Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 114 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. EFFICACY OF PROBIOTIC (PROTOXINE) ON MERCURY-INDUCED NEPHROTOXICITY AND LIPID PEROXIDATION IN RATS Ajwad Awad Muhammad Assumaidaee1,3 Nathera Muhammad Ali1 Suhair Hassan Akutbi1 Ammar Amer Fadhil2 1 Department of Clinical Laboratory Sciences-College of Pharmacy-University of Baghdad, Iraq. 2 Department of Pharmacology and Toxicology-College of Pharmacy-University of Baghdad. 3Corresponding author: drajwadawad@gmail.com ABSTRACT Mercury is the third most dangerous heavy metal and its toxicity causes serious risks to health through unfavorable pathological and biochemical effects of oxidative stress. The aim of the present study was to elucidate the possible protective role of protoxine, a probiotic, on mercuric chloride-induced oxidative stress and histopathological changes in the kidneys of the experimented rats. Adult male Wistar albino rats were daily exposed to mercuric chloride (0.25 mg kg-1) in the diet and water together for 20 days. Treatment with mercuric chloride led to oxidative stress by elevating malondialdehyde (MDA) level and also decreasing superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. Furthermore, mercuric chloride exposure induced histopathological changes characterized by tubulonecrosis in the renal tissue of intoxicated rats. MDA level, SOD, GSH-Px activities and histopathological changes were modulated in concomitantly daily supplementation of protoxine (12 mg rat-1) to mercuric chloride-treated groups. Results from current study suggested mercuric chloride treatment resulted lipid peroxidation, alterations of enzymatic antioxidant defense system and histolopathological changes in the kidneys of male rats. The administration of protoxine proved to be beneficial in ameliorating the mercuric chloride-induced nephrotoxicity. Key words: Mercuric chloride, Oxidative stress, Nephrotoxicity, Probiotic, Histopathology. INTRODUCTION Mercury present in the environment is a well-established toxicant to human and animal health. Exposure to mercury brought harmful effects to human health, but changes resulting from human exposure to mercury only called the attention of the scientific society after the accidents in Japan and Iraq (Bernhoft, 2012). In Iraq, an outbreak mercury poisoning occurred in 1971 when wheat grains were treated with fungicides containing organic mercury. Over 500 people who ate bread made Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 115 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. with contaminated wheat were killed (Bakir et al., 1973). In Japan, in the (Minamata Bay) a tremendous poisonous cases occurred resulting from the deposition of industrial waste with large quantities of mercury. Mercury was then ingested by human through fish intake. Signs and symptoms such as ataxia, speech impairment, visual field constriction, sensory disturbance, deafness, blindness, tremors, involuntary movements, mental retardation, coma, and death were recorded. Infants whose mothers were infected developed mental retardation, peripheral neuropathy, cerebral palsy and blindness. These changes became known as Minamata disease or Russell-Hunter syndrome (Takeuchi et al., 1996 ; Yee and Choi, 1996 ; Gochfeld, 2003). Mercury in the inorganic form such as salts, for example, mercury (II) chloride, primarily affects gastrointestinal tract and kidneys. Since it cannot cross the blood brain barrier easily, mercury salts inflict little neurological damage without continuous or heavy exposure. Mercury (II) salts are usually more toxic than their mercury (I) counterparts because of their greater solubility in water and rapid absorption by the gastrointestinal tract (Lund et al., 1993). Nephrotoxicity is a serious side effect of mercury and is believed to be related to reactive oxygen species in the kidney. It is well known that inorganic mercury causes severe kidney damage after acute and chronic exposure (Monisha et al., 2014). Pretreatment with ZnCl2 abolished mercury-induced delta-ALA-D- inhibition in kidneys. In face of zinc effects to prevent Hg-delta-ALA-D inhibition and to alter Hg-deposition levels in kidney these results suggest that these effects may be partially due to the synthesis of metallothioneins (Peixoto et al., 2003). Recent investigations have proved the crucial role of nutritional antioxidants to prevent the damage