Advancing the use of new approach methodologies for assessing teratogenicity: Building a tiered approach.

Many New Approach Methodologies (NAMs) have been developed for the safety assessment of new ingredients. Research into reproductive toxicity and teratogenicity is a particularly high priority, especially given their mechanistic complexity. Forty-six non-teratogenic and 39 teratogenic chemicals were screened for teratogenic potential using the in silico DART model from the OECD QSAR Toolbox; the devTox quickPredict™ (devTox assay) test and the Zebrafish Embryotoxicity Test (ZET). The sensitivity and specificity were 94.7% and 84.1%, respectively, for the DART tree (83 chemicals), 86.1% and 35.6% for the devTox (81 chemicals) and 77.8% and 76.7% for the ZET (57 chemicals). Fifty-three chemicals were tested in all three assays and when results were combined and based on a "2 out of 3 rule", the sensitivity and specificity were 96.0% and 71.4%, respectively. The specificity of the devTox assay for a sub-set of 43 chemicals was increased from 26.1% to 82.6% by incorporating human plasma concentrations into the assay interpretation. When all 85 chemicals were assessed in a decision tree approach, there was an excellent predictivity and assay robustness of 90%. In conclusion, all three models exhibited a good sensitivity and specificity, especially when outcomes from all three were combined or used in "2 out of 3" or a tiered decision tree approach. The latter is an interesting predictive approach for evaluating the teratogenic potential of new chemicals. Future investigations will extend the number of chemicals tested, as well as explore ways to refine the results and obtain a robust Integrated Testing Strategy to evaluate teratogenic potential.

Ventana sagital paramedial oblicua para el acceso de la rama medial del nervio raquídeo posterior guiado ecográficamente / Ultrasound guided parasagittal obliqua aproach for lumbar medial branch block

Objetivos: el método de abordaje ecográfico más utilizado para el bloqueo del ramo medial del nervio raquídeo posterior (RmNRp) requiere de la utilización de una técnica ecográfica biplanar con punción guiada en plano en ventana transversal, para situar la cánula perpendicular al RmNRp, limitando la realización de radiofrecuencia. La utilización de una ventana ecográfica parasagital oblicua permite el acceso al RmNRp permitiendo situar la cánula de forma paralela al nervio, logrando estímulos sensitivos y motores, y posibilitando la realización de radiofrecuencia térmica para segmentos lumbares por encima de L5.En el presente estudio valoramos la eficacia de este nuevo abordaje ecográfico mediante la comprobación fluoroscópica de la situación de la cánula y la comprobación de la obtención de estímulos sensitivos y motores. Material y métodos: se estudian 31 pacientes diagnosticados de síndrome facetario propuestos para la realización de bloqueos de RmNRp diagnósticos. Describimos cuatro ventanas ecográficas lumbares secuenciales necesarias para incluir los pacientes en el estudio. Una vez obtenidas, se localiza el punto objetivo utilizando una ventana parasagital oblicua para lograr la visualización de la cara externa de la apófisis articular superior y la parte más dorsal de la apófisis transversa. Se realiza punción guiada en plano hasta situar la punta en la unión entre apófisis articular superior y apófisis transversa. Una vez situada la cánula en el objetivo se procede a estímulo sensitivo 50 Hz y motor 2Hz para reposicionar la cánula en caso de no obtenerse estímulos. Una vez obtenido estímulo motor o sensitivo o ambos, se realiza una proyección fluoroscópica oblicua 30° y caudo-craneal para comprobar la localización de la punta de la aguja. Resultados: en el 16% de los pacientes no se pueden identificar las 4 ventanas ecográficas por lo que se aborta el procedimiento. En el 84% restante se consigue situar la aguja en el punto target en el 100% de los casos en el primer intento o con un sólo reposicionamiento de la aguja. Conclusión: la ventana ecográfica parasagital oblicua nos permite una aproximación tangencial al RmNRp lumbar con un alto índice de éxito para situar la cánula de radiofrecuencia de forma similar a cuando utilizamos fluoroscopia. La identificación de las cuatro ventanas ecográficas descritas en el estudio nos permite hacer un cribado de los pacientes tributarios a ecografía para el bloqueo de RmNRp (AU)

