Nitrosation and Nitration of 2-Amino-3-methylimidazo[4,5-f ]quinoline

Chem. Res. Toxicol. 2002, 15, 1059-1068

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Nitrosation and Nitration of 2-Amino-3-methylimidazo[4,5-f ]quinoline by Reactive Nitrogen Oxygen Species Vijaya M. Lakshmi,† Fong Fu Hsu,‡ and Terry V. Zenser*,†,§ VA Medical Center, Division of Geriatric Medicine, and Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63125, and Department of Medicine, Washington University, St. Louis, Missouri 63110 Received February 4, 2002

Both cooked red meat intake and chronic inflammation/infection are thought to play a role in the etiology of colon cancer. The heterocyclic amine 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) is formed during cooking of red meat and may be involved in initiation of colon cancer. Reactive nitrogen oxygen species (RNOS), components of the inflammatory response, contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible chemical transformation of IQ by RNOS. RNOS were generated by various conditions to react with 14C-IQ, and samples were evaluated by HPLC. Myeloperoxidase (MPO)catalyzed reaction was dependent upon both H2O2 and NO2-. This reaction produced an azoIQ dimer and IQ dimer along with two nitrated IQ products identified by ESI/MS. 2-Nitro-IQ was not detected. Product formation was inhibited by 2 mM cyanide. Reduction in nitrated products observed with 100 mM chloride was not altered with 0.5 mM taurine. Nitrated products were also produced by other conditions, ONOO- and NO2- + HOCl, which generate nitrogen dioxide radical. In contrast, conditions which generate N2O3, such as diethylamine NONOate, produced only small amounts of nitrated products with the major product identified by MS and NMR as N-nitroso-IQ. MPO activation of IQ to bind DNA was dependent upon both H2O2 and NO2-. RNOS generated by ONOO- and DEA NONOate also activated IQ DNA binding. The nitrated IQ products were not activated by MPO to bind DNA. In contrast, N-nitroso-IQ was activated to bind DNA by MPO ( NO2-. HOCl activated N-nitroso-IQ, but not IQ. RAW cells produced N-nitroso-IQ and increased amounts of NO2-/NO3-, when incubated with 0.1 mM IQ and stimulated with lipopolysaccharide and interferon gamma. Results demonstrate chemical transformation and activation of IQ by RNOS and activation of its N-nitroso product by biological oxidants, events which may contribute to initiation of colon cancer.

Introduction There is a strong association between diet and cancer (1). During the cooking process, a number of genotoxic heterocyclic amines are formed (2). These amines are derived from amino acids, creatine, and glucose present in meat. The heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)1 is a potent carcinogen, inducing rodent tumors in liver, small and large intestine, pancreas, lung, and mammary gland (2-4), and liver tumors in nonhuman primates (5). In the human diet, consumption of 400 g of cooked lean meat could result in exposure to several micrograms of mutagenic heterocyclic amines (6, 7), which have been detected in urine (8), indicating * To whom correspondence should be addressed at the VA Medical Center (GRECC/11G-JB), St. Louis, MO 63125. Phone: 314/894-6510, Fax: 314/894-6614, E-mail: [email protected]. † VA Medical Center, Division of Geriatric Medicine. ‡ Department of Medicine, Washington University. § Department of Biochemistry, St. Louis University School of Medicine. 1 Abbreviations: IQ, 2-amino-3-methylimidazo[4, 5-f]quinoline; CAD, collisionally activated dissociation; DEA, diethylamine; DETAPAC, diethylenetriaminepentaacetic acid; DMEM, Dulbecco’s modified Eagle’s medium; ESI, electrospray ionization; iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase; NO, nitric oxide; ONOO-, peroxynitrite anion; PHS, prostaglandin H synthase; RNOS, reactive nitrogen oxygen species.

10.1021/tx020008h

their absorption from cooked foods. Dietary heterocyclic amines can be divided into two classes depending on the presence or absence of an aminoimidazo ring. The aminoimidazo ring containing IQ-like compounds are distinguished by the resistance of their amino group to deamination by nitric acid (9). The strong association of cooked red meat intake and colorectal cancer risk (10), the presence of heterocyclic amines in cooked red meat (6, 7), and the initiation of colon cancer by heterocyclic amines (11) suggest that heterocyclic amines present in cooked red meat may be responsible for the increased risk of colon cancer associated with this dietary component. IQ requires metabolic activation to exert its genotoxic effects. IQ is oxidized by cytochrome P-450 1A2, in addition to 1A1 and 1B1, to N-hydroxy-IQ, and a highly selective inhibitor of the 1A2 enzyme increased excretion of dietary heterocyclic amines (12, 13). UDP-glucuronosyltransferases catalyze the formation of N 2- and N 3glucuronides, which are thought to be detoxification products (14). IQ-like amines, perhaps due to the exocyclic amine’s preferential existence as an imine tautomer, are not good substrates for N-acetyltransferases, but their N-OH products are esterified by phase II enzymes (O-acetyltransferase and sulfotransferase) (15-17). These

This article not subject to U.S. Copyright. Published 2002 by the American Chemical Society Published on Web 07/20/2002

