Cytochrome P450 2A-catalyzed metabolic activation of structurally similar carcinogenic nitrosamines: N'-nitrosonornicotine enantiomers, N-nitrosopiperidine, and N-nitrosopyrrolidine

Chem Res Toxicol. 2005 Jan;18(1):61-9. doi: 10.1021/tx0497696.


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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Aryl Hydrocarbon Hydroxylases / metabolism*
  • Carcinogens / chemistry
  • Carcinogens / metabolism*
  • Carcinogens / toxicity
  • Humans
  • Hydroxylation / drug effects
  • Mice
  • Microsomes / drug effects
  • Microsomes / enzymology
  • N-Nitrosopyrrolidine / chemistry
  • N-Nitrosopyrrolidine / metabolism*
  • N-Nitrosopyrrolidine / toxicity
  • Nitrosamines / chemistry
  • Nitrosamines / metabolism*
  • Nitrosamines / toxicity
  • Rats
  • Species Specificity
  • Spodoptera / enzymology
  • Steroid Hydroxylases / metabolism*
  • Structure-Activity Relationship


  • Carcinogens
  • Nitrosamines
  • N-nitrosopiperidine
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • steroid hormone 7-alpha-hydroxylase
  • N-Nitrosopyrrolidine
  • N'-nitrosonornicotine