Multi-scale characterization of Developmental Defects of Enamel and their clinical significance for diagnosis and treatment.

Developmental Defects of Enamel (DDE) such as Dental Fluorosis (DF) and Molar Incisor Hypomineralization (MIH) are a major public health problem. Their clinical aspects are extremely variable, challenging their early and specific diagnosis and hindering progresses in restorative treatments. Here, a combination of macro-, micro- and nano-scale structural and chemical methods, including, among others, Atom Probe Tomography recently applied on tooth enamel, were used to study and compare MIH, DF and healthy teeth from 89 patients. Globally, we show that DF is characterized by an homogenous loss of mineral content and crystallinity mainly disrupting outside layer of enamel, whereas MIH is associated with localized defects in the depth of enamel where crystalline mineral particles are embedded in an organic phase. Only minor differences in elemental composition of the mineral phase could be detected at the nanoscale such as increased F and Fe content in both severe DDE. We demonstrate that an improved digital color measurement of clinical relevance can discriminate between DF and MIH lesions, both in mild and severe forms. Such discriminating ability was discussed in the light of enamel composition and structure, especially its microstructure, organics presence and metal content (Fe, Zn). Our results offer additional insights on DDE characterization and pathogenesis, highlight the potentiality of colorimetric measurements in their clinical diagnosis and provide leads to improve the performance of minimally invasive restorative strategies. STATEMENT OF SIGNIFICANCE: Developmental Defects of Enamel (DDE) are associated to caries and tooth loose affecting billions of people worldwide. Their precise characterization for adapted minimally invasive care with optimized materials is highly expected. Here In this study, first we propose the use of color parameters measured by a spectrophotometer as a means of differential clinical diagnosis. Second, we have used state-of-the-art techniques to systematically characterize the structure, chemical composition and mechanical optical properties of dental enamel teeth affected by two major DDE, Dental Fluorosis (DF) or Molar Incisor Hypomineralization (MIH). We evidence specific enamel structural and optical features for DF and MIH while chemical modifications of the mineral nanocrystals were mostly correlated with lesion severity. Our results pave the way of the concept of personalized dentistry. In the light of our results, we propose a new means of clinical diagnosis for an adapted and improved restoration protocol for these patients.

RESIN INFILTRATION MAY BE A FEASIBLE OPTION TO ESTHETICALLY MASK ENAMEL WHITE SPOT LESIONS.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: Bourouni S, Dritsas K, Kloukos D, Wierichs RJ. Efficacy of resin infiltration to mask post-orthodontic or non-post-orthodontic white spot lesions or fluorosis - a systematic review and meta-analysis. Clin Oral Investig. 2021 Aug;25(8):4711-4719. SOURCE OF FUNDING: University of Bern. TYPE OF STUDY/DESIGN: Systematic review with meta-analysis.

Progress of Signaling Pathways, Stress Pathways and Epigenetics in the Pathogenesis of Skeletal Fluorosis.

Fluorine is widely dispersed in nature and has multiple physiological functions. Although it is usually regarded as an essential trace element for humans, this view is not held universally. Moreover, chronic fluorosis, mainly characterized by skeletal fluorosis, can be induced by long-term excessive fluoride consumption. High concentrations of fluoride in the environment and drinking water are major causes, and patients with skeletal fluorosis mainly present with symptoms of osteosclerosis, osteochondrosis, osteoporosis, and degenerative changes in joint cartilage. Etiologies for skeletal fluorosis have been established, but the specific pathogenesis is inconclusive. Currently, active osteogenesis and accelerated bone turnover are considered critical processes in the progression of skeletal fluorosis. In recent years, researchers have conducted extensive studies in fields of signaling pathways (Wnt/ß-catenin, Notch, PI3K/Akt/mTOR, Hedgehog, parathyroid hormone, and insulin signaling pathways), stress pathways (oxidative stress and endoplasmic reticulum stress pathways), epigenetics (DNA methylation and non-coding RNAs), and their inter-regulation involved in the pathogenesis of skeletal fluorosis. In this review, we summarised and analyzed relevant findings to provide a basis for comprehensive understandings of the pathogenesis of skeletal fluorosis and hopefully propose more effective prevention and therapeutic strategies.

