Sp1-activated FGFR2 is involved in early-life exposure to nickel-induced craniosynostosis by regulating the ERK1/2 signaling pathway.

Nickel, a common environmental hazard, is a risk factor for craniosynostosis. However, the underlying biological mechanism remains unclear. Here, we found that early-life nickel exposure induced craniosynostosis in mice. In vitro, nickel promoted the osteogenic differentiation of human mesenchymal stem cells (hMSCs), and its osteogenic ability in vivo was confirmed by an ectopic osteogenesis model. Further mRNA sequencing showed that ERK1/2 signaling and FGFR2 were aberrantly activated. FGFR2 was identified as a key regulator of ERK1/2 signaling. By promoter methylation prediction and methylation-specific PCR (MSP) assays, we found that nickel induced hypomethylation in the promoter of FGFR2, which increased its binding affinity to the transcription factor Sp1. During pregnancy and postnatal stages, AZD4547 rescued nickel-induced craniosynostosis by inhibiting FGFR2 and ERK1/2. Compared with normal individuals, nickel levels were increased in the serum of individuals with craniosynostosis. Further logistic and RCS analyses showed that nickel was an independent risk factor for craniosynostosis with a nonlinear correlation. Mediated analysis showed that FGFR2 mediated 30.13% of the association between nickel and craniosynostosis risk. Collectively, we demonstrate that early-life nickel exposure triggers the hypomethylation of FGFR2 and its binding to Sp1, thereby promoting the osteogenic differentiation of hMSCs by ERK1/2 signaling, leading to craniosynostosis.

S-Nitrosylation of Septin2 Exacerbates Aortic Aneurysm and Dissection by Coupling the TIAM1-RAC1 Axis in Macrophages.

BACKGROUND: S-Nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in cardiovascular disease. Aortic aneurysm and dissection are high-risk cardiovascular diseases without an effective cure. The aim of this study was to determine the role of SNO of Septin2 in macrophages in aortic aneurysm and dissection. METHODS: Biotin-switch assay combined with liquid chromatography-tandem mass spectrometry was performed to identify the S-nitrosylated proteins in aortic tissue from both patients undergoing surgery for aortic dissection and Apoe-/- mice infused with angiotensin II. Angiotensin II-induced aortic aneurysm model and ß-aminopropionitrile-induced aortic aneurysm and dissection model were used to determine the role of SNO of Septin2 (SNO-Septin2) in aortic aneurysm and dissection development. RNA-sequencing analysis was performed to recapitulate possible changes in the transcriptome profile of SNO-Septin2 in macrophages in aortic aneurysm and dissection. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation were used to uncover the TIAM1-RAC1 (Ras-related C3 botulinum toxin substrate 1) axis as the downstream target of SNO-Septin2. Both R-Ketorolac and NSC23766 treatments were used to inhibit the TIAM1-RAC1 axis. RESULTS: Septin2 was identified S-nitrosylated at cysteine 111 (Cys111) in both aortic tissue from patients undergoing surgery for aortic dissection and Apoe-/- mice infused with Angiotensin II. SNO-Septin2 was demonstrated driving the development of aortic aneurysm and dissection. By RNA-sequencing, SNO-Septin2 in macrophages was demonstrated to exacerbate vascular inflammation and extracellular matrix degradation in aortic aneurysm. Next, TIAM1 (T lymphoma invasion and metastasis-inducing protein 1) was identified as a SNO-Septin2 target protein. Mechanistically, compared with unmodified Septin2, SNO-Septin2 reduced its interaction with TIAM1 and activated the TIAM1-RAC1 axis and consequent nuclear factor-κB signaling pathway, resulting in stronger inflammation and extracellular matrix degradation mediated by macrophages. Consistently, both R-Ketorolac and NSC23766 treatments protected against aortic aneurysm and dissection by inhibiting the TIAM1-RAC1 axis. CONCLUSIONS: SNO-Septin2 drives aortic aneurysm and dissection through coupling the TIAM1-RAC1 axis in macrophages and activating the nuclear factor-κB signaling pathway-dependent inflammation and extracellular matrix degradation. Pharmacological blockade of RAC1 by R-Ketorolac or NSC23766 may therefore represent a potential treatment against aortic aneurysm and dissection.

