Pretreatment of rats with an organophosphorus insecticide, chlorfenvinphos, protects against subsequent challenge with the same compound

14,560-567(1990)

FUNDAMENTALANDAPPLIEDTOXICOLOGY

Pretreatment of Rats with an Organophosphorus Insecticide, Chlorfenvinphos, Protects against Subsequent Challenge with the Same Compound’ TAKANORI

IKEDA,

TAKESHI KOJIMA, MINORU YOSHIDA, HIROAKI SHUJI TSUDA, AND YASUHIKO SHIRASU

TAKAHASHI,

Mitsukaido Laboratories, Institute of Environmental Toxicology, Uchimoriya 4321, Mitsukaido-shi, Ibaraki. 303 Japan

Received Ju1.v5, 1989; accepted November 8, 1989 Pretreatment of Rats with an Organophosphorus Insecticide, Chlorfenvinphos, Protects against Subsequent Challenge with the Same Compound. IKEDA, T., KOJIMA, T., YOSHIDA, M., TAKAHASHI, H., TSUDA, S.. AND SHIRASU, Y. (1990). Fundam. Appi. Toxicol. 14, 560567. A single oral pretreatment of rats with chlorfenvinphos (CVP) reduced toxicity ofthe same compound subsequently administered. This protection occurred 8 hr and became maximal 24 hr after the oral pretreatment at a dose of I5 mg/kg (about half of its LD50). The 24-hr pretreatment with CVP increased the LD50 of CVP threefold, but did not change the type of toxic signs and time to death caused by CVP. The CVP pretreatment did not appreciably change the toxicities of the cholinergic agonists, carbachol and oxotremorine, but significantly increased the toxicity of another organophosphate. dichlorvos. Oral treatment of rats with CVP (15 mg/ kg) inhibited brain acetylcholinesterase (AChE) activity. This inhibition became maximal at 4 hr (about 20% of control) and lasted more than 24 hr after the administration. Twenty-four hours after oral administration of CVP ( I5 mg/kg), the second dose (CVP 30 mg/kg. po) was less effective in inhibiting cholinesterase activities of the brain, erythrocyte, and plasma compared with naive rats treated with the same dose The difference in brain AChE activity between control and CVP pretreatment groups was greater in magnitude than that measured in erythrocytes. CVP concentration in plasma after the oral administration of CVP (30 mg/kg) was decreased by the CVP pretreatment. Area under the concentration vs time curve (AUC) in the CVP-pretreated group was about one-fourth of AUC in the control group. This decrease in the AUC was comparable to the decrease in the toxicity of CVP. Thus, the protection against subsequent CVP challenge may be due to the reduction in the inhibition of brain AChE activity caused by the decrease in plasma CVP concentration. 8 1990societyofToximtogy.

When organophosphates are administered to animals, they cause excessive cholinergic signs and death at high doses because of their anti-cholinesterase properties. These effects of some organophosphates are gradually decreased when sublethal doses of these compounds are repeatedly administered to rats or ’ Presented in part at the 28th Annual Meeting of the Society of Toxicology, February/March 1989, Atlanta, Georgia. 0272-0590/90$3.00 Copyright 0 1990 by the Society ofToxicology. All rights of reproduction in any form reserved.

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mice (Barnes and Denz, 195 1; Rider et al., 1952; Costa et al., 1981). This phenomenon, tolerance to organophosphates, is associated with a decrease in the lethal or sublethal effects of direct cholinergic agonists, carbachol or oxotremorine (Brodeur and DuBois, 1964; McPhillips, 1969; Perrine and McPhillips, 1970; Carson et al., 1973). It is also related to a reduction in the extent of tissue binding of the muscarinic antagonist, t3H]quinuclidinyl benzilate @chiller, 1979; Gazit et al., 1979; Ehlert et al., 1980; Costa et al.,

