Methylmercury Induces Alveolar Macrophages Apoptosis

Abstract

Through the use of a scanning electronic microscope, it was found that alveolar macrophages treated with 10 μM of methylmercury for 24 h showed a decrease of surface microvilli, and those treated with 15 μM of methylmercury underwent deformity and subsequent cell death. To investigate their death patterns, DNA was aspirated from alveolar macrophages and analyzed by electrophoresis. It was discovered that the DNA ladder phenomenon became more obvious as the methylmercury increased in concentration. When 5 mM EGTA was used to eliminate calcium ions, a decrease of the ladder phenomenon was observed. Zinc at 1 mM had a similar inhibitory effect. Moreover, an apoptosis peak was observed on flow cytometry analysis of DNA stained with propidium iodide. Alveolar macrophages stained with Hoechst 33342 demonstrated apoptotic bodies induced by methylmercury. The above data indicate that methylmercury can induce a typical apoptosis in alveolar macrophages. Continuing onto the study of the mechanism of apoptosis as induced by methylmercury in alveolar macrophages, it was discovered that methylmercury could increase the intracellular calcium ion concentration and decrease the pH in alveolar macrophages. To find out which endonuclease was responsible for the methylmercury-induced DNA fragmentation of alveolar macrophages, the nuclear proteins of alveolar macrophages was aspirated and tested under different pH values and in conditions with or without calcium ions, and it was discovered that the endonuclease was calcium dependent without relations to pH values.

Mercury is prevalent in the environment as a result of both natural processes and emissions from anthropogenic sources (Lindberg et al. 2007). However, atmospheric deposition from anthropogenic emissions such as coal power plants is frequently the major source of Hg in aquatic systems (Landis and Keeler 2002). After deposition, inorganic Hg is methylated by microbes, then biomagnified in aquatic food webs. Subsequently, the greatest concentrations of methylmercury (MeHg) are found in piscivorous fish and wildlife (Bjornberg et al. 2005). MeHg is the most toxic form of Hg, and nearly all (95% to 99%) Hg in fish is MeHg (Klaper et al. 2006). The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA), as well as mercuric chloride (HgCl₂) and methylmercury chloride (MeHgCl) was investigated on the cytotoxic potentials from alveolar epithelial lung cell lines in vitro (Reichl et al. 2001).

The alveolar macrophage (AM) is the predominant immune effector cell resident in the alveolar spaces and conducting airways, and it is responsible for activating inflammatory responses sufficient to eliminate the interlopers (Peters-Golden 2004). Due to the phagocytosis of macrophages, macrophages are often exposed to a high concentration of toxic metals and that is cytotoxic to macrophages (Graham et al. 1975). As revealed by many studies (Charnley 2006), mercury or inorganic mercury can induce cell apoptosis, but only few studies focus on the effects of methyl mercury (MeHg) to alveolar macrophages. Therefore, this study aims to explore the apoptosis of alveolar macrophages and its mechanisms due to the cytotoxicity of MeHg.

MATERIALS AND METHODS

Materials

Cell culture media and supplements were purchased from Sigma Chemical (USA). Fetal bovine sera were obtained from the Biological Industries (Israel). All tissue culture plastic wares were purchased from NUNC (Denmark). The fluorescent dye BCECF and Hoechst 33342 were purchased from Molecular Probes (USA). Fura 2-acetoxymethylester was purchased from Sigma Chemical. All other reagents were of analytical grade and obtained from Sigma Chemical.

Animals and Preparation of Rat Alveolar Macrophage

We purchased 200- to 250-g male Wistar rat from the Animal Center of the College of Medicine, National Taiwan University (Taipei, Taiwan). The Animal Research Committee of College of Medicine, National Taiwan University, conducted the study in accordance with the guidelines for the care and use of laboratory animals.

Rats were deeply anesthetized via intraperitoneal injection of methohexital sodium (100 mg/kg body body weight [wt]; Eli Lilly, Sydney, Australia), the tracheae were cannulated, and the lungs were perfused with buffered saline solution A (150 mM NaCl, 5 mM KCl, 2.5 mM sodium phosphate, 10 mM HEPES, 0.2 mM EGTA containing 0.1% glucose, pH 7.4) at 10 ml/min via the pulmonary artery (Mikerov et al. 2005). The lungs were excised, degassed for 1 min, and lavaged with six separate 10-ml volumes of saline, each volume being instilled and withdrawn three times. The lavage fluid was centrifuged at 1000 × g at room temperature for 10 min, and the pellet was resuspended in culture medium (RPMI-1640 containing 23.8 mM NaHCO₃, 10% fetal bovine serum [FBS] [v/v], and 1% [w/v] penicillin and streptomycin) at a density of 1.5 × 10⁵ cells/ml. AMs were plated at a density of 0.75 × 10⁵ cells/cm² in 96-well culture plates (Nalge Nunc International, Rochester, NY) and incubated in a humidified atmosphere (pH 7.4, 95% O₂, 5% CO₂) at 37°C for 16 h. Nonadhered cells were removed by washing the wells with culture medium.

