Micronuclei and other nuclear anomalies in buccal epithelial cells of children with chronic kidney disease.

The objective of this study was to reveal the likely genomic instability in children with chronic kidney disease (CKD) using micronucleus (MN) assay on buccal epithelial cells (BEC). We investigated the frequencies of micronuclei and other nuclear anomalies, such as nuclear buds, binucleated cells, condensed chromatin, and karyorrhectic and pyknotic cells in BEC. Children with CKD were grouped as follows: children in the pre-dialysis (PreD) stage (N=17), children on regular haemodialysis (HD) (N=14), and children who have undergone transplantation (Tx) (N=17). As a control group, twenty age- and gender-matched healthy children were selected. The MN frequency in BEC of all groups of children with CKD was significantly elevated (5- to 7-fold) as compared to the control group (p<0.001). In contrast, the frequencies of nuclear buds were not significantly higher in the study groups compared to the control group. The frequencies of binucleated cells and condensed chromatin cells were significantly higher in all subgroups of children with CKD relative to the control group (p<0.001). Our results show that the BEC of pediatric PreD, HD, and Tx patients with CKD display increased cytogenetic, cytokinetic, and cytotoxic effects. They also point to the sensitivity and usefulness of the BEC MN assay in the assessment of genetic susceptibility of patients with CKD.

Cellular uptake, genotoxicity and cytotoxicity of cobalt ferrite magnetic nanoparticles in human breast cells.

Magnetic nanoparticles (MNPs) have been increasingly used for many years as MRI agents and for gene delivery and hyperthermia therapy, although there have been conflicting results on their safety. In this study, cobalt ferrite magnetic nanoparticles (CoFe-MNPs) were prepared by the co-precipitation method and their surfaces were modified with silica by the sol-gel method. The particle and hydrodynamic sizes, morphology and crystal structure of the bare and silica-coated CoFe-MNPs were evaluated by transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction spectroscopy (XRD) and Fourier transform infrared spectroscopy (FTIR). The size of the bare CoFe-MNPs was in the range 8-20 nm and they were homogeneously coated with 3-4 nm silica shells. The bare and silica-coated CoFe-MNPs were agglomerated at physiological pH. However, the sizes of the agglomerates were below 200 nm both in water and complete medium. The cytotoxic and genotoxic potentials of the bare and silica-coated CoFe-MNPs were evaluated in a metastatic breast cancer cell line, MDA-MB-231, as well as a noncancerous mammary epithelial cell line, MCF-10A, by using XTT cytotoxicity, single-cell gel electrophoresis (comet), and cytokinesis-blocked (CB) micronucleus (CBMN) assays. Characterization studies with TEM, inductively coupled plasma optical emission spectroscopy (ICP-OES) and Prussian blue staining indicated that the CoFe-MNPs were internalized into the cells by energy-dependent endocytosis. The highest amount of uptake was observed in the cancer cells and the uptake of the silica-coated CoFe-MNPs was higher than that of the bare ones in both cell lines. The bare CoFe-MNPs showed higher levels of both cytotoxicity and genotoxicity than the silica-coated CoFe-MNPs. Moreover, the cancer cells seemed to be more susceptible to the CoFe-MNPs' toxicity compared to the noncancerous cells. There was a concentration and time-dependent increase in DNA damage and the micronucleus (MN) frequency, which was statistically significant starting with the lowest concentration of bare CoFe-MNPs (p < 0.05), while no significance was observed below the concentration of 250 µg mL-1 for the silica-coated MNPs. Also, the extent of both DNA damage and MN frequency was much higher in the cancer cells compared to the noncancerous cells. According to our results, the silica coating ameliorated both the cytotoxicity and genotoxicity as well the internalization of the CoFe-MNPs.

Cytogenetic biomonitoring of primary school children exposed to air pollutants: micronuclei analysis of buccal epithelial cells.

There is an increasing attempt in the world to determine the exposures of children to environmental chemicals. To analyze the genotoxic effect of air pollution, micronucleus (MN) assay was carried out in buccal epithelial cells (BECs) of children living in an urban city of Turkey. Children from two schools at urban-traffic and suburban sites were investigated in summer and winter seasons for the determination of BEC-MN frequency (per mille) and frequency of BEC with MN (per mille). The same children were also recruited for lung function measurements within a MATRA project ("Together Towards Clean Air in Eskisehir and Iskenderun") Measured NO2 and SO2 concentrations did not exceed the European Union (EU) limit levels either in urban-traffic or suburban regions. Higher O3 concentrations were measured in the suburban site especially in the summer period. Particulate matter (PM2.5 and PM10) levels which did not differ statistically between two regions were above the EU limits in general. Although BEC-MN frequencies of children living in the suburban sites were higher in general, the difference between two regions was not significant either in the summer or winter periods. BEC-MN frequencies of the urban-traffic children were found to be significantly higher in summer period (mean ± SD, 2.68 ± 1.99) when compared to winter period (1.64 ± 1.59; p = 0.004). On the other hand, no seasonality was observed for the suburban children. Similar results have been obtained in the BEC frequency with MN in our study. In summer, BEC-MN frequencies were significantly increased with the decrease in pulmonary function levels based on forced expiratory flow between 25 and 75% of vital capacity (FEF25-75%) levels (p < 0.05). As a conclusion, children living in urban-traffic and suburban areas in the city of Eskisehir exhibited similar genotoxicity. Seasonal variation in genotoxicity may be interpreted as relatively high ozone levels and increasing time spent at outdoors in the summer.

