RESUMO
Fluoride, a toxic substance, is widely distributed in the environment and causes serious damage to the body. This study was performed to investigate the effects of fluoride on mitochondrial fission in mouse hepatocytes. A total of 48 mice were equally divided into four groups and admisnistered with NaF in drinking water at fluorine ion concentrations of 0, 25, 50 and 100 mg/L for 70 days. The pathomorphology and ultrastructurre of hepatocytes were then observed. The mitochondrial lesion parameters (number, length, width and vacuolization area) are evaluated. The expression of Drp1, Mff, Fis1, MiD49, MiD51 and Dyn2, which are associated with mitochondrial fission, was determined by quantitative real-time PCR and Western blot analysis. Apoptosis was detected by using TUNEL assay. Results showed that fluoride causes notable changes in the pathological morphology of liver tissues and severely damages the ultrastructure of hepatocytes. Damage manifested as nuclear condensation, nuclear membrane breakdown, mitochondrial vacuolation, increased fragmentation, and mitochondrial fission. Moreover, mRNA and protein expression levels were significantly upregulated in the Drp1/Mff signaling pathway. The mRNA expression levels of Cyt c, caspase 9 and 3 markedly increased in the fluoride treated groups in a dose-dependent manner. The percentage of TUNEL-positive nuclei in the liver remarkably increased after fluoride treatment. Overall, the results indicate that excessive fluoride exposure can increase mitochondrial fission via the Drp1/Mff signaling pathway, severely damage the mitochondrial structure, and lead to apoptosis of hepatocytes.
Assuntos
Fluoretos , Proteínas Mitocondriais , Animais , Apoptose , Dinaminas , Hepatócitos , Camundongos , Mitocôndrias , Transdução de SinaisRESUMO
The role of pro-inflammatory cytokines in the toxicity of fluoride to tumor cells was investigated by culturing Hepa1-6 cells in medium containing gradient concentrations of fluoride (0, 0.5, 1, 1.5, 2, 3, 4, and 5 mmol/L). The viability of Hepa1-6 cells was detected via MTT assay. Interleukin (IL)-2, IL-6, tumor necrosis factor (TNF)-α, and IL-1ß levels in the supernatant were determined via an enzyme-linked immunosorbent assay, and the protein expression levels of these enzymes in Hepa1-6 cells were evaluated by immunofluorescence staining. Results showed that the viability of Hepa1-6 cells remarkably decreases after fluoride exposure, especially at concentration of 3, 4, and 5 mmol/L fluoride. Levels of IL-2, TNF-α, and IL-1ß in the supernatant markedly decreased when cells were exposed to fluoride at concentrations of 1 mmol/L or higher. However, levels of TNF-α and IL-1ß substantially increased and IL-2 showed no remarkable change when the fluoride concentration was 0.5 mmol/L. The content of IL-6 remarkably increased with increasing fluoride concentrations up to 2 mmol/L, and then markedly decreased at 3, 4, and 5 mmol/L fluoride; the decreasing trend of IL-6 content under high fluoride exposure is consistent with the decrease in Hepa1-6 cell viability observed at the same concentration. The protein expression levels of IL-2, IL-6, TNF-α, and IL-1ß were in accordance with their contents in the supernatant. In summary, our study demonstrated that fluoride inhibits Hepa1-6 cell growth and results in disorders in the expression and secretion pro-inflammatory cytokines.
