The effects of ozone application on genotoxic damage and wound healing in bisphosphonate-applied human gingival fibroblast cells.

OBJECTIVES: Medication-related osteonecrosis of the jaws (MRONJ) is an extremely therapy-resistant disease involving the jaws especially following bisphosphonate treatment. Bisphosphonates accumulate in bone in concentrations sufficient to be directly toxic to the oral epithelium. Current therapeutic options are inadequate for the prevention and treatment of MRONJ. The aim of this study was to investigate effects of ozone gas plasma therapy on wound healing in bisphosphonate-applied human fibroblasts. MATERIAL AND METHODS: Human primary gingival fibroblasts were cultured. Cytotoxic concentrations (IC50) of bisphosphonates (pamidronate (PAM), alendronate (ALN), and zoledronate (ZOL)) were determined by MTT test. A 60 µg/µl for 30 s of ozone gas plasma application was performed to all experimental culture flasks after drug treatment at 24-h intervals as 3 s/cm2. Genotoxic damages were evaluated by comet assay and wound healing was determined by in vitro scratch assay. RESULTS: PAM, ALN, and ZOL applications caused genotoxic damage on primary human gingival fibroblast DNA. Ozone gas plasma therapy significantly decreased the genotoxic damage (p < 0.05), and this application provided 25, 29, and 27% less genotoxic damage in order of ALN, PAM, and ZOL groups. Ozone gas plasma therapy significantly increased wound healing rates both in postsurgical 24th and 48th hours for all doses of experimental drug groups (p < 0.05). CONCLUSION: The ozone gas plasma application decreased genotoxic damage effect of bisphosphonate usage while improved the wound closure rate on human gingival fibroblasts. CLINICAL RELEVANCE: Ozone gas plasma therapy may be helpful in prevention of gingival healing delay in MRONJ pathogenesis especially when applied simultaneously with surgical intervention.

Two cases with partial trisomy 9: cytogenetic and clinical findings.

Partial trisomy syndrome of chromosome 9 is one of the frequent autosomal trisomies with a well defined phenotype. Here we report two cases with different karyotypes and we aim to compare the phenotypic findings. The first case was an 8.5 months old boy with developmental delay. He had mental and motor retardation, microcephaly, bilateral undescended testes and multiple minor malformations. The karyotype was 46,XY,-7,der(7)t(7;9)(q36;p12) pat. The second case was a 5 year old boy with mental and motor retardation. He had atypical facial appearance with bilateral undescended testes. The karyotype was reported as 47,XY,+del(9)(q22.1 qter)dn[46]/ 46,XY[4]. Partial trisomy 9 syndrome has a wide range of clinical findings depending on the size of the trisomic chromosome segment. Newly diagnosed cases and their chromosome findings will add to the understanding of the syndrome.

Detecting methylation patterns of p16, MGMT, DAPK and E-cadherin genes in multiple myeloma patients.

Multiple myeloma (MM) is a B-cell neoplasia characterized by the clonal proliferation of plasma cells. Besides known genetic abnormalities, epigenetic changes are also known to effect MM pathogenesis. DNA methylation is an epigenetic mechanism that silences genes by adding methyl groups to cytosine-guanine dinucleotides at the promoter regions. In this study, the methylation status of four genes; p16, O6-methyl guanine DNA methyl transferase (MGMT), death-associated protein kinase (DAPK) and E-cadherin (ECAD); at the time of diagnosis was investigated using methylation-specific polymerase chain reaction (MS-PCR). In the 20 cases studied; methylation of the promoter regions of p16, MGMT, DAPK and ECAD genes was detected in 10%, 40%, 10% and 45% of the cases, respectively. In 65% (13/20) of cases, at least one of the genes studied had promoter methylation; while 35% of cases (7/20) had methylated promoters of more than one gene. There was a significant correlation between promoter hypermethylation of MGMT and the presence of extramedullary involvement; but for the other genes no correlation was found regarding disease properties like age, disease stage, clinical course and the presence of lytic bone lesions. Determining the methylation profiles of genes in MM, could lead to a new understanding of the disease pathogenesis and guide the assessment of treatment options.

