Mechanism of Formation of (Deoxy) guanosine Adducts Derived from

Chem. Res. Toxicol. 2009, 22, 1765–1773

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Articles Mechanism of Formation of (Deoxy)guanosine Adducts Derived from Peroxidase-Catalyzed Oxidation of the Carcinogenic Nonaminoazo Dye 1-Phenylazo-2-hydroxynaphthalene (Sudan I) Martin Dracˇ´ınsky´,† Josef Cvacˇka,† Marcela Semanska´,‡ Vaćlav Martıńek,‡ Eva Frei,§ and Marie Stiborova´*,‡ Institute of Organic Chemistry and Biochemistry, V.V.i., Academy of Sciences of the Czech Republic, FlemingoVo n. 6, 166 10 Prague 6, Czech Republic, Department of Biochemistry, Faculty of Science, Charles UniVersity, AlbertoV 2030, 128 40 Prague 2, Czech Republic, and Department of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany ReceiVed February 21, 2009

We investigated peroxidase-mediated oxidation of and the formation of the (deoxy)guanosine adduct by 1-phenylazo-2-hydroxynaphthalene (Solvent Yellow 14, Sudan I), a liver and urinary bladder carcinogen for rodents and a potent contact allergen and sensitizer for humans. Using thin layer chromatography (TLC) and/or high performance liquid chromatography (HPLC) combined with mass and/or nuclear magnetic resonance (NMR) spectrometry, we characterized the structures of two major peroxidase-mediated Sudan I metabolites and those of the adducts of (deoxy)guanosine that are formed during Sudan I oxidation. Peroxidase oxidizes Sudan I to radical species that react with another Sudan I radical to form the Sudan I dimer, or in the presence of (deoxy)guanosine, the oxidized Sudan I can attack the exocyclic amino group of guanine, forming the 4-[(deoxy)guanosin-N2-yl]Sudan I adduct. The reaction product with a second Sudan I radical results in a dimer where the oxygen 2 radical of Sudan I reacted with carbon 1 in the second Sudan I skeleton. The Sudan I dimer is unstable and decomposes spontaneously to the second oxidation product. This compound consists of the 4-oxo-Sudan I skeleton connected via the oxygen of its 2-hydroxyl group and nitrogen of its azo group with carbon 1 of 2-oxonaphthalene, having a unique spironaphthooxadiazine structure. If (deoxy)guanosine is present during the formation of this Sudan I metabolite, an adduct, in which this Sudan I metabolite is bound to the exocyclic amino group of guanine, is generated. This (deoxy)guanosine adduct is again unstable and decomposes spontaneously to the same adduct that is formed by the direct reaction of oxidized Sudan I, the 4-[(deoxy)guanosin-N2-yl]Sudan I adduct. The results presented here are the first structural characterization of Sudan I-(deoxy)guanosine adducts formed during the oxidation of this carcinogen by peroxidase. Introduction Sudan I [1-(phenylazo)-2-hydroxynaphthalene, C.I. Solvent Yellow 14, CAS No: 842-07-9] has been used as a food coloring in several countries (1), but it has been recommended as unsafe because it causes tumors in the liver or urinary bladder in rats, mice, and rabbits, and is considered a possible weak human carcinogen and mutagen (1-7). Besides its carcinogenicity, Sudan I is a potent contact allergen and sensitizer, eliciting pigmented contact dermatitis in humans (8). Nevertheless, it is used to color materials such as hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, shoe and floor polishes, and gasoline (1, 5). Moreover, Sudan I is an important compound, not because it is still used to color these materials but because it is the simplest in a series of dyes and pigments including * To whom correspondence should be addressed. Phone: +420-221951285. Fax: +420-2-21951283. E-mail: [email protected]. † Academy of Sciences of the Czech Republic. ‡ Charles University. § German Cancer Research Center.

Sudan III and Sudan IV that are used in great quantities and occur everywhere in red and orange colored consumer products, foods, and printed matter. Besides Sudan I, Sudan III and Sudan IV have also been found to be weak carcinogens, being classified as category 3 carcinogens by International Agency for Research on Cancer (1, 5, 9). Even though the amounts of these azo dyes used in industry and to color the above materials have not yet been exactly evaluated, their use causes long-term harm in the environment (9) and could result in considerable human exposure (1, 5, 9). Recently, increased attention has been paid to this dye because it has been found as a contaminant of several European foodstuffs, being detected in chilli powder and in chilli-containing food products such as in Pixian douban, Golden Mark guilin chilli sauce, Golden Mark satay sauce, Italian pasta, chilli-snacks, and vegetable sauce (10-16). More recently, the levels of these dyes were evaluated; analysis of a few market samples of turmeric, chilli, and curry powders showed the presence of Sudan I (4.8-12.1 mg/g), Sudan IV (0.9-2.0 mg/ g), and metanil yellow (1.5-4.6 mg/g) in loose turmeric and

10.1021/tx900201q CCC: $40.75  2009 American Chemical Society Published on Web 10/09/2009

