Protective effects of sodium butyrate on fluorosis in rats by regulating bone homeostasis and serum metabolism.

Fluorosis due to high fluoride levels in drinking water profoundly affects the development of human skeletal and dental structures. Sodium butyrate (NaB) has been found to regulate overall bone mass and prevent pathological bone loss. However, the mechanism of NaB action on fluorosis remains unclear. In this study, a rat model of fluorosis induced by 100 mg/L sodium fluoride was used to investigate the impact of NaB on bone homeostasis and serum metabolomics. It was found that NaB significantly reduced the levels of bone resorption markers CTX-Ⅰ and TRACP-5B in fluorosis rats. Moreover, NaB increased calcium and magnesium levels in bone, while decreasing phosphorus levels. In addition, NaB improved various bone microstructure parameters, including bone mineral density (BMD), trabecular thickness (Tb. Th), trabecular bone separation (Tb. SP), and structural model index (SMI) in the femur. Notably, NaB intervention also enhanced the antioxidant capacity of plasma in fluorosis rats. Furthermore, a comprehensive analysis of serum metabolomics by LC-MS revealed a significant reversal trend of seven biomarkers after the intervention of NaB. Finally, pathway enrichment analysis based on differential metabolites indicated that NaB exerted protective effects on fluorosis by modulating arginine and proline metabolic pathways. These findings suggest that NaB has a beneficial effect on fluorosis and can regulate bone homeostasis by ameliorating metabolic disorders.

Rutin mitigates fluoride-induced nephrotoxicity by inhibiting ROS-mediated lysosomal membrane permeabilization and the GSDME-HMGB1 axis involved in pyroptosis and inflammation.

Fluoride is known to induce nephrotoxicity; however, the underlying mechanisms remain incompletely understood. Therefore, this study aims to explore the roles and mechanisms of lysosomal membrane permeabilization (LMP) and the GSDME/HMGB1 axis in fluoride-induced nephrotoxicity and the protective effects of rutin. Rutin, a naturally occurring flavonoid compound known for its antioxidative and anti-inflammatory properties, is primarily mediated by inhibiting oxidative stress and reducing proinflammatory markers. To that end, we established in vivo and in vitro models. In the in vivo study, rats were exposed to sodium fluoride (NaF) throughout pregnancy and up until 2 months after birth. In parallel, we employed in vitro models using HK-2 cells treated with NaF, n-acetyl-L-cysteine (NAC), or rutin. We assessed lysosomal permeability through immunofluorescence and analyzed relevant protein expression via western blotting. Our findings showed that NaF exposure increased ROS levels, resulting in enhanced LMP and increased cathepsin B (CTSB) and D (CTSD) expression. Furthermore, the exposure to NaF resulted in the upregulation of cleaved PARP1, cleaved caspase-3, GSDME-N, and HMGB1 expressions, indicating cell death and inflammation-induced renal damage. Rutin mitigates fluoride-induced nephrotoxicity by suppressing ROS-mediated LMP and the GSDME/HMGB1 axis, ultimately preventing fluoride-induced renal toxicity occurrence and development. In conclusion, our findings suggest that NaF induces renal damage through ROS-mediated activation of LMP and the GSDME/HMGB1 axis, leading to pyroptosis and inflammation. Rutin, a natural antioxidative and anti-inflammatory dietary supplement, offers a novel approach to prevent and treat fluoride-induced nephrotoxicity.

Fluoride promotes the secretion of inflammatory factors in microglia through NLRP3/Caspase-1/GSDMD pathway.

