Coal fly ash nanoparticles induced cytotoxicity and oxidative DNA damage and apoptosis in Chang liver cells

Coal is the main source widely used for electric generation and industrial applications due to its low cost and abundance of this fuel. Exposure to coal fly ash particulate matter (CFA-PM) is a major health concern in developing countries. The in vitro cytotoxicity, oxidative DNA damage and apoptosis of coal fly ash nanoparticles (CFA-NPs) were determined by lactate dehydrogenase (LDH) enzyme, reactive oxygen species (ROS), 4’-6-diamidino-2-phenylindole (DAPI) and expression of apoptosis associated proteins in cultured Chang liver cell lines. The release of LDH was increased based on dose-dependent and time dependent manner in CFA-NPS treated cells. CFA-NPs induced reactive oxygen species (ROS) with increase in dose concentration. The CFA-NPs treated cells showed severe DNA damage and inhibits the cell viability and leads to apoptosis. The apoptotic proteins showed significant changes, with increase in the level of BAX and decrease in the level of B-cell lymphoma 2 (Bcl-2).The studies showed significant amount of toxicity in CFA-NPs treated Chang liver cell lines.


INTRODUCTION
Coal is the major source widely used for electricity generation and industrial applications; due to its low cost and abundance of this fuel.Combustion of coal generates massive amount of ultra fine particles (UFP) formed by mineral transformation during high temperature combustion process.Exposure to coal fly ash particulate matter (CFA-PM) is a major health concern in developing countries like India and china; it is because coal is the main power source used widely.In United States and Europe; the annual production of coal fly ash (CFA) is about 67.7 million tons and 40.4 thou-sand metric tons (Dwivedi et al., 2012).In India, currently there are 82 coal-fired power plants, which consume about 260 million tonnes of coal to produce 100 million tons of fly ash per year (Dwivedi et al., 2012;Manerikar et al., 2008).The CFA emitted from the thermal power plants are controlled by several approaches; such as electrostatic precipitators, particles scrubbers, fabric filters, and mechanical collectors (Helble et al., 2000;Gilmour et al., 2004).The collection efficiency of these technologies is not effective for particles with aero diameter in the range of 0.1 to 1 μm (Helble et al., 2000;Zhuang et al., 2000;Dwivedi et al., 2012).These UFPs are formed as a result of shoot formation, vaporization of material matter and inorganic ash present in the coals.These vaporized particles nucleate to form large number of nano sized particles, which then grows through condensation and coagulation to form accumulation mode of aerosol (Gilmour et al., 2004).The UFP, once generated, are almost totally dispersed in the atmosphere.Due to its extremely small size, these fractions remains airborne for long period of time; which may be inhaled by the coal miners and highly impacted communities and deposited deep into the lungs epithelial cells by diffusion and entering the body through layers of cells lining alveoli of the lung and causing diseases (Costa and Dreher, 1997;Carbone et al., 2009) such as pneumoconiosis.These nanosized particles are smaller than the size of the cells and the cellular organelles, which allows them to penetrate through the basic biological organs and disturbs their normal functions, causing inflammation of tissues and alters the redox balanced towards oxidation and leads to cell death (Buzea et al., 2007).
The ultrafine particles fraction (D < 100 nm) of CFA has highest impact on human health.The greater the surface area of UFP compared with larger particles with the same chemical compositions makes them more environmentally active with respect to the bio uptake and associated health risks (Gilmour et al., 2004;Oberdoerster et al., 2005;Xia et al., 2006).The emission of particulate matter (PM) from CFA is much higher when compared to any other combustion derived nanoparticles.This emission of CFA-PM into the atmosphere causes serious environmental pollution and serious health hazards; it is due to presence of surface bound hydrocarbons and toxic heavy metals (Kalra et al., 1998;Zhou et al., 2005).The toxic heavy metals present in the CFA are Pb, Cr, Hg, Ni, V, Ba, As, Cd, Mo, Se, Zn (Aitken et al., 1984;Dalmau et al., 1990;Okeson et al., 2003).
Several toxicological studies with airborne PM, CFA and oil fly ash (OFA) has been performed.The nanosized particles provide larger surface area for binding of chemicals including transition metals which enters easily into the cells (Donaldson et al., 2004;Gwinn and Vallyathan, 2006;Duffin et al., 2007;Saquib et al., 2012).The in vitro CFA treatment on rat epithelial cells showed acellular generation of hydroxyl radicals and the iron in the CFA induced cytotoxicity and oxidative DNA damage (Van Maanen et al., 1999).Recent studies demonstrated that the transition elements adsorbed to OFA cause damages to DNA, mitochondrial dysfunction and lipid peroxidation in cultured human epithelial cells (Di Pietro et al., 2009;Di Pietro et al., 2011a).The transition metals present in fine PM; generated as a result of combustion induce oxidative DNA damage and inflammation (Di Pietro et al., 2011b).Cellular exposure to PM may result in pulmonary inflammation, epithelial cell damage, increased epithelial permeability and hyperreactivity (Fabbri et al., 1984).
Earlier studies have not considered the CFA particle characteristics and the toxicity of nanosized coal fly ash particles, in terms of cytotoxicity, ROS production and oxidative DNA damage in Chang liver cells.There was only limited information regarding the toxicity of CFA-NPs (Sambandam et al., 2014;Diawedi et al., 2012).In our previous work, the collection, characterization techniques such as scanning electron microscopy-energy dispersive X-ray (SEM-EDAX) analysis, particle size analyzer (PSA) and transmission electron microscope (TEM) analysis of CFA-NPs were performed (Sambandam et al., 2014).The present study elucidates the role of CFA-NPs induced cellular toxicity, generation of ROS, oxidative DNA damage and changes the ratio of apoptotic proteins in cultured human liver cell line (Chang liver cells).Previous studies have used Chang liver cells for assessing the toxicity (Viluksela et al., 1996;Ahamed et al., 2011).Since Chang liver cells were derived from human liver tissue the results can be extrapolated to mammalian models and humans.Thus, Chang liver cells were chosen for assessing the toxicity of CFA-NPs.