Scanning Electron Microscopy (SEM)

For SEM (Ong, Stevens, and Wright 2007), macrophages attached to the surface of the wells were fixed in 1% formaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer for 1 h, washed in the same buffer, postfixed in 1% OsO₄ in the same buffer for 30 min, dehydrated in ethanol, critical-point dried, and coated with gold. Cells were examined with a scanning electron microscope (JOEL JSM-6300).

Estimation of Cytotoxicity

Estimation of cytotoxicity of MeHg on cell viability was determined by means of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Pregel et al. 2007), on cells grown on 96-well plates, after 24 h of treatment. Absorbance at 570 nm was read on a multiwell spectrophotometer (Dynatech MR-5000, CA, USA), with a 655-nm reference filter. In the MTT assay, each MeHg concentration was tested in triplicate to quintuplicate measurements per experiment, and each experiment was carried out at least three times. Percent survival was calculated as the percentage absorbance of the treated wells relative to the untreated wells ± SD (standard deviation of the mean).

Nuclear Condensation Analysis

Cells grown on glass slides were fixed with 4% paraformaldehyde for 15 min at room temperature and washed three times with phosphate-buffered saline (PBS) (Ihara et al. 2007). Fixed cells were permeabilized with 0.5% Triton X-100 in PBS and incubated with 5 μM Hoechst 33342 in PBS for 1 min at room temperature. After washing with PBS, stained nuclei were observed under fluorescent microscope.

DNA Fragmentation Analysis

Fragmentation of DNA was analyzed as described by Ormerod and Collins (1992). After digesting with RNase A (100 μg/ml) at 37°C for 60 min, the soluble DNA prepared from 5 × 10⁶ cells was subjected to electrophoresis in a 1.2% agarose gel and visualized by ethidium bromide staining.

Measurement of Intracellular Ca^{2 +} Concentration [Ca^{2 +}] _i

[Ca2+]_i was measured according to the procedure of Scharff and Foder (1988). Briefly, cells (5 × 10⁶/ml) were incubated for 45 min at 37°C with 2.5 μM fura 2-acetoxymethylester in Krebs’ buffer (145 mM NaCl, 5 mM KCl, 1.2 mM CaCl₂, 1.3 mM MgCl₂, 1.2 mM NaH₂PO₄, 10 mM glucose, and 20 mM HEPES). The cells were then washed twice by centrifugation with Krebs’ buffer. After a 15-min incubation at 37°C to equilibrate them, the cells were centrifuged and resuspended in Krebs’ buffer (2 × 10⁶ cells/ml). The concentration of cytoplasmic free Ca²⁺ was determined by fluorescence measurements at 37°C with continouous stirring in a SLM-AMINCO 8000 spectrofluorometer (USA). Measurements of [Ca²⁺]_i were made from the fluorescence ratio (excitation 340 nm/380 nm, emission 510 nm). The [Ca²⁺]_i could be calculated from a calibration curve using the equation [Ca²⁺]_i = K _d [(R − R _min)/(R _max − R)], where K _d = 224 nM is the apparent dissociation constant for Ca²⁺ and fura 2-AM and R _max and R _min are the maximum and minimum fluorescence ratio obtained by addition of 0.5% Triton X-100 first and further addition of 5 mM EDTA, respectively.

Determination of pH_i

pH_i was measured using the pH-sensitive fluorescent probe 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (Thomas et al. 1979). Cells (2 × 10⁶ cells/ml) were loaded with the probe by incubation with the acetoxymethyl ester form of BCECF (5 μM in RPMI 1640) for 30 min at 37°C. BCECF fluorescence intensity was monitored at excitation wavelengths of 480 nm (peak fluorescence) and 440 nm (isosbestic point) and emission wavelength of 510 nm. The fluorescence intensity ratio (F _peak/F _iso) was calibrated to pH_i using the standard K⁺-nigericin technique (Thomas et al. 1979).

Flow-Cytometric Analysis of DNA Content

Cells were fixed in 50% ethanol and stored at 4°C until analysis, when they were stained with propidium iodide (50 μg/ml), treated with RNase (10 μg/ml), washed in Dulbecco’s phosphate-buffered saline (PBS). The nuclei were analyzed in a FACScan laser flow cytometer (Becton Dickenson, San Jose, CA, USA) as described by Stokke et al. (1993).

