Figure 4.1: DNA Damage Resulting in Multiple Broken Chromosomes

Mutagens

A mutagen (Latin, literally Origin of change) is a physical or chemical agent that changes the genetic information (usually DNA) of an organism and thus increases the frequency of mutations above the natural background. As many mutations cause cancer, mutagens are typically also carcinogens but sometimes mutations occur spontaneously due to DNA replication, repair and recombination.

Agents that Damage DNA

1. Certain wavelengths of radiation
   
   
   
   • Ionizing radiation such as gamma rays and x-rays
     
   • Ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield
   
   
2. Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways.
   
3. Chemicals in the environment
   
   
   
   • Many hydrocarbons, including some found in cigarette smoke
     
   • Some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts
   
   
4. Chemicals used in chemotherapy, especially chemotherapy of cancers

Consequences of Damaged DNA

The biological consequences of individual damaged bases have been deduced by a combination of experimental approaches (Wallace, 2002). The possible consequences of fertilization with sperm possessing abnormal DNA are shown in Figure 4.2.

Pathological Effeccts of Poor DNA Repair (Figure 4.3)

DNA repair rate is an important determinant of cell pathology. In a healthy cell the rate of DNA damage is equal to the rate of repair. Any shift of their balance leads to pathological effects of DNA repair. Experimental animals with genetic deficiencies in DNA repair often show decreased lifespan and increased cancer incidence. For example, mice deficient in the dominant NHEJ pathway and in telomere maintenance mechanisms get lymphoma and infections more often, and consequently have shorter lifespan than wild-type mice similarly, mice deficient in a key repair and transcription protein that unwinds DNA helices have premature onset of aging-related diseases and consequent shortening of lifespan. However, not every DNA repair deficiency creates exactly the predicted effects; mice deficient in the NER pathway exhibited shortened lifespan without correspondingly higher rate of mutation.

Figure 4.2:Consequences of Fertilization with Sperm Possessing Abnormal DNA

Figure 4.3:Pathological Effeccts of Poor DNA Repair

If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis or cancer. Inherited diseases associated with faulty DNA repair functioning result in premature aging, increased sensitivity to carcinogens, and correspondingly increased cancer risk. On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus radiodurans, the most radiation-resistant known organism, exhibit remarkable resistance to the double strand break-inducing effects of radioactivity, likely due to enhanced efficiency of DNA repair and especially NHEJ.

Hereditary Consequences of Damaged DNA

1. Xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and premature aging
   
2. Cockayne syndrome: hypersensitivity to UV and chemical agents
   
3. Trichothiodystrophy: sensitive skin, brittle hair and nails

Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.

Other DNA repair disorders include:

1. Werner’s syndrome: premature aging and retarded growth
   
2. Bloom’s syndrome: sunlight hypersensitivity, high incidence of malignancies (especially leukemias).
   
3. Ataxia telangiectasia: sensitivity to ionizing radiation and some chemical agents

All of the above diseases are often called “segmental progerias” (“accelerated aging diseases”) (Wei et al., 2007) because their victims appear elderly and suffer from aging-related diseases at an abnormally young age.

Other diseases associated with reduced DNA repair function include Fanconi’s anemia, hereditary breast cancer and hereditary colon cancer.

Repairing Damaged Bases (Figure 4.4)

Thankfully, cells have evolved many complex mechanisms to detect and repair DNA damage, and most of the time a cell repairs its damaged DNA without a problem. But, just like the machinery that copies DNA, a cell’s repair machinery is not 100 per cent efficient and not every single error is corrected. Maintenance of genome integrity is essential to minimize heritable mutations and to promote healthy survival of organisms. The DNA damage response (DDR) has evolved to optimize cell survival following DNA damage. It involves the actions of DNA repair proteins together with the “checkpoint” events that slow down or arrest cell-cycle progression while the damage is being removed. The inability to properly face a genotoxic threat leads to genomic instability, and eventually to tumoral transformation.

DNA damage is caused by a variety of sources. The cellular response to damage may involve activation of a cell cycle checkpoint, commencement of transcriptional programs, execution of DNA repair, or when the damage is severe, initiation of apoptosis.

Figure 4.4:DNA Damage Response

The DNA damage response or inappropriate bases can be repaired by several mechanisms such as

1. Direct chemical reversal of the damage
   
2. Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes.

• Base Excision Repair (BER)
  
• Nucleotide Excision Repair (NER)
  
• Mismatch Repair (MMR)

The genotoxicity of the drug like molecule can be tested on the basis of the observations of the different parameters in the experimental animals viz., micronuclei, chromosomal aberrations and sperm aberrations.

Detection of Damaged DNA

Various methods have been developed to detect damaged DNA.

1. UDS: UDS (unscheduled DNA synthesis) most common complementary assay and can be detected e.g. by an elevated incorporation of [3H]–thymidine in to the DNA of cultured mammalian cells during the repair of damage. This can be detected by autoradiography or liquid scintillation counting.
   

   The UDS assay measures repairable DNA damage induced by test article. The UDS assay is customarily conducted in hepatocytes, both in vitro and in vivo. Metabolic activation of the test article is believed to occur in the hepatocytes proximal to DNA, which enhances the sensitivity of mutagen detection. This test has been proposed by the ICH guidelines to be an additional assay for the clarification of equivocal in vitro test.

   
   

   Both in vitro and in vivo UDS assays are most useful in detecting liver carcinogens and less reliable in detecting carcinogens of other organs.

   
   
2. Comet Assay: the comet assay is a relatively simple, but sensitive and well validated tool for measuring strand breaks in DNA in single cells (Wong et al., 2005). In the comet assay, nucleated cells are embedded in low melting point agarose on a microscopic slide, and the membranes and histones are removed by high salt solutions (singh et al., 1988). DNA is unwinds and spills out as a ’halo’ surrounding the nucleoid (Cook et al., 1976). Electrophoresis at neutral, mildly alkaline or strongly alkaline conditions follows the unwinding step (Tice and Strauss, 1995). The comet is visualized by a DNA staining fluorescent dye (Tice et al., 2000) and DNA damage is then scored using either visual or computerized image analysis.

Micronuclei

Micronucleus is the small nucleus that forms whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division or by chromosome fragments that lag at the cell division due to the lack of centromere, damage, or a defect in cytokinesis (Heddle et al., 1991). In newly formed red blood cells in humans, these are known as Howell-Jolly bodies. In normal people and many other mammals, which do not have nuclei in their red blood cells, the micronuclei are removed rapidly by the spleen. Hence high frequencies of micronuclei in human peripheral blood indicate a ruptured or absent spleen. In mice, these are not removed, which is the basis for the in vivo Micronucleus test.

Figure 4.5:Acridine Orange Stained Peripheral Blood Erythrocytes of C. auratus

Arrows; NCE: Normochromatic (mature) erythrocyte; MN-NCE: Micronucleated normochromatic erythrocyte; PCE: Polychromatic (young) erythrocyte with RNA-containing cytoplasm; MN-PCE: Micronucleated polychromatic erythrocyte (Cavas, 2008).

A “micronucleus” is literally a small nucleus. In the micronucleus test, animals are treated with a chemical and then the frequency of micro nucleated cells is determined at some specified time after treatment. If a treated group of animals shows significantly higher frequencies of micro nucleated cells than do the untreated control animals, the chemical is considered to be capable of inducing structural and/or numerical chromosomal damage.

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