Material and Methods [602909]

Material and Methods
Chemicals
Sodium arsenite (NaAsO 2), cadmium chloride (CdCl 2), and
sodium dichromate dehydrate (Na 2Cr2O7.2H 2O) were purchased
fromSigma (St Louis,MO).Chemicalswere dissolved
in sterile distilled water, then filtered with membrane filter
(0.2 μm) tomake 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, 10 0, 1,000 μM); before cell treatments.
Cell lines and Culture Conditions
Non-tumorigenic human prostate epithelial cell (RWPE1)
(ATCC® CRL11609 ™) cell line was purchased from
VACSERA “The Holding Company for Biological Products
and Vaccines, ” Egypt. Itwas cu ltured in a keratinocyte serumfree
medium (K -SFM), supplied with the two additives required
to grow this cell line (bovine pituitary extract, BPE)
and human recombinant epidermal growth factor (EGF)
(Invitrogen (GIBCO), penicillin (100U/ml), and streptomyc in
(100 μg/ml) at 37 °C in 5%CO 2. 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 countedwith
a hemacytometer.The experimentwas repeated twice.
Trypan Blue Test for Cell Viability and Determination
of LC 50
Cells were treated with NaAsO 2, CdCl 2, and Na 2Cr2O7.2H 2O
solutions at different concentrations (as previously mentioned),
then incubated for 48 h to de termine LC 50 of each
chemical using trypan blue exclusion test [ 17]. A concentration
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. LC 50 was found to be 1 μM for CdCl 2 and
Na2Cr2O7.2H 2O, while it was 0.1 μM for NaAsO 2. Diluted
concentrations were tested as the following: 0.1 and 0.01 μM
for CdCl 2 and Na 2Cr2O7.2H 2O, while for NaAsO 2, 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 NaAsO 2 at 0.1, 0.01, and 0.001 μM for 48 h;
group II that was treated with CdCl 2 at 1, 0.1, and 0.01 μMfor
48 h; and group III that was treated with Na 2Cr2O7.2H 2O at 1,
0.1, and 0.01 μMfor 48 h. A matched control group consis ted
of untreated RWPE1 cells was used. Susceptibility to apoptosis
was assessed through DNA fragmentation evaluated by
comet assay and measuring caspase 3/GAPDH gene expression
by semiquantitaive reverse transcriptase- PCR (RTPCR).
PSA gene expression was assessed by
semiquantitaive RT -PCR. Cells were harvested for comet
assay andRNAisolation as follows:
Comet Assay
The comet assay was performed under alkaline conditions.
Briefly, cells were detached by trypsin -EDTA treatment for

5 min and collected by cent rifugation. Cell pellets 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 sl i des were then i mmersed i n pre- chille d lys is s olution
(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
l ysi s, the sl i des were pl aced i n a hori zontal gel el ectrophoresi s
unit 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 electrophoresis,
sl i des were immersed in neutralization buffer (0.4M
Tris-HCl, pH 7.5) and then gently washed three times for
5 min a t 4 °C to re move a lka lis . Fina lly, s lide s we re fixe d in
70% ethanol for 5 min and stored in the dark to dry completely
[18] . Just bef ore i mage anal ysi s, gel s on each sl i de were
stained with 50 ml ethidium bromide (20 mg/ml) and the
slides were examined under a fluorescence microscope
equipped with an excitation filter of 565 nm and a barrier
f i l ter of 590 nm. I mages were captured usi ng a di gi tal camera
and saved as TI FF/JPEG f i l es. A l l steps were perf ormed under
reduced light to minimize DNA damage from ambient ultraviolet
radi ati on.
Images of cells were processed by a computer -assi sted
i mage- anal ysi s system (Comet A ssay I V ) to determi ne the
comet parameters. Resul ts were expressed as tai l l ength (TL ;
the distance that DNA migrated, in μm), percentage of DNA
in ta il (% Ta il; the inte ns ity of migra te d DNA), a nd ta il
moment (TM; tail length×percentage of DNA in tail/100).
RNA Isolation and RT -PCR
Total RNA wa s isolated using AxyPrep Multisource Total
RNA Miniprep kit (Axygen Biosciences, Union City, CA
94587, USA). First -strand complementary (cDNA) synthesis
f rom total RNA and PCR were perf ormed by RT/PCRM aster
Mix. Gold Beads Kits (BIORON The ENZYME Company,
Germany) usi ng caspase 3 -specific primer used the following:
caspase 3 sense, 5 ′-TTCAGAGGGGATCGTTGTAGAA
GTC-3′ and anti sense, 5 ′-CAAGCTTGTCGGCATACTGT
TTCAG -3′ (264 bp fragment) [ 19] . PSA -s pe cific prime rs
yielding 680 bp fragment spanning 4 exons (exon 2, e xon 3,
exon 4, exon 5) are as follows: forward primer, 5 ′-CTCTCG
TGGCAGGGCAGT -3′ and reverse pri mer, 5 ′-AATAGGGG
GTTGATAGGG -3′. A messenger (mRNA ) of gl yceral dehyde
3 phosphate dehydrogenase (G 3PDH) was used as endogenous
external control f or RT -PCR: forwa rd 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 incubated
at 70 °C for 5 min and was pl aced on i ce. The i ncubated
mixture and the forward primer were transferred to the RT/
PCR gold tube, and then the tube was filled to 20 μL byDPEC

DW. The lyophilized pellet was dissolved by vortexing and
was briefly span down. cDNA synthesis reaction was performed
in a thermal cycler (TECHEN TC -312, Model
FTC3102D, Barloworld Scientific Ltd. Stone, Stafford Shire
st 150 SA, UK) by heating the tube for 60 min at 42 °C then
f or 5 mi n at 94 °C f or RTase i nacti vati on.
For caspase 3, PCR was perf ormed by heati ng 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 5min at 94 °C for DNA
denaturation 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 anal yzed by el ectrophoresi s and
ethidium bromide staining on 1.6 % agarose gels, visualized
via a ligh t UV transilluminator (Model TUV -20, OWI S cie ntific,
Inc. 800 242 -5560, France), and photographed under
fixed conditions (the distance, the light, and the zoom). The
resul t photos were anal yzed wi th Sci on I mage® Rel ease A l pha
4.0.3.2. Software for Windows® which performs band
detecti on and conversi on to peaks. A rea under each peak were
calculated in square pixels and used for quantification. Gene
expressi on l evel s were determi ned by cal cul ati ng the rati o
between the square pi xel val ues of the target gene i n rel ati on
to the internal house keeping control gene (GAPDH). Minus
RT controls permitted to rule out genomic contamination.
Si mi l arl y, no products were detected when the RT -PCR step
was carried out with no added RNA, indicating that all reagents
were free of target sequence contamination.
S ta tis tica l Ana lys is
Using the Microsoft Office and MedCalc® programs version
10.0.1, the stati sti cs were summari zed as ari thmeti c mean;
standard deviation for parametric data and range and median
were used for nonparamet ric data. Student t test and M ann
Whi tney test were used to assess the stati sti cal si gni f i cance of
differences. Difference was considered statistically significant
if P<0.05.