Ames test
The Ames test is a widely employed method that uses
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.[3][4][5][6]
General procedure
The Ames test uses several strains of the bacterium
The tester strains are specially constructed to detect either
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.[7] 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 benzo[a]pyrene, are not mutagenic themselves but their metabolic products are.[3]
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.
Ames test and carcinogens
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.
One interesting result from the Ames test is that the dose response curve using varying concentrations of the chemical is almost always linear,
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 the six tests required under the Toxic Substances Control Act (USA).[18]
Limitations
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 between humans and rats that can affect the mutagenicity of the chemicals being tested.[19] 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.[20] An adapted in vitro model has been made for eukaryotic cells, for example yeast.
Mutagens identified in the 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.
Fluctuation method
The Ames test was initially developed using agar plates (the plate incorporation technique), as described above. Since that time, an alternative to performing the Ames test has been developed, which is known as the "fluctuation method". This technique is the same in concept as the agar-based method, with bacteria being added to a reaction mixture with a small amount of histidine, which allows the bacteria to grow and mutate, returning to synthesize their own histidine. By including a pH indicator, the frequency of mutation is counted in microplates as the number of wells which have changed color (caused by a drop in pH due to metabolic processes of reproducing bacteria). As with the traditional Ames test, the sample is compared to the natural background rate of reverse mutation in order to establish the genotoxicity of a substance. The fluctuation method is performed entirely in liquid culture and is scored by counting the number of wells that turn yellow from purple in 96-well or 384-well microplates.
In the 96-well plate method the frequency of mutation is counted as the number of wells out of 96 which have changed color. The plates are incubated for up to five days, with mutated (yellow) colonies being counted each day and compared to the background rate of reverse mutation using established tables of significance to determine the significant differences between the background rate of mutation and that for the tested samples.
In the more scaled-down 384-well plate microfluctuation method the frequency of mutation is counted as the number of wells out of 48 which have changed color after 2 days of incubation. A test sample is assayed across 6 dose levels with concurrent zero-dose (background) and positive controls which all fit into one 384-well plate. The assay is performed in triplicates to provide statistical robustness. It uses the recommended OECD Guideline 471 tester strains (histidine auxotrophs and tryptophan auxotrophs).
The fluctuation method is comparable to the traditional pour plate method in terms of sensitivity and accuracy, however, it does have a number of advantages: it needs less test sample, it has a simple colorimetric endpoint, counting the number of positive wells out of possible 96 or 48 wells is much less time-consuming than counting individual colonies on an agar plate. Several commercial kits are available. Most kits have consumable components in a ready-to-use state, including lyophilized bacteria, and tests can be performed using multichannel pipettes. The fluctuation method also allows for testing higher volumes of aqueous samples (up to 75% v/v), increasing the sensitivity and extending its application to low-level environmental mutagens.[21]
References
- PMID 11113466.
- ^ Charnley G (2002). "Ames Test". Encyclopedia of Public Health. eNotes.com. Archived from the original on 4 February 2009. Retrieved 2014-05-02.
- ^ PMID 4151811.
- ^ PMID 4564203.
- ^ PMID 4577135.
- ^ PMID 165497.
- OCLC 317623365.
- ^ PMID 1061098.
- PMID 373122.
- PMID 318761.
- ^
Hayatsu, Hiroka (1991), Mutagens in Food: Detection and Prevention, ISBN 978-0-8493-5877-7
- ISBN 978-0-470-49919-1.
- PMID 1390264.
- PMID 16135536.
- PMID 1912830.
- PMID 2217209.
- PMID 11562386.
- ISBN 978-0-7099-1044-2.
- PMID 9858674.
- ISBN 978-1-58829-332-9.
- S2CID 23769437.
Further reading
- Phillipson, Caroline E.; Ioannides, Costas (1989-03-01). "Metabolic action of polycyclic aromatic hydrocarbons to mutagens in the Ames test by various animal species including man". Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 211 (1): 147–151. PMID 2493576.
- McKinnell RG (2015-11-06). The Understanding, Prevention and Control of Human Cancer: The Historic Work and Lives of Elizabeth Cavert Miller and James A. Miller. BRILL. ISBN 9789004286801.
- Claxton LD, Umbuzeiro GD, DeMarini DM (November 2010). "The Salmonella mutagenicity assay: the stethoscope of genetic toxicology for the 21st century". Environmental Health Perspectives. 118 (11): 1515–22. PMID 20682480.