Lanqin Shang

Bisphenol A inhibits proliferation and induces apoptosis in micromass cultures of rat embryonic midbrain cells through the JNK, CREB and p53 signaling pathways.

Bisphenol A (BPA) has been widely used in the manufacture of polycarbonate plastic, water bottles and food containers. Previous studies have established that BPA could cause developmental toxicity by inhibiting the proliferation and differentiation of rat embryonic midbrain (MB) cells in vitro. However, the underlying mechanisms have not been well studied yet. In the current study, we examined the proliferation and differentiation of MB cells treated with 10(-12)-10(-4)M BPA and found that only 10(-4)M BPA inhibited proliferation and differentiation. Then, we investigated the cell cycle progression and apoptosis of MB cells; 10(-4)M BPA caused an explicit S phase and G2/M phase arrest in the cell cycle and increased the percentage of apoptotic cells. Moreover, the phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic-AMP response binding protein (CREB) and the expression of some apoptotic regulatory genes were investigated. BPA (10(-4)M) reduced the phosphorylation of C-Jun N-terminal kinase (JNK) and CREB, and increased the mRNA expression level of Bax and p53. Our findings demonstrated that BPA could cause developmental toxicity through anti-proliferation and pro-apoptosis in MB cells. Multiple signaling pathways, such as the JNK, CREB and p53-mitochondrial apoptosis pathways, participate in these effects.

Embryotoxic and Teratogenic Effects of the Combination of Bisphenol A and Genistein on In Vitro Cultured Postimplantation Rat Embryos

The potential teratogenic effects and fetal toxicity of environmental estrogenic endocrine disruptors have become a great concern in recent years, and they have yet to be fully characterized. In the present study, the teratogenic effects of bisphenol A (BPA) and genistein (GEN) on rat embryos during their critical period of organogenesis were investigated using a whole-embryo culture experiment. The combined exposure effects of BPA and GEN were explored using a 4 x 4 full factorial design. Both BPA and GEN produced concentration-dependent inhibition of embryonic development, beginning at 32.0 and 10.0 microg/ml, respectively. Full factorial and isobologram analyses revealed a significant synergistic interaction between BPA and GEN for most end points (12 out of 20 tested), as indicated by the enhanced developmental toxicity of BPA after coexposure with different dose levels of GEN. In particular, serious malformations and a higher abnormal frequency of the central nervous system were induced by the combination of BPA and GEN. Our findings suggest that GEN may be embryotoxic and teratogenic to humans. BPA alone may not be a potential teratogen, but these two estrogenic chemicals have a synergistic effect on embryonic development when present together during the critical period of major organ formation. The current findings suggest that pregnant women should not take soy supplements, but more studies are necessary to provide a conclusive recommendation.

The role of MAPK in the biphasic dose-response phenomenon induced by cadmium and mercury in HEK293 cells

Some studies show that Cd(2+) and Hg(2+) may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells. However, mechanisms underlying this phenomenon are still in puzzle. In this study, we aim at detecting the biphasic effects of Cd(2+) and Hg(2+) on proliferation and apoptosis of human embryonic kidney 293 (HEK293) cells, analyzing the change of the mitogen-activated protein kinase (MAPK) pathways, and discussing the relationship between them. The results demonstrate that Cd(2+) and Hg(2+) can stimulate cell proliferation at lower concentrations (0.05 and 0.5 microM) but inhibit it at higher concentrations (50 and 500 microM). Apoptosis increases at higher concentrations (50 and 500 microM) of Cd(2+) and Hg(2+). While 0.5 microM Cd(2+) and Hg(2+) decrease the JNK phosphorylation, 50 microM Cd(2+) and Hg(2+) increase the JNK and P38 phosphorylation. When HEK293 cells are treated with 20 microM JNK inhibitor or 100 microM ERK1/2 inhibitor, the cell proliferation do not increase significantly at low concentrations (0.05 and 0.5 microM), but still decrease at high concentrations (50 and 500 microM). When HEK293 cells are treated with 20 microM P38 inhibitor, the tendency of cell proliferation is not affected. Data in our study suggests that activation of MAPK pathway may be involved in the biphasic effect induced by Cd(2+) and Hg(2+).

Individual and combined developmental toxicity assessment of bisphenol A and genistein using the embryonic stem cell test in vitro.

