Cadmium Chloride Induces DNA

Damage and Apoptosis of Human Liver

Carcinoma Cells via Oxidative Stress
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Citation Skipper, Anthony, Jennifer N. Sims, Clement G. Yedjou, and Paul B.

Tchounwou. 2016. “Cadmium Chloride Induces DNA Damage and
Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress.”
International Journal of Environmental Research and Public Health
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Article
Cadmium Chloride Induces DNA Damage and
Apoptosis of Human Liver Carcinoma Cells via
Oxidative Stress
Anthony Skipper 1 , Jennifer N. Sims 2 , Clement G. Yedjou 1 and Paul B. Tchounwou 1, *
Received: 7 September 2015; Accepted: 25 November 2015; Published: 2 January 2016
Academic Editor: William Toscano
1 Molecular Toxicology Research Laboratory, NIH-Center for Environmental Health, College of Science,
Engineering and Technology, Jackson State University, 1400 Lynch Street, Box 18540, Jackson, MS 39217,
USA; [email protected] (A.S.); [email protected] (C.G.Y.)
2 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School,
330 Bookline Avenue, Boston, MA 02215, USA; [email protected]
* Correspondence: [email protected]; Tel.: +1-601-979-0777; Fax: +1-601-979-0570

Abstract: Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals.
Many documented studies have shown that cadmium produces various genotoxic effects such as
DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and
several forms of cancer are attributed to overexposure to cadmium. Although there have been
numerous studies examining the effects of cadmium in animal models and a few case studies
involving communities where cadmium contamination has occurred, its molecular mechanisms
of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays
a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver
carcinoma (HepG2 ) cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid
hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was
tested by the means of alkaline single cell gel electrophoresis (Comet) assay. Cell apoptosis was
measured by flow cytometry assessment (Annexin-V/PI assay). The result of MTT assay indicated
that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing
a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant
(p < 0.05) increase of hydroperoxide production, specifically at the highest concentration tested. Data
obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2
cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05)
was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these
in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage,
and programmed cell death in human liver carcinoma (HepG2 ) cells.

Keywords: cadmium chloride; HepG2 cells; cytotoxicity; oxidative stress; DNA damage; apoptosis

1. Introduction
Cadmium is one of the naturally occurring heavy metals. However, it is often used in industry,
and exerts toxic human health effects. It is classified as a human carcinogen by the International Agency
for Research on Cancer and belongs to the group I carcinogens [1]. Cadmium intoxication in humans
usually occurs through inhalation (cigarette smoke) and ingestion (consumption of contaminated water
and food). Acute intoxication of cadmium may lead to liver, lung, and testis damages [2] while chronic
intoxication may result in obstruction of pulmonary disease, disturbance of metabolism, disregulation
of blood pressure, obstruction of kidney function, structure of bones and immune system [1,3,4].

Int. J. Environ. Res. Public Health 2016, 13, 88; doi:10.3390/ijerph13010088 www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2016, 13, 88 2 of 10

Although the mechanism of cadmium induced toxicity is poorly understood, it has been reported that
cadmium causes damage to cells through the generation of reactive oxygen species [5]. Studies using
two-dimensional gel electrophoresis have shown that several stress response systems are expressed in
response to cadmium exposure, including those for heat shock, oxidative stress, stringent response,
cold shock, and Son of Sevenless (SOS) [6–8]. In vivo studies have shown that cadmium modulates
male reproduction in a mice model at a concentration of 1 mg/kg body weight [9]. However, cadmium
is a weak mutagen when compared with other carcinogenic metals [10]. Previous reports revealed that
cadmium affects signal transduction pathways; inducing inositol polyphosphate formation, increasing
cytosolic free calcium levels in various cell types [11], and blocking calcium channels [12,13]. A line
of evidence shows that cadmium alters antioxidant defense mechanisms and increases generation of
reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide [14–16]. Hence,
the present investigation was designed to prove that oxidative stress plays a key role in cadmium
chloride-induced DNA damage and apoptosis of human liver carcinoma (HepG2 ) cells.

