Development Of Skeletal Fluorosis Research Articles

Screening of differentially methylated genes in skeletal fluorosis of rats with different types and involvement of aberrant methylation of Cthrc1

Fluoride is a widespread pollutant in the environment. There is a high risk of developing skeletal fluorosis from excessive fluoride exposure. Skeletal fluorosis has different phenotypes (including osteosclerotic, osteoporotic and osteomalacic) under the same fluoride exposure and depends on dietary nutrition. However, the existing mechanistic hypothesis of skeletal fluorosis cannot well explain the condition's different pathological manifestations and their logical relation with nutritional factors. Recent studies have shown that DNA methylation is involved in the occurrence and development of skeletal fluorosis. DNA methylation is dynamic throughout life and may be affected by nutrition and environmental factors. We speculated that fluoride exposure leads to the abnormal methylation of genes related to bone homeostasis under different nutritional statuses, resulting in different skeletal fluorosis phenotypes. The mRNA-Seq and target bisulfite sequencing (TBS) result showed differentially methylated genes in rats with different skeletal fluorosis types. The role of the differentially methylated gene Cthrc1 in the formation of different skeletal fluorosis types was explored in vivo and in vitro. Under normal nutritional conditions, fluoride exposure led to hypomethylation and high expression of Cthrc1 in osteoblasts through TET2 demethylase, which promoted osteoblast differentiation by activating Wnt3a/β-catenin signalling pathway, and participated in the occurrence of osteosclerotic skeletal fluorosis. Meanwhile, the high CTHRC1 protein expression also inhibited osteoclast differentiation. Under poor dietary conditions, fluoride exposure led to hypermethylation and low expression of Cthrc1 in osteoblasts through DNMT1 methyltransferase, and increased the RANKL/OPG ratio, which promoted the osteoclast differentiation and participated in the occurrence of osteoporotic/osteomalacic skeletal fluorosis. Our study expands the understanding of the role of DNA methylation in regulating the formation of different skeletal fluorosis types and provides insights into new prevention and treatment strategies for patients with skeletal fluorosis.

Effects of fluoride on the proliferation and activation of osteoblasts by regulating methylation of the DNA repair genes MGMT and MLH1

IntroductionFluoride can induce the proliferation and activation of osteoblasts, resulting in skeletal fluorosis progression; however, the specific mechanism is unclear.MethodsCell proliferation was examined using the MTT assay. Flow cytometry was performed to detect the cell cycle distribution. Alkaline phosphatase (ALP) was calculated to evaluate bone formation and turnover. Gene methylation was examined using the MSP assay. mRNA and protein expression levels were assessed using qRT-PCR and Western blot assays.ResultsLow-concentration NaF treatment promoted the cell cycle progression of osteoblasts to S-phase, thus accelerating cell proliferation and activation in a concentration-dependent manner. In addition, the methylation of the MGMT and MLH1 genes was increased, and their mRNA expression was reduced. Furthermore, the DNA methyltransferase inhibitor 5-AZA-dC suppressed cell viability, cell number in S-phase, ALP activity and osteogenesis-related protein levels in osteoblasts treated with low doses of NaF. Meanwhile, 5-AZA-dC suppressed the increase in MGMT and MLH1 gene methylation in osteoblasts treated with low doses of NaF, leading to enhanced expression of MGMT and MLH1 mRNA.ConclusionNaF treatment led to methylation of the DNA repair genes MGMT and MLH1 in osteoblasts, resulting in cell proliferation and activation and causing the development of skeletal fluorosis.

MiR-122-5p Mediates Fluoride-Induced Osteoblast Activation by Targeting CDK4.

Chronic intake of fluoride, existing in the environment, may cause endemic fluorosis, which is characterized by the occurrence of skeletal and dental fluorosis. However, the pathogenesis of fluorosis has not yet been elucidated. Abnormal osteoblast proliferation and activation have a pivotal role in bone turnover disorders which are linked to skeletal fluorosis. MicroRNAs are involved in fundamental cellular processes, including cell proliferation. Based on our previous study, population study and in vitro experiments were designed to understand the effect of miR-122-5p on osteoblast activation in skeletal fluorosis through targeting cyclin-dependent kinase 4 (CDK4). In human populations withcoal-burning type fluoride exposure, the results showed that miR-122-5p was downregulated but CDK4 expression was upregulated and miR-122-5p was negatively correlated with CDK4 expression. Furthermore, in human osteoblasts treated with sodium fluoride, we demonstrated that miR-122-5p mediated osteoblast activation of skeletal fluorosis via upregulation of the CDK4 protein. In support of this, dual-luciferase reporter assay showed that miR-122-5p modulated CDK4 protein levels by targeting its 3'-untranslated region. These findings show, for the first time, that miR-122-5p may be involved in the cause and development of skeletal fluorosis by targeting CDK4.

