Ames Test - Detecting Mutagenicity; and Ethidium Bromide as a case study...

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The Ames Test was developed in the 1970s by Bruce Ames.
This test is a gold standard to determine the mutagenicity of substances.
It is very valuable in the prelinimary studies of potentially carcinogenic and mutagenic compounds.

Mutant strains of the bacterium Salmonella typhimurium (or E.coli) that are histidine auxotrophs (his-), i.e. cannot synthesise histidine from media components and therefore require incorporation of histidine into the media in order to grow, are used.
Apart from the his- mutation, these bacteria also carry mutations that render DNA repair mechanisms defective and cell wall synthesis inefficient. These serve to make the cells highly sensitive to mutation.

The bacteria are subjected to addition of the compound thought to be a mutagen, and then assayed on his- media. The frequency of reverse mutations (resulting in cells that do survive on histidine deficient media) are counted.
This frequency is compared with frequency of spontaneous reverse mutations.
An increase in the number of revertants indicates mutagenicity.

Now, the mammalian liver has several enzymes which bacteria do not.
It is quite possible that these enzymes can act on a harmless substance and convert it to a mutagen.
So, in the procedure of Ames test, extract of rat liver is also added along with the suspected mutagen.

Some Interesting Data from Ames Test ...
SubstanceNo. of Revertants
Naphthalene0
5 mcg Ethidium Bromide1012
condensate from smoke of 1 cigarette18200

For a biotechie who might be reading my blog, we use EtBr routinely in labs right?
A 100ml agarose gel, run with 0.5mcg/ml EtBr may give fewer revertants than one cigarette. But its a fact that during our undergrad course and during research, we handle not one but several gels and we dont keep a count.
A once in a lifetime injection of say, 5mg EtBr may not give you cancer, but large amounts accumulated over extended period of time may be undesirable.

Thats the reason why people tell us to be damn carefull, wear gloves, etc.

References:
http://forums.biotechinstitute.org/showthread.php?t=87
Environmental Biotechnology - Scragg.

For interested people:
http://wps.prenhall.com/wps/media/objects/1143/1171405/15_3.html - animation on Ames test.

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