Multicentre Evaluation Of In Vitro Cytotoxicity Research Articles

The Tenth Anniversary of the Björn Ekwall Memorial Foundation

The Björn Ekwall Memorial Foundation (BEMF) was initiated by the Scandinavian Society for Cell Toxicology in 2001, to honour the memory of Dr Björn Ekwall (1940-2000) and to establish a prize, the Björn Ekwall Memorial Award. The prize is awarded to scientists who have significantly contributed to the field of cell toxicology, and whose work is contributing toward the replacement of animal experiments by alternative toxicity tests. Over the past 10 years, the Björn Ekwall Memorial Award has been presented annually. Björn Ekwall, an outstanding Swedish cell toxicologist, was one of the pioneers in the development and application of alternative methods to animal tests in toxicology. All his scientific work was devoted to in vitro toxicology, and in particular, to the use of cultured human cells for the screening of toxic chemicals. In the middle of the 1980s, he initiated the international Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to evaluate the usefulness of in vitro tests for the estimation of human acute systemic toxicity. To prove his "basal cytotoxicity concept", he established the MEMO database, in which data on the acutely toxic human blood concentrations of drugs and chemicals were collated from the literature and from clinical studies. He also initiated another project, Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). The ideas from the EDIT project, together with those from the MEIC project, became the basis for today's international EU projects, e.g. ACuteTox, Sens-it-iv and ReProTect. In this article, 10 years after the start of the BEMF, the scientific achievements of each of the award winners in the field of in vitro toxicology are presented, together with a brief synopsis of their careers.

Conformational Changes in Proteins In Vitro as a Means of Predicting the Acute Toxicities of Chemicals

Experimental data are presented on ovalbumin denaturation (OD, EC10) and human acetylcholine esterase (AChE) inhibition (IC50) in vitro, following exposure to the chemicals used in the international Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme. Data were obtained for 40 (OD test) and 43 (AChE test) of the 50 MEIC chemicals. These data were compared with similar data from other methods used in the MEIC programme, and good correlations (R2) were obtained with data from MEIC studies on cell lines: 0.80 for human, 0.81 for other animal, and 0.78 for fish cell line IC50 values and AChE values, and 0.76 for human, 0.69 other animal and 0.75 for fish cell line IC50 values and OD values. The correlation increased substantially, if chemicals which freely cross the blood-brain barrier were solely considered, with R2 = 0.90 for human, 0.90 for other animal, and 0.82 for fish cell line IC50 values and AchE values, and 0.87 for human, 0.86 for other animal, and 0.92 for fish cell line IC50 values and OD values, in this case. Such chemicals are the main cause of non-specific depression of the central nervous system (CNS). The AChE IC50 permits a good prediction of human acute toxicity, similar to the IC50 values obtained with human cell lines and the same MEIC chemicals. These results confirm the basal toxicity hypothesis formulated by Björn Ekwall. It is concluded that in vitro methods based on the disruption of the functions of the proteins vital for body operation can be used as an alternative to the cell culture methods, when non-specific toxic effects of chemicals on humans and animals are evaluated.

Quantitative structure–activity–activity and quantitative structure–activity investigations of human and rodent toxicity

Acute toxicity in different biological systems, including humans and rodents in vivo, and human and rodent cell lines in vitro, was investigated. The data were taken from the MEIC (Multicentre Evaluation of In Vitro Cytotoxicity) programme. Quantitative structure–activity–activity relationship (QSAAR) models were developed for the in vivo human and rodent toxicity including a combination of toxicity endpoint and structural descriptors as predictor variables. The human peak blood/serum LC 50 concentrations were most strongly related to human liver cell toxicity, while the in vivo oral human lethal doses were most closely related to the in vivo rodent LD 50 values. The QSAARs included structural descriptors encoding electronic/reactivity properties, presence of H-bond donors, compound aromaticity, and size/shape properties. Quantitative structure–activity relationships (QSARs) were derived by using structural descriptors accounting for molecular hydrophobicity, size and shape, and electronic properties. These models have the potential to provide useful insights in the development of non-animal (in vitro and in silico) methods for predicting human and mammalian toxicity.

