Biological Trace Element Research ISSN 0163-4984 Volume 162 Combined 1-3 Biol Trace Elem Res (2014) 162:95-105 DOI 10.1007/s12011-014-0100-yDNA… [600494]

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Biological Trace Element Research

ISSN 0163-4984
Volume 162
Combined 1-3

Biol Trace Elem Res (2014) 162:95-105
DOI 10.1007/s12011-014-0100-yDNA Fragmentation, Caspase 3 and
Prostate-Specific Antigen Genes Expression
Induced by Arsenic, Cadmium, and
Chromium on Nontumorigenic Human
Prostate Cells
Hend M. Abo El-Atta, Amal A. El-
Bakary, Afaf M. Attia, Ahmed Lotfy,
Shery S. Khater, Ayman Z. Elsamanoudy
& Hussein Abdelaziz Abdalla

123
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DNA Fragmentation, Caspase 3 and Prostate-Specific Antigen
Genes Expression Induced by Arsenic, Cadmium, and Chromium
on Nontumorigenic Human Prostate Cells
Hend M. Abo El-Atta &Amal A. El-Bakary &
Afaf M. Attia &Ahmed Lotfy &Shery S. Khater &
Ayman Z. Elsamanoudy &Hussein Abdelaziz Abdalla
Received: 5 June 2014 /Accepted: 5 August 2014 /Published online: 17 September 2014
#Springer Science+Business Media New York 2014
Abstract Prostate cancer is one of the most common cancers
and the second cause of cancer-related deaths among men.
Metals are recognized as chemical carcinogens where chronicexposures to such metals are implicated in the development of
cancer, including prostate cancer. This in vitro study demon-
strates the relative death sensitivity of prostatic (RWPE-1)cells to arsenic (As), cadmium (Cd), and chromium (Cr) as
environmental pollutants through its apoptotic effects and the
effect of these chemicals on prostate-specific antigen (PSA)gene expression as a marker for their carcinogecity. RWPE-1
cells were divided into three groups that were treated with As,
Cd, and Cr in three replicates, at three different concentrationsfor each metal for 48 h. A control group consisted of untreated
RWPE1 cells was used. Apoptosis was assessed using comet
assay and caspase 3 gene expression; meanwhile, PSA geneexpression was evaluated by semiqualitative real-time PCR
(RT-PCR). One of the novel findings of this study is thatarsenic and cadmium at low concentrations decreased apopto-
sis of RWPE-1 cells in a concentration-dependent manner
while chromium induced significant concentration-dependent increase in apoptosis. Yet, at the highest concen-
trations, apoptosis was rel atively more induced by all
chemicals. Arsenic was the most chemical inhibiting apopto-sis in RWPE-1 cells at low concentration. While at the mod-
erate and highest concentrations, cadmium was the most
inhibiting chemical of RWPE-1 cells ’apoptosis. No distinct
differences between treated and untreated cells for PSA gene
expression were observed. It can be concluded that As and Cd,
at low concentrations, can reduce apoptosis of prostatic cellsin a concentration-dependent manner while chromium in-
duced it; however, all metal salts used in this study did not
induce PSA gene expression.
Keywords RWPE-1 cells .Arsenic .Cadmium .Chromium .
Apoptosis .PSA
Introduction
Metals such as cadmium, nickel, arsenic, beryllium, and chro-
mium VI are recognized as chemical carcinogens that areimplicated in the development of cancer [ 1]. Chronic exposure
to such metals is nearly unavoidable in daily life. Sources of
exposure include airborne particles, soil, water, and subse-
quently food [ 2]a sw e l la so c c u p a t i o n a le x p o s u r e[ 3]. Ciga-
rette smoke is another important source of human exposure[4]. In Egypt, The Nile River is seriously contaminated with
heavy metals as a result of increasing discharge of untreated
industrial wastes and agricultural irrigation by wastewater [ 5].
Multiple studies revealed an association between environmen-
tal exposure to different metals like arsenic [ 6–8], cadmium
[9], and prostate cancer incidence or mortality.H. M. A. El-Atta :A. A. El-Bakary :A. M. Attia
Department of Forensic Medicine and Clinical Toxicology, Faculty
of Medicine, Mansoura University, Mansoura, Egypt
A. Lotfy
Medical Expermintal Research Center, Faculty of Medicine,Mansoura University, Mansoura, Egypt
S. S. Khater
Department of Pathology, Urology and Nephrology Center, Facultyof Medicine, Mansoura University, Mansoura, Egypt
A. Z. Elsamanoudy
:H. A. Abdalla
Department of Medical Biochemistry, Faculty of Medicine,Mansoura University, Mansoura, Egypt
A. Z. Elsamanoudy ( *)
Department of Medical Biochemistry and Molecular Biology,Faculty of Medicine, Mansoura University, El-Gomhoria St.,
Mansoura, Egypt
e-mail: ayman.elsamanoudy@gmail.comBiol Trace Elem Res (2014) 162:95 –105
DOI 10.1007/s12011-014-0100-y
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96 El-Atta et al.

