Associations of cord serum polybrominated diphenyl ether (PBDE) mixture with birth outcomes and mediating role of thyroid function: Evidence from the Sheyang Mini Birth Cohort Study.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs), a series of worldwide applied flame retardants, may influence fetal growth and interfere with thyroid function. The study intended to explore the relationship between in-utero exposure to PBDE mixture and newborn anthropometric indexes and to further examine the potential mediating role of thyroid function. METHODS: Demographics and laboratory measures of 924 mother-infant pairs were obtained from the database of the Sheyang Mini Birth Cohort Study. We applied gas chromatography-mass spectrometry (GC-MS) and electrochemiluminescence immunoassay to measure nine PBDE congeners and seven thyroid function parameters in umbilical cord serum samples, respectively. We fitted generalized linear models and Bayesian kernel machine regression (BKMR) to evaluate associations of lipid-adjusted cord serum PBDEs, as individuals and as a mixture, with newborn anthropometric and cord serum thyroid function parameters. We applied causal mediation analysis to test our hypothesis that thyroid function parameters act as a mediator between PBDEs and birth outcomes. RESULTS: The molarity of cord serum ∑9PBDE had a median value of 31.23 nmol/g lipid (IQR 19.14 nmol/g lipid, 54.77 nmol/g lipid). BDE-209 was the most dominant congener. Birth length was positively associated with both single exposure to BDE-28 and cumulative exposure to PBDEs. Correspondingly, ponderal index (PI) was negatively associated with BDE-28 and the total effects of PBDE mixture. Free triiodothyronine had a negative trend with BDE-209 and PBDE mixture. In the sex-stratified analysis, BDE-153 concentrations were positively correlated with PI among males (ß = 0.03; 95%CI: 0.01, 0.05; P = 0.01) but not among females. Cord serum thyrotropin mediated 14.92% of the estimated effect of BDE-153 on PI. CONCLUSIONS: In-utero mixture exposure to PBDEs was associated with birth outcomes and thyroid function. Thyroid function might act as a mediator in the process in which PBDEs impact the growth of the fetus.

Integrated metabolomic and transcriptomic analysis reveals perturbed glycerophospholipid metabolism in mouse neural stem cells exposed to cadmium.

Cadmium (Cd) is a ubiquitous heavy metal with neurotoxicity. Our previous study reported that Cd could inhibit the proliferation of mouse neural stem cells (mNSCs). However, the underlying mechanisms are obscure. In recent years, the rapid growth of multi-omics techniques enables us to explore the cellular responses that occurred after toxicant exposure at the molecular level. In this study, we used a combination of metabolomics and transcriptomics approaches to investigate the effects of exposure to Cd on mNSCs. After treatment with Cd, the metabolites and transcripts in mNSCs changed significantly with 110 differentially expressed metabolites and 2135 differentially expressed genes identified, respectively. The altered metabolites were mainly involved in glycerophospholipid metabolism, arginine and proline metabolism, arginine biosynthesis, glyoxylate and dicarboxylate metabolism. Meanwhile, the transcriptomic data demonstrated perturbed membrane function and signal transduction. Furthermore, integrated analysis of metabolomic and transcriptomic data suggested that glycerophospholipid metabolism might be the major metabolic pathway affected by Cd in mNSCs. More interestingly, the supplementation of lysophosphatidylethanolamine (LPE) attenuated Cd-induced mitochondrial impairment and the inhibition of cell proliferation and differentiation in mNSCs, further supporting our analysis. Overall, the study provides new insights into the mechanisms of Cd-induced neurotoxicity.

Single-cell transcriptomic analysis reveals the adverse effects of cadmium on the trajectory of neuronal maturation.

Cadmium (Cd) is an extensively existing environmental pollutant that has neurotoxic effects. However, the molecular mechanism of Cd on neuronal maturation is unveiled. Single-cell RNA sequencing (scRNA-seq) has been widely used to uncover cellular heterogeneity and is a powerful tool to reconstruct the developmental trajectory of neurons. In this study, neural stem cells (NSCs) from subventricular zone (SVZ) of newborn mice were treated with CdCl2 for 24 h and differentiated for 7 days to obtain neuronal lineage cells. Then scRNA-seq analysis identified five cell stages with different maturity in neuronal lineage cells. Our findings revealed that Cd altered the trajectory of maturation of neuronal lineage cells by decreasing the number of cells in different stages and hindering their maturation. Cd induced differential transcriptome expression in different cell subpopulations in a stage-specific manner. Specifically, Cd induced oxidative damage and changed the proportion of cell cycle phases in the early stage of neuronal development. Furthermore, the autocrine and paracrine signals of Wnt5a were downregulated in the low mature neurons in response to Cd. Importantly, activation of Wnt5a effectively rescued the number of neurons and promoted their maturation. Taken together, the findings of this study provide new and comprehensive insights into the adverse effect of Cd on neuronal maturation.

