http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, 2015; 37(2): 297–304 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2014.983017
CLINICAL STUDY
Synergistic protective effects of ceftriaxone and ascorbic acid against subacute deltamethrin-induced nephrotoxicity in rats Mohamed M. Abdel-Daim1 and Ashraf El-Ghoneimy2 Faculty of Veterinary Medicine, Pharmacology Department, Suez Canal University, Ismailia, Egypt and 2Faculty of Veterinary Medicine, Pharmacology Department, South Valley University, Qena, Egypt
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1
Abstract
Keywords
Deltamethrin (DLM) is a synthetic class II pyrethroid acaricide and insecticide widely used for veterinary and agricultural purposes. However, its animal and human exposure leads to nephrotoxicity. Our experimental objective was to evaluate protective effects of ceftriaxone and/or ascorbic acid against DLM-induced renal injury in male Wistar albino rats. DLM-treated animals revealed significant alterations in serum biochemical parameters related to renal injury; urea, uric acid and creatinine. There was a significant increase in renal lipid peroxidation and a significant inhibition in antioxidant biomarkers. Moreover, DLM significantly reduced serum acetylcholinesterase (AChE) activity. In addition, It induced serum and kidney tumor necrosis factor-a (TNF-a). Both ceftriaxone and ascorbic acid protect against DLM-induced biochemical alterations in serum and renal tissue when used alone or in combination along with DLMintoxication. Furthermore, both ceftriaxone and ascorbic acid produced synergetic nephroprotective and antioxidant effects. Therefore, it could be concluded that ceftriaxone and/or ascorbic acid administration able to minimize the toxic effects of DLM through their free radical-scavenging and potent antioxidant activity.
Acetylcholinesterase, ceftriaxone, deltamethin, kidney, nephrotoxicity, rats antioxidant,tumor necrosis factor, vitamin C
Introduction Insecticides are extensively used worldwide for control of veterinary, agricultural, and domestic insect pests and disease vectors to maintain human and animal health and enhance the food production. Consequently, the intensive use of pesticides leads to serious ecological hazards and potential health problems, including severe acute, subacute, and chronic cases of animals and human poisonings and therefore, are main reasons of concern1. Human are potentially exposed to insecticides either directly, as manufacturing workers and field applicators (workers in agriculture and in green-houses), or indirectly, through ingestion of contaminated food. In addition, it is likely that a high amount of these chemicals, and their metabolites reach estuaries and rivers via run-off from farmland that are might be toxic to wildlife2. Among pesticides are pyrethroid insecticides, which are synthetic derivatives of the natural pyrethrins, and have replaced the organophosphorus compounds within the last 10–15 years3. Deltamethrin (DLM), a synthetic type II pyrethroid, is commonly used in veterinary, agricultural, and public health practices and highly effective against a broad spectrum of insects. In the veterinary field, it is used for the control of ectoparasites in animals, poultry, and fish, Address correspondence to Mohamed M. Abdel-Daim, Faculty of Veterinary Medicine, Pharmacology Department, Suez Canal University, Ismailia 41522, Egypt. Tel/Fax: +20643207052; E-mail: [email protected], [email protected]
History Received 28 June 2014 Revised 4 September 2014 Accepted 26 September 2014 Published online 19 November 2014
preventing vector-borne diseases. The main important sources of the DLM human and animal exposure are polluted food and water, as it is readily absorbed orally4. Several studies have shown that DLM caused changes in hematological and biochemical parameters as well as oxidative stress status of the experimental animals5,6. While studies describing the protection against DLM-induced injury and oxidative stress are limited5,7. The cells overcome oxidative damage by either expelling of the damaged nucleotides and lipid peroxidation products or directly quenching free radicals via endogenous non-enzymatic and enzymatic antioxidants system8–10. Ceftriaxone (CTX) is a broad-spectrum antimicrobial cephalosporin, resistant to beta-lactamases, with potent activities against gram-positive and gram-negative bacteria11. It is effective against Streptococcus pneumonia, Streptococcus faecalis, Streptococcus pyogenes, Neisseria gonorrhoeae, Brucella melitensis, and Haemophilus influenza11. CTX is reported to inhibit transpeptidase enzymes which are responsible for the last step in bacterial cell wall synthesis12. Other non-antibiotic pharmacodynamic effects of CTX on animal studies demonstrated that it could reduce morphine tolerance and dependence13, ameliorate diabetic hyperalgesia14, and diminish neuropathic pain15. Moreover, CTX attenuated nephrotoxicity in rats induced by cadmium16, cyclosporine A17, tobramycin18, and isepamicin19. Ascorbic acid (AA), vitamin C is a water-soluble vitamin, has a many function in the body and probably the most crucial
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antioxidant in extracellular fluids20. It is an important vitamin and an essential component in the diet of many mammalian species as it cannot be synthesized in the body of these species21. It is highly water soluble and acts as an effective reducing and chelating agent20. AA is the most effective antioxidant in preventing lipid peroxidation induced by peroxyl radicals, and considered as a potent free radical quenching agent22,23. Moreover, it may act in an indirect way by restoring other antioxidants such as the fat-soluble vitamin E20. To the best of our knowledge, the role of CTX and AA, alone or in combination against DLM-induced serum biochemical alteration related to nephrotoxicity as well as renal lipid peroxidation and antioxidant status in rats has not been studied yet. Therefore, the current study was designed to investigate the alterations in serum biochemical parameters related renal damage as well as renal lipid peroxidation and oxidative stress induced by DLM in rats. Moreover, the role of ceftriaxone and/or ascorbic acid in alleviating these DLMinduced hazard effects could be evaluated.
