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Materials and Methds

3.1 Materials

3.1.1 Chemicals and reagents

Chemical Vendor

Acetic acid glacial (CH3COOH) RANKEM, RFCL

Acridine Orange Sigma- Aldrich

Agarose low melting point (LMA) Sigma- Aldrich

Agarose routine Sigma- Aldrich

Ammonium chloride (NH4Cl) Ranbaxy

Boric acid SD-Fine Chem, LR

Citric acid Merck

Cyclophosphamide Sigma-Aldrich

Cytochalasin B Sigma- Aldrich

Dextrose Loba- Chemie

DPX Sigma-Aldrich

Eosin-Y Sigma-Aldrich

Ethanol Merck

Ethidium bromide (EtBr) Sigma-Aldrich

Ethidium diamine tetra acetic acid

(disodium salt) (EDTA)

Sigma-Aldrich

Ethyl methane sulphonate (EMS) Sigma-Aldrich

Fetal bovine serum (FBS) Sigma-Aldrich

Giemsa Stain Sigma-Aldrich

Ham‟s F12 Nutrient medium Gibco

Igepal Sigma-Aldrich

L-Glutamine Gibco

Methanol HPLC grade RANKEM, RFCL

Metronidazole Nicolas Piramal India Ltd.

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Phosphate buffer saline (PBS) Sigma- Aldrich

Potassium bicarbonate (KHCO3) Sigma- Aldrich

Potassium chloride (KCl) Sigma- Aldrich

Potassium hydroxide (KOH) Sigma- Aldrich

Propidium Iodide (PI) Sigma- Aldrich

RNase from bovine pancrease Sigma- Aldrich

Sodium bicarbonate Gibco

Sodium chloride (NaCl) Sigma- Aldrich

Sodium citrate (RM1415) HiMedia

Sodium hydroxide (NaOH) Sigma- Aldrich

Sucrose (194957) SRL

Sulfuric acid (H2SO4) RANKEM, RFCL

Trizma base Sigma- Aldrich

Trypsin-EDTA Gibco

Tween 20 Sigma- Aldrich

Xylene Rankem- RFCL

Antibody markers

Fluorescent marker Vendor

CD71-FITC-antimouse transferine receptor B D Biosciences

CD61-PEantimouse B D Biosciences

Rabbit anti-mouse IgG HRP conjugate Sigma- Aldrich

3.1.2 Equipments

Equipment Vendor

Biochemical Analyzer (Semiautomatic) Erba, Menheim, Germany

Centrifuge (Bench top; 3K-30) Sigma

CO2 incubator Binder

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Cytospin Thermo Shandon, UK

Deep Freezer Thermo

Digital Camera (DS-Fi1) Nikon, Japan

Electrophoresis horizontal submarine system Bio Rad

ELISA reader Thermo electron corporation

Flow cytometer (BD Facs ARIA) BD, Biosciences, California

Flow cytometer (BD Facs CANTO) BD, Biosciences, California

Fluorescent inverted microscope IX-41 Olympus, Japan

Hematology Analyzer Humacount

Leminar flow (Bio safety hood) Esco

Light Microscope (E-200) Nikon, Japan

Magnetic stirrer cum hot plate, RET Ika, Werke-Gmbh, Germany

pH meter, Thermo orion-420 A+ Thermo Orion, USA

Vacuum dryer Thermo Electron,

Weighing balance, ME215P Sartorius AG Gottingen, Germany

3.1.3 Preparations of working solutions

(a) Formaline (10%)

Formaldehyde 10mL

In 90 ml of saline solution.

(b) Phosphate buffered saline (PBS) (1000 mL)

NaCl 7.20 gm

Na2HPO4 1.48 gm

KH2PO4 0.43 gm

Prepared in d.w.

(c) Hypotonic solution (100 mL)

KCl 0.075 M

Prepared in d.w.

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(d) Normal saline (1000 mL)

NaCl (0.91%; w/v) 9 gm

Prepared in d.W.

