Expression of cytochrome P450 2A3 in rat esophagus: relevance to N-nitrosobenzylmethylamine

N-nitrosobenzylmethylamine (NBzMA) must be metabolically activated to exert its carcinogenic potential and is a potent inducer of tumors in the rat es ophagus, The activation is believed to occur in the esophagus. Although the pathways of NBzMA metabolism are well studied, the principal cytochrome P4 50 enzyme(s) (P450) responsible for catalyzing its activation is unknown. S everal preliminary studies have suggested that this enzyme may belong to. t he P450 2A family. We report here that P450 2A3 expressed in a baculovirus system metabolizes NBzMA, predominantly by methylene hydroxylation, To dete rmine whether or not P450 2A3 is present in the rat esophagus, the relative level of P450 2A3 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), The mRNA levels of P450 2A3 were compared with the levels of P450 2A1 and 2A2 mRNA in the esophagus, liver, lung and nasal mu cosa, P450 2A3 mRNA was detected in rat nasal mucosa, lung and esophagus, b ut not in liver, whereas P450 2A1 and 2A2 mRNAs were detected only in the l iver. To determine the relative expression of P450 2A3 in each tissue, quan titative RT-PCR with PCR-MIMICS used as internal standards was performed. T he expression level in the nasal mucosa was by far the greatest. The expres sion in the lung and esophagus was 60- and 1600-fold less, respectively. Us ing antibodies to P450 2A4/5 and P450 2A10/11 a 50 kDa immunoreactive prote in was detected in all three tissues by western blot analysis. This is cons istent with the expression of P450 2A3 in these tissues. However, the amoun t of protein detected in the nasal mucosa was much greater than that in the esophagus or lung. The expression of P450 2A protein was similar in the lu ng and esophagus, The rate of coumarin 7-hydroxylation in cultured rat esop hagus was very low. This is a reaction efficiently catalyzed by P450 2A5, 2 A6 and 2A10, In summary, our results clearly demonstrate the presence of P4 50 2A3 protein and mRNA in the esophagus, but the amounts are low and may n ot be sufficient to account for NBzMA activation in this tissue.