From street to lab: in vitro hepatotoxicity of buphedrone, butylone and 3,4-DMMC.

Synthetic cathinones are among the most popular new psychoactive substances, being abused for their stimulant properties, which are similar to those of amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Considering that the liver is a likely target for cathinones-induced toxicity, and for their metabolic activation/detoxification, we aimed to determine the hepatotoxicity of three commonly abused synthetic cathinones: butylone, α-methylamino-butyrophenone (buphedrone) and 3,4-dimethylmethcathinone (3,4-DMMC). We characterized their cytotoxic profile in primary rat hepatocytes (PRH) and in the HepaRG and HepG2 cell lines. PRH was the most sensitive cell model, showing the lowest EC50 values for all three substances (0.158 mM for 3,4-DMMC; 1.21 mM for butylone; 1.57 mM for buphedrone). Co-exposure of PRH to the synthetic cathinones and CYP450 inhibitors (selective and non-selective) proved that hepatic metabolism reduced the toxicity of buphedrone but increased that of butylone and 3,4-DMMC. All compounds were able to increase oxidative stress, disrupting mitochondrial homeostasis and inducing apoptotic and necrotic features, while also increasing the occurrence of acidic vesicular organelles in PRH, compatible with autophagic activation. In conclusion, butylone, buphedrone and 3,4-DMMC have hepatotoxic potential, and their toxicity lies in the interference with a number of homeostatic processes, while being influenced by their metabolic fate.

Mixtures of 3,4-methylenedioxymethamphetamine (ecstasy) and its major human metabolites act additively to induce significant toxicity to liver cells when combined at low, non-cytotoxic concentrations.

Hepatic injury after 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) intoxications is highly unpredictable and does not seem to correlate with either dosage or frequency of use. The mechanisms involved include the drug metabolic bioactivation and the hyperthermic state of the liver triggered by its thermogenic action and exacerbated by the environmental circumstances of abuse at hot and crowded venues. We became interested in understanding the interaction between ecstasy and its metabolites generated in vivo as users are always exposed to mixtures of parent drug and metabolites. With this purpose, Hep G2 cells were incubated with MDMA and its main human metabolites methylenedioxyamphetamine (MDA), α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA), individually and in mixture (drugs combined in proportion to their individual EC01 ), at normal (37 °C) and hyperthermic (40.5 °C) conditions. After 48 h, viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Extensive concentration-response analysis was performed with single drugs and the parameters of the individual non-linear logit fits were used to predict joint effects using the well-founded models of concentration addition (CA) and independent action (IA). Experimental testing revealed that mixture effects on cell viability conformed to CA, for both temperature settings. Additionally, substantial combination effects were attained even when each substance was present at concentrations that individually produced unnoticeable effects. Hyperthermic incubations dramatically increased the toxicity of the tested drug and metabolites, both individually and combined. These outcomes suggest that MDMA metabolism has hazard implications to liver cells even when metabolites are found in low concentrations, as they contribute additively to the overall toxic effect of MDMA.

Glial cell response to 3,4-(+/-)-methylenedioxymethamphetamine and its metabolites.

3,4-(±)-Methylenedioxymethamphetamine (MDMA) and 3,4-(±)-methylenedioxyamphetamine (MDA), a primary metabolite of MDMA, are phenylethylamine derivatives that cause serotonergic neurotoxicity. Although several phenylethylamine derivatives activate microglia, little is known about the effects of MDMA on glial cells, and evidence of MDMA-induced microglial activation remains ambiguous. We initially determined microglial occupancy status of the parietal cortex in rats at various time points following a single neurotoxic dose of MDMA (20mg/kg, SC). A biphasic microglial response to MDMA was observed, with peak microglial occupancy occurring 12- and 72-h post-MDMA administration. Because direct injection of MDMA into the brain does not produce neurotoxicity, the glial response to MDMA metabolites was subsequently examined in vivo and in vitro. Rats were treated with MDA (20mg/kg, SC) followed by ex vivo biopsy culture to determine the activation of quiescent microglia. A reactive microglial response was observed 72 h after MDA administration that subsided by 7 days. In contrast, intracerebroventricular (ICV) administration of MDA failed to produce a microglial response. However, thioether metabolites of MDA derived from α-methyldopamine (α-MeDA) elicited a robust microglial response following icv injection. We subsequently determined the direct effects of various MDMA metabolites on primary cultures of E18 hippocampal mixed glial and neuronal cells. 5-(Glutathion-S-yl)-α-MeDA, 2,5-bis-(glutathion-S-yl)-α-MeDA, and 5-(N-acetylcystein-S-yl)-α-MeDA all stimulated the proliferation of glial fibrillary acidic protein-positive astrocytes at a dose of 10 µM. The findings indicate that glial cells are activated in response to MDMA/MDA and support a role for thioether metabolites of α-MeDA in the neurotoxicity.

