Objective: To explore the molecular mechanism of cleft palate in mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Methods: The pregnant mice were randomly divided into TCDD-treated group (n=42) and control group (n=42). TCDD-treated group was given by gavage a single dose of TCDD (64 μg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence. The localization and expression of maternally expressed gene3 (MEG3) in mouse embryonic palatal mesenchymal cells was detected by situ hybridization and real-time PCR (RT-PCR). The key protein expressions of transforming growth factor-β (TGF-β)/Smad signaling pathway in mouse embryonic palatal mesenchyme were analyzed by Western blotting. The interaction of MEG3 and TGF-β receptor Ⅰ (TGF-βRⅠ) was examined by RNA binding protein immunoprecipitation (RIP). Results: At GD13 and GD14, compared with the control group, the ratio of BrdU-positive cells in the palatal mesenchyme of TCDD-treated fetuses decreased significantly (GD13, t=6.66, P=0.003; GD14, t=6.56, P=0.003). However, at GD15, the ratio of BrdU-positive cells was significantly increased (t=-5.98, P=0.004). MEG3 was mainly expressed in the nuclei of fetal mouse palatal mesenchymal cells, and the expression of MEG3 in TCDD group was significantly increased at GD13, GD14 and GD15(GD13, t=39.28, P=0.012; GD14, t=18.75, P=0.042; GD15, t=28.36, P=0.045). At GD14, TCDD decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells (p-Smad2, t=9.48, P=0.001;Smad4, t=63.10, P=0.001), whereas the expression of Smad7 was significantly increased at GD14 (t=30.77, P<0.001). The results of the RIP experiment showed that the amount of TGF-βRⅠ-bound MEG3 in mouse embryonic palatal mesenchymal cells in the TCDD group (23.940±1.301) was higher than that in the control group (8.537±1.523)(t=24.55, P<0.001). Conclusions: MEG3 is involved in the suppression of mouse embryonic palatal mesenchymal cell proliferation, functioning at least in part via interacting with the TGF-βRⅠ protein and thereby suppressing Smad signaling in the context of TCDD induced cleft palate.
Animals ; Bromodeoxyuridine ; Cleft Palate/genetics* ; Female ; Mice ; Mice, Inbred C57BL ; Palate/metabolism* ; Polychlorinated Dibenzodioxins/toxicity* ; Pregnancy

OBJECTIVE: The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent organic pollutant, is harmful to the nervous system, but its effects on the brain are still unclear. This study aimed to investigate the effects of TCDD on astrocytes proliferation and underlying molecular mechanism. METHODS: The cell proliferation was measured by EdU-based proliferation assay and PI staining by flow cytometry. Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of signal transducer and activator of transcription 3 (STAT3). RESULTS: C6 cells treated with 10 and 50 nmol/L TCDD for 24 h showed significant promotion of the proliferation of. The exposure to TCDD resulted in the upregulation in the expression levels of phosphorylated protein kinase B (p-Akt), phosphorylated STAT3, and cyclin D1 in a dose- and time-dependent manner. The inhibition of Akt expression with LY294002 or STAT3 expression with AG490 abolished the TCDD-induced cyclin D1 upregulation and cell proliferation. Furthermore, LY294002 suppressed the activation of STAT3. Finally, TCDD promoted the translocation of STAT3 from the cytoplasm to the nucleus, and LY294002 treatment blocked this effect. CONCLUSION TCDD exposure promotes the proliferation of astrocyte cells via the Akt/STAT3/cyclin D1 pathway, leading to astrogliosis.
Animals ; Animals, Newborn ; Astrocytes ; drug effects ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Environmental Pollutants ; toxicity ; Neurotoxins ; toxicity ; Polychlorinated Dibenzodioxins ; toxicity ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats, Sprague-Dawley ; STAT3 Transcription Factor ; metabolism

This study investigated the role of long non-coding RNAs (lncRNAs) in the development of the palatal tissues. Cleft palates in mice were induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression levels of long non-coding RNA H19 (lncRNA H19) and insulin-like growth factor 2 (IGF2) gene were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The rate of occurrence of cleft palate was found to be 100% by TCDD exposure, and TCDD could cause short upper limb, cerebral fissure, webbed neck, and short neck. The expression levels of lncRNA H19 and IGF2 gene specifically showed embryo age-related differences on E13, E14, and E15 in the palatal tissues. The expression levels of lncRNA H19 and IGF2 gene showed an inverse relationship on E13, E14, and E15. These findings demonstrated that lncRNA H19 and IGF2 can mediate the development of mouse cleft palate.
Animals ; Cleft Palate ; chemically induced ; genetics ; pathology ; Female ; Gene Expression Regulation ; Gene Expression Regulation, Developmental ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Palate ; metabolism ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Long Noncoding ; genetics ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the effect of 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD) on the testicular gene expression profile in the testis of mice.

