Chem. Res. Toxicol. 2007, 20, 1211–1217
1211
Glutathione S-Transferase Conjugation of Organophosphorus Pesticides Yields S-Phospho-, S-Aryl-, and S-Alkylglutathione Derivatives Kazutoshi Fujioka and John E. Casida* EnVironmental Chemistry and Toxicology Laboratory, Department of EnVironmental Science, Policy and Management, UniVersity of California, Berkeley, California 94720-3112 ReceiVed April 25, 2007
Pesticide detoxification is a central feature of selective toxicity and safety evaluation. Two of the principal enzymes involved are GSH S-transferases (GSTs) and cytochrome P450s acting alone and together. More than 100 pesticides are organophosphorus (OP) compounds, but with few exceptions, their GSH conjugates have not been directly observed in vitro or in vivo. The major insecticides chlorpyrifos (CP) and diazinon are of particular interest as multifunctional substrates with diverse metabolites, while ClP(S)(OEt)2 and the cotton defoliant tribufos are possible precursors of phosphorylated GSH conjugates. Formation of GSH conjugates by GST with GSH was studied in vitro with and without metabolic activation by human liver microsomes or P450 3A4 with NADPH. Metabolites were analyzed by liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS). Five GSH conjugates were identified from CP and chlorpyrifos oxon (CPO), i.e., GSCP and GSCPO in which the 6-chloro substituent of CP and CPO, respectively, is displaced by GSH; S-(3,5,6-trichloropyridin-2-yl)glutathione; S-(3,5-dichloro-6-hydroxypyridin-2-yl)glutathione; and S-ethylglutathione. GST of a human liver microsomal preparation but not P450 3A4 with GSH metabolized CP to GSCP. With GST and GSH, diazinon and diazoxon gave S-(2-isopropyl-4-methylpyrimidin-6-yl)glutathione and ClP(S)(OEt)2 yielded GSP(S)(OEt)2. With microsomes, NADPH, GST, and GSH tribufos gave GSP(O)(SBu)2. The liver of intraperitoneally treated mice contained GSCP from CP, GSP(S)(OEt)2 from ClP(S)(OEt)2, and GSP(O)(SBu)2 from tribufos. GSP(S)(OEt)2 and GSP(O)(SBu)2 are the first S-phosphoglutathione metabolites observed in vitro and in vivo directly by LC–ESI-MS. Nine other OP pesticides gave only O-dealkylation in the GST/GSH system. GST-catalyzed metabolism joins P450s and hydrolases as important contributors to OP detoxification. Introduction 1
More than 100 commercial organophosphorus (OP) pesticides differ from each other by a variety of substituents designed for optimal pesticidal activity with minimal toxicity to humans and the environment. They are mostly dialkylphosphorylating agents for serine hydrolases with other readily metabolized functionalities leading to selective toxicity. OPs are primarily metabolized by cytochrome P450s and hydrolases, but there is a growing realization that GSH S-transferases (GSTs) also play a role. GSTs metabolize a variety of endogenous and exogenous toxicants by forming GSH conjugates with the compounds directly or their reactive intermediates (1–3). The action of GSTs on OP pesticides can lead to activation or detoxification (4). GST with GSH dealkylates methyl or ethyl groups (e.g., methyl parathion and parathion) and dearylates diazinon and diazoxon * To whom correspondence should be addressed. Phone: (510) 642-5424. Fax: (510) 642-6497. E-mail:
[email protected]. 1 Abbreviations: Bu, butyl; CDNB, 1-chloro-2,4-dinitrobenzene; CP, chlorpyrifos; CPO, chlorpyrifos oxon; ESI, electrospray ionization; Et, ethyl; desGlu, desglutamyl; GSCP and GSCPO, derivatives of CP and CPO, respectively, with the 6-chloro substituent displaced by GSH; GSDCP, S-(3,5-dichloro-6-hydroxypyridin-2-yl)glutathione; GSEt, S-ethylglutathione; GSIMP, S-(2-isopropyl-4-methylpyrimidin-6-yl)glutathione; GSMe, S-methylglutathione; GST, GSH S-transferase; GSTCP, S-(3,5,6-trichloropyridin2-yl)glutathione; iPr, isopropyl; LC–ESI-MS, liquid chromatography–electrospray ionization-mass spectrometry; MSD, mass selective detector; OP, organophosphorus; Ph, phenyl; Pr, propyl; Pyr, pyridinyl; Pyrim, pyrimidinyl; SD, standard deviation; SIM, single-ion monitoring; TCP, 3,5,6trichloro-2-pyridinol; tR, retention time.
