Chem. Res. Toxicol. 1994, 7, 487-494
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Articles Adducts of Dienochlor Miticide with Glutathione, Glutathione S-Transferases, and Hemoglobins Julia A. Fruetel, Susan E. Sparks, Gary B. Quistad, and John E. Casida' Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720 Received December 13,199P
Dienochlor (Pentac) (CloCllo) has been used for 30 years as a miticide with little knowledge of its mode of action or metabolic fate except that it is quickly degraded by rats. This study examines the reactions of dienochlor with GSH and proteins as models for its metabolism and interactions with tissues. Dienochlor reacts rapidly with 1.0 mM GSH in phosphate buffer (pH 7.4) a t 37 "C (t1p 11min) as analyzed by UV/visible spectroscopy and HPLC, yielding a series of more than a dozen adducts. Octachlorofulvalene (ClOCla), a candidate intermediate, also reacts to give the same apparent products (t1/2 < 0.2 min as above); however, its intermediacy in the dienochlor reaction was not established. Isolation and MS analyses characterized two isomeric CloH2Cl(SG)b adducts and a CloH2(SG)6 derivative; these products react further in the presence of GSH to yield two even more polar adducts. Cysteine andN-acetylcysteine alsoreact rapidly with dienochlor whereas GSSG and several non-thiol amino acids are much less reactive. Purified GSH S-transferases (GSTs) and hemoglobins,each from six species of mammals including humans, are extensively labeled in vitro by [14Cldien~chlorto form adducts separable by gel electrophoresis and HPLC. [l4C1Dienochlorreadily derivatizes rat liver GSTs even in cytosol and in the presence of high GSH levels. The potency of dienochlor for inhibition of GST activity is maintained or enhanced upon conversion to GSH adducts. The miticidal action of dienochlor and its toxic effects in mammals, e.g., a mouse ip LDNof 5 mg/kg, may be related to derivatization of thiol proteins. These results suggest that dienochlor will give a complex metabolic profile in mammals since it readily reacts a t physiological pH not only with thiol-containing proteins but also with GSH, leading to the total displacement of all ten chlorines and the introduction of an unprecedented six (or more) glutathionyl substituents into this xenobiotic.
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Introduction Dienochlor (Pentac; 1,1',2,2',3,3',4,4',5,5'-decachlorobi2,4-cyclopentadien-l-y1) is a miticide prepared by catalytic reduction of hexachlorocyclopentadiene (Figure 1)(I,2). It has been used for 30years (3)with registrations restricted to ornamental plants. Dienochlor is stable to acids and bases but is converted to octachlorofulvalene by chemical reducing agents (Figure 1)(4,5). It is also photolabile (6) and undergoes isomerization reactions at high temperatures (7). The mode of action of dienochlor is largely unknown in both mites and mammals, and there are no identified metabolites in any biological system. The acute oral toxicity to rats is quite low (LD50 > 3160 mg/kg) (8); however, the 14C-labeledcompound appears to be poorly absorbed when administered orally since the radiocarbon recoveries at 3-4 days in urine, bile, and tissues are each only 1-276 of the administered dose and most of the remainder is in feces in an unextractable form (9). By analogy with other chlorinated hydrocarbons and elec-
* To whom correspondenceshouldbe addressed,at the Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, 114 Wellman Hall, University of California,Berkeley, CA 94720.Phone: (510)642-5424;FAX (510)6426497; Email:
[email protected]. @Abstractpublished in Advance ACS Abstracts, May 1, 1994.
trophilic compounds (IO),a portion of the metabolic reactions of dienochlor may involve GSH and GSH 5'-transferases (GSTs).' This investigation concerns the reactions of dienochlor with GSH, GSTs, and other proteins and of octachlorofulvalene with GSH. Findings from these model systems and reactions may be applicable to the metabolic fate and mode of action of dienochlor.