"Ecstasy"-induced toxicity in SH-SY5Y differentiated cells: role of hyperthermia and metabolites.

3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a recreational hallucinogenic drug of abuse known to elicit neurotoxic properties. Hepatic formation of neurotoxic metabolites is thought to play a major role in MDMA-related neurotoxicity, though the mechanisms involved are still unclear. Here, we studied the neurotoxicity mechanisms and stability of MDMA and 6 of its major human metabolites, namely α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) and their correspondent glutathione (GSH) and N-acetyl-cysteine (NAC) conjugates, under normothermic (37 °C) or hyperthermic conditions (40 °C), using cultured SH-SY5Y differentiated cells. We showed that MDMA metabolites exhibited toxicity to SH-SY5Y differentiated cells, being the GSH and NAC conjugates more toxic than their catecholic precursors and MDMA. Furthermore, whereas the toxicity of the catechol metabolites was potentiated by hyperthermia, NAC-conjugated metabolites revealed higher toxicity under normothermia and GSH-conjugated metabolites-induced toxicity was temperature-independent. Moreover, a time-dependent decrease in extracellular concentration of MDMA metabolites was observed, which was potentiated by hyperthermia. The antioxidant NAC significantly protected against the neurotoxic effects of MDMA metabolites. MDMA metabolites increased intracellular glutathione levels, though depletion in thiol content was observed in MDMA-exposed cells. Finally, the neurotoxic effects induced by the MDMA metabolite N-Me-α-MeDA involved caspase 3 activation. In conclusion, this study evaluated the stability of MDMA metabolites in vitro, and demonstrated that the catechol MDMA metabolites and their GSH and NAC conjugates, rather than MDMA itself, exhibited neurotoxic actions in SH-SY5Y differentiated cells, which were differently affected by hyperthermia, thus highlighting a major role for reactive metabolites and hyperthermia in MDMA's neurotoxicity.

5-Iodo-2-aminoindan (5-IAI): chemistry, pharmacology, and toxicology of a research chemical producing MDMA-like effects.

In 2010, an internet snapshot of EMCDDA anticipated the presence of 5-iodo-2-aminoindan (5-IAI) within the recreational drug market. In 2011, this compound, a psychoactive derivative of 2-aminoindane, was identified in recreational products sold in the United Kingdom. 5-IAI is a rigid analogue of p-iodoamphetamine producing MDMA-like effects. The aim of this paper is to summarize the clinical, pharmacological, and toxicological information about this new potential drug of abuse.

Recreational drugs, 3,4-Methylenedioxymethamphetamine(MDMA), 3,4-methylenedioxyamphetamine (MDA) and diphenylprolinol, inhibit neurite outgrowth in PC12 cells.

