Mu-Class GSTs Are Responsible for Aflatoxin B1-8,9-Epoxide-Conjugating Activity in the Nonhuman Primate Macaca fascicularis Liver

Charles Wang*, Theo K. Bammler{dagger}, Yingying Guo{dagger}, Edward J. Kelly{dagger} and David L. Eaton{dagger},1

* National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Dr., HFT-100, Jefferson, Arkansas 72079; and {dagger} Department of Environmental Health, University of Washington, 4225 Roosevelt Way NE, #100, Seattle, Washington 98105

Received December 24, 1999; accepted March 3, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B1 (AFB1) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (~200,000 pmol/min/mg) activity toward aflatoxin B1-8,9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB1-induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha-class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (~300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, Mf GSTs were purified from liver tissue and characterized and, Mf mu-class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu-class GST cDNAs, mfaGSTM1 (GenBank accession # AF200709) and mfaGSTM2 (GenBank accession # AF200710), were generated by RT-PCR. CDNA-derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu-class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo- and endo-AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis.

Key Words: aflatoxin; glutathione S-transferase; nonhuman primate; biotransformation; cDNA; mu-class; liver.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aflatoxin B1 (AFB1) is a mycotoxin produced by the common molds Aspergillus flavus and Aspergillus parasiticus (Asao et al., 1963, reviewed in Eaton and Gallagher, 1994; Wilson and Payne, 1994). AFB1 is frequently found as a dietary contaminant in regions of the world where temperature and humidity favor the growth of the mold, and food storage techniques are inadequate (Groopman, 1994Go; Groopman et al., 1988Go; Wilson and Payne, 1994Go). AFB1 is a potent carcinogen in rats (Wogan, 1973Go) and human epidemiological studies have demonstrated that AFB1 exposure is an important risk factor in human hepatocarcinogenesis, especially in populations in which hepatitis B virus infection is endemic (Chen et al., 1997Go; McGlynn et al., 1995Go; Qian et al., 1994Go).

AFB1 requires activation by cytochromes P450 (CYP) to form AFB-8,9 epoxide (AFBO), the ultimate carcinogen that binds to DNA (reviewed in Eaton and Gallagher, 1994). CYP-mediated oxidation of AFB1 to AFBO can produce two stereoisomers, exo- and endo-AFBO (Raney et al., 1992aGo,bGo), but only exo-AFBO binds to DNA at the N7 position of guanine (Iyer et al., 1994Go; Johnson and Guengerich, 1997Go). The metabolic activation of AFB1 by human or rat microsomes produces a mixture of endo- and exo-epoxides, although human microsomes generate predominantly exo-epoxide in vitro at relatively high AFB1 concentrations (Raney et al., 1992aGo,bGo).

Remarkable species differences in susceptibility to AFB1-induced liver cancer have been demonstrated (Eaton and Gallagher, 1994Go; Monroe and Eaton, 1988Go; Ramsdell and Eaton, 1990Go). Rats are very sensitive, whereas mice are highly resistant, to the hepatocarcinogenic effects of AFB1 (Wogan and Newberne, 1967Go), even though mouse liver microsomes form exo-AFBO at a rate slightly higher than rat liver microsomes (Monroe and Eaton, 1987Go). The resistance of mice is due largely, if not exclusively, to the constitutive expression of an alpha-class GST isozyme (mGSTA3-3) that has extraordinarily high conjugating activity (~200,000 pmol/min/mg) toward the reactive intermediate AFBO (Buetler and Eaton, 1992Go; Hayes et al., 1992Go). In contrast, rats do not constitutively express a GST isoform with high AFBO-conjugating activity and are thus sensitive to AFB1-induced heptocarcinogenesis. However, an inducible, alpha-class GST enzyme (rGSTA5-5) with high AFBO conjugating activity (~50,000 pmol/min/mg) can confer resistance to AFB1-induced hepatocarcinogenesis in rats when treated with certain chemicals such as oltipraz, ethoxyquin, and butylated hydroxyanisole (Hayes et al., 1991Go, 1994Go).

In contrast to rodents, the cytosolic fraction from human liver possesses little or no detectable AFBO-conjugating activity (Eaton and Gallagher, 1994Go; Eaton and Ramsdell, 1992Go; Moss and Neal, 1985Go; Slone et al., 1995Go). In addition, purified recombinant human alpha-class GSTs hGSTA1-1 and hGSTA2-2 also lack significant activity toward AFBO (Buetler et al., 1996Go; Johnson et al., 1997Go; Raney et al., 1992bGo). However, human hepatic mu-class GSTs M1a-1a and M2-2 exhibit measurable activity toward AFBO. This activity is directed nearly exclusively toward the endo-AFBO stereoisomer (Raney et al., 1992bGo).

Recent studies from this laboratory found that liver cytosol from the nonhuman primate Macaca fascicularis has significant constitutive hepatic cytosolic GST activity toward the exo-AFBO stereoisomer, although this activity is approximately 100-fold lower than that seen in mouse liver (Eaton and Ramsdell, 1992Go). It is not known which specific GST(s) is (are) responsible for this activity. The objective of this study was to identify the GST(s) in Macaca fascicularis liver that is (are) responsible for this AFBO detoxification activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents.
Aflatoxin B1 (AFB1), reduced glutathione (GSH), NADPH, 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), S-hexylglutathione (SHG), glutathione agarose (GSHA), and S-hexylglutathione agarose (SHGA) were obtained from Sigma Chemical Co. (St. Louis, MO). HPLC solvents were of analytical reagent grade and were obtained from J. T. Baker, Inc. (Phillipsburg, NJ). Nytran-supported nylon transfer and immobilization membranes were obtained from Schleicher & Schuell (Keene, NH). Oligomers were synthesized by Oligos Etc., Inc. (Wilsonville, OR). All other reagents were of analytical reagent grade and were obtained from various commercial sources. The plasmid pBluescript II SK(+) and pET 17b were purchased from Stratagene (La Jolla, CA) and Novagen (Madison, WI), respectively.

Animals and tissues.
Macaca fascicularis liver tissues were obtained from castrated adult males through the tissue acquisition program of the Regional Primate Research Center at the University of Washington. The livers were removed immediately following terminal anesthesia and exsanguination, and were rinsed in ice-cold saline, snap-frozen in liquid nitrogen, and stored at –80°C until homogenization. Hepatic cytosolic fractions were prepared and stored according to methods described previously (Gallagher et al., 1991Go; Ramsdell and Eaton, 1988Go).

Affinity purification of hepatic GSTs from Macaca fascicularis.
Hepatic GST proteins were purified, using a previously described method with the following modifications (Gallagher et al., 1996Go). Liver cytosols (30 ml, 38 mg/ml) were loaded onto an S-hexylglutathione agarose (SHGA) affinity column which was connected to a glutathione agarose (GSHA) affinity column such that the flow-through (FT) from the SHGA column passed directly to the GSHA column. Prior to sample loading, both columns were equilibrated with buffer A [10 mM Tris, pH 7.8; 200 mM NaCl; 1 mM EDTA; 0.5 mM dithiothreitol (DTT)] at 4°C. An initial 100 ml of eluate from the GSHA column was collected to determine the non-retained enzymatic activities. Both SHGA and GSHA columns were washed overnight with >400 ml of buffer A (more than 20 times column volume), and were disconnected before being eluted separately. The GSTs bound to the SHGA column were eluted in buffer A containing 5 mM S-hexylglutathione and 2.5 mM glutathione. The GSTs bound to the GSHA column were eluted with 200 mM Tris, pH 9.0, containing 50 mM GSH. GSTs eluted from the SHGA and the GSHA columns were dialyzed separately in 2 liters of buffer B (25 mM sodium phosphate, pH 7.4; 0.5 mM DTT), changed 4x over 48 h. All of the above procedures were carried out at 4°C. After dialysis, GST samples were aliquoted and stored at –80°C.

