Astrocytic regulatory functions: A possible target for CNS effects of organic solvents

Toxic. in Vitro Vol. 5, No. 5/6, pp. 503-506, 1991 Printed in Great Britain.All rights reserved

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ASTROCYTIC REGULATORY FUNCTIONS: A POSSIBLE TARGET FOR CNS EFFECTS OF ORGANIC SOLVENTS G. EKBLAD-SEKUNDand E. WALUM* Unit of Neurochemistry and Neurotoxicology, Stockholm University, S-106 91 Stockholm, Sweden Abstract--Primary astrocyte cultures from newborn rats were exposed to two model solvents, one chlorinated hydrocarbon (carbon tetrachloride) and one aliphatic hydrocarbon (n-hexane), for 1 hr in a closed chamber system. Both solvents caused a reduction in basal oxygen consumption by approximately 40%. Glutamate-stimulated respiration was reduced to a similar extent. Carbon tetraehloride caused a 100% increase and a 50% decrease, respectively, in the potassium- and A2 receptor-mediated elevation in cAMP levels. On the other hand, n-hexane affected only the basal level of cAMP, causing an eight-fold increase. These results indicate that carbon tetrachloride and n-hexane may interfere with essential astrocyte functions.

Introduction

to be the actual neurotoxic agent (Caprio, 1987). Little attention has been given to n-hexane itself and its ability to produce toxic effects in the absence of a metabolizing system.

Astrocytes serve to maintain adequate intercellular levels of ions, nutrients, metabolic intermediates and precursors in the central nervous system (CNS) (Hertz, 1977; Newman, 1985). Synaptic transmission may be regulated in part through interacting receptor and membrane transport functions (Hansson and R6nnb/ick, 1989). The type of generalized CNS effects seen in organic solvent intoxication may well be due to disturbances in these homoeostatic and regulatory functions. Reports during past years have stressed the neurotoxicological risk of organic solvents (Luse and Wood, 1967; MacFarland, 1986; Stevens and Forster, 1953). Little is known about the primary cellular target for the action of solvents. It therefore seems appropriate to investigate how organic solvents may interact with basic glial functions. The present investigation has been focused on basal and glutamate-stimulated respiratory activity (Peterson, 1985), as well as receptor- (adenosine A2 and adrenergic ill) and potassium-mediated increase in cAMP levels (Hansson, 1989). One chlorinated solvent (carbon tetrachloride) and one aliphatic hydrocarbon (n-hexane) were chosen as model compounds. Carbon tetrachloride is a potent hepatotoxin in both animals and humans. Its equally severe effects on the CNS have largely been neglected. Exposure to acute, high doses of carbon tetrachloride does give symptoms from the CNS such as severe headache, dizziness, blurred vision and constricted visual fields (Stevens and Forster, 1953). The aliphatic hydrocarbon n-hexane causes muscular weakness in the extremities (Schaumburg and Spencer, 1976) by inducing peripheral distal axonopathy, n-Hexane is metabolized to 2,5-hexanedione, which is considered

Materials and Methods

*To whom all correspondence should be addressed. Abbreviations: CNS = central nervous system; GFAP = glial

fibrillary acidic protein; NECA= 5'-(N-ethylcarboxamino)adenosine; PGE: = prostaglandin E:.

The following chemicals were used: n-hexane (pro analysi; Merck, Sp6nga, Sweden), carbon tetrachloride (pro analysi; Merck), 5'-(N-ethylcarboxamido)adenosine (NECA; Sigma, Poole, Dorset, UK), glutamate monosodium salt (Sigma), adenosine deaminase (Boehringer, Mannheim, Germany). Brain cultures, highly enriched in astroglial cells, were prepared according to Booher and Sensenbrenner (1972) from newborn ( < 24 hr) Wistar rats. For astrocyte cultures, cells from one hemisphere were plated in 10 dishes, while for the mixed astrocyte/oligodendrocyte cultures, cells from one hemisphere were plated in five dishes. The cultures were then incubated at 37°C for 28 days. Exposure to organic solvents was carried out using closed airtight chambers (Walum, 1989). In some experiments the chambers were placed in a tube which was rotated in a roller incubator. The cultures were exposed to the two solvents for 1 hr each in the chamber. The solvents were added to the chamber together with a phosphate buffered balanced salt solution to a final concentration of 80 ppm CC14 or 200 ppm n -hexane. Oxygen consumption was determined with a Clark oxygen electrode (Peterson, 1985). The basal level of oxygen consumption was registered during 15 rain. Glutamate was then added to a final concentration of 0.1 mM. The glutamate-induced effect on respiratory activity was determined after another 15 rain. The cAMP content of the cells was determined according to Brown et al. (1972) after pre-incubation with or without organic solvents followed by incubation in the presence of NECA (10-sM, 15rain), isoproterenol (10 -5 M, 15 rain), potassium ions (50 mu, 1 rain) or glutamate (0.1 mu, 15 rain). After completed incubations the cell culture morphology was

