Carcinogenesis, Vol. 21, No. 8, 1559-1566,
August 2000
© 2000 Oxford University Press
Carcinogenesis |
Identification of the human liver microsomal cytochrome P450s involved in the metabolism of N-nitrosodi-n-propylamine
1 Department of Environmental and Industrial Health and
2 Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-0632, USA
The ability of human liver cytochrome P450s to metabolize the environmental carcinogen N-nitrosodi-n-propylamine (NDPA) was investigated. The maximum rate of NDPA depropylation in seven human liver microsomal samples was 1.15 nmol/min/mg (range 0.53–2.60). Troleandomycin, a P450 3A4/5 inhibitor, inhibited depropylation modestly (10–60%) in three of seven samples. Diethyldithiocarbamic acid, a potent 2E1 inhibitor, and a 2E1 inhibitory monoclonal antibody (mAb) inhibited the reaction in all samples (23 to almost 100%). No significant inhibition was observed with the 2C9 inhibitor sulfaphenazole or with mAbs to 3A4, 2A6 and 2D6. The 2C8/9/18/19 mAb inhibited depropylation in one sample by ~25% and ~25% of the activity in another sample could not be accounted for by the inhibitors. Denitrosation of NDPA by three of the microsomal samples exhibited low Km values (51–86 µM) while two of these also had high Km values (2.6 and 4.6 mM). Purified human P450 2B6 and 3A4 and human P450 2A6, 2C8, 2C9 and 2D6 membranes had high Km values relative to their maximum turnover rates and are unlikely to participate in NDPA metabolism at micromolar concentrations. Conversely, purified rabbit 2E1 exhibited Km and Vmax values for depropylation of 52 µM and 13.4 nmol propionaldehyde/min/nmol P450, respectively. Values for denitrosation were 66 µM and 1.44 nmol nitrite/min/nmol P450, respectively. The toxicity of NDPA in transfected human liver epithelial cells expressing 2E1 was dose dependent down to 50 µM. No toxicity was observed in control cells or those expressing 2A6. These results indicate that 2E1 is the major human liver microsomal isoform responsible for NDPA metabolism at low micromolar concentrations. We also show that purified P450s catalyze the denitrosation of NDPA at ~10–20% of the rate of depropylation and Km values for both reactions are the same for each isozyme. This is consistent with the formation of an initial intermediate common to both pathways, presumably an 
-nitrosamino radical.
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