Biomedical subjects

T M Penning

Publications and source records attributed to T M Penning.

At least 91 records · Page 5Linked to original sources

PubMed1989

Synthesis and evaluation of non-steroidal mechanism-based inactivators of 3 alpha-hydroxysteroid dehydrogenase.

Two non-steroidal mechanism-based inactivators for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) of rat liver have been synthesized: 1-(4'-nitrophenyl)-2-propen-1-ol (I), and 1-(4'-nitrophenyl)-2-propyn-1-ol (II). Both of these compounds inactivate homogeneous 3 alpha-HSD in a time- and concentration-dependent manner only in the presence of NAD+. Analysis of the pseudo-first-order inactivation data gave a Kd of 1.2 mM for the allylic alcohol and a t1/2 (time required to promote a 50% loss of enzyme activity) for the enzyme of less than 10 s at saturation. Similar inactivation studies with the acetylenic alcohol gave a Kd of 1.5 mM and a t1/2 for the enzyme of 9.9 min at saturation. The allylic alcohol and acetylenic alcohol are oxidized stereoselectively by the enzyme, yielding a Km of 2.0 mM and a Vmax. of 0.58 mumol/min per mg for the allylic alcohol and a Km of 0.75 mM and a Vmax. of 0.29 mumol/min per mg for the acetylenic alcohol. Effective partition ratios (kcat./kinact.) are low for both alcohols: for the allylic alcohol, 5.3; and for the acetylenic alcohol, 141. H.p.l.c. indicates that the Michael acceptors 1-(4'-nitrophenyl)-2-propen-1-one (III) and 1-(4'-nitrophenyl-2-propyn-1-one (IV) are the products of the enzymic oxidation of the corresponding alcohols. The latter compound (IV) was trapped as its monothioether adducts before h.p.l.c. analysis. The Michael acceptors III and IV inactivate the 3 alpha-HSD in the absence of NAD+ at a rate too high to accurately measure and titrate the enzyme in a stoichiometric manner. Enzyme inactivated by I and NAD+, II and NAD+, III or IV is not re-activated by gel filtration or dialysis, implying a stable covalent bond has been formed between the enzyme and the inactivators. A screen of five other HSDs, and two aliphatic alcohol dehydrogenases, indicates that alcohol I is a selective inactivator of rat liver 3 alpha-HSD. It is concluded that 3 alpha-HSD generates non-steroidal alkylating agents (III and IV) that potently inactivate the enzyme with low effective partition coefficients. This report of non-steroidal mechanism-based inactivators of 3 alpha-HSD may provide a precedent for the development of related compounds to act as suicide substrates of other HSDs.

3-Hydroxysteroid Dehydrogenases↗

PubMed1989

Non-K-region o-quinones as enzyme-generated inactivators of dihydrodiol dehydrogenase.

Homogeneous 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from rat liver cytosol catalyzes the NAD(P)+-dependent oxidation of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons, many of which are proximate carcinogens. These reactions proceed with Km values in the millimolar range to yield highly reactive o-quinones that can be trapped as thioether adducts [Smithgall, T. E., Harvey, R. G., & Penning, T. M. (1988) J. Biol. Chem. 263, 1814-1820]. The enzymatically generated o-quinones, e.g., naphthalene-1,2-dione and benzo[a]pyrene-7,8-dione are potent inhibitors of the dehydrogenase, yielding IC50 values of 5.0 and 10.0 microM, respectively. Naphthalene-1,2-dione was found to be an efficient irreversible inhibitor of the enzyme and can inactivate equimolar concentrations of the dehydrogenase, yielding a t 1/2 for the enzyme of 10 s or less. By contrast (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene promotes a slower inactivation of the dehydrogenase, yielding a Kd of 70 microM and a limiting rate constant that corresponds to a t 1/2 at saturation of 23.2 min. Inactivation by this dihydrodiol has an obligatory requirement for NADP+. Examination of the kcat for the oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene yields a partition ratio for the dihydrodiol of 200,000, suggesting that alkylation from the parent dihydrodiol is a rare occurrence. Benzo[a]pyrene-7,8-dione, which is the product of the enzymatic oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, also promotes a time- and concentration-dependent inactivation of the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗

PubMed1988

Electrophoretic and immunochemical characterization of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues.