2. Materials and Methods

2.1. Chemicals and Test Media

DMEM-F12 containing 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and
1200 mg/L sodium bicarbonate, was supplied by American Type Culture Collection-ATCC (Manassas,
VA, USA), and was used as the growth medium. Costar Company (Cambridge, MA, USA) was the
source for obtaining the ninety six-well plates, while Sigma Chemical Company (St. Louis, MO, USA)
provided reagents such as fetal bovine serum (FBS), penicillin G and streptomycin, phosphate buffered
saline (PBS), G418 and MTT assay kit.

2.2. Cell/Tissue Culture

Human liver carcinoma (HepG2) cells obtained from ATCC were conserved in liquid nitrogen.
During experimentation their containers/vials were gently shaken for 2 min in a water bath at
37 ˝ C, and the content of each vial was transferred to a 25 cm2 tissue culture flask in which
DMEM-F12 medium containing 10% (v/v) fetal bovine serum (FBS), 0.4 mg/mL G418, and 1% (w/v)
penicillin/streptomycin, was added up to a total volume of 10 mL. The cells were examined using an
inverted tissue culture microscope, and incubated for 24 h in a humidified 5% CO2 incubator at 37 ˝ C.
The Trypan blue exclusion test (Life Technologies, Carlsbad, CA, USA) was performed to determine
the cell viability based on the number of live cells counted, using a hemocytometer.

2.3. Assessment of Cell Viability by MTT Assay

HepG2 cells were cultured in enriched DMEM-F12 medium as described above, and 180 µL
aliquots cell suspension (5 ˆ 105 /mL) were pipetted and placed 96-well polystyrene tissue culture
plates, followed by the addition of 20 µL aliquots of stock solutions to make-up six replicates of
final cadmium chloride concentrations of 1, 2, 3, 4, and 5 µg/mL. Control cells received 20 µL of
distilled water. After chemical treatment, HepG2 cells were incubated for 48 h in a humidified 5%
CO2 incubator at 37 ˝ C. After incubation, the MTT assay for cell viability was performed as previously
described [17,18].

2.4. Assessment of Oxidative Stress by Lipid Hydroperoxide Assay

To test the hypothesis that oxidative stress plays a key role in cadmium chloride-induced toxicity to
HepG2 cells, lipid hydroperoxide assay (Calbiochem-Novabiochem, San Diego, CA, USA) was performed and
the production level of hydroperoxide content was estimated in untreated and treated cells. This experiment
was conducted according to the manufacturer’s instructions (Calbiochem-Novabiochem) [19,20], with
few modifications as previously described in our laboratory [21–23].
Int. J. Environ. Res. Public Health 2016, 13, 88 3 of 10

2.5. Assessment of DNA Damage by Comet Assay

The Comet assay was carried out by the method previously described by Collins and his
collaborators [24,25] with some modifications [26]. Briefly, 1 ˆ 106 cells/mL were treated with either
media or cadmium chloride (0, 1, 2, 3, 4, and 5 µg/mL) respectively and incubated in a 5% CO2 at 37 ˝ C
for 48 h. After incubation, the cells were centrifuged, washed with cold PBS, and 1 ˆ 105 cells/mL
counted from the pool of untreated and treated cells were used for performing the comet assay as
previously described [26,27].

2.6. Assessment of Apoptosis by Annexin V/PI Assay

Annexin V FITC/PI assay was performed as described previously [28] to evaluate the apoptotic
effect of cadmium chloride to human liver carcinoma (HepG2 ) cells. Briefly, 2 mL of cells
(1 ˆ 106 cells/mL) were added to each well of 6 plates and treated with 1, 2, 3, 4, and 5 µg/mL
of cadmium chloride for 48 h. Control well plates were also made without cadmium chloride. After
48 h of incubation, 1 ˆ 106 cells/mL were counted and washed in PBS, re-suspended in binding buffer
(10 mM Hepes/NaOH pH 7.4, 140 mM NaCl, 25mM CaCl2 ), and stained with FITC-conjugated annexin
V (Pharmingen, Becton Dickinson Co., San Diego, CA, USA). After staining, the cells were incubated
for 15 min in the dark at room temperature. Cells were re-washed with binding buffer and analyzed
by flow cytometry (FACS Calibur; Becton-Dickinson) using CellQuest software.

