Single oral acute fluoride exposure causes changes in cardiac expression of oxidant and antioxidant enzymes, apoptotic and necrotic markers in male rats.

Several studies have shown that acute fluoride (F(-)) exposure impairs cardiac function, but the molecular mechanism is not clear. In order to study this, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F(-) for 24 h. A significant accumulation of F(-) was found in the serum and myocardium of experimental rats. F(-) treatment causes myocardial necrosis as evident from increased levels of myocardial troponin I, creatine kinase, lactate dehydrogenase and aspartate transaminase. In addition, F(-) induces myocardial oxidative stress via increased reactive oxygen species, lipid peroxidation, protein carbonyl content and nitrate levels along with decreased in the levels of enzymatic (superoxide dismutase 2, catalase, glutathione peroxidase and glutathione s transferase pi class) and non-enzymatic (reduced glutathione) antioxidants. Notably, F(-) triggers myocardial apoptosis through altered Bax/Bcl2 ratio and increased cytochrome c, caspase 3p20 and terminal deoxynucleotidyl transferase dUTP nick end labeled positive cells. An increased cardiac expression of Nox4 and p38α MAPK in F(-) treated rats indicates the oxidative and apoptotic damage. Moreover, ultra-structural changes, histopathological and luxol fast blue staining demonstrates the degree of myocardial damage at subcellular level. Taken together, these findings reveal that acute F(-) exposure causes cardiac impairment by altering the expression of oxidative stress, apoptosis and necrotic markers.

[Expression of sonic hedgehog signaling pathw ay and its inhibition by cyclopamine in rat liver with chronic fluorosis].

OBJECTIVE: To investigate the expression of sonic hedgehog (Shh) signaling pathway in liver fluorosis and to explore related mechanism. METHODS: To establish animal model, 48 normal SD rats (aged 4-5 weeks) were randomly divided into 4 groups (12 each): control group, fluoriosis group, blocking group and blocking control group. After 6 months, the blocking group and blocking control group were injected intraperitoneally once every 2 days for 3 times with 10 mg/kg cyclopamine or dimethysulfoxide, respectively. Rats were sacrificed at the end of the experiment and the fluoride content in urine and liver function was determined. The expression of Shh and Gli1 protein and mRNA in hepatocytes was detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively. RESULTS: The fluoride contents in the urine and the incidence of dental fluorosis increased in the fluoride and blocking control groups as compared with those in the control group, but decreased in the blocking group compared with those of the fluoride and blocking control group. Compared with the control group, the titers of aspartate transaminase (AST) and alanine transaminase (ALT) significantly increased, while the activity of total protein and albumin decreased in the fluoride and blocking control groups. Compared with the fluoride and blocking control groups, the activity of the ALT slightly declined and the AST, total protein and albumin slightly increased in the blocking group. Histologically, the cells were disorganized and swollen with cytoplasmic clearing (balloon cells), compared with the control group. The expression of Shh and Gli1 significantly increased in all but the control group. Compared with the fluoride and blocking control groups, the expression of Shh and Gli1 declined in the blocking group. CONCLUSIONS: The overexpression and cyclopamine inhibition of the Shh signaling pathway are closely related to the content of fluoride in the liver. The Shh signaling pathway plays an important role in the pathogenesis of liver injury caused by fluorosis, suggesting a preventive and therapeutic target of the disease.

[Expression of mRNA and protein of p38, Osx, PI3K and Akt1 in rat bone with chronic fluorosis].

