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, O cells) and subjected (+ ?-glucuronidase) or not (-?-glucuronidase) to ?-glucuronidase hydrolysis. B) Culture medium with CHZ incubated with parental HepG2 cells or recombinant HepG2-CYP2E1 cells without

, ?-glucuronidase hydrolysis prior HPLC-UV analysis. C) Culture medium with CHZ incubated with differentiated parental HepaRG or recombinant HepaRG-CYP2E1 cells without (-?glucuronidase) or with (+?-glucuronidase) ?-glucuronidase hydrolysis prior HPLC-UV analysis. D) Culture medium with CHZ

, parental and CYP2E1 expressing HepG2 and HepaRG cells following HPLC-UV analysis of culture media, without (-?-glucuronidase, left graph) or with (+?glucuronidase, right graph) ?-glucuronidase hydrolysis of media. Statistics: 2 or 3 independent experiments with 6 or 9 independent culture wells, ? p<0.01: OH-CHZ significantly higher in HepaRG and HepaRG-CYP2E1 versus HepG2, ? ? p <0.01: OH-CHZ significantly higher in HH versus HepaRG, HepaRGCYP2E1 and HepG2, ? ? ? p<0.01: OH-CHZ significantly higher in HepG2 CYP2E1 than all others cells. Graph with ?-glucuronidase hydrolysis (+?-glucuronidase), § § p <0.001: OH-CHZ significantly higher in parental HepaRG versus HepG2, § § § p<0.001 OH-CHZ significantly higher in HepG2-CYP2E1, HepaRG CYP2E1 and HH versus HepaRG and HepG2 WT. F)

, Quantification of CHZ-O-and CHZ-N-Glc activities (pmoles/min/mg of total proteins

C. Hepg2 and . Heparg, HepaRG CYP2E1 cells and primary hepatocytes (HH), CHZ-O-Glc significantly higher in HepaRG versus HepG2 CYP2E1 cells and significantly higher in HepaRG-CYP2E1 and primary hepatocytes, vol.01

, Statistics of CHZ-N-Glc graph, § § § p<0.001 CHZ-N-Glc significantly higher in primary hepatocytes (HH) versus HepG2 CYP2E1, and § § p<0.01 parental HepaRG and HepaRG-CYP2E1 cells versus primary hepatocytes (HH) and HepG2 CYP2E1. Figure 3: Conversion of OH-CHZ into CHZ-O-Glc in HepaRG cells and human hepatocytes, versus HepaRG and HepG2 CYP2E1

, A) HPLC-UV chromatograms of the time-course analysis of OH-CHZ conversion into CHZ

O. , by HepaRG cells at 0, 15, 60 and 90 min. B) Quantification in ?M of OH-CHZ (plain lane) and CHZ-O-Glc

C. )-quantification-of and C. , /min/mg of total proteins) after 90 min of 25 incubation of culture medium containing OH-CHZ by HepG2 CYP2E1, HepaRG, HepaRG CYP2E1 cells and primary hepatocytes (HH). Statistics, § § § p<0, CHZ-O-Glc significantly higher in HepaRG, HepaRG-CYP2E1 cells and primary hepatocytes (HH) versus HepG2, vol.001

, Figure 4: Inhibition of CHZ-O-Glc and CHZ-N-Glc production by pentachlorophenol

A. Hplc-uv-detection-of, O. , C. , and C. , Glc in culture media of HepaRG cells after a 90 min incubation with CHZ in absence (0 ?M) or presence of pentacholorophenol (PCP) at 10 and 100 ?M. The 2 main contaminating peaks are indicated (*). B) Quantification of OH-CHZ activities (pmoles/min/mg of total proteins) in culture of primary hepatocytes (HH), parental and CYP2E1 expressing HepaRG cells and HepG2 CYP2E1 cells in absence

, parental and CYP2E1 expressing HepaRG cells in absence and presence of various concentrations of PCP (2.5 to 100 ?M). D) Quantification of CHZ-O-Glc activities (pmoles/min/mg of total proteins) in culture of primary hepatocytes (HH) and parental HepaRG cells in absence and presence of various concentrations of PCP (2.5 to 100 ?M), Glc activities (pmoles/min/mg of total proteins) in culture of primary hepatocytes (HH)

, Production of CHZ-O and CHZ-N-Glc in recombinant HepG2-UGT cells, vol.5

. , CHZ-N-Glc (grey bars) and OH-CHZ (dark bars) in culture medium of recombinant HepG2 cells expressing either UGT1A1, 1A6 or 1A9, and HepG2 cells expression CYP2E1 and UGT1A1, ?M) of CHZ-O-Glc (white bars), pp.1-6

, CHZ (B) and the production of both CHZ-O-Glc and CHZ-N-Glc was analyzed by HPLC and quantified. Statistics, A) Chart for cells incubated with OH-CHZ: § p<0.05 OH-CHZ significantly lower and * p<0.01 CHZ-OGlc significantly higher in cells expressing UGT1A1, 1A6 and 1A9 versus parental/wild type (WT) HepG2 and HepG2-CYP2E1, B) chart for cells incubated with CHZ: * p<0.01 CHZ-OGlc significantly higher in HepG2 cells expressing CYP2E1 and UGT1A1, 1A6 or 1A9 versus all other conditions, vol.29, p.31