Aberrant DNA methylation of Cyclind-CDK4-p21 is associated with chronic fluoride poisoning

Endemic fluorosis is a serious problem in public health, affecting thousands of people. Abnormal proliferation and activation of osteoblasts in skeletal fluorosis lesions play a leading role and osteoblast proliferation is finely regulated by the cell cycle. There are a few reports on fluoride-induced DNA methylation. However, the role of DNA methylation of the cyclin/cyclin-dependent kinase (CDK)/cyclin-dependent kinase inhibitor (CKI) regulatory network in skeletal fluorosis has not been investigated. We used a population study and in vitro experiment to explore the relationship between the pathogenesis of skeletal fluorosis and methylation of Cyclin d1/CDK4/p21. The results showed a positive relationship between fluoride exposure and expression of Cyclin d1/CDK4, and a negative relationship between fluoride exposure and expression of P21. Hypermethylation of p21 was found in the fluoride-exposed population, and low expression of p21 attributed to promoter hypermethylation was confirmed in vitro. However, no changes in methylation levels of Cyclin d1 and CDK4 genes were observed in the population exposed to fluoride and NaF-treated osteoblasts. These results show that methylation of p21 gene has a significant impact on the proliferation of osteoblasts during the development of skeletal fluorosis. The present study was a first attempt to link the methylation of the Cyclin d1/CDK4/p21 regulatory network with osteoblast proliferation in skeletal fluorosis.

Role of fluoride induced epigenetic alterations in the development of skeletal fluorosis

Fluoride is an essential trace element required for proper bone and tooth development. Systemic high exposure to fluoride through environmental exposure (drinking water and food) may result in toxicity causing a disorder called fluorosis. In the present study, we investigated the alteration in DNA methylation profile with chronic exposure (30 days) to fluoride (8 mg/l) and its relevance in the development of fluorosis. Whole genome bisulfite sequencing (WGBS) was carried out in human osteosarcoma cells (HOS) exposed to fluoride. Whole genome bisulfite sequencing (WGBS) and functional annotation of differentially methylated genes indicate alterations in methylation status of genes involved in biological processes associated with bone development pathways. Combined analysis of promoter DNA hyper methylation, STRING: functional protein association networks and gene expression analysis revealed epigenetic alterations in BMP1, METAP2, MMP11 and BACH1 genes, which plays a role in the extracellular matrix disassembly, collagen catabolic/organization process, skeletal morphogenesis/development, ossification and osteoblast development. The present study shows that fluoride causes promoter DNA hypermethylation in BMP1, METAP2, MMP11 and BACH1 genes with subsequent down-regulation in their expression level (RNA level). The results implies that fluoride induced DNA hypermethylation of these genes may hamper extracellular matrix deposition, cartilage formation, angiogenesis, vascular system development and porosity of bone, thus promote skeletal fluorosis.

Aberrant methylation-induced dysfunction of p16 is associated with osteoblast activation caused by fluoride.

Chronic exposure to fluoride continues to be a public health problem worldwide, affecting thousands of people. Fluoride can cause abnormal proliferation and activation of osteoblast and osteoclast, leading to skeletal fluorosis that can cause pain and harm to joints and bones and even lead to permanent disability. Nevertheless, there is no recognized mechanism to explain the bone lesions of fluorosis. In this work, we performed a population study and in vitro experiments to investigate the pathogenic mechanism of skeletal fluorosis in relation to methylation of the promoter of p16. The protein coded by the p16 gene inhibits cdk (cyclin-dependent kinase) 4/cdk6-mediated phosphorylation4 of retinoblastoma gene product and induces cell cycle arrest. The results showed that hypermethylation of p16 and reduced gene expression was evident in peripheral blood mononuclear cells of patients with fluorosis and correlated with the level of fluoride exposure. Studies with cell cultures of osteoblasts revealed in response to sodium fluoride (NaF) treatment, there was an induction of p16 hypermethylation and decreased expression, leading to increased cell proliferation, a longer S-phase of the cell cycle, and development of skeletal fluorosis. Further, the methylation inhibitor, 5-aza-2-deoxycytidine, reversed the p16 hypermethylation and expression in response to NaF. These results reveal a regulatory role of p16 gene methylation on osteoblasts activation during the development of skeletal fluorosis.

