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Disposition and metabolism of [14C]chlorpyrifos in the black imported fire ant, Solenopsis richteri Forel
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
19, 115- 121 (1983)
Disposition and Metabolism of [14C]Chlorpyrifos in the Black Imported Fire Ant, Solenopsis richteri Fore1 JANICE E. CHAMBERS,' W. THOMAS REDWOOD, AND CAROL A. TREVATHAN Department
of Biological
Sciences,
Mississippi
State
University,
Mississippi
State,
Mississippi
39762
Received April 13, 1982; accepted July 19, 1982 The short-term disposition and metabolism of topically administered [Wlchlorpyrifos was assessed in the black imported fire ant (Solenopsis richteri Forel) in the presence and absence of the mixed-function oxidase inhibitor piperonyl butoxide. Chlorpyrifos is readily absorbed into an internal organosoluble fraction which was quickly converted into a water-soluble fraction. The radioactivity was slowly excreted over a 24-hr period. Piperonyl butoxide slowed the conversion of the internal organosoluble radioactivity to the water-soluble fraction. Thin-layer chromatography indicated that piperonyl butoxide slowed the conversion of chlorpyrifos to material remaining at the origin, presumably water-soluble metabolites. The results of acid hydrolysis studies indicated that the water-soluble radioactivity was comprised mainly of conjugates. Although very little chlorpyrifos oxon was recovered in the metabolism experiments, in vitro studies on tire ant head homogenates showed the compound to be an extremely potent anticholinesterase. with an I,, of 4.6 x 10-l” M, while a major metabolite, 3,5,6-trichloropyridinol, was an ineffective acetylchohnesterase inhibitor.
INTRODUCTION
disposition and metabolic patterns of topically applied chlorpyrifos in the black imported fire ant, and (b) to determine how these patterns might be altered in ants treated with piperonyl butoxide, a commonly used insecticide synergist and a known inhibitor of mixed-function oxidases.
The black imported fire ant (Solenopsis richteri Forel) and the red imported tire ant (S. invicta Buren) are considered by some to be pests within the southeastern United States. Although mirex was effective in the past for fire ant control, a less persistent pesticide which is effective as a bait is currently being sought. An effective insecMETHODS ticide must act slowly, allowing the foraging workers sufficient time to transport the bait Chemicals back to the colony. One useful possibility is [2,6J4C]Chlorpyrifos (sp act 12.50 mCi/ the pyridyl phosphorothioate chlorpyrifos mmol) was generously provided by Dow (Dursban; O,O-diethyl-O-3,5,6trichloro-2Chemical Company, Midland, Michigan. pyridyl phosphorothioate). Chlorpyrifos This was dissolved in acetone. Unlabeled has shown some efficacy against fire ants in and 3,5,6 - trichloropyridinol field tests (Dr. Earl Alley, personal com- chlorpyrifos standards for chromatography were domunication). nated by Dow Chemical Company. ChlorOne potential method for slowing the pyrifos oxon (O,O-diethyl-0-3,5,6-trichlorotime course of an insecticide’s lethal action phosphate) was a gift of Dr. would be to alter the rate of metabolism of 2-pyridyl Howard Chambers, Department of Enthe parent compound to activated metabotomology, Mississippi State University. All lites or detoxified products. This project biochemicals were purchased from Sigma was conducted (a) to investigate the normal Chemical Company. All solvents and other 1 To whom all correspondence should be addressed. chemicals were reagent grade. 115 0048-3575183 $3.00 Copyright @ 1983 by Academic Press, Inc. All r&this of reproduction in any form reserved.
116 A. In Vivo Tests
CHAMBERS,
REDWOOD,
AND
TREVATHAN
water prior to counting. The radioactivity of all the above samples was quantified by liquid scintillation counting in a Beckman LS- 100 spectrometer using Aquasolas the liquid scintillation cocktail. The residues of the ants remaining after extraction were heated for 6 hr in Protosol tissue solubilizer at 55’C and were counted using Econofluor as a liquid scintillation cocktail; this was designated the residue fraction. 3. Metabolism. Ethyl acetate extracts from two separate groups of ants were prepared as described above and were evaporated and redissolved in 0.3 ml ethyl acetate. Aliquots of 50 ~1 were applied to silica gel plates with a fluorescent binder (Analtech, silica gel GHLF). Plates were developed to a height of 10 cm in a solvent system consisting of hexane-chloroform-acetone-acetic acid (150:40:9:1). Nonlabeled chlorpyrifos, chlorpyrifos oxon, and 3,5,6 - trichloropyridinol were used as standards and had Rf values of 0.72, 0.31, and 0.06, respectively. Spots corresponding to each of the three standards, the origin, and an area migrating faster than chlorpyrifos were scraped, wetted with 1 ml distilled water, and counted by liquid scintillation using Aquasol-2. A l-ml aliquot of the above-described water fraction was subjected to acid hydrolysis using 100 ~1 concentrated HCl for 15 min in a boiling water bath. This was neutralized with 8.9 mg of sodium bicarbonate and was reextracted with ethyl acetate as described above. The resultant ethyl acetate and water phases were counted in Aquasol-2.
