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Chem. Res. Toxicol. 2003, 16, 1130-1137
Identification of DNA Adducts Derived from Riddelliine, a Carcinogenic Pyrrolizidine Alkaloid Ming W. Chou, Yan Jian, Lee D. Williams, Qingsu Xia, Mona Churchwell, Daniel R. Doerge, and Peter P. Fu* National Center for Toxicological Research, Jefferson, Arkansas 72079 Received April 15, 2003
Riddelliine is a naturally occurring carcinogenic pyrrolizidine alkaloid that produces liver tumors in experimental animals. Riddelliine requires metabolic activation to dehydroriddelliine and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) to exert its toxicity. Previously, 32P-postlabeling HPLC was used to detect a set of eight DHP-derived adduct peaks from DNA modified both in vitro and in vivo. Among these DHP-derived DNA adducts, two were identified as epimers of DHP-2′-deoxyguanosine 3′-monophosphate. In this study, the remaining adducts have been characterized as DHP-modified dinucleotides. A series of dinucleotides, TpGp, ApGp, TpCp, ApCp, TpAp, ApAp, TpTp, and ApTp, were obtained by enzymatic digestion of calf thymus DNA with micrococcal nuclease (MN) and spleen phosphodiesterase (SPD) followed by HPLC separation and structural identification by negative ion electrospray tandem mass spectrometry (ES/MS/MS). Incubation of individual dinucleotides with DHP produced DHP-modified dinucleotide adducts that were also characterized using LC-ES/MS/MS. A parallel analysis of the isolated DHP-modified dinucleotides using 32P-postlabeling recapitulated the series of unidentified adduct peaks that we previously reported from DHP-modified calf thymus DNA in vitro and rat liver DNA in vivo. Intact calf thymus DNA was also reacted with DHP and then digested by MN/SPD under the same conditions. The adduct profile obtained from LC-ES/MS/MS analysis was similar to that observed from the isolated dinucleotides. Structural analysis using LC-ES/MS/MS showed that DHP bound covalently to both 3′- and 5′-guanine, -adenine, and -thymine bases (but not cytosine) of dinucleotides to produce two or more isomers of each DHP-dinucleotide adduct. By comparing adduct formation at dissimilar bases within individual dinucleotides, the relative reactivity of DHP with individual bases was determined to be guanine > adenine ∼ thymine. Identification of the entire set of DHPderived DNA adducts further validates the conclusion that riddelliine is a genotoxic carcinogen and enhances the applicability of these biomarkers for assessing carcinogenic risks from exposure to pyrrolizidine alkaloids.
Introduction
Scheme 1. Oxidative Metabolism of Riddelliine
PAs1 are highly toxic heterocyclic compounds that are common constituents of many plant species around the world (1-4). They cause livestock loss due to liver and pulmonary lesions (4-9) and induce chronic diseases, such as cancer, in experimental animals (2, 10-16). Human foodstuffs, such as herbs, milk, and honey, may also be contaminated by toxic PAs (2-4, 17-19). PAs require metabolic activation in order to exert toxicity (2, 4, 20-22). The pyrrole metabolites (dehydropyrrolizidines) are chemically reactive species responsible for most of the toxic activities of the parent PAs (6, 21, 23-27). The necine product, DHP (Scheme 1), is believed to be the reactive metabolite for retronecine-based macrocyclic PAs, including riddelliine (17, 18, 27, 28). * To whom correspondence should be addressed. Tel: (870)543-7207. Fax: (870)543-7136. E-mail:
[email protected]. 1 Abbreviations: PA, pyrrolizidine alkaloid; MN, micrococcal nuclease; SPD, spleen phosphodiesterase; DHP, (R/S)-6,7-dihydro-7hydroxy-1-hydroxymethyl-5H-pyrrolizine; DHP-3′-dGMP adducts, DHP2′-deoxyguanosine 3′-monophosphate; PNK, cloned T4 polynucleotide kinase; ctDNA, calf thymus DNA.; ES/MS/MS, negative ion electrospray tandem mass spectrometry; NTP, National Toxicology Program; NCTR, National Center for Toxicological Research.
