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Do Environmental Fluoride Exposure and ESRα Genetic Variation Modulate Methylation Modification on Bone Changes in Chinese Farmers? Yanli Zhang,† Hui Huang,† Biao Gong,‡ Leizhen Duan,† Long Sun,‡ Tongkun He,† Xuemin Cheng,† Zhiyuan Li,† Liuxin Cui,† and Yue Ba*,† †

Department of Occupational and Environmental Health, School of Public Health, Zhengzhou University, Zhengzhou, Henan 450001, People’s Republic of China ‡ Kaifeng Center for Disease Prevention and Control, Kaifeng, Henan 475000, People’s Republic of China S Supporting Information *

ABSTRACT: Although increasing evidence suggests that estrogen receptor α (ESRα) genetic variation could modify bone damage caused by environmental fluoride exposure, little is known about epigenetic mechanisms in relation to bone changes. A casecontrol study was conducted among farmers aged 18−55 years in Henan Province, China. X-ray was used to detect bone changes. Methylation status was determined by methylation-specific PCR. Genotypes were identified by Taqman probe and real-time PCR. In this study, we found that methylation status in the promoter region of the ESRα gene was lower in bone change cases than that in controls, which was only observed in male farmers after stratification by gender. Furthermore, methylation level was negatively associated with the urinary fluoride concentration in male farmers. No significant association was found between the distribution of ESRα rs2941740 genotypes and the risk of bone changes. Multivariate logistic regression analysis showed that after adjusting for age and gender, increased serum calcium and methylation status were protective factors for bone changes. No interaction effect was observed between fluoride exposure and ESRα rs2941740 polymorphism on bone changes. In conclusion, the current work suggests that bone changes are associated with methylation status, which might be modulated by fluoride exposure in male farmers. Methylation status and bone changes were not modified by ESRα gene rs2941740 polymorphism in the promoter region.

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bones.10,11 Studies have demonstrated that estrogen receptor α (ESRα) is the main mediator of the estrogenic effects in bone.12 Therefore, the ESRα gene may contribute to bone remodeling and affect bone growth. A major effort to identify ESRα genetic determinants of bone metabolism through studies has shown that many single nucleotide polymorphisms (SNPs) are associated with bone changes,13−15 which together partly explain individual variation. Nevertheless, little is known about the epigenetic mechanism related to bone changes. DNA methylation is an essential epigenetic profile involved in genetic modification16 and occurs almost exclusively at cytosine in the context of CpG dinucleotides. Increasing evidence has suggested that aberrant DNA methylation status could result in various diseases including cancer, autoimmune diseases, and certain genetic diseases.17−19 Both environmental and genetic factors can modulate the degree of DNA methylation.20,21 In many studies, environmental factors influence the establish-

INTRODUCTION Fluoride is naturally present in soil, rocks, and water throughout the world but with higher concentrations in areas marked by pyroclastic activities or geologic uplift.1,2 It is almost always present in the form of compounds of fluoride because of it being a highly reactive element.3 The effects of fluoride on mineralized tissues such as bones and teeth depend on the time of fluoride exposure and the amount of fluoride ingested.4,5 At low doses, fluoride can increase bone hardness and prevent dental caries.6,7 However, chronic intake of excessive environmental fluoride can cause skeletal and dental fluorosis, which is characterized by osteoporosis, along with osteosclerosis, osteopenia, and osteomalacia.8,9 Here, it is of critical importance to note that daily intake of naturally high doses of environmental fluoride is inevitable in many areas of China, making it a major health issue, especially its damage to bone homeostasis. It is well-known that estrogen plays an important role in skeletal development, especially in stimulating osteoblast activity, promoting the deposit of calcium and phosphate in © 2017 American Chemical Society

Received: February 23, 2017 Published: May 15, 2017 1302

DOI: 10.1021/acs.chemrestox.7b00047 Chem. Res. Toxicol. 2017, 30, 1302−1308

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Chemical Research in Toxicology

