Contribution Of OCT1and CYP3A4to The Liver Disposition And Hepatotoxicity Of Pyrrolizidine Alkaloids

Pyrrolizidine alkaloids (PAs) are present in hundreds of plant species, especially the families of Boraginaceae, Conipositae, and Leguminosae. PAs are potentially the most important natural toxicants because of their wide distribution and severe toxicity to animals. About half of the PAs were found to induce hepatotoxicity, genetoxicity, carcinogenicity, and sometimes pneumotoxicity. Toxic PAs are mainly classified into retronecine-type and otonecine-type, among which monocrotaline (MCT) and retrorsine (RTS) are two common hepatotoxic retronecine-type PAs. Most PAs themselves are non-toxic or relatively low-toxic, whereas they become toxic after metabolic activation. It has been demonstrated that the bioactivation of PAs is predominately mediated by cytochrome P450(CYP) enzymes that are mainly located in the liver. Therefore, the liver is the primary target of PAs-induced toxicity and usually experiences the most severe injury. In hepatocytes, CYPs are localized in the endoplasmic reticulum; therefore the influx of PAs is a necessary step for its hepatotoxicity. The liver transport systems may also participate in the liver-specific accumulation of PAs. It has been confirmed that polyspecific organic cation transporters (OCTs) of the SLC22family, including three organic cation transporters (OCT1-OCT3), play an important role in the transport of organic cations, including weak bases and some non-charged compounds. OCT1is highly expressed in rat and human liver and mainly localized in the sinusoidal (basolateral) membrane of hepatocytes. Since some PAs are weak bases and can be partly ionized to organic cations at physiological pH, we deduced that OCTs, especially OCTl, might mediate the transport of some PAs in hepatocytes. The present study investigated the interaction of liver organic cation transporters with four PAs and the role of OCT1in the translocation and toxicity of MCT and RTS.1. Construction of cell model with stable expression of human organic cation transporter1To establish the Madin-Darby canine kidney (MDCK) cell models with stable-expressed wild type and two mutants of human organic cation transporter1(hOCT1). The hOCTl wild-type gene was extracted from human liver, and the two mutants, hOCT1P341L, hOCTlM420del were obtained by site-directed mutagenesis. The plasmids pcDNA3.1(+)-hOCT1, pcDNA3.1(+)-hOCT1P341L, pcDNA3.1(+)-hOCTlM420del were constructed and transfected into MDCK cell line. Several stable transfected clones were obtained by G418screening. The activity of hOCTl was estimated by detecting the accumulation of4-(4-(Dimethylamino)-styryl)-N-methylpyridinium (ASP+, a fluorescent substrate of hOCTl) with or without tetraethylammonium (TEA, an inhibitor/substrate of hOCTl) in monoclones. The mRNA of hOCTl was detected by reverse transcription-polymerase chain reaction (RT-PCR). The function of positive monoclones was confirmed by kinetic studies of hOCT1classical substrates,1-methyl-4-phenylpyridinium (MPP+) and metformin. The RT-PCR results showed high hOCT1mRNA expression in the selected cells comparing with MDCK empty vector cells. The uptake of MPP+and metformin in positive colones was much higher than that in mock cells. The kinetic parameters of metformin in MDCK-hOCTl,-hOCT1P341L,-hOCTlM420dei cells were estimated to be791.5±24.1,779.1±165.3,537.5±62.8(Vmax, pmol/mg protein/min);409.0±55.1,523.2±36.3,913.4±99.1(Km,μmol/L);1.94,1.49,0.59(Clm, Vmax/Km). The developed MDCK-hOCT1,-hOCT1P341L,-hOCTlM420del cells are effective in vitro models for hOCT1substrates and inhibitors screening.2. Interaction of PAs with liver organic cation transportersThe inhibitory effect of four PAs, monocrotaline (MCT), isoline, retrorsine (RTS), senecionine on hOCT1-mediated MPP+uptake was determined in MDCK-hOCT1cells. All the four PAs markedly inhibited the MPP+uptake with the IC50values of5.52μmol/L for MCT,5.35μmol/L for isoline,2.25μmol/L for RTS, and3.50μmol/L for senecionine. The uptake of the four PAs in MDCK-hOCT1and mock cells was also estimated. The uptake of MCT and RTS was significantly higher in MDCK-hOCT1cells than in mock cells, and the uptake in MDCK-hOCT1was significantly