Oxidative metabolism of lung macrophages exposed to sodium arsenite

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Toxicology in Vitro 21 (2007) 1603–1609 www.elsevier.com/locate/toxinvit

Oxidative metabolism of lung macrophages exposed to sodium arsenite Mo´nica A. Palmieri a, Deborah R. Tasat b, Beatriz L. Molinari

c,d,e,*

a b

Biodiversity and Experimental Biology Department, F.C.E.y N., National University of Buenos Aires, Argentina Lung Biology Lab, School of Science and Technology, National University of San Martı´n, Buenos Aires, Argentina c National Research Council, Buenos Aires, Argentina d Radiobiology Department, National Atomic Energy Commission, Buenos Aires, Argentina e L.A.N.A.I.S.-C.N.E.A.-CONICET, National Atomic Energy Commission, Buenos Aires, Argentina Received 14 November 2006; accepted 3 June 2007 Available online 27 June 2007

Abstract Arsenic pollution has become increasingly severe. It occurs as the result of geological processes and different human activities. Arsenic toxicity at the respiratory level occurs mainly by inhalation of products of coal combustion. The aim of this study was to evaluate sodium arsenite (As3+) toxicity in murine alveolar macrophages (AMs) in vitro and its association with the alterations in cell metabolism. No changes in viability, apoptosis or cell area were detected in AMs treated with As3+ concentrations up to 2 lM for 24–96 h. A marked decrease in these end-points was observed for As3+ concentrations ranging from 2.5 lM to 10 lM. Regarding the dynamics of the endo-exocytic process triggered by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell incorporation, no variations were detected for As3+ concentrations lower than 2 lM while higher concentrations markedly modified this response. MTT specific activity, as a measure of cell metabolic activity, was not modified irrespective of the As3+ concentration assayed. However, nitroblue tetrazolium (NBT) specific activity, as a measure of superoxide anion generation, is responsive but only to low As3+ doses. Although this study focuses on lung macrophages, the effects of As3+ described herein may also apply to the response of macrophages residing in other organs. Arsenite modifies the metabolic and the oxidative status of AMs in vitro. When macrophages are in an As3+ rich medium, they exhibit a reduction in respiratory burst levels and lose their intrinsic capacity to respond to toxicants.  2007 Elsevier Ltd. All rights reserved. Keywords: Arsenite; Alveolar macrophages; NBT; MTT; Oxidative metabolism

1. Introduction Arsenic is a common constituent of the earth’s crust. This element is widely distributed and occurs naturally in Abbreviations: AMs, alveolar macrophages; As, arsenic; As3+, sodium arsenite; FCS, fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NBT, nitroblue tetrazolium; O2 , superoxide anion; PBS, phosphate buffer saline; ROS, reactive oxygen species; TOD, total optical density; TPA, 12-O-tetradecanoyl phorbol-13-acetate. * Corresponding author. Address: Radiobiology Department, National Atomic Energy Commission, Buenos Aires, Argentina. Tel.: +54 11 6772 7146; fax: +54 11 6772 7188. E-mail address: [email protected] (B.L. Molinari). 0887-2333/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.06.003

our environment. The exposure of the general population is inevitable and will vary according to local geochemistry and the level of anthropogenic activity. The transport and distribution of arsenic in the environment is complex, due to the many chemical forms in which it may be present and because there is continuous cycling of different forms of arsenic through air, soil and water. In certain geographical areas, there are arsenic-rich strata and this may result in higher levels of arsenic in water due to the weathering of rocks and minerals (USEPA, 1987). On the other hand, much of the arsenic in the atmosphere comes from anthropogenic activity such as fuel combustion, pesticide use, smelting, mining and coal-fired power plant (ATSDR,

