Mutagenesis Assays in Mammalian Cells

Catherine B. Klein1, Limor Broday1, Max Costa1

1 New York University School of Medicine, New York, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.3
DOI:  10.1002/0471140856.tx0303s01
Online Posting Date:  May, 2001
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Mutagenesis assays in mammalian cells are frequently used to complement bacterial mutagenesis assays. This unit describes a mutagenesis assay using either Chinese hamster V79 cells or V79‚Äźderivative gpt transgenic cell line to assess the effects of chemical agents on mammalian cells.

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Table of Contents

  • Basic Protocol 1: Mutagenesis Assays in the V79 and Derived Transgenic Hprt−/GPT+ V79 Cell Lines
  • Support Protocol 1: Toxicity Assay
  • Support Protocol 2: Giemsa Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Mutagenesis Assays in the V79 and Derived Transgenic Hprt−/GPT+ V79 Cell Lines

  • V79 cells (ATCC CCl‐93) or G12 cells (C. Klein, NYU School of Medicine)
  • F‐12 medium/5% FBS (see recipe)
  • F‐12/HAT medium (see recipe)
  • Physiological buffer, 37°C: Earle's balanced salt solution (EBSS; Life Technologies), phosphate‐buffered saline (PBS; appendix 2A), or HEPES‐buffered saline (Life Technologies)
  • Substance to be tested for mutagenicity
  • F‐12 medium/5% FBS (see recipe) containing 10 µg/ml 6‐thioguanine (6‐TG; freshly added)
  • 80‐cm2 tissue culture flasks
  • 100‐mm and 60‐mm tissue culture dishes
  • Additional reagents and equipment for toxicity assay (see protocol 2) and Giemsa staining (see protocol 3)

Support Protocol 1: Toxicity Assay

  • Plates to be stained ( protocol 1 or protocol 2)
  • Giemsa stain (see recipe)
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Literature Cited

Literature Cited
   Aaron, C.S. and Stankowski, L.F. Jr. 1989. Comparison of the AS5/XPRT and the CHO/HPRT assays: Evaluation of 6 drug candidates. Mutat. Res. 223:121‐128.
   Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., Stankowski, L., Theiss, J., and Thompson, E. 1994. Mammalian cell gene mutation assays working group report. Mutat. Res. 312:235‐239.
   Caskey, C.T. and Kruh, G.D. 1979. The HPRT locus. Cell. 16:1‐9.
   Chang, C.C., Castellazzi, M., Glover, T.W., and Trosko, J.E. 1978. Effects of harmon and norharmon on spontaneous and ultraviolet light‐induced mutagenesis in cultures Chinese hamster cells. Cancer Res. 38:4527‐4533.
   Cole, J., Arlett, C.F. 1984. The detection of gene mutations in cultured mammalian cells. In mutagenicity Testing (S. Venitt and J.M. Parry, eds.) pp. 233‐273. IRL Press, Oxford.
   Kargacin, B., Klein, C.B., and Costa, M. 1993. Mutagenic responses of nickel oxides and nickel sulfides in Chinese hamster V79 cell lines at the xanthine‐guanine phosphoribosyl transferase locus. Mutat. Res. 300:63‐72.
   Klein, C.B. and Costa, M. 1997. DNA methylation, heterochromatin and epigenetic carcinogens. Mutat. Res. 386:163‐180.
   Klein, C.B. and Rossman, T.G. 1990. Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis. Environ. Mol. Mutagen. 16:1‐12.
   Klein, C.B., Su, L., Rossman, T.G., and Snow, E.T. 1994. Transgenic gpt+ V79 cell lines differ in their mutagenic response to clastogens. Mutat. Res. 304:217‐228.
   Klein, C.B., Su, L., Singh, J.T., and Snow, E.T. 1997. Characterization of gpt− deletion mutations in transgenic Chinese hamster cell lines. Environ. Mol. Mutagen. 30:418‐428.
   Lee, Y.W., Klein, C.B., Kargacin, K., Salnikow, K., Kitahara, J., Dowjat, K., Zhitkovich, A., Christie, N.T., and Costa, M. 1995. Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: A new model for epigenetic carcinogens. Mol. Cell. Biol. 15:2547‐255.
   Li, A.P., Aaron, C.S., Auletta, A.E., Dearfield, K.L., Riddle, J.C., Slesinski, R.S., and Stankowski, L.F., Jr. 1991. An evaluation of the roles of mammalian cell mutation assays in testing of chemical genotoxicity. Regul. Toxicol. Pharmacol. 14:24‐40.
   Stone‐Wolf, D.S., Klein, C.B., and Rossman, T.G. 1985. HPRT–mutants of V79 cells that revert specifically by base pair substitution and frameshift mutations. Environ. Mutagen. 7:281‐291.
   Tindall, K.R., Stankowski, L.F., Jr., Machanoff, R., and Hsie, A.W. 1986. Analyses of mutation in pSV2gpt‐transformed CHO cells. Mutat. Res. 160:121‐131.
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