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ABSTRACT: Pig 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found
to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11β-HSD type 1,
and the sequence of 11β-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced
from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase
family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region.
The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone
and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression
of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these
results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11β-HSD type 1.
KeywordsPig 11β-HSD type 1-cDNA sequence-H6PDH-Expression in mammalian cells
Molecular and Cellular Biochemistry 04/2012; 338(1):149-156. · 2.06 Impact Factor
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ABSTRACT: The effects of bisphenol A (BPA), an endocrine disruptor, on aromatase (CYP19) expression in human osteoblastic (SV-HFO) and ovarian granulosa-like (KGN) cell lines were examined. CYP19 enzyme activity was suppressed in the presence of BPA in a dose-dependent fashion in both cell lines. CYP19 gene transcript expression, as well as activities of promoter I.4 in SV-HFO and promoter II in KGN, was down-regulated by BPA, suggesting that BPA affects CYP19 at the gene-expression level. These data and the previous finding that BPA induced the down-regulation of promoter I.1 activity within the human placental cell line suggest that there may be a conserved signaling pathway that down-regulates CYP19 expression in response to BPA in both cell lines. Additionally, differences between promoter I.4 and II suggest that there may be cell- and promoter-specific down-regulating mechanisms downstream from the actions of BPA.
Toxicology Letters 02/2012; 210(1):95-9. · 3.23 Impact Factor
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ABSTRACT: We recently established a method for quantitative determination of human catalytically active UDP-glucuronosyltransferases (UGTs) other than UGT2A1 and UGT2B28 by real-time reverse transcription-polymerase chain reaction (RT-PCR), and applied the method to an exhaustive analysis of localization in various human tissues. We report here an additional quantitative determination method targeting UGT2B28. To date, there have been no reports on the distribution of UGT2B28 mRNA expression in human tissues based on quantitative determination. Human UGT2B28 was clearly detected in the breast and adipose tissue. UGT2B28 expression in the breast was comparatively low, about 1.6% of GAPDH mRNA levels, and was less than 5% of normalized (against GAPDH) UGT2B7 and 2B10 mRNA expression levels in the liver. Although the UGT2B28 has 97% identity with UGT2B11 at the cDNA sequence level, the primers constructed for UGT2B28 did not detect UGT2B11. In addition, significant expression of UGT2B11 was detected in the liver, breast and kidney, and was clearly different from the distribution of UGT2B28. Therefore, we conclude that the real-time RT-PCR method established here is very specific for human UGT2B28 isozyme.
Drug metabolism letters. 06/2011; 5(3):202-8.
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ABSTRACT: Pig 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11beta-HSD type 1, and the sequence of 11beta-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region. The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11beta-HSD type 1.
Molecular and Cellular Biochemistry 12/2009; 338(1-2):149-56. · 2.06 Impact Factor
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ABSTRACT: The mechanism for transcriptional suppression of CYP19 by mono-ethylhexyl phthalate (MEHP) in a human ovarian granulosa cell line (KGN) was investigated. It is known that the CYP19 gene transcript in KGN cells predominantly includes exon PII among the 11 alternate exon I sequences. MEHP was found to significantly suppress Forskolin (FSK)-induced CYP19 gene transcription, CYP19 promoter II activity and CYP19 enzyme activity in a dose-dependent manner. Promoter assays using 5'-deleted promoter II reporter constructs suggested that the region important for responsiveness to MEHP exposure includes a putative CRE-like sequence and an SF-1 (NR5A1)/LRH-1 (NR5A2) binding sequence. Meanwhile, MEHP exposure rapidly and transiently induced nuclear receptor 4A (NR4A) mRNA, and gradually and continuously induced gonadotropin-inducible ovarian transcription factor-1 (GIOT-1; ZNF461) mRNA in KGN cells. The ectopic expression of NR4As and GIOT-1 suppressed promoter II activity, while among the NR4As expressed, only Nur77 (NR4A1) secondarily induced GIOT-1 mRNA expression. Based on these results, we believe that induction of the Nur77-GIOT-1 system by MEHP is involved in the transcriptional suppression of the CYP19 gene, and GIOT-1 may attenuate the promoter II activity due to suppression of SF-1 and/or LRH-1 transactivation in KGN cells.
