Changes in ACh levels in the rat brain during subacute administration of diisopropylfluorophosphate

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

w,477-489

(1987)

Changes in ACh Levels in the Rat Brain during Subacute Administration of Diisopropylfluorophosphate D. K. LIM, A. B. PORTER, B. HOSKINS, ANDI.

K. Ho’

Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505

Received December 8.1986; accepted June 1I I98 7 Changes in ACh Levels in the Rat Brain during Subacute Administration of Diisopropylfluorophosphate. LIM, D. K., PORTER, A. B., HOSKINS, B., AND Ho, I. K. (1987). Toxicol. Appl. PharmacoZ. 9fk477-489. Rats were treated with diisopropylfluorophosphate (DFF’) acutely and daily for 14 days. The total, free, and bound acetylcholine (ACh) levels were monitored in striaturn, hippocampus, and frontal cortex after DFP administration. Thirty minutes after daily administration of DFP, the total and free ACh levels were significantly increased and remained constant after each successive dose. The bound ACh levels in striatum and frontal cortex were also significantly increased; however, they were comparable to control levels after the 14th injection of DFP. The total ACh levels 30 min after a challenge dose of 2 mg/kg DFP in saline and DFP subacutely treated rats were significantly increased in hippocampus (34 and 76%) and frontal cortex (49 and 64%) and were not significantly different between the two groups. However, the level of total ACh in striatum was increased less in the tolerant rats (10%) than in the acutely treated rats (36%). The levels of free and bound ACh after acute administration of 2 mp/ kg DFP were markedly increased in three brain regions. After subacute administration, the levels of bound ACh were significantly increased in hippocampus (84%) and frontal cortex (40%); however, that in striatum did not change. The increase in the bound ACh level in the subacute treatment group was less than that in acutely treated rats in all three brain regions; however, the duration of the elevation of the free ACh in striatum was shorter in subacutely treated rats. These results suggest that the presynaptic cholinergic storage sites for ACh might be changed during subacute administration of DFP. o 1987 Academic press hc.

The toxicity of organophosphates is known to be due to their irreversible inhibition of acetylcholinesterase (AChE), which subsequently results in the accumulation of acetylcholine (ACh) at neuroeffector sites. It has also been demonstrated that subacute administration of organophosphates produces tolerance to these compounds as evidenced by the disappearance of some of the toxic symptoms induced by them (Costa and Murphy, 1982; Lim et al., 1983; Overstreet 1973; Russell et al., 1971a,b, 1975). Along with the behav’ To whom requests for reprints should be addressed,

477

ioral tolerance, animals also develop subsensitivity to ACh or cholinergic agonists (Brodeur and Dubois, 1964; Costa et al., 198 1, 1982; McPhillips 1969; Schwab and Murphy, 1981). Receptor alterations induced by subacute administration of organophosphates have been extensively studied, and it has been demonstrated that continuous exposure to organophosphates causes muscarinic recep tors to be down-regulated (Churchill et al., 1984a,b; Costa and Murphy, 1982, 1983; Costa et al., 1981, 1982; Ehlert et al., 1980; Levy, 1981; Sivam et al., 1983; Yamada et al., 1983a,b). Other studies have explored the 0041-008X/87

$3.00

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

LIM ET AL.

presynaptic alteration of ACh availability. Several investigators observed that highaffinity choline uptake was not changed during daily administration of organophosphates (Costa and Murphy, 1982, Russell et al., 1979, 1981). However, Yamada et al. (1983a,b) reported a significant decrease in choline uptake in subacutely treated guinea pigs. Furthermore, it has been reported that during daily administration of organophosphates, the release of ACh is significantly higher in tolerant animals than in the acutely treated animals (Raiteri et al., 198 1; Russell et al., 1985). Although the free ACh was increased more than the bound ACh after administration of organophosphates, it has been shown that levels ofboth free and bound ACh in brain are changed similarly in acutely and subacutely organophosphate-treated animals (Brodeur and Dubois, 1964; Russell et al., 1979, 198 1). Wecker et al. (1977), on the other hand, reported that the increase in the level of bound ACh was significantly different after a single treatment with paraoxon than after subacute treatment. Recently, we have found that presynaptic cholinergic regulatory parameters were involved in the development of tolerance to diisopropylfluorophosphate (DFP) (Lim et al., 1987). To further assess the involvement of presynaptic storage sites in tolerance to organophosphates, studies on the content of ACh after acute and subacute treatment of animals are necessary since few studies have been performed on alterations in ACh levels after subacute administration of DFP. Thus, it was the purpose of this study to determine the effects of acute and subacute administration of DFP on ACh levels in rat brain. Furthermore, the significance of the changes in free and bound ACh levels during and after daily administration of DFP was investigated. METHODS Animals and Chemicals Male Sprague-Dawley rats (Charles River Lab, Wilmington, MA) weighing 175-200 g were used through-

