Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: Toxicol In Vitro. 2011 Sep 3;25(8):1733–1739. doi: 10.1016/j.tiv.2011.08.013

Mitigative action of mono isoamyl 2, 3-dimercaptosuccinate (MiADMS) against cadmium-induced damage in cultured rat normal liver cells

Caroline O Odewumi a,*, Rebecca Buggs a, Veera LD Badisa a, Lekan M Latinwo a, Ramesh B Badisa b, Christopher O Ikediobi c, Selina F Darling-Reed b, Marcia A Owens d
PMCID: PMC3322667  NIHMSID: NIHMS365264  PMID: 21911053

Abstract

Cadmium is non-essential, carcinogenic and multitarget pollutant in the environment. Monoisoamyl 2, 3-dimercaptosuccinate (MiADMS) is an ester of dimercaptosuccinicacid that acts as an antioxidant and chelator. Therefore, the mitigative action of MiADMS on viability, morphology, antioxidative enzymes and cell cycle were studied on rat liver cells treated with cadmium chloride (CdCl2). The cells were treated with 150 μM CdCl2 alone or cotreated with 300 μM MiADMS (concurrently, 2 h or 4 h post CdCl2 treatment) for 24 h. The viability of cells treated with CdCl2 alone was decreased in comparison to the control cells. Cotreatment with MiADMS resulted in an increase in cell viability in comparison to the CdCl2 alone treated cells. The CdCl2 treatment altered the morphological shape of the cells, while cotreatment with MiADMS restored the shape. Antioxidative enzymes activities were decreased in the cells treated with CdCl2 alone, while MiADMS cotreatment resulted in an increase in enzyme activities. The CdCl2 arrested the cells in S phase of the cell cycle. Cotreatment with MiADMS alleviated cell cycle arrest by shifting to G1 phase. These results clearly show the mitigative action of MiADMS on CdCl2 toxicity and may suggest that MiADMS can be used as an antidote against cadmium.

Keywords: Glutathione peroxidase (GPx); Glutathione reductase (GR); Cadmium; Cell cycle; Monoisoamyl 2, 3-dimercaptosuccinate; liver cells

1. Introduction

Cadmium is one of the naturally occurring elements found in rocks, soil and water. It is used commercially for batteries, pigments for coloring plastics, glass, and paints, stabilizers for processing of PVC polymers, and preparation of special alloys. The manufacture and use of these products give rise to high environmental concentration of and exposure to cadmium through atmospheric emissions, liquid effluents, wastewaters, sludges, and solid wastes (Saffron, 2001). Fossil fuel combustion in classical thermal power plants is responsible for 50% of the total cadmium emitted to the atmosphere. Over 500,000 U.S. employees each year work in an environment that potentially exposes them to cadmium (Wittman R, 2002). Cadmium is an abundant, nonessential element and has been classified as one of the most toxic environmental contaminants that are carcinogenic in humans and experimental animals due to its long biological half life of 10-30 years and range of organ toxicity (IARC, 1993, Jarup et al., 1998).

Cadmium enters the human body via three main routes: inhalation, ingestion and dermal. Smoking of a cigarette results in the inhalation of 0.1-0.2 μg of cadmium (WHO, 1992). For the general population, ingestion of contaminated food products is the major route of cadmium exposure. Consumption of vegetables and grains presents the major source of cadmium exposure due to soils amended with phosphate fertilizers, and cadmium-contaminated sewage sludge and irrigation waters (ATSDR, 1999). On average, a person ingests about 30 μg of cadmium per day from food, but only absorbs 1-3 μg of cadmium in the body (ATSDR, 1999). Dermal exposure of cadmium to humans is mostly from the industrial production of cadmium containing products and their use. It is also due to the use of cadmium nanoparticles in the medical field for imaging technique (Zhang et al., 2010). Exposure of cadmium via dermal exposure caused lesions on the skin (hyperkeratosis, acanthosis and scabbing, alopecia and erythema) (Fasanya-Odewumi et. al., 1998). Upon penetration of cadmium into the body, half of the absorbed cadmium highly accumulates in the liver and kidney, causing injuries of different types to the tissue cells and eventually leading to cancer (Klaassen et al., 1999; Waalkes, 2000, Santos et al., 2005).

The mechanism of cadmium toxicity has yet to be fully understood; however, cadmium is known to induce an oxidative stress (Ikediobi et al., 2004; Santos et al., 2005; Latinwo et al., 2006). The antioxidant enzymes play an important role in protecting against reactive oxygen species or free radicals in the body. Cadmium indirectly inhibits the activities of antioxidant enzymes by depleting the SH-group containing biomolecules resulting in the production of reactive oxygen species such as superoxide ion, hydrogen peroxide, and hydroxyl radical (Wasowicz et al., 2001; Casalino et al., 2002; Eybl et al., 2004; Ikediobi et al., 2004; Latinwo et al., 2006). In addition to its action on antioxidant enzymes, cadmium has been reported to cause retardation of the cell cycle progression in Chinese hamster ovary (CHO) and human lung adenocarcinoma cells (Chao and Yang, 2001; Yang et al., 2004).

