original article Wien Klin Wochenschr DOI 10.1007/s00508-015-0736-4
The effects of Nigella sativa oil, thymoquinone, propolis, and caffeic acid phenethyl ester on radiation-induced cataract Elif Demir · Seyithan Taysi · Behcet Al · Tuncer Demir · Seydi Okumus · Oguzhan Saygili · Edibe Saricicek · Ahmet Dirier · Muslum Akan · Mehmet Tarakcioglu · Cahit Bagci
Received: 22 August 2014 / Accepted: 19 January 2015 © Springer-Verlag Wien 2015
Summary Background The aim of this study was to investigate the antioxidant and radioprotective effects of propolis, caffeic acid phenethyl ester (CAPE), Nigella sativa oil (NSO), and thymoquinone (TQ) against ionizing radiationinduced cataracts in lens after total cranium irradiation of rats with single dose of 5-Gy cobalt-60 gamma rays. Methods A total of 74 Sprague–Dawley rats were divided into 8 groups to test the radioprotective effectiveness of Nigella sativa oil, thymoquine, propolis, or caffeic acid phenethyl ester administered by either orogastric tube or intraperitoneal injection. Appropriate control groups were also studied. Results Chylack’s cataract classification was used in the study. At the end of the tenth day, cataracts devel-
Prof. Dr. S. Taysi, PhD () · E. Demir · E. Saricicek · M. Akan · M. Tarakcioglu Department of Medical Biochemistry, Medical School, Gaziantep University, Gaziantep, Turkey e-mail: [email protected] B. Al Department of Emergency Medicine, Medical School, Gaziantep University, Gaziantep, Turkey T. Demir · C. Bagci Department of Physiology, Medical School, Gaziantep University, Gaziantep, Turkey S. Okumus · O. Saygili Department of Ophthalmology, Medical School, Gaziantep University, Gaziantep, Turkey A. Dirier Department of Radiation Oncology, Medical School, Gaziantep University, Gaziantep, Turkey
13
oped in 80 % of the rats in the radiotherapy group. After irradiation, cataract rate dropped to 20 % in NSO, 30 % in propolis, 40 % in CAPE, and 50 % in TQ groups and was limited to grade 1 and grade 2. Cataract formation was observed the least in NSO group and the most in TQ group. In the irradiated (IR) group, superoxide dismutase activity was lower, while glutathione peroxidase and xanthine oxidase activities and malondialdehyde level were higher compared with the other groups. Total superoxide scavenger activity and nonenzymatic superoxide scavenger activity were not statistically significant in IR group compared with the other groups. Conclusions The findings obtained in the study might suggest that propolis, CAPE, NSO, and TQ could prevent cataractogenesis in ionizing radiation-induced cataracts in the lenses of rats, wherein propolis and NSO were found to be more potent. Keywords Oxidative stress · Antioxidant · Lipid peroxidation · Irradiation
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 treatment. In all, 80 % of cancer patients need radiotherapy at some time or other, either for curative or palliative purpose. The radiosensitivity of the normal tissues adjacent to the tumor limits therapeutic gain. The responses of the normal tissues to the therapeutic radiation exposure range from those that cause mild discomfort to others that are life threatening. The speed at which a response develops varies widely from one tissue to another and often depends on the dose of radiation that the tissue received [1–3].
