Genotoxic and genoprotective effects of some antipsychotic drugs, methylphenidate and atomoxetine on human lymphocytes and HepG2 cells.

OBJECTIVE: Aripiprazole, risperidone, atomoxetine, and methylphenidate are drugs commonly prescribed for many psychiatric conditions and can be used alone or in combination in children and adolescents. This study aimed to investigate comparatively the possible genotoxic effects or genoprotective potentials of these drugs on human lymphocytes and HepG2 cells. MATERIALS AND METHODS: Cytotoxicity analysis was performed with the cell viability test on human lymphocytes and HepG2 cells, and half-maximal inhibitory concentration (IC50) values of the drugs were determined, and three different doses (» IC50, ½ IC50, and IC50) were applied for genetic analysis. For the determined doses, cells with and without DNA damage were examined by comet analysis. RESULTS: In lymphocytes, aripiprazole and risperidone increased DNA damage at moderate and maximum doses, whereas atomoxetine increased DNA damage only at the maximum dose. In HepG2 cells, risperidone reduced DNA damage at all doses, while atomoxetine increased DNA damage at all doses. On the other hand, in the DNA-damaged cells induced by hydrogen peroxide (H2O2), DNA damage decreased at all concentrations of all drugs in both lymphocytes and HepG2 cells. CONCLUSIONS: As a result, the genotoxicity of the drugs was found to be dose-dependent, and all drugs showed a genoprotective effect on DNA-damaged cells.

Effects of methylphenidate on femoral bone growth in male rats.

The use of Methylphenidate (MP) can have adverse effects on bone growth and mineralization. This study aimed to investigate the underlying pathophysiology of MP-induced skeletal deficits in growing rats using stereological and immunohistochemical methods. Male rats, aged 4 weeks, were orally treated with MP through an 8-h/day water drinking protocol. The rats (n=30) were randomly divided into three groups: MP-High Dose (30/60 mg/kg/day MP), MP-Low Dose (4/10 mg/kg/day MP), and control (water only). After 13 weeks, the femoral bones were assessed using calliper measurements, dual-energy X-ray absorptiometry, and biomechanical evaluation. The total femur volume, cartilage volume, growth zone volume, and volume fractions were determined using the Cavalieri method. Immunohistochemical analyses were conducted using alkaline phosphatase and anti-calpain antibody staining. Rats exposed to MP exhibited significant reductions in weight gain, femoral growth, bone mineralization, and biomechanical integrity compared to the control group. The total femoral volume of MP-treated rats was significantly lower than that of the control group. The MP-High Dose group showed significantly higher ratios of total cartilage volume/total femoral volume and total growth zone volume/total femoral volume than the other groups. Immunohistochemical evaluation of the growth plate revealed reduced osteoblastic activity and decreased intracellular calcium deposition with chronic MP exposure. The possible mechanism of MP-induced skeletal growth retardation may involve the inhibition of intracellular calcium deposition in chondrocytes of the hypertrophic zone in the growth plate. In this way, MP may hinder the differentiation of cartilage tissue from bone tissue, resulting in reduced bone growth and mineralization.

Dichotomous effect of methylphenidate on microglia and astrocytes: Insights from in vitro and animal studies.

Methylphenidate (MPH) has been used for decades to treat attention-deficit/hyperactivity disorder (ADHD) and narcolepsy. Moreover, several studies have shown that it is subject to misuse, particularly among college students and adolescents, for cognitive enhancement or as a recreational drug. This phenomenon causes concern, and it is critical to clarify better how MPH impacts brain cells. In fact, data has suggested that MPH could result in neuroinflammation and neurodegeneration across several brain regions; however, little is known about the effect of MPH on glial cells. To address this, we used microglia N9 cell line and primary cultures of cortical astrocytes that were exposed to MPH (0.01 - 2 mM), as well as Wistar Kyoto rats (WKY) chronically administered with MPH (1.5 mg/kg/day). Several parameters were analyzed, and we concluded that MPH has no significant direct effect on microglial cells, apart from cell migration impairment. On the contrary, MPH promotes astrogliosis, oxidative/nitrosative stress, and increases proinflammatory cytokine TNF levels by astrocytes, which was concordant with the results obtained in the hippocampus of WKY rats. Overall, the present results suggest that brain cells respond differently to MPH, with a more prominent direct effect on astrocytes when compared to microglia.

