Ames Test

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.

see also Cancer; Carcinogens; Mutagen; Mutagenesis; Mutation; Nucleotide.

David A. Scicchitano

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.

AMES TEST

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.

Gail Charnley

(see also: Cancer; Carcinogen; Carcinogenesis; Toxicology )

Ames test

The Ames test, named for its developer, Bruce Ames, is a method to test chemicals for their cancer-causing properties. It is used by cosmetic companies, pharmaceutical manufacturers, and other industries that must prove that their products will not cause cancer in humans.

Ames, a cancer researcher at the University of California, began development of his method in the late 1950s. He believed an efficient, less-expensive means could be found to screen substances than the cumbersome methods in use. He hit upon the use of bacteria , which could be grown (cultured) cheaply, and grew rapidly so that testing could be completed quickly, yet could indicate the carcinogenic (cancer-causing) potential of many chemicals.

The bacterium used is a strain of Salmonella typhimurium that lacks an enzyme needed to form colonies. The bacterium is grown on agar culture (agar is a gelatin-like substance with nutrients ). The substance to be tested is blotted on a bit of paper and placed on the agar. If the substance is a carcinogen it will cause mutations in the bacterium as the cells divide. The mutant cells will have the enzyme to form colonies. The test can be completed in a day.

Bacterial mutations are the result of DNA damage. Because DNA in bacteria is similar to that in the higher animals, it is assumed the substance also will damage DNA in those animals and cause cell mutations and possibly cancer.

The Ames test is a screening test; it is not the final test that any substance must undergo before being commercially produced. It is designed to detect a cancer-causing agent quickly and inexpensively.

Prior to development of this test the procedure to determine whether a substance was a carcinogen required feeding the test substance to or injecting it into laboratory animals such as rats or mice and then examining them for evidence of tumor formation. Testing took years to complete, hundreds of animals, and millions of dollars.

Any substance that causes bacterial mutation in the Ames test is not given further consideration for development. A substance that does not produce bacterial mutation still must undergo animal testing, but at least the manufacturer has a good idea that it is not a cancer-causing agent.

Ames test

The Ames test, named for its developer, Dr. Bruce Ames, is a method to test chemicals for their cancer-causing properties. It is used by cosmetic companies, pharmaceutical manufacturers, and other industries that must prove their products will not cause cancer in humans.

Ames, a cancer researcher at the University of California, Berkeley, began development of his method in the late 1950s. He believed an efficient, less-expensive means could be found to screen substances than the cumbersome methods in use. His innovation utilized bacteria, which can be grown (cultured) inexpensively and which grow rapidly so that testing can be completed quickly. The test detects chemically-induced mutations, which indicate that the chemical responsible has the potential to be carcinogenic (cancer-causing).

The bacterium used is a strain of Salmonella typhimurium that lacks an enzyme needed to form colonies. The bacterium is grown on agar culture (agar is a gelatin-like substance with nutrients). The substance to be tested is blotted on a bit of paper and placed on the agar. If the substance is a carcinogen it will cause mutations in the bacterium as the cells divide. The mutant cells will have the enzyme to form colonies. The test can be completed in a day.

Bacterial mutations are the result of DNA damage. Because DNA in bacteria is similar to that in the higher animals, it is assumed the substance also will damage DNA in those animals and cause cell mutations and possibly cancer.

The Ames test is a screening test; it is not the final test that any substance must undergo before being commercially produced. It is designed to detect a cancer-causing agent quickly and inexpensively.

Prior to development of this test the procedure to determine whether a substance was a carcinogen required feeding the test substance to or injecting it into laboratory animals such as rats or mice and then examining them for evidence of tumor formation. Testing took years to complete, hundreds of animals, and millions of dollars.

Any substance that causes bacterial mutation in the Ames test is not given further consideration for development. A substance that does not produce bacterial mutation still must undergo animal testing, but at least the manufacturer has a good idea that it is not a cancer-causing agent.

Ames test

A laboratory test developed by biochemist Bruce N. Ames to determine the possible carcinogenic nature of a substance. The Ames test involves using a particular strain of the bacteria Salmonella typhimurium that lacks the ability to synthesize histidine and is therefore very sensitive to mutation . The bacteria are inoculated into a medium deficient in histidine but containing the test compound. If the compound results in DNA damage with subsequent mutations, some of the bacteria will regain the ability to synthesize histidine and will proliferate to form colonies. The culture is evaluated on the basis of the number of mutated bacterial colonies it produced. The ability to replicate mutated colonies leads to the classification of a substance as probably carcinogenic.

The Ames test is a test for mutagenicity not carcinogenicity. However, approximately nine out of 10 mutagens are indeed carcinogenic. Therefore, a substance that can be shown to be mutagenic by being subjected to the Ames test can be reliably classified as a suspected carcinogen and thus recommended for further study.

[Brian R. Barthel ]

RESOURCES

BOOKS

Taber, C. W. Taber's Cyclopedic Medical Dictionary. Philadelphia: F. A. Davis, 1990.

Turk J., and A. Turk. Environmental Science. Philadelphia: W. B. Saunders, 1988.