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Comparative toxicity, absorption, and metabolism of chlorpyrifos and its dimethyl homologue in methyl parathion-resistant and -susceptible tobacco budworms
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
4, 266-274
Comparative Toxicity, Absorption, and Metabolism of Chlorpyrifos and its Dimethyl Homologue in Methyl Parathion-Resistant and -Susceptible Tobacco Budworms1-3 C. J. WHITTEN Cotton
Insects
AND D. L. BULL
Research Laboratory, Agricultural Research United States Department of Agriculture
Received
August
7, 1973 ; accepted
February
Servicr,
5, 1974
Chlorpyrifos (Dowco 179) and its dimethyl homologue, chlorpyrifosmethyl (Dowco 214), were used to study the influence of the O,O-dialkyl group of organophosphorus insecticides on toxicity, absorption, and metabolism among larvae of the tobacco budworm [Heliothis virescens (F.)] from strains that were resistant (R) and susceptible (S) to methyl parathion. In toxicity tests, chlorpyrifos and chlorpyrifosmethyl were more toxic than methyl parathion to 3rd-stage R larvae but less toxic to S larvae. Chlorpyrifosmethyl was more toxic (3-4 X) than chlorpyrifos to both strains of larvae, and the results of absorption studies indicated that the toxicity differential of the homologues may be explained in part by the more rapid absorption of the dimethyl form. Studies of the in viva metabolism of both Dowco compounds indicated that each was degraded mainly by the cleavage of the pyridylphosphate linkage. In vitro tests demonstrated that the NADPH-dependent microsomal oxidases were of primary importance in detoxification, while glutathione (GSH)-dependent mechanisms (aryland alkyltransferases) present in the soluble cell fractions were of lesser importance. 0-dealkylation occurred only with chlorpyrifosmethyl. The R larvae demonstrated greater capability in detoxifying both compounds in the comparative in viva and in vitro studies of metabolism, but the differences were more apparent during the 5th instar than during the 3rd instar.
INTRODUCTION
The development of resistance to methyl parathion in field populations of tobacco budworms, Heliothis virescens (F.), attacking cotton in certain areas has been reported (l-3). Preliminary biochemical studies with organophosphorus insecticide (OP)-susceptible (S) and -resistant (R) 1 In cooperation with the Texas Agricultural Experiment Station, Texas A & M University, College Station, Texas 77840. 2 Mention of pesticide or a propietary product does not constitute recommendation or endorsement by the USDA. 3 From research completed by the senior author in partial fulfillment of Ph.D. degree in entomology from Texas A & M University. 266 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
strains of tobacco budworms suggested that the resistance was related to an increased metabolic activity in R insects ; insecticide absorption and acetylcholinesterase properties of the strains werr similar (4). Further investigations revealed an enhancement of microsomal oxidation, 0-dealkylation (0-demethylation), O-dearylation, and glucosidic conjugation detoxification mechanisms in R larvae (5, 6). Concurrent with the development of resistance has been the search for more effective insecticides. In a recent study with larvae that were exposed to an insecticide film in glass vials, Plapp (7) found that chlorpyrifos (Dowco 179, Dursban) and related compounds were more
CHLORPYRIFOS
AND
ITS
DIMETHYL
toxic than methyl parathion to resistant tobacco budworm larvae. The same study also demonstrated that 0,0-dimethyl insecticides were more toxic than their corresponding O,O-diethyl homologues to the R larvae. Consequently, the purpose of the present study was to compare the topical toxicities of certain OP compounds, and the rates at which chlorpyrifos and its dimethyl homologue chlorpyrifosmethyl (Dowco 214) were absorbed and metabolized by S and R tobacco budworm larvae, and then evaluate the influence of the O,O-dialkyl moiety of the phosphorothioate insecticides on the toxicity and the fate of the compounds in tobacco budworms. METHODS
AND
3IATERIALS
Chemicals Samples of nonradioactive and 14Clabeled (pyridyl ring) chlorpyrifos (9.31 mCi/mmole) and chlorpyrifosmethyl (10.49 mCi/mmole); Dowco 180, diethyl 3,5,6trichloro-Zpyridyl phosphate; fospirate (Dowco 217), dimethyl 3,5,6-trichloro-23,5,6-trichloro-2-pyripyridyl phosphate; dinol; and certain of its rearrangement derivatives were supplied by Dow Chemical Co., Midland, Michigan. Methyl parathion and methyl paraoxon were obtained commercially. The purities of the radiolabeled compounds were greater than 99% as determined by thin-layer chromatography (t,lc); purities of the other insecticidal chemicals used were greater than 95%. Insects The S insects were obtained from a closed culture that had been maintained at College Station, Tex., for several years and the R insects, which were generally resistant to the OP insecticides commonly used in cotton production, were the F,-F, progeny of adults collected at Wcs1ac0, Tcx. Both strains were reared in
HOMOLOGUE
IN
TOBACCO
BUDWORMS
267
the laboratory on a fortified wheat germ diet (8) in continuous light at 27-30°C. Toxicity
Studies
The relative toxicities of chlorpyrifos, Dowco 180, chlorpyrifosmethyl, fospirate, methyl parathion, and methyl paraoxon were established by determining topical LD,, and LD,, values for each compound with 3rd-stage S and R larvae (weight range 25-30 mg) as previously described (4) and in accordance with the standardized procedure recommended by the Entomological Society of America (9). The in vitro anticholinesterase activity of each toxicant was tested against preparations from larvae of each strain by a modified Hestrin calorimetric procedure (10). However, con elusive results were not obtained because the enzyme preparations exhibited very low acetylcholinesterase activity. Absorption
Studies
Third-stage larvae of both strains were treated topically with a sublethal, equimolar concentration (0.15 pg of chlorpyrifosmethyl or 0.18 pg of chlorpyrifos per larvae) of the respective 14C-labeled compounds in acetone solution. Treated larvae were held without food in covered glass containers for the specified times. Then the unabsorbed radioactivity was removed by rinsing the larvae (6 per sample) with acetone, the rinsed larvae were homogenized immediately in distilled water, and the homogenate was partitioned against chloroform (1: 2.5 v/v) to separate the radioactivity into organosoluble and watersoluble fractions. Excreted radioactivity was recovered by scrubbing holding containers with water and rinsing with acetone. Radioassays were made of the external rinse, the partitioned fractions of the homogenate, and the excreta wash. In Vivo Metabolism Fifth-stage S and R larvae were treated by injection of 1.5 pg of x4C-fabeled chlor-
268
WHITTEN
