EURL ECVAM Workshop on New Generation of Physiologically-Based Kinetic Models in Risk Assessment

The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) Strategy Document on Toxicokinetics (TK) outlines strategies to enable prediction of systemic toxicity by applying new approach methodologies (NAM). The central feature of the strategy focuses on using physiologically-based kinetic (PBK) modelling to integrate data generated by in vitro and in silico methods for absorption, distribution, metabolism, and excretion (ADME) in humans for predicting whole-body TK behaviour, for environmental chemicals, drugs, nano-materials, and mixtures. In order to facilitate acceptance and use of this new generation of PBK models, which do not rely on animal/human in vivo data in the regulatory domain, experts were invited by EURL ECVAM to (i) identify current challenges in the application of PBK modelling to support regulatory decision making; (ii) discuss challenges in constructing models with no in vivo kinetic data and opportunities for estimating parameter values using in vitro and in silico methods; (iii) present the challenges in assessing model credibility relying on non-animal data and address strengths, uncertainties and limitations in such an approach; (iv) establish a good kinetic modelling practice workflow to serve as the foundation for guidance on the generation and use of in vitro and in silico data to construct PBK models designed to support regulatory decision making. To gauge the current state of PBK applications, experts were asked upfront of the workshop to fill a short survey. In the workshop, using presentations and discussions, the experts elaborated on the importance of being transparent about the model construct, assumptions, and applications to support assessment of model credibility. The experts offered several recommendations to address commonly perceived limitations of parameterization and evaluation of PBK models developed using non-animal data and its use in risk assessment, these include: (i) develop a decision tree for model construction; (ii) set up a task force for independent model peer review; (iii) establish a scoring system for model evaluation; (iv) attract additional funding to develop accessible modelling software.; (v) improve and facilitate communication between scientists (model developers, data provider) and risk assessors/regulators; and (vi) organise specific training for end users. The experts also acknowledged the critical need for developing a guidance document on building, characterising, reporting and documenting PBK models using non-animal data. This document would also need to include guidance on interpreting the model analysis for various risk assessment purposes, such as incorporating PBK models in integrated strategy approaches and integrating them with in vitro toxicity testing and adverse outcome pathways. This proposed guidance document will promote the development of PBK models using in vitro and silico data and facilitate the regulatory acceptance of PBK models for assessing safety of chemicals

Enhanced Uridine Bioavailability Following Administration of a Triacetyluridine-Rich Nutritional Supplement

Uridine is a therapy for hereditary orotic aciduria and is being investigated in other disorders caused by mitochondrial dysfunction, including toxicities resulting from treatment with nucleoside reverse transcriptase inhibitors in HIV. Historically, the use of uridine as a therapeutic agent has been limited by poor bioavailability. A food supplement containing nucleosides, NucleomaxX®, has been reported to raise plasma uridine to supraphysiologic levels

Desmoglein 3, via an Interaction with E-cadherin, Is Associated with Activation of Src

Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion.To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation.Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation

Detection of microRNA Expression in Human Peripheral Blood Microvesicles

MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis.Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function.This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases