Negative Micronuclei Research Articles

Cyto-Genotoxicity of Tritiated Stainless Steel and Cement Particles in Human Lung Cell Models.

During the decommissioning of nuclear facilities, the tritiated materials must be removed. These operations generate tritiated steel and cement particles that could be accidentally inhaled by workers. Thus, the consequences of human exposure by inhalation to these particles in terms of radiotoxicology were investigated. Their cyto-genotoxicity was studied using two human lung models: the BEAS-2B cell line and the 3D MucilAirTM model. Exposures of the BEAS-2B cell line to particles (2 and 24 h) did not induce significant cytotoxicity. Nevertheless, DNA damage occurred upon exposure to tritiated and non-tritiated particles, as observed by alkaline comet assay. Tritiated particles only induced cytostasis; however, both induced a significant increase in centromere negative micronuclei. Particles were also assessed for their effects on epithelial integrity and metabolic activity using the MucilAirTM model in a 14-day kinetic mode. No effect was noted. Tritium transfer through the epithelium was observed without intracellular accumulation. Overall, tritiated and non-tritiated stainless steel and cement particles were associated with moderate toxicity. However, these particles induce DNA lesions and chromosome breakage to which tritium seems to contribute. These data should help in a better management of the risk related to the inhalation of these types of particles.

Potent aneugenicity of 1-methylpyrene in human cells dependent on metabolic activation by endogenous enzymes.

1-Methylpyrene (1-MP) is a common environmental pollutant and animal carcinogen. After sequential activation by cytochromes P450 and sulfotransferases, it induced gene mutations and micronuclei in mammalian cells. The type of micronuclei formed, entire chromosomes or fragments, was not analysed. In this study, 1-MP and its primary metabolite, 1-hydroxymethylpyrene (1-HMP), were investigated for the induction of centromere-positive and -negative micronuclei in the human hepatoma cell line HepG2 and its derivative C3A, expressing relevant enzymes at higher levels. Under a short-exposure (9h)/long-recovery regime (2 cell cycles in total), 1-MP and 1-HMP provided negative test results in HepG2 cells. However, they induced micronuclei in C3A cells, the effect being blocked by 1-aminobenzotriazole (inhibitor of cytochromes P450s) and reduced by pentachlorophenol (inhibitor of sulfotransferases). Immunofluorescence staining of centromere protein B in the micronuclei revealed purely clastogenic effects under this regime. Unexpectedly, 1-MP and 1-HMP at concentrations 1/5-1/4 of that required for micronuclei formation led to mitotic arrest and spindle aberrations, as detected by immunofluorescence staining of β- and γ-tubulin. Following extended exposure (72h, 2 cell cycles, no recovery), damage to the spindle apparatus and centrosomes was detected at even lower concentrations, with concurrent formation of micronuclei. At low concentrations (1-8µM 1-MP, 0.25-0.5µM 1-HMP), the micronuclei induced were unexceptionally centromere-positive. Thus, the chromosome-damaging mechanism of 1-MP was regime and concentration dependent: potently aneugenic under persistent exposure, while clastogenic at higher concentrations following a short-exposure/long-recovery regime. This is a convincing evidence for the existence of metabolic activation-dependent aneugens.

The repeated cytogenetic analysis of subjects occupationally exposed to nanoparticles: a pilot study.

The application of nanomaterials has been rapidly increasing during recent years. Inhalation exposure to nanoparticles (NP) may result in negative toxic effects but there is a critical lack of human studies, especially those related to possible DNA alterations. We analyzed pre-shift and post-shift a group of nanocomposite researchers with a long-term working background (17.8 ± 10.0 years) and matched controls. The study group consisted of 73.2% males and 26.8% females. Aerosol exposure monitoring during a working shift (involving welding, smelting, machining) to assess the differences in exposure to particulate matter (PM) including nanosized fractions <25-100 nm, and their chemical analysis, was carried out. A micronucleus assay using Human Pan Centromeric probes, was applied to distinguish between the frequency of centromere positive (CEN+) and centromere negative (CEN-) micronuclei (MN) in the binucleated cells. This approach allowed recognition of the types of chromosomal damage: losses and breaks. The monitoring data revealed differences in the exposure to NP related to individual working processes, and in the chemical composition of nanofraction. The cytogenetic results of this pilot study demonstrated a lack of effect of long-term (years) exposure to NP (total frequency of MN, P = 0.743), although this exposure may be responsible for DNA damage pattern changes (12% increase of chromosomal breaks-clastogenic effect). Moreover, short-term (daily shift) exposure could be a reason for the increase of chromosomal breaks in a subgroup of researchers involved in welding and smelting processes (clastogenic effect, P = 0.037). The gender and/or gender ratio of the study participants was also an important factor for the interpretation of the results. As this type of human study is unique, further research is needed to understand the effects of long-term and short-term exposure to NP.