OBJECTIVE: To investigate the expressions of mRNA and protein of p38, Osx, PI3K, Akt1 in the rats bone with chronic fluorosis. METHODS: Dental fluorosis were observed and the fluoride contents in the urine and bone were detected by fluorin-ion selective electrode. The morphologic changes and ultrastructure of rats' bone were observed by light and electronic microscopy. The expressions of protein and mRNA of p38, Osx, PI3K and Akt1 were detected by immunohistochemistry and real-time PCR, respectively. The contents of BALP and BGP in serum were detected by ELISA. RESULTS: The rates of dental fluorosis in the fluorosis rats were increased, and the fluoride contents in bone and urine of the fluorosis rats were increased compared to the control group, the difference was statistically significant (P < 0.05). The bone trabeculae thickness and density and the thickness of bone cortex in fluorosis rats were remarkably increased, the space of bone trabeculae was reduced, and in accordance with the matching morphometrical indices, the difference was statistically significant (P < 0.05) as compared with the control rats. The contents of BALP [(54.61 ± 2.27) U/L] and BGP [(2.38 ± 0.16) µg/L]in the fluoride groups were higher than those in the control group, the difference was statistically significant (P < 0.05). Ultrastructurally, the broadening of the osseouslacuna was observed. The reduced protuberances of the osteocytes, the unclear organelle structure, pyknosis, karyotheca increasation and edged chromatin were also observed. Compared to the control group, the expressions of protein and its mRNA of p38, Osx, PI3K and Akt1 were higher in the fluorosis rats than those in the control rats, and the difference was statistically significant (P < 0.05). There is no any expression of p38, Osx, PI3K and Akt1 in the osteocytes in fluorosis rats. CONCLUSIONS: The over-expression of p38, Osx, PI3K and Akt1 in bone tissue of fluorosis rats may relate to the accumulation of fluorine in the body. The bone injury mainly occur in the stage of the differentiation and proliferation. The upregulation of P38MARK signal path and PI3K/Akt1 signal path may be involved in the pathogenesis of bone injury caused by fluoride.

Efficacy of lycopene against fluoride toxicity in rats.

CONTEXT: Oxidative damage to cellular components such as lipids and cell membranes by free radicals and reactive oxygen species (ROS) is thought to be associated with the development of degenerative diseases. Fluoride intoxication is associated with oxidative stress and altered anti-oxidant defense mechanism. Lycopene is a lipid-soluble powerful anti-oxidant that scavenges free radicals and ROS. OBJECTIVE: This study was extended to investigate lycopene anti-oxidant efficacy in different organs of fluoride-intoxicated rats. METHODS: Twenty-four adult rats were randomly divided into four groups of six animals each. Rats in group I received daily doses of vehicle. Group II rats were given lycopene (10 mg/kg body weight/day), by tubes, dissolved in 0.5 ml of corn oil for 5 weeks. Group III rats were given sodium fluoride (NaF) (10.3 mg/kg body weight/day), by tubes, for 5 weeks. In group IV rats, lycopene was administered 1 h later and NaF was administered for 5 weeks. RESULTS: NaF administration induced oxidative stress as evidenced by elevated levels of lipid peroxidation (51.3, 65.9 and 67.6%) measured as malondialdehyde and total nitrate/nitrite (61.0, 59.7 and 68.9%) in red blood cells, heart and brain tissues. Moreover, significantly decreased reduced glutathione level, total anti-oxidant capacity and superoxide dismutase activity were observed in the examined tissues. The induced oxidative stress and the alterations in anti-oxidant system were normalized by the oral administration of lycopene treatment. CONCLUSION: Lycopene administration could minimize the toxic effects of fluoride indicating its free-radical scavenging and powerful anti-oxidant activities.

Antioxidant and ACE enhancing potential of Pankajakasthuri in fluoride toxicity: an in vitro study on mammalian lungs.

Fluoride toxicity occurs due to high concentrations of fluoride in water sources or anthropogenic causes. The aim of the present study was to investigate the effects of an Ayurvedic drug--Pankajakasthuri (PK)--in relation to fluoride-induced toxicity in mammalian lungs. The results indicated that sodium fluoride increased lipid peroxidation and decreased enzymatic and non-enzymatic antioxidants in a concentration-dependent manner in lungs. The antioxidant potential of the lungs was suppressed maximally at 10 ppm fluoride concentration and PK at all three dose levels (i.e., 100, 200 and 300 µl) decreased fluoride induced lipid peroxidation (p < 0.05) and increased the levels of total ascorbic acid, superoxide dismutase, catalase, reduced glutathione, glutathione peroxidase and FRAP values significantly (p < 0.05) in a dose-dependent manner. When PK was examined for its effects on angiotensin-converting enzyme (ACE) activity, in fluoride-induced toxicity, the ACE activity was found to increase (p < 0.0001) in lung homogenates with all three doses. This study indicates that PK, an Ayurvedic drug, improves mammalian lung function by increasing antioxidant potential and ACE activity under the conditions of fluoride toxicity.

