Structural Biochemistry/Nucleic Acid/Biology of Cancer/Mutagenic Action on Bacteria/Ames test
The Ames test was named after its developer, Bruce Ames, and is a method for determining if a chemical is a mutagen, an agent that causes mutations. This test is used by many cosmetics companies, pharmaceutical companies and other industries to prove that their products will not cause cancer or harmful in that way in humans. The use of this test is based on the assumption that any substance that is mutagenic may also turn out to be a carcinogen, to cause cancer. A positive test shows that the chemical can act as a carcinogen. If the substance is a carcinogen, it will cause mutation in the bacterium as the cell divide. The mutant cells will have the enzyme to form colonies.
In the Ames test, a thin layer of agar containing about 109 bacterial of a specially constructed tester strain of Salmonella is placed on a petri plate. T hese bacteria are unable to grow in the absence of histidine, because a mutation is present in one of the genes for the biosynthesis of this amino acid. The addition of a chemical mutagen to the center of the plate results in many new mutations.
The bacterium used in the test is a strain of Salmonella typhimurium, which carries a defective gene making it unable to synthesize the amino acid histidine from the ingredients in its culture medium. Yet, some of mutations can be reversed with the gene regaining its function. These are able to grow on a medium lacking histine. Since cancer is often linked to DNA damage, the test can also quickly estimate the carcinogenic potentical of a compound as it is hard to determine whether standard carcinogen examination on animals were successful.
A small proportion of them reverse the original mutation, and histidine can be synthesized. These revertants multiply in the absence of an external source of histidine and appear as discrete colonies after the plate has been incubated at 37 Celsius degrees for 2 days. A series of concentrations of a chemical can be readily tested to generate a dose-response curve. These curves are usually linear, which suggests that there is no threshold concentration for mutagenesis.
Some of the tester strains are responsive to base-pair substitutions, whereas others detect deletions or additions of base pairs. The sensitivity of these specially designed strains has been enhanced by the genetic deletion of their excision-repair systems. Potential mutagens enter the tester strains easily because the lipopolysaccaride barrier that normally coats the surface of Salmonella is incomplete in these strains.
A key feature of this detection system is the inclusion of a mammalian liver homogenate. Since some potential carcinogens such as aflatoxin are converted into their active forms by enzyme systems in the liver or other mammalian tissues, bacteria lack these enzymes. So the test plate requires a few milligrams of a liver homogenate to activate this group of mutagens.
This test is extensively used to hep evaluate the mutagenic and carcinogenic risks of a large number of chemicals. This rapid and inexpensive bacterial assay for mutagenicity complements epidemiological surveys and animal tests that are necessarily slower, more laborious, and far more expensive. The Salmonella atest for mutagenicity is an outgrowth of studies of gene-protein relations in bacteria.
The picture shows the Ames test. A suspension of a his- strain of Salmonella typhimurium has been plated with a mixture of rat liver enzymes on agar lacking histidine. The disk of filter paper has impregnated with a carcinogen. The mutagenic effect of the chemical has caused many bacteria to regain to grow without histidine to form the colonies around the disk. The scattered colonies on the disk represents spontaneous revertants.