T.T. Denton et al. / Biochemical Pharmacology 67 (2004) 751–756
753
the Cheng–Prusoff equation. For each assay, the reaction
was a linear function of time for 60 min and of protein
concentration from 0.5 to 2 pmol per reaction well.
final concentrations of 2.5, 4, and 8 mM (b-nicotyrine) and
16, 25, and 50 mM (anatabine) were combined and the
incubations were initiated by the addition of an NADPH-
generating system as described above for a final incubation
volume of 0.2 mL. After incubation of the enzyme for
various time periods in the presence of b-nicotyrine (378)
and anatabine (258), the incubations were terminated by
transferring 10 mL of the reaction mixture to vials at ꢀ788.
In a second step, the percent remaining coumarin 7-hydro-
xylation activity was determined after addition of 190 mL
of a pre-made solution containing the NADPH-generating
system described above and coumarin (i.e. final concen-
tration 3 mM) to the enzyme. After incubation for an
appropriate amount of time, the incubations were termi-
nated by addition of 0.75 mL of CCl COOH/CH CN
2
.3.2. Human CYP2E1 and CYP2B6 assays
To measure CYP2E1 and CYP2B6 functional activity,
isozyme-specific vivid blue substrate O-dealkylation was
used via a modified PanVera Vivid Assay as previously
described [19]. b-Nicotyrine concentrations (i.e. 400, 100,
2
plate (BD Falcon Microtest, Black Flat Bottom) containing
5, 6.3, 1.6, 0.4, 0.1, and 0.02 mM) were added to a 96-well
1
Vivid Substrate (final concentration: CYP2E1 assay,
10 mM; CYP2B6 assay, 5 mM) in 0.2 M potassium phos-
phate buffer (pH 8.0), followed by the appropriate BACU-
LOSOMES (enzyme final concentration: CYP2E1 assay,
5
3
3
nM; CYP2B6 assay, 10 nM). The incubations were
(20:80, w/v) and the formation of 7-hydroxycoumarin
was determined as described above.
initiated by the addition of the NADPH-generating system
for a final incubation volume of 0.2 mL. After a 40-min
incubation at RT, the formation of the fluorescent, O-deal-
kylated metabolite for each isozyme was determined fluor-
ometrically as above at excitation and emissions
wavelengths of 405 and 460 nm, respectively. Apparent Ki
values were determined as described above. For each assay,
the reaction was a linear function of time for 60 min and of
protein concentration from 0.5 to 2 pmol per reaction well.
3. Results and discussion
To examine the inhibitory potency of minor tobacco
alkaloids and nicotine metabolites on human CYP2A6
functional activity, a microtiter plate fluorescence assay
monitoring coumarin 7-hydroxylation was employed [21].
The results are listed in Table 1. Among the nicotine-
related alkaloids and metabolites tested, nornicotine, coti-
2
.3.3. Human CYP3A4 assay
An HPLC enzyme assay for inhibition of human liver
0
0
0
1 ꢀ5
nine, nicotine N-1 -oxide, nicotine D
-iminium ion,
0
microsomal testosterone 6-hydroxylase was done as pre-
viously described [20]. b-Nicotyrine concentrations used
were 44, 15, 4.94, 1.65, and 0.55 mM in the presence of
testosterone (200 mM) and human liver microsomes
N -methylanabasine, and nicotelline failed to inhibit the
formation of 7-hydroxycoumarin by 50% at the highest
concentration tested (i.e. 400 mM). Thus, these compounds
were classified as ‘‘non-inhibitors’’ of CYP2A6. Anaba-
seine, myosmine, and S-norcotinine showed modest inhi-
(0.4 mg of protein). Organic extracts were injected onto
a Hitachi L-7100 system equipped with a Hitachi L-7400
UV detector. Separations were done with an Altex Ultra-
sphere ODS (4.6 mm  250 mm, 5 mm) column. The ana-
lytes were eluted with an isocratic solvent system
consisting of water/acetonitrile/methanol (30:10:60, v/v/
v) at a flow rate of 1.0 mL/min. Testosterone and 6-
hydroxytestosterone were efficiently separated by this
system with retention times of 7.95 and 3.94 min, respec-
tively. Quantification of substrate and metabolite was
determined from peak areas of the chromatogram and
comparison with standard curves. IC50 values were deter-
mined as described above. For each assay, the reaction was
a linear function of time for 60 min and of protein con-
centration from 0.2 to 1 mg of protein per reaction well.
bition of CYP2A6 with K values of 20, 31, and 46 mM,
i
0
respectively. Anabasine and 2,3 -bipyridyl were consider-
ably more potent inhibitors with K values of 5.4 and
i
7.7 mM, respectively, and these K values approached that
i
of nicotine itself (4.4 mM). Two tobacco alkaloids, namely,
b-nicotyrine (0.37 mM), and anatabine (3.8 mM), had Ki
values lower than that of nicotine and b-nicotyrine was the
most potent. b-Nicotyrine (ic50 ¼ 2:2 mM for CYP2A6)
was tested for selectivity among biologically relevant
CYPs by determining the IC50 values for inhibition of
CYP3A4, CYP2E1, and CYP2B6 by high throughput
fluorometric and HPLC assays. The IC50 values for
b-nicotyrine-mediated inhibition of CYP2B6, CYP3A4,
and CYP2E1 were 145:2 ꢁ 13:0 mM, 18:8 ꢁ 2:8 mM, and
1
:8 ꢁ 0:3 mM, respectively. To examine the selectivity of
2.4. Determination of human CYP2A6
mechanism-based inhibition
inhibition, the selectivity ratios of CYP2B6/CYP2A6,
CYP3A4/CYP2A6, and CYP2E1/CYP2A6 were calcu-
lated to be 66.0, 8.5, and 0.8, respectively. The mechanism
of CYP2A6 inhibition by the most potent nicotine minor
alkaloids and metabolites was examined by determining
whether inhibition was time dependent. We evaluated
nicotine, b-nicotyrine, and anatabine for time-dependent
To determine if b-nicotyrine and anatabine are mechan-
ism-based inhibitors of CYP2A6, time-dependent inhibi-
tion of coumarin 7-hydroxylation was determined. First,
microsomes containing 20 pmol CYP2A6 were added to
0
.1 M Tris buffer (pH 7.5). Individual inhibitor studies with
inactivation of CYP2A6. Only nicotine (i.e. kinactivation,