Article Figures & Data
Figures
- Scheme 1.
Chemical structures of NC (A), ERF (B), and E2 (C).
- Fig. 1.
NC-Induced inhibition of EROD activity catalyzed by CYP1 enzymes. CYP1A1, CYP1B1, or CYP1A2 (5 nM) was incubated with ERF (2.0 μM) and NC (0–100 μM) at 37°C for 10 minutes, followed by assessment of remaining enzyme activity. Data represent the mean ± S.D. (n = 3).
- Fig. 2.
Time- and NADPH-dependent inhibition of CYP1A1 (A) and CYP1B1 (B) by NC. CYP1A1 or CYP1B1 (50 nM) was incubated with vehicle (●) and NC (100 μM) in the presence (▲) or absence (■) of NADPH at 37°C for 0, 5, 15, and 30 minutes. Aliquots of the incubation mixtures were transferred to secondary incubation mixtures for determination of residual enzymatic activity.
- Fig. 3.
Kinetic analysis of NC-induced inhibition of EROD activity in a human recombinant CYP1A1 or CYP1B1 system. (A and C) show the velocity (v) vs. ERF concentration (S) plots of activity in the presence of NC at various concentrations as indicated. (B and D) show the Lineweaver-Burk plots for NC-induced inhibition of EROD activity mediated by CYP1A1 and CYP1B1. Data represent the mean ± S.D. (n = 3).
- Fig. 4.
Three-dimensional structural docking of NC with CYP1A1 (A), CYP1B1 (B), and CYP1A2 (C). The nitrogen and oxygen atoms are shown in dark blue and red, respectively. The heme prosthetic group of P450 enzymes is shown in magenta with iron in dark red. The hydrogen bond formation between NC and the amino acid residues of apoprotein of CYP1 enzymes is shown by yellow dashed lines.
- Fig. 5.
NC-induced inhibition of E2 2-hydroxylation (A) and 4-hydroxylation (B) activities catalyzed by CYP1 enzymes. CYP1A1, CYP1B1, or CYP1A2 (10 nM) was incubated with E2 (20 μM) and NC (0–100 μM), followed by assessment of remaining enzyme activity. Data represent the mean ± S.D. (n = 3).
- Scheme 2.
Proposed effect of NC on E2 chemical carcinogenesis. CYP1A1 and 1A2 primarily catalyze the formation of 2-OHE2 (detoxification biomarker). CYP1B1 primarily catalyzes the formation of 4-OHE2 (genotoxic biomarker), which is further oxidized to genotoxic estradiol-3,4-quinone (3,4-E2-Q). NC has been reportedly demonstrated to markedly decrease 2-hydroxylation and relatively increase 4-hydroxylation metabolism of E2, as shown by the green and red arrows.
- Fig. 6.
Kinetic analysis of NC-induced inhibition of E2 hydroxylation activity in a human recombinant CYP1A1 system. (A and C) show the velocity (v) vs. E2 concentration (S) plots of activity in the presence of NC at various concentrations as indicated. (B and D) show the Lineweaver-Burk plots for NC-induced inhibition of E2 hydroxylation activity mediated by CYP1A1. Data represent the mean ± S.D. (n = 3).
- Fig. 7.
Effect of NC on the formation of E2-hydroxylation metabolites (2- and 4-OHE2) in MCF-7 cells. Data represent the mean ± S.D. (n = 3).
Tables
- TABLE 1
NC- and α-NF–induced inhibition of EROD activity of CYP1A1, CYP1B1, and CYP1A2
Data represent the mean ± S.D. (n = 3).
Inhibitor IC50 CYP1A1 CYP1B1 CYP1A2 μM μM μM NC 0.28 ± 0.06 0.32 ± 0.02 >50 α-NF 0.12 ± 0.03 0.06 ± 0.01 0.51 ± 0.12 - TABLE 2
Kinetic parameters of NC-induced inhibition of EROD activity of CYP1A1, CYP1B1, and CYP1A2
Data represent the mean ± S.D. (n = 3).
CYP1 Enzyme EROD Type of Inhibition Ki KI μM μM 1A1 Mixed 0.14 ± 0.04 0.19 ± 0.09 1B1 Competitive 0.47 ± 0.05 - TABLE 3
NC-Induced inhibition of E2 hydroxylation activity of CYP1A1, CYP1B1, and CYP1A2
Data represent the mean ± S.D. (n = 3).
CYP1 Enzyme E2 Hydroxylation Site Km Vmax Vmax/Km Activity Ratio (2-/4-) IC50 IC50 Ratio (2-/4-) μM nmol/min per nanomoles P450 ml/min per nanomoles P450 μM 1A1 2-OHE2 3.38 ± 0.69 6.91 ± 0.33 2.04 255 0.16 ± 0.004 0.64 4-OHE2 28.9 ± 11.5 0.22 ± 0.04 0.008 0.25 ± 0.04 1B1 2-OHE2 2.26 ± 0.30 0.45 ± 0.04 0.20 0.14 35.0 ± 6.44 4.23 4-OHE2 0.87 ± 0.16 1.21 ± 0.12 1.39 8.27 ± 2.40 1A2 2-OHE2 23.7 ± 4.06 50.6 ± 4.93 2.14 55 13.2 ± 5.56 2.47 4-OHE2 24.8 ± 4.78 0.97 ± 0.14 0.04 5.35 ± 0.62 2-/4-, 2-/4-hydroxylation.
- TABLE 4
Kinetic parameters of NC-induced inhibition of E2 hydroxylation activity of CYP1A1, CYP1B1, and CYP1A2
Data represent the mean ± S.D. (n = 3).
CYP1 Enzyme E2 Hydroxylation E2 Hydroxylation Site Type of Inhibition Ki KI μM μM 1A1 2-OHE2 Mixed 0.052 ± 0.007 0.210 ± 0.016 4-OHE2 Competitive 0.26 ± 0.03
