Selenium methylselenocysteine protects human hepatoma HepG2 cells against oxidative stress induced by tert-butyl hydroperoxide
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Selenium methylselenocysteine (Se-MeSeCys) is a common selenocompound in the diet with a tested chemopreventive effect. This study investigated the potential protective effect of Se-MeSeCys against a chemical oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) on human hepatoma HepG2 cells. Speciation of selenium derivatives by liquid chromatography–inductively coupled plasma mass spectrometry depicts Se-MeSeCys as the only selenocompound in the cell culture. Cell viability (lactate dehydrogenase) and markers of oxidative status—concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR)—were evaluated. Pretreatment of cells with Se-MeSeCys for 20 h completely prevented the enhanced cell damage, MDA concentration and GR and GPx activity and the decreased GSH induced by t-BOOH but did not prevent increased ROS generation. The results show that treatment of HepG2 cells with concentrations of Se-MeSeCys in the nanomolar to micromolar range confers a significant protection against an oxidative insult.
KeywordsAntioxidant defences Biomarkers for oxidative stress Dietary antioxidants Selenium compounds
Human exposure to potentially toxic chemicals, either through an occupational environment, or as the result of plant and foodstuff pyrolysis (e.g. tobacco smoke, charbroiled foods) is almost unavoidable. In many instances, increased exposure to these hazardous chemicals, many of which are prooxidants or procarcinogens, is linked to an increased incidence of cardiovascular disease and cancer [1, 2]. This has prompted a search for diets or chemical supplements that might mitigate or prevent the toxic outcome of exposure. There is substantial evidence that antioxidative food components have a protective role against oxidative stress-induced atherosclerosis, degenerative and age-related diseases, cancer and aging [3, 4]. Among dietary compounds considered for chemopreventive activity, selenium showed early and continued promise [1, 2, 4, 5, 6, 7, 8]. Selenium is a trace element essential to human health found in fish, meat and plants such as garlic, onion and broccoli, and a deficiency of this element induces pathological conditions, such as cancer, coronary heart disease and liver necrosis [6, 9]. Garlic is the most popular and well-researched Allium plant that is known to accumulate selenium as selenoamino acid derivatives, including selenium methyl-l-selenocysteine (Se-MeSeCys), one of the major forms of selenium in the diet, and glutamylmethylselenocysteine [9, 10].
Selenium compounds have been widely reported to be effective chemopreventive agents against multiple models of tumorigenesis [4, 5, 6, 7, 8, 11, 12, 13, 14]. These protective effects of selenium seem to be primarily associated with its presence as a cofactor in the enzymes glutathione peroxidase (GPx) and thioredoxin reductase, which are known to protect cellular components from oxidative damage [1, 2]. Although these properties indicate that Se-MeSeCys may favourably affect the antioxidant defence system [1, 2], little is known about the potentially beneficial role of Se-MeSeCys against oxidative damage in vivo, both in cultured cells and in live animals.
Human hepatocarcinoma HepG2 is widely used for biochemical and nutritional studies as a cell culture model of human hepatocytes since they retain their morphology and most of their function in culture . In addition, HepG2 is a reliable model where many dietary antioxidants and conditions can be assayed with minor interassay variations [16, 17, 18, 19, 20]. Previous studies from our laboratory have shown that the plant flavonol quercetin , the olive oil phenol hydroxytyrosol  and a digested coffee melanoidin  elicit a favourable response of the antioxidant defence system in cultured human hepatoma HepG2 cells. Therefore, the potential protective effect of different concentrations of the dietary compound Se-MeSeCys against an oxidative stress chemically induced by a potent prooxidant, tert-butyl hydroperoxide (t-BOOH), has now been tested in cultures of HepG2. Cell integrity and several markers of oxidative status, such as concentration of reduced glutathione (GSH), generation of reactive oxygen species (ROS), evaluation of the activity of the antioxidant enzymes GPx and glutathione reductase (GR) and determination of malondialdehyde (MDA) as a biomarker of lipid peroxidation, were measured to estimate the effect of Se-MeSeCys in cell survival and the response of the antioxidant system of HepG2 to t-BOOH.
