Abstract
Acrylamide is a well-known neurotoxicant and carcinogen. Apart from industrial exposure, acrylamide is also found in different food products. The present study deals with in vivo experiment to test the protective effect of rutin against acrylamide induced toxicity in rats. The study was carried out on female rats with exposure of acrylamide at the dose of 38.27 mg/kg body weight, orally for 10 days followed by the therapy of rutin (05, 10, 20 and 40 mg/kg orally), for three consecutive days. All animals were sacrificed after 24 h of last treatment and various biochemical parameters in blood and tissue were investigated. Histopathology of liver, kidney and brain was also done. On administration of acrylamide for 10 days, neurotoxicity was observed in terms of decreased acetylcholinesterase activity and oxidative stress was observed in terms of increased lipid peroxidation, declined level of reduced glutathione, antioxidant enzymes (superoxide dismutase and catalase) in liver, kidney and brain. Acrylamide exposure increased the activities of serum transaminases, lipid profile, bilirubin, urea, uric acid and creatinine in serum indicating damage. Our experimental results conclude that rutin showed remarkable protection against oxidative DNA damage induced by acrylamide, which may be due to its antioxidant potential.
Keywords: rutin, acrylamide, antioxidant, oxidative stress, liver, kidney, brain
Graphical Abstract
Graphical Abstract.
Introduction
Acrylamide (AA; C3H5NO; Fig. 1) is a low molecular weight hydrophilic monomer, electrophile, versatile chemical, which is polymerized to polyacrylamide in the presence of an initiator. It reacts readily due to the presence of double bond in its structure [1–3]. It is a proven rodent carcinogen. Based on this, the International Agency for Research on Cancer classified it as a group 2A carcinogen in 1994 [4, 5]. It is also categorized as carcinogenic and mutagenic substance by The European Union [6].
Figure 1.
Structure of Acrylamide.
From decades, the primary use of AA was production of polyacrylamide, sewage treatment, in industries and in laboratories. It is also used in cosmetics [7, 8]. AA is also found in cigarette smoke [9]. Before 2002, exposure was mainly occupational. However, in 2002, Swedish Food Administration stated that AA is present in food products specially carbohydrate-rich foods (French fries, potato chips, etc.) which are baked, grilled, roasted at high temperatures (above 120°C; 248°F) [10, 11].
This AA is formed by Maillard reaction through browning of substance. It is present in appreciable amount (up to several mg/kg of foodstuff) in various foodstuffs. It is reported to be hepatotoxic, nephrotoxic, neurotoxic, reproductive toxicant and carcinogenic in animals [12–14].
The presence of significant amount of AA in various starchy foods (especially western diets) which extensively occupy our markets have generated new interest in determining mechanism of hepato, renal, neuro and reproductive toxicity and cancer from AA exposure. It is also reported that AA have significant binding capacity to liver, kidney, brain and erythrocyte [15, 16].
As AA exposure is mainly through diet and is a matter of concern, the possibility of using polyphenolic compounds as a natural antioxidant to prevent oxidative impairment induced by AA has been raised.
Phytochemicals are effective hepato, renal and neuroprotective agents. Rutin (RU) or vitamin P (3, 3′, 4′, 5, 7-pentahydroxyflavone-3-rhamnoglucoside; rutoside; quercetin-3-rutinoside; sophorin; Fig. 2) is an important polyphenolic compound (bioflavonoid of the flavonol-type) [17, 18]. It is found in buckwheat, passion flower, oranges, grapes, lemons, berries, peaches, limes, tea and apple [19, 20]. RU is a glycoside derivative of quercetin and comprises of flavonolic aglycone quercetin and disaccharide rutinose [21]. Among flavonoids, rutin’s bioavailability and free radical scavenging property generally makes it a potential therapeutic agent.
Figure 2.
Structure of Rutin.
Rutin showed varied pharmacological properties like antioxidant, anti-inflammatory, antimicrobial, antifungal, anti-allergic, antihyperglycemic and enhancement of vascular integrity [22–24]. Other than this, it is cardioprotective [25, 26], neuroprotective [27], nephroprotective, hepatoprotective and also showed protection against reproductive toxicity [28]. It suppresses the microglial activation and pro-inflammatory cytokines [29, 30] and is also used in treating clots in arteries and veins. It has multispectrum pharmacological benefits for the treatment of various diseases, such as cancer, diabetes, hypertension and hypercholesterolemia [18].
Various beneficial effects of rutin have been reported, but less scientific information is available on protective effects of rutin against AA-induced damage in liver, kidney and brain of rats. Thus, the present study was designed to explore the therapeutic efficacy of rutin on biochemical and histological integrity in AA intoxicated rats.
