[Report on the ICCVAM workshop on in vitro methods for assessing acute systemic toxicity]. / Ergebnisse des ICCVAM Workshop über in vitro Methoden zur Bewertung der akuten systemischen Toxizität.

It was suggested in the ICCVAM workshop that the Register of Cytotoxicity (RC), using in vitro cytotoxicity data to predict the in vivo starting doses, should be implemented into acute toxicity testing as soon as possible. The validity of the in vitro cytotoxicity data to establish appropriate starting doses for acute toxicity testing will be assessed experimentally. Secondly, in order to replace the use of animals in acute lethality testing a formal validation will be conducted in which the ability to predict rodent LD50 values and toxicity classes from cytotoxicity data will be evaluated.

[Prediction of human lethal concentrations by cytotoxicity data from 50 MEIC chemicals]. / Zur Prädiktion der letalen Konzentration beim Menschen mit Hilfe von Zytotoxitätsdaten von 50 MEIC-Stoffen.

Procedures for predicting human toxicity on the basis of cytotoxicity data for different chemicals are of current interest. The study was designed to clarify the possibility of predicting human toxicity by using the cytotoxicity values IC(50x) of the 50 MEIC chemicals listed in the Registry of Cytotoxicity (RC). All calculations with the data of cytotoxicity and in vivo toxicity were carried out uniformly by using standardised methods with the aim of comparing the results in the literature with each other. The following results were achieved: Firstly, the IC(50x) values in the RC are suited better for predicting human toxicity than IC(50) values determined in cell culture experiments with one cell type and one cytotoxic endpoint. This result is correct for the values of linear regression parameters as well as for the values of the prediction error (PE) defined by Ponsoda et al. (1997). Secondly, by using the geometrical mean of four lethal concentrations (LCx) - calculated from the tabulated lethal concentration (LC), lethal plasma concentration (LPC), clinical lethal concentration (CLC) and forensic lethal concentration (FLC) - the parameters for predicting the human toxicity were slightly improved. Moreover, these comparative examinations demonstrate that in all cases the cytotoxicity data are suited better for predicting acute animal toxicity than for predicting acute human toxicity. The results confirm, once more, the reliability and general validity of RC cytotoxicity data for examinations of different problems in cytotoxicology.

[Interaction of liposomes with vascular endothelial cells]. / Wechselwirkung von Liposomen mit Gefässendothelzellen.

Confluent monolayers (3 days old) of cultured calf aortic endothelial cells (line BKEz-7) were incubated for 30, 60 or 120 min with liposomes (reverse-phase evaporation vesicles) in order to investigate by electron microscopy whether and how they interact. This study demonstrates such interactions on a large scale. Electron micrographs show different ways by which liposomes interact with vascular endothelial cells. The observed mechanisms are adsorption, fusion, endocytosis and a transcellular passage of intact liposomes.

[Is the prediction of LD50 using cell cultures generally valid?]. / Besitzt die Vorhersage der LD50 mit Hilfe der Zellkultur Allgemeingültigkeit?

In this article we use the linear regression model to compare the values of IC50 and LD50 p.o. (mice and rats) taken from the literature and estimated in our laboratory. By this analysis it was found that for different substance classes a similar relationship exists between IC50 and LD50, even if different cell types or cellular activities were used for estimating the IC50 on the one hand and different modes of substance application in animal toxicity tests on the other hand. The minimum and maximum values of the measured LD50 (LD50G) registered in animal experiments deviated from the theoretical LD50 values (LD50T) on the (log transformed) regression line in approximately 77 per cent of the cases by the factor FG less than or equal to log 5 only. The linear correlation between IC50 and LD50 is to be expected only for such substances which do not act specifically via higher levels of integration, cell type specific reactions or metabolites, respectively. These and previously reported results indicate a certain general validity not only for the positive linear correlation between IC50 and LD50 but also for the predicting the LD50 in a relatively narrow dosage range on the basis of in vitro estimated IC50 values. With these results new possibilities were opened for reducing animal experiments to estimate LD50.

