Endemic fluorosis in Henan province, China: ERα gene polymorphisms and reproductive hormones among women.

BACKGROUND AND OBJECTIVES: The aim of this study was to explore the influence of fluoride exposure and ERα gene polymorphisms on reproductive hormone concentrations of women in accordance with endemic fluorosis residence. METHODS AND STUDY DESIGN: A cross sectional study was conducted in Tongxu county, Henan Province, China. A total of 679 women were recruited using cluster sampling and each subject provided fasting blood and an associated urine sample. We measured the concentrations of serum gonadotropin releasing hormone (GnRH), follicle-stimulating hormone, luteinizing hormone, and estradiol and urinary fluoride. RESULTS: In the defluoridation project group (DFPG), serum GnRH was lower in women carrying C/C genotype compared to in those carrying C/T and T/T genotypes of ERα gene rs3798577 (p<0.05). In the endemic fluorosis group (EFG), serum GnRH was lower in women carrying Pp genotype compared to in those carrying PP and pp genotypes of ERα PvuII (p<0.05). Serum GnRH in women from EFG who carried Pp, pp, Xx and xx genotypes in ERα gene PvuII and XbaI was lower than in those in the control group (CG) who carried same genotypes (p<0.05). Furthermore, serum GnRH in women from EFG was significantly lower than in those in CG, regardless of whether the women were carrying C/C, C/T or T/T genotypes of ERα rs3798577 (p<0.05). Serum estradiol concentrations in EFG were significantly lower than in CG when the women were carrying the Pp, Xx and T/T genotypes in ERα gene (p<0.05). CONCLUSION: Interaction of ERα gene and fluoride exposure may influence women's serum reproductive hormone concentrations.

Fluoride Exposure, Follicle Stimulating Hormone Receptor Gene Polymorphism and Hypothalamus-pituitary-ovarian Axis Hormones in Chinese Women.

The effects of fluoride exposure on the functions of reproductive and endocrine systems have attracted widespread attention in academic circle nowadays. However, it is unclear whether the gene-environment interaction may modify the secretion and activity of hypothalamus-pituitary- ovarian (HPO) axis hormones. Thus, the aim of this study was to explore the influence of fluoride exposure and follicle stimulating hormone receptor (FSHR) gene polymorphism on reproductive hormones in Chinese women. A cross sectional study was conducted in seven villages of Henan Province, China during 2010-2011. A total of 679 women aged 18-48 years were recruited through cluster sampling and divided into three groups, i.e. endemic fluorosis group (EFG), defluoridation project group (DFPG), and control group (CG) based on the local fluoride concentration in drinking water. The serum levels of gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were determined respectively and the FSHR polymorphism was detected by real time PCR assay. The results provided the preliminary evidence indicating the gene-environment interaction on HPO axis hormones in women.

Effect of oxidative stress on fluoride-induced apoptosis in primary cultured Sertoli cells of rats.

This study examined the effect of oxidative stress on the apoptosis of Sertoli cells induced by sodium fluoride (NaF). Cell viability, reactive oxygen species, malondialdehyde content, superoxide dismutase activity, mitochondrial membrane potential, and apoptosis were measured after the rat Sertoli cells were exposed to various concentrations of (0, 6, 12, and 24 µg/ml) sodium fluoride in the presence and absence of 2 mM N-acetylcysteine (NAC) for 24 h. The present study showed that decrease in cell viability and excessive oxidative stress were observed in NaF-treated cells. The treatment with NAC restored the decreased cell viability and excessive oxidative stress. Moreover, fluoride exposure decreased mitochondrial membrane potential and increased apoptosis in Sertoli cells. NAC was also found to suppress a loss of mitochondrial membrane potential and the percentage of apoptosis in NaF-treated Sertoli cells. This study proved that oxidative stress probably play a major role in NaF-induced apoptosis of Sertoli cells.

[The influence of high fluoride exposure in drinking water on endocrine hormone in female].

