n this article, we review recent findings related to estrogen exposure and the risk of breast cancer, the mechanisms that may be involved, and the clinical implications of these findings. The weight of evidence indicates that exposure to estrogen is an important determinant of the risk of breast cancer. The mechanisms of carcinogenesis in the breast caused by estrogen include the metabolism of estrogen to genotoxic, mutagenic metabolites and the stimulation of tissue growth. Together, these processes cause initiation, promotion, and progression of carcinogenesis. Insight into the mechanisms of the causation of cancer by estrogen will identify determinants of susceptibility to breast cancer and new targets for prevention and therapeutic intervention.

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Estrogen carcinogenesis in breast cancer.

Parabens are a group of the alkyl esters of p-hydroxybenzoic acid and typically include methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, isopropylparaben, and benzylparabe...

A Review of the Endocrine Activity of Parabens and Implications for Potential Risks to Human Health

Presently, phosphorylation of proteins is the most studied and best understood PTM. However, the analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research. Since not every phosphoprotein is accessible by a certain method and identification of the phosphorylated amino acid residue is required in the majority of cases, various strategies for the detection and localization of phosphorylations have been developed. Identification and localization of protein phosphorylations is mostly done by MS nowadays but phosphoproteins and -peptides are often suppressed in comparison to the unphosphorylated species if measured in complex mixtures. Thus, the isolation of pure phosphopeptide samples is a main task. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and -peptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/pmic.200401289

State-of-the-art in phosphoproteomics.

A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)(3) is presented here. When compared to commercial phosphoprotein-enrichment kits, this method is more selective, more cost effective and easily applicable to method optimization. The use of glutamic and aspartic acid in the loading buffer significantly enhances selectivity. Standard protein mixtures and complex Arabidopsis thaliana leaf protein extracts were tested for efficacy of enrichment. The method can be applied to proteins extracted using either mild or denaturing conditions. The same Al(OH)(3) material is suitable for the enrichment of phosphopeptides out of a tryptic digest of alpha-casein. Peptide phosphorylation was revealed by beta-elimination of phosphate groups. Enrichment and in vivo phosphorylation of A. thaliana leaf proteins were confirmed with Pro-Q diamond stain. Several of the phosphoprotein candidates that were identified by MS are known to be phosphorylated in vivo in other plant species.

Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC).

Quinones represent a class of toxicological intermediates, which can create a variety of hazardous effects in vivo including, acute cytotoxicity, immunotoxicity, and carcinogenesis. In contrast, quinones can induce cytoprotection through the induction of detoxification enzymes, anti-inflammatory activities, and modification of redox status. The mechanisms by which quinones cause these effects can be quite complex. The various biological targets of quinones depend on their rate and site of formation and their reactivity. Quinones are formed through a variety of mechanisms from simple oxidation of catechols/hydroquinones catalyzed by a variety of oxidative enzymes and metal ions to more complex mechanisms involving initial P450-catalyzed hydroxylation reactions followed by two-electron oxidation. Quinones are Michael acceptors, and modification of cellular processes could occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can red...

http://pubs.acs.org/doi/pdf/10.1021/acs.chemrestox.6b00256

Formation and Biological Targets of Quinones: Cytotoxic versus Cytoprotective Effects

Although alpha-cyano-4-hydroxycinnamic acid functions as an excellent matrix for the analysis of most peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry, the ionization of phosphorylated peptides is usually suppressed by nonphosphorylated peptides. As an alternative matrix, 2',4',6'-trihydroxyacetophenone (THAP) with diammonium citrate was found to overcome this problem for the MALDI TOF mass spectrometric analysis of proteolytic digests of phosphorylated proteins. Specifically, the abundances of phosphorylated peptides in tryptic digests of bovine beta-casein and protein kinase C (PKC)-treated mouse cardiac troponin I were enhanced more than 10-fold using THAP during positive ion MALDI TOF mass spectrometry. The protonated molecules of phosphorylated peptides were sufficiently abundant that postsource decay TOF mass spectrometry was used to confirm the number of phosphate groups in each peptide. Finally, tryptic digestion followed by analysis using MALDI TOF mass spectrometry with THAP as the matrix facilitated the identification of a unique phosphorylation site in PKC-treated troponin I.

Enhanced Ionization of Phosphorylated Peptides during MALDI TOF Mass Spectrometry

A non-equilibrium isoelectric focusing method to determine states of phosphorylation of cardiac troponin I: Identification of Ser-23 and Ser-24 as significant sites of phosphorylation by protein kinase C