Materials and Methods
Animals and chemicals
Female albino rats of ‘Wistar’ strain (160 ± 10 g b.w.) were procured from Defence Research & Development Establishment, Gwalior (M.P.). Animals were kept in well-aerated animal house with standard husbandry conditions, temperature (25 ± 2°C), relative humidity (60–70%) and cycle of 14 h light and 10 h dark. Standard pellet diet and water ad libitum were given to rats. Animals were taken care according to the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals Government of India, and experiments were approved by the Institutional Animal Ethics Committee.
AA and Rutin were purchased from Sigma Aldrich Co., USA. All the other chemicals/ reagents required for the study were of high analytical grade and procured from Sigma Aldrich Company (USA), HiMedia, E-Merck (Germany), Loba, Ranbaxy, SRL chemical substances Pvt. Ltd and BDH. All diagnostic kits were acquired from E-Merck.
Experimental protocol
A total number of 42 normal rats were divided into seven groups with six animals in each group. Group I was administered with normal saline and kept as control group. In group II, RU treatment at a dose of 40 mg/kg p.o. and served as per se group. Rats were intoxicated with AA at the dose of 38.27 mg/kg, p.o. for 10 days in groups III–VII in which group III was served as experimental control. In animals of groups IV–VII, treatment of RU was given at the doses of 05, 10, 20 and 40 mg/kg p.o., for three consecutive days. All experimental animals were euthanized after 24 h of last dose. Toxicity was induced by one-third dose of LD50 orally of freshly prepared AA. Dose was selected from our previous study [31].
Group I: Healthy control.
Group II: RU per se (RU at 40 mg/kg p.o.)
Group III: AA (38.27 mg/kg p.o.; one-third of LD50).
Groups IV–VII: AA (as in group III) + RU (5, 10, 20 and 40 mg/kg p.o.)
Assessment of serum biochemical parameters
Blood samples were obtained by puncturing retro-orbital venous sinus [32] and kept at RT for 1 h. These samples were then centrifuged at 3000 rpm for 10 min and isolated serum was stored at −20°C for further assays. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined in obtained serum by Reitman and Frankel [33]. Triglycerides, cholesterol, bilirubin, urea, uric acid and creatinine were measured according to the protocol given in diagnostic kits using a Merck auto-analyser (Micro Lab 200).
Assessment of tissue biochemical parameters
Liver, kidney and brain tissues were immediately excised after necropsy, rinsed in chilled normal saline and blotted to dry for tissue biochemical estimations. Remi Motor Homogenizer (RQ-122) was used to homogenize the tissues using glass tube and Teflon pestle for the assessment of lipid peroxidation (LPO) [34], reduced glutathione (GSH) [35], superoxide dismutase (SOD) [36], catalase (CAT) [37] and acetylcholinesterase (AChE) [38].
Histology
Liver, kidney and brain tissues were collected and immediately fixed in Bouin’s fluid. Paraffin sections of 5 μm thickness were prepared. Then, staining was done with hematoxylin–eosin stain and histopathological changes were observed under light microscope.
Statistical analysis
Data were subjected to statistical analysis through one-way analysis of variance (ANOVA) considering significant at 5% level followed by Student’s t-test considering P ≤ 0.05 [39]. Results are presented as mean ± SE of six animals used in each group.
Percent of protection was calculated by the following formula:
Where X = AA + RU, Y = AA and C = Control.
Results
Serum biochemical observations
Changes in serum enzymes
Administration of AA caused severe increment in the liver marker enzymes (AST, ALT) as compared to control group (Table 1). AST and ALT levels increased by 128% and 357%, respectively. RU at all the four doses (05, 10, 20 and 40 mg/kg) recouped altered enzymatic level, AST (by 38%, 52%, 73% and 78%) and ALT (by 45%, 63%, 75% and 78%), respectively, towards control in a dose-dependent manner. Significant recovery was seen at all the four doses, viz. 05, 10, 20 and 40 mg/kg, when compared to control, but maximum recovery was observed at 20 and 40 mg/kg doses (P ≤ 0.05).
Table 1.
Protective effect of rutin on hepatospecific markers against AA-induced toxicity
| Treatments | AST (IU/l) | ALT (IU/l) |
|---|---|---|
| Control | 61.4 ± 3.39 | 38.9 ± 2.15 |
| RU per se | 68.0 ± 3.75 | 42.0 ± 2.32 |
| AA per se | 140 ± 7.74a | 178 ± 9.84a |
| AA+ RU (5 mg/kg) (% protection) | 110 ± 6.09b(38%) | 114 ± 6.35b(45%) |
| AA+ RU (10 mg/kg) % protection | 99.2 ± 5.48b(52%) | 90.6 ± 5.00b(63%) |
| AA+ RU (20 mg/kg) % protection | 82.2 ± 4.54b(73%) | 73.4 ± 4.05b(75%) |
| AA+ RU (40 mg/kg) % protection | 78.9 ± 4.36b(78%) | 69.0 ± 3.81b(78%) |
| F value (at 5% level) | 32.53c | 95.84c |
Data are mean ± SE; N = 6.
aAA versus Control.
bAA+ therapy versus AA at P ≤ 0.05.
cSignificant at 5% for ANOVA.
