La motivación autodeterminada y la conducta saludable

3.2.1 Preparation of purified glutathionylated BSA

Glutathionylated bovine serum albumin (BSA) was prepared by treating the protein with 10 mM reduced glutathione and 200 mM hydrogen peroxide for 30 minutes at 37°C. The glutathionylated protein was recovered by precipitation with 10 times the sample volume of ice cold acetone for two hours at -20°C and was resuspended in 20 mM Tris pH 8.

3.2.2 Cell culture

Cells (T47-D, HeLa, HEK 293) were purchased from ATCC and were maintained at 37°C, 5% CO2 in RPMI-1640 media (DMEM for HEK 293 cells) supplemented with

10% FBS and 1% glutamine as recommended by ATCC. Cells were maintained at 37°C in a humidified incubator at 5% CO2. Cells were plated at a high density of 5x105 cells

grown to 80% confluency in 6-well plates and treated with 0.5 mM hydrogen peroxide in sterile phosphate buffered saline (PBS) for 60 minutes at 37°C in serum free media followed by lysis in non-reducing lysis buffer as described below.

3.2.3 Animals

Balb/c male mice (aged 8-10 weeks) were maintained at the ANU Biosciences facility under controlled animal room conditions. The animals were fed regular animal diet and water ad libitum.

60

3.2.4 Drug administration

Doxorubicin was diluted in sterile saline and administered intra-peritoneally at a dose of 20 mg/kg. Control animals were injected with equal volume of saline. Animals were regularly monitored and sacrificed at 24 hours. Organs including heart, liver, lungs, brain, kidneys, testes and spleen were harvested immediately in sterile PBS and homogenized in a non-reducing buffer (20 mM Tris pH 8.0, 137 mM NaCl, 1% NP- 40, 2 mM EDTA, 10 mM NEM, 2 mM AEBSF protease inhibitor) using a Dounce homogenizer. No significant weight loss was observed in treated mice during this experiment.

3.2.5 Sample preparation

The steps involved in this assay are shown schematically in Figure 3.1. In this assay of determining protein glutathionylation, free thiols (predominantly GSH) are blocked by the inclusion of 10 mM NEM in the tissue-homogenizing buffer (20 mM Tris pH 8.0, 137 mM NaCl, 1% NP-40, 2 mM EDTA, 10 mM NEM, 2 mM AEBSF protease inhibitor). Tissue homogenates were centrifuged at 20000xg for 20 minutes and the protein concentration of the supernatant was determined by Lowry protein estimation [22]. For a typical assay, a sample of tissue extract containing 100 μg of protein was precipitated with twice the volume of ice cold acetone at -20°C for 60 minutes to remove the alkylated cellular GSH, free thiols and excess NEM. After centrifugation at 20000 x g for 15 minutes, the supernatant was discarded and the precipitated proteins were washed again in ice cold acetone and then re suspended in 40 μl of 0.5 mM Tris-HCL containing 0.1% Triton X-100 at room temperature.

3.2.6 Protein-SSG reduction

The reducing agent TCEP was added to the protein solution at a final concentration of 5 mM to break the P-SSG bonds and elute the protein bound GSH (10 μl 25 mM TCEP at room temperature for 30 minutes). The deglutathionylated protein was then precipitated by the addition of 25 μl 200 mM sulfosalicylic acid. After incubation on ice for 30 minutes, the precipitated protein was removed by repeating the centrifugation step.

3.2.7 Quantification of GSH

Previous studies have shown that at a high pH NDA reacts specifically with GSH to form a highly fluorescent cyclic derivative [313]. Consequently, GSH in the supernatant was reacted with NDA and the fluorescence quantified by comparison with authentic GSH standards that were run in parallel for each experiment. Specifically, 20

61 μl of supernatant was combined with 180 μl of NDA derivatization mix for 30 min in the dark at room temperature. The fluorescence was determined in an Optima FLUOstar Fluorescence plate reader with an excitation wavelength of 485 nm and an emission wave length of 520 nm. The NDA reaction mix was prepared immediately before use by combining 1 ml 10 mM NDA in dimethyl sulfoxide, 7 ml 50 mM tris pH 10, and 1 ml of 0.5 m NaOH with gentle mixing.

Figure 3.1: Step-wise description of the assay developed to determine total protein glutathionylation

62

3.3 Results

3.3.1 Optimization of NDA-based fluorescence assay

To develop and validate the method, commercially available Bovine Serum Albumin was artificially glutathionylated (BSA-SSG). Western blotting with an anti-glutathione antibody confirmed that BSA was strongly glutathionylated by treatment with glutathione and hydrogen peroxide (Figure 3.2A). The excess glutathione was removed by precipitating the glutathionylated albumin with ice cold acetone. Initial experiments using 10 μg of BSA-SSG showed that >95% of the mixed disulphide was reduced by 2 mM TCEP (Figure 3.2B). In subsequent experiments, 5 mM TCEP was used to ensure the reduction had gone to completion. In additional optimization experiments, the quantification of GSH derived from BSA-SSG by incubation with TCEP was found to be linear over a range from 0.05- 1.0 mg/ml BSA (Figure 3.2C). This assay determined the quantity of glutathione bound to BSA-SSG to be 54.3  3.8 nmoles of GSH/mg protein compared with 2.3  3.7 nmoles of GSH/mg protein obtained for normal BSA (Figure 3.2D). To confirm that the method measures reversible glutathionylation, the BSA-SSG was pre-treated with dithiothreitol (DTT). This treatment diminished the level of glutathionylation to background levels. The results were calculated as nmoles GSH/mg protein by reference to a standard curve generated with authentic GSH (Figure 3.2E).

63

Figure 3.2: Optimization of the glutathionylation assay. (A) shows the glutathionylation level detected in recombinant BSA (control) and glutathionylated BSA (BSA-SSG) probed with an anti-glutathione antibody. (B) shows the quantitative measure of NDA-GSH derived fluorescence from BSA-SSG with increasing concentrations of TCEP. (C) shows the linearity of the fluorescence measurements with increasing concentrations of BSA-SSG ( ) and un- glutathionylated control BSA ( ). (D) shows the specificity of the assay for protein bound glutathione by measuring fluorescence levels of BSA-SSG pre and post treatment with DTT. (E) A typical standard curve obtained with authentic GSH.

64

3.3.2 The effect of oxidative stress on cellular glutathionylation

The level of total glutathionylation was determined in human lymphoblastoid cell lines before and after treatment with the glutathionylating agent GSNO. As shown in Figure 3.3A, the basal level of glutathionylation in untreated cells was found to be 0.9 ± 0.45 nmoles/mg protein, which is well within the detection range estimated for this assay (Figure 3.2C). In contrast, one hour after treatment with 1 mM GSNO, the glutathionylation levels had risen to 16.6 ± 2.5 nmoles/mg protein. The experiment was repeated on separate days to indicate the repeatability of the assay and the cellular response. The level of total glutathionylation was determined in several cell lines and significantly higher levels were found in HEK293 cells compared to T47D and HeLa cells (p<0.001). However, treatment with 0.5 mM hydrogen peroxide caused a significant (p<0.001) increase in the level of glutathionylation in all cell lines (Figure 3.3B) (Table 3.1) which is similar to thefindings previously reported [302].

Figure 3.3: Determination of protein glutathionylation in cells

(A) Reproducibility of the assay was determined by measuring the total glutathionylation levels in lymphoblastoid cell lines before and after treatment with 1 mM GSNO on two consecutive days. The mean of individual data points is shown. (B) The assay was exploited to measure glutathionylation levels in basal and oxidative stress conditions in multiple cell lines. The assay consistently detected an increase in total glutathionylation levels in cells treated with hydrogen peroxide across all three cell lines.

65

3.3.3 Quantification of global glutathionylation in mouse tissues

Fresh liver, lung, kidney, heart, brain, spleen, testes and skeletal muscle from normal male BALB/c mice were extracted and glutathionylation was measured. The levels of glutathionylation were found to range from 1-2.5 nmoles GSH/mg protein for each tissue except for significantly higher (p<0.001) levels in skeletal muscle (Figure 3.4). Treatment of mice with the anti-cancer drug doxorubicin (20 mg/kg IP injection) caused an increase in glutathionylation in most tissues but notably caused a highly significant decrease in the glutathionylation of proteins in skeletal muscle which is still not fully understood. Since doxorubicin has been shown to induce oxidative stress [314-316], it was considered a suitable agent to evaluate and compare glutathionylation levels post doxorubicin treatment.

Figure 3.4: Protein glutathionylation levels were measured in mouse tissues pre- and post- treatment with doxorubicin for 24 hours. Data represents mean ± standard error; n=6

Table 3.1:Quantification of protein bound glutathione in cell lines

nmoles GSH/mg of protein

Cell line Untreated H2O2 treated

HEK 293 7.57 ± 0.01 13.33 ± 0.05

T47-D 0.18 ± 0.18 13.61 ± 0.12

66