Prediction of Human Acute Toxicity by the Hep G2/24-hour/Total Protein Assay, with Protein Measurement by the CBQCA Method

In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)- quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r(2) = 0.761 (n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method (r(2) = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.

Flow cytometric methods used as screening tests for basal toxicity of chemicals

Aim of the present study was to evaluate the suitability of flow cytometry to test in vitro effects of toxicants. Flow cytometry offers the possibility to study several parameters simultaneously, e.g. cell cycle modulation, apoptosis and necrosis within the same cell culture. The effects of six compounds (acetaminophen=AAP, isoniazid=INH, digoxin, malathion, paraquat and 2,4-dichlorophenoxy acetic acid=2,4-D) on cell cycle were investigated in HepG2 cells and the induction of apoptosis/necrosis was analyzed by a spectrum of flow cytometric assays in HepG2, AAH-1 and YAC-1 cells. Early indicators of apoptosis––loss of mitochondrial membrane polarization––as well as later events of the apoptotic process––annexin V binding and DNA fragmentation––were studied. The phases of the cell cycle and the occurrence of a sub-G 0 peak of apoptotic cells were determined with propidium iodide staining. The present investigation demonstrated good correlations between results obtained by flow cytometric analyses and the IC50 data of the MEIC (=Multicenter Evaluation of In Vitro Cytotoxicity) study. Regarding the short time required for the tests, the possibility of investigating several parameters of cytotoxicity simultaneously and the ease of performance, flow cytometric analyses are well suited for the pre-screening for toxic effects of chemicals.

Evidence for delayed cytotoxicity effects following exposure of rat hepatoma-derived Fa32 cells: implications for predicting human acute toxicity

The delayed cytotoxicity of the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) reference chemicals was investigated in rat hepatoma-derived Fa32 cells. The cells were treated for 24 h with the test chemicals, and were than further cultured for 5 days in normal culture medium. The cytotoxicity was measured by the neutral red uptake inhibition, and the results were quantified by determining the NI50del. This is the concentration of test compound required to decrease the neutral red uptake with 50% compared with control cells. The results were compared with the acute NI50, the corresponding value measured immediately after 24 h treatment of the cells. On a total of 44 chemicals, nine showed delayed cytotoxicity (NI50del lower than or equal to NI50), 11 a probably delayed, and 24 no delayed cytotoxicity (NI50del more than 1.5×NI50). When the NI50del was compared with human toxicity, a correlation coefficient r 2=0.761 was obtained. For the same series of 44 chemicals this correlation was clearly higher than that for human hepatoma-derived Hep G2 cells ( r 2=0.695). The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Consequently, the delayed cytotoxicity assay on cultured Fa32 cells has the best prediction value so far obtained for the human toxicity.

167 Approaches to the minimisation of dog use in the safety assessment of pharmaceuticals: An industry/animal welfare initiative

The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the increased reactive oxygen species (ROS) formation in rat hepatoma-derived Fa32 cells. ROS formation was measured with 2′,7′-dichlorodihydrofluorescein diacetate as a fluorescent probe. The results were quantified by determining the ROS50. This is the concentration of test compound required to increase the ROS formation with 50% compared with control cells. An extremely high ROS formation was observed with ferrous sulfate. Of a total of 44 chemicals, an increased ROS formation was observed for 24. This was not the case for the 20 other chemicals. When the ROS formation in Fa32 cells was compared with human toxicity, the correlation coefficient was clearly higher than for human hepatoma-derived Hep G2 cells, at least when the extremely sensitive ferrous sulfate was withdrawn from the comparison. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Consequently, the ROS formation assay in Fa32 cells has a high predictive value for human toxicity, with the drawback that only ROS increasing chemicals can be evaluated.

