Drug-metabolizing enzyme activities in HepaRG cells
Determinations of testosterone 6β-hydroxylation (CYP3A4), O-dealkylation of 7-ethoxyresorufin (CYP1A2), chlorzoxazone 6-hydroxylation (CYP2E1), and tolbutamide 4-hydroxylation (CYP2C9) activities were performed on HepaRG cells seeded at low density and cultured for 15 days or 30 days in the presence of 2% DMSO between days 15 and 30, and on differentiated HepaRG cells seeded at high density. Pretreatment with inducers or their vehicle was performed using FCS-free medium with or without added 2% DMSO. The cultures were exposed either 24 h [3-methylcholantrene (3-MC)] or 72 h [rifampicin (RIF), isoniazid (ISO)] to the inducers or their vehicle [untreated (UT)]. Results are expressed as pmol/mg protein/min. Student's t test was applied for statistical studies between UT cells and cells treated with inducer: * p < 0.05, ** p < 0.01, and *** p < 0.001 and between HepaRG cells exposed or not to 2% DMSO during pretreatment with inducers or vehicle: §p < 0.05, §§p < 0.01 and §§§p < 0.001 (GraphPad Prism software).
DMSO | 7-Ethoxyresorufin O-deethylation (CYP1A1/2) | Tolbutamide 4-hydroxylase Activity (CYP2C9) | Chlorzoxazone 6-hydroxylase Activity (CYP2E1) | Testosterone 6β-hydroxylase Activity (CYP3A4) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| UT | 3MC (5 μM) | UT | RIF (25 μM) | UT | ISO (50 μM) | UT | RIF (50 μM) | ||||||
| 15-day culture HepaRG cells seeded at low density | - | Traces | 48.2 ± 3 | 1.0 ± 0.6 | 4.5 ± 3.8 | 31.9 ± 13.2 | 33.4 ± 11.9 | 10.9 ± 4.7 | 129.3 ± 22*** | ||||
| (n = 4) | (n = 5) | (n = 6) | (n = 6) | (n = 6) | (n = 5) | (n = 6) | |||||||
| Differentiated HepaRG cells seeded at low density | - | Traces | 68 ± 5.2 | 6 ± 1.4 | 7.8 ± 1.8 | 131.3 ± 11.2 | 96 ± 12* | 39.7 ± 15 | 212.9 ± 48** | ||||
| (n = 6) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | |||||||
| + | Traces | 154.6 ± 13.7§§§ | 7.5 ± 0.3 | 10.15 ± 0.5** | 140.9 ± 56.5 | 221.5 ± 10§§§ | 576 ± 3§§ | 806 ± 104§§§ | |||||
| (n = 6) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | |||||||
| Differentiated HepaRG cells seeded at high density | - | Traces | 63.1 ± 0.7 | 6.2 ± 1.6 | 13.1 ± 2.6* | 278 ± 8.4 | 197.8 ± 103 | 54.4 ± 10 | 540.1 ± 68*** | ||||
| (n = 6) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | |||||||
| + | Traces | 129 ± 16.3§§§ | 9.8 ± 1.5§ | 11.5 ± 3.5 | 483.1 ± 30.3§§§ | 455.2 ± 65§ | 781.8 ± 85§§§ | 820.5 ± 210 | |||||
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| (n = 6) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | (n = 3) | ||||