Hematological and biochemical evaluation of effect of Mangiferin in Amitraz induced toxicity in rats

Introduction

Parasitism represents a major challenge in veterinary medicine, particularly in hot and humid tropical regions. Among parasitic conditions, ectoparasitism causes substantial reductions in productivity in cattle and other livestock, leading to significant economic losses for farmers. In companion animals, ectoparasite infestations are equally problematic, as they compromise animal welfare and pose public health concerns due to their zoonotic potential (Dantas-Torres and Otranto, 2014) [9] . Formamidines are an important class of ectoparasiticides, with amitraz being the principal compound approved for veterinary use. Amitraz is widely employed for the control of ticks, mites, and lice in cattle, swine, and sheep, and is also used in dogs. The development of formamidine acaricides began with the introduction of chlordimeform in 1963 (Hollingworth, 1976) [14] , followed by several related compounds, of which amitraz remains the most prominent. First synthesized in England in 1969 (Hollingworth, 1976) [14] , amitraz continues to be extensively used, formamidine insecticides currently approved for veterinary applications. Amitraz undergoes hepatic biotransformation, producing several metabolites, among which BTS 27271 is the most pharmacologically active. This metabolite exerts its effects primarily through binding to α2A and α2D adrenergic receptors (Abu-Basha et al., 1999) [1] . In insects, formamidines such as chlordimeform and amitraz act by targeting octopaminergic receptors in the central nervous system. In mammals, however, toxicity is mainly attributed to amitraz acting as an α2-adrenergic receptor agonist and a monoamine oxidase (MAO) inhibitor.

Additional mechanisms have also been reported, including inhibition of histamine receptors, H1 suppression of prostaglandin synthase and adenylyl cyclase, activation of voltage-gated calcium channels, generation of reactive oxygen species (ROS), induction of cell death, and disruption of endocrine function (Evans and Gee, 1980; Chen et al., 2007) [12,33] . Numerous studies have established a link between insecticide exposure and enhanced production of reactive oxygen species, which can cause oxidative damage to vital organs such as the liver, kidneys and brain. Oxidative stress occurs when ROS generation exceeds the capacity of the body's antioxidant defense systems.

Antioxidants play a crucial role in neutralizing free radicals by donating electrons, thereby preventing cellular damage. Maintaining a balance between pro-oxidant and antioxidant mechanisms is essential for health and disease prevention (Srivastava and Dwivedi, 1998; Yu et al., 2008) [30,32] . Both enzymatic antioxidants such as catalase, glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, and glutathione reductase and non-enzymatic antioxidants, including reduced glutathione and total thiols, are vital in protecting cells from oxidative injury. Exposure to pesticides has been shown to significantly alter the activity of these antioxidant systems (Banerjee et al., 2001; Bebe and Panemangalore, 2005) [5,7] . Xanthones are secondary metabolites found in certain higher plants, lichens, and fungi and have attracted considerable interest due to their therapeutic potential and taxonomic significance (Negi et al., 2013) [22] . Structurally and chromatographically similar to flavonoids, xanthones exhibit diverse pharmacological activities, including antiinflammatory, antifungal, antibacterial, antioxidant, and antiviral effects (Saini et al., 2022) [25] . Mangiferin, a xanthone glucoside, is predominantly isolated from the tropical fruit Mangifera indica (mango), native to India and has a molecular weight of 422.34 g/mol. Although mango is its primary source, mangiferin is also present in several other plant species (Jaiswal et al., 2015) [16] . Mangiferin exhibits a broad spectrum of therapeutic properties, including antioxidant, anti-inflammatory, antidiabetic and anticancer activities. Traditionally, it has been used in Indian medicine for the treatment of melancholia and nervous debility (Bhattacharya et al., 1972) [8] . Furthermore, mangiferin is a key active component in over 40 polyherbal formulations used in traditional Chinese medicine (Alduhisa and Demayo, 2019) [4] . Studies have demonstrated that mangiferin enhances glutathione levels by upregulating gamma-glutamyl cysteine synthetase, the ratelimiting enzyme in glutathione synthesis. Its antioxidant action is also associated with the downregulation of NF-κB, resulting in reduced tumor necrosis factor-induced ROS production (Sarkar et al., 2004) [26] . Additionally, mangiferin has been shown to interact directly with catalase, thereby significantly reducing tissue damage (Sahoo et al., 2015) [24] . Considering the toxic potential of amitraz and the antioxidant, anti-inflammatory and anti-apoptotic properties of mangiferin, the present study was designed to evaluate the protective effect of mangiferin against amitraz-induced subacute toxicity in rats, with the following objectives:  To assess hematological and serum biochemical associated with amitraz-induced toxicity in rats.

