2. Contents
• Introduction
• General procedure
• Strains used by Ames
• Use of liver homogenate
• General procedure
• Importance of Ames test
• Limitations of Ames test
3. Introduction
• Mutagens are agents which causes mutation.
• Many chemical mutagens are found to be carcinogenic
• The Ames test (Salmonella typhimurium reverse mutation assay) is
a bacterial short-term test for identification of carcinogens using
mutagenicity in bacteria as an end point.
• All carcinogens are mutagens but some mutagens are not
carcinogens.
• The Ames test was described by Bruce Ames and his group at the
University of California, Berkeley in a series of papers in the 1970s
4. General procedure
• Bacteria (Salmonella typhimurium) is used as a test organism –
cheap and easy to culture.
• Auxotrophic mutant strain of Salmonella typhimurium is used
which lacks genes for histidine biosynthesis and a mutation for
excision repair system.
• Cells in which a back mutation occurs due to the test compound
grows and produce colony.
• Small amount of histidine is added for initial growth
5. • Rat liver extract is added to the medium which provides activation
by cellular enzymes.
• It permit identification of substances that are not directly
mutagenic (or carcinogenic) but are converted into mutagens by
the enzymatic reactions that take place in the liver of animal
6.
7. Strains used by Ames
The deletion through the uvrB region of the chromosome
eliminates the excision repair system for DNA.
The gal and rfa (deep rough) mutations eliminate, to
different extents, the polysaccharide side chain of the LPS
that coats the bacterial surface, making the bacteria more
permeable and completely nonpathogenic.
The TA1535 set (TA1535, TA1536, TA1537, TA1538), which
is rfa and uvrB, is recommended for general testing for
mutagens and carcinogens in vitro, as it is the most
sensitive to mutagenesis.
The TA1975 set is used for examining the effect of repair
on mutagenesis and killing (in comparison to the TA1535
set).
The TA1950'set and TA1530-set are less sensitive to
mutagens in vitro, but may be required for use in the host
mediated assay in which Salmonella strains are incubated
in the peritoneum of a mouse or rat
8. Use of liver homogenates
The active forms of carcinogens such as aflatoxin, polycyclic hydrocarbons,
dimethyl nitrosamine, and various aromatic amines are formed by
mammalian metabolism, in particular by the TPNH-dependent
microsomal enzymes of liver.
The principal limitation of any bacterial system for detecting carcinogens
as mutagens is that bacteria do not duplicate mammalian metabolism in
activating carcinogens. Mammalian-liver homogenates have been used by
Garner et al., to activate aflatoxin B1 to a compound lethal to our bacterial
tester strain lacking excision repair, by Malling to activate dimethyl
nitrosamine to a compound that reverts one of our bacterial tester strains,
and by Slater et al., to activate dimethyl nitrosamine to a compound lethal
for bacteria lacking polymerase I.
9. Preparation of liver homogenate
• Source of Liver
• Male rats (Sprague-Dawley/Bio-1 strain, Horton Animal Laboratories) were maintained on
Purina laboratory chow. A week before they were killed, their drinking water was made 0.1%
in sodium phenobarbital . The rats (250-500 g) were killed by a blow to the head and
cervical dislocation; the liver was removed and placed in a sterile, ice-cold beaker.
• A portion of human liver was obtained from an autopsy of a 77-year-old man who had died 7
hour earlier of heart failure.
• Preparation of Liver Homogenate Fraction "S-9“
• We have used the procedure of Garner et al.,. All steps were performed at 0-4⁰ with cold
and sterile solutions and glassware. The liver (rat livers were 10-25 g each) was washed in
an equal volume of 0.15 M KCl, minced with sterile scissors in three volumes of 0.15 M KCl
(3 ml/g of wet liver), and homogenized with a Potter-Elvehjem apparatus with a Teflon
pestle. The homogenate was centrifuged (Sorvall RC2-B) for 10 min at 9000 X g, and the
supernatant, which we call the S-9 fraction, was decanted and saved. 1 ml of S-9 fraction
contained microsomes from 250 mg of wet liver; the protein concentrations were fairly
constant from preparation to preparation except for the human S-9 fraction which was
about half, perhaps due to difficulties in homogenization because of its fibrous nature.
• The fresh S-9 fractions (rat and human) were distributed in 2-ml portions in small plastic
tubes (2-ml liquid nitrogen storage tubes/4-Shore-USA, La Jolla, Calif.), quickly frozen in
dry ice, and stored at -80° in a Revco freezer. As required, sufficient S-9 fraction was
thawed (at room temperature) and kept in ice; the unused portion was discarded at the end
of the day.
Preparation of liver homogenate
10. Test procedure
• Addition of S-9 mix to the top agar the S-9 Mix contains per ml: 0.3 ml of S-9
fraction, 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM TPN
(triphosphopyridine nucleotide), and 100 mM sodium phosphate (pH 7.4).
• To 2 ml of molten top agar at 45⁰C are added 0.1 ml of the bacterial tester
strain culture (2 to 3 X 109/ml), up to 0.1 ml of a solution (Me2SO or water) of
the compound to be tested, and 0.5 ml of S-9 Mix
• Then the tube is rotated quickly and the contents are poured on the agar plate.
The additions and pouring should take less than a minute. The colonies on the
plates (his+ revertants) are counted after a 2-day incubation at 37°.
14. Importance of Ames test
• Compared to the carcinogenic assays on mice and rats (takes few years) the Ames test
is quick and inexpensive.
• Used in wide variety of fields such as pharmaceutical, chemical, fertilizer, cosmetics,
food and many more industries for testing chemicals for mutagenicity and
carcinogenicity.
• In 1972-77 a compound known as Tris-BP was used as a flame retardant in children’s
polyester pyjamas which was later found to be a potential mutagen in Salmonella and
Drosophila
• And it also interacts with human DNA and damages mammalian chromosomes.
• Before its use was discontinued, more than 50 million children were exposed to the
chemical.
15. Limitations of Ames test
• Salmonella typhimurium is not a perfect model for humans as it is
a prokaryote.
• The sensitivity for some of the known carcinogen is less in the
Ames test.
16. References
1. Ames, B. N. (1971) in Chemical Mutagens: Principles and Methods for their Detection,
ed. Hollaender, A. (Plenum Press, New York), Vol. 1, pp. 267-282.
2. Ames, Bruce N., Frank D. Lee, and William E. Durston. 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
3. Ames, Bruce N., William E. Durston, Edith Yamasaki, and Frank D. Lee. 1973.
“Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for
Activation and Bacteria for Detection.” Proceedings of the National Academy of
Sciences, USA 70: 2281–85.
4. Essential Genetics- a genomics perspective Daniel L. Hartl
5. Genetics Analysis & principles- Robert J. Brooker
