Analysis of melphalan adducts of 2′-deoxynucleotides in calf thymus DNA hydrolysates by capillary high-performance liquid chromatography–electrospray tandem mass spectrometry

In this study a miniaturized LC coupled to electrospray tandem mass spectrometry was used to analyze modifications originating from the interaction between the chemotherapeutic agent melphalan and 2Ј-oligodeoxynucleotides. Low energy CAD product ion spectra gave information about the specificity of melphalan alkylation with regard to certain DNA sequences. These data can be very useful to estimate the risk in the development of secondary leukaemia as a result of a melphalan cure. In the study of the interaction between melphalan and d(GG), differentiation could be made between alkylation on the 5Ј-side and alkylation on the 3Ј-side, because of the presence or absence of the alkylated w 1 fragment in the low energy CAD spectra. In the other di-mers alkylation specificity for the different bases could be observed. Melphalan alkylation occurs in the sequence G Ͼ A Ͼ C Ͼ T. The study of the alkylated d(GGGG) revealed the presence of mainly 5Ј-end alkylation. Furthermore studies were performed which investigated other melphalan treated di-, tetra-, hepta-, and octa-mers. NA is a target for many xenobiotica and endogenous compounds. Electrophiles from either source can interact covalently with DNA with the formation of so called DNA adducts [1]. These adducts are believed to be the cause for an increased risk of cancer. One of these alkylating agents is the nitrogen mustard melphalan currently used as a chemotherapeutic agent in the treatment of malignant melanoma, multiple myeloma, lymphomas and ovarian carcinoma [2]. DNA adduct formation in perfectly healthy cells at the stage of primary cancer treatment might explain the development of a secondary leukaemia afterwards. It is a well known fact throughout literature that melphalan mainly alkylates the N-7 position in a guanine residue [3]. To the best of our knowledge, no information is available on the regioselectivity of melphalan alkylation in 2Ј-oligodeoxynucleotides and/or whole DNA strands. Therefore there were some important issues left as there are the degree of alkylation of G in relation to its position in the sequence, the influence of the neighbors on the degree of alkylation and the fragmentation patterns observed in melphalan modified 2Ј-oligodeoxynucleotides. Furthermore can we detect C, A and/or T adducts and what is the relation with earlier performed experiments on individual 2Ј-deoxynucleotides and -nucleosides [4,5].Electrospray mass spectrometry has become a viable technique for determining both the sequence specificity and the chemical structure of DNA modifications. By using this technology a lot of information was gathered on the adduct formation between potential carcinogens and individual 2Ј-deoxynucleotides (dNMPs) and -nucleosides (dNs). Furthermore modifications in whole DNA strands are studied after enzymatic digestion also resulting in the study of modified dNMPs and dNs [6]. However, this enzymatic digestion eliminates information related to the location of the adduct in the DNA sequence. In this paper we wish to describe a study of th...

supporting

confidence: 81%

mentioning

confidence: 77%

Structural characterization of melphalan modified 2′-oligodeoxynucleotides by miniaturized LC-ES MS/MS

Driessche

Lemière

Dongen

et al. 2004

“…Among different soft desorption-ionization techniques (e.g., MALDI, FAB, TSP), electrospray ionization is now commonly used for DNA adduct analysis, because of the very soft desolvation conditions which avoid fragile bond cleavages. Although negative ESI is widely used for the analysis of unmodified [26,27] as well as modified oligonucleotides [28 -30], the adducts formed between nucleosides and electrophilic reagents are generally analyzed using positive ionization [31][32][33], mainly for better sensitivity reasons. In previous works [34], the resulting products from the reactivity of estradiol-2,3-quinone towards desoxyribonucleosides have been investigated using LC-ESI-MS n .…”

mentioning

confidence: 99%

Investigation of the regio- and stereo-selectivity of deoxyguanosine linkage to deuterated 2-hydroxyestradiol by using liquid chromatography/ESI-ion trap mass spectrometry

