The Antioxidant Effects of Pumpkin Seed Oil on Subacute Aflatoxin Poisoning in Mice ¨ znur Aslan,2 Mu¨rsel Karabacak3 Go¨khan Eraslan,1 Murat Kanbur,1 O 1

Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Erciyes University, Kayseri, Turkey 2

Department of Internal Medicine, Faculty of Veterinary Medicine, Erciyes University, Kayseri, Turkey

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Department of Animal Health, Safiye C¸ıkrıkc¸ıog˘lu Vocational Collage, Erciyes University, Kayseri, Turkey

Received 9 December 2010; revised 27 June 2011; accepted 10 July 2011 ABSTRACT: This study was aimed at the investigation of the antioxidant effect of pumpkin seed oil against the oxidative stress-inducing potential of aflatoxin. For this purpose, 48 male BALB/c mice were used. Four groups, each comprising 12 mice, were established. Group 1 was maintained as the control group. Group 2 was administered with pumpkin seed oil alone at a dose of 1.5 mL/kg.bw/day (1375mg/kg.bw/day). Group 3 received aflatoxin (82.45% AFB1, 10.65% AFB2, 4.13% AFG1, and 2.77% AFG2) alone at a dose of 625 lg/kg.bw/day. Finally, group 4 was given both 1.5 mL/kg.bw/day pumpkin seed oil and 625 lg/kg.bw/day aflatoxin. All administrations were oral, performed with the aid of a gastric tube and continued for a period of 21 days. At the end of day 21, the liver, lungs, kidneys, brain, heart, and spleen of the animals were excised, and the extirpated tissues were homogenized appropriately. Malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were determined in tissue homogenates. In conclusion, it was determined that aflatoxin exhibited adverse effects on most of the oxidative stress markers. The administration of pumpkin seed oil diminished aflatoxin-induced adverse effects. In other words, the values of the group, which was administered with both aflatoxin and pumpkin seed oil, were observed to have drawn closer to the values of the control group. # 2011 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2011. Keywords: aflatoxin; pumpkin seed oil; antioxidant effect; mice

INTRODUCTION Aflatoxins (AF) are highly active organic compounds, belonging to the group of furocoumarolactones, which are composed of a bifuran ring and a 6-membered lactone ring (Dalvi, 1986; Betina, 1989). These toxins are mainly produced by the fungi Aspergillus flavus and Aspergillus paraCorrespondence to: G. Eraslan; e-mail: [email protected] This study was given as a poster presentation at the Third National Veterinary Pharmacology and Toxicology Congress, Aydın, Turkey. Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/tox.20763

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siticus (Gugnani, 2003). Of these species, the first produces AFB1 and B2 and the latter AFB1, B2, G1 and G2. Several factors, including the quality of the product, humidity content, pH value, oxygen and carbon dioxide concentrations and optimum temperature affect aflatoxin synthesis. The most toxic aflatoxin is known as AFB1 (Paster and Bullerman, 1988; Betina, 1989; Dutta and Das, 2001; Bra¨se et al., 2009). Aflatoxins have strong hepatotoxic, immunosuppressive, carcinogenic, mutagenic and terratogenic effects (Dalvi, 1986; Betina, 1989; Farombi et al., 2005; Ortatatli et al., 2005; Paterson and Lima, 2010). The toxic effects of aflatoxins are closely related to the metabolites AFB1-2,3epoxide and AFB1-8,9 epoxide, which are generated

