Transgenic animal technology: alternatives in genotyping and phenotyping

Over the past decade, breakthrough technologies in transgenic animal technology and functional genomics have played a central role in the explosive growth of rodent modeling and in scientific innovation. Various noninvasive alternatives to routine surgical biopsy have been described for genotypic and phenotypic analyses of laboratory animals. A number of options are available to refine or replace potentially painful and invasive procedures ranging from tissue biopsies (including tail biopsies and toe docking) to several blood sampling techniques. Unfortunately, adoption of many non- or minimally invasive alternatives has proven difficult on a number of fronts ranging from historical reservations to procedural expectations and actual experimental productivity. Similarly, a variety of phenotyping considerations have addressed throughput efficiencies and the health and well being of research animals. From an animal welfare perspective, marked increases in laboratory animal populations have accompanied rapid advancements spanning the life sciences. As described for rodent modeling, but with applications across many laboratory animal species, diverse procedural refinements are available that will readily aid in the analysis of whole animal models. Ultimately, non-invasive technologies and complementary refinements have bearing on the quality and reproducibility of data that are reported, as well as of critical importance to the well being and ethical management of animals at all developmental stages: from fetal existence, to the neonatal period, and on through adulthood.     

The contributions of the European cosmetics industry to the development of alternatives to animal testing: dialogue with ECVAM and future challenges

COLIPA (the European Federation of the Cosmetics Industry) represents 24 international companies and 2000 small and medium-sized enterprises. Together with ECVAM, COLIPA has been involved in the development and validation of alternative methods since the beginning of the validation efforts. The work of the Steering Committee on Alternatives to Animal Testing (SCAAT) is based on collaboration between companies, but also with academia, trade associations, the Scientific Committee on Cosmetics and Non-Food Products (SCCNFP), European Commission Directorates General, and ECVAM. Some success has been achieved, but some validation efforts have failed. One lesson is that the search for alternatives requires a lot of humility.     

Integrated decision-tree testing strategies for skin corrosion and irritation with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME recently conducted a research project, sponsored by DEFRA, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This report focuses on how to maximise the use of alternative methods (both in vitro and in silico) for skin corrosion and irritation testing within a tiered testing strategy. It considers the latest developments in in vitro testing, with particular reference to the reconstituted skin models which have now been now been successfully validated and independently endorsed as suitable for both skin corrosivity and irritancy testing within the EU.     

Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles

An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1α and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure). The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing

This report summarizes the proceedings of the September 9-10, 2005 meeting of the Expert Working Group on Hazard Identification and Risk Assessment in Relation to In Vitro Testing, part of an initiative on genetic toxicology. The objective of the Working Group was to develop recommendations for interpretation of results from tests commonly included in regulatory genetic toxicology test batteries, and to propose an appropriate strategy for follow-up testing when positive in vitro results were obtained in these assays. The Group noted the high frequency of positive in vitro findings in the genotoxicity test batteries with agents found not to be carcinogenic and thought not to pose a carcinogenic health hazard to humans. The Group agreed that a set of consensus principles for appropriate interpretation and follow-up testing when initial in vitro tests are positive was needed. Current differences in emphasis and policy among different regulatory agencies were recognized as a basis of this need. Using a consensus process among a balanced group of recognized international authorities from industry, government, and academia, it was agreed that a strategy based on these principles should include guidance on: (1) interpretation of initial results in the "core" test battery; (2) criteria for determining when follow-up testing is needed; (3) criteria for selecting appropriate follow-up tests; (4) definition of when the evidence is sufficient to define the mode of action and the relevance to human exposure; and (5) definition of approaches to evaluate the degree of health risk under conditions of exposure of the species of concern (generally the human). A framework for addressing these issues was discussed, and a general "decision tree" was developed that included criteria for assessing the need for further testing, selecting appropriate follow-up tests, and determining a sufficient weight of evidence to attribute a level of risk and stop testing. The discussion included case studies based on actual test results that illustrated common situations encountered, and consensus opinions were developed based on group analysis of these cases. The Working Group defined circumstances in which the pattern and magnitude of positive results was such that there was very low or no concern (e.g., non-reproducible or marginal responses), and no further testing would be needed. This included a discussion of the importance of the use of historical control data. The criteria for determining when follow-up testing is needed included factors, such as evidence of reproducibility, level of cytotoxicity at which an increased DNA damage or mutation frequency is observed, relationship of results to the historical control range of values, and total weight of evidence across assays. When the initial battery is negative, further testing might be required based on information from the published literature, structure activity considerations, or the potential for significant human metabolites not generated in the test systems. Additional testing might also be needed retrospectively when increase in tumors or evidence of pre-neoplastic change is seen. When follow-up testing is needed, it should be based on knowledge about the mode of action, based on reports in the literature or learned from the nature of the responses observed in the initial tests. The initial findings, and available information about the biochemical and pharmacological nature of the agent, are generally sufficient to conclude that the responses observed are consistent with certain molecular mechanisms and inconsistent with others. Follow-up tests should be sensitive to the types of genetic damage known to be capable of inducing the response observed initially. It was recognized that genotoxic events might arise from processes other than direct reactivity with DNA, that these mechanisms may have a non-linear, or threshold, dose-response relationship, and that in such cases it may be possible to determine an exposure level below which there is negligible concern about an effect due to human exposures. When a test result is clearly positive, consideration of relevance to human health includes whether other assays for the same endpoint support the results observed, whether the mode or mechanism of action is relevant to the human, and - most importantly - whether the effect observed is likely to occur in vivo at concentrations expected as a result of human exposure. Although general principles were agreed upon, time did not permit the development of recommendations for the selection of specific tests beyond those commonly employed in initial test batteries.     

