Glibenclamide Prevents Diabetes in NOD Mice.

Previous work has revealed that Cx36, the sole connexin expressed in the insulin-producing beta cells, enhances the secretion of insulin, and promotes the resistance of beta cells against pro-inflammatory cytokines. In parallel, the anti-diabetic sulphonylurea glibenclamide was shown to promote the assembly and function of Cx36 channels. Here, we assessed whether glibenclamide could protect the insulin-producing cells against conditions mimicking those expected at the onset of type 1 diabetes. We found that the drug 1) protected in vitro the mouse MIN6 cells from the apoptosis and loss of Cx36, which are induced by Th1 cytokines; 2) prevented the development of hyperglycemia as well as the loss of beta cells and Cx36, which rapidly develop with aging in untreated NOD mice; 3) modified the proportion of effector CD4+ and CD8+ T cells in pancreatic draining lymph nodes. The data imply that an early glibenclamide treatment may help protecting beta cells against the autoimmune attack, which triggers the development of type 1 diabetes.

Safety signal detection: the relevance of literature review.

Adverse drug reactions (ADRs) represent an important risk for patients and have a significant economic impact on health systems. ADRs are the fifth most common cause of hospital death, with a burden estimated at 197,000 deaths per year in the EU. This has a societal cost of 79 billion per year. Because of this strong impact in public health, regulatory authorities (RAs) worldwide are implementing new pharmacovigilance legislation to promote and protect public health by reducing the burden of ADRs through the detection of safety signals. Although, traditionally, signal detection activities have mainly been performed based on spontaneous reporting from healthcare professionals and national health RAs, the new pharmacovigilance legislation underlines the relevance of other sources of information (such as scientific literature) for the evaluation of the benefit-risk balance of a certain product. This review aims to highlight the relevance of periodic scientific literature screening in the safety signal detection process. The authors present four practical examples where a safety signal that was detected from a literature report had an impact on the lifecycle of a drug. In addition, based on practical experience of the screening of medical and scientific literature for safety purposes, this article analyses the requirements of the new pharmacovigilance guidelines on literature screening and highlights the need for the implementation of a literature review procedure and the main challenges encountered when performing literature screening for safety aspects.

Clinical and forensic signs related to ethanol abuse: a mechanistic approach.

For good performance in clinical and forensic toxicology, it is important to be aware of the signs and symptoms related to xenobiotic exposure since they will assist clinicians to reach a useful and rapid diagnosis. This manuscript highlights and critically analyses clinical and forensic imaging related to ethanol abuse. Here, signs that may lead to suspected ethanol abuse, but that are not necessarily related to liver disease are thoroughly discussed regarding its underlying mechanisms. This includes flushing and disulfiram reactions, urticaria, palmar erythema, spider telangiectasias, porphyria cutanea tarda, "paper money skin", psoriasis, rhinophyma, Dupuytren's contracture, multiple symmetrical lipomatosis (lipomatosis Lanois-Bensaude, Madelung's disease), pancreatitis-related signs, black hairy tongue, gout, nail changes, fetal alcohol syndrome, seborrheic dermatitis, sialosis and cancer.

The intercellular synchronization of Ca2+ oscillations evaluates Cx36-dependent coupling.

Connexin36 (Cx36) plays an important role in insulin secretion by controlling the intercellular synchronization of Ca(2+) transients induced during stimulation. The lack of drugs acting on Cx36 channels is a major limitation in further unraveling the molecular mechanism underlying this effect. To screen for such drugs, we have developed an assay allowing for a semi-automatic, fluorimetric quantification of Ca(2+) transients in large populations of MIN6 cells. Here, we show that (1) compared to control cells, MIN6 cells with reduced Cx36 expression or function showed decreased synchrony of glucose-induced Ca(2+) oscillations; (2) glibenclamide, a sulphonylurea which promotes Cx36 junctions and coupling, increased the number of synchronous MIN6 cells, whereas quinine, an antimalarial drug which inhibits Cx36-dependent coupling, decreased this proportion; (3) several drugs were identified that altered the intercellular Ca(2+) synchronization, cell coupling and distribution of Cx36; (4) some of them also affected insulin content. The data indicate that the intercellular synchronization of Ca(2+) oscillations provides a reliable and non-invasive measurement of Cx36-dependent coupling, which is useful to identify novel drugs affecting the function of ß-cells, neurons, and neuron-related cells that express Cx36.

