Potential thresholds for genotoxic effects by micronucleus scoring

The concept of thresholds in genotoxicity has been open for debate in the last decades. The micronucleus (MN) test contributed to a large extent in understanding the dose-response relationship for aneugens and clastogens. The threshold concept for aneuploidy is well accepted by the scientific community based on the data and for mechanistic reasons. The concept of threshold for clastogens is still challenging. Acceptance is based on a case-by-case basis together with thorough mechanistic understanding of the different steps from the mutagen-target interactions to MN formation for this class of genotoxicants. This review summarises the significant achievements in the assessment of threshold for genotoxins using the MN test and concludes with an overview of knowledge gaps and recommendation

Comparison of the peripheral blood micronucleus test using flow cytometry in rat and mouse exposed to aneugens after single-dose applications

Detection of clastogenic compounds in the peripheral blood micronucleus test (MNT) in rats is a well-established methodology. However, the results obtained on the induction of micronuclei by aneugens in rat peripheral blood are controversial. Our aim was a comparative evaluation of the peripheral blood flow cytometry MNT in Wistar Han rat and CD1 mouse exposed to three aneugens (vinblastine, vincristine and colchicine) after single-dose applications. In addition, the same compounds were tested in the rat bone marrow MNT. The treatment with vinblastine (0.25, 0.5, 1, mg/kg), vincristine (0.025, 0.05, 0.1 mg/kg) or colchicine (0.7, 1, 1.3 mg/kg) induced no statistically significant increase in MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) in rat peripheral blood. In rat bone marrow, a clear statistically significant increase in MN-PCE was found with vincristine and vinblastine. However, colchicine showed a clear increase in MN-PCE frequency without reaching statistically significant level only at 1 mg/kg. The positive effect in the bone marrow MNT shows that the target organ was exposed to the appropriate concentration levels of the respective aneugens. In mouse, the peripheral blood flow cytometry analysis after the treatment with vinblastine, vincristine and colchicine showed clear statistically significant increase in MN-PCE with all three compounds. The experiments with splenectomized rats treated with vincristine and colchicine were performed and statistically significant increases in MN-PCE were found with 0.05, 0.1, 0.15 mg/kg of vincristine and 0.7 and 1 mg/kg of colchicine. Our results demonstrate that micronucleated cells induced by aneugens are removed from rat peripheral blood by the spleen due to the large size of micronuclei. Based on our data, it is concluded that the flow cytometry peripheral blood MNT after single-dose applications is an appropriate test system for evaluating the genotoxic effects of aneugens in mice. However, in rats peripheral blood MNT aneugen detection might require multiple-dose applications to overwhelm the spleen effec

In vitro genotoxicity testing using the micronucleus assay in cell lines, human lymphocytes and 3D human skin models

The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is simple to score, accurate, applicable in different cell types. In addition, it is predictive for cancer, amenable for automation and allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro micronucleus (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of an OECD guideline became recently available for this test. In this review, we discuss the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes, the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of protocols is highlighted and new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research perspectives including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterial

Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity

Mitomycin C, 5-fluoruracil, colchicine and etoposide tested in the in vitro mammalian cell micronucleus test (MNvit) in the human lymphoblastoid cell line TK6 at Novartis in support of OECD draft Test Guideline 487

The following reference genotoxic agents were tested in the in vitro micronucleus test, at Novartis, Basel, Switezrland. Mitomycin C, 5-fluoruracil, colchicine and etoposide were tested in the human lymphoblastoid cell line TK6, with and without cytokinesis block (in the presence of cytochalasin B). This was done in support of the toxicity measures recommended in the late 2007 version of the draft OECD Test Guideline 487 for the testing of chemicals and were part of an international collaborative work. As toxicity measures, detecting cytostasis and cell death, relative cell counts (RCC), relative increase in cell counts (RICC), and relative population doubling (RPD) were used for treatments in the absence of cytokinesis block, and replication index (RI) or cytokenesis bloked proliferation in the presence of cytokinesis block. All four reference agents were positive in the assay with and without cytokinesis block at concentrations giving approximately 50% toxicity or less as assessed by all of the toxicity measures used. Accordingly, the outcome from this work supports the use of relative population doubling and relative increase in cell counts, as well as relative cell counts, as appropriate measures of toxicity for the non-cytokinesis blocked in vitro micronucleus assay

Analysis of chromosome loss and chromosome segregation in cytokinesis-blocked human lymphocytes: Non-disjunction is the prevalent mistake in chromosome segregation produced by low dose exposure to ionizing radiation

The aim of the present work was to examine in human lymphocytes, firstly, whether in vitro γ-rays as compared with X-rays also induce chromatid malsegregation and at higher frequencies than chromosome loss and, secondly, whether the cytokinesis-blocked micronucleus assay combined with fluorescence in situ hybridization might be useful for the biomonitoring of individuals exposed to ionizing radiation. After irradiation, the relative frequencies of centromere-positive micronuclei decreased from 39.2 at 0.1 Gy to 21.63 at higher doses. There was no statistically significant increase in MNCen + frequencies at doses below 1 Gy (0.1, 0.25 and 0.5 Gy), but a statistically significant increase at 1 (P < 0.05) and 2 Gy (P < 0.001) was observed for all the donors. No significant differences in baseline and γ-ray-induced non-disjunction frequencies for chromosomes 1 (P = 0.9) and 17 (P = 0.8) between individuals were detected. For radiation-induced non-disjunction, lower doses (0.1, 0.25 and 0.5 Gy) of γ-rays did not induce a statistically significant increase in nondisjunction frequencies whereas 1 Gy and above clearly induced a statistically significant increase in the total nondisjunction frequencies for all the donors (P < 0.05 at 1 Gy and P < 0.0001 at 2 Gy). The aneugenic effect of radiation is less clearly dose dependent at the lower doses, suggesting an apparent threshold below which no change could be demonstrated. At high radiation doses the major mechanism for γ-ray-induced aneuploidy is related to chromosome loss through non-disjunction, as has been demonstrated using X-rays, and not through the formation of micronuclei.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Potential thresholds for genotoxic effects by micronucleus scoring