(25 ± 2 C). Food and water were provided ad libitum. Animal handling and experimental design procedures were approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt (the approval no 20147). All precautions were taken into consideration to avoid animal suffering. Rats were kept for 1 week before the beginning of the experiment for acclimatization. Next, rats were randomly divided into seven different groups; eight animals in each. Rats in the first group were administered normal saline and kept as control. The second and third groups were given CTX (100 mg/kg bw SC)24 and ascorbic acid (100 mg/kg bw orally)25, respectively, daily. The fourth group received a daily dose of DLM (2 mg/kg, orally)26. The fifth and sixth groups were given CTX and AA, respectively, at the same dose regimen used for the second and third groups before DLM administration at the same dose and regimen used for the fourth group. The seventh group was given both CTX and AA 1 h before DLM administration (Table 1). All treatments were carried out for 4 weeks. Serum collection and tissue preparation
Materials and methods Chemicals Deltamethrin (ButoxÕ 50 mg/mL) was obtained from Intervet Co., France as a commercial product in clinical formulation for veterinary use. Ceftriaxone (CeftriaxoneÕ vial, 250 mg, 500 mg, and 1 g crystalline powder) was kindly given by Sandoz-Novartis, Egypt. Ascorbic acid was purchased from Adwia Pharmaceuticals, Cairo, Egypt. All kits were purchased from Biodiagnostics, Egypt except, TNF-a from Assay Designs Inc. (Ann Arbor, MI) and AChE from (BioVision Inc., Milpitas, CA). All other chemicals used in the experiment were of analytical grade. Animals and experimental design Fifty six male Wistar rats, weighing 180 ± 20 g, were bought from the Egyptian Organization for Biological Products and Vaccines. Rats were kept in a ventilated animal house of normal light–dark cycle (12 h light/dark) and temperature
At the end of experiment (24 h after the DLM dose), blood samples were collected via retro-orbital plexus. Blood samples were left to clot at room temperature, and centrifuged at 3000 rpm for 15 min. Sera were then separated and stored at 20 C as aliquots for further biochemical analysis. After blood collection, rats were sacrificed by cervical decapitation. Kidneys were rapidly excised from each animal, recovered from surrounding connecting tissue, and washed with 0.9% NaCl solution and distilled water for removal of the blood. Kidneys next were blotted over a piece of filter paper and perfused with a 50 mM sodium phosphate buffer saline (100 mM Na2HPO4/NaH2PO4; pH 7.4) in an ice-containing medium, containing 0.1 mM ethylene di amine tetra acetic acid (EDTA) to remove any clots and blood cells. Subsequently, renal tissues were homogenized in 5–10 mL cold buffer per gram tissue and centrifuged at 5000 rpm for 30 min. The resulting supernatant was then transferred into Eppendorf tubes, and kept at 80 C in a deep freezer until used for various biochemical assays.
Table 1. Experimental design, summary of different rat groups and their treatment. Group
CTX
AA
DLM
Control CTX
– Ceftriaxone at 100 mg/kg body weight daily for 4 weeks –
– –
– –
AA DLM DLM–CTX DLM–AA DLM–CTX–AA
– Ceftriaxone at 100 mg/kg body weight daily for 4 weeks, 1 h before DLM dose – Ceftriaxone at 100 mg/kg body weight daily for 4 weeks, 1 h before DLM dose
Ascorbic acid at 100 mg/kg body weight daily – – Ascorbic acid at 100 mg/kg body weight daily, 1 h before DLM administration Ascorbic acid at 100 mg/kg body weight daily, 1 h before DLM administration
– Deltamethrin 20 mg/kg body weight, daily for 4 weeks Deltamethrin 20 mg/kg body weight, daily for 4 weeks Deltamethrin 20 mg/kg body weight, daily for 4 weeks Deltamethrin 20 mg/kg body weight, daily for 4 weeks
DOI: 10.3109/0886022X.2014.983017
Ceftriaxone and vitamin C against deltamethrin nephrotoxicity
Serum biochemical analysis The sera stored at 20 C were used for estimation of serum renal injury marker products: creatinine, urea, and uric acid were determined according to Larsen 27, Coulombe and Favreau28, and Whitehead et al.29, respectively. Acetylcholinesterase (AChE) was measured according to Ellman et al.30.
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Evaluation of tissue lipid peroxidation and antioxidant enzymes Lipid peroxidation was evaluated by measurement of MDA content in renal tissues according to Mihara and Uchiyama31. Serum nitric oxide (NO) determined according to Green et al.32. The non-enzymatic antioxidant biomarker reduced glutathione (GSH) was assessed according to Beutler et al.33 and glutathione peroxidase (GSH-Px) according to Paglia and Valentine34. The enzymatic antioxidant biomarkers superoxide dismutase (SOD) was evaluated according to Nishikimi et al.35 and catalase (CAT) according to Aebi36. In addition, total antioxidant capacity (TAC) was determined according to Koracevic et al.37. Serum and kidney TNFa assays Serum and renal tissue TNF-a levels were measured using already made kits from R&D Systems (Minneapolis, MN) according to the manufacturer protocol. Briefly, in a microplate, 50 mL of the assay diluent was added to each well. Then, 50 mL of the diluted serum or renal tissue homogenate supernatant samples were added to each well and gently mixed by tapping the plate frame for 1 min then the plate was covered and incubated at room temperature for 2 h. After incubation, each well in the plate was then aspirated and washed five times with the kit washing buffer, then 100 mL substrate solutions were added and incubated at room temperature in dark for 30 min. The optical density was then read at 450 nm using 96-well microtiter plates ELISA Figure 1. Protective effect of ceftriaxone and/ or ascorbic acid against deltamethrin-induced alterations in serum uric acid levels. Note: DLM, Deltamethrin;CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
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reader. TNF-a level was calculated from a standard curve and multiplied by the dilution factor. Statistical analysis Data are presented as mean ± SE. Statistical significance of the data was analyzed using SPSS Program (Statistical Packages for Social Sciences, SPSS Inc., Chicago, IL) version 16. For comparison, One-Way analysis of variance (ANOVA) test followed by Turkey’s range test for post-hoc analysis. Statistical significance was acceptable to a level of p 0.05.
Results Serum biochemical analysis The effects of DLM intoxication as well as the preventive effects of CTX and/or AA on serum renal injury markers; uric acid, urea, and creatinine are shown in (Figures 1–3). Significant increases (p 0.05) in these markers were recorded in DLM intoxicated rats as compared to the untreated control group (332.68, 334.83, and 2266.36, respectively). Pre-treatment with CTX at doses of 100 mg/kg significantly (p 0.05) reduced the serum renal injury markers: uric acid, urea, and creatinine (about 54.51, 60.04, and 42.01%, respectively), while AA pre-treatment at a dose of 100 mg/kg significantly (p 0.05) reduced these biomarkers (about 56.41, 61.41, and 43.46%, respectively) compared with DLM-intoxicated group. Moreover, both CTX and AA were given in combination and significantly (p 0.05) reduced the same parameters more than when each of them used alone (about 31.70, 31.58, and 7.64%, respectively). Moreover, DLM intoxication significantly (p 0.05) reduced (51.84%) AChE as compared to the control rats. At the same time, pretreatment with CTX, AA, or both in combination significantly (p 0.05) increased serum AChE level (154.83, 160.50, and 178.87%, respectively) compared with the DLM-intoxicated rats (Figure 4).
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Figure 2. Protective effect of ceftriaxone and/ or ascorbic acid against deltamethrin-induced alterations in serum urea levels. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
Figure 3. Protective effect of ceftriaxone and/ or ascorbic acid against deltamethrin-induced alterations in serum creatinine levels. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
There were no significant changes in serum markers in rats received CTX or AA only at a dose of 100 mg/kg (second and third groups, respectively) if compared to the normal control (first group), indicating the safety of CTX and AA at the selected doses used in this study (Figures 1–4). Renal lipid peroxidation and antioxidant biomarkers The effects of DLM intoxication as well as preventive effects of CTX and/or AA on renal tissue homogenate lipid peroxidation and antioxidant parameters are shown in Table 2. In DLM-intoxicated group, there was a significant increase (p 0.05) in renal MDA and NO content (373.22 and
252.24%, respectively) was observed compared to the control group. On the other hand, renal GSH, GSH-Px, SOD, CAT, and TAC were significantly (p 0.05) decreased (51.06, 51.52, 42.92, 45.98, and 71.11%, respectively). Concerning DLM–CTX group, renal MDA and NO were significantly (p 0.05) decreased (76.68 and 65.61%, respectively) while GSH, GSH-Px, SOD, CAT, and TAC were significantly (p 0.05) increased (154.24, 152.14, 179.91, 154.17, and 124.12%, respectively) compared to DLM-intoxicated group. Regarding DLM–AA group, renal MDA and NO were significantly (p 0.05) decreased (49.40 and 58.58%, respectively), while GHS, GSH-Px, SOD, CAT, and TAC were significantly (p 0.05) increased (about 185.53, 172.15,
Ceftriaxone and vitamin C against deltamethrin nephrotoxicity
DOI: 10.3109/0886022X.2014.983017
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Figure 4. Protective effect of ceftriaxone and/ or ascorbic acid against deltamethrin-induced alterations in serum acetylcholinesterase (AChE) activity. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
Table 2. Protective effect of ceftriaxone and/or ascorbic acid against deltamethrin-induced alterations in renal tissue malondialdehyde, nitric oxide, and antioxidant biomarkers. Experimental groups Parameters MDA (nmol/g) NO (mmol/g) GSH (mg/g) GSH-Px (U/g) SOD (U/g) CAT (U/g) TAC (mmol/g)
Control a
4.47 ± 0.18 7.56a ± 0.40 8.06a ± 0.31 5.72a ± 0.34 18.22a ± 1.10 0.33a ± 0.03 1.34a ± 0.04
CTX a
4.39 ± 0.19 7.40a ± 0.35 8.29a ± 0.36 6.02a ± 0.30 19.76a ± 1.49 0.33a ± 0.02 1.41a ± 0.06
AA a
4.35 ± 0.18 7.31a ± 0.43 7.68a ± 0.35 6.49a ± 0.25 20.57a ± 1.35 0.34a ± 0.02 1.47a ± 0.07
DLM b
16.67 ± 1.12 19.08b ± 1.36 4.17b ± 0.32 2.95b ± 0.22 7.82b ± 0.57 0.15b ± 0.01 0.95b ± 0.07
DLM–CTX c
12.78 ± 0.48 12.50c ± 0.58 6.43c ± 0.27 4.49c ± 0.30 14.07c ± 0.77 0.23c ± 0.01 1.18c ± 0.05
DLM–AA d
8.24 ± 0.56 11.18c ± 0.69 7.47ac ± 0.30 5.08ac ± 0.40 16.07ac ± 0.82 0.28ac ± 0.02 1.35a ± 0.04
DLM–CTX–AA 5.43a ± 0.20 8.41a ± 0.37 8.19a ± 0.28 5.92a ± 0.25 20.26a ± 0.90 0.32a ± 0.02 1.44a ± 0.05
Notes: Data are expressed as means ± SE (n ¼ 8). DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid; MDA, Malondialdehyde; NO, Nitric oxide; GSH, Reduced glutathione; GSH-Px, Glutathione peroxidase; SOD, Superoxide dismutase; CAT, Catalase; TAC, Total antioxidant capacity. Within the same row, different letters (a–d) means statistical significance at (p50.05).
205.34, 186.67, and 141.55%, respectively). In addition, both CTX and AA were given in combination, significantly (p 0.05) reduced renal MDA and NO level (32.56 and 44.07%, respectively) and increased GHS, GSH-Px, SOD, CAT, and TAC more than when each of them given separately (about 196.40, 200.68, 259.09, 213.33, and 150.98%, respectively; Table 2). Serum and kidney TNF-a assays DLM intoxication significantly (p 0.05) increased both serum and renal tissue TNF-a (531.29 and 842.90%, respectively) compared with control rats (Figures 5 and 6). Pretreatment with CTX significantly (p 0.05) reduced both serum and renal TNF-a levels (42.82 and 40.54%, respectively). Similarly, AA pretreatment induced significant (p 0.05) reduction (about 43.74 and 33.36%, respectively). Moreover, when both CTX and AA were given in combination induced more significant (p 0.05) reduction in these levels (about 21.67 and 14.52%, respectively) compared to DLM-intoxicated rats (Figures 5 and 6).