GST subunit purification.
Both SHGA and GSHA column eluates were further resolved by reverse-phase HPLC to examine GST subunit composition as described by Rowe et al. (1997). An Alltech Macrosphere 300 C4, 4.6 mm x 15 mm, 5 µm bore column was used with Rainin HPLC LD-200 pumps controlled by a Rainin Dynamax HPLC method manager. Solvent A consisted of water containing 0.08% trifluroacetic acid (TFA). Solvent B consisted of acetonitrile containing 0.1% TFA. The gradient conditions were as follows. Injection started at 20% solvent B. At 10 min, solvent B was increased to 40%, held at 40% for 10 min, and then over a period of 40 min was increased to 60%. The flow rate was 1 ml/min. Protein peaks were monitored for absorbance at 214 nm using a Waters LC 480 spectrophotometer. HPLC fractions were collected manually and dried in a Speed-Vac. Dried samples were re-suspended in water immediately prior to SDS/PAGE and Western blotting.

Chromatofocusing of hepatic GSTs from Macaca fascicularis.
As multiple GSTs were present in the SHGA affinity fraction, further GST protein purification was done by chromatofocusing, as described previously with certain modifications (Gallagher et al., 1996Go; Ramsdell and Eaton, 1990Go). Aliquots of the affinity-purified GSTs from the SHGA columns were equilibrated in 25 mM triethylamine (TEA), and then applied to a Mono-P chromatofocusing column (Pharmacia-LKB, Piscataway, NJ) previously equilibrated with 25 mM TEA, pH 10. The column was eluted using a mixture of low molecular weight buffers adapted from a focusing buffer described previously (Ramsdell and Eaton, 1990Go). It was found that a pH range from 8 to 10 provided optimal resolution of alpha and mu class GSTs. The pH gradient was generated with the chromatofocusing buffer groups as follows: 4 min with 25 mM TEA, pH 10, 60 min with a group of buffers consisting of a mixture of low-molecular-weight organic chemicals followed with 30 min of 1 N NaCl. The absorbance of the effluent was monitored at 280 nm and the CDNB activity of the fractions was determined using a standard method (Habig and Jakoby, 1981Go) adapted for 96-well micro-plate reader (Molecular Devices Corp., Menlo Park, CA) in the kinetic mode at a wavelength of 340 nm. Individual peaks were collected and were further evaluated for AFBO-conjugating activity by HPLC as previously described (Monroe and Eaton, 1987Go). All collected peaks were further analyzed by SDS–PAGE and Western blots.

SDS–PAGE and western blotting.
Hepatic cytosolic fractions, SHGA, and GSHA affinity purified GSTs, as well as fractions purified by reverse-phase HPLC and chromatofocusing, were resolved on SDS–polyacrylamide gel (16% acrylamide: 0.09% N,N-bis acrylamide) and gels were either stained with Coomassie brilliant blue or transferred onto Immobilon PVDF membranes (Millipore, Bedford, MA) for Western blot analyses. Nonspecific binding was blocked with 3% (w/v) nonfat-milk powder dissolved in phosphate buffered saline (PBS), pH 7.4. Primary antibodies were incubated in 1% (w/v) nonfat milk for 1 h in PBS containing 0.1% Tween 20. Blots were then incubated with a goat anti-rabbit alkaline phosphatase-conjugated antibody (Bio-Rad, Richmond, CA) for 30 min. Blots were developed using the chemiluminescent substrate CSPD (Tropix, Bedford, MA) according to the manufacturer's recommendations.

RNA isolation.
Frozen liver tissue (0.1 g) was homogenized in 2 ml Trizol reagent (GIBCO BRL, Gaithersburg, MD) with a Polytron tissue mixer. Homogenates were centrifuged at 12,000 x g for 15 min at 4°C. The supernatant was precipitated with isopropanol and collected by centrifugation at 12,000 x g for 10 min. RNA pellets were washed with 75% ethanol, air dried, and re-suspended in 50 µl of Formazol (GIBCO BRL, Gaithersburg, MD), and stored at –80°C. RNAs were precipitated with 2.5 volumes of isopropanol and washed with 75% ethanol, air dried, and resuspended in water prior to RT-PCR reaction.

Primer design and complementary DNA cloning strategy.
All human mu-class GST cDNAs (M1–M5) were aligned using the Clustal software sequence-alignment program. Two sets of primers were designed based on the multiple sequence alignment of human mu-class GST cDNAs at certain conserved regions covering all coding sequences. The primers were synthesized by Oligos Etc. Inc. (Wilsonville, OR). The 5` forward primers were identical for both sets of primers. Primer 1 (5` forward): gcaccaaccagccatatgcccatga. Primer 2 (3` reverse): gtcaggctggcactagtgcagggaa. Primer 3 (3` reverse); acagctcggggctgagctcca. In primer 1, a NdeI (catatg) restriction site was introduced to allow the subsequent PCR products to be cloned directly into the expression vector, pET 17b. Primers 2 and 3 were designed so that they contained a Spe I and Sac I sites respectively to facilitate subsequent cloning of the resulting PCR products.

Initially, cDNAs were synthesized using 50 units of Moloney murine leukemia virus (MMLV) reverse transcriptase, 1 µg total RNA template, 20 units RNase inhibitor (Stratagene, La Jolla, CA), 1 mM dNTP, 1 µg oligo d(T)20 primer and a buffer containing 50 mM Tris–HCl, pH 8.3; 75 mM KCl, and 3 mM MgCl2. The RNA was heated to 65°C for 10 min and immediately cooled on ice before MMLV-RT was added into the reaction mixture. The reverse transcription reaction was performed with a thermal cycler (DNA Engine, MJ Research, Inc., Watertown, MA), using the following temperature profile: (1) 37°C for 60 min; (2) 99°C for 5 min; (3) 5°C for 5 min. Five µl of RT mix was used for the PCR reaction in the presence of either primers 1 and 2 or primers 1 and 3 (1 µM each) in a 100 µl volume containing 1 x Pfu buffer, 200 µM of dNTP, 2.5 units of cloned Pfu polymerase (Stratagene, La Jolla, CA). PCR reactions were done with the same thermal cycler using the following temperature profile: (1) 94°C for 30 s; (2) 55°C (primers 1 and 2) or 60°C (primers 1 and 3) for 15 s; (3) 72°C for 2 min; with 30 cycles from (1) to (3); and (4) 72°C for 10 min.