Ethical considerations regarding parental decisions for termination following prenatal diagnosis of sex chromosome abnormalities.

Termination rates following prenatal diagnosis of sex chromosome abnormalities have been reported to be in a very wide spectrum (12.7-86.5%) in various studies. The different attitudes in management of prenatal diagnosed sex chromosome abnormalities may depend on several factors as the type of the abnormality, the indication for prenatal testing, the number of previous healthy children and whether the pregnancy was assisted or spontaneous. In the current study, we look at prenatal diagnostic procedures carried out in our department over a period of 5 years (2002-2007). We did not detect sex chromosome abnormalities in the 43 cordocenteses and the 26 chorionic villus samples. Among the 1130 amniocentesis patients, 12 cases (1.06%) were diagnosed as having sex chromosome abnormalities. Five (41.67%) of 12 pregnancies with sex chromosome abnormalities were terminated (one case with 47,XXY, one case with 46,X,del(X), and three cases with 45,X karyotype); whereas seven pregnancies (58.33%) continued. Among the factors influencing parents' decision-making, the attitude of the health-care professional giving the post-diagnosis counseling seems to be the most important, next to the socio-economic and educational status of the parents.

Mucinous cystadenoma in a female patient with 45,X/46,XY karyotype.

The mosaic karyotype of 45,X/46,XY has a wide phenotypic spectrum and there are substantial differences between prenatally and postnatally diagnosed cases. The phenotype varies between normal male to classical Turner syndrome. There is a high risk of gonadal tumor development in the dysgenetic gonads of patients with sex chromosome mosaicism. We report a case of a 24-year-old patient with a pelvic mass and amenorrhea referred to our laboratory for karyotyping. Peripheral blood chromosome analysis showed a mosaic karyotype of 45,X[17]/46,XY[83]. The tumor originated from the left ovary and the right ovary was found to be a streak gonad. The uterus was intact. Pathologic examination of the tumor revealed mucinous cystadenoma. Physical examination of the patient showed signs of Turner syndrome, as short stature (145 cm), short neck and asymmetric shoulders. Her mental state was normal. Y chromosome microdeletion screening involving SRY and ZFY genes was performed and no deletion was found. The patient was informed about the condition during the genetic counseling session.

Fryns anophthalmia-plus syndrome with hypoplastic adrenal glands.

We report a family with two consequent sibs with anophthalmia and cleft lip and palate. A 27 year old woman married to her first cousin was counseled for anophthalmia and cleft lip and palate detected during routine fetal ultrasonographic examination on the 23rd week of the pregnancy. Her obstetric history revealed a healthy girl aged 7 years and a boy with anophthalmia and cleft lip and palate who lived for 20 days in the neonatal intensive care unit. The current pregnancy was terminated after the diagnosis, and post mortem examination of the fetus revealed pre-maxilla agenesis, anophthalmia, cerebral ventricular dilatation, adrenal hypoplasia and single umbilical artery. Chromosome analysis resulted in normal karyotypes of the fetus and both parents. The inheritance pattern was regarded as autosomal recessive and the family was informed about the condition and risks during genetic counseling.

The first 2 years of clinical experience with peripheral blood stem cell transplantation for various hematological malignancies: results from a single Baskent University Center.

Autologous stem cell transplantation is the current standard approach for patients with multiple myeloma and relapsed or refractory lymphoma. Nonmyeloablative allogeneic stem cell transplantation has been applied worldwide. We analyzed the results of transplantation activity from 2004 to 2006. Seven evaluable patients younger than 65 years old with stage II/III multiple myeloma were treated with high-dose melphalan therapy (140 mg/m(2)) plus autologous peripheral blood stem cell transplantation. Complete responses or tumor reductions of more than 75% were obtained in all patients. At a median follow-up of 10 months, all patients remained disease-free. Four patients with acute myeloblastic leukemia underwent nonmyeloablative allogeneic peripheral stem cell transplantation. Their median age was 30 years. One patient was refractory and the others were in hematological remission. The patients received fludarabine-based preparative regimens. All patients received fully matched blood from a related donor 2 days after chemotherapy in conjunction with graft-versus-host disease prophylaxis. One refractory patient with >90% engraftment had late autologous reconstitution at 3 months with evidence of relapse. All other patients in remission remained with >90% donor cell engraftment. These patients are disease-free at 13, 10, and 2 months. Toxicity was minimal. These results showed promise due to the minimal toxicity observed with the conditioning regimens which indicated the feasibility of these procedures.

