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Chapter 3 Materials and methods
35
MATERIALS AND METHODS
3.1. Chemicals
HDFa cells were procured from Invitrogen Bioservices, India. Low
Serum Growth Supplement, fetal bovine serum (FBS), human epidermal
growth factor, fibroblast growth factor, heparin, trypsin-EDTA and were
obtained from Invitrogen Bioservices, India. Ferulic acid, monoclonal
antibodies PAb240, anti-TNF !"#$%&-COX-2, anti-iNOS, anti-VEGF, anti-
Bax, anti-Bcl-2, anti- IL-6, '-actin anti-mouse and goat anti-mouse IgG-
HRP polyclonal antibody, 3-(4, 5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H
tetrazolium bromide (MTT), 2,7-diacetyl dichlorofluorescein (DCFH-DA),
rhodamine 123, 1-chloro-2,4-dinitrobenzene (CDNB), 2,4
dinitrophenylhydrazine, 5,50-dithiobis (2-nitro-benzoicacid) (DTNB),
glutathione reductase (GR), hydrogen peroxide (H2O2), nicotinamide
adenine dinucleotide reduced (NADH), nicotinamide adenine
dinucleotide phosphate reduced (NADPH), nitrobluetetrazolium (NBT),
reduced glutathione (GSH), 2-thiobarbituric acid (TBA), trichloroacetic
acid (TCA)were purchased from Sigma chemical Co., St. Louis, MO,
USA.
Radioimmune precipitation assay (RIPA) buffer and bovine serum
albumins (BSA) were purchased from Himedia, Mumbai. The RNeasy
mini kit was purchased from Qiagen, USA. Medox-BioTM Ames test kit
was purchased from Medox Biotech India Pvt. Ltd. Quantitative real-
time polymerase chain reaction teaching kit was purchased from Merk
specialty pvt. Ltd, Mumbai. Low melting agarose (LMPA), normal
melting agarose (NMPA), phosphate-buffered saline (PBS) and all other
chemicals, solvents of analytical grades were obtained from S.D Fine
Chemical, Mumbai and Fisher Inorganic and Aromatic Limited,
Chennai.
Chapter 3 Materials and methods
36
3.2. Evaluation of FA on UVB-induced cellular and molecular changes in HDFa
3.2.1. Culturing of human dermal fibroblasts adult (HDFa) cells
Cultured HDFa cells were incubated at 37 °C, 5% CO2/95% air,
humidified cell culture incubator with supplemented medium 106, low
serum growth supplement, 2% v/v ()%#*" +,-&$)" .)/01!" 2" 3451*"
hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml
+#.&6"(&+/,+*#.%"4/,7%8"(#6%,/!"29"3451*"8):#/&$"#$;"#$%&+&,%&6.< After 7
days the culture was attained approximately 80% confluent and the
cells were harvested with 4 ml of trypsin-EDTA solution. Then the cells
were subcultured for further studies (Ramachandran et al., 2012).
3.2.2. Preparation of FA and mode of administration for HDFa
A stock solution of FA (1 mg/ml) was prepared in 0.05% (v/v)
dimethyl sulfoxide (DMSO). From the stock 10-40 µg/ml was used for
further in vitro studies. 0.05% DMSO was used as a drug control.
3.2.3. Experimental protocol
HDFa cells were divided into six groups as follows:
Chapter 3 Materials and methods
37
3.2.4. Treatment of the HDFa cells
Three test doses (10, 20 and 40 µg/ml) of FA were added to the
grouped HDFa cells thirty minutes before UVB-exposure. Trypan blue
dye exclusion test was carried out to find out the toxicity and suitability
of this concentration of FA for photoprotection studies. Before UVB
exposure, the HDFa cells were washed once with PBS solution. Mock-
irradiated HDFa showed no viability changes over the 30 min period of
incubation.
3.2.5. Irradiation procedure for HDFa cells
HDFa cells were washed twice with PBS and UVB-irradiated in a
thin layer of medium without FBS. A battery of TL 20 W/20 fluorescent
tubes (Heber Scientific, Chennai, India) was used as UVB source, which
possess a wavelength range of 290–320 nm, peaked at 312 nm, and
with an intensity of 2.2 mW/ cm2 for 9 min. The total UVB-radiation
exposure was 19.8 mJ/ cm2, with an average value of 1.52 × 10=>
mJ/cell. Immediately after UVB exposure, the HDFa cells were kept at
37 °C for 4 h at 5% CO2 environment. Irradiated HDFa cells were then
washed with PBS, and transferred to sterile centrifuge tubes for
biochemical analysis (Ramachandran and Prasad 2012).
3.2.6. Preparation of FA for sun protection factor determination
The proposed UV spectrophotometric method of SPF is simple,
rapid, employs low cost reagents and can be used in the in vitro
determination of SPF values in many cosmetic formulations. 100 mg of
FA was dissolved in 100 ml of 0.05% DMSO so the solution of 1mg/ml
was produced. From this 2 ml of solution was withdrawn and diluted to
10 ml with distilled water so the solution concentration 200 3451*"7#."
produced. Then UV reading of FA was taken from wavelength ranging
from 290 to 320 at 5 nm interval and readings were noted down. SPF
was calculated from the formula given by Mansur et al., 1986 by
Chapter 3 Materials and methods
38
utilising values given by Sayre et al., 1979. SPF was calculated three
times and then mean value was taken in consideration.
Where, CF = correction factor = 10, EE = erythemogenic effect of
radiation with wavelength lambda; Abs = absorbance value at lambda
wavelength.
