Possible modulating impact of glutathione disulfide mimetic on physiological changes in irradiated rats

Abstract

Glutathione disulfide mimetic (NOV-002) is a complex of oxidized glutathione (GSSG) formulated with cisplatin at approximately 1000:1 molar ratio. Cisplatin serves to stabilize GSSG but does not assert any therapeutic effect. The objective of this study is to evaluate the impact of NOV-002 on hematological suppression, excessive free radical damage and DNA fragmentation in splenocytes, and metabolite disorders in whole-body γ-irradiated rats. The obtained data revealed that rats treated with 25 mg kg⁻¹ NOV-002 injected intraperitoneally (i.p.) for 5 days after whole-body γ-irradiation (IR) at 6.5 Gy attenuated the decrease of red blood cells, platelets, total white blood cells, absolute lymphocytes and neutrophils counts, hematocrit value, and hemoglobin content. NOV-002 treatment inhibits serum advanced oxidation protein products, malondialdehyde concentrations as well as cholesterol, triglycerides, urea, and creatinine levels, while enhances glutathione content and superoxide dismutase activity and improves DNA fragmentation in splenocytes. These findings provide a better understanding of the NOV-002 modulating impact in whole-body γ-rays-induced hematological toxicities, oxidative stress, and biological disturbances in γ-irradiated rats and could enhance the tolerance to high doses of ionizing IR utilized in radiotherapy.

Keywords

NOV-002 γ-rays oxidative stress DNA fragmentation metabolites

Introduction

Exposure to ionizing radiation is inevitable in nature. However, the most vulnerable populations are either victim of occupational exposure or affected with cancer undergoing routine diagnostic or therapeutic radiation treatment. Radiation generates reactive oxygen species (ROS) such as superoxide anion $(O_{2}^{∙})$ , hydroxyl radical, and hydrogen peroxide (H₂O₂) in the cells.¹ ROS attacks diverse cellular macromolecules such as DNA, lipids, and proteins and induces cell death including apoptosis in vitro or in vivo.² The cells respond to ionizing radiation in variety of ways. The tumor-suppressor protein p53 activates two opposing cellular pathways, in response to DNA damage, one resulting in cell cycle arrest and the other triggering apoptosis. Radiation exposure seems to be able to induce an apoptotic program through ROS generation via activation of caspase 3 pathway.³

Irradiation (IR) at 6 Gy resulted in marked oxidative stress presented by the increase in malondialdehyde (MDA) and decrease in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities.⁴ Also, exposure to γ-rays caused hematological suppression, depletion in the cellularity, viability of bone marrow, and cell viability of spleen.⁵ Although ionizing radiation is a useful tool in a wide variety of fields such as industry, agriculture, military operations, and medicinal studies, especially for cancer therapies, their practical applicability has been limited for many years.⁶ Efforts to develop effective agents to alleviate the harmful effects of ionizing radiation have mounted correspondingly.

Glutathione disulfide mimetic (NOV-002) is an injectable small molecule compound based on oxidized glutathione (GSSG), a naturally occurring substance formulated with cisplatin at approximately 1000:1 molar ratio (Figure 1). The trace cisplatin content of NOV-002 is not sufficient to exert cytotoxicity and improves the bioavailability of NOV-002 in vivo through enhancing GSSG exposure and/or activity or stabilizing the GSSG but does not assert any therapeutic effect.⁷ Even at a relatively high single-dose level compared with that used in clinical studies, NOV-002 had no apparent toxicity in mice.⁸ Initial interest in the drug stemmed from clinical data generated in the Russian Federation where it is approved and marketed under the trade name Glutoxim^®.⁹ NOV-002 is a unique antitumor agent that not only has the ability to inhibit tumor cell proliferation, survival, and invasion but in some settings can also ameliorate cytotoxic chemotherapy-induced hematopoietic and immune suppression.¹⁰

Figure 1.

NOV-002 is composed of the disodium salt of glutathione disulfide in a 1000:1 ratio with cisplatin.

Since NOV-002 is already in clinical trial as a general chemoprotective drug,¹¹ our interest lay in its, as yet undefined, capacity to protect against ionizing IR injuries. The aim of the present study was to evaluate whether NOV-002 in vivo could enhance the tolerance to high doses of ionizing radiation by investigating the characteristics of blood, DNA fragmentation in splenocytes, biological indexes, and oxidative stress parameters in whole-body-irradiated rats at 6.5 Gy.

