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The Effect of Coal Tar Pitch Extract on Nrf2-Keap1/ARE Pathway in BEAS-2B Cells

Author: HaoYanHong
Tutor: WuYongJun；WuYiMing
School: Zhengzhou University
Course: Health Toxicology
Keywords: The Extract of Coal Tar Pitch Nrf2 Keap1 NQO1
CLC: R114
Type: Master's thesis
Year: 2009
Downloads: 48
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Abstract

Coal tar pitch (CTP) is by-product made of coal in coking process. It is mainly used in paint, plastics, rubber and steel production. It has been shown by epidemiological studies that there was a high incidence rate of lung cancer among the people who were exposed to CTP, therefor lung cancer caused by CTP has been defined as occupational tumor.Polycyclic aromatic hydrocarbons (PAHs) are the main components of coal tar pitch. They are recognized carcinogens. Carcinogens are from the biotransformation reactions by the phase I and II metabolic enzymes in vivo. The electrophilic carcinogens are generated by the phase I metabolic enzymes (e.g. cytochrome P450), then combine with DNA and protein to cause the change of oncogenes and tumor suppressor genes. The phase II metabolic enzymes (e.g. glutathione -s-transferase, GST) can enhance the excretion of carcinogens by promoting their hydrophilicity. The phase II metabolic enzymes are induced by the antioxidant response element (ARE) or electrophile response element (EpRE). The activation of Nrf2-Keap1/ARE signal pathway which plays as a "double edged sword" during the carcinogenesis involves in a series of endogenous antioxidant enzymes and proteins. In the normal state or sub-toxic doses of stimulation, Nrf2-Keap1/ARE prevents against the oxidative damage by regulating the expression of detoxification enzymes. But in tumor cells, its expression can’t be inhibited. It also can increase the ability of tolerance and multiplication of tumor cells under oxidative stress, and cause the resistance of the rumor cells to chemotherapeutic drugs. Therefore, the study on Nrf2-Keap1/ARE pathway becomes a hot spot in the field of oxidative stress and cancer prevention.Nrf2-Keap1/ARE system plays an important role in the cell against exogenous or endogenous oxidative stress. The lack and activation obstacle of Nrf2 are related to compound carcinogenesis, drug-induced liver injury, inflammation and apoptosis. It can provide a new idea for the therapy of tumor and inflammatory.It is that Nrf2-Keap1/ARE pathway controlled antioxidation in the cells of mammals. Antioxidative protein and metabolic enymes were regulated via the interaction between Nrf2 and ARE, and it become the hot spot in chemopreventive. In this study,.the BEAS-2B cells were treated with different concentrations of extract of coal tar pitch fume to analyze its effect on the expressions of Nrf2, Keap1 and ARE of BEAS-2B cells. The expressions of Nrf2, Keap1, NQO1 mRNA was measured by reversed transcription polymerase chain reaction (RT-PCR). And the level of Nrf2 protein was detected by western blotting. The study of the effect of CTP fume on Nrf2-Keap1/ARE pathway in BEAS-2B cells will provide a theoretical basis for the carcinogenesis and cancer prevention.Methods:1. Preparation of extract of coal tar pitch fume and the effect on cell proliferation. Coal tar pitch was heated at 400℃, fume was collected by dust samplers, smoke particle on cellulose membrane was dissolved by ethyl acetate, then the solvent was volatilized and condensed by heating, after that, dissolved again by dimethylsulfoxide. Gas chromatography - mass Spectrometry (GC-MS) was used to determine the components of coal tar pitch fume. The proliferation of BEAS-2B exposed to the extract of coal tar pitch was analyzed by MTT.2. Gene expressions of Nrf2, Keap1 and NQO1. BEAS-2B cells were treated at different concentration of CTP extract (0.00, 1.25, 2.50, 5.00, 10.00μg/ml) for 24h. Total RNA of the cells was extracted. GADPH was used as internal control. Gene expressions of Nrf2, Keap1, NQO1 were detected by reversed transcription polymerase chain reaction (RT-PCR).3. Protein expression of Nrf2. BEAS-2B cells were exposed to extract of coal tar patch at doses of 0.00, 1.25, 2.50, 5.00, 10.00μg/ml. Western blotting was applied to examine the protein expression of Nrf2.4. Statistical Analysis. Statistical software was SPSS12.0. Kolmogorov-Smirnov test, Levene test of homogeneity, one-way ANOVA were used to analyze the data (test levelα= 0.05).Results:1. The the analysis of CTP with GC-MS showed that the major elements of CTP are fused-ring aromatic compounds. The cytotoxicity was measured by MTT assay after the BEAS-2B cells were exposed to CTP for 24h. The 1.25μg/ml group increased the proliferation of cells, the difference had a statistics significance (P< 0.05), but the 2.50～10.0μg/ml groups inhibited cells proliferation(P<0.05).2. The BEAS-2B cells exposed to different concentrations of CTP, 0.00、1.25、2.50、5.00、10.00μg/ml for 24h.①The expressions of Nrf2 gene and protein in test groups were significantly lower than the control group (P <0.05).②The expression of test group was significantly higher than the control group (P<0.05).③The expression of NQO1 was higher than the control group in 5.00～10.00μg/ml (P<0.05). The expression of NQO1 gene showed the increased trend with the increase of the concentration of the exposure.Conclusion:1. The low level of the CTP concentration (1.25μg/ml) had hormesis to BEAS-2B cells. With the increase of the CTP concentration, the inhibition of proliferation appeared stronger.2. With the increase of the concentration of CTP, the expressions of Nrf2, NQO1 gene and Nrf2 protein were increased, the level of Keap1 gene was decreased. It was possible that Nrf2-Keap1 might minimize the oxidative damage via induced NQO1. 3. The toxicity of coal tar pitch extract on BEAS-2B cells might be realized through altering the function Nrf2.

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