OBJECTIVETo establish an exonuclease protection mediated polymerase chain reaction (PCR) assay for the non-radioactive, sensitive detection of the binding of protein and DNA.

METHODSThe 1 pmol/L-10 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dissolved in dimethyl sulphoxide (DMSO), was added into 100 microl SD rat hepatic cytosol in vitro, which contained different amount of aromatic hydrocarbon receptors (AhR) and relative proteins, and ligand-AhR-DRE complex were formed in addition to 1 fmol/L-100 nmol/L DNAs probes containing the sequence of DRE. With the digestion of Exonuclease III and S1 nuclease, free DNAs were digested to oligonucleotide and binding DNA remained due to protein (AhR) protection and be amplified by PCR. The results of PCRs were shown by loading on 2% agarose electrophoresis. DMSO was used as negative control and blank control was set up.

RESULTSTarget DNA (285 bp) could be observed in the ligand groups, but not in the control group. The minimal amount of receptor was 2.5 fmol/L and the minimal amount of DNA probes was 2 fmol.

CONCLUSIONSExonuclease protection mediated PCR assay should be a good non-radioactive tool to quantify the interaction of protein and DNA with high sensitivity and simplicity.

Animals ; Aryl Hydrocarbon Receptor Nuclear Translocator ; genetics ; metabolism ; Binding, Competitive ; DNA Probes ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Exonucleases ; metabolism ; Male ; Polymerase Chain Reaction ; methods ; Rats ; Rats, Sprague-Dawley ; Receptors, Aryl Hydrocarbon ; genetics ; metabolism

Zebrafish is widely used as a model organism in the process of drug discovery. It expresses drug metabolizing enzymes like cytochrome P450 (CYP450), uridine 5'-diphospho-glucuronosyltransferase (UGT) and nuclear receptors like pregnane X receptor (PXR), aryl hydrocarbon receptor (AHR), etc. This article summarized the profiles of main drug metabolizing enzymes and nuclear receptors, and reviewed the advances on xenobiotics metabolism in zebrafish.
Animals ; Cytochrome P-450 Enzyme System ; metabolism ; Embryo, Nonmammalian ; drug effects ; Glucuronosyltransferase ; metabolism ; Inactivation, Metabolic ; Pharmaceutical Preparations ; metabolism ; Polychlorinated Dibenzodioxins ; toxicity ; Receptors, Aryl Hydrocarbon ; metabolism ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Receptors, Steroid ; metabolism ; Teratogens ; toxicity ; Xenobiotics ; metabolism ; Zebrafish ; embryology ; metabolism

OBJECTIVETo study the specific binding of the artificial clonal aryl hydrocarbon receptor translocator (ARNT) with the natural aryl hydrocarbon receptor (AhR) and the recolonization by polyclonal antibody. The dose-response relationship with tetrachlo-rodibenzo-dioxin (TCDD) was also studied to develop TCDD detection method and the binding degree related to dose response.

METHODS(1) The target genes including AhR-PAS, AhR-C and ARNT-PAS were amplified by RT-PCR by using the total RNA purified from the liver cells of C57BL/6J mice as templates to construct pGEX-5X1 recombinants. The recombinant plasmids were expressed in E. coli. (2) The rabbits were immuned by the clonal fusion proteins: AhR-PAS, AhR-C to prepare the polyclonal antibody. (3) The natural AhR from the hepatic cytosol of C57BL/6J mice was extracted. The artificial cloning expressed fusion protein:GST-ARNT-PAS and the natural AhR were incubated in different dose of TCDD. The quantity of the heterodimer through affinity adsorption and Western blots were measured.

RESULTS(1) The target proteins including AhR-PAS, AhR-C and ARNT-PAS were successfully cloned and expressed in E. coli. (2) The detection limit of polyclonal antibody AhR-PAS and AhR-C were 5 ng and 1 ng, respectively. (3) The total protein concentration prepared from the liver cells was 60.5 mg/ml. The artificial clonal protein ARNT-PAS could specifically bind to the natural AhR complex with the existence of TCDD. The detection limit of TCDD was 0.25 pmol which was 80 pg approximately.

CONCLUSIONA TCDD detection method based on the aryl hydrocarbon receptor system was established and the detection limit might reach pg grade.

