Kiyoshi Nagata

Tohoku Pharmaceutical University, Japan

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Publications (135)343.83 Total impact

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    ABSTRACT: Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an anti-inflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-Lan-Gen, activated CYP3A4 gene transcription through the activation of the human PXR.
    No preview · Article · Jan 2016
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    ABSTRACT: Purpose: In recent years, a number of natural medicines have been reported to have inductive or inhibitive effects on the activity of drug metabolizing enzymes, upon co-administration with prescribed medicines. However, information regarding natural medicine-drug interactions that influence drug metabolism is limited owing to the lack of efficient screening method for such interactions. Therefore, to understand whether P450 activity is affected by natural medicine in small intestines, we have established frozen recombinant P450-expressing cells infected with human CYP3A4 expressing adenovirus (Ad-CYP3A4) to evaluate the effect of natural medicines on CYP3A4 activity. Methods: Ad-CYP3A4 cells were created by infecting HepG2 cells with Ad-CYP3A4 at 10 multiplicity of infection (MOI) and these cells were stored using cryopreservation medium (fAd-CYP3A4 cells) to obtain long-term consistent data and stable supplies of cells expressing a constant level of CYP3A4 activity. Results: The CYP3A4 activity in fAd-CYP3A4 cells remained unaffected at the end of each frozen period (0, 1, 2, and 6 months). Inhibitory effect on CYP3A4 activity by typical inhibitors (ketoconazole, hyperforin) and natural medicines (Cat's Claw, Devil's Claw, Feverfew, Peppermint Oil, Red Clover, and Siberian Eleuthero) were evaluated. The inhibitors had nearly equal IC50 values in fAd-CYP3A4 cells, Ad-CYP3A4 cells and recombinant CYP3A4 microsomes. Cat's Claw, Peppermint Oil and Siberian Eleuthero inhibited CYP3A4 activity more potently than 0.1 μM ketoconazole in fAd-CYP3A4 cells. Conclusions: In the present study, we have successfully developed a highly reproducible system to evaluate CYP3A4 inhibition in small intestines by natural medicines.
    No preview · Article · Aug 2015 · Journal of Pharmacy and Pharmaceutical Sciences
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    ABSTRACT: There is a large discrepancy between the inter-individual difference in the hepatic expression level of cytochrome P450 (CYP) 3A4 and that of drug clearance mediated by this enzyme. However, the reason for this discrepancy remains largely unknown. As CYP3A4 interacts with UDP-glucuronosyltransferase 2B7 (UGT2B7) to alter its function, the reverse regulation is expected to modulate CYP3A4-catalyzed activity. To address this issue, we investigated whether protein-protein interaction between CYP3A4 and UGT2B7 modulates CYP3A4 function. For this purpose, we co-expressed CYP3A4, NADPH-cytochrome P450 reductase and UGT2B7 using a baculovirus-insect cell system. The activity of CYP3A4 was significantly suppressed by co-expressing UGT2B7, and this suppressive effect was lost when UGT2B7 was replaced with calnexin (CNX). These results strongly suggest that UGT2B7 negatively regulates CYP3A4 activity through a protein-protein interaction. In order to identify the UGT2B7 domain associated with CYP3A4 suppression we generated 12 mutants including chimeras with CNX. Mutations introduced into the UGT2B7 carboxyl terminal trans-membrane helix caused a loss of the suppressive effect on CYP3A4. Thus, this hydrophobic region is necessary for the suppression of CYP3A4 activity. Replacement of the hydrophilic end of UGT2B7 with that of CNX produced a similar suppressive effect as the native enzyme. The data using chimeric protein demonstrated that the internal membrane-anchoring region of UGT2B7 is also needed for the association with CYP3A4. These data suggest that 1) UGT2B7 suppresses CYP3A4 function, and 2) both hydrophobic domains located near the C-terminus and within UGT2B7 are needed for interaction with CYP3A4. The American Society for Pharmacology and Experimental Therapeutics.
