Cytotoxicity Analysis of Human Dental Pulp Stem Cells After Silver Diamine Fluoride Application.

Purpose: The purpose of this study was to evaluate the cytotoxicity of silver diamine fluoride (SDF) to human dental pulp stem cells (hDPSC). Methods: hDPSC were exposed to dilutions of 38 percent SDF ( 10-3, 10-4, and 10-5) and incubated for 24 hours. Cell viability was assessed with colorimetric detection assay at 24 hours. Fresh media was used as a negative control, and 0.1% sodium dodecyl sulfate was used as a positive control. Three independent experiments were performed in triplicate. Cell viability data were analyzed using analysis of variance and Tukey's multiple comparison test. Results: Cells exposed to dilution of 38 percent SDF 10-3 had an average cell viability of 17.0±3.5 (standard deviation) percent. Cells exposed to SDF 10-4 and 10-5 had an average cell viability of 101±2.5 percent and 94±4.4 percent, respectively. Dilution of 10-3 had significantly lower cell viability than the negative control (P<0.001). Dilution of 10-4 and 10-5 SDF had significantly higher cell viability than the positive control (P<0.001) and cells treated with a dilution of 10-3 (P<0.001). Conclusions: Thirty-eight percent silver diamine fluoride was cytotoxic to human dental pulp stem cells at a dilution of 10-3, but not at 10-4 and 10-5. In light of the cytotoxicity of SDF to hDPSC, this in vitro study supports the concern that exposure of the full concentration of 38 percent SDF to the pulp should be avoided.

Biological effects of sodium fluoride varnishes used in remineralisation of enamel: An in vitro study.

AIM: During the last three decades, fluoride varnishes have been recognised as effective strategies for caries prevention in the young-child population and have contributed to a decrease in its prevalence worldwide. The present study aimed to assess in vitro the level of cytotoxicity and genotoxicity in human primary pulp fibroblasts (DPFs) of two NaF varnishes. MATERIALS AND METHODS: Four experimental assays were carried out (MTS, Mitotracker® system [mitochondrial function and morphology], Live/Dead®, and Comet) to assess the morphology, viability, and genotoxicity of two NaF varnishes (Duraphat® and Clinpro White®, both at two different concentrations). The essays were conducted on cultured pulp fibroblasts, grouped in four experimental and two control groups. Collected data were analysed by one-way ANOVA followed by the post hoc Bonferroni test. RESULTS: Some morphological changes of DPFs could be detected after the NaFVs stimulation. Most DPFs incubated in Duraphat (22.6 mg/L) maintained their morphological characteristics, except for a small decrease in cell size and shorter cytoplasmic projections (filopodia); DPFs treated with Clinpro White Varnish (22.6 mg/L) presented a morphology and size similar to the control group. DPFs exposed to Duraphat (113 mg/L) exhibited significant morphological alterations with considerable cell size increases and DPFs treated with Clinpro White Varnish (113 mg/L) showed a slight cell size increase without noticeable morphological anomalies. The Duraphat (22.6 mg/L) and Clinpro White Varnish (22.6 mg/L) groups promoted 31% and 35% cell proliferation, respectively, whereas DPFs proliferation with Duraphat (113 mg/L) decreased up to 59%, and cell proliferation with Clinpro White Varnish (113 mg/L) was similar to that of control. CONCLUSION: All tested varnishes induced changes in the fibroblastic mitochondria. In general, Duraphat was less biocompatible and caused a change in the number of mitochondria compared to Clinpro White Varnish.

The effect of reduced glutathione on the toxicity of silver diamine fluoride in rat pulpal cells.

