- Email: [email protected]
Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes Lene Jørgensen, Johan Van Beek, Søren Lund, Arne Schousboe, Lassina Badolo ∗ Department of Drug Metabolism, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark
a r t i c l e
i n f o
a b s t r a c t
Article history:
Transport proteins control uptake of drugs into the liver (e.g., organic anion transporting
Received 3 July 2006
polypeptide (Oatp)) and excretion of drugs from the liver (e.g., multidrug resistance protein
Received in revised form
1 (Mdr1)). In this study, cryopreserved rat hepatocytes were used to investigate the effect of
5 October 2006
different culture conditions (suspension, conventional culture and sandwich culture) on the
Accepted 1 November 2006
uptake of [3 H]-taurocholate ± probenecid and the efflux of [3 H]-vinblastine ± ketoconazole;
Published on line 15 November 2006
mRNA levels of Oatp1a1, Oatp1a4, Mdr1a and Mdr1b were determined using real-time reverse transcription polymerase chain reaction (RT-PCR) and protein expression of Mdr was
Keywords:
assessed by immunocytochemistry. The uptake of [3 H]-taurocholate was higher in cryopre-
Hepatocytes
served rat hepatocytes maintained in suspension as compared to hepatocytes in culture. A
Cryopreservation
significant time dependent decline in the uptake of [3 H]-taurocholate was noticed from day
Transport proteins
2 to day 4 in conventional and sandwich cultures. [3 H]-taurocholate uptake was significantly
Oatp
reduced using the inhibitor probenecid. Oatp mRNA expression in hepatocytes in suspen-
Mdr1
sion was similar to that of liver, whereas much lower levels were detected in the cultures;
Sandwich culture
this was in accordance with [3 H]-taurocholate uptake results. Mdr1 activity was assessed by
Conventional culture
accumulation of the Mdr1 selective substrate, [3 H]-vinblastine, in hepatocytes using ketoconazole as an inhibitor. The results showed Mdr1 activity in cryopreserved rat hepatocytes in conventional and sandwich cultures. A time dependent increase in Mdr1 activity was noticed from day 2 to day 4. Mdr1 activity was not found using hepatocytes in suspension. Mdr1 mRNA expression was high in cryopreserved hepatocytes from both culture systems. Immunocytochemistry showed the Mdr protein in membranes of hepatocytes in culture as well as in that of hepatocytes in liver sections. In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport activity. Hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to hepatocytes in culture. The presence of Mdr1 in both conventional and sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Drugs given by the most convenient administration route, oral administration, are absorbed through the gastrointestinal tract. Thereafter, they reach the liver via the portal vein
before entering the systemic circulation. For some drugs, transport proteins present in the gastrointestinal tract and in the liver play a significant role in drug absorption and drug bioavailability (Diaz, 2000). Multidrug resistance proteins (Mdr, also known as P-glycoprotein) and organic anion trans-
∗ Corresponding author at: Department of Pharmaceutics and Analytical Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2a, 2100 Copenhagen, Denmark. Tel.: +45 35 30 65 47; fax: +45 35 30 60 10. E-mail address: [email protected] (L. Badolo). 0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.11.003
182
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
porting polypeptides (Oatp) are major members in the group of transport proteins that regulate the disposition of orally administered drugs (Kim, 2002; Beringer and Slaughter, 2005). Oatp participates in the uptake of drugs from the blood into the hepatocytes where they may undergo significant phase I and/or phase II metabolism. Oatp has also been suggested to function as an excreting protein capable of transporting drug and drug metabolites back into the systemic circulation (Chandra and Brouwer, 2004; Beringer and Slaughter, 2005). The interplay between Oatp uptake of drugs into hepatocytes, their subsequent metabolism by hepatic enzymes and further excretion into bile through Mdr efflux system is a powerful detoxification pathway in the body. Besides their presence in the liver and the intestine, Oatp and Mdr are widely distributed in the human body where they play an important role in the disposition and distribution of certain drugs. In the kidneys, Oatp and Mdr1 are involved in renal secretion of xenobiotics; at the blood–brain barrier a range of drugs have limited brain penetration due to Mdr1-mediated efflux. Oatp is also expressed in the blood–brain barrier; this suggests its role in delivery of drugs into the brain and removal of metabolites from the brain (Ayrton and Morgan, 2001; Kim, 2002; Beringer and Slaughter, 2005). Since the important role played by transport proteins in xenobiotic disposition and distribution is now understood, a great body of investigations of their effect on the disposition of new drugs are made by pharmaceutical industries as part of drug discovery screening. Investigations of Oatp activity have mostly been performed in vitro using freshly isolated hepatocytes. There are only very few reports of studies using cryopreserved hepatocytes for the assessment of Oatp transport activity. These studies show that some transport activity is retained in cryopreserved rat and human hepatocytes in suspension (Houle et al., 2003; Shitara et al., 2003). The use of cryopreserved rat hepatocytes for investigation of the functional activity of Mdr1 has not been reported. Moreover, sandwich culture is commonly used to assess the transport activity of Mdr1 and other canalicular transport proteins in freshly isolated rat and human hepatocytes by measurements of canalicular accumulation (Annaert et al., 2001; Hoffmaster et al., 2004; Annaert and Brouwer, 2005). The function of Mdr1 using freshly isolated rat hepatocytes has been demonstrated in conventional culture in a single report (Le Bot et al., 1994). However, this study assessed the transport activity as a decline in doxorubicin accumulation over time in culture as an indication of increased efflux of doxorubicin by Mdr1. The present study is the first, to our knowledge, to investigate the transport activity of Oatp and Mdr1 using cryopreserved rat hepatocytes in suspension, in conventional culture and in sandwich culture by the use of both substrates and inhibitors. Additionally, the protein expression and mRNA expression were investigated. A recent paper published by Bi et al. (2006) investigated MDR1 protein in cryopreserved human hepatocytes. The presence of functional transport proteins in cryopreserved rat hepatocytes will make it possible to investigate these transport proteins for a number of scientists with limited access to freshly isolated hepatocytes. It also allows standardising these studies by using cells from same donors in several experiments and will reduce considerably the number of animals used in these studies.
2.
Materials and methods
2.1.
Materials
Dexamethasone, dimethyl sulfoxide (DMSO), ehthyleneglycolbis(b-amino ethylether)-N,N,N ,N -tetraacetic acid (EGTA), KCl, ketoconazole, Percoll, probenecid, rat tail collagen (type I), triton X-100, trypan blue and -mercaptoethanol were ¨ obtained from Sigma–Aldrich (Brondby, Denmark). Calcium and magnesium free Hanks’ balanced salt solution (HBSS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS), ITS (insulin (1.0 g/L), transferrin (0.55 g/L), selenium (0.67 g/L)), lglutamine 200 mmol/L, penicillin 10,000 U/mL–streptomycin 10,000 g/mL, RLT buffer, RNAlater, RNeasy kit, alexa fluor 488 goat anti-mouse IgG and hoechst 33342 were obtained from Invitrogen (Taastrup, Denmark). [G-3 H]-Taurocholic acid (1.19 Ci/mmol) was obtained from Perkin-Elmer (Hvidovre, Denmark). [G-3 H]-Vinblastine sulfate (6.7 Ci/mmol) was ¨ Denmark). BCA proobtained from GE Healthcare (Hillerod, tein assay kit and vinblastine sulfate were obtained from Bie ¨ & Berntsen (Rodovre, Denmark). DNA-free kit was obtained from Ambion (Huntingdon, United Kingdom). SYBR GREEN PCR Master Mix kit and TaqMan RT-Reagent were obtained from Applied Biosystems (Naerum, Denmark). Bovine serum albumin (BSA) was obtained from Roche (Hvidovre, Denmark). Mouse anti-human P-glycoprotein antibody [C219] was obtained from Abcam (Cambridge, United Kingdom). Tween 20 was obtained from Merck-Schuchardt (Glostrup, Denmark).
2.2.
Methods
2.2.1.
Isolation of rat hepatocytes
Hepatocytes were isolated from female and male Sprague Dawley rats (150–200 g) obtained from Charles River Laboratories (Sulzfeld, Germany). The isolation was performed according to the two-step perfusion method described by LeCluyse et al. (1996). Isolated hepatocytes were used after cryopreservation.
2.2.2.
Cryopreservation of rat hepatocytes
Rat hepatocytes were cryopreserved as described by Le Cam et al. (1976). Isolated hepatocytes were suspended in DMEM medium containing 10% DMSO. The suspension was immediately frozen at −20 ◦ C for 20 min followed by 1 h storage at −80 ◦ C before the storage in liquid nitrogen.
2.2.3.
Thawing of cryopreserved rat hepatocytes
Vials with cryopreserved rat hepatocytes were thawed quickly in a 37 ◦ C water bath. Immediately after thawing, the contents of the vial were emptied into 37 ◦ C DMEM. After centrifugation (52 × g, 3 min, room temperature), the pellet was resuspended in 3 mL/vial of Percoll solution (30% in HBSS, 4 ◦ C) and centrifuged at 115 × g for 5 min at 4 ◦ C. The pellet was resuspended and washed twice in 37 ◦ C DMEM (centrifugation at 52 × g, 3 min, room temperature). The post-thawed viability was determined using the trypan blue exclusion method and was between 63% and 95%.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
2.2.4.
Culture of cryopreserved rat hepatocytes
Hepatocytes were suspended in a DMEM solution containing ITS (1%), penicillin (50 U/mL), streptomycin (50 g/mL), l-glutamine (2 mmol/L) and dexamethasone (1 mol/L). In addition, the medium contained FBS (5%) for the first 24 h. Three hundred and fifty microliters of hepatocyte suspension (106 viable cells/mL) were plated (day 0) in collagen-coated wells (BioCoat collagen I 24-well plates, Becton Dickinsons ¨ (Brondby, Denmark)). Approximately 3 h after plating the hepatocytes, the medium was aspirated and 350 L of DMEM solution was added. Cultured hepatocytes without collagen overlay were defined as hepatocytes in conventional culture. To prepare hepatocytes in sandwich culture, collagen solution (230 L, 0.05 mg/mL, pH 7.4) was added to the hepatocytes 24 h after the hepatocytes were seeded. After addition of the second layer of collagen, the plates were incubated for 90 min at 37 ◦ C to allow the collagen to gel before the addition of DMEM solution up to 350 L. The culture medium was changed on a daily basis and the cultures were used for transport studies after 2, 3 or 4 days.