Gastrodin alleviates bone damage by modulating protein expression and tissue redox state.

Fluorosis is a common disease characterized by disruptions in bone metabolism and enamel development. The production of reactive oxygen species is thought to play an important role in fluorosis. Gastrodin (4-hydroxybenzylalcohol4-O-beta-D-glucopyranoside) has been reported to have antioxidative activity, and so here we examined whether gastrodin has protective effects against oxidative stress and bone tissue toxicity in rats with fluorosis. Wistar rats were given different doses of gastrodin 1 month after fluoride administration, and samples of blood, bone and teeth were collected after 2, 3 and 4 months; glutathione peroxidase glu, CAT and SOD levels in the fluorosis group were lower than those in the control group. Gastrodin treatment in rats ameliorated oxidative stress and fluoride accumulation that were induced by fluoride; treatment with 400 mg·kg-1 gastrodin protected trabecular bone structure and reduced femur and alveolar bone injury in rats with fluorosis. Enhanced expression of cysteinyl aspartate-specific proteinase (caspase) 3, caspase-9 and Bax and decreased expression of Bcl-2 induced by fluoride were also reversed by gastrodin. In summary, the present data suggest that gastrodin, and in particular a dose of 400 mg·kg-1 , can improve the antioxidative capacity of rats, reduce concentration of fluoride in tissues, alleviate bone damage and modulate expression of Bcl-2, Bax, caspase-3 and caspase-9.

Oestrogen receptor Rsa I gene polymorphism in osteoporosis periodontitis patients with or without dental fluorosis.

Background & objectives: : There is a paucity of information on association between dental fluorosis, osteoporosis and periodontitis. The aim of this pilot study was to evaluate oestrogen receptor (ER). Rsa 1: gene polymorphism in osteoporosis periodontitis patients with and without dental fluorosis. Methods: : Twenty one primary osteoporotic patients suffering from periodontitis with dental fluorosis and 20 primary osteoporotic patients suffering from periodontitis without dental fluorosis participated in this study. Periodontitis was diagnosed based on age, gender T-scores using clinical parameters such as plaque scores, gingival bleeding scores and probing pocket depth, clinical attachment level (CAL) and severity of dental fluorosis. DNA was genotyped at the RsaI RFLP (in exon 5) inside the ER gene to study ER Rsa I gene polymorphism in osteoporosis periodontitis patients with and without dental fluorosis. Results: : Patients with dental fluorosis had higher degree of osteoporosis than those without fluorosis. CAL was significantly higher (P <0.05) in those with dental fluorosis compared with those without. Rr heterozygote (21.95%) was observed in patients without fluorosis whereas RR mutant homozygote was absent in both the groups. Rr wild homozygote type was seen more in the patients with fluorosis (51.21%). Significant differences were found in distribution of these genotypes between patients with and without dental fluorosis. Interpretation & conclusions: : This preliminary study showed the presence of ER I gene polymorphism in osteoporosis periodontitis patients without dental fluorosis. Further studies with large sample size are needed to confirm the association shown in this preliminary study.

Evidence of Skeletal Fluorosis at the Ray Site, Illinois, USA: a pathological assessment and discussion of environmental factors.

OBJECTIVE: To carefully assess skeletal lesions in close environment context in order to evaluate whether skeletal fluorosis was present in individuals living in the prehistoric Midwest, USA. MATERIALS: Skeletal remains from minimally 117 individuals recovered from the Ray Site, located in western Illinois (USA) and dated to the Middle/early Late Woodland periods (50 BC-AD 400). METHODS: Macroscopic evaluation of all recovered skeletal elements. RESULTS: Eight individuals display a constellation of abnormal bony changes, including osteosclerosis, a high frequency of fractures, and dental abnormalities. CONCLUSIONS: The osteosclerotic changes along with the naturally high fluoride content of west central Illinois soil and water suggests the presence of skeletal fluorosis. SIGNIFICANCE: This is the first report of skeletal fluorosis from archaeologically recovered human remains from North America. LIMITATIONS: The ambiguous nature of the skeletal changes associated with fluorosis, especially in the less severe stages of the disease, renders determination of the etiology difficult. SUGGESTIONS FOR FURTHER RESEARCH: The continuation of paleopathological investigations of fluoride toxicity within archaeological communities recovered from this region with emphasis on the incorporation of biomedical and environmental data. Furthermore, complementary analyses of the chemical composition and the histological presentation of the skeletons could provide support for this diagnosis.

