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Studies on the Estrogenic Effect and the Biodegradation of Phthalate Esters

Author: YangXue
Tutor: XiongLi
School: Central China Normal University
Course: Biochemistry and Molecular Biology
Keywords: phthalate esters estrogenic activity recombinate yeast screen(YES) mice uterotrophic assays semi-quantitative PCR biodegradation Isolation and identification
CLC: R114
Type: Master's thesis
Year: 2011
Downloads: 26
Quote: 0
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Abstract


Phthalate esters are widely used industrial raw materials that are receiving more and more attention because of their wide distributions in the environment, resistance to degradation and potential estrogenic activities. In this study, firstly, the estrogenic activities of dimethyl phthalate(DMP), diethyl phthalate(DEP), dibutyl phthalate(DBP), di-sec-octyl phthalate (DOP), and butyl benzyl phthalate(BBP) were measured by yeast estrogen screen(YES). Then to examine the mechanism of estrogenic activities of phthalate esters, BBP was injected subcutaneously into 17-18 day-old kunming mice. The increase in uterus wet weight was measureded with uterotrophic assays and the increase in height of the endometrium epithelium was measured by microscopic examination after a HE stain The expression of estrogen receptor a (ERα) and estrogen receptorβ(ERβ) induced by BBP were explored by semi-quantitative RT-PCR. The study further explored the roles of ERαand ERβin estrogenic activity induction by BBP. Finally, two BBP degrading bacterial strains, HS-B1 and HS-B2 were isolated from contaminated sludge to further explore the biodegradation of BBP.The results showed that diethyl phthalate (DEP, 1×10-7-1×10-9 mol/L), dibutyl phthalate (DBP, 1×10-6-1×10-9mol/L), and butyl benzyl phthalate (BBP, 1×10-3-1×10-6 mol/L) were clearly estrogenic but dimethyl phthalate (DMP) and di-sec-octyl phthalate (DOP) showed little evidence of estrogenic activity. The relative potency ratios of DEP, DBP, BBP are 8.85×10-2,2.54×10-2,8.82×10-6, respectively, suggesting that DEP, DBP showed higher estrogenic activity than BBP.The in vivo experiment showed that uterus wet weight, height of endometrium epithelium and ERα(αsubtype) gene expression increased relative to the control. It showed that uterus wet weight reached its highest level in the 2.5ml/kg·bw BBP dosing group and the height of endometrium epithelium reached its highest level in the 1.25ml/kg·bw BBP dosing group. Expression of the ERa gene was significantly different between the 0.25/kg-bw BBP dosing group and the control group. It suggested that the semi-quantitative RT-PCR appeared more sensitive than the other techniques. The ERP here could exhibit an inhibitory action on ERαmediated gene expression and the actions of ERβalso provented the uterus wet weight from increasing. But the ERP expression mechanisms needs further examination relative to BBP estrogenic activity. The molecular level biomarkers seemed to be more sensitive for detecting estrogenic activity.Two BBP degrading bacterial strains, HS-B1 and HS-B2, were isolated from contaminated sludge in the JinZhou River (Hubei Province). The two bacteria were identified as Acinetobacter sp. and Arthrobacter sp., respectively by morphology and 16S rDNA sequence analysis. The degradation of 500 mg/L BBP by the two strains HS-B1 and HS-B2 was 28% and 59% after 24 hours, respectively. The rate of degradation increased to 40% and 75% respectively with the addition of 0.1% LB (24h) suggesting that adding appropriate carbon sources enhances biodegradation. This study is helpful for developing microbial resources for biological remediation purposes. Further studies needed regarding BBP biodegradation.

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