Growth Hormone & IGF Research 24 (2014) 29–34
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Increased genome instability and oxidative DNA damage and their association with IGF-1 levels in patients with active acromegaly Fahri Bayram a, Nazmiye Bitgen b, Hamiyet Donmez-Altuntas b,⁎, Ilkay Cakir a, Zuhal Hamurcu b, Fatma Sahin b, Yasin Simsek a, Gulden Baskol c a b c
Department of Endocrinology and Metabolism, Faculty of Medicine, Erciyes University, Kayseri, Turkey Department of Medical Biology, Faculty of Medicine, Erciyes University, Kayseri, Turkey Department of Biochemistry, Faculty of Medicine, Erciyes University, Kayseri, Turkey
a r t i c l e
i n f o
Article history: Received 11 February 2013 Received in revised form 6 November 2013 Accepted 5 December 2013 Available online 14 December 2013 Keywords: Acromegaly CBMN Cyt assay IGF-1 8-OHdG levels MN frequency
a b s t r a c t Objective: The objectives of this study were to assess cytokinesis-block micronucleus cytome (CBMN Cyt) assay parameters and also oxidative DNA damage in patients with active acromegaly and controls and to assess the relationship between age, serum insulin-like growth factor 1 (IGF-1) levels, pituitary adenoma diameters, 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels and CBMN Cyt assay parameters in patients with active acromegaly. Design: The study population included 30 patients with active acromegaly and 30 age- and sex-matched healthy controls. CBMN Cyt assay parameters in peripheral blood lymphocytes of patients with active acromegaly and controls were evaluated and plasma 8-OHdG levels were measured. Results: Frequencies of micronucleus (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in lymphocytes of patients with acromegaly were found to be significantly higher than those in controls (p b 0.001, p b 0.001, p b 0.001, respectively). The frequencies of apoptotic and necrotic cells in lymphocytes of patients with acromegaly were found to be significantly higher than those in controls (p b 0.001 and p b 0.001 respectively). No statistically significant differences in the number of cells in metaphase, the number of bi-nucleated cells (M2), the number of tri-nucleated cells (M3), the number of tetra-nucleated cells (M4) and nuclear division index (NDI) values were observed between patients and controls (p N 0.05). Plasma 8-OHdG (ng/ml) levels in patients with acromegaly were found to be significantly higher than those in controls (p b 0.005). MN frequency in the lymphocytes of patients with acromegaly increased with elevated serum IGF-1 levels (p b 0.05), whereas the number of NPBs and the frequency of apoptotic cells decreased with elevated serum IGF-1 levels (p b 0.01 and p b 0.05 respectively). Conclusions: Both the increase in chromosomal/oxidative DNA damage and the positive association between MN frequency and serum IGF-1 levels may predict an increased risk of malignancy in acromegalic patients. Long-term follow-up of patients with acromegaly will be necessary to establish the degree of cancer risk in this population. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Acromegaly is a slow-developing disease, with an estimated incidence of 4–5 per million per year and a prevalence of 60 cases per million [1–3]. It is usually caused by pituitary somatotroph adenomas and is associated with an increase in morbidity and mortality often attributed to cardiovascular, cerebrovascular, respiratory and metabolic diseases [4–6]. It is reported that acromegaly patients have an increased risk of developing both benign and malignant tumours, particularly colorectal cancer, and possibly other cancers such as breast, prostate, thyroid and haematological malignancies [7,8]. The aetiology of these cancers remains
⁎ Corresponding author at: Department of Medical Biology, Medical Faculty, Erciyes University, 38039 Kayseri, Turkey. E-mail address: [email protected] (H. Donmez-Altuntas). 1096-6374/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ghir.2013.12.002
uncertain, but they may be related to both growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels [9–14]. However, some epidemiological studies report conflicting findings on cancer risk in acromegaly [15,16]. It is established that IGF-1 is a potent mitogen and anti-apoptotic for a large variety of cells, exerting its mitogenic action by increasing DNA synthesis and stimulating cell cycle progression, whereas the main IGF-binding protein (IGFBP)-3 is a potent inhibitor of IGF-I action, which works in part by binding IGF1 and also by inducing apoptosis through an IGF-1-independent mechanism in target cells [14–18]. It is well known that GH promotes the stimulation of both IGF-1 and IGFBP-3. Because IGF-1 stimulates cell proliferation/growth advantage and IGFBP-3 promotes apoptosis and because GH stimulates both IGF-1 and IGFBP-3 in acromegaly, these factors should balance cell growth regulation [16,18]. However, high levels of IGFBP-3 were associated with an increased risk of premenopausal breast cancer, but not with any other cancer [17].
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F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
The micronucleus (MN) assay and cytokinesis-block micronucleus (CBMN) assay have both been used as methods for evaluating chromosome damage in cultured human lymphocytes because they provide a convenient and reliable index of both chromosome breakage and chromosome loss [19,20]. An important and further development in the CBMN assay is the adoption of the CBMN cytome (CBMN Cyt) assay approach that scores MN and includes other nuclear abnormalities such as nuclear buds (NBUDs) and nucleoplasmic bridges (NPBs), as well as involve frequencies of necrotic and apoptotic cells and the proportion of dividing cells [21,22]. This method allows the measurement of MN as a biomarker of chromosome breakage and/or whole chromosome loss, NPBs as a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair, NBUDs as a biomarker of gene amplification, necrosis and apoptosis as a viability status of cell, and metaphase, anaphase, mono-nucleated, bi-nucleated (BN), and multi-nucleated cells as a mitotic status of cell [21,22]. Oxidative stress is known to potentially cause DNA damage involving point mutations because of base oxidation, single and double strand breaks and genomic instability. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the damaged DNA products caused by reactive oxygen species. 8-OHdG is used as a reliable marker of oxidative DNA damage [23–25]. Our previous study showed that agromegalic patients had increased MN frequency [26]. However, the frequencies of NBPs and NBUDs, which determine DNA damage effects, the proportions of necrotic and apoptotic cells, which determine cytotoxicity and the proportions of mono-, bi- and multi-nucleated cells, and nuclear division index (NDI), which determines cytostasis in patients with acromegaly have not been investigated. The main objectives of this study are to assess the MN frequencies and new criteria such as the frequencies of NPBs and NBUDs, the frequencies of necrotic and apoptotic cells and NDI using CBMN Cyt assay parameters and oxidative DNA damage using the 8-OHdG assay in patients with acromegaly as well as to assess the relationship between age, serum IGF-1 levels, pituitary adenoma diameters, 8-OHdG levels and CBMN Cyt assay parameters, to improve the diagnosis and treatment of acromegaly.
Table 1 Patients profile at study entry. I.D.
Gender
Age (years)
IGF-1 levels (mU/l)
z-SDS IGF-1
Nadir GH after OGTT (μIU/ml)
z-SDS GH
Pituitary adenoma diameters (mm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
M M M M F F F M F F M M F F M M F M F F F F F M F M F M F F
20 24 29 29 30 31 33 35 36 38 38 39 40 41 42 43 46 46 48 49 51 53 53 56 56 57 64 66 74 80
626 987 681 1425 926 1246 534 1225 909 765 859 1600 1125 554 959 343 376 1033 613 907 688 442 1154 816 936 359 389 585 540 736
−0.573 0.543 −0.403 1.897 0.355 1.344 −0.857 1.279 0.302 −0.143 0.148 2.438 0.970 −0.795 0.457 −1.447 −1.345 0.685 −0.613 0.296 −0.381 −1.141 1.059 0.015 0.390 −1.398 −1.305 −0.699 −0.838 −0.233
8.83 14.10 2.04 1.76 9.08 10.53 18.63 4.09 12.73 8.40 – – 24.00 1.08 8.53 1.06 1.05 10.77 4.35 1.93 3.04 1.29 – 10.48 2.33 1.03 1.94 11.77 1.07 6.77
0.34 1.22 −0.78 −0.83 0.38 0.62 1.97 −0.44 0.99 0.27 – – 2.86 −0.94 0.29 −0.95 −0.95 0.66 −0.40 −0.80 −0.62 −0.91 – 0.62 −0.74 −0.95 −0.80 0.83 −0.95 0.00
25 16 11 16 35 8 10 20 30 20 13 17 24 10 7 30 8 30 3 30 5 30 8 6 40 23 15 11 15 6.5
IGF-1: Insulin-like growth factor 1; GH: Growth hormone; OGTT: Oral glucose tolerance test. z-SDS IGF-1 = (IGF-1 of the single patient − mean IGF-1 of total patients) / SD (SD: Standart deviation of total IGF-1). z-SDS GH = (GH of the single patient − mean GH of total patients) / SD (SD: Standart deviation of total GH).