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Fig. 1. Time dependence of stimulated cAMP formation. Astrocytes from newborn rats were cultured for 28 days. The cells were then incubated for 15 min in the presence of 5'-(N-ethylcarboxamino)adenosine (10-s M; II), isoproterenol (10-5M; 4,) or glutamate (0.1 mM; A) or for 1 min in the presence of potassium ions (50 mM, 0). Each point represents the mean + SEM of 5 independent duplicate experiments. examined in a Leitz Diavert microscope. Some cultures were fixed and stained for glial fibrillary acidic protein (GFAP) according to Bignami and Dahl (1974). ResuRs

In accordance with previous observations (Hansson, 1982), cultures seeded at low density formed a monolayer of polygonal cells, with high specific staining for GFAP. Cultures plated at a higher density contained, in addition to the flat monolayer, numerous ( < 1 0 % ) rounded cells with short sparse processes. This population of cells has been identified as oligodendrocytes (McCarthy and deVellis, 1980). Exposure to carbon tetrachloride (80 ppm) or n-hexane (200 ppm) for 1 hr had no effect on the cell morphology. 50 mM solution of potassium ions induced a peak value in cAMP formation after 1 min. Glutamate induced a modest increase in the cAMP level over the first 2.5 min. NECA caused a rapid increase within the first 2.5 min and thereafter a steady but slow increase, whereas isoproterenol induced a maximum level after 2.5min and this level remained stable throughout the incubation time (Fig. 1). Carbon tetrachloride reduced the adenosine A2 receptor-mediated increase in cAMP from 153 to 81 pmol cAMP/mg protein (P < 0.05; Fig. 2). The potassium-induced level, on the other hand, increased from 105 to 221 (P < 0.05; Fig. 2). The adrenergic fl 1 receptor-mediated response in cAMP was not altered by carbon tetrachloride (Fig. 2). In cultures contain-

Iso

NECA

K+

Fig. 2. Effects o f c a r b o n t e t r a c h l o r i d e ( [ ] ) a n d n - h e x a n e ( I )

on cAMP formation in cultured astrocytes from newborn rats; O, unexposed. After pre-incubation for 1 hr with the solvents, isoproterenol (Iso, 10-5 M, 15 min), 5'-(N-ethylcarboxamino)adenosine (NECA, 10-5 M, 15 min) or potassium ions (K +, 50 n'L~, I min) was added to the cultures and the cellular content of cAMP was determined. Each value represents the mean of five independent, duplicate experiments + SEM. ing both astrocytes and oligodendrocytes, subjected to the roller incubation procedure, n-hexane produced a marked increase in the basal cAMP level, from 4.5 to 33 pmol cAMP/mg protein (P < 0.05; Table 1). This latter cAMP increase could not be seen in cultures containing only astrocytes, nor was it possible to reproduce in non-rolling cultures (Table l). Neither receptor- nor the 50mM-K +induced increases in cAMP concentration were altered by addition of n-hexane (Fig. 2). As has been described before (Peterson, 1985), the astrocytes were found to respond to glutamate (0.1 mM) with an elevated oxygen consumption. Both solvents decreased basal as well as glutamate-stimulated respiration (Table 2). Discussion

The results from the present investigation confirm that solvents like carbon tetrachloride and n-hexane exert toxic effects on glial cells. These results also show that there is a marked difference in Table 2. Effects of carbon tetrachloride and n-hexane on the respiratory activity in cultured astrocytes from newborn rats in the absence and presence of glutamate Oxygen (10 2 #l/rain/culture) Solvent

Control

Glutamate (100/zM)

Unexposed Carbon tetrachloride n-Hexane

4.4 ± 0.2 2.9 ± 0.5 2.4 + 0.2

13.5 ± 0.7 9.2 _+ 0.6 10.0 _+ 1.2

Each value represents the mean ± SEM of four experiments.

