Effect of Inhalation Exposure to Nano-Imidacloprid on Liver and Kidney Functions In Male Rats

Pesticide poisoning is a serious global public health issue and is responsible for a sizable number of annual fatalities. This study was designed to examine the potentially harmful effects of adult rats being exposed to imidacloprid (IMD) as a nanoparticle by determining the chronic effect of inhalation of (5,10 and 20) mg/kg/b.w. of nano-imidacloprid for a duration of 60 days. The most important biochemical parameters of the serum liver function parameters were aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase ALP, kidney function [blood urea, creatinine, and urea], and oxidative stress parameters (MDA, GSH, and CAT) in all treated groups when compared to the control group. The exposure caused a highly significant decrease (P≤0.05) for (AST) and (ALT). No significant changes were observed, while a highly significant decrease was recorded for (ALP) in all treatments with nano-imidacloprid compared with the control. On the other hand, a highly significant decrease in blood urea, creatinine, and uric acid was recorded in all groups treated with nano-imidacloprid compared with the control. Oxidative stress parameters revealed a highly significant (P≤0.05) increase in malondialdehyde (MDA) after 60 days of exposure and a decline in reduced glutathione (GSH) and catalase activity CAT.


Introduction
Nanotechnology is rapidly advancing in various fields such as environmental protection, medical treatment and hygiene, as well as designing a variety of biotechnological applications, one of the main goals of applying nanotechnology in agriculture is to reduce the pest problem and increase the efficiency of pesticides [1].The majority of pesticide chemicals are insoluble in aqueous media, which makes it difficult to create formulations that are safe for the environment and apply them effectively.Nanotechnology may develop into a cutting-edge method for creating nano-formulations that will improve the solubility and effectiveness of insoluble pesticides [2].Pesticide dissolving performance could be improved by reducing the size of the pesticide's particles, however this is only noticeable when the particle size is at the nanoscale, Currently, this type of nanoemulsion is the subject of numerous studies, uses, and applications [3].As a model insecticide in this study, Imidacloprid was chosen as the insecticide to be transformed into a nanoemulsion form.Insecticide leaching has been reduced and controlled using a variety of pest control techniques, such as nanosynthesizing for increased efficacy, lowering doses, or even raising efficacy [4].although people have been aware of the serious consequences of pesticide use on humans, their use has continued to rise in developing countries [5].Pesticides developed after the Second World War due to the ubiquity of plant diseases to manage and/or control pests in our society had been widely adopted for industrial, domestic and agricultural purposes and had become a widespread and essential need of human lifestyle today in world.The prevalence of pesticide toxicity in developing countries may be the misuse of the toxic pesticides generally [6].It is imperative to evaluate the potential undesirable effects these pesticides may present the retailers who sell them, agricultural workers, and human's health and well-being due to the myriad number of pesticides used in agriculture today [3].Humans are exposed to pesticides basically in three ways namely; dermal, oral and inhalation [4], and much emphasis has been made on the oral means of exposure and some reports have implicated the oral means to cause multiple organ toxicity [7].

Ethics approval
All study animals were treated and handled in accordance with the necessary biosafety and security protocols.Before beginning this study, the Ethics Committee of the College of Science, Baghdad University, Ministry of Higher Education and Scientific Research, Iraq (No. 984 A.P. on March 14, 2022) accepted the guidelines for the Care and Use of Laboratory Animals and the Rats guide.

Experimental design
The study used 20 adult male rats from Biotechnology College / University of AL-Qassim Green, weighing (250-300g).The rats were kept alive under conditions of humidity (50-60%) and temperature (25-27)°C.The rats were split into four equal groups after five days of acclimation.The first group was designated as the negative control group (T1), followed by the second group (T2), which was administered 5mg/kg/b.w. of nano-imidacloprid for (60) days, the third group (T3), which was administered 10 mg/kg/b.w. of nano-imidacloprid, and the fourth group (T4), which was administered 20 mg/kg/b.w. of nano-imidacloprid for (60) days.

Sample collection and analyses
After (60 days) from treated with nano-imidacloprid, fresh samples of blood were collected from all animals by heart puncture and then extraction of serum which used for evaluation biochemical tests, The serum samples were put in tubes at (-20°C) for biochemical tests.Commercially available colorimetric kits Analytic on Biotechnologies company, (Linear, Spain) were used to measure Serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities according to the methods of [9][10] [11].Commercially available colorimetric kits Analytic on Biotechnologies company, [Linear, Germany] were used to measure Serum urea, creatinine and uric acid according to the methods of [12][13] [14] Commercially available colorimetric kits Analytic on Biotechnologies company, sun long company Eliza kit were used to measure serum MDA, GSH and CAT according to [15], [16].

Statistical Analysis
The statistical analysis system SPSS (2012) was used to analysis the data obtained in this research.The Least Significant Difference (LSD) test was used to evaluate the means.The P value less than 0.0 5 is considered significant.Data are presented as Mean ± Standard Error (S.E.).