3. Results and Discussion

3.1. Inhibition of Cell Viability by Cadmium Chloride

The results of the cytotoxicity of cadmium chloride to human liver carcinoma (HepG2 ) cells are
presented in Figure 1. Data obtained from this assay demonstrated a strong concentration-response
relationship with regard to the cytotoxic of cadmium chloride in HepG2 cells. As indicated in this
Figure, there was a gradual decrease in the viability of HepG2 cells, with increasing concentrations
of cadmium chloride that resulted in a 48 hr-LD50 of 3.6 µg/mL. HepG2 cells exposed to cadmium
chloride concentrations of 1, 2, 3, 4, and 5 µg/mL showed significant mortalities (p < 0.05) compared
to control cells, according to ANOVA Dunnett’s test (Figure 1). Cadmium, a potent toxic metal, has a
high potential to accumulate in the environment. It is very harmful to the environment and to human
beings. The toxicity of cadmium as an industrial pollutant and a food contaminant, and as one of
the major components in cigarette smoke is well established [29]. Cadmium can cause a number of
lesions in many organs, such as the kidney, the testis, the lung, the liver, the brain, the bone, the blood
system [30]. However, the mechanism of toxicity of cadmium is not yet clear. In the present study, we
showed that cadmium chloride at the concentrations of 1, 2, 3, 4, and 5 µg/mL significant caused cell
mortalities (p < 0.05) compared to the control. Consistent with our report, recent study indicated that
cadmium chloride is highly cytotoxic to human lens epithelial cells [31]. In vitro studies also indicated
that cadmium induces cytotoxic effects and free radical-dependent DNA damage in bacteria [32,33],
which causes single-strand DNA damage and disrupts the synthesis of nucleic acids and proteins [34].
Published studies reported that acute ingestion of cadmium causes health adverse effects such as
abdominal pain, burning sensation, nausea, vomiting, salivation, muscle cramps, vertigo, shock, loss of
consciousness and convulsions usually appear within 15 to 30 min [35]. Acute cadmium ingestion can
also cause gastrointestinal tract erosion, pulmonary, hepatic or renal injury and coma, depending on the
route of poisoning [35,36]. Cadmium can cause a number of lesions in many organs, such as the kidney,
the testis, the lung, the liver, the brain, the bone, the blood system [30]. Human exposure to cadmium
is possible through a number of several sources including employment in primary metal industries,
eating contaminated food, smoking cigarettes, and working in cadmium-contaminated work places,
with smoking being a major contributor [1,37]. Other sources of cadmium include emissions from
industrial activities, including mining, smelting, and manufacturing of batteries, pigments, stabilizers,
Int. J. Environ. Res. Public Health 2016, 13, 88 4 of 10

and alloys [38]. Cadmium is also present in trace amounts in certain foods such as leafy vegetables,
potatoes, grains and seeds, liver and kidney, and crustaceans and mollusks [39].

Figure 1. Cytotoxic effect of cadmium chloride on human liver carcinoma (HepG2 ) cells. Cells were
cultured with increasing concentrations (1, 2, 3, 4, and 5 µg/mL) of cadmium chloride for 48 h as
indicated in the Materials and Methods. Cell viability was determined based on the MTT assay. Each
point represents a mean ˘ SD of three experiments with six replicates per concentration. * Significantly
different (p < 0.05) from the control, according to the Dunnett’s test.

3.2. Induction of Lipid Hydroperoxide by Cadmium Chloride

To test whether oxidative stress plays a key role in cadmium chloride-induced toxicity to HepG2
cells, we performed a lipid hydroperoxide assay. As shown in Figure 2, our results indicated
that the treatment of HepG2 cells with cadmium chloride resulted in a significant increase of lipid
hydroperoxide levels, a major degradation product of unsaturated phospholipids and glycolipids.
Similar results have been recorded by Bashandy and collaborators [40]. Our result is also in agreement
with a previous report indicating that cadmium induces formation of superoxide ion and hydrogen
peroxide in HeLa human tumor cells and bovine aorta endothelial cells [41]. Consistent with our
result, previous reports also indicated that lead and cadmium-induced tissue damages through
oxidative stress [42]. Cadmium stimulates the formation of metallothioneins and reactive oxygen
species, thus causing oxidative damage to erythrocytes and various tissues resulting in loss of the
membrane functions [43]. Another study indicated that chronic exposure to cadmium increased lipid
peroxidation and caused inhibition of superoxide dismutase (SOD) activity showing oxidative damage
in liver, kidney, and testes [44]. A previous scientific report showed that treatment with cadmium
causes a more pronounced reduction in intracellular glutathione levels and a significantly higher free
radical accumulation in progenitors [45]. Other reports indicated that cadmium decreases intracellular
glutathione content and activities of cellular antioxidant enzymes, superoxide dismutase, peroxidase
and catalase, leading to the accumulation of ROS and an increase in intracellular oxidative stress in
cadmium exposed CRL-1439 normal rat liver kidney cells [46,47].
Int. J. Environ. Res. Public Health 2016, 13, 88 5 of 10

