Ames Test (Bacterial Reverse Mutation Test): Why, When, and How to Use

Protocol
First Online:
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

The Salmonella typhimurium/mammalian microsome assay is the most widely used short-term test to identify genetic damage. This is used to assess the mutagenic and antimutagenic potential of compounds and mixtures. This assay uses histidine-dependent strains to detect mutations, e.g., substitutions, additions, or deletions of one or several DNA nucleotides reverting originally changed gene sequence of the tester strains. The addition of a mutagenic chemical agent to a plate of cultured cells results in the growth of mutant colonies; the number of such colonies is an indicator of the mutagenic potency of the agent. The Ames test has many advantages, it is a very versatile assay, its different modifications have been developed to determine mutagenic potencies, and it is recommended by several regulatory agencies. This chapter provides a detailed description of how the standard plate incorporation method should be performed, including the experimental design and interpretation of results.

Key words

Ames test Mutagenicity Toxicity Spontaneous revertant Salmonella Standard incorporation plate 
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Notes

Acknowledgments

The authors express their thanks to Dr. Oscar Herrero, Dr. Eduardo de la Peña Jr., Ms. Antonia Martinez, and the Institute of Agricultural Sciences (CSIC).

References

  1. 1.
    Ames BN, McCann J, Yamasaki E (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat Res 31:347–364PubMedCrossRefGoogle Scholar
  2. 2.
    Maron D, Ames BN (1983) Revised methods for the Salmonella mutagenicity test. Mutat Res 113:173–215PubMedCrossRefGoogle Scholar
  3. 3.
    Ames BN, Lee FD, Durston WE (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Natl Acad Sci U S A 70(3):782–786PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Ames BN, Durston WE, Yamasaki E et al (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci U S A 70(8):2281–2285PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Mortelmans K, Riccio ES (2000) The bacterial tryptophan reverse mutation assay with Escherichia coli WP2. Mutat Res 455(1–2): 61–69PubMedCrossRefGoogle Scholar
  6. 6.
    Mortelmans K, Zeiger E (2000) The Ames Salmonella/microsome mutagenicity assay. Mutat Res 455(1–2):29–60PubMedCrossRefGoogle Scholar
  7. 7.
    Claxton LD, Umbuzeiro G, DeMarini DM (2010) The Salmonella mutagenicity assay: the stethoscope of genetic toxicology for the 21st century. Environ Health Perspect 118(11): 1515–1522PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Eaton DL, Gilbert SG (2008) Principles of toxicology. In: Klaassen CD (ed) Casarett and Doull’s toxicology, the basic science of poisons, 7th edn. McGraw-Hill, New York, pp 11–45Google Scholar
  9. 9.
    McCann J, Choi E, Yamasaki E et al (1975) Detection of carcinogens in the Salmonella/microsome test: assay of 300 chemicals. Proc Natl Acad Sci U S A 72:5135–5139PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Mehmet K, Özlem B, Medine G (2012) Salmonella as a unique tool for genetic toxicology, Salmonella—a diversified superbug. In: Kumar Y (ed), ISBN: 978-953-307-781-9. InTech. http://www.intechopen.com/books/salmonella-a-diversified-superbug/salmonella-as-aunique-tool-for-genetic-toxicology. Accessed 15 Nov 2013
  11. 11.
    Kado NY, Langley D, Eisenstadt E (1983) A simple modification of the Salmonella liquid incubation assay: increased sensitivity for detecting mutagens in human urine. Mutat Res 112:25–32CrossRefGoogle Scholar
  12. 12.
    Hughes TJ, Simmons DM, Monteith LG et al (1987) Vaporization technique to measure mutagenic activity of volatile organic chemicals in the Ames/Salmonella assay. Environ Mutagen 9:421–441PubMedCrossRefGoogle Scholar
  13. 13.
    Zeiger E, Anderson B, Haworth S et al (1992) Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals. Environ Mol Mutagen 19(21):1–141Google Scholar
  14. 14.
    Araki A, Noguchi T, Kato F et al (1994) Improved method for mutagenicity testing of gaseous compounds by using a gas sampling bag. Mutat Res 307:335–344PubMedCrossRefGoogle Scholar
  15. 15.
    Tejs S (2008) The Ames test: a methodological short review. Environ Biotechnol 4(1):7–14Google Scholar
  16. 16.
    Dearfield KL, Auletta AE, Cimino MC et al (1991) Considerations in the U.S. Environmental Protection Agency’s testing approach for mutagenicity. Mutat Res 258:259–283PubMedCrossRefGoogle Scholar