Evaluation of 6-PPD quinone toxicity on lung of male BALB/c mice by quantitative proteomics.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ), a transformation product of tyre-derived 6-PPD, has been frequently detected in different environments. After 6-PPDQ exposure, we here aimed to examine dynamic lung bioaccumulation, lung injury, and the underlying molecular basis in male BALB/c mice. After single injection at concentration of 4 mg/kg, 6-PPDQ remained in lung up to day 28, and higher level of 6-PPDQ bioaccumulation in lung was observed after repeated injection. Severe inflammation was observed in lung after both single and repeated 6-PPDQ injection as indicated by changes of inflammatory cytokines (TNF-α, IL-6 and IL-10). Sirius red staining and hydroxyproline content analysis indicated that repeated rather than single 6-PPDQ injection induced fibrosis in lung. Repeated 6-PPDQ injection also severely impaired lung function in mice by influencing chord compliance (Cchord) and enhanced pause (Penh). Proteomes analysis was further carried out to identify molecular targets of 6-PPDQ after repeated injection, which was confirmed by transcriptional expression analysis and immunohistochemistry staining. Alterations in Ripk1, Fadd, Il-6st, and Il-16 expressions were identified to be associated with inflammation induction of lung after repeated 6-PPDQ injection. Alteration in Smad2 expression was identified to be associated with fibrosis formation in lung of 6-PPDQ exposed mice. Therefore, long-term and repeated 6-PPDQ exposure potentially resulted in inflammation and fibrosis in lung by affecting certain molecular signals in mammals. Our results suggested several aspects of lung injury caused by 6-PPDQ and provide the underlying molecular basis. These observations implied the possible risks of long-term 6-PPDQ exposure to human health.

Global analysis of iron metabolism-related genes identifies potential mechanisms of gliomagenesis and reveals novel targets.

AIMS: This study aimed to investigate key regulators of aberrant iron metabolism in gliomas, and evaluate their effect on biological functions and clinical translational relevance. METHODS: We used transcriptomic data from multiple cross-platform glioma cohorts to identify key iron metabolism-related genes (IMRGs) based on a series of bioinformatic and machine learning methods. The associations between IMRGs and prognosis, mesenchymal phenotype, and genomic alterations were analyzed in silico. The performance of the IMRGs-based signature in predicting temozolomide (TMZ) treatment sensitivity was evaluated. In vitro and in vivo experiments were used to explore the biological functions of these key IMRGs. RESULTS: HMOX1, LTF, and STEAP3 were identified as the most essential IMRGs in gliomas. The expression levels of these genes were strongly related to clinicopathological and molecular features. The robust IMRG-based gene signature could be used for prognosis prediction. These genes facilitate mesenchymal transformation, driver gene mutations, and oncogenic alterations in gliomas. The gene signature was also associated with TMZ resistance. HMOX1, LTF, and STEAP3 knockdown in glioma cells significantly reduced cell proliferation, colony formation, migration, and malignant invasion. CONCLUSION: The study presented a comprehensive view of key regulators underpinning iron metabolism in gliomas and provided new insights into novel therapeutic approaches.

SIRT3-dependent mitochondrial redox homeostasis mitigates CHK1 inhibition combined with gemcitabine treatment induced cardiotoxicity in hiPSC-CMs and mice.

Administration of CHK1-targeted anticancer therapies is associated with an increased cumulative risk of cardiac complications, which is further amplified when combined with gemcitabine. However, the underlying mechanisms remain elusive. In this study, we generated hiPSC-CMs and murine models to elucidate the mechanisms underlying CHK1 inhibition combined with gemcitabine-induced cardiotoxicity and identify potential targets for cardioprotection. Mice were intraperitoneally injected with 25 mg/kg CHK1 inhibitor AZD7762 and 20 mg/kg gemcitabine for 3 weeks. hiPSC-CMs and NMCMs were incubated with 0.5 uM AZD7762 and 0.1 uM gemcitabine for 24 h. Both pharmacological inhibition or genetic deletion of CHK1 and administration of gemcitabine induced mtROS overproduction and pyroptosis in cardiomyocytes by disrupting mitochondrial respiration, ultimately causing heart atrophy and cardiac dysfunction in mice. These toxic effects were further exacerbated with combination administration. Using mitochondria-targeting sequence-directed vectors to overexpress CHK1 in cardiomyocyte (CM) mitochondria, we identified the localization of CHK1 in CM mitochondria and its crucial role in maintaining mitochondrial redox homeostasis for the first time. Mitochondrial CHK1 function loss mediated the cardiotoxicity induced by AZD7762 and CHK1-knockout. Mechanistically, mitochondrial CHK1 directly phosphorylates SIRT3 and promotes its expression within mitochondria. On the contrary, both AZD7762 or CHK1-knockout and gemcitabine decreased mitochondrial SIRT3 abundance, thus resulting in respiration dysfunction. Further hiPSC-CMs and mice experiments demonstrated that SIRT3 overexpression maintained mitochondrial function while alleviating CM pyroptosis, and thereby improving mice cardiac function. In summary, our results suggest that targeting SIRT3 could represent a novel therapeutic approach for clinical prevention and treatment of cardiotoxicity induced by CHK1 inhibition and gemcitabine.

