Assessment of Chromium Oxide Nanoparticles Intake in Rattus norvegicus by Primary Renal Function Markers and RBC Architecture

Haematological tests are signifi cant diagnostic tools that are equally valuable as indicators of toxic insult or stress due to xenobiotics and environmental fl uctuations. Present study was designed to investigate alterations in primary renal function markers, pathological changes in kidneys and variations in RBCs shape of male wistar rats due to chromium oxide nanoparticles (Cr2O3NPs) exposure. Cr2O3NPs are transition metal oxide NPs which are widely being used as catalysts, pigments and coating materials. Therefore, toxicological evaluation is fundamental with respect to their increasing applications. In the current study, synthesis of Cr2O3 NPs was accomplished by sol gel method and characterized in sequential manner by electron microscopy (TEM and SEM). TEM analysis revealed size distribution of test NPs in the range 22.50 ± 1.76 nm. SEM represented the morphological features with high homogeneity of sample NPs validating Cr2O3NPs synthesis. Toxicological fi ndings revealed deviations in renal function test of treated rats with respect to the control group indicative of kidney damage. Blood Urea Nitrogen (BUN) was found to be signifi cantly higher (p < 0.05) after 14 days high dose exposure in comparison to control rats. Extensive changes in kidneys architecture were noted after repeated exposures to high dose. Various structural deformations of RBCs including tear drop cells, bite cells, elliptocytes, echinocytes etc were also observed. Results of present investigations, though preliminary but clearly demonstrate that oral administration of Cr2O3 NPs induces biochemical changes consequently leading to alterations in renal function parameters and RBC shapes of exposed rats. Research Article Assessment of Chromium Oxide Nanoparticles Intake in Rattus norvegicus by Primary Renal Function Markers and RBC Architecture Ravish Fatima and Riaz Ahmad* Section of Genetics, Department of Zoology Aligarh Muslim University, Aligarh Dates: Received: 22 December, 2016; Accepted: 18 February, 2017; Published: 21 February, 2017 *Corresponding author: Riaz Ahmad, Doctor, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002 (UP), India, Tel: +91-571-2700920/3445; E-mail:


Introduction
Nanotechnology is a fast -growing research fi eld that has led to many signifi cant scientifi c breakthroughs [1]. It involves the formation and manipulation of particles at 'nanoscale' having novel and improved physico -chemical properties [2][3][4]. Owing to the minuscule magnitudes, nanoparticles (NPs) acquire unique physical, chemical, electrical and magnetic properties. The exclusive characteristics have resulted in increasing synthesis and widespread usage of engineered NPs. Prevalent applications of NPs have thereby conferred them with enormous toxic potential on human health and environment. The NPs being very small in size can pervade the cellular membrane and interfere in the cell's natural processes [5,6]. Entry of NPs via various routes like oral, dermal and inhalation may occur either intentionally or unintentionally, resulting in subjugating normal cell functioning [7,8]. So, safety concerns have arisen regarding the risk assessment of manufactured NPs. Blood plays an integrated and inevitable part of the biological system and haematology can visibly ascertain the diseased or stressed state of animals [9][10][11].
Many workers have studied effects of several heavy metals on haematological parameters in various model organisms [12][13][14][15]. Gardner and Yeuich [16] have established long -term effects of heavy metal on blood chemistry parameters. Jawad et al. [17], advocated that haematological parameters may serve as early biomarkers of toxicity. A study by Ahmad et al. [18], established the correlation between NDMA -induced hepatic fi brosis and changes in clinical blood parameters of wistar rats.
Likewise, serum biochemical indices also play a signifi cant role in monitoring clinical symptoms produced by a toxicant [19]. However, research on haemato -toxic effects due to NPs exposure are limited and such study concerning 'nano' and renal function profi le as indicators of toxicity in wistar rats. These tests are used in examining the response of an organism to toxic insult and can be effi ciently employed for toxicity evaluation of NPs. For this study, rats were exposed to two concentrations of Cr 2 O 3 NPs for multiple durations to study discrepancies in renal function profi le and changes in shape of RBCs due to acute or cumulative dosing. The study may help in curbing nanotoxicity at initial stages by monitoring these parameters in exposed organisms.

