Chlorambucil-Adducts in DNA Analyzed at the Oligonucleotide Level

Chem. Res. Toxicol. 2009, 22, 1435–1446

1435

Chlorambucil-Adducts in DNA Analyzed at the Oligonucleotide Level Using HPLC-ESI MS Dalia Mohamed,† Shereen Mowaka,† Ju¨rgen Thomale,‡ and Michael W. Linscheid*,† Humboldt-UniVersitaet zu Berlin, Department of Chemistry, Applied Analytical and EnVironmental Chemistry, Brook-Taylor-Strasse 2, 12489 Berlin, Germany, and Institute of Cell Biology (Cancer Research), UniVersity of Duisburg-Essen, Essen, Germany ReceiVed March 31, 2009

Chlorambucil (N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating drug belonging to the nitrogen mustard group and is widely used as an anticancer agent. As the antitumor activity of the nitrogen mustards is based on the formation of adducts with genomic DNA, calf thymus DNA-Chlorambucil adducts were the major target in this study. Calf thymus DNA was incubated with Chlorambucil to induce the formation of a wide variety of adducts. Subsequently, enzymatic digestion of the DNA was performed using Benzonase and Nuclease S1 aiming at the production of oligonucleotides. Separation and structure elucidation of the individual DNA-Chlorambucil adducts was achieved using HPLC interfaced to electrospray ionization ion trap mass spectrometry. Both trinucleotide and tetranucleotide Chlorambucil adducts were detected. The majority of the detected trinucleotide adducts involved monofunctional alkylation with guanine being the hotspot for alkylation. Only a few bifunctional trinucleotide adducts both intra- and interstrand cross-links were found. On the contrary, cross-linked adducts were the major detected tetranucleotides in which the intrastrand cross-links predominated over the interstrand cross-links. To a lesser extent, monofunctional guanine alkylated tetranucleotides were detected as well. With MSn experiments, the detailed structures of Chlorambucil adducts of the tri- and tetranucleotides were determined. 1. Introduction Although alkylating agents were the first compounds to be used in cancer chemotherapy, dating from the introduction of the nitrogen mustard in 1946, they still hold a central position in cancer treatment today. Nitrogen mustards (NMs1) were used by themselves at first, but they became quickly a part of effective combinations with radiotherapy and/or other drugs (1). The cytotoxicity of these drugs is thought to be due to alkylation and cross-linking of DNA. The proposed mechanism involves the formation of a reactive aziridinium intermediate with subsequent monofunctional adduct formation. The monofunctional adducts can then react with solvent or another nucleophilic site via the formation of a second aziridinium ion resulting in a cross-link either between two DNA bases or between DNA and proteins (2). Nitrogen mustards show low sequence selectivity for monoalkylation, reacting at almost all available guanines. In addition, monoalkylations by NMs are considered to be genotoxic rather than cytotoxic (3). On the contrary, the lethal interstrand cross-links are limited to DNA sequences that contain two reactive nucleophilic centers within the reach of the mustard (2, 4). Thus, the cytotoxicity of mustards may be related to their ability to form interstrand cross-links, which block DNA replication (5). However, intrastrand lesions can be either promutagenic or lethal (6, 7). Povirk and Shuker * Corresponding author. Humboldt-Universitaet zu Berlin, Department of Chemistry, Brook-Taylor-Str.2, 12489 Berlin, Germany. Phone: 04930-2093 7575. Fax: 049-30-2093 6985. E-mail: [email protected]. † Humboldt-Universitaet zu Berlin. ‡ University of Duisburg-Essen. 1 Abbreviations: NMs, nitrogen mustards; ESI, electrospray ionization; SPE, solid phase extraction; clb, Chlorambucil; NS1, nuclease S1; Ph, phenyl-modified silica-gel; clbx, Chlorambucil which has lost 44 u.

concluded from the mutational spectra generated by NMs that no single lesion is primarily responsible for mustard-induced mutagenesis, as both monofunctional and bifunctional adducts appear to be involved (8). The nitrogen mustard Chlorambucil is an antineoplastic agent used in the treatment of lymphocytic leukemia, ovarian and breast carcinomas, and Hodgkin’s disease (9). In order to improve its clinical antitumor efficiency, Chlorambucil is often administered in combination with prednisolone (10). Therefore, the prednisolone ester of Chlorambucil (prednimustine) was introduced into therapy, and it exhibited distinct advantages over the equivalent dose of its constituents (11). A higher rate of cell death was observed in Vitro, and it proved to be less toxic in ViVo (12, 13). Chlorambucil is extensively metabolized at the butyric acid side chain by β-oxidation, yielding the cytotoxic metabolite phenyl acetic acid mustard, which itself exhibits anticancer activity (14). Chlorambucil preferentially binds to N7 of guanine residues of DNA (5); thus, depurination reactions can take place (15). Additionally, this lesion is subject to imidazole ring-opening, yielding a formamidopyrimidine product, which blocks DNA synthesis in Vitro (16). Furthermore, Chlorambucil can interact with the minor groove of DNA, specifically at N3 of adenine. Wang et al. extensively studied the mutagenesis caused by NMs in the SV40-based shuttle vector pZ189 (17, 18). These studies revealed that Chlorambucil induces mutations predominantly at A·T base pairs, with A·T f T·A transversions being most frequent. It was proposed that these mutations resulted from the thermolabile adenine N3 adducts. Yaghi et al. performed a comparative study between Chlorambucil and its half mustard analogue in terms of mutagenic potency exerted by the two drugs (19). Both drugs are thought to form similar monoadducts,

10.1021/tx900123r CCC: $40.75  2009 American Chemical Society Published on Web 07/22/2009

1436

Chem. Res. Toxicol., Vol. 22, No. 8, 2009

but only the full mustard can form interstrand cross-links. The data suggest that DNA cross-links, although only a minor fraction of the total lesions, dominate the mutagenic spectrum and lead to gross changes at the chromosome level that cannot be readily associated with individual lesions produced by the drug. To date, no detailed product analysis for Chlorambucilnucleotide adducts was available until recently, when Hovinen and co-workers investigated the reaction of Chlorambucil with 2′-deoxyguanosine (20), 2′-deoxyadenosine (21) and 2′-deoxycytidine, 2′-deoxy-5-methylcytidine, and thymidine (22) at physiological pH by HPLC-MS/MS. The authors concluded that the N7 alkylated guanine and the corresponding N7, N7-bisadduct were the major stable dGuo derivatives. In addition, N1 was the principal alkylation site in the case of adenine, while it was suggested that N3 is the major alkylation site for all pyrimidines. However, no information is available on the distribution of Chlorambucil adducts in DNA; thus, investigation of these adducts is the main focus of our studies. To achieve this, reversed phase HPLC interfaced to an electrospray ionization (ESI) ion trap mass spectrometer was used for the separation, detection, and structural characterization of the formed adducts. Here, we present the results for the different Chlorambucil-trinucleotide and -tetranucleotide adducts with respect to their fragmentation behavior, abundance, and biological relevance.