pyrifos or chlorpyrifosmethyl in 1 ~1 of propylene glycol into the body cavity. The treated larvae (2/replicate, 3 replicates/ test) were held in ventilated glass containers for 1 hr and then homogenized in distilled water. Homogenates were partitioned immediately with chloroform (1: 2.5 v/v); the aqueous and organic fractions were radioassayed and the radioactive products in each fraction w-cre resolved by chromatography. Chloroform extracts were analyzed by tic on glass plates coated (0.25 mm thick) wit,h silica gel and a solvent mixture of 90: 10: 4 hexane, dioxane, and acetic acid. Radioactive compounds in the aqueous fraction were resolved by paper chromatography (PC) on Whatman 3-MM paper with a solvent mixture of 40: 9: 1 acetonitrile, water, and ammonium hydroxide. Areas of radioactivity were located by autoradiography and quantified by liquid-scintillat’ion counting. Identifications were based on cochromatography of radioactive products with nonlabeled st’andards that were located calorimetrically by exposure to iodine vapors. The R, values of the known standards have bcrn previously reported (11, 12). In Vitro Metabolism The metabolism of chloropyrifos and chlorpyrifosmethyl was studied in vitro with microsomal and soluble enzymes from homogenates of 5th stage S and R larvae. Enzyme fractions were prepared as previously described (6) and used immediately. The solutions for the reaction with soluble enzymes included 2 larval equivalents of the soluble fraction preparation, 5 pg of either of the 2 labeled compounds, and, in some tests, 4 mg reduced glutathione (GSH) in a final volume of 4 ml of 0.1 M phosphate buffer (pH 7.4) ; microsomal incubation solutions contained 2 larval equivalents of t,he microsomal preparations, 5 pg of labeled insecticide, 8 mg of bovine serum albumin (BSA), and, if included, a cofactor (2.5 mg NADPH or 3 mg GSH) in a final
AND
BULL
volume of 3 ml of 0.1 M phosphate buffer (pH 7.4). (Preliminary experiments indicated that the inclusion of BSA in the incubation mixture enhanced degradation by twofold.) After incubation of open flasks for 2 hr at 3O”C, the reactions were stopped by adding 25 ml of chloroform. Each test was repeated at least three times. The solutions were analyzed as described for the in vivo tests, and the data were corrected for nonenzymic degradation. All radioassays were made at ambient temperature with a liquid scintillator. The appropriate corrections were made for quenching by the use of an internal standard. RESULTS
Toxicity The results of toxicity tests (Table 1) indicated that chlorpyrifos and chlorpyrifosmet,hyl were more toxic than methyl parathion to the R larvae but were less toxic to S larvae. The similarities between the LD,, values for the R and S strains suggested that the relative toxicity of chlorpyrifos and chlorpyrifosmethyl was not influenced by the same mechanisms that contributed to the 20-fold level of resistance in the R strain to methyl parathion. Chlorpyrifosmethyl was more toxic than chlorpyrifos to both strains, but results with the corresponding phosphate (P=O) analogucs did not show a similar pattern. In general, the oxygen analogurs of all insecticides were appreciably more toxic than their unoxidized forms, and the R larvae exhibited a relatively greater tolerance to P=O compounds (especially to methyl paraoxon) than the S larvae. In additional toxicity t’ests, 2 sizes of older larvae (100 and 200 mg each) from each strain were t#reated topically with chlorpyrifos, chlorpyrifosmethyl, or methyl parathion at a dosage of 1 pg of toxicant/lO mg body wt (equivalent to the LDso of methyl parathion of 3rd-stage R larvae). The percentage mortality after 48 hr in-
CHLORPYRIFOS
AND
ITS
DIMETHYL
HOMOLOGUE
TABLE Relative Toxicities of Methyl
Parathion,
Chlorpyrifos,
IN
TOBACCO
269
BUDWORRIS
1 and Their
Chlorpyrifosmethyl,
Oxygen
Analogues
Topical toxicity (pg/g) 3rd-Stage tobacco budworms __
Compound .~-__
LDeo
LDso
-
__~ b
11
S
It
4.8 2.0 79.5 3.0 17.4 1.9
118.4 62.9 66.6 6.3 20.4 9.9
13.5 7.0 573.5 9.3 42.6 5.2
843.3 > 1000.0 703.0 21.4 188.7 77.7
~__-.-
Methyl parathion Methyl paraoxon Chlorpyrifos Dowco 180 Chlorpyrifosmethyl Fospirate -__
Absorption
dicated no difference in susceptibility between the 2 sizes of larvae, but the larger insects were generally more tolerant of the insect,icides than were 3rd-stage larvae : Chlorpyrifos Chlorpyrifosmethyl Methyl parathion
No significant differences were found in rate of absorption by 3rd-stage S and R larvae during 8 hr after treatment with the radiolabeled chlorpyrifos or chlorpyrifosmethyl (Table 2). However, chlorpyrifosmethyl was absorbed at a rate almost twice that of chlorpyrifos; halftimes for the disappearance from the cuticular surface were 2 and 4 hr for chlorpyrifosmethyl and chlorpyrifos, respectively. This qualitative difference in absorption might explain in part the greater toxicity of the dimethyl
S (55) 15; R (80) 20 S (100) 95 ; R (SO) 65 S (100) 95 ; R (50) 5
(Figures in parentheses represent the expect’cd ‘% mortality based upon dosagemortality lines from Brd-stage larvae; the second figure is the actual ‘% mortality). TABLE Absorption
2
and Metabolism
of lVXabeled Chlorpyrifos S and R Tobacco Budworm Larvae
Hours aft,er keatment
and Chlorpyra’fosmethyl (25-30
Ye of recovered dosea External
rinse
by 3rd~Stage
mg Each)
% Internal Water soluble
Int,ernal extract s
.~__ _ ..-~-~~~. Chlorpyrifos 1 4 8
s
It
81.2 z!z 4.6 74.3 zt= 4.1 48.0 zt 6.2 49.0 & 5.6 21.2 zt 1.1 30.4 zk 2.1
Chlorpyrifoamethyl 58.8 f 4.9 1 19.8 f 2.8 4 s 10.6 f 4.0 -. -. *JAverage of four tests.
66.2 + 5.3 21.2 f 6.9 13.5 f 3.6
Ti.
s
It.
13.4 z!z 4.9 38.8 f 6.7 55.0 f 14.8
16.3 f 3.5 36.9 f 5.0 42..5 f 3.8
21.6 f 7.2 22.7 f 6.7 42.8 f 24.8 55.0 ck 9.1 60.5 rt 4.6 80.2 f 6.4
30.1 f 7.9 59.0 zt 7.4 66.4 zt 4.3
24.2 f 4.9 56.1 f 8.0 60.5 zt 5.1
49.2 f 16.9 48.8 zt 7.8 75.1 f 9.3 86.4 f 7.6 83.3 f 7.3 89.6 f 8.6
270
WHITTEN
AND
TABLE Metabolism
of Chlorpyrifos Injection
3
and Chlorpyrijosmethyl of 1.6 pg Toxicant/Larva
Strain
by 6th~Stage Tobacco Budworm Larvae (Average of Four Replicated Tests)
% of recovered P=S
P=O
ChFfos R
82.4 h 4.4 26.1 f 1.2
0.6 f 0.4 8.3 * 1.2
Ch~fosmethyl R
72.6 f 5.5 28.6 zk 4.3
1.3 f 1.0 5.0 * 0.9
1
2.1 z!z 0.6 1.9 f 0.4
BULL
dose as indicated 2
In Vivo Metabolism Fifth-stage larvae of the R strain metabolized both radiolabeled compounds at a much faster rate than did the S larvae (Table 3). The principal metabolito recovered from both treatments from all larvae was 3,5,6-trichloro-2-pyridinol, indicating that cleavage of the pyridylphosphate linkage was the primary detoxification route for both compounds. The unidentified products were recovered in the aqueous extracts and arc assumed to be nontoxic. The det’ection of small quantities of demethyl chlorpyrifosmethyl but no deethyl chlorpyrifos suggests that the 0-dealkylation mechanism in tobacco budworms is relatively specific for dimethyl compounds. The overall metabolism of the 2 dialkyl forms appeared to be equal in R insects while the S larvae detoxified the dimethyl homologur at a slightly faster rate.
compounda. 3
4
5
6
10.7 f 2.2 46.7 r!c 8.8
1.8 f 3.2 f
0.7 1.9
1.2 f 0.7 2.1 f 1.6
2.8 f 1.7 11.4 * 5.X
0.4 * 0.2 2.0 * 1.0
12.5 f 3.4 41.3 i 5.8
4.0 * 1.0 4.6 f 1.7
3.4 * 0.9 2.5 + 0.6
3.8 f 14.2 f
0.3 * 0.0 1.9 f 1.1
* Compound identities are a8 follows: P=S-parent compound; P=O-Oxygen trichloropyridinol; 3-unknown A; 4-unknown B: B-unknown C; 6-unknown b RI valuee of the unknown metabolites in pc system given in Methods are: C, 0.20; unknown D, 0.12.
homologue. Although no appreciable differences were found in rate of detoxification by S and R larvae, both strains apparently degraded the dimethyl form more readily as evidenced by the proportions of water soluble radioactivity partitioned from the internal extracts. (Separate tests indicated that essentially all the radioactivity in the aqueous layer represented nontoxic compounds and that in the organic layer toxic compounds.)