3,4-Methylenedioxymethamphetamine (MDMA) is widely abused as a psychoactive recreational drug. It is well known that MDMA induces neurotoxic damage of serotonergic nerve endings. Although drug abuse is increasing among youths, it is unclear whether recreational drugs affect the development of nerve growth. Thus, the present study examined the effect of recreational drugs, such as MDMA, 3,4-methylenedioxyamphetamine (MDA) and diphenylprolinol, a novel recreational drug with a similar chemical structure as that of psychoactive agent pipradrol, on nerve growth factor (NGF)-induced neurite outgrowth. These recreational drugs induced a dose-dependent cell death in PC12 cells. The IC(50) values of MDMA, MDA, R-diphenylprolinol and S-diphenylprolinol were 4.11 mM, 2.75 mM, 1.00 mM and 0.77 mM, respectively, at 24 hr. To examine the effects of these recreational drugs on NGF-induced neurite outgrowth, PC12 cells were treated with NGF together with MDMA, MDA, S-diphenylprolinol or R-diphenylprolinol at low toxic concentrations. The recreational drugs significantly suppressed neurite outgrowth of PC12 cells induced by NGF. The results suggest that these psychoactive recreational drugs may inhibit neurite growth and thus be implicated in their elicited neurotoxicity.

Cytotoxic effects of 3,4-methylenedioxy-N-alkylamphetamines, MDMA and its analogues, on isolated rat hepatocytes.

The amphetamine-derived designer drugs have been illegally used worldwide as recreational drugs, some of which are known to be hepatotoxic in humans. To compare their cytotoxic effects, 3,4-methylenedioxy-N-methamphetamine (MDMA) and its related analogues, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB), 3,4-(methylenedioxyphenyl)-2-butanamine (BDB) and 2-methylamino-1-(3,4-methylenedioxyphenyl)-propane-1-one (methylone) were studied in freshly isolated rat hepatocytes. MBDB caused not only concentration (0-4.0 mM)- and time (0-2 h)-dependent cell death accompanied by the formation of cell blebs, and the loss of cellular ATP and adenine nucleotide pools, and reduced glutathione levels, but also the accumulation of oxidized glutathione. Of the other analogues examined, the cytotoxicity of MBDB and BDB was greater than that of MDMA and methylone, suggesting that hepatotoxicity is generally induced by these drugs. In addition, DNA damage and the induction of reactive oxygen species were greater after the incubation of hepatocytes with MBDB (2 and 4 mM) than after that with MDMA. In isolated liver mitochondria, MBDB/BDB resulted in a greater increase in the rate of state 4 oxygen consumption than did MDMA/methylone, indicating an uncoupling effect and a decrease in the rate of state 3 oxygen consumption in a concentration dependent manner. Furthermore, MBDB resulted in mitochondrial swelling dependent on the mitochondrial permeability transition (MPT); the effect of MDMA was less than that of MBDB. Taken collectively, these results suggest that (1) the onset of cytotoxicity caused by designer drugs such as MBDB and MDMA is linked to mitochondrial failure dependent upon the induction of the MPT accompanied by mitochondrial depolarization and depletion of ATP through uncoupling of oxidative phosphorylation in rat hepatocytes, and (2) MBDB and MDMA elicit DNA damage, suggesting that nuclei as well as mitochondria are target sites of these compounds.

Synthesis and cytotoxic profile of 3,4-methylenedioxymethamphetamine ("ecstasy") and its metabolites on undifferentiated PC12 cells: A putative structure-toxicity relationship.

The toxicological and redox profiles of MDMA and its major metabolites (MDA, alpha-methyldopamine, N-methyl-alpha-methyldopamine, 6-hydroxy-alpha-methyldopamine, 3-methoxy-alpha-methyldopamine) were studied to establish a structure-toxicity relationship and determine their individual contribution to cell death induction by apoptosis and/or necrosis. The results of the comparative toxicity study, using undifferentiated PC12 cells, strongly suggest that the metabolites possessing a catecholic group are more toxic to the cells than MDMA and metabolites with at least one protected phenolic group. Redox studies reveal that an oxidative mechanism seems to play an important role in metabolite cytotoxicity. Nuclear features of apoptosis and/or necrosis show that most of the metabolites, particularly N-methyl-alpha-methyldopamine, induce cell death by apoptosis, largely accompanied by necrotic features. No significant differences were found between MDMA and the metabolites, concerning overall characteristics of cell death. These results may be useful to ascertain the contribution of metabolism in MDMA neurotoxicity molecular mechanisms.

