Cytotoxic Impact of Zinc Oxide Nanoparticles against ...

International Journal of Pharma Sciences Vol. 4, No. 3 (2014): 560-564 Research Article Open Access

ISSN: 2320-6810

Cytotoxic Impact of Zinc Oxide Nanoparticles against Ehrlich Ascites Carcinoma Cells in Mice Magdy F. Abou El Fatoh1, Mayada R. Farag1, Shafika A.E2, Mohammed A. Hussein3,*, Kamel M.A4 and Salem G.A4 1

Department of Forensic Medicine and Toxicology, Faculty of Veterinary medicine, Zagazig University Department of Histology, Faculty of Veterinary medicine, Zagazig University 3 Department of Biochemistry, Faculty of pharmacy, October 6th University, Egypt 4 Department of Pharmacology, Faculty of Veterinary medicine, Zagazig University 2

* Corresponding author: Mohammed A. Hussein; e-mail: [email protected]

Received: 22 April 2014

Accepted: 05 May 2014

Online: 09 May 2014

ABSTRACT The present study was conducted to evaluate the cytotoxic impact of 5, 7.5 and 10mg/kg b.w zinc oxide nanoparticles (ZNONPS) against Ehrlich Ascites Carcinoma (EAC) Cells bearing mice, through investigation of the total number of EAC life cells, degenerated cells and total tumor cell count as well as some tumor markers (CA and CEA) and histopathological examination of both liver and spleen tissues. Seven days later to EAC implantation showing round cells aggregation with portal area hepatocyte with karyo and cytomegaly besides extra medullary hematopoiesis. The histopathological examination showed that the liver tissues exposed from positive control mice on the same context splenic tissues exposed from positive control mice implanted with EAC. The treated groups by ZNONPS different doses revealed for three weeks increasing of dead cells percentage with reduced volume of ascetic fluid in the peritoneum, reduction in the values of tumor markers (CA and CEA) and reduced total number of EAC cells and the percentage of the life cells as well as intensive lymphoid depletion and presence of clusters of neoplastic cells and megakaryocytes, these lesions were remarkably reduced by co-treatment with ZNONPS.

Keywords: Zinc oxide nanoparticles, tumor cells, Carcinoembryogenic Antigen, Carcino Antigen. INTRODUCTION A nanoparticle is defined as a small particle that behaves as a whole unite in terms of its transport and properties. Nanotechnology concerned with very small molecules (its dimension in the range of 1 to 100nm) that very different from those of the same molecules in large bulk form. Nanoparticles (NPS) are particles [1]. Recently, the metal nanoparticles are available in different sizes and shapes that increase its application in different biological studies [2]. Zinc, an essential metal, is an activator for more than three hundred enzymes in the body [3], and plays a key role in different metabolic pathways including glucose metabolism. Zinc promotes hepatic glycogenesis through its actions on the insulin pathways and thus improves glucose utilization [4]. The mechanism of action of ZnO nanoparticles as antimicrobials function might be from the disruption of cell membrane activity by the release of “Zn+2” ions which can the damage cell membrane and interact with intracellular contents [5]. http://ijps.aizeonpublishers.net/content/2014/3/ijps560-564.pdf

Another possibility could be the induction of intracellular reactive oxygen species, including hydrogen peroxide (H2O2), a strong oxidizing agent harmful to microbial cells [6]. Inhalation of nanoparticles by animal and human courses lung damage [7]. Nanoparticles have been found to be distributable in to the colons, lungs, bone marrow, liver, spleen and the lymphatics [8] and translocated through the circulating lymphatic and nervous system [9, 10]. NPS are cleared in the alveolar region via phagocytosis by macrophages facilitated by chemotactic attraction of alveolar macrophages to the deposition [2]. The present study was aimed to investigate the cytotoxic effect of ZNONPS against Ehrlich ascites carcinoma cells in mice.