The effects of Nigella sativa oil, thymoquinone, propolis, and caffeic acid
1
original article
Eye morbidity is widely observed in patients receiving total-body irradiation prior to bone marrow transplantation or radiotherapy for ocular or head and neck cancers [4]. A deleterious effect of radiation is the production of reactive oxygen species (ROS), which includes superoxide anion (O2•–) and hydroxyl radical (OH•–). These ROS may contribute to radiation-induced cytotoxicity and to metabolic and morphologic changes in humans [5]. Reactive oxygen species are clearly involved in the pathophysiological changes in the eye, and any means of regulating these free radicals can have a huge impact in preventing disease onset in the eye. In the eyes, ROS have been implicated in diseases such as cataracts, uveitis, retrolental fibroplasia, age-related macular degeneration, and neovascular glaucoma [6–8]. Cataract blindness is the major cause of preventable blindness worldwide, especially in the developing countries [7]. It is the opacity of eye lens that interferes with vision. Cataracts are formed in response to a variety of different 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 exposures [1]. Nigella sativa oil (NSO) and thymoquinone (TQ) are reported to possess strong antioxidant properties against oxidative damage induced by a variety of free radicalgenerating agents. Studies had shown that the biological activity of Nigella sativa (NS) seeds is mainly attributed to its essential oil component that is predominantly thymoquinone. Many pharmacological and toxicological studies have reported it to possess diverse pharmacological properties such as antioxidant, hepatoprotective, neuroprotective, antidiabetic, anti-inflammatory, nephroprotective, anticarcinogenic [9–12]. Propolis and caffeic acid phenethyl ester (CAPE), an active component of propolis extract, have immunomodulatory, antitumoral, cytotoxic, antimetastatic, antiinflammatory, and antioxidant properties and have been shown to inhibit lipoxygenase activities as well as suppress lipid peroxidation [13]. All cells in the body are exposed chronically to oxidants from both endogenous and exogenous sources. Cells have developed different antioxidant systems and various antioxidant enzymes to defend themselves against free radical attacks. Superoxide dismutase (SOD) catalyzes the dismutation of the superoxide anion radical (O2•–) into hydrogen peroxide (H2O2) [14, 15]. Hydrogen peroxide is generally considered a major oxidant in cataractogenesis. The major systems degrading H2O2 in the lens involve glutathione peroxidase (GSH-Px), glutathione reductase (GRD), and glutathione (GSH) [16, 17]. Xanthine oxidase (XO) functions in purine and free radical metabolism, which also catalyzes the conversion of xanthine and hypoxanthine to uric acid and the production of O2•– [18]. Efforts to decrease toxicity of irradiation to normal tissue, organs, and cells have led to investigations to identify
cytoprotective agents. Unfortunately, most of radioprotectors possess toxic side effects, which limit their role in medical treatment. For this reason, investigations for effective and nontoxic compounds with radioprotection capability led to increasing interest in naturally occurring antioxidant such as NSO, TQ, CAPE, and propolis. To our knowledge, there is no experimental study that simultaneously investigates the effects of propolis, CAPE, NSO, and TQ supplementations on total (enzymatic plus nonenzymatic) superoxide scavenger activity (TSSA), nonenzymatic superoxide scavenger activity (NSSA), SOD, GSH-Px, XO, and malondialdehyde (MDA), a marker of lipid peroxidation, in the lens tissue of rats receiving ionizing radiation. Therefore, in the present study, we aimed to investigate the effects of these supplementations on oxidant/antioxidant parameters in the lens tissue of rats with or without exposure to total cranium irradiation.
Material and methods Chemicals Acetic acid, caffeic acid phenethyl ester (CAPE), dimethyl sulfoxide (DMSO), thymoquinone, thiobarbituric acid (TBA), sodium dodecyl sulfate (SDS), sodium hydroxide nicotinamide adenine dinucleotide (NADH), n-butanol, hydrogen peroxide (H2O2), xanthine, xanthine oxidase (XO), nitrobluetetrazolium (NBT), and trichloroacetic acid (TCA) were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals and reagents were obtained from the store of the Department of Medical Biochemistry, Gaziantep University Medical Faculty.
