In this article we will discuss about the chemical and physical types of mutagens.
1. Chemical Mutagens:
Singer and Kusmierek (1982) have published an excellent review on chemical mutagenesis.
Some of the chemical mutagens and mutagenesis are given in Table 9.3, and described below:
i. Base Analogues:
A base analogue is a chemical compound similar to one of the four bases of DNA. It can be incorporated into a growing polynucleotide chain when normal process of replication occurs.’ These compounds have base pairing properties different from the bases. They replace the bases and cause stable mutation.
A very common and widely used base analogue is 5-bromouracil (5-BU) which is an analogue of thymine. The 5-BU functions like thymine and pairs with adenine (Fig. 9.6A).
The 5-BU undergoes tautomeric shift from keto form to enol form caused by bromine atom. The enol form can exist for a long time for 5-BU than for thymine (Fig. 9.6B). If 5-BU replaces a thymine, it generates a guanine during replication which in turn specifies cytosine causing G: C pair (Fig. 9.6A).
During the replication, keto form of 5-BU substitutes for T and the replication of an initial AT pair becomes an A: BU pair (Fig. 9.7A). The rare enol form of 5-BU that pairs with G is the first mutagenic step of replication. In the next round of replication G pairs with C. Thus, the transition is completed from AT→GC pair.
The 5-BU can also induce the conversion of GC to AT. The enol form infrequently acts as an analogue of cytosine rather than thymine. Due to error, GC pair is converted into a G: BU pair which in turn becomes an AT pair (Fig. 9.7B). Due to such pairing properties 5-BU is used in chemotherapy of viruses and cancer. Because of pairing with guanine it disturbs the normal replication process in microorganisms.
The 5-bromodeoxyuridine (5-BDU) can replace thymidine in DNA molecule. The 2-amino-purine (2-AP) and 2, 6-di-amino-purine (2, 6-DAP) are the purine analogues. The 2-AP normally pairs with thymine but it is able to form a single hydrogen bond with cytosine resulting in transition of AT to GC. The 2-AP and 2, 6-DAP are not as effective as 5-BU and 5-BDU.
ii. Chemicals Changing the Specificity of Hydrogen Bonding:
There are many chemicals that after incorporation into DNA change the specificity of hydrogen -bonding. Those which are used as mutagens are nitrous oxide (HNO2), hydroxylamine (HA) and ethyl-methane-sulphonate (EMS).
(a) Nitrous Oxide (HNO2):
Nitrous oxide converts the amino group of bases into keto group through oxidative deamination. The order of frequency of deamination (removal of amino group) is adenine > cytosine > guanine.
(b) Deamination of Adenine:
Deamination of adenine results in formation of hypoxanthine, the pairing behaviour of which is like guanine. Hence, it pairs with cytosine instead of thymine replacing AT pairing by GC pairing (Fig. 9.8A).
(c) Deamination of Cytosine:
Deamination of cytosine results in formation of uracil by replacing – NH2 group with -OH group. The affinity for hydrogen bonding of uracil is like thymine; therefore, C-G pairing is replaced by U-A pairing (Fig. 9.8B).
(d) Deamination of Guanine:
Deamination of guanine results in formation of xanthine, the later is not mutagenic. Xanthine behaves like guanine because there is no change in pairing behaviour. Xanthine pairs with cytosine. Therefore, G-C pairing is replaced by X-C pairing.
(e) Hydroxylamine (NH2OH):
It hydroxylates the C4 nitrogen of cytosine and converts into a modified base via deamination which causes to base pairs like thyamine. Therefore, GC pairs are changed into AT pairs.
iii. Alkylating Agents:
Addition of an alkyl group to the hydrogen bonding oxygen of guanine (N7 position) and adenine (at N3 position) residues of DNA is done by alkylating agents. As a result of alkylation, possibility of ionization is increased with the introduction of pairing errors. Hydrolysis of linkage of base-sugar occurs resulting in gap in one chain.
This phenomenon of loss of alkylated base from the DNA molecule (by breakage of bond joining the nitrogen of purine and deoxyribose) is called depurination. Depurination is not always mutagenic. The gap created by loss of a purine can effectively be repaired.
Following are some of the important widely used alkylating agents:
(a) Dimethyl sulphate (DMS)
(b) Ethyl methane sulphonate (EMS) -CH3CH2SO3CH3
(c) Ethyl ethane sulphonate (EES) -CH3CH2SO3CH2CH3
EMS has the specifity to remove guanine and cytosine from the chain and results in gap formation. Any base (A,T,G,C) may be inserted in the gap. During replication chain without gap will result in normal DNA. In the second round of replication gap is filled by suitable base.
If the correct base is inserted, normal DNA sequence will be produced. Insertion of incorrect bases results in transversion or transition mutation. Another example is methyl nitrosoguanidine that adds methyl group to guanine causing it to mispair with thyamine. After subsequent replication, GC is converted into AT transition.
iv. Intercalating Agents:
There are certain dyes such as acridine orange, proflavine and acriflavin which are three ringed molecules of similar dimensions as those of purine pyrimidine pairs (Fig. 9.9). In aqueous solution these dyes can insert themselves in DNA (i.e. intercalate the DNA) between the bases in adjacent pairs by a process called intercalation.