Discussion Reactive oxygen species (ROS) are continuously produced inside the animal and human body as a result of exposure to plenty of exogenous chemicals, toxins, and drugs in our environment and/or endogenous metabolic cascades involving redox enzymes and electron transport mechanism9,38. In normal circumstances, there is balance between the ROS generated and the antioxidants exist, as the ROS produced are neutralized by the endogenous antioxidants8,39. Deleterious effects caused by ROS occur due to an imbalance between the production and inactivation of these species leading to disturbance in normal cellular physiology and different pathological states10,40. Aging process as well as many degenerative diseases, including Alzheimer, diabetes, rheumatoid arthritis, cancer, cataract, coronary heart disease, and stroke occurs due to excessive free radicals’ productions41–45. Deltamethrin, a type II pyrethroid insecticide has been widely used in veterinary, agriculture, and industrial practice, that would be potentially an exposure hazard to animals and
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Figure 5. Protective effect of ceftriaxone and/or ascorbic acid against deltamethrininduced alterations in serum TNF-a levels. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
Figure 6. Protective effect of ceftriaxone and/or ascorbic acid against deltamethrininduced alterations in renal tissue TNF-a levels. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
human as well46. Although several investigations about DLM toxicity have been published, little reports have been explained the preventive agents used against such a toxicity and mechanism of their ameliorative action5,6. In this study, the renal injuries caused by DLM might induce the oxidative stress due to free radical production. DLM intoxication significantly (p 0.05) increased serum renal injury markers; urea, uric acid and creatinine. Moreover, it significantly (p 0.05) reduced serum AchE level. DLM treatment significantly (p 0.05) increased lipid peroxidation through the elevation of kidney MDA level, significantly (p 0.05) decreased kidney enzymatic; SOD, CAT, and
GSH-Px as well as non-enzymatic; GSH antioxidant level. In addition, it significantly (p 0.05) increased both serum and renal tissue TNF-a compared with the control group. All these effects are involved in the cascade of events leading to DLM-mediated kidney oxidative stress and toxicity. This indicates that renal injuries induced by DLM is due to oxidative stress that arises as a result of excessive ROS production, which have been known to attack various cellular molecules, including lipids and causing lipid peroxidation. The antioxidant enzymes’ activities, including the enzymes involved in glutathione metabolism were also disrupted in DLM-intoxicated group, suggesting the involvement of
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DOI: 10.3109/0886022X.2014.983017
Ceftriaxone and vitamin C against deltamethrin nephrotoxicity
oxidative stress in DLM-mediated renal damage. These results are consistent with the literature and point towards the role of ROS in DLM-mediated injury and toxicity. The insecticide, DLM was reported to increase serum urea and creatinine, urinary glucose, and renal tissue MDA, while antioxidant markers were decreased in rats5,6. Another study DLM intoxication reduced AChE in serum and different tissues of rats and many aquatic animals47–49. In addition, It significantly increased the TNF-a in rats serum50. In the current study, the pre-administration of CTX and/or AA at a dose level of 100 mg/kg significantly reduced the serum renal injury markers. Moreover, they reduced the lipid peroxidation in kidney tissues. In addition, there were significant elevations of renal antioxidant enzymes and glutathione levels. Furthermore, they significantly increased serum AChE and reduced serum and kidney TNF-a. Both CTX and ascorbic acid induced synergistic protective effects against DLM-induced serum and tissues’ biochemical alterations. These results were in agreement with many previous literatures, which examined the nephroprotective and antioxidant effects of CTX against many drugs and xenobioticsinduced nephrotoxicity16–19,51. Pre-treatment with AA might play a role in reducing the toxic effect of DLM, and its powerful antioxidant properties seem to mediate such an ameliorative effect, indicated by the normalization of altered hematological and biochemical changes induced by DLM intoxication in rats and catfish51–53. The protective effect of CTX and/or ascorbic acid against DLM-induced oxidative stress in our rat model could be either direct by scavenging ROS and inhibition of lipid peroxidation and/or indirect through the enhancement of the activity SOD and CAT and the enzymatic free radicals’ scavengers in the cells. Therefore, CTX and ascorbic acid could be used in combination to prevent and treat renal injuries, especially those induced by oxidative stress.
Conclusion Oxidative stress plays a major role in DLM-induced nephrotoxicity. Antioxidants have been proven to be effective in preventing DLM-induced toxicity in several previous interventions. CTX and AA are potent antioxidants, which are reported to ameliorate the effect of many known nephrotoxic agents. In this study, DLM exposure resulted in varying degrees of inhibition in antioxidant enzymes, lipid peroxidation induction, and alterations of serum biochemical parameters. CTX and/or AA pre-treatment provided sufficient protection in terms of serum and tissues’ biochemical alteration, activity of antioxidant markers and oxidative stress, especially when given in combination.
Acknowledgements The authors would like to thank Sandoz-Novartis, Egypt, for supplying CeftriaxoneÕ used in our experiment. The company had neither role in designing the experiment nor in the publication process. This research received no grant from any funding agency.
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Declaration of interest The authors declare that there are no conflicts of interest.
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M.M. Abdel-Daim and A. El-Ghoneimy histopathology in male rabbit: Antioxidant role of vitamin C. Food Chem Toxicol. 2005;43(12):1743–1752. Kojo S. Vitamin C: Basic metabolism and its function as an index of oxidative stress. Curr Med Chem. 2004;11(8):1041–1064. Amin KA, Hashem KS. Deltamethrin-induced oxidative stress and biochemical changes in tissues and blood of catfish (Clarias gariepinus): antioxidant defense and role of alpha-tocopherol. BMC Vet Res. 2012;8:45. Li SH, Ryu JH, Park SE, et al. Vitamin C supplementation prevents testosterone-induced hyperplasia of rat prostate by down-regulating HIF-1alpha. J Nutr Biochem. 2010;21(9):801–808. Abdul-Hamid M, Salah M. Lycopene reduces deltamethrin effects induced thyroid toxicity and DNA damage in albino rats. J Basic Appl Zool. 2013;66(4):155–163. Larsen K. Creatinine assay in the presence of protein with LKB 8600 Reaction Rate Analyser. Clin Chim Acta. 1972; 38(2):475–476. Coulombe JJ, Favreau L. A new simple semimicro method for colorimetric determination of urea. Clin Chem. 1963;9:102–108. Whitehead TP, Bevan EA, Miano L, Leonardi A. Defects in diagnostic kits for determination of urate in serum. Clin Chem. 1991;37(6):879–881. Ellman GL, Courtney KD, Andres Jr V, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88–95. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem. 1978; 86(1):271–278. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982;126(1):131–138. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med. 1963;61: 882–888. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967;70(1):158–169. Nishikimi M, Appaji N, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun. 1972; 46(2):849–854. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–126. Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V. Method for the measurement of antioxidant activity in human fluids. J Clin Pathol. 2001;54(5):356–361. Ibrahim A, Abdel-Daim M. Modulating effects of Spirulina platensis against tilmicosin-induced cardiotoxicity in mice. Cell J (Yakhteh). 2015. (Epub ahead of print).
