Biotransformation of Methyl Parathion by Glutathione S-Transferases

Erika L. Abel1,2, Theo K. Bammler1 and David L. Eaton3

Department of Environmental and Occupational Health Sciences and Center for Ecogenetics and Environmental Health, University of Washington, 4225 Roosevelt Way NE, #100, Seattle, Washington 98105

Received November 12, 2003; accepted February 15, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The organo(thio)phosphate esters are one of the most widely used classes of insecticides. Worldwide, organophosphate insecticides (OPs) result in numerous poisonings each year. In insects, glutathione S-transferases (GSTs) play an important role in OP resistance; limited data suggest that GST-mediated O-dealkylation occurs in humans as well. To characterize the capacity of mammalian GSTs to detoxify OPs, we investigated mammalian GST biotransformation of the widely used OP, methyl parathion (MeP). Cytosolic fractions isolated from rat, mouse, and ten individual adult human livers biotransformed 300 µM MeP at rates of 2.36, 1.76, and 0.70 (mean rate) nmol desmethyl parathion/min/mg, respectively. Our study focused on human GSTs; in particular, we investigated hGSTs M1-1 and T1-1, since deletion polymorphisms occur commonly in these genes. However, we found no correlation between hGSTM1/T1 genotypes and MeP O-dealkylation activities of the ten human liver cytosolic samples. We also measured MeP O-dealkylation activities of several purified recombinant GSTs belonging to the alpha (human GSTs A1-1 and A2-2, mouse GSTA3-3, rat GSTA5-5), mu (human GSTs M1a-1a, M2-2, M3-3, M4-4), pi (human GSTP1-1, mouse GSTs P1-1, P2-2), and theta (human GSTT1-1) classes. At 1 mM glutathione and 300 µM MeP concentrations, hGSTT1-1 and hGSTA1-1 exhibited the highest O-dealkylation activities: 545.8 and 65.0 nmol/min/mg, respectively. When expression level and enzymatic activity are considered, we estimate that hGSTA1-1 is responsible for the majority of MeP O-dealkylation in human hepatic cytosol. In target organs such as brain and skeletal muscle, where hGSTT1-1 is expressed, hGSTT1-1-mediated biotransformation of MeP may be important.

Key Words: organo(thio)phosphate esters; glutathione S-transferases; methyl parathion; human; conjugation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The organo(thio)phosphate esters are among the most widely used insecticides because of their efficacy and lack of environmental persistence (Namba, 1971Go). The combination of widespread use and relatively high acute toxicity of organophosphate insecticides (OPs) results in numerous poisonings each year. In Costa Rica alone, 1274 pesticide-related illnesses were reported in 1996 (Leveridge, 1998Go). Of these, 40% were occupationally related, and OPs were the most frequently recorded exposure. Between 1982 and 1985, 215 OP-related illnesses were reported among pesticide applicators in California (Brown et al., 1989Go).

The majority of OPs require cytochrome P450-mediated activation via oxidative desulfuration in order to exert their toxic effects (Fig. 1) (Butler and Murray, 1997Go). The resulting acute toxicity is largely, if not exclusively, due to inhibition of acetylcholinesterases by the oxon metabolites (reviewed in Namba, 1971Go). Most pertinent to manifestation of toxic effects is inhibition of acetylcholinesterase in cholinergic synapses of neurons in the central nervous system and skeletal muscle.



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 1. Biotransformation of MeP (Anderson et al., 1992Go).

 
Cytosolic glutathione S-transferases (GSTs) belong to a super gene family of enzymes whose main function is to catalyze the conjugation of a diverse array of electrophilic compounds with glutathione (reviewed in Eaton and Bammler, 1999Go). In insects, studies suggest that GSTs play an important role in resistance against several classes of insecticides including OPs (Syvanen et al., 1996Go; Wei et al., 2001Go). Methyl parathion (MeP), a widely used OP insecticide, is biotransformed via a glutathione-dependent pathway in rat and mouse liver fractions (Benke et al., 1974Go; Benke and Murphy, 1975Go; Clark et al., 1973Go). In the case of MeP, GSTs catalyze O-dealkylation of the parent compound with formation of S-methylglutathione (reviewed inFukami, 1980Go). O-Demethylation of MeP prevents activation via oxon formation. Radulovic and coworkers have shown that human GSTs expressed in fetal liver and placenta catalyze O-dealkylation of MeP (Radulovic et al., 1986Go, 1987Go). However, the role of GSTs in adult human MeP biotransformation and the GST isoform specificity for MeP O-dealkylation have not been characterized.

Based on primary sequence similarities, the mammalian GSTs can be grouped into at least eight classes termed alpha, kappa, mu, omega, pi, sigma, theta, and zeta (Board et al., 2000Go, 2001Go; Harris et al., 1991Go; Mannervik et al., 1985Go; Meyer et al., 1991Go; Meyer and Thomas, 1995Go; Pemble et al., 1996Go). In this report, we follow the unifying nomenclature system for mammalian GSTs recommended by Mannervik and colleagues (Mannervik et al., 1992Go), according to which the names of the classes are abbreviated by capital letters (e.g., A for alpha, K for kappa, M for mu, O for omega, P for pi, S for sigma, T for theta, and Z for zeta) followed by Arabic numerals indicating the subunit composition, assigned in order of discovery.

The objective of this study was to investigate the ability of mammalian GSTs, in particular human GSTs, to biotransform OPs. Of particular interest are human GSTs M1-1 and T1-1; the genes for both are polymorphic due a common gene deletion. Approximately 50% and 15–20% of Caucasians are homozygous null for GSTM1 and GSTT1, respectively (reviewed in Eaton and Bammler, 1999Go); these individuals lack the hGSTM1 and/or hGSTT1 genes and the corresponding proteins. If hGSTM1-1 and/or hGSTT1-1 played a major role(s) in the O-dealkylation of MeP in vitro, then it could be hypothesized that individuals homozygous for the null alleles would be at heightened risk for MeP poisoning. To address the mechanistic basis for this hypothesis, rat, mouse, and human hepatic cytosolic fractions have been assayed for glutathione-dependent O-demethylation activity toward MeP. Furthermore, we have determined the specific activities of several purified recombinant GSTs toward MeP, including hGSTs M1-1 and T1-1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents.
Methyl parathion was obtained from Chem Service (West Chester, PA) and dissolved in dimethyl sulfoxide (DMSO). DMSO, reduced glutathione, 1-chloro-2,4-dinitrobenzene (CDNB), and p-phenylphenol were purchased from Sigma (St. Louis, MO). HPLC-grade methanol and acetonitrile were obtained from Fisher Scientific (Fair Lawn, NJ) and J.T. Baker (Phillipsburg, NJ), respectively. Tetrabutylammonium phosphate was purchased from Regis Technologies, Inc. (Morton Grove, IL). All other chemicals were of analytical grade or better and obtained from various commercial sources.

Animals.
Adult male Swiss-Webster mice and adult male Sprague-Dawley rats were used. The animals were sacrificed by CO2 narcosis, and the livers excised according to IACUC-approved protocols. The rats served as control animals in unrelated developmental toxicity experiments.

Human liver samples.
Ten human liver samples were obtained from the University of Washington Liver Bank (Dr. Kenneth Thummel). Characteristics of the liver donors are shown in Table 1.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Characteristics of Liver Donors

 
Liver cytosol preparation.
All of the following steps were carried out at 4°C. Frozen liver tissue was thawed in 0.9% NaCl, blot dried, and weighed. Tissue was minced in 1.5 volumes (v/w) of homogenization buffer (0.25 M sucrose, 0.2 mM DTE, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) and homogenized for 10 s. At this step, the rat and mouse livers were pooled according to species, but the individual human livers were kept separate. Each sample was centrifuged at 10,0000 x g for 10 min; the pellet was discarded, and the supernatant was centrifuged at 15,000 x g for 20 min. The subsequent supernatant was centrifuged at 105,000 x g for 60 min. The resulting supernatant (cytosolic fraction) was filtered through gauze to remove lipids. These cytosolic samples were stored at –80°C until used.

CDNB assay.
Human liver cytosols were tested for total hGST protein activity toward the model GST substrate, 1-chloro-2,4-dinitrobenzene (CDNB), as described previously (Habig and Jakoby, 1981Go). All reactions were linear for at least 1 min.

hGSTM1 and hGSTT1 genotyping.
The human liver samples had been previously assayed for hGSTM1 and hGSTT1 genotype by Polymerase Chain Reaction methods (Chen et al., 1996Go). The livers were chosen to ensure an even distribution of genotypes and genotype combinations such that two livers were GST M1+, T1+, three were GST M1+, T1 null, three were GST M1 null, T1+, and two were homozygous null for both GSTM1 and T1.

GST expression and purification.
Expression and purification of hGSTA1-1, hGSTM1a-1a, hGSTM2-2, hGSTM3-3, hGSTM4-4, mGSTA3-3, mGSTP1-1, mGSTP2-2, and rGSTA5-5 were carried out as previously described (Bammler et al., 1995Go). His-hGSTT1-1 construct was a generous gift from Dr. John Hayes (University of Dundee, Dundee, Scotland); expression and purification was performed as previously described (Sherratt et al., 1997Go). cDNA expression constructs for hGSTs M1a, M2, M3, and M4 were generously provided by Dr. Phillip Board (John Curtis School of Medical Research, Australian National University, Canberra ACT 2601, Australia). Purified recombinant hGSTA2-2 was a kind gift from Dr. Chen-Pei D. Tu (Pennsylvania State University, State College, PA).

A hGSTP1 expression construct was generated which allowed purification of recombinant hGSTP1-1 (Abel et al, 2004). Briefly, the cDNA for hGSTP1 was generated by polymerase chain reaction amplification of an existing hGSTP1 cDNA-containing vector (a generous gift from Dr. William Atkins, University of Washington). PCR amplification primers were used that allowed for addition of NdeI and BamHI restriction sites to the 5' and 3' ends of the hGSTP1 cDNA, respectively. Subsequently, this PCR product was ligated into the pCR2.1 vector using a TA cloning kit (Invitrogen Life Technologies, Carlsbad, CA). The vector was propagated in INV{alpha}F' cells. Following BamHI digestion for construct linearization, an NdeI partial digestion allowed for the collection of full-length hGSTP1 cDNA with NdeI and BamHI sticky ends for ligation into the pET17b expression vector (Novagen, Madison, WI).

Protein assays.
Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's microtiter plate protocol. Bovine serum albumin was used as the protein standard.

MeP assay.
The assay for GST-mediated biotransformation of MeP was performed at 37°C in a shaking water bath as previously described (Anderson et al., 1992Go). In a 1.7-ml plastic vial, 50 µL of cytosol or recombinant GST was added to 145 µL incubation buffer (100 mM Tris, pH 7.4) with glutathione (1–10 mM final). The mixture was gently vortexed and prewarmed for 5 min. Following this preincubation, 5 µL of MeP was added to a final concentration of 60–300 µM MeP. Following mixing, the vial was returned to the water bath for either a 10- or 30-min incubation period. Protein concentration and incubation times were modified to ensure that the reaction proceeded within the linear range for the entire incubation period. After the incubation period, the reaction was stopped with 200 µL ice-cold methanol containing 250 µM p-phenylphenol as an internal standard. Immediately following incubation the samples were stored at –20°C. Two negative controls included the replacement of protein with incubation buffer and the elimination of glutathione. Each cytosolic fraction or recombinant GST was assayed in triplicate in each of at least two trials.

Samples were prepared for HPLC/UV analysis by centrifugation at 10,000 x g for 3 min. Subsequently 200 µL of the supernatant was transferred to an HPLC vial.

HPLC protocol.
Reversed-phase HPLC with ion-pairing was used to detect the MeP metabolites and internal standard (Anderson et al., 1992Go). The HPLC instrumentation used for these assays was a Varian-Dynamax model SD-200 with a Varian-Dynamax Method Manager version 1.4.6 integrator. A 4.6 x 250 mm C18 RP column with Econosphere packing, 100 angstrom pore size, 5 µm particle size, and 10% carbon load (Alltech, Deerfield, IL) and a repackable C18 reverse phase guard column (with Alltech Pellicular C18 Refill) were used. The aqueous mobile phase (A) consisted of 0.25 mM tetrabutylammonium phosphate (TBAP, ion-pairing reagent) in water, while the organic phase (B) consisted of 0.25 mM TBAP in 80% acetonitrile. Each phase was brought to pH 3.0 with 10% phosphoric acid. An 18-min gradient was used for metabolite separation and began with 90% A with a flow rate of 1.5 ml per minute and with a column temperature of 40°C. A Shimadzu SIL-6A (Shimadzu, Kyoto, Japan) autosampler delivered injection volumes of 50 µL. The HPLC gradient continued as shown in Table 2.


View this table:
[in this window]
[in a new window]
 
TABLE 2 HPLC Mobile Phase Gradient

 
Detection of the metabolites was performed using a Rainin Dynamax Absorbance Detector Model UV-1 (Rainin, Woborn, MA) at a wavelength of 280 nm. Relative retention patterns were used to identify the metabolites as previously validated with known standards for MeP, desmethyl parathion, methyl paraoxon, and desmethyl paraoxon (Anderson et al., 1992Go). In a standard reaction mixture containing 2.5 mg/ml protein and 300 µM MeP, the detection limit was approximately 0.2 nmol desmethyl parathion/min/mg protein.

hGSTA1 genotyping assay.
Human GSTA1 genotyping was performed as described previously with slight modifications (Coles et al., 2001Go). The hGSTA1/A2 forward primer AF4 (5'-tgt tga ttg ttt gcc tga aat t-3') and hGSTA1 gene-specific reverse primer AR1 (5'-agg acg gtg aca gcg ttt aac-3') were generously provided by Dr. Brian Coles (NCTR, Jefferson, AR). Control DNA standards representing the various genotypes were available in our laboratory. An approximately 480 bp DNA fragment was amplified using 0.2 µM each of the primers AF4 and AR1. Reactions (50 µl) contained approximately 0.1 mg DNA, 0.2 mM of each dNTP, 5 units AmpliTaq DNA polymerase (Applied Biosystems), 1.5 mM MgCl2 and 1x PCR buffer as supplied with the enzyme (Applied Biosystems). Amplification was started at 95°C followed by 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 62°C and 1 min extension at 72°C. A final 7 min extension at 72°C was performed before cooling the reactions to 4°C. The approximately 480 bp amplification product was digested with Ear 1 and electrophoresed in a 1.5% agarose gel.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dealkylation of MeP by Hepatic Cytosolic Fractions
Pooled rodent and individual human hepatic cytosolic fractions were examined for their enzymatic capacity to dealkylate the parent compound MeP (300 µM final MeP concentration) (Fig. 1). Rat liver cytosol showed the highest activity (2.36 nmol/min/mg respectively) followed by mice (1.76 nmol/min/mg) and human samples, which exhibited the lowest mean activity (0.70 nmol/min/mg) (Fig. 2).



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 2. Hepatic cytosolic fraction desmethylation of MeP. Reactions were performed in the presence (1 mM) and in the absence of exogenous reduced glutathione (GSH). The mean and standard deviation of at least two trials performed in triplicate are displayed. BDL = Below the Limit of Detection (0.2 pmol desmethylparathion/min/mg protein)

 
To rule out the possibility that a glutathione-independent biotransformation pathway (i.e., a GST-independent pathway) was predominantly responsible for the O-demethylation of MeP, we included control reactions in which no reduced glutathione was added (Fig. 2). In the absence of exogenous reduced glutathione, the rat, mouse, and human samples exhibited 26, 56, and less than 1% activity, respectively, relative to activity determined in the presence of an additional 1 mM reduced glutathione. The most likely explanation for the substantial activity in the absence of added glutathione is that the hepatic cytosolic fraction still contained sufficient endogenous reduced glutathione to allow a GST-mediated reaction to proceed. This hypothesis is supported by the observation that freshly prepared rodent liver cytosolic fractions showed higher MeP activity without addition of reduced glutathione than did older fractions in which the endogenous glutathione had likely been oxidized to a greater extent. In the presence of added glutathione, MeP O-demethylation activity was virtually the same in the freshly prepared rodent hepatic cytosolic fractions versus the cytosolic fractions that had been previously stored in the freezer (results not shown). The human samples, which showed no activity in the absence of added glutathione, had been stored for several years.

MeP O-Demethylation Activities of Cytosolic Fractions Isolated from Human Livers with Known hGSTM1 and hGSTT1 Genotypes
Activities of the ten cytosolic fractions isolated from individual human livers (livers 106, 108, 109, 110, 113, 118, 124, 126, 131, and 134) showed an almost four-fold interindividual variation in GST-mediated O-dealkylation of MeP, ranging from 0.26 to 0.99 nmol/min/mg. Figure 3 shows MeP activities of individual human samples arranged in groups according to the four possible GSTM1 and GSTT1 genotype combinations. Interestingly, the two samples with the highest activities possess the M1+/ T1–and M1–/ T1+ genotype combinations, suggesting that neither hGSTM1-1 nor hGSTT1-1 are responsible for the majority of MeP activity exhibited by these hepatic cytosolic fractions. Relatively high activity exists both in the absence of hGSTM1-1 and in the absence of hGSTT1-1 protein. In addition, the two samples with both the GSTM1 and GSTT1 genes present (M1+T1+ genotypes) displayed slightly lower activities than the two M1–T1–samples. These findings indicate that no correlation exists between the GSTM1 and/or GSTT1 genotypes and MeP O-demethylation activities of the cytosolic fractions.



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 3. Grouping the ten individual human liver cytosolic samples according to genotype does not reveal a predominant role for hGSTM1-1 or hGSTT1-1 in MeP biotransformation. The mean and standard error of at least two trials performed in triplicate are displayed.

 
To ensure that the samples contained viable GSTs, we measured activity of each of the ten human cytosolic fractions toward the non-isoform-specific GST substrate, CDNB. CDNB activities of the ten samples fell within the expected activity range (0.12 µmol/min/mg [SE:0.01] to 1.94 µmol/min/mg [SE: 0.099]; individual data not shown) (Slone et al., 1995Go).

Enzymatic Capacity of Purified Recombinant GSTs to Dealkylate MeP
In addition to the cytosolic fractions described above, we analyzed a number of purified recombinant cytosolic GSTs belonging to the alpha (human GSTs A1-1 and A2-2, mouse GSTA3-3, rat GSTA5-5), mu (human GSTs M1-1, M2-2, M3-3, M4-4), pi (human GSTP1-1, mouse GSTs P1-1, P2-2), and theta (human GSTT1-1) classes for their capacity to biotransform MeP (Fig. 4). Surprisingly, hGSTT1-1 possessed the highest MeP activity (546 nmol/min/mg), followed by hGSTA1-1 (65.0 nmol/min/mg) and rGSTA5-5 (53.2 nmol/min/mg). In contrast to hGSTA1-1, which shares 95% sequence identity with hGSTA2-2, the MeP O-demethylation activity of the latter was below the detection limit. Human GSTM1-1 also exhibited some capacity to biotransform MeP (10.8 nmol/min/mg), while human and murine pi class GSTs had relatively low activity toward MeP (hGSTP1-1, 2.84 nmol/min/mg, and mGST P1-1, 3.14 nmol/min/mg).



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 4. Purified recombinant GSTs were tested for O-demethylation of MeP at a concentration of 1 mM glutathione. The mean and standard error of at least two trials performed in triplicate are displayed. BDL = Below the Limit of Detection (0.2 pmol desmethylparathion/min/mg protein).

 
All of these specific activities toward MeP were determined at a final concentration of 1 mM glutathione. The Km of alpha, mu, and pi class GSTs for glutathione is approximately 0.1 mM; therefore, the 1 mM reduced glutathione concentration used in the assay was saturating for these enzymes (Meyer, 1993Go). However, Meyer (1993)Go reported that the Km for glutathione of theta class GSTs is approximately 50 times higher (?5 mM). In addition, physiological glutathione concentrations in the liver and other tissues have been estimated to be approximately 5 to 10 mM (Kosower and Kosower, 1978Go). Therefore, we measured hGSTT1-1 MeP activity at those glutathione concentrations. At 5 and 10 mM glutathione, hGSTT1-1 exhibited MeP activities of 705 and 874 nmol/min/mg respectively (Table 3).


View this table:
[in this window]
[in a new window]
 
TABLE 3 Effect of Glutathione Concentration on Desmethylation Activity

 
MeP O-demethylation activity of individual human hepatic samples was also initially measured at 1 mM glutathione. To ensure that we had not underestimated the contribution of hGSTT1-1 to overall MeP activity of human hepatic cytosol, six cytosolic samples (livers 101, 102, 103, 104, 105, 108), each derived from subjects with hGSTM1+hGSTT1+ genotypes, were pooled and assayed for O-demethylation of MeP at 1 versus 5 mM glutathione. We found no significant difference in MeP activity between assays carried out with 1 versus 5 mM glutathione according to Student's test (p < 0.05) (Table 3).

Since hGSTT1-1 and hGSTA1-1 exhibited the highest activities toward MeP, we attempted to conduct kinetic analyses. However, due to limited solubility, we were not able to reach sufficiently high concentrations of MeP to complete kinetic analysis. At 60, 100, and 300 µM MeP, hGSTA1-1 (at 1 mM glutathione) exhibited MeP O-demethylation activities of 24.4, 39.4, and 65.0 nmol/min/mg, whereas hGSTT1-1 (at 10 mM glutathione) biotransformed MeP at rates of 156, 254, and 874 nmol/min/mg, respectively.

Correlation of Human Liver Cytosolic MeP O-Demethylation Activities with the hGSTA1 Genotype
Recently, Coles and colleagues identified a polymorphism in the proximal promoter region of the human GSTA1 gene (Coles et al., 2001Go) and named the corresponding allelic variant hGSTA1*B. In addition, they reported that mean levels of hepatic hGSTA1-1 protein expression correlated significantly with genotype (hGSTA1*A > hGSTA1*B). Therefore, we genotyped the ten human liver samples used in this study for hGSTA1*A and hGSTA1*B. Five samples (131, 126, 124, 110, 108) were heterozygous (hGSTA1*AhGSTA1*B), and three samples (118, 109, 106) were homozygous for hGSTA1*A. One sample (113) was homozygous for hGSTA1*B, and the genotype of one sample (134) could not be identified clearly (Fig. 5). Upon comparison of the mean desmethylation activities corresponding to the three hGSTA1 genotypes using ANOVA (p < 0.05), we found no significant differences between groups. Thus, it is unlikely that a correlation exists between hGSTA1 genotype and MeP activity.



View larger version (59K):
[in this window]
[in a new window]
 
FIG. 5. (a) Ten human liver samples were assayed for hGSTA1*A/*B genotype. *A, *B and het (heterozygous *A/*B) were control DNA samples for the three genotypes. (b) O-demethylation activities toward MeP were compared to hGSTA1 genotype.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
These studies demonstrate that GSTs expressed in human liver have the capacity to O-dealkylate MeP, although the overall hepatic activity is substantially lower than that seen in rodent liver. Using cDNA-expressed human GSTs, there were striking differences in the metabolic capacity of different human GSTs to dealkylate MeP.

Of particular interest to us was the relative importance of hGSTM1-1 and hGSTT1-1, for which gene deletions are common in the human population. The lack of any significant correlation between the hGSTM1 and/or hGSTT1 genotypes and MeP dealkylation activities of hepatic human cytosolic fractions suggests that these GSTs do not contribute significantly to total hepatic GST activity toward MeP (Fig. 3). However, caution needs to be exercised in drawing the general conclusion that hGSTM1-1 and hGSTT1-1 play only minor, if any, roles in O-dealkylation of MeP based upon correlational data alone. While it is clear that individuals with hGSTM1 or hGSTT1 null genotypes lack corresponding hGSTM1-1 or hGSTT1-1 protein, it cannot be assumed that all individuals who possess one or two functional alleles for hGSTM1 and/or hGSTT1 express similar amounts of hGSTM1-1 and/or hGSTT1-1 enzymes in hepatic tissue.

One factor that influences enzyme expression levels is the number of alleles present. The assay we used to genotype the ten human samples (Chen et al., 1996Go) does not distinguish homozygous from heterozygous M1 and/or T1 positive samples and, therefore, does not provide information with respect to the number of GSTM1*A/B or GSTT1*A alleles. In addition, regulation of GST expression is subject to a complex set of developmental, sex, and tissue-specific factors, as well as environmental and dietary parameters (for review see Hayes and Pulford, 1995Go). Therefore, it is possible that human livers with a hGSTM1 and/or hGSTT1 positive genotype express varying levels of the encoded proteins. In such a scenario, it is possible that a correlation between hGSTM1 and hGSTT1 genotypes and MeP activity could be masked by other variables that influence the level of protein expression. This is particularly true if the sample size is small, as is the case in our study.

To clarify this issue, we determined MeP activities of several purified recombinant GSTs, including hGSTs M1-1 and T1-1 (Fig. 4). Surprisingly, purified recombinant hGSTT1-1 exhibited the highest specific activity toward MeP. This finding seems contradictory to the lack of correlation between MeP activities displayed by total hepatic cytosolic samples and their corresponding hGSTT1 genotypes. However, if hepatic hGSTT1-1 is expressed at very low levels compared to a GST that possesses significant MeP activity and also is expressed at much higher levels in human liver, this discrepancy would be explained.

When 1 mM glutathione was used in the assay, hGSTA1-1 biotransformed MeP at a rate of 65.0 nmol/min/mg, whereas hGSTT1-1 displayed approximately eight-fold higher activity (545.8 nmol/min/mg) (Fig. 4). Furthermore, given that the Km for glutathione for hGSTT1-1 is approximately 5 mM (versus 0.1 mM for hGSTA1-1 [Meyer, 1993Go]) and that hepatic concentrations of glutathione are estimated to be 5–10 mM (Kosower and Kosower, 1978Go), a more accurate reflection of the MeP activity of hGSTT1-1 at physiological glutathione concentrations is in the range of 704.8 (measured at 5 mM glutathione) to 874.1 nmol/min/mg (measured at 10 mM glutathione) (Table 3). These activities are approximately 10 to 13-fold higher than that of hGSTA1-1. However, it has been estimated that hGSTA1-1 and hGSTT1-1 account for approximately 2–3% and 0.003% of total hepatic cytosolic protein, respectively (Meyer et al., 1991Go; Rowe et al., 1997Go; van Ommen et al., 1990Go). Thus, hGSTA1-1 is expressed at approximately 670- to 1000-fold higher levels compared to hGSTT1-1. Taking into account both the differences in O-demethylation activities and hepatic expression levels, we estimate that hGSTA1-1 is responsible for more than 97% of total MeP activity afforded by hGSTA1-1 and hGSTT1-1 combined.