2.2.5. [3 H]-Taurocholate uptake and [3 H]-vinblastine efflux in cryopreserved rat hepatocytes in suspension Hepatocytes were suspended in HBSS to obtain a final density of 3 × 106 viable cells/mL for uptake studies in suspension ¨ in a 12-well (Costar, Sigma–Aldrich (Brondby, Denmark)) or a 96-well plate (Nunc (Roskilde, Denmark)). Prior to starting the experiment (three wells per treatment), the hepatocyte suspension was incubated in an incubator at 37 ◦ C for 15 min. The hepatocytes were pre-incubated with either probenecid (1 mmol/L, 5 min; an inhibitor of Oatp), ketoconazole (20 mol/L, 10 min; an inhibitor of Mdr1) or HBSS solution (control wells). For investigation of the effect of the inhibitor concentration on Oatp transport activity three concentrations of probenecid (30 mol/L, 100 mol/L, 1 mmol/L) were used. Uptake studies were initiated by adding substrate – [3 H]-taurocholate (1 mol/L, 1.2 Ci/mL; Oatp substrate) or [3 H]-vinblastine (0.24 mol/L, 0.25 Ci/mL; Mdr1 substrate) – to the hepatocyte suspension. After incubation for 15 min (Oatp) or 30 min (Mdr1), the experiment was stopped by transfer of 100 L of the suspension to 5 mL of ice-cold HBSS, the mixture was immediately centrifuged (2 min, 3345 × g, 4 ◦ C) to separate the hepatocytes from the solution. The cell pellet was treated with 300 L of triton X-100 (0.5%) solution and agitated for 20 min at room temperature to lyse the hepatocytes. An aliquot (200 L) of the cell lysate was used to determine the radioactivity by a liquid scintillation analyzer (Packard TriCarb). BCA protein assay kit was used to determine the protein concentration in the cell lysates according to the manufacturer’s instructions.
2.2.6. [3 H]-Taurocholate uptake and [3 H]-vinblastine accumulation in cryopreserved rat hepatocytes in cultures Hepatocytes (three wells per treatment) cultured for 2, 3 or 4 days in conventional culture or in sandwich culture were rinsed twice and washed with 300 L of HBSS at 37 ◦ C for 10 min. For investigation of Mdr1 either HBSS or calcium and magnesium free HBSS containing 1 mol/L of EGTA was used. After removing the washing solution, the cultures were preincubated with inhibitor as detailed above (Section 2.2.5). For
183
investigation of the effect of inhibitor concentration on Mdr1 transport activity several concentrations of ketoconazole (0.3, 1, 3, 10 and 30 mol/L) were used. Transport studies were initiated by addition of substrates as detailed above (Section 2.2.5). The final incubation volume was 300 L. After incubation for 15 min (Oatp) or 30 min (Mdr1), the transport studies were terminated by removing the incubation solution and washing the wells once with 500 L of ice-cold HBSS. After washing, 300 L of triton X-100 (0.5%) solution was added and the same procedure as for the suspension study was applied (Section 2.2.5).
2.2.7. Oatp1a1, Oatp1a4 and Mdr1 mRNA expression in rat liver, rat spleen, rat lungs and cryopreserved rat hepatocytes in suspension and in culture Cryopreserved hepatocytes in suspension (day 0) or in culture (day 4) were washed twice with KPBS (potassium phosphate buffered saline) and then lysed with RLT buffer. Tissues (liver, spleen and lungs) were obtained from female and male Sprague Dawley rats (150–200 g). Tissue samples were stored in RNAlater containing -mercaptoethanol (10 L/mL) and lysed with RLT buffer. Total RNA was extracted using the RNeasy kit. Purified RNA was then treated with DNase using a DNA-free kit, also according to the manufacturer’s protocol (Invitrogen, Taastrup, Denmark). RNA was reverse transcribed with TaqMan RT-Reagent according to the manufactures protocol (Applied Biosystems, Naerum, Denmark). In brief, 1 g of total RNA was transcribed in a 100 L reaction containing 5.5 mmol/L MgCl2 and random hexamers on a PTC-200 DNA Engine Thermal Cycler (VWR international, Albertslund, Denmark). The program consisted of 10 min annealing at 25 ◦ C, 30 min reverse transcription at 48 ◦ C and 5 min inactivation at 95 ◦ C. The cDNA was quantified using the SYBR GREEN PCR Master Mix kit (Applied Biosystems, Naerum, Denmark). Each reaction contained 2.5 L cDNA of the 100 L RT-product, 300 nmol/L forward and reverse primers, 2.5 mmol/L MgCl2 , 12.5 L master mix and 7 L water in a total volume of 25 L. PCR amplification was run in a 96-well plate format on an iCycler Thermal Cycler equipped with iCycler Optical System (BioRad, Hercules, CA, USA). The program set-up was 10 min at 95 ◦ C, 40 cycles of 15 s at 95 ◦ C/1 min at 60 ◦ C. A melting curve was obtained to verify the measured signal and the product was run on a 2.5% agarose gel to verify the presence of only one amplified band. Quantification was performed as follows: using the iCycler data analysis software (BioRad), the threshold cycle (CT ) was determined for each sample. CT was defined as the cycle at which the level of fluorescence increased linearly. Each sample was run in two reactions, one with the primer set of interest and one with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer set. The mRNA level of the target gene was subsequently expressed as a fraction of the GAPDH level. Primers were designed using DNA-star software package (DNASTAR Inc., Madison, USA) and scrutinized to minimize secondary structures, self-complementarity, optimal melting temperature, etc. All primers were analysed using BLASTn to ensure primer specificity for the gene of interest (www.ncbi.nlm.nih.gov/BLAST/). Primers used were Mdr1a sense (NM 133401): 5 -GGAGGCTTGCAACCAGCATTC-3 , anti-sense: 5 -CTGTTCTGCCGCTGGATTTC-3 . Mdr1b sense (NM 012623): 5 -CCTGGAGGGCGTGGTCAGTATC-3 , anti-
184
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
sense: 5 -GGCGATCGTGGTGGCAAAC-3 . Oatp1a1 sense (NM 17111): 5 -CAACGCAAGACCCAGCAGAATG-3 , antisense: 5 -GCCAGCAACCTTCCCCATTTC-3 . Oatp1a4 sense (NM 18777754): 5 -TCAGGGGGCTTCAATGGCTTAG-3 , anti sense: 5 -GCAATGGGGCATGCACAATTAA-3 .
2.2.8. Mdr protein expression in rat liver and cryopreserved rat hepatocytes in culture Liver tissue from adult Sprague Dawley rats (Harlan Netherlands, Horst, the Netherlands) was snap-frozen on dry ice. Twenty-micrometer sections were cut and processed for immunohistochemistry. Cryopreserved rat hepatocytes were cultured according to Section 2.2.4 with a slight modification: the hepatocytes were cultured on collagen-coated glass ¨ coverslips in a 24-well plate (Costar, Sigma–Aldrich (Brondby, Denmark)). The coverslips were coated with 6 g collagen/cm2 using a collagen type I solution pH 7.4 (incubation at 37 ◦ C, 1 h on a rotator followed by air drying overnight). After 4 days in culture, the hepatocytes on the coverslips were fixed by immersing the coverslips in cold acetone for 10 s. Liver sections were fixed in acetone immediately before use. The samples were incubated in blocking buffer (KPBS (0.1 mol/L) containing 0.1% Tween 20 and 1% BSA) for 10 min at room temperature. Blocking buffer was removed and samples were incubated with primary antibody (mouse anti-human Pglycoprotein antibody [C219]) diluted (1:20) in blocking buffer overnight at 4 ◦ C. Primary antibody was omitted in control slides and coverslips. Samples were washed three times with blocking buffer for 5 min and subsequently incubated in a mixture of secondary antibody (alexa fluor 488-conjugated goat anti-mouse IgG (1:250)) and Hoechst 33342 (1 g/mL) diluted in blocking buffer for 2 h at room temperature. Samples were washed three times with distilled water for 5 min and mounted in Fluoromount (Dako). Immunofluorescence was analysed using a Nikon Eclipse 1000 fluorescence microscope and a ProgRes C14 camera (Jenoptik).
2.2.9.
Data analysis
Results from the transport studies were reported as pmol and normalized to the protein content. The radioactivity was converted from counts per minute to pmol using the specific activity of the radioactive compound. For Mdr1 transport activity the results were reported as fold increase in [3 H]-vinblastine accumulation in the presence of inhibitor (ketoconazole) calculated using results obtained with calcium and magnesium free buffer. The following formula was used:
Fig. 1 – Effect of culture time on the uptake of [3 H]-taurocholate using cryopreserved hepatocytes in suspension, in conventional culture and in sandwich culture. Using suspension the uptake of [3 H]-taurocholate was determined immediately after thawing (n = 4). In addition, the uptake of taurocholate (pmol/(g protein × 15 min)) was investigated after 2 days (n = 1), 3 days (n = 1) or 4 days (n = 8) in both cultures. The error bars on the columns are the S.E.M.
in sandwich culture (days 2–4) is shown in Fig. 1. The results are presented as uptake of taurocholate after 15 min as it was shown that the uptake was not linear in the range of 0–15 min (data not shown). The highest uptake was seen in suspension treated few hours after thawing. Compared with the initial value in suspension (day 0), the uptake of taurocholate decreased, when hepatocytes were cultured in conventional culture and sandwich culture. After 4 days in culture a significant decrease in uptake was noticed in cryopreserved hepatocytes in conventional culture as well as in sandwich culture. In suspension, the inhibitory effect of probenecid on taurocholate uptake increased from 30 mol/L to 1 mmol/L (Fig. 2). The effect using 30 mol/L of probenecid was not significant. However, 100 mol/L of probenecid (p < 0.05) and 1 mmol/L of
Intracellular substrate+Inhibitor Intracellular substrate−Inhibitor Values >1.0 indicate Mdr1 transport activity. When Student’s t-test was used (n ≥ 3 different experiments consisting of triple determinations) for statistical analysis of the results a p-value <0.05 was considered significant.
3.
Results
3.1. Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression The uptake of taurocholate using cryopreserved hepatocytes in suspension (day 0), in conventional culture (days 2–4) and
Fig. 2 – The uptake of [3 H]-taurocholate in cryopreserved hepatocytes in suspension (day 0, n = 4), in conventional culture (day 4, n = 8) and in sandwich culture (day 4, n = 8). The uptake of taurocholate (pmol/(g protein × 15 min)) was determined in the presence and in the absence of 1 mmol/L of the inhibitor probenecid. The error bars on the columns are the S.E.M. Effect of the inhibitor on taurocholate uptake: *** p < 0.001; ** p < 0.01; * p < 0.05.