1. General. Ants were treated according to the methods of Bigley and Vinson (1, 2). Major workers selected from field-collected colonies were used exclusively in these studies. Ants were immobilized by cooling and 0.5 ~1 of an acetone solution containing 0.01 pg [14C]chlorpyrifos was administered topically to the abdomen of each ant. In tests employing piperonyl butoxide, each ant was treated topically with 0.5 ~1 acetone containing 0.25 pg of piperonyl butoxide 1 hr prior to receiving the above dose of [14CJchlorpyrifos. Ants were air dried before being placed in glass petri dishes (4.5 cm in diameter) at room temperature. For the disposition studies ten ants (about 25 mg) were used per treatment group. For study of the time course of chlorpyrifos disposition, ants were sacrificed by freezing at 0, 0.5, 1, 2, 4, 8, or 24 hr after [14C]chlorpyrifos administration; for study of the effect of piperonyl butoxide on [14C]chlorpyrifos disposition, ants were sacrificed at 2 or 4 hr and statistical comparisons were made using Student’s t test. For the metabolism studies, twenty ants (about 60 mg) were used per treatment group, and were sacrificed by freezing at 2 or 4 hr after [14C]chlorpyrifos treatment. 2. Disposition. Hexane and ethyl acetate were tested as extraction solvents, the latter being more effective. Each ant was rinsed with 0.5 ml acetone, and the combined rinses were designated the external fraction. Each group of ants was ground in 2 ml deionized water in a TenBroeck tissue grinder. The homogenate was placed in a glass-stoppered test tube and was extracted Inhibition by shaking with 2 ml of hexane or ethyl B. Acetylcholinesterase acetate. The mixture was centrifuged at Heads of major workers were ground in a 15,600g for 1 min in an Eppendorf centrimortar and pestle in 0.8 M sucrose. The fuge to break the emulsion, yielding two resultant mixture was homogenized further samples, the organic fraction and the water in a TenBroeck tissue grinder. The homogfraction. The treatment dishes were rinsed enate was filtered through cheese cloth and with two l-ml aliquots of acetone. The the filtrate was centrifuged at IOOOg for 5 acetone was evaporated, and the excreted min in a Beckman L2-50 ultracentrifuge fraction was redissolved in 0.5 ml deionized equipped with a type 50 rotor. The clear
DISPOSITION
AND
METABOLISM
supernatant was decanted and diluted to a concentration of 10 heads (equivalent to about 10 mg) per milliliter. This homogenate was then diluted to a concentration of about 0.3 mg/ml (1.5 mg protein/ml) with 0.05 M Tris buffer, pH 7.4. Aliquots were incubated with inhibitors dissolved in ethanol (10 ~1 ethanol/ml) for 15 min prior to introduction of the substrate, acetylthiocholine. Samples were incubated at 30°C for 15 min in a shaking water bath prior to assay by the method of Ellman et at. (3). Protein was quantified by the method of Lowry et al. (4). The inhibitory activities of chlorpyrifos, chlorpyrifos oxon, and 3,5,6trichloropyridinol were studied. Corrections were made for nonspecific esterase activity as assessed by the inhibition of 10m5 M eserine. I,, values were calculated by log-probit regression analyses.
OF
CHLORPYRIFOS
IN
FIRE
ANTS
117
either the external or excreted fractions at 2 or 4 hr following treatment. However, in the ants pretreated with piperonyl butoxide, at 4 hr there was a significantly increased amount of radioactivity in the organic fraction (1.53 _+ 0.05 nmoYg compared to 1.31 +- 0.05 for controls, P < 0.05) and at 2 hr a decreased amount of radioactivity in the water-soluble fraction (0.31 + 0.03 nmol/g compared to 0.48 t 0.04 for controls, P < 0.05). Again, the residue fraction retained only a very small proportion of the radioactivity in both controls and piperonyl butoxide-pretreated ants. B. Metabolism
Although appreciable variation in the metabolic patterns between the two different groups of ants was observed, it was apparent that chlorpyrifos was converted to significant quantities of a material which RESULTS demonstrated the same mobility as 3,5,6 A. Disposition trichloropyridinol and to material remaining The patterns of radioactivity observed in at the origin (Table 1). Insignificant quanants treated with [14C]chlorpyrifos during a tities of radioactivity with mobility corre24-hr period were as anticipated (Fig. 1). sponding to that of chlorpyrifos oxon were The external fraction showed a very rapid detected. Appreciable amounts of radioacdecline within the first 4 hr following treat- tivity were found in an unidentified region ment; from 4 to 24 hr, there was very little migrating faster than chlorpyrifos. Less radioactivity observed in the external frac- radioactivity was found in the chlorpyrifos tion. Organosoluble radioactivity in ho- spot at 4 hr as compared to 2 hr following mogenates was highest at 1 hr following treatment, whereas the reverse was true for treatment, declined rapidly until 4 hr, and the unidentified spot. from 4 to 24 hr demonstrated a very slow In general, pretreatment with piperonyl decline. The water-soluble radioactivity butoxide resulted in a slower metabolism of showed a complementary rapid increase chlorpyrifos. At both 2 and 4 hr after treatearly during the treatment period, with a ment, more radioactivity remained in the plateau between 8 and 24 hr. The radioacchlorpyrifos spot and less at the origin or in tivity in the excreted fraction increased the pyridinol spot in the piperonyl butoxrapidly during the first hour following ide-treated group compared with the corretreatment, and thereafter gradually in- sponding controls. Quantities of radioaccreased during the remainder of the ex- tivity in the group treated with piperonyl perimental period. The amount of radiobutoxide were similar to those found in activity in the residue fraction remained controls. Recovery of radioactivity on all essentially unchanged throughout the ex- chromatograms was 90% or better. perimental period, and comprised only When the water-soluble fractions reabout 2% of the total. maining after ethyl acetate extraction were Piperonyl butoxide pretreatment did not subjected to acid hydrolysis, the majority of alter the amount of radioactivity present in radioactivity was extractable into ethyl
118
CHAMBERS,
REDWOOD,
1 )--cl
AND
TREVATHAN
TIME,HRS
(nmol chlorpyrifos equivalentslg wet wt) observed in internal (organic, water, and residue) and external (external and excreted) fractions ofjke ants treated topically with 0.01 pg [W]chlorpyrifos in acetone. Means and standard errors for four replications are indicated. Standard errors not shown were too small to indicate. FIG.
