Oxidative stress and damage to protein and lipids

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Oxidative damage to protein and lipids is one of the modifications leading to severe failure of their biological functions and even cell death in various tissues especially liver and brain. These damages could be induced by using of different reactive oxygen species (ROS) and meals ions and or combination of both in vitro. In that line, in the present work we investigated the protective effects of EUK-8 and EUK-134, compounds derived from salen, and Catechin, as a standard antioxidant, on two different models of oxidative stress. In the first study, we used from Fe2+/ascorbate model as a well-validated system for production of ROS in the liver homogenates and protective effects of these compounds against this system were evaluated by using various antioxidant assays such as protein carbonyls (PCO) content, protein-bound sulfhydryl (PB-SH) value, lipid peroxidation (LPO) and ROS levels. In second study, to expand our more knowledge about other therapeutic properties of EUK-8 and EUK-134 and regarding the role of oxidized lipid and proteins in formation of the lipofuscin in aging process, the protective capabilities of these compounds against the free-radical damaging effects of hydrogen peroxide (H2O2) on SK-N-MC neuroblastoma cell line by using cell viability restoration and also attenuation of intracellular lipofuscin formation were investigated. Results of the first study indicated that the simultaneous addition of Fe2+/ascorbate and both compounds at different concentrations (5, 10, 25 and 50 µM) to the liver homogenate significantly decreased the extent of PCO, LPO and the rate of ROS formation and increased the levels of PB-SH compared with the control sample. Results of the second study also showed that pretreatment of the cells 25 μM of each of these compounds followed by exposure to H2O2 for 24 hours indicated that compounds were capable of restoring the viabilities of cells relative to the H2O2-treated cells. In addition, each of the compounds significantly reduced the extent of lipofuscin formation among the cells time-dependently. Totally, the results of both studies clearly indicate the importance of EUK-8 and EUK-134 as the potential sources for different antioxidant substances. Since these antioxidants are relatively nontoxic, the present findings suggest that these components might be useful in preventing oxidative damages under various pathological conditions especially liver and neurodegenerative diseases.

Keywords: EUK-8; EUK-134; lipofuscin; liver homogenate; SK-N-MC neuroblastoma cell line; protective effects.

Introduction

Oxidative stress is hallmark a variety of diseases including aging, alzheimer, neurodegeneration, carcinogenesis, coronary heart disease, diabetes and hepatic diseases (Moskovitz et al., 2002; Harman, 1980; Dalle-Donne et al., 2003; Nicholas et al., 2010). This occurrence is results from increase the formation of reactive oxygen species (ROS), or weakening the scavenging system of ROS (Farber et al., 1994; Halliwell., 1999). ROS include hydroxyl (OH), peroxyl (RO2•) and superoxide anion (O2•−) radicals and nonradical species such as hydrogen peroxide (H2O2) are known as toxic oxygen moieties with high reactivity. Despite of the beneficial roles at very low concentration, as they act as a second messenger in some of the signal transudation pathways, these species at the high concentrations can cause oxidative damages to many vital components of the cells (Farber et al., 1994; Halliwell., 1999). The most important mechanisms responsible for the ROS-mediated injuries to cells and tissues mainly include lipid peroxidation, oxidative DNA damage and protein oxidation. Due to relatively high abundance, it is now recognized that proteins and lipids are the main targets for oxidants (Kayali et al., 2007). The role of oxidative lipid and protein damages in the pathophysiology of human diseases is currently a topic of considerable interest as these species has been implicated in a wide spectrum of clinical conditions (Telci et al., 2000; Dalle-Donne et al., 2003). One of the most important implications of these oxidized lipid and proteins is in aging process. Due to this process, cross-linking of proteins with other cellular components, such as 4-hydoxy-2-nonenal (HNE), which are the most typical outcome of ROS over production, leads to the formation and accumulation of aggregates known as lipofuscin (Brunk and Terman, 2002). Lipofuscin as a histological index of aging is mainly made of oxidized protein (30-60%) and lipids (20-50%), and accumulates mostly in post-mitotic cells such as neurons, cardiac myocytes, skeletal muscle fibers, retinal pigments and epithelial cells (Brunk and Terman, 2002; Jung et al., 2007)). Many studies revealed that lipofuscin may induce neurotoxicity through the generation of ROS (Brunk and Terman, 2002; Szweda et al., 2002). In order to counteract these toxic species in aerobic organisms, endogenous antioxidant system [such as enzymatic antioxidants (Superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) and non-enzymatic antioxidants (glutathione, vitamin C, vitamin E and uric acid) antioxidants defenses] has been developed. However, under oxidative stress condition, the efficiency of this system is declined resulting in ineffective scavenging of free radicals (Fridovich, 1999; Yazdanparast et al., 2008). Based on these facts, it is to suggest that a supplement of antioxidants with exogenous source appears to be a good strategy for preventing the overall above-mentioned complications. Besides of antioxidants present in natural substances, scientist have developed a number of synthetic antioxidants, which have either replaced or supplemented natural antioxidants to help attenuate oxidative damage (Ceriello, 2003). These antioxidants can delay and/or prevent the oxidation process through simple or complex mechanisms including suppression of chain initiation, chelating of transitional metal ion catalysts, breakdown of peroxides, inhibition of continued hydrogen abstraction, and radical scavenging (Ames et al., 1993).

