sample="quota" bates="2062555037" isource="pm" decade="1980" class="ui" date="19830100" This proposal, including front page, contains 70 pages. 1. SUMMARY In the present study the influence of "PHENOL" and CATECHOL" on cell to cell communication in the metabolic cooperation (MC) assay with Chinese hamster V79 cells will be investigated. The study includes 3 negative and 8 positive control groups. Negative controls will be untreated, treated with 5 grams/liter dimethyl sulfoxide (DMSO, solvent control) and with 0.16x10E-2 millimoles/liter equivalent to 0.58 milligrams/liter phorbol. Positive controls will be treated with 12-O-tetradecanoylphorbol 12-acetate (TPA) and mainstream whole smoke condensate (MWSC-I) ( ) of the standard reference cigarette type 2R1 respectively. TPA will be administered at the doses 1.6x1oE-4 and 1.6x10E-3 millimoles/liter equivalent to 0.001, 0.01, 0.1 and 1 milligram/liter. MWSC-I will be administerd at 0.001, 0.01, 0.1 and 1 milligram dry condensate/liter. The test substance phenol will be administered at 1.1x10E-4, 1.1x10E-03, 0.011 and 0.11 millimoles/liter equivalent to 0.01, 0.1, 1 and 10 milligrams/liter. Catechol will be administered at 9.0x10E-5, 9.0x10E-4, 9.0x10E-3 and 0.09 millimoles/liter equivalent to 0.01, 0.1 1 and 10 milligrams/liter. For each troup 10 cocultures of V79 wild type and mutant cells and 10 monocultures of mutant cells will be set up. In the cocultures 4x10E5 V79 wild type and 10E2V79 mutant cells will be seeded per dish. In the monocultures 10E2 mutant cells will be seeded per dish. 4 hours after seeding the medium will be aspirated and 6-thioguanine (6-TG) selection medium containing DMSO, reference or test substance will be added. On day 4 after seeding the medium MWSC-I: mainstream smoke condensate collected with an impaction trap will be replaced by 6-TG selection medium without DMSO, reference or test substance. On day 8 after seeding colonies will be fixed with methanol and stained with Giemsa. Colonies will then be counted visually. The study will be performed in 2 experiments, each experiment being performed on a separate day and consisting of 5 cocultures and 5 monocultures per group and dose. Prior to the MC assay a cytotoxicity assay will be performed with the same substances and doses as described above. Of MWSC-I the additional dose of 10 milligrams/liter will be administered. 3 monocultures of 10E2 V79 wild type and 10E2 V79 mutant cells per group and dose will be set up. The procedure will be the same as described for the MC assay. The medium, however, used in the cytotoxicity assay contains no 6-TG. I N B I F O Institut für biologische Forshung GmbH 2 RESPONSIBILITY Study Director and Experimental Conduct: D. Becker Biologist (Diplombiologe) Study Codirector: Dr.rer.nat. R.-A. Walk Biologist (Diplombiologe) and Biochemist Analytical Chemistry: Dr.rer.nat. M. Speck Chemist (Diplomchemicker) Quality Assurance: E. Römer Biologist (Diplombiologe) 3. INTRODUCTION Metabolic cooperation is the transfer of small metabolites between cells which is associated with physical contact between the donor and the recipient cell (Subak-Sharpe et al., 1969). MC can be visualized as the colony forming ability, when Chinese hamster V79 wild type (hypoxanthine-guanine phosphoribosyl transferase positive HGPRT+) cells are seeded together with Chinese hamster V79 mutant (HGPRT-) cells in a medium containing 6-TG. Besides its function in the "Salvage" pathway to synthesize inosine 5'-mono-phosphate by phosphorylation of hypoxanthine, the enzyme HGPRT phosphorylates also directly guanine to guanine-5'-monophosphate. 6-TG is incorporated into nucleotides (see Figure A) instead of guanine causing an inhibition of the proliferation and cell death of HGPRT+ cells in medium containing 6-TG. V79 mutant (HGPRT-) cells proliferate in medium containing 6-TG. When an excess number of V79 wild type cells are cocultured with V79 mutant cells the number of surviving V79 mutant cells is reduced. This reduction of mutant survival is attributable to MC between V79 wild type and mutant cells.. The extent of this reduction is dependent on the number of V79 wild type cells (Yotti et al., 1979). Procedure: MWSC-I washed out of each impaction trap 6 times with approx. 3 ml portions of DMSO repeatedly after sonication (water bath) for approx. 