Increased sperm deoxyribonucleic acid damage leads to poor embryo quality and subfertility of male rats treated with methylphenidate hydrochloride in adolescence.

BACKGROUND: Methylphenidate hydrochloride (MPH) is a psychostimulant widely used in the treatment of attention-deficit hyperactive disorder (ADHD), as well as a performance enhancer, for at least 60 years. Despite the notable effectiveness as a psychostimulant, ADHD is a chronic disorder and has a two-third chance of accompanying the individual throughout life. Long-term use of MPH has been associated not only with an increase in the development of neurodegenerative diseases, but it also causes side effects on male fertility in experimental animals. OBJECTIVES: To investigate whether methylphenidate poses a risk to sperm DNA structure and to the quality of embryos conceived after treatment during adolescence in rats. MATERIALS AND METHODS: Wistar rats at 38 days of age were treated either with 5 mg/kg body weight of MPH, in a single daily dose for 30 days, via gavage or with distilled water-only protocol. Levels of oxidative stress in testicular and epididymal tissues were evaluated. Sperm chromatin quality and acrosome integrity was assessed under flow cytometry. From 107 days of age, animals were mated with untreated females. The effects of the paternal contribution at two different embryo development moments-cleavage stage (2.5 days post coitum) and late gestation (20 days post coitum) -were analyzed. RESULTS: MPH caused high levels of sperm DNA damage, which was reflected in 40% of decrease in early embryo quality and a lower number of live pups at 20 dpc. DISCUSSION: The high level of fragmentation seen in the embryos sired from the MPH group is consistent with the poor chromatin structure of the sperm and does not seem to be a result of oxidative stress in the reproductive tissues. CONCLUSIONS: The results presented here suggest that the subchronic use of MPH during male prepubertal phase may cause long-term subfertility and compromise embryo survival.

Neuroprotective Properties of Minocycline Against Methylphenidate-Induced Neurodegeneration: Possible Role of CREB/BDNF and Akt/GSK3 Signaling Pathways in Rat Hippocampus.

Neurodegeneration is a side effect of methylphenidate (MPH), and minocycline possesses neuroprotective properties. This study aimed to investigate the neuroprotective effects of minocycline against methylphenidate-induced neurodegeneration mediated by signaling pathways of CREB/BDNF and Akt/GSK3. Seven groups of seventy male rats were randomly distributed in seven groups (n = 10). Group 1 received 0.7 ml/rat of normal saline (i.p.), and group 2 was treated with MPH (10 mg/kg, i.p.). Groups 3, 4, 5, and 6 were simultaneously administered MPH (10 mg/kg) and minocycline (10, 20, 30, and 40 mg/kg, i.p.) for 21 days. Minocycline alone (40 mg/kg, i.p.) was administrated to group 7. Open field test (OFT) (on day 22), forced swim test (FST) (on day 24), and elevated plus maze (on day 26) were conducted to analyze the mood-related behaviors; hippocampal oxidative stress, inflammatory, and apoptotic parameters, as well as the levels of protein kinase B (Akt-1), glycogen synthase kinase 3 (GSK3), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF), were also assessed. Furthermore, localization of total CREB, Akt, and GSK3 in the DG and CA1 areas of the hippocampus were measured using immunohistochemistry (IHC). Histological changes in the mentioned areas were also evaluated. Minocycline treatment inhibited MPH-induced mood disorders and decreased lipid peroxidation, oxidized form of glutathione (GSSG), interleukin 1 beta (IL-1ß), alpha tumor necrosis factor (TNF-α), Bax, and GSK3 levels. In the contrary, it increased the levels of reduced form of glutathione (GSH), Bcl-2, CREB, BDNF, and Akt-1 and superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) activities in the experimental animals' hippocampus. IHC data showed that minocycline also improved the localization and expression of CREB and Akt positive cells and decreased the GSK3 positive cells in the DG and CA1 regions of the hippocampus of MPH-treated rats. Minocycline also inhibited MPH-induced changes of hippocampal cells' density and shape in both DG and CA1 areas of the hippocampus. According to obtained data, it can be concluded that minocycline probably via activation of the P-CREB/BDNF or Akt/GSK3 signaling pathway can confer its neuroprotective effects against MPH-induced neurodegeneration.

