Centromere-positive Micronuclei Research Articles

Impact of Extended and Combined Exposure of Bisphenol Compounds on Their Chromosome-Damaging Effect─Increased Potency and Shifted Mode of Action.

Bisphenol (BP) compounds are important environmental pollutants and endocrine disruptors. BPs are capable of inducing DNA/chromosome breaks (clastogenesis, involved in carcinogenesis), which requires activation by human CYP1A1. We hypothesized that combined BPs and extended (from the standard two-cell cycle) exposure may enhance their genotoxicity via modulating CYP enzymes. In this study, individual and combined BPA/BPF/BPS/BPAF and a human hepatoma (HepG2) cell line were used for testing several genotoxicity end points. Exposing for a two-cell cycle period (48 h), each BP alone (0.625-10 μM) was negative in the micronucleus test, while micronucleus was formed under three- (72 h) and four-cell cycle (96 h) exposure; BP combinations further elevated the potency (with nanomolar thresholds). Immunofluorescence analysis of the centromere with formed micronucleus indicated that 48 h exposure produced centromere-negative micronucleus and phosphorylated histone H2AX (γ-H2AX) (evidencing clastogenesis), while extended (72 and 96 h) exposure formed centromere-positive micronucleus and phosphorylated histone H3 (p-H3) (indicating chromosome loss, i.e., aneugenesis); moreover, 1-aminotriabenzotriazole (CYP inhibitor) selectively blocked the formation of centromere-negative micronucleus and γ-H2AX, without affecting that of centromere-positive micronucleus and p-H3. This study suggests that the genotoxicity of BPs is potentiated by combined and extended exposure, the latter being specific for aneuploidy formation, a CYP activity-independent effect.

Chromosome aberrations induced by the non-mutagenic carcinogen acetamide involve in rat hepatocarcinogenesis through micronucleus formation in hepatocytes.

Chromosome aberrations (CAs), i.e. changes in chromosome number or structure, are known to cause chromosome rearrangements and subsequently tumorigenesis. However, the involvement of CAs in chemical-induced carcinogenesis is unclear. In the current study, we aimed to clarify the possible involvement of CAs in chemical carcinogenesis using a rat model with the non-mutagenic hepatocarcinogen acetamide. In an in vivo micronucleus (MN) test, acetamide was revealed to induce CAs specifically in rat liver at carcinogenic doses. Acetamide also induced centromere-positive large MN (LMN) in hepatocytes. Immunohistochemical and electron microscopic analyses of the LMN, which can be histopathologically detected as basophilic cytoplasmic inclusion, revealed abnormal expression of nuclear envelope proteins, increased heterochromatinization, and massive DNA damage. These molecular pathological features in LMN progressed with acetamide exposure in a time-dependent manner, implying that LMN formation can lead to chromosome rearrangements. Overall, these data suggested that CAs induced by acetamide play a pivotal role in acetamide-induced hepatocarcinogenesis in rats and that CAs can cause chemical carcinogenesis in animals via MN formation.

Biomonitoring findings for occupational lead exposure in battery and ceramic tile workers using biochemical markers, alkaline comet assay, and micronucleus test coupled with fluorescence in situ hybridisation.

Manufacture of lead-containing products has long been associated with various health risks. To get an insight into the related genotoxic risks, we conducted a biomonitoring study in 50 exposed workers and 48 matched controls using a battery of endpoints that sensitively detect the extent of genome instability in peripheral blood lymphocytes. The levels of primary DNA damage were estimated with the alkaline comet assay, while cytogenetic abnormalities were determined with the cytokinesis-block micronucleus (CBMN) cytome assay. Additionally, CBMN slides of 20 exposed and 16 control participants were subjected to fluorescence in situ hybridisation (FISH), coupled with pancentromeric probes to establish the incidence of centromere-positive micronuclei, nuclear buds, and nucleoplasmic bridges. Blood lead levels (B-Pb) were measured with atomic absorption spectrometry. To further characterise cumulative effects of occupational exposure, we measured erythrocyte protoporphyrin (EP) concentrations and delta-aminolevulinic acid dehydratase (ALAD) activity in blood. We also assessed the influence of serum folate (S-folate) and vitamin B12 (S-B12) on genome stability. Compared to controls, occupationally exposed workers demonstrated significantly higher B-Pb (298.36±162.07 vs 41.58±23.02), MN frequency (18.71±11.06 vs 8.98±7.50), centromere positive MN (C+ MN) (8.15±1.8 vs 3.69±0.47), and centromere negative MN (C- MN) (14.55±1.80 vs 4.56±0.89). Exposed women had significantly higher comet tail intensity (TI) and length (TL) than control women. Furthermore, workers showed a positive correlation between age and nuclear buds and MN, between MN and years of exposure, and between S-B12 levels and TI and ALAD activity, while a negative correlation was found between TI and B-Pb. These findings suggest that occupational settings in the manufacture of lead-containing products pose significant genotoxic risks, which calls for developing more effective work safety programmes, including periodical monitoring of B-Pb and genetic endpoints.