Early-life exposure to lead changes cardiac development and compromises long-term cardiac function.

Lead (Pb) is widely used in industrial and daily-use consumer products. Early-life exposure may increase the risk of lead-related heart problems in childhood. However, the effects of early-life lead exposure on fetal heart development and long-term cardiac outcomes are unknown. In this study, pregnant ICR mice were exposed to lead acetate trihydrate (50 mg/kg/d) via oral gavage from gestation day 1.5 until offspring weaning. Thereafter, the second hit model was established, two groups of offspring (4 weeks old) were either administered sterile saline or Angiotensin II (Ang II) for 4 weeks until euthanasia. We investigated lead-induced offspring heart damage from embryonic period to adulthood by echocardiographic analysis, pathological H&E staining, and ultrastructural examination, as well as mitochondrial function detection. The results showed early-life lead exposure predisposed offspring mice to decreased ejection fraction, increased left ventricular volume, accompanied by hypertrophy and dilation, cardiomyocyte sarcomere dysplasia, abnormal mitochondrial structure, mitochondrial dysfunction, and decreased expression of key sarcomeric and mitochondrial genes, rendering them more susceptible to cardiac hypertrophy, vascular wall thickening, cardiac fibrosis, apoptosis, and heart failure induced by Ang II infusion. This study elucidates early-life low dose lead exposure compromises cardiac development and exacerbates second hit-induced cardiac pathological responses in adulthood, which furnishes crucial scientific evidence pertaining to the cardiac toxicity and risk evaluation associated with early-life exposure to lead.

Mitochondrial GSNOR Alleviates Cardiac Dysfunction via ANT1 Denitrosylation.

BACKGROUND: The cardiac-protective role of GSNOR (S-nitrosoglutathione reductase) in the cytoplasm, as a denitrosylase enzyme of S-nitrosylation, has been reported in cardiac remodeling, but whether GSNOR is localized in other organelles and exerts novel effects remains unknown. We aimed to elucidate the effects of mitochondrial GSNOR, a novel subcellular localization of GSNOR, on cardiac remodeling and heart failure (HF). METHODS: GSNOR subcellular localization was observed by cellular fractionation assay, immunofluorescent staining, and colloidal gold particle staining. Overexpression of GSNOR in mitochondria was achieved by mitochondria-targeting sequence-directed adeno-associated virus 9. Cardiac-specific knockout of GSNOR mice was used to examine the role of GSNOR in HF. S-nitrosylation sites of ANT1 (adenine nucleotide translocase 1) were identified using biotin-switch and liquid chromatography-tandem mass spectrometry. RESULTS: GSNOR expression was suppressed in cardiac tissues of patients with HF. Consistently, cardiac-specific knockout mice showed aggravated pathological remodeling induced by transverse aortic constriction. We found that GSNOR is also localized in mitochondria. In the angiotensin II-induced hypertrophic cardiomyocytes, mitochondrial GSNOR levels significantly decreased along with mitochondrial functional impairment. Restoration of mitochondrial GSNOR levels in cardiac-specific knockout mice significantly improved mitochondrial function and cardiac performance in transverse aortic constriction-induced HF mice. Mechanistically, we identified ANT1 as a direct target of GSNOR. A decrease in mitochondrial GSNOR under HF leads to an elevation of S-nitrosylation ANT1 at cysteine 160 (C160). In accordance with these findings, overexpression of either mitochondrial GSNOR or ANT1 C160A, non-nitrosylated mutant, significantly improved mitochondrial function, maintained the mitochondrial membrane potential, and upregulated mitophagy. CONCLUSIONS: We identified a novel species of GSNOR localized in mitochondria and found mitochondrial GSNOR plays an essential role in maintaining mitochondrial homeostasis through ANT1 denitrosylation, which provides a potential novel therapeutic target for HF.

