Aging, characterized by progressive changes in cells and tissues of the body, is associated with attenuation of cellular function . Overproduction of Reactive oxygen species (ROS) including free radicals such as superoxide (O2•−), hydroxyl radical (•OH), peroxyl radical (RO2•−) as well as nonradical species such as hydrogen peroxide (H2O2) are accepted as the main modulators of aging . Under oxidative stress condition, H2O2 diffuses into lysosomes (PH ~ 4 - 5) and reacts with iron metal ions released by metalloprotein degradation (Fenton chemistry). This leads to the formation of highly reactive oxygen species such as hydroxyl radicals (•OH) which subsequently oxidize vital macromolecules such as lipids, proteins and nucleic acids. The oxidized products would then react with each other leading to the formation of aggregates known as lipofuscin pigments or age pigments (Fig. 1) [3,4].
Lipofuscin is thus, an intracellular indigestible yellow-brown autofluorescent material composed mainly of oxidized protein (30-58%) and lipid (19-51%) clusters. It is highly resistant to proteolytic degradation and accumulates mostly in post mitotic cells such as neurons [5-7]. The intracellular rate of lipofuscin formation is negatively correlated with the remaining life span of cells and increases with age [7-8]. Regarding the elevated level of polyunsaturated fatty acids in brain, lipofuscin pigments mostly accumulate within this tissue by age .
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However, cells have several antioxidant defense mechanisms to prevent and/or to attenuate the destructive effects of ROS. These defense mechanisms include antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and small molecules such as glutathione and vitamins C and E . The efficiency of the antioxidant defense system is weaken under oxidative stress conditions. Therefore, to back up the system under these conditions, it might be beneficial to use exogenous natural or synthetic antioxidants.
Recently, much attention has been devoted to the phenolic antioxidants of medicinal and dietary plants [11-14]. Yakuchinone B (1-[4'- hydroxy-3'-methoxyphenyl]-7-phenylhept-1-en-3-one) has been characterized in Alpinia oxyphylla from zingiberaceae family . This conjugated 1, 4- enones belongs to chalcone compounds with known diverse biological activities such as anti-inflammatory , antiproliferative , antiviral  and anti-neurodegenerative . To get a better understanding on the structural entities required for free radical scavenging, we evaluated the protective effects of yakuchinone B derivatives (JC1-JC6, table 1) against H2O2-induced damage on SK-N-MC cells in terms of cell viability, intracellular ROS content, MDA and lipofuscin levels and also on the CAT and SOD activities. Our results indicated that yakuchinone B derivatives especially JC4, JC5 and JC6 decreased the extent of apoptosis and ROS levels and increased the activities of SOD and CAT relative to H2O2-treated cells. Antioxidant activity of yakuchinone B derivatives seems to be related to their molecular structure: the presence of a hydroxyl group on ring A (JC1-JC6), presence of ring B (JC3, JC4, JC5 and JC6), presence an alkyl group in ring B (JC4, JC5 and JC6) and the conjugation and resonance effects on rings A and B seemed to be essential for JCs activities.
2. Materials and methods
The cell culture medium (RPMI-1640), fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco BRL (Life Technology, Paisley, Scotland). Cell line was obtained from Pasteur Institute of Iran (Tehran, Iran). The culture plates were obtained from Nunc (Brand products, Denmark). Nicotinamide adenine dinucleotide reduced (NADH), phenazine methosulphate (PMS), glutaraldehyde, nitroblue tetrazolium (NBT), H2O2 and 2-thiobarbituric acid (TBA) were obtained from Merck (Germany). Ethidium bromide, acridine orange and Triton x-100 were purchased from Pharmacia LKB Biotechnology (Sweden). MTT [3-(4, 5-dimethyl tiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] and phenylmethylsulphonyl fluoride (PMSF) were from Sigma Chem. Co (Germany). 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probe (Eugene, Oregon, USA). Ethylenediaminetetraacetic acid (EDTA) was from Aldrich. Lead citrate, Na-cacodylate and uranyl acetate were purchased from PELCO (California, USA). Osmium tetroxide (OsO4) was obtained from TAAB Laboratories Equipment Ltd (Aldermaston, UK). Epoxy (Araldite) was from AGAR scientific LTD. Benzylideneacetophenone derivatives (JC1-JC6) were a generous gift from Dr. Seikwan Oh (Ewha Womans University of korea). JC1-JC6 were dissolved in a minimum amount of dimethyl sulfoxide (DMSO) and then diluted with the culture medium to get the desired concentration. The concentration of DMSO in the culture medium has been kept lower than 0.1% and the control cells has been treated with the vehicle containing the same amount of DMSO.