1. Radioactivity
acetate (Table 2). This was true for both the controls and for the ants treated with piperonyl butoxide. C. Acetylcholinesterase
Inhibition
The specific activity of fire ant head acetylcholinesterase observed was 0.56 A 0.03 mmol substrate hydrolyzed/l5 min/g
protein (five replications). The I,, values calculated for chlorpyrifos and chlorpyrifos oxon for fire ant head acetylcholinesterase were 1.12 x 1OP M (r = 0.98) and 4.5’7 X lo-lo M (r = 0.93), respectively. No acetylcholinesterase inhibition was observed by 3,5,6 - trichloropyridinol when assayed at concentrations up to 1 x 10e5 M.
DISPOSITION
AND
METABOLISM
OF
TABLE Percentage
CHLORPYRIFOS
IN
FIRE
1
of Radioactivity in Chlorpyrifos or Its Metabolites in Ethyl Treated Topically 2 or 4 hr with 0.01 pg [‘4C]Chlorpyrifos
Acetate Extracts in Acetone”
of Fire
2 hr Group 2
ControlC
Piperonyl butoxide
Origin Pyridinol Oxon Chlorpyrifos Unknown Percentage recovery
23.7 2 7.5 14.5 ” 1.8 0.1 t 0.1 42.7 31.2
14.8 f 12.5 f 1.2 * 58.4 2 14.3 2
112.2
101.2
1.7 1.3 0.5 8.0 5.3
Ants
4 hr
Group 1 Regionb
119
ANTS
Control 45.5 6.8 0.1 19.5 22.8
-r- 0.2 % 2.3 2 0.1 ?I 3.1 T 3.1
94.7
Group 1
Piperonyl butoxide 27.2 2.9 0.1 35.1 24.6
2 ” + * k
1.3 0.3 0.1 4.0 2.7
89.9
Piperonyl butoxide
Control 39.1 15.2 0.7 15.2 40.0
+ 1.2 * 1.1 2 0.7
30.2 12.8 0.2 26.8 30.5
” 2.1
110.0
t 2 k + k
3.6 0.0 0.2 6.8 16.8
100.5
0 Data represent percentage of radioactivity recovered from thin-layer chromatograms, mean 2 SEM (two replications). Values without SEM represent one replication. b Region indicates position on chromatogram. Pyridinol corresponds to 3,5,6-trichloropyridinol, oxon to chlorpyrifos oxon, and unknown to a region migrating faster than chforpyrifos. r Control ants were pretreated with acetone and piperonyl butoxide ants were pretreated with 0.25 pg of this inhibitor dissolved in acetone for 1 hr prior to chlorpyrifos administration.
DISCUSSION
Chlorpyrifos is readily absorbed by tire ants following topical administration. The chlorpyrifos migrated rapidly from the exterior of the ants into the interior in an organosoluble form, from which it was quickly converted into a water-soluble form. Chlorpyrifos or its metabolites were gradually excreted. Very little radioactivity became bound in nonextractable residues. Chlorpyrifos can be metabolized by fire
TABLE
Radioactivity
ants, as indicated by the results obtained from thin-layer chromatography. Chlorpyrifos is a relatively weak anticholinesterase, with an I,, in the range of lo-” M. It is converted to significant quantities of 3,5,6trichloropyridinol which does not demonstrate anticholinesterase activity. 3,5,6-Trichloropyridinol, formed via an NADPHdependent microsomal system, is the major chlorpyrifos metabolite in tobacco budworms (Heliothis virescens); no deethylated product was observed in these in-
2
in Acid-Hydrolyzed Water-Soluble Fractions 2 or 4 hr with 0.01 p.g [‘*C]Chlorpyrifos
of Fire Ants in Acetone”
Treated
2 hr Fraction Ethyl acetate Water
Topically
4 hr
Controlb
Piperonyl butoxide
Control
Piperonyl butoxide
88.17 ? 8.57(4) 9.67 k 1.32(4)
50.49 k 8.27(6) 6.73 r 0.93(6)
112.81 k 13.88(6) 14.24 ~fr 1.64(6)
121.40 rt 19.93(5) 16.50 f 2.94(5)
n Data represent radioactivity (pmol chlorpyrifos equivalents/g wet wt) extracted into ethyl acetate or remaining in the water phase following acid hydrolysis. Values are expressed as means 2 SEM (number of replications). b Control ants were pretreated with acetone and piperonyl butoxide ants were pretreated with 0.25 pg of this inhibitor dissolved in acetone for 1 hr.