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39. EL-Sabagh M, Abd Eldaim M, Dahi H, Abdel-Daim M. Effects of Spirulina platensis algae on growth performance, antioxidative status and blood metabolites in fattening lambs. J Agric Sci. 2014; 6(3):92–98. 40. Madkour FF, Abdel-Daim MM. Hepatoprotective and antioxidant activity of Dunaliella salina in paracetamol-induced acute toxicity in rats. Indian J Pharm Sci. 2013;75(6):642–648. 41. Abdel-Daim M, Funasaka Y, Kamo T, et al. Preventive effect of chemical peeling on ultraviolet induced skin tumor formation. J Dermatol Sci. 2010;60(1):21–28. 42. Funasaka Y, Abdel-Daim M, Kawana S, Nishigori C. Effect of chemical peeling on the skin in relation to UV irradiation. Exp Dermatol. 2012;21(Suppl 1):31–35. 43. Abdel-Daim M, Funasaka Y, Kamo T, et al. Effect of chemical peeling on photocarcinogenesis. J Dermatol. 2010;37(10):864–872. 44. Willcox JK, Ash SL, Catignani GL. Antioxidants and prevention of chronic disease. Crit Rev Food Sci Nutr. 2004;44(4):275–295. 45. Eldahshan OA, Abdel-Daim MM. Phytochemical study, cytotoxic, analgesic, antipyretic and anti-inflammatory activities of Strychnos nux-vomica. Cytotechnology. 2014. (Epub ahead of print). 46. Larkin DJ, Tjeerdema RS. Fate and effects of diazinon. Rev Environ Contam Toxicol. 2000;166:49–82. 47. Tuzmen MN, Candan N, Kaya E. The evaluation of altered antioxidative defense mechanism and acetylcholinesterase activity in rat brain exposed to chlorpyrifos, deltamethrin, and their combination. Toxicol Mech Methods. 2007;17(9):535–540. 48. Tu HT, Silvestre F, Meulder BD, Thome JP, Phuong NT, Kestemont P. Combined effects of deltamethrin, temperature and salinity on oxidative stress biomarkers and acetylcholinesterase activity in the black tiger shrimp (Penaeus monodon). Chemosphere. 2012;86(1):83–91. 49. Ensibi C, Hernandez-Moreno D, Miguez Santiyan MP, Daly Yahya MN, Rodriguez FS, Perez-Lopez M. Effects of carbofuran and deltamethrin on acetylcholinesterase activity in brain and muscle of the common carp. Environ Toxicol. 2014;29(4):386–393. 50. El-Gerbed MS. Protective effect of lycopene on deltamethrininduced histological and ultrastructural changes in kidney tissue of rats. Toxicol Ind Health. 2014;30(2):160–173. 51. Abdel-Daim M. Synergistic protective role of ceftriaxone and ascorbic acid against subacute diazinon-induced nephrotoxicity in rats. Cytotechnology. 2014. (Epub ahead of print). 52. Mongi S, Mahfoud M, Amel B, Kamel J, Abdelfattah el F. Protective effects of vitamin C against hematological and biochemical toxicity induced by deltamethrin in male Wistar rats. Ecotoxicol Environ Saf. 2011;74(6):1765–1769. 53. Datta M, Kaviraj A. Ascorbic acid supplementation of diet for reduction of deltamethrin induced stress in freshwater catfish Clarias gariepinus. Chemosphere. 2003;53(8):883–888.
CLINICAL STUDY
Synergistic protective effects of ceftriaxone and ascorbic acid against subacute deltamethrin-induced nephrotoxicity in rats Mohamed M. Abdel-Daim1 and Ashraf El-Ghoneimy2 Faculty of Veterinary Medicine, Pharmacology Department, Suez Canal University, Ismailia, Egypt and 2Faculty of Veterinary Medicine, Pharmacology Department, South Valley University, Qena, Egypt
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1
Abstract
Keywords
Deltamethrin (DLM) is a synthetic class II pyrethroid acaricide and insecticide widely used for veterinary and agricultural purposes. However, its animal and human exposure leads to nephrotoxicity. Our experimental objective was to evaluate protective effects of ceftriaxone and/or ascorbic acid against DLM-induced renal injury in male Wistar albino rats. DLM-treated animals revealed significant alterations in serum biochemical parameters related to renal injury; urea, uric acid and creatinine. There was a significant increase in renal lipid peroxidation and a significant inhibition in antioxidant biomarkers. Moreover, DLM significantly reduced serum acetylcholinesterase (AChE) activity. In addition, It induced serum and kidney tumor necrosis factor-a (TNF-a). Both ceftriaxone and ascorbic acid protect against DLM-induced biochemical alterations in serum and renal tissue when used alone or in combination along with DLMintoxication. Furthermore, both ceftriaxone and ascorbic acid produced synergetic nephroprotective and antioxidant effects. Therefore, it could be concluded that ceftriaxone and/or ascorbic acid administration able to minimize the toxic effects of DLM through their free radical-scavenging and potent antioxidant activity.
Acetylcholinesterase, ceftriaxone, deltamethin, kidney, nephrotoxicity, rats antioxidant,tumor necrosis factor, vitamin C
Introduction Insecticides are extensively used worldwide for control of veterinary, agricultural, and domestic insect pests and disease vectors to maintain human and animal health and enhance the food production. Consequently, the intensive use of pesticides leads to serious ecological hazards and potential health problems, including severe acute, subacute, and chronic cases of animals and human poisonings and therefore, are main reasons of concern1. Human are potentially exposed to insecticides either directly, as manufacturing workers and field applicators (workers in agriculture and in green-houses), or indirectly, through ingestion of contaminated food. In addition, it is likely that a high amount of these chemicals, and their metabolites reach estuaries and rivers via run-off from farmland that are might be toxic to wildlife2. Among pesticides are pyrethroid insecticides, which are synthetic derivatives of the natural pyrethrins, and have replaced the organophosphorus compounds within the last 10–15 years3. Deltamethrin (DLM), a synthetic type II pyrethroid, is commonly used in veterinary, agricultural, and public health practices and highly effective against a broad spectrum of insects. In the veterinary field, it is used for the control of ectoparasites in animals, poultry, and fish, Address correspondence to Mohamed M. Abdel-Daim, Faculty of Veterinary Medicine, Pharmacology Department, Suez Canal University, Ismailia 41522, Egypt. Tel/Fax: +20643207052; E-mail: [email protected], [email protected]
History Received 28 June 2014 Revised 4 September 2014 Accepted 26 September 2014 Published online 19 November 2014
preventing vector-borne diseases. The main important sources of the DLM human and animal exposure are polluted food and water, as it is readily absorbed orally4. Several studies have shown that DLM caused changes in hematological and biochemical parameters as well as oxidative stress status of the experimental animals5,6. While studies describing the protection against DLM-induced injury and oxidative stress are limited5,7. The cells overcome oxidative damage by either expelling of the damaged nucleotides and lipid peroxidation products or directly quenching free radicals via endogenous non-enzymatic and enzymatic antioxidants system8–10. Ceftriaxone (CTX) is a broad-spectrum antimicrobial cephalosporin, resistant to beta-lactamases, with potent activities against gram-positive and gram-negative bacteria11. It is effective against Streptococcus pneumonia, Streptococcus faecalis, Streptococcus pyogenes, Neisseria gonorrhoeae, Brucella melitensis, and Haemophilus influenza11. CTX is reported to inhibit transpeptidase enzymes which are responsible for the last step in bacterial cell wall synthesis12. Other non-antibiotic pharmacodynamic effects of CTX on animal studies demonstrated that it could reduce morphine tolerance and dependence13, ameliorate diabetic hyperalgesia14, and diminish neuropathic pain15. Moreover, CTX attenuated nephrotoxicity in rats induced by cadmium16, cyclosporine A17, tobramycin18, and isepamicin19. Ascorbic acid (AA), vitamin C is a water-soluble vitamin, has a many function in the body and probably the most crucial
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antioxidant in extracellular fluids20. It is an important vitamin and an essential component in the diet of many mammalian species as it cannot be synthesized in the body of these species21. It is highly water soluble and acts as an effective reducing and chelating agent20. AA is the most effective antioxidant in preventing lipid peroxidation induced by peroxyl radicals, and considered as a potent free radical quenching agent22,23. Moreover, it may act in an indirect way by restoring other antioxidants such as the fat-soluble vitamin E20. To the best of our knowledge, the role of CTX and AA, alone or in combination against DLM-induced serum biochemical alteration related to nephrotoxicity as well as renal lipid peroxidation and antioxidant status in rats has not been studied yet. Therefore, the current study was designed to investigate the alterations in serum biochemical parameters related renal damage as well as renal lipid peroxidation and oxidative stress induced by DLM in rats. Moreover, the role of ceftriaxone and/or ascorbic acid in alleviating these DLMinduced hazard effects could be evaluated.
