Ames test

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A test used to determine the mutagenic potential of a substance based on the mutation rate of bacteria that are exposed to the substance.

[After Bruce Ames (born 1928), American biochemist.]

An in vitro test for the ability of chemicals, including potential food additives, to cause mutation in bacteria (the mutagenic potential). Commonly used as a preliminary screening method to detect substances likely to be carcinogenic.

The Ames test is a screening test that is used to help identify chemicals that affect the structure of DNA. The test exposes Salmonella bacteria to chemicals and looks for changes in the way bacteria grow. These changes result from mutations that occur when the structure of DNA is altered in certain places. Many chemicals that cause mutations can cause cancer in animals or in people. When the test was developed, it was thought that most of the chemicals that produce results in the Ames test could also cause cancer. It was hoped that this simple test would be an easy way to find cancer-causing chemicals. Over time, the test was found to be a less reliable predictor of carcino-genesis than had been hoped. Some chemicals that are known to cause cancer do not test positive in the Ames test and some chemicals that test positive do not cause cancer. Nonetheless, the test is still considered an important part of assessing the safety of new chemicals.

The Ames test uses strains of Salmonella that have been altered to make them more susceptible to mutation than normal Salmonella. To perform the test, the altered Salmonella strains are combined in a test tube with the chemical of interest. Because Salmonella bacteria lack the enzymes that animals use to metabolize chemicals, animal liver enzymes are often added to the test tube. That way, the test is able to detect what might happen if the chemical entered a human body. The Salmonella are then transferred to a petri dish to grow for one or two days. The altered Salmonella used for the test require the amino acid histidine to grow, and a positive result in the test is indicated when, in response to mutation, the Salmonella no longer require histidine to grow.

A positive result in an Ames test does not indicate by itself that a particular chemical is capable of causing cancer. It does suggest that a chemical can produce mutations and that more extensive testing is needed to determine whether the chemical is likely to produce cancer in humans. The test is useful as a screening tool for setting priorities because it is an inexpensive and quick way to help single out chemicals that should be targeted for further testing. It is also used in industry as a primary preventive approach to eliminate potential carcinogens early in the process of developing new commercial chemicals.

The test is named for its creator, Dr. Bruce Ames. Its development depended upon basic scientific advances in understanding the role of mutagenesis in chemical carcinogenesis, and its use was fundamental in the understanding of the mechanisms of carcinogenesis.

(SEE ALSO: Cancer; Carcinogen; Carcinogenesis; Toxicology)

— GAIL CHARNLEY

The Ames test is a protocol for identifying mutagenic chemical and physical agents. Mutagens generate changes in DNA. Many mutagenic agents modify the chemical structure of adenine, thymine, guanine, and cytosine, the bases in DNA, changing their base-pairing properties and causing mutations to accumulate during DNA synthesis.

Ethyl methanesulfonate (EMS), for example, is a very potent mutagen. The ethyl group of EMS reacts with guanine in DNA, forming the abnormal base O⁶-ethylguanine. During DNA replication, DNA polymerases that catalyze the process frequently place thymine, instead of cytosine, opposite O⁶-ethylguanine. Following subsequent rounds of replication, the original G:C base pair can become an A:T pair. This changes the genetic information, is often harmful to cells, and can result in disease. Many mutagens cause a wide variety of cancers in humans.

During the 1960s the biologist Bruce Ames developed a test that still carries his name and that is still used as a relatively inexpensive way to assess the mutagenic potential of many chemical compounds. The procedure uses the bacteria Salmonella typhimurium. Wild-type S. typhimurium grows well on agar that contains only minimal nutrients. It can thrive on agar that contains only sugar, ammonium salts, phosphate, sulfate, and some trace metal ions. Amino acids are not needed because the bacteria have genes that encode enzymes that can make all twenty amino acids.

Ames developed strains of S. typhimurium that contain mutations in genes that the bacteria use to make the amino acid histidine. Such his^- strains cannot survive unless histidine is added to their agar. Ames reasoned that mutagenic agents could cause changes in the aberrant gene that encodes the defective his^- enzyme, causing it to revert back to the normal form, encoding the active protein. (The mutagen would likely also cause many other, undetected mutations.) A mutation that returns a function to a mutant is called a reverse mutation. The Ames test measures the ability of his^-S. typhimurium to grow on agar that does not contain histidine. Growth indicates that a reverse mutation has reverted the his^- gene back to an active form.

A typical Ames test involves exposing his^-S. typhimurium to a test agent and then placing the exposed bacteria in petri dishes that contain agar with no histidine. After incubating the dishes, the bacteria that have grown are counted. This number, which reflects the bacteria that undergo a reverse mutation from his^- to his S. typhimurium, is compared to the number of bacteria that undergo reverse mutations when they are not exposed to the agent. If the agent causes too many reverse mutations above those measured as spontaneous, it is considered to be mutagenic.

The Ames test can detect mutagens that work directly to alter DNA. In humans, however, many chemicals are promutagens, agents that must be activated to become true mutagens. Activation, involving a chemical modification, often occurs in the liver as a consequence of normal liver activity on unusual substances. Bacteria such as S. typhimurium do not produce the enzymes required to activate promutagens, so promutagens would not be detected by the Ames test unless they were first activated. An important part of the Ames test also involves mixing the test compound with enzymes from rodent liver that convert promutagens into active mutagens. These potentially activated promutagens are then used in the Ames test. If the liver enzymes convert the agent to a mutagen, the Ames test will detect it, and it will be labeled as a promutagenic agent.

The Ames test is widely used by the pharmaceutical industry to test drugs prior to using them in clinical trials. When a drug is mutagenic in the Ames test, it is usually rejected for further development and will probably not be tested in animals or used therapeutically in humans. The cosmetic industry also uses the Ames test to assess the mutagenic potential of makeup and other hygienic products. The Food and Drug Administration requires companies to perform the Ames test before marketing most drugs or cosmetics.

Bibliography

Ames, Bruce N., and Lois S. Gold. "The Causes and Prevention of Cancer: The Role of Environment." Biotherapy 11 (1998): 205-220.

Mortelmans, Kristien, and Errol Zeiger. "The Ames Salmonella Microsome Mutagenicity Assay." Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis 455 (2000): 29-60.

—David A. Scicchitano

A test for mutagenic substances, in which a strain of Salmonella typhimurium that lacks the enzyme necessary for histidine synthesis is cultured in the absence of histidine and in the presence of the suspected mutagen treated with liver extract. If the substance causes DNA damage resulting in mutations, an increased number of the bacteria will regain the ability to synthesize histidine and will proliferate to form colonies. An important test for detecting potentially carcinogenic agents such as agricultural chemical and food additives.

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