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Microbial Mutagenicity Tests: Strategies and Benefits

Paper Type: Free Essay Subject: Biology
Wordcount: 1898 words Published: 21st May 2018

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Human beings are exposed to an ever-increasing array of chemicals; from food additives, medicines and cosmetics to environmental exposure to pesticides and flame-retardants. It is essential to establish the potential toxicity of these compounds before they are used, and in particular to assess their capacity to cause mutational damage to deoxyribonucleic acid (DNA). Such genotoxicity can cause heritable damage to germline DNA or may increase the likelihood of carcinogenesis. A number of tests are currently employed to assess a chemical’s mutagenicity, including in vitro tests using microbes and mammalian cell cultures and in vivo tests involving animals, most commonly rodents. This review will concentrate on microbial tests.


The basic concept underlying microbial tests is that mutant bacterial strains will undergo further DNA mutation when exposed to mutagenic chemicals. This will cause reversion of the phenotype back to the ‘wild type’. These back mutants are termed revertants. McCann, Ames and colleagues (1975) developed mutant Salmonella typhimurium strains which are unable to synthesise the amino acid histidine (His) and will only survive if it is provided. The bacterial strains are plated out onto histidine-deficient agar and a suspected chemical mutagen is added. Following incubation, the plate is observed for growth; only revertants will have formed colonies, due to their re-acquired ability to synthesise histidine (His+). Spontaneous reversion can also occur and the background level should be determined by plating out each strain onto histidine-deficient agar, without addition of the test substance (Mortelmans & Zeiger 2000). The test substance should produce at least double the number of colonies as the background reversion.

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Ames’ laboratory showed that 90% of chemicals found to be mutagens by a positive test result were known rodent carcinogens: 89% of rodent carcinogens tested were also mutagens. This supported the hypothesis that mutagenicity is a good predictor of carcinogenicity. The relationship between the two types of toxicity is probably not this straightforward and involves several assumptions, as discussed below. More recent experimentation has established a more accurate and reproducible figure of 60-80% for predictivity of carcinogenic potential (Zeiger 2004).

Many strains are in use today in what is now called the Ames test, such as: TA1535, TA1537, TA98 and TA100. Other mutant strains have also been studied, such as TA102, which has been found to confer some advantages over other strains (Levin et al. 1982). Most of the histidine requiring Salmonella mutants have G-C base pairs at the critical site for reversion, whereas TA102 has an A-T pair. This allows mutagens to be detected which are negative for the other strains, so is a useful addition for mutagenicity screening.

Research has also been conducted using mutant strains of other bacteria, most notably Escherichia coli (Mortelmans & Riccio, 2000). The E.coli WP2 reverse-mutation system relies on conversion from trytophan dependence (Trp) to independence (Trp+) and has an A-T base pair, similar to S. typhimurium TA102. It has shown comparable sensitivity to Salmonella assays (Dyrby & Ingvardsen 1983). Despite the usefulness of E.coli assays, the Salmonella/Ames test remains the gold standard. This may be attributed to the fact that the strains and protocols are generally better validated and therefore widely accepted internationally, although many laboratories now use a combination of tests. One example is the work done by Araki and colleagues (2004) to ascertain the mutagenicity of carbon tertrachloride. Results showed that two E.coli strains and S. typhimurium TA98 produced a positive result, whereas TA100, TA1535 and TA1537 did not detect mutagenicity.

Many carcinogens are only biologically active when metabolised in the liver and it is advisable to test for mutagenicity with and without the addition of liver homogenate (S9), which contains microsomes (vesicles encapsulating liver enzymes). Usually S9 is produced from rat liver, but recent research has suggested that human liver preparations may be more accurate for predicting carcinogenicity in humans (Hakura et al., 2005).

Research continues to refine these microbial tests and the principle of back-mutation used to produce assays that are more sensitive, easier and quicker to perform (Reifferscheid et al., 2005), allowing a greater number of chemicals to be screened for mutagenicity and potential carcinogenicity.

Different bacterial species have also been studied, for use in specialised environments: mutant strains of Vibrio harveyi can detect mutagens present in marine samples with greater sensitivity than the gold standard Ames test (Czyz et al. 2002).

Microbial tests, such as the Ames test, are internationally accepted as a well-validated screening method for mutagenicity and potential carcinogenicity of chemicals intended for human use. The Ames test was formally adopted into international guidelines for mutagenicity programmes by the Organisation for Economic Co-operation and Development (OECD) in 1983, and remains there. It is also incorporated into European legislation for chemical testing (EU Directive 67/458/EEC): indicative that the test is generally considered to be an important part of chemical toxicity screening.

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However, the use of such tests to screen for carcinogenic chemicals is based on the assumption that all chemicals that are mutagens are also carcinogenic and vice versa. Although Ames experiments in the 1970s provided support for this hypothesis, further research has revealed that it this is not always true. Zeiger (2001) found that from a database of 172 chemicals that had been shown to be non-carcinogenic in rodent studies, 38 were mutagenic to microbes and also tended to produce mutagenesis in mammalian cell cultures. He also points out that some carcinogens have been found to be non-mutagenic. This arises due to the existence of mechanisms for carcinogenicity, other than direct DNA mutation/genotoxicity. It cannot, therefore, be assumed that a chemical found to be mutagenic by in vitro tests will definitely be carcinogenic. Conversely, a negative result does not guarantee that non-mutagenic chemical will not be carcinogenic in vivo. Although this only applies to a minority of chemicals tested, it should be considered, especially when drawing up toxicity legislation (Botham & Holmes, 2001).

There is also some dispute over the relationship between mutagenicity and actual cancer formation. The presence of mutations in a cell’s DNA is a preliminary step towards cancer formation, but usually requires further damage to trigger uncontrolled cell division (Kumar et al. 2005). Thus, a mutagen increases the probability of cancer formation, but it is not guaranteed to. Despite this, most people would rather not be exposed to chemicals which increase the likelihood of cancer, so the debate is academic. Researchers should consider microbial tests to be a presumptive, rather than definitive test for carcinogenicity.

There has also been some concern that samples containing high levels of amino acids can interfere with the assay, but modification of the protocol may eliminate this (Thompson et al., 2005).

Microbial tests are beneficial for first line mutagenicity screening and support the ‘3Rs’: reducing the number of animal tests required by eliminating highly mutagenic compounds at an early stage.


Microbial mutagenicity tests play a valuable role in chemical screening and are regarded as accurate enough to be incorporated into international regulatory guidelines. Despite a few minor problems with the predictivity of carcinogenicity the Ames test is still regarded as the gold standard by which new in vitro mutagenicity tests are measured.

As further research is conducted microbial tests are now becoming faster, easier and cheaper and seem likely to continue to be widely used.


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