* Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington; University of Great Falls, Great Falls, Montana; and
Department of Biochemistry, University of Washington, Seattle, Washington
Received March 1, 2004; accepted April 13, 2004
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ABSTRACT |
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Key Words: glutathione; transferase; atrazine; human; biotransformation; conjugation.
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INTRODUCTION |
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The metabolic pathways of atrazine in humans have not been fully characterized. In rodents the dominant Phase-I metabolic reaction is cytochrome P450-mediated N-dealkylation (Fig. 1) as demonstrated by urine analysis and liver fraction studies (Hanioka et al., 1999a,b
; Lang et al., 1996
). Phase II biotransformation of atrazine may also occur: several studies demonstrated GSH conjugation of the parent compounds by rat and mouse liver fractions (Egaas et al., 1993
; Timchalk et al., 1990
). In mouse liver, GSH conjugation is likely mediated by a Pi class glutathione S-transferase (GST) (Egaas et al., 1995a
).
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The GSTs are a multigene family of detoxification enzymes that biotransform a wide variety of endogenous and exogenous electrophilic substrates (for review see Hayes and Pulford, 1995). Typically these enzymes catalyze detoxification of substrates by conjugation to GSH. The human GST (hGST) family is composed of at least 8 classes (alpha, A; kappa, K; mu, M; omega, O; Pi, P; sigma, S; theta, T; and zeta, Z) with multiple subfamilies per class (Board et al., 2001
; Eaton and Bammler, 1999
). Although the spectrum of substrates biotransformed by each hGST isoform and the degree to which catalysis occurs is unique, the spectrums overlap in most cases. Several of the human genes for GSTs are known to be polymorphic in the population. For example, 4352% and 1520% of Caucasians are homozygous for gene deletions in the hGSTM1 and hGSTT1 genes, respectively (Eaton and Bammler, 1999
). In addition, functionally relevant single nucleotide polymorphisms (SNP) are found in the coding region of hGSTP1 (Ali-Osman et al., 1997
; Harries et al., 1997
; Watson et al., 1998
).
The purpose of this investigation was to determine the specific role of individual human GSTs in the biotransformation of atrazine. In addition, we have further explored the involvement of GSTs in atrazine biotransformation in mice. To this end, seven recombinant cytosolic hGST isoforms (hGSTM1-1, hGSTM2-2, hGSTM3-3, hGSTM4-4, hGSTA1-1, hGSTP1-1, and his-hGSTT1-1) and two mouse GST (mGST) isoforms (mGSTP1-1 and mGSTP2-2) were expressed and purified. These proteins, along with male CD1 mouse liver and human liver cytosol, have been tested for catalysis of GSH conjugation of atrazine. A secondary purpose of this communication is to relate the generation of an effective hGSTP1-1 expression construct and purification protocol that does not require the use of a histidine tag.
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MATERIALS AND METHODS |
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Liver cytosol preparation. Human liver samples were obtained from the University of Washington Liver Bank (UW School of Pharmacy; Paine et al., 1997). Characteristics of the liver donors are described in Table 2. After excision the liver samples were immediately snap frozen in liquid nitrogen.
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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. Each sample was centrifuged at 10,000 x g for 10 min; the pellet was discarded and the supernatant 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.
Protein Assays
Protein concentrations were approximated 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, and spectrophotometric measurements were made at 590 nm using a Molecular Devices UV Max 96-well plate reader.
GST expression and purification. hGSTA1-1, hGSTM1-1, hGSTM2-2, hGSTM3-3, hGSTM4-4, mGSTP1-1, and mGSTP2-2 were expressed and purified as previously described (Bammler et al., 1995). The authors gratefully acknowledge Dr. Phillip Board (John Curtis School of Medical Research, Australian National University, Canberra ACT 2601, Australia) for providing cDNA expression constructs for hGSTs M1, M2, M3, and M4. Histidine-tagged 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., 1997
).
An hGSTP1 expression construct was generated that allowed purification of native hGSTP1-1. Briefly, the cDNA for hGSTP1 was generated by PCR of an existing hGSTP1 cDNA-containing vector (gift from Dr. William Atkins, University of Washington); PCR amplification primers were used that allowed for addition of NdeI and BamHI sites to the 5' and 3' ends of the hGSTP1 cDNA, respectively (forward primer: 5'GAG AGA GGA GCA TAT GCC GCC CTA CAC CGT GGT C3' and reverse primer: 5'GAG GAG GGA TCC TCA CTG TTT CCC GTT GCC ATT3'). 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 INVF' cells. Following BamHI digestion for construct linearization, an NdeI partial digestion allowed for collection of the full-length hGSTP1 cDNA with NdeI and BamHI sticky ends for ligation into the pET17b expression vector (Novagen, Madison, WI).
Four genotypes for hGSTP1 have been described: hGSTP1*A, hGSTP1*B, hGSTP1*C, and hGSTP1*D (Ali-Osman et al., 1997; Harries et al., 1997
; Watson et al., 1998
). The hGSTP1 expression construct described above corresponded to the hGSTP1*A genotype. Expression constructs for the remaining 3 genotypes were generated using the hGSTP1*A construct and the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The *B and *D alleles were created using the primers (5'CCT CCG CTG CAA ATA CGT CTC CCT CAT CTA C3', 5'GTA GAT GAG GGA GAC GTA TTT GCA GCG GAG G3'), (5'CTA CAC CAA CTA TGA GGT GGG CAA GGA TGA C3', and 5'GTC ATC CTT GCC CAC CTC ATA GTT GGT GTA G3'), respectively (MWG Biotech, High Point, NC). Subsequently, the *C allele was created using the *B primers with the *D construct.
The hGSTP1 polymorphic variant plasmid sequences were confirmed via sequencing. The Big Dye sequencing protocol (ABI, Foster City, CA) was used and was followed by removal of unincorporated dye terminator using Centri-Sep Columns (Princeton Separations, Adelphia, NJ). The following primers were used, which targeted the T7 promoter of pET17b, the T7 terminator of pET17b, the 322340 bp region of hGSTP1, and the 233252 bp region of hGSTP1, respectively: 5'TAA TAC GAC TCA CTA TAG GG3', 5'GCT AGT TAT TGC TCA GCG G3', 5'TAT TTG CAG CGG AGG TCC T3', and 5'GCT CTA TGG GAA GGA CCA GC3'. Following purification, sequencing was performed using an ABI 377 automated sequencer (ABI, Foster City, CA). hGSTP1-1 was then expressed as described previously (Bammler et al., 1995).
Enzyme Assays
Atrazine. The procedure for monitoring GSH-dependent biotransformation of atrazine is a modification of that described by Guddewar and Dauterman (1979). Briefly, the assay was performed as follows: The incubation mixture consisted of 3.0 mM GSH, 30137 µM ring-labeled 14C-atrazine, 0.1 M potassium phosphate buffer (pH 7.4) and the appropriate amount of enzyme. The reaction was performed in a final volume of 1.0 ml and incubated for 3060 min at 37°C. The reaction was stopped by the addition of 1.0 ml chloroform. After vortexing, the two phases were thoroughly separated by 5 min centrifugation at 2500 x g. The radioactivity from aliquots of both phases was determined by liquid scintillation counting until it was established that a vast majority of the radioactivity could be accounted for between the two phases. Further testing included scintillation counting of only the aqueous phase, which reflected the proportion of atrazine that had been GSH-conjugated. All specific activity values were generated within the linear range. Spontaneous reaction rate (no GST control rate) was subtracted during calculation of the specific activity values.
CDNB. The CDNB assay was performed as described previously (Habig and Jakoby, 1981). Briefly, 1 mM CDNB in ethanol, 1 mM GSH, and purified recombinant GST proteins were incubated in 100 mM sodium phosphate buffer at pH 6.5. The final ethanol concentration was 1.67%. CDNB was added to start the reaction. After mixing, the formation of GSH conjugate was monitored at 340 nm at 30°C; the reaction had to be linear for at least one min to be included in the calculation of the specific activity. The measurements were made using a Shimadzu UV-160 spectrophotometer.
GSTM1 and GSTT1 genotyping. The human liver samples had been previously assayed for hGSTM1 and hGSTT1 genotype by polymerase chain reaction methods (Chen et al., 1996).
Molecular Docking Studies
All docking studies were carried out with the molecular modeling program QXP/FLO (McMartin and Bohacek, 1997). Of the various crystal structures of hGSTP1-1 available, PDB 9GSS (Oakley et al., 1997
), which is in complex with S-hexylglutathione, was chosen on the basis of resolution. The binding site model included all protein residues within 10.0 Å of S-hexylglutathione. We first created a model of the docked transition state, also known as the "Sigma" complex. A model of atrazine was covalently attached to the glutathione moiety of S-hexylglutathione (after removal of the hexyl group), with sp3 geometry at the attachment carbon atom. Subsequently, this glutathione conjugate of atrazine was submitted to 300 cycles of an extensive Monte Carlo search procedure, during which the glutathione moiety was kept fixed in its observed pose in the binding site while the fragment consisting of the atrazine moiety and the glutathione cysteine side chain were allowed full conformational flexibility. The procedure was repeated with the ethyl and isopropyl groups of atrazine swapped, since the nucleophilic attack leading to the formation of the transition state may occur on either side of the triazine ring. To investigate the binding mode of the reaction product, a second docking study was carried out in which the chlorine atomic bond was broken, and the sp2 character of the attachment carbon was restored. Subsequently, the structure was energy minimized. With respect to the transition state, a rotation of the triazine ring toward Ile 105 was seen. This is reminiscent of the rotation observed between the hGSTP1-1 crystal structures with Meisenheimer complex (glutathione with 1,3,5-trinitrobenzene) and the product p-bromobenzyl-glutathione (Prade et al., 1997
).
