Editing Ames test

{{Short description|Biological testing method}}
{{about|the test of chemicals for mutagenicity|the purification of metals|Ames process}}
[[File:Ames test.svg|500px|right|thumb|Ames test procedure]]
The '''Ames test''' is a widely employed method that uses [[bacteria]] to test whether a given chemical can cause [[mutation]]s in the [[DNA]] of the test organism. More formally, it is a [[bioassay|biological assay]] to assess the [[mutagenic]] potential of chemical compounds.<ref name="pmid11113466">{{cite journal | vauthors = Mortelmans K, Zeiger E | title = The Ames Salmonella/microsome mutagenicity assay | journal = Mutation Research | volume = 455 | issue = 1–2 | pages = 29–60 | date = November 2000 | pmid = 11113466 | doi = 10.1016/S0027-5107(00)00064-6 | bibcode = 2000MRFMM.455...29M }}</ref> 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.<ref name="urlAmes Test: Encyclopedia of Public Health">{{cite encyclopedia | title = Ames Test | author = Charnley G |year = 2002 | encyclopedia = Encyclopedia of Public Health | publisher = eNotes.com |url=http://www.enotes.com/public-health-encyclopedia/ames-test |archive-url= https://web.archive.org/web/20090204185058/http://www.enotes.com/public-health-encyclopedia/ames-test| archive-date= 4 February 2009 | url-status=dead |access-date=2014-05-02}}</ref>

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]].<ref name="ames" /><ref name="ames 1" /><ref name="ames 2" /><ref name="ames 3" />

== 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 [[auxotrophy|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 [[Translational frameshift|frameshift]] (e.g. strains TA-1537 and TA-1538) or [[point mutation|point]] (e.g. strain TA-1531) [[mutation]]s 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.<ref name="ames 1">{{cite journal | vauthors = Ames BN, Gurney EG, Miller JA, Bartsch H | title = Carcinogens as frameshift mutagens: metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 69 | issue = 11 | pages = 3128–32 | date = November 1972 | pmid = 4564203 | pmc = 389719 | doi = 10.1073/pnas.69.11.3128 | bibcode = 1972PNAS...69.3128A | doi-access = free }}</ref> The tester strains also carry mutations in the genes responsible for [[lipopolysaccharide]] synthesis, making the [[cell wall]] of the bacteria more permeable,<ref name="ames 2">{{cite journal | vauthors = Ames BN, Lee FD, Durston WE | title = An improved bacterial test system for the detection and classification of mutagens and carcinogens | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 70 | issue = 3 | pages = 782–6 | date = March 1973 | pmid = 4577135 | pmc = 433358 | doi = 10.1073/pnas.70.3.782 | bibcode = 1973PNAS...70..782A | doi-access = free }}</ref> and in the [[Nucleotide excision repair|excision repair system]] to make the test more sensitive.<ref name="ames 3">{{cite journal | vauthors = McCann J, Spingarn NE, Kobori J, Ames BN | title = Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 72 | issue = 3 | pages = 979–83 | date = March 1975 | pmid = 165497 | pmc = 432447 | doi = 10.1073/pnas.72.3.979 | bibcode = 1975PNAS...72..979M | doi-access = free }}</ref>

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.<ref>{{cite book | first1 = Leland | last1 = Hartwell | first2 = Michael | last2 = Goldberg | first3 = Leroy | last3 = Hood | first4 = Ann | last4 = Reynolds | first5= Lee | last5 = Silver | name-list-style = vanc | title = Genetics: from genes to genomes|date=2011|publisher=McGraw-Hill | isbn = 978-0-07-352526-6 |edition=4th|location=New York|oclc=317623365}}</ref> 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|benzo[''a'']pyrene]], are not mutagenic themselves but their metabolic products are.<ref name="ames">{{cite journal | vauthors = Ames BN, Durston WE, Yamasaki E, Lee FD | title = Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 70 | issue = 8 | pages = 2281–5 | date = August 1973 | pmid = 4151811 | pmc = 433718 | doi = 10.1073/pnas.70.8.2281 | bibcode = 1973PNAS...70.2281A | doi-access = free }}</ref>

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.

