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Evaluation of chromosome aberrations, sister chromatid exchange and micronuclei in patients with type-1 diabetes mellitus
Mutation Research 676 (2009) 1–4
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Evaluation of chromosome aberrations, sister chromatid exchange and micronuclei in patients with type-1 diabetes mellitus Nilüfer Cinkilic a , Sinem Kiyici b , Serap Celikler a , Ozgur Vatan a , Ozen Oz Gul b , Ercan Tuncel b,∗ , Rahmi Bilaloglu a a b
Uludag University Science and Arts Faculty, Biology Department, Gorukle, Bursa, Turkey Uludag University Faculty of Medicine, Department of Endocrinology and Metabolism, Gorukle, Bursa, Turkey
a r t i c l e
i n f o
Article history: Received 29 September 2008 Received in revised form 16 February 2009 Accepted 20 February 2009 Available online 5 April 2009 Keywords: Type-1 diabetes Chromosome aberrations Sister chromatid exchange Micronucleus formation
a b s t r a c t Oxidative stress-induced DNA damage seems to play a role in the pathogenesis of type-1 diabetes mellitus and its complications. Several in vitro assays have been used to measure the DNA damage produced by oxidative stress. In the present study, we aimed to investigate the frequency of sister chromatid exchange (SCE), chromosomal aberrations (CA) and micronuclei (MN) in type-1 diabetes mellitus patients compared with healthy controls. SCE, CA and MN tests were carried out with the blood-cell cultures from 35 type-1 diabetic patients and 15 healthy, age- and sex-matched control subjects. The mean age of the type-1 diabetic patients was 31.89 ± 10.01 years, with a mean duration of the diabetes of 7.8 ± 6.02 years. The mean level of HbA1c of the type-1 diabetic patients was 8.37 ± 1.36%. Only three (8.5%) patients with type1 diabetes mellitus had an HbA1c level below 7%. Patients with type-1 diabetes mellitus showed a higher frequency of SCE compared with controls (5.44 ± 1.47 and 2.54 ± 0.82, respectively, p < 0.001), but there was no significant correlation between the duration of diabetes, HbA1c and SCE. No significant difference was found in CA or MN frequency in type-1 diabetic patients compared with controls. In conclusion, these results suggest that type-1 diabetes mellitus is a condition with genomic instability characterized by an increased level of SCE. Hyperglycemia-induced oxidative stress may be the underlying factor of the increased SCE frequency. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Oxidative stress plays a crucial role in the cellular and molecular mechanisms of tissue injury in a wide spectrum of disease states [1]. Type-1 diabetes mellitus is a complex multifactorial disorder caused by absolute insulin deficiency due to destruction of the pancreatic -cells [2]. Pancreatic damage caused by oxidative stress has been involved in the pathogenesis of type-1 diabetes mellitus [3]. Systemic oxidative stress is present upon early onset of type-1 diabetes mellitus and becomes stronger over time [1]. Production of reactive oxygen species (ROS) and lipid peroxidation are increased in diabetic patients, especially in those with poor glycemic control and hypertriglyceridemia [4]. Previous studies have clearly shown that ROS, including O2 − , OH• , and H2 O2 , are highly reactive and capable of damaging cellular macromolecules, including proteins, lipids and DNA [5]. During the last decades, several in vitro assays have been developed for measurement of genotoxicity. The sister chromatid
∗ Corresponding author. Tel.: +90 224 2951112; fax: +90 224 4428038. E-mail address: [email protected] (E. Tuncel). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.02.014
exchange (SCE) test, analysis of chromosomal aberrations (CA) and the cytokinesis-block micronucleus (CBMN) assay in lymphocytes are being used extensively to evaluate the presence and extent of chromosomal damage in human populations [6]. There are limited reports on the association between diabetes and the occurrence of genotoxic effects. Sheth et al. [7] reported that there was a significant increase in SCE frequency in type-2 diabetic patients compared with healthy controls. However, there is no knowledge about SCE, CA and micronuclei (MN) in type-1 diabetic patients. Since free radical-induced DNA damage can be measured in vitro, we sought in this study to evaluate type-1 diabetes-associated genotoxicity by use of three cytogenetic assay systems. We evaluated CA, SCE and MN in cultured lymphocytes from individuals with type-1 diabetes mellitus and compared the results with those in healthy controls. 2. Materials and methods 2.1. Subjects A total of 35 patients with type-1 diabetes mellitus and 15 age- and sex-matched healthy control subjects were studied. Patients were recruited from pre-screened type-1 diabetic patients from an endocrinology outpatient clinic of a tertiary referral center. Type-1 diabetes mellitus was clinically defined as a diagnosis made prior to age 30, with a continuous need for insulin therapy within one year of diagnosis
2
N. Cinkilic et al. / Mutation Research 676 (2009) 1–4
and a C-peptide level <0.5 ng/ml. The controls had normal glucose metabolism and none had a family history of diabetes. The use of antioxidant vitamins or drugs that are able to induce the production of ROS was an exclusion criterion. The general characteristics of the study participants and lifestyle factors were collected by means of a questionnaire. Informed consent was obtained from all participants, and the study was performed in accordance with the Declaration of Helsinki and with the approval of the local ethics committee. 2.2. Lymphocyte cultures and cell harvesting Peripheral blood samples were drawn by venipuncture into sodium-heparin tubes. For each donor, two blood cultures were set up for each genotoxicity parameter (SCE, CA and CBMN). A 0.5-ml whole blood sample was added to a culture medium (5 ml) containing RPMI 1640 medium (pH 6.8–7.2), 20% fetal calf serum, 6 g/ml phytohemagglutinin L (PHA-L), 0.5 mg/ml l-glutamine, and antibiotics (100 IU/ml penicillin, 100 g/ml streptomycin) for 72 h (h) at 37 ◦ C. 2.2.1. SCE assay The SCE assay was performed following the addition of 5-bromo-2 deoxyuridine (BrdUrd) at a final concentration of 10 g/ml for the entire incubation period of 72 h. The cultures were carefully shielded from light in all steps until cell harvest. Colcemid (0.2 g/ml) was added 70 h after initiation of all cultures. The cells were harvested and processed through treatments with a hypotonic solution (0.075 M KCl) and fixative (3:1 methanol:glacial acetic acid). Chromosome slides were identified by the fluorescence-plus-Giemsa technique [8]. 