Assuntos
Citocinas , Fluoretos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Fluoretos/toxicidade , Interleucina-1beta , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfaRESUMO
Intestinal microflora plays a key role in maintaining the homeostasis between immune and host health. Here, we reported the fluoride-induced changes of rectal structure and microflora in mice. The morphology of rectal tissue was observed by hematoxylin and eosin staining. The rectal development parameters (the thickness of mucosa, intestinal gland and muscle layer) were evaluated. The proliferation of rectal epithelial cells was evaluated via BrdU labeling. The distribution of goblet, glycoprotein and mast cell were evaluated by specific staining. Rectal microflora was detected using 16S rRNA high-throughput sequencing. The results showed that the rectal structure was seriously damaged and the proliferation of rectal epithelial cells was significantly inhibited by fluoride. The distribution of goblet cells, glycoprotein and mast cells decreased significantly after fluoride exposure. The relative richness of microfloras was changed after fluoride treatment, such as increased Bacteroidetes and decreased Firmicutes. In summary, this study indicated that excessive fluoride damages the intestinal structure, disturbs the intestinal micro-ecology and causes intestinal microflora disorder in mice. Findings mentioned in the present study enrich a new scope for elucidating fluoride toxicity from intestinal homeostasis.
Assuntos
Fluoretos/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Reto/efeitos dos fármacos , Animais , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , RNA Ribossômico 16S , Reto/patologiaRESUMO
A fluoride exposure mouse model is established to evaluate the relationship between mitochondrial respiratory chain complexes and renal dysfunction. Morphological changes in kidney tissues were observed. Renal function and cell proliferation in the kidneys were evaluated. The expression of mitochondrial fusion protein including mitofusin-1 (Mfn1) and optic atrophy 1 (OPA1), and mitochondrial respiratory chain complex subunits, including NDUFV2, SDHA, CYC1 and COX â £, were detected via real-time polymerase chain reaction, immunohistochemistry staining and Western blot, respectively. Results showed that the structures of renal tubule, renal glomerulus and renal papilla were seriously damaged. Renal function was impaired, and cell proliferation was remarkably inhibited by excessive fluoride in kidney. The mRNA and protein expression levels of Mfn1, OPA1, NDUFV2, CYC1 and COX â £ were significantly increased after excessive fluoride exposure. However, the mRNA and protein expression of SDHA significantly decreased. Overall, our findings revealed that excessive fluoride can damage kidney structure, inhibit renal cell proliferation, interfere with the expression of mitochondrial respiratory chain complexes and elevate mitochondrial fusion. Consequently, renal function disorder occurred.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Fluoretos/toxicidade , Doenças Mitocondriais/induzido quimicamente , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Insuficiência Renal/induzido quimicamente , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Transporte de Elétrons , GTP Fosfo-Hidrolases/metabolismo , Rim/patologia , Camundongos , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , RNA Mensageiro/biossínteseRESUMO
Intestinal microflora play an important role in maintaining the homeostasis of the intestinal microenvironment, but fluoride-induced changes in intestinal mechanical barrier and intestinal microflora have not been well studied. Given this paucity of information, this study aims to determine the effects of high fluoride level on intestinal mechanical barrier and intestinal microflora in the cecum of mice. Seventy-two female 21-day-old Kunming mice were randomly assigned to three groups and raised for 70 days. Changes in intestinal pathomorphology and intestinal epithelial cell proliferation were observed by haematoxylin and eosin-staining and Brdu measurement, respectively. The distribution of goblet cells, glycoproteins and mast cells was analysed through Alcian blue and periodic acid-Schiff (AB-PAS) staining, Periodic Acid-Schiff (PAS) staining, and toluidine blue staining. Results showed that excessive fluoride damaged the structure of the cecal tissues, inhibited epithelial cell proliferation and decreased the relative distribution of goblet cells, glycoproteins and mast cells that are involved in defense responses. Intestinal microflora sequencing analysis revealed that the composition of the diversity and composition of intestinal microflora was altered by excessive fluoride based on 16S rRNA amplicon sequencing. The relative abundance of Firmicutes (Pâ¯=â¯0.03174), Bacteroidetes (Pâ¯=â¯0.04462), Actinobacteria (Pâ¯=â¯0.01085) and Spirochacteria (Pâ¯=â¯0.04084) was significantly changed in the fluoride group as compared with the control group. In conclusion, excessive fluoride intake induced intestinal barrier damage, leading to changes in cecal composition, epithelium secretion and intestinal microflora.