2.2. Cell culture and experimental treatment
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Human SK-N-MC neuroblastoma cells, obtained from Pasteur Institute (Tehran, Iran), were cultured at a density of 5-104/ml RPMI 1640 medium supplemented with FBS (10%, v/v), streptomycin (100 μg/ml) and penicillin (100 U/ml) and kept at 37 °C in a 5% CO2 humidified atmosphere. Drug treatments were usually done 24 h after seeding the cells. To induce the oxidative stress, H2O2 was freshly prepared from 8.5 mM stock solution prior to each experiment. SK-N-MC cells were incubated with JC1-JC6 for 3 h before exposure to 300 μM H2O2.
2.3. Cytotoxicity evaluation of JC1-JC6 in SK-N-MC 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 . The cells were seeded in 96-well plates at a concentration of 5-104 cells/ml for 24 h, and then treated with JC1-JC6 at different concentrations (10, 20, 40, 60 and 80 µM). After 24 h, MTT stock solution (10 µl; 5 mg/ml) was applied to each well. After 4 h of incubation, the plates were centrifuged for 15 min at 2500 rpm and the supernatants were aspirated. The formazan crystals in each well were dissolved in 200 µl of DMSO, and the absorbance was measured via ELISA technique at a wavelength of 570 nm. Results were expressed in percentage of MTT reduction relative to the control cell samples, presuming that the absorbance of the control cells was 100%.
2.4. Cytoprotective effect of JC1-JC6 in H2O2-treated SK-N-MC cells
To evaluate the cytoprotective effects of JC1-JC6 against H2O2 damage, SK-N-MC cells were plated at a density of 5-104 cells/ml for 24 h. The cells were treated with 20 µM of JC1-JC6. Three hours later, 300 µM H2O2 was added to the plate followed by incubation for an additional 24 h and 48 h. Cell viability was estimated using the MTT assay and it was expressed in percentage of survival relative to the control cell samples.
2.5. Fluorescence microscopy and detection of apoptotic cells
The SK-N-MC cells were seeded in 24-well plates. After 24 h, the cells were pretreated with 20 µM of each drugs and then were treated with 300 μM H2O2 for a time course of 24 h. Apoptotic cells were characterized morphologically by staining the cells with acridine orange/ethidium bromide (AO/EtBr) followed by fluorescence microscopy inspection . After treatment, cells were washed twice with PBS and adjusted to a cell density of 1-106 cells/ml of PBS and stained with AO/EtBr solution (1:1 v/v) in a final concentration of 100 µg/ml. The nuclear morphology was evaluated by Axoscope 2 plus fluorescence microscope from Zeiss (Germany). The cells with condensed or fragmented nuclei were counted as apoptotic cells. All experiments were repeated three times. In each run, the total numbers of cells along with total number of apoptotic cells were determined in 10 different microscopic fields. The extent of apoptosis was then expressed as a percentage of the total cell count.
