Propolis alleviates aluminium-induced lipid peroxidation and biochemical parameters in male rats

Food and Chemical Toxicology 47 (2009) 1093–1098

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Propolis alleviates aluminium-induced lipid peroxidation and biochemical parameters in male rats Al-Sayeda A. Newairy a, Afrah F. Salama b, Hend M. Hussien a, Mokhtar I. Yousef c,* a

Department of Biochemistry, Faculty of Science, Alexandria University, Alexandria, Egypt Chemistry Department, Biochemistry Section, Faculty of Science, Tanta University, Egypt c Department of Home Economic, Faculty of Specific Education, Alexandria University, 14 Mohamed Amin Shohaib Street, Moustafa Kamel, P.O. Box Roushdi, Alexandria 21529, Egypt b

a r t i c l e

i n f o

Article history: Received 5 November 2008 Accepted 27 January 2009

Keywords: Aluminium chloride Propolis Rats Lipid peroxidation Antioxidant enzymes Biochemical parameters

a b s t r a c t Aluminium is present in many manufactured foods and medicines and is also added to drinking water during purification purposes. Therefore, the present experiment was undertaken to determine the effectiveness of propolis in alleviating the toxicity of aluminium chloride (AlCl3) on biochemical parameters, antioxidant enzymes and lipid peroxidation of male Wistar Albino rats. Animals were assigned to 1 of 4 groups: control; 34 mg AlCl3/kg bw; 50 mg propolis/kg bw; AlCl3 (34 mg/kg bw) plus propolis (50 mg/kg bw), respectively. Rats were orally administered their respective doses daily for 70 days. The levels of thiobarbituric acid reactive substances (TBARS) was increased, and the activities of glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxidase were decreased in liver, kidney and brain of rats treated with AlCl3. While, TBARS was decreased and the antioxidant enzymes were increased in rats treated with propolis alone. Plasma transaminases, lactate dehydrogenase, glucose, urea, creatinine, bilirubin, total lipid, cholesterol, triglyceride and LDL-c were increased, while total protein, albumin and high HDL-c were decreased due to AlCl3 administration. The presence of propolis with AlCl3 alleviated its toxic effects in rats treated with AlCl3. It can be concluded that propolis has beneficial influences and could be able to antagonize AlCl3 toxicity. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Aluminum metal is abundantly present in the earth’s crust. From the environment it gets access to the human body via the gastrointestinal and the respiratory tracts. Aluminium is a constituent of cooking utensils and medicines such as antacids, deodorants and food additives and this has allowed its easy access into the body (Yokel, 2000). The sources of aluminium are especially corn, yellow cheese, salt, herbs, spices, tea, cosmetics, ware and containers. Also, it is present in medicines and is also added to drinking water for purification purposes (Ochmanski and Barabasz, 2000).

Abbreviations: Al, aluminium; AlCl3, aluminium chloride; TBARS, thiobarbituric acid reactive substances; TBA, thiobarbituric acid; GST, glutathione S-transferase; SOD, superoxide dismutase; CAT, Catalase; GSH, reduced glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species; GSH-Px, glutathione peroxidase; GR, glutathione-reductase; NO, nitrogen oxide radical; LPO, lipid peroxidation. AST, aspartate transaminase; ALT, alanine transaminase; LDH, lactate dehydrogenase; HDL-c, high density lipoprotein; LDL-c, low density lipoprotein; GS, glutathione-synthase. * Corresponding author. Tel.: +20 3 5454313; Mobile +20 12 7231691; fax: +20 3 5442776. E-mail address: [email protected] (M.I. Yousef). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.01.032

Aluminium has been proposed as an environmental factor that may contribute to some neurodegenerative diseases, and affects several enzymes and other biomolecules relevant to Alzheimer‘s disease (Domingo, 2006). Also, increased aluminium burdens, can cause neurological symptoms, biochemical responses leading to unhealthy bone metabolism and learning disabilities in children (Zafar et al., 2004). Salts of aluminium may bind to DNA, RNA, inhibit such enzymes as hexokinase, acid and alkaline phosphates, phosphodiesterase and phosphooxydase (Ochmanski and Barabasz, 2000). Strong et al. (1996) found that aluminium exposure caused impairments in glucose utilization, agonist-stimulated inositol phosphate accumulation, free radical-mediated cytotoxicity, lipid peroxidation, reduced cholinergic function, impact on gene expression and altered protein phosphorylation. Yousef (2004) reported that aluminium-induced changes in hemato-biochemical parameters, increased lipid peroxidation and decreased the activities of the antioxidant enzymes in plasma and tissues of male rabbits. Also, Yousef et al. (2005, 2007) demonstrated that AlCl3 caused deterioration in sperm quality, enhancement of free radicals and alterations in antioxidant enzymes in both in vivo and in vitro. The mechanism of aluminium-induced toxicity is that it potentiates the activity of Fe2+ and Fe3+ ions to cause oxidative damage (Xie and Yokel, 1996).

1094

A.-S.A. Newairy et al. / Food and Chemical Toxicology 47 (2009) 1093–1098

Propolis or bee glue is a resinous hive product collected by honey bees from plant exudates and contains more than 160 constituents. Historically it has been used for various purposes, especially as a medicine (Ghisalberti, 1979). Flavonoids are thought to be responsible for many of its biological and pharmacological activities including anticancer (Padmavathi et al., 2006), anti-inflammatory (Paulino et al., 2008), and antioxidant effects (Nieva Moreno et al., 2000; Yousef et al., 2003a, 2003b, 2004a, 2004b). Flavonoids and various phenolic compounds are the most important pharmacologically active constituents in propolis that have been shown to be capable of scavenging free radicals and thereby protecting lipids from being oxidized or destroyed during oxidative damage (Nieva Moreno et al., 2000). Propolis has gained popularity and used extensively in healthy drinks and foods to improve health and prevent diseases such as inflammation, heart disease, diabetes and even cancer (Padmavathi et al., 2006; Paulino et al., 2008). Therefore, oral supplementation with propolis may protect the animals from the harmful effect of aluminium. The role of propolis against aluminium-induced changes in biochemical parameters, lipid peroxidation, and antioxidant enzymes of rats have not so far been studied. Therefore, the present study was carried out to investigate: (1) the alterations in biochemical parameters, free radicals and antioxidant enzymes induced by aluminium chloride in liver, kidney and brain of male rats, (2) the role of propolis in alleviating the negative effects of aluminium chloride, and (3) the effect of propolis alone on the tested parameters.