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esters are thought to undergo heterolytic cleavage of their N-O bond to form nitrenium ion intermediates that attack nucleophilic centers on DNA, C8 and N2 of guanine (18). The mutagenic consequences of these DNA adducts are mainly transversions and frameshift mutations (19). Some studies have reported that individuals with the rapid acetylator genotype (NAT2) have an increased risk for developing colorectal cancer, but this is not a consistent observation (20, 21). Following IQ administration, DNA adducts are found in a variety of rodent and nonhuman primate tissues (22, 23). While there is a considerable amount of evidence that reactive intermediates are formed by hepatic cytochrome P-450s and transported to extrahepatic tissue, activation by other pathways may also occur in extrahepatic tissue (16). Peroxidases, i.e., prostaglandin H synthase (PHS), activate IQ and other heterocyclic amines, as well as aromatic amines (24-26). 2-Nitro-3-methylimidazo[4,5f ]quinoline is a stable mutagenic product of PHScatalyzed IQ metabolism (27). PHS activates IQ to form the same C8 guanine adduct as observed following cytochrome P-450/O-acetyltransferase activation (28, 29). Similarly, PHS and cytochrome P-450 activation of the aromatic amine N-acetylbenzidine yield the same C8 adduct. PHS metabolizes N-acetylbenzidine to 4′-nitro4-acetylaminobiphenyl with N′-hydroxy-N-acetylbenzidine an intermediate in this reaction (30). This intermediate is responsible for the same N-acetylbenzidine C8 guanine adduct observed by both these oxidative pathways, and a structurally similar N-OH intermediate may be involved in IQ activation by these pathways (29). IQ is highly mutagenic for N-acetyltransferase-proficient Salmonella typhimurium strains following oxidation by either cytochrome P-450 or PHS (24, 31). Peroxidases have a wide extrahepatic distribution, including sites of inflammation and injury, and could be responsible for activation of heterocyclic amines at these locations. Chronic inflammation/infection and injury play an important role in several types of cancer (i.e., bladder and colon) (32), which can be initiated by aromatic and heterocyclic amines (2, 33). The risk of colorectal carcinoma increases considerably in patients with chronic ulcerative colitis after 10 years with the accumulative risk for developing cancer 10-13% at 25 years of disease (34, 35). Findings in patients are paralleled in animal models with persistent severe inflammation in the colonic mucosa thought to cause development of colorectal cancer (36, 37). Reactive nitrogen oxygen species (RNOS) derived from the inflammatory response are thought to be responsible for some of the genotoxic effects attributed to this carcinogenic process. Upregulation of inducible nitric oxide synthase (iNOS) during inflammation produces high levels of nitric oxide (NO). NO interacts with either oxygen or superoxide to produce RNOS, including peroxynitrite anion (ONOO-), dinitrogen trioxide (N2O3), and nitrogen dioxide radical (NO2•) (38). Deamination of DNA by RNOS can lead to mispairing, depurination, and strand breakage, contributing to mutagenesis (39, 40). Many of the manifestations of inflammatory bowel disease are consistent with the biological effects of NO (41). High levels of iNOS and 3-nitrotyrosine, a marker of RNOS, are detected in colons from inflammatory bowel disease patients (42, 43) and genotoxic effects of autoxidizing NO have also been reported in both human colorectal tumors and ulcerative colitis patients (44, 45).

Lakshmi et al.

RNOS chemical transformation of the aromatic amine N-acetylbenzidine has recently been demonstrated (46). This carcinogen is both nitrosated and nitrated by RNOS. Whether this observation is limited to diaminobiphenyl carcinogens or can be applied to other aromatic and heterocyclic amines is not known. In view of the associations between cooked meat/inflammation and colon cancer described above, this study was designed to assess the possible chemical transformation of IQ by RNOS. This knowledge will provide a better understanding of the relationship between cooked red meat in the diet, chronic inflammation/infection and injury, and colon cancer.

Experimental Procedures Materials. [2-14C]-IQ (10 mCi/mmol, >98% radiochemical purity), 2-nitro-3-methylimidazo[4,5-f]quinoline, and 6-methylamino-5-nitroquinoline were purchased from Toronto Research Chemicals (Toronto, ON). NaNO2, NaOCl, H2O2, naphthalenediamine hydrochloride, sulfanilamide, nitrate reductase, calf thymus DNA, catalase (bovine liver), ascorbic acid, lipopolysaccharide (serotype 0111:B4), diethylenetriaminepentaacetic acid (DETAPAC), and NaCN were purchased from Sigma Chemical Co. (St. Louis, MO). Murine interferon gamma was purchased from PeproTech, Inc., Rocky Hill, NJ. Diethylamine (DEA) NONOate and myeloperoxidase from human polymorphonuclear leukocytes (180-220 units/mg of protein) were purchased from Calbiochem (San Diego, CA). Alkaline solutions of ONOO- were prepared from acidified NO2- and H2O2, and quantitated spectrophotometrically (302 ) 1.67 mM-1 cm-1) as described (47). Stock solutions were kept at -70 °C. Ultima-Flo AP was purchased from Packard Instruments (Meriden, CT). Caution: 2-Amino-3-methylimidazo[4,5-f]quinoline is carcinogenic to rodents and should be handled in accordance with NIH Guidelines for the Laboratory Use of Chemical Carcinogens (48). Syntheses. 3-Methylimidazo[4,5-f]quinoline was synthesized by heating a solution of 6-methylamino-5-nitroquinoline in formamide with ammonium formate followed by the addition of sodium sulfite as described by Waterhouse and Rapport (49). The CAD tandem mass spectrum of the MH+ at m/z 184 gives major fragment ions at m/z 169, 143, and 116, representing consecutive losses of CH3, C2H2, and HCN, respectively. 2-Hydroxy-3-methylimidazo[4,5-f]quinoline was prepared as a byproduct during the conversion of IQ to 2-nitro-3-methylimidazo[4,5f]quinoline with NO2- as described by Turesky et al. (16). The CAD tandem mass spectrum of the MH+ at m/z 200 yields a prominent ion at m/z 185, arising from loss of CH3, followed by loss of C2H2 or of HCN to give ions at m/z 159 or 158, respectively. An azo-IQ dimer and IQ dimer were made by HOCl oxidation in pH 7.4 phosphate buffer (9). By ESI/MS, this IQ dimer yields the MH+ ion at m/z 395, which produces CAD tandem mass spectrum major fragment ions at m/z 380, 365, 212, 199, and 197. For the azo dimer of IQ, ESI/MS yields the MH+ ion at m/z 393 producing CAD tandem mass spectrum major fragment ions at m/z 391, 378, 377, 376, 224, 211, 199, and 198. Chemical Transformation of IQ by RNOS. 14C-IQ (0.06 mM) was incubated in 100 mM potassium phosphate buffer, containing 0.1 mM DETAPAC in a total volume of 0.1 mL at 37 °C. For incubation with myeloperoxidase, 1 µg/mL peroxidase was added at pH 5.5 and the reaction started by addition of 0.1 mM H2O2. Incubations with DEA NONOate, ONOO-, or HOCl were started immediately following their addition at pH 7.4. The pH was checked after these incubations, and it did not change. Incubation times were 10 min for myeloperoxidase, 1 min for ONOO-, and 30 min for other conditions. Blank values were obtained in the absence of either RNOS generating agent or H2O2. The reactions were stopped by adding 1 µg of catalase to the MPO incubations or 3 mM methionine to ONOO- and

