DZF-Project: Arie Bruinink

Investigation of neurotoxic compounds using serum-free cultures of embryonic chick neuronal retina, brain, RPE and meninges cells

A. Bruinink and coworkers (P. Reiser, J.P. Müller, F. Birchler, C. Sidler)

Inst. Toxicology, ETHZ&Uni Zürich

Current address, e-mail: Empa, Lerchenfeldstr. 5, 9014 St. Gallen, Switzerland


Keywords: In vitro- nerve cells- neurotoxicology – serum-free

Begin and End of the Project: 1990 – 1994

Background and Aim

Background: Cell culture systems may be used to elucidate the toxicomechanisms of compounds of interest which are difficult to assess using in vivo systems. At start of the project no serum-free in vitro system was available to study neurotoxic compounds. Furthermore, at that time nearly all primary in vitro systems used were based on primary mammalian cells.

Aim: The aim of the project was to develop a serum-free in vitro system enabling predictive statements on possible human neurotoxic compounds using primary embryonic chicken cells.

Methods and Results

Methods used and results obtained can be find in the following papers:

Bruinink A and Reiser P (1991) Ontogeny of MAP2 and GFAP antigens in primary cultures of embryonic chick brain: Effect of substratum, oxygen tension, serum and ARA-C. Int. J. Devl. Neurosci. 9, 269-279.

Bruinink A, Zimmermann G and Riesen F (1991) Neurotoxic effects of chloroquine in vitro. Arch. Toxicol. 65: 480-484

Bruinink A, Reiser P, Müller M, Gähwiler BH and Zbinden G (1992) Neurotoxic effects of bismuth in vitro. Toxic. in Vitro 6: 285-293

Bruinink A and Birchler F (1993) Effects of cisplatin and ORG.2766 in chick embryonic cell cultures. Arch. Toxicol. 67: 325-329

Müller JP and Bruinink A (1994) Neurotoxic effects of aluminium on embryonic chick brain cultures. Acta Neuropathol. 88: 359-366

Atterwill CK, Bruinink A, Drejer J, Duarte E, McFarlen-Abdulla E, Nicotera P, Regan C, Rodriguez-Farre E, Simpson M, Smith R, Veronesi B, Vijverberg H, Walum E, Williams DC (1994) In vitro neurotoxicity testing. The report and recommendations of the ECVAM Workshop 3. ATLA 22: 350-362.

Vedani A and Bruinink A (1996) Ochratoxin A und B: Ein dreidimensionales molekulares Modell zur mechanistischen Erklärung ihrer Toxizität. ALTEX 13:124-129.

Zbinden G, Bruinink A and Mitchell DB (1991) Toxicology of Bismuth. In: Proc. 1990 European meeting of the Toxicology Forum, pp. 585-612.

Bruinink A (1992) Serum-free monolayer cultures of embryonic chick brain and retina: Immunoassays of developmental markers, mathematical data analysis and establishment of optimal culture conditions. In: The Brain in Bits and Pieces (Ed. G. Zbinden) pp. 23-50, MTC Verlag, Zollikon.

Bruinink A (1992) Oculotoxicity in vitro. In: In vitro Methods in Toxicology. (Ed.: G.Jolles), pp. 353-366, Academic Press Ltd., London.

Bruinink A, Sidler C and Birchler F (1996) Neurotrophic effects of transferrin on embryonic chick brain and neural retina cell cultures. Int. J. Devl. Neurosci. 14: 785-795

Bruinink A, Rásonyi T and Sidler C (1997) Reduction of ochratoxin A toxicity by heat induced epimerization. In vitro effects of ochratoxins on embryonic chick meningeal and other cell cultures. Toxicol. 118: 205-210

Bruinink A and Sidler C (1997) The neurotoxic effects of ochratoxin-A are cell type specific, are reduced by protein binding but are not affected by l-phenylalanine. Toxicol. Appl. Pharmacol. 146: 173-179

Bruinink A, Faller P, Sidler C, Bogumil R and Vasak M (1998) Growth inhibitory factor (GIF) and zinc affect neural cell cultures in a tissue specific manner. Chem.-Biol. Int. 115: 167-174.

Harry GJ, Billingley M, Bruinink A, Campbell IL, Classen W, Dorman DC, Galli C, Ray D, Smith RA and Tilson HA, (1998) In Vitro Techniques for the assessment of Neurotoxicity. Env. Health Persp. 106  (Suppl. 1): 131-158.

Bruinink A, Rasonyi T and Sidler C (1998) Differences in neurotoxic effects of ochratoxin A, ochracin and ochratoxin-α in vitro. Natural Toxins  6: 173-177

Maier, P., Milosevic, N., and Bruinink, A. (2000) Co-cultures: mimicry of cellular interactions within a tissue and between organs-consequences for toxicity testing. In: Progress in the Reduction, Refinement and Replacement of Animal Experimentation. (eds. M. Balls, AM van Zeller and ME Halder), Elsevier Science BV, Amsterdam, pp 249-256.

Bruinink A., Yu D and Maier P (2002) Short term assay for liver cell activated drugs. Toxicol. in vitro 16, 717-724.

Conclusions and Relevance for 3R

A variety of compounds (e.g. cisplatin, chloroquin, Ara-C, canthaxanthin, aluminium, ochratoxin plus derivates, iron) were evaluated with the system that was developed in latter project. Our results revealed that the model system used have the potential to be predictive regarding the in vivo effects of test compounds on human brain and neural retina. Furthermore, in combination with hepatocytes statements can be made for the neurotoxic potential of organophosphate compounds.

Thus, the present system that was developed with the financial support of the Zbinden-Doerenkamp Foundation may be used not only to elucidate the toxic mechanism of neurotoxic compounds, but the system has also the capacity to deliver data being probably predictive for adverse effects that may be observed in humans after exposure. As a result less animal experiments are needed to make similar statements on the neurotoxic potential of compounds of interest.