Detection and Characterization of DNA Adducts of 3-Methylindole

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Detection and Characterization of DNA Adducts of 3-Methylindole Kelly A. Regal,†,‡ George M. Laws,§ Cai Yuan,| Garold S. Yost,| and Gary L. Skiles*,† Biochemical and Investigative Toxicology and Genetic Toxicology, Department of Safety Assessment, Merck Research Laboratories, WP319, West Point, Pennsylvania 19486, and Department of Pharmacology and Toxicology, 30 South 2000 East, Room 201, University of Utah, Salt Lake City, Utah 84112 Received January 31, 2001

The pneumotoxin 3-methylindole is metabolized to the reactive intermediate 3-methyleneindolenine which has been shown to form adducts with glutathione and proteins. Reported here is the synthesis, detection, and characterization of nucleoside adducts of 3-methylindole. Adducted nucleoside standards were synthesized by the reaction of indole-3-carbinol with each of the four nucleosides under slightly acidic conditions, which catalyze the dehydration of indole3-carbinol to 3-methyleneindolenine. Following solid phase extraction, the individual adducts were infused via an electrospray source into an ion trap mass spectrometer for molecular weight determination and characterization of the fragmentation patterns. The molecular ions and fragmentation of the dGuo, dAdo, and dCyd adducts were consistent with nucleophilic addition of the exocyclic primary amine of the nucleosides to the methylene carbon of 3-methyleneindolenine. The apparent chemical preference of this addition lead primarily to dAdo and dGuo adducts, with substantially less of the dCyd adduct formed. No adduct with dThd was detected. The adducts were purified by HPLC and subsequent NMR analysis of the dGuo and dCyd adducts confirmed the proposed structures. Mass spectral fragmentation of the three adducts produced primarily two ions which were the result of the loss of either the 3-methylindole moiety or the sugar. On a triple quadrupole electrospray mass spectrometer, the neutral loss of the sugar, [M + H - 116]+, was utilized for selected reaction monitoring of the calf thymus DNA adducts, formed by incubations of 3-methylindole with various microsomes (rat liver, goat lung, and human liver). All three adducts were detected from each of the microsomal incubations, following extraction and cleavage of the DNA to the nucleoside level. The dGuo adduct was the primary adduct formed, with smaller amounts of the dAdo and dCyd adducts. Rat hepatocytes incubated with 3-methylindole produced the same three adducts, in approximately the same proportions, while no adducts were detected in untreated hepatocytes. Microsomal incubations in the presence of ([3-2H3]-methyl)indole confirmed the formation and identification of the adducts as well as the fragmentation patterns. These results demonstrate that bioactivated 3-methylindole forms specific adducts with exogenous or intact cellular DNA, and indicates that 3-methylindole may be a potential mutagenic and/or carcinogenic chemical.

Introduction Exposure to 3-methylindole (3MI)1 is the result of tryptophan degradation by ruminal and intestinal microorganisms (1). Absorption and systemic circulation of 3MI are followed by cytochrome P450-mediated bioactivation to multiple reactive intermediates (2-4). Previous characterization of the 3MI-related metabolic pathways indicated that dehydrogenation was the predominant * To whom correspondence should be addressed. E-mail: [email protected]. Phone: (215) 652-5187. Fax: (215) 661-5109. † Biochemical and Investigative Toxicology. ‡ Current address: Merck Research Laboratories, Department of Drug Metabolism, WP75-100, West Point, PA 19486 § Genetic Toxicology. | Department of Pharmacology and Toxicology. 1 Abbreviations: dN, 2′-deoxynucleoside; dAp, 2′-deoxyadenosine 3′monophosphate; dGp, 2′-deoxyguanosine 3′-monophosphate; dCp, 2′deoxycytidine 3′-monophosphate; dTp, thymidine 3′-monophosphate; 3MEI, 3-methyleneindolenine; 3MI, 3-methylindole; 3-2H3-MI, ([3-2H3]methyl)indole; I3COH, indole 3-carbinol; SRM, selected reaction monitoring.

route of P450 metabolism in the presence of goat lung microsomes, leading to the formation of 3MEI (ref 2, Figure 1). Additional reactive intermediates derived from P450-mediated, oxidative pathways include the 2,3epoxide of 3MI (2,3-epoxy-3-methylindoline) and 3-hydroxy-3-methylindolenine (3-5). Because 3-hydroxy-3methyloxindole is the predominant urinary metabolite in all species (5-7) and is believed to be derived from 3-hydroxy-3-methylindolenine, it has been proposed that this reactive intermediate is the predominant one (4). It has also been proposed that the presence of 3-hydroxy3-methyloxindole in human urine may be an indicator of toxicity in humans (8). These reactive intermediates can be generated in both the lung and liver of multiple species (8-10; reviewed in refs 11 and 12). Organ-selective toxicity, however, is seen primarily in the lung (13-15). The P450 2F enzymes are known to be selectively expressed in the lung and are particularly efficient in the generation of 3MEI (16-18).

