Toxic. in Vitro Vol. 9, No. 4, pp. 375-379,1995 0887-2333(%)00028-3 Copyright 0 1995 Elsevicrscience Ltd Printed in Great Britain. All rights reserved 0887-2333/9S $99.50 + 0.00 Effects of Cobalt on Mouse Neuroblastoma Cells Cultured Iiz Vitro G. REPETTO*, P. SANZ and M. REPETTO National Institute of Toxicology, PO Box 863, 41080 Seville, Spain Abstract-To compare the effects of cobalt at different cellular levels, cultured mouse neuroblastoma cells (Neuro-2a) were exposed for 24 hr to cobalt(U) chloride. The following toxicity indicators were assessed: cell proliferation by quantification of total protein content; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage; lysosomal hexosaminidase release; lactate dehydrogenase activity (LDH); mitochondrial succinate dehydrogenase activity (SDH); relative neutral red uptake by lysosomes (RNRU); lysosomal hexosaminidase activity (HEX), and acetylcholinesterase activity (AChE). The effect of cobalt on the various indicators differed. Cobalt was not very toxic to neuroblastoma cell proliferation (EC,, = 200 PM). Cytoplasmic membrane permeability was not specifically increased, and LDH leakage occurred only at high concentrations, prior to the stimulation of HEX activity, an enzyme involved in sphingolipid degradation. In contrast, cobalt was lysosomotropic, with HEX release. The effects on lysosomal function were also studied with the RNRU, showing stimulation at low concentrations and inhibition at high concentrations. Neural AChE was decreased after an initial stimulation at low concentrations. LDH and SDH intracellular activities were both stimulated from low concentrations, mitochondrial SDH activity being the most sensitive marker studied. Metabolic stimulatory effects induced by cobalt were. therefore, more marked than changes in cytoplasmic and lysosomal membrane permeability. Introduction Cobalt is an essential micronutrient for mammals but, apart from its role in vitamin B,2, no other functions for cobalt in human nutrition have been established (Ellinder and Friberg, 1986). The main sources of human exposure to cobalt are (a) occu- pational, since cobalt is used in industry in high-speed tool steel alloys, in jet engines, in cemented carbide cutting tools, in magnets, as a pigment and in cata- lytic applications (Ellinder and F&erg, 1986); (b) clinical, in patients with implanted orthopaedic metal prostheses; and (c) domestic poisoning has also oc- curred, because cobalt chloride may be present in chemistry sets (Everson zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et al., 1988) and in crystal- growing sets sold in supermarkets and toyshops (Mucklow et al., 1990). The human effects of cobalt poisoning comprise cardiomyopathy, allergic der- matitis, hard metal respiratory alterations (Ellinder and F&erg, 1986) and neurological disturbances including loss of sense of smell, peripheral neuritis, optic atrophy, changes in brain electrical activity and nerve deafness (Domingo, 1989; Meechan and Humphrey, 1991). Cobalt can exert a variety of physiological and toxicological effects, but the precise mechanism of *Author for correspondence. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Abbreuiatiom: A&E = acetylcholinesterase, HEX = hexos- aminidase: LDH = lactate dehvdroaenase: RNRU = relative ‘neutral red uptaice; - SDH = succinate dehydrogenase. many activities of cobalt is as yet undisclosed (Domingo, 1989; Stokinger, 1981). For this reason, the aim of this study was to compare the effects of one of the inorganic chemical species of cobalt at different cellular levels. Neuro-2a mouse neuroblastoma cells were exposed for 24 hr in vitro to different concen- trations of cobalt(H) chloride. Toxicity indicators assessed in the in vitro test system were as follows: cell proliferation by quantification of total protein con- tent of the cell culture; cytoplasmic membrane in- tegrity to cytosolic lactate dehydrogenase (LDH) leakage; lysosomal hexosaminidase release; LDH ac- tivity; mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle; lysosomal function evaluated by the relative uptake of neutral red (RNRU) (Repetto and Sanz, 1993); lysosomal hexos- aminidase (HEX) sphingolipid degradation activity, and neuronal acetylcholinesterase (AChE) activity. Materials and Methods Toxicant exposure C1300 cell line, Neuro-2a clone (ATCC CCL 131; Flow, Irvine, UK) was grown in modified Eagle’s medium supplemented with 10% foetal calf serum (Flow). Cells were plated at a density of 10,000 cells/well in 96-well tissue culture plates (Costar). After 24 hr, the culture medium was replaced with 0.2 ml medium containing the test chemical in sol- ution (0, 0.1, 1, 10, 100 and IOOOmg/litre) and incubated for another 24 hr. TIV 9,- 375