Assessment of individual susceptibility to baseline DNA and cytogenetic damage in a healthy Turkish population: evaluation with lifestyle factors.

BACKGROUND: Cytogenetic biomarkers are most frequently used well-established endpoints in human population studies with their sensitivity for measuring exposure to genotoxic agents. They have an important role as early predictors of cancer risk. Identification of individual genotypes of metabolic gene polymorphisms helps to understand the modulation of cancer susceptibility by environmental exposures, such as cigarette smoking and other lifestyle factors. AIM: To evaluate individual susceptibility to chemicals, we determined individual DNA damage related to glutathione S-transferase (GST) genotypes (GSTM1, GSTT1, and GSTP1) in a Turkish population. METHODS: Peripheral blood lymphocytes (PBL) and DNA samples of 127 subjects were analyzed for the presence of DNA damage, using single-cell gel electrophoresis (the Comet assay), and for cytogenetic parameters (chromosomal aberrations [CAs], bleomycin-induced CA, and a cytokinesis-blocked micronucleus assay), and the polymerase chain reaction/restriction fragment length polymorphism method, respectively. RESULTS: Individuals carrying a GSTT1-null allele showed higher frequencies of CA and micronucleus (MN) (p=0.026, p=0.003, respectively), whereas the GSTM1-null and GSTP1 mutant genotypes did not show any differences in cytogenetic parameters. Our findings demonstrated that none of the lifestyle factors (smoking, alcohol drinking, dietary habits, vitamin intake, and physical activity), except for vitamin intake (p=0.002), were significantly associated with the studied cytogenetic parameters. CONCLUSION: Our results suggest that the GSTT1 gene polymorphism may influence the baseline cytogenetic frequency in a healthy population.

Basal damage and oxidative DNA damage in children with chronic kidney disease measured by use of the comet assay.

One consequence of chronic kidney disease (CKD) is an elevated risk for cancer. There is sufficient evidence to conclude that there is an increased incidence of at least some cancers in kidney-dialysis patients. Cancer risk after kidney transplantation has mainly been attributed to immunosuppressive therapy. There are no data evaluating DNA damage in children with CKD, in dialysis patients, or following kidney transplantation. In this study, the comet assay and the enzyme-modified comet assay - with the use of endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) enzymes - were conducted to investigate the basal damage and the oxidative DNA damage as a result of treatment in peripheral blood lymphocytes of children. Children at various stages of treatment for kidney disease, including pre-dialysis patients (PreD) (n=17), regular hemodialysis patients (HD) (n=15), and those that received kidney transplants (Tx) (n=17), comprised the study group. They were compared with age- and gender-matched healthy children (n=20) as a control group. Our results show that the %DNA intensity, a measure of basal damage, was significantly increased in children with CKD (mean ± SD) (5.22 ± 1.57) and also in each of the PreD, HD, and Tx groups [(4.92 ± 1.23), (4.91 ± 1.35), and (5.79 ± 1.94), respectively, vs the healthy children (2.74 ± 2.91) (p<0.001). Significant increases in oxidative DNA damage were only found in the FPG-sensitive sites for the PreD and Tx groups, compared with control and HD groups (p<0.05), suggesting that basal DNA damage was more evident for the PreD, HD, and Tx groups. The findings of the present study indicate a critical need for further research on genomic damage with different endpoints and also for preventive measures and improvements in treatment of pediatric patients, in order to improve their life expectancy.

The HUman MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol.

The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMN(XL) collaborative study. The HUMN(XL) project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74‰ (95% CI 0.52-1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p<0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (≥40cig/day, FR=1.37; 95% CI 1.03-.82) and decreased with daily fruit consumption (FR=0.68; 95% CI 0.50-0.91). The results of the HUMN(XL) project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency.

Micronucleus frequencies in lymphocytes and buccal epithelial cells from patients having head and neck cancer and their first-degree relatives.

