Details of a Researcher - Tamura, Hiroomi

Papers - Tamura, Hiroomi

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ヒト消化管組織を用いたin vitro試験の有用性と課題について考える.

○田村悦臣.

HAB NEWS LETTER (エイチ・エー・ビー研究機構) 16(2) 7-8 2009.10

Research paper (scientific journal), Single Work

Pomegranate juice inhibits sulfoconjugation in the human colon carcinoma cell line, Caco-2.

Saruwatari A, Okamura S, Nakajima Y, Narukawa Y, Takeda Y, Tamura H*.

J Medicinal Food 11(4) 623-628 2008.12

Research paper (conference, symposium, etc.), Joint Work, Accepted

Coffee inhibits the glucuronidation of 1-naphthol in rat syncytiotrophoblast cells.

Ueno M, Nakashima E, Tamura H:.

J Health Sci (Journal of Health Science) 54(6) ( 6 ) 697-703 - 702 2008.12

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 13449702

The placenta acts as a barrier that protects the fetus from xenobiotics in the maternal blood. Phase II conjugation reactions in the placenta are considered to play an important role in this protective process by transferring polar hydroxyl groups and thereby imparting water solubility that facilitates the excretion of xenobiotics. Since coffee consumption during pregnancy is reported to affect fetal growth and development, we examined its effects upon the conjugation reactions in the rat syncytiotrophoblast cell line TR-TBT 18d-1. We detected a high level of glucuronidation for 1-naphthol, a model compound, in both a dose- and culture time-dependent manner in the TR-TBT 18d-1 cells. However, no sulfation was detected in any of our analyses. Coffee was found to inhibit the glucuronidation of 1-naphthol with an IC50 of 4.5% (v/v). In contrast however, caffeine, which is a major bioactive constituent of coffee, did not show any inhibition at doses of up to 100 μM. Coffee was also found to inhibit UDP-glucuronosyl transferase (UGT) activity towards 1-naphthol in vitro to a similar extent [IC50 = 1.5% (v/v)] as in intact cells. Moreover, the expression of the UGT 1A6, which is known to mainly catalyze the glucuronidation of 1-naphthol, was not affected by coffee. If coffee inhibits placental glucuronidation during pregnancy, its consumption would increase the fetal plasma concentrations of harmful xenobiotics, potentially affecting normal fetal growth and development.

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Pomegranate juice inhibits sulfoconjugation in Caco-2 human colon carcinoma cells.

Saruwatari A, Okamura S, Nakajima Y, Narukawa Y, Takeda T, Tamura H

Journal of medicinal food (Journal of Medicinal Food) 11 ( 4 ) 623 - 8 2008.12

Research paper (scientific journal), ISSN 1096-620X

Several fruit juices have been reported to cause food-drug interactions, mainly affecting cytochrome P450 activity; however, little is known about the effects of fruit juices on conjugation reactions. Among several fruit juices tested (apple, peach, orange, pineapple, grapefruit, and pomegranate), pomegranate juice potently inhibited the sulfoconjugation of 1-naphthol in Caco-2 cells. This inhibition was both dose- and culture time-dependent, with a 50% inhibitory concentration (IC50) value calculated at 2.7% (vol/vol). In contrast, no obvious inhibition of glucuronidation of 1-naphthol in Caco-2 cells was observed by any of the juices examined. Punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in Caco-2 cells with an IC50 of 45 μM, which is consistent with that of pomegranate juice. These data suggest that punicalagin is mainly responsible for the inhibition of sulfoconjugation by pomegranate juice. We additionally demonstrated that pomegranate juice and punicalagin both inhibit phenol sulfotransferase activity in Caco-2 cells in vitro, at concentrations that are almost equivalent to those used in the Caco-2 cells. Pomegranate juice, however, shows no effects on the expression of the sulfotransferase SULT1A family of genes (SULT1A1 and SULT1A3) in Caco-2 cells. These results indicate that the inhibition of sulfotransferase activity by punicalagin in Caco-2 cells is responsible for the reductions seen in 1-naphthyl sulfate accumulation. Our data also suggest that constituents of pomegranate juice, most probably punicalagin, impair the enteric functions of sulfoconjugation and that this might have effects upon the bioavailability of drugs and other compounds present in food and in the environment. These effects might be related to the anticarcinogenic properties of pomegranate juice. © 2008 Mary Ann Liebert, Inc.

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Inhibitory effects of various beverages on the sulfoconjugation of 17b-estradiol in human colon carcinoma Caco-2 cells.

Saruwatari A, Isshiki M, Tamura H*.

Biol Pharm Bull 31(11) 2131-2136 2008.11

Research paper (conference, symposium, etc.), Joint Work, Accepted

Inhibitory effects of various beverages on the sulfoconjugation of 17beta-estradiol in human colon carcinoma Caco-2 cells.

Saruwatari A, Isshiki M, Tamura H

Biological & pharmaceutical bulletin (Biological and Pharmaceutical Bulletin) 31 ( 11 ) 2131 - 6 2008.11

Research paper (scientific journal), ISSN 0918-6158

To investigate the possible effects of different beverages in the gastrointestinal tract on the sulfation of estrogen, which is a major hormone and prototype substrate for the human sulfotransferases (SULT), we analyzed the effects of these substances upon the sulfate conjugation of 17β-estradiol (E2) in the human colon carcinoma cell line Caco-2. Sulfoconjugation activity toward E2 was measured by incubating 20 nM E2 with Caco-2 cells in the presence (5% (v/v)) of each beverage. Among the 35 beverages analyzed, four (aronia, blueberry, coffee, and peppermint) exhibited strong inhibitory effects on E2 sulfation within Caco-2 cells (IC50 values ranging from 1.9 to 4.4% (v/v)). These active beverages also strongly inhibited the cytosolic estrogen SULT activity of Caco-2 cells in vitro (IC50 values ranging from 0.18 to 0.3% (v/v)). These inhibitory activities were extractable with ethyl acetate but not hexane or n-butanol, indicating that the molecules responsible are moderately lipophilic. Coffee was found to be the most potent inhibitor but the major constituents of this beverage, caffeic acid, caffeine, and chlorogenic acid, did not show any effects on estrogen SULT activity. Kinetic analyses further indicated that the mode of inhibition by coffee is competitive. A possible relationship between the inhibition of estrogen SULT activity by coffee in the gastrointestinal tract and the reported reduction of colon cancer incidence in women who consume coffee is discussed. © 2008 Pharmaceutical Society of Japan.

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Cloning, Expression, and Characterization of Cytosolic Sulfotransferase Isozymes from Drosophila melanogaster.

Kenji HATTORI, Noriko MOTOHASHI, Izumi KOBAYASHI, Takeshi TOHYA, Mai OIKAWA and Hiro-omi TAMURA*.

Biosci Biotechnol Biochem (Bioscience, Biotechnology and Biochemistry) 72 ( 2 ) 540-547 - 547 2008.02

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 09168451

We have identified four cytosolic sulfotransferase (SULT) homologs in the genome database of Drosophila melanogaster, and have designated these genes dmST1-4. Each of these four isozymes was subsequently classified into a different and novel gene family, as determined by the low amino acid sequence homology (less than 40%) between them, and also toward their vertebrate homologs. The transcripts for these four SULT homologs were detectable at all developmental stages in D. melanogaster. In addition, three of these isozymes, the exception being dmST2, were successfully expressed and purified in Escherichia coli. These recombinant dmST1, 3, and 4 products showed high sulfating activity toward phenolic compounds such as vanillin and 4-nitrophenol, but showed no activity toward typical endogenous substrates such as tyramine and serotonin. DmST4 and dmST3 showed the lowest and highest Km values for 3′-phosphoadenosine-5′-phosphosulfate (PAPS) respectively. DmST4 also showed low but not negligible activity toward 20-hydroxyecdysone.