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chilli samples (14). In addition, there is evidence that ingestion of food products contaminated with Sudan I could lead to exposure in the human gastrointestinal tract to metabolites generated by intestinal bacteria (15). Therefore, the use of Sudan I as an additive in food products has been prohibited in the European Union and in many other countries (17). Nevertheless, the question whether the recent detection of Sudan I and other Sudan I-derived dyes in various food commodities and additional materials is really a serious problem requires further toxicological evaluations by regulatory agencies. Such evaluations should determine the real impact of this Sudan I dye on human health (9-17). Sudan I gives positive results in Salmonella typhimurium mutagenicity tests with S-9 activation (18, 19) and is mutagenic to mouse lymphoma L5178Y TK+/- cells in Vitro, with S-9 activation (18). It is a clastogenic compound, inducing micronuclei in the bone marrow of rats (3). There is also evidence that this compound exhibits genotoxic effects, after its metabolic activation by hepatic P450 and peroxidase enzymes in Vitro, in the rat liver and urinary bladder in ViVo (7, 14, 20-31), and in a human hepatoma cell line, HepG2 (6, 32). While the metabolism of Sudan I is not understood in humans, its metabolism has been characterized in rabbits (33), where it is metabolized primarily in the liver by oxidative or reductive reactions (33). C-Hydroxylated metabolites 1-(4-hydroxyphenylazo)-2-naphthol (4′-OH-Sudan I1) and 1-(phenylazo)-naphthalene-2,6-diol (6-OH-Sudan I) were found to be the major products of Sudan I oxidation in ViVo and excreted in urine (1, 33), and also of its oxidation by rat hepatic microsomes in Vitro (34). Besides the C-hydroxylated metabolites, which are considered detoxication products, the benzenediazonium ion (BDI), formed by microsome-catalyzed enzymatic splitting of the azo group of Sudan I, was found to react with DNA in Vitro (20, 21, 34). The major DNA adduct formed in this reaction has been characterized and identified as the 8-(phenylazo)guanine adduct (21). This adduct was also found in the liver DNA of rats exposed to Sudan I (31). Oxidation of Sudan I with formation of the same C-hydroxylated metabolites and Sudan I-derived DNA adducts was recently demonstrated with human P450 enzymes (22, 35). P450 1A1 is the major enzyme oxidizing Sudan I in human tissues rich in this enzyme, while P450 3A4 is also active in the human liver (22, 35). In addition to microsomal P450 enzymes, Sudan I and its C-hydroxylated metabolites are also oxidized by peroxidases, and as a consequence, DNA, RNA, and protein adducts are formed (23-29, 36). While microsomal P450s were found to be responsible for the activation of Sudan I in human or animal livers (20-22, 35, 37), they play a marginal role in the in ViVo metabolic activation of Sudan I in the urinary bladder because this organ has little or no detectable P450 enzymes. But relatively high levels of peroxidases are expressed in this tissue (38). In the bladder, therefore, peroxidase-catalyzed activation of Sudan I has been suggested, similar to other carcinogens such as aromatic amines (38-41). We have suggested a P450- or peroxidase-mediated activation of Sudan I or a combination of both mechanisms as an explanation for the organ specificity of this carcinogen for the liver and urinary bladder in animals (20-22, 24-26, 35, 37). 1 Abbreviations: APCI, atmospheric pressure chemical ionization; BDI, benzenediazonium ion; CID, collision-induced dissociation; ESI, electrospray-ionization; HRP, horseradish peroxidase; 4′-OH-Sudan I, 1-(4hydroxyphenylazo)-2-naphthol; 6-OH-Sudan I, 1-(phenylazo)-naphthalene2,6-diol; 4′,6-di(OH)-Sudan I, 1-(4-hydroxyphenylazo)-naphthalene-2,6-diol; poly X, polyribonucleotides; COSY, correlation spectroscopy; HSQC, heteronuclear single-quantum correlation; HMBC, heteronuclear multiple bond correlation.

Dracˇ´ınsky´ et al.

Indeed, the 8-(phenylazo)guanine DNA adduct generated from the BDI, the product of P450 activation, was found in the liver of rats treated with Sudan I (31), whereas the physicochemical properties of DNA adducts found in the urinary bladder are identical to those formed by the peroxidase-mediated Sudan I activation that contain the whole molecule of Sudan I (30). Unfortunately, neither the structures of DNA or RNA adducts nor those of the ultimate carcinogens formed by peroxidase from Sudan I are known as yet. This knowledge is, however, crucial for the comparative study with in ViVo products (30, 31). In our former studies with peroxidase activation of Sudan I, we have identified BDI and C-hydroxy derivatives of Sudan I [6-OH-Sudan I and 4′,6-di(OH)-Sudan I] as only minor metabolites, while products of a suggested polymerization of the primarily formed Sudan I radicals were more abundant (23, 36, 42). In the meantime, we have confirmed this for the two major metabolites (43), one is a Sudan I dimer, while the other is the product of secondary, enzyme independent reactions of this Sudan I dimer (43). Plant horseradish peroxidase (HRP) was found in these studies to be an acceptable model for Sudan I oxidation by mammalian enzymes such as nonspecific urinary bladder peroxidases and/or cyclooxygenases (22-25, 31, 42, 44). Even though mammalian and plant peroxidases are structurally different proteins, the oxidation mechanisms are similar because of analogous arrangements of their active sites (36, 38-41, 44). Using the 32P-postlabeling assay (45, 46), we have already analyzed Sudan I-DNA adducts formed by HRP (24-27). Deoxyguanosine was the major target for Sudan I-DNA binding, followed by deoxyadenosine (26). Likewise, guanosine was found to be the major target for peroxidase-activated Sudan I binding in RNA (28). It has been postulated by Eling and coworkers (40) that characterization of peroxidase-mediated adducts derived from carcinogens is exceptionally difficult because of problems in preparing the DNA (or RNA) adducts in sufficient quantities and purity for structural analysis. Indeed, our earlier studies with peroxidase activation of Sudan I in the presence of DNA, tRNA, (deoxy)guanosine 3′-monophosphate, or (deoxy)guanosine 5′-monophosphate did not yield enough adducts for their structural characterization (26, 28). Therefore, the aims of this study were to prepare, isolate, and characterize major deoxyguanosine and guanosine adducts formed by the peroxidase-activated Sudan I in the presence of these (deoxy)nucleosides. Thin layer chromatography (TLC) was utilized to isolate the adducts and mass and nuclear magnetic resonance (NMR) spectrometry to characterize their structure. In addition, we wanted to establish the molecular mechanisms explaining the formation of (deoxy)guanosine adducts generated by Sudan I during its oxidation with peroxidase.

