Monocytes and macrophages form branched cell columns in matrigel: implications for a role in neovascularization

Linear arrays of cells, or cell columns, have been observed in the extracellular matrix prior to neovascularization, but their nature and significance remains elusive. Based on the emerging evidence implicating a role for monocytes and macrophages (MC/MPH) in vasculogenesis, we hypothesized that MC/MPH also can form linear or branched columns, facilitating the co-migration and the spatial arrangement of other cell types. To test this hypothesis, we studied the distribution of MC/MPH effected by chemotactic migration in novel in vitro and in vivo models of development. We induced transversal and lateral migration of THP-1 monocytoid cells in Matrigel in vitro. The effect of this process on co-localization of other micro-objects was assessed using erythrocytes and micron-sized plastic beads. In vivo, we analyzed MC/MPH infiltration in subcutaneously implanted Matrigel plugs containing angiogenic factors and across a microporous filter comprising the wall of a chamber filled with Matrigel, also placed subcutaneously in mice. In vitro, we found that migrating THP-1 cells induced the lasting degradation of Matrigel and produced cell columns, a process amplified by monocyte chemoattractant protein-1 (MCP-1). We also report the co-localization of erythrocytes with THP-1 cells in cell columns. Endothelium-free tunnels containing MC/MPH, neutrophils, or erythrocytes were also observed in the Matrigel-filled chambers. In free subcutaneous Matrigel plugs, we found MC/MPH-based columns harboring isolated Tie-2+ cells (a marker of endothelial progenitor phenotype), as well as fibroblasts, dendritic cells, and adypocytes. Many of these cell columns displayed conspicuous branching. Our data demonstrate formation of branched MC/MPH cell columns in vitro and in vivo, a previously unrecognized pattern of penetration of extracellular matrices by inflammatory cells. Thus, monocytes and macrophages influence the distribution of neovessels as well as their branching points. These cells are the "architects of development," assisting organogenesis, tumorigenesis, and wound healing by patterning the tissular space.     

Human monocyte-derived macrophages and dendritic cells are comparably effective in vitro in presenting HLA class I-restricted exogenous peptides.

Recent experimental data have shown that mice could be immunized efficiently, in particular against cancer, by the injection of antigen-loaded dendritic cells (DC) or macrophages (MPH). In the present work, these two antigen-presenting cells (APC) were prepared in humans from circulating mononuclear cells (MNC). MPH were obtained from MNC that were cultured in hydrophobic plastic bags and purified by elutriation. DC were from the culture of adherent elutriation-purified monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The two APC were prepared in parallel from the same donors and their phenotype and antigen-presenting capacity were compared. DC differed from MPH by a higher expression of HLA-DR and CD23 and a lower expression of CD14, CD64 and of adhesion molecules. DC and MPH were comparably effective in (a) enhancing the mitotic response of autologous lymphocytes to immobilized anti-CD3 (accessory function); (b) presenting melanoma peptides to specific cytotoxic T lymphocyte (CTL) clones; and (c) stimulating the generation of CTL directed against a myxovirus influenza peptide. However, DC were more effective than MPH in inducing the mitotic response of allogeneic peripheral blood leucocytes (PBL), possibly because of their higher expression of HLA class II molecules. In conclusion, DC and MPH prepared from blood MNC did not differ substantially in their ability to activate HLA class I-restricted T-cell responses by exogenous peptide presentation.     

Methylphenidate regulates activity regulated cytoskeletal associated but not brain-derived neurotrophic factor gene expression in the developing rat striatum

