Kinetochore-negative Micronuclei Research Articles

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.

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.

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.

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.

Genomic instability induced by -pinene in Chinese hamster cell line

Here, we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene used in insecticides, solvents and perfumes. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 μM) of α-pinene, indicated a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 μM. This monoterpene caused genomic instability by interfering with mitotic process; in fact, 50% of cells (versus 19% of control cells) showed irregular mitosis with multipolar or incorrectly localised spindles. Cytogenetic analysis demonstrated high-frequency hypodiploid metaphases as well as endoreduplicated cells and chromosome breaks. Clastogenic damage was prevalent over aneuploidogenic damage as demonstrated by the higher proportion of kinetochore-negative micronuclei. Alkaline comet confirmed that monoterpene exposure caused DNA lesions in a concentration-dependent manner. This damage probably arose by increased reactive oxygen species (ROS) production. In order to assess the generation of ROS, the cells were incubated with CM-H(2)DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after α-pinene treatment indicating increased oxidative stress. On the whole, these findings strongly suggest that α-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through ROS production.

DNA damaging properties of single walled carbon nanotubes in human colon carcinoma cells

Single walled carbon nanotubes were studied with respect to cytotoxic and genotoxic properties in cells of the gastrointestinal tract as exemplified for the human colon carcinoma cell line HT29. No effect on cell growth in the sulphorhodamine B assay was observed after 24 h of incubation, whereas growth inhibitory properties were found after 48 and 72 h. After 24 h incubation a decrease of mitochondrial activity (WST-1) was measured (≥0.1 μg/ml), whereas membrane integrity (lactate dehydrogenase) was not affected. In cytotoxic concentrations, the formation of reactive oxygen species and a slight increase of total glutathione and nuclear Nrf2 were observed. However, already in subcytotoxic concentrations substantial DNA damaging effects were found in the alkaline comet assay, which were not associated with enhanced formation of formamidopyrimidine-DNA-glycosylase-sensitive sites. In addition, an increase of kinetochore-negative micronuclei (V79) and phosphorylation of the tumour suppressor protein p53 (HT29) underlined the genotoxic potential of these nanostructures.

Mutagenicity of the mycotoxin alternariol in cultured mammalian cells

The mycotoxin alternariol (AOH) is an important contaminant of fruit and cereal products. Concern about exposure to low levels of AOH was raised after the disclosure that contamination of food with the AOH-producing species Alternaria alternata is associated with oesophagal cancer. Previously we have reported that AOH induces kinetochore-negative micronuclei in cultured Chinese hamster V79 cells. The present study investigates the mutagenicity of AOH at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus in V79 cells and at the thymidine kinase (TK) gene locus in mouse lymphoma L5178Y tk +/− cells (MLC). Concentrations of 10 μM AOH and more gave rise to a significant and concentration-dependent induction of HPRT and TK mutations in V79 cells and in MLC, respectively. The mutagenic potency of AOH was about 50-fold lower than that of the established mutagen 4-nitroquinoline- N-oxide in both cell lines. Discrimination between small and large colonies in the TK assay revealed the predominant induction of small colonies, which are indicative for extensive chromosomal deletions and which correlated with the induction of micronuclei in MLC. The mutagenicity of AOH may have a bearing on the carcinogenicity of this mycotoxin.

Estrogenic and clastogenic potential of the mycotoxin alternariol in cultured mammalian cells

The mycotoxin alternariol (AOH) is found in food and beverages infected by Alternaria alternata. Because consumption of foodstuffs contaminated with A. alternata has been implicated in an elevated incidence of esophageal carcinogenesis, we have investigated the estrogenic potential, the effect on cell proliferation, and the genotoxic effect of AOH in cultured mammalian cells. AOH replaced E2 from isolated human estrogen receptors α and β and increased the level of alkaline phosphatase (ALP) mRNA and the enzymatic activity of ALP in a human endometrial adenocarcinoma cell line (Ishikawa cells). The estrogenicity of AOH was about 0.01% of that of E2. The effects in Ishikawa cells were reversed by the ER antagonist ICI 182,780. Analysis of cell proliferation by flow cytometry and microscopy of Ishikawa and Chinese hamster V79 cells revealed that AOH inhibited cell proliferation by interference with the cell cycle. The genotoxic potential was assessed by the micronucleus (MN) assay and immunochemical differentiation between MN containing whole chromosomes (kinetochore-positive) and DNA fragments (kinetochore-negative) in Ishikawa and V79 cells. AOH induced kinetochore-negative MN in both cell lines. This is the first report on the estrogenic potential, inhibition of cell proliferation and clastogenicity of AOH in Ishikawa and V79 cells in vitro.