MATERIALS AND METHODS Tested compounds Ehrlich ascites carcinoma (EAC) parent line was supplied from National Institute of Cancer, Cairo 560

Magdy F. Abou El Fatoh et al. / Int J Pharma Sci. 2014, 4(3): 560-564

University, Egypt. The zinc oxide (ZNONPS) was purchased from Nanotech Company, Giza, Egypt its size 35nm. All other chemicals were obtained from Sigma (St. Louis, MO, USA). Animals and experimental design Fifty mature male albino mice, apparently healthy (average 45-50 gm b.wt.) were used in this study. The animals were obtained from the laboratory animals house of Faculty of Veterinary Medicine, Zagazig University. Animals were maintained in stain less steel cages with hygienic measures. During the acclimatization period, each animal was raised on a regular diet ad-libitum. The animals were housed at room temperature under 12/12 hr light /dark cycle. Experimental design EAC cells were obtained from the National Cancer Institute, Cairo University. The cells maintained in vivo in Swiss albino mice by subcutaneous transplantation (2x106 cells per mouse) to the animals of all groups except the first group (11). The animals were divided into five groups, 10 of each. The first group was negative control supplied normal food and water. The second group was positive control infected with Ehrlich ascites carcinoma (EAC). The third group was injected with 5mg/kg.b.w. of ZNONPS I.P once daily for 3 weeks after complete solid tumor production.The Fourth group was infected with EAC and injected with ZNONPS 7.5mg/kg. b.w. I.P. once daily for 3 weeks after complete solid tumor production. The fifth group was infected with EAC and injected with ZNONPS 10mg/kg. b.w. I.P once daily for 3 weeks after complete solid tumor production (12). Sampling and analysis EAC cells were collected from moderately growing 7days old donor. The viability was checked by the trypan blue 0.5% exclusion test [13]. The tested animals were sacrificed at 28 days after infected with EAC and collected blood samples for have sting serum for measurement of CEA (Carcino Embryogenic Antigen) and CA (Carcino Antigen) as tumor markers. The liver and spleen were collected and preserved in 10% neutral formalin for histopathological analysis [14].

RESULTS AND DISCUSSION Clinical signs of mice transplanted with EAC Seven days later to intraperitoneal implantation of mice with EAC, there were variable distended abdomen and ascites apparent by naked eye fig. (2) comparing with negative control mice fig. (1). Peritoneal wall of EAC transplanted mice were incised and voluminous ascetic fluid was noticed. Total cells count and viability test Peritoneal ascetic fluid slide smears were stained by trypan blue 0.5% and used for counting of the total life and dead EAC cells by using hemocytometer after the ascetic fluid was stained with trypan blue 0.5% were noticed the life cells nucleus Fig. (3) not accepted blue stain but the dead and the degenerates EAC cells were stained with blue Fig. (4), Table 1. Table 1. Cytotoxic activity of ZNONPS (5, 7.5 and 10mg/kg b.w) on total EAC cells, life cells and degenerated cells in all animals groups. Cells count Groups

Negative control Positive control 71.22+1.25a ZNONPS 65.13+1.28b (5mg/kg.b.w) ZNONPS 42.75+1.65c (7.5mg/kg.b.w) ZNONPS 27.19+1.19d (10mg/kg.b.w) Means with each column carrying significant at (P< 0.5) Means + SE (No. of cells per ml X 105)

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Degenerated EAC cells count 0.72+0.96d

Total tumor cells count

2.77+0.56c

69.45+1.86b

9.11+0.85b

53.0+2.16c

11.26+0.79a

33.25+1.55d

74.0+1.72a

different superscripts are

Effect of ZNONPS on CEA and CA The values of tumor markers (CA and CEA) reduced clearly in the groups treated with ZNONPS than that of tumor bearing one. The 10mg ZNONPS treated group showed the lowest value of these markers indicating that ZNONPS have the efficacy to ameliorate the bad effects induced by tumor. Table 2. Estimation of tumor markers (CEA and CA) for clearing the cytotoxic activity of zinc oxide nanoparticles on the EAC implanted mice groups. Animals Groups

Statistical analysis All analyses utilized SPSS 15.0 statistical package for Windows (SPSS Inc., Chicago, IL) [15]. A one-way analysis of variance (ANOVA) was employed for comparisons of means of the different groups. A pvalue < 0.05 was accepted as statistically significant. All the results were expressed as mean ± SD for ten separate determinations.

