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

ABSTRACT: 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.

SUBMITTER: Liu J

PROVIDER: S-EPMC2517578 | biostudies-other | 2005 Sep

REPOSITORIES: biostudies-other

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

Liu Ju J Li Qian Q Yang Xiaofeng X van Breemen Richard B RB Bolton Judy L JL Thatcher Gregory R J GR

Chemical research in toxicology 20050901 9

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 metabolic ...[more]

PMID: 16167842

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Project description:In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methylcytosine to detect the presence of 5 methylcytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used. A method that detects DNA methylation that is not restricted to specific sequences would provide a useful platform to analyse methylation distribution among the genes in any given phenotypic condition.Availability of additional approaches to examine DNA methylation would lead to increase in our understanding of the methylome and would help define the intrinsic and extrinsic factors which govern it. Microarray based high throughput methods have been extensively used for gene expression analysis. The method described uses a microarray to analyse DNA methylation levels in a gene/cDNA/oligo specific manner. Here, genomic DNA is fragmented and used for hybridization to microarray slides. The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC( 5-methyl cytidine) antibody (monoclonal). The resolution provided by this method is dependent upon the microarray platform used. This method can be adapted to any platform thus enabling seamless integration with existing technology of gene expression analysis

Dataset Information

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

Publications

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

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