Animals and experiments In total, 74 male Sprague–Dawley rats, 10–12 weeks old, weighing 200 ± 25 g at the time of radiation, bred at the Experimental Animal Laboratory, Gaziantep University Medical School, were used for the experiment. The animals were purchased from the Experimental Animal Laboratory, Gaziantep University Medical School, and housed in cages 1 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). This study was approved by the local ethics committee of the Gaziantep University. Rats were randomly divided into eight groups (eight rats from control groups and ten rats from other groups) and placed in separate cages during the study. The groups were as follows: Group A (irradiation (IR) plus NSO group) received both 5 Gy of gamma irradiation as a single dose to total
2 The effects of Nigella sativa oil, thymoquinone, propolis, and caffeic acid
13
original article
cranium and NSO (1 g kg−1day−1) starting 1 h before irradiation dose and continuing for 10 days through an orogastric tube. Group B (IR plus propolis group) received both 5 Gy of gamma irradiation as a single dose to total cranium and propolis (80 mg kg−1 day−1) starting 1 h before irradiation and continuing for 10 days through an orogastric tube. Propolis was dissolved in DSMO just before giving to the rats. Group C (IR plus TQ group) received both 5 Gy of gamma irradiation as a single dose to total cranium and TQ (50 mg kg−1day−1) daily by intraperitoneal (IP) injection starting 30 min before the radiation dose and subsequently daily for 10 days after irradiation. TQ was dissolved in DSMO just before giving to the rats. Group D (IR plus CAPE group) received both 5 Gy of gamma irradiation as a single dose to total cranium and CAPE (10 µmol kg−1day−1, IP) injection starting 30 min before the radiation dose and subsequently daily for 10 days after irradiation. CAPE was dissolved in DSMO just before giving to the rats. The final concentration of DMSO was 0.1 %. Group E IR group received 5 Gy of gamma irradiation as a single dose to total cranium and saline (1 ml kg−1 day−1, IP) injection. Group F1 (the control for A group) received just 1 ml of saline through an orogastric tube and did not receive anything (NSO, TQ, CAPE, or propolis). Group F2 (the control for B, C, and D groups) did not receive NSO, TQ, CAPE, propolis, or irradiation, but received DMSO injections (IP) at an equal volume of that propolis, TQ, and CAPE as dissolved for group B, C, and D, respectively. Group F3 (normal control group) did not receive NSO, TQ, CAPE, propolis, or irradiation. Animal experimentations were carried out in an ethically proper way by following guidelines as set by the ethical committee of the Gaziantep University. Supplementation period was 10 days. Propolis, CAPE, and TQ were dissolved in DMSO just before giving to the rats. Prior to total cranium irradiation, 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 in the IR and the IR plus TQ groups received irradiation via a cobalt-60 teletherapy unit (Picker, C9, Maryland, NY, USA) from a source-to-surface distance of 80 cm by 5 × 5-cm anterior fields, with the total cranium gamma irradiation being a single dose of 5 Gy, while the rats in the control and sham control groups received sham irradiation. 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 opacity classification system, version III (LOCS III), was used in the cataract classification [19]. Before the cataracts were graded, the pupils were
13
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). The amount of cortical cataract (C) was determined by comparing the estimated aggregate of cortical spoking with that seen in five separate photographs. Similarly, the estimated amount of posterior subcapsular cataract (P) was determined by comparing it with another five photographs depicting increasing amounts of posterior subcapsular cataract. At the beginning of the experiment, all rats were examined biomicroscopically and were only included in this study if their lenses had been NO0, NC0, C0, and P0.
Biochemical analysis Ten days after irradiation, all animals were killed by decapitation; their eyes were enucleated; and the lenses were dissected immediately. Lenses were homogenized by an IKA-NERKE (Staufen, Germany) homogenizer in physiological saline solution (20-fold) 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 conducted on this fraction. All he procedures were performed at 4 °C. TSSA and NSSA assays were performed in the samples before and after adding trichloroacetic acid (TCA, 20 %), as described [20, 21]. First, TSSA was measured. In this method, xanthine–xanthine oxidase complex produces superoxide radicals that react with nitroblue tetrazolium (NBT) to form a farmazone compound. TSSA activity was measured at 560 nm by detecting inhibition of this reaction. By using a blank reaction in which all reagents are present except the supernatant sample and by determining the absorbance of the sample and blank, TSSA activity was calculated. Second, NSSA activity was measured in TCA-treated fractions prepared by treating part of the sample with final concentration of 20 % (w/v) TCA solution (to remove all enzymes and proteins), and centrifuging at 5,000 g for 30 min. After the elimination of proteins by this procedure, NSSA activity assaywas performed in the supernatant fraction. SOD activity was calculated as the difference between TSSA and NSSA. One unit of TSSA, NSSA, and SOD was defined as the amount of enzyme protein causing 50 % inhibition in nitroblue tetrazolium reduction rate. Results were expressed as U/mg protein. GSH-Px activity was assayed according to Paglia and Valentina [22]. In this method, GSH-Px catalyzes the oxidation of glutathione in the presence of tert-butyl hydroperoxide. Oxidized glutathione is converted to the reduced form in the presence of glutathione reductase and NADPH, while NADPH is oxidized to NADP. The reduc-