The roles of kinetochore of micronucleus in mitosis of HeLa cells: a live cell imaging study

BackgroundMicronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability. However, the roles of kinetochore of MN in mitosis have not been completely addressed.MethodsThe HeLa CENP B-GFP H2B-mCherry cells are applied to address these questions via the long-term live-cell imaging. In the cells, the kinetochore-positive micronucleus (K+MN) contained CENP B-GFP, while the kinetochore-negative micronucleus (K−MN) did not.ResultsK−MN-bearing cells produced much more chromosome fragments than did MN-free cells. Most of the chromosome fragments eventually merged into K−MNi. K+MN-bearing cells yielded more kinetochore-positive lagging chromosomes (K+LCs) and K+MNi than MN-free cells did. The results suggested the differences in the fates of K+MNi and K−MNi in mitosis. The cycle of K−MN → Chromosome fragment → K−MN may occur in generations of K−MN-bearing cells, while part of K+MNi might reincorporate into the main nucleus. The K+MN-bearing cells prolonged significantly duration of mitosis compared with MN-free cells. The presence of micronuclei, regardless of K−MN and K+MN, enhanced apoptosis cell death. And K+MN-bearing cells were inclined to apoptosis more than K−MN-bearing cells. The results suggested differences in fates between K−MN-bearing and K+MN-bearing cells.ConclusionsKinetochore determined the fates of micronuclei. Kinetochore in micronuclei indirectly prolonged the duration of mitosis. Kinetochore enhanced cytotoxicity of micronuclei. Our data are direct evidences showing the roles of kinetochore of micronucleus in mitosis of HeLa cells.

Markers of oxidative-nitrosative stress induced by artesunate are followed by clastogenic and aneugenic effects and apoptosis in human lymphocytes.

Artesunate (ARS) is a semi-synthetic derivative of artemisinin, used as an outstanding antimalarial drug, which also displays antitumor, anti-inflammatory and immunosuppressive effects. In spite of the numerous reports showing the antitumor activity of ARS, the particular mechanisms associated with its cytotoxicity and genotoxicity in non-neoplastic human cells remain unclear. Here we aimed to verify the specific chromosome damages and the changes in markers of oxidative-nitrosative stress and apoptosis triggered by ARS exposure in human peripheral blood lymphocytes. Cultures were incubated in the presence of ARS and the number of binucleated cells was determined. To discriminate between micronuclei (MN) containing a whole chromosome or an acentric chromosome, the MN test was employed in combination with the fluorescence in situ hybridization assay. Alterations in the levels of superoxide anion (O2- ) and nitric oxide (NO) were measured by the nitroblue tetrazolium and Griess assay, respectively. Changes in the expression of the apoptotic markers were assessed by immunocytochemistry. We found that ARS induced a significant formation of both centromere-positive MN (C+ MN) and centromere-negative MN (C- MN). These alterations were accompanied by an increase in both cellular levels of O2- and total NO production, and a remarkable enhancement in the expression of the apoptotic markers cytochrome c and caspases 8 and 9. Together these findings reveal that ARS induces changes in the oxidative-nitrosative status of human lymphocytes, which are followed by apoptosis and clastogenic and aneugenic effects.

Micronucleus-Centromere Assay to Measure Ionizing Radiation Damage of Low Dose Occupational Exposure

Background: The purpose of the present study was to assess the cytogenetic effect of chronic low dose radiation exposure of nuclear power plant workers using the micronucleus centromere assay. This method allows the differentiation between centromere-positive micronuclei containing whole chromosome and centromere-negative micronuclei containing acentric fragment pointing to clastogenic action of ionizing radiation. Methods: The effect of low dose occupational exposure was estimated in 32 nuclear power plant workers using in situ hybridization (FISH) with the human pancentromeric DNA probe for peripheral blood lymphocytes. Group of 17 persons from administrative staff served as reference control group. Results: Data on the number of micronuclei, centromere-positive micronuclei and centromere-negative micronuclei per 1000 binucleated cells for the workers were significantly higher compared with the control group (p = 0.001, p=0.01 and p=0.002 respectively). No differences in the studied indicators between smokers and non-smokers are observed. No correlation was found between age and frequency of micronuclei, centromere-positive and centromere-negative micronuclei. Conclusions: The slight increase of the micronucleus frequency with the accumulated dose, observed in the group of workers, is mainly due to an enhanced number of centromere-negative micronuclei. Thus, the application of the MN-centromere assay is suitable as the frequency of MNCM−is more useful marker instead of the total number of MN. Keywords: occupational exposure; NPP workers; micronucleus-centromere assay

