Nano Zinc Oxide Induced Cardiovascular Toxicity In Rats

Backgroud and Aim Nano zinc oxide (Nano-ZnO) is a new kind of inorganic materials, widely used in a variety of industries, such as textiles, ceramics, military defense, rubber, cosmetics and animal feed. It can enter human body through respiratory, digestive routes and skin. Toxicological studies have mainly focused on lung, liver and kidney toxicity of nano-ZnO. It has been shown that exposure to nano-ZnO could cause pulmonary inflammation of experimental animals; oral exposure to higher dose of nano-ZnO could cause extensive damage to mouse kidney, liver, lung, small intestine and other organs, and resulted in severe pathological alterations including hemorrhage, congestion, degeneration and necrosis, and nuclei solid shrinkage and fragmentation. So far, there are few studies on cardiovascular toxicity of nano-ZnO.Previous studies have demonstrated that some kinds of nanomaterials, such as titanium dioxide, nano-carbon, nano-nickel could cause cardiovascular damage, but the exact mechanism is unclear. It may through the following ways:1、cause lung and/or systemic inflammatory response, leading to endothelia] dysfunction and endothelial injury, then induce the formation of condensed state, and promote atherosclerosis (AS).2、cause or increase the body’s lipid metabolism disorders and increase the risk of atherosclerosis.3. contact with endothelial cells in the blood circulation directly, induced inflammation or injury, promote thrombosis, affect the stability of the atherosclerotic plaque, lead to deterioration of cardiovascular disease eventually. But there are few research about the cardiovascular toxicity of nano-ZnO, only some studies in vitro found that nano- ZnO can damage human umbilical vein endothelial cells, in view of this research status, carry out nano-ZnO cardiovascular toxicity study is very necessary.Lung inflammation is evaluated commonly using lactate dehydrogenase (LDH) and total protein content; serum total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL) are commonly used in the evaluation of the lipid metabolism of the body; serum tumor necrosis factor-alpha (TNF-a) and heme oxygenase-1 (HO-1) level are commonly used to evaluate the systemic inflammatory response, especially for particulate matter-mediated injury. So in this study we investigated the caodiovascular toxicicy of nano zinc oxide. Wistar rats were intratracheally instilled with different doses of nano-zinc oxide. Then pulmonary inflammation, serum lipid levels, visceral fat accumulation and aortic pathological changes of rats were determined to assess the toxicity of nano zinc oxide on cardiovascular system. It is expected this study will provide some new information on nano-ZnO toxicity.Methods Nano zinc oxide was suspended in phosphate-buffered saline (PBS) in different concentrations,1.25mg/ml,2.5mg/ml,5.0mg/ml, respectively.50 clean-level Wistar rats, male, six-old weeks, were weighed and numbered, and divided into five groups with a random number table:PBS control group (1 ml/kg), low-dose group (1.25mg/kg), medium dose group (2.5mg/kg), high-dose group (5.0mg/kg), and high-fat group (PBS solution lml/kg). Rats were intratracheally instilled with nano -ZnO suspension once a week for a total of 12 weeks. After last instillation, rats were fasted for 12h, with water only. Then, the rats were weighed and anesthetize with 10% chloral hydrate (0.4ml/kg) through intraperitoneal injection. The blood samples collected from heart were tested for levels of serum TC, LDL, HDL, TNF-αand HO-1 using ELISA; epididymal adipose tissue was weighed and its ratio to body weight calculated; aorta was isolated for pathological examination at gross and microscopic levels respectively; BALF was collected for measurement of total proteins with Coomassie brilliant blue method and LDH activity with ELISA. SPSS13.0 software for analysis. The data were analyzed with SPSS 12.0 using oneway-ANOVA to compare the difference between two groups and multi-groups, and the significant level was set at a=0.05.Results1 General condition of instilled ratsWith prolonged exposure, the instilled rats started to show messed fur, poor mental state, and less activity. During the whole experiment, their weights gained slowly. Compared to the rats fed with normal diet, the rats fed with high fat diet have a rapid weight gain and thicker subcutaneous fat in the earlier days after instillation. However, with the progression of exposure rats in the high-fat group appeared slowly to grow, with unhealthy for color and poor mental state. From the 2nd to 7th week after instillation, body weights of rats in high-fat group were heavier than those in other groups (P<0.01); however, from the 9th to the twelfth week, the weights of rats in the high-fat group were lighter than those in the PBS control group, but still heavier than those in nano-zinc oxide group (P<0.01);from the fourth week, the weights in all nano zinc oxide exposure groups were lower than those in the control group (P<0.01).2 Serum lipid and inflammatory cytokines levels:Nano-ZnO induced a dose-dependenet increase in levels of serum TC and LDL. Among all groups, the high-fat group presented the highest levels of serum TC and LDL (P<0.01). In contrast, the levels of serum HDL decreased with the increase in nano-ZnO dose (P <0.01). In addition, levels of serum HDL in the high-fat group was the lowest (P <0.01) among all groups. Levels of serum TNF-αand HO-1 in nano zinc oxide groups were higher than those in PBS control group (P<0.01) and high-fat group; TNF-a levels in high-fat group were higher than those in PBS control group, (P <0.01). HO-1 levels in the nano zinc oxide exposure group were higher than those in the hyperlipidemia group and PBS control group (P<0.01)., There was no significam difference between hyperlipidemia group and PBS control group in levels of serum HO-1 (P>0.05).3 Protein content in BALF:Protein levels in the medium and high nano-ZnO dose groups were higher than those in PBS control group as well as the high-fat group (P <0.01). There were no difference among low nano-ZnO dose group, high-fat group and control group (P> 0.05).4 LDH activity in BALF:LDH activity in BALF of rats exposed to nano zinc oxide were increased in a dose-dependent fashion (P<0.01), and higher than those in control and high-fat groups (P<0.01).5 Ratio of bilateral epididymis adipose tissue weight/body weight:This ratio in medium dose group, high dose group or high-fat group is higher than that in the PBS control group (P<0.01), and in a dose-dependent fashion (P<0.01). The high-fat group showed the highest ratio compared to other groups(P<0.01). 6 Aotic morphological changesMicroscopic alterations:In PBS control group, the aortic endothelial cells demonstrated complete continuous and smooth surface without adhered cells under light microscopy. In addition, it was also seen that suboptimal space was normal, medial smooth muscle cells with regular arrangement, and with no -proliferation In the low and mediam dose group the aterial intimal surface appeared smooth, with a small number of adhered monocytes. The endothelial cells were damaged and smooth muscle cell over-growth to some extent. There was no difference between low and medium ZnO group. Remarkably, we observed thickened vessel wall, damaged smooth endometrial endothelial cells, discontinued or fragmented intimal surface with exposed underlying collagen fibers and adhered monocytes. Penetrated monocytes were also seen in the subendothelial layer. Subendothelial space was enlarged. Spotted internal elastic laminae fracture and medial superficial smooth muscle cell over -growth and derangement was also observed in high-dose group of nano-ZnO and high-fat group. Importantly, the medial smooth muscle cells were seen in the intima layer, indicative of their migtation to the surface.Conclusion Wistar rats was intratracheally instilled with nano -ZnO suspension at dose of 5.0 mg/kg, once a week for a total of 12 weeks, nano-ZnO can induce obvious toxic effect on the cardiovascular system, which may be resulted from oxidative stress, lipid metabolism disorders and aortic pathological damage, and the pathological changes of the aorta mainly is early atherosclerosis kind change.