Efficient Nitroso Group Transfer fromN-Nitrosoindoles to Nucleotides and 2′-Deoxyguanosine at Physiological pH

The endogenous formation ofN-nitrosoindoles is of concern since humans are exposed to a variety of naturally occurring and synthetic indolic compounds. As part of a study to evaluate the genotoxicity ofN-nitrosoindoles, the reactions of three model compounds with purine nucleotides and 2′-deoxyguanosine at physiological pH were investigated. The profiles of reaction products were identical for each of the N-nitrosoindoles and three distinct pathways of reaction could be discerned. These pathways were: (i) depurination to the corresponding purine bases, (ii) deamination, coupled with depurination, to give hypoxanthine and xanthine, and (iii) formation of the novel nucleotide 2′-deoxyoxanosine monophosphate and its corresponding depurination product oxanine in reactions with 2′-deoxyguanosine monophosphate. 2′-Deoxyoxanosine and oxanine were observed in reactions with 2′-deoxyguanosine. Further studies showed that formation of all of these products could be rationalized by an initial transnitrosation step. These results suggest that, in contrast to many other genotoxic N-nitrosocompounds which are known to alkylate DNA, the genotoxicity ofN-nitrosoindoles is likely to arise through transfer of the nitroso group to nucleophilic sites on the purine bases. All of the products resulting from transnitrosation byN-nitrosoindoles are potentially mutagenic. These findings reveal a new pathway for N-nitrosocompounds to exert genotoxicity.

N-Nitrosation frequently transforms innocuous nitrogencontaining compounds into toxic compounds (1). Depending on the structure of the N-substituents the resulting N-nitrosocompounds can either decompose spontaneously to give alkylating intermediates or do so after metabolic activation to ␣-hydroxy derivatives (Fig. 1). Alkylating agents such as alkyldiazonium ions react with DNA to give adducts which can be mutagenic upon replication. Among the many hundreds of nitrogenous compounds that have been studied a number of 3-substituted indoles have been found to produce mutagenic products upon treatment with nitrous acid (2). 3-Substituted indoles occur widely in nature as natural products, such as tryptophan, or the plant growth hormone, indole-3-acetic acid, and this has lead to a concern that endogenous nitrosation of indoles in the acidic environment of the stomach could contribute to the risk of gastric cancer.
Indole-3-acetonitrile (IAN) 1 is a plant growth hormone that is present in various vegetables, notably Chinese cabbage, a common foodstuff in Japan. The mono-N-nitroso derivative, 1-nitrosoindole-3-acetonitrile (NIAN), is a direct acting mutagen toward Salmonella typhimurium TA98 and TA100 and Chinese hamster lung cells (3,4). 32 P-Postlabeling has shown that DNA adducts are formed in vitro and in the gastrointestinal tissues of rats treated with NIAN (5), but no attempt was made to characterize the products. Marked inductions of ornithine decarboxylase and DNA synthesis in rat stomach mucosa have been reported, after administration of NIAN, suggesting that NIAN also has potential tumor promoting activity in carcinogenesis in the glandular stomach (6). Two other indole compounds, 4-methoxyindole-3-acetonitrile and 4-methoxyindole-3-aldehyde have also been isolated from Chinese cabbage as nitrosatable mutagen precursors (7,8). In addition there are around 20 naturally occurring indole compounds, mostly 3-substituted, that have been demonstrated to be mutagenic toward Salmonella strains without S9 mix after nitrite treatment (9 -12).
Synthesis of 3-Substituted Nitrosated Indoles-IAN (Fluka), indole-3-acetamide (Sigma), or indole-3-acetic acid methyl ester (IAAME) (Sigma), dissolved in the minimum amount of acetonitrile (ACN), were reacted with a 15-fold molar excess of aqueous 50 mM nitrous acid (pH 3) at 37°C in the dark for 3 h. Reaction mixtures were extracted 3 times with 3 volumes of dichloromethane and dried over anhydrous sodium sulfate. The resulting filtrates were dried in a stream of nitrogen.
Purification and Characterization of Nitrosated Indoles-The crude products were purified by HPLC using a Gilson-gradient controlled system equipped with either a dual-wavelength 116 Gilson UV detector or an Applied Biosystems Inc., 1000s diode array detector. Analyses were performed using a Hypersil C18 BDS, 5, 250 ϫ 10-mm reversephase Shandon preparative column employing the following elution program: 0 min, 40% B, 20 min, 100% B, 25 min, 100% B, 30 min, 40% B (solvent A water; solvent B methanol) at a flow rate of 3 ml/min with UV detection at 260 and 328 nm. The pure nitrosated indole was dried in a stream of nitrogen to give yellow crystalline product in around 50% yield; the main impurity being unreacted starting material. 1  It was not possible to obtain satisfactory microanalytical results with NIAM and low nitrogen values were consistent with denitrosation (see below).

