Transcript Ames test

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.