Table 1.
Primer sequences.
Fig 1.
Construction and analysis of the 4CYPs-POR MAC vector in CHO cells.
(A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.
Fig 2.
Analysis of HepG2 cells containing the 4CYPs-POR MAC.
(A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.
Fig 3.
Analysis of CYP expression and activity in TC-HepG2 cells.
(A) Reverse transcription-qPCR analysis for the expression of CYPs in TC-HepG2 cells. Each column represents the mean ± S.D. (n = 3). (B) CYP activity analysis in TC-HepG2 cells. The expression and activity levels for each CYP relative to the levels in human primary hepatocytes are shown. Each column represents the mean ± S.D. (n = 3). (C) CYP3A4 inhibition activity in TC-HepG2 cells determined by clarithromycin.
Fig 4.
The 4CYPs-POR MAC stability and sustained transgene regulation in the TC-HepG2.
TC-HepG2 cells were used for the following analyses before and after long-term culturing (50 PDLs) in the presence and absence of selection with G418 (A-C). (A) Genomic PCR analysis of the 4CYPs-POR MAC in TC-HepG2 cells. (B) RT-qPCR analysis for expression of the transgenes and endogenous genes in TC-HepG2 cells. The 4CYPs-POR transgene expression levels at 50 PDLs are standardized to NAT1 expression, and the relative each CYP and POR gene expression in each clone at 0 PDLs was used as control. Each column represents the mean ± S.E. (n = 3). (C) FISH analyses for 4CYPs-POR MAC stability in TC-HepG2 cells (n = 25–30). PDL, population doubling.
Table 2.
Summary of in vitro inhibition data for CYP3A4, CYP2C9, CYP2C19, and CYP2D6 for 20 compounds.
Values represent means ±S.D. of triplicate determinations.
Fig 5.
Relationship between relative CYP activity level and inhibitor concentration.
Relative CYP activity was determined as the ratio of CYP-specific luminescence in the presence of the specific inhibitors to that in the absence of the same inhibitor. Open circles represent the values in cultures treated with the inhibitor and the CYP-specific luminescent substrate simultaneously [direct inhibition (DI)], and closed circles represent the values in cultures pretreated with the inhibitor for 60 min before the addition of the luminescent substrate (time-dependent inhibition). Values represent means ± S.D. (n = 4). *P < 0.05 vs each DI group.
Table 3.
Determination of kinact and Ki,app for metabolism-dependent inhibition of CYP3A4 by macrolides.
Apparent inactivation rates (kobs) were estimated graphically from the slope of the plot for the natural log of enzyme activity remaining after preincubation against preincubation time and were corrected for any loss of activity in the absence of the inhibitor. kinact and Ki,app values were obtained from a double reciprocal plot of kobs against inhibitor concentration. Data represent means ± S.D. of triplicate determinations.
Fig 6.
Inhibitory effects of macrolides on CYP-mediated metabolism in TC-HepG2 cells.
(A), (B), (C) CYP3A-specific metabolism of clarithromycin, erythromycin, and roxithromycin, respectively, in TC-HepG2 cells. (D), (E), (F) CYP3A-specific metabolism of clarithromycin, erythromycin, and roxithromycin, respectively, in human hepatocytes. Macrolides (0–100 μM/L) were preincubated at 37°C for 0–30 min, and the reaction was initiated by the addition of CYP3A-specific substrate for 30 min. (G—I) Apparent inactivation rate (kobs) was estimated graphically from the slope of the plot for the natural log enzyme activity remaining after preincubation against preincubation time and was corrected for any loss of activity in the absence of the inhibitor. kinact and Ki,app values were obtained from a double reciprocal plot of kobs against inhibitor concentration. Closed circles represent TC-HepG2 cells, and opened circles represent parental HepG2 cells. Each symbol represents the average of triplicate determinations.
Fig 7.
Assessment of compound-associated toxicity in TC-HepG2 cells.
Aflatoxin B1 and sterigmatocystin requires CYP-mediated activation to form cytotoxic and DNA-reactive intermediates. Symbols represent means ± S.D. of triplicate determinations.