The Ames test is a biological assay to assess the mutagenic potential of chemical compounds.[1] A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen, since cancer is often linked to mutation. However, a number of false-positives and false-negatives are known.[2] The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound since standard carcinogen assays on rodents is time-consuming (taking around three years to complete) as well as expensive. The procedure is described in a series of papers from the early 1970s by Bruce Ames and his group at the University of California, Berkeley.
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The test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis i.e. it is an auxotrophic mutant, so that they require histidine for growth. The method tests the capability of mutagen in creating mutations that can result in a reversion back to a non-auxotrophic state so that the cells can grow on a histidine-free medium. The tester strains are specially constructed to detect either frameshift (e.g. strains TA-1537 and TA-1538) or point (e.g. strain TA-1531) mutations in the genes required to synthesize histidine, so that mutagens acting via different mechanisms may be identified. Some compounds are quite specific, causing reversions in just one or two strains.[3] The tester strains also carry mutations in the genes responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable,[4] and in the excision repair system to make the test more sensitive.[5] Rat liver extract is optionally added to simulate the effect of metabolism, as some compounds, like benzo[a]pyrene, are not mutagenic themselves but their metabolic products are.[6]
The bacteria are spread on an agar plate with a small amount of histidine. This small amount of histidine in the growth medium allows the bacteria to grow for an initial time and have the opportunity to mutate. When the histidine is depleted only bacteria that have mutated to gain the ability to produce its own histidine will survive. The plate is incubated for 48 hours. The mutagenicity of a substance is proportional to the number of colonies observed.
Early studies by Ames showed that 90% of known carcinogens may be identified via this test.[7] Later studies however showed identification of 50–70% of known carcinogens. The test was used to identify a number of compounds previously used in commercial products as potential carcinogens.[8] Examples include tris(2,3-dibromopropyl)phosphate, which was used as a flame retardant in plastic and textiles as such children's sleepware,[9] and furylfuramide which was used as an antibacterial additive in food in Japan in 1960s and 1970s. Furylfuramide in fact had previously passed animal test, but more vigorous tests after its identification in Ames test showed it to be carcinogenic.[10] Their positive tests resulted in those chemicals being withdrawn from use in consumer products.
One interesting result from Ames test is that the dose response curve in Ames test using varying concentrations of chemical is almost always linear, indicating that there is no threshold concentration for mutagenesis, therefore suggesting that there may be no safe threshold for mutagens or carcinogens. However some proposed that low level of some mutagens may stimulate the DNA repair processes which can mitigate the effect of the mutagens and thus may not be harmful. Bruce Ames himself cautioned against the "hysteria over tiny traces of chemicals that may or may not cause cancer", that "completely drives out the major risks you should be aware of."[11]
Ames test is often used as one of the initial screens for potential drugs to weed out possible carcinogens, and it is one of the eight tests required under Pesticide Act (USA) and one of six tests required under Toxic Substances Control Act (USA).[12]
Salmonella typhimurium is a prokaryote, therefore it is not a perfect model for humans. Rat liver S9 fraction is used to mimic the mammalian metabolic conditions so that the mutagenic potential of metabolites formed by a parent molecule in the hepatic system can be assessed, however there are differences in metabolism and mutagenicity of chemicals between human and rat.[13] The test may therefore be improved by the use of human liver S9 fraction; its use was previously limited by its availability, but it is now available commercially and therefore may be more feasible.[14] An adapted in vitro model has been made for eukaryotic cells, for example yeast.
Mutagens identified in Ames test need not necessarily be carcinogenic, and further tests are required for any potential carcinogen identified in the test. Drugs that contain the nitrate moiety sometimes come back positive for Ames when they are indeed safe. The nitrate compounds may generate nitric oxide, an important signal molecule that can give a false positive. Nitroglycerin is an example that gives a positive Ames yet is still used in treatment today. Nitrates in food however may be reduced by bacterial action to nitrites which are known to generate carcinogens by reacting with amines and amides. Long toxicology and outcome studies are needed with such compounds to disprove a positive Ames test.