EXPERIMENTAL PARASITOLOGY 72, 355-361(1991)

Schistosoma mansoni: Glutathione S-Transferase-Catalyzed Detoxication of Dichlorvos KATHLEEN A. O'LEARY'ANDJAMES W. TRACY~ Departments of Comparative Biosciences and Pharmacology and Environmental Toxicology Center, University of Wisconsin, 2015 Linden Drive West, Madison, Wisconsin 53706-1102, U.S.A. O’LEARY, K. A., AND TRACY, J. W., 1991 Schistosoma mansoni: Glutathione STransferase-Catalyzed Detoxication of Dichlorvos. Experimental Parasitology 72, 355-361. Dialyzed cytosol of adult Schistosoma mansoni worm pairs catalyzed the glutathionedependent O-demethylation of dichlorvos (2,2-dichlorovinyl dimethylphosphate), the active form of the antischistosomal drug metrifonate, to form a thioether conjugate, Smethylglutathione, and desmethyl dichlorvos. The reaction rate was dependent on both time and protein concentration, and no product was formed when either dichlorvos or glutathione was omitted from the reaction mixture. Female worm cytosols were about 2.5-fold more active per milligram of protein that those of males. Partial purification of glutathione Stransferases from male worms by affinity chromatography on glutathione-agarose showed that the reaction could be catalyzed by a preparation containing the three major isoenzymes, but that the unbound fraction, which contains at least one additional form of the enzyme that is particularly active with epoxide substrates, was 16-fold more active toward dichlorvos than the bound fraction. S-Methylglutathione also was formed by S. mansoni worm pairs incubated in the presence but not in the absence of dichlorvos. Because GSH Stransferase-catalyzed metabolism of dichlorvos results in the formation of desmethyldichlorvos, which unlike the parent compound is not an effective acetylcholinesterase inhibitor, the reaction represents a pathway of detoxication in schistosomes. It is the first example of a clinically used schistosomicide shown to be detoxicated by a conjugation pathway. These results raise the possibility that dichlorvos detoxication by S. mansoni may help explain why this species is normally refractory to metrifonate. 0 1991AcademicPress,Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Trematode; Schistosoma mansoni; Glutathione (GSH); Glutathione S-transferase (GST;EC 2.5.1.18); Dichlorvos (2,2-dichlorovinyl dimethylphosphate); metrifonate; Anthelmintic; Organophosphorous insecticide; lChloro-2,4-dinitrobenzene (CDNB); 1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP); 2,4Dinitrophenyl- (DNP-); S-Methylglutathione (GSCH,); Glutathione S-conjugate; Drug metabolism; O-Demethylation; High-pressure liquid chromatography (HPLC).

INTRODUCTION

Glutathione S-transferases (EC 2.5.1.18) are a family of enzymes that serve multiple functions in cellular protection, including the detoxication of electrophiles by thioether conjugate formation with GSH (Mannervik and Danielson 1988). These enzymes are widely distributed in nature, being found in both vertebrates and invertebrates, including schistosomes (Smith et al. i Present address: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706. * To whom correspondence should be addressed.

1986; O’Leary and Tracy 1988; Taylor et al. 1988). Many organisms contain multiple GST isoenzymes that are characterized by distinct, but overlapping substrate speciticities that account for their capacity to catalyze the conjugation of a structurally diverse group of xenobiotics. We have described the purification and characterization of three closely related cytosolic GSTs from Schistosoma mansoni, designated SmGST-1, SmGST-2, and SmGST-3, which comprise 24% of the total soluble protein in the male worm (O’Leary and Tracy 1988). They were purified from adult male worm cytosol by a 355 0014-4894/91$3.00 Copyright0 1991by AcademicPress,Inc. All rightsof reproductionin any form reserved.