Therefore, the dyes are called intercalating agents. The acridines are planer (flat) molecules which can be intercalated between the base pairs of DNA; distort the DNA and results deletion or insertion after replication of DNA molecule. Due to deletion or insertion of intercalating agents, there occur frameshift mutations (Fig. 9.10).
2. Physical Mutagens:
i. Radiations as Mutagens:
Radiation is the most important among the physical mutagens. Radiations damaging the DNA molecules fall in the wavelength range below 340 nm and photon energy above 1 electro-volt (eV). The destructive radiation consists of ultraviolet (UV) rays, X-rays, ү-rays, alpha (α) rays, beta (β) rays, cosmic rays, neutrons, etc. (Fig. 9.11).
Radiation induced damage can be categorized into the three broad types: lethal damage (killing the organisms), potentially lethal damage (can be lethal under certain ordinary conditions) and sub-lethal damage (cells do not die unless radiation reaches to a certain threshold value). The effect of damage is at molecular level.
In a live cell radiation damage to proteins, lipoproteins, DNA, carbohydrates, etc. is caused directly by ionization/excitation, or indirectly through highly reactive free radicals produced by radiolysis of cellular water. DNA stores genetic information’s so a damage to it assumes great dimension. It can perpetuate genetic effects and, therefore, the cellular repair system is largely devoted to its welfare.
When the bacteria are exposed to radiation they gradually lose the ability to develop colonies. This gradual loss of viability can be expressed graphically by plotting the surviving colonies against the gradually increasing exposure time. This dose-response graph is called survival curve. The survival curve of bacteria is given in Fig. 9.12. The survival curve is analysed by a simple mathematical theory called hit theory.
Each organism possesses at least one sensitive site which is known as target site. Radiation photons (particles of light) damage or hit the target site and inactivate the organisms. One can derive the equation based on this theory.
The equations help to calculate the survival curve for many kinds of populations of N identical organisms exposed to dose D of radiation causing damage. The number dN damaged by a dose dD is proportional to the initial population that has not received radiation; hence dN = KN
K is the constant which measures the effectiveness of dose.
Integrating this equation from N = No at D = O we get
N = Noe-KD …(1)
The surviving fraction S = N/No is
S = N/NO = e-KD …(2)
A plot of S virus D gives a straight line with a slope of -K (Fig. 9.12). This type of curves are called exponential or single hit curve. The exponential curve is obtained when the phages are irradiated with X-rays.
If there is a population of different organisms, and each organism consists of at least n sites, each site must be hit to inactivate an organism. Therefore, each organism is hit by n times. The probability of one unit being hit by a dose D is, P = 1-e-KD), so the probability of Pn will be Pn = (1-e-KD)n
The surviving fraction S of the population is 1-pn or S = 1-(1-e-KD)n …(3)
This equation can be expanded as:
S = 1- (1-ne-KD + e-nKD)
At the large value of D, the higher order terms become negligible as compared to Therefore, at high dose D,
S = ne-KD or
In S = In n – KD …(4)
When the equation 3 is plotted for K – 1, various values of n reveals that for small values of D, In S gradually changes (Fig. 9.13). At large value of D, equation 4 dominates and curve becomes linear.
ii. Ultraviolet (UV) Radiation:
UV radiation causes damage in the DNA duplex of the bacteria and phages. The UV rays are absorbed and cause excitation of macromolecules. The absorption maxima of nucleic acid = (280 nm) and protein (260 nm) are more or less similar. The DNA molecule is the target molecule for UV rays but not the proteins. However, absorption spectrum of RNA is quite similar to that of DNA.
The excited DNA leads to cross-linking, single strand breaks and base damage as minor lesion and generation of nucleotide dimer as a major one. Purines are generally more radio – resistant than the pyrimidine of the latter, thymine is more reactive than cytosine.
Hence, the ratio of thymine-thymine (TT), thymine-cytosine (TC), cytosine-cytosine (CC) dimer (Fig. 9.14) is 10:3:3, respectively. A few dimers of TU and UU also appear. The initial step in pyrimidine dimerization is known to be hydration of their 4: 5 bonds.
Formation of thymine-thymine (TT) dimer causes distortion of DNA helix because the thymines are pulled towards one another. The distortion results in weakening of hydrogen-bonding to adenines in the opposing strand. This structural distortion inhibits the advance of replication fork.
iii. The X-Rays:
The X-rays cause breaking of phosphate ester linkages in the DNA. This breakage occurs at one or more points. Consequently, a large number of bases are deleted or rearranged in the DNA molecule.
The X-rays may break the DNA either in one or both strands. If breaks occur in both strands, it becomes lethal. The DNA segment between the two breaks is removed resulting in deletion. Since both the X-rays and UV rays bring about damage in DNA molecule, they are used in sterilization of bacteria and viruses.