INTRODUCTION: Due to its ability to arrest untreated dental caries, silver diamine fluoride (SDF) has been advocated for indirect pulp capping procedures. However, the high concentrations of silver and fluoride in SDF raise concerns about its biocompatibility to pulpal tissues. OBJECTIVES: This study aimed to investigate the effect of SDF on the viability, alkaline phosphatase (ALP) activity, and morphology of pulpal-like cells (RPC-C2A) and to evaluate the influence of reduced glutathione (GSH) on SDF-induced cytotoxicity and deposit formation on dentin. METHODOLOGY: The cytotoxicity of diluted 38% SDF solutions (10-4 and 10-5), with or without the addition of 5 mM or 50 mM GSH, was evaluated at 6 and 24 hours. Cell viability was detected using WST-8 and the effect on ALP activity was performed using an ALP assay kit. Cell morphology was observed using a phase-contrast microscope. Scanning electron microscopy analysis was conducted to evaluate the effect of GSH incorporation or conditioning on SDF-induced deposit formation on dentin discs. Cytotoxicity data were analyzed by two-way analysis of variance (ANOVA) and Tukey post hoc tests (p<0.05). RESULTS: There were significant differences between the groups. The results demonstrated that all tested SDF dilutions caused a remarkable cytotoxic effect, while the addition of GSH prevented SDF-induced damage at 6-hour exposure time in the higher dilution of SDF. Dentin treated with plain SDF or GSH-incorporated SDF solution showed deposit formation with occluded dentinal tubules, unlike the other groups. CONCLUSION: SDF severely disturbed the viability, mineralization-ability, and morphology of pulpal-like cells, while controlled concentrations of GSH had a short-term protective effect against SDF-induced damage. GSH showed an inhibitory effect on SDF-induced dentinal deposit formation. Further research is warranted to evaluate the effect of GSH on caries-arresting, anti-hypersensitivity, and antibacterial functions of SDF.

Temporal Exposure of Dermal Fibroblasts to Silver Diamine Fluoride and Fluorine NMR Quantitation.

OBJECTIVE: Silver diamine fluoride has been advocated as a caries arresting material for Early Childhood Caries (ECC) and has received considerable public attention as the "silver bullet". However, cytotoxicity tests on the current concentrations of Silver Diamine Fluoride (SDF) to soft tissue have not been thoroughly assessed and analyzed at selected time intervals. The level of fluoride that is present within human cells has yet to be quantified. Preliminary SDF toxicity studies in our lab determined exposures of Dermal Fibroblasts to 0.03% SDF for 18 hours resulted in 100% cytotoxicity and complete monolayer loss. Endpoint titration of SDF determined that morphologic cytotoxic effects were ameliorated at input SDF levels lower than 0.002%. Because of the small culture sample volumes, we were unable to effectively assay fluoride concentrations using commercially available assays. In this study we attempted to assess fluoride levels in culture supernatants in a temporal fashion using quantitative Nuclear Magnetic Resonance (NMR). STUDY DESIGN: Dermal Fibroblast (DF) cells were grown in 24 well cluster plates fitted with 0.4 micron TranswellTM inserts to confluency in 0.9mL of DF culture media. Then the DF cells were challenged with 0.1 mL of SDF in sterile water in the Transwell chamber to achieve a final concentration of 0.03% SDF. Cultures were reincubated for 30 minutes, 1, 2, 4 and 8 hours. At the selected time points Transwell inserts were removed. SDF culture media was removed and replaced with fresh media and allowed to re-incubate up to 8 hours. Harvested SDF culture media was centrifuged at 15,000 x g to remove any resulting SDF precipitates and supernatants were harvested and stored at -70°C for fluoride assay. After 8 hours, media was aspirated from all wells and DF cells were fixed and then stained with methylene blue and assessed for cytotoxicity. Harvested supernatants were assessed for fluoride content. While SDF is soluble in pure water, it precipitates instantly in the presence of other media constituents and 0.85% saline. Transwells inserts capture the precipitate but allow soluble SDF and constituents pass through to the cell monolayer. NMR was used to assess SDF (fluoride) prepared in water, in DF media or in normal saline at the same concentrations used in the DF cell studies. The 19F NMR spectra were acquired at 25 °C on an Agilent DD2 500 MHz spectrometer equipped with a 5mm HFX z gradient probe operating at 470.3 MHz for fluorine. For quantitative measurements, all spectra were collected with 64 scans and a delay of 5 seconds. The spectrum width is 220 ppm with offset at resonance of -110 ppm. The processing and analyzing were done by MNOVA. The dataset consists 45371 complex points and is zero-filled to the size of 128k points after applying 5Hz exponential line broadening. The 19F chemical shift was referenced indirectly based on proton chemical shift, which was referenced with respect to the water proton signal of 4.75 ppm at 25°C. RESULTS: Visible DF cell morphology changes begin to appear as early as 1 hour exposure to 0.03% SDF in Transwells and continue with degradation of cell morphology through 8 hours exposure at which point 100% of the cell monolayer is lost. The 8 hour image shows complete cell loss which is consistent with earlier studies using 24 hour exposures at 0.03% concentration. Note that the actual concentration of SDF affecting cell viability is shown in this study to be far lower than the 0.03% input because of the aggregate precipitation captured with the Transwell inserts. In this study, our NMR fluoride assessments showed that only 6-12 % of the input SDF fluoride reaches the lower cell chamber. CONCLUSIONS: Considering that the SDF reagent is applied orally at ~40%, these results warrant more refined testing to identify true lower limit of toxicity end points of SDF. SDF should be utilized only by trained professionals and never contact soft tissue. NMR may be utilized to determine relative amounts of fluoride both in cell culture media and within fluoride exposed cells.

