Lung - Adverse Effects
Sodium fluoride
CAS No. 7681-49-4

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• Note: The following is a limited selection of abstracts from 1994 to present.
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2003. Appl Toxicol. Nov-Dec;23(6):437-46.

Histopathological and biochemical changes in lung tissues of rats following administration of fluoride over several generations.

Aydin G, Cicek E, Akdogan M, Gokalp O.

Suleyman Demirel University Faculty of Medicine, Department of Pathology, Isparta, Turkey.

The possible effects of multigenerational administration of sodium fluoride (NaF) via drinking water on lung tissue morphology and biochemistry and body and lung weight were investigated in second-generation adult male rats. For this purpose we selected 45 Albino adult Wistar rats in nine cages, each of which consisted of four females and one male. Twenty-eight pregnant rats were selected for the experiment, divided into four groups of seven rats given 1 (control group), 10, 50 and 100 mg l(-1) NaF in drinking water during the gestation period. After gestation the rats had 165 pups in total. The mothers received fluoridated water during the lactation period and the offspring of the first generation had access to fluoridated water during the suckling period (21 days) and after the weaning period (30 days) until they became mature and at the start of the second part of the experiment. During this time 23 pups died and 79 female and 63 male first-generation rats survived. These first-generation rats were then used to obtain the second-generation offspring in the same manner as before, which were subjected to the same treatments. At the end of 6 months the rats were sacrificed and autopsied. Serum fluoride levels and the activities of principal antioxidant enzymes were determined in lung tissue samples taken from all groups. In addition, the lung tissues were submitted for histopathological examination. Histological findings showed alveolar congestion, alveolar cell hyperplasia and necrosis, prominent alveolar septal vessels, epithelial desquamation and macrophages in the alveolar spaces in the experimental groups. Additionally, there were inflammatory infiltrations in peribronchial, perivascular, intraparenchymal and respiratory tract lumen; intraparenchymal hyperaemic vessels; respiratory epithelial desquamation and proliferation; intraparenchymal thick valled vessels; parenchymal fibrosis; bronchiolitis; pneumonic and focal emphysematous areas. Furthermore, the lung parenchyma was observed to have a distorted appearance with loss of alveolar architecture. These histopathological findings were more pronounced for the rat groups of 50 and 100 mg l(-1) fluoride. No significant histopathological changes were observed in the rats of the control group. The increased activities of superoxide dismutase (SOD) and reduced glutathione peroxidase (GSH-Px) and the decreased activity of catalase (CAT) in the lung tissues with 10 mg l(-1) fluoride might indicate activation of the antioxidant defence mechanism. The decrease in SOD, GSH-Px and CAT activities with 50 and 100 mg l(-1) fluoride and the increase in thiobarbituric acid-reactive substance levels might be related to oxidative damage that occurred in the lung. This multigenerational evaluation of the long-term effect of different doses of fluoride intake through drinking water on lung damage shows that the lung tissues were damaged, there was emphysema and inflammation of lung parenchyma associated with loss of alveolar architecture and the degree of lung damage seemed to correlate with the increased dosage of fluoride. A similar relationship was observed between the degree of lung damage, body and lung weight and serum fluoride levels according to the fluoride dose. Therefore, these results contribute to a better understanding of chronic fluoride toxicity in lung tissue of second-generation rats, especially via drinking water, and the biochemical findings were in agreement with histological observations. In addition, increased fluoride concentration did not affect reproduction or the number of pups dying but the body weight and lung weight ratios were affected by the high dose of fluoride in a dose-related pattern. Copyright (c) 2003 John Wiley & Sons, Ltd.

PMID: 14635268 [PubMed - in process]

2003. Hum Exp Toxicol Mar;22(3):111-23

Fluoride-induced apoptosis in human epithelial lung cells (A549 cells): role of different G protein-linked signal systems.

Refsnes M, Schwarze PE, Holme JA, Lag M.

Division of Environmental Medicine, Norwegian Institute of Public Health, Geitmyrsvn. 75, PO Box 4404 Nydalen, N-0403 Oslo, Norway.

