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Formation and Reduction of Aryl and Heterocyclic Nitroso Compounds and Significance in the Flux of Hydroxylamines Donghak Kim,† Fred F. Kadlubar,‡ Candee H. Teitel,‡ and F. Peter Guengerich*,† Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Division of Molecular Epidemiology, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received December 22, 2003
Cytochrome P450 (P450) 1A2 and NADPH-P450 reductase (NPR) catalyzed the oxidation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), with consumption of NADPH. The oxidation rate of NADPH by P450 1A2/NPR increased with time in the presence of IQ until depletion of NADPH. This unusual autocatalytic pattern of NADPH oxidation could be rationalized by formation of a nitroso derivative (IQ-NdO) and the subsequent reduction of the hydroxylamine (IQ-NHOH) and IQ-NdO, which would consume more NADPH. The formation of IQ-NHOH and IQ-NdO from IQ was confirmed using HPLC/MS. Reduction of IQ-NHOH and IQ-NdO was NPR-dependent but did not require P450. Autocatalytic NADPH oxidation was also observed in the oxidation of other heterocyclic and arylamines. However, the N-hydroxyl and nitroso oxidation products of 2-aminofluorene and 4-aminobiphenyl were reduced nonenzymatically by NADPH, and NPR did not catalyze the reactions. We simulated the enzymatic kinetic model for possible pathways for IQ metabolism, which included the formation of IQNdO, using some kinetic parameters obtained from the experimental results. In the kinetic model, we could reproduce the similar curvature for NADPH oxidation and the formation of IQ-NdO, and the reduction of IQ-NHOH and IQ-NdO is required to explain the observed results for NADPH oxidation. Our results support a role for nitroso derivatives of HAAs in the unusual autocatalytic NADPH oxidation and may have relevance in terms of possible toxicities of the nitroso derivatives. Both IQ-NHOH and IQ-NdO were mutagenic in a bacterial tester system devoid of P450 and NPR; the mutagenicity of both was decreased by expression of NPR, consistent with the reduction of these compounds observed with purified NPR.
Introduction
Scheme 1. Structures of HAAs and Arylamines
HAAs1
are formed in burned foods as a result of pyrolysis during cooking and are also produced during other pyrolytic reactions, e.g., cigarette smoking (1-3). Many of these compounds are potent bacterial mutagens and potential human carcinogens (1, 4, 5). The HAAs are activated to mutagenic or carcinogenic intermediates by N-oxidation reactions, which are mediated primarily by P450 1A2 but also by P450s 1A1 and 1B1 (6-10). These transformations are the same as those established for the carcinogenic arylamines (11, 12). The resulting N-hydroxy products of HAAs and arylamines can be further activated by acetylation, which yields reactive N-acetoxy esters (13-17), or sulfation [to form reactive sulfuric acid esters (18)]. The reactive ester derivatives yield aryl nitrenium ion species, which react to form DNA adducts (19-21). * To whom correspondence should be addressed. Tel: 615-322-2261. Fax: 615-322-3141. E-mail:
[email protected]. † Vanderbilt University School of Medicine. ‡ National Center for Toxicological Research. 1 Abbreviations: 4-ABP, 4-aminobiphenyl; 2-AF, 2-aminofluorene; APCI, atmospheric pressure chemical ionization; DMSO, dimethyl sulfoxide; HAA, heterocyclic aromatic amine; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; IQ-NHOH, 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline; IQ-NdO, 2-nitroso-3-methylimidazo[4,5-f]quinoline; MeIQ, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NPR, NADPH-P450 reductase; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
