Background Carbendazim (CBZ), a widely used benzimidazole fungicide, has raised increasing concerns regarding the health risks associated with its residues. However, the toxic effects and associated mechanisms of CBZ on the skeletal system have not been reported. Objective To elucidate the effects of carbendazim on osteogenic differentiation and its underlying mechanisms. Methods MC3T3-E1 mouse pre-osteoblastic cells were treated with 1, 10, and 100 μmol·L⁻¹ CBZ for 24 h to examine cell viability, alkaline phosphatase (ALP) activity, bone nodule formation, reactive oxygen species (ROS) level, malondialdehyde (MDA) content, and nitric oxide synthase (NOS) activity. Transcriptomics was used to identify differentially expressed genes (DEGs) in osteoblasts exposed to CBZ. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) were employed to analyze the potential biological pathways of DEGs. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to validate changes in gene and protein expression. Results Exposure to 10 and 100 μmol·L⁻¹ CBZ significantly reduced osteoblast viability, ALP activity, bone nodule formation, and NOS activity, while increasing intracellular ROS levels. CBZ at 100 μmol·L⁻¹ concentration significantly elevated MDA level (P < 0.05). The transcriptomic analysis revealed that 1 μmol·L⁻¹ CBZ treatment resulted in 385 significantly DEGs. The KEGG enrichment analysis revealed that CBZ significantly affects hormone regulation pathways (including parathyroid hormone, growth hormone, dopamine, and oxytocin), mitogen-activated protein kinase (MAPK) and cyclic GMP-dependent protein kinase G (cGMP-PKG) signaling pathways, focal adhesion and adherens junction, as well as the NOD-like receptor signaling pathway and the mRNA surveillance (NMD) pathway. The results of GSEA showed that CBZ significantly inhibited the bile acid metabolism and the Wnt/β-catenin pathway in osteoblasts. The validation results demonstrated that CBZ significantly suppressed the mRNA expression of Wnt3a and β-catenin, as well as the protein expression of Runx2 and Osterix in the Wnt/β-catenin pathway. Conclusion CBZ exposure exhibits potential skeletal toxicity, and its mechanism is through promoting oxidative stress, interfering with the Wnt/β-catenin pathway in osteogenic differentiation, thereby inhibiting the bone formation function of osteoblasts.

Background Toluene and chlorobenzene have been designated as surrogate contaminants in the challenge test for evaluating the safety of recycling processes for food-contact recycled polyethylene terephthalate (rPET). Establishing a reliable analytical method is essential for ensuring the compliant use of rPET and safeguarding food safety. Objective To develop a rapid quantitative method for determining toluene and chlorobenzene in rPET using headspace gas chromatography-mass spectrometry (HS-GC-MS), as part of the challenge test for process safety evaluation. Methods The effects of different chromatographic columns and headspace conditions on detection of target analytes were investigated. Three columns HP-5 ms UI (30 m×0.25 mm×0.25 μm), DB-624 (30 m×0.32 mm×1.8 μm), and VF-WAXms (30 m×0.25 mm×0.25 μm) were compared for separation efficiency and peak shape. Headspace equilibration temperatures (50-100 ℃) and equilibration times (10-30 min) were evaluated to determine the optimal instrumental parameters. The effect of sample grinding on recovery was assessed to select the best pretreatment conditions. The established method was validated for selectivity, linearity, sensitivity, accuracy, and precision, and was subsequently applied to the analysis of 12 rPET samples. Results The target analytes achieved good separation and response within 15 min, under the optimized conditions using an HP-5 ms UI column, a headspace equilibration temperature of 60 ℃ and a 10 min equilibration time. Direct analysis without grinding yielded satisfactory recovery rates. Toluene and chlorobenzene showed excellent linearity (0.0030-5.0 mg·kg⁻¹, R²≥0.999). The limits of detection (LOD) and quantification (LOQ) were 0.00003 mg·kg⁻¹ and 0.00011 mg·kg⁻¹ for toluene respectively, and 0.00011 mg·kg⁻¹ and 0.00037 mg·kg⁻¹ for chlorobenzene respectively. The recoveries of the matrix spike at low and high spiking levels ranged from 88.6% to 110%, with relative standard deviations of 0.52%-4.9% (n=6). Among the 12 rPET samples from different recycling stages, three samples contained detectable levels of toluene (0.196-0.250 mg·kg⁻¹) and chlorobenzene (0.704-0.867 mg·kg⁻¹). Conclusion The proposed method is simple, rapid, highly sensitive, and accurate, making it suitable for determining toluene and chlorobenzene in food-contact rPET. It provides a robust technical approach for the safety evaluation of rPET recycling processes.

Per- and polyfluoroalkyl substances （PFASs） are a new type of persistent organic pollutants with global attention. They have shown multiple toxic effects due to their persistent accumulation in human body through exposure to environmental media such as drinking water， food， atmosphere， and soil. However， the bone toxicity of PFASs has not attracted enough attention. It is believed that the exposure and accumulation of PFASs in human have a significant impact on the bone health， especially hindering the healthy bone development in infants and adolescents， and aggravating the occurrence of bone loss and fracture in the elder populations. This paper will review the research progress of the effects of PFASs exposure on bone health indicators such as bone mineral density， and discuss the mechanisms of PFAS in bone toxicity. This review will provide references for revealing the effects of PFASs exposure on bone health and their toxic mechanisms.

