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Dioxin-related compounds are associated with teratogenic and mutagenic risks in laboratory animals, and result in adverse pregnancy outcomes. However, there were inconsistent results in epidemiology studies. In view of this difference, we conducted a systematic review and meta-analysis to examine this association and to assess the heterogeneity among studies. Comprehensive literature searches were performed to search for relevant articles published in English up to 15 May 2012. In total, we identified 15 studies which included 9 cohort and 6 case control studies. The Cochrane Q test and index of heterogeneity (I(2)) were used to evaluate heterogeneity. In either cohort studies (I(2)=0.89, p<0.0001) or case control studies (I(2)=0.69, p=0.02), significant heterogeneity of risk estimates were observed. Subgroup analyses found no significant increased risk of adverse pregnancy outcome with air dioxin-related compounds exposure (RR=0.99, 95% CI:0.85-1.16), no significant increased risk of spontaneous abortion (SAB) with exposure to food dioxin-related compounds (RR=1.05, 95% CI:0.80-1.37), higher significant risks of low birth weight (LBW) with exposure to food dioxin-related compounds (RR=1.55, 95% CI:1.24-1.94), and higher significant risks of birth defects with maternal solid contaminants dioxin exposure (OR=1.24, 95% CI:1.19-1.29). In conclusion, more evidences are needed to confirm the association between environmental dioxin-related compounds exposure and pregnancy outcome.

Oxidative stress is a recognized factor in nephrotoxicity induced by chronic exposure to inorganic arsenic (As). Grape seed extract (GSE) possesses antioxidant properties. The present study was designed to evaluate the beneficial effects of GSE against arsenic-induced renal injury. Healthy, male Sprague-Dawley rats were exposed to As in drinking water (30 ppm) with or without GSE (100 mg/kg) for 12 months. The serum proinflammatory cytokine levels and mRNA expression levels of fibrogenic markers in the renal tissues were evaluated using enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. The protein expression levels of nicotinamide adenine dinucleotide phosphate (NADPH) subunits, transforming growth factor-β1 (TGF-β1) and phosphorylated Smad2/3 (pSmad2/3) were assessed using western blot analysis. The results demonstrated that cotreatment with GSE significantly improved renal function, as demonstrated by the reductions in relative kidney weight (% of body weight) and blood urea nitrogen, and the increase in the creatinine clearance capacity. GSE attenuated the As-induced changes in the serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β and the mRNA levels of TGF-β1, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and fibronectin (FN) in renal tissue. Furthermore, administration of GSE markedly reduced As-stimulated reactive oxygen species (ROS) production and Nox activity, as well as the protein expression levels of the NADPH subunits (Nox2, p47phox and Nox4). In addition, GSE cotreatment was correlated with a significant reduction in TGF-β/Smad signaling, as demonstrated by the decreased protein levels of TGF-β1 and pSmad2/3 in renal tissue. This study indicated that GSE may be a useful agent for the prevention of nephrotoxicity induced by chronic exposure to As. GSE may exert its effects through the suppression of Nox and inhibition of TGF-β/Smad signaling activation.

We previously showed that atRA (all-trans Retinoic Acid, atRA) inhibites chondrogenesis by downregulation of TGF-beta/Smad signaling. However, the molecular link between RA and TGF-beta signaling is unknown. Using a mouse embryo palate mesenchyme micomass cultures (MMCs) system, we explored interactions of RA and TGF-beta signaling during chondrogenesis. We found that atRA suppressed chondrogenesis and Smad2/3 phosphorylation regardless of the presence of TGF-beta3. Functional assays indicated that TGF-beta3 treatment or co-transfection of expressing Smad2/3 vectors suppressed atRA-induced RARE-tk-Luc activity. Conversely, atRA or RAR-overexpression repressed TGF-beta3-induced transactivation of the TGF-beta-responsive reporter, p3TP-Lux. ChIP assay revealed the binding of the Smad transcriptional co-repressor TGIF (TG-interacting factor, TGIF) to RARbeta promoter in control MMCs, but this association was decreased by the addition of RA and increased by TGF-beta3, respectively. Further examinations revealed that TGIF exerted a pivotal role in regulating crosstalk of RA and TGF-beta signaling, since siRNA knockdown of TGIF partially abolished the ability of atRA to suppress TGF-beta3-induced chondrogenesis, whereas forced expression of TGIF blocked the ability of TGF-beta3 to relieve atRA-mediated the suppression of chondrogenesis. Furthermore, we demonstrated that the effects of atRA on TGF-beta-dependent gene activation and of TGF-beta on RA-dependent gene activation are mediated by TGIF with siRNA to downregulate TGIF. Collectively, these findings indicated a negative functional interplay of RA and TGF-beta signaling mediated by TGIF to modulate chondrogenesis in MMCs., (2009 S. Karger AG, Basel.)