Life cell count

(CEA) Carcinoembryogenic Antigen 0.13+0.03e 9.60+0.56a 7.15+1.35b

(CA) Aarcinoantigen

Negative control 0.19+0.09 e Positive control 20.79+0.07 a ZNONPS (5mg/kg.b.w) 16.95+1.15 b ZNONPS 4.28+1.12c 9.75+1.08 c (7.5mg/kg.b.w) ZNONPS 2.78+1.19d 4.35+1.45 d (10mg/kg.b.w) Means with each column carrying different superscripts are significant at (P< 0.5) Means + SE (No. of cells per ml X 105).

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Effect of ZNONPS on liver and spleen tissue from tumor bearing mice

Figure 1. Negative control mice showing normal abdomen.

Figure 2. Positive control mice showing highly distended abdomen filled with ascetic fluids.

Figure 3. Showing nemerous life cells not acceptes blue stain (Trypan blue stain 0.5%- Bar= 100 n).

Figure 4. The fifth group showing degenerated EAC cells accepted blue stain (Trypan blue stain 0.5 % - Bar =100 m).

Figure 5. Normal liver tissues exposed from negative control mice (H and E) (Bar = 100 µm).

Figure 6. Liver tissues exposed from positive control mice, 7 days later to EAC implantation showing round cells aggregation with portal area, hepatocyte with karyo and cytomegaly besides extra medullary hematopoiesis (H and E) (Bar = 100 µm).

Figure 7. Hepatic tissues exposed from mice induced tumor and treated with ZNONPS (10mg I.P.) showing hepatocyte regeneration and Asterisks. (H and E) (Bar = 100 µm).

Figure 9. Normal spleen tissues exposed from negative control mice. (H and E) (Bar = 100 µm).


Figure 8. Hepatic tissues of mice of 5th group showing hydropic degeneration of hepatocytes and mild mononuclear cells infiltration beside aplastic kuffer cells. (H and E) (Bar = 100 µm).

Figure 10. Splenic tissues exposed from positive control mice implanted with EAC, showing intensive lymphoid depletion and presence of clusters of neoplastic cells and megakaryocytes. (H and E) (Bar = 100 µm).

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Figure 11. Splenic tissues of mice of from positive control mice implanted with EAC, showing lymphoid depletion and presence of tumor masses (arrow) (H and E) (Bar = 100 µm).

Cancers are abnormal cells that compose the cancer tissue when a cell is damaged or altered without repair to its system, the cell is usually die. When such damaged cells do not die and became cancer cells and proliferate with uncontrolled growth, a mass of cancer cells develop. Frequently, cancer cells can break away from this original mass of cells, travel through the blood and lymph system and lodge in other organs where they can again repeat the uncontrolled growth cycle (CandD). ZNONPS have showed decreased mitochondrial function such as generation of ATP due to stoppage of respiratory chain and so no phosphorylation process [16] in marine neuroblastoma [17] and hepatic cells [18]. Although, in vivo studies have not been performed with polyvinyl pyrolidone were shows to penetrate into upper layers of the epidermis in excised human skin in static diffusion cells [19]. The mechanism of action of ZNONPS against bacteria, a few studies have suggested that primary cause of the antibacterial function might be from the disruption of cell membrane activity by the release of Zn++ ions which can destroyed cell membrane and interact with intracellular contents [6]. ZNONPS can be activated by UV and visible light to generate highly reactive oxygen species such as OH, H2O2 and O2- . The negatively charged hydroxyl radicals and super oxides cannot penetrate into cell membrane and are likely to remain on the cell surface, whereas, H2O2 can penetrate into the cells [19]. In present study, we evaluate the cytotoxic activity of zinc oxide nanoparticles on carcinoma cells through noticing the slight distension of the abdomen and a little volume of ascetic fluid in the peritoneum in the treated groups than that of the positive control (tumor bearing group). Increase the total number of EAC life cells, and decrease dead cells count in positive control bearing EAC alone without treatment Figure (3), table (1) which may attributed to a higher mitosis and fewer cell dying which could be attributed to the decrease rate of the natural death mechanisms that occurs in the tumor [20]. The accumulation of ascetic fluid in the peritoneal cavity was either due to (a) a reduced lymphatic recovery system, which is associated with obstruction of the lymphatic by tumor cells, (b) angiogenesis which detected in ascetic tumor bearing peritoneal wall, (c) microvessels hyper-permeability of the peritoneal http://ijps.aizeonpublishers.net/content/2014/3/ijps560-564.pdf

