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Test Methods to Study Genotoxic Compounds
#1
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.
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#2
Methods and regulations on the genotoxicity screening

All living creatures are inseparably associated with chemicals. They are all around us, from food and cosmetics, to the drugs and different industrial products used in everyday life. Safety assessment of the novel compounds is very important as some chemicals are very “aggressive” and could produce devastating effect on the health. Compounds that interact with DNA, changing its structure and integrity are known as genotoxic. Most of those chemicals induce mutagenesis (alter genetic information) or cancerogenesis (tumor induction). Both somatic and germ cells could suffer damage. When germ cells are altered by genotoxic compound, mutagenic effect is seen in the offspring even though it has never been exposed to the genotoxin.

Genotoxic effect could be result of a point mutation, changes in the DNA structure or damage on the chromosome. Tests used for genotoxic screening are discovering the damage on different levels and couple of tests is usually applied to ensure an accurate final result.
In vivo and in vitro screening methods could measure and predict possible toxic effect both in the somatic and germ cells. Some in vivo tests are designed to measure DNA damage that will be passed to the offspring. Examples for that kind of tests are mouse heritable translocation test, mouse specific locus test and rodent dominant lethal test. Mammalian germ cells could be used for assessment of chemical potential to induce heritable damage. The most popular tests of this kind are unscheduled DNA synthesis using testicular cells, spermatogonial chromosome aberration test or oocyte chromosome aberration & aneuploidy test…Commonly used assays for prediction of genotoxicity in somatic cells are mammalian erythrocyte micronucleus test, bone marrow chromosome aberration test, unscheduled DNA synthesis in the liver or mouse spot test. Beside this, in vivo Comet assay and in vivo transgenic mutation assays could be used as well. Also, a lot of tests using in vitro non-animal related alternative methods (such as bacterial and mammalian cell assays) are often performed.

Regulatory agencies and international authorities determine the type and pattern of the testing that should be applied during genotoxic compounds evaluation. Globally respected and recognized organization that establishes standards for various chemical testing is The Organization for Economic Co-operation and Development or OECD. It is established in 1961. when Europe, USA and Canada joined their forces to promote global development. Today, 34 member countries are working on the improvement of the social and economic prosperity of the people around the world by adopting different policies and strategies. OECD assists in defining methods for molecular screening and toxicogenomics that could be applied in a regulatory context. Tests that are adopted and approved by the OECD are used by government, industry and laboratories for the safety assessment of chemicals and/or chemical preparations.

This is the OECD test guidelines for genotoxicity and mutagenicity testing:

1. Bacterial Reverse Mutation Test (Ames Test)
2. Genetic Toxicology: Escherichia coli, reverse assay
3. In Vitro Mammalian Chromosome Aberration Test
4. Mammalian Erythrocyte Micronucleus Test
5. Mammalian Bone Marrow Chromosome Aberration Test
6. In Vitro Mammalian Cell Gene Mutation Test
7. Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster
8. Genetic Toxicology: Rodent Dominant Lethal Test
9. Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells
10. Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
11. Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
12. Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro
13. Mammalian Spermatogonial Chromosome Aberration Test
14. Genetic Toxicology: Mouse Spot Test
15. Genetic Toxicology: Mouse Heritable Translocation Assay
16. Unscheduled DNA Synthesis (UDS) Test with Mouse Liver Cells In Vitro
17. In Vitro Mammalian Cell Micronucleus Test

A lot of money is spent on the research and development of methods that could detect genotoxic compounds quickly and efficiently. Pharmaceutical industry is especially interested for these techniques, because a lot of drugs (around 12%) in the late stage development could show genotoxic potential. They need to be modified and improved before they enter clinical trials. Estimated cost of delaying clinical trials and bringing the drug to the market later than expected could vary from 5 – 30 million dollars.

In vitro toxicity testing is important for one more reason. Animal testing is considered cruel and unethical and it will cease completely as soon as alternative methods become efficient enough. Joined collaboration of scientific methods (in vitro tests) and predictive methods (software based) should become standard in the future safety assessment.
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#3
Genotoxic substances both certain chemical compounds and certain types of radiation.This includes are with DNA .which is not proof of their dangerousness to humans, but does render them potentially mutagenic or carcinogenic.genotoxicity describes a deleterious action on a cell's genetic material affecting its integrity
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