2. Materials and methods 2.1. Patients and controls The study was performed on 30 patients diagnosed with active acromegaly and admitted to the Department of Endocrinology and Metabolism at Erciyes University Medical Faculty from December 2010 to December 2011. Thirty age- and sex-matched healthy controls were also included in the study. The diagnosis of acromegaly was made based on clinical and biochemical findings. Clinical diagnosis was made according to typical disfigurement of the patient related to progressive acral enlargement and modification of facial appearance. The diagnosis of acromegaly was confirmed by measuring GH response to the oral glucose tolerance test (OGTT) and IGF-1 levels. A magnetic resonance imaging (MRI) scan of the pituitary gland is used to locate and detect the size of adenomas (microadenomas being less than 10 mm in diameter and macroadenomas being greater than 10 mm in diameter). Serum IGF-1 levels were also higher than the age- and sex-related reference ranges in acromegalic patients [27,28]. The characteristics of the patients are shown in Table 1. All 30 patients included in the study had been newly diagnosed and had not received any medical or surgical therapy for acromegaly. They all had active disease states, with increased serum IGF-1 levels according to the normal gender and age reference levels and a failure to suppress GH levels after 75-g glucose loading. Twenty-two of the patients had macroadenoma (73.33%) and eight had microadenoma (26.67%). The OGTT was not performed on three of the 30 patients (patient IDs: 11, 12 and 23) because they had diabetes. These three patients were
diagnosed with acromegaly based on clinical findings of high IGF-1 levels, basal GH levels, and an MRI scan of the pituitary. The characteristics of patients in the study were as follows: aged between 20 and 80 years, 17 female and 13 male, seven smokers and 23 non-smokers, body mass index (BMI) between 22.00 and 32.00. No acromegalic patients had dyslipidaemia. Three acromegalic patients (patient IDs: 22, 25 and 30) had hypertension, but these patient symptoms were controlled with diet and non-pharmacologic therapy. The control group was selected from healthy subjects matched for age, gender, BMI, socio-economic status and smoking habits. None of them were known to be receiving any drugs for medical or other reasons or had specific diseases such as hypertension, diabetes mellitus, heart disease and cancer. A standardised questionnaire designed to obtain relevant details of their current health status, history and lifestyle as well as to collect information on past medical history, drug and smoking habits was completed for each patient and control subject. The study excluded patients and control subjects who reported alcohol, tea and/or coffee consumption (more than 3 cups/day), different dietary habits and had a history of occupational and environmental exposure to known genotoxic chemicals. Blood samples were obtained from patients for the measurement of growth hormone (GH), insulin-like growth factor-1 (IGF-1), follicle stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (tT), free testosterone (fT), free thyroxine (fT4), free tri-iodothyronine (fT3), prolactin (PRL), estradiol (e2), adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH) and cortisol. All serum samples were collected in the early morning after an 8- to 10-h fasting period.
F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
Patients who had multiple pituitary-hormone deficiencies and excessive hormone secretion except for GH/IGF-1 were excluded from the study. The local ethics committee approved the study protocol (No. 2010/ 97), and all patients gave written informed consent. The study was conducted in accordance with the Declaration of Helsinki and local laws, depending on which afforded greater protection to the patients.
2.2. Determinations of IGF-1 and GH levels Serum concentrations of IGF-1 and GH were measured by sensitive and specific immunoradiometric assay (Active Non-Extraction IGF-1 IRMA DSL-2800 and Active Growth Hormone IRMA DSL-1900; Diagnostics Systems Laboratories, Webster, TX). For IGF-1, the intra-assay and inter-assay coefficients of variation were 3.9%–7.0% and 4.2%–7.4%, respectively; the International Reference Preparation (IRP) used in the calibration of the IGF-1 assay was WHO 87/518. For GH, the intraassay and inter-assay coefficients of variations were 3.3%–4.3% and 6.3%–6.6%, respectively; the IRP used in the calibration of the GH assay was WHO 80/505/88/624. The use of these kits is one of the procedures recommended for IGF-1 measurements as they have a high sensitivity [29].
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2.5. Determination of 8-OHdG levels Two millilitres of heparinised blood samples for analysis of 8-OHdG was immediately centrifuged at 3000 rpm for 15 min at room temperature. The plasma was then stored in microtubes at −80 °C until it was analysed. The 8-OHdG levels in plasma were measured using an ELISA kit (Catalogue: NWK-8-OHdG02, Northwest Life Science Specialties, LLC, WA, USA) and an intra-assay coefficient of variation of 8-OHdG assay was calculated to be 5.9%. Plasma 8-OHdG levels were expressed in ng/ml. Calibration, curve fitting and data analysis were done according to the instructions of the manufacturer. 2.6. Statistical analysis The data were analysed using the SPSS for Windows statistical package, version 15.0. Differences were considered statistically significant when p values were less than 0.05. Statistical comparisons of CBMN Cyt assay parameters, 8-OHdG levels and serum IGF-1 levels between patients with acromegaly and control subjects were done using a non-parametric Mann–Whitney U test for two independent samples. Spearman's rho correlation analysis was used to assess the relationship between age, serum IGF-1 levels, pituitary adenoma diameters, 8-OHdG levels and CBMN Cyt assay parameters.
2.3. Whole-blood cultures of human lymphocytes 3. Results Antecubital heparinised 3 ml blood samples were taken after informed consent had been obtained from all patients and control subjects. Approximately 0.4 ml of heparinised whole blood samples was cultured for 72 h at 37 °C in 5 ml of peripheral blood karyotyping medium that was supplemented with 1.5% phytohaemagglutinin-M (PHA-M) to stimulate T-lymphocytes (all sources from Biological Industries, Kibbutz Beit Haemek, Israel). To determine intra-individual differences, duplicate cultures were made for each patient and control at the specified time.
2.4. CBMN Cyt assay Forty-four hours after cultures were initiated, cells were blocked from entering cytokinesis by the addition of cytochalasin-B to each culture tube (Sigma-Aldrich, St Louis, MO; final concentration, 3 μg/ml) [22]. The cultures were stopped at 72 h after initiation, treated with hypotonic solution (0.1 mol/l KCl) for 4 min and fixed using two changes of methanol–acetic acid (3:1) [22,26,30]. The fixed cells were spread onto glass slides and stained with 5% Giemsa (Merck) in Sorensen buffer for 10 min. To determine the intra-individual differences, the different slides of the two parallel cultures for each patient and control subject were prepared and evaluated. All slides were scored blindly by a Nikon Alphaphot-2 light optical microscope. A score was obtained for slides from each duplicate culture from two different scorers by identical microscopes. The 1000 BN cells with two macronuclei surrounded by cytoplasm were scored from each patient and control subject, and every subject was analysed for the total number of MN, NBPs and NBUDs per 1000 BN cells to determine DNA damage effects. The frequencies of BN cells containing one or more MN, NBPs and NBUDs were determined. The number of necrotic and apoptotic cells were scored in 1000 mono-nucleated cells to determine cytotoxicity [22]. The number of mono-, bi-, tri- and tetra-nucleated cells per 1000 viable mono-nucleated cells was scored in peripheral blood lymphocytes of all individuals to determine cytostatic effects. NDI has been calculated using the formula: NDI = (M1 + 2 M2 + 3 M3 + 4 M4) / N, where M1–M4 represents the number of cells with 1–4 nuclei and N is the total number of viable cells scored (excluding necrotic and apoptotic cells) [22,31].
