|Year : 2017 | Volume
| Issue : 1 | Page : 262-270
Protective effect of garlic oil on furan-induced damage in the pancreas of adult male rat
Mostafa M El-Habiby, Neveen M El-Sherif, Gehan El-Akabawy, Sara G Tayel MBBCh
Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||03-May-2016|
|Date of Acceptance||28-Jun-2016|
|Date of Web Publication||25-Jul-2017|
Sara G Tayel
Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Menoufia, 32511
Source of Support: None, Conflict of Interest: None
The aim of this study was to investigate the effect of furan on the pancreas of adult male rat and to evaluate the possible protective role of garlic oil (GO).
Furan is carcinogenic in rats and mice and possibly carcinogenic to humans.
Sprague–Dawley rats were divided randomly into four main groups: control, GO (80 mg/kg/day), furan-treated (2 and 8 mg/kg/day), and protected group (furan and GO). All rats were treated orally by a gavage for 5 days per week for 8 weeks. At the end of the experiment, pancreases were subjected to biochemical (measurement of the level of glucose, amylase, lipase, and oxidative stress indices in pancreatic tissue), histological, and immunohistochemical analyses.
Our results showed the toxic effects of furan on the pancreas in adult male albino rats. This was indicated by an increase in pancreatic (amylase and lipase levels) and lipid peroxidation biomarker (malondialdehyde), and a decrease in antioxidant enzymes, including catalase, superoxide dismutase, and glutathione. In addition, histopathological alterations were detected, including mononuclear cellular infiltration, congestion of blood vessels, and cytoplasmic vacuolation of the acinar cells. There were significant increases in the number of inflammatory cells and apoptotic expression, whereas cytochrome P450 2E1 expression was significantly decreased after the administration of furan. Furan-induced toxicity was ameliorated by the coadministration of GO.
The administration of furan-induced biochemical and histopathological changes in the pancreas of adult male albino rats. These changes were improved by the coadministration of GO.
Keywords: furan, garlic oil, pancreas
|How to cite this article:|
El-Habiby MM, El-Sherif NM, El-Akabawy G, Tayel SG. Protective effect of garlic oil on furan-induced damage in the pancreas of adult male rat. Menoufia Med J 2017;30:262-70
|How to cite this URL:|
El-Habiby MM, El-Sherif NM, El-Akabawy G, Tayel SG. Protective effect of garlic oil on furan-induced damage in the pancreas of adult male rat. Menoufia Med J [serial online] 2017 [cited 2019 Aug 21];30:262-70. Available from: http://www.mmj.eg.net/text.asp?2017/30/1/262/211492
| Introduction|| |
Furan is a typical food contaminant, which could exert harmful effects on the health of the human population . It has been detected in a broad variety of foods, particularly coffee, canned meats, and baby food . Furan is used as an intermediate in the synthesis of many chemical and pharmaceutical agents . Concern over furan in foods dates back only to 2004, when a Food and Drug Administration published a report on the occurrence of furan in a number of thermally treated foods; since then, furan has been classified as 'possibly carcinogenic to human' by the International Agency for Research on Cancer . Furan toxicity is linked to cytochrome P450 2E1 (CYP2E1)-mediated bioactivation to a cis-2-butene-1,4-dial (BDA), a chemically reactive α, β-unsaturated dialdehyde, which may covalently bind to cellular nucleophiles. CYP2E1-mediated metabolism can also cause toxicity or cell damage through the production of toxic metabolites, oxygen radicals, and lipid peroxide .
Garlic (Allium sativum) is used widely as an ingredient in many food items; in addition, it has high medicinal value . Among garlic preparations, garlic oil (GO) has been shown to contain more than 30 organic sulfur-containing compounds including diallyl trisulfide, diallyl disulfide, and diallyl sulfide. The antioxidant ability of these compounds is well documented . In addition, they can modulate the activity of several drug-metabolizing enzymes (cytochrome P450s), particularly CYP2E1 .
