Homeopathy (2015) 104, 205e210 Ó 2015 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.homp.2015.02.005, available online at http://www.sciencedirect.com
ORIGINAL PAPER
Effects of Tarantula cubensis D6 on aflatoxin-induced injury in biochemical parameters in rats M€ ursel Karabacak1, G€ okhan Eraslan2,*, Murat Kanbur2 and Zeynep Soyer Sarıca3 1
Erciyes University, Safiye C¸ ıkrıkc¸ıo glu Vacational Collage, Animal Health Department, Kayseri, Turkey Erciyes University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Kayseri, Turkey 3 Erciyes University, Hakan C¸ etinsaya Experimantal Animal Center, Kayseri, Turkey 2
Introduction: Aflatoxins are toxic fungal metabolites that have adverse effects on humans and animals. Tarantula cubensis D6 is used as a homeopathic medicine for different purposes. The present study investigates the effects of Tarantula cubensis D6 on the oxidant-antioxidant balance and some biochemical parameters against exposure to aflatoxin. Methods: Thirty-two Sprague-Dawley female rats were used and evenly divided into four groups. Group 1 served as control. Groups 2, 3, and 4 received 200 ml/kg.bw/day Tarantula cubensis D6 (applied subcutaneously), 400 mg/kg.bw/day total aflatoxin (approximately 80% AF B1, 10% AF B2, 6 %AF G1, and 4%AF G2), and 200 ml/kg.bw/day Tarantula cubensis D6 plus 400 mg/kg.bw/day total aflatoxin, respectively, for 28 days. At the end of 28 days, blood samples and some organs (liver, kidney, brain, and spleen) were taken from all the animals. Oxidative stress markers (MDA, SOD, CAT, GSH-Px) and some biochemical parameters (glucose, triglyceride, cholesterol, BUN, creatinine, AST, ALT and ALP, total protein, albumin) were evaluated in blood samples and tissues. Results: Aflatoxin caused negative changes in all oxidative stress parameters and some biochemical parameters (glucose, triglyceride, cholesterol, creatinine, AST, ALT, ALP, total protein, albumin). Administration of Tarantula cubensis D6 partly alleviated aflatoxin-induced negative changes. Conclusions: Our results indicated that Tarantula cubensis D6 partially neutralized the deleterious effects of aflatoxin. Homeopathy (2015) 104, 205e210.
Key words: Aflatoxin; Tarantula cubensis D6; Oxidative stress; Biochemical parameters; Rat
Introduction Aflatoxins (AF) are secondary metabolites, naturally occurring mycotoxins produced especially by Aspergillus flavus and Aspergillus parasiticus. They are also known as potential contaminants in human and animal food. The aflatoxin species (AFB1, AFB2, AFG1, and AFG2) are
€ € khan Eraslan, Erciyes Universitesi *Correspondence: Go € ltesi, Farmakoloji ve Toksikoloji Anabilim Dalı, Veteriner Faku Kayseri, Turkey. E-mail: [email protected] Received 26 February 2014; revised 23 July 2014; accepted 6 February 2015
visible under ultraviolet light in different colors. Cases of contamination are based on different variables, such as humidity temperature, the amount of fungi in the environment, physical damage, and storage conditions. AFB1 is metabolized to AFB1-exo-8,9-epoxide by the cytochrome P-450 enzyme system. This extremely reactive metabolite binds to DNA guanines to form adducts, causing toxic effect.1e4 The effects of aflatoxins on multiple organs, such as oxidative stress, and other toxicities have been previously described in various experiments on animals.5e10 The homeopathic medicine Tarantula cubensis D6 (syn.: Mygale cubanensis) is nominally prepared from the Cuban tarantula. The spider is a member of the genus Mygale, which consists of large, mouse-shaped hairy tarantulas.
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Venoms of spiders of the genus Loxosceles cause serious arachnidism, a dangerous systemic reaction including renal failure, severe intravascular coagulation, thrombocytopenia, coma, and convulsions.11 TheranekeronÒ is a homeopathic product which contains Tarantula cubensis D6. Some positive effects including such as demercative, regenerative, anti-inflammatory and resorptive effects are defined in many previous studies.12e18 Tarantula cubensis D6 in homeopathic medicine has been used successfully in the treatment of oral lesions in cattle with bluetongue disease,12 canine oral papillomatosis,13 canine mammary tumors,14 indolent ulcers in cats,15 thrombus in the left anterior descending artery,16 foot-andmouth disease in cattle,17 and endometriosis.18 The effects of administration of Tarantula cubensis D6 on aflatoxin-induced biochemical changes in rats were evaluated on the basis of some oxidative stress marker and other biochemical parameters. There are many studies related to aflatoxin intoxication in rats9,19e22 and other animals.5e7,10 Several treatments are used in detoxification of aflatoxin.8,23e26 But their benefit are not satisfactory. Oxidative stress is one mechanism in the formation of aflatoxin intoxication.27 The effects of Tarantula cubensis D6 on cellular oxidative stress and other biochemical parameters in the case of exposure of aflatoxin has not previously been studied. The present study aims at evaluating the effectiveness of Tarantula cubensis D6 against aflatoxin-induced toxications in rats.
Materials and methods Chemicals The chemicals used in the experiments and analysis were purchased from SigmaeAldrich (St Louis, MO, USA) or Merck (Darmstadt, Germany). Tarantula cubensis D6 (TheranekronÒ) was obtained from Richter Pharma AG Wels, Austria. Aflatoxin production Aflatoxin was produced in rice by using the Aspergillus parasiticus (NRLL 2999) strain according to the method of Shotwell et al.28 as modified by Demet et al.29 The extraction/purification and type analyses of produced aflatoxins from rice flour were carried out according to the R-Biopharm RidascreenÒ kit procedure for total aflatoxin and Nabney and Nesbit,30 respectively. The percentages of AFB1, AFB2, AFG1, and AFG2 in extract were found to be approximately 80%, 10%, 6%, and 4%, respectively. The level of total aflatoxin was estimated by means of the R-Biopharm RidascreenÒ total aflatoxin kit and the method suggested by the producer in extract. Following purification of aflatoxin, organic solvent containing aflatoxin was evaporated. Then aflatoxin was solved in dimethysulfoxide/water (4:6, v/v) for using experiment. Animal material Thirty-two 4to 5-week-old (200e250 g) Sprague-Dawley female rats were used and divided evenly Homeopathy
