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CHARGE NUMBER: PROGRAM TITLE: PERIOD COVERED: PROJECT LEADER: WRITTEN BY: DATE OF REPORT: 6906 BIOLOGICAL EFFECTS OF SMOKE July 1-31, 1981. R. A. Pages D. J~. Ayers August 6, 1981 1. YEAST MITOTIC GENE CONVERSION ASSAY11 A. 2-Amino-a-carbol~ine (2AC) As reported previously,2 2AC was two times more active than the spontaneous background in the presence of microsomes (S9). Vary- ing Tevels of S9 were found not to enhance the activity. One concentration of 2AC was tested in the presence of varying concentrations of harman and/or norharman to see if 2AC activity was altered. Harman alone did not enhance 2AC activity. At lower concentrations (P-0.01-0.03 mg/plate), norharman enhanced activity; however, at higher doses (>0.03 mg/plate) inhibition occurred. When both harman and norharman were present, activity was generally depressed. Varying the levels of S9 did not appear to influence the results. B. Quality Assurance Failure to obtain a new working cell stock with a low spontaneous background led to consultation with an outside laboratory. The procedure recommended involved selecting isolated colonies after a series of streakings from overnight cultures. This would replace selecting an isolated colony from an, agar slant. Results from this new method should be available next month. 2. E. COLI (A) PROPHAGE INDUCTION ASSAY12 A series of fractions from a burley IT CSC (prepared by 6908 personnel) were tested in strain BR513. These samples included: Grimmer fractions II, III, and V, the parent CSC, and its acid, base, and neutral fractions. Of these, Grimmer fraction V, and the acid, neutral, and base fractions were weakly active in the plate assay (+S9), but they were not active in the tube assay. In other work, varying the S9 concentration in the tube assay did not lead to detection of activity in the base fraction from 2R1, burley, and LTF-2A CSCs.

Charge Number: 6906 2 August 6, 1981 . Growth characterization of strain BR506 was completed this month. In the tube assay 2R1' and burley CSC and the base fractions of 2R1, burley, and LTF-2A CSCs showed no detectable activity. Additional, experiments are planned in the plate assay wi'th,the CSC base fractions of 14 cigarette types. 3. SALMONELLA/MICROSOME MUTATION ASSAY A. Pyrolysis Studies (with 6908)4 Pyrolysis studies involving 2R1 filler were reported last month.6 In retesting the extracts from the TPM pad, it was found that the extracts were not more active but equal! in activity to the solvent impinger trap pyrolyzates. The pad extracts wil~1: be combined with the pyrolyzates in future studi'es. B. LTF plus Additives (with 6908)`' The normally low activity seen in the CSC from a nitrogen- free, high organic LTF filler (LTF-5E) was greatly increased by the incorpo- ration of 2-aminobutyric acid (2AB) into the filler. The addition of fructose to the LTF-5E + 2AB slightly lowered the CSC activity. In an effort to expand these studies, varying tnole ratios of fructose:2AB (1:1; 1:3; and 3:1) were tested. The results indicated that the addition of fructose did not lower CSC activity. However, when these fillers were heated for 18 hours at 100°C the subsequent CSC activity was reduced. A similar experiment involving higher fructose:2AB ratios is in progress. C. Model Compounds'' Four model compounds (G1u-P-1,9 GIu-P-2,9 Trp-P-1,3 Trp-P-23) were tested in the SaZwrceZZa/microsome assay at three different S9 concentrations (15, 25, and 35 ul/plate). Generally,the results agreed with those reported in the literature:3'9 Glu-P-l was more active than Glu-P-2; Trp-P-2 was more active than Trp-P-1; and Trp-P-2 and Trp-P-1 were more active at lower S9 levels. There were some disagreements, most notably that Glu-P-2 showed much lower activity (lOX) than reported in the l,iterature.9' The reason for this observation was not clear, but will be investigated further. D. Sidestream IT CSC and Mainstream TPM Activity (with 6908)ia A profile of sidestream IT CSC and mainstream TPM activity in TA98 was completed this month. This was necessary to aid in the determi- nation of a better dose-response range for testing these types of smoke samples. The results indicated that an increasedAose level can be used in future testing.

Charge Number: 6906 3 August 6, 1'981 E. Miscellaneousl'0 Four samples were tested'.at the request of J. L. Charles. An additional six samples were tested in two separate experiments at the request of R. D. Carpenter. 4. L5178Y THYMIDINE KINASE MUTATION ASSAY1'S'7 Recent experiments have yielded'cloning efficiencies with an average of 82%.8 Since this was an acceptable average, the cells were cloned after a treatment regimen which mimicked' that experienced during the routine testing of compounds. The results showed a reduced, but still acceptable, cl'oning efficiency (51%). A repeat experiment is in progress. 5. REFERENCES 1'. Ayers, D. J.; Penn, J. M. Notebook No. 7551, pp. 133-134. 2. Drew, S. 6906 Monthly progress report. Monthly Progress Reports 81-172; 1981 July 15. 3. Ishii, K.; Yamazoe, Y.; Kamataki, T.; Kato, R. Metabolic activation of mutagenic tryptophan pyrolysis products by rat liver microsomes. Cancer Research. 40:2596-2600; 1980. 4. McCoy, W.; Drew, S. Notebook No. 7596, pp. 127-134. 5. McCuen, R. W. Notebook No. 7611, pp. 131-135. 6. McKay, C. L. 6908 Monthly progress report. Monthl'y Progress Reports 81-172; 1981 July 15. 7. Meyers, R. M. Notebook No. 7609, pp. 53-54. 8. Penn, J. M. Notebook No. 7669, p. 17. 9. Takeda, K.; Shudo, K.; Okamoto, T.; Nagao, M.; Wakabayashi, K.; Sugimura, T. Effect of inethyl substitution on muta- genicity of 2-amino-dipyrido[1,2-as3',2'-d]imidazole, glu- P-2. Carcinogenesis. 11:889-892; 1980. 10. Rapp, K. E.; Tickle, M. H. Notebook No. 7437, p. 189. 11. Thompson, L. H. Notebook No. 7557, pp. 116-118. 12. Yu, T.; Cunningham, R. Notebook No. 7623, pp. 104-106. nwp P e/. 4Z64--"