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Divergent activities of human glutathione transferases in the bioactivation of azathioprine
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2006 (English)In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 70, no 2, 747-754 p.Article in journal (Refereed) Published
Abstract [en]

Azathioprine is a thiopurine prodrug clinically used for immunosuppression in the treatment of inflammatory diseases and in pharmacological regimens of organ transplantations. Its pharmacological action is based on the release of 6-mercaptopurine, but the biochemical processes underlying this biotransformation have remained obscure. In this investigation, human glutathione transferases (GSTs) from seven distinct classes were assayed with azathioprine. GSTs A1-1, A2-2, and M1-1, all abundantly expressed in human liver, displayed the highest activity among the 14 GSTs tested. The uncatalyzed reaction of azathioprine with glutathione was estimated to be less than 1% of the GST-catalyzed biotransformation. GST M1-1 is polymorphic with a frequently occurring null allele, and GSTs A1-1 and A2-2 show variable expression levels in human subjects, implying significant differences in the rate of 6-mercaptopurine release from azathioprine. Individuals expressing high GST activity are apparently predisposed for adverse reactions to azathioprine treatment, both by promoting excessively high concentrations of free 6-mercaptopurine and its toxic metabolites and by depleting cellular glutathione. These novel aspects of GST-dependent azathioprine biotransformation have not been considered previously.

Place, publisher, year, edition, pages
2006. Vol. 70, no 2, 747-754 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94670DOI: 10.1124/mol.106.025288PubMedID: 16717136OAI: oai:DiVA.org:uu-94670DiVA: diva2:168611
Available from: 2006-08-30 Created: 2006-08-30 Last updated: 2011-07-18Bibliographically approved
In thesis
1. Liquid Chromatography Coupled to Mass Spectrometry: Implementation of Chemometric Optimization and Selected Applications
Open this publication in new window or tab >>Liquid Chromatography Coupled to Mass Spectrometry: Implementation of Chemometric Optimization and Selected Applications
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Liquid chromatography (LC) coupled to mass spectrometry (MS) offers highly selective and sensitive analysis of a wide variety of compounds. However, the use of hyphenated experimental set-ups implies that many parameters may have an effect on the studied response. Therefore, in order to determine optimized experimental conditions it is of vital importance to incorporate systematic procedures during method development. In this thesis, a generic stepwise optimization strategy is proposed that aims at high chromatographic quality, as well as high mass spectrometric response. The procedure comprises (i) screening experiments to identify the most important parameters, (ii) LC studies to ensure sufficient chromatographic separation, (iii) extended infusion experiments in order to maximize precursor signal(s), and in the case of tandem MS (iv) extended infusion experiments to determine optimal conditions for collision induced dissociation and when applicable also ion trap settings. Experimental design and response surface methodology is used throughout the procedure.

Further, the general applicability of LC-MS is demonstrated in this thesis. Specifically, a novel quantitative column-switched LC-MS method for ferrichrome, ferrichrysin and ferricrocin determination is presented. Using the method it was shown how the siderophore content varies with depth in podzolic soil profiles in the north and south of Sweden. The parallel approach using LC coupled to both inductively coupled plasma (ICP) mass spectrometry, and electrospray ionization (ESI) tandem MS is also evaluated as a tool to identify unknown siderophores in a sample. Additionally, different trypsin digestion schemes used for LC-ESI-MS peptide mapping were compared. By multivariate data analysis, it was clearly shown that the procedures tested induce differences that are detectable using LC-ESI-MS. Finally, the glutathione S-transferase catalyzed bioactivation of the prodrug azathioprine was verified using LC-MS.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 201
Keyword
Analytical chemistry, liquid chromatography, mass spectrometry, tandem mass spectrometry, electrospray ionization, inductively coupled plasma, chemometrics, optimization, multivariate data analysis, design of experiments, response surface model, siderophore, azathioprine, peptide, Analytisk kemi
Identifiers
urn:nbn:se:uu:diva-7071 (URN)91-554-6612-5 (ISBN)
Public defence
2006-09-28, Room B:21, BMC, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2006-08-30 Created: 2006-08-30 Last updated: 2009-03-24Bibliographically approved
2. Role of Multiple Glutathione Transferases in Bioactivation of Thiopurine Prodrugs: Studies of Human Soluble Glutathione Transferases from Alpha, Kappa, Mu, Omega, Pi, Theta, and Zeta Classes
Open this publication in new window or tab >>Role of Multiple Glutathione Transferases in Bioactivation of Thiopurine Prodrugs: Studies of Human Soluble Glutathione Transferases from Alpha, Kappa, Mu, Omega, Pi, Theta, and Zeta Classes
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in E. coli. The method is based on spraying monochlorobimane (MCB) directly over bacterial colonies growing on agar. The substrate MCB become fluorescent under UV light, when the bacterial colony contains active GSTs catalyzing the conjugation with endogenous glutathione. Eleven out of twelve GSTs investigated where active with MCB. This method can be used to screen libraries generated from most cytosolic GSTs in the search for proteins with altered functions and structures. Azathioprine (Aza), a thiopurine that has been used clinically for 40 years was investigated with 14 GSTs. Three enzymes showed prominent catalytic activities with Aza and all of them are highly expressed in the liver. We estimated the contribution of the three enzymes GSTs A1-1, A2-2 and M1-1 bioactivation of Aza in the liver and concluded that it was about 2 orders of magnitude more effective than the uncatalyzed reaction. GST bioactivation of Aza could clarify aspects of idiosyncratic reactions observed in some individuals. Two other thiopurine prodrugs, cis-acetylvinylthiopurine (cAVTP) and trans-acetylvinylthioguanine (tAVTG), were investigated with the same 14 GSTs. The results displayed diverse catalytic activities. A mechanism of consecutive reactions was proposed. The studies contribute to knowledge under what conditions the drug should optimally be administered. A study of the same prodrugs with several mutants from the Mu class characterized by a point mutation of a hypervarible residue. We conclude that the effects of the mutations were qualitatively parallel for cAVTP and tAVTG, but they vary significantly in magnitude; steric hindrance may interfere with transition-state stabilization. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 208
Keyword
Biochemistry, Glutathione Transferases, Thiopurines, Prodrug, Bioactivation, Screening Method, Chemotherapy, Functional plasticity, Modulated activity, Biokemi
Identifiers
urn:nbn:se:uu:diva-7102 (URN)91-554-6630-3 (ISBN)
Public defence
2006-09-22, B21, BMC, Husarg 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2006-08-31 Created: 2006-08-31 Last updated: 2011-07-18Bibliographically approved

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