http://informahealthcare.com/cot ISSN: 1556-9527 (print), 1556-9535 (electronic) Cutan Ocul Toxicol, Early Online: 1–6 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15569527.2014.910802
RESEARCH ARTICLE
The radioprotective effect of Nigella sativa on nitrosative stress in lens tissue in radiation-induced cataract in rat
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Seyithan Taysi1, Zainab Khaleel Abdulrahman1,6, Seydi Okumus2, Elif Demir1, Tuncer Demir3, Muslum Akan1, Edibe Saricicek1, Vahap Saricicek4, Adnan Aksoy5, and Mehmet Tarakcioglu1 1
Department of Medical Biochemistry, 2Department of Ophthalmology, 3Department of Physiology, Medical School, Gaziantep University, Gaziantep, Turkey, 4Department of Anesthesiology, Medical School, Gaziantep University, Gaziantep, Turkey, 5Department of Ophthalmology, Medical School, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey, and 6Kirkuk Health Department, Ministry of Health, Irak Abstract
Keywords
Objective: The aim of this study was to investigate the antioxidant and radioprotective effects of Nigella sativa oil (NSO) and thymoquinone (TQ) against ionizing radiation-induced cataracts in lens after total cranium irradiation (IR) of rats with a single dose of 5 gray (Gy). Materials and methods: Seventy-four Sprague-Dawley rats were used for the experiment. The rats were randomly divided into six groups. Group A received total cranium IR plus NSO (1 g kg–1 d–1) orally through an orogastric tube. Group B received total cranium IR plus TQ (50 mgkg–1 d–1) daily by intraperitoneal injection. Group C received 5 Gy of gamma IR as a single dose to total cranium plus 1 ml saline. Group D1 just received 1 ml saline. Group D2 just received dimethyl sulfoxide. Group D3 did not receive anything. Results: At the end of the 10th d, cataract developed in 80% of the rats in IR group only. After IR, cataract rate dropped to 20% and 50% in groups which were treated with NSO and TQ, respectively, and was limited at grades 1 and 2. Nitric oxide synthase activity, nitric oxide and peroxynitrite levels in the radiotherapy group were higher than those of all other groups. Conclusions: The results implicate a major role for NSO and TQ in preventing cataractogenesis in ionizing radiation-induced cataracts in the lenses of rats, wherein NSO were found to be more potent.
Cataract, free radical, irradiation, Nigella sativa, nitric oxide, nitrosative stress
Introduction Radiation therapy has been used effectively in treating head and neck cancers for many decades, which is a common and important tool for cancer treatment1,2. The killing action of ionizing radiation is mainly mediated through the free radicals generated from the radiolytic decomposition of cellular water, including superoxide radical (O 2 ) and hydroxyl radical (OH–), which can cause damage in most major cellular macromolecules such as DNA, proteins, lipids, membrane, etc. These reactions take place in tumor as well as normal cells when exposed to radiation3,4. Eye morbidity is widely observed in patients receiving total-body irradiation prior to bone marrow transplantation or radiotherapy for ocular or head and neck cancers5. Cataract blindness is the major cause of preventable blindness worldwide, especially in the developing countries6. It is the opacity of eye lens that interferes with vision. Cataracts are formed in response to a variety of different
Address for correspondence: Prof. Dr. Seyithan Taysi, Department of Medical Biochemistry, Gaziantep University Medical School, Gaziantep, Turkey. Tel: +90 342 3601617. Fax: +90 342 3600753. E-mail: [email protected]
History Received 18 November 2013 Revised 28 February 2014 Accepted 28 March 2014 Published online 19 June 2014
agents and environmental stresses, and this damage seems in almost all cases to have an oxidative damage component. Although cataract is a known late effect of ionizing radiation exposure, most of the experimental studies have concentrated on single, acute high doses or multiple, fractionated and chronic exposure of radiation3. Nigella sativa (NS), commonly known as black seed, belongs to the Ranunculaceae family. NS contains 430 w/w of fixed oil and 0.40–0.45 w/w of a volatile oil. The volatile oil has been shown to contain 18.4–24% thymoquinone (TQ) and 46% monoterpenes such as p-cymene and a-pinene. Nigella sativa oil (NSO) and seed constituents, in particular TQ, have shown potential medicinal properties in traditional medicine7,8. NSO and TQ, natural main constituent of the volatile oil of NS seeds, are reported to possess strong antioxidant properties against oxidative damage induced by a variety of free radical-generating agents. A lot of pharmacological and toxicological studies have reported it to possess diverse pharmacological properties such as antioxidant, hepatoprotective, neuroprotective, antidiabetic, antiinflammatory, nephroprotective and anticarcinogenic9,10. Efforts to decrease toxicity of irradiation (IR) to normal tissue, organs and cells have led into searching for cytoprotective agents. Unfortunately, most of the
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radioprotectors possess toxic side effects, which limit its role in medical treatment. For this reason, investigations for effective and non-toxic compounds with radioprotection capability led to increasing interest in naturally occurring antioxidant such as NSO and TQ. To our knowledge, there is no experimental study that simultaneously investigates the effects of NSO and TQ supplementations on nitric oxide synthase (NOS) activity, nitric oxide (NO), peroxynitrite (ONOO–) in the lens tissue of rats receiving ionizing radiation. Therefore, in this study, we aimed to investigate the effects of these supplementations on these nitrosative stress parameters in the lens tissue of rats with or without exposure to total cranium IR.
