Effects of cadmium chloride on the cultured human lens epithelial cells.

Abstract

To investigate cadmium chloride cytotoxicity in human lens epithelial cells as well as the mode of cell death and its mechanism.
Cultured human lens epithelial cells were challenged with cadmium chloride. Morphological changes of human lens epithelial cells caused by cadmium chloride exposure were evaluated by microscope. Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny tetrazolium bromice (MTT) assay. To explore the mechanism of cell death, p53 and caspase-8 levels were measured by western blotting.
Microscopic examination indicated that cell death increased after cadmium chloride exposure compared to untreated cells. The MTT assay demonstrated that cadmium chloride significantly decreased cell viability in a dose dependent way. Western blot and quantitative analysis showed that both p53 and caspase-8 increased after cell exposure to cadmium chloride. p53 increased 210% and caspase-8 increased 30% in the experimental group as compared with the control group.
Cadmium chloride induced cytotoxicity and apoptosis in human lens epithelial cells and the mechanism of apoptosis involve an increased expression of p53 and caspase-8.

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Effects of cadmium chloride on the cultured human lens epithelial
cells
Nang-Hee Song, Jae-Woong Koh
Department of Ophthalomology, Chosun University College of Medicine, Gwangju, Republic of Korea
Purpose: To investigate cadmium chloride cytotoxicity in human lens epithelial cells as well as the mode of cell death
and its mechanism.
Methods: Cultured human lens epithelial cells were challenged with cadmium chloride. Morphological changes of human
lens epithelial cells caused by cadmium chloride exposure were evaluated by microscope. Cell viability was evaluated by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny tetrazolium bromice (MTT) assay. To explore the mechanism of cell death,
p53 and caspase-8 levels were measured by western blotting.
Results: Microscopic examination indicated that cell death increased after cadmium chloride exposure compared to
untreated cells. The MTT assay demonstrated that cadmium chloride significantly decreased cell viability in a dose
dependent way. Western blot and quantitative analysis showed that both p53 and caspase-8 increased after cell exposure
to cadmium chloride. p53 increased 210% and caspase-8 increased 30% in the experimental group as compared with the
control group.
Conclusions: Cadmium chloride induced cytotoxicity and apoptosis in human lens epithelial cells and the mechanism of
apoptosis involve an increased expression of p53 and caspase-8.
Cadmium is one of the most notorious heavy metals and
one of the members of the USA Environmental Protection
Agency’s “Priority List of Chemicals,” has been classified by
the International Agency for Research on Cancer as a human
carcinogen [1]. Tobacco smoke is the highest source of
exposure in the general population due to absorption of
cadmium by the lungs [2,3]. Therefore, human exposure to
cadmium is essentially unavoidable. Because of the its
biologically long half–life which has been estimated to be 10–
30 years in humans, cadmium has been demonstrated to cause
pathological changes in organs such as liver, brain, kidney,
and lung [4]. It also accumulates in various ocular tissues such
as the lens, retina, ciliary body, and vitreous humor [5]. Large
amounts of cadmium have been detected in lenses of chronic
smokers who also exhibit early cataract formation [6]. And
increased cadmium levels have also been reported in cataracts
compared to clear human lenses [7-9].
The studies clearly show that there is accumulation of the
heavy metal ion of cadmium in the lens of chronic smokers
which might have a role in cataractogenesis [7-9]. According
to Ramakrishnan et al. [9] 40–80 μM levels of cadmium,
which are approximately the levels found to be present in
cataracts of smokers and
cataractogenesis directly by interaction with lens proteins and
indirectly by its competition with copper, zinc, and selenium
cadmium may hasten
Correspondence to: Jae-Woong Koh, M.D./Ph.D., Department of
Ophthalomology, Chosun University College of Medicine,
Gwangju, Republic of Korea, # 588 Seoseok-dong. Dong–gu,
Gwangju, 501-140, Republic of Korea; Phone: 82-62-220-3190;
FAX: 82-62-225-9839; email: clearcornea@paran.com
and causing a decrease of antioxidants. Recent data show a
fourfold increase in heavy smokers (15.4±0.4 mol/g) and a
nearly threefold increase in light smokers (10.1±0.4 mol/g) as
compared to non-smokers (3.7±0.9 mol/g) [10].