    No preview · Article · Aug 2015 · Molecular pharmacology
  • Keita Inami · Takamitsu Sasaki · Takeshi Kumagai · Kiyoshi Nagata
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    ABSTRACT: The bioavailability of orally administered therapies are often significantly limited in the human intestine by the metabolic activities of Cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). Predicting whether candidate compounds induce CYP3A4 and P-gp is a crucial stage in the drug development process, as drug-drug interactions may result in the induction of intestinal CYP3A4 and P-gp. However, the assay systems needed to evaluate both CYP3A4 and P-gp induction in the intestine are yet to be established. To address this urgent requirement, we used LS174T cells to create two stable cell lines expressing the CYP3A4 or ATP-binding cassette subfamily B member 1 (ABCB1, encoding P-gp) reporter genes. First, we tested these stable cells by treatment with 1α, 25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans-retinoic acid (ATRA), and 9-cis-retinoic acid (9-cis RA) that induce CYP3A4 and P-gp in the intestines. All these compounds significantly increased both CYP3A4 and ABCB1 reporter activities in the stable cell lines. To simultaneously assess induction of CYP3A4 and ABCB1, both stable cells were co-cultivated to measure their reporter activities. The mixed cells showed a significant increase in the CYP3A4 and ABCB1 reporter activities following treatment with 1,25(OH) 2D3, ATRA, and 9-cis RA. These activity levels were maintained after passaging for more than 20 times and following multiple freeze-thaw cycles. These results demonstrate that our established cell lines can be used to simultaneously evaluate CYP3A4 and ABCB1 induction in the intestines, providing a valuable in vitro model for the evaluation of future drug candidates. This article is protected by copyright. All rights reserved.
    No preview · Article · Nov 2014 · Biopharmaceutics & Drug Disposition
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    ABSTRACT: In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.
    Full-text · Article · Aug 2014 · PLoS ONE
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    ABSTRACT: Owing to the popularity of health food consumption for improving health or beauty, the health food market has been steadily growing in Japan in recent years. Accordingly, harmful interactions between health foods and prescription drugs are predicted to increase because patients who are administered prescription drugs are reported to consume more health foods. We aimed to determine the status of health food consumption in patients and consumers visiting pharmacies, and we investigated whether health foods inhibit cytochrome P450 2D6 (CYP2D6) activity in vitro. We conducted a questionnaire survey in pharmacies on the current use of health foods. We received 1,041 responses, and of these respondents, 69.3% consumed health foods, over-the-counter drugs, or foods with health claims, and the use of 249 health foods was confirmed. In addition, a CYP2D6-expressing cell model was constructed using the human hepatoma cell line and the CYP2D6-expressing adenovirus; this model was used to assess the in vitro inhibitory effects of 172 of the 249 products, which were available at drug stores or on the Internet, on CYP2D6 activity by using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. CYP2D6 activity was inhibited by 6 products, including turmeric-, a component having diet effect-, garlic-, and collagen-containing health foods. This study revealed the status of health food utilization and identified the health foods that are currently consumed. Furthermore, we found that 6 products found in these health foods have the potential to inhibit CYP2D6 activity. Our results may provide helpful information to avoid health food-drug interactions during medication use.
    Full-text · Article · Jan 2014
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    ABSTRACT: The use of human induced pluripotent stem (iPS) cells would be of great value for a variety of applications involving drug development studies. Several reports have been published on the differentiation of human iPS cells into hepatocyte-like cells; however, the cells were insufficient for application in drug metabolism studies. In this study, we aimed to establish effective methods for differentiation of human iPS cells into hepatocytes. Two human iPS cell lines were differentiated by addition of activin A, dimethyl sulfoxide, hepatocyte growth factor, oncostatin M, and dexamethasone. The differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes, revealing that the human iPS cells were differentiated into hepatocyte-like cells. Expression of CYP3A4 and UGT1A1 mRNAs increased by treatment with typical inducers of the enzymes, and the response of the cells against the inducers was similar to that of human hepatocytes. Furthermore, the drug-metabolizing activity of CYP3A4, as monitored by testosterone 6β-hydroxylase activity, was elevated by these inducers. In conclusion, we established methods for differentiation of hepatocyte-like cells expressing drug metabolizing activity from human iPS cells. The hepatocyte-like cells derived from human iPS cells will be useful for drug metabolism studies.
