Correlations between bone metabolism biomarkers and fluoride exposure in adults and children.

Increased exposure to fluoride, which notably affects bone metabolism, is a global concern. However, the correlations and sensitivity of bone metabolism to fluoride remain controversial. In this cross-sectional study, 549 children (aged 7-12 years) and 504 adults (≥ 18 years old) were recruited in the high-fluoride areas of the Henan Province. Urinary fluoride (UF) level was determined using a fluoride electrode. Fasting venous blood serum was collected to measure bone metabolism biomarkers. The selected bone metabolism biomarkers for children included bone alkaline phosphatase (BALP), serum alkaline phosphatase (ALP), osteocalcin (OCN), calcitonin (CT), parathyroid hormone (PTH), phosphorus (P5+), and calcium (Ca2+). For adults, the biomarkers included ALP, CT, PTH, ß-CrossLaps (ß-CTX), P5+, and Ca2+. The correlations between UF and bone metabolism biomarkers were analyzed using binary logistic regression, a trend test, a generalized additive model, and threshold effect analysis. Regression analysis indicated a significant correlation between serum OCN, PTH, and UF levels in children aged 7-9 years. Serum OCN, PTH, and BALP contents were significantly correlated with UF in boys (P < 0.05). Furthermore, the interaction between age and UF affected serum P5+ and PTH (P < 0.05). The generalized additive model revealed nonlinear dose-response relationships between P5+, BALP, and UF contents in children (P < 0.05). Serum OCN level was linearly correlated with the UF concentration (P < 0.05). Similarly, a significant correlation was observed between ß-CTX and UF levels in adults. In addition, significant correlations were observed between UF-age and serum Ca2+, ß-CTX, and PTH contents. There was a non-linear correlation between serum Ca2+, P5+, and ß- CTX and UF levels (P < 0.05). Overall, serum OCN, BALP, and P5+ levels can serve as sensitive bone metabolism biomarkers in children, while ß-CTX, P5+, and Ca2+ can be considered fluoride-sensitive bone metabolism biomarkers in adults.

Proteomics Sequencing Reveals the Role of TGF-ß Signaling Pathway in the Peripheral Blood of Offspring Rats Exposed to Fluoride.

The underlying mechanism of fluorosis has not been fully elucidated. The purpose of this study was to explore the mechanism of fluorosis induced by sodium fluoride (NaF) using proteomics. Six offspring rats exposed to fluoride without dental fluorosis were defined as group A, 8 offspring rats without fluoride exposure were defined as control group B, and 6 offspring rats exposed to fluoride with dental fluorosis were defined as group C. Total proteins from the peripheral blood were extracted and then separated using liquid chromatography-tandem mass spectrometry. The identified criteria for differentially expressed proteins were fold change > 1.2 or < 0.83 and P < 0.05. Gene Ontology function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the oeCloud tool. The 177 upregulated and 22 downregulated proteins were identified in the A + C vs. B group. KEGG pathway enrichment analysis revealed that transforming growth factor-ß (TGF-ß) signaling pathway significantly enriched. PPI network constructed using Cytoscape confirmed RhoA may play a crucial role. The KEGG results of genes associated with fluoride and genes associated with both fluoride and inflammation in the GeneCards database also showed that TGF-ß signaling pathway was significantly enriched. The immunofluorescence in HPA database showed that the main expression sites of RhoA are plasma membrane and cytosol, while the main expression site of Fbn1 is the Golgi apparatus. In conclusion, long-term NaF intake may cause inflammatory response in the peripheral blood of rats by upregulating TGF-ß signaling pathway, in which RhoA may play a key role.

T-2 toxin induces mitochondrial dysfunction in chondrocytes via the p53-cyclophilin D pathway.

Kashin-Beck disease is an endemic joint disease characterized by deep chondrocyte necrosis, and T-2 toxin exposure has been confirmed its etiology. This study investigated mechanism of T-2 toxin inducing mitochondrial dysfunction of chondrocytes through p53-cyclophilin D (CypD) pathway. The p53 signaling pathway was significantly enriched in T-2 toxin response genes from GeneCards. We demonstrated the upregulation of the p53 protein and p53-CypD complex in rat articular cartilage and ATDC5 cells induced by T-2 toxin. Transmission electron microscopy showed the damaged mitochondrial structure of ATDC5 cells induced by T-2 toxin. Furthermore, it can lead to overopening of the mitochondrial permeability transition pore (mPTP), decreased mitochondrial membrane potential, and increased reactive oxygen species generation in ATDC5 cells. Pifithrin-α, the p53 inhibitor, alleviated the increased p53-CypD complex and mitochondrial dysfunction of chondrocytes induced by T-2 toxin, suggesting that p53 played an important role in T-2 toxin-induced mitochondrial dysfunction. Mechanistically, T-2 toxin can activate the p53 protein, which can be transferred to the mitochondrial membrane and form a complex with CypD. The increased binding of p53 and CypD mediated the excessive opening of mPTP, changed mitochondrial membrane permeability, and ultimately induced mitochondrial dysfunction and apoptosis of chondrocytes.

