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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 32  |  Issue : 1  |  Page : 41-48

Protective effect of Nigella sativa against cerebral ischemia and sodium valproate-induced hepatotoxicity


Department of Pharmacology and Therapeutics, Faculty of Medicine, Benha University, Benha, Egypt

Date of Submission13-May-2015
Date of Acceptance15-May-2015
Date of Web Publication26-Nov-2015

Correspondence Address:
Yasmin M Ismail
Department of Pharmacology, Faculty of Medicine, Benha University, 3112 Benha
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-208X.170558

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  Abstract 

Background
Nigella Sativa (NS) is one of the traditionally used herb well known for its healing properties. The most of the therapeutic properties of this plant is due to the presence of Thymoquinone (TQ), the major bioactive component of the essential oil. TQ is also a promising dietary chemopreventive agent for the treatment of number of diseases. Very low level of toxicity has been reported through acute and chronic toxicity studies.
Aim
The present study aimed to investigate the anti-ischemic effect of NS using carotid artery occlusion model in rats.This study also aimed to investigate the effect of NS on SVP induced hepatotoxicity.
Method
0Animals given NS and TMZ for 21 days then subjected to 45 min for brain ischemia then reperfusion.Animals administered NS, SVP for 21 days then subjected to serum measurement of serum ALT & AST and liver histopathological examination.
Results
NS significantly reduced the percent of necrosis and reduced the size of cerebral infarction compared to control and enhanced the effect of TMZ on cerebral ischemia.NS produced significant decrease in serum ALT and AST and improvement of histopathlogical picture compared to SVP group.
Conclusion
These results indicate that the NS could have a therapeutic effect against cerebral ischemia.NS has protective effect against SVP induced hepatotoxicity.

Keywords: Cerebral ischemia, hepatotoxicity, Nigella sativa


How to cite this article:
Abd El Latif EA, Sanad RA, Abdallah OM, Ismail YM. Protective effect of Nigella sativa against cerebral ischemia and sodium valproate-induced hepatotoxicity. Benha Med J 2015;32:41-8

How to cite this URL:
Abd El Latif EA, Sanad RA, Abdallah OM, Ismail YM. Protective effect of Nigella sativa against cerebral ischemia and sodium valproate-induced hepatotoxicity. Benha Med J [serial online] 2015 [cited 2017 Sep 19];32:41-8. Available from: http://www.bmfj.eg.net/text.asp?2015/32/1/41/170558


  Introduction Top


Nigella sativa (of the family Ranunculaceae) is a plant that is synonymous with Nigella cretica and is commonly called black cumin. Other names include Kalonji seeds, Ajaji, black caraway seed, and Habbatu Sawda [1] . The medicinal usage of these seeds mostly encompasses diarrhea and abdominal pain, dyslipidemia, asthma and cough, headache, dysentery, renal calculi, infections, obesity, back pain, hypertension, and dermatological problems [2] . The essential oil of the seeds contains a variety of molecules, of which thymoquinone (TQ) is considered as the main component of the entire seed [3] . In folk medicine, it has been traditionally used for a variety of applications, including treatments related to respiratory health, stomach, and intestinal diseases, kidney and liver function, circulatory and immune system support, and for general overall well-being [4] . N. sativa and TQ inhibit nonenzymatic lipid peroxidation [5] . TQ can attenuate picrotoxin-induced seizures, as well as potentiate the protective effects of sodium valproate (SVP) when coadministered [6] . Isolated TQ appears to be relatively safe, with an oral LD 50 of 794.3 mg/kg in rats and 870.9 mg/kg in mice, which is ~100-150 times higher than its therapeutic level. There have been some isolated cases of topical usage of N. sativa causing contact dermatitis, suggesting that it is possible to be allergic to the seed oil [7] . SVP is the sodium salt of valproic acid and is an anticonvulsant used in the treatment of epilepsy, anorexia nervosa, panic attack, anxiety disorder, and bipolar disorder, as well as other psychiatric conditions requiring the administration of a mood stabilizer [8] . SVP is a weak blocker of sodium ion channels; it is also a weak inhibitor of enzymes that deactivate GABA, such as GABA transaminase. It may also stimulate the synthesis of GABA, but the direct mechanism is not known [9] . The present study aimed to investigate the anti-ischemic effect of N. sativa using carotid artery occlusion model in rats. This study also aimed to investigate the effect of N. sativa on SVP-induced hepatotoxicity.