    No preview · Article · Dec 2013 · Drug Metabolism and Pharmacokinetics
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    ABSTRACT: Functional protein-protein interactions between UDP-glucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells which simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of 5-HT, both Km and Vmax were increased by CYP3A4. When CYP3A4 was co-expressed either with UGT1A1 or 1A7, the Vmax for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. The S50 or Km was little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7, and their effects on the interaction with CYP3A4. While the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1*6, the co-expression of CYP3A4 restored the impaired function to a level comparable with the wild-type. Similarly, simultaneous expression of CYP3A4 increased the Vmax of UGT1A7*1 (wild-type) and *2 (R129K and R131K), whereas the same was not observed in UGT1A7*3 (R129K, R131K and W208A). In the kinetics involving different concentrations of UDP-glucuronic acid (UDP-GlcUA), the Km for UDP-GlcUA was significantly higher for UGT1A7*2 and *3 than *1. The Km of UGT1A7*1 and *3 was increased by CYP3A4, whereas *2 did not exhibit any such change. These results suggest that 1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoform-specific manner and 2) non-synonymous mutations in UGT1A7*3 reduce not only the ability of UGT to utilize UDP-GlcUA, but also CYP3A4-mediated enhancement of catalytic activity while CYP3A4 is able to restore the UGT1A1*6 function.
    Preview · Article · Nov 2013 · Drug metabolism and disposition: the biological fate of chemicals
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    ABSTRACT: Human induced pluripotent stem (iPS) cells were differentiated into the endoderm using activin A and were then treated with fibroblast growth factor 2 (FGF2) for differentiation into intestinal stem cell-like cells. These immature cells were then differentiated into enterocyte-like cells using epidermal growth factor (EGF) in 2% fetal bovine serum (FBS). At the early stage of differentiation, mRNA expression of caudal type homeobox 2 (CDX2), a major transcription factor related to intestinal development and differentiation, and leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, was markedly increased by treatment with FGF2. When cells were cultured in medium containing EGF and a low concentration of FBS, mRNAs of specific markers of intestinal epithelial cells, including sucrase-isomaltase, the intestinal oligopeptide transporter SLC15A1/peptide transporter 1 (PEPT1), and the major metabolizing enzyme CYP3A4, were expressed. In addition, sucrase-isomaltase protein expression and uptake of β-Ala-(L)-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA), a fluorescence-labeled substrate of the oligopeptide transporter, were detected. These results demonstrate a simple and direct method for differentiating human iPS cells into functional enterocyte-like cells.
    Full-text · Article · Jul 2013 · Drug Metabolism and Pharmacokinetics
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    ABSTRACT: Human induced pluripotent stem cells (iPSCs) are a valuable source of hepatocytes for applications in drug metabolism studies. However, the current protocols for generating iPSC-derived hepatocyte-like cells (iPSHCs) are still very inefficient, and iPSHCs do not have sufficient hepatocyte-specific features, which include expression of a series of hepatocyte-specific genes, such as those encoding cytochrome P450 (CYP). In this study, we investigated whether introduction of human hepatocyte nuclear factor 6 (HNF6) could modulate the expression of CYP3A4 and other CYP genes in iPSHCs as well as in HepG2 cells, a fetal liver cell line (HFL cells), and in hepatocytes. CYP3A4 mRNA could be detected in iPSHCs, but the expression level was very low compared with those in HepG2 cells and hepatocytes. However, the CYP3A4 mRNA levels markedly increased on introduction of HNF6 and reached one-tenth of those in hepatocytes. We also found that HNF6 introduction increased CYP3A4 gene transcription in HFL cells and HepG2 cells, which have features similar to those of fetal hepatocyte-like cells; however, it did not affect CYP3A4 mRNA expression in hepatocytes. These results suggest that HNF6 plays an important role in the gene regulation of CYP3A4 during development from the fetal period to the postnatal period.