2. Experimental Section Caution: Chlorambucil is highly toxic and should be handled carefully. 2.1. Chemicals and Reagents. All solvents were of HPLC grade. The chemicals were of analytical reagent grade. Chlorambucil, Benzonase, sodium acetate, and triethyl ammonium acetate were purchased from Sigma-Aldrich (Steinheim, Germany). Double stranded calf thymus DNA was obtained from Calbiochem (Darmstadt, Germany). Dimethyl sulphoxide and magnesium acetate were obtained from Merck (Darmstadt, Germany). Calf intestine Alkaline Phosphatase and Nuclease S1 were purchased from Fermentas (St.Leon Rot, Germany). Diethyl ether and ethyl alcohol were obtained from J. T. Baker (Deventer, Netherlands). Ammonium acetate was obtained from Riedel De Haen AG (Seelze, Germany). Solid phase extraction cartridges were purchased from Varian (Middelburg, Netherlands). 2.2. Sample Preparation. DNA adducts of Chlorambucil were prepared by the reaction of Chlorambucil with calf thymus DNA. An aliquot of 250 µL of a DNA stock solution (4 mg/mL in deionized water) was incubated with 200 µL of Chlorambucil stock solution (1 mg/mL in DMSO) for 16 h at 37 °C. Then the reaction mixture was cooled to room temperature and extracted with diethyl ether. Any excess of diethyl ether was removed using a vacuum centrifuge (SpeedVac AES 1000, Savant Instruments, Farmingdale, NY, USA) for 15 min. The treated DNA was precipitated by the addition of 75 µL of 3 M sodium acetate solution (pH 5.2) and 1 mL of ice cold ethanol. The solution was centrifuged at 4 °C and 10,000 g for 15 min using a Sigma 3K30 centrifuge (Sigma laboratory centrifuges, Osterode am Harz, Germany). The resulting DNA pellet was washed twice with 100 µL of aqueous ethanol (70%) and dried. 2.2.1. Enzymatic Digestion of DNA. The dried DNA pellet was redissolved in 400 µL of 2 mM magnesium acetate solution and 25 units of Benzonase were added either alone or followed by 1.75 units of Alkaline Phosphatase. The resulting solution was incubated at 37 °C for 4 h. Afterward, the pH of the solution was adjusted to 4.5 using Nuclease S1 buffer (5× reaction buffer, 200 mM sodiumacetate (pH 4.5 at 25 °C), 1.5 M NaCl, and 10 mM ZnSO4), then 100 units of Nuclease S1 enzyme were added, and the solution was further incubated at 37 °C for 30 min.

Mohamed et al.

2.2.2. Solid Phase Extraction (SPE). SPE was carried out for these samples as follows: 1 mL SPE cartridges were filled with 100 mg of C18 or phenyl modified silicagel sorbent material. The cartridges were rinsed with 3 mL of methanol and 2 mL of 10 mM ammonium acetate solution (pH 5) for cleaning and conditioning. After sample (1 mL) absorption, the cartridges were washed with 4 mL of the ammonium acetate solution to remove unmodified nucleotides, and the modified nucleotides were eluted from the cartridges with 2 mL of methanol. The solvent was removed under vacuum, and the dried residues were redissolved in 250 µL of ammonium acetate solution (10 mM, pH 5). 2.3. Instrumentation. The chromatographic system consisted of an Ultimate pump (LC Packings, Amsterdam, Netherlands) equipped with a calibrator ULT-MIC-1000, an autosampler FAMOS (LC Packings, Amsterdam, Netherlands) equipped with a 5 µL sample loop, a C18 guard column with 5 µm particle size, 1 mm i.d. (Dionex, Idstein, Germany), and a separation column (Discovery Bio Wide Pore) C18 with 1 mm i.d., 10 cm length, 3 µm particle size (Supelco, Sigma-Aldrich, Germany). The flow rate was 50 µL/ min, and the injected volume was 5 µL. Eluent A: 5 mM triethyl ammonium acetate (pH 5.4). Eluent B: 50% 10 mM triethyl ammonium acetate (pH 5.4) and 50% methanol. Gradient program: 0-5 min 88% A, 5-30 min up to 80% B, 30-44 min 80% B, 44-50 min back to 88% A, and 50-60 min equilibration. 2.3.1. Mass Spectrometric Parameters. Measurements were carried out on a LCQ Deca XP (Finnigan, San Jose, CA, USA) in the negative ionization mode. The parameters of the instrument were tuned (using d(GA)) to the following values: 22 arb nitrogensheath gas flow, 4.2 kV sprayer voltage, -33 V capillary voltage, -40 V tube lens offset, 6.75 V multipole 1 offset, 15.5 V multipole 2 offset, 14 V intermultipole lens voltage, 400V p-p octapole RF amplifier, 66 V entrance lens, 250 °C transfer capillary temperature. In full scan mode, a mass range from m/z 300-1500 was scanned. MS/MS experiments (He as collision gas) were carried out using an isolation width of ∆m/z 4, an activation amplitude of 35%, and an activation time of 55 ms.

