Effect of Curcumin on Aflatoxin B 1 –Induced Toxicity in Rats: A Biochemical and Histopathological Study

Objective: The aim of the present study was to investigate the protective effect of curcumin against aflatoxinB 1 (AFB 1 ) induced hepatotoxicity. Materials and Methods: Twenty-eight healthy adult male Wistar rats were divided into four groups. Rats of the first group received basal diet and served as control. Rats in the second group received curcumin orally (15mg/5ml/kg body weight) whereas, rats in the third groups injected with single intraperitoneal injection of AFB 1 (3mg/kg BW). Rats in the fourth group received a combination of second and third groups for five weeks. Results: Biochemical analysis of serum samples indicated a significant increase in aspartate transaminase (AST) and alanine transaminase (ALT) activities and total cholesterol and creatinine concentrations along with significant decrease in protein content of AFB 1 intoxicated rats compared to control group. Oral administration of curcumin along with injected AFB 1 restored AST, ALT, total cholesterol, creatinine and total protein near to control values. Biochemical analysis of liver antioxidants revealed a significant ( P < 0.05 ) reduction in catalase (CAT) and superoxide dismutase (SOD) activities and hepatic reduced glutathione (GSH) content in rats injected with AFB 1 compared to control. On the contrary, oral administration of curcumin along with injected AFB 1 enhanced hepatic CAT and SOD activities and GSH concentration towards the control values, suggesting that curcumin could improve the antioxidant status in AFB 1 induced oxidative stress. The Biochemical findings were supported by histopathology of liver tissues which indicated vacuolar degeneration and necrotizing changes in liver of rats intoxicated with AFB 1 and significant amelioration of these effects in these rats whenever treated with curcumin. Conclusion: Conclusively, oral administration of curcumin along with AFB 1 caused significant inhibition in AFB 1 -induced hepatotoxicity in rats by increasing the concentration of GSH and activation of antioxidant enzymes.


INTRODUCTION
Mycotoxins are toxic metabolites produced by a large number of fungi under a wide range of environmental condition. Many of these fungi invade cereals, nuts and grains that are eventually used in the manufacture of animal feeds. Aflatoxins are secondary metabolites of the moulds Aspergillus flavus, Aspergillus parasiticus, Aspergillus tamarii and Aspergillus nominus [1]. AFB 1 is by far the most potent teratogen, mutagen and hepatocarcinogen of all aflatoxins [2]. The carcinogenic potential of AFB 1 following oral administration has been shown in several animal species, including rodents, nonhuman primates and fish [3]. The biological effects of AFB 1 in animals are related to their level in the feed and to the animal's susceptibility. Epidemiologic, clinical, and experimental studies have revealed that aflatoxins are hepatotoxic, hepatocarcinogenic, and mutagenic [4]. AFB 1 can cause lipid peroxidation in the rat liver, which is closely related to liver cell injury [5,6]. Bosch-Morell et al. [7] have demonstrated the involvement of oxidative stress in retinal detachment by detecting lipid peroxidation products in subretinal fluid of patients undergoing surgery. Medicinal plants and their active principles had received great attention as potentially antiperoxidative agents [8][9][10][11][12][13][14][15]. Turmeric is a perennial herb that grows to a height of three to five feet and is cultivated extensively in Asia (India and China) and other countries with tropical climate. Curcumin, the active ingredient from the spice turmeric is a potent antioxidant and antiinflammatory agent with hepatoprotective, anticarcinogenic and antimicrobial properties [16]. In addition, gene expression of antioxidant enzymes has been up-regulated by curcumin in diabetic rats [15]. Although, the antitoxic effect of curcumin has been investigated [10,12], the literature reports are still contradictory. Therefore, the objective of the present study was to investigate the antitoxic effect of curcumin in AFB 1 intoxicated rats by evaluation of selected serum biochemical parameters, hepatic oxidative stress biomarkers and histopathology of affected liver.