Following
analogue; D.
unknown
l--0-dealkylated d, 0.55;
unknown
1.8 3.1 product;
2-3,5,ti-
B,
unknown
0.45;
In Vitro Metabolism A greater capacity for detoxification among the 5th-stage R larvae was further evidence by the increased degradation of both compounds by microsomal preparations from It larvae (Table 4). Substantial increases in the concentration of 3,5,6trichloro-%-pyridinol recovered from incubation solutions fortified with NADPH was good indication that the NADPH-dependent mixed function oxidases were probably involved in the in vivo metabolism of bot’h compounds. The inclusion of GSH in the incubation mixtures resulted in a 2-fold increase in 0-demcthylation but the convcrsion t,o the pyridinol was virtually muaffected. However, the microsomal pellet, was not washed, so t’hc 0-dralkylase activity may have been influenced by residual soluble enzymes, Microsomal preparations from R larvae appeared to demonstrate a slight preference for the diethyl compound as a more favorable substrate at the substrate concentrat.ion used. However, the presence of metabolitcs other than 3,5,6-trichloro-Zpyridinol in the dimethyl reaction solutions suggests that competition from other enzyme systems could have affected the assay of mixed-function oxidase activity of those solutions. In S microsomal mixtures the dimethyl form was obviously t’he better substrate. Metabolism of the toxicants, especially chlorpyrifosmcthyl, by the soluble enzyme
CHLORPYRIFOS
AND
1TS
DIMETHYL
HOMOLOGUE
TABLE Metabolism
of Chlorpyrifos 6th-Stage Tobacco
IN
TOBACCO
271
BUDWORMS
4
and Chlorpyrijosmethyl Budworms (Average
by Microsomal of at Least Three
Preparations Tests)
of
__---.
_____ Strain
Cofactor
-.-___ Clgorpyrifos s s
% of recovered
___P=S
N&H:
P=O
1
93.3 * 1.2 90.6 ;t 2.6 YO.4 f 2.3
0.4 4 0.1 iti
2 t:;
dose as indicated -
compounda.b
2
-
6.3 z!z 1.8 9.0 * 3.2 8.1zt2.8
_____
4 __.___
..__ 5
-
6 -
-
E
NADPH -
51.5 93.3
*f 5.6 1.8
4.6 0.4 *0.2 f 1.9
--
43.9 6.3 f 8.2 2.0
-
-
R
GSH
91.7
i 3.5
0.4 f 0.3
-
7.9 * 3.4
-
-
89.3 78.8 80.4
f 1.5 f 5.8 f 3.8
0.2 f 0.1 0.4 f 0.3 0.3 * 0.1
3.3 f 2.1 4.4 f 1.1 8.2 f 3.2
5.0 f 2.4 16.4 f 5.1 9.7 f 3.2
-
-
O.YT0.6
-
-
zi!::2 it:
0.4 0.2 * 0.1
3.9 3.1 zk f 1.9 1.8
32.9 5.3 f* 7.6 1.9
0.4 1.3 zk zt 0.1 1.1
-
-
80.6 z!z 5:3
0.3 * 0.2
8.9 f 2.2
7.3 zt 2.8
0.8 zt 0.6
CI$xpyrifosmethyl s s
4 Compounds b Compound
identified in Table 3 was not detected.
The results of these studies demonstrate the influence of the O,O-dialkyl moiety of the pyridyl phosphorothioates tested on toxicity, absorption, and metabolism in TABLE of Chlorpyrifos Tobacco Budworm
o/o of recovered P=S
P=O 0.4 zto.1 0.3 * 0.2
Cl$rpyrifo,s S
G-&
95.8 f 3.1 92.2 ir 2.8
R R
G;sH
91.6 84.7
GSH
Y1.5 f 4.5 75.Y f 5.9
0.3 f 0.1 0.4 * 0.1
(:hH -7
85.6 61.9
0.4 +f 0.2 0.6 0.1
R”
” Compounds 6 Compound
identified in Table 3 W&A no+. detected.
5
and Chlorpyrifosmethyl Homogenates (Average
Cofactor
-
Chprifosmethyl 8
1.1 YO.8
tobacco budworm larvae. In addition, the detoxification mechanisms of the larvae have been further clarified. For example, previous reports (5-7) have suggested the microsomal mixed-function oxidases might be one of the principal detoxification systems contributing to the resistance of tobacco budworms to OP insecticides, but the conclusions were based on the indirect evidence of the increased epoxidation of aldrin to dieldrin by R larvae. The results presented in this paper clearly establish the importance of the microsomal system in the degradation of the 2 Dowco compounds. Results of the i?~ vivo tests demonstrated that 3,5,6-trichloro-2-pyridinol was the
DISCUSSION
Strain
1.0 f0.4
3.
systems was stimulated by the addition of GSH. Preparations from R larvae showed the greatest increase (Table 5). The enhancement by GSH was manifested primarily by increased 0-demethylation and conversion to the unknowns C and D. The absence of deethylated products is further indicat.ion that the specificity of the GSH-dependent alkyltransferases is predominantly in favor of dimethyl esters.
Metabolism
-
3.
f 2.2 f 4.6
f 3.2 7.6
by Soluble of at Least
Enzymes of 5th-Stage Three Tests)
dose as indicated
compounda,
2
1
4
-
2.9 * 0.9 2.8 f 0.8
E
_-
3.5 * 1.1 2.7 f 0.4
1.7 f 0.8 4.7 i 1.7 11.8 3.1 f
1.1 2.9
b
. .-5
6
0.2 i 0.1 1.9 f 0.2
0.4 f 2.2 f
0.9 * 0.3 0.9 * 0.2
1.8 f 5.3 f
1.2 1.8
1.7 l 1.0 5.9 * 3.5
3.5 f 0.6 4.3 * 1.2
:::
1.3 f 6.6 f
0.8 1.6
0.8 zt 0.5 6.7 f 2.1
3.4 *f 0.Y 3.6 1.0
5.0 2.5 zk f 3.8 1.6
13.7 2.7 f 4.7 1.3
3.8 dz 0.9 f 0.1 0.3
2 E
2 0”::
0.2 0.9
272
WHITTEN
predominant metabolite, and the in vitro studies showed that conversion to the pyridinol was mediated primarily by the NADPH-dependent microsomal enzyme system. The activity of the soluble enzymes against chlorpyrifos and chlorpyrifosmethyl appeared to be mainly GSH dependent. An earlier study (6) demonstrated the importance of a GSH-dependent alkyltransferase system in detoxification of a dimethyl phosphate insecticide, and also established the importance of soluble phosphotriesterases in hydrolyzing the ary1phosphat.e linkage of the compound. The present data indicate that these soluble syst’ems may be of only minor importance in the degradation of the phosphorothioate compounds tested. The most apparent response to the addition of GSH to t.he reactions with soluble enzymes was the increased conversion to the unknowns C and D. The biochemical nature of the enhancement is not known, but the possibility exists that the cofactor stimulated the hydrolysis of the pyridylphosphate linkage to produce the substituted-pyridinol residue, which in turn was converted to the unknowns by a secondary react’ion. Smith (13) has reported bhe nonenzymatic conversion of the pyridinol moiety to a series of diols and triols, but the absence of the unknowns in the aqueous phase of the reactions with microsomal enzymes fortified with NADPH suggests that the unknowns were not, formed by this type of a reaction. To test if the unknowns C and D were enzymatic products of the pyridinol, 14C-labeled 3,5,6-trichloro-Zpyridinol was recovered from some of the experiments and incubated with buffer or preparations of soluble enzymes with and without added GSH. The results did not demonstrate> cvidencc of the unknowns being formed, rithrr enzymatically or nonenzymatically, from the pyridinol. Also, the possibility t,hat the unknowns are derivatives of O-dcalkylated products must he discounted hc-
AND
BULL
cause no deethyl metabolites were detected during the study. Thus, it is reasonable to assume that an enzyme system(s) requiring GSH and the complete toxicant molecults is responsible for the formation of the unknowns C and D. Dahm (14) reported the activity of nonoxidat’ive, soluble cnzymes that required GSH in rat’s and in houseflies, Musca donzestica (I,.), in the mrtabolism of W-parathion to O,O-diethyl phosphorothioic acid (DEPTA). In addition, Yang et al. (15) recovered unknown water soluble metabolites attributed to GSH stimulation, and probably not dcalkylated products, in metabolism studies with soluble housefly enzyme systems and 14C-diazinon. Recently, a mctaholitc from diazinon identified as S-(2-isopropyl-4methyl-6-pyrimidinyl) glutathiono was reported from studies involving soluble enzyme preparations from rat liver and from fat body of the American cockroach, Periplaneta mericana (L). (16). The conjugate was formed simultaneously with the cleavage of the pyrimidinyl-phosphate linkage. Therefore, although the evidence is circumstantial, our results suggest t,hat# a similar system may be prcscnt in tobacco budworm larvuc. Other investigations (3, 7, 17) have dcmonstratctd the superiority of dimethyl toxicants over Dhrir diethyl homologues in toxicity t#ests with tobacco budworms, and their superiority has usually been attributed to diffcrcncps in metabolism. However, the results of this study have indicat#cad t#hat the influence of the O,O-dialkyl group on toxicity in tobacco budworms is apparently related to the rate of absorption as well as to t,he rate of metabolism. The faster rat#(h of absorption of the dimethyl ester by thr larvae could explain the greater toxicity of chlorpyrifosmc%hyl evc’n though it was apparently metabolized more rapidly in Y&I by 3rd~stage larvae. The similarity in susceptibility (or t&rante) of 3rd-stage larvae of the S and R strains to each of the Dowco phosphorothioates contrast with the obvious differ-
CHLORPYRIFOS
AND
ITS
DIMETHYL UNKNOWN
HOMOLOGUE
IN
UNKNOWNS
B, C, D
TOBACCO
BUDWORMS
273
A
PARENT
FIG. 1. Proposed metabolic pathways of chlorpyrifos and chlorpyrifosmethyl in tobacco budworms. Dashed lines indicate mechanisms that were not demonstrated but are probable. RI = CH,; Rz = CzHs; 34 = Microsomes + NADPH; (8) = Soluble enzymes; S = Soluble mzymes+ GSH.
ences in degradative potential exhibit’ed by 5th-stage larvae. A possible explanation is that the activity of degradativc cnzymc systems may be diminished during the early stages of growth, thereby decreasing differences in the capacity for detoxification between S and R larval strains. The data from the absorption t’est,s indicat,ed that metabolic differences between 3rdstage S and R larvae were much less than between 5th-stage insects. Also, the ~YLvitro activity of microsomal oxidases in tissues from late instars was greater than that of early instars among larval stages of the cabbage looper, Trichoplusia ni (Htibner) (18)) and the southern armyworm, Prodtm ia erida?ria (Cramer) (19). This evidence suggests that a larva should be able to tolerate relat’ively greater doses (mg toxicant/g body weight) of insecticide as it increased in size. Indeed, such a relationship was reported in tobacco budworms by Gast (20). That the toxicity of chlorpyrifos and chlorpyrifosmethyl was apparently not affected by the mechanism(s) contributing to the resistance to methyl parathion in the R larvae is particularly intriguing. The degradative capability of microsomal enzyme systems has been widely implicated in insecticide resistance
(21), and the results of this study have indicated that both Dowco compounds were detoxified primarily by microsomal oxidases that were particularly enhanced in 5thstage larvae of the R strain. While the activit#y of the microsomal enzyme system present in the younger larvae may have been diminished, the fact remains that the R larvae were resistant to methyl parathion. Thus, a mechanism or a combination of mechanisms other than microsomal oxidases could be involved in the resistance to methyl parathion found in the R strain. Based on the results of this study, tentative pathways for the metabolism of chlorpyrifos and chlorpyrifosmethyl can be constructed (Fig. 1). In the diagram, the unidentified metabolites are depicted as products of primary react’ions although conclusive evidence of this cannot be given. In addition, the nonoxidative hydrolytic syst,cm of the soluble phosphotriesterases and the GSH-dependent alkyltransferase are shown as detoxification mechanisms acting upon the oxidized form of the toxicant. Both systems have been demonstrated in tobacco budworms (6) and could contribute significantly t,o t,he degradation of the phosphate ester.
274
WHITTEN
REFERENCES
1. S. J. Nemec and P. L. Adkisson, Laboratory tests of insecticides for bollworm, tobacco budworm, and boll weevil control, Tes. Agr. Exp. Sta. Progr. Rep. 2674, 5 pp (1969). 2. J. R. Coppedge, W. L. Sterling, R. L. Ridgway, R. E. Kinzer, and C. J. Whitten, Foliar applications of selected insecticides for control of methyl parathion-resistant tobacco budworms, 7’ex. Agr. Exp. Sta. Progr. Rep. 3087, 8 pp (1972). 3. D. A. Wolfenbarger and R. I,. McGarr, Toxicity of methyl parathion, parathion, and monocrotophos applied topically to populations of lepidopteran pests of cotton, J. Econ. Entomol. 63, 1762 (1970). 4. C. J. Whitten and D. L. Bull, Resistance to organophosphorus insecticides in tobacco budworms, J. Econ. Entomol. 63, 1492 (1970). 5. R. L. Williamson and M. S. Schecter, Microsomal epoxidation of aldrin in lepidopterous larvae, Rio&em. Pharmaco(. 19, 1719 (1970). 6. D. L. Bull and C. J. Whitten, Factors influencing organophosphorus insecticide resistance in tobacco budworms, J. Agr. Food Chem. 20, 561 (1972). 7. F. W. Plapp, Jr., Laboratory tests of alternate insecticides for the control of methyl parathion-resistant tobacco budworm larvae, l’ex. Agr. Exp. Sta. Progr. Rep. 3090, 7 pp (1972). 8. P;. 8. Vanderzant, Wheat-germ diets for insects : rearing the boll weevil and the salt-marsh caterpillar, Ann. Entomol. Sot. Amw. 60, 1062 (1967). 9. Second conference on Test Methods for Resistance in Insects of Agricultural Importance : Standard method for detection of insecticide resistance in Hrliolhis zea (Boddie) and H. viwscens (F.), Hull. Entomot. Sot. Amer. 16, 147 (1970). 10. D. R. Simpson, 1). L. Bull, and D. A. Lindquist, A semimicrotechnique for the estimation of cholinesterase activity in boll weevils, Ann. Entomol. Sot. Amer. 57, 367 (1964).
AND
BULL
11. G. N. Smith, B. S. Watson, and F. S. Fischer, The metabolism of [‘4C]O,O-diethyl-0-(3,5,6trichloro-2-pyridyl) phosphorothioate (Dursban) in fish, J. Bon. Entomol. 59, 1464 (1966). 12. G. N. Smith and F. S. Fischer, Use of paper partition and thin-layer chromatography for identification of the active ingredient in Dursban insecticide and its possible metabolites, J. Agr. Food Chem. 15, 182 (1967). 13. G. N. Smith, Ultraviolet light decomposition studies with DURSBAN and 3,.5,6-trichloropyridinol. J. Econ. Entomol. 61, 793 (1968). 14. P. A. Dahm, Some aspects of the metabolism of parathion and diazinon, in “Biochemical Toxicology of Insecticides” (I~. D. O’Brien and I. Yamamoto, Eds.), p. 51, Academic Press New York, 1970. 15. R. S. H. Yang, E. Hodgson, and W. C. Dauterman, Metabolism in vitro of diazinon and diazoxon in susceptible houseflies, J. Agr. Food Chem. 19, 14 (1971). 16. T. Shishido, K. Usui, M. Sato, and J. Fukami, Enzymatic conjugation of diazinon with glutathione in rat and American cockroach, Pest. Biochem. Physiol. 2, 51 (1972). 17. F. W. Plapp, Jr., Insecticide resist,ance in Heliothis: Tolerance in larvae of H. virescens as compared with H. zea to organophosphate insecticides, J. Econ. Entomol. 64, 999 (1971). 18. K. J. Kuhr, Comparative metabolism of carbaryl by resistant and susceptible strains of the cabbage looper, J. &on. Entomol. 64, 1373 (1971). 19. It. I. Krieger and C. F. Wilkinson, Microsomal mixed-function oxidases in insects. I. Localization and properties of an enzyme system effecting aldrin expoxidat,ion in larvae of the southern armyworm (Prod&a eriilania), Riochcm. Pharmacol. 18, 1403 (1969). 20. R. T. Gast, The relationship of weight of lepidopterous larvae to effect.iveness of topically applied insecticides, J. Econ. Entomol. 52, 1115 (1959). 21. E. Hodgson and F. W. Plapp, Jr., Biochemical characteristics of insect microsomes, J. Agr. Food Chcm. 18, 1048 (1970).