Toxicology Letters 10/2009; 191(2-3):353-9. · 3.23 Impact Factor
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ABSTRACT: An exhaustive real-time reverse transcriptase-polymerase chain reaction (PCR) quantification method was used to determine 15 of the catalytically active human UDP-glucuronosyltransferase (UGT) isoforms (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17). The specific primers for respective human UGTs were developed for differential determination. The cDNA derived from the 1A7 isoform was detected in the esophagus, the 1A8 and 1A10 isoforms were detected in the small intestine, and all other isoforms were detected in at least the liver by PCR. In all cases, single bands of the expected size on the agarose gel were confirmed to correspond with the predicted UGT isoform sequences. Each calibration curve showed linearity between the PCR crossing point and the calibrator copy number. The correlation coefficients were greater than 0.9957 with high reproducibility. This exhaustive measurement method was applied to UGT expression in 23 human tissue types. UGT was mostly expressed in the alimentary system and liver. We were surprised to find that extremely high expression in the liver was found for UGT2B4 and UGT2B15, which had, respectively, 8.98 and 4.38 times greater expression than UGT2B7 in the liver. In addition, even though expressed at low levels, several UGT isoforms were expressed in steroidogenic tissues, such as the breast, prostate, heart, and adrenal. Therefore, this quantification method may provide valuable information about the medical efficacy or pharmacokinetic characteristics of a wide variety of UGT-metabolized drugs.
Drug metabolism and disposition: the biological fate of chemicals 11/2008; 37(1):32-40. · 3.74 Impact Factor
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ABSTRACT: The metabolic conversion of morphine to morphine-6-glucuronide (M6G) seems to play a significant role in mediation of the clinical effect of morphine because of the superior analgesic effect of M6G. Therefore, it would be of great interest to clarify the specificity of morphine-6-glucuronidation by UDP glucuronosyltransferase (UGT) isozymes. We investigated the specificity of morphine-6-glucuronidation catalyzed by recombinant human UGT isozymes in microsomes from baculovirus-infected insect cells. The morphine glucuronidation activity of recombinant human UGT isozymes incubated with morphine and UDP-glucuronic acid was determined by high-performance liquid chromatography with a fluorescence detector. Not only UGT2B7, which is well known to catalyze morphine-6-glucuronidation, but also UGT1A1 and 1A8 effectively catalyzed morphine-6-glucuronidation at relatively low morphine concentrations (<100 muM). The kinetics of both isozymes at the low substrate concentrations showed hyperbolic Michaelis-Menten kinetics. However, as the morphine concentration approached 100 muM, morphine-6-glucuronidation activity gradually decreased, and the kinetics closely resembled substrate inhibition Michaelis-Menten kinetic behavior. The K(m) values were 67.9 and 68.1 muM and the K(si) values were 218.9 and 88.0 muM for UGT1A1 and 1A8, respectively. These kinetics are basically different from that of morphine-6-glucuronidation by UGT2B7, which suggested biphasic Michaelis-Menten kinetic behavior. Furthermore, to estimate the contribution of these UGT isozymes in M6G formation in vivo, the expression levels of UGT1A1 and 1A8 mRNA in human liver and intestine were determined by reverse transcription real-time polymerase chain reaction. The results strongly suggest that UGT1A1 and UGT1A8 are isozymes involved in morphine-6-glucuronidation in vivo, as is UGT2B7 in humans.
Drug metabolism and disposition: the biological fate of chemicals 04/2008; 36(4):688-94. · 3.74 Impact Factor
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ABSTRACT: Expression of the human Ke 6 gene, 17beta-hydroxysteroid dehydrogenase type 8, in E. coli and the substrate specificity of the expressed protein were examined. The tissue distribution of mRNA expression of the human Ke 6 gene was also studied using real-time PCR. Human Ke 6 gene was expressed as an enzymatically-active His-tag fusion protein, whose molecular weight was estimated to be 32.5 kDa by SDS-polyacrylamide gel electrophoresis. Expressed human Ke 6 gene effectively catalyzed the conversion of estradiol into estrone. Testosterone, 5alpha-dihydrotestosterone, and 5-androstene-3beta,17beta-diol were also catalyzed into the corresponding 17-ketosteroid at 2.4-5.9% that of estradiol oxidation. Furthermore, expressed enzyme catalyzed the reduction of estrone to estradiol, but the rate was a mere 2.3%. Human Ke 6 gene mRNA was expressed in the various tissues examined, such as brain, cerebellum, heart, lung, kidney, liver, small intestine, ovary, testis, adrenals, placenta, prostate, and stomach. Expression of human Ke 6 gene mRNA was especially abundant in prostate, placenta, and kidney. The levels in prostate and placenta were higher than that in kidney, where it is known to be expressed in large quantities.