185
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
Table 1 – Oatp and Mdr1 mRNA expression in rat tissues and in cryopreserved rat hepatocytes Mean expression of mRNA relative to GAPDH mRNA ± S.E.M.a
Rat tissues or cryopreserved rat hepatocytes
Spleen tissue (n = 3) Lung tissue (n = 3) Liver tissue (n = 3) Hepatocytes in suspension, day 0 (n = 2) Hepatocytes in conventional culture, day 4 (n = 2) Hepatocytes in sandwich culture, day 4 (n = 2) a
Oatp1a1 (×10−6 )
Oatp1a4 (×10−6 )
Mdr1a (×10−3 )
Mdr1b (×10−3 )
3±1 6±4 380 ± 130 360 10 16
39 ± 16 1,800 ± 460 38,000 ± 8,200 24,000 220 240
3.4 ± 0.2 20 ± 4 22 ± 6 Not investigated 250 232
1.9 ± 0.3 93 ± 65 1.6 ± 0.3 Not investigated 654 707
S.E.M. = standard error of the mean.
probenecid significantly inhibited (p < 0.001, 59% inhibition) the uptake of taurocholate using hepatocytes in suspension. After 4 days in both cultures, a significant effect of probenecid (1 mmol/L) was also noticed (conventional culture, p < 0.01, 29% inhibition; sandwich culture, p < 0.05, 29% inhibition). The effect of probenecid was higher on the uptake in hepatocytes in suspension as compared to hepatocytes in culture. In Table 1 the results of the expression Oatp1a1 mRNA and Oatp1a4 mRNA are outlined. Oatp1a1 mRNA and Oatp1a4 mRNA were found at high levels in liver tissue, whereas the levels found in lung and spleen tissues were lower. Using cryopreserved hepatocytes the expression of Oatp1a1 mRNA and Oatp1a4 mRNA was found at high levels in suspension (day 0), which were similar to the levels found in liver tissue. However, low expression of Oatp1a1 mRNA and Oatp1a4 mRNA was found in conventional culture (day 4) and in sandwich culture (day 4).
3.2. Mdr1 transport activity, Mdr1a and Mdr1b mRNA expression and Mdr1 protein expression The accumulation of vinblastine in presence of 20 mol/L ketoconazole as compared to data in absence of ketoconazole was investigated in cryopreserved rat hepatocytes in suspension (day 0), in conventional culture (day 2, 3, 4) and in sandwich culture (day 2, 3, 4). The results (Fig. 3) showed that the intracellular amount of vinblastine was not increased in cryopreserved rat hepatocytes in suspension in presence of the inhibitor ketoconazole. However, vinblastine accumulation increased from day 2 to day 4 in both conventional culture and sandwich culture treated with ketoconazole. The concentration dependent inhibition of the efflux of vinblastine from hepatocytes with ketoconazole was investigated in cryopreserved rat hepatocytes in culture. The results showed that the retention of vinblastine in the hepatocytes increased from 0.3 mmol/L to 10 mmol/L of ketoconazole and levelled off between 10 mmol/L to 30 mmol/L (Fig. 4). In this concentration range, the inhibition of the efflux expressed as an accumulation factor was ranged from a 1.2–fold to a 2.5–fold increase in substrate accumulation for the conventional culture and a 1.2–fold to 2.7–fold increase in substrate accumulation for the sandwich culture. The effect of the inhibitor ketoconazole on day 4 for cultured hepatocytes is illustrated in Fig. 5. A significant increase in intracellular vinblastine accumulation was observed in presence of 20 mmol/L of ketoconazole (conventional culture, p = 10–5 ; sandwich culture, p = 10–6 ). In Table 1 the results of the expression of
Mdr1a mRNA and Mdr1b mRNA are outlined. Mdr1a mRNA and Mdr1b mRNA were detected at higher levels in cultured cryopreserved hepatocytes (day 4) compared to levels found in liver, spleen and lungs. Mdr1a mRNA was higher expressed in lung and liver tissues than in spleen tissue. A higher expression of Mdr1b mRNA was detected in lungs compared with the levels found in spleen and liver. The immunostaining of Mdr is presented in Fig. 6. The Mdr protein was detected in liver sections (positive control) at the membranes between adjacent hepatocytes. The protein was also found in cryopreserved hepatocytes in conventional culture (day 4) and in sandwich culture (day 4); here Mdr was primarily localized at the membranes between the hepatocytes. However, some intracytoplasmic fluorescence was observed in cultured hepatocytes. Omission of the primary antibody resulted in minimal fluorescence. For hepatocytes in conventional culture and in sandwich culture the accumulation of the substrate, vinblastine, was similar in standard buffer and in calcium and magnesium free buffer (see Fig. 7, conventional culture, p = 0.3; sandwich culture, p = 0.7). This suggests that the culture conditions did not allow the formation of tight bile canaliculi.
4.
Discussion
4.1. Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression The present study documented that the uptake of taurocholate into cryopreserved rat hepatocytes in suspension was mediated by transport proteins; the transport activity declined when rat hepatocytes were cultured (Fig. 1). The results from Oatp mRNA investigation correlated with taurocholate update data. mRNA expressions for Oatp1a1 and Oatp1a4 were lower in cryopreserved rat hepatocytes in culture after 4 days as compared to data from cryopreserved rat hepatocytes in suspension (day 0). It is therefore likely that the uptake of taurocholate was related to Oatp transport activity, and that the low uptake into the cultured hepatocytes was caused by a low activity of Oatp as Oatp1a1 and Oatp1a4 are known to mediate the uptake of taurocholate into hepatocytes (Faber et al., 2003). The use of taurocholate, although common in Oatp investigations, has the drawback that it is also a substrate for sodium taurocholate cotransporting polypeptide (Ntcp) and organic anion transporter (Oat), two transport proteins localized at the basolateral membrane. Therefore, transport data based
186
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
only on uptake of taurocholate are not specific. In the present study the inhibition of taurocholate uptake with probenecid rules out the possibility that only Ntcp mediated this uptake as probenecid does not inhibit rat Ntcp (Hagenbuch and Meier, 1994; Sun et al., 2001). The correlation between uptake results and mRNA data suggests that the uptake of taurocholate is mediated by Oatp although the involvement of Oat cannot be ruled out. The collagen top layer used in the sandwich culture was not found to prevent the loss of taurocholate uptake and Oatp mRNA expression. This was in accordance with the reported observations of a general decrease in mRNA expression of basolateral transport proteins, when hepatocytes were cultured for several days in sandwich culture (Luttringer et al., 2002; Richert et al., 2006). Basolateral transport activity was maintained after cryopreservation of rat hepatocytes, as taurocholate uptake into hepatocytes is mediated through basolateral transport proteins. Moreover, it was possible to significantly inhibit the uptake of taurocholate using the inhibitor probenecid. The observation of retained transport activity of the basolateral transport proteins in cryopreserved rat hepatocytes was in accordance with a study of Houle et al. (2003). Houle et al. (2003) investigated three Oatp substrates (taurocholate, estrone-3-sulfate, estradiol-17glucoronide) and found no differences between the transport activities in freshly isolated rat hepatocytes and in cryopreserved rat hepatocytes in suspension, although inhibitors were not used. The present study is, in our knowledge, the first report that documents the uptake of taurocholate in cryopreserved rat hepatocytes using both an Oatp inhibitor (probenecid) and mRNA data for Oatp1a1 and Oatp1a4. The expression of Oatp1a1 mRNA and Oatp1a4 mRNA in suspension was identical to the levels found in liver tissue. However, lower levels of Oatp1a1 mRNA and Oatp1a4 mRNA were found in spleen and in lungs (Table 1). This suggests that the cryopreservation did not affect mRNA expression, as data from rat liver was similar to data from cryopreserved rat hepatocytes. The inhibition of taurocholate (1 mol/L) uptake with probenecid was concentration-dependent and a concentration of 1 mmol/L of probenecid resulted in significant inhibition (ca. 60%) of the uptake of taurocholate in hepatocytes in suspension (Fig. 2). The inhibition of taurocholate uptake in hepatocytes in suspension (day 0) by probenecid, an inhibitor of Oatp and Oat, strongly suggested that part of taurocholate transport into these hepatocytes was mediated through Oatp and/or Oat. The effect of probenecid was much lower (ca. 30% inhibition) on hepatocytes in culture; this suggests a lower transport activity. All uptake data correlated very well with Oatp mRNA results suggesting that Oatp played a role in the uptake of taurocholate in the hepatocytes.
4.2. Mdr1 transport activity, Mdr1a and Mdr1b mRNA expression and Mdr1 protein expression The present study showed that cryopreserved rat hepatocytes regained their canalicular transport activity when hepatocytes were maintained in conventional culture or in sandwich culture for over a 2-day period (Fig. 3). The Mdr1 transport activity
Fig. 3 – Effect of culture time on the effect of ketoconazole on accumulation of [3 H]-vinblastine in cryopreserved hepatocytes in suspension, conventional culture and in sandwich culture. In suspension the transport activity was determined immediately after thawing (n = 1). In cultures, Mdr1 transport activity was investigated after 2 days (n = 1), 3 days (n = 1) and 4 days (n = 5) in the cultures. y-Axis values >1.0 mean that the efflux of [3 H]-vinblastine from cells was inhibited by ketoconazole, a selective inhibitor of Mdr1. If the value on the y-axis ≤ 1.0 no Mdr1 transport activity was observed. The error bars shown on the columns are the S.E.M.
in conventional culture and in sandwich culture was similar. This was confirmed by the Mdr protein expression as determined using immunocytochemistry experiments and Mdr1 mRNA expression using real-time RT-PCR. Mdr1 activity was not found in rat hepatocytes in suspension whether these hepatocytes were freshly isolated (data not shown) or cryopreserved (Fig. 3). No report on the activity of Mdr1 from hepatocytes in suspension is available in the literature. A study by Luttringer et al. (2002) showed that Mdr1a and Mdr1b mRNA levels were below the quantification limit in freshly isolated hepatocytes in suspension and in rat liver. However, when hepatocytes were cultured, mRNA expression for Mdr1b increased significantly (Luttringer et al., 2002). This was similar to data from the present study showing that mRNA expressions for Mdr1a and Mdr1b in cultured rat hepatocytes were higher than that of rat liver (see Table 1). In both sandwich culture and conventional culture, the accumulations of vinblastine in presence of ketoconazole, a known inhibitor of Mdr1 (Achira et al., 1999), were similar for freshly isolated rat hepatocytes (as determined in a single experiment, data not shown) and cryopreserved rat hepatocytes (Figs. 4–6). The set-up of the experiment conducted in this study did not determine the amount of vinblastine effluxed from the hepatocytes. The determination of Mdr1 transport activity was correlated to the accumulation of vinblastine in presence of ketoconazole, a specific inhibitor of Mdr1. This approach has the advantage of not depending on the formation of bile canaliculi. As a consequence the simple conventional culture of hepatocytes was proven to be suitable to investigate the transport of Mdr1 at the canalicular membrane, although no bile canaliculi were formed (Fig. 7). A previous study by Annaert et al. (2001) used biliary excretion index of the fluorescent Mdr1 substrate rhodamine 123 as a measurement of biliary excretion and hence Mdr1 transport activity in freshly isolated rat hepatocytes, which is dependent on functional bile canaliculi. However, tight bile canaliculi may not always be
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
187
Fig. 4 – Effect of inhibitor concentration on the effect of ketoconazole on accumulation of [3 H]-vinblastine in cryopreserved hepatocytes in conventional culture (day 4, n = 3) and in sandwich culture (day 4, n = 3). y-Axis values >1.0 mean that the efflux of [3 H]-vinblastine from cells was inhibited by ketoconazole, a selective inhibitor of Mdr1. If the value on the y-axis ≤ 1.0 no Mdr1 transport activity was observed. The error bars on the graphs are the S.E.M.