Wavelength (nm) EE x I (normalized)
290 0.0150
295 0.0817
300 0.2874
305 0.3278
315 0.0839
320 0.0180
Table 3 Normalized product function used in the calculation of SPF
The values of EE x I are constants. EE – erythemal effect spectrum; I –
solar intensity
3.3. Evaluation of FA on UVB-induced carcinogenesis in Swiss albino mice
3.3.1. Experimental animals
Female Swiss albino mice weighing between 18-20 g were
purchased from the Central Animal House, Rajah Muthiah Medical
College, Annamalai University, Tamilnadu, India. They were accommodated
under standard environmental condition (temperature 25-28 °C 12 h
light/dark cycle and 65-70% humidity) and they were allowed with
standard laboratory feed and water ad libitum at Central Animal House,
Chapter 3 Materials and methods
39
Department of Experimental Medicine, Rajah Muthiah Medical College,
Annamalai University. All studies were carried out in accordance with
Indian National Law of Animal Care and Use (Reg No./160/1999/
CPCSEA) approved by the ethical committee, Annamalai University
(Ethical Clearance No.859 dated 10.01.2012).
3.3.2. Preparation of FA and mode of administration
For in vivo studies, 50 mg/kg b.wt. concentration of FA was
dissolved in 0.05% dimethyl sulfoxide (DMSO) and administered
intraperitoneally (FAIP) and topically (FAT) at a volume of 0.2 ml/kg b. wt.
3.3.3. UVB-irradiation procedure for experimental animals
At least two days before treatment the dorsal portions of the mice
skin were shaven with an electric clipper (Oster A2) followed by the
application of hair removing cream (Anne French, Geoffery Manners,
Bombay, India). Only mice that showed no signs of hair regrowth were
used in the experiments Lukewarm water was used to wash off excess
cream with sorbs.
For exposure to UVB radiations, mice (dorsal portions shaved) were
kept in polypropylene cages and placed under UVB lamps, Philips
TL40W/12 RS lamp (Heber Scientific, Chennai) emitting 312 nm. The
lamp was mounted 20 cm above the table where the mice placed on.
As per the standard photocarcinogenesis protocol, mice were UVB-
irradiated (180 mJ/cm2) three times a week until the end of the
protocol. The mice were then sacrificed by decapitation after the
experimental period and the full thickness of the dorsal skins were
removed for biochemical and molecular analysis. Mice were UVB-
irradiated as described earlier (Vaid et al., 2010).
Chapter 3 Materials and methods
40
3.3.4. Photocarcinogenesis protocol
Swiss albino mice were divided randomly into six separate
treatment groups with 8 mice in each group: Group I mice were not
UVB-exposed or otherwise served as (0.5% DMSO) vehicle controls, mice
in Group II and III were not UVB-exposed but were subjected with
FA (50 mg/kg b. wt. or 1 mg/mouse) intraperitoneally (FAIP) and
topically (FAT) three times per week on their dorsal skin and abdomen.
Mice in Group IV, V and VI were exposed to UVB (180 mJ/cm2/day)
daily for the first 15th - 24th days. One week after interval of the last
UVB-exposure, the mice were UVB-irradiated (180 mJ/cm2) three times
a week until the end of the experiment. Group V and VI were treated
intraperitoneally (FAIP-UVB) and topically (FAT-UVB) with FA
(50 mg/kg b. wt. per mouse or 1 mg per mouse) 1 h before each
exposure to UVB-radiation three times per week. FAIP and FAT
administration was started at least 14 days before the start of UVB-
exposure and on alternative days.
Chapter 3 Materials and methods
41
3.3.5. Effect of FA on UVB-radiation induced lethality (LD50)
Prior to the radioprotective study, whole body survival study was
carried out to fix the optimum dose of FA. The whole body survival
study was conducted according to Satyamitra et al., 2001. For this mice
were categorized in to 6 groups (n=6).
Radiation only Whole body exposure to (180 mJ/cm2/day)
Experimental groups Administration of different concentrations (30, 40, 50, 60, 70 mg/kg b.wt. ) of FA prior to whole body exposure to UVB radiation
3.3.6. Evaluation of tumor formation
During the experimental protocol, UVB-irradiated dorsal skin
area of the mice was examined for papillomas or tumor on a weekly
basis. The dimensions of all the tumors on each mouse were recorded
until their yield and size stabilized. At that time point, the dimensions of
all of the tumors on each mouse were recorded. Tumor volumes were
calculated using the hemiellipsoid model formula: tumor volume = 1/2
?@A5>B" ?*5CB" ?75CB" 8!" 78)/)" *" D" *)$4%8!" 7" D" 7&;%8" #$;" 8" D" 8)&48%!" #."
described earlier. When tumor yield and growth stabilized, the
photocarcinogenesis experiment was terminated and skin and tumor
samples were collected for the analysis of various biomarkers of
interest.
3.3.7. Preparation of skin tissue for histopathological analysis
The skin tissue was dissected out from three mice in each group
and fixed in 10% formalin solution and then dehydrated in ethanol
(50-100%), cleared in xylene and embedded in paraffin wax. Afterwards
thick sections (3-5 µm) were made using microtome and then stained
with hematoxylin and eosin dye for photomicroscopic observation. All
histopathological changes were examined by pathologist.
Chapter 3 Materials and methods
42
3.4. Evaluation of protective effect of FA on UVB-induced cytotoxicity
3.4.1. MTT assay
The cytotoxic effect of UVB-irradiated HDFa was determined
based on the conversion of MTT into formazan crystals by living cells,
which determines mitochondrial activity (Mosmann, 1983).
Principle
This is a colorimetric assay that measures the reduction of yellow
3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by
mitochondrial succinate ehydrogenase or mitochondrial reductase. The
MTT enters the cells and passes into the mitochondria where it is
reduced to an insoluble, coloured (dark purple) formazan product. The
cells are then solubilised with an organic solvent (eg. isopropanol) and
the released, solubilised formazan reagent is measured
spectrophotometrically. Since reduction of MTT can only occur in
metabolically active cells the level of activity is a measure of the viability
of the cells.
Materials
1. 0.5 g/L MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-
tetrazolium bromide)
2. Dimethyl sulfoxide (DMSO)
3. 96 well plate
4. Microplate reader
Chapter 3 Materials and methods
43
Procedure
Cultured HDFa (1x106 cells/ml) were taken into a 96 well plate.
