Determination of glutathione levels and its oxidative state

Disruption of critically important oxidants can impair the redox status of the cell, leading to oxidative stress. The oxidative changes reported range from decreased levels of reduced glutathione (23, 24), «-tocopherol (23), and protein thiols (23), to downregulation of primary antioxidant defence enzymes such as catalase, manganese superoxide dismutase (MnSOD), Cu/Zn superoxide dismutase (Cu/ZnSOD), and thioredoxin (25).

GSH plays a central role in defending cells against radicals and electro-philes (26). The protective role of GSH consists of four components, namely chemical reaction with intracellular targets, enzymatic reduction of peroxides to prevent their conversion into more reactive species, enzymatic detoxification of electrophiles, and maintenance of the redox state of cellular thiols.

The response of cells to certain anti-tumour drugs and to ionizing radiation can be modulated by alteration of intracellular GSH concentration (27). Indeed, GSH has been implicated in the resistance of certain cell lines to oxygen radicals, various toxins, alkylating agents, and other chemosensitizers (28). In addition, GSH levels have been shown to be greater in many neoplasms compared with normal tissue, thus suggesting that resistance to apoptosis may involve altered levels of GSH (29). Moreover, it has recently been shown (30) that Fas ligation results in a rapid and specific efflux of GSH. Severe depletion of GSH will lower the reducing capacity of a cell, enhancing oxidative stress independent of any increase in the production of ROI.

There are a number of methods (e.g. chemical, enzymatic, chromatographic, fluorimetric) which can be used to measure GSH and/or glutathione disulfide (GSSG). Techniques that assay GSH concentration in a cell extract measure an average concentration, usually expressed as nmol/106 cells or on a per mg protein basis. Such measurements are not accurate if there is heterogeneity in the population with respect to the GSH content/cell (27). Solid tumours are very heterogeneous in many aspects and, in fact, micro-environmental factors known to be highly heterogeneous in solid tumours are known to influence cellular GSH content (31). In such cases, flow cytometric analysis using monochlorobimane (mBCl) provides more accurate and informative data (described on p. 151).

GSH can be assayed using an enzymatic procedure, which involves its sequential oxidation by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and reduction by NADPH in the presence of glutathione reductase. The DTNB-glutathione reductase recycling procedure was first described by Tietze (32), and later modified by Griffith (33). GSH is oxidized by DTNB to stoichio-metrically give GSSG and 5-thio-2-nitrobenzoic acid (TNB) (see eqn 1). GSSG is then reduced to GSH by glutathione reductase and NADPH (see eqn 2). The rate of TNB formation can be followed spectrophotometrically (at 412 nm, or 405 nm) and it is proportional to the sum of GSH and GSSG present. The assay can also be monitored at 340 nm (NADPH).

One of the advantages of the enzymatic assay is its ability to measure extremely low GSH levels provided that there is enough starting material. It is also a specific and reliable procedure. It is critical, however, to set up appropriate standards, as the method depends on an accurate standard curve.

Because GSSG is normally present at very low concentrations compared with GSH, the determination of GSSG is often difficult. The above assay can be made specific for GSSG by masking any GSH present with 7V-ethyl-

maleimide (NEM) (32). Although this procedure is effective, residual NEM must be removed as it is a potential inhibitor of glutathione reductase. This process is not only time consuming but also a potential source of error (33). The method described by Griffith (33) uses 2-vinylpyridine (2-VP), a reagent which does not inhibit glutathione reductase and thus need not to be removed. The GSH present in solutions of pH >5.5 is readily derivatized by adding 2 p,l neat 2-VP/100 |xl solution and mixing the solution for 1 min. Depending on the final pH, GSH (up to at least 15 mM) will be fully derivatized after 20-60 min at 25 °C. Acid solutions must be at least partially neutralized (e.g. with triethanolamine, which is very soluble and, due to its pKa, is unlikely to increase the pH to the point where auto-oxidation is rapid) before the reaction of GSH and 2-VP. In some circumstances the 2-VP added is sufficient to raise the pH.

Protocol 4. DTNB-glutathione reductase recycling assay for GSH and GSSG

Reagents3

• stock buffer: 125 mM sodium phosphate, 6.3 mM Na4EDTA, pH 7.5

• DTNB (Sigma), is prepared in stock buffer as a 6 mM stock solution

Method

2. Wash the cells by resuspending in 4 ml of PBS and centrifuging as described in step 1.

3. Lyse the cells in 1.2 ml water.

4. Mix 900 |xl lysate with 100 juul 30% (w/v) SSA, and incubate for 15 min on ice.

5. Centrifuge the sample at 12000 gfor 2 min.

6. In a 1 cm light path cuvette, mix 700 jjlI of NADPH solution (0.21 mM), 100 jd of DTNB (0.6 mM) solution, and 100 |xl GSH sample" (or water) to give a final volume of 1 ml.

7. Equilibrate the cuvette to 30°C (e.g. water bath or incubator), for 15 min.

8. To initiate the assay, add 10 |xl glutathione reductase (0.5 U/ml) and follow the formation of TNB by monitoring the absorbance at 412 nm, until it exceeds 2.0.c

NADPH (Sigma), prepared fresh as a 0.3 mM stock solution, in stock buffer glutathione reductase (Sigma). The yeast enzyme is diluted to 50 U/ml in stock buffer GSH standards (from 100 mM stock solution), are diluted daily in 3% (w/v) SSA

culture by centrifugation (200 g, 5

9. Determine the amount of GSH from a standard curve in which the GSH equivalents are plotted against the rate of change in Aw Express the level of GSH as GSH equivalents (e.g. nmol/10® cells).

" All solutions are stable for two weeks at 0°C, although the NADPH solution forms a precipitate which must be dissolved by warming to room temperature. 6 As a blank, use a sample lacking GSH.

"The linear portion of the curve is usually between 1 and 2 absorbance units.

Protocol 5. DTNB-glutathione reductase recycling assay for GSSG

Reagents3

• 2-VP (Sigma), used undiluted and stored at -20°C. Replace 2-VP if It becomes viscous or brownish

• triethanolamine (Sigma), used undiluted

• stock buffer: 125 mM sodium phosphate, 6.3 mM Na4EDTA, pH 7.5

• DTNB (Sigma), is prepared in stock buffer as a 6 mM stock solution

• NADPH (Sigma), prepared fresh as a 0.3 mM stock solution, in stock buffer

• glutathione reductase (Sigma). The yeast enzyme is diluted to 50 U/ml in stock buffer

• GSSG standards (e.g. 50 mM stock) (Sigma), are diluted daily in 3% (w/v) SSA

Method

1. Harvest the cells (5 x 106) from culture by centrifugation (200 g, 5 min).

2. Wash the cells by resuspending in 4 ml of PBS and centrifuging as described in step 1.

3. Lyse the cells in 1.2 ml water.

4. Mix 900 |xl lysate with 100 jxl 30% (w/v) SSA, and incubate for 15 min on ice.

5. Centrifuge the sample at 12000 gfor 2 min.

6. Add 2 (jlI of 2-VP to a 100 jjlI SSA supernatant aliquot'with gentle agitation.bc

7. Add 6 |i,l (45 (j-mol) of neat triethanolamine to the side of the test tube, above the liquid level, and agitate the tube vigorously.**

8. Check the pH and make sure it is between 6 and 7.®

9. Allow the samples to stand at room temperature for 60 min.

10. In a 1 cm light path cuvette, mix 700 |xl of NADPH solution (0.21 mM), 100 id of DTNB (0.6 mM) solution, GSSG sample (108 m.I), and 92 jjlI water to give a final volume of 1 ml.

11. Equilibrate the cuvette to 30°C (e.g. water bath or incubator), for 15 min.

Ana P. Costa-Pereira and Thomas G. Cotter Protocol 5. Continued

12. To initiate the assay, add 10 |xl glutathione reductase (0.5 U/ml) and follow the formation of TNB by monitoring the absorbance at 412 nm, until it exceeds 2.0.

13. Determine the amount of GSSG from a standard curve.

aAll solutions are stable for two weeks at 0°C, although the NADPH solution forms a precipitate which must be dissolved by warming to room temperature.

'The derivatization procedure should be carried out in a fume hood, as 2-VP has a low vapour pressure and frequent exposure to it might be irritating. cRun a blank containing 2-VP, but no GSSG.

dSince triethanolamine gives a small interference with the assay, the standards should contain an amount of amine equivalent to the samples.

"If the pH inadvertently exceeds 7, redo derivatization on a new sample using less triethanolamine.

Several fluorescent reagents have been developed for determining cellular levels of GSH. However, no probe is without drawbacks in quantitative studies of live cells. The high (up to 10 mM) but variable levels of intracellular GSH make kinetic measurements under saturating substrate conditions difficult (34, 35). The fluorescent reagents designed to measure GSH may react with other intracellular thiols, including proteins in GSH-depleted cells (36). Monochlorobimane (mBCl) is a reagent that reacts non-enzymatically with GSH. It is cell-permeant and non-fluorescent until conjugated. The fluorescent adduct formed between mBCl and GSH can be detected by cytofluoro-

Figure4. GSH levels were mesured in untreated Jurkat cells and in cells treated with 200 ng/ml anti-Fas IgM, for various periods of time. Following anti-Fas treatment there is a decrease in cellular GSH content, which was measured as described in Protocol 6. The data (from triplicate samples) represent mean %GSH±SE of each sample in relation to the control/untreated sample (100% GSH) and results shown are representative of three independent experiments.

Figure4. GSH levels were mesured in untreated Jurkat cells and in cells treated with 200 ng/ml anti-Fas IgM, for various periods of time. Following anti-Fas treatment there is a decrease in cellular GSH content, which was measured as described in Protocol 6. The data (from triplicate samples) represent mean %GSH±SE of each sample in relation to the control/untreated sample (100% GSH) and results shown are representative of three independent experiments.

metry, or by flow cytometry using 50 milliwatt UV excitation and emission integrated above 425 nm. While mBCl will react non-specifically with many different thiols, preferential derivatization of GSH can be achieved by using a low concentration of mBCl (short time incubation), as the reaction with GSH is catalysed by GST and the non-enzymatic reaction is very slow (27).

Although the cytofluorimetric assay is not quantitative, it will allow the comparison of relative GSH content in different samples (e.g. GSH content in cancer cells before and after treatment with a particular chemotherapeutic drug) (see Figure 4). In addition, the assay is reliable, easy, and rapid to perform.

Protocol 6. GSH determination with monochlorobimane (mBCl) by cyt of luoro metry

Equipment and reagents

• mBCl (Molecular Probes), prepared as 10 • PBS mM stock in ethanol. Protect from light and . cytofluorlmeter (e.g. SLM-AMINCO) store at-20°C

Method

1. Harvest 1 x 10® and centrifuge at 200 gfor 5 min at room temperature.

2. Wash the cells by resuspending in 4 ml of PBS and centrifuging as described in step 1.

3. Resuspend the pelleted cells in 2 ml of PBS.

4. Incubate the cells with 50 |xM of mBCl for 15 min at room temperature, in the dark.3

5. Read the fluorescence of the sample with excitation set at 395 nm and fluorescence emission set at 482 nm (see Figure 4).

"As a blank use 2 ml of PBS incubated with 50 p.M mBCl for 15 min at room temperature.

The flow cytometric method was first described by Rice et al. (31). It was shown that the GSH-bismane adduct, quantitated by HPLC, was formed with similar kinetics to the appearance of cellular fluorescence, as quantitated by flow cytometry. In addition, analysis of GSH by flow cytometry showed a strong correlation with parallel GSH analysis by an enzymatic method over a wide range of values for intracellular GSH. Hence, derivatization of cellular GSH with mBCl allows measurement of single-cell GSH content by flow cytometry. The kinetics of the reaction should be determined for each cell type to be sure that the reaction goes to completion, and the stoichiometry of the reaction should also be taken into account (27). For example, if the GSH content of 106 cells is 10 nmol, then at least 10 pM mBCl would be required to derivatize cellular GSH at a density of 106 cells/ml, and the concentration of mBCl would change as derivatization progresses. The assay does not require large numbers of cells but may not be applicable to all cell types (e.g. cells with low levels of glutathione S-transferase (GST). Although the assay may not discriminate very low GSH levels, it will reveal subpopulations and the fractions of cells contained in each, as well as their relative GSH contents.

Protocol 7. Flow cytometric analysis of GSH using monochlorobimane (mBCl)

Equipment and reagents

• mBCl (Molecular Probes), prepared as 10 • flow cytometer equipped with a 50

mM stock in ethanol. Protect from light and milliwatt UV excitation and emission store at -20°C integrated above 425 nm

Method

1. Harvest 1 x 106/ml cells into a standard FACS test tube.

2. Stain the cells with 40 p,M mBCl at room temperature for 5 mln and immediately transfer the cells to ice.

3. Read samples using a flow cytometer with excitation emission integrated above 425 nm. For each sample collect 5000-10000 events.

DL-buthionine-S,i?-sulfoximine (BSO) is a specific inhibitor of GSH synthesis, which has been shown to dramatically reduce GSH levels (28) (see Figure 5). The disruption of antioxidant mechanisms in cells can impair the

Figure 5. gsh levels can be artificially reduced by treating hl60 cells with 100 jig/ml bso (see Protocol 8) for various periods of time. gsh levels were assessed as described in Protocol 6. The data (from triplicate samples) represent mean %gsh±se and the results shown are representative of three independent experiments.

Figure 5. gsh levels can be artificially reduced by treating hl60 cells with 100 jig/ml bso (see Protocol 8) for various periods of time. gsh levels were assessed as described in Protocol 6. The data (from triplicate samples) represent mean %gsh±se and the results shown are representative of three independent experiments.

redox status of the cell, leading to oxidative stress and subsequent apoptosis. For instance, hormone-mediated apoptosis in prostate tissue is preceded by an increase in GST expression (37). McGowan et al. (38) have shown that artificial depletion of GSH significantly increased the levels of peroxide in leukaemic cells and caused subsequent apoptosis.

Protocol 8. Reduction of glutathione levels in cell lines with dl-

buthionine-S,/?-sulfoximine (BSO), a specific inhibitor of GSH synthesis

Reagents

• BSO (Sigma) freshly prepared as a 1 mg/ml stock in cell culture media (e.g. RPM11640) Method

1. Plate the cells in a standard 24-well plate at a density of 5 x 105/ml.

2. Incubate the cells, at 37°C, with varying concentrations of BSO (e.g. 100 M-g/ml), for various time intervals (e.g. 18-24 h), in order to determine the optimal concentration of BSO.

3. Assess the levels of GSH as previously described (see Protocols 4, 6 or 7, and Figure 5).

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