(e) Carnoy’s fixative solution (4 mL)

MeOH 3 mL

CH3COOH 1 mL

Prepared fresh

(f) Alsever’s solution (100 mL)

Dextrose 2.05 gm

Sodium citrate 0.80 gm

Sodium chloride 0.42 gm

Prepared in d.w.

(g) RBC lysis buffer (100 mL)

Ammonium chloride 830 mg

Potassium bicarbonate 100 mg

EDTA 4 mg

Prepared in d.w.

(h) Giemsa staining solution (100 mL)

Giemsa stain 10 mL

Prepared in d.w.

(i) Eosin-Y staining solution (100 mL)

Eosin-Y 100 mg

Prepared in d.w.

(j) Buffer’s for COMET assay

Lysis buffer (500 mL)

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NaCl (2.5 M) 73.05 gm

Disodium EDTA (100 mM) 18.6 gm

Trizma base (10 mM) 0.6 gm

NaOH 4 gm

pH adjusted to 10 using NaOH or HCl

Electrophoresis buffer (500 mL)

Tizma base 5.44 gm

Boric acid 2.76 gm

Disodium EDTA 0.46 gm

pH 8.3-8.4

EtBr Staining Solution (10X stock; 20 µg/mL) (50 mL)

EtBr 10 mg

Prepared in dH2O.

(k) Buffer’s for in vitro micronuclei

Positive control (EMS 10 mL)

EMS 3 mM/ 6 mM

Prepared in dH2O

Ham’s F12 culture medium (complete) (1000 mL)

L-glutamine 300 mg

sodium bicarbonate 2 g

antibiotic/antibiotic solution 10 mL

supplemented with 10% heat-inactivated FBS

Trypsin Solution

Trypsin 0.25%

EDTA 0.02%

Prepared in PBS

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Staining solution-I

Sodium Chloride 292 mg/ 100 mL

Sodium citrate 500 mg/ 100 mL

RNAse 5 mg/ 100 mL

EtBr 6.25 mg/ 50mL

Igepal 150 µl/ 100mL

Above individual stock solutions were prepared in d.w. and 3 mL of each were

mixed at the time of use to prepare 15 mL of staining solution-I.

Staining solution-II

Citric Acid 7.5 gm/ 100 mL

Sucrose 4.275 gm/ 100 mL

EtBr 10 mg/ 50 mL

Above individual stock solutions were prepared in d.w. and 3 mL of each were

mixed with d.w. (6 mL) at the time of use to prepare 15 mL of staining solution-II.

(l) Buffer’s for in vivo micronuclei

Staining solution (100 mL)

Acridine Orange 10 mg

Glacial Acetic Acid 1% v/v

Prepared in d.w.

Washing solution (100 mL)

Glacial Acetic Acid 5% v/v

Prepared in alcohol (abs.)

3.1.4 Animals

Male and female healthy Wistar rats (65–80 gm; ~ 6 weeks old) and adult

BALB/C mice (22-25 gm; 8–10 weeks old) were procured from the Institutional

animal house. They were quarantined for 2 weeks before use in regulated

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environmental conditions (well-ventilated with > 10 air changes/h; 12-h light/dark

photoperiod; temperature 24±2ºC; relative humidity, 60 ± 10%), according to

Committee for the Purpose of Control and Supervision of Experiments on Animals

(CPCSEA) guidelines. The animals were randomized into groups keeping the weight

variation within ± 15% of the mean weight within each sex and were housed in

polycarbonate cages, fed with certified rodent pellet feed (Ashirwad Industries,

Chandigarh, India). Water was available ad libitum. The components of feed are

subjected to routine analysis at the quality control division of this institute and were

not found to vary to any noticeable extent during period of this study. All experimental

protocols were approved by Institutional Animal Ethics Committee and performed as

per the Guidelines for Animal Care as recommended by the Indian National Academy,

New Delhi (1992).

