Biomedical subjects

T M Penning

Publications and source records attributed to T M Penning.

At least 73 records · Page 4Linked to original sources

PubMed1994

Three-dimensional structure of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase: a member of the aldo-keto reductase superfamily.

The 3.0-A-resolution x-ray structure of rat liver 3 alpha-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 alpha-HSD, EC 1.1.1.50) was determined by molecular replacement using human placental aldose reductase as the search model. The protein folds into an alpha/beta or triose-phosphate isomerase barrel and lacks a canonical Rossmann fold for binding pyridine nucleotide. The structure contains a concentration of hydrophobic amino acids that lie in a cavity near the top of the barrel and that are presumed to be involved in binding hydrophobic substrates (steroids, prostaglandins, and polycyclic aromatic hydrocarbons) and inhibitors (nonsteroidal antiinflammatory drugs). At the distal end of this cavity lie three residues in close proximity that have been implicated in catalysis by site-directed mutagenesis--Tyr-55, Asp-50, and Lys-84. Tyr-55 is postulated to act as the general acid. 3 alpha-HSD shares significant sequence identity with other HSDs that belong to the aldo-keto reductase superfamily and these may show similar architecture. Other members of this family include prostaglandin F synthase and rho-crystallin. By contrast, 3 alpha-HSD shares no sequence identity with HSDs that are members of the short-chain alcohol dehydrogenase family but does contain the Tyr-Xaa-Xaa-Xaa-Lys consensus sequence implicated in catalysis in this family. In the 3 alpha-HSD structure these residues are on the periphery of the barrel and are unlikely to participate in catalysis.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

Rat dihydrodiol dehydrogenase: complexity of gene structure and tissue-specific and sexually dimorphic gene expression.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) catalyzes a novel pathway of polycyclic aromatic hydrocarbon (PAH) metabolism in which trans-dihydrodiols (proximate carcinogens) are oxidized to reactive o-quinones which are cytotoxic and genotoxic. In this study, the complementary DNA for rat liver DD was used to examine the structure and regulation of the DD gene. Southern analysis of rat genomic DNA confirmed that DD is a member of the multigene aldo-keto reductase superfamily. Conservative estimates indicate that the rat DD gene is at least 20-25 kilobases in length. Northern analysis showed that the rat liver transcript was 2.4 kilobases whereas the complementary DNA contains an open-reading frame of 966 nucleotides. Primer extension of male and female polyadenylated RNA indicated that the major transcription start sites are only 53 and 54 base pairs upstream from the translation start site, confirming that the RNA has a very long 3'-untranslated region. In male and female tissues, 2.4 kilobase transcripts predominate in liver, small intestine, and lung, which is consistent with a role for the enzyme in PAH metabolism. Transcripts were also detected in male (prostate)- and female (ovary, mammary gland, and uterus)-specific tissues. In the ovary, two transcripts were observed of 2.4 and 1.4 kilobases in length. Using benzenedihydrodiol as a model substrate for PAH trans-dihydrodiols, highest levels of DD activity were observed in the liver and small intestine of both sexes. Enzyme activity is 2.5-fold higher in the female liver versus the male liver. This sexual dimorphism can be explained by increases in the DD mRNA and enzyme protein as measured by dot-blot and immunotitration analyses, respectively. The latter measurements indicate that DD represents 1.0% of the soluble protein in female liver but is only 0.5% of the soluble protein in male liver. Hormonal ablation (ovariectomy and hypophysectomy) abolishes the sexual dimorphism observed in levels of DD mRNA, enzyme protein, and enzyme activity. Administration of estrogens to males is sufficient to establish the female pattern of gene expression. These data indicate that DD gene expression is hormonally regulated, that estrogens exert their effect at the level of the mRNA, and that aldo-keto reductases involved in PAH metabolism may have their expression regulated by female sex hormones.

Alcohol Oxidoreductases↗

PubMed1993

Dihydrodiol dehydrogenase and its role in polycyclic aromatic hydrocarbon metabolism.

