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Research ArticleArticle

Effect of Culture Time on the Basal Expression Levels of Drug Transporters in Sandwich-Cultured Primary Rat Hepatocytes

Eskouhie H. Tchaparian, Jessica S. Houghton, Craig Uyeda, Mark P. Grillo and Lixia Jin
Drug Metabolism and Disposition December 2011, 39 (12) 2387-2394; DOI: https://doi.org/10.1124/dmd.111.039545
Eskouhie H. Tchaparian
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Jessica S. Houghton
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Craig Uyeda
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Mark P. Grillo
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Lixia Jin
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Abstract

Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na+-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction.

Footnotes

  • This work was supported in part by National Institutes of Health National Heart, Lung, and Blood Institute [Training Grant T32-HL07013] (to J.S.H.).

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.111.039545.

  • ↵Embedded Image The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    OATP/Oatp
    organic anion transporting polypeptide
    OCT/Oct
    organic cation transporter
    OAT/Oat
    organic anion transporter 2
    MRP/Mrp
    multidrug resistance-related protein
    P-gp
    P-glycoprotein
    BCRP/Bcrp
    breast cancer resistant protein
    E17BG
    estradiol 17β-d-glucuronide
    PAH
    p-aminohippuric acid
    MPP+
    1-methyl-4-phenylpyridinium
    WEM
    Williams' E medium
    MDR/Mdr
    multidrug resistance protein
    Slc
    solute carrier organic transporter
    Bsep
    bile salt export pump
    NTCP/Ntcp
    Na+-taurocholate cotransporting polypeptide
    DPP IV
    dipeptidyl peptidase IV
    GAPDH
    glyceraldehyde-3-phosphate dehydrogenase
    ZO-1
    zonula occludens protein 1
    RT
    reverse transcription
    PCR
    polymerase chain reaction
    DAPI
    4′,6′-diamidino-2-phenylindole
    PBS
    phosphate-buffered saline
    q
    quantitative
    OTF1
    POU domain, class 2, transcription factor 1.

  • Received March 17, 2011.
  • Accepted August 23, 2011.
  • Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 39 (12)
Drug Metabolism and Disposition
Vol. 39, Issue 12
1 Dec 2011
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Research ArticleArticle

TRANSPORTER EXPRESSION IN PRIMARY RAT HEPATOCYTES

Eskouhie H. Tchaparian, Jessica S. Houghton, Craig Uyeda, Mark P. Grillo and Lixia Jin
Drug Metabolism and Disposition December 1, 2011, 39 (12) 2387-2394; DOI: https://doi.org/10.1124/dmd.111.039545

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Research ArticleArticle

TRANSPORTER EXPRESSION IN PRIMARY RAT HEPATOCYTES

Eskouhie H. Tchaparian, Jessica S. Houghton, Craig Uyeda, Mark P. Grillo and Lixia Jin
Drug Metabolism and Disposition December 1, 2011, 39 (12) 2387-2394; DOI: https://doi.org/10.1124/dmd.111.039545
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