Methylphenidate (MPH) is a psychostimulant drug used to treat attention deficit hyperactivity disorder in children. To explore the central effects of chronic MPH, we investigated the expression of an effector immediate early gene, activity regulated cytoskeletal associated (arc), and the neurotrophin, brain-derived neurotrophic factor (bdnf) in the brain of immature and adult rats following repeated MPH. Prepubertal (postnatal day (PD) 25-38) and adult (PD 53-66) male rats were injected once daily for: a) 14 days with saline or MPH (2 or 10 mg/kg; s.c.) or b) 13 days with saline followed by a single dose of MPH (2 or 10 mg/kg; s.c.). To determine possible long-term effects of MPH, prepubertal rats were allowed a drug-free period of 4 weeks following the 14 days of treatment, and then were given a challenge dose of MPH. We demonstrated, for the first time, that an acute injection of MPH increased levels of activity-regulated cytoskeletal protein (ARC) and arc mRNA in the prepubertal rat striatum and cingulate/frontal cortex. This response was significantly attenuated by chronic MPH. The desensitization in arc expression observed in prepubertal rats persisted in the adult striatum following a later MPH challenge. In contrast to these data we observed little effect of MPH on bdnf expression. We also developed an effective, non-stressful technique to treat freely moving immature rats with oral MPH. Consistent with the results described above, we observed that oral MPH (7.5 and 10 mg/kg) also increased arc expression in the prepubertal rat striatum. However, unlike the effects of injected MPH, repeated oral MPH (7.5 mg/kg) did not alter the normal arc response. This result raises the important possibility that oral doses of MPH that reproduce clinically relevant blood levels of MPH may not down-regulate gene expression, at least in the short term (14 days). We confirmed, using mass spectrometry, that the oral doses of MPH used in our experiments yielded blood levels within the clinical range observed in children. The novel oral administration paradigm that we describe thus provides a clinically relevant animal model to further explore the effects of chronic drug exposure on central gene expression in the developing rat brain.     

Massive pulmonary hemorrhage in newborn infants successfully treated with high frequency oscillatory ventilation.

Massive pulmonary hemorrhage (MPH) in newborn infants is a catastrophic event with a fatal result. The aim of this study was to assess the efficacy of high frequency oscillatory ventilation (HFOV) as a rescue therapy for MPH in newborn infants. Eighteen newborn infants with MPH refractory to conventional mechanical ventilation were treated with HFOV. Changes in oxygenation were assessed using arterial-alveolar oxygen tension ratio (a/APO2) and oxygenation index (OI) during HFOV. The most common underlying disorder of MPH was preterm patent ductus arteriosus (PDA). Thirteen out of 18 (72%) newborn infants with MPH responded to HFOV and survived. Five out of 18 (28%) did not respond to HFOV and died. There were no differences between responders and nonresponders in gestational age, birth weight, pre-HFOV OI, and age of MPH onset. In responders, there was a rapid increase in a/APO2 from 0.18+/-0.04 to 0.40+/-0.08 at 30 minutes after HFOV. There was also significant decrease in OI from 14.9+/-4.7 to 8.1+/-1.5 at 1 hour after HFOV. We conclude that HFOV shows rapid and dramatic improvements and has ultimately life-saving effects in MPH of newborn infants.     

Effects of methylphenidate in ADHD adults on target evaluation processing reflected by event-related potentials

Attention deficit/hyperactivity disorder (ADHD) is a widely examined disease in childhood. There is evidence that the disease responds well to medication with methylphenidate (MPH). The effect of MPH in adults with ADHD is a question that is often raised, because many adults request such therapy today. As yet, there is a lack of studies on event-related potentials (ERPs) and MPH in ADHD adults. In the present study we examined 10 adults diagnosed with ADHD, with and without MPH medication (30 mg daily), by means of ERP in a visual Go/NoGo experiment to gain information about target evaluation processing in these patients and about the effects on these attentional processes during medication with MPH. Visually, there seem to be differences in the frontal electrodes for the N2 and in the parietal electrodes for the P300. But these differences do not achieve statistical significance. There are also no statistically relevant differences for the N1 or the slow wave. MPH does not seem to have an effect on target evaluation processing, when comparing the ERPs of non-medicated and medicated ADHD adults. Especially the P3 does not appear to be modulated by medication with MPH in these ADHD adults, in contrast to findings in ADHD children.     

Methylphenidate increased regional cerebral blood flow in subjects with attention deficit/hyperactivity disorder

The regional cerebral blood flow (rCBF) responses to methylphenidate (MPH) treatment were examined in children with attention deficit/hyperactivity disorder (ADHD). Thirty-two male children, diagnosed with ADHD by the DSM-IV diagnostic criteria, other behavioral assessment scales and neuropsychological battery, were studied using 99mTc-HMPAO-single photon emission computed tomography (SPECT). Subjects were studied before and after MPH treatment. First, using an image subtraction method, we obtained a NDR parametric image of each patient and found increased cerebral blood flow in the frontal lobes, caudate nuclei and thalamic areas after treatment. When the changes in SPECT and clinical response were compared, the matching rate, sensitivity and specificity between them were found to be 77.1, 80.0 and 79.2%, respectively. Second, three transaxial brain slices delineating anatomically defined regions of interest (ROI) at 20, 40, and 60mm above the orbitomeatal line (OML) were used, with the average number of counts for each region of interest normalized to the area of the cerebellar maximal uptake. The left and right prefrontal areas, and caudate and thalamic areas showed significant increases in rCBF after MPH treatment. These findings suggested MPH could affect the function of the fronto-striato-thalamic circuit, which is known as the pathophysiologic site of ADHD and could be used to correct the underlying brain dysfunction of ADHD.     

