Ethanol addictively enhances the in vitro cardiotoxicity of cocaine through oxidative damage, energetic deregulation, and apoptosis

Abstract

Cocaine (COC) is frequently consumed in polydrug abuse settings, and ethanol (EtOH) is the most prominent co-abused substance. Clinical data and experimental evidence suggest that the co-administration of COC with EtOH can be more cardiotoxic than EtOH or COC alone, but information on the molecular pathways involved is scarce. Since these data are crucial to potentiate the identification of therapeutic targets to treat intoxications, we sought to (i) elucidate the type of interaction that occurs between both substances, and (ii) assess the mechanisms implicated in the cardiotoxic effects elicited by COC combined with EtOH. For this purpose, H9c2 cardiomyocytes were exposed to COC (104 µM-6.5 mM) and EtOH (977 µM-4 M), individually or combined at a molar ratio based on blood concentrations of intoxicated abusers (COC 1: EtOH 9; 206 µM-110 mM). After 24 h, cell metabolic viability was recorded by the MTT assay and mixture toxicity expectations were calculated using the independent action (IA) and concentration addition (CA) models. EtOH (EC 305.26 mM) proved to act additively with COC (EC 2.60 mM) to significantly increase the drug in vitro cardiotoxicity, even when both substances were combined at individually non-cytotoxic concentrations. Experimental mixture testing (EC 19.18 ± 3.36 mM) demonstrated that the cardiotoxicity was fairly similar to that predicted by IA (EC 22.95 mM) and CA (EC 21.75 mM), supporting additivity. Concentration-dependent increases of intracellular ROS/RNS and GSSG, depletion of GSH and ATP, along with mitochondrial hyperpolarization and activation of intrinsic, extrinsic, and common apoptosis pathways were observed both for single and combined exposures. In general, the mixture exhibited a toxicological profile that mechanistically did not deviate from the single drugs, suggesting that interventions such as antioxidant administration might aid in the clinical treatment of this type of polydrug intoxication. In a clinical perspective, the observed additive mixture effect may reflect the increased hazards at which users of this combination are exposed to in recreational settings.

A Metabolomic Approach for the In Vivo Study of Gold Nanospheres and Nanostars after a Single-Dose Intravenous Administration to Wistar Rats

“…For ATP determination, 150 µL of the homogenized supernatant was neutralized with the same volume of 0.76 mM KHCO 3 and centrifuged (13,000 rpm, 10 min, 4 °C). The ATP intracellular level was quantified in the neutralized supernatant using a luciferin/luciferase bioluminescent method as previously described [28]. Briefly, 75 µL of luciferin–luciferase reagent (0.15 mM luciferin; 300,000 light units of luciferase from Photinus pyralis (American firefly); 50 mM glycine; 10 mM MgSO 4 ; 1 mM Tris; 0.55 mM EDTA; 1% BSA; pH 7.6; 4 °C; protected from light) was added to 75 µL of neutralized supernatant just before luminescence readings.…”

“…Both total glutathione (GSHt) and oxidized glutathione (GSSG) were quantified using a previously described method [28] of the DTNB-GSSG reductase recycling assay. For the GSSG determination, 10 μL of vinylpyridine was added to 200 µL of all samples, standards and blank, and further shaken during 1 h on ice.…”

Toxicological Evaluation of Luminescent Silica Nanoparticles as New Drug Nanocarriers in Different Cancer Cell Lines

“…Metabolic activity was evaluated by the MTT reduction assay as previously described [27] with slight modifications. Briefly, the colorimetric assay is based on the reduction of the tetrazolium salt MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to insoluble formazan crystals in the presence of mitochondrial enzymes.…”

Association of recreational drug consumption, cardiac toxicity and heart transplantation

“…4 In a recent study, cocaine and ethanol, both together and independently, increased mitochondrial hyperpolarization and activation of common apoptosis pathways in cardiomyocytes. 5 Although in 2015 the European Drug Emergencies Network reported that 35 patients presented with cardiac arrest at emergency departments during a 12-month study period, 1 cardiac arrest and decompensation remain a small fraction of the total volume of emergency admissions following the use of recreational drugs. Moreover, there are no data on the effect of a combination of recreational drugs and alcohol on the cardiac health status of users, but we can speculate that this would increase toxicity and adverse effects.…”