The effects of Nigella sativa oil, thymoquinone, propolis, and caffeic acid
3
original article
tion in the absorbance of NADPH at 340 nm is measured. The absorbance change per minute and the molar extinction coefficient of NADPH were used to calculate GSH-Px activity. GSH-Px activity was expressed as IU/mg protein. XO activity was measured spectrophotometrically by the formation of uric acid from xanthine through the increase in absorbance at 293 nm [23]. XO activity was expressed as U/mg protein. Concentration of MDA, the final product of lipid peroxidation, was determined spectrophotometrically according to a similar method described by Ohkawa et al. [24]. Briefly, a mixture of 8.1 % sodium dodecyl sulfate (SDS; 0.2 ml), 20 % acetic acid (1.5 ml), and 0.9 % thiobarbituric acid (1.5 ml) was added to the mixture to bring the total volume up to 4 ml. This mixture was incubated at 95 °C for 1 h. After incubation, the tubes were left for cooling under cold water and 5 ml of n-buthanol/pyridine (15:1, v/v) was added, followed by mixing up. The samples were centrifuged at 3,500 g for 10 min. The organic phase that accumulated at the top of the tubes was sampled, and sample absorbance values were measured with respect to the blank at 532 nm. The concentration of MDA was calculated using 1.56 × 105 Mcm−1 as molar extinction coefficient. The total thiobarbituric acid-reactive substances were expressed as MDA. Results were expressed as nmol/mg protein. The protein content was determined as described [25]. Biochemical measurements were carried out using a spectrophotometer (Shimadu U 1601, 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 analyzed using post hoc least significance difference test procedure. Acceptable significance was recorded when P values were 0.05). We observed a significant increase of cataract formation in the IR group compared with the control groups (p
The effects of Nigella sativa oil, thymoquinone, propolis, and caffeic acid phenethyl ester on radiation-induced cataract Elif Demir · Seyithan Taysi · Behcet Al · Tuncer Demir · Seydi Okumus · Oguzhan Saygili · Edibe Saricicek · Ahmet Dirier · Muslum Akan · Mehmet Tarakcioglu · Cahit Bagci
Received: 22 August 2014 / Accepted: 19 January 2015 © Springer-Verlag Wien 2015
Summary Background The aim of this study was to investigate the antioxidant and radioprotective effects of propolis, caffeic acid phenethyl ester (CAPE), Nigella sativa oil (NSO), and thymoquinone (TQ) against ionizing radiationinduced cataracts in lens after total cranium irradiation of rats with single dose of 5-Gy cobalt-60 gamma rays. Methods A total of 74 Sprague–Dawley rats were divided into 8 groups to test the radioprotective effectiveness of Nigella sativa oil, thymoquine, propolis, or caffeic acid phenethyl ester administered by either orogastric tube or intraperitoneal injection. Appropriate control groups were also studied. Results Chylack’s cataract classification was used in the study. At the end of the tenth day, cataracts devel-
Prof. Dr. S. Taysi, PhD () · E. Demir · E. Saricicek · M. Akan · M. Tarakcioglu Department of Medical Biochemistry, Medical School, Gaziantep University, Gaziantep, Turkey e-mail: [email protected] B. Al Department of Emergency Medicine, Medical School, Gaziantep University, Gaziantep, Turkey T. Demir · C. Bagci Department of Physiology, Medical School, Gaziantep University, Gaziantep, Turkey S. Okumus · O. Saygili Department of Ophthalmology, Medical School, Gaziantep University, Gaziantep, Turkey A. Dirier Department of Radiation Oncology, Medical School, Gaziantep University, Gaziantep, Turkey
13
oped in 80 % of the rats in the radiotherapy group. After irradiation, cataract rate dropped to 20 % in NSO, 30 % in propolis, 40 % in CAPE, and 50 % in TQ groups and was limited to grade 1 and grade 2. Cataract formation was observed the least in NSO group and the most in TQ group. In the irradiated (IR) group, superoxide dismutase activity was lower, while glutathione peroxidase and xanthine oxidase activities and malondialdehyde level were higher compared with the other groups. Total superoxide scavenger activity and nonenzymatic superoxide scavenger activity were not statistically significant in IR group compared with the other groups. Conclusions The findings obtained in the study might suggest that propolis, CAPE, NSO, and TQ could prevent cataractogenesis in ionizing radiation-induced cataracts in the lenses of rats, wherein propolis and NSO were found to be more potent. Keywords Oxidative stress · Antioxidant · Lipid peroxidation · Irradiation
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 treatment. In all, 80 % of cancer patients need radiotherapy at some time or other, either for curative or palliative purpose. The radiosensitivity of the normal tissues adjacent to the tumor limits therapeutic gain. The responses of the normal tissues to the therapeutic radiation exposure range from those that cause mild discomfort to others that are life threatening. The speed at which a response develops varies widely from one tissue to another and often depends on the dose of radiation that the tissue received [1–3].
