Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats

GENE-39655; No. of pages: 8; 4C: Gene xxx (2014) xxx–xxx

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Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats Janaína Kolling, Emilene B.S. Scherer, Cassiana Siebert, Eduardo Peil Marques, Tiago Marcom dos Santos, Angela T.S. Wyse ⁎ a Laboratório de Neuroproteção e Doenças Neurometabólicas, Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil b Laboratório de Erros Inatos do Metabolismo, Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil

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Article history: Received 7 October 2013 Received in revised form 14 March 2014 Accepted 1 May 2014 Available online xxxx Keywords: Severe hyperhomocysteinemia Oxidative stress Soleus skeletal muscle Creatine

a b s t r a c t Homocystinuria is a neurometabolic disease caused by severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction, being that the pathomechanism is not fully understood. In the present study we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely 2′7′ dichlorofluorescein (DCFH) oxidation, levels of thiobarbituric acid-reactive substances (TBARS), antioxidant enzyme activities (SOD, CAT and GPx), reduced glutathione (GSH), total sulfhydryl and carbonyl content, as well as nitrite levels in soleus skeletal muscle of young rats subjected to model of severe hyperhomocysteinemia. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3–0.6 μmol/g body weight), and/or creatine (50 mg/kg body weight) from their 6th to the 28th days age. Controls and treated rats were decapitated at 12 h after the last injection. Chronic homocysteine administration increased 2′7′dichlorofluorescein (DCFH) oxidation, an index of production of reactive species and TBARS levels, an index of lipoperoxidation. Antioxidant enzyme activities, such as SOD and CAT were also increased, but GPx activity was not altered. The content of GSH, sulfhydril and carbonyl were decreased, as well as levels of nitrite. Creatine concurrent administration prevented some homocysteine effects probably by its antioxidant properties. Our data suggest that the oxidative insult elicited by chronic hyperhomocystenemia may provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function. Creatine prevents some alterations caused by homocysteine. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Homocysteine (Hcy), a sulfur-containing amino acid, is metabolized by remethylation to methionine or by transsulfuration to cysteine via cystathionine (McCully, 2011; Williams and Schalinske, 2010). It is an intermediate for many key processes such as cellular methylation and cellular antioxidant potential. The normal plasma Hcy concentrations vary from 5 to 14 μmol/l. Hyperhomocysteinemia is a systemic condition Abbreviations: DCFH, dichlorodihydrofluorescein diacetate; TBARS, thiobarbituric acid-reactive substances; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GSH, reduced glutathione; Hcy, homocysteine; HCU, homocystinuria; CβS, cystathionine β-synthase; RS, reactive species; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; NADPH, nicotinamide adenine dinucleotide phosphate; O− 2 , superoxide anion; NOS, nitric oxide synthase; GAA, guanidinoacetic acid; DTNB, 5,5′-dithio-bis (2nitrobenzoic acid); EDTA, ethylenediamine tetraacetic acid; ANOVA, one-way analysis of variance. ⁎ Corresponding author at: Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil. E-mail address: [email protected] (A.T.S. Wyse).

and has been attributed to multi-organ pathologies (Veeranki and Tyagi, 2013). It may be classified as mild (15–30 μmol/l), moderate (31–100 μmol/l) and severe (N100 μmol/l) (Mudd et al., 2001; Stead et al., 2001). In some cases, Hcy concentrations can achieve above 100 μM, which is characteristic of classical homocystinuria (HCU), an inborn error of metabolism caused by a deficient of enzyme cystathionine β-synthase activity (CβS, EC 4.2.1.22) (Mudd et al., 2001). Affected patients exhibit plasma concentrations of Hcy that can reach up to 500 μmol/l and usually present clinical and pathologic manifestations that affect the motor system (Huang et al., 2011; Leishear et al., 2012), whose mechanisms are unknown. In addition, it has been observed that hyperhomocysteinemia is associated with muscle dysfunction (Veeranki and Tyagi, 2013). Oxidative stress plays an important role in skeletal muscle damage in hyperhomocysteinemia (Swart et al., 2012). Free radicals are produced as a normal function of cell metabolism, such as mitochondrial bioenergetic, xanthine oxidase, peroxisomes, inflammation processes, phagocytosis, and arachidonate pathways. External factors that help to promote the production of free radicals are smoking, environmental