BIOCHEMISTRY
AND
PHYSIOLOGY
4, 266-274
Comparative Toxicity, Absorption, and Metabolism of Chlorpyrifos and its Dimethyl Homologue in Methyl Parathion-Resistant and -Susceptible Tobacco Budworms1-3 C. J. WHITTEN Cotton
Insects
AND D. L. BULL
Research Laboratory, Agricultural Research United States Department of Agriculture
Received
August
7, 1973 ; accepted
February
Servicr,
5, 1974
Chlorpyrifos (Dowco 179) and its dimethyl homologue, chlorpyrifosmethyl (Dowco 214), were used to study the influence of the O,O-dialkyl group of organophosphorus insecticides on toxicity, absorption, and metabolism among larvae of the tobacco budworm [Heliothis virescens (F.)] from strains that were resistant (R) and susceptible (S) to methyl parathion. In toxicity tests, chlorpyrifos and chlorpyrifosmethyl were more toxic than methyl parathion to 3rd-stage R larvae but less toxic to S larvae. Chlorpyrifosmethyl was more toxic (3-4 X) than chlorpyrifos to both strains of larvae, and the results of absorption studies indicated that the toxicity differential of the homologues may be explained in part by the more rapid absorption of the dimethyl form. Studies of the in viva metabolism of both Dowco compounds indicated that each was degraded mainly by the cleavage of the pyridylphosphate linkage. In vitro tests demonstrated that the NADPH-dependent microsomal oxidases were of primary importance in detoxification, while glutathione (GSH)-dependent mechanisms (aryland alkyltransferases) present in the soluble cell fractions were of lesser importance. 0-dealkylation occurred only with chlorpyrifosmethyl. The R larvae demonstrated greater capability in detoxifying both compounds in the comparative in viva and in vitro studies of metabolism, but the differences were more apparent during the 5th instar than during the 3rd instar.
INTRODUCTION
The development of resistance to methyl parathion in field populations of tobacco budworms, Heliothis virescens (F.), attacking cotton in certain areas has been reported (l-3). Preliminary biochemical studies with organophosphorus insecticide (OP)-susceptible (S) and -resistant (R) 1 In cooperation with the Texas Agricultural Experiment Station, Texas A & M University, College Station, Texas 77840. 2 Mention of pesticide or a propietary product does not constitute recommendation or endorsement by the USDA. 3 From research completed by the senior author in partial fulfillment of Ph.D. degree in entomology from Texas A & M University. 266 Copyright All rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
strains of tobacco budworms suggested that the resistance was related to an increased metabolic activity in R insects ; insecticide absorption and acetylcholinesterase properties of the strains werr similar (4). Further investigations revealed an enhancement of microsomal oxidation, 0-dealkylation (0-demethylation), O-dearylation, and glucosidic conjugation detoxification mechanisms in R larvae (5, 6). Concurrent with the development of resistance has been the search for more effective insecticides. In a recent study with larvae that were exposed to an insecticide film in glass vials, Plapp (7) found that chlorpyrifos (Dowco 179, Dursban) and related compounds were more
CHLORPYRIFOS
AND
ITS
DIMETHYL
toxic than methyl parathion to resistant tobacco budworm larvae. The same study also demonstrated that 0,0-dimethyl insecticides were more toxic than their corresponding O,O-diethyl homologues to the R larvae. Consequently, the purpose of the present study was to compare the topical toxicities of certain OP compounds, and the rates at which chlorpyrifos and its dimethyl homologue chlorpyrifosmethyl (Dowco 214) were absorbed and metabolized by S and R tobacco budworm larvae, and then evaluate the influence of the O,O-dialkyl moiety of the phosphorothioate insecticides on the toxicity and the fate of the compounds in tobacco budworms. METHODS
AND
3IATERIALS
Chemicals Samples of nonradioactive and 14Clabeled (pyridyl ring) chlorpyrifos (9.31 mCi/mmole) and chlorpyrifosmethyl (10.49 mCi/mmole); Dowco 180, diethyl 3,5,6trichloro-Zpyridyl phosphate; fospirate (Dowco 217), dimethyl 3,5,6-trichloro-23,5,6-trichloro-2-pyripyridyl phosphate; dinol; and certain of its rearrangement derivatives were supplied by Dow Chemical Co., Midland, Michigan. Methyl parathion and methyl paraoxon were obtained commercially. The purities of the radiolabeled compounds were greater than 99% as determined by thin-layer chromatography (t,lc); purities of the other insecticidal chemicals used were greater than 95%. Insects The S insects were obtained from a closed culture that had been maintained at College Station, Tex., for several years and the R insects, which were generally resistant to the OP insecticides commonly used in cotton production, were the F,-F, progeny of adults collected at Wcs1ac0, Tcx. Both strains were reared in
HOMOLOGUE
IN
TOBACCO
BUDWORMS
267
the laboratory on a fortified wheat germ diet (8) in continuous light at 27-30°C. Toxicity
Studies
The relative toxicities of chlorpyrifos, Dowco 180, chlorpyrifosmethyl, fospirate, methyl parathion, and methyl paraoxon were established by determining topical LD,, and LD,, values for each compound with 3rd-stage S and R larvae (weight range 25-30 mg) as previously described (4) and in accordance with the standardized procedure recommended by the Entomological Society of America (9). The in vitro anticholinesterase activity of each toxicant was tested against preparations from larvae of each strain by a modified Hestrin calorimetric procedure (10). However, con elusive results were not obtained because the enzyme preparations exhibited very low acetylcholinesterase activity. Absorption
Studies
Third-stage larvae of both strains were treated topically with a sublethal, equimolar concentration (0.15 pg of chlorpyrifosmethyl or 0.18 pg of chlorpyrifos per larvae) of the respective 14C-labeled compounds in acetone solution. Treated larvae were held without food in covered glass containers for the specified times. Then the unabsorbed radioactivity was removed by rinsing the larvae (6 per sample) with acetone, the rinsed larvae were homogenized immediately in distilled water, and the homogenate was partitioned against chloroform (1: 2.5 v/v) to separate the radioactivity into organosoluble and watersoluble fractions. Excreted radioactivity was recovered by scrubbing holding containers with water and rinsing with acetone. Radioassays were made of the external rinse, the partitioned fractions of the homogenate, and the excreta wash. In Vivo Metabolism Fifth-stage S and R larvae were treated by injection of 1.5 pg of x4C-fabeled chlor-
268
WHITTEN