Genotoxic Effects in Human Fibroblasts Exposed to Microwave Radiation.

In the last decades, technological development has led to an increasing use of devices and systems based on microwave radiation. The increased employment of these devices has elicited questions about the potential long-term health consequences associated with microwave radiation exposure. From this perspective, biological effects of microwave radiation have been the focus of many studies, but the reported scientific data are unclear and contradictory. The aim of this study is to evaluate the potential genotoxic and cellular effects associated with in vitro exposure of human fetal and adult fibroblasts to microwave radiation at the frequency of 25 GHz. For this purpose, several genetic and biological end points were evaluated. Results obtained from comet assay, phosphorylation of H2AX histone, and antikinetochore antibody (CREST)-negative micronuclei frequency excluded direct DNA damage to human fetal and adult fibroblasts exposed to microwaves. No induction of apoptosis or changes in prosurvival signalling proteins were detected. Moreover, CREST analysis showed for both the cell lines an increase in the total number of micronuclei and centromere positive micronuclei in exposed samples, indicating aneuploidy induction due to chromosome loss.

The Cycle of Kinetochore-Negative Micronuclei and Chromosome Fragments Occurred in Mitosis of Hela Cells

Objective: Micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability. According to the presence of kinetochores or not, MNi are further classified into kinetochore-negative MNi (K−MNi) and kinetochore-positive MNi (K+MNi), which show the different mechanisms of micronucleus formation. However, the differences in fates of K−MNi and K+MNi have not been completely addressed. The present study aims to address these questions. Methods: Here, HeLa CENP B-GFP H2B-mCherry cells were chosen to distinguish K+MNi and K−MNi in living cells. In the cells, MNi were identified by H2B-mCherry and further classified into K+MNi and K−MNi, i.e. the K+MNi contained CENP B-GFP, while the K−MNi did not. Long-term live-cell imaging was applied in the cells to record the dynamics of cell mitosis, especially K+MNi and K−MNi. Results: Our results show that the presence of K−MN or K+MN did not result in multipolar mitosis. K−MN-bearing cells produced much more chromosome fragments than did MN-free cells. Most of the chromosome fragments eventually merged into K−MNi. K+MN-bearing cells yielded more kinetochore-positive lagging chromosomes (K+LCs) and K+MNi than MN-free cells did. Conclusion: The results suggested the differences in the fates of K+MNi and K−MNi in mitosis. The cycle of K− MN → Chromosome fragment → K−MN may occur in generations of K−MN-bearing cells, while part of K+MNi might reincorporate into the main nucleus.

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

Differences in the origins of kinetochore-positive and kinetochore-negative micronuclei: A live cell imaging study

Micronuclei (MNi) are extensively used to evaluate genotoxicity and chromosomal instability. Classification of kinetochore-negative (K−MNi) and kinetochore-positive micronuclei (K+MNi) improves the specificity and sensitivity of the micronucleus (MN) test; however, the fundamental differences in the origins of K−MNi and K+MNi have not been addressed due to the limitations of traditional methods.In the current study, HeLa CENP B-GFP H2B-mCherry cells were constructed in which histone 2B (H2B) and centromere protein B (CENP B) were expressed as fusion proteins to monomeric Cherry (mCherry) and EGFP, respectively. MNi were identified using H2B-mCherry; K+MN contained CENP B-GFP, while K−MN did not. Long-term live cell imaging was conducted to examine MN formation in the dual-color fluorescent HeLa cells. The results suggested that K−MNi were derived from kinetochore-negative displaced chromosomes (K−DCs), kinetochore-negative lagging chromosomes (K−LCs) and fragments of broken chromosome bridges (CBs) during late mitotic stages. The results also indicated that K+MNi are derived from kinetochore-positive displaced chromosomes (K+DCs), kinetochore-positive lagging chromosomes (K+LCs), and fragments of broken CBs. Different aberrant chromosomes emerged during mitosis at different frequencies and developed into K−MNi and/or K+MNi in the daughter cells at different rates. K+LCs formed K+MNi at a higher frequency than K+DCs, and K−LCs formed K−MNi at a higher rate than K−DCs; however, broken CBs transformed into K−MNi and/or K+MNi.In summary, these results show that K−MNi and K+MNi have different origins in HeLa cells and that each mechanism of MN formation contributes differently to the overall number of K−MNi and K+MNi.