Prenatal diagnosis of mosaicism identified in amniotic fluid cell cultures.

Chromosomal mosaicism in prenatal diagnosis is an important problem to be solved immediately and the probable phenotypic reflections should be explained to the family. We report two numerical and two structural mosaicisms detected in amniocyte cultures. The first fetus had a 47,XY,+mar[10]/46,XY[10] karyotype. The marker chromosome was shown to be derived from chromosome 15 by FISH method. The newborn had intrauterine growth retardation and cerebral thrombosis and died at the 29th day of age. The second fetus had a 45,X[4]/46,XX[26] karyotype. The parents refused cordocentesis and decided to terminate pregnancy in the 21st week. The third case, presented with bilateral large choroid plexus cysts, had a 46,XX, dup(1)(q22-q32)[9]/46,XX[21] karyotype. The parents' karyotypes were normal and the pregnancy was aborted in the 23rd week of gestation. The second structural abnormality was reported as 46,XX,t(6;11)(q23; p13)[3]/46,XX[20]. The mosaicism was detected in only one flask. The parents decided to continue pregnancy and cordocentesis could not be performed due to the fetal and placental position. The baby was born at term. Peripheral blood lymphocyte culture resulted in a 46,XX normal karyotype. Information and risks were explained to all families during genetic counseling. Mosaicism in prenatal diagnosis needs both detailed examination and follow up, since clinical findings depend on the type of abnormality.

The apoptotic effects of mitomycin C on human endometrial cell cultures and reversal of its effects by beta-carotene and folic acid.

Apoptosis is a complex process involving a variety of mechanisms and it has been shown to be a response of cells to various chemical agents including chemotherapeutic ones. We aimed to induce DNA breaks and apoptosis in cultured endometrial stromal cells by mitomycin C (MMC), a chemotherapeutic agent, and also we aimed to observe the effects of beta-carotene and folic acid on MMC-induced apoptosis. Cultured endometrial stromal cells were exposed to MMC for 48 and 72 hours and in order to reverse MMC effects, we added beta-carotene and folic acid to the cultures. DNA fragmentation was observed in all cells. Apoptotic cell ratios and caspase-3 activity were observed to be dependent on exposure time. Ultrastructural examinations revealed positive effects of beta-carotene and folic acid, however they were not sufficient enough to prevent apoptosis in all cells. Beta-carotene profoundy reduced caspase-3 activity whereas folic acid did not seem to have a similar effect. As apoptosis involves several mechanisms, in a cell in which all these mechanisms are triggered, we think that antioxidants and DNA repair agents alone are not enough to reverse all of them.

Conventional and molecular cytogenetic findings of myelodysplastic syndrome patients.

Myelodysplastic syndrome (MDS) involves myeloid cells of the bone marrow, which is important in progressive bone marrow insufficiency. Of all MDS patients, 40%-50% have at least one chromosomal rearrangement. Loss of specific chromosomal regions like 5q- and 7q- are usually the secondary cytogenetic abnormalities associated with MDS. In order to detect chromosome abnormalities associated with MDS, bone marrow samples from 26 patients diagnosed as MDS were obtained prior to chemotherapy. Both conventional cytogenetic analyses and fluorescence in situ hybridisation (FISH) methods were performed and locus-specific probes for 5q and 7q were used. Results obtained were compared. Twenty-one patients had normal karyotypes and four patients had abnormal karyotypes, while in one patient we could not obtain metaphases from cultures. Three patients with normal karyotypes revealed del (5q), two patients had del (7q) and one patient had monosomy (7). A total of 10 of 26 patients had chromosome changes visualised by either conventional or molecular cytogenetics (approximately 38.5%). Our results show that both methods are important in diagnosis and follow up of MDS patients. When used together, conventional cytogenetics and FISH detect clinically significant chromosome abnormalities in MDS patients.

