Shuji Ohno

Determination of mRNA Expression of Human UDP-Glucuronosyltransferases and Application for Localization in Various Human Tissues by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

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.

EffectS Of flaVOnOid phytOChemicalS On cOrtisOl prOductiOn and On actiVitieS Of SterOidOgenic enZymeS in human adrenOCOrtical H295R cellS

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-Burks 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.

Triphenyltin and Tributyltin inhibit pig testicular 17β-hydroxysteroid dehydrogenase activity and suppress testicular testosterone biosynthesis

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.

Flavonoid inhibition of overexpressed human 3β-hydroxysteroid dehydrogenase type II

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.

Mono-(2-ethylhexyl) phthalate (MEHP) induces nuclear receptor 4A subfamily in NCI-H295R cells: A possible mechanism of aromatase suppression by MEHP

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.

Expression in E. coli and tissue distribution of the human homologue of the mouse Ke 6 gene, 17β-hydroxysteroid dehydrogenase type 8

Expression of the human Ke 6 gene, 17β-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 32.5xa0kDa by SDS-polyacrylamide gel electrophoresis. Expressed human Ke 6 gene effectively catalyzed the conversion of estradiol into estrone. Testosterone, 5α-dihydrotestosterone, and 5-androstene-3β,17β-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.

20β-hydroxysteroid dehydrogenase of neonatal pig testis: 3α/β-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme

Abstract Pig testicular 20β-hydroxysteroid dehydrogenase (20β-HSD) has also 3α- and 3β-HSD (3α/β-HSD) activities. The purified 20β-HSD preparation from neonatal pig testes could catalyze the conversion of 5α-dihydrotestosterone (5α-DHT) in the presence of β-NADPH to 5α-androstane-3α, 17β-diol and 5α-androstane-3β,17β-diol at the ratio of 4:3, and the specific 3α/β-HSD activity of 20β-HSD for 5α-DHT was about 10 or 15 times larger than the 20β-HSD activities for 17α-hydroxypregn-4-ene-3,20-dione (17α-hydroxyprogesterone) or progesterone, respectively. The result indicates that the testicular 20β-HSD has high the reversible conversion of various 5α- or 5β-dihydrosteroids which have a 3-carbonyl or 3-hydroxyl group with β-NADP(H) as the preferred cofactor. The enzyme transferred the 4- proS hydrogen of NADPH to the 5α-DHT for both 3α- and 3β-hydroxylation and it was the same as the 20β-hydroxylation of 17α-hydroxyprogesterone. Although the 3α/β-HSD activity has been known to be present in 3α,20β-HSD of Streptomyces hydrogenans , the enzymological properties for 3α/β-HSD activity catalyzed by testicular 20β-HSD were different from the properties for 3α/β-HSD activity catalyzed by prokaryotic 3α,20β-HSD with respect to the specificity of the catalytic reaction and the confactor requirement.

Leydig cells from neonatal pig testis abundantly express 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2 and effectively inactivate cortisol to cortisone

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) isozymes, designated types 1 and 2, catalyze the interconversion of physiologically active glucocorticoids and inactive 11-keto forms. The presence of types 1 and 2 was determined in neonatal pig testis and Leydig cells purified from testes by reverse transcription polymerase chain reaction, Western blotting, and immunohistochemical staining. Type 2 mRNA was expressed at a high level in neonatal pig testis. In particular, in the entire testis, a higher level of type 2 was expressed compared to type 1. Furthermore, these expression levels in the testis were compared with the expression levels of the respective isozymes in pig liver and kidney, which are known to have high levels. Next, the direction of glucocorticoid metabolism in intact Leydig cells was examined, and only oxidation from cortisol to cortisone was detected. Virtually no reduction of cortisone to cortisol was detected. Using a microsomal enzyme preparation from Leydig cells, type 2 exhibited potent oxidation activity, and the activity was higher than the oxidation activity catalyzed by the type 1 isozyme. In kinetic analysis, the K(m) and V(max) for type 1 were 1.36 microM and 0.91 nmol/(h mg), respectively, and 0.38 microM and 1.25 nmol/(h mg), respectively, for type 2. The results of the present study using neonatal pig testis suggest that not only 11beta-HSD type 1 but also type 2, which is abundantly expressed, plays important roles in cortisol inactivation in pig Leydig cells, and furthermore, that excess cortisol will cause glucocorticoid-mediated suppression of testosterone production in even neonatal pig Leydig cells.

Mono-(2-ethylhexyl) phthalate induces NR4A subfamily and GIOT-1 gene expression, and suppresses CYP19 expression in human granulosa-like tumor cell line KGN

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.

Tributyltin chloride suppresses the P450cl7 transcription involved in testosterone production induced by gonadotropin stimulation in cultured pig Leydig cells.

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.