2-Amino-3-methylimidazo[4,5-f]quinoline Metabolism HOCl incubations. Dimethylformamide (0.05 mL) was added to all samples, which were immediately frozen. IQ chemical transformation was assessed by HPLC using solvent system 1 as described below. To assess activation of IQ by binding to DNA, 1 mg/mL DNA was added to the reaction mixtures described above. Calf thymus DNA was purified before use by extracting with phenol and a 24:1 chloroform/isoamyl alcohol mixture. After incubations with RNOS, DNA was precipitated by adjusting the concentration of NaCl to 0.25 M and addition of 2 volumes of ethanol, and left overnight at -20 °C (50). The DNA pellet was washed twice with 70% ethanol and dissolved in water, and the process of precipitation and washing was repeated. The radioactivity of the water-dissolved DNA was determined. The purity and quantity of DNA were assessed by the absorbance at 260 and 280 nm. A A260/A280 ratio of approximately 1.7 was achieved for each sample. Binding is expressed as picomoles per milligram of DNA. Metabolism of IQ by RAW Cells. Mouse macrophage RAW 264.7 cells were obtained from the Washington University Tissue Culture Support Center, St. Louis, MO, on the day of study. Cells were removed from growth flasks with 0.05% trypsin, 0.02% EDTA at 37 °C. Cells were washed 2 times with complete Dulbecco’s modified Eagle’s medium (DMEM) containing 100 units/mL penicillin and 100 µg/mL streptomycin, plated at a concentration of 5 × 106 cells in 35 mm plates with 1 mL of DMEM containing 10% FCS, and incubated under an atmosphere of 95% air and 5% CO2 at 37 °C. After 3 h, nonadherent cells were removed by washing with DMEM, and cells were incubated in the presence or absence of 10 µg/mL lipopolysaccharide and 150 units/mL interferon gamma with 0.1 mM IQ in 3 mL of DMEM without FCS. Media were saved after a 24-h incubation for analysis of NO2-/NO3- and N-NO-IQ. To prepare samples for N-NO-IQ analysis by mass spectrometry, media from several plates were pooled and applied to a Sep Pak column. The 100% methanol fraction was collected, evaporated, and sequentially purified using HPLC solvent system 2 at pH 3.1 and then pH 5.0 as described below. The recovery was estimated to be approximately 10%, using a corresponding media sample spiked with 14C-N-NO-IQ prepared by DEA NONOate nitrosation of IQ. HPLC Analysis of Metabolites. Metabolites were assessed using a Beckman HPLC with System Gold software and a 5 µm, 4.6 × 150 mm C-18 Ultrasphere column attached to a guard column. For solvent system 1, the mobile phase contained 20 mM ammonium formate buffer (pH 3.1) in 8% acetonitrile, 0-2 min; 8-16%, 2-10 min; 16-21%, 13-18 min; 21-35%, 18-23 min; 35-8%, 30-35 min; flow rate 1 mL/min. For solvent system 2, the mobile phase contained either 20 mM ammonium formate buffer (pH 3.1) or 20 mM ammonium acetate buffer (pH 5.0) in 8% acetonitrile, then 8-11%, 0-3 min; 11-13%, 9-18 min; 1335%, 20-25 min; 35-8%, 25-30 min; flow rate 1 mL/min. Radioactivity in HPLC eluents was measured using a FLO-ONE radioactive flow detector. Data are expressed as a percent of the total radioactivity or as picomoles recovered by HPLC. NO2-/NO3- Determination. NO2- and NO3- are degradation products of NO. NO2- is determined with the Griess assay after reduction of NO3- to NO2- with nitrate reductase (51). Absorbance is measured at 546 nm with the concentration of NO2- calculated using a NaNO2 standard curve. Mass Spectral Analysis. Electrospray ionization mass spectrometry (ESI/MS) analyses were performed on a Finnigan TSQ-7000 triple stage quadrupole spectrometer (San Jose, CA) equipped with a Finnigan ESI source and controlled by Finnigan ICIS software operated on a DEC alpha workstation. Samples were loop-injected onto the ESI source with a Harvard syringe pump at a flow rate of 1 µL/min. The electrospray needle and the skimmer were at ground potential, and the electrospray chamber and the entrance of the glass capillary were at 4.5 kV. The heated capillary temperature was 200 °C. For collisionally activated dissociation (CAD) tandem mass spectra, the collision gas was argon (2.3 mTorr), and the collision energy was set at

