Classification of Mutagens and Mutagenicity Testing

Mutagenesis
Mutagenesis is a process by which the genetic

information of an organism is changed in a stable (permanent) manner, resulting in a mutation. It may occur spontaneously in nature or as a result of exposure to mutagens. Mutagens: Mutagens are chemical and physical agents that are capable of producing a mutation. Mutagens include agents such as radiation, chemotherapeutic agents, and many carcinogens.

DNA-Damaging Agents

Hydroxylamine Base analogs Alkylating agents Agents that form DNA adducts DNA intercalating agents DNA crosslinkers Oxidative damage

Categories
DNA-damaging agents can be divided into four major

categories. (1) Direct acting. Carcinogens are intrinsically reactive compounds that do not require metabolic activation by cellular enzymes. Examples include Alkylating agents like N-methyl-Nnitrosourea. (2) Indirect-acting Carcinogens require metabolic activation by cellular enzymes to form the ultimate carcinogenic species that covalently binds to DNA. Examples include Dimethylnitrosamine

(3) Radiation and oxidative DNA damage. occur directly or indirectly. Ionizing radiation produces DNA damage through direct ionization of DNA to produce DNA strand breaks or indirectly via the ionization of water to reactive oxygen species that damage DNA bases. Ultraviolet radiation (UVR) from the sun is responsible for skin cancer. Reactive oxygen species can also be produced by various chemicals and cellular process including respiration and lipid peroxidation. (4) Inorganic agents such as arsenic, chromium and nickel are considered.

Background
In

1927, Hermann Muller first demonstrated mutation with observable changes in the chromosomes can be caused by irradiating fruit flies with X-ray and lent support to the idea of mutation as the cause of cancer.

Types of Mutations
(1) Point mutations Single base pair substitutions that can result in amino acid substitutions in the encoded protein and frame-shift mutations involving the loss or gain of one or two base pairs, resulting in an altered reading frame and gross alterations in the encoded protein.

Point mutations are classified Missense: A missense mutation produces an altered protein in which an incorrect amino acid has been substituted for the correct amino acid. Nonsense Mutations A nonsense mutation is an alteration that produces a stop codon and results in a truncated protein.

 A point mutation can also be characterized based on the

mutagen induced substitution of one base for another within the DNA. When a point mutation produces a substitution of a purine for another purine (i.e., guanine for adenine) or a pyrimidine for another pyrimidine (i.e., thymine for cytosine), the mutation is referred as a transition. If a purine is substituted for a pyrimidine, and vice versa (i.e., thymine for adenine or guanine for cytosine), the mutation is referred to as a transversion.

(2) Chromosome aberrations including chromosomal rearrangement such as, deletion duplication inversion translocation (3) Aneuploidy and polyploidy, Which involve the gain or loss of one or more chromosomes

gross

Mechanisms
Mutagenesis may arise as a result of the presence of

environmental mutagens that induces changes to the DNA. The mechanism by which mutation arises varies according to the causative agent and mutagens that are involved

Spontaneous Hydrolysis
DNA is not entirely stable in aqueous solution.

Under physiological conditions the glycosidic bond may be hydrolyzed spontaneously and 10,000 purine sites in DNA are estimated to be depurinated each day in a cell.

Modification of Bases
Bases may be modified endogenously by normal cellular

molecules. For example DNA may be methylated by Sadenosylmethionine, and glycosylated by reducing sugars. Many compounds, such as aromatic amines, aflatoxin and pyrolizidine alkaloids, may form reactive oxygen species catalyzed by cytochrome P450. These metabolites form adducts with the DNA, which can cause errors in replication. The adducts may also induce conformational changes in the DNA. Some alkylating agents such as N-Nitrosamines may also require the catalytic reaction of cytochrome-P450 for the formation of a reactive alkylation. Alkylation and arylation of bases can cause errors in replication.

Crosslinking
Some alkylating agents may produce crosslinking of

DNA. Some natural occurring chemicals may also promote crosslinking, such as psoralens after activation by UV radiation, and nitrous acid. Interstrand cross-linking is more damaging as it blocks replication and transcription and can cause chromosomal breakages and rearrangements. Some crosslinkers such as cyclophosphamide, mitomycin C and cisplatin are used as anticancer chemotherapeutic.