Molecular and Cellular Biochemistry 03/2008; 309(1-2):209-15. · 2.06 Impact Factor
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ABSTRACT: Phthalate esters are widely used as plasticizers for polyvinylchloride and are suspected of functioning as endocrine disrupters. Di-(2-ethylhexyl) phthalate (DEHP), the most important phthalate ester in commercial use, has been reported to act as a rodent reproductive toxicant. In the present study, we investigated the effects of phthalate esters on aromatase (CYP19) activity and on its gene expression in a human adrenocortical carcinoma cell line, NCI-H295R. Mono-(2-ethylhexyl) phthalate (MEHP), a principle metabolite of DEHP, dose-dependently suppressed aromatase activity and its transcription level. Furthermore, MEHP rapidly and transiently induced transcription of the genes which encode nuclear receptor 4A subfamily members (Nur77, Nurr1 and NOR-1), and up-regulated Nur77 promoter activation and Nur77 protein expression in the cells. MEHP-induced Nur77 transcription was inhibited by bisindolylmaleimide I (protein kinase C inhibitor) and wortmannin (phosphoinositide 3-kinase inhibitor). Finally, ectopic expression of Nur77 markedly suppressed forskolin-induced transcriptional activation of promoters I.3 and II of the CYP19 gene. These results suggest that the suppression of aromatase activity and its transcription level by MEHP exposure to NCI-H295R cells was regulated through the rapid and transient expression of Nur77 gene.
Molecular and Cellular Endocrinology 09/2007; 274(1-2):8-18. · 4.19 Impact Factor
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ABSTRACT: 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) Type3 is an NADPH-dependent membrane-bound enzyme that is specifically expressed in testis and catalyzes the conversion of androstenedione to testosterone. To date, the sequence of Type3 enzymes has been clarified in humans, mice and rats; however, the sequence of the pig enzyme remains unknown. In this study, we determined the cDNA sequence of pig testicular 17beta-HSD Type3. PCR primers for partial pig testicular 17beta-HSD Type3 were designed from rat and human enzyme consensus sequences. Full-length cDNA was obtained by 3'- and 5'-RACE based on partial PCR products. The cDNA coding region was 933 bp in length, which is the same as the human enzyme, and shared 84.7% sequence identity with the human cDNA coding region. The monomer was estimated to have a molecular weight of 34,855 and to contain 310 amino acid residues. The predicted pig amino acid sequence showed 81.9, 75.5 and 72.9% sequence identity with the human, rat and mouse sequences, respectively. To elucidate 17beta-HSD Type3 activity, the expression vector pCMV/pig17beta-HSD3 was established and transfected into human embryo kidney 293 cells. Subsequently, 17beta-HSD activity (androstenedione conversion to testosterone) was strongly detected in cell lysates.
The Journal of Steroid Biochemistry and Molecular Biology 11/2006; 101(2-3):145-50. · 3.05 Impact Factor
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ABSTRACT: We previously reported that tributyltin chloride (TBT) and triphenyltin chloride (TPT) powerfully suppressed human chorionic gonadotropin- and 8-bromo-cAMP-stimulated testosterone production in pig Leydig cells at concentrations that were not cytotoxic [Nakajima Y, Sato Q, Ohno S, Nakajin S. Organotin compounds suppress testosterone production in Leydig cells from neonatal pig testes. J Health Sci 2003;49:514-9]. This study investigated the effects of these organotin compounds on the activity of enzymes involved in testosterone biosynthesis in pig testis. At relatively low concentrations of TPT, 17beta-hydroxysteroid dehydrogenase (17beta-HSD; IC(50)=2.6microM) and cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (IC(50)=117microM) activities were inhibited, whereas cholesterol side-chain cleavage cytochrome P450 and 3beta-HSD/Delta(4)-Delta(5) isomerase activities were less sensitive. Overall, TPT was more effective than TBT. TPT also inhibited both ferredoxin reductase and P450 reductase activities at concentrations over 30microM; however, TBT had no effect, even at 100microM. The IC(50) values of TPT were estimated to be 25.7 and 22.8microM for ferredoxin reductase and P450 reductase, respectively. The inhibitory effect of TPT (30microM) on microsomal 17beta-HSD activity from pig testis was eliminated by pretreatment with the reducing agents dithiothreitol (1mM) and dithioerythritol (1mM). On the other hand, TPT (0.03microM) or TBT (0.1microM) exposure suppressed the testosterone production from androstenedione in pig Leydig cells indicating that these organotins inhibit 17beta-HSD activity in vivo as well as in vitro, and the IC(50) values of TPT and TBT for 17beta-HSD activity were estimated to be 48 and 114nM, respectively. Based on these results, it appears possible that the effects of TBT and TPT are largely due to direct inhibition of 17beta-HSD activity in vivo.
