Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferation and diseases is still lacking. This review reflects on the efforts and results of a large European research network that, from the 1980's until 2004, focused on protection against aneuploidy-inducing factors and tackled the following problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2) aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays to identify aneugenic factors and classify them according to their modes of action; 5) the need for reliable, quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for mechanistic insight into the consequences of aneuploidy for human health. This activity brought together a consortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelines and to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EU research programmes on aneuploidy were having a valuable scientific approach based on well-selected compounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibility and inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascination with the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at the right time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprise benefited from a solid partnership under the lead of an inspired and stimulating coordinator. The research programme elucidated the major modes of action of aneugens, developed scientifically sound assays to assess aneugens in different tissues, and achieved the international validation of relevant assays with the goal of protecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will require additional research, and the study of effects of exposure to multiple agents should become a priority. It is hoped that these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.

1. The Application of Structure Activity Relationships to the Prediction of the Mutagenic Activity of Chemicals Philip Judson 2. Bacterial Mutagenicity Assays: Test Methods David Gatehouse 3. The Mouse Lymphoma Assay Melvyn Lloyd and Darren Kidd 4. Mammalian Cell HPRT Gene Mutation Assay: Test Methods George E. Johnson 5. The In Vitro Mammalian Chromosome Aberration Test Gill Clare 6. The Interpretation and Analysis of Cytogenetic Data Natalie Danford 7. The In Vitro Micronucleus Assay Ann T. Doherty 8. The In Vitro and In Vivo Comet Assays Brian Burlinson 9. Assessment of DNA Interstrand Crosslinks (ICLs) Using the Modified Alkaline Comet Assay Jian Hong Wu and Nigel J. Jones 10. 32P-Postlabelling for the Sensitive Detection of DNA Adducts Nigel J. Jones 11. Methods for the Detection of DNA Adducts Karen Brown 12. The GADD45a-GFP GreenScreen HC Assay Richard M. Walmsley and Matthew Tate 13. Real Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR): Technical Considerations for Gene Expression Analysis Shareen H. Doak and Zoulikha M. Zair 14. Cytogenetic In Vivo Assays in Somatic Cells Ann T. Doherty, Adolph Baumgartner, and Diana Anderson 15. Cytogenetic Methods in Human Biomonitoring: Principles and Uses Raluca A. Mateuca, Ilsa Decordier, and Micheline Kirsch-Volders 16. The Measurement of Induced Genetic Change in Mammalian Germ Cells Ilse-Dore Adler, Francesca Pacchierotti, and Antonella Russo 17. Transgenic Animal Mutation Models: A Review of the Models and How They Function Steve Dean 18. Analysis of Genotoxicity Data in a Regulatory Context Ian de G. Mitchell and David O.F. Skibinski

Sudan-1 and para red are industrial dyes that have been illegally added to some foodstuffs, leading to withdrawal of the adulterated products throughout the UK since 2003. This resulted in international concern that arose because Sudan-1 is classified by International Agency for Research on Cancer as a Category 3 carcinogen. However, little is known about the dose response of this chemical at low, more biologically relevant, doses. The study therefore aimed to characterize the dose response for gene mutation and chromosomal damage induced by two azo dyes, namely Sudan-1 and para red. Gene mutations were analysed using the hypoxanthine phosphoribosyltransferase forward mutation assay and chromosomal damage was measured using the cytokinesis-blocked micronucleus assay. Two cell lines were used in these investigations. These were the AHH-1 cell line, which inducibly expresses CYP1A1, and the MCL-5 cell line derived from a subpopulation of AHH-1 cells that expresses a particularly high level of CYP1A1 activity. The MCL-5 cell line has also been transfected with two plasmids that stably express CYP1A2, CYP2A6 and CYP3A4 and all four of these CYP enzymes are known to metabolically activate Sudan-1. AHH-1 cells were used to investigate the dose response of the azo dyes, and MCL-5 cells were used to see if the dose response changed with increased metabolism. Sudan-1 induced a non-linear dose-response curve for gene mutation and chromosomal damage in AHH-1 cells. The genotoxic activity of Sudan-1 was greatly increased in MCL-5 cells. This indicated that the oxidation metabolites from Sudan-1 were both more mutagenic and more clastogenic than the parent compound. Para red also demonstrated a non-linear dose response for both gene mutation and chromosome damage in AHH-1 cells, and an increase in micronuclei induction was observed after increased oxidative metabolism in MCL-5 cells. Sudan-1 and para red are genotoxic chemicals with non-linear dose responses in AHH-1 but not in MCL-5 cells, and oxidative metabolism increases the genotoxic effect of both compounds.

