Cytotoxic Activity of Pyrovalerone Derivatives, an Emerging Group of Psychostimulant Designer Cathinones.

The growing popularity of novel psychoactive substances (NPS) has aroused the concerns of public health specialists. The pyrovalerone derivatives are a branch of synthetic cathinones, a very popular group of psychostimulant NPS. Despite numerous case reports of fatal intoxications, little is known about the cytotoxicity of these substances. Therefore, this study was aimed to evaluate the toxic properties of pyrovalerone, its highly prevalent derivative 3,4-methylenedioxypyrovalerone (3,4-MDPV) with its two major metabolites (catechol-MDPV and methylcatechol-MDPV) and the structural isomer 2,3-MDPV, together with newer members of the group, i.e., α-pyrrolidinovalerothiophenone (α-PVT) and α-pyrrolidinooctanophenone (PV9), using model human cell lines for neurons (SH-SY5Y), hepatocytes (Hep G2), and upper airway epithelium (RPMI 2650). We found that the first generation pyrovalerones (pyrovalerone, 3,4-MDPV, and 2,3-MDPV) produced a modest decrease of mitochondrial activity in the three examined cell lines, but were active in lower concentrations than methamphetamine used as a reference psychostimulant compound. Since catechol-MDPV displayed greater toxic potential than the parent compound, we suggest that the toxicity of 3,4-MDPV could be attributed to activity of this metabolite. Strikingly, the two new generation pyrovalerones, α-PVT and PV9, seem to be the most potent cytotoxic compounds: both induced highly pronounced mitochondrial dysfunction; the latter also demonstrated significant damage to cell membranes. The reported in vitro toxic activity of pyrovalerone cathinones against different cell types reinforces existing concerns regarding the health risks associated with the intake of these drugs.

Effects of particle size and coating on toxicologic parameters, fecal elimination kinetics and tissue distribution of acutely ingested silver nanoparticles in a mouse model.

Consumer exposure to silver nanoparticles (AgNP) via ingestion can occur due to incorporation of AgNP into products such as food containers and dietary supplements. AgNP variations in size and coating may affect toxicity, elimination kinetics or tissue distribution. Here, we directly compared acute administration of AgNP of two differing coatings and sizes to mice, using doses of 0.1, 1 and 10 mg/kg body weight/day administered by oral gavage for 3 days. The maximal dose is equivalent to 2000× the EPA oral reference dose. Silver acetate at the same doses was used as ionic silver control. We found no toxicity and no significant tissue accumulation. Additionally, no toxicity was seen when AgNP were dosed concurrently with a broad-spectrum antibiotic. Between 70.5% and 98.6% of the administered silver dose was recovered in feces and particle size and coating differences did not significantly influence fecal silver. Peak fecal silver was detected between 6- and 9-h post-administration and <0.5% of the administered dose was cumulatively detected in liver, spleen, intestines or urine at 48 h. Although particle size and coating did not affect tissue accumulation, silver was detected in liver, spleen and kidney of mice administered ionic silver at marginally higher levels than those administered AgNP, suggesting that silver ion may be more bioavailable. Our results suggest that, irrespective of particle size and coating, acute oral exposure to AgNP at doses relevant to potential human exposure is associated with predominantly fecal elimination and is not associated with accumulation in tissue or toxicity.

Electrophysiological profile of vernakalant in an experimental whole-heart model: the absence of proarrhythmia despite significant effect on myocardial repolarization.

