Ames test
Encyclopedia
The Ames test 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
, since cancer
is often linked to mutation
. However, a number of false-positives and false-negatives are known. 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
.
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 an 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) 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. 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. 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.
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.
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. Their positive tests resulted in those chemicals being withdrawn from use in consumer products.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."
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).
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. 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. 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.
Bioassay
Bioassay , or biological standardization is a type of scientific experiment. Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants...
to assess the mutagenic potential of chemical compounds. A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen
Carcinogen
A carcinogen is any substance, radionuclide, or radiation that is an agent directly involved in causing cancer. This may be due to the ability to damage the genome or to the disruption of cellular metabolic processes...
, since cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
is often linked to mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
. However, a number of false-positives and false-negatives are known. 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
Bruce Ames
Bruce Nathan Ames is an American biochemist. He is a professor of Biochemistry and Molecular Biology at the University of California, Berkeley, and a senior scientist at Children's Hospital Oakland Research Institute...
and his group at the University of California, Berkeley
University of California, Berkeley
The University of California, Berkeley , is a teaching and research university established in 1868 and located in Berkeley, California, USA...
.
General Procedure
The test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidineHistidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
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 an non-auxotrophic state so that the cells can grow on a histidine-free medium. The tester strains are specially constructed to detect either frameshift
Translational frameshift
Translational frameshifting or ribosomal frameshifting refers to an alternate process of protein translation. A protein is translated from one end of the mRNA to the other, from the 5' to the 3' end. Normally a protein is translated from a template mRNA with consecutive blocks of 3 nucleotides...
(e.g. strains TA-1537 and TA-1538) or point
Point mutation
A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair...
(e.g. strain TA-1531) mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
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. The tester strains also carry mutations in the genes responsible for lipopolysaccharide
Lipopolysaccharide
Lipopolysaccharides , also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals.-Functions:LPS is the major...
synthesis, making the cell wall
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...
of the bacteria more permeable, and in the excision repair system to make the test more sensitive. Rat liver extract is optionally added to simulate the effect of metabolism
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
, as some compounds, like benzo[a]pyrene, are not mutagenic themselves but their metabolic products are.
The bacteria are spread on an agar
Agar
Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...
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.
Identification of carcinogens
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 as such children's sleepware, and furylfuramideFurylfuramide
Furylfuramide is a synthetic nitrofuran derivative which was widely used as a food preservative in Japan since at least 1965, but withdrawn from the market in 1974 when it was observed to be mutagenic to bacteria in vitro and thus suspected of carcinogenicity...
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. 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."
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
Toxic Substances Control Act of 1976
The Toxic Substances Control Act is a United States law, passed by the United States Congress in 1976, that regulates the introduction of new or already existing chemicals. It grandfathered most existing chemicals, in contrast to the Registration, Evaluation and Authorization of Chemicals ...
(USA).
Limitations
Salmonella typhimurium is a prokaryote, therefore it is not a perfect model for humans. Rat liver S9 fractionS9 fraction
The S9 fraction is the product of a 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...
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. 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. 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
Nitric oxide
Nitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
, 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.