Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet.

In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.

Drug-facilitated sexual assault in youth nightlife contexts in Spain.

OBJECTIVES: This study aimed to provide epidemiological information on drug-facilitated sexual assault in Spanish youth partying, with a focus on prevalence rates and associated sociodemographic factors. STUDY DESIGN: Cross-sectional study. METHODS: Quota sampling was used to recruit 1601 young people aged 18-35 years in Spain from a digital panel. A validated questionnaire on drug-facilitated sexual assault was used to assess five types of lifetime victimisation experiences while partying. Chi-square and the exact Fisher tests were used to describe the prevalence of victimisation, drug use patterns, and perpetrator profiles. Generalised ordered logistic regression was used to explore factors associated with victimisation, analysed by gender. RESULTS: Half of young women and one-quarter of young men had experienced drug-facilitated sexual assault in their lifetime. Female victimisation due to touching and kissing was notably high, whereas men comprised almost half of the victims of more invasive DFSA experiences involving masturbation, penetration, and oral sex. Opportunism prevailed as the assault tactic, consisting of taking advantage of the victims' incapacity derived from voluntary alcohol use. Among women, risk of victimisation was associated with a lower education level, foreign-born status, and being non-heterosexual. Male victimisation risk was highest among non-heterosexual men. CONCLUSIONS: Drug-facilitated sexual violence in youth nightlife contexts is a serious public health issue in Spain, which requires urgent action. Most assaults involve taking advantage of victims who are incapacitated by the effects of voluntary alcohol consumption. This sexual violence primarily affects women with lower educational levels or those who are foreign-born and non-heterosexual men and women.

Anionic markers for the forensic identification of Chemical Ignition Molotov Cocktail composition.

An improved version of the famous Molotov cocktail is the Chemical Ignition Molotov Cocktail (CIMC). This incendiary device contains chemical reagents that enable its self-ignition. The analysis of anions from CIMC residues by capillary electrophoresis (CE) allows the identification of the reagents used to produce the device, and provides forensic analysts with valuable information. Although, sulfate, chlorate, chloride, and perchlorate anions have been recently proposed in the literature as target anions to determine the CIMC composition, the identification of some of them could be controversial due to their presence in the environment. Therefore, the purpose of this study was to identify highly reliable anions capable of indicating the components used to prepare these self-initiated devices. The relationship among the detected anions in CIMC residues and the reagents employed in their elaboration is discussed. Some anions have been proposed as anionic markers of CIMC as incendiary devices. Additionally, the viability of different CIMC compositions was studied.

Brain mitochondrial alterations after chronic alcohol consumption.

The aim of this study was to demonstrate the existence of alterations in glutathione and cholesterol homeostasis in brain mitochondria from alcoholic rats. Glutathione concentration decreased, whereas oxidized glutathione and cholesterol contents increased in these organelles, suggesting the ethanol-induced generation of reactive oxygen species, and the impairment of mitochondrial uptake of glutathione, possibly due to the increase in cholesterol deposition. The release of apoptogenic proteins was increased after stimulating mitochondria from the brain of alcoholic rats with atractyloside. As a conclusion, chronic alcohol consumption might sensitize brain mitochondria to apoptotic stimuli, and promote the subsequent release of apoptotic proteins.

Human Ultraweak Photon Emission: Key Analytical Aspects, Results and Future Trends - A Review.

Living systems emit what is called ultraweak photon emission (UPE). This visually undetectable phenomenon has only been studied in humans for the last 30 years, finding that UPE is a complex process depending on multitude factors. Considering previous literature, this review discusses the current trends in the analysis of in vivo UPE from human beings. To this aim, Analytical Approaches Employed for UPE Measurement section focuses on the analytical techniques employed (photomultipliers and charged coupled device cameras), summarizing analytical conditions and reporting figures of merit reached to date. Then, Human UPE Depending on External Factors and Human UPE Depending on Internal Factors sections address external and internal factors, which have proved to affect UPE, pointing out the important influence on oxidative processes outside and inside the body, and also highlighting some personal states of the individuals affecting UPE. Last section is devoted to give a general view on the goals and achieved up to date regarding UPE measurement, emphasizing some potential applications as well as recommendations which include: use of UPE spectra information together with UPE intensity, larger populations (≈50-100 subjects), further studies on internal states of individuals, and use of statistical tools.