2.6. Measurement of Intracellular ROS
The ROS generation was monitored using 2', 7'- dichlorofluorescein diacetate (DCFH-DA) which readily diffuses into cells .Within the cells, this nonfluorescent dye reacts with intracellular ROS and is converted into dichlorofluorescein (DCF) which is a fluorophor. SK-N-MC cells were cultured in a 12-well plate with 1ml of medium for 24 h. Then, cells were pretreated with drugs and then exposed to 300 µM H2O2. After 24 h incubation, cells were rinsed with serum-free RPMI medium and then, DCFH-DA (10 µM) was added to the cells followed by incubation at 37-C for 1 h. After incubation, cells were washed twice with PBS and the fluorescence intensity was monitored using a Varian-spectrofluorometer, model Cary Eclipse with excitation and emission wavelengths of 485nm and 530 nm, respectively.
2.7. Catalase activity assay
The CAT activity was measured by the method of Aebi  in which the rate of decomposition of H2O2 was determined spectrophotometrically at 240 nm. The enzyme activity was expressed as -10−1 k/mg protein, where k represents the rate constant of the first order reaction of catalase. Protein concentration was determined by the method of Lowry et al .
2.8. Superoxide dismutase activity assay
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The SOD activity was determined according to the method of Kakkar et al , based on the extent inhibition of amino blue tetrazolium formazan formation in the mixture of nicotinamide adenine dinucleotide, phenasine methosulphate and nitroblue tetrazolium (NADH-PMS-NBT). One unit of enzyme activity was defined as the amount of enzyme which caused 50% inhibition of NBT reduction/mg protein.
2.9. Determination of lipid peroxidation
Thiobarbituric acid reactive substances (TBARS), as indicators of lipid peroxidation, were assayed as described in the literature . Briefly, after exposure of SK-N-MC cells to JC1-JC6 for 3 h, cells were exposed to 300 µM H2O2 for 48 h. MDA levels were measured by the double heating method . The method is based on spectrophotometeric measurement of the purple color generated by the reaction of thiobarbituric acid (TBA) with MDA. Briefly, the cells were mixed with 0.5 ml of tricholoroacetic acid (TCA, 10%, w/v) solution followed by boiling in a water bath for 15 min. After cooling to room temperature, the samples were centrifuged at 3000 rpm for 10 min and 0.5 ml of each sample supernatant was transferred to a test tube containing 0.25 ml of TBA solution (0.67%, w/v). Each tube was then placed in a boiling water bath for 15 min. After cooling to room temperature, the absorbance was measured at 532 nm with respect to the blank solution. The concentration of MDA was calculated based on the absorbance coefficient of the TBA-MDA complex (Î = 1.56 - 105 cm−1 M−1) and it was expressed as nmol/mg protein .
2.10. Acid phosphatase assay
The sodium acetate buffer (0.1 M, pH 5; 100 μl), containing 0.1% (vol/vol) Triton X-100 and 10 mM p-nitrophenyl phosphate, was added to each well. The plates were placed in the incubator at 37°C for 3 h. The reaction was stopped by the addition of 10 μl of NaOH (100 mM, pH=10.5) to each well, and the wells were measured via ELISA technique at a wavelength of 405 nm .
2.11. Evaluation of intracellular lipofuscin pigments
Extraction of intracellular lipofuscin was achieved following lysis of each cell sample according to Emig and colleagues procedure with slight modification . The cells (5-104 cells/well) were seeded in 24-well plates for 24 h prior to pretreatments. After pretreatment with 20 µM of each derivative for 3 h, each cell sample was treated with 300 µM H2O2 for 24 h, 48 h 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 centrifugated 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 excitation and emission wavelengthes of 310 and 620 nm, respectively . All experiments were repeated three times and the fluorescence intensities of the samples were then normalized for equal cell numbers.