Effects of curcumin nanoparticle on the histological changes and apoptotic factors expression in testis tissue after methylphenidate administration in rats

The present article sought to evaluate the impact of curcumin-loaded superparamagnetic iron oxide (Fe3O4) nanoparticles (NPs) on the histological variables and apoptotic agents in adult male rats after 3-weeks of methylphenidate (MPH) oral administration (20 mg/kg) versus vehicle therapy on the testis. Twenty-four male rats have been categorized randomly into four groups, in which Group 1 has been chosen as the controls, and Group 2 has been a vehicle and taken the sesame oil as curcumin carrier. Moreover, Group 3 has been taken MPH (20 mg/kg by gavage for 21 consecutive days). Group 4 received MPH plus Curcumin nanoparticles (5.4 mg/100 g) for twenty-one consecutive days. Then, testis histology, apoptosis as well as stereology have been examined. According to the examinations, curcumin nanoparticles are significantly capable of improving the sperms and stereological variables; for example, round spermatid and Leydig cells by enhancing the level of the serum testosterone in comparison with the MPH and vehicle groups. Besides, it was found that the gene expression in inflammation pathways and apoptosis genes largely diminished in the treatment group by curcumin nanoparticles in comparison with the MPH and vehicle groups, also we observed considerable differences for the weight of testes between the examined groups. Therefore, Curcumin effectively inhibited the testis damages and MPH-induced apoptosis, indicating possible protecting features of the Curcumin nanoparticles in opposition to MPH.     

Effects of methylphenidate on aggressive urban children with attention deficit hyperactivity disorder

Determined the efficacy of methylphenidate (MPH) in a clinical population of aggressive, urban children diagnosed with attention deficit hyperactivity disorder (ADHD). In previous studies of prepubertal children with ADHD, MPH has been shown to be effective when compared with placebo. Eighteen inner-city children (ages 6 to 12 years), diagnosed with ADHD and attending a summer treatment program for youth with disruptive behavior disorders, participated in a double-blind placebo trial with assessment data obtained from staff in the program and parents at home. Based on staff ratings of the children's behavior in the program and an academic classroom, the children displayed significant improvements in ADHD symptoms and aggressive behavior with low- and high-dose MPH conditions. At home, parents and guardians reported few significant differences between placebo and MPH on behavior ratings. In both settings, MPH was well tolerated with few side effects found during active drug conditions.     

Effects of methylphenidate on the membrane potential and current in neurons of the rat locus coeruleus

Effects of methylphenidate (MPH), a therapeutic agent used in children presenting the attention deficit hyperactivity disorder (ADHD), on the membrane potential and current in neurons of the rat locus coeruleus (LC) were examined using intracellular and whole cell patch-clamp recording techniques. Application of MPH (30 microM) to artificial cerebrospinal fluid (ACSF) produced a hyperpolarizing response with amplitude of 12 +/- 1 mV (n = 29). Spontaneous firing of LC neurons was blocked during the MPH-induced hyperpolarization. Superfusion of LC neurons with ACSF containing 0 mM Ca(2+) and 11 mM Mg(2+) (Ca(2+)-free ACSF) produced a depolarizing response associated with an increase in spontaneous firing of the action potential. The MPH-induced hyperpolarization was blocked in Ca(2+)-free ACSF. Yohimbine (1 microM) and prazosin (10 microM), antagonists for alpha(2) and alpha(2B/2C) receptors, respectively, blocked the MPH-induced hyperpolarization in LC neurons. Tetrodotoxin (TTX, 1 microM) produced a partial depression of the MPH-induced hyperpolarization in LC neurons. Under the whole cell patch-clamp condition, MPH (30-300 microM) produced an outward current (I(MPH)) with amplitude of 110 +/- 6 pA (n = 17) in LC neurons. The I(MPH) was blocked by Co(2+) (1 mM). During prolonged application of MPH (300 microM for 45 min), the hyperpolarization gradually decreased in the amplitude and eventually disappeared, possibly because of depression of norepinephrine (NE) release from noradrenergic nerve terminals. At a low concentration (1 microM), MPH produced no outward current but consistently enhanced the outward current induced by NE. These results suggest that the MPH-induced response is mediated by NE via alpha(2B/2C)-adrenoceptors in LC neurons. I(MPH) was associated with an increase in the membrane conductance of LC neurons. The I(MPH) reversed its polarity at -102 +/- 6 mV (n = 8) in the ACSF. The reversal potential of I(MPH) was changed by 54 mV per decade change in the external K(+) concentration. Current-voltage relationship showed that the I(MPH) exhibited inward rectification. Ba(2+) (100 microM) suppressed the amplitude and the inward rectification of the I(MPH.) These results suggest that the I(MPH) is produced by activation of inward rectifier K(+) channels in LC neurons.     