Long-term genotoxic effects in the hematopoietic system of prenatally X-irradiated mice

Purpose: To investigate the genotoxic effects of prenatal X-irradiation in mice and the possible presence of late genomic instability.Materials and methods: Pregnant mice were exposed to 0, 1 or 2 Gy at embryonic day 11.5. Blood smears were obtained from pups at birth and on post-natal day 11, 21, 42 and 140. Hematological data (diameter of erythrocytes, percentage of reticulocytes and Granulocyte-to-Lymphocyte ratio [GLR]) and genotoxicity (micronucleated erythrocytes, micronucleated reticulocytes, CREST-positive and negative micronuclei) were assessed.Results: Prenatal irradiation caused perinatal reticulocytosis (which ended on postnatal day 11) and a dose-dependent increase of GLR (indicative of myeloid skewing) on postnatal days 42 and 140. Two temporally distinct genotoxic effects were observed: an early, acute damage (still detectable at birth and soon after) and a late, long-term damage.Conclusions: Increases in micronuclei frequencies and GLR observed from day 42 on are both ascribable to DNA damage. Time of appearance of this late effect may be linked to the shift of hematopoiesis from spleen to bone marrow and to cell-extrinsic factor such as the microenvironment. This study confirms that ionizing radiation can induce long-term genotoxic effects in the hematopoietic system and shows that prenatal irradiation determines genomic instability in blood-forming tissues of adult mice.

Частота поліморфних варіантів генів II фази біотрансформації ксенобіотиків GSTM1 і GSTT1 у новонароджених Івано-Франківської області України

З метою визначення алельного поліморфізму генів глутатіон-S-трансфераз GSTT1 та GSTM1 у здорових новонароджених, недоношених дітей та новонароджених із затримкою внутрішньоутробного розвитку проведено молекулярно-генетичне дослідження пуповинної крові. Для визначення індивідуальної реакції геному новонародженого на можливий токсичний вплив використовували мікроядерний тест. Встановлено, що в усіх новонароджених області частота поліморфного варіанта GSTМ1+ була 43,70 %, частота поліморфного варіанта GSTМ1– — 56,30 %. Для гена GSTT1 частота поліморфного варіанта GSTТ1+ та GSTТ1– була 80,67 та 19,33 % відповідно. Позитивний мікроядерний тест у новонароджених був асоційований із поліморфним варіантом GSTМ1– — 71,95 % випадків порівняно з 21,62 % при негативному мікроядерному тесті. У носіїв поліморфного варіанта GSTТ1– у жодному випадку не було виявлено позитивного мікроядерного тесту, у всіх новонароджених із негативним мікроядерним тестом був виявлений алельний варіант GSTT1+. Для новонароджених із негативним мікроядерним тестом характерне переважання (78,38 %) асоціації генотипів GSTM1+/GSTT1+.

Increased frequency of micronuclei in mononucleated lymphocytes and cytome analysis in healthy newborns as an early warning biomarkers of possible future health risks

Impact of intrauterine development on health risks during adolescence and adulthood still needs to be investigated. The aim of study was to compare genome damage in newborns and mothers using the cytokinesis blocked micronucleus assay, nuclear division index (NDI), and centromere fluorescence in situ hybridization. The study was performed on 92 mothers and their newborns. Results showed that micronucleus frequency in binuclear T-lymphocytes (MNBN) in newborns was significantly lower than in mothers but higher in mononuclear T-lymphocytes (MNMONO). The NDI in the mothers was significantly higher than in the newborns. In newborns with <2500g birth weight, NDI was similar to the mothers’. Mothers have significantly more centromere negative micronuclei than newborns. A significantly higher NDI and MNBN was found in newborns with ≥2 MNMONO/1000 than in newborns with <2 MNMONO/1000. It is suggested that MOMN and NDI might be good candidates for biomarkers of health risks in newborns.

