The Aneugenicity of Ketone Bodies in Colon Epithelial Cells Is Mediated by Microtubule Hyperacetylation and Is Blocked by Resveratrol.

Diabetes mellitus (DM) is considered to be associated with an increased risk of colorectal cancer. Recent studies have also revealed that tubulin hyperacetylation is caused by a diabetic status and we have reported previously that, under microtubule hyperacetylation, a microtubule severing protein, katanin-like (KL) 1, is upregulated and contributes to tumorigenesis. To further explore this phenomenon, we tested the effects of the ketone bodies, acetoacetate and ß-hydroxybutyrate, in colon and fibroblast cells. Both induced microtubule hyperacetylation that responded differently to a histone deacetylase 3 knockdown. These two ketone bodies also generated intracellular reactive oxygen species (ROS) and hyperacetylation was commonly inhibited by ROS inhibitors. In a human fibroblast-based microtubule sensitivity test, only the KL1 human katanin family member showed activation by both ketone bodies. In primary cultured colon epithelial cells, these ketone bodies reduced the tau protein level and induced KL1- and α-tubulin acetyltransferase 1 (ATAT1)-dependent micronucleation. Resveratrol, known for its tumor preventive and tubulin deacetylation effects, inhibited this micronucleation. Our current data thus suggest that the microtubule hyperacetylation induced by ketone bodies may be a causal factor linking DM to colorectal carcinogenesis and may also represent an adverse effect of them that needs to be controlled if they are used as therapeutics.

A new tool for genotoxic risk assessment: Reevaluation of the cytokinesis-block micronucleus assay using semi-automated scoring following telomere and centromere staining.

BACKGROUND: The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS: Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS: The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION: These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.

Aneugen Molecular Mechanism Assay: Proof-of-Concept With 27 Reference Chemicals.

A tiered bioassay and data analysis scheme is described for elucidating the most common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of mitotic kinase(s). To evaluate this strategy, TK6 cells were first exposed to each of 27 presumed aneugens over a range of concentrations. After 4 and 24 h of treatment, γH2AX, p53, phospho-histone H3 (p-H3), and polyploidization biomarkers were evaluated using the MultiFlow DNA Damage Assay Kit. The assay identified 27 of 27 chemicals as genotoxic, with 25 exhibiting aneugenic signatures, 1 aneugenic and clastogenic, and 1 clastogenic. Subsequently, a newly described follow-up assay was employed to investigate the aneugenic agents' molecular targets. For these experiments, TK6 cells were exposed to each of 26 chemicals in the presence of 488 Taxol. After 4 h, cells were lysed and the liberated nuclei and mitotic chromosomes were stained with a nucleic acid dye and labeled with fluorescent antibodies against p-H3 and Ki-67. Flow cytometric analyses revealed that alterations to 488 Taxol-associated fluorescence were only observed with tubulin binders-increases in the case of tubulin stabilizers, decreases with destabilizers. Mitotic kinase inhibitors with known Aurora kinase B inhibiting activity were the only aneugens that dramatically decreased the ratio of p-H3-positive to Ki-67-positive nuclei. Unsupervised hierarchical clustering based on 488 Taxol fluorescence and p-H3: Ki-67 ratios clearly distinguished compounds with these disparate molecular mechanisms. Furthermore, a classification algorithm based on an artificial neural network was found to effectively predict molecular target, as leave-one-out cross-validation resulted in 25/26 agreement with a priori expectations. These results are encouraging, as they suggest that an adequate number of training set chemicals, in conjunction with a machine learning algorithm based on 488 Taxol, p-H3, and Ki-67 responses, can reliably elucidate the most commonly encountered aneugenic molecular targets.

Tributyltin oxide exposure impairs mouse oocyte maturation and its possible mechanisms.

