Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells

Ayene, Iraimoudi S.; Koch, Cameron J.; Tuttle, Steven W.; Stamato, Thomas D.; Perez, Mary Louise; Biaglow, John E.

doi:10.1080/09553000050176289

Cited by 19 publications

(20 citation statements)

References 40 publications

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“…Based on the DTNB reaction of the metabolite of HEDS and our previous high pressure liquid chromatography and electrochemical detector (HPLC/EC) data that identified ME as the only DTNB reactive thiols produced by HEDS bioreduction (Ayene et al, 2000), our current findings demonstrated that ME is the main product produced from HEDS in these human cancer cells (HCT116, HT29, MCF7, MCF10A, SKBR3) and CHO cells. Our results demonstrated that the metabolic conversion of HEDS into DTNB reactive product correlates with the cell number measured by Coulter counter.…”

Section: Discussionsupporting

confidence: 61%

“…In the absence of HEDS, the extracellular medium did not have any detectable amount of mercaptoethanol. These studies also demonstrated that the conversion of HEDS into ME is dependent on the activity of the oxidative pentose phosphate cycle (OPPC), suggesting that this assay could be used for all mammalian cells (Ayene et al, 2008; Ayene et al, 2000; Ayene et al, 2002; Biaglow et al, 2003; Biaglow et al, 2000; Biaglow et al, 2006). There are several biochemical pathways involved in the reduction of HEDS in live mammalian cells, presently below.…”

Section: Discussionmentioning

confidence: 94%

“…We had previously demonstrated using a state of the art HPLC electrochemical detector that live rodent cells convert HEDS into ME in vitro (Ayene et al, 2000). Most importantly, we had demonstrated that the HPLC peak for ME was observed in the extracellular medium only after incubation of rodent and human cells with HEDS (Ayene et al, 2000). In the absence of HEDS, the extracellular medium did not have any detectable amount of mercaptoethanol.…”

Section: Discussionmentioning

confidence: 99%

“…Although we have demonstrated HEDS to ME bioconversion in cell culture using HPLC/EC detection and DTNB assays, our studies did not determine whether HEDS could be used in an assay to quantify cell density or cellular responses to radiation, chemotherapeutic agents and toxins (Ayene et al, 2008; Ayene et al, 2000; Ayene et al, 2002; Biaglow et al, 2003; Biaglow et al, 2000; Biaglow et al, 2006). In this report, we define a HEDS assay that can accurately measure metabolically active live cells and quantify cell death after a cytotoxic insult.…”

Section: Introductionmentioning

confidence: 85%

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Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro

Zhang

Ward

et al. 2012

Toxicology in Vitro

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Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (Phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells.

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Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 85%

See 2 more Smart Citations

Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro

Zhang

Ward

et al. 2012

Toxicology in Vitro

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“…Since the discovery of the importance of glutathione (GSH) and glutathione-dependent pathways in various diseases, xenobiotic detoxification and oxidative stress, several biochemical and high pressure liquid chromatography (HPLC) methods have been used to measure intracellular glutathione (Allen & Bradley, 2011; Ayene, et al, 2000; Jani, Ziogas, Angus, & Wright, 2012; King, Korolchuk, McGivan, & Suleiman, 2004; Melnyk, Pogribna, Pogribny, Hine, & James, 1999; Murias, Rachtan, & Jodynis-Liebert, 2005; Pocernich & Butterfield, 2012; Rao, et al, 1997; Ricketts, Minimair, Yates, & Klaus, 2011; Sekhar, et al, 2011; Wilkins, Kirchhof, Manning, Joseph, & Linseman, 2013). However, the presently available biochemical assays for GSH require preparation of extracts from cells (Ghanizadeh, et al, 2013; Gibson, Korade, & Shelton, 2012; Jenko, Karouna-Renier, & Hoffman, 2012; Lushchak, 2012; Potter, Trappetti, & Paton, 2012; Shungu, 2012; Wright, et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

