List of Chemicals. 1. Medium RPMI 1640 PAA Laboratories. 4. Demecolcine Solution Sigma Aldrich. 8. Mitomycin - C Sigma Aldrich.

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1 List of Chemicals Materials Suppliers 1. Medium RPMI 1640 PAA Laboratories. 2. Lectin (Phytoheamagglutinin PHA) Sigma Aldrich. 3. Heparin Biological Evans. 4. Demecolcine Solution Sigma Aldrich. 5. KCl Ranbaxy chemicals. 6. Trypsin Hi-Media. 7. Giemsa Qualigens. 8. Mitomycin - C Sigma Aldrich. 9. Cytochalasin B Sigma Aldrich. 10. BrdU (5 Bromo 2 deoxyuridine) Sigma Aldrich. 11. Hoechst (Bis - benzamide) Sigma Aldrich. 12. Trans, trans - muconic acid Sigma Aldrich. 13. Vanillic acid Sigma Aldrich. 14. Phenol Cristal Ranbaxy Chemicals aminoantipyrine ACS chemicals. 16. Anhydrous sodium carbonate Sara Fine Chemicals. 17. Sodium bicarbonate Sara Fine Chemicals. 19 P a g e

2 18. Boric acid Ranbaxy Chemicals. 19. Potassium ferricyanide Ranbaxy Chemicals , 6-diamino-2-phenyl-indole (DAPI) Sigma Aldrich. 21. Acridine Orange Sigma Aldrich. 22. Methanol Ranbaxy Chemicals. 23. Ethanol Ranbaxy Chemicals. 24. Glacial Acetic Acid Ranbaxy Chemicals. 25. EDTA Ranbaxy chemicals. 26. Na 2 HPO 4 Ranbaxy chemicals. 27. KH 2 PO 4 Ranbaxy chemicals. 28. NaCl Ranbaxy chemicals. 20 P a g e

3 Blood and urine collection: Before collecting blood, volunteer subjects were informed about the study. They asked to sign an informed consent form and to complete a standardized questionnaire was diploid to obtain necessary data on lifestyles and personal factors like; name, age, sex, address, smoking and other addictions, working place, work experience, working period and medication in language known to them. The trained paramedic collected 5 ml of blood by vain puncture of subject. After pooled out the blood, 3 ml of blood was transferred immediately in sodium heparin containing vacutainer and 2 ml of blood was transferred in EDTA containing vial. Name, age and date were labeled on the vials. After blood collection volunteer subjects were requested for urine collection. Urine samples were collected in white sterile plastic bottles. Minimum 50 ml urine sample from each individual was collected and preserved with 500µl of 6 mol/l hydrochloric acid and stored in deep freezer. 21 P a g e

4 Cytogenetic Method: In this study three cytogenetic endpoints were performed; chromosomal aberrations (CAs), sister chromatid exchanges (SCE) and micronucleus test (MN). From each individual s blood three lymphocyte cultures were seeded in two sets, thus total six lymphocyte cultures were incubated separately and labeled for appropriate end points. Among these two sets, one set was further treated with Mitomycine C (MMC), a known mutagen for positive control. The below table indicates numbers and age of the studied individuals. Groups Group A 1 Group A 2 Group B 1 Group B 2 Individuals Control non smokers Control smokers Non smoker Petrol pump Attendants Smoker Petrol pump Attendants Number of individuals Age range Mean age P a g e

5 Lymphocyte Culture: Lymphocyte cultures were setup by Hungerford (1965) with slight modifications (Gadhia et al., 2004). Heparinized whole blood (0.5 ml) was added to mixture containing 5mL of culture medium RPMI 1640, 0.1ml phytohemagglutinin (Lectin) and 0.05 ml heparin. Then the culture vials were put in HERA cell 150 CO 2 incubator for 71 hrs, at 37 c with 5% CO 2. Then 0.1 ml demecolcine Solution was added at last 2 hours of incubation period to arrest cells at metaphase. The cells were collected by centrifugation, resuspended in a prewarmed hypotonic solution (KCl, M) for minutes and fixed in chilled methanol/acetic acid (3:1, v/v) solution (Carnoy s fixative). Suspensions of cells were prepared after several changes of Carnoy s fixative washes. Then drops of cell suspension were allowed to fall from at least 2.5 feet height on pre chilled and chemically cleaned slides. These slides were air dried on a hot plate at C. All slides were blind coded and labeled soon after assuring about well spread chromosome. 23 P a g e

6 Chromosomal Aberrations: After air dry preparation, slides were stained with 2% Giemsa s stain for 10 mins. From each slide, 100 well spread metaphase chromosomes were scored for chromosome and chromatid type of aberrations. GTG banding: GTG banding means, G-bands produced by trypsin using Giemsa. This banding technique was performed using method of Sebright (1971). Unstained, air dried slides, not more than 4-6 days old slides were incubated at 85 C for one hour in pre heated oven for ageing the slide. After this step slides were allowed to cool at room temperature, then the slides were treated with 0.05% Trypsin solution for 4-15 seconds. The enzyme action was stopped immediately by putting the slide into chilled Phosphate buffer solution. Then the slides were transferred to stain with 5% Giemsa for 4-5 minutes. Differentiation of staining was done by dipping the slides in distilled water containing coupling jar for 3-4 times. Then the slides were air dried and observed under microscope to find good banded metaphase chromosome. G-banded metaphase chromosome captured using Nikon Digital DXM 1200 camera attached to microscope Nikon Eclipse E P a g e

