The Effect of Lipoic Acid on Cyanate Toxicity in Different Structures of the Rat Brain

In biological systems, cyanate shows the highest reactivity with sulfhydryl (SH) groups of proteins (Arlandson et al. 2001; Wisnewski et al. 1999). Since –SH groups are present in active centers of enzymes participating in anaerobic cysteine transformation, e.g., cystathionase (CSE, EC 4.2.1.15) and mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2), and sulfane sulfur transporting enzyme, e.g., rhodanese–thiosulfate sulfurtransferase (TST, EC 2.8.1.1) (Nagahara et al. 1995, 2003), it was hypothesized that cyanate could influence the activity of these enzymes as well as the level of the main cellular antioxidant, glutathione (GSH).

Cysteine, which is formed from exogenous methionine, is both the GSH precursor and the main source of active sulfur in tissues. Sulfur-containing compounds can possess a stably bound sulfur, as that in glutathione and cysteine or labile sulfur, e.g., acid labile, sulfane sulfur (S*), and protein bound sulfane sulfur. S* is a highly reactive sulfur in 0 or −1 oxidation state covalently bound to another sulfur atom. The pool of sulfane sulfur-containing compounds comprises, e.g., polysulfides (R–S–S _n *–S–R), thiosulfate (S₂O₃ ²⁻), and persulfides (R–S–S*H), which are formed during biodegradation of cystine and mixed disulfides (Fig. 1) in the presence of CSE and cystathionine β-synthase (CBS, EC 4.2.1.22). S* plays an important role in cyanide (CN⁻) to thiocyanate (SCN⁻) detoxification catalyzed by TST, MPST, and by CSE (Nagahara et al. 1995, 1999, 2003; Toohey 1989). Most of the labile sulfur is liberated as inorganic sulfides, e.g., H₂S, HS⁻, or S²⁻, 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 indicates a close relation between H₂S and sulfane sulfur. In the brain, hydrogen sulfide (H₂S) fulfills the function of neurotransmitter 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 cystathionine γ-lyase—CSE (Chen et al. 2004; Li et al. 2006; Stipanuk 2004; Toohey 2011). However, the most recent studies have demonstrated that hydrogen sulfide synthesis in the brain tissue is catalyzed mostly by MPST (in the presence of thioredoxin or dihydrolipoic acid) (Shibuya et al. 2009; Mikami et al. 2011) (Fig. 1).

Our earlier studies demonstrated prooxidative properties of cyanate and its inhibitory action on enzymatic activities of sulfurtransferases while lipoic acid [IUPAC name: 5-(1,2-dithiolane-3-yl)pentanoic acid] (Fig. 2) prevented that effect in the rat liver (Sokolowska et al. 2011). Hence, we expected to see similar effect in the rat brain. However, the effect of cyanate on antioxidant enzyme activity and H₂S level was unknown. Since oxidative stress can differently affect antioxidant enzyme activity in various brain regions (Severynovs’ka et al. 2006; Mladenović et al. 2012), we expected to observe diverse effects of cyanate in different brain structures. The effect of cyanate on the organism can be particularly significant in uremia in which plasma level of urea (a cyanate (OCN⁻) and isocyanate (NCO⁻) precursor (Fig. 3) is significantly increased (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 cyanate documented its incorporation into cerebral proteins in the process of S- and N-carbamoylation (Crist et al. 1973; Fando and Grisolia 1974).

Lipoic acid (LA) (Fig. 2), due to its structure, may act as a target of carbamoylation and in this way may protect –SH groups of proteins. In addition, LA is a very strong antioxidant able to quench free radicals and restore the activity of other antioxidants. Since LA participates also in the regulation of sulfane sulfur metabolism, it is probable that it can show a protective action against harmful effects of cyanate in brain structures (Bilska et al. 2008; Smith et al. 2004). These data prompted us to investigate the effect of cyanate and lipoate alone and in combination on anaerobic cysteine transformation, in particular on hydrogen sulfide level and activity of sulfane sulfur synthetic and transport enzymes, and on the concentrations of pro- and antioxidants in different structures of the brain: cortex, striatum, hippocampus, and substantia nigra (SN).

Briefly, the assay was based on the fact that H₂S produced in the brain tissues reacts with Zn(CH₃COO)₂ to form ZnS which than reacts with p-phenylenediamine yielding a fluorescent dye (thionein) in the presence of ferric chloride (FeCl₃). Aliquots of homogenate (125 μl) were mixed with 1 mM Na₂CO₃ (125 μl) and 5 % zinc acetate (125 μl) followed by incubation at room temperature (30 min). Next, p-phenylenediamine (12.5 mM), 40 mM FeCl₃ in 6 M HCl, and H₂O (125 μl) were added. After a 10-min development at room temperature, the samples were centrifuged for 5 min in 10,000×g and fluorescence of the mixture was measured (Ex 600 nm, Em 623 nm). H₂S concentrations were read from a calibration curve prepared from thionein (2.5–25 μM).