Epidemiologically, prostate cancer is one of the most com-
mon cancers and the second cause of cancer-related deaths
among men in the western countries [10]. According to the
National Cancer Institute in Egypt [11], genitourinary system
cancers are considered the second most commonly distributed
cancers (21.3 %) among males, including prostate cancer, and
the third most common cause (2.6 %) of total number of
cancer cases.
The etiolog y of prostate cancer in human s is multi-
factori al and includ es age, ethnicity, environmenta l fac-
tors, and other unkno wn causes [12]. Although rates
today are markedl y highe r than rates observe d three
decades ago, statistic s show that prostat e canc er inci-
dence rates have now stabilized which is though t to
reflec t change s in utilization of prostate -specifi c anti-
gen (PSA ) testing . The basis for these high rates of
abnorma l prostatic growt h is not well understo od de-
spite extensive researc h [13].
Apoptosis, a process that ensure the integrity of tissues and
organs by maintaining the balance with cellular proliferation,
occurs via two main pathways, the extrinsic-/death receptor-
mediated pathway and the intrinsic-/mitochondrial-mediated
pathwa y, the latter of whic h is found to be compro-
mised in most cancer cells [14]. Once triggered , signals
from both pathway s can lead to the activatio n of a
group of cystei ne proteas es known as caspase s and the
initiation of apoptoti c cell death via cleavag e of effector
caspase s (i.e., caspas e 3) [15].
Comet assay is a cheap and simple method that can be used
to assess the genotoxicity of mutagens and carcinogens that
induce DNA damage in addition to DNA fragmentation asso-
ciated with cell death or related to apoptosis [16].
The aim of this in vitro study was to investigate the
relative death sensitivity of prostati c cells (RWPE -1 cells)
to arseni c, cadmium , and chromiu m as environmental
pollut ants through their apoptotic effects as well as carci-
nogenic potentia l of these chemical s through assessme nt
of PSA gene expressio n.

Material and Methods

Chemicals

Sodium arsenite (NaAsO2), cadmium chloride (CdCl2), and
sodium dichromate dehydrate (Na2Cr2O7.2H2O) were pur-
chased from Sigma (St Louis, MO). Chemicals were dissolved
in sterile distilled water, then filtered with membrane filter
(0.2 μm) to make stock solutions at different concentrations as
follows: As concentrations (0.1, 1, 10, 100, 1,000 μM), Cd
concentrations (1, 10, 100, 1,000 μM), and Cr concentrations
(1, 10, 100, 1,000 μM); before cell treatments. Cell lines and Culture Conditions

Non-tumorigenic human prostate epithelial cell (RWPE1)
(ATCC ® CRL 11609™ ) cell line was purc hased from
VACSERA “The Holding Company for Biological Products
and Vaccines,” Egypt. It was cultured in a keratinocyte serum-
free medium (K-SFM), supplied with the two additives re-
quired to grow this cell line (bovine pituitary extract, BPE)
and human recombinant epidermal growth factor (EGF)
(Invitrogen (GIBCO), penicillin (100 U/ml), and streptomycin
(100 μg/ml) at 37 °C in 5 % CO2. Cells were seeded in 6-well
plates in triplicates and allowed to grow for 3 days to reach
60–75 % confluence at which time the cells were detached by
trypsinization and suspended in cell culture media and count-
ed with a hemacytometer. The experiment was repeated twice.

Trypan Blue Test for Cell Viability and Determination
of LC50

Cells were treated with NaAsO2, CdCl2, and Na2Cr2O7.2H2O
solutions at different concentrations (as previously men-
tioned), then incubated for 48 h to determine LC 50 of each
chemical using trypan blue exclusion test [17]. A concentra-
tion of 0.1 μM sodium arsenite was added in this study
because a concentration of 1 μM was found to be lethal for
all cells . LC50 was foun d to be 1 μM for CdCl 2 and
Na2Cr2O7.2H2O, while it was 0.1 μM for NaAsO2. Diluted
concentrations were tested as the following: 0.1 and 0.01 μM
for CdCl2 and Na2Cr2O7.2H2O, while for NaAsO2, 0.01 and
0.001 μM were used.

