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.

Background Per- and polyfluoroalkyl substances (PFASs) are a class of persistent organic pollutants that pose potential health risks to humans. Infants and young children have higher requirements for food safety due to the underdeveloped detoxification and immune systems. Therefore, developing a comprehensive method for determination of PFASs and their novel alternatives in infant complementary food is of great significance. Objective To develop an analytical method using liquid chromatography high-resolution mass spectrometry technology for determination of 55 PFASs in plant- and animal-derived infant complementary fruit purees. Methods Oasis WAX (200 mg, 6 CC) solid-phase extraction columns were used for sample enrichment and purification. The pH of the acetonitrile extract was adjusted using 0%, 1%, 1.5%, and 2% formic acid aqueous solutions to evaluate its impact on the recovery rate of target compounds. Additionally, the impact of a 2 mL methanol wash during the purification process on the recovery of target compounds was assessed to determine the optimal pretreatment conditions. Three types of chromatographic columns—Agilent Poroshell 120 EC-C₁₈, Thermo InfinityLab Poroshell 120 Aq-C₁₈, Acquity Waters BEH-C₁₈, and changes in mobile phase, were compared for their effects on retention time, peak shape, and response of target compounds. The method was validated in terms of selectivity, linear range, detection limit, and precision. The established method was applied to 49 commercial samples of infant complementary fruit purees. Results Adjusting the sample pH using 1.5% formic acid water and incorporating a 2 mL methanol wash during purification achieved satisfactory recovery rates. The target compounds were chromatographically separated using an Agilent Poroshell 120 EC-C₁₈ column with a gradient elution system. The mobile phase consisted of methanol-water (methanol/water: 2/98, v/v) containing 5 mmol·L⁻¹ ammonium formate as mobile phase A, and methanol as mobile phase B. Good separation was achieved within 15 min, resulting in optimal chromatographic peak shapes. The 55 target compounds exhibited good linearity across the standard curve range, with correlation coefficients (R²) greater than 0.99. The method detection limits ranged from 0.02 to 0.05 µg·L⁻¹. In the plant- and animal-based fruit puree samples, the spiked recovery rates ranged from 60% to 112% and 57% to 119%, respectively, with relative standard deviations (RSD) ≤ 30%. A total of 9 traditional PFASs and 5 novel PFASs were positive in 49 samples of infant complementary fruit purees. Conclusion This method enables comprehensive detection of 55 traditional and emerging PFASs, offering wide coverage, high accuracy, and excellent sensitivity. It provides technical support for characterizing contamination by traditional and emerging PFASs in food matrices.

Background With the progression of industrialization, an increasing number of emerging contaminants are entering aquatic environments, posing significant threats to the safety of drinking water. Therefore, establishing a system for identifying unknown hazardous factors and implementing safety warning mechanisms for drinking water is of paramount importance. Among these efforts, non-target screening plays a critical role, but its effectiveness is largely constrained by the scope of coverage of sample pre-treatment methods. Objective To integrate modern chromatography/mass spectrometry techniques with advanced data mining methods to develop a non-discriminatory sample pre-treatment method for comprehensive enrichment of unknown contaminants in drinking water, laying a technical foundation for the discovery and identification of unknown organic hazardous factors in drinking water. Methods A non-discriminatory pre-treatment method based on supramolecular and solid-phase extraction was developed. The final target compounds including 333 pesticides, 194 pharmaceuticals and personal care products (PPCPs), and 59 per- and polyfluoroalkyl substances (PFASs) were used for optimizing the pre-treatment method, confirming its coverage. The impacts of different eluents on the absolute recovery rates of target compounds were compared to select the conditions with the highest recovery for sample pre-treatment. The effects of different supramolecular solvents and salt concentrations on target compound recovery were also evaluated to determine the most suitable solvent and salt concentration. Results The solid-phase extraction elution solvents, supramolecular extraction solvents, and salt concentrations were optimized based on the target compound recovery rates. The optimal recovery conditions were achieved using 2 mL methanol, 2 mL methanol (containing 1% formic acid), 2 mL ethyl acetate, 2 mL dichloromethane, hexanediol supramolecular solvent, and 426 mg salt. The detection method developed based on these conditions showed a good linear relationship for all target compounds in the range of 0.1-100.0 ng·mL⁻¹, with R² > 0.99. The method’s limit of detection ranged from 0.01 ng⁻¹ to 0.95 ng⁻¹, and 95% of target compounds were recovered in the range of 20%-120%, with relative standard deviation (RSD) less than 30%, indicating good precision. Conclusion The combined pre-treatment method of solid-phase extraction and supramolecular solvent extraction can effectively enrich contaminants in drinking water across low, medium, and high polarities, enabling broad-spectrum enrichment of diverse trace contaminants in drinking water. It provides technical support for broad-spectrum, high-throughput screening and identification of organic pollutants in drinking water, and also serves as a reference for establishing urban drinking water public safety warning systems.

