Molecular mechanisms underlaying fluoride-induced neurotoxicity: interplay of antioxidants and endoplasmic reticulum stress-mediated apoptotic pathways in rats.

Abstract

Original abstract online at
https://link.springer.com/article/10.1007/s00210-026-05441-3

Highlights

• Environmentally relevant fluoride exposure induces molecular and structural brain alterations in rats.

• Fluoride exposure impairs the antioxidant enzyme gene expression.

• Chronic fluoride exposure induces neurotoxicity associated with oxidative stress and ER stress pathways.

• ER stress–mediated apoptotic pathways are involved in fluoride-induced neurotoxicity.

• Histopathological alterations were observed in the hippocampus and cerebral cortex of exposed rats.

Fluoride is a naturally occurring compound widely present in soil, water, rocks and is essential to maintain the physiological function and structure of bones and teeth. However, chronic exposure to elevated fluoride levels has been linked to adverse neurological effects. Despite its widespread environmental presence, the molecular mechanisms underlying fluoride-induced neurotoxicity remain incompletely understood. This study aimed to elucidate the effects of fluoride on oxidative stress, endoplasmic reticulum (ER) stress, apoptosis, and associated histopathological alterations in brain tissue. Forty Sprague–Dawley rats were randomly assigned to four groups (n = 10 per group; 5 male + 5 female) and administered sodium fluoride (NaF) in drinking water at concentrations of < 0.5 ppm (control), 50 ppm, 150 ppm, and 300 ppm for 90 consecutive days. The expression of antioxidant genes (SOD1 and GCLC), ER stress, and apoptosis-related genes (XBP1, GRP78, BCL-2, and BAX) was quantified using real-time quantitative PCR (RT-qPCR), and histopathological analysis of the brain tissues was performed. Fluoride exposure caused a dose-dependent downregulation of antioxidant and ER stress–related genes and concurrent upregulation of the pro-apoptotic genes. Histopathological analysis revealed structural damage in hippocampus and cerebral cortex, including neuronal shrinkage, vacuolization, and apoptotic features. These findings indicate that prolonged NaF exposure impairs antioxidant defenses, induces ER stress, and activates apoptotic pathways, thereby contributing to neuronal damage. This study provides mechanistic insights into fluoride-induced neurotoxicity and highlights the need for further research on potential therapeutic strategies targeting oxidative and ER stress pathways.

Data availability

All source data for this work (or generated in this study) are available upon reasonable request.

Acknowledgements

We acknowledge the support of COMSATS University Islamabad (CUI) scholarship, High Education Commission (HEC) Pakistan, National Institute of Health Pakistan (NIH), Shaheed Zulfiqar Ali Bhutto Medical University, and everyone who has helped us during this study. We sincerely acknowledge Mr. Muhammad Tayyab, National Institutes of Health (NIH), Pakistan, for his valuable support in providing the animal house facility and assistance.

Funding

The work was supported by Pakistan High Education Commission (HEC) (grant number NRPU/HEC-14825–2021) and supported by COMSATS University Islamabad (CUI).

Author information

Authors and Affiliations

Department of Biosciences, COMSATS University Islamabad, Islamabad, Pakistan

Ashiru Dahiru, Ismat Nawaz, Muhammad Jadoon Khan, Muhammad Abbas & Syed Ali Musstjab Akber Shah Eqani
Department of Veterinary Physiology and Biochemistry, Usmanu Danfodiyo University, Sokoto, Nigeria

Ashiru Dahiru
Department of Molecular Biology, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan

Syeda Kiran Riaz
Department of Pathology, Pakistan Institute of Medical Sciences, Islamabad, Pakistan

Summaya Sohail Chaudry
Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China

Qingyu Huang

Contributions

Ashiru Dahiru: Methodology, wrote the main manuscript. Ismat Nawaz: Supervision, Conceptualization. Syeda Kiran Riaz: Supervision. Summaya Sohail Chaudry: Histology analysis. Muhammad Jadoon Khan: Supervision. Qingyu Huang: Proofreading. Muhammad Abbas: Experimental lab work/analysis. Syed Ali Musstjab Akber Shah Eqani: Supervision, Conceptualization, Funding. All authors reviewed the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Corresponding authors

Correspondence to Ismat Nawaz or Syed Ali Musstjab Akber Shah Eqani.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary Information

Below is the link to the electronic supplementary material.

