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Patent Application Titled "Methods and Kits for Measuring Toxicity and Oxidative Stress in Live Cells" Published Online

August 19, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventor Ayene, Iraimoudi S. (Newtown Square, PA), filed on March 27, 2014, was made available online on August 7, 2014 (see also Lankenau Institute For Medical Research).

The assignee for this patent application is Lankenau Institute For Medical Research.

Reporters obtained the following quote from the background information supplied by the inventors: "Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

"Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the oxidative pentose phosphate cycle (OPPC). Glucose, a substrate for the OPPC, is required for OPPC mediated detoxification of oxidants/disulfides. Glucose is utilized as a substrate by oxidative pentose phosphate cycle to generate reductants. These reductants are utilized to maintain reduced glutathione homeostasis in mammalian cells when exposed to oxidants/disulfides. Glutathione is a tripeptide consisting of glycine, cysteine and glutamate. The reduced glutathione (GSH) is up to 100 folds higher than the oxidized GSH (GSSG) in mammalian cells under normal conditions.

"Oxidative stress is presently quantified by measuring the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). Oxidized glutathione is the most commonly used biomarker in biomedical research. However, the various biochemical assays currently available require the preparation of tissue extracts and cannot be applied to humans because of their invasiveness. Indeed, while HPLC with electrochemical detection can monitor glutathione with better sensitivity than other biochemical assays, the method still requires tissue or cellular extracts.

"Furthermore, these assays may overestimate the extent of oxidative stress since depletion of GSH measured by biochemical assays may also include oxidation of GSH during lysis of cells and extract preparation. Moreover, none of these assays measures the function of GSH in live cells. In view of the foregoing, it is evident that there is a need for improved oxidative stress assays in live cells."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventor's summary information for this patent application: "In accordance with one aspect of the instant invention, methods of measuring oxidative stress in a live cell are provided. In a particular embodiment, the methods comprise contacting cells with hydroxyethyldisulfide (HEDS) and determining the amount of extracellular mercaptoethanol, wherein the amount of extracellular mercaptoethanol is inversely proportional to the oxidative stress of the cell. In another embodiment, the amount of extracellular mercaptoethanol is directly proportional to the glutathione recycling capacity of the cell.

"According to another aspect of the instant invention, methods of screening for an antioxidant are provided. In one embodiment, the methods comprise a) contacting a first cell with hydroxyethyldisulfide (HEDS); b) determining the amount of extracellular mercaptoethanol produced by the first cell; c) contacting a second cell with a compound; d) contacting the second cell with hydroxyethyldisulfide (HEDS); and e) determining the amount of extracellular mercaptoethanol produced by the second cell; wherein the presence of more extracellular mercaptoethanol from the second cell indicates that the compound is an antioxidant. In still another embodiment, the method comprises a) contacting a cell with a compound; b) contacting the cell with hydroxyethyldisulfide (HEDS); and c) determining the amount of extracellular mercaptoethanol produced by the cell, wherein the presence of more extracellular mercaptoethanol from the cell compared to a standard indicates that the compound is an antioxidant. In another embodiment, the first and second cells are from biological samples and the compound is administered to a subject.

"In yet another aspect, methods of measuring the cytotoxicity of a compound are provided. The methods may comprise contacting cells with the compound, contacting the cells with hydroxyethyldisulfide (HEDS), and determining the amount of extracellular mercaptoethanol for the cells; wherein a decrease in the amount of extracellular mercaptoethanol for the cells compared to cells not exposed to the compound indicates that the compound is cytotoxic. In a particular embodiment, the method comprises obtaining a biological sample from a subject, administering the compound to the subject, and obtaining a second biological sample from the patient. The cells of the first and second biological samples are then contacted with hydroxyethyldisulfide (HEDS) and the amount of extracellular mercaptoethanol is determined.

