The L5178Y/TK Gene Mutation Assay System

  • David E. Amacher


The L5178Y/TK+ ➙ TK assay is one of several recognized mammalian cell systems for estimating the genotoxic potential of environmental or commercial chemicals. In a typical test application, thymidine kinase- competent (TK+) 3.7.2C L5178Y cells are exposed to five or more different concentrations of the test substance for 1–4 hr, maintained 48–72 hr in growth medium, then cloned in soft-agar medium in a single-step selection process to detect mutagen-induced thymidine kinase-deficient (TK) variants. Independent validation studies(11,15,30) have demonstrated that this assay is sensitive to more than 20 known genotoxic chemicals. This short-term mutagenesis assay is a good predictor of mammalian carcinogenicity, with few “false positive” results in those studies where noncarcinogens were tested.(8)


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  1. 1.
    R. Adler and B. R. McAuslan, Expression of thymidine kinase variants is a function of the replicative state of cells,Cell 2, 113–117 (1974).CrossRefGoogle Scholar
  2. 2.
    D. E. Amacher and S. C. Paillet, Induction of trifluorothymidine-resistant mutants by metal ions in L5178Y/TK+/- cells,Mutat. Res. 78, 279–288 (1980).CrossRefGoogle Scholar
  3. 3.
    D. E. Amacher and S. C. Paillet, Trifluorothymidine-resistance and colony size in L5l78Y/TK+/- cells treated with methyl methanesulfonate,J. Cell Physiol. 106, 349–360 (1981).CrossRefGoogle Scholar
  4. 4.
    D. E. Amacher and S. C. Paillet, Hamster hepatocyte–mediated activation of procar- cinogens to mutagens in the L5178Y/TK mutation assay,Mutat. Res. 106, 305–316. (1982).CrossRefGoogle Scholar
  5. 5.
    D. E. Amacher and S. C. Paillet, The activation of procarcinogens to mutagens by cultured rat hepatocytes in the L5178Y/TK mutation assay,Mutat. Res. 113, 77–78. (1983).CrossRefGoogle Scholar
  6. 6.
    D. E. Amacher and G. N. Turner, Promutagen activation by rodent-liver postmitochondrial fractions in the L5178Y/TK cell mutation assay,Mutat. Res. 74, 485–501 (1980).CrossRefGoogle Scholar
  7. 7.
    D. E. Amacher and G. N. Turner, The effect of liver postmitochondrial fraction concentration from Aroclor 1254-treated rats on promutagen activation in L5178Y cells,Mutat. Res. 97, 131–137 (1982).CrossRefGoogle Scholar
  8. 8.
    D. E. Amacher and G. N. Turner, Mutagenic evaluation of carcinogens and non-carcinogens in the L5178Y/TK assay utilizing postmitochondrial fractions (S9) from normal rat liver,Mutat. Res. 97, 49–65 (1982).CrossRefGoogle Scholar
  9. 9.
    D. E. Amacher, and G. N. Turner, The L5178Y/TK gene mutation assay for the detection of chemical mutagens, Ann.NY. Acad. Sci. 407, , 239–252 (1983).CrossRefGoogle Scholar
  10. 10.
    D. E. Amacher, S. C. Paillet, and J. A. Elliott, The metabolism of N-acetyl-2-aminofluorene to a mutagen in L5l78Y/TK+/- mouse lymphoma cells,Mutat. Res. 89, 311–320 (1981).CrossRefGoogle Scholar
  11. 11.
    D. E. Amacher, S. Paillet, and V. A. Ray, Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicological testing,Mutat. Res. 64, 391–406 (1979).CrossRefGoogle Scholar
  12. 12.
    D. E. Amacher, S. C. Paillet, and G. N. Turner, Utility of the mouse lymphoma L5178Y/TK assay for the detection of chemical mutagens, in:Banbury Report 2, Mammalian Cell Mutagenesis: The Maturation of Test Systems (A. W. Hsieet al, eds), pp. 277–293, Cold Spring Harbor Laboratory (1979).Google Scholar
  13. 13.
