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The increasing concerns about greenhouse gas emissions are encouraging the search for efficient combustion technologies for transportation. A valuable strategy consists of tailoring the properties of fuels through addition of additives that might increase the octane number subject to the classification, labeling and packaging regulation of fuel quality. In this context, we present an integrated approach involving a high-throughput screening that relies on selected physicochemical factors of aniline-like compounds, measurements of structural resemblance and susceptibility to participate in chemical reactions with radical species, in conjunction with production viability as well as environmental and toxicological risks. This process led to a final set of representative compounds that were chosen to explore their behavior as anti-knock additives. The suitability of these compounds was determined through assays performed to determine the impact on fuel volatility and RON booster efficiency in conjunction with a critical assessment of their eco/toxicological risk estimated by means of a safety index. This holistic strategy led to the identification of N-methyl-p-anisidine, N',N'-diethyl-2-methyl-p-phenylenediamine and N-nitroso-diphenylamine as promising anti-knock additives. This approach is proposed as an alternative strategy to the unsupervised experimental screening of fuel additives.

The recreational use of MDMA (ecstasy) by pregnant women is associated with impaired neuromotor function in infants, but the Adverse Outcome Pathway behind this effect is not clear yet. We present for the first time the evaluation of developmental neurotoxic (DNT) effects of MDMA in zebrafish embryos. The aim of the study was to determine whether the zebrafish model reproduces the adverse outcome occurring in humans. We have studied the DNT effects of MDMA in zebrafish within a range of 5-250 μM performing different behavioural tests: spontaneous tail-coiling and light-dark locomotor response; after exposing the embryos to 4 different scenarios combining changes in pH, in starting exposure time and exposure duration. In these scenarios we evaluated the effects of MDMA in general embryonic development and compared the concentrations producing them with those inducing specific DNT effects. As a result, we have established the experimental conditions leading to the adverse outcome "lower motor activity" in zebrafish without producing general developmental delay or general toxicity. The experimental condition chosen opens the door to use this model in future mechanistic investigations to better characterize the Adverse Outcome Pathway associated with the adverse effects caused by MDMA prenatal exposure in humans.

Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Aβ42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognition-enhancing anti-AD lead.

The antioxidant effect of compounds is regularly evaluated by in vitro assays that do not have the capability to predict in vivo protective activity or to determine their underlying mechanisms of action. The aim of this study was to develop an experimental system to evaluate the in vivo protective effects of different antioxidant compounds, based on the zebrafish embryo test. Zebrafish embryos were exposed to tert-butyl hydroperoxide (tBOOH), tetrachlorohydroquinone (TCHQ) and lipopolysaccharides from Escherichia coli (LPS), chemicals that are known inducers of oxidative stress in zebrafish. The developmental toxic effects (lethality or dysmorphogenesis) induced by these chemicals were modulated with n-acetyl l-cysteine and N&omega, nitro l-arginine methyl ester hydrochloride, dimethyl maleate and dl-buthionine sulfoximine in order to validate the oxidant mechanism of oxidative stress inducers. The oxidant effects of tBOOH, TCHQ, and LPS were confirmed by the determination of significant differences in the comparison between the concentration&ndash, response curves of the oxidative stress inducers and of the modulators of antioxidant status. This concept was also applied to the study of the effects of well-known antioxidants, such as vitamin E, quercetin, and lipoic acid. Our results confirm the zebrafish model as an in vivo useful tool to test the protective effects of antioxidant compounds.

The aim of this study was to develop a rabbit neurosphere culture to characterize differences in basic processes of neurogenesis induced by intrauterine growth restriction (IUGR). A novel in vitro neurosphere culture has been established using fresh or frozen neural progenitor cells from newborn (PND0) rabbit brains. After surgical IUGR induction in pregnant rabbits and cesarean section 5 days later, neural progenitor cells from both control and IUGR groups were isolated and directly cultured or frozen at −80°C. These neural progenitor cells spontaneously formed neurospheres after 7 days in culture. The ability of control and IUGR neurospheres to migrate, proliferate, differentiate to neurons, astrocytes, or oligodendrocytes was compared and the possibility to modulate their responses was tested by exposure to several positive and negative controls. Neurospheres obtained from IUGR brains have a significant impairment in oligodendrocyte differentiation, whereas no significant differences are observed in other basic processes of neurogenesis. This impairment can be reverted by in vitro exposure of IUGR neurospheres to thyroid hormone, which is known to play an essential role in white matter maturation in vivo. Our new rabbit neurosphere model and the results of this study open the possibility to test several substances in vitro as neuroprotective candidates against IUGR induced neurodevelopmental damage while decreasing the number of animals and resources and allowing a more mechanistic approach at a cellular functional level., Rabbit “Neurosphere Assay.” PND0 rabbit brains are dissected and either frozen or directly cultured until neurosphere formation. “Neurosphere Assay” starts when neurospheres are plated in 8‐chamber slides and maintained under differentiating conditions to evaluate migration and differentiation into neurons, into astrocytes or into oligodendrocytes. Neurospheres were also plated in 96‐well plates under proliferating conditions and evaluated for diameter increase.