2.2.2. Chromosome aberration assay For the CA analysis, we employed the same conditions as specified above, but BrdUrd was not added to the cultures. Chromosome slides were stained with 5% Giemsa solution (pH 6.8) for 15 min. 2.2.3. CBMN assay The CBMN assay was performed following the addition of cytochalasin B at a final concentration of 6 g/ml after 44 h of incubation at 37 ◦ C. The cells were harvested after 72 h and treated with hypotonic solution at room temperature for 10 min. Then, the fixation step with 5:1 methanol:glacial acetic acid was repeated four times. Slides were stained with 8% Giemsa for 15 min [9]. 2.3. Microscopic evaluation 2.3.1. SCE analysis Scoring criteria for SCE were as described by Carrano and Natarajan [10]. A total of 30 well-spread and complete (2n = 46) second-division metaphases per culture were scored for SCE and the frequency of SCE per cell was recorded. 2.3.2. Chromosomal aberrations The analysis of cells with CA was performed on 50 metaphases for each culture and 100 metaphases per individual. Gaps were both included and excluded from total CA counts. CA were classified according to the recommendation of EHC 51 (Environmental Health Criteria) for short-term test for mutagenic and carcinogenic chemicals [11]. For the analyses of the CA, chromatid and isochromatid gaps and breaks, acentric fragments, and exchanges were scored. The numerical CA were not assessed in this study. The mitotic index was also calculated as the number of metaphases in 2000 cells analyzed per culture for each donor. 2.3.3. CBMN analysis We used the protocol described by Fenech and Moorley [9] for CBMN analysis. Only cells with well-preserved cytoplasm were scored. We used the criteria for selection of bi-nucleated cells and identification of MN as reported in the HUMN project website [http://www.humn.org]. Briefly, cells having two distinct nuclei of approximately equal size, which may be connected by a fine nucleoplasmic bridge (NPB), overlapping slightly or touching each other at the edges, were selected. The number of MN (with size varying from 1/16 to 1/3 of the mean diameter of the main nucleus) located within the cytoplasm of bi-nucleated cells were scored and recorded. NPB and nuclear buds (NBUD) per 1000 bi-nucleate cells were also scored and recorded. The numbers of bi-nucleated cells with one, two, three or more MN were recorded. The data for each treatment were expressed as the frequency of MN per 2000 bi-nucleated cells. The nuclear division index (NDI) was calculated as a measure of cytotoxicity according to the following formula:
Table 1 General characteristics of the study participants. Controls
Type-1 diabetic patients
Number of subjects Gender (males/females) Age (years) (mean ± SD) Duration of diabetes (years)
15 9/6 38.76 ± 9.12 –
35 20/15 31.89 ± 10.01 1–22
Smoke habit (n) Non-smokers Smokers
8 7
16 19
Alcohol habit (n) Drinkers Non-drinkers
0 15
0 35
the relative importance of the different explaining variables was done with multiple linear regression analysis. All calculations were performed using the SPSS 13.0 (SPSS Inc., Chicago, IL, USA) and Statistica 6.0 statistical software packages and shown as mean ± standard deviation. A p-value of less than 0.05 was considered to correspond with statistical significance.
3. Results The general characteristics of the study participants are listed in Table 1. The mean age of the type-1 diabetic patients was 31.89 ± 10.01 years, with a mean duration of the diabetes of 7.8 ± 6.02 years (range: 1–22 years). Six diabetic patients (17%) had a family history of type-1 diabetes, and 12 (34%) had a family history of type-2 diabetes. The prevalences of dyslipidemia (13 patients) and thyroid disease (4 patients) were higher in patients with type-1 diabetes mellitus, while none of control subjects had either. All patients were using intensified insulin-therapy regimens with multiple daily injections. Insulin glargine was used as long-acting insulin for basal insulin replacement, whereas insulin lispro and insulin aspartate were used for bolus injections. Four type-1 diabetic patients were taking angiotensinogen-converting enzyme inhibitors for the presence of microalbuminuria and only one patient was using statins. There was no other medication use or chemical substance exposure among the type-1 diabetic patients or the healthy controls. Table 2 shows the frequencies of total CA, types of aberrations and MI in the two study groups. MN and SCE test results of the study participants are shown in Table 3. There were no differences in CA and the frequency of MN between patients and controls. However, patients with type-1 diabetes mellitus showed a higher frequency of SCE (5.44 ± 1.47) than the controls (2.54 ± 0.82) (p < 0.001). NDI, NPB and NBUD per 1000 bi-nucleate cells did not differ significantly when compared with the control group. The frequency of SCE in patients and controls according to age strata and the relationship between frequency of SCE and glucose level are shown in Table 4. Table 2 The frequencies of chromosome aberrations and mitotic index in cultured human lymphocytes from diabetic patients and control groups. Controls
Type-1 diabetic patients
The significance levels of SCE, CA and MN in the cultures of diabetic patients and controls were determined by use of the Mann–Whitney U-test. One-sided tests were found appropriate, considering the hypothesis of the study. Further analysis of
TCA: total chromosome aberrations, TCA-(G): total chromosome aberrations excluding gaps, *: p > 0.05; data are expressed as mean ± standard deviation. 100 lymphocytes were counted per donor for chromosome aberrations.