Chemicals
Curcumin, AFB 1 and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
All other chemicals and buffers were of analytical grade.

Experimental Animals
Twenty-eight healthy adult male inbred Wistar rats weighing between 150-200g were obtained from the laboratory animal house of the Faculty of Veterinary Medicine and Animal Recourses, King Faisal University, Saudi Arabia. They were maintained in accordance with the national guidelines and protocols, approved by the University Animal Ethics Committee, King Faisal University, Saudi Arabia. They were housed in clean and disinfected plastic cages. Commercial basal pelleted diet and water were provided ad libitum. Rats were subjected to natural photoperiod of 12hr light: dark cycle throughout the experimental period (5 weeks). The experimental animals were housed in airconditioned rooms at 21-23ºC and 60-65% of relative humidity. All rats received basal diet for two weeks before the start of the experiment for adaptation and to ensure normal growth and behavior.

Experimental Design
Rats were divided into four groups of 7 rats each (4 animals/cage). Rats of the first group received basal diet and served as normal control (NC). Rats in the second group received curcumin orally (15mg/5ml/kg BW) [15] and labeled as curcumin treated group (CT) whereas, rats in the third groups injected with single i.p injection of AFB 1 dissolved in dimethyl sulphoxide DMSO (3mg/kg BW) [17] and labeled as AFB 1 treated group (AF) for five weeks. Rats in the fourth group received a combination of second and third groups and labeled as AFB 1 + curcumin treated group (AC) for also five weeks.

Sampling and Analysis
At the end of the experiment, rats were sacrificed, anaesthetized by diethyl ether inhalation and blood and liver samples were collected. Serum was separated by centrifugation for 10 min at 1200g and was immediately frozen at -20ºC until the time of analysis.

Assay of oxidative stress biomarker
Portion of the liver was dissected out and trimmed off the attached tissue and stored at -80ºC until used for biochemical analysis of GSH concentration and antioxidant enzyme activities.

Histopathological study
Another portion of liver tissue was collected also and cut in small pieces and immersed in neutral buffered formalin for 24h for histopathological examination. The fixed liver tissue was processed routinely, embedded in paraffin, sectioned, deparaffinized and rehydrated using the standard techniques [18]. The effect of and the ameliorative effect of curcumin was evaluated by assessing the morphological changes in the liver sections stained with hematoxylin and eosin (H and E), using standard techniques.

Statistical Analysis
All the grouped data were statistically evaluated and the significance of changes caused by various treatments was determined using one ways ANOVA. Post hoc tests in the Analysis of Variance (ANOVA), containing one factor (Group) and serum biochemical depe measurements, was used applying GLM Unianova Procedure. Bartlett's, Brown and Forsythe's Tests for Homogeneity of Variance assumptions were reasonably met for the one way ANOVA. The Tables (1-5) shows the significance difference of means and were performed using computer package of the statistical analysis system (SAS) [19].

Selected Biochemical Parameters
The present findings indicated that, AFB treatment (AT) induced significant increase (P<0.05) in total cholesterol, ALT and AST activities ( Table 2) and creatinine concentration (Table 3) however protein contents (Table 1) was significantly decreased (P≤0.05) in serum of rats when compared with the normal control group (NC). Oral administration of curcumin along with injected AFB 1 (AC) restored total cholesterol, ALT, AST, creatinine and total protein near to control values.

Oxidative Stress Biomarkers
The effects of AFB 1 and curcumin (CT) or in combination (AC) on oxidative stress biomarkers of liver tissues of rats are summarized in Table 5. A significant ( reduction of CAT and SOD activities and GSH content were evident in rats injected with AFB (AT) compared to normal control animals (NC). On the contrary, Oral administration of curcumin 66 sectioned, deparaffinized and rehydrated using the standard techniques [18]. The effect of AFB 1 and the ameliorative effect of curcumin was sessing the morphological changes in the liver sections stained with hematoxylin and eosin (H and E), using standard All the grouped data were statistically evaluated and the significance of changes caused by various treatments was determined using one ways ANOVA. Post hoc tests in the Analysis of Variance (ANOVA), containing one factor (Group) and serum biochemical dependent measurements, was used applying GLM-Unianova Procedure. Bartlett's, Brown and Forsythe's Tests for Homogeneity of Variance assumptions were reasonably met for the one 5) shows the significance difference of means and all tests were performed using computer package of the [19].