Materials and methods

Experimental animals

A total of 32 male albino rats (weighing 120–130 g) were obtained from the National Center for Nuclear Research, Atomic Energy Authority, Inchas, Cairo, Egypt. Rats were kept under normal conditions in the temperature range of 18–22°C and fed with rat diet and tap water. All animal treatments were conducted according to the Ethics Committee of the National Research Center and in accordance with the recommendations for the proper care and use of laboratory animals (NIH Publication No. 85-23, revised 1985) in accordance with international ethical considerations.

IR and NOV-002 administration

NOV-002 was purchased from Novelos Therapeutics (Newton, Massachusetts, USA). Rats received 25 mg kg⁻¹ NOV-002¹² injected intraperitoneally (i.p.) for 5 days after whole-body IR.

Whole-body γ-IR was performed using cesium 137 Gammacell 40 biological irradiator at the National Center for Radiation Research and Technology, Nasr City, Cairo, Egypt. Animals were irradiated at an acute single dose of 6.5 Gy delivered at a dose rate of 1 Gy/2.3 min.

Animals were divided into four equal groups (n = 8 per group). The groups include (1) control saline group, (2) γ-rays- irradiated group with 6.5 Gy single dose, (3) NOV-002 group injected i.p. daily for 5 days, and (4) γ-rays IR with 6.5 Gy dose + 25 mg kg⁻¹ NOV-002 intraperitoneal injection daily for 5 days after 2 h of IR group. Experimental animals were sacrificed 10 days post IR.

Blood sample collection and hematological and biochemical assays

Two blood samples were immediately collected by heart puncture. The first sample was collected in heparinized tube (2.25 μL heparin 5 mL⁻¹ blood) for hematological assays. The second blood sample was centrifuged at 1000g for 10 min and the serum was collected and stored at −20°C until analyzed.

Blood samples were investigated using an automated hematology analyzer (BC-2800vet; Mindray, Shenzhen, China). GSH content in blood was measured using spectrophotometry at 412 nm, based on the development of yellow coloration when 5,5′-dithiol-bis(2-nitrobenzoic acid) is added to sulfhydryl compounds.¹³ SOD activity was determined according to the method described by Minami and Yoshikawa based on the generation of $O_{2}^{∙}$ by pyrogallol autoxidation, detection of $O_{2}^{∙}$ by nitroblue tetrazolium formazan color development and by measuring the amount of $O_{2}^{∙}$ generated and scavenged.¹⁴ Lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substances.¹⁵ The method is based on the determination of MDA, an end product of lipid peroxidation, which reacts with thiobarbituric acid in acidic medium to yield a pink-colored trimethine complex exhibiting maximum absorption at 532 nm. Advanced oxidation protein products (AOPP) were measured by spectrophotometry on a microplate reader (DV990BV4.GIO.DE VITA; 990 Win6 Software, Rome, Italy) and were calibrated with chloramine-T (Sigma, St Louis, Missouri, USA) solutions that in the presence of potassium iodide absorb at 340 nm.¹⁶ The chloramine-T absorbance at 340 nm is linear within the range of 0–100 μmol L⁻¹, AOPP concentrations were expressed as micromoles per liter of chloramine-T equivalents.

Serum cholesterol and triglycerides were measured using kits from Spinreact Company (Spain) according to the procedure followed by Richmound¹⁷ and Fossati and Prencipe,¹⁸ respectively. Urea and creatinine were determined using kits according to the procedure described by Young.¹⁹

Determination of DNA fragmentation in spleenocytes

Spleens were removed from rats with scissors and forceps in 0.1 M cold phosphate-buffered saline and passed through a sterilized mesh to obtain single-cell suspensions according to Yuan et al.²⁰ The extent of DNA fragmentation in splenocytes was determined according to the method followed by Sellins and Cohen,²¹ based on the concept that extensively fragmented double-stranded DNA can be separated from chromosomal DNA upon centrifugal sedimentation. The cells were harvested by centrifugation at 200g for 10 min. The pellet was lysed with 0.4 mL hypotonic lysing buffer, and the lysates were centrifuged at 13,000g for 10 min to separate intact DNA from fragmented chromatin. The supernatants contain fragmented DNA. Electrophoresis was carried in 0.75% agarose for 75 min at 90 V. DNA was visualized with ethidium bromide.

Statistical analysis

The results are presented as mean ± SE. Statistical analysis was performed using one-way analysis of variance followed by Duncan’s multiple range test using Statistical Package for Social Sciences Version 15.0 (SPSS Inc., Chicago, Illinois, USA) software for Windows. The value of p < 0.05 was considered statistically significant.

