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

Abstract

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

Dual roles of estrogen metabolism in mammary carcinogenesis

“…Finally, oxidative DNA damage was detected in peripheral leukocytes of postmenopausal women receiving HRT containing conjugated equine estrogens (93). In addition, cyclic stable dC, dG, and dA adducts of 4-OHEN were detected for the first time in part of samples of women with a known history of Premarin-based HRT (88). Detection of stable adducts through reaction with the o-quinones of both endogenous and equine estrogens in human samples raises a question whether the depurinating adducts play a more causative role in estrogen-induced genetic mutation compared to stable ones; however, these data strongly implied that ultimate mutation potential might be altered by DNA repair activities and its kinetics.…”

“…Considering the fact that the redox potentials of 2-OHE1/E2 and 4-OHE1/E2 are similar, the greater carcinogenicity of the 4-OHE1/E2 would come from the experimental results in which 4-OHE1/E2 forms the carcinogenic depurinating DNA adducts than 2-OHE1/E2 at higher levels and 2-OHE1/E2 forms stable adducts. It is important to acknowledge that stable bulky Gua adducts of 4-OHE1/E2 have been detected in human breast tumor tissue (88). Reactive intermediates generated from the redox cycling between catechol equine estrogens and their quinones were shown to induce variety of DNA lesions in vitro, in vivo, and even in human.…”

Induction of Oxidative Stress Responses by Dioxin and other Ligands of the Aryl Hydrocarbon Receptor

“…Rearrangement of estrone 3,4-quinone produces a strongly electrophilic carbonium cation that may undergo a Michael addition reaction with cellular sulfhydrals such as (i.e. glutathione, protein thiols) or by electrophilic addition to DNA purine bases resulting in depurinating adducts (Cavalieri et al, 2000), and ultimately procarcinogenic mutations ( Liehr, 2001;Embrechts et al, 2003). The relative contributions of redox cycling and electrophilic interactions in the oxidative stress response and toxicity have not been firmly established; however, limited evidence suggests that covalent sulfhydral modification by electrophiles is likely to be a greater cytotoxic hazard than transient quinone formation that facilitates disposal from the cell (Buffinton et al, 1989).…”

Analysis of DNA-phosphate adducts in vitro using miniaturized LC-ESI-MS/MS and column switching: Phosphotriesters and alkyl cobalamins

“…The program used was the following: Samples were injected on the pre-column using the auxiliary system C at [15][16][17][18][19][20] L/min. At the same time the binary system A/B pumps 20% Solvent B at a flow of 2-8 L/min over the analytical column.…”

“…During the last decade there has been a large increase in the use of LC coupled to mass-spectrometric detection for direct analysis of DNA-base adducts [14,15]. In addition, applying several new techniques such as miniaturization of LC and column switching enables analysis of low levels of DNA adducts in complex biological matrices [16]. However, there has not been much focus on phosphate adducts in DNA.…”