Fig. 5 – The effect of the inhibitor ketoconazole on intracellular [3 H]-vinblastine accumulation in cryopreserved hepatocytes in conventional culture (day 4, n = 5) and in sandwich culture (day 4, n = 5). The uptake of vinblastine (pmol/(g protein × 30 min)) was determined in the presence and in the absence of 20 mol/L of ketoconazole. The error bars on the columns are the S.E.M. Effect of ketoconazole on vinblastine accumulation: *** p < 0.001.
Fig. 7 – The effect of pre-incubation with a standard buffer and a calcium/magnesium free buffer on [3 H]-vinblastine accumulation in cryopreserved hepatocytes maintained in conventional culture and in sandwich culture (day 4, n = 8). The error bars on the columns are the S.E.M.
Fig. 6 – Mdr protein expression in rat liver (A) and in cryopreserved hepatocytes cultured in conventional culture (B) and in sandwich culture (C), as detected by the monoclonal anti-Pgp antibody C219. Mdr immunoreactivity is visualized using alexa fluor 488-conjugated goat anti-mouse IgG (green). Hoechst 33342 was used to visualize nuclei (blue). Omission of the primary antibody resulted in absence of fluorescence (inserts). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
188
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
formed in hepatocytes in culture. The use of bile canaliculi accumulation of digoxine has also been recently used by Bi et al. (2006) in cryopreserved human hepatocytes. Using immunocytochemistry, it was found that cryopreserved rat hepatocytes in culture express Mdr (Fig. 6). The antibody detected Mdr1a, Mdr1b and Mdr2. Microscopy evaluation revealed that Mdr was primarily localized at the membranes between adjacent hepatocytes after 4 days in both conventional culture and in sandwich culture. This was consistent with the observation of Mdr localization in liver sections, where Mdr was present exclusively at the membranes. Additionally, intracytoplasmic immunoreactivity for Mdr was observed in cultured hepatocytes. Internalized or newly synthesised Mdr protein may account for this staining. Another explanation could be the intracytoplasmic location of Mdr in the Golgi apparatus, as suggested by Molinari et al. (1998) for a human melanoma cell line. Annaert et al. (2001) found an increase in Mdr protein expression (investigated using western blotting) and an increase of biliary excretion index of rhodamine 123 from day 2 to day 4 using freshly isolated rat hepatocytes in sandwich culture. This observation correlates with the time dependent increase of Mdr1 transport activity from day 2 to day 4 found in the present study with cryopreserved hepatocytes. The real-time RT-PCR data showed that Mdr1a and Mdr1b mRNA were present in cryopreserved rat hepatocytes in both cultures. Similar data were obtained in rat liver used as a positive control. High Mdr1a expression was also obtained in lungs whereas in spleen the results were sparse. Mdr1b mRNA expression was higher in rat lungs compared to liver and spleen. The levels of Mdr1a mRNA and Mdr1b mRNA in conventional culture and in sandwich culture were quantitatively higher than those found in the liver tissue. A possible explanation for this difference could be the use of dexamethasone as an additive in the cell culture medium. Luttringer et al. (2002) have shown that 0.1 or 1 mol/L dexamethasone was able to induce Mdr1a mRNA expression. However, Annaert et al. (2001), in a similar experiment did not observe an increase of the efflux activity of Mdr1 towards rhodamine 123. This suggests that dexamethasone-mediated mRNA overexpression in hepatocytes in culture is not followed by a subsequent increase in protein activity. In addition, the presence of non-parenchymal cells in rat liver contributing to the total liver GAPDH mRNA might also explain part of the difference between cultured hepatocytes and liver mRNA data, as the results are expressed relative to GAPDH mRNA values. The results from the present study showed that cryopreserved rat hepatocytes were useful in the investigation of basolateral transport proteins (Oatp) as well as canalicular transport proteins (Mdr). The biology of transport proteins in liver cells is currently under investigation; their importance in the disposition of orally administered drugs is also being established. The possible interplay between uptake transport proteins, drug metabolising enzymes and finally efflux transport proteins is, in our opinion, a powerful tool for the liver to limit the bioavailability of certain drugs. A modern strategy in in vitro ADME (absorption, distribution, metabolism and excretion) evaluation would need to address the transport alongside the metabolism of drug candidates at an early stage of drug development. The first step in the metabolism
of a specific new drug candidate might well require its uptake into hepatocytes by specific transport proteins. Together with metabolising enzymes, transport proteins regulate the hepatic intracellular concentration of drugs; this suggests that the use of isolated hepatocytes to predict drug kinetic and drug toxicity should be performed in cells that are both metabolic competent and express functional transport proteins.
5.
Conclusions
In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport. Cryopreserved rat hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to cryopreserved rat hepatocytes in culture. The presence of Mdr1 in cryopreserved rat hepatocytes cultured in both the conventional and the sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity. However, in cryopreserved rat hepatocytes in suspension, Mdr1 transport activity was not detected. A comparison with rat liver showed that cryopreserved rat hepatocytes in suspension had Oatp mRNA expression similar to that of rat liver. Mdr1 mRNA was higher in cultured cryopreserved rat hepatocytes as compared to results from rat liver; however, Mdr1 protein expression was qualitatively similar in cultured cryopreserved rat hepatocytes and rat liver.
Acknowledgement The study was supported by H. Lundbeck A/S, Valby, Denmark.
references
Achira, M., Suzuki, H., Ito, K., Sugiyama, Y., 1999. Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P450 3A4. AAPS Pharmsci. 1 (4), E18. Annaert, P.P., Turncliff, R.Z., Booth, C.L., Thakker, D.R., Brouwer, K.L., 2001. P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes. Drug Metab. Dispos. 29, 1277–1283. Annaert, P.P., Brouwer, K.L., 2005. Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes. Drug Metab. Dispos. 33, 388–394. Ayrton, A., Morgan, P., 2001. Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica 31, 469–497. Beringer, P.M., Slaughter, R.L., 2005. Transporters and their impact on drug disposition. Ann. Pharmacother. 39, 1097–1108. Bi, Y., Kazolias, D., Duignan, D.B., 2006. Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab. Dispos. 34, 1658–1665. Chandra, P., Brouwer, K.L., 2004. The complexities of hepatic drug transport: current knowledge and emerging concepts. Pharm. Res. 21, 719–735. Diaz, G.J., 2000. Basolateral and canalicular transport of xenobiotics in the hepatocyte: a review. Cytotechnology 34, 225–235.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
Faber, K.N., Muller, M., Jansen, P.L., 2003. Drug transport proteins in the liver. Adv. Drug Deliv. Rev. 55, 107–124. Hagenbuch, B., Meier, P.J., 1994. Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid transporter. J. Clin. Invest. 93, 1326–1331. Hoffmaster, K.A., Turncliff, R.Z., LeCluyse, E.L., Kim, R.B., Meier, P.J., Brouwer, K.L., 2004. P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide. Pharm. Res. 21, 1294–1302. Houle, R., Raoul, J., Levesque, J.F., Pang, K.S., Nicoll-Griffith, D.A., Silva, J.M., 2003. Retention of transporter activities in cryopreserved, isolated rat hepatocytes. Drug Metab. Dispos. 31, 447–451. Kim, R.B., 2002. Transporters and xenobiotic disposition. Toxicology 181/182, 291–297. Le Bot, M.A., Swirsky-Simon, H., Kernaleguen, D., Riche, C., 1994. P-glycoprotein expression and function in rat hepatocytes in culture. Biochem. Pharmacol. 47, 2302–2306. Le Cam, A., Guillouzo, A., Freychet, P., 1976. Ultrastructual and biochemical studies of isolated adult rat hepatocytes prepared under hypoxic conditions, cryopreservation of hepatocytes. Exp. Cell Res. 98, 382–395. LeCluyse, E., Bullock, P., Parkinson, A., Hochman, J., 1996. Cultured rat hepatocytes. In: Borchard, R.T., Smith, P.L.,
189
Wilson, G. (Eds.), Models for Assessing Drug Absorption and Metabolism. Plenum Press, New York, pp. 121–160. Luttringer, O., Theil, F.P., Lave, T., Wernli-Kuratli, K., Guentert, T.W., de Saizieu, A., 2002. Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes. Biochem. Pharmacol. 64, 1637–1650. Molinari, A., Calcabrini, A., Meschini, S., Stringaro, A., Del Bufalo, D., Cianfriglia, M., Arancia, G., 1998. Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells. Int. J. Cancer 75, 885–893. Richert, L., Liguori, M.J., Abadie, C., Heyd, B., Mantion, G., Halkic, N., Waring, J.F., 2006. Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating. Drug Metab. Dispos. 34, 870–879. Shitara, Y., Li, A.P., Kato, Y., Lu, C., Ito, K., Itoh, T., Sugiyama, Y., 2003. Function of uptake transporters for taurocholate and estradiol 17beta-d-glucuronide in cryopreserved human hepatocytes. Drug Metab. Pharmacokinet. 18, 33–41. Sun, A.Q., Arrese, M.A., Zeng, L., Swaby, I., Zhou, M.M., Suchy, F.J., 2001. The rat liver Na+/bile acid cotransporter: importance of the cytoplasmic tail to function and plasma membrane targeting. J. Biol. Chem. 276, 6825–6833.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes Lene Jørgensen, Johan Van Beek, Søren Lund, Arne Schousboe, Lassina Badolo ∗ Department of Drug Metabolism, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark
a r t i c l e
i n f o
a b s t r a c t
Article history:
Transport proteins control uptake of drugs into the liver (e.g., organic anion transporting
Received 3 July 2006
polypeptide (Oatp)) and excretion of drugs from the liver (e.g., multidrug resistance protein
Received in revised form
1 (Mdr1)). In this study, cryopreserved rat hepatocytes were used to investigate the effect of
5 October 2006
different culture conditions (suspension, conventional culture and sandwich culture) on the