Rodent Dental Fluorosis Model: Extraction of Enamel Organ from Rat Incisors.

Chronic fluoride overexposure can cause dental fluorosis. Dental fluorosis is characterized by porous and soft enamel that is vulnerable to erosion and decay. Animal models often contribute to clinical applications by addressing pathogenic questions of disease. To study dental fluorosis, rodent models have been employed because rodent incisors erupt continuously and every stage of enamel development is present along the length of the rodent incisor. Here we present a protocol to induce dental fluorosis in mouse and rat and describe the procedure for extraction of stage specific enamel organ from rat mandibular incisors.

Low-to-moderate fluoride exposure, relative mitochondrial DNA levels, and dental fluorosis in Chinese children.

BACKGROUND: The alteration of mitochondrial DNA (mtDNA) content contributes to many diseases, however, little is known about its effect on the prevalence of dental fluorosis (DF). OBJECTIVES: We conducted a cross-sectional study to investigate the association of low-to-moderate fluoride exposure with relative mtDNA levels in relation to DF in children. METHODS: We recruited 616 resident children, aged 7-13 years, randomly from low-to-moderate fluoride areas in Tianjin, China. We measured the fluoride concentrations in drinking water and urine using the national standardized ion selective electrode method, and determined the relative levels of mtDNA using a quantitative real-time polymerase chain reaction assay. The association among fluoride exposure, relative mtDNA levels, and the prevalence of DF were examined using multivariable linear and logistic regression models. We also performed stratified and mediation analyses. RESULTS: The relative mtDNA levels of participants in the DF group were significantly lower than in the non-DF group (0.95 ±â€¯0.44 vs. 1.12 ±â€¯0.45, P < 0.001). In the adjusted models, we found that a 1 mg/L increment in water fluoride concentration was associated with a 0.10-unit decrease in circulating relative mtDNA levels (95% CI: -0.14, -0.06) and a 2.85-fold increase (95% CI: 2.01, 3.92) in moderate DF prevalence. A 1 mg/L increment in urinary fluoride level was associated with a 0.12-unit decrease in circulating relative mtDNA levels (95% CI: -0.14, -0.09) and a 1.85-fold increase (95% CI: 1.39, 2.39) in moderate DF prevalence. Stratified analysis indicated a weaker positive association of DF prevalence with fluoride exposure, while a stronger inverse relationship with relative mtDNA levels in boys than in girls. Assuming causality, we estimated that circulating mtDNA levels mediated 13.0% (95% CI: 5.2, 28.7%) and 9.6% (95% CI: 4.7, 18.5%) of the estimated effect of a 1 mg/L increment in water fluoride and urinary fluoride on prevalence of moderate DF, respectively. CONCLUSIONS: Gender potentially modifies the associations of DF prevalence with relative mtDNA levels and low-to-moderate fluoride exposure. The reduced circulating mtDNA levels may partly mediate the elevated prevalence of moderate DF in children under such exposure.

The pathogenesis of endemic fluorosis: Research progress in the last 5 years.

Fluorine is one of the trace elements necessary for health. It has many physiological functions, and participates in normal metabolism. However, fluorine has paradoxical effects on the body. Many studies have shown that tissues and organs of humans and animals appear to suffer different degrees of damage after long-term direct or indirect exposure to more fluoride than required to meet the physiological demand. Although the aetiology of endemic fluorosis is clear, its specific pathogenesis is inconclusive. In the past 5 years, many researchers have conducted in-depth studies into the pathogenesis of endemic fluorosis. Research in the areas of fluoride-induced stress pathways, signalling pathways and apoptosis has provided further extensive knowledge at the molecular and genetic level. In this article, we summarize the main results.