Isolation of Macaca fascicularis mu-class GST cDNA clones.
To ensure an adequate amount of cDNAs for the subsequent cDNA cloning and to ensure representative amplification of all mRNAs, 3 rounds of independent PCR reactions were completed for each set of primers. All cDNAs were pooled from 3 rounds of reactions for each set of primers, respectively. PCR amplified cDNAs were resolved on 1% agarose gel containing ethidium bromide and expected bands (~1 kb) were cut from the gel and extracted, using the Qiaex II gel extraction kit (Qiagen, Valencia, CA). The cDNAs synthesized with primers 1 and 2 were cloned into the pET17b, digested with both Nde I and Spe I restriction enzymes. The pET 17b vectors containing the GST cDNA inserts were then transfected into E.coli BL21 for GST recombinant protein expression.

cDNAs synthesized using primers 1 and 3 were initially cloned into pBluescript II SK(-) (Stratagene, La Jolla, CA) digested with Sma I (Stratagene), producing blunted ends. The plasmids were prepared on the white clones and the vectors hosting cDNA inserts were confirmed by DNA sequencing as well as digestions with both Nde I and Sma I restriction enzymes, followed by electrophoresis and ethidium bromide gel staining. The pBluescript II SK(-) containing PCR inserts were then digested with Nde I and Sac I (Boehringer Mannheim, Indianapolis, IN), and the digested cDNA inserts were subcloned into the pET 17b vector for recombinant GST protein expression.

Sequencing.
cDNA clones were sequenced using T3, T7, and nested primers on both strands. Sequencing reactions were done on a thermal cycler using 0.5 µg of plasmid DNA and 8 µl of dye-terminator mix purchased from Perkin-Elmer (Foster City, CA). Sequences were obtained using an ABI377 automatic sequencer in the Biomarker Lab of the Center for Ecogenetics and Environmental Health at the University of Washington. All sequences were analyzed with DNAide (Laboratoire de Biochimie, 91128 Palaiseau cedex, France) and DNA Strider (Service de Biochimie, Cedex, France) software and compared to the human mu-class GST cDNA sequences M1 through M5.

Northern blotting.
Twenty µg of hepatic RNA samples were separated on a 1.25% agarose/formaldehyde gel, blotted onto Nytran membranes, and hybridized with 32P end-labeled oligonucleotide or cDNA probes labeled by random priming. The mfaGSTM1-specific oligo (ggaagtccaataaagtctc) and mfaGSTM2-specific oligo (acttggtttctctcaagg) were synthesized by Oligos Etc. Inc. (Wilsonville, OR). The specificity of mfaGSTM1-specific and mfaGSTM2-specific oligos was examined both by Northern blotting for 4 human mu-class cDNA expressed mRNAs (M1, M2, M3 and M4) and Southern blotting for 5 different primate cDNAs (hGSTM1, hGSTM3, hGSTM4, mfaGSTM1 [GenBank accession # AF200709] and mfaGSTM2 [GenBank accession # AF200710]). Northern blots were hybridized in QuickHyb (Stratagene) solution for 1 h at 68°C with cDNA probes or at 6°C below the Tm of the oligonucleotide probes. After hybridization, blots probed with cDNA probes (mfaGSTA1 and mfaGSTM2) were washed twice at room temperature with 2x SSC buffer and 0.1% (w/v) SDS wash solution followed by a high stringency wash at 60°C with a 0.1x SSC buffer and 0.1% (w/v) SDS solution for 30 min in a hybridization oven (Robbins Scientific Corp., Sunnyvale, CA). Blots probed with oligonucleotides (mfaGSTM1- and mfaGSTM2- specific probes) were washed 2 times with 2x SSC buffer and 0.1% SDS for 20 min at 42°C, followed by an additional wash with the same buffer at 45°C. Autoradiography utilized Kodak X-OMAT AR film.

Mass spectrometry analysis.
Mass spectra of GST subunits were acquired using a delayed extraction matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometer (Voyager DE, Perseption Biosystems, Inc., Framingham, MA) at the Department of Biochemistry of the University of Washington. Protein samples were dissolved in 40% (v/v) acetonitrile containing 0.1% trifluoroacetic acid (TFA), and 1 µl of solution was deposited on MALDI plate along with 1 µl of sinapinic acid matrix, and were air dried and analyzed.

Enzymatic assays.
AFBO-conjugating activity was determined using a method described previously (van Ness et al., 1998Go), in which mouse microsomes were used to generate a racemic mixture of endo and exo AFBO with an initial AFB1 substrate concentration of 128 µM. This concentration of AFB has been found to provide adequate substrate to the microsomal P450 system used to generate the AFBO substrate for the GST assay, such that production of AFBO is not rate-limiting under the conditions of the assay (Monroe and Eaton, 1987Go). AFBO endo- and exo-activities were determined using the method by Raney et al. (1992a), with the following modifications: (1) mouse microsomes were used to generate AFBO with an initial AFB1 substrate concentration of 128 µM and final GSH concentration of 5 mM. (2) 1 mM NADPH was used instead of using an NADPH regenerating system. (3) The gradient was changed, as follows: times 0, 90% Solvent A, 10% Solvent B; 45 min, 20% Solvent B; 47 min, 50% Solvent B; and 56 min, 10% solvent B. Solvent A was 20 mM ammonium acetate, pH 4.0; solvent B was 1:1 acetonitrile:methanol. A 250 x 4.6 mm Econosphere C18 column, heated to 40°C, and a flow rate of 2.0 ml/min was used. Base line resolution of the exo and endo AFBO-GSH conjugates were obtained with these conditions. Both UV (365 nm) and fluorescence (excitation 365, emission 425 nm) detection were used for all assays.

With this AFBO generating system, it is difficult to know what the actual concentration of substrate (AFBO) is because of the very rapid hydrolysis of AFBO in an aqueous environment. However, each assay is repeated with at least 2 different dilutions of GST enzyme to ensure that substrate (AFBO) availability is not rate limiting. If the apparent specific activity of the GST toward endo- or exo-AFBO is significantly higher in the more dilute enzyme solution, the assay is repeated at a lower enzyme concentration. Because mouse liver microsomes form the exo isomer predominately, it is technically difficult to accurately determine AFBO-conjugating activity of a GST which predominantly conjugates the endo isomer. However, because the endo isomer is approximately 15 times more fluorescent than the exo isomer, fluorescence detection provides adequate sensitivity to measure even relatively low levels of GST activity toward the endo-AFBO. All AFBO-conjugating-activity data were repeated at least 4 times under identical conditions, and, therefore, provide reliable estimates of relative AFBO-conjugating activity of these GSTs. Specific activities for GST conjugating activity of both endo and exo AFBO were determined in triplicate, and ratios of the average of the 3 determinations were used to express the relative enantioselectivity of the GST.

Other general enzymatic glutathione S-transferase activities were assayed using 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and ethacrynic acid (ECA) as substrates, according to standard procedures (Habig and Jakoby, 1981Go).

Biohazards and safety precautions.
All primate tissues were handled according to University of Washington biohazard safety protocols for primate tissues. Wastes were treated with bleach prior to disposal. Aflatoxin-contaminated wastes and glassware were also treated with bleach, according to established procedures.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GST Affinity Purification and Activities Recovery
Table 1Go shows the AFBO and CDNB conjugating activities, distribution and recovery from affinity columns. There was 58 pmol/min/mg of AFBO conjugating activity in GSTs eluted from the SHGA column, whereas GSTs eluted from the GSHA column had approximately 4 times this activity (239 pmol/min/mg). GST protein was monitored and selected at OD280. GSTs retained on the SHGA column accounted for 26% of total cytosolic AFBO conjugating activity, whereas GSTs retained on the GSHA column accounted for almost 40% of total AFBO conjugating activity. However, GSTs eluted from the SHGA column accounted for 76% of CDNB activity, whereas GSTs eluted from the GSHA column contained only 11% of total CDNB activity (Table 1Go). AFBO-conjugating activity not retained on either affinity column (initial flow-through; FT) was just above the detection limit of AFBO-conjugating activity (3.3 pmol/min/mg). However, because of the relatively large volume of FT, this accounted for 23% of total cytosolic AFBO-conjugating activity loaded onto the columns. This is likely an over-estimate, as the measured AFBO conjugating activity was close to the detection limit (2 pmol/min/mg), and the FT fraction contained only 1% of total CDNB activity. Overall, there was an 88% recovery in both AFBO and CDNB activities. Interestingly, the AFBO conjugating activity seen in all fractions, including the cytosolic GSTs and the GSTs purified from the SHGA and GSHA columns, was almost exclusively (>95%) directed toward the exo-AFBO stereoisomer (Table 1Go).


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TABLE 1 AFBO and CDNB Activity Distribution and Recovery from Affinity Columns
 
Subunit Analysis by HPLC
GST subunit analysis by HPLC was completed for GSTs eluted from the SHGA (Fig. 1AGo) and GSHA (Fig. 1BGo). Four different peaks (P1–P4) were identified in the GST fraction eluted from the SHGA column, whereas only a single GST subunit peak was found in the eluate from the GSHA column. Therefore, only the SHGA eluate was further subjected to chromatofocusing.



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FIG. 1. Affinity purification of Mf hepatic GSTs. (A) GSTs eluted from the SHGA column were further purified by reverse-phase HPLC using a C4 column to determine subunit composition. (B) GST eluted from the GSHA column was further purified by reverse-phase HPLC using a C4 column. GST protein peaks were monitored at OD 214 nm.

 
Chromatofocusing
GSTs eluted from the SHGA column were further purified by chromatofocusing with a Mono P column. Figure 2Go shows the pH gradient curve from pH 10 to 8.4 (A) and isofocusing chromatogram (B). Five peaks were observed at OD 280 nm, two major peaks (P1 and P3) and three minor peaks (P2, P4, and P5). Peak 3 was split up in 2 fractions named P3-1 and P3-2, whereas only one fraction was collected from each of the other peaks (P1, P2, P4, and P5). To remove any tightly bound protein, the column was eluted with a high salt buffer, resulting in one peak at 78 min. P3-1 and P3-2 shared a pI of 9.4 and had highest AFBO-conjugating activity (Fig. 3Go) among all fractions collected. The GST fraction eluted from the GSHA column was not subject to chromatofocusing as only a single GST peak was identified by reverse-phase HPLC, and Western blotting analyses revealed that it had immunoreactivity exclusively against a mu-class GST polyclonal antibody (see below).



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FIG. 2. Chromatofocusing of affinity-purified Mf hepatic GSTs. (A) pH gradient curve (from pH 10 to 8). (B) Chromatofocusing of GSTs eluted from the SHGA column by Mono P column. GST protein peaks were monitored at OD 280 nm.

 


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FIG. 3. Characteristics of purified Mf hepatic GSTs. (A) SDS–PAGE and Coomassie brilliant blue staining of affinity purified GSTs (eluates from SHGA and GSHA columns) and fractions collected from chromatofocusing column (P1, P2, P3-1, P3-2, P4, and P5), along with 2 primate cDNA-expressed GSTs (mfaGSTM1-1 and hGSTA1-1). (B) Immunoreactivities against 3 different classes of GSTs such as alpha (A), mu (M), and pi (P). (C) AFBO-conjugating activities of each GST as labeled above the top panel. GSHA, GST eluted from the GSHA column; SHGA, GSTs eluted from the SHGA column; P1, P2, P3-1, P3-2, P4 and P5 are peaks collected from Mono P column; mfM1, recombinant mfaGSTM1-1; hA1, recombinant hGSTA1-1.

 
SDS–PAGE and Immunoreactivities of Macaca fascicularis Hepatic GSTs Purified by Chromatography and Chromatofocusing
Figure 3Go shows SDS–PAGE followed by Coomassie brilliant blue staining of affinity purified GSTs and the fractions collected from the chromatofocusing column, along with two recombinant primate GSTs, mfaGSTM1 (see below) and hGSTA1 (panel A). Immunoreactivities of each protein toward antibodies selective for alpha, mu, or pi class GSTs are shown in panel B. AFBO-conjugating activity for each peak is included in panel C. Only a single 25.6 kD band was observed from the GSHA affinity-purified fraction, which cross-reacted only with anti-mu-class antibody. However, there were 2 protein bands from the GSTs purified by the SHGA affinity column (lower band, 25.3 kD; top band, 25.6 kD). The top band reacted with mu-class-specific antibodies, whereas the lower band reacted with alpha-class-specific antibodies, based on Western blotting analyses. There were no peaks that reacted with pi class antibodies.

Only a single band was seen with both P1 and P2 fractions purified by the chromatofocusing column, and it cross-reacted only with anti-alpha-class antibody. These bands had the same mobility as the lower band of the SHGA-column purified GSTs. The P1 and P2 proteins had no GST activity toward AFBO. Both fractions of P3 from the chromatofocusing column contained 2 different protein bands. Western-blot analysis revealed that the top band was a mu-class protein while the lower band belonged to the alpha-class GST family. AFBO-conjugating activity appeared on inspection to be correlated with the intensity of the mu-class band. Similarly, there were 2 different bands on the P4 and P5 peaks eluted from the chromatofocusing column; the upper band was a mu-class GST and the lower band was an alpha-class GST, based on Western-blotting analyses. Substantial AFBO-conjugating activity was also associated with fractions P4 and P5, which contained both alpha- and mu-class immunoreactive proteins.

Complementary DNA Cloning and Sequencing
Eight positive clones were obtained with the first set of primers, and each had an insert of 918 base pairs. Restriction analysis revealed that these clones shared identical patterns (data not shown). DNA sequencing using T3, T7, and nested primers indicated all 8 were identical clones. This clone, referred to as mfaGSTM1, based on the nomenclature recommendations of Mannervik et al (1992), had an open reading frame of 654 base pairs that codes for 218 amino acids (Fig. 4Go). A comparison of the cDNA-derived amino acid sequence with human mu-class GSTs indicated that mfaGSTM1 shares 97% homology with hGSTM4 (Table 2Go and Fig. 4Go). Based on cDNA-derived protein sequences, mfaGSTM1 shares 85%, 84%, 69% and 84% homology with human mu-class GSTs hGSTM1a, hGSTM2, hGSTM3 and hGSTM5 respectively (GenBank accession # for mfaGSTM1 is: AF200709) (Table 2Go).



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FIG. 4. Amino acid sequence alignments of primate mu-class GSTs. Asterisks indicate positions that have a single, fully conserved residue. Double dots indicate positions that have conserved substitutions. Single dots indicate positions that have less conserved substitutions.