Metabolism is required for the expression of ecstasy-induced cardiotoxicity in vitro.

Cardiovascular complications associated with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse have increasingly been reported. The indirect effect of MDMA mediated by a sustained high level of circulating biogenic amines may contribute to the cardiotoxic effects, but other factors, like the direct toxic effects of MDMA and its metabolites in cardiac cells, remain to be investigated. Thus, the objective of the present in vitro study was to evaluate the potential cardiotoxic effects of MDMA and its major metabolites 3,4-methylenedioxyamphetamine (MDA), N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA) using freshly isolated adult rat cardiomyocytes. The cell suspensions were incubated with these compounds in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 4 h. alpha-MeDA, N-Me-alpha-MeDA, and their respective aminochromes (oxidation products) were quantified in cell suspensions by HPLC-DAD. The toxic effects were evaluated at hourly intervals for 4 h by measuring the percentage of cells with normal morphology, glutathione (GSH), and glutathione disulfide (GSSG); intracellular Ca(2+), ATP, and ADP; and the cellular activities of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. No toxic effects were found after exposure of rat cardiomyocytes to MDMA or MDA at any of the tested concentrations for 4 h. In contrast, their catechol metabolites N-Me-alpha-MeDA and alpha-MeDA induced significant toxicity in rat cardiomyocytes. The toxic effects were characterized by a loss of normal cell morphology, which was preceded by a loss of GSH homeostasis due to conjugation of GSH with N-Me-alpha-MeDA and alpha-MeDA, sustained increase of intracellular Ca(2+) levels, ATP depletion, and decreases in the antioxidant enzyme activities. The oxidation of N-Me-alpha-MeDA and alpha-MeDA into the toxic compounds N-methyl-alpha-methyldopaminochrome and alpha-methyldopaminochrome, respectively, was also verified in cell suspensions incubated with these MDMA metabolites. The results obtained in this study provide evidence that the metabolism of MDMA into N-Me-alpha-MeDA and alpha-MeDA is required for the expression of MDMA-induced cardiotoxicity in vitro, being N-Me-alpha-MeDA the most toxic of the studied metabolites.

Hepatotoxicity of 3,4-methylenedioxyamphetamine and alpha-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 alpha-methyldopamine (alpha-MeDA), a major metabolite of MDA, in freshly isolated rat hepatocyte suspensions. The cells were incubated with MDA or alpha-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, alpha-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)-alpha-MeDA and 5-(glutathion- S-yl)-alpha-MeDA were identified by HPLC-DAD/EC within cells incubated with MDA or alpha-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.

Glutathione and N-acetylcysteine conjugates of alpha-methyldopamine produce serotonergic neurotoxicity: possible role in methylenedioxyamphetamine-mediated neurotoxicity.

