Bisphenol A impairs macrophages through inhibiting autophagy via AMPK/mTOR signaling pathway and inducing apoptosis.

Bisphenol A (BPA) is a widespread endocrine disruptor that induces the impairment of immune cells, but the mechanism remains unknown. Macrophages are one of the most important immune cells in innate and adaptive immunity. In this study, we aimed to probe the effects of BPA on the damage of RAW264.7 cells and its mechanisms of action, especially focusing on the relationship between autophagy and apoptosis. Cells were pretreated with 10 mg/L LPS, or added autophagy activator RAPA, autophagy inhibitor 3-MA or Bcl-2 inhibitor ABT-737, then treated with BPA (0, 10, 100 and 200 µmol/L) for 12 h. Results have shown that BPA decreased the cell viability and disrupted secretory function by promoting pro-inflammatory cytokines TNF-α and IL-6 and reducing anti-inflammatory cytokines IL-10 TGF-ß, as well as phagocytic ability. Moreover, autophagy was inhibited by BPA through decreasing p-AMPK/AMPK and increasing p-mTOR/mTOR, and further down-regulating autophagy proteins ATG6, LC3II/I ratio, and up-regulating autophagy flux protein p62. Additionally, BPA significantly increased Bax/Bcl-2 ratio, Caspase-3 expression and apoptosis rate. We found that RAPA ameliorated the cell viability, Bax/Bcl-2 ratio, and macrophage function damage induced by BPA. Intriguingly, ABT-737 might promote ATG6 expression. In summary, our study demonstrated that the effects of BPA on macrophages seemed to be mediated by inhibiting AMPK/mTOR-dependent autophagy and inducing apoptosis via endogenous mitochondrial pathway. Both Bcl-2 and ATG6 were involved in the regulation of apoptosis and autophagy by BPA. These findings provide a broader perspective for understanding the interaction between autophagy and apoptosis in BPA-induced immune cell injury.

The alteration of H4-K16ac and H3-K27met influences the differentiation of neural stem cells.

The neural stem cell therapy provides a promising future for patients with central nerve system damage, thus an insight into its differentiation mechanism is urgently needed. Herein, we aimed to identify various histone modifications and reveal their impact on the differentiation of neural stem cells (NSCs) toward neurons. Firstly, we labeled primary NSCs using the stable isotope labeling with amino acids in cell culture (SILAC) technique. Then we induced these NSCs to differentiate by all-trans retinoic acid (atRA) or SB216763. Next, we identified the alteration of histone modifications in early-differentiated NSCs by mass spectrometry and verified them by Western blot. Interestingly, these modification alterations and phenotype changes were found similar in NSCs induced by the two different drugs. More interestingly, during the differentiation process H3-K27met was significantly up-regulated while H4-K16ac was not altered at the global level but down-regulated in some low-abundance combinatorial codes. We inhibited the methyltransferase of H3-K27 and deacetylase of H4-K16 simultaneously and found the differentiation procedure was obviously delayed. The function of H4-K16ac and H3-K27met in NSCs differentiation would be useful to reveal the differentiation mechanism and valuable for further neural stem cell therapy.

Role of Ca/CaN/NFAT signaling in IL-4 expression by splenic lymphocytes exposed to phthalate (2-ethylhexyl) ester in spleen lymphocytes.

The aims of present study were to investigate the effect of phthalate (2-ethylhexyl) ester (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on Th1/Th2 balance signaling for interleukin 4 (IL-4) expression in splenic lymphocytes, and contribution of MEHP to any hypothesized changes in vitro. Primary splenic lymphocytes were exposed to DEHP/MEHP. ELISA and Western blotting were used to detect proteins. Confocal-microscopy was used to examine nuclear translocation. Nuclear factor of activated T cells (NFAT) DNA binding activity was examined by electrophoretic mobility-shift assay. DEHP significantly increased IL-4 and interferon gamma (IFN-γ) level, and reduced Th1/Th2 ratio (reflected by IFN-γ/IL-4) with 5 µg/L Concanavalin A (ConA) treatment. While MEHP reduced Th1/Th2 ratio (represented by IFN-γ/IL-6). IL-4 mRNA was significantly increased by DEHP but not by MEHP after PMA and Ion treatment. DEHP significantly inhibited NFATp protein in cytosol and nucleus. DEHP augmented nuclear translocation of NFATc in transfected EL4 cells and NFAT DNA-binding activity. DEHP-mediated enhancement of calcium-dependent phosphatase calcineurin (CaN) protein, and NFAT and IL-4 expression were abrogated by calcium antagonist verapamil and CaN inhibitor tarcolimus. Ca(2+)/calmodulin antagonist chlorpromazine significantly suppressed IL-4 and CaN production with no NFAT mRNA change. Our study suggests that DEHP and MEHP impact Th1/Th2 balance by modulating different cytokines. DEHP-affected IL-4 expression through Ca/CaN/NFAT signaling pathway, but no effect was discovered for MEHP.

