mutagen is a physical or chemical agent that permanently changes genetic material, usually DNA, in an organism and thus increases the frequency of mutations above the natural background level. As many mutations can cause cancer in animals, such mutagens can therefore be carcinogens, although not all necessarily are. All mutagens have characteristic mutational signatures with some chemicals becoming mutagenic through cellular processes. The process of DNA becoming modified is called mutagenesis. Not all mutations are caused by mutagens: so-called "spontaneous mutations" occur due to spontaneous hydrolysis, errors in DNA replication, repair and recombination. Discovery The first mutagens to be identified were carcinogens, substances that were shown to be linked to cancer. Tumors were described more than 2,000 years before the discovery of chromosomes and DNA; in 500 B.C., the Greek physician Hippocrates named tumors resembling a crab karkinos (from which the word "cancer" is derived via Latin), meaning crab.[1] In 1567, Swiss physician Paracelsus suggested that an unidentified substance in mined ore (identified as radon gas in modern times) caused a wasting disease in miners,[2] and in England, in 1761, John Hill made the first direct link of cancer to chemical substances by noting that excessive use of snuff may cause nasal cancer.[3] In 1775, Sir Percivall Pott wrote a paper on the high incidence of scrotal cancer in chimney sweeps, and suggested chimney soot as the cause of scrotal cancer.[4] In 1915, Yamagawa and Ichikawa showed that repeated application of coal tar to rabbit's ears produced malignant cancer.[5] Subsequently, in the 1930s the carcinogen component in coal tar was identified as a polyaromatic hydrocarbon (PAH), benzo[a]pyrene. Polyaromatic hydrocarbons are also present in soot, which was suggested to be a causative agent of cancer over 150 years earlier.

1. INTRODUCTION – mutagen and isolation of mutant
⚫ Mutagens are the known agents either physical,
chemical or biological cause mutations by altering the
genotype or gene expression which results in genetic
abnormality.
Not all mutations are caused by mutagens only Induced
mutations were caused by mutagens.
Spontaneous mutations are naturally occurring mutations.
Mutagens causing cancer, are likely to be known as
Carcinogens.
Discovery - The first mutagens to be identified were
Carcinogens.
The mutagenic property of mutagens was first
demonstrated in 1927 by Muller, he discovered that X-
rays can cause genetic mutations in fruit flies.Edger
Altenburg also demonstrated theMutational effect of UV
radiation in 1928.
Charlotte Auerbach and J.M Robson found that mustard
gas can cause mutations in fruit flies.
EFFECTS- At chromosomal level mutagens alter the
structure or number of chromosomes,

2. as deletion, duplication, insertion, translocation,
monosomy and nondisjunction are some of the
chromosomal abnormalities caused by mutagens.
Teratogens- are class of the mutagens which causes
congenital malformations. Eg: X-rays, valporate,
toxoplasma.
Carcinognes -are the class of mutagens which Induces
tumour formation and thus cause cancer.Carcinogenic
agents include X-rays/ UV rays,Aflatoxins, retrovirus etc.
Clastogens -are the class of mutagens responsible for
chromosomal-breakage, deletion, duplication and
rearrangements.
UV rays, bleomycins, HIV virus are common type of
clastogens.
Mutagens also alters the codon, deletes bases, alters
bases, breaks hydrogen bonds, phosphodiester bonds or
changes gene expression.
TYPES OF MUTAGENS
Three different types of common mutations are observed
in nature physical, chemical & biological agents.
Many mutagens are not mutagenic by themselves.
But can form mutagenic metabolites through cellular

3. Process. Such mutagens are called promutagens.
 Physical agents:
Heat and radiation Chemical agents:
 Base anlogues
Intercalating agents
Alkylating agents
Deaminating agents
Metal ions
Biological agents:
Viruses
Bacteria
Transposons.
PHYSICAL MUTAGENS
 RADIATION:
The first mutagenic agent reported in 1920.
ii. Radiations directly damage the DNA or nucleotide
structure which might be either lethal or sub lethal
Radiation causes cross linking of DNA or protein,
chromosomal break, stand break or loss of chromosomes.

