2,3,7,8-Tetrachlorodibenzo-p-dioxin Promotes Proliferation of Astrocyte Cells via the Akt/STAT3/Cyclin D1 Pathway / 生物医学与环境科学（英文）

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.

Expression changes of cell cycle related molecules in palatal tissue of fetal mice with cleft palate induced by TCDD / 中华整形外科杂志

Objective@#The purpose of this study is to investigate the expression change of cell cycle-related molecules in platal tissue of fetal mice with cleft palate, induced by 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD), and to explore the mechanism of cell cycle-related molecules in cleft palate.@*Methods@#In vivo, 48 pregnant mice were randomly divided into TCDD treatment group and control group with Random number table, 24 mice in each group. On the embryonic day 10.5 (E10.5), pregnant mice were orally administrated with TCDD 28 μg/kg (containing 5 μg/ml TCDD of corn oil) in TCDD treatment group. The same volume of corn oil was given to the mice in control group. The pregnant mice in each group were sacrificed on E13.5, E14.5 and E15.5, to collect the fetal palates for analysis. Fetal palates were used to extract total RNA and total protein, so as to detect the expression levels of cell cycle-related molecules, using RT-PCR and western blotting respectively. In vitro, human kidney embryo 293t (HEK293t) cells were treated with different concentrations of TCDD (0.01, 0.1, 0.5 and 1 nmol/L), and cells proliferation activity was detected using MTT assay. Statistical analysis was performed with IBM SPSS 24.0. Kolmogorov-Smimov test was used for normal distribution check, and the distribution was normal. Independent t-test was carried out among two groups. P<0.05 was considered statistically significant.@*Results@#At E13.5, E14.5 and E15.5, the expression level of interferon regulatory factor 6 (Irf6) protein were higher in the control group (1.26 ± 0.13, 1.67 ± 0.14 and 1.42 ± 0.15, respectively) compared to that in the TCDD group (0.81 ± 0.08, 1.04± 0.02 and 0.86 ± 0.12, respectively), on each time point (t value were 2.836, 3.662 and 2.867, respectively; P values were 0.0471, 0.0146 and 0.0241, respectively). The expression level of cyclin-dependent kinase inhibitor 1A (P21) protein on E13.5 and E14.5 of the control group (2.26 ± 0.21, 1.99 ± 0.21)were higher than that in the TCDD group on each time point(1.43 ± 0.12、0.93 ± 0.22), (t value were 3.398 and 3.378; P value were 0.8726 and 0.0273). The expression level of cyclin D1 in the control group (1.00±0.02, 0.94±0.03 and 1.11±0.09, respectively)were higher than that of the TCDD group (0.28±0.01, 0.33±0.06 and 0.88±0.01, respectively) on each time point (t value are were 22.53, 22.35 and 14.27, respectively, P value <0.001, <0.001 and<0.001, respectively). The expression of cyclin E1, cyclin A2, cyclin B1, CDK6, CDK2 and CDK1 in TCDD groups were higher than that of the controls (P<0.05). However, there was no significant difference of cyclin B1 on E13.5 and Cdk2 on E15.5. As treatment with TCDD (0.1 nmol/L) at 1, 2 and 3 days (0.70 ± 0.05, 1.05 ± 0.03 and 1.39 ± 0.04, respectively), the proliferation of HEK293t cells increased compared with the control group (0.49 ± 0.04, 0.98 ± 0.03 and 1.55 ± 0.02, respectively). The differences were statistically significant (t value were 2.829, 1.395 and 2.692, respectively; P value were 0.0198, 0.1320 and 0.0247, respectively).@*Conclusions@#TCDD down-regulates Irf6 and P21, and interferes with the normal expression of cell cycle-associated molecules, which in turn interferes with medial edge epithelia (MEE) cells cycle arrest and proliferation. These indicate that the disorder of spatiotemporal expression of cell cycle-related molecules during palatal development may be involved with the mechanism of TCDD-induced cleft palate..