2.12. Transmission electron microscopy (TEM)
SK-N-MC cells were cultured in a cell culture flask for 24 h. Then, cells were pretreated with JC4 and after 3 h, the cells were exposed to 300 µM H2O2. After 24 h incubation, the cells were fixed in 2.5% glutaraldehyde (0.1 M Na-cacodylate, pH 7.2, 4°C), postfixed in 1% OsO4 (H2O) and embedded in a 2% agar gel (to facilitate further processing) followed with dehydration with ethanol (50-100%) and then embedded in epoxy resin (Araldite). Finally, thin sections were cut with a diamond knife, stained with 2% lead citrate and 2% uranyl acetate (UAc), and examined by a transmission electron microscope (Leo 906, Germany).
2.13. Statistical analyses
Data are expressed as mean ±SD of three independent experiments and statistically analyzed using Student's t-test.Values of p<0.05 were considered significant.
3.1. Effect(s) on cell viability
The toxicities of JC1-JC6 compounds were evaluated based on the viability of SK-N-MC cells exposed to variable concentrations of the analogs using MTT assay. Table 2 indicates that benzylideneacetophenone analogs (JC1-JC6) have slight cytotoxic effects on the cells following a 24 h evaluation time. Based on Table 2, the entire investigations with the derivatives were achieved at doses loss than 20 µM to avoid the cytotoxic effects of the drugs. We evaluated the cytoprotective effect of JC1-JC6 against damage induced in cells by H2O2. For this goal, we pretreated SK-N-MC cells for 3 h with 20 µM of JC1-JC6. Then, the prtreated cells were exposed to 300 µM H2O2 for 24 h and 48 h. As shown in Fig. 2, treatment with H2O2 reduced cell viability by almost 40 and 55%, relative to H2O2-untreated cells, after 24 and 48 h, respectively. However, pretreatment of cells with 20 µM of JC1-JC6 reduced the damaging effects of H2O2 by about 11, 15, 18, 24, 24 and 22% after 24 h and by about 4, 19, 23, 29, 25 and 27% after 48 h, respectively. Regarding these observations, it can be concluded that JC4, JC5 and JC6 at 20 µM concentration scavenging ROS as strongly as catechin at 20 µM.
3.2. Inhibitory effects on apoptosis
As shown in Fig. 3A, the neurite length, as a marker of neuronal aging among H2O2-treated cells, has markedly increased. This is in contrast to the cells which have been pre-treated with JC analogues. For the drug-pretreated cells, homogeneity in shapes and sizes were clearly evident and the cell detachments from the culture plates were significantly lower than the H2O2-treated control cells. To further evaluate the inhibitory effects of benzylideneacetophenone analogs (JC1-JC6) against H2O2 toxicity, we studied the extent of cell death using AO/EtBr double staining technique . In this approach, the non-apoptotic control cells appeared uniformly green; the apoptotic cells showed orange dots in their nuclei corresponding to nuclear DNA fragmentation. The extent of apoptotic cell death for untreated cells was lower than 5%. The number of apoptotic cells increased by 45% upon exposure to H2O2 for 24 h. However, treatment with H2O2 in the presence of JC1-JC6 decreased the extent of apoptotic cells by 4.5, 8.3, 14.5, 17.4, 21.0 and 16.3%, respectively (Fig 3B).
3.3. Deactivation of reactive oxygen species
Exposure of the cells to 300 μM H2O2 caused 165% increase in ROS content relative to H2O2-untreated control cells. Pretreatment of the cells with JC1-JC6 at concentrations of 20 μM diminished the levels of ROS by 38, 14, 50, 77, 56 and 71%, respectively compared to cells exposed only to H2O2 (Fig. 4A). The intracellular ROS level did not varied among cells treated solely with each of the drugs (JC1-JC6, 20 μM, data not shown). Based on our data, JC3, JC4, JC5 and JC6 can certainly be classified as free-radical scavengers.
