Estrogenic Activities of 517 Chemicals by Yeast Two-Hybrid Assay
01 Aug 2000-Journal of Health Science (The Pharmaceutical Society of Japan)-Vol. 46, Iss: 4, pp 282-298
TL;DR: A simple and rapid screening method using the yeast two-hybrid system based on the ligand-dependent interaction of nuclear hormone receptors with coactivators to test the estrogenic activity of chemicals.
Abstract: One of the urgent tasks in understanding endocrine disruptors (EDs) is to compile a list of suspected substances among the huge number of chemicals by using the screening test method. We developed a simple and rapid screening method using the yeast two-hybrid system based on the ligand-dependent interaction of nuclear hormone receptors with coactivators. To date, we have tested the estrogenic activity of more than 500 chemicals including natural substances, medicines, pesticides, and industrial chemicals. 64 compounds were evaluated as positive, and most of these demonstrated a common structure; phenol with a hydrophobic moiety at the para-position without bulky groups at the ortho-position. These results are expected to facilitate further risk assessment of chemicals.
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TL;DR: It is demonstrated that alkyl chain length and hydroxylation of PEs have a significant impact on binding to ERs and peroxisome proliferator-activated receptors.
Abstract: Phthalate esters (PEs) are plasticizers used in many plastic products, food packaging materials, and medical bags. The widespread use of PEs has led to ubiquitous environmental contamination and human-exposure. Several epidemiological studies have indicated that exposure to PEs, especially during the prenatal period, induces adverse effects, including developmental and behavioral abnormalities, suggesting that PEs are endocrine-disrupters. Although the endocrine-disrupting effect of PEs is thought to be estrogen receptor (ER)-mediated, the ER-binding affinity of PEs is quite low, indicating that other mechanisms should be considered. In this review, we demonstrate that alkyl chain length and hydroxylation of PEs have a significant impact on binding to ERs and peroxisome proliferator-activated receptors. In addition, we discuss recent results from our laboratory that suggest additional risks may arise from environmental and metabolic processing of PEs through microbial transformation, microsomal metabolism, and sunlight exposure.
41 citations
TL;DR: A novel rapid ligand in vitro screening method that is easy to use and has high sensitivity, applicable to most nuclear receptors and suitable for high-throughput screening because the entire experimental operation can be carried out on a microplate.
Abstract: Assessment of the risk of human exposure to man-made chemicals that bind to hormone receptors has emerged as a major public health issue. Among hormone receptors, nuclear receptors tend to be targets of xenobiotics because their endogenous ligands are small, fat-soluble molecules. Nuclear receptors are ligand-inducible transcriptional factors and regulate the transcriptional activity of various target genes. At the start of the initiation step of transcription, nuclear receptors interact with coactivators (TIF2, SRC1, ACTR, CBP/p300, etc.) in an agonist-dependent manner. Using the interaction of the nuclear receptor with a coactivator, we have developed a novel rapid ligand in vitro screening method that is easy to use and has high sensitivity. This method, called by us the CoA-BAP system, is applicable to most nuclear receptors and is suitable for high-throughput screening because the entire experimental operation can be carried out on a microplate. We used human TIF2 as a coactivator including LXXLL motifs expressed in Escherichia coli as a fusion protein with BAP and nuclear receptor LBD expressed in E. coli as a fusion protein with GST. On a GSH-coupled microplate these proteins were incubated with chemicals and the protein-protein interactions were detected as alkaline phosphatase activity. To date we have examined seven nuclear receptors (ERalpha/beta, TRalpha, RARalpha/gamma, RXRalpha,and VDR) and confirmed that the method works well.
40 citations
TL;DR: The estrogenic activities of 32 pesticides in agricultural products were evaluated using the E-CALUX assay system developed by Xenobiotic Detection Systems Inc (North Carolina, USA), and it was found that tolclofos-methyl, prothiofos, diazinon, Thiabenclazole (TBZ) and pyriproxyfen had estrogenic activity.