Figure 12. Splenic tissues of mice induced tumor and treated with ZNONPS (10mg I.P.) showing splenic regeneration. (H and E) (Bar = 100 µm).

cavity [21]. On the contrary in treated groups 3, 4 and 5 by zinc oxide nanoparticles by different doses (5, 7.5 and 10mg/kg b.w) respectively, revealed increasing of dead cells percentage with reduced volume of ascetic fluid in the peritoneum, reduction in the values of tumor markers (CA and CEA) and reduced total number of EAC cells and the percentage of the life cells also reduced (Tables 1 and 2). This could be due to the interference with the growth of EAC cells directly during early phase of treatment as in fourth group leading to considerable elimination of EAC cells [22] also animals treated with immuno-stimulants resist in various degrees, subsequent inoculation of tumor cells as evidenced by the reduced tumor take, and prolonged survival of the recipients [23]. While in the fifth group which revealed the beak results in slight distension of the abdomen, little amount of ascetic fluid, reduced values of tumor markers and decreased EAC cells count (Fig. 4) (Table 1) that could be due to improve cellular immune response [24] and antioxidant system [25]. These results were confirmed with examination of peritoneal wash for EAC by trypan blue stain. Films stained revealed numerous life cells not stained blue in positive control group (2nd group). Varying number and degenerated EAC cells stained blue by trypan blue in groups (3-5) and was abundant in 4th and 5th groups. Also, confirmation of the results by matching the tumor markers in the serum which revealed that marked increase in the positive control group and reduction in the ZNONPS treated groups with marked reduction in the 4th and 5th groups. Tumor marker, carcino embryonic antigen (CEA) is a protein found in many types of cells but associated with tumors and the developing fetus. The normal range is < 2.5 ng/ml in an adult human non smoker and < 5.0 ng/ml in a smoker. The CEA was one of the first once fetal antigen to be described. It is a complex glycoprotein of molecular weight 20.000 that is associated with the plasma membrane of tumor cells, from which it may be released into the blood. Although CEA was first identified in colon cancer, and abnormal CEA blood level is specific for colon cancer not for malignancy in general, elevated CEA levels are found in a variety of cancers other than colonic, including 563


pancreatic, gastric, lung, peritoneal and breast cancers. The circulating carcinoantigen (CA) and CEA levels are widely used for surveillance purpose in clinical field. Therefore, higher level of CA may be associated with larger burden of occult disease and poor outcome. All reports showing worse prognosis in patients with high level of CA [26] and CA has been shown to an independent predictor of first recurrence as well as a powerful prognostic indicator in patients with advanced breast cancer [27]. All previous reported data of CEA and CA are in agreement with this study results where they are elevated in positive control group (tumor bearing mice) and decreased in the treated groups by ZNONPS especially in 4th and 5th groups which indicate clinical improvement of the exposed animal due to the anticarcinogenic effect of nanozinc oxide. The aforementioned picture observed earlier in our study, came in harmony and confirmed by the histopathological examination where the liver tissues exposed from positive control mice, 7 days later to EAC implantation showing round cells aggregation with portal area, hepatocyte with karyo and cytomegaly besides extra medullary hematopoiesis where these lesions were regenerated with ZNONPS treatment. On the same context splenic tissues exposed from positive control mice implanted with EAC, showing intensive lymphoid depletion and presence of clusters of neoplastic cells and megakaryocytes, these lesions were remarkably reduced by co- treatment with ZNONPS.

CONCLUSION The zinc oxide nanoparticles had a cytotoxic effect against tumor cells so they could be used as antitumerogenic and anticarcinogenic agent but we need more investigations and field applications.

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