Many mitigating substances that act as chelators (N-acetylcysteine (NAC), diallyl tetrasulfide, picroliv) or antioxidants (selenium, Zinc) have been experimentally proven to provide protection against cadmium toxicity (Wang et al., 2009; Murugavel and Pari, 2007; Yadav and Khandelwal, 2006; Obianime and Roberts, 2009; Jihen et al., 2009). Monoisoamly-2,3-dimercaptosuccinate (MiADMS) which contains disulfhydryl groups is a potent lipophilic metal chelator as well as an antioxidant (Tandon et al., 2003). In this study, we investigated the mitigating action of MiADMS on cadmium-induced cytotoxicity in the cultured rat normal liver CRL-1439 cells on the morphology, viability, antioxidative enzyme activities and cell cycle.

2. Materials and methods

2.1. Materials

F12 K medium(1x), penicillin-streptomycin antibiotic solution (100x), Fetal Bovine Serum (FBS), Trypsin-EDTA solution (1x), phosphate buffer saline (PBS), CdCl2, glutaraldehyde, crystal violet, 5’5-Dithiobis(2-nitrobenzoic acid) (DTNB), NADPH, glutathione reductase, oxidized glutathione, sodium azide, Tris-HCl, Propidium Iodide (PI), RNase, meso-2,3-Dimercaptosuccinic acid (DMSA), isopentyl alcohol, methyl chloride, dimethyl sulfoxide (DMSO), hexane, and cyclohexane were purchased from Sigma-Aldrich company (St. Louis, MO, USA). Potassium phosphate, EDTA, chloroform, D-glucose, SDS, sodium chloride, sodium citrate, and ethyl alcohol were purchased from Thomas Scientific Company (Swedesboro, NJ, USA).

2.2. Synthesis of monoisoamyl-2,3-dimercaptosuccinate

Monoisoamyl-2,3-dimercaptosuccinate (MiADMS) was synthesized in organic chemistry laboratory (Dr. Ikediobi's lab) at Florida A&M University. It was prepared by the controlled esterification of DMSA with isopentyl alcohol in acidic medium according to Jones et al (1992). The purified product was characterized and confirmed using spectral and analytical methods like HPLC, Infra-red and GC-MS before using for treatment studies on the liver cells. MiADMS was stored in the refrigerator (desiccator) to avoid oxidation and thermal decomposition.

2.3. Maintenance of cell line

Rat normal liver CRL-1439 epithelial cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured as per the guidelines supplied. The cells were maintained in F12K medium containing 100 units of penicillin/ml, 100 μg of streptomycin/ml, 2 mM L-glutamine and 10% FBS in T-75 cm2 flasks at 37 °C in a 5% CO2 incubator.

2.4. Treatment of cells with CdCl2

The cells at a density of 10 x 104 cells per well were plated in polystyrene, flat bottom 24-well microtiter plates (Corning Costar, Rochester, NY) in F12K medium containing 10% FBS and allowed to stabilize overnight in a CO2 incubator at 37 °C. Following this, the cells were treated with 0, 50 and 150 μM CdCl2 alone or cotreated with 150 μM CdCl2 and 300 μM MiADMS concurrently, 2, 4 and 6 h post CdCl2 treatment in a final volume of 1 ml per well in triplicate wells for 24 h at 37 °C in a 5% CO2 incubator. In post treatments, cells were first treated with CdCl2. All treatments were repeated at least twice. Cells were also treated with DMSO (0.1% final concentration) since MiADMS was dissolved in DMSO.

2.5. Evaluation of cell viability

At the end of the incubation period, the viability of the cells was evaluated by dye uptake assay according to Badisa et al (2003). After 24 h of treatment, glutaraldehyde (400 μl of 0.25%) was added to each well to give 0.07% final concentration and incubated for 30 min at room temperature to fix the viable cells. Following this, plates were rinsed with water to wash off the dead cells and dried under airflow inside of the laminar hood for 5 min. Crystal violet (400 μl of 0.1%) was added to each well, incubated for 15 min, washed and dried. To solubilize the dye, 1 ml of 0.05 M sodium phosphate solution (monobasic) in 50% ethyl alcohol was added to each well and the plates were read at 540 nm in a plate reader (Bio-Tek EL800 Plate Reader).

2.6. Preparation of enzyme extracts

Crude enzyme extracts were prepared as per the method of Ikediobi et al., (2004). Approximately 3.9 × 106 cells per T-75 flask were plated and stabilized overnight. Following this, the cells were treated with 0, 50 and 150 μM CdCl2 alone or cotreated with 150 μM CdCl2 and 300 μM MiADMS concurrently, 2 and 4 h post CdCl2 treatment in a final volume of 10 ml per flask in triplicate for 24 h at 37 °C in a 5% CO2 incubator. All treatments were repeated at least twice. At the end of the incubation, the cells were trypsinized and pelleted by centrifuging at 2,500 rpm for 5 min. The cell pellets were suspended in 1 ml of 50 mM phosphate buffer (pH 7.0) and homogenized with polytron homogenizer in a glass vial on ice for 1 min at intervals of 15 s. The homogenates were then transferred to eppendorff tubes and centrifuged at 3,000 rpm for 10 min at 4 °C to remove the lysed cell membrane debris. The supernatants were transferred to new tubes and stored at 4 °C for enzyme assay studies.

2. 7. Catalase Enzyme assay

Catalase activity was assayed according to the method of Aebi (1984). The assay total volume (450 μl) contained 50 μl of cell extract and 250 μl of 50 mM phosphate buffer (pH 7.0). The reaction at 37 °C was started by the addition of 150 μl of 30 mM H2O2. The decrease in absorbance at 240 nm was monitored for 1 min in a Beckman DU 7500 spectrophotometer. The enzyme activity was calculated using the extinction coefficient of 0.00394 l mmol-1 mm-1 and the unit of enzyme activity was expressed as mmol H2O2 decomposed per minute.

