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Background Parabens, a widely used class of preservatives, are suspected to be potential obesogens as emerging endocrine disrupting chemicals with reproductive and developmental toxicity. Objective To analyze five urinary parabens (PBs) and estimate the associations of exposure to PBs with adiposity measures in 10-year-old school-age children. Methods A total of 471 school-age children aged 10 years from the Sheyang Mini Birth Cohort were enrolled in this study. A questionnaire survey was conducted to collect socio-demographic information, physical activity, and dietary intake. Weight, height, and waist circumference of children were measured, and age- and sex-adjusted body mass index (BMI-Z score) was calculated. Spot urine samples were collected during the follow-up visits. Urinary concentrations of five PBs including methyl-paraben (MeP), ethyl-paraben (EtP), propyl-paraben (PrP), butyl-paraben (BuP), and benzyl-paraben (BzP) were detected by gas chromatography-tandem mass spectrometry (GC-MS/MS). Generalized linear models (GLMs) and Bayesian kernel machine regression (BKMR) models were applied to estimate associations of individual/overall urinary PBs concentrations with BMI Z-score and waist circumference. Results The positive rates of selected five urinary PBs were in the range from 78.98% to 98.94%. The urinary PBs concentrations (geometric mean) were in the range of 0.31-5.43 μg·L−1. The children's BMI Z-score and waist circumference (mean ± standard deviation) were (0.56±1.40) and (67.62±10.07) cm respectively. The GLMs results showed that the urinary BzP concentration was negatively associated with waist circumference (b=−0.08, 95%CI: −0.14, −0.02; P=0.01). In sex-stratified analysis, the urinary concentration of BzP was negatively associated with BMI-Z score (b=−0.59, 95%CI: −0.88, −0.30; P<0.001) and waist circumference (b=−0.80, 95%CI: −1.23, −0.37; P<0.001) in boys, but not in girls. The BKMR results also found significant negative correlations of urinary BzP concentrations with BMI-Z score and waist circumference, which were consistent with the GLM results. Conclusion The selected 10-year-old children are extensively exposed to PBs in the study area. Furthermore, childhood PBs exposure may have potential impacts on childhood adiposity measures with sex-specific effects.
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Background Cadmium (Cd) is a ubiquitous and toxic heavy metal that can accumulate in human body. Previous studies have shown that Cd exposure can induce neurotoxicity, but the underlying mechanism remains unclear. Objective To investigate the metabolic impacts of multiple doses of Cd on mouse neural stem cells (NSCs), and to explore the potential mechanism and biomarkers of its neurotoxicity. Methods The NSCs were obtained from the subventricular zone (SVZ) of 1-day-old neonatal C57BL/6 mice. The passage 3 (P3) NSCs were exposed to CdCl2 at designed doses (0, 0.5, 1.0, and 1.5 μmol·L−1). The cells were treated with seven replicates, of which one plate was for cell counting. After 24 h of exposure, the intracellular and extracellular metabolites were extracted respectively and then detected by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The orthogonal partial least-squares discriminant analysis (OPLS-DA) was applied to visualize the alterations of metabolomic profiles and to identify the differential metabolites (DMs) based on their variable importance for the projection (VIP) value >1 and P<0.05. The metabolite set enrichment analysis (MSEA) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed to recognize the significantly altered metabolite sets and pathways. The dose-response relationships were established and the potential biomarkers of Cd exposure were identified by 10% up-regulated or 10% down-regulated effective concentration (EC) of target metabolites. Results A total of 1201 metabolites were identified in the intracellular metabolomic samples and 1207 for the extracellular metabolomic samples. The intracellular and extracellular metabolome of Cd-treated NSCs were distinct from that of the control group, and the difference grew more distant as the Cd dosage increased. At 0.5, 1.0, and 1.5 μmol·L−1 dosage of Cd, 87, 83, and 185 intracellular DMs and 161, 176, and 166 extracellular DMs were identified, respectively. Within the significantly changed metabolites among the four groups, 176 intracellular DMs and 167 extracellular DMs were identified. Both intracellular and extracellular DMs were enriched in multiple lipid metabolite sets. Intracellular DMs were mainly enriched in taurine and hypotaurine metabolism, glycerophospholipid metabolism, and glycerolipid metabolism pathways. Extracellular DMs changed by Cd were