The mixture of “ecstasy” and its metabolites is toxic to human SH-SY5Y differentiated cells at in vivo relevant concentrations

The neurotoxicity of “ecstasy” (3,4-methylenedioxymethamphetamine, MDMA) is thought to involve hepatic metabolism, though its real contribution is not completely understood. Most in vitro neurotoxicity studies concern isolated exposures of MDMA or its metabolites, at high concentrations, not considering their mixture, as expected in vivo. Therefore, our postulate is that combined deleterious effects of MDMA and its metabolites, at low micromolar concentrations that may be attained into the brain, may elicit neurotoxicity. Using human SH-SY5Y differentiated cells as dopaminergic neuronal model, we studied the neurotoxicity of MDMA and its MDMA metabolites α-methyldopamine and N-methyl-α-methyldopamine and their correspondent glutathione and N-acetylcysteine monoconjugates, under isolated exposure and as a mixture, at normothermic or hyperthermic conditions. The results showed that the mixture of MDMA and its metabolites was toxic to SH-SY5Y differentiated cells, an effect potentiated by hyperthermia and prevented by N-acetylcysteine. As a mixture, MDMA and its metabolites presented a different toxicity profile, compared to each compound alone, even at equimolar concentrations. Caspase 3 activation, increased reactive oxygen species production, and intracellular Ca²⁺ raises were implicated in the toxic effect. The mixture increased intracellular glutathione levels by increasing its de novo synthesis. In conclusion, this study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at low micromolar concentrations, which represents a more realistic approach of the in vivo scenario, elicited toxicity to human SH-SY5Y differentiated cells, thus constituting a new insight into the context of MDMA-related neurotoxicity.     

Biomimetic electrochemical synthesis of quinol-thioether conjugates: their implication in the serotonergic neurotoxicity of amphetamine derivatives

Injection of 3,4-methylenedioxyamphetamine (MDA) or 3,4-methylenedioxymethylamphetamine (MDMA or ecstasy) directly into the brain fails to reproduce the long-term effects observed after peripheral administration, implying an essential role for systemic metabolites in the development of toxicity. However, the precise identity of the metabolites participating in MDA and MDMA-mediated serotonergic neurotoxicity remains unclear: neither 3,4-alpha-methyldopamine, nor N-methyl-alpha-methyldopamine, major metabolites, produce neurotoxicity following peripheral administration. In vivo, these metabolites are oxidized to the corresponding orthoquinones, that readily react with protein and nonprotein sulphydryls including glutathione (GSH). The resulting quinol-thioether conjugates exhibit a variety of toxicological activities, which can be regulated by intramolecular cyclisation reactions that occur subsequent to oxidation. The ability of quinol-thioether conjugates to redox cycle and produce reactive oxygen species provides a rationale for the potential role of these metabolites in MDA and MDMA neurotoxicity. A biomimetic one-pot synthesis of 5-(GSH-S-yl)-N-Me-alpha-Me-DA involving addition of GSH to the electrogenerated orthoquinone species, is reported to evaluate its in vivo potential neurotoxicity.     

Hepatotoxicity of 3,4-methylenedioxyamphetamine and α-methyldopamine in isolated rat hepatocytes: formation of glutathione conjugates

The amphetamine designer drugs 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) and its N-demethylated analogue 3,4-methylenedioxyamphetamine (MDA or love) have been extensively used as recreational drugs of abuse. MDA itself is a main MDMA metabolite. MDMA abuse in humans has been associated with numerous reports of hepatocellular damage. Although MDMA undergoes extensive hepatic metabolism, the role of metabolites in MDMA-induced hepatotoxicity remains unclear. Thus, the aim of the present study was to evaluate the effects of MDA and -methyldopamine (-MeDA), a major metabolite of MDA, in freshly isolated rat hepatocyte suspensions. The cells were incubated with MDA or -MeDA at final concentrations of 0.1, 0.2, 0.4, 0.8, or 1.6 mM for 3 h. The toxic effects induced following incubation of hepatocyte suspensions with these metabolites were evaluated by measuring cell viability, the extent of lipid peroxidation, levels of glutathione (GSH) and glutathione disulfide (GSSG), the formation of GSH conjugates, and the activities of GSSG reductase (GR), GSH peroxidase (GPX), and GSH S-transferase (GST). MDA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on lipid peroxidation, cell viability, or on the activities of GR, GPX, and GST. In contrast, -MeDA (1.6 mM, 3 h) induced a marked depletion of GSH accompanied by a loss on cell viability, and decreases in GR, GPX and GST activities, although no significant effect on lipid peroxidation was found. For both metabolites, GSH depletion was not accompanied by increases in GSSG levels; rather, 2-(glutathion-S-yl)--MeDA and 5-(glutathion-S-yl)--MeDA were identified by HPLC-DAD/EC within cells incubated with MDA or -MeDA. The results provide evidence that one of the early consequences of MDMA metabolism is a disruption of thiol homeostasis, which may result in loss of protein function and the initiation of a cascade of events leading to cellular damage.     