Este trabajo se fundamenta en la evaluación de la actividad antiinflamatoria de extractos de sofrito de tomate, así como de compuestos estándares de la dieta mediterránea, usando un modelo experimental optimizado basado en larvas de pez cebra. La migración de neutrófilos en larvas de pez cebra de 96 horas post fertilización se indujo mediante una lesión y se potenció añadiéndole lipopolisacárido, dicha migración se visualizó y cuantificó mediante análisis de imagen. El efecto antiinflamatorio del extracto de tomate y de los compuestos utilizados fue correlacionado porcentualmente por la disminución de la migración de los neutrófilos. Los resultados muestran que el extracto de tomate presentó una reducción en la migración de neutrófilos de 40 % respecto al grupo control. Por otra parte, el ácido clorogénico y la cianidina presentes en el sofrito de tomate utilizados como estándares presentaron una disminución de la migración de neutrófilos de un 66,7 % y 62,5 % respectivamente. Estos porcentajes son comparables a los resultados observados en ensayos con drogas antiinflamatorias como la indometacina y piroxicam. Los resultados muestran que el extracto de sofrito de tomate presenta posible actividad antinflamatoria demostrada por la reducción de la migración de neutrófilos, además el modelo se mostró sensible y válido para ser aplicado en matrices alimentarias complejas.

Environmental sciences Europe : ESEU 32(1), 145 (2020). doi:10.1186/s12302-020-00421-7, Published by Springer, Berlin ; Heidelberg

The increasing use of illegal drugs by pregnant women causes a public health concern because it is associated with health risks for mothers and their developing children. One of such drugs is MDMA (3,4-methylenedioxymethamphetamine) or ecstasy due to its high consumption in relevant age and sex groups and its adverse effects on human and rodent developing brains. To Journal Pre-proof 2 thoroughly review the current knowledge on the developmentally neurotoxic potential of MDMA we systematically collected and summarized articles investigating developmental neurotoxicity (DNT) of MDMA in humans and animals in in vivo and in vitro. In addition, we summarized the findings in a putative adverse outcome pathway (AOP). From an initial 299 articles retrieved from the bibliographic databases Web of Science, PubMed and DART, we selected 39 articles according to inclusion/exclusion criteria for data collection after title/abstract and full text screening. Of these 3 where epidemiological studies, 34 where in vivo studies in mice and rats and 2 were in vitro studies. The three epidemiological studies reported from the same longitudinal study and suggested that MDMA exposure during pregnancy impairs neuromotor function in infants. In rat, postnatal exposure towards MDMA also caused locomotor deficits as well as impaired spatial learning that might be associated with decreased serotonin levels in the hippocampus. In vitro MDMA caused cytotoxicity at high concentrations and effects on the serotonergic and neuritogenic alterations at lower concentrations which are in line with some of the in vivo alterations observed. Considering the adverse outcomes of developmental MDMA described in humans and in rodents we summarized the first putative AOP on developmental compound exposure leading to impaired neuromotor function in children. For generation of this AOP, MDMA exposure was taken as a model compound. In addition, we hypothesized a second AOP involving developmental disturbance of the dopaminergic system. However, further in vitro mechanistic studies are needed to understand the molecular initiating event(s) (MIE) triggering the downstream cascades and obtain consistent evidences causally linking the adverse outcome to effects at the cellular, organ and organism level.

The inclusion of a read-out to detect functional consequences of craniofacial alterations in the zebrafish embryotoxicity test will allow to evaluate these alterations which are difficult to assess morphologically, and to detect alterations in cranial nerves functions leading to impairment of jaw movements. In this study we have established an ingestion test in zebrafish larvae younger than 120 hpf. To overcome the challenge of evaluating larvae which still do not present independent feeding behaviour, we have tested the ability of 72, 96 or 102 hpf larvae to ingest food mixed with fluorescent microspheres under several conditions (dark/light, with/without shaking) to find the best experimental set-up for the test. We have included the investigation of two substances as potential positive controls: ketoconazole and tricaine. Ketoconazole 10 μM exposure during development produced significant embryotoxic effects including a characteristic craniofacial alteration pattern consisting in impaired development of brain, nasal cavity, mouth opening and jaw, as well as a significant decrease in food intake. Tricaine exposure at 380 μM during the food availability period significantly decreased the food intake. The method proposed will be a useful alternative tool to animal testing to detect compounds inducing adverse effects on craniofacial development.

The developing cardiovascular system of zebrafish is a sensitive target for many environmental pollutants, including dioxin-like compounds and pesticides. Some polychlorinated biphenyls (PCBs) can compromise the cardiovascular endothelial function by activating oxidative stress-sensitive signaling pathways. Therefore, we exposed zebrafish embryos to PCB126 or to several redox-modulating chemicals to study their ability to modulate the dysmorphogenesis produced by PCB126. PCB126 produced a concentration-dependent induction of pericardial edema and circulatory failure, and a concentration-dependent reduction of cardiac output and body length at 80 hours post fertilization (hpf). Among several modulators tested, the effects of PCB126 could be both positively and negatively modulated by different compounds, co-treatment with &alpha, tocopherol (vitamin E liposoluble) prevented the adverse effects of PCB126 in pericardial edema, whereas co-treatment with sodium nitroprusside (a vasodilator compound) significantly worsened PCB126 effects. Gene expression analysis showed an up-regulation of cyp1a, hsp70, and gstp1, indicative of PCB126 interaction with the aryl hydrocarbon receptor (AhR), while the transcription of antioxidant genes (sod1, sod2, cat and gpx1a) was not affected. Further studies are necessary to understand the role of oxidative stress in the developmental toxicity of low concentrations of PCB126 (25 nM). Our results give insights into the use of zebrafish embryos for exploring mechanisms underlying the oxidative potential of environmental pollutants.