Plasmid DNA Digestion Assay

Purified cells were scraped off the plates with a rubber policeman and collected by centrifugation (Schmidt, Browning, and Markham 2007). The nuclear proteins were extracted sequentially by adding 200 μl of cold hypotonic buffer (10 mM NaH₂PO₄, pH 7.0; 10 mM NaF; 5 mM MgCl₂; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride [PMSF]; 1% NP-40; and 10 μg/ml leupeptin) to the pellets on ice for 20 min, homogenized with a Dounce homogenizer for 10 strokes, and centrifuged at 2000 × g for 10 min at 4°C. The supernatant was the cytosolic fraction. The pellets were resuspended in 100 μl of cold hypertonic buffer (10 mM NaH₂PO₄, pH 7.0; 10 mM NaF; 5 mM MgCl₂; 1mM EDTA; 1 mM PMSF; 1% NP-40; 10 μg/ml leupeptin; and 0.5 M NaCl). Cellular debris was removed by centrifugation for 10 min at 4°C. The supernatant fraction containing nuclear protein was stored at –70°C. The nuclear proteins (3 μg) from cells were incubated with 100 ng supercoiled plasmid (pUC13) in 20 μl of 10 mM Tris-HCl and various concentrations of Ca²⁺ and pH. Each sample was incubated at 37°C for 30 min or the desired time period, and then the DNA was electrophoresed in a 1% agarose gel containing ethidium bromide and photographed by ultraviolet light transillumination.

Statistical Analysis

The data are presented as mean ± SD (n = 6–8). Statistical differences in the dose-response studies were evaluated by Dunnett’s multiple comparison test. Student’s t test was used for the comparison between two groups. p value of <.05 was regarded as significant.

RESULTS

The cytotoxicity of MeHg to alveolar macrophages from rats was first examined by scanning electron microscopy to observe the morphology of alveolar macrophages after treated with different concentrations of MeHg for 24 h. The surface of control alveolar macrophages without MeHg treatment was covered with microvilli (Figure 1A ), which was significantly decreased 24 h after the treatment with 10 μM MeHg (Figure 1B ). The treatment with 15 μM MeHg, however, caused cell distortion (Figure 1C ). The cell viability was determined by MTT test, suggesting 10 μM MeHg was not cytotoxic until the concentration of MeHg increased to 15 μM (Figure 2). MeHg (15 μM) caused a significant decrease to 62.9% ± 6.1% of control respectively and MeHg (20 μM) caused a significant decrease to 50.5% ± 8.5% of control respectively.

In addition to the cell morphology, Hoechst33342 was employed to observe the effects of MeHg to alveolar macrophages. The macrophages were first treated with MeHg for 24 h, fixed by formaldehyde, and stained with Hoechst33342. This revealed concentrated apoptotic bodies after 15 μM MeHg treatment for 24 h (Figure 3C ). Propidium iodide (PI) was then used to stain DNA of the macrophages, followed by the analysis of flow cytometer, indicating MeHg possibly caused hypoploid DNA in the macrophages, and following the increase of MeHg concentration, more alveolar macrophage hypoploid DNA appeared (Table 1). MeHg (15 and 20 μM) caused a significant increase of 11.4 ± 2.3 and 15.8 ± 2.7 times of control, respectively. The nucleus of alveolar macrophages was then extracted and electrophoresd. DNA ladders were resolved that became clearer while the macrophages were treated with a higher concentration of MeHg. When 5 mM EGTA was added to remove calcium ions, the intensity of DNA ladders was lowered, and 1 mM Zinc also showed an inhibitive effect (Figure 4). Hypoploid caused by EGTA (5 mM) inhibit 20 μM MeHg remains 32.5% ± 0.5% and hypoploid caused by 1 mM Zinc inhibit 20 μM MeHg remained 26.2% ± 0.53% (Table 1).

In the study on the mechanism of MeHg-induced apoptosis of alveolar macrophages, MeHg was found to cause the elevation of intracellular calcium ions of the macrophages (Figure 5A ) and the decline of pH value (Figure 5B ). To understand which type of endonucleases caused the MeHg-induced DNA fragmentation in alveolar macrophages, nuclear proteins of alveolar macrophages were extracted and analyzed for endonuclease activity under calcium-free condition and different pH values. Figure 6 shows that the endonuclease is calcium dependent rather than pH dependent.