METHODSTwenty male C57BL/6j mice were randomly divided into normal control group (fed with maize oil) and 3 OCDD groups treated with OCDD by gavage for 30 days at low-, moderate-, and high doses of 1.25×10(-6), 1.25 ×10(-5), and 1.25×10(-4) g/mL, respectively (8 mL/kg daily). The testicular gene expression profiles of the mice were investigated using gene chip technique and compared between OCDD-exposed groups and the control group.

RESULTSCompared with the control group, the mice in low-dose OCDD group showed 1133 differentially expressed genes, including 659 up-regulated and 474 down-regulated ones; in the moderate-dose OCDD group, 978 genes were differentially expressed, including 487 up-regulated and 491 down-regulated ones; in the high-dose group, 895 genes were differentially expressed, including 424 up-regulated and 471 down-regulated ones.

CONCLUSIONThe effect of sub-chronic exposure to OCDD on testicular gene expression profiles in male C57BL/6j mice indicates that the testis is probably the target organ of OCDD.

Animals ; Male ; Mice ; Mice, Inbred C57BL ; Oligonucleotide Array Sequence Analysis ; Polychlorinated Dibenzodioxins ; toxicity ; Testis ; drug effects ; metabolism ; Transcriptome

OBJECTIVEThe present paper aims to investigate the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and N-nitrosodiethylamine (DEN) on tumorigenesis and its potential mechanism.

METHODSThe potentials of TCDD and DEN in separation or in combination to induce malignant transformation were tested in Balb/c 3T3 cells by using a cell transformation assay method. The possible mechanism of observed effects was studied further by adding α-naphthoflavone (α-NF), a competitive binding agent of TCDD, to the Aryl hydrocarbon receptor (AhR) pathway. The mRNA expressions of Cyp1a1 and Cyp2a5 gene in Balb/c 3T3 cells treated by DEN and TCDD in separation or in combination with or without presence of α-NF were measured with fluorescence quantification RT-PCR technique.

RESULTSThe cell transformation frequency (TF) was significantly higher in case of induction with TCDD in combination with DEN, as compared to that with either TCDD or DEN alone. These effects were not inhibited via α-NF. The mRNA expression levels of both Cyp1a1 and Cyp2a5 were enhanced by TCDD treatment alone, but this inducible effect was blocked in cells treated by TCDD and DEN in combination.

CONCLUSIONTCDD and DEN had a significant synergistic effect on tumorigenesis when they were used in combination. AhR pathway may not be the key mechanism of this synergistic effect. Thus, it is necessary to further test the potential mechanism involved in cancer development.

3T3 Cells ; Animals ; Base Sequence ; Carcinogens ; toxicity ; Cell Transformation, Neoplastic ; Cytochrome P-450 Enzyme System ; genetics ; DNA Primers ; Diethylnitrosamine ; toxicity ; Drug Synergism ; Mice ; Mice, Inbred BALB C ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Receptors, Aryl Hydrocarbon ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the mechanism of cleft palate in mice induced by 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD).

METHODSOn gestation day 10 (GD 10), 12 pregnant mice were randomly divided into two groups as the treated group and the control group with 6 mice in each group. The mice in the treated group received intragastric administration with 64 microg TCDD/kg, while the mice in the control group received equivalent corn oil. The embryos were examined under stereomicroscope to detect the incidence of cleft palate on GD 18.5. Another 18 pregnant mice were randomly divided into two groups (treated group and control group) on GD 10 with 9 pregnant mice in each group. Then each group was divided into 3 subgroups: GD 13.5, GD 14.5 and GD 15.5, with 3 pregnant mice in each subgroup. The palatal shelves were dissected from the embryos for RNA and DNA extraction on GD 13.5, GD 14.5 and GD 15.5. At last the expression of Smad 2-4 and Smad 7 mRNA was investigated by RT-PCR, and the TGF-beta3 promoter methylamine levels were investigated by methylation specific PCR (MSP).

RESULTSThe cleft palate mice model was established successfully by exposing pregnant C57BL/6J mice to TCDD. Total frequency of clefts was 100% in TCDD group, and the frequency of clefts was 0 in the control group. The relative expression of Smad 2 mRNA was 0.263 +/- 0.088, 0.296 +/- 0.016 and 0.159 +/- 0.027 in TCDD group, 0.180 +/- 0.042, 0.282 +/- 0.029 and 0.165 +/- 0.018 in control group. The relative expression of Smad 3 mRNA was 0.453 +/- 0.153, 0.551 +/- 0.160 and 0.328 +/- 0.049 in TCDD group, 0.375 +/- 0.126, 0.510 +/- 0.145 and 0.259 +/- 0.035 in control group. The relative expression of Smad 4 mRNA was 0.675 +/- 0.174, 0.577 +/- 0.070 and 0.396 +/- 0.066 in TCDD group, 0.557 +/- 0.138, 0.587 +/- 0.080 and 0.441 +/- 0.054 in control group. The relative expression of Smad 7 mRNA was 0.283 +/- 0.050, 0.320 +/- 0.068 and 0.169 +/- 0.045 in TCDD group, 0.207 +/- 0.043, 0.288 +/- 0.051 and 0.155 +/- 0.040 in control group. There was no significant difference between the TCDD treated mice and the control (P > 0.05). The TGF-beta3 promoters were at the un-methylation state both in the TCDD treated and control group.