Pharmacological and toxicological effects of β-3,4-methylenedioxyamphetamine isomers

Racemic and (R)-methylenedioxyamphetamine (MDA) caused apparent hallucinogenic and amphetamine-like behavioral responses in mice. (S)-MDA never produced apparent hallucinogenic behavior but was more potent than either (±)- or (R)-MDA in eliciting an amphetamine-like behavioral response. The LD50 values of (±)-, (S)-, and (R)-MDA were determined to see if the differential behavioral effects carried over to the toxicities of these drugs. (S)-MDA was the most toxic agent, (R)-MDA was the least toxic, and (±)-MDA was of intermediate toxicity. Some mice died 6 to 18 hr after dosing. Pretreatment with SKF 525-A markedly enhanced the toxicity of (R)-MDA while protecting mice from the toxic effects of (±)- and (S)-MDA. These findings suggest that (R)-MDA metabolism to (R)-α-methyldopamine [(R)-α-MeDA] represents a detoxification mechanism. On the other hand, (S)-α-methyldopamine formed from (±)- or (S)-MDA might be responsible for at least some of the toxic effects observed. Racemic, (S)-, and (R)-MDA caused an initial pressor response in the cat which diminished in amplitude upon repeated administration. This response was blocked by phenoxybenzamine and was greatly reduced by reserpine pretreatment. The MDA isomers also potentiated the rise in blood pressure resulting from norepinephrine. These findings indicate that (±)-, (S)-, and (R)-MDA produce initial pressor responses by an amphetamine-like action. (S)-MDA effects were indistinguishable from those of amphetamine. Repeated administration of (±)- and (R)-MDA resulted in a depressor response after four or five treatments. This nonamphetamine-like effect is postulated to be mediated in part by (R)-α-MeDA formed as a metabolite of (±)- and (R)-MDA in vivo. Low doses of (R)-α-MeDA produced a fall in blood pressure while higher doses and all doses of (S)-α-MeDA increased blood pressure by directly stimulating α receptors.     

In vitro differentiation modifies the neurotoxic response of SH-SY5Y cells

The SH-SY5Y cell line is commonly used for the assessment of neurotoxicity in drug discovery. These neuroblastoma-derived cells can be differentiated into neurons using many methods. The present study has compared 24 of these differentiation methods on SH-SY5Y cells. After morphologic selection of the three most differentiating media (retinoic acid in 10% fetal bovine serum (FBS), staurosporine in 1% FBS medium, and cyclic adenosine monophosphate (cAMP) in B21-supplemented neurobasal medium), cells were analyzed for pan-neuronal and specific neuronal protein expression by fluorescent automated imaging. The response of SH-SY5Y to a set of compounds of known toxicity was examined in these culture conditions performed in 2D, and also in a 3D hyaluronic acid-based hydroscaffold™ which mimics the extracellular matrix. The extent of neuronal markers expression and the sensitivity to neurotoxic compounds varied according to the differentiation medium. The cAMP B21-supplemented neurobasal medium led to the higher neuronal differentiation, and the higher sensitivity to neurotoxic compounds. The culture in 3D modified the neurotoxic response, through a lower sensitivity of cells compared to the 2D culture. The in vitro differentiation environment influences the neurotoxic response of SH-SY5Y cells and thus should be considered carefully in research as well as in drug discovery.     

