Untargeted metabolomic analysis of ischemic injury in human umbilical vein endothelial cells reveals the involvement of arginine metabolism.

OBJECTIVE: In this study, differentially expressed metabolites of vascular endothelial cells were examined to further understand the metabolic regulation of ischemic injury by untargeted metabolomics. METHOD: Human umbilical vein endothelial cells (HUVECs) were selected to construct an ischemia model using oxygen-glucose deprivation (OGD) and 0, 3, 6, and 9 h of treatment. After that, cell survival levels were determined by CCK8 detection. Flow cytometry, ROS detection, JC-1 detection, and western blotting were used to measure apoptosis and oxidative stress in cells. Then, combined with UPLC Orbitrap/MS, we verified the impacted metabolism pathways by western blotting and RT‒PCR. RESULTS: CCK8 assays showed that the survival of HUVECs was decreased with OGD treatment. Flow cytometry and the expression of cleaved caspase 3 showed that the apoptosis levels of HUVECs increased following OGD treatment. The ROS and JC-1 results further suggested that oxidative stress injury was aggravated. Then, combined with the heatmap, KEGG and IPA, we found that arginine metabolism was differentially altered during different periods of OGD treatment. Furthermore, the expression of four arginine metabolism-related proteins, ASS1, ARG2, ODC1 and SAT1, was found to change during treatment. CONCLUSION: Arginine metabolism pathway-related proteins were significantly altered by OGD treatment, which suggests that they may have a potential role in ischemic injury.

circ_0086296 induced atherosclerotic lesions via the IFIT1/STAT1 feedback loop by sponging miR-576-3p.

Extensive inflammation of endothelial cells (ECs) facilitates atherosclerotic lesion formation. Circular RNA (circRNA) participates in atherosclerosis (AS)-related inflammation responses; however, whether and how circ_0086296 regulates atherosclerotic inflammation and lesions have not been investigated. Microarray analysis, quantitative real-time polymerase chain reaction, and fluorescence in situ hybridization assay were performed to detect the expression and location of hsa_circ_0086296 in human carotid artery plaques, aorta of atherosclerotic mice, and human umbilical vein endothelial cells (HUVECs). Sanger sequencing was used to verify the loop structure of circ_0086296. The relationship among circ_0086296, miR-576-3p, IFIT1, STAT1, and EIF4A3 was validated using bioinformatics, luciferase assay, RNA pull-down assay, and RNA immunoprecipitation. The atherosclerosis mouse model was used to evaluate the function of circ_0086296 in vivo. circ_0086296 expression was significantly upregulated in human carotid artery plaques, oxidized low-density lipoprotein (ox-LDL)-treated HUVECs, and the aorta of atherosclerotic mice. Functional analysis indicated that circ_0086296 promotes ECs injury in vitro and atherosclerosis progression in vivo. The mechanism analysis indicated that circ_0086296 sponged miR-576-3p to promote IFIT1-STAT1 expression. Moreover, STAT1 upregulated circ_0086296 expression, forming the circ_0086296/miR-576-3p/IFIT1/STAT1 feedback loop. Notably, inhibition of the circ_0086296/miR-576-3p/IFIT1 axis could block atherosclerotic lesion formation both in vivo and in vitro. Finally, circ_0086296 was overexpressed in exosomes of patients with atherosclerosis and exosomes of ox-LDL-treated ECs. Therefore, the circ_0086296/miR-576-3p/IFIT1/STAT1 feedback loop participates in atherosclerosis progression and contributes to the high circ_0086296 expression observed in the exosomes of serum of patients with atherosclerosis. This study sought to provide a deep understanding of the mechanisms underlying the aberrant EC phenotype in AS.

Fate of chlorpyrifos bound residues in paddy soils: Release, transformation, and phytoavailability.

Chlorpyrifos (CPF) is a widely used organophosphorus insecticide that tends to form bound residues (BRs) in soils. However, the stability and biological activity of CPF-BRs remain to be explored. Facilitated by carbon-14 tracing, this study obtained CPF-BRs initially formed in two typical paddy soils (14C-CPF-BRin), and further investigated their release, transformation and phytoavailability using duckweed (Lemna minor) as a model aquatic organism. Most 14C-CPF-BRin in soils were composed of the parent CPF and its metabolite 3,5,6-trichloro-2-pyridinol (2-OH-TCP), which was mainly formed through reversible entrapment by soil fulvic acids and humin (>80%). At 36 d, 66.67-80.90% of the 14C-CPF-BRin was released, of which only 2-OH-TCP could be released into the water and absorbed by the duckweed, with bioconcentration factors ranging from 247.99 to 324.68 L kg-1. The subsequent metabolism of released 14C-CPF-BRin in duckweed included phase I metabolism from 2-OH-TCP to 4-OH-TCP and phase II metabolism of conjugation of TCP with plant endogenous amino acids. The study suggested that CPF bound residues have high bioavailability in paddy field environments. Given that many pesticides and non-pesticide chemicals share structures analogous to CPF, the findings have important implications for better understanding the environmental and human health risks of man-made chemicals.

Reducing environmental risks of chlorpyrifos application in typical soils by adding appropriate exogenous organic matter: Evidence from a simulated paddy field experiment.

Chlorpyrifos (CPF), as an organophosphate insecticide extensively used in the modern agricultural system, has been gradually banned in many countries due to its reported health risks to organisms, including humans. This study used simulated paddy field experiments and carbon-14 tracing to explore the possibility of reducing environmental risks of chlorpyrifos application through appropriate agronomic practice. Results showed 14C-CPF concentration in rice plants planted in the red soil (RS) was significantly higher than that in black soil (BS) and fluvo-aquic soil (FS). The application of biochar and chicken manure in RS reduced 14C-CPF accumulation in rice plants, and the content of 14C-CPF in rice grains decreased by 25% and 50%, respectively. Adding biochar to all three soils reduced the migration of 14C-CPF, especially in FS with the highest risk of 14C-CPF migration. The addition of chicken manure in FS reduced the migration of 14C-CPF and the total residual amount of 14C-CPF in the soil. In addition, chicken manure treatment increased the formation of 14C-bound residues (BRs) in soils and changed the distribution 14C-BRs in humus. The results indicated that the degree of environmental risks associated with the CPF application varies with soil types and could be reduced by introducing suitable exogenous organic matter into different soils, which is of great significance for guiding the scientific application of chlorpyrifos in agronomic practices.

Combination of high specific activity carbon-14 labeling and high resolution mass spectrometry to study pesticide metabolism in crops: Metabolism of cycloxaprid in rice.

The study of pesticide metabolism in crops is critical for assessing the mode of action and environmental risks of pesticides. However, the study of pesticide metabolism in crops is usually complicated and it is often a daunting challenge to accurately screen the metabolites of novel pesticides in complex matrices. This study demonstrated a combined use of high-specific activity carbon-14 labeling and high-resolution mass spectrometry (HSA-14C-HRMS) for metabolism profiling of a novel neonicotinoid cycloxaprid in rice. By generating the characteristic radioactive peaks on the liquid chromatogram, the use of 14C can eliminate the severe interference of complex matrices and quickly probe target compounds; by producing ion pairs with unique abundance ratios on HRMS, high-specific activity labeling can effectively exclude false matrix positives and promote the elucidation of metabolite structure. The structures of 15 metabolites were identified, three of which were further confirmed by authentic standards. Based on these metabolites, a metabolic profile of cycloxaprid was established, which includes denitrification, demethylation, imidazolidine hydroxylation and ring cleavage olefin formation, oxidation and carboxylation reactions. The strategy of combining high-specific activity 14C labeling and HRMS offers unique advantages and provides a powerful solution for profiling unknown metabolites of novel pesticides in complex matrices, especially when traditional non-labeling methods are not feasible.

Reducing the Environmental Risk of Chlorpyrifos Application through Appropriate Agricultural Management: Evidence from Carbon-14 Tracking.

Chlorpyrifos (CPF) is one of the most critical insecticides in the world. However, many countries are gradually banning its use due to its reported hazardous impacts on humans. This study explored the possibility of reducing the environmental risk of CPF through appropriate agricultural management practices. Results showed that the environmental risk of CPF is lower under drainage conditions because there is more mineralization and less bound residues (BRs) than under submerged conditions. Bioaugmentation significantly enhanced the CPF mineralization and inhibited the formation of CPF-BRs. Biochar adsorbed CPF and thus reduced its bioavailability, but it could not completely eliminate the toxicity of CPF. In addition, bioaugmentation did not significantly affect the native microbial community of CPF-contaminated soil, suggesting its safety in reducing the environmental risk of CPF. The study indicated that the environmental risk of CPF could be reduced by appropriate agricultural management such as water management, bioaugmentation, soil biochar amendment, and selecting suitable soil types.