A validated GC-MS method for ketamine and norketamine in hair and its use in authentic cases.

This work has shown that it is a valid method for determining ketamine, norketamine and amphetamines derivates in hair samples of forensic cases. This method was validated meeting the criteria of sensitivity and accuracy for detecting repeated consumption of ketamine in hair samples of forensic interest, according to the proposed cut-off for ketamine of 0.5 ng/mg. The detection of norketamine allowed discriminating between active uses and external contamination. The assessed method was applied for analyzing 1189 hair samples of judicial interest received in the INTCF along 15 months, obtaining 62 positive in ketamine consumption. This means a 5.2% of positivity. Ketamine consumers present a profile of young age (21-30 years old), polydrug use with consumption of synthetic substances preferably MDMA and, then, amphetamine. As consumer is collective, prone to consume new psychoactive substances, requires special attention due to they show a consumer profile with higher prevalence in MDMA than amphetamine, indicating that ketamine consumers belong to a subgroup with a different profile within the INTCF casuistry. The results of the exercises of the proficiency tests performed satisfactorily in all cases. In conclusion, it is a suitable method also to evaluate the chronic consumption of ketamine, in addition to amphetamines in the same method of analysis.

[Effectiveness of a Decalogue for cardiovascular prevention in diabetics]. / Efectividad del Decálogo de prevención cardiovascular en diabéticos.

INTRODUCTION: To assess the effects of a visual Decalogue aid on the degree of knowledge, control perception and improvement in cardiovascular risk factors (CVRF). MATERIAL AND METHODS: A Primary care randomised non-pharmacological trial of an educational intervention with a parallel control group, and blind evaluation in type 2 diabetic patients. Both groups received an educational intervention on the management of CVRF. The intervention group also received a visual Decalogue aid that showed the level of control patients have over the modifiable CVRF. A total of 50 patients were included in each group in order to identify an improvement of 50% in the multifactorial knowledge of CVRF. All patients received a reminder telephone call at 2 months, with masked evaluation of knowledge and CVRF control perception. In a 6 months visit the level of knowledge and real control of CVRF were re-evaluated. RESULTS: The study included 51 males and 49 females, with mean age of 62.9 years, a mean disease duration of 9.2 years, and low educational level. The level of knowledge, control perception, and real control at baseline was 55%, 80.4%, and 65.9%, respectively. After 2 months the level of knowledge in the Decalogue group increased by 16.5% more than in the conventional education group (73.6% vs. 63.2%; P<.05) and the overestimated control perception improved by 34.5% (P<.001) with no differences between groups, although concordance was better in the Decalogue group. At 6 months there was an overall increase 25.6% (P<.001) in the level of knowledge, with the previous difference between groups levelling off. The final CVRF control improved overall and in the Decalogue group by 6.4% (P<.005) and 9.4% (P<.001), respectively. The SCORE risk significantly decreased overall with no differences between groups. CONCLUSIONS: The educational intervention improves the overall level of knowledge, perception and control of CVRF. The CVRF Decalogue quickly increases the level of knowledge, and decreases the false subjective risk control perception. The benefit, however, becomes equal at 6 months with ongoing education interventions.

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo.