Detection of Urine and Serum Biomarkers. The urinary fluoride levels in samples were detected by a fluoride ion selective electrode (Shanghai Exactitude Instrument Company, China). The levels of serum calcium and serum magnesium were detected using flame atomic absorption spectrometry (Hitachi Z-5000, Japan). Estradiol level in the serum was detected using a chemiluminescence immunoassay (Autobio Labtec Instruments Co. Ltd., Zhengzhou, China). X-ray Diagnosis of Bone. In accordance with the Chinese X-ray Diagnostic Criteria of Endemic fluorosis, X-ray diagnosis was performed to detect the change in the pelvis, forearm including the elbow and wrist joints, and leg including the knee joint by a medical professional in a certified hospital. The spectrum of radiographic bone changes mainly included ground-glass appearance, coarse trabeculae, wide osteoid seams, periosteal changes, lateral bowing, lack of modeling, increased diameter, and more. Methylation Analysis. Genomic DNA was isolated from blood samples using whole-blood genomic DNA miniprep kits (Axygen Biosciences, Union City, CA, USA). DNA samples were treated with sodium bisulfite using an EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA, USA). The promoter sequences of the ESRα gene were searched using UCSC/Ensembl (http://genome.ucsc.edu/ ), and primer sequences were then designed using the methylation primer design software (Methyl Primer Express v1.0). Two pairs of PCR primers were used to conduct methylation-specific PCR (methylated specific primers: L, 5′-CGT AGG TTT ACG GTT AGA TCGG-3′; R, 5′-ATA CAA TAA CAT CAA CGA ACT CGAA3′; unmethylated specific primers: L, 5′-ATG GTT AGA TTG GTT TTT TTT TAGG-3′; R, 5′-ACA TCA ACA AAC TCA AAA ACA CACT-3′). Methylation status was analyzed using methylation-specific PCR (MX3000, Aglient, Santa Clara, CA, USA). PCR amplification was performed in a 15 μL reaction mixture with the following final concentrations: 7.5 μL of 2 × Power SYBR Green PCR Master Mix (Applied Biosystems), 2 μL of primer with a concentration of 1.25 μmol/L each, and 100 ng of bisulfite-treated DNA template with a proximate concentration of 100 μg/mL. PCR conditions were as follows: predegeneration at 95 °C for 10 min, 40 cycles for degeneration at 94 °C for 15 s, annealing at 54 °C for 30 s, and extension at 72 °C for 30 s. Negative controls were set for each experiment. The rate of DNA methylation was calculated using the reference method.34 Genotyping. The rs2941740 SNP was genotyped with TaqMan probe assay at the Applied Biosystems (ABI, 7500 Fast Real-Time PCR system, Foster City, USA) platform. The primers and probes for SNPs were ordered from Applied Biosystems Inc., and the allelic discrimination was measured automatically using Sequence Detection Systems 2.1 software on the 7500 Fast Real-Time PCR system. Realtime PCR reaction was carried out in a 12 μL volume using 0.1 μL of TaqMan probe, 6 μL of Mix, and 100 ng of template DNA. Amplification was obtained by predegeneration at 95 °C for 10 min, 40 cycles for degeneration at 95 °C for 15 s, and annealing at 60 °C for 60 s. Negative and positive controls were set for each experiment. Statistical Analysis. The data were doubly entered into the database independently by different people using Epidata 3.0 software (Epidata 3.0 for windows, Epidata Association Odense, Denmark). Statistical analysis was performed using the SPSS statistical software package, version 21.0 (IBM Corp, Armonk, NY, USA). The DNA methylation level was presented as a percentage, which was logarithmically transformed to approximately normal distribution, and the transformed values were used in data. Differences in age, serum calcium, serum magnesium, estradiol level, urinary fluoride, and methylation status between the case group and control group were examined using t test. The Chi-square test was employed to compare the distributions of ESRα rs2941740 genotypes, sex, smoking, alcohol consumption, prevalence of lumbocrural pain, and joint limitation in different groups. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated using multivariate logistic regression analysis. Levels of significance were established as significant when P ≤ 0.05.

ment and maintenance of DNA methylation modifications, thereby inducing changes in gene expression under the precondition of not changing the DNA sequence and causing phenotypic changes.22,23 Chemical pollutants,24 dietary components,25 and other external factors26 can indeed have enduring effects on the extent of DNA methylation. On the other hand, research in recent years27,28 has indicated that genetic variation is also likely to play an important role in DNA methylation patterns. However, neither the mechanism that DNA methylation is influenced by genetic variation nor the extent is yet clear. Research in recent years has shown that ESRα genetic variations were related to bone changes.29,30 However, to date, only a few studies have addressed the question concerning the association of bone changes with methylation status of the ESRα gene.31,32 Here, we paid attention to the possible impact of DNA methylation status in the promoter region of the ESRα gene on bone changes, as well as the possible modification of rs2941740 polymorphism, a SNP located on the promoter region that results in C/T mutation, on ESRα gene methylation status in the promoter region, and bone changes in Chinese farmers with fluoride exposure. Given that, it is important to consider whether environmental fluoride exposure, rs2941740 polymorphism, or the interaction between environmental fluoride exposure and gene variation could modify methylation status and further modulate bone metabolism. Hence, on the basis of our previous cross-sectional study, we conducted a case-control study to analyze the possible involvement of DNA methylation and rs2941740 polymorphism in the promoter region of the ESRα gene with bone changes in Chinese farmers exposed to fluoride in drinking water.