Tributyltin oxide (TBTO) has been widely used as marine antifouling composition, preservative, biocide, and a stabilizer in plastic industry. Previous studies have indicated that TBTO can cause immunotoxicity as an environmental pollutant. However, little is known about its reproductive toxicity, especially on female oocyte maturation and the underlying mechanisms. In this study, mouse oocytes were cultured with different concentrations of TBTO in vitro, and several crucial events during meiotic maturation were evaluated. We found that the first polar body extrusion rate was significantly reduced, which reflected the disruption of meiotic maturation. The rate of abnormal spindle organization increased significantly, accompanied with a higher rate of chromosome misalignment. In addition, TBTO treatment increased reactive oxygen species generation markedly, which also accelerated the early-stage apoptosis. Moreover, heterogeneous mitochondrial distribution, mitochondrial dysfunction, and higher rate of aneuploidy were detected, which consequently disrupted in vitro fertilization. In conclusion, our results indicated that TBTO exposure could impair mouse oocyte maturation by affecting spindle organization, chromosome alignment, mitochondria functions, oxidative stress, and apoptosis.

Differentiation of Aneugens and Clastogens in the In Vitro Micronucleus Test by Kinetochore Scoring Using Automated Image Analysis.

The in vitro micronucleus test according to OECD Test Guideline 487 (TG 487) is widely used to investigate the genotoxic potential of drugs. Besides the identification of in vitro genotoxicants, the assay can be complemented with kinetochore staining for the differentiation between clastogens and aneugens. This differentiation constitutes a major contribution to risk assessment as especially aneugens show a threshold response. Thus, a novel method for automated MN plus kinetochore (k+) scoring by image analysis was developed based on the OECD TG 487. Compound-induced increases in MN frequency can be detected using the cytokinesis-block (cytochalasin B) method in V79 cells after 24 h in a 96-well format. Nuclei, MN, and kinetochores were labeled with nuclear counterstain and anti-kinetochore antibodies, respectively, to score MN in binuclear or multinuclear cells and to differentiate compound-induced MN by the presence of kinetochores. First, a reference data set was created by manual scoring using two clastogens and aneugens. After developing the automated scoring process, a set of 14 reference genotoxicants were studied. The automated image analysis yielded the expected results: 5/5 clastogens and 6/6 aneugens (sensitivity: 100%) as well as 3/3 non-genotoxicants (specificity: 100%) were correctly identified. Further, a threshold was determined for identifying aneugens. Based on the data for our internally characterized reference compounds, unknown compounds that induce ≥53.8% k+ MN are classified as aneugens. The current data demonstrate excellent specificity and sensitivity and the methodology is superior to manual microscopic analysis in terms of speed and throughput as well as the absence of human bias. Environ. Mol. Mutagen. 60:227-242, 2019. © 2018 Wiley Periodicals, Inc.

In vivo pig-a and micronucleus study of the prototypical aneugen vinblastine sulfate.

The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days -4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen. 59:30-37, 2018. © 2017 Wiley Periodicals, Inc.

A tripartite mode of action approach for investigating the impact of aneugens on tubulin polymerization.

Chemical-induced disruption of the cellular microtubule network is one key mechanism of aneugenicity. Since recent data indicate that genotoxic effects of aneugens show nonlinear dose-response relationships, margins of safety can be derived with the ultimate goal to perform a risk assessment for the support of drug development. Furthermore, microtubule-interacting compounds are widely used for cancer treatment. While there is a need to support the risk assessment of tubulin-interacting chemicals using reliable mechanistic assays, no standard assays exist to date in regulatory genotoxicity testing for the distinction of aneugenic mechanisms. Recently reported methods exclusively rely on either biochemical, morphological, or cytometric endpoints. Since data requirements for the diverse fields of application of those assays differ strongly, the use of multiple assays for a correct classification of aneugens is ideal. We here report a tripartite mode of action approach comprising a cell-free biochemical polymerization assay and the cell-based methods cellular imaging and flow cytometry. The biochemical assay measures tubulin polymerization over time whereas the two cell-based assays quantify tubulin polymer mass. We herein show that the flow cytometric method yielded IC50 values for tubulin destabilizers and EC50 values for tubulin stabilizers as well as cell cycle information. In contrast, cellular imaging complemented these findings with characteristic morphological patterns. Biochemical analysis yielded kinetic information on tubulin polymerization. This multiplex approach is able to create holistic effect profiles which can be individually customized to the research question with regard to quality, quantity, usability, and economy. Environ. Mol. Mutagen. 59:188-201, 2018. © 2017 Wiley Periodicals, Inc.

Surface-modified magnetite nanoparticles act as aneugen-like spindle poison.