    No preview · Article · Jun 2013 · Drug Metabolism and Pharmacokinetics
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    ABSTRACT: Distinctive response patterns of CYP3A4 and CYP3A7 to cobalt chloride (CoCl2) in human fetal liver (HFL) cells were observed and compared with those under hypoxic conditions. The expression levels of CYP3A4 and CYP3A7 mRNAs were decreased by CoCl2 and hypoxia, although significance could not be determined in HFL cells cultured under 3% O-2. The hypoxia-inducible factor-1 alpha (HIF-1 alpha) protein content in HFL cells was significantly increased by CoCl2 and 3% O-2. Transcriptional activities of CYP3A4 and CYP3A7 were not altered by 3% 02 when reporter plasmids containing the promoter region ranging up to about 10 kb and 12 kb upstream, respectively, were transfected into HFL cells, although the activity was significantly suppressed by CoCl2. These results suggested that the mechanisms controlling CYP3A gene expression of HIF-1 alpha chemical stabilizer in fetal hepatocytes might be different from those in adult hepatocytes, and that HIF-1 alpha is not directly involved in regulation of CYP3A4 or CYP3A7 expression.
    No preview · Article · Aug 2012 · Drug Metabolism and Pharmacokinetics
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    ABSTRACT: Human fetal liver (HFL) cells express major drug metabolic enzymes CYP3A4, CYP3A5 and CYP3A7. In the fetal hepatocytes, betamethasone and dexamethasone (DEX) markedly enhanced the expression levels of CYP3A4 and CYP3A7 mRNAs and slightly increased expression level of CYP3A5 mRNA. Interestingly, a high correlation between the CYP3A induction ability and the intensity of anti-inflammatory effect was observed. Human glucocorticoid receptor (GR)-small interfering RNA clearly attenuated the expression level of GR mRNA, and diminished the DEX-stimulated CYP3A4, CYP3A5 and CYP3A7 expression in HFL cells. These findings indicate that GR mediates the induction of CYP3A4 and CYP3A7 expression in human fetal hepatocytes as well as the CYP3A5.
    No preview · Article · May 2012 · Drug Metabolism and Pharmacokinetics
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    ABSTRACT: Distinctive response patterns of CYP3A4 and CYP3A7 to cobalt chloride (CoCl(2)) in human fetal liver (HFL) cells were observed and compared with those under hypoxic conditions. The expression levels of CYP3A4 and CYP3A7 mRNAs were decreased by CoCl(2) and hypoxia, although significance could not be determined in HFL cells cultured under 3% O(2). The hypoxia-inducible factor-1α (HIF-1α) protein content in HFL cells was significantly increased by CoCl(2) and 3% O(2). Transcriptional activities of CYP3A4 and CYP3A7 were not altered by 3% O(2) when reporter plasmids containing the promoter region ranging up to about -10 kb and -12 kb upstream, respectively, were transfected into HFL cells, although the activity was significantly suppressed by CoCl(2). These results suggested that the mechanisms controlling CYP3A gene expression of HIF-1α chemical stabilizer in fetal hepatocytes might be different from those in adult hepatocytes, and that HIF-1α is not directly involved in regulation of CYP3A4 and CYP3A7 expression.
    No preview · Article · Jan 2012 · Drug Metabolism and Pharmacokinetics
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    ABSTRACT: Aryl hydrocarbon receptor (AhR) activators have been shown to induce members of the cytochrome P450 (P450) 1 family. Here we demonstrate that the AhR activators induce CYP3A4 through human pregnane X receptor (PXR). AhR activators, polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased CYP3A4 reporter activity and CYP3A4 mRNA expression in HepG2 cells. The CYP3A4 reporter activity was also increased by treatment with cigarette tar. The increased CYP3A4 reporter activity was clearly knocked down by the introduction of human PXR-small interfering RNA, but not by that of human AhR-small interfering RNA. The CYP3A4 reporter activity enhanced by overexpression of human PXR was further increased by treatment with PAHs and TCDD as well as by treatment with rifampicin. These results suggest that PAHs contained in cigarette smoke induce CYP3A4 in human liver.
