Developing a high-throughput system for the screening of cytochrome P450 1A1--inhibitory polyphenols.

Polycyclic aromatic hydrocarbons (PAH) are established procarcinogens that can be found in our environment. The carcinogenicity of these compounds is initiated by their metabolic intermediates, and the extent of biotransformation determines the amount of reactive intermediates generated. CYP1A1 is a major enzyme that metabolizes PAH into carcinogenic moieties. Since previous studies have shown that increased CYP1A1 activity is associated with a higher cancer risk. Identifying CYP1A1-inhibitory compounds in diet or natural products are of genuine interest for chemoprevention studies. In this project, a stable cell line expressing human CYP1A1 was established for the screening of potential chemopreventive agents. Because of a lacking cellular transport mechanism in assays performed on recombinant protein, an 'in-cell' assay system might be a better estimate at the tissue level. Theaflavins were strong inhibitors of ethoxyresorufin-O-deethylase (EROD) activity when assayed on recombinant human CYP1A1 protein. However, this inhibition was not observed in the CYP1A1-expressing cells. The 'in-cell' IC50 values determined for compounds such as genistein, quercetin, chalcone, etc. were comparable to the values determined in recombinant protein. This 'in-cell' assay has the additional advantages of short sample processing time, and the tedious procedures of protein expression and purification can be waived.