Effect of repeated sodium valproate and topiramate administration on mania­like behaviors induced by methylphenidate in mice.

Novel and effective treatments for mania are needed, and well­validated animal models are important to reach this goal. The psychostimulant­induced hyperactivity is the most frequently animal model of mania used. Although this model is validated pharmacologically using mood stabilizers, data about its predictive validity with negative controls (i.e., drugs that are clinically ineffective in treating mania) are lacking. The present study evaluated the effects of the repeated administration of a clinically effective drug (sodium valproate) and clinically ineffective drug (topiramate) on methylphenidate (MPH)­induced manic­like behaviors in Swiss mice in the behavioral pattern monitor (BPM). Methylphenidate increased locomotor activity and center activity in the BPM. Valproate attenuated the effect of MPH on locomotor and general activity, with no effect on center activity. Topiramate did not affect any MPH­induced manic­like behaviors. Methylphenidate did not change exploratory activity (rearing or nose poking). These results support the predictive validity of MPH­induced hyperactivity for screening antimanic­like drugs.

Effect of Chronic Methylphenidate Treatment in a Female Experimental Model of Parkinsonism.

Methylphenidate (MPH) is the most commonly prescribed drug for the treatment of ADHD in males and females. However, a majority of previous studies investigated the effect of MPH in only males, and little is known regarding consequences of female exposure to MPH. This is unfortunate because the few studies that have been conducted indicate that females have a greater sensitivity to MPH. Previous research in male mice has shown that chronic exposure to MPH causes dopaminergic neurons within the nigrostriatal pathway to be more sensitive to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, estrogen has been shown to protect dopaminergic neurons from MPTP neurotoxicity. Therefore, in this study, we test the hypothesis that chronic MPH exposure in female mice will render dopaminergic neurons in the nigrostriatal pathway more sensitive to MPTP, and that estrogen may play a protective role. Interestingly, proestrus females exhibited greater sensitivity to MPTP, with significantly reduced dopaminergic neurons in the SN and significant increases in DA quinone production. Chronic MPH exposure contributed to GSH depletion, but surprisingly, it did not increase dopamine quinone levels or dopaminergic cell loss. There were no significant differences in anestrus animals, with the exception of a depletion in GSH seen when animals received chronic high-dose (10 mg/kg) MPH followed by MPTP. Thus, estrogen may actually sensitize neurons to MPTP in this model, and chronic MPH may contribute to GSH depletion within the striatum. This study provides insight into how chronic psychostimulant use may affect males and females differently.

Fetal methylphenidate exposure induced ADHD-like phenotypes and decreased Drd2 and Slc6a3 expression levels in mouse offspring.

Methylphenidate (MPD) is used as a first-line treatment for attention-deficit/hyperactivity disorder (ADHD). The number of prescriptions for ADHD patients is increasing, suggesting that the number of fertile women using such medication might be also increasing. The purpose of this study was to clarify the effects of MPD exposure during the fetal period on infant development, behavior, learning, and memory in mice. Expression levels of candidate genes associated with ADHD were also determined in the brain of pups born to MDP-treated dams who were administered MPD orally at a dose of 2.5, 7.5, or 15 mg/kg daily from gestational day 1 to the day before delivery. Offspring aged 6-8 weeks were subjected to the spontaneous locomotor activity, elevated plus-maze, and passive avoidance tests and therapeutic treatments with MPD or atomoxetine. Fetal MPD exposure induced ADHD-like phenotypes, such as hyperactivity and impulsivity, in mouse offspring, which were suppressed by treatment with MPD and atomoxetine. These mice showed decreased Drd2 and Slc6a3 expression levels in the brain, which are often observed in ADHD model animals. Our results suggest that continuous use of MPD during pregnancy induces ADHD phenotypes in the offspring.

Effect of Methylphenidate and buspirone-methylphenidate co-administration on biochemical and hematological parameters in rats: Implications for safe and confrontational use.