Ceramidases (CDases) play a key role in cancer therapy through enhanced conversion of ceramide into sphingosine 1-phosphate (S1P), but their involvement in hepatocarcinogenesis is unknown. Here, we report that daunorubicin (DNR) activated acid CDase post-transcriptionally in established human (HepG2 cells) or mouse (Hepa1c1c7) hepatoma cell lines as well as in primary cells from murine liver tumors, but not in cultured mouse hepatocytes. Acid CDase silencing by small interfering RNA (siRNA) or pharmacological inhibition with N-oleoylethanolamine (NOE) enhanced the ceramide to S1P balance compared to DNR alone, sensitizing hepatoma cells (HepG2, Hep-3B, SK-Hep and Hepa1c1c7) to DNR-induced cell death. DNR plus NOE or acid CDase siRNA-induced cell death was preceded by ultrastructural changes in mitochondria, stimulation of reactive oxygen species generation, release of Smac/DIABLO and cytochrome c and caspase-3 activation. In addition, in vivo siRNA treatment targeting acid CDase reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced DNR therapy. Thus, acid CDase promotes hepatocarcinogenesis and its antagonism may be a promising strategy in the treatment of liver cancer.

Hormone-mediated down-regulation of hepatic glutathione synthesis in the rat.

Our present work characterized the role of hormone-mediated signal transduction pathways in regulating hepatic reduced glutathione (GSH) synthesis. Cholera toxin, dibutyryl cAMP (DBcAMP), and glucagon inhibited GSH synthesis in cultured hepatocytes by 25-43%. Cellular cAMP levels exhibited a lower threshold for stimulation of the GSH efflux than inhibition of its synthesis. The effect of DBcAMP was independent of the type of sulfur amino acid precursor and cellular ATP levels and unassociated with increased GSH mixed disulfide formation or altered GSH/oxidized glutathione ratio. In liver cytosols, addition of DBcAMP and cAMP-dependent protein kinase (A-kinase) inhibited GSH synthesis from substrates (cysteine, ATP, glutamate, and glycine) by approximately 20% which was prevented by the A-kinase inhibitor. However, if only substrates of the second step in GSH synthesis were used (gamma-glutamylcysteine, glycine, and ATP), DBcAMP and A-kinase exerted no inhibitory effect. Phenylephrine, vasopressin, and phorbol ester also inhibited GSH synthesis in cultured cells by approximately 20%, and depleted cell GSH independent of the type of sulfur amino acid precursor. Cellular cysteine level was unchanged despite the significant fall in GSH after glucagon or phenylephrine treatment. Pretreatment with either staurosporine, C-kinase inhibitor, or calmidazolium, a calmodulin inhibitor, partially prevented but, together, completely prevented the inhibitory effect of phenylephrine. The same combination had no effect on the inhibitory effect of glucagon. The effects of hormones were confirmed in both the intact perfused liver and after in vivo administration. Thus, two classes of hormones acting through distinct signal transduction pathways may down-regulate hepatic GSH synthesis by phosphorylation of gamma-glutamylcysteine synthetase.

Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress.

Isolated hepatocytes incubated with [35S]-methionine were examined for the time-dependent accumulation of [35S]-glutathione (GSH) in cytosol and mitochondria, the latter confirmed by density gradient purification. In GSH-depleted and -repleted hepatocytes, the increase of specific activity of mitochondrial GSH lagged behind cytosol, reaching nearly the same specific activity by 1-2 h. However, in hepatocytes from ethanol-fed rats, the rate of increase of total GSH specific radioactivity in mitochondria was markedly suppressed. In in vivo steady-state experiments, the mass transport of GSH from cytosol to mitochondria and vice versa was 18 nmol/min per g liver, indicating that the half-life of mitochondrial GSH was approximately 18 min in controls. The fractional transport rate of GSH from cytosol to mitochondria, but not mitochondria to cytosol, was significantly reduced in the livers of ethanol-fed rats. Thus, ethanol-fed rats exhibit a decreased mitochondrial GSH pool size due to an impaired entry of cytosol GSH into mitochondria. Hepatocytes from ethanol-fed rats exhibited a greater susceptibility to the oxidant stress-induced cell death from tert-butylhydroperoxide. Incubation with glutathione monoethyl ester normalized the mitochondrial GSH and protected against the increased susceptibility to t-butylhydroperoxide, which was directly related to the lowered mitochondrial GSH pool size in ethanol-fed cells.

Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes.

Chronic ethanol feeding selectively impairs the translocation of cytosol GSH into the mitochondrial matrix. Since ethanol-induced liver cell injury is preferentially localized in the centrilobular area, we examined the hepatic acinar distribution of mitochondrial GSH transport in ethanol-fed rats. Enriched periportal (PP) and perivenous (PV) hepatocytes from pair- and ethanol-fed rats were prepared as well as mitochondria from these cells. The mitochondrial pool size of GSH was decreased in both PP and PV cells from ethanol-fed rats either as expressed per 10(6) cells or per microliter of mitochondrial matrix volume. The rate of reaccumulation of mitochondrial GSH and the linear relationship of mitochondrial to cytosol GSH from ethanol-fed mitochondria were lower for both PP and PV cells, effects observed more prominently in the PV cells. Mitochondrial functional integrity was lower in both PP and PV ethanol-fed rats, which was associated with decreased cellular ATP levels and mitochondrial membrane potential, effects which were greater in the PV cells. Mitochondrial GSH depletion by ethanol feeding preceded the onset of functional changes in mitochondria, suggesting that mitochondrial GSH is critical in maintaining a functionally competent organelle and that the greater depletion of mitochondrial GSH by ethanol feeding in PV cells could contribute to the pathogenesis of alcoholic liver disease.

Short wave infrared chemical imaging as future tool for analysing gunshot residues patterns in targets.

This work used chemical imaging in the short-wave infrared region for analysing gunshot residues (GSR) patterns in cotton fabric targets shot with conventional and non-toxic ammunition. It presents a non-destructive, non-toxic, highly visual and hiperspectral-based approach. The method was based on classical least squares regression, and was tested with the ammunition propellants and their standard components' spectra. The propellants' spectra were satisfactorily used (R2 >0.966, and CorrCoef >0.982) for identifying the GSR irrespective of the type of ammunition used for the shooting. In a more versatile approach, nitrocellulose, the main component in the ammunition propellants, resulted an excellent standard for identifying GSR patterns (R2>0.842, and CorrCoef >0.908). In this case, the propellants' stabilizers (diphenilamine and centralite), and its nitrated derivatives as well as dinitrotoluene, showed also high spectral activity. Therefore, they could be recommended as complementary standards for confirming the GSR identification. These findings establish the proof of concept for a science-based evidence useful to support expert reports and final court rulings. This approach for obtaining GSR patterns can be an excellent alternative to the current and traditional chemical methods, which are based in presumptive and invasive colour tests.

PGE1 protection against apoptosis induced by D-galactosamine is not related to the modulation of intracellular free radical production in primary culture of rat hepatocytes.

D-galactosamine (D-GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE1 treatment reduced necrosis and apoptosis induced by D-GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by D-GalN (0-40mM). Also, the present study investigated if PGE1 (1 microM) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H2O2), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of D-GalN concentrations (2.5-10mM) induced apoptosis in association with a moderate oxidative stress. High D-GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with D-GalN (2.5-10 mM)-treated cells. Although PGE1 reduced apoptosis induced by D-GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.

Oxidative stress and alcoholic liver disease.

Toxic substances generated during the metabolism of alcohol in the liver may contribute to the development of alcoholic liver disease. These substances include highly reactive molecules that can destroy vital cell components through a process called oxidation. Cells are protected against oxidation by the action of certain enzymes, vitamins, and other substances, known collectively as antioxidants. An imbalance between oxidants and antioxidants can lead to oxidative stress, characterized by escalating cell damage. Evidence suggests that the major energy-generating structures within cells (i.e., mitochondria) may be especially sensitive to oxidative stress, resulting in diminished energy production. Medications that reduce oxidative stress in mitochondria may ameliorate liver disease.

Analysis of bovine whey proteins in soybean dairy-like products by capillary electrophoresis.