ObjectiveTo explore the biological mechanism of bone loss caused by perfluorooctanoic acid (PFOA) through transcriptomic analysis, and to provide new insights into regulating perfluoroalkyl substances (PFAS) applications and the prevention of hazards affecting bone health. MethodsMouse embryonic osteoblast precursor cells (MC3T3-E1) were exposed to 0.1, 1, 10, and 100 μmol·L^-¹ PFOA for 24 hours to assess the effects on cell viability and alkaline phosphatase (ALP) activity, and to determine the critical concentration of PFOA toxicity. The transcriptome sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs) induced by PFOA. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) were conducted to identify significantly affected gene pathways. Additionally, Seahorse XF metabolic phenotyping and reverse transcription polymerase chain reaction (RT-PCR) were used to validate the key pathways. ResultsExposure to 10 and 100 μmol·L^-¹ PFOA significantly reduced the cell viability and ALP activity of MC3T3-E1 cells. Therefore, the results of transcriptomic analysis for 10 μmol‧L^-1 PFOA exposure found that a total of 80 DEGs were identified, including 32 upregulated genes and 48 downregulated genes. According to GO analysis, PFOA mainly affected cellular components such as mitochondrion and nucleus, molecular functions involving GTPase activity and GTP binding, as well as biological process related to mRNA processing. GSEA identified the downregulation of the β-oxidation of fatty acid pathway in mitochondria. Metabolic phenotyping reserches showed that PFOA indeed reduced mitochondrial aerobic respiration capacity and adenosine triphosphate (ATP) production, and the ratio of ATP production from cellular aerobic respiration to glycolysis was significantly decreased as well. The mRNA expression of glucose metabolism-related genes (GK, G6PD, and CS), as well as fatty acid metabolism-related genes (CPT1A and CPT2), were significantly downregulated. ConclusionPFOA reduces bone formation by inhibiting energy metabolism and β-oxidation of fatty acid pathways in osteoblasts, whihc lays the foundation for revealing the mechanism of PFOA exposure induced bone loss.

ObjectiveTo explore the role of flurochloridone (FLC) on osteogenic differentiation and the potential mechanism of inhibiting bone formation, and to provide new insights into bone health risks associated with FLC pesticide exposure. MethodsNeonatal rat skull differentiation primary osteoblast model was used to investigate the effects of 1, 10 and 100 μmol·L^-1 FLC exposure on cell viability, osteogenic differentiation alkaline phosphatase (ALP) activity, and bone mineralization nodule formation, respectively. The potential mechanism underlying the inhibition of FLC on osteoblast differentiation was analyzed using osteogenic differentiation gene chip technique, and the expression of key genes and proteins in the pathway was validated using reverse transcription polymerase chain reaction (RT-PCR) and protein immunoblotting (Western blot) methods. ResultsExposure to FLC at a concentration of 100 μmol·L⁻¹ reduced cell proliferation, ALP activity, and the formation of mineralized nodules in primary osteoblasts. Gene chip analyses revealed that exposure to 10 μmol·L⁻¹ FLC induced 15 differentially expressed genes (DEGs). Among these, MMP9 and Tnf were up-regulated, while Nkx3⁃2, Tuft1, Bmp2, Col12a1, Pparg, Enam, Igf1, Bmp5, Bmp3, Calcr, Egf, Igfbp3, and Col14a1 were down-regulated. Results of protein-protein interaction analyses and gene ontology enrichment analyses indicated that FLC inhibited the BMP/SMAD pathway involved in osteogenic differentiation. FLC suppressed the protein expression of BMP2 and Osterix, as well as the expression of key genes critical for osteogenic differentiation and ossification, such as BMP2, Runx2, SMAD1, and SMAD5 in the BMP/SMAD pathway. ConclusionFLC affects osteogenic differentiation and bone formation potential by regulating the BMP/SMAD axis and the expression of osteogenic genes, suggesting its potential risk in bone metabolism.