Experimental Procedures Caution: Sudan I is a potent carcinogen and should be handled with care. Wastes must be discarded according to appropriate safety procedures. Chemicals. Chemicals were obtained from the following sources: Sudan I (>99% based on high performance liquid chromatography (HPLC)) from British Drug Houses, Poole, U.K.; deoxyguanosine and guanosine from Roche Mannheim, Germany; homopolyribonucleotides (poly X) from Pharmacia LKB (Uppsala, Sweden); horseradish peroxidase (HRP, isoenzyme C; 300 purporogallin units/ mg protein, 61 guaiacol units/mg protein) from Sigma Chemical Co. (St. Louis, MO, USA), and hydrogen peroxide from Merck (Darmstadt, Germany). All of these and other chemicals were of analytical purity or better. Rat liver tRNA was prepared according to Rogg et al. (47). Incubations. Unless stated otherwise, incubation mixtures used to prepare and characterize Sudan I metabolites contained the

Peroxidase-Catalyzed Formation of (Deoxy)guanosine Adducts by Sudan I

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Figure 1. Semipreparative HPLC of Sudan I metabolites formed by peroxidase. For incubation conditions, see Experimental Procedures. Peaks eluting between 5 and 12 min, solvent front. For 1 and 4, see Scheme 1.

following in a final volume of 70 mL: 10 mM sodium phosphate buffer (pH 8.4), 0.5 µΜ HRP, 100 µM Sudan I, and 200 µΜ hydrogen peroxide. Reactions were initiated by adding Sudan I dissolved in methanol (final concentration of methanol was 1%) (43). After incubation (37 °C, 20 min), the mixtures were extracted with ethyl acetate (2 × 70 mL). The extracts were evaporated, dissolved in a methanol, and separated by TLC or HPLC as described (43). Briefly, silica-gel TLC plates were developed in hexane-diethyl ether (1:3, v/v). Spots of Sudan I metabolites with relative mobilities of 0.21 and 0.19 were extracted with methanol. Alternatively, the products were separated by semipreparative HPLC on a Tessek Separon Hema S 1000 (8.0 × 250 mm) C-18 column. Gradient elution (75%-100% methanol in distilled water) with a flow rate of 0.3-1.5 mL/min, as indicated in Figure 1, was used. Sudan I metabolites were detected at 215, 254, 333, and 480 nm. Recoveries of Sudan I metabolites were around 95%. Two product peaks with tR of 32 and 37 min (Figure 1) were collected, repurified by the same HPLC procedure, and analyzed by mass and/or NMR spectrometry. The incubation mixtures used to analyze the effect of tRNA, poly X, deoxyguanosine, and guanosine on Sudan I oxidation by peroxidase were the same as those described above except that the volume was 1.5 mL, and 0.2 mg of tRNA or poly X, or 0.25-0.5 mM deoxyguanosine or guanosine, was added. After incubation (37 °C, 20 min), the mixtures were extracted with ethyl acetate (2 × 1.5 mL), the extracts evaporated, dissolved in a methanol, and analyzed using the HPLC procedure described above. Incubation mixtures used to modify deoxyguanosine and guanosine by peroxidase-activated Sudan I contained the following in a final volume of 50 mL: deoxyguanosine or guanosine (5 mM), 0.5 mM Sudan I, 1.5 mM hydrogen peroxide, and 0.5 µΜ HRP in 10 mM sodium phosphate buffer (pH 8.4). Reactions were initiated by adding Sudan I dissolved in methanol (final concentration of methanol was 5%). After incubation (37 °C, 20 min), mixtures were extracted twice with ethyl acetate (2 × 30 mL). The extracts were evaporated in a stream of nitrogen to dryness, and residual Sudan I, its metabolites, and the (deoxy)guanosine adducts were separated by TLC on silica gel (Merck, Darmstadt, Germany) in n-butanol/ n-propanol/H2O/NH4OH (14 M) (16:12:10:2, v/v). The separated major adduct zones [Rf of 0.28 (6a) and 0.36 (5a) for guanosine, and of 0.34 (6b) and 0.45 (5b) for deoxyguanosine adducts (see Chart 1)] were scraped from the plates and extracted with methanol/ NH4OH (4 M) (1:1, v/v). The isolated major adducts were repurified 5 times by the same procedure and analyzed by mass-, NMR-, and UV/vis spectrometry. Mass Spectrometry. Spectra of Sudan I metabolites were measured using Esquire 3000 Bruker Daltonics [atmospheric pressure chemical ionization (APCI)] and electrospray ionization

(ESI), positive and negative ionization by Dr. Martin Sˇtıćha (Department of Organic Chemistry, Faculty of Science, Charles University). The mass spectra were internally calibrated using background ions with known elemental composition (43). Mass spectra of Sudan I metabolites as well as deoxyguanosine or guanosine adducts were also recorded using a Thermo Scientific LTQ Orbitrap XL hybrid Fourier transform mass spectrometer equipped with an electrospray ion source and controlled by Xcalibur software. Samples were dissolved in acetonitrile/water/formic acid (50: 50:0.1, v/v) and continually delivered into the ion source at 5 µL/min. For positive ions, spray voltage was set to 6.0 kV, capillary temperature and voltage were 300 °C and 32 V, respectively, and tube lens voltage was maintained at 80 V. MSn fragmentations were carried out in the ion trap using CID and recorded in orbitrap set to a mass resolution of 60 000. The mass scale of the instrument was calibrated using a mixture suggested by the manufacturer (caffeine, Met-Arg-Phe-Ala peptide, and perfluoroalkylphosphazines). NMR Spectrometry. NMR spectra (δ, ppm; J, Hz) were measured on a Bruker Avance II-600 instrument (600.1 MHz for 1 H and 150.9 MHz for 13C) in hexadeuterated dimethyl sulfoxide and referenced to the solvent signals (δ 2.50 and 39.7, respectively). Compound 4 (see Chart 1 and Scheme 1): 13C NMR data are shown in Table 1. 1 H NMR NMR: 6.17 (s, 1 H, H-3), 6.30 (d, 1 H, J3′,4′ ) 10.0, H-3′), 6.91 (m, 2 H, H-10), 7.15 (m, 1 H, H-12), 7.23 (m, 2 H, H-11), 7.52 - 7.62 (m, 4 H, H-6, H-5′, H-6′ and H-7′), 7.73 (m, 1 H, H-7), 7.76 (d, 1 H, J4′,3′ ) 10.0, H-4′), 7.86 (m, 1 H, H-8′), 8.03 (m, 1 H, H-5), 8.13 (m, 1 H, H-8). Ribonucleoside 5a (see 5 in Chart 1 and Scheme 1): 13C NMR: 61.90 (G5′), 70.71 and 70.83 (G3′), 73.56 and 73.87 (G2′), 85.53 and 86.06 (G4′), 87.51 and 87.99 (G1′), 83.55 (C-1′), 102.52 (C3), 119.7 (G5), 121.88 (C-8), 122.40 and 122.49 (C-10), 123.17 (C-3′), 125.63 and 125.82 (C-5), 126.21 and 126.60 (C-12), 128.94 (C-11), 129.02 (C-6), 129.7 (C-4a), 129.91 (C-8′), 130.79 (C-5′), 131.1 (C-4′a and C-8a), 131.50 (C-7′), 131.80 (C-6′), 131.92 (C7), 135.27 (G8), 135.45 (C-8′a), 143.14 (C-9), 146.81 (C-4′), 149.46 and 149.58 (G4), 191.46 and 191.50 (C-2′). 1 H NMR NMR: 3.42 - 3.62 (m, 2 H, G5′), 3.87 (m, 1 H, G4′), 4.03 (m, 1 H, G3′), 4.49 and 4.52 (t, 1 H, JG2′,G1′ ) JG2′,G3′ ) 5.7, G2′), 5.70 (d, 1 H, JG1′,G2′ ) 6.3, G1′), 6.222 and 6.224 (d, 1 H, J3′,4′ ) 10.0, H-3′), 6.40 (bs, 1 H, H-3), 6.87 (m, 2 H, H-10), 7.11 (m, 1 H, H-12), 7.21 (m, 2 H, H-11), 7.51 (m, 1 H, H-7′), 7.54 (m, 2 H, H-5′ and H-6′), 7.61 (m, 1 H, H-6), 7.673 and 7.679 (d, 1 H, J4′,3′ ) 10.0, H-4′), 7.71 (m, 1 H, H-7), 7.78 (m, 1 H, H-8′), 8.02 (bs, 1H, G8), 8.17 (m, 1H, H-8), 8.36 (m, 1 H, H-5). Deoxyribonucleoside 5b (see 5 in Chart 1 and Scheme 1): 13 C NMR NMR: 40.38 (G2′), 62.20 (G5′), 71.25 (G3′), 84.00 and 84.03 (G1′), 88.31 (G4′), 119.44 (G5), 136.96 (G8), 149.20

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Scheme 1. Proposed Mechanism of Peroxidase-Mediated Sudan I Metabolism and Formation of Adducts with (Deoxy)guanosinea

a

* is the new chiral C.

and 149.24 (G4), 154.44 (G2), 165.32 (G6); remaining data are shown in Table 1. 1 H NMR NMR: 2.18 (m, 1 H, G2′a), 2.60 (m, 1 H, G2′b), 3.43 and 3.50 (m, 1 H, G5′), 3.80 (m, 1 H, G4′), 4.30 (m, 1 H, G3′), 6.18 (dd, 1 H, JG1′,G2′a ) 6.1, JG1′,G2′b ) 8.0, G1′), 6.227 and 6.233 (d, 1 H, J3′,4′ ) 10.0, H-3′), 6.44 (bs, 1 H, H-3), 6.88 (m, 2 H, H-10), 7.12 (m, 1 H, H-12), 7.21 (m, 2 H, H-11), 7.50 (m, 1 H, H-7′), 7.54 (m, 2 H, H-5′ and H-6′), 7.62 (m, 1 H, H-6), 7.682 and

7.688 (d, 1 H, J4′,3′ ) 10.0, H-4′), 7.72 (m, 1 H, H-7), 7.79 (m, 1 H, H-8′), 8.04 (bs, 1H, G8), 8.17 (m, 1H, H-8), 8.37 (m, 1 H, H-5), 10.27 (bs, 1 H, G1). Ribonucleoside 6a (see 6 in Chart 1 and Scheme 1): 13C NMR NMR: 60.6 (G5′), 69.5 (G3′), 73.2 (G2′), 84.8 (G4′), 86.1 (G1′), 116.3 (C-10), 119.3 (G5), 121.6 (C-5), 121.7 (C-8), 124.6 (C-12), 125.5 (C-6), 128.7 (C-7), 128.9 (C-11), 142.1 (C-9), 148.1 (G4).



Chart 1. Structures of Metabolites 1 and 4 and Those of (Deoxy)guanosine Adducts 5 and 6 Formed by Sudan I during Its Oxidation by Peroxidase

Table 1.

13

C Chemical Shifts of Sudan I, Metabolite 4, and Adducts 5b and 6ba

carbon

Sudan I

4

1 2 3 4 4a 5 6 7 8 8a 9 10 11 12 1′ 2′ 3′ 4′ 4a′ 5′ 6′ 7′ 8′ 8a′

129.33 169.00 124.13 140.18 128.04 129.12 126.07 129.33 121.52 132.95 145.30 119.17 130.04 128.31

129.98 149.09 108.15 183.96 129.51 125.71 131.92 132.95 121.83 131.65 142.84 122.61 129.03 126.60 83.65 191.46 123.03 147.18 131.05 129.20 130.88 131.57 130.02 135.25

a

1

5b 130.73; 145.84 102.69; 145.56 129.61 125.71 129.06 132.07 122.10 131.11 143.11 122.44; 129.01 126.29; 83.61 191.53; 123.17 146.90 131.11 130.85 131.83 131.54 129.94 135.44

130.78 102.74

122.56 126.31

6b 127.81 171.32 109.84 149.00 122.60 122.16 126.23 129.57 122.16 133.32 143.09 116.60 129.68 125.45

191.59

Guanosine carbons are not shown.

H NMR NMR: 3.58 and 3.62 (m, 1 H, G5′), 3.96 (m, 1 H, G4′), 4.15 (m, 1 H, G3′), 4.57 (m, 1 H, G2′), 4.96 (t, 1 H, JOH,5′ ) 5.5, G5′-OH), 5.20 (d, 1 H, JOH,3′ ) 4.4, G3′-OH), 5.46 (d, 1 H, JOH,2′ ) 5.0, G2′-OH), 5.85 (d, 1 H, J1′,2′ ) 6.1, G1′), 7.25 (m, 1 H, H-12), 7.50 (m, 2 H, H-11), 7.61 (m, 1 H, H-6), 7.69 (m, 1 H, H-7), 7.73 (d, 2 H, J2′,3′ ) 7.9, H-10), 7.90 (s, 1 H, H-3), 8.10 (d, 1 H, J5,6 ) 8.4, H-5), 8.22 (s, 1 H, G8), 8.56 (d, 1 H, J8,7 ) 7.9, H-8), 16.33 (bs, 1 H, 2-OH). Deoxyribonucleoside 6b (see 6 in Chart 1 and Scheme 1): 13C NMR NMR: 40.88 (G2′), 61.65 (G5′), 70.73 (G-3′), 83.30 (G1′), 87.91 (G4′), 119.70 (G5), 137.41 (G8), 148.51 (G4), 156.4 (G6), remaining signals are shown in Table 1.

1 H NMR NMR: 2.34 (ddd, 1 H, Jgem ) 13.0, J2′′,1′ ) 6.2, J2′′,3′ ) 3.3, G2′′), 2.72 (ddd, 1 H, Jgem ) 13.3, J2′,1′ ) 7.8, J2′,3′ ) 6.1, G2′), 3.51 - 3.62 (m, 2 H, G5′), 3.90 (m, 1 H, G4′), 4.42 (m, 1 H, G3′), 4.89 (t, 1 H, JOH,5′ ) 5.5, G5′-OH), 5.33 (d, 1 H, JOH,3′ ) 4.1, G3′-OH), 6.28 (dd, 1 H, J1′,2′ ) 7.8, J1′,2′′ ) 6.2, G1′), 7.22 (m, 1 H, H-12), 7.48 (m, 2 H, H-11), 7.59 (m, 1 H, H-6), 7.66 (m, 1 H, H-7), 7.69 (m, 2 H, H-10), 7.86 (s, 1 H, H-3), 8.13 (m, 1 H, H-5), 8.18 (s, 1 H, G8), 8.52 (d, 1 H, J8,7 ) 7.9, H-8), 16.34 (bs, 1 H, 2-OH).