3. Results To identify DNA modifications at the oligonucleotide level, a combination of Benzonase, Alkaline Phosphatase, and Nuclease S1 (NS1) enzymes was developed for the Chlorambucil (clb)-DNA digestion. Benzonase, an enzyme developed to purify proteins from nucleic acids by cleaving DNA and RNA unspecifically (23), cleaves the DNA into oligonucleotides with chain lengths of 2-8 nucleobases (24). Alkaline Phosphatase removes free 5′ phosphate groups; hence, oligonucleotides lacking the terminal phosphate are produced. NS1 cleaves phosphodiester bonds on single stranded DNA (25). The strand of DNA on the 5′ end of the cleavage site ends with 3′ hydroxyl, whereas the strand of DNA liberated from the 3′ end starts with a 5′ phosphate. The effect of NS1 was controlled by pH and by using adequate concentrations of sodium and zinc ions. Without proper salt concentrations and pH, the enzyme becomes either nonspecific or nonreactive (25). The enzymatic digestion time was optimized to 4 h of Benzonase and 30 min of NS1, resulting in the production of trinucleotide and tetranucleotide adducts (Figure 1A and D). As the enzymatic digestion is not exclusive to the reacted DNA, the nonreacted DNA was also digested, and large amounts of native (unmodified) nucleotides were produced as well. The presence of such unmodified species in high abundance greatly affects the performance of the HPLCMS approach due to insufficient separation and, therefore, introduces difficulties in identification of the adducts. To overcome this problem, SPE was used to remove a great part of the unmodified nucleotides; furthermore, it gave the chance for preconcentration of the modified species. During this work, we used two kinds of SPE cartridges: octadecyl (C18) and

Chlorambucil-Oligonucleotide Adducts

Chem. Res. Toxicol., Vol. 22, No. 8, 2009 1437

Figure 1. BPI chromatograms of m/z 560-640 for the Chlorambucil-DNA samples incubated for 16 h and digested with Benzonase (4 h) and NS1 (30 min) using different SPE cartridges Ph (A), C18 (the time range 21-35 min is ×8 enlarged) (B), and the corresponding mass spectrum of the Chlorambucil-trinucleotide adducts (Ph cartridges) in the time range 21-35 min (C), BPI chromatogram of m/z 700-800 for the same samples using Ph cartridges (D), and the corresponding mass spectrum of the Chlorambucil-tetranucleotide adducts in the time range 20-35 min (E).

phenyl-modified (Ph) silica-gel. Ph cartridges exhibit an added selectivity resulting from the electron density of the aromatic ring with the possibility of π-π exchange effect between the phenyl modified silica-gel with the aromatic moiety of Chlorambucil. Thus, the yield of the alkylated adducts was higher, and

the concentration of the unmodified compounds was greatly reduced upon the use of the Ph cartridges as displayed in Figure 1A and B. 3.1. Monofunctional Chlorambucil-Trinucleotide Adducts. The Chlorambucil-trinucleotide adducts were detected mainly

1438


Mohamed et al.

Figure 2. Comparison between the percentage relative abundance of the different Chlorambucil-trinucleotide adducts formed at guanine, cytosine, and adenine bases (A) and the relative peak areas of the most abundant monoalkylated Chlorambucil-trinucleotide adducts. All adducts possess the hydroxyethyl arm of Chlorambucil (B). The relative peak area is calculated as an average of three experiments with coefficient of variation (as percentage) < 10%. (The position of alkylation is assigned with an asterisk).

as doubly charged species (Figure 1C), where the 2′-hydroxyethyl monoadducts2 were the predominant lesions generating the adducts with the formula [pd(B1B2B3)-clb(OH)]2-. Monoalkylation reaction was restricted to the nucleobases guanine, cytosine, and adenine. Guanine modifications represented about 70% of the total detected adducts, reflecting the preferential alkylation of Chlorambucil to guanine. The high number of guanine adducts was attributed to the detection of several isomers for the same modified compound. Some of these isomers have nearly similar retention times on the HPLC column, resulting in overlapping chromatographic peaks; hence, the MS/ MS data became misleading. However, this problem was solved by removing the 5′ phosphate (trinucleoside diphosphates) with the addition of Alkaline Phosphatase. The absence of the 5′ phosphate decreased the polarities of the different isomers improving their retention and separation. Both the G*NN and NG*N sequences were detected without specific priority for one of these sequences.3 Cytosine was second in alkylation followed by adenine (Figure 2A). Obviously, thymine was not involved in the alkylation reactions as it is considered the least nucleophilic base, hence, the least reactive. However, its presence in the neighborhood of the alkylated bases greatly affected the abundance of the detected adducts. We have shown previously that the ES ionization efficiencies for nucleotides possessing the same number of phosphate residues are very similar (26, 27); thus, peak areas can be applied for comparing sequence preferences. To ascertain the reproducibility of the obtained results, the performed experiments were repeated several times over an extended time period. The most abundant sequences 2 Any MS/MS fragments possessing the hydroxyethyl side arm of Chlorambucil will be referred to as clb(OH). 3 * assigns the alkylation position.

were those in which a thymine base is located on the 3′ end of the sequence as shown in Figure 2B. 3.1.1. Fragmentation Behavior of the Trinucleotide Guanine Adducts. An examination of the fragmentation behavior of the doubly charged trinucleotide or the trinucleoside diphosphate Gua adducts revealed a single major fragmentation pathway. This pathway involved the loss of the alkylated guanine base as a negatively charged ion through N-glycosidic bond cleavage, giving rise to the complementary depurinated backbone fragment as depicted in Figure 3A and B. In the following, a subsequent 3′ C-O bond cleavage for the depurinated nucleotide residue took place, resulting in the production of either the unmodified w2- ion in case of the G*NN adducts or the two alternative negatively charged unmodified fragments w1- and (a2-B2)- for the adducts, possessing the NG*N sequence. Moreover, an ion corresponding to PO3- loss was also observed in the CID spectra of Gua trinucleotide adducts. This specific fragmentation pattern was already observed in our previous work (28) and was also reported in negative ion MS/MS spectra of adducts formed between the naturally occurring antitumor antibiotic hedamycin and the oligonucleotide CACGTG (29). To determine the alkylation site of Chlorambucil on the purine ring, an MS3 experiment was performed for the characteristic ion at m/z 399, which belonged to [Gua-clb(OH)]- (Figure 3E). A characteristic ion was produced at m/z 356 corresponding to a 43 u loss from the precursor ion originating from the elimination of HNCO from the modified base. Thus, both O6 and N1 positions were excluded from being the alkylation sites. Besides, the ions at m/z 381, m/z 248, m/z 176, and m/z 150 were also generated and identified as [M-H-H2O]-, [clb side chain]-, [Gua-CHdCH2]- and [Gua]-, respectively. The observed product ions and the detection of guanine deglycosylation