Material and methods Animals and experiments Fifty-four rats, 10–12 weeks old, weighing 200 ± 25 g, the time of radiation bred at Gaziantep University Medical School, Experimental Animal Laboratory were used for the experiment. The animals were purchased from Gaziantep University Medical School, Experimental Animal Laboratory, and housed in cages one week before the start of the experiments. All animals received humane care in compliance with the guidelines of Gaziantep University Research Council’s criteria. Food and tap water were available ad libitum. The laboratory was windowless with automatic temperature (22 ± 1 C) and lighting controls (14 h light/10 h dark). Rats were randomly divided into six groups (8 rats from control groups and 10 rats from other groups) and placed in separate cages during the study. This study was approved by the local ethics committee of the Gaziantep University. The groups were as follows: Group A: (IR plus NSO group) received both total cranium 5 gray (Gy) of gamma IR as a single dose and NSO (1 g kg–1 d–1) orally through an orogastric tube. NSO supplementation in group A lasted for 10 d. NSO was obtained from Origo Gida Kimya Tarim Urn. San. ve Tic. Ltd. Sti. (Gaziantep, Turkey). Group B: (IR plus TQ group) received TQ (50 mgkg–1 d–1) daily by intraperitoneal (IP) injection starting 30 min before the dose radiation and subsequently daily for 10 d after IR. TQ was dissolved in DSMO just before giving to the rats. Group C: IR group received total cranium 5 Gy of gamma IR as a single dose. Also physiological saline solution (1 mlkg–1 d–1, IP injection) was daily administered for 10 d. Group D1 (control group for group A): control group received neither NSO and TQ nor IR, but received daily physiological saline solution (1 mlkg–1 d–1, orally) for 10 d. Group D2 (control group for group B): Control group did not receive NSO, TQ and IR, but received daily IP injection of dimethyl sulfoxide at an equal volume to that of TQ used in group B for 10 d. Group D3 (normal control group): this group did neither receive NSO, TQ and IR nor oral/IP physiological saline solutions. Prior to total cranium IR, all rats were anesthetized with 80 mg/kg ketamine hydrochloride (Pfizer Ilac, Istanbul, Turkey) and placed on a tray in the prone position. The rats
Cutan Ocul Toxicol, Early Online: 1–6
in the IR and the IR plus TQ groups received IR via a Cobalt60 teletherapy unit (Picker, C9, Maryland, NY) from a source-to-surface distance of 80 cm by 5 5 cm anterior fields, with the total cranium gamma IR being a single dose of 5 Gy, while the rats in the control and sham control groups received sham IR. The dose rate was 0.49 Gy/min. The central axis dose was calculated at a depth of 0.5 cm. Determination of clinical cataract In this study, the lens opacities classification system, version III (LOCS III) was used in the cataract classification11. Before the cataracts were graded, the pupils were dilated with tropicamide (1.0%) and phenylephrine hydrochloride (2.5%) drops, and proparacaine (0.5%) was used as a topical anesthetic. The lenses were graded by slit-lamp biomicroscopy (Keeler PSL Classic, Keeler Ltd., Windsor, UK) as follows: the features of nuclear opacification and brunescence are graded according to one set of six photographs. The brightness of scatter from the nuclear region has been designated nuclear opalescence (NO) and the intensity of brunescence, nuclear color (NC) (Figure 1). The amount of cortical cataract (C) is determined by comparing the estimated aggregate of cortical spoking with that seen in five separate photographs (Figure 2). Similarly, the estimated amount of posterior subcapsular cataract (P) is determined by comparing it with another five photographs depicting increasing amounts of posterior subcapsular cataract (Figure 3). At the beginning of the experiment, all rats were examined biomicroscopically and were only included in this study if their lenses had been NOo–, NC0–, C0– and P0– (Figure 4). Biochemical analysis At the end of the study, all rats were anesthetized with 80 mg/kg ketamine hydrochloride (Pfizer Ilac) and then all animals were killed by decapitation, and their eyes were enucleated, and the lenses were dissected immediately.
Figure 1. Nuclear cataract.
Nigella sativa in irradiation-induced cataract
DOI: 10.3109/15569527.2014.910802
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conducted on this fraction. All of the procedures were performed at 4 C.
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Determination of NOS activity and NO and ONOO– levels
Figure 2. Cortical cataract.
Figure 3. Posterior subcapsular cataract.
NOS activity assay is based on the diazotization of sulfanilic acid by NO at acid pH and subsequent coupling to N-(1-naphthyl) ethylenediamine. To 0.1 ml of sample, 0.2 ml of 0.2 M arginine was added and incubated at 37 C for 1 h. Then, the combination, 0.2 ml of 10 mM HCl, 100 mM sulfanilic acid and 60 mM N-(1-naphthyl) ethylenediamine was added. After 30 min, the absorbance of the sample tube was measured against a blank tube at 540 nm12. Results are expressed as U/mg protein. NO levels in lens tissue were measured using the Griess reagent as previously described13,14. Griess reagent, the mixture (1:1) of 0.2% N-(1-napthyl) ethylenediamine and 2% sulphanilamide in 5% phosphoric acid, gives a red–violet diazo dye with nitrite, and the resultant color was measured at 540 nm. First nitrate was converted to nitrite using nitrate reductase. The second step was the addition of Griess reagent, which converts nitrite to a deep purple azocompound, photometric measurement, the absorbance of 540 nm determines the nitrite concentration. Results were expressed as mmol/g wet tissue. ONOO– assay was determined as described15,16. Ten microliters of sample was added to 5 mM phenol in 50 mM sodium phosphate buffer (pH 7.4) to get a final volume of 2 ml. After 2-h incubation in a dark place at 37 C, 15 mL of 0.1 M NaOH was added and the absorbance, at wavelength of 412 nm, of the samples was immediately recorded. The yield of nitrophenol was calculated from " ¼ 4400/M/cm. Results were expressed as mmol/g wet tissue. The protein content was determined as described17. Biochemical measurements were carried out using a visible/UV spectrophotometer (Shimadzu U 1601, Kyoto, Japan). Statistical analyses Cataract developments in the different treatment groups were compared with Fischer’s exact Chi-square test. Statistical and correlation analyses were undertaken using a one-way variance analysis and Spearman’s rank correlation test, respectively. Following the analysis of variance, the significance of differences between groups was tested using least significant difference multiple range test procedure. Acceptable significance was recorded when p values were 50.05. Statistical analysis was performed with Statistical Package for the Social Sciences for Windows, version 11.5 (SPSS Inc., Chicago, IL).
