Ames test

The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds. A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen, because cancer is often linked to mutation. The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound because standard carcinogen assays on mice and rats are time-consuming (taking two to three years to complete) and expensive. However, false-positives and false-negatives are known. The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds. A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen, because cancer is often linked to mutation. The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound because standard carcinogen assays on mice and rats are time-consuming (taking two to three years to complete) and expensive. However, false-positives and false-negatives are known. The procedure was described in a series of papers in the early 1970s by Bruce Ames and his group at the University of California, Berkeley. The Ames test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis. These strains are auxotrophic mutants, i.e. they require histidine for growth, but cannot produce it. The method tests the capability of the tested substance in creating mutations that result in a return to a 'prototrophic' 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. The tester strains also carry mutations in the genes responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable, and in the excision repair system to make the test more sensitive. Larger organisms like mammals have metabolic processes that could potentially turn a chemical considered not mutagenic into one that is or one that is considered mutagenic into one that is not. Therefore, to more effectively test a chemical compound's mutagenicity in relation to larger organisms, rat liver enzymes can be added in an attempt to replicate the metabolic processes' effect on the compound being tested in the Ames Test. Rat liver extract is optionally added to simulate the effect of metabolism, as some compounds, like benzopyrene, are not mutagenic themselves but their metabolic products are. The bacteria are spread on an agar plate with 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. Mutagens identified via Ames test are also possible carcinogens, and early studies by Ames showed that 90% of known carcinogens may be identified via this test. 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. Examples include tris(2,3-dibromopropyl)phosphate, which was used as a flame retardant in plastic and textiles such as children's sleepwear, 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 the Ames test showed it to be carcinogenic. Their positive tests resulted in those chemicals being withdrawn from use in consumer products. One interesting result from the Ames test is that the dose response curve using varying concentrations of chemical is almost always linear, indicating that there is no threshold concentration for mutagenesis. It therefore suggests that, as with radiations, there may be no safe threshold for chemical mutagens or carcinogens. However some proposed that organisms can tolerate low level of mutagens due to protective mechanisms such as DNA repair, and threshold may exist for certain chemical mutagens. Bruce Ames himself argued against linear dose-response extrapolation from the high dose used in carcinogenesis tests in animal systems to the lower dose of chemicals normally encountered in human exposure, as the results may be false positives due to mitogenic response caused by the artificially high dose of chemicals used in such tests. He also 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'. The 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 the Pesticide Act (USA) and one of six tests required under the Toxic Substances Control Act (USA).