caused by toxic compounds (Ahmed et al., 2010). Probiotics are defined as “live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host. These micro-organisms are normally isolated from intestinal microflora of the intended species and selected based on conditions such as resistance to stomach acids, bile salts, ability to colonize the intestinal harmful microorganisms (Chaucheyras and Durand, 2010 ; Cho et al., 2011 ; Zahra et al., 2017). Some probiotics are also known to have antioxidative properties in human subjects, which may be another important characteristic for mercury toxicity protection. On the basis of these special functions, lactobacilli seem to have potency against mercury toxicity. Alla et al., (2017) found that probiotics can decrease cadmium pollution effectively. This hypothesis was also Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 116 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. proposed in a recent review (Monachese et al., 2012), but to our knowledge, very few studies on the protective effects of lactobacilli against mercury toxicity have been carried out so far and the mechanism of such protection has not been studied yet. Therefore, it is interesting to investigate whether some probiotic like protoxine might play a role in the alleviation and/ or amelioration of mercury toxicity. The current study was designed to determine the antioxidative stress impact of using probiotic (protoxine) on the biological effects of mercurial toxicity in rat model. MATERIALS AND METHODS Animals Eighteen adult male Wister albino rats weighing 250-275 g were used for the present study. They were reared at the animal house of the college of pharmacy, University of Baghdad, Baghdad, Iraq with the approval of animal rights review committee. They were acclimatized for 1 week on normal diet of pelletized rat chow, with water given ad libitum at room temperature (25-28 ᵒC) within a 12:12 hrs. light and dark cycle before the commencement of the experiment. Experimental induction of nephrotoxicity Mercuric chloride was chosen to induce nephrotoxicity in rats. Mercuric chloride was added to the food and water of the experimental rats at a non lethal dose of (0.25 mg HgCl2 kg -1 food or water). Probiotic Material Used PROTOXINE (total viable count= 2.00x1012) a combination of beneficial probiotic microorganisms in dextrose monohydrate: Lactobacillus plantarum Lactobacillus delberueckii spp Bulgaricus PXN39 Lactobacillus acidophilus Lactobacillus rhamnosus Bifidobacterium bifidum Streptococcus salivarius spp Thermophilus Enterococcus faecium Experimental Setup Rats were divided into three groups comprising of six animals each: Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 117 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. Group I: Served as control (Received standard diet and normal tap water). Group II: Received contaminated diet and water with HgCl2 continuously for 21 days. Group III: Received contaminated diet and water with HgCl2 continuously mixed with probiotic (protoxine) in a dose of 12 mg each rat-1 for 20 days. Collection and Processing of Rat tissues At the 0 and 21 days, Blood samples were collected by puncture from the heart in clean test tubes, serum separated by centrifuging the blood samples at 4000 rpm for 5 min to obtain the serum for the estimation of malondialdehyde (MDA) oxidative stress biomarker, GSHP-x (Glutathione peroxidase) and SOD (Superoxide dismutase). Furthermore, at the end of experimental period (21 days), the animals were humanly sacrificed by using 60 mg/kg body weight of sodium pentothal. Kidneys were removed and washed with ice-cold saline and immersed 5 days in 10% neutral buffered formalin, processed in automatic tissue processor and stained with hematoxiline and eosin for histopathologic study. Histopathological Studies Kidneys were fixed for 48 hr in 10% neutral buffered formalin saline. Tissues were embedded in paraffin and sectioned at 5-μm thickness using a rotary microtome. Sections were stained with hematoxylin-eosin (H and E) according to Luna (1968) for light microscopy examination. Biochemical Analysis Determination of MDA by Thiobarbituric acid reactive substances (TBARS): The proteins in 0.15 ml serum were first precipitated by using sulphuric and phosphotungstic acid and then the levels of MDA were measured in these samples. Precipitate obtained incubated with TBA in a water bath at 95 °C for 60 minutes in an environment with oxygen and pH=3.4. The colored complex that occurred was refrigerated to room temperature. Then the complex was taken into n-buthanole phase. At the end, the complex of MDA-(TBA) was measured by using Shimadzu UV-1201V spectrophotometry at 532 nm TEP (1, 1, 3, 3-tetraethoxypropane) was used as standard MDA. Serum activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) of all groups were analyzed on Randox diagnostic’s kits by automated chemistry analyzer. Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 118 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. Statistical Analysis Statistical analysis concluded analysis of variance of mean±SD were performed using the SPSS version 18 statistical package, 2010, (SPSS Inc. Chicago, IL, USA). Data were expressed in as mean ± SEM. P- value of less than 0.05 was considered as statistically significant. RESULTS Oxidative-antioxidative stress biomarkers At day 0 there were no significant statistical differences (p<0.05) between all groups regarding the level of serum malondialdehyde (MDA) and activites of both superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) (Table 1). Table 1. The protective role of protoxine against the toxic effects of mercuric chloride on serum malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in rats at day 0 Values are given as mean ± SEM for 6 rats group-1. Each value not sharing a common letter superscript is significantly different (p<0.05). Whereas, at day 21 there were a significant statistical differences (p<0.05) between both control (Group 1) and HgCl2 +Protoxine (Group 3) comparing to the HgCl2 (Group 2) regarding the estimations of MDA level and SOD activity. Frank and significant statistical differences (p<0.05) in the activity of GSH-Px were seen between all groups at this day (Table 2). Table 2. The protective role of protoxine against the toxic effects of mercuric chloride on serum malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in rats at day 21 Values are given as mean ±SEM for 6 rats group-1. Each value not sharing a common letter superscript is significantly different (p<0.05). Variable Group 1 (Control) N=6 Group 2 (HgCl2) N=6 Group 3 (HgCl2+Protoxine) N=6 MDA (M mol l-1) 0.55 ± 0.19A 0.57 ± 0.21A 0.57 ± 0.11A SOD (µ mg-1 Hb) 11.01 ± 1.26 A 10.90 ± 1.88A 10.13 ± 1.15A GSH-Px (µ mg-1 Hb) 25.35 ± 0.69 A 25.27 ± 0.39 A 24.95 ± 1.61A Variable Group 1 (Control) N=6 Group 2 (HgCl2) N=6 Group 3 (HgCl2+Protoxine) N=6 MDA (M mol l-1) 0.57 ± 0.19A 1.88 ± 0.16B 0.59 ± 0.41A SOD (µ mg-1 Hb) 10.86 ± 1.14 A 6.50 ± 1.17B 9.93 ± 1.15A GSH-Px (µ mg-1 Hb) 24.18 ±0.79 A 12.17 ± 0.49 B 20.45 ± 1.01C Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 119 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. Histopathological findings No histopathological lesions and normal tissue architecture had been seen in the kidneys of the control group (Fig. 1). In Group 2 the histopathological findings show that HgCl2 cause diffuse coagulative necrosis of the distal part of the proximal convoluted tubules, mainly the pars recta and in the outer stripe of outer medulla, respectively. Furthermore, nephropathy was characterized by nephrosis, foci of hydropic tubular degeneration, thickened tubular basement membrane and scattered dilated tubules containing hyaline casts. Massive and diffuse cellular necrosis was observed in the proximal tubules of kidneys from rats treated with HgCl2 alone. The dead cells were in an advanced stage of karyolysis and cytoplasmolysis, fragmentation and dissolution. In addition, the lumen of these tubules were filled with numerous dead cells, fragments of cells, sometimes membrane-bound and debris were shed into the lumen of the tubules with degenerated and desquamated epithelial cells, massive hyaline casts, and very few infiltrated inflammatory cells in the form of mononuclear cells (Fig. 2 and 3). On the contrary, in the treated group with protoxine the damages exclusively, inducing vacuolization of some renal convoluted tubules and rarely early signs of necrosis in few proximal convoluted tubules where seen in limited areas of kidney. There was a significant reduction in lesions caused by epithelial and nuclear changes typically associated with tubular necrosis, observed only in the animals that were continuously treated with protoxine (Fig. 4). This was evidenced by preservation of tubular morphology compared to the group treated with HgCl2 alone. A statistically significant difference was observed in treated animals and controls comparing to the intoxicated group. The kidneys of all treated rats are summarized in (Table 3). Light microscopic examination of kidneys from control and probiotics treated rats showed almost normal architecture to mild structural alterations in renal tissues. Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 120 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. Figure 1. Kidney of rat from control group, normal tissue architecture Figure 2. Kidney of rat from HgCl2 treated group, nephropathy was characterized by nephrosis, foci of hydropic tubular degeneration, thickened tubular basement membrane and scattered dilated tubules containing hyaline casts. A few casts are present in the distal part of the pars recta between the vascular bundles Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 121 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. Figure 3. Kidney of rat from HgCl2 treated group, first part of pars recta showing pyknosis, karyorrhexis, karyolysis, cytoplasmolysis and mild degree of mononuclear cells infiltration as advanced nuclear & cytoplasmic signs of coagulative necrosis Figure 4. Kidney of rat from Group 3 (HgCl2+Protoxine) treated group, a significant reduction in lesions observed only in the animals that were continuously treated with protoxine, there was pronounced preservation of tubular morphology compared to the group treated with HgCl2 alone Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 122 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. Table 3. Histopathologic scoring of renal lesions in kidneys of Group 1 (Control), Group 2 (HgCl2) treated group and Group 3 (HgCl2+Protoxine) treated group at the end of the experimental period 21 days DISCUSSION Current study may be the first to evaluate some of the protective mechanisms of the oral administration of the probiotic (protoxine) as (beneficial microorganism) against mercurial nephrotoxicosis. This study provides biological evidence that protoxine can reduce lipid peroxidation, and reverse deficits in antioxidant defense systems to alleviate subsequent bad consequences of mercurial nephrotoxicosis. Oxygen free radicals are considered to be important mediators of mercury- induced renal failure (Stuart et al., 2006). Characters of mercury nephrotoxicity are oxidative stress and inflammation. Generation of reactive oxygen metabolites may be the basis of a variety of insults, such as lipid peroxidation. The nephrotoxicity of mercury is associated with its accumulation in the renal cortex, which is leading cell damage and death. Treatment with several natural and synthetic antioxidant substances has been extensively studied and shown to be useful for either the prevention or amelioration of nephrotoxicity in experimental rats. Taken together, the results showed that compared to the mice that received mercury only, probiotic treatment can effectively decrease and alleviate renal oxidative stress, and ameliorate renal histopathological changes. Our results suggested that probiotic treatment is more effective against mercuric toxicity than a simple antioxidant treatment due to its special physiological functions and that it can be considered a new dietary therapeutic strategy against nephrotoxicity. Previous studies confirmed that some species of lactic acid bacteria (LAB) including Lactobacillus rhamnosus, L. plantarum, and Bifidobacterium longum are Lesions Group 1 (Control) Group 2 (HgCl2) Group 3 (HgCl2+Protoxine) Nephrosis - + + + + + + + + Pyknosis in the endothelium of the proximal convoluted tubules - + + + + + + + + + + Karyorrhexis - + + + + + + + + + + Karyolysis - + + + + + + + + + Cytoplasmolysis - + + + + + + + + + + Infiltration of inflammatory cells - + + + Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 123 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. capable of binding heavy metals in vitro (Halttunen et al., 2007 ; Halttunen et al., 2008). Furthermore, multiple recent reports confirmed that other probiotics may also be protective against heavy metal toxicity. A mixture of L. rhamnosus Rosell- 11, L. acidophilus Rosell-52 and B. longum Rosell-175 significantly reduced Cd- induced genotoxicity both in vitro using liver tissue culture and in rats (Jama et al., 2012). Frankly, the known histopathological lesions (Haagsma and Pound, 1980) and antioxidant enzyme levels