2011 Wiley Periodicals, Inc.

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through the mediation of cytochrome P450 in the liver. These metabolites are also responsible for the carcinogenicity of aflatoxins. Epoxide derivatives bind to hepatic DNA, cytoplasmic steroid hormone receptors and enzymes by covalent bonds (Kiessling, 1986; Verma et al., 2001; Farombi et al., 2005; Paterson and Lima, 2010). Furthermore, aflatoxins induce lipid peroxidation by means of their effect on both enzymatic and nonenzymatic antioxidants, and thereby, lead to oxidative stress (Shen et al., 1994; Rastogi et al., 2001; Verma and Mathuria, 2008). In fact, mainly AFB1-8,9 epoxide derivatives are responsible for lipid peroxidation and exhibit their effects through different mechanisms. The free radicals generated by aflatoxins affect the enzymatic and nonenzymatic antioxidant systems by means of different mechanisms (Shen et al., 1994, 1995; Verma and Nair, 2001; Verma, 2004). It is known that free radicals generated by aflatoxins are capable of affecting both systems. On the other words, aflatoxin poisoning not only leads to the consumption of certain components (i.e., vitamins and glutathione) of the nonenzymatic defense system, as a result of the effort to eliminate the high level of free radicals generated (Betina, 1989; Beers et al., 1992; Verma et al., 2001; Pimpukdee et al., 2004) but also alters the activity of antioxidant enzymes involved in the enzymatic system (Souza et al., 1999; Verma and Nair, 2001; Choudhary and Verma, 2005; Verma and Mathuria, 2008). Lipid peroxidation is further aggravated by reduced serum Vitamin A and glutathione levels, resulting from the hepatotoxic effect of aflatoxin as well as by its inhibitory effect on protein synthesis (Betina, 1989; Rastogi et al., 2001; Verma et al., 2001; Verma and Mathuria, 2008). The pumpkin (Cucurbita spp.) is known in Europe since the 16th century, and in the course of time, has become the traditional food of southern Austria. Pumpkin seed oil, which is of dark green color, is pleasant-tasting and has a walnut-hazelnut-like flavor. Pharmacodynamically, pumpkin seed oil contains active substances of active nontriacylglycerol origin. Pumpkin seed, if not heat-treated, contains dietary lignans, including secoisolariciresinol, isolariciresinol, and lariciresinol (Siegmund and Murkovic´, 2004; Butinar et al., 2010). The oil content of pumpkin seeds ranges from 42 to 54%. The fatty acid composition of the seeds is mainly made up of palmitic acid (9.5–14.5%), stearic acid (3.1–7.4%), oleic acid (21.0–46.9%), and linoleic acid (35.6–60.8%). These four fatty acids make up  98% of the fatty acid content of pumpkin seeds. The individual percentage of other fatty acids does not exceed 0.5%. Pumpkin seed is rich in vitamin E, and contains high levels of a- and c-tocoferol (Murkovic´ et al., 2004; Stevenson et al., 2007). Although lower than the level found in pumpkin seeds, pumpkin seed oil also contains a high level of c-tocoferol. In addition it also contains a high level of b carotene. The sodium, calcium, magnesium, potassium and phosphor content of pumpkin seed oil are greater than that of pumpkin seeds (Fruhwirth and Hermetter, 2007). Pumpkin seed oil is

Environmental Toxicology DOI 10.1002/tox

also rich in selenium and lutein, and contains phenolic compounds such as tyrosol, vanillic acid, vanillin, ferulic acid, and luteolin (Al-Zuhair et al., 1997; Andjelkovic et al., 2010). As regards its phytosterol content, pumpkin seed oil contains a greater amount of D7-phytosterol, compared to D5-phytosterol (Fruhwirth and Hermetter, 2007). When compared to other kinds of seed oil, pumpkin seed oil contains a higher level of squalene (Nyam et al., 2009). The aim of this study was, first, to investigate the possible effects of aflatoxin on oxidative stress markers in the tissues investigated (liver, kidney, lung, brain, heart, and spleen), when administered at a dose of 625 lg/kg.bw for a period of 21 days. Most importantly, the study aimed to demonstrate the effect of pumpkin seed oil, when administered alone and together with aflatoxin, on oxidative stress parameters in the abovementioned tissues. Thereby, it was aimed to assess whether pumpkin seed oil has any ameliorative effect on aflatoxin-induced lipid peroxidation, thus, on oxidative stress.

MATERIALS AND METHODS Animal Material Forty-eight male 6–8-week-old BALB/c mice were used in the study. The animals were randomly allocated to 4 groups, each comprising 12 mice. The first group was maintained as the control group. Group 2 received pumpkin seed 1 oil (Cucurbita pepo, Arkopharma ) alone at a dose of 1.5 mL/kg.bw/day (1375mg/kg.bw/day). Group 3 was administered with an aflatoxin dose of 625 lg/kg.bw/day in  0.05 mL of vehicle (10% dimethylsulfoxide in water). Finally, group 4 received aflatoxin at a dose of 625 lg/ kg.bw/day and pumpkin seed oil at a dose of 1.5 mL/ kg.bw/day. Aflatoxin and pumpkin seed oil were given directly into the stomach by means of a gastric tube. While aflatoxin was administered in the morning, pumpkin seed oil was administered the same day,  6 h after the administration of aflatoxin. Administrations were continued for a period of 21 days, and all of the animals included in all of the groups were provided with ad libitum commercial mice pellet feed and drinking water. Animals included in the control group, apart from feed and water, were given 0.05 mL of vehicle each day. The animals were housed at a fixed temperature of 22–248C and were grown on a light regime of 12 h light:12 h dark. The protocol of the present study was approved by the Ethics Board for Experimental Animals of Erciyes University.

Aflatoxin Production Aflatoxin was produced in rice using the Aspergillus parasiticus (NRLL 2999) strain in accordance with the modified method of Demet el al. (1995) based on the protocol

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described by Shotwell et al. (1966). The level of total aflatoxin produced in rice was measured using an R-Biopharm Ridascreen1 total aflatoxin kit, in accordance with the manufacturer’s instructions (Anon, 2009). The extraction/ purification of aflatoxin from ground rice and type analysis were performed as described by Robert and Patterson (1975) and Nabney and Nesbitt (1965), respectively. After aflatoxin was extracted and purified, the organic solvent in which it was solubilized was evaporated. Subsequently, aflatoxin was solubilized in dimethylsulfoxide/water. Type analysis revealed that the total aflatoxin was composed of AFB1, AFB2, AFG1, and AFG2 at rates of 82.45%, 10.65% 4.13%, and 2.77%, respectively.

