Time-course of malaoxon-induced alterations in brain ... - Springer Link

Neurochemical Research, Vol. 14, No. 2, 1989, pp. 143-147

Time-Course of Malaoxon-Induced Alterations in Brain Regional Inositol-l-Phosphate Levels in Convulsing and Nonconvulsing Rats Maija-Riitta H i r v o n e n , ~,4 H a n n u K o m u l a i n e n , 2 Leo Palj~rvi, 3 and Kai Savolainen 2

(Accepted September 29, 1988)

The potential of a single dose of malaoxon (26.2 or 39.2 mg/kg i.p.) to produce convulsions and to increase cerebral levels of inositol-l-phosphate (InslP), an intermediate in phosphoinositide (PI) cycle, was followed for 1, 4, or 72 hr. The lower dose of malaoxon did not produce convulsions whereas the higher dose induced convulsions in 60% of the exposed rats. Malaoxon caused a dosedependent, at most 2-fold, increase in brain regional InslP levels in nonconvulsingrats as compared to controls. At the higher dose of malaoxon, in convulsing rats, the InslP-levels increased 4-fold above the control InslP-levels. In nonconvulsing rats, the InslP-levels reached their maximum 14 hr after the administration of malaoxon, whereas in convulsing rats the levels increased for 72 hr. The results suggest that PI-signalling is associated with convulsions produced by malaoxon. KEY WORDS: Malaoxon; convulsions; brain; inositol-l-phosphate.

INTRODUCTION

rat, stimulation of brain cholinergic systems by organophosphates produces convulsions which are accompanied by a marked increase in brain energy (3, 4) and phospholipid (5) metabolism, and accumulation of inositol-l-phosphate (InslP), an intermediate in phosphoinositide (PI) cycle (6-8). Neuronal membrane PIs are hydrolyzed in response to muscarinic receptor activation (9, 10). Facilitated PI hydrolysis results in the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) , a second messenger, which transmits signals from the receptor into the cell by releasing Ca 2+ from nonmitochondrial intracellular stores. This leads to elevation of free intracellular calcium, and subsequent activation of the cell (11, 12). Ins(1,4,5)P3 is sequentially dephosphorylated to InslP and inositol which is used in the resynthesis of PIs (13). InslP accumulation in the tissue has been used as an index for the activity of receptor-coupled PI signalling in vitro (12, 14) and in vivo (8, 15). In the present study, the potential of malaoxon to produce convulsions, and the time-course of malaoxon-

Brain cholinergic systems may play a crucial role in experimentally-induced convulsions as well as in human epilepsy (1, 2). Malaoxon, the active metabolite of the organophosphate insecticide malathion (S-1,2-bis (ethoxycarbonyl) ethyl 0,0-dimethyl phoshorodithioate), inhibits acetylcholinesterase (ACHE), and thereby causes accumulation of brain acetylcholine (ACh). ACh-induced overstimulation of postsynaptic neurones may be the key step in the cascade of events leading to cholinergic convulsions in malaoxon-exposed rats (2). In the 1 Department of Industrial Hygiene, University of Kuopio, P.O. Box 6, SF-70201 Kuopio. 2 Department of Environmental Hygiene and Toxicology, National Public Health Institute, P.O. Box 95, SF-70701 Kuopio. 3 Department of Pathology, University Central Hospital of Kuopio, P.O.B. 6, SF-70211 Kuopio, Finland. 4 To whom to address reprint request, present address: National Public Health Institute, Department of Environmental Hygiene and Toxicology, P.O. Box 95, SF-70701 Kuopio, Finland.

143 0364-3190/89/0200-0143506.00/09 1989PlenumPublishingCorporation

144 induced alterations in brain PI signalling, as expressed by brain InslP concentrations, were examined in malaoxon-exposed convulsing and nonconvulsing rats.