Table I. Effects of n-hexane on cultured astrocytes from newborn rats in the absence and presence of oligodendrocytes cAMP (pmol/mg protein) Control Culture Astrocytes Astrocytes + oligodendrocytes

n-Hexane (200 ppm)

Static

Rolling

Static

Rolling

2.8 + 0.4 (5) 7.4+1.8(4)

2.6 ± 1.5 (7) 4.5__ 1.1 ( l l )

2.2 _+0.4 (5) 8.9_+0.6(4)

5.0 -+ 1.4 (7) 33_+8.201 )

Two different incubation procedures were used, one providing the cells with a high oxygen tension (rolling) and one partially hypoxic (static). Each value represents the mean __ SEM of the number of independent duplicate experiments indicated in parentheses.

Solvent effects on glial cells effects between the two solvents. Carbon tetrachloride was found to enhance the observed high K+-evoked cAMP response, possibly through a general interaction with the plasma membrane integrity (Clemedson et al., 1990). This could result in an increased membrane permeability for ions, thus potentiating the K+-evoked depolarization, which in turn could accentuate the increase in cAMP formation (Nestler and Greengard, 1989; Tsien et al., 1988). Structural interactions with the A2 receptor itself, or with its surrounding lipids, may explain the effect of carbon tetrachloride on the adenosine A2 receptormediated cAMP response. A degree of specificity in this interaction is indicated by the fact that carbon tetrachloride lacked effect on the adrenergic fl 1 receptor-mediated cAMP response. This could be related to molecular differences between the two receptors or differences in their lipid environment. The effect of n-hexane on cAMP formation seems to be primarily correlated with the presence of oligodendrocytes. The elevated basal cAMP level could not arise from the oligodendrocytes, since they constitute only 10% of the cell population. Oligodendrocytes release prostaglandin E 2 (PGEz) (Kaeser et al., 1988), which is known to increase cAMP formation in astrocytes by way of receptor interaction (Hansson, 1988). Synthesis of prostaglandins in oligodendrocytes is dependent on molecular oxygen (Kaeser et al., 1988). In this investigation the elevated cAMP level also seems to be correlated with both oligodendrocytes and access of oxygen. The static incubation model provides a hypoxic situation (Werrlein and Glinos, 1974) as compared with the rolling model (Walum, 1989). It is thus tempting to suggest that the marked inc)-ease in cAMP induced by n-hexane in the astrocytes in the presence of oligodendrocytes and high oxygen tension (Table 1) is due to a PGE 2 release from the oligodendrocytes. One would expect the rolling procedure to increase the exposure of the cells. However, the possibility that n-hexane directly interacts with the astrocyte adenylate cyclase is excluded, since the marked increase in cAMP level was detected only in cultures containing oligodendrocytes. It is also excluded that n-hexane increases the cAMP level through inactivation of phosphodiesterases in the astrocytes; known phosphodiesterase inhibitors (theophylline and isobutylmethylxanthine) were found to have little effect on the cAMP level. Both solvents reduced the repiratory activity, both basal and glutamate-stimulated, to approximately the same degree. Such effects could, if they occurred in vivo, lead to impairment of regulatory processes of vital importance in the CNS (Hansson and Rtnnb/ick, 1989). The glutamate level in the synaptic cleft is to a large extent regulated by the astrocytes through a high-affinity uptake (Hansson, 1988). This uptake depolarizes the astrocytic plasma membrane (Enkvist et al., 1988) and activates the Na+/K ÷ATPase. These events will result in an increase in oxidative phosphorylation and oxygen consumption. Preliminary results indicate that n-hexane reduces the high-affinity uptake of glutamate in cultured astrocytes. Consequently, a decreased uptake of glutamate may result in a lower degree of depolarization and thus a lower demand for ATP.

505

Carbon tetrachloride and n-hexane have thus been found to interfere with some membrane-related processes in cultured astrocytes. On the assumption that these processes are representatively expressed in the in vitro model, one may conclude that the two solvents exert their effects on the CNS, at least in part, by disturbing astrocytic regulatory functions. Acknowledgements--This work was supported by grants from the Swedish Work Environment Fund (Grant No. 84-0142) and the National Swedish Environmental Protection Board (Grant No. 5324210-3).

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