Expression Profiles of Long Non-Coding RNAs in the Articular Cartilage of Rats Exposed to T-2 Toxin.

T-2 toxin could induce bone damage. But there is no specific mechanism about the long non-coding RNAs (lncRNAs) involved in T-2 toxin-induced articular cartilage injury. In this study, 24 SD rats were randomly divided into a control group and a T-2 group, which were administered 4% absolute ethanol and 100 ng/g · bw/day of T-2 toxin, respectively. After treatment for 4 weeks, safranin O/fast green staining identified the pathological changes in the articular cartilage of rats, and immunofluorescence verified the autophagy level increase in the T-2 group. Total RNA was isolated, and high-throughput sequencing was performed. A total of 620 differentially expressed lncRNAs (DE-lncRNAs) were identified, and 326 target genes were predicted. Enrichment analyses showed that the target genes of DE-lncRNAs were enriched in the autophagy-related biological processes and pathways. According to the autophagy database, a total of 23 autophagy-related genes were identified, and five hub genes (Foxo3, Foxo1, Stk11, Hdac4, and Rela) were screened using the Maximal Clique Centrality algorithm. The Human Protein Atlas database indicated that Rela and Hdac4 proteins were highly expressed in the bone marrow tissue, while Foxo3, Foxo1, and Stk11 proteins were reduced. According to Enrichr, etoposide and diatrizoic acid were identified as the key drugs. The real-time quantitative PCR results were consistent with the RNA sequencing (RNA-Seq) results. These results suggested that autophagy was involved in the rat articular cartilage lesions induced by T-2 toxin. The lncRNAs of NONRATG014223.2, NONRATG012484.2, NONRATG021591.2, NONRATG024691.2, and NONRATG002808.2, and their target genes of Foxo3, Foxo1, Stk11, Hdac4, and Rela, respectively, were the key regulator factors of autophagy.

Selenomethionine Antagonized microRNAs Involved in Apoptosis of Rat Articular Cartilage Induced by T-2 Toxin.

T-2 toxin and selenium deficiency are considered important etiologies of Kashin-Beck disease (KBD), although the exact mechanism is still unclear. To identify differentially expressed microRNAs (DE-miRNAs) in the articular cartilage of rats exposed to T-2 toxin and selenomethionine (SeMet) supplementation, thirty-six 4-week-old Sprague Dawley rats were divided into a control group (gavaged with 4% anhydrous ethanol), a T-2 group (gavaged with 100 ng/g·bw/day T-2 toxin), and a T-2 + SeMet group (gavaged with 100 ng/g·bw/day T-2 toxin and 0.5 mg/kg·bw/day SeMet), respectively. Toluidine blue staining was performed to detect the pathological changes of articular cartilage. Three rats per group were randomly selected for high-throughput sequencing of articular cartilage. Target genes of DE-miRNAs were predicted using miRanda and RNAhybrid databases, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway were enriched. The network map of miRNA-target genes was constructed using Cytoscape software. The expression profiles of miRNAs associated with KBD were obtained from the Gene Expression Omnibus database. Additionally, the DE-miRNAs were selected for real-time quantitative PCR (RT-qPCR) verification. Toluidine blue staining demonstrated that T-2 toxin damaged articular cartilage and SeMet effectively alleviated articular cartilage lesions. A total of 50 DE-miRNAs (28 upregulated and 22 downregulated) in the T-2 group vs. the control group, 18 DE-miRNAs (6 upregulated and 12 downregulated) in the T-2 + SeMet group vs. the control group, and 25 DE-miRNAs (5 upregulated and 20 downregulated) in the T-2 + SeMet group vs. the T-2 group were identified. Enrichment analysis showed the target genes of DE-miRNAs were associated with apoptosis, and in the MAPK and TGF-ß signaling pathways in the T-2 group vs. the control group. However, the pathway of apoptosis was not significant in the T-2 + SeMet group vs. the control group. These results indicated that T-2 toxin induced apoptosis, whereas SeMet supplementation antagonized apoptosis. Apoptosis and autophagy occurred simultaneously in the T-2 + SeMet group vs. T-2 group, and autophagy may inhibit apoptosis to protect cartilage. Compared with the GSE186593 dataset, the evidence of miR-133a-3p involved in apoptosis was more abundant. The results of RT-qPCR validation were consistent with RNA sequencing results. Our findings suggested that apoptosis was involved in articular cartilage lesions induced by T-2 toxin, whereas SeMet supplementation antagonized apoptosis, and that miR-133a-3p most probably played a central role in the apoptosis process.