Skeletal fluorosis in Vavuniya District: an observational study

Background: The WHO recommended safe upper limit for fluoride in drinking water is 1.5 mg/l. Groundwater sources in many parts of Sri Lanka often exceed this limit. The high fluoride content of groundwater and high environmental temperatures in Vavuniya District predispose to pre-skeletal fluorosis and skeletal fluorosis in adults. Objectives: To identify residents of Vavuniya District with clinical features of pre-skeletal and skeletal fluorosis; to describe their clinical, biochemical and radiographic features; to determine the fluoride content of blood and urine in individuals with established diagnoses, and of their drinking water. Methods: In 98 volunteers we detected 60 with clinical features of pre-skeletal and skeletal fluorosis. Clinical examination, biochemical and radiographic investigations were performed. Forty four with confounding factors were excluded. The balance 16 had radiographic investigation for fluoride bone disease, and assessment of clinical features for pre-skeletal fluorosis. The radiographic criteria of skeletal fluorosis were trabecular haziness, osteosclerosis, osteophytes, cortical thickening and ligamentous or muscle attachment ossification. All 16 had "spot" samples of 15 ml of venous blood taken for biochemical tests and fluoride estimation; and 30 ml of urine, and water from 16 dug wells for fluoride. Results: The 16 selected (11 males) had BMI between 20.6 and 31.9 kg/m2, and were between 22 and 84 years (x̅ = 59.9 + 20.4). They used water from domestic dug wells for drinking. All had adequate renal function. All serum and urine samples had raised fluoride levels way above the reference ranges for serum (0.02 ­ 0.18 mg/l) and urine (0.6 ­ 2.0 mg/l). The 16 water samples showed a mean fluoride content of 2.90 +0.93 mg/l. Interpretation: In a cohort of 60 individuals in Vavuniya with symptoms suggestive of skeletal fluoride toxicity, 6 had skeletal fluorosis, 10 had pre-skeletal fluorosis, and groundwater sources had fluoride levels much higher than WHO recommended upper limit for drinking water. Residents in Vavuniya are predisposed to pre-skeletal and skeletal fluorosis. All 16 had been misdiagnosed as various types of arthritis.

Trend-analysis of dental hard-tissue conditions as function of tooth age.

OBJECTIVE: This retrospective in-vitro study investigated tooth age effect on dental hard-tissue conditions. METHODS: Unidentified extracted premolars (n = 1500) were collected and their individual age was estimated (10-100 (±10) years old (yo)) using established dental forensic methods Dental caries, fluorosis and tooth wear (TW) were assessed using the International Caries Detection and Assessment System (ICDAS; 0-5 for crown and 0-2 for root), Thylstrup-Fejerskov (TFI; 0-9) and Basic Erosive Wear Examination (BEWE; 0-3) indices, respectively. Staining and color were assessed using the modified-Lobene (MLI) (0-3) and VITA shade (B1-C4) indices, respectively. Relationships between indices and age were tested using regression models. RESULTS: Starting at age ∼10yo, presence of caries increased from 35% to 90% at ∼50yo (coronal), and from 0% to 35% at ∼80yo (root). Caries severity increased from ICDAS 0.5 to 2 at ∼40yo and from ICDAS 0 to 0.5 at ∼60yo for coronal and root caries, respectively. Presence of TW increased from 25% (occlusal) and 15% (smooth-surfaces) to 100% at ∼80yo. TW severity increased from BEWE 0.5 to 2 at ∼50yo (occlusal) and ∼0.3 to 1.5 at ∼50yo (smooth-surfaces). Percentage and severity of fluorosis decreased from 70% to 10% at ∼80yo, and from TFI 1 to 0 at ∼90yo, respectively. Percentage of extrinsic staining increased from 0% to 85% at ∼80yo and its severity increased from MLI 0 to 2 at ∼70yo. Color changed from A3 to B3 at ∼50yo (crown), and from C2 to A4 at ∼85yo (root). CONCLUSIONS: Aging is proportionally related to the severity of caries, TW, staining, and inversely to dental fluorosis. Teeth become darker with age.

Immunohistochemical Localization of Fam83h During Fluorosis-induced Mouse Molar Development.