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Please cite this article as: Kolling, J., et al., Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.005

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pollutants, radiation, drugs, pesticides, industrial solvents and ozone (Carocho and Ferreira, 2013; Lobo et al., 2010). The balance between the production and neutralization of reactive species (RS) by antioxidants is very delicate, and if this balance tends to the overproduction of the RS, the cells start to suffer the consequences of oxidative stress (Wiernsperger, 2003). The main targets of RS are proteins, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) molecules, sugars and lipids (Craft et al., 2012; Halliwell, 2011). It has been suggested that oxidative stress and its consequent oxidative damage may be involved in muscular diseases (Hammouda et al., 2012; Swart et al., 2012). Supporting this notion, a reduction in antioxidant bioavailability along with increased oxidative stress has been reported in both experimental and human conditions. Regarding enzymatic antioxidants, they are divided into primary and secondary enzymatic defenses. The primary defense, is composed of three important enzymes that prevent the formation or neutralize free radicals: superoxide dismutase (SOD) that converts superoxide anions, a highly deleterious radical for all types of cells and tissues, into hydrogen peroxide (H2O2) as a subtract for catalase and molecular oxygen (O2); catalase (CAT) that converts hydrogen peroxide into water and molecular oxygen and has one of the biggest turnover rates known to man, allowing just one molecule of catalase to convert 6 billion molecules of hydrogen peroxide and glutathione peroxidase (GPx), which donates two electrons to reduce peroxides by forming selenoles and also eliminates peroxides as potential substrate for the Fenton reaction (Rahman, 2007). The secondary enzymatic defense includes glutathione reductase and glucose-6-phosphate dehydrogenase. Glutathione reductase reduces glutathione (GSH-antioxidant) from its oxidized to its reduced form, thus recycling it to continue neutralizing more free radicals. Glucose-6-phosphate regenerates NADPH (nicotinamide adenine dinucleotide phosphate) creating a reducing environment (Gamble and Burke, 1984; Ratnam et al., 2006). These two enzymes do not neutralize free radicals directly, but may act through of other endogenous antioxidants. Studies using experimental models and endothelial cells incubated with elevated homocysteine levels indicate that Hcy may promote the formation of reactive oxygen species, especially the superoxide anion (O− 2 ) (Weiss, 2005) by self-oxidation of homocysteine and/or cysteine (Hogg et al., 2006). This increase in intracellular production of free radicals can cause cellular damage (Streck et al., 2003). Moreover, investigators have reported that Hcy can cause changes in the defense system antioxidant (Blundell et al., 1996). Furthermore, studies in our group showed that severe hyperhomocysteinemia causes oxidative stress in other systems, such as cardiovascular (Kolling et al., 2011), hepatic (Matté et al., 2009) and cerebral (Streck et al., 2003). Nitric oxide (NO) is synthesized in the thin endothelial lining of blood vessels by endothelial NO synthase (NOS) (Liu and Fung, 1998). The hallmark of endothelial dysfunction is a reduction in the bioavailability of NO and several studies have demonstrated that the Hcy reduces the availability of endothelial-derived NO (Mujumdar et al., 2001). A decrease in the bioavailability of NO is associated with an increase in leukocyte–endothelial cell interactions (Davenpeck et al., 1994), enhanced platelet adherence and aggregation (Radomski et al., 1990) and with the proliferation of smooth muscle cells (Liu and Fung, 1998). When NO is released by the endothelium, it can modulate blood flow, inflammation and platelet aggregation. Although NO appears to have a number of important physiological roles, it is also a free radical and may be cytotoxic (Ekelund et al., 1999). In addition, it has been shown that exercise increases NO production by vascular endothelial cells of the skeletal muscle (Gielen et al., 2010). Creatine is endogenously synthesized by the liver, kidneys and pancreas from the amino acids glycine and arginine to form guanidinoacetic acid (GAA) and ornithine in a reaction catalyzed by the enzyme L-arginine: glycine amidinotransferase (Deminice et al., 2007; Xu et al., 2010). Next, the irreversible transfer of a methyl group from SAM to GAA is catalyzed by the enzyme SAM: guanidinoacetate

