Abstract
The Caco-2 cell line is a reliable in vitro model for predicting drug intestinal absorption and P-glycoprotein (P-gp)-mediated excretion in humans. Recent in vivo studies suggested the induction of P-gp as a cellular protection tool against paraquat poisoning, through the increase in its pulmonary and intestinal excretion. Thus, the aim of the present work was to evaluate P-gp expression and activity in Caco-2 cells exposed to doxorubicin (a known P-gp inducer) and to correlate these changes with paraquat toxic effects. Cytotoxicity of doxorubicin (0–100 μM) and paraquat (0–1,000 μM) was evaluated for a maximum period of 96 h. In doxorubicin-exposed cells, P-gp expression and transport activity were evaluated by flow cytometry, using a fluorescein isothiocyanate–conjugated antibody and the P-gp fluorescent subtract rhodamine 123, respectively. A significant increase in P-gp expression was observed as soon as 6 h after exposure to 5 μM doxorubicin. P-gp activity also increased after 6 h, but only at higher doxorubicin concentrations (over 50 μM). Paraquat (0–5,000 μM) cytotoxicity was then evaluated with or without previous exposure of the cells to doxorubicin (5–100 μM, a concentration range causing both an increase in P-gp expression and activity). Under P-gp induction, a significant reduction in paraquat cytotoxicity was observed. Furthermore, when these cells were incubated with a specific P-gp inhibitor (UIC2 antibody) the doxorubicin protective effects were blocked, confirming the involvement of P-gp in the reduction in paraquat cytotoxicity. In conclusion, the human Caco-2 cell line model can be used for the study of P-gp induction as an antidotal pathway against substrates of this transporter system.
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Abbreviations
- BCRP:
-
Breast cancer–resistant protein
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- DOX:
-
Doxorubicin
- EDTA:
-
Ethylenediamine tetraacetic acid
- FBS:
-
Fetal bovine serum
- FITC:
-
Fluorescein isothiocyanate
- MTT:
-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
- NEAA:
-
Nonessential aminoacids
- NSAIDs:
-
Nonsteroidal anti-inflammatory drugs
- PBS:
-
Phosphate-buffered saline solution
- P-gp:
-
P-glycoprotein
- PQ:
-
Paraquat
- RHO 123:
-
Rhodamine 123
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Acknowledgments
This work was supported by the Fundação para a Ciência e Tecnologia (FCT) - project [PTDC/SAU-OSM/101437/2008] - QREN initiative with EU/FEDER financing through COMPETE - Operational Programme for Competitiveness Factors. Renata Silva acknowledges FCT for her PhD grant [SFRH/BD/29,559/2006]. Ricardo Dinis-Oliveira acknowledges FCT for his pos-Doc grant [SFRH/BPD/36,865/2007].
Caco-2 cells were kindly provided by Rosário Monteiro from the Biochemistry Department, Faculty of Medicine, University of Porto, Portugal.
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The authors declare that there are no conflicts of interest.
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Silva, R., Carmo, H., Dinis-Oliveira, R. et al. In vitro study of P-glycoprotein induction as an antidotal pathway to prevent cytotoxicity in Caco-2 cells. Arch Toxicol 85, 315–326 (2011). https://doi.org/10.1007/s00204-010-0587-8
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DOI: https://doi.org/10.1007/s00204-010-0587-8