This work aimed at studying the effect of furan on the pancreas of adult male albino rats and to establish the possible protective role of garlic.
| Methods|| |
This study was carried out on 35 adult male Sprague–Dawley albino rats, with an average weight of 200 g, obtained from Helwan Animal House. The animals were maintained (five rats in each cage) in the animal house of the Faculty of Medicine, Menoufia University. The rats were allowed unlimited access to chow and water. All of the ethical protocols for animal treatment were followed and supervised by the animal facilities. All procedures involving the use of the rats were approved by The Animal Care and Use Committee, Faculty of Medicine, Menoufia University.
The animals were divided into four main groups. Group I (control group) included 10 rats subdivided into two equal subgroups: subgroup Ia (negative control) and subgroup Ib (corn oil). Group II included five rats (GO group). Group III (furan-treated group) included 10 rats subdivided into two equal subgroups: subgroup IIIa (low-dose furan) and subgroup IIIb (high-dose furan). Group IV (furan plus GO group) included 10 rats subdivided into two equal subgroups: subgroup IVa (low-dose furan and GO) and subgroup IVb (high-dose furan and GO).
Furan (CAS no. 110-00-9, ≥99% pure) was administered at a dose of 2 and 8 mg/kg dissolved in 4 ml/kg corn oil by a gavage 5 days/week for 8 weeks . GO (El-Capitan, Egypt) was administered at a dose of 80 mg/kg/day by a gavage 5 days/week for 8 weeks . Corn oil, available in the form of an oily solution, was used as a solvent for furan (Sigma–Aldrich Chemical Company, Saint Louis, Missouri, USA) .
Blood samples were collected from the vein of the rat tail and used for the measurement of blood glucose. The blood was centrifuged and plasma was used for the estimation of serum amylase and lipase levels using a spectrophotometer ,.
Histological and immunohistochemical studies
Rats were killed by cervical dislocation. The pancreases were removed immediately, and subdivided into two parts: one part was fixed in 10% formalin saline for 24 h and then processed to obtain paraffin blocks. Sections of 4–6 mm thickness were cut using a microtome and stained with hematoxylin and eosin stain  and immunohistochemical staining  by caspase-3 (1: 500, labvision) and anticytochrome P450 2E1 (CYP2E1, 1: 500; Abcam) antibodies abcam (Cambridge, MA). The other part of the pancreas was homogenized in 1 ml PBS, pH 7.4, and centrifuged at 4500 rpm for 15 min at 4°C, and then the supernatant was obtained and used for the assessment of the oxidative state in pancreatic tissue using a spectrophotometer; malondialdehyde (MDA) , catalase (CAT) , superoxide dismutase (SOD) , and glutathione (GSH) levels were determined .
For histological and immunohistochemical quantitative assessments, five nonoverlapping fields (400×) per se ction were captured randomly by a Lieca Microscope DML B2/11888111 (England) equipped with a Leica camera DFC450. The number of inflammatory cells and immunopositive cells in the fields was obtained from at least three sections/animal and counted using ImageJ software (Image J 1.47v, National Institute of Health, Bethesda, MD, USA) and averaged per field for each animal. The numbers calculated for at least five animals/experimental group were considered for comparison and statistical analyses. This was done at the Anatomy and Embryology Department, Faculty of Medicine, Menoufia University.
Results were expressed as mean ± SEM. One way-analysis of variance, followed by a post-hoc Bonferroni test was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, California, USA). A level of significance of P less than 0.05 was considered to be statistically significant.
| Results|| |
There was no significant difference between the rats in the control group (subgroup Ia, and Ib).