The clinical and pathological features of fluorosis are similar to amelogenesis imperfecta (AI) caused by FAM83H mutations, suggesting that excess fluoride could have effects on the expression of Fam83h. Our previous study found that Fam83h was downregulated by fluorosis induction in ameloblasts; the purpose of this study was to underline the importance of understanding the relationship between fluoride administration and Fam83h expression in vivo. A total of 80 healthy female adult Kunming mice were randomly divided into control group or F group that induced the clinical features of fluorosis. Immunohistochemical staining on sections of the embryo mandible regions was performed at different developmental stages. Mouse primary ameloblast-like cells of the two groups at E13.5, E15.5, and E18.5 were cultured and examined for the expression of Fam83h. The expression of Fam83h in the F group was significantly lower than that in the control group; however, Fam83h was observed clearly in the whole enamel organ in the control group. Our findings shed new light on the potential effects of Fam83h in fluorosis using a mouse model and revealed that high fluoride decreased the expression of Fam83h. This may be one of the reasons for the occurrence of fluorosis.

ClC-7/Ostm1 contribute to the ability of tea polyphenols to maintain bone homeostasis in C57BL/6 mice, protecting against fluorosis.

Epidemiological investigations indicate that certain ingredients in tea bricks can antagonize the adverse effects of fluoride. Tea polyphenols (TPs), the most bioactive ingredient in tea bricks, have been demonstrated to be potent bone-supporting agents. ClC­7 is known to be crucial for osteoclast (OC) bone resorption. Thus, in this study, we investigated the potential protective effects of TPs against fluorosis using a mouse model and explored the underlying mechanisms with particular focus on ClC­7. A total of 40, healthy, 3­week­old male C57BL/6 mice were randomly divided into 4 groups (n=10/group) by weight as follows: distilled water (control group), 100 mg/l fluoridated water (F group), water containing 10 g/l TPs (TP group) and water containing 100 mg/l fluoride and 10 g/l TPs (F + TP group). After 15 weeks, and after the mice were sacrificed, the long bones were removed and bone marrow-derived macrophages were cultured ex vivo in order to perform several experiments. OCs were identified and counted by tartrate­resistant acid phosphatase (TRAP) staining. The consumption of fluoride resulted in severe fluorosis and in an impaired OC function [impaired bone resorption, and a low mRNA expression of nuclear factor of activated T-cells 1 (NFATc1), ATPase H+ transporting V0 subunit D2 (ATP6v0d2) and osteopetrosis­associated transmembrane protein 1 (Ostm1)]. In the F + TP group, fluorosis was attenuated and OC function was restored, but not the high bone fluoride content. Compared with the F group, mature OCs in the F + TP group expressed higher mRNA levels of ClC­7 and Ostm1; the transportation and retaining of Cl­ was improved, as shown by the fluorescence intensity experiment. On the whole, our findings indicate that TPs mitigate fluorosis in C57BL/6 mice by regulating OC bone resorption. Fluoride inhibits OC resorption by inhibiting ClC­7 and Ostm1, whereas TPs attenuate this inhibitory effect of fluoride.

Protective effect of lycopene on fluoride-induced ameloblasts apoptosis and dental fluorosis through oxidative stress-mediated Caspase pathways.

Fluoride is an environmental toxicant and induces dental fluorosis and oxidative stress. Lycopene (LYC) is an effective antioxidant that is reported to attenuate fluoride toxicity. To determine the effects of LYC on sodium fluoride (NaF) -induced teeth and ameloblasts toxicity, rats were treated with NaF (10 mg/kg) and/or LYC (10 mg/kg) by orally administration for 5 weeks; ameloblasts were treated with NaF (5 mM) and/or LYC (2 µM) for 6 h. We found that the concentrations of fluoride, malondialdehyde (MDA) and reactive oxygen species (ROS), gene expressions and activities of Caspase-9 and Caspase-3, and the gene expressions of Bax were significantly decreased, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX), the gene expression of Bcl-2 were significantly increased in the LYC + NaF-treated rats group; concentrations of MDA and ROS, gene expressions and activities of Caspase-9 and Caspase-3, and the gene expression of Bax, and ameloblasts apoptosis rate were significantly decreased, while the activities of SOD and GPX, the gene expression of Bcl-2 were significantly increased in the LYC + NaF-treated ameloblasts group. These results suggest that LYC significantly combated NaF-induced ameloblasts apoptosis and dental fluorosis by attenuation oxidative stress and down-regulation Caspase pathway.

Validation of ICMR index for identification of dental fluorosis in epidemiological studies.

BACKGROUND & OBJECTIVES: The Indian Council of Medical Research (ICMR) formulated a Task Force on dental fluorosis and recommended the subgroup to develop a simplified index for identification and grading of dental fluorosis to be used by the health workers. This study was conducted to pre-test the 'ICMR Index for Dental Fluorosis' in the field to check its reliability and reproducibility. METHODS: A total of 600 photographs were taken, 150 in each grade of fluorosis by screening 14-17 yr school children from eight schools of Hisar (Haryana) and South west Delhi. Eighty photographs were finalized (20 in each grade) before calibration to be used for training of field workers. Calibration exercise was conducted involving the five member survey team on 100 diagnosed cases of dental fluorosis. The members again screened 74 children with dental fluorosis in the field to categorize in to different grades of fluorosis for assessment of inter-examiner reliability. RESULTS: The ICMR criteria showed more difference in agreement in very mild and mild categories during calibration. The inter-examiner reliability (κ) ranged from 0.59-1. The criteria was further modified and inter- examiner reliability (κ) found to be 0.83-0.98 which was almost perfect agreement. INTERPRETATION & CONCLUSIONS: The tool developed by the ICMR to assess dental fluorosis can be used in a field set up by non-dental personnel reliably with high degree of reproducibility.

Chronic Exposure to Bisphenol A Exacerbates Dental Fluorosis in Growing Rats.

Enamel defects resulting from environmental conditions and way of life are public health concerns because of their high prevalence. Because their etiology is unclear, the aim of this study was to analyze the various forms of enamel hypomineralization, and to characterize the genes involved in this process to determine the mechanisms involved in disruptions of amelogenesis. We used bisphenol A (BPA) and fluoride as models; both are commonly encountered in human populations and utilized in dentistry. Wistar rats were chronically exposed to 5 µg/kg/day BPA from day 1 of gestation to day 65 after birth (P65) and 5 mM fluoride from P21 to P65. Resulting enamel defects were comparable to the human enamel pathologies molar incisor hypomineralization (MIH) and dental fluorosis (DF) respectively, and were more severe in rats exposed to both agents than to each agent alone. Large-scale transcriptomic analysis of dental epithelium showed a small group of genes the expression of which was affected by exposure to BPA or NaF. Among the most modulated, many are directly involved in amelogenesis (Amelx, Enam, Klk4, Mmp12, Slc26a4, and Slc5a8), and can be regrouped as forming the "hypomineralization enameloma." Each of these gene expression perturbations may contribute to enamel defects. Exposure to BPA weakens enamel, making it more prone to generate frequent mineralization defects MIH and DF. Our study identifies hypomineralization genes that may enable the use of dental enamel as an early marker of exposure to environmental toxicants because of its unique ability to retrospectively record ameloblast pathophysiology. © 2016 American Society for Bone and Mineral Research.

Grading and quantification of dental fluorosis in zebrafish larva.

OBJECTIVE: The prevalence and severity of dental fluorosis in primary teeth are different from permanent teeth. Previous animal models of dental fluorosis mainly focus on juvenile rats, mice and zebrafish. Our experiment aims to set a dental fluorosis model using zebrafish larva and explore the characteristics of the first generation teeth by fluoride treatment. MATERIALS AND METHODS: After the zebrafish eggs were laid, they were exposed to excess fluoride (19ppm, 38ppm and 76ppm) for five days. The morphological characteristics of first generation teeth were examined by H&E staining, whole-mount alizarin red and alcian blue staining, and scanning electron microscope (SEM) technique. RESULTS: With whole-mount alizarin red and alcian blue staining, the tooth cusps presented red in normal control. 19ppm and 38ppmm fluoride resulted in extensive red staining from tooth cusps to the lower 1/3 of teeth. 76ppm fluoride caused malformed teeth with uneven red staining. H&E staining showed that excess fluoride caused cystic-like changes in 38ppm and 76ppm groups. SEM revealed the dose dependent pathological changes in zebrafish enameloid with fluoride treatment. Based on SEM findings, we set 0-4 dental fluorosis index (DFI) score to label the severity of dental fluorosis. CONCLUSIONS: Excess fluoride presented a dose dependent fluorosis changes in the teeth of zebrafish larva. The DFI scores in our experiment reflect dose dependent fluorosis changes in a good way and will benefit the future research of dental fluorosis.