    No preview · Article · Nov 2011 · Drug Metabolism and Pharmacokinetics
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    ABSTRACT: The mouse cholesterol sulfotransferase St2b2 contributes to epidermal differentiation by biosynthesizing cholesterol sulfate (CS) from cholesterol in the epidermis. 12-O-Tetradecanoylphorbol-13-acetate (TPA) causes epidermal hyperplasia, an abnormal increase in epidermal cell numbers resulting from aberrant cell differentiation and an increase in St2b2 protein levels. The mechanisms underlying enhanced St2b2 expression and the pathophysiologic significance of the increased expression are unclear, however. To verify whether increased St2b2 levels are necessary for TPA-induced epidermal hyperplasia, the effects of St2b2-specific small hairpin RNA (St2b2-shRNA) on hyperplasia were examined in mice. St2b2-shRNA clearly suppressed TPA-induced epidermal hyperplasia and the expression of a marker of epidermal differentiation, involucrin (INV). Interestingly, treating mouse epidermal cells with tumor necrosis factor-alpha (TNFα) increased St2b2 expression. Furthermore, treatment with TNFα-siRNA or anti-TNF receptor antibodies reduced the TPA-induced enhancement of St2b2 expression. Treatment with BAY 11-7082, a specific inhibitor of nuclear factor-kappa B (NF-κB), diminished TPA-induced St2b2 expression. These results suggested that enhancement of St2b2 expression by TPA treatment occurs mainly through the TNFα-NF-κB inflammatory signaling pathway, which in turn leads to increased CS concentrations in epidermal cells and hyperplasia.
    Full-text · Article · Feb 2011 · Biological & Pharmaceutical Bulletin
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    ABSTRACT: Growing evidence indicates that the innate immune system and oxidative stress caused by gut-derived endotoxins play a key role in alcoholic liver disease (ALD). Intracellular mechanisms associated with endotoxin-induced signaling play a crucial role in the initiation and progression of ALD. It is now widely accepted that activation of the innate immune system and increased release of pro-inflammatory cytokines and other mediators play an important role in the development of ALD. Accumulating evidence suggests that alcohol-mediated upregulation of CYP2E1 expression may initiate lipid peroxidation via reactive oxygen species. Non-alcoholic steatohepatitis (NASH) is a liver disease characterized by histopathological features similar to those observed in ALD, but in the absence of significant alcohol consumption. Initial efforts to clarify the mechanisms that promote the progression from steatosis to steatohepatitis somewhat artificially divided disease mechanisms into "first and second hits." This model considered the development of steatosis to be the "first hit," increasing the sensitivity of the liver to the putative "second hit," leading to hepatocyte injury, inflammation, and oxidative stress. We have emphasized the important role of gut-derived bacterial toxins, the innate immune system, and oxidative stress in the common pathogenic mechanism in ALD and NASH progression.
