Alternatives to Laboratory Animals

Groups of large juvenile wolf spiders (Pardosa prativaga) were kept on constant Drosophila melanogaster rations of 0 (starvation), 1, 2, 3.5, 7, 14 or 28 flies per week, or ad libitum feeding. After 3–4 weeks, they were sacrificed and the activities of three biomarker enzyme systems — glutathione S-transferase (GST) with chlorodinitrobenzene as substrate, glutathione peroxidase with H₂O₂ as substrate (GSH-Px[H₂O₂]), or glutathione peroxidase with t-butyl-hydroperoxide as substrate (GSH-Px[TBH]) — were assayed. Two systems (GST and GSH-Px[H₂O₂]) showed decreasing enzyme activity with increasing feeding rate, whereas the variation in GSH-Px(TBH) was independent of feeding rate. Sex and body weight had no influence on enzyme activity.

Established Fa32 cells, derived from a rat hepatoma, were investigated for their glutathione S-transferase (GST) induction capacity, which is an important characteristic of the detoxification capacity in normal liver. The cells were exposed to inducers of drug metabolism for 3 days in complete medium (containing 10% fetal calf serum). Neither dimethyl sulphoxide nor dimethyl formamide could be used as a vehicle to transport the inducers into the cells, because they also interacted with GST. Phenobarbital, butylated hydroxyanisole, allyl isothio-cyanate and dimethyl fumarate (but not fumaric acid) all effectively increased the total specific GST activity. None of the test chemicals produced a very pronounced induction of specific GST subunits, but subunit 2 and subunit 8 were increased more than the others. The effects of inducers of drug metabolism on the GST activity in Fa32 cells are comparable with those in rat liver. These cells can therefore be used as a valuable alternative model for GST-dependent metabolic interactions in rat liver.

To avoid excessive use of pesticides in controlling varroa mites (Varroa jacobsoni) in honey bee (Apis mellifera) colonies, a method for monitoring the population size of the mites was developed. The relationship between the size of mite populations (y) in full-size honey bee colonies and natural mite mortality, measured as the number of mites falling on plastic inserts (drop-down), was investigated in Danish apiaries. The results suggest that a straight linear model (y = + x) describes the relationship between the mite population present in a colony and the calculated daily number of naturally dead mites collected on inserts during either 1-week or 3-week periods before sampling. The parameters of the straight line relationship between the population size and the daily mite drop-down during a 1-week period are: β = 0.0069 and α = -1.858 (r² = 0.77, p < 0.0001). For a 3-week period, the parameters are: β = 0.0063 and α = -0.403 (r ² = 0.83, p < 0.0001). If the model input is adjusted for the brood-rearing pattern of the sampled colonies, i.e. colonies with capped brood cells covering less than one side of a comb in total (2800–3200 cells) are excluded from the input, the fit of the model is improved. In this case, the parameters for the 1-week sampling period are: β = 0.0075 and α = -1.184 (r² = 0.88, p < 0.0001), and the parameters for the 3-week sampling period are: β = 0.0071 and α = -0.864 (r² = 0.91, p < 0.0001).

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.

This paper discusses the limitations of using laboratory animals for direct in vivo observation of the development of renal stones. In fact, the majority of hypotheses related to mechanisms of stone formation have been based on the results of in vitro experiments. The relevance of in vitro experiments that allow the study of urolithiasis depends upon the degree of correspondence between the experimental conditions and those prevailing in the stone-forming kidney in vivo. For this reason, several in vitro experimental systems that attempt to reproduce the conditions found in vivo have been developed in order to study renal stone formation, which have been classified into two main groups: a) models to study papillary stone formation; and b) models to study “sedimentary” stone formation. These models are briefly described in this paper, and the information obtained was compared with that resulting from a study of the fine structure of real human renal calculi, in order to prove the validity of the models. It was concluded that the experimental in vitro models can closely reproduce the renal conditions under which human calculi are developed. This allows important data to be obtained about the aetiology of renal lithiasis, which is of great relevance to the development of effective treatments for this disease. Therefore, experimental in vitro models constitute a clear alternative to the use of laboratory animals.

The Alamar Blue™ reduction assay is used as an indicator of cellular viability in in vitro tests for the prediction of ocular irritancy. Alamar Blue itself has a low cytotoxicity, so repeat exposure and recovery studies can be performed on the same cells. This paper contains the results of a preliminary investigation of interactions between the Alamar Blue dye and a confluent monolayer of epithelial Madin-Darby canine kidney cells. This was performed by using an experimental fluorescence microscope and differential phase confocal microscope designed for studying live samples in vitro. The initiation of Alamar Blue reduction to its fluorescent product did not occur at the same time in all cells, but started in a small number and spread progressively through their immediate neighbours. There was clear localisation of the reduced (fluorescent) Alamar Blue within the nuclei and other organelles.

Monoclonal antibodies (mAbs) are particularly valuable in therapeutics and research. Unfortunately, one of the most familiar methods of producing mAbs, the ascites induction method, causes pain and distress to the animals used. In most cases, non-animal or in vitro alternatives can be employed to reduce or eliminate the use of animals for mAb production. Prohibition of the use of animals in the production of mAbs is recommended, except when the replacement in vitro methods prove to be insufficient, and in a limited number of other well-documented cases, such as an exceptional need for an emergency therapeutic application. A total ban on the use of animals for mAb production is impractical and it is imperative that an appeals process should accompany the prohibition. The need for the establishment of core facilities for in vitro mAb production is emphasised.

This study investigated the effects of some experimental variables on the rate of xenobiotic metabolism in precision-cut rat liver slices. Liver slices of 123 ± 8μm (mean ± SEM of six slices), 165 ± 3μm, 238 ± 6μm and 515 ± 14μm thickness were prepared from male Sprague-Dawley rats, and incubated in RPMI 1640 medium in an atmosphere of 95% O₂/5% CO₂ by using a dynamic organ culture system. Liver slices of all thicknesses metabolised 10μM 7-ethoxycoumarin to total (free and conjugated) 7-hydroxycoumarin in a time-dependent manner. The rate of 7-ethoxycoumarin metabolism was greatest in 165μm thick slices and slowest in 515μm thick slices, being 2.74 ± 0.19pmol/minute/mg slice protein and 0.69 ± 0.07pmol/minute/mg slice protein, respectively. No marked effects on the rate of 7-ethoxycoumarin metabolism in liver slices were observed either by changing the medium to Earle's balanced salt solution (EBSS) or by changing the gas phase to 95% air/5% CO₂. Moreover, the perfusion of rat livers with EBSS at 2–4°C, prior to preparation of tissue cores, did not enhance 7-ethoxycoumarin metabolism in rat liver slices. In this study, the optimal slice thickness was 175μm, with higher rates of 7-ethoxycoumarin metabolism being observed than with 250μm thick slices, which are often used for studies of xenobiotic metabolism. Variable results were obtained with slices of around 100–120μm thickness, which may be attributable to the ratio between intact hepatocytes and cells damaged by the slicing procedure in these very thin slices.