the Polish Chemical Reagent Company (P.O.Ch, Gliwice, Poland).
Cyanate decreased activity of the enzymes participating in the formation and transport of sulfane sulfur: rhodanese (TST), cystathionase (CSE) and mercaptopyruvate sulfurtransferase (MST), which was accompanied by the decrease in sulfane sulfur (S*) level in the rat heart (Table 1). Lipoic acid alone did not affect sulfurtransferase activities. However, when administered in combination with cyanate, it elevated sulfane sulfur level and sulfurtransferase activities (TST, CST and MST) in relation to the cyanate-treated group (Table 1). Cyanate statistically significantly lowered NPSH level; while lipoic acid given alone significantly increased NPSH level in relation to the control. The combined cyanate plus lipoic acid treatment significantly enhanced NPSH level vs. cyanate alone and slightly elevated it vs. control group (Fig. 1). Cyanate treatment was accompanied by a significant increase in ROS and MDA level. In opposite, lipoate alone significantly lowered
Methods 1. Sulfane sulfur (S*) level was assayed by a cold cyanolysis method [43]. 2. Activity of rhodanese (TST) was determined using the method of So¨rbo [30]. 3. MST was assayed with the method of Valentine and Frankenfeld [35]. 4. Cystathionase (CSE) activity was determined by the method of Matsuo and Greenberg [20]. 5. Nonprotein sulfhydryl groups (NPSH) were assayed according to the method of Ellman [26]. 6. Reactive oxygen species (ROS) level was assayed by the method of Bondy and Guo [7]. 7. Determination of lipid peroxidation (MDA) was based on the method of Ohkawa et al. [24]. 8. Catalase activity was determined by the method of Aebi [1].
Fig. 1. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the NPSH level (expressed in nmole/mg of protein) in the rat hearts. Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of differences in post hoc LSD test, DDD ***p < 0.001, *p < 0.05 vs. control (C); p < 0.001 vs. cyanate-treated group (CY).
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Fig. 2. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the level of ROS (expressed in nmole of 2,7-dichlorofluorescein/mg of protein) in the rat hearts. Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of DD p < 0.01 vs. CY group. differences in post hoc LSD test, ***p < 0.001 vs. control (C);
Fig. 5. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the enzymatic activities of glutathione peroxidase (GPx) in the rat hearts. Activity of glutathione peroxidase was expressed in U/mg (mmol of GSH, oxidized in the presence of enzyme during 1 min/mg protein). Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of differences in post hoc LSD test, **p < 0.01, *p < 0.05 vs. control (C) group.
ROS and MDA level. After lipoic acid administration in combination with cyanate, free radical and MDA concentrations dropped to the control level (Figs. 2 and 3). Catalase and GPx activities were raised after cyanate treatment in relation to the control group (Figs. 4 and 5). Lipoic acid alone significantly lowered catalase activity, but did not affect GPx activity. After lipoic acid administered in combination with cyanate, catalase and GPx activities were observed to be unchanged in relation to the cyanate group (Figs. 4 and 5). gGT activity was elevated after cyanate and lowered after lipoic acid treatment in comparison with the control group. Lipoic acid administered in combination with cyanate did not affect gGT activity (Fig. 6). Fig. 6. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the enzymatic activities of gglutamyl transferase (g-GT) expressed in U/mg (mmol of p-nitroaniline formed during 1 min/mg protein) in the rat hearts. Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of differences in post hoc LSD test, ***p < 0.001, *p < 0.05, vs. control (C).
Glutathione transferase activity (GST) declined after cyanate treatment (Fig. 7) while lipoic acid treatment did not change it vs. control group. On the contrary, GST activity was lowered vs. control, but unchanged vs. cyanate group after the combined treatment with cyanate and lipoic acid (Fig. 7). Discussion
Fig. 3. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the level of MDA (expressed in nmole of thiobarbituric acid/mg of protein) in the rat hearts. Data are presented as mean SD, n = 18 for each group. Symbols indicates significance of differences in post DDD hoc LSD test, ***p < 0.001, **p < 0.01, vs. control (C); p < 0.001 vs. CY group.