3.2 Methods

3.2.1 Preparation and chemical characterization of WSF

WSF, the bioactive fraction used in the present study was derived from

aqueous alcoholic (50%) extracts of roots and leaves of W. somnifera (AGB 002) and

the results pertaining to the chemical constituents are given in the appendix (Malik et

al., 2009). In brief finger printing of WSF formulation employing HPLC profile of

isolated chemical markers were carried out by two protocols. According to protocol-I,

HPLC was performed using acetonitrile: water gradient (with varying concentrations

of acetonitrile) and protocol-II involved isocratic resolution employing methanol: H2O

(60:40). Protocol-I resolved the mixture of eight markers where WS1 and WS2

appeared as a common single peak. In protocol-II, besides WS1 and WS2 both WSC

and WSD, in addition were resolved completely. The formulation contains almost

equal quantities of withanolide-A and withaferin-A (Malik et al., 2009).

Experimental methods

3.2.2 Preparation of WSF for oral dosing

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WSF was solubilized in 1% gum acacia and administered orally by gavage

according to body weight. A vehicle (1% gum acacia in distilled water) was used as

control.

3.2.3 Preparation of reference compounds

Reference compounds Cyclophosphamide, EMS and metronidazole were

prepared by dissolving adequate quantity in distilled water to obtain derived

concentration. For solubilizing metronidazole 1% gum acacia was used.

3.2.4 Acute toxicity

Mice were grouped as per the following schedule.

Group WSF

(mg/kg, per oral)

Sighting study Main study

No of mice No of mice

I 5 1 -

II 50 1 -

III 300 1 -

IV 2000 1 5

V Vehicle 1 5

For sighting study, WSF was administered at fixed doses stepwise, as per the

OECD guide line (OECD, 2001). For the main study the highest dose i.e., 2000 mg

was given. Control group received vehicle only. The volume administered was 1 mL/

100 gm body weight.

Mice were kept in Perspex chambers and observed continuously for first two

hours, then half an hour interval for the next six hours for any change in the gross

general behavior and then daily for the next 14 days for mortality. During the

investigation period gross observations were made and body weight recorded. In

addition hematological parameters and biochemical parameters were determined in

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blood collected by retro- orbital sinus puncture. Hematological parameters included

hemoglobin (Hb), red blood cells (RBC), white blood cells (WBC), lymphocytes

(Lym.), neutrophils (Neutr.) which were measured with the aid of an automated

hematology analyzer (Humacount; Human, Weisbaden, Germany). Clotting time (CT)

was measured by collecting approximately 25 µl blood in microhematocrit glass

capillary tube as described by Rubio-PÓO et al., (1998). Bleeding time (BT) was

measured by cutting the tail vein (tail vein bleeding time) as described by Broze

(2001). A transverse incision was made over a lateral tail vein at a position where the

diameter of tail is 2.25- 2.5 mm. the time from the incision to the cessation of bleeding

is recorded as BT. Biochemical parameters included blood glucose (Glu.), urea,

creatinine (CRTN), cholesterol (CHO), triglycerides (TG), uric acid (UA), total

proteins (TPR), bilirubin (BIL), aspartate aminotransferase (AST) and alanine

aminotransferase (ALT) which were evaluated using commercial kits procured from

Bayer Diagnostics (Baroda, India) with the aid of a clinical chemistry analyzer Chem-

7.

At the end of investigation aniomals were euthanized and subjected to

necropsy examination, recording of organ weights. Tissues were preserved in buffered

formaline saline for histopathology. A histopathological assessment of all the vital

organs was made by processing and staining of samples. Tissues about (2.5- 3.0 mm

thick) of the control and treated animals were paraffin embedded and 5 µ thickness

sections were prepared and processed according to standard procedure of Krajian

(1963). The prepared sections were stained with hematoxyline and eosin and slides

were observed by the vetenarian pathologist. Light microscopic examination of

multiple tissue sections was performed and representative images were taken with the

Nikon E200 microscope aided with the imaging software (NIS Element BR 3.0;

Japan).