Prenatal BPA exposure disrupts male reproductive functions by interfering with DNA methylation and GDNF expression in the testes of male offspring rats.

BPA is a ubiquitous environmental endocrine-disrupting chemical, and maternal exposure to BPA is associated with impaired male reproductive functions; however, the mechanisms remain to be elucidated. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in maintaining normal spermatogenesis and fertility. However, the effect of prenatal BPA exposure on GDNF expression and its mechanism in the testis has not been reported. In this study, pregnant Sprague-Dawley rats were respectively exposed to 0, 0.05, 0.5, 5, and 50 mg/kg/day BPA via oral gavage from gestational day (GD) 5 to GD 19, with 6 pregnant rats in each group. ELISA, histochemistry, real-time PCR, western blot, and methylation-specific PCR (MSP) were used to detect the sex hormone levels, testicular histopathology, mRNA and protein expression of DNA methyltransferases (DNMTs) and GDNF, and the promoter methylation of Gdnf in the testes of male offspring at postnatal day (PND) 21 and PND 56. Prenatal BPA exposure increased body weight; decreased sperm counts and serum levels of testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH); and induced testicular histological damage, which indicated the damage of male reproductive function. Prenatal BPA exposure also upregulated Dnmt1 in 5 mg/kg group and Dnmt3b in 0.5 mg/kg group, but down-regulated Dnmt1 in 50 mg/kg group at PND 21. At PND 56, Dnmt1 was significantly increased in 0.05 mg/kg group but decreased in 0.5, 5, and 50 mg/kg groups, Dnmt3a was decreased, and Dnmt3b was markedly increased in 0.05 and 0.5 mg/kg groups but decreased in 5 and 50 mg/kg groups. The mRNA and protein expression levels of Gdnf were decreased markedly in 0.5 and 50 mg/kg groups at PND 21. And the methylation level of Gdnf promoter was significantly increased in 0.5 mg/kg group, but decreased in 5 and 50 mg/kg groups at PND 21. In conclusion, our study indicates that prenatal BPA exposure disrupts male reproductive functions, interferes with the expression of DNMTs, and decreases Gdnf expression in the testes of male offspring. Gdnf expression may be regulated by DNA methylation; however, the detailed mechanism needs to be further investigated.

[Effects of di-(2-ethylhexyl) phthalate exposure on reproductive development and PPARs in prepubertal female rats].

OBJECTIVE: To investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) exposure on pubertal development and reproductive endocrine function in prepubertal female rats and its possible mechanisms. METHODS: 40 female SD rats at 3-week-old were randomly divided into a control group (corn oil) and three exposure groups, which were exposed consecutively for 28 days to DEHP by oral gavage at doses of 50, 150 and 500 mg/(kg x d). The onset of puberty was determined by daily examination for vaginal opening (OV), breast development and the first estrous cycle. By the end of the experiment, the rats were sacrificed during the diestrous stage. The gene expression interrelated with ovary function was detected by real-time PCR. Serum levels of follicle stimulating hormone (FSH) luteinizing hormone (LH), estradiol (E2), progesterone (P4) and testosterone (T) were measured by ELISA. The morphological change of ovaries was observed by HE staining under optical microscope. The expression of PPARgamma protein in ovary cells was measured by immuohistochemistry. RESULTS: The age of vaginal opening was advanced by DEHP exposure in 500 mg/kg group, and the body weight was increased at the time of vaginal opening in 150 and 500 mg/kg groups (P < 0.05). Compared to the control group, the level of T in all DEHP groups was decreased; the levels of FSH and E2 were decreased and the level of P4 was increased in 150 and 500 mg/kg groups (P < 0.05). The gene expression of P4S0Aromin 150 and 500 mg/kg groups was significantly decreased compared to the control group. A significant increase of atretic follicles and a significant reduction of corpora lutea were observed in 150 and 500 mg/kg groups compared to the control group. The expression of PPARgamma protein in granulosa cells of follicle and corpora lutea in 150 and 500 mg/kg groups was higher than that in the control group and 50 mg/kg group (P < 0.05). CONCLUSION: DEHP exposure can affect the pubertal development and reproductive endocrine function in prepubertal female rats, and its possible mechanism may be correlated with PPARgamma.