Alterations in serum metabolites
Significant enhancement in serum metabolites like triglycerides, cholesterol, bilirubin, urea, uric acid and creatinine (P ≤ 0.05) was observed after AA intoxication. Triglycerides, cholesterol and bilirubin increased by 89%, 222% and 500%, respectively (Table 2). Urea, uric acid and creatinine increased by 175%, 563% and 407%, respectively (Table 3). Treatment with RU reversed the values toward baseline level by reducing triglycerides (by 25%, 41%, 60%, 61%), cholesterol (by 38%, 60%, 70%, 72%), bilirubin (by 15%, 20%, 36%, 40%), urea (by 33%, 43%, 55%, 58%), uric acid (by 25%, 29%, 49%, 52%) and creatinine (by 25%, 39%, 60%, 65%) at the dose of 05, 10, 20 and 40 mg/kg, respectively. Percent protection showed maximum recovery at 20 and 40 mg/kg dose (P ≤ 0.05).
Table 2.
Influence of rutin on serum lipid profile and bilirubin
| Treatments | Triglycerides (mg/dl) | Cholesterol (mg/dl) | Bilirubin (mg/dl) |
|---|---|---|---|
| Control | 66.0 ± 3.64 | 41.0 ± 2.26 | 0.30 ± 0.01 |
| RU per se | 68.0 ± 3.75 | 43.0 ± 2.37 | 0.31 ± 0.01 |
| AA per se | 125 ± 6.91a | 132 ± 7.29a | 1.80 ± 0.09a |
| AA+ RU (5 mg/kg) % protection | 113 ± 6.27 (25%) | 97.5 ± 5.38b(38%) | 1.57 ± 0.08 (15%) |
| AA+ RU (10 mg/kg) % protection | 106 ± 5.89 (41%) | 77.5 ± 4.28b(60%) | 1.50 ± 0.08 (20%) |
| AA+ RU (20 mg/kg) % protection | 97.7 ± 5.40b(60%) | 68.1 ± 3.76b(70%) | 1.26 ± .06b(36%) |
| AA+ RU (40 mg/kg) % protection | 97.2 ± 5.37b(61%) | 66.4 ± 3.67b(72%) | 1.20 ± 0.06b(40%) |
| F value (at 5% level) | 19.90c | 61.02c | 88.69c |
Data are mean ± SE; N = 6.
aAA versus Control.
bAA+ therapy versus AA at P ≤ 0.05.
cSignificant at 5% for ANOVA.
Table 3.
Protective effect of rutin on renal specific markers against AA intoxication
| Treatments | Urea (mg/dl) | Uric acid (mg/dl) | Creatinine (mg/dl) |
|---|---|---|---|
| Control | 29.4 ± 1.62 | 0.86 ± 0.04 | 0.14 ± 0.007 |
| RU per se | 31.2 ± 1.72 | 0.87 ± 0.04 | 0.16 ± 0.008 |
| AA per se | 80.8 ± 4.46a | 5.70 ± 0.31a | 0.71 ± 0.039a |
| AA+ RU (5 mg/kg) % protection | 63.9 ± 3.53b(33%) | 4.50 ± 0.24b(25%) | 0.57 ± 0.031b(25%) |
| AA+ RU (10 mg/kg) % protection | 58.9 ± 3.25b(43%) | 4.30 ± 0.23b(29%) | 0.49 ± 0.027b(39%) |
| AA+ RU (20 mg/kg) % protection | 52.6 ± 2.90b(55%) | 3.33 ± 0.18b(49%) | 0.37 ± 0.020b(60%) |
| AA+ RU (40 mg/kg) % protection | 50.9 ± 2.81b(58%) | 3.18 ± 0.17b(52%) | 0.34 ± 0.018b(65%) |
| F value (at 5% level) | 42.24c | 97.81c | 88.06c |
Data are mean ± SE; N = 6.
aAA versus Control.
bAA+ therapy versus AA at P ≤ 0.05.
cSignificant at 5% for ANOVA.
