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Methoxy VO-salen complex: In vitro antioxidant activity, cytotoxicity evaluation and protective effect on CCl4-induced oxidative stress in rats
Food and Chemical Toxicology 47 (2009) 716–721
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Methoxy VO-salen complex: In vitro antioxidant activity, cytotoxicity evaluation and protective effect on CCl4-induced oxidative stress in rats Mohsen Mohammadi, Razieh Yazdanparast * Institute of Biochemistry and Biophysics, University of Tehran P.O. Box 13145-1384, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 20 August 2008 Accepted 19 December 2008
Keywords: Antioxidant Lipid peroxidation Methoxy VO-salen Oxidative stress Protein oxidation ROS
a b s t r a c t Reactive oxygen species (ROS) play crucial roles in normal physiological processes. However, the overproduction of ROS is involved in the onset of many degenerative diseases. Regarding this fact, discovery of new antioxidants is interesting for many research groups. In this study, we evaluated the antioxidant properties of a methoxy VO-salen (MetVO-salen) complex employing various in vitro systems. In addition, the cytotoxic effect of MetVO-salen was assessed based on MTT in treated K562 cells. In an in vivo approach, the protective effect of MetVO-salen against CCl4-induced oxidative stress in rats was also investigated in terms of superoxide dismutase (SOD) and catalase (CAT) activities, as well as in terms of the levels of malondialdehyde (MDA) and glutathione (GSH). Our results indicated that MetVO-salen has an effective capability in scavenging superoxide ðO 2 Þ and hydrogen peroxide (H2O2) radicals in a dose-dependent manner. In vivo results also showed that the administration of MetVO-salen to the CCl4-treated rats caused a significant (222%) increase in SOD activity, a 59% enhancement in GSH content and a 31% decrease in the level of MDA compared to the CCl4-treated control rats. Overall, MetVOsalen appears to be an effective antioxidant and is quite suitable for further biological evaluation. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Exogenous factors such as sunlight, ultraviolet light, ionizing radiation, some chemicals and normal cellular activities lead to the production of reactive oxygen species (ROS). The major types of ROS or ROS-producing species are superoxide anion ðO 2 Þ, hydrogen peroxide (H2O2) and hydroxyl ð OHÞ radicals (Cerutti, 1991). ROS present a paradox in their biological function: on one hand, they prevent diseases by assisting the immune system, mediating cell signaling and playing an essential role in apoptosis. On the other hand, they can damage many biologically active molecules and hence leading to tissue damages and cell death (Finkel and Holbrook, 2000; Martindale and Holbrook, 2002). In cells, there are several antioxidant defense mechanisms that function-
Abbreviations: BHT, butylated hydroxyl toluene; BSA, bovine serum albumin; CAT, catalase; DMSO, dimethylsulfoxide; DTNB, 5,50 dithio-bis (2-nitro benzoic acid); ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GPx, glutathione peroxidase; GSH, glutathione; 4-HNE, 4-hydroxynonenal; i.p., intraperitoneal; LPO, lipid peroxidation; MDA, malondialdehyde; MetVO-salen, methoxy VO-salen; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; NBT, nitroblue tetrazolium; PCO, protein carbonyl; PMS, phenazine methosulphate; ROS, reactive oxygen species; SOD, superoxide dismutase; TBA, 2-thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid; Trolox, 6-hydroxy-2,5,7,8-tetra methyl chroman-2-carboxylic acid. * Corresponding author. Tel.: +98 21 66956976; fax: +98 21 66404680. E-mail address: [email protected] (R. Yazdanparast). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.029
ally assist in preventing the destructive effects of various types of ROS. These defense mechanisms include antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and small molecules such as glutathione (GSH) and vitamins C and E. Deficiency or abnormality in any of these defense mechanisms will lead to overproduction of ROS followed by oxidation of biomolecules such as proteins, lipids and DNA. At normal physiological condition a delicate balance exists between the rate of generation and the consumption of free radicals (Nordberg and Amer, 2001; Duh et al., 1999). In abnormal cases, particularly when the rate of production exceeds the rate of consumption or inactivation of ROS, the biological system is overdosed by ROS with potentially serious health consequences. It is now well accepted that oxidative stress is involved in the pathogenesis of various types of diseases including cancer, coronary heart diseases, aging, diabetes and neurodegenerative diseases (Moskovitz et al., 2002; Pryor, 1991; Lai et al., 2001). Due to wide deleterious effects of ROS on structural and functional integrities of biological systems, a great deal of scientific efforts has been devoted to finding effective free radical scavengers, which are referred to as antioxidants. In this respect, transition metal complexes have notably shown to possess high antioxidative properties (Riley, 1999; Autzen et al., 2003). From this group of compounds, vanadium complexes deserve a special attention. Vanadium is a transition metal and a trace element acting as the catalytic component of several enzymes such as haloperoxidase
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and nitrate reductase, which possess free radical chemistry (Rehder, 2003). Various vanadium complexes (SALENS) have been proposed for potential use as insulin-mimetic agents, and have been shown to have insulin-mimetic properties in animal model systems and cell culture (Sakurai et al., 2002). Besides the well-established antidiabetic effects, vanadium complexes also exhibit antitumor and anti-inflammatory activities (Goc, 2006). On the other hand, it is now well accepted that oxidative stress is involved in the pathogenesis of cancer and diabetes diseases. Since the antioxidative activities of VO-salen complexes have not been studied in in vivo or ex vivo systems; it would certainly be of interest to clarify these properties for MetVO-salen complex using animal (rat) and cell culture (K562 cells) systems, respectively. 2. Materials and methods 2.1. Chemicals Ascorbic acid, 6-hydroxy-2,5,7,8-tetra methyl chroman-2-carboxylic acid (Trolox), trichloroacetic acid (TCA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and ferric chloride were obtained from Sigma (Sigma– Aldrich, Sternheim, Germany). Reduced nicotinamide adenine dinucleotide (NADH), phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), hydrogen peroxide (H2O2), butylated hydroxyl toluene (BHT) and 2-thiobarbituric acid (TBA) were obtained from Merck (Germany). Salicylaldehyde and ethylenediamine were purchased from Fluka (Buchs, Switzerland). All other chemicals used were of analytical grade and were obtained from either Sigma–Aldrich or Merck (Germany). The synthesis and purification of MetVO-salen (Scheme 1) were achieved as reported previously (Kianfar and Mohebbi, 2007). 2.2. In vitro and ex vivo assay models 2.2.1. Hydrogen peroxide scavenging activity The H2O2-scavenging activity of MetVO-salen was studied according to the slightly modified method of Ruch et al. (1989). Different concentrations of MetVO-salen (2.5, 12.5, 25 and 37.5 lM in phosphate buffer, 50 mM, pH 7.4) were added to 2 ml of H2O2 solution (10 mM) in phosphate buffer (50 mM, pH 7.4), and the reaction mixture was incubated at 25 °C for 30 min. The unreacted H2O2 was determined by measuring the absorbance of the reaction mixture at 230 nm with respect to the blank solution (a solution of MetVO-salen in phosphate buffer without H2O2). The percentage of H2O2-scavenging activity of MetVO-salen was calculated using the following equation:
H2 O2 scavenging activity ð%Þ ¼ ½ðA A1 =AÞ 100 where A is the absorbance of the control, and A1 is the absorbance in the presence of MetVO-salen or the standard sample. 2.2.2. Scavenging of superoxide anion in PMS-NADH/ NBT system Superoxide anion-scavenging activity of MetVO-salen was determined based on the method described by Liu (Liu et al., 1997). In the experiment, O 2 anions were generated in 3 ml of Tris–HCl buffer (16 mM, pH8) containing 1 ml of NBT (50 lM), 1 ml of NADH (78 lM) and different concentrations of MetVO-salen. The reaction was initiated by adding 1 ml of PMS solution (10 lM) to the reaction mixture and after 5 min incubation at 25 °C; the absorbance was measured at 560 nm against the corresponding blank. The O 2 anion-scavenging activity, in percentage, was calculated using the following equation:
O 2 anion-scavenging activity ð%Þ ¼ ½ðA A1 =AÞ 100; where A is the absorbance of the control, and A1 is the absorbance in the presence of MetVO-salen or the standard sample. 2.2.3. Antioxidant assay using rat liver homogenate This assay is based on the ability of antioxidants toward the inhibition of lipid peroxidation in rat liver homogenate in the presence of FeSO4 and ascorbate. The extent of lipid peroxidation in the presence and absence of MetVO-salen was eval-
Scheme 1. Chemical structure of MetVO-salen complex.