PROTECTION

AGAINST

198 1). A decrease in the affinity to acetylcholine or the number of acetylcholine receptors was suggested to be the most probable reason for subchronic tolerance to organophosphates (Costa et al., 1982a). Few studies dealing with acute tolerances to organophosphates (i.e., tolerance after a single administration of organophosphate) have been reported. Costa et al. (1982b) reported that a single injection of disulfoton (O, O-diethyl-4[2-(ethylthio)ethyl] phosphorodithioate) makes mice resistant to hypothermic and antinociceptive effects of oxotremorine and the second dose of disulfoton itself. Overstreet et al. (1977a,b) reported that effects of a choline& agonist, pilocarpine, on body temperature and water intake in rats are attenuated by a single injection of diisopropylfluorophosphate (DFP). Our first purpose of the present experiment was to determine whether another organophosphorus insecticide would develop the protection against the toxicity of the same compound after its single administration in rats. For this purpose we chose a P = 0 type organophosphorus insecticide chlorfenvinphos (CVP: 2-chloro-1-(2,4-dichlorophenyl)vinyl diethyl phosphate) as a candidate chemical and compared the toxicity of CVP in rats pretreated with a single dose of CVP with that in naive control rats. After confirmation of the development of protection against subsequent CVP challenge, we investigated the nature and mechanism of this protection using the following parameters: sensitivities to cholinergic agonists and another organophosphate, cholinesterase activities, and CVP concentration in plasma. MATERIALS

AND

METHODS

Animals. Male Fischer 344 rats (7 weeks old, SPF) were obtained from Charles River Japan, Inc., Kanagawa, and acclimatized for 1 week prior to the study. They were maintained in a room controlled under a cycle of 14 hr light-10 hr dark, constant temperature (24 ? 2”C), and constant humidity (55 f 10%). Food and water were freely given.

CHLORFENVINPHOS

561

Chemicals. Chlorfenvinphos (92.7% Shell International Chemical Co. Ltd.. London) and dichlorvos (DDVP) (98.7%, Nippon Chemical Industrial Co. Ltd.. Tokyo) were of technical grades. p-CVP standard solution was purchased from Wako Pure Chemical Industries, Ltd., Osaka. All other chemicals were of the highest grade commercially available. Animal treatments. CVP and DDVP were dissolved in olive oil and were orally administered with a volume of 5 ml/kg of body weight. Control rats were orally treated with the same volume of olive oil. Oxotremorine (Sigma Chemical Co., St. Louis, MO) and carbachol (Wako Pure Chemical Industries, Ltd.) were dissolved in saline and were injected intravenously with a volume of 1 ml/kg of body weight. Acute toxicity. Toxicities were mainly evaluated based on LDSOs because the measurement of lethality is precise, quanta], and unequivocal and enables us to statistically evaluate the difference in the magnitude of toxicity. Clinical signs were also carefully observed. Four to Jive dosages were administered to 5 to IO animals at each dose level. Acetylcholinesterase (AChE) and cholinesterase (ChE) activities measurement. Rats were lightly anesthetized by diethyl ether, and blood was collected from the posterior vena cava in a heparinized tube. After decapitation, the whole brain was quickly removed and weighed. Blood was centrifuged at 2000g for 10 min at 4°C. Plasma was separated and its ChE activity was measured. Erythrocytes were washed once with cold saline. The brain was homogenized in 4 vol of Tris (0.05 M)-NaCl (0.11 M) buffer (pH 7.4) and centrifuged at 12,500g for 10 min at 4OC. A suspension of 20% erythrocytes in saline or the supernatant derived from the brain homogenate was used for the determinations of their respective AChE activities. AChE and ChE activities were measured using an automatic analyzer (Auto Analyzer 11, Technicon Instruments Co., NY). Acetylthiocholine iodide was used as a substrate for AChE activity measurement. Only the physostigmine ( 1O-4 M)-sensitive activity was measured as AChE activity. The substrate butyrylthiocholine iodide was employed for ChE activity measurement. DTNB [5,5’-dithiobis(2-nitrobenzoic) acid] was employed as a chromogenic indicator. Acetylcholinesterase (Type VIS: from electric eel lyophilized powder, Sigma Chemical Co.) and butyrylcholinesterase (Type IV-S: from horse serum lyophilized powder, Sigma Chemical Co.) were used as standard enzymes. One unit of both enzymes should hydrolyze 1.0 pmol acetylcholine and butyrylcholine per minute at pH 8.0 at 37°C. respectively. AChE and ChE activities were expressed as units per gram of wet tissue (brain) or milliliter (plasma and erythrocyte). Determination of CVP concentration in plasma. Plasma was obtained by the same method as for the ChE activity measurement. CVP in plasma was extracted according to the method of Tsuda et al. (1986). Plasma (I

IKEDA ET AL.