Steroids 09/2005; 70(9):645-51. · 2.83 Impact Factor
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ABSTRACT: We previously reported that organotin compounds, such as tributyltin chloride (TBT), dibutyltin dichloride and triphenyltin chloride, strongly suppressed the testosterone production level in isolated neonatal pig testicular Leydig cells at a concentration without cytotoxicity. In this report, the action mechanisms of suppressive effect of the testosterone production by TBT were investigated. TBT (0.1μM) exposure to pig Leydig cells for 4h significantly decreased the intracellular cAMP level stimulated by human chorionic gonadotropin (hCG; 10IU/ml) and also the level stimulated by forskolin (25μM). In the same way, TBT exposure for 6 and 24h significantly decreased the 17α-hydroxylase/17,20-lyase (P450cl7) mRNA level stimulated by hCG. However, TBT exposure did not affect the mRNA levels of other steroidogenic enzymes, such as cholesterol side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ(4)-Δ(5) isomerase, 17β-hydroxysteroid dehydrogenase, and steroidogenic acute regulatory protein, estimated by RT-PCR. These results suggest that TBT exposure inhibits the adenyl cyclase activity of Leydig cells, which in turn suppresses testosterone production due to a decrease in the P450cl7 transcription level induced by decreasing intracellular cAMP levels.
Environmental Toxicology and Pharmacology 07/2005; 20(1):11-7. · 1.47 Impact Factor
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ABSTRACT: Recent progress supports the importance of local estrogen secretion in human bone tissues to increase and maintain bone mineral density. In a previous study, we reported that the expression of aromatase (CYP19) is dexamethasone (Dex)-dependent and oncostatin M (OSM) increases the expression synergistically with Dex. In the present study, we examined the effects of forskolin (FSK) as another potential synergistic factor.
Co-administration of 100 nM Dex and 10 microM FSK increased aromatase activity 4-fold compared with Dex alone. The results of reverse transcriptase (RT)-PCR suggest that the amount of CYP19 gene transcript was also up-regulated by FSK synergistically with Dex, and that promoter I.4, which is not activated by FSK alone, is activated by FSK synergistically with Dex. The results of RT-PCR also suggest that promoter II, which responds to FSK, was not activated even in the presence of FSK in SV-HFO. The promoter I.4 sequence that was transfected into SV-HFO was activated by FSK synergistically with Dex.
Synergistic up-regulation of aromatase activity, CYP19 gene transcript, and promoter I.4 activity were Dex-dependent and not up-regulated by FSK alone. The results of this work may form the basis of bone-specific estrogen-replacement therapy that increases the estrogen concentration in bone tissue only.
European Journal of Endocrinology 05/2005; 152(4):619-24. · 3.42 Impact Factor
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ABSTRACT: The in vitro inhibitory effects of 23 compounds designated as possible endocrine-disrupting chemicals, such as alkylphenols, phthalate esters, phytoestrogens, organotins, and pesticides, on the enzyme activity of the isozymes of human 11beta-HSD, type 1 and 2, were examined. Human liver microsomal 11beta-HSD type 1 was inhibited by 4-nonylphenol (4-NP) and diethylhexyl adipate (IC50 = 49.3 and 49.4 mM, respectively). Further, human kidney microsomal 11beta-HSD type 2 was inhibited by triphenyltin, tributyltin, 4-t-octylphenol, 4-NP, 4-n-octylphenol, 4-n-nonylphenol, and dicyclohexyl phthalate (IC50 = 3.3, 16.5, 8.9, 20.3, 23.5, 26.2 and 46.5 microM, respectively). The other chemicals studied did not significantly inhibit the enzyme at concentrations lower than 50 microM. The expression of mRNA of the isozymes in gonads, accessory genital glands, and adrenal glands were investigated by RT-PCR. 11beta-HSD type 1 was detected in human testis, ovary, and prostate. The level in the adrenal gland(s) was similar to that in the liver, which is known to be expressed in large quantities. In contrast, except for the prostate, only small amounts of the 11beta-HSD type 2 isozyme were detected in these human tissues compared to kidney.