The ability of the in vitro mammalian cell tests currently used to identify genotoxins has been shown to be limited by a high rate of false-positive results, triggering further unnecessary testing in vivo. During an European Centre for the Validation of Alternative Methods workshop on how to improve the specificity of these assays, testing at high concentrations was identified as one possible source of false positives. Thus far, Organisation for Economic Co-operation and Development genotoxicity test guidelines have required testing of chemicals using mammalian cells in vitro should be undertaken to concentrations as high as 10 mM (5000 μg/ml). Recently, a draft revision of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use genotoxicity test guidelines has recommended that testing concentrations should be reduced to 1 mM (500 μg/ml). To assess the impact that this lowering would have on the outcome of in vitro genotoxicity testing, we established a database of 384 chemicals classified as rodent carcinogens and reported Ames test results and the test concentrations that produced positive results in the mouse lymphoma assay (MLA), in vitro chromosome aberration (CA) assay and in vitro micronucleus test. Genotoxicity testing results were illustrated for 229 and 338 compounds in the MLA and in vitro CA assay, respectively. Of these test compounds, 62.5% produced positive results in the MLA, of which 20.3% required testing between 1 and 10 mM. A total of 58.0% produced positive results in in vitro CA assays, of which 25.0% required testing between 1 and 10 mM. If the testing concentration limit for mammalian cell assays was reduced to 1 mM, 24 (6.25%) potential carcinogens would not be detected in any part of the standard in vitro genotoxicity test battery (Ames test, MLA and in vitro CA assay). Further re-evaluation and/or retest of these compounds by Kirkland and Fowler [Kirkland, D. and Fowler, P. (2010) Further analysis of Ames-negative rodent carcinogens that are only genotoxic in mammalian cells in vitro at concentrations exceeding 1 mM, including retesting of compounds of concern. Mutagenesis 25, 539-553] suggest that the current 10 mM top concentration can be reduced without any loss of sensitivity in detecting rodent carcinogens.

The study was concerned with investigating the specific effects of non-DNA reactive oestrogens at low "biologically relevant" doses and the causative role they may play in breast cancer through inducing aneuploidy. A review of previous studies identified a non-random pattern of aneuploidy seen in breast cancers. This information was used to select those chromosomes that undergo copy number changes in breast cancer and chromosomes that appear stable. A panel of centromeric specific probes were selected and centromeric specific fluorescence in situ hybridisation (FISH) was carried out on the human lymphoblastoid cell line, AHH-1, which had been pre-treated with the chemical aneugens 17-beta oestradiol, diethylstilbestrol (DES) and bisphenol-A (BP-A). The results suggest that oestrogens may play a causative role in breast cancer by inducing a specific pattern of aneuploidy similar to that seen in breast carcinomas. 17-beta oestradiol appears to induce changes most similar to those seen in breast tumours, BP-A induces the same pattern but at a lower frequency and DES appears to be less chromosome specific in its act.

A complete hazard and risk assessment of any known genotoxin requires the evaluation of the mutagenic, clastogenic and aneugenic potential of the compound. In the case of aneugenic chemicals, mechanism of action (MOA) and quantitative responses may be investigated by studying their effects upon the fidelity of functioning of components of the cell cycle. These present studies have demonstrated that the plastics component bisphenol-A (BPA) and the natural pesticide rotenone induce micronuclei and modify the functioning of the microtubule organising centres (MTOCs) of the mitotic spindles of cultured mammalian cells in a dose-dependent manner. BPA and rotenone were used as model compounds in an investigation of dose response relationships for the hazard/risk assessment of aneugens. Thresholds of action for the induction of aneuploidy have been predicted for spindle poisons on the basis of the multiple targets, which may need disabling before a quantitative response can be detected. The cytokinesis blocked micronucleus assay (CBMA) methodology was utilised in the human lymphoblastoid cell lines AHH-1, MCL-5 and Chinese hamster V79 cell lines. A no observable effect level (NOEL) at 10.8μg/ml BPA was observed for MN induction. Rotenone showed a small increase in MN induction with the first significant effect at 0.25ng/ml in V79 cells but there was no significant effect in the metabolically competent cell line, MCL-5. For a mechanistic evaluation of the aneugenic effects of BPA and rotenone, fluorescently labelled antibodies were used to visualise microtubules (α-tubulin) and MTOCs (γ-tubulin). The NOELs for tripolar mitotic spindle induction in V79 cells were 7μg/ml for BPA and 80pg/ml for rotenone (concentrations which produced similar changes to mitotic index (M.I.)). Interestingly there was close proximity to the NOEL of 10.8μg/ml BPA for micronucleus (MN) induction in the human lymphoblastoid AHH-1 cell. Multiple MTOCs can therefore be predicted as a possible mechanism for MN induction. The similarity in concentration inducing tripolar mitosis, M.I. and MN changes suggests immunofluorescence analysis to be a useful dose setting assay with emphasis on the mechanism.