Results Figure 4. Clear lens.
Lenses were homogenized by an IKA-NERKE (Staufen, Germany) homogenizer in physiological saline solution (20fold) on ice for 10 s in the first speed level. The homogenate was centrifuged at 10 000 g for 1 h to remove debris. The clear upper supernatant was collected, and all assays were
In this study, the LOCS III was used in the cataract classification11. The lenses were graded by slit-lamp biomicroscopy (Keeler PSL Classic, Keeler Ltd.). All the rat lenses at the beginning were graded as 0. After IR, cataract developed in 80% of the rats in radiotherapy group. After IR, cataract rate dropped to 20% and 50% in groups that were treated with NSO and TQ, respectively, and was limited at grades 1 and 2 (Table 1). NOS activity, levels of NO and ONOO– in the radiotherapy group were significantly higher
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Table 1. Cataract situation in the rat lenses examined by slit-lamp microscopy in all groups. The situation of cataract Kind of cataract
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Group Group Group Group Group Group
Presence
NC
CC
PSC
Absent
Total
2 5 8 0 0 0
0 0 1 – – –
1 2 2 – – –
1 3 5 – – –
8 5 2 8 8 8
10 10 10 8 8 8
A B C D1 D2 D3
Group A: Irradiation (IR) plus Nigella sativa oil (NSO) group; group B: IR plus thymoquinone (TQ) group; group C: IR group only; group D1 (the control group of A group); group D2 (the control group of B group); group D3 (normal control group); NC: nuclear cataract; CC: cortical cataract; and PSC: posterior subcapsular cataract. Table 2. Mean ± SD of nitric oxide synthase activity, nitric oxide and peroxynitrite in lens tissue of rats in all groups. NOS (U/mg protein) Group Group Group Group Group Group
A B C D1 D2 D3
284.11 ± 24.83b,c,e,g 288.79 ± 59.98b,c,f,g 729.20 ± 140.44 202.21 ± 27.46b 198.89 ± 37.61b 187.38 ± 31.01b
NO OONO– (nM/g wet tissue) (mM/g wet tissue) 4.28 ± 0.43b,c,e 4.46 ± 0.44a,d,f,g 5.04 ± 0.35 4.03 ± 0.49b 3.83 ± 0.49b 3.71 ± 0.33b
101.52 ± 14.84b,d 108.90 ± 20.64b,d 172.81 ± 18.85 103.05 ± 32.36b,d 97.31 ± 16.19b,c 62.97 ± 23.69b
a: p50.01, b: p50.0001 versus group C, c: p50.01, d: p50.001 versus group D3, e: p50.05, f: p50.01 versus group D2, g: p50.05 versus group D1. Group A: Irradiation (IR) plus Nigella sativa oil (NSO) group; group B: IR plus thymoquinone (TQ) group; group C: IR group only; group D1 (the control group of A group), group D2 (the control group of B group), group D3 (normal control group): nitric oxide synthase (NOS): nitric oxide (NO): peroxynitrite (ONOO–).
than all other groups (Table 2). In this study, while the cataract ratio was highly observed in radiotherapy group, it decreased in the other groups in which NSO and TQ were given after radiation. NSO was found to be more effective in anticataractogenic effect than TQ. Compared to the IR group, a significant reduction in cataract formation was observed in group A (p ¼ 0.007). As for in group C, the decrease in cataract was not statistically significant (p40.05). We observed a significant increase of cataract formation in the IR group, when compared to the control groups (p50.001).
Discussion As the world’s population ages, cataract-induced visual dysfunction and blindness are on the increase. Cataract is a major cause of blindness and of severe visual impairment leading to bilateral blindness in an estimated 20 million people worldwide. In developing countries, 50–90% of all blindness is caused by cataracts. Pharmacological treatment to prevent human cataracts has so far not been achieved. The exact mechanism of cataract formation is still not very clear2,18. Many studies are being conducted to explore the mechanism of cataractogenesis using various models of cataract and to target crucial steps to halt this process.