Based on those numbers, one would estimate that MeP activity of hepatic cytosolic fractions would be in the range of 1.3 to 1.9 nmol/min/mg, assuming no other GST contributed to that activity. This estimate is in reasonable agreement with the MeP O-demethylation activity we measured in the ten human cytosolic fractions at a concentration of 1 mM glutathione (range of 0.26–0.99 nmol/min/mg; Fig. 3), suggesting that hGSTA1-1 is indeed likely responsible for the majority of MeP activity seen in human liver.

As previously stated, hGSTT1-1 has a relatively high Km for glutathione (?5 mM). To ensure that we had not underestimated the contribution of hGSTT1-1 to overall MeP activity in hepatic cytosolic samples by including only 1 mM glutathione, we pooled six samples that were hGSTT1/hGSTM1 positive and measured MeP activity at both 1 mM and 5 mM glutathione. MeP activity of the pooled cytosolic fractions was not significantly different between those two conditions (Student's t-test; p < 0.05) (Table 3). This is a further indication that, in contrast to hGSTA1-1, hGSTT1-1 does not play a major role in MeP O-demethylation activity in the liver. Importantly, however, hGSTT1-1 is expressed in brain and skeletal muscle, which are major target organs for MeP toxicity (Sherratt et al., 1997Go). Therefore, although hGSTT1-1 does not appear to contribute significantly to hepatic detoxification of MeP in vitro, it could be important in target-tissue biotransformation of MeP. However, whether this is the case in vivo remains to be established.

Interestingly, our finding that hGSTT1-1 biotransforms MeP is consistent with results generated using OP-resistant houseflies. In the resistant insects, the overexpressed GST belongs to the theta class (Zhou and Syvanen, 1997Go). Recently, a theta class GST was cloned from the housefly; the corresponding purified theta class enzyme biotransformed MeP (Wei et al., 2001Go).

Coles and coworkers (2001)Go reported a polymorphism in the proximal promoter region of hGSTA1-1 that correlated with hepatic expression of hGSTA1-1 protein. We genotyped the ten livers used in this study (Fig. 5), but found no correlation between hGSTA1*A/hGSTA1*B genotypes and MeP dealkylation activities. The lack of correlation could be due to the small sample size used in this study; five of our samples were hGSTA1*A/hGSTA1*B heterozygous, three were hGSTA1*A homozygous, and only one sample was hGSTA1*B homozygous (the genotype of one sample could not be clearly identified). While Coles et al. found a correlation between hGSTA1*A/*B genotypes and hepatic hGSTA1-1 protein levels, they also noted much variation of expression among individuals with the same genotype. Several individuals who were homozygous for hGSTA1*A and hGSTA1*B expressed virtually the same amounts of hGSTA1-1 protein in their livers (Coles et al., 2001Go). The hGSTA1*B allelic variant was also identified in an independent study (Bredschneider et al., 2002Go). In contrast to Coles et al., Bredschneider and colleagues did not find a correlation between the hGSTA1*A/hGSTA1*B genotypes and hGSTA1-1 protein levels. It is possible that additional, as yet unidentified, factors may also modulate hepatic expression levels of hGSTA1-1 and therefore undermine the effects of the polymorphism. These inductive or suppressive factors may include variation in diet, therapeutic drug use or disease.

Whether GST-mediated biotransformation plays a significant physiological role in MeP disposition in vivo remains to be established. Certain lines of evidence suggest that GST biotransformation of MeP occurs in vivo in rodents. For example, upon pretreatment of mice with diethyl maleate (DEM), glutathione depletion occurs in every tissue examined, including the brain (Costa and Murphy, 1984Go). This pretreatment also potentiated MeP toxicity (Mirer et al., 1977Go; Sultatos and Woods, 1988Go). In contrast, several other studies suggest that GST-mediated biotransformation of MeP does not occur in vivo. Rat livers perfused in situ did not demonstrate GST-mediated biotransformation (Zhang and Sultatos, 1991Go), and in mouse liver preparations that included microsomes, MeP appeared to be sequestered in the lipid phase and GSH-dependent biotransformation was decreased (Huang and Sultatos, 1993Go). In addition, following glutathione depletion by buthionine sulfoximine or acetaminophen in mice, no potentiation of MeP toxicity occurred (Costa and Murphy, 1984Go; Sultatos and Woods, 1988Go). However, in the case of acetaminophen, glutathione depletion did not occur in the brain, a major target organ for MeP toxicity (Costa and Murphy, 1984Go). Furthermore, these could be species-specific observations.

We have shown that human GSTs A1-1 and T1-1 O-dealkylate MeP in vitro. However, whether GSTs play a role in the detoxification of MeP in humans in vivo remains to be determined.


    ACKNOWLEDGMENTS
 
The authors thank Patricia Stapleton for her excellent technical assistance for RT-PCR and genotyping assays. This work was supported in part by NIEHS Center Grant P30-ES07033, R01 ES05780 and NIA Training Grant AG-00057. The UW Human Liver Tissue bank is supported in part by NIH grant PO1 GM32165.


    NOTES
 
1 TKB and ELA contributed equally to this study. Back

2 Current address: Department of Molecular Carcinogenesis, MD Anderson Cancer Center, Smithville, TX 78957. Back

3 To whom correspondence should be addressed. Fax: (206) 685-4696. E-mail: deaton{at}u.washington.edu


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abel, E. A., Opp, S. M., Verlinde, C. L., Bammler, T. K., and Eaton, D. L. (in press). Characterization of atrazine biotransformation by human Glutathione S-transferases.