MI + 2 × MII + 3 × MIII + 4 × MIV 500
where MI–MIV represent the number of cells with one (I) to four (IV) nuclei per 500 cells, respectively [9].
1.75 0.006 0.002 0.000 0.000 0.000 0.000 0.000 0.009 0.000
4.50 0.003 0.002 0.002 0.000 0.002 0.00 0.000 0.009 0.004
± ± ± ± ± ± ± ± ± ±
2.4. Statistical analysis
NDI =
4.55 0.004 0.003 0.000 0.000 0.000 0.000 0.000 0.007 0.000
± ± ± ± ± ± ± ± ± ±
Mitotic index Chromatid gap Isochromatid gap Chromatid break Isochromatid break Chromatid acentric fragment Isochromatid acentric fragment Chromatid exchange TCA TCA-(G)
2.12* 0.008* 0.003* 0.001* 0.000* 0.001* 0.000* 0.000* 0.012* 0.007*
N. Cinkilic et al. / Mutation Research 676 (2009) 1–4 Table 3 Frequencies of the SCE and MN in patients with type-1 diabetes mellitus and controls. Controls SCE/Cell MNa NPB/1000 BN cells NBUD/1000 BN cells NDI
2.54 1.53 2.20 0.33 1.81
± ± ± ± ±
Type-1 diabetic patients
0.82 0.64 0.86 0.49 0.10
5.44 1.83 2.43 0.49 1.82
± ± ± ± ±
1.47* 1.15 1.33 0.56 0.10
SCE: sister chromatid exchange, MN: micronuclei, a (superscript): micronuclei per 2000 bi-nucleate cells, NPB: nucleoplasmic bridge, NBUD: nuclear bud, NDI: nuclear division index, BN: bi-nucleate cells, *: p < 0.001; data are expressed as mean ± standard deviation. Table 4 Frequencies of SCE per age groups in patients with type-1 diabetes mellitus and controls. Age groups
SCE/cell in controls (n = 15)
SCE/cell in type-1 diabetic patients (n = 35)
p
<25 years 25–44 years ≥45 years
2.84 ± 0.90 (n = 3) 2.30 ± 0.99 (n = 7) 2.72 ± 0.50 (n = 5)
5.27 ± 1.26 (n = 10) 5.56 ± 1.48 (n = 21) 5.25 ± 2.22 (n = 4)
0.007 0.001 0.016
SCE: sister chromatid exchange, n: number of subjects; data are expressed as mean ± standard deviation.
Compared with controls, patients with type-1 diabetes mellitus had a higher frequency of SCE across all age ranges. Mean HbA1c level was 8.37 ± 1.36% and mean serum fasting glucose was 174.34 ± 64.23 mg/dl in type-1 diabetic patients. Out of the 35 patients with type-1 diabetes mellitus, 11 (31%) had an HbA1c level below 8% and only three (8.5%) had an HbA1c level below 7%. The frequency of SCE was 5.27 ± 1.36 in patients with an HbA1c level <8% while it was 5.43 ± 1.42 in patients with an HbA1c level ≥8%. A comparison of the two groups demonstrated no statistically significant difference in the frequency of SCE (p = 0.619) and no correlation between the frequency of SCE, HbA1c and serum glucose levels. According to the multiple linear regression analysis, when SCE was taken as a dependent variable, the duration of diabetes (r = −0.18, p > 0.05) and age (r = −0.077, p > 0.05) were investigated as independent variables. There was no significant correlation between SCE, duration of diabetes and age. We also compared SCE frequency in diabetic patients with and without smoking habit and did not find any significant difference in the frequency of SCE (5.62 ± 1.77, 5.25 ± 1.09, respectively, p > 0.05). 4. Discussion The detection of CA and MN in peripheral blood lymphocytes is commonly used for monitoring human populations exposed to mutagenic agents and for evaluation of genotoxicity [12]. In recent years, several studies have investigated a number of genotoxicity parameters in relation to health and disease [13]. In the present study, there were no differences in CA and MN frequencies in the lymphocytes of type-1 diabetic patients when compared with controls. However, type-1 diabetics showed a significantly higher SCE frequency than the healthy subjects. Sheth et al. [7] previously investigated the occurrence of genotoxic damage in type-2 diabetic patients as evidenced by SCE and CA tests in lymphocytes. The authors reported a higher occurrence of SCE in patients with type-2 diabetes mellitus, but no CA and no changes in the cell-cycle proliferative index. Our current data confirm and expand previous findings by showing that patients with type-1 diabetes mellitus have increased levels of SCE. The hallmark of type-1 diabetes mellitus is the systemic defect in insulin-dependent metabolism. There is evidence to suggest that ROS may play an important role in the pathogenesis of type-1
3
diabetes mellitus [3] and its complications [14,15]. The presence of an increased oxidative stress in diabetic patients has been well documented. Specifically, the hyperglycemic milieu has been suggested to result in an increased production of oxygen-free radicals through glucose auto-oxidation and nonenzymatic glycation of macromolecules, including proteins and DNA [1,16]. Notably, previous studies of oxidative damage in diabetic patients have used several tests, including the comet assay, to investigate DNA damage [4,17–19]. Sardas et al. [4] reported an increase in oxidative DNA damage in the comet assay in both type-1 and type-2 diabetic patients. Moreover, these authors reported that DNA damage as observed in the comet assay was