10.1021/tx0100237 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/27/2001

DNA Adducts of 3-Methylindole

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Figure 1. P450-mediated bioactivation of 3MI, showing oxygenation and dehydrogenation pathways, and trapping of the electrophiles with nucleophilic thiols.

Species selectivity is seen in that goats and cattle are more susceptible than mice, rats and rabbits (1, 19; reviewed in 11 and 12). It is interesting that rabbit lung and goat lung seem to form similar reactive intermediates and glutathione (GSH)/protein adducts but there is a significant difference in the observable toxicity (9, 20). A reactive intermediate of 3MI is known to form adducts with various protein thiols as demonstrated by the isolation of a cysteine adduct from hydrolysates of microsomal proteins, following incubations with 3MI and NADPH (21). Glutathione and mercapturate adducts of 3MI have been isolated and characterized (4, 22) and the levels of protein adduction are believed to inversely correlate with the cellular thiol content (10, 15, 19). Thiol adducts have also been characterized using selective polyclonal antisera, from animals treated with 3MI, and from human lung and liver tissues (23). Thiol adducts (N-acetylcysteine and thiol glycolic acid) on the C-3 methylene group of 3MI and at the C-2 position of the indole moiety have been detected by LC/ MS techniques, with the latter being the predominant adduct (4). While thiol adduction on the outer methylene unit (at C-3 of the indole ring) can only result from interaction with 3MEI, adduction at the C-2 position can occur with all three of the reactive intermediates. The fact that multiple reactive intermediates are formed during the P450-mediated metabolism of 3MI led the authors to postulate that the formation of DNA adducts was possible. In the current studies, initial identification by both MS and NMR of the nucleoside adducts from synthetic reactions led to the successful characterization of the identical adducts generated in both microsomal and hepatocyte incubations with 3MI. This paper describes the detection and characterization of both the synthetic nucleoside adducts and the adducts cleaved from double stranded DNA.

Materials and Methods Chemicals. 3-2H3-MI was synthesized according to the methods of Huijzer et al. (24). I3COH, 3MI, the free nucleosides and nucleotides, RNase T1, isocitric acid (trisodium salt) and isocitrate dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO). RNase 1A, micrococcal nuclease, calf

thymus DNA, nuclease P1, bacterial alkaline phosphatase, phosphodiesterase I (C. adamanteus), and DNase I were obtained from Pharmacia (Piscataway, NJ). Proteinase K and calf spleen phosphodiesterase (spleen exonuclease) were supplied by Boehringer-Mannheim Biochemicals (Indianapolis, IN). Deuterium oxide (D2O; 100% d incorporation) was obtained from Cambridge Isotope Laboratories (Woburn, MA). Rat hepatocytes were provided by Mr. Dave Alberts (Merck Research Labs-West Point). All other chemicals and solvents were of reagent grade. Instrumentation. Purification of 3MI-nucleoside adducts for NMR studies was performed on a Beckman HPLC system equipped with dual 114 solvent pumps, a 421 controller and a Hewlett-Packard model 1040A UV detector (Berkeley, CA; Wilmington, DE, respectively). Proton NMR spectra were recorded on a Varian Unity 500 MHz spectrometer at 24 °C (Palo Alto, CA). The initial MS analysis of the synthetic adducts was performed on an ion trap mass spectrometer (LCQ; Thermoquest, San Jose, CA). Liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis of the DNA adducts via SRM was performed with a Waters liquid chromatography system (Waters, Milford, MA) connected to a Finnigan TSQ 7000 triple quadrupole electrospray MS (Thermoquest). Synthesis of Nucleoside Adducts. I3COH (1 mM) was slightly acidified with 0.1 M phosphate buffer (pH 5-6) (2, 25), in the presence of a 2-fold molar excess of each of the individual nucleosides. After 1 h at room temperature, the reactions were centrifuged at 1500g for 10-15 min to remove the particulate side-product. The adducts were isolated by solid-phase extraction (7 mm/3 mL C18 cartridges; Fisher Scientific, Pittsburgh, PA) and eluted with methanol. Following the evaporation of the solvent, the sample was resuspended in 10 µM EDTA, pH 8, and analyzed by MS. Samples for HPLC purification and NMR analysis were directly injected onto the HPLC following a 2 h incubation under conditions similar to those described above. 3MI-dGuo. 1H NMR (D2O) δ 1.78 (s, 2H, H3-3MI), 2.30 (d,d,d, 1H, 2′R-dGuo), 2.73 (m, 1H, 2′β-dGuo), 3.49 (d,d,d, 2H, 5′-CH2dGuo), 3.90 (d,d, 1H, 4′-dGuo), 4.38 (m, 1H, 3′-dGuo), 6.21 (d,d 1H, 1′-dGuo), 7.03 (d,d, 1H, H5-3MI), 7.12 (d,d, 1H, H6-3MI), 7.27 (s, 1H, H2-3MI), 7.39 (d, 1H, H4-3MI), 7.62 (d, 1H, H7-3MI), 7.78 (s, 1H, H8-dGuo); UV (H2O) λmax 255, 280 nm; electrosprayMS, positive ion, m/z 397 (MH+). 3MI-dA1. 1H NMR not determined; electrospray MS, positive ion, m/z 381 (MH+). 3MI-dCyd. 1H NMR (D2O) δ 1.82 (s, 2H, H3-3MI), 2.22 (d,d,d, 1H, 2′R-dCyd), 2.33 (m, 1H, 2′β-dCyd), 3.67 (d,d,d, 2H, 5′-CH2dCyd), 3.96 (d,d, 1H, 4′-dCyd), 4.34 (m, 1H, 3′-dCyd), 5.42 (s, 1H, H2-3MI), 6.06 (d, 1H, H5-dCyd), 6.19 (d,d 1H, 1′-dCyd), 6.77

