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. Author manuscript; available in PMC: 2016 May 6.
Published in final edited form as: Environ Toxicol. 2014 Jan 13;30(6):704–711. doi: 10.1002/tox.21948

Chemo-protective Effect of Monoisoamyl 2, 3-Dimercaptosuccinate (MiADMS) on Cytokines Expression in Cadmium Chloride Treated Human Lung Cells

Caroline O Odewumi 1, Shiela Fils-Aime 1, Veera LD Badisa 1, Lekan M Latinwo 1, Michael L Ruden 1, Christopher Ikediobi 2, Ramesh B Badisa 3
PMCID: PMC4096614  NIHMSID: NIHMS553057  PMID: 24420767

Abstract

Cadmium is commercially profitable element, but it causes toxicity in humans and animals leading to diseases in various organs. The main route of cadmium exposure to humans is through inhalation. Lungs respond to insult through secretion of cytokines. In this study, the chemo-protective effect of monoisoamyl 2, 3-dimercaptosuccinate (MiADMS) was evaluated on viability and cytokines expression in CdCl2 treated human lung A549 cells by cytokine array. Cells were treated with 0, 50, 75 and 100 μM CdCl2 alone, 300 μM MiADMS alone, and co-treated with 300 μM MiADMS and 75 μM CdCl2 for 24 h. The viability was measured by crystal violet dye. The level of cytokines in the cells’ lysate and cell culture medium was measured using Ray Biotech's Human Cytokine Array 6 in control cells, 75 μM CdCl2 alone and MiADMS co-treated cells. Array results were validated by ELISA kit. The CdCl2 caused a dose dependent decrease in cell viability, while MiADMS co-treatment resulted in a significant increase in viability of CdCl2 treated cells. Morphology of the cells treated with CdCl2 was destroyed, while MiADMS restored the lost shape in CdCl2 treated cells. In addition, the cells co-treated with MiADMS and CdCl2 showed modulation of cytokines expression in comparison to the CdCl2 alone treated cells. The ELISA results showed the similar pattern of cytokine expression as Human Cytokine Array and validated the array results. These results clearly show the chemo-protective effect of MiADMS and suggest that MiADMS can be used as antidote at moderate dose against CdCl2 toxicity.

Keywords: viability; cadmium; cytokine; monoisoamyl 2, 3-dimercaptosuccinate; lung cells

INTRODUCTION

Cadmium is a non-essential, carcinogenic and immunotoxic metal (IARC, 1993; Goering et al., 1995; Dan et al., 2000). It targets many organs in the mammalian system and mostly accumulates in the lungs, liver and kidney after absorption (Habeebu et al., 1998; Rikans and Yamano, 2000). It is a highly reactive metal affecting many human organs depending on the dose, route, and duration of exposure (Chin and Templeton, 1993; Bridges and Zalups, 2005).

Cytokines are a family of proteins that are necessary for optimal functioning of our defense (immune system) and repair systems. They are secreted by a variety of cells in response to injury, infections or toxin insults. Cytokine antibody arrays were very helpful to study many cytokines expression simultaneously in a single experiment. So far, there were no reports of cadmium toxicity studies with cytokine antibody arrays.

Many agents have been used to reverse the toxicity of metals in various organs of mammalian system. External agents such as metals (zinc and selenium); antioxidants agents, such as N-acetyl cysteine, Picroliv, ascorbic acid and internal agents such as metallothionein were studied against cadmium toxicity in various cell lines or animal models (Forrester et al., 2000; Jihen et al., 2008; Odewumi et al., 2011a). Very few studies were reported on the inflammatory action of cadmium in lungs and chemo protective effect of compounds. Hence, it is of great interest to investigate the effect of cadmium on the expression of cytokines that are produced as an inflammatory response of the lung cells to environmental insult.

The monoisoamyl 2,3-dimercapitosccinic acid (MiADMS) is an ester of 2,3-dimercaptosuccinic acid (DMSA) containing two sulfhydryl (SH) groups. This compound is more hydrophilic and less toxic to the cells than other metal chelators (Aposhian and Aposhian, 1990). In addition, MiADMS is an efficient heavy metal mobilizer/metal chelator in soft tissues which contains more sulfhydryl groups that makes it stronger than bioligands (Andersen, 1999; Smith, 2000). In the present study, we investigated the chemo-protective mechanism of Monoisoamyl 2, 3-Dimercaptosuccinate (MiADMS) on the viability and cytokines expression in cadmium chloride treated human lung A549 cells.

