Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
The Ames Salmonella/microsome mutagenicity assay
Introduction
The identification of substances capable of inducing mutations has become an important procedure in safety assessment. Chemicals that can induce mutations can potentially damage the germ line leading to fertility problems and to mutations in future generations. Mutagenic chemicals are also capable of inducing cancer, and this concern has driven most of the mutagenicity testing programs. Mutations can occur as gene (point) mutations, where only a single base is modified, or one or a relatively few bases are inserted or deleted, as large deletions or rearrangements of DNA, as chromosome breaks or rearrangements, or as gain or loss of whole chromosomes.
Gene mutations are readily measured in bacteria and other cell systems when they cause a change in the growth requirements of the cell, whereas chromosome damage in mammalian cells is typically measured by observing the cell's chromosomes under magnification for breaks or rearrangements. The Salmonella typhimurium/microsome assay (Salmonella test; Ames test) is a widely accepted short-term bacterial assay for identifying substances that can produce genetic damage that leads to gene mutations. The test uses a number of Salmonella strains with preexisting mutations that leave the bacteria unable to synthesize the required amino acid, histidine, and therefore unable to grow and form colonies in its absence. New mutations at the site of these preexisting mutations, or nearby in the genes, can restore the gene's function and allow the cells to synthesize histidine. These newly mutated cells can grow in the absence of histidine and form colonies. For this reason, the test is often referred to as a “reversion assay.”
The Salmonella strains used in the test have different mutations in various genes in the histidine operon; each of these mutations is designed to be responsive to mutagens that act via different mechanisms. Additional mutations were engineered into these strains to make them more sensitive to a wide variety of substances.
The Salmonella mutagenicity test was specifically designed to detect chemically induced mutagenesis [7]. Over the years its value as such has been recognized by the scientific community, and by government agencies and corporations. The test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs because there is a high predictive value for rodent carcinogenicity when a mutagenic response is obtained [49], [71], [85], [92]. International guidelines have also been developed (e.g., Organisation for Economic Co-operation and Development (OECD); International Commission on Harmonization (ICH)) for use by corporations and testing laboratories to ensure uniformity of testing procedures prior to submission of data to regulatory agencies for registration or acceptance of many chemicals, including drugs and biocides.
Section snippets
Historical aspects
The Ames Salmonella/microsome mutagenicity assay evolved over the years from the initial screening of a number of histidine mutants which led to the selection of mutants that were highly sensitive to reversion by a variety of chemical mutagens [2], [5], [39], [40], [47]. Because bacteria are unable to metabolize chemicals via cytochromes P450, as in mammals and other vertebrates, a key component for making the bacterial mutagenicity test useful was the inclusion of an exogenous mammalian
Preparation of frozen permanent cultures
For long-term preservation, the Salmonella tester strains should be kept frozen at −80°C (freezer or liquid nitrogen). Upon receipt of the new strains, up to five frozen permanent cultures should be prepared from one single colony isolate that has been purified and checked for its genotypic characteristics (his, rfa, uvrB-bio) and for the presence of plasmids pKM101 and pAQ1, when appropriate. These cultures should be considered the frozen permanent strains and should be accessed only for the
Genetic analysis
It is recommended that the tester strains be analyzed for their genetic integrity and spontaneous mutation rate when frozen cultures are prepared. A strain check should also be performed whenever an experiment is performed. The strain check is usually performed with the nutrient broth overnight cultures in the following way.
The following steps should be followed for a complete strain check.
