Table 1

Metabolic activation of DCBN by CYP2A5, CYP2G1 and mouse liver and OM microsomes

Enzyme preparationAdditionRate of DCBN-protein adduct formation
3 μM DCBN30 μM DCBN
pmol/min/nmol P450
OM microsomesNone248 ± 44462 ± 56
(65 ± 11)1-a (120 ± 15)
Liver microsomesNone3.0 ± 0.313.4 ± 3.1
(1.7 ± 0.2)(5.8 ± 1.8)
CYP2A5BSA 8 23
CYP2G1BSA 8 12
CYP2A5BSA +b 5 18 49
CYP2G1BSA +b 5 20 32
CYP2A5Boiled NMN.D.1-b 116
CYP2G1Boiled NMN.D.59
CYP2A5Boiled NM +b 5 N.D.205
CYP2G1Boiled NM +b 5 N.D.130
CYP2A5Boiled LMN.D.135
CYP2G1Boiled LMN.D.69
CYP2A5Boiled LM +b 5 N.D.250
CYP2G1Boiled LM +b 5 N.D.171

The reaction mixtures contained 50 mM phosphate buffer, pH 7.4, 3 or 30 μM 14C-DCBN, a reconstituted system containing 0.1 μM P450, 0.4 μM P450 reductase and 30 μg/ml dilauroylphosphatidylcholine or microsomal preparations from mouse olfactory mucosa (0.1 mg of protein/ml) or liver (0.2 mg of protein/ml) and 1 mM NADPH. The reactions were carried out at 37°C for 30 min, during which product formation was linear with time. The formation of DCBN-protein adduct from DCBN was determined as described in Materials and Methods. The values reported are the mean ± S.D. (n = 3) for microsomal reactions or the average of two experiments with differences of <10% of the mean for reactions with purified P450s. When indicated, b5 was added at 0.4 μM; BSA was added at 0.5 mg/ml; and liver or nasal microsomes previously boiled for 10 min (LM or NM, respectively) were added at 0.1 mg/ml.

  • 1-a Values in parentheses indicate activities expressed as pmol/min/mg of microsomal protein;

  • 1-b N.D., not determined.