L-arginine Partially Ameliorated Some Biochemical and Oxidative Stress Parameters in Rats Exposed to Imidacloprid Toxicity
Akande MG, Adefioye AE, Fabowale OG, Akumka DD and Ejeh AS
Published on: 2024-06-24
Abstract
Imidacloprid is a neonicotinoid insecticide that is applied extensively for pest control, while L-arginine is an α-amino acid that is important in protein biosynthesis. The purpose of the research was to assess if L-arginine (AR) could improve biochemical and oxidative stress indices in rats exposed to imidacloprid (IM) toxicity. Forty (40) rats were randomly assigned to four groups. They received the treatments by oral gavage for 14 days: distilled water [DW group], imidacloprid (71 mg/kg) (IM group), imidacloprid (71 mg/kg) and L-arginine (100 mg/kg) [IM+AR100 group], imidacloprid (71 mg/kg) and L-arginine (200 mg/kg) [IM+AR200 group]. They were sacrificed after the study ended and the levels of serum proteins, serum enzymes, urea, creatinine and oxidative stress indices were estimated. Significant reductions were recorded in the total protein concentrations of the IM and IM+AR100 groups compared to that of the DW group respectively. Substantial declines occurred in the globulin levels of the IM and IM+AR100 groups relative to the DW and IM+AR200 groups respectively. The activities of hepatic catalase and renal superoxide dismutase of the IM group were significantly reduced relative to the IM+AR200 group respectively. Imidacloprid did not elicit marked toxicity in this investigation. However, L-arginine exerted partial ameliorative impacts on imidacloprid toxicity. Therefore, further research is needed for the exposition of the toxic mechanisms of imidacloprid and bioprotection by L-arginine.
Keywords
L-Arginine; Imidacloprid; Biochemical Parameters; Oxidative Stress; RatsIntroduction
Pesticides are applied for the preclusion, destruction, repulsion, or elimination of insects, rodents, unwanted plants and fungi [1]. Oxidative stresses, intrusion on dopamine transporters, mitochondrial dysfunction and inflammation have been implicated in the lethal effects of pesticides on vertebrates [2]. Neonicotinoids e.g., imidacloprid, acetamiprid, possess substantial potency against various pests, in addition to their moderately reduced mammalian toxicity [3).
Imidacloprid (IM) is a nicotine-mimetic insecticide [4]. It evokes oxidative stress and inflammation through the alteration of antioxidant systems and augmentation of pro-inflammatory cytokine production in non-target organisms [2].
Oxidative stress is an imbalance between pro- and antioxidant species that ensues in molecular and cellular damage [5]. Reactive oxygen species (singlet oxygen, peroxyl radical,etc.) have physiological functions and are typically formed as offshoots of oxygen metabolism [6,7]. In contrast, antioxidants combine with reactive oxygen species and nitrogen species to deactivate the chain reaction preceding the impairment of crucial biomolecules [8].
L-arginine (AR) is an amino acid and it is found in red meat, poultry, fish and dairy [9]. It is required for protein production and is changed to nitric oxide in the body [10,11]. Nitric oxide causes vasodilation for improved blood flow in the body [12]. Additionally, AR suppresses oxidative stress and induces antioxidant responses, promotes glutathione synthesis and stimulates nuclear factor-erythroid 2-related factor 2 (Nrf 2) pathway [13]. It wields its antioxidant activity through the counteraction of free radicals that damage vital cellular components [14].
L-arginine spurs inhibited cholinesterase in invertebrates and vertebrates, and serves as an antidote for poisoning instigated by countless toxicants [15].
The aim of the current research was to find out if L-arginine (AR), a reputed beneficial amino acid, could ameliorate biochemical and oxidative stress parameters in rats exposed to imidacloprid (IM) toxicity.
Materials and Methods
Experimental Animals
The rats used for this study were purchased from the Experimental Animal House of the Faculty of Veterinary Medicine, University of Ibadan, Oyo State, Nigeria. They were pre-conditioned for 14 days before the study started. The animals were housed in cages and in a conducive environment in the Toxicology Experimental Unit of the Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Abuja, Nigeria. They were provided with standard rat chow and water ad libitum.
The research was ratified by the University of Abuja Research Ethics Committee (UAV-22-078). The research, transportation, and care of the animals were carried out in compliance with the stipulations of the National Institute of Health Guide for Care and Use of Laboratory animals [16]. All measures were taken to prevent the experimental animals from suffering.
Chemicals
Imidacloprid marketed as IMIFORCE® (a 100 % solution containing 200 ml of imidacloprid, IM) was purchased from an agrochemical company in Abuja, Nigeria. It was reconstituted in distilled water to a 50 % solution before daily administration to the rats. L-arginine (AR) marketed as PURITAN’S PRIDE® was procured from a pharmacy in Abuja, Nigeria. L-arginine was reconstituted in distilled water to obtain a 100 mg/ml solution before daily administration to the rats.
Subacute Toxicological Study
The experimental animals were weighed and assigned into four different groups (n =10). The DW group received distilled water, while the IM group was treated with imidacloprid at 71 mg/kg (~ 0.25 LD50, where LD50 = 283 mg/kg based on our findings). Imidacloprid and L-arginine were given to the IM+AR100 group at 71 mg/kg and 100 mg/kg successively, while the IM+AR200 group was treated with imidacloprid at 71 mg/kg and L-arginine (200 mg/kg), consecutively.
The rats received the treatments through oral gavage daily for 14 days. Their body weights were evaluated a week prior to the commencement of the research (at week 0), and then weekly (at weeks 1–2). They were observed for evidences of intoxication during the investigation.
Assessment of Some Serum Biochemical Parameters
The rats were sacrificed at the termination of the study and 2 ml of their blood samples were dispensed into plain sample bottles. Their blood samples were allowed to clot and were processed for the assessment of some biochemical indices as described by Akande et al. [17].
Evaluation of Serum Protein Levels
The total protein and albumin levels were determined with a Clinical Chemistry Autoanalyser (Bayer® Germany). The level of serum globulin was calculated by subtracting the serum albumin level from that of total serum protein.
Assessment of the Serum Enzymes Levels and Renal Function Indices
The levels of the serum enzymes [aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH) and creatine kinase (CK)], as well as the concentrations of the renal function indices (urea and creatinine) were estimated with a Bayer Express Plus Clinical Chemistry Autoanalyser (Bayer® Germany).
Evaluation of Some Oxidative Stress Parameters
Preparation of tissue homogenates for the evaluation of oxidative stress parameters
The hepatic (liver) and renal (kidney) samples were cleaned with physiological saline, dried with filter paper and weighed after excision from the experimental animals [17]. Portions of the hepatic and renal samples were mixed with phosphate buffered saline (PBS) (1:10 weight/volume at pH 7.4) and homogenized. Subsequently, the mixtures were kept in ice baths and centrifuged later for the evaluation of the levels of hepatic and renal oxidative stress indices [malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)].
Determination of malondialdehyde levels
The malondialdehyde level as an indication of lipid peroxidation was appraised in the serum, hepatic and renal samples by using a malondialdehyde assay kit (Elabscience Biotechnology Incorporation (EBI), Elabscience® Texas, USA). The method described by Draper and Hadley [18] was deployed. The absorbance was measured with an ultraviolet (UV) spectrophotometer (T80+ UV/VIS spectrometer®, Liicestershire, UK) at 532 nm. The malondialdehyde concentrations in the samples were computed by using the absorbance coefficient of malondialdehyde-thiobarbituric acid complex 1.56 x 105/cm/M and expressed as nmol/mg protein for the hepatic and renal samples. The malondialdehyde levels in the serum samples were expressed as nmol/mL, while the protein contents of the supernatants were appraised according to the method of Dacie and Lewis [19].
Assays of the antioxidant enzymes levels
Superoxide dismutase activity was assessed in the serum, hepatic and renal samples with the SOD assay kit produced by EBI (Elabscience® Texas, USA). The procedure is predicated on the autoxidation of haematoxylin [20]. The catalase (CAT) assay kit manufactured by EBI (Elabscience® Texas, USA) was deployed for the assessment of CAT activity in the serum, hepatic and renal samples. The method used was based on hydrogen peroxide substrate consumption [21]. The level of glutathione peroxidase was analyzed with the NWLSSTM glutathione peroxidase (GPx) activity assay kit (Northwest Life Science Specialties, Vancouver, USA). The basis of the analysis was the oxidation of reduced glutathione to form oxidized glutathione [22].
Statistical Analysis
Data obtained were stated as mean ± standard error of the mean (SEM) and scrutinized with the application of one-way ANOVA followed by Tukey's multiple comparison post hoc test. Graph Pad Prism (version 4.00, Graph Pad Software, California, USA) was utilised. P < 0.05 was considered as statistically significant.
Results
Clinical Observations
The rats in the group administered with imidacloprid (IM) showed some signs of toxicity such as inappetence and lethargy during the investigation. Transient inappetence and hyperaemia around the nostrils were noticed in the IM+AR100 and IM+AR200 groups. However, the DW group did not exhibit signs of toxicity.