Materials and methods
Reagents and samples
Se-MeSeCys (Sigma Chemicals, St. Louis, MO, USA) was dissolved in Milli-Q water. Inorganic selenium solution was obtained by dissolving sodium selenite (Merck, Darmstadt, Germany) in deionized Milli-Q water. Stock solutions of 10 mg/L were stored in the dark at 4 °C and working standard solutions were prepared daily by dilution. For hydride generation atomic fluorescence spectroscopy (AFS) studies, sodium borohydride (Sigma-Aldrich, Steinheim, Germany) was prepared in 0.3% sodium hydroxide. For high-performance liquid chromatography (HPLC)–inductively coupled plasma mass spectrometry (ICP-MS) studies, heptafluorobutyric acid, trifluoroacetic acid and ammonium citrate from Sigma (St. Louis, MO, USA) and methanol (Sharlab, Barcelona, Spain) were used in the different chromatographic mobile phases. For the enzymatic extraction procedure the nonspecific protease XIV (Sigma) was used to prepare the extracts. H2O2 and HNO3 were used for acid digestion of samples. The Bradford reagent was from Bio-Rad Laboratories. All other chemicals, including GR, GSH and oxidized glutathione, NADPH, o-phthaldehyde (OPT), dichlorodihydrofluorescein (DCFH) and 2,4-dinitrophenylhydrazine (DNPH) were purchased from Sigma Chemical Co. Other reagents were of analytical or chromatographic quality.
For total selenium determination, samples were microwave-assisted acid-digested in doubled-walled advanced composite vessels using a 1,000-W microwave sample preparation system microwave oven (CEM, Matthews, NC, USA). A Sonoplus ultrasonic homogenizer (Bandelin, Germany) equipped with a titanium 3-mm-diameter microtip and fitted with a high-frequency generator of 2,200 W at a frequency of 20 kHz was used for the extraction of selenium species. An HP-4500 plus inductively coupled plasma mass spectrometer (Agilent Technologies, Tokyo, Japan), fitted with a Babington nebulizer and a Scott double-pass spray chamber cooled by a Peltier system was used for total selenium determination after chromatographic separation. A PU-2089 HPLC pump (JASCO, Tokyo, Japan) fitted with a six-port injection valve (model 7725i, Rheodyne, Rohner Park, CA, USA) with a 100-μL injection loop was used for the chromatographic experiments. Anion-exchange chromatography was performed using a Hamilton PRP-X100 (Reno, NE, USA) column (10-μm particle size, 250 mm × 4.1-mm inner diameter). Reversed-phase chromatography was performed using a C-18 Gemini column (10-μm particle size, 150 mm × 2.0-mm inner diameter; Phenomenex, Torrance, CA, USA). For molecular weight fractionation, 10-kDa cut-off filters (Millipore, Bedford, MA, USA) and an Eppendorf (Hamburg, Germany) 5804 F34-6-38 centrifuge were used .
Human hepatoma HepG2 cells were maintained in a humidified incubator containing 5% CO2 and 95% air at 37 °C. They were grown in DMEM-F12 medium from Biowhitaker (Innogenetics, Madrid, Spain), supplemented with 2.5% Biowhitaker foetal bovine serum (FBS) and 50 mg/L of each of the following antibiotics: gentamicin, penicillin and streptomycin (all from Sigma, Madrid, Spain). Plates were changed to FBS-free medium before the beginning of the assay. The serum added to the medium favours growth of most cell lines but might interfere in the running of the assays and affect the results. Moreover, fairly good growth of HepG2 cells in FBS-free DMEM-F12 has been observed .
Two sets of experiments were designed for this study: (1) experiments of plain treatment of cells with Se-MeSeCys for 2 or 20 h to test for a direct effect of the selenocompound and (2) experiments of pretreatment of cells with Se-MeSeCys for 2 or 20 h before submitting the cells to an oxidative stress by t-BOOH to test for a protective effect against an oxidative insult. In order to infer the effect of the time of treatment on the different concentrations of Se-MeSeCys, two experimental parameters of short (2-h) and long (20-h) treatment with the compound were selected in accordance with previous studies [17, 18]. Lactate dehydrogenase (LDH), GSH, MDA and ROS were evaluated in both experimental conditions and, in addition, GPx and GR were also determined in experiment 2. The different concentrations of Se-MeSeCys were dissolved in serum-free culture medium. For further details see [18, 19, 20].
Procedure for selenium determination and speciation
Instrumental operating conditions for selenium determination by high performance liquid chromatography (HPLC) inductively coupled plasma mass spectrometry (ICP-MS)
Operating parameters and conditions
10mM citrate buffer, H2O–MeOH (98:2)
0.1% heptafluorobutyric acid; 0.05% trifluoroacetic acid; 2% MeOH
Eluent flow rate
Plasma gas (Ar) flow rate
Auxiliary gas (Ar) flow rate
Carrier gas (Ar) flow rate
77Se, 78Se, 82Se
Evaluation of LDH, GSH and MDA
Cells were plated in 60-mm diameter plates at a concentration of 1.5 × 106 per plate and the assay was carried out 2 days later. Cells were treated as described in the previous section and LDH leakage to the culture medium was estimated from the ratio between the LDH activity in the culture medium and that of the whole cell content [16, 18]. The content of GSH was quantitated by the fluorometric assay of Hissin and Hilf . The method takes advantage of the reaction of GSH with OPT at pH 8.0. Fluorescence was measured at an emission wavelength of 460 nm, with an excitation wavelength of 340 nm. The precise protocol has been described elsewhere [16, 18]. Cellular MDA was analysed by HPLC as its DNPH derivative . Cells were treated as in the LDH assay and then collected. For MDA, values are expressed as nanomoles of MDA per milligram of protein; protein was measured by the Bradford kit.