A mechanistic understanding of carcinogenic genotoxicity is necessary to determine consequences of chemical exposure on human populations and improve health risk assessments. Currently, linear dose-responses are assumed for DNA reactive compounds, ignoring cytoprotective processes that may limit permanent damage. To investigate the biological significance of low-dose exposures, human lymphoblastoid cells were treated with alkylating agents that have different mechanisms of action and DNA targets: methylmethane sulfonate (MMS), methylnitrosourea (MNU), ethylmethane sulfonate (EMS), and ethylnitrosourea (ENU). Chromosomal damage and point mutations were quantified with the micronucleus and hypoxanthine phosphoribosyltransferase forward mutation assays. MNU and ENU showed linear dose-responses, whereas MMS and EMS had nonlinear curves containing a range of nonmutagenic low doses. The lowest observed effect level for induction of chromosomal aberrations was 0.85 microg/mL MMS and 1.40 microg/mL EMS; point mutations required 1.25 microg/mL MMS and 1.40 microg/mL EMS before a mutagenic effect was detected. This nonlinearity could be due to homeostatic maintenance by DNA repair, which is efficient at low doses of compounds that primarily alkylate N(7)-G and rarely attack O atoms. A pragmatic threshold for carcinogenicity may therefore exist for such genotoxins.

OBJECTIVES Because a risk of cancer arising in enterocystoplasties exists, it is necessary to identify which patients are most at risk of tumor formation. The aim of this study was to determine whether rare mutated p53 sequences were more common at the enterovesical anastomosis than in the bladder remnant in patients with a clam ileocystoplasty using the restriction site mutation (RSM) assay. METHODS DNA was extracted from endoscopic biopsies obtained from the ileovesical anastomosis and native bladder remnant (control specimens) of 38 patients with a clam ileocystoplasty. The RSM assay was used to study five known hotspots for mutations of the p53 gene using the restriction enzymes Hha I (codon 175), Taq I (codon 213), Hae III (codon 249/250), and Msp I (codons 248 and 282). The mutational events of p53 were confirmed by sequencing the undigested mutated polymerase chain reaction products identified by RSM analysis. RESULTS We found p53 mutations at the ileovesical anastomosis in 7 of the 38 patients. The mutations were observed at codon 213 (n = 1), codon 248 (n = 3), and codon 250 (n = 3). No p53 mutations were detected in any control specimen. CONCLUSIONS The ileovesical anastomosis is genetically unstable in patients with a clam ileocystoplasty. The p53 mutations identified by the RSM assay at the enterovesical anastomosis could possibly be used as markers of genetic instability to identify patients at risk of developing a tumor. Prospective, randomized longitudinal studies are required to substantiate this hypothesis. UROLOGY 70: 905-909, 2007. (c) 2007 Elsevier Inc.

