Haematobiochemical alterations by lead acetate toxicity and its amelioration with Andrographis paniculata mediated selenium nanoparticles in mice Debbarma K., Ingole R.S.*, Kamdi B.P., Hajare S.W.1, Ingawale M.V.2, Waghmare S.P.3, Ingle P.U.4, Gade A.K.4 Department of Veterinary Pathology, Post Graduate Institute of Veterinary and Animal Sciences, Akola - 444 104 (MS), (Maharashtra Animal and Fishery Sciences University, Nagpur) 1Department of Veterinary Pharmacology & Toxicology, Post Graduate Institute of Veterinary and Animal Sciences, Akola - 444 104 (MS), (Maharashtra Animal and Fishery Sciences University, Nagpur) 2Department of Animal Reproduction, Gynecology and Reproduction, Post Graduate Institute of Veterinary and Animal Sciences, Akola - 444 104 (MS), (Maharashtra Animal and Fishery Sciences University, Nagpur) 3Teaching Veterinary Clinical Complex, Post Graduate Institute of Veterinary and Animal Sciences, Akola - 444 104 (MS), (Maharashtra Animal and Fishery Sciences University, Nagpur) 4Post Graduate Institute of Veterinary and Animal Sciences, Akola - 444 104 (MS), (Maharashtra Animal and Fishery Sciences University, Nagpur), Department of Biotechnology, SGBAU, Amravati *Address for Correspondence: Dr Ranjit S. Ingole, Department of Veterinary Pathology, Akola - 444 104 (MS), (Maharashtra Animal and Fishery Sciences University, Nagpur), E-mail: ingoleranjit@rediffmail.com
Online Published on 03 February, 2023. Abstract Toxicity of lead acetate and its amelioration with Andrographis paniculata mediated selenium nanoparticles on hematobiochemical parameters in mice was studied. Total 32 male Swiss Albino mice were divided into 4 equal groups. Group T1 was served as control, while T2 group was treated with lead acetate @ 20 mg/kg b. wt. T3 group was treated with Andrographis paniculata mediated selenium nanoparticles @ 5mg/kg b.wt and T4 group was treated with lead acetate @ 20mg/kg b.wt + Andrographis paniculata mediated selenium nanoparticles @ 5mg/kg b.wt. The treatment was given for 28 days. T2 group given lead acetate revealed a significant decrease in Hb, PCV, TEC, TLC, and lymphocyte count whereas neutrophil count was significantly increased. Serum total protein and albumin were significantly decreased while serum AST, ALT, creatinine, and BUN were significantly increased in group T2. However, the haematobiochemical values from T4 group showed non significant alteration when compared with control group except Hb. Thus A. paniculata mediated selenium nanoparticles supplementation along with lead acetate showed protective beneficial effect on hematobiochemical parameters during lead toxicity in mice. Top Keywords Andrographis paniculata, Lead acetate, Nanoparticles, Selenium. Top |
Introduction As per the Agency for Toxic Substances and Disease Registry-2003, lead constitutes the second most hazardous substance among the list of 275 toxic substances present in the environment. It is an element that occurs naturally and is found mobilized and redistributed to large extent in the environment by human beings. Lead intoxication is a common cause of domestic animal poisoning all around the world. It`s widespread presence in the environment makes food (65 percent), water (20 percent), and air (15 percent) easily accessible to human and animal bodies1,2. All domestic animals are affected, including cattle, horses, birds/poultry, and dogs, with cattle being the most vulnerable to lead poisoning. Lead metal reaches to animals and humans through various industrial products or byproducts and their wastes such as discarded automotive batteries and engine pump oil, fishing sinkers, drapery weights, sewage sludge, lead mines, rubber industry, insecticides, printing and paint industry3. Among the soft tissues, the liver tissue stores the most lead (33 percent), followed by the renal cortex and medulla. The main site of action of lead is the central nervous system and exposure to lead is related with several neurobehavioral and psychological changes4. |
Selenium is a trace element that is required for a variety of metabolic processes, including oxidative stress protection and immunological functions5. The hepatoprotective and antioxidant role of selenium nanoparticles against lead acetate toxicity has been evaluated in some of the studies that showed the beneficial effects of selenium nanoparticles. It has been observed that selenium nanoparticles administration improved the hepatic antioxidant protection mechanism and decreased cellular sensitivity to DNA fragmentation. |
Andrographis paniculata widely grown in Southern and Southeast Asia, where it’s used to treat infectious and non-infectious diseases and it was commonly utilised before antibiotics were developed. Andrographis paniculata is grown in India during the rainy season. The entire plant can be used for therapeutic purposes. Andrographolide is the main active ingredient in Andrographis paniculata. It has a long history of traditional use in energizing the liver and treating liver problem due to the presence of major active ingredients; ‘andrographolide’ or ‘diterpene lactone’ contributes to hepatoprotective and antioxidant activities6. However, literature reviewed did not reveal information related to experimental studies on use of Andrographis paniculata mediated selenium nanoparticles during lead toxicity and was found to be scanty. Hence the present investigation was carried out to evaluate the ameliorative effect of Andrographis paniculata mediated selenium nanoparticles on hematobiochemical changes during lead acetate toxicity in mice. |