sured using the method of Pearlman and Lee (1974). Plasma concentrations of total lipids were assayed by the method of Knight et al. (1972) and that of cholesterol and triglycerides were determined by the method Carr et al. (1993). High density lipoprotein-cholesterol (HDL-c) and low density lipoprotein-cholesterol (LDL-c) were determined according to the methods of Warnick et al. (1983) and Bergmenyer (1985), respectively. 2.4. Tissue thiobarbituric acid reactive substances and antioxidant enzymes According to the method of Esterbauer and Cheeseman (1990), the extent of lipid peroxidation was measured in terms of thiobarbituric acid reactive substances (TBARS) formation was measured. Plasma and tissue supernatants were mixed separately with 1 ml TCA (20%), 2 ml TBA (0.67%) and heated for 1 h at 100 °C. After cooling, the precipitate was removed by centrifugation. The absorbance of the sample was measured at 535 nm using a blank containing all the reagents except the sample. As 99% TBARS are malondialdehyde (MDA), so TBARS concentrations of the samples were calculated using the extinction co-efficient of MDA, which is 1.56  105 M 1 cm 1. Glutathione S-transferase (GST; EC 2.5.1.18) catalyzes the conjugation reaction with glutathione in the first step of mercapturic acid synthesis. The activity of GST was measured according to the method of Habig et al. (1974). P-nitrobenzylchloride was used as substrate. The absorbance was measured spectrophotometrically at 310 nm using UV-Double Beam spectrophotometer. The catalase enzyme (CAT; EC 1.11.1.6) converts H2O2 into water. The CAT activity in plasma and tissue supernatant was measured spectrophotometrically at 240 nm by calculating the rate of degradation of H2O2, the substrate of the enzyme (Xu et al., 1997). Super oxide dismutase (SOD; EC 1.15.1.1) was assayed according to Misra and Fridovich (1972). The assay procedure involves the inhibition of epinephrine auto-oxidation in an alkaline medium (pH 10.2) to adrenochrome, which is markedly inhibited by the presence of SOD. Epinephrine was added to the assay mixture, containing tissue supernatant and the change in extinction co-efficient was followed at 480 nm in a Spectrophotometer. Glutathione peroxidase (GSH-Px) activity assayed using the method of Chiu et al. (1976) in brain, liver and kidney.

2. Materials and methods 2.1. Chemicals Aluminium chloride (AlCl3) was purchased from Aldrich chemical Company, Milwaukee Wis, USA, while propolis was obtained from Superior Nutrition and Formulation by Jarrow Formulas, Los Angeles, USA. All other chemicals used in the experiment were of analytical grade. The doses of aluminium chloride (AlCl3) and propolis were 34 mg/kg bw and 50 mg/kg bw, respectively. 2.2. Experimental design Forty male Wistar Albino rats (average weight 180–200 g) were used in the present experiment. Animals were obtained from faculty of medicine, Alexandria University, Egypt. The local committee approved the design of the experiments, and the protocol conforms to the guidelines of the National Institutes of Health (NIH). Animals were caged in groups of 5 and given food and water ad libitum. After two weeks of acclimatization, animals were divided into four equal groups. The first group was used as control. While, groups 2, 3 and 4 were orally treated with aluminium chloride (34 mg/kg bw), propolis (50 mg/kg bw) and the combination of aluminium chloride (34 mg/kg bw) and propolis (50 mg/kg bw), respectively. Rats were orally administered their respective doses every day for 70 days. At the end of the experiment, animals were sacrificed by decapitation and brain, liver and kidney were immediately removed. 2.3. Biochemical parameters Rats of each group were euthanized at the end of treatment period. Trunk blood samples were collected from the sacrificed animals and placed immediately on ice. Heparin was used as an anticoagulant and plasma samples were obtained by centrifugation at 860 Xg for 20 min and stored at –60 °C till measurements. Brain, liver and kidney were immediately removed; washed using chilled saline solution. Tissues were minced and homogenized (10% w/v), separately, in ice-cold sodium, potassium phosphate buffer (0.01 M, pH 7.4) containing 1.15% KCl in a Potter–Elvehjem type homogenizer. The homogenate was centrifuged at 10,000 Xg for 20 min at 4 °C, and the resultant supernatant was used for determination of biochemical parameters, assay of antioxidant enzyme and TBARS. Plasma aspartate transaminase (AST; EC 2.6.1.1) and alanine aspartate transaminase (ALT; EC 2.6.1.2) activities were determined with kits from SENTINEL CH. (via principle Eugenio 5–20155 MILAN, Italy). The activity of plasma lactate dehydrogenase (LDH; EC 1.1.1.27) was determined by the method of Martinek (1972). Stored plasma samples were analyzed for total protein by the Biuret method according to Armstrong and Carr (1964). Albumin concentration was determined by the method of Doumas et al. (1977). The concentrations of glucose were determined with kits from Bio systems, S.A. Costa Brava, 30-Barcelona (Spain). Plasma urea and creatinine concentrations were determined by the method of Patton and Crouch (1977) and Henry et al. (1974), respectively. Plasma total bilirubin was mea-

2.5. Statistical analysis Data are expressed as mean values ± SD of ten replicate determinations. Statistical analysis was performed using one-way analysis of variance (ANOVA) to assess significant differences among treatment groups. For each significant effect of treatment, the post-Hoc Tukey’s test was used for comparisons. The criterion for statistical significance was set at p < 0.05 or p < 0.01. All statistical analyses were performed using SPSS statistical version 8 software package (SPSSÒ Inc., USA).