Epigallocatechin gallate (EGCG), the main catechin of green tea, is described to have potential health benefits in several fields like oncology, neurology or cardiology. Currently, it is also under pre-clinical investigation as a potential therapeutic or preventive treatment during pregnancy against developmental adverse effects induced by toxic substances. However, the safety of EGCG during pregnancy is unclear due to its proven adverse effects on neural progenitor cells' (NPCs) migration. As lately several strategies have arisen to generate new therapeutic agents derived from EGCG, we have used the rat ‘Neurosphere Assay’ to characterize and compare the effects of EGCG structurally related compounds and EGCG PEGylated PLGA nanoparticles on a neurodevelopmental key event: NPCs migration. Compounds structurally-related to EGCG induce the same pattern of NPCs migration alterations (decreased migration distance, decreased formation of migration corona, chaotic orientation of cellular processes and decreased migration of neurons at higher concentrations). The potency of the compounds does not depend on the number of galloyl groups, and small structure variations can imply large potency differences. Due to their lower toxicity observed in vitro in NPCs, 4,4′-bis[(3,4,5-trihydroxybenzoyl)oxy]-1,1′-biphenyl and EGCG PEGylated PLGA nanoparticles are suggested as potential future therapeutic or preventive alternatives to EGCG during prenatal period.

Los autores agradecen el soporte a todos los miembros del Grupo de Innovación docente ORFILA, Objetivos. Durante los últimos años, los profesores del Departamento de Farmacología, Toxicología y Química Terapéutica hemos puesto en marcha un conjunto de acciones docentes con el objetivo de promover en los alumnos del grado de Farmacia la mejora de capacidades de integración de los conocimientos de las materias impartidas, así como un modelo de evaluación continuada multidisciplinar y retroactivo que nos permita objetivar su consecución. Métodos. Para desarrollar estas acciones de integración y la posterior evaluación de las mismas, diversas asignaturas del Departamento se han coordinado y se han analizado los conocimientos necesarios para la comprensión de contenidos que se desarrollan en cursos posteriores. Dentro de la asignatura de Toxicología hemos desarrollado un conjunto de acciones que se han concretado fundamentalmente en el diseño, aplicación y, en algunos casos, evaluación de unas actividades de tipología diversa. Resultados. A fin de valorar el éxito de estas acciones, se han comparado los resultados obtenidos en un test de preguntas de integración a principio de curso con los resultados de otro test a final de curso. Los resultados obtenidos en los cuatro ítems comparados muestran una mejora significativa en una pregunta, una mejora relativa no significativa en otras dos y resultados similares en la última. Conclusiones. Podemos concluir que las acciones desarrolladas han conseguido parcialmente los objetivos propuestos. Consideramos que esta herramienta puede ser muy útil para promover una mayor coordinación entre los profesores, en el diseño de material didáctico conjunto y transversal y en modelos integrados de evaluación., Aim. During the last years the professors of the Department of Pharmacology, Toxicology and Therapeutic Chemistry have initiated a set of teaching actions with the aim of promoting the improvement of knowledge integration capacities corresponding to the subjects taught to students of Pharmacy degree; as well as a multidisciplinary and retroactive continuous evaluation model allowing to objectify its achievement. Methods. In order to develop these integration actions and their subsequent evaluation, several subjects of the Department have been coordinated and the knowledge necessary for understanding the contents of following courses have been analyzed. Within the subject of Toxicology, we have developed a set of actions based on the design, application and, in some cases, evaluation of a variety of activities. Results. In order to assess the success of these actions, we have compared the results obtained in a test of integration questions at the beginning of the course with the results of another test at the end of the course. The results obtained in the four items compared show a significant improvement in one question, a maintenance of results in another, and a non-significant relative improvement in the other two. Conclusions. We can conclude that the actions developed have partially achieved the proposed objectives. We consider that this tool can be very useful to promote greater coordination among teachers in the design of joint and transversal teaching materials and in integrated evaluation models., Este trabajo se enmarca en un proyecto de innovación docente de la Universidad de Barcelona (2015PID-UB-031)

The zebrafish embryo is a highly interesting biological model with applications in different scientific fields, such as biomedicine, pharmacology and toxicology. In this study, we used liquid chromatography/electrospray ionisation-linear ion trap quadrupole-Orbitrap-mass spectrometry (HPLC/ESI-LTQ-Orbitrap-MS) to identify the polyphenol compounds in a red wine extract and zebrafish embryos. Phenolic compounds and anthocyanin metabolites were determined in zebrafish embryos previously exposed to the red wine extract. Compounds were identified by injection in a high-resolution system (LTQ-Orbitrap) using accurate mass measurements in MS, MS(2) and MS(3) modes. To our knowledge, this research constitutes the first comprehensive identification of phenolic compounds in zebrafish by HPLC coupled to high-resolution mass spectrometry.

This study evaluates the diet composition of a rural population near a gold mine in the Cajamarca district of Peru. The main consumed items by this population were tubers and cereals, and the mean energy intake (1990 kcal) was shown not to cover the recommended intake values for the male population. The concentrations of As, Cd, Hg, Pb, Zn, Al, Cr and, Cu in drinking water and food samples of items contributing to 91% of this diet (145 samples, 24 different items) were determined and used to calculate their daily intakes for risk assessment. The As, Cd and Pb daily intakes exceeded the limit values established by the European Food Safety Authority (EFSA), entailing serious concerns for the population's health. Moreover, the intake values of As and Pb were shown to be higher, the closer to the gold mine the studied population was.