Methylphenidate (MPH) is a psychostimulant, beneficial in attention deficit hyperactivity disorder (ADHD). Previously it has been shown that MPH-induced locomotor sensitization could be attenuate by buspirone co administration however the effect of chronic MPH and co-administration of MPH-buspirone on biochemical and hematological parameters are unknown. This study is designed to investigate these parameters after long term administration of MPH, Buspirone and their combination in rats. 40 male Wister rats were divided in to 4 groups, and treated with saline, MPH (2mg/kg/day), Buspirone (10mg/kg/day) and MPH-Buspirone co-administration (2mg/kg/day ±10mg/kg/day; respectively) up to six weeks. Administration of MPH significantly increase blood glucose level in saline treated control rats, however co-administration of MPH-buspirone exhibited less effect on blood glucose levels. Serum creatinine levels significantly decreased in all treated groups as compared to control but highly significant results were seen with combination treatment. Co-administration of MPH-buspirone and buspirone treated rats exhibited increased cholesterol and hemoglobin values. All treated groups showed increased values of hematocrit, MCV, MCH and MCHC compared to control group. RBCs and WBC's count were decreased in all treated groups. The platelet count rose significantly by Buspirone and MPH-buspirone administration, while MPH showed decreased platelet count. Thus, results suggested that prolong co-administration of MPH-buspirone is safe and effective for ADHD patients by preventing adverse effects not only on behavioral but also on biochemical and hematological parameter.

Methylphenidate causes cytotoxicity on photoreceptor cells via autophagy.

Methylphenidate (MPH) is used as the first-line treatment for attention-deficit hyperactivity disorder. However, there are concerns that this treatment may be associated with increased risk of retinal damage. This study was to investigate cytotoxicity of MPH on photoreceptor cells and explore its underlying mechanisms. MPH-caused cell toxicity was established in 661 W cells. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromid and lactate dehydrogenase assays. Oxidative stress was measured by the markers: glutathione (GSH) reductase, catalase, and superoxide dismutase activities as well as GSH, reactive oxygen species, and malondialdehyde levels. Gene and protein expression was detected by real-time polymerase chain reaction (PCR) and western blot, respectively. Results showed that MPH decreased 661 W cell viability, increased caspase-3/9 activities, and induced oxidative stress. Furthermore, MPH treatment increased messenger RNA (mRNA) expression of Beclin-1 and microtubule-associated protein 1A/1B-light chain 3B (LC3B) protein expression in 661 W cells, suggesting autophagy was induced. MPH treatment also upregulated p-JAK1/p-STAT1 protein expression. These data demonstrated that MPH could increase oxidative stress in photoreceptor cells to cause cell toxicity via autophagy, providing the scientific rationale for the photoreceptor cell damage caused by the MPH administration.

Stimulants cocktail: Methylphenidate plus caffeine impairs memory and cognition and alters mitochondrial and oxidative status.

Methylphenidate (MPH) is a psychostimulant widely misused to increase wakefulness by drivers and students. Also, MPH can be found in dietary supplements in a clandestine manner aiming to burst performance of physical exercise practitioners. The abusive use of high doses of caffeine (CAF) in these contexts is equally already known. Here, we demonstrate the behavioral, oxidative and mitochondrial effects after acute exposure to high doses of MPH (80 mg/L) and CAF (150 mg/L), alone or associated (80 mg/L + 150 mg/L, respectively). We used zebrafish as animal model due to its high translational relevance. We evaluated the behavioral effects using the Novel Tank Test (NTT), Social Preference Test (SPT) and Y-maze Task and analyzed biomarkers of oxidative stress and activity of mitochondrial respiratory chain complexes. MPH alone induced antisocial behavior. MPH inhibited lipid peroxidation. The association of MPH + CAF presented memory impairment and anxiogenic behavior. In oxidative status, it inhibited lipid peroxidation, increased protein carbonylation and mitochondrial complex II, III and IV activity. Our results demonstrate that MPH and CAF alone negatively impact the typical behavioral of zebrafish. When associated, changes in cognition, memory, oxidative and mitochondrial status are more relevant.

Co-abuse of alprazolam augments the hepato-renal toxic effects of methylphenidate.