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1997). Arsenic is released into the atmosphere and exists mainly adsorbed on particulate matter. These particles are dispersed by the wind and are returned to the earth by wet or dry deposition. The arsenic concentration in industrial and urban areas increases from one hundred to one thousand-fold compared to background levels (Schroeder et al., 1987; Romo-Kro¨ger et al., 1994; WHO, 2001). It has been extensively documented that arsenic exposure is associated with deleterious health effects in humans (Yoshida et al., 2004). The main health problem, arsenicosis, is probably due to the high content of arsenic in drinking water, as reported in Taiwan, Mexico, Chile, Argentina, Thailand, India, Hungary and Romania (Tseng et al., 1968; Cebrian et al., 1983; Smith et al., 1998; Ferreccio and Sancha, 2006; Hopenhayn-Rich et al., 1998; Guha Mazumder, 2003; Lindberg et al., 2006). Another type of arsenicosis is due to occupational exposure in several industries: mining, pesticide, glass, and microelectronics (Gabor and Coldea, 1977; Ng et al., 1998; Palus et al., 2005). Furthermore, residential chronic human exposure to high concentration of arsenic in air due to the use of arsenic-rich coal for cooking, heating, and drying foods was recently reported in China (Liu et al., 2002; Guifan, 2004). Ingestion of contaminated drinking water is the principal source of environmental arsenic exposure while inhalation is the principal route of arsenic exposure in occupational and residential settings. In the human exposed population arsenic is associated with skin melanosis, hyperkeratosis, peripheral vascular disease and high risk of several kinds of cancers (Chen et al., 1997). Skin, bladder, liver and kidney cancers are more frequently associated with arsenic ingestion, while lung cancer is the most frequently found in people exposed to arsenic in air (Chen et al., 1992; Mouro´n et al., 2006). The mechanism of arsenic-induced diseases is still unclear. Studies have suggested that arsenic exerts its toxicity through the generation of reactive oxygen species (Lee et al., 1995; Ramos et al., 1995; Kitchin and Ahmad, 2003; Sakurai et al., 2005) which include hydrogen peroxide and free radicals such as superoxide anion. Thus, new experimental approaches are needed to elucidate the different cell metabolic pathways by which arsenic impairs the normal function of pivotal cells in organs. In the lung, the lining epithelium and alveolar macrophages (AMs) constitute the first line of defence from external injuries. Alveolar macrophages are located at the interface between air and lung tissue, being extensively exposed to high oxygen levels, foreign pathogen organisms and pollutants. Under normal conditions, AMs are key cells of the immune system with major phagocytic ability which, in response to stimulation, release various inflammatory factors and reactive oxygen species (ROS) (Hakim, 1993). It has become increasingly evident that these species can function as second messengers and at low levels can activate signalling pathways resulting in a broad array of physiological responses from cell proliferation to apop-

tosis (Forman and Torres, 2001). An imbalance in the oxidative metabolism of AMs may cause cell damage which can be relevant in the progress of respiratory diseases (Sporn et al., 1990). The study of the effect of sodium arsenite (As3+) on AMs is critical to the knowledge of the potential human hazard of this toxicant in the lung. Bishayi and Sengupta (2003) observed that the viability of bacteria gradually decreased in control macrophages with time, whereas in macrophages of As3+ exposed mice the viability of Staphylococcus aureus gradually increased. As3+ interferes with the ability of macrophages to modulate the immune response, inhibiting cell inflammatory functions. Within this context we evaluated the metabolic alterations of AMs exposed to an As3+-enriched environment, focusing on morphologic and functional cell features and their potential association with arsenic induced diseases. 2. Materials and methods 2.1. Chemicals and reagents Sodium arsenite (As3+), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), 12-O-tetradecanoyl phorbol-13-acetate (TPA), nitroblue tetrazolium (NBT), RPMI-1640, fetal calf serum (FCS), Hoechst 33258 and phosphate buffer saline (PBS) were purchased from Sigma Chemical Co. (St. Louis, MS, USA). 2.2. Animals Two-month-old female SenCar mice were used for harvesting alveolar macrophages. They were bred at the animal house facility of the National Atomic Energy Commission and housed in a controlled environment. Food and drink were provided ad libitum. Institutional and national guidelines for the use and care of laboratory animals were strictly observed. 2.3. Macrophages recruitment and culture Pulmonary AMs from SenCar mice were harvested by broncheoalveolar lavage as described elsewhere (Tasat and de Rey, 1987). The lungs were cannulated and lavaged repeatedly with 10 ml cold PBS (pH 7.2). Lavage fluid was collected and centrifuged at 800 g for 10 min at 4 C. Cells were resuspended in RPMI-1640 medium supplemented with 10% FCS, 100 U/ml penicillin and 100 lg/ml streptomycin. Cell viability was assayed by the trypan blue dye exclusion test (>95%). In all experiments AMs were plated in 35 mm plastic dishes at a density of 4.5–5 · 105 and were incubated for 1 h at 37 C in a 5% CO2. After incubation, cells were carefully washed to remove any non-adherent cells. The remaining attached cells were 95% macrophages. Alveolar macrophages were exposed to increasing concentrations of As3+ (0, 1.25, 2.5, 5 and 10 lM) for 24, 48, 72 or 96 h.