Selected Biochemical Parameters
The present findings indicated that, AFB 1 treatment (AT) induced significant increase l, ALT and AST activities ( Table 2) and creatinine concentration (Table 3) however protein contents (Table 1) was ) in serum of rats when compared with the normal control group (NC). Oral administration of curcumin along with (AC) restored total cholesterol, ALT, AST, creatinine and total protein near to Biomarkers and curcumin either alone (CT) or in combination (AC) on oxidative stress ver tissues of rats are summarized in Table 5. A significant (P<0.05) reduction of CAT and SOD activities and GSH content were evident in rats injected with AFB 1 (AT) compared to normal control animals (NC). On the contrary, Oral administration of curcumin along with AFB 1 (AC) caused significant amelioration in AFB 1 -induced effects observed by decreased CAT and SOD enzymes activities (P≤0.05) and increased reduced glutathione contents (P≤0.001) compared to the AFB treated rats (AT).

Histopathological Examination
Liver of the normal control (NC) and curcumin treated rats (CT) showed central veins surrounded by polygonal cells arranged in regular cords separated from each other by sinusoids (Fig. 1A). The liver of rats intoxicated with AFB 1 (AT) showed distorted lobular architecture and necrobiotic changes ranged from vacuolar degeneration to necrotizing changes. This was associated with mononuclear cell infiltration everywhere (Fig. 1B). However, livers treated with AFB 1 along with curcumin (AC) appeared more or less recovered and have an almost normal architecture (Fig. 1C). aused significant induced effects observed by decreased CAT and SOD enzymes activities ) and increased reduced glutathione ) compared to the AFB 1

Examination
Liver of the normal control (NC) and curcumin treated rats (CT) showed central veins surrounded by polygonal cells arranged in regular cords separated from each other by sinusoids (Fig. 1A). The liver of rats intoxicated distorted lobular architecture and necrobiotic changes ranged from vacuolar degeneration to necrotizing changes. This was associated with mononuclear cell infiltration everywhere (Fig. 1B). However, along with curcumin (AC) ed more or less recovered and have an almost normal architecture (Fig. 1C).