Figure 3. MS/MS spectra of [pd(G*AT)-clb(OH)]2- (A), [pd(AG*T)-clb(OH)]2- (B) at m/z 605.5, and [pd(G*AT)-clbx]2- at m/z 583.5 (C). MS3 spectra of [Gua-clbx]- at m/z 355 (D) and [G-clb(OH)]- at m/z 399 (E). Proposed structure of [Gua-clbx]- at m/z 355 (F). (clbx assigns Chlorambucil which lost 44 u).

products in the reaction mixture pinpointed Chlorambucil alkylation at the N7 position of guanine. An unexpected observation was the detection of guanine adducts with m/z values that do not match theoretically calculated values of known adducts. These compounds were structurally assigned to guanine alkylation due to observing the typical fragmentation pattern of modified guanine species in their MS/MS spectra. In the represented example in Figure 3A and C, a comparison between the CID spectra of the theoretically calculated adduct at m/z 605.5 corresponding to [pd(G*AT)clb(OH)]2- and that of the unknown compound at m/z 583.5 is demonstrated. It was obvious that both compounds are nearly similar and that they possess the same backbone with the sequence pd(AT). Thus, the difference was in the modified fragment, where the alkylated guanine base with hydrolyzed Chlorambucil, which normally corresponds to m/z 399, was replaced by m/z 355 with a shift of 44 u. This mass difference might be attributed to a change in the structure of either Gua or the Chlorambucil side chain. A more detailed structure of this compound was gained by performing an MS3 experiment for the ion at m/z 355 (assigned to [Gua-clbx]-, where clbx is Chlorambucil which has lost 44 u). This further pointed to

guanine alkylation because of the similarity in the obtained fragments with those obtained from the activation of m/z 399 (Figure 3D and E). Hence, it was deduced that the mass difference was produced from the Chlorambucil side chain. It was difficult to give an unambiguous structural assignment for this compound. From our point of view, the 44 u difference might be interpreted as an elimination of C2H4O from the Chlorambucil side arm (Figure 3F). 3.1.2. Fragmentation Behavior of the Trinucleotide Cytosine and Adenine Adducts. The modified cytosine and adenine trinucleotides showed a similar behavior in the ion trap, and they followed the rules of fragmentation developed for the native nucleotides by McLuckey and Habibi-Goudarzi (30). Hence, the sequence specific fragments in combination with the Chlorambucil side chain enabled a straightforward identification of the sequence. The presence of the 5′ phosphate greatly affected the MS/MS spectra of the doubly charged trinucleotide adducts, where the loss of PO3- represented the major fragmentation route. In addition, ions of the w-series and the complementary a-series were detected; the former predominated over the latter. Then, the (a2-B2)- ion was usually observed in moderate abundance (Figure 4A and B). The observation of the

1440


Mohamed et al.

Figure 4. MS/MS spectra of [pd(A*AC)-clb(OH)]2- (A) and [pd(C*AA)-clb(OH)]2- (B) at m/z 590. The m/z range 180-1080 is × 2 enlarged, and the inset shows the EIC of this adduct and MS/MS spectrum of [d(C*AA)-clb(OH)]2- at m/z 550 (C). (The position of alkylation and the modified fragments are assigned with an asterisk).

(a2-B2)- ion in higher abundance than its obvious precursors, a2- and [M-2H-B2]-, indicates that the formation of the (a2B2)- ion either involves a short-lived intermediate not detected in the ion trap or that it was a concerted process (31). Nonsequence specific ions resulting from the elimination of different bases as negatively charged species were also observed. The intensity of these ions was highly dependent on the nature of the lost base and to a lesser extent on its location. As previously described, there is a strong preference for the loss of a charged adenine followed by thymine (30). However, the CID spectra of the doubly charged cytosine and adenine trinucleoside diphosphate adducts were dominated by a loss of B1 as a negatively charged species with the consequent cleavage of the 3′ C-O bond. Thus, the B1-, [M-2H-B1]-, and w2- were usually found in high abundance. The predominance of this pathway was not affected by the alkylation of B1. To a lesser extent, loss of B2 also occurred; consequently, (a2-B2)- and w1- were detected in low abundance (Figure 4C). On the basis of the detailed analysis for the fragmentation data, we could assign N3 as the alkylation site in cytosine. In

all MS/MS spectra of the cytosine adducts, an ion at m/z 291 was generated from the alkylated cytosine fragment [Cytclb(OH)]- at m/z 359 as proved in the MS3 spectrum of the latter ion. A mass difference of 68 u between both ions resulted from the neutral loss of C3H4N2 from the cytosine base, while the detected fragment at m/z 291 belonged to clb-NdCdO. Furthermore, a neutral loss of HNCO from the clb-NdCdO ion producing the Chlorambucil side chain at m/z 248 confirmed this assumption. However, the identification of the alkylation site of the adenine base was more complicated. Activation of the [Adeclb(OH)]- fragment at m/z 383 gave rise to two major ions at m/z 160 and m/z 134 corresponding to [Ade-CHdCH2]- and [Ade]-, respectively (data not shown). However, both ions are considered as site-unspecific ions. It was stated in the literature (21) that the detection of the clb side chain seems to be characteristic for adducts bearing the modification at an endocyclic nitrogen atom. Thus, the absence of this ion might indicate N6 adenine alkylation. 3.2. Bifunctional Chlorambucil-Trinucleotide Adducts. Besides the dominant monoalkylation reaction, bifunctional alky-



Figure 5. MS/MS spectrum of [pd(G∧CA)-clb]2- at m/z 589 (A), MS3 spectrum of [a2*]- ion at m/z 848 (B), and MS/MS spectra of [d(TG∧A)clb]2-/[d(TA∧G)-clb]2- at m/z 556.5 (C) and [d(AG∧G)-clb]2- at m/z 569 (D). The insets show the MS3 spectra of the unmodified backbone fragments of both adducts. (∧ assigns intrastrand cross-link formation between two neighboring bases).