Then the cells were pretreated with different concentration of FA (8, 40,
80 and 160 µM). After 1 h incubation with FA the cells were exposed to
UVB-irradiation. Then the cells were incubated at 5% CO2 and 95% O2
environment at 37 ºC for 24 h. MTT (0.5 mg/ml) was added to the
incubated cells and then further incubated for another 4 h. The cells
7)/)"6)$%/&(04);"(,/"29"1&$"#$;"%8)".0:)/$#%#$%"7#."/)1,-);!"C99"3E"
of DMSO were added into each tubes. Absorbance was measured in a
microplate reader at 540 nm. Images captured under microscope.
Percentage cytotoxicity was calculated as follows:
% Viability = Total number of viable cell
× 100 Total number of viable and nonviable cells
3.4.2. Determination of protective effect of FA on UVB-induced ROS
generation
2’FGH&68*,/,;&8I;/,(*0,/).6)in
diacetate (DCFH-DA) is the cell
permeable fluorescent and
chemiluminescent probe used to direct
measurement of the intracellular ROS
levels. DCFH-DA is a nonpolar dye,
converted into the polar derivative
DCFH by cellular esterases that are
nonfluorescent but switched to highly
fluorescent DCF when oxidized by
intracellular ROS and other peroxides
(Hafer et al., 2008). Accumulation of
DCF in cells may be measured by an
increase in fluorescence at 530 nm
when the sample is excited at 485 nm. The results were expressed as
percentage fluorescence intensities. In addition, the fluorescence cells
Chapter 3 Materials and methods
44
were analyzed using a phase contrast fluorescence microscope with blue
filter (460 nm).
Reagents
1. Phosphate buffered saline (PBS)
2. 2-7-diacetyl dichlorofluorescein diacetate (DCFH-DA)
Procedure
The percentage of ROS levels was estimated in the control, UVB-
irradiated plus FA treated HDFa cells. Briefly, an aliquot of the isolated
cells (8x106 cells/ml) were made up to a final volume of 2 ml in PBS (pH
7.4). Then, 1 ml cell suspension were taken, to which 10 µl DCFH-DA
(10 µM) was added and incubated at 37 °C for 30 min. Then, FA
pretreated and/or UVB-irradiated HDFa were incubated for 30 min in 6
well plates with 10 µM/ml of DCFH-DA in PBS. Finally, cells were
washed thrice with PBS and the fluorescence intensity was recorded using
spectrofluorometer and the images were captured using fluorescence
microscope (460 nm).
3.4.3. Measurement of protective effect of FA on UVB-induced oxidative
damage
The cultured HDFa cells were harvested by trypsinization and
washed twice with phosphate buffered saline (PBS). The cells were
centrifuged at 20,000 x g for 15 min (4 ºC) with 130 mM KCl plus
50 mM PBS containing 10 µM dithiothreitol. Then the supernatant was
collected and used for the biochemical estimations.
3.5. Preparation of skin tissue homogenate
Approximately 250 mg of mice skin tissue samples to be
homogenized with ice cold potassium phosphate buffer (pH of 7.5).
Homogenates (25% wet w/v) were centrifuged at 1000 rpm for 10 min at
4 °C and the supernatant was used for various biochemical estimations.
Chapter 3 Materials and methods
45
3.6. Measurement of lipid peroxidation
3.6.1. Estimation of thiobarbutric acid reactive substances (TBARS)
Principle
The measurement of thiobarbituric acid reactive substances
(TBARS) was carried out as an index of lipid peroxidation and measured
in terms of malondialdehyde (MDA) content by the method outlined by
Niehaus and Samuelsson, (1968). Malondialdehyde (MDA) and other
TBARS were measured by their reactivity with thiobarbituric acid in an
acidic condition to generate pink coloured chromophore which was read
at 535 nm.
Reagents
1. Trichloroacetic acid: 15%
2. Hydrochloric acid: 0.25 N
3. Thiobarbituric acid (TBA): 0.375% in hot distilled water.
4. TBA-TCA-HCl reagent: Solution 1 to 3 was mixed in the ratio of
1:1:1 freshly prepared prior to use.
5. Stock standard: 4.8 molar solution of stock was prepared from
1, 1,'3, 3'tetramethoxypropane purchased commercially.
6. Working standard: Stock solution was diluted to get a concentration of
48 nmol/ml.
Procedure
The HDFa supernatant and mice skin tissue homogenate (0.5 ml)
of sample was diluted with double distilled water (0.5 ml) then 2.0 ml of
TBA-TCA-HCl reagent was added. The mixture was kept in a boiling
water bath for 15 min, after cooling, the tubes were centrifuged at
1000 x g for 10 min and the supernatant was estimated. A series of
standard solution in the concentrations of 2-10 nmol were treated in a
Chapter 3 Materials and methods
46
similar manner. The absorbance were read at 535 nm. The levels of
TBARS were expressed as nmol/mg protein for HDFa and mmol/mg
protein for skin tissue homogenate.
3.6.2. Estimation of lipid hydroperoxides (LPH)
Principle
In the cell suspension and skin tissue homogenate lipid
hydroperoxide was done by the method of Jiang et al. (1992). In this
method oxidation of ferrous ion (Fe2+) under acidic conditions in the
presence of xylenol orange leads to the formation of a chromophore
which was read at 560 nm.
Reagents
Fox reagent: 88 mg butylated hydroxytoluene (BHT), 7.6 mg
xylenol orange and 9.8 mg ammonium iron (II) sulphate were added to
90 ml methanol and 10 ml H2SO4 (250 mM) mixture.
Procedure
Fox reagent (0.9 ml) was mixed with 0.1 ml of the mice skin
homogenate then incubated for 30 min at room temperature and the
absorbance was read at 560 nm.
Lipid hydroperoxides were expressed as nmol/mg protein for
HDFa and mmol/mg protein for mice skin homogenate.
3.7. Determination of antioxidant status
3.7.1. Assay of superoxide dismutase (SOD, EC 1.15.1.1)
Superoxide dismutase in the HDFa cell suspension and skin
tissue homogenate was assayed by the method of Kakkar et al. (1984).