Determination of ROS
Cellular ROS were quantified by the DCFH assay using a microplate reader . After being oxidized by intracellular oxidants, DCFH will become dichorofluorescein (DCF) and this will fluoresce. By quantifying the fluorescence over a period of 90 min, we obtained a fair estimation of the overall oxygen species generated under the different conditions. This parameter gives a very good evaluation of the degree of cellular oxidative stress. The assay has been described elsewhere [16, 18].
Determination of GPx and GR Activity
For the assay of the GPx and GR activity, cells previously treated as in the LDH, GSH and MDA assays were suspended in PBS and centrifuged at low speed for 5 min. Cell pellets were resuspended in 20 mM tris(hydroxymethyl)aminomethane, 5 mM EDTA and 0.5 mM mercaptoethanol, sonicated, and centrifuged at 3,000 g for 15 min. The enzyme activity was measured in the supernatants. The determination of GPx activity is based on the oxidation of GSH by GPx, using t-BOOH as a substrate, coupled to the disappearance of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) by GR . GR activity was determined by following the decrease in absorbance owing to the oxidation of NADPH utilized in the reduction of oxidized glutathione . The methods have been previously described [16, 18]. Protein was measured by the Bradford kit.
Statistical analysis of data was as follows. Prior to analysis the data were tested for homogeneity of variances by the test of Levene. For multiple comparisons, one-way analysis of variance was followed by a Bonferroni test when variances were homogeneous or by a Tamhane test when variances were not homogeneous. The level of significance was P < 0.05. SPSS version 12.0 was used.
Results and discussion
Total selenium content and speciation
Analytical systems developed for the speciation of selenium species employ a powerful liquid chromatograph coupled to a specific detector with a high-efficiency sample introduction system. Nowadays, the most favoured instrument combination for this purpose is ICP-MS coupled with various LC techniques, such as anion-exchange, cation-exchange, reversed-phase, ion-pair, size-exclusion and chiral chromatography. Identification of species is achieved by retention time matching with available standards utilized in a standard addition mode [21, 27, 28]. This analysis has not been previously applied to cell culture studies.
In order to predict the potential bioavailability and metabolism of selenium and its species from selenium-enriched radish, Pedrero et al.  performed an in vitro gastrointestinal process, concluding that the concentration of the species found, selenomethionine and Se-MeSeCys, remains almost unaltered after simulated gastrointestinal digestion. Although other authors have found intense metabolism of Se-MeSeCys to methylseleninic acid in all tissues containing β-lyase enzyme activity , the absence of any structural transformation in the Se-MeSeCys molecule throughout the gastrointestinal tract ensures preservation of the complete biological activity in a strenuous metabolic environment. However, the present findings in cultured live cells should be considered as a first necessary step in research on the protective effects of selenium compounds against oxidative stress, and future research including experiments in live animals should be delineated in order to address the in vivo metabolic fate of this compound.
Total selenium content was measured in cells treated for 24 h with Se-MeSeCys at two different concentrations (10 and 100 μM) to evaluate the uptake capability of selenium by HepG2 cells. The selenium concentration data were validated by applying the method to a certified reference material: bovine liver CRM 185R (1.68 ± 0.14 mg/kg). The result obtained, 1.8 ± 0.2 mg/kg, was in agreement with the reference value. The final concentration of Se-MeSeCys inside the cells remained relatively constant regardless of the selenium dose (13.2 ± 0.2 μg/mL homogenate in cells treated with 10 μM compared with 12.1 ± 1.1 μg/mL in those treated with 100 μM), probably owing to saturation of uptake mechanisms. In fact, in human trabecular meshwork (HTM) cells treated with methylselenic acid, saturation of selenium uptake was observed at the same doses . No amount of any selenium compound was found in either the culture medium or cell homogenate in HepG2 untreated cultures.
Cells are naturally provided with an extensive array of protective enzymatic and nonenzymatic antioxidants that counteract the potentially injurious oxidizing agents [2, 16, 17, 18, 19, 20, 32, 36]. But even this multifunctional protective system cannot completely prevent the deleterious effects of ROS, and consequently oxidatively damaged molecules accumulate in cells. GSH is the main nonenzymatic antioxidant defence within the cell and plays an important role in protection against oxidative stress, as a substrate in GPx-catalysed detoxification of organic peroxides, by reacting with free radicals and by repairing free-radical-induced damage through electron-transfer reactions [2, 16, 17, 18, 19, 20]. It is usually assumed that GSH depletion reflects intracellular oxidation. In contrast, an increase in GSH concentration could be expected to prepare the cell against a potential oxidative insult [16, 17, 18, 19, 20, 38, 39].