N-methyltransferase (Wyss and Kaddurah-Daouk, 2000). Through this reaction, the endogenous synthesis of creatine consumes a considerable number of methyl groups. In humans, creatine synthesis has been reported to account for 70% of Hcy formation (Mudd and Poole, 1975). This amine is naturally found in food, especially meat and fish, although only half of the daily creatine requirement (about 1 g/d) comes from the diet (Persky and Brazeau, 2001). In skeletal muscle of vertebrates, the creatine participates in metabolic reactions within cells and is catabolized in the muscles generating creatinine which is then excreted by the kidney in urine (Terjung et al., 2000). Studies show that the creatine may have a protector and antioxidant role in certain neuromuscular and neurodegenerative diseases (Beal, 2011; Bender et al., 2006; Chung et al., 2007; Sestili et al., 2011). Creatine supplementation has emerged as a promising adjunct therapy in several pathological conditions (Gualano et al., 2011), including muscular diseases (Deminice and Jordao, 2012; Wallimann et al., 1992). Interestingly, a growing body of experimental and clinical literature has suggested that creatine may exert protective effect in diseases, whose mechanisms seem to be associated with oxidative stress (Sestili et al., 2011). In fact, in vitro studies have revealed that the creatine may have antioxidant properties by acting as a scavenger of free radicals, such as superoxide anions and peroxynitrite (Lawler et al., 2002; Sestili et al., 2011). In addition, studies show that the creatine prevents oxidative stress parameter, such as lipid peroxidation, in heart of rats subjected to Hcy (Kolling et al., 2011). In the present study we evaluate the effects of severe hyperhomocysteinemia on parameters namely 2′7′dichlorofluorescein (DCFH) oxidation, levels of thiobarbituric acid-reactive substances (TBARS), antioxidant enzyme activities (SOD, CAT and GPx), reduced glutathione (GSH), total sulfhydryl and carbonyl content, as well as nitrite levels in soleus skeletal muscle of young rats subjected to model of severe hyperhomocysteinemia. We also analyze the role of creatine on the possible biochemical changes observed in this model. Our hypothesis is that the alteration motors caused by Hcy may be associated with the oxidative insult and that creatine might improve such muscle damage since it has been postulated that creatine could act as an antioxidant agent preventing increased oxidative stress. Thus, we investigated the effects of creatine administration alone and in combination with Hcy. 2. Materials and methods 2.1. Ethical approval Male or female Wistar rats (6-day-old) were obtained from the Central Animal House of the Department of Biochemistry, Institute of Basic Science of Health, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Animals were maintained on a 12/12 h light/dark cycle in an air-conditioned constant temperature (22 ± 1 °C) colony room. Rats had free access to a 20% (w/w) protein commercial chow and water. The NIH Guide for the Care and Use of Laboratory Animals (NIH publication no. 80-23, revised 1996) was followed in all experiments and was approved by the Ethical Committee of the Universidade Federal do Rio Grande do Sul, Brazil (#21847). 2.2. Subjects and reagents 2.2.1. Creatine and homocysteine chronic treatment Creatine and D,L-Hcy were dissolved in 0.85% NaCl solution and buffered to pH 7.4. Hcy solution was administered subcutaneously twice a day at 8 h intervals from their 6th to the 28th days of age. Hcy doses were calculated from pharmacokinetic parameters previously determined in our laboratory (Streck et al., 2002). During the first week of treatment, animals received 0.3 μmol Hcy/g body weight. In the second week, 0.4 μmol Hcy/g body weight was administered to the animals, and in the last week the rats received 0.6 μmol Hcy/g body weight. Plasma

Please cite this article as: Kolling, J., et al., Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.005