3.4. Effects on indogenous antioxidant enzymes
To study whether the effect of yakuchinone B derivatives (JC1-JC6) is related to the alteration of intracellular antioxidant status, the activities of CAT and SOD, as the most responsive antioxidant enzymes of the biological system, were determined at 20 µM of each drug (JC1-JC6) among the H2O2-treated cells. As shown in Fig. 4B, 300 μM H2O2 reduced both CAT and SOD activities by 55 and 32%, respectively. However, pre-treatment of the cells with 20 µM of JC1-JC6 increased the CAT activity by 5.3, 10.5, 11.8, 32.6, 18.9 and 32.4% and the dismutase activity by 2.3, 7.2, 8.2, 14.2, 10.0 and 17.1%, respectively relative to cells treated solely with H2O2.
3.5. Inhibition of lipid peroxidation
Biological membranes and intracellular components, which are rich in polyunsaturated fatty acids, can be easily affected by free radicals through peroxidation. The extent of lipid peroxidation significantly increased among the cells exposed to 300 µM H2O2 for 24 h as measured in terms of MDA (Fig. 5A). In SK-N-MC cells treated with H2O2 for 24 h, the level of MDA was 0.65 nmol/mg protein, which was about 4.3- fold higher than that of untreated cells (0.15 nmol/mg protein). As shown in Fig 5A, JC4, JC5 and JC6 effectively inhibited the formation of MDA. The MDA levels in cells pretreated with JC1-JC6 were 0.52, 0.50, 0.54, 0.34, 0.43 and 0.47 nmol/mg protein, respectively. In other words, JC1-JC6 have quenched the formation of MDA by 20.4, 22.7, 16.8, 47.9, 34.2 and 28.1% relative to H2O2-treated cells.
3.6. Effects on the acid phosphatase activity
Acid phosphatase is a stable lysosomal enzyme. Accumulation of lipofuscin in lysosome acts as a trap for acid phosphatase leading to its inactivation. Our results showed a significant decrease (by 38%) in acid phosphatase activity among the H2O2-treated cells. However, pretreatment of the cells with each of the drugs (JC1-JC6) increased the acid phosphatase activity by 1, 8, 12, 20, 19 and 13%, respectively relative to H2O2-trested cells (Fig 5B).
3.7. Influence on lipofuscin formation
Exposure of the cells to 300 μM H2O2 for 24 h, 48h and 72 h caused 85, 158 and 208% increase in the intracellular level of lipofuscin pigments relative to H2O2-untreated control cells, respectively. Pretreatment of the cells with each of the derivatives at a concentration of 20 μM, diminished the formation of lipofuscin pigments by 11, 13, 40, 72, 58 and 61% after 24 h of exposure (Fig 6). Regarding these data, JC drugs can certainly reduced lipofuscin formation due to their free-radical scavenging capabilities as one of their route of action.
3.8. Transmission electron microscopy findings
TEM micrographs of SK-N-MC control cells were devoid of no lipofuscin granules (Fig 7A), After H2O2 treatment, lipofuscin granules with uniformly dense and roughly spherical shapes appeared in the cell cytoplasm. The sizes and distribution of these fluorescent inclusions were consistent with sizes and distributions of the lipid droplets and/or mitochondria (Fig 7B). Pretreatment of the cells with the most active derivative (JC4) resulted in lower lipofuscin content within the cells, as shown in Fig 7C.
It is by now well accepted that free radicals play vital roles in initiation and progression of age-related complications . Hydrogen peroxide, as a pro-oxidant, is capable of penetration into lysosomes, where it interacts with redox sensitive transition metals via fenton reaction and produces hydroxyl radicals . Hydroxyl radicals, in turn, initiate lipid peroxidation with MDA production which will guide the cross-linking of macromolecules such as proteins and the lipid peroxidation products leading to the formation of lipofuscin aggregates . Thus, lipofuscin accumulation is linked to oxidative stress and/or inefficient anti-oxidant defense system. Even liposomal iron overload and mitochondrial dysfunction are considered to play roles in lipofuscin accumulation within the cells . Based on these facts, it is logical to predict that anti-oxidant-based therapeutical approaches would attenuate lipofuscin accumulation via perturbation of free radical chain reactions [3,35].