Materials and Methods

The A total of 40 rats were randomly allocated into five groups, with eight animals in each group. Group I (normal control) received normal saline orally. Group II (positive control) was administered amitraz dose of 80 mg/kg body weight where amitraz of pure form was dissolved in normal saline where normal saline was used as a vehicle. Group III received mangiferin alone at a dose of 30 mg/kg body weight. Group IV was treated with amitraz (80 mg/kg body weight) in combination with mangiferin (30 mg/kg body weight). Group V received amitraz (80 mg/kg body weight) along with a higher dose of mangiferin (60 mg/kg body weight).

Body weight Measurement

The body weight of all the experimental animals were recorded weekly once and the dosing was adjusted accordingly.

Hematology

The blood samples collected in EDTA vials on 14 th and 28 th day of the experiment were subjected for haemoglobin (Hb), total erythrocyte count (TEC), total leucocyte count (TLC), total platelet count, packed cell volume (PCV), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC) estimation using Mindray BC-2800VET auto Haematology auto analyser in the Department of Teaching Veterinary Clinical Complex, Veterinary College, Hassan.

Serum Biochemistry

For serum biochemical parameters analysis, blood was collected on 14 th and 28 th day into serum vacutainers, centrifuged at 3000 rpm for 15 min and serum separated and stored at -20 o C till further analysis. Biochemical estimation for AST, ALT, ALP, Urea, BUN and creatinine levels were analysed using standard laboratory procedure by Robonik, India Semi-automatic Biochemistry Analyzer (Prietest Compact) using reagent kits from Lifechem, Ahmedabad.

Results

General observations

The amitraz was administered orally in rats for 28 days did not result in mortality. However, animals in Group II showed noticeable clinical signs, including roughened hair coats and reduced feed intake compared with Group I. During the last two weeks of treatment, Group II rats exhibited anorexia, lethargy, weakness, abnormal gait, aggressive behavior and dizziness. In contrast, rats in Groups III, IV, and V displayed normal behavior and activity throughout the experimental period. The Body weights were recorded weekly and are presented in Table 1.1, 1.2, 1.3. The rate of body weight gain was significantly influenced by amitraz administration, both alone and in combination with mangiferin. From the second week onward, the mean body weight of rats in Group II was significantly lower compared with Groups I, III, IV, and V. Conversely, Group III animals showed a progressive increase in body weight throughout the study period.

Absolute organ weights

A significant increase in absolute liver weight was observed in Group II compared with Group I. In Groups IV and V, liver weights were significantly reduced relative to Group II, while liver weights in Group III were comparable to those of Group I. Similarly, the absolute kidney weight was significantly higher in Group II than in Group I. In Groups IV and V, kidney weights were significantly lower compared with Group II, whereas a marginal increase was noted in Group III relative to Group I.

Relative organ weights

The relative weights of the liver and kidneys were significantly increased in Group II compared with Group I. In contrast, Groups IV and V showed a significant reduction in relative liver and kidney weights when compared with Group II. No significant differences in relative liver or kidney weights were observed between Group III and Group I.

Hematological parameter

The mean values of hematological parameters with standard error of mean on 14 th and 28 th day are presented in Table 2.1, 2.2, On days 14 and 28, Group II showed a significant decrease in total erythrocyte count (TEC) compared to Groups I, IV, and V. In contrast, TEC levels were significantly higher in Groups IV and V when compared with Group II. TEC levels in Group III were similar to those in Group I. During the same periods, total leukocyte count (TLC) was significantly increased in Group II compared to Group I. However, Groups IV and V showed a significant decrease in TLC levels compared to Group II. No significant difference in TLC levels was observed between Group III and Group I. Hemoglobin (Hb) levels on days 14 and 28 were significantly lower in Group II compared to Group I. In contrast, Hb levels were significantly increased in Groups IV and V relative to Group II. Group III showed Hb levels was comparable to Group I.