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2.4. Macrophage viability Evaluation of macrophage viability in all experimental conditions was performed by means of the trypan blue exclusion test. Data were processed with the GraphPad Prism 5 Demo software. Non-linear regression analysis was performed to calculate the LC50 values and to construct the corresponding dose–response curves. 2.5. Macrophage apoptosis Morphological evaluation of apoptosis was performed on As3+ exposed AMs. Cells were fixed in acetic acid– methanol (1:3) for 10 min and stained with Hoechst 33258 (5 lg/ml PBS) for 20 min. Alveolar macrophages were examined at 460 nm by epifluorescence (Axioplan microscope, Carl Zeiss, Jena, Germany). Morphological features such as pyknosis, nuclear chromatin condensation and marginalization were scored as apoptotic cells (Kerr et al., 1972).

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cells were fixed with paraformaldehyde (4%) and mounted with glycerine. In order to avoid formazan crystallization all measurements were run immediately after fixation. The MTT reaction was evaluated: (a) by light microscopy and (b) by digital image analysis. (a) The percentage of blue formazan-containing cells, either with formazan/endosomes or with formazan/crystals, and of non-reactive cells was determined under phase contrast microscopy (Axioplan microscope, Carl Zeiss, Jena, Germany) according to the criteria employed by Molinari et al. (2005). (b) The amount of formazan formed per unit cell volume was defined as specific MTT reduction activity. Total optical density (TOD) per cell directly quantifies the content of formazan deposited intracellularly after MTT reduction. Specific activity of formazan was calculated by dividing the TOD value by the corresponding cell area (Molinari et al., 2003). Image analysis was performed only on those formazan/endosome-containing cells that had not developed formazan crystals.

2.6. Digital image analysis Image analysis of AMs was performed in keeping with a previous study by our laboratory (Molinari et al., 2000). Cells were observed under an MPM 800 Carl Zeiss microscope (Jena, Germany) using an interference filter (570 nm) and a plan-achromat 40:1/0.75 objective coupled to a DK 7700 SXK camera (Hitachi, Hamburg, Germany). Image intensity was digitized to 8 bits per pixel (256 gray levels) and analyzed with an image analysis system (IBAS-Kontron, Jena, Germany). The software employed allows scanning and evaluation of single cells. It affords data on the total optical density (TOD) of the whole reactive cell. The optical system is set in such a way that once the image is digitized, 1 lm2 of the sample corresponds to an array of 4 · 4 pixels. Thus, the TOD value per cell corresponds to the sum of all optical density values obtained from scanning the whole cell. The TOD of at least 100 NBT or MTT/formazan-containing cells per dish was evaluated (see below under MTT and NBT assays). 2.7. Quantitative measurement of the redox activity (MTT assay) As cells convert MTT to blue formazan by redox activity, the amount of formazan precipitated in the cytoplasm measures the metabolic activity of living cells (Morgan, 1998). MTT assays were performed as previously described (Molinari et al., 2003, 2005). Briefly, at the experimental time-points indicated, media from treated AMs were removed and 1 ml fresh complete growth medium supplemented with 50 ll MTT (4 mg/ml in PBS) was immediately added. As previously demonstrated, incubation in MTT for 45 min was considered adequate to evaluate metabolic changes in these cells (Molinari et al., 2003). At this time