In this respect, transition metal complexes have notably shown to possess high antioxidative properties (Autzen et al., 2003). A number of research groups have developed low molecular weight scavengers which mimic the enzyme functions as SOD and CAT. Among these agents, different derivatives of salen-manganese complexes have shown promising results due to their ability to scavenge a wide range of ROS, namely O2•−, H2O2, ONOO− and RO2• radicals (Baudry et al., 1993; Gonzalez et al., 1995). The SOD-like and free radical scavenging activities of these compounds involves the alternate reduction and oxidation of the Mn center, which cause changes in valence between Mn(III) and Mn(II), much like native SODs. These synthetic complexes also have higher stability and bioavailability related to proteinaceous antioxidant enzymes (Baudry et al., 1993; Sharpe et al., 2002). EUK-8 and EUK-134 (Scheme 1) are two potent members of this group of compounds whose structures and catalytic activities have been described previously (Rong et al., 1999). Both compounds have equivalent SOD activities, but EUK-134 possesses a higher CAT activity (Baudry et al., 1993; Gonzalez et al., 1995). They also decreased the NO production by accelerating the breakdown of numerous NO products to more nonthreatening species (Boucher and Farrell, 1973). In addition, it has shown that these compounds possess therapeutically efficacy in several neurological disorders using animal models (Baudry et al., 1993; Gonzalez et al., 1995; Rong et al., 1999; Sharpe et al., 2002). In order to expend our knowledge about other therapeutic capabilities of these compounds and also to clarify their mechanism of action, the present study was extended to determine the possibility protective effects of EUK-134 and EUK-8 in two different models of oxidative stress in vitro. In the first study, we used of Fe2+/ascorbate model as a well-validated system for induction of oxidative stress in isolated liver homogenates of rats and thereby to evaluate the protective effects of EUK-134 and EUK-8 against this system. In later study, regarding to the role of oxidized lipid and proteins in formation of the lipofuscin in aging process, we evaluated the neuroprotective activity of these compounds against the free-radical damaging effects of H2O2 on SK-N-MC neuroblastoma cell line in terms of cell viability restoration and also attenuation of intracellular lipofuscin formation. Based on the results obtained in this study, the EUK-8 and EUK-134 in different concentrations possessed inhibitory effects against PCO, TBARS and ROS. In addition, pretreatment of the cells with these compounds lead to restoration the viabilities of cells and reduction the extent of lipofuscin formation, relative to the control (H2O2-treated) cells.

Materials and Methods

Materials

Ascorbic acid, catechin, ferrous sulphate (FeSO4), trichloroacetic acid (TCA), 2,4-dinitrophenylhydrazine (DNPH) and Folin-Ciocalteu's reagent (FCR) were obtained from Sigma (St. Louis, MO, USA). 5, 5'-dithiobisnitro benzoic acid (DTNB), hydrogen peroxide (H2O2), butylated hydroxytoluene (BHT) and thiobarbituric acid (TBA) and dimethyl sulfoxide (DMSO) were obtained from Merck Co (Germany). 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular probe (Eugene, Oregon, USA). The cell culture medium (RPMI 1640), penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco BRL (Life technology, Paisley, Scotland). The culture plates were obtained from Nunc (Brand products, Denmark). All other chemicals used were analytical grade.

