Effects of Nano-Fipronil on Male Rats’ Biochemical, Liver, and Renal Functions

Fipronil has been widely employed for crop protection over the past decade because of its low soil stability and insecticidal potency at modest application rates. The present study was designed to investigate the adverse effects of sub-acute exposure to 10 and 20 mg/kg/b. w. of Nano-Fipronil on biochemical blood profile and histological parameters in male rats according to the permissible limits of acceptable daily intake (ADI). The results hematological parameters indicated that Red blood cells (RBCs) and Hemoglobin(H.b) decreased in all treated groups compared with the control. While PLT and white blood cells (WBCs) was significantly increased in all treated groups compared with the control. The toxicity of effects of nano-fipronil pesticides leads to higher levels of liver function parameters (AST, ALT and ALP), kidney function (Blood urea and Creatinin and Uric acid) and Oxidative stress (MDA, GSH and CAT) results demonstrated highly significant differences (P≤0.05) in all groups treated compared with the control.


Introduction
Pesticides are ubiquitous and global statistics have revealed increasing use of these chemicals for the control of pests, Pesticides are usually applied directly to control pests present in both the indoor and outdoor environment where humans spend a considerable amount of time [1].Pesticide overuse leads to inadequate delivery systems and an off waste accumulation, which causes genetic change in the pest species, followed by insecticide resistance.Pesticide drift causes the majority of insecticides to be lost, endangering the ecosystem [2].The utilization of Nanopesticides as "smart delivery systems" for the timely but controlled release of i nsecticides for a chosen time period makes them an attractive alternative.This would lessen the possibility of environmental contamination and the dangers that go along with it.Acute toxicity tests should be carried out since nanoparticles may be non-specifically hazardous both to the targeted and non-targeted organisms [4,5].
Fipronil (FPN) is a phenyl pyrazole, a wide pesticide.It can be utilized to reduce ants, beetles, cockroach, flea, ticks, termite, moles crickets, thrips, rootworms, weevils, and other insects, It is highly efficient by ingestion and produces cerebral stimulation and convulsions in insects, which results in death [6].mechanisms of the insecticides pyrethroids (sodium channel blockers), organophosphates, and carbamates (cholinesterase inhibitors), which appear to be conventional pesticides for which some insects have developed tolerance.FPN was discovered to interfere with GABA-gated channels; FPN impairs nervous system fluid transfer (e.g., chloride ion transit) by targeting the GABA-gated chloride receptor and, at high enough dosages, causes increased neural stimulation, severe paralysis, and insects mortality [7].Pesticide poisons produce both free radicals and reactive oxygen species (ROS), which are extremely reactive and thus can predispose tissues for lipid peroxidation and tissue damage, Enzymatic and nonenzymatic antioxidants are crucial for protecting tissues from the harmful effects of (ROS) and preserving the physiological oxidative equilibrium [8].Animal tissues typically have both enzymatic and non-enzymatic antioxidant activities, such as reduced glutathione (GSH), which are also significant determinants of oxidative stress and toxicity of toxic contaminants [9].In the study was conducted assess the biochemical and hematological changes in male rats exposed to sub-acute nanofipronil.

Fipronil and Preparation Nanoparticles (Liposome)
Fipronil was acquired from the ISAGRO company in the USA.liposome preparation were performed in the College of Veterinary Medicine/University of AL-Qassim green and set as follows: The liposomes were prepared by dried thin lipid film technique as described previously by [10].

Animals and Management
Male albino rats (n=30) almost of the age 3 months and weight ( 250±300 gm ), were obtained from University of Al-Qadisiyah, College of Science, Department of Biology.Rats were kept in plastic boxes (ten rats per box) under standardized living conditions.The rats were acclimatized for one week before the start of the experiment, the rats were divided into three groups the first group was named as negative control group ( NCG), second group (F1) treated group administrated with 10 mg/kg/ b. w. of Nano-Fipronil, third group (F2) treated group administrated with 20 mg/kg/ b. w. of Nano-Fipronil for 3 days.All experiment rats were kept according to the national and global enactments and research protocols placed down by the Ethics Committee about the animal well-being as well as The procedures were carried out in compliance with the "Guide for the Care and Use of Laboratory Animals." of Al-Qasim Green University, College of Environmental Science, Department of Environmental.