7 fluorescence microscope and the automatic karyotyping software VideoTesT - Karyo 3.1 was used for karyotype making. Sister chromatid exchange (SCE): Sister chromatid exchange study was performed by using Wolff and Perry (1974) method, culture was setup and after 24 th hour of incubation 50µl 5-Bromo-2-deoxyuridine (5mg/ml; Sigma) added in SCE labeled culture vials. Then the vials were allowed to incubate in the dark for 48 hrs at 37 C and 5% CO 2 pre adjusted HERA cell 150 CO 2 incubator. Metaphases were blocked during the last 2 h with adding 0.1ml demecolcine. Slide preparation methods were remained same like routine lymphocyte culture. After 2 day of preparation slides were stained in Hoechst (100 µg/ml) in distilled water for 20 min. After rinsing in tap water, the slides were mounted in Sorensen s buffer under a cover glass (edges of cover slip sealed with rubber cement) and exposed to UV light from a Black ray lamp for 12 min on a slide warmer at 60 C. Finally, slides rinsed in ice-cold Sorensen s buffer followed by tap water were stained in 4% Giemsa. Minimum 30 well spread second division M 2 metaphases were scored for calculating sister 25 P a g e

8 chromatid exchange frequencies. The replicative index was calculated by following equation. Raplicative Index (RI) = 1(M 1 ) + 2(M 2 ) + 3(M 3 ) 100 Micronucleus test: Micronucleus test was performed using method of Fenech and Morley (1985) after 44 hours of incubation of lymphocyte culture, 6 µg/ml cytochalcine B was added to the MN labeled culture vials. And then further put in incubator. At 71 th hour of incubation termination process were started with centrifugation. After centrifugation supernatant was poured off. And then the cells were resuspended with mixing chilled hypotonic KCl solution. The mixture then put in refrigerator for 10 minutes. The changes of fixative washes were given until pellet become clear. The slides were prepared by using air-drying method and were stained with 3% Giemsa. For each individual 1000 binucleated cells were scored for micronuclei frequency. 26 P a g e

9 Hematological Analysis: Blood collected in EDTA containing tubes were used for hematological analysis. Different hematological parameters such as hemoglobin (Hb), RBC (Red blood cell) count, WBC (White blood cell) count, Platelet count, differential count and PCV (Packed cell volume) were analyzed in pathological laboratory. Biochemical analysis from Urine: Urinary phenol measurement: Urinary phenol measurement was performed by spectrophotometric method of Yamaguchi and Hayashi (1977). Solution A (90 mg 4-amino antipyrine in 200 ml of 0.05 mol/liter carbonate - bicarbonate buffer) and solution B (0.21 mol/liter boric acid and mol/liter potassium ferricyanide) were prepared fresh before measurement. In clear test tube, 1.5 ml Solution A and 0.2 ml urine were mixed well, then 1.5 ml of solution B added and allowed to stand for 3 minutes to mix well. At 500 ηm, absorbance of standards and samples vs. the absorbance of blank were measured. The concentration of phenol in the urine is calculated as follows: phenol mg/l = [OD of Sample A / OD of Standard (50 mg/l) A] X P a g e

10 Urinary trans, trans - Muconic acid measurement. Urinary trans, trans-muconic acid (tt-ma) was measured by HPLC using method of Lee et al., (1993). Urine samples were sent to the analytical laboratory. Samples were purified by an ion-exchange chromatographic technique. The anion-exchange columns were freshly prepared by packing 4 mm glass tubes with Dowex 1 to a length of 10 mm. To pre-condition the columns they were rinsed twice with 2 ml of deionized water. Urine samples (1 ml) were mixed with 2 ml of 0.5M Tris buffer (ph 10) containing as an internal standard 10 mg /l vanillic acid and allowed to percolate through the preconditioned anion exchange columns. The columns were washed sequentially with 1 ml of 0.2 mm phosphoric acid solution, 1 ml of 0.1M sodium acetate buffer (ph 7) and 1 ml of deionized water. The trans, transmuconic acid was then eluted with 1 ml of an equivolume solution of 1.5M sodium chloride/methanol. The collected eluate was centrifuged at 2000 g for 5 min and 20µl of the upper layer was used for HPLC analysis. A Waters apparatus was used (Waters Corporation, Milford, Massachusetts, USA.). It was equipped with a reverse phase column C18/5 (250 x 4.6 mm), together with a variable wavelength detector. The wavelength used was 265 nm; the 28 P a g e

11 mobile phase was constituted by acetic acid/ methanol/ sodium acetate (1:10:89 by vol.) at ph 3.5. HPLC analysis was performed by isocratic elution at room temperature (20 C±1). Standard curves were prepared by adding known amounts of trans, trans-muconic acid (1.0, 0.75, 0.50, 0.25, 0.125, 0.063, and µg/ml) dissolved in methanol with a fixed amount of Valanic acid (10 mg/ml) dissolved in 1.5 M NaCl and methanol (50:50, v/v). Trans, trans-muconic acid concentrations in urine were expressed as µg ml/l. Statistical analysis : Cytogenetic endpoints, urinary biomarkers and hematological parameters of both groups, controls as well as subjects were employed with Student s t test and ANOVA. Two different variables, phenol against trans, trans-muconic acid were related by correlation coefficient. Microsoft Excel and SPSS (Statistical Package for the Social Sciences) software were used for statistical analysis. 29 P a g e