DANIO RERIO (ZEBRAFISH) AS A MODEL FOR RADIOBIOLOGICAL RESEARCH

Genetic polymorphisms of XRCC1, HOGG1 and MGMT and micronucleus occurrence in Chinese vinyl chloride-exposed workers

The possibility of using the cytokinesis-block micronucleus (MN) assay as a rapid test of chemosensitivity was investigated. SCCVII murine carcinoma cells growing exponentially in vitro were treated for 1 h under aerobic conditions with various concentrations of 11 anticancer agents: mitomycin C, doxorubicin, epirubicin, cisplatin, carboplatin, etoposide, vincristine, 5-fluorouracil, methotrexate, nimustine and dacarbazine. After removing the drugs, cytochalasin B (1.5 micrograms/ml) was added to block cytoplasmic but not nuclear division. At various times during culture, the proportions of binucleate cells (BNC) and multinucleate cells in the total cell population, and the mean number of micronuclei per single BNC were determined. Cell survival was also determined using the standard colony formation assay and was compared with the MN frequency. Maximal percentage of BNC was usually reached at 24-30 h of culture, except for cells treated with doxorubicin and epirubicin, in which it was reached at 30-72 h. All drugs induced formation of micronuclei and dose-response curves for MN frequency were obtained using the data at peak percentage of BNC times. For all drugs, MN frequency increased with concentration, but at the highest concentrations used (which are considered to be overly toxic) the MN frequency was rather lower. This decrease in MN frequency was largely attributable to the decrease in percent BNC. When the data at the highest concentrations of all drugs were excluded, a correlation was found between MN frequency and surviving fraction (r = 0.85). Therefore, it was concluded that the cytokinesis-block MN assay can be used to assess sensitivity of at least some tumor cells to appropriate concentrations of various chemotherapeutic agents.

The micronucleus test (MNT) has shown increased micronuclei (MN) frequencies in BRCA associated and sporadic breast cancer patients, Ataxia telangiectasia and Nijmegen Breakage Syndrome patients, demonstrating a common cellular phenotype of increased radiosensitivity. Some genes, causative of these diseases, have also recently been associated with prostate cancer. In order to investigate if prostate cancer exhibits the cellular phenotype of increased radiosensitivity, we performed MNT analysis on 22 sporadic prostate cancer patients and 43 male controls. We determined the baseline MN frequency, in order to see in vivo chromosomal damage without radiation, and induced (after irradiation with 2 Gy) frequency of MN, both in binucleated cells (BNC) obtained from cultured peripheral blood lymphocytes. An automated image analysis system was used to score the MN employing two different classifiers (Classifier A and B) for detection of BNC. The mean baseline frequencies were 48/43 MN/1000 BNC (A/B) for the controls and 42/50 (A/B) for prostate cancer patients. The induced MN frequencies amounted to 107/111 MN/1000 BNC (A/B) for controls and 111/114 MN/1000 BNC (A/B) for prostate cancer patients. The obtained MN frequencies did not result in a statistically significant difference between unselected cases and controls. However, restricting the analysis to young patients (50–60 years, N = 7) and age-matched controls (N = 7) revealed marginally significant higher MN frequencies in patients. We conclude that increased radiosensitivity is not a property of prostate cancer patients in general.

The genotoxic impact of HIV infection on the oral cavity malignancies is unknown. The aim of this study was to evaluate the effect of HIV infection in micronucleus (MN) frequency on the oral mucosa of HIV+ patients and establish a relationship with early cytogenetic changes in oral carcinogenesis. Thirty HIV+ individuals who are under highly active antiretroviral therapy (HAART) and 30 non-HIV patients were evaluated. Two smears were taken from the lateral border of the tongue and mouth floor and stained by Feulgen. The frequency of MN was examined in 3000 cells per subject under common microscopy. MN analysis showed no significant difference between groups by Mann-Whitney U-test for total MNs (P = 0.178). The presence of single MN was greater in control group with statistical significance (P = 0.009), while in HIV group, multiple MNs were exhibited in higher mean. HIV patients under HAART therapy and low viral load values showed higher frequency of multiple MNs, which, although not statistically significant, may be caused by the action of the Vpr gene, an accessory gene of HIV. These results corroborate the theory of HIV infection cytogenetic damage.