The effect of reduced glutathione on the toxicity of silver diamine fluoride in rat pulpal cells

Abstract Introduction Due to its ability to arrest untreated dental caries, silver diamine fluoride (SDF) has been advocated for indirect pulp capping procedures. However, the high concentrations of silver and fluoride in SDF raise concerns about its biocompatibility to pulpal tissues. Objectives This study aimed to investigate the effect of SDF on the viability, alkaline phosphatase (ALP) activity, and morphology of pulpal-like cells (RPC-C2A) and to evaluate the influence of reduced glutathione (GSH) on SDF-induced cytotoxicity and deposit formation on dentin. Methodology The cytotoxicity of diluted 38% SDF solutions (10-4 and 10-5), with or without the addition of 5 mM or 50 mM GSH, was evaluated at 6 and 24 hours. Cell viability was detected using WST-8 and the effect on ALP activity was performed using an ALP assay kit. Cell morphology was observed using a phase-contrast microscope. Scanning electron microscopy analysis was conducted to evaluate the effect of GSH incorporation or conditioning on SDF-induced deposit formation on dentin discs. Cytotoxicity data were analyzed by two-way analysis of variance (ANOVA) and Tukey post hoc tests (p<0.05). Results There were significant differences between the groups. The results demonstrated that all tested SDF dilutions caused a remarkable cytotoxic effect, while the addition of GSH prevented SDF-induced damage at 6-hour exposure time in the higher dilution of SDF. Dentin treated with plain SDF or GSH-incorporated SDF solution showed deposit formation with occluded dentinal tubules, unlike the other groups. Conclusion SDF severely disturbed the viability, mineralization-ability, and morphology of pulpal-like cells, while controlled concentrations of GSH had a short-term protective effect against SDF-induced damage. GSH showed an inhibitory effect on SDF-induced dentinal deposit formation. Further research is warranted to evaluate the effect of GSH on caries-arresting, anti-hypersensitivity, and antibacterial functions of SDF.

Cytotoxic effects of silver diamine fluoride.

PURPOSE: To investigate the effect of silver diamine fluoride (SDF) and fluoride varnish (FV) on human gingival fibroblasts (HGF) and bacteria. METHODS: HGF cell viability was assessed after exposure to various dilutions of SDF or FV. Hydroxyapatite (HA) discs treated with SDF, FV, or saline were rinsed in artificial saliva for 84 days. HGF were exposed to treated discs and viability assessed fluorescently. Oral bacteria were exposed to treated discs and survival quantified. RESULTS: At 0.01%, SDF was almost 100% cytotoxic to HGF. SDF and FV treated HA discs, induced near-complete cell death after 24 hours of contact. After rinsing FV discs for 21 days, cell survival exceeded 95%. SDF treated discs were toxic to HGF and bacteria after 9 weeks of rinsing. CLINICAL SIGNIFICANCE: SDF and FV can induce cell death. FV lost its cytotoxicity within 3 weeks, while SDF remained cytotoxic even after 9 weeks of rinsing. This research confirms that SDF has long lasting antimicrobial effects at very low concentrations although it does raise concerns regarding cytotoxicity. However, HGF cells are exposed to other cytotoxic substances in dentistry with little, if any, long-term effects.