717
uated based on the extent of thiobarbituric acid reactive substances (TBARS) according to the published method (Mahakunakorn et al., 2004). 2.3. Cytotoxicity evaluations 2.3.1. Cell culture The human leukemia K562 cells were cultured in RPMI-1640 medium supplemented with FBS (10%, v/v), streptomycin (100 lg/ml) and penicillin (100 U/ml). The cells were incubated under 5% CO2 humidified atmosphere at 37 °C. Cell numbers were assessed using a hemocytometer and the abilities of the cells to exclude trypan blue. 2.3.2. Cytotoxicity evaluation of MetVO-salen in K562 cells Cell viability was estimated using the MTT assay. This method is dependent on the conversion of yellow tetrazolium bromide to its purple formazan derivative by mitochondrial succinate dehydrogenase of the viable cells (Mosmann, 1983). The cells were seeded in 96-well plates at a concentration of 105cells/ml for 24 h, and then treated in triplicate with MetVO-salen at different concentrations (2.5, 12.5, 25 and 37.5 lM). After 24 h, MTT stock solution (10 ll; 5 mg/ml) was applied to each well. After 4 h of incubation, the plates were centrifuged for 10 min at 800 g, and the supernatants were aspirated. The formazan crystals in each well were dissolved in 100 ll of dimethylsulfoxide (DMSO), and the absorbance was measured via ELISA technique at a wavelength of 570 nm. 2.3.3. Cytoprotective effect of MetVO-salen in H2O2-treated K562 cells To evaluate the cytoprotective effect of MetVO-salen in H2O2-treated K562 cells, the cells were plated at a density of 105cells/ml for 24 h. The cells were pretreated with various concentrations of MetVO-salen (2.5, 7.5, 12.5 and 17.5 lM). Two hours later, H2O2 (300 lM) was added to the plate followed by incubation for an additional 16 h. Cell viability was estimated using the MTT assay, and the cellular viability was expressed in percentage of survival relative to the control cell samples. 2.4. In vivo assay model 2.4.1. Animals and experimental protocols Male wistar rats, weighting 200–250 g and with an average age of 8 weeks, were purchased from Pasteur institute (Tehran, Iran). They were housed under ambient temperature of 22 ± 2 °C with 60 ± 5% relative humidity and a lighting cycle from 7:00 a.m. to 7:00 p.m. The animals received food and water ad libitum. After the adaptation period, rats were randomly divided into three groups of six animals each: the first group of rats (control group) was administered olive oil intraperitoneally (i.p.) at a dose of 0.2 ml/kg body weight, twice a week, for 4 weeks. The second and third groups of rats were administered CCl4 i.p. at a dose of 0.4 ml/kg body weight (50% CCl4 in olive oil), twice a week, for 4 weeks. The third group of rats was administered, in addition to CCl4, MetVO-salen i.p. at a dose of 0.6 mg/kg body weight, twice a week for the same period. The doses of CCl4 and/ or MetVO-salen were chosen on the basis of preliminary studies. All experiments were carried out according to the guidelines for the care and use of experimental animals approved by state veterinary administration of University of Tehran. 2.4.2. Histopathological studies For histopathological studies, the liver tissues were fixed in 10% formalin neutral solution, and then the blocks were prepared in paraffin followed by getting sections of 5–6 lm in thickness. The specimens were then stained with haematoxylin– eosin solution and observed under a light microscope mainly for inflammatory and necrotic changes with respect to control healthy rats’ livers. 2.4.3. Preparation of liver homogenates and determination of lipid peroxidation All animals were anaesthetized with diethyl ether before killing, and their livers were removed and individually homogenized in phosphate buffer (50 mM, pH 7.4) to give a 10% (w/v) liver homogenate. Each liver homogenate was centrifuged at 5000 g for 15 min. The supernatant was collected and its protein content was determined based on Lowry’s method using crystalline bovine serum albumin for calibration (Lowry et al., 1951). Lipid peroxidation (LPO) was measured by the method of Buege and Aust (1978). The level of LPO in the liver homogenate was measured based on the formation of TBARS. Two millilitres of thiobarbituric acid reagent (15% w/v TCA, 0.375% w/v TBA and 0.25 N HCl) were added to 2 ml of tissue extract. The solution was heated for 15 min in a boiling water bath. After cooling, the precipitate was removed by centrifugation at 1000g for 10 min. Malondialdehyde (MDA) forms adducts with TBA, which is measured spectrophotometrically at 532 nm against a blank containing 50 mM phosphate buffer (pH 7.4) instead of the liver homogenate. The concentration of MDA was calculated based on the absorbance coefficient of the TBA–MDA complex (e = 1.56 105 cm1 M1), and it was expressed as nmol/mg protein. 2.4.4. Assays of antioxidant enzymes of the liver homogenates The activity of catalase (CAT) (EC 1.11.1.6) was assayed by the method of Aebi (1984). The assay volume contained 2 ll of liver homogenate preparation and 2.8 ml of 50 mM phosphate buffer with pH 7.8. The reaction was started by the
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addition of 100 ll of 60 mM H2O2 prepared in 50 mM phosphate buffer (pH 7.4) with an absorbance of 0.5 arbitrary unit. The decomposition of H2O2 was followed by monitoring the decrease in absorbance at 240 nm for 60 s, using a spectrophotometer. The enzyme activity was expressed as k/(s mg protein), where k represents the rate constant of the first-order reaction of CAT. The activity of superoxide dismutase (SOD) (EC 1.15.1.1) was also measured according to the method of Kakkar et al. (1984). This assay was based on the inhibition of amino blue tetrazolium formazan formation in the mixture of NADH, PMS and NBT system. The assay mixture contained 0.1 ml of the supernatant, 1.2 ml of sodium pyrophosphate buffer (52 mM, pH 8.3), 0.1 ml of PMS (186 lM), 0.3 ml of NBT (300 lM) and 0.2 ml of NADH (750 lM). Reaction was started by the addition of NADH. After incubation at 30 °C for 90 s, the reaction was stopped by addition of 0.1 ml of glacial acetic acid. Reaction mixture was stirred vigorously. Colour intensity of the chromogen was measured spectrophotometrically at 560 nm. One unit of enzyme activity corresponds to that amount of enzyme which cause 50% inhibition of NBT reduction/mg protein.
