Ames test

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Ames test procedure Ames test.svg
Ames test procedure

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. [1] 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. [2]

Contents

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 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. [4] The tester strains also carry mutations in the genes responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable, [5] and in the excision repair system to make the test more sensitive. [6]

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. [8] Later studies however showed identification of 50–70% of known carcinogens.[ citation needed ] The test was used to identify a number of compounds previously used in commercial products as potential carcinogens. [9] Examples include tris(2,3-dibromopropyl)phosphate, which was used as a flame retardant in plastic and textiles such as children's sleepwear, [10] and furylfuramide which was used as an antibacterial additive in food in Japan in the 1960s and 1970s. Furylfuramide in fact had previously passed animal tests, but more vigorous tests after its identification in the Ames test showed it to be carcinogenic. [11] 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 the chemical is almost always linear, [8] indicating that there is no threshold concentration for mutagenesis. It therefore suggests that, as with radiation, there may be no safe threshold for chemical mutagens or carcinogens. [12] [13] However, some have proposed that organisms could tolerate low levels of mutagens due to protective mechanisms such as DNA repair, and thus a threshold may exist for certain chemical mutagens. [14] 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. [15] [16] 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". [17]

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

Fluctuation method: 96-well plate Wiki muta2.png
Fluctuation method: 96-well plate
Fluctuation method: 384-well plate Ames MPF 384 plate.jpg
Fluctuation method: 384-well plate

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]

Related Research Articles

Mutagenesis is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures. A mutagen is a mutation-causing agent, be it chemical or physical, which results in an increased rate of mutations in an organism's genetic code. In nature mutagenesis can lead to cancer and various heritable diseases, and it is also a driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller, Charlotte Auerbach and J. M. Robson in the first half of the 20th century.

<span class="mw-page-title-main">Mutagen</span> Physical or chemical agent that increases the rate of genetic mutation

In genetics, a mutagen is a physical or chemical agent that permanently changes genetic material, usually DNA, in an organism and thus increases the frequency of mutations above the natural background level. As many mutations can cause cancer in animals, such mutagens can therefore be carcinogens, although not all necessarily are. All mutagens have characteristic mutational signatures with some chemicals becoming mutagenic through cellular processes.

<span class="mw-page-title-main">Ethidium bromide</span> DNA gel stain and veterinary drug

Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as EtBr, which is also an abbreviation for bromoethane. To avoid confusion, some laboratories have used the abbreviation EthBr for this salt. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle. The high incidence of antimicrobial resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Despite its reputation as a mutagen, tests have shown it to have low mutagenicity without metabolic activation.

Genotoxicity is the property of chemical agents that damage the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, but some genotoxic substances are not mutagenic. The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. Cells prevent expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.

<span class="mw-page-title-main">Phenylbutazone</span> Nonsteroidal anti-inflammatory drug (NSAID)

Phenylbutazone, often referred to as "bute", is a nonsteroidal anti-inflammatory drug (NSAID) for the short-term treatment of pain and fever in animals.

<span class="mw-page-title-main">Auxotrophy</span> Inability to synthesize an organic compound required for growth

Auxotrophy is the inability of an organism to synthesize a particular organic compound required for its growth. An auxotroph is an organism that displays this characteristic; auxotrophic is the corresponding adjective. Auxotrophy is the opposite of prototrophy, which is characterized by the ability to synthesize all the compounds needed for growth.

<span class="mw-page-title-main">Bruce Ames</span> American biochemist (born 1928)

Bruce Nathan Ames is an American biochemist. He is a professor of Biochemistry and Molecular Biology Emeritus at the University of California, Berkeley, and was a senior scientist at Children's Hospital Oakland Research Institute (CHORI). He is the inventor of the Ames test, a system for easily and cheaply testing the mutagenicity of compounds.

<span class="mw-page-title-main">Methylcholanthrene</span> Chemical compound

Methylcholanthrene is a highly carcinogenic polycyclic aromatic hydrocarbon produced by burning organic compounds at very high temperatures. Methylcholanthrene is also known as 3-methylcholanthrene, 20-methylcholanthrene or the IUPAC name 3-methyl-1,2-dyhydrobenzo[j]aceanthrylene. The short notation often used is 3-MC or MCA. This compound forms pale yellow solid crystals when crystallized from benzene and ether. It has a melting point around 180 °C and its boiling point is around 280 °C at a pressure of 80 mmHg. Methylcholanthrene is used in laboratory studies of chemical carcinogenesis. It is an alkylated derivative of benz[a]anthracene and has a similar UV spectrum. The most common isomer is 3-methylcholanthrene, although the methyl group can occur in other places.

<span class="mw-page-title-main">Sudan I</span> Chemical compound

Sudan I is an organic compound, typically classified as an azo dye. It is an intensely orange-red solid that is added to colourise waxes, oils, petrol, solvents, and polishes. Sudan I has also been adopted for colouring various foodstuffs, especially curry powder and chili powder, although the use of Sudan I in foods is now banned in many countries, because Sudan I, Sudan III, and Sudan IV have been classified as category 3 carcinogens by the International Agency for Research on Cancer. Sudan I is still used in some orange-coloured smoke formulations and as a colouring for cotton refuse used in chemistry experiments.

