Arch Toxicol (1990) 64:417-419 Archives of

Toxicology �9 Springer-Verlag 1990

Short communication

The influence of carbaryl on the uptake of (3H)-noradrenaline (3H)-NA by rat hypothalamic slices

Joanna Jablor~ska and Jacek Brzeziffski

Department of Toxicology, Medical Academy Warsaw, 1 Banacha Street, PL-02-097 Warsaw, Poland

Received April 13, 1989/Received after revision November 2, 1989/Accepted November 6, 1989

Abstract. The uptake of (3H)-NA by rat hypothalamic slices was investigated following carbaryl poisoning. The insecticide was administered to rats orally in a single dose of 50% LDs0 and of 20% LDs0, and in repeated dosing for 14 days (5% LD50 daily). The kinetics of (3H)-NA uptake was defined by the modified Lineweaver-Burk plots (Rmax was used instead of Vmax and Km was replaced with t05 max). The results indicated that in all the experiments carbaryl elevated the (3H)-NA uptake by hypothalamic slices in a dosage dependent fashion (insignificantly at the 20% LDso dose level, p >0.05) as evidenced by the in- creased Rmax values. However, carbaryl simultaneously decreased the kinetics of the process in the hypothalamus since I0.5rnax values were enhanced (insignificantly at the 5% LDs0 dose level, p >0.05). Carbaryl produced a dose- dependent increase of t0.Smax values. The changes in the (3H)-NA uptake may be related to the increase of NA level in the hypothalamus following exposure to carbaryl ob- served by other authors.

Key words: Carbaryl - Noradrenaline - Uptake - Hy- pothalamus-Kinetics.

Introduction

Carbaryl, a common representative of the N-methylcarba- mate insecticides has been shown to penetrate through the blood-brain barrier. The residues of carbaryl detected in the brain of rats after acute and chronic exposure (Hassan 1971; Mount et al. 1981).

Changes in EEG have been reported after acute and chronic carbaryl poisoning in humans (Kontek et al. 1973; Dickoff et al. 1987) and experimental animals (Santolucito 1970; Santolucito et al. 1971; Drsi et al. 1974). Moreover, intoxication with this insecticide is known to cause many

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clinical neurological symptoms such as incoordination, ataxia, intention tremor, clonic muscular contraction termi- nating with paraplegia and prostration, and neuromuscular syndrome. Histological studies have shown profound de- generative changes in various regions of the brain (Smalley et al. 1968; Smalley 1970; Vandekar et al. 1971; Branch et al. 1986). Dickoff et al. (1987) observed delayed neuro- toxic effects after ingestion of an acute dose of carbaryl by man.

Apart from these clinical observations, many biochem- ical data indicate that carbaryl affects the mammalian CNS not only by cholinergic mechanism but also by disturbing the physiological balance of other neurotransmitter sys- tems. Some authors have reported increased MAO activity in the rat brain during carbaryl exposure (Hassan et al. 1971; Ray et al. 1984). Hassan et al. (1971) observed correlation between changes in EEG and in the serotonin level in the rat brain.

It has been also shown that carbaryl increased excretion of adrenaline, noradrenaline and 3-methoxy, 4-hydroxy mandelic acid (VMA) in urine and simultaneously decreased NA level in the rat brain (Hassan 1971). Ray et al. (1985) reported that the insecticide induce changes in the striatal catecholamines. An acute intoxication with car- baryl enhanced NA level in the rat hypothalamus (Ray et al. 1984). In our previous study (unpublished data) changes in NA levels in the whole rat brain after acute, subacute and chronic carbaryl poisoning were also noticed.

The aim of this study was to examine the effect of a single acute dose (50% LDs0 and 20% LDs0) and small repeated doses of carbaryl on the kinetics of (3H)-NA uptake by the rat hypothalamic slices. As the recent papers indicate this type of experiments with carbaryl has not been performed so far.