    D. E. Amacher, S. C. Paillet, and I. Zelljadt, Metabolic activation of 3-methylcholan-threne and benzo(a)pyrene to mutagens in the L5178Y/TK assay by cultured embryonic rodent cells,Environ. Mutagen. 4, 109–119 (1982).CrossRefGoogle Scholar
  14. 14.
    D. E. Amacher, G. N. Turner, and J. H. Ellis, Detection of mammalian cell mutagens in urine from carcinogen-dosed mice,Mutat. Res. 90, 79–90 (1981).CrossRefGoogle Scholar
  15. 15.
    D. E. Amacher, S. C. Paillet, G. N. Turner, V. A. Ray, and D. S. Salsburg, Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation,Mutat. Res. 72, 447–474 (1980).CrossRefGoogle Scholar
  16. 16.
    B. N. Ames, W. E. Durston, E. Yamaski, and F. D. Lee, Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection,Proc. Natl. Acad. Sci. USA 70, 2281–2285 (1973).CrossRefGoogle Scholar
  17. 17.
    B. N. Ames, J. McCann, and E. Yamaski, Methods for detecting carcinogens and mutagens withSalmonella/mammalian-microsome mutagenicity test,Mutat. Res. 31, 347–364 (1975).CrossRefGoogle Scholar
  18. 18.
    W. K. Baker, Position–effect variegation,Adv. Gen. 14, 133–169 (1968).CrossRefGoogle Scholar
  19. 19.
    D. Brusick,Principles of Genetic Toxicology, Springer Science+Business Media New York (1980).CrossRefGoogle Scholar
  20. 20.
    M. Caboche, Comparison of the frequencies of spontaneous and chemically-induced 5-bromodeoxy-uridine-resistance mutations in wild-type and revertant BHK-21/13 cells,Genetics 77, 309–322 (1974).Google Scholar
  21. 21.
    D. Clive, Mutagenicity of thioxanthenes (hycanthone, lucanthone and four indazole derivatives) at the TK locus in cultured mammalian cells,Mutat. Res. 26, 307–318 (1974).CrossRefGoogle Scholar
  22. 22.
    D. Clive and G. Hajian, Letter to the editor,Mutat. Res. 89, 250–253 (1981).CrossRefGoogle Scholar
  23. 23.
    D. Clive and M. M. Moore-Brown, The L5178Y/TK mutagen assay system: mutant analysis, in:Banbury Report 2, Mammalian Cell Mutagenesis: The Maturation of Test Systems (A. W. Hsieet al, eds.), pp. 421–429, Cold Spring Harbor Laboratory (1979).Google Scholar
  24. 24.
    D. Clive and J. F. S. Spector, Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells,Mutat. Res. 31, 17–29 (1975).CrossRefGoogle Scholar
  25. 25.
    D. Clive and P. Voytek, Evidence for chemically-induced structural gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells,Mutat. Res. 44, 269–278 (1977).CrossRefGoogle Scholar
  26. 26.
    D. Clive, A. G. Batson, and N. T. Turner, The ability of L5178Y/TK+ mouse lymphoma cells to detect single gene and viable chromosome mutations: Evaluation and relevance to mutagen and carcinogen screening, in:The Predictive Value of Short- Term Screening Tests in Carcinogenicity (G. M. Williamset al., eds.), pp. 103–123, Elsevier/ North-Holland, New York (1980).Google Scholar
  27. 27.
    D. Clive, W. G. Flamm, and M. R. Machesko, Mutagenicity of hycanthone in mammalian cells,Mutat. Res. 14, 262–264 (1972).CrossRefGoogle Scholar
  28. 28.
    D. Clive, W. G. Flamm, and J. B. Patterson, Specific–locus mutational assay systems for mouse lymphoma cells, in:Chemical Mutagens, Principles and Methods for Their Detection, Vol. 3 (A. Hollander, ed.), pp. 79–103, Springer Science+Business Media New York (1973).CrossRefGoogle Scholar
  29. 29.
    D. Clive, W. G. Flamm, M. R. Machesko, and N. J. Bernheim, A mutational assay system using the thymidine kinase locus in mouse lymphoma cells,Mutat. Res. 16, 77–87 (1972).CrossRefGoogle Scholar
  30. 30.