Effects of the Treatments on Body Weight Changes
The body weights of the rats increased in the DW group, underwent fluctuations in the IM group and decreased in the IM+AR100 and IM+AR200 groups during the research (Figure 1). The percentage changes in the body weights of the animals at Week 0 compared to Week 2 were: DW (18 %), IM (7 %), IM+AR100 (18 %) and IM+AR200 (10 %). No significant alteration was recorded in the body weights at Week 0. However, significant elevations (p < 0.05) were detected in the body weights of the DW group compared to those of the IM, IM+AR100 and IM+AR200 groups at Weeks 1 and 2 respectively (Figure 1).
Figure 1: Effects of DW (distilled water), IM [imidacloprid (71 mg/kg)], IM+AR100 [Imidacloprid (71 mg/kg) + L-arginine (100 mg/kg)] and IM+AR200 [Imidacloprid (71 mg/kg)+L-arginine (200 mg/kg)] on the body weights of the rats, n =10 animals per group.
*p < 0.05 DW group versus IM, IM+AR100 and IM+AR200 groups at Weeks 1 and 2 respectively.
Impacts of the Treatments on the Levels of Serum Proteins
The effects of the treatments on the concentrations of serum proteins are depicted in Figure 2. There were significant (p < 0.05) declines in the total protein levels of the IM and IM+AR100 groups relative to that of the DW group. Additionally, substantial (p < 0.05) reductions were noticed in the globulin levels of the IM and IM+AR100 groups relative to those of the DW and IM+AR200 groups respectively.
Figure 2: Effects of DW (distilled water), IM [imidacloprid (71 mg/kg)], IM+AR100 [Imidacloprid (71 mg/kg) + L-arginine (100 mg/kg)] and IM+AR200 [Imidacloprid (71 mg/kg)+L-arginine (200 mg/kg)] on serum protein levels, n =10 animals per group.
TP: aP< 0.05 IM and IM+AR100 groups versus DW respectively
GLB: bP< 0.05 IM and IM+AR100 groups versus DW and IM+AR200 groups respectively
TP: Total Protein
ALB: Albumin
GLB: Globulin
Effects of the Treatments on the Levels of Serum Enzymes and Renal Function Indices
The impacts of the treatments on the levels of serum enzymes and renal function parameters are shown in Table 1. The activity of aspartate aminotransferase was significantly (p < 0.05) elevated in the IM, IM+AR100 and IM+AR200 groups relative to that of the DW group respectively. There was a noteworthy (p < 0.05) upsurge in the creatinine concentration of the IM group in comparison to that of the DW group.
Table 1: Effects of DW (distilled water), IM [Imidacloprid (71 mg/kg)], IM+AR100 [Imidacloprid (71 mg/kg) +Arginine (100 mg/kg)] and IM+AR200 [Imidacloprid (71 mg/kg) +Arginine (200 mg/kg)] on the levels of serum enzymes and renal function indices.
Parameters |
DW |
IM |
IM+AR100 |
IM+AR200 |
ALT (IU/L) |
14.75±0.84 |
16.76±0.62 |
16.01±0.19 |
15.02±0.77 |
AST (IU/L) |
36.84±0.42 |
46.85±0.49* |
46.25±1.01* |
46.11±0.88* |
GGT (IU/L) |
13.25±3.33 |
33.35±11.54 |
19.52±4.11 |
16.72±4.25 |
LDH (IU/L) |
39.62±12.96 |
43.75±18.76 |
43.74±7.68 |
42.92±12.15 |
CK (IU/L) |
30.81±9.66 |
42.91±15.94 |
40.71±11.15 |
40.71±4.30 |
Urea (mg/dl) |
28.95±5.07 |
35.30±1.61 |
33.10±2.57 |
29.88±1.46 |
Creatinine (mg/dl) |
2.07±0.33 |
3.25±0.25** |
2.80±0.22 |
2.71±0.26 |
ALT: Alanine aminotransferase
AST: Aspartate aminotransferase
GGT: Gamma glutamyltransferase
LDH: Lactate dehydrogenase
CK: Creatine Kinase
AST: *p < 0.05 IM, IM+AR100, IM+AR200 groups versus DW group respectively
Creatinine: **p < 0.05 IM group versus DW group
Impacts of the Treatments on the Levels of Serum Oxidative Stress Indices
The impacts of the treatments on the serum oxidative stress indices of the rats are portrayed in Table 2. There was no significant (p > 0.05) alteration in the levels of the parameters among the treatment groups.
