鄰苯二甲酸酯暴露程度與Kiss-1、GPR54基因多型性對性早熟女童之影響研究

Abstract

In recent years, studies have shown the incidence of precocious puberty increased year by year, especially for girls. The epidemiological studies found that phthalate esters (PAEs) exposure may be related to the onset of puberty. PAEs were widely used in PVC plastic products, food containers, personal hygiene and commercial products. The significant estrogenic activities of various PAEs have been observed in different studies. While the PAEs entered the body, it acted as an estrogen analog and bound to estrogen receptor α (ERα). In addiction, several evidences have shown that Kisspeptins were the peptide products of the Kiss1 gene, and its receptor GPR54 (G protein coupled receptor) plays a crucial role in governing reproductive endocrine system and the onset of puberty. Moreover, the ERα regulated the release of GnRH (Gonadotropin -releasing hormone) by interacted with estrogen and therefore played an important role in the feedback loop of Kisspeptin/GPR54 system. In this study, we investigate the association among phthalate exposure, kisspeptin 54 peptide level and Kiss-1/GPR54 gene polymorphism on early onset of girl puberty. The objectives were: (1) to evaluate exposure factor of PAEs in daily life by standardized questionnaires and to assess the internal exposure dose by analyzing of PAEs metabolites in urine; (2) to assess the differences of Kisspeptin54 peptide between two groups; (3) to study the relationship of Kisspeptin54 peptide secretion and degree of sex characteristic development; (4) to assess the relationship of PAEs metabolite concentrations and Kisspeptin54 peptide; (5) to explore the Kiss-1, GPR54 gene sequence variation between case and control, and to assess the protein sequence variation using relevant biological information database. We recruited the precocious puberty girls from the National Cheng Kung University Hospital pediatric clinic as case group, and the control group recruited by voluntary participation of healthy girls. The total of 40 precocious puberty girls and 11 normal girls were recruited in this study. Seven urinary metabolites of PAEs were analysed by HPLC-MS/MS, and the commercial radio immunoassay kit was used for Kisspeptin54 analysis. We found that the higher frequencies and quantities of eating greasy meat, fried foods, beverages, snacks and nutriment, off-premises eating and the usage of PVC plastic wrap were the potential causes of higher phthalate exposure. Futhermore, living in the aged building and less house cleaning may also contribute to higher indoor PAEs exposure. The concentration of seven metabolites of PAEs in urine from precocious puberty girls were higher than normal girls, and the MMP, MBP, MBzP, MEHHP and MEOHP showed significant difference between two groups(MMP:8.35 vs. 4.31 µg/L, P=0.013; MBP: 115.10 vs. 32.12 μg/L , P = 0.0002; MEHHP: 64.86 vs. 27.53 μg/L, P = 0.003; MEOHP: 59.98 vs. 25.73 μg/L, P = 0.003; MBzP: 11.17 vs. 2.79 μg/L, P = 0.0001). After divided all subjects into three development category groups, the MMP, MBP, MBzP, MEHHP and MEOHP concentration were also increased significantly. While adjusted with creatinine, only the MBP and MBzP have show the increased trend (P <0.05). We also calculated the aggregated dose to estimate the internal estrogenic activities of PAEs due to the different ERα binding affinities of PAEs. We found that the aggregated dose of precocious puberty girls (6.47 (2.70-16.75) μg/kg/day) was significantly higher than those of normal girls (2.99 (1.04-10.28) μg/kg/day, P=0.0007). It showed that the higher phthalate exposure in precocious puberty girls may cause more internal estrogenic activites than normal girls. The average Kisspeptin54 concentrations were 2.15 (1.39-2.86) and 1.95 (1.69-2.18) pmol/L (P=0.06) for precocious puberty girls and normal girls, respectively. Moreover, the Kisspeptin54 concentrations were also significantly increased with sexual development. We also found that Kisspeptin54 secretion was significantly correlated with the peak lutenising hormone (LH) concentration analyzed by LHRH test (r=0.4, P= 0.03). The significant correlation between Kisspeptin54 secretion and urinary MMP, MBP and MBzP concentrations (uncorrected for creatinine) were also observed, however, only the correlation between Kisspeptin54 and urinary MBP remained significant after creatinine correction (r=0.537, P =0.0006). The results of gene sequencing showed that 16 and 14 sequence variants in Kiss-1 and GPR54 gene, respectively, were identified in this study. Furthermore, 9 of 16 and 6 of 14 sequence variants in Kiss-1 and GPR54 gene, respectively, were confirmed SNP. Three sequence variants among them could lead to amino acid change in both two genes. For allele and genotype frequency of sequence variants comparison, no significant differences were found between case and control groups. In conclusion, the difference of expression of two genes could be the important index of gene variance. Further studies and more samples are needed to clarify the relationship between precocious puberty and the polymorphism of these genes.