Effect of fluoride on calcium ion concentration and expression of nuclear transcription factor kappa-B ρ65 in rat hippocampus.

The study investigated the neurotoxicity of drinking water fluorosis in rat hippocampus. Just weaning male Sprague-Dawley (SD) rats were given 15, 30, 60 mg/L NaF solution and tap water for 9 months. The calcium ion concentration ([Ca(2+)]) in synaptosomes was measured by double wavelength fluorescence spectrophotometer and the expression level of nuclear transcription factor kappa-B ρ65 (NF-κB ρ65) in hippocampal CA3 region was measured by immunohistochemistry. The results showed that [Ca(2+)] significantly increased (F = 33.218, P < 0.01) in moderate fluoride group compared with the control group, and the expression level of NF-κB ρ65 in CA3 region presented an increasing trend as fluoride concentration increased. These results indicate that increase of synaptosomes [Ca(2+)] and NF-κB ρ65 expression level may be the molecular basis of central nervous system damage caused by chronic fluoride intoxication. NF-κB ρ65 in CA3 region is probably a target molecule for fluorosis.

Elevation of PTH and PTHrp induced by excessive fluoride in rats on a calcium-deficient diet.

Study on the role of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrp) in the process of skeletal fluorosis, involved especially in calcium deficiency, is rare. We evaluated the level of serum PTH and mRNA expression of PTHrp in femur when rats were exposed to excessive fluoride with low-calcium diet. Wistar rats (n = 60) was divided into four groups, a control group, fluoride group, low-calcium group, and low-calcium fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F-/L. The low-calcium diet contained 0.05% calcium. Serum was collected in the first, fourth, eighth, and 12th of phase for the detemination of PTH and Ca(2+). RNA extraction from femora was used to analyze the mRNA express of PTHrp, osteopontin (OPN), and osteocalcin (OCN) after 12 weeks of fluoride dosing. Results showed that serum PTH increased gradually with the extension of fluoride exposure, but Ca2+ decreased, both of which embodied a time-dependent relationship. Cotreatment of excessive fluoride with low-calcium diet largely stimulated the secretion of PTH. The low dietary calcium markedly increased mRNA expression of PTHrp in animals with fluoride treatment. Expression of OPN and OCN significantly increased in the rats treated with excessive fluoride and low-calcium diet. We demonstrated that fluoride by itself affected the body's calcium metabolism and stimulate the secretion of PTH. PTH may play an important role in anabolic effect of excessive fluoride on bone turnover of skeletal fluorosis and calcium deficiency exacerbated the action of PTH and PTHrp on the characteristic bone lesion of fluorosis.

[Fluorosis on expression of nicotinic acetylcholine receptors in protein and gene levels in human SH-SY5Y neuroblastoma cells].

OBJECTIVE: To investigate the influence of fluorosis on nicotinic acetylcholine receptors (nAChRs) in protein and gene levels in SH-SY5Y cells and the mechanism of the receptor modification. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were incubated with different concentrations of fluoride or with antioxidant for 48 hours. The functions of cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and protein oxidation detected by carbonyl content; the alpha3 and alpha7 nAChR subunits in protein level were measured by Western blotting and in mRNA level by RT-polymerase chain reaction (RT-PCR). RESULTS: In high-dose group as compared to the control, the decreased MTT (49%), increased protein oxidation (72%), and lower expression of alpha3 (51%) and alpha7 (47%) nAChR subunit proteins were obviously observed in SH-SY5Y cells. There were no changes in expression of nAChR subunit mRNAs between the cells treated with fluoride and those un-treated in controls. Prior treatment with antioxidant resulted in preventing the decrease of nAChR protein in cells exposed to the high doses of fluoride. CONCLUSION: Fluorosis should result in damage of cells and the declined expression of nAChRs in protein levels, but no influences on gene expression of the receptors in human neuroblastoma neurons. The decreased nAChR proteins might be involved in the mechanism of oxidative stress induced by fluorosis.