2.8. Glutathione peroxidase (GPx) Enzyme assay

Glutathione peroxidase activity was assayed according to the method of Yang et al., (2001). The reaction mixture (500 μl) contained 3.2 mM GSH, 0.32 mM NADPH, 1 unit glutathione reductase (GR), 1 mM sodium azide and 0.82 mM EDTA in 0.16 M Tris-HCl (pH 7.0). The sodium azide was added to the reaction mixture to inhibit endogenous catalase activity. The reaction mixture was incubated with 50 μl of sample at 37 °C for 5 min, and the reaction was started by addition of H2O2 at a final concentration of 100 μM. The rate of NADPH consumption was monitored at 340 nm for 3 min. One unit of GPx activity was defined as the amount of enzyme required to consume 1 μmol of NADPH/min in the coupled assay.

2.9. Glutathione reductase Enzyme assay

Glutathione reductase activity was assayed according to Smith et al (1988). This assay is based on the following reactions:

NADPH+H++GSSG→reductaseglutathioneNADP++2GSH
GSH+DTNB→GSTNB+TNB

Change in absorbance at 412 nm due to the formation of TNB was measured. The assay volume (2 ml) contained 1 ml of 0.2 M potassium phosphate with 1 mM EDTA buffer (pH 7.5), 500 μl of DTNB in 0.01 M phosphate buffer (pH 7.0), 250 μl water, 100 μl of 2 mM NADPH in water, 50 μl cell extract, and 100 μl of 20 mM oxidized glutathione (GSSG). The increase in absorbance at 412 nm was monitored for 3 min in a Beckman DU 7500 Spectrophotometer at 24 °C. The enzyme activity was calculated using the extinction coefficient of TNB (E412 = 13.6 L mmol-1cm-1) One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 μmole NADPH per min.

2.10. Cell cycle analysis by flow Cytometer

Cell proliferation of CdCl2 treated cells and the mitigative activity of MiADMS were examined in rat normal liver cell line, CRL-1439 using FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) according to Badisa et al (2009). In T-25 flask, 1.3 × 106 cells were plated and incubated overnight. The following day, the cells were treated with 0, 25, 50 and 75 μM CdCl2 alone or cotreated with 300μM MiADMS concurrently in triplicate flasks for 24 h in a 5% CO2 incubator at 37 °C. At the end of incubation, the cells were trypsinized and centrifuged at 2,500 rpm for 10 min at room temperature. Each pellet was re-suspended in 100 μl PBS and made singlet cells by passing three times through 25 G needle. The cells were fixed in pre-cooled 95% ethanol (5 ml added in a drop-wise manner to each tube while vortexing) and incubated at 4 °C at least for 24 h. The cells were then harvested and re-suspended in ethanol (100 μl of 95%). The cell suspensions were transferred into BD falcon tubes and were shielded from light. Staining solution (1 ml) containing final concentrations of 1.25 mg/ml ribonuclease A, 1 mg/ml D-glucose and 50 μg/ml PI was added to each tube in the dark. The tubes were incubated at room temperature for 1 h in the dark with occasional stirring. The distribution of cells in each phase was analyzed within 2 h with the FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). In each sample, a total of 10,000 events from the gated subpopulation were analyzed separately. CELLQuest software was used for acquisition and analysis of the data, and the percentage of cells in each phase was determined with ModFit 3.0 software.

2.11. Statistical Analysis

The viability and enzyme assay results were presented as mean ± standard deviation (SD (n=3)). All cadmium treated cells data were presented in percentage value in comparison to the untreated control cells (100%). The data were analyzed for significance by one-way ANOVA, and then compared by Tukey's multiple comparison tests, using GraphPad Prism Software, version 3.00 (San Diego, CA, USA). The test values p < 0.05 and p < 0.01 were considered significant and highly significant in comparison to the respective untreated control or CdCl2 alone.

3. Results

3.1. Mitigative action of MiADMS on CdCl2-induced cytotoxicity

The toxic effects of CdCl2 at different concentrations on various cell lines have been evaluated earlier (Latinwo et al., 2006; Kaplan et al., 2008). However, little is known about the mitigative action of MiADMS on CdCl2-induced toxicity. Therefore, we studied the mitigative action of 300 μM MiADMS (in 1:2 ratio) on 150 μM CdCl2 induced toxicity after 24 h. In this study, we observed that CdCl2 toxicity was dose dependent. As the concentration of the CdCl2 (50, 100 and 150 μM CdCl2) increased, the cell viability decreased to 86.3 ± 3.32, 56.2 ± 2.55 and 38.1 ± 2.97 % respectively (p < 0. 001, Fig. 1) in comparison to the control (100%). Cells were treated with DMSO (0.1%) alone or with MiADMS alone to examine their independent toxicity on cells. The viability of DMSO (solvent used to dissolve MiADMS) treated cells was 102.9 ± 2.26 %, while the viability of 300 μM MiADMS treated cells was 99.6 ± 4.88 %. Both compounds showed no toxicity on the cells’ viability; therefore the toxicity seen on treated cells was mainly from CdCl2. In order to observe a significant mitigative effect role of MiADMS, the cells were cotreated with 300 μM MiADMS at various time intervals (concurrently, 2 h, 4 h, and 6 h post treatment) and 150 μM CdCl2. The mitigative action of MiADMS on cell viability after acute CdCl2 exposure was time dependent. Administration of MiADMS in cotreated cells mitigates the toxicity produced from CdCl2 by increasing cell viability in comparison to cells treated with 150 μM CdCl2 alone (38.1 ± 2.97%). However, the viability within the MiADMS cotreatment groups was significantly (p < 0. 001) decreased as the time of cotreatment increased {83.4 ± 0.57(concurrent), 80.1 ± 4.8 (2 h), 64.35 ± 3.32% (4 h), and 44.3 ± 0% (6 h)} respectively. MiADMS after 6 h of CdCl2 exposure did not significantly (p > 0. 05) increase cell viability. These results clearly demonstrated the mitigative action of MiADMS on CdCl2 induced cell death.

