Original Paper
Cellular Physiology and Biochemistr Biochemistryy
Cell Physiol Biochem 2010;25:241-252
Accepted: October 22, 2009
The Double Edge of Reactive Oxygen Species as Damaging and Signaling Molecules in HL60 Cell Culture Miguel D. Ferrer, Antoni Sureda, Antonia Mestre, Josep A. Tur and Antoni Pons Laboratori de Ciències de l’Activitat Física, Departament de Biologia Fonamental i Ciències de la Salut. Grup de Nutrició Comunitaria i Estrés Oxidatiu, IUNICS, Universitat de les Illes Balears, Palma de Mallorca
Key Words Adaptive response • Gene expression • HL60 • Hormesis • Oxidative damage Abstract Aims: Our aim was to establish the conditions in which reactive oxygen species produce pathological or hormetic effects on HL60 cells. Methods: HL60 cells were treated with either single bouts (1, 10 and 100 µM) or a sustained production (0.1, 1.0 and 10.0 nM/ s) of H2O2. Results: Exposure to 10 and 100 µM H2O2 activated catalase, glutathione peroxidase and glutathione reductase through post-transcriptional mechanisms and induced oxidative modification of proteins. When cells where exposed to sustained H2O2 production, a clear dose-response effect was detected in the activity of the antioxidant enzymes catalase, glutathione peroxidase and Mn-SOD, with higher concentrations of H 2O2 inducing greater enzyme activities. Catalase, HO-1, UCP-3, iNOS and PGC1α expressions were activated after sustained exposure to 1 and 10 nM H 2 O 2/s. Although the antioxidant defenses were activated, oxidative damage appeared in DNA and proteins in cells treated with 1 and 10 nM/s. Conclusions: HL60 cells respond
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to exposure to sustained levels of H2O2 in a doseresponse manner to H2O2 concentration by activating the expression and activity of the antioxidant machinery, although the activation of the antioxidant defenses is not enough to avoid the appearance of oxidative damage. Of the two designs proposed, continuous exposure seems to be more appropriate to study the antioxidant response of HL60 cells to H2O2. Copyright © 2010 S. Karger AG, Basel
Introduction Reactive oxygen species (ROS) are a double-edged sword because they serve as key signal molecules in physiological processes [1-3] but they also play a role in pathological processes such as cachexia [4], atherosclerosis [5], cancer [6] and neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases [7]. The most accepted theory of aging also incorporates the damaging effects of ROS, which are considered to be unavoidable by-products of aerobic metabolism [8]. As in other cases, the dosage is the main variable that influences the side of the double-edge which ROS Dr. Antoni Pons Biescas 241 Laboratori de Ciències de l’Activitat Física Universitat de les Illes Balears Ctra. Valldemossa Km 7.5, E-07122 Palma de Mallorca, Illes Balears (Spain) Tel. +34-971173171, Fax +34-971173184, E-Mail:
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represent at any given moment. The typical reaction to ROS can be described by a bell-shaped curve: low concentrations have a stimulating effect (signaling, receptor stimulation, enzymatic stimulation), while a massive level of ROS inhibits enzyme activity and causes apoptosis or necrosis. The hormesis theory states that biological systems respond with a bell-shaped curve to exposure to chemicals, toxins, and radiation, and this theory has been expanded to include reactive oxygen species [9, 10]. It has been clearly shown that a single bout of exercise above a certain intensity or duration results in increased production of ROS and causes oxidative damage to lipids, proteins, and DNA [11-13]. On the other hand, it is also well established that regular exercise is a preventive measure against oxidative stressrelated diseases including cardiovascular diseases, strokes, and certain cancers [14-16]. In parallel, it has also been pointed out that diet supplementation with some antioxidant nutrients reduces exercise-induced oxidative damage without blocking the cellular adaptation to exercise [17] and dietary supplementation with flavonoids has also been described to reduce the incidence of myocardial infarction [18] and lipid peroxidation [19]. The HL60 cell line was established from a patient with acute promyelocytic leukemia