ESM1 (download DOCX )

(DOCX 597 KB)

About this article

Dahiru, A., Nawaz, I., Riaz, S.K. et al. Molecular mechanisms underlaying fluoride-induced neurotoxicity: interplay of antioxidants and endoplasmic reticulum stress–mediated apoptotic pathways in rats. Naunyn-Schmiedeberg’s Arch Pharmacol (2026). https://doi.org/10.1007/s00210-026-05441-3

References

Aboghazleh R, Boyajian SD, Atiyat A, Udwan M, Al-Helalat M, Al-Rashaideh R (2024) Rodent brain extraction and dissection: a comprehensive approach. MethodsX. https://doi.org/10.1016/j.mex.2023.102516

Article PubMed Google Scholar
Albus U (2012) Guide for the care and use of laboratory animals (8th edn). Lab Anim. https://doi.org/10.1258/la.2012.150312

Article Google Scholar
Aziz SJ, & Zeman-Pocrnich CE (2022) Tissue processing. In Methods in molecular biology, 2422. https://doi.org/10.1007/978-1-0716-1948-3_4
Banala RR, Karnati PR (2015) Vitamin A deficiency: an oxidative stress marker in sodium fluoride (NaF) induced oxidative damage in developing rat brain. Int J Dev Neurosci. https://doi.org/10.1016/j.ijdevneu.2015.08.010

Article PubMed Google Scholar
Barnhart CD, Yang D, Lein PJ (2015) Using the Morris water maze to assess spatial learning and memory in weanling mice. PLoS ONE. https://doi.org/10.1371/journal.pone.0124521

Article PubMed PubMed Central Google Scholar
Cabiscol E, Tamarit J, Ros J (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol 3(1):3–8. https://doi.org/10.1007/s101230000062
Chen R, Zhao LD, Liu H, Li HH, Ren C, Zhang P, Guo KT, Zhang HX, Geng DQ, Zhang CY (2017) Fluoride induces neuroinflammation and alters Wnt signaling pathway in BV2 microglial cells. Inflammation. https://doi.org/10.1007/s10753-017-0556-y

Article PubMed PubMed Central Google Scholar
Dec K, ?ukomska A, Maciejewska D, Jakubczyk K, Baranowska-Bosiacka I, Chlubek D, W?sik A, Gutowska I (2017) The influence of fluorine on the disturbances of homeostasis in the central nervous system. Biol Trace Elem Res. https://doi.org/10.1007/s12011-016-0871-4

Article PubMed Google Scholar
Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride (1997) In Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride. https://doi.org/10.17226/5776
Heindel JJ, Bates HK, Price CJ, Marr MC, Myers CB, Schwetz BA (1996) Developmental toxicity evaluation of sodium fluoride administered to rats and rabbits in drinking water. Fundam Appl Toxicol. https://doi.org/10.1006/faat.1996.0053

Article PubMed Google Scholar
Hetz C, Saxena S (2017) ER stress and the unfolded protein response in neurodegeneration. Nat Rev Neurol. https://doi.org/10.1038/nrneurol.2017.99

Article PubMed Google Scholar
Kinawy AA (2019) Synergistic oxidative impact of aluminum chloride and sodium fluoride exposure during early stages of brain development in the rat. Environ Sci Pollut Res. https://doi.org/10.1007/s11356-019-04491-w

Article Google Scholar
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262

Article CAS PubMed Google Scholar
Lou DD, Guan ZZ, Liu YJ, Liu YF, Zhang KL, Pan JG, Pei JJ (2013) The influence of chronic fluorosis on mitochondrial dynamics morphology and distribution in cortical neurons of the rat brain. Arch Toxicol 87(3):449–457. https://doi.org/10.1007/s00204-012-0942-z

Article CAS PubMed Google Scholar
McPherson CA, Zhang G, Gilliam R, Brar SS, Wilson R, Brix A, Picut C, Harry GJ (2018) An evaluation of neurotoxicity following fluoride exposure from gestational through adult ages in Long-Evans hooded rats. Neurotox Res. https://doi.org/10.1007/s12640-018-9870-x

Article PubMed Google Scholar
Nair A, Jacob S (2016) A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm. https://doi.org/10.4103/0976-0105.177703

