Erik Walum

MEIC—A new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests

A new international project to evaluate the relevance for human systemic and local toxicity of in vitro tests of general toxicity of chemicals has been organized by the Scandinavian Society of Cell Toxicology under the title 1Multicenter Evaluation of In Vitro Cytotoxicity (MEIC). The basic assumptions underlying the project, as well as the practical goals and the design of the program are outlined. The list of the first 50 reference chemicals is presented. The chemicals are an otherwise unbiased selection of compounds with known human acutely lethal dosage and blood concentrations, including LD50-values in the rat or mouse. Most agents also have other data on human toxicity and toxicokinetics, including more extensive animal toxicity data. International laboratories already using or developing in vitro tests of various partial aspects of general toxicity are invited to test the substances, the results of which will be evaluated by us. The predictivity of the in vitro results for both partial and gross human toxicity data will be determined with combined use of univariate regression analysis and soft multivariate modeling. The predictivity of the in vitro results will be compared with the predictivity of conventional animal tests for the same chemicals. Finally, batteries of tests with optimal prediction power for various types of human toxicity will be selected. The need for and possible uses of such batteries are discussed.TABLE 1The First 50 Reference Chemicals of the MEIC Project1. Acetaminophen26. Arsenic trioxide2. Aspirin27. Cupric sulfate3. Ferrous sulfate28. Mercuric chloride4. Diazepam29. Thioridazine HCl5. Amitriptyline30. Thallium sulfate6. Digoxin31. Warfarin7. Ethylene glycol32. Lindane8. Methyl alcohol33. Chloroform9. Ethyl alcohol34. Carbon tetrachloride10. Isopropyl alcohol35. Isoniazid11. 1,1,1-Trichloroethane36. Dichloromethane12. Phenol37. Barium nitrate13. Sodium chloride38. Hexachlorophene14. Sodium fluoride39. Pentachlorophenol15. Malathion40. Verapamil HCl16. 2,4-Dichlorophenoxyacetic acid41. Chloroquine pholphate17. Xylene42. Orphenadrine HCl18. Nicotine43. Quinidine sulfate19. Potassium cyanide44. Diphenylhydantoin20. Lithium sulfate45. Chloramphenicol21. Theophylline46. Sodium oxalate22. Dextropropoxyphene HCl47. Amphetamine sulfate23. Propranolol HCl48. Caffeine24. Phenobarbital49. Atropine sulfate25. Paraquat50. Potassium chloride

Tritiated 2-deoxy-D-glucose as a probe for cell membrane permeability studies

Tritiated 2-deoxy-D-glucose was taken up and phosphorylated by cultured cells of neuronal (NIE 115), glial (138 MG), muscle (L 6) and liver (BRL 123) origin. Upon perfusion the cells slowly released 2-deoxy-D-glucose 6-phosphate. The following values for rate constants, half-lives, and activation energies for the efflux were obtained: NIE 115: 0.0048 min/sup -1/, 143 min, and 72 kJ mol/sup -1/; 138 MG: 0.0013 min/sup -1/, 547 min, and 85 kJ mol/sup -1/; L 6: 0.0022 min/sup -1/, 311 min, and 60 kJ mol/sup -1/; and BRL 123: 0.0013 min/sup -1/, 528 min and 63 kJ mol/sup -1/. When the cultures were perfused with buffer containing Triton X-100 a time- and concentration-dependent increase in the rate of efflux of 2-deoxy-D-glucose 6-phosphate was obtained. It is suggested that 2-deoxy-D-(/sup 3/H)glucose can be used as a probe in studies of general cell membrane permeability changes.

Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery

Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca2+]i) and receptor-activated (carbachol, 0.1 mmol/L) Ca2+ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca2+]i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca2+]i plays a significant role for acrylamide-induced axonopathy.

Comparison of in vivo acute lethal potency and in vitro cytotoxicity of 48 chemicals

The cytotoxicity of 48 compounds included in the MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) list was determined in cultures of rat hepatocytes, McCoy, and MDBK cells. The average minimum concentration of each compound inducing cytotoxicity was measured in each cell type. The cytotoxicity values were then compared with published oral LDS p values for rats and mice. The logarithmic transformation of in vivo toxic doses and the corresponding in vitro cytotoxic concentrations showed a statistically significant correlation between the in vitro and in vivo values. The results show that an accurate in vivo LDS p dose could be predicted from in vitro data for at least 75% of the selected compounds. It is hoped that this finding will not only stimulate others to pursue in vitro technique but will eventually lead to elimination of the in vivo LD50 test.

Reactive gliosis and monoamine oxidase B

A double-staining method was applied to cryosections of human spinal cord from patients who died with amyotrophic lateral sclerosis (ALS) and corresponding controls in order to investigate cellular content of monoamine oxidase B (MAO-B). 3H-L-Deprenyl emulsion autoradiography was used in combination with histochemical methods for the detection of astrocytes and monocytes/microglia. In the ALS spinal cords an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes was demonstrated. No significant 3H-L-deprenyl binding was observed in cells derived from the mesoderm, e.g. monocytes or microglia. Furthermore, a sub-population of reactive astrocytes that contained low levels of MAO-B was observed in spinal sections. These findings were further substantiated by studies performed on primary astrocyte cultures.