The role of metabolism in 3,4-(+)-methylenedioxyamphetamine and 3,4-(+)-methylenedioxymethamphetamine (ecstasy) toxicity

3,4-Methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are ring-substituted amphetamine derivatives with stimulant and hallucinogenic properties. The recreational use of these amphetamines, especially MDMA, is prevalent despite warnings of irreversible damage to the central nervous system. MDA and MDMA are primarily serotonergic neurotoxicants. Because (1) neither MDA nor MDMA produces neurotoxicity when injected directly into brain, (2) intracerebroventricular (i.c.v.) administration of some major metabolites of MDA and MDMA fails to reproduce their neurotoxicity, (3) alpha-methyldopamine (alpha-MeDA) and N-methyl-alpha-MeDA are metabolites of both MDA and MDMA, (4) alpha-MeDA and N-methyl-alpha-MeDA are readily oxidized to the corresponding ortho-quinones, which can undergo conjugation with glutathione (GSH), and (5) quinone thioethers exhibit a variety of toxicologic activities, we initiated studies on the potential role of thioether metabolites of alpha-MeDA and N-methyl-alpha-MeDA in the neurotoxicity of MDA and MDMA. Our studies have revealed that the thioether conjugates stimulate the acute release of serotonin, dopamine, and norepinephrine and produce a behavioral response commensurate with the "serotonin syndrome." Direct injection of the conjugates into rat brain also produces long-term depletions in serotonin (5-HT) concentrations, elevations in GFAP expression, and activation of microglial cells. The data are consistent with the view that thioether metabolites of alpha-MeDA and N-methyl-alpha-MeDA contribute to the neurotoxicity of the parent amphetamines.     

The relationship between core body temperature and 3,4-methylenedioxymethamphetamine metabolism in rats: implications for neurotoxicity

Rationale A close relationship appears to exist between 3,4-methylenedioxymethamphetamine (MDMA)-induced changes in core body temperature and long-term serotonin (5-HT) loss. Objective We investigated whether changes in core body temperature affect MDMA metabolism. Materials and methods Male Wistar rats were treated with MDMA at ambient temperatures of 15, 21.5, or 30°C to prevent or exacerbate MDMA-induced hyperthermia. Plasma concentrations of MDMA and its main metabolites were determined for 6 h. Seven days later, animals were killed and brain indole content was measured. Results The administration of MDMA at 15°C blocked the hyperthermic response and long-term 5-HT depletion found in rats treated at 21.5°C. At 15°C, plasma concentrations of MDMA were significantly increased, whereas those of three of its main metabolites were reduced when compared to rats treated at 21.5°C. By contrast, hyperthermia and indole deficits were exacerbated in rats treated at 30°C. Noteworthy, plasma concentrations of MDMA metabolites were greatly enhanced in these animals. Instrastriatal perfusion of MDMA (100 μM for 5 h at 21°C) did not potentiate the long-term depletion of 5-HT after systemic MDMA. Furthermore, interfering in MDMA metabolism using the catechol-O-methyltransferase inhibitor entacapone potentiated the neurotoxicity of MDMA, indicating that metabolites that are substrates for this enzyme may contribute to neurotoxicity. Conclusions This is the first report showing a direct relationship between core body temperature and MDMA metabolism. This finding has implications on both the temperature dependence of the mechanism of MDMA neurotoxicity and human use, as hyperthermia is often associated with MDMA use in humans. Beatriz Goni-Allo and Brian ó Mathúna contributed equally to this work.     

Zearalenone induced toxicity in SHSY-5Y cells: The role of oxidative stress evidenced by N-acetyl cysteine

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24 h time points. DAPI staining showed apoptotic nuclei at 12 and 24 h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.     

Neurotoxicity of MDMA (ecstasy): the limitations of scaling from animals to humans

Several studies suggest that MDMA-induced acute toxicity and long-term neurotoxicity is dependent on the metabolic disposition of MDMA. Differences in MDMA metabolism among animal species might therefore account for different sensitivities to its neurotoxic effects. The kinetic parameters of enzymes that regulate the formation of neurotoxic metabolites of MDMA differ among species, as does the ability of MDMA to self-inhibit these enzymes and the degree of genetic polymorphisms exhibited by these enzymes. Such features limit allometric scaling across animal models.     