It is widespread of endemic fluorosis in China, and the exposure of excessive fluoride will cause nervous system disease and activate microglia. However, the mechanism of the damage is not clear. It is well-known that NLRP3/Caspase-1/GSDMD pathway, a classic pyroptosis pathway, is widely involved in the occurrence and development of nervous system-related diseases, infectious diseases, and atherosclerotic diseases. This research aimed to explore the molecular mechanism of sodium fluoride on inflammation and pyroptosis in BV2 microglia based on the NLRP3/Caspase-1/GSDMD signaling pathway. BV2 microglia was treated with sodium fluoride at the dose of 0.25, 1, and 2 mmol/L for 24, 48, and 72 h, respectively. Cell viability, cell morphology, lactate dehydrogenase content, and related proteins and genes were examined to investigate if sodium fluoride caused damage to BV2 microglia through the pyroptosis pathway. Dithiolam (5 µmol/L), a pyroptosis inhibitor, was added for further verification. NaF could induced BV2 cells injury in a dose-dependent fashion through disrupting the integrity of cell membranes and increasing IL-1ß via upregulating NLRP3, Caspase-1, and its downstream protein GSDMD. Disulfiram could improve these changes caused by NaF. In conclusion, our results suggested that NLRP3/Caspase-1/GSDMD-mediated classical pyroptosis pathway was involved in fluoride-induced BV2 microglia damage.

Haematological, biochemical, enzymological changes and mitochondrial dysfunction of liver in freshwater climbing perch Anabas testudineus during their acute and chronic exposure to sodium fluoride.

Anthropogenic activities are increasing fluoride concentration in watercourses. The present study focuses on the sublethal toxicity of sodium fluoride during sub-chronic and chronic time periods in the freshwater fish Anabas testudineus. The 96-hour LC50 value for fluoride was found to be 616.50 mg/L. Excessive mucous production and hyper excitability, followed by loss of balance, were seen in fish under acute fluoride exposure. Significant reduction in yield and specific growth rate of fish were assessed at 15, 30 and 45-days exposure intervals. Different bio-indicators like Hepatosomatic-index, Gonadosomatic-index and fecundity were reduced significantly in fish exposed to 10% (61.6 mg/L) and 20% (123.2 mg/L) of 96 h of LC50 values of fluoride in comparison to control. Toxicant concentrations directly correlated with parameter lowering. Fluoride exposure increased plasma glucose, creatinine, AST, and ALT and reduced total RBC, haemoglobin content, Hct (%), plasma protein, and cholesterol. Moreover, fluoride exposure significantly reduces the mitochondrial membrane potential in liver. This may result in metabolic depression, haematological, biochemical, and enzymological stress. The in-silico structural analysis predicts that fluoride may impede cytochrome c oxidase of the electron transport system, hence inhibiting mitochondrial functionality. These findings collectively highlight the urgent need for stringent regulation and monitoring of fluoride levels in freshwater ecosystems, as the subchronic and chronic effects observed in A. testudineus may have broader implications for aquatic ecosystems.

Inactivation of Mst/Nrf2/Keap1 signaling flexibly mitigates MAPK/NQO-HO1 activation in the reproductive axis of experimental fluorosis.

Fluoride induced reprotoxicity through oxidative stress-mediated reproductive cell death. Hence, the current study evaluated the importance of the MST/Nrf2/MAPK/NQO-HO1 signaling pathway in fluorosis-induced reproductive toxicity. For this purpose, the reproductive toxicity of sodium fluoride (NaF) at physiological, biochemical, and intracellular levels was evaluated. In-vivo, NaF at 100 mg/L instigated physiological dysfunction, morphological, stereological, and structural injuries in the gut-gonadal axis of fluorosis mice through weakening the antioxidant signaling, Nrf2/HO-1/NQO1signaling pathway, causing the gut-gonadal barrier disintegrated via oxidative stress-induced inflammation, mitochondrial damage, apoptosis, and autophagy. Similar trends were also observed in-vitro in the isolated Leydig cells (LCs) challenging with 20 mg/L NaF. Henceforth, activating the cellular antioxidant signaling pathway, Nrf2/HO-1/NQO1, inactivating autophagy and apoptosis, or attenuating lipopolysaccharide (LPS) can be the theoretical basis and valuable therapeutic targets for coping with NaF-induced reproductive toxicity.

Proteomics Sequencing Reveals the Role of TGF-ß Signaling Pathway in the Peripheral Blood of Offspring Rats Exposed to Fluoride.