Early exposure to methylphenidate increases fear responses in an aversive context in adult rats

The effects of methylphenidate hydrochloride (MPH) administration during development on fear acquisition and retention in adulthood were examined using classical fear conditioning. Male Sprague-Dawley rats were administered MPH (2 mg/kg) or saline twice daily from postnatal day (PD) 25 to 39, and were trained and tested on PD 81 and 82. During training, shock unconditioned stimulus (US) presentations were explicitly paired with an auditory conditioned stimulus (CS) or occurred unsignaled in the training context. No effect of MPH treatment was found during fear acquisition when shock was signaled or unsignaled during training, but 24 h retention tests in the training context revealed enhanced fear responses in MPH-treated animals that received unsignaled training. These results support recent reports of enhanced anxiety-like behaviors in adult rats caused by early developmental MPH treatment and highlight the need for further research into the long-term effects of developmental exposure to stimulants aimed at pediatric populations.     

Parietal and frontal object areas underlie perception of object orientation in depth

Attention deficit/hyperactivity disorder (ADHD) is a developmental disorder of cognition. Behavioral symptoms of ADHD are inattention, hyperactivity, and impulsivity. We investigated the effects of treadmill exercise and methylphenidate (MPH) on activity and spatial learning memory in relation to dopamine synthesis and brain-derived neurotrophic factor (BDNF) expression using spontaneously hypertensive adult male rats. The rats in the MPH-treated group received 1 mg/kg MPH orally once a day for 28 days. The rats in the treadmill exercise group were made to run on a treadmill for 30 min once a day, five times a week, for 28 days. Activity was determined by an open-field test and spatial learning memory was evaluated by an 8-arm maze test. Immunohistochemistry and Western blotting were conducted to examine the levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, and BDNF. The rats in the ADHD group showed hyperactivity and spatial learning memory deficit. Reduction of TH in the striatum and substantia nigra and BDNF in the hippocampus was observed of the rats in the ADHD group. Treadmill exercise and MPH alleviated the ADHD-induced hyperactivity and spatial learning memory impairment. Expressions of TH and BDNF in the ADHD rats were also increased by both treadmill exercise and MPH. These findings provide a possibility that exercise may be used as an effective therapeutic intervention for ADHD patients as MPH treatment.     

Methylphenidate enhances prepulse inhibition during processing of task‐relevant stimuli in attention‐deficit/hyperactivity disorder

ADHD is characterized by inattention, hyperactivity, and disinhibition, including the inability to screen out distracting stimuli. Prepulse inhibition (PPI) of startle indexes a related gating process and is enhanced during attended compared to ignored stimuli. We predicted that PPI during attended stimuli would be enhanced by the stimulant methylphenidate (MPH) and that this effect would be moderated by baseline PPI. Children with ADHD (n=36) completed a baseline day and a randomized, double‐blind medication trial (placebo vs. sustained release MPH). Bilateral startle eyeblink EMG was measured during a tone discrimination task. MPH enhanced PPI during attended, but not during ignored stimuli. Extending findings that pretreatment functioning moderates stimulant effects on PPI, this effect tended to be inversely related to baseline PPI. These data fit with the clinical literature on ADHD and the hypothesis that MPH enhances interference control for important environmental stimuli.     

