Advances in Medical Sciences 60 (2015) 220–230
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original Research Article
Analysis of oxidative DNA damage and its repair in Polish patients with diabetes mellitus type 2: Role in pathogenesis of diabetic neuropathy Anna Merecz a,*, Lukasz Markiewicz a, Agnieszka Sliwinska a, Marcin Kosmalski b, Jacek Kasznicki b, Jozef Drzewoski b, Ireneusz Majsterek a a b
Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz, Poland Department of Internal Medicine, Diabetology and Clinical Pharmacology, Medical University of Lodz, Lodz, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2014 Accepted 1 April 2015 Available online 13 April 2015
Purpose: Distal symmetric polyneuropathy (DSPN) is common complication of type 2 diabetes (T2DM). In this work we investigated the role of oxidative damage in connection with particular polymorphisms of DNA repair genes and their repair capacity. Material/methods: Materials constitute the peripheral blood of patients with T2DM with and without DSPN and control subjects without disturbance of the carbohydrate fraction. The study of gene polymorphisms which products take part in base excision repair (BER) pathway: 726 Val/Ala adenosine diphosphate ribosyl transferase (ADPRT), 324 His/Glu MutYhomolog (MUTYH) and 148 Asp/Glu human apurinic/apyrimidinic endonuclease (APE) was carried out using restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) method. The study of DNA damage induced by hydrogen peroxide and the efficiency of their repair was carried out using comet assay. Results: None of the 3 polymorphisms were associated with the risk of DSPN. However, in group of patients together with T2DM and T2DM/DSPN 726 Ala ADPRT allele was significantly susceptible to increased risk of T2DM (OR = 1.59; 95% CI: 1.08–2.36). Investigation of DNA damage and repair revealed that T2DM patients have decreased ability to DNA repair. This capacity even drops down in the group of T2DM/DSPN patients compared to subjects with diabetes alone. ADPRT and APE polymorphisms were significantly associated with higher DNA damages (P < 0.05) in heterozygous and mutant homozygous in correlation to homozygous wild type, but for MUTYH polymorphism relation was not confirmed. Conclusions: Pathogenesis of T2DM and development of DSPN may be related to oxidative stress connected with BER gene polymorphisms. ß 2015 Medical University of Bialystok. Published by Elsevier Sp. z o.o. All rights reserved.
Keywords: Diabetes mellitus type 2 Diabetic neuropathy Oxidative stress Oxidative DNA damage Base excision repair
1. Introduction Diabetes mellitus (DM) is considered a civilization disease which affects more than 371 million people in the world [1]. Diabetes mellitus type 2 (T2DM) represents around 90% of all cases of disease and can lead to many complications. One of the most important is diabetic neuropathy which represents around 50% of all patients with T2DM and may affect entire nervous system [2]. This study focuses on patients with distal symmetric polyneuropathy (DSPN). Due to the growing number of DSPN cases
* Corresponding author at: Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Pl. Hallera 1, 90-647 Lodz, Poland. Tel.: +48 042 639 33 06; fax: +48 042 633 11 13. E-mail address: [email protected] (A. Merecz).
it is necessary to understand the pathogenesis and also molecular mechanisms which take part in etiology of that disorder. One of the major hypothesis proposed to explain development of diabetes [3] and its complications [4] including neuropathy is oxidative stress caused by an elevated level of free radicals. Hyperglycemia can induce the production of reactive oxygen species (ROS) through multiple pathways such as mitochondrial superoxide overproduction, elevated polyol pathway flux, increased formation of advanced glycation end products, activation of protein kinase C isoforms and increased hexosamine pathway flux [5]. This oxidative stress is associated with induction of DNA lesions [6] and development of apoptosis in neuron cells [7] but the mechanisms that lead to nervous system damage in diabetes are not fully elucidated. ROS generated DNA damage, such as oxidized bases and strand breaks require repair to maintain genomic integrity. When the lesions have not been removed, the cell can undergo apoptosis or
http://dx.doi.org/10.1016/j.advms.2015.04.001 1896-1126/ß 2015 Medical University of Bialystok. Published by Elsevier Sp. z o.o. All rights reserved.
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
necrosis. In mammalian cells, the base excision repair (BER) pathway is responsible for the repair of oxidative DNA damage. BER has alternative pathways depending on the lesion and protein involved in that process. In the first stage glycosylase, recognizes and cleaves the bond between the sugar and the base, removing the base and creating an apurynic or apyrmidinic site (AP site). Glycosylases may have AP endonuclease and/or AP lyase activity and enable excision DNA and create a single-stranded 50 nick to the AP site and an incision 30 to the AP site. Monofunctional enzymes only demonstrate AP endonuclease activity and require the activity of another protein for incision; bifuncional enzymes perform DNA excision and incision subsequently. Finally, DNA polymerase fills the gap with the correct nucleotide, ligase links the nick and restores the integrity of the helix [8]. Some of the most important proteins involved in this repair pathway are: human apurinic/apyrimidinic endonuclease (APE1), MutYhomolog (MUTYH) and adenosine diphosphate ribosyl transferase (ADPRT). APE1 [9] is a significant multifunctional protein that plays a key role in the repair initiation of the AP site: it is needed for the DNA strand cleavage above the AP site, followed by the creation of a 30 hydroxyl terminus and a 50 -deoxyribose phosphate group flanking the gap. The C-terminus domain is essential for the DNA repair activity. The N-terminal domain has a different function unrelated to DNA repair and is important for the redox activity of APE1. MUTYH [10] is an enzyme of DNA glycosylase activity responsible for removing adenine in the case of 8-hydroxyguanine (8-oxoG) misincorporation or guanine in the case of 1,2-dihydro-2oxoadenine mismatch. The human MutY protein is targeted at both the mitochondria and the nucleus, where it directly interacts with a number of DNA replication and DNA repair proteins, including the DNA sliding clamp PCNA (proliferating cell nuclear antigen), APE1, the ssDNA-binding protein RPA (replication protein A), and the mismatch repair protein 6 (MSH6). The ADPRT [11] gene encodes a zinc-finger DNA-binding protein, the poly(ADP-ribose)polymerase-1 (PARP-1) enzyme involved in the maintenance of genomic stability due to modification of various nuclear proteins by poly(ADP-ribosyl)ation. This process is important for DNA-damage signaling, BER, recombination, genomic stability, and the transcriptional regulation of tumor suppressor genes, such as p53. PARP-1 activation is stimulated by free radicals and oxidants as well as single or double strand breaks. PARP-1 includes three domains, which are responsible for DNA-binding, automodification and catalysis at the COOH terminus. Its action is based on the recognition and binding of DNA single-strand breaks followed by recruitment of other DNA-repair proteins to the damage site. It also serves as an energy source for ligation. A single nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide in a genome is altered. SNPs may cause lower protein activity and impaired ability to fulfill their function [12]. It has been hypothesized that excess ROS production may result in inappropriate BER processes. Genetic
221
polymorphisms in DNA repair genes, which can affect the function of encoded proteins and alter the DNA repair capacity. The present study determines the influence on DNA damage repair capacity of variations of three genetic in polymorphisms in genes whose products take part in the BER repair pathway: APE1 (148 Asp/Glu, rs3136820), MUTYH (324 His/Gln) and ADPRT (762 Val/Ala). The study is based on an analysis of a group of patients with T2DM, with and without coexisting DSPN, as well as subjects with normal glucose metabolism as a reference group. Individuals with reduced effectiveness of repair resulting from the presence of an SNP may exhibit elevated level of ROS, which in turn entail apoptosis in neurons, leading to DM and diabetic neuropathy. In order to explain the role of oxidative stress in the etiology of diabetic neuropathy, the study analyses the association of genetic polymorphisms in the DNA repair genes and the risk of diabetic neuropathy within a Polish population. It assesses the effect of ROS on DNA, the level of DNA damage and repair efficiency in lymphocytes from patients with DM without and with neuropathy in comparison to healthy subjects with respect to occurring polymorphisms. 2. Materials and methods 2.1. Study population The study population consisted of 355 unrelated caucasian subjects residing in the Lodz District, Poland. The subjects were enrolled into three groups, 89 patients with diabetes mellitus type 2 and coexisting diabetic sensory polyneuropathy (T2DM + DSPN), 120 patients with diabetes mellitus type 2 without clinical signs of neuropathy defined as complete lack of symptoms and signs of neuropathy (T2DM), and 146 healthy subjects without T2DM and clinical signs of neuropathy (HS). The inclusion criteria for the experimental groups included a mandatory diagnosis of type 2 diabetes with and without symptoms and signs of neuropathy. The diagnosis of T2DM was based on the American Diabetes Association definition of diabetes [13]. The exclusion criteria included lower limb amputation, psychiatric disorders, cancer or any genetic disease, terminal illness, evidence of peripheral arterial disease, claudication symptoms, alcohol abuse, thyroid disorders, vitamin B12 or folate deficiency, spondyloarthropathy, foot edema, hepatic disease, lumbosacral pathology, toxin exposure including chemotherapeutic agents, diagnosis of neuromuscular disorders, medical or surgical intervention for peripheral nerve pathology, inability to understand or provide inform consent. The control subjects had no known diagnosis of impaired glucose metabolism and neuropathy. All groups were matched for sex, age and diabetic groups were matched for the duration of type 2 diabetes. Characteristic of T2DM patients and controls is given in Table 1. All subjects were recruited from the Department of Internal Disease, Diabetology and Clinical Pharmacology between 2009 and 2012. Medical history was obtained from all subjects, and no one reported present or former
Table 1 Clinical and laboratory characteristics of type 2 diabetes patients (T2DM), diabetic patients with neuropathy (T2DM + DSPN) and healthy control subjects (HS). Parameter
T2DM n = 120
T2DM + DSPN n = 89
HS n = 146
P
Sex (M/F) Age (years) Weight (kg) BMI (kg/m2) A1C (%) GFR (ml/min/1.72 m2)
67/53 63.94 11.83 82.25 18.08 30.47 5.76 9.44 1.68 87.13 31.42
42/47 65.69 11.07 81.68 27.96 28.98 10.35 9.15 1.76 86.81 49.30
77/69 65.11 14.47 81.08 25.27 28.77 6.77 5.50 0.31 84.42 23.14
NS NS NS NS * 0.05). In the case of the ADPRT gene, significant differences between levels of DNA damage were found for ADPRT Val/Ala and Ala/Ala (P < 0.05) compared with the Val/Val genotype in each group of people (HS, T2DM and T2DM + DSPN) for both H2O2 concentrations in both the absence and presence of the Fpg enzyme. The repair capacity of DNA damage was lower in patients (T2DM and T2DM + DSPN) and HS with Val/Ala and Ala/Ala genotype in comparison with Val/Val genotype in the absence and presence of the Fpg enzyme depending on genotype (P < 0.05).
Fig. 4. DNA damage before (I) and after 120 min of repair incubation (II) in lymphocyte in the controls (A), type 2 diabetes (B) and neuropathy (C) patients (n = 36) with respect to the MUTYH genotype. DNA damage (oxidative lesions and strand breakage) was induced by Fpg enzyme and hydrogen peroxide at concentrations of 10 and 20 mM after 10 min incubation at 4 8C and measured as a percentage of the tail DNA in the alkaline comet assay. Data is expressed as mean SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 as compared with homozygous wild type.
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
227
Fig. 5. DNA damage before (I) and after 120 min of repair incubation (II) in lymphocyte in the controls (A), type 2 diabetes (B) and neuropathy (C) patients (n = 36) with respect to the ADPRT genotype. DNA damage (strand breakage) was induced by hydrogen peroxide at concentrations of 10 and 20 mM after 10 min incubation at 4 8C and measured as a percentage of the tail DNA in the alkaline comet assay. Data is expressed as mean SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 as compared with homozygous wild type.
No significant relationship was found between DNA damage values and MUYTH polymorphism distribution in groups subjected to H2O2 application in either the absence or presence of the Fpg enzyme (P > 0.05). In addition, no differences were noted regarding the efficiency of repair in the absence of the Fpg enzyme (P > 0.05) apart from capacity of repair in the present of Fpg enzyme in patients (T2DM and T2DM + DSPN) (P < 0.05). Normal healthy subjects exhibited lower levels of both DNA strand breaks and FPG-sensitive oxidative DNA damage than diabetics. Moreover in T2DM + DSPN patients demonstrated more DNA strand breaks, than the T2DM group. Lower DNA repair efficiency was also observed in the T2DM and T2DM + DSPN groups than in the HS group. 4. Discussion Several lines of evidence indicate that susceptibility to the development of diabetic complications is partly under the control of genetic factors. Therefore analysis of BER gene polymorphisms in a case-control study may represent a valuable opportunity to measure the impact of candidate genes on the development of DSPN, particularly with regard to the role of oxidative stress as a potential mediator of diabetes complications. It will allow identification of the factors involved in neuron death and enable more effective treatment of this disease. Effective repair of DNA damage is based on the coordinated action of many proteins. Any imbalance in their activities will result in the accumulation of oxidative damage and mutation to DNA in the aging tissues. The present study attempts to identify
any association between three gene polymorphisms, 726 Val/Ala ADPRT, 324 His/Glu MUTYH and 148 Asp/Glu APE1, and the presence of diabetes mellitus or diabetic neuropathy. APE1 is a key protein playing a significant role in the processing of abasic sites. Several polymorphisms in the APE gene have been reported [25]. The impact of some polymorphisms on enzyme function has not yet been determined, but the most extensively studied polymorphism is a 148 Asp/Glu (rs3136820). Studies suggest that this polymorphism might be associated with, inter alia, increased risk of lung cancer in Asian population and smokers [26], apoptosis and ulcerative colitis in India population [27], colorectal cancer in a Turkish population [28] and colorectal cancer when accompanied by smoking exposure in Japanese population [29]. It has also been associated with hypersensitivity to ionizing radiation [30]. In human cells, the glycosylase activity of MUTYH is involved in the repair of inappropriate nucleotide mismatch. Very little is known of polymorphism 324 His/Glu however, it appears to play an important role in modifying the risk for lung cancer [31] and colorectal cancer [29] in the Japanese population. ADPRT has been proposed to play a role in DNA repair, recombination, transcription, cell death, cell proliferation and stabilization of the genome. Findings suggest that the levels of poly(ADP-ribosyl)ation by PARP1 might be associated with the cancer risk borne by carriers of the PARP1 Val762Ala polymorphism. The ADPRT Ala762Val polymorphism may, for instance, play a role in the etiology of squamous cell carcinoma of the head and neck [32], and the development of melanoma [33] in Caucasians patients, but may also be involved in the etiology of
228
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
Fig. 6. DNA damage before (I) and after 120 min of repair incubation (II) in lymphocyte in the controls (A), type 2 diabetes (B) and neuropathy (C) patients (n = 36) with respect to the ADPRT genotype. DNA damage (oxidative lesions and strand breakage) was induced by Fpg enzyme and hydrogen peroxide at concentrations of 10 and 20 mM after 10 min. incubation at 4 8C and measured as a percentage of the tail DNA in the alkaline comet assay. Data is expressed as means SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 as compared with homozygous wild type.
breast cancer [34], gastric cancer [35], susceptibility to Alzheimer’s disease [36], and cervical carcinoma [37] in a Chinese population. The presence of polymorphisms is not associated with the risk of T2DM + DSPN. The relationship between the genotypes analyzed in the present study and the susceptibility to diabetes and diabetic neuropathy has not been investigated previously. Our findings indicate that SNP 726 Val/Ala in the ADPRT gene may influence its function and represent one cause of T2DM development. Our results suggest also that the polymorphic variants of this BER gene are not independent risk factors for DSPN. Similarly, no connection has been identified between the other analyzed polymorphisms and the risk of T2DM or DSPN. The efficacy of DNA repair, measured as relative decrease in DNA damage was found to be significantly lower in the patients groups (T2DM and T2DM + DSPN) was significantly lower than in the controls (HS). Lymphocytes from diabetics showed a significantly higher level of DNA damage in comparison with nondiabetics and there were consistent across the dose range. A similar situation was observed regarding the use of specific repair endonucleases which allow DNA lesions to be converted into single strand breaks. A strong connection was found between the ADPRT and APE genes with regard to genotype distribution and DNA damage. The APE variant 148 Asp/Glu genotype displayed significantly higher DNA damage and lower efficiency of repair (P < 0.05) than the APE wild-type genotype. A similar situation could be observed in the case of the ADPRT gene, irrespective of the concentration of H2O2or the presence of the Fpg enzyme: more DNA lesions were found in Val/Ala and Ala/Ala genes than
wild-type Val/Val. For the MUTYH genotypes, the only significant difference observed between the Gln/His and His/His genotypes with the wild-type Gln/Gln genotype was that their DNA repair ability was found to be less effective in the T2DM and T2DM + DSPN patient groups in the presence of the Fpg enzyme. The analysis of the ADPRT polymorphism indicates that the presence of SNP correlates with a significant risk of T2DM, a higher degree of DNA damage and lower capacity of DNA repair in each group of patients and in controls. Our results suggest that the presence of a polymorphism in the ADPRT gene may influence protein action. This is in accordance with literature data, which indicates that this amino acid substitution is responsible for reduced ADPRT activity [38]. Poly-ADP-ribosylation is an important post-transcriptional modification catalyzed by various enzymes, including PARP which uses NAD+ as a substrate for the synthesis and transport of ADPribose units to proteins acceptor. PARP family members have a variety of structural domains, a wide range of functions and are located in different cellular compartments. Among the actions attributed to PARPs are its role in the recognition of DNA damage [39]. Single-stranded DNA breaks occur directly as a part of BER, or indirectly through deoxyribose damage. Polymorphisms in that gene may interfere with the ability of cells to repair damaged DNA. The examination of the APE polymorphism reveals that the Asp/ Glu genotype is associated with greater DNA damage and lower capacity of DNA repair then the Asp/Asp wild-type, apart from in control group, where no difference in DNA repair was noted between genotypes after 120 min. However, previous studies note
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
that this SNP does affect neither the AP endonuclease nor the DNA binding activity of the APE protein [40]. In addition, no connection has been observed between the presence of an SNP and significantly greater risk of T2DM. Hence, it may be possible that this SNP does not change protein activity but contributes to less efficient repair in patients with T2DM. This problem requires additional study. Although the disease process of DSPN is relatively well described, the mechanisms underlying nerve degeneration in diabetic subjects are still poorly understood. A growing consensus indicates that they may be multifactorial. Nerve damage is probably due to a combination of factors: metabolic, neurovascular, autoimmune, mechanical injuries to nerves, inherited traits and life style factors [41]. 5. Conclusions Our results show a prognostic importance for the 726 Val/Ala ADPRT polymorphism in the development of T2DM. There is also a connection between the Val/Ala and Ala/Ala genotypes and the wild-type genotype with regard to degree of DNA damage and efficiency of repair. ADPRT takes part in the BER pathway, which is responsible for removing bases damaged by oxidative stress or single strand breaks. Impaired repair may be responsible for the accumulation of reactive molecules, thus contributing to insulin resistance and decreased insulin secretion in the etiology of T2DM [42] and later complications including neuropathy. Our results confirm also that patients with type 2 diabetes demonstrate greater oxidative DNA damage and impaired DNA repair ability compared to healthy subjects. This is more marked in patients with neuropathy, and it is possible that they are more sensitive to oxidative stress. Our findings indicate that oxidative DNA damage level can be considered as a valuable prognostic marker for T2DM progression. Conflict of interests None declared. Financial disclosure This work was supported by grant N N402 375838 from the Polish Ministry of Science and Higher Education and by grant 50203/5-108-05/502-54-092 from the University of Lodz. References [1] International Diabetes Federation (IDF). Diabetes atlas, 5th ed. Available from: http://www.idf.org/sites/default/files/5E_IDFAtlasPoster_2012_EN.pdf [cited 09.06.14]. [2] WHO. Definition, diagnosis and classification of diabetes mellitus and its complications. Report of WHO consultation. Part 1. Diagnosis and classification of diabetes mellitus. Available from: http://whqlibdoc.who.int/hq/1999/ who_ncd_ncs_99.2.pdf [cited 09.06.14]. [3] Evans JL, Goldfine ID, Maddux BA, Grodsky GM. Are oxidative stress-activated signaling pathways mediators of insulin resistance and beta-cell dysfunction? Diabetes 2003;52(January (1)):1–8. [4] Niedowicz DM, Daleke DL. The role of oxidative stress in diabetic complications. Cell Biochem Biophys 2005;43(2):289–330. [5] Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414(December (6865)):813–20. [6] Dizdaroglu M. Oxidatively induced DNA damage: mechanisms, repair and disease. Cancer Lett 2012;327(December (1–2)):26–47. [7] Greene DA, Stevens MJ, Obrosova I, Feldman EL. Glucose-induced oxidative stress and programmed cell death in diabetic neuropathy. Eur J Pharmacol 1999;375(June (1–3)):217–23. [8] Robertson AB, Klungland A, Rognes T, Leiros I. DNA repair in mammalian cells: base excision repair: the long and short of it. Cell Mol Life Sci 2009;66(March (6)):981–93. [9] Fritz G, Gro¨sch S, Tomicic M, Kaina B. APE/Ref-1 and the mammalian response to genotoxic stress. Toxicology 2003;193(November (1–2)):67–78.
229
[10] Parker AR, Eshleman JR. Human MutY: gene structure, protein functions and interactions, and role in carcinogenesis. Cell Mol Life Sci 2003;60(October (10)):2064–83. [11] Gibson BA, Kraus WL. New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs. Nat Rev Mol Cell Biol 2012;13(June (7)):411–24. [12] Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res 2002;12(March (3)):436–46. [13] American Diabetes Association. Standards of medical care in diabetes—2013. Diabetes Care 2013;36(January (Suppl. 1)):S11–66. [14] England JD, Gronseth GS, Franklin G, Miller RG, Asbury AK, Carter GT, et al. Distal symmetric polyneuropathy: a definition for clinical research. Report of the Amercian Academy of Neurology, the American Association of Electrodiagnostic Medicine, and American Academy of Physical Medicine and Rehabilitation. Neurology 2005;64(January (2)):199–207. [15] Tesfaye S, Boulton AJ, Dyck PJ, Freeman R, Horowitz M, Kempler P, et al. Diabetic neuropathies: update on definitions, diagnostic criteria, estimation of severity, and treatments. Diabetes Care 2010;33(October (10)):2285–93. [16] Rader AJ, Barry TP, Stanley OL. Characteristics of lower extremity pressure sensation impairment in developing diabetic sensory polyneuropathy. Foot Ankle Spec 2008;1(February (1)):39–45. [17] Feldman EL, Stevens MJ, Thomas PK, Brown MB, Canal N, Greene DA. A practical two-step quantitative clinical and electrophysiological assessment for the diagnosis and staging of diabetic neuropathy. Diabetes Care 1994;17(November (11)):1281–9. [18] Abbott CA, Carrington AL, Ashe H, Bath S, Every LC, Griffiths J, et al. The NorthWest Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort. Diabet Med 2002;19(May (5)):377–84. [19] Boulton AJM, Malik RA, Arezzo JC, Sosenko JM. Diabetic somatic neuropathies. Diabetes Care 2004;27(June (6)):1458–86. [20] Meijer JW, Smit AJ, Lefrandt JD, van der Hoeven JH, Hoogenberg K, Links TP. Back to basics in diagnosing diabetic polyneuropathy with the tuning fork. Diabetes Care 2005;28(September (9)):2201–5. [21] Hilz MJ, Axelrod FB, Hermann K, Haertl U, Duetsch M, Neundo¨rfer B. Normative values of vibratory perception in 530 children, juveniles and adults aged 3–79 years. J Neurol Sci 1998;159(August (2)):219–25. [22] American College of Physicians. Available from: http://diabetes.acponline.org/ [cited 09.06.14]. [23] Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175(March (1)):184–91. [24] Klaude M, Eriksson S, Nygren J, Ahnstro¨m G. The comet assay: mechanisms and technical considerations. Mutat Res 1996;363(June (2)):89–96. [25] Xi T, Jones IM, Mohrenweiser HW. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function. Genomics 2004;83(June (6)):970–9. [26] Wei W, He XF, Qin JB, Su J, Li SX, Liu Y, et al. Association between the OGG1 Ser326Cys and APEX1 Asp148Glu polymorphisms and lung cancer risk: a meta-analysis. Mol Biol Rep 2012;39(December (12)):11249–62. [27] Bardia A, Tiwari SK, Gunisetty S, Anjum F, Nallari P, Habeeb MA, et al. Functional polymorphisms in XRCC-1 and APE-1 contribute to increased apoptosis and risk of ulcerative colitis. Inflamm Res 2012;61(April (4)): 359–65. [28] Canbay E, Cakmakoglu B, Zeybek U, Sozen S, Cacina C, Gulluoglu M, et al. Association of APE1 and hOGG1 polymorphisms with colorectal cancer risk in a Turkish population. Curr Med Res Opin 2011;27(July (7)):1295–302. [29] Kasahara M, Osawa K, Yoshida K, Miyaishi A, Osawa Y, Inoue N, et al. Association of MUTYH Gln324His and APEX1 Asp148Glu with colorectal cancer and smoking in a Japanese population. J Exp Clin Cancer Res 2008;27:49. [30] Hu JJ, Smith TR, Miller MS, Mohrenweiser HW, Golden A, Case LD. Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity. Carcinogenesis 2001;22(June (6)):917–22. [31] Miyaishi A, Osawa K, Osawa Y, Inoue N, Yoshida K, Kasahara M, et al. MUTYH Gln324His gene polymorphism and genetic susceptibility for lung cancer in a Japanese population. J Exp Clin Cancer Res 2009;28:10. [32] Li C, Hu Z, Lu J, Liu Z, Wang LE, El-Naggar AK, et al. Genetic polymorphisms in DNA base-excision repair genes ADPRT, XRCC1, and APE1 and the risk of squamous cell carcinoma of the head and neck. Cancer 2007;110(August (4)):867–75. [33] Zhang M, Qureshi AA, Guo Q, Han J. Genetic variation in DNA repair pathway genes and melanoma risk. DNA Repair (Amst) 2011;10(January (1)):111–6. [34] Zhai X, Liu J, Hu Z, Wang S, Qing J, Wang X, et al. Polymorphisms of ADPRT Val762Ala and XRCC1 Arg399Glu and risk of breast cancer in Chinese women: a case control analysis. Oncol Rep 2006;15(January (1)):247–52. [35] Zhang Z, Miao XP, Tan W, Guo YL, Zhang XM, Lin DX. Correlation of genetic polymorphisms in DNA repair genes ADPRT and XRCC1 to risk of gastric cancer. Ai Zheng 2006;25(January (1)):7–10. [36] Liu HP, Lin WY, Wu BT, Liu SH, Wang WF, Tsai CH, et al. Evaluation of the poly(ADP-ribose) polymerase-1 gene variants in Alzheimer’s disease. J Clin Lab Anal 2010;24(3):182–6. [37] Ye F, Cheng Q, Hu Y, Zhang J, Chen H. PARP-1 val762ala polymorphism is associated with risk of cervical carcinoma. PLoS ONE 2012;7(5). http:// dx.doi.org/10.1371/journal.pone.0037446. [38] Wang XG, Wang ZQ, Tong WM, Shen Y. PARP1 Val762Ala polymorphism reduces enzymatic activity. Biochem Biophys Res Commun 2007;354(March (1)):122–6.
230
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
[39] Sousa FG, Matuo R, Soares DG, Escargueil AE, Henriques JA, Larsen AK, et al. PARPs and the DNA damage response. Carcinogenesis 2012;33(August (8)): 1433–40. [40] Hadi MZ, Coleman MA, Fidelis K, Mohrenweiser HW, Wilson DM. Functional characterization of Ape1 variants identified in the human population. Nucleic Acids Res 2000;28(October (20)):3871–9.
[41] Diabetic neuropathies: the nerve damage of diabetes, national diabetes information clearinghouse. Available from: http://diabetes.niddk.nih.gov/dm/ pubs/neuropathies/neuropathies.pdf [cited 09.06.14]. [42] Bloch-Damti A, Bashan N. Proposed mechanisms for the induction of insulin resistance by oxidative stress. Antioxid Redox Signal 2005;7(November–December (11–12)):1553–67.
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original Research Article
Analysis of oxidative DNA damage and its repair in Polish patients with diabetes mellitus type 2: Role in pathogenesis of diabetic neuropathy Anna Merecz a,*, Lukasz Markiewicz a, Agnieszka Sliwinska a, Marcin Kosmalski b, Jacek Kasznicki b, Jozef Drzewoski b, Ireneusz Majsterek a a b
Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz, Poland Department of Internal Medicine, Diabetology and Clinical Pharmacology, Medical University of Lodz, Lodz, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2014 Accepted 1 April 2015 Available online 13 April 2015
Purpose: Distal symmetric polyneuropathy (DSPN) is common complication of type 2 diabetes (T2DM). In this work we investigated the role of oxidative damage in connection with particular polymorphisms of DNA repair genes and their repair capacity. Material/methods: Materials constitute the peripheral blood of patients with T2DM with and without DSPN and control subjects without disturbance of the carbohydrate fraction. The study of gene polymorphisms which products take part in base excision repair (BER) pathway: 726 Val/Ala adenosine diphosphate ribosyl transferase (ADPRT), 324 His/Glu MutYhomolog (MUTYH) and 148 Asp/Glu human apurinic/apyrimidinic endonuclease (APE) was carried out using restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) method. The study of DNA damage induced by hydrogen peroxide and the efficiency of their repair was carried out using comet assay. Results: None of the 3 polymorphisms were associated with the risk of DSPN. However, in group of patients together with T2DM and T2DM/DSPN 726 Ala ADPRT allele was significantly susceptible to increased risk of T2DM (OR = 1.59; 95% CI: 1.08–2.36). Investigation of DNA damage and repair revealed that T2DM patients have decreased ability to DNA repair. This capacity even drops down in the group of T2DM/DSPN patients compared to subjects with diabetes alone. ADPRT and APE polymorphisms were significantly associated with higher DNA damages (P < 0.05) in heterozygous and mutant homozygous in correlation to homozygous wild type, but for MUTYH polymorphism relation was not confirmed. Conclusions: Pathogenesis of T2DM and development of DSPN may be related to oxidative stress connected with BER gene polymorphisms. ß 2015 Medical University of Bialystok. Published by Elsevier Sp. z o.o. All rights reserved.
Keywords: Diabetes mellitus type 2 Diabetic neuropathy Oxidative stress Oxidative DNA damage Base excision repair
1. Introduction Diabetes mellitus (DM) is considered a civilization disease which affects more than 371 million people in the world [1]. Diabetes mellitus type 2 (T2DM) represents around 90% of all cases of disease and can lead to many complications. One of the most important is diabetic neuropathy which represents around 50% of all patients with T2DM and may affect entire nervous system [2]. This study focuses on patients with distal symmetric polyneuropathy (DSPN). Due to the growing number of DSPN cases
* Corresponding author at: Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Pl. Hallera 1, 90-647 Lodz, Poland. Tel.: +48 042 639 33 06; fax: +48 042 633 11 13. E-mail address: [email protected] (A. Merecz).
it is necessary to understand the pathogenesis and also molecular mechanisms which take part in etiology of that disorder. One of the major hypothesis proposed to explain development of diabetes [3] and its complications [4] including neuropathy is oxidative stress caused by an elevated level of free radicals. Hyperglycemia can induce the production of reactive oxygen species (ROS) through multiple pathways such as mitochondrial superoxide overproduction, elevated polyol pathway flux, increased formation of advanced glycation end products, activation of protein kinase C isoforms and increased hexosamine pathway flux [5]. This oxidative stress is associated with induction of DNA lesions [6] and development of apoptosis in neuron cells [7] but the mechanisms that lead to nervous system damage in diabetes are not fully elucidated. ROS generated DNA damage, such as oxidized bases and strand breaks require repair to maintain genomic integrity. When the lesions have not been removed, the cell can undergo apoptosis or
http://dx.doi.org/10.1016/j.advms.2015.04.001 1896-1126/ß 2015 Medical University of Bialystok. Published by Elsevier Sp. z o.o. All rights reserved.
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
necrosis. In mammalian cells, the base excision repair (BER) pathway is responsible for the repair of oxidative DNA damage. BER has alternative pathways depending on the lesion and protein involved in that process. In the first stage glycosylase, recognizes and cleaves the bond between the sugar and the base, removing the base and creating an apurynic or apyrmidinic site (AP site). Glycosylases may have AP endonuclease and/or AP lyase activity and enable excision DNA and create a single-stranded 50 nick to the AP site and an incision 30 to the AP site. Monofunctional enzymes only demonstrate AP endonuclease activity and require the activity of another protein for incision; bifuncional enzymes perform DNA excision and incision subsequently. Finally, DNA polymerase fills the gap with the correct nucleotide, ligase links the nick and restores the integrity of the helix [8]. Some of the most important proteins involved in this repair pathway are: human apurinic/apyrimidinic endonuclease (APE1), MutYhomolog (MUTYH) and adenosine diphosphate ribosyl transferase (ADPRT). APE1 [9] is a significant multifunctional protein that plays a key role in the repair initiation of the AP site: it is needed for the DNA strand cleavage above the AP site, followed by the creation of a 30 hydroxyl terminus and a 50 -deoxyribose phosphate group flanking the gap. The C-terminus domain is essential for the DNA repair activity. The N-terminal domain has a different function unrelated to DNA repair and is important for the redox activity of APE1. MUTYH [10] is an enzyme of DNA glycosylase activity responsible for removing adenine in the case of 8-hydroxyguanine (8-oxoG) misincorporation or guanine in the case of 1,2-dihydro-2oxoadenine mismatch. The human MutY protein is targeted at both the mitochondria and the nucleus, where it directly interacts with a number of DNA replication and DNA repair proteins, including the DNA sliding clamp PCNA (proliferating cell nuclear antigen), APE1, the ssDNA-binding protein RPA (replication protein A), and the mismatch repair protein 6 (MSH6). The ADPRT [11] gene encodes a zinc-finger DNA-binding protein, the poly(ADP-ribose)polymerase-1 (PARP-1) enzyme involved in the maintenance of genomic stability due to modification of various nuclear proteins by poly(ADP-ribosyl)ation. This process is important for DNA-damage signaling, BER, recombination, genomic stability, and the transcriptional regulation of tumor suppressor genes, such as p53. PARP-1 activation is stimulated by free radicals and oxidants as well as single or double strand breaks. PARP-1 includes three domains, which are responsible for DNA-binding, automodification and catalysis at the COOH terminus. Its action is based on the recognition and binding of DNA single-strand breaks followed by recruitment of other DNA-repair proteins to the damage site. It also serves as an energy source for ligation. A single nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide in a genome is altered. SNPs may cause lower protein activity and impaired ability to fulfill their function [12]. It has been hypothesized that excess ROS production may result in inappropriate BER processes. Genetic
221
polymorphisms in DNA repair genes, which can affect the function of encoded proteins and alter the DNA repair capacity. The present study determines the influence on DNA damage repair capacity of variations of three genetic in polymorphisms in genes whose products take part in the BER repair pathway: APE1 (148 Asp/Glu, rs3136820), MUTYH (324 His/Gln) and ADPRT (762 Val/Ala). The study is based on an analysis of a group of patients with T2DM, with and without coexisting DSPN, as well as subjects with normal glucose metabolism as a reference group. Individuals with reduced effectiveness of repair resulting from the presence of an SNP may exhibit elevated level of ROS, which in turn entail apoptosis in neurons, leading to DM and diabetic neuropathy. In order to explain the role of oxidative stress in the etiology of diabetic neuropathy, the study analyses the association of genetic polymorphisms in the DNA repair genes and the risk of diabetic neuropathy within a Polish population. It assesses the effect of ROS on DNA, the level of DNA damage and repair efficiency in lymphocytes from patients with DM without and with neuropathy in comparison to healthy subjects with respect to occurring polymorphisms. 2. Materials and methods 2.1. Study population The study population consisted of 355 unrelated caucasian subjects residing in the Lodz District, Poland. The subjects were enrolled into three groups, 89 patients with diabetes mellitus type 2 and coexisting diabetic sensory polyneuropathy (T2DM + DSPN), 120 patients with diabetes mellitus type 2 without clinical signs of neuropathy defined as complete lack of symptoms and signs of neuropathy (T2DM), and 146 healthy subjects without T2DM and clinical signs of neuropathy (HS). The inclusion criteria for the experimental groups included a mandatory diagnosis of type 2 diabetes with and without symptoms and signs of neuropathy. The diagnosis of T2DM was based on the American Diabetes Association definition of diabetes [13]. The exclusion criteria included lower limb amputation, psychiatric disorders, cancer or any genetic disease, terminal illness, evidence of peripheral arterial disease, claudication symptoms, alcohol abuse, thyroid disorders, vitamin B12 or folate deficiency, spondyloarthropathy, foot edema, hepatic disease, lumbosacral pathology, toxin exposure including chemotherapeutic agents, diagnosis of neuromuscular disorders, medical or surgical intervention for peripheral nerve pathology, inability to understand or provide inform consent. The control subjects had no known diagnosis of impaired glucose metabolism and neuropathy. All groups were matched for sex, age and diabetic groups were matched for the duration of type 2 diabetes. Characteristic of T2DM patients and controls is given in Table 1. All subjects were recruited from the Department of Internal Disease, Diabetology and Clinical Pharmacology between 2009 and 2012. Medical history was obtained from all subjects, and no one reported present or former
Table 1 Clinical and laboratory characteristics of type 2 diabetes patients (T2DM), diabetic patients with neuropathy (T2DM + DSPN) and healthy control subjects (HS). Parameter
T2DM n = 120
T2DM + DSPN n = 89
HS n = 146
P
Sex (M/F) Age (years) Weight (kg) BMI (kg/m2) A1C (%) GFR (ml/min/1.72 m2)
67/53 63.94 11.83 82.25 18.08 30.47 5.76 9.44 1.68 87.13 31.42
42/47 65.69 11.07 81.68 27.96 28.98 10.35 9.15 1.76 86.81 49.30
77/69 65.11 14.47 81.08 25.27 28.77 6.77 5.50 0.31 84.42 23.14
NS NS NS NS * 0.05). In the case of the ADPRT gene, significant differences between levels of DNA damage were found for ADPRT Val/Ala and Ala/Ala (P < 0.05) compared with the Val/Val genotype in each group of people (HS, T2DM and T2DM + DSPN) for both H2O2 concentrations in both the absence and presence of the Fpg enzyme. The repair capacity of DNA damage was lower in patients (T2DM and T2DM + DSPN) and HS with Val/Ala and Ala/Ala genotype in comparison with Val/Val genotype in the absence and presence of the Fpg enzyme depending on genotype (P < 0.05).
Fig. 4. DNA damage before (I) and after 120 min of repair incubation (II) in lymphocyte in the controls (A), type 2 diabetes (B) and neuropathy (C) patients (n = 36) with respect to the MUTYH genotype. DNA damage (oxidative lesions and strand breakage) was induced by Fpg enzyme and hydrogen peroxide at concentrations of 10 and 20 mM after 10 min incubation at 4 8C and measured as a percentage of the tail DNA in the alkaline comet assay. Data is expressed as mean SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 as compared with homozygous wild type.
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
227
Fig. 5. DNA damage before (I) and after 120 min of repair incubation (II) in lymphocyte in the controls (A), type 2 diabetes (B) and neuropathy (C) patients (n = 36) with respect to the ADPRT genotype. DNA damage (strand breakage) was induced by hydrogen peroxide at concentrations of 10 and 20 mM after 10 min incubation at 4 8C and measured as a percentage of the tail DNA in the alkaline comet assay. Data is expressed as mean SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 as compared with homozygous wild type.
No significant relationship was found between DNA damage values and MUYTH polymorphism distribution in groups subjected to H2O2 application in either the absence or presence of the Fpg enzyme (P > 0.05). In addition, no differences were noted regarding the efficiency of repair in the absence of the Fpg enzyme (P > 0.05) apart from capacity of repair in the present of Fpg enzyme in patients (T2DM and T2DM + DSPN) (P < 0.05). Normal healthy subjects exhibited lower levels of both DNA strand breaks and FPG-sensitive oxidative DNA damage than diabetics. Moreover in T2DM + DSPN patients demonstrated more DNA strand breaks, than the T2DM group. Lower DNA repair efficiency was also observed in the T2DM and T2DM + DSPN groups than in the HS group. 4. Discussion Several lines of evidence indicate that susceptibility to the development of diabetic complications is partly under the control of genetic factors. Therefore analysis of BER gene polymorphisms in a case-control study may represent a valuable opportunity to measure the impact of candidate genes on the development of DSPN, particularly with regard to the role of oxidative stress as a potential mediator of diabetes complications. It will allow identification of the factors involved in neuron death and enable more effective treatment of this disease. Effective repair of DNA damage is based on the coordinated action of many proteins. Any imbalance in their activities will result in the accumulation of oxidative damage and mutation to DNA in the aging tissues. The present study attempts to identify
any association between three gene polymorphisms, 726 Val/Ala ADPRT, 324 His/Glu MUTYH and 148 Asp/Glu APE1, and the presence of diabetes mellitus or diabetic neuropathy. APE1 is a key protein playing a significant role in the processing of abasic sites. Several polymorphisms in the APE gene have been reported [25]. The impact of some polymorphisms on enzyme function has not yet been determined, but the most extensively studied polymorphism is a 148 Asp/Glu (rs3136820). Studies suggest that this polymorphism might be associated with, inter alia, increased risk of lung cancer in Asian population and smokers [26], apoptosis and ulcerative colitis in India population [27], colorectal cancer in a Turkish population [28] and colorectal cancer when accompanied by smoking exposure in Japanese population [29]. It has also been associated with hypersensitivity to ionizing radiation [30]. In human cells, the glycosylase activity of MUTYH is involved in the repair of inappropriate nucleotide mismatch. Very little is known of polymorphism 324 His/Glu however, it appears to play an important role in modifying the risk for lung cancer [31] and colorectal cancer [29] in the Japanese population. ADPRT has been proposed to play a role in DNA repair, recombination, transcription, cell death, cell proliferation and stabilization of the genome. Findings suggest that the levels of poly(ADP-ribosyl)ation by PARP1 might be associated with the cancer risk borne by carriers of the PARP1 Val762Ala polymorphism. The ADPRT Ala762Val polymorphism may, for instance, play a role in the etiology of squamous cell carcinoma of the head and neck [32], and the development of melanoma [33] in Caucasians patients, but may also be involved in the etiology of
228
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
Fig. 6. DNA damage before (I) and after 120 min of repair incubation (II) in lymphocyte in the controls (A), type 2 diabetes (B) and neuropathy (C) patients (n = 36) with respect to the ADPRT genotype. DNA damage (oxidative lesions and strand breakage) was induced by Fpg enzyme and hydrogen peroxide at concentrations of 10 and 20 mM after 10 min. incubation at 4 8C and measured as a percentage of the tail DNA in the alkaline comet assay. Data is expressed as means SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 as compared with homozygous wild type.
breast cancer [34], gastric cancer [35], susceptibility to Alzheimer’s disease [36], and cervical carcinoma [37] in a Chinese population. The presence of polymorphisms is not associated with the risk of T2DM + DSPN. The relationship between the genotypes analyzed in the present study and the susceptibility to diabetes and diabetic neuropathy has not been investigated previously. Our findings indicate that SNP 726 Val/Ala in the ADPRT gene may influence its function and represent one cause of T2DM development. Our results suggest also that the polymorphic variants of this BER gene are not independent risk factors for DSPN. Similarly, no connection has been identified between the other analyzed polymorphisms and the risk of T2DM or DSPN. The efficacy of DNA repair, measured as relative decrease in DNA damage was found to be significantly lower in the patients groups (T2DM and T2DM + DSPN) was significantly lower than in the controls (HS). Lymphocytes from diabetics showed a significantly higher level of DNA damage in comparison with nondiabetics and there were consistent across the dose range. A similar situation was observed regarding the use of specific repair endonucleases which allow DNA lesions to be converted into single strand breaks. A strong connection was found between the ADPRT and APE genes with regard to genotype distribution and DNA damage. The APE variant 148 Asp/Glu genotype displayed significantly higher DNA damage and lower efficiency of repair (P < 0.05) than the APE wild-type genotype. A similar situation could be observed in the case of the ADPRT gene, irrespective of the concentration of H2O2or the presence of the Fpg enzyme: more DNA lesions were found in Val/Ala and Ala/Ala genes than
wild-type Val/Val. For the MUTYH genotypes, the only significant difference observed between the Gln/His and His/His genotypes with the wild-type Gln/Gln genotype was that their DNA repair ability was found to be less effective in the T2DM and T2DM + DSPN patient groups in the presence of the Fpg enzyme. The analysis of the ADPRT polymorphism indicates that the presence of SNP correlates with a significant risk of T2DM, a higher degree of DNA damage and lower capacity of DNA repair in each group of patients and in controls. Our results suggest that the presence of a polymorphism in the ADPRT gene may influence protein action. This is in accordance with literature data, which indicates that this amino acid substitution is responsible for reduced ADPRT activity [38]. Poly-ADP-ribosylation is an important post-transcriptional modification catalyzed by various enzymes, including PARP which uses NAD+ as a substrate for the synthesis and transport of ADPribose units to proteins acceptor. PARP family members have a variety of structural domains, a wide range of functions and are located in different cellular compartments. Among the actions attributed to PARPs are its role in the recognition of DNA damage [39]. Single-stranded DNA breaks occur directly as a part of BER, or indirectly through deoxyribose damage. Polymorphisms in that gene may interfere with the ability of cells to repair damaged DNA. The examination of the APE polymorphism reveals that the Asp/ Glu genotype is associated with greater DNA damage and lower capacity of DNA repair then the Asp/Asp wild-type, apart from in control group, where no difference in DNA repair was noted between genotypes after 120 min. However, previous studies note
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
that this SNP does affect neither the AP endonuclease nor the DNA binding activity of the APE protein [40]. In addition, no connection has been observed between the presence of an SNP and significantly greater risk of T2DM. Hence, it may be possible that this SNP does not change protein activity but contributes to less efficient repair in patients with T2DM. This problem requires additional study. Although the disease process of DSPN is relatively well described, the mechanisms underlying nerve degeneration in diabetic subjects are still poorly understood. A growing consensus indicates that they may be multifactorial. Nerve damage is probably due to a combination of factors: metabolic, neurovascular, autoimmune, mechanical injuries to nerves, inherited traits and life style factors [41]. 5. Conclusions Our results show a prognostic importance for the 726 Val/Ala ADPRT polymorphism in the development of T2DM. There is also a connection between the Val/Ala and Ala/Ala genotypes and the wild-type genotype with regard to degree of DNA damage and efficiency of repair. ADPRT takes part in the BER pathway, which is responsible for removing bases damaged by oxidative stress or single strand breaks. Impaired repair may be responsible for the accumulation of reactive molecules, thus contributing to insulin resistance and decreased insulin secretion in the etiology of T2DM [42] and later complications including neuropathy. Our results confirm also that patients with type 2 diabetes demonstrate greater oxidative DNA damage and impaired DNA repair ability compared to healthy subjects. This is more marked in patients with neuropathy, and it is possible that they are more sensitive to oxidative stress. Our findings indicate that oxidative DNA damage level can be considered as a valuable prognostic marker for T2DM progression. Conflict of interests None declared. Financial disclosure This work was supported by grant N N402 375838 from the Polish Ministry of Science and Higher Education and by grant 50203/5-108-05/502-54-092 from the University of Lodz. References [1] International Diabetes Federation (IDF). Diabetes atlas, 5th ed. Available from: http://www.idf.org/sites/default/files/5E_IDFAtlasPoster_2012_EN.pdf [cited 09.06.14]. [2] WHO. Definition, diagnosis and classification of diabetes mellitus and its complications. Report of WHO consultation. Part 1. Diagnosis and classification of diabetes mellitus. Available from: http://whqlibdoc.who.int/hq/1999/ who_ncd_ncs_99.2.pdf [cited 09.06.14]. [3] Evans JL, Goldfine ID, Maddux BA, Grodsky GM. Are oxidative stress-activated signaling pathways mediators of insulin resistance and beta-cell dysfunction? Diabetes 2003;52(January (1)):1–8. [4] Niedowicz DM, Daleke DL. The role of oxidative stress in diabetic complications. Cell Biochem Biophys 2005;43(2):289–330. [5] Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414(December (6865)):813–20. [6] Dizdaroglu M. Oxidatively induced DNA damage: mechanisms, repair and disease. Cancer Lett 2012;327(December (1–2)):26–47. [7] Greene DA, Stevens MJ, Obrosova I, Feldman EL. Glucose-induced oxidative stress and programmed cell death in diabetic neuropathy. Eur J Pharmacol 1999;375(June (1–3)):217–23. [8] Robertson AB, Klungland A, Rognes T, Leiros I. DNA repair in mammalian cells: base excision repair: the long and short of it. Cell Mol Life Sci 2009;66(March (6)):981–93. [9] Fritz G, Gro¨sch S, Tomicic M, Kaina B. APE/Ref-1 and the mammalian response to genotoxic stress. Toxicology 2003;193(November (1–2)):67–78.
229
[10] Parker AR, Eshleman JR. Human MutY: gene structure, protein functions and interactions, and role in carcinogenesis. Cell Mol Life Sci 2003;60(October (10)):2064–83. [11] Gibson BA, Kraus WL. New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs. Nat Rev Mol Cell Biol 2012;13(June (7)):411–24. [12] Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res 2002;12(March (3)):436–46. [13] American Diabetes Association. Standards of medical care in diabetes—2013. Diabetes Care 2013;36(January (Suppl. 1)):S11–66. [14] England JD, Gronseth GS, Franklin G, Miller RG, Asbury AK, Carter GT, et al. Distal symmetric polyneuropathy: a definition for clinical research. Report of the Amercian Academy of Neurology, the American Association of Electrodiagnostic Medicine, and American Academy of Physical Medicine and Rehabilitation. Neurology 2005;64(January (2)):199–207. [15] Tesfaye S, Boulton AJ, Dyck PJ, Freeman R, Horowitz M, Kempler P, et al. Diabetic neuropathies: update on definitions, diagnostic criteria, estimation of severity, and treatments. Diabetes Care 2010;33(October (10)):2285–93. [16] Rader AJ, Barry TP, Stanley OL. Characteristics of lower extremity pressure sensation impairment in developing diabetic sensory polyneuropathy. Foot Ankle Spec 2008;1(February (1)):39–45. [17] Feldman EL, Stevens MJ, Thomas PK, Brown MB, Canal N, Greene DA. A practical two-step quantitative clinical and electrophysiological assessment for the diagnosis and staging of diabetic neuropathy. Diabetes Care 1994;17(November (11)):1281–9. [18] Abbott CA, Carrington AL, Ashe H, Bath S, Every LC, Griffiths J, et al. The NorthWest Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort. Diabet Med 2002;19(May (5)):377–84. [19] Boulton AJM, Malik RA, Arezzo JC, Sosenko JM. Diabetic somatic neuropathies. Diabetes Care 2004;27(June (6)):1458–86. [20] Meijer JW, Smit AJ, Lefrandt JD, van der Hoeven JH, Hoogenberg K, Links TP. Back to basics in diagnosing diabetic polyneuropathy with the tuning fork. Diabetes Care 2005;28(September (9)):2201–5. [21] Hilz MJ, Axelrod FB, Hermann K, Haertl U, Duetsch M, Neundo¨rfer B. Normative values of vibratory perception in 530 children, juveniles and adults aged 3–79 years. J Neurol Sci 1998;159(August (2)):219–25. [22] American College of Physicians. Available from: http://diabetes.acponline.org/ [cited 09.06.14]. [23] Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175(March (1)):184–91. [24] Klaude M, Eriksson S, Nygren J, Ahnstro¨m G. The comet assay: mechanisms and technical considerations. Mutat Res 1996;363(June (2)):89–96. [25] Xi T, Jones IM, Mohrenweiser HW. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function. Genomics 2004;83(June (6)):970–9. [26] Wei W, He XF, Qin JB, Su J, Li SX, Liu Y, et al. Association between the OGG1 Ser326Cys and APEX1 Asp148Glu polymorphisms and lung cancer risk: a meta-analysis. Mol Biol Rep 2012;39(December (12)):11249–62. [27] Bardia A, Tiwari SK, Gunisetty S, Anjum F, Nallari P, Habeeb MA, et al. Functional polymorphisms in XRCC-1 and APE-1 contribute to increased apoptosis and risk of ulcerative colitis. Inflamm Res 2012;61(April (4)): 359–65. [28] Canbay E, Cakmakoglu B, Zeybek U, Sozen S, Cacina C, Gulluoglu M, et al. Association of APE1 and hOGG1 polymorphisms with colorectal cancer risk in a Turkish population. Curr Med Res Opin 2011;27(July (7)):1295–302. [29] Kasahara M, Osawa K, Yoshida K, Miyaishi A, Osawa Y, Inoue N, et al. Association of MUTYH Gln324His and APEX1 Asp148Glu with colorectal cancer and smoking in a Japanese population. J Exp Clin Cancer Res 2008;27:49. [30] Hu JJ, Smith TR, Miller MS, Mohrenweiser HW, Golden A, Case LD. Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity. Carcinogenesis 2001;22(June (6)):917–22. [31] Miyaishi A, Osawa K, Osawa Y, Inoue N, Yoshida K, Kasahara M, et al. MUTYH Gln324His gene polymorphism and genetic susceptibility for lung cancer in a Japanese population. J Exp Clin Cancer Res 2009;28:10. [32] Li C, Hu Z, Lu J, Liu Z, Wang LE, El-Naggar AK, et al. Genetic polymorphisms in DNA base-excision repair genes ADPRT, XRCC1, and APE1 and the risk of squamous cell carcinoma of the head and neck. Cancer 2007;110(August (4)):867–75. [33] Zhang M, Qureshi AA, Guo Q, Han J. Genetic variation in DNA repair pathway genes and melanoma risk. DNA Repair (Amst) 2011;10(January (1)):111–6. [34] Zhai X, Liu J, Hu Z, Wang S, Qing J, Wang X, et al. Polymorphisms of ADPRT Val762Ala and XRCC1 Arg399Glu and risk of breast cancer in Chinese women: a case control analysis. Oncol Rep 2006;15(January (1)):247–52. [35] Zhang Z, Miao XP, Tan W, Guo YL, Zhang XM, Lin DX. Correlation of genetic polymorphisms in DNA repair genes ADPRT and XRCC1 to risk of gastric cancer. Ai Zheng 2006;25(January (1)):7–10. [36] Liu HP, Lin WY, Wu BT, Liu SH, Wang WF, Tsai CH, et al. Evaluation of the poly(ADP-ribose) polymerase-1 gene variants in Alzheimer’s disease. J Clin Lab Anal 2010;24(3):182–6. [37] Ye F, Cheng Q, Hu Y, Zhang J, Chen H. PARP-1 val762ala polymorphism is associated with risk of cervical carcinoma. PLoS ONE 2012;7(5). http:// dx.doi.org/10.1371/journal.pone.0037446. [38] Wang XG, Wang ZQ, Tong WM, Shen Y. PARP1 Val762Ala polymorphism reduces enzymatic activity. Biochem Biophys Res Commun 2007;354(March (1)):122–6.
230
A. Merecz et al. / Advances in Medical Sciences 60 (2015) 220–230
[39] Sousa FG, Matuo R, Soares DG, Escargueil AE, Henriques JA, Larsen AK, et al. PARPs and the DNA damage response. Carcinogenesis 2012;33(August (8)): 1433–40. [40] Hadi MZ, Coleman MA, Fidelis K, Mohrenweiser HW, Wilson DM. Functional characterization of Ape1 variants identified in the human population. Nucleic Acids Res 2000;28(October (20)):3871–9.
[41] Diabetic neuropathies: the nerve damage of diabetes, national diabetes information clearinghouse. Available from: http://diabetes.niddk.nih.gov/dm/ pubs/neuropathies/neuropathies.pdf [cited 09.06.14]. [42] Bloch-Damti A, Bashan N. Proposed mechanisms for the induction of insulin resistance by oxidative stress. Antioxid Redox Signal 2005;7(November–December (11–12)):1553–67.