into four groups. The first group was control. Groups 2, 3, and 4 were given 200 ml/kg.bw/day Tarantula cubensis D6 (TheranekronÒ), 400 mg/kg.bw/day total aflatoxin, and 200 ml/kg.bw/day Tarantula cubensis D6 plus 400 mg/ kg.bw/day total aflatoxin, respectively, for 28 days. Aflatoxin was administered early in the morning via oral gavage. Tarantula cubensis D6 was administered after 6 h the same day by subcutaneous route. Number of animals used in this study was based on earlier aflatoxin studies.8 Determination of oral aflatoxin31,32 and Tarantula cubensis D6 dose,13 previous studies were based on but some modifications were made on application period for Tarantula cubensis D6 considering the severity of aflatoxin intoxication. The protocol of the present study was approved by the Ethics Board for Experimental Animals of Erciyes University. Sample collection and preparations of analysis Blood samples were withdrawn into tubes (without/with anticoagulant) from all animals under a light ether anesthesia. The collected blood samples were centrifuged at 3000 rpm for 10 min at +4 C (Sigma 3K30, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany), and their erythrocytes, plasma and sera were separated. The erythrocytes were washed in saline phosphate buffer (PBS), diluted with equal volumes of PBS and were lysed with ice-cold water.33 Following the collection of blood samples and their deaths, the liver, kidneys, brain and spleen were taken from all the animals. All extracted organs were washed in cold distilled water. Than fat and connective tissues were then removed. The tissues were homogenized with phosphate buffer adjusted to a pH value of 7.4, using a homogenizer (Heidolph, SilentCrusher M) and then the homogenized tissues were centrifuged at 15,000 rpm for 45 min at +4 C, and the supernatant was placed in eppendorf tubes. All samples were stored at 80 C until the analysis. Assessment of oxidative stress parameters Plasma/tissue malondialdehyde (MDA) levels,34,35 blood hemoglobin levels,36 tissue protein levels,37,38 tissue superoxide dismutase (SOD) activities39, catalase (CAT) activities,40 glutathione peroxidase activities (GSH-Px)41 were determined according to the mentioned methods. The MDA results were expressed in units of nmol/ml or nmol/mgprotein. Units of enzymes for tissues SOD, CAT, and GSH-Px were expressed as U/mg protein, U/g protein, and U/g protein, respectively. Units of enzymes for erythrocyte SOD, CAT, and GSH-Px were indicated as U/mgHb, U/ gHb and U/gHb, respectively. All oxidative stress markers and tissue protein levels were measured spectrophotometrically (Helios a, double-beam UV/VIS). Measurement of biochemical parameters in sera Detection of serum glucose, triglyceride, cholesterol, blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT),
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Groups*
Liver
Untreated control Tarant D6 Aflatoxin Afla + Tarant D6 Untreated control Tarant D6 Aflatoxin Afla + Tarant D6 Untreated control Tarant D6 Aflatoxin Afla + Tarant D6 Untreated control Tarant D6 Aflatoxin Afla + Tarant D6 Untreated control Tarant D6 Aflatoxin Afla + Tarant D6
Kidney
Brain
Spleen
Erythrocytes/plasma
MDA (nmol/ml or nmol/mg protein)
SOD (U/mg protein or U/mgHb)
CAT (U/g protein or U/gHb)
GSH-Px (U/g protein or U/gHb)
2.72 0.14a 2.14 0.26a 5.84 0.65b,y 3.50 0.57a 3.90 0.39a 4.29 0.29a 6.07 0.50b,* 5.82 0.62b,* 12.77 0.70a 16.05 0.99b,y 22.34 1.28c,y 20.01 1.06c,y 6.13 0.18a 5.84 0.31a 7.21 0.22b,# 5.79 0.44a 9.78 0.38a 10.05 0.66a 12.37 0.47b,# 11.05 0.44a,b
1.54 0.12a 0.60 0.06b,y 2.02 0.19c,y 1.00 0.11d,y 2.18 0.13a,b 1.78 0.14a 2.79 0.16c,y 2.41 0.19b,c 2.59 0.21a 3.83 0.32b,y 3.76 0.37b,y 4.74 0.36b,y 1.49 0.08a 1.49 0.17a 2.09 0.19b,* 1.36 0.18a 0.80 0.05a 0.77 0.05a 1.12 0.06b,y 0.65 0.04a
164.11 14.43b 79.71 8.23ay 250.72 32.20cy 52.11 6.69a,y 306.63 18.85a,b 277.60 27.18b 365.51 27.11a 248.39 17.24b 24.85 2.80a 34.82 3.74a,b 39.73 5.86c,# 48.42 5.24c,# 30.44 2.10a 19.81 3.27b,y 21.59 2.12b,y 11.47 1.42c,y 74.82 4.75a 72.87 8.51a 143.64 11.60b,y 69.22 4.50a
305.83 25.05a 414.03 35.40a 673.40 49.70b,y 363.86 32.06a 579.29 46.07b 372.31 58.40a,y 738.66 45.03c,y 488.73 57.04a,b 60.66 5.61a 108.19 7.72b,y 116.42 13.21b,y 132.57 12.09b,y 58.03 5.18a 71.58 4.05b,c,# 79.21 2.45c,# 61.94 2.66ab 42.76 4.67a 40.03 3.41a 67.03 4.43b,y 42.20 2.10a
*Group 1, untreated control; group 2, Tarantula cubensis D6 only; group 3, aflatoxin only; group 4, Tarantula cubensis D6 plus aflatoxin. Values not sharing a common superscript letters (aec) differ significantly at p < 0.05. Statistically significant differences obtained for each parameter between groups is indicated by a versus b, c; b versus c. *p < 0.05, #p < 0.01, and yp < 0.001 according to control.
alkaline phosphatase (ALP), total protein, and albumin levels/activities were performed using the Abbott autoanalyzer (Abbott laboratories, Illinois, USA) and same label kits. Statistical analysis Calculations were carried out using the SPSS 10.0 for Windows’ statistical program. Data were expressed with arithmetical means and means of standard errors. Statistical significance between the experimental groups was calculated by one-way analysis of variance (ANOVA). Individual evaluation of different groups was made in accordance with the Duncan multiple range test. The differences were considered as significant at p < 0.05.
Results Tarantula cubensis D6 (Group 2) Compared with the control group, SOD activity increased in brain whereas liver SOD activity decreased
in this group. CAT activity decreased in liver and spleen. GSH-Px activities decreased in kidney, but increased in brain and spleen. Brain MDA levels increased. On the other hand, no significant alterations were observed in the tissues, erythrocyte/plasmas and serums in the remaining oxidative stress parameters and biochemical parameters (Tables 1 and 2). Aflatoxin (Group 3) Compared with the control group, it was observed that liver, kidney, brain, spleen and plasma MDA levels increased in this group. Liver, kidney, brain, spleen and erythrocyte SOD activity increased. Liver, brain, and erythrocyte CAT activity increased, whereas spleen CAT activity had decreased. GSH-Px activity increased in the liver, kidney, spleen and brain, as well as in erythrocytes. Finally, cholesterol, creatinine, AST, ALP, and ALT activities/ levels were increased, whereas a decrease was detected in glucose, triglyceride, total protein, and albumin. On the other hand, the levels/activities of the remaining
Table 2 Effects of Tarantula cubensis D6 and aflatoxin on some biochemical parameters in rat Parameters
Glucose (mg/dL) Triglyceride (mg/dL) Cholesterol (mg/dL) BUN (mg/dL) Creatinine (mg/dL) AST (U/L) ALT (U/L) ALP(U/L) T-Protein (g/dL) Albumin (g/dL)
Groups Untreated control
Tarant D6
215.75 7.12 117.50 9.36a 77.75 5.86a 15.00 0.81 0.55 0.02a 76.80 7.46a 48.25 4.30a 207.50 29.53a 6.25 0.38a 1.02 0.02a
211.80 13.11 122.20 10.96a 81.20 4.10a 17.80 0.86 0.62 0.02ab 88.80 5.16ab 51.60 6.45a 217.00 33.34a 6.08 0.09a 1.12 0.06a
a
Aflatoxin a
Afla + Tarant D6
174.20 8.57 72.00 4.07b,y 98.60 5.95b,* 18.60 1.96 0.68 0.03b,* 105.25 3.96b,* 83.00 8.01b,# 393.20 46.30b,# 3.98 0.25b,y 0.68 0.05b,y b,*
195.20 8.77a,b 85.20 4.87b,y 87.40 4.11a,b 19.40 0.87 0.56 0.02a 81.60 5.20a 51.60 2.67a 336.20 18.48b,# 5.08 0.17c,y 0.80 0.07b,y
Values not sharing a common superscript letters (aec) differ significantly at p < 0.05. Statistically significant differences obtained for each parameter between groups is indicated by a versus b, c; b versus c. *p < 0.05, #p < 0.01, and yp < 0.001 according to control.
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parameters were not altered significantly compared with the controls (Tables 1 and 2). Tarantula cubensis D6 plus aflatoxin (Group 4) Compared with the control group, MDA levels in kidney and brain increased in this group. SOD activity increased in brain but decreased in liver. CAT activity in liver and spleen decreased, whereas CAT activity in brain increased. GSHPx activities in brain increased. In addition, triglyceride, total protein, and albumin levels decreased, whereas ALP activities increased. Compared with the control group, no significant difference was determined in the remaining parameters for this group (Tables 1 and 2). In the other words, mentioned remaining data are similar to the control group.
Discussion Compared to control group, no meaningful changes was observed on liver, kidney, spleen and plasma MDA levels in the only Tarantula cubensis D6-only treated group. But a significant an increase in brain MDA levels shows that Tarantula cubensis D6 causes adverse effect on brain tissue. This may be related to the application procedure and duration. Also, compared to control group, significant increase/decrease was seen in SOD, CAT and GSH-Px activities in some tissues. The target organs for aflatoxicosis are particularly liver and kidney.23e25 No negative effects was found on liver and kidney for indicated doses and periods aspect of MDA levels. Also, there are also no negative effects on biochemical parameters. Previous studies on this subject evaluated, on sheep, after Tarantula cubensis D6 application, the levels/activities of MDA and some biochemical parameters such as AST, ALT, creatin, BUN, albumin and total protein, no negative effects have been seen by Dik et al.42 In addition, another study made by Sardari et al.43 on horses, reveals the same results like Dik et al.42 Hence the present study displays similarity to previous studies. The free radical generating mechanism of aflatoxin is related to the biotransformation of aflatoxin B1 via cytocrome enzymes.44 Additionally, other toxic mechanisms of aflatoxins include consumption of vitamin A45 and glutathione storage,9,27 which are strong radical scavengers. Aflatoxins also cause alterations in some biochemical parameters directly reflected to organ injury, such as injuries involving liver and kidney.8,21,23,26 Results obtained in the current study shows that aflatoxins cause negative effects in biological systems. Compared with the control group, the group that was given only aflatoxin had the highest MDA levels. The results demonstrate that aflatoxins are associated with high level of free radical occurrence in biological systems, but these highly active metabolites are not detoxified via antioxidant defense systems. The antioxidant enzymes analyzed in the samples (SOD, CAT, and GSH-Px) were increased or decreased in animals treated with aflatoxin alone. Alterations in the markers suggest that the activities of antioxidant enzymes are insufficient to compensate for the high level of free radical occurrence against the appliHomeopathy
cation of aflatoxin. Increased MDA levels in the biological materials also support these evaluations. These changes in the activities of the antioxidant enzymes may imply induction or consumption during the transformation of free radicals into less harmful or harmless products. In relevant studies conducted on various animals, aflatoxin has been reported to lead changes in oxidative stress parameters in some biological samples; in a study conducted Alm-Eldeen et al.9 which rats received aflatoxin, an increase in liver TBARS levels and an increase in SOD, CAT and GSH-Px activities are observed. El-Nekeety et al.21 detected a meaningful increase kidney and liver MDA levels after the administration of aflatoxin contained diet in rats. Eraslan et al.10 in which mice applied with aflatoxin, an increase was observed in tissue MDA levels, while SOD, CAT and GSH-Px activities seen two-way alterations such as increase or decrease. Kanbur et al.7 determined that aflatoxin increased in tissue MDA levels, mean while decreased/increased in tissue SOD, CAT and GSH-Px activities. Naaz et al.25 reported that administration of aflatoxin resulted increase kidney MDA levels, while SOD, CAT and GSH-Px activities decreased in kidney. The results obtained in the present study are similar to those previous studies. In this study, the changes in AST, ALT, ALP, glucose, triglyceride and cholesterol levels/activities pointed to the functional disorder mainly of the liver,8,21,26,46,47 in the group treated with only aflatoxin. The changes in the total protein levels and creatinine are also determinative of the development of a disorder in protein synthesis metabolism/catabolism and renal dysfunction.48 In same studies, Yousef et al.49 determined decline by treated with aflatoxin in serum glucose levels in rabbits. ElNeekety et al.21 fed rats with aflatoxin with feed. They implied that AST, ALT, ALP, cholesterol levels/activities had increased. In another study conducted by Mathuria and Verma,23 which mice received aflatoxin determined a decrease in protein level and increase in AST, ALT and creatinine levels/activity. Adedara et al.50 detected an increase AST, ALT and ALP activities after the application of aflatoxin in mice. Alm-Eldeen et al.9 which rats applied with aflatoxin, an increase in AST, ALT and ALP and cholesterol levels/activities was observed. In another study, Casado et al.51 applied mice with aflatoxin with feed and determined such exposure cause to decrease triglyceride level. Such alterations were also observed in this study. Therefore this study exhibits similarity to the previously research. In the group treated with aflatoxin and Tarantula cubensis D6 together, oxidative stress markers displayed positive alterations for some samples, in other words, values were closer to those of the control group. For example, MDA values of the tissues except for brain and kidney tissues are similar to the control group. The primary target of aflatoxin is the liver.2,3,24 In this study, positive changes on liver MDA levels and antioxidant enzyme activities shows that Tarantula cubensis D6 can be used against negative effects of aflatoxin on liver. No study has been published about the antioxidant effects of Tarantula
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cubensis but some clinical studies shows that, a decrease of clinical symptoms and increase epithelisation of oral mucosa in cattle with bluetongue disease, has seen after usage of this compound.12 It compound decreases the tumoral formation in canine oral papillomatosis.13 Additionally, it decreases benign canine mammary tumor in dog.14 Moreover, Lotfollahzadeh et al.17 demonstrated healing of lesions of foot-and-mouth disease in cattle and improvement of general condition. Dolapcıoglu et al.18 found that the usage of Tarantula cubensis has shown significant healing in experimental endometritis of rats. According to earlier studies, Tarantula cubensis as a homeopathic formulation had demercative, regenerative, antiinflammatory and resorptive effects on inflammatory, necrotic and proliferative syndromes.12e19 Free radicals have direct or indirect roles on various inflammatory, necrotic, toxic and proliferative cases.52e56 It is considered that, in these cases Tarantula cubensis 6D may have antioxidant effects. In our study, free radical formation which is the mechanism of aflatoxin intoxication and the possible negative effects of these radicals can clearly be seen from oxidative stress markers. These markers on the Tarantula cubensis D6 treated group, reveals antioxidant effects on liver which is primarily affected by aflatoxin.
Conclusions Aflatoxin caused an imbalance in the antioxidant defense and other biological systems of rats. Oxidative stress and toxic effects of Aflatoxins are partly mitigated in the groups given aflatoxin combined with Tarantula cubensis D6 Tarantula cubensis D6 may have potential as a supportive agent for alleviating the toxic effects of aflatoxin especially liver damage.
Conflict of interests
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References 1 Edlayne G, Simone A, Felicio JD. Chemical and biological approaches for mycotoxin control: a review. Recent Pat Food Nutr Agric 2009; 1: 155e161. 2 Ingawale DK, Mandlik SK, Naik SR. Models of hepatotoxicity and the underlying cellular, biochemical and immunological mechanism(s): a critical discussion. Environ Toxicol Pharmacol 2013; 37: 118e133. 3 Marin S, Ramos AJ, Cano-Sancho G, Sanchis V. Mycotoxins: occurrence, toxicology, and exposure assessment. Food Chem Toxicol 2013; 60: 218e237. 4 Mohd-Redzwan S, Jamaluddin R, Abd-Mutalib MS, Ahmad Z. A mini review on aflatoxin exposure in Malaysia: past, present and future. Front Microbiol 2013; 4: 334. http://dx.doi.org/10.3389/fmicb.2013.00334. 5 Eraslan G, Akdogan M, Yarsan E, S¸ahindokuyucu F, Es¸siz D, Altıntas¸ L. The effects of aflatoxins on oxidative stress in broiler chickens. Turk J Vet Anim Sci 2005; 29: 701e707. 6 Eraslan G, Cam Y, Eren M, Liman BC, Atalay O, Seybek N. Aspects of using N-acetylcysteine in aflatoxicosis and its evaluation
21
22
23
24
25
26
regarding some lipid peroxidation parameters in rabbits. Bull Vet Inst Pulawy 2005; 49: 243e247. Kanbur M, Eraslan G, Soyer Sarıca Z, Aslan O. The effects of evening primrose oil on lipid peroxidation induced by subacute aflatoxin exposure in mice. Food Chem Toxicol 2011; 49: 1960e1964. Akc¸am M, Artan R, Yilmaz A, Ozdem S, Gelen T, Nazıro glu M. Caffeic acid phenethyl ester modulates aflatoxin B1-induced hepatotoxicity in rats. Cell Biochem Funct 2013; 31: 692e697. Alm-Eldeen AA, Mona MH, Shati AA, El-Mekkawy HI. Synergistic effect of black tea and curcumin in improving the hepatotoxicity induced by aflatoxin B1 in rats. Toxicol Ind Health 2013; http:// dx.doi.org/10.1177/0748233713491807. € Karabacak M. The antioxidant efEraslan G, Kanbur M, Aslan O, fects of pumpkin seed oil on subacute aflatoxin poisoning in mice. Environ Toxicol 2013; 28: 681e688. Richardson-Boedler C. The brown spider Loxosceles laeta: source of the remedy Tarentula cubensis? Homeopathy 2002; 91: 166e170. Albay MK, S¸ahinduran S¸, Kale M, Karakum MC¸, Sezer K. Influence of Tarantula cubensis extract on the treatment of the oral lesions in cattle with bluetongue disease. Kafkas Univ Vet Fak Derg 2010; 16: 593e596. Icen H, Sekin S, Simsek A, Kochan A, Tunik S. The Efficacy of Tarantula cubensisi extract (Theranekron) in treatment of canine oral papillomatosis. Asian J Anim Vet Adv 2011; 7: 744e749. G€ ultekin N, Vural MR. The effect of Tarantula cubensis extract applied in pre and postoperative period of canine mammary tumours. JIVS 2007; 2: 13e23. Yardımcı C, Yardımcı B. Bir kedide indolent € ulser olgusu. Ankara € Univ Vet Fak Derg 2008; 55: 65e67. Jain SKA, Patel B, Larsen T, Alexander P, David S. A thrombus in the left anterior descending artery after the use of multiple alternative medicines. Med Pract Rev 2013; 4: 14e16. Lotfollahzadeh S, Alizadeh MR, Mohri M, Dezfouli MRM. The Therapeutic Effect of Tarentula cubensis extract (Theranekron) in foot-and-mouth disease in cattle: a randomised trial in an endemic setting. Homeopathy 2012; 101: 159e164. Dolapcioglu K, Dogruer G, Ozsoy S, et al. Theranekron for treatment of endometriosis in a rat model compared with medroxyprogesterone acetate and leuprolide acetate. Eur J Obstet Gynecol Reprod Biol 2013; 170: 206e210. Yener Z, Celik I, Ilhan F, Bal R. Effects of Urtica diocia L. seed on lipid peroxidation, antioxidants and liver pathology in aflatoxininduced tissue injury in rats. Food Chem Toxicol 2009; 47: 418e424. El-Agamy DS. Comparative effects of curcumin and resveratrol on aflatoxin B(1)-induced liver injury in rats. Arch Toxicol 2010; 84: 389e396. El-Nekeety AA, Mohamed SR, Hathout AS, Hassan NS, Aly SE, Abdel-Wahhab MA. Antioxidant properties of thymus vulgaris oil against aflatoxin-induce oxidative stress in male rats. Toxicon 2011; 57: 984e991. Hathout AS, Mohammed SR, El-Nekeety AA, Hassan NS, Aly SE, Abdel-Wahhab MA. Ability of Lactobacillus Casei and Lactobasillus reuteri to protect against oxidative stress in rat fed aflatoxinscontamineted diet. Toxicon 2011; 58: 179e186. Mathuria N, Verma RJ. Ameliorative effect of curcumin on aflatoxin-induced toxicity in serum of mice. Acta Pol Pharm 2008; 65: 339e343. Choi KC, Chung WT, Kwon JK, et al. Inhibitory effects of quercetin on aflatoxin B1-induced hepatic damage in mice. Food Chem Toxicol 2010; 48: 2747e2753. Naaz F, Abdin MZ, Javed S. Protective effect of esculin against prooxidant B1-induced nephrotoxicity in mice. Mycotoxin Res 2014; 30: 25e32. Nili-Ahmadabadi A, Tavakoli F, Hasanzadeh G, Rahimi H, Sabzevari O. Protective effect of pretreatment with thymoquinone
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against aflatoxin B(1) induced liver toxicity in mice. Daru 2011; 19: 282e287. Alpsoy L, Yalvac ME. Key roles of vitamins A, C, and E in aflatoxin B1-induced oxidative stress. Vitam Horm 2011; 86: 287e305. Shotwell OL, Hesseltine CW, Stubblefield RD, Sorenson WG. Production of aflatoxin on rice. Appl Microbiol 1966; 14: 425e428. Demet O, Oguz H, C¸elik I, Nizamlıo glu F. Pirinc¸te Aflatoksin € Uretilmesi. Vet Bil Derg 1995; 11: 19e23. Nabney J, Nesbitt BF. A spectrophotometric method for determining of the aflatoxins. Analyst 1965; 90: 155e160. Egner PA, Yu X, Johnson JK, et al. Identification of aflatoxin M1N7-guanine in liver and urine of tree shrews and rats following administration of aflatoxin B1. Chem Res Toxicol 2003; 16: 1174e1180. Chen ZY, Yan RQ, Qin GZ, Qin LL. Effect of six edible plants on the development of AFB1-induced gamma-glutamyltranspeptidase-positive hepatocyte foci in rats. Zhonghua Zhong Liu Za Zhi 1987; 9: 109e111. Winterbourn CC, Hawkins RE, Brian M, Carrell RW. The estimation of red cell superoxide activity. J Lab Clin Med 1975; 85: 337e341. Yoshioka K, Kawada K, Shimada T, Mori M. Lipid peroxidation in maternal and cord blood and protective mechanism against activedoxygen toxicity in the blood. Am J Obstet Gynecol 1979; 135: 372e376. Ohkawa H, Ohishi N, Yagi K. Reaction of linoleic acid hydroperoxide with thiobarbituric acid. J Lipid Res 1978; 19: 1053e1057. Fairbanks VF, Klee GG. Biochemical aspect of haematology. In: Tietz NW (ed). Fundamentals of Clinical Chemistry. 3th edn. Philadelphia: WB Saunders, 1987, pp 803e806. Lowry OH, Rosebrough NJ, Farr AL, Randali RJ. Protein measurment with the folin phenol reagent. J Biol Chem 1951; 193: 265e275. Miller GL. Protein determination for large numbers of samples. Anal Chem 1959; 31: 964. Sun Y, Oberley LW, Li Y. A simple for clinical assay of superoxide dismutase. Clin Chem 1988; 34: 497e500. Luck HS. Catalase. In: Bergmeyer HU (ed). Methods of Enzymatic Analysis. London: Academic Press, 1965, pp 885e894. Paglia PE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158e169. Dik B, Er A, Corum O. Effect of alcoholic extract of Tarantula cubensis (TheranekronÒ) on serum thiobarbituric acid-reactive species concentrations in sheep. Eurasian J Vet Sci 2014; 30: 68e71. Sardari K, Mohri M, Sabzevari S, Fathi B. Effects of the TheranekronÒ an alcoholic extract of the tarantula cubensis on he-
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48
49
50
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matology and serum biochemical properties in horses. IJVST 2011; 3: 9e16. Abrar M, Anjum FM, Butt MS, et al. Aflatoxins: biosynthesis, occurrence, toxicity, and remedies. Crit Rev Food Sci Nutr 2013; 53: 862e874. Pimpukdee K, Kubena LF, Bailey CA, Huebner HJ, AfriyieGyawu E, Phillips TD. 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 2004; 83: 737e744. Eraslan G, Essiz D, Akdogan M, et al. Efficacy of dietary sodium bentonite against subchronic exposure to dietary aflatoxin in broilers. Bull Vet Inst Pulawy 2006; 50: 107e112. O guz H, Kurto glu F, Kurto glu V, Birdane YO. Evaluation of biochemical characters of broiler chickens during dietary aflatoxin (50 and 100 ppb) and clinoptilolite exposure. Res Vet Sci 2002; 73: 101e103. Abbes S, Ben Salah-Abbes J, Abdel-Wahhab MA, Ouslati R. Immunotoxicological and biochemical effects of aflatoxins in rats prevented by tunisian montmorillonite with reference to HSCAS. Immunopharmacol Immunotoxicol 2010; 32: 514e522. Yousef MI, Salem MH, Kamel KI, Hassan GA, El-Nouty FD. Influence of ascorbic acid supplementation on the haematological and clinical biochemistry parameters of male rabbits exposed to aflatoxin B1. J Environ Sci Health B 2003; 38: 193e209. Adedara IA, Owumi SE, Uwaifo AO, Farombi EO. Aflatoxin B1 and ethanol co-exposure induces hepatic oxidative damage in mice. Toxicol Ind Health 2010; 26: 717e724. Casado JM, Theumer M, Masih DT, Chulze S, Rubinstein HR. Experimental subchronic mycotoxicoses in mice: individual and combined effects of dietary exposure to fumonisins and aflatoxin B1. Food Chem Toxicol 2001; 39: 579e586. Piechota-Polanczyk A, Fichna J. Review article: the role of oxidative stress in pathogenesis and treatment of inflammatory bowel diseases. Naunyn Schmiedebergs Arch Pharmacol 2014; 387: 605e620. Rodriguez F, Bonacasa B, Fenoy FJ, Salom MG. Reactive oxygen and nitrogen species in the renal ischemia/reperfusion injury. Curr Pharm Des 2013; 19: 2776e2794. Flora SJ, Shrivastava R, Mittal M. Chemistry and pharmacological properties of some natural and synthetic antioxidants for heavy metal toxicity. Curr Med Chem 2013; 20: 4540e4574. Novo E, Cannito S, Paternostro C, Bocca C, Miglietta A, Parola M. Cellular and molecular mechanisms in liver fibrogenesis. Arch Biochem Biophys 2014; 548: 20e37. Mates JM, Segura JA, Alonso FJ, Marquez J. Oxidative stress in apoptosis and cancer: an update. Arch Toxicol 2012; 86: 1649e1665.
