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 esophagus. The activation is believed to occur in the esophagus. Although the pathways of NBzMA metabolism are well studied, the principal cytochrome P450 enzyme(s) (P450) responsible for catalyzing its activation is unknown. Several preliminary studies have suggested that this enzyme may belong to the P450 2A family. We report here that P450 2A3 expressed in a baculovirus system metabolizes NBzMA, predominantly by methylene hydroxylation. To determine 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 mucosa. P450 2A3 mRNA was detected in rat nasal mucosa, lung and esophagus, but not in liver, whereas P450 2A1 and 2A2 mRNAs were detected only in the liver. To determine the relative expression of P450 2A3 in each tissue, quantitative RT-PCR with PCR-MIMICS used as internal standards was performed. The expression level in the nasal mucosa was by far the greatest. The expression in the lung and esophagus was 60- and 1600-fold less, respectively. Using antibodies to P450 2A4/5 and P450 2A10/11 a 50 kDa immunoreactive protein was detected in all three tissues by western blot analysis. This is consistent with the expression of P450 2A3 in these tissues. However, the amount 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 lung and esophagus. The rate of coumarin 7-hydroxylation in cultured rat esophagus was very low. This is a reaction efficiently catalyzed by P450 2A5, 2A6 and 2A10. In summary, our results clearly demonstrate the presence of P450 2A3 protein and mRNA in the esophagus, but the amounts are low and may not be sufficient to account for NBzMA activation in this tissue.
Gopalakrishnan
R
R
Department of Pathology, College of Medicine and Public Health, The Ohio State University, Columbus 43210, USA.
Morse
M A
MA
Lu
J
J
Weghorst
C M
CM
Sabourin
C L
CL
Stoner
G D
GD
Murphy
S E
SE
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015152:Blotting, Northern; D003374:Coumarins / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004128:Dimethylnitrosamine / Q000031:analogs & derivatives / Q000378:metabolism; D004947:Esophagus / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000201:enzymology; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D009297:Nasal Mucosa / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution
D003374:Coumarins; D007527:Isoenzymes; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; C014707:nitrosobenzylmethylamine; C030123:coumarin; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; D004128:Dimethylnitrosamine