out the study. The animals were housed four to a cage with free accessto food and water. DFP (lot No. 84F-0248) was obtained from Sigma (St. Louis, MO); this preparation of DFP was found to have an IC50 (concentration required for 50% inhibition of AChE activity in whole rat brain homogenates) value in vitro of 7 &ml. Ethylhomocholine (EHC) was synthesized by direct reaction of dimethyl-3-amino- 1-propanol and iodoethane according to the method of Potter et al. (1983). Other chemicals and reagents of analytical grade were obtained from commercial suppliers. Administration

of DFP

Freshly prepared solutions of DFP in saline were administered subcutaneously in volumes of 0.1 ml/ 100 g body wt daily between 9 and 11 AM for 13 days. In accordance with our previous studies (Lim et al., 1983; Sivam et al, 1983), the dosage schedule of daily DFP administration was as follows: I st through 3rd day, 1 mg/kg; 4th through 6th day, 0.5 mg/kg; 7th through 13th day, 1 mg/kg; and on the 14th day, either 1 or 2 mg/ kg, SC.The acute DFP-treated rats received daily injections of saline (0.1 ml/100 g) for 13 days. DFP (2 mgJkg, sc) was given to these saline-treated rats 24 hr after the last saline injection. The animals were then divided into four groups and were decapitated at 30 min or 1,6, or 24 hr after the last injection of DFP. Control rats received daily injections of saline for 14 days. They were killed 30 min after the last injection. Each group contained 8 to 10 rats. Determination ofAChE Activity Brain AChE activity was determined according to the method of Ellman et al. ( 196 1). The tissues were homogenized in ice-cold sodium phosphate buffer (0.1 M, pH 8.0) at a concentration of approximately 20 mg wet wt/ ml buffer. Enzyme activity was expressed as nanomoles of acetylthiocholine hydrolyzed/minute/miIligram protein. The protein content of tissue homogenates was determined by the method of Lowry et al. (195 I) using bovine serum albumin as a standard. Determination ofACh Levels Tissue preparation: Free and bound Ach. The methods of Takahashi and Aprison (1964) and Wecker et al. ( 1977) were used, with minor modifications, for tissue preparation in determination of free and bound ACh levels. After the tissue homogenates were centrifuged the supematants and pellets were defined asthe free and bound

ACh LEVELS

479

AND DFP TOLERANCE TABLE 1

EFFEC~OFD~ADMINISTRATIONONAC~EA~~IVI~INTHEDISCRETEREGIONSOFRATBRAINS AChE activity (nmol of acetylthiocholine hydrolyzed/min/mg Treatment

Striatum

I hr Acute Control 306 +52 1 w/k 50 _+ 16 (83) 2 w/kg 10 f 2 (97) Subacute for 13 days, and then Control 291 +23 1 w/kg 3.1 f 0.7 (99) 2 m/kg I .5 + 0.2 (99) 24 hr Control 297 2 16 Acute 1 m&s 66 + 5 (74) 2 wfk 23 f 2 (91) Subacute for 13 days, and then 1 v/kg I9 +- 1 (93) 2 m&g I7 + 2 (94)

protein)

Hippocampus

Frontal cortex

46 k8 8 + 2 (84) 2 +2 (96)

52 + 10 IO + 2 (79) 4 + 0.8(93)

47 *4 0.9 + 0.1 (98) 0.5 -I 0.1 (99)

40 + 6 0.7 AI 0.3 (98) 0.3 f 0.1 (99)

38 fl

44

1 I * 0.5 (70) 6 &0.2(84)

10 * 0.9(77) 4 + 0.4(91)

5 *0.3(87) 6 ?0.5(88)

f

3

4 + 0.5 (91) 4 * 0.1(91)

Note. Animals were treated according to the dosing schedule (see Methods) and killed at the indicated time (I or 24 hr) after the final administration of DFP. Values represent means ? SE of four animals. Numerals in parentheses denote percentage inhibition from the respective control values. p i 0.00 I (all treated groups, compared to control).