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Hcy concentration in rats subjected to this treatment achieved levels similar to those found in homocystinuric patients (Streck et al., 2002). Creatine solution (50 mg/kg of weight body) was injected intraperitoneally, once a day, from their 6th to their 28th days of age (Kolling and Wyse, 2010). Control animals received saline solution in the same volumes as those applied to Hcy-and creatine-treated rats. We chose this dose of creatine because the daily requirement of compound either through diet or endogenous synthesis is suggested to be approximately 2 g/day. A single 5 g oral dose in healthy adults results in a peak plasma creatine level of approximately 120 mg/l at 1–2 h post-ingestion. The elimination half life of creatine is quite short (just less than 3 h), however elevated plasma levels can be maintained by loading small oral doses every 3–6 h (12–24 g total per day) throughout the day (Owen and Sunram-Lea, 2011; Persky et al., 2003; Snow and Murphy, 2003). 2.2.2. Tissue preparation and homogenate preparation Animals were decapitated by decapitation without anesthesia. Soleus skeletal muscle was rapidly removed and dissected on a glass dish over ice for in vivo experiments. For oxidative stress parameter determination, the skeletal muscle was homogenized in 10 volumes (1:10, w/v) of 20 mM sodium phosphate buffer, pH 7.4 containing 140 mM KCl. After the homogenates were centrifuged at 800 ×g for 10 min at 4 °C, the supernatant was again diluted with the same buffer before and taken to biochemical assays. To determine the levels of TBARS, the skeletal muscle was homogenized in a Pyrex tube 1:10 (w/v) in 1.15% KCl. Homogenates were centrifuged at 800 ×g for 10 min at 4 °C, to discard nuclei and cell debris. The pellet was discarded and the supernatant was again diluted with the same buffer before and finally taken to biochemical assays. 2.2.3. 2′7′Dichlorofluorescein (DCFH) oxidation assay Reactive species production was measured following Lebel et al. (1992) method based on 2′7′-dichlorofluorescein (H2DCF) oxidation. Samples (60 μl) were incubated for 30 min at 37 °C in the dark with 240 μl of 100 μM 2′7′-dichlorofluorscein diacetate (H2DCF-DA) solution in a 96 well plate. H2DCF-DA is cleaved by cellular esterases and the resultant H2DCF is eventually oxidized by reactive species presenting in samples. The last reaction produces the fluorescent compound DCF which was measured at 488 nm excitation and 525 nm emission and the results were represented by nmol DCF/mg protein. A calibration curve was performed with purified DCF as standard. 2.2.4. Thiobarbituric acid-reactive substance assay TBARS levels, a measurement of lipid peroxidation, were determined according to Ohkawa et al. (1979). Briefly, homogenates in 1.15% KCl were mixed with 20% trichloroacetic acid and 0.8% thiobarbituric acid and heated in a boiling water bath for 60 min. TBARS levels were determined by the absorbance at 535 nm. The results were reported as nmol of malonaldehyde per mg protein. 2.2.5. Superoxide dismutase assay SOD activity was assayed using SpectraMax M5/M5 Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California, USA). This method for the assay of SOD activity is based on the capacity of pyrogallol to autoxidize, a process highly dependent on superoxide, which is substrate for SOD. The inhibition of autoxidation of this compound occurs in the presence of SOD, whose activity can be then indirectly assayed spectrophotometrically at 420 nm, using a double beam spectrophotometer with temperature control (Marklund, 1985). A calibration curve was performed with purified SOD as standard, in order to calculate the activity of SOD present in the samples. A 50% inhibition of pyrogallol autoxidation is defined as 1 unit of SOD and the specific activity is represented as units/mg protein.