Accepted 1 November 2006
uptake of [3 H]-taurocholate ± probenecid and the efflux of [3 H]-vinblastine ± ketoconazole;
Published on line 15 November 2006
mRNA levels of Oatp1a1, Oatp1a4, Mdr1a and Mdr1b were determined using real-time reverse transcription polymerase chain reaction (RT-PCR) and protein expression of Mdr was
Keywords:
assessed by immunocytochemistry. The uptake of [3 H]-taurocholate was higher in cryopre-
Hepatocytes
served rat hepatocytes maintained in suspension as compared to hepatocytes in culture. A
Cryopreservation
significant time dependent decline in the uptake of [3 H]-taurocholate was noticed from day
Transport proteins
2 to day 4 in conventional and sandwich cultures. [3 H]-taurocholate uptake was significantly
Oatp
reduced using the inhibitor probenecid. Oatp mRNA expression in hepatocytes in suspen-
Mdr1
sion was similar to that of liver, whereas much lower levels were detected in the cultures;
Sandwich culture
this was in accordance with [3 H]-taurocholate uptake results. Mdr1 activity was assessed by
Conventional culture
accumulation of the Mdr1 selective substrate, [3 H]-vinblastine, in hepatocytes using ketoconazole as an inhibitor. The results showed Mdr1 activity in cryopreserved rat hepatocytes in conventional and sandwich cultures. A time dependent increase in Mdr1 activity was noticed from day 2 to day 4. Mdr1 activity was not found using hepatocytes in suspension. Mdr1 mRNA expression was high in cryopreserved hepatocytes from both culture systems. Immunocytochemistry showed the Mdr protein in membranes of hepatocytes in culture as well as in that of hepatocytes in liver sections. In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport activity. Hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to hepatocytes in culture. The presence of Mdr1 in both conventional and sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Drugs given by the most convenient administration route, oral administration, are absorbed through the gastrointestinal tract. Thereafter, they reach the liver via the portal vein
before entering the systemic circulation. For some drugs, transport proteins present in the gastrointestinal tract and in the liver play a significant role in drug absorption and drug bioavailability (Diaz, 2000). Multidrug resistance proteins (Mdr, also known as P-glycoprotein) and organic anion trans-
∗ Corresponding author at: Department of Pharmaceutics and Analytical Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2a, 2100 Copenhagen, Denmark. Tel.: +45 35 30 65 47; fax: +45 35 30 60 10. E-mail address: [email protected] (L. Badolo). 0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.11.003
182
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
porting polypeptides (Oatp) are major members in the group of transport proteins that regulate the disposition of orally administered drugs (Kim, 2002; Beringer and Slaughter, 2005). Oatp participates in the uptake of drugs from the blood into the hepatocytes where they may undergo significant phase I and/or phase II metabolism. Oatp has also been suggested to function as an excreting protein capable of transporting drug and drug metabolites back into the systemic circulation (Chandra and Brouwer, 2004; Beringer and Slaughter, 2005). The interplay between Oatp uptake of drugs into hepatocytes, their subsequent metabolism by hepatic enzymes and further excretion into bile through Mdr efflux system is a powerful detoxification pathway in the body. Besides their presence in the liver and the intestine, Oatp and Mdr are widely distributed in the human body where they play an important role in the disposition and distribution of certain drugs. In the kidneys, Oatp and Mdr1 are involved in renal secretion of xenobiotics; at the blood–brain barrier a range of drugs have limited brain penetration due to Mdr1-mediated efflux. Oatp is also expressed in the blood–brain barrier; this suggests its role in delivery of drugs into the brain and removal of metabolites from the brain (Ayrton and Morgan, 2001; Kim, 2002; Beringer and Slaughter, 2005). Since the important role played by transport proteins in xenobiotic disposition and distribution is now understood, a great body of investigations of their effect on the disposition of new drugs are made by pharmaceutical industries as part of drug discovery screening. Investigations of Oatp activity have mostly been performed in vitro using freshly isolated hepatocytes. There are only very few reports of studies using cryopreserved hepatocytes for the assessment of Oatp transport activity. These studies show that some transport activity is retained in cryopreserved rat and human hepatocytes in suspension (Houle et al., 2003; Shitara et al., 2003). The use of cryopreserved rat hepatocytes for investigation of the functional activity of Mdr1 has not been reported. Moreover, sandwich culture is commonly used to assess the transport activity of Mdr1 and other canalicular transport proteins in freshly isolated rat and human hepatocytes by measurements of canalicular accumulation (Annaert et al., 2001; Hoffmaster et al., 2004; Annaert and Brouwer, 2005). The function of Mdr1 using freshly isolated rat hepatocytes has been demonstrated in conventional culture in a single report (Le Bot et al., 1994). However, this study assessed the transport activity as a decline in doxorubicin accumulation over time in culture as an indication of increased efflux of doxorubicin by Mdr1. The present study is the first, to our knowledge, to investigate the transport activity of Oatp and Mdr1 using cryopreserved rat hepatocytes in suspension, in conventional culture and in sandwich culture by the use of both substrates and inhibitors. Additionally, the protein expression and mRNA expression were investigated. A recent paper published by Bi et al. (2006) investigated MDR1 protein in cryopreserved human hepatocytes. The presence of functional transport proteins in cryopreserved rat hepatocytes will make it possible to investigate these transport proteins for a number of scientists with limited access to freshly isolated hepatocytes. It also allows standardising these studies by using cells from same donors in several experiments and will reduce considerably the number of animals used in these studies.
2.
Materials and methods
2.1.
Materials
Dexamethasone, dimethyl sulfoxide (DMSO), ehthyleneglycolbis(b-amino ethylether)-N,N,N ,N -tetraacetic acid (EGTA), KCl, ketoconazole, Percoll, probenecid, rat tail collagen (type I), triton X-100, trypan blue and -mercaptoethanol were ¨ obtained from Sigma–Aldrich (Brondby, Denmark). Calcium and magnesium free Hanks’ balanced salt solution (HBSS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS), ITS (insulin (1.0 g/L), transferrin (0.55 g/L), selenium (0.67 g/L)), lglutamine 200 mmol/L, penicillin 10,000 U/mL–streptomycin 10,000 g/mL, RLT buffer, RNAlater, RNeasy kit, alexa fluor 488 goat anti-mouse IgG and hoechst 33342 were obtained from Invitrogen (Taastrup, Denmark). [G-3 H]-Taurocholic acid (1.19 Ci/mmol) was obtained from Perkin-Elmer (Hvidovre, Denmark). [G-3 H]-Vinblastine sulfate (6.7 Ci/mmol) was ¨ Denmark). BCA proobtained from GE Healthcare (Hillerod, tein assay kit and vinblastine sulfate were obtained from Bie ¨ & Berntsen (Rodovre, Denmark). DNA-free kit was obtained from Ambion (Huntingdon, United Kingdom). SYBR GREEN PCR Master Mix kit and TaqMan RT-Reagent were obtained from Applied Biosystems (Naerum, Denmark). Bovine serum albumin (BSA) was obtained from Roche (Hvidovre, Denmark). Mouse anti-human P-glycoprotein antibody [C219] was obtained from Abcam (Cambridge, United Kingdom). Tween 20 was obtained from Merck-Schuchardt (Glostrup, Denmark).
2.2.
Methods
2.2.1.
Isolation of rat hepatocytes
Hepatocytes were isolated from female and male Sprague Dawley rats (150–200 g) obtained from Charles River Laboratories (Sulzfeld, Germany). The isolation was performed according to the two-step perfusion method described by LeCluyse et al. (1996). Isolated hepatocytes were used after cryopreservation.
2.2.2.
Cryopreservation of rat hepatocytes
Rat hepatocytes were cryopreserved as described by Le Cam et al. (1976). Isolated hepatocytes were suspended in DMEM medium containing 10% DMSO. The suspension was immediately frozen at −20 ◦ C for 20 min followed by 1 h storage at −80 ◦ C before the storage in liquid nitrogen.
2.2.3.
Thawing of cryopreserved rat hepatocytes
Vials with cryopreserved rat hepatocytes were thawed quickly in a 37 ◦ C water bath. Immediately after thawing, the contents of the vial were emptied into 37 ◦ C DMEM. After centrifugation (52 × g, 3 min, room temperature), the pellet was resuspended in 3 mL/vial of Percoll solution (30% in HBSS, 4 ◦ C) and centrifuged at 115 × g for 5 min at 4 ◦ C. The pellet was resuspended and washed twice in 37 ◦ C DMEM (centrifugation at 52 × g, 3 min, room temperature). The post-thawed viability was determined using the trypan blue exclusion method and was between 63% and 95%.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
2.2.4.
Culture of cryopreserved rat hepatocytes
Hepatocytes were suspended in a DMEM solution containing ITS (1%), penicillin (50 U/mL), streptomycin (50 g/mL), l-glutamine (2 mmol/L) and dexamethasone (1 mol/L). In addition, the medium contained FBS (5%) for the first 24 h. Three hundred and fifty microliters of hepatocyte suspension (106 viable cells/mL) were plated (day 0) in collagen-coated wells (BioCoat collagen I 24-well plates, Becton Dickinsons ¨ (Brondby, Denmark)). Approximately 3 h after plating the hepatocytes, the medium was aspirated and 350 L of DMEM solution was added. Cultured hepatocytes without collagen overlay were defined as hepatocytes in conventional culture. To prepare hepatocytes in sandwich culture, collagen solution (230 L, 0.05 mg/mL, pH 7.4) was added to the hepatocytes 24 h after the hepatocytes were seeded. After addition of the second layer of collagen, the plates were incubated for 90 min at 37 ◦ C to allow the collagen to gel before the addition of DMEM solution up to 350 L. The culture medium was changed on a daily basis and the cultures were used for transport studies after 2, 3 or 4 days.