    D. Clive, K. O. Johnson, J. F. S. Spector, A. G. Batson, and M. M. M. Brown, Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system,Mutat. Res. 59, 61–108 (1979).CrossRefGoogle Scholar
  31. 31.
    D. Clive, R. McCuen, J. F. S. Spector, C. Piper, and K. H. Mavournin, Specific gene mutations in L5178Y cells in culture: A report of the U. S. EPA Gene-Tox Program,Mutat. Res. 115,, 225–251 (1983).CrossRefGoogle Scholar
  32. 32.
    J. Cole and C. F. Arlett, Ethyl methanesulphonate mutagenesis with L5178Y mouse lymphoma cells: A comparison of ouabain, thioguanine and excess thymidine resistance,Mutat. Res. 34, 507–526 (1976).CrossRefGoogle Scholar
  33. 33.
    J. Cole and C. F. Arlett, Methyl methanesulphonate mutagenesis in L5178Y mouse lymphoma cells,Mutat. Res. 50, 111–120 (1978).CrossRefGoogle Scholar
  34. 34.
    E. C. Cox, J. R. White, and J. G. Flaks, Streptomycin action and the ribosome,Proc. Natl. Acad. Sci. USA 51, 703–785 (1964).CrossRefGoogle Scholar
  35. 35.
    J. Davies, L. Gorini, and B. D. Davis, Misreading of RNA codewords induced by aminoglycoside antibiotics,Mol. Pharmacol. 1, 93–106 (1965).Google Scholar
  36. 36.
    J. Dayan, M. C. Crajer, and S. Deguingand, Mutagenic activity of 4 active-principle forms of pharmaceutical drugs. Comparative study of theSalmonella typhimurium microsome test, and the HGPRT and Na+/K+ ATPase systems in cultured mammalian cells,Mutat. Res. 102, 1–12 (1982).CrossRefGoogle Scholar
  37. 37.
    P. B. Dent, S. K. Lia, G. Ettin, and G. B. Cleland, Characterization of an inhibitor of thymidine uptake produced by cultured human melanoma cells,Oncology 35, 235– 241 (1978).CrossRefGoogle Scholar
  38. 38.
    L. Diamond, F. Kruszewski, and W. Baird, Expression time for benzo(α)pyrene-induced 6-thioguanine-resistant mutations in V79 Chinese hamster cells,Mutat. Res. 95, 353–362 (1982).CrossRefGoogle Scholar
  39. 39.
    G. A. Fischer, Studies on the culture of leukemic cellsin vitro, Ann. N. Y. Acad. Sci. 76, 673–680 (1958).CrossRefGoogle Scholar
  40. 40.
    G. A. Fischer, The host-mediated mammalian cell assay,Agents Actions 3/2, 93–98 (1973).Google Scholar
  41. 41.
    G. A. Fischer and A. S. Sartorelli, I. Development, maintenance of assay of drug resistance,Meth. Med. Res. 10, 247–262 (1964).Google Scholar
  42. 42.
    M. Fox and B. W. Fox, Effects of methyl methanesulfonate on the growth of P-388 cellsin vitro and on their rate of progress through the cell cycle,Cancer Res. 27, 1805–1812 (1967).Google Scholar
  43. 43.
    M. Fox and M. Radacic, Adaptational origin of some purine-analogue resistant phenotypes in cultured mammalian cells,Mutat. Res. 49, 275–296 (1978).CrossRefGoogle Scholar
  44. 44.
    C. N. Frantz and H. V. Mailing, Bromodeoxyuridine resistance induced in mouse lymphoma cells by microsomal activation of dimethylnitrosamine,J. Toxicol. Environ. Health 2, 179–187 (1976).CrossRefGoogle Scholar
  45. 45.
    F. M. Griffin, G. Ashland, and R. L. Capizzi, Kinetics of phototoxicity of Fischer’s medium for L5178Y leukemic cells,Cancer Res. 41, 2241–2248 (1981).Google Scholar
  46. 46.
    S. M. Haag and I. G. Sipes, Differential effects of acetone or Aroclor 1254 pretreatment on the microsomal activation of dimethylnitrosamine to a mutagen,Mutat. Res. 74, 431–438 (1980).CrossRefGoogle Scholar
  47. 47.
