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Apr 9, 2009 - [11] S. Walitza, B. Werner, M. Romanos, A. Warnke, M. Gerlach, ... M.D. Shelby, N. Itchon-Ramos, M. Faircloth, G.E. Kissling, A.K. Chris- man, H.
Mutation Research 666 (2009) 44–49

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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres

Absence of cytogenetic effects in children and adults with attention-deficit/hyperactivity disorder treated with methylphenidate Immaculada Ponsa a,b,c , Josep Antoni Ramos-Quiroga d,e , Marta Ribasés d , Rosa Bosch d , Anna Bielsa d , Maria Teresa Ordeig k , Marta Morell a , Rosa Miró b,c , Rafael de Cid a,f,g , Xavier Estivill a,f,g , Miquel Casas d,e , Mònica Bayés a,f,g , Bru Cormand h,i,j , Amaia Hervás k,∗ a

Genes and Disease Program, Center for Genomic Regulation (CRG), UPF, Barcelona, Catalonia, Spain Departament de Biologia Cel.lular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Catalonia, Spain c Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Catalonia, Spain d Department of Psychiatry, Hospital Universitari Vall d’Hebron, Barcelona, Catalonia, Spain e Department of Psychiatry and Legal Medicine, Universitat Autònoma de Barcelona, Catalonia, Spain f CIBER Epidemiología y Salud Pública, Instituto de Salud Carlos III (CRG), Barcelona, Catalonia, Spain g Centro Nacional de Genotipado (CeGen), Barcelona, Catalonia, Spain h Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Catalonia, Spain i CIBER Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Catalonia, Spain j Institut de Biomedicina de la Universitat de Barcelona (IBUB), Catalonia, Spain k Child and Adolescent Mental Health Unit, Hospital Mútua de Terrassa, Rambla de Egara 386-388, 6th floor, 08221 Terrassa, Spain b

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Article history: Received 7 October 2008 Received in revised form 9 March 2009 Accepted 31 March 2009 Available online 9 April 2009 Keywords: ADHD Methylphenidate Cytogenetic effects Children Adult

a b s t r a c t Attention-deficit/hyperactivity disorder (ADHD) is the most common psychiatric condition with onset in childhood, and in more than 50% of cases it persists into adulthood as a chronic disorder. Over five million methylphenidate (MPH) prescriptions were issued in the USA in 2003, mostly for children. A previous report [R.A. El-Zein, S.Z. Abdel-Rahman, M.J. Hay, M.S. Lopez, M.L. Bondy, D.L. Morris and M.S. Legator Cytogenetic effects in children treated with methylphenidate, Cancer Lett. 230 (2005) 284–291.] described the induction of chromosome abnormalities by MPH in children treated for three months, contrary to most of the in vitro and in vivo studies reported since then. We present new relevant information concerning the cytogenetic effects of MPH in children and adults. We include a prospective sample of 12 children and 7 adults with a new diagnosis of ADHD and naive to MPH. We analyzed the cytogenetic effects on peripheral lymphocytes before and three months after starting MPH therapy. The cytogenetic analyses included a cytokinesis-block micronucleus (CBMN) assay, a sister chromatid exchange (SCE) analysis and the determination of chromosome aberrations (CA). Following the same strategy and analyzing the same cytogenetic endpoints that were investigated in the original report [R.A. El-Zein, S.Z. Abdel-Rahman, M.J. Hay, M.S. Lopez, M.L. Bondy, D.L. Morris and M.S. Legator Cytogenetic effects in children treated with methylphenidate, Cancer Lett. 230 (2005) 284–291.], we found no evidence of increased frequency of micronuclei, sister chromatid exchanges or chromosome aberrations induced by MPH in children and adult populations. MPH treatment of children and adults with ADHD resulted in no significant genomic damage (as suggested by the three endpoints studied), results that do not support a potential increased risk of cancer after exposure to MPH. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Methylphenidate (MPH) is a widely used stimulant drug of the central nervous system. It has been proven to be an effective treatment for attention-deficit/hyperactivity disorder (ADHD) [2], a common psychiatric disorder with onset in childhood that con-

∗ Corresponding author. Tel.: +34 937365903; fax: +34 937332317. E-mail addresses: [email protected], [email protected] (A. Hervás). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.03.014

tinues in between 49–66% of cases into adulthood as chronic ADHD with severe limitations on school, work, family and social functioning [2]. ADHD is a very common disorder and occurs in 8–12% of children and in 2–4% of adults [3,4]. Mutagenicity or cell transformation studies with MPH have generally been negative [5–7] but reports of a clastogenic potential in mammalian cells [8,9] and human lymphocytes in vitro [10] have rendered contradictory results on the clastogenic activity of methylphenidate in non-clinical studies. Four studies have analyzed the cytogenetic effects of MPH on peripheral blood lym-

I. Ponsa et al. / Mutation Research 666 (2009) 44–49

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Table 1 Gender, age, MPH dose, treatment, weight and clinical type of ADHD patients. Sample

Gender

Age (years)

MPH dose (mg/day)

Treatment

Weight (kg)

ADHD type

First month

Second month

Third month

ADHD6 ADHD7 ADHD8 ADHD5 ADHD12 ADHD20 ADHD4 ADHD18 ADHD10 ADHD19 ADHD27 ADHD32

Male Male Male Male Female Male Female Male Male Male Female Male

7 7 7 8 9 10 10 11 13 13 13 14

18 15 10 18 15 15 20 15 20 20 18 45

18 15 15 18 15 15 20 15 20 20 18 50

18 20 15 18 15 15 20 20 20 36 18 50

OROS-MPH ir-MPH ir-MPH OROS-MPH ir-MPH ir-MPH ir-MPH ir-MPH ir-MPH ir-MPH + OROS-MPH* OROS-MPH ir-MPH

25 24 38 32 27 32 55 30 54 58 62 77

Combined Combined Combined Combined Inattentive Combined Combined Combined Combined Combined Inattentive Combined

ADHD14 ADHD26 ADHD23 ADHD22 ADHD24 ADHD9 ADHD31

Female Male Male Male Male Male Male

21 27 31 33 39 47 58

54 54 72 54 54 36 54

54 72 90 90 54 72 72

54 72 90 90 54 72 72

OROS-MPH OROS-MPH OROS-MPH OROS-MPH OROS-MPH OROS-MPH OROS-MPH

48 85 119 86 83 79 65

Combined Combined Combined Combined Inattentive Combined Combined

MPH: methylphenidate; ir-MPH: immediate-release methylphenidate; OROS-MPH: extended-release methylphenidate; dotted line divides children and adult groups; ‘*’ ir-MPH during the first and second month and OROS-MPH during the third month after initiation of treatment.

phocytes in children and have shown opposite results [1,11–13]. El-Zein et al. [1] included 12 children treated with therapeutic doses of short-action MPH and after three months of treatment there was an increase in the number of chromosome aberrations, sister chromatid exchanges and micronuclei frequencies in peripheral blood lymphocytes. Significant controversy has arisen from these results [14,15]. Walitza et al. [11] in 2007 studied two samples of children that were assessed for alterations in micronuclei frequency: children with ADHD naive to MPH assessed at one, three or six months after starting MPH treatment, and children with ADHD on MPH treatment for more than six months (chronic group). None of the child groups showed any genomic damage. Two years later the same group published a study with a larger sample and an extended treatment period which added another cell type, buccal mucosa [13].They concluded that no genomic damage, in terms of micronuclei frequency in peripheral lymphocytes or buccal mucosa cells, was detected after MPH treatment. Finally, Witt et al. [12] studied the possible induction of chromosomal damage (chromosome aberrations, micronuclei and sister chromatid exchanges) by methylphenidate or amphetamine-based drugs in the peripheral blood lymphocytes of children after three months of continuous treatment. They concluded that therapeutic levels of MPH do not induce cytogenetic damage in humans. Concern arose because cohort studies showed an association between the frequency of cells with structural chromosomal aberrations or micronuclei in peripheral lymphocytes and the risk of cancer [16]. One study has been published on the association between the use of MPH and cancer [17]. The authors explored a pharmacy database and a cancer registry and found some evidence of an association between uses of MPH in people aged under 20 and an increased risk of lymphocytic leukaemia, although they suggested that this association may also be due to chance alone. Methylphenidate is an effective and widely used treatment for children and adults with ADHD [18]. In 2003 over five million MPH prescriptions were issued in the USA mostly for children who took the drug for several years [19] and in many countries it is the only pharmacological treatment available for ADHD. Any link between MPH and adverse effects requires especially intensive and careful attention, and more clinical cytogenetic studies of children and adults on MPH are very much needed [14]. The present study was conducted in a paediatric and adult population with a new diagnosis of ADHD to explore whether

patients treated with therapeutic doses of short and OROS extended release-acting methylphenidate had an increased rate of cytogenetic abnormalities in lymphocytes. 2. Materials and methods 2.1. Subjects A total of 32 Caucasoid patients that met DSM-IV criteria (Diagnostic and Statistical Manual of Mental Disorder, fourth edition) [20] were recruited between February and March 2006 and consisted of 22 children and 10 adults. The ethics committee of each participating institution approved the study and written informed consent was obtained from all adult subjects, children and their parents. All patients were naive to methylphenidate treatment. Blood samples were collected before and three months after initiation of MPH treatment. During the course of the study 13 subjects stopped taking medication or switched to a non-MPH medication, which means a total clinical sample of 19 ADHD patients completed the study (12 children and 7 adults). Table 1 shows individual phenotypic and therapeutic information for these patients. The average age (± SD) was 10.17 ± 2.62 years in the child group and 36.57 ± 12.59 years in the adult sample. The mean weight (± SD) of the paediatric population was 42.80 ± 17.62 kg and 80.69 ± 21.74 kg for adults. Therapeutic doses of MPH for children ranged from 10 to 50 mg/day with an average dose of 19.08 ± 8.67 mg in the first month, 19.92 ± 9.71 mg in the second month and 22.08 ± 10.40 mg in the third month, and the mean ratio dose of MPH at the third month/weight was 0.54 ± 0.17 mg/kg. In the adult ADHD clinical sample, therapeutic doses of MPH ranged from 36 to 90 mg/day with an average dose of 54.00 ± 10.39 mg in the first month and 72.00 ± 14.70 mg in the second and third month, with a mean ratio dose of MPH at the third month/weight of 0.92 ± 0.18 mg/kg. Nine children were on immediate-release methylphenidate (ir-MPH) and three on extended-release methylphenidate (OROS-MPH) during the first and second month of treatment, while in the third month one patient switched from ir-MPH to OROS-MPH. All adult patients were on OROS-MPH treatment during the study. 2.2. Clinical assessment 2.2.1. Adult ADHD All adult ADHD patients were recruited at a single hospital. The diagnostic interview for ADHD in adults included the Structured Clinical Interview for DSM-IV Axis I and II Disorders (SCID-I and SCID-II) [21] and the Conners’ Adult ADHD Diagnostic Interview for DSM-IV (CAADID Part I and II) [22]. Rating scales to evaluate ADHD symptomatology were the long version of the Conners’ ADHD Rating Scale (self-report form CAARS-S:L and observer form CAARS-O:L) [23], the ADHD Symptom Rating Scale [24], the ADHD Screening Checklist, the Wender Utah Rating Scale (WURS) [25] and the Clinical Global Impressions of ADHD Severity (CGI-ADHD) [26]. Neuropsychological traits were also evaluated using the Wechsler Adult Intelligence Scale (WAIS-III) [27], the Conners’ Continuous Performance Test (CPT) [28], the California Verbal Learning Test (CVLT) [29], the Weschsler Memory Scale Revised (Logical and Visual Memory I-II; WMS-R) [30], the A and B Traces [31]. 2.2.2. Childhood ADHD The same diagnostic procedures were applied in the two hospitals that participated in the study. The diagnosis of ADHD in children was evaluated using the

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I. Ponsa et al. / Mutation Research 666 (2009) 44–49

Table 2 Micronuclei, binucleated lymphocytes with micronuclei, binucleated cells with nucleoplasmic bridges, sister chromatid exchanges and chromosome aberrations before and after three months of methylphenidate treatment. Frequencies are in brackets. Before treatment Sample ADHD6 ADHD7 ADHD8 ADHD5 ADHD12 ADHD20 ADHD4 ADHD18 ADHD10 ADHD19 ADHD27 ADHD32

MNi n (‰a ) 3 (3.00) 7 (7.00) 4 (4.00) 7 (6.95) 0 (0.00) 2 (2.00) 3 (3.00) 6 (6.00) 5 (5.00) 6 (6.00) 7 (7.00) 3 (3.00)

ADHD14 10 (9.85) ADHD26 18 (18.00) ADHD23 19 (19.00) ADHD22 24 (24.00) ADHD24 25 (24.88) ADHD9 10 (10.00) ADHD31 14 (14.00) Children + adults Mean 9.10 SEM 1.70 P

After three months MPH treatment

BNMN n (‰b )

BNNPB n (‰c )

SCE n (d )

CA n (%e )

MNi n (‰a )

BNMN n (‰b )

BNNPB n (‰c )

SCE n (d )

CA n (%e )