MATERIALS AND METHODS

Maintenance of Cell Line

Human lung (A549, catalog number CCL-185) cell line was purchased from the American Type Culture Collection (ATCC). The supplied frozen cells were cultured according to ATCC instructions. The cells were grown in 10 mL of F12K medium containing 100 units of penicillin per mL, 100 μg of streptomycin per ml, 0.025 μg of Amphotericin B per mL, and 10% Fetal Bovine Serum (FBS) in T-75 cm2 tissue culture flasks at 37ºC in a 5% CO2 incubator (Nuaire Inc, Plymouth, MN, USA).

Synthesis of Monoisoamyl meso-2,3-dimercaptosuccinate (MiADMS)

Monoisoamyl meso-2,3-dimercaptosuccinate (MiADMS) was synthesized in the organic chemistry laboratory (Dr. Ikediobi's Lab) at Florida A&M University. In brief, it was prepared by the controlled esterification of Dimercaptosuccinic acid (DMSA) with the corresponding alcohol in acid medium according to Jones et al., 1992 as mentioned in previous report (Odewumi et al., 2011b).

Treatment of Cells

To investigate the chemo-protective effect of MiADMS on human lung cells treated with cadmium chloride, 1 X 105 cells per well were seeded into 24-well culture plate in 900 μL of medium and incubated overnight in a 5% CO2 incubator at 37ºC for stabilization. Following the stabilization, the cells were treated with different concentrations of cadmium chloride (0, 50, 75 and 100 μM CdCl2) or 300 μM MiADMS and 75 μM CdCl2 (co-treatment) in a final volume of 1 mL in triplicate wells and incubated for 24 h at 37°C in a 5% CO2 incubator. Cells incubated with only culture medium without CdCl2 or MiADMS served as the control cells.

Morphology

At the end of the treatment, morphology of untreated (control), cadmium chloride alone treated, and cadmium chloride and MiADMS co-treated lung cells were observed under a phase-contrast microscope and the images were taken with a Kodak digital camera under the Nikon Diaphot phase contrast microscope with 10x objective.

Cell Viability Test

The viability was evaluated by dye uptake assay according to Badisa et al., 2008. Glutaraldehyde (400 μL of 0.25% to make 0.07% final concentration in the well) was added to each well and incubated for 30 min at room temperature to fix the viable cells. Following this, the plates were rinsed with water to wash off the dead cells and dried under airflow inside the laminar hood for 5-10 min. Crystal violet solution (400 μL of 0.1%) was added to each well and incubated for 15 min, followed by several washes and air dried. Crystal violet dye was solubilized in each well with addition of 1 mL of 0.05 M sodium phosphate solution (monobasic) in 50% ethyl alcohol. The culture plates were read at 540 nm in a plate reader (Bio-Tek EL800). The mean O.D. value of the control was taken as 100% and the rest of the other groups were calculated as a percent of the control.

Preparation of Samples for Cytokine Array

To study the various cytokines expression of the cadmium chloride treated cells and the chemo protective effect of MiADMS on cytokines expression in these cells, approximately 3.9 X 106 cells were plated in T-75 cm2 flasks in complete F12K medium and allowed to stabilize overnight. The cells were then treated with 0, 75 μM CdCl2 alone or co-treated with 300 μM MiADMS and 75 μM CdCl2 in triplicate T-75 cm2 flasks. The flasks were incubated for 24 h at 37°C in a 5% CO2 incubator. After 24 h of incubation, the culture medium was transferred to 15 mL falcon tube and stored at -80°C till it was used for cytokine array analysis. The cells were trypsinized, pooled together and pelleted by centrifuging at 2,500 rpm for 5 min. The cell pellet was suspended in 1 mL of 1x cell lysis buffer (from the Cytokine Array Kit) and lysed by homogenization in a vial under ice for 15 s (3x) using a Polytron homogenizer. The homogenate was transferred to an eppendorff tube and centrifuged at 10,000 rpm for 10 min at 4°C to remove the lysed cell membrane debris. The supernatant was transferred to fresh tube and cell lysate was stored at -80°C for use in the cytokine array analysis.

Protein Estimation

The protein concentration was determined by the BCA method (Pierce company) using a BSA standard. Diluted albumin (BSA) standards and working reagent were prepared according to the kit instructions. Different concentrations of each standard and each unknown sample (25 μL) was pipetted in triplicate into appropriately labeled eppendorff tubes with 500 μL of working reagent and mixed well. The tubes were incubated at 37°C for 30 min and then read at 562 nm in a Beckman spectrophotometer. From the standard curve, the protein concentrations of cell lysate were determined.