Serum Biochemistry

The mean values of serum biochemistry parameter values with standard error of mean on 14 th and 28 th day were presented in Table 3.1, 3.2, 3.3 Serum ALT, AST, and ALP levels were significantly elevated in Group II compared to Group I. In contrast, Groups IV and V showed a significant reduction in these enzyme levels relative to Group II. Enzyme levels in Group III remained comparable to those in Group I. Renal function markers, including urea, blood urea nitrogen (BUN) and creatinine, were significantly increased in Group II compared to Group I. However, Groups IV and V exhibited a significant decrease in these parameters when compared with Group II. No significant differences were observed between Group III and Group I.

Discussion

Body weight gain is an important indicator of growth. In the present study, rats were treated with amitraz alone (Group II) showed a significant reduction in both absolute and percentage body weight gain after 28 days compared to the control group (Group I). Although Group III showed an increase in body weight gain, the difference was not statistically significant compared to Group I. These findings are consistent with earlier reports showing reduced body weight gain following amitraz exposure (Ueng et al., 2004; Instiotoris et al., 2007) [31,15] . The reduction in body weight may be due to the metabolic and hormonal effects of amitraz through activation of α2-adrenergic receptors, leading to increased norepinephrine release and elevated metabolism (Del Pino et al., 2015) [11] . Similar effects on body weight have also been reported in fipronil-induced toxicity in rats (Mohan, 2023) [21] . Relative organ weight is a key indicator of organ toxicity. In this study, a significant increase in relative liver weight was observed in Group II compared to Group I, which may be attributed to inflammatory changes and liver enlargement caused by amitraz exposure, as previously reported (Ueng et al., 2004) [31] . Similarly, Group II showed a significant increase in relative kidney weight compared to Group I. This increase may result from inflammatory cell accumulation and altered renal function, consistent with earlier findings ( [31,15,13] . Hematological analysis is commonly used to assess disease conditions and toxic effects caused by chemicals and pesticides (Al-Attar et al., 2010) [3] . Erythrocytes play a vital role in oxygen transport and hemoglobin levels are closely associated with total erythrocyte count (Pasini et al., 2010) [23] . In the present study, Group II showed a significant decrease in TEC and Hb levels, along with an increase in TLC, compared to Group I. These findings are consistent with earlier reports on amitraz toxicity (Barretto et al., 2017; Instiotoris et al., 2007) [6,15] and similar hematological alterations reported in fipronil-induced toxicity (Mohan, 2023) [21] . The reduction in hemoglobin may indicate regenerative anemia due to interference with erythropoiesis, while decreased TEC and increased TLC could be attributed to oxidative stress-induced damage to red blood cells (Barretto et al., 2017; Instiotoris et al., 2007) [6,15] . Similar protective effects of mangiferin on these parameters have also been reported by Mohan (2023) [21] , supporting the present findings. A significant increase in AST, ALT, ALP, creatinine, urea and BUN levels was observed in Group II compared to Group I. These findings are consistent with previous reports showing amitraz-induced liver and kidney toxicity in rodents (Kanbur et al., 2016) [17] . Elevated ALT and AST levels indicate liver cell damage, which may be associated with bile duct hyperplasia, hepatic congestion, Kupffer cell hyperplasia, and altered membrane permeability. Similar hepatocellular changes through histopathological observations. Increased ALP levels, which are present on hepatocyte membranes and bile duct epithelium, serve as a sensitive indicator of toxic liver damage (Ueng et al., 2004) [31] . The rise in urea levels observed in this study supports the findings of Kanbur et al. (2016) [17] and correlates with kidney tissue damage. Elevated BUN and creatinine levels further indicate impaired renal function, likely due to reduced glomerular filtration and tubular dysfunction (Mahmood and Mohammad, 2019; Kanbur et al., 2016) [20,17] . Meanwhile, Mangiferin is a natural xanthone compound known for its antioxidant, anti-inflammatory and protective properties. It reduces toxicity by scavenging free radicals and strengthening antioxidant defense mechanisms (Shashirekha et al., 2015; Ajay et al., 2023) [28,2] . Based on these properties, this study evaluated the protective effect of mangiferin against sub-acute amitraz-induced toxicity in Wistar rats. Groups IV and V showed a significant increase in absolute and percentage body weight gain compared to Group II, indicating that mangiferin reduced amitrazinduced oxidative stress and neurotoxicity. Group III showed values comparable to Group I. Similar protective effects of mangiferin on body weight were reported by Song et al. (2020) [29] . Relative liver and kidney weights were significantly reduced in Groups IV and V compared to Group II, suggesting hepato-and nephroprotective effects of mangiferin. This may be due to inhibition of the NF-κB inflammatory pathway and reduced reactive oxygen species formation (Li et al., 2019) [19] . These findings are consistent with earlier reports on the protective role of mangiferin (Song et al., 2020) [29] .