IQ is one of the mutagenic and carcinogenic HAAs (14). In the course of studies on the rates of IQ oxidation by a P450 1A2/NPR system, we observed unusual patterns of NADPH oxidation. The rate increased with time in the presence of HAAs, in an apparently autocatalytic manner. No oxidation was observed in the absence of enzymes. We ruled out several possible artifacts in repeated experiments, and studies with several HAAs and arylamines (Scheme 1) showed similar phenomena. One potential explanation was Scheme 2, with additional reactions to consume the initial product and regenerate it. This pathway includes generation of a nitroso (or possibly a nitro) product, which is formed by further
10.1021/tx034267y CCC: $27.50 © 2004 American Chemical Society Published on Web 03/05/2004
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Scheme 2. Proposed Mechanism for Redox Cycling of HAAs, Involving Either a P450/NPR Complex (Oxidations) or a NPR Only (Reductions)
oxidation of the N-hydroxy product. A pathway such as this could consume more NADPH as the reaction proceeded and has some possible precedent in the literature of arylamine metabolism (22). Aryl nitroso and hydroxylamine compounds, including some drugs, can undergo reduction reactions in in vivo animal models and also in vitro, with concomitant depletion of thiols (22-25). NADH-cytochrome b5 reductase and cytochrome b5 catalyze reduction of N-hydroxy arylamines (26, 27). Some N-hydroxyl HAAs, including IQ-NHOH, are also reduced by this NADH-dependent system in human liver microsomes (26). Several reports have appeared documenting the conversion of N-hydroxyfluorene (and the N-acetyl derivative) to nitroso products in a radical reaction (28-31). However, the metabolism of HAAs is understood primarily in the context of hydroxylamines, acetylation products, and glucuronides (32, 33). Some of the oxidation products of 3-amino-1-methyl-5H-pyrido-[4,3-b]indole and 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (commonly referred to as Trp-P-2 and Glu-P-1, respectively) had properties consistent with the behavior of nitroso derivatives (but were not characterized by chemical spectroscopy) (34, 35). However, to our knowledge, there have not been any subsequent reports of HAA nitroso derivatives. In this study, we provide evidence for nitroso formation and redox cycling in IQ metabolism by P450 1A2 and NPR. We also consider the possible relevance of this product in terms of contribution to mutagenicity and toxicity.
Experimental Procedures Caution: IQ, MeIQ, MeIQx, PhIP, 2-AF, 4-ABP, and their derivatives are mutagens and/or carcinogens and should be handled with appropriate precautions. Chemicals and Synthesis. IQ, IQ-NHOH, MeIQ, MeIQx, and PhIP were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). 2-AF and 4-ABP were obtained from Sigma Chemical Co. (St. Louis, MO). Other chemicals were of the highest grade commercially available. N-Hydroxy derivatives of 2-AF and 4-ABP were synthesized as described (36). 2-Nitrosofluorene was prepared by the method of Lotlikar et al. (37), and 4-nitrosobiphenyl was synthesized by oxidation of 4-ABP using 3-chloroperoxybenzoic acid. Briefly, 4-ABP (93 mg, 0.55 mmol, dissolved in 2 mL of dry CHCl3) was added to a 3-fold molar excess of 3-chloroperoxybenzoic acid (in 8 mL of dry CHCl3) and set on ice for 10 min. The mixture was then applied to a silica gel (Grade 923) column (4 cm × 20 cm), which was washed with hexane and eluted with hexane:ethyl acetate (20:1, v/v). The eluted yellow band containing crude 4-nitrosobiphenyl was evaporated to dryness, redissolved in ethyl acetate, and applied to a second silica gel column (2 cm × 15 cm). After the column was washed with hexane, 4-nitrosobiphenyl was eluted with benzene:hexane (65:35, v/v). This material was evaporated to dryness and stored at -80 °C (>99% purity by HPLC, vide infra; electron impact mass spectrum M+ at m/z 183).
