MATERIALS AND METHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/46984/3/3.pdf · Swiss albino mice were divided randomly into six separate treatment groups with 8 mice

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.


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:


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


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,


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).


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.


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.


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


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


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.


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


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).


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


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


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


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


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


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.


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.


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.


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


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.


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.


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.


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


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.


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).


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


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


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).


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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MATERIALS AND METHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/46984/3/3.pdf · Swiss albino mice were divided randomly into six separate treatment groups with 8 mice