Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Dec:89:369-78.
doi: 10.1016/j.freeradbiomed.2015.08.015. Epub 2015 Sep 30.

Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling

Maiko Suzuki  1 , Cheryl Bandoski  1 , John D Bartlett  2
Affiliations

Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling

Maiko Suzuki et al. Free Radic Biol Med. 2015 Dec.

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.

Keywords: Ameloblast; Autophagy; Fluorosis; JNK; Oxidative damage; ROS; Sirtuin.

PubMed Disclaimer

Conflict of interest statement

Conflicts of Interest

None.

Figures

Fig. 1
Fig. 1
Fluoride treatment induces ROS production. (A) LS8 cells were treated with 0–10 mM fluoride for 3 h and intracellular ROS was detected with H2DCFDA. Upon oxidation by ROS, the nonfluorescent H2DCFDA is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF). Regression analysis revealed a highly significant positive correlation between fluoride dose and ROS generation (P < 0.001). (B) LS8 cells were treated with 0–500 µM of NOX1/4 inhibitor (GKT137831) for 1 h followed by 5 mM fluoride for 3 h. Intracellular ROS was detected by H2DCFDA. Regression analysis revealed a highly significant negative correlation between NOX1/4 inhibitor dose and ROS generation after fluoride treatment (P < 0.001).
Fig. 2
Fig. 2
Fluoride negatively affects mitochondria function. (A) LS8 cells were treated with 0 or 5 mM fluoride for 6 h and cytochrome-c (12 kDa) levels in the cytosol fraction (Cyto) or in the mitochondrial fraction (Mito) were quantified by western blot analysis. Fluoride treatment released cytochrome c from the mitochondria into the cytosol. α-Tubulin (52 kDa) and VDAC1/porin (31 kDa) were the loading control proteins. The table shows the ratio of cytochrome c/α-tubulin (for cytosol) or cytochrome c/VDAC1 (for mitochondria). *P < 0.05 vs. 0 mM of fluoride. (B) LS8 cells were treated with 0–5 mM fluoride for 6 h and total protein was extracted. UCP2 (dimer: 70 kDa) was detected by western blots and levels increased with increasing concentrations of fluoride. α-Tubulin served as a loading control protein. The table shows the UCP2/α-tubulin ratio. Regression analysis revealed a significant positive correlation between fluoride dose and UCP2 expression (P < 0.05). (C) LS8 cells were treated with 0, 5, or 10 mM fluoride for 6 h and intracellular ATP was measured by a method that utilizes glycerol phosphorylation, which is quantified by colorimetery (570 nm). ATP levels decreased with increasing fluoride concentrations. Data are expressed as mean ± SD. **p < 0.01 vs. 0 mM of fluoride.
Fig. 3
Fig. 3
Fluoride induces SIRT1 phosphorylation (p-SIRT) via JNK signaling. LS8 cells were treated with JNK inhibitor SP600125 (10 or 30 µM) for 1 h prior to 5 mM fluoride treatment for 2 h or 6 h. (A) Total protein was extracted at 2 h and phospho-(p)-JNK (46, 54 kDa), total-(t)-JNK (46, 54 kDa), phospho-(p)-c-Jun (48 kDa), and total-(t)-c-Jun (48 kDa) were quantified by western blot analysis. Note that SP600125 treatment reduced the overall levels of p-JNK and p-c-Jun after exposure to fluoride. (B) Nuclear protein was extracted at 6 h and p-SIRT1 (82 kDa) and total-(t)-SIRT1 (120 kDa) were quantified by western blots. SP600125 treatment decreased p-SIRT1 levels after exposure to fluoride. Histone H3 (17 kDa) was the loading control protein. The tables show the ratios p-JNK/t-JNK, p-c-Jun/t-c-Jun, and p-SIRT1/t-SIRT1. *P < 0.05, **p < 0.01 vs. fluoride treatment alone.
Fig. 4
Fig. 4
Fluoride activates the MAPKKK pathway. LS8 cells were treated with 10 or 100 nM TAK1 (MAP3K7) inhibitor, oxoseaenol (Oxoz), for 1 h prior to 5 mM fluoride treatment for 2 h. (A) Total protein was extracted at 2 h and p-JNK (46, 54 kDa), t-JNK (46, 54 kDa), p-c-Jun (48 kDa), and t-c-Jun (48 kDa) were quantified by western blot procedures. The tables show the ratios p-JNK/t-JNK or p-c-Jun/t-c-Jun. *P < 0.05, **p < 0.01 vs. fluoride treatment alone. These results show that TAK1 inhibition attenuated fluoride-induced JNK phosphorylation.
Fig. 5
Fig. 5
Fluoride activates SIRT1/autophagy through ROS-JNK signaling. LS8 cells were treated with 0, 5, or 10 mM N-acetylcysteine (NAC) for 1 h followed by 10 mM fluoride for 3 h. (A) Intracellular ROS generation was detected by H2DCFDA. Regression analysis revealed a highly significant negative correlation between NAC dose and ROS generation after fluoride treatment (P < 0.001). (B) Cells were treated with 5 or 10 mM NAC for 1 h followed by 5 mM fluoride for 2 h. p-JNK, t-JNK, p-c-Jun, and t-c-Jun were quantified by western blots. NAC treatment reduced p-JNK and p-c-Jun levels after fluoride exposure. α-tubulin (52 kDa) was the loading control protein. (C) Nuclear protein was extracted from cells treated with or without NAC (5 mM) pretreatment for 1 h followed by fluoride (5 mM) treatment for 6 h. p-SIRT1 and t-SIRT1 was quantified by western blots. At 6 h, NAC treatment decreased fluoride-induced p-SIRT1 levels. Histone H3 (17 kDa) was the loading control protein. (D) Cells were treated with 5 mM fluoride with or without 5 mM NAC for 24 h. Total protein was extracted and LC3I (16 kDa) and LC3II (14 kDa) were quantified by western blots. In the fluoride-treated samples, NAC reduced the levels of the autophagy mediator LC3II. Tables show the ratios of p-JNK/t-JNK or p-c-Jun/t-c-Jun (B), p-SIRT1/t-SIRT1 (C), and LC3II/LC3I (D). *P < 0.05, **p < 0.01 vs. fluoride treatment alone.
