Analysis of Three Factors Influencing Ames Test

Salmonella reverse mutation test. The Ames test is currently the internationally recognized test of choice for detecting the mutagenicity and carcinogenicity of new drugs. This test uses the nature of the histidine auxotrophic mutant (His-) of Salmonella typhimurium to produce revertives. Preliminary screening of the mutagenicity of chemical substances. The study found that the concentration of histidine, the concentration of the bacterial solution, and the concentration of S9 will all have an impact on the Ames test. Therefore, these three factors need to be considered when carrying out the Ames test.

S9 concentration

The Salmonella used in the Ames test is a prokaryotic organism and lacks a metabolic activation system. Therefore, the S9 mixture must be added as a metabolic activation system in the Ames test. The activity of the activation system may affect the test results. The biotransformation of many carcinogens depends on the mixed-function oxidase system of intracellular microsomes. Rat liver S9 is a common in vitro activation system. Its main active ingredient is a mixed-function oxidase system. A positive result is obtained without the addition of S9 mixture, indicating that the test substance is a direct mutagen. The positive result was obtained by adding S9 mixture, indicating that the test substance was mutagenic after S9 activation.

Genotoxicity testing not only plays an important role in the initial screening stage of drug development, it can affect the process of drug development to a large extent, but also can reduce the drug risk of clinical trial subjects and drug users after the drug is marketed. Medicilon has established a drug safety evaluation system that complies with international standards. The experimental research follows the guidelines of ICH, CFDA, FDA and OECD, which can provide customers with high-quality data and rapid research cycle preclinical drug safety evaluation services. Dr. Xiaodong Zhang is the vice president of preclinical toxicology of Medicilon. He is systematically and comprehensively responsible for the management of Medicilon’s subject-in-charge (SD) and provides professional technical guidance.

Some researchers used the Ames test to evaluate the potential mutagenicity of protein-modified Enterococcus faecalis. The test substances were divided into 0.05, 0.50, 5.00, 50.00 mg/dish, and a control group was set up. With Salmonella typhimurium mutant strains TA97, TA98, TA100 and TA102 as the test strains, the Ames test was carried out with (+) or without (-) S9, and the number of back-mutant colonies was counted [1]. The results showed that when the protein-modified Enterococcus faecalis was +S9 and -S9, the average number of colonies of the four test bacteria was less than twice that of the negative control group, and there was no dose-response relationship. The Ames test result of protein-modified Enterococcus faecalis was negative, indicating that the test substance is not mutagenic in vitro.

Histidine concentration

Histidine plays an important role in the Ames test. First, the medium used in the Ames test is not completely lacking histidine, but a small amount of histidine is added, so that the auxotrophic bacteria can reproduce for several generations. At the same time, the moss can fix the DNA damage caused by the mutagen during the replication process, and then behave as functional gene bacteria. Only such retromorphic bacteria can further grow into colonies. On the other hand, when the histidine and its precursors (such as polypeptides, proteins, etc.) in the detection system reach a certain amount, it can support the growth and division of His-deficient cells, increase the number of final revertant colonies, and cause false positives. The histidine concentration needs to be discussed when performing the Ames test.

Concentration of bacterial liquid

The concentration of the bacterial solution will have a direct impact on the results of the Ames test. Because the Ames test counts the number of colonies that produce back mutations, too low the amount of bacteria used in the test may lead to incomplete mutagenic performance of the test substance. In order to improve the stability and comparability of the Ames test results, the single factor test results show that histidine concentration, bacterial solution concentration, and S9 concentration are important influencing factors that need to be controlled. Orthogonal experiments show that there may be an interactive relationship between the three influencing factors, and it is not possible to get a better result by arbitrarily combining within a certain limit range. Therefore, a limit range cannot be given for each influencing factor alone.

The Ames test is widely used in the preliminary screening of mutagenic chemicals and can analyze the toxicity of the test substance, providing a reliable test basis for the drug’s clinical use.

[1] Ames test of protein-modified Enterococcus faecalis [J].

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