Protective Role of Edaravone Against Valproic Acid Induced Changes in Skin

Indian Journal of Experimental Biology Vol. 55, May 2017, pp. 286-291

Protective role of Edaravone against valproic acid induced changes in skin

Tugba Tunali-Akbay1*, Burcin Alev1, Sevim Tunali2 , Sehkar Oktay1, Ebru Emekli-Alturfan1, Hazal Ipekci1, Leyla Koc Ozturk1, Refiye Yanardag2 & AysenYarat1 1 Department of Basic Medical Sciences, Faculty of Dentistry, Marmara University, Istanbul, Turkey 2Department of Chemistry, Faculty of Engineering, Istanbul University, Istanbul, Turkey Received 06 April 2015; revised 19 August 2015

Valproic acid (VPA) is an antiepileptic drug known to have some adverse effects on gastrointestinal, neurologic, hematologic and reproductive systems. In this study, we investigated the effect of edaravone (3-methyl-1-phenyl-2- pyrazoline-5-one), a potent free radical scavenger, on skin oxidative and inflammatory changes caused by VPA. Female rats were randomly divided into four groups: control, edaravone (30 mg/kg/day, i.p.), VPA (0.5 g/kg/day, i.p.), and VPA+edaravone (in same dose, i.p.). On the 8th day of experiment, all the animals were fasted overnight and then sacrificed under anesthesia. Malondialdehyde, glutathione, total sialic acid and total protein levels, superoxide dismutase, glutathione- S-transferase, catalase, glutathione peroxidase, Na+/K+-ATPase, myeloperoxidase and tissue factor activities were determined in homogenized skin samples. VPA administration significantly altered the skin oxidant and antioxidant balance which may cause skin cell damage. Edaravone, as an antioxidant, demonstrated its radical scavenger effect, especially on malondialdehyde level and glutathione-S-transferase activity. Edaravone, by preventing inflammatory and oxidative reactions, partly protected the skin against the changes observed after the VPA administration.

Keywords: Adverse drug reaction, Antiepileptic drug, Oxidative damage, Scavenger, Side effects, Tissue Factor Activity

Valproic acid (2-propylvaleric acid, 2-propylpentanoic human and animal models are not uncommon6-9. acid or n-dipropyl acetic acid, VPA) is a well-known In these reports, the generation of oxidative damage is drug for treatment of convulsive seizures related to associated with VPA, as a consequence of VPA epilepsy, as a mood stabilizer and in the treatment of biotransformation6, alterations in glutathione schizophrenia1. The use of VPA can be limited by homeostasis6-8, and/or depletion of co-factors required either loss or lack of efficacy, or by adverse drug for antioxidant defense reactions. The adverse drug reactions associated with Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) the VPA usage include drowsiness, dizziness, is drug that acts as an antioxidative radical scavenger, headache, diarrhea, constipation, heartburn, changes is used in the treatment of cerebrovascular diseases9. in appetite, weight changes, back pain, agitation, In addition, edaravone has been reported to have mood swings, abnormal thinking, memory loss, body protective effects on inflammation-induced pain and tremor, loss of coordination, uncontrollable cisplatin-induced nephrotoxicity in rats9,10. However, movements of the eyes, blurred or double vision, effects of edaravone on skin antioxidant metabolism ringing in the ears, stuffed or runny nose, sore throat after the VPA treatment has not been investigated and hair loss2. VPA is also associated with the adverse until now. Therefore in this study, we evaluated the drug reactions including purple spots on the skin, possible antioxidant effect of edaravone on skin cases of stomatitis, cutaneous leukoclastic vasculitis, following the VPA administration. and psoriasiform eruption3,4. As the skin is constantly under attack by reactive oxygen species (ROS) from Material and Methods both endogenous and exogenous sources5, VPA can All experimental protocols were approved by the disrupt the oxidant and antioxidant balance of the Marmara University Animal Care and Use Committee skin. Studies on the oxidative role of VPA, both in (134.3013 mar). Thirty-seven Sprague Dawley female rats were randomly divided into four groups as ______follows: Control; edaravone (30 mg/kg/day); VPA *Correspondence: Telefax: +90 216 421 16 21/1397 (0.5 g/kg/day); and VPA + edaravone (same dose). E-mail: [email protected](TTA); [email protected] (AY) Edaravone, VPA and VPA+edaravone were given TUNALI-AKBAY et al.: EDARAVONE PROTECTS SKİN AGAINST VALPROIC ACID INDUCED CHANGES 287

