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Radiation-modifying abilities of Nigella sativa and Thymoquinone on radiation-induced nitrosative stress in the brain tissue.

ABSTRACT

To investigate Nigella sativa oil (NSO) and Thymoquinone (TQ) for their antioxidant effects on the brain tissue of rats exposed to ionizing radiation.

Fifty-four male albino Wistar rats, divided into six groups, were designed as group I (normal control group) did not receive NSO, TQ or irradiation; group II (control group of TQ) received dimethyl sulfoxide and sham irradiation; group III (control group of NSO) received saline and sham irradiation; group IV (irradiation plus NSO group) received both 5 Gray of gamma irradiation to total cranium and NSO; group V(irradiation plus TQ group) received both irradiation and TQ; group VI (irradiation alone group) received irradiation plus saline. Alterations in nitric oxide (N[O.sup.*]) and peroxynitrite (ONO[O.sup.-]) levels, and nitric oxide synthase (NOS) enzyme activity were measured by biochemical methods in homogenized brain tissue of rats.

Levels of N[O.sup.*] and ONO[O.sup.-], and enzyme activity of NOS in brain tissue of the rats treated with NSO or TQ were found to be lower than in received IR alone (p < 0.002)

Nigella sativa oil (NSO) and its active component, TQ clearly protect brain tissue from radiation-induced nitrosative stress.

Keywords:

Nigella sativa

Brain

Ionizing radiation

Nitric oxide

Nitrosative stress

Thymoquinone

Introduction

Oxygen-free radicals including reactive oxygen species (ROS) such as superoxide radical ([O.sub.2.sup.*-]) and hydroxyl radical (O[O.sup.*- ]), and reactive nitrogen species (RNS) such as nitric oxide (N[O.sup.*]), peroxynitrite (ONO[O.sup.-]) are produced in humans as a consequence of intracellular metabolic processes and after exposure to genotoxic agents, i.e. ionizing radiation (IR) (Cadenas 1989). IR is an important source in generation of ROS/RNS among many physical/chemical agents and killing action of IR is mainly mediated through oxygen-free radicals (Hall and Giaccia 2006). ROS or RNS mediated oxidative or nitrosative injury plays a crucial role in manifestation of health effects of radiation exposure (Hannig et al. 2000).

Nitric oxide (N[O.sup.*]), a double-edged molecule, is an important biological messenger that plays an important role in the physiology of the central nervous system (CNS) (Yun et al. 1996). N[O.sup.*] is produced from L-arginine by enzyme activity of nitric oxide synthase (NOS) and acts as an important physiological signaling molecule mediating a large variety of cellular functions. In other respects, its overproduction induces cytotoxic and mutagenic effects (Kaynar et al. 2005). When present in excess, N[O.sup.*] can react with [O.sub.2.sup.*-] and produce ONO[O.sup.-] anion which is a powerful oxidant that can cause lipid peroxidation (LPO), inhibit mitochondrial electron transport, oxidize thiol compounds and oxidize and nitrate DNA (Powell et al. 2005; Valko et al. 2006). There is a balance between production and scavenging of ROS/RNS. If the balance changes in support of production of ROS/RNS, oxidative or nitrosative stress takes place and may result in a variety of diseases; cancer, cardiovascular, and neurological diseases (Reuter et al. 2010).

Brain is an important tissue of CNS and IR is a mandatory part in the treatment of brain malignancies. Brain is particularly sensitive to the oxidative and nitrosative injury due to its high content of polyunsaturated fatty acids, relatively low antioxidant capacity, low repair mechanism activity, non-replicating nature of its neuronal cells, and high rate of oxidative metabolic activity and overproduction of ROS/RNS metabolites compared to other organs (Evans 1993). In CNS, oxidative or nitrosative stress results in acute and chronic injury and plays an important role in the pathogenesis of neuronal damage (Facchinetti et al. 1998). Therefore, agents which can protect cellular membranes against IR and ROS/RNS will have potential benefits as radioprotectors, antioxidant and antimutagens (Odin 1997; Stavric 1994).

