Tolerance to the carbamate insecticide propoxur

Toxicology, 21 (1981) 267--278 Elsevier/North-Holland Scientific Publishers Ltd.

TOLERANCE

TO THE CARBAMATE

INSECTICIDE PROPOXUR*,**

LUCIO G. COSTA, H. HAND, B.W. SCHWAB and SHELDON D. MURPHY t

Division of Toxicology, Department of Pharmacology, University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77025 (U.S.A.) (Received May 21st, 1981) (Accepted July 9th, 1981) SUMMARY

Male mice were given the carbamate insecticide p r o p o x u r (2-isopropoxy phenyl methylcarbamate; Baygon® ) in the drinking water at weekly increasing concentrations (from 50 to 2000 ppm), for a period of 6 weeks. At the end of the treatment the LDso for p r o p o x u r was significantly higher in the treated animals as compared with controls. Propoxur-treated animals were also resistant to the hypothermic effect of an acute administration of the same c o m p o u n d . Groups of mice were challenged with the cholinergic agonist carbachol at intervals during the drinking water dosing and at its end. No differences in sensitivity to carbachol acute toxicity were found between control and treated animals. Propoxur-tolerant animals were also n o t resistant to the hypothermic effect of oxotremorine, another cholinergic agonist. [3H]Quinuclidinyl benzilate ([SH]QNB) binding (a measure of muscarinic receptor density and affinity) in forebrain, hindbrain and ileum never differed in control and treated mice. The possibility that repeated administrations of p r o p o x u r induced increased metabolic inactivation was tested by measuring hexobarbital sleeping time and carboxylesterase activity in treated and control mice. No changes in tissue carboxylesterase activities occurred b u t hexobarbital sleeping time was significantly reduced in propoxur treated animals suggesting an induction of hepatic microsomal enzymes. These results suggest that tolerance to p r o p o x u r is not mediated by a decrease of cholinergic receptors, as reported for other acetylcholinesterase inhibitors, b u t possibly by an enhancement of its metabolism. *Part of this study was presented at the 65th annual meeting of the Federation of American Societies for Experimental Biology, Atlanta, GA, April 12--17, 1981. **Supported in part b y grant No. ES 01831 from the National Institute o f Environmental Health Sciences. t T o whom reprint requests should be addressed. Abbreviations: ACHE, acetylcholinesterase; DMSO, dimethylsulfoxide; DTNB, 5,5dithiobis(2-nitrobenzoic)acid; TCA, trichloroacetic acid; [ 3H ] QNB, [ SH ] quinuclidinyl benzilate. 0300-483X/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Scientific Publishers Ltd.

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INTRODUCTION The wide use of insecticides in agriculture and home and garden applications has raised concern regarding the possible effects resulting from chronic exposure. Rider et al. [1] first demonstrated that chronic administration of an organophosphorus insecticide led to the development of tolerance to its toxicity. That is, after the first few days of administration of octamethylpyrophosphoramide, in which the typical signs of organophosphate poisoning were evident, rats almost completely recovered and were more resistant than controls to further administration of the same compound. Organophosphate tolerant animals are also less sensitive than controls to the action of different cholinergic agonists, such as carbachol or oxotremorine [2,3]. Tolerance to organophosphate toxicity can develop under conditions of exposure which do not produce overt cholinergic signs of poisoning [4], a condition which may better represent the chronic exposure of humans to low doses of insecticides. It has been recently demonstrated that tolerance to organophosphorus insecticides is associated with a decrease of muscarinic receptors, as measured by receptor binding techniques with the muscarinic antagonist [3H] QNB [3,5--7]. To our knowledge, there have been no reports regarding the development of tolerance after sub-chronic or chronic exposure to carbamate insecticides. However a few investigators have reported on the development of tolerance to physostigmine and neostigmine, 2 carbamate anticholinesterases used in clinical practice. Simpson [8] and Maayani et al. [9] reported tolerance to physostigmine; similar results were obtained by other investigators [10,11] who did not find any alteration of cholinergic receptors in tolerant animals. In contrast Chang et al. [12] and Costa et al. (unpublished) found a decrease of nicotinic and muscarinic receptors, respectively, in tissues (diaphragm and ileum) of animals tolerant to the carbamate neostigmine. The present study was undertaken to further investigate the possibility of an alteration of cholinergic receptors as a mechanism underlying the development of tolerance to carbamates. The widely used carbamate insecticide propoxur (2isopropoxypheny! methylcarbamate; Fig. 5) was the compound chosen for these experiments. MATERIALS AND METHODS