Chapter 3 Materials and methods
47
Principle
The assay is based on inhibition of the formation of NADH-
phenazine methosulphate, nitroblue tetrazolium formazon. The reaction
is initiated by the addition of NADH to assay mixture. After incubation
for 90 sec, the reaction was terminated by the addition of glacial acetic
acid. The color developed at the end of the reaction was extracted into
n-butanol layer and measured at 520 nm.
Reagents
1. Sodium pyrophosphate buffer: 0.025 M, pH. 8.3
2. Absolute ethanol
3. Chloroform
4. n-butanol
5. Phenazine methosulphate (PMS): 186 mol
6. Nitroblue tetrazolium (NBT): 300 mol
7. NADH: 780 mol
Procedure
The HDFa supernatant/tissue homogenate (0.5 ml) was added to
2.5 ml of ethanol and 1.5 ml of chloroform (chilled reagents were
added). This mixture was shaken for 90 sec at 4 !C and then
centrifuged. The enzyme activity in the supernatant was determined as
follows.
The assay mixture contained 1.2 ml of sodium pyrophosphate
buffer, 0.1 ml of phenazine methosulphate and 0.3 ml of nitroblue
tetrazolium and appropriately diluted enzyme preparation in a total
volume of 3 ml. The reaction was started by the addition of 0.2 ml
NADH. After incubation at 30 !C for 90 sec, the reaction was stopped by
Chapter 3 Materials and methods
48
the addition of 1 ml glacial acetic acid. The reaction mixture was stirred
vigorously and shaken with 4 ml n-butanol. The mixture was allowed to
stand for 10 min; centrifuged and n-butanol layer was separated. The
color density of the chromogen in n-butanol was measured at 510 nm.
A system devoid of enzyme served as control.
The enzyme concentration required to inhibit the chromogen
produced by 50% in one min under standard conditions was taken as
one unit. The specific activity of the enzyme was expressed as units/mg
protein for both HDFa and mice skin homogenate.
3.7.2. Estimation of catalase (CAT, EC 1.11.1.6)
Principle
The catalase activity in the HDFa cell suspension/skin tissue
homogenate was measured quantitatively by the method of Sinha,
(1972). This method is used to quantifying the hydrogen peroxide after
reacting with dichromate in acetic acid. When heated in the presence of
H2O2 dichromate in acetic acid was converted to perchromic acid and
then to chromic acetate. This was read at 620 nm. The catalase
preparation was allowed to split H2O2 for various periods of time. The
reaction was terminated at different time intervals by the addition of
dichromate-acetic acid mixture and the remaining H2O2 as chromic
acetate was determined colorimetrically.
Reagents
1. Phosphate buffer: 0.01 M, pH 7.0
2. Hydrogen peroxide: 0.2 M
3. Dichromate-acetic acid reagent: 1:3 ratio of 5% potassium
dichromate was mixed with glacial acetic acid. From this 1 ml
was diluted again with 4 ml of acetic acid.
4. Standard hydrogen peroxide: 0.2 mM
Chapter 3 Materials and methods
49
Procedure
HDFa cell suspension and mice skin tissue homogenate was
prepared by using PBS. To 0.9 ml of PBS, 0.1 ml of HDFa
suspension/mice skin homogenate and 0.4 ml of hydrogen peroxide
were added. The reaction was arrested after 15, 30, 45 and 60 sec by
adding 2.0 ml of dichromate-acetic acid mixture. The tubes were kept in
a boiling water bath for 10 min, cooled and the color developed was read
at 620 nm. Standards in the concentration range of 20-100 mol were
taken and subjected to the same and proceeded as the test.
The specific activity was expressed as mol of H2O2
consumed/min/mg protein for both HDFa and mice skin homogenate.
3.7.3. Evaluation of glutathione peroxidase (EC 1.11.1.19)
By the method of Rotruck et al. (1973) the activity of glutathione
peroxidase in the cell suspension was evaluated. In the presence of GSH
a known amount of enzyme preparation was allowed to react with H2O2
for a specified time period. Then the remaining GSH content was
measured.
Reagents
1. Tris buffer: 0.4 M, pH 7.0
2. Sodium azide solution: 10 mM
3. TCA : 10%
4. EDTA : 0.4 mM
5. H2O2 solution : 0.2 mM
6. Glutathione solution: 2 mM
Chapter 3 Materials and methods
50
Procedure
0.5 ml of HDFa supernatant/mice skin homogenate was mixed
with 0.2 ml of Tris buffer, 0.2 ml of EDTA, 0.1 ml of sodium azide. To
this mixture, 0.2 ml of GSH followed by 0.1 ml of H2O2 was added,
mixed thoroughly and kept at room temperature for 10 min.
After 10 min, the reaction was arrested by the addition of 0.5 ml of 10%
TCA. The tubes were centrifuged and the supernatant was assayed for
GSH by the method of Ellman (1959). The activity was expressed as
mol of GSH consumed/min/mg protein.
3.7.4. Estimation of reduced glutathione (GSH)
Reduced glutathione in the cell suspension/skin tissue homogenate
was estimated by the method of Ellman (1959). When 5, JG-dithio-bis
(2-nitrobenzoic acid) (DTNB) was added to compounds containing
sulphydryl groups, formation of a yellow colour compound such as 2-
nitro-5-thiobenzoic acid produced by this method.
Reagents
1. Phosphate buffer : 0.1 M, pH 8.0
2. TCA: 5%
3. Ellman’s reagent: 34 mg of DTNB in 10 ml of 0.1% sodium
citrate
4. Disodium hydrogen phosphate: 0.3 M
5. Standard glutathione solution: 10.0 mg/100 ml.
Procedure
HDFa supernatant and tissue homogenate (0.5 ml) was pipette out
and precipitated with 2 ml of 5% TCA. 2 ml of supernatant was taken after
centrifugation and 1 ml of Ellman’s reagent and 4 ml of 0.3 M disodium
hydrogen phosphate were added. The yellow color developed was measured
Chapter 3 Materials and methods
51
at 412 nm. A series of standards (20–100 g) were treated in a similar
manner along with a blank containing 1 ml of buffer. The amount of
glutathione was expressed as expressed µmol/mg protein for HDFa and
mmol/mg protein for mice skin homogenate.
