Studies on Chemically Induced Neoplastic Transformation and Mutation in the BALB/3T3 Cl A31-1-1 Cell Line in Relation to the Quantitative Evaluation of Carcinogens

Abstract

Mutagenesis and neoplastic transformation assays on mammalian cells in culture have been extensively used for quantitative estimates of the activity of carcinogens, in spite of the limitations that such in vitro systems have when compared with in vivo systems for tumor induction. In order to assess the validity of these correlations, a series of studies was undertaken in our laboratory with the BALB/3T3 Cl A31-1-1 mouse embryo cell line. Different carcinogens were found to induce dose-dependent frequencies of transformation, including the direct-acting alkylating agent N-methyI-N'-nitro-N-nitrosoguanidine (MNNG) and carcinogens that were metabolically activated by these cells through different pathways (benzo[a]pyrene, 3-methylcholanthrene, aflatoxin B₁, and benzidine). Their respective level of activity on a molar basis was different from that obtained in standard Salmonella + S9 mutagenesis tests. Studies currently underway indicate the possibility of lowering the serum content in the medium considerably, thereby reducing a major variable in the assay. Methods were established for the induction of ouabain-resistant (oua^r) mutants in these cells. Studies were conducted by applying 30-min MNNG exposures to cells that were synchronized by serum deprivation followed by serum-induced release from growth block. While maximal induction of mutants occurred in the S phase, the transformation frequency remained constant for treatments in G₁ and early or late S. In subsequent studies, cytotoxicity, alkali-labile DNA lesions, oua^r mutations, and neoplastic transformation were analyzed concurrently in this cell line after cells were exposed to two concentrations of MNNG and the exposures were protracted for different time periods (30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). A marked temporal dissociation was found in the exposure times required to induce maximal frequencies of mutations and of transformation. Cytotoxicity increased for periods up to 100-200 min; mutations reached a maximal induction level after a much shorter exposure time (30-60 min); DNA damage detected by alkaline elution was already maximal by 30 min. Transformation frequencies, however, reached maximal levels only after exposure periods 1-3 hr longer than those required for maximal mutation. The ratio of transformation to oua^r mutation frequencies was 3.7 for short treatment times (30-60 min), but it increased to more than 20 for exposure times of 240 min or longer. These studies support the hypothesis that a single gene mutational event is not sufficient to account for the expression of neoplastic transformation.

References

1. Aaronson SA and Todaro GJ (1968). Development of 3T3-like lines from Balb/c mouse embryo cultures: transformation susceptibility to Sv 40. Science 162: 1024–1026.

2. Bertolero F , Kaighn ME , Gonda MA , and Saffiotti U (1984). Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors. Exp. Cell Res. 155: 64–80.

3. Bertolero F , Kaighn ME , and Saffiotti U (1984). Control of growth and differentiation of mouse epidermal keratinocytes (MK cells) by serum-derived factors in serum-free medium. In Vitro 20: 270.

4. Bertram JS and Heidelberger C (1974). Cell cycle dependency of oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine in culture. Cancer Res. 34: 526–537.

5. Bignami M , Ficorella C , Dogliotti E , Norman RL , Kaighn ME , and Saffiotti U (1984). Temporal dissociation in the exposure times required for maximal induction of cytotoxicity, mutation and transformation by N-methyl-N'-nitro-N-nitrosoguanidine in the BALB/3T3 ClA31-1-1- cell line. Cancer Res. 44: 2452–2457.

6. Bignami M and Saffiotti U (1983). Mutagenesis and morphological transformation by N-methyl-N'-nitro-N-nitrosoguanidine in the BALB/3T3 clone A31-1-1 cell line. Carcinogenesis 4: 419–423.

7. Bignami M , Westin EH , Lerman MI , Kakunaga T , Kaighn ME , and Saffiotti U (1984). Activation of different transforming genes in BALB/3T3 cell lines transformed by the same chemical carcinogen, benzo[a]pyrene. Proc. Am. Assoc. Cancer Res. 25: 71.

8. Burki HJ and Aebersold PM (1978). Bromodeoxyuridine-induced mutations in synchronous Chinese hamster cells: temporal induction of 6-thioguanine and ouabain resistance during DNA replication. Genetics 90: 311–321.

9. Cerda-Olmedo E , Hanawalt PC , and Guerola N (1968). Mutagenesis of the replication point by nitrosoguandine and pattern of replication of the Escherichia coli chromosome. J. Mol. Biol. 33: 705–719.

10.

10. Cortesi E , Saffiotti U , Donovan PJ , Rice JM , and Kakunaga T (1983). Dose-response studies on neoplastic transformation of BALB/3T3 Clone A31-1-1 cells by aflatoxin B, benzidine, benzo[a]pyrene, 3-methylcholanthrene, and N-methyl-N'-nitro-N-nitrosoguanidine. Teralogenesis Carcinog. Mutagen. 3: 101–110.'

11.

11. Dawes I and Carter B (1974). Nitrosoguanidine mutagenesis during nuclear and mitochondrial gene replication. Nature 250:709–712

12.

12. Dewey WC , Noel JS , and Dettor CM (1972). Changes in radiosensitivity and dispersion of chromatin during the cell cycle of synchronous hamster cells. Radiat. Res. 52: 373–394.

13.

13. DiPaolo JA , Takano K , and Popescu NC (1972). Quantitation of chemically induced neoplastic transformation of BALB/3T3 cloned cell line. Cancer Res. 32: 2686–2695.

14.

14. Gehly EB and Heidelberger C (1982). The induction of ouar-mutations in nontransformable CVP3SC6 mouse fibroblasts. Carcinogenesis 3: 963–967.

15.