Animals and treatments

Male albino Charles River CD-1 mice, weighing 25--35 g at the beginning of the experiments, were housed in air~onditioned rooms under a constant temperature and light schedule (ambient temperature 25°C, 12 h dark). Food and water were available ad libitum. Propoxur (technical grade 98.8%; Mobay Chemical Corporation, Kansas City, MO) was added to the drinking water in increasing concentrations (from 50 ppm to 2000 ppm) as shown in Fig. 2. Due to the low solubility of propoxur in water, starting from the third week of treatment (300 mg/l), the compound was dissolved in a few

268

milliliters of ethanol (96%) and then diluted with water. The final concentration of ethanol ranged from 0.1% to 0.5%. The same a m o u n t of ethanol was added to the drinking water of the control group. Fresh solutions were made every 3 or 4 days, and water consumption and b o d y weights were checked. Analysis of water samples by high pressure liquid chromatography (HPLC) did not show any spontaneous degradation of propoxur. Each week a group of treated and control animals were challenged with carbachol (Mann Research Laboratories Inc., NY), 4.2 mg/kg, by i.p. injection. Forebrain, hindbrain and ileum were removed for [3H] QNB binding and acetylcholinesterase activity assays. For the LDs0 determinations, p r o p o x u r was dissolved in dimethylsulfoxide (DMSO) and administered orally in a final volume of 1 ml/kg, The LDs0 values were determined by the graphic m e t h o d or Miller and Tainter [ 1 3 ] .

[3H] QNB binding Forebrain, hindbrain and ileum were assayed for [3H] QNB binding by the m e t h o d of Yamamura and Snyder [14]. Tissues were homogenized (1 : 20) in Na2HPO4/KH2PO4 buffer, 0.05 M (pH 7.4 at 4°C) with a Brinkman Polytron homogenizer and centrifuged 3 times at 50 000 g for 10 min. Each time, the supernatant was discarded and the pellet resuspended in the phosphate buffer. Volumes of homogenate equivalent to 100--200 ~g of protein were incubated at room temperature for 1 h in 2 ml of buffer and approximately 0.19 nM [3H] QNB {29.4 Ci/mmol, New England Nuclear, Boston, MA). Atropine sulphate 10 -s M (Nutritional Biochemicals Corporation, Cleaveland, OH} was added to half of the tubes for the estimation of nonspecific binding. Bound radioactivity was separated from free by filtration through Whatman GF/C filters, washed 3 times with 3 ml of ice-cold buffer. The difference in [3H] QNB bound with and without atropine, represents the specific binding. The filters were placed in 10 ml of Liquiscint (National Diagonistic, Somerville, NJ) and c o u n t e d in a Packard Tricarb Scintillation spectrometer at an efficiency of 40%. Each determination was done in triplicate. Proteins were determined by the m e t h o d of L o w r y et al. [15], using bovine serum albumin as a standard.

Hypothermia During the experiments the mice were left free in their cages, except during the temperature measurements. Rectal temperature was taken as an index of b o d y temperature and measured by a thermistor m o u n t e d in a rectal probe connected to a Tele-thermometer (Yellow Springs Instrument Company, Yellow Spring, OH). The flexible thermistor probe was inserted 25 mm deep into the rectum. Two or three control measurements were taken for each mouse during an interval o f 30 min before the injection of p r o p o x u r or oxotremorine. The average value of these measurements was taken as the initial temperature at 0 time. During the temperature measurements the mice were k e p t in a plastic restrainer and the thermistor probe was retained in the rectum until a constant temperature reading was

269

obtained. Propoxur was administered by i.p. injection in DMSO and oxotremorine was given subcutaneously (s.c.) in distilled water. For each experim e n t a group of control mice were given the respective vehicle only.