The concept of thresholds in genotoxicity has been open for debate in the last decades. The micronucleus test contributed to a large extent in understanding the dose response relationship for aneugens and clastogens. The threshold for aneuploidy is well accepted by the scientific community based on the data and for mechanistic reasons. The concept of threshold for clastogens on the other side is still challenging. Acceptance is based on a case by case basis together with thorough mechanistic understanding of the different steps from the mutagen/target interactions to MN formation for this class of genotoxins. This review summarises the significant achievements in the assessment of threshold for genotoxins using the MN test and concludes with an overview of knowledge gaps and recommendations

IN VITRO PRIMARY HUMAN LYMPHOCYTE FLOW CYTOMETRY BASED MICRONUCLEUS ASSAY: SIMULTANEOUS ASSESSMENT OF CELL PROLIFERATION, APOPTOSIS AND MN FREQUENCY.

In order to minimise the number of positive in vitro cytogenetic results which are not confirmed in rodent carcinogenicity tests, biological systems that are p53 and DNA repair proficient should be recommended. Moreover, an appropriate cytotoxicity parameter for top dose selection should be considered. Recent International Conference on Harmonisation draft S2 and Organisation for Economic Co-operation and Development (OECD) 487 guideline accepted the in vitro micronucleus test (MNT) as a valid alternative method for in vitro chromosome aberration test within the in vitro cytogenetic test battery. Since mitosis is a prerequisite for expression of the micronuclei, it is compulsory to demonstrate that cell division occurred, and if possible, to identify the cells that completed mitosis. The OECD guideline recommends the use of a cytokinesis block for the assessment of proliferation in primary T-lymphocytes. The work presented in this manuscript was initiated to develop a novel flow cytometry-based primary human lymphocyte MNT method. This new assay is based on a three-step staining procedure: carboxyfluorescein succinimidyl ester as a proliferation marker, ethidium monoazide for chromatin of necrotic and late apoptotic cells discrimination and 4,6-diaminodino-2-phenylindole as a DNA marker. The proof of principle of the method was performed using genotoxic and non-genotoxic compounds: methyl methanesulfonate, mitomycin C, vinblastine sulphate, cyclophosphamide, sodium chloride and dexamethasone. It has been shown that the new flow cytometry-based primary human lymphocyte MNT method is at least equally reliable method as the standard Cytochalasin B MNT. However, further validation of the assay using a wide selection of compounds with a variety of mechanisms of action is required, before it can be used for regulatory purposes. Moreover, a miniaturisation of the technology may provide an additional advantage for early drug developmen

Automatic Analysis of the Micronucleus Test in Primary Human Lymphocytes Using Image Analysis

The in vitro micronucleus test (MNT) is a well established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and specific parameter. Various automated systems to replace the tedious and time consuming manual slide analysis procedure have been described. Flow cytrometric approaches have been discussed elsewhere. The ROBIAS image analysis system for both automatic cytotoxicity assessment and highly sensitive micronucleus detection in primary human lymphocytes was developed at Novartis, where the assay is used as to confirm positive results obtained in the MNT in TK6 cells which serves as the primary screening system for genotoxicity profiling in early drug development. The comparison of manual with automatic analysis results showed a high degree of concordance for 27 independent experiments conducted for profiling of 12 compounds. For concentration series of Cyclophosphamide (CP) and Carbendazim (MBC), a very good correlation between automatic and manual analysis could be established, both for the relative division index used as cytotoxicity parameter, and for MN scoring in mono- and bi-nucleated cells. Generally, false positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyze 24 slides within 65 hours by fully automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS supporting various assays in genetic toxicology and other biomedical areas

In vitro genotoxic effects of hard metal particles assessed by alkaline single cell gel and elution assays

Hard metals (WC-Co) are made of a mixture of cobalt metal (Co, 5-10%) and tungsten carbide particles (WC, >80%). Excessive inhalation of WC-Co is associated with the occurrence of different lung diseases including an excess of lung cancers. The elective toxicity of hard metal is based on a physico-chemical interaction between cobalt metal and tungsten carbide particles to produce activated oxygen species. The aim of the present study was to assess the genotoxic activity of hard metal particles as compared with Co and WC alone. In human peripheral lymphocytes incubated with Co or WC-Co, a dose- and time-dependent increased production of DNA single strand breaks (ssb) was evidenced by alkaline single cell gel electrophoresis (SCGE) and modified alkaline elution (AE) assays. Addition of 1 M formate, a hydroxyl radical scavenger, had a protective effect against the production of ssb by both WC-Co or Co alone. On the basis of an equivalent cobalt-content, WC-Co produced significantly more ssb than Co. WC alone did not produce DNA ssb detectable by the AE assay, but results obtained with the SCGE assay may suggest that it either allows some uncoiling of the chromatin loops or induces the formation of slowly migrating fragments. Overall, this in vitro study is the first demonstration of the clastogenic property of cobalt metal-containing dusts. The results are consistent with the implication of an increased production of hydroxyl radicals when Co is mixed with WC particles. The SCGE results also suggest that WC may modify the structure of the chromatin, leading to an increased DNA sensitivity to clastogenic effects. Both mechanisms are not mutually exclusive and may concurrently contribute to the greater clastogenic activity of WC-Co dust. This property of WC-Co particles may account for the excess of lung cancers observed in hard metal workers