The potential developmental toxicity of environmental estrogenic endocrine disruptors have become a great concern in recent years. In this study, two typical environmental oestrogen, namely, bisphenol A (BPA) and genistein (GEN) were investigated for potential embryotoxicity using the embryonic stem cell test model. Afterwards, a 4×4 full factorial design and the estimated marginal means plot were performed to assess the combined effects of these two compounds. According to the linear discriminant functions and classification criteria, bisphenol A and genistein were classified as weakly embryotoxic and strongly embryotoxic respectively. As for combined effects, the overall interaction between BPA and GEN on embryonic stem cells (ESCs) differentiation was synergistic at low dosages, however, on ESCs and 3T3 cell proliferation, the predominate action was additive. Considering the actual daily intake of these chemicals, it is concluded that BPA alone might not have adverse reproductive or developmental effects on human being. However, given that BPA and GEN do have synergistic effect at low concentration, they may disturb normal embryo development together, which could result in birth defect and behavioral alterations later in life.

Effects of bisphenol A on the proliferation and cell cycle of HBL-100 cells

Bisphenol A (BPA) is an environmental estrogen that exhibits non-genotoxic carcinogenicity, causing concern globally. The aim was to investigate the effects of BPA on the proliferation and cell cycle of human normal breast cells HBL-100. An improved E-Screen assay was used to study cell proliferation, and flow cytometry was used to study cell cycle phases. Western blot analysis was utilized to detect cell cycle proteins and estrogen receptor α (ERα) expression. The results showed that the highest cell proliferation rate induced by BPA was at 1.0 × 10(-6)mol/L. At 1.0 × 10(-10), 1.0 × 10(-8), and 1.0 × 10(-6)mol/L, BPA promoted more cells to enter into G2/M phase and caused an increase in the expression of cyclinD1 and CDK4. After adding ICI182780 into the system, the promoting effects of BPA on cell proliferation and cell cycle change decreased, but these promoting effects were still significantly greater compared with the solvent control (P<0.05). Regardless of ICI182780 exposure, BPA did not have significant effect on ERα expression. BPA has estrogen-like activity and can stimulate HBL-100 proliferation and cell division through the estrogen receptor pathway. BPA may have other pathways through which it can exert stimulating effects and exhibit non-genotoxic carcinogenicity.

In vitro malignant transformation of human bronchial epithelial cells induced by benzo(a)pyrene.

In this study, the human bronchial epithelial cells (16HBE) were treated five times with 10μM benzo(a)pyrene (BaP), followed by 20 passages culture, and the in vitro BaP-induced malignant transformation of 16HBE cells was established. Five colonies in soft agarose were then amplified and donated as T-16HBE-C1∼5 cells, respectively. T-16HBE-C1∼5 cells can form tumors subcutaneously in nude mice. Histopathological changes in the tumors indicated nests growth, high nuclear-cytoplasmic ratios, coarse and clumped chromatin, numerous and distinctly atypical mitoses, cell necrosis and surrounding normal adipose, muscle and connective tissue immersed. In addition, lung metastasis was observed in nude mice in T-16HBE-C1, 3 and 4 groups. In vitro cell migration assay results indicated that T-16HBE-C2∼5 cells showed much lower migration capabilities than 16HBE cells. Western blotting analysis showed that the expressions of p53 and p-Akt (Ser473) in T-16HBE-C1∼5 cells were significant higher than those in 16HBE cells. Our results demonstrated that BaP could induce the malignant transformation of 16HBE cells, and p53 and p-Akt (Ser473) might play crucial roles in BaP-induced carcinogenesis. The five monoclonal cell lines (T-16HBE-C1∼5) with different migration capabilities could be used as research models for further understanding the mechanisms of BaP-induced carcinogenesis and cell migration.

MAP4K4 deficiency in CD4(+) T cells aggravates lung damage induced by ozone-oxidized black carbon particles.