Preparation of liver homogenate

Male wistar albino rats weighing 200-250 g (purchased from Pasteur Institute, Tehran, Iran) were housed under conventional conditions and were allowed free access to food and water, ad libitum. The rats were anesthetized using diethyl ether and their abdomens were opened and their livers were quickly removed. 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. The livers was then cut into small pieces and homogenized in phosphate buffer (50 mM, pH 7.4) with a homogenizer to give a 10% (w/v) liver homogenate and then centrifuged at 5000 g for 15 min at 4 oC (Beckman). The supernatant was obtained and the protein concentration was determined by the method of Lowry et al (1951) using bovine serum albumin as the standard.

Induction of oxidative stress in liver homogenates

To induce oxidative stress in rat liver homogenates from the oxidant pair Fe2+/ascorbate was used (Ardestani and Yazdanparast, 2007a). The reaction mixture including of 0.5 ml of each liver homogenate, 0.9 ml of phosphate buffer (50 mM, pH 7.4), 0.25 ml of FeSO4 (0.01 mM), 0.25 ml of ascorbic acid (0.1 mM), and 0.1 ml of different concentrations of each compound and/or Catechin as a standard sample,. The reaction mixture was incubated at 37oC for 30 min.

Measurement of ROS levels

The extent of ROS formation in the reaction mixture was measured by following the oxidation of 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) to the highly fluorescent compound, 2′, 7′-dichlorofluorescein (DCF) according to previously published method with slight modification (Ugochukwu and Cobourne, 2003). Each sample composed of 1.85 ml of phosphate buffer (50 mM, pH 7.4) solution, 0.1 ml of liver homogenate, and 50 μl of DCFH-DA solution (10 μM). The samples were incubated in an incubator at 37 °C for 15 min. The ROS levels were measured via the formation of the DCF using a spectrofluorometer (model Cary Eclipse) with the excitation and emission wavelengths at 488 and 525 nm, respectively.

Determination of protein carbonyl content

The extent of protein carbonyls (PCO) were measured by using the method of Reznick and Packer (1994). Based on this method, one milliliter of 10 mM DNPH in 2 M HCl was added to incubated reaction mixture (2 mg protein). Samples were kept for 1 h at room temperature and were vortexed every 15 min. Then, 1 ml of cold trichloroacetic acid (TCA) (10%, w/v) was added to each reaction mixture and centrifuged at 3000 g for 10 min. The protein pellet was washed three times with 2 ml of ethanol/ethyl acetate (1:1, v/v) and dissolved in 1 ml of guanidine hydrochloride (6 M, pH 2.3) and incubated for 10 min at 37 oC while mixing. The carbonyl content was calculated based on the molar extinction coefficient of DNPH (ε=2.2Ã-104 cm−1M−1) and expressed in terms of percentage inhibition.

Determination of protein-bound sulfhydryl groups

The amount of P-SH groups in each liver homogenate were measured according to method of Sedlak and Lindsay (1968) using 5, 5'-dithiobisnitro benzoic acid (DTNB). For total thiol (T-SH) measurement, the reaction mixture comprise of 0.3 ml of incubated liver homogenates , 1.5 ml of Tris buffer (0.2 M, pH 8.2) plus 0.1 ml of 0.01 M DTNB. The reaction mixture with addition of 3.1 ml of absolute methanol was brought to 5.0 ml. After 15 min, each reaction was centrifuged at 3000 g at room temperature for 15 min. The absorbance of each supernatant was read at 412 nm. For non-protein thiol (NP-SH) measurement, an aliquot of 1.7 ml of each incubated homogenate was mixed with 0.3 ml distilled water and 1 ml of 50 % TCA. The samples were shaken intermittently for 10-15 min and centrifuged for 15 min at 3000 g. Two ml of supernatant was mixed with 2 ml of Tris buffer (0.4 M, pH 8.9) and 0.1 ml DTNB. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent blank with no liver homogenate. The experimentally determined molar extinction coefficient at 412 nm was 13,100 in both T-SH and NP-SH procedures. The P-SH groups were calculated by subtracting the NP-SH from T-SH and expressed as nmol/mg protein.