Biochemical Analyses At the end of the trial period, the animals were sacrificed under light ether anesthesia and their liver, kidneys, lungs, brain, heart, and spleen were excised. The internal organs were cleansed from blood in cold distilled water. Later, fat and connective tissue were removed. The extirpated tissues were homogenized in phosphate buffer with the aid of a homogenizer. The supernatant obtained by the centrifugation of tissue homogenates in an ultracentrifuge was used for biochemical analyses. MDA analyses were performed as described by Ohkawa et al. (1978). SOD activity was measured in accordance with the method described by Sun et al. (1988). CAT activity was assessed in compliance with the method of Luck (1965). GSH-Px activity was determined as described by Paglia and Valentine (1967). The protein level of the tissue homogenates was determined according to the modified method of Miller (1959), based on the method described by Lowry et al. (1951). All analyses were performed with minor modifications. Results were expressed in nmol/mg-protein for MDA levels, U/mg-protein for SOD activity, k/g-protein for CAT activity and lmol NADPH1H1/min/g-protein for GSH-Px activity.

Statistical Analysis Calculations were made using the SPSS 13.0 for Windows software. The data obtained was given in arithmetic means and standard deviations. The significance of the differences between the groups was assessed by one-way analysis of variance. The individual assessment of different groups was performed based on P \ 0.05 significance by Duncan’s Multiple Range test.

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Fig. 1. Tissue MDA levels treated with pumpkin seed oil and aflatoxin in mice. (a–c) Data within the united bars are statistically significant (P \ 0.05), if they do not share the same letters. Group 1, control; group 2, pumpkin seed oil; group 3, aflatoxin; group 4, aflatoxin plus pumpkin seed oil.

tered with aflatoxin alone, values tended to be higher than those of the control group, and the MDA levels of all tissues differed significantly from those of the control mice. In the groups, which were given both aflatoxin and pumpkin seed oil, it was observed that values had drawn closer to those of the control group, yet, compared to the controls, the differences were statistically insignificant. On the contrary, when compared with the values of the group, which received aflatoxin alone, the MDA levels of the liver, lung, kidney and spleen tissue differed significantly (Fig. 1).

Tissue SOD Activity In the group, which was given pumpkin seed oil alone, the values for all tissues, excluding lung tissue, were found to be close to those of the control group. It was ascertained that, in the lung tissue, SOD activity was statistically greater than that of the control mice. In the group, which was administered with aflatoxin alone, the values measured for all tissues, excluding lung tissue, had altered significantly, such that the activity of the enzyme decreased in comparison to the control group. The values of the group, which received both aflatoxin and pumpkin seed oil, had incresed, yet, only the values for the lung tissue differed significantly from that of the control group. The values determined in the group, which was administered with aflatoxin alone, differed significantly from the values of the group, which received both pumpkin seed oil and aflatoxin (Fig. 2).

RESULTS Tissue CAT Activity Tissue MDA Levels The tissue MDA levels of the group, which received pumpkin seed oil alone, did not differ significantly from the values of the control group. In the group, which was adminis-

While liver CAT activity did not differ significantly between the groups, statistically significant differences were determined to exist between the groups for CAT activity in the other tissues examined. In the group, which

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decreased in liver tissue, and had increased significantly in lung and heart tissue. GSH-Px activity had decreased significantly in liver, lung, kidney, heart, and spleen tissue in the group given aflatoxin alone. In the group, which was administered with both aflatoxin and pumpkin seed oil, the alterations in GSH-Px activity were observed in the form of increase/decrease, and when compared with the control group, liver, lung, heart, and spleen tissue activities differed significantly. Furthermore, the group administered with both compounds and the group given aflatoxin alone differed from each other significantly for lung, kidney, brain, and heart tissue activities (Fig. 4). Fig. 2. Tissue SOD activities treated with pumpkin seed oil and aflatoxin in mice. (a,b) Data within the united bars are statistically significant (P \ 0.05), if they do not share the same letters.

DISCUSSION

Compared to the control group, in the group administered with pumpkin seed oil alone, enzyme activity had

Lipid peroxidation is described as the peroxidation of unsaturated fatty acids of the cell membrane and organelles by free radicals. Peroxidation is observed as damage to protein and lipid distribution, reduced resistance, and alteration or disruption of the selective permeability of the cell membrane. All of these developments alter intracellular and extracellular ion concentrations and disrupt the function of transporter proteins found in the cell membrane (Esterbauer, 1996; Evans and Halliwell, 2001; Halliwell et al., 2007; Ogino and Wang, 2007; Niki, 2009). Several methods have been developed for the determination of lipid peroxidation products. The most commonly used method is the measurement of MDA levels in biological fluids and tissues (Janero, 1990; Esterbauer, 1996; Mercan, 2004; Michel et al., 2008). Enzymes, including SOD, CAT and GSH-Px, which compose the enzymatic antioxidant defense system of the cell, play an important role in the conversion of free radicals into less harmful or harmless compounds. They are also considered among the major indicators of the course of oxidative stress (Halliwell, 1999; Gutterridge and Halliwell, 2000; Fang et al., 2002; Halliwell et al., 2007). Although a number of studies are available on the oxidative

Fig. 3. Tissue CAT activities treated with pumpkin seed oil and aflatoxin in mice. (a–c) Data within the united bars are statistically significant (P \ 0.05), if they do not share the same letters.

