The Reactive Oxygen Species- and Michael Acceptor-inducible Human Aldo-Keto Reductase AKR1C1 Reduces the alpha ,beta -Unsaturated Aldehyde 4-Hydroxy-2-nonenal to 1,4-Dihydroxy-2-nonene*

Michael E. BurczynskiDagger, Gopishetty R. Sridhar, Nisha T. Palackal, and Trevor M. Penning§

From the Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084

Received for publication, July 26, 2000, and in revised form, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human aldo-keto reductase AKR1C1 (20alpha (3alpha )-hydroxysteroid dehydrogenase) is induced by electrophilic Michael acceptors and reactive oxygen species (ROS) via a presumptive antioxidant response element (Burczynski, M. E., Lin, H. K., and Penning, T. M. (1999) Cancer Res. 59, 607-614). Physiologically, AKR1C1 regulates progesterone action by converting the hormone into its inactive metabolite 20alpha -hydroxyprogesterone, and toxicologically this enzyme activates polycyclic aromatic hydrocarbon trans-dihydrodiols to redox-cycling o-quinones. However, the significance of its potent induction by Michael acceptors and oxidative stress is unknown. 4-Hydroxy-2-nonenal (HNE) and other alpha ,beta -unsaturated aldehydes produced during lipid peroxidation were reduced by AKR1C1 with high catalytic efficiency. Kinetic studies revealed that AKR1C1 reduced HNE (Km = 34 µM, kcat = 8.8 min-1) with a kcat/Km similar to that for 20alpha -hydroxysteroids. Six other homogeneous recombinant AKRs were examined for their ability to reduce HNE. Of these, AKR1C1 possessed one of the highest specific activities and was the only isoform induced by oxidative stress and by agents that deplete glutathione (ethacrynic acid). Several hydroxysteroid dehydrogenases of the AKR1C subfamily catalyzed the reduction of HNE with higher activity than aldehyde reductase (AKR1A1). NMR spectroscopy identified the product of the NADPH-dependent reduction of HNE as 1,4-dihydroxy-2-nonene. The Km of recombinant AKR1C1 for nicotinamide cofactors (Km NADPH ~6 µM, Km(app) NADH >6 mM) suggested that it is primed for reductive metabolism of HNE. Isoform-specific reverse transcription-polymerase chain reaction showed that exposure of HepG2 cells to HNE resulted in elevated levels of AKR1C1 mRNA. Thus, HNE induces its own metabolism via AKR1C1, and this enzyme may play a hitherto unrecognized role in a response mounted to counter oxidative stress. AKRs represent alternative GSH-independent/NADPH-dependent routes for the reductive elimination of HNE. Of these, AKR1C1 provides an inducible cytosolic barrier to HNE following ROS exposure.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxidative stress plays a detrimental role in a number of pathological conditions, including cancer, atherosclerosis, and several neurodegenerative disorders (1-5). One of the main classes of cytotoxic and genotoxic substances produced during oxidative stress is alpha ,beta -unsaturated aldehydes generated endogenously as a break-down product of lipid peroxidation of omega -6 polyunsaturated fatty acids (6). Of these, HNE1 is one of the major unsaturated aldehydes produced and may be more deleterious than the initial production of ROS. This metastable reactive metabolite possesses a longer half-life than ROS and can diffuse from its sites of origin in the cellular membranes. Since HNE is a Michael acceptor, it attacks nucleophilic targets intracellularly and can act as a potent cytotoxin (for a review, see Ref. 6). In vivo, HNE immunoreactive adducts have been detected in atherosclerotic plaques (7), in the degenerating neurons of the substantia nigra affected by Parkinson's disease (8), and in the neurofibrillary tangles associated with Alzheimer's disease (9). HNE is also genotoxic, since it will readily form propano-dGuo adducts, which can lead to promutagenic lesions (10, 11). Related etheno adducts may be derived from the more reactive bifunctional electrophile 4-oxo-2-nonenal (12, 13), and whether this can be metabolically derived from HNE is unknown.

Mammalian cells have developed multiple enzymatic pathways for the detoxification of these lipid peroxidation by-products. The best characterized of these enzymes include the GSTs, aldehyde dehydrogenase, and alcohol dehydrogenase (14, 15). GSTs catalyze conjugation of GSH to HNE via Michael addition at the C-3 carbon, thereby preventing further nucleophilic addition to this toxic compound. Aldehyde dehydrogenase catalyzes the oxidation of HNE to the innocuous 4-hydroxy-2-nonenoic acid, while alcohol dehydrogenase catalyzes reduction of the terminal aldehyde to its alcohol, yielding the unreactive metabolite DHN. Recently two members of the AKR2 superfamily, aldose reductase and FR-1, were also shown to catalyze HNE reduction (16-19), thereby expanding the repertoire of enzyme families that contribute to detoxification of HNE in hepatic and extrahepatic tissues.