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RESULTS |
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To identify GST proteins with activity toward atrazine, we tested our panel of purified recombinant human and mouse GSTs for GSH-dependent biotransformation of atrazine. As shown in Figure 2, two purified recombinant GST proteins demonstrated significant activity toward atrazine. Mouse GSTP1-1 and hGSTP1-1 conjugated atrazine at rates of 7.3-nmol AT-SG/min/mg protein and 7.1-nmol AT-SG/min/mg, respectively. Together with previously reported data (Egaas et al., 1995a; Egaas et al., 1993
), these results provide definitive evidence of GST Pi class-mediated catalysis of GSH conjugation of atrazine in mouse liver cytosol. Other hGST isoforms were capable of catalysis of GSH conjugation to atrazine; however, the rates were miniscule in comparison to hGSTP1-1. hGSTs A1-1, M1-1, and M2-2, which had on average 1.3%, 2.9%, and 1.9% of hGSTP1-1 activity, respectively. Although mGSTP2-2 is highly homologous to mGSTP1-1, mGSTP2-2 demonstrated much less conjugation of atrazine (0.02 nmol/min/mg vs. 7.14 nmol/min/mg). We also determined the activity of our preparation of mGSTP2-2 toward CDNB. Although low, activity was within the anticipated range (mean: 0.05 µmol/min/mg).
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Molecular docking of the transition state of hGSTP1-1 and the atrazine-glutathione conjugate shows that the atrazine moiety aromatic ring and alkyl groups make excellent use of the hydrophobic patches of the H-site, and the aromatic ring is stacked with relation to Tyr 109 (Fig. 3). The amino functions rest parallel on the surface, allowing accessibility for solvation by water. The chlorine atom is situated in a channel known to contain a number of water molecules. Interestingly, this channel also includes the side chain of Arg 14. Both the water molecules and the electropositive potential from the Arg-14 residue might assist the chlorine atom in becoming a leaving group, the second step in the nucleophilic, aromatic substitution reaction. Another residue that may help in stabilizing the developing negative charge of the leaving group is Tyr 109, which also points into the channel. Its phenol function is located only 3.4 Å away from the chlorine atom. The model for the transition state exhibits great similarity to the crystal structure of the Meisenheimer complex (Prade et al., 1997). There, parallel stacking between the aromatic ring and Tyr 109 is seen, and the hydrogen at the sp3 carbon (equivalent to the chlorine atom of atrazine) points roughly in the same direction (i.e., toward the channel). This similarity provides further support for the correctness of the docked model of the transition state complex of GSH-atrazine.
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In order to definitively determine whether the polymorphisms of hGSTP1 affect catalytic activity of the gene products, we expressed and purified hGSTP1*B, hGSTP1*C, and hGSTP1*D proteins in addition to the previously tested hGSTP1*A protein. The hGSTP1*B and hGSTP1*C proteins had altered activity toward the model GST substrate, CDNB, as anticipated and previously demonstrated (Fig. 4b) (Ali-Osman et al., 1997; Watson et al., 1998
). However, no significant differences in the specific activity toward atrazine were noted between the genotypes (Fig. 4a). Two atrazine concentrations were utilized in order to ensure that the ability to demonstrate altered catalytic activity was not dependent upon the substrate concentration tested.
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DISCUSSION |
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Two more potentially relevant residues remain: residues 11 and 105. Both residues are close to the atrazine-glutathione conjugate, especially in the transition state. The Ser11 in mGSTP2-2 (instead of Val) increases the hydrophilic character of the H-site but is not expected to make a dramatic difference in enzyme activity. Also, Ser11 occurs together with Pro12 in the mGSTP2 subunit protein. Because Pro12 appears detrimental to catalysis, the effect of Ser11 by itself is not relevant. The absence of a side chain at position 105 (Glycine) of mGSTP2-2 (as opposed to the Valine at position 105 in mGSTP1-1) deprives the H-site of a significant area of hydrophobic surface and would be expected to have an effect on activity, certainly with larger substrates.
In conclusion, molecular modeling and substrate docking suggest that the poor activity of mGSTP2-2 occurs because of the presence of proline rather than arginine in position 12; the loss of a hydrophobic side chain at position 105 in mGSTP2-2 may also contribute to the lack of activity of mGSTP2-2 toward atrazine. Similar conclusions were reached by Bammler and colleagues (1995) with respect to CDNB activity. Furthermore, a dramatic decrease in activity toward CDNB was demonstrated when Arg12 of hGSTP1-1 was replaced by proline (Bammler, et al., 1995
).
The mGSTP1-1 protein is likely responsible for most of the GSH-dependent metabolism of atrazine by mouse liver cytosol. GSTs are believed to account for up to 4.0% of total cytosolic protein in male DBA/2, C3H/He and C57BL6 mice; as much as 70% of total cytosolic GST protein is believed to be mGSTP1-1 protein (McLellan and Hayes, 1987). If similar percentage values are assumed to apply to male CD1 mouse liver cytosolic protein, then mGSTP1-1 protein would account for approximately 2.8% of cytosolic protein, and the anticipated specific activity for atrazine conjugation would be slightly more than 200 pmol/min/mg protein. This value is in reasonable agreement with the 282-pmol/min/mg protein value revealed in our study for male CD1 mouse liver cytosol catalysis of atrazine conjugation.
GST Pi class biotransformation of atrazine had been previously demonstrated only with liver fractionation studies and correlative evidence (Egaas et al., 1995a; Egaas et al., 1995b
; Egaas et al., 1993
). For example, most male mouse livers contain up to 10-fold higher amounts of GST pi protein as compared to female mouse livers (Hatayama et al., 1986
; McLellan and Hayes, 1987
); GSH conjugation of atrazine is easier to detect in male livers. Furthermore, strain-related differences in hepatic mGST pi content appear to be correlated with atrazine conjugation capacity (Egaas et al., 1995a
; Egaas et al., 1995b
). Rat liver, which does not express high levels of GST pi protein (Satoh et al., 1985
), shows little GSH conjugation of atrazine (Egaas et al., 1993
). Thus, GST pi class protein content is predictive of hepatic atrazine biotransformation.
Human GST-Mediated Conjugation of Atrazine
We have demonstrated for the first time that human GSTP1-1 mediates biotransformation of atrazine. Unlike the mouse, hGSTP1-1 is not highly expressed under normal conditions in human hepatocytes (for review, see Awasthi et al., 1994). Therefore, it is not surprising that human liver cytosolic fraction demonstrated only low-level GSH-dependent biotransformation of atrazine. Due to the lack of GSTpi class protein in rat or human liver, it is unlikely that GSH conjugation reactions would predominate following oral dosing. Although, direct comparison of previous studies of rodent versus human biotransformation is not possible since differing methodologies and routes of exposure have been employed, we predict based upon fundamental similarity in hepatic pi class protein expression that human hepatic clearance of atrazine is more similar to that of rats than that of male mice. Although hepatic GST biotransformation of atrazine would be expected to play only a minor role in atrazine clearance, hGST pi class protein is expressed in other relevant tissues such as skin and lung (Awasthi et al., 1994
; Singhal et al., 1993
). Extrahepatic (skin and lung) biotransformation of atrazine may be very important, as most occupational exposures are likely to occur via inhalation or dermal routes. In line with this idea, measurable levels of atrazine mercapturate metabolites have been detected in human urine following dermal exposure (Lucas et al., 1993
). Furthermore, human placenta contains significant amounts of hGSTP1-1 and may afford metabolic protection to the fetus (for review, see Awasthi et al., 1994
). Overall, our data support a biologically relevant role of hGSTP1-1 in the biotransformation of atrazine in humans, especially following dermal or inhalation exposure, where extrahepatic metabolism may contribute to overall elimination.
Significance of the Human GSTP1 Polymorphism in Atrazine Conjugation
Single nucleotide polymorphisms of the hGSTP1 gene have been described at codon positions 105 and 114 (Ali-Osman et al., 1997). In the referenced study, allele frequencies of the resulting genotypes were as follows: hGSTP1*A (0.70), hGSTP1*B (0.19), hGSTP1*C (0.11) and hGSTP1*D (not detected, rare). Our results suggest that the four variant hGSTP1-1 enzymes exhibit very similar activities toward atrazine. Therefore, it is unlikely that individuals carrying variant forms of the hGSTP1 gene are differently able to clear atrazine, following exposure on the basis of hGSTP1 genotype.
One interesting finding from this study is the discovery that atrazine is a very specific substrate of Pi class GSTs. The hGST isoforms exhibit unique, but often overlapping, substrate specificity (Eaton and Bammler, 1999). For instance, CDNB is a good substrate for hGSTP1-1; however, CDNB is also metabolized by many other hGSTs. The hGSTP1-1 conjugation of atrazine is unusually specific to that isoform; therefore, atrazine could be a useful marker substrate for detection of hGSTP1-1 activity in tissues where multiple GST isoforms are expressed.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at the University of Washington-Roosevelt, 4225 Roosevelt Way, NE, Suite 100, Seattle, WA 98105-6099.
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