Impact of subchronic exposure to triclosan and/or fluoride on estrogenic activity in immature female rats: The expression pattern of calbindin-D9k and estrogen receptor α genes.

This study explored the influence of triclosan (TCS) in the absence and presence of sodium fluoride (NaF) on estrogenic activity and thyroid function of adolescent female rats. The results indicated that the individual exposure to TCS evoked a significant decline in T3 and T4 but the levels of estradiol, FSH, and LH were significantly elevated beside marked up regulation of calbindin-D9k and estrogen α mRNA expression. On the other hand, the single exposure to NaF causes insignificant changes in thyroid hormones, but evoked a trend toward an increase in both estradiol and LH levels. No significant differences in the TSH level were recorded among the experimental groups. The joint exposure to TCS and NaF induced a significant improvement in thyroid and reproductive hormone levels. Overall, these findings revealed that exposure to TCS resulted in significant endocrine and reproductive alterations in immature female rats, while TCS + NaF coexposure resulted in lessening most effects.

Genotoxicity of non-alcoholic mouth rinses: A micronucleus and nuclear abnormalities study with fluorescent microscopy.

AIM: The aim of the present study was to evaluate the genotoxicity of non-alcoholic mouth rinses on buccal epithelial cells using a micronucleus test. METHODS: A total of 105 patients were selected and randomly divided into five groups. Four different mouth rinses and normal saline were given for 2 weeks' duration, and cytological smears were collected before and after exposure. These smears were subjected to micronucleus (MN) and other nuclear abnormalities (ONA) tests using acridine orange stain, and their frequencies were obtained in 500 buccal epithelial cells. The statistical analysis included mean, χ2 -test, analysis of variance, and post-hoc analysis by Bonferroni test. RESULTS: Micronucleated cells (P < .00) and MN (P < .00) were higher in individuals exposed to chlorhexidine (CHX), followed by chlorine dioxide (ClO2 ), potassium nitrate (KNO3 ), and sodium fluoride (NaF), amine fluoride (AmF), and normal saline. ONA were greater (P < .00) in individuals exposed to CHX, followed by ClO2 , AmF, KNO3 , and NaF and normal saline. Overall, the results showed that genotoxic damage was greater in the case of CHX, followed by ClO2 , KNO3 , and NaF, AmF, and normal saline. CONCLUSION: Chronic exposure to mouth rinses can cause genotoxic damage to buccal epithelial cells. Long-term injudicious and inadvertent use of mouth rinses should be discouraged.

Cytotoxicity of three commercial mouthrinses on extracellular matrix metabolism and human gingival cell behaviour.

This study evaluated the effects of commercially available antiseptic mouthrinses on human gingival fibroblast and keratinocyte behaviour and metabolism. Three mouthrinses containing essential oil (EO), chlorhexidine (CHX) and amine fluoride/stannous fluoride (AFSF), were tested in an in vitro study. Human gingival fibroblasts and keratinocytes were washed with 10% or 30% concentration of the commercial mouthrinses and their effects on cell adhesion and proliferation were investigated as well as the specific gene expression of markers involved in oral mucosa metabolism. As markers of cell metabolism, type I and IV collagens, laminin, fibronectin, fibromodulin and integrins were studied with real-time PCR. Moreover, interleukin-1 secretion, one of the major pro-inflammatory cytokines, was evaluated. The results showed that CHX significantly reduced fibroblast and keratinocyte substrate adhesion capacities and CHX and EO inhibited cell proliferation better than AFSF rinse. The gene expression of several matrix components and cell adhesion receptors was downregulated in cells washed with CHX and EO compared with those washed with AFSF rinse. In conclusion, the AFSF mouthrinse does not induce or induces to a lesser extent the onset of irritation and/or cytotoxicity than CHX or EO. These findings and those of future studies will enable us to gain further insight into the clinical significance and effects of commercial mouthrinses. Pending further investigations, clinicians should be aware of the potentially adverse effects of mouthrinses and warn their patients against making improper use of these products.