In this study we analyzed some aspects of the assessment of developmental delay in the zebrafish embryotoxicity/teratogenicity test and explored the suitability of acetylcholinesterase (AChE) activity as a biochemical marker and as a higher throughput alternative to morphological endpoints such as head–trunk angle, tail length and morphological score. Embryos were exposed from 4 to 52 h post-fertilization (hpf) to a selection of known embryotoxic/teratogen compounds (valproic acid, retinoic acid, caffeine, sodium salicylate, glucose, hydroxyurea, methoxyacetic acid, boric acid and paraoxon-methyl) over a concentration range. They were evaluated for AChE activity, head–trunk angle, tail length and several qualitative parameters integrated in a morphological score. In general, the different patterns of the concentration–response curves allowed distinguishing between chemicals that produced growth retardation (valproic and methoxyacetic acid) and chemicals that produced non-growth-delay related malformations. An acceptable correlation between the morphological score, AChE activity and head–trunk angle as markers of developmental delay was observed, being AChE activity particularly sensitive to detect delay in the absence of malformations.

In 2000 and 2006, we determined the dietary intake of hexachlorobenzene (HCB) by the population of Catalonia, Spain. In order to establish the temporal trend in the levels of HCB in foodstuffs, as well as in the dietary exposure to that environmental pollutant, the concentrations were again analyzed by HRGC/HRMS in 65 composite food samples widely consumed by the Catalan population. Food samples were randomly purchased in November–December 2008 in local markets, small stores, supermarkets, and large grocery stores from 12 representative cities from Catalonia. The daily intake of HCB associated with this consumption was estimated for four age groups of the population of Catalonia: children, teenagers, adults and seniors, which were in turn divided according to sex. The highest mean HCB levels in food were detected in oils and fats (0.297 ng/g fw), dairy products (0.225 ng/g fw), and fish and seafood (0.170 ng/g fw). In the 2000 and 2006 surveys, total dietary intakes of HCB were 166.2 and 71.6 ng/day, respectively (or 2.4 and 1.0 ng/kg of body weight per day). In the current study, it was 37.7 ng/day (or 0.54 ng/kg of body weight per day), which means considerable decreases with respect to the previous intakes. According to recommendations of international regulatory organisms, the current dietary intake of HCB should not mean any significant health risk (carcinogenic and non-carcinogenic) for any of the age/gender groups of population here assessed.

Disinfection by-products are contaminants produced during drinking water disinfection. Several DBPs have been implicated in a variety of toxic effects, mainly carcinogenic and genotoxic effects. Moreover, DBPs exposure has also been associated with an increased risk of developmental effects. In this study, the developmental toxicity and genotoxicity of 10 DBPs (four trihalomethanes [THMs], five haloacetic acids [HAAs] and sodium bromate) in the zebrafish embryo model were evaluated. Embryos exposed for 72 hours were observed for different endpoints such as growth, hatching success, malformations and lethality. THMs exposure resulted in adverse developmental effects and a significant reduced tail length. Two HAAs, tribromoacetic acid and dichloroacetic acid, along with sodium bromate were found to cause a significant increase in malformation rate. Chloroform, chlorodibromomethane and sodium bromate produced a weak induction of DNA damage to whole embryos. However, developmental effects occurred at a range of concentrations (20–100 μg/mL) several orders of magnitude above the levels that can be attained in fetal blood in humans exposed to chlorinated water. In conclusion, the teratogenic and genotoxic activity observed by some DBPs in zebrafish reinforce the view that there is a weak capacity of disinfection products to cause developmental effects at environmentally relevant concentrations.

This is the second paper which reports the results obtained from a systematic conformational analysis of the (R)-8-amino-pentacyclo[5.4.0.02,6.03,10.05,9]undecane-8-carboxylic acid monopeptide (Cage monopeptide), using molecular mechanics and ab initio methods. The purpose of this paper is to highlight the performance of force field calculations of a series of amino acids with the ab initio calculations. The series of amino acids, Gly, Ala, Aib and Cage, exhibit a decrease of the conformational space upon an increase of steric factors due to an increase in substitution at the α-carbon. The Ramachandran maps computed in vacuo using the different parameterizations of the Parm94 and Parm96 AMBER force fields, which are not explicitly parameterized for the Cage amino acid, compares favourably with the ab initio results performed at the HF level using the 6-31G* basis set. Analysis of these maps reveal the helical-conformational preferences of the Cage monopeptide, which has the potential to be incorporated in the design of constrained cyclic peptide analogues.

A fast and easy to perform method for the routine determination of aflatoxins in medicinal herbs was developed. The described method involves a single-step extraction with a non-chlorinated solvent, an immunoaffinity clean-up and HPLC with fluorescence detection. Whilst assays with naturally contaminated and with spiked samples of several herbs showed that the recoveries were somewhat low and dependent on the kind of sample and the degree of grinding, the intra-batch reproducibility was good, allowing a reliable quantitation by the standard-addition method. Good linearity, repeatability and accuracy were demonstrated in assays involving several medicinal herbs. The limit of quantivation was of the order of 0.05–0.1 ng/g, being dependent of the species analysed, and the method required no tedious concentration or back-extraction steps. Copyright © 2005 John Wiley & Sons, Ltd.

The conformational profile of the eight stereoisomeric 2-amino-3-phenylnorbornane-2-carboxylic acids (2-amino-3-phenylbicyclo[2.2.1]heptane-2-carboxylic acids) has been assessed by computational methods. These molecules constitute a series of four enantiomeric pairs that can be considered as rigid analogues of either L- or D-phenylalanine. The conformational space of their N-acetyl methylamide derivatives has been explored within the molecular mechanics framework, using the parm94 set of parameters of the AMBER force field. Local minimum energy conformations have been further investigated at the ab initio level by means of the Hartree-Fock and second order Moller-Plesset perturbation energy calculations using a 6-31G(d) basis set. The results of the present work suggest that the bulky norbornane structure induces two kinds of conformational constraints on the residues. On one hand, those of a steric nature directly imposed by the bicycle on the peptide backbone and, on the other hand, those that limit the orientations attainable by the phenyl ring which, in turn, reduces further the flexibility of the peptide backbone. A comparative analysis of the conformational profile of the phenylnorbornane amino acids with that of the norbornane amino acids devoid of the beta-phenyl substituent suggests that the norbornane system hampers the residue to adopt extended conformations in favour of C7-like structures. However, the bicycle itself does not impart a clear preference for any of the two possible C7 minima. It is the aromatic side chain, which is forced to adopt an almost eclipsed orientation, that breaks this symmetry introducing a marked preference for a single region of the (phi, psi) conformational space in each of the phenylalanine norbornane analogues investigated.

