Reduction of aromatic and heterocyclic aromatic N-hydroxylamines by human cytochrome P450 2S1

Abstract

Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals, and there is also strong evidence of some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anticancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions [Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740-1751]. In the study presented here, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs.

Figure 1

Anaerobic incubations of 4-HONH-biphenyl with P450 2S1. Freshly prepared 4-HONH-biphenyl (5 µL of a 10 mM solution in (CH₃)₂SO) was incubated with P450 2S1 (total volume 0.5 mL) at 37 °C for 15 min under anaerobic conditions. (A) 4-HONH-biphenyl in 100 mM sodium HEPES buffer (pH 7.4) containing 1 mM EDTA, incubated at 37 °C for 15 min. (B) 4-HONH-biphenyl incubated with all P450 system components with the exception of P450 2S1. (C) 4-HONH-biphenyl incubated with all components of the P450 2S1 system, with the exception of the NADPH-generating system. (D) 4-HONH-biphenyl incubated with P450 2S1 in the presence of NPR and an NADPH-generating system. (E) Synthetic standard of 4-HONH-biphenyl. UV absorbance was integrated over the range 200–400 nm.

Figure 2

LC-MS spectra of incubation products of 4-HONH-biphenyl with P450 2S1. (A) 4-ABP (t_R 4.6 min). (B) 4-HONH-biphenyl(t_R 5.5 min). (C) 4-NO-biphenyl (t_R 6.8 min). (D) Azoxybiphenyl (8.1 min).

Figure 3

Anaerobic incubations of NO-5F 203 with P450 2S1. Freshly prepared NO-5F 203 (containing trace amounts of 5F 203 and HONH-5F 203, 5 µL of an approximately 2 mM solution in (CH₃)₂SO) was incubated with P450 2S1 (total volume 0.5 mL) at 37 °C for 30 min under anaerobic conditions. (A) NO-5F 203 in 100 mM sodium HEPES buffer (pH 7.4) containing 1 mM EDTA, incubated at 37 °C for 30 min. (B) NO-5F 203 incubated with all P450 system components with the exception of P450 2S1. (C) NO-5F 203 incubated with all components of the P450 2S1 system with the exception of the NADPH-generating system. (D) NO-5F 203 incubated with P450 2S1 in the presence of NPR and an NADPH-generating system. (E) Synthetic standard of NO-5F 203. UV absorbance was integrated over the range 200–400 nm.

Figure 4

Anaerobic incubations of 4-NO-biphenyl with P450 2S1. Freshly prepared 4-NO-biphenyl (containing trace 4-ABP and azoxybiphenyl, 5 µL of an approximately 2 mM solution in (CH₃)₂SO) was incubated with P450 2S1 (total volume 0.5 mL) at 37 °C for 30 min under anaerobic conditions. (A) 4-NO-biphenyl in 100 mM sodium HEPES buffer (pH 7.4) containing 1 mM EDTA, incubated at 37 °C for 30 min. (B) 4-NO-biphenyl incubated with all P450 system components with the exception of P450 2S1. (C) 4-NO-biphenyl incubated with all components of the P450 2S1 system with the exception of the NADPH-generating system. (D) 4-NO-biphenyl incubated with P450 2S1 in the presence of NPR and an NADPH-generating system. (E) Synthetic standard of 4-NO-biphenyl. UV absorbance was integrated over the range 200–400 nm.

Figure 5

Anaerobic incubations of NO₂-IQ and 2-NO₂-naphthalene with P450 2S1. NO₂-IQ (5 µL of a 10 mM solution in (CH₃)₂SO) was incubated with P450 2S1 (total volume 0.5 mL) at 37 °C for 20 min under anaerobic conditions. (A) NO₂-IQ incubated with all P450 system components with the exception of P450 2S1. (B) NO₂-IQ incubated with P450 2S1 in the presence of NPR and an NADPH-generating system. (C) 2-NO₂-naphthalene incubated with all P450 system components with the exception of P450 2S1. (D) 2-NO₂-naphthalene incubated with P450 2S1 in the presence of NPR and an NADPH-generating system. UV absorbance was integrated over the range 200–400 nm.

Figure 6

Binding spectra for titration of P450 2S1 with HONH-5F 203. The calculated K_d was 30 ± 10 µM.

Figure 7

Catalysis of anaerobic reduction of HONH-5F 203 by P450 2S1 as a function of substrate concentration.

Scheme 1

Structures of carcinogenic aromatic and heterocyclic aromatic amines

Scheme 2

Biotransformation pathways of aromatic and heterocyclic aromatic amines

Scheme 3

Reduction of aromatic nitroso and nitro compounds by NADPH and P450 2S1