Ames-Test

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

The principle

Are bacteria (e.g., point mutation ) are no longer by mutation in a gene able to synthesize a particular amino acid (so-called deficient mutants, see auxotrophy ), culture medium not containing a said amino acid (agar ) is applied. Since these bacteria are dependent on this amino acid to the continued existence, they would die or could not grow on this medium deficiency. The amino acid (e.g., histidine) for the synthesis of proteins needed for cell division and thus.

Now it is the bacteria from the potential mutagen by hanging up, for example, a so -soaked filter paper on the breeding ground. Developed after the subsequent incubation so-called bacterial colonies, as individual bacteria are grown and have 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 were re- prototrophic. It is believed that this reversion is most likely attributable to the effect of the added agent, and it is therefore to be a mutagen, which causes a point mutation in a gene. Usually such a reverse mutation also occurs spontaneously even at, however, to a much smaller scale, that is, much less than in the presence of a mutagenic agent.

Most are employed in the Ames test strains of Escherichia coli ( tryptophan auxotrophy ) or Salmonella typhimurium a ( histidine auxotrophy ).

Salmonella typhimurium is, beside the histidine still need two more properties that are for the Ames test of advantage: Firstly, it has a defect in the DNA repair system, so that the resulting mutation can not be corrected; there are almost no restrictions underreporting. In addition, this bacterium has shortened lipopolysaccharide, whereby the membrane is more permeable and potential mutagens are not there already held in whole or in part. Both properties lead to an increase in the explanatory power of the Ames test.

Are also conceivable experiments with biological mutagens ( viruses). Usually, this test is being applied with chemicals.

Application

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

The transferability of the results of the Ames test on humans or other organisms than the bacterial strains used is not given easily. So learn the agent to be tested, particularly in the liver of higher organisms often modifications that only give him the mutagenic properties. Such substances would exist as a false- negative Ames test. The reverse case is also possible, in which the agent is inactivated in the liver loses its mutagenic properties. To account for this, in practice, the agent is previously mixed with a liver extract ( S9- mix). The enzymes contained in the extract simulate taking place in the liver metabolism ( phase 1 reaction ).