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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. ISSN 0022-3549

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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 J Pharm Sci 91:117–128, 2002

Item Type: Article
Subjects: Q Science > Q Science (General)
Q Science > QD Chemistry
Schools: School of Applied Sciences
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Depositing User: Sharon Beastall
Date Deposited: 05 Nov 2010 09:28
Last Modified: 05 Nov 2010 09:28
URI: http://eprints.hud.ac.uk/id/eprint/8957

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