Determination of lipid peroxidation

The level of lipid peroxidation of the rat liver homogenate in the presence and absence of different compounds in various concentrations was evaluated by measuring the product of thiobarbituric acid reactive substances (TBARS) using the method previously described (Bahramikia et al., 2009). After the end of incubation, each reaction was terminated by adding BHT (2% w/v in 95% v/v ethanol) followed by addition of 1 ml of TCA (20% w/v) to the mixture. After centrifugation at 3000 g for 15 min, the supernatant was incubated with 1 ml of thiobarbituric acid (TBA) (0.67%) at 100 oC for 15 min. The color intensity of TBARS/TBA complex was measured and the quantity of TBARS formed was calculated using the absorption coefficient of 1.56 105 cm -1 M-1. The data were calculated from a control measurement of the reaction mixture without the test sample and expressed in terms of percentage inhibition.

Cell culture

SK-N-MC cells were cultured at a density of 5 Ã- 104/ml RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) and incubated at 37 °C in a 5% CO2 humidified atmosphere. Cell numbers and viabilities were assessed using a hemocytometer and the abilities of the cells to exclude trypan blue. After seeding the cells, drug treatments were done 24 h. To induce the oxidative stress, H2O2 was freshly prepared from 8.4 mM stock solution prior to each experiment. SK-NMC cells were incubated with each derivative for 3 h before exposure to 300 μM H2O2.

Cell viability determination

The viability of SK-N-MC neuroblastoma cells was determined using the MTT test (Visitica et al., 1991). After 24 h of seeding of cells (5Ã-104) in 96-well plates, they were treated with different doses (10-100 µM) of compounds EUK-8, EUK-134 and catechin. After the end of this time, 10 µl MTT (5 mg/ml PBS) was added to each well and kept for 4 h. Then, the plates were centrifuged for 15 min at 2500 rpm and the supernatants were discarded and 200 µl DMSO was added to each cell pellet to dissolve Formasan crystals. The absorbance of each sample was recorded at 570 nm with an ELISA reader (Exert 96, Asys Hitch, Ec Austria) after 30 min.

Evaluation of intracellular formation of lipofuscin pigments

Extraction of intracellular of lipofuscin was achieved following lysis of each sample according to procedure with slight modification (Emig et al., 1995). The cells (5Ã-104 cells/well) were seeded in triplicate into 24-well plates for 24 h prior to pretreatments. After pretreatment with each derivative (20 µM) for 3 h, each cell sample was treated with 300 µM H2O2 for 24 h, 48h and 72 h. The attached cells in each well were trypsinized with trypsin- EDTA solution followed by cell counting using a hemocytometer. Each plate was then centrifuged and the cell pellet was washed with PBS, and the cell content was lysed with lysis buffer containing 1% Triton x-100, 1mM EDTA and 1mM PMSF. Each cell lysate was harvested and its fluorescence intensity was monitored on a varian-spectrofluometer (model cary Eclipse} with an excitation wavelength of 310 nm and emission wavelength at 620 nm (Mochizuki et al., 1995). The Fluorescence intensities of the samples were then normalized for equal cell numbers.

Statistical analyses

All data are presented as means ± S.D. The mean values were calculated based on the data taken from at least three independent experiments using freshly prepared reagents. Statistical analyses were performed using student's t-test. The statistical significances were achieved when P< 0.05.

Results

The salen derivatives inhibit the ROS formation in liver homogenates

Fig. 1 shows a significant increase in the fluorescence intensity of DCF in the presence of oxidant pairs as compared to the control. Incubation of each of the compounds and/or Catechin at different concentrations (5, 10, 25 and 50 µM) reduced Fe2+/ascorbate-induced DCF fluorescence (Fig. 1). The highest inhibitory effect was due to EUK-134 and the lowest value observed for EUK-8. These data suggest the potential capacity of compounds to reduce basal ROS production in liver homogenate under oxidative condition.