The present study was aimed at estimating the current (2012) dietary intake of arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) by the population of Catalonia, Spain. The temporal trends with respect to previous surveys, performed in 2000, 2005, and 2008, were also determined. For that purpose, metal concentrations were analyzed in a number of widely consumed foodstuffs. A speciation study was also conducted by experimentally determining the levels of inorganic As (InAs) and methylmercury (MeHg) in the same food items. Furthermore, the dietary intake of those metals and species was calculated both deterministically and probabilistically by considering two food consumption surveys: ENCAT and ENIDE, representative of the Catalan and Spanish populations, respectively. An important temporal decrease of the dietary intake was noted for most elements, irrespective of the age-gender population group. Considering data for a male adult, the current dietary intake of As, InAs, Cd, Hg, MeHg, and Pb was estimated in 216, 2.6, 8.7, 10, 7.3, and 8.4 mu g/day, respectively, being these values lower than the respective provisional tolerable weekly intakes (PTWIs) or benchmark dose lower confidence limits (BMDLs). Moreover, new calculations by means of ENIDE survey indicated similar results to those previously obtained by ENCAT, with the exception of MeHg, whose intake exceeded the maximum recommended values for some part of the population. Although our data are similar to those frequently found in other European countries, the important intake of MeHg, which is linked to the high consumption of fish and shellfish, deserves further investigation.

Human microsomal epoxide hydrolase (mEH) catalyzes a key step in the biotransformation of benzo[a]pyrene that yields the highly mutagenic (+)-anti-7,8-diol-9,10 epoxide (BPDE). Two polymorphisms have been described in the coding region of the mEH gene (EPHX1) that produce two protein variants: 113Tyr-->113His (exon 3) and 139His-->139Arg (exon 4). We performed a case-control study among Northwestern Mediterranean Caucasians to investigate a possible association between these EPHX1 variants and lung cancer risk. Both EPHX1 polymorphisms were analyzed in a group of lung cancer patients (n=176) and in a control group of healthy smokers (n=187). The results showed a significantly decreased risk for the rare homozygous 113His/113His (adjusted odds ratio (OR): 0.44, 95% confidence interval (CI): 0.27-0.71) and 139Arg/139Arg (adjusted OR: 0.55, 95% CI: 0.33-0.91) compared with the major wild-types 113Tyr/113Tyr and 139His/139His, respectively, as the references. Thereafter, we analyzed the EPHX1 variants in combination with three glutathione S-transferase polymorphic genes (GSTM1, GSTT1, and GSTP1) and we found a significant overepresentation of cancer patients with a combination of exon 3 113Tyr/113Tyr EPHX1 and exon 5 105Ile/105Ile GSTP1 (adjusted OR: 2.34, 95% CI: 1.21-4.52). The polymorphic site within the exon 5 of GSTP1 results in a Ile-->Val substitution, and the isoleucine GSTpi isoform has been found in vitro to be less active than the valine isoform towards the conjugation of BPDE. The 113 Tyr/Tyr EPHX1 encodes for a high-activity mEH. Our results agree with these observations in vitro and suggest that a genetically determined combination of a high-activity mEH and a low-activity GSTpi may increase lung cancer risk among smokers.

The conformational profile of the conformationally constrained cyclohexane analogs of phenylalanine (1-amino-2-phenylcyclohexanecarboxylic acids, c6Phe) was assessed using computational methods. For this purpose, the conformational space of the N-acetyl methylamide derivatives of the stereoisomers (2S,3R)c6Phe and (2S,3S)c6Phe was explored by computing their respective Ramachandran maps, and low-energy minima were characterized at molecular mechanics level by means of the amber program, using the parm94 force field set of parameters. In order to assess the performance of the molecular mechanics calculations, each of the low-energy conformations was also investigated further at the ab initio level. Accordingly, the molecular mechanics geometries were used as starting conformations to perform full geometry optimizations at the Hartree-Fock level, using a 6-31G(d) basis set. Analysis of the results revealed that the cyclohexane structure directly induces some restrictions on the backbone, and constrains the orientation of the aromatic side-chain to two narrow regions for each stereoisomer. The conformational profile of these amino acids is then explained on the grounds of the interaction between the rigidly held phenyl ring and the main chain NH and CO groups. The results obtained are in good accordance with the experimental observations.

1-Aminocyclopropanecarboxylic acid (Ac3c) is a constrained α amino acid residue that exhibits peculiar conformational characteristics. The aim of the present study is to provide a deeper understanding of these features to be used as guidance to decide when to choose Ac3c as a building block for the design of peptide and protein surrogates. The whole Ramachandran plot of the Ace-Ac3c-NCH3 dipeptide was investigated at the Hartree–Fock level using a 6-31G(d) basis set and the most favorable structures were assessed on this surface by energy minimization. These results were subsequently used as a reference to generate specific molecular mechanics parameters for Ac3c compatible with the parm94 set of the AMBER force field. The effect of water as a solvent on the conformational profile of the dipeptide was also assessed using the Miertus–Scrocco–Tomasi self-consistent reaction-field model at the Hartree–Fock level using a 6-31G(d) basis set and using the AM1 semiempirical method. The conformational profile of the Ac3c dipeptide can be characterized by two symmetric low-energy regions for values of φ around ±80° with a wide range of values for ψ ranging from −40 to 180°, with the lower areas located at low values of ψ. Solvent effects do not alter the features of the conformational map, but a shift of the two absolute minima to (φ, ψ) values near (±90°, 0°) can be observed. These results are in accord with all experimental evidence and with the known tendency of Ac3c to induce β-turn conformations in peptides.