Chondrogenesis is a critical step in palatogenesis. All-trans retinoic acid (atRA), a vitamin A derivative, is a known teratogenic effector of cleft palate. Here, we evaluated the effects of atRA on the osteo-/chondrogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells. MEPM cells, in a high-density micromass environment, undergo active chondrogenesis in a manner analogous to that of limb-derived mesenchymal cells, and served as a valid model system to investigate the mechanisms regulating chondrogenesis during palatogenesis. atRA-treated MEPM micromass expressed relatively higher levels of osteoblastic gene markers (alkaline phosphatase and collagen type I) and lower levels of chondrocytic gene markers (collagen type II and aggrecan). As transforming growth factor-beta3 (TGF-beta3) is an essential growth factor for chondrogenesis of embryonic mesenchymal cells both in in vivo and in vitro conditions, we thereby explored the effects of atRA on TGF-beta3 signaling pathway. atRA led to an increase in mRNA expression of TGF-beta3 and an instantaneous decrease in TGF-beta type II receptor (TbetaRII) as determined by real-time RT-PCR. Further study showed that atRA inhibited phosphorylation of Smad2 and Smad3 and increased Smad7 expression. Activation of the Smad pathways by transfection with Smad7deltaC mutant or constitutively active TbetaRII retroviral vector abolished atRA-induced inhibition of chondrogenesis as indicated by Alcian blue staining, indicating that Smad signaling is essential for this response. Taken together, these data for the first time demonstrated a role for RA-induced hypochondrogenesis through regulation of the TGF-beta3 pathway and suggested a role for TbetaRII /Smad in retinoid-induced cleft palate.

TGF-beta1/signaling has been shown to be associated with proapoptotic and antimitotic activities in epithelial tissues. Genistein, a major component of soybean isoflavone, has multiple functions resulting in anticancer proliferation. We herein showed that genistein dose-dependently increased TGF-beta1 mRNA expression in mouse colon cancer MC-26 cells. A mouse monoclonal anti-TGF-beta1 neutralizing antibody partially, but not completely, blocked the growth inhibition by genistein. By using adenoviral vector, we demonstrated that Smad7 overexpression attenuated genistein-induced growth inhibition and apoptosis as determined by MTT and apoptosis ELISA. Smad7 overexpression also inhibited upregulation of p21 and caspase-3 activity by geinistein. To further confirm inhibitory effect of genistein in MC-26 cells require TGF-beta1/Smad signaling, we employed Western blot and electrophoretic mobility shift assay to detect formation of Smad-DNA complexes and phosphorylation of Smad2 and Smad3, respectively. Data revealed that genistein induced an evident formation of Smad-DNA complexes and phosphorylation of Smad2 and Smad3, indicating increased TGF-beta1 signaling. Taken together, these findings first provided insights into possible molecular mechanisms of growth inhibition by genistein that required Smad signaling, which could aid in its evaluation for colon tumor prevention.

Objective: To further explore the effects of genistein (GS) on cell proliferation and differentiation in adult human osteoblast., Methods: Human osteoblast from passage 4 were harvested and seeded in phenol red-free DMEM medium containing 5% charcoal-stripped fetal bovine serum (CS-FBS) before the addition of test compounds for 4 days. DNA synthesis was tested by 3H-thymine incorporation and cell cycle was determined by flow cytometry. Collagen protein content and alkaline phosphatase (AKP) activity were examined., Results: After treatment for 48 h, the presence of GS (10(-7) or 10(-6) mol/L) caused a significant increase in DNA content and accumulation at S and G2/M phage in a dose-dependent manner. Additionally, the anabolic effect of GS (10(-7) and 10(-6) mol/L) on the collage content and AKP activity were significantly enhanced in the presence of genistein (10(-7) or 10(-6) mol/L) . The effects of GS on osteoblast were completely abolished in the presence of estrogen receptor antagonist, 4-Hydroxy tamoxifen (TAM) (10(-7) mol/L)., Conclusion: Findings suggest that osteoblast function is promoted by genistein and that the estrogen receptors might be involved in the response, thereby playing an important role in bone remodeling. Presumably, dietary soy products are useful in the prevention of osteoporosis.