There was no significant difference in the blood glucose levels among all the groups studied, suggesting that the endocrine part of the pancreas was not affected by furan toxicity [Figure 1]a. The administration of furan significantly increased the serum amylase and lipase levels in furan-treated rats compared with the control rats (P < 0.001). There was also a significant increase in amylase and lipase levels between high-dose and low-dose furan-treated rats (P < 0.001). Coadministration of GO significantly decreased the levels of amylase (P < 0.001) and lipase in the low-dose and high-dose furan + GO-treated groups compared with the administration of furan alone (P < 0.05 and 0.001, respectively) [Figure 1]b and [Figure 1]c.
|Figure 1: Blood glucose, serum amylase, and lipase levels. °°°P < 0.001, compared with the control, *P < 0.05 and ***P < 0.001 compared with the low-dose furan-treated group, θθθP < 0.001 compared with the high-dose furan-treated group. GO, garlic oil.|
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Oxidative status in pancreatic tissue
Pancreatic MDA level was increased by furan administration compared with control rats (P < 0.001). There was a significant difference in the MDA level between low-dose and high-dose furan-treated groups (P < 0.001). The coadministration of GO alleviated lipid peroxidation induced by furan treatment compared with furan-treated groups (P < 0.001) [Figure 2]a, whereas the administration of furan significantly depleted antioxidant enzyme CAT, SOD, and GSH compared with control rats (P < 0.001). There was a significant statistical difference in CAT and GSH in pancreatic tissue between low-dose and high-dose furan-treated groups (P < 0.01 and 0.001, respectively). GO coadministration improved significantly the activities of CAT, SOD, and GSH in low-dose furan+GO (P < 0.001) and high-dose furan +GO groups (P < 0.001,0.01, and 0.001, respectively)compared with furan-treated groups [Figure 2]b,[Figure 2]c,[Figure 2]d.
|Figure 2: MDA, SOD, CAT, and GSH levels in pancreatic tissue. °°°P < 0.001, compared with the control, **P < 0.01 and ***P < 0.001 compared with the low-dose furan-treated group, ..P < 0.01 and θθθP < 0.001 compared with the high-dose furan-treated group. CAT, catalase; GO, garlic oil; GSH, glutathione; MDA, malondialdehyde; SOD, superoxide dismutase.|
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Control and garlic oil groups
No noticeable histological differences could be detected between the control and the GO groups. In these groups, the pancreas was divided into many small lobules of variable sizes and shapes separated by interlobular connective tissue containing pancreatic duct and blood vessels [Figure 3]a. Each lobule was formed predominantly of the exocrine part: pancreatic acini. Masses of the endocrine part (the islets of Langerhans) were scattered in an irregular manner between the pancreatic acini [Figure 3]b. The acinar cells were pyramidal in shape, with basal rounded vesicular nuclei [Figure 3]c.
|Figure 3: Representative hematoxylin and eosin staining of rat pancreas of different groups. In the control group, (a) multiple pancreatic lobules (L) with interlobular blood vessels (arrowhead) and duct (arrow), the lobule formed the exocrine part; (b) the pancreatic acini (A), and endocrine part; islets of Langerhans (I); (c) the pancreatic acini were lined with pyramidal cells with basal vesicular nuclei (arrow). In the low-dose furan-treated group, (d) interlobular mononuclear cellular infiltration (box); (e) extravasation of blood in the interlobular connective tissue (arrow); (f) dilated congested blood vessels (BV) with perivascular inflammatory infiltration (arrow), and nuclei of some acinar cells appeared dark, small pyknotic (box) – note that the islets of Langerhans appeared to be almost normal (I). In the high-dose furan-treated group, (g) degenerated acini (D) with inflammatory cellular infiltration between them (arrow), congested blood capillaries between the islet cells (arrow head); (h) distortion of acini with cytoplasmic lysis leaving necrotic tissue (box); (i) nuclei of most of acinar cells appeared dark, small pyknotic surrounded by clear zones (curved arrows) multiple vacuoles in the cytoplasm of the acinar cells (arrows). In the low-dose furan + GO-treated group, (j) the pancreatic lobules (L) with (k) interlobular blood vessels were observed to be almost normal (arrow); (l) the acini were with normal vesicular nuclei (arrows). In the high-dose furan + GO-treated group, (m) almost normal architecture of the pancreatic acini (A) and islets of Langerhans (I), congested blood vessel (arrow); (n) extravasation of blood (arrow) were observed; (o) the majority of acinar cells appeared with vesicular nuclei (curved arrow), whereas some pancreatic acinar cells appeared with deeply stained pyknotic nuclei (arrow head) and few cytoplasmic vacuoles (arrow). Inserts show a higher magnification of the boxed regions. Scale bar = 100 μm (a, d, e, j, and m), 50 μm (b, f, g, h, k, and n), and 20 μm (c, i, l, and o). GO, garlic oil.|
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Low-dose furan-treated group: rats received furan at a dose of 2 mg/kg/day
Mononuclear cellular infiltration and extravasation of blood in the interlobular connective tissue [Figure 3]d and [Figure 3]e, congestion of blood vessels with perivascular inflammatory infiltration, and pyknotic nuclei of some acinar cells were detected [Figure 3]f.