Fig. 4. Tissue GSH-Px activities treated with pumpkin seed oil and aflatoxin in mice. (a–c) Data within the united bars are statistically significant (P \ 0.05), if they do not share the same letters.

received pumpkin seed oil alone, only the CAT activity of brain tissue differed significantly from that of the control group, and this difference was observed in the form of decrease. In comparison to the controls, in the group administered with aflatoxin alone, activities had significantly increased in lung, kidney, heart and spleen tissue, while brain CAT activity had decreased significantly. In the group, which was given both aflatoxin and pumpkin seed oil, it was determined that the values had drawn closer to those of the control group with statistical significant differences observed for only lung, brain and heart tissue. As regards the differences between the group administered with both aflatoxin and pumpkin seed oil and the group, which received aflatoxin alone, statistical significance was determined for only kidney and heart tissue (Fig. 3).

Tissue GSH-Px Activity

Environmental Toxicology DOI 10.1002/tox

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stress-inducing potential of aflatoxin and its detoxifications in various animal species (Eraslan et al., 2005b; Adedara et al., 2010; El-Agamy, 2010; Kanbur et al., 2011), to the authors’ knowledge, the use of pumpkin seed oil for the treatment of aflatoxicosis has not been investigated before. Therefore, it is considered that the present study bears originality in that it provides data on a previously untouched issue.

Pumpkin Seed Oil The tissue MDA levels of the trial group administered with pumpkin seed oil alone not differing significantly from the values of the control group, suggests that, when administered at the indicated dose for the indicated time period, pumpkin seed oil does not have any adverse effect on the antioxidant defense system. On the other hand, despite the determination of lung SOD activity having increased, brain CAT activity having decreased and liver GSH-Px activity having significantly decreased and increased significantly in lung and heart tissue, the MDA levels of these tissues not having increased significantly suggests that pumpkin seed oil did not induce any adverse effect on the antioxidant defense system in these tissues. It is considered that, the alterations observed in the activities of the indicated enzymes may be related to the composition of pumpkin seed oil, thus, it is suggested that this oil may have caused physiological alterations in the activity of these enzymes due to its potential of eliminating free radicals generated under normal biological conditions. In relevant research (Al-Zuhair et al., 2000; Nkosi et al., 2006); it has also been reported that pumpkin seed oil/extract/isolate alters the some antioxidant enzyme activity.

Aflatoxin It was observed that, at the dose administered throughout the trial, aflatoxin affected all tissues significantly. The level of MDA, which is an end-product of lipid peroxidation (Janero, 1990; Halliwell, 2007), had increased significantly in all of the tissues examined in the group administered with aflatoxin alone, when compared with the control group. This increase developed most rapidly in liver tissue. Alterations in other tissues displayed similarity. The literature knowledge that aflatoxin causes damage primarily to liver tissue (Betina, 1989; Bra¨se et al., 2009; Kensler et al., 2011), further supports this finding. The significant increase determined in MDA levels is the result of aflatoxin having caused the generation of a high level of free radicals and these free radicals having not been able to be compensated for by the cellular defense system. When not able to be compensated for, free radicals cause the peroxidation of the lipids in the cell membrane, and thereby, lead to the elevation of MDA levels. On the other hand, a second effect of aflatoxin, as previously explained in detail, is the increased

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risk of cell membranes being exposed to the adverse effects of free radicals due to the disruption and major consumption of the components of the nonenzymatic cellular defense system, particularly vitamin A (Evans and Halliwell, 2001; Pimpukdee et al., 2004) and glutathione (Beers et al., 1992; Evans and Halliwell, 2001). The inhibitory effect of aflatoxin on protein synthesis (Betina, 1989; Bra¨se et al., 2009) may cause decrease in the level of transport proteins, particularly those responsible for the transport of certain metals such as copper and iron. This may bring about an increase in the level of the free metal ion, which mediate the Fenton and Haber-Weiss reactions (Kehrer, 2000; Caro and Cederbaum, 2004; Halliwell, 2007). As this accelerates the lipid peroxidation reaction, it is among the reasons of the significant increase observed in MDA levels. The results obtained in the present study for MDA levels have demonstrated that tissues are not affected by aflatoxin at the same degree. For, the rate of alterations observed in MDA levels in comparison to the control group varied. Individually, aflatoxin and/or its metabolites may have also been involved in the increase in MDA levels as they directly affect lipid membranes. As regards the enzymes assessed, the alterations observed in their tissue activities were not uniform. Alterations were bidirectional, in other words, in the form of either increase or decrease. Alterations in the form of decrease can be explained by the consumption of these enzymes during the conversion of free radicals into less harmful or harmless metabolites and/or by the individual effects of aflatoxin and its metabolites. Decrease in GSH-Px activity may have arisen particularly from the aflatoxin-induced alteration of the turnover of glutathione (Beers et al., 1992), which is found in the structure of the enzyme (Caro and Cederbaum, 2004). On the other hand, increase in enzyme activities may be explained as the result of the cellular induction mechanism for the synthesis of a greater amount of enzymes aimed at compensating for the oxidative attack. The alterations observed in enzyme activities were not tissue-specific and the type and degree of the alterations observed in the different tissues were similar. However, based on the significant increase observed in MDA levels, it was clearly understood that, whatever the mechanism of cellular defense was, cell membranes were not able to be protected from peroxidation. In similar studies conducted in mice (Verma and Nair, 2001; Choudhary and Verma, 2005, 2006; Verma and Mathuria, 2008; Adedara et al., 2010; Choi et al., 2010; Kanbur et al., 2011) and other species (Eraslan et al., 2005a, 2005b; Yener et al., 2009), it was also demonstrated that aflatoxin caused significant alterations in the parameters investigated.