The effects of maternal exposure to BPA during pregnancy on the male reproductive system and the testicular microRNA expression profile.

The effect of prenatal bisphenol A (BPA) exposure is increasingly concerned. We investigated the effect of maternal BPA exposure during pregnancy on male offspring and its potential mechanism. Thirty pregnant Sprague Dawley (SD) rats were randomly divided into exposed and control groups. At PND56, the number of sperm, luteinizing hormone, and testosterone in the BPA-exposed group decreased, and testicular tissue structure was damaged in offsprings. At GD20, the miRNA profile in the testis of male offspring was examined and the expression levels of 28 deregulated miRNAs were validated by qRT-PCR. We found that miR-361-5p, miR-203a-3p, and miR-19b-2-5p had significantly different expression levels in the testis. These results suggest that maternal exposure to BPA can lead to differential changes in progeny miRNAs, which will provide direction for future in-depth mechanisms of reproductive injury.

Involvement of NMDAR/PSD-95/nNOS-NO-cGMP pathway in embryonic exposure to BPA induced learning and memory dysfunction of rats.

Bisphenol A (BPA), can lead to learning and memory impairment, but the underlying mechanism is poorly understood. Researchers have indicated that the N-methyl-D-aspartate receptor (NMDAR)/postsynaptic density protein 95 (PSD-95)/neuronal nitric oxide synthase (nNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway greatly contributes to learning and memory process. Pregnant rats were exposed to 0, 0.05, 0.5, 5 and 50 mg/kg/day BPA via oral gavage from gestational day (GD) 5 to GD 19. Morris water maze, transmission electron microscope, western blot, real time PCR, biochemical analysis and ELISA were used to analyze the changes in behavior, synaptic ultrastructure, protein and gene expression of NMDAR, PSD-95, nNOS, together with nNOS activity, NO (Nitrate reductase method) and cGMP levels of the rat pups at different growth stages. Results of this research displayed that exposure to 0.5 mg/kg/day BPA could damage the spatial learning ability of rats at postnatal day (PND) 56. However, spatial memory ability could be affected by exposure to BPA at doses up to 5 mg/kg/day. Moreover, the thickness of the postsynaptic density decreased after exposure to BPA at doses of 5 and 50 mg/kg/day. Levels of NR1, NR2A, PSD-95 protein and mRNA were downregulated to some extent after exposure to BPA, whereas the expression of NR2B increased at GD 20 but decreased at PND 21 and 56. Contrarily, the nNOS expression along with the enzyme activity were promoted after exposure to BPA. Meanwhile, the NO and cGMP levels were suppressed at GD 20 but promoted at PND 21 and 56. In conclusion, these results demonstrated that NMDAR/PSD-95/nNOS-NO-cGMP pathway could be affected by embryonic exposure to BPA, which may involve in the spatial learning and memory dysfunction of rats in later life.

Interaction between CYP1A1/CYP17A1 polymorphisms and parental risk factors in the risk of hypospadias in a Chinese population.