Tissue biochemical observations
Oxidative stress biomarkers
Tables 4 and 5 reveals significant elevation in LPO in terms of increased thiobarbituric acid reactive substance (TBARS) content (by 465%, 553% and 588%) and decrease in reduced GSH (by 35%, 28% and 33%), respectively, in liver, kidney and brain after AA administration. Significant recovery was seen after RU therapy at all the four doses (05, 10, 20 and 40 mg/kg) in LPO (51%, 60%, 75%, 77% for liver, 26%, 41%, 68%, 72% for kidney and 46%, 59%, 71%, 73% for brain) and GSH (41%, 59%, 78%, 80% for liver, 30%, 49%, 78%, 82% for kidney and 27%, 51%, 75%, 79% for brain), respectively. Maximum recovery was observed at both the higher doses, i.e. 20 and 40 mg/kg dose, as shown by ANOVA (P ≤ 0.05).
Table 4.
Therapeutic efficacy of rutin on LPO against AA-induced toxicity
| Treatments | LPO (n mole TBARS/mg protein) | ||
|---|---|---|---|
| Liver | Kidney | Brain | |
| Control | 0.23 ± 0.01 | 0.34 ± 0.01 | 0.33 ± 0.01 |
| RU per se | 0.26 ± 0.01 | 0.38 ± 0.02 | 0.37 ± 0.02 |
| AA per se | 1.30 ± 0.07a | 2.22 ± 0.12a | 2.27 ± 0.12a |
| AA+ RU (5 mg/kg) % protection | 0.75 ± 0.04b(51%) | 1.73 ± 0.09b(26%) | 1.36 ± 0.07b(46%) |
| AA+ RU (10 mg/kg) % protection | 0.65 ± 0.03b(60%) | 1.45 ± 0.08b(41%) | 1.12 ± 0.06b(59%) |
| AA+ RU (20 mg/kg) % protection | 0.49 ± 0.02b(75%) | 0.93 ± 0.05b(68%) | 0.89 ± 0.04b(71%) |
| AA+ RU (40 mg/kg) % protection | 0.47 ± 0.02b(77%) | 0.87 ± 0.04b(72%) | 0.85 ± 0.04b(73%) |
| F value (at 5% level) | 111.5c | 113.4c | 12.1c |
Data are mean ± SE; N = 6.
aAA versus Control.
bAA+ therapy versus AA at P ≤ 0.05.
cSignificant at 5% for ANOVA.
Table 5.
Therapeutic effect of rutin on reduced GSH against AA-induced alterations
| Treatments | Reduced GSH (μ mole/g) | ||
|---|---|---|---|
| Liver | Kidney | Brain | |
| Control | 8.57 ± 0.47 | 7.91 ± 0.43 | 8.17 ± 0.45 |
| RU per se | 8.10 ± 0.44 | 7.85 ± 0.43 | 7.97 ± 0.44 |
| AA per se | 5.57 ± 0.30a | 5.72 ± 0.31a | 5.44 ± 0.30a |
| AA+ RU (5 mg/kg) % protection | 6.80 ± 0.37b(41%) | 6.39 ± 0.35 (30%) | 6.17 ± 0.34 (27%) |
| AA+ RU (10 mg/kg) % protection | 7.35 ± 0.40b(59%) | 6.79 ± 0.37 (49%) | 6.84 ± 0.37b(51%) |
| AA+ RU (20 mg/kg) % protection | 7.91 ± 0.43b(78%) | 7.42 ± 0.41b(78%) | 7.48 ± 0.41b(75%) |
| AA+ RU (40 mg/kg) % protection | 7.98 ± 0.44b(80%) | 7.51 ± 0.41b(82%) | 7.59 ± 0.41b(79%) |
| F value (at 5% level) | 7.085c | 5.147c | 7.639c |
Data are mean ± SE; N = 6.
aAA versus Control.
bAA+ therapy versus AA at P ≤ 0.05.
cSignificant at 5% for ANOVA
Drastic decline (P ≤ 0.05) was observed in SOD and CAT after AA intoxication (Tables 6 and 7). In liver, kidney and brain of rats, 47%, 44% and 39% decrement in SOD level and 52%, 40% and 39% decrease in CAT level were recorded, respectively, as compared to control. Recoupment was observed at all the four doses (05, 10, 20 and 40 mg/kg) of RU in SOD (25%, 54%, 66%, 69% for liver, 32%, 47%, 76%, 78% for kidney and 28%, 48%, 58%, 61% for brain) and CAT (32%, 49%, 74%, 77% for liver, 33%, 39%, 57%, 59% for kidney and 15%, 31%, 60%, 65% for brain), respectively.
Table 6.
