Inhibitory effects of diesel exhaust components and flavonoids on 20alpha-hydroxysteroid dehydrogenase activity in mouse tissues

The inhibitory effects of diesel exhaust components and flavonoids on 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity were examined in cytosolic fractions from the liver, kidney and lung of male mice. 9,10-Phenanthrenequinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), which are contained in diesel exhaust particles (DEPs), potently inhibited 20alpha-HSD activity in liver cytosol. 9,10-PQ also inhibited the enzyme activity in lung cytosol. However, 20alpha-HSD activity in kidney cytosol was little inhibited by 9,10-PQ or 1,2-NQ. Flavonoids such as quercetin, fisetin and kaempferol exhibited high inhibitory potencies for 20alpha-HSD activity in liver cytosol, whereas these flavonoids were poor inhibitors for the enzyme activity in kidney cytosol. It is likely that several diesel exhaust components and flavonoids augment the signaling of progesterone in the liver cells, by potently inhibiting 20alpha-HSD activity in mouse liver cytosol. The possibility that there are distinct enzymes catalyzing 20alpha-HSD activity in the non-reproductive tissues of male mice is also discussed.     

Inhibitory effects of flavonoids on the reduction of progesterone to 20alpha-hydroxyprogesterone in rat liver

The first aim of this study is to characterize the reduction of progesterone in rat liver. Progesterone was mainly reduced to 20alpha-hydroxyprogesterone in the cytosolic fraction of rat liver. The amount of 20alpha-hydroxyprogesterone formed from progesterone in the cytosolic fraction was significantly larger in the males than in the females and this enzyme reaction proceeded not only in the presence of NADPH, but also in the presence of NADH. Furthermore, we attempted to evaluate the inhibitory effects of 15 flavonoids on the NADPH-dependent reduction of progesterone to 20alpha-hydroxyprogesterone in liver cytosol of male rats. The order of the inhibitory potencies was luteolin>apigenin>quercetin>myricetin=fisetin=kaempferol. Other flavonoids exhibited lower inhibitory potencies. Energy-minimized molecular models demonstrated that a planar benzopyrone ring (A and C rings) with a coplanar phenyl ring (B ring) is a structural characteristic determining the inhibitory effects of flavonoids other than isoflavones.     

Inhibitory effects of diesel exhaust components and flavonoids on 20α-hydroxysteroid dehydrogenase activity in mouse tissues

The inhibitory effects of diesel exhaust components and flavonoids on 20α-hydroxysteroid dehydrogenase (20α-HSD) activity were examined in cytosolic fractions from the liver, kidney and lung of male mice. 9,10-Phenanthrenequinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), which are contained in diesel exhaust particles (DEPs), potently inhibited 20α-HSD activity in liver cytosol. 9,10-PQ also inhibited the enzyme activity in lung cytosol. However, 20α-HSD activity in kidney cytosol was little inhibited by 9,10-PQ or 1,2-NQ. Flavonoids such as quercetin, fisetin and kaempferol exhibited high inhibitory potencies for 20α-HSD activity in liver cytosol, whereas these flavonoids were poor inhibitors for the enzyme activity in kidney cytosol. It is likely that several diesel exhaust components and flavonoids augment the signaling of progesterone in the liver cells, by potently inhibiting 20α-HSD activity in mouse liver cytosol. The possibility that there are distinct enzymes catalyzing 20α-HSD activity in the non-reproductive tissues of male mice is also discussed.     

Several distinct enzymes catalyze 20alpha-hydroxysteroid dehydrogenase activity in mouse liver and kidney

The effects of flavonoids and quinones on NADPH- and NADH-dependent 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activities were examined in cytosolic fractions from the liver and kidney of mice. Judging from the data for the inhibition of NADPH- and NADH-dependent 20alpha-HSD activities by flavonoids and quinones, enzyme catalyzing renal NADPH-dependent 20alpha-HSD activity was found to be distinct from enzyme(s) catalyzing hepatic NADPH- and NADH-dependent 20alpha-HSD activities. Sulfobromophthalein (SBP) had little ability to inhibit hepatic NADPH-dependent 20alpha-HSD activity and bromophenol blue (BPB) exhibited a weak activation against the enzyme activity, whereas SBP and BPB were potent and moderate inhibitors, respectively, of hepatic NADH-dependent 20alpha-HSD activity. Thus, enzyme catalyzing hepatic NADPH-dependent 20alpha-HSD activity appeared to be distinct from enzyme catalyzing hepatic NADH-dependent 20alpha-HSD activity. The data for the pH profiles of hepatic NADPH- and NADH-dependent 20alpha-HSD activities also led us to the conclusion. Based on these results, we propose the possibility that several distinct enzymes catalyze NADPH- and NADH-dependent 20alpha-HSD activities in cytosolic fractions from the liver and kidney of mice.     