lation was also observed, and some intra- and interstrand crosslink-adducts were detected in the reaction mixture. Mainly, guanine was involved in the cross-link formation bridged to other nucleobases (except thymine) via Chlorambucil. The structural assignment of these cross-links proved to be less straightforward in comparison to the monofunctional adducts because of their fragmentation pattern in the ion trap. Interpretation of the MSn data depends not only on the presence but also equally on the absence of characteristic or sequencing ions in the spectra. ∧ 3.2.1. Intrastrand Cross-Linked Adducts. 3.2.1.1. [pd(G CA)clb]2- at m/z 589 (I), [pd(G∧CT)-clb]2- at m/z 584.5 (II), and [d(G∧CT)-clb]2- at m/z 544.5 (III)4. The adducts showed two major fragmentation pathways upon their activation (Figure 5A). In the first route, the two glycosidic bonds of guanine and cytosine 4 ∧ indicates intrastrand cross-link formation between two neighboring bases.

are broken, resulting in the two complementary fragments [Guaclb-Cyt]- at m/z 492 and the backbone fragment. The second route was characterized by a 3′ C-O bond cleavage, giving rise to the unmodified w1- ion and the modified [a2*]- fragment. The presence of the unmodified w1- pinpoints the unbound base at the 3′ position. In order to determine the position of guanine and cytosine bases in the sequence, MS3 was carried out for the [a2*]- fragment. Elimination of different neutral species from the sugar phosphate backbone was recorded (Figure 5B). The most abundant fragment resulted from the loss of either 178 u corresponding to pd-H2O (for adducts I and II) or 98 u assigned to d (for adduct III) from the parent ions.5 This elimination most probably occurred due the cleavage of the weaker N-glycosidic bond followed by 3′ C-O 5 pd-H2O refers to the deoxyribose-phosphate moiety with water molecule loss and corresponds to 178 u, d is deoxyribose with water molecule loss and corresponds to 98 u, and p refers to phosphoric acid and corresponds to 98 u.

1442


Mohamed et al.

Figure 6. MS/MS spectrum of [pd(G*G)-clb-dAMP*]2- and [pd(G*A)-clb-dGMP*]2- at m/z 618 (A), MS3 spectrum of [Gua-clb-dAMP*]- at m/z 712 (B), MS3 spectrum of [Gua-clb-dGMP*]- at m/z 728 (C), MS/MS spectrum of [pd(G*T)-clb-dC*MP]2- at m/z 593.5 (D), and MS3 spectrum of [Gua-clb-dC*MP]- at m/z 688 (E). (The position of alkylation is assigned with an asterisk).

bond break. Obviously, the positive charge due to alkylation of the N7 of guanine weakened the N-glycosidic bond at this site and made it more labile. Hence, we assigned the presence of guanine at the 5′ terminus. 3.2.1.2. [d(TG∧A)-clb]2- at m/z 556.5 (I) and [d(AG∧G)2clb] at m/z 569 (II). Activation of both adducts resulted in a small number of fragments; nevertheless, the cross-link formation was ascertained by the detection of the [Gua-clb-Ade]and the [Gua-clb-Gua]- ions together with the backbone fragments in the MS/MS spectra. The absence of the unmodified w1- ion in the MS3 spectrum of the backbone pinpointed the unbound base to the 5′ position (Figure 5C and D). Unfortunately, no fragmentation revealed the relative position of the cross-linked bases in adduct (I); thus, adduct (I) could be either [d(TG∧A)-clb]2- or [d(TA∧G)-clb]2-.

3.2.2. Interstrand Cross-Linked Adducts. 3.2.2.1. [pd(G*G)clb-dAMP*]2- (IA), [pd(G*A)-clb-dGMP*]2- (IB) at m/z 618, and [pd(G*T)-clb-dC*MP]2- at m/z 593.5 (II). The MS/MS spectrum of the ion at m/z 618 indicates two coeluting isomers (Figure 6A). This assumption was based on the detection of the unmodified backbone of dinucleotides, that is, [pdpdGua]at m/z 524 for (IA) and [pdpdAde]- at m/z 508 for (IB), together with the complementary ions of [Gua-clb-dAMP*]- at m/z 712 and [Gua-clb-dGMP*]- at m/z 728 for (IA) and (IB), respectively. Both isomers correspond to dinucleotides with guanine at the 5′ end bridged to the phosphate group of a mononucleotide via Chlorambucil. MS3 data for the backbone showed an unmodified w1- fragment confirming guanine at the 5′ terminus. The most abundant ion (m/z 577) in the MS3 spectrum of the cross-linked fragments corresponds to a base loss followed by



Scheme 1. Main Fragments Observed from MS2 of the Chlorambucil-Tetranucleotide 1,3-Intrastrand and 2,3-Intrastrand Cross-Links and MS3 of the Modified [a3*]- Ion

elimination of a deoxyribose moiety (m/z 461). These consecutive losses can only occur when the base of the mononucleotide does not participate in the cross-link formation. In addition, the presence of the ion at m/z 330 in the MS3 spectrum of [Guaclb-dAMP*]- is an additional evidence for the structural assignment of the mononucleotide part of the adduct (IA) (Figure 6B and C). The third adduct at m/z 593.5 also represented an interstrand cross-link adduct. However, in this case Chlorambucil bridged guanine to cytosine. CID of this adduct gave rise to the two complementary fragments [pdpdThy]- (m/z 499) as the backbone and [Gua-clb-dC*MP]- (m/z 688) representing the modified fragment (Figure 6D). MS3 of the latter ion assured the structural elucidation of the adduct due to the production of the characteristic fragment [Gua-clb-Cyt]- as displayed in Figure 6E. The high abundance of the [Gua-clb-Cyt]- ion might be attributed to the stability of the Watson-Crick base pairs (G···C) in the duplex DNA. 3.3. Chlorambucil-Tetranucleotide Adducts. The Chlorambucil-tetranucleotide adducts were detected as doubly charged species within the mass range of m/z 700-800 (Figure 1E). MSn data for this group of adducts revealed that the modified tetranucleotides belonged mainly to cross-linked adducts of the type 2,3- or 1,3- intrastrand. In addition, interstrand cross-links and a few monofunctional adducts alkylated at the guanine base were detected. The fragmentation pattern of the monofunctional guanine tetranucleotides was analogous to the trinucleotides. The intrastrand cross-linked adducts, however, could be categorized into two fragmentation groups I and II. CID data of group I is characterized by the presence of the unmodified w1- fragment together with its complementary [a3*]- ion, which possessed the cross-linked bases. However, MS3 data of the latter revealed that cross-linking could occur between B1 and B3 (1,3-intrastrand cross-links), group IA, or between B2 and B3 (2,3-intrastrand cross-links), group IB. As shown in Scheme 1, two main steps

