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Effects of N-acetylcysteine amide (NACA), a thiol antioxidant on radiation-induced cytotoxicity in Chinese hamster ovary cells
Wei Wu
a, Linu Abraham
a, Joshua Ogony
a, Richard Matthews
b, Glenn Goldstein
c, Nuran Ercal
a,⁎
aDepartment of Chemistry, Missouri University of Science and Technology, 1870 Miner Circle, Rolla, MO 65409, USA
bBethesda Regional Cancer Center, 1124 N Main St, Sikeston, MO, USA
cDepartment of Surgery, New York University, New York, NY 10010, USA
A B S T R A C T A R T I C L E I N F O
Article history:
Received 14 January 2008 Accepted 21 March 2008
Keywords:
Radiation
N-acetylcysteine amide N-acetylcysteine Chinese hamster ovary Oxidative stress In vitro
Ionizing radiation is known to cause tissue damage in biological systems, mainly due to its ability to produce reactive oxygen species (ROS) in cells. Many thiol antioxidants have been used previously as radioprotectors, but their application has been limited by their toxicity. In this investigation, we have explored the possible radioprotective effects of a newly synthesized thiol antioxidant, N-acetylcysteine amide (NACA), in comparison with N-acetylcysteine (NAC), a commonly used antioxidant. Protective effects of NACA and NAC were assessed using Chinese hamster ovary (CHO) cells, irradiated with 6 gray (Gy) radiation. Oxidative stress parameters, including levels of reduced glutathione (GSH), cysteine, malondialdehyde (MDA), and activities of antioxidant enzymes like glutathione peroxidase, glutathione reductase, and catalase, were measured. Results indicate that NACA was capable of restoring GSH levels in irradiated cells in a dose dependent manner. In addition, NACA prevented radiation-induced loss in cell viability. NACA further restored levels of malondialdehyde, caspase-3 activity, and antioxidant enzyme activities to control levels.
Although NAC affected cells in a similar manner to NACA, its effects were not as significant. Further, NAC was also found to be cytotoxic to cells at higher concentrations, whereas NACA was non-toxic at similar concentrations. These results suggest that NACA may be able to attenuate radiation-induced cytotoxicity, possibly by its ability to provide thiols to cells.
© 2008 Elsevier Inc. All rights reserved.
Introduction
The search for more effective radioprotectors has intensified recently due to increased use of ionizing radiation in radiotherapy for the treatment of malignant tumors. Radiotherapy treatment modality relies on the generation and use of ROS to eradicate tumors (Borek, 2004), and in the process, non-target tissues are also damaged (Mishra, 2002). Therefore, the application of ionizing radiation to the treatment of malignant tumors has been limited by the need to avoid extensive damage to normal tissues (Delanian and Lefaix, 2002).
Radiotherapy for cancer patients can be greatly enhanced by the use of radioprotectors, capable of protecting normal tissues from radiation- induced ROS. Thiol antioxidants, namely, cysteine, glutathione, N- acetylcysteine, and β-mercaptoethylamine (cysteamine) have been shown to protect mice and rats against the harmful effects of radiation (Weiss and Landauer, 2003; Shaheen and Hassan, 1991). However, some of these thiols, such as cysteamine, have been reported to have lethal and behavioral toxicities (Landauer et al., 1988).
N-acetyl-L-cysteine (NAC), a well-known thiol-containing antiox- idant, has had multiple uses in clinics for more than 50 years (Kelly, 1998; Holdiness, 1991; Parcell, 2002; Ziment, 1988; Flanagan and Meredith, 1991). The evidence from both in vitro and in vivo studies indicates that NAC is capable of facilitating intracellular glutathione (GSH) biosynthesis by reducing extracellular cystine to cysteine (Issels et al., 1988), or by supplying sulfhydryl (–SH) groups that can stimulate GSH synthesis and enhance glutathione-S-transferase activity (Nakata et al., 1996; De Vries and De Flora, 1993; De Flora et al., 1985). Additionally, NAC is a potent free radical scavenger as a result of its nucleophilic reactions with ROS (Aruoma et al., 1989).
Therefore, NAC treatment or supplementation may be appropriate for conditions of GSH depletion and free radical formations during oxidative stress. Some studies have also demonstrated that NAC can act as a radioprotective agent against oxidative damage induced by UV, ionizing radiation, and gamma rays (Neal et al., 2003; Murley et al., 2004; Morley et al., 2003; Sridharan and Shyamaladevi, 2002;
Pajonk et al., 2002). However, bioavailability of NAC is very low because its carboxyl group loses its proton at physiological pH, making the compound negatively charged. This renders its passage through biological membranes difficult. N-acetylcysteine amide (NACA) is the modified form of N-acetylcysteine that was designed and synthesized with the possibility that neutralizing the carboxyl group can aid in its
⁎ Corresponding author. Tel.: +1 573 341 6950; fax: +1 573 341 6033.
E-mail address:[email protected](N. Ercal).
