title: In vitro study of P-glycoprotein induction as an antidotal pathway to prevent cytotoxicity in Caco-2 cells.
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.
authors.affiliation: REQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha, 164, 4099-030, Porto, Portugal. rsilva@ff.up.pt
authors.forename: Renata
authors.initials: R
authors.lastname: Silva
authors.forename: Helena
authors.initials: H
authors.lastname: Carmo
authors.forename: Ricardo
authors.initials: R
authors.lastname: Dinis-Oliveira
authors.forename: Anabela
authors.initials: A
authors.lastname: Cordeiro-da-Silva
authors.forename: Sofia Costa
authors.initials: SC
authors.lastname: Lima
authors.forename: Félix
authors.initials: F
authors.lastname: Carvalho
authors.forename: Maria de Lourdes
authors.initials: Mde L
authors.lastname: Bastos
authors.forename: Fernando
authors.initials: F
authors.lastname: Remião
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D000931:Antidotes / Q000378:metabolism*; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004336:Drug Antagonism; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D010269:Paraquat / Q000633:toxicity; D020112:Rhodamine 123
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000931:Antidotes; D005456:Fluorescent Dyes; D020112:Rhodamine 123; D004317:Doxorubicin; D010269:Paraquat