Effect of silver nanoparticles on some blood parameters in rats

The present study aims are investigating the effects of oral administration of AgNPs on some blood the parameters to show potential of toxicity of AgNPs as tool therapy and diagnosis. In this study, 20 healthy male and female rats were used. Randomly, rats were divided into 5 groups depending on the period variable respectively; AgNPs-treated rats' groups for a period of 3 days in concentration 0.2 and 0.4 gm/kg B.W, while the second period after 20 and 30 days after treatment with concentration 0.08 and 0.008 gm/ kg B.W. In addition to a control group. A Manual method was used to measure Hematological parameters. Rats treated with AgNPs showed the significant P<0.05 decrease in Hemoglobin, Packed Cell Volume, Red Blood Cells. Also, Mean Corpuscular Volume; Mean Corpuscular Hb and Mean Corpuscular Hb Concentration. Platelets levels and White Blood Cell Count were After the administration of AgNPs, a protective effect on hematopoietic levels and on Red Blood Cell morphology were shown respectively as it is assessed by Leishman stained blood smears resulted in ovalocytosis at the dose 0.4 gm/kg body weight, while at the dose, 0.08 gm/kg showed echinocytes, acanthocytes, tear drop, stomatocyte and codocyte (target RBCs). Thus, it is concluded that oral administration of AgNPs caused a decreased in the level of Hb concentration, PCV, RBC, MCV, MCH, MCHC, but increase PLTS and WBCs. It means that AgNPs has a toxic effect on blood.


Introduction
It is widely known that Silver nanoparticles (Ag-NP) used as antimicrobial substances (1). The use Ag + ions cause damage to many microorganism (2). Different studies have showed that nanostructures especially nanoparticles, nanorods, nanotubes, cause hemolysis and blood clotting (3). The materials used were different from each other both in size and coating that led to difficulty in holding a comparison. Moreover, a default of suitable control studies done on this subject were not systematic enough which in turn impedes a comprehensive evaluation of AgNPs blood compatibility. Anyhow, it is reported that, due to the large surface area, a significant increase in an in vitro hemolysis was observed with AgNPs that compared with micronsized particles (4). A Complete Blood Count (CBC) explain the type and the numbers of cells in the blood especially RBCs, WBCs and platelets, a CBC helps in diagnosing conditions, such as anemia, infection, and many other disorders (5). Qualifying the AgNPs toxicity, an important step should be considered, i.e. assessing the various hematological parameters such as HCT; Hb; RBCs; WBCs; and PLTs counts (6). In medicine, there is a kind of unawareness of the parameters hematological alterations induced by Nanomaterial. This research, as mentioned earlier, aimed at measuring routine hematological indices and at identifying and interpreting different dimensional hematological parameters to quantify the AgNPs toxicity.

Experimental animals
Twenty male and female of Albino rats 4 weeks old and 45-50 gm body weight were housed in hanging cages and kept at temperature 25±2ºC and 14 hours light and 10 hours dark and were given normal food and tap water. The size of AgNPs which used in this study is100nm were orally administered in rats which were randomly divided into five groups. Each one contains 4 animals.

Experimental design
In group 1 rats treated with AgNPs at concentration 0.2 gm/kg orally given with water and dissection after 3 days. In group 2, rats treated with AgNPs in concentration 0.4 gm/kg orally given with water and dissection after 3 days. In group 3, rats treated with AgNPs in concentration 0.4 gm/kg orally given with water and dissection after 3 days. In group 4, rats treated with AgNPs in concentration 0.4 gm/kg orally given with water and dissection after 3 days. In group 5, rats fed with standard diet and water with any treatment. Blood samples were collected anticoagulant (EDTA) and used for examination of hematological analysis by manual methods. Blood smear was stained with Leishman stain (7).

Statistical analysis
Our data analyzed via the mean ± St. deviation Data were analyzed via using SPSS, the statistical software. One-way ANOVA option was used. Post hoc test was performed using the Duncan's test. Mean values were considered to be statistically significant at P≤0.05 (6).

Results
The present study showed that rats treated with AgNPs developed a difference in Hb, PCV, total WBCs, lymphocyte, mononuclear and granulocyte numbers and RBCs compared with control group. Hb, PCV and red blood cell are higher decrease significantly compared with control animals. Figure 1, showed the Hb in rats after oral administration 0.2, 0.4,0.08 and 0.008 gm/wb of AgNPs compared with the control. Figure 2, showed the hematocrit % or packed cell volume in animals after oral treatment of 0.2, 0.4,0.08 and 0.008 gm/W.B. compared with the group 5.
Most of the WBCs parameters were higher in the treated rats than in the group 5 ones but not significantly, although the lymphocyte percent was significantly higher in the treated compared with control ones, with increasing of lymphocytes percentage accompanied compared with control.
A depletion the majority of monocytes%, neutrophils%, and eosinophils % after treatment with AgNPs compared with the group five. While the account of RBC was decreased in treated animals compared with the control ones. The PLTs count increased after treatment of AgNPs compared with the group 5.
The relationships between the HCT, the HG level, and the RBCs are converted to erythrocyte osisindices through mathematical formulas. The indicator includes MCV, MCH, MCHC and RDW. Figure 2 showed significantly increased in MCV after given 0.2, 0.4,0.08 and 0.008 gm/wb of AgNPs compared with the non-treatment animal (control group), respectively (Tables 1 and 2). The MCV accompanied with the given of treatment of AgNPs significantly increased compared with control. The MCV shows the size of RBCs (Figure 3), while figure 4 showed decrease, RBCs count and increase total WBCs count.