  Materials and methods Top


Materials

Drugs

N. sativa
was supplied as gelatinous capsules of 450 mg (Mepaco Company, Cairo, Egypt). SVP was supplied as tablets, 200 mg (Sanofi Aventis Company, Paris, France). Trimetazidine (TMZ) was supplied as tablets, 20 mg (Global Napi Pharmaceutical Company, Cairo, Egypt).

N. sativa was dissolved in corn oil [3] . SVP and TMZ were dissolved in distilled water [10],[11] .

Animals

Experimental animals (60 rats weighing 250-275 g) were kept six per cage under complete healthy conditions throughout the experiment, including a clean environment, good ventilation, and good nutrition. The animals were housed in groups to acclimatize to laboratory conditions for 3 days before the experiment, such as diet, water, and temperature (22°C). Food and water were freely accessible. The experimental rats were under the care of professional technicians and qualified researchers. The work was approved by the ethical committee (Faculty of Medicine, Benha University).

Methods

Part I: Effect of N. sativa on cerebral ischemia

This procedure was carried out to investigate the effect of N. sativa either singly or in combination with the anti-ischemic agent TMZ. A total of 36 adult male albino rats were used in this study. They were divided into six groups of six animals each: group 1 (normal control animals) received no drugs; group 2 (the corn oil group) received corn oil in volumes comparable to that of tested drugs; group 3 (the saline group) received saline in volumes comparable to that of tested drugs; group 4 (N. sativa-treated cerebral ischemic rats) received N. sativa at 4 mg/kg intraperitoneally for 3 weeks [12] and then was exposed to cerebral ischemia by ligation of carotid artery; group 5 (TMZ-treated cerebral ischemic rats) received TMZ at 2.5 mg/kg [13] for 3 weeks and then was exposed to cerebral ischemia by ligation of carotid artery; and group 6 (TMZ and N. sativa-treated ischemic rats) received both TMZ and N. sativa at the same doses as groups 3 and 4 and then was exposed to cerebral ischemia by ligation of carotid artery.

Brain ischemia

Method of induction of cerebral ischemia
: Cerebral ischemia was induced using the four-vessel-occlusion technique described by Pulsinelli and Briely [14] . Anesthesia was induced with an intraperitoneal injection of ketamine/xylazine (60 and 6 mg/kg, respectively). Following a dorsal neck incision, the first cervical vertebra and alar foraminae were exposed. Vertebral arteries were electrocauterized permanently. On the next day, under brief anesthesia, the common carotid arteries were dissected from the surrounding tissues and temporarily ligated using the microvascular clamps for 20 min. At the end of the ischemic period, the clamps were released and reperfusion restored. Same procedures were applied to the control group, and, instead of TMZ, an equal amount of intraperitoneal saline was administered daily. After 3 days of reperfusion, the animal was killed with a high-dose anesthetic and cervical dislocation. Craniotomy was performed to remove the brain and preserve it in formalin.

Brain samples were removed and placed in 10% formalin. Initially, they were preserved for 72 h, and then 0.5-cm-thick sections were cut and placed in formalin for another 24 h. Using a rat-brain  Atlas More Details [15] , sections including both hippocampuses were cut and placed in paraffin blocks. From these blocks 4-μm-thick sections were cut and stained with hematoxylin and eosin [14] .

Cells that showed karyopyknosis, hyperchromasia, or contour roughness were regarded as delayed ischemic damage, and the proportion of damaged cells were expressed as the percentage of total cells counted.

