title: The toxic effect of fluoride on MG-63 osteoblast cells is also dependent on the production of nitric oxide.
abstract: Some soda-lime-phospho-silicate glasses, such as Hench's Bioglass(®) 45S5, form bone-like apatite on their surface when bound to living bone. To improve their osteointegration for clinical purposes, the fluoride insertion in their structure has been proposed, but we recently showed that fluoride causes oxidative damage in human MG-63 osteoblasts, via inhibition of pentose phosphate oxidative pathway (PPP) and its key enzyme glucose 6-phosphate dehydrogenase (G6PD). In the same cells we have now investigated the role of nitric oxide (NO) in these effects. Fluoride-containing bioactive glasses and NaF caused, as expected, release of lactate dehydrogenase in the extracellular medium, accumulation of intracellular malonyldialdehyde, inhibition of PPP and G6PD: we have now observed that these effects were significantly reverted not only by superoxide dismutase (SOD) plus catalase (scavengers of reactive oxygen species), but also by N-monomethyl l-arginine (l-NMMA, a NOS inhibitor) and 2-phenyl-4,4,5,5,-tetramethylimidazoline-1oxyl 3-oxide (PTIO, a NO scavenger). Moreover the two highest concentrations of both fluoride-containing bioglasses and NaF caused increase of nitrite (a stable derivative of NO) levels in the culture supernatant, which was inhibited by l-NMMA, erythrocytes, PTIO and SOD/catalase, and increase of intracellular NO synthase (NOS) activity. The incubation with bioglasses or NaF increased also the phosphorylation of Ser(1177) in the endothelial NOS isoform. Furthermore, the NO donor spermine NONOate was able to inhibit G6PD activity in vitro, and this effect was partly reverted by PTIO. Therefore our results suggest that most cytotoxic effects of fluoride are mediated by the production of NO: reactive oxygen species are important, causing NOS phosphorylation. We also observed, for the first time, that Tempol, but not SOD/catalase, besides inhibiting the oxidative stress induced by fluoride, also scavenges fluoride ions. For this reason it is not a selective inhibitor of the oxidative effects of fluoride.
authors.affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, Via Santena 5/bis, 10126 Torino, Italy. loredana.bergandi@unito.it
authors.forename: Loredana
authors.initials: L
authors.lastname: Bergandi
authors.forename: Valentina
authors.initials: V
authors.lastname: Aina
authors.forename: Gianluca
authors.initials: G
authors.lastname: Malavasi
authors.forename: Claudio
authors.initials: C
authors.lastname: Morterra
authors.forename: Dario
authors.initials: D
authors.lastname: Ghigo
mesh_terms: D002374:Catalase / Q000378:metabolism; D002460:Cell Line; D003497:Cyclic N-Oxides / Q000494:pharmacology; D005459:Fluorides / Q000633:toxicity*; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology; D010427:Pentose Phosphate Pathway / Q000187:drug effects; D010766:Phosphorylation; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D003497:Cyclic N-Oxides; D007093:Imidazoles; C079391:2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; D009569:Nitric Oxide; D005954:Glucosephosphate Dehydrogenase; D002374:Catalase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase; D005459:Fluorides