Directly assaying potential carcinogens by testing for their ability to form tumors in animals is difficult and expensive. However, in addition to causing tumors in animal cells, most carcinogens are mutagens. Based upon this insight, Bruce Ames and colleagues developed a simple, indirect assay for potential carcinogens. The assay is based upon the reversion of mutations in the histidine (his) operon in the bacterium Salmonella enterica sv Typhimurium. The his operon encodes enzymes required for the biosynthesis of the amino acid histidine. Strains with mutations in the his operon are histidine auxotrophs -- they are unable to grow without added histidine. Revertants that restore the His+ phenotype will grow on minimal medium plates without histidine. This provides a simple, sensitive selection for revertants of his mutants.
The his mutants are mixed with the potential mutagen then plated on minimal medium with a very small amount of histidine. The concentration of histidine used is limiting, so after the cells go through several cell divisions the histidine is used up and the auxotrophs stop growing. However, if the potential mutagen induces His+ revertants during the initial few cell divisions, then each of the resulting revertants will continue to divide and form a colony. The number of colonies produced is proportional to how efficiently the mutagen reverts the original mutation. For example, in the cartoon below two potential mutagens are tested. The potential mutagen A produced a higher frequency of reversion than the control, and thus really is a mutagen and is a likely carcinogen. The potential mutagen B did not produce a higher frequency of reversion than the control -- if this result was obtained for each type of mutation tested, then the results suggest that it is not a mutagen.
An example of some real data is shown in the table below.
Mutagen Number of revertant colonies per plate1
his base substitution mutant his frameshift mutant ________________________________________________________________________________ None 11 25 Diethyl sulfate 14,700 0 ICR-191 13 450 ________________________________________________________________________________
Several different types of his mutants are used to test for different classes of mutagens -- for example, frameshift mutagens will revert a frameshift mutation in his, etc. The Salmonella his mutants used have three additional properties that make them more sensitive to mutagens.
Some chemicals (called pro-mutagens) are not mutagenic unless metabolized to more active derivatives. Liver enzymes that normally detoxify harmful metabolic intermediates are often responsible for the activation of pro-mutagens into mutagens. For example, benzo(a)pyrene is not mutgenic but it is converted by liver enzymes to diolepoxides which are potent mutagens and carcinogens. Therefore, to test for such pro-mutagens, an extract of rat liver enzymes is included in the reversion assay.