Table 2 shows the statistical results and mean ± SD values of age, IGF-1 levels, 8-OHdG levels and CBMN Cyt assay parameters in 30 patients with acromegaly and 30 healthy controls. Pituitary adenoma diameters of patients with acromegaly ranged from 3 to 40 mm (17.74 ± 10.14). The nadir GH levels of patients with acromegaly varied from 1.03 to 24 μIU/ml (6.77 ± 6.03). The serum IGF-1 levels of patients with acromegaly were between 343 and 1600 mU/l (811.27 ± 323.55), while the serum IGF-1 levels of control subjects were between 67 and 297 (212.07 ± 75.06). Serum IGF-1 levels were significantly higher in patients with acromegaly than those in the control subjects (p b 0.001, Table 2).
Table 2 Results of CBMN cyt assay parameters and 8-OHdG levels in patients with acromegaly and control subjects (mean ± SD). Acromegalic patients (n = 30) Age (year) 44.9 ± 14.33 Serum IGF-1 levels (mU/l) 811.27 ± 323.55 MN frequency (%) 1.32 ± 0.49 NPB frequency (%) 4.35 ± 3.18 NBUD frequency (%) 1.51 ± 0.77 Frequency of apoptotic 1.99 ± 0.85 cells (%) Frequency of necrotic cells 6.36 ± 3.21 (%) The number of bi291.17 ± 102.81 nucleated cells (M2) The number of tri16.03 ± 14.18 nucleated cells (M3) The number of tetra7.41 ± 7.84 nucleated cells (M4) Nuclear division index 1.26 ± 0.08 (NDI) 8-OHdG levels (ng/ml) 1.27 ± 0.81
Controls (n = 30) 42.30 212.07 0.78 1.89 0.88 1.16
± ± ± ± ± ±
12.97 75.06 0.45 0.86 0.45 0.71
2.92 ± 1.21
P value
Z value
N0.05 b0.001 b0.001 b0.001 b0.001 b0.001
0.70 6.62 4.57 3.95 3.66 3.92
b0.001 4.85
238.40 ± 114.52 N0.05
1.55
16.73 ± 15.53
N0.05
0.07
10.07 ± 10.44
N0.05
0.89
1.23 ± 0.10
N0.05
0.96
0.67 ± 0.45
b0.005 3.04
IGF-1: Insulin-like growth factor 1; BN cells: Binucleated cells; MN: Micronucleus; NBUD: Nuclear bud; NPB: Nucleoplasmic bridge; 8-OHdG: 8-hydroxy-2′-deoxyguanosine; SD: Standard deviation. Nuclear division index (NDI) = [M1 + 2(M2) + 3(M3) + 4(M4)] / N, where M1–M4 represents the total number of lymphocytes with one to four nuclei scored on 1000 viable cells (excluding necrotic and apoptotic cells; M: The number of nuclei; N: The total number of viable cells scored).
F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
Table 3 shows the Spearman's rho correlation coefficients and significance values for age, serum IGF-1 levels and pituitary adenoma diameters in patients with acromegaly and control subjects with regard to the assessed parameters. 3.1. DNA damage in BN cells of peripheral blood lymphocytes The frequencies of MN, NPBs and NBUDs in patients with acromegaly were all significantly higher than frequencies in control subjects (p b 0.001, p b 0.001, p b 0.001, respectively; Table 2). In patients with acromegaly, increase in MN frequency was positively correlated with serum IGF-1 levels (p b 0.05, r: 0.439, Fig. 1), while increase in NPB frequency was negatively correlated with serum IGF-1 levels (p b 0.01, r: −0.520, Fig. 2). The NPB frequency increased consistently and significantly with age in both patients with acromegaly and control subjects (p b 0.01, r: 0.475 and p b 0.05, r: 0.420, respectively). Increase in the NBUD frequency was associated with age in control subjects but not in patients with acromegaly (p b 0.05, r: 0.429). 3.2. Cytotoxicity in peripheral blood lymphocytes
2,50
Micronucleus (MN) frequency in patients with acromegaly (%)
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2,00
1,50
1,00
0,50 R Sq Linear = 0,215
0,00 250,00
500,00
750,00 1000,00 1250,00 1500,00
Serum IGF-1 levels in patients with acromegaly (mU/l) Fig. 1. Correlation between the serum insulin-like growth factor 1 (IGF-1) levels and micronucleus (MN) frequency of patients with acromegaly (p b 0.05, r: 0.439).
The frequencies of apoptotic and necrotic cells were significantly higher in patients with acromegaly than in control subjects (p b 0.001 and p b 0.001 respectively, Table 2). The frequency of apoptotic cells was negatively related to serum IGF1 levels and pituitary adenoma diameters in patients with acromegaly (p b 0.05, r: − 0.401, Fig. 3 and p b 0.05, r: − 0.413, respectively), whereas the frequency of apoptotic cells was positively correlated with age in patients with acromegaly (p b 0.05, r: 0.414).
3.4. 8-OHdG levels Plasma 8-OHdG (ng/ml) levels were two-fold higher in patients with acromegaly than in control subjects (p b 0.005, Table 2). However, there was no significant correlation among plasma 8-OHdG levels, age, serum IGF-1 levels and pituitary adenoma diameters in both patients with acromegaly and control subjects (p N 0.05, Table 3).
3.3. Cytostasis in peripheral blood lymphocytes 4. Discussion There were no statistically significant differences between patients with acromegaly and control subjects for the number of cells in metaphase, the numbers of cells with two, three or four nuclei (M2–M4) and NDI values (p N 0.05). On the other hand, the correlation between NDI values and age was statistically significant in both patients with acromegaly and control subjects (p b 0.05, r: 0.368 and p b 0.001, r: − 0.554, respectively; Table 3).
Acromegaly is characterised by excessive levels of GH and IGF-1. High concentrations of circulating IGF-1 have been associated with an increased risk of breast, prostate, colorectal cancer and haematological malignancies in humans in several epidemiological studies [9–11,32–35]. IGF-1 mediates growth, apoptosis and metastasis responses in cancer [11].
Table 3 Spearman rho correlation coefficient and significance values for age, pituitary adenoma diameters (mm) and serum IGF-1 levels with assessed parameters in patients with acromegaly (n = 30) and control subjects (n = 30). MN frequency (%) Acromegalic patients Age (years) r p IGF-1 levels (mU/l) r p Pituitary adenoma diameters (mm) r p Control subjects Age (years) r p IGF-1 levels (mU/l) r p
NPB frequency (%)
NBUD frequency (%)
Frequency of apoptotic cells (%)
Frequency of necrotic cells (%)
NDI
0.475⁎⁎ 0.008
0.288 0.123
0.414⁎ 0.023
0.293 0.117
0.439⁎ 0.015
−0.520⁎⁎ 0.003
−0.091 0.634
−0.401⁎ 0.028
−0.114 0.549
−0.135 0.478
0.150 0.430
0.021 0.914
−0.042 0.830
−0.142 0.461
−0.413⁎ 0.026
−0.102 0.598
−0.264 0.166
−0.118 0.541
0.014 0.941
−0.236 0.210
−0.554⁎⁎ 0.001
−0.175 0.355
−0.264 0.159
−0.163 0.390
0.012 0.950
0.309 0.096
0.122 0.522 −0.157 0.407
0.420⁎ 0.021 −0.125 0.511
0.429⁎ 0.018 −0.272 0.146
0.368⁎ 0.045
8-OHdG levels
−0.145 0.445
0.250 0.183
IGF-1: Insulin-like growth factor 1; BN cells: Binucleated cells; MN: Micronucleus; NBUD: Nuclear bud; NPB: Nucleoplasmic bridge; NDI: Nuclear division index; 8-OHdG: 8-hydroxy-2′deoxyguanosine. ⁎ Correlation is significant at the 0.05 level (2-tailed). ⁎⁎ Correlation is significant at the 0.01 level (2-tailed).
