Sex Differences in Liver Toxicity—Do Female and Male Human Primary Hepatocytes React Differently to Toxicants In Vitro?

There is increasing amount of evidence for sex variation in drug efficiency and toxicity profiles. Women are more susceptible than men to acute liver injury from xenobiotics. In general, this is attributed to sex differences at a physiological level as well as differences in pharmacokinetics and pharmacodynamics, but neither of these can give a sufficient explanation for the diverse responses to xenobiotics. Existing data are mainly based on animal models and limited data exist on in vitro sex differences relevant to humans. To date male and female human hepatocytes have not yet been compared in terms of their responses to toxicants. We investigated whether sex-specific differences in acute hepatotoxicity can be observed in vitro at a cellular level by comparing hepatotoxic drug effects in male and female primary human hepatocytes. Significant sex-related differences were found for particular parameters and individual drugs showing that the tested substances are more toxic to female hepatocytes. Moreover, our work demonstrated that high content screening is feasible with pooled primary human hepatocytes in suspension.JRC.I.5-Systems Toxicolog

A biology-based dynamic approach for the modelling of toxicity in cell-based assays. Part I: Fate modelling

There is a need to integrate existing in vitro dose-response data in a coherent framework for extending their domain of applicability as well as their extrapolation potential. This integration would contribute towards the reduction of animal use in toxicology by using in vitro data for quantitative risk assessment; moreover it would reduce costs and time especially when such approaches would be used for dealing with complex human health and ecotoxicological endpoints. In this work, based on HTS (High Throughput Screening) in vitro data, we have assessed the advantages that a dynamic biology-toxicant fate coupled model for in vitro cell-based assays could provide when assessing toxicity data, in particular, the possibility to obtain the dissolved (free) concentration which can help in raking the toxicity potency of a chemical and improve data reconciliation from several sources taking into account the inherent variability of cell-based assays. The results show that this approach may open a new way of analyzing this type of data sets and of extrapolating the values obtained to calculate in vivo human toxicology thresholds.JRC.DG.I.6-Systems toxicolog

Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

JRC.I.5-Systems Toxicolog

Adverse Outcome Pathway-based Screening Strategies for an Animal-free Safety Assessment of Chemicals

Currently the assessment of risk to human health from exposure to manufactured chemicals is mainly based on experiments performed on living animals (in vivo). Substantial efforts are being undertaken to develop alternative solutions to in vivo toxicity testing. Behind these efforts is an emerging paradigm-shift towards systems- and pathway-based approaches that rely on the understanding of biological processes that might be disturbed by the introduction of a chemical into a living organism which may lead to an adverse outcome. This new paradigm based on the Mode-of-Action (MoA) framework postulates that any adverse human health effect caused by exposure to an exogenous substance can be described by a series of causally linked biochemical or biological key events with measurable parameters. The elaboration of mechanistic knowledge through literature research is necessary for a MoA-driven design of integrated testing strategies using in vitro methods for in vivo predictions. The objective of our on-going research is to demonstrate the feasibility of an integrated approach to predict human toxicity following the Adverse Outcome Pathway (AOP) framework. In our publication: ‘Taking a mode-of-action approach to designing a hepatotoxicity screening strategy using the HepaRG cell model and high content imaging’ we reported our experiments related to our design of an in vitro testing strategy to identify chemicals that are potentially hepatotoxic in humans with the purpose of associating them with specific MoA categories and to group them accordingly. This pioneered an innovative way of using data from in vitro experiments to group chemicals based on their MoA, which is likely to be an important step in a toxicity testing strategy. This contribution towards the goal of replacing animals in safety testing was awarded the Lush Science Prize 2012.JRC.I.5-Systems Toxicolog

A human pluripotent carcinoma stem cell-based model for in vitrodevelopmental neurotoxicity testing: Effects of methylmercury, leadand aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After the characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity then was evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial) in the control culture and in the cells exposed to the compounds. The obtained results suggest that lead chloride and aluminum nitrate at low concentrations were toxic primary to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The obtained results suggest that neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of the potential neurotoxic compounds.JRC.I.2-Public Health Policy Suppor

Why women experience more adverse drug reactions than men?

<p>Why women experience more adverse drug reactions than men?</p

Practical use of the Virtual Cell Based Assay: Simulation of repeated exposure experiments in liver cell lines

The Virtual Cell Based Assay (VCBA) was applied to simulate the long-term (repeat dose) toxic effects of chemicals, including substances in cosmetics and personal care products. The presented model is an extension of the original VCBA for simulation of single exposure and is implemented in a KNIME workflow. This work illustrates the steps taken to simulate the repeated dose effects of two reference compounds, caffeine and amiodarone. Using caffeine, in vitro experimental viability data in single exposure from two human liver cell lines, HepG2 and HepaRG, were measured and used to optimize the VCBA, subsequently repeated exposure simulations were run. Amiodarone was then tested and simulations were performed under repeated exposure conditions in HepaRG. The results show that the VCBA can adequately predict repeated exposure experiments in liver cell lines. The refined VCBA model can be used not only to support the design of long term in vitro experiments but also practical applications in risk assessment. Our model is a step towards the development of in silico predictive approaches to replace, refine, and reduce the in vivo repeated dose systemic toxicity studies in the assessment of human safety.JRC.F.3-Chemicals Safety and Alternative Method

Cytotoxicity dose response curves.

<p>5000 human primary hepatocytes pooled from either 12 male donors (M), or 12 pre-menopausal female donors (3F), or 12 post-menopausal female donors (4F) were seeded in 96 well plates and exposed to 8 serial concentrations of Diclofenac (from 13.6 to 1750 uM), Chlorpromazine (from 1.9 to 250 uM), Acetaminophen (from 0.3 to 35 uM), and Verapamil (from 7.8 to 1000 uM). ROS formation (DHE dye), endoplasmic reticulum status (ER tracker red dye), mitochondrial damage (TMRE dye), and plasma membrane permeability (TOTO3 dye) were measured at different time points (30 min, 2h, 3h, 4h, 5h) using the Cellomics ArrayScan VTI platform and the Target Activation Bioapplication v.4. Representative dose-response curves are shown for drugs having a P value<0.05 (M vs 3F vs 4F) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122786#pone.0122786.t003" target="_blank">Table 3</a> (Diclofenac, Chlorpromazine, Acetaminophen, and Verapamil). Three replicates were tested and represented using GraphPad Prism. Results are expressed as mean ±SEM.</p