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combination of affinity chromatography and HPLC chromatofocusing. More recently, a fourth, minor enzyme form, designated SmGST-4, has been resolved from the affinity-purified preparation (O’Leary et al., unpublished observations). At least one other GST, designated SmGST-5, was detected, but not isolated. SmGST-5 can be distinguished from the other four isoenzymes by its lack of affinity for GSHagarose and its distinct substrate specificity. Unlike the other S. munsoni GSTs, SmGST-5 displays very low catalytic activity toward the prototypic substrate, CDNB, but is particularly active toward EPNP, a model epoxide substrate (O’Leary and Tracy 1988). Sm GST-5 activity also is differentially expressed in the two sexes, with specific enzyme activity being over twofold greater in female than in males. We wondered whether besides catalyzing the conjugation of a number of model xenobiotics, S. munsoni GSTs might play a role in the detoxication of antischistosomal drugs. As a prototypic drug we selected dichlorvos, the active form of metrifonate (Nordgren et al. 1978). This selection was based on the fact that one mechanism by which mammals and insects metabolize dichlorvos and other 0,0-dialkyl phosphorous compounds is by conjugation with GSH to form S-alkylglutathione derivatives and the corresponding des-alkyl organophosphate (Dickowsky and Morello 1971; Motoyama and Dauterman 1980). The reaction between dichlorvos and GSH (Fig. 1) represents a detoxication pathway, because the des-alkyl product, unlike the parent organophosphate, is not an effective acetylcholinesterase inhibitor (Motoyama and Dauterman 1980). We report here that dichlorvos is a substrate for S. munsoni GSTs, with the reac-

B

H,CO-P-OCH=CCl, I OCH3

+

GSH

+

tion being preferentially catalyzed by the worm protein fraction containing SmGST-5 activity. Furthermore, the GSH-dependent 0-demethylation of dichlorvos takes place not only in cell-free preparations, but also within intact adult parasites. This represents the first example of a clinically used drug that is detoxicated by conjugation within schistosomes. MATERIALS AND METHODS Femalemice (CF, strain, 18-22g, Sasco,Madison, WI) were exposed subcutaneously (Peters and Warren 1%9) to 200 freshly shed cercariae of a Puerto Rican strain of S. mansoni. Seven to 8 weeks later, parasites were recovered by portal venous perfusion (Duvall and Dewitt 1967) with ice-cold RPM1 medium 1640 (GIBCO, Grand Island, NY) containing 25 mM 2-hydroxyethyl-piperazine-N’-2-ethane sulfonate (Research Organics, Cleveland, OH) buffer, pH 7.4. Parasites were washed several times with cold medium, and as necessary, male and female worms separated while being maintained at 4°C. Adult schistosomes (1 to 2 g wet wt) were homogenized in 4 vol of 10 m&f potassium phosphate buffer, pH 7.0, containing 1 n& Na, EDTA, using a PotterElvehjem tissue grinder. The homogenate was centrifuged at 4°C for 60 min at 47,800g. The supernatant was decanted and was centrifuged at 4°C for 90 min at 105,OCKlg.Both pellets were discarded. The final supematant (cytosol) was dialyzed overnight at 4°C against 1000 vol of homogenization buffer. Dialyzed cytosol was used both for measuring StnGST activity and as the starting material for enzyme purification. SmGSTs were partially purified by atfinity chromatography on thiol-linked GSH-agarose (Sigma, St. Louis, MO) as previously described (O’Leary and Tracy 1988). The unbound protein fraction (Fraction I) was concentrated using a stirred ultratiltration cell fitted with a YM-10 membrane (Amicon, Danvers, MA). Bound proteins were eluted from the column with 5 mM GSH-containing buffer and were concentrated as above to yield an affinity-purified SmGST preparation (Fraction II). SmGST activity toward CDNB (99 + %, Aldrich, Milwaukee, WI) and EPNP (Sigma) was assayed spectrophotometrically at 340 and 360 nm, respectively (Habig et al. 1974). One unit of GST activity is defined as the amount of enzyme catalyzing the formation of 1

P

HSCO-y-OCH=CCl,

+

GSCHQ

OH

FIG. 1. Reaction of dichlorvos with GSH to form desmethyldichlorvos and GSCH,.