Selected Biochemical Parameters
It is well known that, aflatoxin has a harmful and stressful effect on liver tissue. AST and ALT are cytosolic enzymes and are famous biomarkers of liver damage. In the present study, AFB 1 injection (AT) was found to cause an increase in serum ALT and AST activities ( Table 2). These results indicated liver injury and necrosis [20,21]. Whenever liver was injured, levels of hepatic transaminases were significantly increased [22][23][24]. However, administration of curcumin along with injected AFB 1 (AC) showed marked recovery but still beyond the ALT and AST levels ( Table 2) of control group.Regarding the ameliorative effect of curcumin against AFB 1 toxicity, previous reports [25,26] showed a significant hepatoprotective activity of curcumin by lowering the level of serum biomarker enzymes in AFB 1 intoxicated rats. Low total protein level acts as an indicator of the toxic effect of AFB 1 in serum [27]. Aflatoxin is known to impair protein biosynthesis by forming adducts with DNA, RNA and proteins, inhibits RNA synthesis, DNA-dependent RNA polymerase activity and causes degranulation of endoplasmic reticulum [27]. Reduction in protein content (Table 1) observed in the current study (AT) may be attributed to increase in the rate of degeneration of liver tissues as underlined by increasing activities of ALT and AST ( Table 2). The injured liver logically is unable to maintain vital biochemical processes particularly protein biosynthesis. These results are in accordance with previous work [28] which reported a decrease in protein content in skeletal muscle, heart, liver and kidney of aflatoxin-fed animals.
The present results showed that, curcumin treatment along with injected AFB 1 (AC) ameliorates AFB 1 -induced changes in protein contents in the serum of rats ( Table 1). The amelioration in protein contents might be due to increased DNA synthesis and reduction in harmful adduct formation [29]. Authors investigated the inhibitory effects of curcumin, garlic squeeze, grape seed extract, tea polyphenols, vitamin C and vitamin E on nicotine-DNA adduction in vivo. They suggested that these dietary constituents are beneficial in preventing the harmful adduct formation and thus block the potential carcinogenesis induced by nicotine. Similar results described the ameliorative effect of curcumin against AFB 1 induced low protein contents in Broiler chicks [30] and in mice [31]. After biosynthesis of creatine in the liver, it is taken up from the blood by skeletal muscles and converted into creatine phosphate. Creatine and its phosphate are converted spontaneously into creatinine [32]. The significant appearance (P<0.05) of creatinine in the serum of aflatoxin-fed rats indicated the increased transformation of phosphocreatine to creatinine in muscle which might be due to lesser utilization of phosphocreatine in muscular contraction. The elevated creatinine level in AFB 1 treated (AT) rats as observed in the current study, suggests the myotoxic and nephrotoxic effect of AFB 1 in rats [33] which improved upon administration of curcumin.

Oxidative Stress Biomarkers
Oxidative stress was originally defined as the imbalance between prooxidants and antioxidants in biological systems. The significant reduction in the activities of enzymatic antioxidants such as CAT and SOD as well as non-enzymatic antioxidants such as glutathione in the liver of AFB 1 -treated rats (AT) when compared to the NC and CT groups ( Table 5) indicated that AFB 1 induced oxidative stress and subsequent liver damage. SOD protects cells from oxidative damage by converting free radical superoxide to H 2 O 2 and O 2 . The H 2 O 2 produced can then be decomposed enzymatically by CAT. Significant reductions in SOD [27] and CAT [24] have been reported in aflatoxin-fed rat liver. The significant increase of hepatic antioxidant enzymes CAT and SOD activities observed in this study in rats intoxicated with AFB 1 and treated with curcumin (AC) ( Table 5) are in accordance with previous studies which reported that curcumin is a potent inducer of detoxifying enzymes and thereby prevents the toxicity induced by a chemical carcinogen [15,34]. Glutathione has a beneficial effect by virtue of possessing -SH groups. It helps to protect biological membranes, which are readily susceptible to peroxidation. Carcinogens like AFB 1 , which generate epoxides, have been found to conjugate readily with GSH. Lower GSH level would further aggravate the toxic effects of aflatoxin. Many investigators [27,35] have reported significant reduction in glutathione content in aflatoxin-fed rat liver. The impact of curcumin on GSH has been documented [15].

Histopathological Findings
The histopathological findings (Fig. 1) supported the biochemical findings and give evidence of liver damage in rats intoxicated with AFB 1 (AT) . Similar AFB 1 -induced hepatic damage has been reported [36]. The relief of hepatic tissues in AFB 1 intoxicated rats treated with curcumin (AC) is consistent with earlier report [25] which suggested that curcumin but not resveratrol has a hepatoprotective effect against aflatoxin B(1)induced liver injury.

CONCLUSION
The results obtained in this study indicated that, AFB 1 administration induced hepatotoxicity in rats as reflected on elevation of hepatic transaminases, reduction of antioxidant enzymes activities and reduction of glutathione concentration in serum and necrosis of liver tissues. Oral administration of curcumin along with AFB 1 caused significant amelioration in AFB 1 -induced hepatotoxicity in rats by increasing the concentration of GSH and activation of antioxidant enzymes. This suggests that curcumin could improve the antioxidant status in AFB 1 -induced oxidative stress.