Materials and Methods Materials. Dienochlor (A, 330 nm in methylene chloride) was obtained by extracting Pentac miticide (50% wettable powder)with methylene chloride and recrystallizing from hexane. [U-ring-14C]Dienochlor(43.5 mCi/mmol), provided by Sandoz Agro, Inc. (Des Plaines, IL), was purified by silica gel TLC with hexane (R, = 0.7) and recovered by extracting the silica with methylene chloride (radiochemicalpurity 93 % based on TLC). Octachlorofulvalene (A, 386 nm in methylene chloride), prepared by reacting dienochlorwith triisopropylphosphite (Figure 1) (4),was recrystallizedfrom hexane. The purities of dienochlor and octachlorofulvalenewere estimated to be >98 % and >99 % , respectively, based on HPLC (described later) and IR (4). Abbreviations: ES-MS, electrospray mass spectrometry; GS, glutathionyl; GST, GSH S-transferase;LSIMS, liquid secondary ion mass spectrometry;PAGE, polyacrylamide gel electrophoresis;SDS, sodium dodecyl sulfate;TFA, trifluoroacetic acid.
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Figure 1. Synthesis of dienochlor and octachlorofulvalene from hexachlorocyclopentadiene. Tetrachloro-l,4-benzoquinone,an active site-directed irreversible inhibitor of GST (11,12),was from Aldrich (Milwaukee, WI). GSH and GSSG were obtained from Calbiochem (La Jolla, CA) and cysteine was from Aldrich. Sigma (St. Louis, MO) was the source of N-acetylcysteine and the purified proteins examined, Le., hemoglobins, albumin (bovine serum), myoglobin (equine), chymotrypsin (bovine pancreas), and GSTs (which had been purified by affinity chromatography and contained GSH as an impurity). Rat and mouse liver cytosols, as the postmicrosomal supernatants (100000g),werestoredat-70 "C prior touse. Protein concentrations were determined by the procedure of Bradford (13). Instrumentation. HPLC analyses of dienochlor and octachlorofulvalenereactions and reaction products were conducted using a Merck LiChrospher 100 RP-18 (5-pm) reverse-phase column (E. Merck, Darmstadt, Germany) on either a Waters Model 600E solvent delivery system coupled to a Model 994 photodiode array detector or a Hewlett Packard Series 1050 pumping system and Series I1 1040M photodiode array detector interfaced to HPLCSD Chemstation software (DOS series). Proteins derivatized by [14C]dienochlorwere analyzed on the Waters system with a Vydac C4 protein column (Separations Group, Hesperia, CA) ( 5 pm, 0.46 X 5 cm) using acetonitrile/ water/O.l% trifluoroacetic acid (TFA), where an initial 0 % acetonitrile for 5 min was followed by linear gradients of 0-40% acetonitrile over 15 min, 4 0 4 0 % acetonitrile over 10 min, and then 60% acetonitrile for 10 min, all at 1.5 mL/min. The eluent was monitored continuously at 220 nm, and 1.0-min fractions were collected for assay of radioactivity by liquid scintillation counting. UV/visible spectra were collected on a Hewlett Packard 8452A diode array spectrophotometer connected to a constanttemperature water bath. Liquid secondary ion mass spectra (LSIMS) were obtained on a Kratos Analytical MS-50s instrument with samples introduced in a glycerol/thioglycerol (1:l) matrix acidified with 0.1 M hydrochloric acid on a coolable probe. Electrospray mass spectra (ES-MS) were recorded on a VG Bio-Q instrument consisting of an electrospray source and a quadrupole mass analyzer withacetonitrile/water (1:l)containing 1% formic acid as the carrier solvent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) involved adding the reaction mixture of [14C]dienochlor with cytosol or purified protein (GST or hemoglobin) to an equal volume of 2 X loading buffer [2% SDS, 62.5 mM Tris-HC1 (pH 6.8), 15% glycerol, and 100 mM dithiothreitoll and then boiling the samples for 5 min prior to analysis by the method of Laemmli (14).After electrophoresis on a 10% gel in comparison with molecular mass standards, the proteins were stained with Coomassie blue and the gelswere dried and subjected to autoradiography. Reaction Rates of Dienochlor and Octachlorofulvalene with GSH and Amino Acids. A solution of 1 mM GSH or amino acid in 100 mM phosphate buffer (pH 7.4, 1.3 mL) was prewarmed at 37 "C for 1 min. The reaction was initiated by adding dienochlor or octachlorofulvalene (52 nmol) in ethanol (0.2 mL). This level of ethanol (13%)was required to maintain the dienochlor or octachlorofulvalene in solution. Aliquots (0.15 mL), taken during incubation with shaking for 0-30 min at 37 OC, were quickly placed in ice-cold test tubes containing dieldrin (4.8 pg) in ethanol (11pL) as an internal standard. HPLC analyses were performed on 50 pL of each aliquot using acetonitrile/water where an initial 50% acetonitrile for 2 min was followed by a linear gradient to 100 % acetonitrile over 10 min and then 100%