From synapse to network: models of information storage and retrieval in neural circuits

The mechanisms of information storage and retrieval in brain circuits are still the subject of debate. It is widely believed that information is stored at least in part through changes in synaptic connectivity in networks that encode this information and that these changes lead in turn to modifications of network dynamics, such that the stored information can be retrieved at a later time. Here, we review recent progress in deriving synaptic plasticity rules from experimental data and in understanding how plasticity rules affect the dynamics of recurrent networks. We show that the dynamics generated by such networks exhibit a large degree of diversity, depending on parameters, similar to experimental observations in vivo during delayed response tasks.     

Muscle contraction: energy rate equations in relation to efficiency and step-size distance

We derive the energy rate equation for muscle contraction. Our equation has only two parameters m, the maintenance heat rate and 1/S, the shortening heat coefficient. The impulsive model (previously described in earlier papers) provides a physical basis for parameter 1/S as well as for constants a and b in Hill’s force–velocity equation. We develop new theory and relate the efficiency and the step-size distance to our energy rate equation. Correlation between the efficiency and the step-size distance is established. The various numbers are listed in Table 1: we use data from five different muscles in the literature. In summary, our analysis strongly supports the impulsive model as the correct model of contraction.     

"Mutual aid" and "animal dispersion": an historical analysis of alternatives to Darwin

Petr Alekseyevich Kropotkin (1842-1921) and Vero Copner Wynne-Edwards (1906-1997) developed evolutionary theories that emphasized social groups and cooperation rather than the organism-level natural selection of standard Darwinian theory. The most important reason for their alternative interpretations was their experience as field naturalists. Kropotkin and Wynne-Edwards worked in arctic environments and were impressed by aspects of the natural world that differed significantly from those experienced by Darwin or Wallace. These field experiences led to their emphasis on mutual aid and group selection, respectively. Understanding the development of their theories helps to illuminate the continuing debates over evolutionary theory and the current resurgence of interest in group selection.     

Reproductive efficiency and growth of Emex australis in relation to stress

The reaction of Emex australis to different degrees of stress was examined in a pot experiment by varying pot size and in a field experiment by varying density. In pots, the proportion of energy in seeds was more stable to the imposition of stress than in roots and stems; that in roots increasing with increasing stress and that in stems declining. Similarly in the field, there was little difference between densities of 1–32 plants m^?2 in the proportion of seeds in biomass. There were no significant differences in mean individual seed weights in either experiment and all plants set seed. Again in both experiments, high stress plants were more precocious than low stress ones. In pots, this took the form of earlier attainment of maxima in calorific values, ‘reproductive effort’, root, stem and seed weights and in the field, a shorter life cycle and earlier senescence. In pots, estimates of the overall mean reproductive effort were 62% for the low and medium stress plants and 36% for the high stress plants. The results are discussed in terms of the high values for reproductive effort, the plasticity and precocity of E. australis under stress and the implications for weed control.     

A new method to model change in cutaneous blood flow due to mechanical skin irritation part II: parameter identification procedure

Mechanical skin irritation, for example a light scratch with a needle, induces histamine and neuropeptide release on the line of stroke and in the surrounding tissue. Both histamine and neuropeptides are vasodilators. They cause vasodilation by changing the contraction state of the vascular smooth muscles and hence vessel compliance. Smooth muscle contraction state is very difficult to measure in vivo. For that reason we propose in this article an identification procedure to establish an irritation law. The law gives change in vessel compliance as a function of space, time and the intensity of the stroke. We have showed that vessel compliance increases immediately after the stroke not only on the line of stroke, but also in the surrounding tissue. Then, after a short delay, vessel compliance starts decreasing in the surrounding tissue, whereas vessel compliance on the line of stroke keeps increasing. Hence, blood is transported from the surrounding tissue to the line of stroke. In this way, higher blood volume on the line of stroke can be obtained than by only changing vessel compliance locally.     

The '3Rs' model and the concept of alternatives in animal research: a questionnaire survey

It has been 45 years since Russell and Burch first proposed the concept of the '3Rs', yet it remains unclear how those individuals involved in animal research view and implement these concepts. The authors used a questionnaire survey to determine how well-known experts judged issues related to the 3Rs.     

Long-term field metal extraction by Pelargonium: phytoextraction efficiency in relation to plant maturity

The long length of periods required for effective soil remediation via phytoextraction constitutes a weak point that reduces its industrial use. However, these calculated periods are mainly based on short-term and/or hydroponic controlled experiments. Moreover, only a few studies concern more than one metal, although soils are scarcely polluted by only one element. In this scientific context, the phytoextraction of metals and metalloids (Pb, Cd, Zn, Cu, and As) by Pelargonium was measured after a long-term field experiment. Both bulk and rhizosphere soils were analyzed in order to determine the mechanisms involved in soil-root transfer. First, a strong increase in lead phytoextraction was observed with plant maturity, significantly reducing the length of the period required for remediation. Rhizosphere Pb, Zn, Cu, Cd, and As accumulation was observed (compared to bulk soil), indicating metal mobilization by the plant, perhaps in relation to root activity. Moreover, metal phytoextraction and translocation were found to be a function of the metals' nature. These results, taken altogether, suggest that Pelargonium could be used as a multi-metal hyperaccumulator under multi-metal soil contamination conditions, and they also provide an interesting insight for improving field phytoextraction remediation in terms of the length of time required, promoting this biological technique.