were observed in the fragmentation of the modified ions of both groups: the neutral loss of 178 u (pd-H2O) from the 5′ end (1) and the elimination of the unbound base followed by 3′ C-O bond cleavage (2). Fortunately, the sequence of the two steps allows one to identify the position of the cross-linked bases. In group IA adducts, reaction (1) comes first, pointing to the presence of one of the cross-linked bases at the 5′ terminus. Thus, guanine should be at the 5′ end since the N-glycosidic bond of the alkylated guanine is very labile. Then, reaction (2) follows, suggesting the presence of an unbound base at the B2 position. Step (2) creates a fragment corresponding to [Guaclb-pd-H2OB*3]-. However, if this fragment is lost as a neutral, an unmodified ion corresponding to [pdpd-H2OB2]- will be detected. In comparison, activation of the modified fragment of group IB starts with step (2). Hence, the nonalkylated base is positioned on the 5′ end. In the following step, reaction (1) occurs obviously after the depurination of the guanine base at the B2 position producing the same fragment [Gua-clbpd-H2OB*3]-. However, the elimination of latter fragment as a neutral gives rise to the [pdB1pd-H2O]- ion. An example representing both groups IA and IB is the doubly charged adduct at m/z 741. As displayed in its extracted ion chromatogram, two isomers were detected corresponding to [pd(G*TC*A)-clb]2- and [pd(AG∧CT)-clb]2- (Figure 7). In their CID spectra, the earlier discussed steps of fragmentation (1) and (2) are recognizable; hence, the structures of both isomers could be elucidated. With regard to group II adducts, their CID spectra show one major fragmentation pathway, namely, the elimination of the cross-linked bases by N-glycosidic bond cleavages, yielding the unmodified backbone and the cross-linked [Gua-clb-B]- ions. Activation of the unmodified backbone pinpointed this group to 2,3-intrastrand cross-linked adducts. This assignment was based on the observation of reaction sequence (1), a neutral loss of unmodified w1 (unbound base on the 3′ end), followed by

1444


Mohamed et al.

Figure 7. EIC of the intrastrand cross-linked adduct [pd(GTCA)-clb]2- at m/z 741 (A), MS/MS spectrum of [pd(G*TC*A)-clb]2- at m/z 741 (B), MS3 spectrum of the corresponding [a3*]- at m/z 1152 (C), MS/MS spectrum of [pd(AG∧CT)-clb]2- at m/z 741 (D), and MS3 spectrum of the corresponding [a3*]- at m/z 1161 (E). (∧ assigns intrastrand cross-link formation between two neighboring bases, and the position of alkylation is assigned with an asterisk).

Figure 8. MS/MS spectrum of [pd(TG∧AA)-clb]2- at m/z 753 (A) and MS3 spectrum of the unmodified backbone ion at m/z 990 (B). (∧ assigns intrastrand cross-link formation between two neighboring bases; the underlined bases could be in switched position).

step (2) and step (3) as two consecutive losses of pd-H2O (position of B2 and B3 should be unoccupied) in the MS3 spectra of the unmodified backbone. A complete structure elucidation for the adducts in group II was impossible, as every bound base could be positioned either on B2 or B3 as indicated in Figure 8. Besides the intrastrand cross-links, two interstrand crosslinked tetranucleotides were also detected, and their structures are assigned to [pd(G*G)-clb-pd(G*T)]2- and [pd(G*T)-clbpd(G*T)]2-. A general feature in the fragmentation of the interstrand adducts was the depurination of one of the cross-

linked guanines, yielding the unmodified [pdpdB2]- and the modified [Gua-clb-pd(G*T)]- in analogy to the interstrand trinucleotide cross-links.

4. Discussion In light of the previous results, it is clear that there is a great diversity of Chlorambucil-trinucleotide and -tetranucleotide adducts. The majority (84%) of the observed trinucleotides correspond to base alkylated monofunctional adducts. However,


cross-links (80%) represented the main group of adducts generated at the tetramer level. We attribute this difference in adduct observation to the short NS1 digestion time, as the short time and the longer chain lengths allow the cross-links to stay intact for the MS measurement. The more frequent monoalkylation reaction becomes dominant in shorter oligomers. However, it is likely that all types of alkylation of DNA have biological consequences (32). The results obtained here shed some light on the biological significance of the various adducts. Chlorambucil alkylation at the N7 position of guanine does not change the base pairing properties of guanine; however, a rapid depurination of the 7-alkyl guanine occurs. The formed abasic sites could be efficiently repaired by AP endonuclease repair enzymes, but in view of their frequent occurrence in DNA, they may nevertheless contribute to occasional inactivation or mutagenesis of cells (33). Abasic sites exist predominantly as the cyclic acetal, which is in equilibrium with small amounts of the ring opened aldehyde form. The loss of the acidic R-proton adjacent to the carbonyl residue in the aldehyde form of the abasic site can lead to a strand break via elimination of the 3′ phosphate group (34). It was shown already that Chlorambucil shows higher toxicity in an E. coli strain which was lacking the DNase repair enzymes than in the wild type (35). Chlorambucil also induced alkylation at the N3 position of cytosine and obviously at the N6 position of adenine. Although both positions in double stranded DNA are protected from alkylation by Watson-Crick base pairing, it is well known that single stranded regions are temporarily exposed during DNA replication, transcription, and repair. Thus, these sites may become vulnerable to group specific reactants such as alkylating agents, and DNA functions may be obscured (21, 22). It was found that E. coli ALKB functions in DNA repair by oxidative demethylation of 3-methylcytosine (36). Homologous proteins are also present in mammalian cells; however, it is still unknown whether these proteins are able to remove corresponding Chlorambucil adducts from DNA. DNA-DNA (inter or intrastrand) cross-links are generally assumed to be the crucial lesions for cell death. Guanine-adenine cross-links would constitute an effective block to DNA polymerase and would require repair to restore template activity (37). Thus, although the cross-links were detected at low level only in our experiments, it is conceivable that even a single covalent cross-link in DNA out of 3 × 109 base pairs in the human genome would result in cell death (20). The data obtained from this investigation is complementary to our study performed on Melphalan (28) (under similar reaction conditions) and make similarities and differences of the two nitrogen mustards Melphalan and Chlorambucil on DNA evident. The main difference observed at the trimer level was the preferentially alkylated nucleobase. Unexpectedly, Melphalan preferably forms cytosine adducts. This was attributed to the ease of depurination of the guanine adducts. To the contrary, predominance of guanine alkylation was observed with Chlorambucil, in spite of the occurrence of the depurination reaction with guanine-Chlorambucil adducts too. While pd(C*AT) was the adduct of highest abundance following Melphalan alkylation, the most abundant Chlorambucil adduct is pd(AG*T). This observation may be relevant for the explanation of differences found when DNA is incubated with either one of the drug; using Melphalan, DNA is rapidly fragmented; with Chloambucil this does not happen. The reason for that is not clear yet, but investigations along this line are under way. Another difference was the observation of guanine-clb adducts with 44 u less than expected; the absence of these adducts in


Melphalan-DNA hydrolysate are surprising and will be studied in future. Furthermore, interstrand cross-links between guanine and a phosphate residue were detected exclusively with Chlorambucil. However, trinucleotide adducts belonging to both drugs showed analogy in fragmentation behavior in the ion trap. In addition, a preference for triplets with a thymine base at the 3′ terminus was observed with both drugs, which can be explained by preferred adduct formation in the DNA duplex with Gua and Ade in opposite strands and adjacent positions. The nature of the tetranucleotide adducts obtained from both drugs represented two extremely different situations. Melphalan preferentially alkylates the phosphate residues with subsequent monofunctional adduct formation (data not shown (38)), on the other extreme, Chlorambucil reacted bifunctionally with the consequence of cross-link formation. A possible explanation of these differences is that noncovalent interactions occur between the nonalkylating moieties of these drugs and DNA prior to a covalent reaction. Of course, the difference in the structures of both drugs affects these interactions.

5. Conclusions The molecular mechanisms which determine the cellular sensitivity/resistance of tumor cells such as CLL lymphocytes to chemotherapy with Chlorambucil is widely unknown. Therefore, solid information on the structural identity of the major clb-DNA adducts is an indispensable prerequisite to develop suitable analytical tools (e.g., adduct-specific antibodies) for measuring formation and repair rates in primary tumor cells or in experimental systems. Thus, we developed a method for analyzing clb-DNA adducts. In this study, we demonstrated that the combination of Benzonase, Alkaline Phosphatase (4 h), and Nuclease S1 (30 min) are capable of digesting the Chlorambucil treated DNA to produce modified tri- and tetranucleotides. It became evident from previous studies performed in our group that the control of the NS1 digestion time is the crucial factor in obtaining adducts of increased chain lengths (28, 39). By including solid phase extraction prior to analysis, the extraction yield of the adducts was significantly improved, and the large excess of native nucleotides was reduced. Reversed phase HPLC was successful in separating the detected adducts in well identified peaks, especially the less polar nucleosides. In addition, MS/MS was a very helpful tool for the sequence elucidation of adducts and in the assignment of the position of alkylation. From our data, it is evident that investigations at the oligomer level are needed to understand the details of chemistry involved in adduct formation; the digestion to dimers or even monomers camouflages such differences. In the next step, the strategy developed here will be used in conjunction with affinity preconcentration steps to identify and quantify the biologically relevant adducts in ViVo. This data will eventually be used to develop highly specific antibodies for chemotherapy control. Acknowledgment. We are grateful to the Arab Republic of Egypt for a grant to D.M. and to the DFG for financial support to M.L. Dr. Ulrike Hochkirch and Dr. Sebastian Beck are also gratefully acknowledged for supporting and revising the manuscript. Supporting Information Available: Details concerning the characteristic CID fragments and retention time of all the detected [pd(G*B2B3)-clbx]2-adducts, monofunctional Chlorambucil-trinucleotide adducts and Chlorambucil-tetranucleotide intrastrand

1446


cross-linked adducts, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Farmer, P. B. (1987) Metabolism and reactions of alkylating agents. Pharmacol. Ther 35, 301–358. (2) Bauer, G. B., and Povirk, L. F. (1997) Specificity and kinetics of interstrand and intrastrand bifunctional alkylation by nitrogen mustards at a G-G-C sequence. Nucleic Acids Res. 25, 1211–1218. (3) Brendel, M., and Ruhland, A. (1984) Relationships between functionality and genetic toxicology of selected DNA-damaging agents. Mutat. Res. 133, 51–85. (4) Millard, J. T., Raucher, S., and Hopkins, P. B. (1990) Mechlorethamine cross-links deoxyguanosine residues at 5′-GNC sequences in duplex DNA fragments. J. Am. Chem. Soc. 112, 2459–2460. (5) Hemminki, K. (1994) DNA Adducts of Nitrogen Mustards and Ethylene Imines. In DNA Adducts: Identification and Biological Significance; IACR Scientific Publications, Lyon, France; Vol. 125. (6) Balcome, S., Park, S., Quirk Dorr, D. R., Hafner, L., Phillips, L., and Tretyakova, N. (2004) Adenine-containing DNA-DNA cross-links of antitumor nitrogen mustards. Chem. Res. Toxicol. 17, 950–962. (7) Rajski, S. R., and Williams, R. M. (1998) DNA cross-linking agents as antitumor drugs. Chem. ReV. 98, 2723–2796. (8) Povirk, L. F., and Shuker, D. E. (1994) DNA damage and mutagenesis induced by nitrogen mustards. Mutat. Res. 318, 205–226. (9) Chandler, K. J., McCabe, J. B., and Kirkpatrick, D. L. (1994) Highperformance liquid chromatographic method for the separation of chlorambucil and its N-oxide prodrug. J. Chromatogr., B 652, 195– 202. (10) Oppitz, M. M., Musch, E., Malek, M., Ru¨b, H. P., Unruh, G. E., and Loos, U. (1989) Studies on the pharmacokinetics of chlorambucil and prednimustine in patients using a new high-performance liquid chromatographic assay. Cancer Chemother. Pharmacol. 23, 208–212. (11) Konyves, I., Nordenskjold, B., Forshell, G. P., De Schryver, A., and Westerberg-Larsson, H. (1975) Preliminary clinical and absorption studies with prednimustine in patients with mammary carcinoma. Eur. J. Cancer 11, 841–844. (12) Harrap, K. R., Riches, P. G., Gilby, E. D., Sellwood, S. M., Wilkinson, R., and Konyves, I. (1977) Studies on the toxicity and antitumour activity of prednimustine, a prednisolone ester of chlorambucil. Eur. J. Cancer 13, 873–881. (13) Newell, D. R., Calvert, A. H., Harrap, K. R., and McElwain, T. J. (1983) Studies on the pharmacokinetics of chlorambucil and prednimustine in man. Br. J. Clin. Pharmacol. 15, 253–258. (14) Davies, I. D., Allanson, J. P., and Causon, R. C. (1999) Rapid determination of the anti-cancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography-tandem mass spectrometry. J. Chromatogr., B. 732, 173–184. (15) Salmelin, C., and Vilpo, J. (2003) Induction of SOS response, cellular efflux and oxidative stress response genes by chlorambucil in DNA repair-deficient escherichia coli cells (ada, ogt and mutS). Mutat. Res. 522, 33–44. (16) O’Connor, T. R., Boiteux, S., and Laval, J. (1988) Ring-opened 7-methylguanine residues in DNA are a block to in vitro DNA synthesis. Nucleic Acids Res. 16, 5879–5894. (17) Wang, P., Bauer, G. B., Bennett, R. A. O., and Povirk, L. F. (1991) Thermolabile adenine adducts and A.T base pair substitutions induced by nitrogen-mustard analogs in an Sv40-based shuttle plasmid. Biochemistry 30, 11515–11521. (18) Wang, P., Bauer, G. B., Kellogg, G. E., Abraham, D. J., and Povirk, L. F. (1994) Effect of distamycin on chlorambucil-induced mutagenesis in pZ189: evidence of a role for minor groove alkylation at adenine N-3. Mutagenesis 9, 133–139. (19) Yaghi, B. M., Turner, P. M., Denny, W. A., Turner, P. R., O’Connor, C. J., and Ferguson, L. R. (1998) Comparative mutational spectra of the nitrogen mustard chlorambucil and its half-mustard analogue in Chinese hamster AS52 cells. Mutat. Res. 401, 153–164.