Iron oxide nanoparticles are one of the most promising types of nanoparticles for biomedical applications, primarily in the context of nanomedicine-based diagnostics and therapy; hence, great attention should be paid to their bio-safety. Here, we investigate the ability of surface-modified magnetite nanoparticles (MNPs) to produce chromosome damage in human alveolar A549 cells. Compared to control cells, all the applied MNPs increased the level of micronuclei moderately but did not cause structural chromosomal aberrations in exposed cells. A rise in endoreplication, polyploid and multinuclear cells along with disruption of tubulin filaments, downregulation of Aurora protein kinases and p53 protein activation indicated the capacity of these MNPs to impair the chromosomal passenger complex and/or centrosome maturation. We suppose that surface-modified MNPs may act as aneugen-like spindle poisons via interference with tubulin polymerization. Further studies on experimental animals revealing mechanisms of therapeutic-aimed MNPs are required to confirm their suitability as potential anti-cancer drugs.

Chromosomal aberrations, clastogens vs aneugens.

Current anticancer therapy may be one of the most important exogenous sources of exposure to genotoxic agents in US, Japan, and Europe, where approximately 40-55 percent of the population is diagnosed with cancer at a certain point in their life. This review focuses on recent efforts to integrate a novel biomarker, gamma-H2AX, into anticancer drug screening to classify the mode of action (MoA) for genotoxic outcome into clastogenicity and aneugenicity, a distinction that has considerable impact on risk assessment and control strategy. The emerging biomarker gamma-H2AX is applicable to high throughput assay platforms and is therefore changing in vitro mammalian genotoxicity screening from traditional positive/negative selection to MoA elucidation. Because gamma-H2AX is not only a sensitive biomarker for DNA double strand break but is also induced by apoptosis, the key for successful screening is using additional biomarkers of caspase-3 and/or phosphorylated histone H3 to discriminate between relevant and irrelevant elevation of gamma-H2AX. Establishment of a standard methodology and a consensus threshold for its positive criteria will further support the application of gamma-H2AX to drug screening.

Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay.

The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds.

The predominant role of apoptosis in γH2AX formation induced by aneugens is useful for distinguishing aneugens from clastogens.

The phosphorylated form of the histone protein H2AX, called γH2AX, is recognized as a useful biomarker not only for DNA double-strand breaks but also for a wide range of other DNA damage. An increasing number of publications propose γH2AX to be measured when determining genotoxicity, phototoxicity, and the effectiveness of cancer therapy. Because γH2AX is also generated by apoptosis, a γH2AX-assay might assess genotoxic risk incorrectly. The aim of this study was to elucidate the influence of apoptosis on measurements of γH2AX by flow cytometry, with the clastogens mitomycin C (MMC) and etoposide (ETP), and the aneugens vinblastine (VB) and paclitaxel (PT), which do not react directly with DNA. TK6 human lymphoblastoid cells were treated with the clastogens and the aneugens, stained for the apoptotic biomarker caspase-3 and for γH2AX, and then analyzed by flow cytometry. All the test compounds caused a dose-dependent increase of γH2AX-positive (γH2AX+) cells. The γH2AX+ cell population included both caspase-3-positive (γH2AX+/caspase-3+) and caspase-3-negative (γH2AX+/caspase-3-) cells. The increase in γH2AX+ cells after treatment with the aneugens corresponded to the increase in caspase-3+ cells. The increase in γH2AX+/caspase-3- cells after treatment with the clastogens was significant, but there was only a slight increase after treatment with the aneugens. This reflects the fact that the apoptotic pathway of a clastogen starts from DNA damage, whereas that of an aneugen starts from cell-cycle arrest in the M-phase. Therefore, the two pathways contribute differently to apoptosis. Double staining for γH2AX and caspase-3 provided helpful information for the different mechanistic effects of aneugens and clastogens that induce γH2AX.

HSP90 inhibitor CH5164840 induces micronuclei in TK6 cells via an aneugenic mechanism.