3.7.5. Determination of ascorbic acid (vitamin C)
Ascorbic acid in the skin tissue homogenate was measured by the
method of Roe and Kuether, (1943). Mixing of norit and
2, 4 dinitrophenylhydrazine (DNPH) in the presence of thiourea
(a mild reducing agent) the ascorbic acid was converted to
dehydroascorbic acid. When treated with sulphuric acid the coupled
dinitrophenylhydrazine was converted into a red colored compound.
Reagents
1. TCA: 6%
2. 2, 4 DNPH reagent: 2.0 g of DNPH was dissolved in 100 ml of
9 N sulphuric acid. To this, 4.0 g of thiourea was added and
mixed.
3. Acid washed norit
4. Sulphuric acid : 85%
5. Stock ascorbic acid solution: 10 mg of L-ascorbic acid in
100 ml of 4% TCA.
6. Working ascorbic acid solution: 1 in 10 dilution of stock
ascorbic acid solution with 4% TCA to obtain a concentration
of 0.1 mg/ml.
Procedure
To 0.5 ml of sample, 1.5 ml of 6% TCA was added and allowed to
stand for 5 min and centrifuged. To the supernatant, 0.3 g of acid
Chapter 3 Materials and methods
52
washed norit was added, shaken vigorously and filtered. This converts
ascorbic acid to dehydroascorbic acid. 0.5 ml of the filtrate was taken
and 0.5 ml of DNPH was added, stoppered and placed in a water bath at
37 !C for exactly 3 h. Removed, placed in ice-cold water and added 2.5
ml of 85% sulphuric acid drop by drop. The contents of the tubes were
mixed well and allowed to stand at room temperature for 30 min. A set
of standards containing 20-100 g of ascorbic acid were taken and
processed similarly along with a blank containing 2.0 ml of 4% TCA.
The color developed was read at 540 nm. The values were expressed as
mol/mg of protein.
3.7.6. Estimation of "-tocopherol (vitamin E)
By the method of Baker et al. (1980), "–tocopherol in the tissue
homogenate was estimated. Addition of "-tocopherol forms the
reduction of ferric ions to ()//,0."&,$."#$;":/,;06);"%8)"C!"CG";&:I/&;I*"
red colored complex. Absorbance of the chromophore was read at 520
nm.
Reagents
1. Petroleum ether : 60- 80 !C
2. Double distilled ethanol.
3. 2, 2 dipyridyl solution: 0.2% in double distilled ethanol.
4. Ferric chloride solution: 0.5% in double distilled ethanol.
5. Stock standard: 10 mg of "-tocopherol in 100 ml of distilled
ethanol.
6. Working standard: Stock solution was diluted with ethanol to
concentration of 10 g/ml.
Chapter 3 Materials and methods
53
Procedure
To 0.5 ml of mice tissue homogenate, 1.5 ml of ethanol was
added, mixed and centrifuged. The supernatant was evaporated and to
the precipitate, 3.0 ml of petroleum ether, 0.2 ml of 2, 2 dipyridyl
solution and 0.2 ml of ferric chloride solution were added. Mixed well
and kept in dark for 5 min. An intense red color was developed. 4.0 ml
of n-butanol was added to all the tubes and mixed well. Standard
tocopherol in the range of 10-100 g was taken and treated similarly
along with a blank containing only the reagent. The color in the n-
butanol layer was read at 520 nm. The values were expressed as
mol/mg protein.
3.7.7. Estimation of protein
The Lowry et al. (1951) method used to determine the amount of
protein in the enzyme extract.
Principle
Under alkaline condition proteins are react with the Folin’s
phenol reagent to give a blue color complex. This is due to the reduction
of Folin–Ciocalteu reagent and oxidation of aromatic residues
(tryptophan and tyrosine) present in the protein. The concentration of
the reduced Folin’s reagent is measured by absorbance at 640 nm.
Reagents
1. Alkaline copper reagent
Reagent A: 2% sodium carbonate in 0.1 N NaOH.
Reagent B: 0.5% copper sulphate in 1% sodium potassium
tartarate.
Reagent C: 50 ml of reagent A was mixed with 0.5 ml of reagent
B just before use.
Chapter 3 Materials and methods
54
2. Folin’s phenol reagent - Dilute 1:2 with distilled water.
3. Stock standard -100 mg of bovine serum albumin/100 ml of
water.
4. Working standard -10 ml of the stock standard was diluted to
100 ml to get a working standard containing 0.1 mg/100 ml.
Procedure
Mice tissue homogenate/HDFa (0.5 ml) supernatant was mixed
with 0.5 ml of 10% TCA and centrifuged for 10 min. The precipitate was
dissolved in 1.0 ml of 0.1 N NaOH. An aliquot was taken and 4.5 ml of
alkaline copper reagent added and allowed to stand at room
temperature for 10 min. 0.5 ml of Folin’s phenol reagent was added and
the blue color developed was read after 20 min at 640 nm. A standard
curve was obtained with standard bovine albumin.
3.8. Determination of antigenotoxic effect of FA on UVB-induced DNA
damage
3.8.1. Evaluation of DNA damage by single cell gel electrophoresis in HDFa
Single cell gel electrophoresis (SCGE), or comet assay, used to
estimate DNA damage at the single cell level by the method of
Singh et al., 1988.
Cells embedded in agarose on a microscope slide are lysed with
detergent and high salt to form nucleoides containing supercoiled loops
of DNA linked to the nuclear matrix. Electrophoresis at high pH results
in structures resembling comets, observed by fluorescence microscopy.
The intensity of the comet tail relative to the head reflects the number of
DNA breaks. The basis for this is that loops containing a break lose
their supercoiling and become free to extend toward the anode. Cells
containing greater levels of DNA strand break damage generate comets
with more intense tails.