3 min, washings transferred to a 100-ml volumetric flask and filled up to volume with DMSO amount of MWSC-I calculated from weight of impaction trap before and immediately after condensate preparation amount of dry condensate (a) calculated from MWSC-I and water concentration of suspension determination of water concentration: see 5.2.3 The terms "moist condensate" and "dry condensate" are in accordance with DIN 10240 ("Mashinelles Abrauchen von Zigaretten, Bestimmung des feucheten und des trockenen Rauchkondensates"). determination of hydrogen-ion concentration: see 5.2.4 determination of nicotine concentration: see 5.2.5 dilution of MWSC-I suspension: with DMSO to 5 g dry condensate/l storage of MWSC-I suspension: fractioned in 1-ml aliquots, in the dark, in sterile brown glass vials, minus 75 degrees centigrade labeling of the vials: study no., batch no. (a), dry condensate concentration (g/l), date of condensate preparation Scientific version: 15.Jan.81 Text version: 1.Feb.83 5.2.3 Determination of water concentration Principle: titration according to Karl Fischer Time: within 48 h after preparation of MWSC-I suspension Sample material and quantity: MWSC-I/DMSO suspension, 1 ml, 2 determinants/suspension Results expressed in: g/l Equipment: Karl Fischer Titrator E452, Eutsche Metrohm GmbH, D-7024 Filderstadt Batch number consists of cigarette short code, type of condensate, consecutive number, date of condensate preparation, for example: 7A (I)/10/081280 (e. g.: code: 7A, condensate: I, batch no.: 10, date: 8.Dec.80) Chemicals and reagents: Karl Fischer solution no. 9248, methanol, no. 6012, DMSO, no 2950, E. Merck, D-6100 Darmstadt 1 Procedure Titration: 1 mL DMSO mixed with 4 ml methanol in the reaction vessel of the titrator and titrated with Karl Fischer solution to determine the water content of DMSO. Afterwards 1 ml cigarette smoke condensate suspended in DMSO titrated in the same way Computation: titer of the Karl Fischer solution determined by titration of a mixture of 1 ml DMSO and 4 ml methanol with a known amount of water, e. g. 10 mg Detection limit: 0.5 g H20/l Reproducibility (relative standard deviation): 2.8 o/o (10 g H2)/suspension, N = 8) Scientific version: 2.Jun.80 Text version: 7.Jan.83 5.2.4 Determination of hydrogen-ion concentration Principle: electrochemical determination Time: on the day of condensate preparation Sample material and quantity: MWSC-I suspension, 2 ml Results expressed in: pH Equipment: pH meter: PW 9409, glass electrode: CA 1-S, Philip GmbH, D-3500 Kassel Chemicals and reagents: calibration buffer: pH 7.00, no. 9887, pH 4.00, no. 9884, E. Merck, D-6100 Darmstadt 1 Procedure: determination at room temperature after calibration of pH meter with 2 standard buffers Scientific version: 28.Feb.79 Text version: 8.Jul.82 5.2.5 Determination of nicotine concentration Principle: gas chromatography after extraction with dichloromethane computer integration of peak areas Time: immediately after the preparation of the MWSC-I suspension Sample material and quantity: MWSC-I suspension (undiluted and diluted), 1 ml, 2 determinations/suspension Results expressed in: g/l Equipment: gas chromatograph: HP 5710 A, detector: FID, automatic sampler: HP 7671 A, laboratory data system: HP 3351 A, Hewlett-Packard GmbH, D-6000 Franfurt/Main recorder: Servogor 210, Metrawatt GmbH, D-8500 Nürnberg centrifuge: model J-6B, rotor: JS-4.2, Beckman Instruments GmbH, D-8000 Müchen 40 Chemicals and reagents: nicotine, no. 77625, Serva Feinbiochemica GmbH and Co. KG, D-6900 Heidelberg 1 quinoline, no. 802407, dichloromethane, no. 822271, DMSO, no. 2950, sodium hydroxide, no. 5594, sulfuric acid, no. 9074, E. Merck, D-6100 Darmstadt 1 nitrogen, hydrogen, air (synthetic), Linde AG, D-5000 Köln 50 Procedure Extraction: addition of 1 ml of the internal standard solution (0.5 mg quinoline/mL (0.1 mol/l) sulfuric acid), 1 mL sodium hydroxide (200 g/l) and 10 mL dichloromethane to 1 mL MWSC-I suspension, after agitation (5 min) and centrifugation (approx. 7.8x10E3 m/s2 (= 800 x g), 5 min, approx. 