Differentiated thyroid cancers (DTCs) derive from thyroid follicular cells and include papillary and follicular cancers. In patients with DTCs, the initial treatment includes thyroidectomy and radioactive iodine (131-I) therapy. The objective of this study was to examine whether the intensity of DNA damage in peripheral blood lymphocytes (PBLs) of DTC patients depends on the amount of 131-I retained in the selected regions of interest (thyroid and abdominal region) as well as in the whole-body 72 hours after therapy. In addition, the possible influence of other factors that may affect micronuclei (MN) frequency, such as age, gender, smoking habits, and histological type of tumour was analyzed. The study population consisted of 22 DTC patients and 20 healthy donors. Data on the distribution of 131-I were obtained from the whole-body scans. MN frequency and cytokinesis-block proliferation index (CBPI) were measured using cytokinesis-block micronucleus assay. 131-I therapy significantly increased the MN frequency (19.50±6.90 vs. 27.10±19.50 MN) and significantly decreased the CBPI (1.52±0.20 vs. 1.38±0.17) in patients' lymphocytes. There was a clear correlation between the increased MN frequency and 131-I accumulation in the thyroid region in patients without metastases. The MN values did not differ in relation to the factors that could affect MN, such as age, gender, smoking habits, and histological type of tumour. In conclusion, the MN frequency in PBLs of DTC patients without metastases depends on the accumulation of 131-I in the thyroid region and does not depend on the other factors examined.

The carcinogenic effects of low radiation doses have not been fully understood until now. Studies on individuals that are occupationally exposed to low radiation doses can help to address this question. This study assesses the micronucleus (MN) frequencies as indicator of DNA damage in radiation workers that are occupationally exposed to low radiation dose. The influence of single nucleotide polymorphisms (SNPs) in XRCC3 gene on the frequency of micronuclei was also evaluated in this study. The effects of confounding factors of gender, age, and smoking status on MN frequencies was assessed in all samples. A total of 60 subjects consisting of 30 radiation workers from Center of Multipurpose Reactor (CMPR), National Nuclear Energy Agency (NNEA) of Indonesia, and 30 control samples were enrolled in this study. The results showed that the difference between MN frequency in radiation workers and in control samples was not statistically significant [0.019 vs. 0.021; p = 0.549]. Age and smoking status did not affect micronucleus frequencies in all samples ( p = 0.723 and 0.828). Micronucleus frequencies in females were higher compared to males, even though the difference was not significant ( p = 0.3). Radiation workers with variant alleles for XRCC3 olymorphism did not showed higher MN frequencies compared to the controls with the same genotypes. The small numbers of samples with XRCC3 variant alleles found in this study possibly contributed to the insignificant difference of MN frequencies between wild-type allele ( Thr/Thr ) and mutant alleles ( Thr/Met or Met/Met ). Further investigations using larger sample sizes and MN assay in combination with human pan-centromeric probe should be conducted to validate this study results. Other SNP in XRCC3 gene also should be evaluated to find out the association between SNP and MN frequencies.

Genotoxicity potential of soils taken from wastewater irrigation areas and bioremediation sites was assessed using the Vicia faba root tip micronucleus assay. Twenty five soils were tested, of which 8 were uncontaminated soils and taken as the control to examine the influence of soil properties; 6 soils were obtained from paddy rice fields with a history of long-term wastewater irrigation; 6 soils were obtained from bioremediation sites to examine effects of bioremediation; and 5 PAH-contaminated soils were used to examine methodological effects between direct soil exposure and exposure to aqueous soil extracts on micronuclei (MN) frequency ( per thousand) in the V. faba root tips. Results indicate that soil properties had no significant influences on MN frequencies (p > 0.05) when soil pH varied between 3.4 to 7.6 and organic carbon between 0.4% and 18.6%. The MN frequency measured in these control soils ranged from 1.6 per thousand to 5.8 per thousand. MN frequencies in soils from wastewater irrigation areas showed 2- to 48-fold increase as compared with the control. Soils from bioremediation sites showed a mixed picture: MN frequencies in some soils decreased after bioremediation, possibly due to detoxification; whereas in other cases remediated soils induced higher MN frequencies, suggesting that genotoxic substances might be produced during bioremediation. Exposure to aqueous soil extracts gave a higher MN frequency than direct exposure in 3 soils. However, the opposite was observed in the other two soils, suggesting that both exposure routes should be tested in case of negative results from one route. Data obtained from this study indicate that the MN assay is a sensitive assay suitable for evaluating genotoxicity of soils.

