2.1 Materials
Lead (II) chloride and mercury (II) chloride were purchased from Merck & Co., Inc. (Darmstadt, Germany), MTT assay kit (EZcount ™, HiMedia), Annexin V-FITC, Propidium Iodide, Nonyl Acridine Orange (NAO) (Sigma, Missouri, USA), 2′,7′-Dichlorofluorescin diacetate (DCFDA) (Sigma, Missouri, USA), Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), 1X antibiotic-anti-mycotic solution, 1X Trypsin-EDTA solution (Gibco™, Thermofisher Scientific), JC-1 (5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide), Fura-2AM (AcetoxyMethylester), Mito Tracker TM Red FM (Thermo Fisher Scientific, Massachusetts, USA). All other standard chemicals and reagents were purchased from HiMedia Laboratories, Mumbai, India. Antibodies: PLSCR3 (goat polyclonal) (sc-68123), Caspase 8 (mouse monoclonal) (sc-56070), Caspase 3 (mouse monoclonal) (sc-7272), cytochrome c (mouse monoclonal) (sc-13560), VDAC-1 (mouse monoclonal) (sc-390996) were all purchased from Santa Cruz Biotechnology, Inc, Dallas, USA. Bid (rabbit polyclonal) (2002T) (Cell Signaling Technology, Massachusetts, USA). Secondary antibodies conjugated with Horseradish Peroxidase (HRP): anti-mouse (sc-516102), anti-rabbit (sc-2357), anti-goat (sc-2354) were all purchased from Santa Cruz Biotechnology, Inc, Dallas, USA.
2.2 Buffers
1X Phosphate Buffer Saline (PBS) (pH-7.4) − 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl. STM Buffer- 250 mM Sucrose, 50 mM Tris-HCl (pH-7.4), 5 mM MgCl2, 1X protease inhibitor cocktail (HiMedia Laboratories, Mumbai, India). SOL Buffer- 50 mM Tris-HCl (pH-6.8), 1 mM EDTA, 0.5% TritonX-100, 1X protease inhibitor cocktail. NET Buffer- 20 mM HEPES-NaOH (pH-7.9), 1.5 mM MgCl2, 0.5 M NaCl, 0.2 mM EDTA, 20% Glycerol, 1% TritonX-100, 1X protease inhibitor cocktail. RIPA Buffer- 150 mM NaCl, 5 mM EDTA (pH-8.0), 50 mM Tris-HCl (pH-8.0), 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail.
2.3 Cell culture
Human embryonic kidney (HEK 293) cells from American Type Culture Collection (ATCC) were maintained and grown in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/L glucose, L-Glutamine, 110mg/L sodium pyruvate, supplemented with 10% Fetal Bovine Serum (FBS) and 1X antibiotic-anti-mycotic solution (Gibco™, Thermofisher Scientific). HEK 293 cells were cultured in a 25 cm2 cell culture flask and incubated at 37°C in 5% humidified CO2 incubator. Subculture was done once the cells were 90% confluent using 1X Trypsin-EDTA solution (Himedia).
2.4 Cell viability assay
HEK 293 cells were cultured in a 25 cm2 cell culture flask till 90% confluency at 37°C in a 5% CO2 incubator. The cells were then seeded into each well of 96 wells microculture plate at a 104 cells/ml concentration and allowed to attach and grow for 24 h (~ 80% confluency). After the incubation period, the monolayer of cells was washed with phosphate buffer saline (PBS) pH-7.4, and fresh DMEM media were added to the wells. The cells were added with PbCl2 or HgCl2 dissolved in PBS such that their final concentration varied between 2.5 µM to 100 µM and incubated for different time intervals of 12 h and 24 h. Cells incubated with PBS were used as control. MTT assay was used to assess the cell viability after heavy metal exposure. MTT assay is based on the conversion of soluble yellow-coloured tetrazolium salt, [3‐(4, 5‐dimethyldiazol (‐2‐yl)‐2, 5‐diphenyltetrazolium bromide] (MTT), into an insoluble purple‐coloured formazan by viable cells. MTT assay was performed as per the manufacturer's protocol (EZcount ™, HiMedia). Briefly, after the exposure period, MTT prepared in buffer was added to each well at a final concentration of 1 mg/ml and incubated for 4 h. The insoluble formazan crystals were dissolved using the solubilisation buffer provided in the kit. The absorbance was measured at 570 nm using xMark™ microplate spectrophotometer (BIO-RAD). The results were represented as a percentage of control (untreated) cells.