Chem. Res. Toxicol., Vol. 15, No. 8, 2002 1061

Figure 1. HPLC analysis of IQ oxidation by myeloperoxidase or DEA NONOate. 14C-IQ (0.06 mM) was incubated with MPO + 0.1 mM H2O2 + 0.3 mM NO2- for 10 min or with DEA NONOate (2 mM) in a capped tube for 30 min. 22 eV. Product ion spectra were acquired in the profile mode at the scan rate of one scan per 3 s. NMR Analysis. Samples were prepared in 100% dimethyl sulfoxide-d6 (Aldrich gold label) under dry nitrogen purge. About 1 mg of IQ and 0.25 mg of N-NO-IQ were dissolved in dimethyl sulfoxide and transferred into high-quality 5 mm tubes and capped under nitrogen. NMR spectra were acquired on a Varian Inova-500 instrument with the proton resonance frequency at 499.97 MHz with an INVERSE probe (1H 90 pulse of 9 µs). A total of 128 transients were signal-averaged with a recycle delay of 5 s and a 45° tip. The time domain data were processed on a SUN workstation using the VNMR software. A line-broadening parameter of 0.5 Hz was used for the Fourier transformation. Chemical shifts were referenced to TMS at 0.0 ppm.

Results IQ metabolism by different RNOS was assessed by HPLC (Figure 1). With MPO + H2O2, >91% of the radioactivity was recovered as IQ, and distinct metabolite peaks were not detected (not shown). Following addition of NO2- (nitrogen dioxide radical generation), new peaks appeared at 8.7 (NO2-IQ1), 12.6 (NO2-IQ2), and 21 (AzoIQ/IQ-D) min, representing 27, 18, and 24% of the total recovered radioactivity, respectively. The peak at 21 min coeluted with the synthetic standards for the azo-IQ dimer (Azo-IQ) and IQ dimer (IQ-D), while the peaks at 8.7 and 12.6 min were unknown. Elution of the 2-nitro-

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Lakshmi et al. Table 1.

1H

NMR Spectral Parameters for IQ and N-NO-IQa IQ

N-NO-IQ

chemical multiplicity chemical multiplicity assign- proton shift (no. of shift (no. of ment no. (ppm) protons) (ppm) protons) -NHR -N-CH3 -H -H -H -H -H

2 3 4 5 7 8 9

6.55 3.90 7.70 7.56 8.71 7.42 8.54

s (2*) s (3) d (1) dd (1) d (1) dd (1) d (1)

6.46 3.66 7.81 7.61 8.68 7.45 8.70

s (1*) s (3) d (1) dd (1) d (1) dd (1) d (1)

a Coupling constants (Hz) for IQ are J 4,5 ) 9, J8,9 ) 4, and J7,8 ) 4; for N-NO-IQ, values are J4,5 ) 8.5, J8,9 ) 4.0, and J7,8 ) 4.2. Abbreviations are s ) singlet and d ) doublet. *D2O exchangeable protons.

Figure 2. Positive ion ESI product-ion spectra of N-nitrosoIQ obtained from the reaction of IQ with DEA NONOate at collision energies of 25 eV (panel A) and 42 eV (panel B). In panel C, the MH+ ion of the metabolite isolated from stimulated RAW cells incubated with 0.1 mM IQ is illustrated at a collision energy of 42 eV.

3-methylimidazo[4,5-f ]quinoline standard precedes the dimers and was not accompanied by a corresponding peak of radioactivity. When DEA NONOate was used to generate RNOS, i.e., N2O3, the major peak was observed at 10.2 min (N-NO-IQ), representing 45% of the total recovered radioactivity with only minor peaks at 8.7 (4%) and 12.6 (6%) min (Figure 1, bottom panel). These results suggest IQ chemical transformation by different RNOS to different products. To identify the unknown NO2- -dependent metabolites of IQ chemical transformation by MPO, HPLC-purified material was subjected to analysis by both positive and negative mode ESI/MS. The 8.7 min product yields prominent protonated (MH+) and deprotonated (M - H)molecular ions at m/z 244 and 242, respectively, when subjected to ESI/MS. These masses correspond to the addition of a nitro group to IQ. The CAD tandem mass spectrum of MH+ gives ions at m/z 227, 197, and 170, representing consecutive losses of NH3, NO, and HCN, respectively. These results suggest that the 8.7 min product is a nitrated IQ metabolite (NO2-IQ1). The 12.6 min product also yielded both protonated (MH+) and deprotonated (M - H)- molecular ions at m/z 244 and 242, respectively. However, the MH+ ion yields a production spectrum distinguishable from that obtained from the former, suggesting that the 12.6 min product represents an isomeric nitro-IQ (NO2-IQ2). Investigation of the location of the nitro group on each of these IQ metabolites is in progress. The major product of IQ chemical transformation by DEA NONOate was isolated by HPLC and further