However, the mechanism of cigarette smoke-induced
lenticular opacities is poorly understood. The normal single
layer-cuboid shaped lens epithelial cells are essential for
maintaining the metabolic homeostasis and transparency of
the entire lens [11]. If lens epithelial cell viability is required
for transparency, then with lens epithelial cell death, the lens
will become opaque [12]. They contain the highest levels of
enzymes and transport systems in the lens and are the first part
of the lens exposed to environmental insults [13]. Under
normal physiologic conditions, most of these cells have a
relatively long life span [14]. But if such conditions are altered
or disturbed these maintenance functions may be jeopardized,
possibly resulting in opacification of the lens [14].
Recently, both in vitro and in vivo studies have shown
that treatment of adult lens with stress factors induces
apoptosis of lens epithelial cells, which is followed by
cataractogenesis [15-17]. Damaged lens epithelium will be
leaky to calcium. The influx of calcium into the underlying
fiber cells can activate the cellular cysteine protease calpains
and caspase [18,19], which then degrade cytoskeleton
components [19,20] and lens crystallins [21,22]. These
processes eventually lead to crystallin aggregation [23], which
together with other changes such as uptake of water and
electrolytes lead to development of cortical and nuclear
cataract [24,25]. But data on the mechanism of apoptosis in
Molecular Vision 2012; 18:983-988 <http://www.molvis.org/molvis/v18/a103>
Received 5 January 2012 | Accepted 16 April 2012 | Published 19 April 2012
© 2012 Molecular Vision
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human lens epithelium from cataractous lenses are scarce and
conflicting.
We hypothesized that cadmium, a major smoke
constituent, could cause cataractous changes in the lens
through lens epithelial cell damage and explored the
mechanism of apoptosis that occurs in a cultured human lens
epithelial cell line after exposure to cadmium. We also
investigated whether cadmium-induced apoptosis was related
to activation of p53 and caspases-8. p53 can induce apoptosis,
cell cycle control and DNA repair in response to cellular
stress.
METHODS
Culture of cells: The human lens epithelial line CRL 11241
(B-3 cell, ATCC, Rockville, MD) was used for this study.
These cells were cultured in Modified Eagle's Medium
(MEM; Sigma, St Louis, MO), supplemented with 10% fetal
bovine serum (FBS), 100 U/ml penicillin, 100 μ/ml
streptomycin, and 25 μg/ml nystatin, and were cultured at
37 °C in humidified atmosphere, containing 5% CO2. This
research adhered to the tenets of the Declaration of Helsinki
and was approved by the institutional review board (IRB) of
the Chosun Medical School.
Morphological observation of human lens epithelial cells:
Cultured human lens epithelial cells were placed in 6 well
plate (5×104 cells/ml), after 24 h incubation, exposed to
80 μM of cadmium chloride (CdCl2, Catalog No. 202908;
Sigma) for 4 h. The control group and experimental group
were then observed using a contrast-phase microscope (Leica,
Wetzlar, Portugal).
Measuring the effect of cadmium on cell viability in human
lens epithelial cells: Cytotoxicity was determined by an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide; Sigma) assay. The human lens epithelial-B-3 cells
were placed in a 96 well plate (2×104 cell/ml) overnight and
then exposed to various concentrations of cadmium chloride
(0–100 μM) dissolved in PBS. Cells incubated without
cadmium served as control group. Twenty four hours after
incubation with cadmium, cell viability was evaluated using
the MTT assay. In this assay, MTT is reduced to purple
formazan in the mitochondria of living cells. A solubilization
solution is added to dissolve the insoluble purple formazan
product into a colored solution. The MTT assay was
performed using a standard protocol and optical density was
measured at 570 nm using a spectrophotometer.