Reduction of glutathione (GSH) level as well as sulfurtransferase (TST, CSE) and catalase activities (Figs. 4, 6, 7, 8) accompanied by an increase in free radical level (Fig. 10) was observed in almost all rat brain structures after cyanate treatment, through to different degree. It may indicate prooxidative action of cyanate and following carbamoylation of proteins, especially –SH groups of sulfurtransferases and GSH. Cyanate-induced lowering of GSH level of varying magnitude was also reported by Tor-Agbidye et al. (1999) in different mouse brain structures. On the other hand, carbamoylation is corroborated by reports demonstrating cyanate incorporation into cerebral proteins and a direct cyanate reaction with GSH (Crist et al. 1973; Fando and Grisolia 1974; Stark et al. 1960; Wisnewski et al. 1999). The decrease in GSH level, the most notable in the cortex and in the striatum, can also be the result of CSE inhibition in these structures since this enzyme supplies cysteine, a rate-limiting substrate for glutathione synthesis. GSH (γ-glutamylcysteinyl glycine) biosynthesis in brain cells is catalyzed by γ-glutamylcysteine ligase (GCL), which is a rate-limiting enzyme, and glutathione synthetase (GS). On the other hand, GSH is catabolized to glutamate, cysteine, and glycine which can be reused for GSH synthesis catalyzed by the transmembrane enzyme γGT. Therefore, the decrease in γGT activity observed in the cortex can contribute to the drop in GSH level in this structure. Since GSH is the main cellular antioxidant which participates in reactive oxygen species (ROS) scavenging and removal of hydrogen peroxide and organic peroxides, a decrease in its level elevates ROS level. The cyanate-induced increase in ROS level could also be attributed to the decreased level of hydrogen sulfide in the cortex and in the striatum, because H₂S inhibits ROS formation under natural conditions by inhibiting NADH oxidase activity (Samhan-Arias et al. 2009). Since physiological concentration of H₂S also increases the activity of the cystine–glutamate antiporter (Xc⁻) and of γ-glutamylcysteine synthase, the drop in H₂S concentration in the cortex and in the striatum (Fig. 9) can indirectly affect GSH level (Kimura and Kimura 2004; Kimura et al. 2010). According to Chen et al. (2004), CBS is the only enzyme able to produce H₂S in the brain. However, biochemical studies of Linden et al. (2008) evidenced CSE expression and H₂S production in the mouse brain. CSE activity was also present in various regions of the rat brain from a 6-month-old infant at autopsy (Stipanuk 2004). In the hippocampus neither MPST (the main H₂S-producing enzyme) nor CSE activity was changed by cyanate, which was manifested as an unaltered sulfide/H₂S level (Figs. 7, 8, 9). The cyanate-induced lowering of H₂S level in the cortex and striatum correlates with the decrease in CSE activity in both structures and MPST activity in the striatum. The inhibitory effect of cyanate on CSE activity also leads to depletion of the so-called sulfane sulfur pool in the cortex. In addition, the decline in TST and MPST activities (which under physiological conditions are about 5–10 times lower in the brain than in other organs, like the kidney or heart) can increase sensitivity of the brain structures (especially striatum and SN) to the CN⁻ toxicity, leading to the inhibition of cytochrome oxidase, and thus to the decrease in ATP level and neuropathy (de Sousa et al. 2007; Hasuike et al. 2004; Nagahara et al. 1999).

H₂S was shown to increase the activity of neuronal N-methyl-d-aspartate (NMDA) receptor, activating calcium channels and leading to a prolonged enhancement of long-term potentiation (LPT) which is of key significance for some forms of learning and memory (Nagai et al. 2004; Kimura and Kimura 2004). No changes in the hippocampal MPST activity and H₂S level after acute cyanate treatment can indicate undisturbed perceptive processes in rats under these conditions. On the other hand, hippocampal hydrogen sulfide level, unaffected by cyanate, suggests an undisturbed functioning of the Xc⁻ antiporter to supply cystine, and unhindered GSH synthesis in this structure of the brain (Kimura et al. 2010). In that case, the decrease in GSH level, slightly smaller than in other structures (Fig. 9), could be an immediate consequence of its direct carbamoylation by cyanate.

Although the mechanism of catalase (in the cortex and striatum) and GPx (in the cortex) inhibition is not known in detail, there are reports demonstrating a decrease in activities of these enzymes in the rat brain during oxidative stress (Hfaiedh et al. 2012; Bild et al. 2012), and various stress effects on antioxidant enzymes in different brain structures (Mladenovic et al. 2012). However, it would be difficult to compare those results with the present data due to completely different stress conditions.

