Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells

Tran, Christine D.H., Timmins, Peter, Conway, Barbara R. and Irwin, William J. (2002). Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells. Journal of Pharmaceutical Sciences, 91 (1), pp. 117-128.

Abstract

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 μM) 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 μM 1α,25-dihydroxyvitamin D3 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 [3H]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 1α,25-dihydroxyvitamin D3 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. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association.

Publication DOI: https://doi.org/10.1002/jps.1173
Divisions: Life & Health Sciences > Pharmacy
Uncontrolled Keywords: 1α,25-dihydroxyvitamin D,efflux,intestinal cytochrome P450 3A4 (CYP3A4),intestinal P-glycoprotein (P-gp),midazolam metabolism,Drug Discovery,Organic Chemistry,Chemistry(all),Molecular Medicine,Pharmacology,Pharmaceutical Science
Full Text Link: http://onlinelibrary.wiley.com/doi/10.1002/jps.1173/abstract
Related URLs: http://www.scopus.com/inward/record.url?scp=0036144831&partnerID=8YFLogxK (Scopus URL)
Published Date: 2002-01-29
Authors: Tran, Christine D.H.
Timmins, Peter
Conway, Barbara R.
Irwin, William J.

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