Aflatoxin and Pumpkin Seed Oil The MDA levels of the group, which was administered with both aflatoxin and pumpkin seed oil, not significantly

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differing from those of the control group, and in fact the values having drawn closer to those of the control mice suggested that pumpkin seed oil exhibited antiradical and/ or antioxidant effect. Several compounds found in the composition of pumpkin seed oil may induce such effects. Pumpkin seed oil is quite rich in vitamins E and b carotene (Murkovic´ et al., 2004; Stevenson et al., 2007; Fruhwirth and Hermetter, 2007). It is known that, vitamins A and E bind to free radicals and prevent the oxidation of the cell membrane (Zada´k et al., 2009). Their binding to free radicals generated by aflatoxin also prevents the occurrence of lipid peroxidation. Selenium, which is found in the composition of pumpkin seed oil (Al-Zuhair et al., 1997), is incorporated into the structure of the enzyme GSH-Px, and is directly related to its antioxidant activity (Kaya, 2007; Zada´k et al., 2009). This enzyme is involved in the conversion of the hydrogen peroxide generated, into harmless compounds (Esterbauer, 1996; Halliwell et al., 1999, 2007). Selenium and vitamin E have synergistic action, and are also considered to contribute to antioxidant effect in this respect (Fang et al., 2002). The richness of pumpkin seed oil in phenolic compounds is considered among its mechanisms that prevent lipid peroxidation, as these compounds, characteristically, bind strongly to free radicals (Fruhwirth and Hermetter, 2007; Andjelkovic et al., 2010). The potential of phenolic compounds binding to metals, such as iron and copper, which are involved in free radical-generating reactions such as the Haber-Weiss and Fenton reactions, is among the several mechanisms that prevent free radical generation (Senevirathne et al., 2006; Letelier et al., 2010). Furthermore, phytosterols are also reported to have antioxidant effect (Fruhwirth and Hermetter, 2007). Therefore, it is considered that these compounds are involved in the prevention of severe damage to the cell membrane by free radicals. The phytosterols also found in the structure of pumpkin seed oil decrease LDL-cholesterol (Nyam et al., 2009). This effect may also reduce the lipid peroxidation-inducing potential of aflatoxin. Apart from phenols and vitamins, another compound, which protects body lipids containing unsaturated fatty acids as well as the lipid components of cells and organelles from oxidation, is squalene. This compound contains a high level of unsaturated aliphatic hydrocarbons, exhibits its antioxidant effect against unsaturated fatty acids, and is the metabolic precursor of cholesterol (Ryan et al., 2007; Nyam et al., 2009). This compound is considered to be involved in the antioxidant defense system as it protects lipid membranes against the damage of free radicals. As pumpkin seed extract has inhibitory effect on lipid peroxidation catalyzed by lipoxygenase (Xanthopoulou et al., 2009), it is considered that essential fatty acids may also partly contribute to antioxidant efficacy. Furthermore, the inhibitory effect of these fatty acids on the cyclooxygenase pathway (Xanthopoulou et al., 2009), may contribute indirectly to the antioxidant defense system. By means of these mecha-

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nisms, which have been explained in detail, free radicals can be decreased generation or bound. The decrease observed in MDA levels in the present study may have arisen from these mechanisms. MDA levels did not decrease at the same rate in all tissues. The levels measured in spleen tissue were closer to the levels of the control group, when compared to the other tissues investigated. A similar trend was observed in the values measured in the lungs, kidneys and heart. Antioxidant enzyme activities of this group also displayed increase/decrease, and in general, parameters were observed to have drawn closer to the values of the control group. This was attributed to the decrease in the level of free radicals in the biological systems. Although detoxification methods applied in the case of aflatoxicosis have been previously studied (Choi et al., 2010; Kanbur et al., 2011), to the authors’ knowledge, the use of pumpkin seed oil in such cases has not been investigated before. Therefore, the results obtained in the present study were not able to be compared. Similar results were reported in previous studies conducted on the use of different antioxidant compounds in aflatoxicosis in mice (Verma and Nair, 2001; Choudhary and Verma, 2005, 2006; Mathuria and Verma, 2007; Verma and Mathuria, 2008; Choi et al., 2010; Kanbur et al., 2011) and other animal species (Eraslan 2005b; Preetha et al., 2006 Ozen et al., 2009; Yener et al., 2009; ElAgamy, 2010). Furthermore, literature reports are available on the antioxidant effect of pumpkin seed oil in rats (Al-Zuhair et al., 2000; Nkosi et al., 2006).

