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Objective:To study and establish a high-throughput and rapid detection method for fluorine ion content in water based on 96-well plate and microplate reader, and to evaluate its performance.Methods:According to the principle of blue ternary complex formed by reaction of fluorine ion with fluorine reagent and lanthanum nitrate, the absorbance of the complex at the wavelength of 650 nm was proportional to the concentration of fluoride ion, and the content of fluoride ion in water samples was quantitatively determined by microplate reader. The accuracy, precision and detection limit of the method were evaluated by the standard recovery rate method and the reference material method.Results:The linear correlation coefficient of the standard curve of this method was > 0.999; the standard recovery rates of different concentrations were 99.00% - 103.33% in the accuracy experiment of this method, and the average recovery rate was 101.08%. In the standard material method, the measured values are all within the calibration range of the standard solution. The relative standard deviation of water samples with different concentrations was ≤10% in the precision experiment and the detection limit was 0.06 mg/L.Conclusion:The method has good accuracy and precision, low detection limit and small sample consumption, and this method can greatly improve the detection efficiency.
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Objective:To investigate the association of single nucleotide polymorphism at the estrogen receptor 1(ESR1) gene rs1801132 with the risk of brick-tea type skeletal fluorosis.Methods:The typical brick-tea type fluorosis areas in Qinghai, Xinjiang, and Inner Mongolia were selected as the survey sites for a cross-sectional study. An epidemiological questionnaire was conducted by the staffs on the sites for participants older than 16 years, and physical examination and X-ray diagnosis were performed. Brick tea, blood, and urine samples were collected at the same time. The diagnosis of skeletal fluorosis through X-ray was based on the "Diagnostic Criteria for Endemic Skeletal Fluorosis" (WS/T 192-2008); The determination of tea's fluoride and urinary fluoride was performed by fluoride ion-selective electrode method; gene sequencing analysis of rs1801132 locus of ESR1 gene was done by Sequenom MassARRAY flight mass spectrometry system.Results:A total of 994 patients were included in this study. The total prevalence of skeletal fluorosis was 23.9% (238/994). The prevalence of skeletal fluorosis in Tibetans(39.9%, 123/308) was higher than those of Mongolian and Han nationality [22.2% (58/261), 13.4% (57/425), χ 2=20.435, 67.811, P < 0.05]. Based on binary logistic analysis, the daily tea fluoride intake ≤ 3.5 mg, urinary fluoride content ≤1.6 mg/L, and age ≤45 years were used as the reference groups, and then, when the daily tea fluoride intake > 7.0 mg ( OR=2.865, 95% CI: 1.923-4.268), urinary fluoride content > 1.6-3.2 mg/L ( OR=2.368, 95% CI: 1.686-3.326) and > 3.2 mg/L ( OR=3.559, 95% CI: 2.401-5.276), the age > 45-65 years old ( OR=2.361, 95% CI: 1.603-3.477) and > 65 years old ( OR=4.556, 95% CI: 2.845-7.296), the risk of fluorosis was higher than that of the reference group, respectively. When the daily tea fluoride intake was > 3.5-7.0 mg and the level of urinary fluoride was > 1.6-3.2 mg/L, G allele had a protective effect on skeletal fluorosis in Mongolian population (adjusted OR=0.207, 95% CI: 0.044-0.974); when the daily tea fluoride intake was > 3.5-7.0 mg, gender was male group, G allele had a protective effect on skeletal fluorosis in Han population (adjusted OR=0.315, 95% CI: 0.112-0.887). Conclusion:The single nucleotide polymorphism of the rs1801132 locus at the ESR1 gene may be associated with the risk of susceptibility to brick-tea type skeletal fluorosis in Mongolian and Han nationality.