higher in individuals with type-2 diabetes mellitus compared to those with type-1 diabetes. Interestingly, a higher degree of DNA damage was evident in the entire cohort of diabetic patients compared with controls [4]. Under conditions of oxidative stress, damage to cellular biomolecules such as lipids, proteins and DNA occurs. Oxygen-free radicals induce a variety of lesions in DNA, including oxidized bases, abasic sites, DNA strand breaks and formation of cross-links between DNA and proteins [20]. Many of these lesions are cytotoxic and mutagenic. The induction of SCE has been correlated with the primary DNA damage [21]. The SCE assay is an ideal genotoxicity and cytotoxicity assay, since peripheral lymphocytes are easily accessible. Moreover, SCE is much more sensitive as a mutagenic biomarker than CA and MN [22]. Some authors have suggested that ROS may be implicated in the production of high basal SCE frequencies in chromosome-instability syndromes [23]. Oxidative damage to DNA over time can cause changes to both the structure and function of chromosomes. These changes in DNA may lead to cancer and other chronic disease conditions [24,25]. Recently, the genotoxicity of ROS has been well-established [26,27]. In a previous study, the mutagenic effects of ROS were detected in human lymphocytes by use of the SCE technique, and elevated ROS levels were shown to cause an increase in mitotic recombination frequency [28]. We suggest that the increased frequency of SCE in the patients with type-1 diabetes mellitus in this study may be due to the generation of oxygen-derived free radicals occurring in this condition. In conclusion, our current data seem to suggest that an enhanced genomic instability with an increased level of SCE can be a common biochemical feature of patients with type-1 diabetes mellitus. Conflict of interest statement The authors declare that there are no conflicts of interest. References [1] C. Dominguez, E. Ruiz, M. Gussinye, A. Carrascosa, Oxidative stress at onset and in early stages of type 1 diabetes in children and adolescents, Diabetes Care 21 (1998) 1736–1742. [2] G.S. Eisenbarth, Update in type 1 diabetes, J. Clin. Endocrinol. Metab. 92 (2007) 2403–2407. [3] J.W. Baynes, Role of oxidative stress in development of complications in diabetes, Diabetes 40 (1991) 405–412. [4] S. Sardas, M. Yılmaz, U. Oztok, N. C¸akir, A.E. Karakaya, Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation, Mutat. Res. 490 (2001) 123–129. [5] R.K.R. Mantena, O.L.C. Wijburg, C. Vindurampulle, V.R. Bennett-Wood, A. Walduck, G.R. Drummond, J.K. Davies, R.M. Robins-Browne, R.A. Strugnell, Reactive oxygen species are the major antibacterials against Salmonella Typhimurium purine auxotrophs in the phagosome of RAW 264.7 cells, Cell. Microbiol. 10 (5) (2008) 1058–1073. [6] J.D. Tucker, R.J. Preston, Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchange and cancer risk assessment, Mutat. Res. 365 (1996) 147–159. [7] F.J. Sheth, P. Patel, A.D.B. Vaidya, R. Vaidya, J. Sheth, Increased frequency of sister chromatid exchanges in patients with type II diabetes, Curr. Sci. 90 (2006) 236–240.
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[8] P. Perry, S. Wolff, Giemsa method for differential staining of sister chromatids, Nature 251 (1974) 156–158. [9] M. Fenech, A.A. Moorley, Measurement of micronuclei in lymphocytes, Mutat. Res. 147 (1985) 29–36. [10] A.V. Carrano, A.T. Natarajan, Considerations for population monitoring using cytogenetics techniques, ICPEMC Publication No.14, International Commission for Protection Against Environmental Mutagens and Carcinogens, Mutat. Res. 204 (1988) 379–406. [11] IPCS, Guide to Short Term Tests for Detecting Mutagenic and Carcinogenic Chemicals, World Health Organization, Geneva, 1985. [12] A.T. Natarajan, Chromosome aberrations: past, present and future, Mutat. Res. 504 (2002) 3–16. [13] S.C. Schreck, S.A. Latt, Comparison of benzo(alpha)pyrene metabolism and sister chromatid exchange induction in mice, Nature 288 (1980) 407–408. [14] J. Everhard, D. Wright, Diabetes mellitus as a risk factor for pancreatic cancer. A meta-analysis, J. Am. Med. Assoc. 273 (1995) 1605–1609. [15] K. Steenland, S. Nowlin, S. Palu, Cancer incidence in the national-health-and nutrition-survey-I follow up data-diabetes, cholesterol, pulse and physical activity, Cancer Epidemiol. Biomarkers Prev. 4 (1995) 807–811. [16] T.J. Lyons, Glycation and oxidation a role in the pathogenesis of atherosclerosis, Am. J. Cardiol. 71 (1993) B26–B31. [17] D. Anderson, T.W. Yu, J. Wright, C. Ionnides, An examination of DNA strand breakage in the comet assay and antioxidant capacity in diabetic patients, Mutat. Res. 398 (1998) 151–161. [18] Y. Dincer, A. Akc¸ay, Z. Alademir, H. Ilkova, Assessment of DNA base oxidation and glutathione level in patients with type 2 diabetes, Mutat. Res. 505 (2002) 75–81.