Toxicity of amphetamines: an update.

Amphetamines represent a class of psychotropic compounds, widely abused for their stimulant, euphoric, anorectic, and, in some cases, emphathogenic, entactogenic, and hallucinogenic properties. These compounds derive from the ß-phenylethylamine core structure and are kinetically and dynamically characterized by easily crossing the blood-brain barrier, to resist brain biotransformation and to release monoamine neurotransmitters from nerve endings. Although amphetamines are widely acknowledged as synthetic drugs, of which amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are well-known examples, humans have used natural amphetamines for several millenniums, through the consumption of amphetamines produced in plants, namely cathinone (khat), obtained from the plant Catha edulis and ephedrine, obtained from various plants in the genus Ephedra. More recently, a wave of new amphetamines has emerged in the market, mainly constituted of cathinone derivatives, including mephedrone, methylone, methedrone, and buthylone, among others. Although intoxications by amphetamines continue to be common causes of emergency department and hospital admissions, it is frequent to find the sophism that amphetamine derivatives, namely those appearing more recently, are relatively safe. However, human intoxications by these drugs are increasingly being reported, with similar patterns compared to those previously seen with classical amphetamines. That is not surprising, considering the similar structures and mechanisms of action among the different amphetamines, conferring similar toxicokinetic and toxicological profiles to these compounds. The aim of the present review is to give an insight into the pharmacokinetics, general mechanisms of biological and toxicological actions, and the main target organs for the toxicity of amphetamines. Although there is still scarce knowledge from novel amphetamines to draw mechanistic insights, the long-studied classical amphetamines-amphetamine itself, as well as methamphetamine and MDMA, provide plenty of data that may be useful to predict toxicological outcome to improvident abusers and are for that reason the main focus of this review.

Connexins protect mouse pancreatic ß cells against apoptosis.

Type 1 diabetes develops when most insulin-producing ß cells of the pancreas are killed by an autoimmune attack. The in vivo conditions modulating the sensitivity and resistance of ß cells to this attack remain largely obscure. Here, we show that connexin 36 (Cx36), a trans-membrane protein that forms gap junctions between ß cells in the pancreatic islets, protects mouse ß cells against both cytotoxic drugs and cytokines that prevail in the islet environment at the onset of type 1 diabetes. We documented that this protection was at least partially dependent on intercellular communication, which Cx36 and other types of connexin channels establish within pancreatic islets. We further found that proinflammatory cytokines decreased expression of Cx36 and that experimental reduction or augmentation of Cx36 levels increased or decreased ß cell apoptosis, respectively. Thus, we conclude that Cx36 is central to ß cell protection from toxic insults.

Mechanisms underlying the hepatotoxic effects of ecstasy.

3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is a worldwide illegally used amphetamine-derived designer drug known to be hepatotoxic to humans. Jaundice, hepatomegaly, centrilobular necrosis, hepatitis and fibrosis represent some of the adverse effects caused by MDMA in the liver. Although there is irrefutable evidence of MDMA-induced hepatocellular damage, the mechanisms responsible for that toxicity remain to be thoroughly clarified. One well thought-of mechanism imply MDMA metabolism in the liver into reactive metabolites as responsible for the MDMA-elicited hepatotoxicity. However, other factors, including MDMA-induced hyperthermia, the increase in neurotransmitters efflux, the oxidation of biogenic amines, polydrug abuse pattern, and environmental features accompanying illicit MDMA use, may increase the risk for liver complications. Liver damage patterns of MDMA in animals and humans and current research on the mechanisms underlying the hepatotoxic effects of MDMA will be highlighted in this review.

Metabolic interactions between ethanol and MDMA in primary cultured rat hepatocytes.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy), a drug of abuse commonly consumed at rave parties, is often taken in a polydrug abuse scenario, ethanol being one of the most associated drugs. Both MDMA and ethanol are mainly metabolized in the liver with formation of toxic metabolites. Our working hypothesis is that ethanol can modify the metabolism of MDMA through the cytochrome P450 system, and that this effect may be further potentiated by hyperthermia, a well-known consequence of MDMA abuse. To investigate these putative interactions we used primary rat hepatocyte cultures, which were exposed to 300 mM ethanol, 1.6 mM MDMA and the combination of both, at normothermic (36.5 degrees C) and hyperthermic (40.5 degrees C) conditions. After 24 h, the levels of MDA, HMA and HMMA in the cell culture medium were quantified by GC/MS. In addition, we repeated the same experimental design preceded by 1h incubation with 0.18 microM ketoconazole or 150 microM diallyl sulphide (CYP3A and CYP2E1 inhibitors, respectively), to evaluate the putative role of these isoenzymes in the observed effects. The results obtained showed that ethanol exposure increases the formation of some MDMA metabolites such as HMA (1.8 times increase) and MDA (1.5 times increase). This effect was markedly increased under hyperthermic conditions (HMA, MDA and HMMA formation increased 10, 6 and 16 times, respectively) and is mediated, at least partially, by CYP3A and CYP2E1.

Gas chromatography-ion trap mass spectrometry method for the simultaneous measurement of MDMA (ecstasy) and its metabolites, MDA, HMA, and HMMA in plasma and urine.

The investigation of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) abuse requires very robust methods with high sensitivity and wide linearity ranges for the quantification of this drug of abuse and its main metabolites in body fluids. An optimized gas chromatography-ion trap mass spectrometry (GC-IT/MS) methodology with electron impact ionization addressing these issues is presented. The sample preparation involves an enzymatic hydrolysis of urine and plasma for conjugate cleavage, a SPE extraction, and a derivatization process. The method was fully validated in rat plasma and urine. Linearity for a wide concentration range was achieved for MDMA, and the metabolites 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA). Limits of quantification were 2 ng/mL in plasma and 3.5 ng/mL in urine using a Selected Ion Monitoring detection mode. Selectivity, accuracy, precision, and recovery met the required criteria for the method validation. This GC-IT/MS method provides high sensitivity and adequate performance characteristics for the simultaneous quantification of MDMA, MDA, HMA and HMMA in the studied matrices.

GC determination of acetone, acetaldehyde, ethanol, and methanol in biological matrices and cell culture.

A gas chromatography with flame ionization detection method (GC-FID) with direct injection, using a capillary column, was validated to determine ethanol, acetaldehyde, methanol, and acetone in different human matrices, such as whole blood, vitreous humour, and urine, with clinical and forensic interest. This method was also employed to quantify these compounds in cell culture medium, thus being useful in basic research. A good peak resolution was achieved, with linear correlation between concentration and peak areas for all the compounds in all the matrices. The inter- and intra-day precisions of the method were always under 15% and 10%, respectively. The accuracy of the method, calculated as the percentage of the target concentration, was within the acceptable limits. The obtained limits of detection were below 0.85 mg/L for acetaldehyde and below 0.75 mg/L for the other considered compounds. The small injection volume and the high split ratios applied, allied to the high performance of the GC column, resulted in very good peak resolution and high sensitivities. This method is easy to perform, making it suitable for the routine of clinical biochemistry and forensic laboratories.

Synergistic toxicity of ethanol and MDMA towards primary cultured rat hepatocytes.

Ethanol is frequently consumed along with 3,4-methylenedioxymethamphetamine (MDMA; ecstasy). Since both compounds are hepatotoxic and are metabolized in the liver, an increased deleterious interaction resulting from the concomitant use of these two drugs seems plausible. Another important feature of MDMA-induced toxicity is hyperthermia, an effect known to be potentiated after continuous exposure to ethanol. Considering the potential deleterious interaction, the aim of the present study was to evaluate the hepatotoxic effects of ethanol and MDMA mixtures to primary cultured rat hepatocytes and to elucidate the mechanism(s) underlying this interaction. For this purpose, the toxicity induced by MDMA to primary cultured rat hepatocytes in absence or in presence of ethanol was evaluated, under normothermic (36.5 degrees C) and hyperthermic (40.5 degrees C) conditions. While MDMA and ethanol, by themselves, had discrete effects on the analysed parameters, which were slightly aggravated under hyperthermia, the simultaneous incubation of MDMA and ethanol for 24h, resulted in high cell death ratios accompanied by a significant disturbance of cellular redox status and decreased energy levels. Evaluation of apoptotic/necrotic features provided clear evidences that the cell death occurs preferentially through a necrotic pathway. All the evaluated parameters were dramatically aggravated when cells were incubated under hyperthermia. In conclusion, co-exposure of hepatocytes to ethanol and MDMA definitely results in a synergism of the hepatotoxic effects, through a disruption of the cellular redox status and enhanced cell death by a necrotic pathway in a temperature-dependent extent.