3. Results and discussion 3.1. Aluminium chloride The present study was carried out to investigate the protective effects of propolis on aluminium-induced oxidative stress and biochemical alterations in rats. In the aluminium chloride treated rats, the levels of thiobarbituric acid reactive substances (TBARS) were found to be elevated but the activities of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were decreased in liver, kidney and brain (Table 1). These observations are similar to the data reported by Yousef (2004), Yousef et al. (2005, 2007), Nehru and Anand (2005) who indicated that aluminium intake produces oxidative stress. Although aluminium is not a transition metal and therefore cannot initiate peroxidation, many investigations have searched for a correlation between aluminium accumulation and oxidative damage in the body tissues (Cherroret et al., 1995; Wilhelm et al., 1996; Nehru and Anand, 2005). An in vitro study has indicated that aluminium greatly accelerates iron-mediated lipid peroxidation (Xie and Yokel, 1996). In fact, aluminium, a non-redox-active metal, is a pro-oxidant both in vivo and in vitro (Exley, 2004). The primary effects of aluminium on the brain, liver and kidney functions are thought to be mediated via damage to cell membranes. Lipid peroxidation of biological membranes results in the loss of membrane fluidity, changes in membrane potential, an increase in membrane permeability and alterations in receptor functions (Nehru and Anand, 2005). In the present experiment, there was a significant increase in lipid peroxidation after aluminium chloride exposure, measured in terms of TBARS levels in the brain,

1095

A.-S.A. Newairy et al. / Food and Chemical Toxicology 47 (2009) 1093–1098 Table 1 Changes in the levels of TBARS and the activities of GST, GSH-PX, CAT and SOD in liver, kidney and brain of male rats treated with aluminium chloride (AlCl3), propolis and AlCl3 + propolis. Parameter

Experimental groups Control

AlCl3

Propolis

AlCl3 + Propolis

Liver TBARSa GSTb GSH-PXc CATd SODe

32.8 ± 3.05 1.12 ± 0.066 33.3 ± 2.19 52.5 ± 5.81 75.4 ± 4.35

56.2 ± 3.66** 0.67 ± 0.052** 21.3 ± 0.94** 28.8 ± 6.02** 59.5 ± 5.36**

25.2 ± 3.13*## 1.33 ± 0.076## 38.5 ± 2.36*## 64.9 ± 7.32*## 88.9 ± 7.44*##

36.6 ± 2.95# 0.89 ± 0.049*# 29.1 ± 2.27# 42.9 ± 8.32*# 69.1 ± 5.43#

Kidney TBARSa GSTb GSH-PXc CATd SODe

21.5 ± 1.78 0.69 ± 0.068 27.3 ± 1.48 52.8 ± 7.21 32.9 ± 5.23

32.9 ± 1.66** 0.38 ± 0.059** 16.1 ± 0.99** 24.7 ± 6.59** 24.4 ± 4.71**

16.6 ± 0.96*## 0.88 ± 0.069*## 33.6 ± 2.07*## 70.9 ± 4.52**## 40.2 ± 2.99*##

22.8 ± 1.99## 0.52 ± 0.042*# 24.2 ± 1.39# 39.5 ± 5.09*# 28.8 ± 3.42#

Brain TBARSa GSTb GSH-PXc CATd SODe

29.0 ± 1.88 0.39 ± 0.040 24.7 ± 1.47 25.8 ± 6.64 23.7 ± 3.86

40.5 ± 1.47** 0.29 ± 0.018** 14.0 ± 1.41** 18.1 ± 5.88** 16.9 ± 2.65**

20.7 ± 1.21**## 0.45 ± 0.035*## 29.9 ± 1.76*## 31.9 ± 7.01*## 30.1 ± 4.45*##

31.5 ± 2.06# 0.35 ± 0.025# 22.6 ± 1.57## 23.9 ± 5.99## 22.0 ± 2.90##

Values are expressed as means ± SD; n = 10 for each treatment group. Significant difference from the control group at *P < 0.05 and **P < 0.01. Significant difference from the AlCl3-intoxicated group at #P < 0.05 and a TBARS = thiobarbituric acid reactive substances (nmol/g tissue). b GST = glutathione S-transferase (lmol/h/mg protien). c GSH-PX = glutathione peroxidase (U/mg protein). d CAT = catalase (U/mg protein). e SOD = superoxide dismutase (U/mg protein).

##

P < 0.01.

liver and kidney. Nehru and Anand (2005) also reported a significant increase in brain thiobarbituric acid reactive substances after stimulation by aluminium salts. The ionic radii of Al3+ most closely resemble those of Fe3+, therefore the appearance of Al3+ in Fe3+ sites is probable. Aluminium is known to be bound by the Fe3+ carrying protein transferrin thus reducing the binding of Fe2+. The increase in free intracellular Fe2+ causes the peroxidation of membrane lipids and thus causes membrane damage (Nehru and Anand, 2005). The increased lipid peroxidation is, at least in part, due to an inhibition of superoxide dismutase (SOD) and catalase (CAT) activities in the brain, liver and kidney (Table 1). This results in a substantial increase in the rate of phospholipids peroxidation in brain cells, leading to membrane damage and neuron death. SOD presents the first line of defence against superoxide, as it dismutases the superoxide anion to H2O2 and O2 (Nehru and Anand, 2005). Because the SOD enzyme generates H2O2, it works in collaboration with H2O2 removing enzymes. Catalase converts H2O2 to water and oxygen. Catalase is present in the peroxisomes of mammalian cells, and probably serves to destroy H2O2 generated by oxidase enzymes located within these sub cellular organelles (Nehru and Anand, 2005). Orihuela et al. (2005) reported that at high doses, aluminium was able to induce oxidative stress in the intestinal mucosa, as indicated by the significant increase in the concentration of both, oxidized glutathione/reduced glutathione (GSSG/GSH) ratio and TBARS levels. These effects may have been produced owing to concomitant causes. Aluminium might affect the glutathione (GSH) synthesis by decreasing the activity of glutathione-synthase (GS), a non-limiting step of whole reaction, thus leading to a reduced GSH content. Likewise, a slowing down in the conversion of oxidized-to-reduced form of GSH due to the inhibition of glutathione-reductase (GR) by aluminium could explain the increment in GSSG/GSH ratio. On the other hand, it has been demonstrated that aluminium is able to inhibit NADPH-generating enzymes such as