Workshop participants agreed that genotoxicity tests in mammalian cells in vitro produce a remarkably high and unacceptable occurrence of irrelevant positive results (e.g. when compared with rodent carcinogenicity). As reported in several recent reviews, the rate of irrelevant positives (i.e. low specificity) for some studies using in vitro methods (when compared to this "gold standard") means that an increased number of test articles are subjected to additional in vivo genotoxicity testing, in many cases before, e.g. the efficacy (in the case of pharmaceuticals) of the compound has been evaluated. If in vitro tests were more predictive for in vivo genotoxicity and carcinogenicity (i.e. fewer false positives) then there would be a significant reduction in the number of animals used. Beyond animal (or human) carcinogenicity as the "gold standard", it is acknowledged that genotoxicity tests provide much information about cellular behaviour, cell division processes and cellular fate to a (geno)toxic insult. Since the disease impact of these effects is seldom known, and a verification of relevant toxicity is normally also the subject of (sub)chronic animal studies, the prediction of in vivo relevant results from in vitro genotoxicity tests is also important for aspects that may not have a direct impact on carcinogenesis as the ultimate endpoint of concern.In order to address the high rate of in vitro false positive results, a 2-day workshop was held at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy in April 2006. More than 20 genotoxicity experts from academia, government and industry were invited to review data from the currently available cell systems, to discuss whether there exist cells and test systems that have a reduced tendency to false positive results, to review potential modifications to existing protocols and cell systems that might result in improved specificity, and to review the performance of some new test systems that show promise of improved specificity without sacrificing sensitivity.It was concluded that better guidance on the likely mechanisms resulting in positive results that are not biologically relevant for human health, and how to obtain evidence for those mechanisms, is needed both for practitioners and regulatory reviewers.Participants discussed the fact that cell lines commonly used for genotoxicity testing have a number of deficiencies that may contribute to the high false positive rate. These include, amongst others, lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g. Aroclor-induced rat S9), impaired p53 function and altered DNA repair capability.The high concentrations of test chemicals (i.e. 10 mM or 5000 mu g/ml, unless precluded by solubility or excessive toxicity) and the high levels of cytotoxicity currently required in mammalian cell genotoxicity tests were discussed as further potential sources of false positive results. Even if the goal is to detect carcinogens with short in vitro tests under more or less acute conditions, it does not seem logical to exceed the capabilities of cellular metabolic turnover, activation and defence processes. The concept of "promiscuous activation" was discussed. For numerous mutagens, the decisive in vivo enzymes are missing in vitro. However, if the substrate concentration is increased sufficiently, some other enzymes (that are unimportant in vivo) may take over the activation-leading to the same or a different active metabolite. Since we often do not use the right enzyme systems for positive controls in vitro, we have to rely on their promiscuous activation, i.e. to use excessive concentrations to get an empirical correlation between genotoxicity and carcinogenicity. A thorough review of published and industry data is urgently needed to determine whether the currently required limit concentration of 10 mM or 5000 mu g/ml, and high levels of cytotoxicity, are necessary for the detection of in vivo genotoxins and DNA-reactive, mutagenic carcinogens.In addition, various measures of cytotoxicity are currently allowable under OECD test guidelines, but there are few comparative data on whether different measures would result in different maximum concentrations for testing. A detailed comparison of cytotoxicity assessment strategies is needed. An assessment of whether test endpoints can be selected that are not intrinsically associated with cytotoxicity, and therefore are less susceptible to artefacts produced by cytotoxicity, should also be undertaken.There was agreement amongst the workshop participants that cell systems which are p53 and DNA-repair proficient, and have defined Phase 1 and Phase 2 metabolism, covering a broad set of enzyme forms, and used within the context of appropriately set limits of concentration and cytotoxicity, offer the best hope for reduced false positives. Whilst there is some evidence that human lymphocytes are less susceptible to false positives than the current rodent cell lines, other cell systems based on HepG2, TK6 and MCL-5 cells, as well as 3D skin models based on primary human keratinocytes also show some promise. Other human cell lines such as HepaRG, and human stem cells (the target for carcinogenicity) have not been used for genotoxicity investigations and should be considered for evaluation. Genetic engineering is also a valuable tool to incorporate missing enzyme systems into target cells. A collaborative research programme is needed to identify, further develop and evaluate new cell systems with appropriate sensitivity but improved specificity.In order to review current data for selection of appropriate top concentrations, measures and levels of cytotoxicity, metabolism, and to be able to improve existing or validate new assay systems, the participants called for the establishment of an expert group to identify the in vivo genotoxins and DNA-reactive, mutagenic carcinogens that we expect our in vitro genotoxicity assays to detect as well as the non-genotoxins and non-carcinogens we expect them not to detect. (c) 2007 Elsevier B.V. All rights reserved.

The incidence of carcinoma following an enterocystoplasty increases with time and is a major concern after such procedures. The aim of this study was to investigate genetic instability (in the form of numerical chromosomal aberrations) at the enterovesical anastomosis in patients who had undergone a clam ileocystoplasty using fluorescent in-situ hybridisation (FISH). Fluorescent in-situ hybridisation was performed on touch preparation samples prepared from fresh endoscopic biopsies obtained from the enterovesical anastomosis and native bladder remnant (control specimens) of 15 patients who had undergone a clam ileocystoplasty. Fluorescent in-situ hybridisation was also performed on one squamous cell cancer specimen. Significant aneusomic changes were found at the enterovesical anastomosis in all 15 patients. Alterations in chromosome 18 copy number were the most frequent abnormal finding (trisomy 18, n=8; monosomy 18, n=7). Nine patients were monosomic for chromosome 9. Isolated monosomy 8 and trisomy 8 were each found in one patient. The control specimens were all normal. An unusually high incidence of polysomic cells was found in the clam tumour specimen, reflecting the aggressive nature of this cancer. Chromosomal numerical abnormalities occur at the enterovesical anastomosis following a clam ileocystoplasty and chromosome 18 appears to be a particularly good marker of genetic instability. The results of this study indicate that morphologically normal tissue obtained from the enterovesical anastomosis displays evidence of chromosomal instability that may predispose to tumour formation. However, further prospective, blinded, longitudinal studies are required to establish whether predetermined FISH signal patterns in enterocystoplasty cells in urine or obtained by biopsy predict the presence or absence of tumour.