Protective potential of rutin on SOD activity in liver, kidney and brain
| Treatments | SOD (U/min/mg protein) | ||
|---|---|---|---|
| Liver | Kidney | Brain | |
| Control | 64.4 ± 3.56 | 51.4 ± 2.84 | 64.0 ± 3.53 |
| RU per se | 63.0 ± 3.48 | 50.6 ± 2.79 | 63.0 ± 3.48 |
| AA per se | 34.0 ± 1.87a | 28.7 ± 1.58a | 39.0 ± 2.15a |
| AA+ RU (5 mg/kg) % protection | 41.7 ± 2.30b(25%) | 35.9 ± 1.98b(32%) | 45.9 ± 2.53 (28%) |
| AA+ RU (10 mg/kg) % protection | 50.4 ± 2.76b(54%) | 39.3 ± 2.17b(47%) | 50.9 ± 2.81b(48%) |
| AA+ RU (20 mg/kg) % protection | 54.2 ± 2.99b(66%) | 45.9 ± 2.53b(76%) | 53.4 ± 2.95b(58%) |
| AA+ RU (40 mg/kg) % protection | 55.0 ± 3.04b(69%) | 46.5 ± 2.57b(78%) | 54.3 ± 3.00b(61%) |
| F value (at 5% level) | 16.93c | 14.51c | 10.80c |
Data are mean ± SE; N = 6.
aAA versus Control.
bAA+ therapy versus AA at P ≤ 0.05.
cSignificant at 5% for ANOVA.
Table 7.
Protective efficacy of rutin on catalase activity
| Treatments | Catalase (μmole H2O2/min/mg protein) | ||
|---|---|---|---|
| Liver | Kidney | Brain | |
| Control | 68.0 ± 3.75 | 75.7 ± 4.18 | 79.0 ± 4.36 |
| RU per se | 67.0 ± 3.70 | 70.1 ± 3.87 | 75.6 ± 4.18 |
| AA per se | 32.8 ± 1.81a | 45.0 ± 2.48a | 48.4 ± 2.67a |
| AA+ RU (5 mg/kg) % protection | 44.0 ± 2.43b(32%) | 55.1 ± 3.04b(33%) | 53.0 ± 2.92 (15%) |
| AA+ RU (10 mg/kg) % protection | 49.9 ± 2.76b(49%) | 56.9 ± 3.14b(39%) | 58.0 ± 3.20 (31%) |
| AA+ RU (20 mg/kg) % protection | 58.9 ± 3.25b(74%) | 62.6 ± 3.46b(57%) | 66.9 ± 3.69b(60%) |
| AA+ RU (40 mg/kg) % protection | 60.1 ± 3.32b(77%) | 63.3 ± 3.49b(59%) | 68.4 ± 3.78b(65%) |
| F value (at 5% level) | 20.89c | 10.48c | 12.18c |
Data are mean ± SE; N = 6.
aAA versus Control.
bAA+ therapy versus AA at P ≤ 0.05.
cSignificant at 5% for ANOVA.
Lower doses of RU (05 and 10 mg/kg) were also found to be effective in recouping antioxidant enzymes activities; however, most effective restoration was observed at 20 and 40 mg/kg doses when analyzed statistically (P ≤ 0.05).
AChE activity
Results of AChE activity in all groups were recorded in Table 8. The activity of AChE decreased by 73% in AA exposed group when compared with the control group (P ≤ 0.05). This alteration was significantly recouped by the treatment of RU at all four doses (05, 10, 20 and 40 mg/kg) by increasing the AChE activity (24%, 38%, 59% and 64% respectively). Dose of 20 and 40 mg/kg showed maximum recoupment.
Table 8.
Ameliorative effect of rutin on AChE activity
| Treatments | AChE (μmole/min/mg protein) |
|---|---|
| Control | 42.6 ± 2.35 |
| RU per se | 36.0 ± 1.99 |
| AA per se | 11.5 ± 0.64a |
| AA+ RU (5 mg/kg) % protection | 18.9 ± 1.04b(24%) |
| AA+ RU (10 mg/kg) % protection | 23.5 ± 1.29b(38%) |
| AA+ RU (20 mg/kg) % protection | 29.9 ± 1.65b(59%) |
| AA+ RU (40 mg/kg) % protection | 31.4 ± 1.73b(64%) |
| F value (at 5% level) | 50.44c |
Data are mean ± SE; N = 6.
aAA versus Control.
bAA+ therapy versus AA at P ≤ 0.05.
cSignificant at 5% for ANOVA.
Both the highermost doses show almost same pattern of recovery. Thus, dose of RU at 20 mg/kg was selected for further studies. After per se treatment of RU, there were no adverse effects in the blood and tissue biochemical parameters.