Hypospadias (HS) is a common congenital malformation of the genitourinary tract in males and its etiology is viewed as multifactorial, and studies about gene-environment interaction in the etiology of HS are rare. A total of 152 cases and 151 controls were selected in the present study. Information before and during pregnancy from questionnaires finished by mothers of subjects were extracted, and the relating data were analyzed to determine the risk factors of HS. Meanwhile, maternal genomic DNA was genotyped for the single nucleotide polymorphisms (SNPs) of CYP1A1 rs1048943 and CYP17A1 rs4919686. Results of multivariable logistic regression analyses showed that several factors were associated with hypospadias risk. Analysis of the distributions of SNPs in CYP1A1 and CYP17A1 genes showed that the mutant genotype CC (OR = 4.87) of CYP1A1 rs1048943, and mutant genotype CC (OR = 5.82), recessive genotype AC + CC (OR = 2.17) and allele C (OR = 1.77) of CYP17A1 rs4919686 significantly increased the risk of HS. In addition, the additive gene-environment interactions were also found in several models. Several maternal risk factors that are associated with HS risk can interact with CYP1A1/CYP17A1 polymorphisms, which lead to infants vulnerable to occurrence of HS in Chinese populations.

[An analysis of general programs in preventive medicine submitted and funded in NFSC from 2004 to 2006].

OBJECTIVE: To analyze the projects of the general program in preventive medicine submitted to and funded by National Natural Science Foundation of China (NSFC) from 2004 to 2006, and to discuss the disadvantages of these projects and foundation trends. METHODS: 4124 projects submitted and 656 projects supported in 16 fields of preventive medicine between 2004 to 2006 were analyzed statistically. RESULTS: 1082, 1378 and 1664 projects were submitted to NSFC and 199, 210 and 247 projects were financially supported in last three years, respectively, with selecting rate 18.4%, 15.2% and 14.8%, respectively. The number and the selection rate of projects supported in the different study fields from 2004 to 2006 were 15 (7.5%), 24 (11.4%) and 18 (7.3%) in environmental health, 8 (4.0%), 12 (5.7%) and 15 (6.1%) in occupational health, 30 (15.1%), 24 (11.4%) and 31 (12.6%) in nutrition and food hygiene, 3 (1.5%), 4 (1.9%) and 4 (1.6%) in child and adolescent health, 14 (7.0%), 20 (9.5%) and 20 (8.1%) in toxicology, 7 (3.5%), 7 (3.3%) and 2 (0.8%) in social medicine, 17 (8.5%), 22 (10.5%) and 31 (12.6%) in epidemiology, 7 (3.5%), 7 (3.3%) and 10 (4.0%) in endemiology, 2 (1.0%), 5 (2.4%) and 5 (2.0%) in biostatistics, 0 (0%), 2 (1.0%) and 1 (0.4%) in health laboratory technology, 7 (3.5%), 2 (1.0%) and 7 (2.8%) in vector biology, 15 (7.5%), 9 (4.3%) and 16 (6.5%) in parasitology, 0 (0%), 2 (1.0%) and 0 (0%) in disinfection, 58 (29.1%), 51 (24.3%) and 58 (23.5%) in infectious disease, 3 (1.5%), 1 (0.5%) and 5 (2.0%) in sexually transmitted disease, 13 (6.5%), 18 (8.6%) and 24 (9.7%) in dermatology, respectively. The contents of these research subjects reflected that more importance has been attached to the fields of preventive medicine. However, it is necessary that original and innovatory research should be further strengthened, and the field and prospective study should be emphasized in the future. CONCLUSION: Rapid advancement have been made in the fields of toxicology, epidemiology, environmental health, nutrition and food hygiene, and infectious disease, while other areas such as social medicine, health laboratory technology, disinfection and sexually transmitted disease need a rapid advancement. In general, the levels of the projects received and funded have been elevating yearly.

Annexin A10 is a candidate marker associated with the progression of pancreatic precursor lesions to adenocarcinoma.