Cytogenetic abnormalities in serous papillary adenocarcinoma of the ovary.

PURPOSE OF INVESTIGATION: Epithelial ovarian tumors are usually mucinous or serous type, affecting nearly 1% of women during their lifetime. They may be regarded as benign, borderline or invasive according to pathological examination. Karyotypes of the tumor provide critical information about both the genetic predisposition and the stage of the tumor. We aimed to investigate the correlation between karyotype findings and the stage of the serous papillary tumors of the ovary. METHODS: Tissue cultures were set up from 15 serous papillary adenocarcinoma samples of different stages and examined cytogenetically. RESULTS: The most common chromosome abnormalities included both numerical and structural abnormalities of chromosomes 1, 3,6,7,8, 11, 21, 22 and X. CONCLUSION: Karyotypes became more complex, as expected, with the later stages.

Detection of Helicobacter pylori in dental plaque and gastric biopsy samples of Turkish patients by PCR-RFLP.

Helicobacter pylori is regarded as an important pathogen playing a key role in the pathogenesis of peptic ulcer. Different studies about the mode of transmission of the microorganism report conflicting results about dental plaque as the source of H. pylori infection. In the present study we aimed to detect the presence of H. pylori in dental plaque of Turkish patients by polymerase chain reaction (PCR) and if any to do typing by restriction fragment length polymorphism (RFLP) analysis. Fifty dyspeptic patients, to whom upper gastrointestinal endoscopy was performed, were included in the study. Dental plaques were obtained before endoscopic examination. Both dental plaque and gastric biopsy samples were amplified with Ure A and Cag A gene primers. There were no positive dental plaque samples even in the 23 patients whose gastric biopsy specimens were positive. Our findings showed that there is not a correlation between dental presentation of the microorganism and H. pylori gastritis.

C-banding visualized by atomic force microscopy.

C-banding is a method used for studying chromosome rearrangements near centromeres and for investigating polymorphisms. In human chromosomes, the C-bands are located at the centromere of all the chromosomes and the distal long arm of the Y chromosome. In this study, we aimed to detect the structural changes in chromosomes during the stages of C-banding by atomic force microscopy. We observed crater-like structures in the chromosomes after 2xSSC (saline sodium citrate) treatment and measured the relative difference between the heights of chromatid and centromere of the chromosomes. Results showed that the relative difference was 3 nm in chromosomes 1, 9, 16, and Y, whereas in the other chromosomes this value was 11.6 nm. After Giemsa staining, the relative difference increased by a factor of 16 in chromosomes 1, 9, 16, and Y. The other chromosomes showed no such increase, which is in accordance with our suggestion that nonhiston proteins associated with DNA in constitutive heterochromatin can make the constitutive heterochromatin resistant to C-banding.

Effects of estrogen and alendronate on sister chromatid exchange (SCE) frequencies in postmenopausal osteoporosis patients.

OBJECTIVE: To compare two types of osteoporosis treatment for genotoxicity by using sister chromatid exchange (SCE) frequencies. METHOD: Fifty-seven women, aged between 40 and 64 years, composed the population in the study. SCE values of patients under estrogen replacement therapy (ERT) or alendronate therapy were compared to controls who never used any drugs for osteoporosis. RESULT: The difference between the SCE values of women taking ERT and control women was found to be statistically significant (P<0.05). The difference between women taking alendronate and untreated controls was not statistically significant. CONCLUSION: Our results indicate that alendronate does not have genotoxic effects based on SCE frequency, while ERT increases SCE frequencies.

PCR and RFLP analysis for identification and typing of Helicobacter pylori strains isolated from gastric biopsy specimens.

