• Energy Research

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.

Abstract No studies are reported on ethylic biodiesel integrated processes, considering raw materials, reaction optimization and product purification. The present study aims to: i) select key variables for experimental optimization of ethanolysis using a virgin vegetable oil; ii) perform an optimization study using a waste oil; and iii) evaluate the effectiveness of water free purification methods. Sunflower oil ethanolysis was conducted at different temperatures (30 – 80 °C), catalyst concentrations (0.3 – 2 wt.%), reaction times (0.5 – 4 h) and ethanol: oil molar ratios (2:1 – 12:1). Optimization experiments on waste oil ethanolysis were performed at different temperatures (30 – 50 °C) and ethanol: oil molar ratios (6:1 – 12:1), during 1 h and using 1 wt.% catalyst. Quality parameters were measured according to EN 14214. A cation-exchange resin and a ceramic membrane were evaluated for water-free purification. Regarding sunflower oil ethanolysis, when successful, conversion ranged from 75.2 to 97.7 wt.%. Using both oils under optimized conditions (45 °C, 6:1 ethanol:oil molar ratio), a product with a very high purity (> 98.0 wt.%) was obtained after water washing purification. The 0.1 μm ceramic membrane was more effective than the cation-exchange resin, but it was not possible to obtain a good quality product using both methods.

Those who work with cell cultures are certainly familiar-ized with the ritual of sanitizing everything that goesinto the cabinet or into the incubators with 70% ethanol,as is recommended by all the rules of aseptic techniquesand good cell culture practices. In spite of its eVective-ness in preventing microbiological contamination of thecultures, this gesture may have some undesirableconsequences.Probably, the research groups that study the eVects ofethanol on cell cultures are more conscious about therisks of sanitizing all the material with 70% ethanolbecause they need to guarantee that they have ethanol-free control samples. Thus, these groups routinely quan-tify ethanol in the culture medium to con Wrm the ethanolconcentrations used in their studies. However, the scien-tiWc community must be aware that when 70% ethanol isused on cell culture as a surface sanitization process, ifcautions are not taken, the cells will be exposed to highethanol concentrations. These high concentrations areachieved because, under normal cell culture conditions,the cell culture plates have appropriate venting systems(as they should have, otherwise the cells will not be oxy-genated) and generally, both cabinets and incubatorshave an ethanol-saturated atmosphere due to thefrequent sanitization procedures. Without additionalprecautions, ethanol concentrations of 0.5 mM are easilyattained, and depending on the ethanol-saturation of thecabinets or incubators, these concentrations can reachvalues of 5 mM as illustrated in Fig.1, which representsthe ethanol concentrations detected on 14 independentexperiments where no precautions were taken to avoidthe exposure of the medium to ethanol. Both theseconcentrations have proven eVects on cell function(Charness et al. 1994; Smith and Gong 2007). In fact, itis well described that ethanol, as a microsomal and mito-chondrial enzymatic inducer (CYP2E1, ADH-I), caninXuence the metabolism of several endogenous andexogenous compounds causing eventual antagonisms,synergisms or potentiations that will necessarily aVectthe Wnal results of the experiments (Jang and Harris2007).In addition, by itself, ethanol can directly or indirectlyalter the normal cell function, inXuencing cell survival,redox status, receptor functions, mitochondrial homeosta-sis, cell signaling, among others (Wu et al. 2006), and alsoaVecting the activity of infectious agents such as hepatitic Cvirus (Trujillo-Murillo et al. 2007).Thus, some precautions must be taken to minimize this“contamination” with ethanol: 70% ethanol used to sanitizesurface and objects should be completely dry before theXasks or plates are opened and these should not be sprayedwith 70% ethanol between coming out of the cabinet andgoing into the incubator (but obviously, in between, contactwith other surfaces is forbidden).These concerns are crucial for every lab working withcell culture and must be taken into account during the data

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 (EC50 305.26 mM) proved to act additively with COC (EC50 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 (EC50 19.18 ± 3.36 mM) demonstrated that the cardiotoxicity was fairly similar to that predicted by IA (EC50 22.95 mM) and CA (EC50 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.

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.

Chronic ethanol consumption increases oxidative stress, which accounts for the striking neurological changes seen in this condition. Notwithstanding, there is well-documented evidence that polyphenols, present in grape skin and seeds, exhibit a strong antioxidant activity. As red wine is rich in polyphenols, the aim of the present work was to evaluate their putative protective effects on the hippocampal formation by applying biochemical, morphological and behavioral approaches. Six-month old male Wistar rats were fed with red wine (ethanol content adjusted to 20%) and the results were compared with those from ethanol-treated (20%) rats and pair-fed controls. Biochemical markers of oxidative stress (lipid peroxidation, glutathione levels and antioxidant enzyme activities) were assessed on hippocampal homogenates. Lipofuscin pigment, an end product of lipid peroxidation, was quantified in hippocampal cornu ammonis 1 and 3 (CA1 and CA3) pyramidal neurons using stereological methods. All animals were behaviorally tested on the Morris water maze in order to assess their spatial learning and memory skills. In red wine-treated rats, lipid peroxidation was the lowest while presenting the highest levels of reduced glutathione and an induction of antioxidant enzyme activities. Morphological findings revealed that, contrary to ethanol, red wine did not increase lipofuscin deposition in CA1 and CA3 pyramidal neurons. Besides, red wine-treated animals learned the water maze task at a higher rate than ethanol group and had better performance scores by the end of the training period and on a probe trial. Actually, no significant differences were found between pair-fed controls and red wine-treated rats in morphological and behavioral data. Thus, our findings demonstrate that chronic consumption of red wine, unlike the ethanol solution alone, does not lead to a decline in hippocampal-dependent spatial memory. This may be due to the ability of red wine polyphenols to improve the antioxidant status in the brain and to prevent free radical-induced neuronal damage.

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.

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.