2.8. Quantitative measurement of superoxide anion (NBT assay) The intracellular generation of superoxide anion (O2 ) produced by phagocytes during the respiratory burst was evaluated as previously described (Segal, 1974). This active oxygen specie was evidenced by the amount of a blue formazan precipitate in the cell after NBT reduction. This assay was performed by adding NBT (0.1% in PBS) to AMs for 15 min followed by the addition of 10 ll TPA (100 lg/ml acetone) for 45 min at 37 C. Paraformaldehyde (4%) was added to all cultures to stop the NBT reaction and fix the cells. Alveolar macrophages were evaluated by digital image analysis as described above. Results were expressed as specific activity (TOD/area). 2.9. Statistical analysis All the results were expressed as means ± SE. Statistical evaluation of data was performed using analysis of variance (ANOVA), followed by Tukey post hoc test. A probability value of <0.05 was considered as the minimum level of statistical significance. 3. Results 3.1. Macrophage viability and cell size Fig. 1 shows the effect of As3+ on the viability of macrophages determined by the trypan blue dye exclusion test. A dose-dependent reduction in viability by As3+ was observed as from 2 lM. The LC50 calculated for 24, 48, 72 and 96 h in culture in the presence of As3+ were: 4.81 ± 0.25 lM, 4.71 ± 0.29 lM, 2.81 ± 0.41 lM and

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Fig. 1. Effect of As on cell viability. Alveolar macrophages were incubated with various concentrations of As3+ for 24 h (j), 48 h (e), 72 h (m) and 96 h (s). Viability of treated and non-treated alveolar macrophages was determined by the trypan blue dye exclusion test. Each point of the dose–response curves represents the mean of three separate experiments with 3–4 dishes per condition. Results are expressed as mean ± SE. (a) p < 0.05 for 5 and 10 lM versus controls, for all culture times. (b) p < 0.05 for 0.6, 1.2 and 2.5 lM versus controls, only for 96 h culture time.

2.6 ± 0.33 lM, respectively. Higher concentrations (P10 lM) produced a sustained reduction in cell viability. Cell size measurements were also performed in macrophages cultured with increasing concentrations of As3+ as depicted in Fig. 2. With concentrations of up to 2.5 lM no changes in cell area were detected in AMs treated for 24, 48 72 and 96 h. Higher As3+ concentrations induced a significant reduction in cell volume. 3.2. Macrophage apoptosis Morphological analysis showed that most cells exposed to 5 lM of As3+ underwent apoptosis. The percentage of 24 hs 48 hs 72 hs 96 hs

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Fig. 2. Alveolar macrophages area as a function of As3+ concentration. Cells were cultured for 24, 48, 72 and 96 h. At least a hundred cells per dish from three dishes per condition were measured. Experiments were run in triplicate. Results are expressed as mean ± SE. (a) p < 0.05 for 10 lM versus control (24 h); (b)–(d) p < 0.05 for 5 and 10 lM versus control (48 h, 72 h, 96 h).