(5–8). However, knowledge of GSH phosphorylation is less definitive because the metabolites have not been adequately characterized. The first reported phosphorylated GSH conjugate was GSP(S)(Ph)OEt from 4-NO2-PhOP(S)(Ph)OEt (EPN) and GSH incubated with housefly abdomen enzymes characterized primarily by coupling a phenyl-14C-labeled EPN with [35S]GSH since there was no synthetic standard or spectrometric evidence (9). A metabolite of PrSP(O)(OEt)OPh-2,4-Cl2 (prothiofos) was proposed to be GSP(O)(OEt)OPh-2,4-Cl2 (10) but not identified. Four OPs were selected for primary investigation. Chlorpyrifos (CP) is used in large amounts (20 million pounds per year in the United States) with many people exposed. It is activated to chlorpyrifos oxon (CPO) by P450 and undergoes deethylation and dearylation in P450 systems, in human hepatocytes, or in vivo, yielding a large number of metabolites, including GSH conjugates at the 6-position of CP and CPO (GSCP and GSCPO, respectively) (Figure 1) (11–15). Hydrolases also play a role in CPO detoxification (16–18). The third OP examined was ClP(S)(OEt)2 as a reactive model for the large number of diethylphosphorothionate pesticides. Finally, tribufos is a cotton defoliant with annual use in the United States of 4.5 million pounds. Tribufos shows cholinergic action, delayed acute effects, and delayed neurotoxicity (19, 20). It is metabolized by hydrolysis or S-CH2 oxidation at the sulfur or methylene, yielding (BuS)2P(O)OH and (BuS)2P(O)SH, respectively, in urine from experimental animals (21, 22). Tribufos sulfoxide [BuS(O)P(O)(SBu)2] is considered to be the bioactivation
10.1021/tx700133c CCC: $37.00 2007 American Chemical Society Published on Web 07/21/2007
1212 Chem. Res. Toxicol., Vol. 20, No. 8, 2007
Figure 1. GST- and P450-catalyzed metabolism of chlorpyrifos (CP), chlorpyrifos oxon (CPO), diazinon, diazoxon, and ClP(S)(OEt)2. The pyridinyl and pyrimidinyl positions of conjugation are those previously reported for CP (14) and diazinon (8). Although not shown, the desethyl derivatives of GSCP and GSCPO and the desglutamyl derivative of GSCP are also observed.
product of tribufos resulting from P450-mediated oxidation in vitro and in vivo (22–24). The thiocarbamate herbicide EtSC(O)NPr2 (EPTC) serves as a model for tribufos metabolism with formation of a reactive sulfoxide intermediate that couples with GSH in the presence of GST (25, 26). This study considers CP, CPO, ClP(S)(OEt)2, and tribufos metabolism by GST and P450 systems in vitro and/or in vivo. Special attention with CP is given to the diversity of metabolites and with ClP(S)(OEt)2 and tribufos to possible formation of phosphorylated GSH conjugates when incubated with GSH and GST of cytosol and/or a microsomal preparation. Liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) was utilized to identify and quantitate the metabolites with a special emphasis on GSH conjugates.