0024-3205/$– see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2008.03.016
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passage through cell membranes (Fig. 1). One recent study provided evidence that NACA had more efficient membrane permeation than NAC and could replenish intracellular GSH in red blood cells, possibly by disulfide exchange with oxidized glutathione (GSSG) (Grinberg et al., 2005). Further, NACA can be hydrolyzed to give cysteine that can boost the production of endogenous glutathione. This compound was also shown to cross the blood brain barrier, scavenge free radicals, chelate copper, and attenuate myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis in a multiple sclerosis mouse model (Atlas et al., 1999; Offen et al., 2004).
Recent studies have also investigated the ability of NACA to protect mammalian cells from oxidants such as HIV proteins, glutamate and Aβ toxicity (Price et al., 2006, Penugonda et al., 2005; Bartov et al., 2006).
Our study investigates the possible protective effects of NACA in radiation-challenged CHO K1 cells, in comparison to NAC, a previously studied thiol radioprotector. Initial experiments were performed to determine suitable duration of treatment and dosage with the antioxidant. Following this, cells were treated with NACA and NAC prior to radiation challenge of 6 Gy. Assessment of the radioprotective effects of NACA and NAC was done by measuring the parameters (such as GSH, CYS, and MDA levels) in both the treatment and control groups, as well as the activities of antioxidant enzymes, namely, glutathione peroxidase (GPx), glutathione reduc- tase (GR), and catalase (CAT). A comparison was made of all parameters measured in the different treatment groups to identify the antioxidant capable of providing superior radioprotection, with minimal toxic effects.
Materials and methods Materials
Chinese hamster ovary (CHO) K1 cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). The N-(1-pyrenyl)-maleimide (NPM), used as a derivatizing agent for measurement of CYS and GSH, and 1,1,3,3-tetramethoxypropane were purchased from Sigma-Aldrich (Milwaukee, WI, USA). N-acetylcys- teine amide (NACA) was provided by Dr. Glenn Goldstein (David Pharmaceuticals, New York, NY, USA). The HPLC-grade acetonitrile, glacial acetic acid, o-phosphoric acid and water, used for the preparation of mobile phase, were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Bradford reagent was obtained from BioRad (Hercules, CA, USA). Ham's F-12 culture medium, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, and all other chemicals were purchased from Sigma (St. Louis, MO USA). 0.45-µmfilters and 25-cm2 culture flasks were purchased from Advantec MFS, Inc.
(Dublin CA, USA). GR and GPx activity assay kits were purchased from OxisResearch ™, while caspase-3 activity assay kit was purchased from R&D Systems, USA. 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega (Madison, WI, USA).
Culture of Chinese hamster ovary (CHO) cells
Chinese hamster ovary (CHO) K1 cells, were grown in Ham's F- 12 culture medium, supplemented with 10% (v/v) fetal bovine serum (FBS), to which 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin were added. The cells were maintained in a humidified incubator at 37 °C and supplied with 95% O2and 5%
CO2.
Irradiation of cells
The irradiation of the cells was carried out at the Radiation Oncology Department of the Phelps County Regional Medical Center in Rolla, Missouri, using a 9 MeV electron beam generated by a Varian Linear accelerator, model 21 EX (Varian Associates, Walnut Creek, CA, USA). A 20 × 20- or 25 × 25-cmfield was used and output factors were checked once a week. Flatness of thefield was also checked once a week and was maintained within 2%. A 6 Gy radiation dose was used in all studies.
Oxidative stress studies
CHO cells were seeded at a density of 6 × 106perflask (25 cm2) in 5 ml of media. Depending on the particular study, the media was replaced with treatment media containing NAC or NACA and incubated for the required time period. For irradiation studies, the groups include 1) Control (n = 3): received no irradiation and no NACA in media solution; 2) XRT only (n = 3): 6 Gy irradiation and no NACA in media; 3) XRT + NACA (n = 3): 6 Gy irradiation and 1 mM of NACA in media solution; 4) XRT + NAC (n = 3): 6 Gy irradiation and 1 mM of NAC in media solution. The cells were collected and analyzed 24 h after irradiation. The cell pellets were homogenized on ice in serine borate buffer (100 mM Tris–HCl, 10 mM boric acid, 5 mML-serine, 1 mM DETAPAC, pH 7.4). The cell homogenate was used directly for GSH, cysteine, and MDA analysis. For enzyme assays, the homogenate was centrifuged at 10,000 ×g for 10 min at 4 °C and then the supernatant used.
Cell viability determination
The CellTiter 96® AQueous Cell Proliferation assay kit (Promega Corporation, Madison, WI, USA) was used in the cell viability studies. This assay uses the novel tetrazolium compound, MTS, which can be reduced by NADPH or NADH (produced by dehydrogenase enzymes in living cells) into formazan, which is soluble in tissue culture medium (Cory et al., 1991; Berridge et al., 2005). Since the production of formazan is proportional to the number of living cells, the intensity of color produced is a good indicator of the viability of the cells (Cory et al., 1991; Riss and Moravec, 1992). The measurement of the absorbance of the formazan product was carried out at 490 nm using a microplate reader (Fluostar Optima, BMG Labtechnologies. Inc, Durham, NC).