The present in vivo studies evidenced the cyanate-induced decrease in sulfane sulfur (S*) level in the rat heart which was
Fig. 4. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the enzymatic activities of catalase in the rat hearts. Activity of catalase was expressed in U/mg (mmol of H2O2 degraded under the catalytic influence of enzyme/mg protein/min). Data are presented as mean SD, n = 6 for each group. Symbols indicates significance of differences in post hoc LSD test, *p < 0.05 vs. control (C).
Fig. 7. The effect of acute administration of cyanate (CY-200 mg/kg) and lipoate (L50 mg/kg twice), alone and in combination (LCY) on the enzymatic activities of glutathione transferase (GST) in the rat hearts. Activity of glutathione transferase was expressed in U/mg (mmol of 2,4-dinitrofenyl-S-glutathione formed during 1 min/mg protein). Data are presented as mean SD, n = 7 for each group. Symbols indicates significance of differences in post hoc LSD test, **p < 0.01, *p < 0.05 vs. control (C) group.
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associated with a drop in sulfurtransferase activities (Table 1). The decrease in the level of S*-containing compounds observed in our experiments can be explained by a partial inhibition of CSE activity, which also suggests the inhibition of cystathionine biodegradation to cysteine [32]. On the other hand, the drop of cysteine, a rate-limiting amino acid in glutathione synthesis, leads to the decrease in the level of GSH which constitutes about 95% of the pool of non-protein sulfhydryl (NPSH) groups. Further, the drop in GSH level, can be aggravated by a direct reaction of cyanate with –SH groups [31,42]. Theoretically, a harmful effect of cyanate on GSH level could be abolished by gGT activation (Fig. 6), since this enzyme participates in GSH decomposition, promoting de novo synthesis of this compound [21]. However, the concomitant increase in ROS and MDA (Figs. 2 and 3) levels indicates that cyanate enhances pro-oxidant processes in the heart cells. gGT, which catalyzes GSH biodegradation to cysteinylglycine (Cysgly), can be implicated in this process since Cys-gly generates free radicals in the presence of ferric ions. Such action of gGT was confirmed by the presence of this enzyme in atherosclerotic foci and by its participation in low-density lipoprotein cholesterol oxidation. The elevated gGT activity has recently been also accepted as an independent risk factor for death from heart diseases [19]. Besides, the cyanate-induced activation of catalase and GPx (Figs. 4 and 5) can indicate mobilization of cardiomyocytes in oxidative environment. Although a majority of researchers have demonstrated a drop in antioxidant enzyme activities during oxidative stress, a similar compensatory effect, i.e. the increase in GPx activity, was seen in the rabbit heart during daunorubicin-induced oxidative stress [38]. In contrast, the cyanateinduced decrease in GST activity (Fig. 7), which participates in the removal of xenobiotics in the form of glutathione S-conjugates was probably caused by GSH (a GST substrate) depletion. The cyanate-induced decline in S* level and in TST and MST activities may also result in lowering of the ability to detoxify the highly toxic cyanide (CN) to the less toxic thiocyanate (SCN). Since CN inhibits respiratory chain in the mitochondria, impairing ATP production, its removal can be also significant for normal heart function [14,16,23]. CSE and to a lesser extent MST are the major H2S producing enzymes in the cardiovascular system [8,15,32]. Thus, the blockade of these enzymes by cyanate can lead to the limitation of H2S-mediated vasorelaxation and signal transduction. Cyanate may also contribute to pathogenesis of atherosclerosis. Low-density lipoproteins both oxidized (oxLDL) and carbamoylated (cLDL) are taken in by macrophages which transform into foam cells and later give rise to atherosclerotic plaques. Since cLDL promotes also in vitro smooth muscle cell proliferation and apoptosis of endothelial cells, it can be expected that they participate both in the initiation (endothelial cell damage) and progression of atherosclerosis [3,27,40]. Many papers have described ROS scavenging properties of both lipoic acid and dihydrolipoic acid (DHLA), also useful in the prevention of cardiovascular diseases [5,12,28], which was confirmed by our studies, as well. The decrease in ROS and MDA level (Figs. 2 and 3) after the combined treatment with cyanate