RBCs Abnormality
Normal red blood cell has a biconcave disk shape and have an area of pallor center when viewed microscopically ( Figure 5). Different kinds of RBCs anomalies were observed in blood film of rats treated with silver nanoparticles with different doses includes hypochromic normocytic ( Figure 6 Figure (7) showed Hypochromic anemia seen in blood film of rats that are exposed to silver nanoparticles at concentration 0.2 g/kg. Figure (13) showed Macrocytic anemia which the RBCs appears abnormal in size. Figure  (14) showed Rouleaux formation which means stacks either singular or roulea or aggregation of red blood cells, that appear as stacks together in chain.

Discussion
Obtained result on hematology showed an alteration in blood picture and number of red cells parameters. These alterations in red blood cells may indicate effect of the nanoparticles on hemoglobin syntheses during red blood cells maturation during formation in bone marrow (7).
White blood cells have an important role in the immunity of body response, represent the first defense line different types of the neutrophils can indicate an infection, allergic or toxic reaction against drugs or chemicals, (8).
While the RBC account was decreased in treated animals in comparison with the control group. The occurrence of anemia is due to hypoxia and the decrease in RBCs count means that there was an increased destruction of (Red Blood Cells) RBCs which it might be due to the deficiency of iron and cobalamin, or folic acid or it is due to certain chronic diseases, and toxic substances that cause depletion in the account RBC which produce via the bone marrow, the oral administration and intraperitoneal injection of Ag NPs cause toxic effect on RBC (9).
The effect of AgNPs on hemopoietic system lead to decrease in the number of RBCs. Additionally, AgNPs which cause disturbances on the function of immune system (10,11). The slight decrease in the red blood cell and Hb may have been resulted from the suppression of circulating hormone, erythropoietin (a glycoprotein which stimulates the process of erythropoiesis) (12). Reduction in blood concentration of erythropoietin may result in a normochromic, normocytic anemia (13). Although iron deficiency is by far the most common cause, the hypochromic anemia is a generic term for any type of anemia. (14).
Macrocytic RBCs are abnormal caused due to a variety of illnesses. Abnormal red blood cells codocyteare seen in association with some forms of anemia (15). Platelets (thrombocytes) have important role on coagulation of blood so the increase of platelets count lead to thrombus formation with in the blood vessels that promote the progression of atherosclerosis which results from the administration of AgNPs (16).
Hemolysis is the rupture of erythrocytes. This will lead to release of their contents (like hemoglobin) into surroundings the area (17), which failure disturbance decrease in function anemia, jaundice and renal failure (18). Macrocytosis is the first abnormality can be seen in complete blood counts of patients with folate or vitamin B12 deficiency. Tear drop shaped cells may indicate myelofibrosis; anemia decrease in the function of bone marrow in producing which cause by toxin effect. The reason high of plasma proteins, primarily fibrinogen, and globulins is rouleaux formation. It is seen in a variety of infectious and inflammatory disorders associated with polyclonal increases in globulins and/or increased levels of fibrinogen.
Rouleaux formation associated with monoclonal gammopathies can be seen in multiple myeloma and in malignant lymphomas such as Waldenstrom's macroglobulinemia.
The abnormal morphology of BCRs occur due to disturbances in function of bone marrow which caused by pathological processes of this will lead to formation of macrocytic RBC. Otherwise the Hypochromic erythrocytes was occurred due to failure hemoglobinization of erythrocytes in the bone marrow. White stomatocytes which appears bowl shape in three-dimensional views. these cells are appeared in an intermediate form in the transformation of diskocytes to spherocytes. Acanthocytes can be seen in patient suffered from chronic alcoholic liver disease also caused by deficiency of pyruvate kinase form. While the oval shape of RBCs is caused by abnormal proteins of membrane.
This occur due to disorder Infiltration of the bone marrow with disruption of vasculature of the marrow, are associated with the formation of oval and teardrop erythrocytes. Bite cells apparently arise when a phagocyte removes a portion of the red cell along with a Heinz body (19).
Several authors have suggested that silver nanoparticles link to sulfur and phosphorus containing biomolecules like DNA or other biological moieties, so that cause cell toxicity (20). Anther searchers have shown the cytotoxicity of silver nanoparticles related to free radical formation which causes distortion in cell membrane (21). Also, Ag nanoparticles cause low metabolic activity and have toxicological properties (22).
The chemical structure of nanoparticle, shape, size, agglomeration, surface, and function are the factors which effect the biokinetics of silver nanoparticles cytotoxicity effect (23). So, the toxic effect of silver nanoparticles is due to the direct interaction of it with the RBCs which it causes oxidative stress, membrane injury than hemolysis. However, taxological information has other effects on red blood cells (RBCs) and the mechanisms involved remain scant (24,25).
The toxic effects of Ag nanoparticles are hemolysis as membrane injury, lipid peroxidation with fair the production of antioxidant enzyme. The smallest size of AgNPs has ability to induce hemolysis and membrane destruction (24). The results of this study show that the size and the concentration of AgNPs are predisposing factor that affects the RBC by the interaction between erythrocytes and AgNPs.

Conclusion
We concluded that oral administration of AgNPs caused decreased in the level of Hb concentration, PCV, RBC, MCV, MCH, MCHC, but increase PLTS and WBCs. It means that AgNPs has a toxic effect on blood.