Limitations in acceptability, accessibility and affordability of cataract surgical services make it more relevant and important to look into alternative pharmacological measures for treatment of this disorder. Therefore, a lot of effort is being laid on identification of natural compounds that will help to prevent cataractogenesis6. The aim of radiotherapy is to kill cancer cells while causing as little damage as possible to normal cells. Eighty percent of patients with cancer need radiotherapy at some time or other, either for a curative or a palliative purpose. To obtain optimum results, a judicious balance between the total dose of radiotherapy delivered and the threshold limit of the surrounding normal critical tissues is required. To obtain better tumor control with a higher dose, the normal tissues should be protected against radiation injury. Thus, the role of radioprotective compounds is very important in clinical radiotherapy1,8. Aerobic metabolism produces reactive oxygen and nitrogen species (ROS and RNS, respectively) that are normally inactivated through diverse antioxidant mechanisms. Nitrosative stress occurs when intermediates are produced from nitrosate thiol, hydroxy and amine groups. When mammalian cells were exposed to nitrosative stress by addition of NO donor, little toxicity was observed. Under nitrosative stress conditions, nitrosating intermediates have the greatest affinity for thiols, such as glutathione (GSH), suggesting that they are a primary target. Cells depleted of GSH were dramatically more susceptible to toxicity from nitrosative stress. Since these free radicals may also damage normal tissue, the balance between antioxidants and prooxidants is critical for normal function19. However, during oxidative and nitrosative stress conditions, the balance between free radicals and antioxidants shifts toward the former, resulting in diseases such as liver damage, cancer, rheumatoid arthritis, infectious diseases and atherosclerosis and in aging20. Radiation is a known producer of free radicals, contributing to radiation injury in cells and formed in cells. Ionizing radiation damage biological tissues and they produce free radical in aqueous solution, for instance, cell cytoplasm, which in turn can lead to oxidative damage to biological molecules such as nucleic acids, proteins and lipids, leading to cataract. One of the mechanisms proposed to explain lens opacification is the oxidation of crystallins, either by radiation or ROS and RNS. The other mechanism is the formation of opacities with increased calcium release by mitochondrial damage. Ionizing radiation has been shown to enhance the production of these free radicals in cells4,21,22. For this reason, to prevent injury caused by radiation on healthy tissue, many investigations related natural products that have antiviral, anticancer, immunostimulant and antioxidant effects have been constructed. Several studies on radioprotective agent are ongoing. To our knowledge, this is the first report demonstrating lens radioprotection by NSA and TQ. According to our results, NOS activity, NO and ONOO– levels were significantly higher in lens tissues of irradiated rats that were exposed to total cranium IR, compared to other groups. Similar results were obtained in a previous study conducted by Manikandan et al.6. The nitrosative stress
Nigella sativa in irradiation-induced cataract
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DOI: 10.3109/15569527.2014.910802
parameters in the groups receiving IR plus NSO or TQ treatment were significantly decreased compared to the group that received IR only. These results revealed that NSO and TQ clearly decreased the nitrosative stress in lens tissue induced by total cranium IR. NO is synthesized endogenously by the enzyme, NOS, via its precursor arginine. Endogenous NO plays a dual role in specialized tissues and cells, where it is not only an essential physiological signaling molecule mediating various cell functions but also induces cytotoxic and mutagenic effects when present excessively under oxidative stress conditions induced by IR23,24. There are two distinct categories in the chemical biology of NO: direct and indirect effects. Direct effects are those chemical reactions where NO reacts directly with its biological target. The direct effects are very rapid reactions that occur at low NO concentrations and generally involve heme proteins such as guanylate cyclase, cytochrome P450 and hemoglobin, whereas indirect effects are mediated by RNS, which are derived from NO metabolism. It requires that NO is first activated by O or oxygen to form RNS, which then 2 undergoes further reactions with the respective biological target19. Furthermore, NO reacts rapidly with O 2 to form ONOO–, which may be cytotoxic by itself or easily decompose to the highly reactive and toxic OH– and nitrogen dioxide23,24. These RNS are highly reactive with major cellular macromolecules such as DNA, proteins, lipids, membrane, etc., and are thought to be responsible for NOmediated cell death19. Some studies have been reported that NO levels in rats that are exposed to whole-body IR were significantly higher6,25. In this study, NOS activity, NO and ONOO– levels in lens tissue of rats exposed to radiation were also increased before the treatment of rats with NSO and TQ. According to the results, low nitrosative stress parameters in the NS and TQ treatment groups may suggest that NSO and TQ have protective effects against toxicity to IR. Similarly, NS has the potential to reduce toxicity against oxidative stress based on the literature. It is a well-known fact that NSO and TQ have antiinflammatory, antioxidant and antineoplastic effects both in vitro and in vivo studies. Antioxidant/ antiinflammatory effects of these agents have been studied in various disease models, including cancer, sepsis, atherosclerosis, asthma and carcinogenesis. Furthermore, it has been shown that TQ could act as a free radical and superoxide radical scavenger9,10,26.
Conclusion In this study, we have found that NOS activity, NO and ONOO– levels in the radiotherapy group were higher than those of all other groups. This is the first study that simultaneously investigates the effects of NSO and TQ on the nitrosative stress in the lens tissue of the irradiated rats. The results obtained in the study suggest an important role of nitrosative stress in the IR-induced cataractogenesis. We showed that these natural substances clearly appeared to prevent cataractogenesis in IR-exposed lenses by inhibiting RNS generation. NSO and TQ clearly have antioxidant properties and free radical scavenging activities. These are likely to be a valuable drug for protection against gamma-IR
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and/or be used as an antioxidant against cataractogenesis, oxidative/nitrosative stress and other severe side effects occurred in head and neck cancers patients treated with radiotherapy. However, further studies on this subject are needed to implement the use of the natural compounds in clinical protection against ionizing radiation, especially in treatment of patients with cancer exposed to radiotherapy.
Declaration of interest The authors report no declarations of interest. None of the authors has a commercial interest, financial interest and/or other relationship with manufacturers of pharmaceuticals, laboratory supplies and/or medical devices or with commercial providers of medically related services.
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17. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248–254. 18. Yagci R, Aydin B, Erdurmus M, et al. Use of melatonin to prevent selenite-induced cataract formation in rat eyes. Curr Eye Res 2006;31:845–850. 19. Wink DA, Miranda KM, Espey MG. Cytotoxicity related to oxidative and nitrosative stress by nitric oxide. Exp Biol Med (Maywood) 2001;226:621–623. 20. Taysi S, Demircan B, Akdeniz N, et al. Oxidant/antioxidant status in men with Behcet’s disease. Clin Rheumatol 2007;26:418–422. 21. Karslioglu I, Ertekin MV, Kocer I, et al. Protective role of intramuscularly administered vitamin E on the levels of lipid peroxidation and the activities of antioxidant enzymes in the lens of rats made cataractous with gamma-irradiation. Eur J Ophthalmol 2004;14:478–485.