In previous studies, a 20alpha (3alpha )-hydroxysteroid dehydrogenase (AKR1C1), a member of the AKR1C subfamily, was found to be potently induced by Michael acceptors and ROS in human colon and hepatoma cell lines through an apparent antioxidant response element-type mechanism (20-22). However, the physiologic reason for the robust induction of this enzyme by electrophiles and ROS has remained unclear. The HSDs of this subfamily play important roles in steroid target tissues (for a review, see Ref. 23). Human AKR1C1-AKR1C4 are plastic enzymes and display 3-, 17-, and 20-ketosteroid reductase activity and 3alpha -, 17beta -, and 20alpha -hydroxysteroid oxidase activity (24, 25). This plasticity indicates that these enzymes can interconvert potent androgens, estrogens, and progestins with their cognate inactive metabolites and have the potential to regulate occupancy of steroid hormone receptors based on their tissue distribution. In the case of AKR1C1, all of these reactions occurred, but the most catalytically efficient is the interconversion of progesterone to its inactive metabolite, 20alpha -hydroxyprogesterone, suggesting that this is its physiological role (25).

Several HSDs in the AKR1C subfamily also play roles in xenobiotic metabolism by oxidizing PAH trans-dihydrodiol proximate carcinogens to reactive and redox-active o-quinones (26-29). However, the roles of AKR1C1 in steroid and PAH metabolism fail to provide a rationale for its isoform-specific induction by electrophiles and ROS.

In the present studies, we sought to identify an activity that might explain the robust induction of AKR1C1 during oxidative insult. We found that, like its closely related rat homolog AKR1C9 (30), AKR1C1 weakly catalyzed reduction of DHA to regenerate ascorbic acid. However, the low specific activity observed suggested that this was not the ROS counter-response catalyzed by AKR1C1. Subsequent studies revealed that HNE and other alpha ,beta -unsaturated aldehydes produced during lipid peroxidation were substrates of the inducible AKR1C1 isoform and identified DHN as the product of the AKR1C1-catalyzed HNE reduction. HNE was also found to directly induce AKR1C1 expression. The induction of AKR1C1 by HNE and ROS suggests that it may belong to a battery of genes (GST, gamma -glutamylcysteine synthetase, etc.) that mount a counter-response to ROS during periods of electrophilic and oxidative stress.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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Chemicals and Reagents-- Cell culture media and reagents were obtained from Life Technologies, Inc. Homogeneous recombinant AKR proteins, including aldehyde reductase (AKR1A1), were overexpressed in Escherichia coli and purified as described previously (28, 31, 32). beta -NADPH nucleotide cofactor was obtained from Roche Molecular Biochemicals. DHA, alkenals, and 2,3-dimethoxynaphthalene-1,4-dione were purchased from Sigma, and HNE was obtained from Calbiochem. Because HNE will self-polymerize on storage, only new lots of HNE were used in these experiments. All other chemicals used were of the highest grade available.

Chemical Synthesis of 1,4-Dihydroxynonene from 4-Hydroxy-2-nonenal-- DHN was synthesized by NaBH4 reduction of HNE. Briefly, a 4-fold molar excess of NaBH4 (25 µmol) was added dropwise to a solution of 1 mg HNE (6.4 µmol) in 4 ml of anhydrous methanol with continuous stirring over 10 min. Aliquots were removed over time and analyzed by TLC to monitor the progress of the reaction. Reactions were terminated by the addition of 1.0 ml of 0.1 M HCl to decompose unreacted NaBH4. The solution was extracted with 3 × 20 ml of dichloromethane, and the extract was dried over anhydrous sodium sulfate and reduced under vacuum at room temperature. The chemically synthesized DHN was separated from the starting material by TLC, extracted from silica, and resuspended in CDCl3 for subsequent NMR analysis.

Isolation and Characterization of the Product of HNE Reduction-- Large scale enzymatic incubations were conducted in 20 ml of 100 mM potassium phosphate, pH 6.0, containing 3.2 mM NADPH and 320 µM HNE solubilized in 2% methanol. Following the addition of the purified enzyme (100 µg) the reactions were incubated at 37 °C for 4 h and then terminated by extraction of the products with dichloromethane (three 20-ml aliquots). The organic solvent was dried with anhydrous sodium sulfate and then removed under reduced pressure. Following resuspension in dichloromethane, the resulting extract was subjected to TLC separation. DHN was separated from HNE by TLC in acetone-hexane (80:20, v/v). Detection was performed with methanol/sulfuric acid (1:1) reagent followed by heating. TLC plates were analyzed by densitometry on a Uniscan densitometer (Analtech, Newark, DE) to determine the chromatographic profile of HNE standard (Rf = 0.49), chemically synthesized DHN (Rf = 0.20), and the enzymatic product. The enzymatic product was eluted from the silica with methanol, evaporated to dryness, and resuspended in CDCl3 for NMR analysis.