GSTM3 is one of five mu-class genes (M1-M5) belonging to a cluster located in chromosome 1. GSTM3 has been found to be polymorphic in humans with a number of individuals presenting a 3 bp deletion within intron 6 (GSTM3*B). In this study we have addressed the possible role of the GSTM3 polymorphism on lung cancer susceptibility. GSTM3 was genotyped in a group of lung cancer patients (n=176) and in a control group of healthy smokers (n=175). The frequency distribution of GSTM3*A/GSTM3*A, GSTM3*A/GSTM3*B and GSTM3*B/GSTM3*B showed no significant differences between patients and controls. Allelism at GSTM3 was also analysed in combination with the GSTM1 polymorphism. The chi(2) analysis confirmed that GSTM3*B allele is in linkage desequilibrium with GSTM1*A. The over-representation of GSTM1 null detected in previous studies, appeared to be restricted to those individuals with both GSTM1 null and GSTM3*A/GSTM3*A (48.3% in patients versus 36.0% in controls). The application of a second order logistic regression model revealed a significant adjusted odds ratio for the interaction term between GSTM1 null and GSTM3*A/GSTM3*A (OR: 2.14 95% CI 1.08-4.25) suggesting that this combined genotype may increase lung cancer risk. The analysis of transcription factor binding sites near the deleted sequence suggests that the heat-shock transcription factor 1 (HSTF1) could be involved in an enhanced expression of GSTM3*B, thus providing a possible mechanistic basis for a protective role of this allele.

Objectives: The human GSTTP1 gene is polymorphic with an A → G transition in exon 5 causing a replacement 105 Ile→Val in the GSTP1 protein. The two isoforms, encoded by the alleles GSTP1*A and GSTP1*B, respectively, show different catalytic efficiencies towards some carcinogenic epoxides. In this study we have addressed the possible role of the Ile105Val GSTP1 polymorphism in lung cancer susceptibility. Methods: The polymorphic site was genotyped by RFLP in a group of lung cancer patients (n=164) and in two control groups (healthy smokers, n=132; general population, n=200). All patients and controls were Northwestern Mediterranean Caucasians of the same ethnic origin. Results and Conclusions: The cancer patients showed frequencies of GSTP1*A/A; GSTP1*A/B and GSTP1*B/B (50%, 38%, 11%, respectively) very similar to those of both control groups (healthy smokers: 48%, 41%, 11%). After adjusting for age, sex and smoking status, no association was found between the GSTP1*B allele and lung cancer risk (OR: 1.18; 95% CI: 0.67–2.07). The Ile105val GSTP1 polymorphism was also analysed in combination with the GSTM1 and GSTT1 genes. The results showed that allelism at GSTP1 did not increase the risk associated with the GSTM1 or GSTT1 deletions.

In the last years, consumption of organic foods has become increasingly popular. Nevertheless, safety of organic foods is still unclear, and needs to be thoroughly evaluated. Patulin is a mycotoxin mainly present in rotten apples and apple-based products. The aim of this study is to analyse the content of patulin in apple juices and purees derived from organic and conventional production systems, in order to assess the risk to consumers, particularly in children. A total of 93 apple-based products marketed in Catalonia were analysed, 49 of which were derived from conventional and 44 from organic farming. The results showed higher incidence of positive samples and higher concentration of patulin in organic apple purees when comparing with conventional ones. In the case of juices, significant differences were found between conventional and organic samples, but applying a multivariate analysis the type of agriculture did not seem to have a relevant contribution to patulin occurrence, being cloudiness the main factor involved. The estimated daily intake of patulin for infants and young children (0–3 years old), children (4–18 years old) and adults (19–66 years old), were below the provisional maximum tolerable daily intake (PMTDI) of 0.4 μg/kg bw in all scenarios considered.

10.1016/j.fct.2011.06.077 The concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), as well as those of 18 polychlorinated biphenyls (PCBs), including 12-dioxin like (DL)-PCBs, were measured in foodstuffs randomly acquired in Catalonia (Spain) in November-December 2008. A total of 65 composite samples, belonging to various food groups were analyzed by HRGC/HRMS. The dietary intakes of PCDD/Fs and PCBs were subsequently estimated for four age groups of the population of Catalonia: children, teenagers, adults, and seniors, which were in turn divided according to sex. The highest dietary exposure to PCDD/Fs corresponded to fish and seafood (28.0%), dairy products (15.4%), and oils and fats (10.6%), while that of PCBs corresponded to fish and seafood (58.6%), and dairy products (8.9%). In contrast, the lowest contributions of PCDD/Fs and PCBs corresponded to vegetables, fruits and pulses. Concerning the sum of PCDD/Fs plus DL-PCBs, the current total intake expressed in pg WHO-TEQ/kg per day (0.60) showed a notable decreasing trend with respect to those found in previous surveys performed also in Catalonia in 2000 (3.51) and 2006 (1.12pg/kg per day). The current dietary intake of PCDDs plus DL-PCBs is similar or lower than that recently reported in studies performed in a number of regions and countries.

