| It is confirmed that coal tar pitch(CTP) is carcinogenic by epidemiological studies and animal experiments, and lung cancer caused by CTP has been defined as occupational tumor. Due to the complex components, the carcinogenic mechanism of CTP is unclear till now.Mammals have developed adaptive and dynamic programs in the evolutionary process to counteract environmental stress. One of the important antioxidant systems is Keapl-Nrf2/ARE pathway. It is found that it is the main pathway through regulating cellular protection gene expression in response to oxidatie or electrophilic stress which is mainly adjusted by NAD(P)H:benzene quinonoids redox enzyme 1 (NQO1), glutathione S-transferases (GSTs), glutamylcysteine ligase (GCL), gamma-glutamylcysteine synthetase, metallothionein and many other kinds of antioxidant proteins or enzymes to show their antioxidant fuction. ObjectiveBecause of the important role of Keapl-Nrf2/ARE pathway, in this study, the interaction between CTP and Keapl-Nrf2/ARE pathway will be explored to provide feasible strategies and targets for lung cancer early prevention. Methods1 Preparation and analysis of CTP compoundVolatile flue gas of soft CTP from Henan Branch China Aluminum Co. LTD was collected by dust sampler when coal tar pitch was heated to 400℃. The final debris was dissolved in dimethyl sulfoxide (DMSO) to 2 mg/ml. Gas chromatography/mass spectrometry(GC-MS) was used to detect the compositions of CTP.2 Cytotoxicity testBEAS-2B cells activity test was applied to determine CTP extract concentration. Cellstain trypan blue was used to detecting the survial rate of BEAS-2B cells treated with different CTP extract concentrations for 24 h.3 Expressions of gene and protein Real time PCR was used to observe the genes expressions of Keapl, Nrf2 and NQO1 in BEAS-2B cells at the treatment points(3 h,6 h,12 h and 24 h)during different set of experiments. Western blot was used to observe the protein expression levels of Keapl, Nrf2 and NQO1 in BEAS-2B cells after exposued 6 h by different doses of CTP extract, and at the moment, the protein expression levels of Nrf2 in BEAS-2B cells at exposued 3 h,6 h,12 h and 24 h by 5μg/ml of CTP extract were obtainted.4 Statistical analysisSPSS 12.0 software was applied for t test, ANOVA, Prohit regression, linear regression analysis, LSD and SNK. Test level a is 0.05.Results1 Composition analysis of CTP extract by GC-MSThe main components of CTP extract were polycyclic aromatic hydrocarbon (PAHs).The relative percent of PAHs was above 91.0%.2 Cytotoxicity testCTP extract was toxic to BEAS-2B cells. The correlation between the survival rate of BEAS-2B cells and the CTP extract concentrations was calculated with Probit regression. The half inhibition concentration of BEAS-2B cell (IC50) was 8.11μg/ml.3 Expressions of genes and proteinsThere were statistically significant differences in the relative mRNA level of each gene at different time points when BEAS-2B cells were treated with CTP extract. The expressions of Nrf2 mRNA treated by CTP extract were lower than those treated by 6μmol/L ATRA. However, the expression of Nrf2 mRNA treated with 5μg/ml CTP extract alone was higher than that treated with 6μmol/L ATRA(0.5 h) followed by 5μg/ml CTP extract. The expression of NQO1 mRNA trend was similar to Nrf2.Western blot results showed that there was nothing apparent of Keapl protein changes by increasing the concentration of CTP extract, but the expression of NQO1 protein was increased gradually. The level of Nrf2 proteins was highest at 5μg/ml of CTP extract among the test groups, and that of Nrf2 protein was highest for 6 h among time points. ConclusionThe expressions of NQO1 were up-regulated in BEAS-2B cells induced by CTP extract. It is shown that Keapl-Nrf2/ARE pathway may up-regulate cellular protective gene to antagonize the toxicity of CTP. |
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