Multiple mediation effects on association between prenatal triclosan exposure and birth outcomes.

BACKGROUND: Triclosan is a broad-spectrum antimicrobial, and was thought to affect intrauterine development, but the mechanism remains unclear. OBJECTIVE: To explore the association between prenatal triclosan exposure and birth outcomes. METHODS: Based on 726 mother-child pairs from the Sheyang Mini Birth Cohort Study (SMBCS), we used the available (published) data of triclosan in maternal urines, the hormones including thyroid-related hormones, gonadal hormones in cord blood, and adipokines, trimethylamine-N-oxide (TMAO) and its precursors in cord blood to explore possible health effects of triclosan on birth outcomes through assessing different hormones and parameters, using Bayesian mediation analysis. RESULTS: Maternal triclosan exposure was associated with ponderal index (ß = 0.317) and head circumference (ß = -0.172) in generalized linear models. In Bayesian mediation analysis of PI model, estradiol (ß = 0.806) and trimethylamine (TMA, ß = 0.164) showed positive mediation effects, while total thyroxine (TT4, ß = -0.302), leptin (ß = -2.023) and TMAO (ß = -0.110) showed negative mediation effects. As for model of head circumference, positive mediation effects were observed in free thyroxine (FT4, ß = 0.493), TMA (ß = 0.178), and TMAO (ß = 0.683), negative mediation effects were observed in TT4 (ß = -0.231), testosterone (ß = -0.331), estradiol (ß = -1.153), leptin (ß = -2.361), choline (ß = -0.169), betaine (ß = -0.104), acetyl-L-carnitine (ß = -0.773). CONCLUSION: The results indicated triclosan can affect intrauterine growth by interfering thyroid-related hormones, gonadal hormones, adipokines, TMAO and its precursors.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 in rheumatoid arthritis: Longitudinal change after treatment and correlation with treatment efficacy of tumor necrosis factor inhibitors.

BACKGROUND: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) correlates with treatment outcomes in inflammatory bowel disease and rheumatoid arthritis (RA). This study aimed to further evaluate the MALT1 longitudinal change and its relationship with tumor necrosis factor inhibitors (TNFi) response in RA patients. METHODS: Seventy-one RA patients receiving TNFi [etanercept (n = 42) or adalimumab (n = 29)] were enrolled. MALT1 was detected by RT-qPCR in peripheral blood samples of RA patients before treatment (W0), at week (W)4, W12, and W24 after treatment. RA patients were divided into response/non-response, remission/non-remission patients according to their treatment outcome at W24. Meanwhile, MALT1 was also detected by RT-qPCR in 30 osteoarthritis patients and 30 healthy controls (HCs). RESULTS: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 was elevated in RA patients compared with HCs (Z=-6.392, p < 0.001) and osteoarthritis patients (Z = -5.020, p < 0.001). In RA patients, MALT1 was positively correlated with C-reactive protein (rs  = 0.347, p = 0.003), but not other clinical characteristics, treatment history, or current TNFi category. Meanwhile, MALT1 decreased from W0 to W12 in total RA patients (x2  = 86.455, p < 0.001), etanercept subgroup (x2  = 46.636, p < 0.001), and adalimumab subgroup (x2  = 41.291, p < 0.001). Moreover, MALT1 at W24 (p = 0.012) was decreased in response patients compared with non-response patients; MALT1 at W12 (p = 0.027) and W24 (p = 0.010) were reduced in remission patients than non-remission patients. In etanercept subgroup, MALT1 at W24 (p = 0.013) was decreased in response patients compared with non-response patients. In adalimumab subgroup, MALT1 at W24 (p = 0.015) was lower in remission patients than non-remission patients. CONCLUSION: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 reduction after treatment is associated with response and remission to TNFi in RA patients.

MALT1 positively relates to Th17 cells, inflammation/activity degree, and its decrement along with treatment reflects TNF inhibitor response in ankylosing spondylitis patients.

