Changes of DNA repair gene methylation in blood of chronic fluorosis patients and rats.

To investigate the relationship between DNA repair gene methylation and chronic coal-burning fluorosis. The methylation rates of O6-methylguanine-DNA- methyltransferase gene MGMT, a DNA repair gene and mismatch repair gene MutL homolog 1 (MLH1) were analysed by methylation of specific PCR (MSP), and the levels of mRNA in the blood of the chronic fluorosis rats and the patients in the region of endemic coal-burning fluorosis were determined by real-time PCR. The levels of mRNA and protein of MGMT and MLH1 in the liver tissue of the chronic fluorosis rats were determined by real-time PCR and Western blot respectively. The results showed an increased methylation of the MGMT and MLH1 genes in the blood of the patients in the fluorosis region that correlated positively with the severity of fluorosis. The mRNA levels of MGMT and MLH1 genes from the patients in fluorosis region were lower than those of a control group, and also showed a positive correlation with the severity of fluorosis. Both the protein and mRNA levels of MGMT and MLH1 genes from the blood of rats and liver tissue in a fluoride-treated group were lower than those of a control non-fluoride treated group. These results indicate that the degree of methylation of MGMT and MLH1 genes is altered in fluorosis disease, the resulting changed expression of these repair genes may play a role in the liver damage caused by 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.

Enzymatic Activity of Glutathione S-Transferase and Dental Fluorosis Among Children Receiving Two Different Levels of Naturally Fluoridated Water.

This study was conducted to measure the activity of the enzyme glutathione S-transferase (GST) in saliva and to compare the activity of this enzyme in children with and without dental fluorosis in communities with different concentrations of naturally fluoridated water. A total of 141 schoolchildren participated in this cross-sectional study. Children were selected from two communities: one with a low (0.4 ppm) and the other with a high (1.8 ppm) water fluoride concentration. Dental fluorosis was evaluated by applying the Thylstrup and Fejerskov Index (TFI) criteria. Stimulated saliva was obtained, and fluoride concentration and GST activity were measured. The GST activity was compared among children with different levels of dental fluorosis using multinomial logistic regression models and odds ratios (OR). The mean age of the children was 10.6 (±1.03) years. Approximately half of the children showed dental fluorosis (52.5 %). The average GST activity was 0.5678 (±0.1959) nmol/min/µg. A higher concentration of fluoride in the saliva was detected in children with a higher GST activity (p = 0.039). A multinomial logistic regression model used to evaluate the GST activity and the dental fluorosis score identified a strong association between TFI = 2-3 (OR = 15.44, p = 0.007) and TFI ≥ 4 (OR = 55.40, p = 0.026) and the GST activity level, compared with children showing TFI = 0-1, adjusted for age and sex. Schoolchildren with higher levels of dental fluorosis and a higher fluoride concentration in the saliva showed greater GST activity. The increased GST activity most likely was the result of the body's need to inactivate free radicals produced by exposure to fluoride.

LS8 cell apoptosis induced by NaF through p-ERK and p-JNK - a mechanism study of dental fluorosis.

OBJECTIVE: To investigate the possible biological mechanism of dental fluorosis at a molecular level. MATERIAL AND METHODS: Cultured LS8 were incubated with serum-free medium containing selected concentrations of NaF (0 ∼ 2 mM) for either 24 or 48 h. Subcellular microanatomy was characterized using TEM; meanwhile, selected biomolecules were analysed using various biochemistry techniques. Transient transfection was used to modulate a molecular pathway for apoptosis. RESULTS: Apoptosis of LS8 was induced by NaF treatment that showed both time and concentration dependency. The activity of caspase-3, -8, -9 was found to be increased with NaF in a dose-dependent manner. Western blot revealed that the protein expression of p-ERK and p-JNK were decreased, while the expression of p-P38 was increased. Inhibition of the p-ERK and p-JNK pathways resulted in a similar decrease for caspase-3. CONCLUSION: During NaF-induced apoptosis of LS8, p-ERK and p-JNK were closely associated with induction of apoptosis, which might be a mechanism 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.

Coal-burning endemic fluorosis is associated with reduced activity in antioxidative enzymes and Cu/Zn-SOD gene expression.

To study the effect of fluorine on the oxidative stress in coal-burning fluorosis, we investigated the environmental characteristics of coal-burning endemic fluorosis combined with fluorine content surveillance in air, water, food, briquette, and clay binder samples from Bijie region, Guizhou Province, southwest of China. The activities of antioxidant enzymes including copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and level of lipid peroxidation such as malondialdehyde (MDA) were measured in serum samples obtained from subjects residing in the Bijie region. Expression of the Cu/Zn-SOD gene was assessed by quantitative reverse transcriptase PCR (qRT-PCR). Our results showed that people suffering from endemic fluorosis (the high and low exposure groups) had much higher MDA level. Their antioxidant enzyme activities and Cu/Zn-SOD gene expression levels were lower when compared to healthy people (the control group). Fluorosis can decrease the activities of antioxidant enzymes, which was associated with exposure level of fluorine. Down-regulation of Cu/Zn-SOD expression may play an important role in the aggravation of oxidative stress in endemic fluorosis.

Myeloperoxidase activity and its corresponding mRNA expression as well as gene polymorphism in the population living in the coal-burning endemic fluorosis area in Guizhou of China.

The myeloperoxidase (MPO) activity and its corresponding mRNA expression as well as gene polymorphism were investigated in the population who live in the endemic fluorosis area. In the study, 150 people were selected from the coal-burning endemic fluorosis area and 150 normal persons from the non-fluorosis area in Guizhou province of China. The blood samples were collected from these people. The activity of MPO in the plasma was determined by spectrophotometer; the expression of MPO mRNA was measured by employing real-time polymerase chain reaction; DNAs were extracted from the leucocytes in blood and five SNP genotypes of MPO promoter gene detected by a multiplex genotyping method, adapter-ligation-mediated allele-specific amplification. The results showed that the MPO activity and its corresponding mRNA in blood were significantly increased in the population living in the area of fluorosis. The different genotype frequencies of MPO, including -1228G/A, -585T/C, -463G/A, and -163C/T, and the three haplotypes with higher frequencies, including -163C-463G-585T-1228G-1276T, -163C-463G-585T-1228G-1276C, and -163C-463G-585T-1228A-1276T, were significantly associated with fluorosis. The results indicated that the elevated activity of MPO induced by endemic fluorosis may be connected in mechanism to the stimulated expression of MPO mRNA and the changed gene polymorphism.

Assessment of dental fluorosis in Mmp20 +/- mice.

The molecular mechanisms that underlie dental fluorosis are poorly understood. The retention of enamel proteins hallmarking fluorotic enamel may result from impaired hydrolysis and/or removal of enamel proteins. Previous studies have suggested that partial inhibition of Mmp20 expression is involved in the etiology of dental fluorosis. Here we ask if mice expressing only one functional Mmp20 allele are more susceptible to fluorosis. We demonstrate that Mmp20 (+/-) mice express approximately half the amount of MMP20 as do wild-type mice. The Mmp20 heterozygous mice have normal-appearing enamel, with Vickers microhardness values similar to those of wild-type control enamel. Therefore, reduced MMP20 expression is not solely responsible for dental fluorosis. With 50-ppm-fluoride (F(-)) treatment ad libitum, the Mmp20 (+/-) mice had F(-) tissue levels similar to those of Mmp20 (+/+) mice. No significant difference in enamel hardness was observed between the F(-)-treated heterozygous and wild-type mice. Interestingly, we did find a small but significant difference in quantitative fluorescence between these two groups, which may be attributable to slightly higher protein content in the Mmp20 (+/-) mouse enamel. We conclude that MMP20 plays a nominal role in dental enamel fluorosis.

Fluoride alters casein kinase II and alkaline phosphatase activity in vitro with potential implications for dentine mineralization.

Dentine phosphoprotein (DPP), a major non-collagenous acidic protein of dentine, undergoes altered phosphorylation in vivo in the presence of high fluoride concentrations. This has major implications for the altered mineralization patterns found during fluorosis. In dentine, casein kinase II is involved in phosphorylating DPP, and alkaline phosphatase (ALP) is ascribed roles in the dephosphorylation of DPP, increasing the inorganic phosphate at the mineralization front and the removal of pyrophosphate. Here the influence of fluoride in vitro on the activity of purified casein kinase II and ALP and its relation to altered patterns of mineralization were examined. Kinetic analysis showed that casein kinase II activity was completely inhibited at 0.04 M NaF. Vmax when compared to the control assay was significantly decreased (P < 0.0001) between concentrations 4 x 10(-4)-4 x 10(-8) M NaF. Significant changes to the Km (P < 0.0001) were also observed. ALP activity was inhibited by NaF (0.09-9 x 10(-8) M), with Vmax significantly decreased (P < 0.0001) at 0.09 M NaF. Alterations in the activity of these enzymes in the presence of fluoride may in part explain the decreased phosphorylation observed in DPP isolated from fluorotic dentine and may aid understanding of the altered matrix mediated mineralization patterns found during fluorosis.

Fluoride effect on the activity of enamel matrix proteinases in vitro.

Dental fluorosis is common in individuals exposed to different sources of fluoride during tooth development. The mechanism causing this enamel defect is still unknown. Enamel matrix proteinases play a central role in the maturation of dental enamel, and inhibition of these enzymes by fluoride has been one explanation for dental fluorosis. We have investigated the effect of fluoride on the activity of enamel matrix proteinases using a colorimetric assay, casein zymography, and an enamel protein degradation assay. Fluoride (625 microM to 10 mM) inhibited neither the enzymatic activity of the crude matrix extract nor the activity of individual enamel enzymes separated by SDS-PAGE. The proposition that fluoride could directly inhibit enzymes was not confirmed in this study.

[The enzymes of glutamate and organic phosphate metabolism in the saliva in fluorosis (clinical and experimental research)]. / Fermenty metabolizma glutamata i organicheskikh fosfatov v sliune pri fliuoroze (kliniko-éksperimental'noe issledovanie).

Activities of glutamic-oxalacetic transaminase, glutamate dehydrogenase, gamma-glutamyl transferase were reduced in unstimulated pooled saliva of 15-year-old adolescents with endemic fluorosis, vs. those without fluorosis. This point to reduced production of glutamate which is indispensable for bacterial growth. The activity of acid phosphatase in pooled stimulated with 1% pilocarpin saliva of rats fed a water ration with 5 and 20 mg/liter fluorine in comparison with control rats fed water with fluorine concentration of 0.21 mg/liter. Noteworthy that alkaline phosphatase activity was virtually unchanged at fluorine concentrations 5 and 20 mg/liter in the water. The significance of the results as far as it regards the pathogenesis of caries and fluorosis is discussed.