Western blot: Both control groups and experimental groups
(exposed to 60 μM/ml of cadmium chloride) were evaluated
by western blot analysis. Briefly, after cadmium treatment
human lens epithelial cells were washed with Dulbecco’s PBS
1 time and cells were collected. SDS-loading buffer (100 μl;
50mM Tris-HCl ; pH6.8, 2% SDS, 0.1% Bromphenol blue,
10% glycerol) was added to the collected cells and
electrophoresis was conducted. Electrophoresis was done at
100 V in Tris buffer solution (pH8.8, 0.025 M Tris, 0.192 M
glycine, 0.1% SDS) using a 30% polyacrylamide gel. After
protein separation by electrophoresis the proteins were
transferred to nitrocellulose membrane. The membrane was
then blocked in 5% non-fat milk in Tris buffered saline (TBS:
0.1% Tween-20 in pH7.4 Tris-based saline buffer) for 1 h at
room temperature and washed with TBS twice. The
membrane was incubated with primary antibody diluted to
3/1000 in 5% non-fat-milk-TBS. Anti-rabbit polyclonal Anti-
p53 Ab, anti-caspase-8 Ab were used as the primary
antibodies. After washing with TBS 4 times, incubation with
secondary antibody combined in horseradish peroxidase (goat
anti-mouse IgG) diluted to 3/1,000 in 5% non-fat-milk-TBS
was performed for 1 h. Immunoreactive bands were visualized
using an enhanced chemiluminescence light detection kit
(Amersham, Piscataway, NJ). Beta-actin (GeneTex Inc., San
Antonio, TX) was used as an internal control. In all of the
figures with densitometry data, optical density refers to the
integrated density.
Analysis of experimental results: To increase the reliability of
the data, all experiments were repeated 3 times and average
values were calculated. SPSS ver. 10.1 (SPSS Inc., Chicago,
IL) was used to compute routine statistics. The data were
analyzed for significance using repeated measures by two-
way ANOVA, followed by a Duncan’s multiple range test of
post hoc tests, and were expressed as a mean percentage of
the control value plus SEM. A p value <0.05 was considered
significant.
RESULTS
Morphological changes of human lens epithelial cells by
cadmium chloride: Microscopic observation of the human
lens epithelial cells exposed to 80 μM cadmium chloride
revealed marked morphological changes. It looked damaged
in cadmium chloride treated cells (Figure 1).
Effect of cadmium concentration on cell viability: Cadmium
chloride significantly decreased cell viability in a dose
dependent way (Figure 2). Cell viability was 74.5±1.6% in
control group, 68.7±4.1% in 20 μM cadmium chloride,
55.9±3.3% in 40 μM cadmium chloride, 45.1±3.3% in 60 μM
cadmium chloride, 35.4±3.6% in 80 μM cadmium chloride,
and 25.6±2.4% in 100 μM cadmium chloride. Cell viability
decreased to less than 50% in 60 μM cadmium chloride
(p<0.05).
Western blot analysis: Western blot and quantitative analysis
showed that p53 increased in lens epithelial cells after
exposure to 60 μM of cadmium chloride. p53 increased 210%
in the experimental group compared to the control group
(Figure 3). Western blot and quantitative analysis showed that
caspase-8 also increased after exposure to 600 μM of
cadmium chloride. Caspase-8 increased 30% in the
experimental group compared to the control group (Figure 4).
A single band of 53 kDa and 55 kDa corresponding to p53
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protein and caspase 8, respectively, were present in lens
epithelial cell lysates.
DISCUSSION
Cigarette smoke contains 4,000 identified chemical
compounds and of these toxic materials are heavy metals,
particularly cadmium, which go into our system through
inhalation of smoking [26]. It readily pass into the
bloodstream and may accumulate in specific organs [26].
Indeed smoking has long been considered a major source of
several heavy metals in blood and various organs and
cadmium is regarded as one of the “strong carcinogens” in
tobacco smoke [27]. Cadmium has been found in several
studies consistently to transfer into the smoke phase, which
coupled with the fact that the tobacco plant is particularly
efficient in accumulating cadmium from the soil and
translocating most of the metal to the leaves makes this
Figure 2. Effect of cadmium on the cell viability of human lens
epithelial cells. The human lens epithelial cells cells were exposed
to various concentration of cadmium chloride. Twenty four hours
after incubation, cell viability was evaluated by the MTT assay.
Optical density was measured at 570 nm using a spectrophotometer.
Cadmium chloride significantly decreased cell viability in a dose
dependent way.
element the prime focus for particular investigation for any
potential toxic effects [28,29].