2.5 Apoptosis assay
HEK 293 cells were grown in each well of 24 wells microculture plates at a concentration of 2x104 cells/ml and allowed to attach and grow for 24 h at 37°C, 5% CO2. After the incubation period, the monolayer of cells was washed with phosphate buffer saline (PBS) pH-7.4, and fresh DMEM media were added to the wells. Apoptosis was induced by adding PbCl2 or HgCl2 dissolved in PBS such that their final concentrations are 2.5 µM, 5 µM, 10 µM, and 20 µM and incubated further for 12 h, and untreated cells were used as control. Post heavy metal exposure, the cells were washed with phosphate buffer saline (PBS) and harvested by adding trypsin solution. The cells were collected by centrifugation at 1000 rpm for 10 min at 4°C. Harvested cells were then washed once with PBS and with 1x binding buffer (10 mM HEPES pH 7.4; 140 mM NaCl, 2.5 mM CaCl2). The washed cells were then resuspended in binding buffer at a concentration of ~ 1x 106 cells/ml. Annexin V-FITC (Sigma, Missouri, USA) staining was done as per the manufacturer's instructions. Briefly, 5 µl of AnnexinV-FITC (50 µg/ml) was added to the cell suspension and incubated in the dark at room temperature for 10 minutes. After washing with 1x binding buffer, 5 µl of propidium iodide (PI) (100 µg/ml) was added and incubated in ice for a further 10 min in the dark. Cell sorting was performed using BD FACS CANTO II Flow cytometer (BD Biosciences) with FITC and PI dual staining. Results were analyzed using BD FACSuite software (BD Biosciences, USA).
2.6 Intracellular ROS measurement
Intracellular ROS content was measured using 2′,7′-Dichlorofluorescin diacetate (DCFDA). DCFDA, a cell-permeable non-fluorescent probe upon intracellular de-esterification and oxidation, converts into a highly fluorescent 2′,7′-dichlorofluorescein (DCF). The fluorescence of DCF is either visualized or measured to evaluate the intracellular ROS content. Briefly, 1x104 cells were seeded onto a 96-well plate and exposed to either Pb2+ or Hg2+ of different concentrations for 12 h. Untreated cells served as the control for the experiment. 10 mM DCFDA solution was prepared by dissolving in DMSO and from which 10 µM working solution of DCFDA was prepared in DMEM. Cells were washed once with DMEM, and to each well, 200 µl of 10 µM DCFDA solution was added and incubated for 30 minutes at 37°C. After the incubation period, the DCFDA solution was removed, and the cells were washed with DMEM and PBS. Finally, 200 µl of PBS was added to each well, and the images were captured using the fluorescence microscope (Zeiss AX10 microscope). The fluorescence intensity values of the cells were immediately measured using a multi-mode microplate reader (Enspire, PerkinElmer, UK) at Excitation/Emission wavelengths of 485 nm/535 nm.
2.7 Mitochondrial membrane potential measurement
JC-1 (5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide) staining was used to understand the changes in the mitochondrial membrane potential. JC-1 (structure in Fig. 1) exhibits potential-dependent accumulation in mitochondria, dictated by a shift in fluorescence emission wavelength from ~ 535 nm (green) to ~ 600 nm (red). Mitochondrial depolarization was identified by a decrease in the red/green fluorescence intensity ratio. Briefly, 1x104 cells were seeded onto a 96-well plate and are exposed to either Pb2+ or Hg2+ of different concentrations for 12 h. The untreated cells served as the control for the experiment. A stock solution of 10 mM JC-1 dye was prepared by dissolving in DMSO and from which 10 µM working solution of JC-1 was prepared in PBS. The cells were washed with PBS, followed by the addition of 200 µl 10 µM JC-1 solution. After the incubation period (30 minutes at 37°C), the JC-1 dye was removed, and 200 µl PBS was added to the wells, and the images were captured using the fluorescence microscope (Zeiss AX10 microscope). Mitochondrial depolarization was quantified by measuring the ratio of red/green fluorescence intensity ratio. The fluorescent intensities were measured at excitation/emission wavelengths of 550 nm/600 nm (red) and 485 nm/535 nm (green) using a multi-mode microplate plate reader (Enspire, PerkinElmer, UK).