Down-regulation of miR-381-3p inhibits osteogenic differentiation of mouse embryonic palatal mesenchymal cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced cleft palate of fetal mice / 中国修复重建外科杂志

Objective: To investigate the correlation between down-regulation of miR-381-3p and inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal (MEPM) cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate of fetal mice. Methods: Thirty-two pregnant mice were randomly divided into TCDD group and control group, 16 in each group. On embryonic day 10.5 (E10.5), the pregnant mice in TCDD group were orally administrated with TCDD at dosage of 28 μg/kg, while the pregnant mice in control group received equivalent corn oil. The pregnant mice in each group were sacrificed on E13.5 and E14.5, fetal palates were collected for analysis. The expression of miR-381-3p was detected by real-time fluorescent quantitative PCR and the protein expressions of runt- related transcription factor 2 (RUNX2) and osteopontin (OPN) were detected by Western blot. MEPM cells were extracted from fetal palates on E14.5 in control group and passaged. The 3rd passage cells were cultured with TCDD at dosage of 10 nmol/L for 0, 0.5, 1, 2, and 3 days. The expression of miR-381-3p was detected after 0, 0.5, 1, 2, and 3 days and the protein expressions of RUNX2 and OPN were detected after 0, 1, 2, and 3 days. Then, the 3rd passage cells were divided into 4 groups. The MEPM cells were transfected with miR-381-3p inhibitor (inhibitor group), NC inhibitor (NC inhibitor group) and miR-381-3p mimics (mimics group), NC mimics (NC mimics group) for 48 hours, respectively. And the expressions of miR-381-3p and the protein expressions of RUNX2 and OPN were detected. Results: On E13.5 and E14.5, 96 fetal mice in control group and 92 in TCDD group were obtained. The bilateral palates contacted in control group on E14.5, and a gap between the bilateral palates existed in TCDD group. On E13.5 and E14.5, the relative expressions of miR-381-3p and RUNX2 and OPN proteins were significant lower in TCDD group than in control group ( P0.05). The relative expressions of RUNX2 and OPN proteins at 1, 2, and 3 days were significantly lower than that at 0 day ( P<0.05). The relative expressions of miR-381-3p and RUNX2 and OPN proteins significantly lower in inhibitor group than in NC inhibitor group ( P<0.05) and higher in mimics group than in NC mimics group ( P<0.05). Conclusion: Down-regulation of miR-381-3p expression may be associated with inhibition of osteogenic differentiation of MEPM cells in TCDD-induced cleft palate of fetal mice.

Beta-glucan prevents toxic effects of 2, 3, 7, 8-TCDD in terms of oxidative and histopathological damage in heart tissue of rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant which causes severe toxic effects. Despite there is some suggestion concerning with TCDD induced cardiotoxicity such as formation of free radicals, the main mechanism has not been entirely explained. Beta-glucan is known as strong antioxidant matter and can scavenge free radicals. Therefore this study aimed to investigate the protective effects of beta-glucan against TCDD induced cardiotoxicity in rats. In this study, 2-3 months of age and 190-250 g in weight 32 rats were randomly divided into four equal groups (n=8 for each group). Group 1 was control; Group 2 was TCDD group (2 µg/kg/week); group 3 was the beta-glucan group(50 mg/kg/day), and group 4 was TCDD and beta-glucan treatment group. The heart samples were taken from rats after 21 days treatment. The results were shown that Despite TCDD exposure visibly caused to increase (p ≤ 0.001) in TBARS levels, It caused a visible decline in the levels of GSH, CAT, GSH-Px, and SOD. However Beta glucan significantly increased GSH, CAT, GSH-Px, SOD levels and decreased generation of TBARS. Additionally, our histopathological observations were in agreement with the biochemical results. In conclusion, Beta-glucan treatment exhibited protective activity on TCDD induced cardiotoxicity

Identification of differentially expressed proteins of palate tissues by iTRAQ in fetal mice induced by TCDD or retinoic acid / 中华整形外科杂志