Cell treatments and Study Groups

Cells were divided into three test groups: group I that was
treated with NaAsO2 at 0.1, 0.01, and 0.001 μM for 48 h;
group II that was treated with CdCl2 at 1, 0.1, and 0.01 μM for
48 h; and group III that was treated with Na2Cr2O7.2H2O at 1,
0.1, and 0.01 μM for 48 h. A matched control group consisted
of untreated RWPE1 cells was used. Susceptibility to apopto-
sis was assessed through DNA fragmentation evaluated by
comet assay and measuring caspase 3/GAPDH gene expres-
sion by semiquantitaive reverse transcriptase- PCR (RT-
PCR). PSA gene expression was assessed by
semiquantitaive RT-PCR. Cells were harvested for comet
assay and RNA isolation as follows:

Comet Assay

The comet assay was performed under alkaline conditions.
Briefly, cells were detached by trypsin-EDTA treatment for
5 min and collecte d by centri fugation . Cell pellet s were
washed twice with PBS and then suspended in PBS. Fifty
microliters of cell suspension was mixed with 500 μl of 1 %
(w/v) low-melting-point (LMP) agarose dissolved in PBS.

Cell suspension in agarose was then quickly pupated into
comet slides and allowed to set at 4 °C for 10 min in the dark.
The slides were then immersed in pre-chilled lysis solution(2.5 M NaCl, 100 mM sodium-EDTA, and 10 mMTris at pH
10) for 60 min at 4 °C to remove cellular proteins. Following
lysis, the slides were placed in a horizontal gel electrophoresisunit and then electrophoresed in freshly prepared alkaline
electrophoresis buffer (300 mM NaOH and 1 mM EDTA at
pH 13) for 45 min at room temperature. Following electro-phoresis, slides were immersed in neutralization buffer (0.4 M
Tris-HCl, pH 7.5) and then gently washed three times for
5 min at 4 °C to remove alkalis. Finally, slides were fixed in70 % ethanol for 5 min and stored in the dark to dry complete-
ly [18]. Just before image analysis, gels on each slide were
stained with 50 ml ethidium bromide (20 mg/ml) and theslides were examined under a fluorescence microscope
equipped with an excitation filter of 565 nm and a barrier
filter of 590 nm. Images were captured using a digital cameraand saved as TIFF/JPEG files. All steps were performed under
reduced light to minimize DNA damage from ambient ultra-
violet radiation.
Images of cells were processed by a computer-assisted
image-analysis system (Comet Assay IV) to determine the
comet parameters. Results were expressed as tail length (TL;
the distance that DNA migrated, in μm), percentage of DNA
in tail (% Tail; the intensity of migrated DNA), and tailmoment (TM; tail length×percentage of DNA in tail/100).
RNA Isolation and RT-PCR
Total RNA was isolated using AxyPrep Multisource Total
RNA Miniprep kit (Axygen Biosciences, Union City, CA
94587, USA). First-strand complementary (cDNA) synthesisfrom total RNA and PCR were performed by RT/PCR Master
Mix. Gold Beads Kits (BIORON The ENZYME Company,
Germany) using caspase 3-specific primer used the following:caspase 3 sense, 5 ′-TTCAGAGGGGATCGTTGTAGAA
GTC-3 ′and antisense, 5 ′-CAAGCTTGTCGGCATACTGT
TTCAG-3 ′(264 bp fragment) [ 19]. PSA-specific primers
yielding 680 bp fragment spanning 4 exons (exon 2, exon 3,
exon 4, exon 5) are as follows: forward primer, 5 ′-CTCTCG
TGGCAGGGCAGT-3 ′and reverse primer, 5 ′-AATAGGGG
GTTGATAGGG-3 ′. A messenger (mRNA) of glyceraldehyde
3 phosphate dehydrogenase (G
3PDH) was used as endoge-
nous external control for RT-PCR: forward primer, 5 ′-TGAA
GGTCGGAGTCAACG-3 ′and reverse primer, 5 ′-CATGTG
GGCCATGAGGTCCACCAC-3 ′(983-bp fragment) [ 20].
One microgram of template RNA and 30 pmol of reverse
primer were mixed in a sterile tube. The mixture was incubat-
ed at 70 °C for 5 min and was placed on ice. The incubatedmixture and the forward primer were transferred to the RT/
PCR gold tube, and then the tube was filled to 20 μLb yD P E C
DW. The lyophilized pellet was dissolved by vortexing andwas briefly span down. cDNA synthesis reaction was per-
formed in a thermal cycler (TECHEN TC-312, Model
FTC3102D, Barloworld Scientific Ltd. Stone, Stafford Shirest 150 SA, UK) by heating the tube for 60 min at 42 °C then
for 5 min at 94 °C for RTase inactivation.
For caspase 3, PCR was performed by heating for 94 °C for
5 min, 35 cycles of 68 °C for 1 min, and 94 °C for 1 min, with
final extension of 72 °C for 10 min (Winter et al. 2001). PCR
was performed for PSA by heating for 5 min at 94 °C for DNAdenaturation followed by 36 cycles (1 min at 94 °C, 1 min at
57 °C, 1 min at 72 °C) and a final extension for 10 min at
72 °C [ 20].
The PCR products were analyzed by electrophoresis and
ethidium bromide staining on 1.6 % agarose gels, visualized
via a light UV transilluminator (Model TUV-20, OWI Scien-tific, Inc. 800 242-5560, France), and photographed under
fixed conditions (the distance, the light, and the zoom). The
result photos were analyzed with Scion Image® Release Al-pha 4.0.3.2. Software for Windows® which performs band
detection and conversion to peaks. Area under each peak were
calculated in square pixels and used for quantification. Geneexpression levels were determined by calculating the ratio