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22. Ertekin MV, Kocer I, Karslioglu I, et al. Effects of oral Ginkgo biloba supplementation on cataract formation and oxidative stress occurring in lenses of rats exposed to total cranium radiotherapy. Jpn J Ophthalmol 2004;48:499–502. 23. Taysi S, Uslu C, Akcay F, Sutbeyaz MY. Malondialdehyde and nitric oxide levels in the plasma of patients with advanced laryngeal cancer. Surg Today 2003;33:651–654. 24. Taysi S, Polat MF, Sari RA, Bakan E. Serum adenosine deaminase and cytidine deaminase activities in patients with systemic lupus erythematosus. Clin Chem Lab Med 2002;40:493–495. 25. Taysi S, Koc M, Buyukokuroglu ME, et al. Melatonin reduces lipid peroxidation and nitric oxide during irradiationinduced oxidative injury in the rat liver. J Pineal Res 2003;34: 173–177. 26. Woo CC, Kumar AP, Sethi G, Tan KH. Thymoquinone: potential cure for inflammatory disorders and cancer. Biochem Pharmacol 2012;83:443–451.
RESEARCH ARTICLE
The radioprotective effect of Nigella sativa on nitrosative stress in lens tissue in radiation-induced cataract in rat
Cutaneous and Ocular Toxicology Downloaded from informahealthcare.com by University of Laval on 06/30/14 For personal use only.
Seyithan Taysi1, Zainab Khaleel Abdulrahman1,6, Seydi Okumus2, Elif Demir1, Tuncer Demir3, Muslum Akan1, Edibe Saricicek1, Vahap Saricicek4, Adnan Aksoy5, and Mehmet Tarakcioglu1 1
Department of Medical Biochemistry, 2Department of Ophthalmology, 3Department of Physiology, Medical School, Gaziantep University, Gaziantep, Turkey, 4Department of Anesthesiology, Medical School, Gaziantep University, Gaziantep, Turkey, 5Department of Ophthalmology, Medical School, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey, and 6Kirkuk Health Department, Ministry of Health, Irak Abstract
Keywords
Objective: The aim of this study was to investigate the antioxidant and radioprotective effects of Nigella sativa oil (NSO) and thymoquinone (TQ) against ionizing radiation-induced cataracts in lens after total cranium irradiation (IR) of rats with a single dose of 5 gray (Gy). Materials and methods: Seventy-four Sprague-Dawley rats were used for the experiment. The rats were randomly divided into six groups. Group A received total cranium IR plus NSO (1 g kg–1 d–1) orally through an orogastric tube. Group B received total cranium IR plus TQ (50 mgkg–1 d–1) daily by intraperitoneal injection. Group C received 5 Gy of gamma IR as a single dose to total cranium plus 1 ml saline. Group D1 just received 1 ml saline. Group D2 just received dimethyl sulfoxide. Group D3 did not receive anything. Results: At the end of the 10th d, cataract developed in 80% of the rats in IR group only. After IR, cataract rate dropped to 20% and 50% in groups which were treated with NSO and TQ, respectively, and was limited at grades 1 and 2. Nitric oxide synthase activity, nitric oxide and peroxynitrite levels in the radiotherapy group were higher than those of all other groups. Conclusions: The results implicate a major role for NSO and TQ in preventing cataractogenesis in ionizing radiation-induced cataracts in the lenses of rats, wherein NSO were found to be more potent.
Cataract, free radical, irradiation, Nigella sativa, nitric oxide, nitrosative stress
Introduction Radiation therapy has been used effectively in treating head and neck cancers for many decades, which is a common and important tool for cancer treatment1,2. The killing action of ionizing radiation is mainly mediated through the free radicals generated from the radiolytic decomposition of cellular water, including superoxide radical (O 2 ) and hydroxyl radical (OH–), which can cause damage in most major cellular macromolecules such as DNA, proteins, lipids, membrane, etc. These reactions take place in tumor as well as normal cells when exposed to radiation3,4. Eye morbidity is widely observed in patients receiving total-body irradiation prior to bone marrow transplantation or radiotherapy for ocular or head and neck cancers5. Cataract blindness is the major cause of preventable blindness worldwide, especially in the developing countries6. It is the opacity of eye lens that interferes with vision. Cataracts are formed in response to a variety of different
Address for correspondence: Prof. Dr. Seyithan Taysi, Department of Medical Biochemistry, Gaziantep University Medical School, Gaziantep, Turkey. Tel: +90 342 3601617. Fax: +90 342 3600753. E-mail: [email protected]
History Received 18 November 2013 Revised 28 February 2014 Accepted 28 March 2014 Published online 19 June 2014
agents and environmental stresses, and this damage seems in almost all cases to have an oxidative damage component. Although cataract is a known late effect of ionizing radiation exposure, most of the experimental studies have concentrated on single, acute high doses or multiple, fractionated and chronic exposure of radiation3. Nigella sativa (NS), commonly known as black seed, belongs to the Ranunculaceae family. NS contains 430 w/w of fixed oil and 0.40–0.45 w/w of a volatile oil. The volatile oil has been shown to contain 18.4–24% thymoquinone (TQ) and 46% monoterpenes such as p-cymene and a-pinene. Nigella sativa oil (NSO) and seed constituents, in particular TQ, have shown potential medicinal properties in traditional medicine7,8. NSO and TQ, natural main constituent of the volatile oil of NS seeds, are reported to possess strong antioxidant properties against oxidative damage induced by a variety of free radical-generating agents. A lot of pharmacological and toxicological studies have reported it to possess diverse pharmacological properties such as antioxidant, hepatoprotective, neuroprotective, antidiabetic, antiinflammatory, nephroprotective and anticarcinogenic9,10. Efforts to decrease toxicity of irradiation (IR) to normal tissue, organs and cells have led into searching for cytoprotective agents. Unfortunately, most of the
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radioprotectors possess toxic side effects, which limit its role in medical treatment. For this reason, investigations for effective and non-toxic compounds with radioprotection capability led to increasing interest in naturally occurring antioxidant such as NSO and TQ. To our knowledge, there is no experimental study that simultaneously investigates the effects of NSO and TQ supplementations on nitric oxide synthase (NOS) activity, nitric oxide (NO), peroxynitrite (ONOO–) in the lens tissue of rats receiving ionizing radiation. Therefore, in this study, we aimed to investigate the effects of these supplementations on these nitrosative stress parameters in the lens tissue of rats with or without exposure to total cranium IR.