Results

Table 1 shows significant (p < 0.05) decrease in red blood cells (RBCs), platelets counts, hemoglobin (Hb) content, and hematocrit (Hct) values in the γ-irradiated group compared with control group. In γ-irradiated + NOV-002 group, the recovery of the hematological parameters (RBCs, platelets, Hb, and Hct) was significant compared with the irradiated group (Table 1).

Table 1.

Leucocytes, neutrophils, lymphocytes, RBCs, platelets counts, Hb content, and Hct values in different groups.^a

Rat groups	Control	NOV-002	γ-Rays (6.5 Gy)	γ-Rays + NOV-002
Parameters
Leucocytes (×10³ mm⁻³)	10.47 ± 2.12	10.09 ± 1.6	2.52 ± 0.46^b,c	4.67 ± 1.67^b,c,d
Neutrophils (×10³ mm⁻³)	1.70 ± .32	1.45 ± 0.70	0.82 ± 0.03^b,c	1.35 ± 0.15^d
Lymphocytes (×10³ mm⁻³)	8.80 ± 1.64	8.70 ± 1.3	1.70 ± 0.17^b,c	3.23 ± 0.58^b,c,d
RBCs (×10⁶ mm⁻³)	6.40 ± 0.36	6.60 ± 0.25	3.50 ± 0.47^b,c	4.90 ± 0.50^b,c,d
Platelets (×10³ mm⁻³)	972 ± 37	1000 ± 70	570 ± 34^b,c	920 ± 37^c,d
Hb (g dL⁻¹)	13.22 ± 0.39	14.40 ± 0.57	7.41 ± 0.46^b,c	10.16 ± 0.8^b,c,d
Hct (%)	41.63 ± 1.50	43.45 ± 1.40	22.23 ± 1.37^b,c	33.57 ± 2.90^b,c,d

RBCs: red blood cells; Hb: hemoglobin; Hct: hematocrit; NOV-002: glutathione disulfide mimetic.

^aValues are expressed as means ± standard error (n = 8).

^bSignificantly different from control.

^cSignificantly different from NOV-002.

^dSignificantly different from γ-rays.

Total leukocyte count was significantly (p < 0.05) decreased after IR in comparison with the control group. The recovery of total leukocytes in peripheral blood following 25 mg kg⁻¹ NOV-002 injection after IR was significantly high compared with the irradiated group. Furthermore, significant attenuation of blood neutrophils and lymphocytes count was observed in the 25 mg kg⁻¹ NOV-002 + γ-irradiated group (Table 1).

Table 2 shows a significant (p < 0.05) increase in serum MDA and AOPP levels and a significant decrease in blood GSH level and SOD activity in the γ-irradiated group compared with the control group. Injection of 25 mg kg⁻¹ NOV-002 after γ-rays exposure reduced serum MDA and AOPP levels and increased blood GSH level and SOD activity significantly (p < 0.05) compared with the γ-irradiated group after 10 days.

Table 2.

Blood GSH content and SOD and serum MDA and AOPP levels in different groups.^a

Rat groups	Control	NOV-002	γ-Rays (6.5 Gy)	γ-Rays + NOV-002
Parameters
GSH (mg dL⁻¹)	47.23 ± 6.3	49.39 ± 5.5	35.19 ± 3.9^b,c	40.03 ± 3.0^b,c,d
SOD (U mL⁻¹)	5.68 ± 0.3	6.31 ± 0.1	3.43 ± 0.4^b,c	5.33 ± 0.3^c,d
MDA (nmol L⁻¹)	56.30 ± 2.9	54.27 ± 2.2	92.85 ± 4.5^b,c	68.69 ± 3.5^b,c,d
AOPP (μmol L⁻¹)	42.33 ± 3.29	41.53 ± 1.36	52.02 ± 3.31^b,c	44.41 ± 3.48^d

GSH: glutathione; SOD: superoxide dismutase; MDA: malondialdehyde; AOPP: advanced oxidation protein products.

^aValues are expressed as means ± standard error (n = 8).

^bSignificantly different from control.

^cSignificantly different from NOV-002.

^dSignificantly different from γ-rays.

IR with γ rays at 6.5 Gy resulted in increase in serum triglycerides, cholesterol, and urea and creatinine levels. In γ-irradiated + 25 mg kg⁻¹ NOV-002 group, the increase in serum triglycerides, cholesterol, and urea and creatinine levels at 10 days posttreatment was significantly ameliorated (Table 3).

Table 3.