2.2.5. [3 H]-Taurocholate uptake and [3 H]-vinblastine efflux in cryopreserved rat hepatocytes in suspension Hepatocytes were suspended in HBSS to obtain a final density of 3 × 106 viable cells/mL for uptake studies in suspension ¨ in a 12-well (Costar, Sigma–Aldrich (Brondby, Denmark)) or a 96-well plate (Nunc (Roskilde, Denmark)). Prior to starting the experiment (three wells per treatment), the hepatocyte suspension was incubated in an incubator at 37 ◦ C for 15 min. The hepatocytes were pre-incubated with either probenecid (1 mmol/L, 5 min; an inhibitor of Oatp), ketoconazole (20 mol/L, 10 min; an inhibitor of Mdr1) or HBSS solution (control wells). For investigation of the effect of the inhibitor concentration on Oatp transport activity three concentrations of probenecid (30 mol/L, 100 mol/L, 1 mmol/L) were used. Uptake studies were initiated by adding substrate – [3 H]-taurocholate (1 mol/L, 1.2 Ci/mL; Oatp substrate) or [3 H]-vinblastine (0.24 mol/L, 0.25 Ci/mL; Mdr1 substrate) – to the hepatocyte suspension. After incubation for 15 min (Oatp) or 30 min (Mdr1), the experiment was stopped by transfer of 100 L of the suspension to 5 mL of ice-cold HBSS, the mixture was immediately centrifuged (2 min, 3345 × g, 4 ◦ C) to separate the hepatocytes from the solution. The cell pellet was treated with 300 L of triton X-100 (0.5%) solution and agitated for 20 min at room temperature to lyse the hepatocytes. An aliquot (200 L) of the cell lysate was used to determine the radioactivity by a liquid scintillation analyzer (Packard TriCarb). BCA protein assay kit was used to determine the protein concentration in the cell lysates according to the manufacturer’s instructions.
2.2.6. [3 H]-Taurocholate uptake and [3 H]-vinblastine accumulation in cryopreserved rat hepatocytes in cultures Hepatocytes (three wells per treatment) cultured for 2, 3 or 4 days in conventional culture or in sandwich culture were rinsed twice and washed with 300 L of HBSS at 37 ◦ C for 10 min. For investigation of Mdr1 either HBSS or calcium and magnesium free HBSS containing 1 mol/L of EGTA was used. After removing the washing solution, the cultures were preincubated with inhibitor as detailed above (Section 2.2.5). For
183
investigation of the effect of inhibitor concentration on Mdr1 transport activity several concentrations of ketoconazole (0.3, 1, 3, 10 and 30 mol/L) were used. Transport studies were initiated by addition of substrates as detailed above (Section 2.2.5). The final incubation volume was 300 L. After incubation for 15 min (Oatp) or 30 min (Mdr1), the transport studies were terminated by removing the incubation solution and washing the wells once with 500 L of ice-cold HBSS. After washing, 300 L of triton X-100 (0.5%) solution was added and the same procedure as for the suspension study was applied (Section 2.2.5).
2.2.7. Oatp1a1, Oatp1a4 and Mdr1 mRNA expression in rat liver, rat spleen, rat lungs and cryopreserved rat hepatocytes in suspension and in culture Cryopreserved hepatocytes in suspension (day 0) or in culture (day 4) were washed twice with KPBS (potassium phosphate buffered saline) and then lysed with RLT buffer. Tissues (liver, spleen and lungs) were obtained from female and male Sprague Dawley rats (150–200 g). Tissue samples were stored in RNAlater containing -mercaptoethanol (10 L/mL) and lysed with RLT buffer. Total RNA was extracted using the RNeasy kit. Purified RNA was then treated with DNase using a DNA-free kit, also according to the manufacturer’s protocol (Invitrogen, Taastrup, Denmark). RNA was reverse transcribed with TaqMan RT-Reagent according to the manufactures protocol (Applied Biosystems, Naerum, Denmark). In brief, 1 g of total RNA was transcribed in a 100 L reaction containing 5.5 mmol/L MgCl2 and random hexamers on a PTC-200 DNA Engine Thermal Cycler (VWR international, Albertslund, Denmark). The program consisted of 10 min annealing at 25 ◦ C, 30 min reverse transcription at 48 ◦ C and 5 min inactivation at 95 ◦ C. The cDNA was quantified using the SYBR GREEN PCR Master Mix kit (Applied Biosystems, Naerum, Denmark). Each reaction contained 2.5 L cDNA of the 100 L RT-product, 300 nmol/L forward and reverse primers, 2.5 mmol/L MgCl2 , 12.5 L master mix and 7 L water in a total volume of 25 L. PCR amplification was run in a 96-well plate format on an iCycler Thermal Cycler equipped with iCycler Optical System (BioRad, Hercules, CA, USA). The program set-up was 10 min at 95 ◦ C, 40 cycles of 15 s at 95 ◦ C/1 min at 60 ◦ C. A melting curve was obtained to verify the measured signal and the product was run on a 2.5% agarose gel to verify the presence of only one amplified band. Quantification was performed as follows: using the iCycler data analysis software (BioRad), the threshold cycle (CT ) was determined for each sample. CT was defined as the cycle at which the level of fluorescence increased linearly. Each sample was run in two reactions, one with the primer set of interest and one with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer set. The mRNA level of the target gene was subsequently expressed as a fraction of the GAPDH level. Primers were designed using DNA-star software package (DNASTAR Inc., Madison, USA) and scrutinized to minimize secondary structures, self-complementarity, optimal melting temperature, etc. All primers were analysed using BLASTn to ensure primer specificity for the gene of interest (www.ncbi.nlm.nih.gov/BLAST/). Primers used were Mdr1a sense (NM 133401): 5 -GGAGGCTTGCAACCAGCATTC-3 , anti-sense: 5 -CTGTTCTGCCGCTGGATTTC-3 . Mdr1b sense (NM 012623): 5 -CCTGGAGGGCGTGGTCAGTATC-3 , anti-
184
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
sense: 5 -GGCGATCGTGGTGGCAAAC-3 . Oatp1a1 sense (NM 17111): 5 -CAACGCAAGACCCAGCAGAATG-3 , antisense: 5 -GCCAGCAACCTTCCCCATTTC-3 . Oatp1a4 sense (NM 18777754): 5 -TCAGGGGGCTTCAATGGCTTAG-3 , anti sense: 5 -GCAATGGGGCATGCACAATTAA-3 .
2.2.8. Mdr protein expression in rat liver and cryopreserved rat hepatocytes in culture Liver tissue from adult Sprague Dawley rats (Harlan Netherlands, Horst, the Netherlands) was snap-frozen on dry ice. Twenty-micrometer sections were cut and processed for immunohistochemistry. Cryopreserved rat hepatocytes were cultured according to Section 2.2.4 with a slight modification: the hepatocytes were cultured on collagen-coated glass ¨ coverslips in a 24-well plate (Costar, Sigma–Aldrich (Brondby, Denmark)). The coverslips were coated with 6 g collagen/cm2 using a collagen type I solution pH 7.4 (incubation at 37 ◦ C, 1 h on a rotator followed by air drying overnight). After 4 days in culture, the hepatocytes on the coverslips were fixed by immersing the coverslips in cold acetone for 10 s. Liver sections were fixed in acetone immediately before use. The samples were incubated in blocking buffer (KPBS (0.1 mol/L) containing 0.1% Tween 20 and 1% BSA) for 10 min at room temperature. Blocking buffer was removed and samples were incubated with primary antibody (mouse anti-human Pglycoprotein antibody [C219]) diluted (1:20) in blocking buffer overnight at 4 ◦ C. Primary antibody was omitted in control slides and coverslips. Samples were washed three times with blocking buffer for 5 min and subsequently incubated in a mixture of secondary antibody (alexa fluor 488-conjugated goat anti-mouse IgG (1:250)) and Hoechst 33342 (1 g/mL) diluted in blocking buffer for 2 h at room temperature. Samples were washed three times with distilled water for 5 min and mounted in Fluoromount (Dako). Immunofluorescence was analysed using a Nikon Eclipse 1000 fluorescence microscope and a ProgRes C14 camera (Jenoptik).
2.2.9.
Data analysis
Results from the transport studies were reported as pmol and normalized to the protein content. The radioactivity was converted from counts per minute to pmol using the specific activity of the radioactive compound. For Mdr1 transport activity the results were reported as fold increase in [3 H]-vinblastine accumulation in the presence of inhibitor (ketoconazole) calculated using results obtained with calcium and magnesium free buffer. The following formula was used:
Fig. 1 – Effect of culture time on the uptake of [3 H]-taurocholate using cryopreserved hepatocytes in suspension, in conventional culture and in sandwich culture. Using suspension the uptake of [3 H]-taurocholate was determined immediately after thawing (n = 4). In addition, the uptake of taurocholate (pmol/(g protein × 15 min)) was investigated after 2 days (n = 1), 3 days (n = 1) or 4 days (n = 8) in both cultures. The error bars on the columns are the S.E.M.
in sandwich culture (days 2–4) is shown in Fig. 1. The results are presented as uptake of taurocholate after 15 min as it was shown that the uptake was not linear in the range of 0–15 min (data not shown). The highest uptake was seen in suspension treated few hours after thawing. Compared with the initial value in suspension (day 0), the uptake of taurocholate decreased, when hepatocytes were cultured in conventional culture and sandwich culture. After 4 days in culture a significant decrease in uptake was noticed in cryopreserved hepatocytes in conventional culture as well as in sandwich culture. In suspension, the inhibitory effect of probenecid on taurocholate uptake increased from 30 mol/L to 1 mmol/L (Fig. 2). The effect using 30 mol/L of probenecid was not significant. However, 100 mol/L of probenecid (p < 0.05) and 1 mmol/L of
Intracellular substrate+Inhibitor Intracellular substrate−Inhibitor Values >1.0 indicate Mdr1 transport activity. When Student’s t-test was used (n ≥ 3 different experiments consisting of triple determinations) for statistical analysis of the results a p-value <0.05 was considered significant.
3.
Results
3.1. Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression The uptake of taurocholate using cryopreserved hepatocytes in suspension (day 0), in conventional culture (days 2–4) and
Fig. 2 – The uptake of [3 H]-taurocholate in cryopreserved hepatocytes in suspension (day 0, n = 4), in conventional culture (day 4, n = 8) and in sandwich culture (day 4, n = 8). The uptake of taurocholate (pmol/(g protein × 15 min)) was determined in the presence and in the absence of 1 mmol/L of the inhibitor probenecid. The error bars on the columns are the S.E.M. Effect of the inhibitor on taurocholate uptake: *** p < 0.001; ** p < 0.01; * p < 0.05.
