Kazuhiro Shiizaki

Validation of a new yeast-based reporter assay consisting of human estrogen receptors α/β and coactivator SRC-1: application for detection of estrogenic activity in environmental samples.

Endocrine disruptors are exogenous substances that act like hormones in the endocrine system and disrupt the physiologic function of endogenous hormones. In the present study, we established reporter yeast strains (Saccharomyces cerevisiae) expressing human estrogen receptors, ERα or ERβ. These strains contain a reporter plasmid carrying an estrogen responsive element (ERE) upstream of the β‐galactosidase gene, and a plasmid expressing a steroid receptor coactivator, SRC‐1e. Using these reporter strains, we demonstrated dose‐dependent estrogenic activities of different categories of ligands, a natural hormone, 17β‐estradiol (E2); a synthetic drug, diethylstilbestrol (DES); phytoestrogens, genistein, daizein and emodin; and an environmental endocrine disrupter, bisphenol A. EC50 values of E2 for ERα and ERβ are 5.31 × 10−10 and 5.85 × 10−10 M, respectively. We also demonstrated that these yeasts were applicable for measuring estrogenic activities of environmental water samples. Most downstream sites of a river showed similar activity in both ERα and ERβ assays. These yeast strains are useful and convenient for detecting and comparing the estrogenic ligand activities of environmental samples in response to ERα and ERβ.

Establishment of yeast reporter assay systems to detect ligands of thyroid hormone receptors α and β

Thyroid hormones are essential for proper development and differentiation in vertebrates. Recently, concern over the disruption of thyroid hormone homeostasis by industrial chemicals and environmental pollutants has been spreading. To evaluate these chemicals, several bioassays have been developed to detect thyroid hormone ligand activity. Nevertheless, a simple and useful assay is required for the assessment of an enormous number of environmental chemicals. We established yeast reporter assays by expression of full-length thyroid hormone receptor (TRalpha or TRbeta) cDNA and of the TR-dependent reporter gene in yeasts. By additional introduction of the general coactivator SRC-1 cDNA into the yeasts, a higher response to endogenous thyroid hormones, thyroxine (T4), and triiodothyronine (T3) was obtained. The EC50 values for T3 were 35 and 1.5nM for TRalpha and TRbeta assay yeasts, respectively. We tested four chemicals, tetrabromobisphenol A, tetramethylbisphenol A, 2-isopropylphenol, and o-t-butylphenol, which are suspected to have thyroid hormone-disrupting activity. All four chemicals showed agonistic activities in both assay yeasts; however, their activities were weak in comparison with endogenous TR ligands. Antagonist activities of 2-isopropylphenol and o-t-butylphenol were also found in the TRalpha yeast assay. Taken together, these assay yeasts will be powerful tools for assessing TR ligand activity of industrial chemicals and environmental pollutants.

Omeprazole Alleviates Benzo[a]pyrene Cytotoxicity by Inhibition of CYP1A1 Activity in Human and Mouse Hepatoma Cells

Omeprazole is a drug used for treating gastro-oesophageal reflux disease and duodenal ulcers. Omeprazole induces a xenobiotic-metabolizing enzyme, cytochrome P450 1A1 (CYP1A1), as its ligand by aryl hydrocarbon receptor (AhR) activation without binding. CYP1A1-inducible chemicals, such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin, are known to have adverse effects (i.e. carcinogenesis, mutagenesis and malformation). Unlike these typical AhR activators, omeprazole has shown no experimental evidence of carcinogenic activity. The possibility, however, remains that omeprazole may aggravate the effect of environmental carcinogens through CYP1A1 induction. We exposed benzo[a]pyrene and omeprazole simultaneously to human and mouse hepatoma cells to investigate the synergistic effect of these chemicals. Contrary to our prediction, cytotoxicity of benzo[a]pyrene was inhibited by the omeprazole exposure in a dose-dependent manner. Omeprazole did not alter CYP1A1 mRNA and protein levels induced by benzo[a]pyrene. The 7-ethoxy-resorufin-O-deethylase assay revealed that omeprazole inhibited CYP1A1 enzyme activity. Kinetic analysis also demonstrated that it is a competitive inhibitor for CYP1A1. The K(m) value of omeprazole against CYP1A1 activity was 50.1 microM. We conclude that the effects of omeprazole on CYP1A1 involve not only induction through AhR activation but also inhibition of its enzyme activity, and that the protective effect of omeprazole against benzo[a]pyrene cytotoxicity depends on the latter.