acetonitrile for 10 min, all at a flow rate of 1.0 mL/min. Quantitation was by UV/visible spectroscopy [wavelength (nm), t~ (min)]: dienochlor, 220, 14.8; octachlorofulvalene, 386, 16.5; dieldrin, 220, 10.8. Spectroscopic Analyses of t h e Reactions of Dienochlor and Octachlorofulvalene with GSH. Reaction mixtures as above were examined by UV/visible spectroscopy over a period of 30 min at 37 "C. HPLC Analyses of the Reaction Products of Dienochlor and Octachlorofulvalene with GSH. Reaction mixtures as above, following removal of ethanol by rotary evaporation at -25 "C, were analyzed by HPLC with methanol/water/O.l% TFA using linear gradients of 0-45% methanol over 45 min, 45-100% methanol over 10 min, and 100% methanol for 10 min, all at 1.5 mL/min. The eluent was monitored at 430 nm for the reaction products of dienochlor and octachlorofulvalene with GSH, at 220 nm for GSH, GSSG, and dienochlor, and at 386 nm for octachlorofulvalene. Isolation of GSH Conjugates 1-3. Several conjugates of dienochlor with GSH (designated 1-3) were isolated for characterization. Optimized yields of 1 and 2 were obtained by reacting dienochlor (85 nmol) added in ethanol (0.15 mL) with GSH (1pmol) in water (0.85 mL, adjusted to pH 7) for 20 min at 37 "C. To favor the formation of 3, the GSH level was 5 mM in a mixture of water (0.85 mL, pH 7) and ethanol (0.11 mL), to which dienochlor (120 nmol) was added in three equal aliquots in ethanol (0.04 mL) at 10-min intervals with reaction for a total of 30 min at 37 "C. The reaction mixtures were subjected to rotary evaporation at -25 "C to remove the ethanol and immediate HPLC purification under the conditions described for product analysis. The appropriate peaks were collected, and the solvent was removed under vacuum to dryness. The procedure was repeated as required to obtain 100-300 pg for each of 1,2, and 3. They were stored at -20 "C and analyzed by MS and NMR within 48 h to minimize artifacts from decomposition. Reactions of Dienochlor/GSH Conjugates with GSH. Reactions of purified 1or 2 with excess GSH (5mM) were carried out in 10 mM phosphate buffer (pH 7.4) at 37 "C for 15 and 90 min with analysis by HPLC. Reactions of [W]Dienochlor with Rat Liver Cytosol, GSTs, Hemoglobins, and Other Proteins. [l4C1Dienochlor (0.2nmol) was added in ethanol (5pL, injected below the surface) to a solution of GST (50 pg of protein) or rat liver cytosol (200 pg of protein) in 50 mM phosphate buffer (pH 7.4,lOO-pL final volume) in a glass tube. In one study with rat liver GSTs, the GSH level was also varied: 0.05, 0.5, or 5 mM. Following incubation for 1h at 37 "C, the samples were stored refrigerated (up to 1 day without apparent change in composition) before analysisby HPLC (totalsample of 100pL) or SDS-PAGE (aliquot of 40 pL). Inhibition of GST Activity. The effects of dienochlor and octachlorofulvalene on GST activity in mouse liver cytosol were examined both in vitro and in vivo. Tetrachlorobenzoquinone was studied for comparison. The in vitro studies focused on the effects of reacting each inhibitor with varying levels of GSH prior to interaction with GST, thus assessing the inhibitory potencies of the mixtures of GSH reaction products. In the in vitro investigations, endogenous GSH was removed from the cytosol by passage of an aliquot over a Sephadex G-25 column (Pharmacia, Uppsala, Sweden) in 100 mM phosphate buffer (pH 6.5) at 4 "C. Preincubation of inhibitors (50 pM, estimated to be at least 50-fold molar excess over the GST level
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Figure 2. Reaction rates of dienochlor with GSH, cysteine, N-acetylcysteine, and no thiol at pH 7.4 and 37 OC (2.3,4.1,0.8, and