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2.2.6. Catalase assay CAT activity was assayed using SpectraMax M5/M5 Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California, USA). The method used is based on the disappearance of H2O2 at 240 nm in a reaction medium containing 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer pH 7.0, and 0.1–0.3 mg protein/ml. One CAT unit is defined as 1 μmol of hydrogen peroxide consumed per minute and the specific activity is calculated as CAT units/mg protein (Aebi, 1984). 2.2.7. Glutathione peroxidase assay GPx activity was measured using tert-butyl-hydroperoxide as substrate. NADPH disappearance was monitored at 340 nm using SpectraMax M5/M5 Microplate Reader (Molecular Devices, MDS Analytical Technologies). The medium contained 2 mM GSH, 0.15 U/ml GSH reductase, 0.4 mM azide, 0.5 mM tertbutyl-hydroperoxide, and 0.1 mM NADPH. One GPx unit is defined as 1 mmol of NADPH consumed per minute and the specific activity is represented as GPx units/mg protein (Wendel, 1981). 2.2.8. Reduced glutathione content This method is based on the reaction of GSH with the fluorophore o-phtalaldeyde (OPT) after deproteinizing the samples and was measured according to Browne and Armstrong (1998). Initially, metaphosphoric acid was used to deproteinize the samples, which were then centrifuged at 1000 g for 10 min. Briefly, 15 ml of each sample was taken from 200 ml of a mixture containing 15 ml of OPT 1 mg/ml (prepared in methanol) plus 185 ml of 100 mM sodium phosphate buffer pH 8.0 with 5 mM EDTA in a 96-well plates. The assay was allowed to stand in the dark for exactly 15 min. After that, the fluorescence was measured at αex = 350 nm and αem = 420 nm. A calibration curve was also performed with a commercial GSH solution, and the results were calculated as nmol GSH/mg protein (Browne and Armstrong, 1998). 2.2.9. Total sulfhydryl content This assay was performed as described by Aksenov and Markesbery (2001), which is based on the reduction of 5,5′-dithio-bis (2nitrobenzoic acid) (DTNB) by thiols, which in turn become oxidized (disulfide), generating a yellow derivative (TNB) whose absorption is measured spectrophotometrically at 412 nm. Briefly, 50 ml of homogenate was added to 1 ml of PBS buffer pH 7.4 containing 1 mM EDTA. The reaction was started by the addition of 30 ml of 10 mM DTNB and incubated for 30 min at room temperature in a dark room. Results were reported as nmol TNB/mg protein. 2.2.10. Protein carbonyl content Oxidatively modified proteins present an enhancement of carbonyl content (Stadtman and Levine, 2003). In this study, protein carbonyl content was assayed by a method based on the reaction of protein carbonyls with dinitrophenylhydrazine forming dinitrophenylhydrazone, a yellow compound, measured spectrophotometrically at 370 nm. Briefly, 100 ml of homogenate was added to plastic tubes containing 400 ml of 10 mM dinitrophenylhydrazine (prepared in 2 M HCl). This was kept in the dark for 1 h and vortexed each 15 min. After that, 500 ml of 20% trichloroacetic acid was added to each tube. The mixture was vortexed and centrifuged at 20,000 g for 3 min. The supernatant obtained was discarded. The pellet was washed with 1 ml ethanol/ethyl acetate (1:1 v/v), vortexed, and centrifuged at 20,000 g for 3 min. The supernatant was discarded and the pellet re-suspended in 600 ml of 6 M guanidine (prepared in a 20 mM potassium phosphate solution pH 2.3). The sample was vortexed and incubated at 608 °C for 15 min. After that, it was centrifuged at 20,000 g for 3 min and the absorbance was measured at 370 nm (UV) in a quartz cuvette with a Hitachi U-2001 doublebeam spectrophotometer with temperature control (Hitachi High

Please cite this article as: Kolling, J., et al., Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.005

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Technologies America, Inc., Life Sciences Division, Pleasanton, CA). Results were represented as protein carbonyl content (nmol/mg protein). 2.2.11. Determination of nitrite levels Nitrite levels were measured using the Griess reaction; 100 μl of supernatant of heart was mixed with 100 μl Griess reagent (1:1 mixture of 1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water) and incubated in 96-well plates for 10 min at room temperature. The absorbance was measured on a microplate reader at a wavelength of 543 nm. Nitrite concentration was calculated using sodium nitrite standards (Green et al., 1982). 2.2.12. Protein determination Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard. 2.2.13. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test when the F value was significant, as indicated on captions. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software in a PCcompatible computer. Differences were considered statistically significant if p b 0.05. 3. Results Initially, we investigated the effect of chronic administration of Hcy and/or creatine on reactive species production and TBARS in the soleus skeletal muscle of young rats subjected to hyperhomocysteinemia and decapitated at 12 h after the last injection. Fig. 1 shows that Hcy increased the levels of reactive species (A) which was indicated by the 2′,7′-dichlorofluorescein (DCFH) oxidation, [F(3,20) = 3.77; p b 0.05], and also increased the levels of TBARS (B), a measurement of lipid peroxidation, [F(3,20) = 6.91; p b 0.01] in soleus skeletal muscle. Creatine treatment per se did not alter these parameters, but prevented the effects caused by chronic Hcy administration. Next, we evaluated the effect of chronic administration of Hcy and/or creatine on antioxidant enzymes, SOD, CAT and GPx activities in the soleus skeletal muscle of young rats. Fig. 2 shows that Hcy significantly increased the activities of SOD (A) [F(3,20) = 3.85; p b 0.05] and CAT (B) [F(3,20) = 5.97; p b 0.01], but did not alter GPx activity (C) [F(3,20) = 0.304] in the soleus skeletal muscle of young rats. Creatine administration per se also increased these enzymes, but when associated with Hcy it prevents such effects. We also evaluated the effect of chronic administration of Hcy and/or creatine on GSH, sulfhydryl and carbonyl content in the soleus skeletal muscle of young rats subjected to hyperhomocysteinemia. Fig. 3 shows that Hcy decreased significantly GSH (A) [F(3,20) = 6.59; p b 0.001], sulfhydryl (B) [F(3,20) = 7.88; p b 0.01] and carbonyl content (C) [F(3,20) = 12.40; p b 0.001] in the soleus skeletal muscle of young rats decapitated at 12 h after the last injection of Hcy. Creatine administration per se did not alter these parameters, but prevented the effects elicited by Hcy. Finally, we examined the effect of chronic administration of Hcy and/or creatine on nitrite levels in the soleus skeletal muscle of young rats subjected to hyperhomocysteinemia. Fig. 4 shows that Hcy significantly decreased the levels of nitrite [F(3,20) = 5.04; p b 0.01]. Creatine administration per se did not alter nitrite levels, but prevented the effect of Hcy on this parameter. 4. Discussion Muscle and motor impairment, as well as hypotonia are observed in patients with classical HCU, a disease caused by CβS deficiency (Huang et al., 2011; Leishear et al., 2012). CβS is found significantly in the