The Association of LINE-1 Hypomethylation with Age and Centromere Positive Micronuclei in Human Lymphocytes.

Global hypomethylation in white blood cell (WBC) DNA has recently been proposed as a potential biomarker for determining cancer risk through genomic instability. However, the amplitude of the changes associated with age and the impacts of environmental factors on DNA methylation are unclear. In this study, we investigated the association of genomic hypomethylation with age, cigarette use, drinking status and the presence of centromere positive micronuclei (MNC+)—a biomarker for age-dependent genomic instability. Genomic hypomethylation of the repetitive element LINE-1 was measured in WBC DNA from 32 healthy male volunteers using the pyrosequencing assay. We also measured MNC+ with the micronucleus-centromere assay using a pan-centromeric probe. Possibly due to the small sample size and resulting low statistical power, smoking and drinking status had no significant effect on LINE-1 hypomethylation or the occurrence of MNC+. Consequently, we did not include them in further analyses. In contrast, LINE-1 hypomethylation and age significantly predicted MNC+; therefore, we examined whether LINE-1 hypomethylation plays a role in MNC+ formation by age, since genomic hypomethylation is associated with genomic instability. However, LINE-1 hypomethylation did not significantly mediate the effect of age on MNC+. Our data indicate that the repetitive element LINE-1 is demethylated with age and increasing MNC+ frequency, but additional studies are needed to fully understand the relation between genomic DNA hypomethylation, age and genomic instability.

Deoxynivalenol induces cytotoxicity and genotoxicity in animal primary cell culture

Deoxynivalenol (DON), a mycotoxin produced by Fusarium graminearum, is widely found as a contaminant of food. DON is responsible for a wide range of toxic activities, including gastro-intestinal, lymphoid, bone-marrow and cardiotoxicity. But, the complete explorations of toxicity in terms of hepatotoxicity, nephrotoxicity, cytotoxicity and genotoxicity as well have not been documented well. Again, the mechanisms through which DON damages the DNA and promotes cellular toxicity are not well established. Considering the above fact, this research article is focused on the effects of DON-induced toxicities on experimental animal model as well as its effects on cellular level via various toxicological investigations. DON treatment showed cytotoxicity and DNA damage. Further, flow cytometric analysis of hepatocytes showed cellular apoptosis, suggesting that DON-induced hepatotoxicity is, may be partly, mediated by apoptosis. Moreover, significant differences were found in each haematology and clinical chemistry value, either (p > 0.05). No abnormality of any organ was found during histopathological examination. Hence, it can be concluded that DON induces oxidative DNA damage and increases the formation of centromere positive micronuclei due to aneugenic activity.

Genomic Instability in Fanconi Anemia Results from a Combination of Chromosome Mis-Segregation in Mitosis and Unresolved Interphase DNA Damage