Serum biochemical markers, including ALT, AST, ALP, urea, creatinine and BUN, were significantly lowered in Groups IV and V compared to Group II, supporting the protective effects of mangiferin on liver and kidney function (Li et al., 2013) [18] . Hematological parameters such as TEC, Hb, and TLC were also significantly improved in Groups IV and V relative to Group II. These results agree with previous studies showing the hemoprotective and immunoprotective effects of mangiferin, likely through enhanced erythropoiesis and reduced oxidative damage to blood cells (Das et al., 2011; Sellamuthu et al., 2014; Mohan, 2023) [10,27,21] . body weight gain, normalizing relative organ weights, restoring hematological parameters and reducing elevated liver and kidney biochemical markers.  The protective effects of mangiferin are likely mediated through its antioxidant and anti-inflammatory properties, including reduction of oxidative stress and inhibition of inflammatory pathways, thereby preserving hepatic, renal and hematological integrity during amitraz exposure.

Acknowledgement

AcknowledgementI would like to express my sincere gratitude to the Veterinary College, Hassan for granting me the opportunity to carry out my research for the M.V.Sc. program.

References

1. Abu-Basha EA, Yibchok-Anun S, Hopper DL, Hsu WH. Effects of the pesticide amitraz and its metabolite BTS 27271 on insulin and glucagon secretion from the perfused rat pancreas: involvement of α2D adrenergic receptors. Metabolism. 1999;48(11):1461-1469.
2. Ajay HM, Ravikumar C, Sarma MBM, Gowda HK, Ramesh D. Insights into anticancerous, antioxidant and anti-inflammatory effects of mangiferin. Pharm Innov. 2023;12:1576-1582.
3. Al-Attar AM, Al-Taisan WAA. Preventive effects of black seed (Nigella sativa) extract on Sprague Dawley rats exposed to diazinon. Aust J Basic Appl Sci. 2010;4(5):957-968.
4. Alduhisa GU, Demayo CG. Ethnomedicinal plants used by the Subanen tribe in two villages in Ozamis City, Mindanao, Philippines. Pharm. 2019;10(4):28-42.
5. Banerjee SK, Maulik M, Manchanda SC, Dinda AK, Das TK, Maulik SK. Garlic-induced alteration in rat liver and kidney morphology and associated changes in endogenous antioxidant status. Food Chem Toxicol. 2001;39(8):793-797.
6. Barretto ML, de Deus Ferreira A, Pascoal IC, da Silva MBG, de Torres SM, Falcão MVD, et al. Amitraz: pharmacological and toxicological aspects in animals. Med Vet (UFRPE). 2017;11(3):185-191.
7. Bebe FN, Panemangalore M. Pesticides and essential minerals modify endogenous antioxidants and cytochrome P450 in tissues of rats. J Environ Sci Health B. 2005;40(5):769-784.
8. Bhattacharya SK, Sanyal AK, Ghosal S. Monoamine oxidase inhibiting activity of mangiferin isolated from Canscora decussata. Sci Nat. 1972;59:651-651.
9. Dantas-Torres F, Otranto D. Dogs, cats, parasites, and humans in Brazil: opening the black box. Parasit Vectors. 2014;7(1):22-22.
10. Das S, Rao BN, Rao BS. Mangiferin attenuates methylmercury induced cytotoxicity against IMR-32 human neuroblastoma cells by inhibition of oxidative stress and free radical scavenging potential. Chem Biol Interact. 2011;193(2):129-140.
11. Evans PD, Gee JD. Action of formamidine pesticides on octopamine receptors. Nature. 1980;287(5777):6062.
12. Filazi A, Güvenç T, Kum C, Sekkin S. Pathological findings in acute amitraz intoxication in mice. Turk J Vet Anim Sci. 2004;28(5):873-878.
13. Hollingworth RM. Chemistry, biological activity, and uses of formamidine pesticides. Environ Health Perspect. 1976;14:57-69.
