The Ames test is a simple and sensitive assay to determine if a chemical is a mutagen. It was created by Bruce N. Ames, a bacterial geneticist, in 1975 at the University of California, Berkeley. The Ames test assumes than any chemical that is a mutagen, a chemical that causes mutations in the DNA of an organism, may also be a carcinogen, a chemical that induces cancer. While there is a definite correlation between mutagens and carcinogens, a mutagen is not always a carcinogen and vice versa. For example, the steroid hormone diethlystilbestrol is a carcinogen but not a mutagen as it can cause cancer without mutating DNA.
The Ames test uses a special strain of Salmonella typhimurium bacteria. This strain was genetically engineered with a specific mutation in a gene that synthesizes the amino acid histidine. This mutation inactivates the gene and prevents the bacteria from being able to make histidine. Therefore, the bacteria are only able to survive if histidine is present in the liquid or solid media that they grow in. These bacteria are placed onto histidine-free solid media plates along with the chemical of interest. This plate is then incubated at 37 degrees Celsius for several days.
There are two possible outcomes:
- The chemical is a mutagen and will randomly mutate the bacterial DNA. Because the mutations are random it is possible that the mutagen will mutate the inactive histidine-synthesizing gene back to its normal, active form. This mutation is referred to as a back mutation and the resulting bacteria are called revertants. If this happens the bacteria will be able to make histidine and will form colonies on the histidine-free plates.
- The chemical is not a mutagen and does not affect the bacterial DNA. The histidine-synthesizing gene will remain inactive and the bacteria will not form colonies on the plates.
Some chemicals in their original forms are not mutagens or carcinogens. However, these chemicals can become mutagens when they are metabolized in the liver by special enzymes. These chemicals are called promutagens or procarcinogens. In order to test for these chemicals, rat liver extracts are used that contain the metabolizing enzymes. These extracts are placed onto the solid media plates along with the bacteria and the chemical.
The potency of the chemical as a mutagen is quantified by the number of bacterial colonies that form during the test. The unit used in the Ames test is the number of revertants per microgram of the chemical. A chemical that is a more potent mutagen will cause more colonies to form than a weaker mutagen. Potent mutagens can give hundreds to thousands of revertants per microgram. Chemicals that have been shown to be mutagens by the Ames test include ethylene dibromide (EDB), an additive in leaded gasoline, ziram, an anti-fungal agent for crops, and safrole, an artificial root beer flavoring agent. Natural compounds including aflatoxin, a substance found in moldy grain and peanuts, and compounds found in grilled meat also give a positive result. Saccharin, an artificial sweetener many think is a carcinogen, gives a negative Ames test result.
The Ames test is a quick and simple way to test the tens of thousands of chemicals that are produced each year. However, this assay is not necessarily the best representation of a chemical's mutagenicity and potential carcinogenicity in humans. Prokaryotic bacteria and isolated rat liver extracts are certainly not an ideal model of human cells and metabolizing enzymes. Because of this, scientists do not use the Ames test as the only measure of mutagenicity. If a chemical tests positive under the Ames test, subsequent experiments are performed in mice to confirm if the chemical is a mutagen and carcinogen. The Ames test has also been modified to analyze yeast and mammalian cells instead of bacteria to get a better idea of how the chemical will behave in humans.
Sources:
- The World of the Cell, 1996.
- http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AmesTest.html