High-dose furan-treated group: rats received furan at a dose of 8 mg/kg/day
Massive destructive changes were observed, including massive distortion of the general architecture, disorganized pancreatic acini, inflammatory cellular infiltration, and congested blood capillaries between the islet cells [Figure 3]g. Some acini were completely destroyed, leaving only necrotic tissue [Figure 3]h. The cytoplasm of most acinar cells showed multiple vacuoles of variable sizes and shapes, and the nuclei appeared as dark pyknotic and surrounded by clear zones [Figure 3]i.
Low-dose furan+garlic oil-treated group: rats received furan at a dose of 2 mg/kg/day and garlic oil
An improvement was observed in most of the previously mentioned histopathological findings in low-dose furan-treated rats. The pancreatic lobules appeared almost normal [Figure 3]j. The interlobular blood vessels were observed to be of normal size [Figure 3]k. The majority of the acinar cells appeared almost normal, with basal vesicular nuclei [Figure 3]l.
High-dose furan+garlic oil-treated: rats received furan at a dose of 8 mg/kg/day and garlic oil
The pancreatic lobules retained their normal architecture, and islets of Langerhans cells appeared almost normal in shape. Congested blood vessels are still evident [Figure 3]m. Mild extravasation of blood was observed between the acini [Figure 3]n. The acinar cells were observed to be almost normal in shape with vesicular nuclei and some of the acinar cells showed deeply stained pyknotic nuclei, with few cytoplasmic vacuoles detected in the cytoplasm of pancreatic acinar cells [Figure 3]o.
Results of quantitative assessments
Significant increases in the number of inflammatory cells and also in caspase-3 expression were observed after low-dose and high-dose furan administration compared with the control group (P < 0.001). There was a significant increase in the inflammatory cells and caspase-3 expression on high-dose compared with low-dose furan administration (P < 0.001). Cotreatment with GO led to a marked decrease in the inflammatory cells and caspase-3 expression compared with the administration of furan alone (P < 0.001) [Figure 4] and [Figure 5]a, [Figure 5]b, whereas CYP2E1 expression was significantly downregulated in furan-treated rats compared with the control rats (P < 0.001). There was also a significant decrease in CYP2E1 expression on high-dose furan compared with low-dose furan administration (P < 0.05). GO cotreatment significantly inhibited furan-induced decrease in CYP2E1 expression in low-dose and high-dose furan + GO-treated rats compared with furan alone (P < 0.001,0.01, respectively) [Figure 4] and [Figure 5]c.
|Figure 4: Representative immunostaining of rat pancreas of different groups. (a.c) CYP2E1 expression was downregulated in pancreatic acini in both low-dose and high-dose furan-treated rats; (d, e) GO cotreatment inhibited the furan-induced decrease in CYP2E1 expression in the furan. + GO groups; (f.h) caspase-3 immunoreactivity was markedly increased in the acinar cells of the low-dose and high-dose furan-treated rats; (i, j) these increases were reduced in the furan. + GO groups. Inserts show a higher magnification of the boxed regions. Scale bar. = 50 μm. CYP2E1, cytochrome P450. 2E1; GO, garlic oil.|
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|Figure 5: Number of inflammatory cells and the expression of both CYP2E1 and caspase.3 in pancreatic tissue. °°°P < 0.001, compared with the control, *P < 0.05 and ***P < 0.001 compared with the low-dose furan-treated group, θθP < 0. 01 and θθθP. < 0.001 compared with the high-dose furan-treated group. CYP2E1, cytochrome P450. 2E1; GO, garlic oil.|
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| Discussion|| |
The present study has focused on one particular environmental carcinogen: furan. Although a large number of studies have reported detrimental and deleterious effects of furan on the liver, limited numbers of studies have evaluated its effect on the pancreas .
The present study showed that a significant increase in the pancreatic MDA and a decrease in SOD, CAT, and GSH levels were observed in furan-treated groups compared with the control group, indicating that furan-induced oxidative stress in the pancreases of the furan-treated groups. In agreement with the present study, Wang et al.  reported a decrease in the activities of SOD and GSH and an increase in the MDA level in the liver and kidney of furan-treated rats. All these antioxidant enzymes and GSH are used extensively as an index of unbalanced reactive oxygen species (ROS) production and oxidative stress in physiological systems . CYP2E1 is involved in metabolizing and activating many toxicologically important substrates such as furan, mainly localized in the liver . However, it is expressed in pancreatic acinar cells and ductal cells of mouse, rabbit, rats, and humans. This CYP expression makes both cell types more prone to bioactivation of xenobiotics and injury by reactive intermediates . CYP-mediated metabolism of furan into BDA results in cytotoxicity and oxidative stress . In the current study, furan treatment was found to significantly reduce CYP2E1 expression in the pancreatic acini compared with those of the control group. This is in agreement with Hickling et al. , who found that CYP2E1 expression was significantly reduced in hepatocytes after furan treatment. A possible explanation for the decrease in CYP2E1 expression could be the post-translational degradation of CYP2E1 by the ubiquitin-dependent proteasomal degradation system (UPS) . Therefore, it can be concluded that the furan-induced oxidative stress in our study was because of CYP2E1 activation that might be extensively consumed during the process of furan bioactivation and underwent proper post-translational degradation by UPS.
In this study, rats treated with a low dose of furan showed pyknotic nuclei of some acinar cells, vascular congestion, and inflammatory cellular infiltration. There was a significant increase in the number of inflammatory cells after furan administration compared with the control group. These changes were in agreement with Nyska et al. , who indicated that the pancreatic exocrine acini are target tissues of the furans. These changes became more aggravated after high-dose treatment as indicated by massive distortion of the architecture, necrosis of the acinar cells, inflammatory reaction, congestion of the blood vessels in the exocrine part of the pancreas, and congestion of the capillaries between the islet cells. These changes are in agreement with Karacaoglu et al. , who reported that acinar cell necrosis was observed in the exocrine part of the pancreas and vascular congestion in the islets of Langerhans was observed in furan-administered rats. In addition, there was also a significant increase in the number of inflammatory cells on high-dose compared with low-dose furan treatment. The inflammatory reaction observed after furan treatment could be explained by the lipid peroxidation and accumulation of oxygen-derived free radicals .
Furthermore, Wu et al.  reported that furan could induce an inflammatory response by increasing the expression of cytokines and other inflammation-associated genes, such as interlukein (IL)-1b, (IL)-6, and (IL)-10. Cytokines cause induction of the nitric oxide synthase enzyme in epithelial cells and activation of macrophages, which lead to the generation of nitric oxide and its derivatives; the nitric oxide synthase enzyme is involved in the regulation of the rate of perfusion of the pancreatic microvessels. Nitric oxide might exert a direct toxic effect on pancreatic acinar cells, increasing the vascular congestion and vascular permeability.