Fig. 1. Effect of chronic administration of homocysteine, creatine and homocysteine plus creatine on the levels of reactive species which was indicated by the 2′,7′dichlorofluorescein (H2DCF) oxidation and (B) TBARS levels, in soleus skeletal muscle of young rats sacrificed 12 h after the last injection. Data are mean ± S.D. for 5–7 animals in each group. Results are expressed as nmol DCF/mg protein and nmol of TBARS per mg protein, respectively. *p b 0.05; **p b 0.01 compared to control, respectively (Duncan multiple range test). Hcy: homocysteine. Cre: creatine.

muscular tissue (Chen et al., 2010). Elevated levels of Hcy in both plasma and cerebrospinal fluids are associated with a motor neuronal disease that causes muscle degeneration (Valentino et al., 2010; Zoccolella et al., 2008). Studies show that the multiple sclerosis may affect the muscle function and it has been shown that this neurological disease seem to be associated with elevated levels of Hcy in plasma (Zoccolella et al., 2012). Thus, the hyperhomocysteinemia has been associated with muscle impairment. In the present study we investigated some oxidative stress parameters in the soleus skeletal muscle of rats subjected to chemical experimental model of hyperhomocysteinemia, which was developed in our lab (Streck et al., 2002). In this model, plasma Hcy levels (500 μm) in rats were similar to those found in human HCU (Mudd et al., 2001). We also investigated the influence of concurrent administration of creatine on the effect of Hcy on oxidative stress parameters. Results showed that Hcy administration increased 2′7′dichlorofluorescein (DCFH) oxidation, an index of reactive species production, as well as enhanced TBARS levels that reflect the amount of malondialdehyde formation, an end product of lipoperoxidation (Halliwell, 2011). In addition to this, chronic administration of this amino acid increased the activities of SOD and CAT and decreased GSH, sulfhydril and carbonyl content, as well as nitrite levels. Creatine concurrent administration prevented all these effects caused by Hcy. Free radicals seem to be involved in a large number of human diseases. Oxidative stress was observed in some inborn errors of intermediary metabolism owing to the accumulation of toxic metabolites

Please cite this article as: Kolling, J., et al., Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.005

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Fig. 2. Effect of chronic administration of homocysteine, creatine and homocysteine plus creatine on antioxidant enzymes, (A) SOD, (B) CAT and (C) GPx activities in soleus skeletal muscle of young rats sacrificed 12 h after the last injection. Data are mean ± S.D. for 5–7 animals in each group. Results are expressed as units/mg protein. *p b 0.05; **p b 0.01 compared to control, respectively (Duncan multiple range test). SOD: superoxide dismutase; CAT: catalase; GPx: gluthatione peroxidase; Hcy: homocysteine; Cre: creatine.

