The thyroid hormone (TH) system is involved in several important physiological processes, including regulation of energy metabolism, growth and differentiation, development and maintenance of brain ...function, thermo-regulation, osmo-regulation, and axis of regulation of other endocrine systems, sexual behaviour and fertility and cardiovascular function. Therefore, concern about TH disruption (THD) has resulted in strategies being developed to identify THD chemicals (THDCs). Information on potential of chemicals causing THD is typically derived from animal studies. For the majority of chemicals, however, this information is either limited or unavailable. It is also unlikely that animal experiments will be performed for all THD relevant chemicals in the near future for ethical, financial and practical reasons. In addition, typical animal experiments often do not provide information on the mechanism of action of THDC, making it harder to extrapolate results across species. Relevant effects may not be identified in animal studies when the effects are delayed, life stage specific, not assessed by the experimental paradigm (e.g., behaviour) or only occur when an organism has to adapt to environmental factors by modulating TH levels. Therefore, in vitro and in silico alternatives to identify THDC and quantify their potency are needed. THDC have many potential mechanisms of action, including altered hormone production, transport, metabolism, receptor activation and disruption of several feed-back mechanisms. In vitro assays are available for many of these endpoints, and the application of modern ‘-omics’ technologies, applicable for in vivo studies can help to reveal relevant and possibly new endpoints for inclusion in a targeted THDC in vitro test battery. Within the framework of the ASAT initiative (Assuring Safety without Animal Testing), an international group consisting of experts in the areas of thyroid endocrinology, toxicology of endocrine disruption, neurotoxicology, high-throughput screening, computational biology, and regulatory affairs has reviewed the state of science for (1) known mechanisms for THD plus examples of THDC; (2) in vitro THD tests currently available or under development related to these mechanisms; and (3) in silico methods for estimating the blood levels of THDC. Based on this scientific review, the panel has recommended a battery of test methods to be able to classify chemicals as of less or high concern for further hazard and risk assessment for THD. In addition, research gaps and needs are identified to be able to optimize and validate the targeted THD in vitro test battery for a mechanism-based strategy for a decision to opt out or to proceed with further testing for THD.
Methods of in vitro toxicology Eisenbrand, G; Pool-Zobel, B; Baker, V ...
Food and chemical toxicology,
02/2002, Volume:
40, Issue:
2
Journal Article
Peer reviewed
In vitro methods are common and widely used for screening and ranking chemicals, and have also been taken into account sporadically for risk assessment purposes in the case of food additives. ...However, the range of food-associated compounds amenable to in vitro toxicology is considered much broader, comprising not only natural ingredients, including those from food preparation, but also compounds formed endogenously after exposure, permissible/authorised chemicals including additives, residues, supplements, chemicals from processing and packaging and contaminants. A major promise of in vitro systems is to obtain mechanism-derived information that is considered pivotal for adequate risk assessment. This paper critically reviews the entire process of risk assessment by in vitro toxicology, encompassing ongoing and future developments, with major emphasis on cytotoxicity, cellular responses, toxicokinetics, modelling, metabolism, cancer-related endpoints, developmental toxicity, prediction of allergenicity, and finally, development and application of biomarkers. It describes in depth the use of in vitro methods in strategies for characterising and predicting hazards to the human. Major weaknesses and strengths of these assay systems are addressed, together with some key issues concerning major research priorities to improve hazard identification and characterisation of food-associated chemicals.
Abstract Challenges to improve toxicological risk assessment to meet the demands of the EU chemical's legislation, REACH, and the EU 7th Amendment of the Cosmetics Directive have accelerated the ...development of non-animal based methods. Unfortunately, uncertainties remain surrounding the power of alternative methods such as in vitro assays to predict in vivo dose–response relationships, which impedes their use in regulatory toxicology. One issue reviewed here, is the lack of a well-defined dose metric for use in concentration-effect relationships obtained from in vitro cell assays. Traditionally, the nominal concentration has been used to define in vitro concentration–effect relationships. However, chemicals may differentially and non-specifically bind to medium constituents, well plate plastic and cells. They may also evaporate, degrade or be metabolized over the exposure period at different rates. Studies have shown that these processes may reduce the bioavailable and biologically effective dose of test chemicals in in vitro assays to levels far below their nominal concentration. This subsequently hampers the interpretation of in vitro data to predict and compare the true toxic potency of test chemicals. Therefore, this review discusses a number of dose metrics and their dependency on in vitro assay setup. Recommendations are given on when to consider alternative dose metrics instead of nominal concentrations, in order to reduce effect concentration variability between in vitro assays and between in vitro and in vivo assays in toxicology.
Toxicity of a compound for an organism is dependent on the route of exposure, the amount (or concentration), the way in which the compound is taken up, distributes and is eliminated from the organism ...(ADME, kinetics) and the intrinsic properties (reactivity; mode of action, dynamics) of the compound towards the organism. These three elements: exposure, kinetics and dynamics form the basis of hazard and risk evaluations. Developments in our knowledge of the way in which physico-chemical properties of chemicals (on the one side) and physiological processes in the organism (on the other side) determine a compound's toxicity have greatly increased our understanding of toxicological processes and our ability to interpret experimental results. This has now resulted in the development of model systems in which the above-mentioned processes can be described mathematically. Biokinetic modelling is currently of great interest, but the further development of toxicodynamic modelling is equally important. The combination of both allows the estimation of a compound's critical amount/concentration on the critical site of action, which ideally would be the basis for hazard and risk assessments. In vitro systems have been extremely useful in studying the molecular basis of a chemical's biological activity, including its mechanism(s) of toxic action. Other achievements include the prediction of biological reactivity on the basis of a compound's physico-chemical properties and the construction of quantitative structure–activity relationships (QSARs). However, for the incorporation of in vitro-derived data as well as the results of QSARs, kinetic modelling is indispensable. Thus, biokinetic and toxicodynamic modelling are important (if not crucial) tools in toxicological research and there are increasing opportunities to incorporate the results of this work in hazard and risk assessments. Their implementation will allow a much more scientifically-based and a better structured risk assessment, which will be to a much lesser extent relying on animal experimentation.
For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in ...chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.
This paper discusses the role of
in vitro toxicology in hazard and risk assessment. A short historical overview of the development of the field of
in vitro toxicology is given, showing the ...possibilities, as well as the limitation for using
in vitro methods in screening and in studying mechanisms of toxicity. Although the development of the use of
in vitro methods over the last two or three decades is vast, the application in risk assessment is still limited. The need for a more integrated approach, i.e. using
in vitro data not in isolation, but in combination with data on structure–activity and
in silico biokinetic models is discussed. It is foreseen that the role of
in vitro methods in future risk assessments will greatly enhance, also in the light of recent developments in technologies such as genomics and systems biology.
Abstract The aryl hydrocarbon receptor (AhR) is involved in a wide variety of biological and toxicological responses, including neuroendocrine signaling. Due to the complexity of neuroendocrine ...pathways in e.g. the hypothalamus and pituitary, there are limited in vitro models available despite the strong demand for such systems to study and predict neuroendocrine effects of chemicals. In this study, the applicability of the AhR-expressing rat hypothalamic GnV-3 cell line was investigated as a novel model to screen for neuroendocrine effects of AhR ligands using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as reference compound. The qRT-PCR analyses demonstrated the presence of several sets of neurotransmitter receptors in the GnV-3 cells. TCDD (10 nM) altered neurotransmitter signaling by up-regulation of glutamate ( Grik2 ), gamma-amino butyric acid ( Gabra 2 ) and serotonin ( Ht 2C ) receptor mRNA levels. However, no significant changes in basal and serotonin-evoked intracellular Ca2+ concentration (Ca2+ i ) or serotonin release were observed. On the other hand, TCDD de-regulated period circadian protein homolog 1 ( Per1 ) and gonadotropin releasing hormone ( Gnrh ) mRNA levels within a 24-h time period. Both Per1 and Gnrh genes displayed a similar mRNA expression pattern in GnV-3 cells. Moreover, the involvement of AhR in TCDD-induced alteration of Neuropeptide Y ( Npy ) gene expression was found and confirmed by using siRNA targeted against Ahr in GnV-3 cells. Overall, the combined results demonstrate that GnV-3 cells may be a suitable model to predict some mechanisms of action and effects of AhR ligands in the hypothalamus.
Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ...ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD in vitro methods undergo appropriate validation and become accepted in the regulatory arena.
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► Medium concentrations decreased due to uptake into the cells. ► This decrease was dependent on the cell density and the test concentrations used. ► When the medium contains serum, ...the free concentration should be considered as a dose metric. ► The sensitivity ranking of the tested cell systems was dependent on the dose metric used.
The extrapolation of in vitro to in vivo toxicity data is a challenge. Differences in sensitivity between cell systems may be due to intrinsic properties of the cell but also because of differences in exposure. In this study, the cytotoxicity and biokinetics of the antipsychotic chlorpromazine (CPZ) were studied in in vitro assays using different cell types and exposure conditions. Different dose metrics were assessed to express the sensitivity to CPZ.
The biokinetics of CPZ were measured in cell cultures of Balb/c 3T3, Caco-2 and HepaRG cells. Cytotoxicity was measured by Alamar Blue and expressed using different dose metrics, including the nominal, measured total and measured free CPZ medium concentrations.
CPZ was taken up by the cells; the highest amounts in the cell compartments were found in the Caco-2 and HepaRG cells. CPZ was highly protein-bound in the Caco-2 cell medium containing 10% fetal bovine serum, resulting in lower bioavailable exposure concentrations. Moreover, also uptake into the cells strongly influenced the concentration in the medium. The Balb/c 3T3 cells were the most sensitive to the toxic effect of CPZ. The use of different dose metrics influenced the cytotoxicity results found in the three cell types. The data show that in comparing the sensitivity of the tested cell systems, the freely dissolved concentration is a more appropriate dose metric than total concentration in the medium. The ranking in sensitivity of the three cell types for CPZ was dependent on the dose metric used.
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