ORIGINAL PAPER
Effects of Tarantula cubensis D6 on aflatoxin-induced injury in biochemical parameters in rats M€ ursel Karabacak1, G€ okhan Eraslan2,*, Murat Kanbur2 and Zeynep Soyer Sarıca3 1
Erciyes University, Safiye C¸ ıkrıkc¸ıo glu Vacational Collage, Animal Health Department, Kayseri, Turkey Erciyes University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Kayseri, Turkey 3 Erciyes University, Hakan C¸ etinsaya Experimantal Animal Center, Kayseri, Turkey 2
Introduction: Aflatoxins are toxic fungal metabolites that have adverse effects on humans and animals. Tarantula cubensis D6 is used as a homeopathic medicine for different purposes. The present study investigates the effects of Tarantula cubensis D6 on the oxidant-antioxidant balance and some biochemical parameters against exposure to aflatoxin. Methods: Thirty-two Sprague-Dawley female rats were used and evenly divided into four groups. Group 1 served as control. Groups 2, 3, and 4 received 200 ml/kg.bw/day Tarantula cubensis D6 (applied subcutaneously), 400 mg/kg.bw/day total aflatoxin (approximately 80% AF B1, 10% AF B2, 6 %AF G1, and 4%AF G2), and 200 ml/kg.bw/day Tarantula cubensis D6 plus 400 mg/kg.bw/day total aflatoxin, respectively, for 28 days. At the end of 28 days, blood samples and some organs (liver, kidney, brain, and spleen) were taken from all the animals. Oxidative stress markers (MDA, SOD, CAT, GSH-Px) and some biochemical parameters (glucose, triglyceride, cholesterol, BUN, creatinine, AST, ALT and ALP, total protein, albumin) were evaluated in blood samples and tissues. Results: Aflatoxin caused negative changes in all oxidative stress parameters and some biochemical parameters (glucose, triglyceride, cholesterol, creatinine, AST, ALT, ALP, total protein, albumin). Administration of Tarantula cubensis D6 partly alleviated aflatoxin-induced negative changes. Conclusions: Our results indicated that Tarantula cubensis D6 partially neutralized the deleterious effects of aflatoxin. Homeopathy (2015) 104, 205e210.
Key words: Aflatoxin; Tarantula cubensis D6; Oxidative stress; Biochemical parameters; Rat
Introduction Aflatoxins (AF) are secondary metabolites, naturally occurring mycotoxins produced especially by Aspergillus flavus and Aspergillus parasiticus. They are also known as potential contaminants in human and animal food. The aflatoxin species (AFB1, AFB2, AFG1, and AFG2) are
€ € khan Eraslan, Erciyes Universitesi *Correspondence: Go € ltesi, Farmakoloji ve Toksikoloji Anabilim Dalı, Veteriner Faku Kayseri, Turkey. E-mail: [email protected] Received 26 February 2014; revised 23 July 2014; accepted 6 February 2015
visible under ultraviolet light in different colors. Cases of contamination are based on different variables, such as humidity temperature, the amount of fungi in the environment, physical damage, and storage conditions. AFB1 is metabolized to AFB1-exo-8,9-epoxide by the cytochrome P-450 enzyme system. This extremely reactive metabolite binds to DNA guanines to form adducts, causing toxic effect.1e4 The effects of aflatoxins on multiple organs, such as oxidative stress, and other toxicities have been previously described in various experiments on animals.5e10 The homeopathic medicine Tarantula cubensis D6 (syn.: Mygale cubanensis) is nominally prepared from the Cuban tarantula. The spider is a member of the genus Mygale, which consists of large, mouse-shaped hairy tarantulas.
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Venoms of spiders of the genus Loxosceles cause serious arachnidism, a dangerous systemic reaction including renal failure, severe intravascular coagulation, thrombocytopenia, coma, and convulsions.11 TheranekeronÒ is a homeopathic product which contains Tarantula cubensis D6. Some positive effects including such as demercative, regenerative, anti-inflammatory and resorptive effects are defined in many previous studies.12e18 Tarantula cubensis D6 in homeopathic medicine has been used successfully in the treatment of oral lesions in cattle with bluetongue disease,12 canine oral papillomatosis,13 canine mammary tumors,14 indolent ulcers in cats,15 thrombus in the left anterior descending artery,16 foot-andmouth disease in cattle,17 and endometriosis.18 The effects of administration of Tarantula cubensis D6 on aflatoxin-induced biochemical changes in rats were evaluated on the basis of some oxidative stress marker and other biochemical parameters. There are many studies related to aflatoxin intoxication in rats9,19e22 and other animals.5e7,10 Several treatments are used in detoxification of aflatoxin.8,23e26 But their benefit are not satisfactory. Oxidative stress is one mechanism in the formation of aflatoxin intoxication.27 The effects of Tarantula cubensis D6 on cellular oxidative stress and other biochemical parameters in the case of exposure of aflatoxin has not previously been studied. The present study aims at evaluating the effectiveness of Tarantula cubensis D6 against aflatoxin-induced toxications in rats.
Materials and methods Chemicals The chemicals used in the experiments and analysis were purchased from SigmaeAldrich (St Louis, MO, USA) or Merck (Darmstadt, Germany). Tarantula cubensis D6 (TheranekronÒ) was obtained from Richter Pharma AG Wels, Austria. Aflatoxin production Aflatoxin was produced in rice by using the Aspergillus parasiticus (NRLL 2999) strain according to the method of Shotwell et al.28 as modified by Demet et al.29 The extraction/purification and type analyses of produced aflatoxins from rice flour were carried out according to the R-Biopharm RidascreenÒ kit procedure for total aflatoxin and Nabney and Nesbit,30 respectively. The percentages of AFB1, AFB2, AFG1, and AFG2 in extract were found to be approximately 80%, 10%, 6%, and 4%, respectively. The level of total aflatoxin was estimated by means of the R-Biopharm RidascreenÒ total aflatoxin kit and the method suggested by the producer in extract. Following purification of aflatoxin, organic solvent containing aflatoxin was evaporated. Then aflatoxin was solved in dimethysulfoxide/water (4:6, v/v) for using experiment. Animal material Thirty-two 4to 5-week-old (200e250 g) Sprague-Dawley female rats were used and divided evenly Homeopathy
into four groups. The first group was control. Groups 2, 3, and 4 were given 200 ml/kg.bw/day Tarantula cubensis D6 (TheranekronÒ), 400 mg/kg.bw/day total aflatoxin, and 200 ml/kg.bw/day Tarantula cubensis D6 plus 400 mg/ kg.bw/day total aflatoxin, respectively, for 28 days. Aflatoxin was administered early in the morning via oral gavage. Tarantula cubensis D6 was administered after 6 h the same day by subcutaneous route. Number of animals used in this study was based on earlier aflatoxin studies.8 Determination of oral aflatoxin31,32 and Tarantula cubensis D6 dose,13 previous studies were based on but some modifications were made on application period for Tarantula cubensis D6 considering the severity of aflatoxin intoxication. The protocol of the present study was approved by the Ethics Board for Experimental Animals of Erciyes University. Sample collection and preparations of analysis Blood samples were withdrawn into tubes (without/with anticoagulant) from all animals under a light ether anesthesia. The collected blood samples were centrifuged at 3000 rpm for 10 min at +4 C (Sigma 3K30, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany), and