associated with Hg-induced nephrotoxicity in rats were also observed. These findings were confirmed by observation of histopathological lesions such as epithelial desquamation, tubular necrosis, tubular casts, and infiltration of inflammatory cells, indicating that the kidney is very sensitive to Hg toxicity. A significant decrease in oxidative stress (MDA) biomarker and significant increase in antioxidative stress biomarker, however, were observed in the Hg+ probiotic group when compared with the Hg alone group. These data indicate that probiotic at selected dose has exerted a protective effect against Hg nephrotoxicity. The administration protoxine may effectively decreased intestinal Hg absorption and alleviated tissue oxidative stress, reversed renal damage and ameliorated the corresponding histopathological changes. These findings are in the the line of Hemaiswarya et al., (2013) explanations about the mechanism of action of probiotics. The evidence of this study confirms the relationship between oxidative-anti oxidative parameters and histopathological findings, Furthermore, Fermentation breaks down the nutrients in foods by the action of beneficial microorganisms and creates natural chelators that are available to bind toxins and remove them from the body may added as another mode of action to minimize the potential toxic effects of mercurial toxicosis. CONCLUSION The selected combination of beneficial microorganisms (Protoxine) has some significant protective effect on experimental nephrotoxicity induced by HgCl(2) in rats and it is related to inhibition of lipid peroxidation (MDA production), which involves triggering of antioxidative stress master enzymes SOD and GSH-Px too. Based on current findings there is a need to try a more profound investigation into the antioxidant abilities of probiotics and prebiotics based on the available experimental and clinical data. Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 124 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. REFERENCES Ahmed, M., A. Ali, B. Ali, F. Mohammed and M. Hassan. 2010. High fiber probiotic fermented mare's milk reduces the toxic effects of mercury in rats. N. Am. J. Med. 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Sci., 22: 74-84. تأثير إعطاء المسكن الحيوي )البروتوكسين( في تسمم وأكسدة الدهون في كلى الجرذان المعاملة بالزئبق 2عمار عامر فاضل 1سهير حسن القطبي 1ناثرة محمد علي النعيمي 3، 1أجود عواد الصميدعي الصيدلة، جامعة بغداد، العراققسم العلوم المختبرية السريرية، كلية 1 قسم السموم والفارماكولوجين كلية الصيدلة، جامعة بغداد، العراق 2 drajwadawad@gmail.comالمسؤول عن النشر: 3 المستخلص من خالل تاثيراته ةتسبب سميته مخاطرا صحية جدي ذإيشكل الزئبق ثالث اخطر المعادن الثقيلة اهمية ةالحالي ةالتجريبي ةدي التي يحدثها. الهدف من الدراسالمتاتية من عملية الجهد التاكس ةغير المرغوب ةالمرضي وتوكسين على عملية الجهد التاكسدي التي يوفرها اعطاء السابق الحيوي المسمى بر ةهو تخمين دور الحماي من ةبالغ . تضمن البحث استخدام جرذانا ةلمستخدمفي كلى الجرذان ا ةالناشئ ةالمرضي ةوالتغييرات النسيجي Diyala Journal of Agricultural Sciences, 10(Special issue): 114-126, 2018 Assumaidaee et al. 126 1st Scientific Conf., College of Vet. Med. Diyala Univ., 2018 1st Scientific Conf., College of Veter. Medic. Diyala Univ., http://www.agriculmag.uodiyala.edu.iq/ 1st Scientific Conf., College of Veter. ملغم لكل كغم ولمدة 22جنس وستر الباينو التي تم اعطائها كلوريد الزئبق عن طريق الماء والعلف بجرعة لوحده جهدا تاكسديا تمت مالحظته عبر ارتفاع مستوى المالون داي عشرين يوما. سبب كلوريد الزئبق المضاده ةالحيوي ةالديهايد المؤشر الحيوي لعملية اكسدة الدهون وانخفاضا لفعالية بعض الموءشرات االنزيمي ث احدإ فضال عنسوبراوكسيايد دسميوتيز وانزيم الكلوتاثايون بيروكسيديز ـللجهد التاكسدي مثل انزيم ال السابق الحيوي ان المعامله به. احدثت اضافةذمثلت بالنخر النبيبي في كلى الجرت ةتغيرات نسيجية مرضي لوريد الزئبق انخفاضا هاما ملغم لكل حيوان والمستخدم سوية مع ك 12مقدارها ةيومي ةالبروتوكسين بجرع الجهد مع احداث تعديال ايجابيا في هيئة لك ذل ةضادشر الجهد التاكسدي وارتفاعا في اقيام االنزيمات المؤفي م على التاثير السمي لكلوريد الزئبق وقدرته نتائج البحث الحاليأكدت . ةالكلوي ةالمرضي ةاالفات النسيجي به وتغيرات سلبية ةان المعاملذفي كلى الجر ةتغيرات نسيجية مرضيجرائها أنش ةاحداث عملية اكسدة دهني ه ذ. ان االستخدام المرافق للسابق الحيوي البروتوكسين مع كلوريد الزئبق في هفي النظام االنزيمي المضاد ان ذن تناول الجرعة الناشئ ةالكلوي ةاثبتت قدرته على احداث تحسن احصائي هام في وضع السمي ةالتجرب الزئبق. دلمركب كلوري التسمم بالزئبق.البروتوكسين، مضاد أكسدة الدهون، كلى الجرذان، الكلمات المفتاحية: