Progress in Neuro-Psychopharmacology and Biological Psychiatry
DNA damage in rats after treatment with methylphenidate
Introduction
Methylphenidate (MPH) is a frequently prescribed psychostimulant that is widely been used for the treatment of attention-deficit hyperactivity disorder (ADHD) (Kimko et al., 1999, Sunohara et al., 1999, Zuddas et al., 2000, American Academy of Child and Adolescent Psychiatry, 2002). ADHD affects 3% to 6% of school-aged children and adolescents (Goldman et al., 1998, Klein-Schwartz, 2003) and 4% of adults (Faraone et al., 2003), and is characterized by inattention, impulsivity and hyperactivity (Faraone et al., 2003). The symptoms often persist into young adulthood, and long-term consequences include lower educational and occupational achievement and increased risk for developing other psychiatric disorders (Mannuzza et al., 1997, Mannuzza et al., 1998). MPH is a non-catecholamine sympathomimetic drug, and its pharmacological action is related to enhancement of extracellular levels of dopamine in the striatum (Teo et al., 2003, Biederman and Faraone, 2005), by blocking dopamine transporters (Greenhill et al., 2001, Biederman and Faraone, 2005, Volkow et al., 2005). The high prevalence of ADHD and the increased therapeutic use of MPH raises some concerns regarding its long-term side-effects and safety profile (Klein-Schwartz, 2003), such as the potential genotoxic effects of MPH (Klein-Schwartz, 2002).
Prolonged administration of methylphenidate in mice induced liver tumors (Dunnick and Hailey, 1995). However, MPH was non-mutagenic in a Salmonella assay system, suggesting that these effects may be a result to of other pathways such as an increase in cell proliferation (Dunnick and Hailey, 1995). More recently, Teo et al. (2003) showed that d-MPH, l-MPH and d-l-MPH are non-genotoxic using the Ames test (bacterial reverse mutation assay), L5178Y/TK+/− mammalian cells forward gene mutation and mouse bone marrow micronucleus assay.
El-Zein et al. (2005) showed that children treated with MPH for 3 months presented an increase of chromosome aberrations, sister chromatid exchange and micronuclei frequency. In contrast, Suter et al. (2006) conducted a chromosomal aberration study in cultured human peripheral lymphocytes treated with d-l-MPH in concentration up to 10 mM, and observed no structural or numerical chromosome abnormalities.
In the light of the fact that MPH has been approved for human use for over 50 years and the paucity of data regarding its potential genotoxicity, the aim of the present study was to assess an index of early DNA damage using the Comet assay (an index of genotoxicity) and the frequency of micronucleus (an index of mutagenicity) in young and adult rats exposed to acute or chronic treatment with MPH, using as sample: total blood (peripheral damage) and homogenates of striatum and hippocampus (central damage). We also tested whether there is a correlation between peripheral and central DNA damage induced by MPH. The hippocampus and striatum were chosen because of the current hypotheses suggesting that ADHD is associated with dysfunction of dopaminergic circuits, including prefrontal, subcortical regions (e.g. striatum) and limbic regions (e.g. hippocampus) (Castellanos, 1997, Dinn et al., 2001) and because some studies have shown evidence that the striatum is involved in impulsive and attention modulation in ADHD patients (Rubia et al., 2001).
Section snippets
Chemicals
d,l-Methylphenidate hydrochloride (MPH, Ritalin, Novartis Pharmaceutical Inc.), low and normal melting point agarose (Gibco, USA), ethylene-diamine-tetracetic acid (EDTA) (Labsynth Produtos de Laboratório Ltda, SP, Brazil), tris (Labsynth Produtos de Laboratório Ltda, SP, Brazil), triton X-100 (Labsynth Produtos de Laboratório Ltda, SP, Brazil), dimethyl sulfoxide (DMSO) (Labsynth Produtos de Laboratório Ltda, SP, Brazil), ethidium bromide (Sigma, USA), RMPI 1640 medium (Nutricell, Campinas-SP,
Index of DNA damage in total blood
In acute treatment with MPH, there was no difference of DNA damage in young rats (F = 2.81; df = 3, p > 0.05; one-way ANOVA; Fig. 2). In chronic treatment, there was a significant dose-dependent increase of DNA damage in young rats (F = 26.85; df = 3, 19; p < 0.001; one-way ANOVA; Fig. 2A). Chronic treatment with MPH 10 mg/kg induced more DNA damage than acute MPH 10 mg/kg in young rats (t = 4.06; df = 8; p = 0.004; t-test; Fig. 2A).
In adult rats, acute treatment with MPH 10 mg/kg increased DNA damage (F = 14.11;
Discussion
The present study showed that: (1) MPH increased the peripheral index of DNA damage in young and adult rats, which is more pronounced with chronic treatment; (2) the index of DNA damage was more pronounced in the striatum than in hippocampus; (3) neither acute nor chronic MPH treatment increased micronucleus frequency in young or in adult rats; and (4) the index of DNA damage in peripheral blood (peripheral damage) correlated positively with the index of DNA damage in the striatum of young and
Acknowledgements
This study was supported in part by CNPq (Brazil), CAPES (Brazil), FAPESC (Brazil) and FIPE-HCPA (Brazil).
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