Nucleoplasmic bridge (NPB) frequency in patients with acromegaly
F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
14,00 12,00 10,00
R Sq Linear = 0,28
8,00 6,00 4,00 2,00 0,00 250,00
500,00
750,00 1000,00 1250,00 1500,00
Serum IGF-1 levels in patients with acromegaly (mU/l) Fig. 2. Correlation between the serum insulin-like growth factor 1 (IGF-1) levels and nucleoplasmic bridge (NPB) frequency of patients with acromegaly (p b 0.01, r: −0.520).
CBMN assay in peripheral blood lymphocytes is one of the best validated cytogenetic tests for measuring DNA damage, genome instability and cancer risk [36]. CBMN Cyt assay is also a multipurpose method used to measure chromosomal DNA damage (frequencies of MN, NPBs, NBUDs), cytotoxicity (proportions of apoptotic and necrotic cells) and cytostasis (proportions of mono-, bi-, tri- and tetra-nucleated cells; NDI) [37–39]. On the other hand, 8-OHdG levels are also used to determine oxidative DNA damage [24,25]. In our previous study, which used a CBMN assay method, we reported that MN frequency is increased in patients with acromegaly [26]. However, in the present study, MN frequency and all other parameters, including frequency of NPBs and NBUDs, proportion of necrotic and apoptotic cells, NDI and oxidative DNA damage, have been assessed in patients with acromegaly using CBMN Cyt and 8-OHdG assays. Our study shows that the frequencies of MN, NPBs and NBUDs in lymphocytes of patients with acromegaly significantly increased compared with those in the control group, indicating enhanced genome
Frequency of apoptotic cells in patients with acromegaly (%)
4,00
3,00
2,00
1,00
R Sq Linear = 0,156
0,00 250,00
500,00
750,00 1000,00 1250,00 1500,00
Serum IGF-1 levels in patients with acromegaly (mU/l) Fig. 3. Correlation between the serum insulin-like growth factor 1(IGF-1) levels and frequency of apoptotic cells of patients with acromegaly (p b 0.05, r: −0.401).
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instability or chromosomal DNA damage. This finding corroborates the increased MN levels in patients with acromegaly, as reported in our previous study [26]. Indeed, MN frequency may be positively associated with factors such as micronutrient status, occupational or environmental exposures, genetic polymorphisms, lifestyle, smoking and tea and coffee intake [40–42]. However, the patient and control subjects in this study were free from these potential conditions affecting MN frequency. We found that MN frequency increased with elevated serum IGF-1 levels but was not related to pituitary adenoma diameters in acromegalic patients. Elevated serum IGF-1 levels may increase the number of DNA replication errors and induce aneuploidy due to the stimulation of cell proliferation. Thus, the stimulation of cell proliferation resulting from high levels of serum IGF-1 in blood may be responsible for increased MN, NPB and NBUD frequencies in patients with acromegaly. However, our finding of an inverse correlation between NPB frequency and serum IGF-1 levels in patients with acromegaly may also suggest that this increase may be independent of serum IGF-1 levels. Consistent with our findings on chromosomal DNA damage, we found that the frequencies of apoptotic and necrotic cells were higher in patients with acromegaly than in control subjects. Contrary to our findings in the present study, reduced apoptosis has been observed in samples of colonic mucosa (only in a different cell type) of patients with active untreated acromegaly [43]. The higher frequencies of apoptotic and necrotic cells in lymphocytes of patients with acromegaly were an indication of increasing cytotoxicity in these patients. The reason for this increase in apoptotic cells of patients with acromegaly may be explained with increasing DNA damage both by our previous study (of MN frequency) [26] and the present study (of the frequencies of MN, NPBs and NBUDs). The increase in DNA damage in the lymphocytes of patients with acromegaly may result in an increase in apoptotic cells, suggesting that apoptosis may remove DNA-damaged cells. If damaged cells were not removed by apoptosis or necrosis, the surviving cells could result in the propagation of damaged cells and contribute directly to cancer progression. On the other hand, in the present study, the frequency of apoptotic cells in patients with acromegaly is negatively related to serum IGF-1 levels and pituitary adenoma sizes. Moreover, similar to correlation between NPBs and IGF-1 levels, this increase in apoptotic cell frequency of patients with acromegaly may be independent of IGF-1 levels and also pituitary adenoma sizes. The present study, which included NDI results in patients with acromegaly, is the first of its kind in terms of measuring the proliferative status of the viable cell fraction. However, we have found neither a statistically significant difference in NDI values between patients and control groups nor any correlation between NDI values, serum IGF-1 levels and pituitary adenoma sizes in patients with acromegaly. However, this lack of difference in NDI values between the two groups may be related to apoptosis or programmed cell death, induced by increasing DNA damage, in patients with acromegaly. Further studies relating to increased cell proliferation and cell death in patients with acromegaly are required. Another important finding from the present study is that plasma 8-OHdG levels were significantly increased in patients with acromegaly but were not associated with elevated IGF-1 levels. There is only one study regarding oxidative DNA damage in patients with acromegaly. Nishizawa et al. [44] have shown that 8-OHdG levels are significantly increased in patients with acromegaly, and this increase is associated with elevated IGF-1 levels. An increased MN frequency in peripheral blood lymphocytes is generally considered indicative of increased cancer risk in humans. Also, in addition to MN, other DNA damage parameters of the CBMN Cyt assay, such as NPBs and NBUDs, might also improve the information about genomic instability in cancer [36,40]. Both the increase in chromosomal/ oxidative DNA damage and the positive association between MN frequency and serum IGF-1 levels may predict an increased risk of malignancy in acromegalic patients although there is no consensus
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regarding cancer risk in acromegaly. Therefore, long-term follow-up of a large number of patients with acromegaly is necessary. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements This work was supported by Erciyes University Scientific Research Projects Units (Project number: TSD-10-3327). References [1] A. Beckers, Higher prevalence of clinically relevant pituitary adenomas confirmed, Clin. Endocrinol. (Oxf) 72 (2010) 290–291. [2] I.M. Holdaway, Epidemiology of acromegaly, Pituitary 2 (1999) 29–41. [3] G. Lugo, L. Pena, F. Cordido, Clinical manifestations and diagnosis of acromegaly, Int. J. Endocrinol. (2012) 540398(Epub 2012 Feb 1). [4] I.M. Holdaway, R.C. Rajasoorya, G.D. Gamble, Factors influencing mortality in acromegaly, J. Clin. Endocrinol. Metab. 89 (2004) 667–674. [5] A.G. Renehan, B.M. Brennan, Acromegaly, growth hormone and cancer risk, Best Pract. Res. Clin. Endocinol. Metab. 22 (2008) 639–657. [6] P. Chanson, S. Salenave, Acromegaly, Orphanet J. Rare Dis. 25 (2008) 3–17. [7] N. Sekizawa, E. Hayakawa, K. Tsuchiya, et al., Acromegaly associated with multiple tumors, Intern. Med. 48 (2009) 1273–1278. [8] M. Kurimoto, I. Fukuda, N. Hizuka, K. Takano, The prevalence of benign and malignant tumors in patients with acromegaly at a single institute, Endocr. J. 55 (2008) 67–71. [9] S. Yakar, D. Leroith, P. Brodt, The role of the growth hormone/insulin-like growth factor axis in tumor growth and progression: lessons from animal models, Cytokine Growth Factor Rev. 16 (2005) 407–420. [10] H.M. Khandwala, I.E. McCutcheon, A. Flyvbjerg, K.E. Friend, The effects of insulin-like growth factors on tumorigenesis and neoplastic growth, Endocr. Rev. 21 (2000) 215–244. [11] H.I. Yasir, Y. Douglas, Insulin-like growth factor-I and cancer risk, Growth Horm. IGF Res. 14 (2004) 261–269. [12] P.J. Jenkins, Acromegaly and cancer, Horm. Res. 62 (2004) 108–115. [13] S.M. Webb, F. Casanueva, J.A. Wass, Oncological complications of excess GH in acromegaly, Pituitary 5 (2002) 21–25. [14] P. Cohen, D.R. Clemmons, R.G. Rosenfeld, Does the GH–IGF axis play a role in cancer pathogenesis? Growth Horm. IGF Res. 10 (2000) 297–305. [15] S. Loeper, S. Ezzat, Acromegaly: re-thinking the cancer risk, Rev. Endocr. Metab. Disord. 9 (2008) 41–58. [16] S. Melmed, Acromegaly and cancer: not a problem? J. Clin. Endocrinol. Metab. 86 (2001) 2929–2934. [17] A.G. Renehan, M. Zwahlen, C. Minder, S.T. O'Dwyer, S.M. Shalet, M. Egger, Insulin-like growth factor (IGF)-I, IGF binding protein-3, and cancer risk: systematic review and meta-regression analysis, Lancet 363 (2004) 1346–1353. [18] L.L. Corrêa, G.A. Lima, H.B. Paiva, et al., Prostate cancer and acromegaly, Arq. Bras. Endocrinol. Metabol. 53 (2009) 963–968. [19] M. Fenech, The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations, Mutat. Res. 285 (1993) 35–44. [20] M. Fenech, The in vitro micronucleus technique, Mutat. Res. 455 (2000) 81–95. [21] M. Fenech, J. Crott, J. Turner, S. Brown, Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide, Mutagenesis 14 (1999) 605–612.