The effects of object activity distribution on multiplexing multi-pinhole SPECT

We aim to study the effects of activity distribution for multiplexing multi-pinhole (MPH) SPECT. Three digital phantoms, including a hot rod, a cold rod and a cold sphere phantom, were used. Different degrees of multiplexing were obtained by (i) adjusting the MPH pattern for the same 4-pinhole collimator (scheme 1) and (ii) increasing the number of pinholes (scheme 2). Noise-free and noisy projections were generated using a 3D analytical MPH projector based on the same acquisition time. Projections were reconstructed using OS-EM without resolution recovery. Normalized mean-square-error (NMSE), noise, image profiles and signal-to-background ratios (SBR) were assessed. For the hot rod phantom, the NMSE-noise trade-offs slightly improves for multiplexing designs in scheme 2. Substantial artifacts were observed and the NMSE-noise trade-offs slightly worsened for multiplexing designs for the cold phantoms. Resolutions slightly degraded for higher degrees of multiplexing (～39-65%) for the cold rod phantom. For the cold sphere phantom, image profiles showed non-multiplexing designs better emulated the phantom, while ～20% multiplexing performs similarly as compared to non-multiplexing in SBR. Our results indicate that multiplexing can help for sparse objects but leads to a significant image degradation in non-sparse distributions. Since many tracers are not highly specific, and the gain of detection efficiency by allowing multiplexing is fairly offset by image degradations, multiplexing needs to be kept to a minimum for optimum MPH collimator designs.     

Subchronic methylphenidate administration has no effect on locomotion,emotional behavior,or water maze learning in prepubertal mice

Methylphenidate hydrochloride (Ritalin, MPH) is frequently prescribed as a treatment for children with attention deficit hyperactivity disorder (ADHD), yet little research has been conducted to determine its potential long-term neurobehavioral effects. We assessed the effects of subchronic MPH administration (2.5, 5, 10, 20, 40, or 80 mg/kg) on male CD-1 mice treated from 26 to 32 days of age. When tested at 33 days of age in the open field and elevated plus maze, there were no significant differences in spontaneous locomotion, exploration, or fear- and anxiety-related behaviors. Testing from 34 to 37 days of age in a water maze task revealed no significant effects of any dose of MPH on learning in this simple paradigm. While it is difficult to extrapolate directly from these results to clinical effects in humans, our results indicate that preexposure of mice to MPH late in the postnatal developmental period does not appear to alter later behavior. We are currently conducting additional studies to further probe the potential effects of MPH administration during development and to examine various contributing factors including stage of development, duration of MPH administration, complexity of the task used to assess behavioral changes, and type of cognitive process being analyzed (attention, nonspatial working memory, etc.).     

Methylphenidate and MK-801, an N-methyl-d-aspartate receptor antagonist: shared biological properties

Methylphenidate (MPH), a dopamine reuptake inhibitor, is used increasingly to treat attention deficit and hyperactivity disorders in children. Given that dopaminergic mechanisms, contribute to the structural and functional maturation of brain circuitry, consideration of the potential influence of MPH in disrupting such processes seems warranted. Following a similar logic regarding the relevance of glutamate neurotransmission in mediating aspects of brain maturation, we and others have previously utilized in vivo and in vitro studies of the developing rodent brain to establish that MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist has both neuroprotective and pro-apoptotic actions. In this study we used a neonatal murine model of excitotoxin-induced cortical injury to compare such actions between MPH and MK-801, and found that MPH shared some biological properties with MK-801. Specifically, both drugs were neuroprotective against excitotoxic challenge resulting in neonatal brain lesions and in vitro neuronal death, but both drugs also exacerbated programmed neural cell death. However, this profile of action was not shared by the dopamine reuptake blocker GBR-12783, a molecule which like MPH binds to and blocks the dopamine transporter, but which is structurally dissimilar to MPH, suggesting that inhibition of dopamine reuptake alone cannot explain the results from our MPH studies. The implications of our findings are that when studied in our developmental mouse model both drugs demonstrate similar capacities to be either neuroprotective or pro-apoptotic, depending on the specific biologic setting in which they act. Additional studies to identify some potential positive as well as negative consequences of exposure to these drugs during brain development in clinical settings are warranted.     

Acute neurocognitive response to methylphenidate among survivors of childhood cancer: a randomized, double-blind, cross-over trial

OBJECTIVE: To investigate the acute efficacy and adverse side effects of methylphenidate (MPH) among survivors of childhood cancer [acute lymphoblastic leukemia (ALL) or brain tumor (BT)] with learning impairments. METHODS: Participants (N = 122) completed a two-day, in-clinic, double-blind, cross-over trial during which they received MPH (0.60 mg/kg of body weight) and placebo that were randomized in administration order across participants. Performance was evaluated using measures of attention, memory, and academic achievement. RESULTS: A significant MPH versus placebo effect was revealed on a measure of attention, cognitive flexibility, and processing speed (Stroop Word-Color Association Test). Male gender, older age at treatment, and higher intelligence were predictive of better medication response. No significant differences were found for number or severity of adverse side effects as a function of active medication. CONCLUSIONS: MPH shows some neurocognitive benefit and is well tolerated by the majority of children surviving ALL and BT.     

