The oxidation of cyanide (CN-) to a much less toxic thiocyanate (SCN-) is the main in vivo biochemical pathway for CN- detoxification. SCN- is excreted mainly in urine. This study was performed to investigate the effect of lipoic acid (LA) on the urinary excretion of thiocyanate (SCN- ; rhodanate) in rats. Groups of the animals were treated intraperitoneally (i.p.) as follows: group 1: potassium cyanide (KCN) (1 mg/kg); group 2: KCN (1 mg/kg) + LA (100 mg/kg). Urine was collected for 24 h and the pooled samples were examined for SCN- levels. The obtained results indicated that the treatment of animals with potassium cyanide and LA in combination significantly increased the urinary excretion of SCN- in comparison w ith the respective values in the KCN-alone-treated group. It indicates that LA increased the rate of CN- detoxification in rats

Bilska-Wilkosz, Anna

Knutelska, Joanna

Kotańska, Magdalena

Włodek, Lidia B.

Acta Poloniae Pharmaceutica - Drug Research

English

Jagiellonian Univeristy Repository

A cta Poloniae Pharm aceutica -  Drug Research, Vol. 72 No. 1 pp. 49 -5 2 , 2015 ISSN  0001-6837 Polish Pharm aceutical SocietyDRUG BIOCHEMISTRYTHE EFFECT OF LIPOIC ACID ADMINISTRATION ON THE URINARY EXCRETION OF THIOCYANATE IN RATS EXPOSED TO POTASSIUM CYANIDEANNA BILSKA-WILKOSZ1, MAGDALENA DUDEK2, JOANNA KNUTELSKA3and LIDIA WŁODEK1*1Chair of Medical Biochemistry, Jagiellonian University, Medical College,7 Kopernika St., 31-034 Krakow, Poland 2Chair of Pharmacodynamics, 3Department of Pharmacological Screening, Chair of Pharmacodynamics, Jagiellonian University, Medical College, 9 Medyczna St., PL 30-688 Krakow, PolandA bstract: The oxidation of cyanide (CN-) to a m uch less toxic thiocyanate (SCN-) is the m ain in vivo  bio­chem ical pathway for CN- detoxification. SCN-  is excreted mainly in urine. This study was performed to inves­tigate the effect o f lipoic acid (LA) on the urinary excretion of thiocyanate (SCN- ; rhodanate) in rats. Groups o f the animals were treated intraperitoneally (i.p.) as follows: group 1: potassium cyanide (KCN) (1 mg/kg); group 2: KCN (1 mg/kg) + LA (100 mg/kg). Urine was collected for 24 h  and the pooled samples were exam ­ined for SCN-  levels. The obtained results indicated that the treatment o f animals with potassium cyanide and LA in com bination significantly increased the urinary excretion o f SCN-  in comparison w ith the respective val­ues in the KCN-alone-treated group. It indicates that LA increased the rate o f CN-  detoxification in rats.K eyw ords: lipoic acid, dihydrolipoic acid, cyanides, thiocyanates (rhodanates), sulfane sulfurCyanides (CN- ), salts of hydrogen cyanide (HCN), and HCN itself have always been feared as poisons because of their toxic properties. Cyanides are synthesized, excreted and degraded in nature by many species of bacteria, plants and insects. Cyanides have also been used for various purposes in industry. Toxic level of cyanides in the body is generated after administration of certain drugs, like laetrile and sodium nitroprusside. Cyanide targets primarily mitochondrial cytochrome oxidase, there­by causing the inhibition of cellular respiration, acceleration of anaerobic glycolysis, and consequent tissue hypoxia and metabolic lactic acidosis.The oxidation of CN-  to a much less toxic thio- cyanate (the oral LD50 values of cyanide and thio- cyanate in rats are 3 and 854 mg/kg, respectively) (1) is the main in vivo biochemical pathway for CN-  detoxification according to the following reaction:CN-  + S ^  SCN-The conversion of CN-  to SCN-  is catalyzed by sulfurtransferases: rhodanese (EC. 2.8.1.1; TST), 3- mercaptopyruvate sulfurtransferase (EC 2.8.1.2; MST) and cystathionine y-lyase (EC 4.4.1.1; CSE). The role of these enzymes consists in sulfane sulfur transfer to CN- .Sulfane