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Although genotoxicity assays are divided into 3 stages, the final findings they observe are gene mutation, clastogenicity and aneugenicity. Each assay has its own significance and factors including physiochemical nature of test substances and nature of tissues affected by substances are required to consider. Age, sex, smoking and sample preparation influence the outcomes of some assays. Stage 1 tests are composed of in vitro bacterial assays and in vitro mammalian assays. The in vitro bacterial assay alone is not able to cover three main abnormalities of genome. Moreover, some test substances cannot show genotoxicity without stimulating metabolism. Therefore, not only in vitro bacterial assay but also in vitro mammalian assays are recommended to identify genotoxic effects in vitro. However, not all in vitro negative substances can be regarded as non-mutagenic substances because some test substances present positive in vivo assays without showing positive results in vitro assays. Moreover, not all of the substances with positive results in vivo assays reveal positive results in germ cell assays which verify the genotoxic impacts on germ cells exposed to test substances and the potential to pass the genotoxic effect to their future generations. In addition, some of the assays can reveal the link between mutagenicity and carcinogenicity of test substances. Therefore, using only one assay to test substances is not enough to give satisfactory findings that can be achieved by using the combination of genotoxicity assays. Furthermore, using the combination of genotoxicity assays can fulfill the main outcome of determining deleterious effects on genome.
In an industrialized world, people handle substances like drugs and chemicals which have the ability to cause genotoxic effects on them in their daily life. So scientists conduct a lot of studies to develop assays that can be used to detect genotoxic impacts of these substances on human beings. These genotoxicity assays can detect the interaction between test substances and DNA, parts of cells including spindle apparatus, enzymes such as topoisomerases and DNA polymerase and DNA repair systems. All these parts are important for regulating actual information of the genome (United Kingdom Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment, 2000) and genotoxicity is capable of giving deleterious effects on the genome. Moreover, the unclear relationship between mutagenicity and carcinogenicity encourages improving the efficiency of the assays. The end outcomes of genotoxicity include mutational damage to DNA or the production of DNA adducts by directly interacting with these substances or their metabolites. Moreover, genotoxicity also leads to disruption of DNA synthesis and formation of sister chromatid exchange and mitotic recombination in spite of lack of direct evidence of inherited mutation. However, the overall results of genotoxicity are gene mutation like point mutation, deletion, insertion, clastogenicity like sister chromatid exchange and micronuclei and aneuploidy like hyperploidy or polyploidy. Scientists developed genotoxicity assays relying on these three outcomes and a number of assays are developed during decades.
For developing genotoxicity assays, the Committee on Mutagenicity of chemicals in Food, Consumer Products and the Environment (COM) advised to develop a strategy including three stages for assessing the genotoxicity of the substances. To achieve relevant information from the genotoxicity assays, the factors including chemical nature of test substances, their ways of metabolism ,how human beings are exposed to chemicals, how much the exposure give genotoxicity and which organs are targeted from genotoxic effects should be considered. In addition, all these factors are required for choosing relevant assays before conducting stage 1 tests. Among three stages, Stage 1 tests are divided into in vitro bacterial assays and in vitro mammalian assays. In vivo genotoxocity assays form Stage 2 tests and Stage 3 consists of germ cell assays.
Stage 1 tests
In Stage 1 tests, in vitro bacterial assays include the Salmonella typhimurium reverse mutation assay, other S.typhimurium mutants and the Escherichia coli reverse mutation assay. In vitro mammalian assays contain the mouse lymphoma thymidine kinase gene mutation assay, the hypoxanthine guanine phosphoribosyltransferase gene mutation assay, the mammalian chromosome aberration test and the micronucleus test.
In vitro bacterial assay
According to the Organization for Economic Co-operation and Development (OECD) Test Guideline 471(OECD, 1997), five strains of bacteria for example Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP 2 are used to carry out the in vitro bacterial assay by regarding all properties related to the substances. Detecting oxidizing mutagens and cross linking agents are not achieved by using strains of Salmonella typhimurium although Escheria coli WP 2 or Salmonella TA 102 can detect the mutagenic effect of these substances. Moreover, different types of mutations can be detected by using different types of bacterial strains because different bacterial strains are able to give different mutations. For instance, Salmonella typhimurium TA 98 and Salmonella typhimurium TA 1537 cause frame-shift mutations whereas Salmonella typhimurium TA 100, Salmonella typhimurium TA 102 and Salmonella typhimurium TA 1535 cause base pair substitutions. Therefore, using different strains in the in vitro bacterial assay gives a chance to make the preliminary estimation of the mutation status of the test substances.