[19] Y. Dinc¸er, A. Akc¸ay, H. Ilkova, Z. Alademir, G. Ozbay, DNA damage and antioxidant defense in peripheral leukocytes of patients with type I diabetes mellitus, Mutat. Res. 527 (2003) 49–55. [20] M.K. Shigenaga, B.N. Ames, Assays for 8-hydroxy-2 -deoxyguanosine a biomarker of in vivo oxidative DNA damage, Free Radic. Biol. Med. 10 (1991) 211–216. [21] E.M. Parry, J.M. Parry, In vitro cytogenetics and aneuploidy, in: D.H. Philipps, S. Venitt (Eds.), Environmental Mutagenesis, BIOS Scientific Publishers Limited, Oxford, 1995, pp. 121–135. [22] E. Therman, M. Susman, Human chromosomes: structure, behaviour, and effects, 3rd ed., Springer, New York, 1993, pp. 126–134. [23] K.H. Lee, S. Abe, Y. Yanabe, I. Matsuda, M.C. Yoshida, Superoxide-dismutase activity and chromosome damage in cultured chromosome instability syndrome cells, Mutat. Res. 244 (1990) 251–256. [24] M.K. Shigenaga, T.M. Hagen, B.N. Ames, Oxidative damage and mitochondrial decay in aging, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 10771–10778. [25] S. Loft, H.E. Paulson, Cancer risk and oxidative DNA damage in man, J. Mol. Med. 74 (1996) 297–312. [26] H. Tominaga, S. Kodama, N. Matsuda, K. Suzuki, M. Watanable, Involvement of reactive oxygen species (ROS) in the induction of genetic instability by radiation, J. Radiat. Res. (Tokyo) 45 (2004) 181–188. [27] C.L. Limoli, E. Giedzinski, W.F. Morgan, S.G. Swarts, G.D. Jones, W. Hyun, Persistent oxidative stress in chromosomally unstable cells, Cancer Res. 63 (2003) 3107–3111. [28] D.R. Turner, M. Dreimanis, D. Holt, F.A. Firgaira, A.A. Morley, Mitotic recombination is an important mutational event following oxidative damage, Mutat. Res. 522 (2003) 21–26.
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Evaluation of chromosome aberrations, sister chromatid exchange and micronuclei in patients with type-1 diabetes mellitus Nilüfer Cinkilic a , Sinem Kiyici b , Serap Celikler a , Ozgur Vatan a , Ozen Oz Gul b , Ercan Tuncel b,∗ , Rahmi Bilaloglu a a b
Uludag University Science and Arts Faculty, Biology Department, Gorukle, Bursa, Turkey Uludag University Faculty of Medicine, Department of Endocrinology and Metabolism, Gorukle, Bursa, Turkey
a r t i c l e
i n f o
Article history: Received 29 September 2008 Received in revised form 16 February 2009 Accepted 20 February 2009 Available online 5 April 2009 Keywords: Type-1 diabetes Chromosome aberrations Sister chromatid exchange Micronucleus formation
a b s t r a c t Oxidative stress-induced DNA damage seems to play a role in the pathogenesis of type-1 diabetes mellitus and its complications. Several in vitro assays have been used to measure the DNA damage produced by oxidative stress. In the present study, we aimed to investigate the frequency of sister chromatid exchange (SCE), chromosomal aberrations (CA) and micronuclei (MN) in type-1 diabetes mellitus patients compared with healthy controls. SCE, CA and MN tests were carried out with the blood-cell cultures from 35 type-1 diabetic patients and 15 healthy, age- and sex-matched control subjects. The mean age of the type-1 diabetic patients was 31.89 ± 10.01 years, with a mean duration of the diabetes of 7.8 ± 6.02 years. The mean level of HbA1c of the type-1 diabetic patients was 8.37 ± 1.36%. Only three (8.5%) patients with type1 diabetes mellitus had an HbA1c level below 7%. Patients with type-1 diabetes mellitus showed a higher frequency of SCE compared with controls (5.44 ± 1.47 and 2.54 ± 0.82, respectively, p < 0.001), but there was no significant correlation between the duration of diabetes, HbA1c and SCE. No significant difference was found in CA or MN frequency in type-1 diabetic patients compared with controls. In conclusion, these results suggest that type-1 diabetes mellitus is a condition with genomic instability characterized by an increased level of SCE. Hyperglycemia-induced oxidative stress may be the underlying factor of the increased SCE frequency. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Oxidative stress plays a crucial role in the cellular and molecular mechanisms of tissue injury in a wide spectrum of disease states [1]. Type-1 diabetes mellitus is a complex multifactorial disorder caused by absolute insulin deficiency due to destruction of the pancreatic -cells [2]. Pancreatic damage caused by oxidative stress has been involved in the pathogenesis of type-1 diabetes mellitus [3]. Systemic oxidative stress is present upon early onset of type-1 diabetes mellitus and becomes stronger over time [1]. Production of reactive oxygen species (ROS) and lipid peroxidation are increased in diabetic patients, especially in those with poor glycemic control and hypertriglyceridemia [4]. Previous studies have clearly shown that ROS, including O2 − , OH• , and H2 O2 , are highly reactive and capable of damaging cellular macromolecules, including proteins, lipids and DNA [5]. During the last decades, several in vitro assays have been developed for measurement of genotoxicity. The sister chromatid
∗ Corresponding author. Tel.: +90 224 2951112; fax: +90 224 4428038. E-mail address: [email protected] (E. Tuncel). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.02.014
