View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Jagiellonian Univeristy Repository Neurotox Res (2013) 24:345–357 DOI 10.1007/s12640-013-9395-2 ORIGINAL ARTICLE The Effect of Lipoic Acid on Cyanate Toxicity in Different Structures of the Rat Brain Maria Sokołowska • Elzbieta_ Lorenc-Koci • Anna Bilska • Małgorzata Iciek Received: 11 October 2012 / Revised: 27 March 2013 / Accepted: 19 April 2013 / Published online: 27 April 2013 Ó The Author(s) 2013. This article is published with open access at Springerlink.com Abstract Cyanate is formed mostly during nonenzymatic role in these patients contributing to efficient antioxidant urea biodegradation. Its active form isocyanate reacts with defense and protection against cyanate and cyanide toxicity. protein –NH2 and –SH groups, which changes their struc- ture and function. The present studies aimed to investigate Keywords Cyanate Á Lipoate Á Sulfane sulfur Á Hydrogen the effect of cyanate on activity of the enzymes, which sulfide Á Atherosclerosis possess –SH groups in the active centers and are implicated in anaerobic cysteine transformation and cyanide detoxifi- cation, as well as on glutathione level and peroxidative Introduction processes in different brain structures of the rat: cortex, striatum, hippocampus, and substantia nigra. In addition, we In biological systems, cyanate shows the highest reactivity examined whether a concomitant treatment with lipoate, a with sulfhydryl (SH) groups of proteins (Arlandson et al. dithiol that may act as a target of S-carbamoylation, can 2001; Wisnewski et al. 1999). Since –SH groups are prevent these changes. Cyanate-inhibited sulfurtransferase present in active centers of enzymes participating in activities and lowered sulfide level, which was accompa- anaerobic cysteine transformation, e.g., cystathionase nied by a decrease in glutathione concentration and eleva- (CSE, EC 4.2.1.15) and mercaptopyruvate sulfurtransferase tion of reactive oxygen species level in almost all rat brain (MPST, EC 2.8.1.2), and sulfane sulfur transporting structures. Lipoate administered in combination with cya- enzyme, e.g., rhodanese–thiosulfate sulfurtransferase (TST, nate was able to prevent the above-mentioned negative EC 2.8.1.1) (Nagahara et al. 1995, 2003), it was hypothe- cyanate-induced changes in a majority of the examined sized that cyanate could influence the activity of these brain structures. These observations can be promising for enzymes as well as the level of the main cellular antioxi- chronic renal failure patients since lipoate can play a double dant, glutathione (GSH). Cysteine, which is formed from exogenous methionine, is both the GSH precursor and the main source of active sulfur M. Sokołowska (&) Á A. Bilska Á M. Iciek in tissues. Sulfur-containing compounds can possess a stably The Chair of Medical Biochemistry, Jagiellonian University bound sulfur, as that in glutathione and cysteine or labile Medical College, 7, Kopernik Street, 31-034 Krako´w, Poland e-mail: [email protected] sulfur, e.g., acid labile, sulfane sulfur (S*), and protein bound sulfane sulfur. S* is a highly reactive sulfur in 0 or -1 oxi- A. Bilska e-mail: [email protected] dation state covalently bound to another sulfur atom. The pool of sulfane sulfur-containing compounds comprises, M. Iciek 2- e-mail: [email protected] e.g., polysulfides (R–S–Sn*–S–R), thiosulfate (S2O3 ), and persulfides (R–S–S*H), which are formed during biodegra- E. Lorenc-Koci dation of cystine and mixed disulfides (Fig. 1) in the pres- Department of Neuropsychopharmacology, Institute of ence of CSE and cystathionine b-synthase (CBS, EC Pharmacology, Polish Academy of Science, 12, Sme˛tna Street, - 31-343 Krako´w, Poland 4.2.1.22). S* plays an important role in cyanide (CN )to e-mail: [email protected] thiocyanate (SCN-) detoxification catalyzed by TST, 123 346 Neurotox Res (2013) 24:345–357 Fig. 1 Sulfane sulfur (S*) formation by biodegradation of L-cystine during cystathionine synthesis catalyzed by CBS (4); in b- and a,b- to L-thiocysteine, catalyzed by CSE (3) and of homocysteine and elimination reactions of L-cysteine, catalyzed by CSE (3) and CBS cysteine mixed disulfides to L-thiohomocysteine, catalyzed by CBS (4); in the reaction of persulfides (e.g., thiocysteine, thiohomocys- (4). The main pathways of hydrogen sulfide (H2S) formation: by teine) with an excess of cellular reducers (RSH) desulfurization of 3-mercaptopyruvic acid catalyzed by MPST (2); MPST, and by CSE (Nagahara et al. 1995, 1999, 2003; Toohey 1989). Most of the labile sulfur is liberated as inor- - 2- ganic sulfides, e.g., H2S, HS ,orS , in the presence of acids or reducing agents (Toohey 1989, 2011; Ubuka 2002). On the other hand, hydrogen sulfide can be stored in the form of protein bound sulfane sulfur (Shibuya et al. 2009). It indi- cates a close relation between H2S and sulfane sulfur. In the brain, hydrogen sulfide (H2S) fulfills the function of neuro- transmitter