100 Scavenging activity (%)
718
80 60 40 20 0
2.4.5. Estimation of glutathione content of the liver homogenates Reduced glutathione (GSH) forms a characteristic compound with 5, 50 dithiobis 2-nitro benzoic acid (DTNB). The extent of the reaction is followed spectrophotometrically at 412 nm. GSH estimation was achieved based on the method of Sedlak and Lindsay (1968) modified by Moron et al. (1979). GSH was expressed as lg/ mg protein of the liver homogenate. 2.5. Statistical analysis All values are expressed as mean ± S.D. The significance of differences between the means of the treated and untreated groups has been calculated by unpaired Student’s t-test, and P-values lesser than 0.05 were considered significant.
3. Results 3.1. In vitro and ex vivo studies 3.1.1. Scavenging reactive oxygen species in H2O2-mediated systems As shown in Fig. 1, MetVO-salen exhibited relatively strong scavenging activity against reactive species produced by H2O2 in a dose-dependent manner. The free radical-scavenging ability, in term of 50%-inhibition concentration (IC50), was found to be 20.9 lM and that of ascorbic acid, a well-known natural free radical scavenger, under all equal experimental conditions, was found to be 395.9 lM. 3.1.2. Superoxide anion-scavenging activity The O 2 anions are generated in a reaction between PMS, NADH and the molecular oxygen. The generated free radical anions then reduce NBT to form a blue formazon colour with an absorbance band at 560 nm (Nishikimi et al., 1972). Any exogenous compound capable of oxidizing O 2 anions will compete with NBT and slow
80
60
40
20
0 2.5
12.5
25 (568µM)
37.5
Vit C
Fig. 1. Hydrogen peroxide scavenging activity of MetVO-salen at different concentrations (2.5, 12.5, 25 and 37.5 lM). Each value represents the mean ± SD (n = 3).
25
37.5
Vit C (284 µM)
Fig. 2. Superoxide anion-scavenging activity of MetVO-salen in the PMS-NADHNBT model, relative to Vit C, at various concentrations (12.5, 25 and 37.5 lM). Each value represents the mean ± SD (n = 3).
down its reduction. Therefore, this leads to a lower absorbance at 560 nm. According to the results presented in Fig. 2, MetVO-salen is a strong O 2 anion scavenger. The scavenging activity of MetVOsalen reached to 69.77% at a dose of 37.5 lM, while ascorbic acid quenched the reduction of NBT by 69.77% at a dose of 142 lM. Based on these data, it is evident that MetVO-salen is a powerful antioxidant candidate and is suited for further biological evaluation. 3.1.3. Inhibition of lipid peroxidation in rat liver homogenate Addition of FeSO4/ascorbate mixture to an aliquot of the rat liver homogenate significantly increased the extent of TBARS relative to control sample in a 30 min reaction time. However, as shown in Fig. 3, addition of 0.25, 0.5, 0.75 and 1 lM of MetVOsalen to the FeSO4/ascorbate-treated liver homogenate reduced the extent of TBARS by about 17, 37, 80 and 94, respectively, indicating the significant anti-lipid peroxidation activity of MetVOsalen. 3.1.4. Effect(s) of MetVO-salen on cell viability The toxicity of MetVO-salen was evaluated based on the viability of cells exposed to variable concentrations of MetVO-salen using MTT assay. In fact, cytotoxicity is a common test for the pharmaceutical use of new compounds. Fig. 4 indicates that MetVOsalen has a slight cytotoxicity effect (6–8%) on the treated cells in
Inhibition of lipid peroxidation (%)
Scavenging activity (%)
100
12.5
120 100 80 60 40 20 0 0.25
0.5
0.75
1
Trolox
(400µM) Fig. 3. The inhibitory effect of MetVO-salen on lipid peroxidation induced by Fe2+/ ascorbate in rat liver homogenate at different concentrations (0.25, 0.5, 0.75 and 1 lM). Each value represents the mean ± SD (n = 3).
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twice a week for 4 weeks, significantly attenuated the necrotic effects of CCl4 and assisted the restoration of the normal cellular arrangement of the liver (Fig. 6 B and C).
120 100 Cells viability (%)
719
3.2.1. Effect(s) on TBARS level MetVO-salen therapy of CCl4-treated rats restored the TBARS content to the level of the control healthy rats (Fig. 7), further confirming the strong free radical-scavenging activity of MetVO-salen complex.
80 60 40 20
37 .5
25
12 .5
2. 5
0
Fig. 4. Cell viability of human K562 cells treated with different concentrations of MetVO-salen for 24 h relative to the non-treated control cells. Each value represents the mean ± SD (n = 3).
3.2.2. Effect(s) on hepatic antioxidant systems Fig. 8 clearly indicates that MetVO-salen has increased the SOD, but not CAT, activity among the CCl4-treated rats (group III) relative to control rats (group I) and the CCl4-treated control rats (group II). According to the data presented in Fig. 9, MetVO-salen has also increased the level of GSH among the group III rats relative to CCl4-treated rats (group II).
4. Discussion a 24 h evaluation time. Regarding the low cytotoxic effect of MetVO-salen, we then evaluated the protective effect of MetVO-salen against damages induced in cells by H2O2. For this goal, we pretreated the K562 human leukemic cells for 2 h with different concentrations of MetVO-salen (2.5, 7.5, 12.5 and 25 lM). Then, the treated cells were exposed to H2O2 (300 lM) for 16 h. As shown in Fig. 5, treatment with H2O2 reduced cell viability by almost 40% relative to H2O2-untreated cells. However, pretreatment of cells with 7.5 lM of MetVO-salen reduced the damaging effects of H2O2 to about 20%. Regarding these observation, it can be concluded that MetVO-salen is a powerful ROS scavenger with low cytotoxic effects. 3.2. In vivo studies Subcutaneous injection of CCl4 to rats twice a week and for 4 consecutive weeks caused liver damages in rats, which is evident through histopathological evaluations (Fig. 6). In addition, CCl4 treatment enhanced liver MDA level by 57% and decreased the liver SOD activity by 55% and the GSH level decreased by almost 27% relative to normal healthy control rats. Based on Fig. 6, treatment of rats with MetVO-salen at a dose of 0.6 mg/kg body weight,
Cell viability (%)
120 100 **
80 *
60 40 20 0
H2O2 (300µM)
-
+
+
+
+
+
Methoxy VO-salen (µM)
-
-
2.5
7.5
12.5
17.5
Fig. 5. Protective effect of MetVO-salen on H2O2-induced cytotoxicity among K562 cells. K562 cells were pretreated with different concentrations of MetVO-salen (2.5, 7.5, 12.5 and 17.5 lM) for 2 h, and then exposed to 300 lM H2O2 for further 16 h. Cell viability was evaluated by MTT assay. Data were expressed as percent of values in untreated control cells. Each value represents the mean ± SD (n = 3). *Significantly different from control cells (p < 0.001). **Significantly different from H2O2-treated cells (p < 0.001).