ACh, respectively (Wecker et al., 1977; Whittaker et al., 1964; Zimmerman, 1982). Following decapitation of treated rats, heads were cooled to a temperature of approximately 0°C by immersion in liquid nitrogen for 8 sec. The brains were rapidly removed and the striatum, hippocampus, and frontal cortex were dissected out according to the method of Glowinski and Iversen (1966). The tissues were homogenized in 1.2 ml of ice-cold 0.32 M sucrose containing paraoxon (5 X lo-’ M) and CuSO, (20 &ml) to inhibit AChE and choline acetyltransferase, respectively. The tissues were then homogenized for 40 set at 1725 rpm using a tissue homogenizer with a Teflon pestle (Bodine Electric Corp., Chicago, IL). One milliliter of this homogenate was then centrifuged at 100,OOOgfor 1 hr. The resulting supematant was analyzed for free acetylcholine, while the pellet was used in analysis of bound acetylcholine. After centrifugation at 100,OOOgfor I hr, perchloric acid was added to each supematant containing free acetylcholine which also contained 2 nmol of ethylhomocholine (EHC; internal standard) until the concentration was 0.4 M. Pellets obtained from the centrifugation were analyzed for levels of bound ACh. Levels of ACh were determined in pellets and tissues by homogenization in 3 ml of 0.4 M perchloric acid containing the

internal standard and the homogenate was centrifuged for 20 min at 35,000g. After adjustment of the supematant pH to 4.2 with approximately 200 ~1 of 7.5 M potassium acetate, the samples were again centrifuged at 35,000g for 20 min. One hundred microliters of 5 mM tetraethylammonium was then added to each supematam. Ice-cold Reinecke salt solution (3 ml of 2%, w/v) was then added to precipitate ammonium compounds. Precipitation was allowed to proceed on ice for 1 hr, and then the samples were centrifuged at IOOOgfor 10 min at 0°C. The supematants were aspirated and the precipitates were dried under vacuum overnight. On the following day, 5 mM silver tosylate in HPLC-grade acetonitrile was added to the dried precipitates until the pink color disappeared (approximately 1 ml), and the samples were centrifuged for 2 min at IOOOg. The supematants were then transferred to I .5-ml conical polypropylene tubes and evaporated under vacuum. Citrate-phosphate buffer (0.02 M, pH 3.5,200 ~1) wasadded and 20 ~1 was injected into the HPLC system. Protein concentrations of the original homogenate were determined by the method of Lowryetal.(1951). Tissuepreparation: Total ACh. Rats were killed by microwave irradiation (2 set, 3.8 kW, Thermex, Solidyne, Inc., Santa Clara, CA). The discrete areas of brains were

480

LIM ET AL.

rapidly dissected out by the method of Glowinski and Iversen (1966). The tissues were kept frozen at -70°C until analyses. The ACh levels were quantified using high-pressure liquid chromatography (HPLC) with electrochemical detection after using the above procedure (Potter et al., 1983). Protein concentration was determined in tissue samples prepared for total ACh by resuspension in 0.1 N NaOH following the first centrifugation. Liquid chromatography-electrochemical detector sysfem. HPLC analysis was performed using a modification of the method of Potter et al. ( 1983). The chromatography system consisted of a single piston pump (Bioanalytical Systems, West Lafayette, IN) set at 0.8 ml/min for delivery of mobile phase and a lopm particle size BAS acetylcholine column. An Ismatec peristaltic pump (Cole-Parmer Instruments, Chicago, IL) was set at 0.5 ml/min and used for delivery of enzyme solution into the postcolumn reaction coil. A Bioanalytical Systems LC-4B amperometric detector equipped with a platinum electrode set at 0.5 V against a Ag/AgCl reference electrode was used in the determination of hydrogen peroxide formed. Solutions and standards. The mobile phase consisted of 96% 0.01 M sodium acetate (pH 5) 4% acetonitrile, and contained 30 mg/liter sodium octylsulfate. The enzyme reagent solution consisted of 0.2 M sodium phosphate buffer, pH 8.5, filtered through a 0.45rrn Millipore filter, to which was added 1 U/ml of choline oxidase and 2 U/ml of AChE. The pH of the combined mobile phase-enzyme reagent solution in the reaction coil was between 8.0 and 8.1, which is near the optimum of pH 8 for choline oxidase (Ikuta et al., 1977). ACh standards were prepared in 0.02 M citrate-phosphate buffer, pH 3.5. EHC was used as an internal standard. Calibration standards showed linearity between 20 and 250 pmol/20 ~1 of ACh and EHC. The loss of sensitivity between experiments was compensated for by the use of the internal standard, EHC. Statistics Data obtained from the groups which received daily administration of DFP were analyzed by one-way analysis of variance. Comparisons between controls and each treatment group and between two treatment groups at each time point were analyzed by Dunnett’s test and Student’s t test, respectively.