Helicobacter pylori (H. pylori) infection is the most common gastrointestinal tract infection which plays an important role in the ethiopathogenesis of peptic ulcer and gastritis. In recent years, molecular biological methods have been presented for detection of H. pylori in addition to histopathological and microbiological methods. Among these methods, polymerase chain reaction (PCR) and following restriction fragment length polymorphism analyses (RFLP) are highly sensitive methods for diagnosis and follow up of patients. In this present study our aim was to amplify H. pylori urease A and B genes by PCR and perform RFLP analysis. Gastric biopsy specimens from 17 female and 18 male patients were included in the study. Amplified PCR products were subjected to RFLP analysis and typing of the bacteria in pre and posttreatment specimens were performed. H. pylori urease A and B gene amplification was observed in 32 pretreatment samples and in 8 of 21 posttreatment specimens. As a result, PCR is a sensitive method to determine the H. pylori infection. RFLP, which is another effective method in order to demonstrate the reinfection of H. pylori.

The mechanism of G-banding detected by atomic force microscopy.

The morphologic changes occurring in human chromosomes during G-banding by trypsin treatment on the same metaphase were followed with the aid of an atomic force microscope (AFM). It was found that trypsin treatment alone caused a pattern of collapse in the chromosomes that was clearly dependent on the duration of trypsinization. The progressive pattern of collapse first indicated the loss of internal differentiation between chromatids, then bands, and finally all internal structures, except for edges running around the chromosomes' perimeter. When stained with Giemsa, the collapsed chromosomes partly regained their original form, and transverse ridges appeared that correspond to G-positive band regions. However, the treatment of fixed chromosomes with trypsin for 42 s diminished the chromosomal edges, and the z-dimensions could not be measured even with the subsequent application of Giemsa.

Numerical chromosomal abnormalities detected by atomic force microscopy.

The numerical abnormalities of human metaphase chromosomes, fixed according to standard procedures for optical microscopy but not treated for banding, were detected by atomic force microscopy (AFM). High-resolution AFM imaging of chromosomes in trisomy 13, 21, and Klinefelter syndrome can be compared directly with the traditional optical image. The unbanded metaphase chromosomes, including the extra ones in trisomic patients showed a structural pattern very similar to G-banding. Comparison of AFM images with light microscopic data allows the identification of specific chromosomes, and images of chromosomes showing numerical and structural abnormalities can then be analysed.

Gossypol induced apoptosis in the human promyelocytic leukemia cell line HL 60.

In human promyelocytic leukemia cell line HL60, apoptosis was induced by treatment with gossypol that is an inhibitor of protein kinase C. Gossypol acetic acid was added to HL 60 cells at 50, 100, 150 and 200 microM concentrations for six hours. Morphological features of apoptosis as well as internucleosomal DNA fragmentation were evaluated by light microscope, agarose gel electrophoresis and spectrofluorometric quantitation. Our results indicated that with the effective concentrations of gossypol (50 and 100 microM), apoptosis was induced in HL 60 cells.

The in vitro effect of beta-carotene and mitomycin C on SCE frequency in Down's syndrome lymphocyte cultures.

Down's syndrome (DS) has the highest incidence among chromosomal disorders and is a predisposing factor in acute leukemia pathogenesis. DS patients are sensitive to both physical and chemical inducers at the DNA level. Studies on beta-carotene, an antioxidant, suggested the there is a relationship between high beta-carotene diet and reduced tumor incidence in humans indicating that beta-carotene is a chemopreventive agent against cancer. Sister chromatid exchange (SCE) is known as a sensitive parameter among the genotoxicity tests. In this study, we aimed to investigate the in vitro effect of beta-carotene on SCE frequencies in 7 DS patients and 7 healthy controls aged between 0-16 years. A direct leukomogenic agent Mitomycin-C (MMC) was used as a powerful SCE inducer. Addition of MMC to the cultures alone resulted in a significant enhancement of SCE frequencies in both groups when compared to the spontaneous values. In the study, beta-carotene seemed to decrease MMC induced mean SCE/cell values, but did not have an effect on unstimulated cells. As this is a limited study, it is hard to conclude that beta-carotene is a chemopreventive agent in DS patients, although our results seem to support other investigators' reports.