Re: What sort of tests are available to test carcinogenicity?

Hi Jeffrey,

Probably the most widely known and used test of carcinogenicity is the Ames Test. Developed by Bruce Ames in the 1970s, this test is actually a test of the ability of compounds to act as mutagens, which are also largely thought to function as carcinogens (although not all carcinogens are necessarily mutagens). As it was originally described, the Ames Test measures mutation rates in bacteria.

How does it work? Ames used strains of Salmonella enterica (which was known as S. typhimurium in the 70s) for the original test. A bacterial strain that carries a knockout mutation in a particular gene will only grow well when the product of the enzyme encoded by that gene is present in its growth media (e.g., agar). So, for example, if the gene for one of the enzymes that results in the synthesis of histidine is knocked out, those bacteria will require histidine in their media to grow rapidly. In media lacking histidine, there will be very very slow growth as neighboring bacterial cells die, realeasing histidine into the medium, and as bacterial cells recycle their own histidine, but in general, in the absence of histidine, bacterial growth will be very very very slow.

Now, if this gene in the histidine pathway was knocked out with a single point mutation (e.g., resulting in a stop codon), then its function can potentially be restored by another point mutation (aka a back mutation) at the same position, and this is what the Ames Test measures. Because there will be a very very slow rate of bacterial growth, there will be a low (basal) rate of mutation, and occasionally a back mutation restoring gene function.

If a compound is mutagenic, it will encourage the generation of mutations, resulting in a higher mutation rate, and a higher chance that the back mutation will occur, resulting in rapid growth in the descendants of those bacteria that experienced the back mutation.

When bacteria grow, they form colonies, and we can measure the mutagenicity of a compound with the Ames Test by comparing the number of colonies that grow on histidine free media in the presence of the compound to the number of colonies on the histidine free media alone. This gives us a relative value for the mutagenicity, and by extension the carcinogenicity of a compound.

In addition, some refinements of the Ames Test allow us to determine the rates for different kinds of mutations. For example, if the gene is knocked out by a deletion instead of a point mutation, a different mutagen may result in a higher rate of back mutations than one that results in point mutations. This test is called Ames II, and a fancy version of it it is distributed by a Swiss company named Xenometrix. However, I suspect that this test will be too expensive and unnecessarily complex for your needs; you might want to contact the biochemistry or microbiology department in your local university and see if there is a lab that would be willing to assist you in carrying out the Ames Test on your own.

Many other tests based on the principle of the Ames Test have been developed in the intervening 30 years. For example, one test is based on the knockout of a mobility gene in C. elegans (a nematode). With this gene knocked out, the worms cannot move. In the presence of a potent mutagen, the C. elegans worms will develop mobility more frequently than in the absence, and you will be able to count more worm "tracks" left in their media. This test is particularly nice since it is relevant for eukaryotes, rather than prokaryotes.

Now, with regards to your experiment as you have described it, if you use an Ames Test, you are only going to be measuring the mutagenicity of irradiated and pesticide treated fruit RELATIVE to the mutagenicity of organically grown fruit. It sounds like your hypothesis might be that organic fruit will not be mutagenic, while the other two might be, but you should think about expanding your controls, on the off chance that organic fruit turns out to have mutagenic capacity as well. Plants have to protect themselves from pests somehow, and some of them contain mutagens.

The obvious negative control is to add nothing, so that you can determine the basal mutation rate for the organism in your test. You might want to include a positive control as well, using a compound that you know to be mutagenic. You probably have several compounds in your lab at school that are known to be mutagenic; a review of the Materials Saftey Data Sheet (MSDS) for the compounds in lab should help you find them. You should use proper safety precautions of course. In the biochem lab that I TAed in grad school, we had students bring in common household items, and use the Ames Test to determine which of them were mutagenic, and by how much. The winner? Well-done hamburger!

Here are some primary references for the Ames Test. You can find many related references by using the NCBI's PubMED database.

Ames BN, Lee FD, and Durston WE (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proceedings of the National Academy of Sciences USA, 70: 782–786.

Ames BN and McCann J (1976) Detection of carcinogens as mutagens in the Salmonella/ microsome test: Assay of 300 chemicals: Discussion. Proceedings of the National Academy of Sciences USA,73: 950-954.