CHO cells (10,000 cells/well) were seeded in a 96-well plate. After the cells were attached, the original media solution was replaced with a media solution containing three different concentrations each, of NACA or NAC (0.5, 1 and 5 mM). Plain media was used as the control. After 30 min, cells in the radiation groups were exposed to radiation, while cells in the control group did not receive any radiation. Cells were returned to the incubator and maintained at 37 °C, 95% air, and 5% CO2for an additional 24 h after radiation.
Then 20 µl of MTS tetrazolium reagent was added to each well, and the absorbance was read at 490 nm after 1 h of incubation with the MTS reagent. The cytotoxic effects of NACA and NAC were evaluated by incubating CHO cells (10,000 cells/well) with 0.5, 1, 5 or 10 mM NACA or NAC for 24 h, followed by addition of MTS reagent and measurement of absorbance at 490 nm.
Fig. 1. A) Structure of N-acetylcysteine (NAC). B) Structure of N-acetylcysteine amide (NACA).
Apoptosis measurement
The caspase-3 apoptotic assay was performed using a colorimetric substrate, as per the manufacturer's instructions (R&D Systems, Inc.
MN). Briefly, 25 µl of lysis buffer per 1×106cells were was added to each pellet that was collected after treatment. The cell suspension was incubated on ice for 10 min and then centrifuged at 10,000 × g for 3 min. 50 µl of the supernatant, along with 50 µl of the 2× reaction buffer containing 0.1 M DTT and 5 µl of the caspase-3 colorimetric substrate (DEVD-pNA) were added to each well in a 96-well plate. The plate was then incubated for 2 h before the absorbance was measured by a microplate reader at 405 nm.
Determination of GSH and cysteine levels
The levels of GSH and CYS in the cells were determined by RP- HPLC, according to the method developed in our laboratory (Winters et al., 1995). The HPLC system (Thermo Electron Corporation) consisted of a Finnigan Spectra System vacuum membrane degasser (model SCM1000), gradient pump (model P2000), autosampler (model AS3000), and fluorescence detector (model FL3000) with λex=330 nm and λem=376 nm. The HPLC column was a Reliasil ODS- 1 C18 column (5-µm packing material) with 250 × 4.6 mm (Column Engineering, Ontario, CA, USA). The mobile phase was 70% acetonitrile and 30% water and was adjusted to a pH of 2 with acetic acid and o- phosphoric acid. The NPM derivatives of CYS and GSH were eluted from the column isocratically at aflow rate of 1 ml/min. Cell samples were homogenized and centrifuged, and 100 µl of supernatant was added to 130 µl of HPLC-grade water and 750 µl of NPM (1 mM in acetonitrile). The resulting solution was incubated at room tempera- ture for 5 min, and the reaction was stopped by adding 10 µl of 2 N HCl.
The samples were thenfiltered through a 0.45-µm filter and injected onto the HPLC system.
Determination of MDA
The MDA levels were determined according to the method described byDraper et al. (1993). Briefly, 550 µl of 5% tricholoroacetic acid (TCA) and 100 µl of 500 ppm butylated hydroxytoluene (BHT) in methanol were added to 350 µl of the cell homogenates. The mixture was then heated in a boiling water bath for 30 min. After cooling on ice, the mixture was centrifuged, and the supernatant fractions were mixed 1:1 with saturated thiobarbituric acid (TBA). The mixture was again heated in a boiling water bath for 30 min. After cooling on ice a second time, 500 µl of the mixture was extracted with 1 ml of n- butanol and centrifuged to facilitate the separation of phases. The resulting organic layers werefirst filtered through 0.45-µm filters and then injected onto the HPLC system. The concentrations of the TBA– MDA complex in the mixture was determined by using the calibration curve obtained from a 1,1,3,3,-tetraethoxypropane standard solution.
Catalase (CAT) activity assay
The activity of catalase (CAT; EC 1.11.1.6) in the cell supernatant was measured spectrophotometrically at 240 nm following the exponen- tial disappearance of hydrogen peroxide (H2O2; 10 mM) according to the method described by Aebi (1984). The catalase activity is calculated from A60= Ainitiale− ktwhere k, is the rate constant, Ainitial, is the initial absorbance, and A60is the absorbance at 60 s.
Glutathione peroxidase (GPx) activity assay
A glutathione peroxidase (GPx) activity assay (a test kit from OxisResearch, Portland, Oregon, USA) was used to obtain an indirect measure of the activity of GPx. Oxidized glutathione, produced upon reduction of an organic peroxide by GPx, is recycled to its reduced
state by the enzyme glutathione reductase (GR). The oxidation of NADPH to NADP+ is accompanied by decrease in absorbance at 340 nm, providing a spectrophotometric means for monitoring GPx
enzyme activity. The molar extinction coefficient for NADPH is 6220 M− 1 cm− 1 at 340 nm. To assay GPx, a cell homogenate was added to a solution containing glutathione, glutathione reductase, and NADPH. The enzyme reaction was initiated by adding the substrate, tert-butyl hydroperoxide, and the absorbance at 340 nm was recorded. The rate of decrease in the absorbance at 340 nm is directly proportional to the GPxactivity in the sample.