Anderson, P. N., Eaton, D. L., and Murphy, S. D. (1992). Comparative metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes. Fundam. Appl. Toxicol. 18(2), 221–226.[ISI][Medline]

Bammler, T. K., Driessen, H., Finnstrom, N., and Wolf, C. R. (1995). Amino acid differences at positions 10, 11, and 104 explain the profound catalytic differences between two murine pi-class glutathione S-transferases. Biochemistry 34(28), 9000–9008.[ISI][Medline]

Benke, G. M., Cheever, K. L., Mirer, F. E., and Murphy, S. D. (1974). Comparative toxicity, anticholinesterase action and metabolism of methyl parathion and parathion in sunfish and mice. Toxicol. Appl. Pharmacol. 28(1), 97–109.[ISI][Medline]

Benke, G. M., and Murphy, S. D. (1975). The influence of age on the toxicity and metabolism of methyl parathion and parathion in male and female rats. Toxicol. Appl. Pharmacol. 31(2), 254–269.[ISI][Medline]

Board, P. G., Chelvanayagam, G., Jermiin, L. S., Tetlow, N., Tzeng, H. F., Anders, M. W., and Blackburn, A. C. (2001). Identification of novel glutathione transferases and polymorphic variants by expressed sequence tag database analysis. Drug Metab. Dispos. 29(4 Pt. 2), 544–547.[Abstract/Free Full Text]

Board, P. G., Coggan, M., Chelvanayagam, G., Easteal, S., Jermiin, L. S., Schulte, G. K., Danley, D. E., Hoth, L. R., Griffor, M. C., Kamath, A. V., Rosner, M. H., Chrunyk, B. A., Perregaux, D. E., Gabel, C. A., Geoghegan, K. F., and Pandit, J. (2000). Identification, characterization, and crystal structure of the Omega class glutathione transferases. J. Biol. Chem. 275(32), 24798–24806.[Abstract/Free Full Text]

Bredschneider, M., Klein, K., Murdter, T. E., Marx, C., Eichelbaum, M., Nussler, A. K., Neuhaus, P., Zanger, U. M., and Schwab, M. (2002). Genetic polymorphisms of glutathione S-transferase A1, the major glutathione S-transferase in human liver: Consequences for enzyme expression and busulfan conjugation. Clin. Pharmacol. Ther. 71(6), 479–487.[CrossRef][ISI][Medline]

Brown, S. K., Ames, R. G., and Mengle, D. C. (1989). Occupational illnesses from cholinesterase-inhibiting pesticides among agricultural applicators in California, 1982–1985. Arch. Environ. Health 44(1), 34–39.[ISI][Medline]

Butler, A. M., and Murray, M. (1997). Biotransformation of parathion in human liver: participation of CYP3A4 and its inactivation during microsomal parathion oxidation. J. Pharmacol. Exp. Ther. 280(2), 966–973.[Abstract/Free Full Text]

Chen, H., Sandler, D. P., Taylor, J. A., Shore, D. L., Liu, E., Bloomfield, C. D., and Bell, D. A. (1996). Increased risk for myelodysplastic syndromes in individuals with glutathione transferase theta 1 (GSTT1) gene defect. Lancet 347(8997), 295–297.[ISI][Medline]

Clark, A. G., Smith, J. N., and Speir, T. W. (1973). Cross specificity in some vertebrate and insect glutathione- transferases with methyl parathion (dimethyl p-nitrophenyl phosphorothionate), 1-chloro-2,4-dinitro-benzene and s-crotonyl-N- acetylcysteamine as substrates. Biochem. J. 135(3), 385–392.[ISI][Medline]

Coles, B. F., Morel, F., Rauch, C., Huber, W. W., Yang, M., Teitel, C. H., Green, B., Lang, N. P., and Kadlubar, F. F. (2001). Effect of polymorphism in the human glutathione S-transferase A1 promoter on hepatic GSTA1 and GSTA2 expression. Pharmacogenetics 11(8), 663–669.[CrossRef][ISI][Medline]

Costa, L. G., and Murphy, S. D. (1984). Interaction between acetaminophen and organophosphates in mice. Res. Commun. Chem. Pathol. Pharmacol. 44(3), 389–400.[ISI][Medline]

Eaton, D. L., and Bammler, T. K. (1999). Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol. Sci. 49(2), 156–164.[Free Full Text]

Fukami, J. (1980). Metabolism of several insecticides by glutathion S-transferase. Pharmacol. Ther. 10(3), 473–514.[CrossRef][ISI][Medline]

Habig, W. H., and Jakoby, W. B. (1981). Assays for differentiation of glutathione S-transferases. Methods Enzymol. 77, 398–405.[Medline]

Harris, J. M., Meyer, D. J., Coles, B., and Ketterer, B. (1991). A novel glutathione transferase (13–13) isolated from the matrix of rat liver mitochondria having structural similarity to class theta enzymes. Biochem. J. 278(Pt 1), 137–141.[ISI][Medline]

Hayes, J. D., and Pulford, D. J. (1995). The glutathione S-transferase supergene family: Regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30(6), 445–600.[Abstract]

Huang, Y. S., and Sultatos, L. G. (1993). Glutathione-dependent biotransformation of methyl parathion by mouse liver in vitro. Toxicol. Lett. 68(3), 275–284.[CrossRef][ISI][Medline]

Kosower, N. S., and Kosower, E. M. (1978). The glutathione status of cells. Int. Rev. Cytol. 54, 109–160.[Medline]