3.2.5 Chronic toxicity

Experimental groups and treatment- Rats were grouped in different groups and

treated as per the following schedule.

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Group No of rats Treatment (mg/kg, per oral)

I 10 Male

10 Female Vehicle

II 10 Male

10 Female WSF 250

III 10 Male

10 Female WSF 500

IV 10 Male

10 Female WSF 1000

V 10 Male

10 Female Cyclophosphamide (CP) 40

VI 10 Male

10 Female Metronidazole (MTZ; i.p.) 125

Test material WSF was administered once daily for a period of 12 months. One

highest dose (1000 mg/kg) for WSF treatment was selected as limit dose. Thereafter,

concurrent descending doses were selected to define any dose related response and no-

observed-adverse-effect level (NOAEL). CP and MTZ were administered once daily

of 7 days.

Observations

Following observations were made in group I, II, III and IV.

General clinical observations were made at least once a day usually at the

beginning and end of each day. All animals were checked for morbidity or mortality

and for specific toxicological effect. Detailed clinical observation made outside the

home cage, preferably in a standard arena and carefully recorded using scoring system.

Clinical signs of toxicological manifestation include change in skin, fur, eyes, mucous

membranes, occurrence of secretions and excretions, anatomic activities (lacrimation,

piloerection, pupil size, unusual respiratory pattern), change in gait, posture, and

response to handling as well as presence of clonic and tonic movements, stereotypes

(excessive grooming, repetitive circling) and bizarre behavior.

Food and Water Consumption

Food and water consumed in rats was measured daily and weekly consumption

was calculated for first 13 weeks and thereafter on monthly for 12 months.

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Body Weight

Body weight was recorded once a week during the first 13 weeks and then after

4 weeks interval for 52 weeks.

Hematology

Hematological parameters were measured at the end of 3, 6 and 12 months

post-dosing. The parameters include RBC, WBC, Hb, Lym, Neutr., platelet (PLT),

haematocrit (HCT), mean carpuscular volume (MCV), mean carpuscular haemoglobin

(MCH), mean carpuscular haemoglobin concentration (MCHC) were measured with

the aid of an automated hematology analyzer (Humacount; Human, Weisbaden,

Germany). CT and BT were measured as described in section 3.2.4.

Serum biochemistry

Serum biochemistry was evaluated at the end of 3, 6 and 12 month post-

dosing. The parameters included Glu., Urea, CRTN, CHO, TG, UA, TPR, BIL, AST,

ALT, alkaline phosphatase (ALP), albumin (ALB), calcium (Ca), sodium (Na) and

potassium (K) were evaluated using commercial kits as described in section 3.2.4.

Urinalysis

Urinalysis was carried out at the end of 3, 6 and 12 months post-dosing.

Animals were kept in metabolic cages to collect urine sample. Urine examination was

done for volume, specific gravity, color, pH, protein, glucose and other determinants

include ketone, urobilinogen (Urobil), bilirubin (Bil) and occult blood using Multistix

10 SG (Bayer Diagnostics, Baroda, India). In addition of above parameters urinary

sediments, such as pus/epithelial cells, casts and crystals of calcium oxalate were

examined using Nikon E-200 microscope.

Gross necropsy and Organ/Body weight ratio and histopathology

The animals were euthanized by CO2 inhalation at the end of the experiment.

All animals were subjected to full detailed necropsy include careful examination of the

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external surface of the body, all orifices, and the cranial, thoracic and abdominal

cavities. Weights of the vital organs were recorded using analytical balance and then

organ to body weight ratio for each organ calculated. Histopathological examination

was carried out as described in section 3.2.4.

3.2.6 In vivo Chromosome aberration assay

Chromosal aberration assay was performed in Groups I, II, III, IV (WSF-

treated) and V (cyclophosphamide –treated) as shown in section 3.2.5.