Head and neck squamous cell carcinoma is the fifth most common cancer type worldwide. Even though it is known that the most important environmental aetiological factors for head and neck cancer (HNC) development are tobacco and alcohol, genetic susceptibility is also thought to be important. The use of biomarkers of chromosomal damage due to genetic instability in order to predict risk of cancer as well as to identify high-risk individuals is imperative. We have investigated genetic damage in patients having HNC (n = 59) and their first-degree relatives (FDRs) (n = 34) with a biomarker in two different tissues; the micronucleus (MN) test in peripheral blood lymphocytes and in exfoliated buccal cells. The mean (standard deviation) levels of MN frequencies (‰) in lymphocytes of patients, relatives and controls were 27.10 (9.52), 14.09 (5.13) and 9.00 (6.87), respectively. The mean (standard deviation) levels of MN frequencies (‰) in exfoliated buccal cells of patients, relatives and controls were 2.87 (1.16), 1.38 (0.85) and 1.23 (0.93), respectively. Our results indicated that spontaneous genetic damage in lymphocytes of patients having HNC was significantly higher than that of controls (P < 0.01) and thus genetic instability appeared to exist in lymphocytes of cancer patients. Similar findings were obtained for exfoliated buccal cell MN frequencies of cancer patients (P < 0.01). We observed that the FDRs of patients having HNC showed significantly higher chromosomal damage in terms of MN frequencies in lymphocytes when compared with those of controls (P < 0.05), thus reflecting an increased susceptibility to HNC in FDRs. However, for buccal cell MN frequencies, we could not demonstrate enhanced genetic instability in the FDRs of patients having HNC.

Increased micronucleus frequencies in surrogate and target cells from workers exposed to crystalline silica-containing dust.

Mining, crushing, grinding, sandblasting and construction are high-risk activities with regard to crystalline silica exposure, especially in developing countries. Respirable crystalline silica (quartz and cristobalite) inhaled from occupational sources has been reclassified as a human carcinogen in 1997 by the International Agency for Research on Cancer. However, the biological activity of crystalline silica has been found to be variable among different industries, and this has formed the basis for further in vivo/in vitro mechanistic research and epidemiologic studies. This study was conducted for genotoxicity evaluation in a population of workers (e.g. glass industry workers, sandblasters, and stone grinders) mainly exposed to crystalline silica in four different workplaces in Turkey. The micronucleus (MN) assay was applied both in peripheral blood lymphocytes (PBL) as a surrogate tissue and in nasal epithelial cells (NEC) as a target tissue of the respiratory tract. Our study revealed significantly higher MN frequencies in the workers (n = 50) versus the control group (n = 29) (P < 0.001) and indicated a significant effect of occupational exposure on MN induction in both of the tissues. For the NEC target tissue, the difference in MN frequencies between the workers and control group was 3-fold, whereas in peripheral tissue, it was 2-fold. Respirable dust and crystalline silica levels exceeding limit values and mineralogical/elemental dust composition of the dust of at least 70% SiO(2) were used as markers of crystalline silica exposure in each of the workplaces. Moreover, 24% of the current workers were found to have early radiographical changes (profusion category of 1). In conclusion, although the PBL are not primary target cells for respiratory particulate toxicants, an evident increase in MN frequencies in this surrogate tissue was observed, alongside with a significant increase in NEC and may be an indicator of the accumulated genetic damage associated with crystalline silica exposure.

Chromosomal damage in peripheral blood lymphocytes of traffic policemen and taxi drivers exposed to urban air pollution.

Urban air contains a diversity of chemical compounds, some of which are genotoxins. An increased risk of cancer has also been reported in occupations with heavy exposure to traffic-related pollution. The aim of this study was to assess the cytogenetic effects of urban air pollution by analyzing the chromosomal aberration (CA) frequencies in lymphocytes and to estimate the polycyclic aromatic hydrocarbons (PAHs) exposure by measuring urinary 1-hydroxypyrene (1-OHP) levels. A total of 15 traffic policemen and 17 taxi drivers working in the city of Ankara were the exposed groups and 23 healthy men working in the office departments were the control group. The overall mean +/- S.D. values of 1-OHP excretions of traffic policemen, taxi drivers and control subjects were 0.59 +/- 0.40 micromol/mol creatinine, 0.32 +/- 0.25 micromol/mol creatinine and 0.57 +/- 0.36 micromol/mol creatinine, respectively. Urinary 1-OHP levels of non-smoking policemen were significantly greater than those of nonsmoking control subjects (p < 0.05). The overall mean +/- S.D. values for CA frequencies (%) from policemen, taxi drivers and control group were 1.29 +/- 1.59, 1.81 +/- 1.79, and 0.26 +/- 0.73, respectively. There was a significantly greater frequency of CAs in exposed groups relative to the matched control population (p < 0.05; p < 0.01). Age, sex and smoking habits have not influenced the cytogenetic end-point in this study. Our results demonstrate that occupational exposure to urban air pollutants leads to a significant induction of cytogenetic damage in peripheral lymphocytes of traffic policemen and taxi drivers.