identified by ESI CAD tandem mass spectrometry. In the positive-ion mode, the 10.2 min product yields protonated (MH+) ions at m/z 228 (Figure 2, panel A), consistent with observed deprotonated (M - H)- molecular ions at m/z 226, in the negative-ion mode (data not shown). These results suggested that the metabolite is a nitroso derivative of IQ. Structural characterization is further supported by the product-ion spectrum of the MH+ ion at m/z 228, which gives ions at m/z 198 and 197, arising from loss of NO and HNO, respectively. Further loss of CH3 or HCN from m/z 197 gives rise to the m/z 182 or 170 ion, respectively (Figure 2, panel B). This is followed by loss of HCN to yield the ion at m/z 155 (182 - HCN) or 143 (170 - HCN). The structure of the metabolite was further confirmed by NMR spectroscopy (Table 1). The two anticipated metabolites of IQ nitrosation, namely, deaminated IQ, 3-methylimidazo[4,5-f]quinoline and 2-hydroxy-IQ, were not detected in significant amounts during nitrosation of IQ by DEA NONOate. 1 H NMR analysis was used to determine the structure of the major new product (N-NO-IQ) derived from the reaction of IQ with DEA NONOate (Table 1). 1H NMR spectral parameters of IQ were used for comparison. Aromatic protons at positions 4, 5, 7, 8, and 9 of IQ are present in the DEA NONOate product. In addition, the CH3 protons of IQ (3.90 ppm) at position 3 were also detected in the new product (3.66 ppm). While there are two D2O-exchangeable protons at position 2 for IQ (6.55 ppm), there is only one D2O-exchangeable proton in the new product (6.46 ppm). This is consistent with an NH proton. Values for IQ are in agreement with a previous publication (18). Thus, the 1H NMR spectrum of the new product is consistent with nitrosation occurring at the amino group forming N-nitroso-IQ. The stability of N-NO-IQ was assessed at 37 °C. After a 12 h incubation at pH 7.4 in phosphate buffer, only a 6% loss of N-NO-IQ was observed by HPLC. In contrast with 0.1 N HCl, N-NO-IQ was completely hydrolyzed in 30 min. With 0.1 N NaOH, a 43% loss of N-NO-IQ was detected in 30 min. Thus, while N-NO-IQ is unstable in

2-Amino-3-methylimidazo[4,5-f]quinoline Metabolism


Table 2. Chemical Transformation of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) by Different Reactive Nitrogen Oxygen Speciesa products formed (nmol) conditions

NO2-IQ1

N-NO-IQ

NO2-IQ2

MPO + 0.1 mM H2O2 + 0.3 mM NaNO2 -NO2-H2O2 +2 mM NaCN +100 mM NaCl +100 mM NaCl + 0.5 mM taurine 0.3 mM NaNO2 + 0.3 mM HOCl 0.1 mM ONOO0.3 mM ONOO+10 min preincubation before IQ addition 0.2 mM DEA NONOate capped tube 0.2 mM DEA NONOate uncapped tube

1.8 ( 0.08 ND ND 0.3 ( 0.02 0.5 ( 0.06 0.6 ( 0.02 0.1 ( 0.01 0.3 ( 0.03 0.8 ( 0.04 ND 0.05 ( 0.03 ND

ND ND ND ND ND ND ND ND ND ND 0.9 ( 0.04 0.1 ( 0.01

1.1 ( 0.06 ND ND 0.2 ( 0.02 0.3 ( 0.03 0.3 ( 0.01 ND 0.3 ( 0.05 1.1 ( 0.02 ND 0.04 ( 0.01 ND

RNOS NO2

NO2Cl ONOONO,N2O3

a Incubations contained 0.06 mM 14C-IQ, 0.1 mM DETAPAC in 0.1 mL, and were incubated at 37 °C. All incubations were at pH 7.4 with 100 mM potassium phosphate buffer, except those with myeloperoxidase (MPO), which were at pH 5.5. Incubation times were 10 min for MPO, 1 min for ONOO-, and 30 min for other conditions. IQ chemical transformation was analyzed by HPLC, and values represent mean ( SEM (n ) 3-5). Some RNOS thought to be generated by each condition are indicated (38). ND, not detected.

acidic or basic conditions, it is quite stable at physiologic pH. Formation of these RNOS-derived metabolites of IQ was investigated in more detail (Table 2). When 0.06 mM 14 C-IQ was incubated with MPO in the presence of 0.1 mM H2O2 and 0.3 mM NO2-, 1.8 ( 0.08 and 1.1 ( 0.06 nmol of NO2-IQ1 and NO2-IQ2, respectively, were formed. MPO-mediated metabolism required both NO2- and H2O2, and was prevented by 2 mM NaCN, a peroxidase inhibitor. In the presence of physiologic concentrations of chloride (100 mM), a MPO substrate, formation of these products was reduced. Taurine (0.5 mM) did not alter formation of the nitrated products in the presence of chloride, but did reduce formation of dimers (Azo-IQ/ IQ-D) by about 30%. Nitryl chloride (NO2Cl), generated by incubation of NO2- with HOCl (52), elicited a small amount of NO2-IQ1 (0.1 ( 0.01 nmol), which was prevented by 0.5 mM taurine. The major product resulting from the NO2Cl reaction coeluted with the synthetic dimers. A bolus addition of 0.3 mM ONOO- generated amounts of nitrated and dimer products similar to those seen by 0.3 mM NO2- with MPO. Preincubation of ONOO- in buffer for 10 min prior to IQ addition failed to demonstrate product formation. N-NO-IQ was the major product observed with 0.2 mM DEA NONOate (0.9 ( 0.04 nmol). NO2-IQ1 and NO2-IQ2 were minor products observed in the DEA NONOate reaction. Test tubes, which were capped, demonstrated significantly more DEA NONOate chemical transformation of IQ than uncapped tubes. Since the inflammatory milieu may be acidic (53, 54), the MPO response in the presence of NO2- was monitored as a function of pH from pH 4.5 to 7.4 (Figure 3). Optimum formation of the nitrated products was observed at pH 5.5 and 6.0. Product formation decreased from pH 6.0 to 7.4 with nitrated product formation at the limit of detection at pH 7.4. Results are consistent with active metabolism of IQ by MPO in the presence of NO2- during the inflammatory response. The potential for different RNOS to activate IQ to bind DNA and form adducts was assessed. DNA (1 mg/mL) was added to the different reaction conditions described in Table 2 and binding of 14C-IQ evaluated (Table 3). With the complete MPO reaction, 9500 ( 1300 pmol of IQ/mg of DNA was bound. Binding was only 43 ( 4 and 48 ( 4 pmol of IQ/mg of DNA in the absence of H2O2 and NO2-,