Glutathione reductase (GR) activity assay
Glutathione reductase (GR; EC 1.6.4.2) activity was measured spectrophotometrically at 340 nm following the decrease of NADPH using a commercial kit from OxisResearch™ (Portland, Oregon, USA).
This reaction maintains the normal levels of cellular glutathione, essential for keeping the levels of free radicals and organic peroxides down.
Determination of protein
Protein levels of the cell samples were measured by the Bradford method (Bradford, 1976). Concentrated Coomassie Blue (Bio-Rad, Hercules, CA) was diluted 1:5 (v/v) with distilled water, and then
Fig. 2. Time-course results of thiol levels in CHO cells after NACA or NAC treatment: A) GSH levels in cells collected at 0, 0.5, 1, 2, 4 and 6 h after incubation with media containing 1 mM NAC or 1 mM NACA. B) Cysteine levels in cells at indicated time points of incubation with 1 mM NAC or NACA. A rapid rise in cysteine and GSH levels is seen in cells 30 min after incubation with NACA and NAC. Each value is the average of triplicates with error bars indicating standard deviations. GSH levels of cells treated with both NAC and NACA for 30 min or more were significantly higher than control, with pb0.001 as compared to control. Cysteine levels of cells treated with NACA for 30 min or more were significantly higher than controls (pb0.05). Cysteine levels in cells treated with NAC for 1, 2, 4 and 6 h were significantly higher than control group (pb0.05).
2.5 ml of this diluted dye were was added to 50 µl of diluted cell homogenate. The mixture was incubated at room temperature for 10 min and the absorbance measurement was taken at 595 nm using a UV–VIS spectrophotometer. Bovine serum albumin (BSA) was used as protein standard.
Statistical analysis
All reported values were represented as mean ± S.D. of multi- duplicates. The one-way analysis of variance (ANOVA) test was used to analyze the data from the experimental and control groups; p values b0.05 were considered significant.
Results
Time-course studies of thiol levels in CHO cells treated with NAC or NACA
Levels of reduced glutathione and cysteine were measured in CHO cells after treatment with NAC or NACA for different durations (Fig. 2).
A rapid rise in GSH levels was seen in cells incubated with 1 mM NACA for 30 min. GSH levels of cells preincubated with NACA for 1, 2, 4 and 6 h were similar to that of cells pretreated for 30 min. Thus, pretreatment for greater than 30 min resulted in an equivalent increase in GSH levels. A similar trend was also seen in CHO cells treated with 1 mM NAC. Cysteine levels in cells also increased sharply,
30 min after incubation with 1 mM NACA. Treatment with NAC caused a similar, but less rapid rise in cysteine levels. The levels of cysteine were comparable in NAC and NACA treated groups at the 6 h time point. For all further experiments, 30 min was selected as the duration of pretreatment of cells with NACA or NAC, before radiation challenge.
NACA dose response studies on radiation-challenged CHO cells
The effects of various doses of NACA on GSH and cysteine levels in radiation-challenged CHO cells are given inFig. 3. CHO cells were pretreated with different doses of NACA, ranging from 1 mM to 20 mM, for 30 min, followed by a 6 Gy radiation challenge. The cells were collected 24 h later and analyzed for GSH and cysteine levels. The 67% drop in GSH levels, following the radiation challenge, was replenished by pretreatment with 0.5 mM NACA for 30 min. The GSH levels increased in a dose dependent manner with increasing concentrations of NACA. There was a slight drop in the GSH level at the highest concentration of 20 mM that can be attributed either to toxic effects of NACA at such high concentrations, or to saturation and feedback mechanisms in the GSH synthesis pathway. A dose dependent increase was also seen in levels of cysteine in cells treated with NACA, in a manner similar to that seen in GSH levels. Thus, in addition to replenishing GSH and cysteine levels in radiation- challenged cells, NACA was also capable of providing surplus cysteine and GSH in these cells.
Fig. 3. NACA dose response studies in radiation-challenged cells: A) GSH levels. Cells were pretreated with the indicated concentrations of NACA for 30 min, followed by 6 Gy radiation challenge. GSH levels were analyzed in cells collected 24 h after radiation challenge. B) Cysteine levels in cells pretreated with various concentrations of NACA followed by radiation challenge. The radiation challenge is indicated as XRT. Each value is the average of triplicates with error bars indicating standard deviations.⁎pb0.05 as compared to control,
⁎⁎pb0.005 as compared to control,†pb0.005 as compared to XRT-only group,††pb0.0005 as compared to XRT-only group.
