[priyasaravanan_1406]

Genotoxicity, a branch of toxicology is developed to identify the elements or compounds present in the environment having the potential to cause mutation by damaging the DNA. These compounds are also classified under the group of carcinogens, because of their cancer causing property. The necessity to identify the toxic compounds causing mutation is important in various industries like pharmaceutical, agriculture and food as the end user of the products from these industries are humans. As a result various methods were developed to detect and assess the toxic elements. The conventional method of using laboratory animals like mice and rat as test subjects is replaced by newly developed in vitro methods using microorganisms (bacteria) and animal cells. Few of such mostly used testing methods include Ames test, cell line tests and cytogenetic or chromosomal test.

Ames Test: Ames test employs bacteria in detecting the mutagenecity of the test compound. The mostly used species is mutant of Salmonella typhimurium, the ability to produce histidine of this organism is altered by mutating histidine operon gene present in this bacterium. The mutant organism is plated on an agar plate prepared with small amount of added histidine and the test element is placed at the centre of the plate using a filter disc. The toxicity of the compound is assessed by the growth of the bacteria. Initially, the bacterium grows till the presence of added histidine. Later, only those organisms whose mutation is reversed by the test compound, grows by producing histidine. This is qualitative test. The amount of compound required to cause mutation is quantified with the help of dose-response curve.

Later two types of mutations like single base substitution and frame shift mutation were adopted to create mutant organisms with the scope of studying more number of suspected toxic compounds. The sensitivity of the test is enhanced by altering the permeable nature of the bacteria and their cell repair mechanism. The use of bacteria as the test organism to study the mutagenic elements or compounds that are threat to humans has its own limitations. For example, once the compound enters the body, its toxicity is established only after the action of certain enzymes produced in the human body. To overcome this problem, extracts of liver with active enzymes were added along with the test element.

Use of Cell lines: The fact that the testing of Genotoxicity of an element on mammals is more beneficial rather than the use of bacteria, led to the use of mammalian cell lines in vitro. The cell lines derived from the mouse lymphocyte is used and the thymidine kinase heterozygote test is considered as the popular method of toxicity testing. The mouse cell lines are selected based on the mutation occurred on the thymidine kinase gene locus. The mutants are derived by treating the cells with toxic copies of thymidine. The selected cells are grown in the cell culture media by exposing them to the compound under study.

Chromosome test or cytogenetic test: This method involves the use of cell lines. The mutagenic property of the test compound is detected by observing the cells for chromosomal damage. Earlier, the mutagenic property of the test element is assessed by calculating the chromosomal damages like chromosomal breakages, chromosomal exchange, formation of ring chromosome, chromosomal dicentric and chromosomal translocation. Later, due to the difficulty in assessing the toxicity by this method, enumeration of sister chromatid exchange is done to detect the element for its mutagenic property. The increase in the frequency of sister chromatid exchange on exposing to mutagens excited the researchers and they preferred this method than counting the chromosomal aberrations. The sister chromatid are stained to study the type of exchange taken place using the fluorescence Giemsa staining method. In this method the cells are labeled with Bromodeoxyuridine, a thymidine analogue by growing them in the solution containing Bromodeoxyuridine. The staining is done by initial exposure of the chromosomes to fluorochrome, irradiation using ultra violet light and then staining with the Giemsa stain.

The Ames method is less time consuming where the results are obtained within 48 hours whereas the test methods using cell lines to detect the Genotoxicity of a compound takes about 21 days to get the result. This is because of the slow growth rate of the cell lines compared to that of the bacteria. Also methods using cell lines should adopt sophisticated techniques to maintain the cell line.