Regulation of MRP2/ABCC2 and BSEP/ABCB11 Expression in Sandwich Cultured Human and Rat Hepatocytes Exposed to Inflammatory Cytokines TNF-α, IL-6, and IL-1β

Abstract

In the present study MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly downregulated by TNF-␣, IL-6, or IL-1␤. In contrast to mRNA decrease, which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1␤. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-␣ and IL-6, whereas the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-␣ or IL-1␤. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/ Abcb11 protein in liver at 24 h post-treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 h post-LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep and for the first time demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.Drug transporters facilitate the passage of many drugs across cellular barriers during the process of absorption, distribution, metabolism, and elimination. The hepatobiliary excretion of drugs and drug metabolites is one of the primary elimination routes for endogenous and exogenous compounds from blood circulation (1). Multidrug resistance-associated protein 2 (MRP2/ABCC2) 4 and bile salt export pump (BSEP/ABCB11) are localized mainly on the canalicular membrane of hepatocytes and are responsible for elimination of conjugated bilirubin and bile acids as part of the hepatic detoxification process. The transporters are also simultaneously involved in hepatic excretion of diverse drugs and their metabolites into bile. Consequently, functional disruption of MRP2/ABCC25 and BSEP/ ABCB11 may result in drug-induced liver injury (2), which is a major cause of withdrawal of drugs from the market (3).Pharmacokinetics for numerous drugs has been reported to be significantly affected during inflammation (4). Proinflammatory cytokines, such as tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤) and IL-6 have been shown to regulate the expression of hepatic transporters during inflammation in rodents (5-8). Down-regulation of hepatic bile salt influx transporters such as the sodium-dependent taurocholate transporter (NTCP/SLC10A1) by inflammation has been characterized in both rodents and primary...

Impact of Inflammation on Chlorpromazine-Induced Cytotoxicity and Cholestatic Features in HepaRG Cells

“…Discrepant data have been reported in the effects of proinflammatory cytokines on the main canalicular transporters BSEP and MRP2. Thus, transcripts of these two transporters were not affected by LPS in human liver slices (Elferink et al, 2004); by contrast, they were repressed by IL-1b and IL-6 whereas BSEP protein, unlike MRP2, was induced in sandwich-cultured human hepatocytes (Diao et al, 2010). Such discrepancies are likely due to the use of different experimental models and conditions such as cytokine concentrations.…”

Critical Review of Preclinical Approaches to Investigate Cytochrome P450–Mediated Therapeutic Protein Drug-Drug Interactions and Recommendations for Best Practices: A White Paper

“…In vitro effects of proinflammatory cytokines on the dmd.aspetjournals.org expression of various transporters have also been reported (Petrovic et al, 2007). Three proinflammatory cytokines, IL-6, IL-1, and TNF-a, downregulate hepatic transporter expression in hepatocyte sandwich cultures (Diao et al, 2010). IFN-g and oncostatin M (a member of the IL-6 family) also influenced transporter levels in cultured hepatocytes (Donato et al, 1993;Guillen et al, 1998;Le Vee et al, 2009;Chen et al, 2011).…”

“…The bile salt export pump as well as sinusoidal solute carrier uptake transporters are generally suppressed, whereas ATPbinding cassette drug efflux pumps are either unchanged or are up or downregulated depending on the transporter isoform. These changes are observed at mRNA levels for some transporters, and at protein and activity levels for others (Diao et al, 2010). Currently, the clinical relevance of these changes in transporter levels is unclear.…”

Critical Human Hepatocyte-Based In Vitro Assays for the Evaluation of Adverse Drug Effects

“…It is to be noted that recent research findings have demonstrated that in addition to drug metabolizing enzyme activities, uptake and efflux transporters also play critical roles in the manifestation of adverse drug effects 10,45 . Human hepatocyte assays for the evaluation of uptake and efflux transporters have been established and are being applied towards drug development [46][47][48][49][50] . These transporter assays, when applied in conjunction with the assays described in this chapter, should aid the selection of the most appropriate drug candidates for further drug development.…”