In our experimental conditions, addition of Se-MeSeCys did not evoke changes in GSH concentration, whereas treatment of HepG2 cells with 200 μM t-BOOH induced a remarkable decrease in the concentration of GSH indicative of oxidative stress (Fig. 2b). This decrease of GSH induced by t-BOOH was partly (2 h) or completely (20 h) prevented by pretreatment with Se-MeSeCys (Fig. 3b). This result could explain the protected cell integrity reported in the previous section since maintaining GSH concentration above a critical threshold while facing a stressful situation represents an advantage for cell survival.
An important step in the degradation of cell membranes is the reaction of ROS with the double bonds of polyunsaturated fatty acids (PUFAs) to yield lipid hydroperoxides. On breakdown of such hydroperoxides a great variety of aldehydes can be formed; MDA, a three-carbon compound formed by scission of peroxidized PUFAs, mainly arachidonic acid, is one of the main products of lipid peroxidation . Since MDA has been found at elevated levels in various diseases thought to be related to free-radical damage, it has been widely used as an index of lipoperoxidation in biological and medical sciences . However, other than our previous results, reports of determination of MDA levels in cell culture conditions are extremely scant in the literature .
We have established a new method of evaluation of MDA in cultures of human hepatoma cells that is sensitive enough to detect a significant increase in MDA concentration in response to an oxidative stress induced by t-BOOH . By using this method, we found that a 3-h treatment of HepG2 with 200 μM t-BOOH evoked a significant increase of about 35% in the cellular concentration of MDA, indicating damage to cell lipids (Figs. 2c, 3c). In fact, a significant decrease of MDA was observed in cells treated with 1 or 10 μM Se-MeSeCys for 20 h in experiment 1 (Fig. 2c). In addition, the t-BOOH-induced increase of MDA was completely avoided when cells were pretreated for 2 or 20 h with 0.1–10 μM Se-MeSeCys in experiment 2 (Fig. 3c). This protection by Se-MeSeCys against an induced lipid peroxidation in a cell culture has not been previously reported and is in line with previous studies that showed a similar effect by other dietary compounds, including plant polyphenols such as tea catechins [43, 44], quercetin , olive oil hydroxytyrosol , β-carotene or lutein , and Maillard reaction products such as coffee melanoidin  in the same cell line, human hepatoma HepG2.
Accumulation of ROS in several cellular components is thought to be a major cause of molecular injury leading to cell aging and to age-related degenerative diseases such as cancer, brain dysfunction and coronary heart disease [2, 3, 32, 36]. Direct evaluation of ROS yields a very good indication of the oxidative damage to living cells . Based upon the fact that nonfluorescent DCFH crosses cell membranes and is oxidized by intracellular ROS to highly fluorescent DCF , the intracellular DCF fluorescence can be used as an index to quantify the overall oxidative stress in cells [16, 17, 18, 19, 20]. A prooxidant such as t-BOOH can directly oxidize DCFH to fluorescent DCF, and it can also decompose to peroxyl radicals and generate lipid peroxides and ROS, thus increasing fluorescence.
GPx and GR activity
In the defence against oxidative stress, the cellular antioxidant enzyme system plays a crucial role and changes in the activity of antioxidant enzymes can be considered as biomarkers of the antioxidant response [16, 17, 18, 19, 20, 46, 47]. GPx catalyses GSH oxidation to oxidized glutathione at the expense of H2O2 or other peroxides  and GR recycles oxidized glutathione back to GSH [3, 26]; therefore, their activities are essential for the intracellular quenching of cell-damaging peroxide species and the effective recovery of the steady-state concentration of GSH.
The results indicate that at the end of an induced stress period the antioxidant defence system of cells that had been pretreated with Se-MeSeCys has more efficiently returned to a steady-state activity, diminishing, therefore, cell damage and enabling the cell to cope in better conditions with further oxidative insults.
In addition, our results support previous data on the chemoprotective effect of Se-MeSeCys and give more insight into its potential biological activity, showing that concentrations of Se-MeSeCys within the physiological range remain unaltered during the treatment and evoke a favourable response in cellular models. Therefore, Se-MeSeCys may contribute to the protection afforded by fruits, vegetables and plant-derived beverages against diseases for which excess production of ROS has been implicated as a casual or contributory factor.
This work was supported by the grants AGL2000-1314, AGL2004-00302 and CTQ2005-02281 from the Spanish Ministry of Science and Technology (CICYT), and grant CAM-S505/AGR/0312 from the Comunidad Autónoma de Madrid. S.C. has a predoctoral fellowship, R.M. is a postdoctoral fellow and S.R. has a Ramón y Cajal contract, all from the Spanish Ministry of Education.
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