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10.2: Testing for Mutagenic Chemicals in Bacteria and Mice

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  • Ames test

    Ames test is a test for determining if a chemical is a mutagen. It is named for its developer, Bruce Ames. The use of the Ames test is based on the assumption that any substance that is mutagenic for the bacteria used in his test may also turn out to be a carcinogen; that is, to cause cancer. Although, in fact, some substances that cause cancer in laboratory animals (dioxin, for example) do not give a positive Ames test (and vice-versa), the ease and low cost of the test make it invaluable for screening substances in our environment for possible carcinogenicity.

    The bacterium used in the test is a strain of Salmonella typhimurium that caries a defective (mutant) gene making it unable to synthesize the amino acid histidine (His) from the ingredients in its culture medium. However, some types of mutations (including this one) can be reversed, a back mutation, with the gene regaining its function. These revertants are able to grow on a medium lacking histidine.

    Figure 10.2.1 Ames Test

    The picture shows a qualitative version of the Ames test. A suspension of a histidine-requiring (His) strain of Salmonella typhimurium has been plated with a mixture of rat liver enzymes on agar lacking histidine. The disk of filter paper has been impregnated with 10µg of 2-aminofluorene, a known carcinogen. The mutagenic effect of the chemical has caused many bacteria to regain the ability to grow without histidine, forming the colonies seen around the disk. The scattered colonies near the margin of the disk represent spontaneous revertants.

    Many chemicals are not mutagenic (or carcinogenic) in themselves, but become converted into mutagens (and carcinogens) as they are metabolized by the body. This is the reason the Ames test includes a mixture of liver enzymes.

    A large number of chemicals used in agriculture and industry give a positive Ames test. Examples are ethylene dibromide (EDB), which was added to leaded gasoline (to vaporize lead deposits in the engine and send them out the exhaust), and ziram, which is used to prevent fungus disease on crops. Some drugs, such as isoniazid (used to prevent tuberculosis) are mutagenic in this test.

    The potency of AF-2, a food additive once widely-used in Japan, and safrole, a flavoring agent that used to be added to root beers, caused them to be banned. Saccharin, suspected by some of being a carcinogen, does not give a positive Ames test.

    Although most testing has been done on products of the chemical industry, many naturally-occurring substances (like safrole) have been shown to be mutagenic. These include aflatoxin, produced in moldy grain and peanuts (and present in peanut butter at an average level of 2 parts per billion, ppb. Traces of nine different substances that give positive Ames tests have been found in fried hamburger.

    Salmonella typhimurium is a bacterium and thus not a perfect model of the human body (which is why liver enzymes are added to the test). Rapid in vitro tests modeled on the Ames test have been adapted for some eukaryotic cells such as yeast and mammalian cells grown in culture. And thanks to recombinant DNA technology, it is now possible to combine the advantages of rapid in vitro tests like the Ames test with the more realistic conditions of long-term studies in whole animals.

    Mutagen chemicals in mice

    Salmonella typhimurium, the organism used in the Ames test, is a bacterium and thus is not a perfect model of the human body (which is why liver enzymes are added to the test). In fact, some suspected human carcinogens are negative in the Ames test. Rapid in vitro tests modeled on the Ames test have been adapted for some eukaryotic cells such as yeast and mammalian cells grown in tissue culture. But what is needed is a way to combine the speed of in vitro tests like the Ames test with the more realistic conditions in whole animals.

    Thanks to recombinant DNA technology (and the efforts of Stratagene, a biotechnology company), such a test is now available. The test animal is a transgenic mouse which the company calls Big Blue.

    Figure 10.2.2 Blue Mouse

    Big Blue mice are transgenic for a segment of DNA that contains the DNA of bacteriophage lambda, a virus that infects E. coli, and which serves here as the vector for 3 genetic elements from the lac operon of E. coli:

    • the lacI gene
    • the operator of the operon
    • the beta-galactosidase (lacZ) gene

    The Assay

    The transgenic mice are given repeated doses of the suspected carcinogen for a week or two. If the chemical is mutagenic, it will cause random mutations throughout the genome of each mouse cell. If a mutation occurs in either the lacI gene (which encodes the lac repressor) or the operator,

    the gene (lacZ) for beta-galactosidase will no longer be repressed. To detect this,

    • The DNA is extracted from the tissues of the treated mouse.
    • The vector is isolated and used to make functional bacteriophages.
    • E. coli cells are mixed with the bacteriophage and spread on a solid culture medium.
    • The bacteriophages infect and destroy ("lyze") the E. coli cells.
    • This causes clear circular zones, called plaques, to appear in a "lawn" of bacteria.
    • Before they die, cells that have been infected by bacteriophages carrying a mutated lacI or operator will produce beta-galactosidase.
    • This reacts with a substrate in the culture medium turning it blue.
    • Bacteriophages with unmutated genes produce colorless plaques because no beta-galactosidase is synthesized.
    • Count both colorless and blue plaques.
    • The number of blue plaques divided by the total number of plaques gives the mutation frequency.

    Figure 10.2.3 Results of cells dosed with benzopyrene

    The graph shows the results of a test done on the spleen cells of a transgenic mouse that had been dosed with benzopyrene, a known mutagen (and carcinogen). Three days after its last dose, 46 blue plaques were recovered from the spleen sample out of a total of 2,647,040 plaques. This yields a mutation frequency of 1.7 x 10−5 (1.7 in 100,000) — a significant increase over control values.

    Figure 10.2.4 Blue Plaque courtesy of Stratagene

    This photograph shows one mutant (blue) plaque on a lawn of E. coli containing many non-mutant (clear) plaques.