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(d, 1H, H4-3MI), 6.89 (d,d, 1H, H5-3MI), 7.18 (d,d, 1H, H6-3MI), 7.28 (d, 1H, H7-3MI), 7.75 (s, 1H, H6-dCyd); UV (H2O) 238, 276 nm; electrospray-MS, positive ion, m/z 357 (MH+). Microsomal Preparation. Phenobarbital and β-naphthoflavone induction in rats followed the methods of Marshall and McLean (26), Matsushima et al. (27), and Ames et al. (28), respectively. Rat liver and human liver microsomes were prepared according to published procedure (29). The preparation of the goat lung microsomes followed the methods of Ruangyuttikarn et al. (8). In Vitro Generation of DNA Adducts. Microsomes. 3MI or 3-2H3-MI (200 µM) was incubated in the presence of induced, rat liver microsomes ([P450] ) 4 µM) or goat lung microsomes ([P450] ) 1 µM), an NADPH regeneration system (10 mM isocitrate, 1 mM NADP+, 0.5 units of isocitrate dehydrogenase, 9.5 mM MgCl2), 100 mM Tris, pH 7.4, and either calf thymus DNA (150 µg) or the individual nucleosides (2.5 mg each), at 37 °C for 1 h. Potassium phosphate was avoided as a buffering system because it complicated the DNA extraction procedure. Two volumes of acetonitrile were added to the incubations with the free nucleosides, followed by centrifugation to remove the protein. After evaporation of the supernatant under nitrogen, the 3MI adducts were separated from the free nucleosides as described above for the synthetic adducts. The incubations with calf thymus DNA were treated as follows. Contaminant RNA was enzymatically digested (31 units of RNase 1A and 64 units of RNase T1, 37 °C, 1 h), followed by digestion of the protein [10 units of proteinase K, 15.8 mM Tris, pH 7.5, 1% (w/v) SDS and 1 mM EDTA, pH 8, 37 °C, 1 h] and phenol extraction of the DNA (30). The DNA was dissolved in 0.1 mM EDTA (pH 8). The DNA concentration was determined by UV spectroscopy and only the samples with an A260/A230 ratio of 2.2-2.4 were used for the cleavage steps and LC/MS analysis. With a small number of the incubations described above, the incubation was concluded by precipitating the DNA with 70% ethanol three times. After pooling the ethanol washes and evaporating the solvent, the residue was resuspended in 200 µL of methanol and analyzed for depurination products on the TSQ MS (35). Hepatocytes. Rat hepatocytes were prepared via the collagenase perfusion method (31) and resuspended at 1 × 106 cells/ mL in Liebovitz’s L-15 buffer plus glutamine (additionally supplemented with 10% fetal bovine serum; Life Technologies, Grand Island, NY). For each of two treatment groups, control and 3MI-dosed, one 6-well plate was used. Into each well of the control plate, 4 µL of ethanol was added, followed by the addition of 1 × 106 hepatocytes. For the 3MI-treated plate, 4 µL of 1 mM 3MI in ethanol was added, followed by the hepatocytes. The plates were incubated at 37 °C for 3 h, under an atmosphere of 5% CO2/95% air. Then the cells were scraped from the wells and combined so that there were three replicates of 2 × 106 cells, per treatment. The cells were centrifuged (250g, 5 min), resuspended in 5 mL of cold phosphate-buffered saline (no Ca2+ or Mg2+) and pelleted again. The cells were suspended in 400 µL of cold, 10 mM EDTA, pH 8, and treated with RNases and proteinase K, in a scaled-down procedure similar to that described above for the calf thymus DNA. The DNA was extracted as described earlier except that the final pellet was resuspended in 10 µL of water. Since the expected yield of DNA was low, i.e., 55 µg, no UV spectrum was taken in order to conserve sample. The samples were stored at -70 °C. Cleavage of DNA. Three different cleavage protocols were utilized. Digestion of 22.5 µg of DNA in the presence of 30 units of spleen exonuclease and 7.5 units of micrococcal nuclease (37 °C, 2 h) was followed by digestion in the presence of 10 units of nuclease P1 (37 °C, 1-2 h), as described in the methods of Laws et al. (32). A second method involved the incubation of 35 µg of DNA with 6 units of nuclease P1 (37 °C, 2 h) and 1 units of bacterial alkaline phosphatase (37 °C, 1 h) under similar conditions to those described by Douki et al. (33). The third protocol involved cleavage of 22 µg of DNA with 12 units of DNase I (37 °C, 2 h), followed by cleavage with both 0.65 units

Regal et al. of phosphodiesterase I and 1 units of bacterial alkaline phosphatase [37 °C, 2 h (34, 35)]. Nucleoside quantification prior to MS analysis followed the methods of Laws et al. (30). Samples were stored at -70 °C until further analysis. HPLC and NMR. An Ultramex C18 column (250 × 4.6 mm; 5 µm; Phenomenex, Torrance, CA) was used and the eluate was monitored at 254 and 280 nm. The mobile phase consisted of a gradient from 5 to 95% acetonitrile (H2O) over 20 min, at 1 mL/ min. The purified dGuo and dCyd adducts were collected, completely dried and dissolved in D2O just prior to NMR analysis. Proton NMR spectra were recorded under the following conditions: 24 °C; pulse width, 5 s; acquisition time, 2.3 s; relaxation delay, 1 s; observation frequency offset, 500 MHz; spectral width, 6998 Hz, resolution, 0.21 Hz/point. MS and LC/MS. Initial detection of the chemically synthesized 3MI-adducts as well as verification of the fragmentation patterns were performed by infusion (3 µL/min) of the extracted, adducted nucleosides into the LCQ via an electrospray interface. The sheath gas was set at 30 (arbitrary units) while the auxiliary gas was set at 5 (arbitrary units). The capillary temperature was maintained at 180 °C. The spray voltage was set at 4.5 kV. Once the instrument had been optimized for analysis of the 3MI-dGuo adduct, minimal additional enhancement was seen during the optimization of the signal for the dCyd and dAdo adducts. For the LC/MS/MS (SRM) analysis of the DNA adducts, 50 µL of sample were chromatographed on a Supelcosil LC-18S column (150 × 4.6 mm; 5 µm; Supelco, Bellefonte, PA). At a flow rate of 0.5 mL/min, the column was maintained in 4% B for 10 min, followed by a linear gradient to 90% B over the next 10 min and then held for 5 min (solvent B ) 0.25% formic acid, 0.25% acetic acid, 99.5% methanol; solvent A ) 0.25% formic acid, 0.25% acetic acid, 99.5% water). The electrospray voltage of the atmospheric pressure ionization was maintained at 5 kV and the capillary temperature fixed at 240 °C. The sheath gas pressure was 90 (arbitrary units) while the auxiliary gas flow was 30 (arbitrary units). Collision induced dissociation occurred in Q2 with argon as the collision gas (2.3 mT) at a collision offset voltage of 15 eV. SRM experiments were conducted by monitoring the neutral loss of the 2′-deoxyribose [MH - 116]+ from each of the four potential 3MI adducted nucleosides ([3MI-dGuo + H]+, m/z 397.1; [3MI-dAdo + H]+, m/z 381.1; [3MI-dCyd + H]+, m/z 357.1; [3MI-dThd + H]+, m/z 372.1). The total scan time was 2 s. The relative ratios of the different adducts were the same after optimization of ionization parameters and ion optics for the detection of 3MI-dGuo. Some experiments also monitored the neutral loss of the 3′-monophosphate deoxyribose [MH 196]+ from each of the four potential 3MI adducted nucleotides ([3MI-dGp + H]+, m/z 477.1; [3MI-dAp + H]+, m/z 461.1; [3MIdCp + H]+, m/z 437.1; [3MI-dTp + H]+, m/z 452.1). In such instances, the total scan time was 3 s.

Results Characterization of the Synthetic Adducts. Preliminary studies on the LCQ MS demonstrated at least a 10-fold enhancement in sensitivity for the free nucleosides vs free nucleotides (positive ion, data not shown). Detection of the free nucleotides in the negative ion mode did not improve their detection limits and so the free nucleosides were used in the generation of the synthetic, adducted DNA standards. The generation of the reactive intermediate (3MEI) of 3MI from I3COH, under slightly acidic conditions, in the presence of the individual free nucleosides, generated dAdo-, dGuo-, and dCyd-3MI adducts (Figure 2). Only a single adduct was identified with each of these nucleosides. The molecular ion of each adduct indicated that one molecule of 3MI was adducted to each nucleoside. Fragmentation of these adducts resulted in initial losses of either the indole structure [MH - 129]+ or the 2′-deoxyribose ([MH - 116] +; Table



Table 1. Daughter and Granddaughter Ions for the Nucleoside Adducts of 3MI

1

Daughter ions (CID at -15 eV). 2 Granddaughter ions (CID at -20 eV). 3 Imine structures shown; ND indicates not detected.

Figure 2. Reaction of 3MEI (produced by dehydration of I3COH) with the 2′-dN.