Objective@#To explore the common differentially expressed proteins in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin(TCDD) and retinoic acid-induced cleft palate of fetal mice by isobaric tags for relative and absolute quantitation(iTRAQ) combined with mass spectrometry.@*Methods@#Thirty-six pregnant C57BL/6J mice were randomly divided into 3 groups, 12 cases in each group. C57BL/6J pregnant mice were given a gavage of TCDD 28 μg/kg or retinoic acid 80 mg/kg on gestational day 10.5(GD10.5) as experimental groups, while the control group received equivalent corn oil. Anatomical and histological changes of palates in fetal mice were observed on GD17.5. Total proteins were extracted from palates of fetal mice in each group on GD17.5. Differentially expressed proteins were identified in experimental groups as well as in control group by iTRAQ combined with two-dimensional liquid chromatography/tandem mass spectrometry. Western Blot was used for validation of the differentially expressed proteins of Annexin A1 and 14-3-3 sigma. All statistical analyses were measured with SPSS software(version 17.0). Chi-square test was used to compare the incidence of cleft palate. One-way ANOVA was carried out for comparison of the relative expression levels of three groups, homogeneity of variance was analyzed by Levene test, and Turkey HSD test was used for comparison between two groups. P values were judged as significant difference if they were less than 0.05.@*Results@#①Model of cleft palate in fetal mice were successfully established with incidence of cleft palate of 97.1%(68/70)in TCDD group and 98.6%(70/71) in retinoic acid group, respectively(χ2=0.00, P>0.05), without significant difference between two groups. However, they were similar on the phenotype. ② A total of 2 996 proteins were identified. Compared with control group, 75 and 90 differentially expressed proteins were screened out from TCDD group and retinoic acid group respectively. There were 18 differentially expressed proteins in common both in two experimental groups. ③Western Blot assay indicated that the expression of Annexin A1 protein was 0.52±0.11 in control group, while in TCDD group was 0.99±0.34 and in retinoic acid group was 0.98±0.31, with significant difference between any of two experimental groups and control group(P<0.05). The expression of 14-3-3 sigma protein in control group was 0.55±0.15, while in TCDD group was 0.86±0.17 and in retinoic acid group was 0.93±0.13, with significant difference between any of two experimental groups and control group(P<0.05). These results were consistent with the results of iTRAQ experiment.@*Conclusions@#Using iTRAQ technology can quickly and effectively filtrate the common differentially expressed proteins in fetal mice with cleft palate induced by TCDD and retinoic acid. These proteins may have closely related relationship with the occurrence of cleft palate induced by TCDD or retinoic acid.

Induction of Heat Shock Proteins and Antioxidant Enzymes in 2,3,7,8-TCDD-Induced Hepatotoxicity in Rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is an environmental toxicant with a polyhalogenated aromatic hydrocarbon structure and is one of the most toxic man-made chemicals. Exposure to 2,3,7,8-TCDD induces reproductive toxicity, immunotoxicity, and hepatotoxicity. In this study, we evaluated how 2,3,7,8-TCDD-induced hepatotoxicity affect the expression of heat shock proteins and antioxidant enzymes using the real-time polymerase chain reaction (PCR) in rat. 2,3,7,8-TCDD increased heat shock protein (Hsp27, alpha-B-crystallin, Mortalin, Hsp105, and Hsp90s) and antioxidant enzymes (SOD-3, GST and catalase) expression after a 1 day exposure in livers of rats, whereas heat shock protein (alpha-B-crystallin, Hsp90, and GRP78) and antioxidant enzymes (SOD-1, SOD-3, catalase, GST, and GPXs) expression decreased on day 2 and then slowly recovered back to control levels on day 8. These results suggest that heat shock proteins and antioxidant enzymes were induced as protective mechanisms against 2,3,7,8-TCDD induced hepatotoxicity, and that prolonged exposure depressed their levels, which recovered to control levels due to reduced 2,3,7,8-TCDD induced hepatotoxicity.

In Utero Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Affects the Development of Reproductive System in Mouse

PURPOSE: Exposure of male reproductive organs to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) has been reported to cause developmental changes. In this study, we evaluated the effects of in utero TCDD exposure on male reproductive development. MATERIALS AND METHODS: Pregnant C57BL/6 mice were administered a single intraperitoneal injection of TCDD (1microgram/kg) on gestation day (GD) 15. The offspring were examined in the immature stage on postnatal day (PND) 30 and in the mature stage on PND 60. The testes were examined for histological changes, androgen receptor (AR), proliferating cell nuclear antigen (PCNA) and apoptosis following the measurement of morphological changes. RESULTS: Anogenital distance (AGD) and testis weights were reduced by TCDD exposure both on PND 30 and PND 60 while body weights and length of male offspring were not affected by TCDD. The regular sperm developmental stage was impaired with TCDD treatment on PND 30. However, no difference was found between the control group and TCDD groups on PND 60. Simultaneously, the expression of AR was also reduced on PND 30, while it was increased on PND 60 compared with the control group. The expression of PCNA was decreased whereas apoptosis was not affected by TCDD both on PND 30 and PND 60. CONCLUSION: These results suggest that in utero exposure to TCDD influences the development of testes by inhibiting the expression of AR and PCNA. Moreover, the adverse effects of TCDD on male offspring reduced over time.