Part II: Assessment of the hepatoprotective activity of N. sativa against sodium valproate-induced hepatotoxicity

A total of 24 male albino rats were used. They were divided into four groups of six rats each: group 1 (the control group) was not administered any drug; group 2 was administered SVP at a dose of 500 mg/kg; group 3 was administered N. sativa at a dose of 0.2 mg/kg; and group 4 was administered N. sativa at a dose of 0.2 mg/kg and SVP at a dose of 500 mg/kg. All drugs were administered intraperitoneally for 3 weeks.

SVP at a dose of 500 mg/kg/day has been described and proven to be hepatotoxic [16],[17] . For the hepatoprotective effect of N. sativa, the daily dose was calculated to be 0.2 mg/kg/day (intraperitoneal), as already reported by Aniya et al. [18] . The treatment continued for the same duration as for valproate. Rats from the treated and control groups were killed after 21 days. Blood samples were drawn by means of direct cardiac puncture into nonheparinized capillary tubes. Serum was separated by centrifugation for 5 min and stored at −20°C until biochemical assessment of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) was carried out.

Statistical analysis

Data were presented as mean ± SD and analyzed with nonparametric statistics using the Kruskal-Wallis test, followed by the Dunn-post test using GraphPad Prism (California, USA) 5 software at P less than 0.05 (N = 6). For other parameters, data were presented as mean ± SD and analyzed using the one-way analysis of variance test, followed by the Tukey Kramer-post test using GraphPad Prism 5 software at P less than 0.05 (N = 6).


  Results Top


Effect on cerebral infarction

N. sativa showed significant neuroprotective effect on the size of cerebral infarction (2540.17 ± 26 μm 2 ) in comparison with controls (20968.5 ± 13 μm 2 ). No significant difference was found between the control, corn oil (20166 ± 63 μm 2 ), and saline-treated groups (20279 ± 64 μm 2 ) (P > 0.05). Therefore, other groups were compared with the control group. No significant difference was found in the size of cerebral infarction between the N. sativa-treated (2540.17 ± 26 μm 2 ) and the TMZ-treated group (2384.2 ± 31 μm 2 ) (P > 0.05). N. sativa caused significant potentiation of the neuroprotective effect of TMZ (P < 0.05). This has been observed on comparison of the size of cerebral infarction between TMZ+N. sativa-treated group (1039.1 ± 83 μm 2 ) and the TMZ-treated group ([Table 1]).
Table 1 Effect of Nigella sativa on size of cerebral infarction in comparison with trimetazidine


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Histopathological results

The brain of control rats showed necrotic changes in about 50% of neurons. Infarction size was 20968.5 ± 13 μm 2 ([Figure 1]). The corn oil and saline groups showed necrotic changes in about 50% of neurons, and infarction size was 20166.4 ± 63 and 20279.5 ± 64 μm 2 , respectively ([Figure 2]). The brain of N. sativa group rats showed necrotic changes in 10-50% of neurons, and infarction size was 2540.17 ± 26 μm 2 ([Figure 3]). The brain of TMZ group rats showed necrotic changes in 10-50% of neurons, and infarction size was 2384.2 ± 31 μm 2 ([Figure 4]). The brain of N. sativa+TMZ group rats showed necrotic changes in less than 10% of neurons, and infarction size was 2039.1 ± 83 μm 2 ([Figure 5]).
Figure 1 A section of the brain from a control group rat showing necrotic changes in more than 50% of neurons. Area of cerebral infarction. H&E, ×40.



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Figure 2 A section of the brain from corn oil and water group rats showing necrotic changes in more than 50% of neurons.



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Figure 3 A section of the brain from a Nigella sativa-treated rat showing necrotic changes in about 10– 50% of neurons. Arrows are pointing to infarction tissue. H&E, ×40.



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Figure 4 A section of the brain from a trimetazidine-treated rat showing necrotic changes in about 10– 50% of neurons. Arrows are pointing to infarction tissue. H&E, ×40.



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Figure 5 A section of the brain from a Nigella sativa plus trimetazidine-treated rat showing necrotic changes in less than 10% of neurons. Arrows are pointing to infarction tissue. No part lables need to be inserted. H&E, ×40.