In vitro antioxidant assays either measure the free radical-scavenging capability of a test compound or evaluate its anti-lipid peroxidation activity. In this study, we used both approaches to evaluate the antioxidant property of MetVO-salen. Free radicals such as O 2 anions and non-radical derivatives of the oxygen molecule such as H2O2 are usually generated in biological systems. ROS initiate free radical chain reactions that can be interrupted by compounds capable of scavenging radicals. In this way, the antioxidant activity of the test compounds is evaluated. Hydrogen peroxide plays an important role in the immune system and acts either directly or indirectly as a messenger molecule in the inflammation events and cell signaling pathways (Auroma et al., 1989). However, overproduction of H2O2 can lead to cell death (Butterfield and Kanski, 2001). It is thought that H2O2 is a major precursor of highly reactive free radicals involved in the induction of apoptosis among neuronal cells (Ruffels et al., 2004). H2O2 is not very active by itself; however, it can lead to the production of highly reactive hydroxyl ðOH Þ radicals that mediate various types of reactions including oxidative DNA damages. Thus, endogenous and/or exogenous antioxidants would be capable of protecting biological systems that are exposed to high levels of H2O2. Fig. 1 clearly indicates that MetVOsalen is capable of scavenging H2O2 in a dose-dependent manner. The O 2 anion is a precursor of other ROS that have the potential of reacting with biological macromolecules (Okezie, 1998), and it has also been implicated in initiating oxidation reactions associated with aging (Wickens, 2001). The O 2 scavenging effect of MetVO-salen, determined by NBT method, is depicted in Fig. 2. According to this figure, MetVO-salen, in a dose-dependent man ner, suppresses the O 2 radicals. The H2O2- and O2 radical-scavenging activity of MetVO-salen is attributed to its vanadium element. Vanadium can form both cationic and anionic complexes under the pH range of biological systems with the ionization states of V (+4) and V (+5). Therefore, vanadium can act as an electron donor or an electron acceptor. As an electron donor (in its +4 oxidation state), it can reduce H2O2 to H2O and O2 and the vanadium ion will be oxidized to its +5 state, thereby destroying H2O2. On the other hand, in its +5 oxidation state, vanadium can serve as an electron acceptor. In this case, it can stimulate the non-enzymatic oxidation of superoxide anion into molecular oxygen and consequently, destroys the superoxide radicals. In addition, it is known that oxidative stress initiates lipid peroxidation of polyunsaturated fatty acids of cell membranes (Janero, 1990). Lipid peroxidation can affect cell function by accumulating oxidized lipids in the cell membrane. This is believed to interrupt the normal function of cell surface receptors (Marathe et al.,
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Fig. 6. Effect of MetVO-salen on liver architecture in CCl4-treated rats. Liver sections were stained using the haematoxylin–eosin method. (A) Control rats, (B) CCl4-treated rats and (C) CCl4 and MetVO-salen treated rats.
2000). In addition, lipid peroxidation can lead to the production of MDA and 4-hydroxynonenal (4-HNE) with cytotoxic and mutagenic properties. Fig. 3 indicates that MetVO-salen is capable of reducing the extent of TBARS formation (an index of MDA formation) induced by FeSO4-ascorbate system in rat liver homogenate. High free radical-scavenging capability of MetVO-salen makes it an interesting antioxidant for further pharmaceutical evaluations provided that it possesses negligible cytotoxic effects on biological systems. Our toxicity evaluation, based on the MTT assay, indicated that the cytotoxicity of MetVO-salen complex is nil (Fig. 5) among
K562-treated cells. Regarding this property, the protective effect(s) of MetVO-salen complex against H2O2-induced damages to K562 cells was evaluated based on the cell viability. Pretreatment of the cells with MetVO-salen (7.5 lM) for 2 h followed by exposure to H2O2 increased the cell survival from 58% (H2O2-treated cells) to 79% (MetVO-salen and H2O2-treated cells). In other words, at a
7
** group I
6
* **
1.4 MDA (nmol/mg protein)
group II
1.2 1 0.8
5 Activity (Units)
1.6
4 3 2
0.6 0.4
group III
*
**
*
1
0.2
0
0
CAT Group I
Group II
SOD
GroupIII
Fig. 7. Effect of MetVO-salen on hepatic levels of MDA in CCl4-treated rats. Values are mean ± SD for six rats.*Significantly different from group I (P < 0.001). **Significantly different from Group II (P < 0.01). [Control rats (group I), CCl4- treated rats (group II) and CCl4 and MetVO-salen treated rats (group III)].
Fig. 8. Effect of MetVO-salen on hepatic levels of CAT (101 K/mg protein) and SOD (U/mg protein) in CCl4-treated rats. Values are mean ± SD for six rats. * Significantly different from group I (P < 0.05). **Significantly different from group II (P < 0.05). [Control rats (group I), CCl4-treated rats (group II) and CCl4 and MetVOsalen treated rats (group III)].
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14
**
liver GSH (µg/mg protein)
12 10
*
8 6 4 2 0 Group I
Group II
Group III
Fig. 9. Effect of MetVO-salen on hepatic levels of GSH (lg/mg protein) in CCl4treated rats. Values are mean ± S.D for six rats. *Significantly different from group I (P < 0.01). **Significantly different from group II (P < 0.01). [Control rats (group I), CCl4-treated rats (group II) and CCl4 and MetVO-salen treated rats (group III)].
concentration of 7.5 lM, MetVO-salen is capable of protecting the K562 cells against H2O2 damages by almost 20%. Our in vivo antioxidant evaluation results are also parallel to our in vitro observations. According to the present data, the extent of ROS production by the administered CCl4 is significantly quenched by MetVO-salen complex; thereby reducing the extent of liver damages among the CCl4-treated rats relative to normal rats. It is well established that CCl4 causes peroxidation of cell membrane lipids leading to a high level of TBARS production among the exposed rats. The protective effect(s) of MetVO-salen was also evident through higher levels of GSH and SOD activities among the MetVO-salen-treated rats relative to untreated control rats. In summary, this study clearly discloses some of the free radical-scavenging features of MetVO-salen complex. This study makes the background for initiating further research concerning biological evaluation of MetVO-salen complex and/or its other derivatives, as a new class of synthetic antioxidants. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgment The authors appreciate the financial support of this investigation by the research Council of University of Tehran. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Auroma, O.I., Laughton, M.J., Halliwell, B., 1989. Carnosine, homocarnosine and anserine: could they act as antioxidants in vivo? Biochem. J. 264, 863–869.
