Introduction Nanotechnology is engineering at the molecular level. It is the combined term used for a number of technologies, techniques and processes that include the manipulation of matter at the smallest scale (from 1 to 100 nanometres)1. It exploits unique physical, chemical, electrical and mechanical properties. Nanoparticles are particles between 1 and 100 nanometres in size having a surrounding interfacial layer. The interfacial layer comprises of ions, inorganic and organic molecules. Organic molecules coating inorganic nanoparticles are known as stabilizers, capping or passivating agents. The properties of materials differ when their sizes reach the nanoscale. Copper nanoparticles below 50 nanometres are very hard materials that do not show same malleability and ductility as bulk copper2. This size provides them with different physical and chemical properties as compared to materials normally present in the environment3. Liposomes are one of the examples of semi-solid or soft nanoparticles used for administering anticancer drugs and vaccines4. Nanoparticles made from semiconductor materials are called as quantum dots5. These nanoparticles are commonly used in biomedical applications as drug carriers or imaging agents4,6,7. |
Nanomaterials can be composed of many different base substances (carbon, silicon, and metals which include gold, cadmium, aluminium and selenium). Nanomaterials additionally have different shapes: such as nanotubes, nanowires, crystalline structures including quantum dots and fullerenes, particles inside the nanometer length range do arise each in nature and as an incidental by-product of present commercial processes. |
Nanoparticles have outstandingly different physico-chemical properties than their conventional bulk materials and thus present possible threats both medically and environmentally8. With sizes lesser than cellular organelles, nanoparticles can easily enter through basic biological structures9. |
Particularly, particle size and surface area are considered key factors that contribute directly and considerably to toxicity of nanoparticles, with smaller sized nanoparticles showing higher toxic effects due to increased surface area10. Besides size, structure and shape of the nanoparticle also contribute to nanotoxicity. Also, the nanoparticle surface directs the adsorption of ions and biomolecules, thus influencing the cellular responses provoked and thereby promoting nanoparticle induced toxicity11. |
Nanoparticles can either be engineered or found in the environment naturally12. Engineered nanoparticles can exclusively be designed to either target or avoid interactions with the immune system. The engineered nanomaterials occurring in different shapes also induce toxicity depending on their dose, dimension, durability and distribution that is species specific13. |
Copper plays an important role in maintaining the vitality of many enzymes, copper deficiency is associated with the progression of many diseases14. It is an important trace mineral in humans, animals, plants and microbes and acts as a structural component and a cofactor in a variety of biochemical processes including protection against radicals, electron transport chains, hematopoiesis and pigmentation15,16. Copper oxide nanoparticles are one of the most extensively used nanoparticles as a heat transfer fluid because of their outstanding thermal conductivity17. Specifically, copper oxide nanoparticles are useful in the pharmaceutical industry especially for producing anti-microbial fabric treatments or prevention of Escherichia coli and methicillin-resistant Staphylococcus aureus infections18. It has also been employed as a catalyst, as well as being an essential p-type semiconductor in gas sensors, batteries, high-temperature superconductors, solar energy conversion and field emission emitters19. |
They are being utilized in a number of technical applications such as catalysts, solar cells, wood protection, electronics and antimicrobial products19,20,21,22,23. They are also being used as additives in inks24 and coatings in food packaging25. |
