Workshop of the Transatlantic Think Tank for Toxicology (t4)
Title: Biomarkers in in vitro systems, Selection of the most appropriate invitro assays and their use in hazard and risk assessment
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Date and location:
February, 2011, Utrecht, The Netherlands
Goals of the workshop:
This workshop aims at an in-depth exploration of the possibilities to use invitro derived biomarkers for toxicity in the risk assessment process.
The current practice in toxicological risk assessment is to assess human health or environmental risk of chemicals on the basis of clinical or histopathological endpoints in animal studies. This use of these animal models and the distress toxicity testing inevitably evokes on the animals has resulted in extensive discussions on the ethical feasibility of the current process of toxicity testing. Apart from the ethical objections against the use of animals, there is also a scientific motivation for criticising these models. The use of these data to predict the biological activities of compounds in humans is always prone to some degree of uncertainty, due to the differences in kinetics and dynamics between the animal models and man. The apical clinical endpoints do also not take into account the most relevant mechanisms of toxicity.
This has led to a shift in research activities that were mainly focusing on these toxic mechanisms. This is based on the concept of the ability of a chemical to interact with relevant sites or processes in a living organism. Mechanism of toxic action is here defined as being the primary chemico-biological interaction between the compound and a structural moiety in the biological system (viz. in or on a cell, a tissue or an organ). The consequently occurring functional and structural changes within the biological system, including the resulting clinically observable changes in the organism are then collectively referred to as the toxicologically relevant mode of action.
This has resulted in a (re)definition of the paradigm of toxicology: the toxicity of a compound is determined by its effect - or the effect of a bio-activated metabolite - on the most critical target in the biological system. This effect in turn is governed by the dosimetry of the compound– or its metabolite – at the site of action. Depending on the nature of the interaction, this dosimetry can either be described by the course of the concentration over time, or a peak concentration, or a concentration above a certain threshold, etc. These three elements: critical dosimetry, critical compound (viz. parent or metabolite), critical site of action, will have to be the basis of our understanding of the toxicity of a chemical, together with comprehending the physiological relevance of these interactions.
It will be clear that more precise data on the mechanisms and modes of action will not easily be obtained from studying the apical endpoints in animal studies. This has led to the development of in vitro methods.
Over the last decades an increasing number of test systems for evaluating the possible toxicological hazard of chemical compounds have been developed that make use of biological systems on a lower level of organisation than the organism: isolated organs, cell cultures, sub-cellular systems. These in vitro systems have been very useful in studying the mechanism(s) of toxic action. Other important developments have been achieved in the prediction of biological reactivity on the basis of a compound’s physico-chemical properties, such as structure, molecular size, reactive groups, etc. One application of this knowledge is in the construction of structure-activity relationships.
However, the use of in vitro toxicity data for the risk assessment of a chemical highly depends on the relevance of the in vitro derived data and the possibility to use these data in an in vitro-in vivo extrapolation (IVIVE). A difficulty in the use of in vitro systems is the lack of biokinetic considerations in many of these approaches.
Another critical issue is the selection of the relevant in vitro system and the choice of the appropriate biological parameter for the prediction of the most relevant toxicity parameters. For some endpoints there is some experience in prediction the most appropriate endpoint on the basis of structural properties of the compound under study, e.g. by making use of systems such as DEREK, HazardExpert, TOPKAT, and MultiCase, but this is not the case for all endpoints and also not easily quantifiable.
One of the most challenging issues, especially if the need to have a highthroughput for chemical testing is at stake, is the improvement of our possibilities to select the most appropriate biomarker for toxicity. In invitro systems early cellular responses can be studied that may form the basis for a prediction of toxic responses in the in vivo situation. Examples of early cellular responses are: oxidative stress and glutathione homeostasis, cellular stress responses, changes in enzyme activities, cytokine responses, etc. The increasing possibilities to use cell and tissue cultures to measure these biomarkers of effect are now becoming complemented by the potential use of information derived from genomics, transcriptomics and proteomics.