The simultaneous separation of bovine whey proteins [alpha-lactalbumin and beta-lactoglobulin (A+B)] and soybean proteins was performed, for the first time, by capillary electrophoresis. Different experimental conditions were tested. The most suitable consisted of 0.050 M phosphate buffer (pH 8) with 1 M urea and 1.2 mg/ml methylhydroxyethylcellulose, UV detection at 280 nm, 15 kV applied voltage, and 30 degrees C temperature. Quantitation of bovine whey proteins in a commercial powdered soybean milk manufactured by adding bovine whey to its formulation was performed using the calibration method of the external standard. Direct injection of a solution of the powdered soybean milk only enabled quantitation of alpha-lactalbumin in the commercial sample. Detection of beta-lactoglobulin (A+B) required acid precipitation of the solution of the sample in order to concentrate bovine whey proteins in the supernatant prior to the analysis of this protein in the whey obtained. Since alpha-lactalbumin could also be quantitated from the injection of the whey, the simultaneous determination of alpha-lactalbumin and beta-lactoglobulin (A+B) was possible upon acid precipitation of the powdered soybean milk solution. Detection limits obtained were 14 microg/g sol. for alpha-lactalbumin and 52 microg/g sol. for beta-lactoglobulin (A+B) which represent protein concentrations about 60 microg/100 g sample for alpha-lactalbumin and 100 microg/100 g sample for beta-lactoglobulin (A+B).

Rapid enantiomeric separation of polychlorinated biphenyls by electrokinetic chromatography using mixtures of neutral and charged cyclodextrin derivatives.

Electrokinetic chromatography with cyclodextrin derivatives (CD-EKC) was used to achieve the rapid enantiomeric separation of chiral polychlorinated biphenyls (PCBs). Thirteen of the 19 chiral PCBs stable at room temperature were individually separated into their two enantiomers by using 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5) containing carboxymethylated gamma-cyclodextrin (CM-gamma-CD) as pseudostationary phase mixed with beta-cyclodextrin (beta-CD) or permethylated beta-cyclodextrin (PM-beta-CD). Urea was also added to increase the solubility of PCBs and cyclodextrins in the aqueous separation buffer. Several experimental parameters such as the nature, concentration, and pH of the buffer, nature and concentration of the cyclodextrin derivatives used, and the addition of different additives were studied in order to improve the enantiomeric separation. In addition, the effect of some instrumental parameters such as separation temperature and applied voltage was also investigated. PCBs were enantiomerically separated in less than 12 min by using a 50 mM MES buffer (pH 6.5) containing 20 mM CM-gamma-CD, 10 mM beta-CD or 20 mM PM-beta-CD, and 2 M urea at a temperature of 45 degrees C and an applied voltage of 20 kV.

Laser-induced fluorescence detection at 266 nm in capillary electrophoresis. Polycyclic aromatic hydrocarbon metabolites in biota.

The separation of five phenolic polycyclic aromatic hydrocarbon metabolites (hydroxy-PAHs) has been performed by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using a 30 mM borate buffer (pH 9.0) containing 60 mM sodium dodecyl sulfate and varying concentrations of gamma-cyclodextrin (gamma-CD). A concentration of 12.5 mM gamma-CD was found to provide a baseline separation of the five hydroxy-PAHs. We applied conventional fluorescence and laser-induced fluorescence (LIF) detection, using a new, small-size, quadrupled Nd-YAG laser emitting at 266 nm. The best limits of detection, in the low ng/ml range, were achieved using LIF detection. For all analytes, linearity was observed up to ca. 100 ng/ml. As an application, conjugated pyrene metabolites in hepatopancreas samples from the terrestrial isopods Oniscus asellus and Porcellio scaber were separated and detected. Finally, flatfish bile samples from individuals exposed to polluted sediment or crude oil, which were part of an interlaboratory study, were analyzed by CD-MEKC with conventional fluorescence and LIF detection to determine the 1-hydroxypyrene concentrations.