Figure 2. Cadmium chloride-induced lipid peroxidation in human liver carcinoma (HepG2 ) cells. Cells
were incubated for 48 h with increasing concentrations of cadmium chloride (1, 2, 3, 4, and 5 µg/mL).
Lipid hydroperoxide levels were determined as described in Materials and Methods. * Significantly
different (p < 0.05) from the control, according to the Dunnett’s test. Data are representative of three
independent experiments.

3.3. Induction of DNA Damage by Cadmium Chloride

To evaluate the ability of cadmium to trigger genotoxic damage in hepatocytes, HepG2 cells were
treated with different concentrations of cadmium chloride, in the range 0–5 µg/mL for 48 h, and the
degree of DNA damage was quantified by the means of LAI’s Comet Assay Analysis System software
(Loates Associates, Inc. Westminster, MD, USA) after staining with SYBR Green. Our results showed
that cadmium chloride at the concentrations of 1, 2, 3, 4 and 5 µg/mL causes DNA single strand
breaks in HepG2 cells and there is a gradual concentration-response relationship. The representative
comet assay images of control and cadmium chloride-treated HepG2 cells are presented in Figure 3.
This Figure showed a significant increase in the percentage of DNA damage and length of comet tail in
a concentration-dependent manner in human liver carcinoma cells exposed to cadmium chloride.
The percentages of DNA cleavage were as follows: (1.7 ˘ 1.2)%, (2.9 ˘ 1.6)%, (21.9 ˘ 5.5)%, (37.5 ˘ 4.7)%,
(52.4 ˘ 11.4)%, and (66.5 ˘ 18.3)% for 0, 1, 2, 3, 4 and 5 µg/mL of cadmium chloride, respectively
(Figure 4). Similar trend was observed in the mean length of comet tail. The tail lengths of DNA
comets were all longer in cadmium chloride-treated cells compared to the control (p < 0.05) (Figure 4).
Several possible mechanisms may be involved in the induction of DNA damages. With the
currently available data, cadmium chloride seems to have direct genotoxic activity to HepG2 cells at
concentrations relevant tested. Studies have provided evidence that reactive oxygen species (ROS) are
involved in DNA damage induced by carcinogenic metal ion [48]. It has been shown that cadmium
enhances lipid peroxidation in cultured cells and animals [49,50]. Low concentrations of cadmium
binds to proteins, decreases DNA repair [51], activates protein degradation, up-regulates cytokines and
proto-oncogenes such as c-fos, c-jun, and c-myc [52], and induces expression of several genes including
metallothioneins [53], heme oxygenases, glutathione transferases, heat-shock proteins, acute-phase
reactants, and DNA polymerase β [54].
Int. J. Environ. Res. Public Health 2016, 13, 88 6 of 10

Figure 3. Cadmium chloride induced DNA damage in human liver carcinoma (HepG2 ) cells. Cells
were treated for 48 hours with medium (A) supplemented with solvent or 1 (B); 2 (C); 3 (D); 4 (E); and
5 (F) µg/mL cadmium chloride. Representative comet images were analyzed using LAI’s Comet Assay
Analysis System software (Loates Associates, Inc. Westminster, MD, USA).

Figure 4. Comet assay of HepG2 cells showing the percentages of DNA cleavage (Left) and comet tail
lengths (Right) as a function of cadmium chloride concentrations. Each point represents mean ˘ SD of
three independent experiments. * Significantly different (p < 0.05) from the control, according to the
Dunnett’s test.