Direct injection of either 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) or 3,4-(+/-)-methylenedioxyamphetamine (MDA) into the brain fails to reproduce the serotonergic neurotoxicity seen following peripheral administration. The serotonergic neurotoxicity of MDA and MDMA therefore appears to be dependent upon the generation of a neurotoxic metabolite, or metabolites, the identity of which remains unclear. alpha-Methyldopamine (alpha-MeDA) is a major metabolite of both MDA and MDMA. We have shown that intracerebroventricular (icv) injection of 2,5-bis(glutathion-S-yl)-alpha-methyldopamine [2, 5-bis(glutathion-S-yl)-alpha-MeDA] causes decreases in serotonin concentrations in the striatum, cortex, and hippocampus, and neurobehavioral effects similar to those seen following MDA and MDMA administration. In contrast, although 5-(glutathion-S-yl)-alpha-methyldopamine [5-(glutathion-S-yl)-alpha-MeDA] and 5-(N-acetylcystein-S-yl)-alpha-methyldopamine [5-(N-acetylcystein-S-yl)-alpha-MeDA] produce neurobehavioral changes similar to those seen with MDA and MDMA, and acute changes in brain 5-HT and dopamine concentrations, neither conjugate caused long-term decreases in 5-HT concentrations. We now report that direct intrastriatal or intracortical administration of 5-(glutathion-S-yl)-alpha-MeDA (4 x 200 or 4 x 400 nmol), 5-(N-acetylcystein-S-yl)-alpha-MeDA (4 x 7 or 4 x 20 nmol), and 2, 5-bis(glutathion-S-yl)-alpha-MeDA (4 x 150 or 4 x 300 nmol) causes significant decreases in striatal and cortical 5-HT concentrations (7 days following the last injection). Interestingly, intrastriatal injection of 5-(glutathion-S-yl)-alpha-MeDA or 2, 5-bis(glutathion-S-yl)-alpha-MeDA, but not 5-(N-acetylcystein-S-yl)-alpha-methyldopamine, also caused decreases in 5-HT concentrations in the ipsilateral cortex. The same pattern of changes was seen when the conjugates were injected into the cortex. The effects of the thioether conjugates of alpha-MeDA were confined to 5-HT nerve terminal fields, since no significant changes in monoamine neurotransmitter levels were detected in brain regions enriched with 5-HT cell bodies (midbrain/diencephalon/telencephalon and pons/medulla). In addition, the effects of the conjugates were selective with respect to the serotonergic system, as no significant changes were seen in dopamine or norepinephrine concentrations. The results indicate that thioether conjugates of alpha-MeDA are selective serotonergic neurotoxicants. Nonetheless, a role for these conjugates in the toxicity observed following systemic administration of MDA and MDMA remains to be demonstrated, and requires further experimentation.

The neurotoxic effects of methylenedioxymethamphetamine (MDMA) and its metabolites on rat brain spheroids in culture.

Rat whole-brain spheroids were used to assess the intrinsic neurotoxicity of methylenedioxy-methamphetamine (MDMA, Ecstasy) and two of its metabolites, dihydroxymethamphetamine (DHMA) and 6-hydroxy-MDMA (6-OH MDMA). Exposure of brain spheroids to MDMA or the metabolite 6-OH MDMA (up to 500 micromol/L) for 5 days in culture did not alter intracellular levels of glutathione (GSH), glial fibrillary acidic protein (GFAP) or serotonin (5-HT). In contrast, exposure to the metabolite DHMA, which can deplete intracellular thiols, significantly increased GSH levels (up to 170% of control) following exposure to 50 and 100 micromol/L DHMA. There was also a significant reduction in the levels of glial fibrillary acidic protein (GFAP) and GSH by DHMA at the highest concentration tested (500 micromol/L) but there was no effect on 5HT. This may constitute a sublethal neurotoxic compensatory response to DHMA in an attempt to replenish depleted intraneural GSH levels following metabolite exposure. Rat whole-brain spheroids may thus be a useful in vitro model to delineate mechanisms and effects of this class of neurotoxin.

Amphetamines induce apoptosis and regulation of bcl-x splice variants in neocortical neurons.

Amphetamineanalogs have emerged as popular recreational drugs of abuse. The number of reports of these substances producing severe acute toxicity and death is increasing. In 'Ecstasy' -associated deaths, focal necrosis in the liver and individual myocytic necrosis has been reported. Furthermore, serotonergic and dopaminergic neuronal cell damage has been observed in experimental amphetamine intoxication in laboratory animals. Here we demonstrate that subchronic exposure to D-amphetamine, methamphetamine, methylenedioxyamphetamine, and methylenedioxymethamphetamine ('Ecstasy') results in significant neurotoxicity in rat neocortical neurons in vitro. This neuronal cell death is accompanied by endonucleosomal DNA cleavage and differential expression of anti- and proapoptotic bcl-xL/S splice variants. In addition, we observed pronounced induction of cell stress-associated transcription factor c-jun and translation initiation inhibitor p97 after amphetamine treatment. These data support that the neurotoxic effects of different amphetamines are extended to rat neocortical neurons and that apoptotic pathways are involved in amphetamine-induced neurotoxicity.