Hexobarbital sleeping time Hexobarbital sleeping times were measured as described by Kamienski and Murphy [ 1 6 ] . A standard dose of 125 mg/kg of hexobarbital sodium (Winthrop Laboratories, New York, NY) was administered by intraperitoneal (i.p.) injection in distilled water, in a final volume dosage of 5 ml/kg b o d y weight. The mice were placed on their backs during the tests and after initially regaining the righting reflex, they were again placed on their backs. Three consecutive successful attempts to right themselves at 1-min intervals was considered to be recovery of the righting reflex. The time between the loss of righting reflex and the initial righting was recorded and considered as the sleeping time. Acetylcholinesterase and carboxylesterase assays Acetylcholinesterase (ACHE) activity was assayed by the colorimetric m e t h o d of EUman et al. [17] as modified by Benke et al. [18]. An aliquot o f tissue homogenate (equivalent to approx. 1 mg of tissue), 5 ~1 of 1.0 M acetylthiocholine (Sigma Chemical Co.) and 50 pl of 0.1 M 5,5-dithiobis(2nitrobenzoic) acid (DTNB, Sigma Chemical Co.) were added to an appropriate volume of sodium phosphate buffer 0.1 M (pH 8 at 25°C) to make a final volume of 5 ml. The absorbance was read immediately after the addition of the substrate acetylthiocholine and after 30-min incubation at 27°C. The initial absorbance, as well as a reagent blank absorbance were subtracted from the final reading. The change in absorbance during the incubation is due to formation of 5-thio-2-nitrobenzoate from DTNB and thiocholine, the hydrolytic product of acetylthiocholine. AChE activity in experimental animals is expressed as percentage of the activity of control animals included in each experimental series. F o r the determination of carboxylesterase activity, the m e t h o d of Murphy et al. [19] was followed, using a-naphthyl acetate as a substrate. Brain and liver were homogenized in 9 vols. of sodium phosphate buffer 0.1 M (pH 7.4 at 25°C) with a Brinkman Polytron homogenizer. Brain and plasma, 100 pl and 10 pl, respectively, were assayed w i t h o u t further dilution; liver homogenate was diluted 1 : 50 in the same buffer and 100 pl of this last solution were added to 3.5 ml of phosphate buffer, in triplicate. After the addition of 0.5 ml 40% trichloroacetic acid (TCA) to the first tube and 0.4 ml of 0.01 M a-naphthyl acetate to all tubes, the samples were incubated at 37°C for 30 min. The enzymatic reaction was terminated with the addition of 0.5 ml of 40% TCA, the mixture centrifuged at 700 rev./min for 10 min and the absorbance of the clear, supernatant solution was measured at 322 nm. The absorbance of the first tube was subtracted from the mean absorbance of the other 2 respective samples. The a m o u n t of a-naphthol liberated was determined by reference to a standard curve prepared with known quantities

270

of a-naphthol and the carboxylesterase activity is expressed as percentage of control activity.

Statistical analysis Statistical significance was determined by two-tailed Student's t-test on the means or by the analysis of covariance on the regression lines, according to Snedecor and Cochran [20], for the LDs0 determinations. RESULTS

As shown in Fig. 1, an acute administration of propoxur (10 mg/kg i.p.) produced 45--50% inhibition of AChE activity in ileum, brain and diaphragm. The inhibition was maximal after 20 min and no difference between treated and control activities could be detected after 2 h. Body weight gain of mice during the 6-week treatment with propoxur are shown in Fig. 2. Initial body weights were 30.22 + 0.23 (n = 130) (mean + S.E.) for control and 31.21 -+ 0.24 g (n = 130) for experimental animals. Final weights were 40.4 + 0.33 g and 39.28 + 0.37 g for control and experimental mice, respectively (n = 78--80). Student's t-test on the means did n o t reveal any difference between treated and control mice body weights. In contrast, unpretreated animals given the highest concentration {2000 mg/1) of propoxur in the drinking water, showed a drastic loss (more than 10%, P < 0.05) of body

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Fig. 1. Time profile of acetylcholinesterase inhibition by propoxur in mice brain, ileum and diaphragm. Propoxur (10 mg/kg) was administered by i.p. injection in dimethyl sulfoxide. Each point is the mean of at least 4 mice (-+ S.E.). Control values are averaged from 12 animals killed at 0, 60, and 120 rain. Fig. 2. Mice body weights during propoxur treatment. The concentration of propoxur in the drinking water (rag/l) is given in the upper part of the figure. Inset: Body weights of an additional group of 10 unpretreated mice given 2000 mg/l of propoxur in the drinking water for 1 week.

271

weight, after 1 week of treatment (Fig. 2 inset). Water consumption did not differ significantly between treated and control animals during the entire treatment (mean -+ S.E. = 5.05 -+ 0.29 and 5.61 -+ 0.16 ml/day/mouse, respectively). During the treatment no evident signs of AChE inhibitor poisoning were observed. The 2 deaths that occurred among the propoxurtreated mice were n o t accompanied by typical signs of anticholinesterase poisoning. At the end of the drinking water treatment the acute toxicity of p r o p o x u r was tested by determining its oral LDs0. The LDs0 was 25.4 -+ 2.5 mg/kg in the control and 44.5 -+ 3.9 mg/kg (P < 0.05) in the propoxurpretreated animals. Furthermore, propoxur-treated animals were more resistant than controls to the hypothermic effect of an acute dose of propoxur (10 mg/kg i.p.; Fig. 3). In contrast, no difference between propoxur-treated and control mice was found, in their response to an acute dose of the cholinergic muscarinic agonist, oxotremorine (0.1 mg/kg, s.c.; Fig. 4). At the end o f the treatment and at weekly intervals during the treatment, groups of control and propoxur-treated mice were also challenged with an acute toxic dose o f carbachol (4.2 mg/kg, i.p., corresponding to an approximate LDs0), a cholinergic agonist n o t affected by cholinesterase. Table I shows that propoxur-treated mice did n o t differ from controls in their response to carbachol. At the same test intervals, binding of [3H] QNB, a specific muscarinic antagonist, in forebrain, hindbrain and ileum was assayed. There were no significant differences in [3H]QNB binding to muscarinic receptors between treated and control mice (Table II). Furthermore, it is important to note that acetylcholinesterase activity in the propoxur-treated animals

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Fig. 3. Time profile of propoxur induced hypothermia in propoxur pretreated mice. Propoxur was dissolved in dimethyl sulfoxide and administered by i.p. injection at a dose of 10 mg/kg in a final vol. of 0.5 ml/kg of body weight, o, unpretreated animals given the vehicle only; ~, unpretreated animals given propoxur; D, propoxur-pretreated mice given propoxur, a,b,c: significantly different from unpretreated mice given propoxur; P < 0.05, P < 0.02, P < 0.01, respectively. Each point is the mean of 8 mice _+ S.E.

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Fig. 4. Time profiles of oxotremorme induced h y p o t h e r m i a in p r o p o x u r tolerant mice. Oxotremorine was administered s.c. in distilled water at a dose of 0.1 mg/kg (1 ml/kg). Each p o i n t is the m e a n of 6--8 mice -+ S.E. See the legend of Fig. 3 for e x p l a n a t i o n of symbols.

was seldom significantly inhibited, in spite of the rather high doses of prop o x u r administered (Table II). On the basis of these experiments it was concluded that the 6-week treatm e n t with p r o p o x u r had rendered the mice tolerant to p r o p o x u r toxicity b u t n o t more resistant than controls to the action of directly acting cholinergic agonists. Furthermore, [3HI QNB binding assays indicated no alterations of muscarinic cholinergic receptors. The principal identified metabolites of p r o p o x u r are shown in Fig. 5. Hydrolysis, probably mediated by a carboxylesterase or an amidase, and different reactions (hydroxylations, ethercleavage) mediated b y the hepatic mixed function oxidases, appear to be the primary metabolic pathways of p r o p o x u r [21]. Carboxylesterase activity was measured in liver, plasma and brain. The results indicated that carboxylTABLE I A C U T E T O X I C I T Y O F C A R B A C H O L IN P R O P O X U R - T R E A T E D MICE Mice were administered p r o p o x u r in the drinking water at weekly increasing concentrations ( 5 0 - - 2 0 0 0 rag/l). Carbachol, 4.2 mg/kg, was administered b y i.p. injection in distilled water (5 ml/kg). There were 10 mice for each group. Weeks of t r e a t m e n t