Material and methods Animals and experiments Fifty-four rats, 10–12 weeks old, weighing 200 ± 25 g, the time of radiation bred at Gaziantep University Medical School, Experimental Animal Laboratory were used for the experiment. The animals were purchased from Gaziantep University Medical School, Experimental Animal Laboratory, and housed in cages one week before the start of the experiments. All animals received humane care in compliance with the guidelines of Gaziantep University Research Council’s criteria. Food and tap water were available ad libitum. The laboratory was windowless with automatic temperature (22 ± 1 C) and lighting controls (14 h light/10 h dark). Rats were randomly divided into six groups (8 rats from control groups and 10 rats from other groups) and placed in separate cages during the study. This study was approved by the local ethics committee of the Gaziantep University. The groups were as follows: Group A: (IR plus NSO group) received both total cranium 5 gray (Gy) of gamma IR as a single dose and NSO (1 g kg–1 d–1) orally through an orogastric tube. NSO supplementation in group A lasted for 10 d. NSO was obtained from Origo Gida Kimya Tarim Urn. San. ve Tic. Ltd. Sti. (Gaziantep, Turkey). Group B: (IR plus TQ group) received TQ (50 mgkg–1 d–1) daily by intraperitoneal (IP) injection starting 30 min before the dose radiation and subsequently daily for 10 d after IR. TQ was dissolved in DSMO just before giving to the rats. Group C: IR group received total cranium 5 Gy of gamma IR as a single dose. Also physiological saline solution (1 mlkg–1 d–1, IP injection) was daily administered for 10 d. Group D1 (control group for group A): control group received neither NSO and TQ nor IR, but received daily physiological saline solution (1 mlkg–1 d–1, orally) for 10 d. Group D2 (control group for group B): Control group did not receive NSO, TQ and IR, but received daily IP injection of dimethyl sulfoxide at an equal volume to that of TQ used in group B for 10 d. Group D3 (normal control group): this group did neither receive NSO, TQ and IR nor oral/IP physiological saline solutions. Prior to total cranium IR, all rats were anesthetized with 80 mg/kg ketamine hydrochloride (Pfizer Ilac, Istanbul, Turkey) and placed on a tray in the prone position. The rats
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in the IR and the IR plus TQ groups received IR via a Cobalt60 teletherapy unit (Picker, C9, Maryland, NY) from a source-to-surface distance of 80 cm by 5 5 cm anterior fields, with the total cranium gamma IR being a single dose of 5 Gy, while the rats in the control and sham control groups received sham IR. The dose rate was 0.49 Gy/min. The central axis dose was calculated at a depth of 0.5 cm. Determination of clinical cataract In this study, the lens opacities classification system, version III (LOCS III) was used in the cataract classification11. Before the cataracts were graded, the pupils were dilated with tropicamide (1.0%) and phenylephrine hydrochloride (2.5%) drops, and proparacaine (0.5%) was used as a topical anesthetic. The lenses were graded by slit-lamp biomicroscopy (Keeler PSL Classic, Keeler Ltd., Windsor, UK) as follows: the features of nuclear opacification and brunescence are graded according to one set of six photographs. The brightness of scatter from the nuclear region has been designated nuclear opalescence (NO) and the intensity of brunescence, nuclear color (NC) (Figure 1). The amount of cortical cataract (C) is determined by comparing the estimated aggregate of cortical spoking with that seen in five separate photographs (Figure 2). Similarly, the estimated amount of posterior subcapsular cataract (P) is determined by comparing it with another five photographs depicting increasing amounts of posterior subcapsular cataract (Figure 3). At the beginning of the experiment, all rats were examined biomicroscopically and were only included in this study if their lenses had been NOo–, NC0–, C0– and P0– (Figure 4). Biochemical analysis At the end of the study, all rats were anesthetized with 80 mg/kg ketamine hydrochloride (Pfizer Ilac) and then all animals were killed by decapitation, and their eyes were enucleated, and the lenses were dissected immediately.
Figure 1. Nuclear cataract.
Nigella sativa in irradiation-induced cataract
DOI: 10.3109/15569527.2014.910802
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conducted on this fraction. All of the procedures were performed at 4 C.
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Determination of NOS activity and NO and ONOO– levels
Figure 2. Cortical cataract.
Figure 3. Posterior subcapsular cataract.
NOS activity assay is based on the diazotization of sulfanilic acid by NO at acid pH and subsequent coupling to N-(1-naphthyl) ethylenediamine. To 0.1 ml of sample, 0.2 ml of 0.2 M arginine was added and incubated at 37 C for 1 h. Then, the combination, 0.2 ml of 10 mM HCl, 100 mM sulfanilic acid and 60 mM N-(1-naphthyl) ethylenediamine was added. After 30 min, the absorbance of the sample tube was measured against a blank tube at 540 nm12. Results are expressed as U/mg protein. NO levels in lens tissue were measured using the Griess reagent as previously described13,14. Griess reagent, the mixture (1:1) of 0.2% N-(1-napthyl) ethylenediamine and 2% sulphanilamide in 5% phosphoric acid, gives a red–violet diazo dye with nitrite, and the resultant color was measured at 540 nm. First nitrate was converted to nitrite using nitrate reductase. The second step was the addition of Griess reagent, which converts nitrite to a deep purple azocompound, photometric measurement, the absorbance of 540 nm determines the nitrite concentration. Results were expressed as mmol/g wet tissue. ONOO– assay was determined as described15,16. Ten microliters of sample was added to 5 mM phenol in 50 mM sodium phosphate buffer (pH 7.4) to get a final volume of 2 ml. After 2-h incubation in a dark place at 37 C, 15 mL of 0.1 M NaOH was added and the absorbance, at wavelength of 412 nm, of the samples was immediately recorded. The yield of nitrophenol was calculated from " ¼ 4400/M/cm. Results were expressed as mmol/g wet tissue. The protein content was determined as described17. Biochemical measurements were carried out using a visible/UV spectrophotometer (Shimadzu U 1601, Kyoto, Japan). Statistical analyses Cataract developments in the different treatment groups were compared with Fischer’s exact Chi-square test. Statistical and correlation analyses were undertaken using a one-way variance analysis and Spearman’s rank correlation test, respectively. Following the analysis of variance, the significance of differences between groups was tested using least significant difference multiple range test procedure. Acceptable significance was recorded when p values were 50.05. Statistical analysis was performed with Statistical Package for the Social Sciences for Windows, version 11.5 (SPSS Inc., Chicago, IL).
Results Figure 4. Clear lens.
Lenses were homogenized by an IKA-NERKE (Staufen, Germany) homogenizer in physiological saline solution (20fold) on ice for 10 s in the first speed level. The homogenate was centrifuged at 10 000 g for 1 h to remove debris. The clear upper supernatant was collected, and all assays were
In this study, the LOCS III was used in the cataract classification11. The lenses were graded by slit-lamp biomicroscopy (Keeler PSL Classic, Keeler Ltd.). All the rat lenses at the beginning were graded as 0. After IR, cataract developed in 80% of the rats in radiotherapy group. After IR, cataract rate dropped to 20% and 50% in groups that were treated with NSO and TQ, respectively, and was limited at grades 1 and 2 (Table 1). NOS activity, levels of NO and ONOO– in the radiotherapy group were significantly higher
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Table 1. Cataract situation in the rat lenses examined by slit-lamp microscopy in all groups. The situation of cataract Kind of cataract
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Group Group Group Group Group Group
Presence
NC
CC
PSC
Absent
Total
2 5 8 0 0 0
0 0 1 – – –
1 2 2 – – –
1 3 5 – – –
8 5 2 8 8 8
10 10 10 8 8 8
A B C D1 D2 D3
Group A: Irradiation (IR) plus Nigella sativa oil (NSO) group; group B: IR plus thymoquinone (TQ) group; group C: IR group only; group D1 (the control group of A group); group D2 (the control group of B group); group D3 (normal control group); NC: nuclear cataract; CC: cortical cataract; and PSC: posterior subcapsular cataract. Table 2. Mean ± SD of nitric oxide synthase activity, nitric oxide and peroxynitrite in lens tissue of rats in all groups. NOS (U/mg protein) Group Group Group Group Group Group
A B C D1 D2 D3
284.11 ± 24.83b,c,e,g 288.79 ± 59.98b,c,f,g 729.20 ± 140.44 202.21 ± 27.46b 198.89 ± 37.61b 187.38 ± 31.01b
NO OONO– (nM/g wet tissue) (mM/g wet tissue) 4.28 ± 0.43b,c,e 4.46 ± 0.44a,d,f,g 5.04 ± 0.35 4.03 ± 0.49b 3.83 ± 0.49b 3.71 ± 0.33b
101.52 ± 14.84b,d 108.90 ± 20.64b,d 172.81 ± 18.85 103.05 ± 32.36b,d 97.31 ± 16.19b,c 62.97 ± 23.69b
a: p50.01, b: p50.0001 versus group C, c: p50.01, d: p50.001 versus group D3, e: p50.05, f: p50.01 versus group D2, g: p50.05 versus group D1. Group A: Irradiation (IR) plus Nigella sativa oil (NSO) group; group B: IR plus thymoquinone (TQ) group; group C: IR group only; group D1 (the control group of A group), group D2 (the control group of B group), group D3 (normal control group): nitric oxide synthase (NOS): nitric oxide (NO): peroxynitrite (ONOO–).
than all other groups (Table 2). In this study, while the cataract ratio was highly observed in radiotherapy group, it decreased in the other groups in which NSO and TQ were given after radiation. NSO was found to be more effective in anticataractogenic effect than TQ. Compared to the IR group, a significant reduction in cataract formation was observed in group A (p ¼ 0.007). As for in group C, the decrease in cataract was not statistically significant (p40.05). We observed a significant increase of cataract formation in the IR group, when compared to the control groups (p50.001).
Discussion As the world’s population ages, cataract-induced visual dysfunction and blindness are on the increase. Cataract is a major cause of blindness and of severe visual impairment leading to bilateral blindness in an estimated 20 million people worldwide. In developing countries, 50–90% of all blindness is caused by cataracts. Pharmacological treatment to prevent human cataracts has so far not been achieved. The exact mechanism of cataract formation is still not very clear2,18. Many studies are being conducted to explore the mechanism of cataractogenesis using various models of cataract and to target crucial steps to halt this process.