Nigella sativa (NS), commonly known as black seed, belongs to the botanical family of Ranunculaceae and generally grows in Eastern Europe, Middle East and Western Asia. The seeds of NS and their oil have been widely used throughout the world in the treatment of several diseases for centuries. NS contains amino acids, proteins, carbohydrates, both fixed oils (84% fatty acids, including linolenic and oleic acids) and volatile oils, alkaloids, crude fiber, saponins and minerals, such as calcium, iron, sodium and potassium. The volatile component of NS seed, TQ constitutes about 27-57% of the quinone constituents and has been attributed to be the most important active present in the whole seeds or their extracts among the other active ingredients (Ali and Blunden 2003; Salem 2005). Antihistaminic, antihypertensive, analgesic, anti-inflammatory, hypoglycemic, antibacterial, antifungal, antitumour, hepatoprotective, renal protective and antioxidant effects of NS oil (NSO) and TQ have been shown in a large number studies (Ali and Blunden 2003; Burits and Bucar 2000; Padhye et al. 2008).

Besides the known antioxidant properties of NSO and TQ, data on the radiation-protective ability of these agents are limited (Cemek et al. 2006; Rastogi et al. 2010). We hypothesized that NSO and TQ whose antioxidant effects are proven by many studies could protect brain tissue from radiation-induced nitrosative damage. For this reason, we measured the nitrosative biomarkers, N[O.sup.*], NOS and OON[O.sup.-], in the brain tissue of rats with or without exposing of gamma radiation to total cranium with a single dose of 5 Gray (Gy).

Materials and methods

Rats and experiments

Fifty-four male Wistar albino rats, 12-16 weeks old, weighting 220 [+ or -] 25 g at the time of irradiation and bred at Gaziantep University Medical School, department of animal laboratory were used for the experiment. All procedures involving the Wistar albino rats adhered to the ARVO Resolution on the Use of Animals in Research. Animal experimentations were carried out in an ethically proper way by following guidelines as set by the Ethical Committee of the Gaziantep University. The rats were quarantined for at least seven days before irradiation, housed ten to a cage in a windowless laboratory room with automatic temperature (22 [+ or -] 1[degrees]C) and lighting controls (12 h light/12 h dark) and fed with standard laboratory chow and water. The rats were randomly divided into six groups. Control groups included 8 rats and the other groups included 10 rats for each. Group I (normal control group) did not receive NSO, TQor irradiation. Group II (control group of TQ) did not receive NSO, TQ or irradiation, but received sham irradiation and intraperitoneally (i.p.) injections of dimethyl sulfoxide (DMSO) at an equal volume to that of TQ used in group V (the first dose of DMSO was started 30 min before the irradiation and continued during ten days). Group III (control group of NSO) received 1 -ml saline through an orogastric tube and sham irradiation. Group IV (irradiation plus NSO group) received both 5 Gy of gamma irradiation as a single dose to total cranium and NSO (1 g/kg/day) starting 1 h before irradiation and continuing for 10 days through an orogastric tube. Group V (irradiation plus TQ group) received both 5 Gy of gamma irradiation as a single dose to total cranium and TQ(30 mg/kg/day, i.p.) injection starting 30 min before the radiation dose and subsequently daily for 10 days after irradiation. TQ was dissolved in DMSO just before giving to the rats. Group VI (irradiation alone group) received 5 Gy of gamma irradiation as a single dose to total cranium plus 1-ml saline through an orogastric tube. Prior to total cranium irradiation, the rats were anesthetized with 50mg/kg ketamine HCL (Pfizer Inc, Istanbul, Turkey) and placed on a plexiglas tray in the prone position. While the rats in the control groups of II and HI received sham irradiation, the rats in the groups of IV, V, VI were irradiated using cobalt 60 teletherapy unit (Theratron Equinox, MDS Nordion, Kanata, Ontario, Canada) from a source-to-surface distance of 100 cm by 20 cm x 20 cm anterior fields with 5 Gy to the total cranium as a single fraction. The central axis was calculated at a depth of 1 cm. The dose rate was 0.49Gy/min.

Fractination of brain samples

At the end of the study, the rats were anesthetized with 50 mg/kg ketamine i.p. Then an intracardiac withdrawal of blood was performed. Following this process, the rats were sacrificed and their brains were removed. Brain tissues were homogenized by a homogenizer (1KA-NERKE, GmBH & CO. KB D-79219, Staufen, Germany) in isotonic saline (1/10 weight/volume) on ice for 1 min. The supernatant was stored at -80[degrees]C in aliquots for biochemical measurements. N[O.sup.*] and ONO[O.sup.-] levels and enzyme activity of NOS were determined from these supernatants spectrophotometrically.

