Studies On The Estrogenic Effect Of Heavy Metals And Their Mechanism

Environmental estrogens (EEs) mimic the effect of natural estrogen in human or animals. They affect organisms in many aspects and their regulatory networks are also very complex, while our study has focused on their harm to reproductive systems. In this study, firstly, the estrogenic activity of Cd and Hg in vitro were measured by the yeast estrogen screen (YES). Then in order to determine the estrogenic activity of Cd and Hg in vivo, the immatured kunming mice were injected subcutaneously with them for three days. Next the increase of uterus wet weight was detected with uterotrophic assays; the increase of the hight of endometrium epithelium was measured by microscopic examination after HE stain; the influence of Cd and Hg on the expression of estrogen receptorα(ERα) and estrogen receptorβ(ERβ) was investigated by semi-quantitative RT-PCR; finally the data obtained from each experiment was compared respectively to analyze the correlation between them, and inquire the possibility of replacing the time-consuming and laborious assays with easier ones. Additionally, our study further explore the signal transduction path which the heavy metals might participate in, as well as the estrogen receptors'(ERs) response to the 17b-estrogen (E2) and EEs. The results showed that the EC50 value of E2 was 8.0×10-10mol·L-1 and the maximum activity ofβ-galactosidase was 6.48U. And for the Cd at the concentration of 1.0×10-8mol·L-1, the activity ofβ-galactosidase reached the maximum which was 2.60U and 0.40 timed the one induced by E2; for the Hg at the concentration of 5.0×10-7mol·L-1, the activity ofβ-galactosidase reached the maximum which was 0.93U and 0.14 timed the one induced by E2. However it was impossible to calculate the EC50 value withine the test concentration range. The results also indicated that the Cd and Hg had estrogenic activity withine certain concentration range which was their maximum estrogenic concentration even though their EC50 values was impossible to be calculated. If we estimated the estrogenic activity of the heavy metal just by the EC50 value, then it would be hard to calculate their estrogenic activity and the the YES implication will be impeded. Furthermore the EEs assay system in vitro will be improved if the recombinant human estrogen receptor-β(hERβ) gene yeast could be constructed.The results in vivo experiment showed that in the group of Hg 2000μg/kg.bw the Hg showed the heavy metal toxicity while in the others the Hg and Cd took the estrogenic activity. In the uterotrophic assays, the Hg 2000mg/kg.bw and E2 groups showed extremely significant difference with the blank, Cd 930mg/kg.bw showed significant difference and the others failed to show significant difference; in the hight of endometrium epithelium assay, two of the Cd groups showed significant difference while the others showed extremely significant difference with the blank; in the semi-quantitative RT-PCR experiment, both of the ERs expressed stronger in ovary than uterus, while the dose-effect relationship was indistinctive in Cd and Hg groups. Furthermore we found that the hight of endometrium epithelium and the wet weight of uterus were controled by the same factor, therefore we could predict the results of the hight of endometrium epithelium with the ones of uterotrophic assays. However as a kind of pathological examination the former was more sensitive and prophetic than the latter. Meanwhile the expression of ERa and ERβshowed that the correlation between the expression of ERβand uterus wet weight was closer than the one between the expression of ERa and uterus wet weight, which means the EEs assay system containing ERβwould be more excise with the in vivo assay system than ERa once again. But the mRNA assay shoud be improved to be more sensitive and stable to complement the above two in vivo assays. The method was forward and predicting using expression of ERs to measure the EEs' estrogenic activity, however more research should be taken to explore small molecules which could be detected easily as biomarkers of EEs. At the same time, there will be broad application prospects if the ERs could be used as biosensor to monitor EEs in waters on-line.