OBJECTIVE: Methylphenidate (MPH) is a first-line treatment option for attention-deficit hyperactive disorder and narcolepsy. MPH is one of the most abused psychostimulants by the adults and young population to stay awake, perform better, or improve concentration. The scanty reports say that the medical users or abusers mostly consider the administration of benzodiazepines to overcome the adverse effects, i.e., mood- and anxiety-related problems associated with MPH chronic abuse. This work aims to study the effect of alprazolam (ALZ) on MPH-associated adverse effects on liver and kidney. MATERIALS AND METHODS: Female Wistar rats (n = 58) were administered with MPH (10, 20, and 40 mg/kg) and ALZ (5, 10, and 20 mg/kg) alone and in combination for 28 days. Bodyweight, feed intake, and water intake were monitored weekly. Parameters related to liver and renal function, oxidative stress, and histopathology were performed to evaluate the toxic impacts on the liver and kidneys. RESULTS: ALZ, along with MPH, increased the serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatinine, and urea levels. The co-abuse also led to elevated oxidative stress and structural abnormalities in the liver and kidney tissues. CONCLUSION: The co-abuse of ALZ has amplified the hepato-renal toxic effects of MPH. Therefore, it is a significant concern for public safety, and their co-abuse must be restricted and discouraged.

CHRONIC EXPOSURE TO METHYLPHENIDATE-CONTAMINATED WATER ELICITS SOCIAL IMPAIRMENT TO ZEBRAFISH.

Residual contamination of water with MPH represents a severe environmental issue because it can affect non-target animals. Here we describe the behavioral effects in zebrafish after chronic contamination of water containing residues of MPH (0.1875, 1.875 and 3 ug/L). These doses are environmentally relevant since they reflect those found in wastewaters. We evaluated the behavioral effect through the novel tank test (NTT) and social preference test (SPT), and after euthanasia we analyzed oxidative stress parameters. Zebrafish exposed to MPH presented a social impairment, avoiding the conspecifics segment in the social preference test. In addition, MPH in the lowest concentration provoked an anxiolytic effect in the novel tank test. Oxidative stress is not related to these changes. Since the maintenance of an intact behavioral repertoire is crucial for species survival and fitness, our results demonstrate that residual contamination of water by MPH can be a threat to zebrafish, impacting directly to its well-being and survival in the aquatic environment.

Toxicology of long-term and high-dose administration of methylphenidate on the kidney tissue - a histopathology and molecular study.

The present study aims to assess the influences of oral methylphenidate on kidney function and structure versus vehicle treatment in adult male rats. In this study, thirty adult male rats equally into two treatment groups divided randomly, and among them, MPH has been administered for 21 days, at doses of 20 mg/kg, and the control group has received salin. In renal, under the effect of MPH applying quantitative real-time PCR, we analyzed nephrotoxicity-related molecular pathways like autophagy, inflammation, and apoptosis. Moreover, the levels of GSH, CAT, and SOD were investigated as antioxidant enzymes. Afterward, stereological analysis in MPH-treated rats has been performed. Analysis of qPCR displayed inflammation, impaired autophagy, and enhanced apoptosis with histological changes in the kidney's tissue, also an important rise in the antioxidant enzymes' level. Besides, 20 mg/kg of MPH led to a decline in the mean of Bowman's space thickness and renal corpuscle's volume in comparison to the control rats. Collectively, our histological and molecular data implicit that in the kidney region, administrating of MPH evoked discriminative expression alterations in nephrotoxicity-associated signaling cascades, specifically autophagy, inflammation, and apoptosis paired with important damage to kidney tissue.

Differential effects of alprazolam against methylphenidate-induced neurobehavioral alterations.

BACKGROUND: In the previous decade, abuse of several types of prescription drugs, particularly anxiolytics, opioid analgesics, and stimulants has increased significantly worldwide. Methylphenidate (MPH) and Alprazolam (ALZ) are extensively used drugs for the treatment of attention deficit hyperactivity disorder (ADHD) and anxiety disorders, respectively. However, these drugs have a high risk of being misused or abused alone, and their combination in some peculiar cases has shown their deleterious effects. In this study, we evaluated the extent of damage both these drugs (MPH and ALZ) may cause in the brain at different dosages. METHODS: Female Wistar rats were administered with MPH (10, 20, 40mg/kg) and ALZ (5, 10, 20mg/kg) alone and in combination. Following the treatment, neurobehavioral studies were conducted, and later brain tissue was removed for studying the extent of oxidative stress and inflammation in the hippocampus and cortex region of the brain. Further histopathological parameters, along with neurotransmitter levels, were also assessed. RESULTS: Both MPH and ALZ, in combination, enhanced oxidative stress, inflammation, and neurobehavioral alterations in a dose-dependent manner. These toxic effects were associated with histopathological alterations and neurotransmitters levels CONCLUSIONS: In this study, it is found that the combination of psychostimulant (MPH) and depressant (ALZ) tends to enhance toxicity in the brain, and their long-term usage is a significant public health concern. Therefore, their co-administration should be strictly monitored by medical practitioners, and under compulsive circumstances, their use must be restricted to lower doses.