Copper nanoparticles and their oxidized form, copper oxide nanoparticles are extensively used as polymers, plastics and lubricants for metallic covering26. The outstanding thermo physical properties of copper nanoparticles increase their value for applications in the field of electronics technology, such as semiconductors, electronic chips and heat transfer nanofluids26,27. Besides this, synthesis of copper nanoparticles is cost efficient and the synthesized copper nanoparticles exhibit antimicrobial activity against many pathogenic microorganisms. Such advantages of nanoparticles have put them in the highlight as an alternative antimicrobial agent in many biomedical applications 28,29. |
In livestock feed, copper was traditionally used as an additive for its growth promoting effects, but the overuse of copper in the care of livestock led to pollution in the environment30,31. Nano-Cu is considered as being absorbed easily, and benefit to digestion of intestine, hematopoietic function, growth and immunity of animals at the lower doses32,33. Thus, nano-Cu is more widely used in animal feed, because it has more efficiency than normal Cu sources34,35,36. Copper nanoparticles, one of the manufactured nanoparticles, are now industrially produced and available commercially. |
Recently, it has shown great promise as an antibacterial agent37,38,39,40. Copper nanoparticles are used as additives in lubricants, polymers or plastics, metallic coatings41. |
They are also used as bioactive coatings that are capable of inhibiting target microorganisms such as Escherichia coli and Staphylococcus aureus4,2. They are also developed for temperature and pressure sensing43 and as hydrogen catalysts in fuel cells. |
Moreover, the detailed scientific studies on nano-copper using its NOAEL dose are lacking in literature, which will be utilized for future medicinal or other domestic use of nano-copper. Inspite of gaining popularity of nanotechnology in the field of medicine, applications of nanoparticles are restricted due to their potential toxicity and continuing secondary adverse effects44. The tremendous usage of these nanoparticles brings challenges to the environment and to the human life. |
Keeping in view the above facts, the present study is planned on Wistar rats using nano-copper for a period of 90 days so as to observe its clinico pathological effects. |
Top Materials and Methods Six weeks old Wistar Rats of both sexes were procured from Laboratory Animal Resources, Indian Veterinary Research Institute, Izatnagar, Bareilly, India. The rats were kept in separate cages under proper hygienic conditions in Experimental Animal House of Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Pantnagar and were fed standard recommended feed and purified water ad-libitum from 0 day upto 90 days of experiment. Proper light, ventilation and bedding material were provided until the end of the experiment. All rats were acclimatized for 7 days in the Experimental Animal House of the Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Pantnagar. Experiment was conducted with the approval of Institutional Animal Ethics Committee. Preformed copper nanoparticles were procured from Sisco Research Laboratories Pvt. Ltd for the study. Nano-copper was a black colour powder with a molecular weight of 79.54 and 99% assay with a shelf life of upto 60 months when stored at room temperature. The average particle size of the nano-copper was 40 nanometers (nm). Borosil glasswares viz: test tubes, flasks of various volumes and different shapes, beakers, slides, pipettes, petridishes, etc. were soaked in soap water overnight, then washed and rinsed thrice with triple glass distilled water and then dried and sterilized in hot air oven prior to their use. Plasticwares viz: Sterilized micropipette tips, centrifuge tubes and micro centrifuge tubes were used during the study. They were washed with detergent and rinsed thoroughly in tap water. They were finally rinsed in distilled water to eliminate all traces of residue. They were then sterilized in an autoclave at -121°C and 15lbs pressure for 30 minutes to ensure proper sterility. |
The dose of nanomaterial for the study was selected based on No Observable Adverse Effect Level dose (NOAEL) rate of cupric oxide nanoparticles in rats, i.e., 100 mg/kg body weight per day for 90 days, per-os45. |