Role of Glycolysis/Gluconeogenesis and HIF-1 Signaling Pathways in Rats with Dental Fluorosis Integrated Proteomics and Metabolomics Analysis.

Fluoride is widely distributed, and excessive intake will lead to dental fluorosis. In this study, six offspring rats administrated 100 mg/L sodium fluoride were defined as the dental fluorosis group, and eight offspring rats who received pure water were defined as the control group. Differentially expressed proteins and metabolites extracted from peripheral blood were identified using the liquid chromatography tandem mass spectrometry and gas chromatography mass spectrometry, with the judgment criteria of fold change >1.2 or <0.83 and p < 0.05. A coexpression enrichment analysis using OmicsBean was conducted on the identified proteins and metabolites, and a false discovery rate (FDR) < 0.05 was considered significant. Human Protein Atlas was used to determine the subcellular distribution of hub proteins. The Gene Cards was used to verify results. A total of 123 up-regulated and 46 down-regulated proteins, and 12 up-regulated and 2 down-regulated metabolites were identified. The significant coexpression pathways were the HIF-1 (FDR = 1.86 × 10−3) and glycolysis/gluconeogenesis (FDR = 1.14 × 10−10). The results of validation analysis showed the proteins related to fluorine were mainly enriched in the cytoplasm and extrinsic component of the cytoplasmic side of the plasma membrane. The HIF-1 pathway (FDR = 1.01 × 10−7) was also identified. Therefore, the HIF-1 and glycolysis/gluconeogenesis pathways were significantly correlated with dental fluorosis.

Association of AGTR1 gene methylation and its genetic variant in Chinese farmer with hypertension: A case-control study.

The objective was to determine the potential associations of the angiotensin II receptor type 1 (AGTR1) gene polymorphism, methylation, and lipid metabolism in Chinese farmers with hypertension. A case-control study was conducted in Wuzhi county of Henan province in China in 2013 to 2014. A total of 1034 local residents (35-74 years, 386 hypertensive cases, and 648 normotensive subjects) were enrolled in this study. Triglyceride (TG), total cholesterol (TC), high-density lipoprotein, and low-density lipoprotein were measured using automatic chemistry analyzer. The AGTR1 gene promoter methylation level was measured using quantitative methylation-specific polymerase chain reaction method. The single nucleotide polymorphism rs275653 was genotyped with TaqMan probe assay at an applied biosystems platform. The gender, body mass index (BMI), TG, TC, and family history of hypertension in the hypertension group were significantly higher than those in control group (P < .05). No significant difference was observed in the distribution of AGTR1 rs275653 polymorphism in the hypertension and controls (P > .05). The AGTR1 gene methylation in subjects carrying different genotypes was not significantly observed (P > .05). The logistic regression analysis found the AGTR1 gene methylation level was negative correlation with hypertension in the present study (odds ratio, 0.946, 95% confidence interval, 0.896-0.999) through adjusting for age, gender, BMI, education, smoking, alcohol drinking, fruit and vegetable intake, pickles intake, and family history of hypertension. The association of AGTR1 gene hypomethylation and essential hypertension was observed in Chinese farmers; no significant difference was observed in the distribution of AGTR1 rs275653 polymorphism.

Animal models of Kashin-Beck disease exposed to environmental risk factors: Methods and comparisons.