Materials and Methods Chemicals and Biologicals. Structures and abbreviations for most of the compounds considered are shown in Figure 1. Sources were as follows: the OP pesticides, EPTC, and 3,5,6-trichloro-2pyridinol (TCP) from Chem Service (West Chester, PA); GSH, GSTs from equine liver, rat liver, and human placenta, Smethylglutathione (GSMe), 1-chloro-2,4-dinitrobenzene (CDNB), HSCH2CH2OH, ICH2CH2CH2Cl, (EtO)2P(O)OH, (EtO)2P(O)SH, (BuO)2P(O)OH, and ClP(S)(OEt)2 from Sigma-Aldrich (St. Louis, MO); HPLC-grade acetonitrile and water from Fisher Scientific (Pittsburgh, PA); and pooled human liver microsomes and human P450 3A4 from BD Biosciences (Woburn, MA). GSPh-2,4-(NO2)2 was synthesized by reacting CDNB (1 mM) with GSH (6 mM) in the presence of equine GST (0.4 mg/mL protein) at 37 °C for 2 h. Instrumentation. The Agilent 1100 HPLC system coupled with a Kromasil C4 5 µm column (250 mm × 4.6 mm, Supelco, SigmaAldrich) and the Agilent 1100 mass selective detector (MSD) were used for analysis of metabolites. Mobile phases consisted of water (solvent A) and acetonitrile containing 0.05% (v/v) formic acid (solvent B). The program started from 95% solvent A at 0 min with a linear gradient to 10% solvent A at 17 min which was
Fujioka and Casida maintained until the 22 min point with a flow rate of 1 mL/min. The injection volume was 10 µL. The MSD was operated under ESI conditions as follows: nitrogen flow rate, 10 mL/min; nebulizer pressure, 40 psi; drying gas temperature, 300 °C; capillary voltage, 3000 V. The positive mode was normally used for analysis because it gave more daughter ions useful for structural identification. Enzyme Reactions. Incubation mixtures consisted of 6 mM GSH, 1 mM NADPH, GST (80 µg of protein, 10–20 units), and microsomes (100 µg of protein) or P450 3A4 (50 µg of protein) in 200 µL of 100 mM potassium phosphate buffer (pH 7.4). Substrates (100 µM OP or EPTC) were added last in acetone at a final concentration of 1% (v/v). Control samples with appropriate deletions of microsomes, P450 3A4, GST, NADPH, or GSH were incubated under the same conditions. The GSH concentration of 6 mM is typical for that in the cell (+a
[M + Na]
MH
577.2 (100) 593.2 (100) 492.2 (100)
b
599.2 (10) 615.2 (24) 514.2 (13)
nd ndb nae
[MH – 75]+a b
[MH – 146]+a ndb ndb nae
nd ndb ndb
tR (min) 9.8 11.3 12.2
m/z [abundance (%)] butyl phosphate (BuO)2P(O)OH (BuS)2P(O)OH (BuO)2P(O)O(CH2)3Cl (BuS)2P(O)O(CH2)3Cl BuSP(O)(SBu)2
+
MH
211.4 (100) 243.3 (100) 287.4 (43) 319.3 (100) 315.3 (100)
+
[M + Na]
233.3 (37) 265.3 (43) 309.3 (88) 341.3 (96) 337.3 (61)
a See the text for assignment of daughter ions. desglutamyl-GSCP. e Not applicable.
b
Not detected.
reconstituted with a 50% (v/v) acetonitrile/water mixture (400 µL), passed through the Acrodisc filter, and then held at -20 °C. The frozen samples were thawed and subjected to LC with MSD as described above except with both the positive and negative mode.
