ATLA 32.2, June 2004

//ATLA 32.2, June 2004

Editorial: The UK National Centre for the Three Rs: Pathway to Progress or Mere Fig Leaf?

Michael Balls and Robert D. Combes

On 21 May 2004, Lord Sainsbury, Science and Innovation Minister, and Caroline Flint MP, Under-Secretary of State for the Home Department, jointly announced the establishment of a National Centre for the Replacement, Refinement and Reduction of Animals in Research, and a joint press release was issued by the Department of Trade & Industry and the Home Office.1,2
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2017-01-09T06:32:07+00:00

News & Views

ATLA Staff Writer

BUAV Appoints New Chief Executive Officer
Fourth World Congress Proceedings Published
InterNICHE Launches New Polish Life Science and Alternatives Website
The Björn Ekwall Memorial Award 2004
New National Contacts for InterNICHE
InterNICHE Humane Education Award 2003: Successful Applicants Chosen
Intervet Dieter Lütticken Award
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2017-01-09T06:32:07+00:00 Tags: |

ECVAM News & Views

ATLA Staff Writer

ECVAM Task Forces/Steering Groups
ECVAM’s Activities within the Joint Research Centre Enlargement Action
ECVAM Workshops
ECVAM–ICCVAM Collaboration
Seventh Amendment to the Cosmetics Directive
ECVAM’s Activities in Carcinogenicity Testing
Revision of Directive 86/609/EEC
PhD and Post-doctoral Fellowships at ECVAM
ECVAM Publications
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Ischaemia–Reperfusion Injury in the Isolated Haemoperfused Bovine Uterus: An In Vitro Model of Acute Inflammation

Michael Braun and Manfred Kietzmann

Following on from previous studies on dermal inflammation in the isolated perfused bovine udder, a new in vitro model of the isolated haemoperfused bovine uterus was established for studies on acute inflammatory reactions (for example, eicosanoid synthesis and regulation of cyclooxygenase-1 [COX-1] and COX-2) caused by ischaemia–reperfusion (I–R) injury. The organs and blood used in this study were obtained from a slaughterhouse. Within 2 hours of slaughter, uterine perfusion was re-established, by using a mixture of homologous blood and Tyrode solution (4:1). After equilibration, several deposits of arachidonic acid (5mg and 0.1mg) and arachidonylethanolamide (0.1mg) were injected into the myometrial tissue. Tissue biopsies were taken from treated and untreated areas at 180 and 300 minutes after the onset of haemoperfusion, for measuring prostaglandin E2 (PGE2) levels. In addition, the regulation of COX-1 and COX-2 mRNA was investigated by using the reverse transcriptase-polymerase chain reaction. Eicosanoid levels were determined by using an enzyme immunoassay (ELISA). Because both an increase in PGE2 concentration and up-regulation of COX mRNA were observed, the inhibitory effects of dexamethasone, added to the perfusion medium, were studied. Dexamethasone caused a significant decrease in tissue PGE2 production, but did not induce down-regulation of COX-2 mRNA. In conclusion, the isolated haemoperfused bovine uterus was introduced as an in vitro model of acute inflammation, induced by I–R injury. The suitability of the model for investigating anti-inflammatory substances was demonstrated. Use of the isolated haemoperfused bovine uterus in pharmacological research and drug screening may contribute to reducing the number of animals used for testing.
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In Vitro Pollen Tube Growth Reveals the Cytotoxic Potential of the Flavonols, Quercetin and Rutin

Fabiana Antognoni, Elisa Ovidi, Anna Rita Taddei, Gabriella Gambellini and Anna Speranza

Flavonols are phytochemicals widely found in commonly consumed foods. In spite of their beneficial effects on human health, however, cytotoxicity and even suspected genotoxicity have also been reported for the flavonol, quercetin. This points to the need for preventive studies to identify any cytotoxic effects associated with pure flavonol intake. This work was performed with the aim of verifying whether a plant-based in vitro system, the pollen tube, could be used to evaluate the cytotoxic potential of exogenous flavonols. Increasing concentrations of the aglycone, quercetin, and its glycoside, rutin, were assayed with regard to tube growth of kiwifruit pollen, determined by applying the pollen tube growth test protocol. This test, based on the photometric quantification of pollen tube mass production in suspension cultures, has already been applied in the sensitive and reliable toxicological evaluation of a wide range of chemicals. Whereas 60–800μM rutin promoted kiwifruit pollen tube elongation, 10–50μM quercetin strongly inhibited growth, and also produced irreversible malformations, such as screw-like tube growth, abnormal vacuolation, alteration of organelle streaming, and nuclear positioning. Thus, the cytotoxic potentials of the two flavonols have been confirmed to differ. Pollen tubes seem to afford a promising test system for a preventive, rapid in vitro biosafety assessment of antioxidant nutritional supplements, without using laboratory animals.
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The Development of a Standardised Protocol to Measure Squamous Differentiation in Stratified Epithelia, by using the Fluorescein Cadaverine Incorporation Technique