their erythrocytes, plasma and sera were separated. The erythrocytes were washed in saline phosphate buffer (PBS), diluted with equal volumes of PBS and were lysed with ice-cold water.33 Following the collection of blood samples and their deaths, the liver, kidneys, brain and spleen were taken from all the animals. All extracted organs were washed in cold distilled water. Than fat and connective tissues were then removed. The tissues were homogenized with phosphate buffer adjusted to a pH value of 7.4, using a homogenizer (Heidolph, SilentCrusher M) and then the homogenized tissues were centrifuged at 15,000 rpm for 45 min at +4 C, and the supernatant was placed in eppendorf tubes. All samples were stored at 80 C until the analysis. Assessment of oxidative stress parameters Plasma/tissue malondialdehyde (MDA) levels,34,35 blood hemoglobin levels,36 tissue protein levels,37,38 tissue superoxide dismutase (SOD) activities39, catalase (CAT) activities,40 glutathione peroxidase activities (GSH-Px)41 were determined according to the mentioned methods. The MDA results were expressed in units of nmol/ml or nmol/mgprotein. Units of enzymes for tissues SOD, CAT, and GSH-Px were expressed as U/mg protein, U/g protein, and U/g protein, respectively. Units of enzymes for erythrocyte SOD, CAT, and GSH-Px were indicated as U/mgHb, U/ gHb and U/gHb, respectively. All oxidative stress markers and tissue protein levels were measured spectrophotometrically (Helios a, double-beam UV/VIS). Measurement of biochemical parameters in sera Detection of serum glucose, triglyceride, cholesterol, blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT),
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207 Table 1 Effects of Tarantula cubensis D6 and aflatoxin on oxidative stress marker in rat Sample
Groups*
Liver
Untreated control Tarant D6 Aflatoxin Afla + Tarant D6 Untreated control Tarant D6 Aflatoxin Afla + Tarant D6 Untreated control Tarant D6 Aflatoxin Afla + Tarant D6 Untreated control Tarant D6 Aflatoxin Afla + Tarant D6 Untreated control Tarant D6 Aflatoxin Afla + Tarant D6
Kidney
Brain
Spleen
Erythrocytes/plasma
MDA (nmol/ml or nmol/mg protein)
SOD (U/mg protein or U/mgHb)
CAT (U/g protein or U/gHb)
GSH-Px (U/g protein or U/gHb)
2.72 0.14a 2.14 0.26a 5.84 0.65b,y 3.50 0.57a 3.90 0.39a 4.29 0.29a 6.07 0.50b,* 5.82 0.62b,* 12.77 0.70a 16.05 0.99b,y 22.34 1.28c,y 20.01 1.06c,y 6.13 0.18a 5.84 0.31a 7.21 0.22b,# 5.79 0.44a 9.78 0.38a 10.05 0.66a 12.37 0.47b,# 11.05 0.44a,b
1.54 0.12a 0.60 0.06b,y 2.02 0.19c,y 1.00 0.11d,y 2.18 0.13a,b 1.78 0.14a 2.79 0.16c,y 2.41 0.19b,c 2.59 0.21a 3.83 0.32b,y 3.76 0.37b,y 4.74 0.36b,y 1.49 0.08a 1.49 0.17a 2.09 0.19b,* 1.36 0.18a 0.80 0.05a 0.77 0.05a 1.12 0.06b,y 0.65 0.04a
164.11 14.43b 79.71 8.23ay 250.72 32.20cy 52.11 6.69a,y 306.63 18.85a,b 277.60 27.18b 365.51 27.11a 248.39 17.24b 24.85 2.80a 34.82 3.74a,b 39.73 5.86c,# 48.42 5.24c,# 30.44 2.10a 19.81 3.27b,y 21.59 2.12b,y 11.47 1.42c,y 74.82 4.75a 72.87 8.51a 143.64 11.60b,y 69.22 4.50a
305.83 25.05a 414.03 35.40a 673.40 49.70b,y 363.86 32.06a 579.29 46.07b 372.31 58.40a,y 738.66 45.03c,y 488.73 57.04a,b 60.66 5.61a 108.19 7.72b,y 116.42 13.21b,y 132.57 12.09b,y 58.03 5.18a 71.58 4.05b,c,# 79.21 2.45c,# 61.94 2.66ab 42.76 4.67a 40.03 3.41a 67.03 4.43b,y 42.20 2.10a
*Group 1, untreated control; group 2, Tarantula cubensis D6 only; group 3, aflatoxin only; group 4, Tarantula cubensis D6 plus aflatoxin. Values not sharing a common superscript letters (aec) differ significantly at p < 0.05. Statistically significant differences obtained for each parameter between groups is indicated by a versus b, c; b versus c. *p < 0.05, #p < 0.01, and yp < 0.001 according to control.
alkaline phosphatase (ALP), total protein, and albumin levels/activities were performed using the Abbott autoanalyzer (Abbott laboratories, Illinois, USA) and same label kits. Statistical analysis Calculations were carried out using the SPSS 10.0 for Windows’ statistical program. Data were expressed with arithmetical means and means of standard errors. Statistical significance between the experimental groups was calculated by one-way analysis of variance (ANOVA). Individual evaluation of different groups was made in accordance with the Duncan multiple range test. The differences were considered as significant at p < 0.05.
Results Tarantula cubensis D6 (Group 2) Compared with the control group, SOD activity increased in brain whereas liver SOD activity decreased
in this group. CAT activity decreased in liver and spleen. GSH-Px activities decreased in kidney, but increased in brain and spleen. Brain MDA levels increased. On the other hand, no significant alterations were observed in the tissues, erythrocyte/plasmas and serums in the remaining oxidative stress parameters and biochemical parameters (Tables 1 and 2). Aflatoxin (Group 3) Compared with the control group, it was observed that liver, kidney, brain, spleen and plasma MDA levels increased in this group. Liver, kidney, brain, spleen and erythrocyte SOD activity increased. Liver, brain, and erythrocyte CAT activity increased, whereas spleen CAT activity had decreased. GSH-Px activity increased in the liver, kidney, spleen and brain, as well as in erythrocytes. Finally, cholesterol, creatinine, AST, ALP, and ALT activities/ levels were increased, whereas a decrease was detected in glucose, triglyceride, total protein, and albumin. On the other hand, the levels/activities of the remaining
Table 2 Effects of Tarantula cubensis D6 and aflatoxin on some biochemical parameters in rat Parameters
Glucose (mg/dL) Triglyceride (mg/dL) Cholesterol (mg/dL) BUN (mg/dL) Creatinine (mg/dL) AST (U/L) ALT (U/L) ALP(U/L) T-Protein (g/dL) Albumin (g/dL)
Groups Untreated control
Tarant D6
215.75 7.12 117.50 9.36a 77.75 5.86a 15.00 0.81 0.55 0.02a 76.80 7.46a 48.25 4.30a 207.50 29.53a 6.25 0.38a 1.02 0.02a
211.80 13.11 122.20 10.96a 81.20 4.10a 17.80 0.86 0.62 0.02ab 88.80 5.16ab 51.60 6.45a 217.00 33.34a 6.08 0.09a 1.12 0.06a
a
Aflatoxin a
Afla + Tarant D6
174.20 8.57 72.00 4.07b,y 98.60 5.95b,* 18.60 1.96 0.68 0.03b,* 105.25 3.96b,* 83.00 8.01b,# 393.20 46.30b,# 3.98 0.25b,y 0.68 0.05b,y b,*
195.20 8.77a,b 85.20 4.87b,y 87.40 4.11a,b 19.40 0.87 0.56 0.02a 81.60 5.20a 51.60 2.67a 336.20 18.48b,# 5.08 0.17c,y 0.80 0.07b,y
Values not sharing a common superscript letters (aec) differ significantly at p < 0.05. Statistically significant differences obtained for each parameter between groups is indicated by a versus b, c; b versus c. *p < 0.05, #p < 0.01, and yp < 0.001 according to control.
Homeopathy
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parameters were not altered significantly compared with the controls (Tables 1 and 2). Tarantula cubensis D6 plus aflatoxin (Group 4) Compared with the control group, MDA levels in kidney and brain increased in this group. SOD activity increased in brain but decreased in liver. CAT activity in liver and spleen decreased, whereas CAT activity in brain increased. GSHPx activities in brain increased. In addition, triglyceride, total protein, and albumin levels decreased, whereas ALP activities increased. Compared with the control group, no significant difference was determined in the remaining parameters for this group (Tables 1 and 2). In the other words, mentioned remaining data are similar to the control group.
