ABSTRACT
The previous studies from our laboratory reported that benzo(a)pyrene (Bap) influenced efflux transport of rhodamine 123 (Rho-123) by induction of P-glycoprotein (P-gp) in Caco-2 cells. The present study investigated whether induction of P-gp and the enhanced efflux transport of Rho-123 were caused by benzo(e)pyrene (Bep), which has a structure similar to Bap, but is not a carcinogenic compound. In Caco-2 monolayer exposed to 50 microM Bep for 72 h, the ratio of the apparent permeability coefficient (P(app)) of Rho-123 efflux increased significantly compared to that of the control monolayer. Similarly, a significant increase in expression of MDR1 mRNA and of P-gp at the protein level were detected by RT-PCR and by Western blot analysis, respectively, in Caco-2 cells exposed to Bep, compared to that of the control. Caco-2 cells exposed to Bep showed oxidative stress that was detected by fluorescence microscopy using aminophenyl fluorescein. However, the oxidative stress was weaker compared with that of Bap. The cellular GSH content was decreased to 80% or 59% of control cells, respectively, in Caco-2 cells exposed to either Bep or Bap. Our results further show that Bep or Bap-induced P-gp in Caco-2 cells might have been the result of oxidative stress rather than DNA damage.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Benzo(a)pyrene/toxicity , Benzopyrenes/toxicity , Carcinogens/toxicity , Biological Transport, Active/drug effects , Blotting, Western , Caco-2 Cells , Cell Survival/drug effects , DNA Damage/drug effects , Glutathione/metabolism , Humans , Microscopy, Fluorescence , Oxidative Stress/drug effects , Polycyclic Aromatic Hydrocarbons/toxicity , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Rhodamine 123ABSTRACT
The main exposure pathway of benzo[a]pyrene (Bap) for humans is considered to be via the daily diet. The purpose of this study was to investigate the effect of BaP on the intestinal transport of chemicals mediated by P-glycoprotein (P-gp). The intestinal epithelial membrane transport of rhodamine-123 (Rho-123), a substrate of P-gp, was examined using a monolayer of the human Caco-2 cell line grown in transwells. In the monolayer exposed to Bap for 72 h before transport experiments, the ratio of the apparent permeability coefficients (P(app)) of Rho-123 efflux increased compared to that of the control. The permeability of rhodamine-B (Rho-B), not a substrate of P-gp, showed no difference between the monolayers. Treatment with quinidine or cyclosporine A, which are P-gp inhibitors, decreased the P(app) of Rho-123 to the same degree in both monolayers. The transport of Rho-123 was not influenced by the presence of Bap. Thus, Bap seemed not to act directly on the efflux activity of P-gp and be a binding site competitor of Rho-123. In the Caco-2 cells that enhanced the efflux of Rho-123 by the treatment with Bap, an increase in mRNA expression of MDR 1 (P-gp) was confirmed compared to that of control by RT-PCR. Furthermore, Western blot analysis using a monoclonal antibody, C219, demonstrated the increase of P-gp in Caco-2 cells exposed to Bap, compared with controls. It was inferred that Bap exposure induced the expression of P-gp, which led to the observed increase in efflux transport of Rho-123. The possibility was suggested that Bap might affect the disposition of medicines by increasing P-gp expression.