&d~isfoSOmU mUnsoni: DLCHLORVOS Pmole of thioether conjugate per minute under the conditions of the assay. Specific enzyme activity is expressed as units per milligram of protein. Protein concentrations were estimated by a dye-binding method (Bradford 1976), using crystalline bovine serum albumin (Miles Laboratories, Elkhart, IN) as the reference protein. SmGST activity toward dichlorvos, methyl parathion, and methyl iodide was measured by an HPLC assay (Tracy and O’Leary 1991). The reaction was carried out in I.5ml polypropylene centrifuge tubes containing 125 pmole 3-N-morpholinopropanesulfonate (Research Organics) buffer, pH 7.5, 1.25 pmole GSH (Sigma), 1.25 pmole dichlorvos (99%; Chem Service, West Chester, PA), or 0.12 Pmole methyl parathion (98%; Chem Service), or 1.25 pmole iodomethane (99.5%; Aldrich), and enzyme in a total volume of 250 ~1. Control reactions contained an equivalent amount of protein that had been boiled for 10 min. The reaction was initiated by adding 5 pl of xenobiotic substrate stock solution in ethanol and was terminated after incubation at 30°C by adding 25 pJ of ice-cold 60% perchloric acid. Five microliters of 12 mM y-Glu-Gly (Sigma) was added as an internal standard. Next, 125 pl of an ice-cold solution of 2.0 M KOH and 2.4 M KHCO, were added, followed immediately by 200 pl of a freshly prepared 1.5% (v/v) solution of I-fluoro-2,4-dinitrobenzene (Research Organics) in ethanol. The reaction was allowed to proceed overnight at 22°C in the dark. The bright yellow reaction mixtures were filtered through 0.45~pm Nylon 66 filters (Rainin, Woburn, MA) and stored in tightly capped vials at 22°C in the dark. The reaction yields for the DNP derivatives of y-Glu-Gly and GSCH, were 97 f 4 and 95 * 3%, respectively (Tracy and O’Leary 1991). Samples were analyzed using a high-pressure liquid chromatograph (Gilson Medical Electronics, Middleton, WI) fitted with a 3-pm Zorbax C,, column (0.62 cm id. x 8.0 cm; DuPont, Wilmington, DE) that was equilibrated at 1.0 ml/mm with 25% (v/v) acetonitrile (HPLC grade, Baker, Phillipsburg, NJ) in 0.3 M acetic acid (HPLC grade, Baker). Gradient elution was started immediately after sample (5-50 ~1) injection. The acetonitrile concentration was increased linearly over 10 min to 50%. The solvent was maintained at 50% acetonitrile for 2 min and then was returned to initial conditions over 5 min. Under those conditions, elution times were: N-DNP-y-Glu-Gly, 5.2 min; NDNP-GSCH,, 7.0 min; and N,S-(DNP), GSH, 13.1 min. Column effluent was monitored at 365 nm with a Model 757 uv detector (Kratos, Ramsey, NJ), and peak heights were used to quantify the amount of NDNP-GSCH, formed by comparison to a standard curve of synthetic standard (Tracy and O’Leary 1991). With alkyl donor substrates, 1 unit of GST activity is defined as the amount of enzyme catalyzing the for-

357

METABOLISM

mation of 1 nmole of GSCH, per minute under the conditions of the assay. To investigate dichlorvos metabolism by intact 5. munsoni, worm pairs were recovered by perfusion with medium warmed to 37°C. Groups of 25 worm pairs were incubated in 6-well plastic tissue culture plates in 5 ml of supplemented RPMI medium 1640 (Siegel and Tracy 1988) containing 30 PM dichlorvos or 0.1% (v/v, final concentration) ethanol vehicle. After incubation for up to 2 hr at 37°C in a humiditied atmosphere of 5% CO,/95% air, parasites were removed from the medium and were homogenized in 480 ~1of ice-cold 10% (w/v) HCIO,. The homogenate was centrifuged at 4°C for 20 min at 14,OOOg,and 250 PI of the resulting acid-soluble supematant was derivatized with I-fluoro-2,4-dinitrobenzene as described above, except that the internal standard was omitted. RESULTS