Limitations in acceptability, accessibility and affordability of cataract surgical services make it more relevant and important to look into alternative pharmacological measures for treatment of this disorder. Therefore, a lot of effort is being laid on identification of natural compounds that will help to prevent cataractogenesis6. The aim of radiotherapy is to kill cancer cells while causing as little damage as possible to normal cells. Eighty percent of patients with cancer need radiotherapy at some time or other, either for a curative or a palliative purpose. To obtain optimum results, a judicious balance between the total dose of radiotherapy delivered and the threshold limit of the surrounding normal critical tissues is required. To obtain better tumor control with a higher dose, the normal tissues should be protected against radiation injury. Thus, the role of radioprotective compounds is very important in clinical radiotherapy1,8. Aerobic metabolism produces reactive oxygen and nitrogen species (ROS and RNS, respectively) that are normally inactivated through diverse antioxidant mechanisms. Nitrosative stress occurs when intermediates are produced from nitrosate thiol, hydroxy and amine groups. When mammalian cells were exposed to nitrosative stress by addition of NO donor, little toxicity was observed. Under nitrosative stress conditions, nitrosating intermediates have the greatest affinity for thiols, such as glutathione (GSH), suggesting that they are a primary target. Cells depleted of GSH were dramatically more susceptible to toxicity from nitrosative stress. Since these free radicals may also damage normal tissue, the balance between antioxidants and prooxidants is critical for normal function19. However, during oxidative and nitrosative stress conditions, the balance between free radicals and antioxidants shifts toward the former, resulting in diseases such as liver damage, cancer, rheumatoid arthritis, infectious diseases and atherosclerosis and in aging20. Radiation is a known producer of free radicals, contributing to radiation injury in cells and formed in cells. Ionizing radiation damage biological tissues and they produce free radical in aqueous solution, for instance, cell cytoplasm, which in turn can lead to oxidative damage to biological molecules such as nucleic acids, proteins and lipids, leading to cataract. One of the mechanisms proposed to explain lens opacification is the oxidation of crystallins, either by radiation or ROS and RNS. The other mechanism is the formation of opacities with increased calcium release by mitochondrial damage. Ionizing radiation has been shown to enhance the production of these free radicals in cells4,21,22. For this reason, to prevent injury caused by radiation on healthy tissue, many investigations related natural products that have antiviral, anticancer, immunostimulant and antioxidant effects have been constructed. Several studies on radioprotective agent are ongoing. To our knowledge, this is the first report demonstrating lens radioprotection by NSA and TQ. According to our results, NOS activity, NO and ONOO– levels were significantly higher in lens tissues of irradiated rats that were exposed to total cranium IR, compared to other groups. Similar results were obtained in a previous study conducted by Manikandan et al.6. The nitrosative stress
Nigella sativa in irradiation-induced cataract
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DOI: 10.3109/15569527.2014.910802
parameters in the groups receiving IR plus NSO or TQ treatment were significantly decreased compared to the group that received IR only. These results revealed that NSO and TQ clearly decreased the nitrosative stress in lens tissue induced by total cranium IR. NO is synthesized endogenously by the enzyme, NOS, via its precursor arginine. Endogenous NO plays a dual role in specialized tissues and cells, where it is not only an essential physiological signaling molecule mediating various cell functions but also induces cytotoxic and mutagenic effects when present excessively under oxidative stress conditions induced by IR23,24. There are two distinct categories in the chemical biology of NO: direct and indirect effects. Direct effects are those chemical reactions where NO reacts directly with its biological target. The direct effects are very rapid reactions that occur at low NO concentrations and generally involve heme proteins such as guanylate cyclase, cytochrome P450 and hemoglobin, whereas indirect effects are mediated by RNS, which are derived from NO metabolism. It requires that NO is first activated by O or oxygen to form RNS, which then 2 undergoes further reactions with the respective biological target19. Furthermore, NO reacts rapidly with O 2 to form ONOO–, which may be cytotoxic by itself or easily decompose to the highly reactive and toxic OH– and nitrogen dioxide23,24. These RNS are highly reactive with major cellular macromolecules such as DNA, proteins, lipids, membrane, etc., and are thought to be responsible for NOmediated cell death19. Some studies have been reported that NO levels in rats that are exposed to whole-body IR were significantly higher6,25. In this study, NOS activity, NO and ONOO– levels in lens tissue of rats exposed to radiation were also increased before the treatment of rats with NSO and TQ. According to the results, low nitrosative stress parameters in the NS and TQ treatment groups may suggest that NSO and TQ have protective effects against toxicity to IR. Similarly, NS has the potential to reduce toxicity against oxidative stress based on the literature. It is a well-known fact that NSO and TQ have antiinflammatory, antioxidant and antineoplastic effects both in vitro and in vivo studies. Antioxidant/ antiinflammatory effects of these agents have been studied in various disease models, including cancer, sepsis, atherosclerosis, asthma and carcinogenesis. Furthermore, it has been shown that TQ could act as a free radical and superoxide radical scavenger9,10,26.
Conclusion In this study, we have found that NOS activity, NO and ONOO– levels in the radiotherapy group were higher than those of all other groups. This is the first study that simultaneously investigates the effects of NSO and TQ on the nitrosative stress in the lens tissue of the irradiated rats. The results obtained in the study suggest an important role of nitrosative stress in the IR-induced cataractogenesis. We showed that these natural substances clearly appeared to prevent cataractogenesis in IR-exposed lenses by inhibiting RNS generation. NSO and TQ clearly have antioxidant properties and free radical scavenging activities. These are likely to be a valuable drug for protection against gamma-IR
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and/or be used as an antioxidant against cataractogenesis, oxidative/nitrosative stress and other severe side effects occurred in head and neck cancers patients treated with radiotherapy. However, further studies on this subject are needed to implement the use of the natural compounds in clinical protection against ionizing radiation, especially in treatment of patients with cancer exposed to radiotherapy.
Declaration of interest The authors report no declarations of interest. None of the authors has a commercial interest, financial interest and/or other relationship with manufacturers of pharmaceuticals, laboratory supplies and/or medical devices or with commercial providers of medically related services.
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