Cytokine Array Analysis

The cytokines expression was determined in cell lysate and cell culture medium using Ray Biotech's Human Cytokine Antibody Array 6 kit (catalog # AAH-CYT-6). The array procedure was carried out according to manual instructions with minor changes. The membranes were blocked for 30 min and then hybridized with 400 μg of cell lysate protein or 1 mL of cell culture medium for 2 h at room temperature. The membranes were washed with buffer I (3x) and buffer II (2x) for 5 min. Following this, the membranes were incubated with biotin-conjugated primary antibodies for 2 h at room temperature and then washed. Later, membranes were incubated with the HRP-conjugated streptavidin secondary antibodies at room temperature for 2 h, washed again and developed by incubation with detection buffer for 5 min. The chemiluminiscence of the arrays was detected using Alpha Innotech's FluorChem FC2 machine and density of spots was analyzed by AlphaEaseFC software.

ELISA

Ray Bio ELISA kit employs a specific cytokine (IL1- α) antibody coated on a 96-well plate. Each plate well was incubated with 40 μg of cell lysate protein at 4°C for overnight. The wells were washed with wash buffer (4x) and incubated with biotinylated antibody at room temperature for 2 h. Following this, the unbound biotinylated antibodies were washed away from the plate using wash buffer and the wells were incubated with HRP-conjugated streptavidin. Lastly, the wells were washed again and were developed with soluble HRP substrate 3, 3’, 5, 5’-tetramethylbenzidine (TMB) in buffered solution. The stop solution was added and the color intensity was measured at 450 nm in a plate reader (Bio-Tek, Winooski, VT, USA). Cytokine expression level in treatment groups was compared with control cells cytokine expression (100%).

RESULTS

Chemo- Protective Effect of MiADMS on Morphological Changes in CdCl2 Treated A549 Cells

Fig. 1, shows the morphology of A549 human lung alveolar cells treated with 0, 50, 75, and 100 μM cadmium chloride alone, or 300 μM MiADMS alone or co-treated with 75 μM cadmium chloride and 300 μM MiADMS for 24 h. The morphological changes observed in the CdCl2 treated lung A549 cells were dose dependent. The higher the concentration of CdCl2 in the treatment, the more morphological changes were observed. The control cells (Fig. 1A) exhibited triangular epithelium-like extensions. In the cells treated with 50 μM CdCl2, minor change in morphology was observed with few cells showing shortened extension and display of round circular shape (Fig. 1B). In the cells treated with 75 μM CdCl2, the morphology of many cells changed from rod-like to round with either short to no extensions (Fig. 1C). In the cells treated with 100 μM CdCl2, almost all of the cells lost their triangular shape and extensions becoming very small and circular (Fig. 1D). Treatment with 300 μM MiADMS alone did not alter the morphology of the cells (Fig. 1E). The cotreatment of 75 μM CdCl2 treated cells with 300 μM MiADMS (Fig. 1F) restored the cell extensions that were destroyed by cadmium chloride to the normal (control cells’ shape, Fig. 1A). These results clearly show the chemo protective effect of MiADMS on the morphological changes in CdCl2 treated human lung A549 cells.

Fig. 1. Chemo-protective effect of MiADMS on the morphology of CdCl2 treated cells.

Fig. 1

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The cells were treated with CdCl2 (0, 50, 75 and 100 μM) alone, MiADMS (300 μM) alone, or MiADMS and 75 μM CdCl2 for 24 h. The images were taken with a Kodak digital camera under the Nikon Diaphot phase contrast microscope with 10x objective.

Chemo Protective Effect of MiADMS on the Viability of CdCl2 Treated A549 Cells

The viability of the cells treated with CdCl2 (0, 50, 75, 100 μM) or 300 μM MiADMS alone, or co-treated with 75 μM CdCl2 and 300 μM MiADMS for 24 h was shown in Fig. 2. The viability of the cells treated with CdCl2 alone was dose dependent and was significantly decreased to 65.8 ± 3.4%, 43.3 ± 1.5% and 29.2 ± 1.4% respectively in comparison to control cells (100%, Fig. 2). The cell viability of MiADMS alone was measured to see if it may have any toxic effect on the cells. MiADMS didn’t show significant toxic effect on cell viability. When the viability of the cells treated with 75 μM CdCl2 alone (43.3 ± 1.5%) was compared to the co-treated cells (CdCl2 and MiADMS), the viability was increased to 64.8 ± 1.7%. This result clearly shows the chemo-protective effect of MiADMS on the viability of A549 human lung cells treated with CdCl2.

Fig. 2. Chemo-protective effect of MiADMS on the viability of CdCl2 treated cells.

Fig. 2

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The cells were treated with CdCl2 (0, 50, 75 and 100 μM) alone, MiADMS (300 μM) alone or MiADMS and 75 μM CdCl2 for 24 h. The viability was measured by crystal violet assay. *Significantly different compared to control (p < 0.05). #Significantly different compared to CdCl2 alone (p < 0.05).