CONCLUSION The data obtained in the present study demonstrated that, when administered at the indicated dose for the indicated time period, aflatoxin caused severe oxidative damage in all of the tissues examined. Pumpkin seed oil, which was administered with an aim to ameliorate and diminish oxidative damage, was proven to alleviate aflatoxin-induced lipid peroxidation. The ameliorative effect of pumpkin seed oil being similar in all of the tissues examined, demonstrated that it exhibited a systemic effect. When administered alone at the indicated dose for the indicated time period, pumpkin seed oil did not induce any adverse effect on the cellular antioxidant defense system. Therefore, it is considered that pumpkin seed oil can be used for prophylaxis in cases associated with the risk of aflatoxin poisoning. It may be preferred to supplement the daily diet with pumpkin seed oil, as a food additive. Furthermore, pumpkin seed oil can be used in combination with medicinal products for the treatment of aflatoxicosis or for the amelioration of aflatoxin-induced adverse effects. However, attention should be paid to the duration and dose of administration in order to reduce the risk of any harm to the physiological system.

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REFERENCES Adedara IA, Owumi SE, Uwaifo AO, Farombi EO. 2010. Aflatoxin B1 and ethanol co-exposure induces hepatic oxidative damage in mice. Toxicol Ind Health 26:717–724. Al-Zuhair H, Abd EL-Fattah AA, Abd El-Latif HA. 1997. Efficacy of simvastatin and pumpkin seed oil in the management of dietaryinduced hypercholesterolemia. Pharmacol Res 35:403– 408. Al-Zuhair HA, Abd El-Fattah AA, El-Sayed MI. 2000. Pumpkinseed oil modulates the effect of felodipine and captopril in spontaneously hypertensive rats. Pharmacol Res 41:555–563. Andjelkovic M, Camp JV, Trawka A, Verhe´ R. 2010. Phenolic compounds and some quality parameters of pumpkin seed oil. Eur J Lipid Sci Tech 112:208–217. Anon. 2009. R-biopharm Ridascreen1, Enzyme immunoassay for the quantitative analysis of aflatoxin total. Art No: R. 4701. RBiopharm GmbH, Darmstadt, Germany. Beers KW, Glahn RP, Bottje WG, Huff WE. 1992. Aflatoxin and glutathione in domestic fowl (Gallus domesticus)-II. Effects on hepatic blood flow. Comp Biochem Physiol 101: 463–467. Betina V. 1989. Aflatoxins, sterigmatocystins and versicolorins. In: Mycotoxins: Chemical, Biological and Environmental Aspects. Amsterdam-Oxford-New York-Tokyo: Elsevier. pp 115– 150. Bra¨se S, Encinas A, Keck J, Nising CF. 2009. Chemistry and biology of mycotoxins and related fungal metabolites. Chem Rev 109:3903–3990. Butinar B, Bucar-Miklavcic M, Valencic V, Raspor P. 2010. Stereospecific analysis of triacylglycerols as a useful means to evaluate genuineness of pumpkin seed oils: Lesson from virgin olive oil analyses. J Agric Food Chem 58:5227–5234. Caro AA, Cederbaum AI. 2004. Oxidative stress, toxicology, and pharmacology of CYP2E1. Annu Rev Pharmacol Toxicol 44:27–42. Choi KC, Chung WT, Kwon JK, Yu JY, Jang YS, Park SM, Lee SY, Lee JC. 2010. Inhibitory effects of quercetin on aflatoxin B1-induced hepatic damage in mice. Food Chem Toxicol 48:2747–2753. Choudhary A, Verma RJ. 2005. Ameliorative effects of black tea extract on aflatoxin-induced lipid peroxidation in the liver of mice. Food Chem Toxicol 43:99–104. Choudhary A, Verma RJ. 2006. Black tea ameliorates aflatoxininduced lipid peroxidation in the kidney of mice. Acta Pol Pharm 63:307–310. Dalvi RR. 1986. An overview of aflatoxicosis of poultry: Its characteristics, preventation and reduction. Vet Res Commun 10: 429–443. _ Nizamlıog˘lu F. 1995. Pirinc¸te aflatokDemet O, Oguz H, C¸elik I, sin u¨retilmesi. Vet Bil Derg 11:19–23. Dutta TK, Das P. 2001. Isolation of aflatoxigenic strains of Aspergillus and detection of aflatoxin B1 from feeds in India. Mycopathologia 151:29–33. El-Agamy DS. 2010. Comparative effects of curcumin and resveratrol on aflatoxin B(1)-induced liver injury in rats. Arch Toxicol 84:389–396.