3 (3.00) 6 (6.00) 4 (4.00) 7 (6.95) 0 (0.00) 2 (2.00) 3 (3.00) 6 (6.00) 5 (5.00) 6 (6.00) 7 (7.00) 2 (2.00)

1 (1.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 1 (1.00) 0 (0.00) 0 (0.00)

105 (4.20) 100 (4.00) – 152 (6.08) 126 (5.04) 151 (6.04) 123 (4.92) 152 (6.08) 176 (7.04) 154 (6.16) 170 (6.80) 162 (6.48)

0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 1 (2.00) 1 (2.00) 0 (0.00) 1 (2.00) 0 (0.00) 0 (0.00) 1 (2.00) 0 (0.00)

9 (8.97) 6 (5.87) 13 (12.82) 4 (4.00) 6 (5.88) 2 (2.00) 14 (14.00) 4 (4.00) 5 (5.00) 7 (7.00) 10 (10.00) 1 (1.00)

9 (8.97) 5 (4.89) 13 (12.82) 4 (4.00) 5 (4.90) 2 (2.00) 13 (13.00) 4 (4.00) 5 (5.00) 6 (6.00) 7 (7.00) 1 (1.00)

1 (1.00) 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.98) 0 (0.00) 0 (0.00) 0 (0.00) 2 (2.00) 0 (0.00) 2 (2.00) 0 (0.00)

121 (4.84) 124 (4.96) – 153 (6.12) 187 (7.19) 138 (5.52) 168 (6.72) 122 (4.88) 169 (6.76) 145 (5.80) 161 (6.44) 151 (6.04)

0 (0.00) 1 (2.00) 1 (2.00) 1 (2.00) 1 (2.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)

10 (9.85) 18 (18.00) 19 (19.00) 20 (20.00) 25 (24.88) 8 (8.00) 14 (14.00)

0 (0.00) 1 (1.00) 0 (0.00) 0 (0.00) 1 (1.00) 0 (0.00) 0 (1.00)

130 (5.20) 202 (8.08) 233 (9.32) 204 (7.85) 224 (8.96) 282 (11.28) 213 (8.52)

0 (0.00) 1 (2.00) 1 (2.08) 0 (0.00) 1 (2.00) 0 (0.00) 0 (0.00)

18 (17.63) 5 (5.00) 8 (8.00) 31 (31.00) 13 (13.00) 18 (17.98) 9 (9.00)

17 (16.65) 5 (5.00) 8 (8.00) 30 (30.00) 11 (11.00) 18 (17.98) 7 (7.00)

0 (0.00) 0 (0.00) 2 (2.00) 1 (1.00) 1 (1.00) 0 (0.00) 1 (1.00)

188 (7.52) 166 (6.64) 137 (5.27) 185 (6.61) 175 (7.00) 154 (6.16) 158 (6.32)

0 (0.00) 0 (0.00) 1 (2.00) 0 (0.00) 1 (2.00) 0 (0.00) 0 (0.00)

8.70 1.63

0.20 0.10

6.78 0.45

7.40 2.29

9.60 1.63 0.705

8.90 1.59 1.000

0.60 0.18 0.111

6.16 0.19 0.231

6.30 2.19 0.651

MPH: methylphenidate; MNi: micronuclei; BNMN: binucleated cells with micronuclei; BNNPB: binucleated cells with nucleoplasmic bridges; SCE: sister chromatid exchange; CA: chromosome aberrations; SEM: standard error of mean; dotted line divides children and adult groups. a number of MNi per 1000 binucleated cells. b number of binucleated cells with MNi per 1000 binucleated cells. c number of nucleoplasmic bridges per 1000 binucleated cells. d number of SCE per cell. e number of CA per 100 cells.

present and life time versions of the Schedule for Affective Disorders and Schizophrenia for School-age children (K-SADS-PL) [32]. Rating scales used to evaluate ADHD symptoms were the Conners’ Parent Rating Scale (CPRS-48) and the Conners’ Teacher Rating Scale (CTRS-28) [33] Neuropsychological evaluation included the Wechsler Intelligence Scale for Children (WISC-R; WISC IV). Reading and writing performance was evaluated using the TALE, TALEC (Spanish and Catalan version for primary education) [34], and the PROLEC-SE (for secondary education) [35]. Parents completed a general questionnaire including sociodemographic and general health information about their child in addition to the parents’ psychiatric family background. Exclusion criteria for both adults and children included IQ < 70, schizophrenia or other psychotic disorders, ADHD due to mood, anxiety, dissociative or personality disorders, pervasive developmental disorders, adoption, sexual or physical abuse, birth weight