Fanconi anemia (FA) is a complex genetic disorder characterized by bone marrow failure, multiple congenital anomalies, and genomic instability resulting in predisposition to cancer. Disruption of the FA signaling network impairs multiple genome-housekeeping processes, including DNA damage recognition and repair in interphase, DNA replication as well as high-fidelity chromosome segregation during mitosis. Recent data published by several groups, including our work (J Clin Invest 2013; 123: 3839-3847), implicated FA signaling in the control of several cell division events essential for chromosomal stability, including the spindle assembly checkpoint (SAC), centrosome maintenance, resolution of ultrafine anaphase bridges and cytokinesis. Understanding the mechanistic origins of chromosomal instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. However, the relative contribution of the interphase and mitotic events leading to genomic instability in Fanconi anemia has not been systematically evaluated.In this work, we dissected the origins and mechanistic significance of chromosomal instability in Fanconi anemia ex vivo and in vivo. We employed the cytochalasin micronucleus assay to quantify the patterns of spontaneous and chemotherapy-induced genomic lesions in FA-A patient-derived primary fibroblasts and Fancc-/- mouse embryonic fibroblasts (MEFs). In this assay, dividing cells are treated with cytochalasin to inhibit cytokinesis and generate binucleated daughter cells. The presence of micronuclei in the resulting cells is indicative of genomic instability caused by either interphase DNA damage or chromosome mis-segregation. Centromere-negative micronuclei (CNMs) represent chromosomal fragments due to unresolved ds-DNA damage. Centromere-positive micronuclei (CPMs) result from whole-chromosome mis-segregation during mitosis. The frequency of both CPMs and CNMs was significantly increased in FA-deficient human and murine cells compared to gene-corrected isogenic control cells. These results indicate that genomic instability in FA is caused by a combination of interphase DNA damage and disordered mitosis. We confirmed the biological significance of these findings by showing that FA patient cells are hypersensitive to low concentrations of taxol (a spindle checkpoint-activating chemotherapeutic) similarly to mitomycin C (a cross-linking agent). Finally, we found increased frequency of micronuclei in Fancc-/- murine red blood cells compared to age-matched wild-type mice, which indicates that spontaneous chromosome mis-segregation occurs in FA-deficient bone marrow in vivo.Our study supports the emerging model of the FA family of proteins as holistic guardians of the genome during interphase and mitosis (see figure based on F1000Prime Rep. 2014; 6: 23, modified). This model furthers our understanding of genomic instability in Fanconi anemia and FA-deficient cancers, and opens new inroads towards targeted therapeutic interventions in these diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

A comparative study of the aneugenic and polyploidy-inducing effects of fisetin and two model Aurora kinase inhibitors

Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements.

Genome damage in testicular seminoma patients seven years after radiotherapy

Purpose: Testicular seminoma cancer incidence has significantly increased over the last few decades, and although it is successfully treated by radiotherapy, long-term health risks are still unclear. The aim of the study was to show long-term genome damage in patients with seminoma after radiotherapy.Materials and methods: Chromosome aberration (CA) and micronucleus (MN) assays seven years after radiotherapy with a total dose of 25 Gy were conducted in 10 testicular seminoma patients aged 23–49 years and results were compared with 10 healthy control subjects matched for age and smoking status.Results: Although mean CA frequency did not deviate from control values, significantly increased frequencies of dicentrics, double minutes, and ring chromosomes were detected in seminoma patients. MN frequency in binuclear lymphocytes of patients was similar to controls (4.60/1000 vs. 5.82/1000, respectively). Significantly higher MN frequency was detected in mononuclear lymphocytes of patients than in controls (2.55/1000 vs. 0.73/1000, respectively). Average percentage of centromere-positive MN was 62.6% in seminoma patients.Conclusion: This study shows the persistence of unstable CA in seminoma patients seven years after radiotherapy and the relevance of long-term follow up. MN frequency in mononuclear lymphocytes was shown to be relevant biomarker of long-term genome damage.

Genotoxic Properties of Cyclopentenone Prostaglandins and the Onset of Glutathione Depletion

Prostaglandins are endogenous mediators formed from arachidonic acid by cyclooxygenases and prostaglandin synthases during inflammatory processes. The five-membered ring can be dehydrated, and α,β-unsaturated cyclopentenone PGs (cyPGs) are generated. Recent studies have been focused on their potential pharmacological use against inflammation and cancer. However, little is known so far about possible adverse health effects of cyPGs. We addressed the question whether selected cyPGs at a concentration range of 0.1-10 μM exhibit mutagenic and genotoxic properties in the hamster lung fibroblast V79 cell line and whether these effects are accompanied by a depletion of intracellular glutathione (GSH). The cyPGs 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) and prostaglandin A2 (PGA2) significantly induced DNA damage in V79 cells after 1 h of incubation. Furthermore, a more pronounced increase in formamidopyrimidine-DNA glycosylase (FPG) sensitive sites, indicative of oxidative DNA-damage, was observed. The findings on DNA-damaging properties were supported by our results that 15dPGJ(2) acts as an aneugenic agent which induces the amount of kinetochore positive micronuclei associated with an increase of apoptosis. The strong potency of cyPGs to rapidly bind GSH measured in a chemical assay and to significantly reduce the GSH level after only 1 h of incubation may contribute to the observed oxidative DNA strand breaks, whereas directly induced oxidative stress via reactive oxygen species could be excluded. However, after an extended incubation time of 24 h no genotoxicity could be measured, this may contribute to the lack of mutagenicity in the hypoxanthine phosphorybosyltransferase (HPRT) assay. In conclusion, potential in vitro genotoxicity of cyPG and a strong impact on GSH homeostasis have been demonstrated, which may be involved in carcinogenesis mediated by chronic inflammation.