In the present investigation, we attempted to correlate the antioxidant property of several yakuchinone B analogues (JC1-JC6) to the intracellular build up of lipofuscin aggregates. Our cumulative results indicated that the damaging effects of H2O2 on the cells viabilities, morphology and apoptosis were all reversed by pretreating the cells with each of the analogues. For example, the viabilities of the drug-pretreated cells increased by11, 15, 18, 24, 24 and 22% after 24 h and by about 4, 19, 23, 29, 24 and 27% after 48 h for JC1-JC6, respectively relative to H2O2-treated cells (Fig 2). The extent of apoptosis among the drug-pretreated cells decreased by 4.5, 8.3, 14.5, 17.4, 21.0 and 16.3% compared to H2O2-treated cells (Fig 3B). Similar to normal cells, the drug-pretreated cells were mostly attached to while the H2O2-treated cells were mostly detached from the culture plate surface. Based on Fig 4A, H2O2 enhanced the intracellular levels of ROS and this was accompanied with significant depression of CAT and SOD activities as shown in Fig 4B. However, pre-treatments of the cells with a single dose of each of the analogues (JC1-JC6) decreased the intracellular level of ROS and restored the majority of cellular CAT and SOD activities, as it is evident from Fig 4A and 4B, respectively. Superoxide dismutase (SOD) catalyses the dismutation of superoxide anion into oxygen and H2O2 and CAT catalyses the conversion of H2O2 to H2O and molecular oxygen. Repression of SOD and CAT activities by H2O2 (Fig 4B) was accompanied with enhanced intracellular level of lipofuscin as indicated in Fig 6. As mentioned before, lysosome is engaged in lipofuscinogenesis. Accumulation of age pigments within the lysosome might interfere with the activities of lysosomal enzymes . Acid phosphatase is such an enzyme whose activity, upon exposure to H2O2, was significantly declined. However, pre-treatment of the cells with JC3, JC4, JC5 and JC6, followed by exposure to H2O2, led to a pronounced increase in the acid phosphatase activity (Fig 5B).
Transmission electron microscope is an appropriate technique to document the intracellular events under non-physiological conditions. By this technique, the lipofuscin pigments appear as irregularly shaped dark masses mostly in cytoplasm. According to TEM results in Fig7, JC4 has strongly attenuated the accumulation of age pigments.
Our cumulative data clearly documented the yakuchinone B derivatives as free radical scavengers with the following order of activity: JC4> JC6> JC5≥ JC3> JC2> JC1. Previous studies have shown that the phenolic hydroxyl group on the vaniline ring (ring A, table 1) is required for the antioxidant activity of each of the derivatives [3, 37]. The stronger free radical scavenging activity of JC4, JC5 and JC6, relative to JC1, JC2 and JC3, might be attributed to two structural features: the presence of another aromatic ring (ring B, table 1) and the presence of at least one methylene group between the carbonyl group and the aromatic ring B. These structural elements would add up to higher free radical scavenging activity due to the formation of the following intermediates (I and II) for JC4, JC5 and JC6 as additional routes for neutralization of free radicals such as •OH .The other analogues (JC1, JC2 and JC3) can not form either of these intermediates and thus, they will be capable of neutralizing the free radicals only through ring A. This is in contrast to compounds JC4, JC5 and JC6 which would neutralize the free radicals through both rings A and B. Therefore, they have higher anti-oxidant activities .
In summary, the results of this investigation, besides of reconfirming the free-radical hypothesis of aging, clearly indicate that the molecular complications of aging such as intramolecular accumulation of lipofuscin pigments, could be attenuated by balancing the intracellular levels of free radicals to their normal physiological levels. This might be achieved through delicate administration of appropriate exogenous antioxidants.
The authors appreciate the financial support of this investigation by the Research Council of University of Tehran. In addition, the authors thank Mr. N. Pirhajati for his kind assistance in TEM sample preparations.