[Expression of proto-oncogenes c-fos and c-jun in osteoblasts activated by excessive fluoride].

OBJECTIVE: To study expression of proto-oncogenes c-fos and its accompanying gene c-jun in osteoblasts activated by action of excessive fluoride in vivo and in vitro. METHODS: Experimental Wistar rats were exposed to sodium fluoride (NaF) added to their drinking water, and NaF was also added in cell culture supernatant for osteoblast-like cells in vitro. Expression of both mRNA and protein of c-fos and c-jun in bone-tissue of rats with chronic fluorosis and cultured osteoblast-like cells were determined by hybridization in situ, Western blot and immunohistochemistry at varied time periods after exposure. RESULTS: Sodium fluoride could stimulate the proliferation of osteoblast in rats with chronic fluorosis and induce expression of both c-fos and c-jun in all envelops of the spine bone, as compared with its control group. Value of optical absorption in mRNA expression of c-fos and c-jun was 139.63 and 126.37, respectively, in rats with NaF plus high-calcium, significantly lower than that in control group with high-calcium only (107.74 and 117.48, respectively) (P < 0.001). Immunohistochemical analysis showed that protein level of c-fos and c-jun was significantly higher in rats with NaF plus high-calcium than that in control rats with high-calcium only, with values of optical absorption of 139.16, 131.15, 149.98 and 149.19 (P < 0.05), respectively, and protein level of c-fos and c-jun was significantly higher in rats with NaF plus low-calcium than that in control rats with low-calcium only, with values of optical absorption of 117.24, 111.46, 132.46 and 129.79 (P < 0.05), respectively. Western blotting showed that level of protein expression of c-fos and c-jun in periosteal osteoblasts was significantly higher in all rat groups with NaF than that in all control groups, with values of optical absorption of 123.32, 116.60, 115.97 and 108.30, respectively. mRNA expression of c-fos and c-jun in osteoblast-like cells treated with NaF for 12 h increased obviously, and remained at high level 48 h after exposure, with values of optical absorption of 114.80, 161.14, 118.20, and 150.41, respectively, as compared with that in control group (P < 0.001 and P < 0.05). CONCLUSIONS: Exposure to excessive fluoride could stimulate activation and proliferation of both osteoblasts in rats and cultured osteoblast-like cells in vitro, and cause enhanced expression of mRNA and protein of both c-fos and c-jun. Over-expression of c-fos could play an important role in development and proliferation of skeletal lesions in rats with chronic fluorosis.

[Metabolic effect of amaranth oil and impulse hypoxic training under chronic fluoride intoxication and small doses of ionizing radiation]. / Metabolichni efekty oliï amarantu ta impul'snoho hipoksychnoho trenuvannia za umov diï ftorystoï intoksykatsiï ta malykh doz radiatsiï.

Lipid peroxidation and antioxidative defence system in blood, liver and heart tissues, nitric oxide metabolites content in brain tissue of rats under binary action of small-doses of ionizing radiation and fluoride intoxication treated by amaranth oil and interval hypoxic training have been studied. Complex using of amaranth oil and interval hypoxic training result in increase both enzymatic, as nonezymatic links of antioxidant defence in all investigated tissues. It was revealed also enhance of NO system metabolites content in brain gomogenate. In this conditions lipid peroxidation processes in liver and heart tissues normalize comparison with essential increase level LPO under binary action influence. On the basis of obtained results LPO metabolites content we can suppossed that complex using of amaranth oil and interval hypoxic training result in increase of organism adaptative possibility. This complex can be using for binary action of ionizing radiation and fluoride intoxication correction.

[Changes of energy metabolism and concentration of cyclic nucleotides in the kidneys in acute fluoride intoxication and hyperbaric oxygenation]. / Zminy energetychnogo metabolizmy i kontsentratsii tsyklichnykh nukleotydiv u nyrkakh pry gostrii ftorystii intoksykatsii i zastosyvanni giperbarychnoi oksygenatsii.

The energy metabolism and indices of the adenylate-cyclase system in kidneys of rats with acute fluoric intoxication have been studied. It has been determined that the use of hyperbaric oxygenation prevents deep disturbances of energy metabolism in the renal tissue and restricts the increase of adenosine monophosphate concentration. Moreover the survival rate of animals has increased by 30% for the first 24 hours.

