Testing of chemicals for mutagenicity in Salmonella typhimurium is based on the knowledge that a substance that is mutagenic in the bacterium is more likely than not to be a carcinogen in laboratory animals, and thus, by extension, present a risk of cancer to humans. Although about three-fourths of chemicals that are positive in the Ames test are found to be rodent carcinogens, not all substances that cause cancer in laboratory animals are mutagenic in this assay. However, the ease, rapidity (results in 3-4 weeks) and low cost of the test make it an important tool for screening substances for potential carcinogenicity.
Several strains of the S. typhimurium bacterium may be used for testing. Each is genetically different, so using several strains in a test increases the opportunity of detecting a mutagenic chemical. The most frequently used strains are TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. In addition to the Salmonella tester strains, the NTP now routinely employs Escherichia coli strain WP2 uvrA pKM101 as a bacterial tester strain in the Ames test. This E. coli strain is similar in mutagen detection to S. typhimurium strain TA102. All the S. typhimurium bacterial strains used in the Ames test carry a defective (mutant) gene that prevents them from synthesizing the essential amino acid histidine from the ingredients in standard bacterial culture medium. The E. coli strain carries a mutant gene that prevents synthesis of the essential amino acid tryptophan. Therefore, these "tester" strains can only survive and grow on medium that contains excess histidine (or for the E. coli strain, tryptophan). However, in the presence of a mutagenic chemical, the defective genes may be mutated back to the functional state, allowing the bacterium to grow on standard medium that does not contain supplemental histidine or tryptophan. These mutations, which lead to a regaining of normal activity or function, are called "back" or "reverse" mutations and the process is referred to as "reversion." The mutant colonies, which can make histidine or tryptophan, are called "revertants." (There are other mutagenicity assays using other cell-types that measure "forward" mutations, that is, mutations that alter a functional gene in a way that causes a loss, rather than a gain, of function.)
Many chemicals are not mutagenic (or carcinogenic) in their native forms, but they are converted into mutagenic substances by metabolism in the liver. Since bacteria do not have the same metabolic capabilities as mammals, some test protocols utilize extracts of rat or hamster liver enzymes (S9) to promote metabolic conversion of the test chemical. This permits the investigator to determine if a chemical must be metabolized to express mutagenic activity. Some mutagenic chemicals are active with and without metabolism, while others are active only under one condition or the other. Occasionally, other sorts of activation enzymes (e.g., mouse S9) may be employed.
In the standard protocol (preincubation) for conducting the Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium (or E. coli) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen*. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine or tryptophan appear and grow. These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine- or tryptophan- manufacturing gene. The number of colonies is usually counted after 2 days.
Several modifications of the Ames test protocol have been used over the years in special circumstances. These include standard plate incorporation (no preincubation step prior to plating onto Petri dishes), FMN reduction (use of flavin mononucleotide for reduction of test articles such as azo dyes), plate test with volatile liquids (exposure of bacteria in a sealed Petri dish), cecal reduction (use of rat cecal bacteria to provide reduction of azo compounds), and plate tests conducted within a sealed dessicator (gas chamber) for exposure to gaseous substances. The specific test protocol that was used in an Ames test is noted in the description of the assay data.
Spontaneous mutations (those that occur by chance, not by chemical treatment) will appear as colonies on the control petri dishes. If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain of bacteria. The positive control plates are also counted, and the number of mutant colonies appearing on them must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.
Several doses (usually at least 5) of each test chemical and multiple strains of bacteria are used in each experiment. In addition, cultures are set up with and without added liver S9 enzymes at varying concentrations. Therefore, a variety of culture conditions are employed to maximize the opportunity to detect a mutagenic chemical. In analyzing the data, the pattern and the strength of the mutant response are taken into account in determining the mutagenicity of a chemical. All observed responses are verified in repeat tests. If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.
*Positive control chemicals used in NTP Ames tests:
For strains tested in the absence of S9
TA98, 2-nitrofluorene or alternatively,
TA98 and TA1538, 4-nitro-o-phenylenediamine
TA100 and TA1535, sodium azide
TA97 and TA1537, 9-aminoacridine
TA102, mitomycin C
TA104, methyl methanesulfonate
E.coli WP2 uvrA pKM101, methyl methanesulfonate
For strains tested with S9
All strains, 2-aminoanthracene (or occasionally, sterigmatocystin)
The overall assay calls are defined as follows:
A positive response for the assay is defined as a reproducible, dose-related increase in histidine-independent or tryptophan-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose-related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment in any strain/activation combination. There is no minimum percentage or fold-increase required for a chemical to be judged positive or weakly positive.
Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutat Res. 2000 Nov 20;455(1-2):29-60.
Mortelmans K, Riccio ES. The bacterial tryptophan reverse mutation assay with Escherichia coli WP2. Mutat Res. 2000 Nov 20;455(1-2):61-9. Review. PMID: 11113467