NMR Spectroscopy-- 1H NMR spectra were obtained on a Bruker AM-250 spectrometer operating at 250 MHz, using CDCl3 as the solvent. All chemical shifts (delta H) are in ppm downfield from tetramethylsilane. The spectra were referenced to residual protonated solvent residues as internal standards; for CDCl3, delta H = 7.3 ppm. Analysis of HNE gave the following spectral assignments: delta  0.89 (t, 3H, 9-CH3); delta  1.25-1.67 (m, 4H and m, 4H, 5-CH2, 6-CH2, 7-CH2, 8-CH2); delta  3.65-3.76 (m, 1H, 4-CH); delta  5.83-6.05 (d, 1H, 2-CH JAB 3.0 Hz); delta  6.13-6.38 (t, 1H, 3-CH, JBA 1-2 Hz and JBX 1-2 Hz); delta  9.55-9.61 (d, 1H, 1-CHO). Analysis of chemically synthesized DHN and the AKR1C1 product gave the following spectral assignments, which were identical with the previously characterized spectral shifts of DHN (33): delta  0.89 (t, 3H, 9-CH3); delta  1.25-1.69 (m, 10H, 5-CH2, 6-CH2, 7-CH2, 8-CH2, and 2-OH); delta  4.10-4.21 (m, 3H, 1-CH2, 4-CH); delta  5.69-5.90 (m, 2H, 2-CH, 3-CH; gave the expected 10-line hyperfine splitting pattern based on a JAB, JABY, JABZ and JBA, JBX, coupling system). A residual CH2Cl2 shift (delta  = 5.29) was present in the chemically synthesized DHN, and a residual CH3OH shift (delta  = 3.44) was detected in the enzymatic product.

Spectrophotometric Assays and Kinetic Characterization-- Km and kcat values were obtained by varying the substrate concentration at constant saturating cofactor concentration (180 µM NADPH or 2.3 mM NADP+) in 1.0-ml systems containing 100 mM potassium phosphate buffer, pH 7.0, at 37 °C. Initial velocities at each substrate concentration were determined on a Beckman DU640 spectrophotometer by measuring the change in absorbance of pyridine nucleotide at 340 nm (epsilon  = 6220 M-1 cm-1). Actual values of the kinetic constants were determined using ENZFITTER to fit untransformed data (substrate concentration versus initial rate) to a hyperbolic equation to provide estimates of Km and Vmax and their associated S.E. values (34). Km(app) values for nicotinamide cofactors were obtained by varying NADH/NADPH concentration at a constant progesterone concentration (50 µM, limit of solubility) or by varying NAD+/NADP+ concentration at a constant 20alpha -hydroxyprogesterone concentration (50 µM, limit of solubility).

Cell Culture-- HepG2 hepatoma cells (passages 10-30) were maintained in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and were passaged every 4 days at 1:10 dilution. For induction studies, 48 h prior to treatment, 3 × 106 cells were seeded into 100-mm dishes containing fresh medium. Two days later (~50-60% confluency), cells were exposed to HNE. Aliquots (10 µl) of 1000× stock solutions in acetonitrile were added to 10 ml of fresh culture medium, and cells were incubated for the indicated times before harvesting.

RNA Isolation and Northern Analysis of AKR1C mRNA-- Cellular RNA was isolated using the Trizol® reagent. Total RNA (10 µg) was separated by electrophoresis on 1.0% agarose/formaldehyde gels and transferred overnight to Duralon-UV membranes (Stratagene). Membranes were prehybridized in hybridization buffer (50% formamide, 10% dextran sulfate, 1 M NaCl, and 1% SDS) with 100 µg/ml sheared salmon sperm DNA at 42 °C for 2 h. After prehybridization, membranes were hybridized to 107 dpm of an [alpha -32P]dATP probe corresponding to an 855-base pair EcoRI fragment of the human colon DD1 cDNA (pBluescript-hcDD kindly provided by Dr. Paul Ciaccio and Dr. Ken Tew). Signal intensities were measured using the PhosphorImager system (Molecular Dynamics, Inc., Sunnyvale, CA), and blots were exposed to x-ray film at -80 °C overnight. For purposes of normalization, blots were stripped and reprobed with a 780-base pair PstI/XbaI fragment of human GAPDH labeled by random priming as above.

Quantitative Isoform-specific Reverse Transcription PCR of AKR1C1 mRNA-- Total RNA (1 µg) from untreated and HNE-treated HepG2 cells was reverse transcribed into cDNA and subjected to 18 cycles of reverse transcription PCR amplification (94 °C for 45 s (denaturation), 60 °C for 45 s (annealing); 72 °C for 2 min (extension)) using a 1 µM concentration of the following isoform-specific primers: AKR1C1 (forward, 5'-dGTAAAGCTTTAGAGGCCAC-3'; reverse, 5'-dCACCCATGGTTCTTCTCGG-3') and beta -actin amplimers CLONTECH (25). Amplified products from various cycle numbers were fractionated on ethidium bromide-agarose gels and photographed under UV for comparison.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

AKR1C1 and AKR1C3 Possess Low but Detectable Dehydroascorbate Reductase Activities-- ROS can lead to uncontrolled GSH depletion, ascorbic acid oxidation, lipid peroxidation, and DNA damage unless cells mount an effective counter-response. A previous study reported that rat liver 3alpha -HSD (AKR1C9) functions as a dehydroascorbate reductase (30). Thus, human HSDs of the AKR1C subfamily were initially screened for their ability to catalyze the reduction of 1.5 mM DHA as a potential antioxidant activity. Interestingly, only two human isoforms that are induced by ROS and Michael acceptors (AKR1C1 and, to a lesser extent, AKR1C3) catalyzed the reduction of DHA to ascorbic acid. The low specific activities observed for the reduction of DHA (less than 8 nmol/min/mg) at millimolar substrate concentrations suggested that DHA was not the physiological substrate for the inducible AKR1C isoforms during periods of oxidative stress.

