Objective To study the expression of multidrug resistance 1(MDR1)and glutathione S-transferase-π(GST-π) in acquired arsenic tolerant L-02 hepatic cells, and to discuss its relationship with the acquired tolerance to arsenic.
Methods MTT(3-4, 5-dimethy thiazol-2-yl-2, 5-diphenyl tetrazolium)assay was conducted to detect the effects of NaAsO2 exposure in various concentrations(0, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μmol/L)for 24 h on the survival rate of L-02 cells, then the initial dose under which the cell survival rate was 90%-95% was chosen to be added into the medium for culturing experiment L-02 cells(induced cells). L-02 cells grown in medium without arsenic were provided as contro(l normal cells). Induced cells and normal cells were cultured under the same condition for 6 weeks, then the survival rate and LC50 of induced cells and normal cells were detected by MTT assay which can reflect the change of arsenic tolerance. The levels of MDR1 and GST-π mRNA were determined by real-time quantitative PCR, and the expression of P-glycoprotein(P-gp), GST-π was examined by immunohistochemical SABC. Cell survival rate and intracellular concentrations of total arsenic of the cells that were cultured with 10 μmol/L NaAsO2 for 24 h were measured in order to observe the response of the cells to another large dose acute arsenic exposure.
Results After 6 weeks after induction of NaAsO2, LC50 and survival rate of induced cells were significantly higher than that of normal cells(P < .001); expression of the genes for GST-π/MDR1 in induced cells was significantly higher than that in normal cells(P < 0.001). Compared with normal cells, expression of P-pg/GST-π protein in induced cells was significantly higher(P < 0.001). In the re-trial of acute arsenic poisoning, the survival rate of induced cells was significantly higher than that of normal cells(P < 001), the total cellular arsenic content was markedly decreased in induced cells(P < 0.001).
Conclusion L-02 cells possess induced arsenic tolerance phenomenon. Acquired tolerance to arsenic is associated with increased expression of MDR1/GST-π in human hepatocyte cell line. This may provide an experimental basis for the prevention and treatment of arsenic poisoning at the gene level.
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