Characterization of CYP2A6 involved in 3'-hydroxylation of cotinine in human liver microsomes.

Nicotine is primarily metabolized to cotinine, and cotinine is further metabolized to trans-3'-hydroxycotinine in human liver, which is a major metabolite of nicotine in humans. We studied the formation of trans-3'-hydroxycotinine from cotinine in human liver microsomes. trans-3'-Hydroxycotinine formation demonstrated single enzyme Michaelis-Menten kinetics (Km, 234.5 +/- 26.8 MicroM; Vmax, 37.2 +/- 2.4 pmol/min/mg protein). Significant correlation (r = .967, P < .001) between cotinine 3'-hydroxylase activities at low (50 microM) and high (1 microM) cotinine concentrations in 20 human liver microsomes suggested the contribution of a single enzyme to cotinine 3'-hydroxylation. The cotinine 3'-hydroxylase activity correlated significantly with immunoreactive cytochrome P450 (CYP)2A6 contents (r = .756, P < .01) and coumarin 7-hydroxylase activity (r = .887, P < .001). The cotinine 3'-hydroxylase activity was inhibited by coumarin, alpha-naphthoflavone, chlorzoxazone and anti-rat CYP2A1 antibodies. Microsomes of B-lymphoblastoid cells expressing human CYP2A6 exhibited cotinine 3'-hydroxylase activity. The Km value of the expressed CYP2A6 (264.7 microM) was almost identical to that of human liver microsomes. In conclusion, cotinine 3'-hydroxylation appears to be catalyzed solely by CYP2A6 in humans. Cotinine is a candidate for a new substrate for CYP2A6 in humans.