1; Figure 3A), indicating that adduction was via the exocyclic amine portion of the nucleoside. No adduct formation was seen in the presence of dThd, consistent with the conclusion that the primary, exocyclic amine on dGuo, dAdo, and dCyd was the nucleophile involved in adduct formation. The NMR spectra of the synthetic HPLC-purified dGuo adduct (Figure 4A) and the HPLC-purified dGuo adduct

(Figure 4B) from goat lung microsomal incubations with 3MI further supported this conclusion. The chemical shifts for the protons of 3MI and the protons belonging to the nucleoside were identical to the individual standards with the following exceptions. The C-3 methylene protons (integrated as two, not three protons) of 3MI shifted downfield to δ 1.8, indicating that this position was the site of attachment to the base. In addition, the C-2 proton of 3MI exhibited a significant downfield shift to 7.2 ppm. Generation of 3MEI in the presence of an equimolar amount of the four free nucleosides indicated that dAdo and dGuo formed adducts more readily with 3MEI than dCyd (Figure 5). However, dGuo seemed to form the most adducts when 3MEI was generated in the presence of whole, double-stranded DNA (data not shown). In both the ion trap and the triple quadrupole mass spectrometers, the optimization parameters (gas flows, capillary temperature, spray voltage, etc.) were essentially identical for all three of the nucleoside adducts. No additional optimization was done to detect the nucleotide adducts (see below). In Vitro Formation of the 3MI-DNA Adducts. Microsomal incubations in the presence of 3MI and double-stranded DNA or the free nucleosides resulted in adduct formation. The NMR spectrum of the dGuo adduct purified from the goat lung microsomal incubations was essentially identical to the synthetic dGuo adduct, including the respective shifts of the C-3 methylene protons (δ 1.78) and the C-2 proton (δ 7.27) of 3MI (Figure 4, panels A and B). For the dCyd adduct, the proton signal of the C-3 methylene was assigned to the resonance at

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Figure 3. Mass spectra of 3MI-dAdo and 3-2H2-MI-dAdo adducts that were produced by incubation of 3MI and 3-2H3-MI, respectively, with rat hepatic microsomes. LC/MS was accomplished as described in Figure 5.

Figure 4. NMR spectra of (A) synthetic 3MI-dGuo and (B) 3MI-dGuo adducts that were purified by HPLC (tR ) 17.2 min). The synthetic adduct was produced by dehydration of I3COH in the presence of dGuo as described in the Materials and Methods. The adduct in (B) was purified from incubations of 3MI with goat lung microsomes as described in Materials and Methods. dR ) deoxyribose (1′, 2′R, 2′β, 3′, 4′, and 5′-CH2 refer to protons of the sugar).



Figure 5. LC/MS ion chromatogram obtained after the reaction of 3MEI (produced in situ by dehydration of I3COH) with an equimolar mixture of all four nucleosides. The sample was chromatographed on a RP 150 × 4.6 mm C18 column. At 0.5 mL/min, the gradient was as follows (A, 0.25% HCOOH, 0.25% CH3COOH, H2O; B, 0.25% HCOOH, 0.25% CH3COOH, MeOH): 4% B, 10 min; 4 to 90% B over 10 min; 90% B, 5 min. MS analysis was done on the LCQ, via an electrospray source, as described in the Materials and Methods. The relative ion currents were as follows: TIC, 2.76 × 108; 3MI-dAdo, 1.34 × 108; 3MI-dGuo, 6.88 × 107; 3MI-dCyd, 9.41 × 106; 3MI-dThd, 4.04 × 105.

1.82 ppm (Figure 6). The 3MI proton at C-2 shifted downfield, relative to the signal of this proton in 3MI, to 5.42 ppm. The signals at 6.89 and 7.18 were assigned to H-5 and H-6 due to the associated multiplicities (d,d) whereas the signals at 6.77 and 7.28 ppm were assigned to H-4 and H-7. Following extraction and enzymatic cleavage of the DNA from rat hepatic, human hepatic, or goat lung microsomal incubations with 3MI, SRM-MS detection of both the nucleoside and nucleotide adducts indicated that the most significant levels of adduction occurred with dGuo. Smaller amounts of the respective dAdo and dCyd adducts were also formed (Figure 7). Neutral loss scanning provided the same results, in the same relative proportions. Similar incubations with rat hepatic microsomes in the presence of 3-2H3-MI fully confirmed these conclusions, and also verified the interpretation of the MS spectra (Table 1; Figure 3B). Attempts to identify adducts which would result from depurination of the DNA, i.e., N7-guanyl or N7-adenyl adducts, were unsuccessful.

Incubations of rat hepatocytes with 3MI were conducted to determine the extent of adduct formation in whole, viable cells. A 3 h incubation in the presence of 3MI followed by extraction and cleavage of the DNA, produced the same nucleoside adducts (Figure 8), in the same relative proportions, as were observed in microsomal incubations. No adducts were detected from untreated hepatocytes.

