The risky cocktail: what combination effects can we expect between ecstasy and other amphetamines?

Carvalho

et al. 2013

J. Appl. Toxicol.

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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.

Section: Discussionmentioning

confidence: 97%

“…Whether the additivity herein observed extends to other combination ratios awaits further experimental confirmation. However, our previous work using this study model showed that different mixtures of amphetamine drugs, combined at different ratios, always produced additive effects (Dias da Silva et al ., ).…”

Section: Discussionmentioning

confidence: 99%

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

Carvalho

et al. 2013

J. Appl. Toxicol.

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“…As mentioned above, we have previously tested the combined effects of commonly abused amphetamines in an in vitro model using human hepatoma Hep G2 cells. We demonstrated that, even when each component was present at very low concentrations, mixtures of these drugs had significant hepatotoxic effect (Dias da Silva et al ., ). We have now extended our previous research by applying this study model to hyperthermic conditions, which better reflect the physiological environment of the liver in an acute intoxication scenario, owing to the thermogenic action of the drugs and the environmental circumstances of the abuse.…”

Section: Introductionmentioning

confidence: 99%

“…37 °C) to highlight potential changes in cytotoxicity associated with an increase in body temperature. Furthermore, in a similar way to our previous study (Dias da Silva et al ., ), we tested the applicability of two mathematical models, concentration addition (CA) and independent action (IA), with the aim of investigating whether good agreement was still observed with the CA expectations, under the new experimental conditions. Finally, in order to delineate the mechanisms underlying the observed cytotoxicity, we further evaluated the involvement of several stress indicators in the promoted effects, namely the increased formation of reactive oxygen (ROS) and nitrogen (RNS) species, the contents of reduced (GSH) and oxidized (GSSG) glutathione, the loss of mitochondrial membrane potential (Δ ψm ), lipid peroxidation markers and the ATP intracellular levels.…”

Section: Introductionmentioning

confidence: 99%

Combination effects of amphetamines under hyperthermia - the role played by oxidative stress

Carmo

2013

J. Appl. Toxicol.

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Rise in body temperature is a life-threatening consequence of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse. We evaluated the impact of hyperthermia on the cytotoxicity of combinations of MDMA and three other amphetamines, often co-ingested. For this, Hep G2 cells were exposed to MDMA, d-amphetamine, methamphetamine and 4-methylthioamphetamine, individually or combined, at 40.5 °C. The results were compared with normothermia data (37.0 °C). Mixture additivity expectations were calculated by independent action and concentration addition (CA) models. To delineate the mechanism(s) underlying the elicited effects, a range of stress endpoints was evaluated, including quantification of reactive oxygen/nitrogen species (ROS/RNS), lipid peroxidation, reduced/oxidized glutathione (GSH/GSSG), ATP and mitochondrial membrane potential (Δψm) changes. Our data show that, in hyperthermia, amphetamines acted additively and mixture effects were accurately predicted by CA. At 40.5 °C, even slight increases in the concentrations of each drug/mixture promoted significant rises in cytotoxicity, which quickly shifted from roughly undetectable to maximal mortality. Additionally, the increase of RNS/ROS production, decrease of GSH, ATP depletion and mitochondrial impairment were exacerbated under hyperthermia. Importantly, when equieffective cytotoxic concentrations of the mixture and individual amphetamines were compared for all tested stress endpoints, mixture effects did not deviate from those elicited by individual treatments, suggesting that these amphetamines have a similar mode of action, which is not altered in combination. Concluding, our data indicate that amphetamine mixtures produce deleterious effects, even when individual drugs are combined at negligible concentrations. These effects are strongly exacerbated in hyperthermia, emphasizing the potential increased risks of ecstasy intake, especially when hyperthermia occurs concurrently with polydrug abuse.

“…Thus, after exhaustive characterization of the toxicity profile of the single agents, the expected mixture effects were calculated, using the CA and IA approaches, and set as reference, as previously reported Dias da Silva et al 2013b). These concepts are based on general assumptions regarding the mechanisms of action of the mixture constituents and their details have been discussed elsewhere (Kortenkamp 2007).…”

Section: Prediction Of Mixture Effectsmentioning

confidence: 99%

In vitro hepatotoxicity of ‘Legal X’: the combination of 1-benzylpiperazine (BZP) and 1-(m-trifluoromethylphenyl)piperazine (TFMPP) triggers oxidative stress, mitochondrial impairment and apoptosis

Moreira

et al. 2016

Arch Toxicol

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N-Benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP) are two synthetic phenylpiperazine analogues that have been frequently commercialized in combination as an alternative to ecstasy ('Legal X'). Despite reports of several clinical complications following the use of these drugs in association, few studies have been conducted so far to elucidate their combined toxicity. The present study was aimed at clarifying the cytotoxic effects of mixtures of BZP and TFMPP in vitro. Human-derived HepaRG cells and primary rat hepatocytes were exposed to the drugs, individually or combined at different mixture ratios, and cytotoxicity was assessed by the MTT assay. Mixture additivity expectations were calculated by the independent action and the concentration addition (CA) models and compared with the experimental outcomes. To delineate the mechanisms underlying the elicited effects, a range of stress endpoints was evaluated, including oxidative stress, energetic imbalance, and metabolic interactions. It was observed that primary rat hepatocytes are more sensitive than HepaRG cells to the toxicity of BZP (EC 2.20 and 6.60 mM, respectively) and TFMPP (EC 0.14 and 0.45 mM, respectively). For all BZP-TFMPP combinations tested, CA was the most appropriate model to predict the mixture effects. TFMPP proved to act additively with BZP to produce significant hepatotoxicity (p < 0.01). Remarkably, substantial mixture effects were observed even when each drug was present at concentrations that were harmless individually. In primary hepatocytes, a small deviation from additivity (antagonism) was observed toward the upper range of the concentration-response curve. GC/MS data suggest that a metabolic interaction may be at a play, as the mixture favors the metabolism of both substances, to a significant extent in the case of BZP (p < 0.05). Also, our results demonstrate the influence of oxidative stress and energetic imbalance on these effects (increase in RNS and ROS production, decrease in intracellular GSH/GSSG, ATP depletion and mitochondrial Δψm disruption). The present work clearly demonstrates that potentially harmful interactions among BZP and TFMPP are expected when these drugs are taken concomitantly.