Expression of core-binding factor α1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts

To study the effects and importance of fluoride on FBs in the development of extraperiosteal calcification and the ossification of skeletal fluorosis, the presence of the osteogenic phenotype, which is indicated by the expression of core-binding factor α1 (Cbfa1) and osteocalcin (OCN), in an FB cell line (L929) and in osteoblasts (OBs) exposed to fluoride was determined. Fibroblasts and osteoblasts were exposed to different concentrations of fluoride (0, 0.0001, 0.001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻). By using RT-PCR and ELISA, the mRNA levels of Cbfa1 and OCN were measured at 48 h, and the protein levels of Cbfa1 and OCN were measured at 2, 4, 24, 48 and 72 h. The data demonstrated the following: (1) The Cbfa1 protein level in fluoride-treated fibroblasts clearly increased at 48 h in the groups treated with 0.0001, 0.001, 0.1, 1.0 and 20.0 mg/L F⁻. The Cbfa1 protein level of the group treated with 10 mg/L F⁻ at 72 h was higher than that of the control group. The level of Cbfa1 mRNA in the fibroblasts was much higher at 48 h in the group treated with 10.0 mg/L F⁻ than in the control group. (2) The OCN protein level in fluoride-treated fibroblasts was significantly higher than that of the control group in the 0.0001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻ groups at 2 h, and in the 0.001 and 0.1 F⁻ groups at 4 h. A slightly higher level of OCN mRNA in fluoride-treated fibroblasts was also found in the 1.0 and 20.0 mg/L F⁻ groups compared to the control group. (3) The expressions of Cbfa1 and OCN in osteoblasts treated with the same experimental conditions as the fibroblasts were up-regulated by fluoride following the same trend as in the fibroblasts. Our results showed an increase in the expression of Cbfa1 and OCN in fibroblasts and osteoblasts exposed to fluoride and suggested that the osteogenic function of fibroblasts induced by fluoride could play an important role in the development of extraperiosteal ossification during skeletal fluorosis.     

Direct and continuous assay for prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O₂ as cosubstrates, and forms succinate and CO₂ as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.     

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600-620 nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450-460 nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin (Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.     

Proteomic analysis of urine in rats chronically exposed to fluoride

Urine is an ideal source of materials to search for potential disease‐related biomarkers as it is produced by the affected tissues and can be easily obtained by noninvasive methods. 2‐DE‐based proteomic approach was used to better understand the molecular mechanisms of injury induced by fluoride (F^?) and define potential biomarkers of dental fluorosis. Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F^? for 60 days (n = 15/group). During the experimental period, the animals were kept individually in metabolic cages, to analyze the water and food consumption, as well as fecal and urinary F^? excretion. Urinary proteome profiles were examined using 2‐DE and Colloidal Coomassie Brilliant Blue staining. A dose‐response regarding F^? intake and excretion was detected. Quantitative intensity analysis revealed 8, 11, and 8 significantly altered proteins between control vs. 5 ppm F^?, control vs. 50 ppm F^? and 5 ppm F^? vs. 50 ppm F^? groups, respectively. Two proteins regulated by androgens (androgen‐regulated 20‐KDa protein and α‐2μ‐globulin) and one related to detoxification (aflatoxin‐B1‐aldehyde‐reductase) were identified by MALDI‐TOF‐TOF MS/MS. Thus, proteomic analysis can help to better understand the mechanisms underlying F^? toxicity, even in low doses. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:8–14 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20353     

黑龙江省农村饮用水水质健康风险评价

本文以黑龙江省农村饮用水为研究对象,调研分析了该地区农村饮用水水质状况和水质问题,筛查出三种可能造成人体健康风险的典型污染物:砷,氟化物,硝酸盐。在此基础上,选用了USEPA推荐的水质风险健康评价模型对三种污染进行了健康风险评价,评价结果表明砷的健康风险值大于最大可接受限值10-(6a-1),存在较大人体健康风险,氟化物和亚硝酸盐人体健康风险较小。此外,根据水质健康风险评价结果,对保障黑龙江省农村饮用水实质安全提出了初步解决措施。     

Effect of altitude on urinary,plasma and nail fluoride levels in children and adults in Nepal

IntroductionA greater prevalence of dental fluorosis has been reported in higher- versus lower-altitude communities. This study, for the first time, examined several aspects of fluoride metabolism in children, aged 4–5 years, and their parent, living at lower altitude (<78 m) and higher altitude (>1487) areas in Nepal.MethodsThe study assessed total daily fluoride intake (TDFI), 24 h urinary fluoride excretion (UFE), and fluoride concentrations of toe- and finger-nail (FC_toenail, FC_fingernail) in children and parents as well as fluoride concentration of plasma (FC_plasma) in parents. Fractional urinary fluoride excretion (FUFE) was calculated as the ratio between UFE and TDFI. FC_toenail, FC_fingernail and FC_plasma were normalised for TDFI by dividing the variables by TDFI and the ratio was reported as the percentage.ResultsIn total, 89 children and 80 parents took part in the study: 42 children and 41 parents from the lower altitude area; 47 children and 39 parents from the higher altitude area. Fluoride concentration of drinking water was significantly (P < 0.001) higher at lower altitude (0.395 mg F/l) than at higher altitude (0.104 mg F/l). TDFI was significantly (p < 0.001) higher in both children and parents living in lower altitude than those living at higher altitude.There was a statistically significant (p = 0.044) difference in the mean FUFE of children living at lower altitude (53%) and higher altitude (46%). However, no significant difference in FUFE was found between parents living at lower altitude (47%) compared with higher altitude (41%).In both children and parents, no statistically significant differences in normalised FC_toenail, FC_fingernail were found between the two altitude areas. However, normalised FC_plasma was statistically significantly (P = 0.005) higher in parents living at higher altitude (0.15%) compared with those living at lower altitude (0.11%).ConclusionThe results suggest that higher altitude living results in decreased urinary fluoride excretion, and consequently increased fluoride retention in children for a given dose (amount) of fluoride.     