Histological examination
Liver
Figure 3A and B shows normal histological structure of the liver of control rats with well-maintained hepatic lobules along with normal appearance of hepatocytes with prominent nucleus, clear central vein and sinusoidal spaces. Acute exposure of AA caused hepatocytes degeneration with pyknotic nuclei, disturbed cord arrangement, collection of fluid in central vein, necrosis and obliterated sinusoids. Vacuolation was clearly visible (Fig. 3C and D). RU therapy at 5 mg/kg dose showed improvement, but mild degenerative changes were also seen (Fig. 3E and F). Figure 3G and H: At 10 mg/kg dose of RU, mild sinusoidal dilation was seen with regeneration of hepatocytes and reduced necrosis. Figure 3I and J: 20 mg/kg dose of RU was significantly effective in restoring the histological structure of liver. Hepatocytes showed clear sinusoidal spaces with conspicuous nucleus and well-formed hepatic cords. No extensive cytoplasmic vacuolization and necrosis were seen with well-maintained central vein and portal triad. Figure 3K and L: Treatment of RU at 40 mg/kg dose showed well-maintained histoarchitecture and prominent recovery. Hepatocytes, central vein and sinusoidal spaces were found towards normal.
Figure 3(A–L).
Histopathology of Liver (X100 & X400). 3A (X100) & 3B (X400): Photomicrographs of liver of control rat. 3C (X100) & 3D (X400): Liver of AA intoxicated rat. 3E (X100) & 3F (X400): Liver of rat treated with 05 mg/kg RU after AA intoxication. 3G (X100) & 3H (X400): Liver of rat treated with 10 mg/kg RU after AA intoxication. 3I (X100) & 3J (X400): Liver of rat treated with 20 mg/kg RU after AA intoxication. 3K (X100) & 3L (X400): Liver of rat treated with 40 mg/kg RU after AA intoxication. H - Hepatocytes, N - Nucleus, CV - Central Vein, S – Sinusoidal Spaces, HN – Hepatic Necrosis, V- Vacuoles, PN – Pyknotic Nuclei, PT - Portal Triad.
Kidney
Figure 4A and B: Kidney of control rats showed well-formed renal tubules, glomeruli and maintained medullary tubules. The glomeruli were surrounded by narrow Bowman’s space. Figure 4C and D: AA intoxication showed severe degeneration in the renal tubules and necrotic changes in the Bowman’s capsule. Exfoliated nuclei were present in tubules and epithelial linings were distorted. At 05 mg/kg dose of RU, mild recovery was seen with obstructions in the uriniferous tubules (Fig. 4E and F). Figure 4G and H: Dose of 10 mg/kg of RU showed improvement with mild degeneration in the glomeruli. Epithelial cells of the tubules showed improvement. Figure 4I and J: Therapy of RU at 20 mg/kg showed better recovery with almost normal tubules. Bowman’s capsule appeared normal with compact glomeruli. Figure 4K and L: 40 mg/kg dose of RU showed well-maintained glomeruli, Bowman’s capsule and renal tubules. Lumen was wide and clear.
Figure 4(A–L).
Histopathology of Kidney (X100 & X400). 4A (X100) & 4B (X400): Photomicrographs of kidney of control rat. 4C (X100) & 4D (X400): Kidney of AA intoxicated rat. 4E (X100) & 4F (X400): Kidney of rat treated with 05 mg/kg RU after AA intoxication. 4G (X100) & 4H (X400): Kidney of rat treated with 10 mg/kg RU after AA intoxication. 4I (X100) & 4J (X400): Kidney of rat treated with 20 mg/kg RU after AA intoxication. 4K (X100) & 4L (X400): Kidney of rat treated with 40 mg/kg RU after AA intoxication. G – Glomerulus, BC – Bowman’s capsule, T – Renal Tubules, L – Lumen.
Brain
Figure 5A and B: Brain of control rats showed normal cerebral cortex with well-arranged neurons. Figure 5C and D: Exposure to AA caused neuronal degeneration and cytoplasmic vacuolization in the brain of rats. Dark stained degenerated corkscrew-shaped pyramidal cells were observed. Pericellular spaces were also present in pyramidal cells. Figure 5E and F: Mild improvement was seen in the brain of rats of RU (05 mg/kg dose) administered rats; however, mild vacuolization still persists. Figure 5G and H: At 10 mg/kg dose of RU, recovery was seen with improved structure of pyramidal cells. Comparatively less lesions were seen. Figure 5I and J: 20 mg/kg dose of RU showed significant recoupment in the structure of cerebral cortex of rats with prominent and well-arranged neurons with loss of pericellular spaces. At 40 mg/kg dose of RU, neurons were appeared normal with loss of vacuolization. Pyramidal cells were also regular in appearance (Fig. 5K and L).
Figure 5(A–L).
Histopathology of Brain (X100 & X400). 5A (X100) & 5B (X400): Photomicrographs of brain of control rat. 5C (X100) & 5D (X400): Brain of AA intoxicated rat. 5E (X100) & 5F (X400): Brain of rat treated with 05 mg/kg RU after AA intoxication. 5G (X100) & 5H (X400): Brain of rat treated with 10 mg/kg RU after AA intoxication. 5I (X100) & 5J (X400): Brain of rat treated with 20 mg/kg RU after AA intoxication. 5K (X100) & 5L (X400): Brain of rat treated with 40 mg/kg RU after AA intoxication. N – Neurons, PC – Pyramidal Cells, V – Vacuoles, PS – Pericellular Spaces.
