~ Pergamon 0887-2333(94)E0052-U Toxic. in Vitro Vol. 8, No. 4, pp. 609-61 I, 1994 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0887-2333/94$7.00+ 0.00 IN VITRO EFFECTS OF THALLIUM ON MOUSE NEUROBLASTOMA CELLS G. REPETTO*, P. SANZ and M. REPETTO National Institute of Toxicology, PO Box 863. 41080--Sevilla, Spain Abstract--To compare the effects of thallium at different cellular levels, cultured mouse neuroblastoma cells (Neuro-2A) were exposed for 24 hr to thallium(I) acetate. The following toxic indicators were assessed in the in vitro test system: cell proliferation by quantification of total protein content of the culture; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase (LDH) leakage; lysosomal hexosamin- idase release; lactate dehydrogenase activity; mitochondrial succinate dehydrogenase activity; relative neutral red uptake by lysosomes; lysosomal hexosaminidase sphingolipid degradation activity; acetylchol- inesterase activity. The effects of thallium on the various indicators differed. Neural acetylcholinesterase activity was extremely sensitive to TI inhibition. In contrast, hexosaminidase, an enzyme involved in glycosphingolipid degradation, was stimulated prior to cytoplasmic membrane disruption detected as LDH leakage. Relative neutral red uptake was slightly more sensitive than cell growth inhibition and the reduction in hexosaminidase release suggests an interaction with lysosomes. The low degree of sensitivity of cell proliferation, as judged by the protein content of the cultures, may reflect inhibition of protein degradation. LDH glycolytic activity was severely inhibited, but succinate dehydrogenase activity in the citric acid cycle was increased, probably owing to the mitochondrial accumulation of thallium. INTRODUCTION The aim of this study was to compare the effects of thallium on mouse neuroblastoma cells at different cellular levels, including cell proliferation, cyto- plasmic membrane permeability, metabolism, lysosomal and mitochondrial functions and acetyl- cholinesterase activity. Thallium is the most highly toxic cumulative cation (Venugopal and Luckey, 1978). Despite legal restrictions on the use of thal- lium-based rodenticides, its use has been increasing in recent years because of growing resistance to the less toxic dicoumarol-based products. In addition, thal- lium has been found to be a useful biochemical tool (Douglas et al., 1990). Thallotoxicosis still occurs worldwide, therefore, and remains one of the most frequently occurring types of acute toxicity caused by any metal. Concern has also been recently expressed over the chronic consequences of excessive thallium release into areas surrounding cement plants (Manzo and Sabbioni, 1988). Thallium produces a distal (dying back type) and predominantly sensory, rapidly progressive neuropa- thy, with some involvement of the central nervous system. There is considerable evidence to suggest that the intracellular accumulation of thallium in nervous tissue is associated with alterations in biochemical *To whom correspondence should be addressed. Abbreviations: AChE = acetylcholinesterase; ECs0= med- ian effective concentration; HEX=hexosaminidase; LDH = lactate dehydrogenase; RNRU = relative neu- tral red uptake; SDH = succinate dehydrogenase. activities related to transmembrane ion transport, protein metabolism and energy-producing reactions, because of its tendency to form insoluble complexes with riboflavin. However, the precise mechanism of action is not well understood (Manzo and Sabbioni, 1988). MATERIALS AND METHODS Neuro-2A cells were plated at a density of 10,000 cells per well in 96-well tissue culture plates. After 24 hr, the culture medium was replaced with 0.2 ml medium containing thallium(I) acetate in solution (0, 0.1, l, 10, 100 and 1000 mg/litre) and incubated for another 24 hr. Cytotoxic determinations were carried out in the same 96-well tissue culture plates in which exposure took place, as previously described (Repetto et al., 1993). Cell proliferation, as judged by total cellular protein, was quantified in situ using Coomassie Brilliant Blue G-250 as previously de- scribed (Repetto and Sanz, 1993). Other indicators studied were LDH (EC 1.1.1.27) activity and leakage to the medium; hexosaminidase (HEX, N-acetyl-fl-o- hexosaminidase, EC 3.2.1.30) activity and release; relative neutral red uptake (RNRU) (Repetto and Sanz, 1993); succinate dehydrogenase (SDH, EC 1.3.99.1) activity in intact cells, determined by adding 1 mM KCN and antimycin A to the tetrazolium salt incubation medium in order to inhibit other dehydro- genases; acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, EC 3.1.1.7) activity was quantified on intact cells (Repetto et al., 1994). 609