and consists predominantly of promyelocytes [20]. Although it is an undifferentiated cell line it can differentiate into either monocytes/macrophages or granulocytes/neutrophils after a certain stimulus such as phorbol myristate acetate (PMA) or retinoic acid, respectively [21, 22]. Oxidative stress associated to H2O2 exposure was studied in HL60. The exposure of cells to 40-50 µM H2O2 has been shown to induce oxidative damage in DNA, but also to activate the expression of genes involved in NF-κB activation, transcription and DNA methylation along with cytokines and cytokine receptors [23]. The rate of ROS production and the doses the cells are exposed to are crucial to express the adaptive or pathological responses of the cell [3, 24]. Our aim was to evidence the double effects of ROS by establishing the conditions in which ROS produce pathological or hormetic effects on HL60 cells, and to evidence the induction effects of hydrogen peroxide on the expression of antioxidant genes and transcription cofactors. Two situations were assayed for this purpose. The first situation consisted of the exposure of HL60 cells to a bolus of high hydrogen peroxide concentration. In this situation the antioxidant defenses of cells are overwhelmed and cell death is induced. The second situation consisted of the exposure of HL60 cells to a continuous, low production 242
Cell Physiol Biochem 2010;25:241-252
of hydrogen peroxide. In this situation, the stationary state with an excess of hydrogen peroxide leads to a new redox, more oxidized equilibrium in which the antioxidant defenses are activated even though the cellular components are more oxidized. Materials and Methods Cell culture HL60 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 units/ml penicillin, 0.1 ng/ml streptomycin and 2mM Lglutamine, in a humidified atmosphere containing 5% CO2 at 37°C. The cells were cultured into 250 ml tissue culture flasks at the initial concentration of 2 x 105 cells/ml. Cell treatments and experimental design All the treatments were performed in 6-well plates containing 6 x 105 cells/ml. Two experimental designs were performed. In the first experience cells were treated with single bouts of H2O2. The final concentrations of H2O2 used were 1, 10 and 100 µM. Two hours after the addition of H2O2 cells were harvested and washed twice in PBS. In the second experience, cells were incubated for one hour in the presence of glucose and glucose oxidase (GOX) with a controlled GOX activity in order to generate a rate of hydrogen peroxide production of 0.1, 1 and 10 nM H2O2/s [25]. GOX activity was previously determined with 5 mM glucose as substrate with the method described below. Glucose oxidase type X-S from Aspergillus niger (~75% protein, 151,000 U/g solid, Sigma-Aldrich) was added at concentrations 1, 0.1 and 0.01 µg solid/ml to attain the sustained productions of 0.1, 1 and 10 nM H2O2/s, respectively. One hour after the addition of the treatment, cells were washed twice in PBS and incubated for one more hour in culture medium. Total protein concentrations were measured by the method of Bradford [26]. H 2O 2 concentration and glucose oxidase activity determination The change in concentration of H2O2 in the culture medium both in the presence and absence of cells after the addition of 1, 10 and 100 µM H2O2 was monitored colorimetrically using horseradish peroxidase and tetramethylbenzidine (TMB) by measuring the absorbance at 405 nm [27]. H2O2 concentration was calculated with a standard curve of known concentration. H2O2 production by glucose oxidase was also determined with this method using 5 mM glucose as substrate. The kinetic constants (k) for the disappearance of H2O2 were calculated with the following formula:
where Δt = t2 - t1 is measured time interval; A1 is A240 at t = t1; A2 is A240 at t = t2. The kinetic constant was finally corrected and expressed per cells (K/109 cells). Ferrer/Sureda/Mestre/Tur/Pons