exchange (SCE) test, analysis of chromosomal aberrations (CA) and the cytokinesis-block micronucleus (CBMN) assay in lymphocytes are being used extensively to evaluate the presence and extent of chromosomal damage in human populations [6]. There are limited reports on the association between diabetes and the occurrence of genotoxic effects. Sheth et al. [7] reported that there was a significant increase in SCE frequency in type-2 diabetic patients compared with healthy controls. However, there is no knowledge about SCE, CA and micronuclei (MN) in type-1 diabetic patients. Since free radical-induced DNA damage can be measured in vitro, we sought in this study to evaluate type-1 diabetes-associated genotoxicity by use of three cytogenetic assay systems. We evaluated CA, SCE and MN in cultured lymphocytes from individuals with type-1 diabetes mellitus and compared the results with those in healthy controls. 2. Materials and methods 2.1. Subjects A total of 35 patients with type-1 diabetes mellitus and 15 age- and sex-matched healthy control subjects were studied. Patients were recruited from pre-screened type-1 diabetic patients from an endocrinology outpatient clinic of a tertiary referral center. Type-1 diabetes mellitus was clinically defined as a diagnosis made prior to age 30, with a continuous need for insulin therapy within one year of diagnosis
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N. Cinkilic et al. / Mutation Research 676 (2009) 1–4
and a C-peptide level <0.5 ng/ml. The controls had normal glucose metabolism and none had a family history of diabetes. The use of antioxidant vitamins or drugs that are able to induce the production of ROS was an exclusion criterion. The general characteristics of the study participants and lifestyle factors were collected by means of a questionnaire. Informed consent was obtained from all participants, and the study was performed in accordance with the Declaration of Helsinki and with the approval of the local ethics committee. 2.2. Lymphocyte cultures and cell harvesting Peripheral blood samples were drawn by venipuncture into sodium-heparin tubes. For each donor, two blood cultures were set up for each genotoxicity parameter (SCE, CA and CBMN). A 0.5-ml whole blood sample was added to a culture medium (5 ml) containing RPMI 1640 medium (pH 6.8–7.2), 20% fetal calf serum, 6 g/ml phytohemagglutinin L (PHA-L), 0.5 mg/ml l-glutamine, and antibiotics (100 IU/ml penicillin, 100 g/ml streptomycin) for 72 h (h) at 37 ◦ C. 2.2.1. SCE assay The SCE assay was performed following the addition of 5-bromo-2 deoxyuridine (BrdUrd) at a final concentration of 10 g/ml for the entire incubation period of 72 h. The cultures were carefully shielded from light in all steps until cell harvest. Colcemid (0.2 g/ml) was added 70 h after initiation of all cultures. The cells were harvested and processed through treatments with a hypotonic solution (0.075 M KCl) and fixative (3:1 methanol:glacial acetic acid). Chromosome slides were identified by the fluorescence-plus-Giemsa technique [8]. 2.2.2. Chromosome aberration assay For the CA analysis, we employed the same conditions as specified above, but BrdUrd was not added to the cultures. Chromosome slides were stained with 5% Giemsa solution (pH 6.8) for 15 min. 2.2.3. CBMN assay The CBMN assay was performed following the addition of cytochalasin B at a final concentration of 6 g/ml after 44 h of incubation at 37 ◦ C. The cells were harvested after 72 h and treated with hypotonic solution at room temperature for 10 min. Then, the fixation step with 5:1 methanol:glacial acetic acid was repeated four times. Slides were stained with 8% Giemsa for 15 min [9]. 2.3. Microscopic evaluation 2.3.1. SCE analysis Scoring criteria for SCE were as described by Carrano and Natarajan [10]. A total of 30 well-spread and complete (2n = 46) second-division metaphases per culture were scored for SCE and the frequency of SCE per cell was recorded. 2.3.2. Chromosomal aberrations The analysis of cells with CA was performed on 50 metaphases for each culture and 100 metaphases per individual. Gaps were both included and excluded from total CA counts. CA were classified according to the recommendation of EHC 51 (Environmental Health Criteria) for short-term test for mutagenic and carcinogenic chemicals [11]. For the analyses of the CA, chromatid and isochromatid gaps and breaks, acentric fragments, and exchanges were scored. The numerical CA were not assessed in this study. The mitotic index was also calculated as the number of metaphases in 2000 cells analyzed per culture for each donor. 2.3.3. CBMN analysis We used the protocol described by Fenech and Moorley [9] for CBMN analysis. Only cells with well-preserved cytoplasm were scored. We used the criteria for selection of bi-nucleated cells and identification of MN as reported in the HUMN project website [http://www.humn.org]. Briefly, cells having two distinct nuclei of approximately equal size, which may be connected by a fine nucleoplasmic bridge (NPB), overlapping slightly or touching each other at the edges, were selected. The number of MN (with size varying from 1/16 to 1/3 of the mean diameter of the main nucleus) located within the cytoplasm of bi-nucleated cells were scored and recorded. NPB and nuclear buds (NBUD) per 1000 bi-nucleate cells were also scored and recorded. The numbers of bi-nucleated cells with one, two, three or more MN were recorded. The data for each treatment were expressed as the frequency of MN per 2000 bi-nucleated cells. The nuclear division index (NDI) was calculated as a measure of cytotoxicity according to the following formula:
Table 1 General characteristics of the study participants. Controls
Type-1 diabetic patients
Number of subjects Gender (males/females) Age (years) (mean ± SD) Duration of diabetes (years)
15 9/6 38.76 ± 9.12 –
35 20/15 31.89 ± 10.01 1–22
Smoke habit (n) Non-smokers Smokers
8 7
16 19
Alcohol habit (n) Drinkers Non-drinkers
0 15
0 35
the relative importance of the different explaining variables was done with multiple linear regression analysis. All calculations were performed using the SPSS 13.0 (SPSS Inc., Chicago, IL, USA) and Statistica 6.0 statistical software packages and shown as mean ± standard deviation. A p-value of less than 0.05 was considered to correspond with statistical significance.