Procedure

At the end of treatment schedule of chronic toxicity, animals were given

colchicine (400 µL; 4 mg/kg) intraperitoneally and sacrificed after 2 hr. Bone marrow

from the femurs were extracted and chromosome preparation were made according to

the method of Adler (1984) and Savage (1975) with slight modifications as described

in Fig. 2.

3.2.7 In vivo COMET assay

The Comet assay was performed in Groups I, II, III, IV (WSF- treated) and

Group V (cyclophosphamide -treated) as shown in section 3.2.5.

Gel electrophoresis

Whole blood single cell gel elecgtrophoresis was carried out according to the

method of Chandna, (2004) and Dhawan et al., (2001) with minor modifications as

depicted in Fig. 3. Briefly, 50 µL of blood containing EDTA was embedded in 450 µL

of 0.1% agarose and spread over a pre- coated microscopic glass slide. The gel was

covered with a glass cover slip and left to set at 4ºC for 5-10 minutes. Gel embedded

cells were lysed in lysing solution for 20 min at 4ºC, to allow DNA unwinding, and-

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Fig. 2 Schematic flow chart for in vivo bone marrow chromosome aberration assay.

Page | 46

Fig. 3 Schematic flow chart for in vivo COMET assay.

Page | 47

electrophoresed in electrophioresis buffer at 400 mA and 50 V for 20 minutes to

separate DNA. Slides were stained with EtBr staining solution for 20 minutes.

Comet Analysis

Stained slides were then examined with an Olympus fluorescence microscope

IX 41 equipped with an excitation filter (BP 510 nm) and a barrier filter (590 nm). A

computerized imaging analysis system (KOMET 5.5) was used to analyze the amount

of (i) DNA damage, (ii) olive tail moment values, and (iii) tail coefficient variance. A

total of two hundred cells were examined for each sample.

3.2.8 In vivo Micronucleus assay

3.2.8.1 Bone marrow micronucleus assay

Micronucleus assay was performed in all Groups I, II, III, IV (WSF –treated)

and V (cyclophosphamide-treated) as shown in section 3.2.5.

Preparation of bone marrow

Bone marrow was extracted a described in section 3.2.6 and flushed with FBS.

Marrow suspension was prepared as described in Fig. 4.

Fluorescent staining and analysis of micronuclei

Fixed bone marrow smears prepared on the clean glass slides were stained with

acridine orange according to the method of Costa and Costa (2007) as described in

Fig. 4 and examined under fluorescent microscope for the frequencies of

micronucleated polychromatic erythrocytes (MNPCE). Erythropoisis percentage of

polychromatic erythrocytes (PCE) was evaluated over 2000 total erythrocytes

(PCE+NCE) (NCE= Normochromatic erythrocytes). Micronuclei were evaluated on

the basis of number of PCE‟s, MNPCE‟s and MNNCE‟s (micronucleated

normochromatic erythrocytes).

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3.2.8.2 Flow cytometric enumeration of peripheral blood micronuclei

Flow cytometric analysis of micronucleus (micronucleated erythrocytes;

MNRETs) was performed as per the following experimental schedule in Balb/C mice.

Group No. of mice Treatment Dose

(mg/kg; per oral)

I 6 Control

II 6 WSF 250

III 6 WSF 500

IV 6 WSF 1000

V 6 CP 12.5

VI 6 CP 25

VII 6 CP 50

WSF was administered once daily for a period of 28 days. CP was administerd once

daily for 5 days consecutively.

Procedure

At the end of treatment schedule peripheral blood samples were collected by retro-

orbital sinus puncture in EDTA containing tubes. Blood samples were processed

according to the method of Dertinger et al., (2006) as described in Fig. 5.

Briefly, processed blood samples were labeled with antibody markers (FITC-

anti-CD71 and PE-anti-CD61) were added with propidium iodide (PI) and a minimum

of 10000 cells were analyzed in flow cytometer (BD Facs ARIA) enabled with

FACSDiva software.

Gating strategy for MNRET analysis was employed as described in Mouse

Microflow Basic Analysis kit (BD Biosciences).