Objective: This study was designed to investigate the molecular mechanisms of proliferation and apoptosis by environmental estrogens (n-4-nonylphenol, NP; Bisphenol A, BisA; Dibutyl Phthalate, DBP) in ovarian cancer PEO4 cells., Methods: PEO4 cells were maintained in DMEM medium with 10% neonatal bovine serum. Five days before the beginning of experiments, the cells were seeded in phenol red-free DMEM medium containing 5% charcoal dextran-treated FBS. The cells were harvested and seeded in 6-well culture plates or in 75ml flacks. After various concentration of NP, BisA and DBP treatment for 72h, the cells were harvested and detected mRNA and protein expression of PCNA, bcl-2 and bax by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively., Results: 32 x 10(-7) mol/L NP and 32 x 10(-7) mol/L BisA could significantly up-regulate PCNA and bcl-2 mRNA expression and down-regulate the bax mRNA expression, and 32 x 10(-6) mol/L DBP could up-regulate PCNA mRNA expression, but had no effect on bax and bcl-2 mRNA expression. These results were further confirmed by following immunohistochemistry., Conclusion: PCNA, bcl-2 and bax pathway might involve in cell proliferation and apoptosis events by environmental estrogens in ovarian cancer PEO4 cells.

Dietary genistein, a natural flavone compound found in soy, has been proposed to be related to the lower prevalence of breast cancer in Asian women. In order to elucidate the mechanism of chemopreventive effect of genistein so that to provide more scientific basis for dietary intervention for breast cancer in women, the apoptosis in estrogen responsive breast cancer cell lines (MCF-7 and T47D cells) was induced as the study model and the effect of genistein on the apoptosis was observed. The results showed that, functionally similar to the anti-estrogen tamoxifen, genistein induced in a dose- and time-dependent manner the MCF-7 cell apoptosis at concentrations above 20 x 10(-6) mol/L. The findings suggested that the increased isoflavone intake can induce apoptosis in estrogen responsive cells.

The study was designed to explore the estrogenic activity of environmental disrupters [n-4-noniphenol (NP), bisphenol A (bisA) and dibutylphthalate (DBP)]. Estrogen-respective breast cancer cell line T47D was grown in DMEM medium containing 10% bovine serum. Five days before the addition of the test compounds, the cell were washed in phosphate-buffered saline, and the medium was substituted with a phenol red-free DMEM medium containing 5% dextran charcoal-stripped FBS. The respective test compound was added in fresh medium and the control cell received only the vehicle (ethanol). The proliferation of T47D cells was analyzed by MTT assay, 3H-TdR incorporation assay and flow sytometer. The results indicated that, compared with the EtOH control cell, the test compounds (n-4-noniphenol, Bisphenol A and dibutylphthalate), like estradiol, markedly enhanced the proliferation of T47D cell and the metaphase of cell division, and the results showed time-dependent and dose-dependent model. These data showed that the tested chemicals could enhance the proliferation of human cervix cancer cell in vitro. This might hinted that these chemicals possessed estrogenic activity and they might play their estrogen through estrogenic receptor.

The effects of zinc on bone metabolism is explored. A home-made rotating apparatus is used to cultivate fetal mouse front limbs at 16-day gestational age. The activity of alkaline phosphatase(AKP) and histological silce are used to analyze the effects of zinc on bone metabolism. Compared with the control group, the zinc deprived group and Zn2+ 120 mumol/L group have a lower alkaline phosphatase (AKP) activity and the contracted chromatin, membrane rupture and matrix loss are observed in bone cells. The zinc adequate group(Zn2+ concentration are 45 mumol/L and Zn2+ 70 mumol/L respectively) has a higher alkaline phosphatase(AKP) activity, more active bone generation, deeper stain of bone matrix and increased bone trabecula. It is concluded that zinc could affected the bone metabolism and growth; both zinc-deficiency and zinc-excess could alter growth and bone metabolism.

Objective: This study was designed to explore the effects of zinc on bone development., Methods: Forelimbs of mice with 16 d gestation were cultured by a rotating device., Results: Contents of OC and (45)Ca and activities of AKP in the bone tissues cultured at zinc-deficiency media and at media with 120 micro mol/L of Zn(2+) decreased significantly. Synthesis of OC, absorption of calcium and activities of AKP in the bone tissues cultures at media with 45 and 70 micro mol/L of Zn(2+) increased significantly. Radiograph of bone tissues showed that length of long bone cultured at zinc-deficiency media and at media with 120 micro mol/L of Zn(2+) shortened and their density reduced, and those cultured at media with 45 and 70 micro mol/L of zinc increased, as compared with self-control bone. Histological analysis showed the death of bone cells and loss of stroma in the bone tissues cultured at media with 120 micro mol/L of Zn(2+), and active proliferation and differentiation of bone cells, and secretion and synthesis of osteoid increased in the bone tissues cultured at media with 45 and 70 micro mol/L of zinc., Conclusions: Adequate supplementation of zinc could promote formation and development of bone tissues and deficiency or excess of zinc could alter their growth and development and normal metabolism.