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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 137 Cs 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Increasing evidence supports the role of genetic and epigenetic alterations in a wide variety of human diseases, including cancer. Assessment of these alterations is hence essential for estimating the hazardous effects of human exposure to medications. Panobinostat received US Food and Drug Administration's approval in 2015 for treatment of certain tumors and its usefulness as part of a strategy to treat other diseases, such as human immunodeficiency virus infection, is currently investigated. Nevertheless, no data on in vivo genotoxical and epigenotoxical effects of panobinostat are available. The aim of the current study was to assess the genotoxical and epigenotoxical properties of panobinostat in murine bone marrow cells. Molecular mechanisms underlying these alterations were also evaluated. We show that mice treated with panobinostat doses recommended for human developed numerical chromosomal abnormalities, structural chromosomal damage, oxidative DNA damage, and DNA hypomethylation. These effects were dose-dependent. Further, panobinostat altered the expression of 23 genes implicated in DNA damage, as determined by RT² Profiler polymerase chain reaction (PCR) array, and confirmed by quantitative real-time PCR and western blotting. Collectively, these findings indicate that panobinostat exposure induces aneugenicity, clastogenicity, oxidative DNA damage, DNA hypomethylation, and down-regulation of repair gene expression, which may be responsible for panobinostat-induced genotoxical and epigenotoxical effects. Considering the potential toxicity of panobinostat, the medicinal use of panobinostat must be weighed against the risk of tumorigenesis and the demonstrated toxicity profile of panobinostat may support further development of chemotherapeutic treatments with reduced toxicity. Diminishing the metabolic liabilities associated with panobinostat exposure, and simultaneous use of panobinostat with DNA repair enhancers, are examples of strategies for drug design to reduce panobinostat carcinogenicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Theonella sp is an important source of biologically-active 3-alkylpyridine alkaloids (3-APAs) that has shown a wide variety of promising biological effects. In the present work, two new 3-APAs analogues were synthesized based on molecular modeling studies to act as potential antimalarial agents. These theoneladin C analogues, containing the thiocyanate group in their chemical structures, were synthesized and evaluated against Plasmodium falciparum (IC 50 values ranging from 2.3 to 5.5μM). The structural and energetic analysis demonstrated a high chemical affinity of the two analogues for their target, the heme group. However, despite the good antimalarial activity, the compounds exhibited high cytotoxicity and a lack of selectivity for human cell lines. These findings prompted us to evaluate the cytotoxicity of these compounds against human cancer cell lines. In order to better understand the mechanisms responsible for the toxicity, a variety of genotoxicity assays were performed in vitro. One of the compounds assayed presented an interesting selectivity and high toxicity to the human colon cancer cell line RKO-AS45-1. In addition, the results of the micronucleus assay, comet assay, Ames assay and annexin-V/propidium iodide staining showed that the synthetic alkaloids were able to induce chromosomal mis-segregation and trigger cell death by apoptosis. These results demonstrate that the compounds assessed herein may be promising prototypes of anticancer chemotherapeutic agents., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The standard procedure for the micronucleus test in cell lines requires a short exposure (≤0.5 cell cycle) to the test compounds followed by a long recovery (≥1.5 cell cycle), and in case of negative or equivocal results, a second test with extended exposure (≥2 cell cycles) without or with a recovery time. In general the two procedures are advantageous for detecting clastogens and aneugens, respectively. However, whether the recommended procedures apply to micronucleus tests with promutagens in cell lines genetically engineered for expressing biotransformation enzymes has not been identified. In this study, several promutagens dependent on cytochrome P450 (CYP) 2E1 and/or sulfotransferase (SULT) 1A1 were used in the micronucleus test in a Chinese hamster V79-derived cell line expressing human CYP2E1 and SULT1A1 (V79-hCYP2E1-hSULT1A1), with varying exposure/recovery schedules: 3h/21h, 6h/18h, 12h/12h, 18h/6h, and 24h/0h, in comparison with known clastogens and aneugens in V79 control cells. The results showed peaked micronuclei induction by mitomycin C and bleomycin (clastogens) at the 12h/12h schedule, while colchicine and vinblastine (aneugens) showed the strongest effect at 24h/0h. Catechol and trihydroxybenzene (activated by CYP2E1) induced micronuclei most strongly at 6h/18h, whereas somewhat longer exposures were optimal for hydroquinone, another compound activated by CYP2E1. 