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Increased genome instability and oxidative DNA damage and their association with IGF-1 levels in patients with active acromegaly Fahri Bayram a, Nazmiye Bitgen b, Hamiyet Donmez-Altuntas b,⁎, Ilkay Cakir a, Zuhal Hamurcu b, Fatma Sahin b, Yasin Simsek a, Gulden Baskol c a b c
Department of Endocrinology and Metabolism, Faculty of Medicine, Erciyes University, Kayseri, Turkey Department of Medical Biology, Faculty of Medicine, Erciyes University, Kayseri, Turkey Department of Biochemistry, Faculty of Medicine, Erciyes University, Kayseri, Turkey
a r t i c l e
i n f o
Article history: Received 11 February 2013 Received in revised form 6 November 2013 Accepted 5 December 2013 Available online 14 December 2013 Keywords: Acromegaly CBMN Cyt assay IGF-1 8-OHdG levels MN frequency
a b s t r a c t Objective: The objectives of this study were to assess cytokinesis-block micronucleus cytome (CBMN Cyt) assay parameters and also oxidative DNA damage in patients with active acromegaly and controls and to assess the relationship between age, serum insulin-like growth factor 1 (IGF-1) levels, pituitary adenoma diameters, 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels and CBMN Cyt assay parameters in patients with active acromegaly. Design: The study population included 30 patients with active acromegaly and 30 age- and sex-matched healthy controls. CBMN Cyt assay parameters in peripheral blood lymphocytes of patients with active acromegaly and controls were evaluated and plasma 8-OHdG levels were measured. Results: Frequencies of micronucleus (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in lymphocytes of patients with acromegaly were found to be significantly higher than those in controls (p b 0.001, p b 0.001, p b 0.001, respectively). The frequencies of apoptotic and necrotic cells in lymphocytes of patients with acromegaly were found to be significantly higher than those in controls (p b 0.001 and p b 0.001 respectively). No statistically significant differences in the number of cells in metaphase, the number of bi-nucleated cells (M2), the number of tri-nucleated cells (M3), the number of tetra-nucleated cells (M4) and nuclear division index (NDI) values were observed between patients and controls (p N 0.05). Plasma 8-OHdG (ng/ml) levels in patients with acromegaly were found to be significantly higher than those in controls (p b 0.005). MN frequency in the lymphocytes of patients with acromegaly increased with elevated serum IGF-1 levels (p b 0.05), whereas the number of NPBs and the frequency of apoptotic cells decreased with elevated serum IGF-1 levels (p b 0.01 and p b 0.05 respectively). Conclusions: Both the increase in chromosomal/oxidative DNA damage and the positive association between MN frequency and serum IGF-1 levels may predict an increased risk of malignancy in acromegalic patients. Long-term follow-up of patients with acromegaly will be necessary to establish the degree of cancer risk in this population. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Acromegaly is a slow-developing disease, with an estimated incidence of 4–5 per million per year and a prevalence of 60 cases per million [1–3]. It is usually caused by pituitary somatotroph adenomas and is associated with an increase in morbidity and mortality often attributed to cardiovascular, cerebrovascular, respiratory and metabolic diseases [4–6]. It is reported that acromegaly patients have an increased risk of developing both benign and malignant tumours, particularly colorectal cancer, and possibly other cancers such as breast, prostate, thyroid and haematological malignancies [7,8]. The aetiology of these cancers remains
⁎ Corresponding author at: Department of Medical Biology, Medical Faculty, Erciyes University, 38039 Kayseri, Turkey. E-mail address: [email protected] (H. Donmez-Altuntas). 1096-6374/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ghir.2013.12.002
uncertain, but they may be related to both growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels [9–14]. However, some epidemiological studies report conflicting findings on cancer risk in acromegaly [15,16]. It is established that IGF-1 is a potent mitogen and anti-apoptotic for a large variety of cells, exerting its mitogenic action by increasing DNA synthesis and stimulating cell cycle progression, whereas the main IGF-binding protein (IGFBP)-3 is a potent inhibitor of IGF-I action, which works in part by binding IGF1 and also by inducing apoptosis through an IGF-1-independent mechanism in target cells [14–18]. It is well known that GH promotes the stimulation of both IGF-1 and IGFBP-3. Because IGF-1 stimulates cell proliferation/growth advantage and IGFBP-3 promotes apoptosis and because GH stimulates both IGF-1 and IGFBP-3 in acromegaly, these factors should balance cell growth regulation [16,18]. However, high levels of IGFBP-3 were associated with an increased risk of premenopausal breast cancer, but not with any other cancer [17].
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F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
The micronucleus (MN) assay and cytokinesis-block micronucleus (CBMN) assay have both been used as methods for evaluating chromosome damage in cultured human lymphocytes because they provide a convenient and reliable index of both chromosome breakage and chromosome loss [19,20]. An important and further development in the CBMN assay is the adoption of the CBMN cytome (CBMN Cyt) assay approach that scores MN and includes other nuclear abnormalities such as nuclear buds (NBUDs) and nucleoplasmic bridges (NPBs), as well as involve frequencies of necrotic and apoptotic cells and the proportion of dividing cells [21,22]. This method allows the measurement of MN as a biomarker of chromosome breakage and/or whole chromosome loss, NPBs as a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair, NBUDs as a biomarker of gene amplification, necrosis and apoptosis as a viability status of cell, and metaphase, anaphase, mono-nucleated, bi-nucleated (BN), and multi-nucleated cells as a mitotic status of cell [21,22]. Oxidative stress is known to potentially cause DNA damage involving point mutations because of base oxidation, single and double strand breaks and genomic instability. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the damaged DNA products caused by reactive oxygen species. 8-OHdG is used as a reliable marker of oxidative DNA damage [23–25]. Our previous study showed that agromegalic patients had increased MN frequency [26]. However, the frequencies of NBPs and NBUDs, which determine DNA damage effects, the proportions of necrotic and apoptotic cells, which determine cytotoxicity and the proportions of mono-, bi- and multi-nucleated cells, and nuclear division index (NDI), which determines cytostasis in patients with acromegaly have not been investigated. The main objectives of this study are to assess the MN frequencies and new criteria such as the frequencies of NPBs and NBUDs, the frequencies of necrotic and apoptotic cells and NDI using CBMN Cyt assay parameters and oxidative DNA damage using the 8-OHdG assay in patients with acromegaly as well as to assess the relationship between age, serum IGF-1 levels, pituitary adenoma diameters, 8-OHdG levels and CBMN Cyt assay parameters, to improve the diagnosis and treatment of acromegaly.