Fig. 1.

Fig. 1

Open in a new tab

Mitigative action of MiADMS on CdCl2 induced cytotoxicity of rat normal rat liver cells. All values are mean ± standard deviation [S.D. (n = 6)]. Statistically (Tukey's Multiple Comparison Test) different from the control (** p < 0.001) and from the 150 μM cadmium alone (## p < 0.001) were marked.

3.2. Mitigative Action of MiADMS on morphology

The figure 2 shows the mitigating action of MiADMS (concurrent treatment) on the morphology of the normal rat liver cells treated with 150 μM CdCl2 for 24 h. The untreated control cells exhibited triangular shape with extensions (Fig. 2a). The cells treated with MiADMS alone also exhibited similar morphology as the control cells (Fig. 2b) indicating MiADMS did not contribute any morphological alteration to the cells. Cells treated with 150 μM CdCl2 alone lost extensions resulting in forming round shape; an indication that CdCl2 caused morphological alteration to the cells (Fig. 2c). Cotreatment of 300 μM MiADMS with 150 μM CdCl2 treated cells restored the extensions and cell morphology (Fig. 2d). These results clearly demonstrated the mitigating action of MiADMS on the morphology of CdCl2 treated cells.

Fig. 2.

Fig. 2

Open in a new tab

Mitigative action of 150 μM CdCl2 and 300 μM MiADMS on the morphology of the normal rat liver cells. The cells were treated with 150 μM CdCl2 alone or 300 μM MiADMS alone or cotreated with 300 μM MiADMS (concurrent) and 150 μM CdCl2 for 24 h.

3.3. Mitigating action of MiADMS on the status of catalase enzyme

The catalase enzyme activity in CdCl2 treated cells after 24 h and the mitigating action of 300 μM MiADMS is shown in Figure 3. In the 50 μM CdCl2 treated cells, the catalase enzyme activity was increased to 108.2 ± 3.54% in comparison to the control cells (100%). Beyond 50 μM CdCl2, the catalase enzyme activity was decreased in a dose dependent manner. Cells treated with 100 and 150 μM CdCl2 had a significant decrease in catalase activity (52.4 ± 0.42, 17.9 ± 5.37%, P < 0. 001) respectively. The cotreatment of MiADMS (concurrent treatment, 2 h or 4 h post treatment) with the 150 μM CdCl2 treated cells significantly increased (p < 0. 001) the activity of catalase to 71.75± 4.6, 100.45 ± 14.92 and 89.45 ± 15.9% respectively in comparison to CdCl2 alone treated cells (17.9 ± 5.37%). Within the cotreatment groups, the activity of catalase was highest in 2 h post treatment. These results clearly demonstrated the mitigating action of MiADMS and the effect of various time intervals on CdCl2 toxicity of treated cells.

Fig. 3.

Fig. 3

Open in a new tab

Mitigative action of MiADMS on cadmium-induced alteration in catalase activity of rat normal rat liver cells. All values are mean ± S.D. (n = 6). Statistically (Tukey's Multiple Comparison Test) different from the control (** p < 0.001) and from the 150 μM cadmium alone (## p < 0.001) were marked.

3.4. Mitigating action of MiADMS on the status of GPx enzyme

The GPx enzyme activity in CdCl2 treated and cotreated cells with 300 μM MiADMS and CdCl2 after 24 h treatment is shown in Figure 4. In the 50 and 100 μM CdCl2 treated cells, the GPx enzyme activity significantly increased to 121.2 ± 12.8 and 116.8 ± 3.82% respectively (P < 0. 01) in comparison to the untreated control cells (100%). However, in the 150 μM CdCl2 treated cells, the GPx enzyme activity was decreased to 88.1 ± 3.54% (P < 0.001). Cotreatment of MiADMS (concurrent treatment, and 2 h post treatment) with CdCl2 treated cells resulted in a significant increase of GPx activity (p < 0. 001) to 112.4 ± 1.41 and 105.8 ± 7.91 % respectively compared to cells treated with 150 μM CdCl2 alone (88.1 ± 3.54 %). The 4 h post treatment of MiADMS with 150 μM CdCl2 treated cells resulted in significant decrease of GPx enzyme (13.75 ± 4.31%) compared to the 150 μM CdCl2 treated cells (88.1%). These results clearly demonstrated the mitigating action and the effect of various time intervals of MiADMS on cadmium-induced alteration in GPx enzyme activity of CdCl2 treated cells.

Fig. 4.