BACKGROUND: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) facilitates CD4+ T-cell differentiation, immune response, inflammation, and osteoclastogenesis. This study aimed to explore the relation between MALT1 and treatment efficacy to tumor necrosis factor inhibitor (TNFi) in ankylosing spondylitis (AS) patients. METHODS: This study recruited 73 AS patients underwent adalimumab treatment. Peripheral blood mononuclear cell (PBMC) was obtained at Week (W) 0, W4, W8, and W12 after treatment initiation; then, MALT1 was measured using RT-qPCR. Furthermore, PBMC and serum at W0 were proposed to flow cytometry and ELISA for Th1 cells, Th17 cells, IFN-γ, and IL-17A levels measurement. Besides, 20 osteoarthritis patients and 20 healthy controls (HCs) were enrolled to detect MALT1. RESULTS: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 expression was higher in AS patients compared with HCs (p < 0.001) and osteoarthritis patients (p < 0.001). Besides, MALT1 expression was positively linked with CRP (p = 0.002), BASDAI (p = 0.026), PGADA (p = 0.040), ASDASCRP (p = 0.028), Th17 cells (p = 0.020), and IL-17A (p = 0.017) in AS patients, but did not relate to other clinical features, Th1 cells or IFN-γ (all p>0.050). MALT1 was decreased along with treatment only in AS patients with ASAS40 response (p < 0.001), but not in those without ASAS40 response (p = 0.064). Notably, MALT1 expression was of no difference at W0 (p = 0.328), W4 (p = 0.280), and W8 (p = 0.080), but lower at W12 (p = 0.028) in AS patients with ASAS40 response compared with those without ASAS40 response. CONCLUSION: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 positively correlates with Th17 cells, inflammatory, and activity degree; meanwhile, its decrement along with treatment reflects the response to TNF inhibitor in AS patients.

Carbamate pesticides exposure and delayed physical development at the age of seven: Evidence from the SMBCS study.

BACKGROUND: Carbamate pesticides are widely used in agriculture and cause widespread human exposure. The health effect of carbamates on physical development remains unclear. The current study aimed to explore the carbamate's health effect on physical development. METHODS: Prenatal, 3-year-old, 7-year-old urinary carbofuranphenol concentration was measured by gas chromatography tandem mass spectrometry and adjusted by creatinine. Anthropometric indices were measured by standard method and z-score standardized. Generalized linear models (GLM) were using to assess associations between exposure measurements and anthropometric indices. The generalized estimate equation (GEE) was applied to analyze the association between multiperiod exposure and anthropometric indices, and time-interaction terms were used to exam health effect consistency of exposure in each period. Gender-stratified analysis were conducted according to results of gender-interaction terms to identify gender-specific effects. RESULTS: The gender-interaction term of prenatal exposure with height z-score was significant (ß = -0.057; 95% CI: -0.113, -0.001; p = 0.045). The 3-year-old carbofuranphenol level showed negative associations with weight z-score (ß = -0.019; 95% CI: -0.038, -0.000; p = 0.040), height z-score (ß = -0.015; 95% CI: -0.028, -0.001; p = 0.026), chest circumference (ß = -0.086; 95% CI: -0.171, -0.001; p = 0.046), and waist circumference (ß = -0.128; 95% CI: -0.230, -0.026; p = 0.014). No statistically significant trend was found for prenatal and 7-year-old carbofuranphenol levels. In GEEs, carbofuranphenol level was negatively associated with weight z-score (ß = -0.103; 95% CI: -0.195, -0.011; p = 0.027), height z-score (ß = -0.087; 95% CI: -0.152, -0.022; p = 0.008), and chest circumference (ß = -0.472; 95% CI: -0.918, -0.026; p = 0.037). Boy's height z-score was inversely associated with carbamate exposure (ß = -0.140; 95% CI: -0.227, -0.053; p = 0.001). CONCLUSIONS: Prenatal and postnatal carbamate exposure may affect physical developmental process.

Paraquat mediates BV-2 microglia activation by raising intracellular ROS and inhibiting Akt1 phosphorylation.

Microglia is the innate immune cell in central nervous system (CNS) and plays an important role in neuroinflammation. Microglia mediated neuroinflammation is the key factor affecting the development of neurodegenerative diseases. Although there was evidence that paraquat (PQ) could induce inflammatory response, its mechanism was not clear. The present study investigated the mechanisms of PQ-induced inflammatory responses in BV-2 microglia cells, and tried to reveal the role of ROS/Akt1 pathway. The results showed that the cell activation markers (iNOS and CD206) of BV-2 cells were increased after PQ treatment, suggesting that BV-2 microglia were activated. PQ induced the reactive oxygen species (ROS) and inhibited the AKT1 phosphorylation in BV-2 cells. Besides, the M1 markers expression (IL-6, TNF-α and IL-1ß) were significantly increased after PQ treatment, which suggested that PQ induced the increase of M1 phenotype of BV-2 microglia. Pre-treated with NAC (ROS scavenger), the M1 phenotype was decreased while the p-Akt1 was restored compared to PQ stimulation. Furthermore, we built an Akt1(S473E)-overexpression BV-2 cell line. The Akt1 (S473E) partially attenuated the PQ induced increase in M1 phenotype, while ROS did not significantly change. These results indicated that PQ induced BV-2 microglia activation by increased ROS mediated Akt1 activation inhibition, leading to neuroinflammation.

Cadmium inhibits neural stem/progenitor cells proliferation via MitoROS-dependent AKT/GSK-3ß/ß-catenin signaling pathway.

Cadmium (Cd) is a toxic heavy metal widely found in the environment. Cd is also a potential neurotoxicant, and its exposure is associated with impairment of cognitive function. However, the underlying mechanisms by which Cd induces neurotoxicity are unclear. In this study, we investigated the in vitro effect of Cd on primary murine neural stem/progenitor cells (mNS/PCs) isolated from the subventricular zone. Our results show that Cd exposure leads to mNS/PCs G1/S arrest, promotes cell apoptosis, and inhibits cell proliferation. In addition, Cd increases intracellular and mitochondrial reactive oxygen species (ROS) that activates mitochondrial oxidative stress, decreases ATP production, and increases mitochondrial proton leak and glycolysis rate in a dose-dependent manner. Furthermore, Cd exposure decreases phosphorylation of protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK3ß) in mNS/PCs. In addition, pretreatment mNS/PCs with MitoTEMPO, a mitochondrial-targeted antioxidant, improves mitochondrial morphology and functions and attenuates Cd-induced inhibition of mNS/PCs proliferation. It also effectively reverses Cd-induced changes of phosphorylation of AKT and the expression of ß-catenin and its downstream genes. Taken together, our data suggested that AKT/GSK3ß/ß-catenin signaling pathway is involved in Cd-induced mNS/PCs proliferation inhibition via MitoROS-dependent pattern.

Flurochloridone induces Sertoli cell apoptosis through ROS-dependent mitochondrial pathway.

Flurochloridone (FLC), a selective herbicide used on a global scale, has been reported to have male reproductive toxicity which underlying mechanism is still largely unknown. The present study was conducted to determine the effects of FLC on Sertoli cell and explore its mechanism by using normal mouse Sertoli (TM4) cell line. Our data indicate that FLC suppressed proliferation of TM4 cells in a dose- and time-dependent manner. Further studies confirmed that FLC induced apoptosis in TM4 cells, accompanied by reactive oxygen species (ROS) accumulation, intracellular calcium increase, opening of mitochondrial permeability transition pore, depolarization of the mitochondrial membrane potential (MMP) and decrease of adenosine triphosphate (ATP) level. Meanwhile, changes of B-cell lymphoma-2 (Bcl-2) family proteins expression, release of cytochrome c and the activation of caspase-9 and caspase-3 were also confirmed. These results indicate that FLC induces TM4 cells apoptosis through the mitochondrial apoptotic pathway. In addition, pretreatment with ROS scavenger N-acetyl-L-cysteine (NAC), could significantly alleviate FLC-induced TM4 cells apoptosis and MMP depolarization. In conclusion, our results suggested that FLC induced TM4 cells apoptosis and it was regulated by mitochondrial dysfunction and oxidative stresses.

Fluorochloridone induces autophagy in TM4 Sertoli cells: involvement of ROS-mediated AKT-mTOR signaling pathway.