OBJECTIVE: To explore the influence of water fluoride exposure on reproductive hormones in female. METHODS: Cross-sectional study was conducted in seven villages of a county in Henan province by using simple random sampling including high fluoride area, defluoridation project area and control area on April, 2011 based on the preliminary study results of fluoride concentration in drinking water. Women who were born and growth or lived in the village at least 5 years and aged 18-48 years old were recruited using cluster sampling. They were divided into high fluoride group (HFG, 116 subjects), defluoridation project group (DFPG, 132 subjects) and control group (CG, 227 subjects) in accordance with the above areas. All subjects accepted questionnaire and physical checkup. Fasting blood and morning urine samples were collected. The concentration of fluoride in urine was determined by fluoride ion selective electrode method. The serum level of GnRH was detected using enzyme linked immunosorbent assay (ELISA). The serum level of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), estradiol (E2) were determined by chemiluminesence immunoassay (CLIA). RESULTS: The average age was (39.44 ± 7.34), (38.84 ± 8.03), (37.45 ± 7.70) years old in female from DFPG, HFG and CG respectively, there were no significant differences among the three groups (F = 3.02, P = 0.05). The urine fluoride levels were (1.34 ± 1.07), (2.59 ± 1.57), (0.92 ± 0.46) mg/ml in female from DFPG, HFG and CG respectively, there was a significant difference among three groups (F = 105.38, P < 0.01). No significant differences were observed of serum GnRH, LH, T, FSH and E2 among three groups in follicular phase (P > 0.05). The serum levels of E2 in Ovulatory period were 67.73, 58.09, 84.96 pg/ml in female from DFPG, HFG and CG respectively. It was lower in HFG than that in CG (H = 4.00, P < 0.05). The serum levels of T in Ovulatory period were 0.55, 0.45, 0.55 ng/ml in female from DFPG, HFG and CG respectively. It was lower in HFG than that in DFPG (H = 6.47, P < 0.05), but no significant difference was observed between HFG and CG (H = 2.41, P > 0.05). The serum levels of GnRH in Luteal phase were 24.09, 20.16, 23.50 ng/ml in female from DFPG, HFG and CG respectively. It was lower in HFG than that in DFPG (H = 14.14, P < 0.05) and CG (H = 12.53, P < 0.05). The serum level of E2 in luteal phase were 81.47, 64.60, 74.55 pg/ml in female from DFPG, HFG and CG respectively. It was lower in HFG than that in DFPG (H = 5.69, P < 0.05). As for LH, FSH and T, no significant differences were observed among the three groups (P > 0.05 respectively). The abnormal rates of E2 level were 22.73 (30/102), 37.93 (44/72), 20.26 (46/181) in female from DFPG, HFG and CG respectively. The E2 abnormal rate in female from HFG was higher that from DFPG (χ(2) = 6.82, P < 0.05) and CG (χ(2) = 12.38, P < 0.05). CONCLUSION: Fluoride exposure may influence reproductive hormones in female, especially in ovulatory and luteal phase of menstrual cycle.

Influence of polluted SY River on child growth and sex hormones.

OBJECTIVE: To investigate the influence of the polluted SY River on children's growth and sex hormones, and provide scientific data for assessment of the polluted status of the SY River. METHODS: The study areas were selected randomly from the SY River Basin. Lead (Pb), mercury (Hg), arsenic (As), phthalates (DEP, DBP, DMP, DEHP), and bisphenol A (BPA) were measured both in the river water and in the drinking water. School children were selected by cluster sampling (n=154). Physical development indexes (height, weight, bust-circumference, and skinfold thickness) and sex hormones [testosterone (T) and estradiol (E2)] were measured for all the children. RESULTS: The contents of Pb and Hg exceeded Class V standards of surface water quality in each section of the river and other indicators exceeded Class III. Compared to the control area, the concentrations of Pb, Hg, As, BPA, DEP, and DBP in the drinking water were significantly higher than in the polluted area (P<0.05). Children from the control area had significantly lower E2 and T than children from the polluted area (P<0.05). Among anthropometric results, only skinfold thickness had statistically significant difference between the two groups (P<0.05), while the other indexes showed no significant differences between the two groups (P>0.05). CONCLUSION: The drinking water has been polluted by the SY River and affected serum sex hormone levels of children living in the polluted area.