Leveridge, Y. R. (1998). Pesticide poisoning in Costa Rica during 1996. Vet. Hum. Toxicol. 40(1), 42–44.[ISI][Medline]

Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M. K., Warholm, M., and Jornvall, H. (1985). Identification of three classes of cytosolic glutathione transferase common to several mammalian species: Correlation between structural data and enzymatic properties. Proc. Natl. Acad. Sci. U.S.A. 82(21), 7202–7206.[Abstract]

Mannervik, B., Awasthi, Y. C., Board, P. G., Hayes, J. D., Di Ilio, C., Ketterer, B., Listowsky, I., Morgenstern, R., Muramatsu, M., Pearson, W. R., et al. (1992). Nomenclature for human glutathione transferases. Biochem. J. 282(Pt 1), 305–306.[ISI][Medline]

Meyer, D. J. (1993). Significance of an unusually low Km for glutathione in glutathione transferases of the alpha, mu and pi classes. Xenobiotica 23(8), 823–834.[ISI][Medline]

Meyer, D. J., Coles, B., Pemble, S. E., Gilmore, K. S., Fraser, G. M., and Ketterer, B. (1991). Theta, a new class of glutathione transferases purified from rat and man. Biochem. J. 274(Pt 2), 409–414.[ISI][Medline]

Meyer, D. J., and Thomas, M. (1995). Characterization of rat spleen prostaglandin H D-isomerase as a sigma- class GSH transferase. Biochem. J. 311(Pt 3), 739–742.[ISI][Medline]

Mirer, F. E., Levine, B. S., and Murphy, S. D. (1977). Parathion and methyl parathion toxicity and metabolism in piperonyl butoxide and diethyl maleate pretreated mice. Chem. Biol. Interact. 17(1), 99–112.[CrossRef][ISI][Medline]

Namba, T. (1971). Cholinesterase inhibition by organophosphorus compounds and its clinical effects. Bull. World Health Organ. 44(1), 289–307.[ISI][Medline]

Pemble, S. E., Wardle, A. F., and Taylor, J. B. (1996). Glutathione S-transferase class Kappa: Characterization by the cloning of rat mitochondrial GST and identification of a human homologue. Biochem. J. 319(Pt 3), 749–754.[ISI][Medline]

Radulovic, L. L., Kulkarni, A. P., and Dauterman, W. C. (1987). Biotransformation of methyl parathion by human fetal liver glutathione S-transferases: An in vitro study. Xenobiotica 17(1), 105–114.[ISI][Medline]

Radulovic, L. L., LaFerla, J. J., and Kulkarni, A. P. (1986). Human placental glutathione S-transferase-mediated metabolism of methyl parathion. Biochem. Pharmacol. 35(20), 3473–3480.[CrossRef][ISI][Medline]

Rowe, J. D., Nieves, E., and Listowsky, I. (1997). Subunit diversity and tissue distribution of human glutathione S- transferases: Interpretations based on electrospray ionization-MS and peptide sequence-specific antisera. Biochem. J. 325(Pt 2), 481–486.[ISI][Medline]

Sherratt, P. J., Pulford, D. J., Harrison, D. J., Green, T., and Hayes, J. D. (1997). Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse. Comparison of the tissue distribution of GST T1-1 with that of classes Alpha, Mu and Pi GST in human. Biochem J 326(Pt 3), 837–846.[ISI][Medline]

Slone, D. H., Gallagher, E. P., Ramsdell, H. S., Rettie, A. E., Stapleton, P. L., Berlad, L. G., and Eaton, D. L. (1995). Human variability in hepatic glutathione S-transferase-mediated conjugation of aflatoxin B1-epoxide and other substrates. Pharmacogenetics 5(4), 224–233.[ISI][Medline]

Sultatos, L. G., and Woods, L. (1988). The role of glutathione in the detoxification of the insecticides methyl parathion and azinphos-methyl in the mouse. Toxicol. Appl. Pharmacol. 96(1), 168–174.[ISI][Medline]

Syvanen, M., Zhou, Z., Wharton, J., Goldsbury, C., and Clark, A. (1996). Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly. J. Mol. Evol. 43(3), 236–240.[ISI][Medline]

van Ommen, B., Bogaards, J. J., Peters, W. H., Blaauboer, B., and van Bladeren, P. J. (1990). Quantification of human hepatic glutathione S-transferases. Biochem. J. 269(3), 609–613.[ISI][Medline]

Wei, S. H., Clark, A. G., and Syvanen, M. (2001). Identification and cloning of a key insecticide-metabolizing glutathione S-transferase (MdGST-6A) from a hyper insecticide-resistant strain of the housefly Musca domestica. Insect Biochem. Mol. Biol. 31(12), 1145–1153.[CrossRef][ISI][Medline]

Zhang, H. X., and Sultatos, L. G. (1991). Biotransformation of the organophosphorus insecticides parathion and methyl parathion in male and female rat livers perfused in situ. Drug Metab. Dispos. 19(2), 473–477.[Abstract]

Zhou, Z. H., and Syvanen, M. (1997). A complex glutathione transferase gene family in the housefly Musca domestica. Mol. Gen. Genet. 256(2), 187–194.[CrossRef][ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
79/2/224    most recent
kfh118v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (5)
Disclaimer
Request Permissions
Google Scholar
Articles by Abel, E. L.
Articles by Eaton, D. L.
PubMed
PubMed Citation
Articles by Abel, E. L.
Articles by Eaton, D. L.