The underlying mechanism of fluorosis has not been fully elucidated. The purpose of this study was to explore the mechanism of fluorosis induced by sodium fluoride (NaF) using proteomics. Six offspring rats exposed to fluoride without dental fluorosis were defined as group A, 8 offspring rats without fluoride exposure were defined as control group B, and 6 offspring rats exposed to fluoride with dental fluorosis were defined as group C. Total proteins from the peripheral blood were extracted and then separated using liquid chromatography-tandem mass spectrometry. The identified criteria for differentially expressed proteins were fold change > 1.2 or < 0.83 and P < 0.05. Gene Ontology function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the oeCloud tool. The 177 upregulated and 22 downregulated proteins were identified in the A + C vs. B group. KEGG pathway enrichment analysis revealed that transforming growth factor-ß (TGF-ß) signaling pathway significantly enriched. PPI network constructed using Cytoscape confirmed RhoA may play a crucial role. The KEGG results of genes associated with fluoride and genes associated with both fluoride and inflammation in the GeneCards database also showed that TGF-ß signaling pathway was significantly enriched. The immunofluorescence in HPA database showed that the main expression sites of RhoA are plasma membrane and cytosol, while the main expression site of Fbn1 is the Golgi apparatus. In conclusion, long-term NaF intake may cause inflammatory response in the peripheral blood of rats by upregulating TGF-ß signaling pathway, in which RhoA may play a key role.

A new insight into fluoride induces cardiotoxicity in chickens: Involving the regulation of PERK/IRE1/ATF6 pathway and heat shock proteins.

Fluorosis poses a significant threat to human and animal health and is an urgent public safety concern in various countries. Subchronic exposure to fluoride has the potential to result in pathological damage to the heart, but its potential mechanism requires further investigation. This study investigated the effects of long-term exposure to sodium fluoride (0, 500, 1000, and 2000 mg/kg) on the hearts of chickens were investigated. The results showed that an elevated exposure dose of sodium fluoride led to congested cardiac tissue and disrupted myofiber organisation. Sodium fluoride exposure activated the ERS pathways of PERK, IRE1, and ATF6, increasing HSP60 and HSP70 and decreasing HSP90. The NF-κB pathway and the activation of TNF-α and iNOS elicited an inflammatory response. BAX, cytc, and cleaved-caspase3 were increased, triggering apoptosis and leading to cardiac injury. The abnormal expression of HSP90 and HSP70 affected the stability and function of RIPK1, RIPK3, and MLKL, which are crucial necroptosis markers. HSPs inhibited TNF-α-mediated necroptosis and apoptosis of the death receptor pathway. Sodium fluoride resulted in heart injury in chickens because of the ERS and variations in HSPs, inducing inflammation and apoptosis. Cardiac-adapted HSPs impeded the activation of necroptosis. This paper may provide a reference for examining the potential cardiotoxic effects of sodium fluoride.

Nigella sativa oil restores hormonal levels, and endocrine signals among thyroid, ovarian, and uterine tissues of female Wistar rats following sodium fluoride toxicity.

The current study aimed to explore the possible prophylactic and therapeutic effect of Nigella sativa L. oil (NSO) against disruption of endocrine signals and injuries in the thyroid gland, ovary, and uterine tissues induced by sodium fluoride (NaF). Twenty-eight mature female Wistar rats were randomly allocated into four experimental groups (n = 7/group) as follows: control group; NaF group, orally received NaF (20 mg/kg b.wt.) daily; NSO/NaF, orally received NSO (300 mg/kg b.wt.) two weeks before being given NaF and continued throughout the experiment; and NSO+NaF group orally received NSO concurrently with NaF. Our results indicated that NSO restored hormonal balance and suppressed oxidative damage and inflammation. Moreover, the levels of triiodothyronine, thyroxine, thyroid peroxidase, estrogen (E2), progesterone, follicle-stimulating hormone, and luteinizing hormone were elevated, while prostaglandins F2-α and cortisol levels were decreased in NSO treated groups compared to NaF-intoxicated rats. As well, NSO significantly boosted levels of antioxidant molecules, and lowered lipid peroxidation of examined tissues, unlike NaF-treated group. NSO also up-regulated antioxidant enzymes, anti-apoptotic protein, zona pellucida sperm-binding protein, bone morphogenetic protein, and thyroid stimulating hormone, conversely down-regulated inflammatory cytokines, apoptotic proteins, estrogen receptor-α, estrogen receptor-ß, and thyroid stimulating hormone receptors compared to NaF-intoxicated group. Additionally, NSO ameliorated tissue damage of the thyroid gland, ovary, and uterus induced by NaF. -Overall, the prophylactic group (NSO/NaF) performed better antioxidant and anti-inflammatory activities than the treated group almost in all examined tissues, which is reflected by the improvement in the structure of the thyroid, ovarian, and uterine tissues.