The salen derivatives inhibit the PCO formation in liver homogenates

The assessment of PCO content is a widely used marker for oxidative protein modification (Reznick and Packer, 1994). As a shown in Fig. 2, the addition of Fe2+/ascorbate to the liver homogenate significantly increased the extent of PCO formation, compared to the control sample. However, in the presence of both compounds at different concentrations (5, 10, 25 and 50 μM), the extent of PCO significantly reduced. The results were relative to Catechin (Cat) as the positive control. Simultaneous addition of Fe2+/ascorbate and Catechin resulted in a similar inhibitory effect on PCO formation relative to compounds. Based on Fig. 2, the order of inhibition of PCO formation by the four extracts is: EUK-134 > catechin > EUK-8.

The salen derivatives protect the P-SH groups from oxidation in liver homogenates

Fig. 3 shows the changes in the sulfhydryl content of proteins in rat liver homogenates induced by Fe2+/ascorbate system in the presence of the compounds. Addition of oxidant pair to reaction reduced the P-SH contents compared to control sample. However, in the presence of each of compounds at various concentrations (5, 10, 25 and 50 μM) the P-SH content increased to various degrees. The highest protection level was due to EUK-134 and the lowest value observed for EUK-8. A sharp rise in P-SH levels in presence of Catechin, as a positive control, was also observed.

The salen derivatives inhibit the level of TBARS in liver homogenates

Fig. 4 demonstrates oxidative damages induced to lipids in terms of measurement of TBARS levels. The addition of Fe2+/ ascorbate to the liver homogenate for 30 min significantly increased the extent of TBARS formation relative to the control sample. However, as shown in Fig. 4, simultaneous addition of Fe2+/ascorbate and both compounds at different concentrations (5, 10, 25 and 50 µM) to the liver homogenates, resulted in reduction of TBARS values. The highest percent of inhibition was found for the EUK-134 and the lowest activity was found in EUK-8. As the positive control, catechin showed the higher inhibitory effect compared to compounds.

The salen derivatives improve the viabilities of H2O2-treated cells

The toxicity of salen derivatives was evaluated based on the viability of cells exposed to variable concentrations of these compounds using MTT assay.

Fig. 4 indicates that MetVOsalen

(6-8%).

According to Fig. 5A, EUK-8 and EUK-134 in low concentrations (10-25 μM) has a slight cytotoxicity effect and in high doses has a moderate cytotoxicity effect on the treated cells in a 24 h evaluation time. In addition, exposure of the cells to H2O2 at concentrations of 50, 100, 150, 200, 300 and 400 μM caused significant reduction in viability by almost 8, 18, 27, 36, 48, and 57%, respectively (data not shown). Regarding these data, the remaining investigation has been carried out at a H2O2 concentration of 300 μM which induces a viability loss of about 48%. However, pre-treatment of the cells with various concentrations of EUK-8 and EUK-134 (10-100) significantly reduced the damaging effect of H2O2 relative to the control cells treated solely with H2O2, as shown in Fig. 5B. Regarding the high viability of cells at 25 μM dose of both the salen derivatives and catechin, the next investigation was performed at this concentration.

The salen derivatives suppress the lipofuscin formation in H2O2-treated cells

As a shown in Fig. 6, exposure of the cells to 300 μM H2O2 for 24 h, 48 h and 72 h caused 59, 116 and 149% increase in the intracellular level of lipofuscin relative to H2O2-untreated control cells, respectively. Pretreatment of the cells with EUK-8 and EUK-134 at a concentration of 25 μM, diminished the formation of lipofuscin pigments by 32, 29, 46 % and, 24, 33, 27 %, respectively after 24 h, 48 h and 72 h of exposure. Using of the catechin, as the positive control resulted in the similar inhibitory effect on level of the lipofuscin compared to compounds.