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MATERIALS AND METHODS

Study Population. Detailed information regarding our previous cross-sectional study was described elsewhere.33 In brief, three endemic fluorosis villages (EFV) and three nonendemic fluorosis villages (NEFV) were selected in the Tongxu County of Henan Province according to water fluoride concentration, with 1.0 mg/L as the cutoff value (Sanitary Standards for Drinking Water of China). A total of 1131 local residents aged from 18 to 55 years old and who lived in the villages for more than 5 years were recruited via cluster sampling. Among them, 282 eligible subjects were selected by a 25% completely random sampling for the subsequent case-control study which excluded (1) having bone metabolic diseases (thyroid and parathyroid function changes, chronic kidney disease, and Cushing’s syndrome) and (2) taking medicines which could affect bone metabolism (estradiol, calcitonin, calcium preparations, and thyroid hormone). Finally, a total of 252 farmers (144 cases in bone changes and 108 controls, examined by X-ray) participated in the study with a participation rate of 89.36%. The project was approved by the ethical committee of Zhengzhou University. All of the participants signed informed consents. All procedures were performed in accordance with protocols approved by the Human Investigation Committees at Zhengzhou University. Collection of Questionnaires and Biological Samples. As described elsewhere,33 information on demographic factors were obtained by a standard questionnaire, which included socio-economic status, medical condition, marriage status, reproductive history, smoking and alcohol consumption, the main source of heating and cooking fuel, and dietary intake. Fasting blood samples (anticoagulative/nonanticoagulative blood 5 mL) and instant urine samples (no less than 50 mL) were collected from each participant. After centrifugation, serum and white blood cells were separated and frozen at −80 °C for subsequent analyses. Instant urine samples were frozen at −20 °C. 1303

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RESULTS Characteristics of the Study Population. As shown in Table 1, alcohol users were more frequent in the control group Table 1. Background Characteristics of 252 Chinese Farmersa cases(n = 144) male female male female yes no yes no yes no yes no yes no yes no yes no

controls(n = 108)

Age (Years) 39.57 ± 7.81 34.66 ± 9.03 40.26 ± 7.44 34.21 ± 6.93 Gender 51(35.4) 50(46.3) 93(64.6) 58(64.6) Smoking Statusb 34(23.6) 37(34.3) 110(76.4) 71(65.7) Alcohol Consumptionc 21(14.7) 27(25.0) 122(85.3) 81(75.0) Tea-Drinking Habitd 13(9.0) 16(14.8) 131(91.0) 92(85.2) Mineral Supplement 11(7.9) 7(6.5) 129(92.1) 101(93.5) Physical Activitye 126(87.5) 91(84.3) 18(12.5) 17(15.7) Lumbocrural Pain 14(9.7) 9(8.3) 130(90.3) 99(91.7) Joint Activity Limited 27(18.8) 11(10.8) 117(81.3) 97(89.8)

t/χ2

P value

2.923 4.987

0.004 0.05, respectively). Distributions of ESRα Gene Methylation and Biomakers Related to Bone Changes. Significant differences between cases and controls were observed in urinary fluoride (P < 0.05). Compared with the control group, the serum level of magnesium was slightly higher in the case group in female farmers (P < 0.05) (Figure 1A). As we can see from the results, the methylation status in the case group was lower than that in the control group, which was only observed in male farmers (P < 0.05) (Figure 1B). Association of Bone Changes with Methylation Status of the ESRα Gene. We found a negative correlation between methylation level and urinary fluoride in male farmers (P < 0.05) (Table S1 and Figure 2). However, no significant correlation was observed between methylation status and serum calcium, serum magnesium, and estradiol level (P > 0.05, respectively) (Table S1).

Figure 2. Relationship between methylation level of ESRα gene and urinary fluoride in male farmers. Data from the figure, drawn using R language ggplot2 packages, suggest that, with the increase of urinary fluoride, methylation status shows a decreasing trend in male farmers.

Multiple linear regression analysis suggested no significant interaction between urinary fluoride and ESRα gene polymorphism on the methylation status (Table S2). Association between ESRα rs2941740 Genotypes and Bone Changes. In order to investigate if ESRα methylation level and bone changes are associated with ESRα poly1304

DOI: 10.1021/acs.chemrestox.7b00047 Chem. Res. Toxicol. 2017, 30, 1302−1308

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Chemical Research in Toxicology Table 2. Comparison of Genotype Frequencies of ESRα rs2941740 in the Case Group and the Control Group

a

genotypesa

cases (n = 141)

controls (n = 107)

CC CT TT CT+TT C T

80(74.8) 25(23.4) 2(1.8) 27(25.2) 185(86.45) 29(29)

96(68.1) 37(26.2) 8(5.7) 45(31.9) 229(81.21) 53(18.79)

OR (95%CI)b

P value

1.000 1.059(0.545−2.056) 3.144(0.577−17.140) 1.216(0.648−2.282) 1.000 1.333(0.770−2.307)

0.866 0.186 0.542 0.304

Values are expressed as the mean ± SD or number and percentage. bData are adjusted for age, gender, and urinary fluoride.

Table 3. Association of rs2941740 Polymorphism with ESRα Gene Methylation and Related Serum Biomarkersa CC(n = 176)

a

serum Ca(mmol/L) serum Mg(mmol/L) estradiol (pg/mL) methylation (%)

2.69 ± 1.16 0.92 ± 0.25 45.70 ± 43.97 7.75 ± 5.85

serum Ca(mmol/L) serum Mg(mmol/L) estradiol (pg/mL) methylation (%)

2.70 ± 0.85 0.92 ± 0.20 87.74 ± 101.66 9.09 ± 11.66

CT(n = 62) Male 2.65 ± 0.92 0.85 ± 0.36 33.12 ± 13.02 9.43 ± 8.79 Female 2.80 ± 0.96 0.87 ± 0.28 79.81 ± 66.51 7.26 ± 5.21

TT(n = 10)

F

P value

3.10 ± 2.01 0.90 ± 0.21 39.71 ± 10.98 8.36 ± 1.85

0.213 0.565 0.898 0.787

0.808 0.570 0.411 0.458

2.76 ± 0.88 0.77 ± 0.38 77.32 ± 47.47 16.30 ± 27.82

0.176 1.697 0.131 0.460

0.839 0.187 0.877 0.632

Values are expressed as the mean ± SD.

Table 4. Effect of Multifactors on Bone Changes of Farmers by Multivariate Logistic Analysisa

a

dependent variable

independent variable

β

P value

bone changes

serum Ca serum Mg estradiol UF methylation ESRα genotypes CC CT TT UF* CCb UF* TT UF* TT

−0.634 −0.479 −0.001 0.552 −4.986

0.001 0.481 0.737 0.001 0.05). Effect of Multifactors on Bone Changes of Farmers. Multivariate logistic regression analysis, which introduced

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DISCUSSION

In this study, we found that bone changes were related to methylation status in the promoter region of the ESRα gene, which might be modulated by environmental fluoride exposure in male farmers. Furthermore, this relationship might be independent from the modification of ESRα gene rs2941740 polymorphism in the promoter region. Unfortunately, ESRα rs2941740 polymorphism may not be a useful genetic marker for differential risk of bone changes among Chinese farmers. To our knowledge, the present study is the first to explore the association between DNA methylation and rs2941740 polymorphism in the promoter region of the ESRα gene with bone changes in Chinese farmers exposed to environmental fluoride. 1305

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Chemical Research in Toxicology Endemic fluorosis is mainly caused by excessive consumption of fluoride in drinking water in Central China such as in the Henan Province. However, due to the influence of coal consumption against skeletal fluorosis, the fluoride levels in indoor and outdoor air were also determined in the investigated villages, and no differences were found in different villages.35 Epidemiological studies36,37 have suggested that excessive fluoride consumption affects normal bone metabolism, causing fluoride-induced bone damage. This study showed that compared with the control group, the urinary fluoride level was significantly higher in the case group. With a urinary fluoride level of no less than 1.6 mg/L as the cutoff value for higher fluoride burden,38 we observed that 67.9% of subjects with higher fluoride burden experienced bone changes, which was significantly higher than that in subjects with a lower fluoride burden (48.9%) (Table S3) . Our findings are consistent with the adverse skeletal effects of fluoride exposure. Many risk factors have been identified that are related to the development of bone damage. Among those factors, estrogen and calcium deficits are well-known to increase the risk of bone changes.39,40 A growing body of studies41,42 suggest that the combination of fluoride exposure with concomitant estrogen deficit may aggravate bone damage. Also, it is noted that high calcium intake could prevent the skeletal effects of fluoride exposure.43 Although no significant differences were found in serum calcium between the cases and controls, we observed that after adjusting age and gender, as the level of serum calcium increased, the risk of bone tissue changes decreased (Table 4). It is hypothesized that DNA methylation could be involved in the regulation of bone metabolism related gene expression during skeletal development.31,44 More recently, Jordana45 et al. measured methylation status at 22,290 CpG dinucleotides and found that there was a significant overlap of SNPs that could modify both methylation status and gene expression. A review on epigenetic mechanisms in bone has shown that DNA methylation mediates the expression of several genes associated with different stages of bone metabolism.46 In this study, we found that methylation level in the promoter region of the ESRα gene in the case group was lower than that in the control group, which was only observed in male farmers. This association found in our study between methylation status and the risk of bone changes adds new evidence to the relationship between methylation status and bone changes. Moreover, a significant negative correlation was found between methylation level and urinary fluoride concentration in male farmers, which might suggest that methylation status in males were affected by fluoride exposure, which then may interfere with bone metabolism, a result supported in other studies with similar results. Fu and Zhu’s research demonstrated that sodium fluoride may influence methylation modification, thus interfering with the early development of mouse embryos.47,48 On the other hand, some studies demonstrated that the rs2941740 SNP is associated with bone mineral density.49,50 We hypothesize that methylation status and bone changes might be modulated by genetic markers, such as rs2941740 polymorphism of the ESRα gene in the promoter region. However, we did not find the association between ESRα gene rs2941740 polymorphism and methylation status, and bone tissue changes. Moreover, the differences in serum biomarkers related to bone metabolism were also not observed among farmers carrying different genotypes of ESRα rs2941740. Taken