Cathepsin B S-nitrosylation promotes ADAR1-mediated editing of its own mRNA transcript via an ADD1/MATR3 regulatory axis.

Genetic information is generally transferred from RNA to protein according to the classic "Central Dogma". Here, we made a striking discovery that post-translational modification of a protein specifically regulates the editing of its own mRNA. We show that S-nitrosylation of cathepsin B (CTSB) exclusively alters the adenosine-to-inosine (A-to-I) editing of its own mRNA. Mechanistically, CTSB S-nitrosylation promotes the dephosphorylation and nuclear translocation of ADD1, leading to the recruitment of MATR3 and ADAR1 to CTSB mRNA. ADAR1-mediated A-to-I RNA editing enables the binding of HuR to CTSB mRNA, resulting in increased CTSB mRNA stability and subsequently higher steady-state levels of CTSB protein. Together, we uncovered a unique feedforward mechanism of protein expression regulation mediated by the ADD1/MATR3/ADAR1 regulatory axis. Our study demonstrates a novel reverse flow of information from the post-translational modification of a protein back to the post-transcriptional regulation of its own mRNA precursor. We coined this process as "Protein-directed EDiting of its Own mRNA by ADAR1 (PEDORA)" and suggest that this constitutes an additional layer of protein expression control. "PEDORA" could represent a currently hidden mechanism in eukaryotic gene expression regulation.

Endothelial HDAC1-ZEB2-NuRD Complex Drives Aortic Aneurysm and Dissection Through Regulation of Protein S-Sulfhydration.

BACKGROUND: Aortic aneurysm and aortic dissection (AAD) are life-threatening vascular diseases, with endothelium being the primary target for AAD treatment. Protein S-sulfhydration is a newly discovered posttranslational modification whose role in AAD has not yet been defined. This study aims to investigate whether protein S-sulfhydration in the endothelium regulates AAD and its underlying mechanism. METHODS: Protein S-sulfhydration in endothelial cells (ECs) during AAD was detected and hub genes regulating homeostasis of the endothelium were identified. Clinical data of patients with AAD and healthy controls were collected, and the level of the cystathionine γ lyase (CSE)/hydrogen sulfide (H2S) system in plasma and aortic tissue were determined. Mice with EC-specific CSE deletion or overexpression were generated, and the progression of AAD was determined. Unbiased proteomics and coimmunoprecipitation combined with mass spectrometry analysis were conducted to determine the upstream regulators of the CSE/H2S system and the findings were confirmed in transgenic mice. RESULTS: Higher plasma H2S levels were associated with a lower risk of AAD, after adjustment for common risk factors. CSE was reduced in the endothelium of AAD mouse and aorta of patients with AAD. Protein S-sulfhydration was reduced in the endothelium during AAD and protein disulfide isomerase (PDI) was the main target. S-sulfhydration of PDI at Cys343 and Cys400 enhanced PDI activity and mitigated endoplasmic reticulum stress. EC-specific CSE deletion was exacerbated, and EC-specific overexpression of CSE alleviated the progression of AAD through regulating the S-sulfhydration of PDI. ZEB2 (zinc finger E-box binding homeobox 2) recruited the HDAC1-NuRD complex (histone deacetylase 1-nucleosome remodeling and deacetylase) to repress the transcription of CTH, the gene encoding CSE, and inhibited PDI S-sulfhydration. EC-specific HDAC1 deletion increased PDI S-sulfhydration and alleviated AAD. Increasing PDI S-sulfhydration with the H2S donor GYY4137 or pharmacologically inhibiting HDAC1 activity with entinostat alleviated the progression of AAD. CONCLUSIONS: Decreased plasma H2S levels are associated with an increased risk of aortic dissection. The endothelial ZEB2-HDAC1-NuRD complex transcriptionally represses CTH, impairs PDI S-sulfhydration, and drives AAD. The regulation of this pathway effectively prevents AAD progression.

Interactions among maternal smoking, breastfeeding, and offspring genetic factors on the risk of adult-onset hypertension.