Background Per- and polyfluoroalkyl substances (PFAS) are a class of persistent organic pollutants that possess potential toxicity to the human body. The production and utilization of diverse emerging PFAS have resulted in widespread human exposure. Therefore, it is imperative to establish a quantitative methodology encompassing a wide range of PFAS for a comprehensive assessment of human exposure to these compounds. Objective To establish a high-throughput quantitative method for the simultaneous determination of 53 PFAS in human serum based on ultra-high-performance liquid chromatography-Q Exactive high resolution mass spectrometry (UPLC-Q Exactive HRMS). Methods The extraction recoveries of hydrophilic-lipophilic balance (HLB) column, weak anionexchange (WAX) column, and 96-well WAX μElution plate were compared to select the SPE column with the highest recovery. The retention time and peak shape of the target compounds were compared between ACQUITY UPLC BEH C₁₈ column and Accucore aQ column, and the more cost-effective column was chosen. The effects of adding different levels of ammonium formate (0, 2, 5 and 10 mmol·L⁻¹) in mobile phase on peak shape and target response were compared to determine the optimal buffer salt concentration. The optimal spray voltage was obtained by comparing −2 kV and −4 kV. The proposed method was validated from the aspects of selectivity, standard curve, limits of detection, precision, accuracy, and matrix effect. The method was applied to 142 umbilical serum samples. Results The best recovery rate (64%-118%) was achieved by using 96-well WAX μElution plate. The optimal separation and peak shape were obtained by utilizing Accucore aQ column with H₂O-methanol (containing 5 mmol·L⁻¹ ammonium formate) as the mobile phase. Less in-source collision and better target response were observed when the spray voltage was set to −2 kV. All target analytes had a good linearity, with R² > 0.99. The limits of detection ranged from 0.01 to 0.50 μg·L⁻¹, and the recovery ranged from 69% to 127% with the precision less than 26%. A total of 31 PFAS were detected in the 142 actual samples, among which 14 PFAS had a detection frequency over 50%. Perfluorooctanoic acid showed the highest median concentration of 4.16 μg·L⁻¹, followed by 6:2 chlorinated polyfluorinated ether sulfonate and perfluorooctane sulfonates (3.50 μg·L⁻¹ and 1.59 μg·L⁻¹, respectively). Conclusion In this study, we establish a UPLC-Q Excative HRMS method for simutanious determination of 53 PFAS concentrations in serum. This method has the advantages of wide coverage of PFAS, good selectivity, and easy operation, and is suitable for biological detection with a large sample size.

Objective:To investigate the long-term therapeutic effects and safety of renal denervation (RDN) on hypertensive patients with different cardiovascular risks, as well as its impact on adverse events, cardiovascular death and all-cause mortality.Methods:This was a single-center, single-arm, real-world retrospective study. Patients with refractory hypertension who underwent RDN at Tianjin First Central Hospital from July 6, 2011 to December 23, 2015 were enrolled and divided into either a high or intermediate-low risk group based on baseline cardiovascular risk. The treatment responsiveness of hypertensive patients with different cardiovascular stratification to RDN was assessed by comparing the results of office blood pressure, home blood pressure, and 24-h ambulatory blood pressure monitoring at 1, 5, and 11 years after RDN. Long-term safety of RDN was assessed by creatinine, and estimated glomerular filtration rate (eGFR) at 1 and 11 years after RDN. In addition, the total defined daily dose (DDD) of antihypertensive medications and the incidence of long-term adverse events, cardiovascular deaths, and all-cause deaths after RDN were followed up 11 years after RDN in person or by telephone.Results:A total of 62 patients with refractory hypertension, aged (50.2±15.0) years, of whom 35 (56.5%) were male, were included. There were 35 cases in high-risk group and 27 cases in low and medium risk group. The decrease in clinic systolic blood pressure (high risk vs. low-medium risk: (-38.0±15.1) mmHg vs. (-25.0±16.6) mmHg(1 mmHg=0.133kPa), P=0.002), home self-measured systolic blood pressure ((-28.4±12.7) mmHg vs. (-19.7±13.1) mmHg, P=0.011) and clinic systolic blood pressure 11 years after RDN ((-43.0±18.4) mmHg vs. (-27.8±17.9) mmHg, P=0.003) in the high-risk group was significantly higher than that in the low-medium risk group. The differences in heart rate and the decrease in total DDD number of antihypertensive drugs between the two groups were not statistically significant (all P>0.05). Creatinine and eGFR levels in the two groups at 1 and 11 years after RDN were not statistically significant when compared with the baseline values (all P>0.05). The cumulative cardiovascular mortality rate was 1.6% (1/62) and 8.1% (5/62), and the cumulative all-cause mortality rate was 3.2% (2/62) and 11.3% (7/62) at 5 and 11 years after RDN, respectively. The differences in the incidence rate of adverse events, cardiovascular mortality, and all-cause mortality rate between the two groups were not statistically significant (all P>0.05). Conclusions:RDN has long-term antihypertensive effect and good safety. Hypertensive patients who belong to the high-risk stratification of cardiovascular risk may respond better to RDN treatment.

