Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205, USA
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Abstract |
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Abbreviations: AFB1, aflatoxin B1; AFAR, aflatoxin B1-aldehyde reductase; AKR, aldo-keto reductase; CYP450, cytochrome P450; D3T, 1,2-dithiole-3-thione; ECL, enhanced chemiluminescence; GST, glutathione S-transferase; IPTG, isopropyl-thio-ß-D-galactoside; MES, 2-[N-morpholino]ethanesulfonic acid; NTA, nickel nitrilotriacetic acid; ORF, open reading frame; PBS, phosphate-buffered saline; 1x SSC, 0.15 M sodium chloride and 0.015 M sodium citrate pH 7.0.
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Introduction |
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Studies in several experimental animal models have shown that variations in the activities of AFB1-activating and detoxifying enzymes can have a dramatic effect on cancer incidence (11). To extrapolate the work with animal models to human risk assessment, it is important to evaluate the activity of human AFB1-metabolizing enzymes. Human CYP450 and GST enzymes have been expressed as recombinant proteins and their kinetic parameters have been estimated regarding AFB1 metabolism (12,13). Recently, Ireland et al. (14) have cloned a human AFAR, designated AKR7A2, with a specific activity towards AFB1 dihydrodiol of 1.24 ± 0.18 nmol/min/mg, however, kinetic parameters of this enzyme were not reported.
In the present study, we report the isolation of a distinct AFAR cDNA (AKR7A3) from a human liver cDNA library. This protein was expressed in Escherichia coli and used to investigate the catalytic activity of this protein towards several carbonyl-containing substrates, especially AFB1 dihydrodiol. The expression of AKR7A was detected in a series of human liver cytosolic protein samples and in multiple adult RNA tissue samples. The catalytic efficiency of human AKR7A3 was estimated and compared with other AFB1-metabolizing enzymes to determine its relative contribution in the detoxification of AFB1.
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Materials and methods |
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Isolation and characterization of rat and human cDNA clones
A partial rat AKR7A1 cDNA, clone 18.11, was isolated as described (17). The clone 18.11 cDNA sequence began at nucleotide +150, relative to the start codon of the published rat AKR7A1 cDNA (18). To obtain a full-length rat AKR7A1 cDNA, clone 18.11 was used to screen 10 000 colonies of a 1,2-dithiole-3-thione (D3T)-induced rat liver cDNA library (17) by high stringency colony hybridization (19). Clone 18.11 was labeled with [-32P]dCTP (ICN, Costa Mesa, CA) to a specific activity of >108 c.p.m./µg DNA by a random primed labeling method (Boehringer Mannheim, Indianapolis, IN; 20). The hybridization was performed at 65°C for 48 h in a solution containing 5x SSC, 0.5% SDS, 200 µg/ml denatured salmon sperm DNA, 5x Denhardt's solution (1x Denhardt's = 0.2 mg/ml Ficoll, 0.2 mg/ml polyvinylpyrrolidone and 0.2 mg/ml bovine serum albumin), 1 mg/ml skimmed milk powder and 3.2x106 c.p.m./ml labeled DNA probe. After hybridization, the blot was successively washed at 65°C for 1 h in each of: 1x SSC, 1% SDS; 0.5x SSC, 0.5% SDS; 0.1x SSC, 0.1% SDS. After three rounds of screening, eight cDNA clones were colony purified. Plasmid DNA was isolated as described (21) and analyzed by restriction endonuclease mapping with EagI (New England Biolabs, Beverley, MA). To ensure selection of clones which contained the complete coding region, two primers were designed to generate a product which contained the start codon after PCR amplification [primer designation, 5'
3' sequence, nucleotide location relative to first methionine of the published rat AKR7A1 cDNA sequence (GenBank accession no. AF045464) and strand]: P1, 5'-GGATCCTCTCTTACCCGCCACCT-3', 51 to 29, +; P2, 5'-TGTCTTCCCAAACATTGGG-3', 223243, . Of the eight clones analyzed, three independent cDNA clones were selected and sequenced in both directions as described below.