AIM: The most recent European Society of Cardiology (ESC) update on atrial fibrillation has introduced vernakalant (VER) for pharmacological cardioversion of atrial fibrillation. The aim of the present study was to investigate the safety profile of VER in a sensitive model of proarrhythmia. METHODS AND RESULTS: In 36 Langendorff-perfused rabbit hearts, VER (10, 30 µM, n = 12); ranolazine (RAN, 10, 30 µM, n = 12), or sotalol (SOT, 50; 100 µM, n = 12) were infused after obtaining baseline data. Monophasic action potentials and a 12-lead electrocardiogram showed a significant QT prolongation after application of VER as compared with baseline (10 µM: +25 ms, 30 µM: +50 ms, P < 0.05) accompanied by an increase of action potential duration (APD). The increase in APD90 was accompanied by a more marked increase in effective refractory period (ERP) leading to a significant increase in post-repolarization refractoriness (PRR, 10 µM: +30 ms, 30 µM: +36 ms, P < 0.05). Vernakalant did not affect the dispersion of repolarization. Lowered potassium concentration in bradycardic hearts did not provoke early afterdepolarizations (EADs) or polymorphic ventricular tachycardia (pVT). Comparable results were obtained with RAN. Hundred micromolars of SOT led to an increase in QT interval (+49 ms) and APD90 combined with an increased ERP and PRR (+23 ms). In contrast to VER, 100 µM SOT led to a significant increase in dispersion of repolarization and to the occurrence of EAD in 10 of 12 and pVT in 8 of 12 hearts. CONCLUSION: In the present study, application of VER and SOT led to a comparable prolongation of myocardial repolarization. Both drugs increased the PRR. However, VER neither affect the dispersion of repolarization nor induce EAD and therefore did not cause proarrhythmia.

"How high do they look?": identification and treatment of common ingestions in adolescents.

Adolescents have access to a variety of legal or illicit substances that they use to alter their mood or "get high." The purpose of this review is to provide an overview of common substances adolescents use to get high, including the illicit substances synthetic marijuana or "Spice," salvia, MDMA, synthetic cathinones, and 2C-E. Dextromethorphan and energy drinks are easily accessible substances that teenagers abuse. The toxic effects of common ingestions and treatment of overdose is discussed to inform pediatric providers who provide care for adolescents.

Pre-clinical safety evaluation of pyrrolidine dithiocarbamate.

Pyrrolidine dithiocarbamate (PDTC) was examined for its potential in the intranasal treatment of human rhinovirus infections. Prior to clinical testing, a comprehensive non-clinical programme was performed to evaluate the general toxicity of PDTC. The animal experiments included investigations in rodents with study durations ranging from single dose to repeated dosing over a period of 28 days. The routes of administration were intranasal, inhalative, oral and intravenous for single-dose toxicity and pharmacokinetic studies, and intranasal for repeated dose studies. Blood and tissue samples were obtained from PDTC-treated rats to analyse pharmacokinetics and tissue distribution. Accumulation of selected metals due to PDTC treatment was examined in liver, brain, nerves and fat tissues.

Multicomponent T2 analysis of dithiocarbamate-mediated peripheral nerve demyelination.

Standard light microscope histological evaluation of peripheral nerve lesions has been used routinely to assess peripheral nerve demyelination; however, the development of magnetic resonance (MR) methodology for assessing peripheral nerve may provide complementary information, with less expense and in less time than nerve histology methods. In this study, the utility of multicomponent NMR T(2) relaxation analysis for assessing myelin injury in toxicology studies was examined using two dithiocarbamates, N,N-diethyldithiocarbamate (DEDC) and pyrrolidine dithiocarbamate (PDTC), known to produce myelin injury and elevate copper in the nervous system. T(2) analysis was used in conjunction with standard histological methods to assess myelin injury and determine if dithiocarbamate-mediated copper accumulation in peripheral nerve was associated with more severe myelin lesions. Male Sprague-Dawley rats were administered i.p. DEDC for 8 weeks and maintained on either a diet containing normal (13 ppm) or elevated (200 ppm) copper. Another group of male Sprague-Dawley rats was administered oral PDTC and a 200 ppm copper diet, with controls given only the 200 ppm copper diet, for 47 weeks. Following exposures, the morphology of sciatic nerve was evaluated using light microscopy and multicomponent T(2) analysis of excised fixed nerves; and copper levels in sciatic nerve were determined using ICP-AES. Light microscopy demonstrated the presence of a primary myelinopathy in dithiocarbamate-exposed rats characterized by intramyelinic edema, demyelination, and secondary axonal degeneration. Both the nerve copper level and number of degenerated axons, as ascertained by ICP-AES and microscopy, respectively, were augmented by dietary copper supplementation in conjunction with administration of DEDC or PDTC. T(2) analysis revealed a decreased contribution from the shortest T(2) component in multicomponent T(2) spectra obtained from animals administered DEDC or PDTC, consistent with decreased myelin content; and the decrease of the myelin water component was inversely correlated to the levels of nerve copper and myelin lesion counts. Also, the T(2) analysis showed reduced variability compared to histological assessment. These studies support multicomponent T(2) analysis as a complementary method to light microscopic evaluations that may also be applicable to in vivo assessments.