BACKGROUND: Fluorochloridone (FLC), a selective pyrrolidone herbicide, has been recognized as a potential endocrine disruptor and reported to induce male reproductive toxicity, but the underlying mechanism is unclear. The aim of this study was to investigate the mechanism of FLC-induced reproductive toxicity on male mice with particular emphasis on the role of autophagy in mice' TM4 Sertoli cells. METHODS: Adult C57BL/6 mice were divided into one control group (0.5% sodium carboxymethyl cellulose), and four FLC-treated groups (3,15,75,375 mg/kg). The animals (ten mice per group) received gavage for 28 days. After treatment, histological analysis, sperm parameters, the microstructure of autophagy and the expression of autophagy-associated proteins in testis were evaluated. Furthermore, to explore the autophagy mechanism, TM4 Sertoli cells were treated with FLC (0,40,80,160 µM) in vitro for 24 h. Cell activity and cytoskeletal changes were measured by MTT assay and F-actin immunofluorescence staining. The formation of autophagosome, accumulation of reactive oxygen species (ROS), expression of autophagy marker proteins (LC3, Beclin-1 and P62) and AKT-related pathway proteins (AKT, mTOR) were observed. The ROS scavenger N-acetylcysteine (NAC) and AKT agonist (SC79) were used to treat TM4 cells to observe the changes of AKT-mTOR pathway and autophagy. RESULTS: In vivo, it showed that FLC exposure caused testicular injuries, abnormality in epididymal sperm. Moreover, FLC increased the formation of autophagosomes, the accumulation of LC3II/LC3I, Beclin-1 and P62 protein, which is related to the degradation of autophagy. In vitro, FLC triggered TM4 cell autophagy by increasing the formation of autophagosomes and upregulating of LC3II/LC3I, Beclin-1 and P62 levels. In addition, FLC induced ROS production and inhibited the activities of AKT and mTOR kinases. The Inhibition of AKT/mTOR signaling pathways and the activation of autophagy induced by FLC could be efficiently reversed by pretreatment of NAC. Additionally, decreased autophagy and increased cell viability were observed in TM4 cells treated with SC79 and FLC, compared with FLC alone, indicating that FLC-induced autophagy may be pro-death. CONCLUSION: Taken together, our study provided the evidence that FLC promoted autophagy in TM4 Sertoli cells and that this process may involve ROS-mediated AKT/mTOR signaling pathways.

Single-cell RNA sequencing of mouse neural stem cell differentiation reveals adverse effects of cadmium on neurogenesis.

Cadmium (Cd) is a toxic heavy metal and widely exists in the environment. Extensive studies have revealed that Cd exposure can elicit neurotoxicity and potentially interfere with neurogenesis. However, underlying mechanisms by which Cd exposure affects neurogenesis remain unclear. In this study, we performed single-cell RNA sequencing (scRNA-seq) of the differentiated mixture from neonatal mouse Neural Stem Cells (mNSCs) that were exposed to Cd for 24 h and differentiated for 7 days. Our results showed that Cd exposure led to an increase in the differentiation of NSCs into astrocytes while a decrease into neurons. Besides, Cd induced subtype-specific response and dysregulated cell-to-cell communication. Collectively, our scRNA-seq data suggested that Cd had toxic effects on NSCs differentiation at the single-cell level, which offered insight into the potential molecular mechanism of Cd on neurogenesis. Furthermore, our findings provided a new method for assessing the neurodevelopmental toxicity of environmental pollutants.

Umbilical cord serum PBDE concentrations and child adiposity measures at 7 years.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) exist extensively in the environment. Toxicological studies suggested PBDEs may interfere with adipogenic pathways. However, few human evidence addressed PBDE exposures in utero related to childhood adiposity. OBJECTIVE: We assessed associations between PBDEs concentrations in cord serum and childhood adiposity measures at 7 years. METHODS: Among 318 mother-child pairs from Sheyang Mini Birth Cohort Study (SMBCS) in China, nine PBDE congener concentrations were quantified in umbilical cord serum using gas chromatography-negative chemical ionization mass spectrometry (GC-NCI-MS). Anthropometric indicators of children aged 7 years were measured, including weight, height and waist circumference. Age and sex-specific body mass index (BMI) z scores were calculated based on World Health Organization (WHO)'s child growth standards. Multivariate linear and logistic regression models adjusted for putative confounders were performed to examine associations between PBDE congeners and adiposity parameters. RESULTS: BDE-209 was the most abundant congener of PBDEs with a median value of 19.5 ng/g lipid. The geometric mean values of nine PBDE congeners ranged from below limit of detection (LOD) to 18.1 ng/g lipid, and the detection rates were 46.5%~96.5%. Cord serum BDE-153 and BDE-154 concentrations were associated with lower childhood BMI z score (regression coefficient, ß=-0.15, 95% confidence interval: -0.29, -0.02; p=0.02; ß=-0.23, 95%CI: -0.43, -0.03; p=0.03, respectively) and lower waist circumference (ß=-0.75 cm, 95%CI: -1.43, -0.06; p=0.03; ß=-1.22 cm, 95%CI: -2.23, -0.21; p=0.02, respectively), after controlling for potential confounders. Moreover, prenatal BDE-154 exposure was related to a decreased obesity risk of children aged 7 years (odds ratio, OR=0.46, 95%CI: 0.22, 0.94; p=0.03). These effects were only observed among boys in sex-straitified analyses. CONCLUSIONS: Cord serum BDE-153 and BDE-154 concentrations were related to reduced adiposity measures at 7 years of age. Further evidence regarding the impacts of prenatal PBDE exposures on childhood development is warranted.

Associations of melamine and cyanuric acid exposure with markers of kidney function in adults: Results from NHANES 2003-2004.

Higher melamine exposure may increase the risk of kidney stone formation and kidney injury in infants, but little is known about the potential nephrotoxic effects of environmental low-dose melamine and its derivative exposure on kidney function of adults in the general population. Our objective was to assess associations between urinary concentrations of melamine and its derivative, cyanuric acid, and kidney function through analyzing the data from the National Health and Nutrition Examination Survey (NHANES) 2003-2004. Information on 298 participants aged ≥20 years was utilized. Urinary melamine and cyanuric acid levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Estimated glomerular filtration rate (eGFR) and urinary albumin-to-creatinine ratio (UACR) were calculated to reflect kidney function. Covariate-adjusted creatinine standardization concentrations accounting for sex, race, age, race/ethically, and body mass index, was employed to control potential confounding of kidney function. Multivariable linear regression models were conducted to estimate associations of covariate-adjusted creatinine standardization urinary melamine and cyanuric acid concentrations with eGFR and UACR. Log-binomial regression models were performed to estimate risks of impaired kidney function and hypertension associated with urinary melamine and cyanuric acid levels. The geometric mean values of urinary melamine and cyanuric acid concentrations were 1.51 µg/L [95% confidence interval (CI): 1.21 µg/L, 1.89 µg/L] and 5.86 µg/L (95% CI: 5.34 µg/L, 6.44 µg/L), respectively. The median value of estimated daily intake (EDI) for melamine was 0.06 (ranging from undetectable to 1.11) µg/kg body weight/day calculated by urinary concentration and creatinine excretion accounting for sex and body weight. Adults in the fourth quartile of melamine and cyanuric acid exposure had 0.142 mL/min/1.73 m2 (95% CI: -0.271, -0.014) and 0.106 mL/min/1.73 m2 (95% CI: -0.020, 0.006) lower eGFR for melamine and cyanuric acid, respectively, compared to participants in the first quartile of exposure with adjustment for potential confounders. To our best knowledge, this is the first study to report associations between melamine and its derivative and kidney function of the U.S. adults from NHANES 2003-2004. The suggestive evidence revealed that individuals with high melamine exposure had lower eGFR than those with low melamine exposure, although no significant association between melamine and cyanuric acid exposure and markers of kidney function was observed. These findings should be interpreted with caution regarding the possible reverse causality.

Prenatal exposure to mixture of heavy metals, pesticides and phenols and IQ in children at 7 years of age: The SMBCS study.

OBJECTIVE: Prenatal exposure to heavy metals, pesticides and phenols has been suggested to interfere with neurodevelopment, but the neurotoxicity of their mixtures is still unclear. We aimed to elucidate the associations of maternal urinary concentrations of selected chemical mixtures with intelligence quotient (IQ) in children. METHODS: Maternal urinary concentrations of selected heavy metals, pesticide metabolites, and phenols were quantified in pregnant women who participated in the Sheyang Mini Birth Cohort Study (SMBCS) from June 2009 to January 2010. At age 7 years, child's IQ score was assessed using the Chinese version of Wechsler Intelligence Scale for Children (C-WISC) by trained pediatricians. Generalized linear regression models (GLM), Bayesian kernel machine regression (BKMR) models and elastic net regression (ENR) models were used to assess the associations of urinary concentrations individual chemicals and their mixtures with IQ scores of the 7-year-old children. RESULTS: Of 326 mother-child pairs, single-chemical models indicated that prenatal urinary concentrations of lead (Pb) and bisphenol A (BPA) were significantly negatively associated with full intelligence quotient (FIQ) among children aged 7 years [ß = -2.31, 95% confidence interval (CI): -4.13, -0.48; p = 0.013, sex interaction p-value = 0.076; ß = -1.18, 95% CI: -2.21, -0.15; p = 0.025; sex interaction p-value = 0.296, for Pb and BPA, respectively]. Stratified analysis by sex indicated that the associations were only statistically significant in boys. In multi-chemical BKMR and ENR models, statistically significant inverse association was found between prenatal urinary Pb level and boy's FIQ scores at 7 years. Furthermore, BKMR analysis indicated that the overall mixture was associated with decreases in boy's IQ when all the chemicals' concentrations were at their 75th percentiles or higher, compared to at their 50th percentiles. ENR models revealed that maternal urinary Pb levels were statistically significantly associated with lower FIQ scores (ß = -2.20, 95% CI: -4.20, -0.20; p = 0.031). CONCLUSIONS: Prenatal exposure to selected chemical mixtures may affect intellectual performance at 7 years of age, particularly in boys. Pb and BPA were suspected as primary chemicals associated with child neurodevelopment.

