Alternatives to Laboratory Animals

— Human fetal lung fibroblasts (HFL1) were studied in culture to evaluate their potential as a screen for cytotoxicity. The cytotoxic concentrations determined in vitro were compared with established human and animal toxicity data. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 hours, and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxicity data with rodent lethal concentrations and human lethal concentrations obtained from the testing of 50 chemicals in human lung cells, suggests that the experimental IC50 values are as accurate as predictors of human toxicity as the equivalent toxic blood concentrations derived from rodent LD50 tests. In addition, evaluation of the first 15 chemicals reveals no significant differences between results from continuous cell lines of human and rodent origin. Together with a related battery of tests, cell culture procedures have the potential to supplement or replace current animal protocols in screening chemicals for human toxicity.

— The quality of medical devices (including their biocompatibility) is regulated throughout the European Economic Area by Directive 93/42/EEC, which came into effect on 1 January 1995. The CE (conformity assessment) mark placed on the device shows conformity with the essential requirements, which guarantee the desired level of quality. Standards can be used to prove this conformity with the essential requirements. Scientific input is needed to produce and update relevant standards.

— The use of in vitro techniques in fundamental pharmacotoxicological research is widespread, but relatively little progress has been made in applying the knowledge and experience gained to regulatory toxicity testing. This is largely because specific tests of various kinds are required by national and international laws and regulations, before chemicals and products of many types can be manufactured, transported or marketed. These requirements have led to the publication of standardised test guidelines by various regulatory authorities. Although there is an increasing willingness to accept, in principle, the incorporation of non-animal tests and testing strategies into regulatory practice, their relevance and reliability must first be established in recognised validation studies, the outcomes of which must be assessed by independent groups of experts. The formal validation of alternative methods is not progressing satisfactorily. The reasons for this are discussed and some suggestions for improvements are made.

— Seven phosphatase inhibitors were used to investigate the possible importance of phosphatases in the regulation of gap junctional intercellular communication (GJIC). Only the tyrosine phosphatase inhibitor, pervanadate, was found to inhibit GJIC and to alter the band pattern of connexin43 (Cx43) in Western blots. Only the unphosphorylated band of Cx43 was seen after alkaline phosphatase treatment of pervanadate-exposed cells. Application of a tyrosine-specific phosphatase and a phosphotyrosine antibody clearly indicated that pervanadate induced tyrosine phosphorylation in Cx43.

- A cultured epithelial cell line from toad kidney (A6) was used to investigate side-specific toxicity related to the apical (outer) and basolateral (inner) membranes of epithdia. Well-known inhibitors and stimulators of ion transport were used to show that the ion transport proteins are asymmetrically distributed: the apical membrane contains sodium and chloride channels and the basolateral membrane contains Na⁺/K⁺ pumps, N_a⁺/Cl^- co-transporters, potassium channels and receptors for antidiuretic hormone The data demonstrate that the cellular toxicity of chemicals decreases when they are added to the apical side, illustrating that the epithelium acts as a functional barrier. However, the side-specific toxicity was more pronounced for ions and water-soluble molecules than for organic solvents, indicating that A6 epitheha can be used to distinguish between drugs that target specific membrane proteins and those that target membrane lipids. Furthermore, the cell line could be used to pick up chemicals that, at low concentrations inhibit sodium absorption and chloride secretion, without having any effect on cellular toxicity.

— There are several hundred industrial chemicals with neurotoxic potential. The neurotoxic risks of most of these chemicals are unknown. Additional methods are needed to assess the risks more effectively and to elucidate the mechanisms of neurotoxicity more accurately than is possible with the conventional methods.

This paper deals with general tasks concerning the use of in vitro models in the evaluation of neurotoxic risks. It is based on our previous studies with various in vitro models and on recent literature. The induction of glial fibrillary acidic protein in astrocyte cultures after treatment with known neurotoxicants (mercury compounds and aluminium) is discussed in more detail as an important response which can be detected in vitro.

When used appropriately with in vivo tests and with previous toxicological data, in vitro neurotoxicity testing considerably improves risk assessment. The incorporation of in vitro tests into the early stages of risk evaluation can reduce the number of animals used in routine toxicity testing, by identifying chemicals with high neurotoxic potential.

— The objective of the present study was to assess the possible tumour promoting activity of the food mutagen 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), by studying its influence on the expression of three genes considered to be of relevance in the tumour promotion step. The genes were two proto-oncogenes, c-fos and c-myc, and the tumour suppressor gene, p53. We observed that the expression of the c-fos and c-myc genes was induced when human bladder epithelial cells were treated with a standard solution of N-OH-PhIP and dithiothreitol (DTT), previously shown to be genotoxic. However, when cells were treated with DTT alone, the expression of c-fos and c-myc was also transiently induced. We therefore conclude that DTT, and not N-OH-PhIP, induced oncogene expression. Induction of both c-fos and c-myc expression by a reducing agent, DTT, which is frequently used in in vitro toxicology studies, is a novel observation that suggests the need for a cautious interpretation of such studies.