intra-peritoneally (i.p.) for 7 days. VPA was given to coupled with thiobarbituric acid, and the resulting the animals one hour after the edaravone chromophore was extracted into cyclohexanone. The administration every day. On the 8thday of the absorbances of samples were measured at wavelenght experiment, animals were fasted overnight and then of 549 nm and the results were expressed as mg SA/g sacrificed under anesthesia. The skin samples were of protein. taken from the back (dorsal side) of each rat after removing the local fur. After the epidermis was Determination of glutathione peroxidase activity GPx activity of skin tissues were determined by the removed, the skin samples were homogenized in 0.9% 15 NaCl solution. Homogenates [10% (w/v)] were method of Paglia and Valentina . In this method, the prepared. Total protein, lipid peroxidation (LPO), conversion of H2O2 to water is catalyzed by GPx. The glutathione (GSH), sialic acid (SA) levels and generated glutathione disulfide is reduced to GSH glutathione peroxidase (GPx), glutathione-S- with consumption of NADPH by glutathione transferase (GST), superoxide dismutase (SOD), reductase. The decreased absorbance during the catalase (CAT), myeloperoxidase (MPO), sodium oxidation of NADPH to NADP was measured by + + , spectrophotometer at 366 nm. The extinction potassium ATPase (Na /K ATPase) and tissue factor -1 -1 (TF) activities were determined in skin homogenates. coefficient of 6.22 mM cm was used for calculation and results were expressed as U/g protein. Determination of total protein Determination of glutathione S tranferase activity Total protein levels of tissues were determined by 16 the method described by Lowry et al.11. Briefly, in GST catalyzes the conjugation of GSH . The alkali medium proteins are reacted with cupper ions absorbance of mixture at 25C was measured by and reduced by Folin reactive. The absorbance of the spectrophotometer at 340 nm. GST activities of skin tissues were calculated by using the extinction blue colored product was evaluated at 500 nm. Bovine -1 -1 serum albumin was used as a standard and total coefficient of 9.6 mM cm and results were protein levels of skin tissues were used to express the expressed as U/g protein per min. results of the parameters per protein. Determination of superoxide dismutase activity 17 Determination of lipid peroxidation SOD activities were determined by the method Malondialdehyde (MDA), one of the products of based on the ability of SOD to increase the effect of LPO in tissues, was determined as thiobarbituric acid riboflavin-sensitized photooxidation of o-dianisidine. reactive substances according to the methoddescribed The activity of superoxide is generated by by Yagi 12.LPO was expressed in terms of MDA illuminating the reaction mixture which contains equivalents using the extinction coefficient of 1.56 × o-dianisidine dihydrochloride and riboflavin by light 105 M–1cm–1. Results of skin tissues are expressed as of a fluorescent lamp. The oxidation of o-dianisidine, nmol MDA/mg protein. as sensitized by riboflavin, is enhanced by SOD and the increase is linearly dependent on SOD Determination of glutathione concentration. The absorbance of the coloured GSH levels were determined in skin tissues according product is quantified by spectrophotometer at 460 nm. to the method of Beutler13 using metaphosphoric acid for Absorbances at 0 and 8 min of illumunation were protein precipitation and 5-5-dithiobis-2-nitro benzoic measured and the net absorbance was calculated. acid for colour development. The extinction coefficient Bovine SOD (Sigma Chemical Co, S-2515-3000 U) of 1.36 × 104 M–1 cm–1 was used for calculation of GSH was prepared as reference and the results were levels of skin tissues and results were expressed as mg expressed in U/mg protein. GSH per gram of protein. Determination of catalase activity Determination of sialic acid CAT activities of skin tissues were determined by SA levels of skin tissues were determined by the the method of Aebi18 which is based on the 14 thiobarbituric acid method of Warren . Homogenates conversion of hydrogenperoxide (H2O2) to water by were incubated with 0.1 N H2SO4 at 80ºC for 1 h and the effect of CAT enzyme. This conversion was hydrolysate was used for analysis. SA was oxidized observed as a decrease in absorbance measured at with sodium periodate in concentrated phosphoric 240 nm and CAT activity of samples were expressed acid and the product of periodate oxidation was as U/mg protein. 288 INDIAN J EXP BIOL, MAY 2017