Discussion

There are several available models of oxidative stress inducers in cell and tissues varying in kind of the species involved and in the site of formation of those species [Guillouzo, 1998; Groneberg et al., 2002]. Regarding in each model the separately oxidative pathways are involved, they could be valuable to comprehend various pathological conditions. In that line, in the present work we used from two different models of these oxidative stress inducers namely Fe2+/ascorbate model in liver homogenates and H2O2 model in SK-N-MC neuroblastoma cell line, and protective effects EUK-8 and EUK-134, two compounds derived from salen, which are as low molecular weight superoxide dismutase mimetic, and Catechin, as a standard antioxidant on these systems were investigated.

Among the in vitro liver preparations recognized as useful experimental models in toxicology, subcellular fractions such as tissue homogenates and microsomes are useful in studying possible mechanisms of oxidative stress inducers (Gutteridge, 1995; Areias, Rego, Oliveira & Seabra, 2001). Fe2+/ascorbate model is a well-validated system for production of ROS and induction of oxidative stress status in isolated tissues homogenates chiefly liver (Gutteridge, 1995; Areias, Rego, Oliveira & Seabra, 2001; Ardestani and Yazdanparast, 2007a). In this system, a mixture of ascorbate and iron salt plus H2O2 which exit in tissues homogenates, can initiate a Fenton-reaction and produced the highly reactive hydroxyl radicals and other toxic species. They consequently can cause chain-initiation reaction of lipid peroxidation or trigger complex pathways of lipid and protein oxidation (Gutteridge, 1995). Thus the measurement of either level of ROS or the oxidation end products especially lipid and proteins in this system has the potential to determine the amount of oxidative damage. On the other hand, with helping this system, it can be to predict the potential efficiency of antioxidant compounds aimed at reducing the oxidative stress.

Regarding this fact, at first the extent of ROS formation in the reaction mixture of all samples was measured by following the highly fluorescent compound, 2′, 7′-dichlorofluorescein (DCF). The presented data confirmed and guarantied the oxidative stress induction and ROS production in liver homogenates under incubation with Fe2+/ascorbate system. In addition, results clearly indicated that in the presence of each compounds; the DCF fluorescence intensity was reduced. Based on the presented data, it can be concluded that these compounds may scavenge the DCF semiquinone free radical intermediates (oxygen radical) produced during the formation of the fluorescent product DCF.

Aforementioned, a main target of oxidative stress in the cell and tissues is proteins. In the Fe2+/ascorbate model, proteins are known to be damaged by ROS directly and to be targets of secondary modifications by aldehydic products of lipid peroxidation or ascorbate autoxidation. Collectively, all these processes can lead to carbonyl modification in several amino acid residues such as lysine, arginine, proline and threonine residues and/or peptide backbone of proteins and consequently the formation of PCO products (Dean et al., 1997; Stadtman and Levine, 2000; Bahramikia et al., 2009). Protein carbonylation has been associated with important functional alterations in a variety of structural proteins and enzymes. For example, actin carbonylation is a sign of severe functional impairment associated with filament disruption, and occurs at an extent of oxidative insult observed in Alzheimer's disease, inflammatory bowel disease and rat myocardial ischemia (Dalle-Donne et al., 2003). Our results indicated that the compounds EUK-8 and EUK-134 inhibit the PCO production in incubated samples with Fe/ascorbate, as shown in Fig. 2. Regarding the alternate changes in valence between Mn(III) and Mn(II) of these compounds, they might operate in one or more of the following ways including production of reactive aldehyde scavengers, formation of potential complexes of pro-oxidant metals and scavenging the hydroxyl radicals. It has also been shown that both compounds have catalase and superoxide dismutase activities with high scavenging activities against various species of free radicals such as superoxides and hydroxyl radicals, thereby curtailing the quantity of cellular destruction inflicted by protein peroxidation byproducts. The data presented herein provide additional evidences that these compounds suppress oxidative modification of proteins. In that respect, EUK-134 was as potent as Catechin in protecting against PCO formation in the rat liver homogenate model. The results clearly indicate the these compounds as the potential sources for different antioxidant substances.