Figure 1. HPLC/MS chromatogram of synthetic IQ-NdO. IQ-NdO was prepared by ferricyanide oxidation of IQ-NHOH. Briefly, IQ-NHOH (1 mg, 4.7 µmol, dissolved in DMSO) was mixed with K3Fe(CN)6 (20 mg, 60 µmol, dissolved in H2O). After the reaction was stirred for 30 min at room temperature, the product was extracted with 2 volumes of ethyl acetate and dried under N2. The product was characterized by HPLC/MS (Figure 1) and UV spectroscopy (λmax 300 nm). Enzymes. Human P450 1A2 was expressed in Escherichia coli DH5RF′IQ and purified by Ni2+-nitrilotriacetate chromatography as described (38-40). Rat NPR was expressed in E. coli (TOPP 3 strain) containing the plasmid pOR263 and purified as described elsewhere (41). Rat (42) and rabbit (43, 44) P450 1A2 enzymes were purified from liver microsomes as described elsewhere. NADPH Oxidation. NPR was reconstituted with or without purified P450 1A2 enzymes (plus 45 µM L-R-dilauroyl-sn-glycero3-phosphocholine) for some steady state kinetic experiments (45). Reconstituted enzymes (total volume 1.0 mL, with 0.10 µM P450 1A2 and 0.20 µM NPR) were preincubated for 5 min at 37 °C in the presence of IQ, IQ-NHOH, or IQ-NdO (100 µM). Reactions were initiated with the addition of 15 µL of 10 mM NADPH (final concentration 150 µM) and the decrease in A340 was monitored (37 °C) using a Cary14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA). In experiments in which products were generated for analysis, the source of NADPH was a generating system (45). Enzyme Activity Assays and Cytochrome c Reduction. IQ oxidation to IQ-NHOH (by P450) was assayed using a colorimetric assay (18) that was modified for use with enzymatic assays (40). The kinetic parameters for IQ-NHOH reduction by NPR were determined using 0.40 µM NPR with a mixture of 45 µM L-R-dilauroyl-sn-glycero-3-phosphocholine, 100 mM potassium phosphate buffer (pH 7.4), and varying concentrations of IQ-NHOH (10-200 µM). The reactions were initiated by the addition of 0.15 mM NADPH and incubated for 10 min at 37 °C. All reactions were terminated by the addition of 2 volumes of CHCl3:2-propanol (60:40, v/v), and the products were recovered from the organic phase. Cytochrome c reduction (by NPR) was determined as described (46) using ∆550 ) 21 mM-1 cm-1 (47). HPLC and MS. IQ and its enzymatic products were analyzed by HPLC on a 5 µm Ultrasphere C18 column (4.6 mm × 250 mm; Beckman, Fullerton, CA) connected to a SpectroMonitor 3100 UV detector (Milton-Roy, Rivera Beach, FL). Metabolites were eluted at a flow rate of 1.0 mL min-1 using 50 mM NH4CH3CO2 buffer (pH 7.5) containing 30% CH3OH (v/v) as solvent A, 50 mM NH4CH3CO2 buffer (pH 7.5) containing 70% CH3OH (v/v) as solvent B, and 100% CH3OH as solvent C. The percentage of solvent B was increased linearly to 100% over 20 min; the percentage of CH3OH was then increased to 100% over 2 min and held for 13 min. HPLC was also carried out using a Waters 2690 module (Waters Corp., Milford, MA) coupled to a Finnigan TSQ7000 triple quadropole mass spectrometer (Ther-
Cycling of Hydroxylamines
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Figure 2. NADPH oxidation by P450 1A2 (and NPR) in the presence of HAAs. The initial NADPH concentration was 125-150 µM. A340 was measured in the presence of MeIQ, MeIQx, IQ, PhIP, 2-AF, or 4-ABP. moFinnigan, San Jose, CA) equipped with a standard API-1 APCI source running in positive mode. N2 was used as the sheath gas (70 psi); no auxiliary stream was applied. The vaporizer temperature was set to 450 °C, and the corona current was maintained at 5 µA. The capillary was set to 20 V and 230 °C, and the tube lens voltage was set to 60 V. UV absorbance (A300) was recorded in series using an Agilient 1100 series G1314A variable wavelength detector (Agilent, Palo Alto, CA), and the data acquisition and spectral analysis were conducted with Finnigan ICIS software on a Digital Equipment Corp. Alpha workstation. Binding Affinities to P450 1A2 and NPR. Binding affinities of P450 1A2 and NPR for IQ, IQ-NHOH, and IQ-NdO were estimated as described previously (39). The intrinsic fluorescence (tryptophan) of P450 1A2 and NPR was determined by exciting at 295 nm and detecting emission at 340 nm. The decrease of the fluorescence emission intensity at 340 nm was measured as a function of substrate concentration (IQ, IQNHOH, or IQ-NdO). Kinetic Models. Fitting to kinetic models was done using the program DynaFit (48), which is available upon request (http://www.biokin.com;
[email protected]). Examples of files used in this work, with program scripts, are presented in the Supporting Information. Bacterial Mutagenicity Assays. Genotoxicity assays with IQ-NdO and IQ-NHOH were performed using E. coli strain DJ3109pNM12, which bears a lacZ frameshift target on an F′ episome and a plasmid for expression of Salmonella typhimurium acetyl CoA:arylamine N-acetyltransferase (40, 49, 50). E. coli strain DJ3109pNM12pOR was constructed by transforming DJ3109pNM12 with the plasmid pOR for expression of NPR. E. coli strains DJ3109pNM12pOR and DJ3109pNM12 were cultured in TB expression medium at 30 °C. After a 24 h incubation, 50 µL aliquots of the culture were mixed with 5 mL of 0.7% top agar (w/v) containing 100 pmol of IQ-NdO or IQNHOH or only the vehicle DMSO. The mixture was immediately plated on lactose minimal agar. Colonies were counted after 40 h of incubation at 37 °C.