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Eraslan G, Akdog˘an M, Yarsan E, S  ahindokuyucu F, Es siz D, Altıntas L. 2005a. The effects of aflatoxins on oxidative stress in broiler chickens. Turk J Vet Anim Sci 29:701–707. Eraslan G, Cam Y, Eren M, Liman BC, Atalay O, Seybek N. 2005b. Aspects of using N-Acetylcysteine in aflatoxicosis and its evaluation regarding some lipid peroxidation parameters in rabbits. Bull Vet Inst Pulawy. 49:243–247. Esterbauer H. 1996. Estimation of peroxidative damage, a critical review. Pathol Biol 44:25–28. Evans P, Halliwell B. 2001. Micronutrients: Oxidant/antioxidant status. Br J Nutr 85:67–74. Fang YZ, Yang S, Wu G. 2002. Free radicals, antioxidants, and nutrition. Nutrition 18:872–879. Farombi EO, Adepoju BF, Ola-Davies OE, Emerole GO. 2005. Chemoprevention of aflatoxin B1-induced genotoxicity and hepatic oxidative damage in rats by kolaviron, a natural biflavonoid of Garcinia kola seeds. Eur J Cancer Prev 14:207–214. Fruhwirth GO, Hermetter A. 2007. Seeds and oil of the Styrian oil pumpkin: Components and biological activities. Eur J Lipid Sci Technol 109:1128–1140. Gugnani HC. 2003. Ecology and taxonomy of pathogenic aspergilli. Front Biosci 8:346–357. Gutteridge JM, Halliwell B. 2000. Free radicals and antioxidants in the year 2000. A historical look to the future. Ann NY Acad Sci 899:136–147. Halliwell B. 1999. Antioxidant defence mechanisms: From the beginning to the end (of the beginning). Free Radic Res 31:261– 272. Halliwell B. 2007. Biochemistry of oxidative stress. Biochem Soc Trans 35:1147–1150. Janero DR. 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 9:515–540. Kanbur M, Eraslan G, Sarıca Soyer Z, Aslan O. 2011. The effects of evening primrose oil on lipid peroxidation induced by subacute aflatoxin exposure in mice. Food Chem Toxicol 49, 1960– 1964. Kaya S. 2007. Mineral maddeler. In: Kaya S, editor. Veteriner Farmakoloji, fourth edition, Vol. 2, Medisan Yayinevi, Ankara. pp 267–278. Kehrer JP. 2000. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 149:43–50. Kensler TW, Roebuck BD, Wogan GN, Groopman JD. 2011. Aflatoxin: A 50-year odyssey of mechanistic and translational toxicology. Toxicol Sci 20:28–48. Kiessling KH. 1986. Biochemical mechanism of action of mycotoxins. Pure Appl Chem 58:327–338. Letelier ME, Sa´nchez-Jofre´ S, Peredo-Silva L, Corte´s-Troncoso J, Aracena-Parks P. 2010. Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects. Chem Biol Interact 188:220–227. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurment with the folin phenol reagent. J Biol Chem 193:265–275. Luck H, 1965. Catalase. In: Bergmeyer H, editor. Methods of Enzymatic Analysis. New York: Academic Press. pp 885–894.

Environmental Toxicology DOI 10.1002/tox

8

ERASLAN ET AL.

Mathuria N, Verma RJ. 2007. Curcumin ameliorates aflatoxininduced lipid peroxidation in liver, kidney and testis of mice-an in vitro study. Acta Pol Pharm 64:413–416. Mercan U. 2004. Toksikolojide serbest radikallerin o¨nemi. YYU Vet Fak Derg 15:91–96. Michel F, Bonnefont-Rousselot D, Mas E, Drai J, The´rond P. 2008. Biomarkers of lipid peroxidation: Analytical aspects. Ann Biol Clin (Paris) 66:605–620. Miller GL. 1959. Protein determination for large numbersof samples. Anal Chem 31:964. Murkovic´ M, Piironen V, Lampi AM, Kraushofer T, Sontag G. 2004. Changes in chemical composition of pumpkin seeds during roasting process for production of pumpkin seed oil (Part 1: non-volatile compounds). Food Chem 84:359–365. Nabney J, Nesbitt BF. 1965. A spectrophotometric method for determining the aflatoxins. Analyst 90:155–160. Niki E. 2009. Lipid peroxidation: Physiological levels and dual biological effects. Free Radic Biol Med 47:469–484. Nkosi CZ, Opoku AR, Terblanche SE. 2006. Antioxidative effects of pumpkin seed (Cucurbita pepo) protein isolate in CCl4induced liver injury in low-protein fed rats. Phytother Res 20:935–940. Nyam KL, Tan CP, Lai OM, Long K, Che Man YB. 2009. Physicochemical properties and bioactive compounds of selected seed oil. LWT-Food Sci Technol 42:1396–1403. Ogino K, Wang DH. 2007. Biomarkers of oxidative/nitrosative stress: An approach to disease prevention. Acta Med Okayama 61:181–189. Ohkawa H, Ohishi N, Yagi K. 1978. Reaction of linoleic acid hydroperoxide with thiobarbituric acid. J Lipid Res 19:1053– 1057. Ortatatli M, Og˘uz H, Hatipog˘lu F, Karaman M. 2005. Evaluation of pathological changes in broilers during chronic aflatoxin (50 and 100 ppb) and clinoptilolite exposure. Res Vet Sci 78:61–68. Ozen H, Karaman M, C ¸ ig˘remis  Y, Tuzcu M, Ozcan K, Erdag˘ D. 2009. Effectiveness of melatonin on aflatoxicosis in chicks. Res Vet Sci 86:485–489. Paglia PE, Valentine WN. 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70:158–169. Paster N, Bullerman LB. 1988. Mould spoilage and mycotoxin formation in grains as controlled by physical means. Int J Food Microbiol 7:257–265. Paterson RR, Lima N. 2010. Toxicology of mycotoxins. EXS 100:31–63. Pimpukdee K, Kubena LF, Bailey CA, Huebner HJ, AfriyieGyawu E, Phillips TD. 2004. Aflatoxin-induced toxicity and depletion of hepatic vitamin A in young broiler chicks: Protection of chicks in the presence of low levels of NovaSil PLUS in the diet. Poult Sci 83:737–744. Preetha SP, Kanniappan M, Selvakumar E, Nagaraj M, Varalakshmi P. 2006. Lupeol ameliorates aflatoxin B1-induced peroxidative hepatic damage in rats. Comp Biochem Physiol C Toxicol Pharmacol 143:333–339.