The effects of Nigella sativa oil, thymoquinone, propolis, and caffeic acid
1
original article
Eye morbidity is widely observed in patients receiving total-body irradiation prior to bone marrow transplantation or radiotherapy for ocular or head and neck cancers [4]. A deleterious effect of radiation is the production of reactive oxygen species (ROS), which includes superoxide anion (O2•–) and hydroxyl radical (OH•–). These ROS may contribute to radiation-induced cytotoxicity and to metabolic and morphologic changes in humans [5]. Reactive oxygen species are clearly involved in the pathophysiological changes in the eye, and any means of regulating these free radicals can have a huge impact in preventing disease onset in the eye. In the eyes, ROS have been implicated in diseases such as cataracts, uveitis, retrolental fibroplasia, age-related macular degeneration, and neovascular glaucoma [6–8]. Cataract blindness is the major cause of preventable blindness worldwide, especially in the developing countries [7]. It is the opacity of eye lens that interferes with vision. Cataracts are formed in response to a variety of different 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 exposures [1]. Nigella sativa oil (NSO) and thymoquinone (TQ) are reported to possess strong antioxidant properties against oxidative damage induced by a variety of free radicalgenerating agents. Studies had shown that the biological activity of Nigella sativa (NS) seeds is mainly attributed to its essential oil component that is predominantly thymoquinone. Many pharmacological and toxicological studies have reported it to possess diverse pharmacological properties such as antioxidant, hepatoprotective, neuroprotective, antidiabetic, anti-inflammatory, nephroprotective, anticarcinogenic [9–12]. Propolis and caffeic acid phenethyl ester (CAPE), an active component of propolis extract, have immunomodulatory, antitumoral, cytotoxic, antimetastatic, antiinflammatory, and antioxidant properties and have been shown to inhibit lipoxygenase activities as well as suppress lipid peroxidation [13]. All cells in the body are exposed chronically to oxidants from both endogenous and exogenous sources. Cells have developed different antioxidant systems and various antioxidant enzymes to defend themselves against free radical attacks. Superoxide dismutase (SOD) catalyzes the dismutation of the superoxide anion radical (O2•–) into hydrogen peroxide (H2O2) [14, 15]. Hydrogen peroxide is generally considered a major oxidant in cataractogenesis. The major systems degrading H2O2 in the lens involve glutathione peroxidase (GSH-Px), glutathione reductase (GRD), and glutathione (GSH) [16, 17]. Xanthine oxidase (XO) functions in purine and free radical metabolism, which also catalyzes the conversion of xanthine and hypoxanthine to uric acid and the production of O2•– [18]. Efforts to decrease toxicity of irradiation to normal tissue, organs, and cells have led to investigations to identify
cytoprotective agents. Unfortunately, most of radioprotectors possess toxic side effects, which limit their role in medical treatment. For this reason, investigations for effective and nontoxic compounds with radioprotection capability led to increasing interest in naturally occurring antioxidant such as NSO, TQ, CAPE, and propolis. To our knowledge, there is no experimental study that simultaneously investigates the effects of propolis, CAPE, NSO, and TQ supplementations on total (enzymatic plus nonenzymatic) superoxide scavenger activity (TSSA), nonenzymatic superoxide scavenger activity (NSSA), SOD, GSH-Px, XO, and malondialdehyde (MDA), a marker of lipid peroxidation, in the lens tissue of rats receiving ionizing radiation. Therefore, in the present study, we aimed to investigate the effects of these supplementations on oxidant/antioxidant parameters in the lens tissue of rats with or without exposure to total cranium irradiation.