Dietary copper enhances the peripheral myelinopathy produced by oral pyrrolidine dithiocarbamate.

The neurotoxic hazard of a dithiocarbamate is influenced by route of exposure and acid stability of the dithiocarbamate. As an example, oral administration of the acid labile dithiocarbamate N,N-diethyldithiocarbamate (DEDC) causes a central-peripheral axonopathy thought to result from acid-promoted decomposition to CS2 in the stomach. In contrast, parenteral administration of DEDC, which bypasses the acidic environment of the stomach, causes a primary demyelination that is thought to be mediated through the intact parent dithiocarbamate. The relative acid stability of pyrrolidine dithiocarbamate (PDTC) suggests that a significant portion of a dose can be absorbed intact following oral exposure with the potential to produce a primary myelin injury. The present study was performed to characterize the neurotoxicity of PDTC and evaluate the possible role of copper in dithiocarbamate-mediated demyelination. Male Sprague Dawley rats were administered PDTC in drinking water and given either a normal- or high-copper diet for 18, 47, or 58 weeks. Examination of peripheral nerve by light microscopy and electron microscopy at the end of exposures revealed primary myelin lesions and axonal degeneration in the PDTC groups, with a significant increase in the severity of several lesions observed for the PDTC, high-copper group relative to the PDTC normal-copper diet. ICP-AES metal analysis determined that the PDTC groups had significantly increased brain copper, and at 58 weeks a significant increase in copper was seen in the sciatic nerve of PDTC high-copper animals relative to PDTC normal-copper diet animals. Although RP-HPLC analysis could not detect globin alkylaminocarbonyl cysteine modifications analogous to those seen with parenteral DEDC, LC/MS/MS identified (pyrrolidin-1-yl carbonyl)cysteine adducts on PDTC-exposed rat globin. These findings are consistent with previous studies supporting the ability of acid-stable dithiocarbamates to mediate myelin injury following oral exposure. The greater severity of lesions associated with dietary copper supplementation and elevated copper levels in nerve also suggests that perturbation of copper homeostasis may contribute to the development of myelin lesions.

Increases in cortical glutamate concentrations in transgenic amyotrophic lateral sclerosis mice are attenuated by creatine supplementation.

Several lines of evidence implicate excitotoxic mechanisms in the pathogenesis of amyotrophic lateral sclerosis (ALS). Transgenic mice with a superoxide dismutase mutation (G93A) have been utilized as an animal model of familial ALS (FALS). We examined the cortical concentrations of glutamate using in vivo microdialysis and in vivo nuclear magnetic resonance (NMR) spectroscopy, and the effect of long-term creatine supplementation. NMDA-stimulated and Ltrans-pyrrolidine-2,4-dicarboxylate (LTPD)-induced increases in glutamate were significantly higher in G93A mice compared with littermate wild-type mice at 115 days of age. At this age, the tissue concentrations of glutamate were also significantly increased as measured with NMR spectroscopy. Creatine significantly increased longevity and motor performance of the G93A mice, and significantly attenuated the increases in glutamate measured with spectroscopy at 75 days of age, but had no effect at 115 days of age. These results are consistent with impaired glutamate transport in G93A transgenic mice. The beneficial effect of creatine may be partially mediated by improved function of the glutamate transporter, which has a high demand for energy and is susceptible to oxidative stress.