Experiment Design The rats for experimental research were randomly divided into two groups viz. group 1 and group 2. Group 1 was kept as control and consisting of 20 rats. Group 2 was nano copper treated and comprising of 15 rats. The rats in group 2 (G2) were further randomly divided into 3 groups consisting of 5 rats each which were sacrificed at 30th, 60th and 90th day post treatment while 5 rats of group 1(G1) were sacrificed at 0, 30, 60 and 90 days. The rats in group 1 (G1) were provided with standard diet and water while the rats in group 2 (G2) along with standard feed and water were additionally given copper nanoparticles mixed in distilled water at NOAEL dose rate of 100mg/ kg body weight/day (Lee et al., 2016) from 0 day of experiment till 90 days post treatment (DPT), per-os. Examination of Blood For this purpose, blood samples were collected from each group of rats at different time intervals as per the experimental protocols. Collection of Blood Samples The blood samples were collected from each group of rats retroorbitally at 0, 30, 60 and 90 days of interval. The blood samples were transported to lab in ice packs in sterile conditions. Blood samples for haematological analysis were collected in EDTA containing vials and in heparin vials for lymphocyte blastogenesis assay at 0, 30th, 60th and 90th day post treatment. Separation of Serum For serum collection, the fresh blood without anticoagulant was collected in test tubes and was kept in slanting position at room temperature for 20 to 30 minutes followed by overnight refrigeration. The samples were then subjected to centrifugation at 3000 rpm for 30 minutes. Centrifugation of the sample results into division of blood into 3 components; the lowermost being red blood cells, buffy coat and then topmost layer of serum. The serum was then collected in a separate eppendorf tube. The serum was stored at - 20°C in deep freezer until completion of biochemical analysis. Haematological Parameters Haemoglobin Concentration Blood from rats in both the control and test groups was collected in EDTA vials at 0, 30, 60 and 90 days interval. The haemoglobin concentration was then evaluated using Sahli’s haemoglobinometer under the standard protocol and was expressed as gm/dl. Total Leucocyte Count The blood samples from both the groups were taken and then using haemocytometer, WBC’s diluting fluid and Neubauer’s chamber, the total leucocyte counts were performed. The leucocytes were counted at 10X magnification in the microscope at 4 primary corner squares of the Neubauer’s chamber and was expressed as 103/μl of blood. It was calculated by multiplying the total number of cells counted in 4 primary squares by 50. Differential Leucocyte Count For differential count, blood smear on the slide was prepared for each blood samples of the group. The blood smear was then allowed to air dry and was then fixed using methanol for 5 minutes. After fixing of the smear, the slide was flooded with Giemsa stain. The slides were then allowed to stand for 45 minutes to 1 hour. Then the slides were rinsed using distilled water. The slides were air dried and then observed under oil immersion in microscope. Differential leucocyte count was performed as per the standard protocol in a zig-zag manner upto a total of 200 leucocytes. Mean lymphocyte and neutrophil counts were calculated. Cells count were presented in percent46. Absolute Lymphocyte Count and Absolute Neutrophil Count Among the absolute counts, absolute lymphocyte (ALC) and absolute neutrophil counts (ANC) were calculated. Absolute lymphocyte count and absolute neutrophil count were calculated from the total leucocyte count and differential leucocyte count based on the following formula. ALC (103/μl) = % lymphocyte X TLC (103/μl) /100 ANC (103/μl) = % neutrophil X TLC (103/μl) /100