Vitamin D may assist the UPR against sodium fluoride-induced damage by reducing RIPK1, ATG5, BECN1, oxidative stress and increasing caspase-3 in the osteoblast MC3T3-E1 cell line.

BACKGROUND: Out of all measure systemic exposure to fluorides can cause defect of skeletal and dental fluorosis. Endoplasmic reticulum (ER) stress is caused by fluorine-induced oxidative stress and importance of vitamin D in its prevention is not known enough in bone cells. This study was carried out to investigate fluorine-induced oxidative stress, ER stress, and death pathways and the effect of vitamin D on them. METHODS: MC3T3-E1 mouse osteoblast cell line was used as the material of the study. The NaF and vitamin D concentrations were determined by the MTT assay. NaF treatments and vitamin D supplementation (pre-add, co-add, and post-add) was administered in the cell line at 24th and 48th hours. The expression of the genes in oxidative stress, ER stress, and death pathways was determined using RT-qPCR and Western blotting techniques. RESULTS: Vitamin D significantly reduced mRNA expression levels of SOD2, CYGB, ATF6, PERK, IRE1, ATG5 and BECN1 whereas caused an increase in levels GPX1, SOD1, NOS2 and Caspase-3 in MC3T3-E1 mouse osteoblast cell line of NaF-induced. In addition, GPX1, SOD1, ATF6, PERK, IRE1, BECN1, Caspase-3 and RIPK1 protein levels were examined by Western blot analysis, and it was determined that vitamin D decreased IRE1 and PERK protein levels, but increased GPX1, SOD1, ATF6 and Caspase-3 protein levels. CONCLUSION: The findings of the study suggest that vitamin D has protective potential against NaF-induced cytotoxicity reasonably through the attenuation of oxidative stress, ER stress, ATG5, IRE1 and by increasesing caspase-3 in vitro conditions.

Morphological changes and Mitochondrial alterations on Cardiomyocytes exposed to Fluoride.

OBJECTIVE: To evaluate the morphological changes of cardiomyocytes exposed to different sodium fluoride (NaF) concentrations, as well as to evaluate the behavior of the mitochondria. METHODS: Rat H9c2 cardiomyocytes were exposed to NaF at concentrations of 0.5 to 5 mmol/L. The morphology and number of mitochondria in these cells were monitored, and the calcium ion (Ca2+) concentration was determined. RESULTS: Morphological changes were evident in the cells treated with different NaF concentrations, and both the number of mitochondria and the Ca2+ concentration decreased in a dose-dependent manner. CONCLUSION: Sodium fluoride induced morphological damage in cardiomyocytes, decreases the Ca2+ concentration and mitochondrial number.

Neuroprotective effect by naringin against fluorosis-induced neurodegeneration in adult Wistar rats.