Microsomal 20 alpha-hydroxysteroid dehydrogenase activity for progesterone in human placenta

Placental 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity was studied in order to evaluate the mechanism of continuation of pregnancy and initiation of labor. The placentas obtained at various gestational weeks were homogenized and fractionated into "nuclear", "mitochondrial", "microsomal" and "supernatant" fractions. Each fraction was incubated with 14C-progesterone and a hydrogen donor. Enzymatic activity was measured by the conversion of progesterone to 20 alpha-dihydroprogesterone. The highest activity of 20 alpha-HSD for progesterone was found to be localized in "microsomal" fraction. The Km constant of 20 alpha-HSD was 4.5 X 10(-6)M for progesterone in "microsomal" fraction. It was found that placental microsomal 20 alpha-HSD required NADPH as well as NADH. 20 alpha-HSD activity for progesterone increased as gestational weeks advanced. The addition of DHA-sulfate and DHA inhibited 20 alpha-HSD activity for progesterone significantly, suggesting that the steroid produced by the feto-placental unit may be involved in the metabolism of progesterone in human placenta.     

Sex-related expression of 20alpha-hydroxysteroid dehydrogenase mRNA in the adult mouse.

The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone into its inactive form, 20alpha-hydroxyprogesterone. To gain information about the exact sites of 20alpha-HSD mRNA expression, we performed in situ hybridization using a (35)S-labeled cRNA probe in tissues of adult mice of both sexes. 20alpha-HSD mRNA was expressed in both male and female gonads. In the ovary, high expression was found in luteal cells of corpora lutea, while much lower expression could be detected in granulosa cells of growing follicles. In the testis, a specific hybridization signal was detected only in Leydig cells. In the female reproductive tract, 20alpha-HSD mRNA was found in the epithelial cells of the uterine cervix. In the adrenal cortex, only the zona reticularis exhibited specific radiolabeling, the expression being very high in the female and very low in the male. In the skin, specific labeling was restricted to sebaceous glands, the hybridization signal being much higher in the female than in the male. In the liver, 20alpha-HSD mRNA was found in hepatocytes, with a higher degree of expression in the female. In the kidney, specific labeling was observed in the epithelial cells of distal convoluted tubules, the signal being also much more striking in the female than in the male. In non-reproductive tissues, it clearly appears that the expression of 20alpha-HSD mRNA is higher in the female than in the male, suggesting that 20alpha-HSD may play an important role in reducing the intracellular concentration of progesterone originating from the circulation at a much higher level in the female.     

Purification and properties of human hepatic 3 alpha-hydroxysteroid dehydrogenase.

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5 beta-[3H]dihydrocortisol (5 beta-DHF) as substrate. Electrophoretically homogeneous enzyme was obtained using a procedure that involved ammonium sulfate precipitation and three successive column chromatography steps: DEAE-cellulose, hydroxylapatite and Blue-Sepharose. The enzyme is a monomer since the native molecular weight was found to be 37,000, using a calibrated Sephadex G-75 column, and the denatured subunit molecular weight was determined to be 38,500, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme had a pI of 5.6-5.9. The 3-ketosteroids: cortisol, testosterone, progesterone and androstenedione, were not substrates for 3 alpha-HSD indicating that a saturated 4,5 double bond was required for substrate activity. The conformation at the 5 position, however, did not influence substrate activity since 5 alpha- and 5 beta-DHF and 5 alpha-dihydrotestosterone were all reduced at similar rates. The purified enzyme preferred NADPH to NADH as a cofactor and showed a broad peak of activity in the pH range of 6.8-7.4. The apparent Km for 5 beta-DHF was 18 microM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4 degrees C. Freezing and thawing of the enzyme resulted in a large loss of activity during early stages of the purification. This is the first report of the purification to homogeneity of 3 alpha-HSD from human tissue.     

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.     

Exocyclic-keto reductase activities for progesterone and S-warfarin in hepatic microsomes from adult male rats.

Hepatic microsomes from adult male rats representing six inbred strains catalyzed quantitatively significant, NADPH dependent reductions of progesterone to the 20 beta (20R) alcohol and S-warfarin to its 11S-OH product. Microsomes from mature females and immature rats of both sexes were essentially devoid of these activities. Two strains of rat evidenced about 21% of these activities compared with the other strains and both activities were 25-81% repressed by treatment of rats with phenobarbital (PB). An excellent linear correlation was demonstrated for the two activities considering sex, age, NADPH much greater than NADH preference, PB-repression and strain differences. However, detergent latency (71%) and resistance to trypsinolysis were only observed for the keto-reductase activity with S-warfarin. Microsomes also catalyzed the reduction of progesterone to its 20 alpha-OH derivative but this activity preferred NADH greater than NADPH, was induced 2.7-fold by PB and was essentially independent of age, sex and animal strain. Furthermore, unlike the 20 beta-OH activity, this reduction was resistant to proteolytic inactivation.     

Microsomal carbonyl reductase responsible for reduction of 4-phenyl-3-buten-2-one in rats

trans-4-Phenyl-3-buten-2-one (PBO), a flavoring additive, was transformed to the carbonyl-reduced product, trans-4-phenyl-3-buten-2-ol (PBOL) by rat liver microsomes, but not by liver cytosol, in the presence of NADH or NADPH. PBOL formed was identified by comparison with an authentic sample. The reductase activity was not inhibited by quercitrin, an inhibitor of cytosolic carbonyl reductase. The carbonyl reduction product of PBO by liver microsomes was identified as the R-enantiomer of PBOL by HPLC analysis. Rat blood also exhibited the carbonyl reductase activity in the presence of NADH or NADPH, but to a lesser extent.     

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.     

Hormonal regulation of male-specific 20beta-hydroxysteroid dehydrogenase with carbonyl reductase-like activity present in kidney microsomes of rats.

Progesterone, 17alpha-hydroxyprogesterone, cortisone and cortisol, which are C(21)-steroids with a ketone group at the 20-position, potently inhibited the activity of enzyme acetohexamide reductase (AHR) responsible for the reductive metabolism of acetohexamide in kidney microsomes of male rats. Furthermore, progesterone was a competitive inhibitor of AHR. In the case of progesterone usage as the substrate, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) activity was much higher than 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity in kidney microsomes of male rats. These results indicate that AHR present in kidney microsomes of male rats, functions as 20beta-HSD with carbonyl reductase-like activity. In male rats, both testectomy and hypophysectomy decreased the renal microsomal 20beta-HSD activity, but the decreased enzyme activities were increased by the treatment with testosterone propionate (TP). We propose the possibility that TP treatment regulates the renal microsomal 20beta-HSD activity by acting directly on the kidney of male rats. This is supported from the fact that when TP was given to ovariectomized and hypophysectomized female rats, the male-specific 20beta-HSD activity was detected in their kidney microsomes.     

Reduction of tertiary amine N-oxides by liver preparations: function of aldehyde oxidase as a major N-oxide reductase

Reduction of tertiary amine N-oxides to the corresponding amines by liver preparations was investigated with imipramine N-oxide and cyclobenzaprine N-oxide under anaerobic conditions. Rabbit liver cytosol in the presence of an electron donor of aldehyde oxidase exhibited a significant N-oxide reductase activity which is comparable to the activity of the liver microsomes supplemented with NADPH. Rabbit liver aldehyde oxidase also exhibited the N-oxide reductase activity in the presence of its electron donor, indicating that the activity observed in the liver cytosol is due to this cytosolic enzyme. Furthermore, the tertiary amine N-oxide reductase activity of liver cytosols from rats, mice, hamsters and hogs was demonstrated by comparison with that of liver microsomes from these mammalian species.     

NAD (P) H-dependent reduction of nicotinamide N-oxide by an unique enzyme system consisting of liver microsomal NADPH-cytochrome C reductase and cytosolic aldehyde oxidase

NAD (P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide.     

The mobilization of ferritin iron by liver cytosol. A comparison of xanthine and NADH as reducing substrates.

Considerable evidence suggests that the release of iron from ferritin is a reductive process. A role in this process has been proposed for two hepatic enzymes, namely xanthine oxidoreductase and an NADH oxidoreductase. The abilities of xanthine and NADH to serve as a source of reducing power for the enzyme-mediated release of ferritin iron (ferrireductase activity) were compared with turkey liver and rat liver homogenates. The maximal velocity (Vmax.) for the reaction with NADH was 50 times greater than with xanthine; however, the substrate concentration required to achieve half-maximal velocity (Km) was 1000 times less with xanthine than with NADH. NADPH could be substituted for NADH with little loss in activity. Dicoumarol did not inhibit the reaction with NADH or NADPH, demonstrating that the ferrireductase activity with those substrates was not the result of the liver enzyme 'DT-diaphorase' [NAD(P)H dehydrogenase (quinone)]. A flavin nucleotide was required for ferrireductase activity with rat and turkey liver cytosol when xanthine, NADH or NADPH was used as the reducing substrate. FMN yielded twice the activity with NADH or NADPH, whereas FAD was twice as effective with xanthine as substrate. Kinetic comparisons, differences in lability and partial chromatographic resolution of the ferrireductase activities with the two types of reducing substrates strongly indicate that the ferrireductase activities with xanthine and NADH are catalysed by separate enzyme systems contained in liver cytosol. Complete inhibition by allopurinol of the ferrireductase activity endogenous to undialysed liver cytosol preparations and the ability of xanthine to restore equivalent activity to dialysed preparations indicate that the source of reducing power for the endogenous activity is xanthine. These studies suggest that xanthine, NADH or NADPH can serve as a source of reducing power for the enzyme-mediated reduction of ferritin iron, with a flavin nucleotide serving as the shuttle of electrons from the enzymes to the ferritin iron.     

Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.     

Participation of cytochrome b5 in CMP-N-acetylneuraminic acid hydroxylation in mouse liver cytosol

The activity of CMP-N-acetylneuraminic acid hydroxylase, that converts CMP-N-acetylneuraminic acid (CMP-NeuAc) to CPM-N-glycolylneuraminic acid (CMP-NeuGc), in mouse liver was determined by a newly developed HPLC method using non-radioactive CMP-NeuAc as a substrate. The activity was detected in the cytosol fraction but not in the microsomal fraction. Either NADH or NADPH was used as an electron donor by the cytosol enzyme, but NADH was much more efficiently used than NADPH. An antibody against cytochrome b5 markedly reduced the CMP-NeuAc hydroxylase activity when added to incubation mixture containing either NADH or NADPH as an electron donor. These data led us to postulate the following electron transport system, which is involved in the CMP-NeuAc hydroxylation in mouse liver cytosol: (formula; see text) where X, Y, and Z are components supposedly involved.     

Ubiquinone redox cycle as a cellular antioxidant defense system

Ubiquinone (UQ) reductase responsible for reduction of non-mitochondrial UQ was investigated in rats toward demonstrating an antioxidant role of UQ. In the liver, most of cellular UQ-10 reductase activity was attributable to a novel NADPH-UQ reductase in cytosol. The enzyme was not inhibited by dicumarol and rotenone, and had a Km of 19 microM for NADPH and 307 microM for NADH at the optimum pH 7.4. The enzyme was purified 300-fold to apparent homogeneity from the liver cytosol by an affinity chromatographic method. The purified enzyme reduced UQ-10 in lecithin liposomes, and protected the liposomes from lipid peroxidation. Furthermore, supplementation of rats with UQ-10 was observed to increase the enzyme level in their livers without affecting levels of other antioxidant factors. The observations suggested that cytosolic NADPH-UQ reductase is responsible for cellular UQ redox cycle as an endogenous antioxidant.     

Functional and immunological relationships between metyrapone reductase from mouse liver microsomes and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni.

3 Alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from Pseudomonas testosteroni was shown to reduce the xenobiotic carbonyl compound metyrapone (MPON). Reversely, MPON reductase purified from mouse liver microsomes and previously characterized as aldehyde reductase, was competitively inhibited by 3 alpha-HSD steroid substrates. For MPON reduction both enzymes can use either NADH or NADPH as co-substrate. Immunoblot analysis after native and SDS gel electrophoresis of 3 alpha-HSD gave a specific crossreaction with the antibodies against the microsomal mouse liver MPON reductase pointing to structural homologies between these enzymes. In conclusion, there seem to exist structural as well as functional relationships between a mammalian liver aldehyde reductase and prokaryotic 3 alpha-HSD. Moreover, based on the molecular weights and the co-substrate specificities microsomal mouse liver MPON reductase and Pseudomonas 3 alpha-HSD seem to be members of the short-chain alcohol dehydrogenase family.