Chronic exposure to ethanol exacerbates MDMA-induced hyperthermia and exposes liver to severe MDMA-induced toxicity in CD1 mice.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is an amphetamine derivative drug with entactogenic, empathogenic and hallucinogenic properties, commonly consumed at rave parties in a polydrug abuse pattern, especially with cannabis, tobacco and ethanol. Since both MDMA and ethanol may cause deleterious effects to the liver, the evaluation of their putative hepatotoxic interaction is of great interest, especially considering that most of the MDMA users are regular ethanol consumers. Thus, the aim of the present study was to evaluate, in vivo, the acute hepatotoxic effects of MDMA (10mg/kg i.p.) in CD-1 mice previously exposed to 12% ethanol as drinking fluid (for 8 weeks). Body temperature was continuously measured for 12h after MDMA administration and, after 24h, hepatic damage was evaluated. The administration of MDMA to non pre-treated mice resulted in sustained hyperthermia, which was significantly increased in ethanol pre-exposed mice. A correspondent higher increase of hepatic heat shock transcription factor (HSF-1) activation was also observed in the latter group. Furthermore, MDMA administration resulted in liver damage as confirmed by histological analysis, slight decrease in liver weight and increased plasma transaminases levels. These hepatotoxic effects were also exacerbated when mice were pre-treated with ethanol. The activities of some antioxidant enzymes (such as SOD, GPx and Catalase) were modified by ethanol, MDMA and their joint action. The hepatotoxicity resulting from the simultaneous exposure to MDMA and ethanol was associated with a higher activation of NF-kappaB, indicating a pro-inflammatory effect in this organ. In conclusion, the obtained results strongly suggest that the consumption of ethanol increases the hyperthermic and hepatotoxic effects associated with MDMA abuse.

Effect of chronic ethanol exposure on the hepatotoxicity of ecstasy in mice: an ex vivo study.

3,4-Methylenedioxymethamphetamine (MDMA) is frequently consumed at "rave" parties by polydrug users that usually take this drug in association with ethanol. In addition, many young people are repeatedly exposed to ethanol, which likely leads to tolerance phenomena. Both compounds are metabolized in the liver, with formation of hepatotoxic metabolites, which gives high relevance to the evaluation of their putative toxicological interaction. Therefore, the aim of this study was to evaluate the toxicity induced by 0.8 and 1.6 mM MDMA to freshly isolated hepatocytes obtained from ethanol-treated mice whose tap drinking water was replaced by a 5% ethanol solution for 1 week and, afterwards, by a 12% ethanol solution for 8 weeks (ethanol group) comparatively to non-treated animals (non-ethanol group). The hepatocytes were incubated under normothermic and hyperthermic conditions in order to simulate in vitro the hyperthermic response induced in vivo by MDMA, a condition that has been recognized as a life-threatening effect associated with MDMA exposure and implicated in its hepatotoxicity. Six mice treated under the same protocol as the ethanol group were used for histological analysis, and compared to non-ethanol-treated animals. The pre-treatment of mice with ethanol caused a significant decrease in the hepatocytes yield in the isolation procedure comparatively to the non-ethanol group, which can be explained by an increase in collagen deposition along the hepatic parenchyma as observed in the histological analysis. The initial cell viability of hepatocytes suspensions was similar between ethanol and non-ethanol groups. However, the ethanol group showed a higher GSH oxidation rate, which was enhanced under hyperthermia. Additionally, a concentration-dependent MDMA-induced loss of cell viability and ATP depletion was observed for both groups, at 41 degrees C. In conclusion, the repeated treatment with ethanol seems to increase the vulnerability of freshly isolated mice hepatocytes towards pro-oxidant conditions, as ascertained by the increase in collagen deposition, lower hepatocyte yield and decreased glutathione levels. However, MDMA toxicity to the isolated hepatocytes was independent of ethanol pre-treatment, while significantly dependent on incubation temperature.

Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPalpha.

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPalpha showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPalpha binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.