[Prediction of LD50 values by cell culture]. / Vorhersage von LD50-Werten mit der Zellkultur.

Alternative approaches to toxicity tests with animals-more precisely defined as complementary methods-serve for reducing the number of animal experiments. The determination of cytotoxicity with cell cultures could be an important complementary method to get more information about a general basal toxicity of a substance in an organism during the course of an acute toxicity experiment. We employed the endothelial cell line BKEz-7 in a general (i.e. nonspecific) cytotoxicity test. As a quantitative marker of cytotoxic effects we determined the inhibition concentration for a fifty per cent reduction of the cell number per culture (IC50). the IC50 values of 28 compounds were compared with the corresponding LD50 values in the acute toxicity experiments. In these comparisons we included the determination of the lethality index (LI = IC50/LD50), the correlation analyses and the application of a single linear regression model. The following results were obtained. Between the IC50 and LD50 values a positive correlation exists at the significance level 1 - alpha = 0.95. After plotting the regression line in a coordinate system, a novel graphic prediction of LD50 estimations is possible with a relatively high accuracy. On the basis of tenfold steps of concentrations a new scale of cytotoxicity was attributed to the internationally employed classification of LD50. In the cytotoxicity test with the cell line BKEz-7 a broad spectrum of substance classes can be tested. The cytotoxicity test and the comparison of IC50 and LD50 p.o. as described here contribute to reduce the number of animals in the acute toxicity experiments.

[Effect of a proliferation- stimulating preparation from bovine brain on cultured calf aortic endothelial cells]. / Zur Wirkung eines proliferationsfördernden Rinderhirn-Präparates auf Endothelzellkulturen aus Kälberaorten.

A preparation from bovine brain (HS-3) stimulates the proliferation of a calf aortic cell line (BKEz-7) in concentrations between 50-200 micrograms/ml. It is especially effective at low inoculum cell density, it economizes fetal serum and is very efficient as additive for the improvement of inadequate sera. Important questions for the practical use as the properties of the preparation with regard to durability and sterile filtration have been investigated.

The membrane potential of pig aortic endothelial cells.

Membrane potential and cell number of endothelial cells from pig aorta (line BSEz-3) were determined in vitro in different phases of growth. We found different membrane potentials for mononuclear (-8.6 mV) and multinuclear (-21.9 mV) endothelial cells. In both cases the maximum membrane potential occurred on the 6th day after seeding (during the exponential growth phase). The fraction of multinuclear endothelial cells was about 2.1% in the total culture. The membrane potentials of pig aortic endothelial cells are similar to our earlier values obtained from calf aortic endothelial cells.

[Establishment and characterization of the pig aortic endothelial cell line BSEz-3 in a serum-reduced medium]. / Etablierung und Charakterisierung der Schweineaorten-Endothelzellinie BSEz-3 in einem serumreduzierten Nähr-medium.

The cell line BSEz-3 was established in a serum-reduced medium MEMPAS. The cells were isolated and cultivated under the same conditions as the calf aortic endothelial cell line BKEz-7. With regard to cell isolation and cell density in the stationary phase, BSEz-3-cells show characteristic differences from BKEz-7-cells. For the isolation of BSEz-3-cells an enzymatic-mechanical method was more successful than a mechanical technique only. The low saturation density in the stationary phase with an average number of 50,000 cells/cm2 of glass surface area is considered as a strong contact inhibition of cell proliferation. As a cell type specific marker the BSEz-3-cells contain factor-VIII-antigen and possess angiotensin-converting enzyme activity. For a practical use of the BSEz-3-cells in drug research we give recommandations regarding cell inoculum density for subcultivation and cryo-conservation in ampoules.

Metabolism of [1-14C]-arachidonic acid by cultured calf aortic endothelial cells: evidence for the presence of a lipoxygenase pathway.