Dialyzed cytosol prepared from adult S. worm pairs catalyzed the GSHdependent O-demethylation of dichlorvos, resulting in formation of GSCHj. Product formation was dependent on time and schistosome protein concentration (Fig. 2). At a protein concentration of 0.5 mg/ml, the munsoni

‘“A

TIME

(min)

FIG. 2. Time course of GSCH, formation

from dichlorvos catalyzed by dialyzed Schistosoma mansuni cytosol. Complete reaction mixtures contained 7.5 pmole GSH, 7.5 pmole dichlorvos, and either (0) 0.75 mg or (A) 0.38 mg of adult worm pair cytosol in a total volume of 1.5 ml. The control reaction (m) contained 0.75 mg of protein that had been inactivated by boiling for 10 min. At the indicated times, 250~pl samples were withdrawn and GSCHS formation was determined by HPLC as described under Materials and Methods. Data represent the means 2 SD for three separate experiments. In some cases the error bars were smaller than the data points.

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O’LEARY AND TRACY

enzymatic rate of GSCH, formation was 8to lo-fold greater than the nonenzymatic rate. No product was detected when either dichlorvos or GSH was omitted from the reaction. Adult worm cytosol also catalyzed the formation of GSCH, from other methyl donor substrates. Specific activities with dichlorvos, methyl parathion, and iodomethane were 11.3, 7.8, and 127 nmol/ min/mg protein, respectively (average value of two different cytosolic preparations). A comparison of dialyzed cytosols of separated male and female schistosomes showed that GST specific activity toward dichlorvos was about 25fold greater in females than in males (Table I). That ratio was nearly the same as that observed with the model epoxide substrate, EPNP. In contrast, male worm cytosol was about threefold more active toward CDNB than female worm cytosol. The values for CDNB and EPNP were comparable to those found previously (O’Leary and Tracy 1988). The similar gender-specific distribution of EPNP and dichlorvos conjugating activity suggested that dichlorvos metabolism might be catalyzed by SmGST-5, the form of the enzyme that is excluded from the GSH-agarose column (O’Leary and Tracy 1988). To test this hypothesis, cytosol of male parasites was fractionated by chromatography on GSH-agarose and the activity of the unbound (Fraction I) and the bound (Fraction II) fractions was compared with

CDNB, EPNP, and dichlorvos (Table II). The bulk of the CDNB conjugating activity was associated with Fraction II, whereas EPNP conjugating activity was exclusively recovered in Fraction I. Dichlorvos conjugating activity was detected in both fractions, but was ldfold greater in Fraction I than in Fraction II. To determine whether the metabolism of dichlorvos also occurs in vivo, S. mansoni worm pairs were incubated in supplemented RPM1 1640 containing 30 $t4 dichlorvos and 0.1% ethanol vehicle. Parasites incubated in the presence of the drug did not attach to the plastic dish and appeared paralyzed when first observed after 15 min. The presence of 0.1% ethanol alone had no apparent effect on either worm motility or attachment to plastic. Analysis of the acid-soluble parasite fraction revealed that GSCH, was formed only in organisms incubated in the presence of dichlorvos. Conjugate formation was time dependent, with about twofold more GSCH, being found in worms incubated for 2 hr than after 1 hr. In three separate experiments, the amount of GSCH, formed after 2 hr of inTABLE II Fractionation of Dialyzed Male Schistosoma rnansoni Cytosol by GSH Affinity Chromatography Fraction Cytosol Fraction I

EPNP

1.12 (100%) 0.03

0.23 (100%) 0.32 (107%) 60.001 (0%)

(2%)

Fraction II TABLE I SmGST Activity in Dialyzed Cytosols of Male and Female Schistosoma mansoni

CDNB

24.1 (97%)

Dichlorvos (log (8%) 0.15

(6%)