Alison C. Gray, Joanne Malton and Richard H. Clothier

Fluorescein cadaverine (FC) incorporation into cornified envelopes during squamous differentiation in stratified epithelia acts as a fluorescent substitute for endogenous transglutaminase substrates that can be visualised and quantified. The FC incorporation technique has been used to evaluate squamous differentiation in keratinocytes cultured in a medium that stimulates differentiation and in response to modulation by chemicals. A Standard Operating Procedure for the measurement of squamous differentiation is required as part of the prevalidation procedure for in vitro assays. In the present study, keratinocytes were isolated from the epidermis of 34 human donors. Cellular metabolic activity (resorufin production), total protein (kenacid blue uptake) and transglutaminase activity (FC incorporation) were measured in 87 and 21 independent runs at 6 and 12 days, respectively. Analysis of the control data showed that the cultures had a mean resorufin production that decreased over 12 days. This was inversely related to FC incorporation, which increased over 12 days. Mean protein concentration was reduced over the 12 days, but not in analyses that used donors for whom both 6-day and 12-day data were available. This information was used to define the normal limits within which the data should fall (mean ± 1 SD). Data sets falling outside the normal limits performed statistically no differently from the normal responders, in experiments conducted to investigate the effects of chemicals on the modulation of squamous differentiation in keratinocytes. This was demonstrated by using compounds that modify transglutaminase expression and/or activity. All-trans retinoic acid significantly inhibited FC incorporation, but stimulated resorufin production at 1 × 10–7M and above. Nicotine significantly up-regulated both FC incorporation and resorufin production at 125μg/ml. Hence, it was concluded that this robust assay approach, in which keratinocytes from a range of donors respond predictably to the test chemicals employed, did not justify the limitations that would be imposed by setting criteria that eliminated all data lying outside the normal range.
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Drug Metabolism by Cultured Human Hepatocytes: How Far Are We from the In Vivo Reality?

Xavier Ponsoda, M. Teresa Donato, Gabriela Perez-Cataldo, Maria José Gómez-Lechón and José V. Castell

The investigation of metabolism is an important milestone in the course of drug development. Drug metabolism is a determinant of drug pharmacokinetics variability in human beings. Fundamental to this are phenotypic differences, as well as genotypic differences, in the expression of the enzymes involved in drug metabolism. Genotypic variability is easy to identify by means of polymerase chain reaction-based or DNA chip-based methods, whereas phenotypic variability requires direct measurement of enzyme activities in liver, or, indirectly, measurement of the rate of metabolism of a given compound in vivo. There is a great deal of phenotypic variability in human beings, only a minor part being attributable to gene polymorphisms. Thus, enzyme activity measurements in a series of human livers, as well as in vivo studies with human volunteers, show that phenotypic variability is, by far, much greater than genotypic variability. In vitro models are currently used to investigate the hepatic metabolism of new compounds. Cultured human hepatocytes are considered to be the closest model to the human liver. However, the fact that hepatocytes are placed in a microenvironment that differs from that of the cells in the liver raises the question of to what extent drug metabolism variability observed in vitro actually reflects that in the liver in vivo. This issue has been examined by investigating the metabolism of the model compound, aceclofenac (an approved analgesic/anti-inflammatory drug), both in vitro and in vivo. Hepatocytes isolated from programmed liver biopsies were incubated with aceclofenac, and the metabolites formed were investigated by HPLC. The patients were given the drug during the course of clinical recovery, and the metabolites, largely present in urine, were analysed. In vitro and in vivo data from the same individual were compared. There was a good correlation between the in vitro and in vivo relative abundance of oxidised metabolites (4´-OH-aceclofenac + 4´-OH-diclofenac; Spearman’s ρ = 0.855), and the hydrolysis of aceclofenac (diclofenac + 4´-OH-aceclofenac + 4´-OH-diclofenac; ρ = 0.691), while the conjugation of the drug in vitro was somewhat lower than in vivo. Globally, the metabolism of aceclofenac in vitro correlated with the amount of metabolites excreted in urine after 16 hours (ρ = 0.95). Overall, although differing among assays, the in vitro/in vivo metabolism data for each patient were surprisingly similar. Thus, the variability observed in vitro appears to reflect genuine phenotypic variability among the donors.
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Peer Review of Validation Studies: An Assessment of the Role of the OECD by Reference to the Validation of the Uterotrophic Assay for Endocrine Disruptors