Objectives: The most used ultrasound guided medial branch block method requires the use of a biplane ultrasound technique. Using this technique the cannula is positioned not parallel to the medial branch by limiting their use to perform conventional radiofrequency. Using a sonographic parasagittal obliqua view allows placing the cannula parallel to the nerve, achieving sensory and motor stimuli and making possible radiofrequency above L5 lumbar segments. In the present study we determined the effectiveness of this new approach by using fluoroscopy and by obtention of sensory and motor stimuli. Material and methods: Thirty-one patients diagnosed with lumbar facet joint pain proposed for diagnostic medial branch blocks were studied. We describe four lumbar sequential sonographic views necessary to include patients in the study. Once obtained, the target point is located using a parasagittal oblique sonographic view to achieve the external face of the superior articular process and the more dorsal part of the transverse process. Puncture is performed guided in plane to place the tip at the junction between superior articular process and transverse process. Once the cannula located in the target proceeds to sensory stimulus 50 Hz and Motor 2 Hz to perform a cannula repositioning in case there were no stimuli. Once obtained either motor or sensory stimulus a caudal to craneal 30° oblique projection is performed by fluoroscopy to check the location of the needle tip. Results: In 16% of patients cannot identify the four ultrasound views so the procedure is aborted. In the remaining 84% is achieved by placing the needle into the target point at 100% of the cases on the first attempt or by once a repositioning of the needle. Conclusion: Ultrasound oblique parasagittal view allows us a tangential approach to the lumbar medial branch with a high rate of success to position the radiofrequency cannula similar to when using fluoroscopy. The sonographic identification of the four windows described in the study allows us to screening tributary patient for ultrasound medial branch blocks (AU)

Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines.

The development of in vitro genotoxic assays as an alternative method to animal experimentation is of growing interest in the context of the implementation of new regulations on chemicals. However, extrapolation of toxicity data from in vitro systems to in vivo models is hampered by the fact that in vitro systems vary in their capability to metabolize chemicals, and that biotransformation can greatly influence the experimental results. Therefore, much attention has to be paid to the cellular models used and experimental conditions. Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic ubiquitous pollutants. Human exposure to PAHs is mainly from food origin. In this study, a detailed analysis of the biotransformation capabilities of three human cell lines commonly used for in vitro testing (HepG2, ACHN and Caco-2) was undertaken using 3 model PAHs (benzo(a)pyrene [B(a)P], fluoranthene [FLA] and 3-methylcholanthrene [3-MC]). Concomitantly the genotoxicity of these PAHs was investigated in different cell lines, using a new genotoxic assay (H2AX) in 96-well plates. The metabolic rates of B(a)P, FLA and 3-MC were similar in HepG2 and Caco-2 cell lines, respectively, though with the production of different metabolites. The ACHN cell line was shown to express very limited metabolic capabilities. We demonstrated that the PAHs having a high metabolic rate (B(a)P and 3-MC) were genotoxic from 10(-7) molar in both HepG2 and Caco-2 cells. The present study shows that H2AX measurement in human cell lines competent for the metabolism, is an efficient and sensitive genotoxic assay requiring less cells and time than other currently available tests.

Characterisation of bioactive compounds in infant formulas using immobilised recombinant estrogen receptor-alpha affinity columns.

In this study, the use of recombinant estrogen receptor alpha (ERalpha)-based affinity columns was reported, for the isolation and the identification of estrogenic substances present in complex matrices, focusing on bioactive compounds present in foodstuff. The capability of affinity columns to trap high, but also low-affinity radio-labelled ligands (17beta-estradiol, genistein and bisphenol A) was demonstrated. Three pooled samples of infant formulas (milk-based, hypoallergenic and soy-based formulas for infants aged 0-4 months) from a EU market basket were prepared by the CASCADE Network of Excellence. After determining the estrogenic activity of these food samples, human recombinant ERalpha ligand binding domain (LBD) based affinity columns combined with suitable analytical methods (high resolution LC-MS/MS) were used to identify the bioactive compounds present in the soy-based formula extract, namely phytoestrogens (genistein and daidzein) involved in the agonistic activity measured. Incubations of genistein with liver microsomes were carried out and the extracts analysed following the same protocol, demonstrating that hERalpha affinity columns can also be used for trapping active metabolites. This approach combining bioluminescent cell lines with this useful tool based on hERalpha-LBD affinity columns thus allowed the purification and the concentration of both known and unknown estrogenic ligands prior to investigation of their structure using LC-MS.

A dominant hepatitis B virus population defective in virus secretion because of several S-gene mutations from a patient with fulminant hepatitis.