CONCLUSIONIt suggests that TCDD could induce a stable formation of cleft palate, but it is not through the TGF-beta/Smad signaling nor through the modification of TGF-beta3 promoter methylation.

Animals ; Cleft Palate ; chemically induced ; DNA Methylation ; Female ; Mice ; Mice, Inbred C57BL ; Polychlorinated Dibenzodioxins ; toxicity ; Pregnancy ; Promoter Regions, Genetic ; Signal Transduction ; Smad Proteins ; metabolism ; Teratogens ; toxicity ; Transforming Growth Factor beta3 ; 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

Animals ; Brain ; drug effects ; metabolism ; Calcium-Transporting ATPases ; metabolism ; Female ; Mice ; Mice, Inbred Strains ; Polychlorinated Dibenzodioxins ; toxicity ; Sodium-Potassium-Exchanging ATPase ; metabolism

OBJECTIVETo study HaCaT-keratinocyte cell lines, a chosen model of human epidermis in an attempt to analyze the mRNA expression of AhR and TGF-alpha induced by TCDD METHODS: Semi-quantitative reverse transcription PCR-technique was used for assaying the relative levels of AhR and TGF-alpha mRNA of HaCaT-cells during the proliferation period when the cells were cultured for 24 hours.

RESULTSRelative level of the AhR-transcripts (corrected with beta-actin) decreased with the elevated concentration of TCDD and the relevant coefficient between the proliferation rate and concentration was -0.548, and the differences among all groups were significant (F =4.124, P =0.021). The vehicle control was respectively compared with 7 x 10(-10) mol/L (0.0620 +/- 0.0085) and 7 x 10(-9) mol/L (0.0518 +/- 0.0194) group, significantly different from the control group (0.1138 +/- 0.0227) (t = -3.48, P <0.05; t = -4.17, P <0.01), the expression amount being 55% and 45% of the control. Relative levels of TGF-alpha mRNA tended to increase with the elevated concentration with the significant coefficient of 0.695 (P < 0.01), and the differences among all groups were significant (F = 15.789, P =0.000). In two higher concentration group 7 x 10(-10) mol/L (0.1474 +/- 0.0390) and 7 x 10 (-9) mol/L (0.2133 +/- 0.0364), their relative expression amount of TGF-alpha mRNA was 2.6-fold, 3.8-fold of the control group (0.0561 +/- 0.0100) respectively. Further analysis for the relevant relationship between the amounts of the AhR mRNA and TGF- alpha mRNA showed a highly negative correlation, the coefficient being - 0.561 (P <0.05).

CONCLUSIONSTCDD down-regulate the expression of AhR and up-regulate the expression of TGF-alpha. A strong negative correlation between AhR and TGF-alpha expression is found.

Cell Line ; Epidermis ; cytology ; Gene Expression ; Humans ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Messenger ; genetics ; Receptors, Aryl Hydrocarbon ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Toxicity Tests ; Transforming Growth Factor alpha ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of the environmental carcinogenic factor-TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) on cell apoptosis and gene regulation of insulin-like growth factor binding protein 6 (IGFBP-6) in osteogenic sarcoma (SaOS-2) cells.

METHODSThe SaOS-2 cells were cultured with TCDD (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) mol/L) for 24 hours. The MTT reduction assay and flow cytometry were used to measure the cell proliferation and the cell apoptosis in TCDD-treated SaOS-2 cells. The Nitrophenol phosphate salt method was used to measure activity of alkaline phosphatase (ALP) in SaOS-2 cells. The IGFBP-6 mRNA and protein in SaOS-2 cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blotting analysis.

RESULTSSaOS-2 cell proliferation was up-regulated with TCDD (1 x 10(-9), 1 x 10(-8), and 1 x 10(-7) mol/L) about 20%, 47% and 93% (18.4 +/- 4.5, 22.5 +/- 3.6 and 29.4 +/- 4.2), respectively. The synthesis of ALP was up-regulated about 28%, 95%, and 142% (1.12 +/- 0.28, 1.58 +/- 0.14 and 1.96 +/- 0.17), respectively (P < 0.05). The cell apoptosis was down-regulated in dose-dependent biological manner about 5%, 26% and 52%, respectively (P < 0.05). The expression of IGFBP-6 mRNA and protein was decreased in 1 x 10(-7) mol/L TCDD-treated SaOS-2 cells about 76% and 72% (P < 0.05).

CONCLUSIONTCDD at low concentration may have the negative effect on cell apoptosis and down-regulation on gene expression of IGFBP-6 in SaOS-2 cells.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; metabolism ; Osteosarcoma ; metabolism ; pathology ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Messenger ; genetics