Heat-shock protein 90 (HSP90) is a promising druggable target for therapy of conditions including cancer, renal disease, and chronic neurodegenerative diseases. Despite the possible beneficial effects of HSP90 inhibitors, some of these agents present a genotoxicity liability. We have examined the mode of action of micronucleus formation in TK6 cells by a novel and highly specific HSP90 inhibitor, CH5164840, by means of an in vitro micronucleus test with fluorescence in situ hybridization (FISH), γH2AX staining to detect DNA damage, and microscopic observation of chromosomal alignment in mitotic cells. The percentage of centromere-positive micronuclei induced by CH5164840 (FISH analysis) was significant, but the percentage of centromere-negative ones was not, suggesting that induction of micronuclei was due to a mechanism of aneugenicity rather than DNA reactivity. This conclusion was further supported by the result of co-staining γH2AX and the apoptosis marker caspase-3; the predominant elevation of apoptotic γH2AX rather than non-apoptotic γH2AX indicated little involvement of DNA-reactivity mechanisms. Microscopic observation revealed asymmetric spindle microtubules and chromosomal misalignment of metaphase cells. These data indicated that CH5164840 causes spindle dysfunction that induces micronuclei. The risk/benefit ratio must be considered in the development of HSP90 inhibitors.

Aneugenic effects of epirubicin in somatic and germinal cells of male mice.

The ability of the antineoplastic agent epirubicin to induce aneuploidy and meiotic delay in the somatic and germinal cells of male mice was investigated by fluorescence in situ hybridization assay using labeled DNA probes and BrdU-incorporation assay. Mitomycin C and colchicine were used as positive controls for clastogen and aneugen, respectively, and these compounds produced the expected responses. The fluorescence in situ hybridization assay with a centromeric DNA probe for erythrocyte micronuclei showed that epirubicin is not only clastogenic but also aneugenic in somatic cells in vivo. By using the BrdU-incorporation assay, it could be shown that the meiotic delay caused by epirubicin in germ cells was approximately 48 h. Disomic and diploid sperm were shown in epididymal sperm hybridized with DNA probes specific for chromosomes 8, X and Y after epirubicin treatment. The observation that XX- and YY-sperm significantly prevailed over XY-sperm indicates missegregation during the second meiotic division. The results also suggest that earlier prophase stages contribute less to epirubicin-induced aneuploidy. Both the clastogenic and aneugenic potential of epirubicin can give rise to the development of secondary tumors and abnormal reproductive outcomes in cured cancer patients and medical personnel exposed to epirubicin.

Aneuploidogenic effects and DNA oxidation induced in vitro by differently sized gold nanoparticles.

Gold nanoparticles (Au NPs) are used in many fields, including biomedical applications; however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms is available. For this reason, experiments in human primary lymphocytes and murine macrophages (Raw264.7) were performed exposing cells to spherical citrate-capped Au NPs with two different nominal diameters (5 nm and 15 nm). The proliferative activity, mitotic, apoptotic, and necrotic markers, as well as chromosomal damage were assessed by the cytokinesis-block micronucleus cytome assay. Fluorescence in situ hybridization with human and murine pancentromeric probes was applied to distinguish between clastogenic and aneuploidogenic effects. Our results indicate that 5 nm and 15 nm Au NPs are able to inhibit cell proliferation by apoptosis and to induce chromosomal damage, in particular chromosome mis-segregation. DNA strand breaks were detected by comet assay, and the modified protocol using endonuclease-III and formamidopyrimidine-DNA glycosylase restriction enzymes showed that pyrimidines and purines were oxidatively damaged by Au NPs. Moreover, we show a size-independent correlation between the cytotoxicity of Au NPs and their tested mass concentration or absolute number, and genotoxic effects which were more severe for Au NP 15 nm compared to Au NP 5 nm. Results indicate that apoptosis, aneuploidy, and DNA oxidation play a pivotal role in the cytotoxicity and genotoxicity exerted by Au NPs in our cell models.

Effects of DNA damage and short-term spindle disruption on oocyte meiotic maturation.

DNA damage has recently been shown to inhibit or delay germinal vesicle breakdown (GVBD) in mouse oocytes, but once meiosis resumes, DNA-damaged oocytes are able to extrude the first polar body. In this study, using porcine oocytes, we showed that DNA damage did not affect GVBD, but inhibited the final stages of maturation, as indicated by failure of polar body emission. Unlike mitotic cells in which chromosome mis-segregation causes DNA double-strand breaks, meiotic mouse oocytes did not show increased DNA damage after disruption of chromosome attachment to spindle microtubules. Nocodazole-treated oocytes did not display increased DNA damage signals that were marked by γH2A.X signal strength, but reformed spindles and underwent maturation, although aneuploidy increased after extended nocodazole treatment. By using the mouse for parthenogenetic activation studies, we showed that early cleavage stage embryos derived from parthenogenetic activation of nocodazole-treated oocytes displayed normal activation rate and normal γH2A.X signal strength, indicating that no additional DNA damage occured. Our results suggest that DNA damage inhibits porcine oocyte maturation, while nocodazole-induced dissociation between chromosomes and microtubules does not lead to increased DNA damage either in mouse meiotic oocytes or in porcine oocytes.