EXPERIMENTAL PROCEDURE Animals. Adult male Han/Wistar rats (National LaboratoryAnimal Center, University of Knopio, Kuopio, Finland) weighing 200250 g were used. The rats were on a 12 hr artificial light/dark rhythm (lights on from 7 a.m. to 7 p.m.) and housed in stainless steel cages. The rats had free access to food and tap water, and were allowed to acclimatize for 7 days to the environment prior to the experiments. Chemicals.Inositol-2-phosphate(Ins2P)was fromSigma (St. Louis, MO, U.S.A.). Tri-Sil Z, a mixture of trimethylsilyl-imidazole in dry pyridine (1.5 mEq/ml) was from Pierce (Rockford, IL, U.S.A.). Purified inositol-l-phosphate (InslP) as cyclohexylammoniumsalt (99.5% pure) was a generous gift from Dr. W.R. Sherman, Washington University, St. Louis, M.O.U.S.A. Malaoxon (97% pure) was from Riedel-de Haen AG, (Hannover, FRG). ExperimentalDesignand SamplePreparation.Rats were injected (i.p.) with saline (control) or malaoxon (26.2 or 39.2 mg/kg; these i.p. doses correspond to 0.6 and 0.9 x LDsovalues of malaoxon in this rat strain). After the administration of malaoxon, rats were followed for signs of organophosphate intoxication (salivation, respiratory depression, fasciculations, tremors) for 1, 4, or 72 hr, and then decapitated. Special attention was paid to malaoxon-inducedclonic or tonic-elonic convulsions. After decapitation, the brains were quickly removed and frozen in liquid Freon 12 (-70~ The frozen brains were manually cut into coronal sections, about 1 mm in thickness, and mounted on histological glass slides. Before cutting, the brains were allowed to warm up to about -10~ in a cold room (+4~ to make the brains easier to cut. The slides were quickly reimmersed in liquid Freon 12 to attach the sections to the slides, and stored in airtight plastic bags at - 70~ until further processed. Severalbilateral samples from the frozen coronal brain sections were collected at -20~ by using a needle (i.d. 1.2 mm) punch. The samples were taken from the piriform cortex, frontal cortex, thalamus, caudate, hippocampus and cerebellum. The brain areas were identified using a standard stereotaxic atlas (16). Bilateral samples from each brain region were lyophilized for 48 h at - 53~ after the addition of 500 pmol of Ins2P used as an internal standard. Dry sampleswere weighed at room temperature and derivatized without further extraction of the samples. They were allowed to react with 100 Ixl of Tri-Sil Z overnightat room temperature after heating the mixture in a water bath for 10 min at 60-80~ The samples were analyzed immediately after the derivatizatiou on the following day (17). Gas Chromatography of Inositol-l-Phosphate. The derivatized tissue samples were analyzedwith Perkin Elmer Sigma 2000 gas chromatograph on a fused silica SE-30 capillary column (length 15 m; i.d. 0.32 ram; film thickness (0.25 Ixm)with a flame ionization detector. The quantitation was made by Perkin Elmer LCI-100 computing integrator. The operation conditions were as follows: injector temperature 240~ oven temperature program 180~ to 230~ 7~ 230~ to 250~ 30~ and detector temperature 270~ Nitrogen (1.5 ml/ min) was used as carrier gas. Attenuation range was 4-32 (17). StatisticalAnalysis.The data were analyzedusing one-wayanalysis of variance and Duncan's Multiple Range test. Independent means were compared using Student's t-test. P < 0.05 was considered statistically significant.

Hirvonen, Komulainen, Palj/irvi, and Savolainen RESULTS

Behavioral Changes. The lower dose of malaoxon (26.2 rng/kg) did not produce convulsions, but cholinergic signs, i.e., salivation, fasciculations, and tremors, appeared within 10-20 min after the malaoxon injection. The higher dose of malaoxon (39.2 mg/kg) produced tonic-clonic convulsions in 60% of the exposed rats. Malaoxon-induced intoxication usually progressed to tonic-clonic convulsions within 60 rain after the malaoxon injection. The convulsions were associated with Straub tail and preceded by extreme salivation and tremors (5-10 and 10-20 rain after the malaoxon injection, respectively). The lethality of convulsing rats was 28%, and in these rats, death took place within 10-20 min after the malaoxon injection. Nonconvulsing rats exposed to the higher dose of malaoxon had also peripheral cholinergic signs (salivation, chewing, fasciculations, tremors, and respiratory depression) beginning 15 rain after the injection, but no deaths occurred. Brain Regional Inositol-l-Phosphate Concentrations. The regional concentrations of InslP in saline treated control rats were 200-250 ixmol/kg dry brain weight (Figures 1 and 2). In nonconvulsing rats, malaoxon caused a dose-dependent increase in InslP levels, particularly in the cortex, which levelled off 1-4 hours after the administration of malaoxon. In convulsing rats, malaoxon produced a marked increase of InslP levels in all brain regions which lasted at least for 72 hours. At the lower dose of malaoxon (26.2 mg/kg), which did not produce convulsions, the concentrations of InslP were not yet markedly changed at one hour after the administration of malaoxon, but there was a 34-67% increase in the regional InslP levels four hours after the injection. The increase was, however, statistically significant only in the frontal cortex (Figures I and 2). At 72 hr after the administration, InslP levels were still significantly elevated in the frontal cortex but not in other brain regions (Figs. 1 and 2). At the higher dose of malaoxon (39.2 mg/kg), the InslP levels were always markedly higher in convulsing than in nonconvulsing rats. The differences in InslP levels between convulsing and nonconvulsing rats were usually most striking at 72 h. One hour after malaoxon administration, in nonconvulsing rats, a 17-58% increase in brain regional InslP concentrations occurred, but the change was statistically significant only in the frontal cortex (Figures 1 and 2). In convulsing rats, at the same time point, the InslP levels were elevated 7%216% above the level of controls in analyzed brain regions (Figures 1 and 2). Four hours after the higher dose of malaoxon, the

Malaoxon-Induced Alterations in Inositol-l-Phosphate F R O N T A L CORTEX

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Fig. 1. The time-course of malaoxon-induced alterations in cerebral inositol-l-phosphate (InslP) levels in the frontal cortex, piriform cortex and caudate of convulsing (C) and nonconvulsing (NC) rats. The i.p. doses of malaoxon were 26.2 and 39.2 mg/kg corresponding to 0.6 and 0.9 • LD50 doses of malaoxon, respectively. Malaoxon-induced alterations in cerebral InslP levels were followed for 1, 4, or 72 h. * P