Ames Test

Ames test is a biological assay widely used for the identification and evaluation of mutagenic potential in chemical compounds. Using bacteria, this test examines whether a specific chemical has the potential to cause mutations in DNA of the organism being tested. Mutations represent changes in the genetic makeup of the organism. They can lead to various biological consequences, including carcinogenesis. As such, Ames test aims to determine which chemicals possess mutagenic activity. This is done by observing whether they trigger mutations in bacterial samples.

The principle of Ames test

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Ames test is a powerful tool for examining the mutagenic potential of a chemical, as a positive result means that the chemical may act as a carcinogen. If the test bacteria acquire mutations, the colony frequency observed is would be significantly higher than the one in negative control cultures. While traditional carcinogen assays in mammals are time-consuming and costly, Ames test is quick and convenient. This makes it a very advantageous test for the estimation of carcinogenic potential of a compound.

The test uses specific strains of bacteria, such as Salmonella typhimurium and Escherichia coli, that exhibit specific mutations. These mutations cause the bacteria to be unable to produce amino acids. Thus, when the bacteria are cultured in specific chemicals, histidine-encoding gene mutations may occur. This results in the bacteria regaining the ability to synthesise histidine. The phenomenon is known as reverse mutation, where bacteria go from His- to His+.

Dose response curve examples

Applications of Ames test

Not only can Ames test be used for the identification of revert mutations in bacterial strains, it can also be used for detecting mutagenicity of various environmental samples. Among these are drugs, dyes, reagents, cosmetics, wastewater, pesticides, and other substances that are readily solubilized in a liquid suspension.

Ames test and carcinogens

The mutagens identified by Ames test have the potential to be carcinogens. Early studies by Bruce Ames, the developer of this method, demonstrated that around 90% of known carcinogens can be identified with this method. Later studies demonstrated that the detection rate is more around 50%-70% of known carcinogens. When used on commercial products, the test led to the identification of several potential carcinogens. Due to majority of the dose-response curves of the test appearing linear, thus exhibiting no threshold, it was suggested that low levels of mutagens may be tolerable due to protective mechanisms such as DNA repair.

General procedure and method

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Ames test used in practice involve strains of Salmonella typhimurium with mutations in genes related to histidine synthesis. The strains are auxotrophic mutants. This means that they need histidine for growth but are not able to synthesize it themselves. The substance being tested is examined for its ability to cause mutations that revert the bacteria back to a prototrophic state, which allows them to grow in a histidine-free media.

Special tester strains can help detect the type of the mutations, which can either be frameshift or point mutation. This helps with the identification of mutagens that act through different mechanisms. Since larger organisms, such as mammals, have metabolic processes that can change the chemical's mutagenic potential, rat liver enzymes can be used to simulate the metabolic effects. This is especially useful if the compound itself is not mutagenic but its metabolites are.

Ames test involves the spread of bacteria on an agar plate containing small amount of histidine. This enables the growth of the bacteria and presents an opportunity for mutations. As the histidine becomes depleted, only bacteria that acquired mutations are able to grow further, as they now possess the ability to produce their own histidine. Once the bacteria is on agar plates, the plates are incubated for 48 hours. The mutagenicity of the substance is determined based on the colony count. It is important to set up a control plate that does not contain any histidine to determine for comparison.

Ames test method

Interpretation of results

The mutagenic potential of a chemical is proportional to the number of colonies observed. If a high number of colonies appears on the test plate when compared to the control plate, the chemical is mutagenic. It is possible that few colonies will appear on the control plate. This is the result of spontaneous mutations within the histidine-encoding gene and is considered a normal background noise in this experiment.

Limitations of Ames test

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As Salmonella typhimurium is a prokaryote and may not represent the perfect model for humans. Therefore, rat liver S9 fraction is used to mimic the specific mammalian metabolic conditions. However, the differences between humans and rats can have an effect on the mutagenicity assessment. Therefore, the use of human S9 fractions could improve the test's accuracy, as they are now commercially available. On top of that, not all mutagens identified by Ames test are carcinogens, warranting additional tests to confirm this characteristic.

False-positives are an issue in many microbiological tests and the Ames test is not exception. This includes certain compounds, such as the ones containing nitrate moiety. Nitrate compounds can trigger a positive Ames test as they generate nitric oxide, even if the substance is safe. In such cases, extended toxicology and outcome studies are needed to validate the Ames test results.

Fluctuation method

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Fluctuation method is an alternative to the traditional agar-plate Ames test. By counting the number of wells that change their colour from purple to yellow, the technique uses liquid culture and pH indicators to investigate the mutagenicity. The sensitivity and accuracy of fluctuation method is comparable with the traditional test. However, it does offer multiple advantages such as reducing the sample requirements, a simple colorimetric endpoint, and quicker processing.

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