Study design and sample size
The study was carried out with an observational cross-sectional survey design and performed from 1-Jan-2016 to 28-Feb-2017. The sample size was calculated by the PASS software. The calculation formula is below:
$$\mathrm{n}=\frac{2\overline{p}\ \overline{q}{\left({Z}_{\alpha }+{Z}_{\beta}\right)}^2}{{\left(P1-P2\right)}^{2.}}$$
P1 = 0.2, P2 = 0.4, α = 0.05, Z0.05 = 1.96, β = 0.9, Zβ = 1.28, nmin = 110.
Study of subjects
The fluoride concentration of drinking water was high in the Feng County, Xuzhou City, Jiangsu Province, China, where dental fluorosis was very common. However, there was no data available regarding the cognitive function of the individuals who lived in this area. In this study, a total of 272 subjects from the high fluoride drinking water community (water fluoride concentration > 2 mg/L) of Feng County, Xuzhou City, Jiangsu Province, China, were randomly enrolled in this study. And a total of 172 subjects, from the normal fluoride drinking water community (water fluoride concentration < 0.8 mg/L) of Suining County, Xuzhou City, Jiangsu Province, China, were randomly chosen as the control group. These two counties have a similar culture, lifestyle and economic development level. Individuals with cerebral ischaemia, brain tumours and psychiatric disorders were excluded. This study was approved by the Ethics Committee of Affiliated Xuzhou Oriental Hospital of Xuzhou Medical University, China.
General information
Socio-demographic and personal information was collected from each subject. These data included gender, age, education, alcohol drinking, smoking, dental status, hypertension, hyperlipidaemia, diabetes and family history (psychiatric diseases or dementia). Age was categorized into 3 groups as follows: 60–69 years of age, 70–80 years of age and older than 80 years of age. Education was categorized into the following groups: illiterate, primary school, middle school, and high school or higher. Dental status was categorized as follows: dental fluorosis, normal and dentures.
Cognitive function tests
The Montreal Cognitive Assessment-Basic (MoCA-B) and AD-8 were used to investigate the cognitive functions of the subjects. The MoCA-B had excellent validity in screening for mild cognitive impairment in poorly educated older adults regardless of literacy [25]. The AD-8 is an 8-item informant-based questionnaire, which was designed to detect changes in the fields of memory, orientation, judgement and executive function [26].
Blood sampling and pretreatment
A total of 4 tubes of venous blood (5 ml each) were collected and centrifuged at 5000 rpm/min for 5 min. Sera were stored at − 80 °C. These samples were used for testing the mRNA level of DKK1, fluoride concentration, SOD, GSH, malondialdehyde (MDA) concentration and apolipoprotein E (APOE) gene polymorphism.
Biochemical tests
Blood fluoride concentration was measured using the method of fluoride ion selective electrode method. Briefly, different concentrations of Fluoride Standard Liquid reagent were used to make a standard line, and then the concentration of the blood samples was adjusted according to the standard line.
The different oxidative stress status was evaluated by measuring levels of SOD, GSH and MDA according to the manufacturer’s instructions in the reagent kit (Nanjing, Jiancheng, China).
The mRNA level of DKK1 was measured using qRT-PCR. Briefly, total human blood RNA was isolated with Trizol reagent (ProbeGene, China), and the concentration was measured using ultraviolet spectrophotometry. Reverse transcription was achieved using the cDNA Synthesis Kit (ProbeGene, China), and qRT-PCR amplification was performed using the SYBR-Green Master mix (Probegene MQ051, China) with the following amplification conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 2 min. The amplification primer sequences were F: 5′- TCA TAG CAC CTT GGA TGG GTA TTC - 3′, and R: 5′- TTG GAC CAG AAG TGT CTA GCA CAA - 3′. The results were analysed using the ABI2720 PCR System (Applied Bio-systems, USA).
For analysis of APOE gene polymorphisms (also known as genotype), genomic DNA was extracted from collected venous blood samples using a commercial kit (QIAamp DNA Blood Mini Kit, Qiagen, Shanghai, China) beforehand. Then, the potentially mutated positions in 112 (rs429358) and 158 (rs7412) of the APOE gene were conducted by a detection kit (GeneChip Assay, Sinochip, Zhuhai, China) based on genomic DNA. First, all samples were amplified with the Verti™ DX Thermal Cycler (Life Technologies, Singapore) (45 cycles, 94 °C for 30 s and 65 °C for 45 s); then the amplified products were assayed by the fully automated GeneChip detection system (Sinochip, Zhuhai, China). All genotyping results were obtained from the GeneChip automated analysis system.
Statistical analysis
Socio-demographic factors and numeration data were analysed using the chi-square (χ2) test. The nonparametric rank sum test was used to compare the quantitative data between two groups. The degree of association between fluoride-induced cognitive impairment and risk factors was analysed using binary logistic regression analysis. Bivariate correlation analysis was performed using Spearman correlation analysis. SSPS16.0 software was used to analyse all data. A p-level of 0.05 was considered statistically significant.