    No preview · Article · Dec 2010 · Drug Metabolism and Pharmacokinetics
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    ABSTRACT: Malondialdehyde-modified low-density lipoprotein (MDA-LDL) and oxidized LDL (Ox-LDL), which accelerate the pathogenesis of arteriosclerosis, are thought to be involved in parthenogenesis caused by smooth muscle cell proliferation. In this study, we investigated the suppression mechanism of polycyclic aromatic hydrocarbons (PAHs) on the growth of an MDA-LDL-induced human acute monocyte leukemia suspension cell line (THP-1 cells). We found that PAHs suppressed MDA-LDL-induced THP-1 cell growth. Cotreatment with benzo[a]pyrene (BaP) or 3-methylchoranthrene (3-MC) decreased MDA-LDL-induced THP-1 cell growth, whereas treatment with benzo[e]pyrene (BeP) or pyrene, which is not a ligand for the arylhydrocarbon receptor (AhR), did not decrease THP-1 cell growth. Our findings clearly demonstrated that THP-1 cell growth, which was suppressed by PAHs, was restored by the addition of alpha-naphtoflavone, which is a partial antagonist to AhR. Moreover, it was shown that cotreatment with MDA-LDL and BaP markedly induced the expression of human cytochrome P4501A1 (hCYP1A1) messenger ribonucleic acid (mRNA) and significantly induced the expressions of p53 and p21 mRNAs. In support of these findings, AhR small interfering RNA suppressed the induced level of p21 mRNA and by BaP and the overexpression of hCYP1A1 significantly induced levels of p21 mRNA. On the other hand, the uptake rate of [(14)C]BaP into cells was increased more significantly by cotreatment with MDA-LDL than by treatment with [(14)C]BaP alone. These results strongly suggest that the suppression of MDA-LDL-induced THP-1 cell growth is caused by the increased uptake of PAHs, which strongly activate the AhR signal pathway accompanying DNA damage.
    No preview · Article · Apr 2010 · The Journal of Toxicological Sciences
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    ABSTRACT: Human CYP1A1 and CYP1A2 genes are in a head-to-head orientation on chromosome 15 and are separated by a 23-kb intergenic space. To our knowledge, this is the first report on a stable cell line that contains the 23-kb full-length regulatory region and is able to simultaneously assess the transcriptional activation of CYP1A1 and CYP1A2 genes. The stable cell line that constitutively expresses the reporter activities was constructed by inserting the dual reporter plasmid containing the 23-kb region between the CYP1A1 and CYP1A2 genes into the chromosome. Transcriptional activation of the CYP1A1 and CYP1A2 genes was measured simultaneously using luciferase (Luc) and secreted alkaline phosphatase (SEAP) activities, respectively. To demonstrate the utility of the stable cell line, CYP1A1/1A2 induction by the majority of compounds previously identified as CYP1A1/1A2 inducers was measured. The results clearly show that all compounds caused induction of reporter activities. In addition to assessing transcriptional activation of the CYP1A1 and CYP1A2 genes by measuring reporter activities, we determined the intrinsic CYP1A1 and CYP1A2 mRNA levels by treating them with the same compounds. The results suggest that this stable cell line may be used to rapidly and accurately predict CYP1A1/1A2 induction.
    No preview · Article · Jan 2010 · Drug Metabolism and Pharmacokinetics
  • Hiroyuki Suzuki · Takeshi Kumagai · Kiyoshi Nagata
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    ABSTRACT: Polycyclic aromatic carbons (PAHs) are common environmental pollutants. Exposure to air pollutants with PAHs has been suspected to be associated with the occurrence of pulmonary diseases. Benzo[a]pyrene (BaP), a carcinogenic model compound of PAHs found in cigarette smoke and air pollutants, has been extensively studied. BaP is a potent inducer for CYP1A1, which play important roles in the genotoxicity of BaP. Metabolic activation of BaP by CYP1A1 is required to cause its toxic, mutagenic, and carcinogenic effects. Furthermore, PAHs have also been reported to induce atherosclerosis.Generally, pathogenesis of atherosclerosis has been accelerated through malondialdehyde-modifided low density lipoprotein (MDA-LDL) and oxidized LDL. MDA-LDL was reported to be involved in atherogenesis causing smooth muscle cell proliferation which may be generated by the uptake of MDA-LDL by macrophages via scavenger receptors. However, the association between PAHs and MDA-LDL, which have also been reported to induce atherosclerosis, has not been established. Recently, we found that PAHs suppressed MDA-LDL-induced human acute monocyte leukemia suspension cell line (THP-1 cell) growth.In this study, we investigated the suppression mechanism by PAHs in MDA-LDL-induced THP-1 cell growth. Co-treatment with BaP or 3-methylchoranthrene decreased the MDA-LDL-induced THP-1 cell growth, but, benzo[e]pyrene or pyrene that is not ligand for the arylhydrocarbon receptor (AhR) did not decrease. The suppression of THP-1 cell growth was clearly restored by the addition of -naphtoflavone which is a partial antagonist to AhR. Co-treatment with MDA-LDL and BaP markedly induced the expression of CYP1A1 mRNA. These results predicted that activated BaP caused DNA damage and consequently increase tumor suppressor protein p53. As a result, the proliferation inhibitor protein p21 might be induced. As expected, co-treatment with BaP and MDA-LDL significantly induced expressions of p53 and p21. These findings strongly suggest that the MDA-LDL-induced THP-1 cell growth is suppressed by induction of p21 through DNA damage caused by activated metabolites of PAHs. In this presentation, the interplay between PAHs and MDA-LDL, which are risk factors for atherosclerosis, are discussed.