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Autzen, S., Korth, H.G., Boese, R., Groot, H., Sustmann, R., 2003. Studies of pyridinylcontaining 14-membered macrocyclic copper (II) complexes. Eur. J. Inorg. Chem. 2003, 1401–1410. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Butterfield, D.A., Kanski, J., 2001. Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins. Mech. Ageing. Dev. 122, 945–962. Cerutti, P.A., 1991. Oxidant stress and carcinogenesis. Eur. J. Clin. Invest. 21, 1–5. Duh, P.D., Du, P.C., Yen, G.C., 1999. Action of methanolic extract of mung bean hulls as inhibitors of lipid peroxidation and non-lipid oxidative damage. Food Chem. Toxicol. 37, 1055–1061. Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247. Goc, A., 2006. Biological activity of vanadium compounds. Cent. Eur. Biol. 1, 314– 332. Janero, D.R., 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic. Biol. Med. 9, 515–540. Kakkar, P., Das, B., Viswanathan, P.N., 1984. A modified spectrophotometric assay of superoxide dismutase. Ind. J. Biochem. Biophys. 21, 130–132. Kianfar, A.H., Mohebbi, S., 2007. Synthesis and electrochemistry of vanadium (IV) schiff base complexes. J. Iran. Chem. Soc. 4, 215–220. Lai, L.S., Chou, S.T., Chao, W.W., 2001. Studies on the antioxidative activities of Hsian-tsao (Mesona procumbens Hemsl) Leaf Gum. J. Agric. Food Chem. 49, 963– 968. Liu, F., Ooi, V.E., Chang, S.T., 1997. Free radical scavenging activities of mushroom polysaccharide extracts. Life Sci. 60, 763–771. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Mahakunakorn, P., Tohda, M., Murakami, Y., Matsumoto, K., Watanabe, H., 2004. Antioxidant and free radical-scavenging activity of Choto-san and its related constituents. Biol. Pharm. Bull. 27, 38–46. Marathe, G.K., Harrison, K.A., Murphy, R.C., Prescott, S.M., Zimmerman, G.A., McIntyre, T.M., 2000. Bioactive phospholipid oxidation products. Free Radic. Biol. Med. 28, 1762–1770. Martindale, J.L., Holbrook, N.J., 2002. Cellular response to oxidative stress: signaling for suicide and survival. J. Cell. Physiol. 192, 1–15. Moskovitz, J., Yim, M.B., Chock, P.B., 2002. Free radicals and disease. Arch. Biochem. Biophys. 397, 354–359. Moron, M.S., Depierre, J.N., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione-S-transferase activity in rat lung and liver. Biochim. Biophys. Acta 582, 67–78. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55– 63. Nishikimi, M., Appaji, N., Yagi, K., 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 46, 849–854. Nordberg, J., Amer, E.S.J., 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Med. 31, 1287–1312. Okezie, I.A., 1998. Free radicals, oxidative stress, and antioxidants in human health and disease. J. Am. Oil Chem. Soc. 75, 199–212. Pryor, W.A., 1991. The antioxidant nutrients and disease prevention: what do we know and what do we need to find out? Am. J. Clin. Nutr. 53, 391–393. Rehder, D., 2003. Biological and medicinal aspects of vanadium. Inorg. Chem. Commun. 6, 604–617. Riley, D.P., 1999. Functional mimics of superoxide dismutase enzymes as therapeutic agents. Chem. Rev. 99, 2573–2588. Ruch, R.J., Cheng, S.J., Klaunji, J.E., 1989. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant. Carcinogenesis 10, 1003–1008. Ruffels, J., Griffin, M., Dickenson, J.M., 2004. Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: role of ERK1/2 in H2O2-induced cell death. Eur. J. Pharmacol. 483, 63–173. Sakurai, H., Kojima, Y., Yoshikawa, Y., Kawabe, K., Yasui, H., 2002. Antidiabetic vanadium (IV) and zinc (II) complexes. Coor. Chem. Rev. 226, 187–198. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein bound and non-protein sulfhydryl groups in tissue with Ellmans reagent. Anal. Biochem. 25, 293–305. Wickens, A.P., 2001. Ageing and the free radical theory. Respir. Physiol. 128, 379– 391.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Methoxy VO-salen complex: In vitro antioxidant activity, cytotoxicity evaluation and protective effect on CCl4-induced oxidative stress in rats Mohsen Mohammadi, Razieh Yazdanparast * Institute of Biochemistry and Biophysics, University of Tehran P.O. Box 13145-1384, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 20 August 2008 Accepted 19 December 2008
Keywords: Antioxidant Lipid peroxidation Methoxy VO-salen Oxidative stress Protein oxidation ROS
a b s t r a c t Reactive oxygen species (ROS) play crucial roles in normal physiological processes. However, the overproduction of ROS is involved in the onset of many degenerative diseases. Regarding this fact, discovery of new antioxidants is interesting for many research groups. In this study, we evaluated the antioxidant properties of a methoxy VO-salen (MetVO-salen) complex employing various in vitro systems. In addition, the cytotoxic effect of MetVO-salen was assessed based on MTT in treated K562 cells. In an in vivo approach, the protective effect of MetVO-salen against CCl4-induced oxidative stress in rats was also investigated in terms of superoxide dismutase (SOD) and catalase (CAT) activities, as well as in terms of the levels of malondialdehyde (MDA) and glutathione (GSH). Our results indicated that MetVO-salen has an effective capability in scavenging superoxide ðO 2 Þ and hydrogen peroxide (H2O2) radicals in a dose-dependent manner. In vivo results also showed that the administration of MetVO-salen to the CCl4-treated rats caused a significant (222%) increase in SOD activity, a 59% enhancement in GSH content and a 31% decrease in the level of MDA compared to the CCl4-treated control rats. Overall, MetVOsalen appears to be an effective antioxidant and is quite suitable for further biological evaluation. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Exogenous factors such as sunlight, ultraviolet light, ionizing radiation, some chemicals and normal cellular activities lead to the production of reactive oxygen species (ROS). The major types of ROS or ROS-producing species are superoxide anion ðO 2 Þ, hydrogen peroxide (H2O2) and hydroxyl ð OHÞ radicals (Cerutti, 1991). ROS present a paradox in their biological function: on one hand, they prevent diseases by assisting the immune system, mediating cell signaling and playing an essential role in apoptosis. On the other hand, they can damage many biologically active molecules and hence leading to tissue damages and cell death (Finkel and Holbrook, 2000; Martindale and Holbrook, 2002). In cells, there are several antioxidant defense mechanisms that function-
Abbreviations: BHT, butylated hydroxyl toluene; BSA, bovine serum albumin; CAT, catalase; DMSO, dimethylsulfoxide; DTNB, 5,50 dithio-bis (2-nitro benzoic acid); ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GPx, glutathione peroxidase; GSH, glutathione; 4-HNE, 4-hydroxynonenal; i.p., intraperitoneal; LPO, lipid peroxidation; MDA, malondialdehyde; MetVO-salen, methoxy VO-salen; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; NBT, nitroblue tetrazolium; PCO, protein carbonyl; PMS, phenazine methosulphate; ROS, reactive oxygen species; SOD, superoxide dismutase; TBA, 2-thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid; Trolox, 6-hydroxy-2,5,7,8-tetra methyl chroman-2-carboxylic acid. * Corresponding author. Tel.: +98 21 66956976; fax: +98 21 66404680. E-mail address: [email protected] (R. Yazdanparast). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.029
ally assist in preventing the destructive effects of various types of ROS. These defense mechanisms include antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and small molecules such as glutathione (GSH) and vitamins C and E. Deficiency or abnormality in any of these defense mechanisms will lead to overproduction of ROS followed by oxidation of biomolecules such as proteins, lipids and DNA. At normal physiological condition a delicate balance exists between the rate of generation and the consumption of free radicals (Nordberg and Amer, 2001; Duh et al., 1999). In abnormal cases, particularly when the rate of production exceeds the rate of consumption or inactivation of ROS, the biological system is overdosed by ROS with potentially serious health consequences. It is now well accepted that oxidative stress is involved in the pathogenesis of various types of diseases including cancer, coronary heart diseases, aging, diabetes and neurodegenerative diseases (Moskovitz et al., 2002; Pryor, 1991; Lai et al., 2001). Due to wide deleterious effects of ROS on structural and functional integrities of biological systems, a great deal of scientific efforts has been devoted to finding effective free radical scavengers, which are referred to as antioxidants. In this respect, transition metal complexes have notably shown to possess high antioxidative properties (Riley, 1999; Autzen et al., 2003). From this group of compounds, vanadium complexes deserve a special attention. Vanadium is a transition metal and a trace element acting as the catalytic component of several enzymes such as haloperoxidase