Quasi-irreversible inhibition of enolase of Streptococcus mutans by flouride

Abstract Fluoride at concentrations greater than 0.01 mM was found to be a quasi-irreversible inhibitor of enolase of permeabilized cells of Streptococcus mutans GS-5 and also of isolated yeast enolase. The inhibition appeared to be of the type that has been described for P-ATPases, but was not dependent on added Al³⁺ or Be²⁺ ions. Fluoride inhibition of enolase was not reversed by repeatedly washing the permeabilized cells in chilled fluoride-free medium but could be reversed by the product, phosphoenolpyruvate, or by very high levels of the substrate, 2-phosphoglycerate. Irreversible inhibition of glycolysis was not evident after fluoride treatment of intact cells, washing to remove unbound or loosely bound fluoride and addition of glucose, presumably because intracellular levels of phosphoenolpyruvate were sufficiently high to preclude irreversible fluoride inhibition of enolase.     

氟对正畸儿童牙面菌斑变形链球菌的影响

目的:观察和探讨2%氟化钠对正畸患者牙面菌斑内细菌总数及变形链球菌数的影响.方法:选择34例正畸儿童,分为两组,试验组涂布2%氟化钠;对照组不做处理.分别于戴用矫治器前,戴入后1月采集上颌牙唇颊面菌斑,测定菌斑中细菌总数及变形链球菌数.结果:对照组戴用后1月,细菌总数及变形链球菌数较戴用前明显增加(P＜0.01).对照组与试验组戴用后1月相比,试验组细菌总数及变形链球菌数明显少于对照组(P＜0.05).结论:戴用固定矫治器后,牙面菌斑内细菌总数及变形链球菌数较戴用前增加,应用2%氟化钠可明显抑制正畸患者口腔内变形链球菌数,减少龋坏发生.     

Arginine metabolism of the Antarctic Bivalve <Emphasis Type="Italic">Laternula elliptica</Emphasis> (King &; Broderip, 1831): an ecophysiological approach

The potential aerobic ATP-generating pathway and the argininolytic capacity of the Antarctic bivalve Laternula elliptica in its main tissues were measured by the specific activity of the enzymes malate dehydrogenase (MDH), citrate synthase (CS) and arginase. The kidney showed the major potential for aerobic ATP-generating pathway and argininolytic capacity. High levels of CS and MDH activities indicated that renal tissue can be involved in activities that require a lot of energy such as excretion of metabolic end products, amino acids catabolism or even gluconeogenic activities related to inter-tissue metabolism. The fact that kidneys are the main site for arginase activity is very unusual for mollusks and could be related to the living habits of L. elliptica. Genetic expression of the L. elliptica renal arginase could be controlling the levels of l-arginine and forming urea in the excretory organ, which may not have its physiological functions directly affected by the seasonal retraction of its siphons. Compared to the bivalve Dreissena polymorpha, renal arginase of L. elliptica is more resistant to inhibition by copper and cadmium. This could be related to naturally high levels of these metals in the Antarctic marine environment and its bioaccumulation in the renal tissue of L. elliptica, as a probable advantage to its environmental adaptation. Different from other Antarctic animals that feed on Krill, the arginase of L. elliptica is much more sensitive to fluoride inhibition. However, diet composition of L. elliptica would be expected to be variable site to site and its high sensitivity to fluoride inhibition may be a matter of concern in areas near ornithogenic soils subjected to ice-melting processes.     

Fluoride and environmental health: a review

The relationship between environmental fluoride and human health has been studied for over 100 years by researchers from a wide variety of disciplines. Most scientists believe that small amounts of fluoride in the diet can help prevent dental caries and strengthen bones, but there are a number of adverse affects that chronic ingestion at high doses can have on human health, including dental fluorosis, skeletal fluorosis, increased rates of bone fractures, decreased birth rates, increased rates of urolithiasis (kidney stones), impaired thyroid function, and lower intelligence in children. Chronic occupational exposure to fluoride dust and gas is associated with higher rates of bladder cancer and variety of respiratory ailments. Acute fluoride toxicity and even death from the ingestion of sodium fluoride pesticides and dental products have also been reported. The distribution of fluoride in the natural environment is very uneven, largely a result of the geochemical behavior of this element. Fluorine is preferentially enriched in highly evolved magmas and hydrothermal solutions, which explains why high concentrations are often found in syenites, granitoid plutonic rocks, alkaline volcanic, and hydrothermal deposits. Fluoride can also occur in sedimentary formations that contain fluoride-bearing minerals derived from the parent rock, fluoride-rich clays, or fluorapatite. Dissolved fluoride levels are usually controlled by the solubility of fluorite (CaF₂); thus, high concentrations are often associated with soft, alkaline, and calcium-deficient waters. Although much is known about the occurrence and health effects of fluoride, problems persist in Third World countries, where populations have little choice in the source of their drinking water and food. However, even in developed nations, fluoride ingestion can exceed the recommended dose when sources other than drinking water are ignored.