10.1021/tx030018y
Chronic exposure to riddelliine induces liver hemangiosarcomas in male and female F344 rats and male
This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society Published on Web 08/27/2003
DNA Adducts Derived from Riddelliine
B6C3F1 mice and hepatocellular adenomas and carcinomas in male and female rats (29). Oxidative metabolism of riddelliine in vitro produces DHP and riddelliine N-oxide (see Scheme 1; 4, 20, 29); however, in rats and mice, riddelliine N-oxide and a hydrolytic product, retronecine, were the only circulating metabolites observed, presumably because the highly reactive DHP can bind to macromolecules (e.g., proteins) in the blood (30). Our previous studies suggested that riddelliine is a genotoxic carcinogen because a set of eight DHP-derived DNA adducts was detected in rats treated with riddelliine (20, 31). Although two of the adduct peaks were fully characterized as epimers of DHP-3′-dGMP (20, 31), the other six DHP-derived DNA adduct peaks were not identified. In this study, we have characterized the six adducts as DHP-derived dinucleotides that resulted from incomplete enzymatic hydrolysis.
Materials and Methods Materials. Riddelliine, obtained from the NTP, was previously characterized (17). Retronecine, DHR, and DHP-3′-dGMP were prepared in our laboratory as previously described (20, 28, 31). ctDNA, nuclease P1, MN, and SPD were purchased from the Sigma Chemical Co. (St. Louis, MO). PNK was obtained from U.S. Biochemical Corp. (Cleveland, OH). [γ-32P]Adenosine 5′-triphosphate (γ-32P]ATP) (specific activity > 7000 Ci/mmol) was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). All solvents were HPLC grade. Preparation of DHP-Modified DNA. DHP-modified ctDNA was prepared by reacting DHP (5 mg in 0.5 mL of methanol) with ctDNA (117 mg dissolved in 59 mL of 20 mM K2CO3 buffer, pH 8.0) at 60 °C for 2 h. After the reaction, the solution was washed three times with chloroform/amyl alcohol (24:1) and the modified ctDNA was precipitated by adding 2 volumes of cold 2-propanol, washed with 70% ethanol, dissolved in Tris-EDTA buffer, and stored at -78 °C. Riddelliine-modified ctDNA was obtained by incubating riddelliine with ctDNA in the presence of rat liver microsomes as previously described (30). Riddelliine-modified rat liver DNA was obtained from rats treated by gavage with riddelliine conducted by the NTP (20). Briefly, the rat, from a study using 72 rats, was orally gavaged (5 days/week) with 1.0 mg/kg/day of riddelliine beginning at weaning and continuing until sacrifice at 3 months, after the first dosing day. After sacrifice, liver tissue was collected, stored at -78 °C, and shipped from the NTP to NCTR for DNA adduct analysis. Rat liver DNA was isolated according to the method of Beland et al. (32). MN/SPD Digestion of DHP-Modified DNA. Fifty milligrams of DHP-modified ctDNA was dissolved in 150 mL of 20 mM sodium succinate buffer (pH 6) containing 10 mM calcium chloride and the DNA digesting enzymes MN/SPD (78 units/ mg DNA of MN and 4 units/mg DNA of SPD) in a 500 mL Erlenmeyer flask and incubated at 37 °C for 20 min. After the incubation, 100 mL of methanol was added to inhibit further digestion. The mixture was then concentrated under reduced pressure and stored at -70 °C prior to LC-ES/MS analysis. MN/ SPD digestion of liver DNA from rats treated with riddelliine was performed similarly. MN/SPD Digestion of Unmodified ctDNA. Fifty milligrams of ctDNA was enzymatically hydrolyzed at 37 °C for 20 min with MN (78 U/mg DNA) and SPD (4 U/mg DNA) in a 150 mL solution of 20 mM sodium succinate and 10 mM calcium chloride (pH 6). The mixture was then concentrated to about 20-30 mL under reduced pressure, and the resulting digested nucleotides were separated by reversed-phase HPLC equipped with an ODS column (10 mm × 250 mm, Prodigy 5 µm ODS, Phenomenex), eluted with a linear gradient of 10% methanol in 10 mM triethylammonium acetate (TAA buffer, pH 4.5) to 20% methanol in the TAA buffer for 40 min at a flow rate of 5 mL/min. Analysis of the digestion mixture using LC-ES/MS/
Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1131 MS showed that eight dinucleotides were present in addition to the four mononucleotides. HPLC fractions eluting after the four mononucleotides were collected and individual dinucleotide fractions from multiple injections (each injection, 0.5 mL) were combined and concentrated in vacuo. Further purification of each dinucleotide fraction was conducted by using a Prodigy 5 µm ODS column (10 mm × 250 mm, Phenomenex), eluted with 15% methanol in 20 mM ammonium acetate buffer (pH 7.0) isocratically. The dried individual dinucleotides were weighed and redissolved in 20 mM K2CO3 buffer, pH 8.0, to the final solution of 3 mg/mL. For reaction, DHP was added to each of the individual dinucleotides in an equal amount (in weight) of solution, and the reaction mixture was incubated at 60 °C with stirring for 2 h. After incubation, each resulting DHP-modified dinucleotide was concentrated in vacuo and analyzed using LCES/MS/MS. LC-MS Analysis. The HPLC separation of unmodified dinucleotide fractions from MN/SPD-digested ctDNA was performed using a Columbus C18 column (5 µm, 2.0 mm × 250 mm, Phenomenex) and a linear mobile phase gradient from 5 to 50% (v/v) acetonitrile in aqueous ammonium acetate (25 mM, pH 7) over a 60 min period at a flow rate of 200 µL/min. The entire column effluent was directed into the electrospray probe after flowing through a UV detector to monitor absorption at 260 nm. The LC separations of DHP-modified nucleotides were performed similarly. Typically, an Alliance 2790 liquid handling system (Waters Associates, Milford, MA) was used but in some cases a Dionex GP40 (Sunnyvale, CA) pump was substituted. A Quattro Ultima triple quadrupole mass spectrometer (Micromass, Manchester, U.K.) equipped with an electrospray interface was used with an ion source temperature of 150 °C. Negative product ion scans and multiple reaction monitoring (MRM) were obtained from collision-induced dissociation of selected (M - H)- ions using a cone voltage between 37 and 40 V and collision energies between 24 and 31 eV using MS/MS. The collision gas was Ar at pressures between 2 and 4 × 10-3 mbar. The mass spectrometer was calibrated over the mass range m/z 85-1200 using a solution of polyethylene glycols. Background-subtracted mass spectra were obtained by averaging spectra across the respective chromatographic peak and subtracting average background spectra immediately before and after this peak. 32P-Postlabeling/HPLC Analysis. The 32P-postlabeling/ HPLC of DHP-derived DNA adduct from (i) DHP-modified ctDNA, (ii) riddelliine-modified rat liver DNA, and (iii) DHPmodified nucleotide fractions was conducted according to the method previously developed in our laboratory (31). Ten micrograms of DNA was digested with MN/SPD for 20 min, and the digested DHP-derived DNA adducts were enriched by nuclease P1 treatment, followed by 32P-postlabeling and HPLC analysis. Instrumentation. A Waters HPLC system consisted of a model 600 controller and a model 996 photodiode array detector, and a pump was used for the separation and purification of reaction mixtures, metabolite mixtures, and DHP-derived DNA adducts. For radiochromatography analysis of the 32P-postlabeled reaction mixtures, an HPLC system with on-line detection of both radioactivity and UV absorbance was conducted consisting of a solvent gradient programer (Waters model 680), two HPLC pumps (Waters 510), a radio-chromatography detector (Hewlett-Packard FLO-ONE/Beta A-500) equipped with a diverter, and an autosampler (Waters 717).
Results 32P-Postlabeling/HPLC Analysis of DHP-Derived DNA Adducts. Figure 1 shows the HPLC profiles of the 32 P-postlabeled DHP-derived DNA adducts formed from (i) reaction of DHP with ctDNA (Figure 1A), (ii) metabolism of riddelliine by rat liver microsomes in the presence of ctDNA (B), and (iii) liver DNA of rats treated with riddelliine (Figure 1C). As previously reported, a set of eight DHP-derived DNA adducts, designated as P1-P8,
1132 Chem. Res. Toxicol., Vol. 16, No. 9, 2003
Chou et al. Table 1. LC-ES/MS Analysis of MN/SPD-Catalyzed Dinucleotide Formation from ctDNA peak
HPLC retention time (min)
(M - H)-
(M + H)+
5′f3′ sequence
D1 D2 D3 D4 D5 D6 D7 D8
13.8 16.5 18.1 20.3 22.4 23.3 27.2 29.9
650 659 610 619 634 643 625 634
652 661 612 621 636 not done not done 636
TpGp ApGp TpCp ApCp TpAp ApAp TpTp ApTp
Scheme 2. Proposed Mass Spectral Fragmentation Reactions of Dinucleotidesa
Figure 1. 32P-postlabeling/HPLC analysis of DHP-derived DNA adducts obtained from (A) reacting DHP with ctDNA, (B) ctDNA incubated with riddelliine in a microsome-mediated system, (C) liver DNA from a female F344 rat treated by gavage with riddelliine 1.0 mg/kg/day 5 days/week beginning at weaning and continuing until sacrifice at 3 months (20), and (D) synthetic DHP-3′-dGMP standards. For HPLC conditions, see the Materials and Methods.