Fig. 4

Open in a new tab

Mitigative action of MiADMS on cadmium-induced alteration in GPx activity of rat normal rat liver cells. All values are mean ± S.D. (n = 6). Statistically (Tukey's Multiple Comparison Test) different from the control (* p < 0.05) and from the 150 μM cadmium alone (# p < 0.05, ## p < 0.01 and ### p < 0.001) were marked.

3.5. Mitigating action of MiADMS on the status of GR enzyme

Figure 5 shows the mitigating action of 300 μM MiADMS on the GR enzyme activity in CdCl2 treated cells after 24 h treatment. The effect of CdCl2 on the GR enzyme activity was dose dependent; as the CdCl2 concentration increased the GR enzyme activity decreased. In the 50 μM CdCl2 treated cells, the GR enzyme was significantly increased to 185.6 ± 3.18 % (P < 0. 01), while it was significantly decreased to 65.2 ± 6.08 and 12.4 ± 0.28 % (P < 0. 01) respectively in 100 and 150 μM CdCl2 treated cells in comparison to the untreated control cells (100%). In the cotreated cells with MiADMS (concurrent, 2 h and 4 h post treatment) and 150 μM CdCl2, the GR activity was significantly (p < 0. 001) increased to 122.8 ± 9.62, 157.1 ± 9.62 and 78.6 ± 5.16 % respectively, compared to cells treated with 150 μM CdCl2 alone (12.4 ± 0.28 %). Within the cotreatment groups, the GR activity was significantly increased in 2 h post treatment compared to 4 h post treatment. These results clearly demonstrated the mitigating action of MiADMS and the effect of dosing time on CdCl2-induced alteration in GR enzyme activity of CdCl2 treated cells.

Fig. 5.

Fig. 5

Open in a new tab

Mitigative action of MiADMS on cadmium-induced alteration in GR activity of rat normal rat liver cells. All values are mean ± S.D. (n = 6). Statistically (Tukey's Multiple Comparison Test) different from the control (** p < 0.001) and from the 150 μM cadmium alone (## p < 0.001) were marked.

3.6. Mitigating action of MiADMS on cell cycle arrest

The mitigating action of MiADMS on cells treated with CdCl2 was examined in rat normal liver cells. Liver cells were either treated without CdCl2 (0 μM CdCl2) which served as the control, or treated with various CdCl2 concentrations (25, 50 and 75 μM CdCl2), and/or cotreated with all CdCl2 concentrations and 300 μM MiADMS for 24 h followed by flow cytometer analysis. The figure 5 shows the flow analysis data of 10,000 cells (events). In cells treated with cadmium chloride alone, the population of cells in G1 phase decreased compared to all other treatments. However, in cells cotreated with MiADMS at all CdCl2 concentrations, the cell population in G1 phase was similar to control. Significant (p<0.001) cell arrest was observed in S phase of cells treated with CdCl2 alone at all concentrations compared to control and MiADMS cotreated cells. These results indicated that MiADMS mitigated CdCl2 toxicity by inhibiting the cell cycle arrest at all CdCl2 concentrations, shifting it to G1 phase.

4. Discussion

Cadmium induced cell damage has been demonstrated earlier in our laboratory by the increased DNA damage, lipid peroxidation and inhibition of antioxidative enzymes in rat liver cells (Fasanya-Odewumi et al., 1998; Ikediobi et al., 2004; Latinwo et al., 2006). Many compounds that act as chelators [N-acetylcysteine (NAC), diallyl tetrasulfide, picroliv] or antioxidants (selenium, Zinc) have been experimentally proven to provide protection against cadmium toxicity (Wang et al., 2009; Murugavel and Pari, 2007; Yadav and Khandelwal, 2006; Obianime and Roberts, 2009; Jihen et al., 2009). In this present study, we used monoisoamyl-2, 3-dimercaptosuccinate (MiADMS) which is a potent lipophilic metal chelator to investigate the mitigating action against CdCl2-induced cytotoxicity in rat liver cells. The cells were either treated with CdCl2 alone or cotreated with MiADMS (concurrently, 2, 4 and 6 h post-treatment of cadmium) in order to study the mitigative action and time of treatment of MiADMS against CdCl2-induced cytotoxicity. The cytotoxicity was evaluated by a simple and reproducible crystal violet dye staining assay (Badisa et al., 2003). From our previous results, it was shown that the LD50 of cadmium chloride in CRL 1439 rat liver cells was 125 μM, but in the present study, we used 150 μM CdCl2 for better visualization of the MiADMS mitigative action. The CdCl2 at 50, 100 and 150 μM treatments had decreased the viability to 86.3 ± 3.32, 56.2 ± 2.55 and 38.1 ± 2.97% respectively (p < 0. 001, Fig. 1) in comparison to control (100%). The MiADMS provided similar protection in concurrent cotreated and 2 h post-treated cells (83.4 ± 0.57, 80.1 ± 4.8% Fig 1) compared to the 4 or 6 h post-treated with less protection against cadmium chloride toxicity on viability study ( 64.35 ± 3.32 and 44.3 ± 0% Fig. 1). However, the 4 or 6 h post-treated with MiADMS increased the cell viability to 64.35 ± 3.32, and 44.3 ± 0% respectively when compared to the 150 μM CdCl2 treated cells (38.1 ± 2.97%). The time of MiADMS treatment on cadmium chloride treated cells was shown to affect its mitigative action on the cell viability in this study. As the delay in time of post-treatment with MiADMS increased, the cell viability was decreased. This may be due to the toxicity caused by the cadmium prior to the MiADMS treatment. In this case, MiADMS may be acting as a chelator by immobilizing cadmium and also as an antioxidant by removing the free radicals induced by cadmium from the site of deleterious oxidation reactions.