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗

PubMed1988

Oxidation of the trans-3,4-dihydrodiol metabolites of the potent carcinogen 7,12-dimethylbenz(a)anthracene and other benz(a)anthracene derivatives by 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase: effects of methyl substitution on velocity and stereochemical course of trans-dihydrodiol oxidation.

The homogeneous 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase of rat liver cytosol catalyzes the NADP-dependent oxidation of a wide variety of polycyclic aromatic trans-dihydrodiols and has been implicated in their detoxification (T.E. Smithgall, R.G. Harvey, and T.M. Penning, J. Biol. Chem., 261:6184-6191,1986). This study examined the influence of methyl groups on the velocity and stereochemical course of enzymatic benz(a)anthracene (BA) trans-dihydrodiol oxidation. The racemic trans-3,4-dihydrodiols of BA and 7-methylbenz(a)anthracene (7-MBA) were completely consumed by the purified dehydrogenase, indicating that both stereoisomers are substrates. However, 50% of the ( +/- )-trans-3,4-dihydrodiols of 12-methylbenz(a)anthracene (12-MBA) and 7,12-dimethylbenz(a)-anthracene (DMBA) were oxidized, suggesting that only one stereoisomer is utilized in each case. At low substrate concentrations, enzymatic oxidation of the trans-3,4-dihydrodiols of BA, 12-MBA, and DMBA followed simple first-order kinetics. By contrast, oxidation of the 3,4-dihydrodiol of 7-MBA was of higher order, due to differences in the rate of oxidation of each stereoisomer. Rate constants estimated for each reaction indicate that the non-bay-region methyl group at position 7 has a greater enhancing effect on the rate of oxidation than the bay-region methyl group at position 12 (10- versus 4-fold, respectively). The 3,4-dihydrodiol of DMBA, which possesses both non-bay- and bay-region methyl groups, is oxidized more than 30 times faster than the unmethylated parent hydrocarbon. The absolute stereochemistry of the preferentially oxidized dihydrodiols was assigned by circular dichroism spectrometry. For the 3,4-dihydrodiols of DMBA and 12-MBA, the stereoisomer oxidized has the 3S,4S configuration. A large negative Cotton effect was observed in the circular dichroism spectrum of the 7-MBA 3,4-dihydrodiol which remained at the end of the rapid phase of oxidation of this racemic substrate, indicating that the dehydrogenase displays partial stereochemical preference for the 3S,4S enantiomer. These results suggest that methylation of BA at C-7 greatly enhances the oxidation of the 3S,4S-dihydrodiol, while the presence of a bay-region methyl group at C-12 completely blocks the oxidation of the 3R,4R-stereoisomer. Rapid, stereoselective oxidation of methylated polycyclic aromatic trans-dihydrodiols by this route in vivo may significantly influence their carcinogenicity.

3-Hydroxysteroid Dehydrogenases↗

PubMed1988

Spectroscopic identification of ortho-quinones as the products of polycyclic aromatic trans-dihydrodiol oxidation catalyzed by dihydrodiol dehydrogenase. A potential route of proximate carcinogen metabolism.

The homogeneous dihydrodiol dehydrogenase of rat liver cytosol catalyzes the NADP-dependent oxidation of polycyclic aromatic trans-dihydrodiols, a reaction that may suppress their carcinogenicity provided the products of the reaction are noncarcinogenic. This report demonstrates that the products of naphthalene and benzo[a]pyrene trans-dihydrodiol oxidation are electrophilic o-quinones, which arise via autoxidation of catechols produced from the dihydrodiols by the action of dihydrodiol dehydrogenase. Oxidation of the trans-1,2-dihydrodiol of naphthalene or the 7,8-dihydrodiol of benzo[a]pyrene by the homogeneous rat liver dehydrogenase in 50 mM glycine at pH 9.0 led to the formation of multiple products by TLC, none of which co-migrated with the corresponding o-quinone standards. An identical result was obtained when these standards were incubated with buffer alone, suggesting that o-quinones were formed enzymatically from the dihydrodiols, and then underwent addition reactions with the glycine buffer. In subsequent reactions, the o-quinones formed from the enzymatic oxidation of the trans-dihydrodiols of naphthalene and benzo[a]pyrene were trapped by conducting the reactions in phosphate buffer containing 2-mercaptoethanol. The products of these reactions were identified by 500 MHz nmr and electron impact mass spectrometry as adducts of the 1,2-quinone of naphthalene (m/e M+ = 234) and the 7,8-quinone of benzo[a]pyrene (m/e M+ = 358), which contained mercaptoethanol as a thioether at C-4 and C-10, respectively. Kinetic analysis of the reactivity of the 1,2-quinone of naphthalene showed that the cellular nucleophiles, cysteine and glutathione, react very rapidly with the quinone. The 7,8-quinone of benzo[a]pyrene also reacted with glutathione and cysteine to form water-soluble metabolites, but did not react with adenosine or guanosine. These results suggest that o-quinones formed by enzymatic dihydrodiol oxidation may be effectively scavenged by cellular nucleophiles, resulting in their detoxification.

Alcohol Oxidoreductases↗

PubMed1987

Prostaglandin dehydrogenase activity of purified rat liver 3 alpha-hydroxysteroid dehydrogenase.

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from rat liver cytosol displays 9, 11, and 15-hydroxyprostaglandin dehydrogenase activity. Using [14C]-PGF2 alpha as substrate the products of this reaction were separated by TLC and identified by autoradiography as PGE2 and PGB2. The purified enzyme catalyzes this reaction at a rate 200 times faster than cytosol. This corresponds to the rate enhancement observed when the enzyme is purified from cytosol using androsterone (a 3 alpha-hydroxysteroid) as substrate and suggests that it may represent a major 9-hydroxyprostaglandin dehydrogenase in this tissue. Although the 3 alpha-HSD has many properties in common with the 9-hydroxyprostaglandin dehydrogenase of rat kidney, rat kidney contains no protein that is immunodetectable with polyclonal antibody raised against the purified 3 alpha-HSD.

3-Hydroxysteroid Dehydrogenases↗

PubMed1987

Affinity-labelling of the anti-inflammatory drug and prostaglandin-binding site of 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol with 17 beta- and 21-bromoacetoxysteroids.

The homogeneous 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol binds prostaglandins with low micromolar affinity at its active site and is competitively inhibited by the non-steroidal and steroidal anti-inflammatory drugs [Penning, Mukharji, Barrows & Talalay (1984) Biochem. J. 222, 601-611]. To examine the portion of this binding site that accommodates the glucocorticoid side chain, we have synthesized 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone (BrDHT) and 21-bromoacetoxydesoxycorticosterone (BrDOC) as affinity-labelling agents. Both these agents promote rapid inactivation of the purified enzyme in a time- and concentration-dependent manner. Analyses of the inactivation progress curves gave estimates of Ki for the inactivators and half-life (t1/2) for the enzyme at saturation (tau) as follows: Ki = 33 microM and tau = 18 s for BrDHT, and Ki = 10 microM and tau = 203 s for BrDOC. Under initial-velocity conditions BrDHT and BrDOC act as competitive inhibitors, yielding Ki values identical with those measured in the inactivation experiments. Both indomethacin and prostaglandin E2 can protect the enzyme from inactivation, yielding Ki values for these ligands consistent with those measured independently by competitive-inhibition studies. These data confirm that the bromoacetoxysteroids label the active site, which is coincident with the prostaglandin- and anti-inflammatory-drug-binding site. Neither gel filtration nor extensive dialysis restores activity to the enzyme inactivated with either affinity-labelling agent. Use of radioactive BrDHT or BrDOC, in which either the steroid portion is labelled with 3H or the bromoacetate portion is labelled with 14C, indicates that inactivation is accompanied by a stoichiometric incorporation of 0.7-1.0 molecules of inhibitor per enzyme monomer. The linkage that forms between the dehydrogenase with either [14C]BrDHT or [14C]BrDOC is stable to acid and base treatment. Complete acid hydrolysis of the enzyme inactivated with [14C]BrDHT, followed by amino acid analyses, indicates that 87% of the radioactivity is eluted with carboxymethylcysteine. An almost identical result is obtained with [14C]BrDOC, where at least 75% of the radioactivity is eluted with this amino acid. Thus BrDHT and BrDOC alkylate at least one reactive cysteine residue at the active site that may be of functional importance in binding the glucocorticoid side chain.

3-Hydroxysteroid Dehydrogenases↗

PubMed1987

Radiochemical detection of dihydrodiol dehydrogenase: distribution of the enzyme in male Sprague-Dawley rat tissues and its sensitivity to inhibition by indomethacin and 6-medroxyprogesterone acetate.

Dihydrodiol dehydrogenase (EC 1.3.1.20) catalyzes the NADP+-dependent oxidation of (-)-7R,8R-dihydroxy-dihydro-benzo(a)pyrene and (+)-7S,8S-dihydroxy-dihydro-benzo(a)pyrene, which are potent proximate carcinogens (Smithgall, Harvey, and Penning, J. Biol. Chem., 261: 6184-6191, 1986). Using benzenedihydrodiol [(+)-trans-1,2-dihydroxy-3,5-cyclohexadiene] as a model substrate for these reactions, dihydrodiol dehydrogenase can be assayed in rat liver cytosol by measuring the change in absorbance of the pyridine nucleotide. This method lacks the sensitivity to detect the enzyme in extrahepatic tissues. Here we describe a sensitive radiochemical assay for dihydrodiol dehydrogenase in which the oxidation of benzenedihydrodiol to pyrocatechol is coupled to O-methylation catalyzed by catechol-O-methyltransferase (EC 2.1.1.6). In this manner the pyrocatechol formed in the oxidation step can be radiolabeled using S-adenosyl[methyl-3H]methionine as methyl donor. The resulting tritiated product, guaiacol, is readily extracted into toluene and quantified by scintillation counting. Using S-adenosyl[methyl-3H]methionine at a specific activity of 0.1 microCi/nmol, the assay provides a 5000-fold increase in sensitivity over the existing spectrophotometric method. The radiochemical assay was validated by comparing the Km and Vmax values obtained for the 40-75% (NH4)2SO4 fraction of rat liver cytosol with those measured spectrophotometrically. There was close agreement between values determined radiochemically (Km = 0.77 +/- 0.11 mM, Vmax = 2.14 +/- 0.13 nmol/min/mg protein) and determined spectrophotometrically (Km = 0.96 +/- 0.10 mM, Vmax = 6.31 +/- 0.50 nmol/min/mg protein). Using the radiochemical method, dihydrodiol dehydrogenase activity was detected in extrahepatic sites of polycyclic aromatic hydro-carbon metabolism: lung greater than small intestine greater than testis greater than bladder greater than prostate. Specific activities varied over 50-fold (0.866-0.017 nmol/min/mg protein) and did not show a strict inverse correlation with organ susceptibility to PAH-induced chemical carcinogenesis. Four tissues predominantly concerned with trans-dihydrodiol oxidation (liver, lung, small intestine, and testis) contain dihydrodiol dehydrogenase which is potently inhibited by indomethacin, two of these tissues (liver and small intestine) contain dehydrogenase sensitive to inhibition by 6-medroxyprogesterone acetate. These observations suggest that indomethacin and 6-medroxyprogesterone acetate may prevent the oxidation of trans-dihydrodiol proximate carcinogens in major tissues involved in their further metabolism and activation.

Alcohol Oxidoreductases↗

PubMed1986

Active-site directed inactivation of rat ovarian 20 alpha-hydroxysteroid dehydrogenase.

Rat ovarian 20 alpha-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition by catalysing the reduction of progesterone to give the progestationally inactive steroid 20 alpha-hydroxyprogesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestational maintaining agents, including the epimeric allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol and the related vinyl ketone 1-(3 beta-hydroxy-5 alpha-androstan-17 beta-yl)-2-propen-1-one. The vinyl ketone inactivates rat ovarian 20 alpha-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarose column chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivation plots gave a Ki of 2.0 microM for the inhibitor and a t1/2 for the enzyme of 20 s at saturation. These data indicate that the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis in the presence or absence of a competing nucleophile nor gel filtration reserves the inactivation, suggesting that a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20 alpha-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocity measurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketone can behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivation experiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrast the allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol are neither substrates nor inhibitors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivation observed with the vinyl ketone suggests that this compound may be useful as a progestational maintaining agent.

20-Hydroxysteroid Dehydrogenases↗

PubMed1986

Indomethacin and glucocorticoid metabolism in rat liver cytosol.

3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol catalyzes the second step in glucocorticoid metabolism, namely the NADPH-dependent reduction of 5 beta-dihydrocortisol to tetrahydrocortisol. The purified enzyme is potently inhibited by the nonsteroidal anti-inflammatory drugs [Penning and Talalay, Proc. natn. Acad. Sci. U.S.A. 80, 4504 (1983)], and the consequences of this inhibition on hepatic glucocorticoid metabolism are now examined. Analyses of the specific activities for the reduction of 5 beta-dihydro-cortisone catalyzed by homogeneous preparations of 3 alpha- and 3 beta-hydroxysteroid dehydrogenases of rat liver cytosol indicated that this steroid was predominantly reduced to the 3 alpha-hydroxysteroid (tetrahydrocortisone). Whether this reaction was catalyzed by the purified 3 alpha-hydroxysteroid dehydrogenase or unprocessed cytosol, it was potently inhibited by indomethacin (IC50 = 0.6 microM). In the presence of 30 microM indomethacin, the rate of reduction of 5 beta-dihydro-glucocorticoids was 16 times smaller than in the absence of drug. Measurement of 5 beta-reductase activity in rat liver cytosol indicated that it was 25-30 times less active than the 3 alpha-hydroxysteroid dehydrogenase. In the presence of 30 microM indomethacin, enough residual dehydrogenase may exist to reduce 5 beta-dihydro-glucocorticoids as they are formed. This was confirmed by incubating the supernatant fraction obtained from the 10,000 g centrifugation of a rat liver homogenate, with [1,2-3H]cortisol plus NADPH in the presence and absence of drug. At the end of the incubation, cortisol metabolites were analyzed by TLC and the results expressed as CL:CM ratio (cortisol left:cortisol metabolized). It was found that no change in this ratio occurred in the presence of the drug. Moreover, the addition of trapping quantities of 5 beta-dihydrocortisol to incubations containing 30 microM indomethacin were insufficient to promote an increase in cortisol levels as reflected by a rather small increase in the CL:CM ratio. Thus, nonsteroidal anti-inflammatory drugs cannot effect a rise in hepatic glucocorticoid levels in vitro, and do not promote a "glucocorticoid saving effect" in this organ.

3-Hydroxysteroid Dehydrogenases↗

PubMed1986

Regio- and stereospecificity of homogeneous 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons.

The homogeneous 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol is indistinguishable from dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol dehydrogenase EC 1.3.1.20), Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). Examination of the substrate specificity of the purified dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons indicates that the enzyme will catalyze the NAD(P)-dependent oxidation of trans-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, 5-methylchrysene, and benzo[a]pyrene under physiological conditions. Comparison of the utilization ratios Vmax/Km indicates that benzenedihydrodiol and the trans-1,2- and trans-7,8-dihydrodiols of 5-methylchrysene were most efficiently oxidized by the purified dehydrogenase, followed by the trans-7,8-dihydrodiol of benzo[a]pyrene and the trans-1,2-dihydrodiols of phenanthrene, chrysene, and naphthalene. The purified enzyme appears to display rigid regio-selectivity, since it will readily oxidize non-K-region trans-dihydrodiols but will not oxidize the K-region trans-dihydrodiols of phenanthrene and benzo[a]pyrene. The stereochemical course of enzymatic dehydrogenation was investigated by circular dichroism spectrometry. For the trans-1,2-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, and 5-methylchrysene, the dehydrogenase preferentially oxidized the (+)-[S,S]-isomer. Apparent inversion of this stereochemical preference occurred with the trans-7,8-dihydrodiol of 5-methylchrysene, as the (-)-enantiomer was preferentially oxidized. No change in the sign of the Cotton Effect was observed following oxidation of the racemic trans-7,8-dihydrodiol of benzo[a]pyrene, suggesting that both stereoisomers of this compound were substrates. Large-scale incubation of the [3H]-(+/-)-trans-7,8-dihydrodiol of benzo[a]pyrene with the purified dehydrogenase resulted in greater than 90% utilization of this potent proximate carcinogen, suggesting that the enzyme utilizes both the (-)-[R,R] and the (+)-[S,S]-stereoisomers, which confirms the circular dichroism result. These data show that dihydrodiol dehydrogenase displays the appropriate regio- and stereospecificity to catalyze the oxidation of both the major and minor non-K-region trans-dihydrodiols that arise from the microsomal metabolism of benzo[a]pyrene in vivo.

3-Hydroxysteroid Dehydrogenases↗

PubMed1986

Inhibition of trans-dihydrodiol oxidation by the non-steroidal anti-inflammatory drugs.

The homogeneous dihydrodiol dehydrogenase of rat liver cytosol (EC 1.3.1.20) reduces the mutagenicity of benzo[a]pyrene in the Ames test, suggesting that the enzyme may detoxify the trans-dihydrodiol proximate carcinogens formed from this compound. This report directly demonstrates for the first time that the purified dehydrogenase catalyzes the NADP-dependent oxidation of the potent proximate carcinogen [1,3-3H](+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at physiological pH. An initial velocity of 1.8 nmol [3H]trans-dihydrodiol oxidized/min/mg protein was observed at a substrate concentration of 20 microM. This potential detoxification reaction was potently inhibited by the nonsteroidal anti-inflammatory drug indomethacin, yielding an IC50 value of 10 microM. At higher drug concentrations (30 microM), the inhibition persisted for many hours. In an extension of this work, benzenedihydrodiol (trans-1,2-dihydroxy-3,5-cyclohexadiene) and naphthalenedihydrodiol (trans-1,2-dihydroxy-1,2-dihydronaphthalene) were synthesized as models of the scarce proximate carcinogen trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and examined as substrates. The Km for benzenedihydrodiol was 1.74 mM and the Vmax was 530 nmol substrate oxidized/min/mg protein, while the Km for naphthalenedihydrodiol was 10.71 mM and the Vmax was 268 nmol oxidized/min/mg protein; the former compound was oxidized to catechol. Eight different non-steroidal anti-inflammatory drugs were found to inhibit the oxidation of these model trans-dihydrodiols, yielding IC50 values comparable to or lower than peak plasma concentrations observed in man. These results suggest that therapeutically relevant concentrations of the non-steroidal anti-inflammatory drugs may inhibit the oxidation of trans-dihydrodiol proximate carcinogens by this route.

3-Hydroxysteroid Dehydrogenases↗

PubMed1985

Purification and properties of 3 alpha-hydroxysteroid dehydrogenase from rat brain cytosol. Inhibition by nonsteroidal anti-inflammatory drugs and progestins.

The 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat brain cytosol has been purified to apparent homogeneity. The purification procedure involves six successive steps, includes one affinity chromatography, and yields enzyme which displays a 1,550-fold enhancement in specific activity. The homogeneous enzyme has a Km of 8.0 microM for 5 alpha-dihydrotestosterone, a Vmax of 1.3 mumol of 3 alpha-androstanediol formed per h/mg of protein, and displays a preference for NADPH. It appears to be the major activity responsible for the reduction of 5 alpha-dihydrotestosterone in this tissue and may play a pivotal role in brain androgen metabolism. The homogeneous enzyme has several properties in common with the 3 alpha-hydroxysteroid dehydrogenase purified from rat liver cytosol (Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). It is a monomer with a molecular weight of 31,000, it has a pI of 5.5, and it is potently inhibited by the nonsteroidal anti-inflammatory drugs (IC50 value for indomethacin = 2.0 microM). The potency of inhibition observed for the brain enzyme parallels that observed for cyclooxygenase: indomethacin greater than fenamates greater than l-methylpyrrole acetic acids greater than arylpropionic acids greater than salicylates greater than acetaminophen. Examination of a variety of steroidal contraceptives as modulators of the dehydrogenase indicates that ethinylestradiol is a very poor inhibitor (IC50 = 100 microM), while 6-medroxyprogesterone acetate (Provera) is an extremely potent inhibitor (IC50 = 0.2 microM). The possibility exists that brain androgen metabolism may be altered by the nonsteroidal anti-inflammatory drugs and synthetic progestins.

3-Hydroxysteroid Dehydrogenases↗

PubMed1985

Potent inhibition of mammalian progesterone synthesis by 2 alpha-cyanoprogesterone.

2 alpha-Cyanoprogesterone potently inhibits the conversion of [3H]pregnenolone into progesterone catalysed by bovine corpora lutea, bovine adrenal cortex and human term placenta microsomes (microsomal fractions), yielding IC50 (concentration causing 50% inhibition) values of 66 nM, 120 nM and 700 nM respectively. By contrast, it is an exceedingly poor inhibitor of the isomerization of pregn-5-ene-3,20-dione, yielding IC50 values between 50 and 70 microM. On this basis, 2 alpha-cyanoprogesterone would appear to be an extraordinarily selective inhibitor of the 3 beta-hydroxysteroid dehydrogenase. Dixon plots indicate that it is a very-tight-binding competitive inhibitor of the corpus-luteum enzyme, yielding a Ki of 15 nM. In the bovine adrenal cortex and human placenta the steroid is less potent and inhibits the dehydrogenase non-competitively with Ki values of 150 nM and 1.0 microM respectively. Thus 2 alpha-cyanoprogesterone inhibits the corpus-luteum dehydrogenase with substantial selectivity. Because of its high affinity for the ovarian enzyme, the presence of low-micromolar concentrations of 2 alpha-cyanoprogesterone can promote a complete cessation of progesterone synthesis in corpora-lutea microsomes for several hours. Since this effect is observed in the presence of saturating concentrations of pregnenolone (50 microM), it is predicted that this inhibitor may be even more potent in vivo. 2 alpha-Cyanoprogesterone displays very low affinity for the human progesterone receptor, yielding a Kd of 600 nM as against a Kd of 1.6 nM for progesterone. It is suggested that 2 alpha-cyanoprogesterone may be a selective inhibitor of ovarian progesterone synthesis and may act as an effective anti-gestational agent in vivo.

3-Hydroxysteroid Dehydrogenases↗

PubMed1985

Indomethacin-sensitive 3 alpha-hydroxysteroid dehydrogenase in rat tissues.

The purified 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol is potently inhibited by the nonsteroidal anti-inflammatory drugs in rank-order of their therapeutic potency, i.e. by micromolar concentrations that would inhibit cyclooxygenase [T. M. Penning, and P. Talalay, Proc. natn. Acad. Sci. U.S.A. 80, 4504 (1983)]. In the present study, indomethacin-sensitive 3 alpha-hydroxysteroid dehydrogenase is shown to exist in seven rat tissues, including those that require androgens for growth (e.g. prostate) and those that rapidly metabolize prostaglandins (e.g. lung). Thus, the reduction of 5 alpha-dihydrotestosterone catalyzed by prostatic cytosol was potently inhibited by indomethacin (IC50 = 10 microM), while the reduction of 5 beta-dihydrocortisone catalyzed by liver, lung and testis was more sensitive to inhibition by this drug (IC50 1-3 microM). These data suggest that, under conditions in which cyclooxygenase is inhibited, androgen and cortisone metabolism may be affected. A surprising feature is that the specific activity of the indomethacin-sensitive dehydrogenase was higher in the lung than in tissues that are hormonally responsive (e.g. prostate and testis).

3-Hydroxysteroid Dehydrogenases↗

PubMed1985

Irreversible inhibition of delta 5-3-oxosteroid isomerase by 2-substituted progesterones.

2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates the isomerase after direct nucleophilic attack at the electropositive 2-position, and that tautomerization plays no role in the inactivation event. By contrast, 2-mercaptoethanol offers no protection against the inactivation mediated by 2-hydroxymethyleneprogesterone, and under the conditions of inactivation this compound appears to exist in the semi-enolized form.

Binding Sites↗

PubMed1985

Inhibition of 5 beta-dihydrocortisone reduction in rat liver cytosol: a rapid spectrophotometric screen for nonsteroidal anti-inflammatory drug potency.

The purified NAD(P)-linked 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol is powerfully inhibited by the nonsteroidal anti-inflammatory drugs in rank-order of their therapeutic potency. This observation has now been developed into a rapid screen for predicting the potency of these agents. The screening method requires the preparation of a rat liver cytosol and access to a recording spectrophotometer. By monitoring the NADPH-linked reduction of 5 beta-dihydrocortisone catalyzed by these cytosols in the presence of nonsteroidal anti-inflammatory drugs, a high correlation between their log IC50 values for this reaction with their recommended daily dose in humans can be obtained (r = 0.960; n = 10; p greater than 0.0001). This correlation scans four orders of magnitude and demonstrates the value of this method for screening the potency of these drugs. This method is superior to traditional predictive methods (e.g., measurement of cyclooxygenase inhibition), since this assay measures the change in absorbance of a pyridine nucleotide over 5 min and is considerably more rapid than the extraction, separation, and quantitation of prostaglandins. The method does not require enzyme purification, and the cytosol, which can be prepared in a matter of hours, can be stored at -80 degrees C and used routinely for screening purposes.

3-Hydroxysteroid Dehydrogenases↗

PubMed1985

Sex differences in indomethacin-sensitive 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol.

The 3 alpha-hydroxysteroid:nicotinamide adenine dinucleotide (phosphate) oxidoreductase (EC 1.1.1.50) of rat liver cytosol is indistinguishable from trans-1,2-dihydrobenzene-1,2-diol dehydrogenase (EC 1.3.1.20) (T.M. Penning, I. Mukharji, S. Barrows, and P. Talalay, Biochem. J., 222: 601-611, 1984) and has been implicated in the detoxification of ultimate carcinogens (H. R. Glatt et al., Science (Wash. DC), 215: 1507-1509, 1982). Using trans-1,2-dihydroxy-3,5-cyclohexadiene as a model substrate for trans-dihydrodiol proximate carcinogens, this study shows that the specific activity of 3 alpha-hydroxysteroid:nicotinamide adenine dinucleotide (phosphate) oxidoreductase is 2-fold higher in the 40-75% ammonium sulfate fraction prepared from female rat liver cytosol than in similar fractions prepared from males. Comparable differences were also observed for the nicotinamide adenine dinucleotide-dependent oxidation of 5 alpha-androstan-3 alpha-ol-17-one. Chromatofocusing of these cytosolic fractions separated the bulk of the protein from the dehydrogenase, which eluted as a single peak at pH 5.4. Examination of the protein profiles indicates that twice as much protein coeluted with the enzyme from female rat liver cytosol, suggesting that induction is responsible for the sex difference in enzyme activity. These differences were abolished by ovariectomy, while administration of a single dose of estradiol 3-sulfate (100 micrograms) to ovariectomized rats restored enzyme activity to within 90% of normal female levels. These findings suggest that ovarian estrogen is a natural inducer of rat liver 3 alpha-hydroxysteroid:nicotinamide adenine dinucleotide (phosphate) oxidase reductase/trans-1,2-dihydrobenzene-1,2-diol dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