Identification of the Major Hepatic DNA Adduct Formed by the Food

The corresponding nitro compound 2-nitro-9H-pyrido[2,3-b]indole (NαC) was prepared and shown to be a direct acting mutagen in the Salmonella assay, w...
0 downloads 0 Views 126KB Size
Download PDF
1192

Chem. Res. Toxicol. 1997, 10, 1192-1197

Identification of the Major Hepatic DNA Adduct Formed by the Food Mutagen 2-Amino-9H-pyrido[2,3-b]indole (ArC) Wolfgang Pfau,* Christian Schulze,† Tomoyuki Shirai,§ Rhyohei Hasegawa,§ and Ulrike Brockstedt Department of Toxicology and Environmental Medicine of the Fraunhofer Society and Center of Molecular Neurobiology, Hamburg University Medical School, Grindelallee 117, 20146 Hamburg, Germany, and Department of Pathology, Nagoya City University Medical School, Nagoya 467, Japan Received July 9, 1997X

2-Amino-9H-pyrido[2,3-b]indole (ARC) is among the most prevalent heterocyclic amines detected in grilled or panfried meat; it was shown to be carcinogenic in mice, to induce preneoplastic foci in rat liver, and to form covalent DNA adducts in vitro and in vivo. The corresponding nitro compound 2-nitro-9H-pyrido[2,3-b]indole (NRC) was prepared and shown to be a direct acting mutagen in the Salmonella assay, while the amino compound required external metabolic activation with rat liver homogenate (S9). When ARC was incubated with S9 in the presence of calf thymus DNA, one major DNA adduct spot was detected upon 32Ppostlabeling analysis. This adduct comigrated on ion-exchange TLC and reversed-phase HPLC with the major adduct detected in primary hepatocytes treated with ARC. In DNA isolated from livers of male F344 rats treated with 800 and 160 ppm, the formation of the same major adduct was observed with relative adduct levels of 20.6 ( 9.6 and 1.4 ( 1.1 adducts/108, respectively, as determined with the butanol extraction variant of the 32P-postlabeling assay. No DNA adducts were detected in liver DNA from rats treated with 32 ppm ARC or control animals. The major adduct spot was eluted and hydrolyzed and the modified base characterized by chromatographic and UV spectral comparison with a synthetic standard synthesized from acetylated guanine N3-oxide and ARC. Electrospray mass spectrometry and 1H- and 13C-NMR spectroscopy provided further evidence for the major adduct as N2-(guanin-8-yl)-2-amino-9Hpyrido[2,3-b]indole. ARC is formed especially in high-temperature preparation of food and may contribute considerably to the human carcinogenic risk that might be imposed by heterocyclic amines.

Introduction Carcinogenic heterocyclic amines are detected at parts per billion levels in cooked food and tobacco smoke (1, 2). 2-Amino-9H-pyrido[2,3-b]indole (ARC)1 was originally identified as a mutagenic product of pyrolysis of soy protein (3) and was subsequently detected in cooked and grilled food. It is one of the most prevalent of these compounds occurring in cooked beef, poultry, and fish at levels up to 180 ppb and in tobacco smoke (42 ng/ cigarette) (3-5). Although it failed to induce tumors in one study when rats were treated with up to 800 ppm ARC in the diet (1), high incidences (97% in female and 39% in male CDF mice) of tumors in the liver and the vascular system occurred in mice fed a diet supplemented with 800 ppm ARC (6). Furthermore, this compound was active in a number of short-term genotoxicity tests both in vitro and in vivo (2). ARC is generally considered to belong to the group of food mutagens, although the mutagenic activity of ARC * To whom correspondence should be addressed. † Center of Molecular Neurobiology, Hamburg University Medical School. § Nagoya City University Medical School. X Abstract published in Advance ACS Abstracts, September 15, 1997. 1 Abbreviations: ARC, 2-amino-9H-pyrido[2,3-b]indole; ARC-Gua, N2-(guanin-8-yl)-2-amino-9H-pyrido[2,3-b]indole; CID, collision-induced dissociation; CT-DNA, calf thymus DNA; ESI-MS, electrospray ionization mass spectrometry; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; NRC; 2-nitro-9H-pyrido[2,3-b]indole; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine.

S0893-228x(97)00118-5 CCC: $14.00

in Salmonella typhimurium is several orders of magnitude lower in comparision to the aminoimidazoazarenes (1, 2). However, genotoxicity in mammalian test systems caused by ARC is as strong as for the related heterocyclic amines (6-8). ARC has been shown to induce chromosomal abberations in Chinese hamster ovary cells in vitro and to induce mutations in Drososphila melanogaster (2). In an in vivo medium-term bioassay for rapid detection of liver carcinogens and promoters, glutathione S-transferase placental form-positive foci were induced in livers of rats treated with doses as low as 32 ppm ARC in the diet (8). Similar to other polycyclic and heterocyclic amines, ARC is metabolically activated by hepatic cytochrome P450 1A2 to the corresponding hydroxylamine. Among ring-hydoxylated products, this was the major metabolite detected in an in vitro study using rodent and human microsomes (9). Recently, we were able to identify the DNA adduct formed by the 3-methyl derivative 2-amino-3-methyl-9Hpyrido[2,3-b]indole as N2-(deoxyguanosin-8-yl)-2-amino3-methyl-9H-pyrido[2,3-b]indole (10). In the present paper we describe the analysis of DNA adducts formed by ARC in vitro in primary rat hepatocytes and in vivo in the liver of male F344 rats treated with ARC using butanol-extraction-enhanced 32P-postlabeling analysis. The major adduct was characterized by chromatographic comparison on both ion-exchange TLC and reversedphase HPLC with the reaction product of ARC with calf © 1997 American Chemical Society

ARC DNA Adducts

thymus DNA in the presence of rat liver homogenate (S9). Full structural characterization was achieved by chemical synthesis of the modified base using an alternative route. Further indications for the pathway of metabolic activation of ARC were obtained by mutagenicity testing of ARC and the nitro derivative NRC with the Salmonella tester strain YG 1019 overexpressing a bacterial acetyltransferase.

Materials and Methods Caution: The following chemicals are hazardous and should be handled carefully: Mo(CO)6, trifluoroacetic acid, and trifluoroacetic anhydride; ARC and NRC may be carcinogenic. Synthesis of NrC. 2-Nitro-9H-pyrido[2,3-b]indole (NRC) was prepared in analogy to a protocol by Hashimoto (11) with minor modifications: A mixture of 1100 µL of trifluoroacetic anhydride and 150 µL of trifluoroacetic acid was cooled to 0 °C, and 330 µL of aqueous hydrogen proxide (30%) was added followed by 39 mg of molybdenum hexacarbonyl. To this mixture was added a precooled solution of 40 mg of ARC in 3.5 mL of dichloromethane and 160 µL of trifluoroacetic acid at 0 °C, and the mixture stirred at room temperature. The reaction was monitored on silica gel TLC plates. The yellow reaction product [Rf ) 0.9 in CH2Cl2:ethyl acetate 4:1 (v/v)] was quickly formed, and when the fluorescent amino compound (Rf ) 0.01) had disappeared, the reaction was stopped by cautious addition of 5 mL of saturated potassium carbonate solution. Extraction into CH2Cl2 and evaporation afforded the crude product that was purified by flash column chromatography on silica gel with CH2Cl2. The product was characterized by electron impact mass spectrum (m/z 213 (M+) and UV/vis spectrum (λmax ) 251, 288, 335 nm) in agreement with the proposed structure. Synthesis of N2-(Guanin-8-yl)-2-amino-3-methyl-9H-pyrido[2,3-b]indole. In analogy to a protocol described by Hashimoto et al. (11), guanine N3-oxide (11.2 mg) was suspended in 2 mL of dimethyl sulfoxide:dimethylformamide (3/1, v/v) and cooled to 0 °C, and acetic anhydride (6 µL) was added. After vigorous stirring for 10 min, ARC (2.3 mg, 11 µmol) was added, and stirring continued overnight at room temperature. The mixture was concentrated under reduced pressure to 100 µL, deionized water (1 mL) was added, and the precipitate was isolated by centrifugation, redissolved in 0.1 N hydrochloric acid, and analyzed by HPLC. Analysis of Modified Guanine. The major DNA adduct spot obtained upon 32P-postlabeling analysis was excised, and 1 mL of hydrochloric acid (1 N) was added. After 60 min the supernatant was extracted with n-butanol (200 µL), the organic solvent was evaporated, and the residue was redissolved in methanol. Analyses were performed with a HPLC gradient system HP 1050M equipped with a diode array detector (Hewlett Packard, Waldbronn, Germany) on an Alltima ODS column (250 × 4 mm i.d.; Alltech Assoc., Deerfield, IL) with a linear gradient of acetonitrile (5-60% at 40 min) in water. Mass Spectrometry. Electrospray mass spectra were recorded on a MAT 95 (Finnigan MAT, Bremen, Germany) sector field mass spectrometer equipped with an electrospray ion source. The mass spectrometer was set to a resolution of 2000, scanned from mass 50 to 700 in 2.8 s for molecular weight determination, and scanned from mass 50 to 350 for skimmer CID (capillary skimmer voltage 180 V). Samples were dissolved in acetic acid, diluted with 80% methanol, and introduced into the ion source by injecting 10 µL aliquots into a constant carrier flow (3 µL/min) of 0.1% formic acid in 80% methanol. In Vitro Incubation. According to published methods (12) a solution of ARC (5 µmol in 100 µL of DMSO) was added to a mixture of 8 mg of NADPH, 5 mg of MgCl2, 1 mg of CT-DNA, and 150 µL of S9 (5.4 mg of protein) in 1 mL of sodium phosphate buffer (pH 7.4, 0.1 M). After incubation for 60 min the reaction was stopped by extraction with n-butanol (500 µL). DNA was precipitated in the cold from the aqueous phase by addition of sodium chloride and 2-ethoxyethanol.

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1193 Treatment of Primary Hepatocytes. Primary rat hepatocytes were isolated from male Wistar rats with collagenase according to published procedures (13). Cells were incubated with ARC (Toronto Research Chemicals, Toronto, Canada) at a final concentration of 100 µM for 24 h at 37 °C. Hepatocytes were harvested by scraping and centrifugation; the DNA was isolated by the phenol extraction procedure as described by Gupta (14). Treatment of Animals. Groups of three 6-week old male F344 rats (Charles River Japan Inc., Atsugi) were treated for 2 weeks with ARC added in 2% corn oil to a powdered basal diet (Oriental MF, Oriental Yeast Co., Tokyo) at 800, 160, or 32 ppm. Control animals received the basal diet with 2% corn oil. All animals were killed after 2 weeks; the livers were excised, immediately frozen, stored, and shipped at -80 °C. DNA was isolated by phenol extraction (14), precipitated with sodium chloride and ethanol at -20 °C, redissolved in sodium chloride/ sodium citrate (0.15 mM/0.015 mM), and stored at -80 °C. 32P-Postlabeling Analysis. Aliquots of DNA (5 µg), isolated from rat liver, hepatocytes, or modified calf thymus DNA, were analyzed in duplicate by the 32P-postlabeling method using the butanol extraction procedure following published protocols (15). TLC. The labeling mix was applied to poly(ethylenimine) cellulose plates (10 × 10 cm) with a wick (10 × 7 cm) of Whatman no. 17 paper stapled to the top. Multidirectional chromatography was performed as described elsewhere (10, 16). Autoradiography was performed at -80 °C using intensifying screens. HPLC. Analysis of 32P-labeled adducts was performed according to the method of Pfau and Phillips (17) with modifications (18): Adduct spots were excised from PEI plates after fourdirectional TLC or from the origins after one-directional TLC, and the adducts were eluted with pyridinium formate (4 M, pH 4.0). Aliquots were injected onto a Zorbax phenyl-modified reversed-phase column (250 × 4.6 mm i.d., particle size 5 µm) and eluted at a flow rate of 1.0 mL/min with a linear gradient of B (water/acetonitrile, 35/65 vol %) in buffer A (0.5 M NaH2PO4/0.5 M H3PO4, pH 2.0) as follows: for 5 min 15% B, linearly increasing to 30% B at 35 min and 50% B at 45 min. Quantification. Determination of adduct levels was accomplished by Cerenkov counting of excised adduct spots and published calculation procedures (15) taking into account the specific activity of the [γ-32P]ATP batch used in the experiment. Specific activities were determined by labeling of deoxyadenosine-3′-phosphate as described (16) and varied between 1200 and 3100 Ci/mmol. Mutagenicity Assays. Reversion to prototrophy using S. typhimurium histidine auxotrophic strain YG 1019 was measured essentially as described by Maron and Ames (19) with modifications according to Yahagi et al. (20). This tester strain, overexpressing the bacterial acetyltransferase, was developed by Watanabe et al. (21) and was kindly donated to us by P. D. Josephy. Rat liver homogenate (S9) from aroclor-pretreated rats was obtained from Organon, Heidelberg, Germany. Each concentration was assessed in triplicate in the preincubation assay on two different occasions.