Note. Dialyzed cytosol (20 mg protein) was chromatographed on GSH-agarose (O’Leary and Tracy Units Male Substrate Female 1988).Fraction I consisted of proteins that did not bind CDNB pmole/midmg 1.05 2 0.07 0.33 + 0.03 to the column, including SmGST-5, whereas Fraction pmole/min/mg 0.31 f 0.04 0.78 + 0.05 II consisted primarily of SmGST-1, -2, and -3. Values EPNP 8.5 + 0.9 Dichbrvos nmolelminlmg 22.0 2 4.3 given are specific enzyme activities (pmole/min/ mg protein for CDNB and EPNP, nmole/min/mg proNote. Values given are the meansf SD of three independent sampleswith each sampleassayedin duplicate. Values tein for dichlorvos). Numbers in parentheses are the for dichlorvoshave beencorrectedfor nonenzymaticGSCH, percentage of total enzyme activity recovered in each formation. fraction.

~chistosoma mansoni:

DICHLORVOS

METABOLISM

359

purified, it is quite unstable (Meyer et al. 1984), and this has precluded extensive studies of its physicochemical and catalytic DISCUSSION properties. Our preliminary results suggest S. mansoni have the enzymatic capacity that SmGST-5 activity also is labile to metabolize dichlorvos, the active form of (O’Leary and Tracy, unpublished observametrifonate. Reiner (198 1) reported that tions). Because this isoenzyme had not parasite nonspecific esterase catalyzed hy- been purified, we cannot exclude the posdrolysis of the dichlorvos ester bond to sibility that more than one GST is present in the unbound fraction. form O,O-dimethylphosphate and dichloroUnder the assay conditions used, GST acetaldehyde. We have now demonstrated specific activity of unfractionated male the existence of a second pathway of dichlorvos metabolism, namely GSH- worm cytosol toward CDNB was about 140-fold greater than toward dichlorvos dependent O-demethylation to form GSCH, and desmethyldichlorvos. This re- (Table II). One might be tempted to infer action is catalyzed by SmGSTs and occurs from these data that dichlorvos is a poor not only in cell-free preparations, but also GST substrate. It must be borne in mind, in intact schistosomes incubated in the however, that direct comparisons of catalytic efficiency (k,,&,,J for different subpresence of a concentration of dichlorvos (30 pJ4) that was about loo-fold greater strates cannot be made in protein mixtures than the typical therapeutic plasma concen- containing multiple GST isoenzymes. Furtrations in humans treated with a standard thermore, even when such comparisons are dosage of metrifonate (Nordgren et al. made using a single purified enzyme, a low 1980). k,,,/K,,, value cannot be equated with a lack Although GSCH, formation was cata- of biological relevance of the reaction catlyzed by an affinity-purified preparation of alyzed. GSTs are generally thought to enSmGST, composed principally of the isoen- counter most xenobiotic substrates in vivo zymes, SmGST-1, -2, and -3, the fraction of at concentrations that are well below satuworm cytosol that is excluded from the ration, yet the corresponding GSH SGSH affinity column was much more ac- conjugates and/or mercapturic acids can be tive. That fraction contains an additional found in tissues and in urine (Sies and Ketform of the enzyme that we have desig- terer 1988). It is now apparent that schistosomes can nated SmGST-5. SmGST-5 appears to share several properties with rat liver GST metabolize antischistosomal drugs. Niridaisoenzyme E, including a lack of affinity for zole, a nitrothiazole derivative, undergoes GSH-agaroses (Meyer et al. 1984). Like enzymatic nitroreduction within adult S. SmGST-5, rat GST isoenzyme E displays mansoni to form reactive drug species that minimal catalytic activity toward the proto- bind covalently to parasite macromolecules type GST substrate CDNB, but substantial (Tracy et al. 1983). That niridazole metabactivity toward a model epoxide, EPNP, olism is essential for efficacy is supported and toward iodomethane (Habig et al. by the observation that a structural ana1972). The ratio of specific activity with logue, 4’-methylniridazole, which has no idomethane to that with dichlorvos is in fact antischistosomal activity, is not a substrate similar in the unbound protein fractions of for schistosome nitroreductase and does not bind covalently to parasite macromoleboth rat liver (12: 1; Tracy and O’Leary 1991) and S. mansoni cytosol (11:l). Al- cules (Tracy et al. 1983). Hycanthone, anthough rat liver GST isoenzyme E had been other antischistosomal agent, also appears cubation corresponded to 3.3 ? 0.9% of the drug dose.