Cytotoxic effect and electrophysiological activity of (S)-bgugaine, an alkylpyrrolidine alkaloid against MRC-5 fibroblasts.

(+)-S-bgugaine [1], is an alkaloid prepared by enantioselective synthesis. This alkaloid is an isomer of R-bgugaine [2], an alkaloid isolated from Arisarum vulgare, an Araceae toxic plant of Morocco. The cytotoxic effect and the electrophysiological activity of (+)-S-bgugaine [1] against MRC-5 fibroblasts of (+)-S-bgugaine 1, were studied. (+)-S-bgugaine [1] showed a cytotoxic potential at 40 microg/ml against these MRC-5 cells. The electrophysiological study on MRC-5 cells was carried out using the technique of patch-clamp and showed that the activity of compound 1 involved a reduction of outward potassic current at the concentration of 100 microM (28.1 microg/ml) and was accentuated by 200 microM (56.2 microg/ml). In this study we show that S-bgugaine [1], decreases the outward potassic current.

Bgugaine, a pyrrolidine alkaloid from Arisarum vulgare, is a strong hepatotoxin in rat and human liver cell cultures.

Toxicity of bgugaine, a pyrrolidine alkaloid extracted from the tubers of Arisarum vulgare, was studied in three different liver cell culture models: (1) the rat hepatocyte primary culture; (2) a liver epithelial cell line; and (3) the human hepatoblastoma cell line HepG2. Cytotoxicity was evaluated by LDH release, MTT reduction and MDA production. DNA fragmentation was analysed by flow cytometry or DNA gel-electrophoresis. In hepatocyte and epithelial cell cultures, drug toxicity appeared at 30 microM and was evaluated by an increase in LDH release, a decrease in MTT reduction and a higher level of MDA production. Bgugaine concentrations lower than 30 microM did not induce changes in these parameters. In HepG2 cells, bgugaine treatment also induced LDH release at concentrations of 40 and 50 microM. DNA fragmentation, analysed in the HepG2 cell line by flow cytometry, was observed in cultures exposed to 50 microM bgugaine. However, using DNA gel-electrophoresis, we demonstrated that lower bgugaine concentrations (10, 20 and 30 microM) also induced DNA damage. Our results show that: (1) bgugaine induces an important hepatotoxicity; (2) bgugaine toxicity is not mediated by a metabolic derivative; and (3) bgugaine induces a significant DNA damage. Therefore, our data suggest that the alkaloid bgugaine contained in Arisarum vulgarae may be involved in the toxicologic symptoms observed after consumption of this plant tubers by humans and animals.

Inhibition of glutamate uptake induces progressive accumulation of extracellular glutamate and neuronal damage in rat cortical cultures.

It is known that neurons exposed to high concentrations of glutamate degenerate and die. The clearance of this amino acid from the extracellular space depends on their active transport by Na(+)-dependent high-affinity carriers. In the present study we tested whether inhibition of glutamate transport in mixed glial/neuronal cortical cultures induces accumulation of extracellular glutamate and whether such increase results in cell damage. Three inhibitors of glutamate transport were used: L-trans-pyrrolidine-2,4-dicarboxylate (PDC), DL-threo-beta-hydroxyaspartate (THA), and dihydrokainate (DHK). Cell damage was assessed by light microscopy observations, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and leakage of lactate dehydrogenase. PDC induced a significant concentration- and time-dependent neuronal damage, whereas pure glial cultures were not affected. A good correlation was found between this damage and elevations of glutamate concentration in the medium. These effects of PDC were similar in glutamine-free medium and in medium supplemented with glutamine. THA induced identical cell damage and elevations of extracellular glutamate to those produced by PDC, while DHK did not affect at all any of these parameters. PDC- and THA-induced toxicity was protected by the N-methyl-D-aspartate receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-(a,d)cyclohepten-5,10-imine maleate but not by the non-N-methyl-D-aspartate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline.

Mutagenicity of nitroxyl compounds: structure-activity relationships.