Membrane lesions in cultured mouse neuroblastoma cells exposed to metal compounds.

Tritiated 2-deoxy-D-glucose (dGlc) was rapidly taken up into cultured mouse neuroblastoma C1300 cells (clone 41A3). Upon perfusion the preloaded cultures slowly released radioactivity as [3H] 2-deoxy-D-glucose-6-phosphate ([3H]dGlc-6-P) (rate const. = 0.017 min-1) from a pool corresponding to 74% (t1/2 = 41 min) of the total radioactivity incorporated. Destruction of the plasma membrane of the cells by means of Triton X-100 (1.0%) resulted in a rapid and total release of the radioactivity. CH3HgCl, HgCl2, (C2H5)3SnCl and K2Cr2O7 all caused an increase in the passive cell membrane permeability to [3H]dGlc-6-P. A membrane toxic concentration (MTC) was defined as the concentration of the tested metal compound giving rise to an increase in the relative efflux from 1.0 to 1.2 during 60 min perfusion. Using this MTC-value, the membrane toxicity of the compounds could be ranked in the following order: CH3HgCl (MTC = 9 x 10(-7) M) greater than HgCl2 (MTC = 6 x 10(-6) M) greater than (C2H5)3SnCl (MTC = 3 x 10(-4) M) greater than K2Cr2O7 (MTC = 7 x 10(-4) M). Since this differential toxicity is in accordance with other reports it is concluded that 2-deoxy-D-glucose (dGlc) may be used together with 41A3 cells to screen metal compounds for their membrane toxicity.

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.

On the application of cultured neuroblastoma cells in chemical toxicity screening.

The acute toxic action of a number of common chemicals was tested by their ability to cause detachment of cultured mouse neuroblastoma C1300 cells. A TD25 value was obtained by graphic estimation of the concentration needed to cause 25% of the total cell number to detach. These TD25 values were compared with LD50 values obtained from the literature, and they were found to correlate with a coefficient of 0.86. For six of the tested substances-diuron, butylated hydroxytoluene, benzidine, cyclophosphamide, Na2SeO3, and KCN-a very poor correlation was obtained. These diverging results could be ascribed to deficiencies in the neuroblastoma cell detachment test and emphasize the necessity for combined in vitro test procedures.

Cellular methods for identification of neurotoxic chemicals and estimation of neurotoxicological risk.

This review critically addresses key aspects of neurotoxicity that can be assessed by using in vitro test procedures and batteries. Such test schemes must most probably be hierarchical and multi-optional in order to be able to cope with the large number of possible mechanisms of neurotoxicity. Although the regenerative capacity of the nervous system is low, lesions can be compensated for by a number of cellular dynamic functions (e.g. intracellular calcium sequestration, membrane-bound ion transport systems and increases in the rates of energy metabolism and protein synthesis). Therefore, cellular tests included in primary screens of a multiple system should be based on determinations of cell physiological parameters rather than on measurements of single biochemical reactions. A general neurotoxicity test system for the determination of critical neurotoxic concentrations is suggested to include, as a first step, the assessment of basal cytotoxicity in a human neuroblastoma cell line. In a second step, differential cytotoxicity is assayed in highly developed primary cultures of neuronal and non-neuronal cells. In order to find out whether the compound is likely to produce axonopathy, a test procedure in mouse neuroblastoma cells is carried out. Toxicokinetic information is obtained from hepatocyte/target cell and endothelial cell/astrocyte co-cultures. To disclose alterations in cell physiology, studies of cell respiration, protein synthesis, membrane permeability and calcium homoeostasis are suggested. When information from these test steps is evaluated together with available data on in vivo toxicity, toxicokinetics and physical/chemical parameters, it may be necessary to proceed to more neuronal specific determinations or mechanistically oriented studies. If a consistent pattern of effects or non-critical and critical concentrations is found, the toxicokinetic distribution over the blood-brain barrier must be considered in relation to actual in vivo blood concentrations if estimates of neurotoxic risk are to be made.

GROWTH AND MORPHOLOGY OF NEURONAL CELL LINES CULTURED IN PERFUSION

SummaryTo optimize culture conditions and gain a more reliable culturing system for studies of metabolic properties of neuronal cells, a simplified perfusion chamber was developed. It consists of two parts: a perfusion block and a standard plastic culture dish. To confirm the suitability of this chamber for continuous culturing of anchorage-dependent cells, the growth and morphology of the four neuronal cell lines glioma C6 and glioma 138MG, neuroblastoma C1300, clones N1E115 and N18 were followed for 4 d using both traditional and perfusion techniques. A marked increase in growth and a decrease in the degree of morphological differentiation were obtained with the latter technique compared to the former.