Frontiers in Genetics (Jun 2015)
Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay
Abstract
The cytokinesis-block micronucleus cytome (CBMN) assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells. The endpoints possible to be measured are micronuclei (MN), a biomarker of chromosome breakage and/or whole chromosome loss, nucleoplasmic bridges (NPB), a biomarker of DNA misrepair and/or telomere end-fusions, and nuclear buds (NBUD), a biomarker of elimination of amplified DNA and/or DNA repair complexes. Cytostatic effects are measured via the proportion of mono-, bi- and multinucleated cells and cytotoxicity via necrotic and/or apoptotic cell ratios (Fenech, 2006, 2007). The CBMN assay has become one of the most commonly used methods for assessing chromosome breakage and loss in human lymphocytes both in vivo and ex vivo (Fenech et al., 1999a; 1999b). Because of its reliability and good reproducibility, the CBMN assay has become one of the standard cytogenetic tests for genetic toxicology testing in human and mammalian cells (Fenech & Crott, 2002; Fenech, 2007) and has been extensively used to evaluate the presence and the extent of chromosome damage in human populations exposed to genotoxic agents in various occupational settings, in the environment, or as a consequence of lifestyles (Bonassi et al., 2011). The use of the CBMN assay in in vitro genetic toxicology testing is well established and in fact it has become an accepted standard method to assess the genotoxic hazard of chemicals which led to the development of a special guideline by the Organization for Economic Cooperation and Development (OECD), the OECD 487 guideline (Kirsch-Volders et al., 2014). The CBMN assay is an effective tool for the study of cellular and nuclear dysfunction caused by in vitro or in vivo aging, micronutrient deficiency or excess, genotoxins exposure and genetic defects in genome maintenance. It is also fruitful in the emerging fields of nutrigenomics and toxicogenomics and their combinations, as it becomes increasingly clear that nutrient status also impacts on sensitivity to exogenous genotoxins (Fenech, 2005, 2007). Many results obtained by this assay indicate the potential predictive value of the CBMN assay with respect to cancer risk and validate its use as a test for detecting nutritional, environmental and genetic factors that are potentially carcinogenic. Also it is used by pharmaceutical industry, human biomonitoring of genotoxic exposures and its increasing application in preventive medicine and nutrition and the increased investment in the automation of the CBMN assay are indicative of the increasing importance of this test (Fenech, 2007). The comet assay or single-cell gel electrophoresis (SCGE) is a simple, sensitive method for detecting DNA-strand breaks. Cells embedded in agarose on a microscope slide are lysed with detergent and 2.5 M NaCl and fresh Triton X-100 to remove membranes and soluble cell constituents, including most histones, leaving the DNA, still supercoiled and attached to a nuclear matrix, as a nucleoid. A break in one strand of a DNA loop is enough to release the supercoiling, and during electrophoresis the relaxed loops are able to extend towards the anode (Fairbairn et al., 1995; Collins et al., 1997; Moller et al., 2000; Azqueta et al., 2009; Collins & Dusinska, 2009). Electrophoresis causes DNA loops containing breaks to move towards the anode, forming “comets” when stained and visualised by fluorescence microscopy. The relative content of DNA in the tail indicates the frequency of breaks (Gedik et al., 2002; Kumaravel & Jha, 2006; Collins & Dusinska, 2009). DNA strand breaks can originate from the direct modification of DNA by chemical agents or their metabolites; from the processes of DNA excision repair, replication, and recombination; or from the process of apoptosis. Direct breakage of the DNA strands occurs when ROS interact with DNA. In what refers to alkaline labile sites, those can be generated by depurination of an adducted base of the nucleotide and a subsequent conversion of the abasic site to a strand break detected by alkaline treatment (pH above 13.1) (Moller et al., 2000). This assay was adapted to measure oxidised purines and oxidised pyrimidines by the incubation of the nucleoids with bacterial DNA repair enzymes (Azqueta et al., 2009), including formamidopyrimidine DNA glycosylase (FPG), which recognizes the oxidised purine 8-OHdG, Endonuclease III do detect oxidised pyrimidines, T4 endonuclease V to detect UV-induced pyrimidines dimmers, AlkA (3-methyladenine DNA glycosylase) for alkylated bases, or uracil DNA glycosylase, which removes misincorporated uracil from DNA (Collins & Dusinska, 2009). Comet assay has become one of the standard methods for assessing DNA damage, with a wide range of applications, namely in genotoxicity testing, human biomonitoring and molecular epidemiology, as well as fundamental research in DNA damage and repair (Collins, 2004, 2009). This assay is useful for evaluating xenobiotic impacts based on its use of small cell samples, and its ability to evaluate DNA damage in non-proliferation cells such as lymphocytes. In addition, the ability to obtain sufficient numbers of cells for analysis from different tissues, for instance lymphocytes and buccal cells provides a relatively non-invasive procedure for analysis (Valverde & Rojas, 2009a). Two samples were formed – the group of those occupationally exposed to antineoplastic drugs (n=46) and the non-exposed group (n=46). Significant differences (Mann-Whitney test, p 0.05) were found between subjects with and without exposure. There were positive significant correlations between endpoints evaluated by the same technique. Regarding CBMN assay results, there was a positive correlation between NPB and NBUDs (r=0.362, p=0.013). As for the comet assay, % DNA in the tail and DNA oxidative damage, were correlated (r = 0.325, p=0.002). However, correlations across the two assays – CBMN and comet assays –were not significant. The comet assay and the CBMN assay detect genotoxic effects caused by different mechanisms. The comet assay identifies still reparable injuries such as single and double-strand DNA breaks, alkali labile lesions that are converted to strand breaks under alkaline conditions and single-strand breaks associated with incomplete excision repair sites; whereas the CBMN assay detects injuries that survive at least one mitotic cycle and reflect unrepaired fixed DNA damage (Villarini et al., 2012). It is considered that, for chronic exposures, micronuclei test express cumulative facts whereas comet assay provides information on recent exposures (Laffon et al., 2005). Comet assay used in biomonitoring studies reflect the current exposure (over the previous few weeks) and the actual levels of DNA damage present in white blood cells at the moment of blood sampling. The comet assay is able to sensitively reveal early, still repairable, moderate DNA damage, and can therefore furnish useful information on early effects induced by occupational exposure to low doses of xenobiotics (Kopjar et al., 2009) being recommended to monitor population chronically exposed to genotoxic agents combined with CBMN assay (Maluf & Erdtmann, 2000; Kopjar & Garaj-Vrhovac, 2001; Rekhadevi et al., 2007; Cornetta et al., 2008; Cavallo et al., 2009; Kopjar et al., 2009). A combination of cytogenetic tests and the comet assay in biomonitoring studies makes it possible to compare the relative sensitivities of the two test systems and, therefore, gives us a possible clue about the fraction of the DNA damage detected by the comet assay that will lead to fixed mutations (Milic et al., 2010). The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005) study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009), the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005).
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