Assessment of the role of alpha-methylepinine in the neurotoxicity of MDMA.

To assess the potential involvement of metabolism of 3,4-methylenedioxymethamphetamine (MDMA) to the catechol alpha-methylepinine in producing serotonergic neurotoxicity, we attempted to correlate aspects of this reaction with the neurotoxicity profile of MDMA. In contrast to the stereoselectivity of S-(+)-MDMA in causing persistent declines in rat brain 5-hydroxyindole levels, no stereochemical component to the metabolic reaction was apparent. Rat liver microsomes generated a significantly greater amount of alpha-methylepinine than did mouse microsomes, but similar amounts of metabolite were produced by brain microsomes from the two species. Formation of alpha-methylepinine by hepatic, but not brain, microsomes was inhibited by SKF 525A and induced by phenobarbital, possibly indicating a tissue specificity in cytochrome P-450-dependent metabolism of MDMA. To directly assess whether alpha-methylepine is a likely mediator of MDMA neurotoxicity, the compound was administered intracerebroventricularly. No persistent declines in biogenic amines or their metabolites were observed one week following treatment. These data suggest that alpha-methylepinine alone is not responsible for the neurotoxic effects of MDMA.     

Lost in translation: preclinical studies on 3,4-methylenedioxymethamphetamine provide information on mechanisms of action, but do not allow accurate prediction of adverse events in humans

3,4-Methylenedioxymethamphetamine (MDMA) induces both acute adverse effects and long-term neurotoxic loss of brain 5-HT neurones in laboratory animals. However, when choosing doses, most preclinical studies have paid little attention to the pharmacokinetics of the drug in humans or animals. The recreational use of MDMA and current clinical investigations of the drug for therapeutic purposes demand better translational pharmacology to allow accurate risk assessment of its ability to induce adverse events. Recent pharmacokinetic studies on MDMA in animals and humans are reviewed and indicate that the risks following MDMA ingestion should be re-evaluated. Acute behavioural and body temperature changes result from rapid MDMA-induced monoamine release, whereas long-term neurotoxicity is primarily caused by metabolites of the drug. Therefore acute physiological changes in humans are fairly accurately mimicked in animals by appropriate dosing, although allometric dosing calculations have little value. Long-term changes require MDMA to be metabolized in a similar manner in experimental animals and humans. However, the rate of metabolism of MDMA and its major metabolites is slower in humans than rats or monkeys, potentially allowing endogenous neuroprotective mechanisms to function in a species specific manner. Furthermore acute hyperthermia in humans probably limits the chance of recreational users ingesting sufficient MDMA to produce neurotoxicity, unlike in the rat. MDMA also inhibits the major enzyme responsible for its metabolism in humans thereby also assisting in preventing neurotoxicity. These observations question whether MDMA alone produces long-term 5-HT neurotoxicity in human brain, although when taken in combination with other recreational drugs it may induce neurotoxicity.     

Is hyperthermia the triggering factor for hepatotoxicity induced by 3,4-methylenedioxymethamphetamine (ecstasy)? An in vitro study using freshly isolated mouse hepatocytes

The consumption of 3,4-methylenedioxymethamphetamine (ecstasy; MDMA) may cause hepatocellular damage in humans, a toxic effect that has been increasing in frequency in the last few years, although the underlying mechanisms are still unknown. The metabolism of MDMA involves the production of reactive metabolites which form adducts with intracellular nucleophilic sites, as is the case with glutathione (GSH). Also, MDMA administration elicits hyperthermia, a potentially deleterious condition that may aggravate its direct toxic effects. Thus, the objective of this study was to evaluate the extent of MDMA-induced depletion of GSH, induction of lipid peroxidation and loss of cell viability in freshly isolated mouse hepatocytes under normothermic conditions (37 degrees C) and to compare the results with the effects obtained under hyperthermic conditions (41 degrees C). By itself, hyperthermia was an important cause of cell toxicity. A rise in incubation temperature from 37 degrees C to 41 degrees C caused oxidative stress in freshly isolated mouse hepatocytes, reflected as a time-dependent induction of lipid peroxidation and consequent loss of cell viability (up to 40-45%), although the variations in GSH and GSSG levels were similar to those under normothermic conditions. MDMA (100, 200, 400, 800 and 1600 microM) induced a concentration- and time-dependent GSH depletion at 37 degrees C but had a negligible effect on lipid peroxidation and cell viability at this temperature. It is particularly noteworthy that hyperthermia (41 degrees C) potentiated MDMA-induced depletion of GSH, production of lipid peroxidation and loss of cell viability (up to 90-100%). It is therefore concluded that hyperthermia potentiates MDMA-induced toxicity in freshly isolated mouse hepatocytes.     

