We have genetically engineered V79 cells (Chinese hamster lung fibroblasts) by cDNA encoding the rat liver UDP-glucuronosyltransferase (UGT) isoforms, UGT1A6 and UGT2B1. These proteins are involved in the glucuronidation of toxic substances, such as phenols (carcinogens, pesticides) and carboxyl drugs (nonsteroidal anti-inflammatory drugs).
Upon transfection of the cells, and selection of the clones by antibiotics, high production of catalytically active proteins was achieved. The expression was stable over time, thus allowing a continuous supply of recombinant enzymes.
These two metabolic competent cells, which express each a distinct UGT isoform have been used in this study in order to determine if they catalysed the glucuronidation of catechols.
They can also be potentially used for other purposes, such as bioavailability, drug metabolism and toxicity of drugs and chemicals, as well as drug-drug interactions. Additionally, heterologous expression of UGTs provides an opportunity to study the structure and the function of these membrane proteins.
V79 cells were co-transfected with a eukaryotic expression vector carrying the cDNA encoding the rat liver UGT1A6 or UGT2B1 and a plasmid encoding the Geneticin resistance gene. Upon transfection, the cells were incubated in a medium containing Geneticin in order to select the recombinant clones.
The expression of the recombinant enzymes was monitored by measurement of the activity with 1-naphthol or ketoprofen, as substrates and by determining the amount of protein by Western blot with specific antibodies. The most producing and stable clones were selected and stored in liquid nitrogen. Membrane enriched fractions were prepared from cellular homogenate by differential ultracentrifugations. These microsomes were used to investigate the in vitro glucuronidation of catechols by the recombinant UGT1A6 and UGT2B1.