Determination of myeloperoxidase activity Results MPO is a natural constituent of primary granules Administration of VPA to the rats did not of neutrophils. Enzymatic activity of MPO was significantly change the skin total protein. MDA level 19 determined by the method of Wei and Frenke increased, though insignificantly, in the VPA which contains a solution of tissue homogenate, administered group compared to the control groupt. phenol, H2O2 and 4-AAP as colour generating Similarly, VPA did not significantly change GSH substance. One unit of enzyme activity was defined levels and GPx, SOD and CAT activities when as the amount of the MPO present per gram of compared with the control group (Table 1 and Fig. 1). protein which caused a change in absorbance SA levels, TF and MPO activities significantly per minute at 460 nm and 37C. The results were increased with the administration of VPA (Table 2). expressed as U/g protein. VPA also decreased the Na+/K+-ATPase activity (Table 2). Determination of Na+/K+-ATPase activity Edaravone administration to VPA group, + + Na K ATPase activity was measured by the significantly decreased MDA level and TF activities 20 method of Ridderstap & Bonting . The reaction (Table 1). It also significantly decreased MPO level medium for total standard reaction mixtures contained (Table 1) in VPA administered group. GST activity 5 mM KCI, 150 mM NaCl, 2.5 mM MgCl2 and significantly increased with the edavarone 0.1 mM EDTA in 600 mL of 20 mM imidazole administration to the VPA administered group (Table 1 buffer. The ghost suspension was added in a volume of 0.5 mL and the mixture was placed in a water bath Table 1—Skin MDA level and SOD and CAT activities C C+E VPA VPA+E at 37C for 10 min. The reaction was started with the (n=6) (n=6) (n=6) (n=6) addition of 2.5 mM of disodium ATP in a volume of Mean/SD Mean/SD Mean/SD Mean/SD

0.1 mL. At the end of 1 h, the tubes were placed in MDA 1,89/ 2,30 / 1,36 / an ice bath. To stop the reaction, 1 mL of 15% (nmolMDA/ mg p) 1,80/ 0,36 0,31 0,37 0,35b trichloroacetic acid was added. Pi released by the SOD (U/mg p) 4,12/ 3,32 3,40 3,57/ reaction was measured. Na+K+ ATPase activities 0,06 0,66 0,37 0,72 CAT (U/mg p) 36,00/ 37,33/ 35,49/ 36,00 / were expressed as µmol of Pi per milligram of 6,07 7,95 4,14 8,72 protein per hour. [C, Control; C+E, Control+Edaravone; VPA, Valproic Acid; VPA+E, Valproic acid+Edaravone; p, protein; MDA, Determination of tissue factor activity malondialdehyde; SOD, superoxide dismutase; CAT, catalase. TF activities of skin tissues were determined by Values are given as mean/SD, SD standard deviation p: protein h: 21 hour. b P <0,05 significantly different from VPA] Quick’s one stage method . Pooled plasma collected from healthy subjects was used and all reagents were Table 2—Skin TP, SA levels and TF, Na+/K+ -ATPase, MPO activities brought to 37ºC, which is the reaction temperature, C C+E VPA VPA+E before admixture. TF activity of skin tissues were (n=6) (n=6) (n=6) (n=6) Mean/SD Mean /SD Mean/SD Mean/SD performed by mixing 0.1 mL homogenate with 0.1 mL of plasma, then the clotting reaction being TP 2,08/ 2,63/ 2,61/ 2,78/ ((mg/g wet tissue) 0,34 0,48 0,56 0,38 started in addition of 0.1 mL of 0.02 M CaCl2. The SA 13,60/ 17,50/ 39,32/ 46,47/ clotting time is inversely proportional to the TF (mg/g p) 3,85 4,24 4,75 a, c 9,36 a,c activity, and for that reason the increase of the TF 122,8/ 101,5/ 70,83/ 126,7/ clotting time is a manifestation of decreased TF (sec) 33,55 24,19 21,94 a 21,07b + + activity. TF results were expressed as seconds. Na /K -ATPase 0,28/ 0,26/ 0,15/ 0,21/ (µmol Pi/mg p h) a, c 0,05 0,07 0,03 0,06 MPO (U/g p) 3,20/ 2,70/ 5,92/ 3,99/ Statistical analysis 0,64 0,65 1,64 a, c 1,16b Statistical analyses were carried out using [C, Control; C+E, Control+Edaravone; VPA, Valproic Acid; GraphPad Prism 5 (GraphPad Software, San Diego, VPA+E, Valproic acid+Edaravone ; TP, total protein; SA, sialic CA, USA). All data were expressed as mean ± SD acid; TF, tissue factor; Na+/ K+ -ATPase, sodium potassium (standard deviation). Groups of data were compared ATPase; MPO, myeloperoxidase; s. seconds; Pi, inorganic using ANOVA followed by Tukey’s multiple phosphate; p, protein; h, hour. Values are given as mean/SD, SD standard deviation. a P <0,05 significantly different from C; comparison tests. Values of P <0.05 were regarded as b P <0,05 significantly different from VPA; and significant. c P <0,05 significantly different from C+E] TUNALI-AKBAY et al.: EDARAVONE PROTECTS SKİN AGAINST VALPROIC ACID INDUCED CHANGES 289