==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.<ref name="ames75">{{cite journal | vauthors = McCann J, Choi E, Yamasaki E, Ames BN | title = Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 72 | issue = 12 | pages = 5135–9 | date = December 1975 | pmid = 1061098 | pmc = 388891 | doi = 10.1073/pnas.72.12.5135 | bibcode = 1975PNAS...72.5135M | doi-access = free }}</ref>  Later studies however showed identification of 50–70% of known carcinogens.{{Citation needed|date=March 2012}}  The test was used to identify a number of compounds previously used in commercial products as potential carcinogens.<ref>{{cite journal | vauthors = Ames BN | title = Identifying environmental chemicals causing mutations and cancer | journal = Science | volume = 204 | issue = 4393 | pages = 587–93 | date = May 1979 | pmid = 373122 | doi = 10.1126/science.373122 | url = http://legacy.library.ucsf.edu/documentStore/e/p/c/epc74e00/Sepc74e00.pdf | author-link1 = Bruce Ames | jstor = 1748159 | bibcode = 1979Sci...204..587A }}</ref>  Examples include [[tris(2,3-dibromopropyl)phosphate]], which was used as a flame retardant in plastic and textiles such as children's sleepwear,<ref>{{cite journal | vauthors = Prival MJ, McCoy EC, Gutter B, Rosendranz HS | title = Tris(2,3-dibromopropyl) phosphate: mutagenicity of a widely used flame retardant | journal = Science | volume = 195 | issue = 4273 | pages = 76–8 | date = January 1977 | pmid = 318761 | doi = 10.1126/science.318761 | bibcode = 1977Sci...195...76P }}</ref> 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.<ref>
{{Citation
  | last = Hayatsu
  | first = Hiroka
  | title = Mutagens in Food: Detection and Prevention
  | publisher = [[CRC Press]]
  | year = 1991
  | pages = 286 pages
  | url = https://books.google.com/books?id=eQyMCWRIVf4C&q=carcinogen+preservative+(furylfuramide%7Caf2)&pg=RA1-PA1
|isbn = 978-0-8493-5877-7 }}</ref>  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,<ref name="ames75"/> indicating that there is no threshold concentration for mutagenesis. It therefore suggests that, as with radiation, there may be [[Linear no-threshold model|no safe threshold]] for chemical mutagens or carcinogens.<ref>{{cite book | first = Andrew | last = Teasdale | name-list-style = vanc | title = Genotoxic Impurities: Strategies for Identification and Control |publisher=Wiley-Blackwell |year=2011 |isbn=978-0-470-49919-1 }}</ref><ref name="linear">{{cite journal | vauthors = Tubiana M | title = The carcinogenic effect of exposure to low doses of carcinogens | journal = British Journal of Industrial Medicine | volume = 49 | issue = 9 | pages = 601–5 | date = September 1992 | pmid = 1390264 | pmc = 1039303 | doi = 10.1136/oem.49.9.601 }}</ref>  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.<ref>{{cite journal | vauthors = Jenkins GJ, Doak SH, Johnson GE, Quick E, Waters EM, Parry JM | title = Do dose response thresholds exist for genotoxic alkylating agents? | journal = Mutagenesis | volume = 20 | issue = 6 | pages = 389–98 | date = November 2005 | pmid = 16135536 | doi = 10.1093/mutage/gei054 | doi-access = free }}</ref>  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.<ref>{{cite journal | vauthors = Forman D | title = Ames, the Ames test, and the causes of cancer | journal = BMJ | volume = 303 | issue = 6800 | pages = 428–9 | date = August 1991 | pmid = 1912830 | pmc = 1670593 | doi = 10.1136/bmj.303.6800.428 }}</ref><ref>{{cite journal | vauthors = Ames BN, Gold LS | title = Chemical carcinogenesis: too many rodent carcinogens | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 87 | issue = 19 | pages = 7772–6 | date = October 1990 | pmid = 2217209 | pmc = 54830 | doi = 10.1073/pnas.87.19.7772 | bibcode = 1990PNAS...87.7772A | doi-access = free }}</ref>  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".<ref>{{cite journal | vauthors = Twombly R | title = Federal carcinogen report debuts new list of nominees | journal = Journal of the National Cancer Institute | volume = 93 | issue = 18 | pages = 1372 | date = September 2001 | pmid = 11562386 | doi = 10.1093/jnci/93.18.1372 | doi-access = free }}</ref>