mainly enriched in glycerophospholipid metabolism, steroid hormone biosynthesis, and cysteine and methionine metabolism pathways. Among intracellular DMs, 125 metabolites were fitted with dose-response relationships, of which 108 metabolites showed linear changes with the increase of Cd dosage. And 134 metabolites were fitted with dose-response relationships among extracellular DMs, of which 86 metabolites showed linear changes. The intracellular DMs with low EC values were hypotaurine, ethanolamine, phosphatidylethanolamine, and galactose, while the extracellular DMs with low EC values were acetylcholine and 1,5-anhydrosorbitol. Conclusion Cd treatment can significantly alter the intracellular and extracellular metabolome of mouse NSCs in a dose-dependent manner. The neurotoxicity of Cd may be related to glycerophospholipid metabolism. Acetylcholine, ethanolamine, and phosphatidylethanolamine involved in glycerophospholipid metabolism pathway might be potential biomarkers of Cd-induced neurotoxicity.
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Background Flurochloridone (FLC) is toxic to male reproduction and can induce apoptosis of testicular tissue and supporting cells under oxidative stress. Of particular concern is whether nuclear factor-erythrocyte 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway and nuclear factor kappa B (NFκB) signaling pathway participate this process. Objective To observe apoptosis of testicular tissue and sertoli TM4 cells and alterations of Nrf2/HO-1 and NFκB signaling pathways in mice treated with FLC in vivo/in vitro. Methods (1) Animal experiment. Testis samples were harvested from male C57BL/6 mice after 28-day FLC (0, 3, 15, 75, and 375 mg·kg−1 per day) exposure via oral route. Malondialdehyde (MDA) and superoxide dismutase (SOD) in homogenate of testicular tissue were measured by colorimetry. Apoptosis of testicular tissue was evaluated by TUNEL staining. Expression and distribution of Nrf2 and NFκB were detected by immunohistochemistry. Protein expression levels of Nrf2, HO-1, NAD(P)H: quinone oxidoreductase 1 (NQO1), NFκB, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), and phosphorylated recombinant inhibitory subunit of nuclear factor kappa-B alpha (P-IκBα) in testicular tissue homogenate were determined by Western blotting. (2) Cell experiment. TM4 cell lines were treated with 40, 80, 120, 160, and 200 μmol·L−1 FLC for 6 h, and cell viability was detected by CCK-8. After 6 h exposure to 40, 80, and 160 μmol·L−1 FLC, the apoptosis rate was detected by flow cytometry, and the protein expression levels of Nrf2, HO-1, NQO1, NFκB, IKKβ, and IκBα were detected by Western blotting. Results (1) Animal experiment. Apoptosis occurred in the interstitial and basal parts of spermatogenic tubules in male C57BL/6 mice after 28 days of oral FLC exposure. Compared with the control group, the MDA level in testicular tissue of the 375 mg·kg−1 FLC-treated group was significantly increased (P<0.05), and the SOD activity was significantly decreased (P<0.05). After 375 mg·kg−1 FLC exposure, apoptosis occurred in the interstitial and basal parts of spermatogenic tubules. The results of immunohistochemistry showed the expression of Nrf2 and NFκB in the interstitium and basal part of spermatogenic tubules of the treated groups. Compared with the control group, the protein levels of Nrf2, NQO1, P-IκBα, NFκB, and IKKβ in the 15, 75, and 375 mg·kg-1 groups were significantly increased (P<0.001), and the HO-1 protein level was significantly increased in the 375 mg·kg−1 group (P<0.001). (2) Cell experiment. Compared with the control group, the TM4 cell viabilities in the 40, 80, 120, 160, and 200 μmol·L−1 FLC-treated groups significantly decreased (P<0.01). The apoptosis rates were significantly increased (P<0.05), and the apoptosis rates increased from 5.7% in the control group to 7.4%, 9.4%, and 11.7% in the 40, 80, and 160 μmol·L−1, respectively. The Nrf2 protein level in the 40 μmol·L−1 group was significantly increased (P<0.01), while the levels significantly decreased in the 80 and 160 μmol·L−1 groups (P<0.01). The HO-1 protein levels in the 40, 80, and 160 μmol·L−1 groups were significantly increased (P<0.01). The level of NQO1 protein in the 40 μmol·L−1 group was significantly increased (P<0.01). The NFκB protein levels were significantly increased in the 80 and 160 μmol·L−1 groups (P<0.001). The IκBα protein levels were significantly decreased in all treated groups (P<0.001). The IKKβ protein had no significant change. Conclusion FLC induces testicular tissue apoptosis, and the process affects Nrf2/HO-1 signaling pathway and NFκB signaling pathway. The in vitro study confirms that FLC could induce apoptosis of TM4 cells and activate Nrf2/HO-1 and NFκB signaling pathways.
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Background Paraquat (PQ) is a widely used herbicide that exerts neurotoxicity. The effects of PQ on neural stem cells (NSCs) through microglia mediated neuroinflammation remain limitedly studied. Objective To investigate the effects of PQ on the proliferation and neurogenesis of NSCs through neuroinflammation mediated by microglia. Methods Microglial cell lines (BV2 cells) and primary NSCs were used. BV2 cells were exposed to 0, 1, 3.3, 10, 33, and 100 μmol·L−1 of PQ for 6 h followed by viability assessment. The highest PQ concentration that had no effect on cell viability was selected as the final exposure concentration (33 μmol·L−1). To exclude the direct effect of PQ on NSCs, after the BV2 cells were cultured in complete medium containing 33 μmol·L−1 PQ for 6 h, the BV2 culture medium was replaced by NSCs complete medium without PQ for 24 h. The concentration of interleukin-1β (IL-1β) in supernatant was detected by enzyme-linked immune sorbent assay. Besides, in order to detect the effects of IL-1β on NSCs proliferation and neurogenesis, NSCs isolated from hippocampus of adult mice were cultured in the supernatant obtained above and divided into four groups: control supernatant + control antibody, control supernatant + IL-1β neutralizing antibody (10 ng·mL−1), PQ supernatant + control antibody, PQ supernatant + IL-1β neutralizing antibody (10 ng·mL−1). Proportion of Ki67-positive NSCs was detected by flow cytometry (FCS) and immunofluorescence after 24 h culture, and neurogenesis was detected by FCS and immunofluorescence after 3-7 d of culture. Results The IL-1β concentration in the supernatant of BV2 cells was significantly increased after the 33 μmol·L−1 PQ exposure compared with the control group (t=3.020, P<0.05). After the NSCs were cultured with the supernatant of PQ-treated BV2 cells, the proportion of Ki67-positive NSCs (t=9.129, P<0.01) and the proportion of newborn neurons (t=4.638, P<0.01) were significantly decreased compared to the control group. After neutralizing IL-1β, the proportion of Ki67-positive NSCs (t=22.05, P<0.01) and the proportion of newborn neurons (t=11.09, P<0.01) were significantly higher than those in the un-neutralized group. The results of immunofluorescence detection also showed that after neutralizing IL-1β secreted by 33 μmol·L−1 PQ-treated BV2 cells, the number of Ki67-positive NSCs and the number of newborn neurons were significantly higher than those in the un-neutralized group. Conclusion The secretion of IL-1β by microglia is increased after PQ treatment, resulting in a decrease in the proliferation and neurogenesis of NSCs. These results suggest that neuroinflammation is involved in NSCs damage caused by PQ.