Biomarkers Measurements
At the end of the study, fresh samples of blood were collected from heart puncture, and then the rats were sacrificed by cervical dislocation.Hematological parameters used for evaluation of complete blood count (CBC) and Serum was isolated and stored at -20°C until for analysis of liver function biomarkers that includes measurement of enzymes (ALT, AST, ALP) as serum biomarkers were conducted in accordance with the information provided in commercially available kit (Linear , Spain) according to the methods of [11][12][13] and kidney function biomarkers that includes measurement of enzymes ( urea , creatinine , uric acid ) as serum biomarkers were conducted in accordance with the information provided in commercially available kit (Linear, Germany) according to the methods of [14][15][16].Oxidative stress (MDA, GSH and CAT) were assessed spectrophotometrically according to the methods of [17][18][19].

Statistical Analysis
To find the impact of several groups on study parameters, the statistical analysis system-SPSS (2012) application was employed.In this study, the Least Significant Difference (LSD) test was employed to compare means.P values lower than 0.05 are regarded as significant.The presentation of data is as Mean Standard Deviation (S.D.).

Results and Discussion
Rats treated to the FPN at various doses for 30 days showed no signs of death.Several investigations in mice, rats, and humans, indicated that the FPN may be able to cause a particular neurotoxicity [23].
Table 1.Effect of fipronil and Nano-fipronil on hematological parameters of male rats.Each value is a mean and standard deviation (n=16), *significant at P ≤ 0.05.

Groups
Fipronil and Nano-fipronil were administered for 30 days, which caused hematological changes.Rats treated with the FPN-IMI mixture had significantly lower levels of white blood cells and platelets than the control group, but their red blood cell count and hemoglobin levels decreased (P ≤ 0.05).
An increase in the rate of hemoglobin oxidation or a decrease in the rate of hemoglobin synthesis may be the cause of a reduction in the hemoglobin concentration.Because iron is needed for the this synthesis and is standard in relation from vegetable sources and stored ferritin [20].it is possible that exploratory rats' decreased overall food intake and lack of additional iron supplies are to blame for their rats' iron deficiency, which is necessary for the synthesis of hemoglobin.Reduced H.b concentration may also be linked to smaller red blood cells or a barrier to the synthesis of heme in the bone marrow [21].
Although it can also happen after some parasite infections or bone tumors, a rise in white blood cells are typically an indication of an inflammatory response, most frequently brought on by an infected.Any flaws in the structure, quantity, or stabilization of platelets can resulting in bleeding or thrombotic diseases, which would ultimately result in mortality.High platelet counts could result in venous and arterial thrombosis.The signs and symptoms differ on where the thrombosis occurs [22].
According to the current study's findings, sub-chronic exposure to the FPN and nano-FPN damages the liver and kidneys in animals treated more than control rats, as evidenced by a chemical rise in the levels of the indicator serum enzymes AST, ALT, and ALP as well as creatinine, uric acid, and total protein.Liver function enzymes including AST, ALT, and ALP are primarily used to assess hepatic impairment.In the metabolism and manufacture of amino acids, aspartate transaminase (AST and ALT) are crucial.They carry out the activities of detoxification, the biochemistry, and the production of energy components for several vital purposes and they serve as particular markers of liver disease [24][25][26].
The rise in these enzymes may very well be caused by liver dysfunction, which would disrupt their manufacture and change the permeable of the liver membrane [27].Several investigations done on different pesticides provide support for the findings [28][29][30][31].Treatment of rats with FPN had considerably higher serum levels of uric acid and creatinine [28,30].Higher uric acid concentrations may be caused by purine and pyrimidine breakdown or by increased production or inability to excrete, and a rise in creatinine levels are thus an indicator of compromised kidney function [32,33].Further research showed that rats exposed to several pesticides had increased serum concentrations of urea, creatinine, and uric acid [32].Table 2. Effect of fipronil and Nano-fipronil on liver function parameters of male rats.Each value is a mean and standard deviation (n=16), *significant at P ≤ 0.05.