Material and methods Chemicals Acetic acid, caffeic acid phenethyl ester (CAPE), dimethyl sulfoxide (DMSO), thymoquinone, thiobarbituric acid (TBA), sodium dodecyl sulfate (SDS), sodium hydroxide nicotinamide adenine dinucleotide (NADH), n-butanol, hydrogen peroxide (H2O2), xanthine, xanthine oxidase (XO), nitrobluetetrazolium (NBT), and trichloroacetic acid (TCA) were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals and reagents were obtained from the store of the Department of Medical Biochemistry, Gaziantep University Medical Faculty.
Animals and experiments In total, 74 male Sprague–Dawley rats, 10–12 weeks old, weighing 200 ± 25 g at the time of radiation, bred at the Experimental Animal Laboratory, Gaziantep University Medical School, were used for the experiment. The animals were purchased from the Experimental Animal Laboratory, Gaziantep University Medical School, and housed in cages 1 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). This study was approved by the local ethics committee of the Gaziantep University. Rats were randomly divided into eight groups (eight rats from control groups and ten rats from other groups) and placed in separate cages during the study. The groups were as follows: Group A (irradiation (IR) plus NSO group) received both 5 Gy of gamma irradiation as a single dose to total
2 The effects of Nigella sativa oil, thymoquinone, propolis, and caffeic acid
13
original article
cranium and NSO (1 g kg−1day−1) starting 1 h before irradiation dose and continuing for 10 days through an orogastric tube. Group B (IR plus propolis group) received both 5 Gy of gamma irradiation as a single dose to total cranium and propolis (80 mg kg−1 day−1) starting 1 h before irradiation and continuing for 10 days through an orogastric tube. Propolis was dissolved in DSMO just before giving to the rats. Group C (IR plus TQ group) received both 5 Gy of gamma irradiation as a single dose to total cranium and TQ (50 mg kg−1day−1) daily by intraperitoneal (IP) injection starting 30 min before the radiation dose and subsequently daily for 10 days after irradiation. TQ was dissolved in DSMO just before giving to the rats. Group D (IR plus CAPE group) received both 5 Gy of gamma irradiation as a single dose to total cranium and CAPE (10 µmol kg−1day−1, IP) injection starting 30 min before the radiation dose and subsequently daily for 10 days after irradiation. CAPE was dissolved in DSMO just before giving to the rats. The final concentration of DMSO was 0.1 %. Group E IR group received 5 Gy of gamma irradiation as a single dose to total cranium and saline (1 ml kg−1 day−1, IP) injection. Group F1 (the control for A group) received just 1 ml of saline through an orogastric tube and did not receive anything (NSO, TQ, CAPE, or propolis). Group F2 (the control for B, C, and D groups) did not receive NSO, TQ, CAPE, propolis, or irradiation, but received DMSO injections (IP) at an equal volume of that propolis, TQ, and CAPE as dissolved for group B, C, and D, respectively. Group F3 (normal control group) did not receive NSO, TQ, CAPE, propolis, or irradiation. Animal experimentations were carried out in an ethically proper way by following guidelines as set by the ethical committee of the Gaziantep University. Supplementation period was 10 days. Propolis, CAPE, and TQ were dissolved in DMSO just before giving to the rats. Prior to total cranium irradiation, 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 in the IR and the IR plus TQ groups received irradiation via a cobalt-60 teletherapy unit (Picker, C9, Maryland, NY, USA) from a source-to-surface distance of 80 cm by 5 × 5-cm anterior fields, with the total cranium gamma irradiation being a single dose of 5 Gy, while the rats in the control and sham control groups received sham irradiation. 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 opacity classification system, version III (LOCS III), was used in the cataract classification [19]. Before the cataracts were graded, the pupils were
13
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). The amount of cortical cataract (C) was determined by comparing the estimated aggregate of cortical spoking with that seen in five separate photographs. Similarly, the estimated amount of posterior subcapsular cataract (P) was determined by comparing it with another five photographs depicting increasing amounts of posterior subcapsular cataract. At the beginning of the experiment, all rats were examined biomicroscopically and were only included in this study if their lenses had been NO0, NC0, C0, and P0.
