http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, 2014; 24(8): 603–607 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2014.956913
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RESEARCH ARTICLE
Cytogenetic biomonitoring in mucopolyssacharosis I, II and IV patients treated with enzyme replacement therapy Joice Marques Guilheiro1, Marcelo Donizetti Chaves1, Ana Maria Martins2, Daniel Araki Ribeiro1, and Vania D’Almeida1,3 1
Department of Biosciences, 2Department of Pediatrics and 3Department of Psychobiology, Federal University of Sao Paulo, UNIFESP, SP, Brazil
Abstract
Keywords
Background and objectives: The aim of this study was to evaluate genotoxicity and mutagenicity in peripheral blood and buccal mucosal cells in mucopolysaccharidosis (MPS) I, II or VI patients. Methods: A total of 12 patients with MPS type I, II and VI attended at the Institute of Genetics and Inborn Errors of Metabolism treated with enzyme replacement therapy (ERT) and 10 healthy control volunteers were included in this study. Mechanically exfoliated cells from cheek mucosa (left and right side) were used to micronucleus test and single cell gel (comet) assay in peripheral blood cells. Results: The results of this study detected the presence of genetic damage in peripheral blood for all individuals with MPS treated with ERT, regardless of type of MPS as depicted by tail moment results. In addition, an increased number of micronucleated cells were found in buccal cells of MPS type II patients. It was also observed an increase of other nuclear alterations closely related to cytotoxicity as depicted by the frequency of pyknosis, karyolysis and karyorrhexis in buccal mucosa cells of MPS VI patients (p50.05). Conclusion: Taken together, such results demonstrate that metabolic alterations induced by the enzymatic deficiency characteristic of MPS associated with ERT therapy can induce genotoxicity and mutagenicity in peripheral blood and buccal mucosa cells, respectively. This effect appears to be more pronounced to MPS II.
Comet assay, genetic damage, micronucleus test, mucopolyssacharodosis
Introduction Mucopolysaccharidosis (MPS) is a genetically heterogeneous group of metabolic disorders caused by a deficiency or absence of enzymes responsible for the lysosomal degradation of glycosaminoglycans (GAGs). In this context, GAGs are able to accumulate in lysosomes, extracellular matrix and body fluids (Neufeld & Muenzer, 2001). Clinical presentation of MPS could include some symptoms and characteristics such as short stature, facial dysmorphism, skeletal deformities, pulmonary dysfunction, joint stiffness and contractures, myocardial hypertrophy, neurological symptoms, mental retardation and potentially major adverse consequences for speech, language and cognitive development (Perenc, 2013). Nowadays, MPS has been treated with enzyme replacement therapy (ERT) (Desnick, 2004). The ERT has demonstrated clinical improvements of somatic manifestations
Address for correspondence: Daniel Araki Ribeiro, DDS, PhD, Departamento de Biocieˆncias, Universidade Federal de Sa˜o Paulo – UNIFESP, Av. Ana Costa 95, Santos – SP, 11060-001 Brazil. Phone/Fax: +55 13 38783823. E-mail: [email protected]; daribeiro@ pesquisador.cnpq.br
History Received 24 June 2014 Revised 14 August 2014 Accepted 16 August 2014 Published online 5 September 2014
as well as increase the quality of life (Tomatsu et al., 2013). This treatment comprises administration of a recombinant enzyme to reduce and prevent the accumulation of substrates specific for each disorder (Desnick, 2004). Among types of MPS described in the literature, MPS I is one of the most common lysosomal storage disease, with a reported incidence of per 100 000 population in Europe (Nelson, 1997). This autosomal recessive disorder is due to a deficiency or default in enzyme activity the alphaL -iduronidase with subsequent accumulation of the upstream metabolites as heparan (HS) and dermatan sulfates (DS) (Campos & Monaga, 2012). Similarly, the MPS II shows accumulation DS and HS and compromises the architecture and function of cells and organs, but this rare X-linked recessive disorder is due to a deficiency iduronate-2-sulfatase (I2S) (Christianto et al., 2013). MPS type VI, a deficiency of N-acetylgalactosamine-4-sulfatase deficiency or arylsulfatase B lead a cellular accumulation of DS as well (Valayannopoulos et al., 2010). The strategies for risk assessment and therapeutic handle are more effective with screening for potential genotoxicity and/or mutagenicity of MPS patients treated with ERT by the use of some assays, for example, the bacterial gene mutation assay, the mammalian cell gene mutation assay and
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
604
J. M. Guilheiro et al.
mouse lymphoma assays or chromosomal aberration test. However, these methods are typically laborious and timeconsuming or require highly trained technicians to accurately read and interpret slides. For this purpose, a great deal of enthusiasm was raised by the application of the micronucleus test to uncultured exfoliated cells (Belie¨n et al., 1995). Micronucleus arises from acentric fragments or whole chromosomes, which are not included into the main nuclei of the daughter cells. The formation of micronuclei can be induced by substances that cause chromosome breakage (clastogens) as well as by agents that affect the spindle apparatus (aneugens) (Belie¨n et al., 1995). According to Tolbert et al. (1992), the specificity of the test to detect genotoxic and cytotoxic effects is increased by recording other degenerative nuclear alterations indicative of cell death, besides the micronucleus. Among them, pyknosis, karyolysis and karyorrhexis are suitable for this purpose. To evaluate the magnitude of DNA damage, the comet assay is a relatively new, rapid, simple and reliable biochemical technique for evaluating DNA damage in mammalian cells (Tice et al., 2000). This technique includes embedding cells in agarose gel onto microscope slides, incubating them with the test compound and then lysing the cells with detergent and high salts (Tice et al., 2000). During electrophoresis under alkaline conditions, cells with damaged DNA display increased rates of DNA migration to the anode. The increase in DNA migration rate is caused by the formation of smaller fragments of DNA caused by double-strand breaks, single-strand breaks and alkali-labile sites. Smaller fragments of DNA migrate farther in the electric field compared with intact DNA and the cellular lysates thus resembles a ‘‘comet’’ with brightly fluorescent head and a tail region. In this study, we investigated the genotoxicity and mutagenicity induced by MPS in patients treated with ERT by means of comet assay and micronucleus test. To monitor cytotoxic effects, pyknosis, karyolysis and karyorrhexis were also evaluated in this setting.
Materials and methods Subjects The subjects of this study comprised a total of 12 patients with MPS type I, II or VI with age between 3 and 30 years old assisted at the Institute of Genetics and Inborn Errors of Metabolism with clinical and laboratory diagnosis, confirmed by direct analysis of a-L-iduronidase, I2S and arylsulfatase B activities (Mu¨ller et al., 2010) and urine GAGs (Cohen et al., 1977). All patients were submitted to ERT. The individual characteristics of all patients are demonstrated in Tables 1 and 2. A total of 10 healthy donors were used in this study. Three donors were female being 7, 11 and 22, and seven were male donors being 6, 7, 8, 21, 23, 25 and 30-years old. Each person was interviewed about possible confounding factors and was excluded from this study when there was a history of smoking or cancer, previous radio- or chemotherapy, use of therapeutic drugs, exposure to diagnostic X-rays during the past six months, intensive sportive activities during the last week and high alcohol consumption. All donors gave informed consent to participate in this study, and the study was approved by the
Toxicol Mech Methods, 2014; 24(8): 603–607
Table 1. Individual characteristic of all patients used in this study. Patients #
Age (years)
Gender
MPS I 1 2 3 4 Mean ± SD
6 11 25 26 17 ± 10.0
F F M F
MPS II 5 6 7 8 Mean ± SD
9 5 30 5 12.2 ± 11.9
M M M M
MPS VI 9 10 11 12 Mean ± SD
3 8 24 14 12.25 ± 9.0
M M M F
M ¼ male; F ¼ female. Table 2. Classification of patients according type of MPS, age of diagnosis, time of therapy (ERT) and frequency of therapy sessions.
Patient #
Frequency of Time of enzyme Age of therapy sessions diagnosis replacement therapy (weekly or bimonthly) (in months) (in months)
MPS I 1 4 2 7 3 24 4 72 Mean ± SD 26.7 ± 31.4
60 72 24 12 42 ± 28.5
W B W W
MPS II 5 60 6 19 7 84 8 60 Mean ± SD 55.7 ± 26.9
24 24 73 24 36.2 ± 24.5
W W W W
MPS VI 9 24 10 48 11 84 12 26 Mean ± SD 45.5 ± 27.8
12 9 20 59 25 ± 23.1
W W W W
Ethical Committee for Human Research, Universidade Federal de Sao Paulo, SP, Brazil (Protocol 1647/10). Micronucleus test in oral mucosa cells Exfoliated oral mucosa cells were collected from buccal mucosa. After rinsing the mouth with tap water, cells were obtained by scraping the right/left cheek mucosa with a disposable wood spatula. Cells were transferred to a tube containing saline solution (NaCl at 0.9% concentration in distilled water), centrifuged (800 rpm) for 5 min, fixed in 3:1 methanol/acetic acid and dropped onto pre-cleaned slides. Later, the air-dried slides were stained using the Feulgen/FastGreen method (Belie¨n et al., 1995) and examined under a light microscope at 400 magnification. A total of 1000 cells
DNA damage in mucopolyssacharydosis
DOI: 10.3109/15376516.2014.956913
were scored from each person from control and exposed groups as described elsewhere (Buajeeb et al., 2007). Micronucleus as well as pyknosis, karyolysis and karyorrhexis were identified as described elsewhere (Tolbert et al., 1992). Results were expressed in percentage (%). Such analysis was established in a previous study conducted by our research group (Angelieri et al., 2007).
Table 3. DNA damage (tail moment) in peripheral blood cells from MPS patients and treated with ERT. Groups
n
DNA damage (mean ± SD)
Control MPS I MPS II MPS VI
10 4 4 4
0.7 ± 1.2 4.7 ± 1.4a 5.3 ± 1.3a 3.2 ± 1.4a
a
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Single cell gel (comet) assay The protocol used for peripheral blood cells followed the guidelines outlined by Tice et al. (2000) with some modifications. Briefly, a volume of 5 mL from peripheral blood were added to 120 mL 0.5% low-melting-point agarose at 37 C, layered onto a pre-coated slide with 1.5% regular agarose and covered with a coverslip. Prior to electrophoresis, the slides were left in alkaline buffer (pH413) for 20 min and then electrophoresed for another 20 min at 0.7 V/cm and 300 mA. After electrophoresis, the slides were neutralized in 0.4 M Tris-HCl (pH 7.5), fixed in absolute ethanol and stored until analysis on a fluorescent microscope at 400 magnification. To measure DNA damage, two image analysis system parameters were considered: tail intensity (% migrated DNA) and tail moment (the product of the tail length and the fraction of DNA in the comet tail) (Tice et al., 2000). Tail moment is a virtual measure calculated by the computerized image analysis system considering both the length of DNA migration in the comet tail and the tail intensity.