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Protective effect of N. sativa against valproate-induced hepatotoxicity

Administration of toxic dose of SVP caused statistically significant (P < 0.05) increase in liver enzymes (ALT 142.22 ± 14 and AST 355.5 ± 30) in comparison with the control (ALT 35.5 ± 8 and AST 100.1 ± 21). Elevated liver enzymes decreased significantly (P < 0.05) in the group that received both SVP and N. sativa (ALT 39.5 ± 10 and AST 106.17 ± 19).

Liver enzymes

The effect of N. sativa on liver function enzymes compared with valproate-induced hepatotoxicity is shown in [Table 2].
Table 2 The effect of Nigella sativa on liver function enzymes compared with valproate-induced hepatotoxicity


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Histological results

The liver of control rats showed normal histological architecture. The hepatic lobules showed central veins from which the hepatocytes radiated in the form of cords. These cords were separated by blood sinusoids ([Figure 6]). The liver of rats from the SVP intoxication group showed focal areas of mononuclear cellular infiltrations. The hepatocytes of the affected area appeared as cords without clear cell boundaries ([Figure 7]).
Figure 6 A section showing normal histological architecture in control group rats. The hepatic lobules show central veins from which the hepatocytes radiate in the form of cords. These cords are separated by blood sinusoids. The arrow is pointing to central vein. H&E, ×40.



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Figure 7 A section showing congestion and dilation of blood sinusoids and central vein in the valproate-intoxicated group. The liver shows focal areas of mononuclear cellular infiltrations. No hepatocyte boundaries. Arrow is pointing to congestion and dilation of blood sinosoids and central vein. H&E, ×40.



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The liver of rats that received N. sativa revealed more or less normal hepatic architecture ([Figure 8]). On combination of N. sativa with SVP, the liver showed many hepatocytes with small well-circumscribed vacuoles (microvesicular steatosis). Kupffer cells appeared swollen and hypertrophied. Hepatocytes of some rats showed strong acidophilic cytoplasm and small, darkly stained nuclei ([Figure 9]).
Figure 8 A section showing the effect of Nigella sativa oil on the liver of rats, revealing more or less normal hepatic architecture. Arrow is pointing to normal central vein. H&E, ×40.



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Figure 9 A section showing the effect of combination of Nigella sativa oil with sodium valproate, revealing many hepatocytes with small well-circumscribed vacuoles (microvesicular steatosis). Kupffer cells appear swollen and hypertrophied. H&E, ×100.



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Hepatocytes of some rats showed strong acidophilic cytoplasm and small, darkly stained nuclei.


  Discussion Top


Many pharmacological investigations have proved the potential therapeutic effects of N. sativa seed, as well as its oil, through various studies [19] . Generation of free radicals may be the basis of many neurological and neurodegenerative disorders, such as ischemia-reperfusion and seizures [20] . N. sativa is a herbal drug that phytochemically confirms to have antioxidant properties, which suppress reactive oxygen and nitrogen species formation. This scavenging reactive oxygen and nitrogen species play a major role in protecting the antioxidant defense system [21] . The pathogenesis of neuronal damage in response to brain ischemia has been widely studied. A number of observations indicate that a series of events occur in response to a transient ischemic insult and attendant reperfusion, which precede and is necessary for eventual neuronal death. Among these are a decrease in the production of ATP, which results in energy failure [22] . In cerebral ischemia, particularly after reperfusion, free radical production is elevated, thus disrupting the endogenous antioxidant system [23] . Free-radical-induced lipid peroxidation produces cytotoxic aldehydes, including malondialdehyde (MDA), 4-hydroxynonenal, and acrolein [24] .

It was already reported that N. sativa decreased lipid peroxidation and increased the antioxidant defense system activity [25] . The results obtained in the present investigation suggest that N. sativa decreases cerebral ischemia-reperfusion injury-induced pathological stress in the rat model. The size of cerebral infarction and percentage of necrotic changes had significantly decreased in the N. sativa group in comparison with the control group. These findings confirm the results of previous studies that proved that the active constituent of N. sativa has an anti-ischemic effect [26] . The exact mechanism of action of anti-ischemic activities is not clear. N. sativa seed has a variety of activities, including antioxidant [12],[27] , lipoxygenase and cyclooxygenase inhibitory activities [28] , and a decreasing effect on intracellular calcium in mast cells [29] . The efficacy of TMZ has recently been investigated in reducing reperfusion damage in the myocardium [30] . In our findings, pretreatment with TMZ for 21 days in animals subjected to 15 min of global cerebral ischemia has exhibited better neurologic outcomes. As regards infarction size and necrotic changes, there was insignificant difference between the N. sativa-pretreated and TMZ-pretreated groups. A combination of N. sativa with TMZ produced reduction in the size of cerebral infarction and decreased neurological damage to 10%. Thus, N. sativa produced enhancement in the anti-ischemic effect of TMZ. To our knowledge, no previous studies have been conducted comparing N. sativa with TMZ. TMZ significantly reduces free radical production [31] .

Several studies have shown that the antioxidant compounds and free radical scavengers inhibit lipid peroxidation caused by free radicals and excitatory amino acid-induced neuronal injury following ischemia [32] . Gupta et al. 2004 [33] showed that rats pretreated with N. sativa for 7 days showed improvement in motor performance tests. These findings suggested that the improvement in the motor performance test could be due to reduction in the ischemic lesion. Our finding confirmed the results of a previous study by Al-Omar et al. [34] , in which chloroform and petroleum extracts of N. sativa significantly reduced the infarct size when compared with middle cerebral artery (MCA) occluded rats. Thus, pretreatment with both extracts of N. sativa showed neuroprotection. Reports from previous in-vivo studies indicated that N. sativa can protect the brain, through its antioxidant activities, from oxidative stress resulting from lipid peroxidation in transient global ischemia to the brain [35] . El-Abhar et al. [36] showed that N. sativa has a marked protective effect against ischemia reperfusion injury in gastric mucosa. It has been reported that N. sativa seeds exhibit an inhibitory effect on nitric oxide production [37] . Excessive production of oxygen free radicals has been reported in ischemic reperfused liver leading to tissue damage [38] . It has been shown in many studies that supplementation of free radical scavengers is helpful in reducing hepatic ischemia-reperfusion-induced tissue damage [39] . N. sativa has been identified as a potent antioxidant acting as a free radical scavenger [40] . Therefore, it should not be surprising that N. sativa pretreatment has a protective effect on hepatic ischemia reperfusion injury in rats.

SVP is a simple fatty acid largely used as an anticonvulsant. The structure facilitates the interaction of SVP with cell membranes, which may participate in both its therapeutic and adverse effects. SVP is extensively metabolized by the liver through glucuronic acid conjugation, mitochondrial β-oxidation and cytosolic ω-oxidation to produce multiple metabolites. As the clinical usage of SVP increased, reports of its hepatotoxicity began to appear. This hepatotoxicity ranged from mild increase in aminotransferase enzyme in 15-30% of patients to liver cell failure and death in some patients. The mechanism by which SVP caused hepatotoxicity was poorly understood [41],[42] . This study reinforced the previous results [43],[44] . El-Gharieb et al. 2010 [44] reported that administration of SVP caused significant elevation in liver enzymes. In contrast, Lee et al. [45] reported that ALT and AST did not change with oral administration of SVP. This might be because of different route and/or dose of drug administration. N. sativa has protective effect against SVP-induced hepatotoxicity, which has been demonstrated by the prevention of elevation in AST and ALT in the group that received both N. sativa and SVP compared with the group that received SVP alone, in which there was marked elevation in both ALT and AST. Our findings confirm the results of a previous study, which demonstrated the protective effect of N. sativa against hepatotoxicity induced by anticancer drug cyclophosphamide (CTX). Toxicity related to anticancer drugs is usually associated with significant hepatotoxicity due to the alteration in ALT and lipid peroxidation. The data presented here show that CTX treatment is associated with disregulation of liver function, as shown by increases in AST and ALT levels. However, these effects can be ameliorated after treatment with N. sativa or TQ, indicating their protective effects against CTX-induced toxicity [46] .