FIG. 2. Structures of 3-substituted nitrosated indoles described in this study.
mg in 250 l of ACN) were incubated with solutions of dGp or dpG (0.5 mg: 1.44 mol in 250 l of 10 mM Tris-HCl buffer, pH 7.4) as described for reactions of dAp with NIAN.
Reactions of dpA and dpG with NIAM and NIAAME-NIAM (4.8 mg in 250 l of ACN) or NIAAME (4.65 mg in 250 l of ACN) were reacted with dpA or dpG (0.5 mg in 250 l of 10 mM Tris-HCl buffer, pH 7.4) as described for reactions of dpA with NIAN. Both these concentrations correspond to a 15 M fold excess of nitrosated indole over nucleotides.
HPLC Analyses of Reaction Mixtures-HPLC-UV analyses were performed using a Hypersil C18 BDS, 5, 250 ϫ 4.6-mm reverse-phase Shandon analytical column on the Gilson gradient-controlled system equipped with either a dual-wavelength 116 Gilson UV detector or to obtain UV spectra, an Applied Biosystems Inc., 1000s diode array detector. 100-l injection volumes of aqueous reaction mixture were analyzed at a flow rate of 1 ml/min, using the following elution program: 0 min, 0% B, 25 min, 20% B, 35 min, 50% B, 40 min, 0% B (solvent A: 50 mM ammonium formate, pH 5.4; solvent B: methanol). Column eluants were monitored at 260 and 290 nm. For the separation of NIAN, NIAM, or NIAAME extracts after reaction, 30-l aliquots were analyzed at a flow rate of 1 ml/min using the elution program described for the purification of the nitrosated indoles. Column eluants were monitored at 260 and 328 nm. The fractions corresponding to reaction products were collected from multiple HPLC runs, pooled together, and dried down in a centrifugal vacuum evaporator for further analysis by ESI-MS. Reaction products were identified from their UV spectra, obtained by diode array analysis, and retention times which were compared with those of the authentic standards. Further evidence for structural assignment was obtained from the ESI-MS and ESI-MS-MS spectra of the pooled fractions.
Characterization of Reaction Products by Mass Spectrometry-Offline ESI-MS and ESI-MS-MS characterization of reaction products was carried out using a VG Autospec-Ultima Q. Dried fractions previously collected from the HPLC were resuspended in 50:50 ACN/water and inserted into the ESI via 20-l "loop" injection or continuous infusion, at a flow rate of typically 8 l/min. The cone voltage was in the range 8 -23 V and full scan mass spectra were obtained by scanning from m/z 1650 to 50 at a scan speed of 10 s/decade. ESI-MS-MS product ion spectra were obtained by selecting the desired precursor ion with MS1 and allowing collision induced dissociation to occur in the collision cell using air as the target gas, typically at 10% transmission of the precursor ion with a collision energy of 48 eV. The resulting product ions were analyzed in MS2.

Synthesis and Characterization of a Series of 3-Substituted
Nitrosated Indoles-NIAN, NIAM, and NIAAME (Fig. 2) were obtained as crystalline products by treatment of the parent compounds with acidified nitrite followed by HPLC purification. Spectral data agreed well with data previously published by Wakabayashi et al. (3). NIAN, NIAM, and NIAAME are isomeric compounds. The E/Z isomerization of the NO group is reflected in the nmr data, affecting principally the 2-H, 7-H, and -CH 2 -resonances; essentially the indole nucleus remains the same for the series of nitrosated indoles. The ratio of the two isomers was consistent for all three compounds at approximately 2:1. The disappearance of the -NH signal (as -NNO is produced) is evident in all nmr spectra. Satisfactory microanalytical data could not be obtained for NIAM, despite repeated preparations, with precautions taken to reduce denitrosation. However, spectral data for NIAM were consistent with that obtained for NIAN and NIAAME.
Reaction of Nitrosated Indoles with dAp and dpA-Reaction of NIAN with dpA at pH 7.4 in buffered 50% aqueous acetonitrile yielded 3 products not seen in control incubations, hypoxanthine, adenine (Ade), and N 6 -acetyl adenine with retention times of 7.7, 11.4, and 15.5 min, respectively (Fig. 3). Identification of hypoxanthine was based on comparison of the retention time and UV spectrum with authentic hypoxanthine analyzed under the same conditions. When ESI-MS was performed on this fraction, a molecular ion with m/z 137 (M ϩ H) ϩ was observed. Identification of adenine was again based on comparison of the retention time and UV spectrum with adenine standard. When ESI-MS was performed a main molecular ion with m/z 136 (M ϩ H) ϩ was observed. A molecular ion with m/z 332 (M ϩ H) ϩ was also observed suggesting the presence of unreacted dpA. Adenine and dpA co-elute under the HPLC conditions described. The identification of reaction product N 6acetyl adenine was based on ESI-MS results. A molecular ion with m/z 178 (M ϩ H) ϩ was observed for this fraction. When ESI-MS was performed under conditions promoting cone voltage induced dissociation, a fragment ion with m/z 136 corresponding to protonated adenine was observed, confirming the structure as an adenine adduct. When deuterated ACN was used as co-solvent, a molecular ion with m/z 181 (M ϩ H) ϩ was observed for the fraction, corresponding to N 6 -acetyl (d 3 )-adenine. All 3 reaction products increased in concentration with accompanying increases in the molar ratio of NIAN to dpA. Analogous reaction products with similar dose-related responses were seen with reactions of NIAM and NIAAME with dpA and NIAN with dAp (data not shown).
Reactions of Nitrosated Indoles with dpG and dGp and dGuo-Reaction of NIAN with dpG at pH 7.4 in buffered 50% aqueous acetonitrile yielded 6 products, guanine (Gua), xanthine, oxanine, 2Ј-deoxyoxanosine-5Ј-monophosphate (dpO), N 2 -acetyldeoxyguanosine-5Ј-monophosphate (N 2 -AcdpG), and N 2 -acetylguanine (N 2 -AcGua), not seen in control incubations, with retention times of 7.8, 8.7, 11.4, 12.1, 19.6, and 20.1 min, respectively (Fig. 4). Identification of reaction products guanine and xanthine was based on comparison of retention times, UV spectra, and ESI-MS results compared with standards analyzed using the same systems and conditions. The identification of reaction products N 2 -acetyldeoxyguanosine-5Ј-monophosphate and N 2 -acetylguanine was based on ESI-MS results and comparison of retention time and UV spectrum for authentic N 2 -acetylguanine. A molecular ion with m/z 194 (M ϩ H) ϩ was observed for the fraction corresponding to N 2 -acetylguanine. When conditions were employed to promote cone voltage induced dissociation, a fragment ion with m/z 152 corresponding to protonated guanine was observed, confirming the structure as a guanine adduct. Replacement of acetonitrile in the reaction mixture by the deuterated solvent gave a molecular ion with m/z 197 (M ϩ H) ϩ for this fraction confirming it as N 2 -(d 3 )-acetylguanine. Similarly, ESI-MS analysis for the fraction corresponding to N 2 -acetyldeoxyguanosine-5Ј-monophosphate afforded a molecular ion with m/z 388 (M ϩ H) Ϫ and again by exchanging the solvent as described above, an increase of 3 units to m/z 391 (M ϩ H) Ϫ was observed for the molecular ion.
The concentration of xanthine increased with accompanying increases in the molar ratio of NIAN to dpG, while the concentration of guanine decreased as conversion to reaction products occurred. The results indicate that depurination occurs independently of the other pathways and is more evident at the higher molar ratios. This is illustrated by the dose-response behavior of the N 2 -acetyl adducts. The formation of N 2acetyldeoxyguanosine-5Ј-monophosphate increased with an increase in the molar ratio of NIAN to dpG up to a ratio of 10:1. As the molar ratio increased to 20:1, the concentration of the product decreased in the system, and an increase in the depurinated adduct N 2 -acetylguanine was observed.
The identification of reaction products 2Ј-deoxyoxanosine-5Јmonophosphate and the depurination product oxanine (Fig. 5) was based on comparison of retention time, UV spectra, and ESI-MS results when compared with oxanine derived from the hydrolysis of the N-glycosidic bond of authentic 2Ј-deoxyox-anosine (dOxo, 13). dOxo was hydrolyzed as described by Suzuki et al. (14). dOxo (0.37 mM) was incubated in 0.1 M acetate buffer at pH 4.0 for 4 h. At hourly intervals, 100-l aliquots were injected onto the Gilson gradient-controlled HPLC system exactly as described previously for the analysis of aqueous reaction mixtures. As the dOxo peak (retention time ϭ 19.9 min) decreased, a new peak (retention time ϭ 11.4) appeared in the chromatogram, corresponding to oxanine; max 240, 287 nm (8% MeOH). The UV spectra of the novel products are very similar (Fig. 4): oxanine, max 240, 287 nm (8% MeOH); dpO, max 245, 288 nm (8.5% MeOH). Thus the retention time and UV spectrum of the hydrolysis product of dOxo was identical to that of reaction product oxanine. Upon ESI-MS analysis of the HPLC fraction corresponding to dpO, a molecular ion with m/z 347 (M ϩ H) Ϫ was observed (Fig. 6a). ESI-MS-MS analysis produced fragment ions with m/z 151, 79 (PO 3 ) Ϫ , 97 (H 2 PO 4 ) Ϫ , and 195 (C 5 H 8 PO 6 ) Ϫ (Fig. 6b). These results alone would sensibly suggest the formation of 2Ј-deoxyxanthosine-5Ј-monophosphate, however, the retention time and UV spectrum of the reaction product are not consistent with this suggestion. ESI-MS analysis on the fraction corresponding to oxanine resulted in a molecular ion with m/z 153 (M ϩ H) ϩ , corresponding to the same mass as xanthine and suggesting this product as the depurination product of dpO. The replacement of the N atom at N-1 of guanine with an O atom increases the relative molecular mass by one and coincidentally oxanine has the same mass as xanthine and 2Ј-deoxyoxanosine-5Ј-monophosphate has the same mass as 2Ј-deoxyxanthosine-5Ј-monophosphate. The dose-response behavior of these novel reaction products was similar to that found for the N-acetyl adducts previously described, lending further support to their identification. Analogous reaction products with similar dose-related responses were seen with reactions of NIAM and NIAAME with dpG and NIAN with dGp (data not shown). Reaction of NIAN, NIAM, or NIAAME with dGuo at pH 7.4 yielded the same 4 products, guanine, xanthine, oxanine, and N 2 -acetylguanine with the same retention times as described for reactions of nitrosated indoles with dpG or dGp. The absence of reaction products 2Ј-deoxyoxanosine-5Ј-monophosphate and N 2 -acetyldeoxyguanosine-5Ј-monophosphate is expected, as these are nucleotide adducts. All products exhibited analogous dose-response relationships to those described above.
Decomposition of Nitrosated Indoles in the Reaction System-When diethyl ether extracts of reaction mixtures were analyzed by HPLC, denitrosation was the major pathway of decomposition for all the nitrosated indoles. N-Nitrosoindoles gave a positive result in the Liebermann test (15) for a nitrosamine or N-nitroso compound, without the addition of acid and at room temperature. Under the usual conditions of the test, the nitrosamine is warmed with phenol and acid. The nitrosating species is liberated from the nitrosamine and nitrosates phenol to form -nitrosophenol. Another molecule of phenol combines with -nitrosophenol to form indophenol which is colored red. Under alkaline conditions, the red indophenol yields a blue indophenol anion. When reactions were repeated in the presence of azide ion, a scavanger of nitrosating agents (azide ion: N-nitrosoindole, 1:1 molar ratio), the formation of any reaction products was completely inhibited (data not shown).

DISCUSSION
Identical nucleotide reaction products, which are structurally independent of the 3-substituted nitrosated indoles, were seen for reactions of nitrosated indoles with nucleotides and dGuo. Modification via depurination, deamination, and the formation of 2Ј-deoxyoxanosine monophosphate and the depurination product oxanine were apparent. N-Acetyl adducts were also observed, with the source of acetyl groups being the co-solvent used in the monophasic reaction system. All these pathways of modification can be rationalized by a transnitrosation mechanism.
Depurination is probably catalyzed by N-nitrosation at the N-7 atom of guanine or adenine residues and/or the N-3 atom of adenine, imparting a destabilizing positive charge on the pu-rine ring system. Cleavage of the N-glycosidic bond neutralizes this charge and gives the depurination products. The corresponding N-nitrosopurine is then rapidly hydrolyzed to generate the observed base (Fig. 7).
The formation of deamination products can be explained by transnitrosation to exocyclic amino groups of purine bases. Nitrogen is readily displaced from the purine diazonium ion generated via transnitrosation. In this case, simple hydrolysis affords the deamination product, namely hypoxanthine from adenine and xanthine from guanine (Fig. 8).