Discussion The characterization of the synthetic, 3MI-adducted nucleosides led to several conclusions. The generation of 3MEI, in the presence of the free nucleosides, resulted in the attachment of one molecule of 3MI to dAdo, dGuo, and dCyd. One adduct was seen for each of these nucleosides, although there was always an additional uncharacterized minor peak within the same m/z window as the main dGuo adduct throughout all of the experiments. The apparent chemical preference led primarily to dAdo adduction, followed by dGuo and dCyd. Attempts to isolate large quantities of these three adducts for NMR

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Figure 6. NMR spectrum of 3MI-dCyd that was purified by HPLC (tR ) 16.2 min) from incubations of 3MI with goat lung microsomes, as described in Materials and Methods. dR ) deoxyribose (1′, 2′R, 2′β, 3′, 4′, and 5′-CH2 refer to protons of the sugar).

Figure 7. Mass spectra of 3MI-DNA adducts from microsomal incubations of calf thymus DNA with 3MI. HPLC separation, after DNA isolation and hydrolysis, was performed as described for Figure 5, except for C, which utilized a slower gradient (20 min from 4 to 90% B). SRM experiments were conducted by monitoring either the neutral loss of the 2′-deoxyribose [MH - 116]+ from each of the four potential 3MI adducted nucleosides ([3MI-dGuo + H]+, m/z 397.1; [3MI-dAdo + H]+, m/z 381.1; [3MI-dCyd + H]+, m/z 357.1; [3MI-dThd + H]+, m/z 372.1) OR the neutral loss of the 3′-monophosphate 2′deoxyribose [MH - 196]+ from each of the four potential 3MI adducted nucleotides ([3MI-dGp + H]+, m/z 477.1; [3MI-dAp + H]+, m/z 461.1; [3MI-dCp + H]+, m/z 437.1; [3MI-dThd + H]+, m/z 452.1). The relative ion currents for each channel are as follows: (A) 3MI-dAdo, 4.2 × 103; 3MI-dGuo, 1.5 × 104; 3MIdCyd, 4.3 × 102; 3MI-dThd, 4.6 × 101; (B) 3MI-dAp, 4.0 × 101; 3-MI-dGp, 3.9 × 103; 3MI-dCp, 2.5 × 101; 3MI-dTp, 4.9 × 101; (C) 3MI-dAdo, 2.0 × 102; 3MI-dGuo, 9.9 × 103; 3MI-dCyd, 1.8 × 102; 3MI-dThd, 1.4 × 101. (1) The DNA was cleaved with micrococcal nuclease, spleen exonuclease and nuclease P1. (2) The DNA was cleaved with DNase I, phosphodiesterase I and bacterial alkaline phosphatase.

analysis were complicated because the 3MI-nucleoside linkage was acid-labile. The reaction which initially showed the preference for dAdo adduction changed with time, i.e. days, to produce approximately equivalent amounts of the dAdo, dGuo, and dCyd adducts, presumably due to equilibration of adduction, in the presence of trace acid, prior to breakdown of the adducts (data not shown). This acid lability along with the fact that no dThd adduct was seen suggested that a Michael-like reaction between 3MEI and the exocyclic amines on dAdo, dGuo, and dCyd was responsible for the formation of the 3MI adducts. This is analogous to the conclusions of Shen et al. (35) and is supported by the NMR spectra in Figures 4 and 6. Attachment of the 3MI moiety cannot occur at the N7 position of dGuo, since this process would form an adduct which would lose the deoxyribose. The sugar was detected in the NMR and MS spectra. While

we cannot definitively rule out adduction via the N1 position on dGuo, the data support adduct formation at the exocyclic basic amine site. Comparison of the adducts formed with dAdo and dCyd, which do not possess a secondary amine, also indicated the involvement of the exocyclic, primary amine of dGuo as the nucleophile involved in 3MI adduction. The daughter ions of all three adducts result primarily from the neutral loss of either the 3MI moiety or the sugar. Further fragmentation led to loss of the remaining 3MI or the sugar in addition to an ion with a residual methylene unit attached to the free base, presumably via the exocyclic, primary amine. The loss of the sugar moiety was used for the SRM-MS detection of the analogous adducts from microsomal and hepatocyte incubations. The propensity of intact, calf thymus DNA to form adducts with the reactive intermediates of 3MI, as well


Figure 8. Electrospray MS spectra of the DNA adducts isolated from incubations of 3MI with rat hepatocytes. The nucleoside adducts were obtained by cleavage of the isolated DNA with DNase I, phosphodiesterase I and bacterial alkaline phosphatase as described in Materials and Methods. HPLC conditions were similar to those described in Figure 5 except that a slower gradient was used (20 min from 4 to 90% B). SRM analysis of the adducts was described in Figure 7. The relative ion currents are as follows: 3MI-dAdo, 5.5 × 102; 3MI-dGuo, 5.5 × 103; 3MI-dCyd, 2.9 × 102; 3MI-dThd, 3.3 × 101.