RNA-seq analysis of testes from flurochloridone-treated rats.

Flurochloridone (FLC) is a widely used herbicide in developing countries. Although the testes are a target organ for FLC in rats, the adverse effects of FLC on testes have not been fully elucidated. To clarify them, we performed RNA-seq analysis using the testes of FLC-treated rats from our previous subchronic toxicity tests. Unilateral testes of three male rats from solvent control groupand three FLC-treated groups (3 mg/kg, 31.25 mg/kg and 125 mg/kg) were used for RNA extraction. A poly A selection protocol coupled with an Illumina TruSeq RNA-Seq library protocol was used to construct RNA-Seq libraries. Principal component analysis (PCA), differentially expressed gene (DEG) analysis, and hierarchical clustering analysis (HCA) were conducted using R. Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to understand the biological characteristics of the DEGs using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The results indicated that many up-regulated DEGs were enriched in pathways associated with testicular injury, such as mitogen-activated protein kinase (MAPK) signaling, lysosome and focal adhesion. Many down-regulated DEGs were enriched in pathways associated with testicular reproduction function, such as sexual reproduction, spermatogenesis and germ cell development. Moreover, we confirmed the oral no-observed-adverse-effect level (NOAEL) of 3 mg/kg in subchronic toxicity test, because the overall testicular gene expression in 3 mg/kg FLC-treated group was similar to that of the solvent control group. In 31.25 mg/kg and 125 mg/kg groups, DEGs revealed that testicular injury was related to oxidative stress.

Sex-Specific Differences in Cognitive Abilities Associated with Childhood Cadmium and Manganese Exposures in School-Age Children: a Prospective Cohort Study.

To examine sex-specific associations of neonatal and childhood exposure to eight trace elements with cognitive abilities of school-age children. The association between exposure and effects was assessed among 296 school-age children from a population-based birth cohort study, who had manganese (Mn), cadmium (Cd), and lead (Pb) exposure measured in cord blood and chromium (Cr), manganese, cobalt (Co), copper (Cu), arsenic (As), selenium (Se), cadmium, and lead exposure quantified in spot urine. Cognitive abilities were assessed using the Wechsler Intelligence Scale for Children-Chinese Revised (WISC-CR). Generalized linear models were performed to analyze associations of intelligence quotient (IQ) with trace element concentrations in cord blood and urinary trace element levels. General linear models were used to evaluate association between exposure fluctuation and children's IQ. Urinary Cd concentrations were negatively associated with full-scale IQ (ß = - 3.469, 95% confidence interval (CI) - 6.291, - 0.647; p = 0.016) and performance IQ (ß = - 4.012, 95% CI - 7.088, - 0.936; p = 0.011) in girls; however, neonatal Cd exposure expressed as Cd concentrations in cord blood was in inverse associations with verbal IQ (ß = - 2.590, 95% CI - 4.570, - 0.609; p = 0.010) only in boys. Positive association between urinary Mn concentrations and performance IQ (ß = 1.305, 95% CI 0.035, 2.575; p = 0.044) of children was observed, especially in girls. In addition, inverse association of urinary Cu concentrations with verbal IQ (ß = - 2.200, 95% CI - 4.360, - 0.039; p = 0.046) was only found in boys. Childhood Cd exposure may adversely affect cognitive abilities, while Mn exposure may beneficially modify cognitive abilities of school-age children, particularly in girls.

N-acetylcysteine alleviated paraquat-induced mitochondrial fragmentation and autophagy in primary murine neural progenitor cells.