Biochemical Assays Total Serum Protein Measurements of total serum protein was done by Modified Biuret method47, using a kit from Erba Diagnostics Mannheim Ltd. Baddi, Distt. Solan (H.P.), India and the results were expressed as gram per deciliter (g/dl). The procedure given with the kit was followed. Standard, test and blank samples were made and the OD was then taken using UV-Vis spectrophotometer at 546 nm. Serum Albumin Serum albumin concentration was determined by Bromocresol Green (BCG) method48, using a kit from Erba Diagnostics Mannheim Ltd. Baddi, Distt. Solan (H.P.), India and the results were expressed as gram per deciliter (g/dl). The procedure given with the kit was followed. Standard, test and blank samples were made and the OD was then taken using UV-Vis spectrophotometer at 630 nm. Serum Globulin The serum globulin content was calculated by subtracting the values of serum albumin from the respective values of total serum protein. The serum globulin was calculated by the following formula: Serum globulin (g/dl) = Total serum protein (g/dl) -Serum albumin (g/dl) Serum Gamma Globulin For the determination of serum gamma globulin, a solution of ammonium sulphate ((NH4)2SO4) and sodium chloride (NaCl) (19.5 % and 2.03 %) was prepared. 5.7 ml of this solution was then put in a centrifuge tube and overlaid with 0.3 ml of clear serum. After mixing gently, the mixture was kept on an ice-bath for 15 minutes followed by centrifugation at 1250g for 10 minutes. The precipitate was obtained, which was dissolved in 0.2 ml of normal saline solution and process of precipitation was repeated. Finally, all the precipitates were dissolved in 2 ml of normal saline solution and then 5 ml of biuret reagent was added. The mixture was then kept at room temperature for 10 minutes and then the optical density (OD) was read at 555 nm in UV-Vis spectrophotometer49. Serum Aspartate Aminotransferase (AST) ERBA kit procured from Erba Diagnostics Mannheim Ltd. Baddi, Dist. Solan (HP), India was used for detection of serum aspartate aminotransferase (AST) or serum glutamic oxaloacetic transaminase (SGOT)51 following the procedure given with the kit. Standard, test and blank samples were made and the OD was then taken using UV-Vis spectrophotometer at 340 nm. Serum Creatinine Serum creatinine concentration was determined by modified Jafte’s reaction method50, using a kit from Erba Diagnostics Mannheim Ltd. Baddi, Dist. Solan (HP), India, and the results were expressed as milligram per deciliter (mg/dl). The procedure given with the kit was followed. Standard, test and blank samples were made and the OD was then taken using UV-Vis spectrophotometer at 505 nm. Statistical Analysis The data generated during the experiment was statistically analysed by using standard statistical procedures52 with the help of SPSS software. The collected data was analysed by one-way ANOVA. Top Results Mean Haemoglobin Concentration Mean haemoglobin (Hb) of experimental rats in different groups at different time intervals are expressed in g/dl and presented in Table 1 and Fig. 1. Mean haemoglobin concentration (Mean ±SE) of control rats were 14.80±1.31, 15.16±0.45, 14.40±0.53 and 15.90±1.00 at 0, 30th, 60th and 90th DPT, respectively. Mean haemoglobin concentration (Mean ±SE) of copper nanoparticles treated rats were 14.80±1.31, 15.10±0.53, 13.70±0.56 and 15.50±1.12 at 0, 30th, 60th and 90th DPT, respectively. There was decrease of 0.39%, 4.86% and 2.52% in the mean haemoglobin concentration of treated group as compared to control rats at 30th, 60th and 90th DPT, respectively. The decrease in mean haemoglobin concentration of treated rats was found to be statistically significant than control rats at 60th DPT. Packed Cell Volume Mean packed cell volume (PCV) of experimental rats in different groups at different time intervals is expressed in percent and presented in Table 1 and Fig. 2. Mean packed cell volume (Mean ±SE) of control rats were 46.40±3.93, 45.40±1.12, 44.10±1.60 and 49.10±3.00 at 0, 30th, 60th and 90th DPT, respectively. Mean packed cell volume (Mean ±SE) of treated rats were 46.40±3.93, 47.10±1.62, 43.10±1.68 and 48.50±3.35 at 0, 30th, 60th and 90th DPT, respectively. There was increase of 3.74% in the mean packed cell volume of treated rats as compared to control rats at 30th DPT. There was decrease of 2.27% and 1.22% in the mean packed cell volume of treated rats as compared to control rats at 60th