Cadmium exemplifies the double edge nature of many
toxic substances [30]. On the one hand, it can act as a mitogen,
stimulate cell proliferation, inhibit apoptosis, inhibit DNA
repair, and promote cancer in several tissues. On the other
hand, it causes tissue damage by inducing cell death [31].
According to Templeton and Liu [31], the concentration
dependence of the effects of cadmium is an important factor
for cell death. There was also a report that low dose exposure
is related to various types of apoptotic cell death and high dose
exposure is related to necrosis [31]. That is, depending on the
Figure 3. Effects of cadmium chloride on the expression of p53 in
human lens epithelial cells. Western blot and quantitative analysis
showed that p53 increased after exposure to 60 μM of cadmium
chloride. p53 increased 210% in the experimental group as compared
to the control group.
Figure 1. Morphological changes of
human lens epithelial cells after
exposure to cadmium
Photograph of the human lens epithelial
cell control group (A) and the
experimental group
epithelial cells exposed to 80 μM of
cadmium chloride; B). Compared to
untreated cells, microscopy analysis
indicated that cell damage increased
after cadmium chloride exposure.
chloride.
(human lens
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exposure conditions, cadmium may induce either necrosis or
apoptosis in mammalian cells.
Cadmium is a direct enzyme poison. Cadmium inhibits
plasma membrane calcium channels and Ca2+ ATPase groups
and hence can inhibit enzymes. It can exerts toxic effect
[32]. Cadmium is also a potent oxidative stress factor [33].
Oxidative stress occurs when the levels of pro-oxidants
exceed the ability of the cell to respond through antioxidant
defense [33]. Oxidative stress is a crucial event in activation
of apoptotic mechanisms [33]. Cadmium induces excessive
reactive oxygen species (ROS) generation and it may alter the
structure and function of proteins, lipids and DNA, besides
activating various signaling pathways which collectively
cause apoptosis [34,35]. One of the major effects that
oxidative stress induces is the death of lens epithelial cells
[36,37]. According to Kalariya et al. [38] Cadmium can
increase ROS levels in human lens epithelial cells and
weakens antioxidative reactions by inhibiting the action of
peroxide removal enzymes. Thus, both oxidative damage and
direct toxicity induced by cadmium appear to play major roles
in cataract formation. Recently, cadmium was found to induce
DNA fragmentation, a biochemical hallmark of apoptosis, in
cultured renal cells, hepatocytes and human T-cells [39].
Therefore, the various toxicities of cadmium are thought
to be caused by the induction of apoptosis. Cadmium
modulates protein kinase, phosphatase activities and
tranascription factors and mitogen-activated protein kinase
(MAPK) [40,41]. Mitochondria, caspases and ROS pathways
Figure 4. Effects of cadmium chloride on the expression of caspase-8
in human lens epithelial cells. A representative western blot and
quantitative analysis showed that caspase-8 increased after exposure
to 60 μM of cadmium chloride. Caspase-8 increased 30% in the
experimental group as compared to the control group.
all seem to palys role in cadmium induced apoptosis [42]. It
is conceivable that cadmium may induce different apoptotic
pathways in different cell types depending on the exposure
conditions. But the apoptotic pathway induced by cadmium
remains controversial. A large number of studies have
demonstrated that cadium-induced activation of the MAPK
pathway leads to apoptosis in various cell types [34,35,38].
According karariya et al., the activation of MAPK pathway
along with ROS generation and apoptosis in human lens
epithelial cells could collectively damage the lens epithelial
layer which could make the lens vulunerable to develop
opacity [38]. Toxic metals have been reported to induce the
generation of reactive oxygen species, which may target the
mitochondrial membrane, triggering one or more of the
intrinsic, mitochondrial apoptotic pathways leading to
activation of pro-caspases-9 and-3 [43]. However, ROS are
also thought to play a role in the Fas receptor-mediated,
extrinsic apoptotic pathway via c-jun N- terminal kinase
(JNK) mediated induction of FasL or Fas expression [44].