Excitotoxic lesions to the prefrontal cortex of Sprague-Dawley rats do not impair response matching

In previous studies, chronic low-dose methylphenidate (MPH) administration during early development has been shown to increase emotional responding in adulthood. However, most studies employed male subjects, which generally show enhanced fear relative to females in laboratory tests of anxious behaviors. The present study examined the sex-dependent effects of MPH treatment on innate and learned fear behaviors. Rats were treated for 4 weeks from periadolescence through early adulthood with oral MPH. In open field testing, females showed greater levels of activity than males, and MPH (5mg/kg) decreased locomotion relative to control and 2mg/kg treatment in both sexes. In contextual fear conditioning, females exhibited less freezing than males at all retention intervals. Both sexes treated with 5mg/kg MPH showed increased fear to the shock context, although MPH treatment did not interfere with contextual discrimination in either sex. Upon reexposure to the shock context at 24h, only females treated with 5mg/kg MPH exhibited increases in freezing. MPH treatment did not disrupt extinction of contextual fear (48 h post-conditioning) in either sex. These findings illustrate subtle sex differences in the effects of prolonged MPH exposure on fear behaviors, and highlight the need to examine further the underlying mechanisms in both sexes.     

Effects of ADHD therapeutic agents,methylphenidate and atomoxetine,on hippocampal neurogenesis in the adolescent mouse dentate gyrus

Methylphenidate (MPH) and atomoxetine (ATX) are commonly used as attention-deficit/hyperactivity disorder (ADHD) therapeutic agents. In the present study, we investigated the effects of MPH and ATX on cell proliferation and neuronal differentiation in the dentate gyrus (DG) of the adolescent mouse by 5-bromo-2′-deoxyuridine (BrdU) and doublecortin (DCX) immunohistochemistry. BrdU-positive (⁺) cells, DCX⁺ cells and BrdU⁺/NeuN⁺ neurons (BrdU⁺ cells with NeuN immunoreaction) were easily detected in the subgranular zone (SGZ) of the DG in the vehicle-, MPH- and ATX-treated groups. Among them, only in the 10 mg/kg MPH-treated group, the numbers of BrdU⁺, DCX⁺ and BrdU⁺/NeuN⁺ cells were significantly increased compared to those in the vehicle-treated group. In addition, brain-derived neurotrophic factor (BDNF) level was significantly increased in 10 mg/kg MPH-treated group, not in the other experimental groups, compared to the vehicle-treated group. These results indicate that MPH, not ATX, can enhance cell proliferation and neuroblast differentiation in the SGZ of the DG via increasing BDNF level.     

Pharmacokinetics, dose-range, and mutagenicity studies of methylphenidate hydrochloride in B6C3F1 mice

Methylphenidate hydrochloride (MPH) is one of the most frequently prescribed pediatric drugs for the treatment of attention deficit hyperactivity disorder. In a recent study, increased hepatic adenomas were observed in B6C3F1 mice treated with MPH in their diet. To evaluate the reactive metabolite, ritalinic acid (RA) of MPH and its mode of action in mice, we conducted extensive investigations on the pharmacokinetics (PK) and genotoxicity of the drug in B6C3F1 mice. For the PK study, male B6C3F1 mice were gavaged once with 3 mg/kg body weight (BW) of MPH and groups of mice were sacrificed at various time points (0.25-24 hr) for serum analysis of MPH and RA concentrations. Groups of male B6C3F1 mice were fed diets containing 0, 250, 500, 1,000, 2,000, or 4,000 ppm of MPH for 28 days to determine the appropriate doses for 24-week transgenic mutation studies. Also, the micronucleus frequencies (MN-RETs and MN-NCEs), and the lymphocyte Hprt mutants were determined in peripheral blood and splenic lymphocytes, respectively. Mice fed 4,000 ppm of MPH lost significant BW compared to control mice (P < 0.01). There was a significant increase in the average liver weights whereas kidneys, seminal vesicle, testes, thymus, and urinary bladder weights of mice fed higher doses of MPH were significantly lower than the control group (P < or = 0.05). There was no significant increase in either the Hprt mutant frequency or the micronucleus frequency in the treated animals. These results indicated that although MPH induced liver hypertrophy in mice, no genotoxicity was observed.