Abstract: The estrogenic activities of 32 pesticides in agricultural products were evaluated using the E-CALUX assay system developed by Xenobiotic Detection Systems Inc (North Carolina, USA). This system utilizes human ovarian carcinoma cells (BG1) stably transfected with an estrogen-responsive luciferase reporter gene plasmid. It was found that tolclofos-methyl, prothiofos, diazinon, Thiabenclazole (TBZ) and pyriproxyfen had estrogenic activity. Several pesticides are often present in agricultural products. Therefore the estrogenicity of the mixtures of two kinds of pesticides was evaluated. The activity of diazinon/tolclofos-methyl, pyriproxyfen/prothiofos and TBZ/o-phenylphenol (OPP) was increased up to 1.2-5.3 fold. On the other hand, chlorfluazuron, imazalil and chlorfenapyr had anti-estrogenic activity. Further, to evaluate the change in the estrogenic activity of pesticide metabolites, an experimental system was established using a rat S9 mixture. Metabolites of permethrin and OPP had no estrogenic activity, but they had weak activity after the metabolism. On the other hand, the metabolites of TBZ exhibited less estrogenic activity than the original compounds.
40 citations
Additional excerpts
...Of these 3 kinds, there is no estrogenic activity even in the yeast TwoHybrid test using recombinant yeast cells expressing the human estrogen receptor gene and a coactivator(TIF2) (Nishihara et al. 2000)....
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TL;DR: By the proposed method, 2-hydroxyfluorene and 1-hydroxypyrene were identified in airborne particulates and their concentrations were determined for the first time.
Abstract: This paper describes a highly sensitive and selective method for the determination of hydroxylated polycyclic aromatic hydrocarbons (PAHs-OH) in airborne particulates. PAHs-OH were extracted from airborne particulates with ethanol by ultrasonication. The extractant was further cleaned up by solid phase extraction (SPE) with an aminopropylsilica cartridge, and subsequently analyzed by reversed-phase high-performance liquid chromatography with fluorescence detection. 2-Hydroxy-1-acetonaphthone was used as an internal standard. By the proposed method, 2-hydroxyfluorene and 1-hydroxypyrene were identified in airborne particulates and their concentrations were determined for the first time.
39 citations
TL;DR: Data indicate that fenvalerate, in this experimental model, interfered with initial development of the male reproductive system, but that these effects on sperm production or fertility did not persist into adulthood.
Abstract: The aim of this study was to determine the consequent reproductive developmental and immunotoxic effects due to exposure to fenvalerate during pregnancy and lactation in male offspring of maternal-treated rats Pregnant rats were treated daily by oral gavage with 40 or 80 mg/kg of fenvalerate or corn oil (vehicle, control), from d 12 of pregnancy to d 21 of lactation Immune and reproductive developmental effects were assessed in male offspring at postnatal days (PND) 40 (peripuberty), 60 (postpuberty), and 90 (sexual maturity) Treatment with the higher dose (80 mg/kg) resulted in convulsive behavior, hyperexcitability, and mortality in 45% of the dams Fenvalerate was detected in the fetus due to placental transfer, as well as in pups due to breast-milk ingestion, persisting in male offspring until PND 40 even though pesticide treatment was terminated on PND 20 However, fenvalerate did not produce marked alterations in age of testicular descent to the scrotum and prepucial separation, parameters indicative of puberty initiation In contrast, at puberty, there was a reduction in testicular weight and sperm production in male offspring of maternal-treated rats At adulthood, the sperm counts and fertility did not differ between control and treated groups Testosterone levels were not changed at any time during reproductive development Similarly, no apparent exposure-related effects were detected in the histological structures of the lymphohematopoietic system Data indicate that fenvalerate, in this experimental model, interfered with initial development of the male reproductive system, but that these effects on sperm production or fertility did not persist into adulthood There was no apparent evidence that fenvalerate altered testosterone levels or produced a disruption in male endocrine functions
39 citations
Cites result from "Estrogenic Activities of 517 Chemic..."
...In contrast, other researchers demonstrated that pyrethroids at low concentrations in vitro did not produce estrogenic or antiandrogenic effects (Gaido et al., 1997; Nishihara et al., 2000; Saito et al., 2000; Sumida et al., 2001)....
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References
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Book•
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01 Jan 1996
TL;DR: The cause of disruptions in animal breeding cycles, accompanied by increases in birth defects, sexual abnormalities and reproductive failure, is traced to the pervasive presence in the environment of chemicals that mimic hormones and trick the reproductive system.
Abstract: For years, scientists have noticed disruptions in animal breeding cycles, accompanied by increases in birth defects, sexual abnormalities and reproductive failure. Humans are not immune either, with sperm counts dropping by as much as 50% in recent decades and with women seeing a rise in hormone-related cancers, endometriosis and other disorders. This book traces the cause of these aberrations and diseases to the pervasive presence in the environment of chemicals that mimic hormones and trick the reproductive system. The conclusions are as obvious as they are inescapable - unless we make vital changes in the way we manufacture and employ the artefacts of our "good life", there will be no life at all.
917 citations