The main etiological mechanism for Kashin-Beck disease (KBD) is deep chondrocyte necrosis induced by environmental risk factors (ERFs). The scholars have conducted several epidemiological, cellular, and animal model studies on ERFs. Gradually, four etiological hypotheses have been formed, including water of organic poisoning hypothesis represented by fulvic acid (FA), biogeochemical hypothesis represented by selenium (Se) deficiency, food mycotoxin poisoning hypothesis represented by T-2 toxin poisoning and compound etiology theory hypothesis. The animal models of KBD have been replicated based on the previous etiological hypotheses. The different species of animals (monkey, rat, dog, pig, chicken, and rabbit) were treated with different ERFs interventions, and the clinical manifestations and pathological changes of articular cartilages were observed. The animals in the experimental group were fed with endemic water, endemic grain, low nutrition, thallium sulfate, FA, Se, T-2 toxin, and iodine. The dose of thallium sulfate was 1154 µg/d; the doses range of FA were 5, 50, 150, 200, and 211 mg/kg; the doses range of Se were 0.00035, 0.00175, 0.005, 0.02, 0.031, 0.1, 0.15, 0.314, 0.5, and 10 mg/kg; the doses range of T-2 toxin were 40, 100, 200, 600, 1000, 1500, 3000, 6000, and 9000 ng/g; and the doses range of iodine were 0.04, 0.18, and 0.4-0.5 µg/g. The sample size ranged from 9 to 230 depending on the interventions and grouping; the follow-up duration ranged from 1 week to 18 months. Moreover, the methods and comparisons of different animal models of KBD had been summarized to provide a useful basis for studying the pathogenesis of KBD. In conclusion, the rhesus monkeys administrated endemic water and grain were susceptible animals to replicate KBD. The rats treated with T-2 toxin combined with Se/nutrition deficiency could be a suitable and widely used animal model.

Role of the hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure.

To identify the role of the Hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure. Environmental response genes (ERGs) of bone injury induced by fluoride exposure were obtained from the Comparative Toxicogenomics Database, and annotated by STRING for KEGG pathway enrichment analysis. The CCK-8 kit was used to measure the proliferation of ATDC5 cells. The malondialdehyde (MDA), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-PX) levels in ATDC5 cells were measured using oxidative stress detection kit. Western blot analysis was used to measure the p-MST1/2, p-LATS1/2, and p-YAP/YAP1 expression levels in the Hippo pathway and the COL2A1, ACAN and MMP13 expression levels in the cartilage matrix. Localizations of YAP1 and COL2A1 proteins in chondrocytes were performed using cell immunofluorescence. Continuous data from the multiple groups were compared using one-way analysis of variance, and then the differences between groups were tested with Dunnett's t-test, with the test level α = 0.05. The 145 ERGs of bone injury induced by fluoride exposure were identified, and KEGG enrichment analysis revealed Hippo signaling pathways significantly related to bone injury. A CCK-8 assay revealed that the viability of the ATDC5 cells was significantly decreased with increased fluorine concentration. The MDA content in 20 mg/L sodium fluoride (NaF) exposure group was significantly higher than that in the control group, the T-SOD, T-AOC and GSH-PX activities in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group (P < 0.05). Western blot results showed the protein levels of p-MST1/2, p-LATS1/2 and p-YAP1 in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group, while the YAP1 protein level in 20 mg/L NaF group was significantly higher than that in the control group. The COL2A1 and ACAN proteins in 20 mg/L NaF group were significantly decreased, while the MMP13 protein level in 15 and 20 mg/L NaF groups were significantly increased (P < 0.05). It was observed that the expression of YAP1 protein expression level in the cytoplasm decreased with the increased fluoride exposure, whereas that the expression level of YAP1 protein in the nucleus increased. Fluoride inhibited the proliferation of ATDC5 cells, induced oxidative stress, inhibited the activity of the Hippo pathway, and eventually led to cartilage matrix degradation.

Statistical analysis of Chinese phonemic contrast.

Two phonemes that may induce minimal pairs constitute a phonemic contrast. Some phonemic contrasts may disappear for various reasons, which, nevertheless, does not seem to seriously impede linguistic communication. Does it mean that the disappeared phonemic contrasts are unimportant? In our study, we calculated the proportions (here termed degree of contrast) of minimal pairs to the words in which the two contrastive phonemes occur and explored the role of phonemic contrasts in the phonemic combinations. The degree of contrast of phonemes reflects the relation between phonemes. Our results indicate that (1) the average degree of contrast of Chinese phonemes declines exponentially with the increase in the number of syllables, rapidly approaching zero; (2) the average degree of contrast of Chinese consonants that differ from each other in only one distinctive feature and of the consonants that are absent in some Chinese dialects is significantly higher than that of other consonants; (3) the degree of contrast of Chinese consonants that differ from each other in only one distinctive feature is not significantly different from that of the consonants absent in some Chinese dialects; (4) Chinese phonemic combinations exhibit high degree of sparsity, which increases exponentially with the number of syllables and rapidly approaches 1. All these results show that the high degree of sparsity and the low degree of contrast of human languages not only leave enough room for new words, new dialects and new languages to appear but also contribute to effective and reliable communication, because a few phonemic mistakes are not likely to cause wrong decoding (sound recognition) and failed communication.