Results Incubation of ARC with rat liver homogenate in the presence of DNA resulted in one major DNA adduct spot as analyzed by butanol extraction/32P-postlabeling analysis with ion-exchange TLC analysis. Relative adduct levels were 792 ( 46 adducts/108 nucleotides (Figure 1A). Similarly, the treatment of primary rat hepatocytes with ARC (100 µM) resulted in one major DNA adduct upon 32P-postlabeling analysis with conventional ionexchange TLC (Figure 1B) similar to published data (7). The relative DNA adduct level was 62 ( 26 adducts/108 nucleotides when the butanol extraction procedure was applied. A similar pattern of adducts was detected upon 32P-postlabeling analysis of DNA isolated from the liver

1194 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

Pfau et al.

Figure 2. Radiochromatograms of HPLC analyses of 32Plabeled adducts from (A) rat hepatocytes following incubation with ARC and (B) the liver of an ARC-treated rat. 32P-Labeled adducts were analyzed on a Zorbax phenyl-modified silica gel column with a gradient of acetonitrile in 0.5 M phosphate buffer (pH 2.0) as described in Materials and Methods.

Figure 1. Autoradiographic representations of butanol-extraction-enhanced 32P-postlabeling/ion-exchange TLC analyses of DNA (A) modified with ARC in vitro in the presence of rat liver S9, (B) from rat hepatocytes following incubation with ARC (100 µM), and (C) from the liver of an ARC-treated rat (800 ppm).

of Fischer 344 rats treated with ARC (Figure 1C). Relative adduct levels were 20.6 ( 9.6 adducts/108 for rats treated with 800 ppm ARC and 1.4 ( 1.1/108 for animals treated with 160 ppm ARC; no DNA adducts were detectable in hepatic DNA from F344 rats treated with 32 ppm ARC or in DNA isolated from the livers of control animals (not shown). Using HPLC analysis of 32P-labeled nucleotide bisphosphate adducts (18), the major adducts derived from the different hepatic systems had identical retention times. The major adduct observed in ARC-modified DNA eluted at 38:00 min upon reversed-phase HPLC analysis and appeared as one homogeneous product (Figure 2). Upon addition of 1 N hydrochloric acid to the excised adduct spot, the adduct was hydrolyzed and the modified base dissolved. Following extraction into n-butanol the modified base was analyzed by reversed-phase HPLC analysis on C-18 modified silica gel with a gradient of acetonitrile in water. Apart from an early eluting peak (unmodified bases) one peak was observed with a retention time of 24.14 min and a characteristic UV spectrum with maxima of absorption at 249, 290, and 360 nm (Figure 3A). In order to characterize the structure of the modified base and thus the adduct, we synthesized guanine modified at the C8-position with ARC by chemical reac-

Figure 3. HPLC analyses of (A) the acid hydrolysate of the adduct spot shown in Figure 1A derived from 32P-postlabeling analysis of DNA modified with ARC and rat liver homogenate and (B) the reaction product guanine N3-oxide with ARC monitoring absorbance at 360 nm. Inserts show online DADUV/vis absorption spectra of the peaks eluting at 24.2 min.

tion of ARC with acetylated guanine N3-oxide [similar to a protocol reported by Hashimoto et al. (11)]. The reaction

ARC DNA Adducts

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1195 Table 1.