DNA replication occurs in all lamina positive micronuclei, but never in lamina negative micronuclei

A micronucleus is a small nucleus-like structure found in the cytoplasm of dividing cells that suffered from genotoxic stress. It is generally hypothesised that micronuclei content is eventually lost from cells, though the mechanism of how this occurs is unknown. If DNA located within the micronucleus is not replicated, it may explain the loss of micronuclei content. Because there had been no compelling evidence for this issue, we have addressed whether DNA located within the micronucleus is replicated this issue. Pulse labelling of bromodeoxyuridine revealed that DNA synthesis takes place in a portion of micronuclei that contain nuclear lamin B protein. By using iodine 3'-deoxyuridine/chlorodeoxyuridine double labelling, we found that all micronuclei containing lamin B are replicated during one cell cycle, whereas micronuclei lacking lamin B are never replicated. This result suggests that the content of lamin B-negative micronuclei is lost during cell division. Furthermore, we simultaneously visualised sites of DNA synthesis, lamin B and the extrachromosomal double minutes chromatin, which contain amplified oncogenes. We found that although the replication timing of double minutes was generally preserved in micronuclei, at times it differed greatly from the timing in the nucleus, which may perturb the expression of the amplified oncogenes. Taken together, these findings uncovered the DNA replication occurring inside micronuclei.

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.

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C−MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C−MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.

Micronuclei in lymphocytes of uranium miners of the former Wismut SDAG

We studied micronucleus frequencies in former German uranium miners of the Wismut SDAG (Sowjetisch-Deutsche Aktiengesellschaft). Various other groups were analyzed for comparison (individuals with lung tumors or lung fibrosis, controls). We had shown previously that micronucleus frequencies were not different among the various groups. Differences were observed, however, when centromere-positive and -negative micronuclei were distinguished. In the analyses presented here, we looked for the effects of smoking habits, alcohol consumption, vitamin uptake, chronic diseases, allergies, doing sports, γ-GT (gamma-glutamyltranspeptidase), lymphocyte numbers, CEA (carcinoembryonic antigen), X-ray diagnostics, computer tomographies, and scintigraphies. With the exception of more than one scintigraphy carried out during the last four months before micronucleus analysis, none of the factors mentioned above significantly affected micronucleus numbers. One result deserves specific attention: individuals with low percentages of binucleated lymphocytes after in vitro cytochalasin B exposure showed higher micronucleus frequencies than those individuals with high percentages of binucleated cells. The same result was obtained for various other populations that we monitored in the past.

Differential effectiveness of solar UVB subcomponents in causing cell death, oncogenic transformation and micronucleus induction in human hybrid cells

Purpose : (1) To determine the biological effectiveness of two solar ultraviolet (UVB) spectra with different lower wavelength thresholds for oncogenic transformation and micronucleus induction in CGL1 cells; (2) to investigate whether the action spectra for short- and long-term effects are similar; and (3) to investigate possible links between transformation and other delayed effects. Material and methods : Two spectra were derived from a solar UV simulator by using two filters: the first transmitted radiation with 5 > 284 nm, the second with 5 > 293 nm. The resulting spectra have the same UVA, but different UVB components (5 between 284 and 320 nm, 19 W m m 2, and 5 between 293 and 320 nm, 13 W m m 2) . CGL1 cells were irradiated with 466 J m m 2 with 5 > 284 nm and 1582 J m m 2 with 5 > 293 nm. These doses were approximately equilethal. The endpoints examined were oncogenic transformation, and centromere-positive and -negative micronucleus frequencies in the directly irradiated cells and in transtheir progeny. Results : At equilethal doses, the oncogenic transformation frequency in the directly irradiated cells was greater by a factor of at least 7 for 5 > 284 nm irradiation compared with 5 > 293 nm. The micronucleus induction frequency was also significantly higher with the 5 > 284 spectrum. Consistent with our previous findings, no delayed micronucleus formation was found in the progeny of cells exposed to 5 > 293 nm, while a threefold elevation above controls was seen in the progeny of cells exposed to 5 > 284 nm irradiation. This was also the case for formation of micronuclei with a centromere. Conclusions : It was found that: (1) for equilethal doses the 5 > 284 nm spectrum was more biologically effective than the 5 > 293 nm spectrum for induction of oncogenic transformation and micronucleus formation; and (2) the higher effectiveness of the 5 > 284 nm spectrum found at equilethal doses for delayed effects in the progeny of irradiated cells resembles that found for transformation. The results suggest that the UVB action spectrum for cell killing is different from that of some delayed effects, and from that of transformation.