HIPPOCAMPUS

I

0

1

7

3 DAYS

11

14

OF TREATMENT

FIG. 1. Effect of daily injection of DPP on ACh levels in striatum, hippocampus, and frontal cortex. Animals were treated according to the dosing schedule described under Methods and killed 30 min after the final injection. Each value is the mean + SE for four to five determinations. Stars denote significant differences from control values. (*p < 0.05 and **p < 0.01).

toxicity within 6 hr after the administration. Rats which received daily administration of DFP exhibited similar cholinergic overactivity for a period of 9 days; thereafter the symp toms gradually disappeared. Efects of DFP AChE Activity

Administration

on Brain

AChE activity was significantly inhibited during daily administration of DFR (Table 1). One hour after the last administration of 1 mg/kg DFP greater than 80 and 98% of AChE activity was inhibited in acutely and subacutely treated rats, respectively. AChE activity returned to 30 and 10% of the control value 24 hr after the 1 mg/kg treatment. Eflects of DFP Administration on Total ACh Leveis in Three Brain Regions

RESULTS Effects of DFP Administration

on Behaviors

Rats which received a single injection of 2 mg/kg DFP exhibited overt parasympathetic

The total ACh levels in the control rats were 636 f 45,346 f 12, and 252 f 20 pmol/ mg protein in the striatum, hippocampus, and frontal cortex (mean + SE, n = 13 to 15), respectively (Fig. 1). These values are compa-

ACh LEVELS

481

AND DFF’ TOLERANCE HIPP~~~AMPUS

1000

--

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acute

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600

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200

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Time,

hrs.

Time, hrs.

FIG. 2. Effect of a challenge dose, 2 mgjkg, of DFP on ACh levels in striatum, hippocampus, and frontal cortex in acutely and subacutely treated rats. Animals were treated according to the dosing schedule described under Methods and killed at the indicated time after the administration of 2 mgjkg DFP, sc. Each value is the mean + SE for four to five determinations. Stars denote significant differences from control values and asterisks denote significant differences between two groups (*p < 0.05).

rable to those reported in the literature (Khandelwal et al., 1981). In general, during subacute treatment, ACh levels were increased 27% in the striatum after the first day and 2 1% in the hippocampus 30 min after an injection of DFF’, beginning with the first day (Fig. 1). The significant increase (45%) in ACh levels in the frontal cortex was not observed until the seventh day. There were no significant differences in ACh levels in any area examined 30 min after an injection. As shown in Fig. 2, acute administration of 2 mg/kg DFP resulted in a significant increase in ACh levels (36-49%) in all three regions 30 min af?er the treatment. Thereafter, the ACh level returned to the control levels by 1 and 6 hr in the striatum and cortex, respectively. However, the level in the hippocampus was still significantly higher (27%) than the control level at 24 hr. The levels of ACh were significantly increased in the hippocampus (76%) and fron-

tal cortex (60%) 30 min after the additional high dose of 2 mg/kg DFP in subacutely treated rats. However, ACh was increased only 10% in the striatum. Thereafter, the ACh levels rapidly returned to the control level. Furthermore, the ACh level in the striaturn was only 40% of the controls 6 hr after treatment. The levels of ACh after the challenge dose of DFP in acutely and subacutely treated rats were significantly different at the 30-min and 6-hr time points in the striatum and at 6 hr in the hippocampus. However, in the frontal cortex, there was no difference between the two treatments. Total ACh levels after the seventh daily injection of DFP were also measured and significant increases in total ACh levels in the hippocampus and frontal cortex were observed 30 min after this injection (Table 2). The elevated levels in the hippocampus remained for the 24 hr following the injection.