Figure 3. pH profile of myeloperoxidase metabolism of IQ with 0.3 mM NO2-. Samples were incubated at pH 4.5, 5.5, 6.0, 6.4, 6.8, or 7.4. After 10 min, reactions were stopped and samples analyzed by HPLC as illustrated in Figure 1. Table 3. Activation of

14C-IQ

by RNOS To Bind DNAa

conditions

DNA binding (pmol/mg of DNA)

MPO + 0.1 mM H2O2 + 0.3 mM NaNO2 -H2O2 -NO20.2 mM DEA NONOate -DEA NONOate 0.3 mM ONOO+10 min preincubation before IQ addition

9500 ( 1300 43 ( 4 48 ( 4 324 ( 28 28 ( 3 867 ( 80 38 ( 2

a

DNA (1 mg/mL) was added to incubations containing 0.06 mM 0.1 mM DETAPAC in 0.25 mL, and incubated at 37 °C for 30 min. All incubations were at pH 7.4 with 100 mM potassium phosphate buffer, except those with myeloperoxidase which were at pH 5.5. Activation was assessed as binding to DNA, and values represent mean ( SEM (n ) 3). 14C-IQ,

respectively. Thus, binding of 14C-IQ to DNA requires both NO2- and H2O2. Significantly less DNA binding was observed with DEA NONOate. However, the amount bound was more than 10-fold greater with (324 ( 28) than without (28 ( 3) DEA NONOate. More 14C-IQ bound to DNA with ONOO- (867 ( 80) than DEA NONate. These results are consistent with different RNOS activating IQ to bind DNA. The potential for products of IQ chemical transformation by RNOS to become activated and bind DNA was assessed. Both nitrated and nitrosated 14C-products of

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Table 4. Activation of 14C-IQ and Its Metabolites by RNOS and HOCl To Bind DNAa conditions MPO + 0.06 mM H2O2 + 0.3 mM NaNO2 IQ -NO2N-NO-IQ -NO2NO2-IQ1 -NO2NO2-IQ2 -NO2HOCl IQ N-NO-IQ

DNA binding (pmol/mg of DNA) 2787 ( 188 ND 824 ( 70 472 ( 38 ND ND ND ND ND 969 ( 44

a Purified 14C-products of IQ (0.04 mM) RNOS metabolism were incubated for 20 min with myeloperoxidase (pH 5.5) or 0.1 mM HOCl (pH 7.4) reaction mixtures containing 1 mg/mL DNA and 0.1 mM DETAPAC in 0.25 mL at 37 °C. Values for individual experiments are expressed minus blanks, which were obtained in the absence of either H2O2 or HOCl. Activation was assessed as binding to DNA, and values represent mean ( SEM (n ) 3-4). ND, not detected

IQ were tested (Table 4). One experiment assessed activation by MPO. Binding was assessed in the presence or absence of NO2-. While significant binding for IQ depended upon the presence of NO2-, N-NO-IQ demonstrated significant binding in both the presence and absence of NO2-. About twice as much N-NO-IQ bound in the presence (824 ( 70 pmol/mg of DNA) than in the absence (472 ( 38 pmol/mg of DNA) of NO2-. The nitrated products of IQ chemical transformation by RNOS were not activated by MPO to bind DNA. Another experiment evaluated activation by HOCl. HOCl did not activate IQ to bind DNA. In contrast, significant activation of N-NO-IQ occurred with HOCl (969 ( 44 pmol/mg of DNA). Thus, the nitrosated product of IQ metabolism can be activated to bind DNA by different biological systems. RAW cells (macrophage) were used to assess the formation of RNOS-derived IQ metabolites in an intact cell system associated with the inflammatory response. Cells were incubated with 0.1 mM IQ in the presence or absence of lipopolysaccharide and interferon gamma. The latter agents are known to increase iNOS expression and the accompanying increase in NO (55). Media NO2-/NO3levels increased from 1.4 ( 0.01 to 22.4 ( 1.1 nmol/106 cells following a 24 h incubation with lipopolysaccharide and interferon gamma. The media from these stimulated cells were purified by HPLC and analyzed by mass spectrometry for N-NO-IQ. N-NO-IQ is present in media with elevated levels of NO2-/NO3-. This is evidenced by a prominent protonated (MH+) molecular ion at m/z 228 (data not shown), which yields a product-ion spectrum (Figure 2, panel C) identical to that obtained with DEA NONOate nitrosation of IQ (Figure 2, panel B). N-NOIQ was not detected in media from cells incubated in the absence of lipopolysaccharide and interferon gamma. These results are consistent with an intact cell mediated inflammatory response producing the nitrosated product of IQ via a RNOS pathway.

Discussion This is the first demonstration of RNOS-mediated metabolism and activation of a heterocyclic amine. IQ was shown to be metabolized by several different RNOS,

Lakshmi et al.