    C. Heidelberger, Chemical carcinogenesis, chemotherapy: Cancer’s continuing core challenges—G. H. A. Clower memorial lecture,Cancer Res. 30, 1549–1569 (1970).Google Scholar
  48. 48.
    T. Hirakawa, N. Nemoto, M. Yamada, and S. Takayama, Metabolism of benzo(α)pyrene and the related enzyme activities in hamster embryo cells,Chem. Biol. Interact. 25, 189–195 (1979).CrossRefGoogle Scholar
  49. 49.
    J. Hozier, J. Sawyer, M. Moore, B. Howard, and D. Clive, Cytogenetic analysis of the L5178Y/TK+/- TK–/- mouse lymphoma mutagenesis assay system,Mutat. Res. 84, 169–181 (1981).CrossRefGoogle Scholar
  50. 50.
    E. Huberman and C. A. Jones, The use of liver cell cultures in mutagenesis studies,Ann. N. Y. Acad. Sci. 349, 264–272 (1980).Google Scholar
  51. 51.
    E. D. Jacobson, K. Krell, M.J. Dempsey, M. H. Lugo, O. Ellingson, and C. W. Hench II, Toxicity and mutagenicity of radiation from fluorescent lamps and a sunlamp in L5178Y mouse lymphoma cells,Mutat. Res. 51, 61–75 (1978).CrossRefGoogle Scholar
  52. 52.
    E. M. Jensen, R. J. LaPolla, P. E. Kirby, and S. R. Haworth,In vitro studies of chemical mutagens and carcinogens. Stability studies in cell culture medium,J. Natl. Cancer Inst. 59, 941–944 (1977).Google Scholar
  53. 53.
    D. Henssen and C. Ramel, Relationship between chemical damage of DNA and mutations in mammalian cells. I. Dose–response curves for the induction of 6-thioguanine-resistant mutants by low doses of monofunctional alkylating agents, x-rays and UV radiation in V79 Chinese hamster cells,Mutat. Res. 73, 339–347 (1980).CrossRefGoogle Scholar
  54. 54.
    M. M. Jotz and A. D. Mitchell, Effects of 20 coded chemicals on the forward mutation frequency at the thymidine kinase locus in L5178Y mouse lymphoma cells, in:Progress in Mutation Research, Evaluation of Short-Term Tests for Carcinogenesis, (F. J. deSerres and J. A. Ashby, eds.) Vol. 1 pp. 580–593, Elsevier/North-Holland, New York (1981).Google Scholar
  55. 55.
    R. W. Kapp, Jr. and B. E. Eventoff, Mutagenicity of dimethyl sulfoxide (DMSO):In vivo cytogenetics study in the rat,Teratogen. Carcinogen. Mutagen. 1, 141–145 (1980).CrossRefGoogle Scholar
  56. 56.
    S. Kit, Thymidine kinase, DNA synthesis and cancer,Mol Cell. Biochem. 11, 161–182 (1976).CrossRefGoogle Scholar
  57. 57.
    R. E. Kouri, R. Kiefer, and E. M. Zimmerman, Hydrocarbon–metabolizing activity of various mammalian cells in culture,In Vitro 10, 18–25 (1974).CrossRefGoogle Scholar
  58. 58.
    T. Kuroki, G. Malaveille, C. Drevon, C. Piccoli, M. Macleod, and J. K. Selkirk, Critical importance of microsome concentration in mutagensis assay with V79 Chinese hamster cells,Mutat. Res. 63, 259–272 (1979).CrossRefGoogle Scholar
  59. 59.
    D. Y. Lai, S. C. Myers, Y. Woo, E. J. Greene, M. A. Friedman, M. F. Argus, and J. C. Areos, Role of dimethylnitrosamine-demethylase in the metabolic activation of dimethylnitrosamine,Chem. Biol. Interact. 28, 107–126 (1979).CrossRefGoogle Scholar
  60. 60.