Dihydrodiol dehydrogenase(s) (DD) have been implicated in the detoxication of proximate (trans-dihydrodiol) and ultimate carcinogenic (anti-diol-epoxide) metabolites of polycyclic aromatic hydrocarbons (PAHs). These activities are catalyzed by soluble hydroxysteroid dehydrogenases and/or by aldehyde reductases. Molecular cloning indicates tha these enzymes have a high degree of sequence identity with members of the aldo-keto reductase super family. Substrate specificity studies indicate that non-K-region trans-dihydrodiols are the preferred substrates and that anti-dio-epoxides are not oxidized by the enzyme. The products of the DD reaction are transient catechols which auto-oxidize to PAH-o-quinones. As a consequence of this auto-oxidation superoxide anion, hydrogen peroxide and semiquinone radicals are generated. Studies on the biotransformation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene indicate that in subcellular fractions from uninduced rat liver, DD plays a significant role in the metabolism of this proximate carcinogen. Thus, the formation of benzo[a]pyrene-7,8-dione is only superseded by the formation of tetraols which are derived from the anti-diol epoxide of benzo[a]pyrene [anti-BPDE;(+/-)-anti-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene]. PAH-o-quinones produced by DD can inactivate the enzyme. These PAH-o-quinones also vary in their reactivity towards cellular nucleophiles, their cytotoxicity and their genotoxicity. Non-bay region and methylated bay-region PAH-o-quinones generated by DD are the most reactive Michael acceptors, and are also the most cytotoxic in hepatoma cells. Cytotoxicity results from the 1e- redox-cycling of the PAH-o-quinone, concomittant production of superoxide anion and a subsequent alteration in redoxstate. PAH-o-quinones are also genotoxic thus [3H]-benzo[a]pyrene-7,8-dione readily forms deoxyguanosine-adducts with native calf-thymus DNA, i.e., to the same extent as anti-BPDE. The cytotoxic and genotoxic properties of PAH-o-quinones suggest that DD may initiate a hitherto unrecognized pathway of PAH activation.

Alcohol Oxidoreductases↗

PubMed1993

Reactivity of benzo[a]pyrene-7,8-dione with DNA. Evidence for the formation of deoxyguanosine adducts.

Polycyclic aromatic hydrocarbon (PAH) o-quinones are products of the dihydrodiol dehydrogenase-catalyzed oxidation of trans-dihydrodiols which are proximate carcinogens. The PAH o-quinones are highly reactive molecules and have the potential to alkylate DNA. In this study, the reactivity of [3H](+-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene ([3H] (+/-)-anti-BPDE), [3H]benzo[a]pyrene-7,8-dione ([3H]BPQ) and [3H](+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene ([3H](+/-)-B[a]P-diol) with DNA were compared. (+/-)-anti-BPDE reacted equally well with native, deproteinated and deproteinated/sheared calf thymus DNA. In each case DNA adducts were formed which upon digestion to deoxyribonucleosides comigrated on reverse-phase (RP)-HPLC with adducts synthesized by reacting (+/-)-anti-BPDE with oligo-p(dG)10. (+/-)-anti-BPDE also reacted with plasmid (pGEM-3) DNA to yield multiple adducts one of which comigrated with the (+)-anti-BPDE-deoxyguanosine adduct. Under identical conditions [3H]BPQ reacted preferentially with native calf thymus DNA but displayed low reactivity with deproteinated and deproteinated/sheared calf thymus DNA. RP-HPLC analysis of deoxyribonucleoside-BPQ adducts indicated that the predominant adduct formed comigrated with a standard synthesized by reacting BPQ with oligo-p(dG)10. BPQ also reacted with pGEM-3 DNA to yield multiple adducts one of which comigrated with the BPQ-deoxyguanosine adduct. Reactions between [3H]BPQ and poly(dA), poly(dT), poly(dC) and oligo-p(dG)10 indicated that BPQ preferentially formed deoxyguanosine adducts. In this study, [3H]BPQ and [3H](+/-)-anti-BPDE covalently labeled native calf thymus DNA to an equal extent, however, less [3H]BPQ was recovered as deoxyguanosine adducts. By contrast, no covalent modification of calf thymus DNA, pGEM-3 DNA or oligonucleotides was observed with [3H](+/-)-B[a]P-diol. These studies indicate that BPQ has the potential to be genotoxic in vitro; that reactivity is heightened in the presence of protein or circular DNA and that the major adduct formed is a deoxyguanosine adduct.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1992

Polycyclic aromatic hydrocarbon (PAH) ortho-quinone conjugate chemistry: kinetics of thiol addition to PAH ortho-quinones and structures of thioether adducts of naphthalene-1,2-dione.

Polycyclic aromatic hydrocarbon (PAH) o-quinones are products of an NADP+ dependent oxidation of non-K-region trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase (EC 1.3.1.20). Since these PAH o-quinones could be detoxified by non-enzymatic or enzymatic conjugation with cellular thiols, their reactivity with 2-mercaptoethanol, cysteine and glutathione (GSH) was examined by ion-pair reverse phase high pressure liquid chromatography (RP-HPLC). Second-order rate constants for the addition of these thiols to naphthalene-1,2-dione (NPQ) in water ranging from 4.9 x 10(3) - 1.1 x 10(4) min-1 M-1 and the reactions were complete within 10 min. When these reactions were conducted at near physiological pH (50 mM potassium phosphate buffer pH 7.0), the rate constants increased by 2-orders of magnitude. When benzo[a]pyrene-7,8-dione (BPQ) was substituted in these reactions the second-order rate constants decreased by 2-3 orders of magnitude and the reactions took several hours to reach completion. The decrease in reactivity can be explained by the presence of the bay region in BPQ. Methylation influenced the reactivity of PAH o-quinones with GSH and the following order of reactivity was observed: 7,12-dimethyl-benz[a]anthracene-3,4-dione (7,12-DMBAQ) >> 12-methyl-BAQ, 7-methyl-BAQ and BAQ >> BPQ. Of these quinones 7,12-dimethyl-BAQ was almost equi-reactive with NPQ. This suggests that methyl substitution in the bay and peri regions enhances reactivity with GSH. Using NPQ as a model for other PAH o-quinones, N-acetyl-L-cysteine, L-cysteine and GSH conjugates of NPQ were synthesized and characterized by [1H]- and [13C]NMR. Evidence for Michael type 1,4-addition products was obtained in which the resultant adduct could exist as either a catechol or o-quinone. By contrast, L-cysteine was able to form adducts via S- or N-attack and N-attack gave a purple p-iminoquinone. There was no evidence for the formation of bis-N-acetyl-L-cysteinyl-, bis-glutathionyl adducts or phenolic coupled products. The toxicity of thiol conjugates of NPQ remains to be explored.

Benzopyrenes↗

PubMed1992

Hormonal regulation of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase in rat liver cytosol.

In rat liver, dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities are catalyzed by the same protein. This study shows that estrogen and growth hormone can induce the enzyme in hypophysectomized rats. This implies that estrogen can exert an effect on hepatic steroid and carcinogen metabolism in the absence of the anterior pituitary.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

Contribution of dihydrodiol dehydrogenase to the metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in fortified rat liver subcellular fractions.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) purified to homogeneity from rat liver cytosol will catalyze the NAD(P)(+)-dependent oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-diol) to yield benzo[a]pyrene-7,8-dione (BPQ). To verify that BPQ is a metabolite of B[a]P-diol in rat liver, an S100 fraction was supplemented with NAD+ and NADP+, and the formation of BPQ was followed by reverse-phase HPLC. The identity of BPQ was established by co-chromatography with an authentic standard (under different solvent conditions) and by RP-HPLC using a diode-array detector which established that the metabolite shared spectral identity with BPQ. The formation of BPQ in the S100 fraction was blocked by either a competitive inhibitor (indomethacin) or a suicide substrate [1-(4-nitrophenyl)-propen-1-ol] for DD, indicating that BPQ was being formed by this enzyme. To assess the contribution of DD to the metabolism of [3H]B[a]P-diol, subcellular fractions obtained from uninduced rat liver were fortified with co-factors to optimize the activity of enzymes that would compete for this proximate carcinogen. Under these conditions, S100 fractions fortified with NAD+ and NADP+ metabolized 25% of the B[a]P-diol, producing 731 +/- 154 pmol of BPQ. In contrast, rat liver microsomes fortified with an NADPH generating system metabolize 75% of the B[a]P-diol producing 2614 +/- 379 pmoles of benzo[a]pyrene-tetrahydrotetrols. Rat liver homogenates (S10) fortified with either uridine diphosphoglucuronic acid or phosphoadenosine phosphosulfate produced 180 +/- 56 and 95 +/- 31 pmoles of conjugates respectively, which were recovered as B[a]P-diol after treatment of the aqueous phase with either beta-glucuronidase or aryl sulfatase. Of the metabolites analyzed BPQ was formed in the second largest amount. These studies show that in uninduced rat liver DD may play a significant role in the metabolism of B[a]P-diol. The metabolic fate of BPQ remains to be determined.