Human AKR7A-related cDNA clones were isolated from an oligo(dT)-primed adult human liver cDNA library (catalogue no. A550-07; Invitrogen, San Diego, CA). The rat AKR7A1 cDNA clone was used to screen 10 000 colonies of the human cDNA library by low stringency colony hybridization (22). The hybridization was performed at 50°C for 36 h as described above. After hybridization, the blot was washed twice in 2x SSC, 1% SDS at 50°C for 1 h and then once in 2x SSC, 0.1% SDS at 50°C for 1 h. After three rounds of screening, 24 cDNA clones were colony purified. Plasmid DNA was isolated and analyzed by restriction endonuclease mapping with EcoRI and NotI (New England Biolabs). Three of the longest independent clones were selected for DNA sequence analysis. The 21 clones not sequenced were further analyzed by restriction endonuclease mapping with restriction sites unique to the previously isolated human AFAR (AKR7A2), XmnI and NheI (New England Biolabs).
DNA sequencing strategy and analysis
The rat AKR7A1 cDNA sequence was determined using primers specific to the SP6 and T7 sides of the vector plasmid, pcDNAII (Invitrogen), by the dideoxy chain termination method as implemented (Sequenase v.2.0; US Biochemicals, Cleveland, OH) using [-35S]dATP (ICN). When overlapping sequence could not be obtained on both DNA strands, specific sense and anitsense oligonucleotide primers were used: rP1, 5'-AATTGCCACCAAGGCCTGC-3', 206223, +; rP3, 5'-TGGAGACTGAGCTCTTCC-3', 522539, +; rP2, 5'-AGGGTTGAAGGCGTAGAACC-3', 565584, ;rP4, 5'-AAGGCAGATGTATCTTAGCGG-3', 977997, .
The initial sequence of the human AKR7A-related cDNA clones was determined using a primer specific to the T7 side of the pcDNA3 polylinker (Invitrogen) and then six additional primers were used to determine the complete DNA sequence in both orientations by automated sequencing using fluorescent dye terminators (Protein/Peptide/DNA Laboratory, Department of Biological Chemistry, Johns Hopkins University School of Medicine): hP2, 5'-AAATTGATACCAAGGCC 3', 218234, +; hP4, 5'-CTGTACCCTCTGCAAGA-3', 459475, +; hP6, 5'-GGATGTACCACCACTCACA-3', 809825, +; hP7, 5'-CGTGTCTATCTCGGTGTG-3', 118135, ; rP2, 5'-AGGGTTGAAGGCGTAGAACC-3', 578597, ; hP9, 5'-TGTTACAGAAGAGCCTTGG-3', 10181136, . DNA sequences obtained from independent cDNA clones were assembled using the GeneWorks program (IntelliGenetics, Mountain View, CA).
Rat (AKR7A1) and human (AKR7A3) AFAR protein expression constructs
The rat AKR7A1 and human AKR7A3 were subcloned into pTrcHis expression vectors (Invitrogen) such that the expressed protein had an N-terminal hexa-histidine (His6) peptide tag, thereby allowing affinity purification by metal chelate affinity chromatography.
The preparation of the rat AKR7A1 expression construct (herein referred to as the His6-AKR7A1 plasmid) involved a two step ligation strategy. Initially, a 1.2 kb BamHIXhoI AKR7A1 cDNA fragment (region 25 to 1176, GenBank accession no. AF045464) was subcloned into the BamHI and HindIII sites of the pTrcHisA plasmid using T4 DNA ligase to initially ligate the cohesive BamHI sites, T4 DNA polymerase to convert the HindIII and XhoI ends to blunt ends and T4 DNA ligase to join these ends. The plasmid was transformed into E.coli DH5- cells, creating AKR7A1.pTrc1. Next, two primers were designed: rpTrc1-P1, 5'-CGCGGATCCCAAGCCCGGCCTGC-3', 3 to 20, +, which would introduce a unique BamHI restriction site (italics) to be fused in-frame with the His6-tag without altering the original rat AKR7A1 amino acid sequence; rpTrc1-P2, 5'-GTGACATCTCCAGCTGGAACC-3', 262282, . This primer pair amplified a 285 bp fragment that contained an internal AvrII restriction site (position +59) by PCR. This 285 bp fragment was digested with BamHI and AvrII, separated in a low melting point agarose gel and then ligated into the BamHI and AvrII sites of AKR7A1.pTrc1 according to the instructions of FMC Bioproducts, to produce plasmid His6-AKR7A1. This final construct was sequenced using primer rP2 to confirm the proper orientation of the His6-AKR7A1 plasmid and to ensure a correct sequence of nucleotides.
Preparation of the human AKR7A3 expression construct (herein referred to as the His6-AKR7A3 plasmid) involved a similar two step ligation strategy. Initially, a 1.2 kb EcoRINotI AKR7A3 cDNA fragment (region 25 to 1176, GenBank accession no. AFO40639) was subcloned into the EcoRI and HindIII sites of the pTrcHisC plasmid using T4 DNA ligase to initially ligate the cohesive EcoRI sites, T4 DNA polymerase to convert the HindIII and NotI sites to blunt ends and then T4 DNA ligase to join the ends. The plasmid was transformed into E.coli DH5- cells, creating AKR7A3.pTrc1. Next, two primers were designed: hpTrc1-P1, 5'-GCCGTGAATTCCCGG-3', 7 to 15, +, which would introduce a unique EcoRI restriction site (italics) to be fused in-frame with the His6-tag without altering the original human AKR7A3 amino acid sequence; hpTrc1-P2, 5'-ACTCAGTGTCTGTTCCACC-3', 378396, . The primer pair amplified a 395 bp fragment that contained an internal AatII restriction site (position +287) by PCR. The 395 bp fragment was then digested with EcoRI and AatII, separated in a low melting point agarose gel, NuSieve® GTG (FMC Bioproducts, Rockland, ME) and ligated into the EcoRI and AatII sites of AKR7A3.pTrc1, to produce plasmid His6-AKR7A3. This final construct was sequenced using primer rP2 to confirm proper orientation of the His6-AKR7A3 plasmid and to ensure a correct sequence of nucleotides.
Expression of rat AKR7A1 and human AKR7A3 protein
Escherichia coli transformed with either rat His6-AKR7A1 or human His6-AKR7A3 constructs were inoculated into LuriaBertani medium (1.0% w/v bacto-tryptone, 0.5% w/v bacto-yeast extract, 0.5% w/v NaCl) containing 100 µg/ml ampicillin and grown overnight at 37°C with orbital shaking at 200 r.p.m. This culture (5 ml) was used to inoculate 300 ml of medium and grown at 37°C, with orbital shaking at 200 r.p.m., until the cells were in mid log phase (A600 = 0.70.9). Protein expression was induced by the addition of isopropyl-thio-ß-D-galactoside (IPTG) to a final concentration of 1 mM. After 3.5 h, 1 ml of the cell suspension was pelleted by centrifugation, resuspended and boiled for 5 min in 200 µl SDS sample dilution buffer (50 mM TrisHCl, pH 6.8, 1.5% 2-mercaptoethanol, 2% SDS, 0.1% bromophenol blue and 10% glycerol). A 15 µl sample lysate was analyzed by SDSPAGE (12% acrylamide), to confirm induction of the expressed proteins.
For production of active protein, His6-tagged proteins from the soluble fraction of the bacterial lysates were purified under non-denaturing conditions as described, with modification (23). Escherichia coli was induced with 0.1 mM IPTG, as this concentration would reduce expression levels and prevent incorporation of protein into inclusion bodies and thereby increase solubility of protein in the cytoplasm. After 3 h, this culture was collected in 50 ml aliquots by centrifugation at 2300 g for 15 min at 4°C. These cell pellets were resuspended in 5 ml of ice-cold buffer (50 mM Tris, pH 8.0, 1 mM EDTA and 10 mM 2-mercaptoethanol) and lysozyme was added to a final concentration of 0.5 mg/ml. The suspension was rocked for 30 min at 4°C and lysed by two freeze/thaw cycles, alternating in liquid nitrogen and a 37°C water bath. The thawed and broken cells were supplemented to a final concentration of 300 mM NaCl and 10 mM MgCl2, incubated for 45 min with constant rocking and subsequently centrifuged for 30 min at 26 400 g (23). The supernatant was applied to a 5 ml nickel nitrilotriacetic acid (NTA)agarose column (Qiagen, Chatsworth, CA), which had been pre-equilibrated at 4°C with 50 mM imidazole in NTA buffer [50 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 6.0, 300 mM NaCl, 50 µM EDTA, 1 mM MgCl2 and 10 mM 2-mercaptoethanol] that provided optimal pH conditions for elution of proteins using an imidazole step gradient (23). Removal of background proteins was achieved using an imidazole step gradient of 1 column vol of 50 mM imidazole, 3 column vol of 75 mM imidazole and 1 column vol of 100 mM imidazole in NTA buffer at 4°C. His6-tagged proteins were eluted from the column with 250 mM imidazole in NTA buffer, collected in 1 ml fractions and analyzed by SDSPAGE. Ten milliliters of eluted protein was injected into a Slide-A-Lyzer dialysis cassette (Pierce, Rockford, IL) according to the manufacturer's instructions and dialyzed against 2 l of 25 mM MES, pH 6.0, 300 mM NaCl, 50 µM EDTA for 8 h and then against 2 l of 0.1 M sodium phosphate, pH 6.8, 150 mM NaCl for 8 h at 4°C. Protein concentrations were measured using the method of Bradford (24) (Bio-Rad Laboratories, Hemel Hempstead, UK). Bovine serum albumin was used as a standard (Pierce).
