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Oxidative stress biomarkers in four Bloom syndrome (BS) patients and in their parents suggest in vivo redox abnormalities in BS phenotype
Clinical Biochemistry 40 (2007) 1100 – 1103
Oxidative stress biomarkers in four Bloom syndrome (BS) patients and in their parents suggest in vivo redox abnormalities in BS phenotype Adriana Zatterale a , Frank J. Kelly b , Paolo Degan c , Marco d'Ischia d , Federico V. Pallardó e , Rita Calzone a , Christina Dunster b , Ana Lloret e , Paola Manini d , Ozgur Coğulu f , Kaan Kavaklı f , Giovanni Pagano g,⁎ a Department of Genetics, “Elena d’Aosta” Hospital, I-80136 Naples, Italy Pharmaceutical Science Research Division, King’s College London, SE1 9NH, UK c Italian National Cancer Institute, IST, I-16132 Genoa, Italy Department of Organic Chemistry and Biochemistry, “Federico II” Naples University, I-80126 Naples, Italy e Department of Physiology, University of Valencia, E-46010 Valencia, Spain f Ege University Hospital, Department of Paediatrics, TK-35100 Bornova, Izmir, Turkey g Italian Association for Fanconi Anemia Research, I-80133 Naples, Italy b
d
Received 8 April 2007; received in revised form 4 June 2007; accepted 7 June 2007 Available online 30 June 2007
Abstract Objective: To evaluate an association of Bloom syndrome (BS) phenotype with an in vivo prooxidant state. Methods: The following endpoints were measured in 4 BS patients, their 6 parents, and 78 controls: a) leukocyte and urinary 8-hydroxy-2′deoxyguanosine (8-OHdG); b) blood glutathione (GSSG and GSH), c) plasma levels of some plasma antioxidants (uric acid, UA, ascorbic acid, AA, α- and γ-tocopherol), and of glyoxal (Glx) and methylglyoxal (MGlx). Results: Leukocyte 8-OHdG levels were significantly increased in the 4 BS patients vs. 40 controls (p = 0.04), while the urinary 8-OHdG levels were non-significantly increased in BS patients. Glutathione disulfide levels and GSSG/GSH ratio were significantly decreased in BS patients vs. 44 controls (p = 0.02). The plasma levels of UA in BS patients were significantly increased vs. 24 controls (p = 0.005). No significant alterations were found in the in the plasma levels of Glx, MGlx, AA, and tocopherol. No changes in the tested parameters were found in the BS heterozygotes. Conclusion: This report shows a significant increase in oxidative DNA damage in leukocytes and in plasma UA levels from 4 BS patients. Should these data be confirmed in more extensive BS patient groups, an involvement of oxidative stress in the clinical BS phenotype might be suggested. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Bloom syndrome; Oxidative stress; 8-hydroxy-2′-deoxyguanosine; Glutathione; Uric acid; Ascorbic acid; Glyoxal; Methylglyoxal
Introduction Bloom syndrome (BS, OMIM #210900) is a very rare autosomal recessive disorder, caused by mutations in the BLM gene encoding the DNA helicase RecQ protein-like-3 and located to 15q26.1 [1,2]. The clinical BS phenotype is ⁎ Corresponding author. Associazione Italiana per la Ricerca sull'Anemia di Fanconi (AIRFA), Piazza Bovio 14, I-80133 Naples, Italy. Fax: +39 0825 511146. E-mail address: [email protected] (G. Pagano).
characterized by hypersensitivity to sunlight with facial telangiectasia in butterfly midface pattern, severe growth retardation, immunodeficiency, and very high frequency of malignancies of several cell types and sites [3,4]. The cytogenetic analysis of BS cells shows multiple non-specific chromosomal breakages with interchanges (mostly between homologous chromosomes), and a marked increase in sister chromatid exchanges (SCE) [5–7]. The current opinion on BS attributes the disease to defective DNA repair as the BLM protein belongs to the RecQ family of helicases, and has similarity to the Werner syndrome protein (WRN) and to the
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.06.003
A. Zatterale et al. / Clinical Biochemistry 40 (2007) 1100–1103
1101
France) supplemented with autologous plasma, glutamine, penicillin/streptomycin and stimulated with phytohemagglutinin. Each culture was set up in duplicate; in one of them 5′bromodeoxyuridine (BrdU) (Sigma, St. Louis, MO, USA) at a final concentration of 1 mM was added to obtain SCE differential staining; in the other one BrdU was added 7 h before harvesting the cells to obtain RBA chromosome banding. Culture harvesting and slide preparation were performed following standard procedures. To study the spontaneous chromosomal instability, 50 RBA banded metaphases from each patient were analyzed and scored for numerical and structural chromosome abnormalities, taking into account any kind of rearrangements and breakages, except for gaps (achromatic areas less than a chromatid in width). The results were given as percentage of aberrant cells (cells with breakages and/or rearrangements). The SCE rate was scored as mean number of chromatid exchanges in 50 cells per patient. In order to avoid any bias due to inter-laboratory differences in cell culture techniques and/or in evaluating chromosomal instability, all the diagnoses were performed in the same laboratory (R.C., A.Z.).
product of the yeast gene SGS1, suggesting that the proteins play similar roles in metabolism, namely by interacting with topoisomerases [8–10]. Another line of studies suggested that the BS phenotype might be related to oxidative stress based on redox abnormalities in cultured cells from BS patients [11–16]. However, to date no report is available, to the best our knowledge, evaluating any in vivo abnormalities in oxidative stress biomarkers in BS patients or BS heterozygotes. The present investigation, as a part of a more extensive study of oxidative stress in some cancer-prone genetic diseases [17–19], was conducted on four BS patients (two siblings and two unrelated patients) and their six parents (obligate heterozygotes), in the hypothesis that an in vivo prooxidant state may be associated to the BS phenotype. Materials and methods Study population Four patients with BS, one male and three females aged 8 to 20 years (14.0 ± 4.9 years), were enrolled in Naples, Italy (unrelated patients NI and GM) and in Izmir, Turkey (sibling patients MK and AK), whose clinical data are summarized in Table 1. Diagnosis was based on clinical and cytogenetic data [3–5]. Also enrolled were 6 BS heteroygotes (three parental couples), aged 34 to 49 years (42.2 ± 5.2 years), and a total of 78 unrelated controls that were grouped in two age classes, i.e. 52 healthy donors (30 males and 22 females) aged 6 to 25 years (12.7 ± 5.2 years) and 26 donors (10 males and 16 females) aged 34 to 47 years (38.5 ± 5.4 years) corresponding to patient and heterozygote age classes, respectively. All the enrolled persons, or the parents/guardians of minors, received appropriate information about the study and signed their consent along with the approval of the Ethical Committees at two institutions (ASL Napoli 1, and Cardarelli Hospital, Naples, Italy), in accordance with the Helsinki Declaration of 1975, as revised in 1983. Recruitment required a 50-mL urine sample and drawing 15 mL of heparinized peripheral blood. Each sample from any patient, parent, or control was tagged with random numbers and analyzed in blind fashion.
Analytical procedures Sample processing and analytical measurements of: i. 8OHdG in DNA from leukocytes and from urine, ii. glutathione from whole blood, iii. plasma levels of UA, AA, and α- and γ-T were carried out as described previously [17]. Statistical analyses To test normal distribution of samples we used the Levene Test. To compare 2 means we used Mann–Whitney test for nonnormal distributions. The level of significance was set at α = 0.05. All statistical analysis were performed using SPSS 13.0 software. Results The levels of leukocyte 8-OHdG in the four BS patients were increased vs. 40 controls in the same age range (4.8 ± 1.7 vs. 2.5 ± 1.0 mol 8-OHdG × 106/mol dG, p = 0.04) (Table 2). The leukocyte 8-OHdG levels in the six BS heterozygotes were superimposable with the data from 24 controls in the same age range. The urinary levels of 8-OHdG in the four BS patients displayed a non-significant increase vs. 9 control data (7.0 ± 1.7
Cytogenetic diagnosis To determine spontaneous chromosomal instability and SCE rate, heparinized blood samples of the 4 patients were incubated for 72 h at 37 °C in RPMI 1640 medium (Eurobio, Les Ulis, Table 1 Main clinical data in the enrolled BS patients Patients
Patients' age at sample collection (years)
Body weight (g) at birth
Facial erythema
Cafèau-lait spots
Growth retardation
Mental retardation
Parents' consanguinity
Other
Mean SCE no./cell
Chromosomal instability as aberrant cells %
NI (twin) a GM MK AK
15 3 13 8
1350a 1640 1350 1250
+ + + +
− + − −
+ + + +
± − ± ±
+ − + +
Congenital renal cystis
58 98 71 66
4 13 24 8
a
Normal twin: 3900.
Recurrent infections Recurrent infections
1102
A. Zatterale et al. / Clinical Biochemistry 40 (2007) 1100–1103
Table 2 Glutathione levels in BS patients and their parents vs. control donors in the respective age ranges
Table 4 Levels of plasma antioxidants in BS patients and their parents vs. control donors in the respective age ranges
Status
Status
(n) TG (μM)
BS patients 4 Controls 1 44 (5 to 25 years) BS parents 6 Controls 2 17 (30 to 50 years)
GSSG (μM)
3730 ± 1779 4192 ± 1542
GSH (μM)
GSSG/GSH × 100
24.2 ± 17.4⁎# 3682 ± 1751 0.6 ± 0.3§O 66.7 ± 40.5 4058 ± 1554 2.1 ± 2.0
3984 ± 1588 77.4 ± 59.2 3379 ± 1574 101.9 ± 104.0
3829 ± 1632 2.7 ± 2.9 3228 ± 1594 2.9 ± 2.6
GSSG: BS patients vs. Controls 1 ⁎p = 0.01; vs. parents #p = 0.038. GSSG/GSH × 100: BS patients vs. Controls 1 §p = 0.02; vs. parents Op = 0.038.
vs. 5.2 ± 1.3), whereas the BS heterozygotes failed to display any significant changes in urinary 8-OHdG levels vs. the data from 8 controls in the same age range (Table 2). As shown in Table 3, the analyses of plasma antioxidants showed that UA plasma levels in the four BS patients were significantly increased vs. 24 controls in the same age range (163 ± 37 vs. 98 ± 32 μM) (p = 0.005), whereas the concentrations of AA, α- and γ-tocopherol were unchanged (Table 3). Also unchanged were the plasma levels of Glx and MGlx in BS patients vs. controls (data not shown). No differences were detected in the levels of any plasma antioxidants in the six BS heterozygotes vs. the data from 13 controls in the same age range. As shown in Table 4, the four BS patients displayed a significant decrease in the GSSG levels in blood samples (24.2 ± 17.4 μM vs. 66.7 ± 40.5 μM) vs. 44 controls in the same age range (p = 0.01), and vs. the six BS heterozygotes (p = 0.038). The GSSG/GSH × 100 ratio in BS patients was also decreased vs. control values (0.6 ± 0.3 vs. 2.1 ± 2.0, p = 0.02) and vs. heterozygote values (2.7 ± 2.9, p = 0.04). The six BS heterozygotes displayed superimposable glutathione levels vs. 17 controls in the same age range (Table 4). Discussion An established consensus relates the BS-associated genetic defect with impaired DNA metabolism, since the defective gene, BLM, encodes the RECQL3 protein, belonging to the RECQ family of helicases, required for the maintenance of
Table 3 Levels of 8-hydroxy-2′-deoxyguanosine in leukocytes and urine from BS patients and their parents vs. control donors Status
(n)
Leukocyte 8-OHdG (mol × 106/mol dG)
(n)
Urinary 8-OHdG (nmol 8-OHdG/mmol creatinine)
BS patients Controls 1 (5 to 25 years) BS parents Controls 2 (30 to 50 years)
4 40
4.8 ± 1.7⁎ 2.5 ± 1.0
4 9
7.0 ± 1.7 5.2 ± 1.3
6 24
3.3 ± 1.7 3.5 ± 1.7
5 8
3.7 ± 1.5 4.5 ± 1.9
⁎p = 0.04.
(n)
Uric acid (μM)
Ascorbic acid (μM)
α-Tocopherol (μM)
γ-Tocopherol (μM)
BS patients 4 Controls 1 24 (5 to 25 years) BS parents 6 Controls 2 13 (30 to 50 years)
163 ± 37⁎ 98 ± 32
20.7 ± 11.1 20.5 ± 9.7
25.5 ± 4.0 25.2 ± 4.7
1.3 ± 0.6 1.1 ± 0.7
136 ± 28 127 ± 46
25.3 ± 6.0 25.9 ± 9.9
32.3 ± 10.7 30.9 ± 9.0
1.8 ± 1.9 1.0 ± 1.0
⁎p = 0.005.
genomic stability in organisms ranging from bacteria to humans and including RECQL2 (mutated in Werner syndrome), RECQL4 (mutated in Rothmund-Thomson syndrome), and the yeast proteins Sgslp and Rqh1p [1,10]. Furthermore, BLM protein has been functionally related to the gene products associated with two other chromosomal instability syndromes, Fanconi anemia (FA) and Ataxia telangiectasia (AT) that are commonly associated with deficiencies in DNA repair [9,10,20–23]. In the presence of this established opinion, another line of early studies has pointed to a role for oxidative stress as a component of the metabolic deficiencies associated with BS phenotype. Cerutti et al. [11] reported excess clastogenic factor in plasma from BS patients, and cultured BS cells were found to express excess superoxide dismutase [12,13] and increased chemiluminescence [14], i.e. excess formation of reactive oxygen species (ROS) vs. normal cells. This finding of excess chemiluminescence might be regarded as analogous to the data obtained in freshly drawn WBC from FA patients, which correlated with accumulation of oxidative DNA damage and to spontaneous chromosomal instability [24]. Poot et al. related cell kinetic abnormalities in BS cells to those observed in normal cells exposed to products of lipid peroxidation, or hyperoxia, or redox-cycling xenobiotics [15]. The same group reported on the concentration-dependent G2 arrest of BS cells exposed to bromodeoxyuridine (BrdU), a recognized radiosensitizer enhancing ROS-mediated growth inhibition [16]. This line of studies was confined to investigations on cell lines, except for the early report by Cerutti et al. [11] who studied clastogenic factor in plasma from BS patients. Thus, the last known report in this line of studies dates back to 1993 [14]. To the best of our knowledge, the data obtained in the present study represent the first attempt to verify any redox abnormalities in blood and urine freshly drawn from BS patients and heterozygotes. In spite of the scanty number of patients, accounted for by the extreme rarity of BS, the results suggest the occurrence of an in vivo prooxidant state worth being reassessed in more extensive numbers of BS patients. A cellular accumulation of oxidative DNA damage appears to be increased, as assessed by WBC 8-OHdG levels, whereas 8OHdG urinary excretion (resulting from in vivo removal of damaged DNA bases) fails to show any decrease vs. controls, as might be expected from a defective maintenance of DNA integrity, but rather suggests proficient in vivo DNA repair
A. Zatterale et al. / Clinical Biochemistry 40 (2007) 1100–1103
mechanisms. The observed increase in uric acid plasma levels is also consistent with an in vivo redox abnormality, similar to the increased UA levels in plasma from Werner syndrome and Down syndrome patients [18,19]. While UA is per se an antioxidant, its production results from xanthine oxidase (XO) [25], the enzyme that produces UA from hypoxanthine and xanthine, also produces superoxide and hydrogen peroxide as by-products [26]. In turn, one might speculate that SOD overexpression in BS cells [12,13] might play a role in the XOdriven production of UA. An unexpected finding was the decrease in glutathione disulfide along with a decrease in the GSSG/GSH ratio, which might suggest a compensatory mechanism in response to oxidative stress, as observed in Down syndrome and in Ataxia telangiectasia patients [19,27]. This finding, and a candidate compensatory mechanism might rely, again, on SOD overexpression in BS cells [12,13]. This attempt of interpretation, however, requires further studies as the asset of SOD, catalase and other redox-related activities in BS patients that has not yet been assessed, to the best of our present knowledge. Together, the previous literature from in vitro studies and the present findings on blood cells, plasma and urine from BS patients concur to suggest a role of oxidative stress in BS phenotype. If an in vivo redox imbalance were confirmed in more extensive groups of BS patients, these findings might shed light into BS phenotype, with possible consequences in patients' clinical management. Acknowledgments The present study, a part of the EUROS (European Research on Oxidative Stress) Project, was supported by the European Commission, DG XII, contract # BMH4-CT98-3107, and by the Italian Association for Fanconi Anaemia Research (AIRFA). Thanks are due to the Contributors to the EUROS Project, who were as follows: Clinicians contributing to patient and control recruitment: Maria A. Pisanti, Anna Saviano; Sample processing and analyses: Paolo Ciavolino, Francesca Gallucci, Virginia Rossi, Emilia Vuttariello; Cytogenetic analyses: Vincenzo Altieri, Antonio Lioniello, Giuseppe Peperna. References [1] Ellis NA, German J. Molecular genetics of Bloom's syndrome. Hum Mol Genet 1996;5:1457–63. [2] Karow JK, Chakraverty RK, Hickson ID. The Bloom's syndrome gene product is a 3′–5′ DNA helicase. J Biol Chem 1997;272:30611–4. [3] Bloom D. The syndrome of congenital telangiectatic erythema and stunted growth. J Pediatr 1966;68:103–13. [4] German J. Bloom's syndrome. XX. The first 100 cancers. Cancer Genet Cytogenet 1997;93:100–6. [5] German J, Crippa LP, Bloom D. Bloom's syndrome. III. Analysis of the chromosome aberration characteristic of this disorder. Chromosoma 1974;48:361–6. [6] Chaganti RSK, Schönberg S, German J. A manyfold increase in sister
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25] [26] [27]
1103
chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci U S A 1974;71:4508–12. German J, Schönberg S, Louie E, Chaganti RSK. Bloom's syndrome. IV. Sister chromatid exchanges in lymphocytes. Am J Hum Genet 1977;29: 248–55. Bischof O, Kim SH, Irving J, Beresten S, Ellis NA, Campisi J. Regulation and localization of the Bloom syndrome protein in response to DNA damage. J Cell Biol 2001;153:367–80. Imamura O, Fujita K, Itoh C, Takeda S, Furuichi Y, Matsumoto T. Werner and Bloom helicases are involved in DNA repair in a complementary fashion. Oncogene 2002;21:954–63. von Kobbe C, Karmakar P, Dawut L, Opresko P, Zeng X, Brosh Jr RM, et al. Colocalization, physical, and functional interaction between Werner and Bloom syndrome proteins. J Biol Chem 2002;277:22035–44. Cerutti P, Emerit I, Hirschi M, Zbinden I. Bloom's syndrome: a deficiency in the detoxification of active oxygen species? (Abstract) Eur. J Cancer Clin Oncol 1981;17:938. Nicotera TM, Notaro J, Notaro S, Schumer J, Sandberg AA. Elevated superoxide dismutase in Bloom's syndrome: a genetic condition of oxidative stress. Cancer Res 1989;49:5239–43. Lee KH, Abe S, Yanabe Y, Matsuda I, Yoshida MC. Superoxide dismutase activity and chromosome damage in cultured chromosome instability syndrome cells. Mutat Res 1990;244:251–6. Nicotera T, Thusu K, Dandona P. Elevated production of active oxygen in Bloom's syndrome cell lines. Cancer Res 1993;53:5104–7. Poot M, Hoehn H, Nicotera TM, Rudiger HW. Cell kinetic evidence suggests elevated oxidative stress in cultured cells of Bloom's syndrome. Free Radic Res Commun 1989;7:179–87. Poot M, Rudiger HW, Hoehn H. Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome. Mutat Res 1990;238:203–7. Pagano G, Degan P, d'Ischia M, Kelly FJ, Pallardó FV, Zatterale A, et al. Gender- and age-related distinctions for the in vivo prooxidant state in Fanconi anaemia patients. Carcinogenesis 2004;25:1899–909. Pagano G, Zatterale A, Degan P, d'Ischia M, Kelly FJ, Pallardó FV, et al. In vivo prooxidant state in Werner syndrome patients. Free Radic Res 2005;39:529–33. Pallardó FV, Degan P, d'Ischia M, Kelly FJ, Zatterale A, Calzone R, et al. Higher age-related prooxidant state in young Down syndrome patients indicates accelerated aging. Biogerontology 2006;7:211–20. Ababou M, Dutertre S, Lecluse Y, Onclercq R, Chatton B, Amor-Gueret M. ATM-dependent phosphorylation and accumulation of endogenous BLM protein in response to ionizing radiation. Oncogene 2000;19:5955–63. Beamish H, Kedar P, Kaneko H, Chen P, Fukao T, Peng C, et al. Functional link between BLM defective in Bloom's Syndrome and the Ataxiatelangiectasia-mutated protein. ATM J Biol Chem 2002;277:30515–23. Hirano S, Yamamoto K, Ishiai M, Yamazoe M, Seki M, Matsushita N, et al. Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM. EMBO J 2005;24:418–27. Meetei AR, Sechi S, Wallisch M, Yang D, Young MK, Joenje H, et al. A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome. Mol Cell Biol 2003;23:3417–26. Degan P, Bonassi S, De Caterina M, Korkina LG, Pinto L, Scopacasa F, et al. In vivo accumulation of 8-hydroxy-2′-deoxyguanosine in DNA correlates with release of reactive oxygen species in Fanconi's anaemia families. Carcinogenesis 1995;16:735–42. Hochstein P, Hatch L, Sevanian A. Uric acid: functions and determination. Methods Enzymol 1984;105:162–6. Link EM, Riley PA. Role of hydrogen peroxide in the cytotoxicity of the xanthine/xanthine oxidase system. Biochem J 1988;249:391–9. Degan P, d'Ischia M, Pallardó FV, Zatterale A, Brusco A, Calzone R, et al. Glutathione levels in blood from Ataxia Telangiectasia patients suggest in vivo adaptive mechanisms to oxidative stress. Clin Biochem 2007;40: 666–70.
Oxidative stress biomarkers in four Bloom syndrome (BS) patients and in their parents suggest in vivo redox abnormalities in BS phenotype Adriana Zatterale a , Frank J. Kelly b , Paolo Degan c , Marco d'Ischia d , Federico V. Pallardó e , Rita Calzone a , Christina Dunster b , Ana Lloret e , Paola Manini d , Ozgur Coğulu f , Kaan Kavaklı f , Giovanni Pagano g,⁎ a Department of Genetics, “Elena d’Aosta” Hospital, I-80136 Naples, Italy Pharmaceutical Science Research Division, King’s College London, SE1 9NH, UK c Italian National Cancer Institute, IST, I-16132 Genoa, Italy Department of Organic Chemistry and Biochemistry, “Federico II” Naples University, I-80126 Naples, Italy e Department of Physiology, University of Valencia, E-46010 Valencia, Spain f Ege University Hospital, Department of Paediatrics, TK-35100 Bornova, Izmir, Turkey g Italian Association for Fanconi Anemia Research, I-80133 Naples, Italy b
d
Received 8 April 2007; received in revised form 4 June 2007; accepted 7 June 2007 Available online 30 June 2007
Abstract Objective: To evaluate an association of Bloom syndrome (BS) phenotype with an in vivo prooxidant state. Methods: The following endpoints were measured in 4 BS patients, their 6 parents, and 78 controls: a) leukocyte and urinary 8-hydroxy-2′deoxyguanosine (8-OHdG); b) blood glutathione (GSSG and GSH), c) plasma levels of some plasma antioxidants (uric acid, UA, ascorbic acid, AA, α- and γ-tocopherol), and of glyoxal (Glx) and methylglyoxal (MGlx). Results: Leukocyte 8-OHdG levels were significantly increased in the 4 BS patients vs. 40 controls (p = 0.04), while the urinary 8-OHdG levels were non-significantly increased in BS patients. Glutathione disulfide levels and GSSG/GSH ratio were significantly decreased in BS patients vs. 44 controls (p = 0.02). The plasma levels of UA in BS patients were significantly increased vs. 24 controls (p = 0.005). No significant alterations were found in the in the plasma levels of Glx, MGlx, AA, and tocopherol. No changes in the tested parameters were found in the BS heterozygotes. Conclusion: This report shows a significant increase in oxidative DNA damage in leukocytes and in plasma UA levels from 4 BS patients. Should these data be confirmed in more extensive BS patient groups, an involvement of oxidative stress in the clinical BS phenotype might be suggested. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Bloom syndrome; Oxidative stress; 8-hydroxy-2′-deoxyguanosine; Glutathione; Uric acid; Ascorbic acid; Glyoxal; Methylglyoxal
Introduction Bloom syndrome (BS, OMIM #210900) is a very rare autosomal recessive disorder, caused by mutations in the BLM gene encoding the DNA helicase RecQ protein-like-3 and located to 15q26.1 [1,2]. The clinical BS phenotype is ⁎ Corresponding author. Associazione Italiana per la Ricerca sull'Anemia di Fanconi (AIRFA), Piazza Bovio 14, I-80133 Naples, Italy. Fax: +39 0825 511146. E-mail address: [email protected] (G. Pagano).
characterized by hypersensitivity to sunlight with facial telangiectasia in butterfly midface pattern, severe growth retardation, immunodeficiency, and very high frequency of malignancies of several cell types and sites [3,4]. The cytogenetic analysis of BS cells shows multiple non-specific chromosomal breakages with interchanges (mostly between homologous chromosomes), and a marked increase in sister chromatid exchanges (SCE) [5–7]. The current opinion on BS attributes the disease to defective DNA repair as the BLM protein belongs to the RecQ family of helicases, and has similarity to the Werner syndrome protein (WRN) and to the
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.06.003
A. Zatterale et al. / Clinical Biochemistry 40 (2007) 1100–1103
1101
France) supplemented with autologous plasma, glutamine, penicillin/streptomycin and stimulated with phytohemagglutinin. Each culture was set up in duplicate; in one of them 5′bromodeoxyuridine (BrdU) (Sigma, St. Louis, MO, USA) at a final concentration of 1 mM was added to obtain SCE differential staining; in the other one BrdU was added 7 h before harvesting the cells to obtain RBA chromosome banding. Culture harvesting and slide preparation were performed following standard procedures. To study the spontaneous chromosomal instability, 50 RBA banded metaphases from each patient were analyzed and scored for numerical and structural chromosome abnormalities, taking into account any kind of rearrangements and breakages, except for gaps (achromatic areas less than a chromatid in width). The results were given as percentage of aberrant cells (cells with breakages and/or rearrangements). The SCE rate was scored as mean number of chromatid exchanges in 50 cells per patient. In order to avoid any bias due to inter-laboratory differences in cell culture techniques and/or in evaluating chromosomal instability, all the diagnoses were performed in the same laboratory (R.C., A.Z.).
product of the yeast gene SGS1, suggesting that the proteins play similar roles in metabolism, namely by interacting with topoisomerases [8–10]. Another line of studies suggested that the BS phenotype might be related to oxidative stress based on redox abnormalities in cultured cells from BS patients [11–16]. However, to date no report is available, to the best our knowledge, evaluating any in vivo abnormalities in oxidative stress biomarkers in BS patients or BS heterozygotes. The present investigation, as a part of a more extensive study of oxidative stress in some cancer-prone genetic diseases [17–19], was conducted on four BS patients (two siblings and two unrelated patients) and their six parents (obligate heterozygotes), in the hypothesis that an in vivo prooxidant state may be associated to the BS phenotype. Materials and methods Study population Four patients with BS, one male and three females aged 8 to 20 years (14.0 ± 4.9 years), were enrolled in Naples, Italy (unrelated patients NI and GM) and in Izmir, Turkey (sibling patients MK and AK), whose clinical data are summarized in Table 1. Diagnosis was based on clinical and cytogenetic data [3–5]. Also enrolled were 6 BS heteroygotes (three parental couples), aged 34 to 49 years (42.2 ± 5.2 years), and a total of 78 unrelated controls that were grouped in two age classes, i.e. 52 healthy donors (30 males and 22 females) aged 6 to 25 years (12.7 ± 5.2 years) and 26 donors (10 males and 16 females) aged 34 to 47 years (38.5 ± 5.4 years) corresponding to patient and heterozygote age classes, respectively. All the enrolled persons, or the parents/guardians of minors, received appropriate information about the study and signed their consent along with the approval of the Ethical Committees at two institutions (ASL Napoli 1, and Cardarelli Hospital, Naples, Italy), in accordance with the Helsinki Declaration of 1975, as revised in 1983. Recruitment required a 50-mL urine sample and drawing 15 mL of heparinized peripheral blood. Each sample from any patient, parent, or control was tagged with random numbers and analyzed in blind fashion.
Analytical procedures Sample processing and analytical measurements of: i. 8OHdG in DNA from leukocytes and from urine, ii. glutathione from whole blood, iii. plasma levels of UA, AA, and α- and γ-T were carried out as described previously [17]. Statistical analyses To test normal distribution of samples we used the Levene Test. To compare 2 means we used Mann–Whitney test for nonnormal distributions. The level of significance was set at α = 0.05. All statistical analysis were performed using SPSS 13.0 software. Results The levels of leukocyte 8-OHdG in the four BS patients were increased vs. 40 controls in the same age range (4.8 ± 1.7 vs. 2.5 ± 1.0 mol 8-OHdG × 106/mol dG, p = 0.04) (Table 2). The leukocyte 8-OHdG levels in the six BS heterozygotes were superimposable with the data from 24 controls in the same age range. The urinary levels of 8-OHdG in the four BS patients displayed a non-significant increase vs. 9 control data (7.0 ± 1.7
Cytogenetic diagnosis To determine spontaneous chromosomal instability and SCE rate, heparinized blood samples of the 4 patients were incubated for 72 h at 37 °C in RPMI 1640 medium (Eurobio, Les Ulis, Table 1 Main clinical data in the enrolled BS patients Patients
Patients' age at sample collection (years)
Body weight (g) at birth
Facial erythema
Cafèau-lait spots
Growth retardation
Mental retardation
Parents' consanguinity
Other
Mean SCE no./cell
Chromosomal instability as aberrant cells %
NI (twin) a GM MK AK
15 3 13 8
1350a 1640 1350 1250
+ + + +
− + − −
+ + + +
± − ± ±
+ − + +
Congenital renal cystis
58 98 71 66
4 13 24 8
a
Normal twin: 3900.
Recurrent infections Recurrent infections
1102
A. Zatterale et al. / Clinical Biochemistry 40 (2007) 1100–1103
Table 2 Glutathione levels in BS patients and their parents vs. control donors in the respective age ranges
Table 4 Levels of plasma antioxidants in BS patients and their parents vs. control donors in the respective age ranges
Status
Status
(n) TG (μM)
BS patients 4 Controls 1 44 (5 to 25 years) BS parents 6 Controls 2 17 (30 to 50 years)
GSSG (μM)
3730 ± 1779 4192 ± 1542
GSH (μM)
GSSG/GSH × 100
24.2 ± 17.4⁎# 3682 ± 1751 0.6 ± 0.3§O 66.7 ± 40.5 4058 ± 1554 2.1 ± 2.0
3984 ± 1588 77.4 ± 59.2 3379 ± 1574 101.9 ± 104.0
3829 ± 1632 2.7 ± 2.9 3228 ± 1594 2.9 ± 2.6
GSSG: BS patients vs. Controls 1 ⁎p = 0.01; vs. parents #p = 0.038. GSSG/GSH × 100: BS patients vs. Controls 1 §p = 0.02; vs. parents Op = 0.038.
vs. 5.2 ± 1.3), whereas the BS heterozygotes failed to display any significant changes in urinary 8-OHdG levels vs. the data from 8 controls in the same age range (Table 2). As shown in Table 3, the analyses of plasma antioxidants showed that UA plasma levels in the four BS patients were significantly increased vs. 24 controls in the same age range (163 ± 37 vs. 98 ± 32 μM) (p = 0.005), whereas the concentrations of AA, α- and γ-tocopherol were unchanged (Table 3). Also unchanged were the plasma levels of Glx and MGlx in BS patients vs. controls (data not shown). No differences were detected in the levels of any plasma antioxidants in the six BS heterozygotes vs. the data from 13 controls in the same age range. As shown in Table 4, the four BS patients displayed a significant decrease in the GSSG levels in blood samples (24.2 ± 17.4 μM vs. 66.7 ± 40.5 μM) vs. 44 controls in the same age range (p = 0.01), and vs. the six BS heterozygotes (p = 0.038). The GSSG/GSH × 100 ratio in BS patients was also decreased vs. control values (0.6 ± 0.3 vs. 2.1 ± 2.0, p = 0.02) and vs. heterozygote values (2.7 ± 2.9, p = 0.04). The six BS heterozygotes displayed superimposable glutathione levels vs. 17 controls in the same age range (Table 4). Discussion An established consensus relates the BS-associated genetic defect with impaired DNA metabolism, since the defective gene, BLM, encodes the RECQL3 protein, belonging to the RECQ family of helicases, required for the maintenance of
Table 3 Levels of 8-hydroxy-2′-deoxyguanosine in leukocytes and urine from BS patients and their parents vs. control donors Status
(n)
Leukocyte 8-OHdG (mol × 106/mol dG)
(n)
Urinary 8-OHdG (nmol 8-OHdG/mmol creatinine)
BS patients Controls 1 (5 to 25 years) BS parents Controls 2 (30 to 50 years)
4 40
4.8 ± 1.7⁎ 2.5 ± 1.0
4 9
7.0 ± 1.7 5.2 ± 1.3
6 24
3.3 ± 1.7 3.5 ± 1.7
5 8
3.7 ± 1.5 4.5 ± 1.9
⁎p = 0.04.
(n)
Uric acid (μM)
Ascorbic acid (μM)
α-Tocopherol (μM)
γ-Tocopherol (μM)
BS patients 4 Controls 1 24 (5 to 25 years) BS parents 6 Controls 2 13 (30 to 50 years)
163 ± 37⁎ 98 ± 32
20.7 ± 11.1 20.5 ± 9.7
25.5 ± 4.0 25.2 ± 4.7
1.3 ± 0.6 1.1 ± 0.7
136 ± 28 127 ± 46
25.3 ± 6.0 25.9 ± 9.9
32.3 ± 10.7 30.9 ± 9.0
1.8 ± 1.9 1.0 ± 1.0
⁎p = 0.005.
genomic stability in organisms ranging from bacteria to humans and including RECQL2 (mutated in Werner syndrome), RECQL4 (mutated in Rothmund-Thomson syndrome), and the yeast proteins Sgslp and Rqh1p [1,10]. Furthermore, BLM protein has been functionally related to the gene products associated with two other chromosomal instability syndromes, Fanconi anemia (FA) and Ataxia telangiectasia (AT) that are commonly associated with deficiencies in DNA repair [9,10,20–23]. In the presence of this established opinion, another line of early studies has pointed to a role for oxidative stress as a component of the metabolic deficiencies associated with BS phenotype. Cerutti et al. [11] reported excess clastogenic factor in plasma from BS patients, and cultured BS cells were found to express excess superoxide dismutase [12,13] and increased chemiluminescence [14], i.e. excess formation of reactive oxygen species (ROS) vs. normal cells. This finding of excess chemiluminescence might be regarded as analogous to the data obtained in freshly drawn WBC from FA patients, which correlated with accumulation of oxidative DNA damage and to spontaneous chromosomal instability [24]. Poot et al. related cell kinetic abnormalities in BS cells to those observed in normal cells exposed to products of lipid peroxidation, or hyperoxia, or redox-cycling xenobiotics [15]. The same group reported on the concentration-dependent G2 arrest of BS cells exposed to bromodeoxyuridine (BrdU), a recognized radiosensitizer enhancing ROS-mediated growth inhibition [16]. This line of studies was confined to investigations on cell lines, except for the early report by Cerutti et al. [11] who studied clastogenic factor in plasma from BS patients. Thus, the last known report in this line of studies dates back to 1993 [14]. To the best of our knowledge, the data obtained in the present study represent the first attempt to verify any redox abnormalities in blood and urine freshly drawn from BS patients and heterozygotes. In spite of the scanty number of patients, accounted for by the extreme rarity of BS, the results suggest the occurrence of an in vivo prooxidant state worth being reassessed in more extensive numbers of BS patients. A cellular accumulation of oxidative DNA damage appears to be increased, as assessed by WBC 8-OHdG levels, whereas 8OHdG urinary excretion (resulting from in vivo removal of damaged DNA bases) fails to show any decrease vs. controls, as might be expected from a defective maintenance of DNA integrity, but rather suggests proficient in vivo DNA repair
A. Zatterale et al. / Clinical Biochemistry 40 (2007) 1100–1103
mechanisms. The observed increase in uric acid plasma levels is also consistent with an in vivo redox abnormality, similar to the increased UA levels in plasma from Werner syndrome and Down syndrome patients [18,19]. While UA is per se an antioxidant, its production results from xanthine oxidase (XO) [25], the enzyme that produces UA from hypoxanthine and xanthine, also produces superoxide and hydrogen peroxide as by-products [26]. In turn, one might speculate that SOD overexpression in BS cells [12,13] might play a role in the XOdriven production of UA. An unexpected finding was the decrease in glutathione disulfide along with a decrease in the GSSG/GSH ratio, which might suggest a compensatory mechanism in response to oxidative stress, as observed in Down syndrome and in Ataxia telangiectasia patients [19,27]. This finding, and a candidate compensatory mechanism might rely, again, on SOD overexpression in BS cells [12,13]. This attempt of interpretation, however, requires further studies as the asset of SOD, catalase and other redox-related activities in BS patients that has not yet been assessed, to the best of our present knowledge. Together, the previous literature from in vitro studies and the present findings on blood cells, plasma and urine from BS patients concur to suggest a role of oxidative stress in BS phenotype. If an in vivo redox imbalance were confirmed in more extensive groups of BS patients, these findings might shed light into BS phenotype, with possible consequences in patients' clinical management. Acknowledgments The present study, a part of the EUROS (European Research on Oxidative Stress) Project, was supported by the European Commission, DG XII, contract # BMH4-CT98-3107, and by the Italian Association for Fanconi Anaemia Research (AIRFA). Thanks are due to the Contributors to the EUROS Project, who were as follows: Clinicians contributing to patient and control recruitment: Maria A. Pisanti, Anna Saviano; Sample processing and analyses: Paolo Ciavolino, Francesca Gallucci, Virginia Rossi, Emilia Vuttariello; Cytogenetic analyses: Vincenzo Altieri, Antonio Lioniello, Giuseppe Peperna. References [1] Ellis NA, German J. Molecular genetics of Bloom's syndrome. Hum Mol Genet 1996;5:1457–63. [2] Karow JK, Chakraverty RK, Hickson ID. The Bloom's syndrome gene product is a 3′–5′ DNA helicase. J Biol Chem 1997;272:30611–4. [3] Bloom D. The syndrome of congenital telangiectatic erythema and stunted growth. J Pediatr 1966;68:103–13. [4] German J. Bloom's syndrome. XX. The first 100 cancers. Cancer Genet Cytogenet 1997;93:100–6. [5] German J, Crippa LP, Bloom D. Bloom's syndrome. III. Analysis of the chromosome aberration characteristic of this disorder. Chromosoma 1974;48:361–6. [6] Chaganti RSK, Schönberg S, German J. A manyfold increase in sister
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25] [26] [27]
1103
chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci U S A 1974;71:4508–12. German J, Schönberg S, Louie E, Chaganti RSK. Bloom's syndrome. IV. Sister chromatid exchanges in lymphocytes. Am J Hum Genet 1977;29: 248–55. Bischof O, Kim SH, Irving J, Beresten S, Ellis NA, Campisi J. Regulation and localization of the Bloom syndrome protein in response to DNA damage. J Cell Biol 2001;153:367–80. Imamura O, Fujita K, Itoh C, Takeda S, Furuichi Y, Matsumoto T. Werner and Bloom helicases are involved in DNA repair in a complementary fashion. Oncogene 2002;21:954–63. von Kobbe C, Karmakar P, Dawut L, Opresko P, Zeng X, Brosh Jr RM, et al. Colocalization, physical, and functional interaction between Werner and Bloom syndrome proteins. J Biol Chem 2002;277:22035–44. Cerutti P, Emerit I, Hirschi M, Zbinden I. Bloom's syndrome: a deficiency in the detoxification of active oxygen species? (Abstract) Eur. J Cancer Clin Oncol 1981;17:938. Nicotera TM, Notaro J, Notaro S, Schumer J, Sandberg AA. Elevated superoxide dismutase in Bloom's syndrome: a genetic condition of oxidative stress. Cancer Res 1989;49:5239–43. Lee KH, Abe S, Yanabe Y, Matsuda I, Yoshida MC. Superoxide dismutase activity and chromosome damage in cultured chromosome instability syndrome cells. Mutat Res 1990;244:251–6. Nicotera T, Thusu K, Dandona P. Elevated production of active oxygen in Bloom's syndrome cell lines. Cancer Res 1993;53:5104–7. Poot M, Hoehn H, Nicotera TM, Rudiger HW. Cell kinetic evidence suggests elevated oxidative stress in cultured cells of Bloom's syndrome. Free Radic Res Commun 1989;7:179–87. Poot M, Rudiger HW, Hoehn H. Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome. Mutat Res 1990;238:203–7. Pagano G, Degan P, d'Ischia M, Kelly FJ, Pallardó FV, Zatterale A, et al. Gender- and age-related distinctions for the in vivo prooxidant state in Fanconi anaemia patients. Carcinogenesis 2004;25:1899–909. Pagano G, Zatterale A, Degan P, d'Ischia M, Kelly FJ, Pallardó FV, et al. In vivo prooxidant state in Werner syndrome patients. Free Radic Res 2005;39:529–33. Pallardó FV, Degan P, d'Ischia M, Kelly FJ, Zatterale A, Calzone R, et al. Higher age-related prooxidant state in young Down syndrome patients indicates accelerated aging. Biogerontology 2006;7:211–20. Ababou M, Dutertre S, Lecluse Y, Onclercq R, Chatton B, Amor-Gueret M. ATM-dependent phosphorylation and accumulation of endogenous BLM protein in response to ionizing radiation. Oncogene 2000;19:5955–63. Beamish H, Kedar P, Kaneko H, Chen P, Fukao T, Peng C, et al. Functional link between BLM defective in Bloom's Syndrome and the Ataxiatelangiectasia-mutated protein. ATM J Biol Chem 2002;277:30515–23. Hirano S, Yamamoto K, Ishiai M, Yamazoe M, Seki M, Matsushita N, et al. Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM. EMBO J 2005;24:418–27. Meetei AR, Sechi S, Wallisch M, Yang D, Young MK, Joenje H, et al. A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome. Mol Cell Biol 2003;23:3417–26. Degan P, Bonassi S, De Caterina M, Korkina LG, Pinto L, Scopacasa F, et al. In vivo accumulation of 8-hydroxy-2′-deoxyguanosine in DNA correlates with release of reactive oxygen species in Fanconi's anaemia families. Carcinogenesis 1995;16:735–42. Hochstein P, Hatch L, Sevanian A. Uric acid: functions and determination. Methods Enzymol 1984;105:162–6. Link EM, Riley PA. Role of hydrogen peroxide in the cytotoxicity of the xanthine/xanthine oxidase system. Biochem J 1988;249:391–9. Degan P, d'Ischia M, Pallardó FV, Zatterale A, Brusco A, Calzone R, et al. Glutathione levels in blood from Ataxia Telangiectasia patients suggest in vivo adaptive mechanisms to oxidative stress. Clin Biochem 2007;40: 666–70.