glucose 6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase. Since, NADPH is shown to be a main factor for the GSH regeneration, the decreased GSH level could be also ascribed to insufficient supply of NADPH. Besides, aluminium is able to diminish the activity of enzymes related to cell antioxidant defense, such as super oxide dismutase, catalase and GSH-peroxidase, in brain and liver. Therefore, aluminium could indirectly contribute to unbalance redox equilibrium in the enterocyte (Orihuela et al., 2005). In the present experiment, aluminium chloride-induced free radicals and inhibited the enzymes involved in antioxidant defence, that is SOD, catalase, GST and GSH-Px, which function as blockers of free radical processes. We observed a significant decrease in the enzymes in all tested tissues of treated rats. The results are in accordance with Nehru and Anand (2005) who observed a significant decrease in the activities of SOD and catalase in brain after aluminium treatment. The decrease in both enzyme activities could be the result of a reduced synthesis of the enzyme proteins as a result of higher intracellular concentrations of aluminium (Nehru and Anand, 2005). Gaskill et al. (2005) reported that releasing of transaminases (AST and ALT) and lactate dehydrogenase (LDH) from the cell cytosol can occur secondary to cellular necrosis. The activity of AST is significantly increases in such cases and escapes to the plasma from the injured hepatic cells. In addition, ALT level is of value also indicating the existence of liver diseases, as this enzyme is present in large quantities in the liver. It increases in serum when cellular degeneration or destruction occurs in this organ (Hassoun and Stohs, 1995). In the present study, the activities of AST, ALT and LDH were significantly increased in plasma (Table 2) of rats treated with aluminium chloride (AlCl3) and this is in agreement with our previous studies in rabbits (Yousef, 2004). This may be due to the leakage of these enzymes from the liver cytosol into the blood stream and/or liver dysfunction and disturbance in the biosynthesis of these enzymes with alteration in the permeability of liver membrane takes place. Also, Wilhelm et al. (1996) reported that aluminium exposure can result in aluminium accumulation in the liver and this metal can be toxic to the hepatic tissue at high concentrations. The present results revealed that aluminium chloride decreased (P < 0.05) plasma total protein and albumin, while increased glucose, urea, creatinine and bilirubin as compared to control (Table 3). The decline in plasma total protein after treatment with aluminium chloride (AlCl3) was mainly due to the decrease in albumin (Table 3). The inhibitory effect of AlCl3 on protein profile is in agreement with the finding of Yousef (2004). Although the intestine regulates the uptake of amino acids, the liver is of major importance because it regulates protein metabolism. So, the significant decrease in the concentrations of total proteins in rats treated with AlCl3 particularly the albumin could be attributed on the one hand to an under nutrition and on the other hand to a reduction of the protein synthesis in the liver (Cherroret et al., 1995). Also, their

Table 2 Changes in the activities of plasma aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) of male rats treated with aluminium chloride (AlCl3), propolis and AlCl3 + propolis. Enzyme (U/L)

AST ALT LDH

Experimental groups Control

AlCl3

Propolis

AlCl3 + propolis

39.7 ± 4.3 27.3 ± 2.11 720 ± 30.1

59.3 ± 8.96** 60.8 ± 2.51** 1124 ± 60.1**

26.9 ± 3.38*## 21.56 ± 2.05*## 692 ± 25.7*##

42.8 ± 3.72## 38.5 ± 1.77*# 823 ± 40.1*#

Values are expressed as means ± SD; n = 10 for each treatment group. Significant difference from the control group at *P < 0.05 and **P < 0.01. Significant difference from the AlCl3-intoxicated group at #P < 0.05 and

##

P < 0.01.

1096

A.-S.A. Newairy et al. / Food and Chemical Toxicology 47 (2009) 1093–1098

Table 3 Plasma biochemistry of male rats treated with aluminium chloride (AlCl3), propolis and AlCl3 + propolis. Parameter (g/dl)

Total protein (g/dl) Albumin (g/dl) Glucose (mg/dl) Urea (mg/dl) Creatinine (mg/dl) Bilirubin (mg/dl)

Experimental groups Control

AlCl3

Propolis

AlCl3 + Propolis

7.4 ± 0.36 4.4 ± 0.28 81 ± 7.16 25.1 ± 2.33 0.52 ± 0.103 0.56 ± 0.06

5.8 ± 0.34** 3.5 ± 0.22* 124 ± 5.25** 35.4 ± 2.83* 0.99 ± 0.119** 0.97 ± 0.116**

7.5 ± 0.32## 4.7 ± 0.28*# 82 ± 7.88## 23.8 ± 2.62# 0.49 ± 0.099## 0.52 ± 0.104##

7.0 ± 0.21# 4.1 ± 0.17# 88 ± 4.27## 27.2 ± 1.87# 0.74 ± 0.097*# 0.62 ± 0.123#

Values are expressed as means ± SD; n = 10 for each treatment group. Significant difference from the control group at *P < 0.05 and **P < 0.01. Significant difference from the AlCl3-intoxicated group at #P < 0.05 and

##

P < 0.01.

