The source of NADPH-dependent cytosolic 3-hydroxysteroid dehydrogenase (3-HSD) activity is unknown to date. This important reaction leads e.g. to the reduction of the potent androgen 5␣-dihydrotestosterone (DHT) into inactive 3-androstanediol (3-Diol). Four human cytosolic aldo-keto reductases (AKR1C1-AKR1C4) are known to act as non-positional-specific 3␣-/ 17-/20␣-HSDs. We now demonstrate that AKR1Cs catalyze the reduction of DHT into both 3␣-and 3-Diol (established by 1 H NMR spectroscopy). The rates of 3␣-versus 3-Diol formation varied significantly among the isoforms, but with each enzyme both activities were equally inhibited by the nonsteroidal anti-inflammatory drug flufenamic acid. In vitro, AKR1Cs also expressed substantial 3␣[17]-hydroxysteroid oxidase activity with 3␣-Diol as the substrate. However, in contrast to the 3-ketosteroid reductase activity of the enzymes, their hydroxysteroid oxidase activity was potently inhibited by low micromolar concentrations of the opposing cofactor (NADPH). This indicates that in vivo all AKR1Cs will preferentially work as reductases. Human hepatoma (HepG2) cells (which lack 3-HSD/⌬ 5-4 ketosteroid isomerase mRNA expression, but express AKR1C1-AKR1C3) were able to convert DHT into 3␣-and 3-Diol. This conversion was inhibited by flufenamic acid establishing the in vivo significance of the 3␣/3-HSD activities of the AKR1C enzymes. Molecular docking simulations using available crystal structures of AKR1C1 and AKR1C2 demonstrated how 3␣/3-HSD activities are achieved. The observation that AKR1Cs are a source of 3-tetrahydrosteroids is of physiological significance because: (i) the formation of 3-Diol (in contrast to 3␣-Diol) is virtually irreversible, (ii) 3-Diol is a pro-apoptotic ligand for estrogen receptor , and (iii) 3-tetrahydrosteroids act as ␥-aminobutyric acid type A receptor antagonists.Two classes of 3-hydroxysteroids, i.e. the ⌬ 5 -3-hydroxysteroids and the fully saturated 3-tetrahydrosteroids, represent pivotal intermediates in steroid hormone metabolism. In steroidogenic glands, ⌬ 5 -3-hydroxysteroid precursors are converted into ⌬ 4 -3-ketosteroids to produce active steroid hormones (1, 2), whereas 3-ketosteroid reduction of 5␣/5-dihydrosteroids into 3-tetrahydrosteroids is an important catabolic step in steroid hormone transformation.Human steroid hormone target tissues like the prostate express membrane attached and/or cytosolic 3␣-HSD 1 and 3-HSD activity (3-9). One key example of the catabolic function of these HSDs is the 3-ketosteroid reduction of the potent androgen 5␣-dihydrotestosterone (DHT, 17-hydroxy-5␣-androstan-3-one) into the inactive androgens 5␣-androstane-3␣,17-diol (3␣-Diol; Fig. 1) and 5␣-androstane-3,17-diol (3-Diol) (10 -12). In vivo, the formation of 3-Diol is virtually irreversible, whereas 3␣-Diol can be converted back to DHT via 3␣-hydroxysteroid oxidase activity (13-17). Reformation of DHT from 3-Diol is prevented, because 3-Diol is either irreversibly hydroxylated at the C-6 and/or C-7 position or ...
Human aldo-keto reductases (AKRs) regulate nuclear receptors by controlling ligand availability. Enzymes implicated in regulating ligand occupancy and trans-activation of the nuclear receptors belong to the AKR1C family (AKR1C1-AKR1C3). Nuclear receptors regulated by AKR1C members include the steroid hormone receptors (androgen, estrogen, and progesterone receptors) and the orphan peroxisome proliferator-activated receptor (PPAR␥). In human myeloid leukemia (HL-60) cells, ligand access to PPAR␥ is regulated by AKR1C3, which diverts PGD 2 metabolism away from J-series prostanoids (Desmond et al., 2003). Inhibition of AKR1C3 by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), caused PPAR␥-mediated terminal differentiation of the HL-60 cells. To discriminate between antineoplastic effects of NSAIDs that are mediated by either AKR1C or cyclooxygenase (COX) isozymes, selective inhibitors are required. We report a structural series of N-phenylanthranilic acid derivatives and steroid carboxylates that selectively inhibit recombinant AKR1C isoforms but do not inhibit recombinant COX-1 or COX-2. The inhibition constants, IC 50 , K I values, and inhibition patterns were determined for the NSAID analogs and steroid carboxylates against AKR1C and COX isozymes. Lead compounds, 4-chloro-N-phenylanthranilic acid and 4-benzoyl-benzoic acid for the N-phenylanthranilic acid analogs and most steroid carboxylates, exhibited IC 50 values that had greater than 500-fold selectivity for AKR1C isozymes compared with COX-1 and COX-2. Crystallographic and molecular modeling studies showed that the carboxylic acid of the inhibitor ligand was tethered by the catalytic Tyr55-OH 2 ϩ and explained why A-ring substituted N-phenylanthranilates inhibited only AKR1C enzymes. These compounds can be used to dissect the role of the AKR1C isozymes in neoplastic diseases and may have cancer chemopreventive roles independent of COX inhibition.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and procarcinogens that require activation by host metabolism. Metabolic activation of PAHs by AldoKeto-Reductases (AKRs) leads to formation of reactive and redox active o-quinones, which may cause oxidatively generated DNA damage. Spectrophotometric assays showed that NADPH caused PAH o-quinones to enter futile redox-cycles, which result in the depletion of excess cofactor. Copper (II) amplified NADPH-dependent redox-cycling of the o-quinones. Concurrent with NADPH oxidation, molecular oxygen was consumed, indicating the production of ROS. To determine whether PAH o-quinones can cause 8-oxo-dGuo formation in salmon testis DNA, three pre-requisite experimental conditions were satisfied. Quantitative complete enzymatic hydrolysis of DNA was achieved, adventitious oxidation of dGuo was eliminated by the use of chelex and desferal, and basal levels of less than 2.0 8-oxo-dGuo/10 5 dGuo were obtained. The HPLC-ECD analytical method was validated by spiking the DNA with standard 8-oxo-dGuo and demonstrating quantitative recovery. HPLC-ECD analysis revealed that in the presence of NADPH and Cu(II), submicromolar concentrations of PAH o-quinones generated > 60.0 8-oxo-dGuo adducts/10 5 dGuo. The rank order of 8-oxo-dGuo generated in isolated DNA was NP-1,2-dione > BA-3,4-dione > 7,12-DMBA-3,4-dione > BP-7,8-dione. The formation of 8-oxo-dGuo by PAH o-quinones was concentrationdependent. It was completely or partially inhibited when catalase, tiron or a Cu(I) specific chelator, bathocuproine were added, indicating the requirement for H 2 O 2 , O 2 − and Cu(I), respectively. Methional which is a copper-hydroperoxo complex (Cu(I)OOH) scavenger also suppressed 8-oxodGuo formation. By contrast, mannitol, sodium benzoate and sodium formate, which act as hydroxyl radical scavengers, did not block its formation. Sodium azide which can act as both a hydroxyl radical and 1 O 2 scavenger abolished the formation of 8-oxo-dGuo. These data showed that the production of 8-oxo-dGuo was dependent on Cu(II) /Cu(I) catalyzed redox cycling of PAH o-quinones to
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