as the relative reactivity with the four bases of the double stranded helix, was initially analyzed following the generation of 3MEI in the presence of calf thymus DNA. Three cleavage protocols were utilized and compared for their ability to cleave adducted DNA to the nucleoside level. The combination of micrococcal nuclease, spleen exonuclease and nuclease P1 is believed to selectively avoid cleavage of the phosphate from the adducted bases while the nonadducted DNA is cleaved to the nucleoside level (36). However, variations in the chemical properties of the reactive intermediates, as well as the length of cleavage time and the amount of nuclease P1, can affect the ratio of adducted nucleotides to adducted nucleosides. Cleavage of the 3MI-adducted DNA, under these conditions, led to the detection of dAdo, dGuo, and dCyd (nucleoside) adducts as well as dGp adducts. While the results are not quantitative, guanine was believed to be the preferred site of adduction within calf thymus DNA due to the magnitude of the peaks for the dGuo and dGp adducts relative to those seen for the other adducted bases. When identical DNA samples were cleaved with a combination of nuclease P1 and bacterial alkaline phosphatase or DNase I, phosphodiesterase I, and bacterial alkaline phosphatase, the expected nucleoside adducts were seen and the relative amount of the 3MI-dGuo adduct was 4-55-fold greater than the respective dAdo and dCyd adducts. Thus, the innate chemical reactivity of 3MEI with double stranded calf thymus DNA led primarily to dGuo adducts. No difference was seen in the relative peak heights of the adducts between SRM and neutral loss scanning. However, conclusions about the relative affinity of 3MEI for dGp, compared to the other nucleotides from intact DNA must be made cautiously, because the recoveries, pH stabilities, or relative ioniza-


tion efficiencies of each adduct were not determined. In addition, incomplete enzyme digestion of adducted DNA or instabilities of the adducts to the digestion conditions could have influenced the relative amounts of the nucleoside adducts. Three sources of microsomal cytochrome P450 (goat lung, rat liver, and human liver) were used to generate one or more of the reactive species of 3MI, in the presence of calf thymus DNA. The goat is particularly susceptible to 3MI-induced toxicity and the lung is the target organ (15). Rodents are less susceptible to this toxicity. The potential toxicity in humans, as a result of tryptophan metabolism in the gut (37, 38) or exposure to cigarette smoke (39-41), is poorly understood. However, metabolism and covalent adduction of radiolabeled 3MI intermediates have been shown to be P450-mediated in human lung and liver (8). Calf thymus DNA was incubated in the presence of 3MI and the various microsomal sources of P450. The purified and cleaved DNA was analyzed by SRM-MS. Once again, dGuo, dAdo, and dCyd adducts were detected and dGuo was the primary site of 3MI attachment. NMR analysis of the purified adducts formed by goat lung microsomal incubations with 3MI and the pure dNs, verified that the outer methylene unit at C-3 provides the linkage to the base, presumably through the exocyclic amine. Incubations performed in the presence of 3-2H3-MI gave the same three nucleoside adducts, in the same proportions. While these analyses were not quantitative, the total amount of nucleosides on column was approximately the same. The overall extent of adduct formation in the presence of 3-2H3-MI seemed to be lower relative to the levels seen with 3MI, presumably due to primary deuterium isotope effects of hydrogen abstraction from the perdeuterated methyl group of 3MI (2). Electrophilic adduct formation of 3MEI to the base portion of dGuo, dAdo, and dCyd is believed to occur via a Michael-like nucleophilic attack of the primary, exocyclic amine of these three bases on the reactive intermediate. Further evidence for this exocyclic linkage in the 3MI-DNA adducts was seen in the granddaughter ion which retains a methylene unit attached to the free base (Table 1). The analogous adducts, formed in the presence of bioactivated 3-2H3-MI, led to granddaughter ions which were larger by two mass units, which presumably were the result of nucleophilic attack on the outer methylene unit, at the C-3 position of the deuterated analogue of 3MEI. No fully labeled (d3) oxygenated adducts or fragments resulted from the incubations with 3-2H3-MI. Since involvement of 2,3-epoxy-3-methylindoline or 3-hydroxy-3-methylindolenine would lead to C-2 linked adducts (Scheme 1), it is believed that nucleophilic addition of the exocyclic primary amine(s) occurred via attack on the outer methylene unit at C-3 of 3MEI, in both naked DNA/microsomal incubations and whole cells. Thus, the proposed “predominant” reactive intermediate, i.e., 3-hydroxy-3-methylindolenine (4, 5), does not appear to be the reactive intermediate leading to DNA adduction in the in vitro systems utilized here. For all of the characterized reactive intermediates of 3MI, an sp2 carbon is the preferred site of nucleophilic attack. Thus, it is understandable that there would be conjugation with thiols and water, i.e., soft nucleophiles (42). DNA, however, falls into the category of hard nucleophiles and, thus, would not be expected to form adducts to the same intermediates as the free thiols. One