In the present study, possible mechanisms involved in fluoride-induced apoptosis in a human epithelial lung cell line (A549) were examined. Sodium fluoride (NaF) induced apoptosis in the A549 cells, with a maximum at 5-7.5 mM after 20 hours of exposure. The number of cells with plasma membrane damage (PI-positive cells) increased moderately up to 5 mM, but markedly at 7.5 mM. Deferoxamine (an Al3+ chelator) almost completely prevented these NaF-induced responses, which may suggest a role for G protein activation. The apoptotic effect was partially reduced by the PKA inhibitor H89. NaF induced a weak but sustained increase in PKC activity, whereas the PKC activator TPA induced a transient effect. TPA, which enhanced the NaF-induced PKC activity, was not apoptotic when added alone, but facilitated the NaF-induced apoptosis and the increase in PI-positive cells. PKC downregulation induced by TPA pretreatment almost completely prevented the NaF-induced apoptosis and the increase in PI-positive cells. Pretreatment with the PKC inhibitor GF109203X, which abolished the PKC activity after 3 hours, enhanced the NaF-induced apoptosis. KN93 (a CaM kinase II inhibitor) and W7 (a calmodulin inhibitor) seem to reduce the apoptotic effect of NaF, whereas BAPTA-AM (a Ca2+ chelator) was without effect. The tyrosine kinase inhibitor genistein also markedly reduced the NaF-induced apoptosis, whereas the PI-3 kinase inhibitor wortmannin augmented the response. In conclusion, the present results suggest that NaF induces an apoptotic effect and an increase in PI-positive A549 cells via similar mechanisms, involving PKC, PKA, tyrosine kinase and Ca2+-linked enzymes, whereas PI-3 kinase seems to exert a counteracting effect.

PMID: 12723891 [PubMed - in process]

• Note: The following is the article, without graphs, as it appeared in this journal. We present this as PubMed did not provide an abstract.

Annals of the New York Academy of Sciences 973:218-220 (2002)

Involvement of Protein Kinase C in Fluoride-Induced Apoptosis in Different Types of Lung Cells


Division of Environmental Medicine Norwegian Institute of Public Health, N-0403 Oslo, Norway Address for correspondence: Dr. M. Refsnes, Division of Environmental Medicine, Norwegian institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway. Voice: +4722042533; fax: +4722042686.

INTRODUCTION The lung is a target for fluoride-induced toxicity. Fluoride is known to induce apoptosis in different cell types. We previously showed that sodium fluoride (NaF) induced apoptosis in a human epithelial lung cell line (A549) and in epithelial type 2 cells isolated from rat lung, the type 2 cells being most sensitive. Involvement of different MAP kinases was also demonstrated. In the present study, the ability of NaF to induce apoptosis in rat alveolar macrophages and A549 cells was compared, and the role of protein kinase C (PKC) in the apoptotic process was examined.

METHODS A549 cells, presumably originating from human type 2 cells, were cultured as previously described. Primary rat alveolar macrophages were isolated by bronchoalveolar lavage. The macrophages were cultured in RPMI 1640 medium, with antibiotics and 5% fetal bovine serum for 1 hour at a density of 1.5 x 106 cells per milliliter, and the attached macrophages were used for further studies. Both cell types were exposed to NaF and assessed for PKC activity and apoptosis. In some experiments the cells were pretreated with TPA (100 nM) for 20 hours to down-regulate PKC or with the PKC inhibitor GF109203X (20 M) for 1 hour and further incubated with NaF. The PKC activity was measured by a commercial assay (Amersham). Apoptosis was measured by flow cytometry.

RESULTS AND DISCUSSION NaF induced more marked apoptosis in macrophages than in A549 cells and at much lower concentrations. This may reflect the difference between cells from different species, but more conceivably reflects the use of primary lung cells versus an established tumor cell line. Furthermore, NaF induced a slight, but significant increase in PKC activity in both cell types. PKC down-regulation induced by TPA pretreatment prevented these increases, but more importantly it strongly reduced basal PKC activity. PKC down-regulation almost completely prevented NaF-induced apoptosis in both cell types, suggesting that PKC may allow NaF-induced apoptosis. GF109203X inhibited PKC activity to the same extent as did TPA pretreatment . Surprisingly, GF109203X increased the apoptosis of A549 cells and was without effect on macrophages . The reason for the lack of an inhibitory effect is unclear, but it may indicate that the effect of GF109203X is too transient to suppress apoptosis or that GF109203X cannot inhibit a specific PKC isoform crucial for the apoptotic response. In conclusion, NaF induced apoptosis in both rat alveolar macrophages and A549 cells via mechanisms that involved PKC.