Estrogenic and genotoxic potential of equol and two hydroxylated metabolites of Daidzein in cultured human Ishikawa cells

The soy isoflavone daidzein (DAI) is known to undergo metabolism to equol (EQO) and to 3′-hydroxy-DAI (3′-HO-DAI) and 6-hydroxy-DAI (6-HO-DAI) in humans. In order to better understand the implications of soy diets for human health, the hormonal and genotoxic activities of these DAI metabolites were studied in cultured human endometrial carcinoma cells. When the estrogenicity was tested by cell-free binding to recombinant human estrogen receptor (ER) α and β as well as by the induction of enzyme activity and gene expression of alkaline phosphatase (ALP) in Ishikawa cells, the ranking order was EQO > DAI > 3′-HO-DAI > 6-HO-DAI. All compounds had a higher affinity to ERβ than to ERα. No significant anti-estrogenic effects of the DAI metabolites were observed in the cells at non-cytotoxic concentrations. The in vitro genotoxicity was assessed by analyzing effects on cell cycle distribution and cell morphology as well as the induction of micronuclei (MN). EQO caused a slight increase in G1 and decrease in S phase of the cell cycle, and slightly but significantly induced kinetochore-positive as well as kinetochore-negative MN and an elevated proportion of abnormal mitotic spindles. 3′-HO-DAI, but not 6-HO-DAI, induced kinetochore-negative MN. The observation that major human metabolites of DAI exhibit estrogenic and genotoxic potential may be of relevance for the safety evaluation of diets containing soy isoflavones.

Aneugenic and clastogenic effects of doxorubicin in human lymphocytes.

Doxorubicin, a benzanthroquinone anticancer agent, was examined for its effect on micronucleus induction in cultured human lymphocytes. A statistically significant dose-dependent increase in micronucleus frequency (P<0.001) in binucleated cells was seen and an increase in the kinetochore-positive (P<0.001) and kinetochore-negative micronuclei (P<0.001) was observed. An increase was also observed in the number of necrotic cells, but the frequency of apoptotic cells remained almost constant. This confirms that doxorubicin is both clastogenic and aneugenic.

Induction of kinetochore-positive and kinetochore-negative micronuclei in CHO cells by ELF magnetic fields and/or X-rays.

To test the genotoxic effects of extremely low frequency (ELF) magnetic fields, the induction of micronuclei by exposure to ELF magnetic fields and/or X-rays was investigated in cultured Chinese hamster ovary (CHO) cells, using the cytokinesis block method. Micronuclei derived from acentric fragments or from whole chromosomes were evaluated by immunofluorescent staining using anti-kinetochore antibodies from the serum of scleroderma (CREST syndrome) patients. A 60 Hz ELF magnetic field at 5 mT field strength was applied, either before or after 1 Gy X-ray irradiation or without additional X-ray irradiation. No statistically significant difference in the frequency of micronuclei in CHO cells was observed between a sham exposure (no exposure to an ELF magnetic field) and a 24 h ELF magnetic field exposure. Exposure to an ELF magnetic field for 24 h before X-ray irradiation or for 18 h after X-ray irradiation did not affect the frequency of X-ray-induced micronuclei. However, the number of kinetochore-positive micronuclei was significantly increased in the cells subjected to X-ray irradiation followed by ELF magnetic field exposure, but not in the cells treated with ELF magnetic field exposure before X-ray irradiation, compared with exposure to X-rays alone. The number of spontaneous kinetochore-positive and kinetochore-negative micronuclei was not affected by exposure to an ELF magnetic field alone. Our data suggest that exposure to an ELF magnetic field has no effect on the number of spontaneous and X-ray-induced micronuclei. However, ELF magnetic field exposure after but not before X-ray irradiation may somehow accelerate X-ray-induced lagging of whole chromosomes (or centric fragments) in CHO cells.

Induction of aneuploidy by nickel sulfate in V79 Chinese hamster cells

The ability of nickel sulfate (NiSO 4) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100–400 μM NiSO 4 for 24 h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay. Aneuploid cells were induced in a significant fraction of the cell population 24–48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO 4. The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose. These results indicate that NiSO 4 has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.

Mechanisms involved in the induction of aneuploidy: the significance of chromosome loss

The induction of aneuploidy by physical and chemical agents using different test systems was evaluated. The effect of X-rays, caffeine, acetaldehyde, ethanol, diethylstilbestrol, propionaldehyde, and chloral hydrate was studied by chromosome counting in Chinese hamster embryonic diploid cells. Aneugenic ability of cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride, nickel chloride, and nickel sulfate was assessed by means of anaphase-telophase analysis in Chinese hamster ovary cells. Chromosome counting in human fibroblasts (MRC-5 cell line) was employed to evaluate the effect of cacodilic acid, cadmium chloride, cadmium sulfate, and potassium dichromate. Finally, the induction of kinetochore-positive and kinetochore negative micronuclei by cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride, and nickel chloride was studied using CREST antibodies. When the effect of different agents was determined by chromosome counting, an increase of hypoploid but not of hyperploid cells was observed. Anaphase-telophase analysis showed that metal salts increased the frequency of lagging chromosomes. This finding has been confirmed by the increment of kinetochore-positive micronuclei using CREST antibodies. Therefore, chromosome loss could be considered as the main cause of induced aneuploidy.

Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts

Numerous reports have shown that oxidative stress is involved in arsenite-induced genetic damage. Arsenite is also a potent inducer of heme oxygenase (HO)-1. To understand whether HO-1 could function as a cellular antioxidant and protect cells from arsenite injury, the effects of tin-protoporphyrin (SnPP), a competitive inhibitor of HO-1, on arsenite-induced genetic damage were examined in human skin fibroblasts (HFW). In the present study, we found that SnPP at 100 μM significantly potentiated arsenite-induced cytotoxicity, DNA strand breaks (assayed by alkaline single cell gel electrophoresis(SCGE)), and chromatid breaks. Although arsenite alone mainly induced kinetochore-plus micronuclei (K +-MN), SnPP only synergistically enhanced kinetochore-negative micronuclei (K −-MN). The increase in K −-MN by SnPP cotreatment was consistent with the increase in DNA strand breaks and chromatid breaks caused by SnPP. However, at higher arsenite doses, K +-MN was significantly reduced by SnPP. Pretreatment of HFW cells with hemin, an inducer of HO-1, significantly attenuated the cytotoxicity of arsenite. Therefore, the present results suggest that HO-1 induction by arsenite plays certain roles in protecting cells from arsenite-induced injury.

Dibenzo[a,l]Pyrene Induction of Erythrocyte Micronuclei in A/J and P53-Deficient Mice

Male A/J and C57Bl/6 background p53+/+, p53+/- and p53−/- mice were treated with dibenzo[a,l]pyrene (DB[a,l]P), and micronucleus (MN) frequencies were measured in erythrocytes from bone marrow and peripheral blood. MN were also evaluated with an antikinetochore antibody to distinguish whether they were derived from chromosome breakage or from chromosome missegregation. Treatment of A/J mice with 6 mg/kg DB[a,l]P, and harvest of marrow erythrocytes 48 and 72 hrs later, resulted in statistically significant increases in kinetochore-negative MN levels (2.8x and 5.5x control levels, respectively). Treatment of p53+/+ and p53−/- mice with 18 mg/kg DB[a,l]P, and harvest of marrow erythrocytes 48 hrs later, resulted in statistically significant increases in kinetochore-negative MN frequencies (1.9x and 4.2x control levels, respectively). Our results indicate that DB[a,l]P induces moderate levels of chromosome breakage without dose-dependence in erythrocytes, and that p53 protein plays a protective role.

Effects of exposure protocols on induction of kinetochore-plus and -minus micronuclei by arsenite in diploid human fibroblasts

Arsenic, widely distributed in the environment, is a potent human carcinogen. Arsenite genotoxicity has been observed in a variety of cells and animal systems. However, the underlying mechanism is not completely clear. In this study, human fibroblasts (HFW) were treated with 1.25–10 μM arsenite for 24 h (low dose and long exposure) and 5–80 μM for 4 h (high dose and short exposure), and the arsenite accumulation, cytotoxicity, and micronucleus (MN) induction were examined. By these two different protocols, HFW cells showed equivalent levels of arsenite accumulation, but exhibited different kinetics of cell killing and different types of MN generation. Arsenite induced mainly kinetochore-positive MN (K +-MN) in HFW cells by low dose exposure whereas mainly kinetochore-negative MN (K −-MN) was induced by high dose exposure. Catalase reduced both K +- and K −-MN induced by these two exposure protocols. Except for the case of K +-MN induction by the high dose exposure protocol, N-acetyl-cysteine (NAC) in both low and high dose protocols was also shown to effectively reduce arsenite-induced MN. The present results imply that oxidative stress is involved in arsenite-induced MN in diploid human fibroblasts. However, different protocols for arsenite exposure may result in different cellular damage.

Micronuclei in lymphocytes and exfoliated buccal cells of postmenopausal women with dietary changes in folate