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Changes in the Expression of Cytochrome P450 Genes in Hemin-Induced Differentiated K562 Cells.

Nagai F, Shimizu M, Sakamoto T, Kobayashi T, Tamura H*.

Biol. Pharm. Bull (Biological and Pharmaceutical Bulletin) 30 (10) ( 10 ) 1954-1957 - 7 2007.10

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 0918-6158

We have previously reported the expression of CYP genes in human myeloblastic and lymphoid cell lines, and the induction of the CYP3A4 and GSTP1 genes by oxidative stress in the human erythroleukemia cell line, K562. To further elucidate the role of drug metabolizing enzymes in hematogenesis, we have characterized the expression of CYP genes in hemin-induced differentiated K562 cells. After incubation with 50 μM hemin for 3 d, the expression of CYP1A1 and CYP3A4 genes was induced by 2.5- and 3.5-fold, respectively. In contrast, the CYP1B1 and CYP2E1 genes were downregulated in these cells to below 10% of the control levels. Moreover, these changes correlated with the hemin dose and culture time. Metabolism of midazolam, a probe substrate for CYP3A4, in the differentiated K562 cells increased by 2-folds, suggesting that the induction of CYP3A4 activity is consistent with the mRNA level. If these changes in the CYP expression profile in hematopoietic cells occurred, the susceptibility to xenobiotics and/or the therapeutic drugs of the cells might be influenced, and it also affects the metabolism of endogenous substrates, such as steroids and prostaglandins. © 2007 Pharmaceutical Society of Japan.

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Changes in the gene expression patterns of the cytochrome P450s in selenite-induced cataracts in the rat.

Nakamura K, Fujiki T, Tamura H:.

J Health Sci (Journal of Health Science) 53(3) ( 3 ) 347-350 - 350 2007.06

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 13449702

A single injection of an overdose of selenite is known to induce cataracts in young rat eyes. In our current study, we have investigated the gene expression profiles of the cytochrome P450s (CYPs) in the ocular tissues of rats developing such selenite-induced cataracts. Seven days after the injection of selenite, the expression levels of the CYP1A1 and CYP2E1 genes in the lenses of these animals increased and a slight reduction in the expression of the CYP3A gene family could be observed in the extralenticular tissues. When pantethine, an antioxidant, was administered 30 min before selenite injection, the extent of the CYP gene expression changes diminished. These results suggest that selenite-induced oxidative stress may play a role in both the induction and downregulation of CYP genes. In addition, the elevated expression of CYP1A1 and CYP2E1 in the lens may underlie the enhancement of cataract development induced by selenite.

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Cloning and Expression of a Novel Sulfotransferase with Unique Substrate Specificity from Bombyx mori.

Hattori K, Hirayama M, Suzuki H, Hamamoto H, Sekimizu K, Tamura H*.

Biosci Biotechnol Biochem (Bioscience, Biotechnology and Biochemistry) 71 ( 4 ) 1044-1051 - 1051 2007.04

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 09168451

We identified a cDNA encoding a putative cytosolic sulfotransferase (SULT) by searching the expressed sequence tag database of Bombyx mori, and subsequently obtained the full-length cDNA for this gene via rapid amplification of cDNA ends (RACE). We designated this gene bmST1, and showed by sequence analysis that it belongs to a novel SULT family. The tissue specificity of bmST1 mRNA expression was examined in fifth instar larvae by reverse transcriptase-polymerase chain reaction (RT-PCR), and transcripts were detectable in the silk gland, gut, fat body, and Malpighian tube. A recombinant form of bmST1 was then expressed using a gluthathione S-transferase (GST) gene fusion system, and it was purified from Escherichia coli. Purified bmST1 did not exhibit sulfating activity toward SULT substrates such as 4-nitrophenol, vanillin, hydroxysteroids, or monoamines. Surprisingly, however, recombinant bmST1 showed considerable activity toward 4-nitrocatechol and also gallate esters, although the catechins are not sulfated by this enzyme.

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A developed determination of midazolam and 1′-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: Application of human pharmacokinetic study for measurement of CYP3A activity

Shimizu M., Uno T., Tamura H., Kanazawa H., Murakami I., Sugawara K., Tateishi T.

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences) 847 ( 2 ) 275 - 281 2007.03

Research paper (scientific journal), ISSN 15700232

This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1′-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1 ml of alkalinized plasma using n-hexane-chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C18, 50 mm × 2.0 mm i.d.). The mobile phase for separation consisted of 10 mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2 ml/min. The drift voltage was 100 V. The sampling aperture was heated at 120 °C and the shield temperature was 260 °C. The total time for chromatographic separation was less than 16 min. The validated concentration ranges of this method were 0.25-50 ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40 ng/ml. The limits of quantification were 0.25 ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1 mg) and a single-oral administration (2 mg) of subtherapeutic MDZ dose. © 2006 Elsevier B.V. All rights reserved.

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A developed determination of midazolam and 1'-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: Application to human pharmacokinetic study for measurement of CYP3A activity.

Shimizu M, Uno T*, Tamura HO, Kanazawa H, Murakami I, Tateishi T.

J Chromatogr B 847 (2) 275-281 2007.03

Research paper (conference, symposium, etc.), Joint Work, Accepted

Metabolism of bisphenol A in the rat syncytiotrophoblast cell line, TR-TBT 18d-1.

Ikeda Y, Nakashima E, Kanazawa H, Tamura H*.

J Health Sci (Journal of Health Science) 53 ( 1 ) 146-150 - 150 2007.02

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 13449702

TR-TBT 18d-1 is a syncytiotrophoblast cell line established from placenta of the transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen (Tg-rat). Previously we have reported high level expressions of drug metabolizing enzymes in the TR-TBT cells. To elucidate the ability of metabolizing xenobiotics in the TR-TBT cells, we measured the metabolism of bisphenol A (BPA) as a model compound in the TR-TBT 18d-1 cells. It was found that BPA was metabolized and secreted as a monoglucuronide conjugate in a manner proportional to the incubation time and the BPA concentration. To elucidate the possible effect of green tea consumption on the placental metabolism, we investigated the effect of green tea on the metabolism of BPA in the TR-TBT cells. Green tea inhibited the conjugation reaction of BPA at the low concentration (IC50=5%), however, epigallocathechin gallate (EGCG) showed only weak inhibition, suggesting another active component existence. These results suggest that TR-TBT cells are useful for the model system of rat syncytiotrophoblasts which act as a barrier to protect fetus from harmful xenobiotics.

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Expression profiling of drug metabolizing enzymes in rat syncytiotrophoblast cell lines.

Ikeda Y, Nakashima E, Tamura H*.

J Health Sci (Journal of Health Science) 52 ( 5 ) 633-636 - 636 2006.10

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 13449702

TR-TBT-18d-1 and -18d-2 are syncytiotrophoblast cell lines established from the placenta of a transgenic rat harboring a temperature-sensitive simian virus 40 large T-antigen (Tg-rat). To explore the functional roles of drug metabolizing enzymes in the syncytiotrophoblasts of the placenta, we analyzed the gene expression of both cytochrome P450 (CYP) and phase II conjugation enzymes in these TR-TBT cell lines. A relatively high level of expression of CYP1A1 and CYP2C11 was detectable in the 18d-1 cells whereas CYP2E1 was more strongly expressed in the 18d-2 cells. A moderate degree of expression of CYP2B2 and CYP3A, and low expression of CYP1A2, was observed in both cell lines, but no CYP3A2 was detectable in either. Relatively high levels of SULT2 family gene expression were also evident in these TR-TBT cells, although the SULT1 family genes were found to be either absent or present at only moderate levels. Strong expression of both the UGT1 and UGT2 subfamily, and also the glutathione S-transferase (GST) subfamily, was also observed in both cell lines. These results suggest that TR-TBT cells are a useful model system for the study of syncytiotrophoblasts, which act as a protective fetal barrier against harmful xenobiotics.

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Turmeric and curcumin modulate the conjugation of 1-naphthol in Caco-2 cells.

Naganuma M, Saruwatari A, Okamura S, Tamura H*.

Biol Pharm Bull (Biological and Pharmaceutical Bulletin) 29 ( 7 ) 1476-1479 - 9 2006.06

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 0918-6158

Turmeric, the powdered dry rhizome of the Curcuma longa plant, and curcumin, the major anti-oxidant constituent of turmeric, have been shown to possess chemopreventive activity. To elucidate the possible interaction of turmeric and curcumin with conjugation reactions, which in many cases are involved in the activation of procarcinogens, we measured their effects in the conjugation of 1-naphthol in Caco-2 cells, a human colon carcinoma cell line, within a 24 h period. Turmeric exhibits inhibitory activity toward both sulfo- and glucuronosyl conjugations of 1-naphthol at approximately the same levels (IC50=0.24 and 0.29 mg/ml, respectively). Curcumin inhibits sulfo-conjugation at lower concentrations (IC50=9.7 μg/ml), but only showed weak inhibition toward glucuronosyl conjugation of 1-naphthol in Caco-2 cells. In addition, turmeric was found to strongly inhibit in vitro phenol sulfotransferase (SULT) activity and demonstrate moderate inhibitory properties against UDP-glucuronosyl transferase (UGT) activity in Caco-2 cells (IC50=0.17 mg/ml and 0.62 mg/ml, respectively). Curcumin also strongly inhibits in vitro phenol sulfotransferase activity with an IC 50 of 2.4 μg/ml. Moreover, and in contrast to the moderate inhibition of UGT activity by turmeric and curcumin, both induce the expression of the UGT1A1 and UGT1A6 genes, revealed by real-time PCR analysis. These findings are indicative of a possible interaction of both turmeric and curcumin with conjugation reactions in the human intestinal tract and colon. This in turn may affect the bioavailability of therapeutic drugs and toxicity levels of environmental chemicals, particularly procarcinogens. © 2006 Pharmaceutical Society of Japan.

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A novel sulfotransferase abundantly expressed in the dauer larvae of Caenorhabditis elegans.

Hattori K, Inoue M, Inoue T, Arai H, Tamura H*.

J Biochem (Journal of Biochemistry) 139 ( 3 ) 355-362 - 362 2006.03

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 0021924X

We have isolated a gene (clone Y113G7A.11) from Caenorhabditis elegans (C. elegans), that we have designated as ceST1, and which is the only member of the cytosolic sulfotransferase (SULT) gene family present in the genome of this organism. We identified the SULT motifs of ceST1 based upon their deduced amino acid sequence, and subsequently expressed the ceST1 cDNA in Escherichia coli and characterized its enzymatic properties. The recombinant protein showed sulfation activity for 4-nitrophenol and 2-naphthol substrates, but did not catalyze the sulfation of either monoamines or hydroxysteroids. Another compound sulfated by ceST1 is bisphenol A, which is known to stimulate germ cell proliferation in C. elegans. SULT activity was not detected in the cytosol of C. elegans, probably due to heat labile inhibitors. The ceST1 protein was detectable in the cytosol of C. elegans using anti-sera raised against recombinant ceST1, and transcripts could also be detected throughout the developmental stages. Moreover, high levels of ceST1 expression were evident at both the embryonic and adult stages and were augmented in dauer larva. These findings suggest that this sulfotransferase either forms part of a defence system against xenobiotics or regulates germ cell proliferation in C. elegans. © 2006 The Japanese Biochemical Society.

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Changes in the gene expression of drug metabolizing enzymes during cataractogenesis in Shumiya cataract rats.

Nakamura K, Oka M, Takehana M, Kobayashi S, Tamura H*.

J Health Sci (Journal of Health Science) 51 ( 6 ) 734-737 - 737 2005.12

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 13449702

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in both the nuclear and perinuclear regions of 10-11 week old animals. The expression of drug metabolizing enzymes, including cytochrome P450 (CYP) and conjugation enzymes, was investigated in the ocular tissues of SCRs during cataractogenesis. Significantly high levels of gene expression were detected for CYP1A1, CYP2B2, CYP2C11 and CYP2E1 in normal lenses, whereas CYP1A1 and CYP2C11 were at lower levels in the cataractous lenses. The gene expression profiles of conjugation enzymes were also compared between normal and cataractous ocular tissues in which a moderate reduction in ST1A1 and ST1B1 expression, and an extensive loss of UGT1A1 expression, were observed in the SCR animals that had developed cataracts. In contrast, the elevated expression of the GSTA family of genes was evident in cataractous lenses. The reduced expression of several drug metabolizing enzymes in the cataractous ocular tissues might therefore be related to cataractogenesis via the accumulation of endogenous or exogenous toxic substances.

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Age, gender and region-specific differences in drug metabolizing enzymes in rat ocular tissues.

Nakamura K, Fujiki T, Tamura H*.

Exp Eye Res 81 710-715 2005.12

Research paper (conference, symposium, etc.), Joint Work, Accepted

Age, gender and region-specific differences in drug metabolising enzymes in rat ocular tissues

Nakamura K., Fujiki T., Tamura H.

Experimental Eye Research (Experimental Eye Research) 81 ( 6 ) 710 - 715 2005.12

Research paper (scientific journal), ISSN 00144835

Previously we have reported our analyses of cytochrome P450 (CYP) expression in rat ocular tissues and in our current study we have extended these analyses to the different stages of growth in both male and female rats. Additionally, we have examined the expression levels of the different conjugation enzymes, sulfotransferases (SULTs), UDP-glucuronosyl transferases (UGTs) and glutathione S-transferases (GSTs), in ocular tissues. In 5-week-old animals, the CYP genes, CYP2B2 and CYP3A1, were abundantly expressed in the lens, with higher CYP1A1 expression detectable in the extra-lenticular tissues, of both genders. However, the expression of CYP1A2 and CYP2E1 in female ocular tissues was more extensive than in male. Moreover, in females the expression of the phenol-sulfating SULTs, ST1A1, ST1B1 and ST1C1, was more abundant in the extra-lenticular tissues, whereas the levels of the hydroxysteroid-sulfating SULTs, ST2A1 and ST2A5, predominated in the lens. The expression levels of the UGTs, UGT1A1, UGT1A6 and UGT2B1, were also higher in the female extra-lenticular tissues but the expression of the GSTA subfamily (GSTA1 and GSTA2) was very weak in the ocular tissues of both genders. The overall gene expression profiles were found to change with the age of the animals (from 3 to 8 weeks) and, in general, the expression levels of the CYPs and SULTs declined with age, whereas the levels of the UGTs and GSTs increased. These results demonstrate that the expression profiles of drug metabolising enzymes show both region- and gender-specific patterns in rat ocular tissues. © 2005 Elsevier Ltd. All rights reserved.

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ポリエチレンテレフタレート再生材中のホルムアルデヒド,アセトアルデヒドおよびオリゴマーの分析.

大門由佳, 河村葉子*, 六鹿元雄, 田村悦臣, 棚元憲一.

食品衛生学雑誌 46 46 2005.10

Research paper (conference, symposium, etc.), Joint Work, Accepted

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