1-Hydroxymethylpyrene (activated by SULT1A1) and 1-methylpyrene (activated sequentially by CYP2E1 and SULT1A1) produced the highest response with the 18h/6h treatment regimen. Moreover, mitotic arrest by 1-hydroxymethylpyrene was observed in V79-hCYP2E1-hSULT1A1 cells but not in V79 cells, and 1-methylpyrene arrested mitosis in V79-hCYP2E1-hSULT1A1 more strongly than in V79 cells. Our study suggests that intracellular bioactivation of promutagens may not delay the induction of micronuclei in the present model, and 1-methylpyrene and 1-hydroxymethylpyrene may be activated to mitosis-arresting metabolites., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although inter-laboratory validation efforts of the in-vivo micronucleus (MN) assay based on flow cytometry (FCM) have taken place in the EU and US, none have been organized in China. Therefore, an inter-laboratory study that included eight laboratories in China and one experienced reference laboratory in the US was coordinated to validate the in-vivo FCM MicroFlow(®) method to determine the frequency of micro-nucleated reticulocytes (MN-RETs) in rat blood. Assay reliability and reproducibility were evaluated with four known genotoxicants, and the results obtained with the FCM method were compared with the outcome of the traditional evaluation of bone-marrow micronuclei by use of microscopy. Each of the four chemicals was tested at three sites (two in China and the one US reference laboratory). After three consecutive daily exposures to a genotoxicant, blood and bone-marrow samples were obtained from rats 24h after the third dose. MN-RET frequencies were measured in 20,000 RET in blood by FCM, and micro-nucleated polychromatic erythrocyte (MN-PCE) frequencies were measured in 2,000 PCEs in bone marrow by microscopy. For both methods, each genotoxicant was shown to induce a statistically significant increase in the frequency of MN after treatment with at least one dose. Where more doses than one caused an increase, responses occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM-based MN-RET vs microscopy-based MN-PCE measurements (eight experiments, 200 paired measurements) was 0.723, indicating a high degree of correspondence between methods and compartments. The rs value for replicate FCM MN-RET measurements performed at the eight collaborative laboratories was 0.940 (n=200), and between the eight FCM laboratories with the reference laboratory was 0.933 (n=200), suggesting that the automated method is very well transferable between laboratories. The FCM micronucleus analysis method is currently used in many countries worldwide, and these data support its use for evaluating the in-vivo genotoxic potential of test chemicals in China., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of this study was to perform the first transferability assessment in China of the micronucleus (MN) scoring method based on three-colour flow cytometry (FCM). This was accomplished for both rat bone-marrow and peripheral blood specimens following exposure to a variety of genotoxic and non-genotoxic chemicals, whereby micronucleus induction was measured both with FCM and with traditional microscopy. In an initial study, rats were treated with vehicle or cyclophosphamide (CP) for 2 consecutive days by oral gavage, and blood and bone marrow were sampled at 24 h after the second treatment. Staining with acridine orange (AO) of methanol-fixed slides was used for microscopical analysis and 2000 reticulocytes (RET) or polychromatic erythrocytes (PCE) were scored for MN frequency. The erythrocytes in the remaining bone-marrow cell suspensions were eluted on cellulose columns. The eluted bone marrow as well as the peripheral blood cells was fixed, incubated and analyzed by FCM. In another experiment, the performance of the FCM-MN method was further evaluated with five clastogens (urethane, 5-fluorouracil, mitomycin C, methylmethane sulfonate and 6-thioguanine), two aneugens (vincristine sulfate and colchicine) and two non-genotoxic new drugs (compounds A and B), whose results were negative in the routine mouse-micronucleus test (MNT). The MN frequencies in rat peripheral blood induced by the positive chemicals were found to be lower than the frequencies in rat bone-marrow by both scoring methods. However, a high level of agreement for the MN frequencies in both compartments was obtained. Good correspondence between the two analysis methods was also achieved. These data provide support that the three-colour FCM method is more rapid and objective than manual microscopy, while yielding comparable data. It further supports the premise that rat peripheral blood may be an alternative to rat bone marrow in the MNT., (Copyright © 2013 Elsevier B.V. All rights reserved.)

When characterizing the genotoxicity of chemicals that induce micronuclei, it is practical to be able to classify the chemicals as aneugens or clastogens. This classification gives information on the mechanistic properties of chemicals and is indispensable for setting the threshold safety margins for genotoxicity in pharmaceutical development. A widely used method for detecting aneugens is fluorescence in situ hybridization (FISH) but, even though the rat is an experimental animal generally used in preclinical studies in drug development, DNA probes that hybridize to all the centromeres of rat chromosomes have not yet been established. In the present study, in addition to the previously known satellite I sequence, we identified two novel satellite sequences, satellite II and satellite III, from the rat genome database. DNA probes with a mixture of these satellite DNA sequences were used to establish a FISH method for pan-centromeric staining of rat chromosomes. To confirm the feasibility of the method, vinblastine (VBS) and mitomycin C (MMC) were administered to rats as a typical aneugen and clastogen, respectively. Micronucleated polychromatic erythrocytes (MNPCE) from bone marrow were enriched by sorting in flow cytometry and subjected to the FISH method. As a result, the ratio of centromere-positive MNPCE increased in VBS-treated rats but not in MMC-treated ones. Since the FISH method using the novel DNA probes clearly discriminates the aneugens from the clastogens, we suggest this method as a useful tool for providing mechanistic information for micronucleus induction in vivo., (Copyright © 2013 Elsevier B.V. All rights reserved.)