Three piperidinoxyl radicals were found to be directly mutagenic in Salmonella typhimurium TA 100, one pyrrolidinoxyl compound had weaker activity, and two other pyrrolidinoxyl derivatives did not produce an increase of the spontaneous revertants. The tester strain TA 100 was selected in preliminary tests for its higher sensitivity compared to TA 98 and TA 102. The mutagenic activity of the three active compounds was abolished by partial reduction with ascorbic acid, suggesting that the mutagenicity was linked to the free radical nature of these compounds, and reduced in the presence of a cofactor supplemented rat liver subcellular fraction. The mutagenicity of the tested compounds was correlated to the resistance of the nitroxyl spin labels to reduction: the more reactive radicals were found to possess higher mutagenic activity.

The organotypic tissue culture model of corticostriatal system used for examining amino acid neurotoxicity and its antagonism: studies on kainic acid, quinolinic acid and (-) 2-amino-7-phosphonoheptanoic acid.

Organotypic cultures of caudate nucleus and frontal cerebral cortex, either alone or in combination with each other, have been used to evaluate and compare the neurotoxic effects of two dicarboxylic amino acids, kainic acid (KA) and the tryptophan metabolite quinolinic acid (QUIN). Both of these agents can induce specific post-synaptic degeneration in cultures in which a complement of well-developed mature synapses exists. The neurotoxic effects of QUIN can be blocked by the synthetic anti-convulsant agent (-)2-amino-7-phosphonoheptanoic acid [( -]APH), but neurotoxicity of KA cannot. These studies support the candicacy of QUIN as an endogenous neurotoxin with properties similar to KA. Furthermore, the studies demonstrate the usefulness of the organotypic nerve tissue culture model as a research tool for examining certain neurodegenerative phenomena and for identifying neurotoxic amino acids as well as compounds which may antagonize amino acid neurotoxicity.

Failure of folic acid derivatives to mimic the actions of kainic acid in brain in vitro or in vivo.

The ability of three derivatives of folic acid, N-5-methyltetrahydrofolic acid (MTHF), tetrahydrofolic acid (THF) and dihydrofolic acid (DHF) to mimic the actions of kainic acid (KA) in a number of in vitro and in vivo systems known to be sensitive to KA was examined. None of the three folate derivatives at 100 microM concentration significantly inhibited the specific binding of [3H]-KA to striatal membranes although 2 microM L-glutamate produced a 40% inhibition. None of the three folate derivatives stimulated the formation of cyclic GMP in cerebellar slices incubated in vitro although KA (0.5 mM) increased cyclic GMP levels by 2.5-fold. Whereas intrahippocampal injection of 2.3 nmoles of KA produces prolonged abnormalities of the EEG, limbic-type seizures and a characteristic pattern of neuronal degeneration in the hippocampal formation and related structures, intrahippocampal injection of a 100-fold greater dose of THF caused only minor and transient EEG abnormalities, no overt seizures and a highly restricted lesion. Whereas intrastriatal injection of 5.6 nmoles of KA caused a profound reduction in the specific activities of choline acetyltransferase and glutamate decarboxylase, markers for striatal intrinsic cholinergic and GABAergic neurons, 50-fold greater doses of MTHF did not affect either enzyme although this high dose of THF did cause a significant 33% reduction in choline acetyltransferase activity. These findings support the suggestion that THF may have weak neurotoxic effects in brain but indicate that the actions of this compound and the related MTHF and DHF are not mediated through KA-specific receptors.

Effects of kainic acid applied to the ventral surface of the medulla oblongata on vasomotor tone, the baroreceptor reflex and hypothalamic autonomic responses.