Aortic calcification in chronic fluoride poisoning: biochemical and electronmicroscopic evidence.

Fluoride is known to cause ectopic calcification. The biochemical mechanism(s) involved in the initiation of calcification is not understood and the accompanying ultrastructural changes remain to be elucidated. Therefore, certain relevant parameters have been investigated in the aorta of rabbits administered fluoride, 10 mg NaF/kg body wt, every 24 hr for 17 and 24 months. The significant findings are: (i) degeneration of smooth muscle fibers in the tunica media of the aorta, (ii) presence of electron-dense granules in the mitochondria and on the inner surface of the plasma membrane of smooth muscle cells, (iii) presence of matrix vesicles with electron-dense deposits, (iv) enhanced calcium content and the Ca/P ratio, and (v) increased total glycosaminoglycan (GAG) content with reduced dermatan sulfate. The presence of electron-dense granules in the mitochondria, on the plasma membrane and matrix vesicles is suggestive of the process of calcification. The enhanced calcium content as well as the Ca/P ratio supports the view that the aorta is undergoing mineralization. The total GAG is enhanced, possibly due to an increase in the content of GAGs other than isomers of chondroitin. The observation that conveys an important message is that the dermatan sulfate normally known to exist in high concentrations in soft tissues begins to decrease as the process of calcification sets in. This perhaps would hold true and may serve as an index in the process of ectopic calcification.

Experimental fluorosis in sheep: alleviating effects of aluminum.

Five groups of 4 sheep were given for 33 mo a daily oral dose of 0, 1.9 or 4.7 mg fluoride (F)/kg body weight with or without 13.5 mg aluminum (Al)/kg. In all treated animals the general health status was disturbed and osteo-dental symptoms appeared while F levels were increased in teeth, bones and organs. In sheep given the higher F dose, pathologic lesions were observed in kidney and liver. Most disturbances were dose-related and alleviated by simultaneous administration of aluminum sulfate, mainly for the lower F dose.

Hip fracture incidence not affected by fluoridation. Osteofluorosis studied in Finland.

Iliac crest biopsies were taken from patients with hip fracture from a low-fluoride area (less than 0.3 ppm), from an area with fluoridated drinking water (1.0-1.2 ppm), and from a high-fluoride area (greater than 1.5 ppm). Fluoride content analysis and histomorphometry of bone were performed. The hip fracture incidence during 1972-1981 was studied in the same areas. The fluoride content of the bone samples correlated with drinking water fluoride. In patients with hip fracture, both osteomalacia and osteoporosis were common. In the high-fluoride area also osteofluorosis was found in many patients. Osteofluorosis may occur if the fluoride content of trabecular bone exceeds 4,000 ppm and either the volumetric density of osteoid or the osteoid-covered trabecular bone surface is abnormally increased. There was no difference in incidence of hip fracture in the three areas.

Alleviation of fluorine toxicity in starting turkeys and chicks with aluminum.

Aluminum (Al) compounds were evaluated as fluorine (F) toxicity alleviators in starting broiler chicks and turkeys. Added F levels from NaF ranged from 0 to 1000 ppm, whereas Al levels varied from 0 to 800 ppm. Al was fed either as Al2O3 or Al2 (SO4)3.18H2O. When fed as the sulphate salt, 800 ppm of Al completely prevented the toxic effect of at least 1000 ppm of F. Al2O3 was not effective as an alleviator of fluorine toxicity. When the mode of action of Al2(SO4)3.18H2O against F toxicity was studied in colostomized turkeys it was apparent that F absorption occurred but was probably less efficient than previously reported in ruminants. Al significantly (P less than .05) reduced F absorption in turkeys. Urinary F levels were: 2.4 ppm in birds fed a control diet (26 ppm F), 17.8 ppm in birds fed a diet with 1000 ppm F, and 6.7 ppm in birds fed the high F diet with 800 ppm Al as the sulphate salt. In addition, data from this study indicated that starting broiler chicks were more tolerant (800 ppm F) than starting turkeys (600 ppm F) to fluorine toxicosis.