was obtained both in vitro and in vivo (20, 31). Figure 1D shows that the two 32P-peaks derived from the synthetic epimeric DHP-3′-dGMP adducts were identical to P4 and P6 in all DHP-derived DNA adduct samples. MN catalyzes cleavage of both DNA and RNA to yield 3′-nucleotides through its exo- and endo-5′-phosphodiesterase activities. The enzyme catalyzes preferential endohydrolysis of the DNA at sites rich in deoxyadenylate or thymidylate. SPD is an exonuclease cleaving from 5′ to 3′. The sizes of resulting 3′-nucleotides are highly dependent upon the enzyme concentration and the incubation time. Our previous study demonstrated that because the DHP-modified mononucleotides are easily destroyed by MN/SPD, the levels of DHP-derived DNA adducts were markedly affected by the amount of MN/ SPD used for digestion (31). Optimal DHP-DNA adduct formation, as measured using 32P-postlabeling, was obtained with 78 mU of MN and 4 mU of SPD (31). However, even under optimal conditions, the DHP-3′dGMP epimers (P4 and P6) composed less than 15% of the total adducts. MN/SPD Digestion of Unmodified Nucleotide Fractions. MN/SPD-digested unmodified ctDNA was prepared. On the basis of LC-MS analysis, the nucleotide fractions with the retention times 3.8, 6.0, 8.0, and 10.9 min were identified as 3′-dCMP, 3′-dGMP, 3′-dTMP, and 3′-dAMP, respectively. The other nucleotide fractions designated as D1-D8 were collected and were analyzed by LC-ES/MS. The HPLC retention times and the mass spectral analysis of the identified eight dinucleotides are shown in Table 1. The sequences were determined unambiguously through the characteristic fragment ions produced by the proposed fragmentation reactions shown in Scheme 2. A representative negative product ion mass spectrum is shown for 5′-ApTp in Figure 2A. The 5′-base of these dinucleotides was either A or T but never G or C; all four 3′-bases were observed. Characterization of DHP-Modified Dinucleotide Fractions. After each dinucleotide fraction was isolated individually and reacted with DHP, the resulting DHPmodified fraction was analyzed by LC-ES/MS. Table 2 shows the precursor dinucleotides, the corresponding
a
Note: dribose ) 2′-deoxyribose and p ) PO4-. Table 2. LC-ES/MS Analysis of DHP-Dinucleotide Adducts Formed by Reaction of DHR with Isolated Dinucleotidesa
dinucleotide precursor (5′-3′) TpGp (D1)b ApGp (D2)b TpCp (D3)b ApCp (D4)b TpAp (D5)b ApAp (D6)b TpTp (D7)b ApTp
(D8)b
RT (min)
(M - H)-
19.2, 21.7 22.65, 24.0 24.6, 26.2 37.4, 39.0, 41.5 35.7, 36.6 36.3, 39.2, 40.6 36.1, 41.1, 43.0 35.5, 36.5, 37.8 36.3, 37.1 37.8, 39.0 39.7, 40.5 41.2-45.0
785 794 745 754 769 778 760 769
adduct sequence
no. of isomers
TpGp-DHP ApGp-DHP DHP-TpCp DHP-ApCp DHP-TpAp TpAp-DHP DHP-ApAp ApAp-DHP DHP-TpTp TpTp-DHP ApTp-DHP DHP-ApTp
2 2 2 3 2 3 3 3 2 2 2 3
a Individual dinucleotides were isolated and reacted with DHP prior to analysis using negative product ion tandem MS (see Figure 3, left panel, for a representative example). The adduct sequence was determined using the characteristic fragmentation reactions shown in Scheme 2. b The HPLC chromatographic peak retention times are shown in Table 1.