 

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TABLE 2 Amino Acid Sequence Similarities of Primate Mu-class GSTs
 
cDNAs generated with a second set of primers produced four positive clones. DNA sequencing indicated that all four clones were identical, yet different from mfaGSTM1. This clone, named mfaGSTM2, has a 989-base pair insert with 654 base pairs of open reading frame that also codes for 218 amino acids (Fig. 4Go). Sequence analysis suggests that mfaGSTM2 is a homolog of hGSTM2 (Table 2Go and Fig. 4Go), as it shares 96% amino acid sequence identity with hGSTM2 (Table 2Go). In addition, cDNA-derived amino acid sequence comparison indicated that mfaGSTM2 shares 86%, 68%, 86%, and 84% similarities with human mu-class GSTs hGSTM1a, hGSTM3, hGSTM4 and hGSTM5 (Table 2Go). mfaGSTM2 shares 86% amino acid sequence identity with mfaGSTM1.

Characteristics of Recombinant Macaca fascicularis mu-Class GSTs
MfaGSTM1-1 and mfaGSTM2-2 were expressed in bacteria and purified by glutathione affinity chromatography (McHugh et al., 1996Go). Table 3Go shows activity characterization of these two recombinant GST proteins. mfaGSTM1-1 had no AFBO conjugating activity, although it had detectable conjugating activity toward CDNB (0.34 µmol/min/mg) as well as some activity toward ECA (0.1 µmol/min/mg). In contrast, mfaGSTM2-2 had substantial conjugating activity toward AFBO (333 pmol/min/mg). However, in contrast to what was observed for affinity-purified GSTs from Macaca fasicularis liver, this activity was directed almost exclusively toward the endo-AFBO stereoisomer. mfaGSTM2-2 had somewhat lower CDNB (207 µmol/min/mg) but higher DCNB-conjugating activity (4.9 µmol/min/mg) than its human homologue hGSTM2-2 (295 µmol/min/mg and 2 µmol/min/mg, respectively) (Table 3Go).


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TABLE 3 Specific Activities of Primate cDNA Expressed Mu-class GSTs
 
Messenger RNA Expression in the Macaca fascicularis Liver and Other Tissues
To assess expression levels of the mfaGSTM1 and mfaGSTM2 genes, specific oligonucleotide probes were used for Northern-blot analysis. mfaGSTM2 was expressed in the 4 Macaca fascicularis livers examined (Fig. 5Go). In addition, mfaGSTM2 mRNA was also found at a low level in the duodenum, but was barely detectable in kidney and lung in the one animal examined (Fig. 5Go). The mfaGSTM2 message was approximately 1.3 kb in size.



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FIG. 5. Northern-blot analysis of mfaGST mRNA in different Mf tissues. (A) Northern blot using mfaGSTM2-specific oligomer probe. (B) Northern blot using mfaGSTM2 cDNA probe. (C) Northern blotting using mouse ß-actin cDNA probe. L, liver (1 to 4); K, kidney; Lu, lung; Duo, duodenum.

 
In contrast to mfaGSTM2, mfaGSTM1 expression was not detected in any of the 4 tissues examined, including liver (data not shown). Interestingly, when the mfaGSTM2 cDNA was used as a probe in Northern-blot analysis, 2 distinct bands were detected, suggesting that at least 2 different mu-class GSTs are expressed in Mf liver and duodenum. The size of the lower-molecular-weight band was estimated to be 1.3 kb and was most likely identical to the message detected with the mfaGSTM2-specific oligonucleotide probe (Fig. 5Go).

Mass Analysis of mfaGSTs by Protein Mass Spectrometry
Because some of the results above suggested that the hepatic mfaGST protein with AFBO-conjugating activity, isolated by GSHA affinity chromatography, might not be the same as the recombinant mfaGSTM2-2 protein, the molecular masses of these and other GST subunits were determined by MALDI-TOF mass spectrometry, with an accuracy of 0.1%. Overall, masses obtained by protein mass spectrometry were very consistent with peptide sequence-deduced mass. The concordance was particularly good for mfaGSTM1 and mfaGSTM2 cDNA expressed subunits (mfaGSTM1: 25430 Dalton estimated by MS vs. 25429 Dalton deduced from the cDNA sequence; mfaGSTM2: 25556 Dalton determined by MS vs. 25560 Dalton deduced from the cDNA sequence). However, there was an 81-Da difference between the GSHA column purified GST and the recombinant mfaGSTM2-2 subunit, based on MW determined by MS.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Guengerich and colleagues have demonstrated previously that mu-class GSTs, rGSTM2-2 and rGSTM2-3, and hGSTM1a-1a purified from rat and human liver respectively, can conjugate both exo- and endo-AFB1 epoxides, with the endo-stereoisomer being the preferred substrate (Guengerich et al., 1998Go; Johnson et al., 1997Go; Raney et al., 1992aGo,bGo). Consistent with these findings, this study clearly showed that mu-class GSTs are largely, if not exclusively, responsible for cytosolic hepatic AFBO-conjugating activity in the non-human primate Macaca fascicularis. The mu-class enzyme GSHA-GST purified from liver tissue exhibited a specific activity of 239 pmol/min/mg toward AFBO (Table 1Go). In contrast, 2 partially purified alpha-class GSTs (peaks P1, P2, in Fig. 3Go) lacked AFBO-conjugating activity. In addition, the partially purified fractions P3-1, P3-2, P4, and P5 obtained by SHGA affinity chromatography followed by chromatofocusing exhibited AFBO-conjugating activities of 140, 280, 70, and 140 pmol/min/mg respectively (Fig. 3Go). Immunoblot analyses showed that each of these fractions contained a mixture of alpha- and mu-class GSTs. In addition, based on visual inspection of the immunoblot band intensitities, AFBO-conjugating activity appeared to correlate with the intensity of the mu-class, but not the alpha-class bands. Together, these results suggest that mu-class GSTs are responsible for the majority of the AFBO-conjugating activity in Macaca fascicularis liver. However, in this study the activities of theta and zeta class GSTs were not evaluated, and it is possible that either of these forms could contribute to AFBO-conjugating activity seen in cytosolic fractions. They could not, however, contribute to the GST-conjugating activity isolated from the affinity columns as neither form is retained on GSH- or GSHA-affinity columns.

Interestingly, in contrast to rat and human mu-class GSTs (Guengerich et al., 1998Go; Johnson et al., 1997Go; Raney et al., 1992aGo,bGo), but similar to the rodent alpha-class GSTs mGSTA3-3 and rGSTA5-5 (Buetler and Eaton, 1992Go; Hayes et al., 1991Go, 1992Go, 1994Go), it appears that mu-class GST(s) purified from Macaca fascicularis liver preferentially conjugate the exo-AFB1 epoxide (Table 1Go). It is very difficult to compare quantitatively the AFBO-conjugating activity between the study of Raney et al. (1992b) and this study, because the assays were carried out under very different conditions, and the results were expressed in different activity units (Raney et al., 1992bGo). Nevertheless, these findings suggest that the Mfa hepatic mu-class GST(s) GSHA-GST present in the GSHA affinity-purified fraction may be the only known primate GST(s) to date that has (have) high and selective activity toward the exo-AFBO stereoisomer.

Identification and characterization of the homologous mu-class GST(s) in humans may have important implications in the prevention of AFB1-induced hepatocarcinogenesis, as it is known that only the exo-AFBO isomer reacts with DNA (Iyer et al., 1994Go; Johnson and Guengerich, 1997Go; Raney et al., 1992bGo). Although Mf mu-class GST purified by the GSHA column catalyzes the formation of the exo-AFBO-glutathione conjugate (AFBO-SG), it has to be pointed out that this activity is approximately 2 orders of magnitude lower than that of rodent alpha-class GSTs, mGSTA3-3 and rGSTA5-5 (Buetler and Eaton, 1992Go; Hayes et al., 1991Go, 1992Go, 1994Go).

To further characterize the mu-class GST(s) with high activity toward exo-AFBO, we attempted to clone the corresponding cDNA by RT-PCR from a Macaca fascicularis liver sample. Two different mu-class GST cDNAs containing complete open reading frames were obtained and named mfaGSTM1 and mfaGSTM2, based on the recommendations by Mannervik et al. (1992). Analysis of cDNA-deduced amino acid sequences revealed that mfaGSTM1 and mfaGSTM2 are most likley homologs of the human mu-class GSTs, hGSTM4 and hGSTM2, respectively, as they share 97% and 96% identities with these proteins respectively (Table 2Go).

Similar to its human homolog, bacterially expressed recombinant mfaGSTM1-1 had no detectable AFBO-conjugating activity, although it was active toward the classic GST substrate, CDNB (0.34 µmol/min/mg). In contrast, recombinant mfaGSTM2-2 had significant conjugating activity toward AFBO (333 pmol/min/mg) (Table 3Go). Paradoxically, in contrast to the purified native mu-class GST, GSHA-GST, (GSHA column-purified GST), which displayed almost exclusively exo activity, the cDNA expressed recombinant mfaGSTM2-2 exhibited almost exclusively endo activity (Tables 1 and 3GoGo).

Interestingly, the human GSTM2 cDNA was initially isolated from myoblasts (Vorachek et al., 1991Go), and it is significantly expressed in brain and heart, in addition to muscle (Rowe et al., 1997Go). Human GSTM2-2 displays unique and high catalytic activity toward reactive quinones of endogenous catecholamines such as dopaminochrome, produced by oxidation of dopamine, and it has been suggested that hGSTM2-2 may play some cytoprotective role against endogenous oxidative tissue damage in the brain (Baez et al., 1997Go). In contrast to hGSTM2, which is barely detectable in human liver (Hussey and Hayes, 1993Go; Rowe et al., 1997Go), mfaGSTM2 was significantly expressed in Macaca fascicularis liver, but similar to its human counterpart, mfaGSTM2 was barely detectable in either kidney or lung (Rowe et al., 1997Go). However, it should be noted that there can be large interindividual differences in expression of GST isoforms in different tissues, and only a few human livers have been evaluated for the presence of hGSTM2.

Although mfaGSTM2-2 shares 96% sequence identity with hGSTM2-2, it exhibited 30% lower activity toward CDNB and 2.5-fold higher activity toward DCNB than hGSTM2-2 (Table 3Go). Comparison of the amino acid sequences revealed that mfaGSTM2 and hGSTM2 differ by 8 amino acids at positions 25, 90, 94, 104, 107, 113, 129, and 130 (Fig. 4Go). Only one out of the 8 differences is non-conservative: at position 25, aspartic acid is found in hGSTM2, whereas a glycine occupies this position in mfaGSTM2. It is possible that the amino acid difference at position 25 or any of the remaining 7, alone or in combinations, may be responsible for the difference of CDNB activities between mfaGSTM2-2 and hGSTM2-2. Several studies have demonstrated that single-site changes in GSTs can profoundly influence the catalytic properties, substrate specificity, and stereoselectivity (Bammler et al., 1995Go; Bjornestedt et al., 1995Go; Boehlert and Armstrong, 1984Go; Cobb et al., 1983Go; Hu et al., 1997aGo,bGo,cGo; Nanduri et al., 1996Go; Shan and Armstrong, 1994Go; Zimniak et al., 1994Go).

MfaGSTM1 (the homolog of human M4), isolated by RT-PCR from the Macaca fascicularis liver, was not detected in liver, kidney, lung, or duodenum (data not shown), suggesting that mfaGSTM1-1 is either not expressed in these tissues or is expressed at extremely low levels. The human GSTM4 cDNA was initially cloned from a human testis cDNA library (Ross and Board, 1993Go). Comstock et al. (1993) detected hGSTM4 mRNA at various levels in many human tissues including heart, placenta, lung, brain, liver, skeletal muscle, pancreas, testis, cerebral cortex, uterus, and ovary. In addition, a more recent study indicated that hGSTM4 subunit is actually a minor mu-class GST form that is expressed at extremely low levels in all tissues except testis, which is rich in almost all human mu-class GSTs (M1 through M3 and M5) (Rowe et al., 1997Go). Similar to hGSTM4-4, recombinant mfaGSTM1-1 exhibited no AFBO and low CDNB activities (Table 3Go) (Ross and Board, 1993Go).

This study raises the question of whether recombinant mfaGSTM2-2 is identical to the native GST, GSHA-GST, obtained by GSHA affinity purification from the same liver. Immunoblot analysis identified both enzymes as GSTs belonging to the mu class. However, differences in enantioselectivity toward exo- versus endo-AFBO suggest that the cloned mfaGSTM2-2 protein is not identical to that purified from the liver. Furthermore, there was an 81-Dalton difference in the molecular weight determined by MS between recombinant mfaGSTM2-2 and the native GSHA-GST. Finally, the cDNA-expressed mfaGSTM2-2 protein had 10 times higher CDNB activity than did GSHA-GST.

While it is highly unlikely, it is possible that there was a second, but highly similar GST subunit in the GSHA affinity column-purified GST(s) that could not be separated from the major subunit by reverse phase HPLC. This contamination from a second GST could have altered the molecular weight of the GSHA column purified GST. Alternatively, it is possible that a single site mutation was introduced into mfaGSTM2 during RT-PCR cloning that resulted in an alteration of the enantioselectivity toward AFBO and catalytic activity toward CDNB. This hypothesis is supported by recent studies demonstrating that single-site changes in GSTs and P450s are responsible for differences in enzyme catalytic efficiency, substrate specificity and stereoselectivity or enantioselectivity (Bammler et al., 1995Go; Bjornestedt et al., 1995Go; Boehlert and Armstrong, 1984Go; Cobb et al., 1983Go; Hu et al., 1997aGo,bGo,cGo; Hu et al., 1998Go; Lindberg and Negishi, 1989Go; Nanduri et al., 1996Go; Shan and Armstrong, 1994Go; Zimniak et al., 1994Go).

It should be pointed out that the design of forward primer (primer 1, see Materials and Methods for sequence) was based on human mu-class GST cDNA sequences and included the first 10 highly conserved nucleotides of the coding regions of hGSTM1, hGSTM2, and hGSTM5. Therefore, the theoretical possibility exists that the first 2 codons following the initiator methionine were biased in mfaGSTM2. However, this possibility seems unlikely because the first 3 codons are conserved in the human mu-class GST cDNAs hGSTM1, hGSTM2, and hGSTM5. In the unlikely event that the 2 codons following the initiator methionine in the RT-PCR-generated clone mfaGSTM2 were indeed different from the actual cDNA, it is still highly unlikely that they would account for the differences in stereoselectivity towards exo- and endo-AFBO and the approximately 10-fold difference in CDNB activity exhibited by recombinant mfaGSTM2-2 and the native GSHA-GST purified from tissue.

Together our data strongly suggest that (a) mfaGSTM2-2 and GSHA-GST are 2 distinct enzymes, and (b) the cDNA encoding GSHA-GST still remains to be cloned.

In summary, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-SG activity found in Macaca fascicularis liver. In addition, our data suggest that at least two distinct mu-class GSTs exhibiting this activity are expressed constitutively in the liver of this species. One of the enzymes, GSHA-GST, preferentially conjugates exo-AFBO, whereas the other, mfaGSTM2-2, almost exclusively metabolizes the endo isomer. To date, none of the known human mu-class GSTs exhibit predominant activity toward the ultimate genotoxic AFB1 metabolite exo-AFBO. Identification of a potential human homolog of GSHA-GST would be relevant to the planning and design of chemointervention strategies to reduce AFB1-induced liver cancer in highly exposed populations, because it is currently uncertain whether induction of known human GSTs with little or no activity toward the epoxide of AFB1 will be effective at lowering AFB1 genotoxicity.


    ACKNOWLEDGMENTS
 
This project was supported by NIH grants R01ES-05780, P30ES-07033 and P30RR00166. The authors wish to thank Dr. Terrance Kavanagh for his scientific advice and comments. The authors also would like to acknowledge Dennis Slone and Collin White for their superior technical assistance.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (206) 685-4696. Email: deaton{at}u.washington.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asao, T., Buchi, G., Abdel-Kader, M. M., Chang, S. B., Wick, E., and Wogan, G. N. (1963). Aflatoxins B and G. J. Am. Chem. Soc. 85, 1706–1707.

Baez, S., Segura-Aguilar, J., Widersten, M., Johansson, A. S., and Mannervik, B. (1997). Glutathione transferases catalyze the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes. Biochem. J. 324, 25–28.[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, 9000–9008.[ISI][Medline]

Bjornestedt, R., Tardioli, S., and Mannervik, B. (1995). The high activity of rat glutathione transferase 8–8 with alkene substrates is dependent on a glycine residue in the active site. J. Biol. Chem. 270, 29705–29709.[Abstract/Free Full Text]

Boehlert, C. C., and Armstrong, R. N. (1984). Investigation of the kinetic and stereochemical recognition of arene and azaarene oxides by isozymes A2 and C2 of glutathione S-transferase. Biochem. Biophys. Res. Commun. 121, 980–986.[ISI][Medline]

Buetler, T. M., Bammler, T. K., Hayes, J. D., and Eaton, D. L. (1996). Oltipraz-mediated changes in aflatoxin B1 biotransformation in rat liver: implications for human chemointervention. Cancer Res. 56, 2306–2313.[Abstract]

Buetler, T. M., and Eaton, D. L. (1992). Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues. Cancer Res. 52, 314–318.[Abstract]

Chen, C. J., Yu, M. W., and Liaw, Y. F. (1997). Epidemiological characteristics and risk factors of hepatocellular carcinoma. J. Gastroenterol. Hepatol. 12, S294–308.[ISI][Medline]

Cobb, D., Boehlert, C., Lewis, D., and Armstrong, R. N. (1983). Stereoselectivity of isozyme C of glutathione S-transferase toward arene and azaarene oxides. Biochemistry 22, 805–812.[ISI][Medline]

Comstock, K. E., Johnson, K. J., Rifenbery, D., and Henner, W. D. (1993). Isolation and analysis of the gene and cDNA for a human mu-class glutathione S-transferase, GSTM4. J. Biol. Chem. 268, 16958–16965.[Abstract/Free Full Text]

Eaton, D. L., and Gallagher, E. P. (1994). Mechanisms of aflatoxin carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 34, 135–172.[ISI][Medline]

Eaton, D. L., and Ramsdell, H. H. (1992). Species and diet-related differences in aflatoxin biotransfromation. In Handbook of Applied Mycology: Mycotoxins in Ecological Systems, (D. Bhatnagar, E. B. Lillehoj, and D. K. Arora, Eds.), pp. 157–182. Marcel Dekker, New York.

Gallagher, E. P., Kedderis, G. L., and Di Giulio, R. T. (1991). Glutathione S-transferase-mediated chlorothalonil metabolism in liver and gill subcellular fractions of channel catfish. Biochem. Pharmacol. 42, 139–145.[ISI][Medline]

Gallagher, E. P., Stapleton, P. L., Slone, D. H., Schlenk, D., and Eaton, D. L. (1996). Channel catfish glutathione S-transferase isoenzyme activity toward (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide. Aq. Toxicol. 34, 135–150.[ISI]

Groopman, J. D. (1994). Molecular dosimetry methods for assessing human aflatoxin exposure. In Toxicology of Aflatoxins: Human Health, Veterinary and Agricultural Significance, (D. L. Eaton and J. D. Groopman, Eds.), pp. 259–280, Academic Press, New York.

Groopman, J. D., Cain, L. G., and Kensler, T. W. (1988). Aflatoxin exposure in human populations: measurements and relationship to cancer. CRC Crit. Rev. Toxicol. 19, 113–145.[ISI]

Guengerich, F. P., Johnson, W. W., Shimada, T., Ueng, Y. F., Yamazaki, H., and Langouet, S. (1998). Activation and detoxication of aflatoxin B1. Mutat. Res. 402, 121–128.[ISI][Medline]

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

Hayes, J. D., Judah, D. J., McLellan, L. I., Kerr, L. A., Peacock, S. D., and Neal, G. E. (1991). Ethoxyquin-induced resistance to aflatoxin B1 in the rat is associated with the expression of a novel alpha-class glutathione S-transferase subunit, Yc2, which possesses high catalytic activity for aflatoxin B1–8,9-epoxide. Biochem. J. 279, 385–398.[ISI][Medline]

Hayes, J. D., Judah, D. J., Neal, G. E., and Nguyen, T. (1992). Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1–8,9-epoxide. Biochem. J. 285, 173–180.[ISI][Medline]

Hayes, J. D., Nguyen, T., Judah, D. J., Petersson, D. G., and Neal, G. E. (1994). Cloning of cDNAs from fetal rat liver encoding glutathione S-transferase Yc polypeptides. The Yc2 subunit is expressed in adult rat liver resistant to the hepatocarcinogen aflatoxin B1. J. Biol. Chem. 269, 20707–20717.[Abstract/Free Full Text]

Hu, X., Ji, X., Srivastava, S. K., Xia, H., Awasthi, S., Nanduri, B., Awasthi, Y. C., Zimniak, P., and Singh, S. V. (1997a). Mechanism of differential catalytic efficiency of two polymorphic forms of human glutathione S-transferase P1–1 in the glutathione conjugation of carcinogenic diol epoxide of chrysene. Arch. Biochem. Biophys. 345, 32–38.[ISI][Medline]

Hu, X., O'Donnell, R., Srivastava, S. K., Xia, H., Zimniak, P., Nanduri, B., Bleicher, R. J., Awasthi, S., Awasthi, Y. C., Ji, X., and Singh, S. V. (1997b). Active-site architecture of polymorphic forms of human glutathione S-transferase P1–1 accounts for their enantioselectivity and disparate activity in the glutathione conjugation of 7-beta,8-alpha-dihydroxy-9-alpha,10-alpha-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Biochem. Biophys. Res. Commun. 235, 424–428.[ISI][Medline]

Hu, X., Pal, A., Krzeminski, J., Amin, S., Awasthi, Y. C., Zimniak, P., and Singh, S. V. (1998). Specificities of human glutathione S-transferase isozymes toward anti-diol epoxides of methylchrysenes. Carcinogenesis 19, 1685–1689.[Abstract]

Hu, X., Xia, H., Srivastava, S. K., Herzog, C., Awasthi, Y. C., Ji, X., Zimniak, P., and Singh, S. V. (1997c). Activity of four allelic forms of glutathione S-transferase hGSTP1–1 for diol epoxides of polycyclic aromatic hydrocarbons. Biochem. Biophys. Res. Commun. 238, 397–402.[ISI][Medline]

Hussey, A. J., and Hayes, J. D. (1993). Human mu-class glutathione S-transferases present in liver, skeletal muscle, and testicular tissue. Biochim. Biophys. Acta 1203, 131–141.[ISI][Medline]

Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994). DNA adduction by the potent carcinogen aflatoxin B1: Mechanistic studies. J. Am. Chem. Soc. 116, 1603–1609.[ISI]

Johnson, W. W., and Guengerich, F. P. (1997). Reaction of aflatoxin B1 exo-8,9-epoxide with DNA: kinetic analysis of covalent binding and DNA-induced hydrolysis. Proc. Natl. Acad. Sci. U.S.A. 94, 6121–6125.[Abstract/Free Full Text]

Johnson, W. W., Ueng, Y. F., Widersten, M., Mannervik, B., Hayes, J. D., Sherratt, P. J., Ketterer, B., and Guengerich, F. P. (1997). Conjugation of highly reactive aflatoxin B1 exo-8,9-epoxide catalyzed by rat and human glutathione transferases: estimation of kinetic parameters. Biochemistry 36, 3056–3060.[ISI][Medline]

Lindberg, R. L., and Negishi, M. (1989). Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue. Nature 339, 632–634.[ISI][Medline]

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, 305–306.[ISI][Medline]

McGlynn, K. A., Rosvold, E. A., Lustbader, E. D., Hu, Y., Clapper, M. L., Zhou, T., Wild, C. P., Xia, X. L., Baffoe Bonnie, A., Ofori Adjei, D., et al. (1995). Susceptibility to hepatocellular carcinoma is associated with genetic variation in the enzymatic detoxification of aflatoxin B1. Proc. Natl. Acad. Sci. U.S.A. 92, 2384–2387.[Abstract]

McHugh, T. E., Atkins, W. M., Racha, J. K., Kunze, K. L., and Eaton, D. L. (1996). Binding of the aflatoxin-glutathione conjugate to mouse glutathione S-transferase A3–3 is saturated at only one ligand per dimer. J. Biol. Chem. 271, 27470–27474.[Abstract/Free Full Text]

Monroe, D. H., and Eaton, D. L. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and mouse. Toxicol. Appl. Pharmacol. 90, 401–409.[ISI][Medline]

Monroe, D. H., and Eaton, D. L. (1988). Effects of modulation of hepatic glutathione on biotransformation and covalent binding of aflatoxin B1 to DNA in the mouse Toxicol. Appl. Pharmacol. 94, 118–127.[ISI][Medline]

Moss, E. J., and Neal, G. E. (1985). The metabolism of aflatoxin B1 by human liver. Biochem. Pharmacol. 34, 3193–3197.[ISI][Medline]

Nanduri, B., Hayden, J. B., Awasthi, Y. C., and Zimniak, P. (1996). Amino acid residue 104 in an alpha-class glutathione S-transferase is essential for the high selectivity and specificity of the enzyme for 4-hydroxynonenal. Arch. Biochem. Biophys. 335, 305–310.[ISI][Medline]

Qian, G. S., Ross, R. K., Yu, M. C., Yuan, J. M., Gao, Y. T., Henderson, B. E., Wogan, G. N., and Groopman, J. D. (1994). A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People's Republic of China. Cancer Epidemiol. Biomarkers Prev. 3, 3–10.[Abstract]

Ramsdell, H. S., and Eaton, D. L. (1988). Modification of aflatoxin B1 biotransformation in vitro and DNA binding in vivo by dietary broccoli in rats. J. Toxicol. Env. Health 25, 269–284.[ISI][Medline]

Ramsdell, H. S., and Eaton, D. L. (1990). Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B1–8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide. Toxicol. Appl. Pharmacol. 105, 216–225.[ISI][Medline]

Raney, K. D., Coles, B., Guengerich, F. P., and Harris, T. M. (1992a). The endo-8,9-epoxide of aflatoxin B1: a new metabolite. Chem. Res. Toxicol. 5, 333–333.[ISI][Medline]

Raney, K. D., Meyer, D. J., Ketterer, B., Harris, T. M., and Guengerich, F. P. (1992b). Glutathione conjugation of aflatoxin B1 exo- and endo-epoxides by rat and human glutathione S-transferases. Chem. Res. Toxicol. 5, 470–478.[ISI][Medline]

Ross, V. L., and Board, P. G. (1993). Molecular cloning and heterologous expression of an alternatively spliced human mu-class glutathione S-transferase transcript. Biochem. J. 294, 373–380.[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, 481–486.[ISI][Medline]

Shan, S., and Armstrong, R. N. (1994). Rational reconstruction of the active site of a class-mu glutathione S-transferase. J. Biol. Chem. 269, 32373–32379.[Abstract/Free Full Text]

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, 224–233.[ISI][Medline]

van Ness, K. P., McHugh, T. E., Bammler, T. K., and Eaton, D. L. (1998). Identification of amino acid residues essential for high aflatoxin B1–8,9-epoxide conjugation activity in alpha class glutathione S-transferases through site-directed mutagenesis. Toxicol. Appl. Pharmacol. 152, 166–174.[ISI][Medline]

Vorachek, W. R., Pearson, W. R., and Rule, G. S. (1991). Cloning, expression, and characterization of a class-mu glutathione transferase from human muscle, the product of the GST4 locus. Proc. Nat'l. Acad. Sci. 88, 4443–4447.[Abstract]

Wilson, D. M., and Payne, G. A. (1994). Factors affecting aspergillus flavus-group infection and aflatoxin contaminatin of crops. In The Toxicology of Aflatoxins: Human Health, Veterinary, and Agricultural Significance, (D. L. Eaton and J. D. Groopman, Eds.), pp. 309–325. Academic Press, New York.

Wogan, G. (1973). Aflatoxin carcinogenesis. In Methods of Cancer Research, (H. Busch, Ed.), pp. 309–344, Academic Press, New York.

Wogan, G. N., and Newberne, P. M. (1967). Dose-response characteristics of aflatoxin B1 carcinogenesis in the rat. Cancer Res. 27, 2370–2376.[ISI][Medline]

Zimniak, P., Nanduri, B., Pikula, S., Bandorowicz-Pikula, J., Singhal, S. S., Srivastava, S. K., Awasthi, S., and Awasthi, Y. C. (1994). Naturally occurring human glutathione S-transferase GSTP1–1 isoforms with isoleucine and valine in position 104 differ in enzymic properties. Eur. J. Biochem. 224, 893–899.[Abstract]