Article PubMed PubMed Central Google Scholar
Niu Q, Chen J, Xia T, Li P, Zhou G, Xu C, Zhao Q, Dong L, Zhang S, & Wang A (2018) Excessive ER stress and the resulting autophagic flux dysfunction contribute to fluoride-induced neurotoxicity. Environ Pollut 233. https://doi.org/10.1016/j.envpol.2017.09.015
Nowicka B (2022) Heavy metal–induced stress in eukaryotic algae-mechanisms of heavy metal toxicity and tolerance with particular emphasis on oxidative stress in exposed cells and the role of antioxidant response. Environ Sci Pollut Res. https://doi.org/10.1007/s11356-021-18419-w

Article Google Scholar
Pan Y, Lu P, Yin L, Chen K, He Y (2015) Effect of fluoride on the proteomic profile of the hippocampus in rats. Z Naturforsch C J Biosci. https://doi.org/10.1515/znc-2014-4158

Article PubMed Google Scholar
Park SM, Kang TI, So JS (2021) Roles of XBP1s in transcriptional regulation of target genes. Biomedicines. https://doi.org/10.3390/biomedicines9070791

Article PubMed PubMed Central Google Scholar
Ran LY, Xiang J, Zeng XX, Tang J, Dong YT, Zhang F, Yu WF, Qi XL, Xiao Y, Zou J, Deng J, Guan ZZ (2021) Integrated transcriptomic and proteomic analysis indicated that neurotoxicity of rats with chronic fluorosis may be in mechanism involved in the changed cholinergic pathway and oxidative stress. J Trace Elem Med Biol. https://doi.org/10.1016/j.jtemb.2020.126688

Article PubMed Google Scholar
Ren C, Li HH, Zhang CY, Song XC (2022) Effects of chronic fluorosis on the brain. Ecotoxicol Environ Saf. https://doi.org/10.1016/j.ecoenv.2022.114021

Article PubMed Google Scholar
Rzeuski R, Chlubek D, Machoy Z (1998) Interactions between fluoride and biological free radical reactions. Fluoride 31(1):43–45
Sampias C, Rolls G (2021) Leica Biosystems, H&E staining overview: a guide to best practices. https://www.leicabiosystems.com/knowledge-pathway/he-staining-overview-a-guide-to-best-practices/
Shivarajashankara YM, Shivashankara AR, Bhat PG, Rao SH (2001) Effect of fluoride intoxication on lipid peroxidation and antioxidant systems in rats. Fluoride 34(2):108–113
Tonetto S, Weikop P, Brudek T, Thomsen M (2023) Behavioral and biochemical effects of alcohol withdrawal in female C3H/HeNRj and C57BL/6JRj mice. Front Behav Neurosci. https://doi.org/10.3389/fnbeh.2023.1143720

Article PubMed PubMed Central Google Scholar
Valdez-Jiménez L, Soria Fregozo C, Miranda Beltrán ML, Gutiérrez Coronado O, Pérez Vega MI (2011) Effects of the fluoride on the central nervous system. Neurología (English Edition). https://doi.org/10.1016/s2173-5808(11)70062-1

Article Google Scholar
Varner JA, Jensen KF, Horvath W, Isaacson RL (1998) Chronic administration of aluminum-fluoride or sodium-fluoride to rats in drinking water: alterations in neuronal and cerebrovascular integrity. Brain Res. https://doi.org/10.1016/S0006-8993(97)01336-X

Article PubMed Google Scholar
Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science. https://doi.org/10.1126/science.1209038

Article PubMed PubMed Central Google Scholar
Zhang CY, Chen R, Wang F, Ren C, Zhang P, Li Q, Li HH, Guo KT, Geng DQ, Liu C (2017) EGb-761 attenuates the anti-proliferative activity of fluoride via DDK1 in PC-12 cells. Neurochem Res. https://doi.org/10.1007/s11064-016-2115-6

Article PubMed PubMed Central Google Scholar
Zuo H, Chen L, Kong M, Qiu L, Lü P, Wu P, Yang Y, Chen K (2018) Toxic effects of fluoride on organisms. Life Sci. https://doi.org/10.1016/j.lfs.2018.02.001

Article PubMed Google Scholar

Research Studies

Study Tracker

Molecular mechanisms underlaying fluoride-induced neurotoxicity: interplay of antioxidants and endoplasmic reticulum stress-mediated apoptotic pathways in rats.

Abstract