Materials and methods

Animal and treatment. The experiments were carried out on male albino rats of the Wistar strain (180-200 g) given standard chow and water ad lib. The animals were maintained in 12h light cycle. All rats had been

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acclimated to the new environment for at least one week prior to testing. Carbaryl produced by Organika-Azot in Jaworzno (technical grade 99.9%) was used in all the experiments: the oral LDs0 value for male rats was 850 mg/kg as calculated according to the Weil method (Weil 1952). The experiments were performed in the following manner. To achieve acute intoxication carbaryl was given in doses: a single dose of 50% LDso and of 20% LDso while the rats were sacrificed by decapitation after 4 h. In the repeated low-dose intoxication experiment carbaryl was given in a daily dose 5% LDs0 for 14 days and 24 h after the last dose the rats were sacrificed. Carbaryl was suspended in soya bean oil and was adminis- tered per os intraventricullary. Control animals received the vehicle alone. The rat brains were rapidly removed and the hypothalamus was dissected according to the method of Glowinski and Iversen (1966).

The uptake of (3H)-NA by hypothalamic slices. Experiments were per- formed according to Snyder et al. (1968) and Carmichael et al. (1973).

Isolated rat's brains were placed in a cold (temp. 0 ~ C) Krebs-Ringer phosphate buffer (pH = 7.4) saturated with oxygen (118 mM NaC1; 15.9 mM Na2HPO4; 4,7 mM KCI; 1.8 mM CaCI2; 1.2 mM MgSO4; 1.3 mM EDTA; 5.6 mM glucose; 1.7 mM ascorbic acid). The hy- pothalamic slices approx. 0.5 mm thick were cut on an ice-cold glass plate in a drop of Krebs-Ringer buffer (t = 0~ The slices were weighed, placed individually into 25 ml flasks (approx. 50 mg wet weight in each) and preincubated for 5 min in 4.8 ml Krebs Ringer's buffer. The incubation medium was saturated with oxygen and the flasks were shaken at 70 to 80 strokes)/min in a water bath at 37 ~ C. After the preincubation period 0.1 ml of (3H)-NA (1 gCi/ml) solution/(L - 7 - (3H)-NA hydrochloride: 10.9 Ci/mmol. Radiochemical Centre Amer- sham. UK) and 0.l ml of unlabelled NA (1 ktg/ml) solution (L-nor- adrenaline-l-hydrogen tartrate; Sigma Chemical Co. St. Louis, MO USA) were added to the incubation medium giving a final concentration of NA 10 -7 M.

Samples were incubated for 5-60 min. At the end of the incubation period, the flasks were rapidly cooled up to 0 ~ C and their contents were transferred to 10-ml polyethylene tubes and centrifuged at 0 ~ C 3000 • g for 10 min. After decantation, the pellet was washed 3 times with 5 ml Krebs-Ringer buffer and centrifuged for 2 min. 1 ml of medium was added to 10 ml of dioxane scintillation fluid (4 g PPO, 200 mg POPOP, 60 g naphthalene, 100 ml methanol, 20 ml ethylene glycol dilute up to 1000 ml of dioxane). The tissue was dried on filter paper and digested for 2 h at 50* C in 1.5 ml solubiliser (Nuclear Chicago Solubiliser, NCS). After dissolving 0.068 ml glacial acetic acid and 10 ml toluene scintilla- tion fluid (6 g PPO, 75 mg POPOP in 1000 ml toluene) were added to each sample and radioactivity was determined in a liquid scintillation spectrometer (Nuclear Chicago Isocap 300).

The noradrenaline uptake into hypothalamic slices was calculated according to the formula:

R = dpm/g wet wt. of tissue dpm/ml medium

Data analysis. Statistical evaluation were carried out using Stu- dent's t-test. The kinetic analysis of the (3H)-NA uptake was performed by Lineweaver-Burk plots modified as follows:

Vmax was replaced by Rmax (max R value while t ~ oo) Km was replaced by to.s max (time needed to achieve 50% Rm~x value).