Collection and characterization of CFA-NPs
CFA particles were collected from the electrostatic precipitator (ESP) of coal fired boilers located in the Thermal Power station.The collected CFA-NPs were characterized using SEM-EDAX, PSA and TEM analysis.

Cell culture
Chang liver (human liver) cell lines were purchased from National Centre for Cell Science (NCCS), Pune, India.Cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), D-Glucose and antibiotics at 37°C under the humidified atmosphere containing 5% CO 2 in SLIM CELL incubator.

Determination of lactate dehydrogenase (LDH)
The release of LDH, an indicator of plasma membrane integrity, was measured using LDH release quantification (Fischer scientific, India) Cytotoxicity Assay Kit, in accordance with manufacturer's instructions.The percent of LDH released from the cells was determined by units/mg of protein.Release of cytosolic enzyme, lactate dehydrogenase (LDH), in the cell culture medium is indicative of cell membrane damage.Briefly, Chang liver cells (2 × 10 4 cells/well) were treated with various concentrations of (12.5 to 1000 μg/ml) CFA-NPs and incubated for 24 and 72 h at 37°C.Upon completion of the incubation, 50 μl of the upper medium were collected from each well.The untreated cells were then lysed with a cell lysis solution (10% Triton X 100) for 40 min at room temperature and the lysate collected.LDH activity was measured in a 96-well plate with triplicates of each group at an absorbance of 490 nm and with the reference wavelength at 630 nm.The total protein was estimated using the method reported by Lowry et al. (1951).

Determination of reactive oxygen species (ROS)
Intracellular ROS production was quantified by using fluorescent probe DCFH-DA according to previously described method (Cheng et al., 2004).For deduction of intracellular ROS, Chang liver cells were seeded into 96-well plates and incubated for 24 h at 37°C in CO 2 incubator.DMEM was replaced by PBS with glucose (5.5 mM) and the cells were treated with 1 µM of 2, 7dichlorofluorescindiacetate (CM-H2 DCFH-DA) for 30 min at 37°C in dark.The fluorescence was recorded at 495 nm excitation and 530 nm emissions by a fluorescence microscope (Labomed, India).The generation of ROS was quantified using image J software (Version.2.1).