Dental Fluorosis and Catalase Immunoreactivity of the Brain Tissues in Rats Exposed to High Fluoride Pre- and Postnatally.

This study evaluated dental fluorosis of the incisors and immunoreactivity in the brain tissues of rats given chronic fluoride doses pre- and postnatally. Female rats were given drinking water with 0, 30 or 100 ppm fluoride ad libitum throughout gestation and the nursing period. In addition, 63 male offspring were treated with the same water regimens as the mothers after weaning and were followed for 1, 3 or 5 months. The upper and lower incisors were collected, and all teeth were examined under a stereomicroscope and scored by two blinded examiners using a modified rodent enamel fluorosis index. Cortical, hippocampal and cerebellar brain samples were evaluated morphologically and immunohistochemically. All fluoride-treated pups were born with low body weight (p = 0.001). All animals from the fluoride groups had enamel fluorosis with defects of various degrees. The increase in the dental fluorosis scores in the fluoride treatment groups was significant (p < 0.01). The catalase immunoreactivity in the 30- and 100-ppm fluoride groups was significantly higher than that in the controls after 1, 3 and 5 months (p < 0.001). In conclusion, this study showed that rats with dental fluorosis had catalase immunoreactivity in the brain tissues, which may reflect the neurobehavioral toxicity of fluoride.

3D scanning electron microscopy applied to surface characterization of fluorosed dental enamel.

The enamel surfaces of fluorotic teeth were studied by scanning electron stereomicroscopy. Different whitening treatments were applied to 25 pieces to remove stains caused by fluorosis and their surfaces were characterized by stereomicroscopy in order to obtain functional and amplitude parameters. The topographic features resulting for each treatment were determined through these parameters. The results obtained show that the 3D reconstruction achieved from the SEM stereo pairs is a valuable potential alternative for the surface characterization of this kind of samples.

Developmental and Post-Eruptive Defects in Molar Enamel of Free-Ranging Eastern Grey Kangaroos (Macropus giganteus) Exposed to High Environmental Levels of Fluoride.

Dental fluorosis has recently been diagnosed in wild marsupials inhabiting a high-fluoride area in Victoria, Australia. Information on the histopathology of fluorotic marsupial enamel has thus far not been available. This study analyzed the developmental and post-eruptive defects in fluorotic molar enamel of eastern grey kangaroos (Macropus giganteus) from the same high-fluoride area using light microscopy and backscattered electron imaging in the scanning electron microscope. The fluorotic enamel exhibited a brownish to blackish discolouration due to post-eruptive infiltration of stains from the oral cavity and was less resistant to wear than normally mineralized enamel of kangaroos from low-fluoride areas. Developmental defects of enamel included enamel hypoplasia and a pronounced hypomineralization of the outer (sub-surface) enamel underneath a thin rim of well-mineralized surface enamel. While the hypoplastic defects denote a disturbance of ameloblast function during the secretory stage of amelogenesis, the hypomineralization is attributed to an impairment of enamel maturation. In addition to hypoplastic defects, the fluorotic molars also exhibited numerous post-eruptive enamel defects due to the flaking-off of portions of the outer, hypomineralized enamel layer during mastication. The macroscopic and histopathological lesions in fluorotic enamel of M. giganteus match those previously described for placental mammals. It is therefore concluded that there exist no principal differences in the pathogenic mechanisms of dental fluorosis between marsupial and placental mammals. The regular occurrence of hypomineralized, opaque outer enamel in the teeth of M. giganteus and other macropodids must be considered in the differential diagnosis of dental fluorosis in these species.