Biochemical Test

Ames Test Principle, Procedure, Result

What is Ames Test? The Ames test is a biological experiment used to evaluate the potential mutagenicity of chemical substances. As cancer...

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Ames Test Principle, Procedure, Result
Ames Test Principle, Procedure, Result

What is Ames Test?

The Ames test is a biological experiment used to evaluate the potential mutagenicity of chemical substances. As cancer is frequently associated with DNA damage, the test also acts as a rapid assay to estimate the carcinogenic risk of a chemical, as the traditional rat assays for carcinogenicity take years to complete and are costly. In a series of papers from the early 1970s, Bruce Ames and his team at the University of California, Berkeley, describe the technique.

  • Using histidine auxotrophs of Salmonella typhimurium (which lacks the ability to biosynthesise histidine due to a mutation in an important gene) or tryptophan auxotrophs of Escherichia coli, this assay evaluates the possible carcinogenic effect of substances.
  • Not every mutagen is also a carcinogen, but many mutagenic substances are also carcinogenic (capable of causing cancer in humans and other animals); hence, the fact that a compound is mutagenic to bacteria is an indication of potential hazard.
  • Salmonella strains that have undergone mutations cannot develop and form colonies in the absence of histidine.
  • When these mutant bacterial cells are exposed to substances with mutagenic properties, the mutation may be reversed, allowing the bacterial cells to grow on media without histidine.
  • Mutagenic chemicals identified by the Ames test can be evaluated for their propensity to cause cancer in animals.
  • The Ames Test combines a bacterial revertant mutation assay with a modelling of mammalian metabolism to generate a highly sensitive test for environmental mutagenic substances.
  • A homogenate of rat liver is used to make a metabolically active extract (S9). The extract is mixed with a strain of his- Salmonella bacteria; without histidine, the bacteria cannot thrive in minimum media (control result).
  • The homogenate and bacterial strain are mixed with a chemical suspected of being mutagenic (X). Substance X is a mutagen since the production of revertant colonies implies that some his- bacteria have transformed (reverted) to his+.
  • Different bacterial strains are susceptible to distinct mutation types.
  • Initially, the reversion assay was conducted without a liver homogenate. Unlike toxicity, mutagenicity is not caused by the consumption of a suspicious drug, but rather by its accumulation and that of its breakdown products within the body.
  • The utilisation of a liver homogenate simulates the metabolic breakdown of the suspected mutagen in a mammalian system and more precisely predicts the mutagenicity of items consumed by humans.
  • Sodium nitrate (NaNO3), which naturally occurs in smoked foods such as bacon, hot dogs, ham, etc., is not carcinogenic.
  • However, following interaction with HCl in the stomach, it is transformed to nitrous acid (HNO2), which the Ames Test has shown to be a potent mutagen.
  • In the 1970s, Bruce Ames (1928 -) and his undergraduate students at UC Berkeley tested a huge number of commercial products in student laboratories.
  • Numerous common products, such as hairspray and food colouring, were deemed mutagenic and removed from the market.
  • Ames also demonstrated that many mutagenic chemicals are also carcinogenic, providing an early indication that alterations in the DNA sequence linked to cancer.

Principle of Ames Test

  • Salmonella typhimurium histidine auxotrophs are distributed on a culture medium lacking histidine in the presence of the putative mutagen.
  • As the media lacks a critical ingredient, histidine in this example, auxotrophic/mutant strains are unable to thrive, even if a vast number of cells are dispersed across the medium.
  • However, if a particular bacterial strain could return to its natural type (known as back mutants or revertants), it would form colonies and proliferate.
  • The presence of mutagenicity in the test chemical is validated if the reversion rate increases in the presence of a suspected mutagen (with proof of the development of additional colonies in the presence of a suspected mutagen).
  • The greater the number of such colonies, the greater the putative mutagen’s mutagenic potential.
  • Using animal models, compounds identified as mutagenic by the Ames test are evaluated for their possible carcinogenic effects.
  • Tryptophan auxotrophs of Escherichia coli can be utilised to conduct the Ames test; however, in a medium devoid of amino acid, tryptophan must be employed.
Ames Test Principle, Procedure, Result
Ames Test Principle, Procedure, Result

Test organism

Ames test uses several strains of bacteria (Salmonella, E.coli) that carry mutation. Eg A particular strain of Salmonella Typhimurium carry mutation in gene that encodes histidine. So it is an auxotrophic mutant which loss the ability to synthesize histidine (an amino acid) utilizing the ingredients of culture media. Those strains are known as His- and require histidine in growth media.

Culturing His- salmonella is in a media containing certain chemicals, causes mutation in histidine encoding gene, such that they regain the ability to synthesize histidine (His+) This is the reverse mutation. Such chemicals responsible to revert the mutation is actually a mutagen. So, this Ames test is used to test mutagenic ability of varieties of chemicals.

Procedure of Ames Test

  1. Isolate a Salmonella Typhimurium strain that is auxotrophic for histidine. (ie. His-ve)
  2. Prepare a test suspension of his-positive Salmonella Typhimurium in a simple buffer containing the test chemical (eg. 2-aminofluorene). Add a small dose of histidine as well. Small amounts of histidine are required for bacterial growth. Once histidine is low, only bacteria with the mutation to synthesis histidine are able to form colonies.
  3. Also create a control suspension of His-ve Salmonella Typhimurium that does not include any test chemicals.
  4. At 37°C, incubate the suspensions for 20 minutes.
  5. Prepare two agar plates on which to disperse the suspension.
  6. Incubate the plates for 48 hours at 37°C.
  7. Count the number of colonies on each plate after 48 hours.
Ames Test Principle, Procedure, Result
Ames Test Principle, Procedure, Result

Result Interpretation

  • Mutagenicity of substances is proportional to the observed number of colonies.
  • If there are more colonies on the test plate compared to the control, then these chemicals are considered mutagens.
  • Very few colonies are also visible on the control plate. This could be the result of a spontaneous mutation in the hisidine-encoding gene.

Uses of Ames Test

  • The practical application of the Ames test is to identify chemical mutagens that are carcinogenic to humans and animals and cause mutations. Some of the food additives (AF-2) and flavouring agents (Safrole) are both mutagenic and carcinogenic.
  • The anti-tuberculosis medicine isoniazid is also a mutagen.
  • Ames test accepted for testing mutagens using eukaryotic cell culture, yeast cell, and animal model. Since Salmonella is not the ideal organism for testing human mutagens, it cannot be used as a test organism. Certain compounds are originally not mutagens for humans, but during metabolism they become such (acted upon by body enzymes). For instance, sodium nitrate (NaNO3) is not mutagenic until it reacts with HCL in the stomach to generate nitrogen oxide (HNO2) (a potent mutagen).
  • A highly sensitive Ames test can find Suitable mutations in vast populations of bacteria.
  • It is a mutagenicity test, not a carcinogenicity test. However, more than ninety percent of the mutagens found by the Ames test are cancer-causing.
  • It is a test for bacterial reverse mutation. Therefore, bacteria’s faulty gene can be transformed into a functional gene.

Limitations

  • Some compounds that cause cancer in laboratory animals (such as dioxin) do not yield a positive Ames test (and vice versa). 
  • The Ames assay is comprised of Salmonella typhimurium strains and is therefore not an ideal model for humans.

Sterilization (safety considerations while working with Salmonella)

  • As S. typhimurium is a dangerous bacteria, it is prudent to always take precautions and follow basic biosafety procedures, such as using pipettes with plugs, sterilising all contaminated material with 70% ethanol, and autoclaving it.
  • A biosafety cabinet is required for the handling of chemicals and pathogens. Before and after usage, the cabinet must be sanitised with 70% ethanol and 15 minutes of ultraviolet light.
  • To prevent against chemical exposure, gowns, eyeglasses, and gloves must be used.
  • All contaminated materials (such as test tubes, pipettes, and pipette tips, gowns, and gloves) must be adequately autoclaved prior to disposal.

References

  • Föllmann, W., Degen, G., Oesch, F., & Hengstler, J. G. (2013). Ames Test. Brenner’s Encyclopedia of Genetics, 104–107. doi:10.1016/b978-0-12-374984-0.00048-6 
  • Hengstler, J. G., & Oesch, F. (2001). Ames Test. Encyclopedia of Genetics, 51–54. doi:10.1006/rwgn.2001.1543 
  • Jain, A. K., Singh, D., Dubey, K., Maurya, R., Mittal, S., & Pandey, A. K. (2018). Models and Methods for In Vitro Toxicity. In Vitro Toxicology, 45–65. doi:10.1016/b978-0-12-804667-8.00003-1 
  • Vijay U, Gupta S, Mathur P, Suravajhala P, Bhatnagar P. Microbial Mutagenicity Assay: Ames Test. Bio Protoc. 2018 Mar 20;8(6):e2763. doi: 10.21769/BioProtoc.2763. PMID: 34179285; PMCID: PMC8203972.
  • Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutat Res. 2000 Nov 20;455(1-2):29-60. doi: 10.1016/s0027-5107(00)00064-6. PMID: 11113466.
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