data indicated a very high accumulation of aluminium in the hepatic tissues of adult rats treated with aluminium. As early as 1972, Berlyne et al. (1972) observed high levels of aluminium in the hepatic tissues of rats exposed to high aluminium concentrations, leading to the proposal of a direct toxic action of aluminium ions in the liver, particularly a reduction of the protein synthesis. Also, the observed decrease in plasma proteins (Table 3) could be attributed in part to the damaging effect of AlCl3 on liver cells as confirmed by the increase in the activities of plasma AST, ALT and LDH (Table 2). The increased glucose production and decreased glucose utilization would lead to hyperglycemia. The increase in blood glucose (Table 3) may indicate disrupted carbohydrate metabolism due to enhanced breakdown of liver glycogen, possibly mediated by increase in adrenocorticotrophic and glucagon hormones and/or reduced insulin activity (Raja et al., 1992). Also, oxidative stress (OS) has been suggested as a major pathogenic link to both insulin resistance and b cell dysfunction. Oxidative stress causes structural damages to the pancreatic islets with the formation of amyloid proteins, which not only prevents the release of insulin into the circulation, but also destroys the insulin-secreting b cells (Hayden, 2002). The elevation in plasma urea and creatinine levels in AlCl3-treated rats is considered as a significant marker of renal dysfunction (Table 3), and this is supported by the finding of Mahieu et al. (2005), who reported that alterations in serum urea may be related to metabolic disturbances (e.g. renal function, cation–anion balance). In addition, Katyal et al. (1997) reported that aluminium has been implicated in the pathogenesis of several clinical disorders, such as renal dysfunction. The increase in urea concentrations in plasma of animals treated with aluminium may be due to its effect on liver function, as urea is the end-product of protein catabolism and this is confirmed by the decrease in plasma proteins (Table 3) and/or referred to liver dysfunction as proven by the increase in plasma AST, ALT and LDH activities (Table 2). The increase in plasma total bilirubin (Table 3) may result from decreased liver uptake, conjugation or increased bilirubin production from haemolysis (El-Sharaky et al., 2007). The increase in plasma total bilirubin concentrations in rats treated with AlCl3 (Table 3) are in accordance with the previous study of Yousef (2004). Thomas et al. (2004) found that the induction rate in serum bilirubin was associated with free radical production and this is in accordance with the present results (Table 1). Also, the elevation in plasma bilirubin concentration could be due to the onset of periportal necrosis. The present results also showed that the activities of AST, ALT and LDH were significantly increased in plasma (Table 2) of rats treated with AlCl3 and this is an indication to liver damage. The present data indicated that plasma total lipids, cholesterol, triglycerides and LDL-c were significantly increased by aluminium chloride (AlCl3) treatment, while HDL-c levels were decreased (Table 4) and this is in accordance with the results reported by

Yousef (2004). The increase in plasma lipids due to aluminium administration indicates a loss of membrane integrity. This was further confirmed when AlCl3 treatment resulted in a significant effect on the various membrane-bound enzymes in terms of increased activities of plasma AST, ALT and LDH (Table 2). Also, Wilhelm et al. (1996) reported that AlCl3 exposure resulted in aluminium accumulation in the liver and this may lead to disturbance of lipid metabolism and an elevation of serum cholesterol. 3.2. Propolis The present study has been conducted to evaluate the curative effect of propolis against AlCl3 induced oxidative stress and dysfunction in brain, liver, and kidney. Although certain compounds have been tested for the detoxification of aluminium (Yousef, 2004; Yousef et al., 2005, 2007), there is no previous study carried out with propolis. Some authors have underlined the occurrence of alterations in enzyme activities and TBARS levels upon the administration of propolis. Jasprica et al. (2007) reported that propolis caused reduction in TBARS levels and increase in SOD, GSH-Px, and CAT activities. Also, propolis is able to induce hepatoprotective effects on paracetamol induced liver damage in mice (Nirala et al., 2008a,b). Taken together, these findings constitute evidence that the antioxidative properties of the propolis contribute to the prevention of damage induced by AlCl3 in rats. Our results indicated an increased TBARS levels in the liver, kidney and brain in response to AlCl3 treatment, implying the increased oxidative damage to the tissues. Propolis treatments returned the increased TBARS levels back to the control levels (Table 1), this result is in accordance with the findings that propolis extract induced reduction of the increased TBARS concentrations in serum of rats treated with galactoseamine (Nirala et al., 2008a,b). In the present work, AlCl3 caused oxidative stress and consequently decreased the activities of the antioxidant enzymes (GST, GSH-PX, SOD, and CAT). After treatment of rats with AlCl3 plus propolis the activities of these antioxidant enzymes were normalized to their control values (Table 1). Jasprica et al. (2007) showed that propolis and related flavonoids exercise their activity through the scavenging of hydroxyl, superoxide free radicals, and lipid peroxides. The antioxidant activities of propolis and its polyphenolic/flavonoid components are related to their ability to chelate metal ions and scavenge singlet oxygen, superoxide anions, proxy radicals, hydroxyl radicals and peroxynitrite (Ferrali et al., 1997). Also, our previous studies (Yousef et al., 2003a,b) showed that using isoflavones in combination with cypermethrin minimized its hazardous effects and this may be attributed to the vital role of isoflavone as antioxidant. Moreover, the results of Yousef et al. (2004a,b) concluded that isoflavone dosages (2.5 or 5 mg/kg body weight), which is more than two or four times to the amounts consumed (40 mg) in many Eastern nations, have beneficial effect on plasma lipid and lipoprotein concentrations, and antioxidant

1097

A.-S.A. Newairy et al. / Food and Chemical Toxicology 47 (2009) 1093–1098 Table 4 Plasma lipid and lipoprotein profiles of male rats treated with aluminium chloride (AlCl3), propolis and AlCl3 + propolis. Lipid profile (mg/dl)

TL Cholesterol TG HDL-c LDL-c

Experimental groups Control

AlCl3

Propolis

AlCl3 + propolis

465 ± 12.4 151 ± 6.2 80.3 ± 6.29 27.4 ± 1.98 107.2 ± 7.08

626 ± 15.9** 183 ± 10.0** 107.7 ± 10.71* 19.3 ± 1.83* 142.2 ± 10.81**

403 ± 13.4*## 124 ± 5.6*## 60.9 ± 5.15*## 39.7 ± 2.11*# 89.2 ± 5.51*##

529 ± 16.6*# 155 ± 8.28## 89.0 ± 4.29*# 27.2 ± 1.75# 110.9 ± 7.23##

Values are expressed as means ± SE; n = 10 for each treatment group. Significant difference from the control group at *P < 0.05 and **P < 0.01. Significant difference from the AlCl3-intoxicated group at #P < 0.05 and ##P < 0.01. TL = total lipids, TG = triglycerides, HDL-c = high density lipoprotein-cholesterol, LDL-c = low density lipoprotein-cholesterol.

