Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv Role of fluoride induced epigenetic alterations in the development of skeletal fluorosis Atul P. Daiwile a , Prashant Tarale a , Saravanadevi Sivanesan a, ⁎ , Pravin K. Naoghare b , Amit Bafana c , Devendra Parmar d , Krishnamurthi Kannan a a Health and Toxicity Cell, CSIR-National Environmental Engineering Research Institute (NEERI), Nagpur 440020, India b Environmental Impact and Sustainability Division, CSIR-National Environmental Engineering Research Institute (NEERI), Nagpur 440020, India c Director's Research Cell, CSIR-National Environmental Engineering Research Institute (NEERI), Nagpur 440020, India d Developmental Toxicology Division, CSIR-Indian Institute of Toxicology Research (IITR), Lucknow 226001, India ARTICLE INFO Keywords: DNA methylation Fluorosis Bone formation Fracture repair Extracellular matrix ABSTRACT 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 morpho- genesis/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-regula- tion 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. 1. Introduction Fluoride is a vital element for human and intake of low dosages of fluoride (below 1 mg/L) is beneficial for normal bone and tooth de- velopment. However, it has been observed that systemic high and chronic exposure of fluoride above the WHO guideline level of 1.5 mg/L leads to adverse health effect (Daiwile et al., 2015, 2018; Kanduti et al., 2016; Dutta et al., 2017). Contaminated drinking water is a major source of fluoride exposure to human population (Narsimha and Sudarshan, 2017a, 2017b). Natural and Anthropogenic activities are the two main sources of fluoride contamination of ground water and water bodies (Ranjan and Ranjan, 2015; Thivya et al., 2017). Geo- graphical areas rich in mineral rocks like fluorspar, cryolite, fluorite and fluorapatites have higher fluoride concentration (Ground Water Quality in Shallow Aquifers of India, 2010). Weathering of these mi- nerals rocks, formation of salts with Calcium and Magnesium, and leaching into groundwater serve as the major natural contributor of fluoride contamination (Thivya et al., 2017). Anthropogenic sources such as industrial effluent, household products, mining, surface runoff of mineral mixture, animal feed supplements and agrochemicals release fluoride in aquatic system, and thus pose a threat to the human health (Ranjan and Ranjan, 2015). Globally 200 million people from approximately 20 countries are at risk due to consumption of drinking water having high concentration of fluoride (Thivya et al., 2017). In India around 177 districts distributed in 20 states have high concentration of fluoride in ground water, posing https://doi.org/10.1016/j.ecoenv.2018.11.035 Received 5 August 2018; Received in revised form 21 October 2018; Accepted 9 November 2018 Abbreviations: BACH1, BTB Domain and CNC Homolog 1; BMP1, Bone Morphogenetic Protein 1; ECM, Extracellular Matrix; METAP2, Methionine Aminopeptidase 2; MMP11, Matrix Metallopeptidase 11; CHG, Cytosine H is A, C or T Guanine; CHH, Cytosine H is A, C or T; CpG, Cytosine Phosphate Guanine; NaF, Sodium Fluoride; WGBS, Whole Genome Bisulfite Sequencing ⁎ Corresponding author. E-mail address: ss_devi@neeri.res.in (S. Sivanesan). Ecotoxicology and Environmental Safety 169 (2019) 410–417 0147-6513/ © 2018 Elsevier Inc. All rights reserved. T