562

ml) was applied to a C- I8 column (C- 18 Preseps, Gasukuro Kogyo Inc., Tokyo) and washed with 3 ml distilled water. CVP was eluted with 2 ml of diethyl ether. The eluate was evaporated at about 40°C and the residue was dissolved in acetone. The CVP concentration in the acetone solution was measured with a Hewlett-Packard 5890A gas chromatograph equipped with a nitrogenphosphorus detector and a capillary column (Hi CapCBPI-W12-100, Shimazu Corp., Kyoto). Flow rates of helium, hydrogen, nitrogen, and air were 3.5. 15, 150, and 15 ml/min, respectively. Temperatures of the column, detector, and injection port were 180. 250, and 300°C. respectively. A standard curve was determined with a b-CVP standard solution. Recovery of CVP from water solutions containing 3% bovine serum albumin was 95.0 + 25.4% (mean + SD, n = 4). CVP concentration was not adjusted by this recovery. The lower limit of detection of this method was 0.00 1 mg/liter CVP. Data an&sis. LD5Os and the significance of differences in LD5Os between the control and the pretreatment groups were determined by the method of Litchfield and Wilcoxon (1949). The area under the plasma concentration vs time curves (AUC) was determined by a trapezoidal method. Student’s f test was used to determine the significance of differences in ChE activities or CVP concentration between the control and the pretreatment groups.

RESULTS Acute Toxicity of CVP

,

1

048

,

‘I

24 Time

48 (hr)

FIG. I. Effect of CVP pretreatment on the oral LD50 of the same compound subsequently administered. The LD50 of CVP was determined 4, 8, 24, and 48 hr after CVP oral pretreatment (15 mg/kg). The 0 time point represents the LD50 of nonpretreated rats. Each point and bar represents LD50 and its 95% confidence limit, respectively. Significance of differences in LDSOs between nonpretreated and pretreated rats was determined by the method of Litchfield and Wilcoxon (1949) (*p < 0.05).

treatment with vehicle (olive oil) did not change the oral toxicity of CVP. Vehicle-pretreated rats were used as the controls in the following experiments. Oral LDSOs of CVP measured 24 hr after oral pretreatments of rats with 7.5 and 15 mg/kg CVP were 2.2 and 3.1 times higher than that of the control group with statistical significances (p < 0.05), respectively. The LD50 in the 15 mg/kg pretreatment group was significantly (17 < 0.05) higher than that in the 7.5 mg/kg pretreatment group. The type of toxic signs and times to death of the CVP-pretreated rats were not different from those of the control rats.

Rats administered CVP by gavage showed typical anti-cholinesterase signs consisting of salivation, fasciculation, lacrimation, tremors, irregular respiration, and prostration. Toxic signs were noted from 1 hr but almost disappeared 24 hr after the administration. All deaths were observed between 2 hr and 1 day after the administration. The oral LD50 of CVP was 34.6 mg/kg of body weight. Oral LD5Os of CVP were determined 4, 8, 24, and 48 hr after oral pretreatment with Carbachol, Oxotremorine, and DDVP ToxicCVP at a dose of 15 mg/kg (about half of its ities oral LD50). This pretreatment (15 mg/kg) evoked no obvious toxic signs except for a To ascertain whether this protection small degree of weight loss (3.7% of initial against subsequent CVP challenge is related weight) 24 hr after the treatment. Figure 1 to changes in acetylcholine receptors, cholinshows the LDSOs of CVP after the pretreatergic agonists, carbachol and oxotremorine, ment. The LD50 of CVP was increased at 8 toxicities were examined. Another organohr after the pretreatment and became maxiphosphate, DDVP, was used to examine inmal (threefold) at 24 hr. The 24-hr oral pre- volvement of acetylcholine release in the pro-

PROTECTION

AGAINST TABLE

EFFECT

OF CHLORFENVINPHOS

PRETREATMENT Dose

Test substance“ Carbachol (iv)