Reproductive Development and Pulmonary CYP1A1 Levels in Mice Offspring Exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) During Lactation / 环境与健康杂志

Objective To explore the effect of lactational TCDD exposure alone on growing development and cytochrome P4501A1(CYP1A1) in mice offspring. Methods Mature Kunming mice (clean animal,No.604017) were divided to 2 treatment groups (40 and 20 ?g /kg bw TCDD),2 vehicle controls and 1 normal control. There were 3 maternal mice and 25-28 pups in each group. Maternal mice were administered TCDD by intraperitoneal injection in three times on post-parturition days 1,3,5,and the mice offspring were exposed to TCDD by maternal milk. The changes of body weight and the development of reproductive system were observed. The pups were sacrificed on postnatal days (PND) 35,and CYP1A1 expression in the lungs of mice offspring were measured by immunohistochemistry. Results The average body weight of mice offspring in 2 TCDD treatments decreased significantly from PND14 (P

Pathologic Comparative Studies on the Protective Effects by Panax Ginseng and Panax Quinquefolium for Treating 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced Toxicity in Male Rats

BACKGROUND: Panax ginseng is known to decrease the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced testicular toxicity. Thus, we aimed to reveal the differences between Panax ginseng and Panax quinquefolium extract for their effects on TCDD-induced toxicity. METHODS: Forty rats were divided into four groups; the control group, the TCDD only group, the TCDD plus Panax ginseng group, and the TCDD plus Panax quinquefolium-treated groups. Ginseng extract was given orally to rats from day one to twenty-one. TCDD was intraperitoneally administered to rats at a single dose of 50 microgram/kg on the seventh day. The pathologic changes were then examined. The changes of body weight, cholesterol and GOT in the serum were also examined. RESULTS: The TCDD toxicity was prominent in the thymus, liver and testis. The thymus showed atrophy and an inverse pattern of lymphocyte density in the cortex and medulla. The liver revealed central necrosis with fatty changes. On electron microscopy, the seminiferous tubules showed destruction of the spermatogonia, clear spaces or vacuolar changes and degeneration in the Sertoli cells or germ cells. The above mentioned TCDD-induced changes were reduced in the rats that were administered with Panax ginseng, whereas Panax quinquefolium did not reduce these changes. CONCLUSION: The protective effects of Panax ginseng on the TCDD-induced toxicity were more effective than those of Panax quinquefolium.

A Case of Incidental Epidermolytic Hyperkeratosis Occurring Normal Looking Skin Adjacent to Folliculitic Papules: In Veterans Who Participated in Vietnam War

On histological examination, an epidermolytic hyperkeratosis was observed adjacent to follicular papules on the back of a 53-year-old man. It has been reported that incidental epidermolytic hyperkeratosis occur either within various lesion (epidermal neoplasm, melanocytic neoplasm, scars, and inflammatory conditions) or in the normal skin adjacent to the lesion. This patient participated in the Vietnam War for 2 years, and had had contact with defoliants. He was treated for multiple peripheral neuropathies and cerebral infarcts. In keratinocytes, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; Agent Orange) contained in defoliating agents is associated with altered patterns of keratinocyte differentiation. So, as a cause of incidental epidermolytic hyperkeratosis, defoliant contact could be suspected.

The Neutralizing Effect of Panax Ginseng for Toxicities in the Survival, Sperm Quality, Pregnancy and F1 Generation of Guinea Pigs Exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin / 대한비뇨기과학회지