A cyanobacterial toxin, microcystin-LR, induces apoptosis of sertoli cells by changing the expression levels of apoptosis-related proteins.

Toxic cyanobacterial blooms in freshwater have been considered as threats to human health. Microcystins are a family of cyclic polypeptides produced by cyanobacteria and are toxic to plants and animals. Microcystin-LR (MC-LR) is the most toxic variant among the microcystin family and could cause oxidative stress in various organs, including the reproduction system. The aim of this study was to investigate the effect of MC-LR on apoptosis of Sertoli cells that play an essential role in the development and maturation of sperm cells. Sertoli cells were isolated from healthy immature rats and cultured with MC-LR. The viability of Sertoli cells was decreased after treatment with MC-LR at 10 µg/ml for 24 h (P < 0.05). Moreover, the MC-LR-treated cells exhibited condensed chromatin and fragmented nuclei, features of apoptosis, as judged by Hoechst 33258 staining. We also analyzed the mRNA and protein levels of three apoptosis-related genes, p53, bax and bcl-2, using reverse transcription-polymerase chain reaction and Western blot analyses, respectively. Both p53 and bax function as promoters of apoptosis, while bcl-2 is an apoptotic suppressor. The mRNA and protein expression levels of p53 and bax were increased in Sertoli cells treated with MC-LR at 10 µg/ml compared with the control group (P < 0.05), while the bcl-2 protein levels were decreased in cells treated with MC-LR at 10 µg/ml (P < 0.05). Moreover, caspase-3 activity that is involved in the induction of apoptosis was significantly increased in Sertoli cells treated with MC-LR. These results indicate that MC-LR induces apoptosis of Sertoli cells.

Serum calciotropic hormone levels, and dental fluorisis in children exposed to different concentrations of fluoride and iodine in drinking water.

BACKGROUND: High fluoride exposure can result in dental fluorosis. Fluoride and iodine are coexistent in the drinking water of areas in China and may affect the prevalence of dental fluorosis and osteogenesis. The aim of this study was to investigate the relationship between serum calciotropic hormone level, and dental fluorisis in children exposed to different concentrations of fluoride and iodine in drinking water. METHODS: A pilot study was conducted in three villages located in the Kaifeng and Tongxu counties of Henan Province, China in 2006. Children aged 8 to 12 years, born and raised in the three villages were recruited. The fluoride levels in the samples of urine from these children were detected by fluoride ion selective electrode. Calcitonin and osteocalcin levels in the serum, and serum calcium were measured by radioimmunassay and flame atomic absorption spectrometry, respectively. RESULTS: Fluoride levels in urine were significantly lower in children from control area (CA) as compared with those from the high fluoride & iodine areas (HFIA) and the high fluoride area (HFA) (P < 0.05 respectively), and no statistically significant difference was found between the children from HFIA and HFA. Additionally, calcitonin levels in the serum were significantly lower in children from CA and HFA as compared with that from HFIA (P < 0.05 respectively), and osteocalcin levels in the serum was lower in children from CA than those from HFIA (P < 0.05). No statistically significant difference in serum osteocalcin concentrations was found between children from HFA and HFIA. CONCLUSION: This study provides an evidence that iodine exposure may modify the serum calciotropic hormone levels related to fluorine exposure.

[Study on the relationship between ER Rsa I gene polymorphism and children's dental fluorosis].