Chapter 3 Materials and methods
55
Reagents
1. Normal melting point agarose: 1%
2. Low melting point agarose: 0.8%
3. Lysis solution: 7.3 g of sodium chloride, 1.8 g of EDTA and 0.06 g of
Tris base was mixed with 35 ml of distilled water. After mixing,
0.06 g of sodium hydroxide, 1.0 ml of triton X-100 was added and
the solution was made up to 50 ml with distilled water.
4. Electrophoresis buffer: 6.0 g of sodium hydroxide and 186 mg of
EDTA was dissolved in 500 ml of distilled water. The solution was
stored in the refrigerator 1 h before electrophoresis.
5. Neutralising buffer: 0.4 M Tris-HCl, pH 7.4.
6. Staining solution: Ethidium bromide (20 µg/ml).
Procedure
Frosted slides were prepared by pouring 3-5 ml of 1% normal
agarose over frosted glass slides (Gold Coin Microslides, Blue Label
Scientifics). It was allowed to dry at room temperature and placed in
hot-air oven at 70-80 !C for 30 min.
Freshly suspended HDFa cells (50 µl) were mixed with 200 µl in
0.8% low-melting point agarose (LMPA) was cast on to frosted
microscopic slides, immediately covered with cover slip and kept for
10 min in ice box to solidify. Then, the cover slip was removed and a top
layer of 100 µl of LMPA was added and the slides were again cooled for
10 min. The cells were then lysed by immersing the slides in the lysis
buffer for 1 h at 4 !C. After lysis, slides were placed in a horizontal
electrophoresis tank. The unit was filled with alkaline electrophoresis
buffer to a level of 0.25 cm above the slides. The cells were exposed to
the alkaline electrophoresis buffer for 30 min to allow DNA unwinding.
Electrophoresis was conducted in a cold condition for 20 min at
25 V or 300 mA. After electrophoresis, the slides were placed
Chapter 3 Materials and methods
56
horizontally and neutralised with Tris-HCl buffer. Finally, 50 µl of
ethidium bromide was added to each slide and analysed using a
fluorescence microscope. To prevent additional DNA damage, all steps
were conducted under dim light or in the dark.
Twenty five images were randomly selected from each sample and
were examined at 40x magnification in a fluorescence microscope
connected to a personal computer-based image analysis system. Images
were captured with a digital camera with networking capability and
analyzed by image analysis software, CASP (Konca et al., 2003). The
relative amount of DNA appearing in the tail of the comet (percent tail
DNA), tail length and tail moment (% tail DNA x tail length) were linearly
related to DNA break frequency. In each sample a minimum of 200 cells
was counted and cells having DNA damages were expressed as
percentage.
3.8.2. Analysis of antimutagenic effect of FA against UVB-induced mutagenesis
In the early 1983’s Maron and Ames was originally developed the
mutagenesis test. The single base level DNA genetic damage was
determind by this assay with using an Escherichia coli AB1157 test
strain. This strain has a single mutation (His-) that has turned off
histidine biosynthesis. So the bacteria requires exogenous histidine to
survive and will starve to death if grown without this essential nutrient
(auxotrophy). During a mutagenic event, E.coli AB1157 can undergo a
reverse mutation turning the essential gene back grown in the absence
of histidine (His- to His+).
Materials
1. Autoclave, centrifuge, incubator, electronic balance, pH meter.
2. Measuring cylinder, conical flask, beaker, test tubes, Petri plates and pipettes.
3. Micropipettes, tips, microfuge tubes and stand.
Chapter 3 Materials and methods
57
Procedure
The E. coli strains (His-) were incubated for 24 h at 37 °C in LB broth
with 0.5% NaCl. After UVB-irradiation and/or FA treatment cell
suspensions were serially diluted using PBS and the dilutions were spread
plated on minimal soft agar plates and incubated at 37 ºC for 48-72 h in an
incubator. Number of reverted colonies present in the sample was
enumerated after plating and number of Colony Forming Units was
calculated (CFUs/ml). A sample was considered to be mutagenic when the
numbers of revertant colonies were more than the non-irradiated control
yield.
Calculation
No. of colonies No. of CFUs /ml = Dilution factor
No. of CFUs/ ml after UV irradiation Percentage of survival = X 100 No. of CFUs/ml before UV irradiation
3.8.3. Analysis of mitochon !"#$%&!#'()*)+!#'*%,-&*'&"#$%./0m) in HDFa
The changes in ( m in different treatment groups were observed
microscopically and determined spectrofluorometrically using fluorescent
dye Rh 123 (Bhosle et al., 2005). To the treated and control HDFa,
1 l of rhodamine-123 (5 mmol) was added and kept in the incubator for
30 min. The cells were then washed with PBS and observed with a
fluorescence microscope using blue filter (450-490 nm). Polarized
mitochondria emit orange-red fluorescence and depolarized mitochondria
emit green fluorescence.
Chapter 3 Materials and methods
58
Reagents
1. 1% PBS.
2. Fluorescent probe – Rh-123.
Stock - mg/ml in 1% PBS.
Working – 10 µl from stock and made up to 1 ml with PBS.
Procedure
After incubation with treatment compounds for 24 h, 1 µl of
fluorescent dye, Rh-123 (5 mM) was added to HDFa cells and kept in
the incubator for 15 min. At the end of the incubation period, cells were
washed with PBS and observed under a fluorescence microscope using
blue filter (450-490 nm). The fluorescence intensity was measured
?KEx/KEm=490 nm/530 nm) by spectrofluorometer (Shimadzu, USA). The
results were expressed as arbitrary units (a.u) of the fluorescence
intensity (FI).
3.8.4. Evaluation of apoptotic morphological changes by acridine orange/
ethidium bromide dual staining
Ethidium bromide (EtBr) is a cyclic planar membrane
impermeable molecule that binds between the stacked base-pairs of
relaxed DNA. Apoptotic cells uptake EtBr and emits red/orange
fluorescence under 550 nm. Acridine orange is a DNA selective and
membrane permeable fluorescent cationic dye freely enters normal cell
nuclei and emits green fluorescence under 525 nm
(Lakshmi et al., 2008).