The genotoxicity and cytotoxicity assessments of andrographolide in vitro

Andrographolide is a major phytoconstituent present in Andrographis paniculata, a plant used in traditional medicines in Asia for various ailments. This tropical shrub was reported to possess various pharmacological activities and has been marketed around the world including Europe, however the toxicological data especially potential genotoxicity assessment on the phytocompound is still lacking. This study was performed to assess the ability of andrographolide to induce chromosomal changes using the in vitro cytokinesis-blocked micronucleus assay with immunofluorescent labelling of kinetochores in metabolically-competent AHH-1 and MCL-5 human lymphoblastoid cell lines. Various cytotoxicity endpoints were also evaluated in this study. Andrographolide was found to cause a weak increase in micronuclei induction at 10–50μM in both AHH-1 and MCL-5 cell lines, respectively which were within the historical range. Kinetochore analysis revealed that the micronuclei induced in MCL-5 cells due to andrographolide exposure originated via an aneugenic mechanism that was indicated by the relatively higher but non-significant percentage of kinetochore positive micronuclei compared to negative control. Andrographolide also elicited a dose-dependent cellular cytotoxicity, with cells dying primarily via necrosis compared to apoptosis. Here we report that andrographolide was not genotoxic at the doses tested and it induces dose-dependent necrosis in vitro.

Does formaldehyde induce aneuploidy?

Formaldehyde (FA) was tested for a potential aneugenic activity in mammalian cells. We employed tests to discriminate between aneugenic and clastogenic effects in accordance with international guidelines for genotoxicity testing. The cytokinesis-block micronucleus test (CBMNT) in combination with fluorescence in situ hybridisation (FISH) with a pan-centromeric probe was performed with cultured human lymphocytes and the human A549 lung cell line. FA induced micronuclei (MN) in binuclear cells of both cell types under standard in vitro test conditions following the OECD guideline 487. FISH analysis revealed that the vast majority of induced MN were centromere negative, thus indicating a clastogenic effect. A similar result was obtained for MN induced by γ-irradiation, whereas the typical aneugens colcemid (COL) and vincristine (VCR) predominantly induced centromere-positive MN. Furthermore, COL and VCR clearly enhanced the MN frequency in mononuclear lymphocytes in the CBMNT, whereas such an effect was not observed for γ-irradiation and FA. In experiments with the Chinese hamster V79 cell line, the aneugens COL and VCR clearly increased the frequency of tetraploid second division metaphases, whereas FA did not cause such an effect. Altogether, our results confirm the clastogenicity of FA in cultured mammalian cells but exclude a significant aneugenic activity.

Micronucleus frequencies in peripheral blood lymphocytes of children with chronic kidney disease

One of the crucial adverse effects of chronic kidney disease (CKD) and its treatment is an elevated cancer risk. There are no data on cytogenetic effects in children with CKD or children undergoing dialysis or those who have received a transplant. In this study, cytogenetic effects in children with CKD in pre-dialysis (PreD) stage, on regular haemodialysis (HD) and transplanted (Tx) compared with a control group of healthy children has been investigated using the cytokinesis-blocked micronucleus (CBMN) assay and fluorescence in situ hybridisation (FISH) combined with CBMN (CBMN-FISH) in peripheral blood lymphocytes. The results revealed a significant increase (P < 0.001) in micronucleus (MN) frequencies [mean ± SD (n)] in the PreD, HD and Tx groups versus the control group [CBMN assay; 9.19 ± 2.61 (16), 9.07 ± 4.86 (15), 6.12 ± 5.33 (17) versus 1.60 ± 0.99 (20), respectively]. Moreover, centromere negative micronucleus (C- MN) and centromere positive micronucleus (C+ MN) frequencies were significantly higher in each subgroup children (PreD, HD and Tx) than in the control group (P < 0.01) although children in Tx group had lower C- MN frequencies than PreD and lower C+ MN frequencies than PreD and HD groups (P < 0.05). Additionally, MN frequencies in mononuclear cells, nucleoplasmic bridges and nuclear buds in binucleated cells were increased in children with CKD (P < 0.001, P < 0.001, P > 0.05, respectively). The nuclear division index significantly decreased in Tx group relative to the control, PreD and HD groups (P < 0.001). Associations between cytogenetic parameters and creatinine or blood urea nitrogen were found (P < 0.05). To provide longer and better life expectancy of children with CKD and treatment modes, further research is needed to better understand and avoid these effects.