The Prediction of Human Acute Systemic Toxicity by the EDIT/MEIC In Vitro Test Battery: The Importance of Protein Binding and of Partitioning into Lipids

The aim of the two studies presented in this paper was to further improve the predictability of the original Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) in vitro test battery for acute systemic toxicity. In the first study, whether a protein-free cytotoxicity assay could improve the prediction of human acute systemic toxicity was investigated. The cytotoxicity of 39 MEIC reference chemicals was measured by the neutral red uptake inhibition test after 30 minutes in phosphate-buffered saline (PBS), with hepatoma-derived Fa32 cells. The results were compared with the corresponding values obtained in complete culture medium, including 10% fetal calf serum. Mercuric chloride and hexachlorophene were much more cytotoxic in PBS, as was the case, to a lesser extent, for seven other chemicals. Potassium cyanide and eight other chemicals were less cytotoxic in PBS than in complete culture medium, probably because of poor physiological conditions. The correlation between the cytotoxicity measured in PBS and human acute toxicity was rather low, but became of the same order as for other assays, when mercuric chloride and hexachlorophene were withdrawn from the comparison. In the second study, modelling of human lethal blood concentrations by using the results of the three cell line tests of the original MEIC test battery were complemented by logP (octanol-water partition coefficient) values. The introduction of logP into the modelling did not improve the correlations, but some improvement of both R(2) and Q(2) was obtained by expanding the logP values with logP(2) values. The highest R(2) (0.84) and Q(2) (0.80) values were obtained for a model in which both experimental and calculated (ambiguous) logP values were used. When only experimental logP values were used, the corresponding values were 0.80 and 0.78. These two studies showed that including protein binding and the partition of chemicals in the MEIC in vitro test battery is important, in order to improve the predictability of the results obtained.

Use of the PFGE Assay for Studies of DNA Breakage Induced by Toxic Chemicals

The relevance of the pulsed field gel electrophoresis (PFGE) assay for the estimation of the DNA damaging effects of chemicals was studied. Four chemicals were randomly chosen from the list of 50 Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) reference chemicals with known human acute systemic toxicity: acetylsalicylic acid, paracetamol, ethylene glycol and sodium chloride. Human fibroblasts (VH-10) were used as a model system. For the estimation of cytotoxic effect, cell monolayers were treated with chemicals for 24 hours. Cloning efficiency (colony-forming ability) at different concentrations of the test chemicals was estimated, and the 50% inhibitory concentration (IC50) was determined. The IC50 values obtained demonstrated a correlation with human lethal blood concentrations. The induction of DNA double-strand breaks, measured by PFGE as the fraction of activity released, was detected after treatment with paracetamol. However, the other three chemicals tested mainly induced DNA degradation.

Serum albumin binding at cytotoxic concentrations of chemicals as determined with a cell proliferation assay

The aim of the present study was to measure the influence of albumin binding on cytotoxic concentrations of chemicals and to determine binding parameters which can be used for quantitative in vitro–in vivo extrapolations. Protein binding parameters were determined from cytotoxic potencies measured with Balb/c 3T3 cells cultured in the presence of 18 and 600 μM bovine serum albumin (BSA). A subset of 27 chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) project was investigated. At 18 μM BSA the EC 50-values ranged from 2.54 μM (As(III)) to 527 mM (ethylene glycol). Increasing the BSA concentration either decreased the cytotoxic potency (12 compounds) by factors up to 34 (pentachlorophenol), had no effect (14 compounds), or increased the cytotoxicity (paraquat). Calculated molar ratios of binding ranged from 0.05 (Hg 2+) to 4.8 moles per mole albumin (acetylic salicylic acid). At 18 μM BSA fractional binding of most of these compounds was low (<25%) but increased up to ≥90% (hexachlorophene, mercuric chloride, thioridazine, pentachlorophenol) at 600 μM BSA. The results obtained in general were compatible with available protein binding data and can be used to calculate equipotent concentrations of chemicals in biological systems containing different albumin concentrations.