Mohamed et al. (20) Haapala, E., Hakala, K., Jokipelto, E., Vilpo, J., and Hovinen, J. (2001) Reactions of N, N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2′-deoxyguanosine. Chem. Res. Toxicol. 14, 988– 995. (21) Florea-Wang, D., Haapala, E., Mattinen, J., Hakala, K., Vilpo, J., and Hovinen, J. (2003) Reactions of N, N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2′-deoxyadenosine. Chem. Res. Toxicol. 16, 403–408. (22) Florea-Wang, D., Haapala, E., Mattinen, J., Hakala, K., Vilpo, J., and Hovinen, J. (2004) Reactions of N, N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2′-deoxycytidine, 2′-deoxy-5methylcytidine, and thymidine. Chem. Res. Toxicol. 17, 383–391. (23) Schrader, W., and Linscheid, M. (1997) Styrene oxide DNA adducts: in vitro reaction and sensitive detection of modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass spectrometry. Arch. Toxicol. 71, 588–595. (24) Janning, P., Schrader, W., and Linscheid, M. (1994) A new mass spectrometric approach to detect modifications in DNA. Rapid Commun. Mass Spectrom. 8, 1035–1040. (25) Bartolini, W. P., Bentzley, C. M., Johnston, M. V., and Larsen, B. S. (1999) Identification of single stranded regions of DNA by enzymatic digestion with matrix-assisted laser desorption/ionization analysis. J. Am. Soc. Mass Spectrom. 10, 521–528. (26) Siethoff, C., Feldmann, I., Jakubowski, N., and Linscheid, M. (1993) Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry. J. Mass Spectrom. 34, 421–426. (27) Liao, Q., Chiu, N. H., Shen, C., Chen, Y., and Vouros, P. (2007) Investigation of enzymatic behavior of benzonase/alkaline phosphatase in the digestion of oligonucleotides and DNA by ESI-LC/MS. Anal. Chem. 79, 1907–1917. (28) Mohamed, D., and Linscheid, M. (2008) Separation and identification of trinucleotide-melphalan adducts from enzymatically digested DNA using HPLC-ESI MS. Anal. Bioanal. Chem. 392, 805–817. (29) Iannitti, P., Sheil, M. M., and Wickham, G. (1997) High sensitivity and fragmentation specificity in the analysis of drug-DNA adducts by electrospray tandem mass spectrometry. J. Am. Chem. Soc. 119, 1490–1491. (30) Mcluckey, S. A., and Habibigoudarzi, S. (1993) Decompositions of multiply-charged oligonucleotide anions. J. Am. Chem. Soc. 115, 12085–12095. (31) Wang, Z., Wan, K. X., Ramanathan, R., Taylor, J. S., and Gross, M. L. (1998) Structure and fragmentation mechanisms of isomeric T-rich oligodeoxynucleotides: a comparison of four tandem mass spectrometric methods. J. Am. Soc. Mass Spectrom. 9, 683–691. (32) Singer, B. (1976) All oxygens in nucleic acids react with carcinogenic ethylating agents. Nature 264, 333–339. (33) Lindahl, T. (1982) DNA repair enzymes. Annu. ReV. Biochem. 51, 61–87. (34) Gates, K. S., Nooner, T., and Dutta, S. (2004) Biologically relevant chemical reactions of N7-alkylguanine residues in DNA. Chem. Res. Toxicol. 17, 839–856. (35) Salmelin, C., and Vilpo, J. (2002) Chlorambucil-induced high mutation rate and suicidal gene downregulation in a base excision repairdeficient Escherichia coli strain. Mutat. Res. 500, 125–134. (36) Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T., and Sedgwick, B. (2002) Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419, 174–178. (37) Osborne, M. R., and Lawley, P. D. (1993) Alkylation of DNA by melphalan with special reference to adenine derivatives and adenineguanine cross-linking. Chem. Biol. Interact. 89, 49–60. (38) Mohamed, D. (2009) Analysis of DNA adducts caused by alkylating cytostatic drugs using mass spectrometry, Ph.D. Thesis, Humboldt Universitaet zu Berlin, Berlin, Germany. (39) Mowaka, S., and Linscheid, M. (2008) Separation and characterization of oxaliplatin dinucleotides from DNA using HPLC-ESI ion trap mass spectrometry. Anal. Bioanal. Chem. 392, 819–830.

TX900123R

Chlorambucil-Adducts in DNA Analyzed at the Oligonucleotide Level

Recommend Documents