DHP-modified nucleotide sequences, the number of isomeric products formed, the LC retention times, and the (M - H)- ions observed. Reaction of DHP was observed to occur with G, A, and T but not with C. The relative reactivity of individual bases within a dinucleotide with DHP could be approximated by comparing relative adduct formation at the 5′- or 3′-base. For example, G appeared to be more reactive than T because only TpGpDHP was observed from reaction of TpGp with DHP; G appeared to have greater reactivity than A because only ApGp-DHP adducts were observed. Also, T and A appeared to have comparable reactivity based on the formation of both T-DHP and A-DHP adducts from TpAp and ApTp. Figure 2 shows representative negative product ion mass spectra, with proposed fragment ion assign-
DNA Adducts Derived from Riddelliine
Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1133
Figure 2. Negative product ion electrospray mass spectra for (A) ApTp and its products upon reaction with DHP, (B) ApTp-DHP, and (C) DHP-ApTp.
ments, derived from isomeric adducts formed from ApTp by reaction of DHP at either base. The fragment ions observed were sufficient for unambiguous characterization of the dinucleotide sequence and the position of DHP modification (see Scheme 2 and Figure 2). The LC-ES/MS/MS analysis of DHP-modified dinucleotides revealed the presence of numerous apparent isomeric products. In the two dinucleotides containing a common base (ApAp, TpTp), DHP adducts were formed with either the 5′- or the 3′-base. No evidence was observed for either incorporation of more than one DHP moiety into a dinucleotide or DHP binding to multiple nucleotides (i.e., no intrachain or interchain cross-linking). Among these nucleotides, two isomers were observed for all thymine- and guanine-based adducts, and three isomers were observed for adenine-derived DHP adducts (see Table 2 and Figure 3, right panel). The individual DHP-modified dinucleotides were also 32 P-postlabeled and analyzed by HPLC following the conditions described in Figure 1C for the analysis of the eight DHP-modified DNA adducts formed from liver of rats fed riddelliine (31). Comparison of the adduct profile from 32P-postlabeling/HPLC with that formed in vivo (Figure 1C) showed that identical DHP-modified dinucleotides P1, P2, P3, P5, P7, and P8 were formed (Figure 4). As shown in Figure 4, multiple isomeric DHP-modified dinucleotides eluted in P7 and P8.
Characterization of DHP-DNA Adducts from DHP-Modified ctDNA. To investigate possible differences in reactivity between DHP and bases contained in intact DNA, as opposed to individual dinucleotides, ctDNA was reacted with DHP, purified, and hydrolyzed using identical MN/SPD hydrolysis conditions. The resulting DHP-modified dinucleotides were then analyzed using LC-ES/MS/MS. In this experiment, the total amount of DHP-modified dinucleotides was much lower than from the isolated dinucleotide reactions. Therefore, LC-ES/MS/ MS was used in the MRM mode to maximize sensitivity using the most abundant transitions identified previously from the isolated DHP-modified dinucleotides. At least two MRM transitions, including both structure specific (e.g., DHP base) and common (e.g., deoxyribose phosphate) transitions, were monitored to maximize specificity of detection of the individual adducts. Table 3 shows the adducts identified, the range of retention times for each set of isomeric forms, the respective (M - H)- ion, and the product ions monitored. Figure 3 (left panel) shows an example of the MRM chromatograms selected for the set of isomeric adducts derived from DHP modification of either adenine or thymine base in ApTp and TpAp in intact ctDNA. For comparison, the corresponding mass chromatograms from product ion scans from the reaction of DHP with either TpAp or ApTp are also displayed in Figure 3 (right panel). In general, products
1134 Chem. Res. Toxicol., Vol. 16, No. 9, 2003
Chou et al.