In the present study, caspase-3 expression was significantly upregulated in some acinar cells of the low-dose furan-treated group and became more obvious in most acinar cells after high-dose furan treatment. This was in agreement with Hickling et al. , who showed that apoptosis increased in the liver throughout the treatment period of furan. Apoptosis might be induced by the activation of initiator procaspase-12, which is activated by cleavage under endoplasmic reticulum stress conditions because of oxidative stress, and can then activate caspases-9 and caspases-3, which induce apoptosis .
In this study, there was a dose-dependent increase in the serum lipase and amylase levels in furan-treated groups compared with the control group. Similar results were reported by Gill et al. , who found that 2-methylfuran (furan derivatives) caused a significant increase in the lipase level that was dose dependent, suggesting that the pancreas, particularly the exocrine part, may have been affected by furan. Antony et al. explained that these enzymes are synthesized by pancreatic cells, which, when subjected to injury, release the enzymes into blood circulation and lead to an increase in the enzyme level in serum.
The present study showed that there were no significant changes in the levels of glucose compared with the control group; the same result was obtained by Gill et al. , suggesting that there was no effect of function of the islets of Langerhans in our study.
GO treatment induced significant increases in the activities of GSH, SOD, and CAT, with a concomitant reduction in MDA levels. In agreement with the present study, Kiruthiga et al. postulated that the administration of garlic can modulate the oxidative stress and improve the antioxidant system through the antioxidant abilities of GO constituents: diallyl disulfide and diallyl trisulfide. Our study showed that GO cotreatment significantly inhibited furan-induced decreases in CYP2E1 expression. This result was in agreement with Zhang et al. , who found that GO cotreatment significantly suppressed the decreases in CYP2E1 and explained that GO cotreatment might suppress the activation of UPS. The modulation of CYP2E1 expression might offer another explanation for the detected protective role of GO on furan-induced oxidative stress, in addition to its antioxidant ability.
Histological study of pancreatic tissue of animals of (furan+GO) group showed a remarkable improvement in the pancreatic lobular architecture; however, some pancreatic acinar cells appeared to have deeply stained pyknotic nuclei and extravasation of blood was still evident in the high-dose furan+GO group and there was a significant decrease in the number of inflammatory cells compared with furan-treated rats. The histopathological beneficial effects promoted by GO could be attributed to improved antioxidant activity, which could potentially result in a reduction in membrane lipid peroxidation, with subsequent prevention of free radicals-induced damage . Our results showed that the immunoreactivity for caspase-3 of group IV (furan+GO) showed a significant improvement as most acinar cells showed negative cytoplasmic immunoreactivity for caspase-3 compared with the furan-treated group; this was in agreement with Santra et al. , who reported that the antiapoptotic effect of garlic could be because of its ability to counteract the increase in ROS and the subsequent depolarization of the mitochondrial membrane as it has been reported that ROS increase and loss of mitochondrial membrane permeabilization could lead to apoptosis.
The present study showed that there were significant decreases in serum amylase and lipase levels of group IV (furan+GO) compared with furan-treated groups. Ohaeri  explained that the decrease in the levels of the enzymes in rats treated with GO is a result of improvements in histopathological alterations.
| Conclusion|| |
Our results indicate that furan exerts a toxic effect on the pancreas, mainly the exocrine part, by exerting oxidative stress. GO coadministration exerted conspicuous modulating effects and was capable of overcoming the oxidative stress induced by furan through its antioxidant properties and its ability to modulate CYP2E1 expression. These results highlight the importance of the consumption of natural vegetables and also contribute toward the understanding of the beneficial effects of functional foods in the prevention of furan-induced oxidative damage.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]