Objective:The aim of this study was to determine micronucleus (MN) frequencies in exfoliated cervical cells and peripheral blood lymphocytes of women with polycystic ovarian syndrome (PCOS).Materials and Methods:Fifteen patients with PCOS and 11 healthy control patients were included in the study. Cervical smears and peripheral blood were collected from all patients. Specimens were analyzed for MN frequencies and compared between the groups. In addition to MN, other nuclear anomalies connected with both genotoxicity and cytotoxicity were evaluated.Results:The MN frequencies in cervical smear and peripheral blood lymphocytes were compared in patients with PCOS and normal controls. There was no statistically significant difference between the groups regarding micronucleus frequency in peripheral blood lymphocytes (p=0.239). The mean MN scores in exfoliated cervical cells of patients with PCOS and normal controls were 1.19±0.57 and 0.74±0.34, respectively. The difference regarding micronucleus frequencies in cervical cells was statistically significant between the groups (p=0.032).Conclusion:Although study group is small, our study results support that there is an increased micronucleus frequency in cervical exfoliated cells of PCOS patients; this is a determinant of genetic hazard in the disease.

Lansium domesticum (Meliaceae) is an economic plant grown in the Southern, Eastern and Northern parts of Thailand. Fruit of L. domesticum (LD) is very popular in Thailand and commonly called ‘Longkong’. The aim of this study was to investigate anti-oxidant activity and genotoxicity property of L. domesticum fruits, particularly the skins and seeds. The air-dried samples of skin (SK) and seed (SD) were extracted with 50 and 95% ethanol. The ethanolic extracts were partitioned between dichloromethane (DCM) and 50% aqueous ethanol. The aqueous phase was further extracted with ethyl acetate (EA) to yield a total of 12 fractions. Their anti-oxidant capacity was determined on superoxide anion (O2-∙) scavenging by photochemiluminescence (PCL) assay, of both lipid (ACL) and water (ACW) soluble substance systems. The deoxyribose assay was also performed to assess their hydroxyl radicals (OH-∙) scavenging activity. Our results suggested that skin of L. domesticum fruits possessed higher antioxidant potential than seeds, particularly when extracted with 50% ethanol and partitioned with ethyl acetate, which had high potential both hydrophilic and lipophilic antioxidant substances. The genotoxicity effect of ethanolic extract of L. domesticum was investigated in TK6 human lymphoblasts using cytokinesis-blocked micronucleus (CBMN) assay. Mitomycin C (MMC) served as a positive control and results were analyzed on micronucleus (MN) frequencies in binucleated cells (BNC). We found that there was no significant increase in MN frequencies in L. domesticum-treated cells whereas MMC-treated cells clearly exhibited a marked increase in MN frequency (40-72 MN/1000 BNC). It indicated that the ethanolic extracts of L. domesticum seeds were non- genotoxic to TK6 cells.

Determination of toxic effects of lead acetate on different sizes of zebra fish (Danio rerio) in soft and hard water

Purpose: To investigate (1) the radiosensitivity of B versus T lymphocytes with respect to micronucleus (MN) induction and (2) the possible application of the B cell MN assay for biological dosimetry of individuals after acute exposure to low doses of ionizing radiation. Materials and methods: MN analysis was performed in T and B lymphocytes of six healthy volunteers exposed in vitro to gamma -ray doses ranging from 0.05 Gy to 1 Gy. For the MN assay on B cells, peripheral blood mononuclear cells were cultured and stimulated with pokeweed mitogen (PWM). Afterwards the B lymphocytes (characterized by the CD20 phenotype) were separated with the FACSort flow cytometer and the number of MN in the sorted binucleate cells was scored. For T lymphocytes the standard MN protocol was applied. Results: The number of spontaneous and radiation induced MN were significantly higher in B lymphocytes compared to T lymphocytes in the low dose range up to 1 Gy. An analysis of the present data showed that when the spontaneous MN frequencies are not known, doses from 0.08 Gy could be detected with the B cell MN assay while the conventional MN assay only allowed detection of doses 0.25 Gy. However, in contradiction to the linear-quadratic dose-response for T cells, for B cells the initial steep increase of the MN yield with the very low dose was followed by a flattening of the curve towards higher doses. Conclusion: This study shows that B lymphocytes express a high number of MN for doses up to 1 Gy gamma -rays reflecting the highly radiosensitive behaviour of B cells. The results also point to the possible application of the B-cell MN assay for individual dose assessment. When blood samples can be taken within 24 h after acute accidental overexposure, the B-cell MN assay can be performed but only as a supplementary test to the conventional MN assay.

The genotoxic potential of hexachlorocyclohexane (HCH) isomers (α-, β-, and γ-) which are organochlorine pesticides was tested in peripheral blood lymphocyte cultures from two donors by using the cytokinesis-block micronucleus assay. Micronucleus (MN) frequency, binucleated cells with micronucleus (BNMN), and cytokinesis-blocked proliferation index (CBPI) were determined as genotoxic and cytotoxic endpoints. At the concentration ranges tested (12.5–100 μg.L −1), all HCH isomers induced dose-dependent cytotoxic effects, γ-HCH being the most toxic. This isomer was also able to induce significant increase in MN frequency and BNMN cells indicating a genotoxic potential at 50 and 100 μg.L −1. The genotoxic test of β-HCH showed a positive induction of MN and BNMN cells at the highest concentration of 100 μg.L −1 and a significant cytotoxicity at 50 μg.L −1. Under the experimental condition used, α-HCH was unable to induce any significant increase in MN frequency confirming that α-HCH is a non-genotoxic agent.

Cytogenetic biomarkers, such as micronuclei in peripheral blood or oral mucosa, are widely used for evaluation of exposure to genotoxins or carcinogens. Tobacco is one of the strongest carcinogens, responsible for development of different types of cancers. The aim of this study was to assess the genotoxicity of cigarette consumption in young smokers and to correlate results of cytogenetic analysis in peripheral blood lymphocytes and exfoliated buccal cells. The study was conducted on samples taken from 43 smokers and 44 non-smokers, young individuals from Bosnia and Herzegovina. Significantly higher frequency of micronuclei in peripheral blood lymphocytes was observed in smokers (p < 0.05). No significant correlations were found for age, duration and intensity of smoking, and frequency of micronuclei in lymphocytes. Significantly higher frequency of degenerated (apoptotic) buccal cells was also revealed in smokers (p < 0.05). The frequency of apoptotic cells in smokers was significantly influenced by the age of participants (F = 8.649; p < 0.01) and duration of smoking (F = 5.389; p < 0.05). Results of cytogenetic analysis conducted in peripheral blood and exfoliated buccal cells are in significant positive correlation, indicating complementarities of those analyses.

Alcohol abuse greatly increases the risk of various malignancies, including cancer of the liver and digestive tract. Although it is thought that this may be due, at least partially, to the mutagenic properties of ethanol, little is known about the genotoxic effects of ethanol in humans. We investigated the chromosomal damage in lymphocytes from 20 alcoholics and 20 controls using the micronucleus (MN) assay combined with fluorescence in situ hybridization (FISH) with a pancentromeric DNA probe capable of differentiating centromere positive (C+) from centromere negative (C-) MN. The frequency of MN in binucleate lymphocytes was significantly higher in alcoholics than in controls (12.0 +/- 5.4 and 7.6 +/- 1.6, respectively; P: < 0.05). FISH revealed significantly higher frequencies of C+ MN in alcoholics than in controls (8.2 +/- 4.8 and 3.4 +/- 1.4, respectively; P: < 0.05). In the alcoholics, no association was found between years of alcohol abuse and frequency of MN or C+ MN. However, age influenced MN and C+ MN frequency both in alcoholics and controls. These results indicate that alcohol abuse may well induce chromosome loss in humans, suggesting a possible aneugenic mechanism of alcohol. This effect could contribute to the health hazards related to alcoholism such as cancer risk.

Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions