Ames test

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The Ames test is a test procedure to identify (chemical) mutagens . The method was developed by Bruce Ames (University of California, Berkeley, USA).

The principle

There are bacteria , by mutation (z. B. point mutation ) in a gene is no longer capable of a certain amino acid to synthesize (so-called deficient mutants, see auxotrophy ), not containing a said amino acid medium ( agar ) is applied. Since these bacteria are dependent on this amino acid to survive, they would die or could not reproduce on this deficient medium. The amino acid (e.g. histidine) is necessary for the synthesis of proteins and thus for cell division.

The bacteria are now exposed to the potential mutagen , for example by placing a filter paper soaked with it on the nutrient medium. If so-called bacterial colonies form after the subsequent incubation , individual bacteria have grown and regained the ability to synthesize the corresponding amino acid. These are so-called revertants , in which the point mutation leading to auxotrophy was reversed in a gene - they became prototrophic again . It is assumed that this reverse mutation is very likely to be attributed to the effect of the added agent and that it is therefore a mutagen that causes a point mutation in a gene. As a rule, such a reverse mutation also occurs spontaneously by itself, but on a much smaller scale, that is to say much less frequently than in the presence of a mutagenic agent.

In the Ames test, bacterial strains of Escherichia coli ( tryptophan auxotrophy) or Salmonella typhimurium ( histidine auxotrophy) are usually used.

In addition to its need for histidine, Salmonella typhimurium is characterized by two other properties that are advantageous for the Ames test: On the one hand, it has a defect in the DNA repair system, so that the mutation that has arisen cannot be repaired; there is, so to speak, no unreported number. In addition, this bacterium has shortened lipopolysaccharides , which means that the membrane is more permeable and potential mutagens are not completely or partially blocked there. Both properties lead to an increase in the informative value of the Ames test.

Experiments with biological mutagens ( viruses ) are also conceivable . Usually, however, this test is used with chemicals.

application

The test is recognized by the OECD and is used, for example, in pharmaceutical research to test potential drugs for mutagenicity . The Ames test is also used as a basic requirement in the chemical assessment under REACH . The procedure is quick (about a week), cheap and easy. More than 3500 substances (as of 2006) were tested with it; the test is also well suited to investigate unknown mixtures.

The transferability of the results of the Ames test to humans or organisms other than the bacterial strains used is not necessarily given. The agent to be tested, for example, often undergoes modifications, particularly in the liver of higher organisms, which give it its mutagenic properties. Such substances would pass the Ames test as false negative. The reverse is also possible, in which the agent is inactivated in the liver and loses its mutagenic properties. To take this into account, in practice the agent is first mixed with a liver extract ( S9 mix ). The enzymes contained in the extract simulate the metabolism taking place in the liver ( phase 1 reaction ).

literature

  • Bruce N. Ames , Joyce McCann, Edith Yamasaki: Methods for Detecting Carcinogens and Mutagens with the Salmonella / Mammalian-Microsome Mutagenicity Test. In: Mutation Research . Vol. 31, No. 6, December 1975, pp. 347-364, PMID 768755 .

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