Aerobic incubation of NIC with rabbit hepatic microsomes in the presence of NADPH resulted in the formation of seven metabolites. The major product of NIC metabolism in rabbit liver microsomes was COT. COT formation at 50 μM NIC increased linearly for up to 30 min in the presence of 0.2–1 mg/ml microsomal protein and a constant concentration (2 mg/ml) of cytosolic protein (data not shown). Cytosolic protein was used as the source of aldehyde oxidase which is required for the first step in COT formation. The concentration of NIC was varied and the COT formation data were fit to a single enzyme Michaelis-Menten model which generated the following parameter estimates: Km = 13.2 ± 4.0 μM and Vmax = 978 ± 89.2 pmol/min/mg protein. Cytosolic protein concentrations from 1–10 mg/ml did not significantly alter NIC metabolism (data not shown). The mean rates of COT formation in rabbit liver microsomes are shown in . The rates of COT formation were similar in hepatic microsomes from nonpregnant and pregnant rabbits. COT formation rates were at least twice the rate of the other metabolites.
Profile of nicotine (A) and cotinine (B) metabolism in rabbit liver microsomes
Greater than 75% of the starting NIC could be accounted for by the appearance of seven metabolites in hepatic microsomes. In addition to COT, only nicotine-N’-oxide and nornicotine were present in significant amounts (16% and 12% of all metabolites in non-pregnant animals, respectively). Four other metabolites of NIC were also detected: norcotinine, cotinine-N’-oxide, 3’-hydroxycotinine, and 5’-hydroxycotine (). The results from a quantitative assessment of COT metabolism in rabbit liver microsomes are presented in . COT was metabolized to four metabolites. Norcotinine, cotinine-N’-oxide, and 5’-hydroxycotinine were formed in similar amounts and there were no differences in formation rates between liver microsomes from non-pregnant or pregnant rabbits. The rate of formation of 3’-hydroxycotinine was only a fraction of the rate of formation of norcotinine, cotinine-N’-oxide or 5’-hydroxycotinine.
COT formation from NIC was also compared in rabbit adult liver, fetal liver, and placental microsomes (). The rate of COT formation was 752 ± 227 pmol/min/mg protein in liver microsomes from non-pregnant rabbits and was much higher than corresponding rates in placental and fetal liver microsomes. In placental microsomes, NIC metabolism to COT was only seven percent of that in liver (53 ± 17.2 pmol/min/mg protein) and COT formation was barely detectable in fetal liver (14.3 ± 7.1 pmol/min/mg protein).
Figure 2 Cotinine formation in microsomes from rabbit adult non-pregnant and pregnant liver, placenta and fetal liver. Microsomal proteins (1 mg/ml for adult livers and placenta for 30 min or 2 mg/ml for fetal liver for 60 min) were incubated at 37°C with (more ...)
Total P450 content was similar in liver microsomes of pregnant and non-pregnant animals (11.1 ± 4.12 vs. 10.4 ±1.95 nmol/mg protein, respectively). The CYP content in the rabbit placental microsomes (3.49 ± 1.99 nmol/mg protein) was only 30% of that in pregnant rabbit liver microsomes and that in fetal liver microsomes (0.96 ± 0.68 nmol/mg protein) was 9% of that in pregnant rabbit liver microsomes.
Western blots of hepatic microsomes with an antibody against human CYP2A6 were used to compare the levels of CYP2A immunoreactive proteins in liver microsomes from non-pregnant and pregnant rabbits. Unfortunately, the cross-reactivity of this antibody with the rabbit CYPs has not been characterized but this antibody is highly specific for human CYP2A6 and shows no cross-reactivity with CYP1A, CYP1B, CYP2B, CYP2C, CYP2D or CYP3A human isoforms. The human CYP2A6 antibody also detects CYP2E1 with a lower mobility than CYP2A6 (Gentest product information). Western blot detection of rabbit liver microsomes with this antibody results in two distinct immunoreactive proteins and we are assuming that CYP2A6 is the upper band (). The level of both immunoreactive proteins showed significant intra-rabbit variability, but there was no apparent difference in the level of expression between pregnant and non-pregnant rabbit liver microsomes.
Figure 3 Immunoblotting of CYP2A6 immunoreactive proteins in non-pregnant and pregnant rabbit liver. Microsomal proteins (50 μg) were separated on a 8% SDS-polyacrylamide gel, transferred to nitrocellulose and blotted with antisera against human CYP2A6. (more ...)