Neither VPA, nor VPA + edaravone did not significantly change SOD, GPx and CAT activities and GSH level in the skin. The skin GST activity significantly decreased in VPA group compared to the control and the edaravone administered control group. Edaravone significantly increased skin GST activity in VPA administered group. In the glutathione metabolism, the net effect of edaravone was found on skin GST activity after the VPA administration. GST catalyze the conjugation of GSH, via a sulfhydryl group, to electrophilic centers on a wide variety of substrates in order to make the compounds more 28 soluble . It has been reported that GST null genotype

Fig. 1—Skin GSH level, GST and GPx activities in all groups. increases the susceptibility of skin cancer and also [C, Control; C+E, Control+Edaravone; VPA, Valproic Acid; shows lower enzymatic activity than GST positive VPA+E, Valproic acid+Edaravone. GSH, glutathione; GST, genotype29. These studies suggest that lower GST glutathione S transferase and GPx, glutathione perooxidase. activity possibly induces cell damage that causes skin Values are given as mean±SD, SD standard deviation, p: protein. a, b & c: significantly different from C, VPA and C+E group. cancer. Similarly, VPA induced skin damages Level of significance, P <0,05] observed here might be attributed to the decreased GST activity after the VPA administration. As and Fig. 1). Edaravone administration to the control edaravone markedly increased the GST activity after group also significantly increased GSH and GPx level the VPA administration, the usage of edaravone may (Fig. 1). Edaravone also increased GPx level in VPA prevent the skin disorders observed after the VPA administered group but this increase was not treatment. significant. TF, also known as thromboplastin or Factor III, is a

cell membrane component. As TF is a labile protein, Discussion its activity can be changed by the disturbances in The antiepileptic drug, valproic acid (VPA) membrane composition, heating and pH changes30. In treatment may cause some skin diseases such as the present study, the VPA administration stomatitis, cutaneous leukoclastic vasculitis, and significantly increased skin TF activity when psoriasiform eruption. Here, we investigated the compared to the control group. Increase in skin TF possible effects of VPA and/or edaravone on skin, in activity may be relevant to the skin damage seen terms of oxidative stress and tissue damage. As following VPA administration. Asero et al.30 too the rates of skin rash with VPA are very low22 as observed an increased TF expression in the skin of the compared to another antiepileptic drug lamotrigine23, patients with urticaria. They revealed that if the mechanism of skin rashes under the VPA therapy autoantibodies, complement, and mast cell-derived still unknown. There are reports on the antiepileptic factors such as histamine, tryptase, and/or different drug, sodium valproate increasing the lipid cytokines are responsible for activation of endothelial peroxidation in patients receiving it24-26 . Chang & cells causing TF expression, then the observed Abbott26 also showed that oxidative stress has a activation of the extrinsic coagulation pathway would potential role on sodium valproate-induced be a secondary consequence of urticaria and they hepatotoxicity. On the other hand, Verrotti et al.27 recommended using anticoagulants in patients with found that sodium valproate therapy does not cause urticaria. Cadroy et al.31 demonstrated the activation oxidative stress in epileptic children who remained of coagulation by free radicals and also the fact that non-obese during the treatment. polymorphonuclear leukocytes modulates the In the present study, as a marker of lipid production of TF by monocytes via the release of free peroxidation, MDA level was determined. The VPA radicals. In the present study, the skin TF activity was administration did not significantly change the skin used as an indicator of tissue damage, which MDA and as an antioxidant edaravone administration increased due to the VPA administration, whereas to VPA group significantly decreased the MDA level. edaravone administration to the VPA group 290 INDIAN J EXP BIOL, MAY 2017

significantly decreased the TF activity. The increased activity in skin could be due to the skin damages TF activity following the VPA administration can be caused by the VPA treatment. attributed to the skin cell damage, and the normalized Conclusion TF activity with edaravone administration to the free Increased skin SA, TF and MPO activities and radical scavenger effect of the edaravone. decreased GST and Na+/K+-ATPase after valproic Sialic acid (SA) is the generic term of a family, acid (VPA) administration are symptoms of the which is the acetylated derivative of neuraminic acid. damaged skin cells subsequent to the VPA treatment. These are suggested to be major participants in many Edaravone by preventing the inflammatory and biological functions. 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