482

LIM ET AL. TABLE 2

CHANGES

OF ACh LEVELS IN THE

Total ACh” Control 30 mm 1 hr 6 hr 24 hr Bound ACh Control 30 min 1 hr 6 hr 24 hr Free ACh Control 30 min 1 hr 6hr 24 hr

DISCRETE REGIONS OF RAT BRAIN AFTER 7th DAILY ADMINISTRATION OF DFP Striatum

Hippocampus

Frontal cortex

636 -t 65 705k 13 618271 437 + 67: 308 f 20*

363 f 23 462 t 31* 436 + 58* 411+45* 416f24”

328 462 255 202 211

289+ 353 + 342 t 166 + 240 t

13 27* 28 17*** 12*

184k 10 201+21 194 + 19 178k21 168 t 19

135+ 278 + 273 + 195 f 234 k

49+ 289 + 290 + 76+ 61+

4 17*** 12*** 8 9

88k 9 202 f 38* 134 + 12* 101 * 19 135+37

+ k f + f

26 33* 14* 18* 12*** 6 20** 24** 14* 16**

’ pmol/mg protein. Note. Animals were treated according to the dosing schedule (see Methods) for 6 days and killed at the indicated time after the administration of DFP, 1 mg/kg at 7th day. Values represent means + SE of four or five animals. *p < 0.05, **p < 0.0 1, and ***p < 0.00 1 compared to the respective control value.

On the other hand, levels of ACh were significantly reduced in the striatum and frontal cortex 6 and 24 hr after the injection.

Efects of DFP Administration on Levels of Free and Bound ACh Figure 3 shows that 30 min after daily administration of DFP, the level of bound ACh in the striatum was significantly increased after 1,3, and 7 injections, but was comparable to the control level after the 1 lth injection. The level of bound ACh in frontal cortex was maximally increased after the 7th administration (p < 0.05), but after the 14th administration it was not significantly different from the control level. The level of bound ACh in the hippocampus was significantly increased after the 1 lth injection; however, there was no significant difference between levels in the

hippocampus after the earlier treatments. The levels of free ACh in the striatum and hippocampus were significantly increased 30 min after the 1st (94%) and 3rd (97%) injections. The level of free ACh was maximally increased after the 7th injection in the striaturn (480%). In both areas, free ACh was found to be elevated at all time points after the first injection of DFP. The levels of both bound and free ACh were markedly increased 30 min after a single dose of 2 mg/kg DFP in the striatum (Fig. 4) hippocampus (Fig. 5) and frontal cortex (Fig. 6). Thereafter, the bound ACh level gradually returned to the control level in the hippocampus and the frontal cortex; however, it was still significantly higher in the striatum (68%) 24 hr after administration. Also, the free ACh levels in the striatum and hippocampus were twice the control levels 24 hr after administration.

483

ACh LEVELS AND DFF’ TOLERANCE

500’ C Z % 400. ‘; z 2:

300,

K r”5:

z s m

200,

100.

T

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0

1

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HIPPOCAMPUS coRTu(

7 DAYS OF TREATMENT

11

14

300

3

7

11

14

DAYS OF TREATMENT FIG. 3. Effect of daily injection of DF? on bound and free ACh levels in striatum, hippocampus, and frontal cortex. Animals were treated according to the dosing schedule described under Methods and killed 30 min after the final injection. The discrete areas were homogenized and centrifuged for the separation of free and bound ACh asdescribed under Methods. Each value is the mean -t SE for four to five determinations. Stars denote significant differences from the control values (*p < 0.05 and **p < 0.01).

In the subacutely treated rats, the levels of bound ACh were significantly increased in the hippocampus (84%) (Fig. 5) and frontal cortex (40%) (Fig. 6), but not in the striatum

30 min after the additional high dose of 2 mg/ kg DFF’. Thereafter, levels of bound ACh decreased to the control level in the hippocampus and frontal cortex. The levels of bound

484

LIM ET AL. STRIATUM

80

STRIAllJM

-4 acute . . . 0. e´

Time.

hrs.

0

0.5

1

6

Time,

24

hrs.

FIG. 4. Effect of a challenge dose of 2 mg/kg DFP on bound and free ACh levels in striatum in acutely and subacutely treated rats. Animals were treated according to the dosing schedule described under Methods and killed at the indicated time after the administration of 2 mg/kg DFF’, sc. The discrete areas were homogenized and centrifuged for the separation of free and bound ACh as described under Methods. Each value is the mean + SE for four to five determinations. Stars denote significant differences from control values and asterisks denote significant differences between two groups (*p < 0.05 and **p < 0.01).