Serum urea, creatinine, triglycerides, and cholesterol levels in different animal groups.^a

Rat groups	Control	NOV-002	γ-Rays (6.5 Gy)	γ-Rays + NOV-002
Parameters
Urea (mg dL⁻¹)	54.02 ± 6.01	51.02 ± 5.01	67.02 ± 7.01^b,c	57.5 ± 6.30^c,d
Creatinine (mg dL⁻¹)	5.00 ± 0.17	4.70 ± 0.20	5.90 ± 0.30^b,c	5.30 ± 0.30^c,d
Triglycerides (mg dL⁻¹)	45.05 ± 2.6	44.69 ± 3.4	74.69 ± 4.2^b,c	62.59 ± 5.2^b,c,d
Cholesterol (mg dL⁻¹)	85.09 ± 5.6	84.09 ± 4.4	115.24 ± 4.3^b,c	72.29 ± 2.9^b,c,d

NOV-002: glutathione disulfide mimetic.

^aValues are expressed as means ± standard error (n = 8).

^bSignificantly different from control.

^cSignificantly different from NOV-002.

^dSignificantly different from γ-rays.

DNA fragmentation in splenocytes

Figure 2 represents black and white photograph of agarose gel stained with ethidium bromide under ultraviolet radiation. The control group at lane (A) and NOV-002 group at lane (C) showed normal DNA with no fragmentation or damage recorded; both groups have neither fragmentation nor necrosis. While γ-irradiated group at lane (B) showed clear DNA damage, fragmentation, and necrosis as DNA traveled as smear from top of the gel to the bottom. Treatment of rats with NOV-002 after γ-IR reveals significant improvement of DNA fragmentation in splenocytes as shown in lane (D).

Figure 2.

NOV-002 impact on splenocytes DNA fragmentation induced by γ-rays. Lane A: control group; lane B: γ-irradiated group; lane C: NOV-002 group; and lane D: γ-irradiated + NOV-002 group. NOV-002: glutathione disulfide mimetic; DNA: deoxyribonucleic acid.

Discussion

The present results show that γ-IR at 6.5 Gy induced significant depressions in RBCs, platelets, total WBCs, lymphocyte and neutrophil counts, Hct value, and Hb content, besides clear DNA damage, fragmentation and necrosis in DNA of splenocytes. These findings corroborate previous results.^{5

–22,23} Fragmentation of DNA into kilobase size fragments appears to be an early event in apoptosis, preceding the complete digestion of DNA into multiples of nucleosomal size fragments.²⁴

Wang et al.²⁵ reported that exposure of C57BL/6 mice to 6.5 Gy induced a quantitative and qualitative reduction of hematopoietic stem cells (HSC), resulting from induction of senescence and impairment of HSC self-renewal via activation of the specific cellular pathways. This is complicated by thrombocytopenia and concomitant hemorrhages, besides effects in adaptive immune system resulting from apoptosis of lymphocytes and deficient lymphopoiesis.²⁶ The present data show that i.p. treatment of NOV-002 cause improvement in RBCs, platelets, total WBCs, absolute lymphocytes and neutrophils counts, Hct value, and Hb content as well as improvement of DNA fragmentation in splenocytes. The results corroborate the findings of Diaz-Montero et al.²⁷ that NOV-002 treatment at a clinically comparable dose regimen attenuated cyclophosphamide-induced reduction in bone marrow hematopoietic stem and progenitor cells and a significant improvement in hematopoietic and immune functions in mice, suggesting that the myeloproliferative effects of NOV-002 may be related to recovery rather than acute protection.

It is well documented that the redox balance, particularly involving thiols such as GSH influences aspects of myeloproliferation, hematopoietic progenitor cell mobilization, and immune response.²⁸ NOV-002 has been reported to produce multiple intracellular changes indicative of alterations in redox balance against the backdrop of stimulating the rate of cell proliferation.²⁹ NOV-002 treatment also increased tolerance to chemotherapy, improved hematological and immune parameters, normalized kidney/liver toxicity markers, improved quality of life through activated p38, c-Jun-NH₂-kinase, and extracellular signal-regulated kinase and increased in phosphorylation of three proteins that have previously been linked with hematopoiesis.⁷

In the current study, significant inhibition of blood GSH content and SOD activity as well as significant elevation of MDA and AOPP levels were observed in γ-irradiated rats. Depletion of blood GSH level and SOD activity may be due to their utilization by the enhanced production of ROS.³⁰ $O_{2}^{∙}$ is one of the strongest oxidants and plays a central role as a source of many ROS. SOD is an important antioxidative enzyme catalyzing the conversion of $O_{2}^{∙}$ to H₂O₂.