15. Gerchman LL and Ludlum DB (1973). The properties of O6-methylguanine in templates for RNA polymerase. Biochim. Biophys. Acta 308: 310–316.

16.

16. Goth-Goldstein R and Burki HJ (1980). Ethylnitrosourea-induced mutagenesis in asynchronous and synchronous Chinese hamster ovary cells. Mutat. Res. 69: 127–137.

17.

17. Grisham JW , Greenberg DS , Kaufman DG , and Smith GJ (1980). Cycle-related toxicity and transformation in 10T1/2 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine. Proc. Nat. Acad. Sci. U.S.A. 77: 4813–4817.

18.

18. Grisham JW , Greenberg DS , Smith GJ , and Kaufman DG (1979). Temporary culture in isoleucine-free medium enhances transformation of 10T1/2 cells by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Biochem. Biophys. Res. Commun. 87: 969–975.

19.

19. Hildebrand CE and Tobey RA (1975). Cell cycle-specific changes in chromatin organization. Biochem Biophys. Res. Commun. 63: 134–139.

20.

20. Kaighn ME and Lechner JF (1984). Cell separation by biological methods. In: Cell Separation: Methods and Selected Applications. TG Pretlow, II and TP Pretlow (eds). Academic Press, New York, pp. 285–306.

21.

21. Kakunaga T (1974). Requirement for cell replication in the fixation and expression of the transformed state in mouse cells treated with 4-nitroquinoline-1-oxide. Int. J. Cancer 14: 736–742.

22.

22. Kakunaga T (1975) The role of cell division in the malignant transformation of mouse cells treated with 3-methylcholanthrene. Cancer Res. 35: 1637–1642.

23.

23. Kakunaga T and Crow JD (1980). Cell variants showing differential susceptibility to ultraviolet light-induced transformation. Science 209: 505–507.

24.

24. Konze-Thomas B , Hazard RM , Maher VM , and McCormick JJ (1982). Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation. Mutat. Res. 94: 421–434.

25.

25. Lever JE and Seegmiller JR (1976). Quabain-resistant human lymphobastoid lines altered in the (Na+/K+)-dependent ATPase membrane transport system. J. Cell. Physiol. 88: 343–352.

26.

26. Lo KY and Kakunaga T (1982). Similarities in the formation and removal of covalent DNA adducts in benzo[a]pyrene-treated BALB/3T3 variant cells with different induced transformation frequencies. Cancer Res. 42: 2644–2650.

27.

27. Marquardt H (1974). Cell cycle dependence of chemically induced malignant transformation in vitro. Cancer Res. 34:1612–1615

28.

28. McCormick PJ and Bertram JS (1982). Differential cell cycle phase specificity for neoplastic transformation and mutation to ouabain resistance induced by N-methyl-N'-nitro-N-nitrosoguanidine in synchronized C3H10T1/2 Cl8 cells. vProc. Natl. Acad. Sci. U.S.A. 79: 4342–4346.

29.

29. Orkin SH and Littlefield JW (1971). Nitrosoguanidine mutagenesis in synchronized hamster cells. Exp. Cell Res. 66: 69–74.

30.

30. Peterson AR , Randolph JR , Peterson H , Spears CP , and Heidelberger C (1981). Oncogenic transformation and mutation of C3H/10T1/2 clone 8 mouse embryo fibroblasts by alkylating agents. Cancer Res. 41: 3095–3099.

31.

31. Randazzo R , Sermonti G , Carere A , and Bignami M (1973). Comutation in Streptomyces. J. Bacteriol. 113: 500–501.

32.

32. Saffiotti U (1970). Experimental respiratory tract carcinogenesis and its relation to inhalation exposures. In: Inhalation Carcinogenesis, Technical Report Series No. 18, MG Hanna, P Nettesheim, and JR Gilbert (eds). United States Atomic Energy Commission, Oak Ridge, Tennessee, pp. 27-54.

33.

33. Saffiotti U (1973). Metabolic host factors in carcinogenesis. IARC Sci. Publ. 7: 243–252.

34.

34. Saffiotti U , Rice JM , and Donovan P (1979). Interactions of multiple carcinogens at low levels of exposure: preliminary mutagenicity studies using the Ames Salmonella system. In: Short Term Tests for Prescreening of Potential Carcinogens. L Santi and S Parodi (eds). Instituto Scientifico per lo Studio e la Cura dei Tumori, Genoa, pp. 99–109.

35.

35. Shipley GD and Ham RG (1980). Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of Swiss 3T3 cells. In Vitro 17: 656–670.

36.

36. Shipley SD and Ham RG (1983). Control of entry of Swiss 3T3 cells into S phase by fibroblast growth factor under serum-free conditions. Exp. Cell Res. 146: 261–270.

37.

36a. Shipley SD and Ham RG (1984). Personal communication.

38.

37. Smith G , Grisham JW , and Kaufman DG (1981). Cycle-dependent removal of certain methylated bases from DNA of 10T1/2 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine. Cancer Res. 41: 1373–1378.

39.

38. Tong C , Fazio M. and Williams GM (1980). Cell cycle-specific mutagenesis at the hypoxanthine phosphoribosyltransferase locus in adult rat liver epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 77: 7377–7379.

40.

39. Vergara R and Terzi M (1980). Studies of mutagenicity in quiescent and proliferating hamster cell populations. Cell Biol. Int. Rep. 4: 303–312.

41.

40. Yang LL , Maher VM , and McCormick JJ (1982). Relationship between excision repair and the cytotoxic and mutagenic effect of the ‘anti’ 7,8-diol 9,10-epoxide of benzo[a]pyrene in human cells. Mutat. Res. 94: 435–447.