Quantification of biliary excretion, sinusoidal excretion and diffusion of 5(6)-carboxy-2',-7'-dichlorofluorescein (CDF) in hepatocytes isolated from Sprague Dawley, Wistar and Mrp2 (TR-) deficient rats

L.C.J. Ellis, M.H. Grant, G.M. Hawksworth, R.J. Weaver

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 ± 0.8 μM (SD), 9.0 ± 3.1 μM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 ± 291.5 μM (SD), 69.2 ± 36.2 μM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 ± 0.5) and vesicles (h = 1.6 ± 0.2) expressing Mrp2 and from SD (h = 1.6 ± 0.4) and Wistar (h = 4.0 ± 0.6) hepatocytes suggests transport involves more than one binding site. In TR− hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 ± 36.0 μM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.
LanguageEnglish
Pages1165–1175
Number of pages11
JournalToxicology in Vitro
Volume28
Issue number6
Early online date4 Jun 2014
DOIs
Publication statusPublished - Sep 2014

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Rats
Hepatocytes
Membranes
Binding Sites
Pharmaceutical Preparations
Wistar Rats
Kinetics
Hepatobiliary Elimination
5(6)-carboxy-2',7'-dichlorofluorescein
Liver

Keywords

  • biliary excretion
  • sinusoidal excretion
  • dichlorofluorescein

Cite this

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title = "Quantification of biliary excretion, sinusoidal excretion and diffusion of 5(6)-carboxy-2',-7'-dichlorofluorescein (CDF) in hepatocytes isolated from Sprague Dawley, Wistar and Mrp2 (TR-) deficient rats",
abstract = "Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 ± 0.8 μM (SD), 9.0 ± 3.1 μM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 ± 291.5 μM (SD), 69.2 ± 36.2 μM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 ± 0.5) and vesicles (h = 1.6 ± 0.2) expressing Mrp2 and from SD (h = 1.6 ± 0.4) and Wistar (h = 4.0 ± 0.6) hepatocytes suggests transport involves more than one binding site. In TR− hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 ± 36.0 μM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.",
keywords = "biliary excretion, sinusoidal excretion, dichlorofluorescein",
author = "L.C.J. Ellis and M.H. Grant and G.M. Hawksworth and R.J. Weaver",
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doi = "10.1016/j.tiv.2014.05.010",
language = "English",
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Quantification of biliary excretion, sinusoidal excretion and diffusion of 5(6)-carboxy-2',-7'-dichlorofluorescein (CDF) in hepatocytes isolated from Sprague Dawley, Wistar and Mrp2 (TR-) deficient rats. / Ellis, L.C.J.; Grant, M.H.; Hawksworth, G.M.; Weaver, R.J.

In: Toxicology in Vitro, Vol. 28, No. 6, 09.2014, p. 1165–1175.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Quantification of biliary excretion, sinusoidal excretion and diffusion of 5(6)-carboxy-2',-7'-dichlorofluorescein (CDF) in hepatocytes isolated from Sprague Dawley, Wistar and Mrp2 (TR-) deficient rats

AU - Ellis, L.C.J.

AU - Grant, M.H.

AU - Hawksworth, G.M.

AU - Weaver, R.J.

PY - 2014/9

Y1 - 2014/9

N2 - Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 ± 0.8 μM (SD), 9.0 ± 3.1 μM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 ± 291.5 μM (SD), 69.2 ± 36.2 μM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 ± 0.5) and vesicles (h = 1.6 ± 0.2) expressing Mrp2 and from SD (h = 1.6 ± 0.4) and Wistar (h = 4.0 ± 0.6) hepatocytes suggests transport involves more than one binding site. In TR− hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 ± 36.0 μM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.

AB - Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 ± 0.8 μM (SD), 9.0 ± 3.1 μM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 ± 291.5 μM (SD), 69.2 ± 36.2 μM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 ± 0.5) and vesicles (h = 1.6 ± 0.2) expressing Mrp2 and from SD (h = 1.6 ± 0.4) and Wistar (h = 4.0 ± 0.6) hepatocytes suggests transport involves more than one binding site. In TR− hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 ± 36.0 μM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.

KW - biliary excretion

KW - sinusoidal excretion

KW - dichlorofluorescein

UR - http://www.sciencedirect.com/science/journal/00062952

U2 - 10.1016/j.tiv.2014.05.010

DO - 10.1016/j.tiv.2014.05.010

M3 - Article

VL - 28

SP - 1165

EP - 1175

JO - Toxicology in Vitro

T2 - Toxicology in Vitro

JF - Toxicology in Vitro

SN - 0887-2333

IS - 6

ER -