Robert D. Combes

The involvement of the OECD in managing the validation of the rat uterotrophic assay for endocrine disruptors, and in organising the peer review of the results of this study, has been assessed and compared with the many conclusions and recommendations in several published reports of international workshops on validation, and information in guidance documents, produced by the European Centre for the Validation of Alternative Methods (ECVAM), the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the OECD itself. It is concluded that the OECD has not followed the recommendations for full transparency and independence of the peer-review process. This is based on the fact that it has published a draft guidance document that differs from the report of a recent OECD workshop on validation, in such a way as to give the OECD the flexibility to fully control the peerreview process and, in so doing, to avoid full transparency. Comparison of the timing of the organisation of workshops by the OECD and the progression of the validation study, together with the fact that a draft test guideline for the assay was written before completion of the peer review, suggest that the OECD has given a higher priority to the expedition of the validation and regulatory acceptance of the uterotrophic assay than it has to good scientific and logistical practice. This severely undermines its credibility in the validation process, so, in order for the OECD to be rightly perceived as an honest broker, it is recommended that the OECD should play no role in the validation of new or revised tests, until after they have been successfully validated, peer reviewed, and endorsed by the appropriate authorities, and are ready for test guideline development. With regard to the on-going OECD validation studies of other in vivo assays for endocrine disruptors, the OECD should take immediate steps to ensure full independence and transparency of their peer review.
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The Use of Non-human Animals in Research: A Guide for Scientists

W.M.S. Russell

“It is a truism, though one that cannot too often be repeated, that we owe to animal experimentation many if not most of the benefits of modern medicine and countless advances in fundamental scientific knowledge”. This statement, from the first page of the 1959 book on humane experimental technique by the late Rex Burch and myself (1), is the subject of the first part of this new Royal Society booklet (2).
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2017-01-09T06:32:36+00:00 Tags: |

The Case for Taking Account of Metabolism when Testing for Potential Endocrine Disruptors In Vitro

Robert D. Combes

Legislation in the USA, Europe and Japan will require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Such chemicals are known as endocrine disruptors (EDs), and will require extensive testing as part of the new European Union Registration, Evaluation and Authorisation of Chemicals (REACH) system for the risk assessment of chemicals. Both in vivo and in vitro tests are proposed for this purpose, and there has been much discussion and action concerning the development and validation of such tests. However, to date, little interest has been shown in incorporating metabolism into in vitro tests for EDs, in sharp contrast to other areas of toxicity testing, such as genotoxicity, and, ironically, such in vitro tests are criticised for not modelling in vivo metabolism. This is despite the existence of much information showing that endogenous and exogenous steroids are extensively metabolised by Phase I and Phase II enzymes both in the liver and in hormonally active tissues. Such metabolism can lead to the activation or detoxification of steroids and EDs. The absence of metabolism from these tests could give rise to false-positive data (due to lack of detoxification) or false-negative data (lack of activation). This paper aims to explain why in vitro assays for EDs should incorporate mammalian metabolising systems. The background to ED testing, the test methods available, and the role of mammalian metabolism in the activation and detoxification of both endogenous and exogenous steroids, are described. The available types of metabolising systems are compared, and the potential problems in incorporating metabolising systems into in vitro tests for EDs, and how these might be overcome, are discussed. It is recommended that there should be: a) an assessment of the intrinsic metabolising capacity of cell systems used in tests for EDs; b) an investigation into the relevance of using the prostaglandin H synthase system for metabolising EDs; and c) a feasibility study into the generation of genetically engineered mammalian cell lines expressing specific metabolising enzymes, which could also be used to detect EDs.
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