Animals ; Cells, Cultured ; Limit of Detection ; Liver Extracts ; chemistry ; Mice ; Mice, Inbred C57BL ; Polychlorinated Dibenzodioxins ; analysis ; Rabbits ; Receptors, Aryl Hydrocarbon ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease II (Exo III) digestion. With Exo III treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.
Binding Sites ; Cytochrome P-450 CYP1A1/*genetics ; Cytosol/metabolism ; DNA-Binding Proteins/chemistry ; Exodeoxyribonucleases/chemistry ; Liver/*metabolism ; Polymerase Chain Reaction ; Rats, Sprague-Dawley ; Receptors, Aryl Hydrocarbon/*chemistry ; Tetrachlorodibenzodioxin/*analogs & derivatives ; Tetrachlorodibenzodioxin/chemistry

Dioscin has a wide range of biological effects and broad application prospects. However the studies concerning the toxicology and mechanism of dioscin is small. This article is to study the hepatotoxicity of dioscin and the effect of dioscin treatment on expression of aryl hydrocarbon receptor (AhR) mRNA and CYP1A mRNA and protein in HepG2 cells in vitro. Dioscin 0.5-32 µmol · L(-1) exposed to HepG2 cells for 12 h, cell viability was examined by CCK-8 assay and the release rate of lactate dehydrogenase (LDH) was to evaluate cell membrane damage. HepG2 cells morphologic changes were quantified by inverted Microscope, and the effect on production of reactive oxygen species (ROS) was detected by flow cytometry. The mRNA expression of CYP1A and AhR was evaluated by RT-RCR. The protein expression of CYP1A1 was detected by western blot. The cell viability was significantly inhibited after HepG2 cells were exposed to dioscin 0.5-32 µmol · L(-1). Compared with the control, the LDH release rate and ROS were significantly increased. The expression of CYPlA and AhR mRNA was increased. The expression of CYP1Al protein was increased after dioscin treatment, and resveratrol, an AhR antagonist, could downregulate the expression of CYP1A1. It follows that large doses dioscin has potential hepatotoxicity. The possible mechanism may be dioscin can active aryl hydrocarbon receptor (AhR) and induce the expression of CYP1A.
Cell Survival ; drug effects ; Chemical and Drug Induced Liver Injury ; etiology ; Cytochrome P-450 CYP1A1 ; genetics ; Diosgenin ; analogs & derivatives ; toxicity ; Hep G2 Cells ; Humans ; L-Lactate Dehydrogenase ; secretion ; RNA, Messenger ; analysis ; Reactive Oxygen Species ; metabolism ; Receptors, Aryl Hydrocarbon ; genetics

OBJECTIVETo explore the toxic mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by studying the induction of cytochrome P4501A1 (CYP1A1) and aryl hydrocarbon receptor (AHR) mRNA in liver of TCDD-treated SD rats.

METHODSThirty female SD rats were randomly divided into control group and 5 exposure groups, every group had 5 rats. The animals were treated i.p. with 0.01, 0.1, 1, 10, 50 microg TCDD/kg BW. AHR and CYP1A1 mRNA expression were analyzed by RT-PCR after 24 h.

RESULTSThe contents of AHR and CYP1A1 mRNA were increased in all exposure groups except the 0.01 microg TCDD/kg BW group. AHR mRNA content was significantly increased in 50 microg TCDD/kg BW group (P<0.05); CYP1A1 mRNA contents were significantly increased in all exposure groups (P<0.05) but not 0.01 microg TCDD/kg BW group. There were dose-response relationship between TCDD doses and AHR, CYP1A1 gene expression.

CONCLUSIONBoth AHR and CYP1A1 gene in liver of TCDD-treated SD rats can be induced 24 h after exposure and CYP1A1 gene is more inducible than AHR gene.

Animals ; Cytochrome P-450 CYP1A1 ; genetics ; Dose-Response Relationship, Drug ; Female ; Gene Expression Regulation ; drug effects ; Liver ; drug effects ; metabolism ; Polychlorinated Dibenzodioxins ; toxicity ; Rats ; Rats, Sprague-Dawley ; Receptors, Aryl Hydrocarbon ; genetics

This paper is to report the development of a high-throughput in vitro system to screen hPXR/CAR mediated CYP2B6 drug inducers, and the application of it into the quick determination of induction activity toward CYP2B6 by various commonly used traditional Chinese medicines (TCMs) extract. Dual reporter gene assays were performed. The hPXR/CAR expression vectors and the reporter vector pGL3-CYP2B6-Luc involved in the distal and proximal promoters of CYP2B6 were co-transfected into HepG2 cells. Relative luciferase activities in cell lysate were analyzed after 48 h treatment of blank vehicle or drugs to determine the induction activity toward CYP2B6 by various commonly used TCMs extract. The positive hPXR/hCAR activators rifampicin and CITCO were applied to make sure that the reporter gene model was successfully established. Then 5 kinds of commonly used TCM extracts and 1 herbal compound were successfully investigated, some were found to activate hPXR or hCAR and therefore have the potential to induce CYP2B6 enzyme. This is the first domestic article to report the hCAR3-mediated CYP2B6 induction model and the establishment of a reporter gene system for hPXR/CAR-mediated CYP2B6 induction can be an effective and systemic in vitro method to investigate the drug inducers of CYP2B6 and to explain the mechanism involved.
Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Cytochrome P-450 CYP2B6 ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Genes, Reporter ; Genetic Vectors ; Hep G2 Cells ; High-Throughput Screening Assays ; Humans ; Luciferases ; genetics ; metabolism ; Oximes ; pharmacology ; Plants, Medicinal ; chemistry ; Plasmids ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Receptors, Steroid ; genetics ; metabolism ; Rifampin ; pharmacology ; Thiazoles ; pharmacology ; Transfection