Formation of reactive oxygen species in rat hepatoma-derived Fa32 cells to predict human toxicity

The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the increased reactive oxygen species (ROS) formation in rat hepatoma-derived Fa32 cells. ROS formation was measured with 2′,7′-dichlorodihydrofluorescein diacetate as a fluorescent probe. The results were quantified by determining the ROS50. This is the concentration of test compound required to increase the ROS formation with 50% compared with control cells. An extremely high ROS formation was observed with ferrous sulfate. Of a total of 44 chemicals, an increased ROS formation was observed for 24. This was not the case for the 20 other chemicals. When the ROS formation in Fa32 cells was compared with human toxicity, the correlation coefficient was clearly higher than for human hepatoma-derived Hep G2 cells, at least when the extremely sensitive ferrous sulfate was withdrawn from the comparison. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Consequently, the ROS formation assay in Fa32 cells has a high predictive value for human toxicity, with the drawback that only ROS increasing chemicals can be evaluated.

Comparisons of human-cell-based and submitochondrial particle bioassay responses to the MEIC compounds in reference to human toxicity data

Toxicity results from submitochondrial particle (SMP) bioassays were compared to results from multiple human-cell-based assays to evaluate the SMP tests’ ability to indicate cellular toxicity. Correlation analyses between cell-based and SMP responses were conducted on a series of diverse chemicals of human toxicologic interest chosen in the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) study and suggest a high degree of ordering. The r 2 correlation coefficient obtained when comparing the log-transformed SMP results to the average cellular response for all compounds was 0.75 ( n=42). When specific mitochondrial inhibitors (to which SMP arc extremely sensitive) and toxic metals (which SMP modeled poorly) were removed, the correlation improved to 0.91 ( n=34). When the SMP assay of each individual cell-based assay was compared to the average toxic response of all the cell-based assays for these 34 compounds, the SMP r 2 was greater than the median r 2 of the cell-based assays, indicating its ability to predict cell-based toxic responses with a high degree of accuracy. Comparisons of the SMP data to the human toxicity data are similar to the cell line assays, where removal of the specific mitochondrial toxicants and metals greatly improves the relationship.

Development of an in vitro test battery for the estimation of acute human systemic toxicity: An outline of the EDIT project. Evaluation-guided Development of New In Vitro Test Batteries.

The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.

In vitro toxicity: mechanisms, alternatives and validation - a report from the 19th annual scientific meeting of the Scandinavian Society for Cell Toxicology.

The Scandinavian Society for Cell Toxicology (SSCT) has arranged annual scientific meetings since 1983. These workshops were the forum for the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. Along with the MEIC programme, which was completed in 1998, a wide range of topics relating to cytotoxicity have been discussed. The meetings have also given an opportunity for graduate students and young scientists to present their work to an international audience. At the same time, experts in the fields of in vitro toxicity have been invited as speakers. The 19th SSCT scientific meeting, which was held at Sørup Manor in Ringsted, Denmark, was no exception. The meeting consisted of four sessions: mechanisms of toxicity; environmental toxicological testing; alternatives to animal experiments; and validation of in vitro tests.

Morphology of cell injury: An approach to the EDIT programme by the use of tobacco pollen tubes. Evaluation-guided Development of New In Vitro Test Batteries.

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubulin immunofluorescence and rhodamin-phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.

Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity.

The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells. The adhered cells were seeded and then treated and the adhering cells were treated simultaneously upon seeding. Five of the 44 test chemicals were twofold more toxic in adhering cells; ethylene glycol was 28-fold more toxic and mercuric chloride was 5.2-fold more toxic than in adhered cells. The cytotoxicity of dithiothreitol was altered in the same way as that of ethylene glycol, probably by interacting with calcium. When the neutral red uptake inhibition was compared with human toxicity, the correlation coefficient for adhering cells was almost identical to that obtained previously in human hepatoma-derived Hep G2 cells and slightly higher for adhered cells. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. An obviously better correlation was obtained when the strong intoxicant mercuric chloride was withdrawn from the comparison, both for the adhered and the adhering cells. Altogether, the results can be integrated very well with the basal cytotoxicity concept.