<span class="mw-page-title-main">Methyl carbamate</span> Chemical compound

Methyl carbamate (also called methylurethane, or urethylane) is an organic compound and the simplest ester of carbamic acid (H2NCO2H). It is a colourless solid.

<span class="mw-page-title-main">3,3',5,5'-Tetramethylbenzidine</span> Chemical compound

3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays (ELISA). TMB is a white solid that forms a pale blue-green liquid in solution with ethyl acetate. TMB is degraded by sunlight and by fluorescent lights.

<span class="mw-page-title-main">SYBR Green I</span> Dye used for molecular genetics

SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DNA-dye-complex best absorbs 497 nanometer blue light and emits green light. The stain preferentially binds to double-stranded DNA, but will stain single-stranded (ss) DNA with lower performance. SYBR Green can also stain RNA with a lower performance than ssDNA.

The S9 fraction is the product of an organ tissue homogenate used in biological assays. The S9 fraction is most frequently used in assays that measure the metabolism of drugs and other xenobiotics. It is defined by the U.S. National Library of Medicine's "IUPAC Glossary of Terms Used in Toxicology" as the "Supernatant fraction obtained from an organ homogenate by centrifuging at 9000 g for 20 minutes in a suitable medium; this fraction contains cytosol and microsomes." The microsomes component of the S9 fraction contain cytochrome P450 isoforms and other enzyme activities. The cytosolic portion contains the major part of the activities of transferases. The S9 fraction is easier to prepare than purified microsomes.

The Environmental Mutagenesis and Genomics Society (EMGS) is a scientific society "for the promotion of critical scientific knowledge and research into the causes and consequences of damage to the genome and epigenome in order to inform and support national and international efforts to ensure a healthy, sustainable environment for future generations."

<span class="mw-page-title-main">SOS chromotest</span>

The SOS chromotest is a biological assay to assess the genotoxic potential of chemical compounds. The test is a colorimetric assay which measures the expression of genes induced by genotoxic agents in Escherichia coli, by means of a fusion with the structural gene for β-galactosidase. The test is performed over a few hours in columns of a 96-well microplate with increasing concentrations of test samples. This test was developed as a practical complement or alternative to the traditional Ames test assay for genotoxicity, which involves growing bacteria on agar plates and comparing natural mutation rates to mutation rates of bacteria exposed to potentially mutagenic compounds or samples. The SOS chromotest is comparable in accuracy and sensitivity to established methods such as the Ames test and is a useful tool to screen genotoxic compounds, which could prove carcinogenic in humans, in order to single out chemicals for further in-depth analysis.

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<span class="mw-page-title-main">2-Aminofluorene</span> Chemical compound

2-Aminofluorene (2-AF) is a synthetic arylamine. It is a white to tan solid with a melting point of 125-132 °C. 2-AF has only been tested in controlled laboratory settings thus far. There is no indication that it will be tested in industrialized settings. There is evidence that 2-aminofluorene is a carcinogen and an intercalating agent that is extremely dangerous to genomic DNA that potentially can lead to mutation if not death. Furthermore, it has been suggested that 2-aminofluorene can undergo acetylation reactions that causes these reactive species to undergo such reactions in cells. Several experiments have been conducted that have suggested 2-aminofluorene be treated with care and with an overall awareness of the toxicity of this compound.

Benzo(<i>c</i>)fluorene Chemical compound

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<span class="mw-page-title-main">Glycidamide</span> Chemical compound

Glycidamide is an organic compound with the formula H2NC(O)C2H3O. It is a colorless, oil. Structurally, it contains adjacent amides and epoxide functional groups. It is a bioactive, potentially toxic or even carcinogenic metabolite of acrylonitrile and acrylamide. It is a chiral molecule.

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Francesco De Lorenzo is a physician and politician, member of the Italian Liberal Party. He was born in Naples. He was minister of health (1989–1993) in the Government of Italy. He served in the cabinet of Prime Minister Bettino Craxi (1986–1987), Giulio Andreotti (1989–1992) and Giuliano Amato (1992–1993). He served in the Chamber of Deputies of Italy in Legislature IX (1983–1987), Legislature X (1987–1992) and Legislature XI (1992–1994).