Among short term in vitro assays, the Salmonella test developed by Ames can give approximately 90% accuracy of detecting carcinogens as mutagens which damage DNA (Sufian et al, 2007). In this test, one of the Salmonella strains is used as a maker for DNA destroyed by test substance. Moreover, mammalian liver extracts including S9 fraction from human liver or ratsâ€™ liver are necessary to activate metabolism of the test substance in which metabolism can influence the mutagenic effects. Metabolic activation of the test substance is a major part for some agents (Ames et al, 1973; Cooper and Grover1990; Guengerich and Shimada, 1991 and Guengerich, 2000) which cannot be described as mutagens without changing to the mutagenic forms. Bacteiraâ€™ strains consist of mutated histidine operons and some strains which have uvr mutation which eliminates DNA repair system and rfa mutation that changes the properties of the bacterial cell walls and increase the permeability of cell walls to substances are regarded as standard strains and these strains improve the detection power of damaged DNA.
However, it is found that different outcomes result from using rat or human liver S9 fractions because of different chemical structures of test substances (Guengerich and Shimada, 1991and Guengerich, 2000). For instance, most of the polycyclic hydrocarbons revealed mutagencity by using rat S9 fraction but no mutagenicity was detected with human S9 fractions. Different mutagenicity assessed by using rat or human S9 fraction may be due to differences in the quantity of S9 fraction, the amount of metabolizing enzymes, the quality of S9 fraction and the chemical composition of test substances.
In vitro mammalian assay
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) assay
HPRT mutation arises from cells exposed to genotoxic agents. 6-thioguanine changes to thioguanine monophosphate by HPRT enzyme and this effect can kill normal cells. So cells with mutated HPRT gene can survive with treatment of 6-thioguanine. This assay detects HPRT mutation in a human cell line which is exposed to morphine in vitro (Schafer et al, 1994). This also identifies point mutation, small deletions and large deletions and is used as positive controls for quality control.
Mouse lymphoma thymidine kinase (TK) gene mutation assay
In this assay, thymidine kinase phosphorylates trifluorothimidine and the phosphorylated trifluorothimidine becomes incorporated into DNA and is toxic. Frequencies of mutants are used as indicatiors to clarify test substances as genotoxic agents. The consequences of mutations including point mutations, deletions, translocation, rearrangement, aneuploidy, mitotic recombination and gene conversion are determined by this assay. Moreover, this assay can identify substances like acrylamide with negative bacterial reverse mutation assay as genotoxic agents. Therefore, it can be used as a standard genotoxicity test compared to other tests in developing new drugs. Two versions of the assay include micro-well method and soft agar method. Growth conditions and positive controls influence the quality of the assay and these factors should be considered when the assay is undertaken (Moore et al, 1999).
Micronuclei occur in 2 forms: (1) micronuclei with chromosomal fragments caused by direct DNA breakage, replication on a damaged DNA template and inhibition of DNA synthesis (2) micronuclei with whole chromosomes caused by failure of a mitotic spindle, kinetochore, or other parts of mitotic apparatus or by damage to chromosomal sub-structures, alterations in cellular physiology and mechanical disruption. This test can detect both structural and chromosomal abnormalities. Moreover, aneuploidy produced only by chromosome loss is detected by the bone marrow micronucleus assay. The Micronucleus assay can be conducted not only by using peripheral blood lymphocyte but also by using various tissues depending on the action of the exposing agent. For example, micronuclei numbers significantly increase in nasal mucosal cells exposed to formaldehyde compared to non-exposed groups (Ying et al, 1997; Tienko-Holland et al, 1996 and Ballarin et al, 1992). Like nasal mucosal cells, a significant increase in the level of micronuclei is found in exfoliated urothelial cells after exposing them to arsenic and smoking (Burgaz et al, 1995 and Moore et al, 1997). Studies show that there is a strong association with micronuclei frequency and the aging process (Bolognesi et al, 1997). An increased number of micronuclei in females verify that sex also has a role in determining the micronuclei frequency (Bonassi et al, 1995). Conversely, personal habits like smoking have no impact on micronuclei frequency in spite of one study with a significant relationship between micronuclei and smoking (Cruz et al, 1994). Therefore, age, sex and personal habit influence the outcomes of genotoxicity of agents. These factors needed to be considered to verify that the increase number of micronuclei really occur by means of genotoxic agents. There is no relevant study of relationship between micronuclei formation and carcinogensis.