2 3 4 5 6

% Mortality Control

Treated

90 90 100 100 100

70 90 100 :100 100

273

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1 1 5 3 ± 25 1 2 2 2 ± 42 1203± 28 1 0 1 6 ± 81 1 0 5 8 ± 118

Control 1 0 9 6 ± 33 1 3 2 6 ± 36 1 3 1 0 ± 54 996±64 1 1 1 8 ± 52

Treated 90± 3 100+3 100 ± 2 8 0 ± 7* 95±4

341± 318± 361± -375± 21

11 10 27

Control 309±10 3 2 5 ± 15 372+16 -3 4 8 ± 11

Treated

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[3H ]Q N B Bound a

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Hindbrain

Forebrain

a f m o l e s / m g p r o t e i n ± S.E. b p e r c e n t a g e o f c o n t r o l activity ± S.E. *P < 0.05.

2 3 4 5 6

Weeks o f treatment

86+4 100± 5 94±3 97 ± 3 92 ± 3

AChE b

98± 6 102± 2 121± 9 9 6 ± 18 139± 9

Control

117± 9 1 1 6 ± 13 1 3 2 ± 16 79 ± 2 1 1 7 ± 11

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[3H ]Q N B Bound a

Ileum

94 ± 7 92±4 70±6" 90±8 91±8

AChE b

T h e mice were t r e a t e d w i t h p r o p o x u r in t h e d r i n k i n g w a t e r for a p e r i o d o f 6 w e e k s a t w e e k l y increasing c o n c e n t r a t i o n s ( 5 0 - - 2 0 0 0 mg/1). Six t o 10 m i c e were used for each d e t e r m i n a t i o n .

[ 3H ] Q N B B I N D I N G A N D A C h E A C T I V I T Y IN P R O P O X U R - T R E A T E D MICE

T A B L E II

T A B L E HI H E X O B A R B I T A L S L E E P I N G TIME A N D C A R B O X Y L E S T E R A S E MICE C H R O N I C A L L Y T R E A T E D WITH P R O P O X U R

A C T I V I T Y IN

Mice were given p r o p o x u r in the drinking water for 6 weeks according to the schedule of Fig. 2. The results are the m e a n ± S.E. (n) H e x o b a r b i t a l sleeping t i m e (min)

Control Treated

47.37 + 4.38 (12) 32.70 ± 1 . 4 6 " (12)

Carboxylesterase activity (% of controls) Liver

Plasma

Brain

100 ± 6 (5) 111 ± 11 (5)

100 ± 22 (5) 134 ± 23 (5)

100 ± 8 (9) 115 ± 3 (9)

*P < 0.005.

esterase activity was n o t significantly changed b y p r o p o x u r administration in the drinking water (Table III). However, hexobarbital sleeping time was significantly decreased in the propoxur-treated animals (Table III). This suggests, indirectly, that mixed function oxidases which detoxify p r o p o x u r may have been induced. In order to see if an induction of hepatic microsomal enzymes could reduce p r o p o x u r toxicity, groups of mice were treated with phenobarbital (100 mg/kg, i.p.) for 3 days. On the fourth day the animals were challenged with p r o p o x u r (10 mg/kg, i.p.) and rectal temperature and AChE inhibition were recorded 20 min after the injection. The hypothermic effect of p r o p o x u r was significantly lower in the phenobarbital pretreated mice (AT = --2.88 + 0.33°C and --1.82 + 0.13°C for control and pretreated animals, respectively; n = 5; P < 0.05). Diaphragm AChE activity CHEMICAL STRUCTURE

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Fig. 5. P r o p o x u r ( 2 - i s o p r o p o x y p h e n y l m e t h y l c a r b a m a t e ) , its principal identified m e t a bolites and their relative p o t e n c y for inhibiting h u m a n plasma cholinesterase (Data adapted f r o m O o n n i t h a n and Casida [21 ] ).