pyrifos or chlorpyrifosmethyl in 1 ~1 of propylene glycol into the body cavity. The treated larvae (2/replicate, 3 replicates/ test) were held in ventilated glass containers for 1 hr and then homogenized in distilled water. Homogenates were partitioned immediately with chloroform (1: 2.5 v/v); the aqueous and organic fractions were radioassayed and the radioactive products in each fraction w-cre resolved by chromatography. Chloroform extracts were analyzed by tic on glass plates coated (0.25 mm thick) wit,h silica gel and a solvent mixture of 90: 10: 4 hexane, dioxane, and acetic acid. Radioactive compounds in the aqueous fraction were resolved by paper chromatography (PC) on Whatman 3-MM paper with a solvent mixture of 40: 9: 1 acetonitrile, water, and ammonium hydroxide. Areas of radioactivity were located by autoradiography and quantified by liquid-scintillat’ion counting. Identifications were based on cochromatography of radioactive products with nonlabeled st’andards that were located calorimetrically by exposure to iodine vapors. The R, values of the known standards have bcrn previously reported (11, 12). In Vitro Metabolism The metabolism of chloropyrifos and chlorpyrifosmethyl was studied in vitro with microsomal and soluble enzymes from homogenates of 5th stage S and R larvae. Enzyme fractions were prepared as previously described (6) and used immediately. The solutions for the reaction with soluble enzymes included 2 larval equivalents of the soluble fraction preparation, 5 pg of either of the 2 labeled compounds, and, in some tests, 4 mg reduced glutathione (GSH) in a final volume of 4 ml of 0.1 M phosphate buffer (pH 7.4) ; microsomal incubation solutions contained 2 larval equivalents of t,he microsomal preparations, 5 pg of labeled insecticide, 8 mg of bovine serum albumin (BSA), and, if included, a cofactor (2.5 mg NADPH or 3 mg GSH) in a final
AND
BULL
volume of 3 ml of 0.1 M phosphate buffer (pH 7.4). (Preliminary experiments indicated that the inclusion of BSA in the incubation mixture enhanced degradation by twofold.) After incubation of open flasks for 2 hr at 3O”C, the reactions were stopped by adding 25 ml of chloroform. Each test was repeated at least three times. The solutions were analyzed as described for the in vivo tests, and the data were corrected for nonenzymic degradation. All radioassays were made at ambient temperature with a liquid scintillator. The appropriate corrections were made for quenching by the use of an internal standard. RESULTS
Toxicity The results of toxicity tests (Table 1) indicated that chlorpyrifos and chlorpyrifosmet,hyl were more toxic than methyl parathion to the R larvae but were less toxic to S larvae. The similarities between the LD,, values for the R and S strains suggested that the relative toxicity of chlorpyrifos and chlorpyrifosmethyl was not influenced by the same mechanisms that contributed to the 20-fold level of resistance in the R strain to methyl parathion. Chlorpyrifosmethyl was more toxic than chlorpyrifos to both strains, but results with the corresponding phosphate (P=O) analogucs did not show a similar pattern. In general, the oxygen analogurs of all insecticides were appreciably more toxic than their unoxidized forms, and the R larvae exhibited a relatively greater tolerance to P=O compounds (especially to methyl paraoxon) than the S larvae. In additional toxicity t’ests, 2 sizes of older larvae (100 and 200 mg each) from each strain were t#reated topically with chlorpyrifos, chlorpyrifosmethyl, or methyl parathion at a dosage of 1 pg of toxicant/lO mg body wt (equivalent to the LDso of methyl parathion of 3rd-stage R larvae). The percentage mortality after 48 hr in-
CHLORPYRIFOS
AND
ITS
DIMETHYL
HOMOLOGUE
TABLE Relative Toxicities of Methyl
Parathion,
Chlorpyrifos,
IN
TOBACCO
269
BUDWORRIS
1 and Their
Chlorpyrifosmethyl,
Oxygen
Analogues
Topical toxicity (pg/g) 3rd-Stage tobacco budworms __
Compound .~-__
LDeo
LDso
-
__~ b
11
S
It
4.8 2.0 79.5 3.0 17.4 1.9
118.4 62.9 66.6 6.3 20.4 9.9
13.5 7.0 573.5 9.3 42.6 5.2
843.3 > 1000.0 703.0 21.4 188.7 77.7
~__-.-
Methyl parathion Methyl paraoxon Chlorpyrifos Dowco 180 Chlorpyrifosmethyl Fospirate -__
Absorption
dicated no difference in susceptibility between the 2 sizes of larvae, but the larger insects were generally more tolerant of the insect,icides than were 3rd-stage larvae : Chlorpyrifos Chlorpyrifosmethyl Methyl parathion
No significant differences were found in rate of absorption by 3rd-stage S and R larvae during 8 hr after treatment with the radiolabeled chlorpyrifos or chlorpyrifosmethyl (Table 2). However, chlorpyrifosmethyl was absorbed at a rate almost twice that of chlorpyrifos; halftimes for the disappearance from the cuticular surface were 2 and 4 hr for chlorpyrifosmethyl and chlorpyrifos, respectively. This qualitative difference in absorption might explain in part the greater toxicity of the dimethyl
S (55) 15; R (80) 20 S (100) 95 ; R (SO) 65 S (100) 95 ; R (50) 5
(Figures in parentheses represent the expect’cd ‘% mortality based upon dosagemortality lines from Brd-stage larvae; the second figure is the actual ‘% mortality). TABLE Absorption
2
and Metabolism
of lVXabeled Chlorpyrifos S and R Tobacco Budworm Larvae
Hours aft,er keatment
and Chlorpyra’fosmethyl (25-30
Ye of recovered dosea External
rinse
by 3rd~Stage
mg Each)
% Internal Water soluble
Int,ernal extract s
.~__ _ ..-~-~~~. Chlorpyrifos 1 4 8
s
It
81.2 z!z 4.6 74.3 zt= 4.1 48.0 zt 6.2 49.0 & 5.6 21.2 zt 1.1 30.4 zk 2.1
Chlorpyrifoamethyl 58.8 f 4.9 1 19.8 f 2.8 4 s 10.6 f 4.0 -. -. *JAverage of four tests.
66.2 + 5.3 21.2 f 6.9 13.5 f 3.6
Ti.
s
It.
13.4 z!z 4.9 38.8 f 6.7 55.0 f 14.8
16.3 f 3.5 36.9 f 5.0 42..5 f 3.8
21.6 f 7.2 22.7 f 6.7 42.8 f 24.8 55.0 ck 9.1 60.5 rt 4.6 80.2 f 6.4
30.1 f 7.9 59.0 zt 7.4 66.4 zt 4.3
24.2 f 4.9 56.1 f 8.0 60.5 zt 5.1
49.2 f 16.9 48.8 zt 7.8 75.1 f 9.3 86.4 f 7.6 83.3 f 7.3 89.6 f 8.6
270
WHITTEN
AND
TABLE Metabolism
of Chlorpyrifos Injection
3
and Chlorpyrijosmethyl of 1.6 pg Toxicant/Larva
Strain
by 6th~Stage Tobacco Budworm Larvae (Average of Four Replicated Tests)
% of recovered P=S
P=O
ChFfos R
82.4 h 4.4 26.1 f 1.2
0.6 f 0.4 8.3 * 1.2
Ch~fosmethyl R
72.6 f 5.5 28.6 zk 4.3
1.3 f 1.0 5.0 * 0.9
1
2.1 z!z 0.6 1.9 f 0.4
BULL
dose as indicated 2
In Vivo Metabolism Fifth-stage larvae of the R strain metabolized both radiolabeled compounds at a much faster rate than did the S larvae (Table 3). The principal metabolito recovered from both treatments from all larvae was 3,5,6-trichloro-2-pyridinol, indicating that cleavage of the pyridylphosphate linkage was the primary detoxification route for both compounds. The unidentified products were recovered in the aqueous extracts and arc assumed to be nontoxic. The det’ection of small quantities of demethyl chlorpyrifosmethyl but no deethyl chlorpyrifos suggests that the 0-dealkylation mechanism in tobacco budworms is relatively specific for dimethyl compounds. The overall metabolism of the 2 dialkyl forms appeared to be equal in R insects while the S larvae detoxified the dimethyl homologur at a slightly faster rate.
compounda. 3
4
5
6
10.7 f 2.2 46.7 r!c 8.8
1.8 f 3.2 f
0.7 1.9
1.2 f 0.7 2.1 f 1.6
2.8 f 1.7 11.4 * 5.X
0.4 * 0.2 2.0 * 1.0
12.5 f 3.4 41.3 i 5.8
4.0 * 1.0 4.6 f 1.7
3.4 * 0.9 2.5 + 0.6
3.8 f 14.2 f
0.3 * 0.0 1.9 f 1.1
* Compound identities are a8 follows: P=S-parent compound; P=O-Oxygen trichloropyridinol; 3-unknown A; 4-unknown B: B-unknown C; 6-unknown b RI valuee of the unknown metabolites in pc system given in Methods are: C, 0.20; unknown D, 0.12.
homologue. Although no appreciable differences were found in rate of detoxification by S and R larvae, both strains apparently degraded the dimethyl form more readily as evidenced by the proportions of water soluble radioactivity partitioned from the internal extracts. (Separate tests indicated that essentially all the radioactivity in the aqueous layer represented nontoxic compounds and that in the organic layer toxic compounds.)