The change in cell morphology observed in 150 μM CdCl2 treated cells resulted in the loss of membrane integrity that eventually led to cell death (Fig. 2c). This result was consistent with our previous work (Ikediobi et al., 2004) and others (Newairy et al., 2007; Murugavel and Pari 2007; Yadav and Khandelwal 2006) which revealed that cadmium induced lipid peroxidation. Addition of MiADMS to the treatment, restored cell morphology (Fig. 2d), and this observation was consistent with earlier studies which indicated that MiADMS prevented cadmium or lead-induced toxicity in liver, renal, and brain in rats (Saxena and Flora, 2004; Tandon et al, 2002 and 2003).

Cadmium exposure in vitro or in vivo altered several stress response genes involved in the production of antioxidant enzymes which play an important role in the cellular defense mechanism against oxidative stress. Catalase and GR enzymes activity were decreased drastically in the cells treated with 150 μM CdCl2 alone, while the enzymes activity were increased in cotreatment with MiADMS (concurrent, 2 or 4 h post treatment) groups (Figs. 3 and 5). The result shown here signifies the time response of MiADMS among the cotreated cells. Cotreatment beyond 2 h resulted in the decrease of these enzymes activity. A similar pattern of result was observed in viability and enzymes data. More cells observed in 2 h MiADMS post treatment resulted in more enzymes activity and the fewer cells observed in 4 h MiADMS post-treatment resulted in the less enzymes activity. This result is consistent with earlier reports where MiADMS reversed lead or cadmium induced oxidative stress in rats (Tandon et al., 2002, 2003).

Glutathione peroxidase is another enzyme involved in protection of cells during oxidative stress. In this study, GPx activity was increased in cells treated with 50 and 100 μM CdCl2 alone compared to the control cells (Fig. 4) which indicates the cells’ response. In cells treated with 150 μM CdCl2 alone, the GPx activity level decreased compared to other CdCl2 alone treated and control cells which indicate an intensive ROS generation by CdCl2. Cotreatment of 150 μM CdCl2 with 300 μM MiADMS restored the GPx activity in concurrent and 2 h post treatment but not in 4 h post treatment. Based on these results the mitigative action of MiADMS might be due to the reaction of sulfhydryl groups of MiADMS with metals or free oxygen radicals or with highly reactive byproducts of lipid peroxidation produced by CdCl2. These results clearly show that the time of dosing plays an important role in mitigating action of MiADMS on the status of GPx activity.

Cell cycle analysis showed interesting results when mitigative action of MiADMS was examined in rat normal liver cells treated with different CdCl2 concentrations. Compared to the control cells, cells treated with CdCl2 alone had an increase in cell population in S phase which indicates the cell arrest with CdCl2 treatment. The cell arrest was shifted to G1/G0 phase when cotreated with MiADMS which indicates a mitigative action of MiADMS by inhibition of cell arrest (Fig. 6). This result was supported by previous report (Gaofeng et al., 2008) which showed the cell arrest in S phase of lung embryo fibroblast cells treated with a low dose of cadmium (0.9 – 1.5 μM). In another study with CdCl2, the cell arrest was observed in G1 in kidney distal epithelial cells (Bjerregaard, 2007). Hence, cadmium's effect on cell cycle depends on the type of cells, cadmium concentration and duration of the treatment. Inhibition of cells treated with CdCl2 in S phase may be as a result of its interference with genes associated with cell growth as reported in earlier studies (Joseph et al., 2001; Spruill et al., 2002). The shifting of the arrest to G0/G1 from S phase by MiADMS may be as a result of MiADMS inhibiting the signal-regulated kinase (ERK) activation responsible for cell proliferation as seen in study with NAC (Kim et al., 2005).

Fig. 6.

Fig. 6

Open in a new tab

Mitigative action of MiADMS on cadmium-induced alteration in cell cycle phases of rat normal rat liver cells. The cells were treated with 0, 25, 50, 75 μM CdCl2 alone or cotreated with 300 μM MiADMS (concurrent) for 24 h. All values are mean ± S.D. (n = 6). Statistically (Tukey's Multiple Comparison Test) different from the control (** p < 0.01) and from the respective cadmium alone (# p < 0.05 and ## p < 0.01) were marked.

In conclusion, this study clearly showed that cotreatment with MiADMS in CdCl2 treated rat normal liver cells (CRL-1439) mitigates the cadmium toxicity by increased cell viability, restored cells morphology, inhibition of cell arrest and increased antioxidative enzymes.

Acknowledgements

This work was supported by grants from the National Institutes of Health, National Center for Research Resources (NIH NCRR 003020-21) and DOE-HBGI P 031B40108-08.

Footnotes

Conflicts of interest

The authors would like to state that there are no conflicts of interest related to this work.