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and nitrate reductase, which possess free radical chemistry (Rehder, 2003). Various vanadium complexes (SALENS) have been proposed for potential use as insulin-mimetic agents, and have been shown to have insulin-mimetic properties in animal model systems and cell culture (Sakurai et al., 2002). Besides the well-established antidiabetic effects, vanadium complexes also exhibit antitumor and anti-inflammatory activities (Goc, 2006). On the other hand, it is now well accepted that oxidative stress is involved in the pathogenesis of cancer and diabetes diseases. Since the antioxidative activities of VO-salen complexes have not been studied in in vivo or ex vivo systems; it would certainly be of interest to clarify these properties for MetVO-salen complex using animal (rat) and cell culture (K562 cells) systems, respectively. 2. Materials and methods 2.1. Chemicals Ascorbic acid, 6-hydroxy-2,5,7,8-tetra methyl chroman-2-carboxylic acid (Trolox), trichloroacetic acid (TCA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and ferric chloride were obtained from Sigma (Sigma– Aldrich, Sternheim, Germany). Reduced nicotinamide adenine dinucleotide (NADH), phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), hydrogen peroxide (H2O2), butylated hydroxyl toluene (BHT) and 2-thiobarbituric acid (TBA) were obtained from Merck (Germany). Salicylaldehyde and ethylenediamine were purchased from Fluka (Buchs, Switzerland). All other chemicals used were of analytical grade and were obtained from either Sigma–Aldrich or Merck (Germany). The synthesis and purification of MetVO-salen (Scheme 1) were achieved as reported previously (Kianfar and Mohebbi, 2007). 2.2. In vitro and ex vivo assay models 2.2.1. Hydrogen peroxide scavenging activity The H2O2-scavenging activity of MetVO-salen was studied according to the slightly modified method of Ruch et al. (1989). Different concentrations of MetVO-salen (2.5, 12.5, 25 and 37.5 lM in phosphate buffer, 50 mM, pH 7.4) were added to 2 ml of H2O2 solution (10 mM) in phosphate buffer (50 mM, pH 7.4), and the reaction mixture was incubated at 25 °C for 30 min. The unreacted H2O2 was determined by measuring the absorbance of the reaction mixture at 230 nm with respect to the blank solution (a solution of MetVO-salen in phosphate buffer without H2O2). The percentage of H2O2-scavenging activity of MetVO-salen was calculated using the following equation:
H2 O2 scavenging activity ð%Þ ¼ ½ðA A1 =AÞ 100 where A is the absorbance of the control, and A1 is the absorbance in the presence of MetVO-salen or the standard sample. 2.2.2. Scavenging of superoxide anion in PMS-NADH/ NBT system Superoxide anion-scavenging activity of MetVO-salen was determined based on the method described by Liu (Liu et al., 1997). In the experiment, O 2 anions were generated in 3 ml of Tris–HCl buffer (16 mM, pH8) containing 1 ml of NBT (50 lM), 1 ml of NADH (78 lM) and different concentrations of MetVO-salen. The reaction was initiated by adding 1 ml of PMS solution (10 lM) to the reaction mixture and after 5 min incubation at 25 °C; the absorbance was measured at 560 nm against the corresponding blank. The O 2 anion-scavenging activity, in percentage, was calculated using the following equation:
O 2 anion-scavenging activity ð%Þ ¼ ½ðA A1 =AÞ 100; where A is the absorbance of the control, and A1 is the absorbance in the presence of MetVO-salen or the standard sample. 2.2.3. Antioxidant assay using rat liver homogenate This assay is based on the ability of antioxidants toward the inhibition of lipid peroxidation in rat liver homogenate in the presence of FeSO4 and ascorbate. The extent of lipid peroxidation in the presence and absence of MetVO-salen was eval-
Scheme 1. Chemical structure of MetVO-salen complex.
717
uated based on the extent of thiobarbituric acid reactive substances (TBARS) according to the published method (Mahakunakorn et al., 2004). 2.3. Cytotoxicity evaluations 2.3.1. Cell culture The human leukemia K562 cells were cultured in RPMI-1640 medium supplemented with FBS (10%, v/v), streptomycin (100 lg/ml) and penicillin (100 U/ml). The cells were incubated under 5% CO2 humidified atmosphere at 37 °C. Cell numbers were assessed using a hemocytometer and the abilities of the cells to exclude trypan blue. 2.3.2. Cytotoxicity evaluation of MetVO-salen in K562 cells Cell viability was estimated using the MTT assay. This method is dependent on the conversion of yellow tetrazolium bromide to its purple formazan derivative by mitochondrial succinate dehydrogenase of the viable cells (Mosmann, 1983). The cells were seeded in 96-well plates at a concentration of 105cells/ml for 24 h, and then treated in triplicate with MetVO-salen at different concentrations (2.5, 12.5, 25 and 37.5 lM). After 24 h, MTT stock solution (10 ll; 5 mg/ml) was applied to each well. After 4 h of incubation, the plates were centrifuged for 10 min at 800 g, and the supernatants were aspirated. The formazan crystals in each well were dissolved in 100 ll of dimethylsulfoxide (DMSO), and the absorbance was measured via ELISA technique at a wavelength of 570 nm. 2.3.3. Cytoprotective effect of MetVO-salen in H2O2-treated K562 cells To evaluate the cytoprotective effect of MetVO-salen in H2O2-treated K562 cells, the cells were plated at a density of 105cells/ml for 24 h. The cells were pretreated with various concentrations of MetVO-salen (2.5, 7.5, 12.5 and 17.5 lM). Two hours later, H2O2 (300 lM) was added to the plate followed by incubation for an additional 16 h. Cell viability was estimated using the MTT assay, and the cellular viability was expressed in percentage of survival relative to the control cell samples. 2.4. In vivo assay model 2.4.1. Animals and experimental protocols Male wistar rats, weighting 200–250 g and with an average age of 8 weeks, were purchased from Pasteur institute (Tehran, Iran). They were housed under ambient temperature of 22 ± 2 °C with 60 ± 5% relative humidity and a lighting cycle from 7:00 a.m. to 7:00 p.m. The animals received food and water ad libitum. After the adaptation period, rats were randomly divided into three groups of six animals each: the first group of rats (control group) was administered olive oil intraperitoneally (i.p.) at a dose of 0.2 ml/kg body weight, twice a week, for 4 weeks. The second and third groups of rats were administered CCl4 i.p. at a dose of 0.4 ml/kg body weight (50% CCl4 in olive oil), twice a week, for 4 weeks. The third group of rats was administered, in addition to CCl4, MetVO-salen i.p. at a dose of 0.6 mg/kg body weight, twice a week for the same period. The doses of CCl4 and/ or MetVO-salen were chosen on the basis of preliminary studies. All experiments were carried out according to the guidelines for the care and use of experimental animals approved by state veterinary administration of University of Tehran. 2.4.2. Histopathological studies For histopathological studies, the liver tissues were fixed in 10% formalin neutral solution, and then the blocks were prepared in paraffin followed by getting sections of 5–6 lm in thickness. The specimens were then stained with haematoxylin– eosin solution and observed under a light microscope mainly for inflammatory and necrotic changes with respect to control healthy rats’ livers. 2.4.3. Preparation of liver homogenates and determination of lipid peroxidation All animals were anaesthetized with diethyl ether before killing, and their livers were removed and individually homogenized in phosphate buffer (50 mM, pH 7.4) to give a 10% (w/v) liver homogenate. Each liver homogenate was centrifuged at 5000 g for 15 min. The supernatant was collected and its protein content was determined based on Lowry’s method using crystalline bovine serum albumin for calibration (Lowry et al., 1951). Lipid peroxidation (LPO) was measured by the method of Buege and Aust (1978). The level of LPO in the liver homogenate was measured based on the formation of TBARS. Two millilitres of thiobarbituric acid reagent (15% w/v TCA, 0.375% w/v TBA and 0.25 N HCl) were added to 2 ml of tissue extract. The solution was heated for 15 min in a boiling water bath. After cooling, the precipitate was removed by centrifugation at 1000g for 10 min. Malondialdehyde (MDA) forms adducts with TBA, which is measured spectrophotometrically at 532 nm against a blank containing 50 mM phosphate buffer (pH 7.4) instead of the liver homogenate. The concentration of MDA was calculated based on the absorbance coefficient of the TBA–MDA complex (e = 1.56 105 cm1 M1), and it was expressed as nmol/mg protein. 2.4.4. Assays of antioxidant enzymes of the liver homogenates The activity of catalase (CAT) (EC 1.11.1.6) was assayed by the method of Aebi (1984). The assay volume contained 2 ll of liver homogenate preparation and 2.8 ml of 50 mM phosphate buffer with pH 7.8. The reaction was started by the
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addition of 100 ll of 60 mM H2O2 prepared in 50 mM phosphate buffer (pH 7.4) with an absorbance of 0.5 arbitrary unit. The decomposition of H2O2 was followed by monitoring the decrease in absorbance at 240 nm for 60 s, using a spectrophotometer. The enzyme activity was expressed as k/(s mg protein), where k represents the rate constant of the first-order reaction of CAT. The activity of superoxide dismutase (SOD) (EC 1.15.1.1) was also measured according to the method of Kakkar et al. (1984). This assay was based on the inhibition of amino blue tetrazolium formazan formation in the mixture of NADH, PMS and NBT system. The assay mixture contained 0.1 ml of the supernatant, 1.2 ml of sodium pyrophosphate buffer (52 mM, pH 8.3), 0.1 ml of PMS (186 lM), 0.3 ml of NBT (300 lM) and 0.2 ml of NADH (750 lM). Reaction was started by the addition of NADH. After incubation at 30 °C for 90 s, the reaction was stopped by addition of 0.1 ml of glacial acetic acid. Reaction mixture was stirred vigorously. Colour intensity of the chromogen was measured spectrophotometrically at 560 nm. One unit of enzyme activity corresponds to that amount of enzyme which cause 50% inhibition of NBT reduction/mg protein.