Methylphenidate affects task-switching and neural signaling in non-human primates.

RATIONALE: Low doses of psychostimulants such as methylphenidate (MPH), which increase extracellular dopamine and norepinephrine by inhibiting their reuptake, are the most commonly used treatment for attention deficit hyperactivity disorder (ADHD). Therapeutic doses of these drugs may improve focused attention at the expense of hindering other cognitive functions, including the ability to adapt behavior in response to changing circumstances-cognitive flexibility. Cognitive flexibility is thought to depend on proper operation of the prefrontal cortex (PFC) and is also linked to reward processing, which is dopamine-dependent. Additionally, reward outcome signals have been recorded from the PFC. OBJECTIVES: This study tested the hypothesis that therapeutic doses of MPH impair cognitive flexibility and that this impairment in performance resulted from interference in reward signals within the PFC. METHODS: Four rhesus monkeys were given therapeutically relevant doses of oral MPH (0, 3, and 6 mg/kg) while performing an oculomotor switching task to evaluate its effect on task performance. Single-unit recordings in the PFC of two monkeys were taken before and after MPH administration during task performance. RESULTS: The results show that MPH does hinder switching task performance, an effect that was correlated with a reduction in the amplitude of outcome signals found in the discharges of some neurons in the PFC. CONCLUSIONS: Methylphenidate impaired task-switching performance, which can be used as a measure of cognitive flexibility. This detriment may result from degraded outcome signaling within the PFC. This study has implications for the use of MPH in the treatment of ADHD.

Effects of sub-chronic methylphenidate on risk-taking and sociability in zebrafish (Danio rerio).

Attention deficit hyperactive disorder (ADHD) is the most common psychiatric disorder in children affecting around 11% of children 4-17 years of age (CDC 2019). Children with ADHD are widely treated with stimulant medications such as methylphenidate (Ritalin®). However, there has been little research on the developmental effects of methylphenidate on risk-taking and sociability. We investigated in zebrafish the potential developmental neurobehavioral toxicity of methylphenidate on these behavioral functions. We chose zebrafish because they provide a model with extensive genetic tools for future mechanistic studies. We studied whether sub-chronic methylphenidate exposure during juvenile development causes neurobehavioral impairments in zebrafish. Methylphenidate diminished responses to environmental stimuli after both acute and sub-chronic dosing. In adult zebrafish, acute methylphenidate impaired avoidance of an approaching visual stimulus modeling a predator and decreased locomotor response to the social visual stimulus of conspecifics. Adult zebrafish dosed acutely with methylphenidate demonstrated behaviors of less retreat from threatening visual stimuli and less approach to conspecifics compared with controls. In a sub-chronic dosing paradigm during development, methylphenidate caused less robust exploration of a novel tank. In the predator avoidance paradigm, sub-chronic dosing that began at an older age (28 dpf) decreased activity levels more than sub-chronic dosing that began at earlier ages (14 dpf and 21 dpf). In the social shoaling task, sub-chronic methylphenidate attenuated reaction to the social stimulus. Acute and developmental methylphenidate exposure decreased response to environmental cues. Additional research is needed to determine critical mechanisms for these effects and to see how these results may be translatable to neurobehavioral toxicity of prescribing Ritalin® to children and adolescents.

4-Fluoromethylphenidate: Fatal Intoxication Involving a Previously Unreported Novel Psychoactive Substance in the USA.

