title: Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures.
abstract: The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported. Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200 μM concentration range). Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 μM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress.
authors.affiliation: Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 2, I-53100 Siena, Italy. Electronic address: giustarini@unisi.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Giustarini
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, I-53100 Siena, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Galvagni
authors.affiliation: Bioscience Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, via Piero Maroncelli 40, Meldola 47014, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Tesei
authors.affiliation: Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, via Piero Maroncelli 40, Meldola 47014, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Farolfi
authors.affiliation: Bioscience Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, via Piero Maroncelli 40, Meldola 47014, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Zanoni
authors.affiliation: Bioscience Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, via Piero Maroncelli 40, Meldola 47014, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Pignatta
authors.affiliation: Department of Biosciences, Università degli Studi di Milano, via Celoria 26, I-20133 Milan, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Milzani
authors.affiliation: Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.
authors.forename: Ilaria M
authors.initials: IM
authors.lastname: Marone
authors.affiliation: Department of Biosciences, Università degli Studi di Milano, via Celoria 26, I-20133 Milan, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Dalle-Donne
authors.affiliation: Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.
authors.forename: Romina
authors.initials: R
authors.lastname: Nassini
authors.affiliation: Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 2, I-53100 Siena, Italy.
authors.forename: Ranieri
authors.initials: R
authors.lastname: Rossi
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism*; D006801:Humans; D009369:Neoplasms / Q000473:pathology*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011506:Proteins; D005978:Glutathione; D005033:Ethylmaleimide; D019803:Glutathione Disulfide
keywords: Cell cultures; Glutathione; Glutathione disulfide; S-glutathionylated proteins