1H-

and13C-NMR Resonance Frequencies of ArC-Gua, ArC, and Guaninea ARC

ARC-Gua

6.17 (2H) s, br 6.48 (1H) d 7.21 (1H) t 7.33 (1H) t 7.47 (1H) d 7.96 (1H) d 8.18 (1H) d 11.2 (1H) s, br

10.35 (1H) s, br 6.95 (1H) d 7.18 (1H) t 7.35 (1H) t 7.50 (1H) d 8.03 (1H) d 8.45 (1H) d 11.80 (1H) s

Gua

1H-NMR

Figure 4. Proposed structure and fragmentation pattern of N2(guanin-8-yl)-2-amino-9H-pyrido[2,3-b]indole upon collisioninduced dissociation.

product was identical to the modified base from ARCmodified DNA as judged by retention time (24.2 min) and DAD-UV/vis absorbance spectrum (insert of Figure 3B). This product was purified by means of micropreparative reversed-phase HPLC and analyzed by electrospray mass spectrometry (22). The mass spectrum of this compound shows an ion at m/z 333, corresponding to the protonated molecular ion, and indicates Mr ) 332, as expected for the proposed structure shown in Figure 4 (C16H12N8O). Further confirmation of this structure by fragmentation of the molecule was achieved by collision-induced dissociation (CID). The fragment ion spectrum for ARC-Gua produced by CID in the ion source region of the electrospray source (skimmer CID) is dominated by an ion at m/z 167, indicative of ARC (loss of the exocyclic amino group). Ions found were normalized to m/z 333 (m/z, relative intensity): 333 (MH+, 100), 209 (38), 194 (18), 182 (10), 167 (90), 140 (75), and 113 (41). This pattern can be explained by fragmentations of the purine moiety with charge retention at the ARC as indicated in Figure 4. The occurrence of a fragment ion at m/z 209 confirms the binding of the ARC to a guanine carbon atom. This fragmentation pattern is in agreement with the proposed linkage at the C8 of guanine via the exocyclic amino group of ARC. The structural characterization of the guanine adduct was further corroborated by 1H- and 13C-NMR data (400 MHz, DMSO-d6) summarized in Table 1. The 1H-NMR spectrum showed signals for all ring protons of the ARC moiety but no resonance for the guanine H8 atom. In Table 1 the 13C-NMR resonance frequencies of ARC and guanine are shown for comparison. Most striking is the loss of the C8 resonance in the in-depth experiment, indicating that no proton is bound to guanine-C8 in the modified guanine. Mutagenic activity of ARC and its nitro derivative NRC was compared using the Salmonella preincubation assay (19, 20). In analogy to other heterocyclic amines ARC requires the addition of an external metabolizing system (rat liver homogenate) to exert its mutagenic activity (14). Compared to the tester strain TA 1538 the mutagenic activity was 5.3-fold increased in the S. typhimurium tester strain YG 1019 that was shown to overexpress the bacterial acetyltransferase (21). The nitro derivative MeNRC was a direct mutagen, and addition of rat liver homogenate reduced the mutagenic activity observed (Figure 5).

Discussion The major adducts formed by heterocyclic or polycyclic aromatic amines have generally been shown to have a structure with the amine binding via its exocyclic amino

N2′-H2 C3′-H C6′-H C7′-H C8′-H C5′-H C4′-H N9′-H C8-H N2-H2 N9-H 13C-NMR C2′ C3′ C4′ C5′ C6′ C7′ C8′ C10′ C11′ C12′ C13′ C2 C4 C5 C6 C8

7.7 (2H) s, br 11.58 (1H) s, br 152.9 111.3 119.2 119.7 123.8 130.7 102.0 159.5 122.9 105.6 137.9 100.2 148.4 150.7 153.8 138.8

8.9 (1H) s 7.4 (2H) s, br 11.9 s, br

151.5 112.2 120.6 120.9 126.1 132.1 104.2 154.2 121.7 103.8 132.2 108.3 150.6 154.0 155.9 137.9

a Spectra were recorded in DMSO-d on a Bruker 500 MHz 6 B-ACS 120 spectrometer. Signals (br, broad) and multiplicities (s, singlet; d, doublet; t, apparent triplet) are in accordance with published data (9). 13C-NMR resonances in bold were also observed in an in-depth experiment (some assignments are arbitrary).