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Fig. 4 Schematic flow chart for in vivo bone marrow micronuclei assay.

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Fig. 5 Schematic flow chart for in vivo micronuclei assay in peripheral blood using

flow cytometry.

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3.2.9 Sperm abnormality assay

Sperm abnormality assay was performed in Groups I, II, III, IV (WSF –treated)

and VI (metronidazole-treated) as shown in section 3.2.5.

Procedure

Epididymis was excised by laparoctomy. Sperm suspension was prepared by

mincing the epididymis as depicted in Fig. 6. The filtered suspension was stained with

1% eosin-Y for 30 min according to the procedure of Narayana et al. (2002). The

morphological abnormalities in sperm head shape and other changes were enumerated

by the method of Wyrobek and Bruce (1975) using Nikon E200 light microscope at

100X magnification.

For each animal, 1000 sperms were assessed for morphological damage and

data were presented as percentage incidence of total abnormalities. These included

coiled/ folded, flagellum with ansa, bent at cephalocaudal region, amorphous head,

double headed, double tailed, hookless flagellum, coiled with microcephali and

multiple abnormalities.

3.2.10 In vitro Genotoxicity study

Cell Culture

Chinese hamster ovary (CHO; wild type) cells were obtained from the National

Center for Cell Sciences, Pune, India, and maintained in complete Ham‟s F12 nutrient

medium in 25 cm2 flasks at 37ºC and humidified atmosphere of 5% CO2 in air.

Passaging of the cells was done four times a week. Experiments were performed with

cells cultivated not longer than twenty passages to avoid mutations. Exponentially

growing cultures were used for the study. Cells were counted using haemocytometer.

The viability of cells was determined by standard trypan blue exclusion test. A visual

count was made of the number of live and dead cells using haemocytometer following

staining with trypan blue (0.4% in PBS) and percentage of live vs. dead cells was

determined. Cell with >95% viability were selected for the studies. CHO cells were

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disaggregated using 1 ml of 0.25% trypsin solution and when all the cells were

detached, trypsin was inactivated with 2 mL complete medium. The cells were

centrifuged at 250 g for 10 min. Supernatant was discarded and pellet resuspended in

complete medium to give 106 cells/ml suspensions. This suspension was then used for

plating cells in 12 well plates (40,000 cells/well) for micronuclei assay.

3.2.10.1 Flow cytometric analysis of micronuclei (MN) in CHO cells

Procedure

In vitro micronucleus assay in CHO cells using flow cytometry was carried out

as depicted in Fig 7. WSF was treated at varying concentrations ranging from 25-150

µg/ mL. The reference compound ethyl methane sulfonate (EMS) was tested at conc.

of 6 mM.

Samples were analyzed with a BD flow cytometer. A log scale was used to

register DNA and side scatter (SSC) signals. Strategies for acquiring flow cytometric

data, including configuration of regions and gating logic, were as described by Wagner

et al., (2003). Briefly G1-phase nuclei were stored around channel 2000 and

micronuclei were counted in the region between 5 and 40% of the DNA content of G1-

phase nuclei. A minimum of 20,000 events per sample were analyzed.

3.2.10.2 Cytokinesis block micronuclei (CBMN) assay

Procedure

CBMN assay was carried out according to the method of Fenech (2000) as

depicted in Fig 8. CHO Cells were treated with different concentrations (25, 50, 75,

100, 125 and 150 µg/ml) of test material for 6 hour. DMSO 1% and EMS 6mM were

served as vehicle control and positive controls, respectively. After exposure, cells were

washed with medium and grown further for 18 h in 1 mL of fresh complete HF12

medium containing Cytochalasin-B (final concentration 3 µg/ mL medium) in a CO2

incubator at 5% CO2.

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Fig. 6 Schematic flow chart for sperm abnormality assay.

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Fig. 7 Schematic flow chart for in vitro micronucleus assay in CHO cells using flow

cytometry.