Salivary fluoride concentrations and fluoride ingestion following application of preparations containing high concentration of fluoride.

The aim of the study was to assess salivary fluoride concentrations and ingestion of fluorides after five different techniques of topical fluoride treatment. Ten volunteers received applications of fluoride gel, toothpaste, and foam. Fluoride concentrations were determined using ion-selective fluoride electrode in the samples of unstimulated saliva before and after procedures. The amounts of fluoride applied, recovered from the mouth, and retained in the mouth were calculated for each treatment. It was proved that fluoride ingestion following tray application of fluoride foam was significantly lower than ingestion following tray applications of fluoride gel (p < 0.01). The use of limited amounts of fluorides on a tray resulted in similar fluoride retention and lower ingestion comparing to the method which involves a large portion of fluorides followed by rinsing. Tooth brushing resulted in high salivary retention rates per amount of fluorides used in the procedure. These data provide initial concept about the possible advantages of some methods of topical fluoride application over others.

Evaluation of a natural resin-based new material (Shellac F) as a potential desensitizing agent.

OBJECTIVES: To evaluate the cytotoxicity of Shellac F, a new fluoride varnish, and its effects on human dentin hydraulic conductance. METHODS: Shellac F was compared to another fluoride varnish (Duraphat) and a fluoride containing desensitizing agent (Isodan). The cytotoxicity test was performed on human gingival fibroblasts and through dentin slice on human pulp fibroblasts. The hydraulic conductance (Lp) was recorded by fluid filtration with a Flodec device under a constant pressure (15 cm H2O). The treated surface of the dentin disks and their sections were also investigated with SEM. RESULTS: The cytotoxicity test on gingival fibroblasts revealed that Duraphat was the least cytotoxic material, followed by Shellac F then Isodan. With dentin slice interposition, a lower level of cytotoxicity was obtained. All of them showed a lower cytotoxicity decreasing on further dilutions (p<0.001). The measurement of hydraulic conductance showed that all materials resulted in a significant decrease in dentin permeability after 24h comprising between 60 and 76%, but there was no statistically significant difference among the materials. This decrease was still over 50% of the initial values after 7 days for all three materials. SEM investigation showed dentin tubules covered with a thick layer of Shellac F or Duraphat whilst no material was observed on dentin surfaces treated with Isodan. SIGNIFICANCE: Shellac F showed an adequate cellular compatibility and a significant effect on human dentin hydraulic conductance. This indicates that the new material is safe and seems to be effective as a potential desensitizing agent.

Efetividade da aplicação semestral de verniz fluoretado no controle da cárie dentária em pré-escolares: resultados após 12 meses de acompanhamento / Effectiveness of bi-annual fluoride varnish application in the control of dental caries in preschool children: results after 12 months of follow-up