10 degrees centigrade), injection of 1 ul of the lower phase into the gas chromatograph Gas chromatography Column: 2 m x 1/8 inch outer diameter, stainless steel Column packing: 100 g/kg Apiezon L and 100 g/kg potassium hydroxide on Chromosorb W-AW DMCS ( ), 80 to 100 mesh AW: acid washed, DMCS: treated with dimethyldichlorosilane staining: on day 8 aspiration of medium, fixation of colonies with methanol, 3 ml/dish for 5 min, staining of colonies with Giemsa, 1 ml/dish for 2 min, rinsing of colonies 1 time with bidistilled water, 3 times with tap water evaluation: counting of colonies visually survival (o/o) = CT/CU x 100 CT: number of colonies, treated culture CU: number of colonies, untreated culture Scientific Version: - ( ) Text version: 1.Feb.83 will be given in the report 5.7 Determination of Population Doubling Time, Growth Curve Principle: determination of the number of cells on 10 subsequent days Time Sampling: - Determination: on 10 subsequent days Sample material and quantity: wild type and V79 mutant cells, dependent on the day of determination Results expressed in: h Equipment: centrifuge tubes: no 2070, conical graduated, polypropylene diameter: 30 mm, length: 115 mm, Falcon, Div. Becton and Dickinson GmbH, D-6900 Heidelberg-Wieblingen culture vials: tissue culture flask, growth areas 25 cm2, no. 163371, Nunc GmbH, D-6200 Wiesbaden micro vials : type "Eppendorf", polypropylene, no. 3810, Netheler and Hinz GmbH, D-2000 Hamburg 65 centrifuge: Digifuge GL, rotor: 03350, Heraues-Christ GmbH, D-3360 Osterode incubator: Cytoperm 8080/8100124, carbon dioxide, humidity and temperature regulation, W. C. Heraues GmbH, D-6450 Hanau inverted microscope D, objectives: Ph2 Plan 16, Ph2 Plan 25, microscope KM, objective: Ph2 Plan 16, hemocytometer chamber according to Neubauer, no. 422903, Carl Zeiss, D-7082 Oberkochen hood: type Biogard Baker, No. B 60-112, Labotect, D-3406 Bovenden Chemicals and reagents Chemicals: Dulbecco's Modification of Eagle's Medium (DMEM), no. 12-332-54, fetal bovine serum, filter sterilized mycoplasma and virus screened, no. 29-101-54 (Lot no. ( )), MEM 50-fold amino acids (AA), no. 16-011-49, MEM 100-fold non-essential amino acids (NEAA), no. 16-810-49, trypsin EDTA, no 16-891-49, phosphate-buffered saline (PBS), no. 18-604-49, sodium pyruvate, no. 16-820-49, Flow Laboratories GmbH, D-5309 Meckenheim glutamine, no. 22942, streptomycin, no. 35500, penicillin, no. 31749, Serva Feinbiochemica GmbH and Co. KG, D-6900 Heidelberg 1 sodium chloride, no. 6400, E. Merck, D-6100 Darmstadt 1 Trypan Blue, no. 1B187, Chroma Gesellschaft Schmidt and Co., D-7000 Stuttgart-Untertürkheim Reagents: cell culture medium: DMEM with 4 mmol/1 glutamine, 1x10E5 U pencillin/1, 10 mg streptomycin/1, will be given in the report. 10 ml NEAA/1, 20 ml AA/1, 1 mmol/1 sodium pyruvate and 30 g/l fetal bovine serum, final pH: 7.4, final osmolality: 330 mmol/kg Trypan Blue solution: 5 g Trypan Blue/1 saline Procedure: handling under sterile conditions day 1: 20 culture flasks each for wild type and mutant cells filled with 5 ml medium, seeding of 3x10E4 wild type and mutant cells respectively per flask incubation at 37 degrees centigrade days 2 to 11: splitting and determination of the number of cells of 2 wild type and mutant creatures respectively (1) splitting: medium taken off, monolayer washed 1 time with cold trypsin (3 ml), addition of 3 ml cold trypsin, incubation for 5 m min at 37 degrees centigrade, detachment of the cells from the flask bottom by mechanical shock, addition of 3 ml medium to stop trypsinization, cell suspension transferred into a centrifuge tube, centrifugation at 4905 m/s2 (=500 x g) for 5 min, supernatant discarded and pelleted cells resuspended in an appropriate volume (5 to 10 ml) medium (2) determination of the number of cells: sample of the cell suspension mixed with equal volume of Trypan Blue solution in a micro vial, cells counted after 5 min of incubation at 0 degrees centigrade, unstained cells: viable, stained cells: dead, cells in 4-mm3 squares are counted. days 4 and 8: aspiration of medium and addition of 5 ml fresh medium Calculation: formulaname Scientific version: - ( ) Text version: 1.Feb.83 will be given in the report 6. STORAGE OF MATERIALS AND RECORDS Remains of substances examined, protocols and records are stored in our archives for at least 5 years, they can be claimed by the client. Stained colonies are stored at least 3 months after the delivery of the report. 7. REPORTING Report language: English Report concept: The report will contain the following parts: 1 SUMMARY 2 RESPONSIBILITY 3 INTRODUCTION 4 SUBSTANCES EXAMINED 5 METHOD 6 STORAGE OF MATERIALS AND RECORDS 7 RESULTS AND DISCUSSION 8 REFERENCES FORMS