Nazmiye Bitgen

Evaluation of the genotoxicity and cytotoxicity in the general population in Turkey by use of the cytokinesis-block micronucleus cytome assay

The cytokinesis-block micronucleus cytome (CBMN-Cyt) assay was originally developed as an ideal system for measuring DNA damage, cytostasis and cytotoxicity. The objective of the present study is to simultaneously evaluate the background levels of micronuclei (MN), nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division index (NDI) in the peripheral blood lymphocytes of non-occupationally exposed, healthy subjects living in the city of Kayseri in Turkey. We used the CBMN-Cyt assay, taking into account factors - age, gender, and smoking habits - that might affect MN frequency and also other CBMN-Cyt assay parameters. Ninety-six healthy subjects (48 female and 48 male) were selected with ages varying between 21 and 60 years. The parameters, except for the number of binucleated (BN) cells with NPBs, showed no statistically significant difference between smokers and non-smokers. There were significant differences between female and male groups in MN frequency (higher in females) and in the number of NPBs (lower in females), while the other parameters were not significantly different between genders. The correlations between years of age and MN frequency, number of NPBs and the frequency of necrotic cells were statistically significant, while the correlations between the years of age and the other parameters were not. The results of the correlation analysis between years of smoking and MN frequency were positive, although no statistically significant correlation was found between the years of smoking and the other parameters. Among the smokers, no correlation was found either between the pack-years of smoking and the parameters assessed in this group. The results of the present study provide evidence of increasing MN frequency, number of NPBs and frequency of necrotic cells with increasing age in the peripheral blood lymphocytes of healthy individuals and influencing MN frequency and number of NPBs by gender.

Evaluation of genotoxicity, cytotoxicity and cytostasis in human lymphocytes exposed to patulin by using the cytokinesis-block micronucleus cytome (CBMN cyt) assay

Patulin (PAT) is a fungal secondary metabolite commonly present in apples and apple products. In the present study, PAT was evaluated for its genotoxic, cytotoxic and cytostatic effects to human peripheral blood lymphocytes by using the cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Lymphocyte cultures were treated with PAT at the following concentrations, 0.1, 0.3, 0.5, 1.0, 2.5, 5.0, and 7.5 μM, as well as 0.5 μM mitomycin c (MMC) as a positive control and dimethyl sulfoxide (DMSO) as a vehicle control. PAT was found to induce nucleoplasmic bridges (NPBs) at 5.0 and 7.5 μM concentrations (P < 0.05), apoptotic cells at 0.1, 1.0, 5.0 μM (P < 0.05), 7.5 μM concentrations (P < 0.01) and necrotic cells at 0.3 and 2.5 μM (P < 0.05), 5.0 and 7.5 μM (P < 0.01) concentrations in human lymphocytes. The 2.5, 5.0, and 7.5 μM PAT concentrations also led to a clear decrease in the nuclear division index (NDI) (P < 0.05). PAT caused a significant dose-dependent increase in the number cells of NPBs, in the frequency of apoptotic and necrotic cells, and a significant dose-dependent decrease in the NDI values in lymphocytes. These results indicate that PAT at high concentrations is genotoxic, cytotoxic and cytostatic in cultured human lymphocytes.

Micronucleus evaluation in mitogen-stimulated lymphocytes of patients with acromegaly

Acromegaly is a syndrome characterized by a sustained elevation of circulating growth hormone and insulin-like growth factor-1 (IGF-1). Insulin-like growth factor-1 is a potent mitogen and has a role in the transformation of normal cells to malignant cells. This study aims to evaluate the spontaneous micronucleus (MN) frequency by using the cytokinesis-block MN assay to determine genetic damage in the lymphocytes of patients with acromegaly. The study was carried out in 20 patients who had active acromegaly and in 20 age- and sex-matched healthy controls. The MN values were measured in binucleated cells obtained from mitogen-stimulated lymphocytes of patients and control subjects. The distribution of binucleated cells with 1, 2, 3, or more MNs was also measured. We found significantly higher MN frequency values in the lymphocytes of acromegalic patients than in those of the control subjects (2.23 ± 0.68 vs 1.03 ± 0.54, P = .001). The MN frequency increased with increasing IGF-1 levels of acromegalic patients (P = .036, R = 0.47). We observed that the number of binucleated cells with 2 MNs was higher for the majority of patients with acromegaly than for control subjects. Furthermore, the receiver operating characteristic curve (area under the curve = 0.914, P < .0001) was calculated to assess the discriminative power of the MN frequency. Our results indicate that increased MN frequency in the lymphocytes of patients with acromegaly may reflect genomic instability and this increased MN frequency may be associated with elevated levels of circulating growth hormone and IGF-1.

