| At present, organic chemicals for global daily use has been more than100000, most of which are not of the safety test. After being used, organic chemicals enter the environment via various ways, and most of them remain in the water environment as their parent forms or the products, and then induce the water pollution. However, the evaluation of water quality is dominated more by physical and chemical analysis. The measurement results do not accurately reflect the current situation of water pollution and the pollutants toxicity. Toxicology is an important way to evaluate the toxicity of the water pollutants, but the traditional toxicological methods can not meet the requirements of toxicity evaluation of the hazardous chemicals and new compounds, which increase dramatically. Therefore, the development of simple, rapid, sensitive and low-cost toxicological methods, which also reflects the biological properties of the test organism, has become a hot issue in the field of environmental monitoring.As a new technique, the in vitro cell electrochemical method has shown a great promise in the detection of the cell viability due to its intrinsic advantages, such as simple instrumentation, high sensitivity, rapidness, low-cost, label-free, non-toxicity and reutilization of the samples. Therefore, the evaluation of the toxicity of chemicals using the cell electrochemical method can provide a new technique and method for the evaluation of the safety of the water environment and the toxicity of the environmental pollutants.In this paper, the voltammetric responses of human breast cancer (MCF-7) cells and human cervical carcinoma (HeLa) cells as the model cell were detected by using electrochemical detection method, and the concentration of the electrochemical active species of the cells was analysed by high performance liquid chromatographic (HPLC) method to explain the mechanism and the origin of the voltammetric response. Then a two-signal cell electrochemical detection method and the in-situ cell electrochemical detection method were established. With these methods, the cell growth curve and the effect of the inhibitors on cell growth were described and evaluated. Also the reliability of these methods in the cell viability evaluation was investigated. Finally the cytotoxicity of chlorophenol pollutants on the model cells were studied by using the in-situ cell electrochemical method. Based on the voltammetric response of the fragmented cell suspension, a novel in-situ cell electrochemical method, which could evaluate the cytotoxicity of environmental pollutants and was simpler, more rapid, reliable and objective, was suggested.The main results are concluded as follows:1. Establishment of a two-signal cell electrochemical system to evaluate the cell viability. For expanding the response potential of cell electrochemical detection and studying the other source of the cell electrochemical response, except guanine and xanthine (signal Ⅰ), a mixed system consisted of Multi-walled carbon nanotubes and ionic liquid was scraped coating on the bare glassy carbon electrode, and the potential window of the electrochemical detection system was expanded. Then the electrochemical response behaviors of MCF-7cells were studied, and an visible irreversible oxidation peak (signal Ⅱ) was found after+1.0V for the first time, which was given by the oxidation of adenine and hypoxanthine in the cytoplasm. On this basis, a two-signal electrochemical detection method was established, and the cell growth curve was tracked and described by this electrochemical method. Also the effect of genistein on the MCF-7cell growth was studied, and the IC50values wrer calculated. The conventional MTT assay was used to verify the reliability of the two-signal electrochemical method. The results showed that the proposed two-signal electrochemical method could accurately describe the cell growth state. Compared with the MTT assay, the result based on signal Ⅰ was more accurate and the IC50values using the signal Ⅰ as the index was lower than that measured by MTT assay. All the above showed that the developed electrochemical method based on MCF-7cells voltammetric behavior was more sensitive than the traditional MTT assay to some extent in the evaluation of the cell viability.2. Building the evaluating system of the cell viability by the in-situ electrochemical method. A novel and simple in-situ electrochemical method was developed by using the human breast cancer (MCF-7) cells and the human cervical carcinoma (HeLa) cells as models. It broke through traditional patterns of cell pretreatment and electrochemical detection, integrated the cell culture, pretreatment and detection in the culture dish. The electrochemical responses of the selected cells, which were given by the oxidation of guanine and xanthine in the cytoplasm, were much higher than that obtained from the conventional electrochemical method, which implies the sensitivity of the electrochemical detection system was significantly improved. Additionally, the in-situ electrochemical method was applied to the detection of the evaluation of the effect of cyclophosphamide monohydrate on cell viability of MCF-7cells and HeLa cells. The results showed that when the dose or the treatment time of cyclophosphamide monohydrate increased, the peak currents of the in-situ fragmented cell suspensions reduced significantly, which showed the decline of the cell viability, the dosage-dependent and time-dependent could be observed. The reliability of the in-situ cell electrochemical method was verified by comparing with the MTT assay. Both the results obtained from both the methods were essentially consistent, and the IC50value obtained from the electrochemical method was much lower. Also the in-situ electrochemical method simplifies operation process, reduces experiment cost and time, and avoids trypsin usage. All the results suggested the in-situ electrochemical method was simpler, more rapid, reliable and objective than the conventional electrochemical method. 3. Building an in-situ cell electrochemical method for the study of the cytotoxicity of chlorophenol pollutants. The effect of pentachlorophenol (PCP),2,4,6-trichlorophenol (TCP) and2,4-dichlorophenol (DCP) on the growth of MCF-7cells and HeLa cells was studied by using the above established in-situ cell electrochemical method. The cytotoxicity of chlorophenols pollutants was evaluated and the IC50values were calculated. The results showed that the three chlorophenols pollutants had a treatment time-dependent or dose-dependent toxicity on MCF-7cells and HeLa cells, that is, with the increase of the concentrations of chlorophenols, the cell viability decreased continuously. Verified by the traditional MTT assay, the in-situ cell electrochemical method for the study of the environmental pollutants toxicity was reliable. Both the results obtained from both the methods were essentially consistent, the order of the cytotoxicity was as follow:PCP>TCP> DCP. The results suggested the evaluation of the cytotoxicity of environmental pollutants by the in-situ cell electrochemical method was more sensitive, credible and objective. Meanwhile, the result showed that HeLa cells were more sensitive and suitable than MCF-7cells in the cytotoxicity study of environmental pollutants. |
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