and 90th DPT, respectively. However, these different data were not statistically significant. Total Leucocyte Count Mean total leucocyte count (TLC) values of experimental rats in different groups at different time intervals are expressed in 103/μl of blood and presented in Table 1 and Fig. 3. Mean total leucocyte count (Mean ±SE) of control rats were 11.91±1.92, 15.54±2.35, 17.10±2.35 and 18.21±4.27 at 0, 30th, 60th and 90th day post-treatment (DPT), respectively. Mean total leucocyte count (Mean ±SE) of treated rats were 11.91±1.92, 19.66±1.93, 11.66±1.93 and 17.45±2.45 at 0, 30th, 60th and 90th DPT, respectively. There was an increase of 26.51% in the mean total leucocyte count of treated rats as compared to that of control rats at 30th DPT. However, there was decrease of 31.81% and 5.05% in the mean total leucocyte count of treated rats as compared to that of control rats at 60th and 90th DPT, respectively. The decrease in mean total leucocyte count of treated rats was found to be statistically significant than control rats at 60th DPT. There was significant difference in the mean TLC of treated rats between 0 and 30th DPT, 30th and 60th DPT. Absolute Lymphocyte Count Mean absolute lymphocyte count of experimental rats are expressed in 103/μl and shown in Table 1 and Fig. 4. Mean absolute lymphocyte count (Mean ±SE) of control rats were 10.34±1.74, 12.21±2.16, 12.72±1.90 and 15.80±3.82 at 0, 30th, 60th and 90th day post-treatment (DPT), respectively. Mean absolute lymphocyte count (Mean ±SE) of treated rats were 10.34±1.74, 13.10±2.50, 8.14±0.78 and 15.09±2.09 at 0, 30th, 60th and 90th DPT, respectively. In treated rats, an increase of 7.29% was observed in mean absolute lymphocyte count at 30th DPT as compared to control rats. In treated rats, decrease of 36.01% and 4.49% were observed in mean absolute lymphocyte count at 60th and 90th DPT as compared to control rats, respectively; it was not statistically significant. Absolute Neutrophil Count Absolute neutrophil count (ANC) values of experimental rats in two groups are shown in Table 1 and Fig. 5 and expressed in 103/μl Mean absolute neutrophil count (Mean±SE) of control rats were 1.08±0.20, 2.66±0.41, 2.77±0.73 and 1.44±0.39 at 0, 30th, 60th and 90th day post-treatment (DPT), respectively. Mean absolute neutrophil count (Mean ±SE) of treated rats were 1.08±0.20, 4.99±1.26, 2.54±0.93 and 1.73±0.39 at 0, 30th, 60th and 90th DPT, respectively. Absolute neutrophil count was found to be decreased by 8.30% at 60th DPT and it was found to be increased by 87.59% and 20.14% at 30th and 90th DPT, respectively in nano-copper treated rats. There was significant difference in the mean ANC of treated rats between 0 and 30th DPT, 30th and 60th DPT, 30th and 90th DPT. Differential Leucocyte Count Differential leucocyte count (DLC) was done to assess the different leucocyte counts like lymphocytes, neutrophils, monocytes, eosinophils and basophils. The variation in counts of different white blood cells over the entire experimental period is described below. Lymphocytes Table 1 and Fig. 6 shows mean lymphocyte count of experimental rats in two groups at different time intervals and is expressed in percent. Mean lymphocyte count (Mean ±SE) of control rats were 86.60±0.93, 77.80±2.87, 73.60±2.87 and 86.80±2.01 at 0, 30th, 60th and 90th day post-treatment (DPT), respectively. Mean lymphocyte count (Mean ±SE) of treated rats were 86.60±0.93, 65.20±9.32, 72.00±9.32 and 86.60±0.51 at 0, 30th, 60th and 90th DPT, respectively. There was decrease of 17.05%, 2.17% and 0.23% in the mean lymphocyte count of treated rats as compared to controls at 30th, 60th and 90th DPT, respectively. There was significant difference in the mean lymphocyte count of treated rats between 0 and 30th DPT, 30th and 90th DPT. Neutrophils Table 1 and Fig. 7 shows mean neutrophil count of experimental rats in two groups at different time intervals and is expressed in percent. Mean neutrophil count (Mean ±SE) of control rats were 9.20±0.97, 17.60±1.86, 16.60±1.86 and 7.80±1.16 at 0, 30th, 60th and 90th DPT, respectively. Mean neutrophil count (Mean ±SE) of treated rats were 9.20±0.97, 28.60±8.74, 20.20±8.74 and 