0

0 15

Dichlorvos

(PO)

563

I OF CARBACHOL,

OXOTREMORINE,

AND

DICHLORVOS

of

pretreatmentb (w/k)

15 Oxotremorine (iv)

ON LDSOs

CHLORFENVINPHOS

0

15

LD50’ (m&z)

Ratio of LDSOs’

Sloped

0.243 (0.232-0.253)’ 0.230 (0.220-0.240) 1.22 (1.15-1.29) 1.53 (1.47-1.59) 97.5 (88.6-107) 38.1 (33.5-43.4)

1.10 (1.04-1.15) 1.09 (1.04-1.13) 1.10 (1.05-1.16) 1.07 (1.02-1.11) 1.24 (1.15-1.34) 1.29 (1.16-1.51)

0.95 (0.89-1.01) 1.25* (1.16-1.34) 0.39* (0.33-0.46)

a Test substance was administered via the route in parentheses 24 hr after the pretreatment. b Chlorfenvinphos was administered orally. LD50 and the significance of differences were determined by the method of Litchheld and Wilcoxon (1949). d (LD84/LD50 + LDSO/LD16)/2. ’ LD50 of 15 mg/kg pretreated group/LDSO of 0 mg/kg pretreated group. /Values in parentheses are 95% confidence limits. * Significantly changed (p < 0.05) by I5 mg/kg pretreatment.

tection. The results are presented in Table 1. The 24-hr oral pretreatment of rats with CVP ( 15 mg/kg) did not alter the iv LD50 of carbachol. The iv LD50 of oxotremorine was significantly but slightly increased by the pretreatment. The po LD50 of DDVP was significantly decreased by the pretreatment. Excess cholinergic signs such as salivation, tremors, chromodacryorrhea, dyspnea, and cyanosis were observed in rats injected with oxotremorine and carbachol. DDVP induced the anti-cholinesterase signs similar to those of CVP. These toxic signs were not changed by the CVP pretreatment.

stored but was still inhibited 48 hr after the treatment (Fig. 2). AChE activities of brain and erythrocyte and ChE activity of plasma were measured 0, I, 2, 4, and 6 hr after oral administrations of CVP (30 mg/kg) in control groups and 24-hr

*m%+-

Eflects on AChE and ChE Activities

48

Time (hr)

When CVP (15 mg/kg) was orally administered to rats, the brain AChE activity was maximally inhibited at 4 hr after the treatment. The AChE activity was gradually re-

FIG. 2. Brain AChE activities after oral administration of CVP

(15 mg/kg).

Data

are presented

as means

f SD (n

= 5-7). The control value (100%) was 2.75 + 0.2 1 units/g wet tissue (mean ? SD, n = 7).

1KEDA ET AL.

564

Brain

0

1

4

2

6

group after the CVP administration was not lower than that in the control group. Figure 4 illustrates brain AChE activity after the oral administrations of CVP in control groups and 24-hr pretreatment groups with CVP ( 15 mg/kg, po) at 4 hr when the activity was most prominently inhibited by the second dose. Inhibition of brain AChE activity by CVP was dose dependent in both the groups. At a dose of 15 mg/kg or higher, the AChE activity of the CVP pretreatment group was significantly higher than that of the control group. CVP Concentration

% o 0I, 0

, , 1

2

4 Time (hr)

6

FIG. 3. Effect of CVP pretreatment on brain and erythrocyte AChE activities and plasma ChE activity. Rats were pretreated with vehicle (0) or CVP ( I5 mg/kg) (0) 24 hr before second administration of CVP (30 mg/kg). The AChE and ChE activities were measured 0. I, 2, 4, and 6 hr after the second administration. Data are presented as means +- SD (?I = 4-5). The control values (100%) for brain, erythrocyte, and plasma were 2.56 ? 0.16. 1.87 +- 0.2 1, and 0.46 f 0.03 units/g wet tissue or ml (mean f SD, n = 4) respectively. *p < 0.05, **p c: 0.0 1, and ***p < 0.00 I compared to corresponding vehicle pretreatment group by Student’s t test.