Groups
The development of lipid peroxidation as a result of the FPN therapy in male rats' liver and kidneys demonstrated that the treatment had generated oxidative stress (LPO).LPO has been linked to the pathophysiology of numerous liver and kidney ailments and is known to compromise the integrity of cellular membranes [34].It has been employed as an indicator for pesticide-induced oxidative stress and proposed among the possible molecular pathways behind pesticide-induced toxicity [35].Higher synthesis of reactive oxygen metabolites, particularly hydroxyl radicals, may be the cause of higher levels of malondialdehyde (MDA), a lipid peroxidation, which is formed in the liver and kidney of rats given FPN [36].Tukhtaev et al. [38] found that elevated LPO in the liver of pregnant rats and their pups after longterm exposure to low doses of FPN.Studies currently available show that LPO in animals was increased by pesticides.By changing the enzyme activity linked to antioxidant defense systems in the liver and kidney of male rats, FPN therapy elevated oxidative stress [35,37].Because they are free radical scavengers, both enzymatic and non-enzymatic antioxidants participate actively in the defense against oxidative cell injury and collaborate to avoid the effect of ROS in tissues [39].
In the present study, the decreased in Oxidative stress (MDA, GSH and CAT) are regarded as the first line of defense against lipid peroxidation to cell macromolecules.This is because MDA speeds up the dismutation of superoxide anion into a less reactive molecule (H2O2), which GSH and CAT then use to create water and oxygen quickly [40,41].
In the current investigation, the excess production of O2•, which quickly converted to H2O2 by SOD and to water by CAT, could be the cause of the decreased oxidative stress activity in the kidneys and the liver in the rats exposed to FPN.According to earlier research, oxidative stress in animals' liver and kidneys is reduced by pesticides like fipronil [45].Higher amounts of lipid peroxidation and oxidative stress are caused by cells producing more reactive oxygen species (ROS) as a result of FPN [48].According to reports, FPN caused oxidative stress in C. carpio which was demonstrated by changes in antioxidative enzymes and high amounts of lipid peroxidation [44].MDA, GSH, and CAT, three enzymes associated with oxidative stress, are crucial for the detoxification of xenobiotics like pesticides and the transport of metabolites inside of cells [45].These enzymes have been linked to the possibility of producing reactive intermediates, especially when GSH levels in the cells are reduced, which could have toxicological effects [46,47].These enzymes have been linked to the possibility of producing reactive intermediates, especially when GSH levels in the cells are reduced, which could have toxicological effects [46,47].
In the current investigation, animals exposed to FPN had considerably lower GST activity and GSH levels in their liver and kidney.Because of cell damage and the death of healthy cells that can response to the oxidative stress, the antioxidant enzyme concentrations in the liver and kidney organs decreased.Moreover, it can be a result of reactive oxygen species damage and the FPN's inadequate capacity for detoxification.Table 4. Effect of fipronil and Nano-fipronil on oxidative stress parameters of male albino rats.Each value is a mean and standard deviation (n=16), *significant at P ≤ 0.05.

Conclusion
All treated groups had lower RBCs and H.b than the control.All treated groups had higher PLT and WBCs than the control.The toxicity of nano-fipronil pesticides increases liver function parameters (AST, ALT, and ALP), kidney function (Blood urea, Creatinin, and Uric acid), and oxidative stress (MDA, GSH, and CAT).All treated groups showed highly significant differences (P ≤ 0.05) compared to the control.

Table 3 .
Effect of fipronil and Nano-fipronil on renal function parameters of male rats.