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Detection of aflatoxin B1 hepatotoxicity using PXB-cells®
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
Figure 1. Separation and detection of 15 isomeric SCFAs using 0.1% formic acid in water and isopropanol as mobile phases on a sub-2 mm high strength silica chromatographic column (100 mm).
P125 DETECTION OF AFLATOXIN B1 HEPATOTOXICITY USING PXB-CELLS® Chihiro Yamasaki 1, Yuko Ogawa 1, Yutaka Kageyama 1, Seigo Sanoh 2, Yuji Ishida 1,3, Chise Tateno 1, 3. 1 PhoenixBio Co. Ltd., Higashi-Hiroshima, Japan; 2 Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan; 3 Liver Research Project Center, Hiroshima University, Hiroshima, Japan We have generated PXB-mice® (PhoenixBio Co., Ltd., Japan) with humanized livers by transplanting human hepatocytes into cDNA-uPA/SCID mice (Tateno et al., 2015). Approximately 1.5e2 x 108 human hepatocytes (PXBcells®) are obtained from a PXB-mouse by the collagenase perfusion method. Fresh human hepatocytes can attach on cell plates confluently and can be maintained for at least 4 weeks. Since the hepatitis B virus (HBV) infected the PXB-cells efficiently (Ishida et al., 2015), HBV-infected PXB-cells have been used for in vitroefficacy tests of anti-HBV agents. At the last meeting, we showed that PXB-cells cultured on type I collagen-coated plates showed high expression of cytochrome P450 (CYP), UDP-glucuronosyltransferase, and transporter genes for at least 4 weeks and retained CYP3A and transporter activities. In the present study, we tried to detect the toxicity of 0.014-10 mM aflatoxin B1 (AFB1), a mycotoxin metabolized by CYP3A4 and known to be toxic for human hepatocytes, using PXB-cells by WST-1 analysis at 1, 4, and 7 days, and a real-time cell analysis using iCELLigence (ACEA Biosciences Inc., San Diego, CA, USA) for 6 days. These two methods allowed detection of dose-dependent toxicity of AFB1. CYP inhibitors, 1-aminobenzotriazole (ABT) and ketoconazole, were added to AFB1-treated PXB-cells. CYP3A activity decreased in ABT- and ketoconazole-treated PXB-cells. Analysis using iCELLigence showed amelioration of AFB1 toxicity in the ABT- and ketoconazole-treated PXB-cells. These results suggest that iCELLigence is useful for detecting the toxicity of AFB1 and metabolites, and inhibitory effects of CYP inhibitors in PXB-cells. In conclusion, PXB-cells are useful for metabolism and toxicity tests. 1. Tateno et al., Generation of novel chimeric mice with humanized livers by using hemizygous cDNA-uPA/SCID mice. PLoS One. 2015; 10: e0142145 and Ishida et al., Novel robust in vitro hepatitis B virus infection model using fresh human hepatocytes isolated from humanized mice. Am J Path. 2015; 85: 1275-1285. P126 EXPANDED PRIMARY HUMAN HEPATOCYTES ENABLE METABOLISM STUDIES INCLUDING CYP2D6 DEPENDENT PATHWAYS (UPCYTE® HEPATOCYTES) Astrid Noerenberg 1, Nils Runge 1, Werner Schroth 2. 1 upcyte technologies GmbH, Hamburg, Germany; 2 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart, Germany
S63
Primary human hepatocytes have limitations for drug testing due to the low availability of human liver tissue. To address this, we have improved our upcyte technology enabling primary human hepatocytes to proliferate up to 40 doublings whilst still retaining a highly metabolic competent phenotype when cultured at confluence. The resulting cells are called “upcyte hepatocytes” and enable studies of metabolic clearance, hepatotoxicity and drug-drug interactions. Here, we tested upcyte hepatocytes from donor 653-03 and its genetically modified, CYP2D6 expressing derivative (653-03-2D6) for their enzymatic activities of CYP1A2, 2C8, 2B6, 2C19, 2D6, 2C9, 3A4 using a substrate cocktail incubation and Triple Quad MS analysis. In addition 75 ADME related genes were quantified using Fluidigm Taqman array (Biomark). upcyte hepatocytes show high basal activities of phase I enzymes which are inducible in the range of 2 to 23 fold by prototypical inducers rifampicin (CYP3A4, CYP2C8, CYP2C9, CYP2C19) and phenobarbital (CYP2B6) indicating functional PXR/CAR mediated CYP induction. On the level of gene expression, induction of CYP gene expression followed the same pattern as observed based on activities. Overall, there was no difference in the global regulatory gene expression pattern comparing both cell strains. upcyte hepatocytes from donor 653-03 express a number of endogenous CYP enzymes yet show low levels (5 pmol/mL) of CYP2D6 activity. In the 653-03-2D6 cell strain, CYP2D6 is stably expressed with a basal activity of over 1600 pmol/mL. In comparison, HepaRG cells almost completely lack CYP2D6 activity with less than 1 pmol/mL. In addition, this study shows that the recombinant expression of CYP2D6 does not largely influence other CYP activities, thus enabling complex metabolic profile analysis of CYP2D6 dependent drugs such as the breast cancer antiestrogen tamoxifen. In conclusion, our data shows that upcyte hepatocytes due to their possibility of expansion (up to 12x10^9 cells per donor) combined with long-term flexible expression of adult hepatic phenotypes constitute novel in vitro liver cell models for CYP2D6-dependent and independent metabolism and toxicity analysis in pharmaceutical drug development. P127 USE OF EXPANDED PRIMARY HUMAN HEPATOCYTES (UPCYTE® HEPATOCYTES) TO PREDICT TOXICOLOGICAL OUTCOMES Astrid Noerenberg 1, Nils Runge 1, Gahl Levy 2. 