together, ESRα gene rs2941740 polymorphism may not be obviously associated with bone tissue changes. In consideration of the differences in individual susceptibility to the environment, we analyzed the interaction between fluoride exposure and ESRα gene rs2941740 polymorphism and bone changes. The result showed that no interaction was observed. Curiously, the effects of serum calcium, urinary fluoride, and methylation level on bone tissue changes were statistically significant. Serum calcium and methylation levels of the ESRα gene were protective factors for bone tissue. In other words, an increase in the level of serum calcium and the methylation level of ESRα, led to a decrease in the risk of bone tissue changes. It supports that skeletal fluorosis was always closely related to calcium deficiency.51,52 Previous studies have shown that ESRα gene methylation plays a critical role in human bone metabolism, although the evidence is limited. In this study, we found an inverse association between methylation levels in the promoter region of the ESRα gene and bone changes, and an increased methylation status might reduce the risk of bone changes. However, this was inconsistent with some existing research. Lambertini et al.53 demonstrated that methylation status could mediate ESRα gene expression with a consequent possible loss of osteoblastic function. Lv et al.54 found that methylation degree in the promoter region of the ESRα gene in postmenopausal women was significantly higher than that in premenopausal women, and in vitro data showed that homocysteine could induce hypermethylation of the promoter region and reduce ESRα mRNA transcription. Several potential reasons might explain the differences. First, this may be due to differences in the selected ESR gene methylation site. Second, the methylation status could influence bone changes, which was modified by fluoride exposure. Third, besides osteoporosis, bone changes also included osteosclerosis, osteopenia, and osteomalacia in this study. It may also be responsible for the different results. Considering that the most important limitation of this study is the relatively small sample size, it is essential to replicate these findings with larger sample sizes in further studies. Complete information, a better control of confounding factors such as age, gender, genetic background, living habit, and dietary structure would make the results acceptable. Bone metabolism is likely involved in many complex pathways and could be affected by various genes. Therefore, further studies should focus on a wide variety of SNP sites of the ESR gene and epigenetic markers of candidate genes influencing bone mass.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00047. Correlation between methylation status of the ESRα gene and the related biomarkers; interaction between urinary fluoride and ESRα gene polymorphism on methylation status in the promoter region of the ESRα gene; and comparison of prevalence in the different fluoride exposure groups (PDF) 1306

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AUTHOR INFORMATION

Corresponding Author

*School of Public Health, Zhengzhou University, 100 Science Rd., Henan 450001, China. Phone: 86-150-3601-8960. Fax: 86371-6778-1868. E-mail: [email protected]. ORCID

Yue Ba: 0000-0002-9659-7993 Funding

This work was supported by the National Science Foundation of China (81673116, 81072247) and Henan Department of Science and Technology, China (162300410272). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We express our sincere thanks to all individuals who volunteered to participate in this study and the numerous members of the Zhengzhou University School of Public Health, Kaifeng and Tongxu Center for Disease Control and Prevention.

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ABBREVIATIONS ESRα, estrogen receptor α; SNP, single nucleotide polymorphism REFERENCES

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DOI: 10.1021/acs.chemrestox.7b00047 Chem. Res. Toxicol. 2017, 30, 1302−1308

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DOI: 10.1021/acs.chemrestox.7b00047 Chem. Res. Toxicol. 2017, 30, 1302−1308