185
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
Table 1 – Oatp and Mdr1 mRNA expression in rat tissues and in cryopreserved rat hepatocytes Mean expression of mRNA relative to GAPDH mRNA ± S.E.M.a
Rat tissues or cryopreserved rat hepatocytes
Spleen tissue (n = 3) Lung tissue (n = 3) Liver tissue (n = 3) Hepatocytes in suspension, day 0 (n = 2) Hepatocytes in conventional culture, day 4 (n = 2) Hepatocytes in sandwich culture, day 4 (n = 2) a
Oatp1a1 (×10−6 )
Oatp1a4 (×10−6 )
Mdr1a (×10−3 )
Mdr1b (×10−3 )
3±1 6±4 380 ± 130 360 10 16
39 ± 16 1,800 ± 460 38,000 ± 8,200 24,000 220 240
3.4 ± 0.2 20 ± 4 22 ± 6 Not investigated 250 232
1.9 ± 0.3 93 ± 65 1.6 ± 0.3 Not investigated 654 707
S.E.M. = standard error of the mean.
probenecid significantly inhibited (p < 0.001, 59% inhibition) the uptake of taurocholate using hepatocytes in suspension. After 4 days in both cultures, a significant effect of probenecid (1 mmol/L) was also noticed (conventional culture, p < 0.01, 29% inhibition; sandwich culture, p < 0.05, 29% inhibition). The effect of probenecid was higher on the uptake in hepatocytes in suspension as compared to hepatocytes in culture. In Table 1 the results of the expression Oatp1a1 mRNA and Oatp1a4 mRNA are outlined. Oatp1a1 mRNA and Oatp1a4 mRNA were found at high levels in liver tissue, whereas the levels found in lung and spleen tissues were lower. Using cryopreserved hepatocytes the expression of Oatp1a1 mRNA and Oatp1a4 mRNA was found at high levels in suspension (day 0), which were similar to the levels found in liver tissue. However, low expression of Oatp1a1 mRNA and Oatp1a4 mRNA was found in conventional culture (day 4) and in sandwich culture (day 4).
3.2. Mdr1 transport activity, Mdr1a and Mdr1b mRNA expression and Mdr1 protein expression The accumulation of vinblastine in presence of 20 mol/L ketoconazole as compared to data in absence of ketoconazole was investigated in cryopreserved rat hepatocytes in suspension (day 0), in conventional culture (day 2, 3, 4) and in sandwich culture (day 2, 3, 4). The results (Fig. 3) showed that the intracellular amount of vinblastine was not increased in cryopreserved rat hepatocytes in suspension in presence of the inhibitor ketoconazole. However, vinblastine accumulation increased from day 2 to day 4 in both conventional culture and sandwich culture treated with ketoconazole. The concentration dependent inhibition of the efflux of vinblastine from hepatocytes with ketoconazole was investigated in cryopreserved rat hepatocytes in culture. The results showed that the retention of vinblastine in the hepatocytes increased from 0.3 mmol/L to 10 mmol/L of ketoconazole and levelled off between 10 mmol/L to 30 mmol/L (Fig. 4). In this concentration range, the inhibition of the efflux expressed as an accumulation factor was ranged from a 1.2–fold to a 2.5–fold increase in substrate accumulation for the conventional culture and a 1.2–fold to 2.7–fold increase in substrate accumulation for the sandwich culture. The effect of the inhibitor ketoconazole on day 4 for cultured hepatocytes is illustrated in Fig. 5. A significant increase in intracellular vinblastine accumulation was observed in presence of 20 mmol/L of ketoconazole (conventional culture, p = 10–5 ; sandwich culture, p = 10–6 ). In Table 1 the results of the expression of
Mdr1a mRNA and Mdr1b mRNA are outlined. Mdr1a mRNA and Mdr1b mRNA were detected at higher levels in cultured cryopreserved hepatocytes (day 4) compared to levels found in liver, spleen and lungs. Mdr1a mRNA was higher expressed in lung and liver tissues than in spleen tissue. A higher expression of Mdr1b mRNA was detected in lungs compared with the levels found in spleen and liver. The immunostaining of Mdr is presented in Fig. 6. The Mdr protein was detected in liver sections (positive control) at the membranes between adjacent hepatocytes. The protein was also found in cryopreserved hepatocytes in conventional culture (day 4) and in sandwich culture (day 4); here Mdr was primarily localized at the membranes between the hepatocytes. However, some intracytoplasmic fluorescence was observed in cultured hepatocytes. Omission of the primary antibody resulted in minimal fluorescence. For hepatocytes in conventional culture and in sandwich culture the accumulation of the substrate, vinblastine, was similar in standard buffer and in calcium and magnesium free buffer (see Fig. 7, conventional culture, p = 0.3; sandwich culture, p = 0.7). This suggests that the culture conditions did not allow the formation of tight bile canaliculi.
4.
Discussion
4.1. Taurocholate uptake and Oatp1a1 and Oatp1a4 mRNA expression The present study documented that the uptake of taurocholate into cryopreserved rat hepatocytes in suspension was mediated by transport proteins; the transport activity declined when rat hepatocytes were cultured (Fig. 1). The results from Oatp mRNA investigation correlated with taurocholate update data. mRNA expressions for Oatp1a1 and Oatp1a4 were lower in cryopreserved rat hepatocytes in culture after 4 days as compared to data from cryopreserved rat hepatocytes in suspension (day 0). It is therefore likely that the uptake of taurocholate was related to Oatp transport activity, and that the low uptake into the cultured hepatocytes was caused by a low activity of Oatp as Oatp1a1 and Oatp1a4 are known to mediate the uptake of taurocholate into hepatocytes (Faber et al., 2003). The use of taurocholate, although common in Oatp investigations, has the drawback that it is also a substrate for sodium taurocholate cotransporting polypeptide (Ntcp) and organic anion transporter (Oat), two transport proteins localized at the basolateral membrane. Therefore, transport data based
186
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
only on uptake of taurocholate are not specific. In the present study the inhibition of taurocholate uptake with probenecid rules out the possibility that only Ntcp mediated this uptake as probenecid does not inhibit rat Ntcp (Hagenbuch and Meier, 1994; Sun et al., 2001). The correlation between uptake results and mRNA data suggests that the uptake of taurocholate is mediated by Oatp although the involvement of Oat cannot be ruled out. The collagen top layer used in the sandwich culture was not found to prevent the loss of taurocholate uptake and Oatp mRNA expression. This was in accordance with the reported observations of a general decrease in mRNA expression of basolateral transport proteins, when hepatocytes were cultured for several days in sandwich culture (Luttringer et al., 2002; Richert et al., 2006). Basolateral transport activity was maintained after cryopreservation of rat hepatocytes, as taurocholate uptake into hepatocytes is mediated through basolateral transport proteins. Moreover, it was possible to significantly inhibit the uptake of taurocholate using the inhibitor probenecid. The observation of retained transport activity of the basolateral transport proteins in cryopreserved rat hepatocytes was in accordance with a study of Houle et al. (2003). Houle et al. (2003) investigated three Oatp substrates (taurocholate, estrone-3-sulfate, estradiol-17glucoronide) and found no differences between the transport activities in freshly isolated rat hepatocytes and in cryopreserved rat hepatocytes in suspension, although inhibitors were not used. The present study is, in our knowledge, the first report that documents the uptake of taurocholate in cryopreserved rat hepatocytes using both an Oatp inhibitor (probenecid) and mRNA data for Oatp1a1 and Oatp1a4. The expression of Oatp1a1 mRNA and Oatp1a4 mRNA in suspension was identical to the levels found in liver tissue. However, lower levels of Oatp1a1 mRNA and Oatp1a4 mRNA were found in spleen and in lungs (Table 1). This suggests that the cryopreservation did not affect mRNA expression, as data from rat liver was similar to data from cryopreserved rat hepatocytes. The inhibition of taurocholate (1 mol/L) uptake with probenecid was concentration-dependent and a concentration of 1 mmol/L of probenecid resulted in significant inhibition (ca. 60%) of the uptake of taurocholate in hepatocytes in suspension (Fig. 2). The inhibition of taurocholate uptake in hepatocytes in suspension (day 0) by probenecid, an inhibitor of Oatp and Oat, strongly suggested that part of taurocholate transport into these hepatocytes was mediated through Oatp and/or Oat. The effect of probenecid was much lower (ca. 30% inhibition) on hepatocytes in culture; this suggests a lower transport activity. All uptake data correlated very well with Oatp mRNA results suggesting that Oatp played a role in the uptake of taurocholate in the hepatocytes.
4.2. Mdr1 transport activity, Mdr1a and Mdr1b mRNA expression and Mdr1 protein expression The present study showed that cryopreserved rat hepatocytes regained their canalicular transport activity when hepatocytes were maintained in conventional culture or in sandwich culture for over a 2-day period (Fig. 3). The Mdr1 transport activity
Fig. 3 – Effect of culture time on the effect of ketoconazole on accumulation of [3 H]-vinblastine in cryopreserved hepatocytes in suspension, conventional culture and in sandwich culture. In suspension the transport activity was determined immediately after thawing (n = 1). In cultures, Mdr1 transport activity was investigated after 2 days (n = 1), 3 days (n = 1) and 4 days (n = 5) in the cultures. y-Axis values >1.0 mean that the efflux of [3 H]-vinblastine from cells was inhibited by ketoconazole, a selective inhibitor of Mdr1. If the value on the y-axis ≤ 1.0 no Mdr1 transport activity was observed. The error bars shown on the columns are the S.E.M.
in conventional culture and in sandwich culture was similar. This was confirmed by the Mdr protein expression as determined using immunocytochemistry experiments and Mdr1 mRNA expression using real-time RT-PCR. Mdr1 activity was not found in rat hepatocytes in suspension whether these hepatocytes were freshly isolated (data not shown) or cryopreserved (Fig. 3). No report on the activity of Mdr1 from hepatocytes in suspension is available in the literature. A study by Luttringer et al. (2002) showed that Mdr1a and Mdr1b mRNA levels were below the quantification limit in freshly isolated hepatocytes in suspension and in rat liver. However, when hepatocytes were cultured, mRNA expression for Mdr1b increased significantly (Luttringer et al., 2002). This was similar to data from the present study showing that mRNA expressions for Mdr1a and Mdr1b in cultured rat hepatocytes were higher than that of rat liver (see Table 1). In both sandwich culture and conventional culture, the accumulations of vinblastine in presence of ketoconazole, a known inhibitor of Mdr1 (Achira et al., 1999), were similar for freshly isolated rat hepatocytes (as determined in a single experiment, data not shown) and cryopreserved rat hepatocytes (Figs. 4–6). The set-up of the experiment conducted in this study did not determine the amount of vinblastine effluxed from the hepatocytes. The determination of Mdr1 transport activity was correlated to the accumulation of vinblastine in presence of ketoconazole, a specific inhibitor of Mdr1. This approach has the advantage of not depending on the formation of bile canaliculi. As a consequence the simple conventional culture of hepatocytes was proven to be suitable to investigate the transport of Mdr1 at the canalicular membrane, although no bile canaliculi were formed (Fig. 7). A previous study by Annaert et al. (2001) used biliary excretion index of the fluorescent Mdr1 substrate rhodamine 123 as a measurement of biliary excretion and hence Mdr1 transport activity in freshly isolated rat hepatocytes, which is dependent on functional bile canaliculi. However, tight bile canaliculi may not always be
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
187
Fig. 4 – Effect of inhibitor concentration on the effect of ketoconazole on accumulation of [3 H]-vinblastine in cryopreserved hepatocytes in conventional culture (day 4, n = 3) and in sandwich culture (day 4, n = 3). y-Axis values >1.0 mean that the efflux of [3 H]-vinblastine from cells was inhibited by ketoconazole, a selective inhibitor of Mdr1. If the value on the y-axis ≤ 1.0 no Mdr1 transport activity was observed. The error bars on the graphs are the S.E.M.