Liver function parameters
Table (1) explain the results of the effect of imidacloprid and nano-imidacloprid on the alternation in liver function enzyme for albino rats after 60 days of treatment, There was a highly significant (p<0.05)increase in AST, ALT and ALP level in all groups treated when compared with the control group.Results of this study indicated that inhalation imidacloprid for 60 days causes pathophysiological changes in the liver, These changes are likely caused by damage to the hepatocytes' membrane permeability, which allows this enzyme and pesticide to leak out and disrupt the normal structure of the hepatocellular membrane.Exposure to pesticides lead to liver damage and leakage of cytosolic enzymes from hepatocytes and other body organs into blood [17].These enzymes are important and critical enzymes in the biological processes as the breakdown of amino acids into α keto-acids and are responsible for the detoxification process, metabolism, and biosynthesis of energetic macromolecules for different essential functions [18].The current findings are consistent with [19] which found that continuous imidacloprid exposure promotes inflammation in the liver of rats, this inflammation also resulted in a considerable increase in the levels of the enzymes ALT, ALP, and AST in the blood plasma of rats receiving high doses of imidacloprid.according to [20] the increase in ALT enzyme in plasma, which is a sign of liver malfunction or injury, is not unrelated to the fact that the enzyme spilled into the bloodstream from the liver cytosol.However, according to [21], ALP is frequently used to evaluate the integrity of the plasma membrane.Based on this premise, our current investigation found that persistent inhalation of these pesticides increased the tissue activity of ALP.

Renal function parameters
Statistical analysis of the results of the effect of imidacloprid and nano-imidacloprid on renal function parameters in male rats after 60 days of treatment.Table (2) shows there were highly significant (P<0.05)increases in urea, creatinine, and uric acid levels in all treated groups as compared with the control group.The results of the present study indicated that male rat's inhalation of nano-imidacloprid caused an increase in the kidney function enzymes blood urea, creatinine, and uric acid levels as compared to the control group.Renal function markers such as urea, creatinine and uric acid are used to assess the functional capacity of the kidney of experimental animals, increased levels are indicative of a possible defective functional condition [22].An increase in uric acid content in the tissue is an indication of an increase in purine metabolism that remains targeted by imidacloprid and nanoimidacloprid, This may be due to could be because creatinine is a metabolic product of creatine phosphate dephosphorylation of muscle, Studies [21] have demonstrated that plasma creatine concentration is a good marker of glomerular filtration rate.The elevation in creatinine levels may indicate possible changes in kidney function [23].As the kidney became impaired for any reason as case of pesticide poisoning, the creatinine level in the blood will rise due to poor clearance by the kidney or due to damaged functioning nephrons and impaired renal function [24].The present results are in agreement with [25] which found a significant increase in creatinine in male rats' plasma after being treated with a pesticide.

Oxidative stress parameter
Table (3) revealed the results of the effect of imidacloprid and nano-imidacloprid on oxidative stress parameters in rats treated with pesticide for 60 days.Data in Table (3) show a highly significant (p<0.05)increase in MDA level in all treated groups when compared with the control group as well as a highly significant (p<0.05)decrease in catalase and GSH levels in all treated groups as compared with control group.In the present study, imidacloprid significantly induced MDA and decreased another vital antioxidants in the liver at a high dose rate.High level of MDA in the liver suggested the production of oxidative metabolites or free radicals during hepatic metabolism and this might be due to the progressive nature of free radical chain reaction, Increased level of MDA in tissues supports the results of [26] and [27].the decrease in activity of GSH and CAT in might be due to the consumption of this enzyme in O 2 -to H 2 O 2 .A similar decrease in the activity of GSH and CAT in animals was also reported with different pesticides namely which showed decreased GSH activity in rats [28,29 and 30].
The second barrier is provided by GSH because of its lower H 2 O 2 and the third by catalase [31].GSH is localized mainly in the cytosol and mitochondria of the liver, so this organ can be accepted as source of this enzyme.Therefore, decreased GSH and CAT activity in the liver might be due to oxidative inactivation of the enzyme protein because of the accumulation of insecticide in the liver [32] and [26].

Conclusion
Based on the results of the present research, it is apparent that nano-imidacloprid influenced the levels of kidney enzyme activity [blood urea, creatinine, and uric acid], the functions of the liver [AST, ALT, and ALP], and oxidative stress parameters [MDA, GSH, and CAT] in all treated groups when compared to the control group.

Table 1 .
Effect of inhalation of imidacloprid and nano-imidacloprid on liver function parameters of rats after 60 days of treatment

Table 2 .
Effect of inhalation of imidacloprid and nano-imidacloprid on kidney function parameters of rats after 60 days of treatment.

Table 3 .
Effect of inhalation of imidacloprid and nano-imidacloprid on oxidative stress parameters of rats after 60 days of treatment.