Fig. 6
Fig. 6
Fluoride induces DNA damage. LS8 cells were treated with 0–5 mM fluoride for 6 h. Total protein was extracted and the DNA double-strand break marker phosphorylated H2AX (γH2AX, 15 kDa) was quantified by western blots. Fluoride treatment at 3 and 5 mM increased the level of γH2AX. α-Tubulin was the loading control protein. The table shows the γH2AX/α-tubulin ratio. Regression analysis revealed a highly significant positive correlation between fluoride dose and γH2AX expression (P < 0.01).
Fig. 7
Fig. 7
NAC treatment increases the levels of fluoride-induced γH2AX and cleaved caspase-3. (A) LS8 cells were treated with or without 5 or 10 mM NAC for 1 h prior to 0 or 5 mM fluoride treatment for 6 h. Total protein was extracted and γH2AX was quantified by western blot analysis. NAC treatment significantly increased the amount of fluoride-induced γH2AX. (B) Cleaved-caspase-3 (17 kDa) was also quantified by western blots after treatment with 0, 5, or 10 mM NAC and 0 or 5 mM fluoride for 6 or 18 h. NAC treatment increased the levels of fluoride-induced cleaved caspace-3 at 6 h, but no significant difference was observed at 18 h. α-Tubulin was the loading control protein. Tables show the ratios of γH2AX/α-tubulin (A) or Cleaved-caspase-3/α-tubulin (B). *P < 0.05, **p < 0.01 vs. fluoride treatment alone.
Fig. 8
Fig. 8
Fluoride-induced mitochondrial and DNA damage in rodent enamel organs. Rodents were provided water ad libitum containing 0, 50, 100, or 125 ppm fluoride as sodium fluoride for 6 weeks. Left panels show immunohistochemical (IHC) staining performed on paraffin sections from rat incisors in the secretory (SEC) or maturation (MAT) stage of enamel development. Right panels show immunofluorescent staining (IF) performed on paraffin sections from rat (A–D) or mouse (E) maturation-stage incisors. (A) Identification of cytochrome-c released into the ameloblast cytoplasm. Right panels show maturation-stage enamel organs stained with DAPI to locate cell nuclei (blue), the mitochondria marker VDAC1 (red), and cytochrome-c (Cyto-c) stained green. Note that fluoride treatment caused release of cytochrome-c during the maturation stage of enamel development. (B) Identification of 8-oxoguanine in enamel organs stained with DAPI, VDAC1, or 8-oxoguanine (8-OXO). 8-Oxoguanine located primarily outside the nucleus in fluoride-treated secretory and maturation stage incisors. (C–E) Maturation-stage incisors stained with DAPI and phospho-(p)-JNK (C), phospho-(p)-ATM (D), or histone γH2AX (E). p-JNK, p-ATM, and γH2AX staining increased in fluoride-treated maturation stage ameloblasts in a dose-dependent manner. Scale bar represents 10 µm. Brackets denote ameloblasts.
Fig. 9
Fig. 9
Schematic summary depicting fluoride-induced oxidative damage and adaptive response. Fluoride induces ROS generation that elicits JNK/c-Jun signaling. The ROS-mediated JNK/c-Jun pathway induces oxidative damage, mitochondrial damage, DNA damage, and apoptosis. Conversely, fluoride activates SIRT1/autophagy as an adaptive response through the ROS-mediated JNK/c-Jun pathway to protect cells from fluoride-induced oxidative damage.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. CDC. Recommendations for using fluoride to prevent and control dental caries in the United States. Centers for Disease Control and Prevention. MMWR. Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports/Centers for Disease Control. 2001;50:1–42. - PubMed
    1. Dean HT, Elvove E. Some Epidemiological Aspects of Chronic Endemic Dental Fluorosis. American journal of public health and the nation’s health. 1936;26:567–575. - PMC - PubMed
    1. Boivin G, Chavassieux P, Chapuy MC, Baud CA, Meunier PJ. Skeletal fluorosis: histomorphometric analysis of bone changes and bone fluoride content in 29 patients. Bone. 1989;10:89–99. - PubMed
    1. Zager RA, Iwata M. Inorganic fluoride. Divergent effects on human proximal tubular cell viability. The American journal of pathology. 1997;150:735–745. - PMC - PubMed
    1. Thrane EV, Refsnes M, Thoresen GH, Lag M, Schwarze PE. Fluoride-induced apoptosis in epithelial lung cells involves activation of MAP kinases p38 and possibly JNK. Toxicological sciences: an official journal of the Society of Toxicology. 2001;61:83–91. - PubMed

Publication types

MeSH terms

  NODES
chat 2
Note 3
twitter 2