Determination of NOS activity and N[O.sup.*] and ONO[O.sup.-] levels

NOS activity assay is based on the diazotization of sulfanilic acid by N[O.sup.*] at acid pH and subsequent coupling to N-(l-naphthyl) ethylenediamine. To 0.1 ml of sample, 0.2 ml of 0.2 M arginine was added and incubated at 37[degrees]C for 1 h. Then, the combination, 0.2 ml of 10mM HCl, 100mM sulfanilic acid, and 60mM N-(1-naphthyl) ethylenediamine was added. After 30 min, the absorbance of the sample tube was measured against a blank tube at 540 nm (Durak et al. 2001). Results are expressed as U/mg protein. N[O.sup.*] levels in brain tissue were measured using the Griess reagent as previously described (Bories and Bories 1995; Moshage et al. 1995). Griess reagent, the mixture (1:1) of 0.2% N-(1-naphthyl) 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 [micro]mol/g wet weight. Peroxynitrite assay was determined as described (Al-Nimer et al. 2012; Vanuffelen et al. 1998). Ten microliters of samples 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[degrees]C, 15 [micro]l of 0.1 M NaOH was added and the absorbance, at wavelength of 412 nm, of the samples were immediately recorded. The yield of nitrophenol was calculated from [epsilon] = 4400 [M.sup.-1] [cm.su.-1]. Results were expressed as [micro]mol/g wet weight. The protein content was determined as described (Bradford 1976). Biochemical measurements were carried out using a spectrophotometer (Shimadu U 1601, Japan).

Statistical analyses

Analyses were conducted using Statistical Package for the Social Sciences (SPSS, version 18) software. Data were analyzed with one-way analysis of variance (ANOVA) followed by a post hoc test (LSD alpha) for multiple comparisons. Data were expressed as mean [+ or -] standard deviation (SD) and p values <0.05 was considered to be statistically significant.

Results

Enzyme activity of NOS and levels of N[O.sup.*] and ONO[O.sup.-] of the six groups are presented in Table 1. Levels of N[O.sup.*] and ONO[O.sup.-], and enzyme activity of NOS in brain tissue of the rats in the irradiation alone group (group VI) were found to be higher than all of the other groups (p< 0.001). In other words, levels of N[O.sup.*] and ONO[O.sup.-], and enzyme activity of NOS in brain tissue of the rats treated with NSO (group IV) or TQ (group V) were found to be lower than irradiation alone group (p< 0.002). No statistical difference was detected between the control groups (I, II and III) and the groups treated with NSO (group IV) or TQ (group V), with respect to the levels of N[O.sup.*] and ONO[O.sup.-], and enzyme activity of NOS in brain tissue of the rats (p> 0.05).

There was no statistically significant difference between the groups treated with NSO and TQ. in terms of nitrosative stress biomarkers.

Discussion

Currently herbal preparations are becoming popular because of their beneficial effects with fewer side effects compared to synthetic/semi-synthetic drugs. In the present study, antioxidant role of NSO and TQ in the radiation-injured brain tissue of rats were investigated. Although strong antioxidant properties of NSO and TQ are proven in a large number of studies, to our knowledge, this is the first investigation which studied the effects of these agents on radiation-induced nitrosative stress in brain tissue. In the present study, we found that N[O.sup.*] and ONO[O.sup.-] levels, and the enzyme activity of NOS in the rats treated with NSO or TQ were significantly lower compared to the rats received irradiation alone. The results of the present study support the research hypothesis that the systemic administration of NSO and TQ would reduce the nitrosative damage in irradiated brain tissue.

Ionizing radiation (IR) is one of the most common therapies for treating human cancers. IR interacts with living cells and produces different cytotoxic effects that are mediated through the production of ROS/RNS (Schaue et al. 2002). Cumulative results involving animals exposed to either non-lethal or lethal doses of X-radiation show that the biological effects of the IR are dependent on the radiation dose and post-irradiation time (Shechmeister and Fishman 1955). A judicious balance should be between the total dose of IR received and the threshold limit of the normal tissue near the tumor. Normal tissue should be protected against the side effects of radiation to obtain better tumor control with a higher dose (Nair et al. 2001).

Organisms have adaptive responses to oxidative or nitrosative stress by activating of genes encoding defensive enzymes, transcription factors, and structural proteins (Dalton et al. 1999). In response to each environmental change such as exposing to IR, the genes encoding the translation apparatus and its regulator are remarkably coordinated. Since the ability of the antioxidant defense system against ROS/RNS is feeble in the brain compared to other tissues, the brain cells are vulnerable to death against ROS/RNS. Therefore, a decrease in the antioxidant enzyme capacity of the brain tissue could result in ROS/RNS accumulation during IR.