Cytotoxicity of NAC and NACA in CHO cells
Fig. 4displays the results of cytotoxicity studies of NACA and NAC in CHO cells. Cells were incubated with 0.5, 1, 5 and 10 mM NAC or NACA for 24 h, after which MTS reagent was added and absorbance was measured at 490 nm. Reagent controls and cell controls were included. These results clearly indicate that NAC is highly toxic to cells, even at the lowest concentration of 0.5 mM used in this experiment.
Further decrease in cell viability was seen with an increasing concentration of NAC, with a complete loss of viability at 10 mM. In contrast, 0.5 and 1 mM NACA treated groups had cell viability comparable to that of the control. However, higher concentrations of 5 and 10 mM NACA appear to be somewhat cytotoxic to cells.
Nevertheless, the toxic effects of NAC in CHO cells appear far more severe than those of NACA.
Protective effects of NACA and NAC in radiation-challenged cell
In order to study the protective effects of thiol antioxidants in radiation-induced cytotoxicity, CHO cells were pretreated with 0.5, 1 and 5 mM NAC or NACA, followed by a 6 Gy radiation exposure.Fig. 5 depicts the effects of NACA and NAC on radiation-induced cytotoxicity, respectively. The 6 Gy radiation challenge caused a 45% loss in cell viability, as compared to that of the control group. Pretreatment with NACA, however, clearly aided in preventing loss of cell viability.
Although 0.5 mM NACA had the ability to protect the cells, there was a greater protective effect when higher concentrations of NACA were used. There was not a large difference in the effects of 1 and 5 mM NACA, since, at both concentrations, cell viability was restored to about 71% of the untreated control. At 0.5 mM concentration, NAC had a similar effect in preventing cell death, as did a 0.5 mM concentration of NACA. However, at higher concentrations of NAC treatment, there was a significant decrease in cell viability. It appears that, as opposed to protecting cells from radiation-induced cytotoxicity, NAC was, in fact, causing toxic effects. The cell viability of 1 and 5 mM NAC treated groups was even lower than that of the XRT-challenged group, indicating that, although NAC can provide some protection at lower concentrations, it is cytotoxic at higher concentrations.
Radiation-induced apoptosis and protection by antioxidants
Caspase-3 activity in cells is a good indicator of the apoptotic activity in these cells. An increase in caspase-3 activity corresponds to increased apoptosis. Fig. 6 represents caspase-3 activity in cells receiving 6 Gy radiation and compares the effects of NACA and NAC
treatment before a radiation challenge on apoptotic activity. There was a significant increase in caspase-3 activity in radiation-challenged cells, pointing to the role of apoptosis in the loss of cell viability in these cells. Cells pretreated with 1 mM NACA and NAC had caspase-3 activity levels comparable to those of the control group. Thus, NACA and NAC appear to have the ability to protect cells from radiation- induced cytotoxicity, possibly due to their ability to prevent apoptosis.
Oxidative stress studies in radiation-challenged cells
NACA and, to some extent NAC, appear to provide protection to CHO cells against radiation-induced cytotoxicity. Since these thiols have antioxidant properties, various oxidative stress parameters were analyzed to determine if the protective effects of NACA and NAC are due to their ability to alleviate oxidative stress. For this purpose, GSH, cysteine and MDA levels were measured in radiation- challenged cells, as well as in cells pretreated with 1 mM NAC or NACA for 30 min.
Fig. 4. Cytotoxicity of NACA and NAC in CHO cells: In thisfigure, a comparison has been made of the effects of identical concentrations of NAC and NACA on the viability of CHO cells. CHO cells were incubated with various concentrations of NACA or NAC for 24 h and cell viability was assessed using MTS assay. A decrease in absorbance at 490 nm indicates decreased cell viability. Each value is the average of triplicates with error bars indicating standard deviations.⁎pb0.005 as compared to control group,
⁎⁎pb0.0001 as compared to control,†pb0.005 as compared to NACA group of the same concentration.
Fig. 5. Protective effects of NACA and NAC in radiation-induced cytotoxicity: viability of cells pretreated with NACA or NAC before XRT treatment. NACA pretreatment for 30 min has clearly aided in preventing radiation-induced loss of cell viability. 1 and 5 mM NACA appear to have a greater effect in preventing loss of cell viability, although 0.5 mM NACA is also capable of providing a protective effect, though not to the extent that higher concentrations do. 0.5 mM NAC protected the cells to an extent similar to that provided by 0.5 mM NACA. As the concentration of NAC increased, however, there appeared to be a severe loss of cell viability. In fact, the viability of the 1 and 5 mM NAC treated groups was even lower than that of the XRT-only group. Each value is the average of triplicates with error bars indicating standard deviations.⁎pb0.05 as compared to control group,
⁎⁎pb0.0005 as compared to control,†pb0.005 as compared to XRT treated group,
††pb0.0005 as compared to XRT treated group.
