The European ACuteTox project: a modern integrative in vitro approach to better prediction of acute toxicity.

ACuteTox (optimization and prevalidation of an in vitro test strategy for predicting human acute toxicity) is an integrated European Union project under the Sixth Framework Programme with the aim of demonstrating that animal tests for acute systemic toxicity currently used for regulatory purposes could be replaced by a combination of alternative assays. Validated alternative test methods are urgently required for safety toxicology testing of chemicals, cosmetics, and drugs.

EDIT: A New International Multicentre Programme to Develop and Evaluate Batteries of In Vitro Tests for Acute and Chronic Systemic Toxicity.

The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) programme provided a battery of three basal cytotoxicity tests with a good (R2 = 0.77) prediction of human acute lethal blood concentrations. The predictive power of this battery would be considerably improved by the addition of new supplementary in vitro tests. The development of these new tests will be facilitated by a close coupling of test development to evaluation. The Cytotoxicology Laboratory, Uppsala (CTLU), is therefore inviting all interested in vitro toxicologists to take part in the Evaluation-guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). All EDIT activities (subprojects) will be designed on a case-by-case basis, but will follow a common pattern. The CTLU will use the accumulated MEIC/EDIT data, and its experience from the previous MEIC evaluation, to suggest priority areas, i.e. the need for certain in vitro toxicity data/tests as supplements to existing in vitro models/batteries on human systemic toxicity. Detailed research programmes corresponding to these areas will be published on the Internet. The CTLU will also try to raise funds for some projects and will coordinate multilaboratory studies. Interested laboratories developing or already using priority tests are encouraged to join the subprojects and to test specific sets of substances (usually sets of MEIC reference chemicals) in their new assays. The CTLU will provide adequate human reference data and will also evaluate results as single components of complex models, together with the laboratory conducting the test. At present, ten priority areas have been identified: a) repeat dose toxicity in vitro; b) urgent mechanistic information from in vitro studies of protein denaturation, morphology of cell injury, differential toxicity between various rapidly measured endpoints (10-60 minutes) and 24-hour cytotoxicity, toxicity to aerobic cells, and discrimination between rapid and slow cytotoxic mechanisms; c) in vitro tests on vitally important, specific receptor toxicity in humans; d) excitatory cytotoxicity; e) reversibility of cell toxicity; f) in vitro tests on passage across the blood-brain barrier; g) in vitro tests on absorption in the gut; h) protein binding in vitro; i) in vitro tests on distribution volumes (Vd); and j) in vitro tests on biotransformation to more-toxic metabolites (hepatocytes plus target cells). This paper gives a short presentation of the rationale for each subproject and reports on ongoing activities.

Overview of the Final MEIC Results: I. The In Vitro--In Vitro Evaluation.

In the MEIC study, the first 30 reference chemicals were tested in 82 in vitro toxicity assays while the last 20 chemicals were tested in 67 assays. To increase understanding of the performance of in vitro toxicity tests, these two subsets of results were compared by principal components analyses (PCA) combined with a "random probe" analysis of five key methodological factors, that is, the results from all pairs of methods which were similar in all other respects than the analysed factor were systematically compared by linear regression. This paper is an overview of these published comparisons, and also includes a new "random probe" analysis of another segment of the same MEIC results, namely tests of all 50 reference chemicals by 61 of the methods. A PCA indicated high general similarity (around 80%) of all the results from the 61 methods. According to the new "random probe" analysis, this similarity must depend on the high correlation of results from assays with different cell types (mean R(2) 0.81) and/or different viability endpoints (mean R(2) 0.85). Main factors contributing to the 20% dissimilarity of results were different exposure times and the use of phylogenetically distant test objects in the non-analogous ecotoxicological assays. As expected, the new analysis of the 61 methods gave roughly similar results as the previous "random probe" analyses of the other two segments of the data. Findings support the basal cytotoxicity concept and will improve future in vitro toxicity testing. This testing will probably need assays with varied exposure times. As judged from the similarity of the results, simple assays with cell lines may replace complicated primary culture systems for many testing purposes (e.g. screening).

Biotransformation of carbon tetrachloride in cultured neurons and astrocytes.