9.40±1.03 at 0, 30th, 60th and 90th DPT, respectively. There was increase of 62.5%, 21.69% and 20.51% in the mean neutrophil count of treated rats as compared to controls at 30th, 60th and 90th DPT, respectively. Monocytes Table 1 and Fig. 8 shows mean monocyte count of experimental rats in two groups at different time intervals and is expressed in percent. Mean monocyte count (Mean±SE) of control rats were 1.60±0.40, 2.00±0.32, 5.20±0.32 and 1.40±0.75 at 0, 30th, 60th and 90th DPT, respectively. Mean monocyte count (Mean±SE) of treated rats were 1.60±0.89, 3.00±0.45, 5.00±1.14 and 2.00±1.09 at 0, 30th, 60th and 90th DPT, respectively. There was increase of 50% and 11.11% in the mean monocyte count of treated rats as compared to controls at 30th and 90th DPT, respectively. However, there was decrease of 3.85% in the mean monocyte count of treated rats as compared to controls at 60th DPT. However, these different data were not statistically significant. Eosinophils Table 1 and Fig. 9 shows mean eosinophil count of experimental rats in two groups at different time intervals and is expressed in percent. Mean eosinophil count (Mean±SE) of control rats were 2.00±0.55, 1.40±0.40, 4.40±2.04 and 3.80±0.86 at 0, 30th, 60th and 90th DPT, respectively. Mean eosinophil count (Mean±SE) of treated rats were 2.00±0.55, 2.60±1.29, 2.80±1.11 and 2.00±0.55 at 0, 30th, 60th and 90th DPT, respectively. There was increase of 85.71% in the mean eosinophil count of treated rats as compared to controls at 30th DPT. However, there was decrease of 36.36% and 47.37% in the mean eosinophil count of treated rats as compared to controls at 60th DPT and 90th DPT, respectively. However, these different data were not statistically significant. Basophils Table 1 and Fig. 10 shows mean basophil count of experimental rats in two groups at different time intervals and is expressed in percent. Mean basophil count (Mean±SE) of control rats were 0.60±0.40, 0.40±0.40, 0.20±0.20 and 0.20±0.20 at 0, 30th, 60th and 90th DPT, respectively. Mean basophil count (Mean±SE) of treated rats were 0.60±0.40, 0.60±0.40, 0±0 and 0±0 at 0, 30th, 60th and 90th DPT, respectively. There was increase of 50% in the mean basophil count of treated rats as compared to controls at 30th DPT. However, there was decrease of 100% and 100% in the mean basophil count of treated rats as compared to controls at 60th DPT and 90th DPT, respectively. However, these different data were not statistically significant. Total Serum Protein Mean total serum protein values of experimental rats in control and treated groups at different time intervals are expressed in g/dl and presented in Table 2 and Fig. 11. Mean total serum protein (Mean ±SE) of control rats were 5.41±0.27, 5.30±0.24, 7.43±0.33 and 7.89±0.69 at 0, 30th, 60th and 90th DPT, respectively. Mean total serum protein (Mean ±SE) of treated rats were 5.41±0.27, 5.63±0.24, 6.64±0.73 and 4.78±1.52 at 0, 30th, 60th and 90th DPT, respectively. Total serum protein was found to be decreased by 10.63% and 39.42% at 60th DPT and 90th DPT, respectively. However, total serum protein was found to be increased by 6.23% at 30th DPT. The increase in mean total serum protein of treated rats was found to be statistically significant than control rats at 30th DPT. Serum Albumin Mean serum albumin values of experimental rats in control and treated groups at different time intervals are expressed in g/dl and presented in Table 2 and Fig. 12. Mean serum albumin (Mean ±SE) of control rats were 2.49±0.20, 2.67±0.17, 2.75±0.15 and 2.81±0.17 at 0, 30th, 60th and 90th DPT, respectively. Mean serum albumin (Mean ± SE) of treated rats were 2.49±0.20, 2.64±0.03, 3.07±0.08 and 2.07±0.69 at 0, 30th, 60th and 90th DPT, respectively. Serum albumin was found to be decreased by 1.12% and 26.33% at 30th and 90th DPT, respectively and serum albumin was found to be increased by 11.64% at 60th DPT. However, these different data were not statistically significant. Serum Globulin Mean serum globulin values of experimental rats in control and treated groups at different time intervals are expressed in g/dl and presented in Table 2 and Fig. 13. Mean serum globulin (Mean ±SE) of control rats were 2.92±0.40, 2.63±0.11, 4.69±0.27 and 5.08±0.61 at 0, 30th, 60th and 90th DPT, respectively. Mean serum globulin (Mean±SE) of treated rats were 2.92±0.40, 2.99±0.26, 3.57±0.80 and 2.71±0.85 at 0, 30th, 60th and 90th DPT, respectively. Mean serum globulin was found to be increased by 13.69% at 30th DPT. However, it was found to be decreased by 23.88% and 46.65% at 60th and 90th DPT, respectively. There was no significant difference in the mean serum globulin of control and treated rats at 0, 30th, 60th and 90th DPT, respectively. Also, there was no significant difference within days in the mean serum globulin of treated rats. Serum Gamma Globulin Mean serum gamma globulin values of experimental rats in control and treated groups at different time intervals are expressed in g/dl and presented in Table 2 and Fig. 14. Mean serum globulin (Mean ± SE) of control rats were 0.33±0.05, 0.59±0.11, 0.48±0.14 and 0.88±0.16 at 0, 30th, 60th and 90th DPT, respectively. Mean serum gamma globulin (Mean ± SE) of treated rats were 0.33±0.05, 0.91±0.004, 0.93±0.15 and 0.75±0.18 at 0, 30th, 60th and 90th DPT, respectively. Mean serum gamma globulin was found to be increased by 54.24% and 93.75% at 30th and 60th DPT, respectively. However, it was found to be decreased by 14.77% at 90th DPT. The increase in mean serum gamma globulin of treated rats was found to be statistically significant than control rats at 30th and 60th DPT, respectively. There was significant difference in the mean serum gamma globulin of treated rats between 0 and 30th DPT, 0 and 60th DPT, 0 and 90th DPT, 60th and 90th DPT, respectively. Serum Aspartate Aminotransferase (AST) Mean AST values of experimental rats in control and treated groups at different time intervals are expressed in IU/L and presented in Table 2 and Fig. 15. Mean AST (Mean ±SE) of control rats were 18.04±4.85, 27.23±5.26, 58.36±7.73 and 28.65±10.14 at 0, 30th, 60th and 90th DPT, respectively. Mean AST (Mean ±SE) of treated rats were 18.04±4.85, 12.73±2.12, 36.08±5.50 and 55.44±2.28 at 0, 30th, 60th and 90th DPT, respectively. Mean AST was found to be decreased by 53.25% and 38.18% at 30th and 60th DPT, respectively. However, it was found to be increased by 93.51% at 90th DPT. The mean AST values of treated rats were found to be statistically significant as compared to control rats at 30th, 60th and 90th DPT, respectively. There was significant difference in the mean AST values of treated rats between 0 and 60th DPT, 0 and 90th DPT, 30th and 60th DPT, 30th and 90th DPT, 60th and 90th DPT, respectively. Serum Creatinine Mean serum creatinine values of experimental rats in control and treated groups at different time intervals are expressed in mg/dl and presented in Table 2 and Fig. 16. Mean serum creatinine (Mean ± SE) of control rats were 2.79±0.65, 2.13±0.98, 0.37±0.04 and 0.74±0.04 at 0, 30th, 60th and 90th DPT, respectively. Mean serum creatinine (Mean ± SE) of treated rats were 2.79±0.65, 0.53±0.09, 0.41±0.05 and 0.82±0.07 at 0, 30th, 60th and 90th DPT, respectively. Mean serum creatinine was found to be decreased by 75.12% at 30th DPT. However, it was found to be increased by 10.81% and 10.81% at 60th and 90th DPT, respectively. There was significant difference in the mean serum creatinine of treated rats between 0 and 30th DPT, 0 and 60th DPT, 0 and 90th DPT, respectively. Top Discussion The present study of various haematological parameters revealed decrease in the mean haemoglobin, mean packed cell volume, mean total leucocyte count, mean absolute lymphocyte count and mean lymphocyte count in nano-copper treated group as compared to control group. There was an increase observed in absolute neutrophil count and mean neutrophil count. These observations are in agreement with one study53. The decrease in Hb and PCV levels observed in this study may be due to the dysfunction of haematopoietic system induced by nanoparticles or due to the haemolytic effect induced by the release of oxidative stress products following the exposure to copper nanoparticles. Decrease in total leucocyte count and absolute lymphocyte count is mainly due to the lymphocytotoxic effect of nano-copper, which may lead to immune dysfunction. The elevation of neutrophil count in the present study might be due to the compensatory mechanism of the body due to decrease in lymphocyte counts. The present study of various biochemical parameters revealed decrease in total serum protein (TSP), serum albumin (SA), serum globulin (SG) and serum gamma globulin (SGG) in nano-copper treated group as compared to control group. |