References

  1. ↑ Mortelmans K, Zeiger E (November 2000). "The Ames Salmonella/microsome mutagenicity assay". Mutation Research. 455 (1–2): 29–60. doi:10.1016/S0027-5107(00)00064-6. PMID   11113466.
  2. ↑ Charnley G (2002). "Ames Test". Encyclopedia of Public Health. eNotes.com. Archived from the original on 4 February 2009. Retrieved 2014-05-02.
  3. 1 2 Ames BN, Durston WE, Yamasaki E, Lee FD (August 1973). "Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection". Proceedings of the National Academy of Sciences of the United States of America. 70 (8): 2281–5. Bibcode:1973PNAS...70.2281A. doi: 10.1073/pnas.70.8.2281 . PMC   433718 . PMID   4151811.
  4. 1 2 Ames BN, Gurney EG, Miller JA, Bartsch H (November 1972). "Carcinogens as frameshift mutagens: metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens". Proceedings of the National Academy of Sciences of the United States of America. 69 (11): 3128–32. Bibcode:1972PNAS...69.3128A. doi: 10.1073/pnas.69.11.3128 . PMC   389719 . PMID   4564203.
  5. 1 2 Ames BN, Lee FD, Durston WE (March 1973). "An improved bacterial test system for the detection and classification of mutagens and carcinogens". Proceedings of the National Academy of Sciences of the United States of America. 70 (3): 782–6. Bibcode:1973PNAS...70..782A. doi: 10.1073/pnas.70.3.782 . PMC   433358 . PMID   4577135.
  6. 1 2 McCann J, Spingarn NE, Kobori J, Ames BN (March 1975). "Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids". Proceedings of the National Academy of Sciences of the United States of America. 72 (3): 979–83. Bibcode:1975PNAS...72..979M. doi: 10.1073/pnas.72.3.979 . PMC   432447 . PMID   165497.
  7. ↑ Hartwell L, Goldberg M, Hood L, Reynolds A, Silver L (2011). Genetics: from genes to genomes (4th ed.). New York: McGraw-Hill. ISBN   978-0-07-352526-6. OCLC   317623365.
  8. 1 2 McCann J, Choi E, Yamasaki E, Ames BN (December 1975). "Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals". Proceedings of the National Academy of Sciences of the United States of America. 72 (12): 5135–9. Bibcode:1975PNAS...72.5135M. doi: 10.1073/pnas.72.12.5135 . PMC   388891 . PMID   1061098.
  9. ↑ Ames BN (May 1979). "Identifying environmental chemicals causing mutations and cancer" (PDF). Science. 204 (4393): 587–93. Bibcode:1979Sci...204..587A. doi:10.1126/science.373122. JSTOR   1748159. PMID   373122.
  10. ↑ Prival MJ, McCoy EC, Gutter B, Rosendranz HS (January 1977). "Tris(2,3-dibromopropyl) phosphate: mutagenicity of a widely used flame retardant". Science. 195 (4273): 76–8. Bibcode:1977Sci...195...76P. doi:10.1126/science.318761. PMID   318761.
  11. ↑ Hayatsu, Hiroka (1991), Mutagens in Food: Detection and Prevention, CRC Press, pp. 286 pages, ISBN   978-0-8493-5877-7
  12. ↑ Teasdale A (2011). Genotoxic Impurities: Strategies for Identification and Control. Wiley-Blackwell. ISBN   978-0-470-49919-1.
  13. ↑ Tubiana M (September 1992). "The carcinogenic effect of exposure to low doses of carcinogens". British Journal of Industrial Medicine. 49 (9): 601–5. doi:10.1136/oem.49.9.601. PMC   1039303 . PMID   1390264.
  14. ↑ Jenkins GJ, Doak SH, Johnson GE, Quick E, Waters EM, Parry JM (November 2005). "Do dose response thresholds exist for genotoxic alkylating agents?". Mutagenesis. 20 (6): 389–98. doi: 10.1093/mutage/gei054 . PMID   16135536.
  15. ↑ Forman D (August 1991). "Ames, the Ames test, and the causes of cancer". BMJ. 303 (6800): 428–9. doi:10.1136/bmj.303.6800.428. PMC   1670593 . PMID   1912830.
  16. ↑ Ames BN, Gold LS (October 1990). "Chemical carcinogenesis: too many rodent carcinogens". Proceedings of the National Academy of Sciences of the United States of America. 87 (19): 7772–6. Bibcode:1990PNAS...87.7772A. doi: 10.1073/pnas.87.19.7772 . PMC   54830 . PMID   2217209.
  17. ↑ Twombly R (September 2001). "Federal carcinogen report debuts new list of nominees". Journal of the National Cancer Institute. 93 (18): 1372. doi:10.1093/jnci/93.18.1372. PMID   11562386.
  18. ↑ Farmer PB, Walker JM (2006). The Molecular Basis of Cancer. Krieger Publishing Company. ISBN   978-0-7099-1044-2.
  19. ↑ Hakura A, Suzuki S, Satoh T (January 1999). "Advantage of the use of human liver S9 in the Ames test". Mutation Research. 438 (1): 29–36. doi:10.1016/s1383-5718(98)00159-4. PMID   9858674.
  20. ↑ Hakura A, Suzuki S, Satoh T (2004). "Improvement of the Ames test using human liver S9 preparation". In Yan Z, Caldwell G (eds.). Optimization in Drug Discovery: in vitro methods. Methods in Pharmacology and toxicology. Humana Press. ISBN   978-1-58829-332-9.
  21. ↑ Bridges BA (November 1980). "The fluctuation test". Archives of Toxicology. 46 (1–2): 41–4. doi:10.1007/BF00361244. PMID   7235997. S2CID   23769437.

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