activities in rabbits. Fuliang et al. (2004) showed that propolis elevates GSH-PX, SOD, GST and catalase activities. Therefore, by increasing the activities of antioxidant enzymes, flavonoids from propolis reduce the levels of free radicals and ROS and increase the production of molecules capable of protecting against oxidative stress. Kanbura et al. (2009) found decrease in the plasma and tissue (liver, kidney and brain) malondialdehyde (MDA) levels, and increase/decrease in the antioxidant enzyme parameters (SOD, CAT, and GSH-Px) of animals that were administered propolis in association with propetamphos, in comparison to the group that was administered propetamphos alone. The primary mechanism of this effect of propolis may involve the scavenging of free radicals that cause lipid peroxidation. The other mechanism may comprise the inhibition of xanthine oxidase, which is known to cause free radicals to be generated, by propolis. Studies exist, which report xanthine oxidase to be inhibited by propolis (Harris et al., 2000). Among other studies that demonstrate the mechanisms responsible for the antiradical and antioxidant activities of propolis, in a trial conducted by Matsushige et al. (1995), propolis has been determined to exhibit antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the superoxide radical by means of xanthine/xanthine oxidase and NADH/phenazine reactions. The present data indicated that treatment with propolis alone decreased the activities of AST, ALT and LDH in plasma. The present results showed that propolis decreased the increased levels of AST, ALT and LDH in plasma of rats treated with AlCl3 (Table 2), indicating that propolis tended to prevent damage and suppressed the leakage of enzymes through cellular membranes. This result is in accordance with the findings that propolis induced reduction of the increased activity of AST and ALT concentrations in plasma of rats treated with galactoseamine (Nirala et al., 2008a,b). Treatment with AlCl3 plus propolis decreased the plasma glucose levels, compared to the rats treated with AlCl3 (Table 3). This suggests that propolis can control blood glucose and modulate the metabolism of glucose (Fuliang et al., 2004). Treatment with propolis alone did not cause any significant change in urea and creatinine levels (Table 3). Also, Sforcin et al. (2002) reported that treatment of rats with propolis does not induce kidney damage came from urea and creatinine determinations. While, treatment with AlCl3 plus propolis returned the increased levels of creatinine and urea back to the control levels (Table 3), this result is in accordance with the findings that propolis dramatically reversed the alterations induced by beryllium in all the biochemical variables, including creatinine and urea, more towards control (Nirala et al., 2008a,b). Also, propolis treatment significantly attenuated the AlCl3-mediated increase in plasma total bilirubin level (Table 3).This result is in agreement with the findings that propolis reverses acetaminophen induced bilirubin levels (Nirala and Bhadauria, 2008). This effect may be related to the antioxidant properties of propolis.

Our study showed that treatment of rats with AlCl3 plus propolis decreased total lipids, cholesterol, triglycerides and low density lipoprotein-cholesterol (LDL-c), and increased high density lipoprotein-cholesterol (HDL-c) levels compared to AlCl3-intoxicated group (Table 4). These results are in agreement with Fuliang et al. (2004), who found that oral administration of propolis significantly lowered total cholesterol, triglycerides, LDL-c, very low density lipoprotein-cholesterol (VLDL-c) in serum of rats; and to increased serum levels of HDL-c. Also, Kolankaya et al. (2002) found that propolis significantly decreased cholesterol and triglycerides. Some studies suggested that propolis can act in several ways to lower plasma LDL-bound cholesterol. First, uptake of cholesterol in the gastrointestinal tract could be inhibited; second, LDL-c could be eliminated from the blood via LDL receptor; and finally, the activity of cholesterol-degrading enzymes, namely cholesterol-7-hydroxylase could be increased. It has been suggested that propolis decreased total cholesterol and LDL-c, while increased HDL-c due to absorption, degradation or elimination of cholesterol. Moreover, other studies showed that propolis reduced cholesterol and increased HDL-c, indicating that it may be mobilizing cholesterol from extra hepatic tissues to the liver where it is catabolised (Kolankaya et al., 2002; Fuliang et al., 2004). Alves et al. (2008) reported that the hypocholesterolemic effect of propolis is the result of a direct effect on liver or an indirect effect through thyroid hormones, since thyroid hormones affect reactions in almost all the pathways of lipid metabolism. 4. Conclusion Aluminium has adverse effects on human health. Our results reported that aluminium chloride (AlCl3) capable of caused marked alterations in some biochemical parameters induced oxidative damage and inhibited the activities of antioxidant enzymes. While, propolis administered in combination with aluminium minimized its hazards. In addition, propolis alone proved to be beneficial in decreasing the levels of free radicals and lipids, and increasing the activities of the antioxidant enzymes. Consequently, the exposure to aluminium should be reduced and attention paid to sources of aluminium in foods, water and personal-care products. Furthermore, using diets rich in propolis could be beneficial in alleviating aluminium toxicity. Conflict of interest statement The authors declare that there are no conflicts of interest. References Alves, M.J.Q.F., Mesquita, F.F., Sakaguti, M., Tardivo, A.C., 2008. Hypocholesterolemic effect of propolis’ caffeic acids. Revista Brasileira de Plantas Medicinais 10, 100– 105.