For antibody production, rat His6-AKR7A1 protein was purified by affinity chromatography with NTAagarose using a pH step gradient as described (25). Approximately 1 mg of affinity-purified protein was then separated by SDSPAGE and the region of the gel containing the desired protein was excised following copper staining (25). Proteins were electroeluted from the gel slices in 25 mM Tris base, 192 mM glycine, 0.01% SDS (Bio-Rad Laboratories) dialyzed against 0.5x phosphate-buffered saline (PBS), 0.05% SDS. Dialyzed samples were concentrated in centricon-10 microconcentrators (Amicon, Beverley, MA) according to the manufacturer's instructions, analyzed by SDSPAGE and stored at 20°C. The purified protein was used to immunize two male New Zealand White rabbits as previously described (25). The amount of protein antigen used for immunization per rabbit was as follows: primary immunization, a gel slice containing 195 µg protein; first boost, 200 µg protein in 800 µl 0.5x PBS; second boost, 100 µg protein in 400 µl 0.5x PBS; 1 month extension boost, 100 µg protein in 400 µl 0.5x PBS. All animal procedures were carried out by Spring Valley Laboratories (Sykesville, MD).
Enzyme assays and kinetic studies
Carbonyl-reducing activity was determined by monitoring the oxidation of NADPH at 340 nm ( = 6270/M/cm) (26), using a Beckman DU-7 UV/visible spectrophotometer with a light path of 1 cm. Standard incubation mixtures consisted of 100 mM sodium phosphate buffer, pH 6.6 or pH 7.4, 0.2 mM NADPH, 810 µg of purified recombinant protein and various amounts of substrate dissolved in methanol or acetonitrile in a final volume of 1 ml. Enzymatic activity of AKR7A recombinant proteins was not inhibited by methanol or acetonitrile at concentrations of <4%, as confirmed by solvent controls. Baseline measurements were recorded without the addition of recombinant protein. The reaction was initiated by addition of either recombinant rat AKR7A1 or human AKR7A3 and was measured for 4 min at 25°C. Kinetic constants were derived from the computer generated best fit to the MichaelisMenten equation for three or four separate experiments using Enzfitter, a non-linear regression data analysis program (Biosoft, Cambridge, MA).
Metabolite production and analytical procedures
Incubation mixtures consisted of 10 µg recombinant human AKR7A3 protein, 100 mM sodium phosphate buffer, pH 7.4, 5 mM MgCl2, 1 mM NADP+, 15 mM glucose 6-phosphate, 1 U/ml glucose 6-phosphate dehydrogenase and AFB1 dihydrodiol in a total volume of 1 ml. Reaction mixtures were incubated at 37°C for 2 h and terminated by adding 10 µl of formic acid. The post-incubation reaction mixtures were applied to activated Waters C18 Sep-Pak Cartridges (Millipore, Milford, MA), washed twice with 4 ml water and then eluted with 2 ml 100% ethanol. The eluates were concentrated under argon gas.
A Finnigan LCQ liquid chromatography mass spectrometry system was used to perform electrospray ionization mass spectrometry in positive ion mode to confirm the identity of the AFB1 dialcohol. A Thermal Systems Products HPLC was used to provide a constant flow of 200 µl/min to a YMC ODS J-sphere M-80 column (2x250 mm). A gradient starting at 4% acetonitrile, 2% methanol and finishing at 13% acetonitrile, 12% methanol over 60 min was used to separate the aflatoxin B1 metabolites. A buffer consisting of 0.1% formic acid, pH 2.5, was used throughout the run. The HPLC column was maintained at 55°C and the column effluent directed through a UV detector (365 nm) and into the electrospray ionization interface on the mass spectrometer. The instrument was scanned from 200 to 600 a.m.u. at 1 s/scan. A collision energy of 22% was used for collision reaction monitoring during MS/MS analysis.
Immunoblot analysis
Human liver samples were obtained as discarded surgical specimens from the Johns Hopkins Hospital Department of Pathology. Cytosolic protein fractions were prepared (27) and analyzed (25) as described previously. Cytosolic samples were solubilized in sample dilution buffer, separated by denaturing 12% SDSPAGE and electrophoretically transferred to a nitrocellulose membrane (Hybond ECL; Amersham, Arlington Heights, IL). Incubation with primary antibody (rabbit anti-rat AKR7A1 serum from the final bleed diluted 1:10 000 (for AKR7A1) and 1:8 000 (for AKR7A3) was for 1 h at room temperature. Bound antibody was detected by incubation with a horseradish peroxidase-linked secondary antibody (goat anti-rabbit IgG diluted 1:30 000) (Promega, Madison,WI). Immunoreactive protein was detected by enhanced chemiluminescence (ECL) (Supersignal System; Pierce). Amounts of AKR7A-related proteins were estimated using purified rat (2, 4, 8 and 16 ng) and human (10, 20, 40 and 80 ng) recombinant proteins as standards. The density values of the ECL signals were determined using a Fujix BAS1000 Bio-imaging analyzer (Fujiphoto Film Co., Stamford, CT). For the selected exposure (1 min) the linearity of the ECL signals for the rat and human standard curves corresponds to r values of 0.95 and 0.99 and limits of detection of 2 and 10 ng, respectively.
RNA analysis of human tissues samples
Total cellular RNA (10 µg) was isolated from human liver and analyzed as described previously (17). After hybridization, the blot was washed twice in 1x SSC, 0.1% SDS at 50°C for 30 min. The hybridization signals were visualized by autoradiography using Kodak X-Omat AR film (Kodak Eastman Co., Rochester, NY) with a Cronex Lightning PlusTM intensifying screen (Dupont, Wilmington, DE) for 12 days at 80°C.
Human tissue poly(A)+ mRNA blots were obtained from Clontech (Palo Alto, CA). All specimens were from normal Caucasians. Samples represent either single or pooled RNA and were not matched for sex or age. No information was available concerning smoking history, diet or other potential environmental exposures. Northern blot analysis was performed according to the manufacturer's instructions, using human cDNA probes for AKR7A3 and ß-actin (Clontech). The membranes were washed twice in ExpressHyb solution (Clontech) at 50°C for 40 min and the signals were detected by autoradiography. ß-Actin was used as a control for RNA integrity and the transfer and hybridization procedures. Because levels of this RNA vary between tissues, it is not an accurate control for sample loading (28).
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Results |
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In addition to the complete sequencing of three clones, we characterized the 21 clones not sequenced by restriction endonuclease mapping, using two sites unique to human AKR7A2 (13), XmnI and NheI (Figure 2A). Nineteen of these clones had patterns indicative of the human AKR7A3 cDNA sequence. Two clones, designated c.14 and c.21, showed patterns indicative of AKR7A2 (13). As a follow-up, the complete cDNA sequences of c.14 and c.21 proved to be identical to the reported human AKR7A2 cDNA sequence (Figure 2
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Expression of rat AKR7A1 and human AKR7A3 proteins in E.coli
To study their activities, recombinant rat AKR7A1 and human AKR7A3 proteins were expressed as His6-tagged fusion proteins in E.coli. Bacterial cultures induced with IPTG yielded high expression of both rat and human His6-AKR7A proteins (Figure 3, lanes 3 and 6, respectively) when compared with uninduced cultures (Figure 3
, lanes 2 and 5, respectively). Rat and human His6-AKR7A proteins were purified by affinity chromatography, yielding ~2 mg fusion protein/300 ml bacterial culture. The purities of these preparations were >95%, as judged by SDSPAGE and Coomassie brilliant blue staining; the apparent molecular weight of each protein (His6-AKR7A1, 40 kDa and His6-AKR7A3, 42 kDa) was as expected (Figure 3
, lanes 4 and 7, respectively).
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Expression of AFAR in human liver
To characterize the rabbit polyclonal antibodies produced using recombinant rat AKR7A1 protein as the antigen, we performed immunoblot experiments using hepatic cytosols prepared from control or D3T-treated rats (17). On average, the level of AKR7A-immunoreactive protein was increased 18-fold in the hepatic samples from treated animals (Figure 4A, lanes 46), relative to control samples (Figure 4A
, lanes 13). Using the recombinant protein as a standard, the mean ± SE concentration of AKR7A-immunoreactive protein in control cytosols was determined to be 0.3 ± 0.1 µg/mg total cytosolic protein. Since the recombinant human AKR7A3 protein showed strong crossreactivity towards antibodies raised against the recombinant rat AKR7A1 protein (data not shown), we used these antibodies to determine whether AKR7A is constitutively expressed in adult human liver. Immunoblot analyses of cytosolic protein fractions prepared from liver samples of eight individuals were performed; four of these are presented in Figure 4B
. A 37 kDa protein band was detected, consistent with the calculated molecular weight of the deduced amino acid sequences of the known human AKR7A proteins. Based on the recombinant AKR7A3 standards, the amount of this AFAR-related protein in liver samples from the eight individuals examined ranged from 0.3 to 0.8 µg/mg, with a mean ± SE of 0.5 ± 0.1 µg/mg cytosolic protein. The expression of AKR7A-related mRNA in these eight individual liver samples was further characterized by northern RNA analysis. The same four representative samples are presented (Figure 5
). Overall, the levels of AKR7A-related mRNA expression varied and was poorly correlated (r = 0.2) with AKR7A-related protein amounts.
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Discussion |
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Using AKR7A1, we isolated cDNA clones corresponding to the previously characterized human AKR7A2 (14) and to human AKR7A3, which was previously unknown. By aligning the segments of these human cDNA sequences corresponding to their protein coding regions, we observed that all of the differences between their predicted amino acid sequences resulted from nucleotide differences in the third (wobble) codon position. Many silent nucleotide differences were also observed. The distribution of these silent and coding nucleotide differences throughout the AKR7A2 and the AKR7A3 sequences indicates that these distinct cDNAs are likely the products of independent genes rather than mRNA splice variants of a single gene.
In the initial characterization of human AKR7A2, fractionation of human liver cytosol by anion exchange chromatography on Q Sepharose resulted in multiple peaks containing carbonyl reductase activity (14). This observation is consistent with our identification of a second human member of the AKR7A superfamily. Further studies will be required in order to determine the enzyme multiplicity and substrate specificity of the human AKR7A family.
A previous analysis of RNA expression, using an AKR7A2 cDNA probe, identified AKR7A2-related mRNA in liver and several other organs, with especially strong hybridization signals observed for kidney, pancreas, small intestine and skeletal muscle (14). Although the tissue RNA expression reported in this current study, using an AKR7A3 cDNA probe, is in good agreement with the previous study, the high homololgy of the AKR7A2 and AKR7A3 cDNA sequences (80%) indicates that neither study utilized methods to discriminate specific AKR7A2 and AKR7A3 mRNAs. Further studies using sequence-specific oligonucleotide probes will be required in order to determine the relative expression of each gene and their responses to administration of D3T, ethoxyquin and other chemoprotective agents. Since the amino acid sequences of the AKR7A2 and AKR7A3 proteins do not differ by any stretch of residues greater than four, it appears unlikely that specific antibodies can be produced. However, substrate and inhibitor specificity attributes may help to determine the relative expression of these proteins in different tissues.
Previously, it was shown by others (32) that the apparent Km and Vmax values for a variety of carbonyl-containing substrates were comparable for the recombinant AKR7A1 and the native form, purified from rat liver. Although we did not purify the native AKR7A proteins, the kinetic parameters of our recombinant AKR7A1 enzyme, determined for 4-nitrobenzaldehyde and 9,10-phenanthrenequinone, are in good agreement with the literature values (32). The purified recombinant AKR7A3 was also shown to be active towards several model substrates for AKRs. Kinetic analysis of the recombinant AKR7A3 showed a 4-fold lower apparent Km for 4-nitrobenzaldehyde relative to AKR7A1; the apparent Km values for 9,10-phenanthrenequinone were similar (Table I). The apparent Km value of AKR7A3 for 4-nitrobenzaldehyde is considerably lower than that determined for AKR7A2 (14), while that for 9,10-phenanthrenequinone is similar.
The reactive AFB1 8,9-oxide metabolite plays a central role in the formation of cellular marcromolecular adducts (Figure 1). It is well known that the C8 position of the unstable, AFB1 exo-8,9-epoxide isomer forms a covalent bond with the N7 of guanine bases in DNA, which, if not repaired, can lead to mutations (46). Another pathway, which follows from the spontaneous hydrolysis of the epoxide to form AFB1 dihydrodiol, is the reversible base-catalyzed conversion of AFB1 dihydrodiol to the ring opened oxyanionic form, AFB1 dialdehyde (31). The concentration of these two metabolites in vivo at equilibrium is not known. However, the relative amount of AFB1 dialdehyde formed from solutions of AFB1 dihydrodiol over a range of pH values has been determined (31); the AFB1 dialdehyde metabolite exists at pH > 6.0. AFB1 dialdehyde forms Schiff bases with lysine residues in proteins at physiological pH (68). As a consequence, the potential of this dialdehyde tautomer of AFB1 dihydrodiol to interact with lysine residues in critical cellular proteins, thereby altering their function, has been considered to contribute to AFB1-induced cytotoxicity. Evidence to support this hypothesis comes largely from in vitro studies, where incubation of AFB1 dihydrodiol with isolated reticulocytes results in direct inhibition of protein synthesis (33). However, additional studies will be required to directly correlate the effects of protein binding, impairment of cellular function and toxicity caused by the dialdehydic form of AFB1 dihydrodiol.
Since previous studies did not report the apparent Km and Vmax values for the reduction of AFB1 dihydrodiol, we determined these values for AKR7A1 and AKR7A3 (Table I). At the pH optimum for activity of these enzymes with model substrates (pH 6.6), both AKR7A1 and AKR7A3 had similar kinetic rates for AFB1 dihydrodiol, although AKR7A3 had a 3-fold lower apparent Km. AKR7A3 had a 2-fold higher Vmax in incubations with AFB1 dihydrodiol at pH 7.4 compared with pH 6.6. This outcome is consistent with the observation of Johnson et al. (31) showing that the relative amount of the dialdehydic form of AFB1 dihydrodiol in solution is pH dependent and increases under basic conditions (31). The reported rate for AKR7A2 (1.2 nmol/min/mg at pH 7.4; 14) is much lower than the Vmax determined for AKR7A3 (340 nmol/min/mg). However, the study of AKR7A2 did not report the concentration of AFB1 dihydrodiol used, which may have been low.
Investigations of the catalytic activities of AFB1-metabolizing enzymes is of interest in determining the sensitivity of humans to the toxic and carcinogenic effects of AFB1. For example, several cytosolic GSTs are known to play critical roles in the detoxification of AFB1 by enzymatically conjugating the AFB1 8,9-oxide with reduced glutathione. Induction of GSTs by chemopreventive agents can profoundly affect the metabolism and disposition of AFB1 in vivo, both in rats (34) and humans (35). In addition, the 52-fold greater GST activity towards aflatoxin in mice than rats appears to be the basis for the resistance of mice to the hepatocarcinogenic actions of this toxin (36). The catalytic efficiencies of AKR7A1 and AKR7A3 were determined in this study and compared with kinetic values derived from several other studies for specific recombinant rat and human CYP450s (12) and GSTs (13) involved in AFB1 metabolism (Table II). The available information suggests that both AKR7A1 and AKR7A3 have activities comparable with other AFB1-metabolizing enzymes, and that these AKRs have the potential to catalyze the reduction of AFB1 dihydrodiol to AFB1 dialcohol in the milieu of various competing pathways of AFB1 metabolism in vivo. Further comparative studies of these enzymes, singly and in combination, can be used to probe the relative contribution of these potentially competing metabolic pathways to the balance of activation and detoxification of AFB1.
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Acknowledgments |
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Notes |
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References |
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