3. Results The general characteristics of the study participants are listed in Table 1. The mean age of the type-1 diabetic patients was 31.89 ± 10.01 years, with a mean duration of the diabetes of 7.8 ± 6.02 years (range: 1–22 years). Six diabetic patients (17%) had a family history of type-1 diabetes, and 12 (34%) had a family history of type-2 diabetes. The prevalences of dyslipidemia (13 patients) and thyroid disease (4 patients) were higher in patients with type-1 diabetes mellitus, while none of control subjects had either. All patients were using intensified insulin-therapy regimens with multiple daily injections. Insulin glargine was used as long-acting insulin for basal insulin replacement, whereas insulin lispro and insulin aspartate were used for bolus injections. Four type-1 diabetic patients were taking angiotensinogen-converting enzyme inhibitors for the presence of microalbuminuria and only one patient was using statins. There was no other medication use or chemical substance exposure among the type-1 diabetic patients or the healthy controls. Table 2 shows the frequencies of total CA, types of aberrations and MI in the two study groups. MN and SCE test results of the study participants are shown in Table 3. There were no differences in CA and the frequency of MN between patients and controls. However, patients with type-1 diabetes mellitus showed a higher frequency of SCE (5.44 ± 1.47) than the controls (2.54 ± 0.82) (p < 0.001). NDI, NPB and NBUD per 1000 bi-nucleate cells did not differ significantly when compared with the control group. The frequency of SCE in patients and controls according to age strata and the relationship between frequency of SCE and glucose level are shown in Table 4. Table 2 The frequencies of chromosome aberrations and mitotic index in cultured human lymphocytes from diabetic patients and control groups. Controls
Type-1 diabetic patients
The significance levels of SCE, CA and MN in the cultures of diabetic patients and controls were determined by use of the Mann–Whitney U-test. One-sided tests were found appropriate, considering the hypothesis of the study. Further analysis of
TCA: total chromosome aberrations, TCA-(G): total chromosome aberrations excluding gaps, *: p > 0.05; data are expressed as mean ± standard deviation. 100 lymphocytes were counted per donor for chromosome aberrations.
MI + 2 × MII + 3 × MIII + 4 × MIV 500
where MI–MIV represent the number of cells with one (I) to four (IV) nuclei per 500 cells, respectively [9].
1.75 0.006 0.002 0.000 0.000 0.000 0.000 0.000 0.009 0.000
4.50 0.003 0.002 0.002 0.000 0.002 0.00 0.000 0.009 0.004
± ± ± ± ± ± ± ± ± ±
2.4. Statistical analysis
NDI =
4.55 0.004 0.003 0.000 0.000 0.000 0.000 0.000 0.007 0.000
± ± ± ± ± ± ± ± ± ±
Mitotic index Chromatid gap Isochromatid gap Chromatid break Isochromatid break Chromatid acentric fragment Isochromatid acentric fragment Chromatid exchange TCA TCA-(G)
2.12* 0.008* 0.003* 0.001* 0.000* 0.001* 0.000* 0.000* 0.012* 0.007*
N. Cinkilic et al. / Mutation Research 676 (2009) 1–4 Table 3 Frequencies of the SCE and MN in patients with type-1 diabetes mellitus and controls. Controls SCE/Cell MNa NPB/1000 BN cells NBUD/1000 BN cells NDI
2.54 1.53 2.20 0.33 1.81
± ± ± ± ±
Type-1 diabetic patients
0.82 0.64 0.86 0.49 0.10
5.44 1.83 2.43 0.49 1.82
± ± ± ± ±
1.47* 1.15 1.33 0.56 0.10
SCE: sister chromatid exchange, MN: micronuclei, a (superscript): micronuclei per 2000 bi-nucleate cells, NPB: nucleoplasmic bridge, NBUD: nuclear bud, NDI: nuclear division index, BN: bi-nucleate cells, *: p < 0.001; data are expressed as mean ± standard deviation. Table 4 Frequencies of SCE per age groups in patients with type-1 diabetes mellitus and controls. Age groups
SCE/cell in controls (n = 15)
SCE/cell in type-1 diabetic patients (n = 35)
p
<25 years 25–44 years ≥45 years
2.84 ± 0.90 (n = 3) 2.30 ± 0.99 (n = 7) 2.72 ± 0.50 (n = 5)
5.27 ± 1.26 (n = 10) 5.56 ± 1.48 (n = 21) 5.25 ± 2.22 (n = 4)
0.007 0.001 0.016
SCE: sister chromatid exchange, n: number of subjects; data are expressed as mean ± standard deviation.