Protein precipitation In Vitro as a measure of chemical-induced cytotoxicity: an EDIT sub-programme.

As a priority area of the Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT) programme, an in vitro protein precipitation (PP) assay was used on the 50 reference chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to confirm and extend the MEIC results. Dose-response curves were generated for only 30 of the chemicals, and the concentrations causing 10% (EC10) and 50% (EC50) protein precipitation versus the positive control were chosen as endpoints. The number of chemicals with a positive response increased to 46 when a new endpoint, the minimum effect concentration (MEC) that induces protein precipitation with respect to the negative control, was used. When the results were correlated with in vitro cytotoxicity in human cell lines, a similarly good correlation was found between the various endpoints of the PP assay at 5 hours and the 24-hour IC50 average cytotoxicity in human cell lines, even though the number of chemicals included in the correlation was larger for the MEC. Using the prediction error, the endpoint that gave the best correlation between the PP assay and human cell cytotoxicity was once more found to be the 5-hour MEC, and this was chosen for the PP assay. The sensitivity of the PP assay is lower than that of the in vitro cell-line cytotoxicity assay, possibly due to its shorter exposure period and because precipitation is the ultimate event in the sequence of a protein disturbance. It is expected that earlier denaturation steps would give better sensitivity. However, this simple, inexpensive and rapid assay could be useful in the early stages of testing chemicals.

Fuzzy clustering analysis of the first 10 MEIC chemicals

In this paper, we discuss the classification results of the toxicological responses of 32 in vivo and in vitro test systems to the first 10 MEIC chemicals. In this order we have used different fuzzy clustering algorithms, namely hierarchical fuzzy clustering, hierarchical and horizontal fuzzy characteristics clustering and a new clustering technique, namely fuzzy hierarchical cross-classification. The characteristics clustering technique produces fuzzy partitions of the characteristics (chemicals) involved and thus it is a useful tool for studying the (dis)similarities between different chemicals and for essential chemicals selection. The cross-classification algorithm produces not only a fuzzy partition of the test systems analyzed, but also a fuzzy partition of the considered 10 MEIC (multicentre evaluation of in vitro cytotoxicity) chemicals. In this way it is possible to identify which chemicals are responsible for the similarities or differences observed between different groups of test systems. In another way, there is a specific sensitivity of a chemical for one or more toxicological tests.

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.

Gill epithelial cells as tools for toxicity screening—comparison between primary cultures, cells in suspension and epithelia on filters

In the search for suitable cell models that could be used in a test battery for toxicity screening purposes, we have focused on gill epithelial cells from rainbow trout. Gill epithelial cells are attractive models, since the gill is the primary target and uptake site for many toxicants in the water (Evans, D.H., 1987. Environ. Health Perspect. 71, 54–58). The aim of this study was to compare the sensitivity of gill epithelial cells in different test systems: suspensions, primary cultures and epithelia on filters in order to find out which system would be most suitable in a test battery for toxicity screening. The toxicity of the first 30 reference chemicals from the MEIC (multicenter evaluation of in vitro cytotoxicity) project (Bondesson, I., Ekwall, B., Hellberg, S. et al., 1989. Cell Biol. Toxicol. 5, 331–347) to gill epithelial cells in primary culture was tested using the fluorescent viability probe calcein-AM. Ten of the chemicals were also tested on cells suspended after 6–8 days in culture and five of the chemicals were tested on cells cultured on permeable filters. Decrease in transepithelial resistance was used as the endpoint for cells cultured on filters. The results were compared with toxicity data previously obtained by using freshly isolated gill epithelial cells in suspension. There was no significant difference in sensitivity of gill epithelial cells in suspension, primary cultures or resuspended cells. This observation strongly supports the use of cells in suspension in a test battery for toxicity screening purposes. Cultured epithelia on filters proved to be less suitable for screening purposes due to the considerable variation in resistance between epithelia even from the same fish.