| Pollution of environmental hormone (EH) in the aquatic environment has drawn much attention around the world. Yellow River is the second largest river in China and one of the largest rivers in the world. It flows across 9 provinces in north China, from west to east with mainstream 5464 km long and drainage area 795 thousand km2.It is also the very important water resource for industry, agriculture and people's living. The Lanzhou Reach belongs to upper reaches of Yellow River. Dense wastes discharge along the river in Lanzhou Reach makes the pollution very serious, among those pollutants, EH and heavy metals in the aquatic environment have been getting increasing attention.This study focused on variation, distribution and pollution level of the environmental EH in Lanzhou Reach of Yellow River in China so as to explore possible EH sources, their environmental behaviors and health effects. Furthermore, the extracted EH from Lanzhou reach of Yellow River were carried out to examine their cytotoxicity on human breast cancer MCF-7 cells by observing cell growth, cell cycles and apoptosis. Finally, the influences of the extracted organic pollutants on telomerase expression in MCF-7 cells were investigated and the relationship between apoptosis and free cytoplasmic calcium of cells concentration ([Ca2+]i) was explored in order to understand the cytotoxicity of the extracted organic pollutants.In this study,solid phase extraction (SPE),gas chromatography-mass spectrophometry (GC-MS) and gas chromatograph (GC-FID) and atomic fluorescence were applied to detect the EH and heavy metals such as Cd2+, Pb2+ and Hg2+ in the aquatic environment, respectively. The organic pollutants were extracted by SPE from Lanzhou reach of the Yellow River. The 50% inhibitory concentration (IC50) at 48h was measured with MTT method. Cell cycles and apoptosis were performed by flow cytometry. Telomerase activity expression was measured by the telomeric repeat amplification protocol (TRAP) method and [Ca2+]i concertration was detected by the laser confocal microscope method.The main research results in this study were as follows: (1)Totally,89 organic pollutants were determined including 6 priority pollutants by U.S. Environmental Protection Agency (EPA) and 6 priority pollutants by China.(2)10 EH were detected from the Lanzhou reach of Yellow River including naphthalene (NP), phenanthrene, di-n-butyl-phthalate (DBP), di-(2-ethyl)hexyl phthalate (DEHP) and benzothiazole with a detection possibility of 20%,40%,100%,100% and 20%, respectively.(3)The concentrations of naphthalene, phenanthrene, DBP and DEHP range from 0.180-0.260,0.117-0.320,1.480-13.890,1.040-109.930ug·L-1 in Lanzhou Reach of yellow River. It should be noted that the detected concentrations of phenanthrene, DBP and DEHP at some sampling sites exceeded the Chinese surface water quality standard (GB3838-2002) and EPA standard (1991).(4)Heavy metal Pb2+ was detected at all the sampling sites with a range of 0.012-0.032mg·L-1but Hg2+ and Cd2+ were below the detection limit.(5)The concentrations of the extracted organic pollutants at Zhongshan Bridge (the pollutive section)sampling site, Shichuan Bridge(the self-purification section) sampling site and Xincheng Bridge(the clean section) sampling site were 0.58mg·L-1,0.45mg·L-1 and 0.33mg·L-1 respectively. MCF-7 cells in vitro were treated with the extracted organic pollutants at three sections for 48h. No significant inhibition were observed for the extracted organic pollutants at three sections in the range of 0-0.50mg·L-1; However, the cellular proliferation inhibition of MCF-7 was observed in a range of 1.00-5.00mg·L-1 as compared to the control group; especially the cellular proliferation inhibition rate (CPIR) of the pollutive section was higher than those at other sections. There was a possitive correlation between CPIR of MCF-7 and the extracted organic pollutants concentrations (p<0.05) with correlation coefficients (r2) of 0.903, 0.766 and 0.820 respectively at three sections. Then the IC50 of MCF-7 cells treated with the extracted organic pollutants at the pollutive section, the self-purification section and the clean section was 3.37±0.02mg·ml-1,4.03±0.01mg·ml-1,4.34±0.03mg·ml-1, respectively. It showed that the inhibition in the pollutive section was the highest among three sections under the same concentration and there was significant difference at the pollutive section (p<0.05). Cellular proliferation inhibition rate of MCF-7 was inclining in the range of 10.00-40.00mg·ml-1 as compared to the control group, especially at the pollutive section.(6)Flow cytometry analysis showed that MCF-7 apoptosis rate was significantly increased with the extracted organic pollutants concentrations at different sections for 48h with a apoptosis rate of 31.43±3.00% (the pollutive section),17.63±0.47% (the clean section),13.37±1.43% (the self-purification section),and 8.10±0.25% (the control group),respectively; There were significant differences (p<0.05) between each group, especially the difference was more significant at the pollutive section, as compared to the control group(p<0.01). The cell cycles distribution revealed that the mean percentage of G1 phase (84.47±3.63) at the clean section was the highest, secondly the self-purification section and the pollutive section. Compared to the control group, a significant difference (p<0.01) was found; The mean percent of G1 was all over 55% at three sections, which indicated that cells were accumulated in the G1 phase.(7)Compared to the control group, telomerase expression declined in each group after MCF-7 cells were treated by the organic pollutants for 24h,48h and 72h respectively at different sections (p<0.01) with a time-dependent manner (p<0.05).(8)[Ca2+]i concentrations in MCF-7 cells treated by the extracted organic pollutants for 24h, 48h and 72h respectively were measured by laser scanning microscope(LSCM). The results showed that [Ca2+]i concentrations were increased in each group as compared to the control group within the different time. Furthermore, [Ca2+]i concentration was decreased in a time-dependent manner. It was also found that there is more significant change between the calcic group and non-calcium ion cells extracellular (p<0.05)The following conclusions can be summarized.(1)Organic pollutants discharge from the local petrochemical industry results in the serious aquatic pollution in Lanzhou Reach of Yellow River.89 organic pollutants were found including 12EH,6 priority pollutants by U.S. Environmental Protection Agency (EPA) and 6 priority pollutants by China.(2)The main environmental endocrine disruptors in Lanzhou Reach of Yellow River are di-n-butyl-phthalates (DBPs) and Pb2+(3)Water pollution in each section was all seriously.The main source of EH is the petroleum chemistry industry and agricultural source is minor relatively.(4) The extracted organic pollutants in Lanzhou reach of the Yellow River inhibit MCF-7 cellular proliferation through blocking cell cycles and inducing apoptosis.(5)The extracted organic pollutants in Lanzhou reach of the Yellow River may inhibit the expressions of telomerase in vitro MCF-7 cells, but the inhibition is declining with the time, the mechanism needs further exploration.(6)The extracted organic pollutants may increase the intracellular [Ca2+]i concentration in MCF-7 cells, but the increas is declining with the time...
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