Halil Demirtas

Condensed chromatin surface and NORs surface enhancement in mitogen-stimulated lymphocytes of Down syndrome patients

Mitogen-stimulated lymphocytes of 20 Down syndrome (DS) patients with regular trisomy 21 contain more condensed chromatin surface (11.28 +/- 2.64 % of the total nuclear surface: mean +/- SD) and more nucleolus organiser regions surface (13.21 +/- 3.45 %) than that of 12 healthy controls: (8.84 +/- 2.23 and 9.12 +/- 2.33 %, reciprocally). The source of this peculiarity has been investigated. A computer program was designed for the planimetric measurement of the condensed chromatin surface (CCs)/ total nuclear surface(TNs) and the nucleolus organiser regions surface (NORss) /TNs proportions in interphase nuclei. CCs/TNs and NORss/TNs of 100 maximally activated nuclei (MANs) were measured for each patient and control case. The difference was found highly significant (P<0.01). Nuclei with a diameter of >/= 17 micrometer measured on the slide (in flattened state) were considered as maximally activated nuclei (MANs). NORss/TNs enhancement and fluorescent in situ hybridisation (FISH) studies in MANs of DS patients indicate that this phenomenon is due to the over-expression (or lack of downregulative mechanism) of NORs (rDNA) to some extent, including the NOR of the supernumerary chromosome 21. No statistical difference was observed between 12 healthy controls and 5 Robertsonian translocation type of DS Patients (where the two involved NORs are missing) when the two parameters were considered.

Micronucleus frequency in the oral mucosa and lymphocytes of patients with Behçet's disease

Behçets disease (BD) is a chronic, multisystemic, inflammatory disorder characterized mainly by recurrent oral and genital aphthous ulcerations and uveitis. Our study aimed to determine the genetic damage in patients with BD. The micronucleus (MN) frequency was counted in peripheral lymphocytes and exfoliated cells of the patients with BD. MN analysis was performed in peripheral lymphocytes of 30 patients with BD and in 20 healthy controls by the cytokinesis‐block method, and on uncultured cells of the oral cavity in 10 patients and 9 healthy controls. We found significantly higher MN rates in lymphocytes of the patients than the control subjects (P = 0.000). There were no significant differences between the patients with or without treatment (P = 0.860). The MN frequency in exfoliated cells of the patients was higher than in those of healthy controls (P = 0.013), and there was no significant difference between the exfoliated cells of the treated and untreated patients (P = 0.201). Our results indicate that genetic damage may play a secondary but important part in the aetiology of BD and that treatment with colchicine does not induce MN.

Evaluation of the Nucleolar Organizer Regions in Alzheimer’s Disease

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder in middle and late age. Ribosomal RNA (rRNA) genes are located in the nucleolus (nucleolar organizer regions = NORs). There are increased deposits of β-amyloid protein in the brains of the patients with AD and aged individuals with Down’s syndrome (DS). The β-amyloid gene is located in the acrocentric chromosome 21 that is responsible for rRNA synthesis. Therefore, it is possible that there is a relationship between ribosomal genes and AD. Objective: To investigate the activities of ribosomal genes of AD patients by comparing the activities of NORs in AD patients and healthy controls with the silver-staining method. Methods: NOR surface/the total nucleus surface proportions in interphase nuclei, and silver stainability and satellite association (SA) of acrocentric chromosomes in the metaphases of cultivated lymphocytes of 20 AD patients and 20 healthy controls (10 elderly and 10 young) were evaluated. Results: A decrease in NOR surface/total nucleus surface proportions has been observed in the interphase nucleus of AD patients when compared with elderly controls (p = 0.035). When compared with the sizes of Ag+ segments of acrocentric chromosomes of AD patients and control groups, the Ag-staining size 1 of the chromosome 22 of AD patients was found to be more increased than that of the young controls (p = 0.018). There was no statistically significant difference between AD patients and control groups regarding the number of Ag+ acrocentric chromosomes, Ag+ chromosome 21 and SA frequency (p > 0.05). It has been found that there is only a slight increase in the total number of chromosomes in SA in AD patients when compared with elderly controls (p = 0.05). Conclusion: The decrease in NOR surface/total nucleus surface proportions of AD patients may indicate a reduction in the activity of the ribosomal genes of these patients.

NOR expression increases on metaphase chromosomes of down syndrome lymphocytes in concordance with mitogen concentration in culture medium

Regulation of nucleolus organizer region (NOR) expression in trisomy 21 (Down syndrome [DS]) cells is not fully explained. This work compared NOR expression on metaphase chromosomes in gradiently stimulated lymphocytes from DS patients with those from healthy controls.

Micronucleus frequencies in workers exposed to lead, zinc, and cadmium

Micronuclei (MN) in blood lymphocytes were determined in 31 male workers occupationally exposed to lead (Pb), zinc (Zn), and cadmium (Cd) and 20 control workers matched for age and smoking habits. Exposed workers have higher MN mean values than control workers (p<0.01). In exposed workers, blood Pb concentrations were also significantly higher than in control workers (p<0.001), but the mean concentrations of Zn and Cd in the blood were not statistically significant compared to the controls (p>0.05). These results suggest that lead may be genotoxic and the human lymphocyte micronucleus test can be used to assess genotoxic effects that result from occupational exposures.