leading to excessive free radical production. The field of free radicals and antioxidants is fundamental to aerobic life. Aerobes constantly make reactive species, but modulate their actions by synthesizing antioxidants. This balance allows some reactive species to perform useful functions while minimizing oxidative damage (Halliwell, 2011). Some authors have demonstrated an association between hyperhomocysteinemia and free radical formation (Huang et al., 2001; Streck et al., 2003). Hcy acts as a potent oxidizing agent of SH-group by reactive species production, such as O− 2 and H2O2, during its auto-oxidation

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Fig. 3. Effect of chronic administration of homocysteine, creatine and homocysteine plus creatine on (A) GSH, (B) sulfhydryl and (C) carbonyl content in the soleus skeletal muscle of young rats sacrificed 12 h after the last injection. Data are mean ± S.D. for 5–7 animals in each group. Results are expressed in nmol/mg protein, nmol TNB/mg protein, nmol/mg protein, respectively. ***p b 0.001, **p b 0.01, *p b 0.05 compared to control (Duncan multiple range test). GSH: glutathione; Hcy: homocysteine. Cre: creatine.

(Dayal et al., 2004; Faraci and Lentz, 2004). In this context, the reduction of plasma Hcy levels by creatine supplementation is associated with a significant reduction of plasma lipid peroxidation biomarkers (Deminice and Jordao, 2012). A negative correlation between plasma creatine and TBARS levels in the plasma has been shown, suggesting that it may act as an antioxidant (Lawler et al., 2002; Sestili et al., 2011). In addition, studies show that the creatine may be cytoprotector independent of the antioxidant status of the enzymatic defenses, indicating a direct interaction between creatine and oxidant and/or free radicals. Studies also show that the creatine supplementation reduces plasma Hcy levels in rats (Deminice and Jordao, 2012; Stead et al., 2001; Taes et al., 2003). In agreement with the studies that show an antioxidant effect of creatine, in the present study we demonstrated that

Please cite this article as: Kolling, J., et al., Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.005

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Fig. 4. Effect of chronic administration of homocysteine, creatine and homocysteine plus creatine on the nitrite levels in the soleus skeletal muscle of young rats sacrificed 12 h after the last injection. Data are mean ± S.D. for 5–7 animals in each group. Results are expressed in μM/mg protein. **p b 0.01 compared to control (Duncan multiple range test). Hcy: homocysteine. Cre: creatine.

the creatine can act as an antioxidant, preventing the lipid peroxidation (Kolling and Wyse, 2010) and the increase in RS production. Regarding the enzymatic antioxidant defenses, we observed that chronic hyperhomocysteinemia provoked a significant increase in SOD and CAT activities, but did not alter GPx activity in rat skeletal muscle. The activities of these enzymes increased in the same proportion and we did not observed an imbalance in the SOD/CAT ratio. Creatine concurrent administration prevented the increase in SOD and CAT activities caused by Hcy. Evidences show that the continued presence of low concentrations of RS is able to induce upregulation of antioxidant enzymes, as a cellular strategy of adaptation to oxidative stress (Halliwell, 2011). Therefore, based on this data and in the results of the present study, it is conceivable to suggest that high Hcy levels could lead to generation of superoxide radical in muscle tissue, as well as the increase in SOD activity. Superoxide is dismutated by SOD with consequent generation of H2O2, which is in turn reduced by CAT. Then, the increase in the activities of these antioxidant enzymes in skeletal muscle of rats observed in the present study may be a consequence from tissue adaptation to sustained production of RS, mainly superoxide and hydrogen peroxide, which are substrates scavenged by SOD and CAT, respectively. It would be interesting to know if the reversal of abnormal Hcy metabolism could reduce the pathological events caused by the compromised cellular antioxidant potential. RS can have multiple targets in any given time based on the site and amount of its production. Hence, relevance of Hcy-mediated changes via RS production and muscle malfunction need to be studied to understand muscle weakness associated with hyperhomocysteinemia (Veeranki and Tyagi, 2013). Therefore, the unchanged levels of reactive species (assessed by DCFH assay) and the increase in lipid peroxidation (detected by TBARS) in rat skeletal muscle submitted to Hcy administration could be attributed to an effective detoxification of RS by enzymatic antioxidant defense as demonstrated by increased SOD and CAT activities. This putative role of Hcy in the upregulation of antioxidant enzymes could be a consequence a compensatory mechanism to protect the skeletal muscle from oxidative injury caused by Hcy. We also measured the levels of GSH, a major antioxidant in muscles (Martensson and Meister, 1989). Our result showed that chronic hyperhomocysteinemia decreased GSH levels and creatine prevented this effect. It has been demonstrated that a severe depletion in the levels of muscular GSH may compromise the mitochondrial function and cause muscle degeneration (Marí et al., 2009). Moreover, levels of Hcy and