Differential Expression of Cytokines in the Cells’ Lysate and Cell Culture Medium of Cells Treated with CdCl2 alone and Co-treated Cells with MiADMS

The various cytokines level in the lysate of the control, 75 μM CdCl2 alone, and co-treated cells with 300 μM MiADMS and 75 μM CdCl2 were measured using Ray Biotech Arrays. In the lysate of CdCl2 alone treated cells, 17 cytokines were significantly up regulated (up regulation is considered as the expression level 30% above control) and 15 cytokines were significantly down regulated (down regulation is considered as the expression level 30% below control) in comparison to control (100%). The up regulated cytokines in the lysate of CdCl2 alone treated cells were eotaxin-2, FGF-6, FGF-7, Fit-3 ligand, fractalkine, GCP-2, GDNF, GM-CSF, IFN-γ, IGF-1, IGFBP-1, IL-1α, IL-1β, IL-1ra, IL-3, IL-4, IL-5, IL-10, IL-13, IL-15, IL-16, and light (Figs. 3A, B). Contrasting results were observed when the level of the cytokines expressed in the lysate of CdCl2 alone treated cells was compared to co-treated cells with MiADMS and CdCl2. All up regulated cytokines in the lysate of CdCl2 alone treated cells were down regulated in the lysate of co-treated cells. The cytokines that were down regulated in the lysate of CdCl2 alone cells were CNTF, EGF, eotaxin, IGFBP-2, IGFBP-4, MCP-3, MCP-4, MDC, MIG, MIP-1δ, MIP-1 α, NT-3, PARC, PDGFBB, and TGF-β3 (Fig. 4). Similar to the up regulated cytokines level result, when the level of the cytokines in the lysate of co-treated cells was compared to the lysate of the CdCl2 alone treated cells, all the down regulated cytokines in the lysate of CdCl2 alone treated cells were increased in the lysate of the co-treated cells (Fig. 4).

Fig. 3. Up-regulated cytokines expression in the cell lysate of 75 μM CdCl2 alone treated cells and their alteration in MiADMS and 75 μM CdCl2 co-treated cells.

Fig. 3

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The cells were treated with CdCl2 (0, 75 μM) alone, or MiADMS and 75 μM CdCl2 for 24 h. The cells were lysed and cytokines expressions in the cell lysate were measured by Ray Biotech Human Cytokine arrays 6. *Significantly different compared to control (p < 0.05). #Significantly different compared to CdCl2 alone (p < 0.05).

Fig. 4.

Fig. 4

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Down-regulated cytokines expression in the cell lysate of 75 μM CdCl2 alone treated cells and their alteration in MiADMS and 75 μM CdCl2 co-treated cells. The cells were treated with CdCl2 (0, 75 μM) alone, or MiADMS and 75 μM CdCl2 for 24 h. The cells were lysed and cytokines expressions in the cell lysate were measured by Ray Biotech Human Cytokine array 6. *Significantly different compared to control (p < 0.05). #Significantly different compared to CdCl2 alone (p < 0.05).

In addition to the cytokine levels measured in the lysate, level of the cytokines in the cell culture medium of the above mentioned treatments were also measured using the same method as lysate. In the CdCl2 alone treated cell culture medium, 13 cytokine levels were significantly up regulated and 4 cytokines were significantly down regulated in comparison to the control cells cell culture medium (Figs. 5, 6). In the cell culture medium of the co-treated cells with MiADMS and CdCl2, 8 (IL-13, leptin, MCP-3, rantes, SCF, SDF-1, TARC, and TGF-β1) cytokine levels out of the 13 cytokines that were up regulated in the CdCl2 alone treatment were decreased and 3 cytokine (GM-CSF, IL-5, and IL-6) levels were increased (Fig. 5). The levels of angiogenin, IGFBP-4, IGF-1 and NAP-2 cytokines in the cell culture medium of CdCl2 alone treated cells were significantly down regulated in comparison to the cell culture medium of the control cells, while they were up regulated in the cell culture medium of co-treated cells with MiADMS and CdCl2 (Fig. 6). These results clearly show the chemo-protective effect of MiADMS on the level of various cytokines expressed in the CdCl2 treated lung cells.

Fig. 5.

Fig. 5

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Up-regulated cytokines expression in the cell culture medium of 75 μM CdCl2 alone treated cells and their alteration in MiADMS and 75 μM CdCl2 co-treated cells. The cells were treated with CdCl2 (0, 75 μM) alone, or MiADMS and 75 μM CdCl2 for 24 h. The cells were lysed and cytokines expressions in the cell lysate were measured by Ray Biotech Human Cytokine array 6. *Significantly different compared to control (p < 0.05). #Significantly different compared to CdCl2 alone (p < 0.05).

Fig. 6.