Fig. 5 – The effect of the inhibitor ketoconazole on intracellular [3 H]-vinblastine accumulation in cryopreserved hepatocytes in conventional culture (day 4, n = 5) and in sandwich culture (day 4, n = 5). The uptake of vinblastine (pmol/(g protein × 30 min)) was determined in the presence and in the absence of 20 mol/L of ketoconazole. The error bars on the columns are the S.E.M. Effect of ketoconazole on vinblastine accumulation: *** p < 0.001.
Fig. 7 – The effect of pre-incubation with a standard buffer and a calcium/magnesium free buffer on [3 H]-vinblastine accumulation in cryopreserved hepatocytes maintained in conventional culture and in sandwich culture (day 4, n = 8). The error bars on the columns are the S.E.M.
Fig. 6 – Mdr protein expression in rat liver (A) and in cryopreserved hepatocytes cultured in conventional culture (B) and in sandwich culture (C), as detected by the monoclonal anti-Pgp antibody C219. Mdr immunoreactivity is visualized using alexa fluor 488-conjugated goat anti-mouse IgG (green). Hoechst 33342 was used to visualize nuclei (blue). Omission of the primary antibody resulted in absence of fluorescence (inserts). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
188
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
formed in hepatocytes in culture. The use of bile canaliculi accumulation of digoxine has also been recently used by Bi et al. (2006) in cryopreserved human hepatocytes. Using immunocytochemistry, it was found that cryopreserved rat hepatocytes in culture express Mdr (Fig. 6). The antibody detected Mdr1a, Mdr1b and Mdr2. Microscopy evaluation revealed that Mdr was primarily localized at the membranes between adjacent hepatocytes after 4 days in both conventional culture and in sandwich culture. This was consistent with the observation of Mdr localization in liver sections, where Mdr was present exclusively at the membranes. Additionally, intracytoplasmic immunoreactivity for Mdr was observed in cultured hepatocytes. Internalized or newly synthesised Mdr protein may account for this staining. Another explanation could be the intracytoplasmic location of Mdr in the Golgi apparatus, as suggested by Molinari et al. (1998) for a human melanoma cell line. Annaert et al. (2001) found an increase in Mdr protein expression (investigated using western blotting) and an increase of biliary excretion index of rhodamine 123 from day 2 to day 4 using freshly isolated rat hepatocytes in sandwich culture. This observation correlates with the time dependent increase of Mdr1 transport activity from day 2 to day 4 found in the present study with cryopreserved hepatocytes. The real-time RT-PCR data showed that Mdr1a and Mdr1b mRNA were present in cryopreserved rat hepatocytes in both cultures. Similar data were obtained in rat liver used as a positive control. High Mdr1a expression was also obtained in lungs whereas in spleen the results were sparse. Mdr1b mRNA expression was higher in rat lungs compared to liver and spleen. The levels of Mdr1a mRNA and Mdr1b mRNA in conventional culture and in sandwich culture were quantitatively higher than those found in the liver tissue. A possible explanation for this difference could be the use of dexamethasone as an additive in the cell culture medium. Luttringer et al. (2002) have shown that 0.1 or 1 mol/L dexamethasone was able to induce Mdr1a mRNA expression. However, Annaert et al. (2001), in a similar experiment did not observe an increase of the efflux activity of Mdr1 towards rhodamine 123. This suggests that dexamethasone-mediated mRNA overexpression in hepatocytes in culture is not followed by a subsequent increase in protein activity. In addition, the presence of non-parenchymal cells in rat liver contributing to the total liver GAPDH mRNA might also explain part of the difference between cultured hepatocytes and liver mRNA data, as the results are expressed relative to GAPDH mRNA values. The results from the present study showed that cryopreserved rat hepatocytes were useful in the investigation of basolateral transport proteins (Oatp) as well as canalicular transport proteins (Mdr). The biology of transport proteins in liver cells is currently under investigation; their importance in the disposition of orally administered drugs is also being established. The possible interplay between uptake transport proteins, drug metabolising enzymes and finally efflux transport proteins is, in our opinion, a powerful tool for the liver to limit the bioavailability of certain drugs. A modern strategy in in vitro ADME (absorption, distribution, metabolism and excretion) evaluation would need to address the transport alongside the metabolism of drug candidates at an early stage of drug development. The first step in the metabolism
of a specific new drug candidate might well require its uptake into hepatocytes by specific transport proteins. Together with metabolising enzymes, transport proteins regulate the hepatic intracellular concentration of drugs; this suggests that the use of isolated hepatocytes to predict drug kinetic and drug toxicity should be performed in cells that are both metabolic competent and express functional transport proteins.
5.
Conclusions
In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport. Cryopreserved rat hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to cryopreserved rat hepatocytes in culture. The presence of Mdr1 in cryopreserved rat hepatocytes cultured in both the conventional and the sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity. However, in cryopreserved rat hepatocytes in suspension, Mdr1 transport activity was not detected. A comparison with rat liver showed that cryopreserved rat hepatocytes in suspension had Oatp mRNA expression similar to that of rat liver. Mdr1 mRNA was higher in cultured cryopreserved rat hepatocytes as compared to results from rat liver; however, Mdr1 protein expression was qualitatively similar in cultured cryopreserved rat hepatocytes and rat liver.
Acknowledgement The study was supported by H. Lundbeck A/S, Valby, Denmark.
references
Achira, M., Suzuki, H., Ito, K., Sugiyama, Y., 1999. Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P450 3A4. AAPS Pharmsci. 1 (4), E18. Annaert, P.P., Turncliff, R.Z., Booth, C.L., Thakker, D.R., Brouwer, K.L., 2001. P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes. Drug Metab. Dispos. 29, 1277–1283. Annaert, P.P., Brouwer, K.L., 2005. Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes. Drug Metab. Dispos. 33, 388–394. Ayrton, A., Morgan, P., 2001. Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica 31, 469–497. Beringer, P.M., Slaughter, R.L., 2005. Transporters and their impact on drug disposition. Ann. Pharmacother. 39, 1097–1108. Bi, Y., Kazolias, D., Duignan, D.B., 2006. Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab. Dispos. 34, 1658–1665. Chandra, P., Brouwer, K.L., 2004. The complexities of hepatic drug transport: current knowledge and emerging concepts. Pharm. Res. 21, 719–735. Diaz, G.J., 2000. Basolateral and canalicular transport of xenobiotics in the hepatocyte: a review. Cytotechnology 34, 225–235.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 181–189
Faber, K.N., Muller, M., Jansen, P.L., 2003. Drug transport proteins in the liver. Adv. Drug Deliv. Rev. 55, 107–124. Hagenbuch, B., Meier, P.J., 1994. Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid transporter. J. Clin. Invest. 93, 1326–1331. Hoffmaster, K.A., Turncliff, R.Z., LeCluyse, E.L., Kim, R.B., Meier, P.J., Brouwer, K.L., 2004. P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide. Pharm. Res. 21, 1294–1302. Houle, R., Raoul, J., Levesque, J.F., Pang, K.S., Nicoll-Griffith, D.A., Silva, J.M., 2003. Retention of transporter activities in cryopreserved, isolated rat hepatocytes. Drug Metab. Dispos. 31, 447–451. Kim, R.B., 2002. Transporters and xenobiotic disposition. Toxicology 181/182, 291–297. Le Bot, M.A., Swirsky-Simon, H., Kernaleguen, D., Riche, C., 1994. P-glycoprotein expression and function in rat hepatocytes in culture. Biochem. Pharmacol. 47, 2302–2306. Le Cam, A., Guillouzo, A., Freychet, P., 1976. Ultrastructual and biochemical studies of isolated adult rat hepatocytes prepared under hypoxic conditions, cryopreservation of hepatocytes. Exp. Cell Res. 98, 382–395. LeCluyse, E., Bullock, P., Parkinson, A., Hochman, J., 1996. Cultured rat hepatocytes. In: Borchard, R.T., Smith, P.L.,
189
Wilson, G. (Eds.), Models for Assessing Drug Absorption and Metabolism. Plenum Press, New York, pp. 121–160. Luttringer, O., Theil, F.P., Lave, T., Wernli-Kuratli, K., Guentert, T.W., de Saizieu, A., 2002. Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes. Biochem. Pharmacol. 64, 1637–1650. Molinari, A., Calcabrini, A., Meschini, S., Stringaro, A., Del Bufalo, D., Cianfriglia, M., Arancia, G., 1998. Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells. Int. J. Cancer 75, 885–893. Richert, L., Liguori, M.J., Abadie, C., Heyd, B., Mantion, G., Halkic, N., Waring, J.F., 2006. Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating. Drug Metab. Dispos. 34, 870–879. Shitara, Y., Li, A.P., Kato, Y., Lu, C., Ito, K., Itoh, T., Sugiyama, Y., 2003. Function of uptake transporters for taurocholate and estradiol 17beta-d-glucuronide in cryopreserved human hepatocytes. Drug Metab. Pharmacokinet. 18, 33–41. Sun, A.Q., Arrese, M.A., Zeng, L., Swaby, I., Zhou, M.M., Suchy, F.J., 2001. The rat liver Na+/bile acid cotransporter: importance of the cytoplasmic tail to function and plasma membrane targeting. J. Biol. Chem. 276, 6825–6833.