Intact cultured calf aortic endothelial cells from a 10th-14th subculture rapidly metabolize exogenous [1-14C]-arachidonic acid by three different routes: i) incorporation into triglycerides and phosphilipids in a ratio of about 2:1, ii) formation of lipoxygenase metabolites (12-hydroxy-5,8,10,14-eicosatetraenoic acid) and iii) formation of cyclooxygenase metabolites (6-keto-PG F1 alpha and PG F2 alpha). From analyses by thin-layer chromatography and high-pressure liquid chromatography it was established that the main lipoxygenase metabolites in intact cells are 12-hydroxy-5,8,10,14-eicosatetraenoic acid and a compound proposed to be (a) dihydroxyeicosatetraenoic acid(s). In frozen and thawed cells the incorporation of arachidonic acid into cellular lipids is abolished, whereas the lipoxygenase pathway is strongly enhanced. Under these conditions the cells produce predominantly 15-hydroxy-5,8,11,13-eicosatetraenoic acid in addition to 12-hydroxy-5,8,10,14-eicosatetraenoic acid. The formation of lipoxygenase products was inhibited by heating the cells or by preincubation with nordihydroguaiaretic acid or BW 755 degrees C, whereas indomethacin was without effect. The formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid by intact cells was inhibited by 5,8,11-eicosatriynoic acid. Indomethacin and acetylsalicylic acid inhibited the formation of cyclooxygenase metabolites. 15Ls-hydroxy-5,8,11,13-eicosatetraenoic acid was incorporated into cellular lipids, but not dihydroxyeicosatetraenoic acids. Exogenous [3H]-labelled prostacyclin and TxB2 were not incorporated but were metabolized to less polar products.

[Effect of growth factor preparations on the proliferation of in vitro cultivated aortic endothelial cells]. / Wirkung von Wachstumsfaktor-Präparationen auf die Proliferation in vitro kultivierter Aorten-Endothelzellen.

A functionally and structurally well-defined calf aortic endothelial cell line has been used to characterize tissue preparations with mitogenic activity by means of cell proliferation kinetics. The following preparations were tested: An extract from corpora lutea (pig), produced by stepwise ammonium sulfate precipitation (CL-S3), a fraction obtained from CL-S3 extract by ion exchange chromatography (CM-S4), and a chorioallantoic membrane preparation (CAM preparation). Under optimal cultivation conditions 100 micrograms CL-S3, 5 micrograms CM-S4, or 100 micrograms CAM preparation per ml medium increased the cell number and growth rate of the endothelial cells in approximately like manner after an incubation period of 48 hours. Compared with CL-S3 these results demonstrate a 20 fold enrichment of the mitogenic activity in the CM-S4 fraction. Under suboptimal cultivation conditions (i.e. a low cell number at the seeding and a reduced nutritive quality) 100 micrograms CL-S3 per ml medium stimulated the cell proliferation up to 12 times at the end of a 6-days-cultivation-period. These results are discussed in connection with the use of these preparations in the angiogenesis research and with respect to the application of selected preparations in cell culture media in order to reduce the serum concentrations.

Membrane potential of vascular mono- and multinuclear endothelial cells cultured in vitro.

Membrane potential (-19.1 mV) and fraction (about 1%) of multinuclear endothelial cells from calf aorta (in vitro) were determined and compared with mononuclear cells (-8.2 mV).

[Effect of blood and tissue factors on the migration of cultured endothelial cells of calf aorta]. / Wirkung von Blut- und Gewebefaktoren auf die Migration von kultivierten Endothelzellen der Kälberaorta.

Fractions of extracts of porcine corpora lutea, of bovine brain with fibroblast growth factor activity and whole blood serum versus plasma derived serum were tested using Boyden technique and agarose migration with regard to their influence on the migration behaviour of endothelial cells of calf aortas. All factors showed a concentration dependent stimulation of random migration of the cells. Only whole blood serum versus plasma derived serum had chemotactic activity. Thus it must be supposed that vascularization processes will be only favoured by the first mentioned two factors whereas angiogenesis will be possibly triggered by factors released from blood platelets during blood coagulation.