120
CHAMBERS,
REDWOOD,
sects in either in viva or in vitro metabolism studies (5). Negligible quantities of chlorpyrifos oxon were detected. Apparently this metabolite is formed in very small quantities and/or it is extremely labile; however, it is a highly effective anticholinesterase with an I,, of about 5 x lo-lo M. This is a much more potent fire ant acetylcholinesterase inhibitor than some other common phosphates; methyl paraoxon, paraoxon, and isopropyl paraoxon have I,, values of 1.40 x lo-*, 6.25 x 10pg, and 1.13 x 1O-5 M, respectively (Dr. Howard Chambers, personal communication). Activation of chlorpyrifos to its oxon is required for insecticidal activity, but because of the latter’s potency, very little such activation may be required to elicit toxic action. A significant amount of radioactivity was detected in a spot chromatographing faster than chlorpyrifos. This spot remains unidentified since no standard was available corresponding to this region’s mobility. A major metabolite of chlorpyrifos excreted in rats was 3,5,6 - trichloro - 2 - pyridylphosphate (6), but, because of the lack of chromatography standard, it is unknown whether this metabolite is formed in fire ants. The majority of the water-soluble metabolites appear to be conjugates since most of the radioactivity in the water-soluble fraction could be reextracted into ethyl acetate following acid hydrolysis. The nature of the putative conjugates was not elucidated. Piperonyl butoxide has been shown to affect the metabolism of the juvenile hormone mimic, methoprene, in fire ants (2). In the present study, piperonyl butoxide slowed the conversion of the chlorpyrifos to presumed water-soluble metabolites as indicated by the decreased amount of material remaining at the origin on TLC analysis of the metabolites. Although the watersoluble metabolites appear to be mainly conjugates, they may well be the product of some mixed-function oxidase-mediated
AND
TREVATHAN
metabolite(s). The amount of pyridinol formed also appeared to be reduced by piperonyl butoxide treatment. Piperonyl butoxide did not significantly affect the rate of chlorpyrifos absorption or excretion, nor did it consistently alter the conversion of chlorpyrifos to the unidentified metabolite. Piperonyl butoxide also did not alter the amount of radioactivity which could be reextracted into ethyl acetate following acid hydrolysis of the water-soluble fraction. Numerous attempts to observe mixedfunction oxidase or glutathione S- transferase activity in vitro met with limited success. A variety of preparation techniques were tested, including grinding, sonication, and various degrees of centrifugation. Mixed-function oxidase activities investigated were p-nitroanisole Odemethylase, aminopyrine N-demethylase (7), aniline hydroxylase (8), and NADPHcytochrome c reductase (9). Glutathione S-transferaseiwas investigated with 3,4 dichloronitrobenzene as the substrate (10). Slight activity was observed with all assays except aniline hydroxylase, but since activity was so low, it did not seem worthwhile to extensively pursue in vitro studies. Insect preparations, in general, are difficult to assay in vitro because of such complications as eye pigment interference and the presence of endogenous inhibitors and proteases (11). The protease inhibitor, phenylmethylsulfonyl fluoride, was consistently included in fire ant preparation media to prevent excessive proteolysis. Ants were divided into anterior and posterior regions to eliminate possible eye pigment interference and abdominal preparations did demonstrate somewhat higher enzyme activities than did those from head plus thorax preparations. Nevertheless, in the in viva studies the effect of piperonyl butoxide as well as the production of uncharacterized water-soluble metabolites suggest the presence in fire ants of both mixed-function oxidation and conjugation pathways.
DISPOSITION
AND
METABOLISM
OF
CHLORPYRIFOS
REFERENCES
1. W. S. Bigley and S. B. Vinson, Degradation of [14C]-methoprene in the imported fire ant, Solenopsis invicta, Pestic. Biochem. Physiol. 10, 1 (1979). 2. W. S. Bigley and S. B. Vinson, Effects of piperonyl butoxide and DEF on the metabolism of methoprene by the imported tire ant, Solenopsis invicta Buren, Pestic. Biochem. Physiol. 10, 14 (1979). 3. G. L. Ellman, K. D. Courtney, K. Andres, Jr., and R. M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88 (1961). 4. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Biot. Chem. 193, 265, (1951). 5. C. J. Whitten and D. L. Bull, Comparative toxicity, absorption, and metabolism of chlorpyrifos and its dimethyl homologue in methyl parathion-resistant and -susceptible tobacco budworms, Pestic. Biochem. Physiol. 4,266(1974). 6. G. N. Smith, B. S. Watson, and F. S. Fischer, Investigations on Dursban insecticide. Metabolism of [?I] O,O-diethyl O-3,5,6-trichloro-2-
7.
8.
9.
10.
11.
IN
FIRE
ANTS
121
pyridyl phosphorothioate in rats, J. Agric. Food Chem. 15, 132 (1%7). C. L. Litterst, T. E. Gram, E. G. Mimnaugh, P. Leber, D. Emmerling, and R. I. Freudenthal, A comprehensive study of in vitro drug metabolism in several laboratory species, Drag Metah. Dispos. 4, 203 (1976). R. Kato and J. R. Gillette, Effect of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats, J. Pharmacol. Exp. Ther. 150, 279 (1%5). B. S. S. Masters, C. H. Williams, Jr., and H. Kamin, The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver, in “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman, Eds.), Vol. 10, p. 565, Academic Press, New York, 1967. W. H. Habig, M. J. Pabst, and W. B. Jakoby, Glutathione S-transferases: The first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249, 7130 (1974). C. F. Wilkinson, The use of insect subcellular components for studying the metabolism of xenobiotics, in “Xenobiotic Metabolism: In Vitro Methods” (G. D. Paulson, D. S. Frear, and E. P. Marks, Eds.), p. 249, Amer. Chem. Sot. New York, 1979.
