Stable expression of human cytochrome P450 2D6*10 in HepG2 cells

Abstract

Aim: Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established.

Methods: Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from human liver tissue and cloned into pGEM-T vector. cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells. Expression of mRNA was validated by RT-PCR. Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernatant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC).

Results: The cloned cDNA had 4 base differences, e.g. 100 C-T, 336 T-C, 408 C-G and 1 457 G-C, which resulted in P34S, and S486T amino acid substitutions, and two samesense mutations were 112 F and 136 V compared with that reported by Kimura et al (GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele. The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31 +/- 0.19 nmol/min(-1)/mg(-1) S9 protein (n=3), but was undetectable in parental HepG2 cells.

Conclusion: cDNA of human CYP2D6*10 can be successfully cloned. A cell line, HepG2-CYP2D6*10, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.

Figure 1

Electrophoresis identification of pGEM-CYP2D6 and pREP9-CYP2D6 recombinants. Lane 1: Marker (λ/EcoR I and Hind III), 2: PCR products of CYP2D6 (1.543 kb), 3: Recombi-nant of pGEM-CYP2D6 digested by Xho I and BamH I, 4: pGEM-T vector (3 kb), 5: Recombinant of pREP9-CYP2D6 digested by Xho I and BamH I, 6: pREP9 vector (10.5 kb).

Figure 2

Identification of CYP2D6 mRNA expression in HepG2-CYP2D6 and HepG2 cells by RT-PCR with beta-actin as internal control. Lane 1: 1 kb ladder marker, 2: RT-PCR prod-ucts of HepG2 cells showing a 462 bp of beta-actin, 3: RT-PCR products of HepG2-CYP2D6 cells showing a 462 bp of beta-actin and 1.5 kb of CYP2D6.

Figure 3

Representative chromatograms of metabolites in supernatant. 10 μL of supernatant was injected into a Water HPLC equipped with a Shimadzu RF-535 fluorescence detector. A CLC phenyl column (15 cm×4.5-mm i.d.) was used to sepa-rate the metabolites. The mobile phase consisted of a mixture of 30% acetonitrile, 1% acetic acid, and 0.05% triethylamine in water. The flow rate through the column at 25 °C was 0.75 ml·min^-1. The excitation and emission wavelengths of the fluorescence de-tector were 285 nm and 310 nm, respectively. The retention times for dextrophan and dextromethorphan were 6.5 min and 16.8 min, respectively. The retention time of an unidentified metabolite was 8.9 min.