O presente estudo teve como objetivos avaliar se a aplicação de verniz fluoretado com periodicidade semestral em crianças pré-escolares reduz o número de crianças com lesões de cárie em dentina na dentição decídua, diminui a incidência de lesões de cárie em esmalte e dentina, está inversamente associado à ocorrência de dor e abscesso dentário e produz quaisquer efeitos adversos. A população de estudo consistiu de 200 crianças na faixa etária de 12 a 48 meses, recrutadas em uma unidade de saúde pública da cidade do Rio de Janeiro, as quais foram alocadas aleatoriamente nos grupos teste (verniz fluoretado Duraphat®) e controle (verniz placebo). Para o registro da incidência de cárie, as crianças foram examinadas na linha de base e a cada seis meses, durante um ano, por dois odontopediatras previamente treinados e calibrados (Kappa=0,85). A ocorrência de dor, abscesso e efeitos adversos foi verificada a partir de entrevistas com os responsáveis. Os participantes, os seus responsáveis, os operadores e os examinadores desconheciam a qual grupo cada criança pertencia. No final do período de acompanhamento, 71 crianças do grupo teste e 77 do grupo controle foram avaliadas. Constatou-se que, nos grupos teste e controle, o número de crianças com novas lesões de cárie em dentina foi igual a 13 e 20 (teste Qui-quadrado, p=0,34) e que a média do incremento de cárie considerando apenas lesões em dentina (c3eos) foi de 1,1(dp=3,4) e de 1,4(dp=2,8), respectivamente (teste de Mann-Whitney, p=0,29). Uma criança apresentou dor de dente e abscesso dentário e outras duas crianças apresentaram apenas dor de dente. Todas pertenciam ao grupo teste. Com relação aos efeitos adversos, encontrou-se que uma criança pertencente ao grupo controle relatou ardência na cavidade bucal após a aplicação do placebo e que o responsável por um participante do grupo teste sentiu-se incomodado com a coloração amarelada dos dentes da criança após a aplicação do verniz fluoretado. ....

The aims of this study were to evaluate whether the application of fluoride varnish in preschool children at six-month intervals reduces the number of children with dentin caries and decreases the incidence of enamel and dentinal caries lesions in the primary dentition. We also sought to assess whether this intervention is inversely associated with the occurrence of tooth abscess and dental pain and to describe any possible side effects. The study population comprised 200 children, 12 to 48 months of age. They were enlisted in a public health center in the city of Rio de Janeiro and were randomly assigned to the test group (Duraphat® fluoride varnish) or the control group (placebo varnish). To record caries incidences, the children were examined at the baseline and every six months, during the period of one year, by two trained and calibrated pediatric dentists (Kappa=0.85). The presence of pain, abscess and side effects were recorded through interviews with the children’s caregivers. The children, their caregivers, the operators and the examiners did not know which group each child belonged. At the end of the follow-up period, 71 children of test group and 77 of the control group were analyzed. We found that 13 children in the test group and 20 children in the control group presented new dentinal caries lesions (Chi-square test, p= 0.34) and that the mean caries increment at the dentin level was 1.1(dp= 3.4) and 1.4 (dp=2.8) in the test and the control groups respectively (Mann-Whitney test, p=0,29). One child reported dental pain and tooth abscess, and two other children reported only dental pain. All three children belonged to the test group. One child reported a burning sensation in the mouth following application of the placebo varnish and one parent of a child in the test group felt upset about the yellowish discoloration of the child’s teeth after the application of the fluoride varnish. We concluded that the six-monthly ...(AU)

Prevention. Part 7: professionally applied topical fluorides for caries prevention.

This paper reviews the use of professionally applied topical fluorides (PATF) in caries prevention. PATFs are indicated for children and adults with one or more decayed smooth surfaces and/or those who are at high caries risk. Frequency of administration depends on the patient's caries risk, and is usually every 6 months. The effectiveness of fluoride varnish and gel applications has been well established in caries prevention trials involving permanent teeth. Although both types are effective, varnish may be preferred because it is easier to apply, reduces the risk of fluoride over-ingestion, and has greater patient acceptance. Fluoride foams are similar products to gels, but have not been tested clinically. The use of in-office two-part rinses is not recommended because they have not been proven effective. A cleaning, or prophylaxis, is not necessary before the application of topical fluoride for caries prevention. In conclusion, when used appropriately, PATFs are a safe, effective means of reducing caries risk among high-risk populations.

