Spectral modification and catalytic inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2A6, and 2A13 by four chemopreventive organoselenium compounds

Abstract

Several organoselenium compounds including benzyl selenocyanate (BSC), 1,2-phenylenebis(methylene)selenocyanate (o-XSC), 1,3-phenylenebis(methylene)selenocyanate (m-XSC), and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) have been shown to prevent cancers caused by polycyclic aromatic hydrocarbons (PAHs) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in experimental animals; these chemical carcinogens are activated by human P450 1 and 2A family enzymes, respectively, to carcinogenic metabolites. In this study, we examined whether these selenium compounds interact with and inhibit human P450 1 and 2A enzymes in vitro. Four organoselenium compounds induced reverse Type I binding spectra with P450 1A1, 1A2, and 1B1 and Type I binding spectra with P450 2A6 and 2A13. The spectral dissociation constants (K(s)) for the interaction of P450 1B1 with these chemicals were 3.6-5.7 μM; the values were lower than those with seen with P450 1A1 (19-30 μM) or 1A2 (6.3-13 μM). The K(s) values for Type I binding of P450 2A13 with m-XSC and BSC were both 0.20 μM; the values were very low compared to those for the interaction of P450 2A6 with m-XSC (5.7 μM) and BSC (2.0 μM). Four selenium compounds directly inhibited 7-ethoxyresorufin O-deethylation activities catalyzed by P450 1A1, 1A2, and 1B1 with IC(50) values <1.0 μM, except for the inhibition of P450 1A2 by BSC (1.3 μM). Coumarin 7-hydroxylation activities of P450 2A13 were more inhibited by four selenium compounds than those of P450 2A6, with IC(50) values of 0.22-1.4 μM for P450 2A13 and 2.4-6.2 μM for P450 2A6. Molecular docking studies of the interaction of four organoselenium compounds with human P450 enzymes suggest that these chemicals can be docked into the active sites of these human P450 enzymes and that the sites of the selenocyanate functional groups of these chemicals differ between the P450 1 and 2A family enzymes.

Figure 1

Chemical structures of selenium compounds used in this study.

Figure 2

Spectral interaction of BSC with P450 1A1 (A and D), p-XSC with P450 1A2 (B and E), and m-XSC with P450 1B1 (C and F). Absolute spectra (A, B, and C) and difference spectra (D, E, and F) were recorded between 350 and 500 nm.

Figure 3

Spectral interaction of BSC with rat liver P450 1A1 (A), rat liver 1A2 (B and D), and rabbit liver P450 1A2 (C and E). BSC (at concentrations of 1.25, 2.5 5.0, 10, 20, 40, and 80 µM) were added to the buffer with 1.5 µM rat P450 1A1 or 1A2 or 1.0 µM rabbit P450 1A2 and the spectra were recorded between 350 and 700 nm for rat P450 1A1 (A) and 350 and 500 nm for rat and rabbit P450 1A2 (B and C). The difference spectra of interaction of P450s with selenium compounds are shown in Figures 3D and 3E.

Figure 4

Spectral interaction of BSC with P450 2A6 (A, and B) and P450 2A13 (D and E). Chemicals (at concentration of 0.078–160 µM) were added to the buffer with or without 1 µM each P450 and the spectra were recorded between 350 and 700 nm (A and D). The difference spectra of interaction of P450s with selenium compounds are shown in Figures 4B and 4E. The concentration dependent interaction of BSC with P450 2A6 and 2A13 are shown in Figures 4C and 4F, respectively.

Figure 5

Time-dependent changes in the formation of resorufin by incubating 7-ethoxyresorufin with P450 1B1 in the absence (Control) and in the presence of 0.0083 µM, 0.033 µM, 0.083 µM, 0.33 µM, and 0.83 µM m-XSC in Figure 5A and in the presence of 0.083 µM o-XSC, 0.083 µM m-XSC, 0.083 µM p-XSC, and 0.083 µM BSC in Figure 5C. E. coli membranes expressing P450 1B1 and NADPH-P450 reductase were mixed with 7-ethoxyresorufin and chemical inhibitors, and the reactions were started by adding NADPH. The semi-logarithmic plots of the percent relative activity (activities with vs without inhibitors) are shown in different concentrations of m-XSC (B) and 0.083 µM each of o-, m-, and p-XSC and BSC (D).

Figure 6

Effects of preincubation time on the inhibition of P450 1B1-dependnet EROD activities by m-XSC. E. coli membranes expressing P450 1B1 and NADPH-P450 reductase were first incubated without (Control) or with 0.033 µM, 0.083 µM, and 0.33 µM m-XSC in the presence of NADPH. 7-Ethoxyresorufin was added at incubation time of 0 min (A, without preincubation), 2 min (B, with preincubation for 2 min), and 4 min (C, with preincubation for 4 min) and the formation of resorufin was determined as a function of time.

Figure 7

Docking simulation of interaction of m-XSC with P450 1A1 (A), 1A2 (B), and 1B1 (C). The heme group of the P450 is shown at the lower part of each of the figures and the amino acid residues that may interact with m-XSC are presented. In the figure, oxygen, nitrogen, sulfur, selenium, and iron are colored with red, blue, yellow, dark yellow, and light blue, respectively. Heme and m-XSC are shown in thick lines and colored with red and gray, respectively. The distances (shown in lines with green) between the N-atom in the −CH₂SeCN moieties of m-XSC and the Fe- atom in P450 1A1, 1A2, and 1B1 are also shown in part 7D, 7E, or 7F, respectively.

Figure 8

Docking simulation of m-XSC with P450 2A6 (A) and 2A13 (B). The distance between the N-atom in the −CH₂SeCN moieties of m-XSC and the Fe-atom in P450 2A6 or 2A13 is also shown in part 8C or 8D, respectively. Other details are the same as in the legend to Figure 7.