In vitro mammalian chromosome aberration test
This test is one of the tests which can detect structural chromosome aberrations like chromosome gaps, chromosome breaks and chromatic exchange. The Chromosomal aberration is categorized into two groups which are chromosome type chromosomal aberration and chromatid type chromosomal aberration. Aneugenic agents can also be detected by using this test. Increases in chromosomal aberrations with aging highlight that age should be considered to perform the test. Although cancer risk elevates with increase in structural chromosomal aberrations, there is no study which shows the association with increased cancer risks and aneuploidy. To analyze genotoxicity of substances by using micronuclei and chromosomal aberration assays, the outcomes depend on level of substances, duration, and frequency of exposure and clastogenic mechanism involved. Moreover, sample timing is also important to give informative results. Detection of chromosomal aberration in peripheral blood lymphocyte can reveal genetic damage in precursor cells for carcinogenic process in target tissue and this method is useful in assessing genetic risks in health surveillance programs. Moreover, some studies show the association between chromosomal aberration frequency and increased cancer risk (Nordic Study Group on the Health Risk of Chromosome Damage. An inter-Nordic prospective study and A Nordic data 1990; Hagmar et al 1994; Bonassi et al, 1995 and Bolognesi et al,1997 ) especially haematological malignancies( Bonassi, et al, 1995) in which assays detecting micronuclei and sister chromatic exchanges gave no conclusive results.
In vitro positive substances are followed by in vivo tests to assess the mutagenic status in vivo and to confirm that they are mutagenic. Moreover, in vivo tests are recommended for the in vitro negative substances which are considered as highly or moderately harmful to human beings. Ambiguous results should be solved by additional in vitro tests to confirm positive or negative mutagenicity. Moreover, using only in vitro bacterial assays are not enough to prove that the test substance is mutagenic because a substance like acrylamide which has no mutagenic effects in bacterial assays but the HPRT assay and mouse lymphoma assay show that acrylamide is mutagenic.
Stage 2 tests
Any substances which are positive in in vitro tests are required to perform in in vivo tests because factors like toxicokinetics, metabolism, chemical reactivity, lack of absorption, and inability of the active metabolite to reach DNA, rapid detoxification and elimination are needed to be considered. These factors influence on the activities of in vitro and in vivo tests. Therefore, in vivo tests should be performed to confirm positive results from in vitro tests. However, unreasonable results and unnecessary sacrifice of animals can be avoided by careful selection of in vivo tests. Stage 2 tests consist of micronucleus test in erythropoietic cells( described in Stage 1), metaphase analysis in vivo test (described in Stage 1), transgenic animal assays, 32p postlabelling assay, convalent binding assay, comet assay and liver unscheduled DNA assay.
Comet assay is useful in measuring the amount of DNA damage and repair in individual cells ( Collins, 2004) by detection single ( Tice et al, 2000) and double strand breaks ( Olive, Banath, 1993), DNA-DNA/DNA-protein cross links ( Merk, Speit, 1999) or changes of DNA repair(Collins et al, 2003). For undergoing this assay, not only blood leukocytes but also other different cell types including gastric and nasal cells can be used to detect DNA damage relying on how cells contact with chemicals and the ways used to collect cells. Moreover, this assay is capable of observing how much damaged cells occur within cell population that seems to be undamaged. However, this test has no ability to detect mutagens without strand breaks or alkaline labile lesions. Positive DNA damage results are obtained in analyzing 28 studies of environmental exposure in human by using Comet assay and 71% of these studies correlate with other biomarkers (Mahara Valverde, Emilio Rojas, 2009). The Comet assay is useful in monitoring not only for environmental substances but also for occupational substances such as chemical, biological or physical agents which give deleterious effects on human health. Studies show that the Comet assay can observe DNA damage induced by occupational substances including volatile organic compounds, poly aromatic hydrocarbons (Binkova et al, 1996), asbestos (Zhao, 2006), anti-neoplastic drugs (Laffon et al, 2005), anaesthetics (Sardas et al, 1998), radiation (Aroutiounian 2006), metals (Wilhelm et al, 2007) or pesticides (Perez-Maldonado et al, 2006). Using the comet and FISH assay simultaneously develop Comet FISH technique in which cancer relevant genes like APC, KRAS and Tp 53( Glei et al, 2007) or telomerase ( Arutyunyan et al, 2005) can be detected. Moreover, the combination of these two tests also identifies the sites between normal and damaged ones. Both of these assays may become suitable assays which can assess individual sensitivity to cancer resulted from different DNA repair mechanisms of individuals. Furthermore, Instable p53 and HER/2-neu genes in breast cancer cell lines can be revealed by Comet-FISH assay (Tirukalikundram et al, 2005). Moreover, identifying genotoxic effect in any tissue can be simply done by this assay (McGregor and Anderson, 1999).