ACh in the striata of subacutely treated rats were significantly less than those in the striata of control rats (50% of the control) 6 and 24 hr after treatment. The increases in the levels of bound ACh in these three regions from subacutely treated rats were less than those in regions from acutely treated animals 30 min to 6 hr after treatment. However, 24 hr after treatment, the levels of bound ACh in regions from the two groups were the same except in the striatum. The free ACh level was also markedly increased in the striatum (600%) (Fig. 4) and hippocampus (180%) (Fig. 5) 30 min after 2 mg/kg of DFP, in subacutely treated rats. However, unlike the acute treatment, the free ACh returned to the control level 24 hr after treatment.

The increase in the level of free ACh in the striatum of the subacutely treated group was significantly higher than that of the acutely treated group 30 min after administration; however, the level was lower 6 and 24 hr after administration. There was no significant difference in the level of the free ACh in hippocampus between the two treatments. During daily administration, the level of bound ACh after the seventh administration of DFF’ was significantly increased at 1 hr and decreased at 6 and 24 hr in the striatum. There was no difference in the hippocampus. The ratio of free ACh to bound ACh was significantly increased 30 min after both treatments in the striatal area (Fig. 7). Although the ratios between the two treatments were similar 24 hr after the challenge dose,

485

ACh LEVELS AND DFP TOLERANCE 300

5001

HIPPOCAMPUS

i,

HIPPOCAMPUS

24

6

Time,

1

hrs. 0

0.5

1

6

Time,

3

hrs.

FIG. 5. Effect of a challenge dose of 2 mg/kg DF’P on bound and free ACh level in hippocampus in and subacutely treated rats. The experimental protocol was same as in Fig. 4.

acutely

the ratio in the subacutely treated rats was significantly higher than that in the acutely treated rats prior to this 24-hr time point. DISCUSSION It has been demonstrated that behavioral tolerance to DFP develops when brain AChE

i

1 6

0 0.5 1 Time,

24

hrs.

FIG. 6. Effect of a challenge dose of 2 mg/kg DFP on bound ACh level in frontal cortex in acutely and subacutely treated rats. The experimental protocol was same as that in Fig. 4.

activity is below 30% of the control activity (Glow et al., 1966; Russell et al., 1969). The AChE activity in our subacutely treated rats was only 10% of the control value 24 hr after the final dose of DFP. The present results show that the degrees of increase in total ACh and bound ACh are significantly different in rats treated acutely and subacutely with DIP. However, the elevation in the level of free ACh is the same after the two treatments. Our results reveal that in striatum, the total ACh level was increased by 36% 30 min after acute administration, whereas after subacute treatment there was no change. Although there were no differences between effects of acute and subacute treatments in hippocampus and frontal cortex 30 min after injections, the elevated ACh level in subacutely treated rats declined more rapidly than that in acutely treated rats. The level of bound ACh after acute treatment increased significantly in striatum. However, after subacute treatment, bound ACh was comparable to the control level. Also, in the hippocampus and frontal cortex, the elevation of bound ACh

486

LIM ET AL.

%x-r-------

24

Time. t-vi.

FIG. 7. Comparison of the ratios of free and bound ACh levels after a challenge dose of 2 mg/kg DFP in striaturn in acutely and subacutely treated rats. Ratios were calculated from data on the same animals as in Fig. 4. Stars denote significant differences from control values and asterisks denote significant differences between two groups (*p < 0.05 and **p < 0.01).

(40-80%) after subacute treatment was much less than that after acute treatment ( 120%). Our results suggest that tolerance development to DFP is, in part, due to attenuation of the elevation in the level of bound ACh during daily administration of DFP. It has been reported that organophosphates markedly increase the total ACh levels in synapses by the inhibition of ChE activity (Brodeur and Dubois, 1964; Fonnum and Gottormsen, 1969; Harris et al., 1978, 1982; Russell et al., 198 1; Shih, 1982; Stitcher et al., 1978; Wecker et al., 1977) and that the increase in the total ACh level is almost entirely due to a rise in extracellular ACh after acute administration of organophosphates (Brodeur and Dubois, 1964; Fonnum and Gottormsen, 1969). It has also been reported that free ACh of rat brain is elevated to approximately the same extent by each successive dose of organophosphate, but that levels of bound ACh do not change (Brodeur and Dubois, 1964; Russell et al., 1979, 198 1). However, Wecker et al. (1977) reported that the increase of total ACh in rats treated chronically with paraoxon was less than half of that in acutely treated rats, and that although free ACh levels were increased to the same extent by both treatments, the increase of bound ACh in rats treated acutely with paraoxon