Figure 5. Dose-response curves of mutagenic activity of ARC and the corresponding nitro derivative NRC in the S. typhimurium strain YG 1019 with (+S9) and without (-S9) addition of rat liver S9 mix.

group to the C8-position of guanine (23). Here we pursued several lines of evidence indicating that the major adduct formed by ARC in rat liver in vivo and in vitro in primary rat hepatocytes has a corresponding structure. As for similar aromatic and heterocyclic amines, the hydroxylamine is believed to be the metabolite of ARC that leads to the formation of covalent DNA adducts (9, 24). Mutagenicity data indicate that ARC

1196 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

might be metabolically activated via this conventional route. Whereas the amine required external metabolic activation, [shown by Yoshida et al. (3)], the nitro compound was a direct-acting mutagen in the Ames assay. This is in analogy to related heterocyclic amines where the corresponding nitro derivatives have been shown to be direct mutagens (2). Furthermore, compared to the mutagenic activity of ARC in the Salmonella strain TA 1538 (1), mutagenicity was increased by a factor of 5.3 in YG 1019, a tester strain overexpressing the bacterial acetyltransferase (21). This indicates that O-acetylation of the putative hydroxylamine might play a role in the metabolic activation of ARC similar to IQ, MeARC, and MeIQx. Using the butanol extraction enrichment procedure of the 32P-postlabeling assay (15), we detected one major adduct spot in DNA modified with ARC in the presence of rat liver S9, in primary rat hepatocytes treated with ARC, and in hepatic DNA from F344 rats treated subchronically with 800 or 160 ppm ARC added to the feed. The identity of the major adducts formed in these three hepatic systems was confirmed by HPLC analysis with online radioactivity detection using a chromatographic system that has been shown to be suited to separate HAderived adducts (10, 17, 18). It has been demonstrated that the butanol extraction variant of the 32P-postlabeling assay is more suitable for the analysis of adducts derived from aromatic amines than the nuclease P1 enhancement procedure (25). When we applied the nuclease P1 method to ARC-modified DNA, the adduct recovery was reduced to about 25% (data not shown). However, even the butanol extraction recovers only about 10% of the DNA adducts as has been shown for 4-aminobiphenyl-modified DNA (D. H. Phillips and J. J. Castegnaro, personal communication). If this applies to ARC-derived adducts as well, DNA adduct levels reported here may be an underestimation of true levels of modification. While being among the most sensitive methods for the analysis of bulky DNA adducts, a major drawback of the 32 P-postlabeling assay is the lack of structural information that can be obtained from these analyses. Thus it has been suggested that the 32P-postlabeling assay is being combined with spectroscopic techniques. Here we report a convenient procedure to analyze DNA adduct spots by HPLC/DAD-UV/vis spectroscopic analysis. By acid hydrolysis of aromatic amine-modified DNA, the modified bases are liberated from the DNA backbone, as shown 30 years ago by Kriek et al. (26) for the carbon analog of ARC, 2-aminofluorene. This simple procedure allows further analysis of adduct spots following the highly sensitive 32P-postlabeling analysis and may, especially in combination with mass spectrometric techniques, lead toward identification of unknown adducts that are readily detected by 32Ppostlabeling in human tissues. The detection limit of UV detection is several orders of magnitude higher as compared to 32P-postlabeling analysis. Therefore, detection of ARC-Gua was achieved only for DNA highly modified with ARC in vitro. Hashimoto et al. (11) have shown that reaction of heterocyclic amines with acetylated guanine N3-oxide yields a guanine base modified at the C8-position with the heterocyclic amine. Indeed, we were able to show that the major reaction product of guanine N3-oxide with ARC was identical to the hydrolysis product of the major hepatic DNA adduct spot observed upon 32P-postlabeling

Pfau et al.

