Grouping of chemicals into mode of action classes by automated effect pattern analysis using the zebrafish embryo toxicity test.

A central element of high throughput screens for chemical effect assessment using zebrafish is the assessment and quantification of phenotypic changes. By application of an automated and more unbiased analysis of these changes using image analysis, patterns of phenotypes may be associated with the mode of action (MoA) of the exposure chemical. The aim of our study was to explore to what extent compounds can be grouped according to their anticipated toxicological or pharmacological mode of action using an automated quantitative multi-endpoint zebrafish test. Chemical-response signatures for 30 endpoints, covering phenotypic and functional features, were generated for 25 chemicals assigned to 8 broad MoA classes. Unsupervised clustering of the profiling data demonstrated that chemicals were partially grouped by their main MoA. Analysis with a supervised clustering technique such as a partial least squares discriminant analysis (PLS-DA) allowed to identify markers with a strong potential to discriminate between MoAs such as mandibular arch malformation observed for compounds interfering with retinoic acid signaling. The capacity for discriminating MoAs was also benchmarked to an available battery of in vitro toxicity data obtained from ToxCast library indicating a partially similar performance. Further, we discussed to which extent the collected dataset indicated indeed differences for compounds with presumably similar MoA or whether other factors such as toxicokinetic differences could have an important impact on the determined response patterns.

Species-specific developmental toxicity in rats and rabbits: Generation of a reference compound list for development of alternative testing approaches.

For regulatory information requirements, developmental toxicity testing is often conducted in two mammalian species. In order to provide a set of reference compounds that could be used to explore alternative approaches to supersede testing in a second species, a retrospective data analysis was conducted. The aim was to identify compounds for which species sensitivity differences between rats and rabbits are not caused by maternal toxicity or toxicokinetic differences. A total of 330 compounds were analysed and classified according to their species-specific differences. A lack of concordance between rat and rabbit was observed in 24% of the compounds, of which 10% were found to be selective developmental toxicants in one of the species. In contrast to previously published analyses the presented comparison is based entirely on publically data allowing validating and comparing alternative approaches for developmental toxicity testing. Furthermore, this list could be useful to identify mechanisms leading to species differences.

Assessment of developmental delay in the zebrafish embryo teratogenicity assay.

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.

Quality assessment of river waters using risk indexes for substances and sites, based on the COMMPS procedure.

The well established risk assessment COMMPS methodology (Fraunhofer Institut, Schmallenberg, Germany) developed at a European basin scale to identify priority pollutants based on their exposure and effects, has been applied at a local scale along a double step process: (a) first, the corresponding adapted list of priority pollutants has been obtained from monitoring data at a regional scale; (b) second, a new site pollution risk index has been developed for the relative comparison of the chemical pollution status of the investigated geographical region. As a case study, representative of the Mediterranean area, the process has been applied to 17 Catalan rivers (NE Spain), using the monitoring data collected between 1997 and 2006 in 207 different sampling sites. After an appropriate selection procedure, 52 parameters were finally used for the calculation of the local priority substance and site risk indexes. From the exposure point of view the most relevant substances at the Catalan scale were volatile organohalogen compounds (VOX) and volatile aromatic hydrocarbons (toluene and xylene), whereas the priority substance risk index points to dibromomethane (volatile organohalogen compound) and benzo-a-anthracene (PAH) as the most concerning compounds. The local and European priority substance indexes have been compared, showing minor differences. Finally, site risk indexes have been calculated and utilized for comparison of the chemical status of different sites. They can be conveniently displayed in quality geographical maps and are considered a valuable tool for the environmental management and risk assessment of the region under study.

MDMA (ecstasy) delays pubertal development and alters sperm quality after developmental exposure in the rat.

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.

La coordinación entre profesores de fisiología y toxicología: un caso práctico en la Facultad de Farmacia de la Universidad de Barcelona / Physiology and toxicology teachers’ coordination. A case study in the Faculty of Pharmacy, University of Barcelona

El aprendizaje es un proceso continuo que no debería finalizar una vez aprobada una determinada asignatura. En cualquier estudio universitario hay muchas materias que, para su comprensión, requieren de conocimientos adquiridos previamente en otras. En los estudios de Farmacia del plan 2002, los profesores de toxicología habían constatado que los estudiantes de dicha asignatura no recordaban conceptos básicos cursados en asignaturas de semestres anteriores. La asignatura de toxicología necesita para su comprensión conocimientos de, entre otras materias, fisiología y fisiopatología. Por esta razón se planteó la necesidad de hacer una actuación conjunta entre los profesores de Fisiología y Toxicología.Los objetivos de este proyecto fueron: a) Identificación de los contenidos fisiológicos y fisiopatológicos que los alumnos deben conocer para el seguimiento de la asignatura de toxicología. b) Unificación terminológica. c) Realización de un conjunto de preguntas básicas sobre estos contenidos. d) Detección, a través de estas preguntas, de los temas o grupos de temas con porcentajes más altos de respuestas incorrectas. e) Detectar los temas de fisiología y fisiopatología en los que hay que hacer más hincapié para favorecer el seguimiento de toxicología. En esta comunicación se describe la experiencia y los resultados obtenidos(AU)

Learning is a continuous process that should be still performed once a particular subject has been passed. In the university, many subjects require prior knowledge of others subjects for better understanding. During the Pharmacy curriculum of 2002, toxicology teachers observed that students did not seem to remember the basic concepts presumably acquired in previous semesters. For example, for the toxicology subject, students should have basic knowledge of physiology and pathophysiology. For this reason, a joint action among physiology and toxicology teachers was considered.The objectives of this project were: a) Identifying the physiological and pathophysiological aspects that students should know to follow the toxicology course. b) Reaching agreement over the common terminology. c) Executing a set of basic questions about these physiological and pathophysiological aspects. d) Detecting through these questions, the topics with a highest percentage of incorrect answers. e) Identifying which physiology and pathophysiology topics should be emphasized to encourage students to follow the toxicology subject. This communication describes the experience and outcomes of this project(AU)

Analysis of 5-hydroxymethylfurfural in foods by gas chromatography-mass spectrometry.

A new, simple and selective method for the analysis of 5-hydroxymethylfurfural (HMF) in foods by gas chromatography coupled to mass spectrometry (GC-MS) is proposed. Several derivatising procedures based on the formation of an HMF silylated derivative using different reagents were studied. Among the derivatising reagents examined, N,O-bis-trimethylsilyltrifluoroacetamide (BSTFA) provided the best derivatisation yield. Sample clean-up was also optimised, using either liquid-liquid extraction with dichloromethane or solid-phase extraction (SPE) with several commercially available cartridges, and the best results were obtained using ENV+ cartridges. Quality parameters such as day-to-day and run-to-run precision (RSD<10%), linearity (between 25 and 700 ng g(-1)) and detection limit (6 ng g(-1)) were established. This method was successfully applied to the analysis of HMF content in several Spanish food samples from a local market, such as jam, honey, orange juice and bakery products.