Reduction of 4-Hydroxy-2-nonenal by Multiple AKR Family Members-- One of the most common cytotoxic products of lipid peroxidation is HNE. Like prostaglandins, HNE is derived from arachidonic acid and possesses several structural characteristics in common with prostaglandins (Fig. 1). HNE (200 µM) was thus screened as a substrate for several human AKR1C subfamily members that share high sequence identity with bovine prostaglandin F synthase, which is involved in prostaglandin metabolism (Fig. 2). The ROS- and Michael acceptor-inducible AKR1C1 (20alpha (3alpha )-HSD) and its closely related (>98% amino acid identity) AKR1C2 (type 3 3alpha -HSD, bile acid-binding protein) homolog possessed the highest specific activities for HNE reduction of the six homogeneous recombinant AKRs studied. AKR1C3 (type 2 3alpha -HSD) and AKR1A1 (aldehyde reductase) possessed intermediate specific activities for HNE reduction, while the major 3alpha -HSD activities in human and rat liver, AKR1C4 (type 1 3alpha -HSD) and AKR1C9 (rat 3alpha -HSD), respectively, possessed low activity toward HNE. Of these enzymes, AKR1C3 has recently been shown to be identical to human prostaglandin F synthase (35). In this comparison, AKR1C1 had a specific activity that was 3 times greater than that seen with AKR1A1. In previous studies, AKR1C1 was shown to have a specific activity 300 times less than AKR1A1 (36), raising the issue as to why this discrepancy exists. In the earlier studies, assays were conducted with nonphysiologic amounts of HNE (4 mM). This led to a comparison of the catalytic efficiencies of AKR1C1 and AKR1A1 for HNE and an examination of whether HNE could inactivate either enzyme by Michael addition.



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Fig. 1.   Prostaglandins and 4-hydroxy-2-nonenal arise from arachidonic acid. Enzymatic and nonenzymatic processes convert arachidonic acid to prostaglandins and HNE, respectively.



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Fig. 2.   Reduction of 4-hydroxy-2-nonenal by AKRs. Homogeneous recombinant AKR1 enzymes (2 µg) were incubated in cuvettes containing 200 µM HNE as described under "Experimental Procedures." Reactions were initiated by the addition of enzyme. No detectable rate was observed in the minus cofactor and minus enzyme controls. Specific activities are expressed in nmol/min/mg.

Catalytic Efficiency of AKR1C1 for HNE-- Determination of kinetic constants for AKR1C1 at pH 7.0 and 37 °C revealed a Km of 34 µM and a kcat of 8.8 min-1 for HNE. The catalytic efficiency for HNE (kcat/Km = 0.27 × 106 M-1 min-1) observed with AKR1C1 was considerably higher than that anticipated with AKR1A1. AKR1A1 was not saturable at 1 mM HNE, and in fact v versus [S] plots were entirely linear, indicating that under the conditions of the highest substrate concentrations employed the velocity was still pseudo-first order. Thus, the catalytic efficiency is not measurable and must be considerably lower than that observed with AKR1C1. Preincubation of AKR1C1 and AKR1A1 with 4 mM HNE (under assay conditions reported in Ref. 36) led to a time-dependent inactivation of AKR1C1 only (data not shown). Thus, an explanation for the inadvertently high specific activity reported for AKR1A1 can be explained by its lack of saturation and lack of time-dependent inactivation, both of which are observed with AKR1C1 at 4 mM HNE. Importantly, we find that AKR1C1 is not inactivated in the presence of NADP+. Since cofactor protects AKR1C1 against this event, no inactivation occurs over the time course of the enzyme assay used to generate the steady-state kinetic parameters. When the catalytic efficiency of AKR1C1 for HNE is compared with its values for the NADP(H)-dependent oxidation/reduction of various physiological (20alpha -hydroxysteroids, prostaglandins) and xenobiotic (PAH trans-dihydrodiols) substrates (Table I), HNE is reduced with a catalytic efficiency similar to that of 20alpha -hydroxysteroids, which are the presumed substrates of AKR1C1 in vivo.


                              
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Table I
Comparison of catalytic efficiencies of AKR1C1 for various PAH trans-dihydrodiols, steroids, and 4-hydroxy-2-nonenal

Substrate Specificity of AKR1C1 for alpha ,beta -Unsaturated Aldehydes of Increasing Chain Length-- To investigate the specificity of the AKR1C1-catalyzed reduction of HNE, a series of unsaturated aldehydes (alkenals) of increasing chain length were also screened as substrates of AKR1C1. Calculation of catalytic efficiencies revealed that kcat/Km increased over 20-fold as the length of the carbon chain increased from 5 to 9 carbons (Table II). The predominant effects were on Km and were reflected in kcat/Km. Interestingly, the presence of the 4' hydroxyl group on 2-nonenal raises the Km ~5-fold, while kcat remained unchanged.


                              
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Table II
Substrate specificity of AKR1C1 for unsaturated aldehydes of increasing chain length
Assays were performed in 100 mM potassium phosphate buffer, pH 7.0, at 25 °C in the presence of 180 µM NADPH.

Km of Recombinant Human AKR1C1 for NADP+ and NADPH Cofactors-- To determine the nucleotide cofactor specificity of AKR1C1-catalyzed reduction of HNE, the ability of the enzyme to catalyze NADH- versus NADPH-dependent reduction of HNE was assessed (Fig. 3). AKR1C1 catalyzed HNE reduction with a 100-fold increase in activity using NADPH as cofactor compared with NADH. Kinetic constants of AKR1C1 for NAD(P)(H) were generated using 20alpha -hydroxyprogesterone and progesterone as a reversible oxidation/reduction substrate pair (Table III). AKR1C1 appears to be specific for the triphosphate-containing nicotinamide cofactors, with Km constants for NADP+ and NADPH in the low micromolar range. Since AKR1C1 fails to oxidize DHN using NADP+ as cofactor (data not shown), AKR1C1 appears to be a unidirectional NADPH-specific alpha ,beta -unsaturated aldehyde reductase. This suggests that AKR1C1 is primed to catalyze the NADPH-dependent reduction of alpha ,beta -unsaturated aldehydes.



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Fig. 3.   Specificity of AKR1C1 for NADPH. Using a standard spectrophotometric assay, increasing concentrations of homogeneous recombinant AKR1C1 (0.01-0.05 mg) were incubated with 200 µM HNE in the presence of 180 µM NAD(P)H. Initial rates for each enzyme concentration with each cofactor were calculated and converted to total activity in nmol/min.


                              
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Table III
Km constants of homogeneous recombinant AKR1C1 for nicotinamide cofactors

Identification of 1,4-Dihydroxy-2-nonene as the Product of the AKR1C1-dependent Reduction of 4-Hydroxy-2-nonenal-- Since members of the AKR1 family can possess either double bond reductase or aldehyde reductase activities (37), it was unclear whether AKR1C1 was catalyzing the reduction of the Delta 2-double bond or the terminal aldehyde in HNE or both. To distinguish between these possibilities, DHN was chemically synthesized, and its chromatographic profile was compared with that of the enzymatic product of the AKR1C1-catalyzed reaction. Thin layer chromatographic separation revealed that the AKR1C1-derived product (Fig. 4C) possessed an Rf value (0.20) identical to that of chemically synthesized DHN (Fig. 4B), and the products of both reactions coeluted when the products were mixed (Fig. 4D).



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Fig. 4.   Chromatographic identity of the product formed by the reduction of HNE catalyzed by AKR1C1 with the chemically synthesized DHN. HNE (320 µM) was incubated with NADPH (3.2 mM) in the presence of AKR1C1 (0.5 mg) until the reaction was 50% complete. The products of the reaction were then extracted and separated by TLC, and Rf values were compared with those obtained for the chemically synthesized DHN. A, HNE starting material. B, HNE and chemically synthesized DHN standards. C, AKR1C1-dependent reaction at 50% completion. D, co-chromatography of the AKR1C1 product and DHN.

To establish the structure of the AKR1C1-derived metabolite, the enzymatic product was extracted, isolated by TLC, and subjected to NMR spectroscopy (Fig. 5). The product of the enzymatic reaction gave identical chemical shifts to those assigned to the synthetic standard (Fig. 5, compare B and C). Importantly, there was loss of the aldehydic proton (delta  ~9.5 ppm) and retention of the vinylic protons of the Delta 2-double bond (delta  ~5.8 ppm), yielding a 10-line hyperfine splitting pattern characteristic of a JAB, JABY, JABZ and JBA, JBX, coupling system (Fig. 5, B and C), establishing the identity of the enzymatic product as DHN.



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Fig. 5.   Confirmation that DHN is the enzymatic product of AKR1C1 reduction of HNE by NMR spectroscopy. The product of the AKR1C1-dependent reaction was extracted, subjected to TLC purification, and then analyzed by high field proton NMR. In the NMR spectrum of both chemically and enzymatically synthesized DHN, note especially the loss of the aldehydic proton at ~10 ppm and the retention of the vinylic protons at ~5.8-6.2 ppm with new resonances, which gave the predicted 10-line splitting pattern for a JAB, JABY, JABZ and JBA, JBX, coupling system. A, HNE starting material. B, chemically synthesized DHN standard. C, enzymatically synthesized product. (The inset in A shows the structure of HNE and the splitting patterns for its methine protons; the inset in B shows the structure of DHN and the splitting patterns for its vinylic protons.)

Induction of Human AKR1C1 by HNE-- Since human AKR1C1 is inducible by extracellular ROS (H2O2), intracellular ROS (2,3-dimethoxynaphthalene-1,4-dione) and exogenous Michael acceptors (ethacrynic acid), we also sought to determine whether the ROS-derived, endogenous Michael acceptor HNE could also stimulate the expression of AKR1C1. Exposure of HepG2 cells to HNE for 24 h caused a concentration-dependent induction of AKR1C mRNA expression as measured by Northern blot analysis (Fig. 6A). Equal loading of total RNA was verified by reprobing the blot for the expression of GAPDH. A concurrent time-dependent induction of AKR1C enzyme activity was observed in HepG2 cell lysates following HNE treatment as measured by 1-acenapthenol oxidation (Fig. 6B).



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Fig. 6.   Induction of AKR1C mRNA and enzymatic activity by HNE. A, Northern blot analysis of AKR1C mRNA. HepG2 cells (3 × 106) were seeded into 10-cm dishes, and 1 day later they were replaced with serum-free medium. Following serum withdrawal for 24 h, HNE (0-100 µM) was added in a final concentration of 0.1% ethanol. At various time points, total RNA was harvested. RNA (5 µg) from the various treatments was electrophoresed, transferred to membranes, and sequentially analyzed for AKR1C and GAPDH expression as described under "Experimental Procedures." Only the levels of the AKR1C transcript are shown. B, induction of AKR1C1 enzymatic activity by HNE. HepG2 cells were exposed to 10 µM HNE, and lysates were prepared over time and assayed for 1-acenapthenol oxidation (22). Protein content for purposes of normalization was determined by the Bradford method. Data are expressed as -fold induction of AKR1C1-specific activity relative to the time 0 control.

To verify that the ROS- and Michael acceptor-inducible AKR1C1 was responsible for the global increases in AKR1C mRNA caused by HNE, first strand cDNA libraries from untreated and HNE-treated HepG2 mRNA were PCR-amplified with isoform-specific primers for AKR1C1 and beta -actin (Fig. 7). Isoform-specific semiquantitative reverse transcription PCR demonstrated that AKR1C1 was the AKR isoform induced following a 6-h exposure of HepG2 cells to 10 µM HNE. By contrast, primer pairs for AKR1C3, an isoform that shows modest induction by ROS, failed to detect induction of this isoform by HNE (data not shown). In previous experiments, we have shown that AKR1A1 is not induced by ROS or Michael acceptors (22).



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Fig. 7.   Isoform-specific induction of the ROS- and Michael acceptor-inducible AKR1C1 isoform by HNE. HepG2 cells were treated with Me2SO (-) or 10 µM HNE (+), and total RNA was harvested after 6 h. RNA (1 µg) from each sample was reverse-transcribed into cDNA and then subjected to linear PCR amplification (12-24 cycles) using specific primer pairs for AKR1C1 and beta -actin. PCRs were electrophoresed and visualized with ethidium bromide under UV light.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxidative stress induces the expression of a battery of human enzymes by signaling to antioxidant response elements/electrophilic response elements present in the 5'-flanking region of their respective genes. Regulated genes include GSTs (38, 39), gamma -glutamylcysteine synthetase (40, 41), metallothionein (42, 43), and NQO1 (44, 45). The proteins encoded by these inducible genes often represent either phase II (de)toxification enzymes (NQO1, GST) or enzymes involved in antioxidant defense (gamma -glutamylcysteine synthetase, metallothionein). The pattern of induction of AKR1C1 by monofunctional and bifunctional inducers and oxidative insult strongly implies that AKR1C1 is another member of the antioxidant response element/electrophilic response element-inducible battery of genes (22). However, the reason for the robust induction of AKR1C1 by GSH-depleting electrophiles (e.g. ethacrynic acid) and/or ROS was unclear. Based on these observations, we determined whether AKR1C1 and/or other closely related human AKRs might either 1) regenerate endogenous antioxidants in the cell or 2) detoxify endogenous toxic metabolites generated during oxidative stress.

Several antioxidant vitamins are protective during oxidative stress, including alpha -tocopherol (46) and ascorbic acid (47, 48). The closely related 3alpha -HSD from rat liver (AKR1C9) was previously shown to catalyze the reduction of DHA to ascorbic acid (30). Since such an activity could contribute to the regeneration of an endogenous antioxidant in cells, we determined whether any human AKRs might also catalyze the reduction of DHA to ascorbic acid. Interestingly, DHA reduction was identified as an enzymatic activity of the inducible AKR1C1 and AKR1C3 isoforms. However, the low specific activity (~8 nmol/min/mg) observed at high (1.5 mM) concentrations of DHA made it unlikely that DHA represents a physiologically relevant substrate of AKR1C1 in vivo.

By contrast, HNE appears to be a physiological substrate of AKR1C1 with a catalytic efficiency similar to that of the 20alpha -hydroxysteroids. The various AKR1C isoforms displayed significant variability in their ability to catalyze HNE reduction, illustrating a notable dissociation of this function within the AKR1C subfamily. It is not surprising that HNE is also a substrate of AKR1C2, since AKR1C1 and AKR1C2 are 98% identical, with only a single conservative amino acid substitution within the active site. AKR1C1 may be preferentially induced by ROS and Michael acceptors over AKR1C2, which has a slightly higher specific activity for two reasons. First, in liver, AKR1C2 (bile acid-binding protein) will be saturated with bile acids, which act as potent inhibitors of AKR1C2 but are far weaker inhibitors of AKR1C1 (49, 50). Thus, AKR1C1 can continue to eliminate HNE in the presence of normal bile acid concentrations. As an aside, under conditions of cholestatic liver damage and/or bile acid duct ligation (51, 52), hepatic HNE metabolism is impaired, and this may result from inhibition of AKR1C1 by elevated bile acids. Second, AKR1C1 undergoes time-dependent inactivation by HNE and may serve a sacrificial role to eliminate HNE at high concentrations. Induction of AKR1C1 may be necessary to replenish AKR1C1 under these conditions.

AKR1C1 also demonstrated specificity for other alpha ,beta -unsaturated aldehydes. The catalytic efficiency of AKR1C1 increased over 20-fold as the length of the hydrocarbon backbone increased from five to nine carbons. This is almost entirely due to a decrease in Km and possibly reflects increased interaction of the hydrophobic substrate with hydrophobic residues predicted to line the substrate binding site (53).

The Km value of AKR1C1 for HNE (34 µM) is substantially lower than those of isozymes I and II of alcohol dehydrogenase, 250-1430 and 100 µM, respectively (54), which have long been considered to be the sole source of reductive HNE metabolism in many tissues. These Km values are important when it is considered that under normal conditions the HNE concentration is <1.0 µM but may rise significantly and approach concentrations of 100 µM under conditions of oxidative stress. These arguments would suggest that the Km constant for AKR1C1 is more favorable to handle physiological concentrations of HNE than that observed with isozymes I and II of alcohol dehydrogenase. The Km of AKR1C1 for HNE is, however, similar to that reported for the HNE-specific GST isoform GSTA4-4 (15) and the NADH-specific human class IV alcohol dehydrogenase (55). This warrants a comparison of the catalytic efficiencies of these enzymes to determine which is most important in HNE elimination.

Catalytic efficiencies of GSTA4-4 are 500-fold greater than that observed with AKR1C1. Although GSTA4-4 has been characterized in human fetal brain, it has been argued that this isozyme is ubiquitously expressed in a manner similar to its rodent counterparts (15). In addition, GST-catalyzed conjugation of HNE has been described in human liver, heart, cornea, and retina (56). Thus, GST-catalyzed conjugation of GSH to HNE is undoubtedly an important elimination pathway. However, under conditions of oxidative stress, there is a concomitant decrease in GSH leading to a change in redox state (increased GSSG:GSH) making non-GSH-dependent pathways an important second barrier to the toxic effects of HNE. Importantly, under conditions of oxidative stress (H2O2) and drugs that deplete GSH (e.g. ethacrynic acid), AKR1C1 can be induced up to 10-fold, and this induction may be a component of an effective counter-response to this stress.

The recently described human class IV alcohol dehydrogenase possesses a kcat which is 2 orders of magnitude higher than that of the AKR1C1 isoform, suggesting that this enzyme is the better catalyst (57). However, the cytosolic concentrations of nicotinamide cofactors favor NAD+ over NADH and NADPH over NADP+, suggesting that NADPH-dependent reductases (i.e. AKRs) may be "primed" with the appropriate cofactor for HNE reduction. Calculation of the Km constant of AKR1C1 for NADPH (Km NADPH) demonstrates that AKR1C1 will be saturated with NADPH under physiological conditions and will probably possess sufficient affinity for HNE to contribute to its overall reductive elimination. Together, these observations suggest an important role of AKR1C1 in the elimination of the toxic HNE.

To date, the only other enzymes with a similar low Km constant for HNE are other AKR1 family members, aldose reductase and FR-1, which gave Km values of 22 and 9 µM, respectively (17-19). By contrast, AKR1A1 is not saturatable with these high concentrations of HNE (4 mM) and is unlikely to play a significant role in the reductive elimination of this metabolite. AKR1C1 is expressed at relatively high levels in multiple tissues associated with xenobiotic metabolism/oxidative stress, including lung, liver, and mammary gland (25). Importantly, AKR1C1 is the only known AKR with a low Km for HNE that is robustly induced by ROS and by agents that deplete GSH.

Given the multiplicity of potential HNE-derived metabolites, it was important to determine the identity of the product of the AKR1C1-dependent reduction of HNE. Certain AKR1 family members function as double bond reductases, and a single point mutation (H117E) is sufficient to introduce a double bond reductase activity into AKR1C9 (37). Whether AKR1C1 can function as a double bond reductase is unknown. Chromatographic separation and subsequent NMR spectroscopy confirmed the structure of the enzymatic product to be that of DHN. These findings verified that human AKR1C1 catalyzes reduction of the terminal aldehyde in HNE. Other experiments demonstrated that AKR1C1 does not function as an oxidoreductase with respect to HNE; it fails to oxidize DHN to HNE (Fig. 8). Thus, AKR1C1 is expected to yield a net accumulation of the innocuous metabolite DHN within the cell.



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Fig. 8.   AKR1C1 catalyzed reduction of HNE to DHN. The role of AKR1C1 in the reductive elimination of HNE is shown.

DHN is a well characterized metabolite of HNE in multiple tissues in vivo. DHN appears to represent between 10 and 20% of the total metabolic fate of HNE in hepatocytes, enterocytes, and in perfused kidney (58-60). It is likely that while alcohol dehydrogenases catalyze NADH-dependent reduction of HNE, various members of the AKR superfamily are responsible for the fraction of NADPH-dependent reduction of HNE in these and other tissues. Indeed, recent reports have identified aldose-reductase and/or closely related oxidoreductases as major activities involved in the detoxification of HNE in cardiac tissue homogenates (61).

HNE was also shown to be an inducer of AKR1C1. HNE has also been reported to weakly induce both aldose reductase and GST-P (3- and 1.7-fold, respectively), two other enzymes that are capable of catalyzing its metabolic elimination (62, 63). Thus, in addition to its cytotoxic and genotoxic properties, HNE appears to act as a toxic "second messenger," which induces the expression of enzymes involved in its own metabolism. Recently, HNE present in oxidized LDL was shown to be sufficient to stimulate/activate the EGF receptor present in endothelial cells (64). It is unknown whether this is the mechanism whereby HNE exerts its induction of AKR1C1 expression. Genestein, but not equimolar concentrations of the inactive analog dadzien, inhibits the ability of the redox-cycling quinone 2,3-dimethoxynaphthalene-1,4-dione to induce AKR1C1 mRNA levels in HepG2 cells.3 However, it is unclear whether this inhibition is mediated by the nonspecific antioxidant properties of genestein (65) or by a specific inhibitory effect on the EGFR.

Finally, the identification of HNE as a substrate of the ROS-inducible AKR1C1 isoform may explain, at least in part, its closely associated pattern of regulation with certain GST isoforms by GSH-depleting agents and GST inhibitors (20-22). While GST undoubtedly represents an important pathway in the detoxification of HNE, studies in H2O2-resistant hamster fibroblasts imply that GSH-dependent processes are not the sole determinants of H2O2 resistance (66). The studies presented here as well as by others (17-19) imply that members of the AKR superfamily act as a cytosolic barrier to the products of lipid peroxidation in the absence of GSH. Of these, AKR1C1 is the isoform that is induced by ROS and HNE as the counter-response to oxidative stress is mounted.


    FOOTNOTES

* This research was supported by National Institutes of Health Grants CA55711 and CA39504 (to T. M. P.) and a Pharmaceutical Research and Manufacturers of America Foundation Advanced Predoctoral Fellowship (to M. E. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a Bristol Myers Squibb Young Investigator Award for a preliminary account of this work at the 90th Annual Meeting of the American Association for Cancer Research, Philadelphia, PA, April 10-14, 1999. Present address: R. W. Johnson Pharmaceutical Research Inst., Route 202, P.O. Box 300, Raritan, NJ 08869.

§ To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Tel.: 215-898-1186; Fax: 215-573-2236; E-mail: penning@pharm.med.upenn.edu.

Published, JBC Papers in Press, November 11, 2000, DOI 10.1074/jbc.M006655200

2 The nomenclature for the aldo-keto reductase superfamily was proposed by Jez et al. and adopted at the 8th International Symposium on Enzymology and Molecular Biology of Carbonyl Metabolism in Deadwood, June 29 to July 3, 1996 (16). It is also described on the AKR superfamily home page on the World Wide Web.

3 M. E. Burczynski and T. M. Penning, unpublished data.


    ABBREVIATIONS

The abbreviations used are: HNE, 4-hydroxy-2-nonenal; AKR, aldo-keto reductase; ADH, alcohol dehydrogenase; DHA, dehydroascorbate; DHN, 1,4-dihydroxy-2-nonene; GST, glutathione S-transferase; HSD, hydroxysteroid dehydrogenase; 3alpha -HSD, 3alpha -hydroxysteroid dehydrogenase (EC 1.1.1.213: A-face specific); 20alpha -hydroxyprogesterone, 20alpha -hydroxypregn-4-en-3-one; PAH, polycyclic aromatic hydrocarbon(s); NQO1, NAD(P)H:quinone oxidoreductase 1; ROS, reactive oxygen species; PCR, polymerase chain reaction.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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