BIOCHEMISTRY
AND
PHYSIOLOGY
19, 115- 121 (1983)
Disposition and Metabolism of [14C]Chlorpyrifos in the Black Imported Fire Ant, Solenopsis richteri Fore1 JANICE E. CHAMBERS,' W. THOMAS REDWOOD, AND CAROL A. TREVATHAN Department
of Biological
Sciences,
Mississippi
State
University,
Mississippi
State,
Mississippi
39762
Received April 13, 1982; accepted July 19, 1982 The short-term disposition and metabolism of topically administered [Wlchlorpyrifos was assessed in the black imported fire ant (Solenopsis richteri Forel) in the presence and absence of the mixed-function oxidase inhibitor piperonyl butoxide. Chlorpyrifos is readily absorbed into an internal organosoluble fraction which was quickly converted into a water-soluble fraction. The radioactivity was slowly excreted over a 24-hr period. Piperonyl butoxide slowed the conversion of the internal organosoluble radioactivity to the water-soluble fraction. Thin-layer chromatography indicated that piperonyl butoxide slowed the conversion of chlorpyrifos to material remaining at the origin, presumably water-soluble metabolites. The results of acid hydrolysis studies indicated that the water-soluble radioactivity was comprised mainly of conjugates. Although very little chlorpyrifos oxon was recovered in the metabolism experiments, in vitro studies on tire ant head homogenates showed the compound to be an extremely potent anticholinesterase. with an I,, of 4.6 x 10-l” M, while a major metabolite, 3,5,6-trichloropyridinol, was an ineffective acetylchohnesterase inhibitor.
INTRODUCTION
disposition and metabolic patterns of topically applied chlorpyrifos in the black imported fire ant, and (b) to determine how these patterns might be altered in ants treated with piperonyl butoxide, a commonly used insecticide synergist and a known inhibitor of mixed-function oxidases.
The black imported fire ant (Solenopsis richteri Forel) and the red imported tire ant (S. invicta Buren) are considered by some to be pests within the southeastern United States. Although mirex was effective in the past for fire ant control, a less persistent pesticide which is effective as a bait is currently being sought. An effective insecMETHODS ticide must act slowly, allowing the foraging workers sufficient time to transport the bait Chemicals back to the colony. One useful possibility is [2,6J4C]Chlorpyrifos (sp act 12.50 mCi/ the pyridyl phosphorothioate chlorpyrifos mmol) was generously provided by Dow (Dursban; O,O-diethyl-O-3,5,6trichloro-2Chemical Company, Midland, Michigan. pyridyl phosphorothioate). Chlorpyrifos This was dissolved in acetone. Unlabeled has shown some efficacy against fire ants in and 3,5,6 - trichloropyridinol field tests (Dr. Earl Alley, personal com- chlorpyrifos standards for chromatography were domunication). nated by Dow Chemical Company. ChlorOne potential method for slowing the pyrifos oxon (O,O-diethyl-0-3,5,6-trichlorotime course of an insecticide’s lethal action phosphate) was a gift of Dr. would be to alter the rate of metabolism of 2-pyridyl Howard Chambers, Department of Enthe parent compound to activated metabotomology, Mississippi State University. All lites or detoxified products. This project biochemicals were purchased from Sigma was conducted (a) to investigate the normal Chemical Company. All solvents and other 1 To whom all correspondence should be addressed. chemicals were reagent grade. 115 0048-3575183 $3.00 Copyright @ 1983 by Academic Press, Inc. All r&this of reproduction in any form reserved.
116 A. In Vivo Tests
CHAMBERS,
REDWOOD,
AND
TREVATHAN
water prior to counting. The radioactivity of all the above samples was quantified by liquid scintillation counting in a Beckman LS- 100 spectrometer using Aquasolas the liquid scintillation cocktail. The residues of the ants remaining after extraction were heated for 6 hr in Protosol tissue solubilizer at 55’C and were counted using Econofluor as a liquid scintillation cocktail; this was designated the residue fraction. 3. Metabolism. Ethyl acetate extracts from two separate groups of ants were prepared as described above and were evaporated and redissolved in 0.3 ml ethyl acetate. Aliquots of 50 ~1 were applied to silica gel plates with a fluorescent binder (Analtech, silica gel GHLF). Plates were developed to a height of 10 cm in a solvent system consisting of hexane-chloroform-acetone-acetic acid (150:40:9:1). Nonlabeled chlorpyrifos, chlorpyrifos oxon, and 3,5,6 - trichloropyridinol were used as standards and had Rf values of 0.72, 0.31, and 0.06, respectively. Spots corresponding to each of the three standards, the origin, and an area migrating faster than chlorpyrifos were scraped, wetted with 1 ml distilled water, and counted by liquid scintillation using Aquasol-2. A l-ml aliquot of the above-described water fraction was subjected to acid hydrolysis using 100 ~1 concentrated HCl for 15 min in a boiling water bath. This was neutralized with 8.9 mg of sodium bicarbonate and was reextracted with ethyl acetate as described above. The resultant ethyl acetate and water phases were counted in Aquasol-2.