1.HIRANO, S. & M. ANDO. 1996. Apoptotic cell death following exposure to fluoride in rat alveolar macrophages. Arch. Toxicol. 70: 249-251.
2.LOWETH, A., G.T. WILLIAMS, J.H. SCARPELLO & N.G. MORGAN. 1996. Heterotrimeric G-proteins are implicated in the regulation of apoptosis in pancreatic b-cells. Exp. Cell. Res. 229: 69-76.
3.THRANE, E.V., M. REFSNES, G.H. THORESEN, et al. 2001. Fluoride-induced apoptosis in epithelial lung cells involves activation of MAP kinases p38 and possibly JNK. Toxicol. Sci. 61: 83-91.- Free Full Text.

Toxicology 2001 Oct 15;167(2):145-58

Mechanisms in fluoride-induced interleukin-8 synthesis in human lung epithelial cells.

Refsnes M, Thrane EV, Lag M, Hege Thoresen G, Schwarze PE.

Department of Environmental Medicine, National Institute of Public Health, PO Box 4404, Nydalen, N-0403, Oslo, Norway.

Sodium fluoride (NaF) has previously been reported to induce a strong IL-8 response in human epithelial lung cells (A549) via mechanisms that seem to involve the activation of G proteins. In the present study the signal pathways downstream of the G proteins have been examined. NaF induced a weak, but sustained increase in PKC activity. In contrast, the PKC activator TPA induced a relatively strong, but transient effect and augmented the NaF-induced PKC activity. TPA induced a marked IL-8 response compared to NaF. PDB, another PKC activator, was less effective, but augmented the IL-8 response to NaF. Pretreatment with TPA for 20 h, or the PKC inhibitor GF109203X for 1 h, abolished the basal and NaF-induced PKC activities and partially prevented the NaF-induced IL-8 response. Inhibition of the MAP kinase p38 by SB202190 partially reduced the IL-8 response to NaF, whereas a reduction in ERK activity by PD98059 led to an increased response. The NaF-induced IL-8 response was weakly augmented by the PKA stimulator forskolin and the G(i) inhibitor pertussis toxin. The PKA inhibitor H89 seemed to reduce the NaF-induced IL-8 response, but the measured effect was not statistically significant. BAPTA-AM, KN93 and W7, that inhibit Ca(2+)-linked effects, did not affect the IL-8 response. Furthermore, the tyrosine kinase inhibitor genestein, the PI-3 kinase inhibitor wortmannin and phosphatase inhibition were without effects. In conclusion, the data suggest that NaF-induced increase of IL-8 in A549 cells involved PKC- and p38-linked pathways, whereas an ERK-dependent pathway counteracted the response. Tyrosine kinases, Ca(2+)-linked pathways, PI-3 kinase, PKA and phosphatase inhibition seem to play no or minor roles in the fluoride-induced IL-8 response.

PMID: 11567778 [PubMed - indexed for MEDLINE]

Arch Toxicol 1996;70(3-4):249-51

Apoptotic cell death following exposure to fluoride in rat alveolar macrophages.

Hirano S, Ando M.

Regional Environment Division, National Institute for Environmental Studies, Ibaraki, Japan.

Since inhaled fluoride is implicated in the acute respiratory failure, cytotoxic effects of fluoride on alveolar macrophages, primary target cells of inhaled toxicants, were investigated. The LC50 of sodium fluoride was estimated to be 0.41 mM, while 1 mM sodium chloride, bromide and iodide had virtually no effects on the viability of alveolar macrophages. Photomicroscopic observation revealed that nuclei of the fluoride-exposed alveolar macrophages were fragmented. The ladder formation was observed when DNA isolated from fluoride-exposed alveolar macrophages was electrophoresed in agarose gel. These results suggest that cytotoxicity of fluoride is associated with apoptosis in rat alveolar macrophages.

PMID: 8825685 [PubMed - indexed for MEDLINE]

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