Table 2: Effects of DW (distilled water), IM [imidacloprid (71 mg/kg)], IM+AR100 [imidacloprid (71 mg/kg) + arginine (100 mg/kg)] and IM+AR200 [imidacloprid (71mg/kg)+arginine (200 mg/kg)] on the levels of serum oxidative stress parameters in male Wistar rats.
Parameters |
DW |
IM |
IM+AR100 |
IM+AR200 |
Serum MDA (nmol/Ml) |
1.12±0.28 |
2.14±0.46 |
2.11±0.28 |
1.84±0.31 |
Serum SOD (IU/L) |
76.96±1.98 |
72.42±5.57 |
83.79±3.64 |
86.07±1.76 |
Serum CAT (IU/L) |
15.73±0.22 |
15.44±2.09 |
16.05±0.78 |
16.13±0.67 |
Serum Gpx (IU/L) |
16.30±2.69 |
16.16±2.70 |
18.13±3.83 |
19.99±1.33 |
MDA: Malondialdehyde
SOD: Superoxide dismutase
CAT: Catalase
GPx: Glutathione peroxidase
Effects of the Treatments on the Levels of Hepatic Oxidative Stress Parameters
The effects of the treatments on the levels of hepatic oxidative stress parameters are portrayed in Table 3. There was a significant (p < 0.05) reduction in the activity of hepatic catalase in the IM group compared to that of the IM+AR200 group.
Table 3: Effects of DW (distilled water), IM [imidacloprid (71 mg/kg)], IM+AR100 [imidacloprid (71 mg/kg) + arginine (100 mg/kg)] and IM+AR200 [imidacloprid (71mg/kg)+arginine (200 mg/kg)] on the levels of hepatic oxidative stress parameters in male Wistar rats.
Parameters |
DW |
IM |
IM+AR100 |
IM+AR200 |
Hepatic MDA (nmol/mg protein) |
11.69±2.48 |
20.5±3.41 |
17.14±3.03 |
13.77±2.03 |
Hepatic SOD (IU/L) |
87.77±4.51 |
82.09±9.95 |
94.6±14.41 |
130.7±33.28 |
Hepatic CAT (IU/L) |
227±4.34 |
203.5±11.6* |
232.7±25.71 |
278.7±16.47 |
Hepatic GPx (IU/L) |
76.19±6.67 |
63.98±9.29 |
76.25±5.49 |
88.32±9.81 |
*p < 0.05 IM group versus IM+AR200 group for hepatic catalase (CAT)
MDA: Malondialdehyde
SOD: Superoxide dismutase
CAT: Catalase
GPx: Glutathione peroxidase
Impacts of the Treatments on the Levels of Renal Oxidative Stress Parameters
The impacts of the treatments on the levels of renal oxidative stress parameters are depicted in Table 4. There was a considerable (p < 0.05) reduction in the activity of renal superoxide dismutase in the IM group in comparison to that of the IM+AR200 group.
Table 4: Effects of DW (distilled water), IM [imidacloprid (71 mg/kg)], IM+AR100 [imidacloprid (71 mg/kg) +arginine (100 mg/kg)] and IM+AR200 [imidacloprid (71mg/kg) +arginine (200 mg/kg)] on the levels of renal oxidative stress parameters in male Wistar rats.
Parameters |
DW |
IM |
IM+AR100 |
IM+AR200 |
Renal MDA (nmol/mg protein) |
6.54±1.48 |
7.66±2.1 |
7.06±1.91 |
6.53±1.84 |
Renal SOD (IU/L) |
106.3±4.23 |
90.38±4.7* |
107.4±2.52 |
172.2±35.32 |
Renal CAT (IU/L) |
280.5±4.41 |
264.4±6.4 |
296.8±11.37 |
320.1±24.31 |
Renal GPx (IU/L) |
106.6±5.62 |
88.97±7.8 |
106±7.27 |
122.2±12.63 |
*p < 0.05 IM group versus IM+AR200 group for renal superoxide dismutase (SOD)
MDA: Malondialdehyde
SOD: Superoxide dismutase
CAT: Catalase
GPx: Glutathione peroxidase
Discussion
In this research, the calculated oral LD50 of IM used was 283 mg/kg, indicating that the commercial grade of IM administered was moderately toxic [23]. The oral LD50 value of IM in rats is 450 mg/kg [24]. In this study, the dosage of IM administered to the rats for two weeks was ~ 0.25 LD50 i.e., 71 mg/kg. The rats in the IM group showed signs of toxicity which included inappetence and lethargy. These signs, as well as others such as increased salivation, tremor and diarrhoea were reported by Vohra et al. [24] in female rats administered with IM at a dosage of 10 and 20 mg/kg for 60 days. Likewise, other investigators [25,26] observed inappetence, lethargy and dyspnoea in rodents treated with IM. It was surmised that the clinical signs exhibited by the rats in this investigation may be indicative of imidacloprid’s effect on the nicotinic receptors.