Numerous xenobiotics, including therapeutics agents, are substrates for bioactivation to electrophilic reactive intermediates that may covalently modify biomolecules. Selective estrogen receptor modulators (SERMs) are in clinical use for long-term therapy of postmenopausal syndromes and chemoprevention and provide a potential alternative for hormone replacement therapy (HRT). Raloxifene, in common with many SERMs and other xenobiotics, is a polyaromatic phenol that has been shown to be metabolically bioactivated to electrophilic and redox active quinoids. Nucleic acid and glutathione adduct formation have been reported, but little is known about protein covalent modification. A novel COATag (covert oxidatively activated tag) was synthesized in which raloxifene was linked to biotin. The COATag was reactive toward a model protein, human glutathione-S-transferase P1-1, in the presence but not the absence of monooxygenase. The covalent modification of proteins in rat liver microsomal incubations was NADPH-dependent implicating cytochrome P450 oxidase. The COATag facilitated isolation and identification of covalently modified microsomal proteins: cytosolic glucose regulated protein (GRP78/BiP), three protein disulfide isomerases, and microsomal glutathione S-transferase 1. Oxidative metabolism of raloxifene produces reactive intermediates of sufficient lifetimes to covalently modify proteins in tissue microsomes, behavior anticipated for other polyaromatic phenol xenobiotics that can be tested by the COATag methodology. The combined use of a COATag with a simple biotin-linked electrophile (such as an iodoacetamide tag) is a new technique that allows quantification of protein covalent modification via alkylation vs oxidation in response to xenobiotic reactive intermediates. The identification of modified proteins is important for defining pathways that might lead alternatively to either cytotoxicity or cytoprotection.

Analysis of protein covalent modification by xenobiotics using a covert oxidatively activated tag: raloxifene proof-of-principle study.

Numerous xenobiotics, including therapeutics agents, are substrates for bioactivation to electrophilic reactive intermediates that may covalently modify biomolecules. Selective estrogen receptor modulators (SERMs) are in clinical use for long-term therapy of postmenopausal syndromes and chemoprevention, and provide a potential alternative for hormone replacement therapy (HRT). Raloxifene, in common with many SERMs and other xenobiotics, is a polyaromatic phenol that has been shown to be metabolically bioactivated to electrophilic and redox active quinoids. Nucleic acid and glutathione adduct formation has been reported, but little is known about protein covalent modification. A novel COATag (covert oxidatively activated tag) was synthesized in which raloxifene was linked to biotin. The COATag was reactive towards a model protein, human glutathione-S-transferase P1-1, in the presence but not absence of monooxygenase. The covalent modification of proteins in rat liver microsomal incubations was NADPH-dependent implicating cytochrome P450 oxidase. The COATag facilitated isolation and identification of covalently modified microsomal proteins: cytosolic glucose regulated protein (GRP78/BiP); three protein disulfide isomerases; and microsomal glutathione S-transferase 1. Oxidative metabolism of raloxifene produces reactive intermediates of sufficient lifetime to covalently modify proteins in tissue microsomes, behaviour anticipated for other polyaromatic phenol xenobiotics that can be tested by the COATag methodology. The combined use of a COATag with a simple biotin-linked electrophile (such as an iodoacetamide tag) is a new technique that allows quantification of protein covalent modification via alkylation versus oxidation in response to xenobiotic reactive intermediates. The identification of modified proteins is important for defining pathways that might lead alternatively to either cytotoxicity or to cytoprotection.

Analysis of Protein Covalent Modification by Xenobiotics using a Covert Oxidatively Activated Tag

Redox and/or electrophilic metabolites formed during estrogen metabolism may play a role in estrogen carcinogenesis. 4-Hydroxyequilenin (4-OHEN) is the major phase I catechol metabolite of the equine estrogens equilenin and equilin, which are components of the most widely prescribed estrogen replacement formulation, Premarin. Previously, we have found that 4-OHEN rapidly autoxidized to an o-quinone in vitro and caused toxic effects such as the inactivation of human detoxification enzymes. 4-OHEN has also been shown to be a substrate for catechol-O-methyltransferase (COMT) in human breast cancer cells. In the present study, we demonstrated that 4-OHEN was not only a substrate of recombinant human soluble COMT in vitro with a K(m) of 2.4 microM and k(cat) of 6.0 min(-)(1) but it also inhibited its own methylation by COMT at higher concentrations in the presence of the reducing agent dithiothreitol. In addition, 4-OHEN was found to be an irreversible inhibitor of COMT-catalyzed methylation of the endogenous catechol estrogen 4-hydroxyestradiol with a K(i) of 26.0 microM and a k(2) of 1.62 x 10(-)(2) s(-)(1). 4-OHEN in vitro not only caused the formation of intermolecular disulfide bonds as demonstrated by gel electrophoresis, but electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry also showed that 4-OHEN alkylated multiple residues of COMT. Peptide mapping experiments further indicated that Cys33 in recombinant human soluble COMT was the residue most likely modified by 4-OHEN in vitro. These data suggest that inhibition of COMT methylation by 4-OHEN might reduce endogenous catechol estrogen clearance in vivo and further enhance toxicity.

Xiaofeng Yang

Papers

Enhanced Ionization of Phosphorylated Peptides during MALDI TOF Mass Spectrometry

A non-equilibrium isoelectric focusing method to determine states of phosphorylation of cardiac troponin I: Identification of Ser-23 and Ser-24 as significant sites of phosphorylation by protein kinase C

Analysis of protein covalent modification by xenobiotics using a covert oxidatively activated tag: raloxifene proof-of-principle study.

Analysis of Protein Covalent Modification by Xenobiotics using a Covert Oxidatively Activated Tag

Catechol estrogen 4-hydroxyequilenin is a substrate and an inhibitor of catechol-O-methyltransferase