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Fig. 8 Schematic flow chart for CBMN assay

Page | 56

Cells were harvested (500 µl of trypsin; 0.125%) and washed with PBS and

then resuspended in incomplete culture medium. This suspension loaded in cytofunnel

(250 µl) and centrifuged in cytospin (Thermo Shadon, UK). Slides were then air dried

and fixed in MeOH. Slides further kept overnight for air drying. Slides were stained

with 10% Giemsa and 1000 binucleate cells from each concentration scored according

to the protocol of Shukla et al., (2011b). A cytokinesis block proliferation index

(CBPI) was further calculated from 500 cells/ concentrations as recommended in the

OECD guideline No. 487 (OECD, 2007) as follows: CBPI = (No. of mononucleate

cells + 2 X No. of binucleate cells + 3 X No. of multinucleate cells) / Total No. of

cells.

3.2.11 Effect of WSF on immune response

Collection of sheep RBC (SRBC)

Sheep red blood cells (SRBC) were collected asceptically from jugular vein of

sheep at weekly intervals and stored in cold sterile Alsever‟s solution. SRBC collected

in Alsever's solution were centrifuged and washed 3 times with sterile normal saline

(0.9 % NaCl w/v).

Immunization

Mice were immunized by injecting 0.2 mL of SRBC suspension (5 x 109 cells)

i.p. on day 0, and segregated into 5 groups of 6 mice each and treated as indicated

below. The dose volume was 0.2 ml. Control group animals received vehicle only.

Group No. of mice Treatment

(mg/kg p.o.)

I. 6 -

II. 6 WSF 250

III. 6 WSF 500

IV. 6 WSF 1000

V 6 CP 50

Page | 57

Test material was administered for 21 days consecutively and CP was

administered daily from day -5 to day 0.

3.2.11.1 Determination of serum antibody (anti-SRBC) titre

Blood samples were collected by retro-orbital plexus on day 7 (for primary

antibody titre) and day 14 (for secondary antibody titre). Serum was separated and

antibody levels were determined by the hemagglutination technique (Gupta et al,

2006). Briefly, equal volumes of individual serum samples of each group were pooled.

Two- fold dilutions of pooled serum samples were made in 25 μL of normal saline in a

micro-titration plate to which was added 25 μL of 1% suspension of SRBC in saline.

After mixing, the plates were incubated at room temperature for 1 h and examined

under the microscope. The reciprocal of the highest dilution of the test serum

producing agglutination was taken as the antibody titre.

3.2.11.2 Determination of IgG titre.

SRBC specific IgG antibody response in the serum of control and treatment

groups were measured by ELISA method as described by Biella et al., (2008) with

slight modifications. In brief, SRBC antigen, in aliquots of a 5 μg/mL suspension in

PBS, pH 7.2 (150 μL/well), was incubated overnight at 4ºC in high-binding

microplates (Costar, New York, USA). Prior to each subsequent step, plates were

washed three times with PBS-T (0.05% Tween 20). Unbound antigen was blocked

with 5 % FCS/ PBS at 37 ºC for 1h. 100 μl of diluted serum samples (diluted in PBS-

T; 1:8) were added to the wells. The plates were then incubated for 1 h at 37 ºC,

followed by three times of washing. Aliquots of 100 μl of rabbit anti-mouse IgG

horseradish peroxidase conjugate (1:1000) were added to each well. Plates were

Page | 58

further furnished to incubate for another hour at 37 ºC. After washing, the peroxidase

activity was assayed by adding 100 μL of substrate solution (10 mg of OPD). The

plates were incubated for 20 min at 37 ºC in the dark, and enzyme reaction was

terminated by adding 50 μL/well 2 N H2SO4. The O.D. was measured in ELISA reader

at 450 nm.

3.3 Statistics

Data were expressed as mean± S.E. and analyzed using ANOVA followed by

Student-Newman-Keuls test for multiple comparisons. Results were considered

significant at p<0.05.