BACKGROUND: Previous studies have reported that maternal smoking during pregnancy and breastfeeding may affect the occurrence of hypertension, but whether early life factors modify the impact of the offspring's genetic risk on hypertension is still unknown. The aim of this study was to investigate the relationships among maternal smoking and breastfeeding with adult-onset hypertension and the modified impact of offspring genetic susceptibility. METHODS: This study included 437,185 participants from the UK Biobank who were initially free of hypertension and provided a prospective cohort of individuals aged 40 to 69 years. The association of maternal smoking during pregnancy and breastfeeding with hypertension was examined by using the Cox regression model. Then, a polygenic risk score (PRS) for hypertension was used to test the gene-environmental interaction on hypertension. RESULTS: During a median follow-up period of 8.7 years, a total of 68,148 cases of hypertension were identified in this study. The hazard ratios (HRs) and 95% confidence intervals (CIs) of hypertension for maternal smoking and breastfeeding were 1.11 (1.09, 1.13) and 0.96 (0.94, 0.98), respectively. However, no evidence of an interaction between maternal smoking and breastfeeding was observed. Across all levels of genetic risk, including high genetic risk, maternal smoking and nonbreastfeeding had higher hypertension hazards than nonmaternal smoking and breastfeeding, respectively. The adjusted HRs (95% CIs) of hypertension were 1.80 (1.73, 1.87) in those who had high genetic predisposition plus maternal smoking and 1.67 (1.60-1.74) in those with nonbreastfeeding and high genetic risk. There were significant additive interactions between maternal smoking or breastfeeding and genetic factors on the incidence of hypertension. CONCLUSIONS: Maternal smoking and nonbreastfeeding were associated with a higher risk of hypertension in adulthood and may attenuate the risk of hypertension related to genetic factors. These results suggested that adherence to nonmaternal smoking and breastfeeding was associated with a lower risk of hypertension among participants with all gradients of genetic risk.

Understanding gilteritinib resistance to FLT3-F691L mutation through an integrated computational strategy.

FMS-like tyrosine kinase 3 (FLT3) serves as an important drug target for acute myeloid leukemia (AML), and gene mutations of FLT3 have been closely associated with AML patients with an incidence rate of ~ 30%. However, the mechanism of the clinically relevant F691L gatekeeper mutation conferred resistance to the drug gilteritinib remained poorly understood. In this study, multiple microsecond molecular dynamics (MD) simulations, end-point free energy calculations, and dynamic correlated and network analyses were performed to investigate the molecular basis of gilteritinib resistance to the FLT3-F691L mutation. The simulations revealed that the resistant mutation largely induced the conformational changes of the activation loop (A-loop), the phosphate-binding loop, and the helix αC of the FLT3 protein. The binding abilities of the gilteritinib to the wild-type and the F691L mutant were different through the binding free energy prediction. The simulation results further indicated that the driving force to determine the binding affinity of gilteritinib was derived from the differences in the energy terms of electrostatic and van der Waals interactions. Moreover, the per-residue free energy decomposition suggested that the four residues (Phe803, Gly831, Leu832, and Ala833) located at the A-loop of FLT3 had a significant impact on the binding affinity of gilteritinib to the F691L mutant. This study may provide useful information for the design of novel FLT3 inhibitors specially targeting the F691L gatekeeper mutant.

Hsp90 S-nitrosylation at Cys521, as a conformational switch, modulates cycling of Hsp90-AHA1-CDC37 chaperone machine to aggravate atherosclerosis.