1098

A.-S.A. Newairy et al. / Food and Chemical Toxicology 47 (2009) 1093–1098

Armstrong, W.D., Carr, C.W., 1964. Estimation of serum total protein. In: Physiological Chemistry Laboratory Directions, third ed. Burges Publishing Co., Minneapolis, MN, USA. Bergmenyer, H.U., 1985. Methods of Enzymatic Analysis, vol. VIII, third ed, pp. 154– 160. Berlyne, G.M., Ben Ari, J., Knopf, E., Yagil, R., Weinberger, G., Danovitch, G.M., 1972. Aluminum toxicity in rats. Lancet 1, 564–568. Carr, T., Andressen, C.J., Rudel, L.L., 1993. Enzymatic determination of triglyceride, free cholesterol and total cholesterol in tissue lipid extracts. Clin. Chem. 26, 39– 42. Cherroret, G., Capolaghi, B., Hutin, M.F., Burnel, D., Desor, D., Lehr, P.R., 1995. Effects of postnatal aluminum exposure on biological parameters in the rat plasma. Toxicol. Lett. 78, 119–125. Chiu, D.T.Y., Stults, F.H., Tappel, A.L., 1976. Purification and properties of rat lung soluble glutathione peroxidase. Biochim. Biophys. Acta 445, 558–566. Domingo, J.L., 2006. Aluminium and other metals in Alzheimer’s disease: a review of potential therapy with chelating agents. J. Alzheimer Dis. 10, 331–341. Doumas, B.T., Watson, W.A., Biggs, H.G., 1977. Albumin standards and the measurement of serum albumin with bromocresol green. Clin. Chem. Acta 31, 87–96. El-Sharaky, A.S., Newairy, A.A., Badreldeen, M.M., Eweda, S.M., Sheweita, S.A., 2007. Protective role of selenium against renal toxicity induced by cadmium in rats. Toxicology 235, 185–193. Esterbauer, H., Cheeseman, K.H., 1990. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 186, 407–421. Exley, C., 2004. The pro-oxidant activity of aluminum. Free Rad. Biol. Med. 36, 380– 387. Ferrali, M., Signorini, C., Caciotti, B., 1997. Protection against oxidative damage of erythrocytes membrane by the flavinoid quercetin and its relation to iron chelating activity. FEBS Lett. 416, 123–129. Fuliang, H.U., Hepburn, H.R., Xuan, H., Chen, M., Daya, S., Radloff, S.E., 2004. Effects of propolis on blood glucose, blood lipid and free radicals in rats with diabetes mellitus. Pharmacol. Res. 104, 147–152. Gaskill, C.L., Miller, L.M., Mattoon, J.S., Hoffmann, W.E., Burton, S.A., Gelens, H.C.J., Ihle, S.L., Miller, J.B., Shaw, D.H., Cribb, A.E., 2005. Liver histopathology and liver serum alanine aminotransferase and alkaline phosphatase activities in epileptic dogs receiving phenobarbital. Vet. Pathol. 42, 147–160. Ghisalberti, E.L., 1979. Propolis: A review. Bee World 60, 59–84. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130– 7139. Harris, S.R., Panaro, N.J., Thorgeirsson, U.P., 2000. Oxidative stress contributes to the anti-proliferative effects of flavone’s acetic acid on endothelial cells. Anticancer Res. 20, 2249–2254. Hassoun, E.A., Stohs, S.J., 1995. Comparative studies on oxidative stress as a mechanism for the fetotoxic of TCDD, endrin and lindane in C57BL/6J and DBA/ 2J mice. Teratology 51, 186–192. Hayden, M.R., 2002. Islet myeloid, metabolic syndrome and the natural progressive history of type 2 diabetes mellitus. J. Orthod. Pract. 3, 126–138. Henry, R.J., Cannon, D.C., Winkelman, W., 1974. Clinical Chemistry Principals and Techniques, 11th ed. Happer and Row Publishers. 1629 pp. Jasprica, D., Mornar, A., Debelijak, Z., Smolcic-Bubalo, A., Medic- Saric, M., Mayer, L., Romic, Z., Bucan, K., Balog, T., Sobocanec, S., Sverko, V., 2007. In vivo study of propolis supplementation effects on antioxidative status and red blood cells. J. Ethnopharmacol. 110, 548–554. Kanbura, M., Eraslan, G., Silici, S., 2009. Antioxidant effect of propolis against exposure to propetamphos in rats. Ecotoxicol. Environ. Safety 72, 909–915. Katyal, R., Desigan, B., Sodhi, C.P., Ojha, S., 1997. Oral aluminium administration and oxidative injury. Biol. Trace Elem. Res. 57, 125–130. Knight, J.A., Anderson, S., Rawle, J.M., 1972. Chemical basis of the sulphaphosphovanillin reaction estimating total serum lipids. Clin. Chem. 18, 199– 202. Kolankaya, D., Selmano, G., Sorkun, K., Bekir, S., 2002. Protective effects of Turkish propolis on alcohol-induced serum lipid changes and liver injury in male rats. Food Chem. 87, 213–217. Mahieu, S., Millen, N., Gonzalez, M., Carmen Contini, M.D., Elias, M.M., 2005. Alterations of the renal function and oxidative stress in renal tissue from rats chronically treated with aluminium during the initial phase of hepatic regeneration. J. Inorg. Biochem. 99, 1858–1864. Martinek, R.G., 1972. A rapid ultraviolet spectrophotometric lactic dehydrogenase assay. Clin. Chem. Acta 40, 91–99. Matsushige, K.I., Kusumoto, T., Yamamoto, Y., Kadota, S., Namba, T., 1995. Quality evaluation of propolis. 1. A comparative study on radical scavenging effects of propolis and vespae nidus. J. Trad. Med. 12, 45–53. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170–3175.