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O'LEARYANDTRACY

to undergo metabolism within the parasite to a reactive intermediate (Cioli et al. 1985). That hycanthone metabolism relates to its efficacy is suggested by the observation that hycanthone-resistant strains of S. mansoni appear unable to catalyze the reaction (Cioli et al. 1985). In both instances, drug metabolism by the parasite results in toxic activation. Niridazole and hycanthone are thus examples of prodrugs that are metabolically activated within the target organism to give the actual toxic drug form. Alternatively, metabolism of a drug by the parasite can result in its inactivation. Clearly, conjugation of dichlorvos with GSH within S. mansoni represents a detoxication pathway. This finding is significant, because it is the first example of an antischistosomal drug that is metabolically inactivated by conjugation within the parasite. It seems likely that other drugs will be found to be detoxicated by parasite enzymatic defense mechanisms. Despite extensive clinical use of metrifonate for treatment of schistosomiasis hematobium, its mechanism of action and the basis of its species-specific activity remain obscure. Dichlorvos is a potent inhibitor of schistosome cholinesterase (Bueding et al. 1972). However, the species difference in drug susceptibility cannot be attributed to differential cholinesterase inhibition, because the bimolecular rate constant for dichlorvos inhibition is nearly the same in both S. mansoni and S. hematobium (Reiner 1981). Organophosphates do inhibit other enzymes that contain an essential serine residue at the active site. Thus, it is possible that species differences in dichlorvos susceptibility are due to inhibition of another serine enzyme that has yet to be identified. One hypothesis attributes the difference to differences in the anatomical location within the host of S. hematobium compared with S. mansoni (Forsyth and Rashid 1967). Under drug influence, S. mansoni are reversibly shifted to the liver, whereas S. hematobium are irreversibly

shifted to the lungs. This “lung-shift” hypothesis is supported by animal experiments (James et al. 1972) and by some indirect evidence from studies on human patients with mixed infections (Doehring et al. 1986). However, there has been no direct demonstration that the lung shift occurs in humans. Our results suggest still a third possible explanation. S. mansoni could be resistant to dichlorvos by virtue of a greater enzymatic capacity compared with that of S. hematobium to detoxicate the drug by conjugation with GSH. Direct comparisons of S. mansoni and S. hematobium GST activity toward dichlorvos are needed to test this hypothesis. ACKNOWLEDGMENTS This work was supported by NIH Grant AI22520 from the United States-Japan Cooperative Medical Sciences Program. K.A.O. was supported by NIH Training Grant ES07015. This is contribution No. 231 of the Environmental Toxicology Center, University of Wisconsin-Madison. REFERENCES BRADFORD,M M. 1976.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254. BUEDING, E., Lm, C. L., AND ROGERS,S. H. 1972. Inhibition by metrifonate and dichlorvos of cholinesterases in schistosomes. British Journal of Pharmacology 46, 48W87. CIOLI, D., PICA-MATTOCCIA,L., ROSENBERG,S., AND ARCHER, S. 1985. Evidence for the mode of antischistosomal action of hycanthone. Life Sciences 37, 161-167. DICOWSKY,L., AND MORELLO,A. 1971. Glutathionedependent degradation of 2,Zdichlorovinyl dimethyl phosphate (DDVP) by the rat. Life Sciences 10, 1031-1037. D~EHRING, E., POGGENSEE,II., AND FELDMEIER, H. 1986. The effect of metrifonate in mixed Schistosoma haematobium and Schistosoma mansoni infections in humans. American Journal of Tropical Medicine and Hygiene 35, 323-329. DUVALL, R. H., AND DEWITT, W. D. 1%7. An improved perfusion technique for recovering adult schistosomes from laboratory animals. American Journal of Tropical Medicine and Hygiene 16, 483486.