Endothelial dysfunction is the initial process of atherosclerosis. Heat shock protein 90 (Hsp90), as a molecular chaperone, plays a crucial role in various cardiovascular diseases. Hsp90 function is regulated by S-nitrosylation (SNO). However, the precise role of SNO-Hsp90 in endothelial dysfunction during atherosclerosis remains unclear. We here identified Hsp90 as a highly S-nitrosylated target in endothelial cells (ECs) by biotin switch assay combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The elevation of SNO-Hsp90 was observed in atherosclerotic human and rodent aortas as well as in oxidized LDL (oxLDL)-treated ECs. Inhibition of inducible nitric oxide synthase (iNOS) or transfection with Hsp90 cysteine 521 (Cys521) mutation plasmid decreased the level of SNO-Hsp90 in oxLDL-cultured ECs. Coimmunoprecipitation and proximity ligation assay demonstrated that SNO-Hsp90 at Cys521 suppressed the interaction between Hsp90 and activator of Hsp90 ATPase activity 1 (AHA1), but promoted the association of Hsp90 and cell division cycle 37 (CDC37). Hsp90 Cys521 mutation increased endothelial nitric oxide synthase (eNOS) activity and inhibited nuclear factor kappa-B (NF-κB) signaling, thereby increasing nitric oxide (NO) bioavailability and alleviating endothelial adhesion, inflammation and oxidative stress in oxLDL-treated ECs. Also, administration of endothelial-specific adeno-associated viruses of Cys521-mutated Hsp90 significantly mitigated vascular oxidative stress, macrophage infiltration and atherosclerosis lesion areas in high fat diet-fed ApoE-/- mice. In conclusion, SNO-Hsp90 at Cys521, that serves as a conformational switch, disrupts Hsp90/AHA1 interaction but promotes recruitment of CDC37 to exacerbate atherosclerosis.

Association between urinary cadmium concentrations and liver function in adolescents.

Evidence from previous studies has shown that exposure to cadmium (Cd) is associated with cardiovascular disease, kidney disease, and osteoporosis, but the effects of Cd on liver toxicity in adolescents are unclear. The data of 4411 adolescents who participated in the US The National Health and Nutrition Examination Survey (NHANES) during 1999-2016 was analyzed. Liver function was indicated by the levels of alanine aminotransferase (ALT) and aspartate amino transferase (AST). The associations between the levels of urinary Cd and liver function were evaluated using multivariate logistic regression models adjusted for covariates. The results showed that the odds ratios of ALT and AST in the highest quartiles of urinary Cd were 1.40 (95% confidence interval [CI], 1.07-1.82) and 1.64 (95% CI, 1.10-2.44), respectively, compared with the lowest quartiles, which were similar to using urinary creatinine as the covariate. We also found linear regression of associations of urinary Cd with elevated ALT and AST levels in boys. In addition, one augmented urinary Cd concentration unit (Log10) was associated with a 0.04-mg/dL increase in C-reactive protein and a 0.53-mg/dL decrease in HDL cholesterol in the fully adjusted model. Our results add novel evidence that exposure to Cd might be positively associated with indicators of liver injury, indicating the potential toxic effect of Cd exposure on the adolescent liver. Further confirmatory studies are needed.

A systematic comparison of the developmental vascular toxicity of bisphenol A and its alternatives in vivo and in vitro.

Due to the potential toxicity of bisphenol A (BPA), several bisphenols (BPs), including bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF), have been gradually used as its main substitutes, and the levels of these alternatives in different environmental media have been constantly increasing. Although some previous studies have shown that bisphenol substitutes have similar or greater acute toxicity and estrogenic effects than BPA, comparative studies on the cardiovascular toxicity of BPs have not been evaluated. In this study, the developmental vascular toxicity of BPA and three predominant substitutes (BPF, BPS and BPAF) were evaluated using zebrafish embryos and human vascular endothelial cells (HUVECs). BP exposure at a sublethal concentration of 1/10 96 h median lethal concentration (96 h-LC50) significantly hindered intersegmental vessel (ISV) growth, delayed common cardinal vein (CCV) remodeling and decreased subintestinal vessels (SIVs) in Tg (fli1:EGFP) zebrafish embryos. Meanwhile, the results of the endothelial tube formation assay showed that in vitro angiogenesis was inhibited by BP exposure. Mechanistically, BP exposure increased oxidative stress characterized by a significant decrease in superoxide dismutase (SOD) and catalase (CAT) activity, accompanied by increased levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in both zebrafish and HUVECs. Therefore, the vascular toxicity and oxidative stress potency of the BPs were compared and evaluated, ranking as follows: BPAF > BPF > BPA > BPS. To the best of our knowledge, the present work, for the first time, systematically provides direct evidence for BPA and its alternatives on developmental vascular toxicity in vitro and in vivo. Therefore, these findings will provide insight into the rational and safe application of BPA substitutes.

Maternal smoking during pregnancy is risk factor for gallbladder disease in offspring during adulthood: a prospective study from UK Biobank.