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Fig. 3. Percentage of apoptotic cells. Alveolar macrophages were incubated with increasing As3+ concentrations for 24, 48, 72 and 96 h. Apoptotic cells were judged by cellular morphological changes as revealed by Hoechst dye. Bars represent the mean ± SE of three separate experiments. Results represent one of three independent experiments. (a) p < 0.05 for 2.5 lM versus control (96 h) and (b) p < 0.05 for 5 lM versus control (96 h).

apoptotic cells varied from 40% to 80% as a function of culture time (Fig. 3). For lower concentrations apoptotic values remained below 20%. 3.3. Redox state (MTT and NBT assays) 3.3.1. Dynamic process of formazan/endosomes and formazan/crystals formation The metabolic process of MTT incorporation and reduction in AMs is evidenced by the intracellular formation of endosomes full of reduced tetrazolium and the subsequent emergence of formazan crystals at the cell surface. The formation of formazan/endosomes and formazan/ crystals in As3+-treated AMs is shown in Fig. 4. The percentage of formazan/endosome, formazan/crystal containing cells and of non-reactive cells remained unchanged for As3+ concentrations lower than 2 lM. In the presence of increasing As3+ concentrations, the formazan/endosome fraction steadily decreased concomitantly with the rise in non-reactive cells. In addition, cells with formazan/crystals always remained below 20% irrespective of the As3+ concentration employed. The pattern depicted in this figure corresponds to AMs exposed for 24 h. Similar responses were observed for all the time points assayed (data not shown). 3.3.2. Specific activity of redox reactions by image analysis Fig. 5 shows specific MTT reduction activity expressed as total optical density per cell area (TOD/cell area) of endosome containing cells. In keeping with Molinari et al. (2003) specific MTT reduction activity of non-treated macrophages rose as a function of culture–time (24– 96 h). This response was not significantly modified when AMs were cultured with increasing concentrations of As3+.

NBT Specific Activity (TOD/area)

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Fig. 4. Kinetics of MTT formazan exocytosis. Percentage of cells with formazan–endosomes (•), non-reactive cells (j) and cells with formazan– crystals (D) incubated with increasing As3+ concentrations for 24 h. Each point represents the average ± SE of three experiments. Similar responses were observed for assays performed at 48, 72 and 96 h. For fraction (•) (a) p < 0.05 for 5 and 10 lM versus control. For fraction (j) (b) p < 0.05 for 5 and 10 lM versus control.

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Fig. 5. Specific MTT-reduction activity. Effect of increasing As3+ concentration on specific MTT-reduction activity of alveolar macrophages cultured for 24, 48, 72 and 96 h. At least a hundred cells per dish from three dishes per condition were measured. Values are mean ± SE of three experiments. Increasing As3+ concentrations failed to induce statistically significant changes in MTT-reduction activity.

3.3.3. Generation of superoxide anion Non-treated AMs showed an increasing specific NBT reduction activity (TOD/cell area) as a function of culture time probably due to the effect of cell surface adherence during the respiratory burst (Mondal et al., 2000; Robinson and Badwey, 1994). AMs are responsive to low As3+ doses showing a drop in specific activity of about 1.6 fold for 48 h and 72 h and 4.3 fold for 96 h (Fig. 6). For concentrations above 2.5 lM the ability to respond showed few variations regardless of the culture time. 4. Discussion Exposure to arsenic is an environmental risk for the population at large and particularly for those that live in areas with high arsenic levels in drinking water or in air.

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Fig. 6. Specific NBT-reduction activity. Effect of increasing As3+ concentration on specific NBT-reduction activity of alveolar macrophages cultured for 24, 48, 72 and 96 h. At least a hundred cells per dish from three dishes per condition were measured. Values are mean ± SE of three experiments. NBT-reduction activity higher than control: (a) p < 0.05 for 1.2 lM versus control for 24 h culture time. NBT-reduction activity lower than control: (b) p < 0.05 for 10 lM versus control for 48 h culture time, (c) p < 0.05. 1.2, 2.5, 5 and 10 lM versus control for 72 h culture time, (d) p < 0.05. 5 and 10 lM versus control for 96 h culture time.

