title: Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling.
abstract: Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through c-Jun N-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125 ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0 ppm fluoride). These results suggest that fluoride-induced ROS generation causes mitochondrial damage and DNA damage, which may lead to impairment of ameloblast function. To counteract this impairment, SIRT1/autophagy is induced via JNK signaling to protect cells/ameloblasts from fluoride-induced oxidative damage that may cause dental fluorosis.
authors.affiliation: Department of Mineralized Tissue Biology, The Forsyth Institute & Harvard School of Dental Medicine, 245 First Street, Cambridge, MA.
authors.forename: Maiko
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: Department of Mineralized Tissue Biology, The Forsyth Institute & Harvard School of Dental Medicine, 245 First Street, Cambridge, MA.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Bandoski
authors.affiliation: Department of Mineralized Tissue Biology, The Forsyth Institute & Harvard School of Dental Medicine, 245 First Street, Cambridge, MA. Electronic address: jbartlett@forsyth.org.
authors.forename: John D
authors.initials: JD
authors.lastname: Bartlett
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D005459:Fluorides / Q000494:pharmacology*; D007124:Immunoenzyme Techniques; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects*; D010710:Phosphates / Q000494:pharmacology*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D056564:Sirtuin 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D010710:Phosphates; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C012980:fluorophosphate; D048031:JNK Mitogen-Activated Protein Kinases; C470545:Sirt1 protein, mouse; D056564:Sirtuin 1; D005459:Fluorides
keywords: Ameloblast; Autophagy; Fluorosis; JNK; Oxidative damage; ROS; Sirtuin