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(as percent of control) after p r o p o x u r administration was 55 +- 4 in control and 72 + 6 in the phenobarbital pretreated mice (n = 5; P < 0.05). DISCUSSION Administration of the carbamate insecticide p r o p o x u r in the drinking water for 6 weeks, at weekly increasing concentrations, did not affect normal weight gain. However, unpretreated animals, given the last and highest weekly concentration of dose (2000 ppm) showed a marked drop in b o d y weight within 1 week. Furthermore, the LDs0 of p r o p o x u r in propoxur-pretreated mice was significantly higher than in controls, and experimental animals were resistant to the hypothermic effect of a single acute i.p. administration of propoxur. These 3 observations provided strong evidence that propoxurpretreated mice had developed tolerance to propoxur acute toxicity. Propoxur tolerant animals, however, were never subsensitive to the lethal action of the cholinergic agonist carbachol. [3HI QNB binding in brain and ileum also was not different in control and pretreated mice. Moreover, propoxur-pretreated mice did not show any resistance to the hypothermic effect of oxotremorine, a specific muscarinic cholinergic agonist. Thus, although the animals were tolerant to p r o p o x u r toxicity, no indications of alteration of cholinergic receptors were found. These results differ from those reported after chronic treatment With organophosphates [3,5--7] and the carbamate drug neostigmine [12, Costa, L.G. et al., unpublished]. In those studies, a reduction in muscarinic receptor binding was observed in the tissues of tolerant animals. Failure to obtain evidence of altered cholinergic receptor sensitivity suggests that another mechanism must be responsible for the development of tolerance to propoxur toxicity. One hypothesis was that the animals developed tolerance by an induction of p r o p o x u r metabolism. The principal metabolites of p r o p o x u r were identified by Oonnithan and Casida [21] and are shown in Fig. 5. The 2 principal pathways are an hydrolysis, probably mediated by a carboxylesterase and different oxidative reactions (hydroxylations, ether-cleavage) that are catalyzed by hepatic mixed function oxidases [ 2 2 ] . Determinations of carboxylesterase activities in differe n t tissues did n o t show any significant increase in the propoxur-tolerant animals. However, hexobarbital sleeping time was significantly reduced. This is usually considered a good indication of induced microsomal enzyme systems [ 2 1 ] . It appears likely, therefore, that an induction of propoxur metabolism is responsible for the development of tolerance to propoxur. Although the evidence for this conclusion is only indirect, it is consistent with the findings of Neskovic [24] who reported that male rats, fed the insecticide carbaryl (2000 ppm) for 60 days, had an increase in c y t o c h r o m e P-450 in the liver and were also more resistant to the acute toxicity of p r o p o x u r (the LDso increased 1.34-fold). Our findings that a pretreatment with phenobarbital decreases p r o p o x u r toxicity in mice also suggests that the development of tolerance is due to an induction of propoxur detoxify-