Development of yeast reporter assay for screening specific ligands of retinoic acid and retinoid X receptor subtypes

INTRODUCTION Retinoic acids are essential for embryonic development, tissue organization, and homeostasis and act via retinoic acid receptors (RARs) that form heterodimers with retinoid X receptors (RXRs). Human RARs and RXRs include the three subtypes α, β, and γ, which have varying distributions and physiological functions among human tissues. Recent reports show that subtype-specific binding of several chemicals to RARs or RXRs may lead to endocrine disruption. To evaluate these ligand-like chemicals, convenient assay systems for each receptor subtype are required. METHODS We developed reporter assay yeasts to screen ligands for RXR subtype receptor homodimers. To screen RAR ligands, yeasts were engineered to express RAR subtypes with defective RXRα, which fails to bind to coactivators because of its shortened c-terminus. RESULTS These assay yeasts were validated using known RXR- and RAR-specific ligands and subtype-specific responses were clearly shown. Subtype-specific ligand activities of the suspected chemical RAR or RXR ligands o-t-butylphenol, triphenyltin chloride, tributyltin chloride, and 4-nonylphenol were determined. DISCUSSION The present assay yeasts may be valuable tools for subtype-specific assessments of unidentified environmental ligand chemicals and receptor-specific pharmaceuticals.

Identification of Amino Acid Residues in the Ligand-Binding Domain of the Aryl Hydrocarbon Receptor Causing the Species-Specific Response to Omeprazole: Possible Determinants for Binding Putative Endogenous Ligands

Omeprazole (OME) induces the expression of genes encoding drug-metabolizing enzymes, such as CYP1A1, via activation of the aryl hydrocarbon receptor (AhR) both in vivo and in vitro. However, the precise mechanism of OME-mediated AhR activation is still under investigation. While elucidating species-specific susceptibility to dioxin, we found that OME-mediated AhR activation was mammalian species specific. Moreover, we previously reported that OME has inhibitory activity toward CYP1A1 enzymes. From these observations, we speculated that OME-mediated AhR target gene transcription is due to AhR activation by increasing amounts of putative AhR ligands in serum by inhibition of CYP1A1 activity. We compared the amino acid sequences of OME-sensitive rabbit AhR and nonsensitive mouse AhR to identify the residues responsible for the species-specific response. Chimeric AhRs were constructed by exchanging domains between mouse and rabbit AhRs to define the region required for the response to OME. OME-mediated transactivation was observed only with the chimeric AhR that included the ligand-binding domain (LBD) of the rabbit AhR. Site-directed mutagenesis revealed three amino acids (M328, T353, and F367) in the rabbit AhR that were responsible for OME-mediated transactivation. Replacing these residues with those of the mouse AhR abolished the response of the rabbit AhR. In contrast, substitutions of these amino acids with those of the rabbit AhR altered nonsensitive mouse AhR to become sensitive to OME. These results suggest that OME-mediated AhR activation requires a specific structure within LBD that is probably essential for binding with enigmatic endogenous ligands.

Modulation of benzo[a]pyrene-DNA adduct formation by CYP1 inducer and inhibitor

Benzo[a]pyrene (BaP) is a well-studied pro-carcinogen that is metabolically activated by cytochrome P450 enzymes. Cytochrome P4501A1 (CYP1A1) has been considered to play a central role in the activation step, which is essential for the formation of DNA adducts. This enzyme is strongly induced by many different chemical agents, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which binds to the aryl hydrocarbon receptor (AhR). Therefore, AhR activators are suspected to have the potential to aggravate the toxicity of BaP through the induction of CYP1A1. Besides, CYP1A1 inhibitors, including its substrates, are estimated to have preventive effects against BaP toxicity. However, strangely, increased hepatic BaP–DNA adduct levels have been reported in Cyp1a1 knockout mice. Moreover, numerous reports describe that concomitant treatment of AhR activators reduced BaP–DNA adduct formation. In an experiment using several human cell lines, TCDD had diverse modulatory effects on BaP–DNA adducts, both enhancing and inhibiting their formation. In this review, we focus on the factors that could influence the BaP–DNA adduct formation. To interpret these complicated outcomes, we propose a hypothesis that CYP1A1 is a key enzyme for both generation and reduction of (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), the major carcinogenic intermediate of BaP. Conversely, CYP1B1 is thought to contribute only to the metabolic activation of BaP related to carcinogenesis.

Construction of sensitive reporter assay yeasts for comprehensive detection of ligand activities of human corticosteroid receptors through inactivation of CWP and PDR genes

INTRODUCTION The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are members of the nuclear receptor superfamily and ligand-dependent transcription factors, whose major ligands are glucocorticoid and mineralocorticoid, so-called corticosteroids. The corticosteroids are a class of substances that include steroid hormones naturally produced in the adrenal cortex of vertebrates and analogues of these hormones that are synthesized in industry. They are involved in a wide range of physiological processes including stress and immune responses, and the regulation of carbohydrate metabolism, protein catabolism, sodium homeostasis, and inflammation. These substances are potential environmental contaminants because they are clinically consumed in large amounts worldwide. To develop a simple and sensitive bioassay to detect corticosteroids, we newly established reporter assay yeasts expressing human GR and MR. METHODS Ligand responses of the established assay yeasts were improved by forced expression of a human transcription coactivator SRC-1e. Further enhancement of the responses was achieved by inactivating the CWP and PDR genes that encode cell wall mannoproteins and plasma membrane efflux pumps, respectively, which may be attributable to an increased intracellular concentration of ligands. RESULTS These new assay yeasts were more responsive to both natural and synthetic agonist ligands than the conventional assay yeasts. They detected both agonistic and antagonistic activities of mifepristone, spironolactone, and eplerenone in a receptor-selective manner. They also detected ligand activities contained in oral pharmaceutical tablets and human urine. DISCUSSION This assay system will be a valuable tool to detect agonists as well as antagonists of corticosteroid receptors, in the fields of drug discovery and the assessment of environmental pollutants.

