[Histochemical demonstration of beta-glucuronidase and leucine aminopeptidase in cultivated aortic endothelial cells (author's transl)]. / Zum histochemischen Nachweis von beta-Glucuronidase und Leucinaminopeptidase in kultivierten Gefässendothelzellen.

With the aid of histochemical techniques, the activity of beta-GD, LAP, NADH-D, and LDH of in vitro cultivated calf aortic endothelial cells were compared with the activity of these enzymes in different tumours in vivo. The relative high level of beta-GD, NADH-D, and LDH in the endothelial cells is comparable with the increased activity of these enzymes in invasive growing tumours. The results were discussed in connection with a possible enzymolytic effect during the invasive growth of capillary endothelial cells in course of neovascularization in vivo.

[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.

[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.

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 a chorioallantoic membrane preparation on the proliferation of cultivated vascular endothelial cells and mouse embryo fibroblasts]. / Die Wirkung einer Chorioallantoismembran-Präparation auf die Proliferation kultivierter Gefässendothelzellen und Mäuseembryofibroblasten.

We investigated whether the chorioallantoic membrane (CAM) has growth stimulating capacity for in vitro cultivated calf aortic endothelial cells and mouse embryo fibroblasts. Pieces of chorioallantoic membranes of 12 days incubated hen eggs were collected in aqua bidest. and lyophilized. After further mechanical dispersion the preparations were added to the culture medium. In the range of 10 to 100 micrograms per ml medium CAM-preparations stimulate the proliferation of both cell types significantly, whereas 500 micrograms/ml exhibit cytotoxic effects. The relatively simple manner of preparation in connection with the use of cultivated cells may be a new and simple way for testing different tissues and organs with respect to their proliferation modulating properties.

[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.

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.

[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.

DNA synthesis in cultures of calf aortic endothelial cells treated with corpus luteum extract: its inhibition by 3H-thymidine.

In cultures of calf aorta endothelial cells treated with extracts from corpora lutea a paradoxical effect occurred: Cell proliferation was regularly stimulated, but DNA synthesis was more or less inhibited. This inhibition was related to the presence of 3H-thymidine and could be reduced by lowering te 3H-thymidine concentration in the incubation medium. To decide whether the molar concentration or the activity of the 3H-thymidine was responsible for this effect, unlabelled thymidine was added to increase the molar thymidine concentration. A mixture of unlabelled thymidine and 3H-thymidine (ratio 200:1) added to the medium at various concentrations stimulated the DNA synthesis in a dose-dependent was up to 4.1 x 10(-6) M. Furthermore, nonradioactive thymidine slightly promoted the proliferation of calf aorta endothelial cells up to 10(-6) M independently of the presence or absence of corpus luteum extract. It is concluded that the radioactivity of 3H-thymidine is responsible for this effect. Internal 3H-beta radiation may perturb cell cycle progression after incorporation of this radioactive precursor into the cells. This view is supported by the fact that the inhibitory effect can be reduced by adding unlabelled thymidine, i.e. by lowering the specific activity of 3H-thymidine. On the other hand, increasing concentrations of growth promoting factors which stimulate the uptake of 3H-thymidine render the cells more sensitive to this effect.

[Electrophysiological studies of in vitro cultured endothelial cells from the calf aorta]. / Elektrophysiologische Untersuchungen an in vitro kultivierten Endothelzellen aus Kälberaorten.

The relation between membrane potential (MP) and cell number was studied on cells of a calf aortic endothelial cell line. The intracellular recording of membrane potentials was made with glass microelectrodes. From the 2nd to the 6th day of cultivation the membrane potential increased with the exponentially increasing cell number from --2.8 mV to --9.9 mV. Thereafter the membrane potential decreased. The membrane potential reached its maximum only 1 day after the cell culture had reached its stationary growth phase.

[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.

[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.

[The effect of a synthetic tripeptide nervous tissue cultured in vitro]. / Uber die Wirkung eines synthetischen Tripeptids auf in vitro kultiviertes Nervengewebe.

Explants from chick embryo PNS (ganglion trigeminale) and from CNS of embryonal rats (hippocampus) and dissociated cells from chick embryo cerebral hemispheres were cultivated in maximow chambers in the presence of various concentrations of placental serum and of a chemically synthesized tripeptide Gly-His-Lys. 1. The presence of tripeptide in the nutrient medium with a low concentration of serum did not compensate the outgrowth of nerve fibers, that take place in the growth medium. 2. In the presence of tripeptide in the nutrient medium with low concentration of serum the index of growth area increased significantly. 3. Within the first days in cell cultures 0,01 microgram tripeptide pro ml medium stimulated the outgrowth of neuronal processes. 4. The experiments indicated, that the tripeptide did not replace the serum. The possible role of tripeptide as a system in controlling neuron-glial ratio in vitro is discussed.