Establishment of the transformants expressing human cytochrome P450 subtypes in HepG2, and their applications on drug metabolism and toxicology

Transformants with stable expression of a series of human cytochrome P450 ( CYP) subtypes in the human hepatic cell line, HepG2, were established. Thes e transformants are designated Hepc/1A1.4. Hepc/1A2.9, Hepc/2A6L.14. Hepc/2 B6.68. Hepc/2C8.46, Hepc/2C9.1, Hepc/2C19.12, Hepc/2D6.39, Hepc/2E1.3-8 and Hepc/3A4.2-30, which stably expressed human CYP1A1, CYP1A2, CYP2A6, CYP2B6 , CYP2C8, CYP2C9, CYF2C19, CYP2D6, CYP2E1 and CYP3A4, respectively. The exp ression of the CYP subtypes in the transformants was confirmed by both dete rmination of enzyme activities and the reverse transcriptase polymerase cha in reaction (RT-PCR) procedure. The apparent K-m values of the expressed CY P subtypes for their specific substrates were close to those of human liver microsomes. In addition to their CYP activities, these transformants retai ned glucuronide- and sulfate-conjugating activities. Furthermore, the activ ities of CYP2C9, CYP2D6 and CYP3A4 were inhibited by their specific inhibit ors. The cytotoxicity of acetaminophen (APAP), cyclophosphamide (CPA) and b enz[a]anthracene (BA) were analyzed by CYP-expressing transformants. The cy totoxicity depended on the expression of CYP subtypes: and increased in a d ose-dependent manner. These results show the metabolic activation of APAP, CPA and BA by the specific CYP subtypes expressed in the transformants and demonstrate the usefulness of these transformants for in vitro metabolic an d toxicological studies in human liver. (C) 2001 Elsevier Science Ltd. All rights reserved.