Discussion
Nowadays, AA is one of the major environmental public health issues as its presence has been reported in various food products. As AA is electrophilic in nature, it can react with nucleophilic compounds containing SH, NH2 and OH groups. AA gets converted to its genotoxic form, glycidamide, by epoxidation reaction via CYP2E1 and ultimately forms Hb and GA-DNA adducts. AA is eliminated through the body by conjugating with GSH and forms mercapturic acid through GST; hence, we have checked GSH level in our study. AA cause impairment to liver, kidney, brain and testes [40, 41].
There are developing evidences, which suggests oxidative stress-induced hepatic, renal and brain injury. To prevent oxidative DNA damage caused by several chemicals or environmental factors, antioxidant role of phytochemicals is also reported [42, 43].
To reduce AA risks, phytoceuticals like polyphenols are used. RU is one of the polyphenolic flavonoids, which has antioxidant property and reported beneficial effects in various disease conditions such as cancer, liver, kidney and brain injury [44, 45].
AA is formed in foods by Maillard reaction with multiple step reactions. Polyphenols like antioxidants can alter the Maillard reaction by interfering in these steps. These may affect reactive carbonyl pool, asparagine, intermediates or the final product AA as well [46].
In this study, the main aim was to investigate the antitoxic role of rutin against AA-induced toxicity in rats.
This study demonstrated the protective effect of RU by various biochemical and histological observations. For the observation of pathological changes, histological sections of liver, kidney and brain were prepared.
Transaminase and phosphates are important enzymes in biological processes. As a consequence of damage in cell membrane integrity, these enzymes are released into the blood stream. Estimation of these serum enzymes is a remarkable marker of hepatocellular damage [47–49]. Dysfunction of these enzymes causes various biochemical damage and cell injury. In present study, the enhanced activities of these enzymes are a sign of liver damage induced by toxin, which was demonstrated by histopathological observations. The bipolar nature of AA is the reason for the increase in the activities of these serum transaminases enzymes. Hypothesis recorded by Abdel-Daim et al. [50] supported these results, which showed increase in serum transaminases indicated the liver dysfunction. Treatment of the animals with RU showed protection against AA-induced hepatic damage. The observed reduced level of AST and ALT in RU-treated animals was most probably due to its ability to repair the liver tissue damage and to stabilize the plasma membrane.
AA-induced nephrotoxicity is characterized by an increase in serum urea, uric acid and creatinine. These are important indicators of renal damage in clinical findings [51]. These findings are also substantiated by the histopathology of kidney. Elevated levels of these serum metabolites point towards alterations in the glomerular filtration rate due to damage in the filtering compartments of kidney. Our results are in accordance with the previously reported results. Increase in serum urea, creatinine and uric acid levels in rats was shown by Elhelaly et al. [52] and Teodor et al. [53], which might be either because of excessive rate of production or damage in filtering compartments or by the degradation of purines and pyrimidines. Therapy of RU was effective in maintaining cellular function of kidney by exerting their antioxidative effect and by modulating key enzymes, which consequently brought serum urea, uric acid and creatinine level towards control.
Lipid profile was ascertained by observing the level of triglycerides and cholesterol in serum. AA intoxication significantly increased the level of lipid profile, which may be due to the presence of trans-fat in high amount in fried foods and most bakery items or due to the failure of normal uptake, conjugation and excretion by the damaged hepatic parenchyma. This trend of lipid profile level is in accordance with the studies by Shrivastava et al. [54] and Alanazi et al. [55]. RU therapy declined these levels in rat serum towards control. This protective property of flavonoids to lower the level of serum triglycerides, cholesterol and lipid peroxides may be due to their ability to chelate minerals.
From the results of the present study, bilirubin level was increased after AA intoxication in experimental animals. This may be either from the production of bilirubin more than normal in liver or by the impairment or blockage of excretory ducts of the liver due to which liver is unable to excrete the normal amounts of bilirubin. Our results are in concurrence with the results of Rivadeneyra-Domínguez et al. [15] and Khan et al. [56]. RU offered significant protection proven by the declined level of bilirubin.
Reduced GSH is one of the components of glutathione peroxidase system. It is tripeptide non-enzymatic biological antioxidants present in the liver and one of the essential compounds for maintaining cell integrity [57]. GSH is recognized as a protective compound within the body, detoxifying many xenobiotics or their metabolites through several mechanisms. It acts as a substrate for the H2O2 removing enzyme glutathione peroxidase and for dehydroascorbate reductase. It is a powerful reducing agent, which disturbs the radical chain production of LPO.