100 Scavenging activity (%)
718
80 60 40 20 0
2.4.5. Estimation of glutathione content of the liver homogenates Reduced glutathione (GSH) forms a characteristic compound with 5, 50 dithiobis 2-nitro benzoic acid (DTNB). The extent of the reaction is followed spectrophotometrically at 412 nm. GSH estimation was achieved based on the method of Sedlak and Lindsay (1968) modified by Moron et al. (1979). GSH was expressed as lg/ mg protein of the liver homogenate. 2.5. Statistical analysis All values are expressed as mean ± S.D. The significance of differences between the means of the treated and untreated groups has been calculated by unpaired Student’s t-test, and P-values lesser than 0.05 were considered significant.
3. Results 3.1. In vitro and ex vivo studies 3.1.1. Scavenging reactive oxygen species in H2O2-mediated systems As shown in Fig. 1, MetVO-salen exhibited relatively strong scavenging activity against reactive species produced by H2O2 in a dose-dependent manner. The free radical-scavenging ability, in term of 50%-inhibition concentration (IC50), was found to be 20.9 lM and that of ascorbic acid, a well-known natural free radical scavenger, under all equal experimental conditions, was found to be 395.9 lM. 3.1.2. Superoxide anion-scavenging activity The O 2 anions are generated in a reaction between PMS, NADH and the molecular oxygen. The generated free radical anions then reduce NBT to form a blue formazon colour with an absorbance band at 560 nm (Nishikimi et al., 1972). Any exogenous compound capable of oxidizing O 2 anions will compete with NBT and slow
80
60
40
20
0 2.5
12.5
25 (568µM)
37.5
Vit C
Fig. 1. Hydrogen peroxide scavenging activity of MetVO-salen at different concentrations (2.5, 12.5, 25 and 37.5 lM). Each value represents the mean ± SD (n = 3).
25
37.5
Vit C (284 µM)
Fig. 2. Superoxide anion-scavenging activity of MetVO-salen in the PMS-NADHNBT model, relative to Vit C, at various concentrations (12.5, 25 and 37.5 lM). Each value represents the mean ± SD (n = 3).
down its reduction. Therefore, this leads to a lower absorbance at 560 nm. According to the results presented in Fig. 2, MetVO-salen is a strong O 2 anion scavenger. The scavenging activity of MetVOsalen reached to 69.77% at a dose of 37.5 lM, while ascorbic acid quenched the reduction of NBT by 69.77% at a dose of 142 lM. Based on these data, it is evident that MetVO-salen is a powerful antioxidant candidate and is suited for further biological evaluation. 3.1.3. Inhibition of lipid peroxidation in rat liver homogenate Addition of FeSO4/ascorbate mixture to an aliquot of the rat liver homogenate significantly increased the extent of TBARS relative to control sample in a 30 min reaction time. However, as shown in Fig. 3, addition of 0.25, 0.5, 0.75 and 1 lM of MetVOsalen to the FeSO4/ascorbate-treated liver homogenate reduced the extent of TBARS by about 17, 37, 80 and 94, respectively, indicating the significant anti-lipid peroxidation activity of MetVOsalen. 3.1.4. Effect(s) of MetVO-salen on cell viability The toxicity of MetVO-salen was evaluated based on the viability of cells exposed to variable concentrations of MetVO-salen using MTT assay. In fact, cytotoxicity is a common test for the pharmaceutical use of new compounds. Fig. 4 indicates that MetVOsalen has a slight cytotoxicity effect (6–8%) on the treated cells in
Inhibition of lipid peroxidation (%)
Scavenging activity (%)
100
12.5
120 100 80 60 40 20 0 0.25
0.5
0.75
1
Trolox
(400µM) Fig. 3. The inhibitory effect of MetVO-salen on lipid peroxidation induced by Fe2+/ ascorbate in rat liver homogenate at different concentrations (0.25, 0.5, 0.75 and 1 lM). Each value represents the mean ± SD (n = 3).
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twice a week for 4 weeks, significantly attenuated the necrotic effects of CCl4 and assisted the restoration of the normal cellular arrangement of the liver (Fig. 6 B and C).
120 100 Cells viability (%)
719
3.2.1. Effect(s) on TBARS level MetVO-salen therapy of CCl4-treated rats restored the TBARS content to the level of the control healthy rats (Fig. 7), further confirming the strong free radical-scavenging activity of MetVO-salen complex.
80 60 40 20
37 .5
25
12 .5
2. 5
0
Fig. 4. Cell viability of human K562 cells treated with different concentrations of MetVO-salen for 24 h relative to the non-treated control cells. Each value represents the mean ± SD (n = 3).
3.2.2. Effect(s) on hepatic antioxidant systems Fig. 8 clearly indicates that MetVO-salen has increased the SOD, but not CAT, activity among the CCl4-treated rats (group III) relative to control rats (group I) and the CCl4-treated control rats (group II). According to the data presented in Fig. 9, MetVO-salen has also increased the level of GSH among the group III rats relative to CCl4-treated rats (group II).