1. General. Ants were treated according to the methods of Bigley and Vinson (1, 2). Major workers selected from field-collected colonies were used exclusively in these studies. Ants were immobilized by cooling and 0.5 ~1 of an acetone solution containing 0.01 pg [14C]chlorpyrifos was administered topically to the abdomen of each ant. In tests employing piperonyl butoxide, each ant was treated topically with 0.5 ~1 acetone containing 0.25 pg of piperonyl butoxide 1 hr prior to receiving the above dose of [14CJchlorpyrifos. Ants were air dried before being placed in glass petri dishes (4.5 cm in diameter) at room temperature. For the disposition studies ten ants (about 25 mg) were used per treatment group. For study of the time course of chlorpyrifos disposition, ants were sacrificed by freezing at 0, 0.5, 1, 2, 4, 8, or 24 hr after [14C]chlorpyrifos administration; for study of the effect of piperonyl butoxide on [14C]chlorpyrifos disposition, ants were sacrificed at 2 or 4 hr and statistical comparisons were made using Student’s t test. For the metabolism studies, twenty ants (about 60 mg) were used per treatment group, and were sacrificed by freezing at 2 or 4 hr after [14C]chlorpyrifos treatment. 2. Disposition. Hexane and ethyl acetate were tested as extraction solvents, the latter being more effective. Each ant was rinsed with 0.5 ml acetone, and the combined rinses were designated the external fraction. Each group of ants was ground in 2 ml deionized water in a TenBroeck tissue grinder. The homogenate was placed in a glass-stoppered test tube and was extracted Inhibition by shaking with 2 ml of hexane or ethyl B. Acetylcholinesterase acetate. The mixture was centrifuged at Heads of major workers were ground in a 15,600g for 1 min in an Eppendorf centrimortar and pestle in 0.8 M sucrose. The fuge to break the emulsion, yielding two resultant mixture was homogenized further samples, the organic fraction and the water in a TenBroeck tissue grinder. The homogfraction. The treatment dishes were rinsed enate was filtered through cheese cloth and with two l-ml aliquots of acetone. The the filtrate was centrifuged at IOOOg for 5 acetone was evaporated, and the excreted min in a Beckman L2-50 ultracentrifuge fraction was redissolved in 0.5 ml deionized equipped with a type 50 rotor. The clear
DISPOSITION
AND
METABOLISM
supernatant was decanted and diluted to a concentration of 10 heads (equivalent to about 10 mg) per milliliter. This homogenate was then diluted to a concentration of about 0.3 mg/ml (1.5 mg protein/ml) with 0.05 M Tris buffer, pH 7.4. Aliquots were incubated with inhibitors dissolved in ethanol (10 ~1 ethanol/ml) for 15 min prior to introduction of the substrate, acetylthiocholine. Samples were incubated at 30°C for 15 min in a shaking water bath prior to assay by the method of Ellman et at. (3). Protein was quantified by the method of Lowry et al. (4). The inhibitory activities of chlorpyrifos, chlorpyrifos oxon, and 3,5,6trichloropyridinol were studied. Corrections were made for nonspecific esterase activity as assessed by the inhibition of 10m5 M eserine. I,, values were calculated by log-probit regression analyses.
OF
CHLORPYRIFOS
IN
FIRE
ANTS
117
either the external or excreted fractions at 2 or 4 hr following treatment. However, in the ants pretreated with piperonyl butoxide, at 4 hr there was a significantly increased amount of radioactivity in the organic fraction (1.53 _+ 0.05 nmoYg compared to 1.31 +- 0.05 for controls, P < 0.05) and at 2 hr a decreased amount of radioactivity in the water-soluble fraction (0.31 + 0.03 nmol/g compared to 0.48 t 0.04 for controls, P < 0.05). Again, the residue fraction retained only a very small proportion of the radioactivity in both controls and piperonyl butoxide-pretreated ants. B. Metabolism
Although appreciable variation in the metabolic patterns between the two different groups of ants was observed, it was apparent that chlorpyrifos was converted to significant quantities of a material which RESULTS demonstrated the same mobility as 3,5,6 A. Disposition trichloropyridinol and to material remaining The patterns of radioactivity observed in at the origin (Table 1). Insignificant quanants treated with [14C]chlorpyrifos during a tities of radioactivity with mobility corre24-hr period were as anticipated (Fig. 1). sponding to that of chlorpyrifos oxon were The external fraction showed a very rapid detected. Appreciable amounts of radioacdecline within the first 4 hr following treat- tivity were found in an unidentified region ment; from 4 to 24 hr, there was very little migrating faster than chlorpyrifos. Less radioactivity observed in the external frac- radioactivity was found in the chlorpyrifos tion. Organosoluble radioactivity in ho- spot at 4 hr as compared to 2 hr following mogenates was highest at 1 hr following treatment, whereas the reverse was true for treatment, declined rapidly until 4 hr, and the unidentified spot. from 4 to 24 hr demonstrated a very slow In general, pretreatment with piperonyl decline. The water-soluble radioactivity butoxide resulted in a slower metabolism of showed a complementary rapid increase chlorpyrifos. At both 2 and 4 hr after treatearly during the treatment period, with a ment, more radioactivity remained in the plateau between 8 and 24 hr. The radioacchlorpyrifos spot and less at the origin or in tivity in the excreted fraction increased the pyridinol spot in the piperonyl butoxrapidly during the first hour following ide-treated group compared with the corretreatment, and thereafter gradually in- sponding controls. Quantities of radioaccreased during the remainder of the ex- tivity in the group treated with piperonyl perimental period. The amount of radiobutoxide were similar to those found in activity in the residue fraction remained controls. Recovery of radioactivity on all essentially unchanged throughout the ex- chromatograms was 90% or better. perimental period, and comprised only When the water-soluble fractions reabout 2% of the total. maining after ethyl acetate extraction were Piperonyl butoxide pretreatment did not subjected to acid hydrolysis, the majority of alter the amount of radioactivity present in radioactivity was extractable into ethyl
118
CHAMBERS,
REDWOOD,
1 )--cl
AND
TREVATHAN
TIME,HRS
(nmol chlorpyrifos equivalentslg wet wt) observed in internal (organic, water, and residue) and external (external and excreted) fractions ofjke ants treated topically with 0.01 pg [W]chlorpyrifos in acetone. Means and standard errors for four replications are indicated. Standard errors not shown were too small to indicate. FIG.