Fig. 6

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Down-regulated cytokines expression in the cell culture medium of 75 μM CdCl2 alone treated cells and their alteration in MiADMS and 75 μM CdCl2 co-treated cells. The cells were treated with CdCl2 (0, 75 μM) alone, or MiADMS and 75 μM CdCl2 for 24 h. The cells were lysed and cytokines expressions in the cell lysate were measured by Ray Biotech Human Cytokine array 6. *Significantly different compared to control (p < 0.05). #Significantly different compared to CdCl2 alone (p < 0.05).

Validation of Array Results by ELISA

The ELISA assay was used to validate the data of cytokines expression analysis by the array. IL-1α cytokine was one of the highly regulated cytokines in the cytokines expression analysis by the array. In the cell lysate of 75 μM CdCl2 alone treated cells, the cytokine IL-1α expression increased to 281.7 ± 10.4% in comparison to control cells (100%, Fig. 7), while in the cells co-treated with MiADMS and 75 μM CdCl2, the expression of IL-1α was decreased to 141.7 ± 7.6% (Fig. 7). The similar pattern of expression was observed earlier in the array (Fig. 3A). This result clearly validates the data of cytokines expression analysis by the array.

Fig. 7.

Fig. 7

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ELISA confirmation of IL-1α expression in the cell lysate of 75 μM CdCl2 alone treated and MiADMS and 75 μM CdCl2 co-treated cells. The cells were treated with CdCl2 (0, 75 μM) alone, or MiADMS and 75 μM CdCl2 for 24 h. The cells were lysed and cytokine expression in the cell lysate was measured by Ray Biotech Human IL-1α Cytokine ELISA kit. *Significantly different compared to control (p < 0.05). #Significantly different compared to CdCl2 alone (p < 0.05).

DISCUSSION

The prevalent exposure of humans due to the increased level of cadmium in the environment has prompted the testing of various chemicals that can ameliorate the effects of cadmium toxicity and their mode of action in cell lines or animal models. Previously, we reported various studies on cadmium toxicity, and the protective effect of different antioxidant or chelating compounds that reduce the toxic effect of cadmium on the liver cells (Forrester et al., 2000; Badisa et al., 2008; Odewumi et al., 2011a; Odewumi et al., 2011b). Since inhalation is the main route of the cadmium exposure in the humans due to cigarette smoke and the fumes containing cadmium that was released into the environment from various industries. Further, cadmium toxicity has been linked to lung cancer and various pulmonary diseases (Kirschvink et al., 2005; Nawrot et al., 2006). Hence in this report, we studied the chemo protective of MiADMS against cadmium toxicity in the lung cells. Human lung A549 cells were chosen because they display many differentiated features of lung alveolar cells and was used by many researchers in the cadmium toxicity studies (Schwerdtle et al., 2010; Kundu et al., 2011).

In our previous studies with liver cells, we studied the chemo-protective effect of MiADMS against CdCl2 toxicity at the concurrent and 2, 4 or 6 h post-treatment period on the viability and found that concurrent MiADMS treatment showed higher protection on the viability and decreased with increase of post-treatment time (Odewumi et al., 2011b). It was also noticed that the tetrazolium salts react with MiADMS or N-acetyl cysteine and showed the positive color (data not shown) and therefore crystal violet dye was used for the viability assay. Hence, in this report, we studied the chemo-protective effect of MiADMS at the concurrent or same time against CdCl2 toxicity in the lung cells. The viability and morphology of the cells cotreated with cadmium chloride and MiADMS showed an increase in viability as well as restoration of the morphology in comparison to cells treated with 75 μM CdCl2 alone (Figs. 1, 2). These results clearly showed the chemo-protective role of MiADMS against cadmium chloride toxicity in lung cells. The chemoprotective effect of MiADMS is due to its ability to act as a metal chelator by binding to the cadmium or as an antioxidant (Jones et al., 1992, Odewumi et.al. 2011b). This observation is consistent with previous studies which showed MiADMS preventing cadmium and lead induced toxicity in the liver, renal, and brain of rats or in rat liver cells (Tandon et al., 2002; Swaran et al., 2003; Tandon et al., 2003; Odewumi et al., 2011b).