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 [[Federal Insecticide, Fungicide, and Rodenticide Act|Pesticide Act]] (USA) and one of the six tests required under the [[Toxic Substances Control Act of 1976|Toxic Substances Control Act]] (USA).<ref>{{cite book |title=The Molecular Basis of Cancer | first1 = Peter B | last1 = Farmer | first2 = John M | last2 = Walker | name-list-style = vanc |url=https://books.google.com/books?id=t9ENAAAAQAAJ |publisher=Krieger Publishing Company |year=2006 |isbn=978-0-7099-1044-2 }}</ref>

==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.<ref>{{cite journal | vauthors = Hakura A, Suzuki S, Satoh T | title = Advantage of the use of human liver S9 in the Ames test | journal = Mutation Research | volume = 438 | issue = 1 | pages = 29–36 | date = January 1999 | pmid = 9858674 | doi = 10.1016/s1383-5718(98)00159-4 | bibcode = 1999MRGTE.438...29H }}</ref>  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.<ref>{{cite book | title=Optimization in Drug Discovery: in vitro methods | series=Methods in Pharmacology and toxicology | editor1-first=Zhengyin | editor1-last=Yan | editor2-first=Gary | editor2-last=Caldwell | name-list-style = vanc | publisher=Humana Press | year=2004 | isbn=978-1-58829-332-9 | first1 = Atsushi | last1 = Hakura | first2 = Satoshi | last2 = Suzuki | first3 = Tetsuo | last3 = Satoh | chapter=Improvement of the Ames test using human liver S9 preparation }}</ref>  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==
[[File:Wiki muta2.png|300px|right|thumb|Fluctuation method: [[96-well plate]]]]
[[File:Ames MPF 384 plate.jpg|thumb|300px|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 [[microplate]]s 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.<ref>{{cite journal | vauthors = Bridges BA | title = The fluctuation test | journal = Archives of Toxicology | volume = 46 | issue = 1–2 | pages = 41–4 | date = November 1980 | pmid = 7235997 | doi = 10.1007/BF00361244 | bibcode = 1980ArTox..46...41B | s2cid = 23769437 }}</ref>

== References ==
{{reflist|32em}}

== Further reading ==
{{refbegin|32em}}
*{{Cite journal|date=1989-03-01|title=Metabolic action of polycyclic aromatic hydrocarbons to mutagens in the Ames test by various animal species including man|journal=Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis|language=en|volume=211|issue=1|pages=147–151|doi=10.1016/0027-5107(89)90115-2|issn=0027-5107|last1=Phillipson|first1=Caroline E.|last2=Ioannides|first2=Costas|pmid=2493576|bibcode=1989MRFMM.211..147P }}
*{{Cite book|url=https://books.google.com/books?id=Zm3sCgAAQBAJ&q=the+ames+test+gelboin&pg=PA72|title=The Understanding, Prevention and Control of Human Cancer: The Historic Work and Lives of Elizabeth Cavert Miller and James A. Miller|last=McKinnell|first=Robert Gilmore | name-list-style = vanc |date=2015-11-06|publisher=BRILL|isbn=9789004286801|language=en}}
* {{cite journal | vauthors = Claxton LD, Umbuzeiro GD, DeMarini DM | title = The Salmonella mutagenicity assay: the stethoscope of genetic toxicology for the 21st century | journal = Environmental Health Perspectives | volume = 118 | issue = 11 | pages = 1515–22 | date = November 2010 | pmid = 20682480 | pmc = 2974687 | doi = 10.1289/ehp.1002336 | bibcode = 2010EnvHP.118.1515C }}
{{refend}}

{{Genotoxicity}}

[[Category:Applied genetics]]
[[Category:Biochemistry detection reactions]]
[[Category:Laboratory techniques]]
[[Category:Toxicology tests]]