Abstract
Liver plays a major role in drug metabolism that was carried out by a family of critical enzymes; cytochrome P450 (CYP450). In this study, cell lines with high expression level of CYP450 are established based on Huh7 cells: Huh7-1A2-I-E cells expressing CYP1A2, Huh7-2E1 cells expressing CYP2E1, and Huh7-3A4-I-E cells expressing CYP3A4. To achieve this, we constructed recombinant lentiviral particles, containing a single promoter encoding CYP1A2 or CYP3A4 followed by an internal ribosome entry site (IRES) to permit the translation of enhanced green fluorescence protein (EGFP). Such a design has greatly facilitated the selection of stable cell lines because the translations of CYP450 and EGFP proteins would be based on a single bi-cistronic mRNA. The stable CYP450-expressing cells were evaluated as a cell-based model for identification of CYP450 inhibitors and for studies of cytotoxicity resulted from CYP-mediated drug metabolism. Treatment of stable cells with aflatoxin B1 showed that cells with CYP1A2 or CYP3A4 expression were much sensitive to aflatoxin B1 and the cellular toxicity of aflatoxin B1 in cells was prevented by CYP450 inhibitors (CYP1A2: furafylline; CYP3A4: ketoconazol). In addition, acetaminophen was also shown as a toxic substrate in Huh7-2E1 cells. Thus, we confirm that the CYP450-expressing cells have CYP450 characteristics based on the expressed protein level, the detectable enzyme activity, and the CYP450-mediated cytotoxicity study. Furthermore, a collection of approximately 200 drugs were screened using this system, and them could be separated into active metabolites, toxic metabolites, and inactive metabolites according to the cell-based screening based on the cells carrying the CYP450 metabolic activity. Several previously unidentified CYP1A2 inhibitors such as evoxine and berberine were also identified in this study. To enhance the intercellular CYP450 enzyme activity of stable cells, we attempted to modify the cellular characteristics using the genetic engineering approach and to improve the protein expression with the help of the small molecular compounds in culture medium. Cytochrome b5, cytochrome b5 reductase, cytochrome P450 reductase, and baculovirus IE2 (the activator of CMV promoter) were used to increase the enzyme activity. Results showed that cytochrome b5 and baculovirus IE2 enhanced the CYP450 enzyme activity in non-liver cells (293FT and Vero), but it was ineffective in Huh7 cells. Neither did cytochrome b5 reductase nor cytochrome P450 reductase help to enhance the enzymatic activity of CYP450 in liver and non-liver cells. Besides, valproic acid could increase the CYP450 enzyme activity when it was included in the culture medium. Overall, results from this thesis showed that the CYP450-expressing system can be effectively employed to study drug metabolism and to facilitate the establishment of hepatocyte-like cells in the future.