4. Discussion a 24 h evaluation time. Regarding the low cytotoxic effect of MetVO-salen, we then evaluated the protective effect of MetVO-salen against damages induced in cells by H2O2. For this goal, we pretreated the K562 human leukemic cells for 2 h with different concentrations of MetVO-salen (2.5, 7.5, 12.5 and 25 lM). Then, the treated cells were exposed to H2O2 (300 lM) for 16 h. As shown in Fig. 5, treatment with H2O2 reduced cell viability by almost 40% relative to H2O2-untreated cells. However, pretreatment of cells with 7.5 lM of MetVO-salen reduced the damaging effects of H2O2 to about 20%. Regarding these observation, it can be concluded that MetVO-salen is a powerful ROS scavenger with low cytotoxic effects. 3.2. In vivo studies Subcutaneous injection of CCl4 to rats twice a week and for 4 consecutive weeks caused liver damages in rats, which is evident through histopathological evaluations (Fig. 6). In addition, CCl4 treatment enhanced liver MDA level by 57% and decreased the liver SOD activity by 55% and the GSH level decreased by almost 27% relative to normal healthy control rats. Based on Fig. 6, treatment of rats with MetVO-salen at a dose of 0.6 mg/kg body weight,
Cell viability (%)
120 100 **
80 *
60 40 20 0
H2O2 (300µM)
-
+
+
+
+
+
Methoxy VO-salen (µM)
-
-
2.5
7.5
12.5
17.5
Fig. 5. Protective effect of MetVO-salen on H2O2-induced cytotoxicity among K562 cells. K562 cells were pretreated with different concentrations of MetVO-salen (2.5, 7.5, 12.5 and 17.5 lM) for 2 h, and then exposed to 300 lM H2O2 for further 16 h. Cell viability was evaluated by MTT assay. Data were expressed as percent of values in untreated control cells. Each value represents the mean ± SD (n = 3). *Significantly different from control cells (p < 0.001). **Significantly different from H2O2-treated cells (p < 0.001).
In vitro antioxidant assays either measure the free radical-scavenging capability of a test compound or evaluate its anti-lipid peroxidation activity. In this study, we used both approaches to evaluate the antioxidant property of MetVO-salen. Free radicals such as O 2 anions and non-radical derivatives of the oxygen molecule such as H2O2 are usually generated in biological systems. ROS initiate free radical chain reactions that can be interrupted by compounds capable of scavenging radicals. In this way, the antioxidant activity of the test compounds is evaluated. Hydrogen peroxide plays an important role in the immune system and acts either directly or indirectly as a messenger molecule in the inflammation events and cell signaling pathways (Auroma et al., 1989). However, overproduction of H2O2 can lead to cell death (Butterfield and Kanski, 2001). It is thought that H2O2 is a major precursor of highly reactive free radicals involved in the induction of apoptosis among neuronal cells (Ruffels et al., 2004). H2O2 is not very active by itself; however, it can lead to the production of highly reactive hydroxyl ðOH Þ radicals that mediate various types of reactions including oxidative DNA damages. Thus, endogenous and/or exogenous antioxidants would be capable of protecting biological systems that are exposed to high levels of H2O2. Fig. 1 clearly indicates that MetVOsalen is capable of scavenging H2O2 in a dose-dependent manner. The O 2 anion is a precursor of other ROS that have the potential of reacting with biological macromolecules (Okezie, 1998), and it has also been implicated in initiating oxidation reactions associated with aging (Wickens, 2001). The O 2 scavenging effect of MetVO-salen, determined by NBT method, is depicted in Fig. 2. According to this figure, MetVO-salen, in a dose-dependent man ner, suppresses the O 2 radicals. The H2O2- and O2 radical-scavenging activity of MetVO-salen is attributed to its vanadium element. Vanadium can form both cationic and anionic complexes under the pH range of biological systems with the ionization states of V (+4) and V (+5). Therefore, vanadium can act as an electron donor or an electron acceptor. As an electron donor (in its +4 oxidation state), it can reduce H2O2 to H2O and O2 and the vanadium ion will be oxidized to its +5 state, thereby destroying H2O2. On the other hand, in its +5 oxidation state, vanadium can serve as an electron acceptor. In this case, it can stimulate the non-enzymatic oxidation of superoxide anion into molecular oxygen and consequently, destroys the superoxide radicals. In addition, it is known that oxidative stress initiates lipid peroxidation of polyunsaturated fatty acids of cell membranes (Janero, 1990). Lipid peroxidation can affect cell function by accumulating oxidized lipids in the cell membrane. This is believed to interrupt the normal function of cell surface receptors (Marathe et al.,
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Fig. 6. Effect of MetVO-salen on liver architecture in CCl4-treated rats. Liver sections were stained using the haematoxylin–eosin method. (A) Control rats, (B) CCl4-treated rats and (C) CCl4 and MetVO-salen treated rats.
2000). In addition, lipid peroxidation can lead to the production of MDA and 4-hydroxynonenal (4-HNE) with cytotoxic and mutagenic properties. Fig. 3 indicates that MetVO-salen is capable of reducing the extent of TBARS formation (an index of MDA formation) induced by FeSO4-ascorbate system in rat liver homogenate. High free radical-scavenging capability of MetVO-salen makes it an interesting antioxidant for further pharmaceutical evaluations provided that it possesses negligible cytotoxic effects on biological systems. Our toxicity evaluation, based on the MTT assay, indicated that the cytotoxicity of MetVO-salen complex is nil (Fig. 5) among
K562-treated cells. Regarding this property, the protective effect(s) of MetVO-salen complex against H2O2-induced damages to K562 cells was evaluated based on the cell viability. Pretreatment of the cells with MetVO-salen (7.5 lM) for 2 h followed by exposure to H2O2 increased the cell survival from 58% (H2O2-treated cells) to 79% (MetVO-salen and H2O2-treated cells). In other words, at a
7
** group I
6
* **
1.4 MDA (nmol/mg protein)
group II
1.2 1 0.8
5 Activity (Units)
1.6
4 3 2
0.6 0.4
group III
*
**
*
1
0.2
0
0
CAT Group I
Group II
SOD
GroupIII
Fig. 7. Effect of MetVO-salen on hepatic levels of MDA in CCl4-treated rats. Values are mean ± SD for six rats.*Significantly different from group I (P < 0.001). **Significantly different from Group II (P < 0.01). [Control rats (group I), CCl4- treated rats (group II) and CCl4 and MetVO-salen treated rats (group III)].
Fig. 8. Effect of MetVO-salen on hepatic levels of CAT (101 K/mg protein) and SOD (U/mg protein) in CCl4-treated rats. Values are mean ± SD for six rats. * Significantly different from group I (P < 0.05). **Significantly different from group II (P < 0.05). [Control rats (group I), CCl4-treated rats (group II) and CCl4 and MetVOsalen treated rats (group III)].
M. Mohammadi, R. Yazdanparast / Food and Chemical Toxicology 47 (2009) 716–721
14
**
liver GSH (µg/mg protein)
12 10
*
8 6 4 2 0 Group I
Group II
Group III
Fig. 9. Effect of MetVO-salen on hepatic levels of GSH (lg/mg protein) in CCl4treated rats. Values are mean ± S.D for six rats. *Significantly different from group I (P < 0.01). **Significantly different from group II (P < 0.01). [Control rats (group I), CCl4-treated rats (group II) and CCl4 and MetVO-salen treated rats (group III)].
concentration of 7.5 lM, MetVO-salen is capable of protecting the K562 cells against H2O2 damages by almost 20%. Our in vivo antioxidant evaluation results are also parallel to our in vitro observations. According to the present data, the extent of ROS production by the administered CCl4 is significantly quenched by MetVO-salen complex; thereby reducing the extent of liver damages among the CCl4-treated rats relative to normal rats. It is well established that CCl4 causes peroxidation of cell membrane lipids leading to a high level of TBARS production among the exposed rats. The protective effect(s) of MetVO-salen was also evident through higher levels of GSH and SOD activities among the MetVO-salen-treated rats relative to untreated control rats. In summary, this study clearly discloses some of the free radical-scavenging features of MetVO-salen complex. This study makes the background for initiating further research concerning biological evaluation of MetVO-salen complex and/or its other derivatives, as a new class of synthetic antioxidants. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgment The authors appreciate the financial support of this investigation by the research Council of University of Tehran. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Auroma, O.I., Laughton, M.J., Halliwell, B., 1989. Carnosine, homocarnosine and anserine: could they act as antioxidants in vivo? Biochem. J. 264, 863–869.
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