PURPOSE: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most potent environmental pollutants, is known to disrupt the endocrine, immune, and reproductive system. This study was carried out to investigate the effect of a panax ginseng water extract (PG-WE) on the survival rate, sperm quality, and fertility impaired by TCDD. MATERIALS AND METHODS: Eighty male guinea pigs were divided into 8 groups. The normal control group received the vehicle and saline. TCDD was intraperitoneally injected at a single dose of 1microgram/kg. A PG-WE was administered at 100 or 200mg/kg/ day 1wk prior to (P groups) or subsequent to (C groups) TCDD-exposure for 12 and 10 weeks, respectively. The G groups received the vehicle and the PG-WE of 100 or 200mg/kg/day, respectively. The parameters for the male guinea pigs were assessed for 40 weeks. The effects on the F1 generation were assessed at a growth period of F1. RESULTS: All single TCDD-treated group animals died within 18 days and the survival rate of the PG-WE-treated groups increased in a dose dependant manner. Forty to 70% of the P and C groups survived until the 40th week and reached sexual maturation. The death rate of the progeny born from the PG-WE-treated groups was significantly lower than that in the NC group (14.3%). The M/F ratio of the F1 generation in the P and C groups had higher female birth ratio. The sperm number and morphology showed no significant differences among the groups. The PG-WE increased the sperm motility in the guinea pigs exposed to TCDD. CONCLUSIONS: Panax ginseng is a useful agent that can neutralize endocrine disrupters and environmental pollutants, and help maintain a high sperm quality after a growth period.

Teratological effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): induction of cleft palate in the ddY and C57BL/6 mouse

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic halogenated aromatic hydrocarbon, is a teratogen to induce cleft palate when exposed during the pregnancy. There are inter-strain differences in the sensitivity to cleft palate induced by TCDD and other chemicals including polychlorinated terphenyls (PCTs). The C57BL/6 mouse and the ddY mouse had been shown to be different in the induction of cleft palate following the treatment of PCTs, which attempts us to evaluate the TCDD-induced cleft palate in two mouse strains to understand the mechanism through which TCDD and PCTs induce cleft palate. This study evaluated the induction of cleft palate in the fetuses of ddY and C57BL/6 mice after subcutaneous treatment of TCDD on gestation day (GD) 10.5-14.5 or oral treatment on GD 8.5-13.5. Our results clearly showed that ddY mice, a susceptible strain to PCTs-induced cleft palate, are resistant to the induction of cleft palate by TCDD comparably to the high susceptibility of C57BL/6 mice, suggesting a different teratological mechanism between TCDD and PCTs. In addition, at the low doses, our study supported the concept of "window effect" of TCDD on around GD 12 for the induction of cleft palate in C57BL/6 and ddY mice.

Effect of organosulfur compounds on the expression of UDP-glucuronosyltransferase and thyroid hormone level in TCDD-treated rats

In this study, we investigated the effect of organosulfur compounds including, diallyl sulfide (DAS), garlic extract, and dially disulfide (DADS) on inducible expression of UDP-glucuronosyltransferase (UGT1) and thyroid hormone level in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats. We measured UGT1 gene expression in rat liver and lung tissues after treatments. We found that a correlation was found to exist between UGT1 gene expression and active T-4 concentrations. The total concentration of plasma T-4 decreased in TCDD-alone treated male and female rats compared to control animals (TCDD-alone < control). Plasma T-4 concentration in TCDD + DAS, TCDD + garlic extract and TCDD + DADS treated groups were moderately higher than in TCDD alone animals, but lower than control animals. TCDD + DAS, TCDD + garlic extract and TCDD + DADS moderately increased T-4 glucuronidation and moderately reduced total T-4. The nonthyroidal nature of thyroid hormone reduction observed in this study extends the evidence for microsomal enzyme inducers as indirect thyroid tumor promoters in rats. Furthermore, the results of present study suggest protective effects of DAS, garlic extract, or DADS on TCDD-induced changes.

Protein Expression Profile in Serum of Male Rats Chronically Exposed to Low-level 2,3,7,8-tetrachlorodibenzo-p-dioxin / 环境与健康杂志

Objective To explore the chronic effect of low levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) exposure on the protein expression profile in the serum of rats and to get the further understanding of the molecular mechanism of TCDD toxicity.Methods Thirty-two male SD rats were randomly divided into three TCDD exposed groups treated with TCDD at the doses of 875,350 and 140 ng /kg by gavage,once a week,for 29 consecutive weeks respectively and one control group treated with single corn oil.The proteins in the serum were separated and analyzed by two-dimensional gel electrophoresis and mass spectrometry,the histopathological examination of livers was performed at the same time.Results TCDD induced significant fatty degeneration and necrosis in the livers.Several interesting volume-altered protein spots were identified.Among these proteins,Bal-647 was down-regulated,apolipoprotein E was up-regulated by TCDD and fetuin-like protein was only expressed in the group of 875 ng/kg.Conclusion The differentially expressed proteins may contribute to a better understanding of the toxicity induced by TCDD and may be used as the biomarkers of effect for TCDD exposure.