Environmental sciences: an international journal of environmental physiology and toxicology 02/2005; 12(4):219-30.
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ABSTRACT: Besides residue of the catalytic triad that is conserved in the short-chain dehydrogenase/reductase (SDR) superfamily, a Cys side chain reportedly plays functional roles in NADP-dependent 15-hydroxyprostaglandin dehydrogenase and human carbonyl reductase (CR). The three-dimensional structure of porcine 3alpha/beta,20beta-hydroxysteroid dehydrogenase, also known as porcine testicular carbonyl reductase, demonstrates the proximity of the Cys 226 side chain to the bound NADP. However, no clear explanation with respect to the basis of the catalytic function of the Cys residue is yet available. By chemical modification, point mutation, and kinetic analysis, we determine that two Cys residues, Cys 149 and Cys 226, are involved in the enzyme activity. Furthermore, we found that pretreatment with NADP markedly protects the enzyme from inactivation by 4-(hydroxyl mercury) benzoic acid (4-HMB), thereby confirming that Cys 226 is involved in binding of the cofactor. On the basis of the tertiary structure of 3alpha/beta,20beta-HSD, the possible roles of Cys residues, especially that of Cys 226, in enzyme action and in the binding of cofactor NADPH are discussed.
The Journal of Steroid Biochemistry and Molecular Biology 05/2004; 88(4-5):393-8. · 3.05 Impact Factor
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ABSTRACT: The inhibitory effects of various flavonoids on human 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase type II (3beta-HSD type II), overexpressed in baculovirus, were investigated, and the structure-inhibition relationship was examined. The isoflavone derivatives daidzein, genistein, formononetin and biochanin A inhibited 3beta-HSD type II activity at a concentration of 10 microM and of these, genistein was the most potent inhibitor. 6-Hydroxyflavone (6-HF), a synthetic flavone, also strongly inhibited 3beta-HSD activity but 5-HF, 7-HF and other natural flavones were less potent. Energy minimization structures of the flavonoids, as produced using MOE software, showed that isoflavones and flavones have an almost flat A-C ring structure, and that flavonoids that acted as inhibitors had similar steric structures to DHEA. Genistein, 6-HF and cyanoketone, which is known as a typical 3beta-HSD inhibitor, were found to act as competitive inhibitors with K(i) values of 0.12 microM, 0.19 microM and 0.67 nM, respectively. Furthermore, the LUMO (lowest unoccupied molecular orbital (LUMO)) values, as calculated using WinMOPAC (Fujitsu, Japan), of the inhibitors were correlated with the IC(50) values (r2 = 0.84). From these results, it appears that inhibitory effects of flavonoids are due to the combination of steric structure and electron affinity between the active center of 3beta-HSD type II and the flavonoid molecule.
The Journal of Steroid Biochemistry and Molecular Biology 03/2004; 88(2):175-82. · 3.05 Impact Factor
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ABSTRACT: The phytochemical flavonoid genistein has been shown to act as a potent competitive inhibitor of human adrenocortical 3beta-hydroxysteroid dehydrogenase and cytochrome P450 21-hydroxylase activities in vitro [J. Steroid Biochem. Molec. Biol. 2002; 80: 355-363]. In the present study, we evaluated the effects of large amounts of genistein continuously administered to weanling rats, particularly on steroidogenesis at the pubertal stage in vivo. Serum concentrations of free and total genistein were significantly higher in the 40 mg/kg genistein administration group when compared with the control group. In genistein administered rats, adrenal weight was significantly higher. Furthermore, a clear expansion of cells was observed in hematoxylin and eosin stained tissue at the zona fasciculata and zona reticularis of the adrenal cortex. However in the testis, no differences in weights or histologic changes were observed. Serum corticosterone concentration significantly decreased to 50% of control levels by 40 mg/kg genistein administration and testosterone also tended to decrease with this dose of genistein. On the other hand, although serum follicle stimulating hormone was unchanged, adrenocorticotropic hormone and luteinizing hormone levels increased with genistein administration. These results suggest a significant effect of genistein on steroidogenesis in the adrenal gland and testis of rats, and this effect appeared to be more evident on steroid production in adrenals than in testis in vivo.