INTRODUCTION AND OBJECTIVES: Gallbladder disease is a common disease with high prevalence. Majority of gallbladder disease is due to gallstone. Though genetics are believed to play a role in its pathogenesis, the contribution of environmental pressures in early life to the development of this disease in adulthood has not been ever investigated. This study aimed to clarify the risk of maternal smoking exposure in association with gallbladder disease in adulthood. The interaction of maternal smoking and own smoking during adulthood on this association was studied as well. PATIENTS AND METHODS: A total of 286,731 eligible participants from the UK Biobank population-based cohort were included. Multivariable Cox regression analysis were used to examine the HR and 95% CI with adjustment for covariates. RESULT: During a median of 8.8 years follow-up, 7110 incident cases of gallbladder disease including 6800 (95.6%) gallstone were identified. Maternal smoking was associated with increased risk of incident total gallbladder disease (HR = 1.13; 95%CI: 1.06 - 1.21; P = 0.0002) as well as gallstones (HR = 1.13; 95%CI: 1.06 -1.21; P = 0.0003) in adulthood. Compared with those who were neither exposed to maternal smoking nor own smoking, subjects adherence to no smoking during adulthood but having maternal smoking exposure still had increased risk of total gallbladder disease (HR = 1.21; 95%CI: 1.1-1.34, P=0.0001) and gallstones (HR = 1.21; 95%CI: 1.1-1.35, P=0.0001). CONCLUSION: The present study using large prospective cohort data from UK Biobank, for the first time, demonstrated maternal smoking exposure bringing elevated risk of incident total gallbladder disease/gallstone in adulthood.

Perfluorooctane sulfonate interferes with non-genomic estrogen receptor signaling pathway, inhibits ERK1/2 activation and induces apoptosis in mouse spermatocyte-derived cells.

Perfluorooctane sulfonate (PFOS) is a widespread persistent organic pollutant. Both epidemiological survey and our previous in vivo study have revealed the associations between PFOS exposure and spermatogenesis disorder, while the underlying mechanisms are far from clear. In the present study, GC-2 cells, a mouse spermatocyte-derived cell line, was used to investigate the toxic effects of PFOS and its hypothetical mechanism of action. GC-2 cells were treated with PFOS (0, 50, 100 and 150 µM) for 24 h or 48 h. Results demonstrated that PFOS dose-dependently inhibited cell viability, induced G0/G1 cell cycle arrest and triggered apoptosis, which might be partly explained by the decrease in cyclin D1, PCNA and Bcl-2 protein expression; increase in Bax protein expression; and activation of caspase-9, -3. In addition, PFOS did not directly transactivate or repress estrogen receptors (ERs) in gene reporter assays, whereas the protein levels of both ERα and ERß were significantly altered and the downstream ERK1/2 phosphorylation was inhibited by PFOS. Furthermore, pretreatment with specific ERα agonist PPT (1 µM) significantly attenuated the above PFOS-induced effects while specific ERß agonist DPN (1 µM) accelerated them. These results suggest that PFOS may induce growth inhibition and apoptosis via non-genomic estrogen receptor/ERK1/2 signaling pathway in GC-2 cells, which provides a novel insight regarding the potential role of ERs in mediating PFOS-triggered spermatocyte toxicity.

HDL cholesterol: A potential mediator of the association between urinary cadmium concentration and cardiovascular disease risk.

Evidence from previous studies has shown that exposure to metals is associated with cardiovascular disease (CVD). However, the association between metal mixtures and CVD risk and the potential mechanisms in epidemiologic studies remain unclear. The data of 14,795 adults who participated in the U.S. National Health and Nutrition Examination Survey (NHANES) 1999-2016 were analyzed. Multivariate logistic regression was performed to investigate the associations between urinary metal levels and CVDs. Weighted quantile sum (WQS) regression was performed to examine the effects of mixed metals on CVDs. Multivariate linear regression and mediation analysis were conducted to explore the associations between metals and blood lipids. Urinary cadmium (Cd) was significantly associated with an increased total CVD risk and with individual CVD risk. The odds ratio (OR) for CVD in the highest quartile of the WQS index was 1.43 (95% confidence interval [CI]: 1.19, 1.71). One augmented urinary Cd concentration unit (Log10) was associated with a 0.93 mg/dL decrease in HDL cholesterol, a 1.34 mg/dL increase in LDL cholesterol and a 1.30 mg/dL increase in total cholesterol in the fully adjusted model. Mediation analysis showed that HDL cholesterol mediated 4.91% of the association between urinary Cd and the prevalence of CVD. Our findings suggest that urinary Cd and metal mixtures were significantly and positively associated with CVD. The downregulation of HDL cholesterol might play a significant role in mediating Cd exposure-associated CVD risk increases.