Role of fluoride induced histone trimethylation in development of skeletal fluorosis

Chronic exposure to fluoride has been associated with the development of skeletal fluorosis. Limited reports are available on fluoride induced histone modification. However, the role of histone modification in the pathogenesis of skeletal fluorosis is not investigated. In the present study, we have investigated the role of fluoride induced histone modification on fluorosis development using human osteosarcoma (HOS) cell line. The expression of histone methyltransferases (EHMT1 and EHZ2) and level of global histone trimethylation (H3K9 and H3K27) have been assessed and observed to be increased significantly after fluoride exposure (8 mg/L). EpiTect chromatin immunoprecipitation (CHIP) qPCR Array (Human TGFβ/BMP signaling pathway) was performed to assess the H3K9 trimethylation at promoter regions of pathway-specific genes. H3K9 ChIP PCR array analysis identified hyper H3K9 trimethylation in promoter regions of TGFBR2 and SMAD3. qPCR and STRING analysis was carried out to determine the repressive epigenetic effect of H3K9 trimethylation on expression pattern and functional association of identified genes. Identified genes (TGFBR2 and SMAD3) showed down-regulation which confirms the repressive epigenetic effect of promoter H3K9 hyper trimethylation. Expression of two other vital genes COL1A1 and MMP13 involved in TGFBR2-SMAD signaling pathway was also found to be down-regulated with a decrease in expression of TGFBR2 and SMAD3. STRING analysis revealed functional association and involvement of identified genes TGFBR2, SMAD3, COL1A1 and MMP13 in the collagen and cartilage development/morphogenesis, connective tissue formation, bio-mineral tissue development, endochondral bone formation, bone and skeletal morphogenesis. In conclusion, present investigation is a first attempt to link fluoride induced hyper H3K9 tri-methylation mediated repression of TGFBR2 and SMAD3 with the development of skeletal fluorosis.

Chronic Alcohol Consumption and the Development of Skeletal Fluorosis in a Fluoride Endemic Area of the Ethiopian Rift Valley

This study compared the occurrence of skeletal fluorosis in chronic consumers of locally brewed alcoholic beverages and their matched controls in the Ethiopian Rift Valley. The study revealed that chronic alcohol consumers developed severe forms of crippling skeletal fluorosis quite early in life. The controls were either symptom-free or exhibited mild forms of the fluorosis. The study showed that crippling skeletal fluorosis was directly associated with the large volumes of the locally brewed beer and honey-mead consumption on a daily basis. Chemical analysis of the alcoholic beverages showed that high concentration of fluoride which was much higher than the fluoride in the water was used for the brewing process. From this study one would conclude that in communities residing in high fluoride areas, there should be awareness creation campaigns to point out the relationship of excessive consumption of locally brewed alcoholic drinks and skeletal fluorosis. Regulations should also be put in place to require producers of local alcoholic beverages to use low fluoride water for brewing.

Effect of Fluoride on Insulin Level of Rats and Insulin Receptor Expression in the MC3T3-E1 Cells

Studies on the role of insulin and insulin receptor (InsR) in the process of skeletal fluorosis, especially in osteogenic function, are rare. We evaluated the effect of increasing F⁻ doses on the marker of bone formation, serum insulin level and pancreatic secretion changes in vivo and mRNA expression of InsR and osteocalcin (OCN) in vitro. Wistar rats (n = 50) were divided into two groups, i.e. a control group and fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F⁻/L. The fluoride ion-selective electrode measured the fluoride concentrations of femurs. The alkaline phosphatase (ALP), OCN, insulin and glucagon of serum were tested to observe the effect of fluoride action on them. Meantime, the pancreas pathological morphometry analysis via β cells stained by aldehyde fuchsin showed the action of fluoride on pancreas secretion. MC3T3-E1 cells (derived from newborn mouse calvaria) were exposed to varying concentrations and periods of fluoride. The mRNA expression of InsR and OCN was quantified with real-time PCR. Results showed that 1-year fluoride treatment obviously stimulated ALP activity and OCN level along with increase of bone fluoride concentration of rats, which indicated that fluoride obviously stimulated osteogenic action of rats. In vitro study, the dual effect of fluoride on osteoblast function is shown. On the other hand, there was a significant increase of serum insulin level and a general decrease of glucagon level, and the histomorphometry analysis indicated an elevated insulin-positive area and increase in islet size in rats treated with fluoride for 1 year. In addition, fluoride obviously facilitated the mRNA expression of InsR in vitro. To sum up, there existed a close relationship between insulin secretion and fluoride treatment. The insulin signal pathway might be involved in the underlying occurrence or development of skeletal fluorosis.

Neurology of endemic skeletal fluorosis

Endemic skeletal fluorosis is widely prevalent in India and is a major public health problem. The first ever report of endemic skeletal fluorosis and neurological manifestation was from Prakasam district in Andhra Pradesh in the year 1937. Epidemiological and experimental studies in the endemic areas suggest the role of temperate climate, hard physical labor, nutritional status, presence of abnormal concentrations of trace elements like strontium, uranium, silica in water supplies, high fluoride levels in foods and presence of kidney disease in the development of skeletal fluorosis. Neurological complications of endemic skeletal fluorosis, namely radiculopathy, myelopathy or both are mechanical in nature and till date the evidence for direct neurotoxicity of fluoride is lacking. Prevention of the disease should be the aim, knowing the pathogenesis of fluorosis. Surgery has a limited role in alleviating the neurological disability and should be tailored to the individual based on the imaging findings.