Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells.
The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 microM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 microM 1alpha,25-dihydroxyvitamin D(3) to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213 +/- 54.35%) compared to that of parent cells (100 +/- 6.05%). Furthermore, they retained significantly less [(3)H]vincristine sulphate (p < 0.05), a P-gp substrate, after efflux (272.89 +/- 11.86 fmol/mg protein) than the parent cells (381.39 +/- 61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1alpha,25-dihydroxyvitamin D(3) treatment was quantified to be 76.2 +/- 7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0 +/- 20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0 +/- 20.0 to 1057.0 +/- 46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics.
Tran
Christine D H
CD
Pharmaceutical Sciences Research Institute, Aston Pharmacy School, Aston University, Aston Triangle, Birmingham B4 7ET, UK.
Timmins
Peter
P
Conway
Barbara R
BR
Irwin
William J
WJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000502:physiology*; D000042:Absorption / Q000502:physiology; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D018938:Caco-2 Cells / Q000187:drug effects / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000502:physiology*; D004317:Doxorubicin / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D006801:Humans; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000502:physiology*; D014750:Vincristine / Q000493:pharmacokinetics; D015262:Xenobiotics / Q000493:pharmacokinetics*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D015262:Xenobiotics; D014750:Vincristine; D004317:Doxorubicin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human