DISCUSSION

The extent to which mercury in the environment poses a risk to human health has been under debate for a number of years (Mergler et al. 2007). Mercury (Hg) is a naturally occurring element that is ubiquitous in the Earth’s core, crust, soils, oceans, and atmosphere. Mercury can exist in elemental, inorganic, and organic forms, but almost all of the global mercury pool is elemental (Lindberg 2007). One of the organic forms, methylmercury, is the form of current toxicologic concern. Mercury can and does change its chemical form and move among different environmental media, with the global amount of mercury remaining the same, but our understanding of its environmental behavior is still evolving. Human activity has made substantial amounts of mercury bioavailable that would not be under natural conditions (Poulain et al. 2007). Mercury is usually released into the environment in its inorganic forms. Mercury vapor can be transported globally, whereas other forms are generally deposited relatively close to their sources. Inorganic mercury deposited in sediment can be converted to organic methylmercury by microorganisms (a conversion that does not take place in the human body) (Karahalil, Rahravi, and Ertas 2005).

Many studies have shown that MeHg causes cytotoxicity in various cells, including rat cerebellar granule cells (Limke, Bearss, and Atchison 2004), human oligodendroglial cells (Issa et al. 2003), human and rat lung cells (Reichl et al. 2001), and human lymphocytes and monocytes (Shenker et al. 1997). Further studies demonstrated that MeHg killed cells by inducing apoptosis (Issa et al. 2003). However, the cytotoxic effect and toxicological mechanism of MeHg on alveolar macrophage have not been established.

MeHg was found in this experiment to cause the decrease of pH value and uprise of calcium ions (Figure 5), which can cause apoptosis. In addition to that, MeHg also resulted in DNA ladders (Figure 4) and hypoploid DNA (Table 1) as well as apoptotic bodies (Figure 3). Based on these experimental results, MeHg could lead to apoptosis, and the endonuclease, which fragmentizes the DNA of alveolar macrophage, is dependent on calcium ions rather than acidity (Figure 6). However, with regards to whether it will at the same time cause necrosis phenomenon will require further studies, because increase in calcium or fall in pH may also cause necrosis. Necrosis is also one of the ways that MeHg can cause death in cells. In our previous research, MeHg can cause neutrophils to have necrosis phenomenon but cannot exclude necrosis phenomenon in this research. MeHg-induced increases in [Ca²⁺]_i may contribute to granule cell necrosis (Marty and Atchison 1998).

Apoptosis or programmed cell death describes a genetically determined elimination of cells (Vauzour et al. 2007). The process is initiated by a death signal that tilts the balance between pro- and antiapoptotic factors. Apoptotic cells undergo well-ordered morphologic and molecular alterations, including cytoskeletal rearrangement, nuclear membrane collapse, chromatin condensation, DNA fragmentation, cell shrinkage, plasma membrane blebbing, and formation of apoptotic bodies. Necrosis, another form of cell death, involves a different manner of cell killing. Necrosis is called ‘accidental’ cell death, differentiating it from programmed death. It is the destructive, disorderly process that occurs when cells are exposed to serious physical or chemical insult or as a result of virus-induced cytolysis. Necrosis begins when the ability of the cell to maintain homeostasis is impaired, leading to an influx of water and extracellular electrolytes. Intracellular organelles, notably mitochondria, swell and the cell ruptures, liberating the cytoplasmic contents. The DNA is fragmented in a random fashion and appears as a smear on electrophoretic gels without the appearance of the DNA fragment laddering characteristic of apoptosis (Abdel-Latif et al. 2006). Apoptosis is important in developmental biology, in remodeling of tissues during repair, and in the progression of some diseases (Jha et al. 2007).

Apoptosis is mediated by a variety of intracellular enzymes, among which are endonucleases that catalyze the internucleosomal fragmentation of DNA, which is one of the hallmarks of apoptotic death (Wan and Tsang 2007; Bortner and Cidlowski 2007; Pedreira et al. 2006). Many scientists believed that apoptosis is an active process, and associated with the synthesis or activation of one or several types of endogenous Ca²⁺/Mg²⁺-dependent endonucleases. When the concentration of zinc ions increases, these endonucleases can be inhibited (Gabriel 1971; Waring, Kos, and Mullbacher 1991; Waring 1990; Waring et al. 1990). Targeted programmed cell death by cytotoxic T cells is also associated with the elevation of calcium ion level. Ionomycin can also increase the level of calcium ions, and further cause cell death (Allbritton et al. 1989). Furthermore, the apoptosis of immature rat thymus gland cells due to glucocorticoids (McConkey et al. 1989) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (McConkey et al. 1988) are also related to the increase of cytosolic calcium ion level. The inhibition of the rise of calcium concentration can prevent DNA fragmentation. From the aforementioned findings, MeHg causes the apoptosis of alveolar macrophages through the elevation of calcium level. To conclude the important findings of this study, first, MeHg can result in the apoptosis of alveolar macrophages. The mechanism is that after MeHg increases intracellular calcium concentration, an endonuclease is activated, leading to DNA fragmentation to form DNA ladders. This phenomenon is different from the cell death caused by other heavy metals.