inhibited by OCT1inhibitors, suggesting MCT and RTS are substrates of hOCTl. The Km values were25.0±6.7μmol/L,23.6±3.0μmol/L; the Vmax were266.0±63.9pmol/mg protein/min,209.9±69.3pmol/mg protein/min for MCT and RTS respectively. The uptake of isoline and senecionine showed no obvious difference between MDCK-hOCT1and mock cells.To confirm whether other transporters localized in the hepatocytes were involved in the transport of MCT or RTS, MDCK or LLC-PK1cells stably expressing hOCT3, human multidrug and toxin extrusion (hMATE) transporter1, multidrug resistance1(MDR1, P-gp), or breast cancer resistance protein (BCRP) were used to investigate the interaction of MCT and RTS with these transporters. The results showed that the transporters described above showed weak or no significant contribution to the translocation of MCT and RTS.Primary cultured rat hepatocytes (PRCH) were used to investigate the role of OCT1in the liver disposition of MCT and RTS. The inhibitors of OCT1, quinidine,(+)-tetrahydropalmatine ((+)-THP), obviously inhibited the uptake of MCT and RTS in PRCH, and attenuated the viability reduction and LDH release of the PRCH caused by RTS or MCT. The above results suggested OCT1mediates the hepatic uptake of MCT and RTS and may play an important role in MCT or RTS induced-hepatotoxicity.3. Establishment of MDCK cells with single-or double-expression of hOCTl andCYP3A4Cell models with double-, triple-, and quadruple-transfected transporters and enzymes have been recognized as useful tools for the understanding of drug disposition in liver. OCT1and CYP3A4are both predominantly expressed in the liver, and may both participate in the disposition of their common substrates, which may include MCT or RTS. Cell models with stably single-and double-expression of hOCT1and/or CYP3A4were constructed here. The single transfected cells (MDCK-hOCTl, MDCK-pcDNA3.1(+)) were further transfected with pcDNA3.1(+)-Hygro-CYP3A4or pcDNA3.1(+)-Hygro empty vector. Stable clones were selected with media containing700μg/mL G418and400μg/mL hygromycin B, and then the positive single clones were selected based on Western blot and quantitative Real-time PCR. The function of hOCTl in the positive clones was validated by ASP+and TEA (substrate/inhibitor of hOCT1), while the activity of CYP3A4was confirmed by a P450-Glo CYP3A4Assay (Luciferin-IPA, a sensitive substrate of CYP3A4). Four transfectants named MDCK-mock, MDCK-hOCTl, MDCK-CYP3A4, MDCK-hOCTl-CYP3A4were constructed. The uptake of ASP+in MDCK-hOCT1and MDCK-hOCTl-CYP3A4cells were about16-fold of that in mock cells, and the activity of CYP3A4in MDCK-CYP3A4and MDCK-hOCTl-CYP3A4cells was about66-fold of that in mock cells. These results demonstrated that all the transfected cells expressed respective proteins, and the cell lines could be applied to investigate the function of hOCT1and CYP3A4.4. The contribution of hOCTl and CYP3A4in RTS-induced toxicityConsidering that CYP3A4may be responsible for the bioactivation of RTS, and OCT1plays an important role in the liver uptake of RTS, hOCTl and/or CYP3A4single-and double-expressing cells were used to determine the contribution of OCT1and CYP3A4to the toxicity of RTS. Mock, MDCK-hOCTl, MDCK-CYP3A4, MDCK-hOCTl-CYP3A4cells were treated with different concentrations of RTS for different periods. The toxic effect of RTS was measured by morphological analysis, MTT assays, and cell cycle distribution analysis using flow cytometry. The results showed that MDCK-CYP3A4cells showed a time-and concentration-dependent injury after being exposed to RTS, while mock and MDCK-hOCTl cells were not affected, indicating that CYP3A4-mediated metabolism was responsible for the RTS induced toxicity. Furthermore, RTS exhibited a more severe toxicity in the OCT1/CYP3A4double-transfected cells compared to all other cells, suggesting that OCT1also played an important role in RTS induced cytotoxicity. In addition, the following toxic effects of RTS on MDCK-CYP3A4and MDCK-hOCTl-CYP3A4cells were observed:cell and nuclei enlargement, inhibition of cell growth, and the cell cycle analysis showed that RTS induced G2/M phase arrest. The understanding of the mechanism for RTS toxicity will help us develop methods to prevent the liver injury induced by RTS.