Recently, it was demonstrated that toxic metal- induced
apoptosis in cultured murine podocytes through the extrinsic
Fas-associated death domain protein (FADD) capsase 8
pathway, rather than the mitochondrial apoptotic pathway
[44].
Apoptosis is an important mechanism to maintain
homeostasis in multicellular organisms and is a series of
controlled processes that selectively removes damaged cells
without injuring surrounding tissues [45]. Apoptosis is a
normal morphogenetic process of lens development [46].
During development, apoptosis is necessary for lens vesicle
formation and detachment [46]. And apoptosis helps to
remove damaged epithelial cells or aberrantly differentiated
lens cells [46]. Suppression or enhancement of developmental
apoptosis because of genetic mutations and manipulations, or
environmental conditions causes formation of abnormal
lenses or absence of the ocular lens [46]. It is a very
sophisticated operative process and the mechanism can be
largely divided into an external mechanism and internal
mechanism [47]. The signal in the external mechanism
(receptor–dependent pathway) starts from death receptors,
such as Fas or tumor necrosis factor-α (TNF-α) receptors, and
is passed to caspase-8 which activates caspase-3, the caspase
that directly triggers apoptosis [47]. This external signal can
cause apoptosis through the internal mechanism by increasing
transcription of B-cell lymphoma protein-2 (Bcl-2) family
proteins and stimulating mitochondria at the same time [47].
The internal mechanism (mitochondria–dependent pathway)
activates caspase-9 through apoptotic protease activating
factor-1 (Apaf-1) by cytochrome C being secreted in
mitochondria and this causes death of cells by activating
caspase-3 and Bcl-2 family protein regulates apoptosis by
causing secretion of cytochrome C by adjusting the
permeability of mitochondria [48]. Caspases can be broadly
dived into two groups: initiator caspases, such as
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caspases-8,-9 and −12, whose main function is to activate
downstream caspases, and executor caspases, such as
caspases-3,-6 and −7, which are a responsible for degradation
of cellular proteins [49]. Caspase-8, encoded by the CASP8
gene, is an initiator caspase of the death receptor pathway, as
well as a target of the caspase-3 downstream pathway of
mitochondria, and is composed of 60 amino acids of NH2-
terminal death effector domain (DED), that facilitates
caspase-8-FADD direct interaction. Depending on the cellular
context, this results in different outcomes [49]. Cell death
induced through the p53 pathway is executed by the caspase
proteinases [49].
In this study, the toxicity of cadmium in human lens
epithelial cells was measured by MTT method after culturing
the cells in medium containing 20 μM 40 μM, 60 μM, 80 μM,
and 100 μM of cadmium chloride. It was confirmed that
cytotoxicity increased significantly
concentrations. To explore the mechanism of the apoptotoic
process, the expression of p53 and caspase-8, a potent
mediator of apoptosis, were examined. Western blot analysis
revealed that protein expression levels of p53 and caspase 8
increased by 210% and 30%, respectively, in the group
exposed to cadmium compared to the control group. Thus,
apoptosis in the human lens epithelial cells is related to p53
and caspase-8 expression. Based on the result above,
cadmium affects cytotoxicity and death of human lens
epithelial cells via a p53 dependent pathway and activation of
caspase-8. How cadmium activates caspase-8 is not clear, but
it’s clear that cadmium has an effect on caspase-8 protein
levels, suggesting that the death receptor pathway might
contribute appreciably to the observed cadmium induced
apoptosis. Since it is difficult to obtain normal human
materials, cultured models have been used to further examine
the relationship between apoptosis and cataractogenesis. In
this study, we confirmed that cadmium induced apoptosis
occurs in cultured human lens epithelial cells, but great
caution should be useded in transferring this finding to the
human sitiuation. because there are a few difference in lens
composition between them, cultured human lenses have a very
low level of crystallins.
In conclusion, we studied the effects of cadmium on the
viability of human lens epithelial cells and showed that
cadmium caused significant decline in the viability of human
lens epithelial cells in a dose dependent manner. Cadmium
also induced p53 and caspase-dependent apoptosis of human
lens epithelial cells, a potential cause of human lens opacity.
ACKNOWLEDGMENTS
This work was supported by a grant from the Clinical
Medicine Research Insititute of the Chosun University
Hospital (2011).
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