Following
analogue; D.
unknown
l--0-dealkylated d, 0.55;
unknown
1.8 3.1 product;
2-3,5,ti-
B,
unknown
0.45;
In Vitro Metabolism A greater capacity for detoxification among the 5th-stage R larvae was further evidence by the increased degradation of both compounds by microsomal preparations from It larvae (Table 4). Substantial increases in the concentration of 3,5,6trichloro-%-pyridinol recovered from incubation solutions fortified with NADPH was good indication that the NADPH-dependent mixed function oxidases were probably involved in the in vivo metabolism of bot’h compounds. The inclusion of GSH in the incubation mixtures resulted in a 2-fold increase in 0-demcthylation but the convcrsion t,o the pyridinol was virtually muaffected. However, the microsomal pellet, was not washed, so t’hc 0-dralkylase activity may have been influenced by residual soluble enzymes, Microsomal preparations from R larvae appeared to demonstrate a slight preference for the diethyl compound as a more favorable substrate at the substrate concentrat.ion used. However, the presence of metabolitcs other than 3,5,6-trichloro-Zpyridinol in the dimethyl reaction solutions suggests that competition from other enzyme systems could have affected the assay of mixed-function oxidase activity of those solutions. In S microsomal mixtures the dimethyl form was obviously t’he better substrate. Metabolism of the toxicants, especially chlorpyrifosmcthyl, by the soluble enzyme
CHLORPYRIFOS
AND
1TS
DIMETHYL
HOMOLOGUE
TABLE Metabolism
of Chlorpyrifos 6th-Stage Tobacco
IN
TOBACCO
271
BUDWORMS
4
and Chlorpyrijosmethyl Budworms (Average
by Microsomal of at Least Three
Preparations Tests)
of
__---.
_____ Strain
Cofactor
-.-___ Clgorpyrifos s s
% of recovered
___P=S
N&H:
P=O
1
93.3 * 1.2 90.6 ;t 2.6 YO.4 f 2.3
0.4 4 0.1 iti
2 t:;
dose as indicated -
compounda.b
2
-
6.3 z!z 1.8 9.0 * 3.2 8.1zt2.8
_____
4 __.___
..__ 5
-
6 -
-
E
NADPH -
51.5 93.3
*f 5.6 1.8
4.6 0.4 *0.2 f 1.9
--
43.9 6.3 f 8.2 2.0
-
-
R
GSH
91.7
i 3.5
0.4 f 0.3
-
7.9 * 3.4
-
-
89.3 78.8 80.4
f 1.5 f 5.8 f 3.8
0.2 f 0.1 0.4 f 0.3 0.3 * 0.1
3.3 f 2.1 4.4 f 1.1 8.2 f 3.2
5.0 f 2.4 16.4 f 5.1 9.7 f 3.2
-
-
O.YT0.6
-
-
zi!::2 it:
0.4 0.2 * 0.1
3.9 3.1 zk f 1.9 1.8
32.9 5.3 f* 7.6 1.9
0.4 1.3 zk zt 0.1 1.1
-
-
80.6 z!z 5:3
0.3 * 0.2
8.9 f 2.2
7.3 zt 2.8
0.8 zt 0.6
CI$xpyrifosmethyl s s
4 Compounds b Compound
identified in Table 3 was not detected.
The results of these studies demonstrate the influence of the O,O-dialkyl moiety of the pyridyl phosphorothioates tested on toxicity, absorption, and metabolism in TABLE of Chlorpyrifos Tobacco Budworm
o/o of recovered P=S
P=O 0.4 zto.1 0.3 * 0.2
Cl$rpyrifo,s S
G-&
95.8 f 3.1 92.2 ir 2.8
R R
G;sH
91.6 84.7
GSH
Y1.5 f 4.5 75.Y f 5.9
0.3 f 0.1 0.4 * 0.1
(:hH -7
85.6 61.9
0.4 +f 0.2 0.6 0.1
R”
” Compounds 6 Compound
identified in Table 3 W&A no+. detected.
5
and Chlorpyrifosmethyl Homogenates (Average
Cofactor
-
Chprifosmethyl 8
1.1 YO.8
tobacco budworm larvae. In addition, the detoxification mechanisms of the larvae have been further clarified. For example, previous reports (5-7) have suggested the microsomal mixed-function oxidases might be one of the principal detoxification systems contributing to the resistance of tobacco budworms to OP insecticides, but the conclusions were based on the indirect evidence of the increased epoxidation of aldrin to dieldrin by R larvae. The results presented in this paper clearly establish the importance of the microsomal system in the degradation of the 2 Dowco compounds. Results of the i?~ vivo tests demonstrated that 3,5,6-trichloro-2-pyridinol was the
DISCUSSION
Strain
1.0 f0.4
3.
systems was stimulated by the addition of GSH. Preparations from R larvae showed the greatest increase (Table 5). The enhancement by GSH was manifested primarily by increased 0-demethylation and conversion to the unknowns C and D. The absence of deethylated products is further indicat.ion that the specificity of the GSH-dependent alkyltransferases is predominantly in favor of dimethyl esters.
Metabolism
-
3.
f 2.2 f 4.6
f 3.2 7.6
by Soluble of at Least
Enzymes of 5th-Stage Three Tests)
dose as indicated
compounda,
2
1
4
-
2.9 * 0.9 2.8 f 0.8
E
_-
3.5 * 1.1 2.7 f 0.4
1.7 f 0.8 4.7 i 1.7 11.8 3.1 f
1.1 2.9
b
. .-5
6
0.2 i 0.1 1.9 f 0.2
0.4 f 2.2 f
0.9 * 0.3 0.9 * 0.2
1.8 f 5.3 f
1.2 1.8
1.7 l 1.0 5.9 * 3.5
3.5 f 0.6 4.3 * 1.2
:::
1.3 f 6.6 f
0.8 1.6
0.8 zt 0.5 6.7 f 2.1
3.4 *f 0.Y 3.6 1.0
5.0 2.5 zk f 3.8 1.6
13.7 2.7 f 4.7 1.3
3.8 dz 0.9 f 0.1 0.3
2 E
2 0”::
0.2 0.9
272
WHITTEN
predominant metabolite, and the in vitro studies showed that conversion to the pyridinol was mediated primarily by the NADPH-dependent microsomal enzyme system. The activity of the soluble enzymes against chlorpyrifos and chlorpyrifosmethyl appeared to be mainly GSH dependent. An earlier study (6) demonstrated the importance of a GSH-dependent alkyltransferase system in detoxification of a dimethyl phosphate insecticide, and also established the importance of soluble phosphotriesterases in hydrolyzing the ary1phosphat.e linkage of the compound. The present data indicate that these soluble syst’ems may be of only minor importance in the degradation of the phosphorothioate compounds tested. The most apparent response to the addition of GSH to t.he reactions with soluble enzymes was the increased conversion to the unknowns C and D. The biochemical nature of the enhancement is not known, but the possibility exists that the cofactor stimulated the hydrolysis of the pyridylphosphate linkage to produce the substituted-pyridinol residue, which in turn was converted to the unknowns by a secondary react’ion. Smith (13) has reported bhe nonenzymatic conversion of the pyridinol moiety to a series of diols and triols, but the absence of the unknowns in the aqueous phase of the reactions with microsomal enzymes fortified with NADPH suggests that the unknowns were not, formed by this type of a reaction. To test if the unknowns C and D were enzymatic products of the pyridinol, 14C-labeled 3,5,6-trichloro-Zpyridinol was recovered from some of the experiments and incubated with buffer or preparations of soluble enzymes with and without added GSH. The results did not demonstrate> cvidencc of the unknowns being formed, rithrr enzymatically or nonenzymatically, from the pyridinol. Also, the possibility t,hat the unknowns are derivatives of O-dcalkylated products must he discounted hc-
AND
BULL
cause no deethyl metabolites were detected during the study. Thus, it is reasonable to assume that an enzyme system(s) requiring GSH and the complete toxicant molecults is responsible for the formation of the unknowns C and D. Dahm (14) reported the activity of nonoxidat’ive, soluble cnzymes that required GSH in rat’s and in houseflies, Musca donzestica (I,.), in the mrtabolism of W-parathion to O,O-diethyl phosphorothioic acid (DEPTA). In addition, Yang et al. (15) recovered unknown water soluble metabolites attributed to GSH stimulation, and probably not dcalkylated products, in metabolism studies with soluble housefly enzyme systems and 14C-diazinon. Recently, a mctaholitc from diazinon identified as S-(2-isopropyl-4methyl-6-pyrimidinyl) glutathiono was reported from studies involving soluble enzyme preparations from rat liver and from fat body of the American cockroach, Periplaneta mericana (L). (16). The conjugate was formed simultaneously with the cleavage of the pyrimidinyl-phosphate linkage. Therefore, although the evidence is circumstantial, our results suggest t,hat# a similar system may be prcscnt in tobacco budworm larvuc. Other investigations (3, 7, 17) have dcmonstratctd the superiority of dimethyl toxicants over Dhrir diethyl homologues in toxicity t#ests with tobacco budworms, and their superiority has usually been attributed to diffcrcncps in metabolism. However, the results of this study have indicat#cad t#hat the influence of the O,O-dialkyl group on toxicity in tobacco budworms is apparently related to the rate of absorption as well as to t,he rate of metabolism. The faster rat#(h of absorption of the dimethyl ester by thr larvae could explain the greater toxicity of chlorpyrifosmc%hyl evc’n though it was apparently metabolized more rapidly in Y&I by 3rd~stage larvae. The similarity in susceptibility (or t&rante) of 3rd-stage larvae of the S and R strains to each of the Dowco phosphorothioates contrast with the obvious differ-
CHLORPYRIFOS
AND
ITS
DIMETHYL UNKNOWN
HOMOLOGUE
IN
UNKNOWNS
B, C, D
TOBACCO
BUDWORMS
273
A
PARENT
FIG. 1. Proposed metabolic pathways of chlorpyrifos and chlorpyrifosmethyl in tobacco budworms. Dashed lines indicate mechanisms that were not demonstrated but are probable. RI = CH,; Rz = CzHs; 34 = Microsomes + NADPH; (8) = Soluble enzymes; S = Soluble mzymes+ GSH.