(25 ± 2 C). Food and water were provided ad libitum. Animal handling and experimental design procedures were approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt (the approval no 20147). All precautions were taken into consideration to avoid animal suffering. Rats were kept for 1 week before the beginning of the experiment for acclimatization. Next, rats were randomly divided into seven different groups; eight animals in each. Rats in the first group were administered normal saline and kept as control. The second and third groups were given CTX (100 mg/kg bw SC)24 and ascorbic acid (100 mg/kg bw orally)25, respectively, daily. The fourth group received a daily dose of DLM (2 mg/kg, orally)26. The fifth and sixth groups were given CTX and AA, respectively, at the same dose regimen used for the second and third groups before DLM administration at the same dose and regimen used for the fourth group. The seventh group was given both CTX and AA 1 h before DLM administration (Table 1). All treatments were carried out for 4 weeks. Serum collection and tissue preparation
Materials and methods Chemicals Deltamethrin (ButoxÕ 50 mg/mL) was obtained from Intervet Co., France as a commercial product in clinical formulation for veterinary use. Ceftriaxone (CeftriaxoneÕ vial, 250 mg, 500 mg, and 1 g crystalline powder) was kindly given by Sandoz-Novartis, Egypt. Ascorbic acid was purchased from Adwia Pharmaceuticals, Cairo, Egypt. All kits were purchased from Biodiagnostics, Egypt except, TNF-a from Assay Designs Inc. (Ann Arbor, MI) and AChE from (BioVision Inc., Milpitas, CA). All other chemicals used in the experiment were of analytical grade. Animals and experimental design Fifty six male Wistar rats, weighing 180 ± 20 g, were bought from the Egyptian Organization for Biological Products and Vaccines. Rats were kept in a ventilated animal house of normal light–dark cycle (12 h light/dark) and temperature
At the end of experiment (24 h after the DLM dose), blood samples were collected via retro-orbital plexus. Blood samples were left to clot at room temperature, and centrifuged at 3000 rpm for 15 min. Sera were then separated and stored at 20 C as aliquots for further biochemical analysis. After blood collection, rats were sacrificed by cervical decapitation. Kidneys were rapidly excised from each animal, recovered from surrounding connecting tissue, and washed with 0.9% NaCl solution and distilled water for removal of the blood. Kidneys next were blotted over a piece of filter paper and perfused with a 50 mM sodium phosphate buffer saline (100 mM Na2HPO4/NaH2PO4; pH 7.4) in an ice-containing medium, containing 0.1 mM ethylene di amine tetra acetic acid (EDTA) to remove any clots and blood cells. Subsequently, renal tissues were homogenized in 5–10 mL cold buffer per gram tissue and centrifuged at 5000 rpm for 30 min. The resulting supernatant was then transferred into Eppendorf tubes, and kept at 80 C in a deep freezer until used for various biochemical assays.
Table 1. Experimental design, summary of different rat groups and their treatment. Group
CTX
AA
DLM
Control CTX
– Ceftriaxone at 100 mg/kg body weight daily for 4 weeks –
– –
– –
AA DLM DLM–CTX DLM–AA DLM–CTX–AA
– Ceftriaxone at 100 mg/kg body weight daily for 4 weeks, 1 h before DLM dose – Ceftriaxone at 100 mg/kg body weight daily for 4 weeks, 1 h before DLM dose
Ascorbic acid at 100 mg/kg body weight daily – – Ascorbic acid at 100 mg/kg body weight daily, 1 h before DLM administration Ascorbic acid at 100 mg/kg body weight daily, 1 h before DLM administration
– Deltamethrin 20 mg/kg body weight, daily for 4 weeks Deltamethrin 20 mg/kg body weight, daily for 4 weeks Deltamethrin 20 mg/kg body weight, daily for 4 weeks Deltamethrin 20 mg/kg body weight, daily for 4 weeks
DOI: 10.3109/0886022X.2014.983017
Ceftriaxone and vitamin C against deltamethrin nephrotoxicity
Serum biochemical analysis The sera stored at 20 C were used for estimation of serum renal injury marker products: creatinine, urea, and uric acid were determined according to Larsen 27, Coulombe and Favreau28, and Whitehead et al.29, respectively. Acetylcholinesterase (AChE) was measured according to Ellman et al.30.