Novel mixed-ligand copper(I) complexes: role of diimine ligands on cytotoxicity and genotoxicity.

Novel tetrahedral copper(I) mixed-ligand complexes of the type [Cu(X)(N(∩)N)(PCN)], 3-10, where X = Cl or Br, N(∩)N = 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen), 5,6-dimethyl-1,10-phenanthroline (dmp), and dipyrido-[3,2-d:2',3'-f]-quinoxaline (dpq), and PCN = tris-(2-cyanoethyl)phosphine, have been synthetized and characterized by NMR, ESI-MS, and X-ray diffraction on two representative examples, [CuCl(phen)(PCN)]·DMF (5·DMF) and [CuBr(dpq)(PCN)]·2DMF (10·2DMF). Cu(I) complexes were evaluated for their in vitro antitumor properties against a panel of human cancer cell lines, including cisplatin- and multidrug-resistant sublines. The most effective complex, [CuCl(dpq)(PCN)] (9), exhibited nanomolar cytotoxicity toward both sensitive and resistant cancer cells, but it significantly inhibited the growth of cultured normal cells. In vitro DNA assays and single cell gel electrophoresis revealed that 9 induced DNA fragmentation resulting in cell apoptosis. In parallel, fluorescence in situ hybridization (FISH) micronucleus assay attested high levels of genotoxicity following treatment of peripheral blood lymphocytes with complex 9, suggesting that the potential risk posed by diimine metal complexes should be carefully reconsidered.

Induction of a whole chromosome loss by colcemid in human cells elucidated by discrimination between FISH signal overlap and chromosome loss.

Aneuploidy is a change in the number of chromosomes and an essential component in tumorigenesis. Therefore, accurate and sensitive detection of aneuploidy is important in screening for carcinogens. In vitro micronucleus (MN) assay has been adopted in the recently revised International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S2 guideline and can be employed to predict both clastogenic and aneugenic chromosomal aberrations in interphase cells. However, distinguishing clastogens and aneugens is not possible using this assay. The Organization for Economic Co-operation and Development (OECD) guideline TG487 therefore recommends the use of centromere/kinetochore staining in micronuclei to differentiate clastogens from aneugens. Here, we analyzed numerical changes of a specific chromosome in cytokinesis-blocked binucleated cells by fluorescence in situ hybridization (FISH) using the specific centromere probe in human lymphoblastoid TK6 cells treated with aneugens (colcemid and vincristine) or clastogens (methyl methanesulfonate [MMS] and 4-nitroquinoline-1-oxide [4-NQO]). Colcemid and vincristine significantly increased the frequencies of nondisjunction and loss of FISH signals, while MMS and 4-NQO slightly increased only the frequency of loss of FISH signals. The loss of FISH signals of a specific chromosome from two to one per nucleus implies either a loss of a whole chromosome or an overlap of two signals. To distinguish a chromosome loss from signal overlap, we investigated the number of FISH signals and the fluorescent intensity of each signal per nucleus using a probe specific for whole chromosome 2 in binucleated TK6 cells and primary human lymphocytes treated with colcemid and MMS. By discriminating between chromosome loss and FISH signal overlap, we revealed that colcemid, but not MMS, induced a loss of a whole chromosome in primary lymphocytes and TK6 cells.

A mode-of-action approach for the identification of genotoxic carcinogens.