Lungs react to airway insults through inflammation response by secreting inflammatory mediators such as cytokines (Borošková et al., 2007; Kataranovski et al., 2009; Låg et al., 2010). The induction of cytokines expression by cadmium chloride in lung cells and the alteration of cytokine levels in cells co-treated with MiADMS and cadmium chloride were investigated using Ray Biotech antibody membrane arrays. The Human Antibody Array 6 was used to quantify the expression of 60 different cytokines simultaneously in the lysate and medium of the control cells, cells treated with cadmium chloride alone and co- treated with MiADMS and cadmium chloride for 24 h. It was found that in the lysate of cells treated with cadmium chloride alone, level of 22 cytokines that were associated with proliferation like fit-3, FGF-6, FGF-7, IGF-1, IGFBP-1 and inflammatory immune response like eotaxin-2, fractalkine, and anti-inflammatory cytokine like IL-10 were highly up regulated and level of 15 cytokines that were associated with chemotaxis like MCP-3, MCP-4, MDC, MIG, MIP-1α, and MIP-1δ and regulators of growth factors like IGFBP-2, IGFBP-4 and PARC were highly down regulated (Figs. 3, 4). Furthermore, from the above data, we can hypothesize that the high expression of growth factors and anti-inflammatory cytokines in the cadmium chloride alone treated cells was to resist the cadmium chloride toxicity. In addition, chemokines which are responsible for inflammatory response and protection of the cells against tissue damage were highly down regulated in the cadmium alone treated cells which suggest the cells damage due to cadmium toxicity. Interestingly, the up regulated cytokines in the cadmium chloride alone treated cells were down regulated in the cells co-treated with MiADMS and cadmium chloride and vice-versa. This result clearly showed the chemo-protective mechanism of MiADMS which was reflected in the cytokine expression levels in the co-treated cells with MiADMS and CdCl2. The cytokines expressed in the cadmium chloride alone treated cells were similar to the cytokines expressed in chronic treatment reported in earlier study (Feghali et al., 1997). A study done using low concentration of cadmium (3-6 μM) in lung cells reported alterations in inflammation related genes expression and an increased release of IL-6 and MIP-2/CXCL2 from the epithelial cells and MIP-2, IL-1beta and TNF-alpha from alveolar macrophages (Låg et al., 2010). Our present study is the first report to investigate the effect of cadmium and the chemo-preventive effect of MiADMS on various cytokines expression at the protein level by antibody array.

IL-1α is one of the highly regulated cytokine in cadmium chloride alone treated cells and was selected to validate the array results. It plays specific role in the onset of inflammatory process that regulates expression of other cytokines and chemokines in the cells against infection or insult (Barksby et al., 2007; Grivennikov et al., 2006). The result of ELISA experiment validated the result obtained from Cytokine Antibody Array analysis with the same pattern of IL-1α cytokine expression in the cadmium chloride alone and co-treated cells (Fig. 3A, 7).

CONCLUSIONS

In conclusion, our present study showed that MiADMS compound increased the viability and restored the morphology of lung cells treated with cadmium chloride. The cytokine array analysis showed that cadmium chloride altered the expression of various inflammatory cytokines that were important for inflammatory response in the presence of insult and cotreatment with MiADMS reversed the altered cytokines expression. The present study clearly demonstrated the chemo protective effect of MiADMS in the human lung A549 cells treated with cadmium chloride.

Acknowledgments

We acknowledge the financial support of Department of Education for the Grant # DOEHBGI P 031B40108-08 and National Institutes of Health, National Center for Research Resources for the Grant # G12RR03020, G12D007582.