Table 1. Primers and conditions used in Real Time PCR. Cell viability Cell viability was measured using the MTT method [28]. Briefly, cells were treated with all treatments (single bouts and continuous H 2O 2 production) for 1 hour. After a washing procedure with PBS (to remove the remaining H2O2 or glucose oxidase), MTT was added to each well (0.5 mg/ml) and cells were incubated for 4 h at 37ºC. The plates were then centrifuged and the supernatant discarded. Tetrazolium crystals were resuspended in DMSO and the absorbance was measured at 570/620 nm. Enzymatic determinations Catalase (CAT) activity was measured by the spectrophotometric method of Aebi using H2O2 as substrate [29]. Glutathione reductase (GRd) activity was measured by the Goldberg and Spooner spectrophotometric method using oxidized glutathione as the substrate [30]. Glutathione peroxidase (GPx) activity was measured using the spectrophotometric method of Flohé and Gunzler [31]. This assay required H2O2 and NADPH as substrates and glutathione reductase as enzyme indicator. Superoxide dismutase (SOD) activity was measured using a xanthine/xanthine oxidase system to generate the superoxide anion. This anion produced the reduction of cytochrome c, which was monitored at 550 nm. The superoxide dismutase in the sample removed the superoxide anion and produced an inhibition of the cytochrome c reduction. MnSOD was achieved after specific inhibition of Cu-ZnSOD with 5 mM potassium cyanide [32]. All activities were determined with a Shimadzu UV-2100 spectrophotometer at 37ºC. mRNA gene expression mRNA expressions were determined by real time RT-PCR. For this purpose, mRNA was isolated by extraction with Tripure Isolation Reagent (Roche). cDNA was synthesized from 1 µg total RNA using reverse transcriptase with oligo-dT primers. Quantitative PCR was performed using the LightCycler instrument (Roche Diagnostics) with DNA-master SYBR Green I. The primers used are shown in Table 1. For all PCRs there Antioxidant Response to ROS Exposure in HL60 Cells
was one cycle at 95°C for 10 min, followed by 40 cycles at the conditions shown in Table 1. The relative quantification was performed by standard calculations considering 2(-ΔΔCt). Basal mRNA levels of control samples were arbitrarily referred to as 1. The expression of the target gene was normalized with respect to ribosomal 18S. Hydrogen peroxide production H 2 O 2 production was measured using 2,7dichlorofluorescin-diacetate (DCFH-DA) as indicator [33]. A stock solution of DCFH-DA (2.05 mM) in ethanol was prepared, and stored at -20°C until analysis. DCFH-DA (61.6 µM) in PBS was added to a 96-well microplate containing 50 µl cell suspension (containing about 6 x 105 cells) The fluorescence (Ex, 480 nm; Em 530 nm) was recorded at 37°C for 30 min in FLx800 Microplate Fluorescence Reader (Bio-tek Instruments, Inc.) by punctual ultraviolet light exposures and emission readings every minute (30 total readings). Nitrite determination Nitrite levels were determined in the culture medium by the acidic Griess reaction using a spectrophotometric method [34]. Cell samples were centrifuged for 10 min at 900xg at 4°C. Supernatants were collected and deproteinized with acetone and kept overnight at -20°C. Samples were centrifuged for 10 min at 15000xg at 4ºC, and supernatants were recovered. A 96well plate was loaded with the samples or standard nitrite solutions (100 µl) in duplicate. 50 µl sulfanilamide (2% w/v) in 5% HCl was added to each well, and 50 µl N-(1-napthyl)ethylenediamine (0.1% w/v) in water was then added. Absorbance was measured at 540 nm following an incubation of 30 min. Nitrite concentration was calculated with a standard curve of known concentration. Protein carbonyl determination Protein carbonyl derivatives were determined by an immunological method using the OxyBlotTM Protein Oxidation Detection Kit (Chemicon International) following the Cell Physiol Biochem 2010;25:241-252
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Fig. 1. Changes in H2O2 concentrations with time. The change in concentration of H2O2 in the culture medium both in the presence ( ) and absence ( ) of cells after the addition of (A) 100 µM and (B) 10 µM H2O2 was monitored colorimetrically using horseradish peroxidase and tetramethylbenzidine (TMB). ( ) represents LN [H2O2] in the presence of cells. The kinetic constants (k) for the disappearance of H2O2 were 15.8 and 12.2 k/10 9 cells after the addition of 10 and 100 µM H 2 O 2, respectively. Statistical analysis: One-way ANOVA. (*) Significant differences vs t = 0 s. (†) Significant differences vs t = 900 s. (‡) Significant differences vs t = 1800 s, p