Blood sampling
Whole blood sample was collected at the end of each doseduration exposure. 5 ml of blood sample was withdrawn from the heart of each rat by direct cardiac puncture through sterilized syringe into EDTA vials and clot activator vials for RBC shape and serum study, respectively. The blood samples drawn from animals were used for preparing permanent smears of RBCs.
For observation of deformation in RBCs shape, slides were fi xed in methanol for 12 -15 min, stained with Giemsa for 8 -10 min and fi xed in DPX. The stained slides were then randomly selected for rheological studies and observed on Nikon ECLIPSE E200 microscope at 40 X magnifi cation. For renal function test, serum was separated by centrifugation at 3000 rpm for 10 min and kept at -80°C until used for biochemical estimations.
Activities of kidney function (BUN i.e. blood urea nitrogen, uric acid, creatinine, Na + , K + ) were measured in the serum of rats by an automatic biochemical analyzer (Cobas Mira Plus, Roch Diagnsotics, Germany).

Data and statistical analysis
All observations were replicated thrice for varied observations and the data are expressed as mean ± SEM.
Statistical analyses were performed using Graph Pad Prism software version 3.02. Groups' variance was analyzed by one way analysis of variance (ANOVA) followed by post hoc Tukey to test for signifi cant difference between the groups. The level of signifi cance was set as p < 0.05 (*).

Preparation and characterization
The size -distribution of Cr 2 O 3 NPs was determined to be 22.50 ± 1.76 nm in diameter by TEM, illustrated in Figure 1

RBC Shape
Although quantitative assessment was not performed but various deformities in shape of RBCs like tear drop red cells, acanthocytes, echinocytes, bite cells, elliptocytes and spherocytes were observed extensively in exposed rats. These structural distortions in RBCs were noted to increase with dose -duration exposure of Cr 2 O 3 NPs (Figure 2).     continual exposure. Thus, in our study, aberrant expression in renal function profi le (BUN, creatinine, uric acid, Na + and K + levels) of treated rats clearly pinpoints injury to the kidney of treated rats as compared to control rats. This study shows that functional activity of kidneys happens to be disturbed due to Cr 2 O 3 NPs exposure which resulted in the leakage of some of the investigated renal parameters. In general, renal function biomolecules have been reported to spill over into the serum upon exposure to toxic materials [24][25][26]. Furthermore, it can be hypothesized that Cr 2 O 3 NPs exposure may lead to dysfunction of renal haemopoiesis as the kidney function is found to be impaired in treated rats. Considering the biological clearance function of kidneys, deviations of serum parameters from normal control was well anticipated. Multiplicity of structural deformations observed in RBCs of rats might be due to stress of NPs exposure and may indicate towards onset of apoptosis which is a matter of critical concern. These fi ndings clearly signify that after oral administration, Cr 2 O 3 NPs get effi ciently absorbed in the digestive tract and ultimately released into blood as evidenced by deformed RBCs and altered renal profi le. Some previous studies have also indicated that nano-sized particles may possibly cross the small intestine by persorption and further distribute into blood, brain, lungs, heart, kidneys, spleen, liver, intestine and stomach [27,28]. NPs deposition in vital organs or tissues could induce cellular damage [29]. It is generally agreed that upon ingestion NPs can be absorbed and that absorption increases with decreasing particle size [30].Structural changes observed in renal tissues are indicative of kidneys being the main target organs of Cr 2 O 3 NPs toxicity that may have occurred through the absorption of these NPs from digestive tract and their subsequent circulation in blood.

Renal function markers
Obviously possibilities of detrimental effects to other organs cannot be ignored, once NPs come into circulation, but this study is limited to investigate the effect of Cr 2 O 3 NPs on kidneys only. Thus, we can say that administered Cr 2 O 3 NPs deposited in kidneys may have induced cellular damage via ROS formation which resulted in leakage of investigated parameters into the bloodstream [31][32][33]. The present results taken together strongly indicate kidney damage as well as variations in RBCs shape that may further enhance other metabolic disturbances and cause disease. This study is preliminary on potential haemato -toxicological changes on oral intake of Cr 2 O 3 NPs that may serve as useful information to determine the safety regulations against Cr 2 O 3 NPs toxicity. Future studies are warranted to explore the exact molecular mechanism of this renal toxicity as well as damage to other tissue/organ types.

Conclusions
In conclusion, variations in RBCs shape and alterations of renal function markers are a consequence of Cr 2 O 3 NPs induced toxicity in rats. This investigation may be helpful to monitor and regulate Cr 2 O 3 NPs toxicology by accumulating information on the type of toxicities generated by these NPs in vitro or in vivo. Development of friendly synthetic processes for NPs, such as green synthesis, may be a possible alternative and hope in regressing NP induced-toxicity. From this experimental study, it is suggested that primary toxic potential of Cr 2 O 3 NPs shall be assessed before its use in various industrial sectors using blood and renal function parameters.