The measurement of proteins sulfhydryl (P-SH) groups is another useful approach for checking oxidative state of biological system. The antioxidant role of these groups might be due to their scavenging activity and thereby, protecting the cellular constituents against free radical attacks (Telci et al., 2000; Bahramikia et al., 2009). Regarding the high susceptibility of P-SH groups to oxidation by free radicals, consumption of antioxidants under the oxidative stress state seems crucial for protecting the functional sulfhydryl groups of proteins (Stadtman and Levine, 2000). The increased of P-SH content by compounds EUK-8 and EUK-134 in our study can be due to chelation of redox active metals, as well as to the trapping of some ROS.

ROS are not only strongly associated with protein oxidation but also are capable of the quick initiation of lipid peroxidation process as by abstracting hydrogen atoms from unsaturated fatty acids (Aruoma, 1998; Kappus, 1991; Dean et al., 1997). Based on different evidences, oxidative modification of proteins night also occurs by reactions with aldehydes produced during lipid peroxidation (Traverso et al., 2004). In that regard, end products of lipid peroxidation such as MDA and 4-HNE as well as products from polyunsaturated fatty acids cause protein damages (Refsgaard et al., 2000). It is established that MDA, 4-HNE and others lipid peroxidation products can oxidize protein thiols or incorporate carbonyl groups into polypeptide chains or cause covalent cross linking of different protein molecules or fragmentation of polypeptide chains leading to impairment of protein functions (Traverso et al., 2004; Dean et al., 1997).

In the present study, exposure of rat liver homogenate to the ascorbate and Fe3+ oxidation system uniformly resulted in an increase in lipid peroxidation levels. However, treatments with compounds significantly decreased TBARS contents. Based on data, it can be concluded that these compounds by decreasing lipid peroxidation may be effective in preventing oxidative protein damages which are believed to occur under oxidation processes.

In addition to the above mentioned events, it has also been demonstrated the oxidative damages provoked by free radicals to lipid and proteins, to play a significant role in aging process in different organs especially brain (Stadtman and Levin, 2000). Brain, because of the high level of fatty acids and low antioxidant capacity compared with other organs, has incredible susceptibility to the damaging effects of ROS (Andersen, 2004). In aging process, antioxidant levels diminish with age, therefore the lipid and proteins appears to be a most targets for oxidative damage. Regarding to the important role of oxidized lipid and proteins in aging process, the second study was extended to the neuroprotective activity of these compounds against the free-radical damaging effects of H2O2 on SK-N-MC neuroblastoma cell line. Hydrogen peroxide, as the main source of hydroxyl free radicals, has been used as another model for induction of oxidative stress and ROS production in vitro. During these precesses, large amounts of H2O2 may enter the lysosomal compartment resulting in the formation of highly reactive hydroxyl and iron-centered radicals and/or both of which endanger the stability of lysosomes. In addition, hydroxyl free radicals can induce the transcription factor NF-Ò›B which can trigger stress factor responses (Schreck et al., 1991; Behl, 1997). These events consequently lead to oxidative damages to lipid, proteins and carbohydrates. One of the subsequent the lysosome instability is the formation of the intralysosomal material as lipofuscin. Lipofuscin as an age pigment primarily composed of oxidatively modified protein and lipid degradation residues. Our data showed that salen derivatives were capable of increasing the viability of H2O2-treated cells. In addition, results indicated that EUK-8 and EUK-134 compounds were capable of decrease the lipofuscin formation, which this decrease can be probably due to their antioxidant activities. These results confirmed the potential antioxidative and protective properties of these compounds in the first study and also performed studies by other researchers on their antioxidant activities in several other models of oxidative stress.

In conclusion, the obtained data presented in both studies indicated that EUK-8 and EUK-134 are able to ameliorate oxidative damages to proteins and lipids which induced by free radical generating Fe2+/ascorbate and H2O2 systems. In addition, the results in H2O2 model in SK-N-MC neuroblastoma cells indicated that anti-oxidant therapy with salen derivatives resulted in lower intracellular accumulation of lipofuscin pigments. These protective effects collectively could be attributed to antioxidant activity of these compounds in addition to the suppression of lipid peroxidation. Since these antioxidants are relatively nontoxic, the present findings suggest that these components might be useful in preventing oxidative damages under various pathological conditions especially liver and neurodegenerative diseases.

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