Due to the great number of congeners, the use of the correct nomenclature for PCBs is a difficult task, and unfortunately errors in the assignment of chemical denominations are not unusual. We suggest in this paper to correctly apply the rules initially introduced by Ballschmiter and Zell in 1980, which differ from those of the IUPAC. A simple algorithm in BASIC was created to help researchers in this field to denominate all the 209 congeners by their correct Ballschmiter and Zell and/or IUPAC systematic names.

The human health risks derived from the multipathway/multipollutant exposure to various chemicals were assessed in an area with significant petrochemical activity (Tarragona, Catalonia, Spain). Environmental exposure to several Persistent Organic Pollutants (POPs) (PCDD/Fs, PCBs, PCNs, and PAHs) and metals (As, Cd, Cr, Hg, Mn, Pb, and V) was determined and compared with the dietary intake of these pollutants. The mean environmental exposure to organic pollutants ranged from 6.36 x 10(-6) ng WHO-TEQ kg(-1) day(-1) to 3.34 ng kg(-1) day(-1) for PCDD/Fs and PAHs, respectively. In turn, the minimum and maximum values of environmental exposure to metals corresponded to Cd (9.35 x 10(-8) mg kg(-1) day(-1)) and Mn (8.72 x 10(-5) mg kg(-1) day(-1)), respectively. Among the environmental exposure pathways, dermal absorption and soil ingestion were the most important pathways for POPs and metals, respectively. However, this exposure was notably lower than the dietary intake of these contaminants, with percentages of2% for most of them. Considering cumulative effects, the current concentrations of micropollutants do not mean significant additional non-carcinogenic and carcinogenic human health risks. Notwithstanding, in order to consider the synergistic/antagonistic effects according to the target organ or mode-of-action, the development of alternative methodologies of risk assessment are necessary for a more accurate evaluation.

3,4-Methylenedioxymethamphetamine, MDMA or "ecstasy" is consumed mainly by young population at childbearing age. Therefore, there may be a risk of exposure of some pregnant women. The effects of the developmental exposure to MDMA on the sexual development and long-term sexual behaviour/fertility were assessed in Sprague-Dawley rats. MDMA was administered subcutaneously at 0 (control), 0.5, 5 and 10 mg/kg to female rats once a day, three consecutive days a week during 10 weeks, including gestation and lactation. The male offspring was evaluated for sexual maturation and mated with untreated sexually receptive females to evaluate the mating and pregnancy rates. Hormonal, haematological, biochemical, histological, genotoxicological and testicular and sperm parameters were also evaluated. A significant higher incidence of DNA damage in sperm and interstitial oedema in testes was found. There was also a significant and dose-related decrease in sperm count and a significant decrease in sperm motility at all doses. A significant delay in preputial separation onset in all treated groups was observed. This study reports by the first time an alteration of spermatogenesis after in utero and lactation MDMA exposure in the rat.

3,4-Methylenedioxymethamphetamine (MDMA or "ecstasy") is consumed mainly by young population. For this reason, it is especially relevant to take into consideration the effects on the reproductive system. The influence of MDMA on the fertility and reproduction of the male rat was assessed in this study. MDMA was administered subcutaneously at 0 mg/kg (control), 0.5 mg/kg, 5 mg/kg and 10 mg/kg to SD male rats once a day, 3 consecutive days a week during 12 weeks, simulating human weekend associated consumption. Hormonal, haematological, biochemical, histological, genotoxicological and testicular and sperm parameters were evaluated in half of the rats. The remaining animals were mated with untreated sexually receptive females to evaluate the mating and pregnancy rates. A significantly higher incidence of DNA damage in Comet Test in sperm, tubular degeneration and interstitial oedema in testes was found. At all doses tested, sperm motility, morphology, mating and pregnancy rates, and number of implantation sites were not affected. This study fills the existing gap of knowledge about the chronic effects of MDMA in reproductive function using a realistic experimental design. Taking into account the higher sensitivity of human males, some concerns about the effects on the reproductive health still remain.

Polychlorinated biphenyls (PCBs) are a class of aryl halides widely distributed in the environment. Although production has virtually ceased, and most industrial applications (capacitors, transformers, hydraulic fluids, lubricants, etc) are severely restricted, they are one of the most ubiquitous and persistent environmental pollutants. Several toxic effects caused by PCBs have been described including: liver enlargement, hepatomegalocytosis, acne, lymphoid atrophy, immunosuppression, tumor promotion, porphyria, etc (Reggiani, 1982; McConneU, 1980). This work continues our previous reports about organochlorine residues in human tissues of some Spanish populations, that showed high levels of some residues, specially of hexachlorobenzene (HCB) (To-Figueras et al, 1986; Camps et al, 1988). PCBs pattern and concentration in the adipose tissue of the inhabitants of an urban and industrial area (Barcelona) were determined.

Two polymorphic sites of the microsomal epoxide hydrolase gene (EPHX1, 113Tyr--113His, 139His--139Arg) and four glutathione S-transferase genes (GSTM1, GSTM3, GSTP1, GSTT1) were genotyped in a group of patients with larynx cancer (N=204) and in a group of healthy controls (N=203), all Spanish caucasians. After adjusting for gender, age, and tobacco smoking, none of the polymorphisms alone were found to be associated with larynx cancer risk. The analysis of EPHX1/GST combinations, however, showed a significant over-representation of patients with a combination of 113Tyr/113Tyr EPHX1 and 105Ile/105Ile GSTP1 (adjusted odds ratio (OR): 1.95; 95% confidence interval (CI): 1.02-3.78). The calculation of the predicted epoxide hydrolase (EH) activity also showed an increased risk for the individuals with both predicted high activity EH and 105Ile/105Ile GSTP1 (OR: 2.90; 95% CI: 1.10-7.67). These results on larynx cancer tend to confirm a former study on lung cancer (Cancer Lett. 173 (2001) 155) suggesting the existence of an interaction between variants of EH and GSTpi, both enzymes being involved in the metabolism of aromatic hydrocarbons, that may increase susceptibility to tobacco-related cancers.