sulfur is a divalent sulfur atom (-S-) covalently bound to another sulfur atom (R-S-S- H). The elemental sulfur (S8) and outer sulfur atom of thiosulfate (S=SO3- ) exhibit also sulfane sulfur properties. The sulfane sulfur-containing compounds are endogenous m etabolites formed from cysteine. Cysteine is synthesized from the essential amino acid methionine to a lim ited extent and for this reason, it is called a sem i­essential amino acid. Cysteine and m ethionine are only two of the twenty amino acids normally pres­ent in proteins and belong to the sulfur-containing amino acids (SAA). Thiocyanate is excreted mainly in urine.* Corresponding author: e-mail: mbwlodek@ cyf-kr.edu.pl4950 ANNA BILSKA-WILKOSZ et al.Lipoic acid (R)-5-(1,2-dithiolan-3-yl)pentanoicacid, LA, C8H14O2S2), a naturally occurring dithiolcompound, is a cofactor for a number of multienzy-matic complexes involved in energy metabolism.Dihydrolipoic acid (6,8-dimercaptooctanoic acid,DHLA, C8H16O2S2) is the reduced form of LA.Exogenous LA has been shown to exhibit pharma-cological activities (2ñ6). Literature data indicatethat the LA/DHLA system participates also in sul-fane sulfur metabolism. Namely, it was shown thatDHLA serves as a sulfane sulfur acceptor in theTST- and MST-catalyzed sulfane sulfur transfer. Inthese reactions DHLA hydropersulfide is formed,from which sulfane sulfur is released in the form ofhydrogen sulfide (H2S) and LA is produced con-comitantly (7, 8).Thus, it can be assumed that LA may beinvolved in cyanide detoxification reactions in thebody. This study was undertaken to investigate forthe first time the effect of LA administration on theurinary excretion of SCNñ in rats exposed to CNñ. EXPERIMENTALAnimals The experiments were carried out on maleWistar rats (300ñ350 g). Animals were housed inmetabolic cages for urine collection, in a room at aconstant temperature of 20 ± 2OC with a naturallight-dark cycle. They had free access to standardpellet diet and water. Groups consisted of 6 animalseach. The pharmaceutical with the commercialname Thiogamma, which contains LA as an activesubstance was used in our experiments as a sourceof LA. All procedures were approved by the EthicsCommittee for Animal Research in KrakÛw (licenseno. 44/2012). Groups were treated as follows: Group 1: KCN(1 mg/kg b.w., i.p.). Group 2: KCN (1 mg/kg b.w.;i.p.) + LA (100 mg/kg b.w.; i.p.). Urine was collected for 24 h after drug injectionand the pooled samples were examined for SCNñlevels. MethodsDetermination of thiocyanate level in the urine ofrats The content of SCNñ was determined by themethod of Goldstein (9). SCNñ present in the urinereacts with Fe3+ ions to form the colored compoundFe(SCN)3, the concentration of which is measuredspectrophotometrically at a wavelength of λ = 450 nm.SCNñ concentration is calculated based on a standardcurve prepared for potassium thiocyanate (KSCN).Statistical analysis Data are presented as the means ± SEM. The sta-tistical significance of the differences between themeans was analyzed using Studentís t-test. A levelof p ≤ 0.001 was adopted to indicate statistical sig-nificance.RESULTS The results presented in Table 1 indicated that24-h urinary excretion of SCNñ was significantlyhigher in rats which were administered KCN andLA in combination in comparison with the grouptreated with KCN alone (4.86 µmol/24 ± 0.23 h vs.2.5 µmol/24 ± 0.37 h, respectively).DISCUSSION AND CONCLUSIONSThe obtained results showed that LA increasedCNñ detoxification rate in rats, which indicates thatthis compound can act as a CNñ antidote. It wasconfirmed also by studies of other authors. M¸llerand Krieglstein reported that one hour of preincuba-tion with 1 µM DHLA reduced damage of neuronsfrom chick embryo telencephalon caused by 1 mMsodium cyanide (10). Abdel-Zaher et