Effect of GBR 12909 and fluoxetine on the acute and long term changes induced by MDMA ('ecstasy') on the 5-HT and dopamine concentrations in mouse brain

We examined the long term effect of 3,4 methylenedioxymethamphetamine (MDMA, 10, 20 and 30 mg/kg, i.p.) on the cerebral 5-hydroxytryptamine (5-HT) and dopamine content in Swiss Webster mice. Three injections of MDMA (20 or 30 mg/kg, i.p.) given 3 h apart produced a marked depletion in the striatal content of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) 7 days later. None of the doses administered altered the concentration of 5-HT or its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in several brain areas. Pre-treatment with the dopamine uptake inhibitor GBR 12909 (10 mg/kg, i.p.), 30 min before each of the three MDMA (30 mg/kg, i.p.) injections, completely prevented the long term loss in the striatal catechol concentrations. However, GBR 12909 (10 mg/kg, i.p.) not only failed to prevent the acute effects induced by MDMA (30 mg/kg x 3, i.p.) on dopamine metabolism 30 min later, but in fact potentiated them. The 5-HT uptake inhibitor, fluoxetine (10 mg/kg, i. p.) failed to prevent both the acute and long term dopaminergic deficits. MDMA (30 mg/kg x 3) altered the body temperature of the mice biphasically, producing a rapid hyperthermia followed by prolonged hypothermia. In contrast, MDMA (20 mg/kg x 3) produced an initial hypothermia followed by hyperthermia. The present experiments therefore appear to rule out any direct relationship between the neurotoxic effects of MDMA and its acute effects on body temperature in mice. Fluoxetine administered 30 min before each MDMA (30 mg/kg) injection prevented these temperature changes, while GBR 12909 was without effect. This suggests that the neuroprotective effect of GBR 12909 against MDMA-induced neurotoxicity is not directly related to its ability to inhibit the MDMA-induced acute effects on dopamine metabolism or alter the MDMA-induced temperature change. The data illustrate major differences in the neurotoxic profile of MDMA in mice and rats.     

Neuronal effects of 4-t-Butylcatechol: a model for catechol-containing antioxidants

Many herbal medicines and dietary supplements sold as aids to improve memory or treat neurodegenerative diseases or have other favorable effects on the CNS contain a catechol or similar 1,2-dihydroxy aromatic moiety in their structure. As an approach to isolate and examine the neuroprotective properties of catechols, a simple catechol 4-t-Butylcatechol (TBC) has been used as a model. In this study, we investigated the effects of TBC on lipopolysaccharide (LPS)-activated microglial-induced neurotoxicity by using the in vitro model of coculture murine microglial-like cell line HAPI with the neuronal-like human neuroblastoma cell line SH-SY5Y. We also examined the effects of TBC on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. TBC at concentrations from 0.1-10 microM had no toxic effect on HAPI cells and SH-SY5Y cells, and it inhibited LPS (100 ng/ml)-induced increases of superoxide, intracellular ROS, gp91(Phox), iNOS and a decrease of HO-1 in HAPI cells. Under coculture condition, TBC significantly reduced LPS-activated microglia-induced dopaminergic SH-SY5Y cells death. Moreover, TBC (0.1-10 microM) inhibited 6-OHDA-induced increases of intracellular ROS, iNOS, nNOS, and a decrease of mitochondria membrane potential, and cell death in SH-SY5Y cells. However, the neurotoxic effects of TBC (100 microM) on SH-SY5Y cells were also observed including the decrease in mitochondria membrane potential and the increase in COX-2 expression and cell death. TBC-induced SH-SY5Y cell death was attenuated by pretreatment with NS-398, a selective COX-2 inhibitor. In conclusion, this study suggests that TBC might possess protective effects on inflammation- and oxidative stress-related neurodegenerative disorders. However, the high concentration of TBC might be toxic, at least in part, for increasing COX-2 expression.     