References

  1. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–126. doi: 10.1016/s0076-6879(84)05016-3. [DOI] [PubMed] [Google Scholar]
  2. 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: 1999. pp. 242–259. [Google Scholar]
  3. Badisa RB, Darling-Reed SF, Joseph P, Cooperwood S, Latinwo LM, Goodman CB. Selective cytotoxic activities of two novel synthetic drugs on human breast carcinoma MCF-7 cells. Anticancer Res. 2009;29:2993–2996. [PMC free article] [PubMed] [Google Scholar]
  4. Badisa RB, Tzakou O, Couladis M, Pilarinou E. Cytotoxic activities of some Greek Labiatae herbs. Phytother Res. 2003;17:472–476. doi: 10.1002/ptr.1175. [DOI] [PubMed] [Google Scholar]
  5. Bjerregaard H. Effects of cadmium on differentiation and cell progression in cultured Xenopus kidney distal epithelial (A6) cells. Altern Lab Anim. 2007;35:343–348. doi: 10.1177/026119290703500302. [DOI] [PubMed] [Google Scholar]
  6. Casalino E, Calzaretti G, Sblano C, Landriscina C. Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicol. 2002;179:37–50. doi: 10.1016/s0300-483x(02)00245-7. [DOI] [PubMed] [Google Scholar]
  7. Chao JI, Yang JL. Opposite roles of ERK and p38 mitogen-activated proteinkinases in cadmium-induced genotoxicity and mitotic arrest. Chem Res Toxicol. 2001;14:1193–1202. doi: 10.1021/tx010041o. [DOI] [PubMed] [Google Scholar]
  8. Eybl V, Kotyzova D, Bludovska M. The effect of curcumin on cadmium-induced oxidative damage and trace elements level in the liver of rats and mice. Toxicol Lett. 2004;151:79–85. doi: 10.1016/j.toxlet.2004.02.019. [DOI] [PubMed] [Google Scholar]
  9. Fasanya-Odewumi C, Latinwo LM, Ikediobi CO, Gilliard L, Sponholtz G, Nwoga J, Stino F, Hamilton N, Erdos GW. The genotoxicity and cytotoxicity of dermally-administered cadmium: effects of dermal cadmium administration. Int. J. Mol. Med. 1998;1:1001–1006. doi: 10.3892/ijmm.1.6.1001. [DOI] [PubMed] [Google Scholar]
  10. Gaofeng J, Lei X, Shizhen S, Changcai Z, Qing W, Ling Z, Lei W. Effects of long-term low –dose cadmium exposure on genomic DNA Methylation in human embryo lung fibroblast cells. Toxicol. 2008;244:49–55. doi: 10.1016/j.tox.2007.10.028. [DOI] [PubMed] [Google Scholar]
  11. Ikediobi CO, Badisa VDL, 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]
  12. 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]
  13. Jarup L, Berglund M, Elinder CG, Nordberg G, Vahter M. Health effects of cadmium exposure – a review of the literature and a risk estimate. Scand J Work Environ Health. 1998;24:1–51. [PubMed] [Google Scholar]
  14. Jihen H, Imed M, Fatima H, Abdelhamid K. Protective effects of selenium (Se) and zinc (Zn) on cadmium (Cd) toxicity in the liver of the rat: effects on the oxidative stress. Ecotoxicol Environ Saf. 2009;72(5):1559–1564. doi: 10.1016/j.ecoenv.2008.12.006. [DOI] [PubMed] [Google Scholar]
  15. Jones M, Singh P, Gale G, Smith A, Atkins L. Cadmium Mobilization in Vitro by Intraperitoneal or Oral Administration of Monoalkyl Esters of meso-2,3-Dimercaptosuccinic Acid in the Mouse. Pharmacol Toxicol. 1992;70:336–343. doi: 10.1111/j.1600-0773.1992.tb00483.x. [DOI] [PubMed] [Google Scholar]
  16. Joseph P, Muchnok TK, Klishis ML, Roberts JR, Antonini JM, Whong WZ, Ong T. Cadmium-induced cell transformation and tumorigenesis are associated with transcriptional activation of c-fos, c-jun, and c-myc proto-oncogenes: role of cellular calcium and reactive oxygen species. Toxicol Sci. 2001;61(2):295–303. doi: 10.1093/toxsci/61.2.295. [DOI] [PubMed] [Google Scholar]
  17. Kaplan M, Atakan IH, Aydoğdu N, Aktoz T, Ozpuyan F, Seren G, Tokuç B, Inci O. Influence of N-acetylcysteine on renal toxicity of cadmium in rats. Pediatr Nephrol. 2008;23:233–241. doi: 10.1007/s00467-007-0696-7. [DOI] [PubMed] [Google Scholar]
  18. Kim J, Kim SH, Johnson VJ, Sharma RP. Extracellular signal-regulated kinase –signaling-dependent G2/M arrest and cell death in murine macrophages by cadmium. Environ Toxicol Chem. 2005;24(12):3069–3077. doi: 10.1897/04-503r3.1. [DOI] [PubMed] [Google Scholar]
  19. Klaassen CD, Lui J, Choudhuri S. Metallothionein: an intercellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol. 1999;39:267–294. doi: 10.1146/annurev.pharmtox.39.1.267. [DOI] [PubMed] [Google Scholar]
  20. Latinwo LM, Badisa VDL, Ikediobi CO, Odewumi CO, Lambert AT, Badisa RB. Effect of cadmium-induced oxidative stress on antioxidative enzymes in mitochondria and cytoplasm of CRL-1439 rat liver cells. Int. J. Mol. Med. 2006;18(3):477–481. [PubMed] [Google Scholar]
  21. Murugavel P, Pari L. Effects of diallyl tetrasulfide on cadmium-induced oxidative damage in the liver of rats. Hum exp toxicol. 2007;26(6):527–534. doi: 10.1177/0960327107073810. [DOI] [PubMed] [Google Scholar]