Pentachlorothiophenol, a sulphur derivative of the widespread environmental pollutant hexachlorobenzene (HCB) has been detected and quantified in the urine of a human general population with high body burden of HCB. The sulphur derivative was analysed by GLC-MS as pentachlorothioanisole (PCTA) after hydrolysis and methylation of the respective conjugate and was found in 100% of the samples ( n = 40) with a mean concentration of 1.85 ± 0.98 ng ml-1 (X ± s.d., range 0.58-4.50 ng ml-1). No correlation with urinary pentachlorophenol (PCP) and no sex-related differences were found. The derivative may originate from the biotransformation of HCB stored in tissues and may be a useful maker of HCB metabolism in humans.

We have synthesized the model dipeptides Piv-l-Pro-c6Phe-NHiPr, incorporating each of the two cis cyclohexane analogues of phenylalanine: (S,S)- and (R,R)-1-amino-2-phenylcyclohexanecarboxylic acid. Their structural analysis has been carried out in solution by 1H NMR and FTIR absorption spectroscopy and in the solid state by X-ray diffraction. In weakly polar chlorinated solvents, the (S,S)c6Phe-containing dipeptide mainly accommodates a type I β-turn, whereas the (R,R) residue shows a greater propensity to βII-folding. This behavior does not differ significantly from that exhibited by the analogous dipeptides containing l- and d-Phe. However, the l-Pro-l-Phe sequence has been shown to undergo a βI-to-βII transition in the presence of a strong solvating medium, such as DMSO, or in the crystalline state. Interestingly, Piv-l-Pro-(S,S)c6Phe-NHiPr, incorporating its cyclohexane analogue with χ1 fixed at +60°, retains the βI-folded structure under these conditions. Theoretical calculations, supported by the experimental data, indicate that a c6Phe-NH to aromatic π-orbitals interaction has an important influence on the observed β-folding preferences., Financial support from the Ministerio de Educación y Cultura (PB97-0998 and acción integrada HF97-46) and Ministère des Affaires Etrangères (action intégrée 97039) is gratefully acknowledged.

The integration of chemicals into the environment by human activities can represent a serious risk to environmental and human health. Many chemical products like endocrine-disrupting chemicals and pharmaceuticals have been found in surface waters, and they can enter waterways via discharge from wastewater treatment plants (WWTPs) and later from drinking water treatment plants (DWTPs). It would be convenient to evaluate the treatment system performance, to avoid their arrival to water consumption. Fish embryo is an attractive model for risk assessment of chemicals in waters since they offer the possibility to perform small-scale, high-throughput analyses.

The present work reports the results of a conformational study performed on seven unnatural amino acid residues and on its natural precursor, investigated by means of computational methods at the molecular mechanics level. Amino acid residues selected for the present study are derivatives of l-phenylalanine substituted at the α and/or β carbons. This series is composed of different linear analogs, including α-methyl, β-methyl and β-phenyl substituted with different stereochemistry. Analysis of the Ramachandran maps of the corresponding dipeptides in vacuo reveals their conformational preferences, to be used as guidance for the synthesis of constrained peptide analogs with desired conformational propensities. The available conformational space for every dipeptide is also analysed.

blood donors from the general population (n 5 192). All patients and controls were Northwestern Mediterranean controversial since the initial phenotyping studies showed a Caucasians. The results show that the GSTM1 null genotype clear association between class μ GST deficiency and lung (GSTM1*0/GSTM1*0) was slightly over represented in the cancer risk (5,6) but the later genotyping studies have shown lung cancer patients (frequency of 58%; OR: 1.40, 95% discrepant results (12,13). Furthermore, some studies on CI: 0.74‐2.61, referred to healthy smokers). The histological bladder cancer have suggested that the possible protective type most clearly modified was small cell carcinoma (fre- effect of the alleles GSTM1*A and GSTM1*B may not be quency of 62.2%, OR: 1.91, CI: 0.78‐4.69). The subdivision identical (14) whereas others have found that GSTM1*A/B of the patients with one or two copies of the GSTM1 heterozygosity, but not GSTM1*Aor GSTM1*B, was protective gene according to a GSTM1*A, GSTM1*B or GSTM1*A/B among skin cancer patients with multiple basal cell carcinomas genotype (frequencies of 28.2 %, 11.2%, 2.5% respectively) (11). This has lead some authors to suggest that a model based revealed no significant differences between the cases and on the hypothesis that GSTM1*0 alone confers increased risk both control groups. The frequency of the deleted GSTT1 may be too simplistic and that the role of the different GSTM1 genotype among the lung cancer patients (24%) was not alleles and of other genes of the same family such as GSTM3 significantly increased (OR: 1.08, CI: 0.57‐2.05, referred should be taken into account (15). to healthy smokers). The results showed that 14.4% of the Another polymorphic gene of the same family is GSTT1 patients presented homozygous deletion of both GSTT1 which encodes for a class θ GST that catalyzes the conjugation and GSTM1 (12.5% among healthy smokers) suggesting of halomethanes in human erythrocytes (16,17). Substrates of no potentiation between null genotypes for lung cancer risk. θ GST include industrial chemicals such as methyl chloride, methyl bromide, dichloromethane, ethylene oxide and diepoxybutane, a reactive metabolite of 1,3-butadiene. Indi