Application of an excitotoxic amino acid, kainic acid, to the ventral medullary surface just caudal to the trapezoid bodies (at Feldberg and Guertzenstein's glycine-sensitive area) led to the following observations. (1) Blood pressure began to rise within 25 s and by 10 min rose to high levels (200-240 mm Hg). Blood pressure subsequently fell to levels at or approaching those of a spinal animal. (2) Sympathetic vasomotor activity became insensitive to baroreceptor inhibition shortly after the peak in blood pressure, and the cardioinhibitory action of the reflex was enhanced during this time. (3) The autonomic effects of hypothalamic stimulation were differentially affected--pupillary dilatation and retraction of the nictitating membranes were unaffected, while the increases in blood pressure and renal nerve activity were blocked. (4) Recovery from these effects was observed on two occasions, when the animals were infused with a pressor agent and allowed to survive beyond 6 h after the kainic acid application. These results support the view that vasomotor tone is dependent upon the activity of relatively superficial cells in the ventral medulla. We further suggest that baroreceptor inhibition of sympathetic vasomotor activity acts via these cells and that descending hypothalamic autonomic pathways are organized at this level in terms of separate end organs.

Effects of kainic acid injection and cortical lesion on ornithine and aspartate aminotransferases in rat striatum.

The effects of cortical lesions and intrastriatal kainic acid injections on various striatal enzyme activities were investigated. Ornithine aminotransferase decreased concomitantly with glutamate uptake in decorticated and chronic kainic acid-treated rats. It was also decreased in acute kainic acid-lesioned striatum where glutamate uptake was unaffected. Aspartate aminotransferase, however, decreased only after acute kainic acid treatment. Results for glutamate uptake, glutamate decarboxylase, and choline acetyltransferase were in agreement with previous findings. These results suggest that ornithine may act as a precursor for glutamate in nerve terminals, although the nonspecific localization does not allow ornithine aminotransferase to be a convenient biochemical marker. The decrease in aspartate aminotransferase is thought to be due to the widespread cell degeneration after acute kainic acid. Aspartate aminotransferase activities were also found to be reduced in the frontal cortex, caudate nucleus and putamen of Huntington's disease brains.

Kainic acid neurotoxicity; effect of systemic injection on neurotransmitter markers in different brain regions.

Systemic injection of kainic acid (12 mg/kg) induces necrosis and neuronal degeneration in several brain regions. The most pronounced effects were observed in the piriform cortex, amygdaloid complex, hippocampus and septum. A good correlation between morphological changes and changes in some neurotransmitter markers was observed in these 4 areas. High affinity uptake of L-glutamate, as well as glutamate decarboxylase and choline acetyltransferase activities were reduced in the piriform cortex and amygdaloid complex whereas in the hippocampus and septum only the first two markers were reduced. No morphological changes or decrease in any of these neurotransmitter markers were observed in striatum or globus pallidus. A pronounced neuronal degeneration could be demonstrated in lateral thalamus and geniculate body, but this degeneration was not accompanied by any decrease in the transmitter markers tested.

Selective effects of kainic acid on diencephalic neurons.

Injections of kainic acid (KA) into the lateral hypothalamus (LH) produce neuronal loss in this region without apparent damage to medial forebrain bundle fibers passing through the area. Cellular destruction is not limited to the LH; the neuronal loss in the thalamic reticular nucleus, the subthalamic nucleus and zona incerta is more extensive than that in the LH. Since all of the nuclei of ventral thalamic origin except the ventral lateral geniculate nucleus (VLGN) were destroyed by LH injections of KA, we sought to determine whether this nucleus also is sensitive to KA. Injections directly into the VLGN produce total neuronal loss here as well as in the thalamic reticular nucleus, zona incerta and subthalamic nucleus. Other areas showing cell loss are the dorsal LGN, medial geniculate, lateral portion of the ventrobasal complex and midline thalamic nuclei. Injections of KA into medial hypothalamus adjacent to the suprachiasmatic nucleus produced no neuronal degeneration. In addition, no neuronal loss was noted in medial hypothalamic nuclei lying adjacent to areas of LH in which KA was injected. Therefore, the sensitivity of diencephalic nuclei appears to range from highly sensitive regions such as derivatives of ventral thalamus and midline thalamic nuclei to regions of moderate sensitivity such as the LH, geniculate nuclei and ventrobasal thalamic nucleus, to regions resistant to KA toxicity such as the suprachiasmatic nucleus and other nuclei and areas of medial hypothalamus.