The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization

AbstractIn human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84–86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter

Induction and repression effects on CYP and transporter protein abundance by azole mixture uptake in rat liver

Detection of mixture effects is a major challenge in current experimental and regulatory toxicology. Robust markers are needed that are easy to quantify and responsive to chemical stressors in a broad dose range. Several hepatic enzymes and proteins related to drug metabolism like cytochrome-P-450 (CYP) enzymes and transporters have been shown to be responsive to pesticide active substances in a broad dose range and are therefore good candidates to be used as markers for mixture toxicity. Even though they can be well quantified at the mRNA level, quantification on the protein level is challenging because most of these proteins are membrane bound. Here we report the development of mass spectrometry-based assays using triple-x-proteomics (TXP) antibodies in combination with targeted selected ion monitoring (tSIM) to quantify changes of protein levels due to exposure to mixtures of pesticide active substances. Our results indicate that changes on the protein level of CYP1A1, ABCB2, ABCC3 are in line with observations on the mRNA and enzyme activity level and are indicative of mixture effects. Therefore, the tests are promising to reveal effects by chemical mixture effects in toxicological studies in rats

Cumulative dietary risk characterisation of pesticides that have chronic effects on the thyroid

A retrospective chronic cumulative risk assessment of dietary exposure to pesticide residues, supported by an uncertainty analysis based on expert knowledge elicitation, was conducted for two effects on the thyroid, hypothyroidism and parafollicular cell (C‐cell) hypertrophy, hyperplasia and neoplasia. The pesticides considered in this assessment were identified and characterised in the scientific report on the establishment of cumulative assessment groups of pesticides for their effects on the thyroid. Cumulative exposure assessments were conducted through probabilistic modelling by EFSA and the Dutch National Institute for Public Health and the Environment (RIVM) using two different software tools and reported separately. These exposure assessments used monitoring data collected by Member States under their official pesticide monitoring programmes in 2014, 2015 and 2016 and individual consumption data from 10 populations of consumers from different countries and different age groups. This report completes the characterisation of cumulative risk, taking account of the available data and the uncertainties involved. For each of the 10 populations, it is concluded with varying degrees of certainty that cumulative exposure to pesticides that have the chronic effects on the thyroid mentioned above does not exceed the threshold for regulatory consideration established by risk managers

Guidance on dermal absorption

This guidance on the assessment of dermal absorption has been developed to assist notifiers, users of test facilities and Member State authorities on critical aspects related to the setting of dermal absorption values to be used in risk assessments of active substances in Plant Protection Products (PPPs). It is based on the ‘scientific opinion on the science behind the revision of the guidance document on dermal absorption’ issued in 2011 by the EFSA Panel on Plant Protection Products and their Residues (PPR). The guidance refers to the EFSA PPR opinion in many instances. In addition, the first version of this guidance, issued in 2012 by the EFSA PPR Panel, has been revised in 2017 on the basis of new available data on human in vitro dermal absorption for PPPs and wherever clarifications were needed. Basic details of experimental design, available in the respective test guidelines and accompanying guidance for the conduct of studies, have not been addressed but recommendations specific to performing and interpreting dermal absorption studies with PPPs are given. Issues discussed include a brief description of the skin and its properties affecting dermal absorption. To facilitate use of the guidance, flow charts are included. Guidance is also provided, for example, when there are no data on dermal absorption for the product under evaluation. Elements for a tiered approach are presented including use of default values, data on closely related products, in vitro studies with human skin (regarded to provide the best estimate), data from experimental animals (rats) in vitro and in vivo, and the so called ‘triple pack’ approach. Various elements of study design and reporting that reduce experimental variation and aid consistent interpretation are presented. A proposal for reporting data for assessment reports is also provided. The issue of nanoparticles in PPPs is not addressed. Data from volunteer studies have not been discussed since their use is not allowed in EU for risk assessment of PPPs

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term 'nanoplex'. Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with ζ potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVβ was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD50 values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a ζ potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVβ plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 µM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering

Seroepidemiological study on the spread of SARS-CoV-2 in Germany:

The SARS-CoV-2 coronavirus has spread rapidly across Germany. Infections are likely to be under-recorded in the notification data from local health authorities on laboratory-confirmed cases since SARS-CoV-2 infections can proceed with few symptoms and then often remain undetected. Seroepidemiological studies allow the estimation of the proportion in the population that has been infected with SARS-CoV-2 (seroprevalence) as well as the extent of undetected infections. The ‘CORONA-MONITORING bundesweit’ study (RKI-SOEP study) collects biospecimens and interview data in a nationwide population sample drawn from the German Socio-Economic Panel (SOEP). Participants are sent materials to self-collect a dry blood sample of capillary blood from their finger and a swab sample from their mouth and nose, as well as a questionnaire. The samples returned are tested for SARS-CoV-2 IgG antibodies and SARS-CoV-2 RNA to identify past or present infections. The methods applied enable the identification of SARS-CoV-2 infections, including those that previously went undetected. In addition, by linking the data collected with available SOEP data, the study has the potential to investigate social and health-related differences in infection status. Thus, the study contributes to an improved understanding of the extent of the epidemic in Germany, as well as identification of target groups for infection protection

Statement on Advancing the Assessment of Chemical Mixtures and their Risks for Human Health and the Environment

The number of anthropogenic chemicals, manufactured, by-products, metabolites and abiotically formed transformation products, counts to hundreds of thousands, at present. Thus, humans and wildlife are exposed to complex mixtures, never one chemical at a time and rarely with only one dominating effect. Hence there is an urgent need to develop strategies on how exposure to multiple hazardous chemicals and the combination of their effects can be assessed. A workshop, “Advancing the Assessment of Chemical Mixtures and their Risks for Human Health and the Environment” was organized in May 2018 together with Joint Research Center in Ispra, EU-funded research projects and Commission Services and relevant EU agencies. This forum for researchers and policy-makers was created to discuss and identify gaps in risk assessment and governance of chemical mixtures as well as to discuss state of the art science and future research needs. Based on the presentations and discussions at this workshop we want to bring forward the following Key Messages: We are at a turning point: multiple exposures and their combined effects require better management to protect public health and the environment from hazardous chemical mixtures. Regulatory initiatives should be launched to investigate the opportunities for all relevant regulatory frameworks to include prospective mixture risk assessment and consider combined exposures to (real-life) chemical mixtures to humans and wildlife, across sectors. Precautionary approaches and intermediate measures (e.g. Mixture Assessment Factor) can already be applied, although, definitive mixture risk assessments cannot be routinely conducted due to significant knowledge and data gaps. A European strategy needs to be set, through stakeholder engagement, for the governance of combined exposure to multiple chemicals and mixtures. The strategy would include research aimed at scientific advancement in mechanistic understanding and modelling techniques, as well as research to address regulatory and policy needs. Without such a clear strategy, specific objectives and common priorities, research, and policies to address mixtures will likely remain scattered and insufficient

Seroepidemiologische Studie zur bundesweiten Verbreitung von SARS-CoV-2 in Deutschland: Studienprotokoll von CORONA-MONITORING bundesweit (RKI-SOEP-Studie)

Das Coronavirus SARS-CoV-2 hat sich in kurzer Zeit bundesweit ausgebreitet. In den Meldedaten der Gesundheitsämter zu laborbestätigten Infektionsfällen ist von einer Untererfassung des Infektionsgeschehens auszugehen, da Infektionen häufig unentdeckt bleiben, zum Beispiel weil sie symptomarm verlaufen. In seroepidemiologischen Studien kann der Bevölkerungsanteil mit durchgemachter SARS-CoV-2-Infektion (Seroprävalenz) wie auch der Umfang unentdeckter Infektionen abgeschätzt werden. In der Studie CORONA-MONITORING bundesweit (RKI-SOEP-Studie) werden Bioproben und Befragungsdaten in einer deutschlandweiten Bevölkerungsstichprobe des Sozio-oekonomischen Panels (SOEP) erhoben. Den Teilnehmenden werden Materialien zur selbstständigen Gewinnung einer Trockenblutprobe aus Kapillarblut des Fingers und einer Abstrichprobe aus Mund und Nase sowie ein Fragebogen postalisch zugesendet. Die zurückgesendeten Proben werden auf SARS-CoV-2-IgG-Antikörper und SARS-CoV-2-RNA zur Identifikation einer durchgemachten oder aktuellen Infektion untersucht. Die eingesetzten Methoden ermöglichen es, auch solche SARS-CoV-2-Infektionen zu erkennen, die bislang unentdeckt blieben. Durch die Verknüpfung mit bereits vorhandenen SOEP-Daten hat die Studie das Potenzial, auch soziale und gesundheitsbezogene Unterschiede im Infektionsstatus zu untersuchen. So kann die Studie zu einem verbesserten Verständnis des Ausmaßes der Epidemie in Deutschland wie auch zur Identifikation von Zielgruppen für den Infektionsschutz beitragen