Quantitative global sensitivity analysis of a biologically based dose-response pregnancy model for the thyroid endocrine system

A deterministic biologically based dose-response model for the thyroidal system in a near-term pregnant woman and the fetus was recently developed to evaluate quantitatively thyroid hormone perturbations. The current work focuses on conducting a quantitative global sensitivity analysis on this complex model to identify and characterize the sources and contributions of uncertainties in the predicted model output. The workflow and methodologies suitable for computationally expensive models, such as the Morris screening method and Gaussian Emulation processes, were used for the implementation of the global sensitivity analysis. Sensitivity indices, such as main, total and interaction effects, were computed for a screened set of the total thyroidal system descriptive model input parameters. Furthermore, a narrower sub-set of the most influential parameters affecting the model output of maternal thyroid hormone levels were identified in addition to the characterization of their overall and pair-wise parameter interaction quotients. The characteristic trends of influence in model output for each of these individual model input parameters over their plausible ranges were elucidated using Gaussian Emulation processes. Through global sensitivity analysis we have gained a better understanding of the model behavior and performance beyond the domains of observation by the simultaneous variation in model inputs over their range of plausible uncertainties. The sensitivity analysis helped identify parameters that determine the driving mechanisms of the maternal and fetal iodide kinetics, thyroid function and their interactions, and contributed to an improved understanding of the system modeled. We have thus demonstrated the use and application of global sensitivity analysis for a biologically based dose-response model for sensitive life-stages such as pregnancy that provides richer information on the model and the thyroidal system modeled compared to local sensitivity analysis

The aggregatibacter actinomycetemcomitans heat shock protein GroEL interacts directly with human peripheral blood T cells

Heat shock family protein GroEL of Aggregatibacter actinomycetemcomitans (Aa) has antigenic properties. We previously demonstrated that A. actinomycetemcomitans GroEL-like protein affects human CD4 T cells by converting them into IL-10 and IFNg double cytokine producing Tbet+ Th1 cells. The objective of this study was to investigate whether or not AaGroEL communicates with T cells directly. To do this, sorted cells from peripheral blood mononuclear cells were stimulated with AaGroEL for 48 h. Flow cytometry was used to measure soluble and intracellular cytokine expression in the cell cultures and detect TLR2 expression on the surface of T cells. Expression of six different soluble cytokines was evaluated by CBA assay. To determine whether AaGroEL affects CD3+ T cells directly or not, purified CD3+ T cells or CD14+ cells were cultured with AaGroEL separately, and the quantity of soluble cytokine was measured. Results showed that sorted CD3+ cells produced soluble IL-6, TNFα-and IFNγ cytokines. Additionally, the intracellular cytokine staining data showed that AaGroEL-stimulated CD3+ cells were also TNFα-and IFNγ-positive. Moreover, AaGroEL-responsive T cells slightly increased their TLR2 expression. These findings suggest that CD3+ T cells produce cytokines in response to AaGroEL protein without requirements for other cells, such as CD14+ monocytes.Scientific and Technological Research Council of Turkey (TUBITAK 106T417

Mechanistic understanding of P-gp mediated transport and inhibition kinetics across a confluent monolayer of MDCKII-hMDR1 cells using a mass action kinetic model

The purpose of this work is to investigate several aspects of transporter-mediated interactions in a physiologically relevant model system and to gain a clear mechanistic understanding of the same using a mass action kinetic model. We have first shown that the parameters determining the potency of an inhibitor, IC50 & KI are not the same for P-gp and that IC50 overestimates KI. The quantitative relationship between them is derived and the physical mechanism of overestimation, explained. This sensitive modeling tool was further used for the kinetic identification of other transporters in addition to P-gp in several confluent cell monolayer systems. It was shown that a simple IC50 plot has more than one inhibition events convolved in them and the model was used to separate the individual contributions. This is of clinical relevance for the accurate assessment of transporter mediated drug-drug interaction risks. We have also derived an exact equation for measuring the passive permeability coefficients of compounds that show significant drug loss. Such measurements would help us carefully delineate the passive component from the total transport and describe the P-gp mediated transport of these compounds unambiguously. In summary, such an understanding of drug -transporter relations in vitro obtained as a part of this thesis work is valuable for making accurate predictions of drug disposition in vivo, aiding the ability to make informed decisions during the several stages of drug discovery and development.Ph.D., Biological Sciences -- Drexel University, 201

Pediatric Exposures to Ionizing Radiation: Carcinogenic Considerations

Children are at a greater risk than adults of developing cancer after being exposed to ionizing radiation. Because of their developing bodies and long life expectancy post-exposure, children require specific attention in the aftermath of nuclear accidents and when radiation is used for diagnosis or treatment purposes. In this review, we discuss the carcinogenic potential of pediatric exposures to ionizing radiation from accidental, diagnostic, and therapeutic modalities. Particular emphasis is given to leukemia and thyroid cancers as consequences of accidental exposures. We further discuss the evidence of cancers that arise as a result of radiotherapy and conclude the review with a summary on the available literature on the links between computer tomography (CT) and carcinogenesis. Appropriate actions taken to mitigate or minimize the negative health effects of pediatric exposures to ionizing radiation and future considerations are discussed