ObjectiveTo establish a non-targeted screening method for emerging contaminants in drinking water based on high-resolution mass spectrometry and apply it to actual water samples. MethodsA total of 9 drinking water samples collected from 3 reservoirs in Shanghai were purified and concentrated by HLB solid phase extraction column， then separated and analyzed by liquid chromatography high-resolution mass spectrometer and gas chromatography high⁃resolution mass spectrometer. The acquired data were analyzed by Thermo Tracefinder， Excel and other software combined with mzCloud and NIST databases. The methodology was verified with representative compound standards. Pesticide and perfluorinated compounds were taken as examples to analyze their pollution status. ResultsA non-targeted analysis strategy based on liquid chromatography and gas chromatography tandem high-resolution mass spectrometry was established. The pollution level of 20 kinds of pesticides and 4 kinds of perfluorinated compounds identified in 9 drinking water samples were higher in the Huangpu River than in the Yangtze River estuary. ConclusionThe established non-targeted screening method by high-resolution mass spectrometry can detect potential emerging contaminants in drinking water without relying on the standards， which provides a powerful technical means for water quality monitoring and risk assessment.

Background The production and consumption of high polar pesticides in China are the largest in the world. Therefore, it is urgent to develop a method with fast analysis, large flux, and high accuracy to determine the residues of these pesticides in food. Objective To establish a method for the determination of eight highly polar pesticides [chlormequat, paraquat, difenzoquat, cyromazine, propamocarb, glyphosate, (aminomethyl)-phosphonic acid, and glufosinate] in vegetables and fruits by ultra-performance liquid chromatography-tandem mass spectrometry. Methods After comparing various types of hydrophilic interaction liquid chromatography (HILIC) columns, and optimizing pH value and buffer concentration of mobile phase, effective chromatographic retention and separation of selected eight pesticides were achieved. Based on the optimization of mass spectrometry under chromatographic conditions, a multiple reaction monitoring (MRM) channel of target compounds was established. In the sample pretreatment, through optimization of water content, extraction solvent, and purification method, a final MRM mode of ultra-performance liquid chromatography-tandem mass spectrometry was used for detection, and the isotope internal standard method was used for quantification. The accuracy and the precision of the method were evaluated using recovery and relative standard deviation. The established method was applied to detect 57 samples of retail vegetables and fruits to investigate the adaptability of the proposed method and the residual levels of selected high polar pesticides. Results For positive ion electrospray ionization (ESI⁺) detection, we chose Sielc Obelisc R as chromatographic column, and 20 mmol·L⁻¹ ammonium formate solution (pH=3±0.05) and acetonitrile as mobile phase; for negative ion electrospray ionization (ESI⁻) detection, we chose Shodex Asahipak NH2P-50 2D as chromatographic column, and 5 mmol·L⁻¹ ammonium acetate solution (pH=11±0.05) and acetonitrile as mobile phase to obtain good chromatographic separation and peak shape. Under the optimal conditions of sample water content standardization, using 2% acidified methanol as extraction solvent, and C18 dispersed solid phase extraction purification, the linearity ranges of five analytes (chlormequat, paraquat, difenzoquat, cyromazine, and propamocarb) and three analytes [glyphosate, (aminomethyl)phosphonic acid, and glufosinate] were 1.00-100 μg·L⁻¹ and 5.00-500 μg·L⁻¹ (both correlation coefficients>0.999) respectively, the detection limits were 0.002-0.010 mg·kg⁻¹, and the limits of quantification (LOQ) were 0.005-0.025 mg·kg⁻¹. At three spiked levels (LOQ, 2LOQ, and 5LOQ), the recoveries were in the range of 85.3%–113.2%, and the relative standard deviations were 1.5%–9.5% (n=6). Three target pesticides (chlormequat, cyromazine, and propamocarb) were detected in 57 samples of retail vegetables and fruits, and the residue of chlormequat in cowpea exceeded the maximum residue limit. Conclusion The established method of HILIC combined with ultra-performance liquid chromatography-tandem mass spectrometry and isotopic internal standard quantification has the characteristics of simplicity, stability, and easy operation, which is suitable for rapid screening and quantitative detection of selected eight high polar pesticide residues in large quantities of vegetables and fruits, and provides technical support for monitoring and risk assessment of high polar pesticide residues.

Objective To investigate the protective effect of menatetrenone (MK₄) on the osteoblasts in oxidative stress, and to clarify the anti-osteoporosis mechanism of MK₄. Methods Mouse osteoblasts (MC3T3-E1) induced by hydrogen peroxide (H₂O₂) was used. Cell viability, ALP activity and the area of bone nodule were observed. The level of ROS was detected by DCFH-DA, mitochondrial membrane potential by JC-1, apoptosis rate by annexin V-FITC/PI, and the expression of FoxO1, FoxO3, SOD, bcl-2 and bax by RT-PCR. Results Menatetrenone at 10 μmol/L significantly increased the proliferation of osteoblasts stimulated by H₂O₂, ALP activity, bone nodule formation area, cell membrane potential, the antioxidant SOD and transcription factors FoxO1 and FoxO3 mRNA expression. In the meantime, the elevated malondialdehyde and reactive oxygen species level in cells induced by H₂O₂, the apoptosis rate and the mRNA expression level of bax/Bcl-2 were significantly reduced. Conclusion menatetrenone can protect osteoblasts from oxidative damage by regulating FoxO pathway and reduce osteoblasts apoptosis by up regulating the proportion of Bcl-2/bax.