and lipoic acid, together with the increase in GSH level (Fig. 1) and restoration of normal catalase activity (Fig. 4) indicates that lipoic acid has a normalizing effect on the antioxidant status in cardiomyocytes. The increased CSE activity which elevates the level of cysteine, a GSH precursor, may contribute to that effect. The parallel increase in TST and MST activities (Table 1) probably promotes recovery of cyanide detoxifying abilities in cardiomyocytes and by increasing of S* level, further increases their antioxidant power. It should be emphasized that the S*-containing compounds are stronger antioxidants than thiol compounds, including GSH. The ability of lipoic acid to restitute the activity of TST and other sulfurtransferases possessing S* binding domain
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may be attributed to the fact that it can be an acceptor of S* from their active centers [39]. Cystathionase and mercaptopyruvate sulfurtransferase activation by the combined cyanate and lipoic acid treatment can elevate the level of H2S, which shows antioxidant properties and acts as a vasodilator, and this can contribute to the improvement of heart function. It was corroborated, for instance, by the alleviation of ventricular dysfunction and inhibition of myocardial apoptosis in the developing heart failure model after the administrations of NaHS, a H2S precursor [41]. In addition, lipoate ability to chelate Fe2+ cations can lead to the diminution of pro-oxidant action of cysteinylglycine. Further, DHLA can suppress LDL oxidation and can repair oxidative protein damage, thereby contributing to reconstruction of the damaged vascular endothelium [5]. Since lipoate is a target of carbamoylation, its administration can decrease LDL carbamoylation thus lowering the risk of atherosclerosis progression. Conclusions Cyanate showed pro-oxidant action in cardiomyocytes which was manifested by the elevation of ROS and MDA level and gGT activity in parallel with the decrease in the main cellular antioxidant GSH and the pool of sulfane sulfur (S*)-containing compounds. It was accompanied by the decreased activity of sulfurtransferases and the increased activities of antioxidant enzymes, i.e. catalase and GPx which can suggest adaptation of cardiomyocytes under oxidative conditions. Lipoic acid administered in combination with cyanate lowered ROS and MDA to the control level thus restoring physiological GSH and S* concentrations and sulfurtransferase activities, which could result in the increase in H2S level. It suggests a possibility to recover the capability of cyanide detoxification and vasorelaxation in the rat heart after lipoic acid treatment. In addition, the ability of lipoic acid to maintain H2S homeostasis can be decisive for the heart function since it was proven that the treatment with the H2S precursor, NaHS could protect cardiomyocytes of patients with heart problems from apoptosis. Conflict of interest The authors declare that there are no conflicts of interest. Funding No financial support for the conduct of the research and preparation of the article. References [1] Aebi H. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysis, vol. 77. Weinheim: Verlag: Chemic Academic Press, Inc.; 1981. p. 325. [2] Arlandson M, Decker T, Roongta VA, Bonilla L, Mayo KH, MacPherson JC, et al. Eosinophil peroxidase oxidation of thiocyanate. J Biol Chem 2001;276:215–24. [3] Asci G, Basci A, Shah SV, Basnakian A, Toz H, Ozkahya M, et al. Carbamylated low-density lipoproteins induced proliferation and increases adhesion molecule expression of human coronary artery smooth muscle cells. Nephrology 2008;13:480–6. [4] Beddie C, Webster CE, Hall MB. Urea decomposition facilitated by a urease model complex: a theoretical investigation. Dalton Trans 2005;21:3542–51. [5] Biewenga G, Haenen G, Bast A. The pharmacology of the oxidant lipoic acid. Gen Pharmacol 1997;29:315–31. [6] Bilska A, Dudek M, Iciek M, Kwiecien´ I, Sokołowska-Jez˙ewicz M, Filipek B, et al. Biological actions of lipoic acid associated with sulfane sulfur metabolism. Pharmacol Rep 2008;60:225–32. [7] Bondy SC, Guo SX. Effect of ethanol treatment on indices of cumulative oxidative stress. Eur J Pharmacol 1994;270:349–55. [8] Chen X, Jhee KH, Kruger WD. Production of the neuromodulator H2S by cystathionine b-synthase via the condensation of cysteine and homocysteine. J Biol Chem 2004;279:52082–86.
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