using different incubation conditions. Conditions which generate NO2•, such as NO2- + myeloperoxidase + H2O2; ONOO-; NO2- + HOCl, produced two nitrated and dimer products of IQ. NMR characterization has determined NO2-IQ1 to have the nitro group in the 5 position and NO2-IQ2 to be an N-NO2, while IQ-D is substituted in the 5 position. MPO metabolism was dependent upon the presence of NO2- and was inhibited by the peroxidase inhibitor cyanide. Cl- is a physiologic substrate for MPO whose product is HOCl. NaCl at 100 mM, a physiological concentration, reduced formation of the nitrated IQ products, but this reduction was not altered by taurine, a scavenger of HOCl. Thus, the reduction in IQ metabolism observed with NaCl is not due to the HOCl formed, but rather to substrate competition. Nitryl chloride produced only a small amount of nitrated IQ product, with the major products being IQ dimers. ONOO- was quite effective in nitrating IQ. N-NO-IQ was the primary product observed with DEA NONOate, a NO donor. N2O3, a nitrosating species, is thought to be responsible for the chemical transformation of IQ observed with DEA NONOate. Small amounts of nitrated IQ products are also observed with DEA NONOate. All results are consistent with metabolism of IQ by RNOS and with different RNOS transforming IQ to distinct products. DNA binding of N-OH-IQ correlates with N-OH-IQ mutagenesis (56) and was used in the present study to determine the genotoxic potential of RNOS metabolism of IQ. Oxidative activation of IQ by MPO required NO2-. This is consistent with the results in Table 2 demonstrating that IQ metabolism by MPO also required NO2-. This suggests that IQ is not a substrate for MPO, but rather is oxidized by RNOS derived from this reaction, i.e., NO2•. ONOO- and DEA NONOate also activated IQ. NO2• is a possible intermediate responsible for ONOO- oxidative activation of IQ. Thus, several different RNOS appear capable of activating IQ to genotoxic products. Purified nitrated and nitrosated products of IQ metabolism were assessed for their genotoxic potential. Each compound was tested for binding to DNA following oxidation by MPO in the presence and absence of NO2-. The two nitrated products of IQ were not activated by MPO. N-NO-IQ was activated by MPO in both the presence and absence of NO2-. This is consistent with the nitrosated product being a substrate for MPO. Oxidation of N-NO-IQ by HOCl also resulted in activation. In contrast to N-NO-IQ, IQ was not activated by HOCl or by MPO in the absence of NO2-. Thus, IQ activation appears to depend on RNOS, while N-NO-IQ activation occurs with several different biological oxidizing agents. Activation of IQ by the NO donor may be the result of in situ N-NO-IQ formation. Activation of IQ can occur with other peroxidases, such as PHS. PHS activation does not require NO2- (24), and the major product is 2-nitro-3-methylimidazo[4,5-f]quinoline (27), which is not detected in the MPO reaction. The presence of this oxidation product in PHS reactions could allow for its autoreduction to N-OH-IQ, which would then bind DNA. Alternatively, PHS peroxygenase metabolism of N-acetylbenzidine to N′-hydroxy-N-acetylbenzidine and then 4′-nitro-4-acetylaminobiphenyl may also exist for IQ with a reactive N-OH-IQ intermediate formed. Either mechanism could account for the same adduct being observed with cytochrome P-450 and PHS activation (28, 29) and the high mutagenicity observed with N-acetyltransferase-proficient Salmonella typhimurium strains


following oxidation by either enzyme system (24, 31). Lactoperoxidase and horseradish peroxidase, in the absence of NO2-, were both shown to activate IQ to form the same adduct as PHS (28). We have detected 2-nitro3-methylimidazo[4,5-f ]quinoline as a major product of horseradish peroxidase IQ metabolism (not shown), and this could also occur with lactoperoxidase. Thus, while IQ appears to be a substrate for these peroxidases, it is not a substrate for MPO. Neutrophil activation of IQ is initiated by phorbol ester (28, 57). Azide, an MPO inhibitor, but not other enzyme pathway inhibitors, prevented DNA adduct formation. While this suggests neutrophil MPO involvement, azide is also a potent inhibitor of nitrosation (58), and this reaction pathway may be indicated. Direct activation of IQ by MPO was not assessed in the neutrophil experiments. The identities of the adducts formed by RNOS activation of IQ and N-NO-IQ have not been determined, but may represent unknown adducts detected by 32P-postlabeling in colon and other tissues (22, 57, 59). RNOS transform the aromatic amine N-acetylbenzidine to distinct products (46). All conditions, which primarily formed nitrated IQ products, converted Nacetylbenzidine to 3′-nitro-N-acetylbenzidine. Spermine NONOate, another NO donor, transformed N-acetylbenzidine to 4-acetylaminobiphenyl and 4′-OH-4-acetylaminobiphenyl. These nitrosation products were thought to be derived from an unstable N-nitroso intermediate which loses water, forming a labile diazonium ion. For this reason, deaminated IQ and 2-hydroxy-IQ were expected as products of DEA NONOate chemical transformation. Instead, a stable N-nitroso product of IQ was formed. This is the major difference observed between RNOS metabolism of the primary aromatic amine Nacetylbenzidine and heterocyclic amine IQ. This difference may be related to the reactivity of the IQ amino group, which favors an imine structure and contributes to its lack of N-acetylation compared to aromatic amines (15). The RNOS responsible for IQ chemical transformation were not determined. Two different RNOS acting by two different mechanisms are generally considered for nitration. The nitronium ion (NO2+) is known to nitrate aromatic rings by electrophilic aromatic substitution. However, activated aromatic rings, such as phenols, proceed via an electron-transfer mechanism involving radical combination reactions. The nitration of tyrosine (60) and acetaminophen (61) is attributed to NO2•. NO2• is thought to combine with a phenoxyl radical to form the nitrated product. Formation of a tyrosine dimer and of a dimer and trimer of acetaminophen during these reactions supports the role of a phenoxyl radical in nitration (60, 61). For N-acetylbenzidine, a radical intermediate is thought to be present during 3′-nitro-Nacetylbenzidine formation (46). Previous studies have reported the azo dimer to be an oxidation product of IQ (9). While this dimer was observed in our incubations, an IQ dimer was also detected. The presence of both dimers during the nitration of IQ is consistent with an IQ radical combining with NO2• to form the nitrated products. N-NO-IQ formation is attributed to the potent nitrosating agent N2O3. N2O3 is the major autoxidation product of NO in aqueous solution and is expected to be the major product formed by the NO donor DEA NONOate (58). The nitrosonium ion, NO+, is thought to be