Técnica de spray para aplicaçöes tópicas de soluçöes fluoretadas / Topical application of fluoride solutions by spray technique (II)

Foram selecionadas 422 crianças (de 6 a 10 anos), as quais receberam diferentes aplicaçöes de soluçöes fluoretadas (FFA 1,23 por cento F, FFA 0,5 por cento F ea associaçäo de uma soluçäo saturada de DCPD com a FFA 0,5 por cento), sob a forma de um jato de spray devidamente balanceado (30 libras de pressäo e emissäo de 10ml de soluçäo para cada aplicaçäo). As crianças receberam uma aplicaçäo anual dessas soluçöes e foram observadas durante 24 meses. A análise dos resultados indicam que: a) um provável ingestäo das soluçöes aplicadas foi detectada em média em apenas 19 por cento das crianças. b) os efeitos tóxicos decorrentes da ingestäo involuntária da soluçäo fluoretada foram mínimos (2 a 3 por cento dos casos com ingestäo). Concluindo, esses resultados associados a simplicidade do equipamento (manipulaçäo e baixo custo de produçäo), a facilidade de aplicaçäo técnica, aos benefícios preventivos, a inocuidade do spray e a aceitaçäo pelas crianças, permitem indicá-la como uma técnica adequada para uso em saúde pública

[Cytotoxicity and genotoxicity of fluorides in human mucosa and lymphocytes]. / Zytotoxizität und Genotoxizität von Fluoriden in humaner Mukosa und Lymphozyten.

BACKGROUND: Fluorides are widely used in dental health products and drinking water, due to their beneficial effects in caries-prophylaxis and -treatment. Nevertheless, irritation of the gingiva and oropharyngeal mucosa as well as in gastric mucosa is observed since neither local nor systemic application is restricted to the teeth. These effects may partly be attributed to a known cytotoxicity of fluorides. Whether fluorides also have genotoxic effects on human mucosa or lymphocytes as a possible factor in tumor initiation was investigated in this study. MATERIAL AND METHODS: Human oropharyngeal epithelial cells and peripheral lymphocytes were incubated after single cell preparation with the aminefluoride Olaflur at concentrations of 2 ppm, 21 ppm, 35 ppm, 71 ppm and 213 ppm. The extent of cytotoxicity was investigated using the trypan blue exclusion test. Following incubation, electrophoresis for migration of DNA fragments, fluorescence staining and digital image analysis according to a standard protocol of the single cell microgel electrophoresis assay (Comet assay) followed. DNA damage was characterized using the Olive Tail Moment (OTM). RESULTS: For fluoride concentrations of 2 ppm to 35 ppm, non vital cells of less than 10% could be shown. After incubation with 71 ppm and 213 ppm Olaflur, there were 15% and 43% of damaged cells, respectively. Weak genotoxic effects on mucosal cells as well as on lymphocytes could be demonstrated at all concentrations tested. In fluoride concentrations of 213 ppm genotoxicity increased to max. OTM-levels of 23. CONCLUSIONS: Beside the cytotoxic effect of fluorides, also a minor genotoxic impact on human mucosa and on peripheral lymphocytes could be demonstrated using the Comet assay. Further investigations are warranted to examine fluorides in a model allowing for repeated or long term incubations on structurally intact human mucosa in vitro. Such a model will help to distinguish between DNA damage that may be repaired successfully and other impairments that may show an additive character in repetitive or chronic exposure in vivo.

Potential of 4 per cent silver fluoride to induce fluorosis in rats: clinical implications.

The Health Department in Western Australia uses a 40 per cent silver fluoride (AgF) solution for prevention and treatment of dental caries in children. Analysis of this solution has revealed high fluoride concentrations (75,000-120,000 mg/L), raising concerns of potential toxicity and prompting investigation of clinical protocols utilizing low-strength AgF in an animal model. A single topical application of 4 per cent AgF solution to Sprague-Dawley rats resulted in moderate to severe localized fluorosis in 24 per cent of animals. In a second experiment, caries was induced in rats aged 19 days; six weeks later, between one-four carious molar teeth from each rat were treated with 4 per cent AgF (atraumatic technique). A generalized form of fluorosis developed in the continually growing incisors of less than 10 per cent of animals which had one or two carious teeth treated, and in 70-90 per cent of rats which received AgF to either three or four carious teeth. These results confirm the potential of a 4 per cent AgF solution to induce fluorosis and support previous recommendations that AgF at its empirical concentration of 40 per cent should be withdrawn from clinical use.