It may be due to functional and structural damage caused by copper dissociated from copper nanoparticles into liver, kidney and spleen which is consistent with the observations of another study45. However, there was an elevated level of serum creatinine and liver function enzyme i.e., Aspartate aminotransferase (AST) in nano-copper treated group as compared to that of control group. This observation is consistent with the data obtained in one study53. This might be due to increase in production of free radicals and beginning of reactive oxygen species (ROS) reactions, that causes damage to hepatocytes in liver and increase the level of liver enzymes due to tissue destruction and releasing these enzymes into the blood stream. These findings on the consequence of copper nanoparticles exposure suggest that they cause substantial damage to the liver and kidneys. |
Top Conclusion The present study can be concluded that the nano-copper in NOAEL dose is exerting its deleterious effects on vital organs/tissues/systems of the body that may lead to immunopathology, hepatopathy, nephropathy, neuropathy and may adversely affect the reproductive health of animals. Further studies should be carried out in different animal models using varied doses and increased duration of copper nanoparticles to exactly find out the clinicopathological alterations. |
Top Acknowledgements I would like to extend my sincere gratitude to Dr.RS Chauhan sir for his guidance and assistance in selection of the nanoparticle and planning of the research work. I am also very thankful to Dr. Diksha, Dr. Shodhan and Dr. Neha for their assistance during the entire research work. |
Top Figures | Fig. 1.: Mean haemoglobin concentration in g/dl of experimental rats at different time intervals
|  | |
| | Fig. 2.: Mean packed cell volume in % of experimental rats at different time intervals
|  | |
| | Fig. 3.: Mean total leucocyte count in 103/μl of experimental rats at different time intervals
|  | |
| | Fig. 4.: Mean absolute lymphocyte count in 103/μl of experimental rats at different time intervals; *Alphabetical letters (a and b) indicate significant difference within days in treated group
|  | |
| | Fig. 5.: Mean absolute neutrophil count in 103/μl of experimental rats at different time intervals
|  | |
| | Fig. 6.: Mean lymphocyte count in % of experimental rats at different time intervals.
? ? ? ? ?* indicates significant difference (P<0.05) between groups at a particular DPT (DPT= Day Post-Treatment) whereas alphabetical letters (a and b) indicate significant difference within days in treated group;
|  | |
| | Fig. 7.: Mean neutrophil count in % of experimental rats at different time intervals
|  | |
| | Fig. 8.: Mean monocyte count in % of experimental rats at different time intervals
|  | |
| | Fig. 9.: Mean eosinophil count in % of experimental rats at different time intervals
|  | |
| | Fig. 10.: Mean basophil count in % of experimental rats at different time intervals
|  | |
| | Fig. 11.: Mean total protein in g/dl of experimental rats at different time intervals.
|  | |
| | Fig. 12.: Mean serum albumin in g/dl of experimental rats at different time intervals.
|  | |
| | Fig. 13.: Mean serum globulin in g/dl of experimental rats at different time intervals
|  | |
| | Fig. 14.: Mean serum gamma globulin in g/dl of experimental rats at different time intervals; *indicates significant difference (P<0.05) between groups at a particular DPT (DPT= Day Post-Treatment) whereas alphabetical letters (a, b and c) indicate significant difference within days in treated group
|  | |
| | Fig. 15.: Mean AST in IU/L of experimental rats at different time intervals
|  | |
| | Fig. 16.: Mean Creatinine in mg/dl of experimental rats at different time intervals
|  | |
|
Tables |