4. Iv. At the molecular level it causes deletion of bases or
DNA strand bases.
X-RAY RADIATION: X-rays are one of the most
common types of ionizing radiation used in many of the
medical practices.
At molecular level the lethal dose of X-ray (350 -
500rems)breaks phosphodiester bonds between
DNA and thus results in strands breakage. It causes
multiple strand breakage. If strand breakage occurs in
both the strands, it becomes lethal for cell.
UV-rays: It is non ionizing type of radiation having less
energyIn it.
The major causes of UV radiation are base deletion
Strand breakage, cross linking and generation of
nucleotide dimers. UV induced mutations are dimer
formation, thymine Thymine dimer & thymine-cytosine
dimers are
Commonly formed as the lesions block replication as
Well as transcription.
Formation of pyrimidine dimerization cause distortion in
structure of DNA and prevents Formation of replication
fork.
CHEMICAL MUTAGENS

5. BASE ANALOGS: They are similar to the bases of
DNA-purine and pyrimidines or structurally resemble the
DNA bases.
Bromouracil and aminopurine are two common base
analogs incorporated in DNA instead of normal Bases.
Bromouracil pairs with adenine as like the thymine And
causes mutation.
Aminopurines cause AT to GC or GC to AT transition
During replication.
ALKYLATING AGENTS:
Mutagens, types of mutations AnuKiruthika
Ethylnitrosourea, mustard gas and vinyl chloride are
common alkylating agents, add alkyl group to the DNA
and damages it. The agents cause base pairing errors by
increasing
Ionization and produces gaps in DNA strand.
Alkylated purine bases are removed by the
Phenomenon called depurination.
Common alkylating agents are: Methylhydrazine,
Temozolomide, Busulfan, Thio-TEPA, Dimethyl
sulphate, Ethyl ethane sulphate.
 INTERCALATING AGENTS:

6. Ethidium bromide, proflavine, acridine orange are
Few intercalating agents. The molecules intercalate
between the bases od
DNA & disrupt its structure.
If incorporated during replication, it can cause Frameshift
mutation. It may also block transcription.
 METAL IONS: Nickel, chromium, cobalt, cadmium
are common metal ions that cause mutations.
 The metal ions work by producing ROS(reactive
oxygen species), hindering DNA repair pathway.
Cause DNA hypermethylation or may directly
damage DNA.
 OTHER CHEMICAL MUTAGENS:
ROS, benzene, synthetic rubber and rubber products,
sodium azide, aromatic amines, deaminating agents etc
are other mutagens which create different mutations
BIOLOGICAL MUTAGENS
Viruses, bacteria and transposon are biological mutagens.
VIRUS: Virus insert their DNA into our genome and
disrupt the normal function of DNA or a gene.
Once it inserts DNA, the DNA is replicated transcribed
and translated viral protein instead of our own protein.
Eg: HIV.

7. BACTERIA: Some bacteria can cause inflammation. It
provokes DNA damage and DNA breakage.
TRANSPOSONS: They are non coding DNA sequences,
jumps from one place to another in a genome and
influence function of genes,
ISOLATION OF MUTANTS:
Mutation occurring in microorganism can be Detected
and efficiently isolated from the parent organism of other
mutants. While studying we must be aware of wild type
characters of an organism,so the mutants can Easily
detected.
 In bacteria and other haploid microorganism, the
detection system are straight forward because any
new allele should be observed immediately.
 In albino mutation, the detection is very simple. It
requires only change in colour of bacterial colony.
The other detection systems are rather complex.
SOME DETECTION METHODS
 Replica plating technique. Resistance selection
method.
 Substrate utilization method.
 Ames method
REPLICA PLATINGTECHNIQUE:

8.  Joshua and Esther Ledgerberg (1952) developed a
New technique called replica plating. This technique
is used to detect auxotrophic mutants and wild type
strains on the basis of ability to grow in the absence
of amino acids.
 Also this test is used to demonstrate the presence of
antibiotic resistance in bacterial cultures prior to
exposure of antibiotic
STEPS INVOLVED
 Generate the mutants by treating a culture with a
mutagen e.g.nitrosoguanidine. Inoculate a plate
containing complete growth medium and incubate it
at proper temperature. Both Wild type and mutant
survivors will from complete medium
 This plate containing complete medium is called
master plate.
 Prepare a piece of sterile velvet and gently on the
upper surface of the master plate to pick up bacterial
cell from each colony.
 As pressed the master plate, again gently press the
velvet on the replica plates containing complete
medium in one set and lacking cine in only leucine in
the other set. Thus, the bacterial cells are transferred
in replica plates in the same position as in master
plate. Incubate the plates and compare the replica

9. plate with master plate for bacterial colony not
growing on replica plate..
2.RESISTANCE SELECTION METHOD
This is another method used for isolation of Mutants.
Generally the wild the wild type cells not resistant either
to antibiotics or bacteriophage. • Therefore, it is possible
to grow the bacterium in the Presence of agent.
 This method is applied for isolation of mutants
resistant to chemical compounds that can be
amended in agar, phage resistant mutants.
3.SUBSTRATE UTILIZATION METHOD:
 This method is employed in the selection of bacteria.
Several bacteria utilize only a few carbon sources.
The cultures are plated on to medium
containingAlternate carbon sources.
 Any colony that grows on medium can use the
Substrate and are possibly mutants. These can be
Isolated.
 Sugar utilization mutants are also isolated by Means
of color indicator plates
 . EMB medium is used for this purpose.This medium
contain lactose sugar as carbonSource and complete
mixture of amino acids.

10.  Therefore both lactose wild type and lactose mutant
cells can grow and form colonies on EMB agar
plates.
 The lac+ cells catabolize lactose and secrete acids,
therefore the pH of the medium decreases. This will
result in staining of colony to dark purple.
 On the other hand, Lac- cells are unable to utilize
Lactose and use some of the amino acids as carbon
Source.
 After utilization of amino acid, ammonia is produced
that increases the pH and de colorize the dye
resulting in white colony.
Ames test In 1974 Bruce Ames
 developed a method for evaluating the potential of
chemical to cause cancer, known as Ames test.
 Ames test is based on the principle that both Cancer
and mutations results from the damage of DNA, and
results of experiments have Demonstrated that 90%
of known carcinogen areAlso mutagens.
 Several species of salmonella typhimurium are
employed. Each strain contains a different mutation
in the operon histidine biosynthesis.
STEPS OF AMES TEST:

11.  Prepare the culture of Salmonella histidine
Auxotrophs (His-).
 Mix the bacterial cells and test substance( mutagen)
in dilute molten top agar with a small amount of
histidine in one set, and control with cmplete
medium plus large amount of histidine.
 Pour the molten mix on the top of minimal agar
plates and incubate at 37°C for 2-3 days.
 Until histidine is depleted all the His- cells will grow
In the presence of test mutagen.
 When the histidine is completely exhausted only the
revertants will grow on the plate. 。
 The number of spontaneous revertants is low,
Whereas the number of revertant induced by
Carcinogen is quite high.
 High number of colonies represent the greater
Mutagenicity.
 A mammalian liver extract is added to the above
Molten top agar before plating. The extract converts
the carcinogen in to electrophilic derivatives which
will soon react with DNA molecule.
 In natural way it is occurs in mammalian system
When foreign particle are metabolized in the liver.
Bacteria does not have metabolizing capacity,

12. therefore, the liver extract is added to this test, to
promote transformation.