Induction of micronuclei by 7 H-dibenzo[ c, g]carbazole and its tissue specific derivatives in Chinese hamster V79MZh1A1 cells

The clastogenicity/aneugenicity of N-heterocyclic polycyclic aromatic pollutant 7 H-dibenzo[ c, g]carbazole (DBC) and its two synthetic derivatives N-methyl DBC (MeDBC) and 5,9-dimethyl DBC (diMeDBC) was evaluated in the genetically engineered Chinese hamster V79 cell line V79MZh1A1 with stable expression of human cytochrome P4501A1 and in the parental V79MZ cell line without any cytochrome P450 activity. While none of the three carbazoles changed significantly the level of micronuclei in the parental V79MZ cells, a variable, but statistically significant rise of micronucleus frequencies was assessed in V79MZh1A1 cells. DBC induced dose-dependent increase in the number of micronuclei at harvest times of 24 and 48 h and MeDBC at sampling time of 48 h in V79MZh1A1 cells in comparison to untreated cells, however, no significant time-dependent increase in micronucleus frequencies was found. The use of the antikinetochore immunostaining revealed that DBC and MeDBC induced approximately equal levels of both kinetochore positive (C+) and kinetochore negative (C−) micronuclei. DiMeDBC, a strict hepatocarcinogen, did not manifest any effect on micronucleus induction in V79MZh1A1 cells. These studies suggest that genetically engineered Chinese hamster V79 cell lines expressing individual CYP cDNAs are a useful in vitro model for evaluation the role of particular cytochromes P450 in biotransformation of DBC and its tissue and organ specific derivatives.

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.

Cytotoxic and genotoxic effect of inhibitor of vulcanisation N-cyclohexylthiophthalimide in a battery of in vitro assays

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells. The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations <7 μg/ml. Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 μg/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 μg/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with moderately damaged DNA were more common than cells with heavily damaged DNA. Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially. After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA. Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death.

Low persistence of radiation-induced centromere positive and negative micronuclei in cultured human cells

The micronucleus (MN) assay is widely used both in genetic toxicology and in the biomonitoring of human populations. Lymphocytes, cell lines, and bone marrow and epithelial cells are usually employed as target systems in such studies. However, little effort has been done to assess the persistence of MN in highly proliferative cells. To study the behaviour of MN containing whole chromosomes or acentric fragments, we have performed a time course experiment on the persistence of γ-ray (3 Gy) induced MN in a human lymphoblastoid cell line. The frequency and content of MN were analyzed 1, 3, 7, 14, and 56 days after irradiation by pancentromeric fluorescence in situ hybridization (FISH). We observed a clear induction of both centromere positive and negative MN at completion of the first mitotic division. The frequency of both types of MN drastically declined to basal levels 7 days after irradiation with an identical kinetics. We therefore conclude that centromere positive and negative MN are highly unstable upon cell division, indicating that the MN assay could not be a good biomarker of DNA damage induced by acute treatments in highly proliferative cells. The implication of our findings in biomonitoring and in genotoxicity studies is discussed.

Delayed cytotoxic and genotoxic effects in a human cell line following X-irradiation

Background: In order to clarify the relationship between delayed reproductive death and radiation-induced genomic instability, the colony-forming efficiency of surviving, irradiated human squamous carcinoma cells and centromere positive as well as centromere negative micronuclei in surviving progeny were examined. Materials and methods: Colony-forming ability and micronucleus (MN) frequency in binucleated cells 24h after the addition of cytochalasin B during 2 weeks of post-irradiation growth were determined in a squamous cell carcinoma cell line (SCL-II) of human origin. In addition, centromeres in micronuclei were detected using FISH. Results: In the human epithelial cell line used for these experiments, delayed reproductive death was pronounced and persisted for at least 2 weeks after irradiation. Although there is evidence for an increased rate of centromere positive micronuclei, but not of centromere negative micronuclei, arising during the first week of post-irradiation proliferation, this decreases later while the rate of delayed reproductive death remains elevated. Conclusion: In the studied cell line, the observed delayed reproductive death is not closely related to the investigated criteria of radiation-induced genomic instability. This casts doubt on the common assumption that delayed reproductive death is a direct manifestation of radiation-induced genomic instability.