and vasodilator. It was believed earlier that the formation of the main pool of hydrogen sulfide in the brain was catalyzed by CBS, while in the periphery by cystathio- nine c-lyase—CSE (Chen et al. 2004; Li et al. 2006; Stipanuk 2004; Toohey 2011). However, the most recent studies have Fig. 2 The structure of oxidized (LA) and reduced (DHLA) form of lipoic acid demonstrated that hydrogen sulfide synthesis in the brain tissue is catalyzed mostly by MPST (in the presence of thi- oredoxin or dihydrolipoic acid) (Shibuya et al. 2009; Mikami that effect in the rat liver (Sokolowska et al. 2011). Hence, et al. 2011) (Fig. 1). we expected to see similar effect in the rat brain. However, Our earlier studies demonstrated prooxidative properties the effect of cyanate on antioxidant enzyme activity and of cyanate and its inhibitory action on enzymatic activities H2S level was unknown. Since oxidative stress can dif- of sulfurtransferases while lipoic acid [IUPAC name: ferently affect antioxidant enzyme activity in various brain 5-(1,2-dithiolane-3-yl)pentanoic acid] (Fig. 2) prevented regions (Severynovs’ka et al. 2006; Mladenovic´ et al. 123 Neurotox Res (2013) 24:345–357 347 2012), we expected to observe diverse effects of cyanate in cysteine transformation, in particular on hydrogen sulfide different brain structures. The effect of cyanate on the level and activity of sulfane sulfur synthetic and transport organism can be particularly significant in uremia in which enzymes, and on the concentrations of pro- and antioxi- plasma level of urea (a cyanate (OCN-) and isocyanate dants in different structures of the brain: cortex, striatum, (NCO-) precursor (Fig. 3) is significantly increased hippocampus, and substantia nigra (SN). (Beddie et al. 2005; Estiu and Merz 2007; Vanholder et al. 2003). This compound can also affect brain tissue because both, in vitro and in vivo studies with 14C radiolabeled Materials and Methods cyanate documented its incorporation into cerebral proteins in the process of S- and N-carbamoylation (Crist et al. Animals 1973; Fando and Grisolia 1974). Lipoic acid (LA) (Fig. 2), due to its structure, may act as The experiments were carried out on male Wistar rats a target of carbamoylation and in this way may protect –SH weighing *250 g. The animals were kept under standard groups of proteins. In addition, LA is a very strong anti- laboratory conditions and were fed a standard diet. All oxidant able to quench free radicals and restore the activity experiments were carried out in accordance with the of other antioxidants. Since LA participates also in the National Institutes of Health Guide for the Care and Use of regulation of sulfane sulfur metabolism, it is probable that Laboratory Animals and with approval of the Bioethics it can show a protective action against harmful effects of Commission as compliant with the Polish Law (21 August cyanate in brain structures (Bilska et al. 2008; Smith et al. 1997) (permission no. 645, 23.04. 2009). Animals were 2004). These data prompted us to investigate the effect of assigned to four groups, containing 7 animals each. Groups cyanate and lipoate alone and in combination on anaerobic were treated as follows. Group 1 0.9% NaCl O.9% NaCl 0.9% NaCl sacrifice 03060 150 min Group 2 0.9% NaCl cyanate 0.9% NaCl sacrifice 03060 150 min Group 3 lipoate 0.9% NaCl lipoate sacrifice 03060 150 min 123 348 Neurotox Res (2013) 24:345–357 Group 4 lipoate cyanate lipoate sacrifice 03060 150 min Fig. 3 Urea breakdown to O urease ammonia (NH3) and carbon urease O H2 O C H N-C + - dioxide (CO ) catalyzed by CO 2 + 2NH 3 H2 O + + H 2O 2 + NH 4 HCO 3 + 2NH3 2 O- urease, and to isocyanate H2 N NH2 -) - (NCO and cyanate (OCN ) urea carbamate CO2 (nonenzymatic and enzymatic elimination) nonenzymatic (urease) NH3 -N=C=O isocyanate -O -C N cyanate Cyanate (KCNO) was administered intraperitoneally nitrobenzoic acid (DTNB), NADPH, mercaptopyruvic acid (i.p.) at a dose of 200 mg/kg b.w. The dose of cyanate was sodium salt L-homoserine, pyridoxal 50-phosphate mono- chosen based on the data presented in the article by Tor- hydrate, 3-methyl-2-benzothiazolinone hydrazone hydro- Agbidye et al. (1999). The dose of lipoic acid (LA) 50 mg/kg chloride monohydrate, and lactic dehydrogenase (LDH), b.w. i.p. twice was chosen on the basis of our earlier studies potassium cyanate (KNCO), glutathione reduced form, (Bilska et al. 2008). Efficacy of this dose was confirmed by glutathione reductase, and a-lipoic acid sodium salt were literature data (Micili et al. 2013; Shay et al. 2009). provided by Sigma Chemical Co. (St. Louis, MO, USA). Animals were sacrificed 2.5 h after the first injection, Formaldehyde, ferric chloride (FeCl3), thiosulfate, and all because there had to be an interval between preventive lipoate the other reagents were obtained from the Polish Chemical administration and cyanate dose, and then the next therapeutic Reagent Company (P.O.Ch, Gliwice, Poland).