between the square pixel values of the target gene in relation
to the internal house keeping control gene (GAPDH
). Minus
RT controls permitted to rule out genomic contamination.
Similarly, no products were detected when the RT-PCR stepwas carried out with no added RNA, indicating that all re-
agents were free of target sequence contamination.
Statistical Analysis
Using the Microsoft Office and MedCalc® programs version
10.0.1, the statistics were summarized as arithmetic mean;
standard deviation for parametric data and range and medianwere used for nonparametric data. Student ttest and Mann
Whitney test were used to assess the statistical significance of
differences. Difference was considered statistically significantifP<0.05.
Results
Comet AssayFigure 1shows representative photographs captured by fluo-
rescence microscope for RWPE-1 treated with chemicalscompared to a control group of untreated cells.
As shown in Table 1, arsenic induced significant decrease
in comet assay parameters at all concentrations compared tountreated control cells. However, the group that was treated
with the moderate concentration caused a significant decrease
in the comet tail compared to cells treated with the lowAr, Cd, and Cr Effects on Nontumorigenic Human Prostate Cell 97
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concentration. Meanwhile, the high concentration caused in-
crease in comet parameters compared to cells treated with the
moderate concentration.
Similarly, cadmium induced significant decrease in comet
assay parameters at all concentrations in comparison to un-
treated control group. However, there is significant decrease in
the comet tail length in the cells treated with the moderate andhigh concentrations compared to cells treated with the lowconcentration. There was significant increase in tail intensity
and tail moment in the cells treated with the high concentra-
tion compared to cells treated the moderate concentration
(Table 2).
On one hand, compared to untreated control cells, chromi-
um induced insignificant increase in tail length and moment at
the low concentration but these changes were significant at thehighest concentration. Meanwhile, it induced significant
a bFig. 1 Comet assay for RWPE-1
cells ×200. aUntreated control
cells. bTreated cells
Table 1 Comet assay parameters and caspase 3/GAPDH mRNA gene expression in arsenic-treated RWPE-1 cells ’groups at different concentrations
Groups Control Arsenic
Low concentration (0.001 μM) Moderate concentration (0.01 μM) High concentration (0.1 μM)
Tail length
Median (range) 44.90 (15.6 –151.3) 23.91 (9.3 –58.1) 18.79 (7.3 –48.3) 30.26 (8.3 –165.9)
P<0.0001* P<0.0001* P<0.001*
P1<0.01* P1<0.01*
P2<0.0001*
Tail intensityMedian (range) 14.01 (0.03 –99.8) 1.95 (0.04 –90.6) 4.79 (0.01 –32.4) 2.72 (0.03 –99.7)
P<0.001* P<0.0001* P<0.01*
P1=0.80 P1=0.51
P2=0.62
Tail momentMedian (range) 2.82 (0.01 –71.8) 0.26 (0.01 –24.4) 0.69 (0.01 –4.7) 0.53 (0.01 –79.6)
P<0.0001)* P<0.0001* P<0.0001*
P1=0.31 P1=0.32
P2=0.83
Caspase 3/GAPDH mRNAMean±SD 0.71±0.04 0.42±0.06 0.21±0.04 0.56±0.07
P<0.0001)* P<0.0001* P<0.0001*
P1<0.0001)* P1<0.0001)*
P2<0.0001)*
P, test groups vs. control group; P1, moderate and high concentrations vs. low concentration; P2, moderate vs. high concentrations
*statistically significant98 El-Atta et al.
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decrease in tail intensity and moment at the moderate concen-
tration compared to untreated control cells. On the other hand,
comparing the moderate to the low concentrations, chromiuminduced significant decrease in tail intensity and tail moment;
meanwhile, it induced significant increase in tail length and
moment at the high concentration compared to the moderateconcentration (Table 3).
Comparing the effects of the three metals to each other,
arsenic had the most powerful decreasing effect on the cometassay parameters at the low concentration while cadmium was
the most powerful to do so at the moderate and high concen-
trations. Meanwhile, chromium was found to be the leastsignificantly affecting the comet assay parameters (insignifi-
cant increase) at low and moderate concentrations while at the
highest concentrations, chromium was the most significantlyincreasing chemical for comet assay parameters denoting
induction of apoptosis (Tables 4,5,a n d 6).
RT-PCR for Caspase 3/GAPDH Gene Expression
There was significant decrease in caspase 3/GAPDH expres-
sion in RWPE-1 cells after exposure to arsenic and cadmium
that was most evident at the moderate concentrationscompared to untreated control cells. Chromium induced
dose-related significant increase in caspase 3/GAPDH expres-
sion (Tables 1,2,a n d 3). RWPE-1 cells were more resistant to
arsenic-induced caspase 3/GAPDH expression compared to
cadmium and to both compared to chromium at low and
moderate concentrations. At the highest concentration,RWPE-1 cells were the most resistant cadmium-induced cas-
pase 3/GAPDH expression compared to arsenic and chromi-
um (Tables 4,5,a n d 6and Fig. 2).
RT-PCR for PSA Gene Expression
mRNA expression of prostatic-specific antigen (PSA) gene is