Nitric oxide (N[O.sup.*]), an oxygen-free radical produced by NOS, is an important small molecule that is required in several biochemical pathways and regulate several steps of the inflammatory process. The interaction of N[O.sup.*] with [O.sub.2.sup.*-] has both protective (microbial killing, neutralizing [O.sub.2.sup.*-]) and toxic effects by the formation of the ONO[O.sup.-], which is now generally considered more toxic species than N[O.sup.*], [O.sub.2.sup.*-], and O[H.sup.*] (Kendall et al. 2001). To minimize undesired effects, its production must be tightly controlled; however, this balance can be corrupted in certain pathophysiologic states (Dijkstra et al. 1998; McCaVerty 2000; Singer et al. 1996). NOS is an enzyme that produces NO and activity of NOS can be stimulated by IR in injured cells resulting in generation of large amounts of N[O.sup.*] (Mikkelsen and Wardman 2003; Taysi et al. 2003; Tsuji et al. 2000). In the present study, when rats were exposed to a single dose (5 Gy) total cranial gamma irradiation, N[O.sup.*] levels and the enzyme activity of NOS significantly increased in the brain tissue. The results of the present study are in agreement with the aforementioned studies performed on different tissues in rats. Differently from these studies, we determined ONO[O.sup.-] level which is a known power oxidant molecule, produced by radiation-induced changes in the nitrosative stress, may play a role in DNA strands break, lipid peroxidation, and protein modification and also has been shown to be particularly involved in neuronal cell injury and contribute to the pathogenesis of neurodegeneration (Floyd 1999; Ischiropoulos and Beckman 2003). We found ONO[O.sup.-] levels which indicate nitrosative stress to be increased in irradiation alone group compared to the groups treated with NSO or TQ.

For many centuries, NS has been widely used as a traditional medicine for a wide range of illnesses. NS has been confirmed to have antioxidant properties by scavenging ROS/RNS (Hosseinzadeh et al. 2007), and by suppressing formation of them, so play a major role in the antioxidant defense system (Arts and Hollman 2005; Moyers and Kumar 2004). Many chemical components of NS such as flavonoids, fatty acids, sterols, and other volatile oils are responsible for its antioxidant effect. NSO and volatile component of NS seed, TQ were found to improve antioxidant capacity induced by several agents in different animal tissues by suppressing oxidative/nitrosative stress, N[O.sup.*] overproduction and inducible NOS expression. TQ dithymoquinone, and thymol were tested for their free radical scavenging effects against several ROS; it was reported that thymol played role as singlet oxygen quencher, while TQand dithymoquinone showed superoxide dismutase-like activity (Kruk et al. 2000). TQis known to be a scavenger of several ROS, including [O.sub.2.sup.*-] and O[H.sup.*-] (Badary et al. 2003; Burits and Bucar 2000) and was shown to prevent cellular membrane from LPO (Hosseinzadeh et al. 2007).

It has been reported that NSO downregulates C[Cl.sub.4]-induced inducible NOS mRNA and N[O.sup.*] production in rat liver (Ibrahim et al. 2008), aqueous extract of NS inhibits N[O.sup.*] production (Mahmood et al. 2003) and TQ suppresses N[O.sup.*] production and inducible NOS expression (El-Mahmoudy et al. 2002) in lipopolysaccharide stimulated rat peritoneal macrophages. Also, NSO and TQwere found to inhibit gentamicin- (Yaman and Balikci 2010) and cyclosporine (Uz et al. 2008)-induced oxidative stress and N[O.sup.*] overproduction in rat kidney.

Abdel-Zaher et al. (2011) reported that NSO can protect brain against tramadol-induced tolerance and dependence in mice through blockade of N[O.sup.*] overproduction and oxidative/nitrosative stress induced by the drug. Fathy and Nikaido (2013) have shown the chemopreventive effects of NS that protect from diethylnitrosamine-induced hepatocarcinogenesis in rats by inhibition of the NOS pathway.

Umar et al. (2012) have demonstrated the antiarthritic ability of TQ in collagen induced arthritis. They found that TQ significantly suppressed the increase of LPO products, N[O.sup.*], myeloperoxidase activity, enhanced the activity of antioxidant enzymes and eliminated the accumulation and activation of polymorphonuclear cells and maintained the homeostasis in the cytokine imbalance. Gilhotra and Dhingra (2011) investigated the role of GABAergic and nitriergic modulation in the antianxiety effect of TQ. in mice under unstressed and stressed conditions and demonstrated that TQ decreased plasma nitrite, a stable metabolite of N[O.sup.*], in stressed mice and showed anxiolytic effects.