was significantly higher than that of subacutely treated rats. Our results are generally consistent with those of Wecker et al. ( 1977). Furthermore, the ratio of free to bound ACh in striata of tolerant rats was significantly higher than that of acutely treated rats 30 min and 1 hr after the challenge dose of DFP. This implies an increase in release of ACh and/or a decrease in synthesis of ACh. The synthesis of ACh by choline acetyltransferase is regulated by the availability of choline, ACh, and CoA (Haga and Noda, 1973; Simon et al., 1976; Yamamura and Snyder, 1973). Many investigators have reported that choline acetyltransferase is not affected by either acute or chronic administration of organophosphates (Russell et al., 1975; Sivam et al., 1984; Stavinoha et al., 1969; Wecker et al., 1977). It has also been reported that repeated administration of DFP significantly decreased the high-affinity choline uptake (Yamada et al., 1983a,b) and increased the release of ACh (Raiteri et al., 198 1) due to down-regulation of presynaptic choline& autoreceptors (Lim et al., 1987). Therefore, the DFP-induced decrease in high-affinity choline uptake might be expected to result in decreased synthesis and increased release of ACh, further attenuating the elevation of bound ACh. The observed decrease in the elevation of the level of bound ACh after the daily injections as well as after the challenge dose of DIP might be due to adaptive changes in the ACh availability to presynaptic function. The elevation of free ACh in DFP-tolerant rats should be counterbalanced by the down-regulation of muscarinic receptors. It has been reported that free ACh refers to ACh contained in the synaptic space and partially to that in the cell sap of cell bodies and in broken synaptosomes, whereas bound ACh refers to ACh contained in the synaptosomes. Furthermore, bound ACh is compartmented into labile and stable ACh, which refer to the cytoplasmic ACh sequestered within the synaptosomes and to ACh bound to synaptic

ACh LEVELS

487

AND DFP TOLERANCE

vesicles or contained with them, respectively (Whittaker et al., 1964; Zimmerman, 1982). Although the level of free ACh itself does not totally represent neuronal activity, the comparison of the levels of free ACh among treatment groups could explain, at least in part, the neuronal activities. The release of ACh from cholinergic nerve terminals is believed to be that which is released from the synaptic vesicles by exocytosis. However, Dunant and Israel (1985) have suggested that the release of ACh is derived directly from the cytoplasmic ACh. If there is an equilibrium between cytoplasmic and vesicular ACh, the attenuated elevation of bound ACh after subacute administration of DFP suggests decreased availability of the ACh. Kobayashi et al. ( 1980) have reported that both ACh compartments in rat brain are increased by an acute administration of dichlorvos; however, it is unclear which compartment is more related to the adaptation process in DFP-tolerant animals. Further studies on the changes of ACh levels in these two compartments in the presynaptic sites are needed to fully elucidate the mechanisms of development of tolerance to DFP. Our results also suggest that the changes in the bound ACh levels during daily administration are different for each brain region. Shih ( 1982) reported regionally differentiated changes in ACh levels in brain after acute administration of soman. AChE activities are equally inhibited after subacute administration of DFP (Sivam et al., 1983), although striatal regions exhibit the highest turnover of ACh (Choi et al., 1973; Racagni et al., 1976). It is clear that additional studies are needed to determine whether the adaptational changes in the levels of bound ACh in different areas are dependent on the dose schedule, the ACh turnover rate, or the involvement of other neurotransmitter systems. It is generally agreed that animals tolerant to organophosphates are subsensitive to ACh due to the down-regulation of muscarinic receptors (Churchill et al., 1984a,b; Costa et al., 1981,

1982; Costa and Murphy, 1982, 1983; Ehlert et al., 1980; Levy, 1981; Sivam et al., 1983; Yamada et al., 1983a,b). Along with the down-regulation of muscarinic receptors, our results suggest that the change in the ACh level is also affected by daily administration of DFP and that the presynaptic sites in cholinergic neurons of the brain are involved in the tolerance development to organophosphates. ACKNOWLEDGMENTS This work was supported by Contract DAMD 17-8% C-5036 from the U.S. Army Medical Research and Developmental Command and NIH Biomedical Research Support Grant 2-S07RR05386.

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