Gonca Cakmak Demircigil

The HUman MicroNucleus project on eXfoLiated buccal cells (HUMNXL): 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.

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.

Assessment of cytogenetic damage in lymphocytes and in exfoliated nasal cells of dental laboratory technicians exposed to chromium, cobalt, and nickel

Dental laboratory technicians may be exposed to metal alloys that are used in the production of crowns, bridges and removable partial dentures. These alloys consist of 35-65% cobalt, 20-30% chromium, 0-30% nickel, and small amounts of molybdenum, silica, beryllium, boron and carbon. The aim of this study was to assess whether dental technicians are occupationally exposed to chromium, cobalt and nickel, by analyzing urinary excretion levels of these metals and to investigate the genotoxic effects of occupational exposure associated with dental prostheses production operations by analyzing cytokinesis-blocked micronucleus (CB-MN) frequencies in peripheral lymphocytes and micronucleus (MN) frequencies in exfoliated nasal cells from 27 dental laboratory technicians and 15 control subjects. The differences in the urinary excretion of metals between technicians and controls were statistically significant. The mean (+/-S.D.) CB-MN frequencies ( per thousand ) in peripheral lymphocytes were 4.00 (+/-2.98) among the dental technicians and 1.40 (+/-1.30) among the controls, a statistically significant difference (P<0.005). The mean (+/-S.D.) MN frequencies ( per thousand ) in nasal cells were 3.50 (+/-1.80) among the dental technicians and 1.19 (+/-0.53) among the controls, which was also a statistically significant difference (P<0.005). There was a significant correlation between duration of exposure and MN frequencies in lymphocytes (r=0.642, P<0.01), but not in nasal cells of technicians. Our data reveal that in vivo exposure to chromium, nickel and cobalt metals is evident and that this occupational exposure may contribute to the observed genotoxic damage in two types of cells, e.g. lymphocytes and exfoliated nasal cells. However, it cannot be determined which compound(s) are responsible for the genotoxic damage observed in this study.

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.

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.

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.

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.

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.

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

Abstract 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.