Results NADPH Oxidation by P450 1A2 in the Presence of HAAs and Arylamines. Preliminary experiments indicated that the oxidation rate of NADPH by a P450 1A2/NPR system increased with time in the presence of MeIQ, a model HAA that we have used as a promutagen in studies involving molecular breeding studies with P450 1A2 (40, 51). Four different HAAs and two arylamines showed patterns of increasing rates of NADPH oxidation (Figure 2). Of the six different amines, IQ and PhIP showed the most dramatic increases of NADPH oxidation rate (more than 10 times the initial rate after 4 min).
Figure 3. Steady state kinetics of IQ N-hydroxylation by P450 1A2 (in the presence of NPR and an NADPH-generating system). Each point represents the mean ( SD of triplicate assays. The estimated parameters are kcat ) 3.9 ( 0.1 min-1, Km ) 12 ( 1 µM, and kcat/Km ) 0.32 ( 0.03.
With IQ, A340 decreased in an autocatalytic manner until the depletion of NADPH (Figure 2). In the case of the arylamines 2-AF and 4-ABP, the changes in rate were less marked. N-Hydroxylation of IQ by P450 1A2 and NPR. Rates of IQ N-hydroxylation by purified P450 1A2 (in the presence of NPR) were measured, using a colorimetric assay that we had previously developed (22) and subsequently modified for increased sensitivity with reduced volumes (40) (Figure 3). Steady state kinetic analysis, using the initial reaction velocities, yielded an apparent kcat for IQ-NHOH formation of 3.9 ( 0.1 min-1, Km of 12 ( 1 µM, and catalytic efficiency (kcat/Km) of 0.32 min-1 µM-1. Characterization of Enzymatic Products of IQ N-Hydroxylation. Potential pathways of IQ oxidation are shown in Scheme 2. The enzymatic products of IQ N-hydroxylation were characterized using HPLC/MS (Figure 4). The major product was IQ-NHOH, shown to be identical with authentic commercial IQ-NHOH (tR ) 7.44 min). A second product was detected with a later retention time (tR ) 12.2 min) and identified as IQ-NdO by cochromatography with synthetic material and by its mass spectrum (Figure 4A, inset). NADPH Oxidation by IQ-NdO and IQ-NHOH. Incubation of IQ-NdO with NADPH and NPR yielded extensive conversion to IQ-NHOH and some IQ (Figure 5). The NADPH oxidation rate was measured with various concentrations of IQ-NdO in the presence of NPR (Figure 6A). Steady state kinetic analysis yielded the parameters kcat ) 49 ( 6 min-1 and Km ) 22 ( 7 µM.
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Steady state kinetic parameters for reduction of IQNHOH by NPR and NADPH were determined using HPLC analysis (Figure 6B): kcat ) 2.7 ( 0.2 min-1, Km ) 190 ( 23 µM, catalytic efficiency (kcat/Km) ) 0.014 min-1 µM-1. The NADPH oxidation rates by IQ-NdO and IQNHOH were also measured in the absence of NPR, and the nonenzymatic reactions were very slow as compared to NPR-assisted oxidation (