Background Perfluorinated and polyfluoroalkyl substances (PFAS), a large group of emerging pollutants, are ubiquitous in the ecological environment. Their multiple organ toxic effects on human body are reported. Understanding the exposure level of PFAS in cord serum and associated influencing factors can provide scientific evidence for studying maternal and newborn health effects and risk regulation. Objective To explore the exposure levels of PFAS in cord serum and potential impact factors. Methods This study was based on the maternal and infant database and the cord serum sample bank of the Sheyang Mini Birth Cohort Study (SMBCS) established in 2009. A self-designed questionnaire was used to collect information on sociodemographic characteristics, living environment, and lifestyle of mothers during pregnancy. A total of 1097 cord serum samples were measured utilizing ultra-high performance liquid chromatography-Q-Exactive Orbitrap high-resolution mass spectrometry. Multiple linear regression models were used to identify influencing factors related to PFAS exposure level in cord serum. Results A total of 38 types of PFAS were detected in the cord serum samples, including 20 perfluorocarboxylic acids (PFCA), 14 perfluorosulfonic acids (PFSA), and 4 other types of PFAS. The detection rates of 14 PFAS compounds were above 50%. The detection rates of perfluoro-1-hexanesulfonic acid (PFHxS), perfluoro-1-octanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUdA) were in the range of 99.18%-100.00%. The median concentrations of PFOA and 6∶2 chlorinated polyfluorinated ether sulfonic acid (6∶2CI-PFESA) were higher than those of the other compounds, which were 3.76 µg·L⁻¹ and 2.61 µg·L⁻¹, respectively. The correlation coefficients among cord serum PFAS were in the wide range of 0.0911-0.8429 (P < 0.05). The associations between PFHxS and perfluoro-1-heptanesulfonic acid (PFHpS) (r=0.8429, P < 0.05), PFOS and PFHpS (r=0.7012, P < 0.05), and PFUdA and perfluorododecanoic acid (PFDoA) (r=0.7875, P < 0.05) were positive. The proportions of PFCA and PFSA in the cord serum samples were 60.1% and 38.4%, respectively. PFOA and 6∶2 Cl-PFESA were the predominant compounds in the cord serum samples, whose proportions were 37.8% and 19.3%, respectively. The concentrations of specific types of cord serum PFAS had varied associations with the following demographic variables of pregnant women: age, parity, education level, gestational age, and occupation. The multiple linear regression analysis found that the concentrations of 8 PFAS (PFHxS, PFHpS, PFNA, PFOS, PFDA, PFUdA, PFDoA, and 6∶2 CI-PFESA) in umbilical cord serum were lower in pregnant women aged 25 years and older than in those under the age of 25 years; compared to pregnant women with education levels at or below junior middle school, those with education levels at or above high school had lower concentrations of perfluoroheptanoic acid (PFHpA); the concentration of PFHpA in cord serum was negatively correlated with household annual income; multiparous women had higher concentrations of PFNA, PFUdA, and PFDoA than primiparous women; compared to mothers engaged in manual labor, the concentration of perfluoro-1-butanesulfonic acid (PFBS) in umbilical cord serum was lower in mothers engaged in mental labor, while the concentrations of PFBS and PFOA were lower and the concentrations of perfluorotridecanoic acid (PFTrDA) and 8∶2 chlorinated polyfluorinated ether sulfonic acid (8∶2CI-PFESA) were higher in mothers of other occupational types; the concentrations of umbilical cord serum PFOA, PFOS, PFUdA, and PFDoA were positively correlated with the gestational age of pregnant women, while N-methylperfluoro-1-octanesulfonamido acetic acid (N-MeFOSAA) was negatively correlated with the gestational age of pregnant women; PFTrDA was negatively correlated with the birth weight of newborns; compared with those with insufficient gestational weight gain, those with adequate gestational weight gain had lower PFDoA, while those with excess gestational weight gain had higher N-MeFOSAA. Conclusion PFAS compounds exposure is common in the mothers and infants in the study area. PFOA and 6∶2 CI-PFESA are the two main compounds in cord serum. Cord serum PFAS are associated with several demographic characteristics of the pregnant women. Future research needs to pay more attention to PFAS alternatives and other emerging contaminants.

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.