Determining the degree of toxicity of this agent in target organs or systems is crucial for evaluating the risk of exposure to human health. The analysis of the in vitro response of AMs to different concentrations of As3+ by means of several cell redox metabolism assays show a new insight into the behavior of these cells when challenged by a frequent pollutant of large areas of the world. Alveolar macrophages are very sensitive to changes in environmental conditions, releasing several proinflammatory factors when they are challenged by various agents (Bannai et al., 1991; Issekutz and Issekutz, 1993). The effects of pollutants on the production of lipid peroxides and other harmful molecules that alter cellular functions have been extensively investigated in AMs. In order to evaluate the response of AMs to As3+, representative parameters which are pivotal to the evaluation of cell pathology were assayed. In particular, newly developed end-points can shed light on the oxidative metabolism by means of in vitro analysis of target cells. When culture AMs were submitted to increasing concentrations of As3+, a distinct behaviour was observed. For lower doses (<2 lM) cell homeostasis regulated their response maintaining cell viability, apoptosis and cell size similar to non-treated cells. However, higher doses up to 5 lM reduced cell viability to 50% (LC50) in keeping with previous results reported by Sakurai et al. (1998). Cell size reduction was also observed with a concomitant increase in the percentage of apoptotic cells, indicating a clear cytotoxic effect of As3+ on macrophages (Bortner and Cidlowski, 2002; Hessler et al., 2005). Concentrations above 5 lM produced larger changes in these end-points. It is generally assumed that cytotoxicity depends on the concentration of the toxic agent and the duration of

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exposure. However, in our in vitro model this only holds true for the higher doses (5 and 10 lM). For the lower As3+ concentrations viability only depends on dose, with no significant association with culture time. MTT and NBT are assays in which tetrazolium salts are stoichiometrically reduced to insoluble formazan. However, as previously reported by our laboratory (Molinari et al., 2003, 2005), these assays evaluate different cell processes: MTT measures the metabolic activity of living cells while NBT evaluates the production of superoxide anion during the respiratory burst (Molinari et al., 2000; Muller et al., 1989). The metabolic activity, represented herein by the endoexocytic mechanism, which is one of the central processes of macrophages, was evaluated by means of the MTT assay: cells exhibiting a normal endo-exocytic activity gradually turn formazan–endosomes into abundant formazan crystals protruding on their surfaces. This normal mechanism is modified when macrophages are treated with different agents. Likewise, this process is altered when macrophages are treated with increasing concentrations of As3+. The declining number of formazan endosome-containing cells at the expense of a concomitant increase in dead cells reveals a clear reduction in AMs metabolic activity. This mechanism resembles that already described for macrophages cultured in the presence of different concentrations of genistein (Molinari et al., 2003, 2005). Several studies have suggested that arsenic exerts its toxicity through the generation of reactive oxygen species (Lee et al., 1995; Flora, 1999; Pi et al., 2002; Kannan and Flora, 2006). The NBT test proved to be an adequate tool to monitor oxidative stress, particularly superoxide anion generation, during the respiratory burst (Basu et al., 2001; Pineda-Zavaleta et al., 2004). Arsenite modifies the oxidative state of AMs in vitro. Thus, when macrophages are in an As3+ rich medium, they exhibit a reduction in respiratory burst levels and lose their intrinsic capacity to respond to toxicants. This effect is detrimental to the individual’s health. The cytotoxic effect and the altered release of superoxide anion by As3+ as a proinflammatory mediator may cause immunological disorders. Arsenic-exposed cells cannot optimally release free oxygen radicals and their capacity to eradicate bacteria from organs and blood is partially or completely inhibited (Bishayi and Sengupta, 2003). This may be a key process in severe inflammatory response in chronic arsenicosis patients. Although this study focuses on lung macrophages, the effects of As3+ described herein may also apply to the response of macrophages residing in other organs. The As3+-induced function impairment in macrophages can affect different organs and systems mostly altering the immunosurveillance function of these cells and rendering the organism susceptible to the development of multiple pathologies, e.g. arsenic exposure via drinking water and inhalation is associated with increased risks of lung, skin, bladder and liver neoplasias. Furthermore non-specific chronic inflammation of the upper airways has been demon-

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