Schistosoma

munsoni:

DICHLORVOS METABOLISM

FORSYTH,D. M., AND RASHID, C. 1%7. Treatment of urinary schistosomiasis with trichlorphone. Lancet 2, 909-912. HABIG, W. H., PABST, M. J., AND JAKOBY, W. B. 1974. Glutathione S-transferases: The first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130-7139. JAMES, C., WEBBE, G., AND PRESTON,J. M. 1972. A comparison of the susceptibility to metrifonate of Schistosoma haematobium, S. mattheei, and S. mansoni in hamsters. Annals of Tropical Medicine and Parasitology 66, 467474. MANNERVIK, B., AND DANIELSON, U. H. 1988. Glutathione transferases: Structure and catalytic activity. CRC Critical Reviews in Biochemistry 23, 283337. MEYER, D. J., CHRISTODOULIDES, L. G., TAN, K. H., AND KETTERER, B. 1984. Isolation, properties, and tissue distribution of rat liver glutathione transferase E. FEBS Letters 173, 327-330. MOTOYAMA, N., AND DAUTERMAN, W. C. 1980. Glutathione S-transferases: Their role in the metabolism of organophosphorous insecticides. In “Reviews in Biochemical Toxicology” (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), Vol. 2, pp. 49-69. Elsevier, New York. NORDGREN, I., BERGSTROM,M., HOLMSTEDT, B., AND SANDOZ, M. 1978. Transformation and action of metrifonate. Archives of Toxicology 41, 3141. NORDGREN,I., HOLMSTEDT,B., BENGTSSON,E., AND FINKEL, Y. 1980. Plasma levels of metrifonate and dichlorvos during treatment of schistosomiasis with Bilarcil. American Journal of Tropical Medicine and Hygiene 29, 426430. O’LEARY, K. A., AND TRACY, J. W. 1988.Puritication of three cytosolic glutathione S-transferases from adult Schistosoma mansoni. Archives of Biochemistry and Biophysics 264, l-12.

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PETERS, P. A., AND WARREN, K. S. 1%9. A rapid method of infecting mice and other laboratory animals with Schistosoma mansoni: Subcutaneous injection. Journal of Parasitology 55, 558. REINER, E. 1981. Esterases in schistosomes: Reaction with substrates and inhibitors. Acta Pharmacologic et Toxicologic 49, Suppl. 5, 72-78. SIEGEL,D. A., AND TRACY, J. W. 1988. Effect of pairing in vitro on the glutathione level of male Schistosoma mansoni. Journal of Parasitology 74, 524531. SIES, H., AND KETTERER, B. (EDs.) 1988. “Glutathione Conjugation: Mechanisms and Biological Significance.” Academic Press, London. SMITH, D. B., DAVERN, K. M., BOARD, P. G., TIU, W. U., GARCIA, E. G., AND MITCHELL, G. F. 1986. &f, 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proceedings of the National Academy of Sciences USA 83, 8703-8707. TAYLOR, J. B., VIDAL, A., TORPIER, G., MEYER, D. J., ROITSCH,C., BALLOUL, J.-M., SOUTHAN,C., SONDERMEYER,P., PREMBLE, S., LECOCQ, J.-P., CAPRON,A., AND KETTERER,B. 1988.The glutathione transferase activity and tissue distribution of a cloned M, 28K protective antigen of Schistosoma mansoni. EMBO Journal 7, 465-472. TRACY, J. W., CA~O, B. A., AND WEBSTER,L. T. 1983. Reductive metabolism of niridozole by adult Schistosoma mansoni: Correlation with covalent drug binding to parasite macromolecules. Molecular Pharmacology 24,291-299. TRACY, J. W., AND O’LEARY, K. A. 1991. Analysis of glutathione S-transferase-catalyzed S-alkylglutathione formation by high-performance liquid chromatography. Analytical Biochemistry 193, l-5. Received 28 August 1990; accepted with revision 4 January 1991.