ences in degradative potential exhibit’ed by 5th-stage larvae. A possible explanation is that the activity of degradativc cnzymc systems may be diminished during the early stages of growth, thereby decreasing differences in the capacity for detoxification between S and R larval strains. The data from the absorption t’est,s indicat,ed that metabolic differences between 3rdstage S and R larvae were much less than between 5th-stage insects. Also, the ~YLvitro activity of microsomal oxidases in tissues from late instars was greater than that of early instars among larval stages of the cabbage looper, Trichoplusia ni (Htibner) (18)) and the southern armyworm, Prodtm ia erida?ria (Cramer) (19). This evidence suggests that a larva should be able to tolerate relat’ively greater doses (mg toxicant/g body weight) of insecticide as it increased in size. Indeed, such a relationship was reported in tobacco budworms by Gast (20). That the toxicity of chlorpyrifos and chlorpyrifosmethyl was apparently not affected by the mechanism(s) contributing to the resistance to methyl parathion in the R larvae is particularly intriguing. The degradative capability of microsomal enzyme systems has been widely implicated in insecticide resistance
(21), and the results of this study have indicated that both Dowco compounds were detoxified primarily by microsomal oxidases that were particularly enhanced in 5thstage larvae of the R strain. While the activit#y of the microsomal enzyme system present in the younger larvae may have been diminished, the fact remains that the R larvae were resistant to methyl parathion. Thus, a mechanism or a combination of mechanisms other than microsomal oxidases could be involved in the resistance to methyl parathion found in the R strain. Based on the results of this study, tentative pathways for the metabolism of chlorpyrifos and chlorpyrifosmethyl can be constructed (Fig. 1). In the diagram, the unidentified metabolites are depicted as products of primary react’ions although conclusive evidence of this cannot be given. In addition, the nonoxidative hydrolytic syst,cm of the soluble phosphotriesterases and the GSH-dependent alkyltransferase are shown as detoxification mechanisms acting upon the oxidized form of the toxicant. Both systems have been demonstrated in tobacco budworms (6) and could contribute significantly t,o t,he degradation of the phosphate ester.
274
WHITTEN
REFERENCES
1. S. J. Nemec and P. L. Adkisson, Laboratory tests of insecticides for bollworm, tobacco budworm, and boll weevil control, Tes. Agr. Exp. Sta. Progr. Rep. 2674, 5 pp (1969). 2. J. R. Coppedge, W. L. Sterling, R. L. Ridgway, R. E. Kinzer, and C. J. Whitten, Foliar applications of selected insecticides for control of methyl parathion-resistant tobacco budworms, 7’ex. Agr. Exp. Sta. Progr. Rep. 3087, 8 pp (1972). 3. D. A. Wolfenbarger and R. I,. McGarr, Toxicity of methyl parathion, parathion, and monocrotophos applied topically to populations of lepidopteran pests of cotton, J. Econ. Entomol. 63, 1762 (1970). 4. C. J. Whitten and D. L. Bull, Resistance to organophosphorus insecticides in tobacco budworms, J. Econ. Entomol. 63, 1492 (1970). 5. R. L. Williamson and M. S. Schecter, Microsomal epoxidation of aldrin in lepidopterous larvae, Rio&em. Pharmaco(. 19, 1719 (1970). 6. D. L. Bull and C. J. Whitten, Factors influencing organophosphorus insecticide resistance in tobacco budworms, J. Agr. Food Chem. 20, 561 (1972). 7. F. W. Plapp, Jr., Laboratory tests of alternate insecticides for the control of methyl parathion-resistant tobacco budworm larvae, l’ex. Agr. Exp. Sta. Progr. Rep. 3090, 7 pp (1972). 8. P;. 8. Vanderzant, Wheat-germ diets for insects : rearing the boll weevil and the salt-marsh caterpillar, Ann. Entomol. Sot. Amw. 60, 1062 (1967). 9. Second conference on Test Methods for Resistance in Insects of Agricultural Importance : Standard method for detection of insecticide resistance in Hrliolhis zea (Boddie) and H. viwscens (F.), Hull. Entomot. Sot. Amer. 16, 147 (1970). 10. D. R. Simpson, 1). L. Bull, and D. A. Lindquist, A semimicrotechnique for the estimation of cholinesterase activity in boll weevils, Ann. Entomol. Sot. Amer. 57, 367 (1964).
AND
BULL
11. G. N. Smith, B. S. Watson, and F. S. Fischer, The metabolism of [‘4C]O,O-diethyl-0-(3,5,6trichloro-2-pyridyl) phosphorothioate (Dursban) in fish, J. Bon. Entomol. 59, 1464 (1966). 12. G. N. Smith and F. S. Fischer, Use of paper partition and thin-layer chromatography for identification of the active ingredient in Dursban insecticide and its possible metabolites, J. Agr. Food Chem. 15, 182 (1967). 13. G. N. Smith, Ultraviolet light decomposition studies with DURSBAN and 3,.5,6-trichloropyridinol. J. Econ. Entomol. 61, 793 (1968). 14. P. A. Dahm, Some aspects of the metabolism of parathion and diazinon, in “Biochemical Toxicology of Insecticides” (I~. D. O’Brien and I. Yamamoto, Eds.), p. 51, Academic Press New York, 1970. 15. R. S. H. Yang, E. Hodgson, and W. C. Dauterman, Metabolism in vitro of diazinon and diazoxon in susceptible houseflies, J. Agr. Food Chem. 19, 14 (1971). 16. T. Shishido, K. Usui, M. Sato, and J. Fukami, Enzymatic conjugation of diazinon with glutathione in rat and American cockroach, Pest. Biochem. Physiol. 2, 51 (1972). 17. F. W. Plapp, Jr., Insecticide resist,ance in Heliothis: Tolerance in larvae of H. virescens as compared with H. zea to organophosphate insecticides, J. Econ. Entomol. 64, 999 (1971). 18. K. J. Kuhr, Comparative metabolism of carbaryl by resistant and susceptible strains of the cabbage looper, J. &on. Entomol. 64, 1373 (1971). 19. It. I. Krieger and C. F. Wilkinson, Microsomal mixed-function oxidases in insects. I. Localization and properties of an enzyme system effecting aldrin expoxidat,ion in larvae of the southern armyworm (Prod&a eriilania), Riochcm. Pharmacol. 18, 1403 (1969). 20. R. T. Gast, The relationship of weight of lepidopterous larvae to effect.iveness of topically applied insecticides, J. Econ. Entomol. 52, 1115 (1959). 21. E. Hodgson and F. W. Plapp, Jr., Biochemical characteristics of insect microsomes, J. Agr. Food Chcm. 18, 1048 (1970).