Table 1 Patients profile at study entry. I.D.
Gender
Age (years)
IGF-1 levels (mU/l)
z-SDS IGF-1
Nadir GH after OGTT (μIU/ml)
z-SDS GH
Pituitary adenoma diameters (mm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
M M M M F F F M F F M M F F M M F M F F F F F M F M F M F F
20 24 29 29 30 31 33 35 36 38 38 39 40 41 42 43 46 46 48 49 51 53 53 56 56 57 64 66 74 80
626 987 681 1425 926 1246 534 1225 909 765 859 1600 1125 554 959 343 376 1033 613 907 688 442 1154 816 936 359 389 585 540 736
−0.573 0.543 −0.403 1.897 0.355 1.344 −0.857 1.279 0.302 −0.143 0.148 2.438 0.970 −0.795 0.457 −1.447 −1.345 0.685 −0.613 0.296 −0.381 −1.141 1.059 0.015 0.390 −1.398 −1.305 −0.699 −0.838 −0.233
8.83 14.10 2.04 1.76 9.08 10.53 18.63 4.09 12.73 8.40 – – 24.00 1.08 8.53 1.06 1.05 10.77 4.35 1.93 3.04 1.29 – 10.48 2.33 1.03 1.94 11.77 1.07 6.77
0.34 1.22 −0.78 −0.83 0.38 0.62 1.97 −0.44 0.99 0.27 – – 2.86 −0.94 0.29 −0.95 −0.95 0.66 −0.40 −0.80 −0.62 −0.91 – 0.62 −0.74 −0.95 −0.80 0.83 −0.95 0.00
25 16 11 16 35 8 10 20 30 20 13 17 24 10 7 30 8 30 3 30 5 30 8 6 40 23 15 11 15 6.5
IGF-1: Insulin-like growth factor 1; GH: Growth hormone; OGTT: Oral glucose tolerance test. z-SDS IGF-1 = (IGF-1 of the single patient − mean IGF-1 of total patients) / SD (SD: Standart deviation of total IGF-1). z-SDS GH = (GH of the single patient − mean GH of total patients) / SD (SD: Standart deviation of total GH).
2. Materials and methods 2.1. Patients and controls The study was performed on 30 patients diagnosed with active acromegaly and admitted to the Department of Endocrinology and Metabolism at Erciyes University Medical Faculty from December 2010 to December 2011. Thirty age- and sex-matched healthy controls were also included in the study. The diagnosis of acromegaly was made based on clinical and biochemical findings. Clinical diagnosis was made according to typical disfigurement of the patient related to progressive acral enlargement and modification of facial appearance. The diagnosis of acromegaly was confirmed by measuring GH response to the oral glucose tolerance test (OGTT) and IGF-1 levels. A magnetic resonance imaging (MRI) scan of the pituitary gland is used to locate and detect the size of adenomas (microadenomas being less than 10 mm in diameter and macroadenomas being greater than 10 mm in diameter). Serum IGF-1 levels were also higher than the age- and sex-related reference ranges in acromegalic patients [27,28]. The characteristics of the patients are shown in Table 1. All 30 patients included in the study had been newly diagnosed and had not received any medical or surgical therapy for acromegaly. They all had active disease states, with increased serum IGF-1 levels according to the normal gender and age reference levels and a failure to suppress GH levels after 75-g glucose loading. Twenty-two of the patients had macroadenoma (73.33%) and eight had microadenoma (26.67%). The OGTT was not performed on three of the 30 patients (patient IDs: 11, 12 and 23) because they had diabetes. These three patients were
diagnosed with acromegaly based on clinical findings of high IGF-1 levels, basal GH levels, and an MRI scan of the pituitary. The characteristics of patients in the study were as follows: aged between 20 and 80 years, 17 female and 13 male, seven smokers and 23 non-smokers, body mass index (BMI) between 22.00 and 32.00. No acromegalic patients had dyslipidaemia. Three acromegalic patients (patient IDs: 22, 25 and 30) had hypertension, but these patient symptoms were controlled with diet and non-pharmacologic therapy. The control group was selected from healthy subjects matched for age, gender, BMI, socio-economic status and smoking habits. None of them were known to be receiving any drugs for medical or other reasons or had specific diseases such as hypertension, diabetes mellitus, heart disease and cancer. A standardised questionnaire designed to obtain relevant details of their current health status, history and lifestyle as well as to collect information on past medical history, drug and smoking habits was completed for each patient and control subject. The study excluded patients and control subjects who reported alcohol, tea and/or coffee consumption (more than 3 cups/day), different dietary habits and had a history of occupational and environmental exposure to known genotoxic chemicals. Blood samples were obtained from patients for the measurement of growth hormone (GH), insulin-like growth factor-1 (IGF-1), follicle stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (tT), free testosterone (fT), free thyroxine (fT4), free tri-iodothyronine (fT3), prolactin (PRL), estradiol (e2), adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH) and cortisol. All serum samples were collected in the early morning after an 8- to 10-h fasting period.
F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
Patients who had multiple pituitary-hormone deficiencies and excessive hormone secretion except for GH/IGF-1 were excluded from the study. The local ethics committee approved the study protocol (No. 2010/ 97), and all patients gave written informed consent. The study was conducted in accordance with the Declaration of Helsinki and local laws, depending on which afforded greater protection to the patients.
2.2. Determinations of IGF-1 and GH levels Serum concentrations of IGF-1 and GH were measured by sensitive and specific immunoradiometric assay (Active Non-Extraction IGF-1 IRMA DSL-2800 and Active Growth Hormone IRMA DSL-1900; Diagnostics Systems Laboratories, Webster, TX). For IGF-1, the intra-assay and inter-assay coefficients of variation were 3.9%–7.0% and 4.2%–7.4%, respectively; the International Reference Preparation (IRP) used in the calibration of the IGF-1 assay was WHO 87/518. For GH, the intraassay and inter-assay coefficients of variations were 3.3%–4.3% and 6.3%–6.6%, respectively; the IRP used in the calibration of the GH assay was WHO 80/505/88/624. The use of these kits is one of the procedures recommended for IGF-1 measurements as they have a high sensitivity [29].
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2.5. Determination of 8-OHdG levels Two millilitres of heparinised blood samples for analysis of 8-OHdG was immediately centrifuged at 3000 rpm for 15 min at room temperature. The plasma was then stored in microtubes at −80 °C until it was analysed. The 8-OHdG levels in plasma were measured using an ELISA kit (Catalogue: NWK-8-OHdG02, Northwest Life Science Specialties, LLC, WA, USA) and an intra-assay coefficient of variation of 8-OHdG assay was calculated to be 5.9%. Plasma 8-OHdG levels were expressed in ng/ml. Calibration, curve fitting and data analysis were done according to the instructions of the manufacturer. 2.6. Statistical analysis The data were analysed using the SPSS for Windows statistical package, version 15.0. Differences were considered statistically significant when p values were less than 0.05. Statistical comparisons of CBMN Cyt assay parameters, 8-OHdG levels and serum IGF-1 levels between patients with acromegaly and control subjects were done using a non-parametric Mann–Whitney U test for two independent samples. Spearman's rho correlation analysis was used to assess the relationship between age, serum IGF-1 levels, pituitary adenoma diameters, 8-OHdG levels and CBMN Cyt assay parameters.