Discussion Compared to control group, no meaningful changes was observed on liver, kidney, spleen and plasma MDA levels in the only Tarantula cubensis D6-only treated group. But a significant an increase in brain MDA levels shows that Tarantula cubensis D6 causes adverse effect on brain tissue. This may be related to the application procedure and duration. Also, compared to control group, significant increase/decrease was seen in SOD, CAT and GSH-Px activities in some tissues. The target organs for aflatoxicosis are particularly liver and kidney.23e25 No negative effects was found on liver and kidney for indicated doses and periods aspect of MDA levels. Also, there are also no negative effects on biochemical parameters. Previous studies on this subject evaluated, on sheep, after Tarantula cubensis D6 application, the levels/activities of MDA and some biochemical parameters such as AST, ALT, creatin, BUN, albumin and total protein, no negative effects have been seen by Dik et al.42 In addition, another study made by Sardari et al.43 on horses, reveals the same results like Dik et al.42 Hence the present study displays similarity to previous studies. The free radical generating mechanism of aflatoxin is related to the biotransformation of aflatoxin B1 via cytocrome enzymes.44 Additionally, other toxic mechanisms of aflatoxins include consumption of vitamin A45 and glutathione storage,9,27 which are strong radical scavengers. Aflatoxins also cause alterations in some biochemical parameters directly reflected to organ injury, such as injuries involving liver and kidney.8,21,23,26 Results obtained in the current study shows that aflatoxins cause negative effects in biological systems. Compared with the control group, the group that was given only aflatoxin had the highest MDA levels. The results demonstrate that aflatoxins are associated with high level of free radical occurrence in biological systems, but these highly active metabolites are not detoxified via antioxidant defense systems. The antioxidant enzymes analyzed in the samples (SOD, CAT, and GSH-Px) were increased or decreased in animals treated with aflatoxin alone. Alterations in the markers suggest that the activities of antioxidant enzymes are insufficient to compensate for the high level of free radical occurrence against the appliHomeopathy
cation of aflatoxin. Increased MDA levels in the biological materials also support these evaluations. These changes in the activities of the antioxidant enzymes may imply induction or consumption during the transformation of free radicals into less harmful or harmless products. In relevant studies conducted on various animals, aflatoxin has been reported to lead changes in oxidative stress parameters in some biological samples; in a study conducted Alm-Eldeen et al.9 which rats received aflatoxin, an increase in liver TBARS levels and an increase in SOD, CAT and GSH-Px activities are observed. El-Nekeety et al.21 detected a meaningful increase kidney and liver MDA levels after the administration of aflatoxin contained diet in rats. Eraslan et al.10 in which mice applied with aflatoxin, an increase was observed in tissue MDA levels, while SOD, CAT and GSH-Px activities seen two-way alterations such as increase or decrease. Kanbur et al.7 determined that aflatoxin increased in tissue MDA levels, mean while decreased/increased in tissue SOD, CAT and GSH-Px activities. Naaz et al.25 reported that administration of aflatoxin resulted increase kidney MDA levels, while SOD, CAT and GSH-Px activities decreased in kidney. The results obtained in the present study are similar to those previous studies. In this study, the changes in AST, ALT, ALP, glucose, triglyceride and cholesterol levels/activities pointed to the functional disorder mainly of the liver,8,21,26,46,47 in the group treated with only aflatoxin. The changes in the total protein levels and creatinine are also determinative of the development of a disorder in protein synthesis metabolism/catabolism and renal dysfunction.48 In same studies, Yousef et al.49 determined decline by treated with aflatoxin in serum glucose levels in rabbits. ElNeekety et al.21 fed rats with aflatoxin with feed. They implied that AST, ALT, ALP, cholesterol levels/activities had increased. In another study conducted by Mathuria and Verma,23 which mice received aflatoxin determined a decrease in protein level and increase in AST, ALT and creatinine levels/activity. Adedara et al.50 detected an increase AST, ALT and ALP activities after the application of aflatoxin in mice. Alm-Eldeen et al.9 which rats applied with aflatoxin, an increase in AST, ALT and ALP and cholesterol levels/activities was observed. In another study, Casado et al.51 applied mice with aflatoxin with feed and determined such exposure cause to decrease triglyceride level. Such alterations were also observed in this study. Therefore this study exhibits similarity to the previously research. In the group treated with aflatoxin and Tarantula cubensis D6 together, oxidative stress markers displayed positive alterations for some samples, in other words, values were closer to those of the control group. For example, MDA values of the tissues except for brain and kidney tissues are similar to the control group. The primary target of aflatoxin is the liver.2,3,24 In this study, positive changes on liver MDA levels and antioxidant enzyme activities shows that Tarantula cubensis D6 can be used against negative effects of aflatoxin on liver. No study has been published about the antioxidant effects of Tarantula
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cubensis but some clinical studies shows that, a decrease of clinical symptoms and increase epithelisation of oral mucosa in cattle with bluetongue disease, has seen after usage of this compound.12 It compound decreases the tumoral formation in canine oral papillomatosis.13 Additionally, it decreases benign canine mammary tumor in dog.14 Moreover, Lotfollahzadeh et al.17 demonstrated healing of lesions of foot-and-mouth disease in cattle and improvement of general condition. Dolapcıoglu et al.18 found that the usage of Tarantula cubensis has shown significant healing in experimental endometritis of rats. According to earlier studies, Tarantula cubensis as a homeopathic formulation had demercative, regenerative, antiinflammatory and resorptive effects on inflammatory, necrotic and proliferative syndromes.12e19 Free radicals have direct or indirect roles on various inflammatory, necrotic, toxic and proliferative cases.52e56 It is considered that, in these cases Tarantula cubensis 6D may have antioxidant effects. In our study, free radical formation which is the mechanism of aflatoxin intoxication and the possible negative effects of these radicals can clearly be seen from oxidative stress markers. These markers on the Tarantula cubensis D6 treated group, reveals antioxidant effects on liver which is primarily affected by aflatoxin.
Conclusions Aflatoxin caused an imbalance in the antioxidant defense and other biological systems of rats. Oxidative stress and toxic effects of Aflatoxins are partly mitigated in the groups given aflatoxin combined with Tarantula cubensis D6 Tarantula cubensis D6 may have potential as a supportive agent for alleviating the toxic effects of aflatoxin especially liver damage.
Conflict of interests
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The authors have no conflicts of interests. 20
References 1 Edlayne G, Simone A, Felicio JD. Chemical and biological approaches for mycotoxin control: a review. Recent Pat Food Nutr Agric 2009; 1: 155e161. 2 Ingawale DK, Mandlik SK, Naik SR. Models of hepatotoxicity and the underlying cellular, biochemical and immunological mechanism(s): a critical discussion. Environ Toxicol Pharmacol 2013; 37: 118e133. 3 Marin S, Ramos AJ, Cano-Sancho G, Sanchis V. Mycotoxins: occurrence, toxicology, and exposure assessment. Food Chem Toxicol 2013; 60: 218e237. 4 Mohd-Redzwan S, Jamaluddin R, Abd-Mutalib MS, Ahmad Z. A mini review on aflatoxin exposure in Malaysia: past, present and future. Front Microbiol 2013; 4: 334. http://dx.doi.org/10.3389/fmicb.2013.00334. 5 Eraslan G, Akdogan M, Yarsan E, S¸ahindokuyucu F, Es¸siz D, Altıntas¸ L. The effects of aflatoxins on oxidative stress in broiler chickens. Turk J Vet Anim Sci 2005; 29: 701e707. 6 Eraslan G, Cam Y, Eren M, Liman BC, Atalay O, Seybek N. Aspects of using N-acetylcysteine in aflatoxicosis and its evaluation
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25