The ability of brain neuronal cells to metabolize carbon tetrachloride (CCl(4)) has been studied in an attempt to explain earlier observed toxic effects of CCl(4) on these cells. The expression of cytochrome P-450, the glutathione (GSH) content and the activity of glutathione-S-transferase (GST) were measured in cultured neurons and astrocytes from chick embryo cerebral hemispheres. The metabolism of CCl(4) in the neuron and astrocyte cultures was also assessed by determining the formation of: CCl(2) in membrane preparations of these cells. In the membrane fractions of neurons and astrocytes, no measurable levels of cytochrome P-450 were observed. Nevertheless, neurons as well as astrocytes had a capacity for the metabolism of CCl(4). The metabolic capacity of the neurons was significantly greater than that of the astrocytes. The neuron cultures had a higher initial content of GSH and a higher control activity of GST than had the astrocytes. Neither the GSH level nor GST activity were significantly affected in the neuron cultures after exposure to CCl(4). In astrocyte cultures 2 mm CCl(4) slightly depleted the GSH level and significantly induced GST activity. At 3 mm CCl(4), GSH was depleted by 30% and by more than 50% at 4 mm CCl(4). It can be concluded that the metabolic activation of CCl(4) was higher in neurons than in astrocytes. This can explain the earlier observation of CCl(4)-induced lipid peroxidation in cultured neurons. Moreover, neuron GSH was not able to protect these cells against CCl(4)-induced peroxidative damage. In the astrocytes, on the other hand, GSH and GST appeared to have a role in detoxification of CCl(4).

Glutathione content, glutathione transferase activity and lipid peroxidation in acrylamide-treated neuroblastoma N1E 115 cells.

Acrylamide is a well known neurotoxic compound that produces central and peripheral distal axonopathy. Degenerative changes of this type can be induced in the neuroblastoma cell line C 1300, clone N1E 115 and have been extensively studied in our laboratory particularly with regard to the interference by acrylamide with cellular metabolism. In the present study the mechanism of acrylamide-induced neurite degeneration in N1E 115 cells is elucidated further. Acrylamide concentrations were selected that were not cytotoxic but caused an increasingly severe neurite degeneration. The rate of protein synthesis was decreased in a concentration-dependent manner in response to acrylamide exposure (0-2.5 mm). Detoxification of acrylamide in vivo occurs mainly through conjugation with glutathione, (GSH) both non-enzymatically and enzymatically by glutathione S-transferases (GST). Cells grown in the presence of acrylamide showed a concentration-dependent decrease in GSH content. At the highest acrylamide concentration tested this was accompanied by an increased GST activity. Despite the reduced level of GSH and possible impaired protection of the plasma membrane against oxidative stress no elevated level of lipid peroxidation could be observed in the acrylamide-treated cells.

Principles for the validation of in vitro toxicology test methods.

This review addresses key aspects of validation of in vitro toxicity test methods as they have been presented by different authors during the last decade. The MEIC study is presented as a practical case in which several of these aspects have been considered.

Differential effect of carbon tetrachloride on the cell membranes of neurons and astrocytes.

Primary cultures of neurons and astrocytes from chick embryos were used to study the effect of CCl4 on the plasma membranes. The cultures were exposed to 0, 1, 2, 3 or 4 mM CCl4 in a closed chamber system for 30 or 60 minutes. The effects of the exposure were examined by means of scanning electron microscopy and by measuring the degree of lipid peroxidation. In neuron cultures the presence of 1 mM CCl4 for 60 min caused holes in the plasma membranes and led to a swelling of the cell bodies. At 4 mM CCl4 the membranes were totally destroyed, leaving cytoskeletal elements visible. In astrocyte cultures, on the other hand, no effects up to 2 mM were observed. At 4 mM some cells had rounded up, but the membranes were still intact. These data correspond very well with the results that neurons, in contrast to astrocytes, show a concentration-dependent increase in lipid peroxidation. The results from this study may indicate that the mechanism of action of CCl4 is different in neurons and astrocytes.

A combined in ovo-in vitro system for studies of volatile compounds on brain development: differential effects of carbon tetrachloride on neurones and astrocytes.

A method for studies of organic solvent effects on the development of neurones and astrocytes is described. Chick embryos in ovo were exposed to a low concentration of carbon tetrachloride vapours. After the exposure, primary cultures of neurones and astrocytes were prepared from the brain hemispheres of the embryos. Toxic effects of the solvent were analyzed in the cultures during the course of cellular differentiation as changes in cell growth and morphology, respiratory activity, and biochemical properties characteristic of each cell type. The development of neurones from embryos exposed from day 5 to day 8 to 25 p.p.m. of carbon tetrachloride was not affected by the exposure. On the other hand, growth rate and respiratory activity of astrocytes from embryos exposed from day 11 to day 14 to 25 p.p.m. of CCl4 were affected by the exposure, whereas no morphological changes or changes in the cyclic AMP levels could be observed. Consequently, low concentrations of carbon tetrachloride seem to affect the development of astrocytes rather than that of neurones in our experimental system.