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Background Flurochloridone (FLC) can induce apoptosis in Sertoli cells, but the specific mechanism remains unknown. Objective To investigate the testicular cell apoptosis in mice as well as apoptosis and activation of endoplasmic reticulum stress in TM4 cell line induced by FLC through in vivo and in vitro study designs respectively, and study the role of inosital-requiring enzyme 1α (IRE1α)-c-Jun N-terminal kinase (JNK) signaling pathway in the process of FLC-induced apoptosis in TM4 cells through intervention study design. Methods Testicular tissues were collected from male C57BL/6 mice which were treated with 3, 15, 75, and 375 mg·（kg·d）−1 FLC by oral perfusion for 28 d. Apoptosis was observed by TUNEL staining, and the levels of apoptosis-related proteins were detected by Western blotting, including B-cell lymphoma-2 (Bcl-2), Bcl-2 interacting mediator of cell death (Bim), and Bcl-2 associated X protein (Bax). In the in vitro study, TM4 cells were treated with different concentrations of FLC (40, 80, and 160 μmol·L−1) for 6 h, then apoptosis rate was detected by flow cytometry, and the levels of apoptosis-related proteins (Bcl-2, Bim, and Bax) and endoplasmic reticulum stress-related proteins [glucose regulated protein 78 (GRP78), phosphorylated-protein kinase R like endoplasmic reticulum kinase (p-PERK), activating transcription factor 6 (ATF6), phosphorylated-inosital-requiring enzyme 1α (p-IRE1α), and phosphorylated-JNK (p-JNK)] were measured by Western blotting. In the intervention study, TM4 cells were pretreated with IRE1α phosphorylation inhibitor 4μ8C and JNK phosphorylation inhibitor SP600125 for 6 h, then treated with 160 μmol·L−1 FLC for 6 h. The levels of apoptosis-related proteins and endoplasmic reticulum stress-related proteins were measured by Western blotting, and cell viability was detected by cell counting kit-8. Results After the male C57BL/6 mice orally exposed to FLC for 28 d, apoptosis occurred in the seminiferous tubule. The protein expression level of Bcl-2, apoptosis inhibitor, was decreased in the 75 and 375 mg·(kg·d)−1 groups (P<0.05), and the protein expression levels of Bim and Bax, apoptosis promoters, were increased in the 75 and 375 mg·(kg·d)−1 groups respectively (P<0.05). The percentages of apoptotic cells in the 0, 40, 80, and 160 μmol·L−1 FLC groups were 2.7%±0.2%, 4.8%±1.3%, 9.4%±0.3%, and 13.2%±0.2%, respectively, increased significantly compared with the control group (P<0.05). The protein expression level of Bcl-2 also was decreased in the 160 μmol·L−1 FLC group (P<0.05), while the levels of Bim and Bax were increased in both of the 80 and 160 μmol·L−1 groups (P<0.05). The expression levels of endoplasmic reticulum stress-related proteins (GRP78, p-PERK, ATF6, p-IRE1α, and p-JNK) were increased (P<0.05) or showed a rising trend in TM4 cells. Pre-treatment with 4μ8C (25 and 50 μmol·L−1) and SP600125 (10 and 20 μmol·L−1) significantly down-regulated the protein expression levels of GRP78, p-IRE1α, p-JNK, and Bax induced by FLC (P<0.05) or in a downward trend. Both of the inhibitors alleviated the decreased cell viability induced by FLC (P<0.05) or in alleviating fashion. Conclusion FLC could induce apoptosis in mice testis and TM4 cell apoptosis through activating endoplasmic reticulum stress and IRE1α-JNK signaling pathway.