Environmental Toxicology DOI 10.1002/tox

Rastogi R, Srivastava AK, Rastogi AK. 2001. Long term effect of aflatoxin B1 on lipid peroxidation in rat liver and kidney: Effect of picroliv and silymarine. Phytother Res 15:307–310. Roberts BA, Patterson DSP. 1975. Detection of twelve mycotoxins in mixed animal feedstuffs, using a novel membrane clean up procedure. J Assoc Off Anal Chemists 58:1178–1181. Ryan E, Galvin K, O’Connor TP, Maguire AR, O’Brien NM. 2007. Phytosterol, squalene, tocopherol content and fatty acid profile of selected seeds, grains, and legumes. Plant Foods Hum Nutr 62:85–91. Senevirathne M, Kim SH, Siriwardhana N, Ha JH, Lee KW, Jeon YJ. 2006. Antioxidant potential of Ecklonia cava on reactive oxygen species scavenging, metal chelating, reducing power and lipid peroxidation inhibition. Food Sci Tech Int 12:27–38. Shen HM, Ong CN, Shi CY. 1995. Involvement of reactive oxygen species in aflatoxin B1-induced cell injury in cultured rat hepatocytes. Toxicology 99:115–123. Shen HM, Shi CY, Lee HP, Ong CN. 1994. Aflatoxin B1-induced lipid peroxidation in rat liver. Toxicol Appl Pharmacol 127:145–150. Shotwell OL, Hesseltine CW, Stubblefield RD, Sorenson WG. 1966. Production of aflatoxin on rice. Appl Microbiol 14:425–428. Siegmund B, Murkovic´ M. 2004. Changes in chemical composition of pumpkin seeds during the roasting process for production of pumpkin seed oil (Part II: Volatile compounds). Food Chem 84:367–374. Souza MF, Tome AR, Rao VS. 1999. Inhibition by the bioflavonoid ternatin of aflatoxin B1-induced lipid peroxidation in rat liver. J Pharm Pharmacol 51:125–129. Stevenson DG, Eller FJ, Wang L, Jane JL, Wang T, Inglett GE. 2007. Oil and tocopherol content and composition of pumpkin seed oil in 12 cultivars. J Agric Food Chem 55:4005–4013. Sun Y, Oberley LW, Li Y. 1988. A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500. Verma RJ. 2004. Aflatoxin Cause DNA Damage. Int J Hum Genet 4:231–236. Verma RJ, Mathuria N. 2008. Curcumin ameliorates aflatoxininduced lipid peroxidation in liver and kidney of mice. Acta Pol Pharm 65:195–202. Verma RJ, Nair A. 2001. Ameliorative effect of vitamin E on aflatoxin induced lipid peroxidation in the testis of mice. Asian J Androl 3:217–221. Verma RJ, Shukla RS, Mehta DN. 2001. Amelioration of cytotoxic effects of aflatoxin by vitamin A: An in vitro study of erythrocytes. Toxicol in Vitro 15:39–42. Xanthopoulou MN, Nomikos T, Fragopoulou E, Antonopoulou S. 2009. Antioxidant and lipoxygenase inhibitory activities of pumpkin seed extracts. Food Res Int 42:641–646. Yener Z, Celik I, Ilhan F, Bal R. 2009. Effects of Urtica dioica L. seed on lipid peroxidation, antioxidants and liver pathology in aflatoxin-induced tissue injury in rats. Food Chem Toxicol 47:418–424. Zada´k Z, Hyspler R, Ticha´ A, Hronek M, Fikrova´ P, Rathouska´ J, Hrnciarikova´ D, Stetina R. 2009. Antioxidants and vitamins in clinical conditions. Physiol Res 58:13–17.