2.3. Whole-blood cultures of human lymphocytes 3. Results Antecubital heparinised 3 ml blood samples were taken after informed consent had been obtained from all patients and control subjects. Approximately 0.4 ml of heparinised whole blood samples was cultured for 72 h at 37 °C in 5 ml of peripheral blood karyotyping medium that was supplemented with 1.5% phytohaemagglutinin-M (PHA-M) to stimulate T-lymphocytes (all sources from Biological Industries, Kibbutz Beit Haemek, Israel). To determine intra-individual differences, duplicate cultures were made for each patient and control at the specified time.
2.4. CBMN Cyt assay Forty-four hours after cultures were initiated, cells were blocked from entering cytokinesis by the addition of cytochalasin-B to each culture tube (Sigma-Aldrich, St Louis, MO; final concentration, 3 μg/ml) [22]. The cultures were stopped at 72 h after initiation, treated with hypotonic solution (0.1 mol/l KCl) for 4 min and fixed using two changes of methanol–acetic acid (3:1) [22,26,30]. The fixed cells were spread onto glass slides and stained with 5% Giemsa (Merck) in Sorensen buffer for 10 min. To determine the intra-individual differences, the different slides of the two parallel cultures for each patient and control subject were prepared and evaluated. All slides were scored blindly by a Nikon Alphaphot-2 light optical microscope. A score was obtained for slides from each duplicate culture from two different scorers by identical microscopes. The 1000 BN cells with two macronuclei surrounded by cytoplasm were scored from each patient and control subject, and every subject was analysed for the total number of MN, NBPs and NBUDs per 1000 BN cells to determine DNA damage effects. The frequencies of BN cells containing one or more MN, NBPs and NBUDs were determined. The number of necrotic and apoptotic cells were scored in 1000 mono-nucleated cells to determine cytotoxicity [22]. The number of mono-, bi-, tri- and tetra-nucleated cells per 1000 viable mono-nucleated cells was scored in peripheral blood lymphocytes of all individuals to determine cytostatic effects. NDI has been calculated using the formula: NDI = (M1 + 2 M2 + 3 M3 + 4 M4) / N, where M1–M4 represents the number of cells with 1–4 nuclei and N is the total number of viable cells scored (excluding necrotic and apoptotic cells) [22,31].
Table 2 shows the statistical results and mean ± SD values of age, IGF-1 levels, 8-OHdG levels and CBMN Cyt assay parameters in 30 patients with acromegaly and 30 healthy controls. Pituitary adenoma diameters of patients with acromegaly ranged from 3 to 40 mm (17.74 ± 10.14). The nadir GH levels of patients with acromegaly varied from 1.03 to 24 μIU/ml (6.77 ± 6.03). The serum IGF-1 levels of patients with acromegaly were between 343 and 1600 mU/l (811.27 ± 323.55), while the serum IGF-1 levels of control subjects were between 67 and 297 (212.07 ± 75.06). Serum IGF-1 levels were significantly higher in patients with acromegaly than those in the control subjects (p b 0.001, Table 2).
Table 2 Results of CBMN cyt assay parameters and 8-OHdG levels in patients with acromegaly and control subjects (mean ± SD). Acromegalic patients (n = 30) Age (year) 44.9 ± 14.33 Serum IGF-1 levels (mU/l) 811.27 ± 323.55 MN frequency (%) 1.32 ± 0.49 NPB frequency (%) 4.35 ± 3.18 NBUD frequency (%) 1.51 ± 0.77 Frequency of apoptotic 1.99 ± 0.85 cells (%) Frequency of necrotic cells 6.36 ± 3.21 (%) The number of bi291.17 ± 102.81 nucleated cells (M2) The number of tri16.03 ± 14.18 nucleated cells (M3) The number of tetra7.41 ± 7.84 nucleated cells (M4) Nuclear division index 1.26 ± 0.08 (NDI) 8-OHdG levels (ng/ml) 1.27 ± 0.81
Controls (n = 30) 42.30 212.07 0.78 1.89 0.88 1.16
± ± ± ± ± ±
12.97 75.06 0.45 0.86 0.45 0.71
2.92 ± 1.21
P value
Z value
N0.05 b0.001 b0.001 b0.001 b0.001 b0.001
0.70 6.62 4.57 3.95 3.66 3.92
b0.001 4.85
238.40 ± 114.52 N0.05
1.55
16.73 ± 15.53
N0.05
0.07
10.07 ± 10.44
N0.05
0.89
1.23 ± 0.10
N0.05
0.96
0.67 ± 0.45
b0.005 3.04
IGF-1: Insulin-like growth factor 1; BN cells: Binucleated cells; MN: Micronucleus; NBUD: Nuclear bud; NPB: Nucleoplasmic bridge; 8-OHdG: 8-hydroxy-2′-deoxyguanosine; SD: Standard deviation. Nuclear division index (NDI) = [M1 + 2(M2) + 3(M3) + 4(M4)] / N, where M1–M4 represents the total number of lymphocytes with one to four nuclei scored on 1000 viable cells (excluding necrotic and apoptotic cells; M: The number of nuclei; N: The total number of viable cells scored).
F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
Table 3 shows the Spearman's rho correlation coefficients and significance values for age, serum IGF-1 levels and pituitary adenoma diameters in patients with acromegaly and control subjects with regard to the assessed parameters. 3.1. DNA damage in BN cells of peripheral blood lymphocytes The frequencies of MN, NPBs and NBUDs in patients with acromegaly were all significantly higher than frequencies in control subjects (p b 0.001, p b 0.001, p b 0.001, respectively; Table 2). In patients with acromegaly, increase in MN frequency was positively correlated with serum IGF-1 levels (p b 0.05, r: 0.439, Fig. 1), while increase in NPB frequency was negatively correlated with serum IGF-1 levels (p b 0.01, r: −0.520, Fig. 2). The NPB frequency increased consistently and significantly with age in both patients with acromegaly and control subjects (p b 0.01, r: 0.475 and p b 0.05, r: 0.420, respectively). Increase in the NBUD frequency was associated with age in control subjects but not in patients with acromegaly (p b 0.05, r: 0.429). 3.2. Cytotoxicity in peripheral blood lymphocytes
2,50
Micronucleus (MN) frequency in patients with acromegaly (%)
32
2,00
1,50
1,00
0,50 R Sq Linear = 0,215
0,00 250,00
500,00
750,00 1000,00 1250,00 1500,00
Serum IGF-1 levels in patients with acromegaly (mU/l) Fig. 1. Correlation between the serum insulin-like growth factor 1 (IGF-1) levels and micronucleus (MN) frequency of patients with acromegaly (p b 0.05, r: 0.439).
The frequencies of apoptotic and necrotic cells were significantly higher in patients with acromegaly than in control subjects (p b 0.001 and p b 0.001 respectively, Table 2). The frequency of apoptotic cells was negatively related to serum IGF1 levels and pituitary adenoma diameters in patients with acromegaly (p b 0.05, r: − 0.401, Fig. 3 and p b 0.05, r: − 0.413, respectively), whereas the frequency of apoptotic cells was positively correlated with age in patients with acromegaly (p b 0.05, r: 0.414).
3.4. 8-OHdG levels Plasma 8-OHdG (ng/ml) levels were two-fold higher in patients with acromegaly than in control subjects (p b 0.005, Table 2). However, there was no significant correlation among plasma 8-OHdG levels, age, serum IGF-1 levels and pituitary adenoma diameters in both patients with acromegaly and control subjects (p N 0.05, Table 3).
3.3. Cytostasis in peripheral blood lymphocytes 4. Discussion There were no statistically significant differences between patients with acromegaly and control subjects for the number of cells in metaphase, the numbers of cells with two, three or four nuclei (M2–M4) and NDI values (p N 0.05). On the other hand, the correlation between NDI values and age was statistically significant in both patients with acromegaly and control subjects (p b 0.05, r: 0.368 and p b 0.001, r: − 0.554, respectively; Table 3).
