Serotonergic Neurotoxic Metabolites of Ecstasy Identified in Rat Brain

Abstract

The selective serotonergic neurotoxicity of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) depends on their systemic metabolism. We have recently shown that inhibition of brain endothelial cell ␥-glutamyl transpeptidase (␥-GT) potentiates the neurotoxicity of both MDMA and MDA, indicating that metabolites that are substrates for this enzyme contribute to the neurotoxicity. Consistent with this view, glutathione (GSH) and N-acetylcysteine conjugates of ␣-methyl dopamine (␣-MeDA) are selective neurotoxicants. However, neurotoxic metabolites of MDMA or MDA have yet to be identified in brain. Using in vivo microdialysis coupled to liquid chromatography-tandem mass spectroscopy and a high-performance liquid chromatography-coulometric electrode array system, we now show that GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA are present in the striatum of rats administered MDMA by subcutaneous injection. Moreover, inhibition of ␥-GT with acivicin increases the concentration of GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA in brain dialysate, and there is a direct correlation between the concentrations of metabolites in dialysate and the extent of neurotoxicity, measured by decreases in serotonin (5-HT) and 5-hydroxyindole acetic (5-HIAA) levels. Importantly, the effects of acivicin are independent of MDMA-induced hyperthermia, since acivicin-mediated potentiation of MDMA neurotoxicity occurs in the context of acivicin-mediated decreases in body temperature. Finally, we have synthesized 5-(N-acetylcystein-S-yl)-N-methyl-␣-MeDA and established that it is a relatively potent serotonergic neurotoxicant. Together, the data support the contention that MDMA-mediated serotonergic neurotoxicity is mediated by the systemic formation of GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA (and ␣-MeDA). The mechanisms by which such metabolites access the brain and produce selective serotonergic neurotoxicity remain to be determined.Although the selectivity of (Ϯ)-3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and (Ϯ)-3,4-methylenedioxyamphetamine (MDA) for the serotonergic system in rats and humans is firmly established, the mechanism(s) involved are not fully understood. In rats, MDMA is cleared mainly by hepatic metabolism by N-demethylation to form MDA. MDMA and MDA are further O-demethylenated to 3,4-dihydroxymethamphetamine (N-methyl-␣-methyldopamine; N-Me-␣-MeDA) and 3,4-dihydroxyamphetamine (␣-methyldopamine; ␣-MeDA), respectively. N-Me-␣-MeDA and ␣-MeDA are highly redox-unstable catechols and are conjugated with sulfate and glucuronic acid. Both catechols can also be rapidly oxidized to their corresponding orthoquinones and form adducts with glutathione (GSH) and other thiol-containing compounds (Lim and Foltz, 1988;Hiramatsu et al., 1990). Alternatively, N-Me-␣-MeDA and ␣-MeDA can be O-methylated to form 4-hydroxy-3-methoxymethamphetamine (3-O-Me-N-Me-␣-MeDA) or 4-hydroxy-3-methoxyamphetamine (3-O-Me-␣-MeDA), respectively.

Electrophysiologic Changes in Ventral Midbrain Dopaminergic Neurons Resulting from (+/−) -3,4-Methylenedioxymethamphetamine (MDMA—“Ecstasy”)

“…To understand the psychostimulant effects of MDMA, our results, obtained at the level of midbrain DA perikaria, need to be integrated with the actions of the drug at the axon terminals, which are largely responsible for the net DA efflux in the central nervous system. Furthermore, additional serotoninergic effects of MDMA's hepatic metabolites in vivo (Jones et al 2005) that could participate in the overall action of the drug should be considered.…”

Actions of 3,4-methylenedioxymethamphetamine (MDMA) on cerebral dopaminergic, serotonergic and cholinergic neurons

“…Data from microdialysis studies that support the conclusion that MDMA induces oxidative stress include the findings that neurotoxic regimens of MDMA increase the formation of reactive oxygen species (e.g., hydroxyl radicals), as well as reactive nitrogen species (e.g., nitric oxide) (Colado et al, 1997;Shankaran et al, 1999a, Shankaran et al, 1999b. Furthermore, the acute activation of the DA and 5-HT transporters by MDMA or a toxic metabolite of MDMA appears necessary for MDMAinduced hydroxyl radical formation (Shankaran et al, 1999a, b;Camarero et al, 2003;Jones et al, 2005). Thus, activation of the DA and 5-HT transporters appears critical in linking the acute stimulatory effects of MDMA to the long-term neurotoxic effects of this drug of abuse.…”

A developmental comparison of the neurobehavioral effects of ecstasy (MDMA)

“…Interestingly, direct administration of MDMA into the rat brain does not cause enduring neurochemical changes [43] suggesting that peripheral metabolism is necessary and that one or more metabolites cause neurotoxic effects. The identity of the presumed "neurotoxic metabolite" is uncertain as it has been difficult to identify a single metabolite with the same neurochemical profile as MDMA [68]. Due to this complexity, many investigators have focused their efforts on quantifying the levels of MDMA and its primary demethylated metabolite, methylenedioxyamphetamine (MDA).…”