Inflammatory responses of rat alveolar macrophages following exposure to fluoride.

Inhalation exposure to fluoride compounds has been associated with respiratory failure. We have addressed effects of fluoride on alveolar macrophages and lung responses to intratracheally (i.t.) instilled fluoride in rats. I.t. instillation of fluoride at doses of 200 and 400 microg F/rat caused significant polymorphonuclear leukocyte (PMN) infiltration in the rat lung at 20 h post-administration, while 100 microg fluoride did not recruit a significant number of PMNs in the alveolar space. Total RNA was extracted from the lung lavage cells obtained from 5 h post i.t. instillation and mRNA levels of chemokines and proinflammatory cytokines were semi-quantitatively evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). I.t. instillation of fluoride significantly enhanced mRNA expression of cytokines such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory proteins-1alpha and -2. Fluoride-induced augmentation in IL-1beta mRNA expression was also examined by Northern hybridization following in vitro exposure of alveolar macrophages to fluoride. However, the enhancement of IL-1beta mRNA expression following in vitro exposure to fluoride was observed only at 500 microM, a dose higher than the 50% lethal concentration (LC(50)). Non-specific adhesion of alveolar macrophages to the plastic dish was significantly increased following in vitro exposure to fluoride. The fluoride-induced non-specific adhesion was significantly reduced by anti-CD18, suggesting that beta(2) integrin played a role in the increase of adherence. Those results suggest that fluoride activates alveolar macrophages, enhances the production of chemokines and proinflammatory cytokines, and causes PMN infiltration in the lung.

Usefulness of the assessment of urinary enzyme leakage in monitoring acute fluoride nephrotoxicity.

A single oral dose of sodium fluoride (NaF) in aqueous solution was given to male Wistar rats. Twenty-four-hour urine samples were collected and examined to evaluate fluoride-induced acute renal damage. The following parameters were measured in 24-h urine: urine volume and urinary excretion of fluoride, N-acetyl-beta-d-glucosaminidase (NAG), alpha-glutathione-S-transferase (alpha-GST), and creatinine (CR). Fluoride exposure produced specific, dose-dependent changes of these parameters. Significant increases of fluoride and fluoride-induced polyuria were observed. NAG as specific marker of proximal convoluted tubule (PCT) function showed a significant increase when the lowest dose of fluoride was administered. At this minimal dose, alpha-GST, a specific marker for the S3 segment, did not show a significant increase but presented the strongest relationship (r = 0. 83) to fluoride dose. No significant changes were measured for CR excretion, which showed a low correlation coefficient (r = 0.36) to administered fluoride. The specific differences in the increase pattern of these parameters show that the PCT is more susceptible to damage by low-dose fluoride than the S3 segment or the glomerulus. We concluded that both NAG and alpha-GST are useful for the diagnosis of fluoride-induced acute nephrotoxicity. Proper evaluation of these urinary indices may be of help to establish the site and extent of kidney injury in acute fluoride toxicity.

Influence of chronic fluorosis on membrane lipids in rat brain.

Brain membrane lipid in rats were analyzed after being fed either 30 or 100 ppm fluoride for 3, 5, and 7 months. The protein content of brain with fluorosis decreased, whereas the DNA content remained stable during the entire period of investigation. After 7 months of fluoride treatment, the total brain phospholipid content decreased by 10% and 20% in the 30 and 100 ppm fluoride groups, respectively. The main species of phospholipid influenced by fluorosis were phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. The fatty acid and aldehyde compositions of individual phospholipid classes were unchanged. No modifications could be detected in the amounts of cholesterol and dolichol. After 3 months of fluoride treatment, ubiquinone contents in brain were lower; however, at 7 months they were obviously increased in both groups of fluoride treatment. The results demonstrate that the contents of phospholipid and ubiquinone are modified in brains affected by chronic fluorosis and these changes of membrane lipids could be involved in the pathogenesis of this disease.