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Scheme 1. Adducts of 3MI Showing the Retention of Deuterium Atoms from 3-2H3-MI

resonance structure for 3MEI places a positive charge on the outer methylene unit at C3 of the indole and this structure could potentially react with hard nucleophiles such as those found in DNA. This mechanism is in agreement with the retention of two deuterium atoms on the dAdo and dGuo adducts following incubations in the presence of 3-2H3-MI (Table 1). Numerous epoxide intermediates have been shown to form DNA adducts, including exo-aflatoxin B1-8,9-epoxide (43), diepoxybutane (44), styrene oxide (45), and the diol epoxides of some of the polycyclic aromatic hydrocarbons, namely benzo[a]pyrene (46), benzo[c]phenanthrene (47), dibenz[a,j]anthracene (48), and benzo[g]chrysene (49). However, nucleophilic attack of the DNA on the epoxide intermediate of 3MI is inconsistent with the retention of only two deuterium atoms in the dAdo and dGuo adducts. Incubations with hepatocytes were conducted to determine if the reactive species of 3MI generated within the cell would have access to the nuclear DNA. The 3-h time point was chosen based on previous work in which positive controls (precocine and allyl alcohol) were known to elicit a toxic response.2 Figure 8 demonstrates the presence of the dGuo- and dAdo-3MI adducts while the dCyd-3MI adduct, if present, was below the limits of detection. The dGuo-3MI adduct was again present in significantly larger amounts than the dAdo adduct. Since 3MI is formed in vivo as a result of tryptophan metabolism in the gut, it had been postulated that rat liver hepatocytes might contain background levels of 3MI-DNA adducts. In general, it is known that there are background levels of DNA adducts which may or may not play a physiological role in mutagenesis and carcinogenesis (50, 51). However, no 3MI adducts were detected in the control hepatocytes (via SRM or neutral loss scanning). It is well-known that there is a link between adduct formation of cellular DNA and mutagenicity as well as carcinogenicity. DNA adducts have been detected in susceptible organs (50, 51). However, the exact mechanisms which lead from initiation to the obvious endpoints are less certain. Presumably, the mutagen/carcinogen or one of its metabolites binds covalently to the nuclear DNA, resulting in damage which may then lead, either through misrepair or subsequent miscoding, to either inheritable changes in the affected sections of DNA or selective clonal expansion of the altered cells (52, 53). In addition, there may be a link between DNA adduction

and cytotoxicity (54) or DNA adduction and apoptosis (55, 56). Preliminary evidence has shown that 3MI can cause apoptosis in human lung cells at very low concentrations (57). However, the role played by DNA alkylation by 3MI in apoptosis or cell cycling has not been established. Cytotoxicity is believed to be related to protein and GSH adducts while mutagenesis and carcinogenesis are believed to be the result of DNA adduction. 3MI is known to be cytotoxic in the lung following metabolic activation. It is also known that there is extensive GSH depletion and some protein adduction. While there is no obvious link with carcinogenesis, i.e., tumors, the possibility remains that 3MI-DNA adducts may play a role in the known toxicity, possibly through apoptotic mechanisms. Very little is known about the mutagenic/carcinogenic potential of 3MI. Preliminary studies3 have shown that 3MI is modestly mutagenic to the salmonella/microsome mutagenicity test in strain TA 100 after bioactivation by goat lung microsomes. However, to our knowledge, no long-term carcinogenicity studies have been conducted with 3MI. Since cigarette smoke contains reasonably high concentrations of 3MIsup to 1.4 µg/cigarette (41)sit seems reasonable to speculate that 3MI contributes substantially to cigarette-induced lung cancer in humans. Fecal concentrations of 3MI in humans has been shown to be as high as 150 µg/g feces (38). However, a conclusion that 3MI causes lung, colon, or other types of cancer in humans is speculative at this time, and this hypothesis certainly requires additional experimental evidence. In conclusion, generation of 3MEI in the presence of the individual nucleosides led to a single molecule adducted to dGuo, dAdo, and dCyd. The innate chemical preference led primarily to dAdo adduction while this preference appeared to shift to dGuo adduction in double stranded DNA. On the basis of the evidence obtained by NMR and MS, the proposed mechanism for the adduction is a Michael-like nucleophilic addition of the exocyclic amine(s) of dGuo, dAdo, and dCyd on the outer methylene unit at the 3 position of 3MEI. Goat lung microsomes, induced rat liver microsomes and human liver microsomes each formed the reactive species of 3MI that led to all three adducts. In addition, demonstration of the formation of these same adducts in rat hepatocytes demonstrated that 3MEI had access to the nuclear DNA of intact cells. These results support the hypothesis that

2 Dave Alberts, Investigative Toxicology, Merck, personal communication.

3 Dr. Charlene McQueen, University of Arizona, Tucson, AZ, personal communication.


3MI may be a mutagenic and/or carcinogenic chemical, although verification of this hypothesis requires significant additional experimental evidence.

Acknowledgment. The authors wish to thank M. V. Reddy (Genetic Toxicology, Merck) for his helpful discussions regarding DNA cleavage, Dave Alberts (Investigative Toxicology, Merck) for contributing the rat hepatocytes and Amy Loughlin (Biochemical Tox., Merck) for answering questions regarding the LCQ. This work was supported in part by a grant (HL13645) from the National Heart, Lung, and Blood Institute of the National Institutes of Health (G.S.Y.). A grant (RR06262) from the National Institutes of Health was used to purchase 500 MHz NMR instrumentation for the Health Sciences NMR Facility at the University of Utah.

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Detection and Characterization of DNA Adducts of 3-Methylindole

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