As the main composition of combustion, black carbon (BC) is becoming more and more noticeable at home and abroad. Ozone-oxidized black carbon (oBC) was produced through aging of ozone, one of the near-surface pollutants, to black carbon. And oBC was found to be more oxidation and cell toxicity when compared with BC. Besides, as a key cell of immunity, whether CD4(+) T cell would involve in lung inflammation induced by particular matter is still unclear. This study aims to observe the effect of oBC on lung damage in mice and discuss how the functional MAP4K4 defect CD4(+) T cells (conditional knockout of MAP4K4) presents its role in this process. In our study, MAP4K4 deletion in CD4(+) T cells (MAP4K4 cKO) could increase cell number of macrophages, lymphocytes and neutrophils in bronchoalveolar lavage fluid (BALF) exposed to oBC. MAP4K4 deletion in CD4(+) T cell also affected CD4(+) T cell differentiation in mediastinal lymph nodes after oBC stimulation. The number of CD4(+) IL17(+) T cell increased obviously. The levels of IL-6 mRNA of lung in MAP4K4 cKO mice was higher than that in wild type mice after exposed to oBC, while the level of IL-6 in BALF had the same trend. Histological examination showed that MAP4K4 deletion in CD4(+) T cells affected lung inflammation induced by oBC. Results indicated that MAP4K4 cKO in CD4(+) T cells upgraded the level of inflammation in lung when exposed to oBC, which may be connected to the CD4(+) T cell differentiation and JNK, ERK and P38 pathways.

The skeletal developmental toxicity of chlormequat chloride and its underlying mechanisms

Chlormequat Chloride (CCC), a widely used plant growth regulator, could decrease body weight in animals; however, the mechanism has not been well studied. This study was designed to evaluate the skeletal development toxicity of CCC on pubertal male Sprague-Dawley (SD) rats and to investigate whether CCC impacts the development of chondrocyte, osteoblast and osteoclast through growth hormone (GH) and insulin like growth factor 1 (IGF-I). Rats from 23 to 70 on postnatal days were exposed to CCC daily by gavage at doses of 0, 75, 150, and 300mg/kg bw/d. The results showed that the size of femurs and tibias, bone mineral density and biomechanical parameters were significantly decreased in the 300mg/kg bw/d group compared with the control group. The concentration of osteocalcin (OCN) and C-terminal telopeptide of type I collagen (CTX-I) in blood in the 150mg/kg bw/d group was also changed. The mRNA expression ratio of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in 150 and 300mg/kg bw/d group was increased. Histological analysis of proximal and distal epiphyseal plates of the right femurs showed that both the proliferative zone and hypertrophic zone narrowed in CCC-treated groups. The concentration of IGF-I in blood was reduced with an increase in exposure doses of CCC. The mRNA expression of growth hormone receptor (GHR) in tibia was decreased in the CCC-treated group. The results indicated that CCC might indirectly impact the formation and activation of chondrocytes, osteoblasts and osteoclasts because of the decline of GHR and IGF-I, leading to skeletal development damage.

The effects of chlormequat chloride on the development of pubertal male rats

Chlormequat Chloride (CCC) is a plant growth regulator that is widely applied in agriculture. Previous studies have shown that long-term exposure of CCC could decrease body weight in animals. However, the underlying mechanisms have not been studied. In this study, CCC was administered to rats daily by gavage on postnatal days 23-60 at doses of 0, 75, 150 and 300mg/kg bw/d. The results showed that body weight and the length of the right femur were significantly decreased in the 300mg/kg bw/d group. Histological analysis of proximal growth plates of the right femurs showed narrowed proliferative zones and hypertrophic zones in CCC-treated groups. The mRNA expression of growth hormone, growth hormone receptor and insulin like growth factor 1 were decreased in the CCC-treated group. The results indicated that CCC may affect the expression of growth hormone and insulin-like growth factor 1 and subsequently cause a decrease in body weight and bone length.

The research of genetic toxicity of β-phellandrene

β-Phellandrene, a plant extract, can be used as natural pesticides and synthetic materials. As a factor that human may be exposed to, the toxicity information about β-phellandrene is scared at present. This study focused on the genetic toxicity of β-phellandrene. The genetic toxicity of β-phellandrene was evaluated by micronucleus test, comet assay, Ames test, and chromosomal aberration test. In this study, 2850, 1425, 712.5mg/kg β-phellandrene were used in vivo experiments (comet assay and micronucleus test). For Ames test, pure β-phellandrene and different concentrations were used in the experiment. According to the results of cell viability assay (MTT test), the concentration of chromosomal aberration test was formulated. The result of comet assay showed that β-phellandrene can significantly induce DNA damage at the dosage of 1425 and 2850mg/kg. While the results of Micronucleus test and chromosome aberration test showed that β-phellandrene does not lead to apparently genetic toxicity on chromosome level. Ames tests suggest that β-phellandrene had the ability to increase gene mutation with or without S9 mixture. So, it could be drawn that β-phellandrene would have certain genetic toxicity, and the toxicity is reflected as DNA strand breaks and mutation. This study filled the lack of genetic toxicity study of β-phellandrene, and enriched information for risk assessment for β-phellandrene.