Results LC–ESI-MS Characteristics of GSH Conjugates and Butyl Phosphates (Table 1). The parent ions, fragmentation patterns, and retention times (tR) were used to characterize the metabolites. All GSH conjugates gave the parent ion (MH+) as the base peak, accompanied by [M + Na]+ and several daughter ions: [MH – 129]+ from neutral loss of pyroglutamate (29), [MH – 146]+ from loss of pyroglutamate and ammonia, and usually [MH – 75]+ from loss of glycine. The [MH – 129]+ daughter ion was prominent with all GSH conjugates except those from desethyl derivatives (desEtGSCPO and desEtGSCP). The molecular weights of the GSH conjugates determine the stability of the parent ions, at least in part, since they were inversely correlated with the intensity of the sum of daughter ions relative to the parent ion (R2 ) 0.77, p < 0.001, n ) 14). Butyl phosphates exhibited a parent ion (MH+), [M + Na]+, and a [MH – 56]+ daughter ion, which represented a loss of butene via McLafferty rearrangement (30), sometimes with loss of two butene [MH – 112]+. Chloropropyl derivatives gave the isotopic parent ion [MH + 2]+ (not shown) and another daughter ion [MH – 76]+ which represented loss of chloropropene. The tR values were generally greater for the GSH conjugates with phosphorus or aryl moieties (9.9–12.5 min) than those with only alkyl substituents (4.0–6.3 min). The desEt and desglutamyl (desglu) hydrolyzed GSH conjugates exhibited shorter tR than the compounds without the substituent removed. In Vitro Metabolites of CP, CPO, and TCP. CP, CPO, and TCP were incubated with one of both of human liver
[MH – 112]+a b
nd 131.2 (15) 175.3 (100) ndb ndb c
[MH – 56]+a
[MH – 76]+a
tR (min)
155.3 (45) 187.3 (50) 231.3 (51) 263.2 (16) ndb
nae nae ndb 243.3 (15) nae
11.3 14.3 17.8 19.4 21.2
See Figure 1 for structures.
d
Desethyl-GSCPO, desethyl-GSCP, and
microsomes (P450) and GST in the presence of GSH with or without NADPH under standard conditions (Table 2). No attempt was made to optimize the reaction parameters. CP gave three major metabolites [GSCP, GSTCP, and S-ethylglutathione (GSEt)] and one minor metabolite (GSDCP) on incubation with GST and GSH, with similar site specificity for equine and rat liver sources (Table 2 and Figure 2), and complete dependence in each case on added GSH (data not shown). The GSCP: GSTCP:GSEt ratio with equine GST was 1.0:1.2:0.9 at 37 °C (Table 2) and 1.0:0.7:0.3 at 25 °C (Figure 2), indicating a possible temperature effect on metabolic formation or stability which was not studied further. When human liver microsomes or placental GST were used, CP gave only GSCP (47 or 1%, respectively) at 37 °C (Table 2). When incubated with human liver microsomes, NADPH, and the equine GST/GSH system, CP gave GSCP as the only significant metabolite (63%), presumably formed by an endogenous GST in the microsomal preparation (Table 2 and Figure 3). Addition of microsomes to the equine GST/GSH system reduced the extent of CP loss and GSH conjugate formation except for GSCP, suggesting that this conjugate and its metabolites might inhibit GST. For a direct test of this hypothesis, CP (100 µM) and its metabolites marginally inhibited equine GST (8 ( 3%) on preincubation with GSH (5 mM) for 2 h at 37 °C but not without preincubation. Under the preincubation condition, GSCP (0.4 µM), GSTCP (0.4 µM), GSEt (0.2 µM), and (EtO)2P(O)SH (0.3 µM) were formed but GSDCP was not detected; i.e., these metabolite levels cause marginal or no GST inhibition. CPO gave GSCPO, GSEt, GSTCP, GSDCP, and TCP with equine and rat liver enzymes (Table 2). When human liver microsomes or placental GST was used, CPO gave TCP as a major metabolite and GSCPO as a minor metabolite. TCP was
1214 Chem. Res. Toxicol., Vol. 20, No. 8, 2007
Fujioka and Casida
Table 2. Metabolism of Chlorpyrifos, Chlorpyrifos Oxon, and Trichloropyridinol by Human Liver Microsomes and GST Systems in the Presence of GSH with or without NADPH metabolite yield (%) (n ) 3)a
components CP
CPO
TCP
GST
microsomes
CP
CPO
GSCP
GSCPO
GSEt
GSTCP
GSDCP
TCP
equine – equine rat placenta equine – equine rat placenta equine – equine
+ + + + + +
20 (5)b 53 (15) 39 (24) 49 99 – – – – – – – –