In vitro metabolism, glutathione conjugation, and CYP isoform specificity of epoxidation of 4-vinylphenol

4-Vinylphenol (4VP) has been identified as a minor urinary metabolite of styrene in rat and human volunteers. This compound has been shown to be more hepatotoxic and pneumotoxic than both styrene and styrene oxide at lower doses in rats and mice. To explore the possible toxicity mechanism of 4VP, the current study was conducted to investigate the metabolism of 4VP, the glutathione (GSH) conjugation of the metabolites of 4VP and its cytochrome P(450) (CYP) specificity in epoxidation in different microsomes in vitro. Incubations of 4VP with mouse lung microsomes afforded two major metabolites which were identified as 4-(2-oxiranyl)-phenol of 4VP (4VPO) and 4VP catechol. 4VPO was found to react with GSH to form GSH conjugate and 4VP catechol was found to further be metabolized to electrophilic species which react with GSH to form the corresponding 4VP catechol GSH conjugates. Relative formation rates for those GSH conjugates and the regioisomer formation of 4VPO-GSH conjugates with both inhibitors of CYP 2F2 and CYP 2E1 in microsomal incubation condition were also investigated. This present study provides better insight on the lung toxicity seen with 4VP, the toxic metabolite of commercial styrene.     

The toxicity of N-methyl-alpha-methyldopamine to freshly isolated rat hepatocytes is prevented by ascorbic acid and N-acetylcysteine

In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity. The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes. Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h. To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds). The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST. In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities. For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation. Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA. The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes. Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.     

Serotonergic neurotoxicity of 3,4-(+/-)-methylenedioxyamphetamine and 3,4-(+/-)-methylendioxymethamphetamine (ecstasy) is potentiated by inhibition of gamma-glutamyl transpeptidase.

Reactive metabolites play an important role in 3,4-(+/-)-methylenedioxyamphetamine (MDA) and 3,4-(+/-)-methylenedioxymethamphetamine (MDMA; ecstasy)-mediated serotonergic neurotoxicity, although the specific identity of such metabolites remains unclear. 5-(Glutathion-S-yl)-alpha-methyldopamine (5-GSyl-alpha-MeDA) is a serotonergic neurotoxicant found in the bile of MDA-treated rats. The brain uptake of 5-GSyl-alpha-MeDA is decreased by glutathione (GSH), but sharply increases in animals pretreated with acivicin, an inhibitor of gamma-glutamyl transpeptidase (gamma-GT) suggesting competition between intact 5-GSyl-alpha-MeDA and GSH for the putative GSH transporter. gamma-GT is enriched in blood-brain barrier endothelial cells and is the only enzyme known to cleave the gamma-glutamyl bond of GSH. We now show that pretreatment of rats with acivicin (18 mg/kg, ip) inhibits brain microvessel endothelial gamma-GT activity by 60%, and potentiates MDA- and MDMA-mediated depletions in serotonin (5-HT) and 5-hydroxylindole acidic acid (5-HIAA) concentrations in brain regions enriched in 5-HT nerve terminal axons (striatum, cortex, hippocampus, and hypothalamus). In addition, glial fibrillary acidic protein (GFAP) expression increases in the striatum of acivicin and MDA (10 mg/kg) treated rats, but remains unchanged in animals treated with just MDA (10 mg/kg). Inhibition of endothelial cell gamma-GT at the blood-brain barrier likely enhances the uptake into brain of thioether metabolites of MDA and MDMA, such as 5-(glutathion-S-yl)-alpha-MeDA and 2,5-bis-(glutathion-S-yl)-alpha-MeDA, by increasing the pool of thioether conjugates available for uptake via the intact GSH transporter. The data indicate that thioether metabolites of MDA and MDMA contribute to the serotonergic neurotoxicity observed following peripheral administration of these drugs.     

In vitro metabolism,glutathione conjugation,and CYP isoform specificity of epoxidation of 4-vinylphenol

1. 4-Vinylphenol (4VP) has been identified as a minor urinary metabolite of styrene in rat and human volunteers. This compound has been shown to be more hepatotoxic and pneumotoxic than both styrene and styrene oxide at lower doses in rats and mice. To explore the possible toxicity mechanism of 4VP, the current study was conducted to investigate the metabolism of 4VP, the glutathione (GSH) conjugation of the metabolites of 4VP and its cytochrome P₄₅₀ (CYP) specificity in epoxidation in different microsomes in vitro.

2. Incubations of 4VP with mouse lung microsomes afforded two major metabolites which were identified as 4-(2-oxiranyl)-phenol of 4VP (4VPO) and 4VP catechol. 4VPO was found to react with GSH to form GSH conjugate and 4VP catechol was found to further be metabolized to electrophilic species which react with GSH to form the corresponding 4VP catechol GSH conjugates. Relative formation rates for those GSH conjugates and the regioisomer formation of 4VPO-GSH conjugates with both inhibitors of CYP 2F2 and CYP 2E1 in microsomal incubation condition were also investigated.

3. This present study provides better insight on the lung toxicity seen with 4VP, the toxic metabolite of commercial styrene.

     

Effects of Salicylate on 3,4-Methylenedioxymethamphetamine (MDMA)-Induced Neurotoxicity in Rats

The drug 3,4-methylenedioxymethamphetamine (MDMA) is a serotonergic neurotoxicant that causes hyperthermia and depletion of serotonin (5-HT) and 5-hydroxy-indole-3-acetic acid (5-HIAA) in the central nervous system. Formation of neurotoxic metabolites of MDMA, e.g., 2,4,5-trihydroxy-methamphetamine and 2,4,5-trihydroxyamphetamine, involves hydroxyl and/or superoxide free radicals. The present study was designed to determine whether the hydroxyl free-radical-trapping agent salicylate could provide protection against MDMA neurotoxicity in rats. In the acute studies, sodium salicylate (12.5–400 mg/kg, calculated as free acid) was injected interperitoneally (IP) 1 h before subscutaneous (SC) injections of MDMA (20 mg/kg as base). In the chronic studies, sodium salicylate (3.1–100 mg/kg) was injected IP 1 h before repeated SC injections of MDMA (10 mg/kg as base, twice daily, at 0830 and 1730 h for 4 consecutive days). Repeated MDMA administration depleted contents of 5-HT and 5-HIAA in the frontal cortex, hippocampus and striatum. Coadministration of salicylate plus MDMA did not significantly alter MDMA-induced depletion of 5-HT and 5-HIAA in these tissues. Thus, salicylate, a hydroxyl free-radical-trapping agent, does not protect against MDMA-induced hyperthermia and depletion of 5-HT and 5-HIAA. These observations suggest that MDMA-induced neurotoxicity may occur mainly through the production of superoxide or other radicals rather than hydroxyl free radicals. Salicylate actually potentiated MDMA-induced hyperthermia and lethality, findings that might be of clinical relevance. Published by Elsevier Science Inc.     

A review of the mechanisms involved in the acute MDMA (ecstasy)-induced hyperthermic response

The predominant severe acute adverse effect following ingestion of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) by recreational users is hyperthermia which can induce other associated clinical problems and occasionally death. There is no pharmacologically specific treatment. MDMA also induces dose-dependent hyperthermia in experimental animals. This review examines the consequences of MDMA administration on body temperature in humans and rodents. In rats hyperthermia results primarily from dopamine release and is influenced by dose, ambient temperature and other housing conditions. The response is increased in rats with a prior MDMA-induced neurotoxic lesion of 5-hydroxytryptamine (5-HT) nerve endings. Increased MDMA-induced locomotor activity appears to play no role in the hyperthermic response. However, the size of the acute hyperthermic response plays a major role in determining the severity of the subsequent neurotoxicity. These results suggest that any MDMA-induced hyperthermic response will be enhanced in hot, crowded dance club conditions and that ingesting the drug in such conditions increases the possibility of subsequent cerebral neurotoxic effect.