Fluorosis is widespread in several areas of the world and including India leading to dental and skeletal fluorosis as well as neurological manifestations. With a limited number of treatment options available, we have tried to address the issue with a nutraceutical such as naringin which is an alkaloid derived from the citrus fruit. Naringin is a potent antioxidant and has neuroprotective action which can counteract the redox imbalance induced by sodium fluoride ingestion. Neurological effects of fluorosis were evaluated in Wistar rats by open field test (OFT) and novel object recognition test (NORT) along with lipid peroxidation (LPO) and glutathione estimation in brain homogenate and cresyl violet staining of CA3 neurons in the hippocampus. Animals were divided into groups namely, normal, vehicle, fluoride, naringin 100 mg/kg bd.wt group and fluoride with naringin (FLU-NAR) group. Fluorosis was induced by providing 100 ppm of sodium fluoride ad libitum in drinking water for 30 days and prophylactic treatment of naringin for 15 days per oral. OFT, NORT and forced swim test showed significant (P ≤ 0.05) changes in the FLU-NAR group as compared to the fluoride group indicating behavioral changes in the fluoride group and positive changes in the FLU-NAR group with attenuation of stress, fear, hyperactivity and memory impairment. The decrease in LPO and increase in glutathione levels in the treatment group compared to the fluoride group were supported by histological improvement as compared to the fluoride group. Prophylactic treatment of naringin showed its possible neuroprotective effect, thus giving an alternative treatment strategy to deal with neurological manifestations of fluorosis.

PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

Chronic fluoride exposure can cause developmental neurotoxicity, however the precise mechanisms remain unclear. To explore the mechanism of mitophagy in fluoride-induced developmental neurotoxicity, specifically focusing on PRKAA1 in regulating the PINK1/Parkin pathway, we established a Sprage Dawley rat model with continuous sodium fluoride (NaF) exposure and an NaF-treated SH-SY5Y cell model. We found that NaF exposure increased the levels of LC3-Ⅱ and p62, impaired autophagic degradation, and subsequently blocked autophagic flux. Additionally, NaF exposure increased the expression of PINK1, Parkin, TOMM-20, and Cyt C and cleaved PARP in vivo and in vitro, indicating NaF promotes mitophagy and neuronal apoptosis. Meanwhile, phosphoproteomics and western blot analysis showed that NaF treatment enhanced PRKAA1 phosphorylation. Remarkably, the application of both 3-methyladenosine (3-MA; autophagy inhibitor) and dorsomorphin (DM; AMPK inhibitor) suppressed NaF-induced neuronal apoptosis by restoring aberrant mitophagy. In addition, 3-MA attenuated an increase in p62 protein levels and NaF-induced autophagic degradation. Collectively, our findings indicated that NaF causes aberrant mitophagy via PRKAA1 in a PINK1/Parkin-dependent manner, which triggers neuronal apoptosis. Thus, regulating PRKAA1-activated PINK1/Parkin-dependent mitophagy may be a potential treatment for NaF-induced developmental neurotoxicity.

Effects of Treadmill Exercise on Liver Apoptosis in Fluoride-Exposed Mice.

Hepatotoxicity induced by excessive fluoride (F) exposure has been extensively studied in both humans and animals. Chronic fluorosis can result in liver apoptosis. Meanwhile, moderate exercise alleviates apoptosis caused by pathological factors. However, the effect of moderate exercise on F-induced liver apoptosis remains unclear. In this research, sixty-four three-week-old Institute of Cancer Research (ICR) mice, half male and half female, were randomly divided into four groups: control group (distilled water); exercise group (distilled water and treadmill exercise); F group [100 mg/L sodium fluoride (NaF)]; and exercise plus F group (100 mg/L NaF and treadmill exercise). The liver tissues of mice were taken at 3 months and 6 months, respectively. Hematoxylin-eosin (HE) staining and situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) results showed that nuclear condensation and apoptotic hepatocytes occurred in the F group. However, this phenomenon could be reversed with the intervention of treadmill exercise. The results of QRT-PCR and western blot displayed NaF- induced apoptosis via tumor necrosis factor recpter 1 (TNFR1) signaling pathway, while treadmill exercise could restore the molecular changes caused by excessive NaF exposure.

Nrf2/PINK1-mediated mitophagy induction alleviates sodium fluoride-induced hepatic injury by improving mitochondrial function, oxidative stress, and inflammation.

Mitophagy has distinct functions, which can lead to either protection or damage of tissues. Though current evidence indicated that NaF triggers mitophagy, the role and regulation of mitophagy in sodium fluoride (NaF)-induced liver injury still remain unclear. Therefore, we exployed the cell and mouse models and confirmed that NaF treatment activates mitophagy. Knocking down PTEN-induced putative kinase protein 1 (PINK1) expression attenuated mitophagy and increased the degree of mitochondrial impairment, oxidative stress, and apoptosis in NaF-treated HepG2 cells. In vivo experiments indicated that PINK1 deficiency weakened NaF-induced mitophagy. Moreover, PINK1-deficient mices aggravated NaF-induced hepatic mitochondrial injury, oxidative stress, and inflammation in livers, evidenced by the increased number of abnormal mitochondria, decreased adenosine triphosphate (ATP) and glutathione (GSH) levels, elevated reactive oxygen species (ROS) and malondialdehyde (MDA) content, enhanced hepatic macrophage infiltration and inflammatory cytokine levels. Notably, NaF exposure activated Nrf2 signaling both in vitro and in vivo. Nrf2 siRNA transfection blocked the upregulation of PINK1 expression and the induction of mitophagy in NaF-treated HepG2 cells. Also, ML385 (Nrf2 inhibitor) partially blocked the upregulation of PINK1 expression caused by NaF in mice livers. To sum up, the present study provided the demonstration that Nrf2/PINK1-mediated mitophagy activation offers a hepatoprotective effect by inhibiting NaF-induced mitochondrial dysfunction, oxidative stress, and inflammation.

Contribution of Oxidative Stress, Apoptosis, Endoplasmic Reticulum Stress and Autophagy Pathways to the Ameliorative Effects of Hesperidin in NaF-Induced Testicular Toxicity.

The ameliorative effects of hesperidin (HES) on the toxicities created by sodium fluoride (NaF) in the testes tissue of rats were studied via oxidative stress, apoptosis and endoplasmic reticulum (ER) stress pathways. The animals were divided into five distinct groups (7 rats in each group). Group 1 was control group, group 2 received NaF-only (600 ppm), group 3 received HES-only (200 mg/kg bw); group 4 received NaF (600 ppm)+HES (100 mg/kg bw) and group 5 received NaF (600 ppm)+HES (200 mg/kg bw) for 14 days. NaF-induced testes tissue damage by reducing activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and levels of glutathione (GSH), and increasing lipid peroxidation levels. NaF treatment significantly downregulated the mRNA levels of SOD1, CAT and GPx. NaF supplementation caused apoptosis in the testes by upregulating p53, NFkB, caspase-3, caspase-6, caspase-9, and Bax and downregulating Bcl-2. Furthermore, NaF caused ER stress via increasing mRNA transcript levels of PERK, IRE1, ATF-6 and GRP78. NaF treatment led to autophagy via upregulation of Beclin1, LC3A, LC3B and AKT2. In testes tissue, however, co-treatment with HES at doses of 100 and 200 mg/kg significantly reduced oxidative stress, apoptosis, autophagy and ER stress. Overall, the findings of this study suggest that HES may help to reduce testes damage caused by NaF toxicity.

TFE3-mediated impairment of lysosomal biogenesis and defective autophagy contribute to fluoride-induced hepatotoxicity.

Excessive fluoride exposure can cause liver injury, but the specific mechanisms need further investigation. We aimed to explore the role of impaired lysosomal biogenesis and defective autophagy in fluoride-induced hepatotoxicity and its potential mechanisms, focusing on the role of transcription factor E3 (TFE3) in regulating hepatocyte lysosomal biogenesis. To this end, we established a Sprague-Dawley (SD) rat model exposed to sodium fluoride (NaF) and a rat liver cell line (BRL3A) model exposed to NaF. The results showed that NaF exposure diminished liver function and led to apoptosis as well as autophagosome accumulation and impaired autophagic degradation. In addition, NaF exposure caused compromised lysosome biogenesis and decreased lysosomal degradation, and inhibited TFE3 nuclear translocation. Notably, the mTOR inhibitors rapamycin (RAPA) and Ad-TFE3 promoted lysosomal biogenesis and enhanced lysosomal degradation function. Furthermore, RAPA and Ad-TFE3 reduced NaF-induced apoptosis by alleviating impaired autophagic degradation. In conclusion, NaF impairs lysosomal biogenesis by inhibiting TFE3 nuclear translocation, decreasing lysosomal degradation function, resulting in impaired autophagic degradation, and ultimately inducing apoptosis. Therefore, TFE3 may be a promising therapeutic target for fluoride-induced hepatotoxicity.

The inhibition of TRPML1/TFEB leads to lysosomal biogenesis disorder, contributes to developmental fluoride neurotoxicity.

Fluoride is capable of inducing developmental neurotoxicity; regrettably, the mechanism is obscure. We aimed to probe the role of lysosomal biogenesis disorder in developmental fluoride neurotoxicity-specifically, the regulating effect of the transient receptor potential mucolipin 1 (TRPML1)/transcription factor EB (TFEB) signaling pathway on lysosomal biogenesis. Sprague-Dawley rats were given fluoridated water freely, during pregnancy to the parental rats to 2 months after delivery to the offspring. In addition, neuroblastoma SH-SY5Y cells were treated with sodium fluoride (NaF), with or without mucolipin synthetic agonist 1 (ML-SA1) or adenovirus TFEB (Ad-TFEB) intervention. Our findings revealed that NaF impaired learning and memory as well as memory retention capacities in rat offspring, induced lysosomal biogenesis disorder, and decreased lysosomal degradation capacity, autophagosome accumulation, autophagic flux blockade, apoptosis, and pyroptosis. These changes were evidenced by the decreased expression of TRPML1, nuclear TFEB, LAMP2, CTSB, and CTSD, as well as increased expression of LC3-II, p62, cleaved PARP, NLRP3, Caspase1, and IL-1ß. Furthermore, TRPML1 activation and TFEB overexpression both restored TFEB nuclear protein expression and promoted lysosomal biogenesis while enhancing lysosomal degradation capacity, recovering autophagic flux, and attenuating NaF-induced apoptosis and pyroptosis. Taken together, these results show that NaF promotes the progression of developmental fluoride neurotoxicity by inhibiting TRPML1/TFEB expression and impeding lysosomal biogenesis. Notably, the activation of TRPML1/TFEB alleviated NaF-induced developmental neurotoxicity. Therefore, TRPML1/TFEB may be promising markers of developmental fluoride neurotoxicity.

L-arginine and lisinopril supplementation protects against sodium fluoride-induced nephrotoxicity and hypertension by suppressing mineralocorticoid receptor and angiotensin-converting enzyme 3 activity.

Sodium fluoride (NaF) is one of the neglected environmental toxicants that has continued to silently cause toxicity to both humans and animals. NaF is universally present in water, soil, and atmosphere. The persistent and alarming rate of increase in cardiovascular and renal diseases caused by chemicals such as NaF in mammalian tissues has led to the use of various drugs for the treatment of these diseases. The present study aimed at evaluating the renoprotective and antihypertensive effects of L-arginine against NaF-induced nephrotoxicity. Thirty male Wistar rats (150-180 g) were used in this study. The rats were randomly divided into five groups of six rats each as follows: Control, NaF (300 ppm), NaF + L-arginine (100 mg/kg), NaF + L-arginine (200 mg/kg), and NaF + lisinopril (10 mg/kg). Histopathological examination and immunohistochemistry of renal angiotensin-converting enzyme (ACE) and mineralocorticoid receptor (MCR) were performed. Markers of renal damage, oxidative stress, antioxidant defense system, and blood pressure parameters were determined. L-arginine and lisinopril significantly (P < 0.05) ameliorated the hypertensive effects of NaF. The systolic, diastolic, and mean arterial blood pressure of the treated groups were significantly (P < 0.05) reduced compared with the hypertensive group. This finding was concurrent with significantly increased serum bioavailability of nitric oxide in the hypertensive rats treated with L-arginine and lisinopril. Also, there was a significant reduction in the level of blood urea nitrogen and creatinine of hypertensive rats treated with L-arginine and lisinopril. There was a significant (P < 0.05) reduction in markers of oxidative stress such as malondialdehyde and protein carbonyl and concurrent increase in the levels of antioxidant enzymes in the kidney of hypertensive rats treated with L-arginine and lisinopril. The results of this study suggest that L-arginine and lisinopril normalized blood pressure, reduced oxidative stress, and the expression of renal ACE and mineralocorticoid receptor, and improved nitric oxide production. Thus, L-arginine holds promise as a potential therapy against hypertension and renal damage.