Compared with controls, patients with type-1 diabetes mellitus had a higher frequency of SCE across all age ranges. Mean HbA1c level was 8.37 ± 1.36% and mean serum fasting glucose was 174.34 ± 64.23 mg/dl in type-1 diabetic patients. Out of the 35 patients with type-1 diabetes mellitus, 11 (31%) had an HbA1c level below 8% and only three (8.5%) had an HbA1c level below 7%. The frequency of SCE was 5.27 ± 1.36 in patients with an HbA1c level <8% while it was 5.43 ± 1.42 in patients with an HbA1c level ≥8%. A comparison of the two groups demonstrated no statistically significant difference in the frequency of SCE (p = 0.619) and no correlation between the frequency of SCE, HbA1c and serum glucose levels. According to the multiple linear regression analysis, when SCE was taken as a dependent variable, the duration of diabetes (r = −0.18, p > 0.05) and age (r = −0.077, p > 0.05) were investigated as independent variables. There was no significant correlation between SCE, duration of diabetes and age. We also compared SCE frequency in diabetic patients with and without smoking habit and did not find any significant difference in the frequency of SCE (5.62 ± 1.77, 5.25 ± 1.09, respectively, p > 0.05). 4. Discussion The detection of CA and MN in peripheral blood lymphocytes is commonly used for monitoring human populations exposed to mutagenic agents and for evaluation of genotoxicity [12]. In recent years, several studies have investigated a number of genotoxicity parameters in relation to health and disease [13]. In the present study, there were no differences in CA and MN frequencies in the lymphocytes of type-1 diabetic patients when compared with controls. However, type-1 diabetics showed a significantly higher SCE frequency than the healthy subjects. Sheth et al. [7] previously investigated the occurrence of genotoxic damage in type-2 diabetic patients as evidenced by SCE and CA tests in lymphocytes. The authors reported a higher occurrence of SCE in patients with type-2 diabetes mellitus, but no CA and no changes in the cell-cycle proliferative index. Our current data confirm and expand previous findings by showing that patients with type-1 diabetes mellitus have increased levels of SCE. The hallmark of type-1 diabetes mellitus is the systemic defect in insulin-dependent metabolism. There is evidence to suggest that ROS may play an important role in the pathogenesis of type-1
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diabetes mellitus [3] and its complications [14,15]. The presence of an increased oxidative stress in diabetic patients has been well documented. Specifically, the hyperglycemic milieu has been suggested to result in an increased production of oxygen-free radicals through glucose auto-oxidation and nonenzymatic glycation of macromolecules, including proteins and DNA [1,16]. Notably, previous studies of oxidative damage in diabetic patients have used several tests, including the comet assay, to investigate DNA damage [4,17–19]. Sardas et al. [4] reported an increase in oxidative DNA damage in the comet assay in both type-1 and type-2 diabetic patients. Moreover, these authors reported that DNA damage as observed in the comet assay was higher in individuals with type-2 diabetes mellitus compared to those with type-1 diabetes. Interestingly, a higher degree of DNA damage was evident in the entire cohort of diabetic patients compared with controls [4]. Under conditions of oxidative stress, damage to cellular biomolecules such as lipids, proteins and DNA occurs. Oxygen-free radicals induce a variety of lesions in DNA, including oxidized bases, abasic sites, DNA strand breaks and formation of cross-links between DNA and proteins [20]. Many of these lesions are cytotoxic and mutagenic. The induction of SCE has been correlated with the primary DNA damage [21]. The SCE assay is an ideal genotoxicity and cytotoxicity assay, since peripheral lymphocytes are easily accessible. Moreover, SCE is much more sensitive as a mutagenic biomarker than CA and MN [22]. Some authors have suggested that ROS may be implicated in the production of high basal SCE frequencies in chromosome-instability syndromes [23]. Oxidative damage to DNA over time can cause changes to both the structure and function of chromosomes. These changes in DNA may lead to cancer and other chronic disease conditions [24,25]. Recently, the genotoxicity of ROS has been well-established [26,27]. In a previous study, the mutagenic effects of ROS were detected in human lymphocytes by use of the SCE technique, and elevated ROS levels were shown to cause an increase in mitotic recombination frequency [28]. We suggest that the increased frequency of SCE in the patients with type-1 diabetes mellitus in this study may be due to the generation of oxygen-derived free radicals occurring in this condition. In conclusion, our current data seem to suggest that an enhanced genomic instability with an increased level of SCE can be a common biochemical feature of patients with type-1 diabetes mellitus. Conflict of interest statement The authors declare that there are no conflicts of interest. References [1] C. Dominguez, E. Ruiz, M. Gussinye, A. Carrascosa, Oxidative stress at onset and in early stages of type 1 diabetes in children and adolescents, Diabetes Care 21 (1998) 1736–1742. [2] G.S. Eisenbarth, Update in type 1 diabetes, J. Clin. Endocrinol. Metab. 92 (2007) 2403–2407. [3] J.W. Baynes, Role of oxidative stress in development of complications in diabetes, Diabetes 40 (1991) 405–412. [4] S. Sardas, M. Yılmaz, U. Oztok, N. C¸akir, A.E. Karakaya, Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation, Mutat. Res. 490 (2001) 123–129. [5] R.K.R. Mantena, O.L.C. Wijburg, C. Vindurampulle, V.R. Bennett-Wood, A. Walduck, G.R. Drummond, J.K. Davies, R.M. Robins-Browne, R.A. Strugnell, Reactive oxygen species are the major antibacterials against Salmonella Typhimurium purine auxotrophs in the phagosome of RAW 264.7 cells, Cell. Microbiol. 10 (5) (2008) 1058–1073. [6] J.D. Tucker, R.J. Preston, Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchange and cancer risk assessment, Mutat. Res. 365 (1996) 147–159. [7] F.J. Sheth, P. Patel, A.D.B. Vaidya, R. Vaidya, J. Sheth, Increased frequency of sister chromatid exchanges in patients with type II diabetes, Curr. Sci. 90 (2006) 236–240.
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