Chlorpyrifos-induced dysregulation of synaptic plasticity in rat hippocampal neurons.

Chlorpyrifos (CPF) is a widely used organophosphorus pesticide. Increasing evidence has shown that exposure to CPF in early life might induce neurodevelopmental disorders, but the pathogenesis remains uncertain. Synaptic plasticity plays a crucial role in neurodevelopment. This study aimed to investigate the effect of CPF on synaptic plasticity in hippocampal neurons and establish the cellular mechanism underlying these effects. Using CPF-exposed rat and primary hippocampal neurons model, we analyzed the impact of CPF on the synaptic morphology, the expression level of a presynaptic protein, a postsynaptic protein and ionotropic glutamate receptors (iGluRs), as well as the effects on the Wnt/ß-catenin pathway. We found that the synapses were shortened, the spines were decreased, and the expression of synaptophysin (Syp), postsynaptic density-95 (PSD-95), GluN1, GluA1 and Wnt7a, as well as active ß-catenin in primary hippocampal neurons was decreased. Our study suggests that CPF exposure induced dysregulation of synaptic plasticity in rat hippocampal neurons, which might provide novel information regarding the mechanism of CPF-induced neurodevelopmental disorders.

Chlorpyrifos residue level and ADHD among children aged 1-6 years in rural China: A cross-sectional study.

Background: Attention-deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders in childhood and is caused by both genetic and environmental factors. As genetic factors are nonmodifiable, environmental factors have attracted increasing attention. Objective: To investigate the relationships between urinary chlorpyrifos (CPF) levels, blood micronutrient levels, and ADHD prevalence in children living in rural areas of China. Methods: This cross-sectional study collected data on CPF exposure (according to urinary levels), blood micronutrient levels, and ADHD prevalence in children aged 1-6 years in rural China. The CPF levels were determined by mass spectrometry. Blood levels of micronutrients, including zinc, iron, calcium, copper, magnesium, and vitamin D, were measured by professional detection kits. ADHD was diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition. Descriptive statistics and univariate analysis were conducted using SPSS 21.0, and path analysis was conducted using Mplus 8.0. Results: Of the 738 children who met the eligibility criteria, 673 children (673/738, 91.2%) were included in the final analysis. Baseline questionnaires and urine samples were collected from all 673 subjects. A total of 672 children provided blood samples for micronutrient testing, and 651 completed the ADHD assessment. Approximately one-fifth of children (144/673, 21.4%) had detectable levels of CPF in their urine, and 6.9% (45/651) were diagnosed with ADHD. Path analysis showed that the total effect of CPF exposure on ADHD risk was 0.166 (P < 0.05), with a direct effect of 0.197 (P < 0.05) and an indirect effect of -0.031 (P < 0.05) via vitamin D. The mediating effect of urinary CPF levels on ADHD risk via vitamin D was 18.67%. Conclusion: Higher levels of CPF exposure are associated with higher risk of ADHD. Additionally, increasing vitamin D levels may have a beneficial effect on the relationship between CPF exposure and ADHD risk. Our findings highlight the importance of modifying environmental factors to reduce ADHD risk and provide insight into future ADHD interventions.

Erratum to "Diagnostic performance of serum CK-MB, TNF-α and hs-CRP in children with viral myocarditis".

[This corrects the article DOI: 10.1515/biol-2019-0005.].

Knowledge and behavior regarding pesticide use: a survey among caregivers of children aged 1-6 years from rural China.

Little is known about pesticide exposure risks for children in rural areas in China, many of whom have been left behind by migrant workers. To survey caregivers of children in rural China and assess their pesticide use and disposal methods, the measures were used to protect the children and their perceptions of the adverse effects of pesticides on children's health. Three or four villages in each of Xinhua, Gongan and Sansui Counties in China were selected by random cluster sampling. The main caregivers of children aged 1-6 years were surveyed in face-to-face interviews. The questionnaire used was adapted from the World Health Organization "Exposure to Pesticides: Standard Protocol" survey and similar studies. The study included 464 caregivers (mean age, 46.4 years), who were most commonly the children's grandparents (65.3%). Among the caregivers, 41.9% were educated to middle school level or higher, 45.4% had a household income < 297 USD, and 29.7% had received education/training about pesticide use/adverse health effects in children. The score for caregivers' knowledge of the adverse effects of pesticides on children's health was higher in those who had received education/training (5.1 ± 2.6 vs. 3.4 ± 2.5, P < 0.001). Factors associated with unsafe behaviors during pesticide use included grandparent as the caregiver (odds ratio [OR] 0.551; 95% confidence interval [95% CI] 0.368-0.824; P = 0.004), annual income < 297 USD (OR 0.580; 95% CI 0.395-0.853; P = 0.006), and insufficient health-related education/training (OR 0.436; 95% CI 0.286-0.665; P < 0.001). Improved education and training are needed to promote the safe use of pesticides by caregivers of children in rural China.

Protective mechanism of Taxifolin for chlorpyrifos neurotoxicity in BV2 cells.

Chlorpyrifos (CPF) is an organophosphorus pesticide that can damage the central nervous system in children upon exposure. Taxifolin (Tax) exerts protective effects against neurotoxins; however, the mechanism has not yet been illustrated. The current study used BV2 cells to investigate the protective mechanism underlying the organophosphorus pesticide taxifolin on CPF-induced neurotoxicity, which might present a therapeutic potential for the prevention and treatment of the nervous system diseases in children. BV2 cells were randomly divided into 4 groups: DMSO, CPF, Tax, and Tax + CPF. The viability, morphocytology, oxidative stress, inflammatory reaction, and autophagocytosis have been studied in the cells using Western blot analysis, CCK-8 assay, enzyme-linked immunosorbent assay, and immunofluorescence to estimate the level of LC3 II. As a result, CPF was found to exert a significant toxic effect on BV2 cells that was characterized by rounded cell body, atrophic synapse, poor adhesion, cell aggregation, inflammation, oxidative reaction, and autophagy. Tax treatment has a protective effect on CPF-induced neurotoxicity via downregulation of ROS, TNF-α, IFN-γ, and p62 levels and increased LC3 II level, which in turn, improved the viability and activity of BV2 cells. This phenomenon suggested that Tax can reduce the inflammation and oxidative stress and promote autophagy. Furthermore, the current study suggested that the protective mechanism of Tax on CPF-induced BV2 cell toxicity was via up-regulation of pAMPK level and activation of Nrf2/HO-1 signaling pathway.

Diagnostic Performance of Serum CK-MB, TNF-α and Hs-CRP in Children with Viral Myocarditis.

OBJECTIVE: The purpose of this study was to investigate the diagnostic performance of serum CK-MB, TNF-α and hs-CRP in children with viral myocarditis (VMC). METHODS: Fifty-six children with confirmed diagnosis of VMC were included in this study. Of the included 56 cases, 25 subjects were in acute and other 31 were in the recovery stage. A contemporaneous control group of 22 children were included for comparison. The serum concentration of CK-MB, TNF-α and hs-CRP were examined in both VMC and control groups. RESULTS: The serum concentration of CK-MB, TNF-α and hs-CRP were 31.77±9.48 (UL), 143.11±23.27 (ng/L) and 8.10±1.94(mg/L) for acute stage VMC; 12.72±4.99 (UL), 83.15±13.35 (ng/L) and 4.07±1.12 (mg/L) for recovery stage VMC; 8.11±3.20 (UL), 68.27±12.55 (ng/L) and 2.56±1.27 (mg/L) for control group respectively; The serum concentration of CK-MB, TNF-α and hs-CRP were significantly different between acute stage VMC, recovery stage VMC and control groups (p<0.05); Significant positive correlation between CK-MB and hs-CRP were found in acute stage VMC (r=0.54, p=0.01) and recovery stage VMC (r=0.37, p=0.04). Using serum CK-MB, TNF-α and hs-CRP as the reference, the differential diagnosis sensitivity for acute and recovery stage VMC were 87.10 (70.17-96.37)%, 87.10 (70.17-96.37)% and 77.42 (58.90-90.415)%; The specificity were 92.00 (73.97-99.02)%, 96.00 (79.65-99.90)% and 100.00 (86.28-100.00)% respectively. CONCLUSION: Serum concentration of CK-MB, TNF-α and hs-CRP in children with VMC were significant increased especially in acute stage, which can be used as biomarkers for VMC diagnosis.

Evaluation of the effects of chlorpyrifos combined with lipopolysaccharide stress on neuroinflammation and spatial memory in neonatal rats.

Chlorpyrifos (CPF) may weaken the immune defenses of children, making them vulnerable to opportunistic bacterial infection. CPF combined with bacterial infection is a potential problem for children during their childhood development. However, there is a lack of studies on the joint effects of these two factors on children. Here, we assessed the effects of CPF combined with lipopolysaccharide (LPS) on the inflammation and development of the nervous system. In this study, the cell toxicity of CPF plus LPS in cultured astrocytes, and the pathogenic effects of CPF plus LPS in neonatal rat models were observed. The hydrogen (H2)-inhalation was used for treatment to explore its therapeutic potential. We found that CPF plus LPS activated the astrocyte, which increased the expressions of HMGB1, TLR4, and p-NF-κB p65, while H2-inhalation reduced the expressions (p < 0.05). We also found that CPF plus LPS induced long-lasting spatial memory deficits throughout brain maturation. However, H2-inhalation improved rat performance in these behavioral experiments (p < 0.05). In conclusion, the sub-toxic concentration of CPF did not cause a significant damage in short term, but induced a severe long-term damage to the brain when combined with LPS. H2-inhalation reduced the neuronal damage and behavioral abnormalities caused by CPF and LPS exposure.

Effects of dimethyl sulfoxide on the morphology and viability of primary cultured neurons and astrocytes.

BACKGROUND: Dimethyl sulfoxide (DMSO) is a widely used solvent and vehicle for in vivo and in vitro administration of test compounds. Effects of DMSO independent of the test compound, such as in studies examining morphological plasticity or neurotoxic responses, may lead to spurious results. AIM: To investigate effects of DMSO concentration ([DMSO]) on morphology and survival of primary cultured neurons and astrocytes. METHODS: Primary cultured neurons and astrocytes were treated with 0.25%-10.00% [DMSO] for 12-48h. Viable cell number and morphology were compared to untreated cultures using the CCK-8 assay and phase-contrast microscopy. Expression levels of the neuronal marker NeuN and astrocyte marker glial fibrillary acidic protein (GFAP) were determined by immunofluorescence and western blotting. RESULTS: A [DMSO]≤0.50% had no effect on neuronal number or NeuN expression up to 24h, while ≥1.00% induced a progressive and dramatic loss of both viability and NeuN expression even after 12h. Brief (12h) exposure to ≤1.00% DMSO had no effect on astrocytes survival or GFAP expression, while ≥5.00% significantly reduced both at all exposure durations. In contrast to neurons, exposure to 0.50% and 1.00% DMSO for 24 or 48h enhanced astrocytes proliferation and GFAP expression. Astrocytic processes were maintained at 0.50% and 1.00% DMSO, while neurons exhibited marked neurite retraction at ≥0.50%. CONCLUSION: A [DMSO]≥0.5% markedly disrupts neuronal morphology and reduces viability, even after brief exposure. In astrocytes, 0.50% and 1.00% DMSO appear to induce reactive gliosis. For treatment of neural cells, [DMSO] should be ≤0.25% to obviate spurious vehicle effects.

SGA as a Risk Factor for Cerebral Palsy in Moderate to Late Preterm Infants: a System Review and Meta-analysis.

Small for gestational age (SGA) is an established risk factor for cerebral palsy (CP) in term infants. However, there is conflicting data on the association between SGA and CP in moderate to late preterm infants. The aim of the article was to explore the relationship between SGA and CP in the moderate to late preterm infants and its strength by meta-analysis. We performed a system search in OVID (EMBASE and MEDLINE) and WANFANG from inception to May 2016. The study-specific risk estimates were pooled using the random-effect model. A total of seven studies were included in the meta-analysis, consisting of three cohort and four case-control studies. A statistically significant association was found between SGA and CP in moderate to late premature infants (OR: 2.34; 95% CI: 1.43-3.82). The association were higher in the several subgroups: 34-36 week gestational age (OR: 3.47; 95% CI: 1.29-9.31), SGA < 2SDs (OR: 3.48; 95% CI: 1.86-6.49), and malformation included in CP (OR: 3.00; 95% CI: 1.71-5.26). In moderate to late premature infants, SGA is a convenient and reliable predictor for CP. More studies are needed to explore the underlying mechanisms between SGA and CP association.

The effect of HMGB1 on sub-toxic chlorpyrifos exposure-induced neuroinflammation in amygdala of neonatal rats.

Chlorpyrifos (CPF), one of organophosphorus pesticides (OPs), is associated with developmental neurotoxicity. Inflammatory response is closely related with CPF-induced neurotoxicity. The present study aimed at exploring whether sub-toxic CPF exposure on neonatal rats results in neuroinflammation that mediated by HMGB1/TLR4/NF-κB signaling pathway in the amygdala. The neonatal rats were subcutaneously injected with 5mg/kg CPF for 4 consecutive days (postnatal day 11-14) with or without HMGB1 inhibitor, glycyrrhizin. We assessed the levels of pro-inflammatory cytokines at 12, 24, and 72 h after CPF exposure. The role of HMGB1 on neuroinflammation in sub-toxic exposure during brain development was studied. CPF-treated neonatal rats exhibited a significant increase in the expression of pro-inflammatory cytokines, such as IL-6, TNF-α and HMGB1, and a significant increase in the activation of NF-κB in the amygdala after CPF exposure. Inhibited HMGB1 reduced the release of IL-6 and TNF-α, and inhibited activation of NF-κB. Our findings indicate that CPF exposure on developmental brain might induce the activation of neuroinflammation mediated by HMGB1/TLR4/NF-κB pathway in the amygdala.

Neonatal chlorpyrifos exposure induces loss of dopaminergic neurons in young adult rats.

Increasing epidemiological and toxicological evidence suggests that pesticides and other environmental exposures may be associated with the development of Parkinson's disease (PD). Chlorpyrifos (CPF) is a widely used organophosphorous pesticide with developmental neurotoxicity. Its neurotoxicity, notably on the monoamine system, suggests that exposure of CPF may induce dopaminergic neuronal injury. We investigated whether neonatal exposure to CPF contributes to initiation and progression of dopaminergic neurotoxicity and explored the possible underlying mechanisms. The newborn rats were administrated 5 mg/kg CPF subcutaneously from postnatal day (PND) 11 to PND 14 daily. The effect of CPF on dopaminergic neurons, microglia, astrocyte, nuclear factor-κB (NF-κB) p. 65 and p. 38 mitogen-activated protein kinase (MAPK) signaling pathways was analyzed in the substantia nigra of rats at 12h, 24h, 72 h, 16d and 46 d after exposure. CPF-treated rats exhibited significant reduction of dopaminergic neurons at 16d and 46 d after exposure, and a significant increase in the expression of microglia and astrocytes in the substantia nigra after CPF exposure. Intense activation of NF-κB p. 65 and p. 38 MAPK inflammatory signaling pathways was observed. Our findings indicate that neonatal exposure to CPF may induce long-term dopaminergic neuronal damage in the substantia nigra mediated by the activation of inflammatory response via NF-κB p. 65 and p. 38 MAPK pathways in the nigrostriatal system.

PINK1/Parkin-mediated mitophagy alleviates chlorpyrifos-induced apoptosis in SH-SY5Y cells.

Chlorpyrifos (CPF) is one of the most widely used organophosphorous insecticides. There are links between CPF exposure and neurological disorders. Mitochondrial damage has been implicated to play a key role in CPF-induced neurotoxicity. Mitophagy, the selective autophagic elimination of mitochondria, is an important mitochondrial quality control mechanism. However, the role of mitophagy in CPF-induced neurotoxicity remains unclear. In this study, CPF-caused mitochondrial damage, role and mechanism of mitophagy on CPF-induced neuroapoptosis were extensively studied by using SH-SY5Y cells. We showed that CPF treatment caused mitochondrial fragmentation, excessive ROS generation and mitochondrial depolarization, thus led to cell apoptosis. Moreover, CPF treatment also resulted in increased colocalizaton of mitochondria with LC3, decreased levels of mitochondrial proteins, PINK1 stabilization and mitochondrial accumulation of Parkin. These data suggested that CPF treatment induced PINK1/Parkin-mediated mitophagy in SH-SY5Y cells. Furthermore, knockdown of Parkin dramatically increased CPF-induced neuroapoptosis. On the other hand, overexpression of Parkin markedly alleviated CPF-induced SH-SY5Y cell apoptosis. Together, these findings implicate a protective role of PINK1/Parkin-mediated mitophagy against neuroapoptosis and that enhancing mitophagy provides a potential therapeutic strategy for CPF-induced neurological disorders.