Nuclear staining with DAPI fluorescent dye
Apoptosis of nuclei was detected by 4'-6-diamidino-2-phenylindole (DAPI) staining assay.DAPI staining was performed as described earlier by Lou et al. (2013).Briefly, the Chang liver cell lines were seeded onto glass slides and treated with various concentrations of CFA-NPs (control, 50, 100, 250, 500 μg/ml) for 24 h.Untreated and treated cells were rinsed with phosphate buffered saline (PBS), fixed with ice-cold 10% trichloroacetic acid, and further washed with cold 70, 80, 90% and absolute ethanol.The cells were permeabilized with Triton-X (10% v/v) and stained with 1 µg/ml DAPI for 3 min.To reduce the background, the stained cells were washed with PBS, cover-slipped with 90% glycerol and observed under a fluorescence microscope (Labomed-Carl zeiss Lens with blue filter Olympus India).The image was quantified using image J software (Version.2.1) for scoring the DNA damage.

RNA extraction and cDNA synthesis
The RNA extraction and cDNA synthesis was performed by following the procedure described by Amirkhiz et al. (2013).The Chang liver cells were cultured and mRNA was synthesized for gene expression studies.Total RNA was extracted using RNX-plus solutions according to the manufacturer's protocol.Purified RNAs were dried and dissolved in 50 μl DEPC-water.Then the cDNAs was transcribed from mRNAs as decribed further.Prior to cDNA synthesis, the extracted mRNA was quantified using nanodrop UV spectrophotometer.The 20 µl reaction mixture contains 3 µg of mRNA and 15 µl of AcccuPower RT PreMix tubes.The mixture tube were loaded in thermal cycler apparatus under the following conditions: cDNA synthesis at 42°C for 60 min and 94°C for 5 min to inactivate RT-ase and terminate the reaction.

Semi-quantitative RT-PCR PCR.
The volume of each reaction was considered to be 20 µl.The Ampliqon master mix that is 2× was used to make reaction mixture as follows: 2 µl of each cDNA sample was added to 10 µl of master mix then 1 µl of each forward and reverse primers (10 mM) of Bcl-2 forward 5'-CTCGTCGCTACCGTCGTGACTTCG-3'; reverse 5'-CAGATGCC GGTTCAGGTACTCAGTC-3'; BAX forward Sense primer: 5'-CATGTTTTCT GACGGCAACTTC-3' reverse primer: 5'-AGGGCCTTGAGCACCAGTTT-3' and GAPDH forward 5'-TTACTCCTTGGAGGC CATGTGGGCC-3' reverse 5'-ACTGCCACCCAGAAGACTGTGGATGG-3' gene for internal control and 6 µl of distilled, deionised, sterile water were added to make the final volume of 20 µl and MgCl 2 concentrationsof 1.5 mM.In the end, one drop of mineral oil was added to the top of each sample to avoid evaporation of samples.Then microtubes were put in the thermal cycler apparatus under the following conditions: initial denaturation at 94°C for 4 min, followed by 35 amplification cycles, each consisting of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, with an additional extension step at the end of the procedure at 72°C for 5 min.All RT-PCR products were visualized by electrophoresis through 2% agarose gel followed by ethidium bromide staining.

Characterization of CFA-NPs
The CFA-NPs characterization and its effects were studied briefly in our previous work (Sambandam et al., 2014).The composition of CFA varies with the origin and the quality of the coal.The morphological analysis of CFA-NPs showed spherical shaped particles.The EDAX analysis revealed the presence of several major elemental constituents such as A1, Fe, Ca, C, S, Si, Mg, Na, Mo and other trace elements such as Zn, Rh, Ar, Cd, K, Ba, Ti, V, Nd, Cr, Mn, Co, Ti, Pb, Se, Sr were also associated with the particle surfaces.The particle size analysis of CFA-NPs showed the uniform distribution of CFA-NPs with an average size about 50 nm.The transmission electron microscopy (TEM) image of CFA-NPs shows the presence of spherical shaped particles with the size range varying from 9 to 24 nm.The average size of the collected CFA particles ranges below the PM 0.1 which was confirmed by PSA and TEM analysis (results not shown).