Figure 3. Characterization of representative DHP-modified dinucleotides from either isolated dinucleotides or intact ctDNA. Product ion chromatograms of selected transitions that characterize DHP adducts of TpAp and ApTp produced by reaction of DHP with the isolated dinucleotides (either TpAp or ApTp, right panel) or MRM chromatograms of the same adducts derived from DHP modification of intact ctDNA (left panel). The transitions monitored and the associated structural assignments are listed at the right of each chromatogram. Table 3. LC-ES/MS Analysis of DHP-Dinucleotide Adducts Formed by Reaction of DHR with Intact ctDNAa adduct sequence (5′-3′) (no. of isomers) TpGp-DHP (3) DHP-TpCp (2) ApGp-DHP (4) DHP-ApGp (4) DHP-ApCp (4) DHP-TpAp (2) TpAp-DHP (3) DHP-ApAp (4) ApAp-DHP (4) TpTp-DHR (2) DHP-TpTp ApTp-DHP (2) DHP-ApTp (6)
RT (min)
(M - H)precursor ion
product ions
34-37 32-34 36-40 42-47 44-49 46-47 43-47 45-50 43-44 45-46 not observed 46-47 43-52
785 745 794 794 754 769 769 778 778 760 NA 769 769
463, 125, 195 386, 195, 260, 125 195, 134 346, 269 306, 269, 195 410, 330, 260, 134 269, 195, 125 269, 330, 195, 134 269, 330, 195, 134 321, 260, 195, 125 NA 410, 330, 260, 195 401, 321, 269, 195, 125
a Intact ctDNA was reacted with DHP prior to analysis using MRM (see Figure 3, right panel, for a representative example).
of DHP modification were similar whether the reaction was carried out with individual dinucleotides or intact DNA with some exceptions. The differences seen in retention times for the dinucleotide-derived vs intact DNA-derived adducts (i.e., consistently longer retention for DNA-derived) were attributed to differences in matrix
(a purified dinucleotide vs hydrolyzed DNA), different injection volume (200 vs 500 µL), and different configurations of the HPLC injection systems. Four isomers of DHP-ApGp were observed only in the DNA reaction (Table 3) and two isomers of DHP-TpTp were formed with the isolated dinucleotide (Table 2), but none were observed from DNA. Comparison of the data in Tables 2 and 3 shows that in many cases, fewer isomers were observed from the reaction of DHP with isolated dinucleotides than those formed with intact DNA. This is exemplified by the chromatograms shown in Figure 3. Although three peaks were observed for DHP-ApTp derived from the isolated dinucleotide reaction, reaction of DHP with DNA produced six such peaks. While the specificity of full product ion scans is certainly greater than MRM, the use of structurally complementary MRM transitions does give the highest possible specificity and sensitivity needed for analysis of limited amounts of dinucleotide adducts derived from intact DNA.
Discussion We previously reported the detection using 32P-postlabeling HPLC of a common set of eight DHP-derived
DNA Adducts Derived from Riddelliine
Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1135
Scheme 3. Proposed Metabolic Activation Pathway for Riddelliine Leading to DNA Adduct Formation
DNA adducts from either DHP-modified ctDNA in vitro or from the liver of rats treated with riddelliine in vivo (20, 31). A dose-response relationship was obtained between the liver tumor incidence and the level of the total DHP-derived DNA adducts (33). These results suggest that riddelliine induces liver tumors in rats through a genotoxic mechanism and the eight DHPderived DNA adducts are likely to contribute to the liver tumor development. This was the first time that formation of PA-derived DNA adducts was definitively linked with induction of liver tumors in laboratory rodents. The formation of eight DHP-DNA adducts was not limited to rodent liver because similar adduct profiles were also found in lung, kidney, and blood DNA of rats and mice treated with riddelliine (34, 35). The DHP-derived DNA adduct formation was cell type specific; that is, the DNA adduct levels in rodent liver endothelial cells, the cells of origin for hemangiosarcomas, were significantly greater than the levels in parenchymal cells (33). This cellular adduct
profile, which was observed in rats and mice, was consistent with the preferential induction of liver hemagiosacomas by riddelliine (28, 33). Both DHP and dehydroriddelliine are bifunctional alkylating agents capable of binding to DNA; however, only DHP-derived DNA adducts were detected by 32Ppostlabeling/HPLC in an in vitro metabolic incubation of riddelliine in the presence of ctDNA or in the liver DNA of rats treated with riddelliine. Furthermore, for the DHP adducts with dGMP, only substitution at the 7-position of DHP was observed (i.e., there was no substitution at the 9-position of DHP). Although dehydro-PAs are considered obligatory oxidative metabolites of PAs (36, 37), their high reactivity in aqueous solution leads to rapid hydrolysis and formation of DHP as the predominant ultimate reactive intermediate that binds to DNA during the metabolism of riddelliine in vitro or in vivo (20, 31). However, the formation of DHP-derived DNA adducts through the reactive dehydroriddelliine intermediates cannot be excluded. A proposed metabolic activation
1136 Chem. Res. Toxicol., Vol. 16, No. 9, 2003
Figure 4. 32P-postlabeling chromatograms of epimeric DHP3′,5′-dG-bisphosphate adducts (top panel) or DHP-modified ctDNA (bottom panel) with assignment of individual peaks to the respective DHP-modified dinucleotide.
