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Detecting Mutagens and Carcinogens. introduction - Increased number of chemicals used and present as environmental contaminats, testes for the mutagenicity of these substances become important. - Mutagenicity provides an initial screening for these hazardous agents.
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Detecting Mutagens and Carcinogens
introduction - Increased number of chemicals used and present as environmental contaminats, testes for the mutagenicity of these substances become important. - Mutagenicity provides an initial screening for these hazardous agents. - One simple method for screening large numbers of substances for mutagenicty is areversion test.
Simple tests - Using auxotrophic mutants of bacteria. - Potential mutagen is added to solid growth media. - Known numbers of mutant bacterium are plated. - The number of revertant colonies that arise is counted. - Simple testes of this type fail to demonstrate the mutagenicity of an number of potent carcinogenic . - Why??
- Some substances are not directly mutagenic (carcinogenic), but are converted to active compounds by enzymatic reactions that occur in the liver of animals. - The enzymes are contained in acomponant of liver cells called “microsomal fraction”. - Addition of microsomal fraction of the rat liver to growth medium as activation system has been used to extend the sensitivity and usfulness of the reversion test system. - The use of microsomal is the basis of the “Ames test” for carcinogens.
Ames test - Histidine requiring (His-) mutants of the bacterium Salmonella typhimurium are used to test for reversion to (His+). - The bacterial strains have been made more sensitive to mutagenesis by the incorporation of mutations that inactivate the repair system and make the cells more permeable to foreign molecules.
- Rat-liver Microsomal fraction is spread on the agar surface and bacteria are plated. - Paper disc saturated with distilled water (as control) is placed in the center of the plate. - Paper saturated with solution of the compound being tested is placed in the center of the plate. - The test compound diffuses outward from the disc. - If substance is amutagen colonies form. Highly effective mutagen, colonies will be present all over the surface of the medium. Weak mutagen, colonis will form very near the disc. The procedure is highly sensitive and permits the detection of very weak mutagens.
- The Ames test has now been used with thousands of substances and mixtures such as industrial chemicals, food additives, pesticides, hair dyes and cosmetics. - As a result of these tests many industries have refer mutated their products for example changed the formulation of many hair dyes and cosmetics. - A few percent of more than 300 substances known from animal experiments to be carcingens failed to increase the reversion frequency in the Ames test.
Dovert test - Using of lamda phage to screen for carcinogens - Dovert test utilizes E.coli strains lysogenic for phage lamda. - Many carcinogens cause DNA damage and are inducing agent. - The induction can be detected quite simply.
- One lysogenic cell is mixed with 108 bacteria and put on agar (as plaque). - The lysogen which is not induced by agent forms microcolony in the bacterial lawn. - In the late development of the colony a cell will be induced. - This not usually produce plaque because depletion of nutrients in the agar limits cell growth, so released phage cant multiply.
- If an inducing agent (carcinogen) is in the agar . - Lysogenic cell will be induced to make phage at the time all of the bacteria in the agar begin to grow, so that a plaque will form. - Effectiveness of inducing agent can be measured by the fraction of cells yielding plaques. - Like the Ames test, uses liver extracts in the agar to activate carcinogens. - Devort test is often more sensitive, requiring lower concentration of carcinogens. - Ames test and devort test are being used to examine a very large number of industrial chemical and food additives.
Using mice to test for chemical mutagens - Salmonella typhimurium, the bacterium used in the Ames test, is a prokaryote and this is not aperfect model of human body. - In fact, some suspected human carcinogens are negative in the Ames test. - Rapid in vitro tests modeld on the Ames test have been adapted for som eukaryotic cells such as yeast and mammalian cells grown in tissue culture.
This photograph shows one mutant (blue) plaque on a lawn of E. coli containing many non-mutant (clear) plaques.
Rec Assay - bacterial DNA damage or repair tests measure DNA damage which is expressed as cell killing or growth inhibition of repair deficient bacteria in deficient strains. - these tests used as indication of the interaction of chemical with genetic material. - measure differences in chemically induced cell killing between wild type strains with full repair and mutant strains deficient in one or more the enzymes which govern repair of damaged DNA.
Strain selection:- Bacillus subtilis rec(H17/M45). Metabolic activation:- fraction prepared from the liver of rat treated with enzyme inducing agent. Control groups:- 1- negative control:- Chlramphenicol is an example. 2- positive control:- Mitomycin c is an example.
Test method:- 1- test performed on solid medium (diffusion test):- strain of bacteria spread on the surface of the agar and the test chemical placed on a filter disc on the surface of the agar . 2- test performed in liquid culture (suspension test):- bacterial suspensions treated with dose of chemical . Incubation condition:- incubation period should be for 18 -24hr at 37c.
Treatment of results:- 1- diffusion assays:- result should be expressed in diameters of zones of growth inhibition in mm. Suspension assays: determined by the absence of visible growth in liquid medium and the later is determined by plating dilutions onto semisolid media.