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<p><b>OBJECTIVE</b>To investigate the role of CCL20/CCR6/Th17 axis in vascular invasion and metastasis of primary hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Expression levels of CCL20 mRNA in the normal human liver cell line L-02, and human hepatocellular carcinoma cell lines Hep3B, Huh7 and HepG2 were quantified by using SYBR green real time PCR. CCL20 secretions from these cell lines were quantified by using ELISA. The chemotactic effect of HCC cell line Hep3B on human peripheral blood mononuclear cells was determined by using transwell chemotaxis assay. Pre-therapy serum levels of IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-17, IL-23, IFN-γ, TNF-α and CCL20 in 93 patients with HCC were measured by using 9-plex array and ELISA. All the patients were chronic hepatitis B virus associated HCC, and 51 cases were those with vascular invasion and metastasis (metastasis group) and 42 cases were not (non-metastasis group). CCL20 and CCR6 mRNA expressions in the HCC and tumor-adjacent tissues were determined by using SYBR Green real time PCR in 41 patients, among them, 20 cases were from the group of patients with metastasis and 21 cases were from the group of patients without metastasis. The CCL20 expression was further determined by immunohistochemistry.</p><p><b>RESULTS</b>The HCC cell lines expressed and secreted higher amount of CCL20, which effectively recruited CCR6(+) T cells. Pre-therapy serum levels of CCL20 in 93 HCC patients were (38.2 ± 28.4)pg/ml, significantly increased than those with benign hepatic hemangiomas [(7.8 ± 17.8)pg/ml, P < 0.01]. In addition, the serum levels of CCL20 were positively correlated with the tumor diameters in HCC patients (r = 0.32, P = 0.0018). CCL20 was dominantly expressed in the cytoplasm in HCC cells, and it was also expressed by some infiltrating immune cells. The mRNA expression levels of CCL20 of the tumor tissues were significantly higher than that in the tumor-adjacent tissues (P < 0.05). Multivariate logistic regression analysis showed that serum levels of IL-17 and CCL20 were independent risk factors of metastasis in HCC patients (P < 0.05 for both). CCL20 mRNA showed no statistically significant differences between patients with metastasis and without metastasis in both tumor tissues and tumor-adjacent tissues (P > 0.05 for both). But the patients with metastasis showed significantly higher expressions of CCR6 both in their tumor [5.75 (1.79, 19.13)]and tumor-adjacent tissues [7.99 (4.49, 19.54)] than those with non-metastasis [1.69 (0.76, 2.87) and 3.58 (1.84, 4.32), P < 0.05 for both].</p><p><b>CONCLUSION</b>CCL20/CCR6/Th17 axis may promote vascular invasion and metastasis hepatocellular carcinoma.</p>
Subject(s)
Humans , Bile Duct Neoplasms , Carcinoma, Hepatocellular , Metabolism , Chemokine CCL20 , Metabolism , Interleukin-10 , Metabolism , Interleukin-17 , Metabolism , Interleukin-23 , Metabolism , Interleukin-6 , Metabolism , Interleukin-8 , Metabolism , Leukocytes, Mononuclear , Liver Neoplasms , Metabolism , RNA, Messenger , Th17 Cells , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of Wnt signaling pathway on paraquat (PQ)induced PC12 cells damage.</p><p><b>METHODS</b>Using PC12 cells, in this study CCK8 assay was used to detect the effect of cell viability. The cell apoptosis and cell cycle was detected by flow cytometry. The real-time polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of Wnt pathway key genes including Fzd1, Dvl2 and β-catenin and downstream genes including Bax, Bcl2, Survivin, Cyclin D1 and C-myc.</p><p><b>RESULT</b>Compared with the control, PC12 cells viability in 50.00 and 100.00 µmol/L PQ treatment groups were obviously decreased, the cell cycle S phase arrest, and cell apoptosis increased (P<0.05). The 25.00, 50.00 and 100.00 µmol/L PQ treatment groups mRNA expression of Wnt pathway key genes including Fzd1, Dvl2 and β-catenin and downstream genes including apoptosis suppressor genes (Bcl-2 and survivin)and cyclin gene (Cyclin D1) were downregulated (P<0.05). The mRNA expression of pro-apoptosis gene (Bax) and cyclin gene (C-myc) were upregulated (P<0.05).</p><p><b>CONCLUSION</b>It suggested that PQ can activate Wnt pathway to regulate downsteam genes expression, resulting in PC12 cell cycle arrest and apoptosis.</p>