Acromegaly is characterised by excessive levels of GH and IGF-1. High concentrations of circulating IGF-1 have been associated with an increased risk of breast, prostate, colorectal cancer and haematological malignancies in humans in several epidemiological studies [9–11,32–35]. IGF-1 mediates growth, apoptosis and metastasis responses in cancer [11].
Table 3 Spearman rho correlation coefficient and significance values for age, pituitary adenoma diameters (mm) and serum IGF-1 levels with assessed parameters in patients with acromegaly (n = 30) and control subjects (n = 30). MN frequency (%) Acromegalic patients Age (years) r p IGF-1 levels (mU/l) r p Pituitary adenoma diameters (mm) r p Control subjects Age (years) r p IGF-1 levels (mU/l) r p
NPB frequency (%)
NBUD frequency (%)
Frequency of apoptotic cells (%)
Frequency of necrotic cells (%)
NDI
0.475⁎⁎ 0.008
0.288 0.123
0.414⁎ 0.023
0.293 0.117
0.439⁎ 0.015
−0.520⁎⁎ 0.003
−0.091 0.634
−0.401⁎ 0.028
−0.114 0.549
−0.135 0.478
0.150 0.430
0.021 0.914
−0.042 0.830
−0.142 0.461
−0.413⁎ 0.026
−0.102 0.598
−0.264 0.166
−0.118 0.541
0.014 0.941
−0.236 0.210
−0.554⁎⁎ 0.001
−0.175 0.355
−0.264 0.159
−0.163 0.390
0.012 0.950
0.309 0.096
0.122 0.522 −0.157 0.407
0.420⁎ 0.021 −0.125 0.511
0.429⁎ 0.018 −0.272 0.146
0.368⁎ 0.045
8-OHdG levels
−0.145 0.445
0.250 0.183
IGF-1: Insulin-like growth factor 1; BN cells: Binucleated cells; MN: Micronucleus; NBUD: Nuclear bud; NPB: Nucleoplasmic bridge; NDI: Nuclear division index; 8-OHdG: 8-hydroxy-2′deoxyguanosine. ⁎ Correlation is significant at the 0.05 level (2-tailed). ⁎⁎ Correlation is significant at the 0.01 level (2-tailed).
Nucleoplasmic bridge (NPB) frequency in patients with acromegaly
F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
14,00 12,00 10,00
R Sq Linear = 0,28
8,00 6,00 4,00 2,00 0,00 250,00
500,00
750,00 1000,00 1250,00 1500,00
Serum IGF-1 levels in patients with acromegaly (mU/l) Fig. 2. Correlation between the serum insulin-like growth factor 1 (IGF-1) levels and nucleoplasmic bridge (NPB) frequency of patients with acromegaly (p b 0.01, r: −0.520).
CBMN assay in peripheral blood lymphocytes is one of the best validated cytogenetic tests for measuring DNA damage, genome instability and cancer risk [36]. CBMN Cyt assay is also a multipurpose method used to measure chromosomal DNA damage (frequencies of MN, NPBs, NBUDs), cytotoxicity (proportions of apoptotic and necrotic cells) and cytostasis (proportions of mono-, bi-, tri- and tetra-nucleated cells; NDI) [37–39]. On the other hand, 8-OHdG levels are also used to determine oxidative DNA damage [24,25]. In our previous study, which used a CBMN assay method, we reported that MN frequency is increased in patients with acromegaly [26]. However, in the present study, MN frequency and all other parameters, including frequency of NPBs and NBUDs, proportion of necrotic and apoptotic cells, NDI and oxidative DNA damage, have been assessed in patients with acromegaly using CBMN Cyt and 8-OHdG assays. Our study shows that the frequencies of MN, NPBs and NBUDs in lymphocytes of patients with acromegaly significantly increased compared with those in the control group, indicating enhanced genome
Frequency of apoptotic cells in patients with acromegaly (%)
4,00
3,00
2,00
1,00
R Sq Linear = 0,156
0,00 250,00
500,00
750,00 1000,00 1250,00 1500,00
Serum IGF-1 levels in patients with acromegaly (mU/l) Fig. 3. Correlation between the serum insulin-like growth factor 1(IGF-1) levels and frequency of apoptotic cells of patients with acromegaly (p b 0.05, r: −0.401).
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instability or chromosomal DNA damage. This finding corroborates the increased MN levels in patients with acromegaly, as reported in our previous study [26]. Indeed, MN frequency may be positively associated with factors such as micronutrient status, occupational or environmental exposures, genetic polymorphisms, lifestyle, smoking and tea and coffee intake [40–42]. However, the patient and control subjects in this study were free from these potential conditions affecting MN frequency. We found that MN frequency increased with elevated serum IGF-1 levels but was not related to pituitary adenoma diameters in acromegalic patients. Elevated serum IGF-1 levels may increase the number of DNA replication errors and induce aneuploidy due to the stimulation of cell proliferation. Thus, the stimulation of cell proliferation resulting from high levels of serum IGF-1 in blood may be responsible for increased MN, NPB and NBUD frequencies in patients with acromegaly. However, our finding of an inverse correlation between NPB frequency and serum IGF-1 levels in patients with acromegaly may also suggest that this increase may be independent of serum IGF-1 levels. Consistent with our findings on chromosomal DNA damage, we found that the frequencies of apoptotic and necrotic cells were higher in patients with acromegaly than in control subjects. Contrary to our findings in the present study, reduced apoptosis has been observed in samples of colonic mucosa (only in a different cell type) of patients with active untreated acromegaly [43]. The higher frequencies of apoptotic and necrotic cells in lymphocytes of patients with acromegaly were an indication of increasing cytotoxicity in these patients. The reason for this increase in apoptotic cells of patients with acromegaly may be explained with increasing DNA damage both by our previous study (of MN frequency) [26] and the present study (of the frequencies of MN, NPBs and NBUDs). The increase in DNA damage in the lymphocytes of patients with acromegaly may result in an increase in apoptotic cells, suggesting that apoptosis may remove DNA-damaged cells. If damaged cells were not removed by apoptosis or necrosis, the surviving cells could result in the propagation of damaged cells and contribute directly to cancer progression. On the other hand, in the present study, the frequency of apoptotic cells in patients with acromegaly is negatively related to serum IGF-1 levels and pituitary adenoma sizes. Moreover, similar to correlation between NPBs and IGF-1 levels, this increase in apoptotic cell frequency of patients with acromegaly may be independent of IGF-1 levels and also pituitary adenoma sizes. The present study, which included NDI results in patients with acromegaly, is the first of its kind in terms of measuring the proliferative status of the viable cell fraction. However, we have found neither a statistically significant difference in NDI values between patients and control groups nor any correlation between NDI values, serum IGF-1 levels and pituitary adenoma sizes in patients with acromegaly. However, this lack of difference in NDI values between the two groups may be related to apoptosis or programmed cell death, induced by increasing DNA damage, in patients with acromegaly. Further studies relating to increased cell proliferation and cell death in patients with acromegaly are required. Another important finding from the present study is that plasma 8-OHdG levels were significantly increased in patients with acromegaly but were not associated with elevated IGF-1 levels. There is only one study regarding oxidative DNA damage in patients with acromegaly. Nishizawa et al. [44] have shown that 8-OHdG levels are significantly increased in patients with acromegaly, and this increase is associated with elevated IGF-1 levels. An increased MN frequency in peripheral blood lymphocytes is generally considered indicative of increased cancer risk in humans. Also, in addition to MN, other DNA damage parameters of the CBMN Cyt assay, such as NPBs and NBUDs, might also improve the information about genomic instability in cancer [36,40]. Both the increase in chromosomal/ oxidative DNA damage and the positive association between MN frequency and serum IGF-1 levels may predict an increased risk of malignancy in acromegalic patients although there is no consensus
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F. Bayram et al. / Growth Hormone & IGF Research 24 (2014) 29–34
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