2. mutagens  Humans and other animals are surrounded by a variety of chemical substance both naturally occurring as well as synthetic, that have the potential to act as mutagens. Some of these substances are in the food we eat, others in the air we breathe, and still others can be absorbed through the skin or via other contact. DNA base sequence  Mutagens act in a variety of ways but they all have the ability to alter the DNA base sequence (e.g. recall point mutations, frameshift mutations, etc.) within the genome. most (though not all) mutagens have the potential to act as carcinogens  Cancer researchers and clinical oncologists would likely agree that most (though not all) mutagens have the potential to act as carcinogens and can play a role in the induction of neoplastic cell growth seen in many cancers.

3. Ames test  The Ames test is a biological assay to assess the mutagenic potential of chemical compounds.  A positive test indicates that the chemical might act as a carcinogenic (although a number of false- positives and false-negatives are known).  The test also serves as a quick assay to estimate the carcinogenic potential of a compound since it is difficult to ascertain whether standard carcinogen assays on rodents were successful.

4. Bruce Ames  The procedure is described in a series of papers from the early 1970s by Bruce Ames and his group at the University of California, Berkeley.  Bruce Ames and his undergraduate students tested large numbers of commercial products in student labs at UC Berkeley when the test was first introduced in the 1970s.  Many common items such as hairspray and food colours were discovered to be mutagenic and were withdrawn from the market. Ames also established that many mutagenic compounds are also carcinogenic.

5. Salmonella typhimurium histidine auxotrophic  The test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis i.e. it is an auxotrophic mutant, so that they require histidine for growth.  The variable being tested is the mutagen's ability to cause a reversion to growth on a histidine-free medium.

6.  The disk of filter paper has been impregnated with 10µg of 2-aminofluorene, a known carcinogen.  The mutagenic effect of the chemical has caused many bacteria to regain the ability to grow without histidine, forming the colonies seen around the disk. The scattered colonies near the margin of the disk represent spontaneous revertants.

7. lipopolysaccharide  The tester strains also carry mutations in the genes responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable, and in the excision repair system to make the test more sensitive.

8. benzo[a]pyrene  Rat liver extract is optionally added to simulate the effect of metabolism, as some compounds, like benzo[a]pyrene, are not mutagenic themselves but their metabolic products are.  sodium nitrate (NaNO 3 ), HCl nitrous acid (HNO 2 ),  sodium nitrate (NaNO 3 ), which occurs naturally in smoked meat such as bacon, hot dogs, ham, etc., is not itself mutagenic, too. However, when acted upon by HCl in the stomach,it is converted to nitrous acid (HNO 2 ), which has been demonstrated to be a powerful mutagen by the Ames Test.

9.  The bacteria are spread on an agar plate with a small amount of histidine. This small amount of histidine in the growth medium allows the bacteria to grow for an initial time and have the opportunity to mutate.  When the histidine is depleted only bacteria that have mutated to gain the ability to produce its own histidine will survive.

11.  The plate is incubated for 48 hours.  The mutagenicity of a substance is proportional to the number of colonies observed.

12.  A large number of chemicals used in agriculture and industry give a positive Ames test. Examples are ethylene dibromide (EDB), which is added to leaded gasoline (to vaporize lead deposits in the engine and send them out the exhaust), and ziram, which is used to prevent fungus disease on crops. Some drugs, such as isoniazid (used to prevent tuberculosis) are mutagenic in this test.  The potency of AF-2, a food additive once widely- used in Japan, and safrole, a flavoring agent that used to be added to root beers, caused them to be banned. Saccharin, suspected by some of being a carcinogen, does not give a positive Ames test.

13.  Although most testing has been done on products of the chemical industry, many naturally-occurring substances (like safrole) have been shown to be mutagenic. These include aflatoxin, produced in moldy grain and peanuts and present in peanut butter at an average level of 2 parts per billion, PPB.  Traces of nine different substances that give positive Ames tests have been found in fried hamburger.

14.  As Salmonella is a prokaryote, it is not a perfect model for humans. An adapted in vitro model has been made for eukaryotic cells, for example yeast structure.  The original test also doesn't count for metabolites that are formed by in the hepatic system. Modified tests can include liver S9 fraction to help recreate the system and observe whether the parent molecule's metabolites formed in the hepatic system are positive.  Further tests would be needed to determine the specific metabolite that causes a positive Ames to further any drugs development.

15.  Drugs that contain the nitrate moiety sometimes come back positive for Ames when they are indeed safe. Nitroglycerin is an example that gives a positive Ames yet is still used in treatment today.  The conditions of the Ames test are dosed at very high concentrations and with nitrate compounds that can potentially generate nitric oxide (NO), an important signal molecule, will give a false positive.  Long toxicology and outcome studies are needed with such compounds to disprove a positive Ames test.

16.  Salmonella typhimurium is a bacterium and thus not a perfect model of the human body (which is why liver enzymes are added to the test).  Rapid in vitro cells grown in culture  Rapid in vitro tests modeled on the Ames test have been adapted for some eukaryotic cells such as yeast and mammalian cells grown in culture. recombinant DNA technology  And thanks to recombinant DNA technology, it is now possible to combine the advantages of rapid in vitro tests like the Ames test with the more realistic conditions of long-term studies in whole animals.

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