biomarkers

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The Use of Biomarkers as Alternatives to Current Animal Tests on Food Chemicals1

Krys Bottrill

Recent developments in biomarkers relating to the interrelationship of diet, disease and health were surveyed. Most emphasis was placed on biomarkers of deleterious effects, since these are of greatest relevance to the subject of this review. The area of greatest activity was found to be that relating to biomarkers of mutagenic, genotoxic and carcinogenic effects. This is also one of the major areas of concern in considerations of the beneficial and deleterious effects of dietary components, and also the area in which regulatory testing requires studies of the longest duration. A degree of progress has also been made in the identification and development of biomarkers relating to certain classes of target organ toxicity. Biomarkers for other types of toxicity, such as immunotoxicity, neurotoxicity, reproductive toxicity and developmental toxicity, are less developed, and further investigation in these areas is required before a comprehensive biomarker strategy can be established. A criticism that recurs constantly in the biomarker literature is the lack of standardisation in the methods used, and the lack of reference standards for the purposes of validation and quality control. It is encouraging to note the growing acknowledgement of the need for validation of biomarkers and biomarker assays. Some validation studies have already been initiated. This review puts forward proposals for criteria to be used in biomarker validation. More discussion on this subject is required. It is concluded that the use of biomarkers can, in some cases, facilitate the implementation of the Three Rs with respect to the testing of food chemicals and studies on the effects of diet on health. The greatest potential is seen to be in the refinement of animal testing, in which biomarkers could serve as early and sensitive endpoints, in order to reduce the duration of the studies and also reduce the number of animals required. Biomarkers could also contribute to establishing a mechanistic basis for in vitro test systems and to facilitating their validation and acceptance. Finally, the increased information that could result from the incorporation of biomarker determinations into population studies could reduce the need for supplementary animal studies. This review makes a number of recommendations concerning the prioritisation of future activities on dietary biomarkers in relation to the Three Rs. It is emphasised, however, that further discussions will be required among toxicologists, epidemiologists and others researching the relationship between diet and health.
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2017-01-09T06:26:45+00:00 Tags: , , , , |

Alternative Approaches and Tests in Ecotoxicology: A Review of the Present Position and the Prospects for Change, Taking into Account ECVAM’s Duties, Topic Selection and Test Criteria

Colin H. Walker

The objectives of this review are to summarise the present position concerning the use of vertebrates in ecotoxicity testing, giving particular attention to tests that cause suffering, and to discuss in some detail, alternatives to them, and the prospects for change. The report has been written with the objectives of the European Centre for the Validation of Alternative Methods (ECVAM) in mind, and some recommendations for action have been made at the end of the discussion section. The first section of the review describes the present requirements within the European Union for the ecotoxicity testing of industrial chemicals in general, and for pesticides in particular, and the very limited documentation of the tests that are actually carried out. The next four sections describe the many different assays and systems used to evaluate the harmful effects of chemicals on free-living organisms and natural populations, and the extent to which they might be suitable alternatives to vertebrate toxicity tests that cause suffering. Attention is drawn to certain assays and strategies that can already be used as satisfactory alternatives, and thus provide the basis for short-term change. Included here are nondestructive assays on vertebrates which are available for certain types of chemicals, and which provide a direct and relatively uncomplicated approach to the problem. Other approaches are described which still require development, but hold considerable promise in the longer term. The growth of knowledge in the broad field of biochemical toxicology and the development of related technologies should lead to the development of better and more-sophisticated alternatives in the future. In vitro assays employing vertebrate cell systems are of particular interest here. The last section of the review deals with conclusions and recommendations. The recommendations are made with a view to the activities and responsibilities of ECVAM.
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A Review of In Vitro Modelling Approaches to the Identification and Modulation of Squamous Metaplasia in the Human Tracheobronchial Epithelium

Alison C. Gray, Julie D. McLeod and Richard H. Clothier

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein–cadaverine (FC) into bis(γ-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.
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Ecotoxicity Testing: Science, Politics and Ethics

Colin H. Walker

Animal welfare organisations have long been concerned about the use of animals for ecotoxicity testing. Ecotoxicity testing is a necessary part of the statutory risk assessment of chemicals that may be released into the environment. It is sometimes also carried out during the development of new chemicals and in the investigation of pollution in the field. This review considers the existing requirements for ecotoxicity testing, with particular reference to practices in the European Union, including the recent REACH system proposals, before discussing criticisms that have been made of existing practices for environmental risk assessment. These criticisms have been made on scientific and ethical grounds, as well as on questions of cost. A case is made for greater investment in the development of alternative testing methods, which could improve the science, as well as serving the cause of animal welfare. It has frequently been suggested that the statutory requirements for environmental risk assessment are too rigid and bureaucratic. A case is made for flexibility and the greater involvement of scientists in the risk assessment procedure, in the interests of both improved science and improved animal welfare.
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