carcinogenecity

1. AMES TEST
K.Krupa sagar
17M71S0105
M.Pharmacy 1st year
Annamacharya college of pharmacy.

2. WHAT IS AMES TEST ?
◦It is a test to determine the
mutagenic activity of chemicals by
observing whether they cause
mutations or NOT

3. ◦ MUTATIONS: any sudden permanent change in
the sequence of DNA
◦ Any agents that cause mutations are called “
MUTAGEN’S.”
◦ Mutagens may be : I) chemical agents
ex:1-4,dichlorobenzene
1-3, dichloro-2 propanol
II) physical agents
ex:
by sun
By microwaves
By X-rays

4. INTRODUCTION
◦ AMES TEST is widely employed method used to
test whether a given chemical can cause
mutations in DNA in test organism.
◦ It is also called “BACTERIAL REVERSE
MUTATION ASSAY”.
◦ It is based on the Principle of back mutation or
reverse mutation.

5. ◦ It is used to estimate the carcinogenic potential
of a compound
◦ There are many standard carcinogen assay's on
mice and rat’s but they are time consuming
(nearly 2-3 yrs) and expensive with false-positive
and false-negative results.
◦ But AMES TEST serves as quick and convenient
assay.

6. HISTORY
◦ AMES test was brought forwarded by
BRUCE AMES in 1970.
◦ He is a professor in biochemistry department in the
University of California.
◦ He developed this method as the previous methods
or assay’s are expensive and time consuming.

7. GENERAL PROCEDURE
◦ Several strains of bacterium Salmonella typhimurium, carries
mutations in genes involving in histidine(amino acid)
synthesis.
◦ The strains are auxotrophic mutants h- (defective organism
or deficiency mutant) i.e., they require histidine for growth
as they cannot produce it.
◦ Ames test tests the capability of the test substance
creating mutations that results in prototrophic state h+ i.e.,
the strains can grow on the histidine free medium.

8. Isolate an auxotrophic S.thypimurium for
histidine
Prepare a test suspension(-H) in a plain
buffer and add small amounts of histidine.
[NOTE :small amount of histidine is
necessary of initial growth of bacteria]
Also prepare (-H) control suspension i.e.,
without test chemicals.
Incubate the suspensions at 37°C for
20mins.

9. Spread the suspensions on
two different agar plates.
Again incubate the plates 37°C
for 48hours.
After 48hrs count the colonies
in each plate .the mutagenicity
of chemicals is proportional to
number of colonies observed.

12. ◦ Examples of chemicals that gives positive responses to
Ames test :
 2-aminofluorene
 Ethylene dibromide(EDC)
 Ziram
 Safrole
 Saccharine
 Aflotoxin

13. AMES TEST & CARCINOGENS
◦ Mutagens identified via Ames test are possible carcinogens, early
studies showed 90% of known carcinogens.
◦ Later studies showed 50-70% carcinogens.

14. ◦ The dose response curve obtained in Ames test is almost always
LINEAR ,indicates that there is no threshold concentrations for
Mutagenesis => indicates there may be no safe threshold level for
chemical mutagens or carcinogens.

15. IMPORTANCE
◦ Ames test is one of the test which screens for potential chemical
carcinogens.
◦ Under PESTICIDE ACT (USA), there are 8 tests and Ames test is
one of them.
◦ Similarly, TOXIC SUBSTANCES CONTROL ACT (USA). There are 6
tests and Ames test is also one of them.

16. LIMITATIONS
◦ Salmonella typhimurium is a bacterium and thus not a
perfect model of human body(which is why liver enzymes
are added to the test)
◦ In vitro model of eukaryotic cells has been adapted like
yeast and mammalian cells grown in culture.

18. ◦Why are liver
extracts are used in
AMES TEST ?

19. Why are liver extracts are
used in AMES TEST ?
◦Liver enzymes may
activate some
innocuous compounds,
making them mutagenic

20. ◦WHICH BACTERIA
GROW ON THE
CULTURE PLATE IF
AMES TEST IS POSITIVE?

21. WHICH BACTERIA GROW ON THE
CULTURE PLATE IF AMES TEST IS
POSITIVE?
◦ his+ prototroughs
the bacteria used in the Ames test to evaluate
mutagenicity are his – auxotrophs. If the Ames
test is positive, these bacteria have reverted
back to wild type and are his+ prototrophs.