In present study, we found the nitrosative biomarkers such as N[O.sup.*] and ONO[O.sup.-] levels and the enzyme activity of NOS decreased in irradiated brain tissue of rats treated with NSO or TQ compared to the irradiation alone group. With respect to NO levels and NOS activity, results of the present study are in agreement with the studies as mentioned above. The findings of the present study support the antioxidant properties of NSO and TQ in radiation-injured brain tissue.

Although biochemical analyses have been suggested that NSO and TQexhibit radiation-protective effects against nitrosative damage in the brain tissue of irradiated rats, limitation of this study is lack of histological evaluation which may support this data.

In conclusion, we found increased nitrosative stress in brain tissue of irradiated rats in comparison to the other groups. This is the first study that concurrently investigates the effects of NSO and TQ on the nitrosative stress in the brain tissue of the irradiated rats. We showed that these natural substances clearly prevent from nitrosative stress in radiation-injured brain tissue by inhibiting free radical generation or scavenging ROS/RNS. These agents are likely to be valuable drugs for protection against harmful effects of 1R and/or be used as an antioxidant against nitrosative stress and other severe side effects occurred in patients with head and neck cancers treated with radiotherapy. However, additional pharmacological and toxicological studies are required to support these findings.

http://dx.doi.org/10.1016/j.phymed.2013.10.023

ARTICLE INFO

Article history:

Received 17 August 2013

Received in revised form 24 September 2013

Accepted 17 October 2013

Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgements

We thank all the staff of the Department of Radiation Oncology, Department of Biochemistry and Department of Physiology for their contributions.

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Adem Ahlatci (a), Abdurahman Kuzhan (a), *, Seyithan Taysi (b), Omer Can Demirtas (c), Hilal Eryigit Alkis (a), Mehmet Tarakcioglu (b), Ali Demirci (a), Derya Caglayan (a), Edibe Saricicek (d), Kadir Cinar (e)

(a) Department of Radiation Oncology, Gaziantep University, Medical School, Gaziantep, Turkey

(b) Department of Biochemistry and Clinical Biochemistry, Gaziantep University, Medical School, Gaziantep, Turkey

(c) Department of Biophysics, Gaziantep University, Medical School, Gaziantep, Turkey

(d) Department of Biochemistry, Dr. Ersin Arslan State Hospital, Gaziantep, Turkey

(e) Department of Neurosurgery, Sehitkamil State Hospital, Gaziantep, Turkey

* Corresponding author at: Gaziantep University. School of Medicine, Department of Radiation Oncology. Oncology Hospital, Kizilhisar Koyu, 27310 Sahinbey, Gaziantep, Turkey. Tel.: +90 342 4720711x6309; fax: +90 342 4720718.

E-mail address: aJtuzhan@hotmail.com (A. Kuzhan).
Table 1
Enzyme activity of NOS and levels of NO* and ONO[O.sup.-] of the
groups.

Croups       NOS mean [+ or -] SD        NO* mean [+ or -] SD
             (U/mg protein)              ([micro]mol/g wet weight)

Group I       0.73 [+ or -] 0.22          8.24 [+ or -] 1.51
Group II      0.84 [+ or -] 0.15          8.01 [+ or -] 1.07
Group III     0.87 [+ or -] 0.12          8.81 [+ or -] 1.21
Group IV      0.98 [+ or -] 0.12          8.33 [+ or -] 0.89
Group V       0.82 [+ or -] 0.18          7.47 [+ or -] 1.38
Group VI      1.26 [+ or -] 0.26 (a,b)   10.20 [+ or -] 0.96 (a,c)

Croups       OON[O.sup.-] mean [+ or -] SD
             ([micro]mol/g wet weight)

Group I       92.80 [+ or -] 2.08
Group II      92.73 [+ or -] 3.23
Group III     97.31 [+ or -] 16.19
Group IV      102.7 [+ or -] 18.73
Group V       99.22 [+ or -] 39.99
Group VI     170.14 [+ or -] 18.39 (a,c)

Croup 1: normal control group, II: control group of TQ, III: control
group of NSO, IV: irradiation plus NSO group, V: irradiation plus TQ
group, VI: irradiation alone group, SD: standard deviation.

(a) p< 0.001 as compared to other groups.

(b) p<0.002 as compared to groups of IV, V.

(c) p < 0.001 as compared to groups of IV, V.
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Author:Ahlatci, Adem; Kuzhan, Abdurahman; Taysi, Seyithan; Demirtas, Omer Can; Alkis, Hilal Eryigit; Tarakc
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Apr 15, 2014
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