1 upcyte technologies GmbH, Hamburg, Germany; 2 Mircroliver Technologies Lab, The Hebrew University of Jerusalem, Jerusalem, Israel Primary human hepatocytes have limitations for drug testing due to the low availability of human liver tissue. To address this, we have developed a novel technique which causes primary human hepatocytes to proliferate up to 40 population doublings producing 10^13 to 10^16 cells from a single human hepatocyte isolate whilst still retaining a metabolic competent polarized phenotype after oncostatin M removal in confluent cultures. The resulting cells are named “upcyte hepatocytes.” To assess the utility of upcyte hepatocytes for toxicity screening, we used a library of known hepatotoxins and similarly structured control compounds. First, we compared the 24-h acute TC50 toxicity profile of six compounds in differentiated upcyte hepatocytes, primary human hepatocytes and HepG2 cells. A permutation test showed that the TC50 toxicity profile of differentiated hepatocytes for these six compounds was not significantly different from that of primary hepatocytes (P ¼ 0.466; n ¼ 4), whereas the HepG2 profiles were significantly different (P ¼ 0.03; n ¼ 4). We then measured TC50 values of 23 additional compounds in differentiated hepatocytes derived from four donors and found an excellent correlation between all donors of upcyte and primary human hepatocytes (R^2 ¼ 0.99). Second, we compared the toxicological profile of proliferating and differentiated upcyte hepatocytes. The TC50 of nine known hepatotoxic drugs clustered into two groups. One group (aflatoxin B1, acetylsalicylic acid and acetaminophen) showed no difference between proliferating and differentiated cells. The second group, which included six of the nine hepatotoxic compounds, showed significantly higher toxicity in differentiated upcyte hepatocytes than in proliferating upcyte hepatocytes, suggesting that metabolic activation was required for the toxic effect. Our results demonstrate that expandable upcyte hepatocytes from multiple genotypes show toxicity profiles similar to those of primary human
Figure 1. Separation and detection of 15 isomeric SCFAs using 0.1% formic acid in water and isopropanol as mobile phases on a sub-2 mm high strength silica chromatographic column (100 mm).
P125 DETECTION OF AFLATOXIN B1 HEPATOTOXICITY USING PXB-CELLS® Chihiro Yamasaki 1, Yuko Ogawa 1, Yutaka Kageyama 1, Seigo Sanoh 2, Yuji Ishida 1,3, Chise Tateno 1, 3. 1 PhoenixBio Co. Ltd., Higashi-Hiroshima, Japan; 2 Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan; 3 Liver Research Project Center, Hiroshima University, Hiroshima, Japan We have generated PXB-mice® (PhoenixBio Co., Ltd., Japan) with humanized livers by transplanting human hepatocytes into cDNA-uPA/SCID mice (Tateno et al., 2015). Approximately 1.5e2 x 108 human hepatocytes (PXBcells®) are obtained from a PXB-mouse by the collagenase perfusion method. Fresh human hepatocytes can attach on cell plates confluently and can be maintained for at least 4 weeks. Since the hepatitis B virus (HBV) infected the PXB-cells efficiently (Ishida et al., 2015), HBV-infected PXB-cells have been used for in vitroefficacy tests of anti-HBV agents. At the last meeting, we showed that PXB-cells cultured on type I collagen-coated plates showed high expression of cytochrome P450 (CYP), UDP-glucuronosyltransferase, and transporter genes for at least 4 weeks and retained CYP3A and transporter activities. In the present study, we tried to detect the toxicity of 0.014-10 mM aflatoxin B1 (AFB1), a mycotoxin metabolized by CYP3A4 and known to be toxic for human hepatocytes, using PXB-cells by WST-1 analysis at 1, 4, and 7 days, and a real-time cell analysis using iCELLigence (ACEA Biosciences Inc., San Diego, CA, USA) for 6 days. These two methods allowed detection of dose-dependent toxicity of AFB1. CYP inhibitors, 1-aminobenzotriazole (ABT) and ketoconazole, were added to AFB1-treated PXB-cells. CYP3A activity decreased in ABT- and ketoconazole-treated PXB-cells. Analysis using iCELLigence showed amelioration of AFB1 toxicity in the ABT- and ketoconazole-treated PXB-cells. These results suggest that iCELLigence is useful for detecting the toxicity of AFB1 and metabolites, and inhibitory effects of CYP inhibitors in PXB-cells. In conclusion, PXB-cells are useful for metabolism and toxicity tests. 1. Tateno et al., Generation of novel chimeric mice with humanized livers by using hemizygous cDNA-uPA/SCID mice. PLoS One. 2015; 10: e0142145 and Ishida et al., Novel robust in vitro hepatitis B virus infection model using fresh human hepatocytes isolated from humanized mice. Am J Path. 2015; 85: 1275-1285. P126 EXPANDED PRIMARY HUMAN HEPATOCYTES ENABLE METABOLISM STUDIES INCLUDING CYP2D6 DEPENDENT PATHWAYS (UPCYTE® HEPATOCYTES) Astrid Noerenberg 1, Nils Runge 1, Werner Schroth 2. 1 upcyte technologies GmbH, Hamburg, Germany; 2 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart, Germany
S63
Primary human hepatocytes have limitations for drug testing due to the low availability of human liver tissue. To address this, we have improved our upcyte technology enabling primary human hepatocytes to proliferate up to 40 doublings whilst still retaining a highly metabolic competent phenotype when cultured at confluence. The resulting cells are called “upcyte hepatocytes” and enable studies of metabolic clearance, hepatotoxicity and drug-drug interactions. Here, we tested upcyte hepatocytes from donor 653-03 and its genetically modified, CYP2D6 expressing derivative (653-03-2D6) for their enzymatic activities of CYP1A2, 2C8, 2B6, 2C19, 2D6, 2C9, 3A4 using a substrate cocktail incubation and Triple Quad MS analysis. In addition 75 ADME related genes were quantified using Fluidigm Taqman array (Biomark). upcyte hepatocytes show high basal activities of phase I enzymes which are inducible in the range of 2 to 23 fold by prototypical inducers rifampicin (CYP3A4, CYP2C8, CYP2C9, CYP2C19) and phenobarbital (CYP2B6) indicating functional PXR/CAR mediated CYP induction. On the level of gene expression, induction of CYP gene expression followed the same pattern as observed based on activities. Overall, there was no difference in the global regulatory gene expression pattern comparing both cell strains. upcyte hepatocytes from donor 653-03 express a number of endogenous CYP enzymes yet show low levels (5 pmol/mL) of CYP2D6 activity. In the 653-03-2D6 cell strain, CYP2D6 is stably expressed with a basal activity of over 1600 pmol/mL. In comparison, HepaRG cells almost completely lack CYP2D6 activity with less than 1 pmol/mL. In addition, this study shows that the recombinant expression of CYP2D6 does not largely influence other CYP activities, thus enabling complex metabolic profile analysis of CYP2D6 dependent drugs such as the breast cancer antiestrogen tamoxifen. In conclusion, our data shows that upcyte hepatocytes due to their possibility of expansion (up to 12x10^9 cells per donor) combined with long-term flexible expression of adult hepatic phenotypes constitute novel in vitro liver cell models for CYP2D6-dependent and independent metabolism and toxicity analysis in pharmaceutical drug development. P127 USE OF EXPANDED PRIMARY HUMAN HEPATOCYTES (UPCYTE® HEPATOCYTES) TO PREDICT TOXICOLOGICAL OUTCOMES Astrid Noerenberg 1, Nils Runge 1, Gahl Levy 2. 1 upcyte technologies GmbH, Hamburg, Germany; 2 Mircroliver Technologies Lab, The Hebrew University of Jerusalem, Jerusalem, Israel Primary human hepatocytes have limitations for drug testing due to the low availability of human liver tissue. To address this, we have developed a novel technique which causes primary human hepatocytes to proliferate up to 40 population doublings producing 10^13 to 10^16 cells from a single human hepatocyte isolate whilst still retaining a metabolic competent polarized phenotype after oncostatin M removal in confluent cultures. The resulting cells are named “upcyte hepatocytes.” To assess the utility of upcyte hepatocytes for toxicity screening, we used a library of known hepatotoxins and similarly structured control compounds. First, we compared the 24-h acute TC50 toxicity profile of six compounds in differentiated upcyte hepatocytes, primary human hepatocytes and HepG2 cells. A permutation test showed that the TC50 toxicity profile of differentiated hepatocytes for these six compounds was not significantly different from that of primary hepatocytes (P ¼ 0.466; n ¼ 4), whereas the HepG2 profiles were significantly different (P ¼ 0.03; n ¼ 4). We then measured TC50 values of 23 additional compounds in differentiated hepatocytes derived from four donors and found an excellent correlation between all donors of upcyte and primary human hepatocytes (R^2 ¼ 0.99). Second, we compared the toxicological profile of proliferating and differentiated upcyte hepatocytes. The TC50 of nine known hepatotoxic drugs clustered into two groups. One group (aflatoxin B1, acetylsalicylic acid and acetaminophen) showed no difference between proliferating and differentiated cells. The second group, which included six of the nine hepatotoxic compounds, showed significantly higher toxicity in differentiated upcyte hepatocytes than in proliferating upcyte hepatocytes, suggesting that metabolic activation was required for the toxic effect. Our results demonstrate that expandable upcyte hepatocytes from multiple genotypes show toxicity profiles similar to those of primary human