SHORT COMMUNICATION, C Basic & Clinical Pharmacology & Toxicology 2005, 96, 249–250. Printed in Denmark . All rights reserved Copyright C ISSN 1742-7835 Short Communication Expression of MRP2 and MDR1 and Other Hepatic Markers in Hepatocytes in situ and WRL 68 Cells in vitro Dana Cizkova 1 , Jaroslav Mokry 1 , Stanislav Micuda 2 , Jan Osterreicher 3 and Jirina Martinkova 2 1 Department of Histology and Embryology, 2 Department of Pharmacology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, and 3 Department of Radiobiology, Purkyne Military Medical Academy in Hradec Kralove, Czech Republic Human hepatocytes cultured in vitro are broadly used for the study of hepatocytic properties. Primary hepatocyte cultures have several known disadvantages: heterogenicity, rapid loss of the hepatocytic phenotype, limited replicating capacity etc. Therefore, human adult or foetal hepatic cell lines represent suitable models for many different studies (Gutierrez-Ruiz et al. 1994). We performed a comparative study of phenotypic properties of hepatocytes in situ and in vitro with aims to define a difference between a) the foetal and adult liver tissue and b) human foetal hepatocytes in situ and WRL 68 cells in vitro. We chose markers feasible for immunophenotypisation of hepatocytes and their detec- tions were performed in the liver tissue of a 14-week-old and 19-week-old human foetus, in the adult human liver and in foetal human hepatocytes of the WRL 68 cell line. We analysed the expression levels and distribution patterns of MRP2 and MDR1 transporters, intermediate filaments pan-cytokeratin and cytokeratin 18, plasma proteins albu- min and a-foetoprotein and also the specific hepatocyte marker OCH1E5. MRP2 and MDR1 represent ABC trans- porters localised at the canalicular membrane domain of hepatocytes (Hooiveld et al. 2001; König et al. 2003). Formalin-fixed and paraffin-embedded sections were de- waxed and treated to suppress endogenous peroxidase activ- ity and re-establish an original conformation of epitopes. Following the incubation with primary antibody, secondary biotinylated antibodies and streptavidin-horseradish peroxi- dase were used to visualise antigen-binding sites. In several sections, the intensity of immunostaining was enhanced with the use of Catalysed Signal Amplification. Cells har- vested from culture were fixed with acetone and methanol, permeabilised and processed for immunofluorescence using secondary antibody conjugated to Cy3 fluorochrome. MRP2 was weakly expressed in the canalicular mem- Author for correspondence: Dana Cizkova, Department of His- tology and Embryology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Simkova 870, 50038 Hradec Kralove, Czech Republic (fax π420 495 816 376, e-mail cizkovad/lfhk.cu- ni.cz). brane of differentiating hepatocytes in the liver of 14-week- old foetus. In 19-week-old foetus and in the adult, this transporter was detected in canalicular membranes of hepatocytes and interestingly, also in apical membrane of cholangiocytes. The expression pattern of MDR1 was al- most identical. A weak signal appeared in canalicular mem- brane of hepatocytes in the liver of 14-week-old foetus and a stronger one in the older examined foetus and in the adult in canalicular membranes of hepatocytes and in apical membrane of cholangiocytes. In WRL 68 cells, MRP2 and MDR1 immunopositivity was not noticed after 3 days in vitro, their signal appeared after 5–6 days of cultivating and 9 days after the passage these two transporters were fully expressed. An immunoreactivity localised to the plasma- lemma and a weak punctuate signal in the cytoplasm of WRL 68 cells indicated that these hepatocytes cultivated in vitro were still not polarised. We did not find any distinct difference in the expression of pan-cytokeratin and cytoker- atin 18 in all examined liver tissues. These intermediate filaments were strongly detected in the cytoplasm of cholan- giocytes and weakly in hepatocytes. Their immunopositivity in WRL 68 cells showed their filamentous structure. A granular staining pattern of human hepatocyte marker OCH1E5 was observed in the cytoplasm of hepatocytes in the liver of foetuses of both developmental stages as well as in the adult liver and in WRL 68 cells. The intensity of immunostaining of albumin in the cytoplasm of hepatocytes increased with maturation of these elements. On the con- trary, the highest levels of a-foetoprotein expression were detected in 14-week-old and in 19-week-old foetus in the cytoplasm of hepatocytes and an extremely weak signal was visible in the same localization in the adult liver. Albumin and a-foetoprotein were expressed in the cytoplasm of WRL 68 cells. MRP2 expression in the human foetal liver tissue may be a noteworthy finding, because immunohistochemical detec- tions of this transporter in the foetal liver were not studied in detail. The immunopositivity for MRP2 in the adult liver tissue in the apical membrane of cholangiocytes forming interlobular bile ducts represent the other finding, which