Fig. 6. Effect of NACA on caspase-3 activity in radiation-challenged cells: Radiation- challenged cells had increased caspase-3 activity, indicating that 6 Gy irradiation causes cell death, partly by the process of apoptosis. Pretreatment with 1 mM NACA and NAC prevented apoptosis, since the caspase-3 activity of these groups was similar to that of the control group. Each value is the average of triplicates with error bars indicating standard deviations.⁎pb0.005 compared with the control groups,†pb0.005 compared with the XRT-only groups.
Fig. 7shows the effects of NACA and NAC on GSH and CYS levels in radiation-challenged CHO cells. Irradiation with 6 Gy caused a significant decrease in the levels of both GSH and cysteine in cells.
Both NACA and NAC, when given 30 min before radiation, were able to replenish the GSH and CYS levels in radiated CHO cells back to control levels. NACA treated groups had more GSH than the NAC treated groups, although cysteine levels in both of these groups were almost identical. These results show that pretreatment with thiol antiox- idants can replenish GSH loss caused by irradiation.
Lipid peroxidation is a consequence of oxidative stress, and can usually be estimated using levels of malondialdehyde (MDA), a product of lipid peroxidation.Table 1represents MDA levels in cells challenged with 6 Gy radiation as well as those pretreated with 1 mM NAC or NACA. Radiation appeared to cause significant lipid peroxida- tion in CHO cells, as seen by the increase in levels of MDA. NACA treatment was able to reverse the MDA levels back to control levels.
NAC had a similar ability to reduce the elevated MDA levels in radiated cells.
Antioxidant enzyme activities in radiation-challenged CHO cells
Table 2 displays the effects of NACA and NAC on the levels of antioxidant enzymes, including catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) in radiation-challenged CHO cells. XRT challenge significantly decreased levels of glutathione reductase in CHO cells. Exposure to NACA, prior to irradiation, prevented the decrease in GR activity, as NACA pretreated cells had levels of GR
activity similar to those of the control group. NAC treated groups also had GR activity that was higher than that of the XRT-challenged group, although NAC was not able to increase the activity back to control levels. Radiation challenge caused a decrease in GPxactivity. NACA treated groups had significantly higher GPxactivity than radiation- treated groups, although the levels did not revert completely back to control levels. In a similar manner, NAC was also capable of increasing the activity of GPx.
Effects of irradiation on the oxidative status of CHO cells is also reflected in the decrease in catalase activity in XRT-challenged cells.
NACA and NAC were both capable of partially reversing the loss of catalase activity. This effect was more significant in the NACA treated groups. The levels however could not be bought back all the way to control levels.
Discussion
Early studies have shown that the most effective radioprotective compounds are those that contain a sulfhydryl group (–SH) at the end of a 2 or 3 carbon chain (Maisin, 1998). Another desirable property for these compounds is water solubility, to facilitate administration in animals and humans. A number of sulfhydryl compounds have been previously investigated for their radioprotective properties. Their application, however, has been limited by their toxic side effects in both animal and cell models (Cairnie, 1983; Held and Melder, 1987).
The search therefore continues tofind an ideal radioprotector with the desired structural properties and minimal toxicity.
It is thought that the radioprotective effects of sulfhydryl compounds are due to their ability to scavenge free radicals, thus providing protection from radiation-induced oxidative stress (Murray and McBride, 1996). N-acetylcysteine is one such compound that is a well-known sulfhydryl-containing antioxidant whose role in radio- protection has been explored in several studies (Neal et al., 2003;
Murley et al., 2004; Morley et al., 2003). The antioxidant property of N-acetylcysteine can be attributed to its ability to provide cysteine and other precursors of glutathione synthesis, as well as its ability to directly scavenge free radicals (Kelly, 1998). According to several studies, NAC on the one hand acts as an antioxidant, but on the other hand, it can also act as a prooxidant, resulting in cytotoxicity and oxidative stress (Held and Biaglow, 1994; Sprong et al., 1998). N- acetylcysteine amide (NACA) is the amide form of N-acetylcysteine, designed and synthesized in order to be more cell permeable and lipophilic than NAC (Offen et al., 2004; Grinberg et al., 2005). A number of recent studies have also shown that NACA is indeed an excellent antioxidant in numerous cases of oxidant injury (Price et al., 2006; Penugonda et al., 2005; Bartov et al., 2006). In this study, we have attempted to evaluate the possible protective role played by NACA in radiation-induced oxidative stress, with NAC being used as a reference antioxidant.
The cellular antioxidant, reduced glutathione plays a major role in protecting cells against radiation-induced oxidative stress, and its levels are subject to change depending on the extent of radiation exposure (Bump and Brown, 1990). Glutathione and cysteine levels Fig. 7. GSH and cysteine levels in XRT-challenged CHO cells: CHO cells were pretreated
with either plain media (CTR) or with 1 mM NACA and NAC, followed by exposure to 6 Gy radiation. Oxidative stress parameters were analyzed in cells 24 h later. Radiation challenge caused a sharp decrease in glutathione levels. Pretreatment with NAC and NACA brought back the GSH levels to the control levels. The NACA treated groups had higher GSH levels than the NAC treated groups, although this difference was not significant. Cysteine levels were seen to decrease in radiation-challenged groups as well, with NACA and NAC restoring cysteine content to control group levels. Each value is the average of triplicates with error bars indicating standard deviations.⁎pb0.005 compared with the control groups,†pb0.005 compared with the XRT-only groups.