There is increasing evidence that certain pathogenic hepatitis B virus (HBV) variants may play a role in the pathogenesis of fulminant hepatitis (FHB). Recently, we isolated from a patient with fulminant recurrent hepatitis B after liver transplantation variants with enhanced replication competence and a possible defect in viral particle secretion. Both viral features may have contributed to the severity of the disease. The aim of this study was to prove the secretion defect of these variants, to analyze the consequences, and to identify the responsible viral mutations. The variant genomes and appropriate wild-type/variant hybrid genomes were functionally characterized after transfection in human hepatoma cells. Two cloned genomes and the polymerase chain reaction (PCR)-amplified mixture of full-length genomes showed a block in viral particle secretion. This was caused by a combination of amino acid changes in the S-protein including the mutation G145R frequently emerging after hyperimmunoglobulin treatment. The mutations induced retention of the surface proteins in an endoplasmic reticulum (ER)-like compartment, but no intracellular accumulation. These data provide evidence for the in vivo existence of a dominant HBV population with a severe defect in viral particle secretion caused by mutations in the S-gene. This viral phenotype in combination with the enhanced replication competence may have contributed to the fulminant clinical course of the infection.

The incidence of breastfeeding in our environment.

AIMS: To establish the incidence of breastfeeding and its persistence after three and six months; to identify the factors which might modify attitudes towards breastfeeding. METHODS: An interview, administered to mothers of term born infants of adequate weight for gestational age, after a delivery of one sole foetus, between 1st March and 31st May 1997. Mothers were asked about: previous pregnancies, children and type of feeding; during this pregnancy, their intentions regarding feeding; supervision of pregnancy, and the information received regarding breastfeeding; their work situation, and educational level. Questions regarding the neonatal period referred to the kind of lactation initiated, information received, and the use of a pacifier and supplements. A two-part poll was conducted by telephone after 3 months and after 6 months, enquiring about what kind of feeding was used, the reason for change (if any), who had provided information regarding the change, degree of satisfaction, and work situation. Three hundred and twenty-nine polls were completed, accounting for 70% of the births. RESULTS: During pregnancy, 91% of mothers had intended to breastfeed, and this figure was maintained at birth. A trend towards breastfeeding was observed: 74% of those who had fed earlier children artificially started breastfeeding this time, compared with 7% of mothers who changed from breastfeeding to artificial feeding. After three months, 57% of mothers continued to breastfeed their child, and 24% after six months. High educational level exerted a positive influence on the duration of breastfeeding (OR = 1.7; p = 0.03) and the giving of supplements had a negative effect (OR = 0.4; p = 0.04). The fact that mothers work outside the home did not modify the duration of breastfeeding. CONCLUSIONS: The rate of breastfeeding is high (91%). Among mothers with previous children, there is a greater trend to change from artificial feeding to breastfeeding (74% vs 7%; p < 0.002). The mother's educational level is the most important positive factor, and the early giving of supplements the most important negative factor.

[Variations in post-small bowel resection gastric acid hypersecretion and hyper-gastrinemia after vagotomy and antrectomy. Experimental study in rat (author's transl)]. / Variations amenées par la vagotomie et l'antrectomie sur l'hyper-sécrétion gastrique et l'hyper-gastrinémie post-résection du grêle. Etude expérimentale chez le rat.

Resection of the small bowel provokes gastric acid hypersecretion and hyper-gastrinemia. Various studies have been conducted to assess the effects of vagotomy and antrectomy on post-resection gastric hyperacidity, both on basal levels and those after stimulation with histamine and pentagastrin. Similar studies involved hypergastrinemia levels. A total of 100 Sprague-Dawley rats were divided into 6 groups : controls (C). massive small bowel resection (R), vagotomy plus pyloroplasty (VP). antrectomy (A), vagotomy plus pyloroplasty and bowel resection (VPR), and antrectomy plus bowel resection (AR). - It was demonstrated that vagotomy is the most effective method for reducing gastric acid hypersecretion after small bowel resection. Antrectomy provokes diminution of gastric acid secretion in animals without bowel resections, but neither this nor gastrinemia levels are significantly altered in operated animals. Synergistic action of the mechanisms provoking post-resection and post-vagotomy hyper-gastrinemia is observed when they occur in the same group of animals, the incidence being higher than that observed after vagotomy or resection alone. Gastrinemia levels increased after VPR or reduced after AR cannot be differentially transposed to gastric secretion in these two groups of animals, demonstrating that, under these conditions, blood gastrin levels are not the principal factor involved in gastric secretion control.