2.8 Intracellular calcium measurement
Fura-2AM (AcetoxyMethylester) (Thermo Fisher Scientific), a cell-permeable calcium indicator, was used for intracellular calcium measurement. A stock solution of Fura-2, AM dye was prepared by dissolving in DMSO and from which 5 µM working solution was prepared in 1XPBS. HEK 293 cells were treated with PbCl2 or HgCl2 (2.5 µM, 5 µM, 10 µM, and 20 µM) for 12 h, washed with PBS and then the cells were loaded with 5 µM Fura-2AM in the PBS for 30 min at 37°C in the dark. Excess Fura-2AM was removed by washing twice with PBS. The cells were added with PBS and incubated for another 30 min before measuring the fluorescence using a multi-mode microplate reader (Enspire, PerkinElmer, UK). Fluorescence intensity was measured at an emission wavelength of 510 nm by using a pair of excitation wavelengths at 340 nm and 380 nm and subsequently expressed as the ratio of light excited at 340 nm to that at 380. Changes in the mitochondrial calcium levels were visualized by probing for mitochondria and calcium subsequently. The cells were stained with 0.1 µM Mito Tracker™ red in the PBS for 30 min at 37°C in the dark. Excess stain was removed by washing twice with PBS, followed by staining with Fura-2AM as described above. The images were captured using the fluorescence microscope (Zeiss AX10 microscope).
2.9 Nonyl Acridine Orange (NAO) staining
CL distribution within mitochondrial membrane was estimated using NAO dye. NAO interacts with diacidic phospholipids and forms dimers. Therefore the fluorescence emission shifts from 535 nm (green fluorescence-monomeric NAO) to 640 nm (red fluorescence-dimeric NAO). The heavy metal exposed and control cells were fixed using 1% formaldehyde (in PBS) and incubated with the various concentrations of NAO for 15 min at room temperature as described earlier [32, 34, 37]. The cells were washed twice with cold PBS and resuspended in PBS at a final concentration of 1x104. The fluorescence intensity of the cells was measured using a multi-mode microplate reader (Enspire, Perkin Elmer, UK) with excitation at 490 nm and emission at 640 nm.
2.10 Sub-cellular fractionation
Sub-cellular fractionation was done as described earlier [38]. Cells were harvested at 1000 rpm in STM Buffer. The harvested cells were vortexed at maximum speed for 15 seconds. After a brief incubation period on ice for 30 minutes, the cell suspension was subjected to centrifugation at 800xg for 15 minutes. The supernatant fraction was collected and spun again for additional 10 minutes at 800xg to remove any debris. The collected supernatant was spun at 11,000xg for 10 minutes, and the supernatant (S1) and the pellet fractions (P1) were separated. The collected supernatant (S1) was precipitated in 100% cold acetone at -20°C for an hour and centrifuged at 12,000xg for 5 minutes. The collected pellet fraction upon resuspension in STM buffer comprised the cytosolic fraction. The P1 pellet fraction was resuspended in STM buffer and spun at 11,000 xg for 10 minutes. The pellet (P2) fraction collected was resuspended in SOL buffer and sonicated at 30% amplitude on ice for three cycles of 10 seconds pulse with 30 seconds pause. The sonicated pellet is comprised of the mitochondrial fraction.
2.11 Western Blotting
The protein samples (30 µg) were separated by a 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Sigma). The membrane was probed using primary antibodies for PLSCR3, caspase 8, caspase 3, VDAC-1, β-actin, Bid, and cytochrome c. The probed proteins were then visualized by adding specific secondary HRP-conjugated antibodies followed by Western ECL substrate (Clarity™, Bio-Rad Laboratories). The images were visualized using the VersaDoc TM imaging system (Bio-Rad Laboratories). The band intensities were calculated using Image Lab 5.0 software (Bio-Rad Laboratories).
2.12 Statistical analysis
Statistical analysis of the data was done by One-way ANOVA followed by Dunnett's comparison test using Prism 5.0 Graphpad software (San Diego, CA, USA) unless otherwise mentioned. Significance was acknowledged when probability values p < 0.05 were observed. The analyses corresponding to individual experiments are presented in the respective figure legends.