A Normal and Biotransforming Model of the Human Bronchial Epithelium for the Toxicity Testing of Aerosols and Solubilised Substances

Zoë C. Prytherch and Kelly A. BéruBé

In this article, we provide an overview of the experimental workflow by the Lung and Particle Research Group at Cardiff University, that led to the development of the two in vitro lung models — the normal human bronchial epithelium (NHBE) model and the lung–liver model, Metabo-Lung™. This work was jointly awarded the 2013 Lush Science Prize. The NHBE model is a three-dimensional, in vitro, human tissue-based model of the normal human bronchial epithelium, and Metabo-Lung involves the co-culture of the NHBE model with primary human hepatocytes, thus permitting the biotransformation of inhaled toxicants in an in vivo-like manner. Both models can be used as alternative test systems that could replace the use of animals in research and development for safety and toxicity testing in a variety of industries (e.g. the pharmaceutical, environmental, cosmetics, and food industries). Metabo-Lung itself is a unique tool for the in vitro detection of toxins produced by reactive metabolites. This 21st century animal replacement model could yield representative in vitro predictions for in vivo toxicity. This advancement in in vitro toxicology relies on filter-well technology that will enable a wide-spectrum of researchers to create viable and economic alternatives for respiratory safety assessment and disease-focused research.
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Alternative Methods for Skin Irritation Testing: the Current Status

Philip A. Botham, Lesley K. Earl, Julia H. Fentem, Roland Roguet and Johannes J.M. van de Sandt

The ECVAM Skin Irritation Task Force was established in November 1996, primarily to prepare a report on the current status of the development and validation of alternative tests for skin irritation and corrosion and, in particular, to identify any appropriate non-animal tests for predicting human skin irritation which were sufficiently well-developed to warrant ECVAM supporting their prevalidation/validation. The task force based its discussions around the proposed testing strategy for skin irritation/corrosion emanating from an OECD workshop held in January 1996. The following have been reviewed: a) structure-activity and structure-property relationships for skin corrosion and irritation; b) the use of pH and acid/alkaline reserve measurements in predicting skin corrosivity; c) in vitro tests for skin corrosion; d) in vitro tests for skin irritation (keratinocyte cultures, organ cultures, and reconstituted human skin models); and e) human patch tests for skin irritation. It was apparent that, although several promising candidate in vitro tests for skin irritation (for example, reconstituted human skin methods, and human and animal skin organ culture methods) were under development and evaluation, a test protocol, a preliminary prediction model and supporting data on different types of chemicals were only available for a method employing EpiDermTM. Thus, it is proposed that this EpiDerm test undergoes prevalidation during 1998. In addition, since it was felt preferable to be able to include other in vitro tests in such a prevalidation study, it is recommended that a “challenge” be set to anyone interested in taking part. This involves submitting data on ten test chemicals selected by the task force, obtained according to a standard protocol with a preliminary prediction model, for review by the task force by 31 May 1998.
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Detection of Electrophysiological Indicators of Neurotoxicity in Human and Rat Brain Slices by a Three-Dimensional Microelectrode Array

Rüdiger Köhling, Raffaella Melani, Uwe Koch, Erwin-Josef Speckmann, Milena Koudelka-Hep, Pierre Thiébaud and Maurizio Balestrino

Electrophysiological techniques for the assessment of in vitro neurotoxicology have several advantages over other currently-used methods (for example, morphological techniques), including the ability to detect damage at a very early stage. Novel recording techniques based on microelectrode arrays are available, and could improve recording power. In this study, we investigated how a three-dimensional microelectrode array detects the electrophysiological endpoints of neurotoxicity. We conclude that electrophysiology sensitively reveals neurotoxic actions, and that three-dimensional microelectrode arrays could be proposed for use in neurotoxicology as recording tools that allow easy and sensitive multisite recording, from both rodent and human brain tissue.
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Immunotoxicology: Opportunities for Non-animal Test Development

Emanuela Corsini and Erwin L. Roggen

At present, several animal-based assays are used to assess immunotoxic effects such as immunosuppression and sensitisation. The use of whole animals, however, presents several secondary issues, including expense, ethical concerns and relevance to human risk assessment. There is a growing belief that non-animal approaches can eliminate these issues without impairing human safety, provided that biological markers are available to identify the immunotoxic potentials of new chemicals to which humans may be exposed. Driven by the 7th Amendment to the EU Cosmetics Directive, the new EU policy on chemicals (the REACH system), proposals to update the European legislation on the protection of animals used in research, and emerging visions and strategies for predicting toxicity, such in vitro methods are likely to play a major role in the near future. The realisation that the immune system can be the target of many chemicals, resulting in a range of adverse effects on the host’s health, has raised serious concerns from the public and within the regulatory agencies. Hypersensitivity and immunosuppression are considered the primary focus for developing in vitro methods in immunotoxicology. However, in vitro assays to detect immunostimulation and autoimmunity are also needed. This review of the state-of-the-art in the field of in vitro immunotoxicity, reveals a lack of cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way.
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Alternatives for Lung Research: Stuck Between a Rat and a Hard Place

Kelly A. BéruBé

The respiratory system acts as a portal into the human body for airborne materials, which may gain access via the administration of medicines or inadvertently during inhalation of ambient air (e.g. air pollution). The burden of lung disease has been continuously increasing, to the point where it now represents a major cause of human morbidity and mortality worldwide. In the UK, more people die from respiratory disease than from coronary heart disease or non-respiratory cancer. For this reason alone, gaining an understanding of mechanisms of human lung biology, especially in injury and repair events, is now a principal focus within the field of respiratory medicine. Animal models are routinely used to investigate such events in the lung, but they do not truly reproduce the responses that occur in humans. Scientists committed to the more robust Three Rs principles of animal experimentation (Reduction, Refinement and Replacement) have been developing viable alternatives, derived from human medical waste tissues from patient donors, to generate in vitro models that resemble the in vivo human lung environment. In the specific case of inhalation toxicology, human-oriented models are especially warranted, given the new REACH regulations for the handling of chemicals, the rising air pollution problems and the availability of pharmaceutically valuable drugs. Advances in tissue- engineering have made it feasible and cost-effective to construct human tissue equivalents of the respiratory epithelia. The conducting airways of the lower respiratory system are a critical zone to recapitulate for use in inhalation toxicology. Three-dimensional (3- D) tissue designs which make use of primary cells, provide more in vivo-like responses, based on the targeted interactions of multiple cell types supported on artificial scaffolds. These scaffolds emulate the native extracellular matrix, in which cells differentiate into a functional pulmonary tissue. When 3-D cell cultures are employed for testing aerosolised chemicals, drugs and xenobiotics, responses are captured that mirror the events in the in situ human lung and provide human endpoint data.
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