2,5-Bis-(glutathion-S-yl)-alpha-methyldopamine, a putative metabolite of (+/-)-3,4-methylenedioxyamphetamine, decreases brain serotonin concentrations.

3,4-(+/-)-Methylenedioxyamphetamine (MDA) and 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) are serotonergic neurotoxicants. However, when injected directly into brain, MDA and MDMA are not neurotoxic, suggesting that systemic metabolism plays an important role in the development of neurotoxicity. The nature of the metabolite(s) responsible for MDA- and MDMA-mediated neurotoxicity is unclear. alpha-Methyldopamine is a major metabolite of MDA and is readily oxidized to the o-quinone, followed by conjugation with glutathione (GSH). Because the conjugation of quinones with GSH frequently results in preservation or enhancement of biological (re)activity, we have been investigating the role of quinone-thioethers in the acute and long-term neurochemical changes observed after administration of MDA. Although intracerebroventricular (i.c.v.) administration of 5-(glutathion-S-yl)-alpha-methyldopamine (4 x 720 nmol) and 5-(N-acetylcystein-S-yl)-alpha-methyldopamine (1 x 7 nmol) to Sprague-Dawley rats produced overt behavioral changes similar to those seen following administration of MDA (93 mumol/kg, s.c.) they did not produce long-term decreases in brain serotonin (5-hydroxytryptamine, 5-HT) concentrations. In contrast, 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine (4 x 475 nmol) decreased 5-HT levels by 24%, 65% and 30% in the striatum, hippocampus and cortex, respectively, 7 days after injection. The relative sensitivity of the striatum, hippocampus and cortex to 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine was the same as that observed for MDA; the absolute effects were greater with MDA. The effects of 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine were also selective for serotonergic nerve terminal fields, in that 5-HT levels were unaffected in regions of the cell bodies. Because 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine caused long-term depletion in 5-HT without adversely affecting the dopaminergic system, it also mimics the selectivity of MDA/MDMA. The data imply a possible role for quinone-thioethers in the neurobehavioral and neurotoxicological effects of MDA/MDMA.

Transformation of dopamine and alpha-methyldopamine by NG108-15 cells: formation of thiol adducts.

The catecholamines, alpha-methyldopamine (alpha-MeDA) and dopamine (DA), have been implicated in 3,4-(methylenedioxy)amphetamine (MDA) toxicity. The toxicity and metabolic fate of alpha-MeDA, a metabolite of MDA, and DA, a neurotransmitter released by MDA administration, were examined in NG108-15 cells. Both catechols were found to accumulate intracellularly into NG108-15 cells. alpha-MeDA was about 4 times more toxic than DA in the cells. The depletion of glutathione (GSH) by buthionine sulfoximine (BSO) resulted in a drastic increase (10 times) in the alpha-MeDA mediated toxicity while the toxicity of DA was enhanced by 2 times. DA was largely metabolized to dihydroxyphenylacetic acid (DOPAC) and, to a smaller extent, formed an adduct with GSH. alpha-MeDA was primarily metabolized to a GSH adduct. alpha-MeDA was also metabolized to a product which was identified as the cysteinyl adduct. These adducts were identified by HPLC coelution with authentic standards. The GSH and cysteinyl adducts are presumably formed through conjugation of the thiols with intermediary quinone oxidation products of DA and alpha-MeDA. Previous studies indicate that alpha-MeDA is significantly more toxic than DA, especially under conditions of GSH depletion. The results of this study suggest that alpha-MeDA toxicity may occur through cytoplasmic accumulation and oxidation to a reactive quinone species followed by reaction with vital thiol functions or generation of reactive oxygen species. Cytoplasmic DA levels, on the other hand, appear to be significantly lower due to MAO metabolism and vesicular storage, and therefore, DA appears less likely to form conjugates with thiol groups or participate in possible redox cycling.