LPO can cause different diseases by the peroxidation of membrane lipids. Increased TBARS after AA intoxication indicated enhanced LPO due to failure of the antioxidant defense mechanism. Increased LPO is a result of GSH depletion to certain critical levels. AA is an electrophile, which reacts with –SH group of GSH and forms GSH conjugates. In this study, GSH content decreased due to its increased utilization and LPO was increased. The result of the current study was consistent with the observed effects of [58]. Treatment of RU hinders AA-induced reactive oxygen species due to its antioxidant nature and showed beneficial protective effects by upregulating the levels of GSH and decreased the level of LPO to normal. Polyhydroxylated substitutions on rings A and B, a 2, 3-double bond, a free 3-hydroxyl substitution and a 4-keto moiety are responsible for anti-peroxidative properties of rutin [59].
CAT, SOD and peroxidase are the antioxidant enzymes, which comprise a mutually supportive defensive consortium against free radicals. SOD occurs in a considerably high amount in liver, kidney, brain, heart and erythrocytes cells. AA-administered rats showed decreased activity of SOD and CAT in liver, kidney and brain. This decrement may have occurred due to the increased LPO or inactivation of enzymes by crosslinking with malondialdehyde [60]. This study was in accordance with other studies, which showed that AA caused significant decrease in SOD and CAT activity in liver, kidney and brain [61–63]. Treatment with RU alleviates the activities of SOD and CAT. It stimulates production of antioxidant enzymes (SOD, CAT) and reduced the oxidative stress.
AA induced oxidative stress by the generation of free radicals. AChE receptors were blocked due to free radicals. This caused decrement in the activity of AChE in the brain of rats. This is in accordance with the study of Farouk et al. [7] and Shrivastava et al. [64]. There may be two reasons of declined activity, one is direct binding of AA or GA with –SH group of the enzyme and the second is oxidation of the cysteine in the enzyme by ROS [65]. In our previous study, AA-induced neurotoxicity was supported by the morphological symptoms like hind limb splaying, dragging of back legs and limb weakness [31]. Treatment with RU at different doses restored AChE activity in brain of rats. RU at a dose of 20 and 40 mg/kg was significantly effective showing that RU has strong neuroprotective effects. RU has 4-oxo group and the 2, 3 double bond in the C ring, which may be responsible for its neuroprotective property [66]. Histological observations basically support the results obtained from biochemical assays.
Conclusion
This study concludes that RU helps in attenuating the AA induced ailments. This efficacy may not only due to its antioxidant activity but also due to the presence of functional groups in its structure or other mechanisms like precipitating asparagine, which is involved in the formation of AA through Maillard reaction and other constituents. This study would provide a useful information to aware people about the health risk of dietary AA exposure and to use natural antioxidants to lower this risk. From potential challenges for food safety, research and risk management point of view, more investigation is warranted to know the molecular mechanisms of action behind the efficacy of RU against AA-induced damage.
Authors’ contributions
C.U. conceived and planned the experiments under the guidance of S.S. C.U., M.S.R., A.J., D.Y., S.S. and N.S. carried out experiments and aided in processing and analyzing of experimental data. C.U. performed calculations, interpreted the results, prepared figures and tables and drafted the manuscript. S.S. helped in managing and execution of the work. S.S. verified the results and supervised the work. All authors approved the final manuscript.
Acknowledgement
The author thanks Jiwaji University for providing lab facility to carry out this work.
Contributor Information
Chhavi Uthra, Reproductive Biology and Toxicology Laboratory, UNESCO-Trace Element Satellite Centre, School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, 474011, India.
Mohd Salim Reshi, Toxicology and Pharmacology Lab, Department of Zoology, School of Biosciences and Biotechnology, Baba Ghulam Shah Badshah University, Rajouri, Jammu & Kashmir, 185234, India.
Amita Jaswal, Reproductive Biology and Toxicology Laboratory, UNESCO-Trace Element Satellite Centre, School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, 474011, India.
Deepa Yadav, Reproductive Biology and Toxicology Laboratory, UNESCO-Trace Element Satellite Centre, School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, 474011, India.
Sadhana Shrivastava, Reproductive Biology and Toxicology Laboratory, UNESCO-Trace Element Satellite Centre, School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, 474011, India.
Neelu Sinha, Reproductive Biology and Toxicology Laboratory, UNESCO-Trace Element Satellite Centre, School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, 474011, India.
Sangeeta Shukla, Reproductive Biology and Toxicology Laboratory, UNESCO-Trace Element Satellite Centre, School of Studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, 474011, India.
Funding
The author thanks University Grants Commission, New Delhi (F.4-1/2006(BSR)/7-97/2007(BSR), 26, June, 2012) for financial assistance.
Conflict of interest statement
The authors declare no conflict of interest.
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