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 Modern robotic technology is used to perform the Ames test today, but the fundamental procedure remains the same. Each square represents a single well in a plastic growth plate. Mutated Salmonella (yellow) are recorded by computer-driven detectors. Courtesy of Xenometrix, Inc., a subsidiary of Discovery Partners International, Inc. 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.