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Objective To explore the effects of different doses of sodium fluoride (NaF) on cartilage lesion and expression of interleukin-6 (IL-6) in serum and cartilage tissue of Balb/c mice.Methods Sixty-four 5-week-old male Balb/c mice were divided into 4 groups based on body weight via the random number table method and 16 mice were in each group.The mice in control group were fed with distilled water,and experimental animals in low,middle and high fluoride groups were fed with distilled water containing NaF 25,50 and 100 mg/L,respectively.The mice were weighed once a week and fed for three months to establish the drinking water fluorosis model.The fluoride contents in spine were detected via the fluorin-ion selective electrode method.The pathological changes in articular cartilage and epiphyseal plate cartilage were observed through optical microscope.The levels of serum IL-6 and souble IL-6 receptor (sIL-6R) were detected via the enzyme-linked immunosorbent assay.The expression of IL-6 protein in articular cartilage and epiphyseal plate cartilage was examined by immunohistochemistry.Results From the sixth week of the experiment,compared with other 3 groups,the body weight of high fluoride group decreased significantly (all P ＜ 0.05);from the seventh week,compared with control and low fluoride groups,the body weight of middle fluoride group decreased significantly (all P ＜ 0.05);throughout the experiment,compared with control group,the body weight of low fluoride group had not changed significantly (all P ＞ 0.05).The fluoride contents of bone in control group,low fluoride group,middle fluoride group and high fluoride group were (842.46 ± 89.27),(1 705.05 ± 105.76),(2 614.17 ± 156.10) and (3 444.58 ± 233.69) mg/kg,respectively.The differences between groups were statistically significant (F =309.716,P ＜ 0.05),and fluoride contents of bone increased with increase of fluoride doses (all P ＜ 0.05).Under optical microscope,the cartilage tissue of control group was normal,while articular cartilage and epiphyseal plate cartilage showed different degrees of cartilage ossification in fluorosis mice and the changes increased with the increase of fluoride doses.The levels of serum IL-6 in control group,low fluoride group,middle fluoride group and high fluoride group were (5.98 ± 1.43),(7.54 ± 2.16),(5.25 ± 1.97) and (6.31 ±-1.36) ng/L,respectively.The differences between groups were statistically significant (F =3.840,P ＜ 0.05),low fluoride group was significantly higher than control group (P ＜ 0.05),and middle fluoride group was significantly lower than low fluoride group (P ＜ 0.05).The levels of serum slL-6R in control group,low fluoride group,middle fluoride group and high fluoride group were (0.83 ± 0.20),(0.93 ± 0.23),(0.82 ±0.27) and (0.92 ± 0.28) μg/L,respectively.The differences between groups were not statistically significant (F =0.738,P ＞ 0.05).Immunohistochemical results showed that articular cartilage full-layer cells in each group expressed IL-6 protein especially in the middle layer of chondrocytes,while IL-6 protein only expressed in hypertrophic chondrocytes of epiphyseal plate cartilage.Comparing with other groups,IL-6 positive cells were the most and had the deepest staining in low fluoride group.Conclusions Different doses of NaF could not only cause cartilage lesion,but also change the expression of IL-6 in serum and cartilage tissue of Balb/c mice.The results indicate that IL-6 may be involved in the cartilage lesion caused by fluoride.
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Objective To investigate the effects of fluoride on trabecular bone of the tibia and lumbar in BALB/c mice.Methods Totally 64 four-week-old BALB/c mice were randomly divided into 4 groups by weight,16 per group:control group (treated with distilled water) and 3 sodium fluoride (NaF) exposure groups (treated with NaF at 25,50 and 100 mg/L F-),respectively.At 12 weeks,mice were killed and blood,two hind limbs and lumbar were collected.Bone fluoride content and incidence rates of dental fluorosis were determined.Serum content of alkaline phosphatase (AKP) and acid phosphatase (ACP) were detected by micro enzyme labeled method.The ultrastructure of osteoblasts and osteoclasts in lumbar were observed via transmission electron microscope.The pathological changes of the trabecular bone of the tibia and the lumbar were observed under optical microscope,the percentage of trabecular area (％Tb.Ar) was measured with Image-Pro Plus (IPP) software.Results Bone fluoride contents of low,middle and high fluoride groups [(1 828.62 ± 102.93),(3 308.27 ± 185.63),(4 933.36 ± 301.16) mg/kg] were higher than that of the control group [(775.23 ± 92.56) mg/kg,all P ＜ 0.05].The incidences of dental fluorosis in the 4 groups were 0(0/16),47％(7/15),93％(14/15) and 100％(16/16),respectively;the difference was statistically significant (x2 =27.23,P ＜ 0.05).In middle and high fluoride groups,serum AKP [(18.30 ± 1.99),(24.50 ± 3.14) king unit/100 ml] and ACP [(11.97 ± 1.73),(11.31 ± 1.46) king unit/100 ml] were significantly higher than those of control [(14.63 ± 1.21),(9.07 ± 1.47) king unit/100 ml,respectively,all P ＜ 0.05].Under the electron microscope,osteoblast had developed organelles in each fluoride group,rough endoplasmic reticulum,Golgi body,and mitochondria were abundant,and nucleolus was obvious in the osteoblast.Osteoclast was rich in mitochondria,ruffled border clear and distributed phagocytic vacuoles in low fluoride group and middle fluoride group.Compared with the control group (17.03 ± 3.73),HE staining of tibia ％Tb.Ar in high fluoride group (28.79 ± 8.26) was significantly increased (P ＜ 0.05).The lumbar spine ％Tb.Ar in low,middle and high fluoride groups (15.87 ± 2.59,18.28 ± 0.89,21.99 ± 1.81) were higher than that of the control group (12.06 ± 1.76,all P ＜ 0.05].Conclusions BALB/c mice could be used as a model of skeletal fluorosis.Osteoblast and osteoclast are activated in BALB/c mice with skeletal fluorosis.Bone formation is more obvious than bone resorption and bone mass is increased.What is more,bone mass has increased more significantly in the lumbar spine of mice.
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Objective To investigate the relationship between Cytochrome P-450 1A1 (CYP1A1) gene polymorphism and the ethnic differences to brick-tea fluorosis and the gene-environment interaction.Methods Inhabitants over the age of 16 years old in Inner Mongolia,Qinghai and Xinjiang were investigated.The questionnaire survey included basic information,dietary survey and total fluoride intake,and peripheral venous blood was collected.The CYP1A1 gene single nucleotide polymorphism (SNP) genotyping was determined using mass spectrometry;the diagnosis of skeletal fluorosis was based on the X-ray method;combined genetic factors with environmental factors,the interaction of gene-environment was analyzed.Results In the 1 414 copies of whole blood samples (308 Tibetans,290 Kazakhs,261 Mongolians,425 Han people,130 Russians),CYP1A1 genes rs1048943 sites were typed into AA,AG and GG genotypes,and gene distribution met Hardy-Weinberg equilibrium (P ＞ 0.05).The frequencies of genotypes AA,AG and GG in Tibetans were 55.8％ (172/308),37.3％ (115/308) and 6.8％ (21/308),respectively;the frequencies of the three genotypes in Kazakhs were 69.7％ (202/290),27.6％ (80/290) and 2.8％ (8/290),respectively;the frequencies of the three genotypes in Mongolians were 60.5％ (158/261),36.0％ (94/261) and 3.4％ (9/261),respectively;the frequencies of the three genotypes in Han people were 60.9％ (259/425),33.6％ (143/ 425) and 5.4％ (23/425),respectively;the frequencies of genotypes in Russians were 72.3％ (94/130),26.9％ (35/130) and 0.8％ (1/130),respectively;the differences of the three genotype frequencies between different ethnic groups were statistically significant (x2 =24.757,P ＜ 0.05).The skeletal fluorosis detection rates in different ethnic from high to low were Tibetans (39.94％,123/308),Kazakhs (33.79％,98/290),Mongolians (22.22％,58/261),Han people (13.41％,57/425) and Russians (8.46％,11/130),and the differences were statistically significant (x2 =100.156,P＜ 0.05).Skeletal fluorosis detection rates of different genotypes were AA (24.18％,214/885),AG/GG (25.14％,133/529),the difference was not statistically significant between the groups (x2 =0.165,P ＞ 0.05).After the ethnic stratification,the differences were also not statistically significant (P ＞ 0.05).Only in the group of Tibetans whose urine fluoride level was 1.6-3.2 mg/L and Mongolians under age 45 were found that the G gene was one of the risk factors in skeletal fluorosis [OR =2.035,95％ CI (1.003-4.128);OR =5.602,95％CI (1.461-21.479)];G gene might be a protective factor in the Mongolians aged 45 years and over [OR =0.422,95％CI(0.190-0.938)].Conclusion This study does not find a positive correlation between CYP1A1 gene polymorphism and the ethnic differences to bricktea fluorosis.