Annexins are a multigene family of calcium and phospholipid-binding proteins that play important roles in calcium signaling, cell motility, differentiation and proliferation. Our previous mass spectrometry-based proteomics study revealed that annexin A10 (ANXA10) was uniquely overexpressed in pancreatic CD24+ adenocarcinoma cells that were dissected from clinical PDAC tissues but was absent in CD24- adjacent normal cells. The correlation between ANXA10 expression and the progression of pancreatic cancer remains unknown. In this study, we performed an immunostaining assay to evaluate ANXA10 expression in 155 primary human tissue specimens, including normal pancreas, chronic pancreatitis (CP), pancreatic adenocarcinoma (PDAC), pancreatic intraepithelial neoplasia (PanIN, the most important precursor of PDAC), and intraductal papillary mucinous neoplasm (IPMN). The immunostaining result showed that ANXA10 was significantly overexpressed in PanINs, IPMNs, and PDACs but negative in normal pancreas and the majority of chronic pancreatitis tissues. Statistical analysis revealed that ANXA10 expression was significantly associated with PDAC and its precursor lesions (p<0.0001). Abundant ANXA10 expression was predominantly present in pancreatic ductal epithelial cells of PanINs, IPMNs, and tumor cells of PDACs. Since PDAC develops through a series of PanINs which in turn arise from pancreatic ducts, the consistent overexpression of ANXA10 in ductal epithelial cells in PanINs and PDACs but negative in normal pancreatic ducts suggests that ANXA10 could serve as a potential marker indicating the presence of PDAC at its earliest precancerous stages. Double immunostaining of ANXA10 and CD24 showed that there was a large overlap between these two markers in PDAC and high-grade neoplasia lesions. The statistical analysis showed that the coexpression of ANXA10 and CD24 was significantly correlated with the progression of pancreatic precursor lesions towards PDACs.

CD90 and CD24 Co-Expression Is Associated with Pancreatic Intraepithelial Neoplasias.

Thy-1 (CD90) has been shown to be a potential marker for several different types of cancer. However, reports on CD90 expression in pancreatic intraepithelial neoplasia (PanIN) lesions are still limited where PanINs are the most important precursor lesion of pancreatic ductal adenocarcinoma (PDAC). Herein, we investigate candidate markers for PanIN lesions by examining the distribution and trend of CD90 and CD24 expression as well as their co-expression in various stages of PanINs. Thirty cases of PanINs, which were confirmed histopathologically and clinically, were used to evaluate protein expression of CD90 and CD24 by immunofluoresence double staining. CD90 was found to be mainly expressed in stroma around lesion ducts while not observed in acini and islets in PanINs. CD90 also showed increased expression in PanIN III compared to PanIN III. CD24 was mainly present in the cytoplasm and membrane of pancreatic ductal epithelia, especially in the apical epithelium of the duct. CD24 had higher expression in PanIN III compared with PanIN IIIIII or PanIN III. CD90 was expressed around CD24 sites, but there was little overlap between cells that expressed each of these proteins. A correlation analysis showed that these two proteins have a moderate relationship with PanIN stages respectively. These results suggest that co-expression of CD90 and CD24 may have an important role in the development and progression of PanINs, which is also conducive to early detection and treatment of PDAC.

Protein Markers Associated with an ALDH Sub-Population in Colorectal Cancer.

ALDH has been shown to be a marker that denotes a sub-population of cancer stem cells in colorectal and other cancers. This sub-population of cells shows an increased risk for tumor initiation, metastasis, and resistance to chemotherapy and radiation resulting in recurrence and death. It is thus essential to identify the important signaling pathways related to ALDH1+ CSCs in colon cancer. The essential issue becomes to isolate pure sub-populations of cells from heterogeneous tissues for further analysis. To achieve this goal, tissues from colorectal cancer Stage III patients were immuno-stained with ALDH1 antibody. Target ALDH1+ and ALDH1- cells from the same tissue were micro-dissected using Laser Capture Microdissection (LCM). Captured cells were lysed and analyzed using LC-MS/MS where around 20,000 cells were available for analysis. This analysis resulted in 134 proteins which were differentially expressed between ALDH1+ and ALDH1- cells in three patient sample pairs. Based on these differentially expressed proteins an IPA pathway analysis was performed that showed two key pathways in cell to cell signaling and organismal injury and abnormalities. The IPA analysis revealed ß-catenin, NFκB (p65) and TGFß1 as important cancer-related proteins in these pathways. A TMA validation using immunofluorescence staining of tissue micro-arrays including 170 cases was used to verify that these key proteins were highly overexpressed in ALDH1+ cells in colon cancer tissues compared to ALDH1- cells.