The increase in GSH level after the combined administration of cyanate and lipoate was the greatest in those structures where it was decreased the most by cyanate, i.e., in the cortex and in the striatum, which can indicate a stimulating lipoate effect during cyanate-induced oxidative stress. Both LA and its reduced form DHLA easily cross the blood–brain barrier thus creating redox system with a low redox potential (E _o = −0.29 V), capable of regeneration of other cellular antioxidants, including GSH and cysteine (CSH) (by the reduction of respective disulfides: GSSG and CSSC) (Biewenga et al. 1997; Shay et al. 2009). It was confirmed by literature reports indicating the increase in GSH concentration in cell cultures and animal tissues after lipoate administration (Busse et al. 1992; Shay et al. 2009; Wessner et al. 2006). CSE activation after lipoate + cyanate administration, observed in the cortex, striatum, and to the greatest degree in the hippocampus, could lead to elevation of the concentration of cysteine, a GSH precursor (Fig. 1). According to Smith et al. (2004), there are two paths by which LA/DHLA can increase GSH level: (1) boosting of cysteine availability by extracellular cystine reduction and (2) facilitation of cystine uptake by enhancement of Xc⁻ transport system (cystine/glutamate antiporter) expression. The recent hypothesis suggests that LA increases total antioxidant capacity of cells by induction of antioxidant response element (ARE)-regulated gene transcription, including those encoding γ-glutamylcysteine ligase, an enzyme participating in the first stage of GSH synthesis. ARE region can also partially regulate of Xc⁻ system contributing to transport of cystine, which is the main substrate for GSH production (Shay et al. 2009).

Lipoate was also able to restore sulfutransferase activities (Figs. 7, 8) in all structures under study (except for MPST activity in the hippocampus which was not changed by cyanate). Lipoate’s ability to restitute activity of rhodanese (and probably other sulfurtransferases possessing S* binding domain, e.g., MPST and CSE) (Toohey 2011) can be attributed to the fact that the reduced form of lipoate can be an acceptor of sulfane sulfur from the rhodanese active center (Villarejo and Westley 1963) (Fig. 11). It is accompanied by H₂S formation, confirmed by its increased cortical level after lipoate administration (Fig. 9), which could lead to γ-glutamylcysteine synthetase and cystine transporter (Xc⁻) activation and acceleration of GSH synthesis (Kimura and Kimura 2004). Since hydrogen sulfide more increases cysteine than cystine transport, and parallely cysteine uptake by ASC transporter is faster than cystine uptake by Xc⁻ system, it appears that ASC-involving mechanism can be decisive for elevation of GSH synthesis by H₂S (Kimura et al. 2010; Smith et al. 2004). To sum up three factors could contribute to the reduction of ROS level after the combined administration of cyanate and lipoate: (1) the increase in GSH level (in all studied structures), (2) the increase in the activity of antioxidant enzymes: catalase (in the cortex and striatum) and GPx (in the cortex), and (3) the increase in H₂S concentration (in the cortex), which inhibits ROS formation (Samhan-Arias et al. 2009). All these factors may be decisive for antioxidant protection of neurons in the cortex and striatum. Additional protection can be obtained due to LA-induced reestablishment of the activities of sulfurtransferases, involved in the production of strongly antioxidant sulfane sulfur. Thus, lipoate administered in combination with cyanate was able to restore cyanide detoxifying capabilities to brain cells. The protective effect of LA administrated in a rat sciatic nerve injury model is another confirmation of restorative lipoate action on the nervous tissue (Ranieri et al. 2010). The increased level of oxidized and carbamoylated low density lipoproteins (LDL) and proteins, detected in atherosclerotic plaques and cholesterol deposits, can suggest a role of both cyanate and oxidative stress in pathogenesis of atherosclerosis and cerebral stroke (Asci et al. 2008; Shah et al. 2008; Wang et al. 2007). Since LA decreased ROS level (Fig. 10) and concomitantly might act as a target for carbamoylation, thus lowering cLDL level, it is able both to alleviate the symptoms of neuropathy observed in CRF patients and to decrease the risk of cerebral stroke. Although cyanate toxicity after acute treatment is relatively low, long-term effects of its action can be much more serious (Alter et al. 1974).

This article presents the studies demonstrating that the treatment of rats with cyanate alone lowered GSH level and raised reactive oxygen species (ROS) level in all structures of the brain. Besides, cyanate was shown to inhibit TST and CSE activities, to decrease sulfide/hydrogen sulfide level in all structures except for the hippocampus, and to inhibit the activities of MPST (in the striatum and substantia nigra), catalase (in the striatum and cortex), and peroxidase (in the cortex). This indicates that cyanate inhibits anaerobic cysteine transformation and shows prooxidant action in almost all structures of the brain. From among the above-mentioned changes, lipoate administered in combination with cyanate was able to correct ROS and GSH levels, as well as activities of sulfurtransferases, catalase, and peroxidase. It indicates that lipoate can prevent prooxidant cyanate action and cyanate-induced inhibition of enzymes engaged in anaerobic cysteine transformation and cyanide detoxication as well as can activate antioxidant enzymes. These observations can be promising for prophylaxis and alleviation of symptoms of cerebral stroke and neurodegenerative diseases especially in chronic renal failure patients since lipoate can play a dual role in these patients contributing to efficient antioxidant defense and protection against cyanate and cyanide toxicity.