A bioactive probe for glutathione-dependent antioxidant capacity in breast cancer patients: Implications in measuring biological effects of arsenic compounds

Zhang

Jefferson

et al. 2014

Journal of Pharmacological and Toxicological Methods

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Introduction Glutathione, a major cellular non-protein thiol (NPSH), serves a central role in repairing damage induced by cancer drugs, pollutants and radiation and in the detoxification of several cancer chemotherapeutic drugs and toxins. Current methods measure glutathione levels only, which require cellular extraction, rather than the glutathione recycling dependent antioxidant activity in intact cells. Here, we present a novel method using a bioactive probe of the oxidative pentose phosphate cycle, termed the OxPhos™ test, to quantify glutathione recycling dependent antioxidant activity in whole blood and intact human and rodent cells without the need for the isolation and cytoplasm extraction of cells. Methods OxPhos™ test kit (Rockland Immunochemicals, USA), which uses hydroxyethyldisulfide (HEDS) as a probe for the oxidative pentose phosphate cycle, was used in these studies. The results with OxPhos™ test kit in human blood and intact cells were compared with total thiol and high pressure liquid chromatography/electrochemical detection of HEDS metabolism. Results The OxPhos™ test measured glutathione-dependent antioxidant activity both in intact human and rodent cells and breast cancer patient’s blood with a better correlation coefficient and biological variability than the thiol assay. Additionally, human blood and mammalian cells treated with various arsenicals showed a concentration-dependent decrease in activity. Discussion The results demonstrate the application of this test for measuring the antioxidant capacity of blood and the effects of environmental pollutants/toxins. It opens up new avenues for an easy and reliable assessment of glutathione-dependent antioxidant capacity in various diseases such as stroke, blood borne diseases, infection, cardiovascular disease and other oxidative stress related diseases and as a prognostic indicator of chemotherapy response and toxicity. The use of this approach in pharmacology/toxicology including screening drugs that improve the glutathione-dependent antioxidant capacity and not just the glutathione level is clinically relevant since mammalian cells require glutathione dependent pathways for antioxidant activity.

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Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: Mechanism and implication

Ayene

Biaglow

Kachur

et al. 2007

J of Cellular Biochemistry

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More than 400 million people are susceptible to oxidative stress due to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Protein glutathionylation is believed to be responsible for loss of protein function and/or cellular signaling during oxidative stress. To elucidate the implications of G6PD deficiency specifically in cellular control of protein glutathionylation, we used hydroxyethyldisulfide (HEDS), an oxidant which undergoes disulfide exchange with existing thiols. G6PD deficient (E89) cells treated with HEDS showed a significant increase in protein glutathionylation compared to wild-type (K1) cells. In order to determine whether increase in global protein glutathionylation by HEDS leads to loss of function of an important protein, we compared the effect of HEDS on global protein glutathionylation with that of Ku protein function, a multifunctional DNA repair protein, using a novel ELISA. E89 cells treated with HEDS showed a significant loss of Ku protein binding to DNA. Cellular protein thiol and GSH, whose disulfide is involved in protein glutathionylation, were decreased by HEDS in E89 cells with no significant effect in K1 cells. E89 cells showed lower detoxification of HEDS, that is, conversion of disulfide HEDS to free sulfhydryl mercaptoethanol (ME), compared to K1 cells. K1 cells maintained their NADH level in the presence of HEDS but that of E89 cells decreased by tenfold following a similar exposure. NADPH, a cofactor required to maintain reduced form of the thiols, was decreased more in E89 than K1 cells. The specific role of G6PD in the control of such global protein glutathionylation and Ku function was further demonstrated by reintroducing the G6PD gene into E89 (A1A) cells, which showed a normal phenotype.

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Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells

Abstract: These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.

Cited by 19 publications

References 40 publications

Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro

Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro

A bioactive probe for glutathione-dependent antioxidant capacity in breast cancer patients: Implications in measuring biological effects of arsenic compounds

Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: Mechanism and implication

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