Ames Test and Eurofins’ Ames Fluctuation Assay
The identification of compounds capable of inducing mutations is crucial in safety assessment, since mutagenic compounds can potentially damage the germ line and also induce cancer. Gene mutations can easily be measured in bacteria, where they cause a change in nutrient dependent growth. The Ames test, which is conducted using Salmonella typhimurium, is a widely used bacterial assay for the identification of compounds that can produce gene mutations, and it shows a high predictive value with rodent carcinogenicity tests.
The Ames test typically uses five strains of Salmonella with pre-existing mutations that render the bacteria unable to synthesize the essential amino-acid histidine, and, as a result, cannot grow in histidine-free medium. If a compound induces mutations in these particular genes, it can restore gene function, allowing the cells to regain the capacity to synthesize histidine and therefore grow in its absence ("reversion assay"). The Salmonella strains have different mutations in various genes in the histidine operon, and are designed to be responsive to mutagenic compounds that act through different mechanisms.
Eurofins' Ames Fluctuation Assay
Eurofins offers a miniaturized screening version of the Ames test that requires a very small amount of compound (typically 5 mg) compared with the regulatory test. The Ames fluctuation assay is performed in 384-well plates using four Salmonella strains, TA98, TA100, TA1535 and TA1537. TA98 and TA1537 detect frameshifts. TA100 detects base-pair substitutions leading to missense mutations. TA1535 detects base-pair substitutions and is used to identify certain mutagenic compounds which are not detected by TA98 and TA100. While the original Ames test is carried out by plating bacteria onto selective agar plates, the Ames fluctuation assay is carried out in liquid culture using 384-well plates. The bacterial plates are incubated with the test compounds for 96 hours, after which bacterial growth is measured spectrophotometrically using a pH indicator that changes color in response to the acidification of the media due to bacterial growth.
Table 1. Gene mutations detected in Ames
|Salmonella strain||DNA target||His mutation||Reversion event|
|TA1537||Near CCC run||hisC3076||Frameshifts|
Metabolic activation is achieved by using rat liver S9 fraction. To prevent false negatives due to bacteriocidal or bacteriostatic effects, a bacterial cytotoxicity assay is conducted in parallel with the Ames fluctuation assay.
Compounds are typically tested in four bacterial strains with and without S9, at four concentrations (default concentrations are 5, 10, 50 and 100 μM; higher customized concentrations are also available upon request) with n = 48 wells. A bacterial cytotoxicity test is conducted in parallel at 8 concentrations (with 100 μM as the highest concentration) and n = 3 wells. Four reference compounds (quercetin, streptozotocin, aminoanthracene and aminoacridine) are included in all assays.