BY: RAHUL ANDHARIA (MSIWM001)

Introduction:

  • Mutagenic potential of any compound can be assessed by using AMES test. This test can be also called as test for mutagenicity (refers to compounds or substances causing mutations).
  • Whether a given chemical can cause mutations in the DNA of test organism or not bacteria are utilized in this test.
  • To determine whether the chemical at hand is a mutagen or not, this test was developed in the year 1970 by Bruce N Ames.
  • Examples of mutagens: certain chemicals like Ethidium bromide, which is a carcinogenic agent, UV radiations and X-rays can be called as potential mutagens.
  • If the testing substance yields positive result, then it indicates that the substance is a potential carcinogen or mutagen.

Objective of the test:

  • This test is specifically employed to test mutagenic activity of chemicals.
  •  The test is employed in sample bacteria to study mutations which it confers and to identify the potential mutagen and its pattern of causing mutations.
  • This test is also utilized in testing plumbing products in contact with drinking water, using the reversion system of Salmonella typhimurium histidine (His).

Principle of Ames test:

  • Strains of bacteria carrying a particular mutation are used in Ames test. Strains of bacteria like E.coli, salmonella are used.
  • Histidine or tryptophan operon of salmonella or E.coili is induced with point mutations (change in single base) which make the bacteria deprived of producing the corresponding amino acid.
  • Organisms grow only when histidine or tryptophan is supplied, and this is because of the induced point mutations.
  • Media containing certain chemicals is used to culture His-salmonella which results in mutation in histidine encoding gene, making them able to regain the capacity to synthesize histidine. (His+).
  • This can be explained as, when base substitution or frame shifts (shift in the reading frame) occurs within the gene, it causes reversion to amino acid prototrophy due to reverse mutations.
  • The bacteria obtained from the reverted culture will then be able to grow in histidine- or tryptophan- deficient media.
  • By exposing amino acid requiring organisms to different chemical concentrations and selecting for reversion event, samples mutagenic potential can be thoroughly assessed.
  • For selection purpose media lacking the specific amino acids (histidine and tryptophan) are used to allow growth of only those cells that have undergone prototrophy reversion to histidine and tryptophan to grow and survive.
  • If the reversion is seen in the test sample used, it indicated that the substance used for testing is a mutagen. 

Procedure:

  1. Isolation of auxotrophic strain (strain which lacks ability to synthesize an essential compound or nutrient) from Salmonella for histidine. (His-).
  2. Test suspension is prepared with histidine negative (His-) Salmonella in a plain buffer along with the test chemical to be used. Small amount of histidine is also required to be added to the mixture. (histidine added in smaller amounts helps in the growth of bacteria. As histidine is applied in smaller amounts, once histidine (his) depletes only those bacteria mutated to gain the ability to synthesize histidine will form the colonies).
  3. Control isprepared with his- salmonella without adding test chemicals.
  4. Suspensions are incubated at temperatures of 37 degrees, which is the temperature set inside incubator for 20 minutes.
  5. After 20min, the suspension is taken and is spread on the agar plates. (Perform this under laminar hood.)
  6. Colonies can be observed after 48 hours. Count the number of colonies in each of the agar plates.

Interpretation of results:

  • Chemical mutagenicity is proportional to number of colonies observed.
  • Comparing to control, if there are larger number of colonies, then result can be interpreted as, the chemicals used for testing are potential mutagens.
  • If less number of colonies is observed, probably this could be due to point mutations which occurred spontaneously on histidine encoding gene.

Applications:

  • Screens chemicals that are potential carcinogens or mutagens. Example- AF-2 which is a food additive commonly known as Furylfuramide. This was used as food additives but later was withdrawn in the year 1974 from the market after been identified as mutagenic to bacteria in-vitro.
  • Ames test has the ability to detect mutants from a larger population of bacteria with higher sensitivity.
  • This test is commonly known as test for mutagenicity and not as Carcinogenicity but most of the mutagens, almost above 90% detected by Ames test are known to cause cancer.
  • The defective gene of bacteria can be mutated into functional gene, as Ames is a bacterial reverse mutation assay.
  • Several samples like dye, drugs, reagents, cosmetics are been tested using Ames test to detect mutagens present in them.

Merits:

  • Low cost affordable assay.
  • The assay is simple, rapid and less time consuming.
  • Can detect mutants with high sensitivity even from a very large population.

Limitations:

  • Some Cancer causing substances in laboratory animals does not give positive result for Ames test. Example- Dioxins, highly toxic chemical compounds.
  • This assay cannot be a perfect model for humans, as strains used are of Salmonella Typhimurium.

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