Serum nickel is associated with craniosynostosis risk: Evidence from humans and mice.

BACKGROUND: To date, few studies have explored the effects of exposure to metal mixtures on adverse developmental outcomes, and no reported studies have linked metal exposure to craniosynostosis (CS). The purpose of this study is to investigate the association between metal exposure and the risk of CS by conducting epidemiological and experimental studies. METHODS: Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the concentrations of 6 metals (chromium [Cr], nickel [Ni], tin [Sn], arsenic [As], thallium [Tl], and lead [Pb]) in serum samples from 174 CS patients and 85 control individuals. Non-syndromic patients with isolated sagittal suture closure were selected as the case group, and healthy children matched by sex and age were selected as controls. Bayesian kernel machine regression (BKMR) models were used to account for joint metal effects. Multiple logistic regression analysis was used to explore the association between metal concentration and CS occurrence, with adjustment for potential confounders. During pregnancy, mice were exposed to Ni (0, 0.05, or 0.1 g/kg/day) until weaning, and the widths of the sutures and shapes of the skull were analysed by micro-CT 3D imaging and histological analysis. MC3T3-E1 cells were treated with Ni (0, 0.005, or 0.05 µg/mL) for 72 h. Alkaline phosphate (ALP) staining and Alizarin red staining were performed to observe the development of osteoblasts. The expression levels of osteoblast-related genes were also detected. RESULTS: A positive association between the metal mixture and CS risk was observed based on population data; the Ni group had the highest conditional posterior inclusion probability (PIP), at 0.8416, and in the fully adjusted model, the highest Ni exposure level had a more significant association with CS (coefficient = 2.65, 95% CI: 0.29, 5.02) than the lowest Ni exposure level. The mean widths of the sagittal sutures in mice were 8.8 ± 0.6 mm in the control group, 8.0 ± 0.8 mm in the 0.05 g/kg/day group and 6.8 ± 0.4 mm in the 0.1 g/kg/day group. After Ni exposure, ALP gene expression in skull tissue was increased, and ALP activity was increased in MC3T3-E1 cells. Moreover, increased collagen content in mouse skull sections and elevated osteocalcin (OCN) expression in MC3T3-E1 cells were observed in the Ni-treated groups compared to the control group. CONCLUSIONS: This study is the first to provide evidence that increased serum Ni was associated with an increased risk of CS. Early life exposure to Ni promoted osteogenesis during skull growth, which may contribute to the development of CS.

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to ß-hexachlorocyclohexane (ß-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with ß-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

In vitro evaluation of the hepatic lipid accumulation of bisphenol analogs: A high-content screening assay.

Bisphenol A (BPA) has a variety of adverse effects on human health; therefore, BPA analogs are increasingly used as replacements. Notably, recent studies have revealed that BPA exposure induced hepatic lipid accumulation, but few studies are available regarding the similar effects of other bisphenol analogues (BPs). Thus, in the present study, a high-content screening (HCS) assay was performed to simultaneously evaluate the hepatic lipid accumulation of 13 BPs in vitro. The BPs induced lipid deposition in HepG2 cells ranking as below: 4,4'-thiodiphenol (TDP) < bisphenol S (BPS) < 4,4'-dihydroxybenzophenone (DHBP) < tetrabromobisphenol A (TBBPA) < tetrachlorobisphenol A (TCBPA) < bisphenol E (BPE) < bisphenol F (BPF) < bisphenol B (BPB) < bisphenol AF (BPAF) < bisphenol A (BPA) < bisphenol C (BPC) < tetramethylbisphenol A (TMBPA) < bisphenol AP (BPAP). Meanwhile, Oil Red O staining and triacylglycerol detection further validated the lipid accumulation elicited by the latter 8 BPs, which exhibited the more significant effects on lipid deposition. Mechanistically, significantly increased expressions of genes involved in fatty acid synthesis and nuclear receptors and decreased levels of genes associated with fatty acid ß-oxidation were observed under BPs treatment. Therefore, the present work is the first to systematically provide direct evidence for BPs-induced hepatic lipid accumulation in vitro via HCS, which can be helpful for safety assessments of BPs.