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ing e n z y m e s ; h o w e v e r , a d d i t i o n a l m e t a b o l i s m studies are r e q u i r e d t o d i r e c t l y p r o v e this h y p o t h e s i s . A n o t h e r q u e s t i o n c o n c e r n s t h e r e a s o n w h y a decrease o f cholinergic r e c e p t o r s was f o u n d in o r g a n o p h o s p h a t e t o l e r a n t animals [ 3 , 5 - - 7 ] a n d n e o s t i g m i n e t o l e r a n t a n i m a l s [ 1 2 , Costa, L.G. et al., u n p u b l i s h e d ] , b u t n o t in m i c e t o l e r a n t t o t h e a n t i c h o l i n e s t e r a s e c a r b a m a t e p r o p o x u r . A differe n c e in t h e m e c h a n i s m u n d e r l y i n g t o l e r a n c e , b e t w e e n o r g a n o p h o s p h a t e s a n d c a r b a m a t e s , s e e m s u n l i k e l y since a d e c r e a s e o f b o t h n i c o t i n i c [ 1 2 ] a n d m u s c a r i n i c [ C o s t a , L.G. et al., u n p u b l i s h e d ] r e c e p t o r s was f o u n d in animals c h r o n i c a l l y t r e a t e d w i t h t h e c a r b a m a t e n e o s t i g m i n e . An e x p l a n a t i o n m a y be f o u n d b y e x a m i n i n g t h e d i f f e r e n c e in d u r a t i o n o f A C h E i n h i b i t i o n a f t e r t h e a d m i n i s t r a t i o n o f d i f f e r e n t A C h E inhibitors. T h e h a l f - t i m e o f A C h E a c t i v i t y r e c o v e r y a f t e r an a c u t e dose o f t h e o r g a n o p h o s p h a t e d i s u l f o t o n , giving m o r e t h a n 50% i n h i b i t i o n o f brain A C h E in rats, is a l m o s t 60 h [ 2 5 ] . Seven h o u r s a f t e r an a c u t e a d m i n i s t r a t i o n o f n e o s t i g m i n e (0.2 m g / k g , i.p.) to m i c e , i l e u m a n d d i a p h r a g m A C h E activity was still 50% i n h i b i t e d [Costa, L.G. et al., u n p u b l i s h e d ] . In c o n t r a s t t h e d u r a t i o n o f i n h i b i t i o n o f ACHE, f o l l o w i n g a c u t e a d m i n i s t r a t i o n o f p r o p o x u r , is c o n s i d e r a b l y shorter. Figure 1 s h o w s t h a t p r o p o x u r ( 1 0 m g / k g ) p r o d u c e d 4 5 - - 5 0 % i n h i b i t i o n o f A C h E in brain, i l e u m a n d d i a p h r a g m o f m i c e . O n l y 2 h later, h o w e v e r n o significant (P > 0.05) d i f f e r e n c e in A C h E activity c o u l d be d e t e c t e d b e t w e e n c o n t r o l a n d p r o p o x u r - i n j e c t e d animals. In v i t r o studies, using d i f f e r e n t cell c u l t u r e s y s t e m s , d e m o n s t r a t e d t h a t t h e decrease o f [3H] Q N B b i n d i n g a f t e r incubat i o n w i t h a cholinergic a g o n i s t is a t i m e - d e p e n d e n t p h e n o m e n o n [ 2 6 , 2 7 ] . In o u r in vivo s i t u a t i o n t h e fast r e c o v e r y o f A C h E a c t i v i t y a f t e r p r o p o x u r m a y n o t a l l o w a c e t y l c h o l i n e t o a c c u m u l a t e at t h e r e c e p t o r level f o r a suffic i e n t t i m e a n d m a y a c c o u n t f o r the l a c k o f cholinergic s u b s e n s i t i v i t y in t h e p r o p o x u r tolerant mice. REFERENCES 1 J.A. Rider, L.E. Ellinwood and J.M. Coon, Proc. Soc. Exp. Biol. Med., 81 (1952) 455. 2 J. Brodeur and K.P. DuBois, Arch. Int. Pharmacodyn., 149 (1964) 560. 3 L.G. Costa, B.W. Schwab, H. Hand and S.D. Murphy, Toxicol. Appl. Pharmacol. (1981) in press. 4 B.W. Schwab and S.D. Murphy, J. Toxicol. Environ. Health, in press. 5 F.J. Ehlert, N. Kokka and A.S. Fairhurst, Mol. Pharmacol., 17 (1980) 24. 6 F.J. Ehlert, N. Kokka and A.S. Fairhurst, Bioehem Pharmacol., 29 (1980) 1391. 7 B.W. Schwab, H. Hand, L.G. Costa and S.D. Murphy, Neurotoxicology, (1981) in press. 8 L.L. Simpson, Psychopharmacol. (Berl.), 38 (1974) 145. 9 S. Maayani, Y. Egozi, I. Pinchasi and M. Sokolovski, Psychopharmacology, 55 (1977) 43. 10 D.H. Overstreet and A. Schiller, Soc. Neurosci. Abstr., 9 (1979) 658. 11 I. Pinchasi, S. Maayani and M. Sokolovski, Biochem. Pharmacol., 26 (1977) 1671. 12 C.C. Chang, T.F. Chen and S.T. Chuang, J. Physiol., 230 (1973) 613. 13 L.C. Miller and M.L. Tainter, Proc. Soc. Exp. Biol. Med., 57 (1944) 216. 14 H.I. Yamamura and S.H. Snyder, Proc. Nail. Acad. Sci. USA, 71 (1974) 1725.

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