OBJECTIVE: To explore the distribution of ER Rsa I genotype in children who lived in the areas with or without high fluoride, and evaluate the relationship between ER Rsa I gene polymorphism and children's dental fluorosis. METHODS: Children aged 8 to 12 years, born and raised in high fluoride areas and control areas in two counties of Henan Province were recruited. The Rsa I marker of ER gene was genotyped in 237 children composed of both dental fluorosis cases and controls by PCR-RFLP procedure. Urine fluoride was detected with fluoride ion selective electrode method. RESULTS: The frequency distribution of ER Rsa I genotype was rr 60.81% (45/74), Rr 27.02% (20/74), RR 12.16% (9/74) in children with fluorosis; rr 73.91% (51/69), Rr 20.29% (14/69), RR 5.80% (4/69) in children without fluorosis from high fluoride areas, and rr 63.83% (60/94), Rr 34.04% (32/94), RR 2.13% (2/94) in the children without fluorosis from control areas respectively. There were no significant differences in the three groups (P>0.05), but children carrying allele R of ER Rsa I had a significantly increased risk of dental fluorosis (OR=1.821, 95% CI: 1.013-3.274) compared to children carrying the allele r in endemic fluorosis areas. CONCLUSION: Although no significant difference was found in ER Rsa I genotype between cases and non-dental fluorosis in endemic fluorosis areas, children carrying R allele of ER Rsa I had a higher risk compared to children carrying r allele, and the further study is needed.

[Research in the relation between telomerase reverse transcriptase expression in spermatogenic cells and serum levels of estradiol of fluorotic rats].

OBJECTIVE: To study the relation between telomerase reverse transcriptase (TERT) activity expression in spermatogenic cells and serum levels of estradiol of fluorotic rats. METHODS: We randomly divided thirty SD male rats into control group, low-dose group and high-dose group, then inject sodium fluoride (0, 10, 20 mg/kg bw) into celiac of rats. We respectively observed changes of estrogen and TERT using methods of radioimmunoassay, in situ hybridization. In addition, we observed the quality of spermatozoa. RESULTS: The level of estrogen, the expression of telomerase and the number and the livability of the spermatozoon in low-dose and high-dose fluorotic rats were lower than those of control rats (P < 0.05). Therefore,the above indexes decreased with the increase of dosage. In addition, sperm aberration of each fluorotic group was higher than control group (P < 0.05). And it increased with the increase of dosage. The content of E2 in serum of different fluoride treatment groups was positively correlated with the expression of telomerase in seminiferous tubule significantly, respectively (low-dose fluoride treatment groups, r = 0.941, P < 0.01, high-dose fluoride treatment groups, r = 0.929, P < 0.01). CONCLUSION: NaF possibly damaged the male reproductive system by the approach of E2/ER-TERT-spermatozoon, relation between TERT expression in spermatogenic cells and serum levels of estradiol is positive correlation.

[Relationship between spermatogenic cell apoptosis and serum estradiol level in rats exposed to fluoride].

OBJECTIVE: To observe the relationship between spermatogenic cell apoptosis and serum estradiol level in rats exposed to fluoride. METHODS: A total of 30 male Wistar rats were allocated into six groups randomly. The six experimental groups were 28-day control group, 28-day low-dose fluoride treatment group, 28-day high-dose fluoride treatment group, 38-day control group, 38-day low-dose fluoride treatment group, and 38-day high-dose fluoride treatment group. The fluorosis model was acquired by subcutaneous injection of NaF solution. The content of NaF in testis was measured by using fluorine selective electrode. The serum estradiol level was radioimmunochemically detected. And the apoptotic spermatogenic cells were quantitatively measured by TUNEL. RESULTS: The content of NaF in testis and the ratio of apoptotic spermatogenic cell in fluoride treatment groups significantly increased with increased experimental dosage and prolonged experimental period (P < 0.05). Meanwhile, the serum estradiol level significantly decreased (P < 0.05), which was negatively correlated with the content of NaF in testis as well as the ratio of apoptotic spermatogenic cell (P < 0.05). CONCLUSION: Excessive fluoride could lead disturbance to serum estradiol level during some range of dose and time, which is an important factor to spermatogenic cell apoptosis.