CFA-NPs effects on LDH
The cytotoxic effect and cell lysis was determined by LDH quantification was shown in Figure 1.The CFA-NPs treated Chang liver cells showed an increase in the level of LDH release in cell supernatant.It reached up to 15.13 and 16.23 U/mg during 24 and 48 h incubation at higher dose concentration.It was observed that, there is a gradual release in the level of LDH in all the CFA-NPs treated concentrations.Thus the release of LDH was observed in time-and dose dependent process.The increased LDH levels and LDH mediated cell death are due to CFA-NPs toxicity.The increase in the concentration of CFA-NPs treated cells showed increase in the release of LDH levels.The cytoplasmic enzyme release was exponential at higher dose treated CFA-NPs concentrations and the release of these enzymes starts even at initial concentration of CFA-NPs treated cells.Diabate et al. (2011) reported that the release of LDH was increased when treated with MAF98 fly ash and decreased against CBl4 NPs.The toxic effect differs, due to chemical nature and place of origin.The release of LDH from the cells were measured in mice; after intratracheal instillation of 25 and 100 µg of ultrafine coal fly ash showed significant increase after 4 h exposure when compared with the control (Gilmour et al., 2004).

CFA-NPs effects on ROS production
Intracellular ROS generation induced by CFA-NPs was The generation of ROS represents an early and sensitive cellular response to PM (Valko et al., 2006).Nel et al. (2006) reported that production of ROS is one of the primary mechanisms of nanoparticles toxicity; it results in oxidative stress, inflammation and damaged cell membranes and DNA.Rapid generation of ROS to an extent may overwhelm the antioxidant defence system and results in oxidation, and therefore destruction of cellular biomolecules, such as DNA, leading to heritable mutations (Risom et al., 2005;Peters et al., 2006).ROS generation and induction of oxidative stress may be due to particle surface or metals associated on the surface (Diabate et al., 2011).Dwivedi et al. (2012) showed that the Fe (III) associated CFA-NPs showed a dose dependent increase in the level of generation of ROS in PBMN cells.Fly ash treatment on rat lung epithelial cells showed increase in the generation of ROS in dose dependent manner; which may be due to insoluble compounds present in the fly ash (Diabate et al., 2011).

DAPI assay
DNA morphological changes in cells undergoing apoptosis (condensation and fragmentation of DNA) can be monitored via DAPI-staining method.DAPI displays enhanced blue fluorescence upon selective binding to double stranded DNA.The nuclear staining assay was used to determine the morphological changes of apoptosis in Chang liver cells.The DAPI staining of CFA-NPs treated cells showed significant induction of nuclear rounding, chromosomal alterations and nuclear membrane shrinkage.In liver cells at concentration 500 μg/ml of CFA-NPs treated cells it increased up to 3.2 fold in the level of nuclear modification, and damage compared to control cells was shown in Figure 3.The DAPI dye stains the nuclear DNA of cells and indicates the morphological changes in the nuclei of treated cells undergoing apoptosis.The changes in the nuclei and DNA damage was observed in liver cells of all tested concentrations.At higher concentration of CFA-NPs treated cells showed up to 3.5 folds of DNA damage when compared to control cells.There was significant damage of DNA observed with increase in concentration of CFA-NPs treated cells.Upadhyay et al. (2003) showed that the PM induced the dose-dependent DNA damage and apoptosis in Alveolar epithelial cells.The PM [benzo[a]pyrine adsorbed on carbon black (CB+BaP)] treatment on cultured macrophages cells showed a time dependent expression and release of TNF-α and also induced cell death by DNA damage (Upadhyay et al., 2003).The blastocytes isolated from rats showed concentration dependent increase in the rate of apoptosis when compared to the control groups on treatment with various increasing concentrations of diesel exhaust particles (Januario et al., 2010).