al. demonstrat-ed that intraperitoneal injection of KCN into miceproduced clonic and tonic seizures. The severity ofconvulsions was dose-dependent. The ED50 value ofKCN was 4.8 mg/kg. In contrast, animals adminis-tered LA at a dose of 100 mg/kg before KCN injec-tion showed the increase in CNñ ED50 values from4.8 mg/kg to 9.4 mg/kg (10). The same authors alsodemonstrated that LA (25, 50 and 100 mg/kg) givento mice 15 min before KCN injection increased theCNñ LD50 values (based on 24 h mortality) from 6mg/kg to 6.2, 7.2 and 11.2 mg/kg, respectively (11).It has also been reported that LA protects choliner-gic cells against sodium nitroprusside neurotoxicity(12). Therefore, it is a plausible proposal that LAplays a role in cyanide detoxification process.However, further studies are necessary to explainthe mechanisms of these processes. Swenne et al.exposed normal and protein-malnourished rats to thecyanogenic compound acetonitrile (CH3CN); thatstudy indicated that urinary excretion of SCNñ waslower in rats fed a low-protein diet compared to ratsfed a normal-protein diet (13). Analysis of the abovedata suggests that there may be some analogy withthe present results. Namely, when rats are exposedto CNñ the urinary SCNñ excretion is higher in ani-mals, which were fed a normal protein diet and inthose which were administered LA. It indicates thatEffect of lipoic administration on the urinary excretion of... 51LA acted in that case like a normal-protein diet. Theliterature data indicate that a low-protein dietincreases CNñ toxicity, because the concentrationsof sulfane sulfur compounds are dependent on theavailability of SAA from dietary protein (14). Tor-Agbidye et al. proposed that SAA deficiency mightbe a risk factor for human neurological diseasesamong protein-poor populations subsisting on cas-sava (Manihot esculenta), a cyanophoric plant (15). In 1981, Westley found that purified bovine liverTST had a high turnover rate of almost 20,000/minin vitro, which meant that 1 mole of TST could con-vert 20,000 moles of CNñ to SCNñ per 1 minute.This high turnover rate means that the availability ofsulfane sulfur compounds, rather than the TSTactivity, is the rate limiting factor for CNñ conver-sion to SCNñ (16). Therefore, it appears that all the data warranthypothesis that LA increases CNñ to SCNñ trans-formation rate by elevation of the level of sulfanesulfur compounds in the body. It was confirmed alsoby data from Bilska et al. studies, which demon-strated for the first time in vivo that the level of sul-fane sulfur compounds in the heart, liver and kidneyof rats given LA at a dose of 100 mg/kg/24 h for 8days was significantly higher than in the control LA-free group (17). The same experiments indicatedthat LA elevated also TST activity in several tissuesof rats (17). However, in the light of the presentknowledge, LA sulfur is not a sulfane sulfur, andTST does not participate in its formation. However,on the other hand, Frankenberg indicated that theadministration of TST together with sulfane sulfurcompounds had a good antidotic effect against CNñpoisoning in mice, yet, when sulfane sulfur com-pounds were given without TST, the antidotic effectwas weaker (18).So, based on literature data, it can be hypothe-sized that, apart from TST, LA also elevates activi-ty of MST and/or CSE (Fig. 1). It is known that CSEand MST belong to enzymes which are directlyinvolved in the generation of sulfane sulfur com-pounds. MST is also known to be implicated in theCNñ to SCNñ conversion reaction. As for CSE, invivo studies indicated that the inhibition of thisenzyme by L-propargylglycine increased the toxici-ty of CNñ (19). It can indicate that, like for MST, theCSE-catalyzed reactions lead both to the productionof sulfane sulfur compounds, and to CNñ to SCNñFigure 1. A proposal of