Effects of carbon tetrachloride on embryonic development studied in the post-implantation rat embryo culture system and in chick embryos in ovo.

The effects of carbon tetrachloride on embryonic development were investigated in a mammalian and a non-mammalian system. In the former, whole-rat embryos, taken at 9.5 days of gestation, were exposed in vitro to different concentrations of CCl(4) (10, 100, 300, 600 and 1000 mug/ml) in rat serum with or without a rat liver microsomal activating system (S-9 mix). In the latter system, chick embryos in ovo were exposed to different concentrations of CCl(4) vapour (25, 35 and 75 ppm). When studied in the whole-rat embryo culture system without metabolic activation, concentrations of up to 300 mug CCl(4)/ml had no effect on the overall development. Concentrations -600 mug CCl(4)/ml affected the somite number, growth and morphology of the embryos, which can be interpreted as general toxicity. In the presence of S-9 mix, toxicity occurred at concentrations -300 mug/ml. In ovo exposure to CCl(4) showed that 25 ppm caused a decrease in the number of somites. At 35 ppm, CCl(4) induced further toxicity, as indicated by reduced somite number and growth and increased malformation rates. The results indicate that effects on morphogenic events appeared in both systems at concentration levels that also affected the overall development and that, independently of the choice of species or route of administration, CCl(4) has no potential to induce specific malformation patterns. The presence of a metabolic system in the rat embryo cultures approximately doubled the toxicity of CCl(4).

Amphetamine as a protective agent against oxygen-induced convulsions in mice.

Male mice exposed to hyperbaric oxygen (HBO) at 4.5 ATA O2 exhibit a number of toxic symptoms including convulsions, diminished respiration and an acoustic reaction controlled by the central nervous system. To study whether stimulation of the nervous system could offer protection against the convulsions, mice were injected i.p. with various doses of d-amphetamine before HBO. At a dose of 1.0 mg X kg-1 of d-amphetamine the mice could stay at 5 ATA O2 without convulsions about three times as long as those injected with saline only. At high doses, 4 and 8 mg X kg-1, there was a weak protective effect or the time to convulsions was shortened. Amphetamine increases the release of dopamine in the brain and it is possible that the mechanisms of protection against HBO induced convulsions are involved in that process. The degree of protection, however, depends on the dose; therefore, it also is supposed that amphetamine in low doses acts on the autoreceptors with a presynaptic effect, which in this case is protective against the convulsions without affecting the respiration.

The respiratory response to microwaves.

Male mice were irradiated with microwaves of 2450 MHz at a power intensity of 1, 10, and 100 mW/cm2 and a dose of 300 mW.min/cm2. An intensity of 100 mW/cm2 for 3 min causes thermal effects. The rectal temperature increased 2 degrees C, and the respiratory minute volume decreased during the first 2 min. After that, it increased rapidly and fluctuated until the irradiation was finished, when the minute volume decreased to a value lower than normal. A new method has been used for studying the effects on the nervous system with the help of tone pulses and the respiratory minute volume, an acoustic reaction. This reaction is affected at the very moment the irradiation starts and remains for some minutes after irradiation is finished. During an irradiation of 10 mW/cm2 in 30 min, both the minute volume and the acoustic reaction are decreased. The rectal temperature does not change. 1mW/cm2 during 300 min does not change the minute volume nor the rectal temperature but decreases the acoustic reaction 4 and 5 h after irradiation has started. In the present experiments, it is possible to evaluate the influence of heat and the heat regulation with the help of the respiratory minute volume and rectal temperature. The effects on the conditioned acoustic reaction indicate that the nervous system is involved independently of the created heat and the thermal regulatory processes during and after irradiation with microwaves of 2450 MHz and at intensities of 1, 10, and 100 mW/cm2.

Dynamic factors influencing the production of lung injury in rabbits subjected to blunt chest wall impact.

Anesthetized rabbits were exposed to blunt impact on the right chest wall. The controlled impact was delivered with a special machine equipped with a captive piston driven by a freely falling weight. Chest wall deflections were recorded in order to study the effects of magnitude and rate of deformation of the thorax on the resulting lung injury. The ranges of magnitude and rate of deformation corresponded to an inward deflection of the chest wall of about 5-60% of the lateral diameter of the chest, and to a velocity of the chest wall of about 2-20 m/s. Impulses delivered to the chest and intrathoracic pressures were also studied. Severe hemorrhages in the lungs were produced at velocities of the chest wall exceeding about 10 m/s. For velocities of the chest wall below about 5 m/s, lethal injuries were produced without severe hemorrhages in the lungs.