qualitatively investigated by RT-PCR in RWPE-1 cells which
show no distinct differences in treated and untreated cells in all
studied groups as shown in Fig. 3.
Discussion
One of the most important findings of this study is that arsenic
and cadmium at low concentrations decreased apoptosis ofTable 2 Comet assay parameters and caspase 3/GAPDH mRNA gene expression in cadmium-treated RWPE-1 cells ’groups at different concentrations
Groups Control Cadmium
Low concentration (0.01 μM) Moderate concentration (0.1 μM) High concentration (1 μM)
Tail length
Median (range) 44.90 (15.6 –151.3) 26.85 (7.8 –185.5) 17.08 (9.7 –30.8) 18.06 (4.4 –54.2)
P<0.0001* P<0.0001* P<0.0001*
P1<0.0001* P1=0.0001*
P2=0.28
Tail intensityMedian (range) 14.01 (0.03 –99.8) 1.71 (0.01 –96.8) 0.42 (0.01 –25.2) 3.82 (0.17 –95.6)
P<0.0001* P<0.0001* P<0.01*
P1=0.01* P1=0.14
P2=0.001*
Tail momentMedian (range) 2.82 (0.01 –71.8) 0.38 (0.01 –40.1) 0.11 (0.01 –2.9) 0.38 (0.02 –21.6)
P<0.0001* P<0.0001* P<0.0001*
P1=0.05 P1=0.96
P2<0.01*
Caspase 3/GAPDH mRNAMean±SD 0.71±0.04 0.49±0.05 0.24±0.06 0.41±0.03
P<0.0001* P<0.0001* P<0.0001*
P1<0.0001)* P1<0.01*
P2<0.001*
P, test groups vs. control group; P1, moderate and high concentrations vs. low concentration; P2, moderate vs. high concentrations
*statistically significantAr, Cd, and Cr Effects on Nontumorigenic Human Prostate Cell 99
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Table 3 Comet assay parmeters and caspase 3/GAPDH mRNA gene expression in chromium treated RWPE-1 cells ’groups at different concentrations
Groups Control Chromium
Low concentration (0.01 μM) Moderate concentration (0.1 μM) High concentration (1 μM)
Tail length
Median (range) 44.90 (15.6 –151.3) 29.29 (6.8 –164.9) 38.56 (2.9 –154.2) 59.3 (10.7 –196.2)
P=0.228 P=0.465 P<0.01*
P1=0.38 P1=0.001*
P2=0.01*
Tail intensityMedian (range) 14.01 (0.03 –99.8) 8.20 (0.1 –99.8) 3.00 (0.01 –99.8) 4.88 (0.02 –99.9)
P=716 P<0.01* P=0.250
P1=0.01* P1=0.43
P2=0.13
Tail moment
Median (range) 2.82 (0.01 –71.8) 1.00 (0.03 –131.4) 0.51 (0.01 –76.19) 1.26 (0.01 –110.5)
P=0.557 P<0.001* P=0.649
P1<0.01* P1=0.86
P2=0.01*
Caspase 3/GAPDH mRNA
Mean±SD 0.71±0.04 0.95±0.06 1.31±0.21 2.31±0.43
P>0.05 P<0.0001* P<0.0001*
P1<0.05 P1<0.0001*
P2<0.0.001*
P, test groups vs. control group; P1, moderate and high concentrations vs. low concentration; P2, moderate vs. high concentrations
*statistically significant
Table 4 Comet assay parameters and caspase 3/GAPDH mRNA gene expression in RWPE-1 cells ’groups treated with arsenic, cadmium, and
chromium at low concentrations
Groups Control Lowest concentrations
Arsenic (0.001 μM) Cadmium (0.01 μM) Chromium (0.01 μM)
Tail length
Median (range) 44.90 (15.6 –151.3) 23.91 (9.3 –58.1) 26.85 (7.8 –185.5) 29.29 (6.8 –164.9)
P<0.0001* P<0.0001* P=0.228
P1=0.221 P1<0.01*
P2=0.283
Tail intensityMedian (range) 14.01 (0.03 –99.8) 1.95 (0.04 –90.6) 1.71 (0.01 –96.8) 8.20 (0.1 –99.8)
P<0.001* P<0.0001* P=716
P1=0.849 P1<0.001*
P2<0.0001*
Tail momentMedian (range) 2.82 (0.01 –71.8) 0.26 (0.01 –24.4) 0.38 (0.01 –40.1) 1.00 (0.03 –131.4)
P<0.0001)* P<0.0001* P=0.557
P1=0.320 P1<0.001*
P2<0.001*
Caspase 3/GAPDH mRNA
Mean±SD 0.71±0.04 0.42±0.06 0.49±0.05 0.95±0.06
P<0.0001* P<0.0001* P<0.0001*
P1<0.05 P1<0.001*
P2<0.0001*
P, test groups Vs. control group; P1, arsenic vs. chromium- and cadmium-treated groups; P2, chromium- vs. cadmium-treated groups
*statistically significant100 El-Atta et al.
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RWPE-1 cells in a concentration-dependent manner. Yet, at
the highest concentrations, apoptosis was relatively more
induced by all chemicals that may be due to excess DNAdamage. Arsenic was the most chemical inhibiting apoptosis
in RWPE-1 cells at low concentration. While at the moderate
and highest concentrations, cadmium was the most inhibitingchemical of RWPE-1 cells ’apoptosis.
Arsenic was reported to produce similar results on RWPE-
1 and WPE-stem cells (derived from the RWPE-1 cell lineafter two consecutive cycles of single cell cloning) [ 7,21–23]
although concentration and duration of exposure may differ.
Arsenic as 5 μM sodium arsenite (a relatively nontoxic, but
environmentally relevant level) transformed RWPE-1 cell line
and WPE-stem cells into cancerous cells when exposed for30 weeks [ 21] and 18 weeks [ 7], respectively. Even when
applied as low as100 pg/ml to RWPE-1 cells for 3 months, cells
were transformed into malignant epithelial cells [ 23]. Levels
and duration of exposure in the present study are far lower than
previously reported. As transf ormation of diploid human cells
with carcinogenes in vitro to cancerous cells takes about 6 m[24]; thus, the reported relatively higher concentrations trans-
formed RWPE-1 cells into cancerous cells within relatively
shorter periods probably due to higher concentrations.Clinical and experimental evidence indicate that carcino-
genesis is a lengthy, multi-step process involving damage to
genes regulating normal cell growth and division (initiation).However, if repair does not occur and the damage to DNA is
in the location of a gene that regulates cell growth and prolif-
eration, DNA repair, or a function of the immune system, thenthe cell is more prone to becoming cancerous. Inhibition of
apoptosis can be an essential step in carcinogenesis. So,
inhibition of apoptosis in the present study by such low dosesof arsenic and cadmium over a relatively short period may be
just the first step in the carcinogenic process. This is augment-
ed by previously reported interaction of arsenic with signal
transduction systems, tumor suppressor functions, and DNA
repair systems [ 25,26]. Oxidative stress is thought to be an
important mechanism in arsenic-induced carcinogenicity [ 27].
Controversially, Singh et al. [ 22] found that chronic exposure
to arsenic caused increased cell survival by decreasing theexpression of pro-apoptotic genes.
Although, a relation between cadmium exposure and pros-
tate cancer seems to be supported by laboratory and epidemi-ologic studies [ 28], the specific molecular events associated
with cadmium-induced transformation are still elusive. While
Chen et al. [ 29] could not prove that cadmium is a predictor ofTable 5 Comet assay parameters and caspase 3/GAPDH mRNA gene expression in RWPE-1 cells ’groups treated with arsenic, cadmium, and
chromium at moderate concentrations
Groups Control Moderate concentrations
Arsenic (0.01 μM) Cadmium (0.1 μM) Chromium (0.1 μM)
Tail length
Median (range) 44.90 (15.6 –151.3) 18.79 (7.3 –48.3) 17.08 (9.7 –30.8) 38.56 (2.9 –154.2)
P<0.0001* P<0.0001* P=0.465
P1=0.432 P1<0.0001*
P2<0.0001*
Tail intensityMedian (range) 14.01 (0.03 –99.8) 4.79 (0.01 –32.4) 0.42 (0.01 –25.2) 3.00 (0.01 –99.8)
P<0.0001 P<0.0001* P<0.01*
P1<0.01* P1=0.309
P2<0.001*
Tail momentMedian (range) 2.82 (0.01 –71.2) 0.69 (0.01 –4.7) 0.11 (0.01 –2.9) 0.51 (0.01 –76.19)
P<0.0001* P<0.0001* P<0.001*
P1<0.01* P1=0.281
P2<0.01*
Caspase 3/GAPDH mRNAMean±SD 0.71±0.04 0.21±0.04 0.24±0.06 1.31±0.21
P<0.0001* P<0.0001* P<0.0001*
P1>0.05 P1<0.001*
P<0.0001*
P, test groups vs. control group; P1, arsenic vs. chromium- and cadmium-treated groups; P2, chromium- vs. cadmium-treated groups
*statistically significantAr, Cd, and Cr Effects on Nontumorigenic Human Prostate Cell 101
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prostate cancer, cadmium was reported not only to induce
mitogenic signaling and interfere with p53-mediated cell cyclecontrol, but it has been shown to impair almost all major DNA
repair pathways [ 30].
In accordance with the present results, Achanzar et al. [ 31]
found that chronic exposure of RWPE-1 cells to 10 μMc a d –
mium, a concentration very near to the estimated range in the
prostates of people with no known occupational exposure tocadmium, for 8 weeks, showed malignant transformation. As
the human prostate progressively accumulates cadmium with
age [ 32], continuous exposure of prostatic epithelial cells to
cadmium in vivo is a likely scenario hence the potential forcarcinogenesis [ 31]. Higher concentrations of cadmium com-
parable with observed in vivo levels in exposed humans (10 –
30μM) induced apoptosis in RWPE-1 cells in a dose-
dependent manner but induced resistance to apoptosis in
CTPE cells (cadmium-transformed derivatives of RWPE-1)[9]. In the same time, low doses of cadmium for a short period
of time induced both apoptosis and atypical hyperplasic pro-
liferative lesions in rat prostate [ 12].
Another important finding of the present study was that
chromium effects were not uniform especially at low and
moderate concentrations; thus, the tail moment was chosenas a conclusive result tool in this study since it is calculatedTable 6 Comet assay parameters and caspase 3/GAPDH mRNA gene expression in RWPE-1 cells ’groups treated with arsenic, cadmium, and
chromium at high concentrations
Groups Control Highest concentrations
Arsenic (0.1 μM) Cadmium (1 μM) Chromium (1 μM)
Tail length
Median (range) 44.90 (15.6 –151.3) 30.26 (8.3 –165.9) 18.06 (4.4 –54.2) 59.3 (10.7 –196.2)
P<0.001* P<0.0001* P<0.01*
P1<0.0001* P1<0.0001*
P2<0.0001*
Tail intensityMedian (range) 14.01 (0.03 –99.8) 2.72 (0.03 –99.7) 4.88 (0.02 –99.9) 3.82 (0.17 –95.6)
P<0.01* P=0.250 P<0.01*
P1=0.065 P1=0.424P2=0.367
Tail momentMedian (range) 2.82 (0.01 –71.2) 0.53 (0.01 –79.6) 0.38 (0.02 –21.6) 1.41 (0.01 –110.5)
P<0.0001* P<0.0001* P=0.649
P1=0.811 P1<0.01*
P2<0.01 *
Caspase 3/GAPDH mRNAMean±SD 0.71±0.04 0.56±0.07 0.41±0.03 2.31±0.43
P>0.05 P<0. 05 P<0.0001*
P1>0.05 P1<0.001*
P<0.0001*
P, test groups vs. control group; P1, arsenic vs. chromium- and cadmium-treated groups; P2, chromium- vs. cadmium-treated groups
*statistically significant
Fig. 2 Detection of caspase 3 mRNA by RT-PCR in RWPE-1 cells.
Untreated control cells ( lane 1 ), arsenic-treated cells at concentrations 0.1,
0.01, and 0.001 μM(lanes 2 ,3,a n d 4, respectively), cadmium-treatedcells at concentrations 1, 0.1, and 0.01 μM(lanes 5 ,6,a n d 7, respective-
ly), and chromium-treated cells at concentrations 1, 0.1, and 0.01 μM
(lanes 8 ,9,a n d 10,r e s p e c t i v e l y )102 El-Atta et al.
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Ar, Cd, and Cr Effects on Nontumorigenic Human Prostate Cell 103

Fig. 3 Detection of prostate-specific antigen (PSA) mRNA by RT-PCR
in RWPE-1 cells. Untreated control cells (lane 1), arsenic-treated cells at
concentrations 0.1, 0.01, and 0.001 μM (lanes 2, 3, and 4, respectively), cadmium-treated cells at concentrations 1, 0.1, and 0.01 μM (lanes 5, 6,
and 7, respectively), and chromium-treated cells at concentrations 1, 0.1,
and 0.01 μM (lanes 8, 9, and 10, respectively)

from both tail length and intensity. At the lowest con-
centration , chromi um induc ed apoptosis but at moderate
concentration , apoptosis was inhibite d. Meanwhile , chro-
mium significantly induced apoptosis at the highest con-
centration . In spite of the prostate being one of the target
organs for occupationa l and environme ntal chromium
exposure , there is very limite d informatio n on the ad-
verse effect of it on the prostate [33]. Howeve r, Huvinen
and Pukkala [34] found that the incidence of prostate
cance r was significantl y incre ased amon g Finnish
ferroc hromiu m and stainless steel prod uction workers
through 1967– 2011. Chromium metabolite s were found
to be direct genotoxic through generation of reactive
oxyg en species [35]. Also , chromi um was reporte d to
cause DNA repair inhibiti on throug h a competiti on inhi-
bitio n mechanis m as sugges ted by Salniko w and
Zhitk ovich [36]. Wise et al. [37] concluded that exposure
to chromiu m may induc e epithelia l proliferatio n and
DNA damage in target organs that can explain inhibition
of apoptosis at the moderate concentr ation encou ntered
in the presen t study.
Prostate -specific antigen is a glycoprotein that is
expresse d by both norma l and neoplastic prostate tissue
[38]. Elevated serum PSA concentration is alway s asso-
ciate d with certai n pathologica l chan ges in the prostate
includi ng malignan t metaplasi a and usually indicates
prostati c epithelial damage [39]. In the present study,
PSA gene expressio n meas ured by RT-PCR was found
to be not statisticall y differen t betwee n treate d and
untreated cells. In accordance with this result , González
et al. [40] failed to prove an association between total
prostatic -specifi c antige n (tPSA) and arsenic.van
Wijnga arden et al. [41] and Pizent et al. [42] also failed
to prove an associatio n betw een PSA levels and both
blood and urinary cadmium levels. Contr oversiall y,
Achan zar et al. [21] found that RWPE -1 cells stained
positi ve for human PSA after exposur e to arseni c for
29 weeks while De Coste r et al. [43] and Wu et al. [44]
declared an associatio n betwee n PSA level s and both
blood and urinary cadmiu m levels. A si gnificant posi-
tive correlatio n betwee n urinar y chromiu m and tPSA
was found in work ers chronically exposed to chromi um
but a negativ e correlatio n was found with free PSA (whic h represe nts the matured PSA protein whic h has
been inactivate d by interna l proteolyti c cleavage) in the
same workers [33].
Absence of PSA overexpression in the present study could
be explained by short period of exposure or minimal cellular
damage as evidenced by inhibition of apoptosis in all groups
compared to control.