pathway of riddelliine and the DHP-DNA adduct formation is shown in Scheme 3. Although we previously reported that 32P-postlabeling/ HPLC detected eight DNA adduct peaks in vitro or in vivo, only two were fully characterized as the epimic DHP-3′-dGMP adducts and the structures of the others were not determined (20, 31). The present study extends our previous research by identifying the remaining adducts as DHP-modified dinucleotides. These dinucleotide adducts result from incomplete enzymatic hydrolysis of DNA that occurred under the optimal conditions for adduct quantification reported previously (31). The adduct profile formed upon reaction of DHP with either individual purified dinucleotides or intact ctDNA was similar but not identical. Furthermore, 32P-postlabeling/ HPLC analysis of either source of DHP dinucleotide adducts recapitulated the same series of peaks observed from rats treated with riddelliine. It was not possible to analyze directly the adducted dinucleotides from riddelliine-treated rat liver DNA by LC-ES/MS/MS because the quantities available were insufficient. The relative reactivity of individual bases in the dinucleotides with DHP could be assessed directly from the adduct structures determined from LC-ES/MS/MS. The reactivity decreased in the order guanine > adenine ∼ thymine. No reaction was observed with cytosine. These results are consistent with previous published reports that dehydroretronecine produced DHP adducts with all nucleosides and mononucleotides derived from guanine, adenine, thymine, and uridine but not cytidine and 5′-dCMP (38, 39). Reaction of DHP with isolated dinucleotides produced two isomeric forms by binding to guanine or thymine and three isomeric forms upon binding to adenine (see Table 2). The results for G, and probably T, are consistent with previous studies in which two epimeric adducts were characterized from the reaction of the 7-position of DHP
Chou et al.
with either 3′-dGMP (31) or dG (38, 39). However, the formation of three isomers of DHP-dA residues suggests that reaction occurred at more than one nucleophilic site on adenine (e.g., N1, N3, N6, or N7). Although no DNA adduct resulting from reaction at the 9-position of DHP has been characterized, this possibility cannot be excluded for the dinucleotide adducts described here. Reaction of DHP with intact ctDNA produced an even more complex mixture of purine adduct isomers (see Table 3). Up to four apparent isomers of DHP-dG-containing adducts were observed and as many as six apparent isomers of DHP-dA-containing adducts were observed; however, two isomers of DHP-T-containing adducts were also observed from intact DNA. These results suggest that the three-dimensional structure of intact doublestranded DNA confers additional reactivity to the purine bases, which contain multiple nucleophilic centers, but not to thymine. This notion is consistent with the results of Pereira et al. (40) from an investigation of DNA alkylation and cross-linking properties of dehydromonocrotaline with a 375 bp EcoRI-Bam HI fragment of pBR322. The results of that study were consistent with covalent binding of DHP to guanine residues at both the N7-position of DNA, based on piperidine lability, and the N2-position based on stability to thermal and piperidine treatments and with the earlier structural characterization of the DHP adduct with dG (38, 39). The small amounts of available samples limited the options for structural determination techniques to MS, which has limited ability to differentiate isomers. Future spectroscopic investigations (e.g., circular dichroism and 1H NMR) will be required for more complete structural assignments.
Acknowledgment. This research was supported in part by an Interagency Agreement No. 224-93-0001 between the FDA/NCTR and the NIEHS/NTP. Through this agreement, Q.X., J.Y., and L.D.W. were supported by appointments to the Postgraduate Research Program at the NCTR administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Food and Drug Administration.
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