pretreatment groups with CVP (15 mg/kg, PO). The results are shown in Fig. 3. These activities were still inhibited 24 hr after the CVP pretreatment. The second CVP only slightly inhibited these activities so that AChE activities of brain and erythrocyte in the CVP pretreatment group were significantly higher than those in the control group 2,4, and 6 hr after the second administration. The difference in brain AChE activity between the control and the CVP pretreatment groups was greater than in erythrocyte AChE. Plasma ChE activity in the CVP pretreatment

in Plasma

CVP concentration in plasma was measured 1,2,3,4, and 6 hr after oral administrations of CVP (30 mg/kg) in control groups and 24-hr pretreatment groups with CVP ( 15 mg/kg, PO). CVP in plasma was not detected 24 hr after the CVP pretreatment. The CVP

Dose

(mg/kd

FIG. 4. Dose-inhibition relationship ofbrain AChE activity. Rats were pretreated with vehicle (open column) or CVP ( 15 mg/kg) (shaded column) 24 hr before second administrations ofCVP (7.5, 15.30, and 60 mg/kg). Rats were euthanized 4 hr after the second administration and brain AChE activities were measured. Data are represented as means * SD (n = 4-S). The control value (100%) was 2.0 1 t 0. IO units/g wet tissue (mean & SD, n = 5). ***p < 0.00 I compared to corresponding vehicle pretreatment group by Student’s I test. The dosage of 60 mg/kg in the vehicle-pretreated group was not conducted because all rats were expected to die 4 hr after the administration.

PROTECTION

AGAINST

Time (hr) FIG. 5. Effect ofCVP pretreatment on CVP concentration in plasma. Rats were pretreated with vehicle (0) or CVP (15 mg/kg) (0) 24 hr before the second administration of CVP (30 mg/kg). CVP concentration in plasma was measured 1,2,3,4, and 6 hr after the second administration. Data are presented as means + SE (n = 4-6). *p < 0.05 compared to corresponding vehicle pretreatment group by Student’s f test.

concentration in the CVP-pretreated group was lower than that in the control group at each time point and was significantly lower at 1 hr (Fig. 5). The AU& hr of CVP in control and CVP pretreatment groups were 0.419 and 0.119 mg hr/liter, respectively, such that the CVP pretreatment decreased AUC of CVP administered subsequently to 28% of control. DISCUSSION This study demonstrated that protection against the toxicity of CVP was produced by a single oral pretreatment to rats with the same compound at a dose of about one-fourth or one-half LD50. This protection was characterized by a very rapid development (8 hr after the pretreatment) and a greater increase in the LD50 (threefold increase). The mechanism of tolerance to many organophosphates after their repeated administration has been considered to be the result of down-regulation of postsynaptic acetylcholine receptors (Costa et al., 1982a). The decrease in acetylcholine release from the nerve terminal has been suggested as the mecha-

CHLORFENVINPHOS

565

nism for paraoxon tolerance (Thomsen and Wilson, 1986). The down-regulation has been proposed in the acute tolerance to disulfoton since the mice injected with a single dose of disulfoton are subsensitive to the effects of oxotremorine and have fewer numbers of binding sites of [3H]oxotremorine-M (Costa et al., 1982b). A single injection of DFP also reduces the binding site of the muscarinic antagonist N-[3H]methylscopolamine (Cioffi and El-Fakahany, 1986) and the effects of pilocarpine (Overstreet et al., 1977a,b). If the CVP pretreatment had reduced the number of acetylcholine receptors or release of acetylcholine, the toxicities of the cholinergic agonists and other organophosphates would have been decreased. The CVP pretreatment significantly reduced the oxotremorine toxicity. The reduction of oxotremorine toxicity was, however, very small. The carbachol toxicity was not changed by the CVP pretreatment. The development of the protection against the toxicity of CVP was faster than that of the down-regulation and the subsensitivity to the cholinergic agonists reported in disulfoton and DFP, which are observed 24 hr or later after the injections. In addition to these observations, the toxicity of DDVP was increased by the CVP pretreatment. This increase might be due to decreased AChE activity resulting from the CVP pretreatment. Although the possibility that the CVP pretreatment altered the metabolism or other disposition of these chemicals could not be discarded, these results suggest that the down-regulation of acetylcholine receptors or the decreased acetylcholine release may not be enough for the mechanism of the protection. Brain AChE inhibition was reported to be the mode of toxic actions of CVP (Hutson and Hathway, 1967). CVP inhibited brain AChE activity rather irreversibly. When rats became tolerant to CVP, their brain AChE activities were still inhibited to about 30 (8 hr)-70% (48 hr) of control. Increases in brain AChE activity by the CVP pretreatment was