Fluoride Exposure Provokes Mitochondria-Mediated Apoptosis and Increases Mitophagy in Osteocytes via Increasing ROS Production.

Fluoride is a persistent environmental pollutant, and its excessive intake causes skeletal and dental fluorosis. However, few studies focused on the effects of fluoride on osteocytes, making up over 95% of all bone cells. This study aimed to investigate the effect of fluoride on osteocytes in vitro, as well as explore the underlying mechanisms. CCK-8, LDH assay, fluorescent probes, flow cytometry, and western blotting were performed to examine cell viability, apoptosis, mitochondria changes, reactive oxygen species (ROS) and mitochondrial ROS (mtROS), and protein expressions. Results showed that sodium fluoride (NaF) exposure (4, 8 mmol/L) for 24 h inhibited the cell viability of osteocytes MLO-Y4 and promoted G0/G1 phase arrest and increased cell apoptosis. NaF treatment remarkably caused mitochondria damage, loss of MMP, ATP decrease, Cyto c release, and Bax/Bcl-2 ratio increase and elevated the activity of caspase-9 and caspase-3. Furthermore, NaF significantly upregulated the expressions of LC-3II, PINK1, and Parkin and increased autophagy flux and the accumulation of acidic vacuoles, while the p62 level was downregulated. In addition, NaF exposure triggered the production of intracellular ROS and mtROS and increased malondialdehyde (MDA); but superoxide dismutase (SOD) activity and glutathione (GSH) content were decreased. The scavenger N-acetyl-L-cysteine (NAC) significantly reversed NaF-induced apoptosis and mitophagy, suggesting that ROS is responsible for the mitochondrial-mediated apoptosis and mitophagy induced by NaF exposure. These findings provide in vitro evidence that apoptosis and mitophagy are cellular mechanisms for the toxic effect of fluoride on osteocytes, thereby suggesting the potential role of osteocytes in skeletal and dental fluorosis.

Excessive Lysosomal Stress Response and Consequently Impaired Autophagy Contribute to Fluoride-Induced Developmental Neurotoxicity.

Fluoride can cause developmental neurotoxicity; however, the precise mechanism has yet to be determined. We aimed to explore the possible role and mechanism of fluoride-induced developmental neurotoxicity, specifically the significance of the lysosomal stress response. As an in vivo model, Sprague Dawley rats were exposed to sodium fluoride (NaF) from embryo to 2 months of age. We found that NaF caused autophagic flux blockage and apoptosis in the rat hippocampus. These results were validated in human neuroblastoma (SH-SY5Y) cells in vitro. In addition, in SH-SY5Y cells, NaF hindered autophagosome-lysosome fusion, decreased lysosomal degradation, and elevated lysosomal pH, which is the most prominent hallmark of a lysosomal stress response. Interestingly, rapamycin promoted autophagosome-lysosome fusion, effectively restoring autophagic flux and reducing apoptosis. Notably, bafilomycin A1, a lysosomal lumen alkalizer, unsurprisingly exacerbated the NaF-induced increase in lysosomal pH and decreased lysosomal degradability, as well as enhanced apoptosis of SH-SY5Y cells. In conclusion, our results suggest that NaF exposure initiates excessive lysosomal stress response, resulting in elevated lysosomal pH, decreased lysosomal degradation, and blocked autophagic flux, which leads to neuronal apoptosis. Thus, the lysosomal stress response may be a promising target for the prevention and treatment of fluoride-induced developmental neurotoxicity.