Debrauwer

Rathahao

Jouanin

et al. 2003

From previous studies on the reactivity of estradiol 2,3-quinone towards deoxyribonucleosides, it was demonstrated that several isomeric adducts were formed. Although adduction on steroid ring A or B has been evidenced using sequential MS n experiments, in some cases attachment positions are difficult to identify unambiguously. In this work, 2-hydroxyestradiol labeled with deuterium at various positions [6␤ (1); 6␣-7␣ (2); 6␣-6␤-7␣ (3)] have been used. Isomeric adduct differentiation could be achieved using LC-ESI-MS n . The m/z shift of the quasi-molecular ions as well as the fragmentation pathways suggested that adduction could occur on both C6 and C9 sites of the steroid B ring: Nucleophilic attack of the base on the C6 position of the steroid led to major adducts and addition of the base on the activated C9 site gave minor adducts that were found to be unstable. LC-MS n experiments carried out under deuterated medium provided information about some fragmentation processes by studying the m/z shift of fragment ions: (1) the loss of deoxyribose from the quasi-molecular ions took place according to a process involving a deuterium transfer from the deoxyribose alcohol function; (2) the cleavage of the steroid-base linkage involved a deuterium transfer from the hydroxy group of the catechol and likely occurred via the formation of an ion-dipole complex. The model studies conducted in this work provide new information on the fragmentation mechanisms of covalent adducts formed from estrogen quinones and deoxyguanosine, the most reactive DNA base. Besides, the first unequivocal characterization of adducts involving the steroid C9 position is shown by using deuterium labeled estrogen quinones. (J Am Soc Mass Spectrom 2003, 14, 364 -372)

“…The typical SRM transition for the benzo[a]pyrene adduct was found in the literature [50]. The [MH] ϩ Ͼ [BH 2 ] ϩ transition is a well known fragmentation pattern for (2'-deoxy)nucleosides because of the preferred cleavage of the anomeric bond [26,27,33,43,44,51,52]. For a CV of 40 V and a CE of 15 eV the loss of the 2'-deoxyribofuranosyl group from the protonated molecule [MH] ϩ was the main fragmentation pattern for all our reference compounds.…”

Section: Mass Spectrometric Detectionmentioning

confidence: 93%

“…The coupling of a miniaturized 2-DLC system to tandem mass spectrometry is a powerful approach enabling the analysis of low quantities of DNA-adducts present in a matrix of mainly unmodified 2'-deoxynucleosides [33,[42][43][44][45][46][47][48][49].…”

Section: Lc-ms Analysis Of Standard Dna Adductsmentioning

confidence: 99%

Detection of estrogen DNA-adducts in human breast tumor tissue and healthy tissue by combined nano LC-nano ES tandem mass spectrometry

Embrechts

Lemière

Dongen

et al. 2003

For the first time estrogen DNA-adducts were identified in DNA human breast tumor tissue using nano-LC coupled to nano-Electrospray Tandem Mass Spectrometry. Normal breast tissue was analyzed analogously. The data obtained in the five breast tumor and five adjacent normal tissue samples were compared qualitatively, but no straightforward difference was observed. Prior to LC-MS analysis the DNA was enzymatically hydrolyzed to a nucleoside pool. The DNA-hydrolysates were directly injected onto a column switching system developed for on-line sample clean-up and subsequent analysis of the DNA-adducts. In four patients using Premarin, DNA-adducts of 4-hydroxy-equilenin (4OHEN) were detected. All except three samples contained DNA-adducts from 4-hydroxy-estradiol or 4-hydroxy-estrone. Also DNA isolated from eight alcohol fixed and paraffin embedded breast tumor tissue showed the presence of different estrogen DNA-adducts. Worthwhile mentioning is the presence of adducts responding to m/z 570 Ͼ m/z 454 transition. This is a well-known SRM-transition indicative for the presence of the 2'-deoxyguanosine (dGuo) adduct of Benzo H ormone treatment is widespread for women of all ages. In the United States, for instance, 30% of post-menopausal women use hormone eeplacement therapy (HRT) [1], e.g., Premarin. Studies in which the development of breast and endometrial cancer was associated with estrogen therapy [2][3][4][5][6] were supported by a more recent follow-up study in which it was demonstrated that post-menopausal women have an increased risk of breast cancer when using estrogens, especially in combination with progestin [7]. In animals, too, a relationship between the administration of estrogens and the development of cancer was shown [8].The carcinogenic properties of estrogens are explained by direct stimulation of cell proliferation via estrogen receptor mediated mechanisms [9,10] and by mechanisms based on metabolic activation [11][12][13][14], leading to DNA damage such as oxidative damage [15][16][17][18] and the formation of DNA-adducts [19 -28], which can cause mutations and induce cancer [29].The latter pathways are the result of metabolic activation of estrogens by the cytochrome P-450 system leading to 2-and 4-hydroxy derivatives. The 2-hydroxy estrogens are excreted in the urine as a result of their fast transformation to water-soluble compounds [13,14]. The 4-hydroxy form, however, has a longer half-life