605
p50.05 when compared to control group.
Table 4. Frequency of micronucleus in buccal mucosa cells from MPS patients treated with ERT. Groups
n
Micronucleus % (mean ± SD)
Control MPS I MPS II MPS VI
10 4 4 4
0.04 ± 0.04 0.02 ± 0.07 0.2 ± 0.07a 0.07 ± 0.01
a
p50.05 when compared to control group.
Table 5. Frequency (%) of cellular death (pyknosis, karyolysis and karyorrhexis) parameters of buccal mucosa cells from MPS patients treated with ERT. Groups
n
Pyknosis
Karyolysis
Karyorrhexis
Total
Control MPS I MPS II MPS VI
10 4 4 4
12.3 ± 2 12.2 ± 5.5 16 ± 4.5 16.5 ± 2.4
3.0 ± 1.7 4.1 ± 2.3 3.4 ± 3.4 9.8 ± 4
4.8 ± 1.6 4.2 ± 2.0 4.3 ± 1.4 7.8 ± 7.1
20.5 ± 8.5 20.6 ± 9.1 23.7 ± 11.3 34.2 ± 13.4a
a
p50.05 when compared to control group.
Statistical methods Values obtained from the comet assay (tail moment) were evaluated by one-way analysis of variance (ANOVA) followed by post-hoc analysis – Tukey test. Differences from micronucleus assay were statistically analyzed using the conditional test for comparing proportions in situations in which events are rare using SigmaStat software, version 1.0 (Jadel Scientific, Chicago, IL). The level of statistical significance was set at 5%.
Results In this study, statistically significant differences (p50.05) for DNA damage in MPS I patients treated with ERT was found in peripheral blood cells. In the same way, MPS II patients treated with ERT were able to induce genetic damage in peripheral blood cells. MPS IV and treated with ERT-induced DNA damage in peripheral blood cells. Overall, MPS associated with ERT induces genetic damage in peripheral blood cells. Such findings are listed in Table 3. Regarding micronucleus test, an increased number of micronucleated cells was observed in individuals presenting MPS II and treated with ERT with statistically significant differences (p50.05) when compared to control group. MPS I treated with ERT did not show remarkable differences between groups (Table 4). In a similar manner, MPS IV patients treated with ERT did no induce an increase of micronucleated cells in buccal mucosa cells. These results are listed in Table 4.
In addition, it was observed an increase of other nuclear alterations closely related to cytotoxicity as depicted by the frequency of pyknosis, karyolysis and karyorrhexis in buccal mucosa cells of MPS VI patients treated with ERT (p50.05). MPS I patients treated with ERT did not show statistically significant differences in cytotoxicity parameters when compared to control group (p40.05). Moreover, MPS II patients treated with ERT did not induce an increase of cytotoxicity in buccal mucosa cells. Such findings are listed in Table 5.
Discussion The goal of this study was to investigate genotoxicity and mutagenicity in MPS patients treated with ERT using the alkaline comet assay in peripheral blood cells and the micronucleus test in oral exfoliated cells. In Brazil, although there are no official data on the incidence of MPS, Coelho et al. (1997) reported that this disorder represented 54.5% lysosomal storage disease being MPS I and VI showed the highest frequency, followed by MPS II. For this reason, we chose MPS I, II and VI in this setting. To the best of our knowledge, the approach has not been addressed so far. The alkaline comet assay was chosen in this study because it is a rapid test and high sensitivity used to evaluated DNA breaks, especially single and double-strand breaks, alkali labile sites, DNA-DNA and DNA protein crosslink in individual cells and oxidative DNA base injury (Tice et al., 2000). Our results demonstrated that all types of MPS, even in
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term of ERT, were able to induce genetic damage in peripheral blood cells. Filippon et al. (2011) have demonstrated greater DNA damage in pretreatment MPS II patients when compared to controls. ERT led to a significant decrease in levels of DNA damage when compared to pretreatment, but did not reach control values (Filippon et al., 2011). An earlier study conducted by our research group has revealed that MPS I-induced genetic damage in mice peripheral blood cells (Noguti et al., 2011). This instability detected in MPS patients seems to be related to high susceptibility to oxidative stress (Pereira et al., 2008; Terman & Kurz, 2006). Although these mechanisms are not completely understood, it is known that the accumulation of metabolites (Ribas et al., 2010; Sitta et al., 2009) such as GAGs may increase the oxidative imbalance arising to direct or indirect enhancement of free radical formation or causing a decrease in the antioxidant activity of tissues (Wajner et al., 2004). The typical accumulation of GAGs found in MPS may lead to excessive production of reactive oxygen species, which can react with and damage proteins, DNA and RNA (Ribas et al., 2010). However, variation mechanisms, side health effects due to the storage of GAGs, age of diagnosis as well as beginning ERT may explain the discrepant values found for quantification of levels of DNA damage. Further studies are necessary to elucidate the issue. Micronucleated cell indexes may reflect genomic damage (He et al., 2000). The detection of an elevated frequency of micronuclei in a given population indicates increased risk of cancer. Our results demonstrated that the micronucleus frequencies were significantly different between control and MPS II patients treated with ERT in human buccal mucosa cells. By contrast, Noguti et al. (2011) did not find increased micronucleated cells in murine model of MPS I. Considering that 490% of all human cancers are of epithelial origin (Kujan et al., 2006) and the oral mucosa is in the close contact with the environment, we assumed that MPS II associated with ERT is able to induce genomic damage as a result of chromosomal breakage or loss in oral mucosa cells. To monitor cytotoxic effects, the frequencies of karyorrhexis, karyolysis and pyknosis were evaluated into this experimental design. MPS VI patients showed increased cytotoxicity in oral mucosa cells, suggesting that the cellular response to MPS VI and ERT could represent a cytotoxic stimulus. It is important to stress that cytotoxicity interferes with micronucleus induction since some micronucleated cells are inevitably lost after cytotoxic injury confirming, therefore, our present findings. Some studies have assumed that repeated exposure to cytotoxicants can result in chronic cell injury, compensatory cell proliferation, hyperplasia and ultimately tumor development (Swenberg, 1993). In fact, a correlation between cell proliferation and induction of cancer is assumed (Sugano et al., 2001). Probably, proliferation may increase the risk of mutations within target cells, and also be important in selective clonal expansion of (exogenously or endogenously) initiated cells from preneoplastic foci as far as malignant tumors (Mally & Jagetia, 2002). Besides the power of the statistical analysis as a critical factor for the determination of an effect, various additional explanations (including seasonal and regional differences) for the reported discrepancies have been
Toxicol Mech Methods, 2014; 24(8): 603–607
proposed (Jagetia et al., 2001). Particularly, some confounding factors are important to be considered to human cytogenetic studies. Viruses, alterations in the immune system, failures in DNA repair system and inter-individual variations have already been associated with increased frequencies of chromosome aberrations. Furthermore, an age-related increase of micronuclei has been postulated (Torres-Buga´rin et al., 2003). Due to the complexity for collecting data from experimental group, it was not possible to correlate the frequency of micronucleated cells as well as cytotoxicity with age and gender in this setting. In conclusion, the results of the present study suggest that MPS associated with ERT induces genetic damage in peripheral blood cells and buccal mucosa cells. This effect appears to be more pronounced in MPS II patients. Since DNA damage and cellular death are considered to be prime mechanisms during chemical carcinogenesis, these data may be relevant in risk assessment for protecting human health and preventing carcinogenesis in these disease patients. However, further elucidation in forthcoming studies is welcomed.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. D. A. R. and V. D’A. are recipients of the CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) fellowship. This study was supported by CNPq and FAPESP (Fundac¸a˜o de Amparo a Pesquisa do Estado de Sao Paulo).
References Angelieri A, Oliveira GR, Sannomiya EK, Ribeiro DA. (2007). DNA damage and cellular death in oral mucosa cells of children who have undergone panoramic dental radiography. Ped Radiol 37:561–5. Belie¨n JA, Copper MP, Braakhuis BJ, et al. (1995). Standardization of counting micronuclei, definition of a protocol to measure genotoxic damage in human exfoliated cells. Carcinogenesis 16:2395–400. Buajeeb W, Kraivaphan P, Amornchat C, Triratana T. (2007). Frequency of micronucleated exfoliated cells in oral lichen planus. Mutat Res 627:191–6. Campos D, Monaga M. (2012). Mucopolysaccharidosis type I, current knowledge on its pathophysiological mechanisms. Metab Brain Dis 27:121–9. Christianto A, Watanabe H, Nakajima T, Inazu T. (2013). Idursulfase enzyme replacement therapy in an adult patient with severe Hunter syndrome having a novel mutation of iduronate-2-sulfatase gene. Clin Chim Acta 423:66–8. Coelho JC, Wajner M, Burin MG, et al. (1997). Selective screening of 10,000 high-risk Brazilian patients for the detection of inborn errors of metabolism. Eur J Pediatr 156:650–4. Cohen DM, Mourao PA, Dietrich CP. (1977). Differentiation of mucopolysaccharidoses by analyses of the excreted sulfated mucopolysaccharides. Clin Chim Acta 80:555–62. Desnick RJ. (2004). Enzyme replacement and enhancement therapies for lysosomal diseases. J Inherit Metab Dis 27:385–410. Filippon L, Wayhs CA, Atik DM, et al. (2011). DNA damage in leukocytes from pretreatment mucopolysaccharidosis type II patients, protective effect of enzyme replacement therapy. Mutat Res 721: 206–10. He JL, Chen LF, Jin LF, Jin HY. (2000). Comparative evaluation of the in vitro micronucleus test and the comet assay for the detection of genotoxic effects of X-ray radiation. Mutat Res 469:223–31. Jagetia GC, Jayakrishnan A, Fernandes D, Vidyasagar MS. (2001). Evaluation of micronuclei frequency in the cultured peripheral blood
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lymphocytes of cancer patients before and after radiation treatment. Mutat Res 491:9–16. Kujan O, Oliver RJ, Khattab A, et al. (2006). Evaluation of a new binary system of grading oral epithelial dysplasia for prediction of malignant transformation. Oral Oncol 42:987–93. Mally A, Jagetia JK. (2002). Non-genotoxic carcinogens, early effects on gap junctions, cell proliferation and apoptosis in the rat. Toxicology 180:233–48. Mu¨ller KB, Rodrigues MD, Pereira VG, et al. (2010). Reference values for lysosomal enzymes activities using dried blood spots samples – a Brazilian experience. Diagn Pathol 5:65. Nelson J. (1997). Incidence of the mucopolysaccharidoses in Northern Ireland. Hum Genet 101:355. Neufeld EF, Muenzer J. (2001). The mucopolysaccharidoses. In Scriver CR, Sly WS, ed. The metabolic and molecular bases of inherited disease. New York: McGraw Hill, 3421–52. Noguti J, Pereira VG, Martins AM, et al. (2011). Genomic instability in blood cells from murine model of mucopolysaccharidosis type I. J Mol Histol 42:575–8. Pereira VG, Martins AM, Micheletti C, D’Almeida V. (2008). Mutational and oxidative stress analysis in patients with mucopolysaccharidosis type I undergoing enzyme replacement therapy. Clin Chim Acta 387: 75–9. Perenc L. (2013). Anthropometric characteristics of four Polish children with mucopolysaccharidosis. BMC Res Notes 6:246. Ribas GS, Manfredini V, de Marco MG, et al. (2010). Prevention by L-carnitine of DNA damage induced by propionic and L-methylmalonic acids in human peripheral leukocytes in vitro. Mutat Res 702: 123–8.
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Sitta A, Manfredini V, Biasi L, et al. (2009). Evidence that DNA damage is associated to phenylalanine blood levels in leukocytes from phenylketonuric patients. Mutat Res 679:13–16. Sugano N, Minigeshi T, Kawamoto K, Ito K. (2001). Nicotine inhibits UV-induced activation of the apoptotic pathway. Toxicol Lett 125: 61–5. Swenberg JA. (1993). Cell proliferation and chemical carcinogenesis, conferences summary and future directions. Environ Health Perspect 101:153–8. Terman A, Kurz T, Gustafsson B, Brunk UT. (2006). Lysosomal labilization. IUBMB Life 58:531–9. Tice RR, Agurell E, Anderson D, et al. (2000). Single cell gel/comet assay, guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–21. Tolbert PE, Shy CM, Allen JW. (1992). Micronuclei and other nuclear anomalies in buccal smears, methods development. Mutat Res 271: 69–77. Tomatsu S, Fujii T, Fukushi M, et al. (2013). Newborn screening and diagnosis of mucopolysaccharidoses. Mol Genet Metab 110:42–53. Torres-Buga´rin O, Ventura-Aguilar A, Zamora-Pilar A, et al. (2003). Evaluation of cisplatin +5-FU, carboplatin +5-FU, and ifosfamide + epirubicine regimens using the micronuclei test and nuclear abnormalities in buccal mucosa. Mutat Res 539:177–86. Valayannopoulos V, Nicely H, Harmatz P, Turbeville S. (2010). Mucopolysaccharidosis VI. Orphanet J Rare Dis 5:5. Wajner A, Michelin K, Burin MG, et al. (2004). Biochemical characterization of chitotriosidase enzyme, comparison between normal individuals and patients with Gaucher and with Niemann-Pick diseases. Clin Biochem 37:893–7.
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RESEARCH ARTICLE
Cytogenetic biomonitoring in mucopolyssacharosis I, II and IV patients treated with enzyme replacement therapy Joice Marques Guilheiro1, Marcelo Donizetti Chaves1, Ana Maria Martins2, Daniel Araki Ribeiro1, and Vania D’Almeida1,3 1
Department of Biosciences, 2Department of Pediatrics and 3Department of Psychobiology, Federal University of Sao Paulo, UNIFESP, SP, Brazil
Abstract
Keywords
Background and objectives: The aim of this study was to evaluate genotoxicity and mutagenicity in peripheral blood and buccal mucosal cells in mucopolysaccharidosis (MPS) I, II or VI patients. Methods: A total of 12 patients with MPS type I, II and VI attended at the Institute of Genetics and Inborn Errors of Metabolism treated with enzyme replacement therapy (ERT) and 10 healthy control volunteers were included in this study. Mechanically exfoliated cells from cheek mucosa (left and right side) were used to micronucleus test and single cell gel (comet) assay in peripheral blood cells. Results: The results of this study detected the presence of genetic damage in peripheral blood for all individuals with MPS treated with ERT, regardless of type of MPS as depicted by tail moment results. In addition, an increased number of micronucleated cells were found in buccal cells of MPS type II patients. It was also observed an increase of other nuclear alterations closely related to cytotoxicity as depicted by the frequency of pyknosis, karyolysis and karyorrhexis in buccal mucosa cells of MPS VI patients (p50.05). Conclusion: Taken together, such results demonstrate that metabolic alterations induced by the enzymatic deficiency characteristic of MPS associated with ERT therapy can induce genotoxicity and mutagenicity in peripheral blood and buccal mucosa cells, respectively. This effect appears to be more pronounced to MPS II.
Comet assay, genetic damage, micronucleus test, mucopolyssacharodosis
Introduction Mucopolysaccharidosis (MPS) is a genetically heterogeneous group of metabolic disorders caused by a deficiency or absence of enzymes responsible for the lysosomal degradation of glycosaminoglycans (GAGs). In this context, GAGs are able to accumulate in lysosomes, extracellular matrix and body fluids (Neufeld & Muenzer, 2001). Clinical presentation of MPS could include some symptoms and characteristics such as short stature, facial dysmorphism, skeletal deformities, pulmonary dysfunction, joint stiffness and contractures, myocardial hypertrophy, neurological symptoms, mental retardation and potentially major adverse consequences for speech, language and cognitive development (Perenc, 2013). Nowadays, MPS has been treated with enzyme replacement therapy (ERT) (Desnick, 2004). The ERT has demonstrated clinical improvements of somatic manifestations
Address for correspondence: Daniel Araki Ribeiro, DDS, PhD, Departamento de Biocieˆncias, Universidade Federal de Sa˜o Paulo – UNIFESP, Av. Ana Costa 95, Santos – SP, 11060-001 Brazil. Phone/Fax: +55 13 38783823. E-mail: [email protected]; daribeiro@ pesquisador.cnpq.br
History Received 24 June 2014 Revised 14 August 2014 Accepted 16 August 2014 Published online 5 September 2014
as well as increase the quality of life (Tomatsu et al., 2013). This treatment comprises administration of a recombinant enzyme to reduce and prevent the accumulation of substrates specific for each disorder (Desnick, 2004). Among types of MPS described in the literature, MPS I is one of the most common lysosomal storage disease, with a reported incidence of per 100 000 population in Europe (Nelson, 1997). This autosomal recessive disorder is due to a deficiency or default in enzyme activity the alphaL -iduronidase with subsequent accumulation of the upstream metabolites as heparan (HS) and dermatan sulfates (DS) (Campos & Monaga, 2012). Similarly, the MPS II shows accumulation DS and HS and compromises the architecture and function of cells and organs, but this rare X-linked recessive disorder is due to a deficiency iduronate-2-sulfatase (I2S) (Christianto et al., 2013). MPS type VI, a deficiency of N-acetylgalactosamine-4-sulfatase deficiency or arylsulfatase B lead a cellular accumulation of DS as well (Valayannopoulos et al., 2010). The strategies for risk assessment and therapeutic handle are more effective with screening for potential genotoxicity and/or mutagenicity of MPS patients treated with ERT by the use of some assays, for example, the bacterial gene mutation assay, the mammalian cell gene mutation assay and
Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
604
J. M. Guilheiro et al.
mouse lymphoma assays or chromosomal aberration test. However, these methods are typically laborious and timeconsuming or require highly trained technicians to accurately read and interpret slides. For this purpose, a great deal of enthusiasm was raised by the application of the micronucleus test to uncultured exfoliated cells (Belie¨n et al., 1995). Micronucleus arises from acentric fragments or whole chromosomes, which are not included into the main nuclei of the daughter cells. The formation of micronuclei can be induced by substances that cause chromosome breakage (clastogens) as well as by agents that affect the spindle apparatus (aneugens) (Belie¨n et al., 1995). According to Tolbert et al. (1992), the specificity of the test to detect genotoxic and cytotoxic effects is increased by recording other degenerative nuclear alterations indicative of cell death, besides the micronucleus. Among them, pyknosis, karyolysis and karyorrhexis are suitable for this purpose. To evaluate the magnitude of DNA damage, the comet assay is a relatively new, rapid, simple and reliable biochemical technique for evaluating DNA damage in mammalian cells (Tice et al., 2000). This technique includes embedding cells in agarose gel onto microscope slides, incubating them with the test compound and then lysing the cells with detergent and high salts (Tice et al., 2000). During electrophoresis under alkaline conditions, cells with damaged DNA display increased rates of DNA migration to the anode. The increase in DNA migration rate is caused by the formation of smaller fragments of DNA caused by double-strand breaks, single-strand breaks and alkali-labile sites. Smaller fragments of DNA migrate farther in the electric field compared with intact DNA and the cellular lysates thus resembles a ‘‘comet’’ with brightly fluorescent head and a tail region. In this study, we investigated the genotoxicity and mutagenicity induced by MPS in patients treated with ERT by means of comet assay and micronucleus test. To monitor cytotoxic effects, pyknosis, karyolysis and karyorrhexis were also evaluated in this setting.
Materials and methods Subjects The subjects of this study comprised a total of 12 patients with MPS type I, II or VI with age between 3 and 30 years old assisted at the Institute of Genetics and Inborn Errors of Metabolism with clinical and laboratory diagnosis, confirmed by direct analysis of a-L-iduronidase, I2S and arylsulfatase B activities (Mu¨ller et al., 2010) and urine GAGs (Cohen et al., 1977). All patients were submitted to ERT. The individual characteristics of all patients are demonstrated in Tables 1 and 2. A total of 10 healthy donors were used in this study. Three donors were female being 7, 11 and 22, and seven were male donors being 6, 7, 8, 21, 23, 25 and 30-years old. Each person was interviewed about possible confounding factors and was excluded from this study when there was a history of smoking or cancer, previous radio- or chemotherapy, use of therapeutic drugs, exposure to diagnostic X-rays during the past six months, intensive sportive activities during the last week and high alcohol consumption. All donors gave informed consent to participate in this study, and the study was approved by the
Toxicol Mech Methods, 2014; 24(8): 603–607
Table 1. Individual characteristic of all patients used in this study. Patients #
Age (years)
Gender
MPS I 1 2 3 4 Mean ± SD
6 11 25 26 17 ± 10.0
F F M F
MPS II 5 6 7 8 Mean ± SD
9 5 30 5 12.2 ± 11.9
M M M M
MPS VI 9 10 11 12 Mean ± SD
3 8 24 14 12.25 ± 9.0
M M M F
M ¼ male; F ¼ female. Table 2. Classification of patients according type of MPS, age of diagnosis, time of therapy (ERT) and frequency of therapy sessions.
Patient #
Frequency of Time of enzyme Age of therapy sessions diagnosis replacement therapy (weekly or bimonthly) (in months) (in months)
MPS I 1 4 2 7 3 24 4 72 Mean ± SD 26.7 ± 31.4
60 72 24 12 42 ± 28.5
W B W W
MPS II 5 60 6 19 7 84 8 60 Mean ± SD 55.7 ± 26.9
24 24 73 24 36.2 ± 24.5
W W W W
MPS VI 9 24 10 48 11 84 12 26 Mean ± SD 45.5 ± 27.8
12 9 20 59 25 ± 23.1
W W W W
Ethical Committee for Human Research, Universidade Federal de Sao Paulo, SP, Brazil (Protocol 1647/10). Micronucleus test in oral mucosa cells Exfoliated oral mucosa cells were collected from buccal mucosa. After rinsing the mouth with tap water, cells were obtained by scraping the right/left cheek mucosa with a disposable wood spatula. Cells were transferred to a tube containing saline solution (NaCl at 0.9% concentration in distilled water), centrifuged (800 rpm) for 5 min, fixed in 3:1 methanol/acetic acid and dropped onto pre-cleaned slides. Later, the air-dried slides were stained using the Feulgen/FastGreen method (Belie¨n et al., 1995) and examined under a light microscope at 400 magnification. A total of 1000 cells
DNA damage in mucopolyssacharydosis
DOI: 10.3109/15376516.2014.956913
were scored from each person from control and exposed groups as described elsewhere (Buajeeb et al., 2007). Micronucleus as well as pyknosis, karyolysis and karyorrhexis were identified as described elsewhere (Tolbert et al., 1992). Results were expressed in percentage (%). Such analysis was established in a previous study conducted by our research group (Angelieri et al., 2007).
Table 3. DNA damage (tail moment) in peripheral blood cells from MPS patients and treated with ERT. Groups
n
DNA damage (mean ± SD)
Control MPS I MPS II MPS VI
10 4 4 4
0.7 ± 1.2 4.7 ± 1.4a 5.3 ± 1.3a 3.2 ± 1.4a
a
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Single cell gel (comet) assay The protocol used for peripheral blood cells followed the guidelines outlined by Tice et al. (2000) with some modifications. Briefly, a volume of 5 mL from peripheral blood were added to 120 mL 0.5% low-melting-point agarose at 37 C, layered onto a pre-coated slide with 1.5% regular agarose and covered with a coverslip. Prior to electrophoresis, the slides were left in alkaline buffer (pH413) for 20 min and then electrophoresed for another 20 min at 0.7 V/cm and 300 mA. After electrophoresis, the slides were neutralized in 0.4 M Tris-HCl (pH 7.5), fixed in absolute ethanol and stored until analysis on a fluorescent microscope at 400 magnification. To measure DNA damage, two image analysis system parameters were considered: tail intensity (% migrated DNA) and tail moment (the product of the tail length and the fraction of DNA in the comet tail) (Tice et al., 2000). Tail moment is a virtual measure calculated by the computerized image analysis system considering both the length of DNA migration in the comet tail and the tail intensity.
605
p50.05 when compared to control group.
Table 4. Frequency of micronucleus in buccal mucosa cells from MPS patients treated with ERT. Groups
n
Micronucleus % (mean ± SD)
Control MPS I MPS II MPS VI
10 4 4 4
0.04 ± 0.04 0.02 ± 0.07 0.2 ± 0.07a 0.07 ± 0.01
a
p50.05 when compared to control group.
Table 5. Frequency (%) of cellular death (pyknosis, karyolysis and karyorrhexis) parameters of buccal mucosa cells from MPS patients treated with ERT. Groups
n
Pyknosis
Karyolysis
Karyorrhexis
Total
Control MPS I MPS II MPS VI
10 4 4 4
12.3 ± 2 12.2 ± 5.5 16 ± 4.5 16.5 ± 2.4
3.0 ± 1.7 4.1 ± 2.3 3.4 ± 3.4 9.8 ± 4
4.8 ± 1.6 4.2 ± 2.0 4.3 ± 1.4 7.8 ± 7.1
20.5 ± 8.5 20.6 ± 9.1 23.7 ± 11.3 34.2 ± 13.4a
a
p50.05 when compared to control group.
Statistical methods Values obtained from the comet assay (tail moment) were evaluated by one-way analysis of variance (ANOVA) followed by post-hoc analysis – Tukey test. Differences from micronucleus assay were statistically analyzed using the conditional test for comparing proportions in situations in which events are rare using SigmaStat software, version 1.0 (Jadel Scientific, Chicago, IL). The level of statistical significance was set at 5%.
Results In this study, statistically significant differences (p50.05) for DNA damage in MPS I patients treated with ERT was found in peripheral blood cells. In the same way, MPS II patients treated with ERT were able to induce genetic damage in peripheral blood cells. MPS IV and treated with ERT-induced DNA damage in peripheral blood cells. Overall, MPS associated with ERT induces genetic damage in peripheral blood cells. Such findings are listed in Table 3. Regarding micronucleus test, an increased number of micronucleated cells was observed in individuals presenting MPS II and treated with ERT with statistically significant differences (p50.05) when compared to control group. MPS I treated with ERT did not show remarkable differences between groups (Table 4). In a similar manner, MPS IV patients treated with ERT did no induce an increase of micronucleated cells in buccal mucosa cells. These results are listed in Table 4.