  22. Newairy AA, El-Sharaky AS, Badreldeen MM, Eweda SM, Shewita SA. The hepatoprotective effects of selenium against cadmium toxicity in rats. Toxicol. 2007;242(1-3):23–30. doi: 10.1016/j.tox.2007.09.001. [DOI] [PubMed] [Google Scholar]
  23. Obianime AW, Roberts II. Antioxidants, cadmium-induced toxicity, serum biochemical and the histological abnormalities of the kidney and testes of the male Wistar rats. Niger J Physiol Sci. 2009;24(2):177–185. doi: 10.4314/njps.v24i2.52910. [DOI] [PubMed] [Google Scholar]
  24. Saffron L. Australia cuts cadmium in food. Environ Health perspect. 2001;109:A158. doi: 10.1289/ehp.109-a158b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Santos EJ, Herrmann AB, Ribeiro AS, Curtius AJ. Determination of Cd in biological samples by flame AAS following on-line preconcentration by complexation with O, O-diethyldithiophosphate and solid phase extraction with Amberlite XAD-4. Talanta. 2005;65(2):593–597. doi: 10.1016/j.talanta.2004.07.025. [DOI] [PubMed] [Google Scholar]
  26. Saxena G, Flora SJS. Lead-Induced oxidative stress and hematological alterations and their response to combined administration of calcium disodium EDTA with a thiol chelator in rats. J Biochem Molecular Toxicol. 2004;18:221–233. doi: 10.1002/jbt.20027. [DOI] [PubMed] [Google Scholar]
  27. Smith IK, Vierhell TL, Thorne CA. Assay of glutathione reductase in crude tissue homogenates using 5,5’-dithiobis(2-nitrobenzoic acid). Anal Biochem. 1988;175:408–413. doi: 10.1016/0003-2697(88)90564-7. [DOI] [PubMed] [Google Scholar]
  28. Spruill MD, Song B, Whong WZ, Ong T. Proto-oncogene amplification and overexpression in cadmium-induced cell transformation. J Toxicol Environ Health A. 2002;65(24):2131–244. doi: 10.1080/00984100290071379. [DOI] [PubMed] [Google Scholar]
  29. Tandon SK, Singh S, Prasad S, Srivastava 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]
  30. Tandon SK, Singh S, Prasad S, Srivastava S, Khandekar K, Dwivedi VK, Chatterjee M, Mathur N. Reversal of Lead-Induced Oxidative Stress by Chelating Agent, Antioxidant, or Their Combination in the Rat. Toxicol Lett. 2003;145:211–217. doi: 10.1016/s0378-4274(03)00265-0. [DOI] [PubMed] [Google Scholar]
  31. Thornton I. Sources and pathways of cadmium in the environment. In: Nordberg GF, Herver RFM, Alessio L, editors. Cadmium in the Human Environment: Toxicity and Carcinogenicity. International Agency for Research on Cancer; Lyon: 1992. pp. 149–162. [Google Scholar]
  32. Waalkes MP. Cadmium carcinogenesis in review. J. Inorg. Biochem. 2000;79:241–244. doi: 10.1016/s0162-0134(00)00009-x. [DOI] [PubMed] [Google Scholar]
  33. Wang L, Chen D, Cao J, Liu Z. Protective effect of N-acetylcysteine on experimental chronic cadmium nephrotoxicity in immature female rats. Hum Exp Toxicol. 2009;28(4):221–229. doi: 10.1177/0960327109102365. [DOI] [PubMed] [Google Scholar]
  34. Wasowicz W, Gromadzinska J, Rydzynski K. Blood concentration of essential trace elements and heavy metals in workers exposed to lead and cadmium. Int J Occup Med Environ Health. 2001;14:223–229. [PubMed] [Google Scholar]
  35. Wittman R, Hu H. Cadmium exposure and nephropathy in a 28-year-old female metals worker. Environ Health Perspect. 2002;110:1261–1266. doi: 10.1289/ehp.021101261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. World Health Organization (WHO) Environmental Health Criteria 134 - Cadmium International Programme on Chemical Safety (IPCS) Monograph. 1992.
  37. Yaday N, Khandelwal S. Effect of Picroliv on cadmium-induced hepatic and renal damage in the rat. Hum Exp Toxicol. 2006;25(10):581–591. doi: 10.1177/096032706072455. [DOI] [PubMed] [Google Scholar]
  38. Yang PM, Chiu SJ, Lin KA, Lin LY. Effect of cadmium on cell cycle progression in Chinese hamster ovary cells. Chem Biol Interact. 2004;149:125–136. doi: 10.1016/j.cbi.2004.08.001. [DOI] [PubMed] [Google Scholar]
  39. Yang Y, Cheng JZ, Singhaal SS, Saini M, Pandya U, Awasthi S, Awaathi YC. Role of glutathione S-transferases in protection against lipid peroxidation: over expression of hGSTA2-2 in K562 cells protects against hydrogen peroxide induced apoptosis and inhibits JNK and caspase 3 activation. J Biol Chem. 2001;276:19220–19230. doi: 10.1074/jbc.M100551200. [DOI] [PubMed] [Google Scholar]
  40. Zhang HL, Li YQ, Wang JH, Li XN, Lin S, Zhao YD, Luo QM. Special method to prepare quantum dot probes with reduced cytotoxicity and increased optical property. J Biomed Opt. 2010;15(1):015001. doi: 10.1117/1.3291999. [DOI] [PubMed] [Google Scholar]

RESOURCES