responsible for nitrosation reactions catalyzed by NO via N2O3 (38), such as the reaction of DEA NONOate with IQ to produce N-NO-IQ. Substantially more DEA NONOate chemical transformation of IQ was observed with 20 mM than 100 mM phosphate buffer, and with 100 mM ammonium acetate instead of 100 mM phosphate buffer (not shown). Similar effects of buffer salts have been reported for nitrosation of 2,3-diaminonaphthylene by NO (58). Nitrosation of amines can occur with NO2- at acidic pH (62). However, acidic conditions in our incubations, i.e., pH 5.5, did not initiate this nonenzymatic reaction (Table 2, MPO conditions without NO2- or H2O2). Furthermore, the IQ amino group has been demonstrated to be resistant to this type of nitrosation (9). RNOSderived products of IQ can serve as biomarkers for specific RNOS, identifying their presence and allowing for evaluation of parameters, which modulate their effects. N-NO-IQ formation was much larger when the reaction was conducted in capped rather than uncapped test tubes. This was attributed to higher concentrations of aqueous NO being attained in capped than uncapped tubes. Higher concentrations of NO would translate to higher concentrations of N2O3 with increased amounts of nitrosated product formed. In a separate DEA NONOate experiment similar to those described in Table 2, the amount of N-NO-IQ formed was 1 ( 0.05 and 0.2 ( 0.01 nmol with and without caps, respectively. The amount of NO2-/NO3- in these tubes was 34 ( 0.5 and 25 ( 0.7 nmol with and without caps, respectively. Thus, the 0.8 nmol increase in N-NO-IQ corresponded to more than a 10-fold increase (9 nmol) of NO (NO2-/NO3-). NO is a colorless gas at room temperature and regarded as relatively insoluble in aqueous solution. Due to the volatility and diffusibility of NO, its autoxidation and reaction with target molecules are not as extensive as one might expect (63). This means that if a solution containing NO is exposed to a gas interface which does not contain NO, the NO in solution will readily volatilize to the gas phase. Experiments with cells have demonstrated that diffusion of NO out of a cell is more rapid than its reaction within the cell (64). As a result, a considerable amount of NO that reacts within a cell from which it is produced does so after having entered from the outside. Preventing NO escape with an impermeable barrier by capping test tubes dramatically increases its nitrosating ability. To assess the feasibility of the nitrosation reaction occurring with intact cells, macrophages, components of the inflammatory response, were selected for study. The ability of these cells to increase iNOS expression with various stimuli has been studied (55). A 16-fold increase in NO2-/NO3- was observed after a 24 h treatment of RAW cells with lipopolysaccharide, a constituent of bacteria cell membrane, and a cytokine. A previous study has reported that IQ attenuates increased NO2-/NO3formation by lipopolysaccharide-stimulated RAW cells (65). Concentrations of IQ from 0.05 to 0.5 mM were used, with 0.1 mM causing only slight inhibition of NO2-/NO3formation. In the current study, a significant reduction in NO2-/NO3- formation by 0.1 mM IQ was not detected during the 24 h incubation period. The concentration of IQ thought to inhibit iNOS is not likely to be achieved during in vivo exposure. Stimulated cells incubated with 0.1 mM IQ contained significant amounts of N-NO-IQ in their media. Mass spectral analysis demonstrated that

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Figure 4. Illustrated are possible routes of metabolism and activation of IQ via inflammation and oxidative stress. Mediators of inflammation (endotoxin and cytokines), oxidant stress (H2O2), and injury induce the synthesis of iNOS, increasing NO synthesis from L-arginine (L-Arg). NO produces RNOS by reacting with superoxide (O2-•) to form peroxynitrite anion (ONOO-) or with oxygen (O2) to form nitrogen dioxide radical (NO2•) and dinitrogen trioxide (N2O3). NO2-, a product of N2O3 metabolism, is oxidized to RNOS by myeloperoxidase (MPO). N2O3 transforms IQ to N-NO-IQ. N-NO-IQ is activated by different oxidants and IQ by RNOS to bind and damage DNA.

cell-derived material was identical to the synthetic N-NOIQ standard (Figure 2, panel C). The stability of N-NOIQ at physiologic pH and its accumulation in RAW cell media suggest that it would accumulate in biologic systems in vivo. N-NO-IQ can be activated by biological oxidizing agents, some of which are present in the inflammatory milieu, contributing to the carcinogenic response. Based on these experiments, we hypothesize that RNOS formed in the colon by various conditions, including inflammatory bowel disease, react with IQ (heterocyclic amines) present due to digestion of cooked red meat (Figure 4). These reactions result in activation of IQ to bind DNA and/or conversion of IQ to N-NO-IQ, which could then be activated to bind DNA, initiating carcinogenesis. While activation of IQ required RNOS, N-NOIQ activation did not. In colon, RNOS can be derived from a variety of cell types. In addition to macrophage, human colon epithelial cells can be stimulated to synthesize NO in response to oxidant stress or cytokines (66, 67). Our results demonstrate that stimulated macrophages exposed to IQ synthesize NO and produce N-NO-IQ. Future studies in animals treated to induce colitis and exposed to IQ could further test this hypothesis.

Acknowledgment. We thank Priscilla DeHaven and Cindee Rettke for excellent technical assistance. This work was supported by the Department of Veterans Affairs (T.V.Z.) and by National Cancer Institute Grant CA72613 (T.V.Z.). Mass spectrometry was performed at the Mass Spectrometry Resource Center, Washington University School of Medicine, through NIH Grants RR00954 and AM-20579. 1H NMR analysis was performed by Dr. Narayana Mysore, Shell Chemicals, subsidiary of Shell Oil Co., Houston, TX.

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Nitrosation and Nitration of 2-Amino-3-methylimidazo[4,5-f ]quinoline

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