Table 1
Effects of NACA and NAC on MDA levels in XRT-challenged CHO cells
Groups MDA levels
(nmol/100 mg protein)
Control 9 ± 0.7
XRT 13 ± 0.6⁎⁎
XRT + NACA 9 ± 0.9†
XRT + NAC 11 ± 0.5⁎,†
Cells were pretreated with 1 mM NACA or NAC for 30 min before radiation challenge.
XRT treated cells received 6 Gy irradiation. Each value is the average of triplicates.
*pb0.05 as compared to control groups, **pb0.005 as compared to control, and
†pb0.005 compared to XRT-only group.
Table 2
Effects of NACA and NAC on antioxidant enzyme activities in XRT-challenged CHO cells
Groups GR levels GPxlevels Catalase levels
(mU/mg protein) (mU/mg protein) (U/mg protein)
Control 37.6 ± 3.0 35.8 ± 1.3 0.048 ± 0.002
XRT 19.6 ± 0.5⁎⁎ 26.1 ± 1.1⁎⁎ 0.022 ± 0.003⁎⁎
XRT + NACA 36.0 ± 3.0†† 31.1 ± 0.8⁎,†† 0.033 ± 0.003⁎⁎,††
XRT + NAC 22.1 ± 3.6⁎ 30.4 ± 2.2⁎,† 0.030 ± 0.004⁎⁎,†
CHO cells were incubated in media containing 1 mM NACA or NAC for 30 min, followed by 6 Gy irradiation. Each value is the average of triplicates. *pb0.05 as compared to control group, **pb0.001 compared to control group,†pb0.05 compared to XRT-only group, and††pb0.005 as compared to XRT-only group.
were initially analyzed in CHO cells incubated with 1 mM NACA or NAC for different incubation durations. This was done in an attempt to arrive at the most effective duration of antioxidant treatment. The greatest rise in glutathione and cysteine levels was seen within 30 min of incubation with either NAC or NACA. It was therefore decided to pretreat cells for 30 min prior to being exposed to radiation. Several other studies have also followed a similar regimen of pretreatment with antioxidant before radiation exposure (Iordanov and Magun, 1999; Grdina et al., 1995). Next, glutathione and cysteine contents were analyzed in cells exposed to 6 Gy radiation alone, or pretreated with various concentrations of NACA. The decrease in glutathione content in radiation-treated cells was countered effectively by pretreatment with a range of concentrations of NACA. However, it is also important that the concentration of NACA selected for treatment of cells is not toxic to the cells. This was evaluated using the MTS cytotoxicity assay. N-acetylcysteine was seen to be toxic to cells at concentrations as low as 0.5 mM, while N-acetylcysteine amide began to exhibit toxicity only at concentrations of 5 mM and above. This is consistent with previous investigations, which established the toxicity of NAC (Maisin, 1998). The same concentrations of NACA that are non- toxic to CHO cells were found to be effective in replenishing the levels of both GSH and CYS. The lowered toxicity of NACA thus makes it a more suitable candidate for use at higher concentrations, as compared to its predecessors such as cysteamine, NAC, and other previously- used thiol radioprotectors.
After comparing results of cytotoxicity studies and thiol replenishment studies, an antioxidant concentration of 1 mM was selected as being appropriate for comparing the effects of NAC and NACA on radiation-mediated oxidative stress. Both antioxidants had a similar capability in replenishing glutathione and cysteine levels that had been depleted by radiation treatment. Free radicals produced by ionizing radiation also attack lipids in biological mem- branes, leading to the formation of a number of degradation pro- ducts. Poly unsaturated fatty acids (PUFAs) found in membranes are especially susceptible to radicals, producing byproducts such as malondialdehyde (MDA) (Karbownik and Reiter, 2000). Thus, as an additional index of oxidative stress, levels of MDA were analyzed in irradiated CHO cells, with or without antioxidant pretreatment. It was seen that radiation induced a significant elevation in MDA levels in CHO cells. This is consistent with other studies that reported elevated levels of thiobarbituric acid reactive substances (TBARS) and MDA content in cell models exposed to ionizing radiation (Benderitter et al., 2003; Prasad et al., 2005). In one of these studies, TBARS was seen to be elevated in cultured human lymphocytes irradiated with radiation doses as low as 1 to 4 Gy (Prasad et al., 2005). According to the results we obtained, NACA was able to return the elevated levels of MDA back to control levels. NAC was also partially effective in preventing lipid peroxidation, although not to the extent of NACA. The concentration and dose of antioxidant treatment used here appear suitable for combating radiation-induced oxidative stress, as evi- denced by their ability to prevent glutathione and cysteine depletion, as well as lipid peroxidation.
The results of our cell viability studies showed that NACA had better abilities to prevent radiation-induced cytotoxicity as compared to NAC. When the cells were treated with a range of concentrations (0.5 to 5 mM) of NACA and NAC, only the lowest concentration of NAC (0.5 mM) offered some protection to the cells, as reflected by the magnitude of the absorbance of the MTS assay, while all concentra- tions (0.5 to 5 mM) of NACA offered significant protection to the cells.
This finding suggests that NACA holds a brighter promise in the ongoing search for better radioprotectors. Higher concentrations of NACA used in our investigation resulted in higher cell viability.
Previous investigations have reported a decrease in cell viability caused by ionizing radiation (Wright et al., 1998). The highly toxic hydroxyl radicals produced by ionizing radiation, attack the DNA molecules, causing single and double strand breaks. Additionally, the
ROS also cause lipid peroxidation and protein oxidation. These deleterious changes impact the cellular functions negatively, resulting in cell death, thereby lowering cell viability. The improved cell viability with the treatment of modest concentrations of NACA implies that this thiol could, in the future be used extensively in radiotherapy situations.
The results of our caspase-3 activity experiments in radiation- challenged cells showed that radiation significantly elevated the caspase-3 activity in CHO cells (1.6 times that of the control caspase-3 levels), and NACA (1 mM) and NAC (1 mM) treatments were both significantly effective in returning the caspase-3 activity to near control levels. Apoptosis is a programmed cell death, and involves the systematic disassembly of a cell. The process of apoptosis is mediated by a family of cysteine proteases called caspases (Thornberry and Lazebnik, 1998; Degterev et al., 2003), and among them, caspase-3, existing as a proenzyme, can become activated during the cascade of events associated with apoptosis (Alnemri et al., 1996); therefore, the level of caspase-3 activity is a good indicator of apoptosis. Apoptosis can be induced by ROS formed from ionizing radiation (Shinomiya, 2001; Verheij and Bartelink, 2000; Feinendegen, 2002). The ROS produced by ionizing radiation damages the mitochondrial membrane leading to the release of cytochrome c from the mitochondria into the cytosol, activating the caspases and triggering apoptosis (England and Cotter, 2005). Free radical scavengers, such as thiol antioxidants, vitamin E and beta-carotene have been found to attenuate apoptosis, and exhibit radioprotection in irradiated human lymphoblastic MOLT-3 cells (Ortmann et al., 2004; Lee and Park, 2003). In our investiga- tion, both NACA and NAC were found to protect the cells from radiation-induced apoptosis by controlling the levels of caspase-3, the enzyme that triggers programmed cell death. It is interesting to note here, that the same concentration of NAC (1 mM) that was found to be cytotoxic to cells, was also capable of preventing apoptosis. A similar observation was made in another study, where it was noted that NAC, at a concentration of 10 mM, did not protect against radiation-induced cytotoxicity, but was capable of comple- tely inhibiting a radiation-induced increase in cleaved caspase-3 levels (Samuni et al., 2004).
Other than antioxidant molecules like glutathione, mammalian cells also contain specific antioxidant enzymes that catalyze reactions designed to remove free radicals and other oxidant species. Catalase is one such enzyme found in peroxisomes that catalyzes the removal of hydrogen peroxide. According to our studies, radiation significantly decreased the activity of catalase, possibly due to oxidation of sulfhydryl groups of the enzyme active sites, or due to other structural and functional changes induced in the enzyme by ionizing radiation (Zigman et al., 1996). Both NAC and NACA were capable of returning catalase activity to control levels. The increase in catalase activity may help protect cells by aiding in increased removal of hydrogen peroxide. Two other enzymes investigated in our studies are glutathione peroxidase (GPx) and glutathione reductase (GR). GPx utilizes reduced glu- tathione to reduce hydrogen peroxide to water, while GR replenishes GSH. A decrease was seen in the activities of both GPx
and GR as a result of irradiation. Treatment of the cells with NACA restored GPxand GR activities to near control levels. However, NAC was comparatively less effective in bringing the activities of these enzymes to control levels. Previous investigations have shown that exposure to ionizing radiation does decrease activities of antioxidant enzymes, while antioxidants that help restore these activities have a protective effect on the cells (Karbownik and Reiter, 2000).
It can be concluded from our studies, that NACA is indeed superior to NAC as an antioxidant thiol radioprotector. The effectiveness of NACA in in vitro models renders that further investigation is warranted in vivo models, to determine its effectiveness in protecting normal cells in animals subjected to irradiation. NACA thus holds
promise in being used as a supplement in situations that require protection from ionizing radiation.
Acknowledgements
The authors would like to thank Dr. Viswanathan Subbaratnam from the Department of Radiation Oncology at the Phelps County Regional Medical Center for his help with irradiation of cells. The authors would also like to acknowledge Barbara Harris for carefully editing this manuscript.
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