Folate deficiency is associated with anemia, birth defects, cancer and neuropsychiatric disorders. The purpose of this study was to determine if a moderate folate deficiency during controlled changes in folate intake would affect chromosomal damage in lymphocytes and buccal cells. A study of nine healthy postmenopausal women volunteers (age 49–63 years) was carried out in a metabolic unit (baseline week with folate intake of 195 μg/day, five-week depletion at 56 μg/day, and gradual repletion including four weeks at 111 μg/day, 11 days at 286 μg/day and 9 days at 516 μg/day). Plasma folate, vitamin B-12, and homocysteine were measured weekly. Cytogenetic damage was assessed by scoring micronucleus (MN) frequency in lymphocytes and buccal cells three times: (1) at the beginning of the study, (2) at the end of depletion, and (3) after repletion. The MN frequency increased in binucleated lymphocytes, as well as in all lymphocytes, after depletion ( p=0.037), and later decreased following repletion ( p=0.028). Both kinetochore-positive and kinetochore-negative MN were increased after depletion ( p=0.015 and 0.028), but after repletion only the change in kinetochore-positive MN was statistically significant ( p=0.048). The main variables affecting MN were: (1) vitamin B-12 level, (2) plasma folate level, and (3) baseline frequency of MN. The MN frequency in exfoliated buccal cells was decreased after dietary supplementation of 516 μg/day folate ( p=0.010). Thus, low folate, without clinical symptoms of anemia, results in higher levels of cytogenetic damage in both the blood and oral cavity of postmenopausal women.

Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo.

Molybdenum is an essential element in plants and animals as a cofactor for enzymes. Molybdenum trioxide is used in metallurgical processes, in cosmetics as a pigment, and in contact lens solution, yet limited information is available on molybdenum genotoxicity. In the present study the micronucleus (MN) assay in human lymphocytes and mouse bone marrow and the dominant lethal assay in mice were used to assess the genotoxic effects of molybdenum salts in vitro and in vivo. Two salts of molybdenum were tested in whole blood cultures. Ammonium molybdate was more potent than sodium molybdate in causing a dose-dependent decrease in viability and replicative index and an increase in MN formation in binucleated lymphocytes (P < 0.001). A dose-response in both kinetochore-positive MN (caused by chromosome lagging) and kinetochore-negative MN (associated with chromosome breakage) was observed. Based on the results of a toxicity study of sodium molybdate, two doses, 200 and 400 mg/kg, were assessed in the bone marrow MN assay in mice (two i.p. injections 24 and 48 hr prior to euthanasia). A modest but statistically significant increase in MN frequency in polychromatic erythrocytes was observed (P < 0.05). The same treatment protocol was used to analyze dominant lethality. A dose-dependent increase in postimplantation loss represented mostly by early resorptions was observed the first week after treatment (P = 0.003). These preliminary data suggest that sodium molybdate induces dominant lethality at the postmeiotic stage of spermatogenesis. Overall, molybdenum salts produced moderately positive results both in vitro in human cells and in vivo in mice.

Induction of kinetochore positive and negative micronuclei in V79 cells by N-methyl- N′-nitro- N-nitrosoguanidine: the protective effect of the pyridoindole antioxidant stobadine

The induction of micronuclei by N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and their reduction by the cardioprotective synthetic antioxidant, stobadine were studied in hamster V79 cells cultured in vitro. The micronuclei derived from acentric fragments or from whole chromosomes were evaluated with the help of an immunofluorescent staining using antikinetochore antibodies from the serum of scleroderma (CREST syndrome) patients. Our results showed that MNNG (0.5 μg/ml) induced mainly kinetochore-negative micronuclei. At 6, 24 and 48 h after MNNG treatment, we measured a 2.7-, 4.3- and 7.0-fold increase, respectively, of kinetochore-negative micronuclei over the controls. The increase of kinetochore-positive micronuclei was rather low and represented at 6, 24 and 48 h, respectively 0.9-, 1.8- and 2.6-fold increases over the controls. Stobadine decreased the level of kinetochore-negative micronuclei at 6, 24 and 48 h to approximately one-half; the frequency of kinetochore-positive micronuclei was reduced only at 6 h. We suppose that the antioxidant stobadine reduces the induction of micronuclei by MNNG by scavenging of MNNG-induced highly reactive OH radicals which cause chromosomal damage.

Genotoxicity of quercetin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes

Quercetin, a mutagenic flavonoid widely distributed in edible plants, was studied for the induction of micronuclei (MN). We have carried out the MN assay in bone marrow polychromatic erythrocytes in mice, in cytokinesis-blocked human lymphocytes and in cytokinesis-blocked V79 cells. MN assay in vitro was performed in the presence and in the absence of S9. To further extend the study, an antikinetochore antibody (CREST staining) was used to distinguish MN containing whole chromosomes (kinetochore positive) from those containing acentric fragments (kinetochore negative). When tested in vivo quercetin failed to induce micronuclei, a result which is in agreement with other published reports. When tested in vitro in V79 cells quercetin clearly induces micronuclei in the absence of S9 and also in the presence of S9 for the highest dose used. When tested in vitro in human lymphocytes quercetin shows a significant induction of micronuclei in the absence and in the presence of S9. The presence of s9 compared to its absence is not significant for any of the systems used. Both in the presence and absence of S9, quercetin appears to behave as a clastogenic agent in human lymphocytes inducing a significant majority of kinetochore-negative MN.