— As part of a developmental study on theophylline and pentoxifylline, these drugs were tested for possible cytotoxic, mutagenic and clastogenic effects on V79 hamster cells and human lymphocytes cultured in vitro. After the short-term treatment of V79 cells with theophylline and pentoxifylline, the cells were relatively resistant to the toxic effects of these methylxanthines. Generally, only high concentrations of theophylline or pentoxifylline had toxic effects on exposed V79 cells. Short-term treatment of V79 cells with theophylline or pentoxifylline did not induce 6-thioguanine resistant mutations in either the presence or absence of S9 fractions. However, in the absence of an S9 fraction, elevated levels of single-strand breaks in DNA were induced. Both methylxanthines caused clastogenic effects in human lymphocytes and hamster V79 cells after long-term exposure. We suggest that theophylline and pentoxifylline are clastogenic but not genotoxic, and that the molecular mechanism for the induction of single-strand breaks in DNA and the induction of chromosomal aberrations in cells treated with theophylline and pentoxifylline is not the induction of lesions in the DNA but the inhibition of DNA chain elongation.

— Several defence mechanisms against the toxicity of triethyllead (TEL) in cultures of glial cells have been studied. In contrast to cultures of glioma C₆ cells, primary cultures of glial cells generated a resistance to the toxicity of TEL, which increased with the age of the cultures.

When the time of exposure of glial cells was extended from 24 hours to 6 days and the cells were exposed to increasing doses of TEL, the activities of several metabolic enzymes were stimulated by concentrations of TEL over 10^-7M. Among these were glucose-6-phosphate-dehydrogenase and acid phosphatase. On the other hand, lactate dehydrogenase activity was not significantly affected under these experimental conditions.

When the cultures were exposed to 10 ⁸M TEL, the cellular calcium content increased and the potassium content fell. This indicates that the activation of metabolic enzymes is a response to early events in the toxic process, which include disturbances in calcium metabolism. When glial cells were incubated with lead and selenium simultaneously, the uptake of lead into the cells was reduced, and the cellular calcium content was increased by selenium.

— The impact of computer-assisted learning (CAL) packages on the use of animals in university teaching has been investigated in universities in the UK and abroad. The pilot study has focused on two issues: a) academic staff perceptions of the usability of CAL packages designed to offer an alternative to animal practicals in physiology and pharmacology; and b) whether the use of such programs has led to a reduction in the number of animals used. A questionnaire survey of purchasers of a minimum of three commercially available programs, which offer an alternative approach to traditional laboratory experiments, was conducted. The study found that in most departments the packages were used in a staff-supervised learning situation, to either replace or support a practical class. Their use saved academic and non-academic staff time, and they were considered to be less expensive and an effective and enjoyable mode of student learning. It was also clear that their use had contributed to a significant reduction in animal use.

— A new method was developed for the direct determination of glutathione reductase (GOR) activity in Vero cells cultured in microtitre plates, avoiding cell-free extract preparation. The cells in each well were washed twice with phosphate-buffered saline, lysed with Triton X-100, and assayed in 0.1M potassium phosphate, pH 7.0. After subtracting oxidase activity, which increased with NADPH concentration, the net GOR activity was similar at different oxidised glutathione (GSSG) and NADPH concentrations, thus confirming enzyme saturation. The optimised GOR assay used 2.5mM GSSG and 0.12mM NADPH; 5mM EDTA was also added to prevent the enzyme from redox inactivation. The GOR activity was directly proportional to the number of cells per well for a wide range of cell densities, thus supporting the assay's validity for use with cultured cells.

The effects on GOR activity of three chemicals which induce oxidative stress, namely, paraquat, iron (II) chloride and iron (III) chloride, were examined to validate the assay under experimental conditions. The specific enzymatic activity increased to 357% of untreated control activity in 5mM paraquat-treated cells, and to 407% of control activity in cells exposed to 7.5mM iron (II) chloride. By contrast, activity decreased to 56% of control activity in cells exposed to 5mM iron (III) chloride. In conclusion, the changes in GOR activity detected in Vero cells confirm that the new assay is suitable for routine in vitro screening of toxicants capable of inducing oxidative stress.

— Several concentrations of carbocaine hydrochloride, a local anaesthetic, were applied to isolated porcine corneas and stromas for periods of 1, 2, 3 and 4 hours. Increases in opacity (manifested as decreases in the intensity of a beam of light passed through the cornea from the epithelium to the endothelium), hydration (determined by the loss of water on drying to constant weight), and thickness (measured with a micrometer screw gauge) were determined. Small, time-related increases in opacity, associated with increases in hydration and thickness, were observed in control preparations and in preparations treated with 0.5–2% carbocaine hydrochloride over a 4-hour incubation period. However, only those increases resulting from incubation for 4 hours with 2% carbocaine hydrochloride differed significantly from control values. Higher concentrations (8% and 16%) also caused opacity, but decreased hydration and thickness, probably because of the hypertonicity of these solutions.