Characterization and Comparison of Nicotine and Cotinine Metabolism in Vitro and in Vivo in DBA/2 and C57BL/6 Mice

DBA/2 and C57BL/6 are two commonly used mouse strains that differ in response to nicotine. Previous studies have shown that the nicotine-metabolizing enzyme CYP2A5 differs in coumarin metabolism between these two strains, suggesting differences in nicotine metabolism. Nicotine was metabolized to cotinine in vitro by two enzymatic sites. The high-affinity sites exhibited similar parameters ( K m , 10.7 ± 4.8 versus 11.4 ± 3.6 μM; V max , 0.58 ± 0.18 versus 0.50 ± 0.07 nmol/min/mg for DBA/2 and C57BL/6, respectively). In vivo, the elimination halflives of nicotine (1 mg/kg, s.c.) were also similar between DBA/2 and C57BL/6 mice (8.6 ± 0.4 versus 9.2 ± 1.6 min, respectively); however, cotinine levels were much higher in DBA/2 mice. The production and identity of the putative cotinine metabolite 3′-hydroxycotinine in mice was confirmed by liquid chromatography/mass spectrometry/mass spectrometry. The in vivo half-life of cotinine (1 mg/kg, s.c.) was significantly longer in the DBA/2 mice compared with the C57BL/6 mice (50.2 ± 4.7 versus 37.5 ± 9.6 min, respectively, p < 0.05). The in vitro metabolism of cotinine to 3′-hydroxycotinine was also less efficient in DBA/2 than C57BL/6 mice ( K m , 51.0 ± 15.6 versus 9.5 ± 2.1 μM, p < 0.05; V max , 0.10 ± 0.01 versus 0.04 ± 0.01 nmol/min/mg, p < 0.05, respectively). Inhibitory antibody studies demonstrated that the metabolism of both nicotine and cotinine was mediated by CYP2A5. Genetic differences in Cyp2a5 potentially contributed to similar nicotine but different cotinine metabolism, which may confound the interpretation of nicotine pharmacological studies and studies using cotinine as a biomarker.