Ames test

The Ames test is a biological assay to assess the mutagenic potential of chemical compounds. A positive test indicates that the chemical might act as a carcinogen (although a number of false-positives and false-negatives are known). ["Ames Test." Encyclopedia of Public Health. Ed. Lester Breslow. Gale Cengage, 2002. eNotes.com. 2006. as of 29 Aug, 2008 ] As cancer is often linked to DNA damage, the test also serves as a quick assay to estimate the carcinogenic potential of a compound since it is difficult to ascertain whether standard carcinogen assays on rodents were successful. The procedure is described in a series of papers from the early 1970s by Bruce Ames and his group at the University of California, Berkeley.

General procedure

The test uses several strains of the bacterium "Salmonella typhimurium" that carry mutations in genes involved in histidine synthesis, so that they require histidine for growth. The variable being tested is the mutagen's ability to cause a reversion to growth on a histidine-free medium. The tester strains are specially constructed to have both frameshift and point mutations in the genes required to synthesize histidine, which allows for the detection of mutagens acting via different mechanisms. Some compounds are quite specific, causing reversions in just one or two strains. [cite journal | author= Bruce N. Ames, E. G. Gurney, James A. Miller, and H. Bartsch | title= Carcinogens as Frameshift Mutagens: Metabolites and Derivatives of 2-acetylaminofluorene and other Aromatic Amine Carcinogens | journal= PNAS | year= 1972 | volume= 69| pages= 3128–2132 | url = http://www.pnas.org/cgi/content/full/69/11/3128 | doi= 10.1073/pnas.69.11.3128 | pmid= 4564203] The tester strains also carry mutations in the genes responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable, [cite journal | author= Bruce N. Ames, Frank D. Lee, and William E. Durston | title= An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens | journal= PNAS | year= 1973 | volume= 70 | pages= 782–6 | url = http://www.pnas.org/cgi/content/full/70/3/782 | doi= 10.1073/pnas.70.3.782 | pmid= 4577135] and in the excision repair system to make the test more sensitive. [cite journal | author= Joyce McCann, Neil E. Spingarn, Joan Kobori, and Bruce N. Ames | title= Detection of Carcinogens as Mutagens: Bacterial Tester Strains with R Factor Plasmids | journal= PNAS | year= 1975 | volume= 72 | pages= 979–83 | url = http://www.pnas.org/cgi/content/full/72/3/979 | doi= 10.1073/pnas.72.3.979 | pmid= 165497] Rat liver extract is added to simulate the effect of metabolism, as some compounds, like benzopyrene, are not mutagenic themselves but their metabolic products are. [cite journal | author= Bruce N. Ames, William E. Durston, Edith Yamasaki, and Frank D. Lee | title= Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection | journal= PNAS | year= 1973 | volume= 70 | pages= 2281–5 | url = http://www.pnas.org/cgi/content/full/70/8/2281 | doi= 10.1073/pnas.70.8.2281 | pmid= 4151811]

The bacteria are spread on an agar plate with a small amount of histidine. This small amount of histidine in the growth medium allows the bacteria to grow for an initial time and have the opportunity to mutate.When the histidine is depleted only bacteria that have mutated to gain the ability to produce its own histidine will survive. The plate is incubated for 48 hours. The mutagenicity of a substance is proportional to the number of colonies observed.

Problems

As Salmonella is a prokaryote, it is not a perfect model for humans. An adapted "in vitro" model has been made for eukaryotic cells, for example yeast structure.

References