Subject(s)
Animals , Rats , Adaptor Proteins, Signal Transducing , Metabolism , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Cell Cycle , Cell Survival , Dishevelled Proteins , Down-Regulation , Flow Cytometry , Frizzled Receptors , Metabolism , Gene Expression , PC12 Cells , Paraquat , Toxicity , Phosphoproteins , Metabolism , Receptors, Neurotransmitter , Metabolism , Wnt Signaling PathwayABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of fluorochloridone (FLC) exposure on the testes of adult Sprague-Dawley (SD) rats.</p><p><b>METHODS</b>Forty male SD rats were randomly divided into four groups. These groups, each of 10 male rats, were separately given FLC by gavage at a dose of 0 (control), 30, 150, or 750 mg/kg once daily for 28 d. The oxidative stress biomarkers in the testes were measured by spectrophotometry. The pathological changes in testicular tissues were evaluated under the light and electric microscopes. The cauda epididymal sperm count was determined. The testicular toxicity of FLC was assessed accordingly.</p><p><b>RESULTS</b>Compared with the control group, the 750 mg/kg FLC group had significantly lower testicular weight and organ coefficient, epididymal weight, and cauda epididymal sperm count (P < 0.05 or P < 0.01), the 150 and 750 mg/kg FLC groups had significantly increased malonaldehyde content (P < 0.05 or P < 0.01), each exposed group had a significantly reduced glutathione (GSH) level (P < 0.05 or P < 0.01), the 750 mg/kg FLC group had significantly reduced activities of superoxide dismutase (SOD), catalase (CAT), GSH peroxidase, GSH S-transferase (GSH-ST), and GSH reductase (GSH-GR) (P < 0.05 or P < 0.01), the 150 mg/kg FLC group showed significant decreases in the activities of all antioxidant enzymes except GSH-GR (P < 0.05 or P < 0.01), and the 30 mg/kg FLC group showed significant decreases in the activities of SOD and CAT (P < 0.05 or P < 0.01). Furthermore, seminiferous epithelial degeneration, Sertoli cell vacuolization, spermatogenic cell loss, and nuclear damage were observed under the light and electronic microscopes in the 150 and 750 mg/kg FLC groups.</p><p><b>CONCLUSION</b>FLC could damage the testes of adult rats by inducting oxidative stress. This research provided clues and directions for further exploration of the mechanism of FLC testicular toxicity.</p>
Subject(s)
Animals , Male , Rats , Oxidative Stress , Pyrrolidinones , Toxicity , Rats, Sprague-Dawley , Sperm Motility , Testis , Metabolism , PathologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of paraquat on microRNA expressions in PCl2 cells, and to explore the regulatory mechanism of bcl-2.</p><p><b>METHODS</b>We used PC12 cells as a popular in vitro cell model system for characterizing the dopaminergic neuron. After 24 h treatment with different concentrations of PQ (0, 62.5 ümol/L), expression difference of microRNA was detected by microarray and examined by real-time quantitative PCR (RT-PCR). Cell apoptosis was analyzed with flow cytometry (FCM) and the relative levels of miR-34a, miR-Let-7e were measured by RT-RCR following the PCl2 cells treatment with 0, 62.5, 125, 250, 500, 1000 ümol/L PQ. Meanwhile, the protein expression of bcl-2 was evaluated by western blot according to forecasting targets analysis databases.</p><p><b>RESULTS</b>Cell viability decreased and cell apoptosis increased with increasing PQ concentrations (from 125 to 1000 ümol/L) in a dose-dependent manner (P < 0.05 or P < 0.01). MiRNA microarray showed that after 62.5 ümol/L PQ treatment, 11 miRNAs were significantly up-regulated while 8 miRNAs were down-regulated compared with control (P < 0.01). We chose miR-34a, miR-Let-7e which appeared most remarkable changes in microarray to examine by RT-PCR. It revealed that the level of miR-34a gradually ascended while miR-Let-7e declined after PQ treatment, which are accordant to the microarray results. The protein expression of bcl-2 treated with PQ significantly decreased compared with control and presented a negative correlation with the expression of miR-34a (P < 0.05 or P < 0.01).</p><p><b>CONCLUSION</b>The alteration of miRNAs expression may be involved in the neurotoxicity of PQ. Especially, mir-34a negatively regulated the level of bcl-2, and thus plays a key role in PQ-induced cell apoptosis.</p>