Increased Chromosomal and Oxidative DNA Damage in Patients with Multinodular Goiter and Their Association with Cancer

Thyroid nodules are a common clinical problem worldwide. Although thyroid cancer accounts for a small percentage of thyroid nodules, the majority are benign. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) levels are a marker of oxidative stress and play a key role in the initiation and development of a range of diseases and cancer types. This study evaluates cytokinesis-block micronucleus cytome (CBMN-cyt) assay parameters and plasma 8-OHdG levels and their association with thyroid nodule size and thyroid hormones in patients with multinodular goiter. The study included 32 patients with multinodular goiter and 18 age- and sex-matched healthy controls. CBMN-cyt assay parameters in peripheral blood lymphocytes of patients with multinodular goiter and controls were evaluated, and plasma 8-OHdG levels were measured. The micronucleus (MN) frequency (chromosomal DNA damage), apoptotic and necrotic cells (cytotoxicity), and plasma 8-OHdG levels (oxidative DNA damage) were significantly higher among patients with multinodular goiter. Our study is the first report of increased chromosomal and oxidative DNA damage in patients with multinodular goiter, which may predict an increased risk of thyroid cancer in these patients. MN frequency and plasma 8-OHdG levels may be markers of the carcinogenic potential of multinodular goiters and could be used for early detection of different cancer types, including thyroid cancer.

Increased micronucleus, nucleoplasmic bridge, nuclear bud frequency and oxidative DNA damage associated with prolactin levels and pituitary adenoma diameters in patients with prolactinoma

Prolactinoma is the most common pituitary tumor. Most pituitary tumors are benign, but they often are clinically significant. We investigated cytokinesis-block micronucleus cytome (CBMN cyt) assay parameters and oxidative DNA damage in patients with prolactinoma to assess the relations among age, prolactin level, pituitary adenoma diameter and 8-hydroxy-2’-deoxyguanosine (8-OHdG) level in patients with prolactinoma. We investigated 27 patients diagnosed with prolactinoma and 20 age- and sex-matched healthy controls. We measured CBMN cyt parameters and plasma 8-OHdG levels in peripheral blood lymphocytes of patients with prolactinoma and controls. The frequencies of micronucleus (MN), nucleoplasmic bridge, nuclear bud, apoptotic and necrotic cells, and plasma 8-OHdG levels in patients with prolactinoma were significantly greater than controls. MN frequency was correlated positively with age, prolactin levels and pituitary adenoma diameters in patients with prolactinoma. The increased chromosomal and oxidative DNA damage, and the positive correlation between MN frequency, prolactin levels and pituitary adenoma diameters may be associated with increased risk of cancer in patients with prolactinoma, because increased MN frequency is a predictor of cancer risk.

Investigation of genome instability in patients with non-alcoholic steatohepatitis

AIM To evaluate the occurrence of micronucleus (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in the mitogen-stimulated lymphocytes of patients with non-alcoholic steatohepatitis (NASH). METHODS The study was performed in 25 (9 females, 16 males) patients newly diagnosed with NASH, and 25 healthy subjects of similar ages and genders were used as a control group. None of the controls was known to be receiving any drugs for medical or other reasons or using alcohol. Hepatosteatosis was further excluded by abdominal ultrasound imaging in the control group. The numbers of MN, NPBs and NBUDs scored in binucleated (BN) cells were obtained from the mitogen-stimulated lymphocytes of patients and control subjects. Statistical comparisons of the numbers of BN cells with MN, NPBs and NBUDs and ages between the patients with NASH and control subjects were performed. RESULTS The mean ages of the patients and the control group were 41.92 ± 13.33 and 41.80 ± 13.09 years (P > 0.05), respectively. The values of the mean body mass index (BMI), HOMA-IR, hemoglobin, creatinin, aspartate aminotransferase, alanine aminotransferase, triglyceride, high density lipoprotein, and low density lipoprotein were 31.19 ± 4.62 kg/m(2) vs 25.07 ± 4.14 kg/m(2), 6.71 ± 4.68 vs 1.40 ± 0.53, 14.73 ± 1.49 g/dL vs 14.64 ± 1.30 g/dL, 0.74 ± 0.15 mg/dL vs 0.80 ± 0.13 mg/dL, 56.08 ± 29.11 U/L vs 16.88 ± 3.33 U/L, 92.2 ± 41.43 U/L vs 15.88 ± 5.88 U/L, 219.21 ± 141.68 mg/dL vs 102.56 ± 57.98 mg/dL, 16.37 ± 9.65 mg/dL vs 48.72 ± 15.31 mg/dL, and 136.75 ± 30.14 mg/dL vs 114.63 ± 34.13 mg/dL in the patients and control groups, respectively. The total numbers and frequencies of BN cells with MN, NPBs and NBUDs, which were scored using the CBMN cytome assay on PHA-stimulated lymphocytes, were evaluated in the patients with NASH and control group. We found significantly higher numbers of MN, NPBs and NBUDs in the BN cells of patients with NASH than in those of the control subjects (21.60 ± 9.32 vs 6.88 ± 3.91; 29.28 ± 13.31 vs 7.84 ± 3.96; 15.60 ± 5.55 vs 4.20 ± 1.63, respectively, P < 0.0001). CONCLUSION The increased numbers of MN, NPBs and NBUDs observed in the lymphocytes obtained from patients with NASH may reflect genomic instability.