REFERENCES

  1. Agency for Toxic Substances and Disease Registry (ATSDR) Toxicological Profile for cadmium. U.S. Department of Health and Human Services, Public Health Service; Atlanta, GA: 2009. pp. 1–512. [Google Scholar]
  2. Andersen O. Principles and recent developments in chelation treatment of metal intoxication. Chem Rev. 1999;99:2683–2710. doi: 10.1021/cr980453a. [DOI] [PubMed] [Google Scholar]
  3. Aposhian HV, Aposhian MM. Meso-2,3-Dimercaptosuccinic Acid: Chemical, Pharmacological and Toxicological properties of an Orally Effective Metal chelating Agent. Annu Rev Pharmacol Toxicol. 1990;30:279–306. doi: 10.1146/annurev.pa.30.040190.001431. [DOI] [PubMed] [Google Scholar]
  4. Badisa VL, Latinwo LM, Odewumi CO, Ikediobi CO, Badisa RB, Brooks-Walter A, Lambert AT, Nwoga J. Cytotoxicity and stress gene microarray analysis in cadmium- exposed CRL-1439 normal rat liver cells. Int J Mol Med. 2008;22:213–219. [PubMed] [Google Scholar]
  5. Barksby HE, Lea SR, Preshaw PM, Taylor JJ. The expanding family of interleukin 1 cytokines and their role in destructive inflammatory disorders. Clin Exp Immunol. 2007;149:217–225. doi: 10.1111/j.1365-2249.2007.03441.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borošková Z, Dvorožňáková E. The effect of cadmium on the immune behaviour of guinea pigs with experimental ascariasis. J Helminthology. 2007;71(2):139–146. [PubMed] [Google Scholar]
  7. Bridges CC, Zalups RK. Molecular and ionic mimicry and the transport of toxic metals. Toxicol Appl Pharmacol. 2005;204(3):274–308. doi: 10.1016/j.taap.2004.09.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chin TA, Templeton DM. Protective elevations of glutathione and metallothionein in cadmium-exposed mesangial cells. Toxicology. 1993;77:145–156. doi: 10.1016/0300-483x(93)90145-i. [DOI] [PubMed] [Google Scholar]
  9. Dan G, Lall SB, Rao DN. Humoral and cell mediated immune response to cadmium in mice. Drug Chemi Toxicol. 2000;23(2):349–360. doi: 10.1081/dct-100100120. [DOI] [PubMed] [Google Scholar]
  10. Feghali CA, Wright TM. Cytokines in acute and chronic inflammation. Frontiers in Bioscience. 1997;2:12–26. doi: 10.2741/a171. [DOI] [PubMed] [Google Scholar]
  11. Forrester LW, Latinwo LM, Fasanya-Odewumi C, Ikediobi CO, Abazinge MD, Mbuya O, Nwoga J. Comparative studies of cadmium-induced single strand breaks in female and male rats and the ameliorative effect of selenium. Int J Mol Med. 2000;6(4):449–452. doi: 10.3892/ijmm.6.4.449. [DOI] [PubMed] [Google Scholar]
  12. Goering PL, Waalkes MP, Klaassen CD. Goyer RA, Cherias M, editors. Toxicology of cadmium. Toxicology of metals: Biochemical Aspects. Handbook of Experimental Pharmacology. 1995;15(9):189–214. [Google Scholar]
  13. Grivennikov SI, Kuprash DV, Liu ZG, Nedospasov SA. Intracellular signals and events activated by cytokines of the tumor necrosis factor super family: from simple paradigms to complex mechanisms. Int Rev Cytol. 2006;252:129–161. doi: 10.1016/S0074-7696(06)52002-9. [DOI] [PubMed] [Google Scholar]
  14. Habeebu SS, Liu J, Klaassen CD. Cadmium-induced apoptosis in mouse liver. Toxicol Appl Pharmacol. 1998;149:203–209. doi: 10.1006/taap.1997.8334. [DOI] [PubMed] [Google Scholar]
  15. Horiguchi H, Mukaida N, Okamoto S, Teranishi H, Kasuya M, Matsushima K. Cadmium induces interleukin-8 production in human peripheral blood mononuclear cells with the concomitant generation of superoxide radicals. Lymphokine Cytokine Research. 1993;12:421–428. [PubMed] [Google Scholar]
  16. Hyun JS, Satsu H, Shimizu M. Cadmium induces interleukin-8 production via NF- Kappa B activation in the human intestinal epithelial cell, Caco-2. Cytokine. 2007;37:26–34. doi: 10.1016/j.cyto.2007.02.011. [DOI] [PubMed] [Google Scholar]
  17. Ikediobi CO, Badisa VL, Ayuk-Takem LT, Latinwo LM, West J. Response of antioxidant enzymes and redox metabolites to cadmium-induced oxidative stress in CRL- 1439 normal rat liver cells. Int J Mol Med. 2004;14(1):87–92. [PubMed] [Google Scholar]
  18. International Agency for Research on Cancer (IARC) Monographs on the evaluation of the carcinogenic risks to humans, Beryllium, Cadmium, Mercury and exposures in the glass manufacturing industry. IARC Scientific Publications; Lyon, France: 1993. pp. 119–238. [PMC free article] [PubMed] [Google Scholar]
  19. Jihen EH, Imed M, Fatima H, Abdelhamid K. Protective effects of selenium (Se) and zinc (Zn) on cadmium toxicity in the liver and kidney of the rat: Histology and Cd accumulation. Food Chem Toxicol. 2008;46:3522–3527. doi: 10.1016/j.fct.2008.08.037. [DOI] [PubMed] [Google Scholar]