566

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ET AL.

thus ruled out for a mechanism of the protec- against CVP toxicity (Hutson and Wright, tion. 1980). Thus, the induction of metabolism of The typical anticholinesterase signs caused CVP by the pretreatment could be an explaby CVP were not changed by the CVP pre- nation of the reduced plasma CVP concentreatment. CVP pretreatment decreased the tration. Most organophosphates, however, inhibition of brain AChE activity caused by have been reported to be inhibitors of mfo acthe next dose of CVP, in which time course tivity (Conney et al., 1967; Stevens et al., and dose dependency of the inhibition of 1972a; Uchiyama et al., 1975). The induction brain AChE activity seemed parallel to those of mfo caused by CVP has not been reported. of development of the toxic signs in both the The onset of protection appeared to be too control and the CVP pretreatment groups. rapid to be caused by induction of hepatic mfo because even potent inducers require These results suggest that the CVP pretreatmore than 1 day for their effects to become ment does not alter the site of action of CVP maximal (Sipes and Gandolfi, 1986). Thus, and that the decreased inhibition of brain AChE activity could be related to the de- there has been no favorable evidence that the induction of its own metabolism would be crease in toxicity to CVP. The plasma CVP concentration in the the reason for the decrease in the plasma CVP CVP pretreatment group was lower than that concentration so far, but the effects of CVP in the control group at each time point and on mfo activities and its own metabolism rewas significantly lower at 1 hr. The AUC of main to be examined. Absorption and elimiCVP in the CVP pretreatment group was nation processes also depend on other facabout one-fourth of the control group. This tors, for example, hepatic blood flow. Furdecrease in the AUC was comparable to the thermore, the distribution process, for decrease in the toxicity of CVP. Thus, this example, distribution between plasma and concomitant decrease in plasma CVP con- tissues, is one of the important factors for a centration may lead to the decreased inhibidecrease in AUC. These factors also remain tion of brain AChE, and then the decrease in to be clarified. the toxicity of CVP. The time lag between the In conclusion, CVP pretreatment reduced peak of CVP concentration (1 hr) and the the toxicity of the same compound adminismaximal inhibition of AChE (4 hr) after the tered subsequently. It may be due to the obCVP treatment may be due to the irreversibilserved reduction in the brain AChE inhibiity of AChE inhibition caused by this chem- tion caused by the decrease in plasma CVP ical. concentration. The decrease in the AUC caused by the CVP pretreatment could result from alterREFERENCES ation of absorption, distribution, or elimination processes. Metabolism is concerned with both absorption and elimination processes. BARNES, J. M., AND DENZ, F. A. (1951). The chronic toxicity of pnitrophenyl diethyl thiophosphate Significant induction of hepatic mixed func(E.605): A long-term feeding experiment with rats. J. tion oxidases (mfo) have been observed when Hyg. 49,430-44 1. some organophosphates are administered re- BRODEUR, J., AND DuBors, K. P. (1964). Studies on the peatedly (Stevens et al., 1972b; Moroi et al.. mechanism of acquired tolerance by rats to O,O-di1976; Fabacher et al., 1980). CVP is mainly ethyl S-2-(ethylthio)ethyl phosphorodithioate (Di-Syston). Arch. Irtt. Pharmacodyn. 149,560-570. metabolized by hepatic mfo (Hutson et al., CARSON, V. G., JENDEN, D. J., AND RUSSELL, R. W. 1967; Hutson and Logan, 1986). Pretreat(1973). Changes in peripheral cholinergic systems folments with hepatic mfo inducers such as phelowing development of tolerance to the anticholinesnobarbital and dieldlin increase the metaboterase diisopropylfluorophosphate. To.xicol. Appl. lism of CVP (Donninger, 197 l), and protect Pharmacol. 26,39-48.

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