    R. Langenbach, S. Nesrow, A. Tompa, R. Gingell, and C. Kuszynski, Lung and liver cell-mediated mutagenesis systems: Specificities in the activation of chemical carcinogens,Carcinogenesis 2, 851–858 (1981).CrossRefGoogle Scholar
  61. 61.
    J. W. Littlefield, The periodic synthesis of thymidine kinase in mouse fibroblasts,Biochim. Biophys. Acta 114, 398–403 (1966).CrossRefGoogle Scholar
  62. 62.
    D. Matheson and B. Creasy, Use of the L5178Y (TK+/-) mouse lymphoma cell line coupled with anin vitro microsomal enzyme activation system to study chemical promutagens,Mutat. Res. 38, 400–401 (1976).CrossRefGoogle Scholar
  63. 63.
    S. McMillan and M. Fox, Failure of caffeine to influence induced mutation frequencies and the independence of cell killing and mutation induction in V79 Chinese hamster cells,Mutat. Res. 60, 91–107 (1979).CrossRefGoogle Scholar
  64. 64.
    M. L. Meltz and J. T. MacGregor, Activity of the plant flavanol quercetin in the mouse lymphoma L5178Y TK+/- mutation, DNA single-strand break, and Balb/c 3T3 chemical transformation,Mutat. Res. 88, 317–324 (1981).CrossRefGoogle Scholar
  65. 65.
    G. E. Moore, R. E. Gerner, and H. A. Franklin, Culture of normal human leukocytes,J. Am. Med. Assoc. 199, 519–524 (1967).CrossRefGoogle Scholar
  66. 66.
    M. M. Moore-Brown and D. Clive, The L5178Y/TK+/- mutagen assay system:In situ results, in:Banbury Report 2, Mammalian Cell Mutagenesis: The Maturation of Test Systems (A. W. Hsie,et al, eds.), pp. 71–88, Cold Spring Harbor Laboratory (1979).Google Scholar
  67. 67.
    M. M. Moore–Brown, D. Clive, B. E. Howard, A. G. Batson, and K. O. Johnson, The utilization of trifluorothymidine (TFT) to select for thymidine kinase–deficient (TK–/–) mutants from L5l78Y/TK+/- mouse lymphoma cells,Mutat. Res. 85, 363–378 (1981).CrossRefGoogle Scholar
  68. 68.
    M. M. Moore-Brown and B. E. Howard, Quantitation of small colony trifluorothym- idine-resistant mutants of L5178Y/TK+/- mouse lymphoma cells in RPMI-1640 medium,Mutat. Res. 104, 287–294 (1982).CrossRefGoogle Scholar
  69. 69.
    M. Nagao and T. Sugimura, Molecular biology of the carcinogen, 4-nitroquinoline- 1-oxide, in:Advances in Cancer Research (G. Kleinet al., eds.), Vol. 23, pp. 131–169, Academic Press, New York (1976).Google Scholar
  70. 70.
    H. Nottebrock and R. Then, Thymidine concentrations in serum and urine of different animal species and man,Biochem. Pharmacol. 26, 2175–2179 (1977).CrossRefGoogle Scholar
  71. 71.
    NTP Technical Bulletin #6, p. 6, National Institute of Environmental Health Sciences (January 1982).Google Scholar
  72. 72.
    NTP Technical Bulletin #8, pp. 6–7, National Institute of Environmental Health Sciences (July 1982).Google Scholar
  73. 73.
    F. Oesch, D. Raphael, H. Schwind, and H. R. Glatt, Species differences in activating and inactivating enzymes related to the control of mutagenic metabolites,Arch. Toxicol. 39, 97–108 (1977).CrossRefGoogle Scholar
  74. 74.
    T. Ong, B. Slade, and F. J. de Serres, Mutagenicity and mutagenic specificity of metronidazole and niridazole inNeurospora crassa, J. Environ. Pathol Toxicol. 2, 1109–1118 (1979).Google Scholar
  75. 75.
    K. A. Palmer, Characteristics of thymidine kinse in L5178Y mouse lymphoma cell lines TK+/+ P4, TK+/- p4.3.4, TK+/- 3.7.2, and TK-/- p4.3.Diss. Abstr. Int. B 41, 4382-B (1981).Google Scholar
  76. 76.