⋅ Histidine dependence (his): streak a loopful of the culture across a GM agar plate supplemented with an
Spontaneous control values
Each tester strain has a characteristic spontaneous mutant frequency. There is usually some day-to-day and laboratory-to-laboratory variation in the number of spontaneous revertant colonies. Choice of solvent may also affect the spontaneous mutant frequency [46]. Each laboratory has a characteristic range of revertant colonies for each strain which is referred to as “historical control values”. The spontaneous mutant frequency obtained when the strain check is performed should be compared to
Toxicity determination
Toxicity determination in the Ames Salmonella test requires the evaluation of characteristics of the final population on the GM agar plate after the 48-h incubation instead of a quantitative survival determination. These characteristics are:
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thinning of the background lawn which may be accompanied by a decrease in the number of revertant colonies
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absence of background lawn (i.e., complete absence of growth)
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presence of pinpoint non-revertant colonies (generally in conjunction with an absence of
Top agar
The top agar, consisting of 0.6% agar and 0.6% NaCl, is one of the most critical medium components in the Ames test because it contains the trace amount of histidine (0.05 mM) for limited growth. It also contains biotin at a concentration of 0.05 mM which is in excess of what is needed for the growth of the Salmonella strains. Because the His− bacteria stop growing when the histidine is depleted, the final population of His− bacteria is dependent on the histidine concentration. In turn, the
Standard plate incorporation assay
Concept: The standard plate incorporation assay consists of exposing the tester strain(s) to the test chemical directly on a minimal glucose agar plate (GM plate) usually in the presence and absence of a metabolic activation system (see Fig. 2). The different components are first added to sterile test tubes containing 2 ml of molten top agar supplemented with limited histidine and biotin. It is important to maintain the top agar at a temperature between 43°C and 48°C and to minimize prolonged
Important steps to follow for all assay procedures
⋅ The cofactors for preparing the S-9 mix are the same for each of the assay protocols, with the exception of the reductive metabolism assay. The preparation of the cofactors is described in the section: Recipes for reagents and media.
⋅ It is important to quickly swirl the plates after addition of the top agar to the surface of the GM agar plates to ensure an even distribution of the top agar which contains the bacteria, test chemical and S-9 mix or buffer.
⋅ When the plates are inspected after
Experimental design
The Ames Salmonella test is a versatile assay as evidenced by the different procedural variations that are available, in addition to the numbers of strains and types of metabolic activation systems that can be used. Before initiating an Ames test, a number of critical parameters will need to be evaluated.
Counting of histidine revertant colonies
After the plates are removed from the incubator, the colonies are counted and the results are expressed as revertant colonies per plate. For this purpose, an electronic counter is a convenient way to count the colonies, especially for strains TA100 and TA97 which usually have a spontaneous background above 100 colonies/plate. However, hand-counting is required when strains TA102 and TA104 are used because of the high number of spontaneous revertant colonies, usually above 200 colonies/plate.
Aseptic technique
It is important that basic bacteriological laboratory procedures be used to minimize exposure to the Salmonella tester strains. Surface areas must be properly disinfected before and after use. Though wild-type S. typhimurium can cause diarrhea and food poisoning, the gal and rfa (deep rough) mutations that are present in all the Salmonella tester strains described here eliminate to different levels, the polysaccharide side chain of the LPS layer that coats the bacterial surface which makes the
Available databases
There are a couple of databases publicly available through the internet. The Environmental Mutagen Information Center (EMIC) database can be entered through http://toxnet.nlm.nih.gov/servlets/simple-search?1.25.0.3876. In this database, information can be retrieved regarding the test organisms used and endpoints examined.
The other database is that of the U.S. National Toxicology Program's genetic toxicology testing program. Results have been published on more than 1500 chemicals, which are
Troubleshooting
Following are some problems that are frequently encountered and a brief description of their probable causes and suggested solutions.PROBLEM: The spontaneous revertant count is too low Cause: (1) Toxicity is associated with a new batch of agar (2) The wrong tester strain may have been used (3) Too little histidine was added to the top agar Solution: (1) Each new batch of agar should be checked for toxicity prior to using it on a routine basis (2) Double check the identity of the strain, and if needed,
Vogel–Bonner (VB salts) medium E (50×)
Use: salts for the GM agar platesIngredients Per liter Warm distilled water (about 50°C) 650 ml Magnesium sulfate (MgSO4·H2O) 10 g Citric acid monohydrate 100 g Potassium phosphate, dibasic, anhydrous (K2HPO4) 500 g Sodium ammonium phosphate (Na2NH2PO4·4H2O) 175 g
Add the above ingredients in the order indicated to warm water in a 2-l flask making sure that each salt is dissolved thoroughly by stirring on a magnetic stirrer before adding the next salt. It takes about 1 h to dissolve all ingredients. Adjust
Equipment and supplies
The following items are needed for performing the mutagenicity protocols.
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