Results Peroxidase-Mediated Sudan I Oxidation. When Sudan I was incubated with peroxidase and hydrogen peroxide, two product peaks with tR of 32 and 37 min were separated by semipreparative HPLC with UV/vis monitoring at 215, 254, 333, and 480 nm (see peaks detected at 480 nm in Figure 1 and compounds 1 and 4 in Chart 1). Recently, these two Sudan I metabolites have been partially characterized (43). Here, we suggest the mechanism of their formation catalyzed by peroxidase. If oxidation products of Sudan I were separated by TLC on silica gel, an additional six minor products, undetectable by HPLC, were found (36, 42, 43), and three of them have already been characterized to be BDI, 6-OH-Sudan I, and 4′,6-di(OH)Sudan I, but the instability of the other products precluded their structural characterization (23, 36, 42). When Sudan I metabolites 1 and 4 (Chart 1) were purified by HPLC, we found that metabolite 1 is also unstable and reacts spontaneously to metabolite 4. We therefore combined the TLC and HPLC separation procedures (43) and have obtained these two major Sudan I metabolites in amounts and purities sufficient for their structural characterization. Structural Characterization of Sudan I Metabolites 1 and 4. The Sudan I metabolite 1 (Chart 1) was analyzed by mass spectrometry, in the positive ion mode. Compound 1 provided a singly charged protonated molecule [M + H]+ at m/z 495.1821. The calculated elemental composition C32H23N4O2

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was in agreement with the assumed structure of 1 (a Sudan I dimer). Moreover, ion peaks at m/z 417, formed by elimination of a phenyl ring from a Sudan I dimer, m/z 389, generated from the Sudan I dimer by elimination of a diazo-naphthol group, m/z 247 (the ion of Sudan I), m/z 172 (the ion of diazo-naphthol), and m/z 159 (the ion of naphthol containing one nitrogen) (43) are fragments that could be assigned to compound 1 (Chart 1). Because metabolite 1 is highly unstable and decomposes spontaneously to metabolite 4, compound 1 could not be isolated in quantities sufficient for its NMR characterization. Metabolite 4 (Chart 1) provided a singly charged protonated molecule [M + H]+ at m/z 405.1220, and the calculated elemental composition C26H17O3N2 was in agreement with the assumed structure of 4. The 1H NMR spectrum showed one singlet at 6.17 ppm, two doublets with a coupling constant of 10.0 Hz, a set of signals of N-substituted aniline, and two sets of signals of ortho-disubstituted benzene derivatives. No exchangeable protons were detected by the addition of a drop of D2O to the sample. The most interesting signals in the 13C NMR spectra were two carbonyl carbons (C-2′ at 191.5 and C-4 at 184.0 ppm) and a quaternary carbon atom (C-1′ at 83.7 ppm). The structure of compound 4 was elucidated from 2D NMR spectra [H,H-correlation spectroscopy (COSY), H,C-heteronuclear single-quantum correlation (HSQC), and H,C-heteronuclear multiple bond correlation (HMBC)]. The molecule contains two naphthalene fragments and one N-substituted aniline. By combining 1D and 2D NMR techniques, 1H and 13 C chemical shifts were completely assigned. In the HMBC spectrum of 4, we observed among others strong cross-peaks corresponding to three bond correlations among H3 and C1 and C4, weak two bond correlations H3-C1 and H3-C4a, weak four bond correlations H3-C5, strong three bond correlation H3′C1′ and H3′-C4′a, weak two bond correlations H3′-C2′, weak four bond correlations H3′-C8′a′, and strong three bond correlations H8′-C1′, H4′-C2′, H5-C4, and H8-C1. Compound 4 is a spiro compound with one chiral center (quaternary carbon 1′). From the metabolic pathway of Sudan I proposed below, it follows that compound 4 should be a racemic mixture of both enantiomers. This was confirmed after adding a chiral NMR shift reagent, (-)-2,2,2-trifluoro-1-(9-anthryl)-ethanol, when the signals split into two sets corresponding to the two antipodes. Peroxidase-Mediated Formation of Sudan I-(Deoxy)guanosine Adducts. In the presence of nucleophiles such as tRNA, poly X, guanosine, or deoxyguanosine in the reaction mixture used for Sudan I oxidation by peroxidase, the levels of the two major Sudan I metabolites 1 and 4 (determined by HPLC) decreased significantly (data not shown). This finding indicates that the generation of these metabolites competes with the binding of reactive Sudan I intermediates to nucleophiles, thereby forming the adducts 5a and 6a with guanosine and 5b and 6b with deoxyguanosine (Chart 1 and Scheme 1). Adducts 5a/5b are unstable under ambient temperature and during purification, and react spontaneously to adducts 6a/6b. Both sets of adducts were characterized by UV/vis (Figure 2), mass, and 1 H- and 13C NMR-spectrometry. Structural Determination of Sudan I-(Deoxy)guanosine Adducts (Compounds 5 and 6). Adduct 5 (Chart 1) (with guanosine 5a or deoxyguanosine 5b) had hydrogen and carbon chemical shifts and HMBC correlations similar to those of compound 4. Furthermore, signals of guanine and (deoxy)ribose were present in the NMR spectra. H8 of guanine and its correlation to carbons C4 and C5 were also found. Biggest changes in chemical shifts with respect to compound 4 exhibited


Figure 2. UV/vis absorption spectra of Sudan I, Sudan I-guanosine, and Sudan I-deoxyguanosine adducts 6a/6b (a) and 5a/5b (b). Spectra of compounds (dissolved in methanol) were measured using a HewlettPackard 8452 diode array spectrophotometer.