Transgenic Animal assay
The assay uses a foreign gene constructed with a target gene and shuttle vector and it can detect gene mutation like point mutation, small insertions and deletions depending on the dose level. Although it is not a widely used assay to detect mutation, it has the ability to detect systemic and local mutagenic effects. Moreover, it can detect mutation in every organ. Commercial models for these assays are Big BlueTM and MutaTM mouse.
32p postlabelling assay and convalent binding assay
Premutagenic defects can be detected by using 32-p postlabelling assay and convalent binding assay. Radiolabelling of test substances is not required in the former assay (Phillips et al, 1993) whereas the latter one needs radiolabelling for testing substances. Although data resulting from these assays are complicated to interpret, these assays are useful for verifying mechanisms in vivo compared with other data.
Liver Unscheduled DAN synthesis (UDS) assay
This assay is used to detect the genotoxic effect of substances including dimethylnitrosamine, 2-nitropropane, 2,4-dinitrotolune, 3-methyldiaminobenzanthracene and dimethylaminophenylazobenzthiazole (Tweats, 1994). These substances give positive outcomes in vitro tests but result negative in bone marrow micronucleus test. The final status revealed by this assay is DNA damage and subsequent repair in liver which is the main site of metabolism. Therefore, this assay is suitable for substances that need metabolites to give genotoxic effects on cells.
Positive in vivo substances are regarded as in vivo somatic cell mutagens and these substances are needed to undergo germ cell tests because almost all somatic cell mutagens cannot be regarded as germ cell mutagens. Additional in vivo assays are required for negative in vivo substances which are positive in vitro studies. Substances which are negative in both in vitro and in vivo are considered as negative in vivo mutagens. For inconclusive results, the reason is low sample amount and may be corrected by elevating the amount of sample.
Stage 3 tests (Germ cell Assays)
Germ cell assays are required for investigating whether the agents that have the ability to cause genotoxic impacts on somatic cells in vivo can give the genotoxic effects on germ cells and their offspring. A number of germ cell assays are discovered but the relevant assays should be carefully chosen to avoid the unnecessary use of animals. The germ cell assays are categorized into 2 groups. The first group detects mutagenicity in germ cells and contains transgenic animal assay(described in Stage 2), expanded simple tandem repeat assay (ESTR), mammalian spermatogonial chromosome aberration assay, comet assay(described in Stage 2), Comet-FISH (fluorescence in situ hybridization) assay (described in Stage 2) and DNA adduct assay(described in Stage 2). The second group consists of ESTR assay, dominant lethal assay, mouse visible specific locus assay, mouse biochemical specific locus assay and mouse heritable translocation assay. The tests in second group are used to verify whether mutagenic effects of animals exposed to chemicals can be passed to their offspring.
Expanded Simple Tandem Repeat Assay (ESTR)
The assay is useful for monitoring human neurodegenerative disorder like Fragile X syndrome and Friedreich ataxia (FRDA) which are related to genetic instability. Moreover, exposure to chemicals including aphidicolin, araC and caffeine can determine the rate of simple repeat expansion. Furthermore, this assay becomes a suitable method to detect genetic instability caused by environmental hazards.
Dominant lethal Assay
Dominant lethal assay can observe the toxic effect of substances like triethylenemelamine, cyclophosphamide and ethyl methanesulphonate which give no abnormal effects on the gamete but have lethal effect on the fertilized egg or developing embryo. The end results of lethal effects are structural and numerical abnormalities and mutations and the results should be statistically significant. Therefore, evaluation of the results from the assay should be considered depending on biological and statistical significances.
Mouse specific locus test
The alteration of genetic information of the species which are generated from animals exposed to mutagenic chemicals is assessed by mouse specific locus test. Changes in mouse coat color and other visible features of mouse strain can be detected. This test has been widely used in assessing genetic hazards due to environmental agents.
Mouse biochemical specific locus test
This test can observe altered protein resulting from gene mutation by means of electrophoretic methods. Mouse heritable translocation test is also used to detect the mutagenic impact on the offspring which are generated from animals exposed to mutagenic chemicals. However, there is no relevant test which can observe the numerical chromosomal aberration of the offspring.
