Identification of angiotensin I in a cyclostome, Lampetra fluviatilis.

Angiotensin I (ANG I) was isolated from incubates of plasma and kidney extracts of the river lamprey, Lampetra fluviatilis, using eel vasopressor activity as an assay during purification. Its sequence was Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Thr-Leu as determined by the sequence analysis and mass spectrometry. The sequence was confirmed by identity of the elution profile with the synthetic peptide in two different reverse-phase columns of high-performance liquid chromatography. Lamprey ANG I produced dorsal-aortic pressor responses in L. fluviatilis but the rise was very small in comparison to that produced by angiotensin II. Angiotensin III produced an even bigger increase. It was not possible to demonstrate a difference in response to Asn(1) (lamprey) ANG I and Asp(1) (human) ANG I. The present study directly demonstrated the presence and biological activity of the renin-angiotensin system in the most primitive extant vertebrates, the cyclostomes. Thus the renin-angiotensin system is a phylogenetically old hormonal system that is present throughout the vertebrates.

The Third FRAME Toxicity Committee: working toward greater implementation of alternatives in toxicity testing.

FRAME (the Fund for the Replacement of Animals in Medical Experiments; http://www. frame.org.uk) is a scientific charity, which has, for over 30 years, been advocating and conducting its own research on the application of the Three Rs (reduction, refinement and replacement) to animal experimentation. FRAME develops and validates scientifically based replacement alternative methods to facilitate their acceptance by scientists and regulators. As part of these activities, FRAME established a FRAME Toxicity Committee in 1979, and a report of its work was published in 1982, and discussed in the proceedings of a subsequent meeting, published in 1983. A Second Toxicity Committee formed in 1988, reported its work in 1990, which was discussed in the proceedings of a subsequent conference, published in 1991. The work of these committees was extremely successful and influential in laying the foundation for later activities in alternatives research. A Third FRAME Toxicity Committee was formed in 1999, since much progress had been achieved in the previous decade, especially with regard to the successful validation of several non-animal replacement methods and the start of their regulatory acceptance. Moreover, some new test methods are on the point of being validated, and many new techniques and discoveries are impacting on toxicity testing. Also, interest in reduction and refinement in toxicology has increased. However, there is considerable scope and need for the further implementation of the Three Rs in toxicity testing, especially due to recent plans for the large-scale testing of high-production volume, hormonally-active and existing chemicals, and the increasing use of transgenic animal models. The new committee comprises 18 experts from industry, academia, animal welfare, legislative and regulatory bodies, with one observer from the UK Government Home Office. The main objective is to review progress made in the application of the Three Rs in the development and safety evaluation of medicines, biologicals, cosmetics, agrochemicals and other products, as well as industrial chemicals, and to make recommendations as a basis for further sensible progress according to sound scientific and ethical criteria. The main committee is to be augmented by several working parties that will focus on specific scientific issues: 1) targeted risk assessment versus hazard identification; 2) data sharing; 3) endocrine disruption; and 4) carcinogenicity testing. The Committee is also to publish a status report on the current situation regarding alternatives in toxicity testing, based on the recommendations of the Second Toxicity Committee, and will organise a conference to discuss its overall conclusions and recommendations.

Preclinical safety evaluation using nonrodent species: an industry/welfare project to minimize dog use.

This review of the dog, the primary nonrodent species used in toxicology, and its use in the safety evaluation of pharmaceuticals, provides data on the number used in particular projects in an effort to establish a baseline from which some minimization can be measured. Opportunities for reduction and replacement, as identified by a European Industry/Welfare Steering Group, are discussed. The three distinct areas of potential approaches to minimize dog use are categorized as industrial cooperation/data sharing, achieving best practice in study design, and assessing the need for a particular study. The Steering Group prioritized the approaches based on the impact on the number of animals used, the impact on the welfare of the remaining animals, the potential for industry's acceptance of the scientific approach, the potential for regulators' acceptance of the validated approach, and the time/cost of evaluation or implementation. Examples of each category are presented, and the work needed to facilitate industry/regulatory change is discussed.

The implications of microarray technology for animal use in scientific research.

Microarray technology has the potential to affect the number of laboratory animals used, the severity of animal experiments, and the development of non-animal alternatives in several areas scientific research. Microarrays can contain hundreds or thousands of microscopic spots of DNA, immobilised on a solid support, and their use enables global patterns of gene expression to be determined in a single experiment. This technology is being used to improve our understanding of the operation of biological systems during health and disease, and their responses to chemical insults. Although it is impossible to predict with certainty any future trends regarding animal use, microarray technology might not initially reduce animal use, as is often claimed to be the case. The accelerated pace of research as a result of the use of microarrays could increase overall animal use in basic and applied biological research, by increasing the numbers of interesting genes identified for further analysis, and the number of potential targets for drug development. Each new lead will require further evaluation i n studies that could involve animals. In toxicity testing, microarray studies could lead to increases in animal studies, if further confirmatory and other studies are performed. However, before such technology can be used more extensively, several technical problems need to be overcome, and the relevance of the data to biological processes needs to be assessed. Were microarray technology to be used in the manner envisaged by its protagonists, there need to be efforts to increase the likelihood that its application will create new opportunities for reducing, refining and replacing animal use. This comment is a critical assessment of the possible implications of the application of microarray technology on animal experimentation in various research areas, and makes some recommendations for maximising the application of the Three Rs.