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Metabolomic profiling unravels DNA adducts in human breast that are formed from peroxidase mediated activation of estrogens to quinone methides.

Gaikwad NW - PLoS ONE (2013)

Bottom Line: Surprisingly quinone methides were found to be stable with t1/2 of 20.8 and 4.5 min respectively.Incubation of estrogens with lactoperoxidase (LPO) and H2O2 resulted in formation of respective quinone methides (E1(E2)-QM).The error prone repair of the damaged DNA can result in mutation of critical genes and subsequently cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition and Department of Environmental Toxicology, University of California Davis, Davis, California, United States of America.

ABSTRACT
Currently there are three major hypotheses that have been proposed for estrogen induced carcinogenicity, however exact etiology remains unknown. Based on the chemical logic, studies were undertaken to investigate if estrogens could generate quinone methides in an oxidative environment which then could cause DNA damage in humans. In presence of MnO2 estrogens were oxidized to quinone methides. Surprisingly quinone methides were found to be stable with t1/2 of 20.8 and 4.5 min respectively. Incubation of estrogens with lactoperoxidase (LPO) and H2O2 resulted in formation of respective quinone methides (E1(E2)-QM). Subsequent addition of adenine to the assay mixture lead to trapping of E1(E2)-QM, resulting in formation of adenine adducts of estrogens, E1(E2)-9-N-Ade. Targeted ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based metabolomic analysis of the breast tissue extracts showed the presence of adenine adducts of estrogens, E1(E2)-9-N-Ade, along with other estrogen related metabolites. Identity of E1(E2)-N-Ade in LPO assay extracts and breast tissue extracts were confirmed by comparing them to pure synthesized E1(E2)-9-N-Ade standards. From these results, it is evident that peroxidase enzymes or peroxidase-like activity in human breast tissue could oxidize estrogens to electrophilic and stable quinone methides in a single step that covalently bind to DNA to form adducts. The error prone repair of the damaged DNA can result in mutation of critical genes and subsequently cancer. This article reports evidence for hitherto unknown estrogen metabolic pathway in human breast, catalyzed by peroxidase, which could initiate cancer.

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Related in: MedlinePlus

Breast tissue samples were homogenized and extracted as described in Materials and Methods.UPLC–MS/MS chromatogram of standard E1(E2)-9-N-Ade (A, C) and breast tissue extract (B, D).
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pone-0065826-g006: Breast tissue samples were homogenized and extracted as described in Materials and Methods.UPLC–MS/MS chromatogram of standard E1(E2)-9-N-Ade (A, C) and breast tissue extract (B, D).

Mentions: Finally, targeted UPLC-MS/MS based metabolomic analysis was performed on human breast tissues to investigate total estrogen anabolic/catabolic pathways using total 42 steroids and estrogen related reference standards. The average levels of steroids and estrogen related compounds measured from ten breast tissue samples are presented in table 1. Since estrone and estradiol are constantly inter-converting, we have combined estrone and estradiol values of all the derivatives (Table 1). The GSH conjugates of estrogen quinones are further converted to cysteine and N-acetyl-cysteine via the mercapturic acid biosynthesis pathway [25]. Hence we have combined all the values of 2-catechol conjugates and 4-catechol conjugates (Table 1) [21], [25]. From cursory examination of the metabolic profile, presence and identification of E1(E2)-9-N-Ade in the breast tissue extracts appears to be most significant (Fig. 6). Synthetic E1-9-N-Ade and E2-9-N-Ade as well as the breast tissue extracts showed a peak (404.3>268.96) at Rt of ∼6.4 min (Fig. 6 C&D) and a peak (406.3>271.02) at Rt of ∼6.0 min (Fig. 6 A&B), unequivocally confirming the presence of E1-9-N-Ade (#41) and E2-9-N-Ade (#42). The formation of E1(E2)-9-N-Ade could be possible only through metabolism of estrogens to quinone methide followed by its interaction with adenine from DNA.


Metabolomic profiling unravels DNA adducts in human breast that are formed from peroxidase mediated activation of estrogens to quinone methides.

Gaikwad NW - PLoS ONE (2013)

Breast tissue samples were homogenized and extracted as described in Materials and Methods.UPLC–MS/MS chromatogram of standard E1(E2)-9-N-Ade (A, C) and breast tissue extract (B, D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675060&req=5

pone-0065826-g006: Breast tissue samples were homogenized and extracted as described in Materials and Methods.UPLC–MS/MS chromatogram of standard E1(E2)-9-N-Ade (A, C) and breast tissue extract (B, D).
Mentions: Finally, targeted UPLC-MS/MS based metabolomic analysis was performed on human breast tissues to investigate total estrogen anabolic/catabolic pathways using total 42 steroids and estrogen related reference standards. The average levels of steroids and estrogen related compounds measured from ten breast tissue samples are presented in table 1. Since estrone and estradiol are constantly inter-converting, we have combined estrone and estradiol values of all the derivatives (Table 1). The GSH conjugates of estrogen quinones are further converted to cysteine and N-acetyl-cysteine via the mercapturic acid biosynthesis pathway [25]. Hence we have combined all the values of 2-catechol conjugates and 4-catechol conjugates (Table 1) [21], [25]. From cursory examination of the metabolic profile, presence and identification of E1(E2)-9-N-Ade in the breast tissue extracts appears to be most significant (Fig. 6). Synthetic E1-9-N-Ade and E2-9-N-Ade as well as the breast tissue extracts showed a peak (404.3>268.96) at Rt of ∼6.4 min (Fig. 6 C&D) and a peak (406.3>271.02) at Rt of ∼6.0 min (Fig. 6 A&B), unequivocally confirming the presence of E1-9-N-Ade (#41) and E2-9-N-Ade (#42). The formation of E1(E2)-9-N-Ade could be possible only through metabolism of estrogens to quinone methide followed by its interaction with adenine from DNA.

Bottom Line: Surprisingly quinone methides were found to be stable with t1/2 of 20.8 and 4.5 min respectively.Incubation of estrogens with lactoperoxidase (LPO) and H2O2 resulted in formation of respective quinone methides (E1(E2)-QM).The error prone repair of the damaged DNA can result in mutation of critical genes and subsequently cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition and Department of Environmental Toxicology, University of California Davis, Davis, California, United States of America.

ABSTRACT
Currently there are three major hypotheses that have been proposed for estrogen induced carcinogenicity, however exact etiology remains unknown. Based on the chemical logic, studies were undertaken to investigate if estrogens could generate quinone methides in an oxidative environment which then could cause DNA damage in humans. In presence of MnO2 estrogens were oxidized to quinone methides. Surprisingly quinone methides were found to be stable with t1/2 of 20.8 and 4.5 min respectively. Incubation of estrogens with lactoperoxidase (LPO) and H2O2 resulted in formation of respective quinone methides (E1(E2)-QM). Subsequent addition of adenine to the assay mixture lead to trapping of E1(E2)-QM, resulting in formation of adenine adducts of estrogens, E1(E2)-9-N-Ade. Targeted ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based metabolomic analysis of the breast tissue extracts showed the presence of adenine adducts of estrogens, E1(E2)-9-N-Ade, along with other estrogen related metabolites. Identity of E1(E2)-N-Ade in LPO assay extracts and breast tissue extracts were confirmed by comparing them to pure synthesized E1(E2)-9-N-Ade standards. From these results, it is evident that peroxidase enzymes or peroxidase-like activity in human breast tissue could oxidize estrogens to electrophilic and stable quinone methides in a single step that covalently bind to DNA to form adducts. The error prone repair of the damaged DNA can result in mutation of critical genes and subsequently cancer. This article reports evidence for hitherto unknown estrogen metabolic pathway in human breast, catalyzed by peroxidase, which could initiate cancer.

Show MeSH
Related in: MedlinePlus