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce drug transporter genes such as the ATP-binding cassette G member 2 (ABCG2), which contributes to multidrug resistance. We investigated the effect of TCDD pretreatment on drug transporters induction from cancer cells of various origins. Cell viabilities after treatment of cisplatin were measured to evaluate acquiring cisplatin resistance by TCDD. Acquring cisplatin resistance was found only in cisplatin senstivie cancer cells including gastric SNU601, colon LS180, brain CRT-MG and lymphoma Jurkat cells which showed a significant increase in cell viability after combined treatment with TCDD and cisplatin. High increase of ABCG2 gene expression was found in SNU601 and LS180 cells with a mild increase in the expression of the ABCC3, ABCC5,and SLC29A2 genes in SNU601 cells, and of major vault protein (MVP) in LS180 cells. The AhR inhibitor kaempferol suppressed the upregulation of ABCG2 expression and reversed the TCDD-induced increase in cell viability in LS180 cells. However, in CRT-MG cells, other transporter genes including ABCC1, ABCC5, ABCA3, ABCA2, ABCB4, ABCG1, and SLC29A1 were up-regulated. These findings suggested the acquiring cisplatin resistance by TCDD associated with cancer cell-type-specific induction of drug transporters.
ATP-Binding Cassette Transporters/genetics/*metabolism ; Cell Line, Tumor ; Cell Survival/drug effects ; Cisplatin/*pharmacology ; Drug Resistance, Neoplasm/drug effects ; Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; Humans ; Jurkat Cells ; K562 Cells ; Kaempferols/pharmacology ; Multidrug Resistance-Associated Proteins/genetics/metabolism ; Neoplasm Proteins/genetics/*metabolism ; RNA, Messenger/metabolism ; Receptors, Aryl Hydrocarbon/metabolism ; Tetrachlorodibenzodioxin/*pharmacology ; Up-Regulation/*drug effects ; Vault Ribonucleoprotein Particles/genetics/metabolism

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce drug transporter genes such as the ATP-binding cassette G member 2 (ABCG2), which contributes to multidrug resistance. We investigated the effect of TCDD pretreatment on drug transporters induction from cancer cells of various origins. Cell viabilities after treatment of cisplatin were measured to evaluate acquiring cisplatin resistance by TCDD. Acquring cisplatin resistance was found only in cisplatin senstivie cancer cells including gastric SNU601, colon LS180, brain CRT-MG and lymphoma Jurkat cells which showed a significant increase in cell viability after combined treatment with TCDD and cisplatin. High increase of ABCG2 gene expression was found in SNU601 and LS180 cells with a mild increase in the expression of the ABCC3, ABCC5,and SLC29A2 genes in SNU601 cells, and of major vault protein (MVP) in LS180 cells. The AhR inhibitor kaempferol suppressed the upregulation of ABCG2 expression and reversed the TCDD-induced increase in cell viability in LS180 cells. However, in CRT-MG cells, other transporter genes including ABCC1, ABCC5, ABCA3, ABCA2, ABCB4, ABCG1, and SLC29A1 were up-regulated. These findings suggested the acquiring cisplatin resistance by TCDD associated with cancer cell-type-specific induction of drug transporters.
ATP-Binding Cassette Transporters/genetics/*metabolism ; Cell Line, Tumor ; Cell Survival/drug effects ; Cisplatin/*pharmacology ; Drug Resistance, Neoplasm/drug effects ; Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; Humans ; Jurkat Cells ; K562 Cells ; Kaempferols/pharmacology ; Multidrug Resistance-Associated Proteins/genetics/metabolism ; Neoplasm Proteins/genetics/*metabolism ; RNA, Messenger/metabolism ; Receptors, Aryl Hydrocarbon/metabolism ; Tetrachlorodibenzodioxin/*pharmacology ; Up-Regulation/*drug effects ; Vault Ribonucleoprotein Particles/genetics/metabolism

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-alpha production. However, the signaling pathway of TCDD that leads to TNF-alpha expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-alpha expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-alpha in a dose- and time-dependent manner. Alpha-Naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-alpha at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-alpha expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-alpha expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-alpha expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with alpha-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-alpha production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-alpha mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as alpha-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-alpha production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-alpha.
Animals ; Benzoflavones/pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Enzyme Activation ; Genistein/pharmacology ; Hazardous Substances/*toxicity ; Humans ; MAP Kinase Signaling System/drug effects/physiology ; Macrophages/*metabolism ; Mice ; Phosphorylation ; Pyrimidines/pharmacology ; Quinazolines/pharmacology ; RNA, Messenger/metabolism ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptors, Aryl Hydrocarbon/antagonists & inhibitors ; Signal Transduction ; Tetrachlorodibenzodioxin/*toxicity ; Tumor Necrosis Factor-alpha/*biosynthesis ; src-Family Kinases/antagonists & inhibitors/metabolism