Capturing the applicability of in vitro-in silico membrane transporter data in chemical risk assessment and biomedical research

Costs, scientific and ethical concerns related to animal tests for regulatory decision-making have stimulated the development of alternative methods. When applying alternative approaches, kinetics have been identified as a key element to consider. Membrane transporters affect the kinetic processes of absorption, distribution, metabolism and excretion (ADME) of various compounds, such as drugs or environmental chemicals. Therefore, pharmaceutical scientists have intensively studied transporters impacting drug efficacy and safety. Besides pharmacokinetics, transporters are considered as major determinant of toxicokinetics, potentially representing an essential piece of information in chemical risk assessment. To capture the applicability of transporter data for kinetic-based risk assessment in non-pharmaceutical sectors, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) created a survey with a view of identifying the improvements needed when using in vitro and in silico methods. Seventy-three participants, from different sectors and with various kinds of expertise, completed the survey. The results revealed that transporters are investigated mainly during drug development, but also for risk assessment purposes of food and feed contaminants, industrial chemicals, cosmetics, nano materials and in the context of environmental toxicology, by applying both in vitro and in silico tools. However, to rely only on alternative methods for chemical risk assessment, it is critical that the data generated by in vitro and in silico methods are scientific integer, reproducible and of high quality so that they are trusted by decision makers and used by industry. In line, the respondents identified various challenges related to the interpretation and use of transporter data from non-animal methods. Overall, it was determined that a combined mechanistically-anchored in vitro-in silico approach, validated against available human data, would gain confidence in using transporter data within an animal-free risk assessment paradigm. Finally, respondents involved primarily in fundamental research expressed lower confidence in non-animal studies to unravel complex transporter mechanismsJRC.F.3-Chemicals Safety and Alternative Method

Membrane transporter data to support kinetically-informed chemical risk assessment using non-animal methods: Scientific and regulatory perspectives

Humans are continuously exposed to low levels of thousands of industrial chemicals, most of which are poorly characterised in terms of their potential toxicity. The new paradigm in chemical risk assessment (CRA) aims to rely on animal-free testing, with kinetics being a key determinant of toxicity when moving from traditional animal studies to integrated in vitro-in silico approaches. In a kinetically informed CRA, membrane transporters, which have been intensively studied during drug development, are an essential piece of information. However, how existing knowledge on transporters gained in the drug field can be applied to CRA is not yet fully understood. This review outlines the opportunities, challenges and existing tools for investigating chemical-transporter interactions in kinetically informed CRA without animal studies. Various environmental chemicals acting as substrates, inhibitors or modulators of transporter activity or expression have been shown to impact TK, just as drugs do. However, because pollutant concentrations are often lower in humans than drugs and because exposure levels and internal chemical doses are not usually known in contrast to drugs, new approaches are required to translate transporter data and reasoning from the drug sector to CRA. Here, the generation of in vitro chemical-transporter interaction data and the development of transporter databases and classification systems trained on chemical datasets (and not only drugs) are proposed. Furtheremore, improving the use of human biomonitoring data to evaluate the in vitro-in silico transporter-related predicted values and developing means to assess uncertainties could also lead to increase confidence of scientists and regulators in animal-free CRA. Finally, a systematic characterisation of the transportome (quantitative monitoring of transporter abundance, activity and maintenance over time) would reinforce confidence in the use of experimental transporter/barrier systems as well as in established cell-based toxicological assays currently used for CRA.JRC.F.3-Chemicals Safety and Alternative Method

Membrane transporter data to support kinetically-informed chemical risk assessment using non-animal methods Scientific and regulatory perspectives

International audienceHumans are continuously exposed to low levels of thousands of industrial chemicals, most of which are poorly characterised in terms of their potential toxicity. The new paradigm in chemical risk assessment (CRA) aims to rely on animal-free testing, with kinetics being a key determinant of toxicity when moving from traditional animal studies to integrated in vitro-in silico approaches. In a kinetically informed CRA, membrane transporters, which have been intensively studied during drug development, are an essential piece of information. However, how existing knowledge on transporters gained in the drug field can be applied to CRA is not yet fully understood. This review outlines the opportunities, challenges and existing tools for investigating chemical-transporter interactions in kinetically informed CRA without animal studies. Various environmental chemicals acting as substrates, inhibitors or modulators of transporter activity or expression have been shown to impact TK, just as drugs do. However, because pollutant concentrations are often lower in humans than drugs and because exposure levels and internal chemical doses are not usually known in contrast to drugs, new approaches are required to translate transporter data and reasoning from the drug sector to CRA. Here, the generation of in vitro chemical-transporter interaction data and the development of transporter databases and classification systems trained on chemical datasets (and not only drugs) are proposed. Furtheremore, improving the use of human biomonitoring data to evaluate the in vitro-in silico transporter-related predicted values and developing means to assess uncertainties could also lead to increase confidence of scientists and regulators in animal-free CRA. Finally, a systematic characterisation of the transportome (quantitative monitoring of transporter abundance, activity and maintenance over time) would reinforce confidence in the use of experimental transporter/barrier systems as well as in established cell-based toxicological assays currently used for CRA

Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

<div><p>We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC₅₀ measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.</p></div