HDFa cells exhibiting typical changes of apoptosis i.e. nuclear
condensation, membrane blebbing etc. were stained with AO/EtBr and
% apoptotic cells were calculated as a function of total number of AO
stained cells present in the field.
Chapter 3 Materials and methods
59
Reagents
1. Methanol: glacial acetic acid (3:1 ratio)
2. Phosphate buffered saline (PBS)
3. Acridine orange (AO): Ethidium bromide (EtBr) (1:1 ratio)
4. 6 well plate
Procedure
HDFa cells were seeded in 6-well plate (1x106/well) and
incubated in CO2 incubator for 24 h. The cells were fixed with
methanol:glacial acetic acid (3:1) for 30 min at room temperature. Then,
the cells were washed in PBS, and stained with 1:1 ratio of AO/EtBr.
Stained cells were immediately washed again with PBS and viewed
under a fluorescence microscope. The number of cells showing features
of apoptosis was counted as a function of the total number of cells
present in the field.
3.9. Western blot analysis for proinflammatory markers expression
The western blot analysis was carried out for TNF- !"LMN-2 and
IL -6 expressions in FA plus UVB-irradiated HDFa cells and mice skin
tissue by the method of Towbin et al. (1979). The results were
$,/1#*&O);"%,"'-actin gene expression.
Principle
Following the protein estimation, the samples were separated
using SDS-PAGE gel electrophoresis and the separated molecules are
blotted onto a polyvinylidene fluoride (PVDF) membrane. After blocking,
the primary antibody was added and allowed to bind to the protein
followed by washing (which removes nonspecifically bound antibody);
then an enzyme-labeled secondary antibody was added, to detect the
primary antibody. The location of the secondary antibody was
Chapter 3 Materials and methods
60
determined by adding an appropriate substrate for the enzyme conjugated
to the secondary antibody.
Reagents
1. Acrylamide stock: 30% acrylamide, 0.PQ" R!RG-methylene
bisacrylamide.
2. Separating gel buffer: 2.25 M Tris, 0.6% sodium dodecylsulfate
(SDS), pH 8.8
3. Sample buffer: 0.063 M Tris, 2% SDS, 10% sucrose, 0.01%
bromophenol blue, pH 6.8
4. 10% ammonium per sulfate.
5. N,N,N',N'-tetramethylethylenediamine (TEMED).
6. Running gel buffer (5X): 0.25 M Tris, 0.5% SDS, 1.92 M glycine
30.3 g Tris 5.0 g SDS and 144.1 g glycine were dissolved in 700
ml of distilled water. pH was adjusted to 8.3 with conc. HCl and
diluted to 1 L with distilled water. The working running buffer
was prepared by making a 1:5 dilution of the stock 5X buffer
with distilled water.
7. Water-saturated isobutanol.
8. '-Mercaptoethanol.
9. Staining and fixing solution:
2.5 g coomassie brilliant blue R250 was dissolved in 1 L
solution containing methanol, acetic acid and distilled water in
the ratio 5:1:4.
10.Destaining solution: 100 ml of absolute methanol and 100 ml of
glacial acetic acid were mixed with 800 ml of distilled water.
Chapter 3 Materials and methods
61
Procedure
Treated HDFa cells were washed with PBS and detached using
0.25% trypsin/EDTA solution. Cell suspensions were centrifuged and
the pellets/mice skin homogenate were lysed with an ice-cold lysis RIPA
buffer containing a protease inhibitor cocktail (Sigma–Aldrich, St. Louis,
MO, USA) for 30 min. The lysate was centrifuged at 4°C at 13,000 rpm
for 10 min and the supernatant was used to determine protein
concentration using Nanodrop 2000 (Thermo Scientific, USA).
Transfer of proteins to membrane
L)**" )S%/#6%." 6,$%#&$&$4" J9" 34" ,(" :/,%)&$." 7)/)" .0+T)6%);" %,"
electrophoresis on 12% SDS-PAGE gel and transferred to a
PVDF membrane using transblot semi-dry apparatus (Biorad, USA).
Before assembling the transfer system, soaked PVDF membrane in
methanol for 10 min and blotting papers in cold transfer buffer.
Prepared sandwich, blotting paper, membrane, gel and blotting paper,
were placed in the transfer apparatus and few drops of transfer buffer
was added and subjected to an electric current 20 V for 1 h under cold
condition. After the transfer, the sandwich was removed from the
transfer system. Membrane was stained with 0.5% ponceau in
1% acetic acid to confirm equal loading.
PVDF membranes were blocked with non-fat milk (5% (w/v) for
6 h and then incubated overnight with TNF- , COX-2 and IL-6
antibodies (Sigma-Aldrich, USA), in blocking solution at 37 °C. Then the
membranes were washed with TBST thrice with 10 min interval and
incubated with secondary antibody (diluted 1:2000) in blocking solution
for 2 h at 37 °C. Then, the PVDF membranes were washed with TBST
thrice with 10 min interval and the developed bands were detected
using a DAB solution. The images were acquired by Image Studio
software (LI-COR, USA).
Chapter 3 Materials and methods
62
3.10. Quantitative RT-PCR
3.10.1. Analysis of expression of DNA repair enzymes and peroxisome
proliferator activated receptor
Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) for
the mRNA expression levels of XRCC1, GADD45 , hOGG1, ATM and
PPAR- ! in HDFa were performed using Cell Direct One-Step qRT-PCR
SYBR green detection kit (Invitrogen, Carlsbad, CA) in a Realplex
Mastercycler (Eppendrof, Indianapolis, CN). 18S rRNA house keeping
gene expression were used for normalization.
Reaction mixture
S. No PCR reaction mixtures Volume
1 Super script IIIRT/platinum Taq mix !"#
2 2x PCR reaction mixer $%!"#
3 %!"&!'()*+),!-)./0 1 "l
4 %!"&!)010)20!-)./0) !"#
5 SYBR Green $!"#
6 Template RNA sample $!"#
Primers
XRCC1 FP: %3-CTGGGGAGTAGGACGTCAGTGCTG-43
567!%3-GGCTTGCGGCACCACCCCATAGAGC-43
Human "#$$%&'
867!%3-CTAGCCGTGGCAGGAGCAGC-43
567!%3- TGAGCAGCTTGGCCGCTTCG-43
Human hOGG1 867!%3-CCTCCTCCCCTTCCCTTCAACCAAG-43
567!%3-TTGGCCCACACGAGGTCCAGA-43
PPAR-' 867!%3-GCCTGTCTGTCGGGATGT-43
567!%3-GGCTTCGTGGATTCTCTTG-43
PPAR-!
FP: %3-GCCCTTTGGTGACTTTATGGA-43
567!%3-GCAGCAGGTTGTCTTGGATG-43
18S RNA 867%3-AGGAATTCCCAGTAAGTGCG-43
567%3-GCCTCACTAAACCATCCAA-43
Chapter 3 Materials and methods
63
Procedure
The total RNA was extracted from the HDFa cells using RNeasy Mini
kit (Qiagen, USA) as per the protocol recommended by the
manufacturer. The mRNA expression of XRCC1, "#$$%&'( hOGG1, ATM
and PPAR- ! in HDFa was determined by using real-time PCR, as
described previously (Ponchel et al., 2003). RNA purification and
quantity was analyzed by nanodrop 2000 (thermoscientific, USA).
Experiments were run in triplicate to conform amplification integrity.
Manufacturer-synthesized primer pairs were used to measure the mRNA
expression. For cDNA synthesis, PCR cyclic condition used were 25 °C
for 10 min; 42 °C for 50 min; 75 °C for 15 min. The cyclic condition
used for cDNA amplification was 95 °C for 2 sec; 55 °C for 15 sec; and
68 °C for 20 sec, as prescribed by the primer’s manufacturer. The
expression levels of genes were normalized to 18S mRNA expression
level. The cyclic threshold for positivity of real-time PCR was determined
based on negative controls. The calculations for determining the relative
level of gene expression were made using the cycle threshold
(Ct) method. The mean Ct values from triplicate measurements were
used to calculate the expression of the target gene using the 29::;<
formula.
:;<= Ct values of 18S rRNA – values of Ct gene of interest
::;<=!:;<!('!>(?<)(#!– :;<!('!2+/-#0
RQ= 29::;<
3.11. Immunohistochemistry
3.11.1. Analysis of iNOS, VEGF, mutated p53, Bcl-2 and Bax in
photocarcinogenic mice skin
Immunohistochemistry is the localization of antigens in tissue
sections by the use of labeled antibodies that causes the antigens in the
tissue to light up under a light microscope. 5 µM thick tissue sections
Chapter 3 Materials and methods
64
were fixed with formaldehyde and embedded with paraffin for further
analysis or long-term storage. Sections were deparaffinised with xylene
followed by rehydrated with 100% ethanol, 90% ethanol, 80% ethanol
and 70% ethanol and wash the sections with double distilled water.
Sections were incubated in antigen retrieval buffer for 15 min and then
allowed to cool to room temperature for 20 min. The sections were then
blocked with blocking buffers include 0.5% BSA in PBS for 20 min and
treated with 5% normal horse serum and 1% BSA in PBS for 2 h at
room temperature. The samples were then incubated overnight with
primary antibodies at 37 °C. Both primary and secondary antibodies are
diluted into a buffer to help stabilize the antibody, promote the uniform
dissemination throughout the sample and discourage nonspecific
binding. After overnight incubation the sections were washed with PBS.
Then the sections were rinsed with rinse buffer (0.5% BSA in PBS) for
three times. Finally sections were incubated with secondary antibodies
for 2 h. Then the samples were viewed and images captured by
fluorescent microscope.
3.12. Molecular docking
Molecular docking was performed on Red Hat Enterprise Linux EL 5
workstation using Maestro (Schrodinger LLC 2009, USA). GLIDE 5.5
searches were performed for understanding docking interactions
between FA and 66@5ABC. All the molecular modeling was carried out
using OPLS AA (Optimized Potential Liquid Simulation for All Atom)
force field (Glide 2009). PyMOL (DeLano WL, 2002) software was
employed for the analysis of hydrogen bond interactions. Hydrophobic
interactions were analyzed between protein and ligand using Ligplot
software (Wallace AC, 1995).
Ligprep 2.3 module (Schrödinger, USA) was employed for FA
-)0-+)+<.(?D! EF0! <F)00! ,./0?2.(?+#! >)G2<+#! 2<)H><H)0! ('! 66@5! ABC!
(PDB Id: 1K7L/ PDB Id: 3DZY) and Cox-2 (PDB Id: 6COX) were
downloaded from the Protein Data Bank (PDB) (http://www.rcsb.org).
Chapter 3 Materials and methods
65
Protein preparation wizard of Schrodinger’s was used for PPA5ABC!+?,!
COX-2 preparation. Non-hydrogen atoms were minimized until the
average root mean square deviation reached default value of 0.3 Å.
Sitemap 2.3 was used to understand binding site in the ligand binding
,(/+.?!IJKLM!('!<F0!66@5ABC!+?,!;NO-2 (Schrodinger Suite 2009).
Induced fit docking (IFD) was performed to predict ferulic acid
binding modes and 2<)H><H)+#!/(10/0?<2!.?!<F0!JKL!)0P.(?!('!66@5ABC!
and COX-2 using Glide and Prime modules. The prepared proteins were
loaded in the workstation and the Grid values were calculated about
20 Å in order to cover all the active site amino acids. The Vander Waal’s
radii of nonpolar amino acids and ligand atoms were scaled by a default
value of 0.50. About 20 conformational images were created and
analyzed for the best conformational pose based on the docking score
and glide energy.
3.13. Statistical Analysis
All the values were expressed as means of six (n=6) determinations.
The data were statistically analyzed using one-way analysis of variance
(ANOVA) on SPSS (statistical package for social sciences) and the group
means were compared by Duncan’s Multiple Range Test (DMRT). The
results were considered statistically significant if the P <0.05 levels.
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