with multidirectional TLC analysis, with identical retention on reversed-phase HPLC and superimposable UV/ vis spectra, as shown in Figure 3. Thus, we propose the structure of the major adduct formed by ARC as being N2-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole. This structure was confirmed by mass spectrometric analysis. CID fragmentation resulted in a pattern of fragments that concurred with the proposed structure, especially a prominent signal at m/z 209 corresponding to a cyanamide fragment, thus proving the binding of MeARC amino group to a C atom of the guanine moiety. A similar pattern of fragmentation was recently described by Rindgen et al. (27) for the C8-guanine adduct of PhIP analyzed by electrospray CID. A corresponding pattern of fragmentation was observed upon CID analysis of the MeARC-C8-guanine adduct (10). In a preliminary abstract King and Kadlubar reported on the major DNA adduct formed by the N-hydroxy derivative of ARC as being a desoxyguanosine adduct (28). Most recently Okonogi et al. (29) reported on the mutational spectra induced by ARC in the lacI gene in the colon of Big Blue mice. In accordance with the major adduct being a deoxyguanosine derivative, the majority of mutations (>90%) was detected at GC base pairs. Similar to PhIP and MeIQ [both have been shown to produce mainly C8-guanine adducts (29, 30)] base substitutions were predominant with GC f TA transversions being the most common mutation. Research in this area has focused on aminoimidazoazarenes with high specific bacterial mutagenic activity (IQ) or those that induce tumors in rats with relatively high incidence (PhIP) (31-33). Recent findings by Raza et al. (9) suggest that ARC might be more relevant to human carcinogenicity. Since ARC is among the most prevalent HA in cooked foods (5) and is formed not only in heated meat but also in vegetarian food and cigarette smoke, the cumulative dose may contribute considerably to human cancer risk. We are currently investigating if the low doses to which man is exposed might lead to ARCinduced DNA adducts in human tissues.

Acknowledgment. S. typhimurium strain YG 1019 was a generous gift from P. D. Josephy (University of Guelph, Ontario). This work was supported by the DFG (Grant Pf 283/2) and the Stiftung Verhalten und Umwelt, Munich.

References (1) Sugimura, T. and Sato, S. (1983) Mutagens-carcinogens in foods. Cancer Res. 43 (Suppl.) 2415a-2421s. (2) Eisenbrand, G., and Tang, W. (1993) Food-borne heterocyclic amines, chemistry, formation, occurrence and biological activities. A literature review. Toxicology 84, 1-82. (3) Yoshida, D., Matsumoto, T., Yoshimura, and Matsuzaki, T. (1978) Mutagenicity of amino-R-carbolines in pyrolysis products of soybean globulin. Biochem. Biophys. Res. Commun. 83, 915-920. (4) Matsumoto, T., Yoshida, D., and Tomita, H. (1982) Determination of mutagens, amino-R-carbolines in grilled foods and cigarette smoke condensate. Cancer Lett. 12, 105-110. (5) Layton, D. W., Bogen, K. T., Knize, M. G., Hatch, F. T., Johnson, V. M., and Felton, J. S. (1995) Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis 16, 39-52. (6) Ohgaki, H., Matsukura, N., Morino, K., Kawachi, T., Sugimura, T., and Takayama, S. (1984) Carcinogenicity in mice of mutagenic compounds from glutamic acid and soybean globolin pyrolysates. Carcinogenesis 5, 815-819. (7) Yamashita, K., Umemoto, A., Grivas, S., Kato, S., Sato, S., and Sugimura, T. (1988) Heterocyclic amines-DNA adducts analyzed by 32P-postlabeling method. Nucleic Acids Res. Symp. Ser. 14, 111-114.

ARC DNA Adducts (8) Hasegawa, R., Shirai, T., Hakoi, K., Takaba, K., Iwasaki, S., Hoshiya, T., Ito, N., Nagao, M., and Sugimura, T. (1991) Synergistic enhancement of glutathione S-tranferase placental formpositive hepatic foci development in diethylnitrosamine-treated rats by combined administration of five heterocyclic amines at low doses. Jpn. J. Cancer Res. 82, 1378-1384. (9) Raza, H., King, R. S., Squires, R. B., Guengerich, F. P., Miller, D. W., Freeman, J. P., Lang, N. P., and Kadlubar, F. F. (1996) Metabolism of 2-amino-R-carboline - A food-borne heterocyclic amine mutagen and carcinogen by human and liver microsomes and by human cytochrome P4501A2. Drug Metab. Dispos. 24, 395-400. (10) Pfau, W., Brockstedt, U., Schulze, C., Neurath, G., and Marquardt, H. (1996) Characterization of the major DNA adduct formed by the food mutagen 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeARC) in primary rat hepatocytes. Carcinogenesis 17, 2727-2732. (11) Hashimoto, Y., Shudo, K., and Okamoto, T. (1982) Modification of DNA with potent mutacarcinogenic Glu-P1 isolated from a glutamic acid pyrolysate: Structure of the modified nucleic acid base and initial chemical event caused by the mutagen. J. Am. Chem. Soc. 104, 7636-7640. (12) Pfau, W., Hughes, N. C., Grover, P. L., and Phillips, D. H. (1992) HPLC analysis of DNA adducts formed by benzo[b]fluoranthene. Cancer Lett. 65, 159-167. (13) Williams, G. M. (1977) Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37, 1845-1851. (14) Gupta, R. C. (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. U.S.A. 81, 6943-6947. (15) Gupta, R. C. (1985) Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts. Cancer Res. 45, 5656-5662. (16) Reddy, M. V. and Randerath, K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551. (17) Pfau, W., and Phillips, D. H. (1991) Improved reversed-phase high-performance liquid chromatographic separation of 32Plabeled nucleoside 3′,5′-bisphosphate adducts of polycyclic aromatic hydrocarbons. J. Chromatogr. 570, 65-76. (18) Pfau, W., Brockstedt, U., So¨hren, K. D., and Marquardt, H. (1994) 32P-Postlabeling analysis of DNA adducts formed by food-derived heterocyclic amines: evidence for incomplete hydrolysis and a procedure for adduct simplification. Carcinogenesis 15, 877-882. (19) Maron, D. M., and Ames, B. (1983) Revised methods for the Salmonella mutagenicity test. Mutat. Res. 64, 159-165. (20) Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T., and Okada, M. (1977) Mutagenicity of N-nitrosamines in Salmonella. Mutat. Res. 48, 121-130. (21) Watanabe, M., Ishidate, M., and Nohmi, T. (1990) A sensitive method for the detection of mutagenic nitroarenes and aromatic

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1197

(22) (23)

(24)

(25) (26)

(27)

(28)

(29)

(30)

(31) (32) (33)

amines: New derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels. Mutat. Res. 234, 337-348. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64-71. Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerba¨ck, D., and Bartsch, H., Eds. (1994) DNA Adducts Identification and Biological Significance, IARC Scientific Publications, Lyon, France. Nagao, M., Fujita, Y., Wakabayashi, K., and Sugimura, T. (1983) Ultimate forms of mutagenic and carcinogenic heterocyclic amines produced by pyrolysis. Biochem. Biophys. Res. Commun. 114, 626-631. Gupta, R. C., and Earley, K. (1988) 32P-Adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis 9, 1687-1693. Kriek, E., Miller, J. A., Juhl, U., and Miller, E. C. (1967) 8-(N2-Fluorenylacetamido)guanosine, an arylamidation reaction product of guanosine and the carcinogen N-acetoxy-n-2-fluorenylacetamide in neutral solution. Biochemistry 6, 177-182. Rindgen, D., Turesky, R. J., and Vouros, P. (1995) Determination of in vitro formed DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine using capillary liquid chromatography/electrospray ionization/tandem mass spectrometry. Chem. Res. Toxicol. 8, 1005-1013. King, R. S., Doerge, D., Lang, N. P. and Kadlubar, F. F. (1997) Metabolic activation of N-hydroxy-2-amino-R-carboline (N-OHARC) and identification of the ARC-nucleotide adducts formed in vitro and in vivo. Proc. Am. Assoc. Cancer Res. 38, Abstract 2270. Okonogi, H., Ushijima, T., Zhang, X. B., Heddle, J. A., Suzuki, T., Sofuni, T., Felton, J. S., Tucker, J. D., Sugimura, T., and Nagao, M. (1997) Agreement of mutational characteristics of heterocyclic amine on lacI of the Big Blue mouse with those in tumor related genes in rodents. Carcinogenesis 18, 745-748. Turesky, R. J. (1994) DNA adducts of heterocyclic amines, arylazides and nitroquinoline-1-oxide. In DNA Adducts - Identification and Biological Significance (Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerba¨ck, D., and Bartsch, H., Eds.) pp 217-228, IARC Scientific Publications, Lyon, France. Felton, J. S., and Knize, M. G. (1991) Occurrence, identification, and bacterial mutagenicity of heterocyclic amines in cooked food. Mutat. Res. 259, 205-217. Weisburger, J. H. (1993) Heterocyclic amines in cooked foods: possible human carcinogens. Cancer Res. 53, 2422-2424. Snyderwine, E. G. (1994) Some perspectives on the nutritional aspects of breast cancer research. Food derived heterocyclic amines as etiologic agents in human mammary cancer. Cancer 74, 1070-1077.

TX9701182