Dimerization
UV radiation promotes the formation of a cyclobutyl

ring between adjacent thymines, resulting in the formation of pyrimidine dimers. DNA polymerase may help bypass these lesions in an error-free manner; however, individuals with defective DNA repair function, such as sufferers of Xeroderma pigmentosum, are sensitive to sunlight and may be prone to skin cancer.

Intercalation Between Bases

The planar structure of chemicals such as ethidium

bromide and proflavine allows them to insert between bases in DNA. This insert causes the DNA's backbone to stretch and makes slippage in DNA. Forward slippage will result in deletion mutation, while reverse slippage will result in an insertion mutation.

Backbone Damage
Ionizing radiations may produce highly reactive free

radicals that can break the bonds in the DNA. Double-stranded breakages are especially damaging and hard to repair, producing translocation and deletion of part of a chromosomes. Oxidative stress may also generate highly reactive oxygen species that can damage the DNA. Incorrect repair of other damages induced by the highly reactive species can also lead to mutations.

Insertional Mutagenesis
Transposon and virus may insert DNA sequence into

coding region or functional elements of a gene and result in inactivation of the gene.

Error in Replication
While most mutagens produce effects that ultimately

result in error in replication, some mutagens may affect directly the replication process. Base analog such as 5-bromouracil may substitute for thymine in replication. Some metals such as cadmium, chromium, and nickel may alter the fidelity of DNA replication.

Mutagenicity Tests
Test for Gene Mutations in Bacteria AmesTest The Ames test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis i.e. it is an auxotrophic mutant, so that they require histidine for growth. The method tests the capability of mutagen in creating mutations that can result in a reversion back to a prototrophic state so that the cells can grow on a histidine-free medium. The tester strains are specially constructed to detect either frameshift or point mutations

Test for Chromosomal Aberrations in Mammalian Cells In Vitro The chromosome aberration assay in cultured cells has been widely used for many years, and it has proved to be a useful and sensitive test for detection of genotoxic agents. The damage is scored by microscopic examination of chromosomes in mitotic metaphase cells. Tests are carried out with and without extrinsic metabolic activation.

In-vivo Genetic Assays The positive result found in bacteria can be additionally studied in a system that has the complex eukaryotic chromosomal structure. This structural complexity also allows the possibility of detection of mutations arising through mechanisms that cannot occur in the simple bacterial genome. Suitable tests include those using mammalian cells designed to detect induction of mutations at specific loci.

In vivo and in vitro micronucleus Assay The micronucleus test is used for detection of damage to the chromosomes or the mitotic apparatus induced by chemicals. An in vitro micronucleus assay using cultured cells has been developed. This assay is more easily scored than the chromosome aberration assay and utilizes relatively small amounts of testarticle, thus requiring less time to make an assessment of mutagenic potential of a chemical.

Comet Assay
An Alternative Tool to Assess DNA Damage and DNA Repair The single cell gel electrophoresis (Comet assay) can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. Comet assay detects DNAstrand breaks which , when subjected to electrophoresis, will result in migration of DNA fragments out of the nucleus to form the tail of a comet-like structure. The extent of migration of DNA fragments is an indication of DNA damage that can be quantified.

The Effects of Mutations

Somatic mutations occur in non-reproductive cells

and won't be passed onto offspring. The only mutations that matter to large-scale evolution are those that can be passed on to offspring. These occur in reproductive cells and are called germ line mutations.

How can gene mutations affect health and development? When a mutation alters a protein that plays a critical role in the body, it can disrupt normal development or cause a medical condition. A condition caused by mutations in one or more genes is called a genetic disorder. In some cases, gene mutations are so severe that they prevent an embryo from surviving until birth. It is important to note that genes themselves do not cause diseasegenetic disorders are caused by mutations that make a gene function improperly.

References
RANG and DALEs Pharmacology, Sixth edition

www.currentseparations.com
http://evolution.berkeley.edu A Text Book of Modern Toxicology, 3rd edition By

Ernest Hodgson