The mechanism for the formation of dOxo from dGuo by nitrous acid or nitric oxide has been reported (14,16). By using guanosine and its methyl derivatives, Suzuki et al. (14) demonstrated that reaction at N-2 to give the diazonium ion is followed by cleavage of the N-1-C-6 bond and that the exocyclic amino nitrogen of dOxo originates from the imino nitrogen (N-1) of dGuo. Consequently, the mechanism for the formation of dOp/dpO and oxanine would logically follow the same route starting with transnitrosation to N-2 of dGp/dpG or dGuo (Fig. 8).
The transnitrosating potential of the three substituted nitrosoindoles is further highlighted by the formation of N-acetyl adducts. The replacement of ACN with d 3 -ACN resulted in the formation of d 3 -acetylated products, thus confirming the cosolvent as the source of acetyl groups. One possible explanation involves a slow hydrolysis step of acetonitrile to acetamide followed by transnitrosation to the amino nitrogen to produce reactive acetyldiazonium ion. However, when acetamide was added to the reaction mixtures at varying concentrations, no increase in the amount of acetylated products was observed, suggesting that this pathway may be at best, a minor pathway. Direct transnitrosation to the tertiary nitrogen atom of ACN followed by hydrolysis may be the more likely pathway and there is some evidence for this possibility (17).
When reactions were carried out using methanol and ethanol as co-solvents, no reaction products were seen. The rate of denitrosation was slightly higher in alcohols than in ACN, and it is likely that the free nitrosating agent was used up in the formation of volatile alkyl nitrites. Reaction of alcohol with nitrous acid is used as the route for their preparation (18). The procedure depends on the fact that the alkyl nitrite has a lower boiling point than the alcohol and can be distilled out from the equilibrium mixture.
It would appear therefore, that most, if not all of the products formed by reaction of N-nitrosoindoles with isolated purine nucleotides and deoxyguanosine, are the result of transnitro- sation, that is, the ready transfer of the nitroso group to nucleophilic nitrogen atoms in the purines. In the case of guanine this results in formation of a diazonium ion at C-2. The solvolysis product of this diazonium ion to give xanthine is a well known reaction in purine chemistry but it is only recently that a more profound consequence of this pathway has been discovered. The interesting observations by Suzuki et al. (13) that treatment of dGuo, oligodeoxynucleotides, and DNA with nitrous acid resulted in the formation of dOxo due to a rearrangement of the C-2-diazonium ion (14) (Fig. 8), have important mutagenic implications. The presence of an oxygen atom in place of N-1 of guanine is likely to have a profound effect on Watson-Crick base pairing and recent results on the misincorporation of 2Ј-deoxyoxanosine triphosphate into oligonucleotides suggests that it is likely to be a potent mutagenic lesion (19). Interestingly, dOxo is considerably more stable to depurination than dGuo suggesting that it is also likely to be a persistent lesion in the absence of a specific repair pathway (20). Interestingly, the riboside of oxanine, oxanosine, is a natural product with a range of biological activities (21,22).
A further consequence of the formation of purine diazonium ion in double-stranded DNA is that this may lead to the formation of interstrand cross-links by displacement of nitrogen by the exocyclic amino group of guanine on the opposing strand (23). Current studies are directed at investigating the relevance of this pathway for N-nitrosoindoles. In postlabeling studies such a G-G dimer would probably appear as a bulky adduct and this may explain the results of Yamashita et al. (5) who observed adducts formed by reaction of NIAN with calf thymus DNA.
Transnitrosation has been invoked, without any direct evidence, to explain direct-acting mutagenicity of N-nitrosoindoles (24). The mechanism of denitrosation of N-nitrosoindoles under weakly acidic or neutral conditions (pH 4 -7) has been studied and may involve intramolecular transfer of the nitroso group to C-3 of the indole prior to transnitrosation of weakly basic amines and other nucleophiles (25)(26)(27). The results presented in this paper demonstrate that, at neutral pH, N-nitrosoindoles transfer the nitroso group to nucleophilic sites on DNA bases resulting in depurination, deamination, and the formation of the novel products dOp/dpO and oxanine. All of these processes are potentially mutagenic events if they occur in DNA (28,29). These observations represent a new pathway for N-nitrosocompounds, exemplified by the N-nitrosoindoles, to exert genotoxicity mediated by transfer of the nitroso group to DNA bases. In the wider perspective, this pathway may be operative for many agents which release nitric oxide.