    A. R. Peterson, DNA synthesis, mutagenesis, DNA damage and cytotoxicity in cultured mammalian cells treated with alkylating agents,Cancer Res. 40, 682–683 (1980).Google Scholar
  77. 77.
    P. G. W. Plagemann and D. P. Richey, Transport of nucleosides, nucleic acid bases, choline and glucose by animal cells in culture,Biochim. Biophys. Acta 344, 263–305 (1974).CrossRefGoogle Scholar
  78. 78.
    E. H. Postel and A. J. Levine, Studies on the regulation of deoxypyrimidine kinases in normal, SV40-transformed and SV40- and adenovirus-infected mouse cells in culture,Virology 63, 404–420 (1975).CrossRefGoogle Scholar
  79. 79.
    P. Reyes and C. Heidelberger, Fluorinated pyrimidines XXV. The inhibition of thymidylate synthetase from Ehrlich ascites carcinoma cells by pyrimidine analogs,Biochem. Biophys. Acta 103, 177–179 (1965).Google Scholar
  80. 80.
    A. M. Rogers and K. C. Back, Comparative mutagenicity of hydrazine and 3 methylated derivatives in L5178Y mouse lymphoma cells,Mutat. Res. 89, 321–328 (1981).CrossRefGoogle Scholar
  81. 81.
    J. V. Soderman,CRC Handbook of Identified Carcinogens and Noncarcinogens: Carcinogenicity-Mutagenicity Database, CRC Press, Boca Raton, Florida (1982).Google Scholar
  82. 82.
    W. Suter, J. Brennand, S. McMillan, and M. Fox, Relative mutagenicity of antineoplastic drugs an other alkylating agents in V79 Chinese hamster cells, independence of cytotoxic and mutagenic responses,Mutat. Res. 73, 171–181 (1980).CrossRefGoogle Scholar
  83. 83.
    A. D. Tates and E. Kriek, Induction of chromosomal aberrations and sister–chromatid exchanges in Chinese hamster cellsin vitro by some proximate and ultimate carcinogenic arylamide derivatives,Mutat. Res. 88, 397–410 (1981).CrossRefGoogle Scholar
  84. 84.
    W. G. Thilly, J. G. DeLuca, E. E. Furth, H. Hoppe, D. A. Kaden, J. J. Krolewski, H. L. Liber, T. R. Skopek, S. A. Slapikoff, R. J. Tizard, and B. W. Penman, Gene–locus mutation assays in diploid human lymphoblast lines, in:Chemical Mutagens, Principles and Methods for Their Detection Vol. 6 (F, J. de Serres and A. Hollaender, eds.), pp. 331–364, Plenum, Press, New York (1980).CrossRefGoogle Scholar
  85. 85.
    S. Thornton and M. Hite, Inhibitory effect of high S-9 concentrations on activation of hydrocarbons in the L5178Y mouse lymphoma assay, 10th Annual Meeting of the Environmental Mutagen Society, New Orleans, Louisiana (1979).Google Scholar
  86. 86.
    A. Toliver, E. H. Simon, and P. T. Gilham, On the mechanism of 5-bromouracil inhibition of DNA synthesis and cell division,Exp. Cell Res. 53, 506–518 (1968).CrossRefGoogle Scholar
  87. 87.
    J. Unger and J. B. Guttenplan, Kinetics of benzo(α)pyrene-induced mutagensis in a highly sensitiveSalmonella/microsome assay,Mutat. Res. 77, 221–228 (1980).CrossRefGoogle Scholar
  88. 88.
    A. A. Van Zeeland and J. W. I. M. Simons, The effect of calf serum on the toxicity of 8-azaguanine,Mutat. Res. 27, 135–138 (1975).CrossRefGoogle Scholar
  89. 89.
    F. J. Wiebel, S. Brown, H. L. Waters, and J. K. Selkirk, Activation of xenobiotics by monooxygenases: Cultures of mammalian cells as an analytical tool,Arch. Toxicol. 39, 133–148 (1977).CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1984

Authors and Affiliations

  • David E. Amacher
    • 1
  1. 1.Drug Safety EvaluationPfizer Central ResearchGrotonUSA

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