Plasma membrane fluidity and permeability are directly affected by radiation-induced lipid damage.³¹ Free radicals attack biomolecules such as fatty acid component of membrane lipids, proteins, and DNA, leading to lipid peroxidation, AOPP, strand breaks, and ultimately cell death³² as confirmed by fragmentation and necrosis of DNA in splenocytes as well as elevation of MDA and AOPP in the present study. Free radicals destroy the cell membranes, enhance cholesterol release, and increase lipid peroxidation.³³ On the other hand, NOV-002 injection daily for 5 days attenuates γ-IR-induced MDA and AOPP increment and restores the GSH level and SOD activity, implicating an antioxidant effect of this NOV-002.

Intraperitoneal injection of glutathione mimetic (tricyclodecan-9-yl-xanthogenate, D609) protects brain mitochondria against oxidative stress induced in vitro by different oxidants. In vivo delivery of D609 showed significant protection against protein oxidation, lipid peroxidation, and cytochrome c release in gerbil brain mitochondria.³⁴ Intravenous injection of 50 mg kg⁻¹ D609 to mice for 10 min prior to total body IR (6.5 and 8.5 Gy) protected the mice from IR-induced lethality. Thus, these results indicate that D609 is a potent antioxidant and has the ability to inhibit IR-induced cellular oxidative stress.³⁵

In general, the pleiotropic pharmacological effects of NOV-002 are attributable to the glutathione disulfide component of the drug, and modulation of cellular redox balance is a feature central to NOV-002s mechanism of action.⁷ NOV-002 uptake is highest in the liver, kidney, and immuno- and hematopoiesis organs. After the drug is reduced by natural cellular metabolic systems, its metabolites, such as reduced GSH, can undergo intracellular degradation to its constituent amino acids that can be used as protein construction material or participate in cellular energy metabolism. Oxidation of essential cysteine residues in plasma proteins may reflect in vivo effects of NOV-002 that outlast increased plasma levels of GSH or GSSG.⁸ It is generally accepted that GSSG does not passively cross the cell membrane.³⁶ Thus, the effects of NOV-002 on cells may be mediated via cell surface targets. NOV-002 treatment of HL-60 cells reduced cell surface thiol content through oxidative modification of cell surface proteins. Since NOV-002 inhibits cell surface protein disulfide isomerase activity, this enzyme may represent a cell surface target for this drug.³⁷

In the current study, lipid metabolites (triglycerides and cholesterol) were elevated in serum of irradiated animals, which could be attributed to acceleration of other pathways of cholesterol formation in the liver and other tissues. Free radicals destruct cell membranes and enhanced cholesterol release and increase lipid peroxidation,³³ which comes in agreement with Salama and El-Fatih.²² In addition, IR induced hyperlipidemia through cell membrane destruction, enhancement of lipid metabolism, cholesterol release, and triglyceride synthesis.³⁸ The increase of triglycerides may be attributed to the inhibition in lipoprotein lipase activity.³⁹ NOV-002 treatment alleviated hyperlipidemia induced by γ-IR via its free radical scavenging and membrane stabilizing ability as confirmed by attenuation of MDA and AOPP marked increments and restoration of the GSH level and SOD activity.

Serum urea and creatinine levels in 6.5 Gy γ-irradiated animals were increased. Radiation-induced oxidative stress in the kidney was associated with a significant increase in the level of serum urea and creatinine. It could be referred to increased ammonia formed by deamination of amino acids in the liver converted to urea.⁴⁰ The impaired detoxification function of the liver by IR could also contribute in the increase of urea in the blood.⁴¹ Serum creatinine elevation might be attributed to the interaction of ionizing radiation with the sites of biosynthesis.⁴²

NOV-002 treatment attenuated the kidney function disturbance induced by γ-IR. NOV-002-treated (25 mg kg⁻¹ i.p. for 5 days) mice had significantly lower levels of plasma creatinine compared with cisplatin-treated mice (15 mg kg⁻¹, i.p. single dose) alone. Moreover, NOV-002 protected the kidneys from cisplatin-mediated proximal tubule damage, including dilation of tubules and the presence of protein casts. Preclinical results showed that NOV-002 enacts protection against cisplatin-induced nephrotoxicity through improving in vivo kidney functions and reducing morbidity and mortality in the mice.¹²

In conclusion, NOV-002 reduces hematological toxicities, serum lipid peroxidation, triglycerides, cholesterol, AOPP, urea, and creatinine and improves blood GSH level and SOD activity induced by γ-rays exposure thus restrains oxidative stress and prevents DNA fragmentations. The present study provides details about the effect of NOV-002 in rats exposed to whole-body γ-IR, suggesting that it could be a potential radioprotective pharmaceutical agent for radiotherapy complications.

Footnotes

Acknowledgment

The authors are grateful to the Department of Radiation Biology, National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt for providing support.

Conflict of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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