The current study revealed that intraperitoneal administration of a toxic dose of SVP for 21 days caused focal areas of liver cell degeneration, which were accompanied by occasional eosinophilia of hepatocytes, mononuclear cellular infiltration, prominent microvesicular steatosis (which referred to a variant form of hepatic fatty infiltration), remark cell lines of hepatocytes, and Kupffer cell enlargement. These findings confirmed the results of others [47] . There was an elevation in serum ALT as well. This is parallel to the results of Loscher and Nau [16] . Addition of N. sativa to SVP protected against SVP-induced hepatotoxicity indicated by inhibition of serum ALT and improvement of the histopathological picture compared with SVP alone.

As the mechanism by which SVP causes hepatic damage is uncertain, hepatotoxicity was suspected to result from the formation of toxic SVP metabolites [48] . A possible mechanism of SVP-induced hepatotoxicity was that it caused depression of free radical scavenging enzyme activities [49] . The cause of liver cell injury was due to decreased plasma and tissue carnitine [50] . Another cause for liver cell injury might be due to decreased activity of complex IV of the respiratory chain and/or depletion of hepatic pool of glutathione. N. sativa protects against toxic damage caused by free radical generation. One of the first noticeable morphological changes in SVP-treated livers was the accumulation of cytoplasmic fat [51] . This might be due to the accumulation of esterified SVP and failure of lipid secretion, which were due to interference with vesicular movement, which might lead to fat accumulation. A second study mentioned that SVP interfered with the mitochondrial inner membrane. The loss of that membrane integrity might interfere with the release of calcium from internal stores. This calcium could be a contributor to the secretory problems caused by SVP, either directly affecting the movement of the vesicles along microtubules or indirectly through a second messenger system; thus, lipid accumulation was not only due to overproduction but also due to inability to be secreted at a rate to match the production [52] . Microvesicular steatosis, which is marked in SVP-associated liver injury, possibly occurs through reactive intermediates that interfere with the process of fatty acid β-oxidation, which inhibit key enzymes in β-oxidation cycle, or through idiosyncrasy for production of toxic SVP metabolites [53] . Oda et al. [54] demonstrated acidophilic degeneration observed with light microscopy. Kupffer cell hypertrophy might be due to lipid deposition in their cytoplasm [55] . Administration of SVP and N. sativa caused some improvement in the histological changes at the light microscopic level. At the light microscopic level, hepatocytes revealed less microvesicular steatosis. Hepatocytes did not return completely to the control pattern. This might be due to the short duration of N. sativa. The findings of the present study indicate that N. sativa has hepatoprotective activity. Daba and Abdel-Rahman [56] tested TQ in isolated rat hepatocyte as a protective agent against butylhydroperoxide toxicity. Moreover, our findings confirmed the results of previous study by Al-Gharably et al. [57] , in which pretreatment with N. sativa protected against hepatotoxicity induced by CCL 4 . This hepatoprotective effect is demonstrated through significant prevention of any increase in ALT and AST. Therefore, it can be mentioned that N. sativa behaves as an antioxidant and protects liver against the detrimental effects of SVP. The antioxidant action of N. sativa and TQ may explain the protective effect of these agents against hepatotoxic models in vivo and in vitro [58] , as well as against liver fibrosis and cirrhosis [27] .


  Conclusion Top


The result of this study revealed that N. sativa has a protective effect against cerebral ischemia, which is proved by reduction in the percentage of necrosis and decrease in infarction size in the N. sativa-treated group in comparison with the control group. Moreover, N. sativa enhanced the anti-ischemic effect of TMZ. N. sativa also has a protective effect against SVP-induced hepatotoxicity, which is proved by the reduction in elevated serum ALT and AST and improvement in histopathological picture in the N. sativa-treated group in comparison with the control group.

Acknowledgements

Nil.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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