Alcohol Oxidoreductases↗

PubMed1991

Development of affinity labeling agents based on nonsteroidal anti-inflammatory drugs: labeling of the nonsteroidal anti-inflammatory drug binding site of 3 alpha-hydroxysteroid dehydrogenase.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their effect by inhibiting the target enzyme cyclooxygenase (prostaglandin H2 synthase); however, little is known about the peptides comprising its NSAID binding site. Hydroxyprostaglandin dehydrogenases also bind NSAIDs, but their NSAID binding sites have not been well characterized. Using existing synthetic strategies, we have incorporated the bromoacetoxy affinity labeling moiety around the perimeter of two potent NSAIDs, indomethacin and mefenamate, a N-phenylanthranilate. The compounds synthesized were 1-(4-(bromoacetamido)benzyl)-5-methoxy-2-methylindole-3-acetic acid (1), 3-(2-(2-bromoacetoxy)ethyl)-1-(4-chlorobenzyl)-5-methoxy-2-methylindole (2), 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (3), N-(3-(bromoacetamido)phenyl)-anthranilic acid (4), and N-(4-(bromoacetamido)phenyl)anthranilic acid (5). To access whether these compounds have general utility in labeling NSAID binding sites, the compounds were evaluated as affinity labeling agents for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from rat liver cytosol. This enzyme displays 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase activity, is inhibited potently by NSAIDs, and is homologous to bovine lung prostaglandin F synthase. Compounds 1-5 were shown to affinity label the NSAID binding site of 3 alpha-HSD. They inactivated 3 alpha-HSD through an E.I complex in a time- and concentration-dependent manner with t1/2 values ranging from seconds to hours. Ligands that compete for the active site of 3 alpha-HSD (NAD+ and indomethacin) afforded protection against inactivation, and the inactivators could demonstrate competitive kinetics against 3 alpha-hydroxysteroid substrates by forming an E.NAD+.I complex.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗

PubMed1991

The kinetic mechanism catalysed by homogeneous rat liver 3 alpha-hydroxysteroid dehydrogenase. Evidence for binary and ternary dead-end complexes containing non-steroidal anti-inflammatory drugs.

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase that is potently inhibited at its active site by non-steroidal anti-inflammatory drugs (NSAIDs). Initial-velocity and product-inhibition studies performed in either direction at pH 7.0 are consistent with a sequential ordered Bi Bi mechanism in which pyridine nucleotide binds first and leaves last. This mechanism is supported by fluorescence titrations of the E-NADH complex, and by the failure to detect the binding of either [3H]androsterone or [3H]androstanedione to free enzyme by equilibrium dialysis. Dead-end inhibition studies with NSAIDs also support this mechanism. Initial-velocity studies with indomethacin show that this drug is an uncompetitive inhibitor against NAD+, but a potent competitive inhibitor against androsterone, indicating the ordered formation of an E.NAD+.indomethacin complex. Calculation of the individual rate constants reveals that the binding and release of pyridine nucleotide is rate-limiting and that isomerization of the central complex is favoured in the forward direction. Equilibrium dialysis experiments with [14C]indomethacin reveal the presence of two abortive NSAID complexes, a high-affinity ternary complex corresponding to E.NAD+.indomethacin (Kd = 1-2 microM for indomethacin) and a low-affinity binary complex corresponding to E.indomethacin (Kd = 22 microM for indomethacin). Since indomethacin has a low affinity for free enzyme, the formation of this abortive binary complex does not complicate kinetic measurements which are made in the presence of NAD+, but may contribute to the inhibition of the enzyme by NSAIDs. Using either pro-R-[4-3H]NADH or pro-S-[4-3H]NADH as cofactor, radiolabelled androsterone was formed only when the pro-R-[4-3H]NADH was used, confirming that purified 3 alpha-HSD is a Class A dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗

PubMed1991

Cloning and sequencing of the cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase.

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase which will terminate androgen action by converting 5 alpha-dihydrotestosterone to 3 alpha-androstanediol. It is identical to dihydrodiol dehydrogenase and it can function as a 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase. Its reactions are potently inhibited by the nonsteroidal anti-inflammatory drugs (NSAIDs). A cDNA (2.1 kilobases) for 3 alpha-HSD was cloned from a rat liver cDNA expression library in lambda gt11. Portions of the cDNA insert which contained an internal EcoRI site were subcloned into pGEM3, and dideoxysequencing revealed that the cDNA contains an open reading frame of 966 nucleotides which encode a protein of 322 amino acids with a monomer Mr of 37,029. The identity of this clone was confirmed by locating two tryptic peptides and two endoproteinase Lys-C peptides from purified 3 alpha-HSD within the nucleotide sequence. The amino acid sequence of rat liver 3 alpha-HSD bears no significant homology with 3 beta-, 17 beta- or 11 beta-hydroxysteroid dehydrogenases but has striking homology with bovine lung prostaglandin F synthase (69% homology at the amino acid level and 74% homology at the nucleotide level) which is a member of the aldehyde/aldose reductase family. This sequence homology supports previous correlates which suggest that in rat 3 alpha-HSD may represent an important target for NSAIDs. The nucleotide sequence also contains three peptides that have been identified by affinity labeling with either 3 alpha-bromoacetoxyandrosterone (substrate analog) or 11 alpha-bromoacetoxyprogesterone (glucocorticoid analog) to comprise the active site (see accompanying article (Penning, T. M., Abrams, W. R., and Pawlowski, J. E. (1991) J. Biol. Chem. 266, 8826-8834]. The sequence data presented suggests that 3 alpha-HSD, prostaglandin F synthase, and aldehyde/aldose reductases are members of a common gene family.

3-Hydroxysteroid Dehydrogenases↗

PubMed1991

Affinity labeling of 3 alpha-hydroxysteroid dehydrogenase with 3 alpha-bromoacetoxyandrosterone and 11 alpha-bromoacetoxyprogesterone. Isolation and sequence of active site peptides containing reactive cysteines; sequence confirmation using nucleotide sequence from a cDNA clone.

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) of rat liver cytosol is potently inhibited at its active site by nonsteroidal anti-inflammatory drugs (NSAIDs). Using 3 alpha-bromoacetoxy-5 alpha-androstan-17-one (BrAnd, a substrate analog) and 11 alpha-bromoacetoxyprogesterone (Br11P, a glucocorticoid analog) as affinity-labeling agents, kinetic evidence was obtained that these agents alkylate this site. Inactivation of 3 alpha-HSD with either [14C]BrAnd or [14C] Br11P led to the incorporation of 1 mol of affinity-labeling agent per enzyme monomer. Complete acid hydrolysis of 3 alpha-HSD radiolabeled with either agent followed by amino acid analysis led to the identification of [14C]carboxymethylcysteine indicating that [14C]BrAnd and [14C]Br11P covalently tag discrete reactive cysteine(s) at the enzyme active site. Trypsin digestion of [14C]BrAnd-inactivated 3 alpha-HSD followed by peptide mapping led to the purification of a single radiolabeled peptide (3A1) which gave the following sequence: H2N-Ser-Ile-Gly-Val-Ser-Asn-Phe-Asn-X-Arg-CO2H. Identical experiments on [14C] Br11P-inactivated 3 alpha-HSD led to the purification of three radiolabeled peptides (11P1-11P3). The major radiolabeled peptide (11P1) had an identical sequence to 3A1 which was tagged with [14C]BrAnd. The minor radiolabeled peptides had the following sequences: H2N-Ser-Lys-Asp-Ile-Ile-Leu-Val-Ser-Tyr-X-Thr-Leu-Gly-Ser-Ser-Arg-CO2H (11P2) and H2N-Ser-Pro-Val-Leu-Leu-Asp-Asp-Pro-Val-Leu-X-Ala-Ile-Ala-Lys-CO2H (11P3). In each peptide group X was identified as carboxymethylcysteine. Alignment of the peptide sequences with the primary structure of 3 alpha-HSD, deduced from its cDNA clone, assigned peptide 11P1 to residues 162-171, peptide 11P2 to residues 208-223, and peptide 11P3 to residues 232-246 of the amino acid sequence. The reactive cysteines correspond to Cys170, Cys217, and Cys242. We propose that Cys170 labeled by BrAnd may lie within the catalytic pocket of the enzyme. By contrast the 11 alpha-bromoacetoxy group in Br11P labeled several reactive cysteines which may be involved in the binding of glucocorticoids and NSAIDs.

3-Hydroxysteroid Dehydrogenases↗

PubMed1991

Isolation and partial characterization of a full-length cDNA clone for 3 alpha-hydroxysteroid dehydrogenase: a potential target enzyme for nonsteroidal anti-inflammatory drugs.

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD; EC 1.1.1.50) of rat liver cytosol is a monomeric (Mr 34000) NAD(P)+ dependent oxidoreductase which displays 9-, 11- & 15-hydroxyprostaglandin dehydrogenase activity. The enzyme is potently inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that 3 alpha-HSD may be a target enzyme for NSAIDs. A monospecific, polyclonal anti-sera raised against the purified enzyme was used to screen a lambda gt11 expression library and oligonucleotide probes complementary to the 5' and 3' ends of immunopositive clones were used to isolate a 2.1 kb full-length cDNA. Digestion of the full-length cDNA with Eco RI generated two fragments of 1.1 and 1.0 kb in length. Both fragments were subcloned into pGEM3 and partially sequenced. The 1.1 kb fragment contains the C-terminus of 3 alpha-HSD which was confirmed by an in-frame stop codon and comparison of the predicted amino acid sequence to peptide sequence obtained from two endo lys-C peptides of 3 alpha-HSD. The 1.0 kb fragment is 5' to the 1.1 kb fragment and is sufficient in length to contain the remainder of the entire open reading frame for 3 alpha-HSD. Dideoxysequencing reveals significant sequence homology with bovine lung prostaglandin PGF2 alpha synthase. These findings support the role 3 alpha-HSD in inflammation and suggest that hydroxysteroid dehydrogenases, hydroxyprostaglandin dehydrogenases and prostaglandin F2 alpha synthase may be members of a common gene family.

3-Hydroxysteroid Dehydrogenases↗

PubMed1991

Clues to the development of mechanism-based inactivators of 3 alpha-hydroxysteroid dehydrogenase: comparison of steroidal and nonsteroidal Michael acceptors and epoxides.

A series of steroidal and nonsteroidal Michael acceptors that represent reaction products for 3 alpha-hydroxysteroid dehydrogenase were synthesized and evaluated as potential enzyme-generated inactivators. Introduction of exocyclic olefins either at C-2 or C-6 produced inhibitors with high affinity for the enzyme (0.05 to 5.0 microM). However, despite this affinity, none of these compounds produced time-dependent inactivation of the enzyme. By contrast, analogs based on 1-phenyl-2-propen-1-one were stoichiometric inactivators of the enzyme and ease of turnover of the parent latent Michael acceptor depended on the presence of an electron-withdrawing substituent at the para position. A series of steroidal and nonsteroidal epoxides in which the oxiranyl oxygen could be substituted for the 3-ketone (the acceptor carbonyl of a steroid substrate) were also synthesized and evaluated as potential mechanism-based inactivators. Steroidal 2 alpha,3 alpha-, and 3 alpha,4 alpha-epoxides as well as 3 alpha- and 3 beta-spiroepoxides did not bind to the enzyme and were unable to cause enzyme inactivation in either the presence or absence of pyridine nucleotide. In contrast, nitrostyrene oxides produced time-dependent inactivation, the rate of which was governed by the presence of an electron withdrawing group at the para position. These data indicate that the design of mechanism-based inactivators for 3 alpha-hydroxysteroid dehydrogenase requires the incorporation of electron-withdrawing groups adjacent to the latent enzyme-activated group and, as a result, the turnover and/or reactivity of these compounds is increased. Moreover, these compounds can be modeled on nonsteroids.

3-Hydroxysteroid Dehydrogenases↗

PubMed1991

Hormonal control of Leydig cell differentiation.

Leydig cell progenitors contain significant concentrations of androgen receptors. When the metabolism of DHT to 3 alpha-DIOL is blocked, DHT stimulates testosterone production by Leydig cell progenitors, most probably via an androgen receptor dependent mechanism. Rapid metabolism by 3 alpha-HSD may limit the potency of exogenous DHT to stimulate differentiation of Leydig cell progenitors in vitro. Insulin-like growth factor-I enhances androgen production by purified immature Leydig cells. The elevated sensitivity of immature Leydig cells versus adult Leydig cells to IGF-I stimulation indicates that this peptide hormone has a role in their differentiation during puberty.

Androgens↗

PubMed1991

Mechanism based inhibition of hydroxysteroid dehydrogenases.

Steroid hormone action can be regulated not only at the receptor level but also by the enzymes that are responsible for the synthesis and degradation of biologically active steroids. Traditionally the pharmacological intervention of steroid hormone action has focused on the development of steroidal and nonsteroidal hormone receptor agonists and antagonists with appropriate pharmacokinetics. Recently, the development of selective inhibitors/inactivators of steroid metabolizing enzymes has gained momentum. This review will concentrate on the development of mechanism-based inhibitors for one class of steroid hormone transforming enzymes, the hydroxysteroid dehydrogenases.

Animals↗

PubMed1990

Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3 alpha-hydroxysteroid dehydrogenase.

The non-steroidal allylic and acetylenic alcohols 1-(4'-nitrophenyl)prop-2-en-1-ol (I) and 1-(4'-nitrophenyl)prop-2-yn-1-ol (II) are oxidized by homogeneous 3 alpha-hydroxysteroid dehydrogenase to the corresponding alpha beta-unsaturated ketones 1-(4'-nitrophenyl)prop-2-en-1-one (III) and 1-(4'-nitrophenyl)prop-2-yn-1-one (IV), which then inactivate the enzyme selectively with high affinity; low effective partition ratios are observed for the parent alcohols [Ricigliano & Penning (1989) Biochem. J. 262, 139-149]. Inactivation of 3 alpha-hydroxysteroid dehydrogenase by compound (I) displays an NAD+ concentration optimum. Scavenging experiments indicate that the enzyme-generated inactivators (III) and (IV) alkylate the enzyme via a release-and-return mechanism. Several lines of evidence suggest that compounds (III) and (IV) covalently modify the NAD(P)(+)-binding site. First, micromolar concentrations of NAD(P)H offer substantial protection against enzyme inactivation mediated by Michael acceptors (III) and (IV). In these protection studies Kd measurements for NAD(P)H approached those measured by fluorescence titration of free enzyme. Secondly, under initial-velocity conditions compounds (III) and (IV) act essentially as competitive inhibitors of NAD+ binding, and as mixed competitive or non-competitive inhibitors against androsterone binding. Thirdly, enzyme inactivated with either compound (III) or compound (IV) fails to bind to NAD+ affinity columns (e.g. Affi-gel Blue). Under the same conditions of chromatography native enzyme and enzyme affinity-labelled at the steroid-binding site with 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone is retained on the affinity column. A kinetic scheme that represents the inactivation of the homogeneous dehydrogenase by the enzyme-generated alkylators (III) and (IV) is presented.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Characterization of dihydrodiol dehydrogenase in human liver and lung.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) will oxidize non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons (PAHs), a reaction that can suppress the formation of PAHs) anti-diol epoxides or ultimate carcinogens. Using benzenedihydrodiol [(+/-)-trans-1,2-dihydroxy-3,5-cyclohexadiene] as a model substrate for trans-dihydrodiol metabolites of PAHs, 23 human liver and eight human lung samples were examined for enzyme activity. In human liver, enzyme activity could be measured spectrophotometrically and specific activities ranged from 0.16 to 6.1 nmol benzenedihydrodiol oxidized min/mg protein. Western blot analysis of human liver cytosol using rabbit anti-rat DD serum detected two bands of mol. wts 34,000 and 27,000. The former mol. wt is identical to that observed for the homogeneous rat liver enzyme. Gel-filtration experiments indicate that human liver DD activity elutes as a single peak and co-elutes with the purified rat liver enzyme, suggesting that the lower mol. wt species may be an artefact of degradation. Preparations of the human liver enzyme required NADP- for activity and were in general, insensitive to inhibition by dicoumarol, indomethacin and 6-medroxyprogesterone acetate. These properties distinguish the enzyme from alcohol dehydrogenase, quinone reductase and rat liver DD. In human lung, DD activity was barely detectable using a sensitive radiochemical assay in which the oxidation of benzenedihydrodiol to catechol is linked to catechol-O-methyl transferase using [3H]S-adenosyl methionine as methyl donor. Specific activities were approximately 1000th of that observed for human liver and ranged from 1 to 4 pmol benzenedihydrodiol oxidized/min/mg protein. Western blot analysis of lung cytosol detected three bands of mol. wts 34,000, 31,000 and 28,000. The relatively high levels of DD in human liver suggest that this enzyme may play an important role in PAH detoxication in this organ, while the low levels of DD in lung may contribute to the susceptibility of this tissue to PAH-induced carcinogenesis.

Adolescent↗

PubMed1989

Characterization of dihydrodiol dehydrogenase in rat H-4IIe hepatoma cells.

Dihydrodiol dehydrogenase (EC 1.3.1.20) catalyzes the NADP+-dependent oxidation of a variety of trans-dihydrodiol proximate carcinogens, a reaction that may suppress their carcinogenicity. Using benzenedihydrodiol [(+)-trans-1,2-dihydroxy-3,5-cyclohexadiene] as a substrate, this enzyme can be detected spectrophotometrically in rat H-4IIe hepatoma cells with a specific activity similar to that observed in rat liver cytosol. The hepatoma cell enzyme is potently inhibited by 6-medroxy-progesterone acetate (IC50 = 38 nM) and indomethacin (IC50 = 3.5 microM). These cells contain 3 alpha-hydroxysteroid dehydrogenase which is also sensitive to inhibition by the same two drugs. Chromatofocusing of hepatoma cell lysates indicates that both dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities coelute with a pI = 5.8. Western blot analysis of hepatoma cell lysates, using rabbit anti-rat 3 alpha-hydroxy-steroid/dihydrodiol dehydrogenase serum detects a single immunoreactive species with a Mr 34,000. Using this antiserum it was possible to immunotitrate both these enzyme activities in H-4IIe lysates. Exposure of confluent cells to either 10 microM benz[a]anthracene or 10 microM dexamethasone, which are known inducers in H-4IIe cells of aryl-hydrocarbon hydroxylase and tyrosine aminotransferase respectively, failed to elevate dihydrodiol dehydrogenase activity. The following agents also failed to induce dihydrodiol dehydrogenase activity: phenobarbital, ethoxyquin, phenolic anti-oxidants, testosterone, estradiol-17 beta, and growth hormone. Since the hepatoma cell enzyme has properties in common with the purified rat liver enzyme (which is identical to 3 alpha-hydroxysteroid dehydrogenase) including, Mr, pI, immunoreactivity, and sensitivity to drug inhibition, this cell line represents a useful system for studying the role of dihydrodiol dehydrogenase in the further metabolism of trans-dihydrodiols. Interestingly, the enzyme does not appear to be under the control of known inducers of phase I and phase II drug metabolizing enzymes.

Alcohol Oxidoreductases↗