Life Sciences 01/2004; 74(6):733-42. · 2.53 Impact Factor
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ABSTRACT: The in vitro effects of exposure to tributyltin chlo-ride (TBT), dibutyltin dichloride (DBT), and triphenyl-tin chloride (TPT) on testosterone production in iso-lated Leydig cells from neonatal pig testes were exam-ined. These organotin compounds strongly suppressed human chorionic gonadotropin (10 IU/ml) or 8-bromo-cAMP (0.1 mM) induced testosterone production when the cells were exposed to nontoxic concentrations. The suppression of testosterone production was dose–de-pendent over the concentration range of 0.03–0.3 M for TBT and DBT, and 0.01–0.3 M for TPT. These effective concentrations for the suppression of testoster-one production were lower than that of the cytotoxic concentrations of the organotin compounds in Leydig cells and were equivalent to the accumulated concen-tration levels that were previously reported in marine organisms.
Journal of Health Science. 01/2003; 49:514-519.
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ABSTRACT: Inhibitory effects of flavonoid phytochemicals, flavones, flavonols and isoflavones on cortisol production were examined in human adrenal H295R cells stimulated with di-buthylyl cAMP. In addition, the inhibitory effects of these chemicals on the activity of P450scc, 3beta-HSD type II (3beta-HSD II), P450c17, P450c21 and P45011beta, steroidogenic enzymes involved in cortisol biosynthesis, were examined in the same cells. Exposure to 12.5 microM of the flavonoids 6-hydroxyflavone, 4'-hydroxyflavone, apigenin, daidzein, genistein and formononetin significantly decreased cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8 and 11.3%, respectively), and biochanin A significantly decreased cortisol production (by 47.3%) at a concentration of 25 microM without any significant cytotoxic effects or changes in cell number. Daidzin, the 7-glucoside of daidzein, did not alter cortisol production by H295R cells at concentrations over 10 microg/ml (24 microM). Daidzein-induced reduction of cortisol production by H295R cells was not inhibited by the estrogen receptor antagonist ICI 182,780. The flavonoids 6-hydroxyflavone, daidzein, genistein, biochanin A and formononetin strongly and significantly inhibited microsomal 3beta-HSD II activity at concentrations from 1 to 25 microM, and I(50) values were estimated to be 1.3, 2, 1, 0.5 and 2.7 microM, respectively. In addition, these flavonoids significantly inhibited microsomal P450c21 activity at 12.5 and/or 25 microM. In addition, 6-hydroxyflavone inhibited activity of microsomal P450c17 and mitochondrial P45011beta at 12.5 and/or 25 microM. Results of Lineweaver-Burk's plot analysis indicate that daidzein is a competitive inhibitor of the activity of 3beta-HSD II and P450c21. K(m) and V(max) values of 3beta-HSD II for DHEA were estimated to be 6.6 microM and 328pmol/minmg protein, respectively. K(m) and V(max) values of P450c21 for progesterone were estimated to be 2.8 microM and 16pmol/minmg protein, respectively. K(i) values of 3beta-HSD II and P450c21 for daidzein were estimated to be 2.9 and 33.3 microM, respectively.
The Journal of Steroid Biochemistry and Molecular Biology 04/2002; 80(3):355-63. · 3.05 Impact Factor
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ABSTRACT: Porcine testicular carbonyl reductase (PTCR) belongs to the short chain dehydrogenases/reductases (SDR) superfamily and catalyzes
the NADPH-dependent reduction of ketones on steroids and prostaglandins. The enzyme shares nearly 85% sequence identity with
the NADPH-dependent human 15-hydroxyprostaglandin dehydrogenase/carbonyl reductase. The tertiary structure of the enzyme at
2.3 Å reveals a fold characteristic of the SDR superfamily that uses a Tyr-Lys-Ser triad as catalytic residues, but exhibits
neither the functional homotetramer nor the homodimer that distinguish all SDRs. It is the first known monomeric structure
in the SDR superfamily. In PTCR, which is also active as a monomer, a 41-residue insertion immediately before the catalytic
Tyr describes an all-helix subdomain that packs against interfacial helices, eliminating the four-helix bundle interface conserved
in the superfamily. An additional anti-parallel strand in the PTCR structure also blocks the other strand-mediated interface.
These novel structural features provide the basis for the scaffolding of one catalytic site within a single molecule of the
enzyme.
Journal of Biological Chemistry 05/2001; 276(21):18457-18463. · 4.77 Impact Factor