the mechanism of LA action in processes leading to the formation of sulfane sulfur compounds: thiosulfate, thio-cysteine and thiocystine, and in the CNñ to SCNñ transformation. TST ñ thiosulfate: cyanide sulfurtransferase (EC 2.8.1.1; rhodanese).MST ñ 3-mercaptopyruvate:cyanide sulfurtransferase (EC 2.8.1.2). CSE ñ cystathionine γ-lyase (EC 4.4.1.1; cystathionase) Table 1. The urinary excretion of SCNñ in rats treated with KCNin the absence or presence of LA.Treatment SCN- [µmol/24 h] KCN 2.50 ± 0.37  KCN + LA 4.86a ± 0.23  aStudentís t-test, significance p < 0.001 in comparison with KCNgroup52 ANNA BILSKA-WILKOSZ et al.conversion. Already in the 1960s, it was document-ed that the products of CSE-catalyzed cleavage ofcystine (Cys-S-S-Cys) produced sulfane sulfurdonors for TST (20, 21). In summary, this paper clearly demonstrated thatthe treatment of rats with KCN and LA in combina-tion significantly increased the urinary excretion ofSCNñ in comparison with the respective values inthe KCN-alone-treated group, which confirms thehypothesis suggesting that LA plays a role in theCNñ detoxification process. These results suggestthat biological activity of LA may be connected withsulfane sulfur metabolism (Fig. 1). It points topotential new pharmacological properties of LA.However, confirmation of this hypothesis requiresfurther studies.AcknowledgmentsThis work was supported by statutory funds ofthe Faculty of Medicine and Faculty of Pharmacy,Jagiellonian University Medical College, KrakÛw,Poland. The authors have declared no conflict of interest.REFERENCES1. Beasley D.M., Glass W.I.: Occup. Med. 48, 427(1998). 2. Gorπca A., Huk-Kolega H., Piechota A.,Kleniewska P., Ciejka E., Skibska B.:Pharmacol. Rep. 63, 84 (2011).3. Lyzogub V.H., Altunina N.V., BondarchukO.M.: Lik. Sprava 7-8, 20 (2011).4. Rochette L., Ghibu S., Richard C., Zeller M.,Cottin Y., Vergely C.: Mol. Nutr. Food Res. 57,114 (2013).5. Sa≥at K., Moniczewski A., Librowski T.: MiniRev. Med. Chem. 13, 335 (2013). 6. Zygmunt M., Dudek M., Bilska-Wilkosz A.,Bednarski M., Mogilski S., Knutelska J., SapaJ.: Acta Pol. Pharm. Drug Res. 70, 899 (2013).7. Mikami Y., Shibuya N., Kimura Y., NagaharaN., Ogasawara Y., Kimura H.: Biochem. J. 439,485 (2011). 8. Villarejo M., Westley J.: J. Biol. Chem. 238,4016 (1963).9. Goldstein F.: J. Biol. Chem. 187, 523 (1950).10. M¸ller U., Krieglstein J.: J. Cereb. Blood FlowMetab. 15, 624 (1995).11. Abdel-Zaher A.O., Abdel-Hady R.H., AbdelMoneim W.M. Salim, S.Y.: Exp. Toxicol.Pathol. 63, 161 (2011).12. Bielarczyk H., Gul S., Ronowska A., Bizon-ZygmaÒska D., Pawe≥czyk T., Szutowicz A.: J.Neurochem. 98, 1242 (2006). 13. Swenne I., Eriksson U.J., Christoffersson R.,Kagedal B., Lundquist P., Nilsson L., Tyllesk‰rT., Rosling H.: Fundam. Appl. Toxicol. 32, 66(1996).14. Cliff J., Lundquist P., Martenson J., Rosling H.,Sˆrbo B.: Lancet 2, 1211 (1985).15. Tor-Agbidye J., Palmer V.S., Lasarev M.R.,Craig A.M., Blythe L.L., Sabri M.I., SpencerP.S.: Toxicol. Sci. 50, 228 (1999). 16. Westley J.: Methods Enzymol. 77, 285 (1981).17. Bilska A., Dudek M., Iciek M., KwiecieÒ I.,Soko≥owska-Jeøewicz M., Filipek B., W≥odekL.: Pharmacol. Rep. 60, 225 (2008). 18. Frankenberg L.: Arch. Toxicol. 45, 315 (1980).19. Porter D.W., Nealley E.W., Baskin S.I.:Biochem. Pharmacol. 52, 941 (1996). 20. Cavallini D., De Marco C., Mondovi B., MoriB.G.: Enzymologia 22, 161 (1960).21. Szczepkowski T.W., Wood J.L.: Biochim.Biophys. Acta 139, 469 (1967).Received: 10. 12. 2013

The effect of lipoic acid administration on the urinary excretion of thiocyanate in rats exposed to potassium cyanide

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The effect of lipoic acid administration on the urinary excretion of thiocyanate in rats exposed to potassium cyanide

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