  20. Jones MM, Singh PK, Gale GR, Smith AB, Atkins LM. Cadmium mobilization in vivo by intraperitoneal or oral administration of monoalkyl esters of meso-2,3-dimercaptosuccinic acid in mouse. Pharmacol Toxicol. 1992;70:336–343. doi: 10.1111/j.1600-0773.1992.tb00483.x. [DOI] [PubMed] [Google Scholar]
  21. Kataranovski M, Mirkova I, Belij S, Nikolic M, Zolotarevski L, Ciric D, Kataranovski D. Lungs: Remote inflammatory target of systemic cadmium administration in rats. Environ Toxicol Pharmacol. 2009;28:225–231. doi: 10.1016/j.etap.2009.04.008. [DOI] [PubMed] [Google Scholar]
  22. Kayama F, Yoshida T, Elwell MR, Luster MI. Cadmium-induced renal damage and proinflammatory cytokines: possible role of IL-6 in tubular epithelial cell regeneration. Toxicol Applied Pharmacol. 1995;134:26–34. doi: 10.1006/taap.1995.1165. [DOI] [PubMed] [Google Scholar]
  23. Kirschvink N, Vincke G, Fievez L, Onclinx C, Wirth D, Belleflamme M, Louis R, Cataldo D, Peck MJ, Gustin P. Repeated cadmium nebulizations induce pulmonary MMP-2 and MMP-9 production and emphysema in rats. Toxicology. 2005;211:36–48. doi: 10.1016/j.tox.2005.02.012. [DOI] [PubMed] [Google Scholar]
  24. Kundu S, Sengupta S, Chatterjee S, Mitra S, Bhattacharyya A. Cadmium induces lung inflammation independent of lung cell proliferation: a molecular approach. J Inflammation. 2009;6:19. doi: 10.1186/1476-9255-6-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kundu S, Sengupta S, Bhattacharyya A. EGFR upregulates inflammatory and proliferative responses in human lung adenocarcinoma cell line (A549), induced by lower dose of cadmium chloride. Inhal Toxicol. 2011;23(6):339–348. doi: 10.3109/08958378.2011.572931. [DOI] [PubMed] [Google Scholar]
  26. Låg M, Rodionov D, Ovrevik J, Bakke O, Schwarze PE, Refsnes M. Cadmium- induced inflammatory responses in cells relevant for lung toxicity: Expression and release of cytokines in fibroblasts, epithelial cells and macrophages. Toxicol Lett. 2010;193(3):252–260. doi: 10.1016/j.toxlet.2010.01.015. [DOI] [PubMed] [Google Scholar]
  27. Nawrot T, Plusquin M, Hogervorst J, Roels HA, Celis H, Thijs L, Vangronsveld J, Van Hecke E, Staessen JA. Environmental exposure to cadmium and risk of cancer: A prospective population-based study. Lancet Oncology. 2006;7:119–126. doi: 10.1016/S1470-2045(06)70545-9. [DOI] [PubMed] [Google Scholar]
  28. Odewumi CO, Badisa VL, Le UT, Latinwo LM, Ikediobi CO, Badisa RB, Darling-Reed SF. Protective effects of N-acetylcysteine against cadmium-induced damage in cultured rat normal liver cells. Int J Mol Med. 2011;27(2):243–248. doi: 10.3892/ijmm.2010.564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Odewumi CO, Buggs R, Badisa VL, Latinwo LM, Badisa RB, Ikediobi CO, Darling-Reed SF, Owens MA. Mitigative action of monoisoamyl-2,3-dimercaptosuccinate (MiADMS) against cadmium-induced damage in cultured rat normal liver cells. Toxicol In Vitro. 2011;25(8):1733–1739. doi: 10.1016/j.tiv.2011.08.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rikans LE, Yamano T. Mechanisms of cadmium-mediated acute hepatotoxicity. J Biochem Mol Toxicol. 2000;14:110–117. doi: 10.1002/(sici)1099-0461(2000)14:2<110::aid-jbt7>3.0.co;2-j. [DOI] [PubMed] [Google Scholar]
  31. Schwerdtle T, Ebert F, Thuy C, Richter C, Mullenders LH, Hartwig A. Genotoxicity of soluble and particulate cadmium compounds: impact on oxidative DNA damage and nucleotide excision repair. Chem Res Toxicol. 2010;23(2):432–442. doi: 10.1021/tx900444w. [DOI] [PubMed] [Google Scholar]
  32. Smith DR, Woolard D, Luck ML, Laughlin NK. Succimer and the reduction of tissue lead in juvenile monkeys. Toxicol Appl Pharmacol. 2000;166:230–240. doi: 10.1006/taap.2000.8973. [DOI] [PubMed] [Google Scholar]
  33. Swaran J, Mehta A. Haematological, hepatic and renal alterations after repeated oral and intraperitoneal administration of Monoisoamyl DMSA. II. Changes in female rats. J Appl Toxicol. 2003;23:97–102. doi: 10.1002/jat.890. [DOI] [PubMed] [Google Scholar]
  34. Tandon SK, Singh S, Prasad S, Srivasta S, Siddiqui M. Reversal of lead-induced oxidative stress by chelating agent, antioxidant, or their combination in the rat. Toxicol Lett. 2002;90:61–66. doi: 10.1006/enrs.2002.4386. [DOI] [PubMed] [Google Scholar]
  35. Tandon SK, Singh S, Prasad S, Khandekar K, Dwivedi VK, Chatterjee M, Mathur M. Reversal of lead-induced oxidative stress by chelating agent, antioxidant, or their combination in the rat. Toxicol Lett. 2003;145(3):211–217. doi: 10.1016/s0378-4274(03)00265-0. [DOI] [PubMed] [Google Scholar]

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