1. Radioactivity
acetate (Table 2). This was true for both the controls and for the ants treated with piperonyl butoxide. C. Acetylcholinesterase
Inhibition
The specific activity of fire ant head acetylcholinesterase observed was 0.56 A 0.03 mmol substrate hydrolyzed/l5 min/g
protein (five replications). The I,, values calculated for chlorpyrifos and chlorpyrifos oxon for fire ant head acetylcholinesterase were 1.12 x 1OP M (r = 0.98) and 4.5’7 X lo-lo M (r = 0.93), respectively. No acetylcholinesterase inhibition was observed by 3,5,6 - trichloropyridinol when assayed at concentrations up to 1 x 10e5 M.
DISPOSITION
AND
METABOLISM
OF
TABLE Percentage
CHLORPYRIFOS
IN
FIRE
1
of Radioactivity in Chlorpyrifos or Its Metabolites in Ethyl Treated Topically 2 or 4 hr with 0.01 pg [‘4C]Chlorpyrifos
Acetate Extracts in Acetone”
of Fire
2 hr Group 2
ControlC
Piperonyl butoxide
Origin Pyridinol Oxon Chlorpyrifos Unknown Percentage recovery
23.7 2 7.5 14.5 ” 1.8 0.1 t 0.1 42.7 31.2
14.8 f 12.5 f 1.2 * 58.4 2 14.3 2
112.2
101.2
1.7 1.3 0.5 8.0 5.3
Ants
4 hr
Group 1 Regionb
119
ANTS
Control 45.5 6.8 0.1 19.5 22.8
-r- 0.2 % 2.3 2 0.1 ?I 3.1 T 3.1
94.7
Group 1
Piperonyl butoxide 27.2 2.9 0.1 35.1 24.6
2 ” + * k
1.3 0.3 0.1 4.0 2.7
89.9
Piperonyl butoxide
Control 39.1 15.2 0.7 15.2 40.0
+ 1.2 * 1.1 2 0.7
30.2 12.8 0.2 26.8 30.5
” 2.1
110.0
t 2 k + k
3.6 0.0 0.2 6.8 16.8
100.5
0 Data represent percentage of radioactivity recovered from thin-layer chromatograms, mean 2 SEM (two replications). Values without SEM represent one replication. b Region indicates position on chromatogram. Pyridinol corresponds to 3,5,6-trichloropyridinol, oxon to chlorpyrifos oxon, and unknown to a region migrating faster than chforpyrifos. r Control ants were pretreated with acetone and piperonyl butoxide ants were pretreated with 0.25 pg of this inhibitor dissolved in acetone for 1 hr prior to chlorpyrifos administration.
DISCUSSION
Chlorpyrifos is readily absorbed by tire ants following topical administration. The chlorpyrifos migrated rapidly from the exterior of the ants into the interior in an organosoluble form, from which it was quickly converted into a water-soluble form. Chlorpyrifos or its metabolites were gradually excreted. Very little radioactivity became bound in nonextractable residues. Chlorpyrifos can be metabolized by fire
TABLE
Radioactivity
ants, as indicated by the results obtained from thin-layer chromatography. Chlorpyrifos is a relatively weak anticholinesterase, with an I,, in the range of lo-” M. It is converted to significant quantities of 3,5,6trichloropyridinol which does not demonstrate anticholinesterase activity. 3,5,6-Trichloropyridinol, formed via an NADPHdependent microsomal system, is the major chlorpyrifos metabolite in tobacco budworms (Heliothis virescens); no deethylated product was observed in these in-
2
in Acid-Hydrolyzed Water-Soluble Fractions 2 or 4 hr with 0.01 p.g [‘*C]Chlorpyrifos
of Fire Ants in Acetone”
Treated
2 hr Fraction Ethyl acetate Water
Topically
4 hr
Controlb
Piperonyl butoxide
Control
Piperonyl butoxide
88.17 ? 8.57(4) 9.67 k 1.32(4)
50.49 k 8.27(6) 6.73 r 0.93(6)
112.81 k 13.88(6) 14.24 ~fr 1.64(6)
121.40 rt 19.93(5) 16.50 f 2.94(5)
n Data represent radioactivity (pmol chlorpyrifos equivalents/g wet wt) extracted into ethyl acetate or remaining in the water phase following acid hydrolysis. Values are expressed as means 2 SEM (number of replications). b Control ants were pretreated with acetone and piperonyl butoxide ants were pretreated with 0.25 pg of this inhibitor dissolved in acetone for 1 hr.