14. Institóris L, Banfi H, Lengyel Z, Papp A, Nagymajtényi L. A study on immunotoxicological effects of subacute amitraz exposure in rats. Hum Exp Toxicol. 2007;26(5):441-445.
15. Jaiswal S, Ramesh K, Kapusetti G, Ray AK, Ray B, Misra N. Mangiferin as chain transfer agent: effect on molecular weight of poly(methyl methacrylate) and polystyrene. Polym Bull. 2015;72:1407-1416.
16. Kanbur M, Siliğ Y, Eraslan G, Karabacak M, Soyer Sarıca Z, Şahin S. The toxic effect of cypermethrin, amitraz and their combinations in rats. Environ Sci Pollut Res. 2016;23(6):5232-5242.
17. Li HW, Deng JG, Du ZC, Yan MS, Long ZX, Pham TPT, et al. Protective effects of mangiferin in subchronic developmental lead-exposed rats. Biol Trace Elem Res. 2013;152:233-242.
18. Li M, Wu C, Guo H, Chu C, Hu M, Zhou C. Mangiferin improves hepatic damage-associated molecular patterns, lipid metabolic disorder and mitochondrial dysfunction in alcohol hepatitis rats. Food Funct. 2019;10(6):3514-3534.
19. Mahmood MB, Mohammed ZT. Determination of LD50 and acute toxicity effects induced by amitraz in rats. Bas J Vet Res. 2019;18(2):344-352.
20. Mohan. Effect of mangiferin on fipronil-induced toxicity in rats. Karnataka Veterinary, Animal and Fisheries Sciences University, Nandinagar, Bidar, Karnataka; 2023.
21. Negi JS, Bisht VK, Singh P, Rawat MSM, Joshi GP. Naturally occurring xanthones: chemistry and biology. J Appl Chem. 2013;13:1-10.
22. Pasini EM, Lutz HU, Mann M, Thomas AW. Red blood cell membrane proteomics Part II: comparative proteomics and RBC pathophysiology. J Proteomics. 2010;73(3):421-435.
23. Sahoo BK, Zaidi AH, Gupta P, Mokhamatam RB, Raviprakash N, Mahali SK, et al. A natural xanthone increases catalase activity but decreases NF-κB and lipid peroxidation in U-937 and HepG2 cell lines. Eur J Pharmacol. 2015;764:520-528.
24. Saini N, Lather V, Gahlawat SK. Exploring phytochemicals from Himalayan medicinal plants as novel therapeutic agents. Anti-Cancer Agents Med Chem. 2022;22(9):1674-1698.
25. Sarkar A, Sreenivasan Y, Ramesh GT, Manna SK. β-Dglucoside suppresses tumor necrosis factor-induced activation of nuclear transcription factor κB but potentiates apoptosis. J Biol Chem. 2004;279(32):33768-33781.
26. Sellamuthu PS, Arulselvan P, Kamalraj S, Fakurazi S, Kandasamy M. Protective nature of mangiferin on oxidative stress and antioxidant status in tissues of streptozotocin-induced diabetic rats. Int Sch Res Notices. 2013;2013:534-541.
27. Shashirekha MN, Mallikarjuna SE, Rajarathnam S. Status of bioactive compounds in foods with focus on fruits and vegetables. Crit Rev Food Sci Nutr. 2015;55(10):1324-1339.
28. Song Y, Liu W, Tang K, Zang J, Li D, Gao H. Mangiferin alleviates renal interstitial fibrosis in streptozotocin-induced diabetic mice through regulating the PTEN/PI3K/Akt signaling pathway. J Diabetes Res. 2020;2020:356-373.
29. Srivastava MK, Dwivedi UN. Salicylic acid modulates glutathione metabolism in pea seedlings. Journal of Plant Physiology. 1998;153(3-4):409-414.
30. Ueng TH, Hung CC, Wang HW, Chan PK. Effects of amitraz on cytochrome P450-dependent monooxygenases and estrogenic activity in MCF-7 human breast cancer cells and immature female rats. Food Chem Toxicol. 2004;42(11):1785-1794.
31. Yu X, Shao XG, Sun H, Li YN, Yang J, Deng YC, et al. Activation of cerebral peroxisome proliferatoractivated receptor gamma exerts neuroprotection by inhibiting oxidative stress following pilocarpineinduced status epilepticus. Brain Res. 2008;1200:146158.
32. Chen G, Held IM. Phase speed spectra and the recent poleward shift of Southern Hemisphere surface westerlies. Geophysical Research Letters. 2007 Nov;34(21).