cysteine are able of modulating the muscle damage (Veeranki and Tyagi, 2013). Hcy is the intermediate in the transsulfuration and remethylation pathways and its production may influence the synthesis and function of GSH (Prudova et al., 2006). Studies showed that a decrease in plasma GSH may cause a modulation in the methyl balance and transsulfuration pathway, affecting the redox status of the cells. Thus, in our study the creatine supplementation could have reduced the levels of plasma Hcy by modulating the methyl balance and the bioavailability of Hcy for the remethylation and transsulfuration processes, in turn influencing GSH synthesis. Mutations in the gene of the enzyme CβS that cause hyperhomocysteinemia and also cysteine deficiency were reported (Majors et al., 2002). Cysteine is critical for the production of GSH, furthermore in transsulfuration enzymes compromise the antioxidant potential of the muscle cells, resulting in reduced GSH content in skeletal muscles, which could enhance the sensitivity to oxidative stress, and lethal muscular atrophy (Ishii et al., 2010). This depletion of muscular glutathione levels probably causes muscle disorders, because it is a major nonenzymatic antioxidant in this tissue. Two markers of protein oxidation in skeletal muscle of rats were also investigated: sulfhydryl content, which is employed to verify protein damage to sulphydryl groups and carbonyl content, formed mainly by oxidation of side chains of some amino acid residues (Dalle-Donne et al., 2003). In the present study we observed a decrease in these parameters, suggesting oxidative damage to proteins in soleus skeletal muscle of rats subjected to chronic hyperhomocysteinemia. Regarding nitrite levels, Hcy significantly decreased this parameter. An important signaling pathway of NO is represented by the reaction with thiol groups. The spontaneous reaction of NO with thiol groups of the Hcy itself and/or O− 2 , metabolite generated by multiple processes, including the auto-oxidation of Hcy, may also reduce the availability of NO synthesized (Lang et al., 2000; MacCarthy et al., 2001). NO production was reported in muscular dystrophies (Villanueva and Giulivi, 2010) because the blood flow to muscle cells is typically regulated by NO. Hcy diminishes the bioavailability of NO through uncoupling of nitric oxide synthase (NOS) increasing RS production. In the presence of elevated RS, NO reacts with RS and generates peroxynitrite limiting NO signaling (Steed and Tyagi, 2011). High concentrations of Hcy might compromise NO signaling in muscular vessels and result in fatigue, ischemia and reduced physical endurance (Veeranki and Tyagi, 2013). Creatine administration presents protective role, preventing oxidative stress induced by Hcy administration (Balestrino et al., 2002; Caretti et al., 2010; Kolling et al., 2013; Sestili et al., 2011). Several investigators have suggested that the antioxidant effects of creatine can be attributed to indirect antioxidant mechanisms such as hydration, membrane stability and improved energy capacity of the cell (Persky and Brazeau, 2001; Wyss and Schulze, 2002). In this context, we have shown that the creatine prevent some alterations in energy metabolism of skeletal muscle of rats (Kolling et al., 2013). In conclusion, we showed that chronic Hcy administration increased the 2′7′dichlorofluorescein (DCFH) oxidation and TBARS levels, indexes of production of reactive species and lipoperoxidation, respectively. Antioxidant enzyme activities, SOD and CAT were also increased, but GPx activity was not altered. GSH, sulfhydril and carbonyl contents were decreased, as well as nitrite levels. Beyond what has been reported, our results also showed that creatine administration prevented RS production and other oxidative stress parameters induced by Hcy probably by its protective role, including antioxidant properties. Based on these findings, we suggest that creatine may be used as adjunctive therapy for ameliorating the symptoms associated with oxidative insult that can be found in patients with hyperhomocysteinemia. Conflict of interest All authors declare no financial/commercial conflicts of interest.

Please cite this article as: Kolling, J., et al., Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.005

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Please cite this article as: Kolling, J., et al., Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.005