120
CHAMBERS,
REDWOOD,
sects in either in viva or in vitro metabolism studies (5). Negligible quantities of chlorpyrifos oxon were detected. Apparently this metabolite is formed in very small quantities and/or it is extremely labile; however, it is a highly effective anticholinesterase with an I,, of about 5 x lo-lo M. This is a much more potent fire ant acetylcholinesterase inhibitor than some other common phosphates; methyl paraoxon, paraoxon, and isopropyl paraoxon have I,, values of 1.40 x lo-*, 6.25 x 10pg, and 1.13 x 1O-5 M, respectively (Dr. Howard Chambers, personal communication). Activation of chlorpyrifos to its oxon is required for insecticidal activity, but because of the latter’s potency, very little such activation may be required to elicit toxic action. A significant amount of radioactivity was detected in a spot chromatographing faster than chlorpyrifos. This spot remains unidentified since no standard was available corresponding to this region’s mobility. A major metabolite of chlorpyrifos excreted in rats was 3,5,6 - trichloro - 2 - pyridylphosphate (6), but, because of the lack of chromatography standard, it is unknown whether this metabolite is formed in fire ants. The majority of the water-soluble metabolites appear to be conjugates since most of the radioactivity in the water-soluble fraction could be reextracted into ethyl acetate following acid hydrolysis. The nature of the putative conjugates was not elucidated. Piperonyl butoxide has been shown to affect the metabolism of the juvenile hormone mimic, methoprene, in fire ants (2). In the present study, piperonyl butoxide slowed the conversion of the chlorpyrifos to presumed water-soluble metabolites as indicated by the decreased amount of material remaining at the origin on TLC analysis of the metabolites. Although the watersoluble metabolites appear to be mainly conjugates, they may well be the product of some mixed-function oxidase-mediated
AND
TREVATHAN
metabolite(s). The amount of pyridinol formed also appeared to be reduced by piperonyl butoxide treatment. Piperonyl butoxide did not significantly affect the rate of chlorpyrifos absorption or excretion, nor did it consistently alter the conversion of chlorpyrifos to the unidentified metabolite. Piperonyl butoxide also did not alter the amount of radioactivity which could be reextracted into ethyl acetate following acid hydrolysis of the water-soluble fraction. Numerous attempts to observe mixedfunction oxidase or glutathione S- transferase activity in vitro met with limited success. A variety of preparation techniques were tested, including grinding, sonication, and various degrees of centrifugation. Mixed-function oxidase activities investigated were p-nitroanisole Odemethylase, aminopyrine N-demethylase (7), aniline hydroxylase (8), and NADPHcytochrome c reductase (9). Glutathione S-transferaseiwas investigated with 3,4 dichloronitrobenzene as the substrate (10). Slight activity was observed with all assays except aniline hydroxylase, but since activity was so low, it did not seem worthwhile to extensively pursue in vitro studies. Insect preparations, in general, are difficult to assay in vitro because of such complications as eye pigment interference and the presence of endogenous inhibitors and proteases (11). The protease inhibitor, phenylmethylsulfonyl fluoride, was consistently included in fire ant preparation media to prevent excessive proteolysis. Ants were divided into anterior and posterior regions to eliminate possible eye pigment interference and abdominal preparations did demonstrate somewhat higher enzyme activities than did those from head plus thorax preparations. Nevertheless, in the in viva studies the effect of piperonyl butoxide as well as the production of uncharacterized water-soluble metabolites suggest the presence in fire ants of both mixed-function oxidation and conjugation pathways.
DISPOSITION
AND
METABOLISM
OF
CHLORPYRIFOS
REFERENCES
1. W. S. Bigley and S. B. Vinson, Degradation of [14C]-methoprene in the imported fire ant, Solenopsis invicta, Pestic. Biochem. Physiol. 10, 1 (1979). 2. W. S. Bigley and S. B. Vinson, Effects of piperonyl butoxide and DEF on the metabolism of methoprene by the imported tire ant, Solenopsis invicta Buren, Pestic. Biochem. Physiol. 10, 14 (1979). 3. G. L. Ellman, K. D. Courtney, K. Andres, Jr., and R. M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88 (1961). 4. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Biot. Chem. 193, 265, (1951). 5. C. J. Whitten and D. L. Bull, Comparative toxicity, absorption, and metabolism of chlorpyrifos and its dimethyl homologue in methyl parathion-resistant and -susceptible tobacco budworms, Pestic. Biochem. Physiol. 4,266(1974). 6. G. N. Smith, B. S. Watson, and F. S. Fischer, Investigations on Dursban insecticide. Metabolism of [?I] O,O-diethyl O-3,5,6-trichloro-2-
7.
8.
9.
10.
11.
IN
FIRE
ANTS
121
pyridyl phosphorothioate in rats, J. Agric. Food Chem. 15, 132 (1%7). C. L. Litterst, T. E. Gram, E. G. Mimnaugh, P. Leber, D. Emmerling, and R. I. Freudenthal, A comprehensive study of in vitro drug metabolism in several laboratory species, Drag Metah. Dispos. 4, 203 (1976). R. Kato and J. R. Gillette, Effect of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats, J. Pharmacol. Exp. Ther. 150, 279 (1%5). B. S. S. Masters, C. H. Williams, Jr., and H. Kamin, The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver, in “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman, Eds.), Vol. 10, p. 565, Academic Press, New York, 1967. W. H. Habig, M. J. Pabst, and W. B. Jakoby, Glutathione S-transferases: The first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249, 7130 (1974). C. F. Wilkinson, The use of insect subcellular components for studying the metabolism of xenobiotics, in “Xenobiotic Metabolism: In Vitro Methods” (G. D. Paulson, D. S. Frear, and E. P. Marks, Eds.), p. 249, Amer. Chem. Sot. New York, 1979.