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regarding some lipid peroxidation parameters in rabbits. Bull Vet Inst Pulawy 2005; 49: 243e247. Kanbur M, Eraslan G, Soyer Sarıca Z, Aslan O. The effects of evening primrose oil on lipid peroxidation induced by subacute aflatoxin exposure in mice. Food Chem Toxicol 2011; 49: 1960e1964. Akc¸am M, Artan R, Yilmaz A, Ozdem S, Gelen T, Nazıro glu M. Caffeic acid phenethyl ester modulates aflatoxin B1-induced hepatotoxicity in rats. Cell Biochem Funct 2013; 31: 692e697. Alm-Eldeen AA, Mona MH, Shati AA, El-Mekkawy HI. Synergistic effect of black tea and curcumin in improving the hepatotoxicity induced by aflatoxin B1 in rats. Toxicol Ind Health 2013; http:// dx.doi.org/10.1177/0748233713491807. € Karabacak M. The antioxidant efEraslan G, Kanbur M, Aslan O, fects of pumpkin seed oil on subacute aflatoxin poisoning in mice. Environ Toxicol 2013; 28: 681e688. Richardson-Boedler C. The brown spider Loxosceles laeta: source of the remedy Tarentula cubensis? Homeopathy 2002; 91: 166e170. Albay MK, S¸ahinduran S¸, Kale M, Karakum MC¸, Sezer K. Influence of Tarantula cubensis extract on the treatment of the oral lesions in cattle with bluetongue disease. Kafkas Univ Vet Fak Derg 2010; 16: 593e596. Icen H, Sekin S, Simsek A, Kochan A, Tunik S. The Efficacy of Tarantula cubensisi extract (Theranekron) in treatment of canine oral papillomatosis. Asian J Anim Vet Adv 2011; 7: 744e749. G€ ultekin N, Vural MR. The effect of Tarantula cubensis extract applied in pre and postoperative period of canine mammary tumours. JIVS 2007; 2: 13e23. Yardımcı C, Yardımcı B. Bir kedide indolent € ulser olgusu. Ankara € Univ Vet Fak Derg 2008; 55: 65e67. Jain SKA, Patel B, Larsen T, Alexander P, David S. A thrombus in the left anterior descending artery after the use of multiple alternative medicines. Med Pract Rev 2013; 4: 14e16. Lotfollahzadeh S, Alizadeh MR, Mohri M, Dezfouli MRM. The Therapeutic Effect of Tarentula cubensis extract (Theranekron) in foot-and-mouth disease in cattle: a randomised trial in an endemic setting. Homeopathy 2012; 101: 159e164. Dolapcioglu K, Dogruer G, Ozsoy S, et al. Theranekron for treatment of endometriosis in a rat model compared with medroxyprogesterone acetate and leuprolide acetate. Eur J Obstet Gynecol Reprod Biol 2013; 170: 206e210. Yener Z, Celik I, Ilhan F, Bal R. Effects of Urtica diocia L. seed on lipid peroxidation, antioxidants and liver pathology in aflatoxininduced tissue injury in rats. Food Chem Toxicol 2009; 47: 418e424. El-Agamy DS. Comparative effects of curcumin and resveratrol on aflatoxin B(1)-induced liver injury in rats. Arch Toxicol 2010; 84: 389e396. El-Nekeety AA, Mohamed SR, Hathout AS, Hassan NS, Aly SE, Abdel-Wahhab MA. Antioxidant properties of thymus vulgaris oil against aflatoxin-induce oxidative stress in male rats. Toxicon 2011; 57: 984e991. Hathout AS, Mohammed SR, El-Nekeety AA, Hassan NS, Aly SE, Abdel-Wahhab MA. Ability of Lactobacillus Casei and Lactobasillus reuteri to protect against oxidative stress in rat fed aflatoxinscontamineted diet. Toxicon 2011; 58: 179e186. Mathuria N, Verma RJ. Ameliorative effect of curcumin on aflatoxin-induced toxicity in serum of mice. Acta Pol Pharm 2008; 65: 339e343. Choi KC, Chung WT, Kwon JK, et al. Inhibitory effects of quercetin on aflatoxin B1-induced hepatic damage in mice. Food Chem Toxicol 2010; 48: 2747e2753. Naaz F, Abdin MZ, Javed S. Protective effect of esculin against prooxidant B1-induced nephrotoxicity in mice. Mycotoxin Res 2014; 30: 25e32. Nili-Ahmadabadi A, Tavakoli F, Hasanzadeh G, Rahimi H, Sabzevari O. Protective effect of pretreatment with thymoquinone
Homeopathy
Tarantula and aflatoxin M Karabacak et al
210
27 28 29 30 31
32
33
34
35 36
37
38 39 40 41
42
43
against aflatoxin B(1) induced liver toxicity in mice. Daru 2011; 19: 282e287. Alpsoy L, Yalvac ME. Key roles of vitamins A, C, and E in aflatoxin B1-induced oxidative stress. Vitam Horm 2011; 86: 287e305. Shotwell OL, Hesseltine CW, Stubblefield RD, Sorenson WG. Production of aflatoxin on rice. Appl Microbiol 1966; 14: 425e428. Demet O, Oguz H, C¸elik I, Nizamlıo glu F. Pirinc¸te Aflatoksin € Uretilmesi. Vet Bil Derg 1995; 11: 19e23. Nabney J, Nesbitt BF. A spectrophotometric method for determining of the aflatoxins. Analyst 1965; 90: 155e160. Egner PA, Yu X, Johnson JK, et al. Identification of aflatoxin M1N7-guanine in liver and urine of tree shrews and rats following administration of aflatoxin B1. Chem Res Toxicol 2003; 16: 1174e1180. Chen ZY, Yan RQ, Qin GZ, Qin LL. Effect of six edible plants on the development of AFB1-induced gamma-glutamyltranspeptidase-positive hepatocyte foci in rats. Zhonghua Zhong Liu Za Zhi 1987; 9: 109e111. Winterbourn CC, Hawkins RE, Brian M, Carrell RW. The estimation of red cell superoxide activity. J Lab Clin Med 1975; 85: 337e341. Yoshioka K, Kawada K, Shimada T, Mori M. Lipid peroxidation in maternal and cord blood and protective mechanism against activedoxygen toxicity in the blood. Am J Obstet Gynecol 1979; 135: 372e376. Ohkawa H, Ohishi N, Yagi K. Reaction of linoleic acid hydroperoxide with thiobarbituric acid. J Lipid Res 1978; 19: 1053e1057. Fairbanks VF, Klee GG. Biochemical aspect of haematology. In: Tietz NW (ed). Fundamentals of Clinical Chemistry. 3th edn. Philadelphia: WB Saunders, 1987, pp 803e806. Lowry OH, Rosebrough NJ, Farr AL, Randali RJ. Protein measurment with the folin phenol reagent. J Biol Chem 1951; 193: 265e275. Miller GL. Protein determination for large numbers of samples. Anal Chem 1959; 31: 964. Sun Y, Oberley LW, Li Y. A simple for clinical assay of superoxide dismutase. Clin Chem 1988; 34: 497e500. Luck HS. Catalase. In: Bergmeyer HU (ed). Methods of Enzymatic Analysis. London: Academic Press, 1965, pp 885e894. Paglia PE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158e169. Dik B, Er A, Corum O. Effect of alcoholic extract of Tarantula cubensis (TheranekronÒ) on serum thiobarbituric acid-reactive species concentrations in sheep. Eurasian J Vet Sci 2014; 30: 68e71. Sardari K, Mohri M, Sabzevari S, Fathi B. Effects of the TheranekronÒ an alcoholic extract of the tarantula cubensis on he-
Homeopathy
44
45
46
47
48
49
50
51
52
53
54
55
56
matology and serum biochemical properties in horses. IJVST 2011; 3: 9e16. Abrar M, Anjum FM, Butt MS, et al. Aflatoxins: biosynthesis, occurrence, toxicity, and remedies. Crit Rev Food Sci Nutr 2013; 53: 862e874. Pimpukdee K, Kubena LF, Bailey CA, Huebner HJ, AfriyieGyawu E, Phillips TD. 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 2004; 83: 737e744. Eraslan G, Essiz D, Akdogan M, et al. Efficacy of dietary sodium bentonite against subchronic exposure to dietary aflatoxin in broilers. Bull Vet Inst Pulawy 2006; 50: 107e112. O guz H, Kurto glu F, Kurto glu V, Birdane YO. Evaluation of biochemical characters of broiler chickens during dietary aflatoxin (50 and 100 ppb) and clinoptilolite exposure. Res Vet Sci 2002; 73: 101e103. Abbes S, Ben Salah-Abbes J, Abdel-Wahhab MA, Ouslati R. Immunotoxicological and biochemical effects of aflatoxins in rats prevented by tunisian montmorillonite with reference to HSCAS. Immunopharmacol Immunotoxicol 2010; 32: 514e522. Yousef MI, Salem MH, Kamel KI, Hassan GA, El-Nouty FD. Influence of ascorbic acid supplementation on the haematological and clinical biochemistry parameters of male rabbits exposed to aflatoxin B1. J Environ Sci Health B 2003; 38: 193e209. Adedara IA, Owumi SE, Uwaifo AO, Farombi EO. Aflatoxin B1 and ethanol co-exposure induces hepatic oxidative damage in mice. Toxicol Ind Health 2010; 26: 717e724. Casado JM, Theumer M, Masih DT, Chulze S, Rubinstein HR. Experimental subchronic mycotoxicoses in mice: individual and combined effects of dietary exposure to fumonisins and aflatoxin B1. Food Chem Toxicol 2001; 39: 579e586. Piechota-Polanczyk A, Fichna J. Review article: the role of oxidative stress in pathogenesis and treatment of inflammatory bowel diseases. Naunyn Schmiedebergs Arch Pharmacol 2014; 387: 605e620. Rodriguez F, Bonacasa B, Fenoy FJ, Salom MG. Reactive oxygen and nitrogen species in the renal ischemia/reperfusion injury. Curr Pharm Des 2013; 19: 2776e2794. Flora SJ, Shrivastava R, Mittal M. Chemistry and pharmacological properties of some natural and synthetic antioxidants for heavy metal toxicity. Curr Med Chem 2013; 20: 4540e4574. Novo E, Cannito S, Paternostro C, Bocca C, Miglietta A, Parola M. Cellular and molecular mechanisms in liver fibrogenesis. Arch Biochem Biophys 2014; 548: 20e37. Mates JM, Segura JA, Alonso FJ, Marquez J. Oxidative stress in apoptosis and cancer: an update. Arch Toxicol 2012; 86: 1649e1665.