Top Materials and Methods Biogenic Synthesis of Nanoparticles The fresh leaves of Andrographis paniculata were carefully washed with distilled water, cut into small pieces, dried for few days at room temperature and powder was prepared. Thirty grams of dried leaves powder was extracted with 750 ml of ethanol and boiled for 1 hour under reflux conditions and extract was filtered through Whatmann filter paper No. 1. The extract prepared was further used for biogenic synthesis of Andrographis paniculata mediated selenium nanoparticles (APSeNPs) as per standard methodology. Briefly, for synthesis of APSeNPs, 300 ml of 0.05 M sodium selenite was prepared in distilled water, 150 ml (1:5) A. paniculata extract and 90 ml distilled water was added to it. This was kept on magnetic stirrer at 500 rpm and 60 ml of 0.05M Ascorbic acid was added drop wise using micropipette. The APSeNPs are synthesized as a dark red coloured precipitate. The precipitate was collected by centrifugation at 15000 rpm for 10 min in Beckman Coulter’s ultracentrifuge (Allegra 64 R)7. The resulting APSeNPs powder was used for further experimental studies. Experimental Design The experiment was duly approved by Institutional Animal Ethics Committee of the Post Graduate Institute of Veterinary and Animal Sciences, Akola (PGIVAS, Akola/IAEC/312/09/2000/21, Date: 6th August, 2021). Thirty two healthy male albino mice of 2-3 months old having 25-30 gm body weight was procured from M/s-Kusum Lifesciences A-13, Wasmat MIDC, Dist-Hingoli 413512, MH, India (CPCSEA Reg No: 2117/P0/RcBiBt/S/20/CPCSEA). The experimental animals were acclimatized for 10 days before the start of experiment under standard hygienic and managemental conditions in Small Laboratory Animal House at Department of Veterinary Pharmacology and Toxicology, PGIVAS, Akola. After acclimatization, animals were randomly allotted into four equal groups (Group T1, T2, T3, and T4). Each group comprised of 8 male Swiss Albino mice. All the animals were given identification mark. Group T1 was served as negative control and was given saline water orally by gavages for 28 days, while, group T2 was orally gavaged with lead acetate @ 20 mg/kg b. wt once daily for 28 days. Group T3 was orally gavaged with APSeNPs @ 5 mg/kg b.wt once daily for 28 days. Group T4 was orally gavaged with lead acetate @ 20 mg/kg b.wt + APSeNPs @ 5 mg/kg b.wt daily for 28 days. Hematobiochemical Observations On 29th day of experiment blood sample from randomly selected six animals was collected from the retro-orbital plexus using a capillary tube under anesthesia with appropriate dose of Thiopentone sodium given intraperitonially. Blood obtained in K2EDTA (1-2 mg/ml) was used for hematological analysis, and blood collected in clot activator was used for separation of serum for further biochemical estimations. Thin blood smears were prepared from a fresh blood for all the mice for differential leucocyte counts. Immediately after collection of blood, hematological and serum enzyme tests were estimated using standard method and biochemical kits. Top Results Characterization of Nanoparticles Characterization of Andrographis paniculata mediated selenium nanoparticles revealed synthesis of stable selenium nanoparticles in colloidal form, amprphous nature with nano sized particles ranging from 50-150 nm having a concentration of 1.03x109 particles/mL. Hematological Observations At the end of the experiments, T2 group mice given lead acetate @ 20 mg/kg b.wt revealed a significant decrease in Hb, PCV, TEC, TLC. However TEC values revealed non significant differences between T2 and T3 group. T2 group showed significant decrease in lymphocyte count, whereas neutrophil count was found to be significantly increased compared to the control group animals, while, animals given lead acetate along with nanoparticles showed significant increased Hb, PCV and TEC when compared to the lead acetate positive control group and showed a non significant differences with that of control group suggested a beneficial effect of APSeNPs during lead toxicity in mice (Table 1). Erythrocytic indices of control and different treatment groups revealed non significant differences. The present findings of TLC in lead acetate group (T2) revealed significantly decreased value, when compared to control group and group T3, but differ non significantly with that of group T4 (Table 2). There was no significant difference between group T3, T4 and the control group. However, animals given lead acetate along with APSeNPs revealed a non-significant increase when compared to lead acetate group and differ non significantly with the control group suggesting some beneficial effects of A. paniculata mediated selenium nanoparticles during lead toxicity. Animals given lead acetate alone showed significant increased neutrophil count compared to control group and group T4 