3.4. Induction of Apoptosis by Cadmium Chloride

To gain insight into the mechanism of cadmium chloride-induced apoptosis, we examined
the modulation of phosphatidylserine externalization in HepG2 cells. We observed that cadmium
chloride induces cellular apoptosis of HepG2 cells in a concentration-dependent manner, showing a
gradual increase of annexin positive cells in cadmium chloride-treated cells compared to the control
(Figure 5). Figure 6 shows the percentages of both annexin V and PI positive cells were (10.3 ˘ 3.2)%,
(14.4 ˘ 5.6)%, (21.4 ˘ 4.6)%, (30.5 ˘ 2.8)%, (43.2 ˘ 7.5)%, and (52.5 ˘ 9.4)% in 0, 1, 2, 3, 4, and 5 µg/mL
cadmium chloride, respectively.
The effect of cadmium chloride was more pronounced at 5 µg/mL (p < 0.05) compared to the
control cells. We observed that the percentage of annexin positive cells increased gradually (p < 0.05)
with increasing cadmium chloride concentrations and reached a maximum of (52.5 ˘ 9.4)% cell death
upon 48 h of exposure. Cadmium can affect cell proliferation and differentiation, cell cycle progression,
DNA synthesis and repair, apoptosis and other cellular activities [55,56]. Recently, several reports have
shown that cadmium can induce apoptosis of many tissues and cells both in vivo and in vitro including
cells of the respiratory system, the testis, the kidney, the liver, and the immune system [57–61].
Int. J. Environ. Res. Public Health 2016, 13, 88 7 of 10

Figure 5. Representative flow cytometry analysis data from annexin V/PI assay. The histograms show
a comparison of the distribution of annexin V/PI negative cells (M1) and annexin V/PI positive cells
(M2) after 48 h exposure to cadmium chloride. A = control; B = 1 µg/mL; C = 2 µg/mL; D = 3 µg/mL;
E = 4 µg/mL; and F = 5 µg/mL.

Figure 6. Annexin V and PI positive cells. Cells were exposed to different concentrations of cadmium
chloride as described in the Materials and Methods. * Significantly different (p < 0.05) from the control,
according to the Dunnett’s test.

In addition to the apoptotic effects of cadmium, other reports have indicated that cadmium
chloride decreases the viability of HepG2 cells and increases lactate dehydrogenase leakage, DNA
damage, malondialdehyde, and antioxidant enzymes activities [62,63]. Additionally, a significant
decrease in ATP production and increase in ROS levels in cadmium chloride-treated HepG2 cells was
observed at all concentrations tested. A significant decrease in GSH/GSSG ratio was also found. These
effects were reported to be attenuated when cells were co-exposed to N-acetylcysteine (NAC) and
cadmium chloride [62,63].

4. Conclusions
The present in vitro study demonstrated that cadmium chloride exposure gradually decreases the
viability of HepG2 cells; increases lipid hydroperoxide levels resulting from reactive oxygen species
formation; induces DNA damage and triggers apoptosis of HepG2 cells through phosphatidylserine
externalization. In summary, these findings suggest that oxidative stress plays a role in cadmium
chloride-induced cyto/genotoxicity and apoptosis of HepG2 cells, especially at higher level of
exposure (4 and 5 µg/mL) where CdCl2 -treated cells show a statistically significant difference in
Int. J. Environ. Res. Public Health 2016, 13, 88 8 of 10

lipid hydroperoxide concentrations compared to control cells. This study therefore provides insight
into the mechanism underlying cadmium chloride-induced toxicity and apoptosis of HepG2 cells.

Acknowledgments: The research described in this publication was made possible in part by a grant from the
National Institutes of Health (NIH/NIMHD-G12MD007581) through the RCMI-Center for Environmental Health
at Jackson State University; and in part by the Mississippi INBRE grant (NIH/NIGMS-P20GM103476).
Author Contributions: Anthony Skipper, Clement G. Yedjou, and Paul B. Tchounwou conceived, designed, and
drafted the manuscript. Jennifer N. Sims participated in the implementation of the study, acquisition, analysis
and interpretation of data. All authors read and approved the final draft of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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