In addition, it was observed an increase of other nuclear alterations closely related to cytotoxicity as depicted by the frequency of pyknosis, karyolysis and karyorrhexis in buccal mucosa cells of MPS VI patients treated with ERT (p50.05). MPS I patients treated with ERT did not show statistically significant differences in cytotoxicity parameters when compared to control group (p40.05). Moreover, MPS II patients treated with ERT did not induce an increase of cytotoxicity in buccal mucosa cells. Such findings are listed in Table 5.
Discussion The goal of this study was to investigate genotoxicity and mutagenicity in MPS patients treated with ERT using the alkaline comet assay in peripheral blood cells and the micronucleus test in oral exfoliated cells. In Brazil, although there are no official data on the incidence of MPS, Coelho et al. (1997) reported that this disorder represented 54.5% lysosomal storage disease being MPS I and VI showed the highest frequency, followed by MPS II. For this reason, we chose MPS I, II and VI in this setting. To the best of our knowledge, the approach has not been addressed so far. The alkaline comet assay was chosen in this study because it is a rapid test and high sensitivity used to evaluated DNA breaks, especially single and double-strand breaks, alkali labile sites, DNA-DNA and DNA protein crosslink in individual cells and oxidative DNA base injury (Tice et al., 2000). Our results demonstrated that all types of MPS, even in
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J. M. Guilheiro et al.
term of ERT, were able to induce genetic damage in peripheral blood cells. Filippon et al. (2011) have demonstrated greater DNA damage in pretreatment MPS II patients when compared to controls. ERT led to a significant decrease in levels of DNA damage when compared to pretreatment, but did not reach control values (Filippon et al., 2011). An earlier study conducted by our research group has revealed that MPS I-induced genetic damage in mice peripheral blood cells (Noguti et al., 2011). This instability detected in MPS patients seems to be related to high susceptibility to oxidative stress (Pereira et al., 2008; Terman & Kurz, 2006). Although these mechanisms are not completely understood, it is known that the accumulation of metabolites (Ribas et al., 2010; Sitta et al., 2009) such as GAGs may increase the oxidative imbalance arising to direct or indirect enhancement of free radical formation or causing a decrease in the antioxidant activity of tissues (Wajner et al., 2004). The typical accumulation of GAGs found in MPS may lead to excessive production of reactive oxygen species, which can react with and damage proteins, DNA and RNA (Ribas et al., 2010). However, variation mechanisms, side health effects due to the storage of GAGs, age of diagnosis as well as beginning ERT may explain the discrepant values found for quantification of levels of DNA damage. Further studies are necessary to elucidate the issue. Micronucleated cell indexes may reflect genomic damage (He et al., 2000). The detection of an elevated frequency of micronuclei in a given population indicates increased risk of cancer. Our results demonstrated that the micronucleus frequencies were significantly different between control and MPS II patients treated with ERT in human buccal mucosa cells. By contrast, Noguti et al. (2011) did not find increased micronucleated cells in murine model of MPS I. Considering that 490% of all human cancers are of epithelial origin (Kujan et al., 2006) and the oral mucosa is in the close contact with the environment, we assumed that MPS II associated with ERT is able to induce genomic damage as a result of chromosomal breakage or loss in oral mucosa cells. To monitor cytotoxic effects, the frequencies of karyorrhexis, karyolysis and pyknosis were evaluated into this experimental design. MPS VI patients showed increased cytotoxicity in oral mucosa cells, suggesting that the cellular response to MPS VI and ERT could represent a cytotoxic stimulus. It is important to stress that cytotoxicity interferes with micronucleus induction since some micronucleated cells are inevitably lost after cytotoxic injury confirming, therefore, our present findings. Some studies have assumed that repeated exposure to cytotoxicants can result in chronic cell injury, compensatory cell proliferation, hyperplasia and ultimately tumor development (Swenberg, 1993). In fact, a correlation between cell proliferation and induction of cancer is assumed (Sugano et al., 2001). Probably, proliferation may increase the risk of mutations within target cells, and also be important in selective clonal expansion of (exogenously or endogenously) initiated cells from preneoplastic foci as far as malignant tumors (Mally & Jagetia, 2002). Besides the power of the statistical analysis as a critical factor for the determination of an effect, various additional explanations (including seasonal and regional differences) for the reported discrepancies have been
Toxicol Mech Methods, 2014; 24(8): 603–607
proposed (Jagetia et al., 2001). Particularly, some confounding factors are important to be considered to human cytogenetic studies. Viruses, alterations in the immune system, failures in DNA repair system and inter-individual variations have already been associated with increased frequencies of chromosome aberrations. Furthermore, an age-related increase of micronuclei has been postulated (Torres-Buga´rin et al., 2003). Due to the complexity for collecting data from experimental group, it was not possible to correlate the frequency of micronucleated cells as well as cytotoxicity with age and gender in this setting. In conclusion, the results of the present study suggest that MPS associated with ERT induces genetic damage in peripheral blood cells and buccal mucosa cells. This effect appears to be more pronounced in MPS II patients. Since DNA damage and cellular death are considered to be prime mechanisms during chemical carcinogenesis, these data may be relevant in risk assessment for protecting human health and preventing carcinogenesis in these disease patients. However, further elucidation in forthcoming studies is welcomed.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. D. A. R. and V. D’A. are recipients of the CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) fellowship. This study was supported by CNPq and FAPESP (Fundac¸a˜o de Amparo a Pesquisa do Estado de Sao Paulo).
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