The developing brain is uniquely vulnerable to toxic chemical exposures. Studies indicate that neural stem cell (NSC) self-renewal is susceptible to oxidative stress caused by xenobiotics. However, the impact of antioxidants on NSC self-renewal and the potential mechanisms remain elusive. In this study, primary murine neural progenitor cells (mNPCs) from the subventricular zone were used as a research model. In addition, paraquat (PQ) was used to elicit oxidative stress and N-acetylcysteine (NAC) was used as a powerful antioxidant. mNPCs were treated with 80 µm PQ for 24 hours with or without 4 hours of NAC pretreatment. Our results showed that PQ treatment increased intracellular reactive oxygen species production, decreased cell viability and DNA synthesis, and promoted cell apoptosis. Meanwhile, pretreatment with NAC alleviated PQ-induced cytotoxicity in mNPCs. To elucidate the mechanisms further, we found that NAC pretreatment prevented PQ-induced reactive oxygen species production, mitochondrial fragmentation and autophagy in mNPCs. NAC-pretreated cells showed increased anti-apoptotic protein Bcl-2 and decreased pro-apoptotic protein Bax expression. Similarly, NAC pretreatment increased p-mTOR and decreased LC3B-II protein expression. Moreover, NAC decreased mitophagy related mRNA Pink1 and Parkin expression. Taken together, our results suggested that the antioxidant NAC treatment significantly attenuated PQ-induced mNPC self-renewal disruption through decreasing autophagy and salvaging mitochondrial morphology. These findings revealed a potential mechanism for neurological treatment relating to antioxidant and suggested potentially relevant implications for PQ-related neurodegenerative disorders. Thus, our study also provided insight into therapeutic strategies for the neurotoxic effects of oxidative stress-associated toxicants.

Fluorochloridone induces primary cultured Sertoli cells apoptosis: Involvement of ROS and intracellular calcium ions-mediated ERK1/2 activation.

Fluorochloridone (FLC) is a widely used pyrrolidone selective herbicide and reported to induce testis injuries in male rats, but the underlying mechanism is largely unknown. In the present study, primary-cultured Sertoli cells were exposed to FLC at the concentration of 0-10.00µM to study the mechanism of FLC-induced apoptosis. The roles of ROS, intracellular calcium, endoplasmic reticulum (ER), and ERK1/2 were looked at with ROS scavenger N-acetyl-cysteine (NAC), intracellular calcium chelator BAPTA-AM, ER calcium depleting agent thapsigargin (TG), and ERK1/2 inhibitor U0126, respectively. FLC induced dose-dependent apoptosis increase as well as the elevation in levels of ROS, intracellular calcium, and ERK1/2 activation. FLC treatment led to constantly increasing apoptotic rates and ERK1/2 activation over time, while inversed-V shaped change tendencies of ROS and intracellular calcium levels were observed. FLC-induced ROS generation disrupted the intracellular calcium homeostasis by attacking the ER, and the elevated intracellular calcium levels resulted in ERK1/2 over-phosphorylation and consequently promoted Sertoli cell apoptosis. Taken together, ROS and intracellular calcium-mediated ERK1/2 activation led to FLC-induced Sertoli cell apoptosis.

CCR6+ B lymphocytes responding to tumor cell-derived CCL20 support hepatocellular carcinoma progression via enhancing angiogenesis.

BACKGROUND & AIMS: Different immune cells in tumor microenvironment shape tumor progression. CCL20 over-expression was reported as one of the "stemness" trait in TP53 mutated hepatocellular carcinoma (HCC). We aimed to understand the effect of CCL20 on HCC progression. METHODS: In two HCC cohort patients (n=95, n=85 respectively), serum CCL20 concentration was quantified by using ELISA. Expressions of CCL20 and CCR6 in 41 paired HCC tumor and adjacent non-tumor tissues were determined by quantitative Real-Time PCR, confirmed by immunohistochemistry (CCL20) or by flow cytometry analysis (CCR6). Chemotaxis of splenocytes or purified CD19+ B cells to tumor cell-derived CCL20, and angiogenesis of different CD19+ B subtypes responding to tumor cell-derived CCL20 were measured in vitro. H22 murine hepatoma cells were inoculated into immunocompetent or immunodeficient SCID mice, tumor growth and metastasis were monitored after the mice were treated with anti-CCL20 neutralizing antibody or depleted B cells by anti-CD20. RESULTS: Elevation of pretherapy serum CCL20 in HCC patients and increase of CCR6 expression in HCC tissues were closely associated with tumor metastasis and disease poor prognosis. In HCC tissues, CCL20 expression was positively correlated with CCR6 (R2 =0.3134, P=0.0002), and CCR6 was exclusively identified in tumor infiltrated immune cells. CD19+CD5+ B lymphocytes expressed higher CCR6, responded to tumor cell-derived CCL20 and enhanced angiogenesis in vitro. Neutralizing CCL20 activity in immunocompetent mice, not in SCID mice, attenuated tumor incidence, restrained tumor growth and distal metastasis. Tumor angiogenesis was significantly inhibited after CCL20 activity was blockade. In addition, inhibiting B lymphocyte infiltration into tumor mileum also attenuated tumor growth. CONCLUSIONS: Tumor cell-derived CCL20 interacts with CCR6 highly expressed CD19+CD5+ B cells, to promote HCC progression, which might be via enhancing angiogenesis.