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Evaluation of tissue lipid peroxidation and antioxidant enzymes Lipid peroxidation was evaluated by measurement of MDA content in renal tissues according to Mihara and Uchiyama31. Serum nitric oxide (NO) determined according to Green et al.32. The non-enzymatic antioxidant biomarker reduced glutathione (GSH) was assessed according to Beutler et al.33 and glutathione peroxidase (GSH-Px) according to Paglia and Valentine34. The enzymatic antioxidant biomarkers superoxide dismutase (SOD) was evaluated according to Nishikimi et al.35 and catalase (CAT) according to Aebi36. In addition, total antioxidant capacity (TAC) was determined according to Koracevic et al.37. Serum and kidney TNFa assays Serum and renal tissue TNF-a levels were measured using already made kits from R&D Systems (Minneapolis, MN) according to the manufacturer protocol. Briefly, in a microplate, 50 mL of the assay diluent was added to each well. Then, 50 mL of the diluted serum or renal tissue homogenate supernatant samples were added to each well and gently mixed by tapping the plate frame for 1 min then the plate was covered and incubated at room temperature for 2 h. After incubation, each well in the plate was then aspirated and washed five times with the kit washing buffer, then 100 mL substrate solutions were added and incubated at room temperature in dark for 30 min. The optical density was then read at 450 nm using 96-well microtiter plates ELISA Figure 1. Protective effect of ceftriaxone and/ or ascorbic acid against deltamethrin-induced alterations in serum uric acid levels. Note: DLM, Deltamethrin;CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
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reader. TNF-a level was calculated from a standard curve and multiplied by the dilution factor. Statistical analysis Data are presented as mean ± SE. Statistical significance of the data was analyzed using SPSS Program (Statistical Packages for Social Sciences, SPSS Inc., Chicago, IL) version 16. For comparison, One-Way analysis of variance (ANOVA) test followed by Turkey’s range test for post-hoc analysis. Statistical significance was acceptable to a level of p 0.05.
Results Serum biochemical analysis The effects of DLM intoxication as well as the preventive effects of CTX and/or AA on serum renal injury markers; uric acid, urea, and creatinine are shown in (Figures 1–3). Significant increases (p 0.05) in these markers were recorded in DLM intoxicated rats as compared to the untreated control group (332.68, 334.83, and 2266.36, respectively). Pre-treatment with CTX at doses of 100 mg/kg significantly (p 0.05) reduced the serum renal injury markers: uric acid, urea, and creatinine (about 54.51, 60.04, and 42.01%, respectively), while AA pre-treatment at a dose of 100 mg/kg significantly (p 0.05) reduced these biomarkers (about 56.41, 61.41, and 43.46%, respectively) compared with DLM-intoxicated group. Moreover, both CTX and AA were given in combination and significantly (p 0.05) reduced the same parameters more than when each of them used alone (about 31.70, 31.58, and 7.64%, respectively). Moreover, DLM intoxication significantly (p 0.05) reduced (51.84%) AChE as compared to the control rats. At the same time, pretreatment with CTX, AA, or both in combination significantly (p 0.05) increased serum AChE level (154.83, 160.50, and 178.87%, respectively) compared with the DLM-intoxicated rats (Figure 4).
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Figure 2. Protective effect of ceftriaxone and/ or ascorbic acid against deltamethrin-induced alterations in serum urea levels. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
Figure 3. Protective effect of ceftriaxone and/ or ascorbic acid against deltamethrin-induced alterations in serum creatinine levels. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
There were no significant changes in serum markers in rats received CTX or AA only at a dose of 100 mg/kg (second and third groups, respectively) if compared to the normal control (first group), indicating the safety of CTX and AA at the selected doses used in this study (Figures 1–4). Renal lipid peroxidation and antioxidant biomarkers The effects of DLM intoxication as well as preventive effects of CTX and/or AA on renal tissue homogenate lipid peroxidation and antioxidant parameters are shown in Table 2. In DLM-intoxicated group, there was a significant increase (p 0.05) in renal MDA and NO content (373.22 and
252.24%, respectively) was observed compared to the control group. On the other hand, renal GSH, GSH-Px, SOD, CAT, and TAC were significantly (p 0.05) decreased (51.06, 51.52, 42.92, 45.98, and 71.11%, respectively). Concerning DLM–CTX group, renal MDA and NO were significantly (p 0.05) decreased (76.68 and 65.61%, respectively) while GSH, GSH-Px, SOD, CAT, and TAC were significantly (p 0.05) increased (154.24, 152.14, 179.91, 154.17, and 124.12%, respectively) compared to DLM-intoxicated group. Regarding DLM–AA group, renal MDA and NO were significantly (p 0.05) decreased (49.40 and 58.58%, respectively), while GHS, GSH-Px, SOD, CAT, and TAC were significantly (p 0.05) increased (about 185.53, 172.15,
Ceftriaxone and vitamin C against deltamethrin nephrotoxicity
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Figure 4. Protective effect of ceftriaxone and/ or ascorbic acid against deltamethrin-induced alterations in serum acetylcholinesterase (AChE) activity. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
Table 2. Protective effect of ceftriaxone and/or ascorbic acid against deltamethrin-induced alterations in renal tissue malondialdehyde, nitric oxide, and antioxidant biomarkers. Experimental groups Parameters MDA (nmol/g) NO (mmol/g) GSH (mg/g) GSH-Px (U/g) SOD (U/g) CAT (U/g) TAC (mmol/g)
Control a
4.47 ± 0.18 7.56a ± 0.40 8.06a ± 0.31 5.72a ± 0.34 18.22a ± 1.10 0.33a ± 0.03 1.34a ± 0.04
CTX a
4.39 ± 0.19 7.40a ± 0.35 8.29a ± 0.36 6.02a ± 0.30 19.76a ± 1.49 0.33a ± 0.02 1.41a ± 0.06
AA a
4.35 ± 0.18 7.31a ± 0.43 7.68a ± 0.35 6.49a ± 0.25 20.57a ± 1.35 0.34a ± 0.02 1.47a ± 0.07
DLM b
16.67 ± 1.12 19.08b ± 1.36 4.17b ± 0.32 2.95b ± 0.22 7.82b ± 0.57 0.15b ± 0.01 0.95b ± 0.07
DLM–CTX c
12.78 ± 0.48 12.50c ± 0.58 6.43c ± 0.27 4.49c ± 0.30 14.07c ± 0.77 0.23c ± 0.01 1.18c ± 0.05
DLM–AA d
8.24 ± 0.56 11.18c ± 0.69 7.47ac ± 0.30 5.08ac ± 0.40 16.07ac ± 0.82 0.28ac ± 0.02 1.35a ± 0.04
DLM–CTX–AA 5.43a ± 0.20 8.41a ± 0.37 8.19a ± 0.28 5.92a ± 0.25 20.26a ± 0.90 0.32a ± 0.02 1.44a ± 0.05
Notes: Data are expressed as means ± SE (n ¼ 8). DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid; MDA, Malondialdehyde; NO, Nitric oxide; GSH, Reduced glutathione; GSH-Px, Glutathione peroxidase; SOD, Superoxide dismutase; CAT, Catalase; TAC, Total antioxidant capacity. Within the same row, different letters (a–d) means statistical significance at (p50.05).