Investigation of micronucleus frequencies in lymphocytes of inhabitants environmentally exposed to chrysotile asbestos

Abstract Exposure to asbestos minerals has been associated with a wide variety of adverse health effects including lung cancer, pleural mesothelioma, and cancer of other organs. Many of the regions of Turkey have asbestos deposits. People in Doğanlı village – one of these regions – have been environmentally exposed to chrysotile asbestos since they were born. In this study the effects of asbestos on micronucleus (MN) frequencies of inhabitants exposed to chrysotile asbestos have been examined. Thirty subjects who had been environmentally exposed to chrysotile asbestos and living in Doğanlı village, and 25 controls were studied to assess the MN frequency. The control group was selected from healthy individuals with no exposure to asbestos and living in similar geographic conditions to Doğanlı village. Peripheral blood samples were collected from each subject and cultured for MN assay. Cytochalasin-B was added to lymphocyte cultures for evaluation of MN in binucleated (BN) cells. The differences between those exposed to chrysotile asbestos and controls were not statistically significant in terms of BN cells with MN (p > 0.05). There was not a significant relationship between MN frequencies and age, sex, smoking, both in chrysotile asbestos-exposed subjects and in controls (p > 0.05). Although the detection of calcified pleural plaques found in the inhabitants has indicated environmental exposure to chrysotile asbestos, our results show that chrysotile asbestos was not an inducer of MN in subjects exposed to chrysotile asbestos.

AgNOR increase in buccal epithelial cells of trisomy 21 infants

The aim of this study is to compare the Argilophilic Nucleolus Organizer Regions (AgNORs) level between Down syndrome (DS) patients and controls in a tissue sharing the same embryonic origin with the central nervous system and compare the results with those obtained recently by us from DSs lymphocytes. For this, buccal desquamating epithelial cells well known as the ectodermic origin were used. Since the AgNOR staining intensity is an indicator of the ribosomes biosynthesis rate, comparison of the image analysis values of the AgNOR area/total nuclear area (NORa/TNa) in buccal desquamating epithelial cells of DS patients and controls provided a plausible conclusion about the regulation/deregulation of the rRNA genes (rDNA) in these cells of DS babies/infants. The (NORa/TNa) proportion was calculated using an in-house computer program. Fifty buccal desquamating cells were analysed for each individual to determine the average NORa/TNa value per individual. In contrast to healthy controls, NORa/TNa proportion value of buccal epithelial cells from DS patients found significantly higher than that of the controls: (4.08+/-1.16)% and (2.13+/-0.55)%, respectively. This 92% increase is far higher than the expected value due to the extra rRNA genes on the extra-chromosome 21. Finally DS babies/infants exhibit very higher AgNOR expression increase in their buccal epithelial cells compared to controls. This is the first study that is available on the comparison of AgNOR expression levels in buccal epithelial cells between DS infants and their controls.

Age-Dependent Decreases in Mitogen-Stimulation Level and RNA Content in Peripheral Blood Mononuclear Cells of Down Syndrome Patients

The aim of the present study was to determine whether or not the phytohemagglutinin (PHA)‐activated proliferation and average RNA content in peripheral blood mononuclear cells (PBMC) of Down syndrome (DS) patients change with age.

Do Non-Steroidal Anti-Inflammatory Drugs Induce Sister Chromatid Exchanges in T Lymphocytes?

The genetic toxicity of non-steroidal anti-inflammatory drugs was investigated using the sister chromatid exchange technique in cultured human lymphocytes. A total of 48 patients were treated with non-steroidal anti-inflammatory drugs (ibuprofen, ketoprofen, naproxen, indomethacin, diclofenac or acetylsalicylic acid) for 2 weeks. The average numbers of sister chromatid exchanges in cultured lymphocytes from the patients, before and after treatment with these drugs, did not differ significantly (P > 0.05). These results indicate that treatment with non-steroidal anti-inflammatory drugs for 2 weeks does not induce sister chromatid exchanges in T lymphocytes.

AgNOR status in Down's syndrome infants and a plausible phenotype formation hypothesis

Downs syndrome (DS) or trisomy 21 is the most frequent genetic birth defect associated with mental retardation. Although DS has been known for more than a 100 years and its chromosomal basis recognized for half a century (1959), the underlying patho-mechanisms for the phenotype formation remain elusive and cannot be fully explained by simple gene dosage effect. The general consensus is that the extra chromosome 21 genes perturb the global metabolism of the body cells. Our experiments show that the most prominent metabolic perturbation occurs during ribosome biogenesis in the cells of DS babies/infants. In humans, ribosomal RNA (rRNA) gene families or nucleolar organizer regions (NORs) are localized at the secondary constriction (on the satellite stalks) of five pairs of acrocentric chromosomes (13, 14, 15, 21 and 22) and their activities are evaluated specifically either in metaphase or interphase through a procedure known as AgNOR or silver staining. Our successive AgNOR studies, supported by RNA and nuclear protein measurement, show that cells from DS infants produce more ribosomes than expected, accounting for the extra set of active rRNA gene family (1/6-1/11) situated on the extra chromosome 21. Thus, the presence of an extra chromosome 21 stimulates a global increase in ribosome biogenesis in cooperation with other NOR-bearing chromosomes, causing unnecessary rRNA and ribosomal proteins synthesis compared to controls. Following the description of NORs, AgNOR, AgNOR-proteins, AgNOR measurement and our experimental results, we propose that the extra RNA and protein synthesis can cause a fundamental handicap to DS infants, contributing to the formation of DS phenotypes, due to the wasted energy in producing unnecessary macromolecules, including energy (GTP)-dependent transport of the excessive ribosomes from the nucleus to the cytoplasm.