Influence of GSH S-transferase on the mutagenicity induced by dichloromethane and 1,2-dichloropropane

It has been suggested that dichloromethane (DCM) and 1,2-dichloropropane (DCP) are responsible for occupational cholangiocarcinoma. Dihaloalkanes are metabolically activated by GSH S-transferase theta1 (GSTT1) to yield products such as episulfonium ions. However, whether the GSTT1-mediated step of these dihaloalkanes is related to occupational cholangiocarcinoma is not known. In the present study, we investigated the influence of GSTT1 activation on the mutagenicity of DCM and 1,2-DCP using GSTT1-expressing Salmonella typhimurium TA100 (TA100-GST). Since the mutagenicity of DCM was significantly increased in TA100-GST compared with mock control (TA100-pCTC), GSTT1 is thought to be involved in the mutagenicity of DCM. Mutation spectrum analysis on the hisG gene revealed that C:G to A:T transversions were the predominant form observed in DCM-treated TA100-pCTC. However, C:G to T:A transitions were dramatically increased in TA100-GST. We also analysed the DCM-DNA adduct, N2-GSH-Me-dG, and formation of N2-GSH-Me-dG was increased in TA100-GST compared with TA100-pCTC. On the other hand, 1,2-DCP did not increase the numbers of revertants in TA100-GSTT1. In mutation spectrum analysis, C:G to T:A transitions was predominant in both TA100-pCTC and TA100-GSTT1. These findings suggest that GSTT1 has little involvement in DCP mutagenicity, and other mechanisms might be more important for bioactivation and consequent genotoxicity. Clarification of the mechanisms underlying the development of DCM- and/or 1,2-DCP-related human cholangiocarcinoma may help establish risk assessment and prevention strategies against occupational cancer.

Microbial metabolites of omeprazole activate murine aryl hydrocarbon receptor in vitro and in vivo.

Omeprazole (OME), a proton pump inhibitor used to treat gastritis, is also an aryl hydrocarbon receptor (AhR) activator. OME activates AhR in human hepatocytes and hepatoma cells, but not in mice in vivo or in vitro. We recently discovered that this species-specific difference results from a difference in a few amino acids in the ligand-binding domain of AhR. However, OME activates both mouse and human AhRs in the yeast reporter assay system. Nevertheless, the cause of this discrepancy in OME responses remains unknown. Here, we report that CYP1A1 mRNA expression in mouse cecum was elevated after OME administration, although the mouse is regarded as an OME-unresponsive animal. Using the yeast reporter assay system with human and murine AhRs, we found AhR agonist–like activity in the cecal extracts of OME-treated mice. We speculated that OME metabolites produced by cecal bacteria might activate murine AhRs in vivo. In high-performance liquid chromatography (HPLC) analysis, AhR agonist–like activity of cecal bacterial culture and cecal extracts were detected at the same retention time. AhR agonist–like activity was also detected in the HPLC fractions of yeast culture media containing OME. This unknown substance could induce reporter gene expression via mouse and human AhRs. The agonist-like activity of the OME metabolite was reduced by concomitant α-naphthoflavone exposure. These results indicate that a yeast-generated OME metabolite elicited the response of mouse AhR to OME in the yeast system, and that bacterial OME metabolites may act as AhR ligands in human and mouse intestines.

Development of yeast reporter assays for the enhanced detection of environmental ligands of thyroid hormone receptors α and β from Xenopus tropicalis

Thyroid hormones (THs) are involved in the regulation of metabolic homeostasis during the development and differentiation of vertebrates, particularly amphibian metamorphosis, which is entirely controlled by internal TH levels. Some artificial chemicals have been shown to exhibit TH-disrupting activities. In order to detect TH disruptors for amphibians, we herein developed a reporter assay using yeast strains expressing the thyroid hormone receptors (TRs) α and β together with the transcriptional coactivator SRC-1, all of which were derived from the frog Xenopus tropicalis (XT). These yeast strains responded to endogenous THs (T2, T3, and T4) in a dose-dependent manner. They detected the TR ligand activities of some artificial chemicals suspected to exhibit TH-disrupting activities, as well as TR ligand activity in river water collected downstream of sewage plant discharges, which may have originated from human excrement. Moreover, the responses of XT TR strains to these endogenous and artificial ligands were stronger than those of yeast strains for human TRα and β assays, which had previously been established in our laboratory. These results indicate that the yeast reporter assay system for XT TRα and β is valuable for assessing TR ligand activities in environmental samples that may be particularly potent in amphibians.