but differ non significantly with the animals given APSeNPs only. However, T4 group reported non significant difference with that of the control group. When compared with the T1 group, group T2 showed significantly decreased lymphocyte count. However, group T4 showed significant increased lymphocyte count compared to group T2 and differ non significantly with that of group T1. The mean values of monocyte, eosinophil and basophil revealed a non significant difference between control and different treatment groups. Biochemical Observations The serum total protein and albumin revealed significant differences between control and treatment groups, however, serum globulin showed non significant differences (Table 3). Significant decreased serum total protein and albumin was observed in lead acetate group when compared with control group and group T3. However, the value in group T4 showed non-significant differences with that of lead acetate group but was observed to be numerically increased and also found comparable with that of the control group suggesting mild improvement towards the control group. Mice given lead acetate @ 20 mg/kg b.wt. revealed significant increased serum AST and ALT level when compared to control group T1, group T3 and group T4 (Table 4). Group T3 showed non significant difference with that of the control group. However, group T4 revealed a significant decreased value when compared to lead acetate group and differ non significantly with group T1 indicating the hepatoprotective effect of A. paniculata mediated APSeNPs might be due to the presence of flavonoids and terpenoids in the plant. The present finding of T2 group revealed significant increased serum creatinine and BUN level when compared to that of group T1, T3 and T4 (Table 4). T3 group recorded a non-significant differences when compared to group T1. However, group T4 revealed a significant declined value of creatinine and BUN when compared to lead acetate group and differ non significantly with group T1 and T3. Top Discussion The present study was undertaken to evaluate the ameliorative effect of APSeNPs in hematobiochemical parameters during lead toxicity in male mice. The hematological findings of decreased Hb, PCV and TEC in lead toxicated group are in accordance with earlier report recorded during lead toxicity in rat and mice8,9,10. Lead directly affects the hematopoietic system by restraining the formation of Hb by inhibiting various important enzymes involved in the heme synthesis pathway particularly the enzyme Aminolevulinic Acid Dehydratase (ALAD) might be the reason for significant decreased Hb, PCV and TEC level in lead treated mice in present investigation11. The significant reduction in Hb, PCV and TEC suggested that lead directly reduces the life span of circulating RBC by increasing the fragility of cell membranes which attributed to the anemia. The use of lead acetate in the erythroid tissue culture medium has shown that lead nearly inhibits the proliferation of erythroid lineage and perturbs cell development and hemoglobin synthesis12. Findings of increased TEC in mice given lead acetate along with A. paniculata mediated selenium nanoparticles is in accordance with earlier studies who reported effect of Andrographis paniculata during lead toxicity in broilers and suggested dose dependant increase in TEC value might be due to erythrocyte building capacity of Andrographis paniculata due to presence of iron in the plant13,14. Although the values of TEC revealed non significant differences between T2 and T3 group but the values were numerically increased in group T3. |
The present findings in group T4 can be corroborates with the earlier study of gentamycin toxicity in mice and its amelioration with selenium nanoparticles suggesting strong antioxidant activity of selenium nanoparticles in chelating free radicals15. Reduced ROS level in bone marrow by 37.80% pretreatment with Nano selenium might be the reason for significant increase in TEC in group T416. Also additive antioxidant properties of A. paniculata and selenium nanoparticles may show ameliorative effect during lead toxicity in mice. Decreased MCV and MCH are the other concordant hematological changes recorded in group given lead aetate, however present observations revealed non significant differences with that of control and other treatment group9. Inconsistent observations recorded with respect to MCV, MCH and MCHC findings, since some studies recorded lead induced microcytic hypochromic anemia in lead induced toxicity. |
Contrary to the present finding, previous studies recorded leukocytosis in lead toxicated group and suggested that leukocytosis may be linked to the inflammatory effect of lead on lymphatic organs11,17. Present finding of leukopenia in T2 group mice is in agreement with earlier finding on lead toxicity in rat and mice and suggested toxic effect of lead on bone marrow and progenitor cells decreasing the production of leucocytes10,18,19. Decrease in TLC might be due to decreased production from germinal center of lymphoid organ11. The present finding of increase in TLC from T4 group is in accordance with earlier studies who also observed increase in TLC when treated with selenium nanoparticles against lead and gentamycin induced toxicity, respectively in rat and mice and suggested potent antioxidant activity in chelating free radicals and protect cell from oxidative threat15,20. The significant increase in neutrophil count in T2 group during lead toxicity at different dose level and for different dose durations and suggested that it might be due to inflammatory condition produced by lead in different organs17,18,19. |
In similar type of study significant increase in leukocyte along with increase in the percent value of neutrophil in rats given nanoselenium in lead toxicated group was recorded and suggested lowest activity of catalase and increased SOD activity because of nanoselenium might be the reason20. |
Contrary to the present findings, significant increased lymphocyte count was recorded during lead toxicity in rat17. Similarly decreased lymphocyte count during lead toxicity in laboratory animals corroborates the earlier findings and suggested direct toxic action of lead acetate on leucopoiesis in lymphoid organ and this might be the reason for present observation in group T218,19. Significant increased lymphocyte count was recorded in rats given nanoselenium against lead toxicity22 and is in agreement with the present finding in group T420. The present result thus suggested that Andrographis paniculata mediated selenium has an anti-inflammatory and antioxidant additive status in overcoming the effects of leucopenia caused by lead intoxication. Monocyte, eosionophil and basophil count revealed within the normal range and showed non significant differences between control and treatment groups, however contrary to this monocytosis has been reported by previous workers during lead toxicity in rat and mice and suggested inflammatory changes in multiple organs by lead toxicity. |
Significant reduction in total protein level is in agreement with the similar type of study13 might be possibly due to lead binding to plasma protein and their removal through detoxification process which alters a large number of enzymes and disrupts protein synthesis in hepatocytes. Liver synthesizes proteins and albumin, the present finding of a decrease in total protein and albumin level in the liver attributed to the change in protein and free amino acid metabolism and their synthesis in the liver10. Lead is known to bind to the sulhydryl groups of enzymes containing cysteine and found to form complexes with amino acids and protein21. Degenerative changes reported in the liver of the T2 group might be the reason for decreased protein and amino acid metabolism which ultimately decreased albumin synthesis and total protein synthesis. Increase in protein level in group T4 suggested APSeNPs as strong antioxidants that can help in decreasing the negative effects of hepatic toxicity by scavenging reactive oxygen species (ROS)22. High plasma protein binding capacity has lower elimination half-life, as well as mean residence time of andrographolide, which resulted in hepatoprotection suggested it might be the reason for a significant increase to the tissues and improve the activities of hepatic antioxidant enzymes25. This corroborates with the present findings in group T4 treated with A. paniculata mediated selenium nanoparticles due to their antioxidant ability which repairs the hepatic injury also restores the cellular permeability and membrane-stabilizing activity which helps in healing parenchyma and the regeneration of hepatocytes. Significant decreased serum AST and ALT level given selenium nanoparticles during hepatotoxicity using different drugs and suggested hepatoprotective effect of selenium nanoparticles because of their antioxidant properties and its enhancing ability to eliminate free radicals from liver cells caused by lead toxicity26. The present finding of T2 group are in accordance with previous studies who also recorded increased in urea level against lead toxicity in rat and suggested might be due to oxidative damage lead to lipid peroxidation and cellular damage of kidney9,10. Elevation of serum urea and creatinine in lead acetate group reflecting renal impairment in kidneys in which oxidative damage might be a primary cause of lead toxicity due to lipid peroxidation and cellular damage. Decrease in creatinine and urea level given selenium nanoparticles against gentamycin toxicity in mice and suggested selenium nanoparticles easily penetrate the cells with the help of a capping agent, inhibiting ROS generation and protecting cells from oxidative dangers15. As a result, the presence of nanosized selenium particles enhanced and recovered changes in kidney indicators. Thus A. paniculata mediated selenium nanoparticles showed protective beneficial effect on hematobiochemical parameters during lead toxicity in mice. |
How to cite this article : Debbarma, K., Ingole, R.S., Kamdi, B.P., Hajare, S.W., Ingawale, M.V., Waghmare, S.P., Ingle, P.U. and Gade, A.K. 2022. Haematobiochemical alterations by lead acetate toxicity and its amelioration with Andrographis paniculata mediated selenium nanoparticles in mice. Indian J. Vet. Pathol., 46(4) : 327-332. Top Tables | Table 1.: Mean haematological values of Hb, PCV and TEC in control and treatment groups.
| | GROUP | Hb (g/dL) | PCV (%) | TEC (106/cumm) | MCV (fL) | MCH (pg) | MCHC (g/dl) | | T1 | 12.53±0.44a | 33.08±1.42a | 5.51±0.33a | 61.59±5.73 | 23.26±1.83 | 38.03±0.78 | | T2 | 10.03±0.34c | 27.96±1.33b | 3.63±0.28c | 80.16±8.69 | 28.17±2.85 | 36.17±1.93 | | T3 | 13.33±0.48a | 36.07±1.78a | 4.53±0.39bc | 81.64±6.18 | 30.15±1.83 | 37.31±1.93 | | T4 | 11.26±0.38b | 33.47±1.14a | 4.73±0.13ab | 70.81±2.12 | 23.92±1.24 | 33.87±1.60 | | P value | ** | ** | ** | NS | NS | NS | | CD (0.05) | 1.14 | 4.25 | 0.90 | - | - | - |
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| Values indicate mean ± S.E. Mean values with common alphabet as superscript do not differ significantly. | NS = Non-significant | Significant at 5% | Significant at 1% | | | Table 2.: Mean haematological values related to leucocytes in control and different groups.
| | GROUP | TLC (103/mm3) | Neutrophil (%) | Lymphocyte (%) | Monocyte (%) | Eosinophil (%) | Basophil (%) | | T1 | 8.46±0.23a | 35.00±2.50b | 62.50±2.37a | 1.16±0.16 | 1.33±0.21 | 0.00±0.00 | | T2 | 6.58±0.73b | 45.50±2.71a | 51.50±3.15b | 1.33±0.21 | 1.5±0.22 | 0.16±0.16 | | T3 | 8.56±0.33a | 45.00±3.20a | 52.33±3.28b | 1.66±0.33 | 0.83±0.16 | 0.16±0.16 | | T4 | 7.95±0.40ab | 33.50±2.15b | 63.50±2.48a | 1.50±0.34 | 1.16±0.16 | 0.33±0.21 | | P value | * | ** | ** | NS | NS | NS | | CD (0.05) | 1.37 | 7.88 | 8.27 | - | - | - |
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| Values indicate mean ± S.E. Mean values with common alphabet as superscript do not differ significantly. | NS = Non-significant | Significant at 5% | Significant at 1% | | | Table 3.: Serum total protein, albumin and globulin level in the control and different treatment groups.
| | GROUP | Total Protein (g/dl) | Albumin (g/dl) | Globulin (g/dl) | | T1 | 6.51±0.26a | 5.54±0.37a | 0.97±0.27 | | T2 | 5.00±0.38b | 4.25±0.31b | 0.74±0.15 | | T3 | 6.16±0.42a | 5.34±0.28a | 0.82±0.19 | | T4 | 5.85±0.27ab | 5.02±0.12ab | 0.82±0.21 | | P value | ** | ** | NS | | CD (0.05) | 1.01 | 0.85 | - |
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| Values indicate mean ± S.E. Mean values with common alphabet as superscript do not differ significantly. | NS = Non-significant | Significant at 5% | Significant at 1% | | | Table 4.: Serum AST, ALT, creatinine and BUN profile in different groups.
| | GROUP | AST (IU/L) | ALT (IU/L) | Creatinine (mg/dL) | BUN (mg/dL) | | T1 | 84.76±3.18bc | 36.7±1.11b | 0.25±0.02b | 26.35±1.07b | | T2 | 173.13±7.53a | 48.37±2.13a | 0.77±0.09a | 63.41±7.48a | | T3 | 77.93±4.51c | 38.52±1.19b | 0.40±0.05b | 22.49±0.63b | | T4 | 96.43±3.83b | 40.6±1.42b | 0.37±0.02b | 30.04±0.97b | | P value | ** | ** | ** | ** | | CD (0.05) | 14.89 | 4.49 | 0.17 | 11.28 |
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| Values indicate mean ± S.E. Mean values with common alphabet as superscript do not differ significantly. | NS = Non-significant | Significant at 5% | Significant at 1% | |
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