Nehru, B., Anand, P., 2005. Oxidative damage following chronic aluminium exposure in adult and pup rat brains. J. Trace Elem. Med. Biol. 19, 203–208. Nieva Moreno, M.I., Isla, M.I., Sampietro, A.R., Vattuone, M.A., 2000. Comparison of the free radical – scavenging activity of propolis from several regions of Argentine. J. Ethnopharmacol. 71, 109–114. Nirala, S.K., Bhadauria, M., Shukla, S., Agrawal, O.P., Mathur, A., Li, P.Q., Mathur, R., 2008a. Pharmacological intervention of tiferron and propolis to alleviate beryllium-induced hepatorenal toxicity. Fundam. Clin. Pharmacol. 22, 403–415. Nirala, S.K., Bhadauria, M., 2008. Propolis reverses acetaminophen induced acute hepatorenal alterations: a biochemical and histopathological approach. Arch. Pharmacol. Res. 31, 451–461. Nirala, S.K., Bhadauria, M., Shukla, S., Agrawal, O.P., Mathur, A., Li, P.Q., Mathur, R., 2008b. Pharmacological intervention of tiferron and propolis to alleviate beryllium-induced hepatorenal toxicity. Fundam. Clin. Pharmacol. 22, 403–415. Ochmanski, W., Barabasz, W., 2000. Aluminium-occurrence and toxicity for organisms. Przegl. Lek. 57, 665–668. Orihuela, D., Meichtry, V., Pregi, N., Pizarro, M., 2005. Short-term oral exposure to aluminium decreases glutathione intestinal levels and changes enzyme activities involved in its metabolism. J. Inorg. Biochem. 99, 1871–1878. Padmavathi, R., Senthilnathan, P., Chodon, D., Sakthisekaran, D., 2006. Therapeutic effect of paclitaxel and propolis on lipid peroxidation and antioxidant system in 7, 12 dimethyl benz (a) anthracene-induced breast cancer in female Sprague Dawley rats. Life Sci. 78, 2820–2825. Patton, C.J., Crouch, S.R., 1977. Spectrophotometric and kinetics investigation of the Berthelot reaction for determination of ammonia. Anal. Chem. 49, 464–469. Paulino, N., Abreu, S.R., Uto, Y., Koyama, D., Nagasawa, H., Hori, H., Dirsch, V.M., Vollmar, A.M., Scremin, A., Bretz, W.A., 2008. Anti-inflammatory effects of a bio available compound, Artepillin C, in Brazilian propolis. Eur. J. Pharmacol. 587, 296–301. Pearlman, F.C., Lee, R.T.Y., 1974. Detection and measurement of total bilirubin in serum, with use of surfactants as solubilizing agents. Clin. Chem. 20, 447–453. Raja, M., Al-Fatah, A., Ali, M., Afzal, M., Hassan, R.A., Menon, M., Dhami, M.S., 1992. Modification of liver and serum enzymes by paraquat treatment in rabbits. Drug Metab. Drug Inter. 10, 279–291. Sforcin, J.M., Novell, E.L.B., Funarl, S.R.C., 2002. Seasonal effect of Brazilian propolis on seric biochemical variables. J. Venom. Anim. Toxins 8, 1–6. Strong, M.J., Garruto, R.M., Joshi, J.G., Mundy, W.R., Shafer, T.J., 1996. Can the mechanisms of aluminum neurotoxicity be integrated into a unified scheme? J. Toxicol. Environ. Health 48, 599–613. Thomas, W., Sedlak, M.D., Solomon, H., Snyder, M.D., 2004. Bilirubin benefits: Cellular protection by a biliverdin reductase antioxidant cycle. Paediatrics 113, 1776–1782. Warnick, G.R., Benderson, V., Albers, N., 1983. Selected methods. Clin. Chem. 10, 91– 99. Wilhelm, M., Jaeger, D.E., Schull-Cablitz, H., Hafner, D., Idel, H., 1996. Hepatic clearance and retention of aluminium: studies in the isolated per fused rat liver. Toxicol. Lett. 89, 257–263. Xie, C.X., Yokel, R.A., 1996. Aluminum facilitation of iron mediated lipid per oxidation is dependent on substrate, pH and aluminum and iron concentrations. Arch. Biochem. Biophys. 327, 222–226. Xu, J.B., Yuan, X.F., Lang, P.Z., 1997. Determination of catalase activity and catalase inhibition by ultraviolet spectrophotometry. Chinese Environ. Chem. 16, 73–76. Yokel, R.A., 2000. The toxicology of aluminum in the brain: a review. Neurotoxicology 21, 813–828. Yousef, M.I., 2004. Aluminum-induced changes in hemato-biochemical parameters, lipid per oxidation and enzyme activities of male rabbits: Protective role of ascorbic acid. Toxicology 199, 47–57. Yousef, M.I., El-Demerdash, F.M., Al-Salhen, K.S., 2003a. Protective role of isoflavones against the toxic effect of cypermethrin on semen quality and testosterone levels of rabbits. J. Environ. Sci. Health Part B 38, 463–478. Yousef, M.I., El-Demerdash, F.M., Kamel, K.I., Al-Salhen, K.S., 2003b. Changes in some haematological and biochemical indices of rabbits induced by isoflavones and cypermethrin. Toxicology 189, 223–234. Yousef, M.I., El-Morsy, A.M.A., Hassan, M.E., 2005. Aluminium-induced deterioration in reproductive performance and seminal plasma biochemistry of male rabbits: Protective role of ascorbic acid. Toxicology 215, 97–107. Yousef, M.I., Esmail, A.M., Baghdadi, H.H., 2004a. Effect of isoflavones on reproductive performance, testosterone levels, lipid per oxidation and seminal plasma biochemistry of male rabbits. J. Environ. Sci. Health Part B 39, 819–833. Yousef, M.I., Kamel, I.K., El-Guendi, I.E., El-Demerdash, F.M., 2007. An in vitro study on reproductive toxicity of aluminium chloride on rabbit sperm: the protective role of some antioxidants. Toxicology 239, 213–223. Yousef, M.I., Kamel, I.K., Esmail, A.M., Baghdadi, H.H., 2004b. Antioxidant activities and lipid lowering effects of isoflavone in male rabbits. Food Chem. Toxicol. 42, 1497–1503. Zafar, T.A., Teegarden, D., Ashendel, C., Dunn, M.A., Weaver, C.M., 2004. Aluminium negatively impacts calcium utilization and bone in calcium-deficient rats. Nutr. Res. 24, 234–259.