[Effects of benzo(a)pyrene on the expression of gamma-glutamate-cysteine ligase: a primary study with rat alveolar epithelial cells].

Objective: To study the effect of benzo(a)pyrene [B(a)P] on the expression of gamma-glutamate-cysteine ligase (gamma-GCS) in rat alveolar epithelium cells (CCL-149 cell line).

Methods: Rat alveolar cells of the line CCL-149 were cultured and exposed to B(a)P of the concentrations of 0, 100, 500, and 5000 microg/L respectively for 24, 36, 48, and 72 hours respectively. Then the A values was measured to observe the influence of B(a)P on the growth of the CCL-149 cells. Another CCL-149 cells were exposed to B(a)P of the concentration of 200 microg/L for 6 h, then the nuclear protein was extracted. Electrophoretic mobility shift assays (EMSA) and antibody supershift assay were used to observe the specific binding of aryl hydrocarbon receptor nuclear translocator (ARNT) to the E-box element. CCL-149 cells were cultured to 95% confluent and transfected with GCLC-luc and GCLC-delE-box-luc for 6 h, exposed to B(a)P of different concentrations (2, 20, and 200 microg/L) for 2, 6, 12, or 24 h, then the cells were harvested and the luciferase activity was measured. Cells treated with DMSO were used as negative control group.

Results: B(a)P of the concentrations over 5000 microg/L significantly influenced the growth of the CCL-149 cells (all P < 0.01). Treated with B(a)P induced binding of AHR/ARNT to E-box element was significantly increased by treatment of B(a)P. Treatment of B(a)P of different concentrations at different time points did not significantly influence the luciferase activity (all P > 0.05).

Conclusion: B(a)P induces the binding of ARNT to the E-box element on the gamma-GCS gene, but this interaction between E-box and ARNT seems not to have effect on the gene expression of gamma-GCS.