Effects Of Organic Pollutants On Cell Cycle And Telomerase Activity Of MCF-7 In Lanzhou Reach Of The Yellow River

Objective: The organic pollutants were extracted in Lanzhou reach of the Yellow River in this study, the cellular proliferation inhibition of MCF-7 cells, cell cycles, telomerase expression and free cytoplasmic calcium of cells ([Ca²⁺]i) concentration were observed with the extracted organic pollutants treated MCF-7 cells, the mechanisms and effects of the extracted organic pollutants on cell toxicity were investigated.Methods: Water samples were collected from three sections (the pollution section is Zhongshan bridge, the self-purification section is Shichuan bridge and the clean section is Xincheng bridge) in Lanzhou reach of the Yellow River and the trace organic pollutants were concentrated in samples by Solid-Phase Extraction (SPE) method. The proliferation of MCF-7 cells treated by different concentrations of the extracted organic pollutants for 48h at three sections by MTT assay, the 50% inhibitory concentrations (IC₅₀) of the extracted organic pollutants groups were measured. Based on the IC₅₀, assessment of 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.Results:(1) Analysis of the extracted organic pollutants on MCF-7 cells proliferation by MTT assaySPE method was used to extract the organic pollutants in Zhongshan, Shichuan and Xincheng bridge at three sections, the concentration of three sections is 46.00mg·ml^-1, 35.80mg·ml^-1 and 26.40mg·ml^-1 respectively. Compared with the control group, in the range of 0-0.50mg·ml^-1 no significant inhibitory effects was observed at three sections with the MTT detection, while in the range of 1.00-5.00mg·ml^-1 the cellular proliferation inhibitory effects of MCF-7 were observed, there was a linear correlation between CPIR of MCF-7 and different concentration (P<0.05), with correlation coefficients (r²) is 0.903, 0.766 and 0.820 respectively, the IC50 in Zhongshan, Shichuan and Xincheng bridge were 3.37±0.02mg·ml^-1, 4.03±0.01mg·ml^-1, 4.34±0.03mg·ml^-1 respectively. It showed that the inhibitory effect in Zhongshan bridge was highest among three sections under the same concentration (P<0.05). In the range of 10.00-40.00mg·ml^-1 the cellular proliferation inhibitory effects of MCF-7 declined, especially in Zhongshan bridge (P<0.01).(2) Analysis of MCF-7 cell cycles and apoptosis in 48hCompared with the control group, the flow cytometry analysis showed that the apoptosis rates in MCF-7 cells [31.43±3.00% (Zhongshan bridge), 17.63±0.47% (Xincheng bridge) and 13.37±1.43% (Shichuan bridge)] increased significantly after exposure to the extracted organic pollutants at the concentration of half IC₅₀ for 48h (P<0.01). There have significant effects when compared with treated groups (P<0.05). The cell cycles distribution revealed that the mean percentage of G₁ phase (84.47±3.63) in Xincheng bridge was the highest, secondly Shichuan bridge and Zhongshan bridge as compared with the control group (P<0.01). The mean percent of G₁ was more than 55% at three sections, so cells were accumulated in the G₁ phase.(3) Influences of the extracted organic pollutants on telomerase activityCompared with the control group, telomerase expression declined in the organicpollutants group at three sections (P<0.01); telomerase expression had declined in a time-dependent manner (P<0.05).(4) Changes of free intracellular calcium ([Ca²⁺]i) concentrationThe [Ca²⁺]i concentrations were detected after the extracted organic pollutants treated MCF-7 cells for 24h, 48h and 72h by Laser scanning microscope (LSCM), it showed that [Ca²⁺]i concentrations were increased in each group as compared with the control group in different times. Furthermore, [Ca²⁺]i concentration was decreased in a time-dependent manner. It was also found that there have significant changes between the calcic groups and non-calcium groups (P<0.05). When the D-Hank's buffer was changed into Hank's buffer, [Ca²⁺]i was detected again, the increasing of cytoplasm [Ca²⁺]i had been inhibited. It suggested that the increasing was not derived from the flow of extracellular calcium. Conclusions:The extracted organic pollutants in Lanzhou reach of the Yellow River can lead to cells impairmed, apoptosis or death, the probable mechanisms were as follows:(1) The extracted organic pollutants in Lanzhou reach of the Yellow River inhibit MCF-7 cellular proliferation through blocking cell cycles and inducing apoptosis. (2)Expressions of telomerase are detected in human breast cancer MCF-7 cells by PCR-TRAP-silver staining method. The extracted organic pollutants may inhibit their expressions in vitro MCF-7 cells in Lanzhou reach of the Yellow River, but the inhibition is declining with the time, the mechanism needs further exploration. (3) The [Ca²⁺]i concentration in MCF-7 cells increased by the extracted organic pollutants with treated time it declined.