Solubilization of 17β-hydroxysteroid dehydrogenase from rat liver microsomes

The conditions for the solubilization of 17β-hydroxysteroid dehydrogenase from a rat liver microsomal preparation with the non-ionic detergent Triton X-100 were studied. The recoveries of 17β-hydroxysteroid dehydrogenase activity and of proteins in the solubilized form were determined as a function of detergent concentration, of pH and temperature, of incubation time and of saline concentration. The soluble fraction obtained under the optimal conditions contained 80% of the proteins and 75% of the enzymatic activity of initial microsomes. The presence of Triton X-100 in the solubilized proteins was not essential for enzyme activity.     

Inhibition of human and rat 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances

Perfluoroalkylated substances (PFASs) including perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) have been classified as persistent organic pollutants and are known to cause reduced testosterone production in human males. The objective of the present study was to compare the potencies of five different PFASs including PFOA, PFOS, potassium perfluorooctane sulfonate (PFOSK), potassium perfluorohexane sulfonate (PFHxSK) and potassium perfluorobutane sulfonate (PFBSK) in the inhibition of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in the human and rat testes. Human and rat microsomal enzymes were exposed to various PFASs. PFOS and PFOSK inhibited rat 3β-HSD activity with IC₅₀ of 1.35 ± 0.05 and 1.77 ± 0.04 μM, respectively, whereas PFHxSK and PFBSK had no effect at concentrations up to 250 μM. All chemicals tested weakly inhibited human 3β-HSD activity with IC₅₀s over 250 μM. On the other hand, PFOS, PFOSK and PFOA inhibited human 17β-HSD3 activity with IC₅₀s of 6.02 ± 1.02, 4.39 ± 0.46 and 127.60 ± 28.52 μM, respectively. The potencies for inhibition of 17β-HSD3 activity were determined to be PFOSK > PFOS > PFOA > PFHxSK = PFBSK for human 17β-HSD3 activity. There appears to be a species-dependent sensitivity to PFAS-mediated inhibition of enzyme activity because the IC₅₀s of PFOS(K) for inhibition of rat 17β-HSD3 activity was greater than 250 μM. In conclusion, the present study shows that PFOS and PFOSK are potent inhibitors of rat 3β-HSD and human 17β-HSD3 activity, and implies that inhibition of steroidogenic enzyme activity may be a contributing factor to the effects that PFASs exert on androgen secretion in the testis.     

Effects of phthalates on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities in human and rat testes

The 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3β-HSD and 17β-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5μM for human 3β-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7μM for rat 3β-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2μM) and rat (IC(50)s, 30.24 and 9.1μM) 17β-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3β-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17β-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3β-HSD and 17β-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.     

Molecular cloning and expression of 17β-hydroxysteroid dehydrogenase type 2 gene in Hu sheep

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the NADP+-dependent oxidation of the most potent estrogen 17β-estradiol into the weak estrogen estrone, and the conversion of testosterone to androstenedione. It has been reported that 17β-HSD2 was expressed in many tissues in human, rats, however, the full-length sequence of 17β-HSD2 gene and its expression in ewe were still unknown. In this study, we cloned the full-length cDNA sequence and investigated mRNA differential expression in 28 tissues of 12 adult Hu-Sheep which were fed with high- and low- dietary intake. The 1,317 bp full-length cDNA sequence was first cloned. The coding region was 1,167 bp in length, and the monomer was estimated to contain 389 amino acid residues. It shares high AA sequence identity with that of bos Taurus (96.13 %), sus scrofa (77.06 %), canis lupus familiaris (70.44 %), Callithrix jacchus (65.72 %), Nomascus leucogenys (65.46 %), pan troglodytes (65.21 %), human (64.69 %), mus musculus (58.35 %), and a comparatively lower identity to danio rerio (37.85 %). 17β-HSD2 gene was high expressed in gastrointestinal (GI) tract, liver, but weakly expressed in other tissues. No detected expression was examined in lung. 17β-HSD2 gene expression was significantly difference in rumen, omasum, duodenum, cecum, hypophysis after high- and low- dietary intake. Results from the present study suggested that 17β-HSD2 plays a crucial role in almost all tissues protecting against excessive levels of active steroid hormone, and GI tract maybe an important steroid hormone metabolizing organ in Hu-Sheep. This present study is the first to provide the primary foundation for further insight into this ovine gene.     

Inactivation of rat ovarian 20α-hydroxysteroid dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides, varying in chain length from N-ethylmaleimide and N-butyl to N-octyl, inclusive, was shown to effectively inactivate rat ovarian 20α-hydroxysteroid dehydrogenase at pH 7.7, 25 °C. The apparent second-order rate constants for inactivation were observed to increase with increasing chain length of the N-alkylmaleimide used. Positive chain length effects were also indicated by the K_d values for N-alkylmaleimides calculated from double-reciprocal plots resulting from the saturation kinetics observed in the inactivation reactions. The maximum rate constant for inactivation at enzyme saturation was 0.3 min^?1 for each maleimide studied. NADP-and coenzyme-competitive inhibitors such as 3-aminopyridine adenine dinucleotide phosphate and various adenosine derivatives protected the enzyme against maleimide inactivation, whereas no protection was observed with the steroid substrate, 20α-hydroxypregn-4-en-3-one. The pH profile for maleimide inactivation indicated the involvement of an enzyme functional group with a pK_a near 8.0. Sulfhydryl modification was also indicated by fluorescein mercuric acetate inactivation and titration experiments. Inactivation of the enzyme by a lysine-modifying reagent exhibited a pH profile differing from that observed in the maleimide inactivation process. It is proposed that N-alkylmaleimides inactivate the enzyme through covalent modification of sulfhydryl groups located in a nonpolar region of the enzyme.     

The physiological significance of 11β-hydroxysteroid dehydrogenase in the rat lung

We have examined the interconversion of cortisone (E) and cortisol (F) in rat lung homogenate and microsomal fraction and in the isolated rat lung perfused with Krebs bicarbonate solution containing 4.5% albumin. In the perfused lung the apparent K_m was 5.1 μM E and the V_max was 9nmol·g⁻¹ · min⁻¹. The ability of the lung to reduce E to F was enhanced both by 7 days prior exposure of the rat to an ambient temperature of 2°C and by starvation of the rat for 3 days. The activity was inhibited by adrenalectomy and castration of 7 days duration. Whereas little steroid oxidation occurred in the perfused lung, preparations of lung homogenatcs and microsomal fraction readily reduced or oxidised the 11-position of the corticoid molecule depending on the preponderance of either NADPH or NADP, respectively. We conclude, that the predominance of the reductive reaction in the whole rat lung under physiological conditions reflects the very active pentose-phosphate shunt in the lung, which produces NADPH. We suggest that this ability of the lung to activate E to F may exert a fine control over the arterial concentrtion of unbound, physiologically active, 11-hydroxylated steroid.     

3α-Hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

Summary The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3-hydroxysteroid and the 3-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6.During the entire estrous cycle, 3-hydroxysteroid dehydrogenase and 3-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Antagonism of the estradiol-mediated repression of microsomal 3β-hydroxysteroid dehydrogenase activity in rat liver by antiestrogenic substances

5α-Dihydrotestosterone, 17-hydroxyprogesterone caproate, 2-methoxyestrone and a number of nonsteroidal antiestrogens (clomiphene citrate, nafoxidine hydrochloride, tamoxifen, MER-25) were tested for their ability to block estradiol-mediated repression of the androgen-dependent 3β-hydroxy-steroid dehydrogenase activity of male rat liver. With the exception of 5α-dihydrotestosterone, which induced activity in females, none of these substances affected 3β-hydroxy-steroid dehydrogenase activity when administered alone to otherwise untreated male and female rats. Tamoxifen (100 or 500 μg/day) was the only substance which prevented a decrease in enzyme activity when given simultaneously with estradiol (5 μg/day). The estradiol-mediated decrease in activity was not antagonized by a 100-fold higher dose of androgen (5α-dihydrotestosterone, 0.5 mg/day), demonstrating the potent antiandrogenic effect of estradiol on this hepatic androgen-dependent enzyme activity.     

Molecular cloning and developmental expression of rat fibroblast growth factor receptor 3

Fibroblast growth factors (FGFs) are involved in the control of a variety of biological functions including regulation and differentiation of various cell types. Furthermore, they play important roles in the processes of regeneration, angiogenesis, and chemotaxis. The family of FGF receptors (FGFRs) comprises four members, FGFR-1 to -4, which exist in several differentially expressed splice variants. Except for FGFR-3, primary structures and expression of the three other FGFRs have been described in the rat system. Although expression studies with heterologous probes of FGFR-3 from mice have been performed in the rat system, these analyses were limited and the complete set of receptors has not yet been revealed. To understand the developmental functions of FGFR-3, it is important to elucidate the expression pattern in embryos of different stages. In this study, we have isolated a cDNA of FGFR-3 from rat brain. Expression analyses by RT-PCR of adult rat revealed expression in several tissues, however, expression levels were highest in lung and brain. During embryonic development, FGFR-3 displays a diffuse expression in most tissues at embryonic day 14 (E14), as observed by in situ hybridization experiments. In E18 the expression pattern is more restricted, showing strong signals in spinal cord, dorsal root ganglia, cortex, chondrocytes, and endothelial cells. The temporal and spatial pattern of FGFR-3 expression suggests specific functions in several tissues during development.     

Structural assessment and identification of 11β-hydroxysteroid dehydrogenase type 1 inhibitors

Communicated by Ramaswamy H. Sarma     

Cloning and expression of cDNA encoding hamster liver 3-hydroxyhexobarbital/17β(3α)-hydroxysteroid dehydrogenase 1

Using RACE techniques we have cloned and sequenced one of the hamster liver 3-hydroxy-hexobarbital dehydrogenases which catalyze not only cyclic alcohols but also 17β-hydroxy-steroids and 3α-hydroxysteroids. The gene specific primers to 3-hydroxyhexobarbital dehydrogenase 1 (G2) were synthesized on the basis of its partial peptide sequences. The sequence of full length cDNA generated by 3′- and 5′-RACE PCR consisted of 1225 nucleotides including an open reading frame of 972 nucleotides encoding a protein of 323 amino acids. The deduced amino acid sequence matched exactly with the partial peptide sequences of hamster liver 3-hydroxyhexobarbital dehydrogenase 1 (G2). The sequence showed 84.5% identity to mouse liver 17β-dehydrogenase(A-specific), and 74–76% identity to human liver bile acid binding protein/3α-hydroxysteroid dehydrogenase (DD2), human liver 3α-hydroxysteroid dehydrogenase type I (DD4) and type II (DD3), and rabbit ovary 20α-hydroxysteroid dehydrogenase. The protein contains catalytic residues of aldo-keto reductases, Asp50, Tyr55, Lys84, His117. These results suggest that the hamster liver 3-hydroxyhexobarbital/17β(3α)-hydroxysteroid dehydrogenase belongs to aldo-keto reductase superfamily. The insert containing the full-length cDNA of 3-hydroxyhexobarbital dehydrogenase and vector specific overhang produced by PCR was annealed with pET-32 Xa/LIC vector. The plasmid was transformed into BL21 (DE3) cells containing pLysS. The recombinant enzyme was induced 1 mM IPTG. The expressed enzyme was produced as fusion protein and purified by nickel chelating affinity chromatography followed by POROS CM column chromatography and superdex 75 gel filtration. Molecular weight of the recombinant enzyme fused thioredoxin and his•tag was about 55 000 and that was 35 000 after Factor Xa protease treatment. The recombinant enzyme dehydrogenated 3-hydroxy-hexobarbital, 1-acenaphthenol, 2-cyclohexen-1-ol, testosterone, glycolithocholic acid as well as the native enzyme purified from hamster liver.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.     

Characterization of rat 17β-hydroxysteroid dehydrogenase type 1 gene and mRNA transcripts

In the present study, the gene encoding rat 17β-hydroxysteroid dehydrogenase type 1 (rHSD17B1 gene) was cloned and characterized. Like the analogous human gene (hHSD17B1), rHSD17B1 contains six exons and five introns spanning approximately 2.2 kb. The identity between the exons and introns of the two genes ranges from 58% to 82% and 42% to 57%, respectively. In contrast to hHSD17B1, rHSD17B1 is not duplicated. The cap site for rHSD17B1 was localized to position −41 upstream of the ATG translation initiation codon. Sequence comparison of the first 200 bp upstream of the cap site showed 72% identity between the human and rat HSD17B1 genes, including a conserved GC-rich area. Further upstream, no significant identity between the two genes was observed and several, cis-acting elements known to modulate the expression of hHSD17B1 are not conserved in the rat gene. Rat HSD17B1 unlike hHSD17B1 with two cap sites, possesses two polyadenylation signals, thus resulting in two mRNAs.     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

The inhibition of human and rat 11β-hydroxysteroid dehydrogenase 2 by perfluoroalkylated substances

11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) regulates active glucocorticoid access to glucocorticoid and mineralocorticoid receptors by metabolizing it to an inactive form. Perfluoroalkylated substances (PFASs) are man-made polyfluorinated compounds that are widely used and persistent in the environment. We tested the inhibitory potencies of four PFASs including perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexanesulfonate (PFHxS) and perfluorobutane sulfonate (PFBS) on human and rat 11β-HSD2. PFOS was a potent inhibitor of both human (IC(50)=48 nM) and rat (IC(50)=293 nM) 11β-HSD2 activities. The potencies for the inhibition of human and rat 11β-HSD2 activities were PFOS>PFOA>PFHxS>PFBS. PFASs showed competitive inhibition of both human and rat 11β-HSD2 activities. This observation indicates that PFOS is a potent endocrine disruptor for glucocorticoid metabolism. Article from the Special issue on Targeted Inhibitors.     

Structure-dependent inhibition of human and rat 11β-hydroxysteroid dehydrogenase 2 activities by phthalates

Phthalates are diesters of phthalic acid and an alcohol moiety. Phthalates have been classified as endocrine disruptors and have a broad range of effects with unknown mechanisms. Some of the effects of phthalate are consistent with disruptions of normal glucocorticoid homeostasis, and in particular, with defective function of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). In the present study, we tested 12 phthalate diesters and four monoesters for the inhibition of human and rat kidney 11β-HSD2. We examined the modes of inhibition and looked for a relationship between the potency for inhibition and the chemical structures. Of the phthalate diesters we tested, dipropyl phthalate (DPrP) and di-n-butyl phthalate (DBP) significantly inhibited both human and rat 11β-HSD2 activities. The IC₅₀s were 85.59 μM for DPrP and 13.69 μM for DBP when calculated for rat 11β-HSD2. As diesters, 8 of the phthalates did not affect 11β-HSD2 enzyme activity. Compared to the diesters that were inhibitory, the 8 non-inhibitory phthalates, had either fewer carbons, that is 1 or 2 carbons in the alcohol moiety, or more carbons, 5–10, as a branched or unbranched chain in the alcohol moeity. However, phthalates could be inhibitors with six carbons in the alcohol moiety if the carbons were cyclized, as in dicyclohexyl phthalate (DCHP), which inhibited rat 11β-HSD2 with an IC₅₀ of 32.64 μM. Thus, whether a phthalate is an inhibitor may reflect the size and shape of the compound. Although the diesters are the compounds used in manufacturing and present as environmental contaminants, it is the monoester metabolites that are detected in human serum and urine. We showed that mono (2-ethylhexyl) phthalate (MEHP) significantly inhibited human (IC₅₀ = 110.8 ± 10.9) and rat (121.8 ± 8.5 μM) 11β-HSD2 activity even though its parent compound, di(2-ethylhexyl) phthalate (DEHP) did not. MEHP was a competitive inhibitor of 11β-HSD2 enzymatic activity. We conclude that phthalates of a certain size act as competitive inhibitors.     

Molecular cloning,sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet ₂-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney ₂-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5-untranslated region. Southern-blot analysis with a human platelet ₂-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain ₂-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A⁺) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the ₂-adrenergic antagonist [³H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the _2A subclass. We conclude that the rat brain cDNA encodes a new ₂-adrenergic receptor subtype that may be brain-specific.Abbreviations G protein guanine nucleotide-binding proteins - cA2-47 ₂-adrenergic receptor cDNA from rat brain - SSC (1X SSC contains 0.15 M NaCl, 15 mM Na₃citrate, pH 7.0)     

Purification and comparative properties of isoenzymes of nicotinamide–adenine dinucleotide phosphate–isocitrate dehydrogenase from rat heart and liver

1. Rat liver and heart major isoenzymes of NADP-isocitrate dehydrogenase have each been purified about 100-fold by a combination of ammonium sulphate fractionation and chromatography on ion-exchange cellulose and their properties compared. 2. The properties were similar in respect of pH, inhibition by Hg(2+) and Michaelis constants for isocitrate and NADP. 3. Some of the properties of the isoenzymes were different. 4. The heart isoenzyme was activated about 210% by 0.8m-ammonium sulphate whereas the liver isoenzyme was unaffected. The heart isoenzyme showed greater sensitivity to inactivation by heat (30 degrees C for 30min), whereas the liver isoenzyme was more sensitive to inactivation by p-chloromercuribenzoate and by Cu(2+). 5. The Michaelis constants with 3-acetylpyridine-adenine dinucleotide phosphate showed a twofold difference between liver and heart isoenzyme. 6. The differential sensitivity to heat and its mainly non-cytoplasmic location may be an explanation of the failure of plasma isocitrate dehydrogenase activity to increase after a myocardial infarction.