C atoms in positions 2, 3, and 4 (see Table 1). Compound 5 is a 1:1 mixture of two diastereoisomers. The new chiral center in position 1′ can have both configurations, and indeed, both possible stereoisomers of compound 5 were formed. This is manifested by the splitting of some signals in the 1H and 13C spectra into two sets of equal intensity. In the mass spectra, the isolated adducts 5 provided singly charged protonated molecules [M + H]+ at m/z 670.2046 (5a) and m/z 654.2100 (5b). The calculated elemental composition, C36H28N7O7 (5a) and C36H28N7O6 (5b), was in good agreement with the structures of 5. Under MS/MS conditions (Figure 3), both ions eliminated a neutral molecule of sugar, leaving ion m/z 538.1622 (C31H20 N7O3). Further fragmentation of this ion gave the same fragments for 5a and 5b. MS3 spectra (fragmentation of m/z 538) showed two ions at m/z 305.0783 (C15H9N6O2) and 234.0914 (C16H12NO) formed by cleavage of two bonds in the oxadiazine ring. Further fragmentation (MS4) of m/z 305 gave m/z 262.0724 (C14H8N5O) after elimination of HNCO. In addition, the signal at m/z 171.0553 (C10H7N2O) was found, presumably formed by elimination of hypoxanthine. Fragmentation of m/z 234 (MS4) provided m/z 216.0808 and 206.0965 after the loss of H2O and CO, respectively. The CID fragmentation of 5a/5b is shown in Figure 3. Adduct 6 (Chart 1) (with guanosine 6a or deoxyguanosine 6b adduct) had hydrogen and carbon chemical shifts similar to those of Sudan I, indicating the presence of its ring system



Figure 3. CID fragmentation of peroxidase-mediated Sudan I-(deoxy)guanosine adducts 5a/5b.

Figure 4. CID fragmentation of peroxidase-mediated Sudan I-(deoxy)guanosine adducts 6a/6b.

in compound 6. This finding was further supported by an absorption maximum of the (deoxy)guanosine adducts 6a/ 6b at 480 nm (Figure 2), which is typical for the spectrum of Sudan I. Furthermore, the signals in the NMR spectra of guanine and (deoxy)ribose showed that the carbon 4 of the Sudan I molecule was attached to the guanosine. No crosspeak between hydrogens in positions 5 and the singlet at 7.90 (6a)/7.86 (6b) was observed in the spectrum obtained by nuclear Overhauser enhancement spectroscopy (NOESY) (a strong cross-peak between H-4 and H-5 is observed in the NOESY spectrum of Sudan I). The singlet at 7.90/7.86 ppm was assigned to be the signal of hydrogen H-3. In the HMBC spectrum, there are cross-peaks between hydrogen H-3 and carbon atoms C-1 and C-4a (the usual three bond couplings observed in substituted benzenes). The binding position of Sudan I to the guanosine moiety was determined as follows: the saccharide had typical signals of all hydrogen and carbon atoms in the spectra, and guanine H-8 was also observed (and its typical HMBC correlation to carbon atoms C-4 and C-5). We considered three possible substitution sites: O6, N1, or N2 in the guanine residue. The O6 substitution was excluded because no downfield shift (3-5 ppm) of the C-2, C-4, and C-6 resonances compared to that of guanosine was observed (48, 49). The O6 substitution was also excluded because the UV absorbance maxima of adducts 5a and 6a (dissolved in 30% methanol) shifted to higher wavelengths with increasing pH (pH 7.0-10.0), similar to the shift in the guanosine spectrum (Figure 1S in Supporting Information). If polar hydrogen on N1 or O6 of the keto or enol tautomeric form of guanine was substituted, the UV absorbance maxima of N1- or O6-modified guanosine would be insensitive to such pH changes. Adduct 5a elicits a shift to 246 nm, which is identical to that of guanosine, and adduct 6a to a similar wavelength of 241 nm (Figure 1S in Supporting Information). These findings indicate the presence of unmodified N1 and O6 in both adducts and support their structures determined by NMR and MS/MS spectrometry. The mass spectra of adducts 6 showed singly charged protonated molecules [M + H]+ at m/z 530.1778 (6a) and m/z 514.1830 (6b). The calculated elemental composition, C26H24N7O6 (6a) and C26H24N7O5 (6b), was in good agreement with the structure of 6. Under the MS/MS conditions (Figure 4), both ions eliminated a neutral molecule of sugar, resulting in m/z 398.1358 (C21H16N7O2). Further fragmentation of this ion gave the same fragments for 6a and 6b. MS3 spectra (fragmentation of m/z 398) showed m/z 305.0783 (C15H9N6O2), presumably formed by the elimination of aniline. The loss of H2O and CO gave small signals at m/z 380.1255 (C21H14N7O) and 370.1411 (C20H16N7O), respectively. Elimination of guanine yielded m/z 247.0867 (C16H11N2O). In the next MS

step, m/z 305 was fragmented (MS4). A prominent peak at m/z 262.0724 (C14H8N5O) was observed, and it could be explained by the elimination of HNCO. Hence, N1 and O6 in guanine are not substituted, and therefore, the Sudan I metabolite is bound to the exocyclic N2 of guanine. The CID fragmentation of 6a/6b is shown in Figure 4.

Discussion The results found in the present work and in former studies (28, 42, 43) demonstrate that Sudan I is oxidized by peroxidase. Upon peroxidase-mediated oxidation, Sudan I radicals are formed (28, 42, 43) (Scheme 1). The fate of these radicals depends on the environment in which they exist. We have found that the Sudan I-derived radicals (i) form additional metabolites (refs 36, 42, 43, and the present work); (ii) react with NADH and ascorbate (24, 25, 36); and (iii) react with SH groups of glutathione (reducing Sudan I radicals with the formation of a thiyl radical) (26, 36). Sudan I oxidized by peroxidase also reacts with nucleophilic centers in DNA (24, 26, 30), RNA (25, 28), proteins (36), (deoxy)ribonucleosides (the present work), and ribonucleoside phosphates (32) to form potentially toxic adducts in Vitro and in ViVo. We demonstrate herein that the formation of two Sudan I metabolites by peroxidase-catalyzed reactions proceeds via a radical mechanism. With the results found in this study, we here suggest a possible mechanism for these reactions as well as that for the peroxidase-mediated Sudan I-(deoxy)ribonucleoside adduct formation. The proposed mechanism is depicted in Scheme 1. First, peroxidase forms Sudan I free radicals, which might react further via a variety of reactions. The Sudan I radical reacts with another Sudan I radical, resulting in the formation of azo compound 1 (one of the two major Sudan I metabolites). If (deoxy)guanosine is present in the reaction mixture, oxidized Sudan I can attack the exocyclic amino group of a guanine residue, forming compound 6, the 4-[(deoxy)guanosin-N2-yl]Sudan I adduct (Scheme 1). Because of the instability of metabolite 1, its structure could not be determined by NMR spectrometry. However, the structure of this Sudan I metabolite was assumed by mass spectrometry and by using the structural identification of the second Sudan I metabolite (4), which is the product of metabolite 1 degradation and rearrangement. Metabolite 1 is the Sudan I dimer, in which the two Sudan I skeletons are connected via the oxygen of one Sudan I molecule and carbon 1 of the second Sudan I molecule. Aliphatic azo compounds are unstable, and the C-N bond is easily subjected to homolytic cleavage forming two radicals. The Sudan I dimer 1 is thus homolytically cleaved to radical 2 and a benzenediazonium radical, which decomposes to molec-

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ular nitrogen and a phenyl radical, which abstracts a hydrogen radical from water to form benzene. Radical 2 can react intramolecularly to form radical 3, which may react with oxygen to form a hydroperoxy radical, which can be decomposed to compound 4 (the second Sudan I metabolite, Scheme 1). Reaction of radical 3 with water (50) and subsequent oxidation to compound 4 might also be possible. Compound 4 is formed without stereochemical preference (peroxidase forms the starting radical, but is not further involved in the reaction pathway); therefore, both configurations of the new chiral center in position 1′ are equally probable and occur in a ratio of 1:1. Compound 4 has a unique spironaphthooxadiazine structure, which has never been observed before. If a suitable nucleophile such as guanine or (deoxy)guanosine is present in the reaction mixture, it can react with cation 3′ (oxidized radical 3), and after the next oxidation, imine 5 (adduct 5a/5b) appears. Compound 5 is converted to compound 6, the 4-[(deoxy)guanosin-N2-yl]Sudan I adduct (6a/6b). Hence, this conversion is the second pathway of adduct 6 formation. We suggest several mechanisms for the transformation of adducts 5 to adducts 6; three of which are shown in Figure 2S in Supporting Information. It should be noted that the Sudan I metabolites analyzed here are much less likely to be formed physiologically than in the in Vitro system because many nucleophilic molecules are present in cells to scavenge the Sudan I reactive species. Indeed, during Sudan I oxidation by peroxidase in the presence of (deoxy)guanosine, the formation of adducts runs parallel to a decrease in the generation of Sudan I metabolites. Such a decrease was also observed when other nucleophiles such as tRNA and poly X were added to the incubation mixtures. Hence, the formation of adducts between Sudan I reactive species and these nucleophilic compounds seems to be the preferred reaction under physiological conditions. In conclusion, the results of this study are the first report on the molecular mechanism of the formation of adducts of Sudan I with (deoxy)guanosine during the oxidation of this carcinogen with peroxidase, utilizing the structural characterization of these adducts. Studies that are in progress in our laboratory will show which of the (deoxy)guanosine adducts prepared and characterized as standards in this work are formed in DNA/RNA in Vitro (24-26, 28) and in ViVo (30). Acknowledgment. This research was supported in part by Grant Agency of the Czech Republic (grants 303/09/0472 and 203/ 09/0812), Ministry of Education of the Czech Republic (grant MSM0021620808), and Academy of Sciences of the Czech Republic (Research projects Z4 055 0506 and KJB 400 550 903). We thank Dr. Martin Sˇtıćha for the mass spectrometric analyses. Supporting Information Available: Difference spectra of guanosine and Sudan I-derived guanosine adducts 5 and 6 measured as transitions from pH 7.0 to 10.0 and possible mechanisms of the transformation of adducts 5 to 6. This material is available free of charge via the Internet at http:// pubs.acs.org.


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Mechanism of Formation of (Deoxy) guanosine Adducts Derived from

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