    No preview · Conference Paper · Aug 2009
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    ABSTRACT: Glucuronidation is one of the major pathways to eliminate SN-38,an active metabolite of irinotecan. Although UDP-glucuronosyltransferase 1A1 (UGT1A1)has been considered to primarily contribute to this metabolism, UGT1A9 is alsoknown to catalyze SN-38 glucuronidation. We have previously demonstrated thatcytochrome P450 3A4 (CYP3A4) alters the regio-selectivity of UGT2B7-catalyzedmorphine glucuronidation [1]. Further, the J-helix orsurrounding region of CYP3A4 is suggested to be involved in the interactionwith UGT2B7 [2]. Another group has shown that CYP3A4 interacts with members ofthe UGT1A subfamily as well as with UGT2B7. However, the effects of CYP3A4 onUGT1A1/9-catalyzed SN-38 glucuronidation have not been elucidated. To addressthis issue, we investigated whether hepatic UGT1A1/9-catalyzed SN-38glucuronidation is altered by simultaneous expression of CYP3A4. When CYP3A4was co-expressed with UGT1A1 or 1A9 in Sf-9 cells, their activities weresignificantly elevated compared with the corresponding UGT single expressionsystem. The enhancement in SN-38 conjugation by CYP3A4 was much higher forUGT1A9 than for UGT1A1. The Kms for the UGT1A1/9-mediatedreaction remained unchanged even with co-expressed CYP3A4. In contrast, the Vmaxvalues for both UGTs were increased by co-expression with CYP3A4,although the effect on UGT1A1 catalysis was not large. Thus, the intrinsicclearance (Vmax/Km) wasincreased 7-fold by dual expression compared with UGT1A9 single expression.These results suggest that CYP3A4 interacts with UGT1A9 to markedly affect theefficiency of detoxification of SN-38. It has been reported that hepatic CYP3A4levels vary 40-fold. This work suggests that the CYP3A4-dependent facilitation ofSN-38 glucuronidation is, at least partially, a factor affecting an individual'scapacity to eliminate SN-38. Refs: 1) Takeda et al, Mol. Pharmacol. 67:665 (2005); 2) Takeda et al, Mol. Pharmacol., in press.
    No preview · Conference Paper · Aug 2009

Publication Stats

3k Citations
343.83 Total Impact Points

Institutions

  • 2006-2014
    • Tohoku Pharmaceutical University
      Japan
  • 1997-2011
    • Tohoku University
      • • Graduate School of Pharmaceutical Sciences
      • • Department of Life and Pharmaceutical Science
      Miyagi, Japan
  • 1981-2007
    • Kyushu University
      • • Graduate School of Pharmaceutical Sciences
      • • Faculty of Pharmaceutical Sciences
      Hukuoka, Fukuoka, Japan
  • 2003
    • Taisho Pharmaceutical
      Edo, Tōkyō, Japan
  • 1989-1996
    • Keio University
      • School of Medicine
      Edo, Tōkyō, Japan
  • 1989-1994
    • National Heart, Lung, and Blood Institute
      베서스다, Maryland, United States
  • 1987-1990
    • National Institutes of Health
      • Laboratory of Human Carcinogenesis
      베서스다, Maryland, United States