The (±)-threo-4-fluoromethylphenidate (4F-MPH) is a fluorinated analog of the prescription central nervous system stimulant medication, methylphenidate. This novel psychoactive substance was first detected in drug paraphernalia at the Miami-Dade County Medical Examiner Department Toxicology Laboratory in 2016 but was not detected in a biological specimen until 2018. Limited literature is available on 4F-MPH, with predominate literature being published out of Europe, and no known toxicities reported in the USA. Post-mortem specimens were screened using both gas chromatography mass spectrometry and liquid chromatography ion trap mass spectrometry (LC-Ion Trap-MSn). In addition, a validated method for the quantification of 4F-MPH was developed using liquid chromatography-tandem mass spectrometry (LC-MS-MS), with a linear range of 0.01-0.500 mg/L and acceptable validation criteria including precision, bias, carry-over, linearity and endogenous/exogenous interferences. In addition to the detection of 4F-MPH, 3-methoxy-PCP, amphetamine, methamphetamine, 6-monoacetylmorphine, morphine, codeine and tetrahydrocannabinol were also identified in the decedent. A single source of blood was collected (femoral vein) and quantified in all blood tubes used for collection, with concentrations varying from 0.012 to 0.05 mg/L. Additional specimens available for screening included gastric contents and urine. An additional peak having the same targeted ions and transitions as 4F-MPH was identified in both the LC-Ion Trap-MSn screening procedure and the LC-MS-MS quantitative procedure. This peak suggests the presence of a structural isomer, possibly (±)-erythro-4-fluoromethylphenidate, which cannot be confirmed due to there being no available certified reference material. This case report presents the first time that 4F-MPH was detected in a decedent, as well as the first time 4F-MPH has been listed in the official cause of death of a decedent in Florida.

Nootropic drugs: Methylphenidate, modafinil and piracetam - Population use trends, occurrence in the environment, ecotoxicity and removal methods - A review.

Pharmaceuticals which originally were designed to treat people with neurological and psychiatric conditions, e.g. Alzheimer's disease or attention deficit hyperactivity disorder (ADHD), are nowadays often misused by students as a 'brain doping' substances. These substances are known as nootropic drugs, smart drugs or cognitive enhancers, as they increase memory, attention and concentration of healthy individuals. Since they are easily available illicitly, their consumption is observed to be growing. Currently, these pharmaceuticals started gaining researchers' attention, especially since they have been recently detected in wastewater, surface water and even drinking water. This review summarises the current state of knowledge on nootropic drugs in terms of their population use trends and ethics, occurrence in the environment and detection techniques, toxicity and removal methods, in example of methylphenidate, modafinil and piracetam - three most popular nootropics. It points out that the main sources of knowledge on cognitive enhancers illicit use are often inconsistent questionnaires, which are not supported by wastewater analysis to become more veracious. Simultaneously, the studies concerning toxicity and removal methods of nootropic drugs are still limited and in many cases environmentally irrelevant. Although the prescription rules has been subjected to more strict control in developed countries, regulatory frameworks with regard to their ecosystem occurrence are still lacking and should be introduced. Moreover, the use of environmentally relevant concentrations in toxicity studies should be a standard, leading to proper ecotoxicity risk assessment. Based on this review, it is recommended to routinely monitor nootropics and their metabolites in waste- and surface waters.

Effects of methylphenidate on the aggressive behavior, serotonin and dopamine levels, and dopamine-related gene transcription in brain of male Nile tilapia (Oreochromis niloticus).

The occurrence of pharmaceuticals in the aquatic environment has increased considerably in the last decades, causing negative biochemical, physiological, and behavioral effects in aquatic organisms. In this study, we evaluated the effects of methylphenidate (MPH) on the aggressive behavior, dopamine-related gene transcript levels, monoamine levels, and carboxylesterase transcript levels and activity in the brain of male Nile tilapia (Oreochromis niloticus). Carboxylesterase activity was also measured in the liver and gills. Fish were exposed for 5 days to MPH at 20 and 100 ng L-1. Fish exposed to 100 ng L-1 of MPH showed increased aggressiveness and decreased dopamine (DA) and serotonin (5-HT) levels. No changes were observed in plasma testosterone levels and in the transcript levels of D1 and D2 dopamine receptors, dopamine transporter (DAT), and carboxylesterase 2 (CES2). Exposure to 100 ng L-1 of MPH caused a decrease in the transcript levels of carboxylesterase 3 (CES3) and an increase in tyrosine hydroxylase (TH), while exposure to 20 ng L-1 of MPH increased the transcript levels of D5 dopamine receptor. Carboxylesterase activity was unchanged in the brain and liver and increased in the gills of fish exposed to 20 ng L-1. These results indicate that MPH at 100 ng L-1 increases aggressiveness in Nile tilapia, possibly due to a decrease in 5-HT levels in the brain and alterations in dopamine levels and dopamine-related genes.