Influence of serum micronutrients on the incidence of kinetochore-positive or -negative micronuclei in human peripheral blood lymphocytes

The possible contribution of some selected serum micronutrients (β-carotene, vitamins B12 and C, folic acid and α-tocopherol) to spontaneous chromosomal damage was investigated in human peripheral blood lymphocytes from 33 non-smoking healthy donors by the cytokinesis-block micronucleus assay. Labelling of micronuclei with antikinetochore serum was used to discriminate between kinetochore-positive and -negative micronuclei and thus between micronuclei which arise from whole chromosome loss and those which arise from chromosome breaks. Simple correlation analysis showed that age was significantly associated with the increased frequency of micronucleated cells, and this age-related increase in these cells was due to the increase in cells with both kinetochore-positive and -negative micronuclei. Serum micronutrient levels had no apparent significant effects on incidence of micronucleated cells except for the weak positive correlation between vitamin B12 levels and frequency of kinetochore-positive micronucleated cells. Multiple regression analysis with age and serum micronutrient levels as independent variables showed that (a) age was the most influential variable for the frequency of micronucleated cells, (b) the serum vitamin C level was associated with increased frequency of spontaneous micronucleated cells, and this increase was mainly due to the increase in cells with kinetochore-positive micronuclei, and (c) the serum folic acid level was significantly and negatively related to the frequencies of cells with both kinetochore-positive and -negative micronuclei. To avoid the predominant age-effect, we also performed separate multiple regression analysis with age-adjusted frequency of micronucleated cells as dependent variable. The results from this analysis again showed a significant and positive effect of serum vitamin C level on age-adjusted frequency of kinetochore-positive micronucleated cells, while marginal negative effect of folic acid on age-adjusted frequency of total micronucleated cells (P<0.06) and kinetochore-positive micronucleated cells (P<0.051) was detected. These results suggest that age and serum vitamin C are definitely variables for frequencies of spontaneous chromosome loss, and that serum folic acid is perhaps another important micronutrient which influence the frequency of spontaneous chromosomal damage.

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.

A study of the aneugenic activity of trichlorfon detected by centromere-specific probes in human lymphoblastoid cell lines

The potential of the pesticide trichlorfon to induce mitotic aneuploidy has been investigated in genetically engineered human lymphoblastoid cell lines. Trichlorfon failed to induce micronuclei in the AHH-1 and MCL-5 cell lines when treated in media at normal cell culture pH (pH 7.3). Under a treatment pH of 5.5, trichlorfon exposures resulted in the induction of both chromosome loss and chromosome non-disjunction as measured by fluorescence in situ hybridisation (FISH) using a pan-centromeric probe for all human centromeres and centromere probes specific for chromosomes 2, 7 and 18. At treatment concentrations greater than 20 μg/ml trichlorfon also induced structural chromosome damage resulting in the production of centromere negative micronuclei.

Immunofluorescent and confocal laser cytometric analyses of centromeres in V79 cells

Previously, a modified anticentromere antibody (ACA) technique was established in the V79 Chinese hamster lung cells to simulataneously analyze chromosome damage and aneuploidy induced by various agents. Using this method, cyclophosphamide (CP) was evaluated further in the presence and absence of S9 activation for micronucleus/aneuploidy induction. The specific binding nature of ACA to the centromeric region was also analyzed using a confocal scanning laser cytometry. The results indicated that CP was primarily a clastogen and S9 activation was required for its activity. Vinblastine, the positive control for aneuploidy, produced predominantly centromere containing micronuclei and the addition of S9 was not required for its activity. X-radiation, the positive control for clastogenicity, predominantly produced centromere negative micronuclei confirming its clastogenicity. An evaluation of centromeric region under the standard fluorescence microscope indicated that ACA generally binds to most centromeric regions in a cell. However, by confocal imaging it was found that ACA binds to the central core proteins of the centromere region and not to the peripheral proteins.