Animals ; Dioxins ; toxicity ; Humans ; Liver ; drug effects ; Polychlorinated Dibenzodioxins ; toxicity

OBJECTIVEThe toxicology of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has been studied mainly with regard to the carcinogenicity of its metabolites, but its phototoxicity is not well understood. Although some studies have indicated the lethal phototoxicity of TCDD, this study was designed to investigate its effect on SPC-A1 cells.

METHODSSPC-A1 cells were cultured in 1640 medium and treated with 10 nmol/L, 0.1 micromol/L, 1 micromol/L TCDD for either 24 h or 96 h at each concentration. SPC-A1 cells were co-cultured with TCDD at different concentrations. Then the cell morphology, DNA fragment electrophoresis, and cell cycle were analyzed by flow cytometry, and enzyme assays were used to observe the effect of TCDD on the morphology, growth rate, and enxyme change of SPC-A1 cells.

RESULTSWith the increasing concentrations of TCDD and prolongation of culture time, the morphology of SPC-A1 cells was changed from round shape to spindle, and the ability of SPC-A1 cells to adhere to wall was decreased. With debris emitted around the cells, the morphologic changes included reduction in cell volume. Nuclear chromatin condensation and PI were observed. With the increasing concentrations of TCDD, DNA ladder occurred. After treatment with TCDD, extraction of cancer cells exhibited typical DNA fragmentation, and flow cytometry analysis showed apoptosis in a dose-dependent manner. As the concentration of TCDD rose from 10 nmol/L to 1 micromol/L, the ratio of apoptotic cells increased from 10.76% to 21.82%.

CONCLUSIONSTCDD has in vitro cytotoxicity on SPC-A1 cells, and the cytotoxicity is positively related to its concentration and culture time. TCDD may inhibit the growth and proliferation of SPC-A1 cells through the pathway of apoptosis introduction.

Cell Line, Tumor ; DNA Fragmentation ; Environmental Pollutants ; toxicity ; Humans ; Polychlorinated Dibenzodioxins ; toxicity

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

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

Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Epidermis ; cytology ; drug effects ; Humans ; Mice ; Polychlorinated Dibenzodioxins ; adverse effects ; toxicity ; Rats ; Skin Diseases ; chemically induced

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

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

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 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