205.34, 186.67, and 141.55%, respectively). In addition, both CTX and AA were given in combination, significantly (p 0.05) reduced renal MDA and NO level (32.56 and 44.07%, respectively) and increased GHS, GSH-Px, SOD, CAT, and TAC more than when each of them given separately (about 196.40, 200.68, 259.09, 213.33, and 150.98%, respectively; Table 2). Serum and kidney TNF-a assays DLM intoxication significantly (p 0.05) increased both serum and renal tissue TNF-a (531.29 and 842.90%, respectively) compared with control rats (Figures 5 and 6). Pretreatment with CTX significantly (p 0.05) reduced both serum and renal TNF-a levels (42.82 and 40.54%, respectively). Similarly, AA pretreatment induced significant (p 0.05) reduction (about 43.74 and 33.36%, respectively). Moreover, when both CTX and AA were given in combination induced more significant (p 0.05) reduction in these levels (about 21.67 and 14.52%, respectively) compared to DLM-intoxicated rats (Figures 5 and 6).
Discussion Reactive oxygen species (ROS) are continuously produced inside the animal and human body as a result of exposure to plenty of exogenous chemicals, toxins, and drugs in our environment and/or endogenous metabolic cascades involving redox enzymes and electron transport mechanism9,38. In normal circumstances, there is balance between the ROS generated and the antioxidants exist, as the ROS produced are neutralized by the endogenous antioxidants8,39. Deleterious effects caused by ROS occur due to an imbalance between the production and inactivation of these species leading to disturbance in normal cellular physiology and different pathological states10,40. Aging process as well as many degenerative diseases, including Alzheimer, diabetes, rheumatoid arthritis, cancer, cataract, coronary heart disease, and stroke occurs due to excessive free radicals’ productions41–45. Deltamethrin, a type II pyrethroid insecticide has been widely used in veterinary, agriculture, and industrial practice, that would be potentially an exposure hazard to animals and
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Figure 5. Protective effect of ceftriaxone and/or ascorbic acid against deltamethrininduced alterations in serum TNF-a levels. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
Figure 6. Protective effect of ceftriaxone and/or ascorbic acid against deltamethrininduced alterations in renal tissue TNF-a levels. Note: DLM, Deltamethrin; CTX, Ceftriaxone; AA, Ascorbic acid. Data are expressed as means ± SE (n ¼ 8). Different letters means statistical significance at p50.05.
human as well46. Although several investigations about DLM toxicity have been published, little reports have been explained the preventive agents used against such a toxicity and mechanism of their ameliorative action5,6. In this study, the renal injuries caused by DLM might induce the oxidative stress due to free radical production. DLM intoxication significantly (p 0.05) increased serum renal injury markers; urea, uric acid and creatinine. Moreover, it significantly (p 0.05) reduced serum AchE level. DLM treatment significantly (p 0.05) increased lipid peroxidation through the elevation of kidney MDA level, significantly (p 0.05) decreased kidney enzymatic; SOD, CAT, and
GSH-Px as well as non-enzymatic; GSH antioxidant level. In addition, it significantly (p 0.05) increased both serum and renal tissue TNF-a compared with the control group. All these effects are involved in the cascade of events leading to DLM-mediated kidney oxidative stress and toxicity. This indicates that renal injuries induced by DLM is due to oxidative stress that arises as a result of excessive ROS production, which have been known to attack various cellular molecules, including lipids and causing lipid peroxidation. The antioxidant enzymes’ activities, including the enzymes involved in glutathione metabolism were also disrupted in DLM-intoxicated group, suggesting the involvement of
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DOI: 10.3109/0886022X.2014.983017
Ceftriaxone and vitamin C against deltamethrin nephrotoxicity
oxidative stress in DLM-mediated renal damage. These results are consistent with the literature and point towards the role of ROS in DLM-mediated injury and toxicity. The insecticide, DLM was reported to increase serum urea and creatinine, urinary glucose, and renal tissue MDA, while antioxidant markers were decreased in rats5,6. Another study DLM intoxication reduced AChE in serum and different tissues of rats and many aquatic animals47–49. In addition, It significantly increased the TNF-a in rats serum50. In the current study, the pre-administration of CTX and/or AA at a dose level of 100 mg/kg significantly reduced the serum renal injury markers. Moreover, they reduced the lipid peroxidation in kidney tissues. In addition, there were significant elevations of renal antioxidant enzymes and glutathione levels. Furthermore, they significantly increased serum AChE and reduced serum and kidney TNF-a. Both CTX and ascorbic acid induced synergistic protective effects against DLM-induced serum and tissues’ biochemical alterations. These results were in agreement with many previous literatures, which examined the nephroprotective and antioxidant effects of CTX against many drugs and xenobioticsinduced nephrotoxicity16–19,51. Pre-treatment with AA might play a role in reducing the toxic effect of DLM, and its powerful antioxidant properties seem to mediate such an ameliorative effect, indicated by the normalization of altered hematological and biochemical changes induced by DLM intoxication in rats and catfish51–53. The protective effect of CTX and/or ascorbic acid against DLM-induced oxidative stress in our rat model could be either direct by scavenging ROS and inhibition of lipid peroxidation and/or indirect through the enhancement of the activity SOD and CAT and the enzymatic free radicals’ scavengers in the cells. Therefore, CTX and ascorbic acid could be used in combination to prevent and treat renal injuries, especially those induced by oxidative stress.
Conclusion Oxidative stress plays a major role in DLM-induced nephrotoxicity. Antioxidants have been proven to be effective in preventing DLM-induced toxicity in several previous interventions. CTX and AA are potent antioxidants, which are reported to ameliorate the effect of many known nephrotoxic agents. In this study, DLM exposure resulted in varying degrees of inhibition in antioxidant enzymes, lipid peroxidation induction, and alterations of serum biochemical parameters. CTX and/or AA pre-treatment provided sufficient protection in terms of serum and tissues’ biochemical alteration, activity of antioxidant markers and oxidative stress, especially when given in combination.
Acknowledgements The authors would like to thank Sandoz-Novartis, Egypt, for supplying CeftriaxoneÕ used in our experiment. The company had neither role in designing the experiment nor in the publication process. This research received no grant from any funding agency.
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Declaration of interest The authors declare that there are no conflicts of interest.
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