"According to still another aspect, kits for practicing the methods of the instant invention are provided. In a particular embodiment, the kits comprise a) 5,5-dithiobis 2-nitrobenzoic acid (DTNB); b) hydroxyethyldisulfide (HEDS); and c) sulfosalicyclic acid buffer (SSA). The kits may also comprise at least one of: DTNB buffer, HEDS buffer, 96-well plate(s), and reaction buffers containing glucose-6-phosphate dehydrogenase, thioredoxin reductase, thioredoxin or other antioxidants.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIGS. 1A and 1B demonstrate the conversion of HEDS into mercaptoethanol (ME) by human cells as determined by High Performance Liquid Chromatography (HPLC)/electrochemical analysis. FIGS. 1A and 1B are HPLC chromatograms obtained from the extracellular medium after 2 hours incubation of human cells with (FIG. 1B) and without (FIG. 1A) HEDS. FIG. 1A shows only a solvent peak (peak 1) for extracellular medium from cells incubated for 2 hours without HEDS. FIG. 1B shows a solvent peak (peak 1), a mercaptoethanol (ME) peak (peak 2), and a HEDS peak (peak 3) for extracellular medium from cells incubated for 2 hours with HEDS.

"FIGS. 2A and 2B depict the measurement of arsenite induced oxidative stress in human cells in vitro as measured by HPLC/electrochemical analysis of extracellular medium samples. HPLC chromatograms for human cells incubated with 0 (FIG. 2A) and 10 .mu.M (FIG. 2B) arsenite for 24 hours in vitro are provided. FIGS. 2A and 2B show a solvent peak (peak 1), a ME peak (peak 2), and a HEDS peak (peak 3) for extracellular medium from human cells incubated with HEDS for 2 hours after 24 hours treatment with and without arsenite. The results show that 10 .mu.M arsenite (FIG. 2B) decreased the ME peak (peak 2) by almost 70% with a corresponding increase in the HEDS peak (peak 3) compared to the cells not treated with arsenite (FIG. 2A). Chromatograms without HEDS for these samples showed only a solvent peak.

"FIG. 3 is a graph of HEDS conversion measured by the HEDS/DTNB assay of the instant invention of the extracellular medium from human HCT116 cells treated with different concentrations of arsenite. The results in FIG. 3 show an arsenite concentration dependent decrease in HEDS conversion by human cells incubated with arsenite for 24 hours. HEDS conversion was measured immediately after the 24 hours incubation of cells with arsenite.

"FIG. 4 is a graph of arsenite toxicity measured by Coulter Counter Analysis of cells harvested six days after arsenite treatment. The results in FIG. 4 demonstrate an arsenite concentration dependent decrease in the survival of human HCT116 cells incubated with arsenite for 24 hours. The cell growth was measured six days after removing the arsenite from the extracellular medium by washing the cells three times with fresh growth medium.

"FIGS. 5A-5C are graphs of the application of the assay of the instant invention compared to other commercially available assays (WST-1 and XTT) used for cell survival in tissue culture medium. The figure shows a linear cell density dependent conversion of HEDS better than the commercially available assays (WST-1 and XTT) measured 16 hours after plating the cells in a 96 well microtiter plate. Cells tested: HCT116 cells (FIG. 5A), MCF10A cells (FIG. 5B), and MCF7 cells (FIG. 5C).

"FIG. 6 is a graph showing the application of the assay of the instant assay to quantify the toxicity of cisplatin in human cells in a 96 well platform. FIG. 6 demonstrates that the instant assay can be used to measure the toxicity induced by cisplatin in human cells. The results show a ciplatin dose dependent decrease in HEDS conversion measured 5 days after overnight incubation of cells with micromolar (0, 10, 20, 30, 40, or 50 .mu.M) concentrations of cisplatin.

"FIGS. 7A-7C are graphs of the total antioxidant capacity of various cell densities of three cell lines: Jurkat (FIG. 7A), CCRF-CEM (FIG. 7B), and T98G (FIG. 7C)."

For more information, see this patent application: Ayene, Iraimoudi S. Methods and Kits for Measuring Toxicity and Oxidative Stress in Live Cells. Filed March 27, 2014 and posted August 7, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2766&p=56&f=G&l=50&d=PG01&S1=20140731.PD.&OS=PD/20140731&RS=PD/20140731

Keywords for this news article include: Antioxidants, Anions, Phosphates, Biochemical, Glutathione, Biochemistry, Dehydrogenase, Oligopeptides, Electrochemical, Mercaptoethanol, Phosphoric Acids, Protective Agents, Sulfhydryl Compounds, Enzymes and Coenzymes, Lankenau Institute For Medical Research.

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Source: Life Science Weekly


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