Conclusion and Recommendations

Results of the present study suggest that arsenic and
cadmiu m at low concentration s induced significant
concentration -dependent reductio ns in apoptosis in
RWPE -1 cells while chromium increas ed it. How ever,
at the highes t concentration, apoptosis was relatively
more induc ed by all chemi cals. Arsenic was the most
powerfu l chemic al inhibitin g apoptosis at low conc en-
tration , while at the moderat e and highest concentra-
tions, cadmiu m was the most powerful inhibitin g chem –
ical of RWPE -1 cells’ apoptosis . Thes e results give a
reason for furthe r attention to the occupatio nal and
environmental exposure to these metals that carry the
risk of cancer prostate . We recommen d studyin g the
effects of these metal s on RWPE -1 cells for longer
duration s and increasing doses for subsequent in vivo
study in the future.

Limitations of the Study

This study has some limitations; first, It did not evalu-
ate the synergistic effect of metals whic h simul ate actual
environmenta l condition s on prostatic cells as metals
were tested separatel y. Another limitatio n that may be
added is the study duration and its effects on the results
of PSA expressio n in our study, as a chose n marker or
indicator for the carcinogenic potentia l of those toxic
metals. Thus, a further study with longer duration of
exposure may confir m or reject our finding s regarding
PSA expressio n.

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