Cytochrome P450 2A-catalyzed metabolic activation of structurally similar carcinogenic nitrosamines: N'-nitrosonornicotine enantiomers, N-nitrosopiperidine, and N-nitrosopyrrolidine.
N'-Nitrosonornicotine (NNN) and N-nitrosopiperidine (NPIP) are potent esophageal and nasal cavity carcinogens in rats and pulmonary carcinogens in mice. N-Nitrosopyrrolidine (NPYR) induces mainly liver tumors in rats and is a weak pulmonary carcinogen in mice. These nitrosamines may be causative agents in human cancer. alpha-Hydroxylation is believed to be the key activation pathway in their carcinogenesis. P450 2As are important enzymes of nitrosamine alpha-hydroxylation. Therefore, a structure-activity relationship study of rat P450 2A3, mouse P450 2A4 and 2A5, and human P450 2A6 and 2A13 was undertaken to compare the catalytic activities of these enzymes for alpha-hydroxylation of (R)-NNN, (S)-NNN, NPIP, and NPYR. Kinetic parameters differed significantly among the P450 2As although their amino acid sequence identities were 83% or greater. For NNN, alpha-hydroxylation can occur at the 2'- or 5'-carbon. P450 2As catalyzed 5'-hydroxylation of (R)- or (S)-NNN with Km values of 0.74-69 microM. All of the P450 2As except P450 2A6 catalyzed (R)-NNN 2'-hydroxylation with Km values of 0.73-66 microM. (S)-NNN 2'-hydroxylation was not observed. Although P450 2A4 and 2A5 differ by only 11 amino acids, they were the least and most efficient catalysts of NNN 5'-hydroxylation, respectively. The catalytic efficiencies (kcat/Km) for (R)-NNN differed by 170-fold whereas there was a 46-fold difference for (S)-NNN. In general, P450 2As catalyzed (R)- and (S)-NNN 5'-hydroxylation with significantly lower Km and higher kcat/Km values than NPIP or NPYR alpha-hydroxylation (p <0.05). Furthermore, P450 2As were better catalysts of NPIP alpha-hydroxylation than NPYR. P450 2A4, 2A5, 2A6, and 2A13 exhibited significantly lower Km and higher kcat/Km values for NPIP than NPYR alpha-hydroxylation (p <0.05), similar to previous reports with P450 2A3. Taken together, these data indicate that critical P450 2A residues determine the catalytic activities of NNN, NPIP, and NPYR alpha-hydroxylation.
Wong
Hansen L
HL
University of Minnesota, The Cancer Center, 420 Delaware Street SE, Mayo Mail Code 806, Minneapolis, Minnesota 55455, USA.
Murphy
Sharon E
SE
Hecht
Stephen S
SS
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002273:Carcinogens / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D051379:Mice; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D009242:N-Nitrosopyrrolidine / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D009602:Nitrosamines / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D051381:Rats; D013045:Species Specificity; D018411:Spodoptera / Q000201:enzymology; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002273:Carcinogens; D009602:Nitrosamines; C002743:N-nitrosopiperidine; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C029009:steroid hormone 7-alpha-hydroxylase; D009242:N-Nitrosopyrrolidine; C008655:N'-nitrosonornicotine