The chemicals which give negative results in germ cell tests are not regarded as germ cell mutagens. On the other hand, the chemicals which give positive results in germ cell tests are noted as germ cell mutagen. For the conditions in which the numbers of offspring that inherit mutations from their ancestor should be considered, dominant lethal test, mouse heritable translocation assay, gene mutation in ESTR assay and specific locus tests are required to examine whether the mutagenic effects pass through generations.
Genotoxic agents give adverse effects not only on DNA itself but also on the components of gene regulating parts like the spindle apparatus, enzymes including topoisomerase and DNA polymerase and DNA repair systems. These results lead to impairment in DNA synthesis, formation of chromosome abnormalities like sister chromatid exchange. The final outcomes of genotoxic agents are gene mutation, clastogenicity and aneugenicity. There is a strategy consisting of 3 stages to detect these 3 outcomes. Each stage has its own significance and factors to consider giving ideal information. Prior to undergoing these tests, the chemical composition of test substances, their status in solutions, how the cells are exposed to these substances and the concentration of substances contacted with cells are considered. The reason why these factors are taken into account is due to the fact that they influence the outcomes of tests.
In in vitro bacterial assays, preliminary estimation of types of mutations can be done by using different strains with different mutations. In addition, metabolic activation plays an important role in assessing genotoxic impacts of substances which are needed to pass through metabolism to be mutagenic. However, different mutagenic effects are observed by using different types of liver extracts (eg. human S9 fraction and rat S9 fraction). In spite of being a useful test in assessing genotoxicity, the in vitro bacterial test does not cover all 3 outcomes. Therefore, in vitro mammalian assays are required. Some substances like acrylamide is nonmutagenic in bacterial assay while positive mutagenic effect of this substance is found in the HPRT and mouse lymphoma assay (Dearfield, et al, 1988 and Hashimoto, et al, 1985). Moreover, in vitro bacterial assays are not able to reveal abnormalities in chromosomal structure and number which can be detected by the in vitro chromosome aberration test and micronucleus test. The outcomes of substances can be altered by factors including the level of test substance, exposure time, frequency of exposure and clastogenic mechanisms involved. Therefore, these factors should be taken into consideration when genotoxicity is analysed by the in vitro chromosomal aberration assay and micronucleus assay. Furthermore, it can be found that age, sex and personal habit like smoking can change the outcomes of these assays.
Besides Stage 1 test, in vivo somatic assays are necessary to be conducted because in vitro mutagenic effects of substances may not be seen in vivo. The underlying reasons may be due to the absence of absorption, distribution, elimination and metabolism which are fundamental limitations of in vitro tests. Therefore, in vivo somatic assays are important to confirm in vitro mutagenic substances. Among these assays, comet assay can detect genotoxicity by using different tissues relying on types of tissues exposed. In addition, Comet-FISH assay is capable of revealing a relationship between tumor formation and genotoxicity. Like the comet assay, there is a clear connection between a positive chromosome aberration assay and cancer incidence while there is no study which is a strong revealing the relationship between micronucleus assay and cancer. Before conducting in vivo assays, factors concerned with test substances, tissues exposed and mechanisms involved are clearly clarified to avoid irrelevant information and unnecessary scarifice of animals.
Furthermore, germ cell tests are also required because substances which are positive in germ cell tests are regarded as somatic cell mutagens. On the other hand, some substances which give positive in somatic cell assays cannot show positive in germ cell assays. Using only one test cannot reveal gene mutation, clastogenicity and aneugenicity. Therefore, a combination of appropriate tests can detect mutated effects of gene, abnormal structures of chromosome and abnormalities in chromosome number.
Although there are many assays to identify genotoxic effects of substances, no definite test is developed to cover three end points (gene mutation, clastogenicity and aneuploidy). The underlying reason may be due to the fact that some genotoxic agents change the genome by means of gene mutation while some change genome by chromosomal aberrations without gene mutation. Moreover, the outcomes of the assays can be influenced by age, sex, personal habits like smoking and sample preparation. Furthermore, chemical structures as well as pharmacokinetic properties of test substances should be taken into account prior to use of the assays because these factors can give unreasonable conclusions.
To sum up, the final observations of substances which give deleterious effects on genome are gene mutation, abnormalities in number and structure of chromosomes. Although there are many assays to detect genotoxic effects, no single assay can fulfill these three observations. Moreover, each assay has its own significance and factors needed to be considered before conducting the assay. In addition, each assay should be chosen by considering physical and chemical properties of test substances and the nature of assays to avoid unfavorable outcomes and unnecessary animal sacrifice. Therefore, genotoxic effects of substances can be successfully examined by combination of appropriate in vitro, in vivo somatic and germ cell assays.