  17. 17.
    Kramers PGN, Knaap AGAC, van der Heijden CA et al (1991) Role of genotoxicity assays in the regulation of chemicals in The Netherlands: considerations and experiences. Mutagenesis 6: 487–493PubMedCrossRefGoogle Scholar
  18. 18.
    FDA (US Food and DruGg Administration) (1993) Toxicological principles for the safety assessment of direct food additives and color additives used in food “Redbook II” [Draft]. FDA, Center for Food Safety and Applied Nutrition, Washington, DCGoogle Scholar
  19. 19.
    HPBGC (Health Protection Branch Genotoxicity Committee Guidelines; Canada) (1993) The assessment of mutagenicity. Health Protection Branch mutagenicity guidelines. Environ Mol Mutagen 21:15–37CrossRefGoogle Scholar
  20. 20.
    Kirkland DJ (1993) Genetic toxicology testing requirements: official and unofficial views from Europe. Environ Mol Mutagen 21: 8–14PubMedCrossRefGoogle Scholar
  21. 21.
    OECD 471 (1997) Guideline for testing of chemicals. Bacterial reverse mutation test. Organization for Economic Cooperation and Development, Paris, Adopted 26 May 1983, last updated 21st JulyGoogle Scholar
  22. 22.
    Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) (2006)Google Scholar
  23. 23.
    Houk VS (1992) The genotoxicity of industrial wastes and effluents. Mutat Res 277:91–138PubMedCrossRefGoogle Scholar
  24. 24.
    Wilkinson RG, Gemski P Jr, Stocker BAD (1972) Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J Gen Microbiol 70:527–554PubMedCrossRefGoogle Scholar
  25. 25.
    Walker GC, Dobson PP (1979) Mutagenesis and repair deficiencies of Escherichia coli umuC mutants are suppressed by the plasmid pKM101. Mol Gen Genet 172:17–24PubMedCrossRefGoogle Scholar
  26. 26.
    Mahon G, Green M, Middleton B et al (1989) Analysis of data from microbial colony assays. In: Kirkland DJ (ed) UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report: part III. Statistical evaluation of mutagenicity test data. Cambridge University Press, Cambridge, pp 28–65Google Scholar
  27. 27.
    Ong TM, Mukhtar CR, Wolf CR et al (1980) Differential effects of cytochrome P450-inducers on promutagen activation capabilities and enzymatic activities of S9 from rat liver. J Environ Pathol Toxicol 4:55–65PubMedGoogle Scholar
  28. 28.
    Barrueco C, de la Peña E (1988) Mutagenic evaluation of pesticides captan, folpet, captafol, dichlofluanid and related compounds with the mutants TA102 and TA104 of Salmonella typhimurium. Mutagenesis 3(6):467–480PubMedCrossRefGoogle Scholar
  29. 29.
    Barrueco C, Herrera A, de la Peña E (1991) Mutagenic evaluation of trichlorfon using different assay methods with Salmonella typhimurium. Mutagenesis 6(1):71–76PubMedCrossRefGoogle Scholar
  30. 30.
    Zeiger E, Mortelmans K (1999) The Salmonella (Ames) test for mutagenicity. Curr Protoc Toxicol Unit 3.1:3.1.1–3.1.29. Doi:  10.1002/ 0471140856.tx0301s00
  31. 31.
    Ansari MI, Malik A (2009) Genotoxicity of agricultural soils in the vicinity of industrial area. Mutat Res 673:124–132PubMedCrossRefGoogle Scholar
  32. 32.
    Líman R, Eren Y, Akyil D, Konuk M (2012) Determination of mutagenic potencies of aqueous extracts of Thermopsis turcica by Ames test. Turk J Biol 36:85–92Google Scholar
  33. 33.
    Pillco A (2011) Evaluación toxicológica de mezclas complejas mediante ensayos alternativos a la experimentación animal de genotoxicidad, mutagenicidad y ecotoxicidad (Toxicological evaluation of complex mixtures employing genotoxicity, mutagenicity and ecotoxicity assays as an alternative to animal testing) Doctoral thesis, Autonomous University of Madrid, p 173Google Scholar
  34. 34.
    Rank J, Nielsen MH (1998) Genotoxicity testing of wastewater sludge using the Allium cepa anaphase-telophase chromosome aberration assay. Mutat Res 418:113–119PubMedCrossRefGoogle Scholar
  35. 35.
    Monarca S, Feretti D, Zerbini I et al (2002) Soil contamination detected using bacterial and plant mutagenicity test and chemical analyses. Environ Res Sect A 88:64–69CrossRefGoogle Scholar
  36. 36.
    Sandoval AM (2008) Ensayo de mutagenicidad con la bacteria Salmonella typhimurium. Prueba de Ames (Mutagenicity assay with Salmonella typhimurium. Ames test). In: Ramirez P, Mendoza A (eds) Ensayos toxicológicos para la evaluación de sustancias químicas en agua y suelo, 1st edn. La experiencia en México, MéxicoGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Laboratory of Environmental MutagenesisSpanish National Research Council (CSIC)MadridSpain

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