The kinetic parameters were calculated by a least squares curve fitting procedure.

Results

In the acute study at a dose of 50% LD50 the value of Rmax increased to 152% (p <0.001), but s imul taneously carbaryl inhibi ted the kinetics of the uptake because t0.5max was elevated to about 220% as compared to control (p <0.001). The kinetic analysis of the results at 20% LD50 dose level demonstrated an increase in the uptake expressed as the elevat ion of Rmax value to 146%, though the increase was insignif icant (p >0,05). S imul taneous ly the rate of the process was inhibi ted because t0.5max value increased to 152% of that observed in controls. That change was als0 nons igni f icant (p >0.05).

After repeated exposure to carbaryl (5% LDs0) the uptake of (3H)-NA by hypothalamic slices was also in- creased. The value of Rmax was elevated to 136% of the control value (p <0.01). The kinetics of N A uptake by hypothalamic slices in the exper iment was found to remain unchanged. The value of t0.5max was increased by only 11% (p >0.05), which means that the t ime needed to achieve the dynamic balance of the uptake process remained unchanged.

Discussion

The mechan i sm of neurochemical action of carbaryl on the N A uptake in the hypothalamus was studied. This brain region is responsible for different, physiological ly impor. tant, regulatory processes in the body. In the recent opinion the studies of neurotransmit ters uptake, as a sensitive in- dicator of alteration in the brain funct ion, are more impor- tant than measur ing the changes of their concentrat ion in the whole brain.

In this respect our results indicate that acute as well as repeated low-dose intoxicat ion with carbaryl significantly elevate the (3H)-NA uptake by hypothalamic slices in a dosage dependent fashion (Table 1). It is evident in the elevat ion of Rmax values. However, at the same time car- baryl decreased the kinetics of this process in the hy- pothalamus. The rate of achieving the dynamic balance state was decreased, because t0.5max value (time needed l0 achieve 50% m a x i m u m of uptake) was enhanced. Carbaryl produced a dose-dependent increase of tO.5max values (Table 1).

Judging from our kinetic experiments , the uptake process resembles an enzymat ic reaction. The changes ol3- served do not seem to result f rom the s t imulat ion of active transport across neuronal membrane . If such were the case,

Table 1. Kinetic parameters Rmax and t0.5 max calculated for (3H)-NA uptake by hypothalamic slices at all dosage levels of carbaryl

Dose Group Rmax % Control t0.5 (max/min) % Control

50% LD5o Control 6.90 + 0.30 5.72 ___ 1.12 Carbaryl 10.52+_ 1.61"* 152.46 12.55+- 2.52** 219.40

20% LDs0 Control 9.09 + 1.90 9.82 + 1.78 Carbaryl 13.33 + 3.45 146.64 14.96+- 4.02 152.34

5% LDs0 Control 13.33 +_ 1.73 18.08 + 2.35 for 14 days Carbaryl 18.18___ 1.97" 136.38 20.04+- 2.61 110.84

Each value is the mean ___ SD of 4 -6 experiments * P <0.01, ** P <0.001

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according to the principle governing the method employed, kinetic curves should have crossed in one point the axis of ordinates. The uptake process modified by carbaryl is of the Michaelis-Menten type, which has a clearly marked maximum and fixed equilibrium dynamic state between the tissue and the incubation medium.

Ray et al. (1984) showed that administration of car- baryl in acute dose (200 and 400 mg/kg) caused a signifi- cant increase in hypothalamus NA level. The kinetic anal- ysis presented in this study has revealed the increase of (3H)-NA uptake expressed as the elevation of Rmax value. This alteration of Rmax may explain, at least partially, the increase of NA level in the hypothalamus as a result of enhanced NA uptake.

In conclusion, it may be stated that carbaryl produced significant, dose-dependent changes in the uptake of (3H)- NA, but the mechanism underlying this action remains unknown.

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