Distinguishing between clastogens and aneugens is vital in cancer risk assessment because the default assumption is that clastogens and aneugens have linear and non-linear dose-response curves, respectively. Any observed non-linearity must be supported by mode of action (MOA) analyses where biological mechanisms are linked with dose-response evaluations. For aneugens, the MOA has been well characterised as disruptors of mitotic machinery where chromosome loss via micronuclei (MN) formation is an accepted endpoint used in risk assessment. In this study we performed the cytokinesis-block micronucleus assay and immunofluorescence mitotic machinery visualisation in human lymphoblastoid (AHH-1) and Chinese Hamster fibroblast (V79) cell lines after treatment with the aneugen 17-ß-oestradiol (E2). Results were compared to previously published data on bisphenol-A (BPA) and Rotenone data. Two concentration-response approaches (the threshold-[Td] and benchmark-dose [BMD] approaches) were applied to derive a point of departure (POD) for in vitro MN induction. BMDs were also derived from the most sensitive carcinogenic endpoint. Ranking comparisons of the PODs from the in vitro MN and the carcinogenicity studies demonstrated a link between these two endpoints for BPA, E2 and Rotenone. This analysis was extended to include 5 additional aneugens, 5 clastogens and 3 mutagens and further concentration and dose-response correlations were observed between PODs from the in vitro MN and carcinogenicity. This approach is promising and may be further extended to other genotoxic carcinogens, where MOA and quantitative information from the in vitro MN studies could be used in a quantitative manner to further inform cancer risk assessment.

Comparison of the aneugenic properties of nocodazole, paclitaxel and griseofulvin in vitro. Centrosome defects and alterations in protein expression profiles.

We have comparatively investigated the aneugenic activity of two anticancer drugs, nocodazole (NOC) and paclitaxel (PTX), and the antifungal griseofulvin with promising role in cancer treatment (GF), which affect microtubule dynamics in different ways. The comparison was achieved in HFFF2 human fibroblasts, MCF-7 human breast cancer cells and C2C12 mouse myoblasts, and focused on three issues: (i) induction of chromosome delay by estimation of MN frequency using CREST analysis; (ii) disturbance of spindle organization with Aurora-A/ß-tubulin immunofluorescence; and (iii) alterations in the expression of Aurora-A, ß- and γ-tubulin by western blotting. They induced chromosome delay, provoked metaphase arrest and promoted microtubule disorganization, reflecting their common characteristic of generating aneuploidy. In particular, NOC induced mainly monopolar metaphases, although PTX induced only multipolar metaphases. GF generated different types of abnormal metaphases, exhibiting cell specificity. Additionally, NOC decreased the expression of Aurora-A and ß-tubulin, while the opposite held true for PTX and GF. γ-Tubulin expression was not modulated owing to NOC treatment, whereas PTX and GF increased γ-tubulin expression. Our findings throw a light on the manifestation of the aneugenicity of the studied compounds through centrosome proliferation/separation and protein expression, reflecting their different effects on microtubule dynamics.

Aneugenic effects of the genistein glycosidic derivative substituted at C7 with the unsaturated disaccharide.

Genistein, due to its recognized chemopreventive and antitumour potential, is a molecule of interest as a lead compound in drug design. Recently, we found that the novel genistein derivative, [7-O-(2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl)-(1 → 4)-(6-O-acetyl-hex-2-ene-α-D-erythro pyranosyl)genistein, named G21, induced aberrations in mitotic spindle formation. In the presented study, we investigated the properties of G21 relevant to its genotoxic activity. The inhibition of topoisomerase IIα activity was evaluated in decatenation assay and immunoband depletion assay, the covalent DNA-topoisomerase IIα complexes and histone É£H2AX were detected immunofluorescently. Genotoxic effects of the tested compounds were assessed in micronucleation assay. The presence of centromeres in the micronuclei and the multiplication of centrosomes were evaluated in fluorescence immunolabelled specimens. The inhibition of tubulin polymerization was measured spectrophotometrically. We found that both tested drugs were able to inhibit topoisomerase II activity; however, G21, in contrast to genistein, blocked this enzyme at the concentration far exceeding cytotoxic IC(50). We also found that both compounds caused micronucleation in DU 145 prostate cancer cells, but in contrast to genistein, G21 exhibited aneugenic activity, manifested by the presence of centromeres in micronuclei formed in cells treated with the drug. Aneugenic properties of G21 resulted from the inhibition of tubulin polymerization and centrosome disruption, not observed in the presence of genistein. The study supports and extends our previous observations that the mechanisms of cytotoxicity of genistein and its new glycosidic derivative-G21 are significantly different.