Biochemical analysis Ten days after irradiation, all animals were killed by decapitation; their eyes were enucleated; and the lenses were dissected immediately. Lenses were homogenized by an IKA-NERKE (Staufen, Germany) homogenizer in physiological saline solution (20-fold) 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 conducted on this fraction. All he procedures were performed at 4 °C. TSSA and NSSA assays were performed in the samples before and after adding trichloroacetic acid (TCA, 20 %), as described [20, 21]. First, TSSA was measured. In this method, xanthine–xanthine oxidase complex produces superoxide radicals that react with nitroblue tetrazolium (NBT) to form a farmazone compound. TSSA activity was measured at 560 nm by detecting inhibition of this reaction. By using a blank reaction in which all reagents are present except the supernatant sample and by determining the absorbance of the sample and blank, TSSA activity was calculated. Second, NSSA activity was measured in TCA-treated fractions prepared by treating part of the sample with final concentration of 20 % (w/v) TCA solution (to remove all enzymes and proteins), and centrifuging at 5,000 g for 30 min. After the elimination of proteins by this procedure, NSSA activity assaywas performed in the supernatant fraction. SOD activity was calculated as the difference between TSSA and NSSA. One unit of TSSA, NSSA, and SOD was defined as the amount of enzyme protein causing 50 % inhibition in nitroblue tetrazolium reduction rate. Results were expressed as U/mg protein. GSH-Px activity was assayed according to Paglia and Valentina [22]. In this method, GSH-Px catalyzes the oxidation of glutathione in the presence of tert-butyl hydroperoxide. Oxidized glutathione is converted to the reduced form in the presence of glutathione reductase and NADPH, while NADPH is oxidized to NADP. The reduc-
The effects of Nigella sativa oil, thymoquinone, propolis, and caffeic acid
3
original article
tion in the absorbance of NADPH at 340 nm is measured. The absorbance change per minute and the molar extinction coefficient of NADPH were used to calculate GSH-Px activity. GSH-Px activity was expressed as IU/mg protein. XO activity was measured spectrophotometrically by the formation of uric acid from xanthine through the increase in absorbance at 293 nm [23]. XO activity was expressed as U/mg protein. Concentration of MDA, the final product of lipid peroxidation, was determined spectrophotometrically according to a similar method described by Ohkawa et al. [24]. Briefly, a mixture of 8.1 % sodium dodecyl sulfate (SDS; 0.2 ml), 20 % acetic acid (1.5 ml), and 0.9 % thiobarbituric acid (1.5 ml) was added to the mixture to bring the total volume up to 4 ml. This mixture was incubated at 95 °C for 1 h. After incubation, the tubes were left for cooling under cold water and 5 ml of n-buthanol/pyridine (15:1, v/v) was added, followed by mixing up. The samples were centrifuged at 3,500 g for 10 min. The organic phase that accumulated at the top of the tubes was sampled, and sample absorbance values were measured with respect to the blank at 532 nm. The concentration of MDA was calculated using 1.56 × 105 Mcm−1 as molar extinction coefficient. The total thiobarbituric acid-reactive substances were expressed as MDA. Results were expressed as nmol/mg protein. The protein content was determined as described [25]. Biochemical measurements were carried out using a spectrophotometer (Shimadu U 1601, 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 analyzed using post hoc least significance difference test procedure. Acceptable significance was recorded when P values were 0.05). We observed a significant increase of cataract formation in the IR group compared with the control groups (p