The successful validation of the in vitro micronucleus assay by the SFTG now provides the opportunity for this highly cost effective assay to be used to screen chemicals for their ability to induce both structural (clastogenic) and numerical (aneugenic) chromosome changes using interphase cells. The use of interphase cells and a relatively simple experimental protocol provides the opportunity to greatly increase the statistical power of cytogenetic studies on chemical interactions. The application of molecular probes capable of detecting kinetochores and centromeres provides the opportunity to classify mechanisms of micronucleus induction into those which are primarily due to chromosome loss or breakage. When a predominant mechanism of micronucleus induction has been shown to be based upon chromosome loss then further investigation can involve the determination of the role of non-disjunction in the induction of aneuploidy. The binucleate cell modification of the in vitro micronucleus assay can be combined with the use of chromosome specific centromere probes to determine the segregation of individual chromosomes into daughter nuclei. The combination of these methods provides us with powerful tools for the investigation of mechanisms of genotoxicity particularly in the low dose regions.

In this series of experiments, a novel protocol was developed whereby gastric cells were collected using endoscopic cytology brush techniques, and prepared, such that interphase fluorescence in situ hybridization (FISH) could be performed. In total, 80 distinct histological samples from 37 patients were studied using four chromosome probes (over 32 000 cells analysed). Studies have previously identified abnormalities of these four chromosomes in upper GI tumours. Using premalignant tissues, we aimed to determine how early in Correa's pathway to gastric cancer these chromosome abnormalities occurred. Aneuploidy of chromosomes 4, 8, 20 and 17(p53) was detected in histologically normal gastric mucosa, as well as in gastritis, intestinal metaplasia, dysplasia and cancer samples. The levels of aneuploidy increased as disease severity increased. Amplification of chromosome 4 and chromosome 20, and deletion of chromosome 17(p53) were the more common findings. Hence, a role for these abnormalities may exist in the initiation of, and the progression to, gastric cancer. Helicobactor pylori infection was determined in premalignant tissue using histological analysis and PCR technology. Detection rates were comparable. PCR was used to subtype H. pylori for CagA status. The amplification of chromosome 4 in gastric tissue was significantly more prevalent in H. pylori-positive patients (n=7) compared to H. pylori-negative patients (n=11), possibly reflecting a role for chromosome 4 amplification in H. pylori-induced gastric cancer. The more virulent CagA strain of H. pylori was associated with increased disease pathology and chromosomal abnormalities, although numbers were small (CagA+ n=3, CagA− n=4). Finally, in vitro work demonstrated that the aneuploidy induced in a human cell line after exposure to the reactive oxygen species (ROS) hydrogen peroxide was similar to that already shown in the gastric cancer pathway, and may further strengthen the hypothesis that H. pylori causes gastric cancer progression via an ROS-mediated mechanism.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder characterized by premature ageing in childhood and serves as a valuable model for the human ageing process in general. Most recently, point mutations in the lamin A (LMNA) gene on chromosome 1q have been associated with the disease, however how these mutations relate to the complex phenotype of HGPS remains to be established. It has been shown that fibroblasts from HGPS patients are frequently resistant to immortalization with telomerase (hTERT), consistent with the idea that the loss of a dominant acting HGPS gene is a pre-requisite for immortalization. In this study we report the first detailed cytogenetic analysis of hTERT-immortalised HGPS cell lines from three patients and one corresponding primary fibroblast culture. Our results provide evidence for a cytogenetic mosaicism in HGPS with a distinctive pattern of chromosome aberrations in all the HGP clones. Chromosome 11 alterations were observed at a high frequency in each immortalised HGPS cell line but were also present at a lower frequency in the corresponding primary cells. Moreover, we were able to identify the 11q13→q23 region as a potential site of breakage. Our results are therefore consistent with a role of chromosome 11 alterations in the escape from senescence observed in HGPS cells. In addition to this defined rearrangement, we consistently observed complex chromosomal rearrangements, suggesting that HGPS displays features of chromosomal instability.