The effect of CFA-NPs on BAX/Bcl-2 ratio in chang liver cells
The ratio of BAX-to-Bcl-2 protein can determine the susceptibility of the cell apoptosis.Changes in the regulation of mRNA on treatment with CFA-NPs were examined by reverse transcriptase PCR in order to determine cell death by altering the ratio between Bcl-2 and BAX.The Figure 4 showed treatment on Chang liver cells with various concentrations of CFA-NPs showed a significant dose-dependent decrease in the level of expression of Bcl-2 and increase in the level of BAX when compared to the control.A densitometric analysis of the bands showed that CFA-NPs resulted in a dose dependent increase in the BAX/Bcl-2 ratio.The alteration in the expression of BAX and Bcl-2 leads to high changes in the regulation of BAX/Bcl2 which is an important factor in determining the cell's vulnerablity to apoptosis.These two proteins play a prominent role in the regulation of apoptosis.The Bcl-2 mRNA levels were down regulated at 100 and 200 μg/ml of CFA-NPs treated concentrations.The significant upregulation of pro-apototic gene (BAX) expression may result in altering mitochondria permeability.It is a key regulator of mitrochondirial damage in lung cells (Lee et al., 2012).All the tested mRNA gene expressions were compared with internal control gene expression (β-actin).Ahamed et al. (2011) showed that cells exposed to ZnO nanorods showed significant decrease in the level of apoptotic mRNA of Bcl-2 and increase in the level of pro-apoptotic protein; suggesting that these two proteins could be the excellent biomarker to apoptotic response to NPs (Zhu et al., 2007).Previous reports findings demonstrated that metal oxide NPs have greater potential to induce apoptotic cell death (Ahamed et al., 2010;Nel et al., 2006;Gojova et al., 2007).

Conclusion
We demonstrated that CFA-NPs induced cytotoxicity, oxidative stress, DNA damage and apoptosis in Chang liver cells.The toxicity of CFA-NPs depends on size of the particles and chemical composition.CFA-NPs with surface adsorbed heavy metals can act as a cellular and DNA toxicant, capable of inducing oxidative stress, DNA damage and cell death.The expression of pro-apoptotic protein BAX were upregulated, and the antiapototic protein Bcl-2 were down regulated by CFA-NPs.These suggest that the CFA-NPs induced apoptosis in Chang liver cells, which acts as an indicating event in triggering process for carcinogenesis and mutagenesis.Considering the level of toxicty at cellular levels, the population exposed to CFA-NPs are being subjected to major health effects.Primary exposure to CFA-NPs induces oxidative damage in the cells; on prolonged exposure to this CFA-NPs may cause deadly diseases.It is necessary to investigate the potential risk of CFA-NPs to human health by further implementing in vivo toxicity studies to divulge the general mechanisms of organ toxicity.The population affected by these CFA-NPs should be treated with appropriate drugs.The future studies should be focussed on further evaulating the toxicty of CFA-NPs using different challenge studies in cell models.The flavonoids can be suggested as a drug for its protective and curative effects against CFA-NPs toxicity.

Figure 1 .
Figure 1.Cytotoxicity effects of CFA-NPs on the LDH enzyme.Chang liver cells treated with various concentrations (12.5, 25, 50, 100, 250, 500 and 1000 µg/ml) of CFA-NPs showed concentration and time dependent increase in the relase of LDH enzyme.

Figure 2 .
Figure 2. The level of intracellular ROS in CFA-NPS treated Chang liver cells showed concentration dependent increase in the level of ROS (a -Control, b, c, d -12.5, 250 and 1000 µ/ml of CFA-NPs treated cells).

Figure 3 .
Figure 3.The various concentrations of CFA-NPs treated DAPI stained cells showed morphological changes and nuclear condensation in Chang liver cells (A -control, B, C, D, E -fluorescent microscopy images of various concentration of CFA-NPs treated DAPI stained Chang liver cells (50,100, 250 and 500 µg/ml), F-graphical representation of various concentrations of CFA-NPs treated DAPI stained Chang liver cells).

Figure 4 .
Figure 4.The various concentrations of CFA-NPs induced alteretion in expression of BAX and Bcl-2 protein (Lane 1 -control, Lane 2, 3, 4 -various concentrations (50, 100 and 200 µg/ml) of CFA-NPS treated changliver cells expressed alteration in level of BAX and Bcl-2.The expression were compared with the β-actin served as a control.