Mutagenic Chemicals Can Be Detected by Reversion

Testing chemicals for tumor formation in animals is expensive and time-consuming. However, since cancer is due to DNA alterations, most carcinogens are in fact also mutagens. Consequently, chemicals suspected of being carcinogenic are routinely screened for possible mutagenic effects by testing against bacteria. The Ames test makes use of multiple strains of the bacterium Salmonella typhimurium carrying well characterized mutations in the genes for histidine synthesis. It is used routinely by industry and government agencies to screen food colorings and preservatives, cosmetics such as hair dyes, and many other industrial chemicals for possible mutagenic effects.

Mutants of Salmonella typhimurium carrying mutations in the his genes can no longer make the amino acid histidine and cannot grow unless given histidine. When large numbers of these mutant bacteria are placed on growth medium lacking histi-dine, just a handful of colonies appear. These are revertants, and since reversions are merely mutations back to the original state, the frequency of reversion is also increased by mutagenic agents. To test a suspect chemical, samples of Salmonella his mutants are mixed with the agent and then plated onto minimal medium with just a trace of histi-dine. The amount of histidine added is growth-limiting. Therefore the bacteria can only divide a few times and run out of histidine before making visible colonies. If the added

Ames test Test for mutagenic activity that makes use of bacteria suppressor tRNA A mutant tRNA that recognizes a stop codon and can insert an amino acid when it reads a stop codon on the mRNA

Screening for reversion using well characterized mutations allows the detection of mutagenic chemicals.

chemical does induce mutation then His+ revertants will be formed during these few cell divisions, then each resulting revertant can grow into a visible colony. Different types of original mutations, for example, base changes or frameshift mutations, are used to screen for different classes of mutagenic agents.

The Salmonella typhimurium strains used in practice for mutagen testing have several alterations that make them more sensitive to mutagens. Firstly, they carry mutations that make the bacterial outer membrane more permeable to large and/or hydrophobic molecules. Secondly a variety of alterations have been made to inactivate bacterial DNA repair mechanisms (see Ch. 14). For example, the uvrB gene may be deleted to eliminate excision repair of DNA.

Certain chemicals (pro-mutagens) are only mutagenic after metabolic conversion to active derivatives. In animals this is usually due to liver enzymes such as cytochrome P450 that are intended to detoxify harmful chemicals by oxidation. When testing for pro-mutagens, an extract containing such rat liver enzymes is mixed with the bacteria in the Ames test. Recently, genes for some variants of human cytochrome P450 have been cloned and successfully expressed in the Salmonella strains used for mutagen testing. The resulting bacteria synthesize the liver enzymes internally and are much more sensitive in their response to pro-mutagens.

From a practical viewpoint, the problem is not so much causing mutations as finding and isolating the ones desired.

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