L-arginine (AR) is an amino acid and a precursor to nitric oxide (NO) [27]. When AR is consumed, it is converted to citrulline and NO by the enzyme, nitric oxide synthase. Subsequently, the NO produced by nitric oxide synthase disperses into the vascular smooth muscle cells, thereby ensuing in their relaxation and dilation [28] This results in elevated perfusion of the tissues which is termed “hyperaemia” [29]. In the current research, hyperaemia of the nares was noticed in the IM+AR100 and IM+AR200 groups.
There was a decline in the body weights of the experimental animals in the IM group at Week 1 compared to Week 0, and this was followed by an upsurge in the body weights at Week 2 relative to Week 1. In an investigation conducted by Hassanen and his colleagues [30), IM caused a significant decrease in the body weights of rats and this was associated with the toxic effects of the pesticide on the organs of the rats that might have impeded food consumption and nutrients absorption [31,32,33]. Conversely, the consistent decrease in the body weights in the IM+AR100 and IM+AR200 groups may suggest that AR has anorectic effects and this may be linked with the role of gut hormones [34].
In our investigation, a significant reduction was recorded in the concentration of total protein of the IM group. This outcome may suggest that IM elicited hepatic injury in the rats. Similarly, Hassanen et al. [30] reported a substantial decline in total protein level in rodents exposed to IM. According to [35], this finding may be caused by a diminution in albumin synthesis in response to hepatocellular damage. Moreover, the globulin concentration of the IM group was significantly reduced in the present investigation, and this is in consonance with the result obtained by Hassanen et al. [30]. Contrariwise, the globulin level was remarkably ameliorated in the IM+AR200 group, suggesting a dose-dependent effect of AR in this study. This desirable impact of AR may be associated with its protective properties in biological system [36,37].
Furthermore, a significant upsurge was observed in the AST activity in the IM, IM+AR100 and IM+AR200 groups relative to the DW group, respectively. In agreement with our findings in the current study, other researchers have reported significant elevations in ALT and AST activities in rats [38,39]. Amplified activities of ALT and AST have been linked to hepatic damage as a result of heightened cell membrane absorptivity [40]. On the other hand, non-significant reductions were recorded in the ALT and AST levels in the IM+AR100, IM+AR200 groups in relation to the IM group. In contrast, ALT and AST levels were partially and significantly decreased in male Sprague-Dawley rats treated with 500 mg/kg of AR twice a day for 7 days in a research conducted by Ozsoy et al [41]. This outcome was attributed to the inhibition of hepatic damage in acute cholestasis by the stimulation of inducible nitric oxide synthase expression and nitric oxide synthesis through the administration of AR to the rats.
An insignificant elevation was observed in the urea concentration, while there was a significant upsurge in the creatinine concentration in the IM-treated rats. Also, Hassanen et al. [30] stated that IM evoked a noteworthy increase in serum urea and creatinine levels in rats and these results may be suggestive of the nephrotoxic potential of IM. On the contrary, AR did not elicit an overt attenuation of the potential nephrotoxic effects of IM in this study. However, Abdelhalim et al. [42] affirmed that AR (200 mg/kg) decreased creatinine levels in rodents exposed to gold nanoparticles for 7 days probably because of its antioxidant and anti-inflammatory properties biological systems [36,37].
Furthermore, there were slight elevations in the serum, hepatic and renal MDA levels, including significant reductions in the levels of hepatic CAT and renal SOD of the IM group. This indicated that IM did not evoke profound oxidative stress in this study. Contrariwise, other investigators affirmed that IM increased ROS production and lipid peroxidation in the serum, hepatic and renal tissues [43,44,45]. On the other hand, marginal declines were recorded in the serum, hepatic and renal MDA concentrations in the AR-treated groups, while only the hepatic CAT and renal SOD activities were significantly improved in the IM+AR200 group, thereby suggesting a slight dose-dependent impact of AR in this regard. Arginine supplementation has been shown to markedly decrease the MDA concentration and amplify the activities of GPx and SOD, as well as total oxidative capacity in the plasma of pigs [46]. These results may suggest that AR supplementation could be efficacious for the relief of oxidative stress [47,48] reported that AR may reduce the quantity of free oxygen radicals and inflammation status in rats, thereby impeding tissue damage. However, the lack of prominent antioxidant impacts of AR in the current research, compared to other investigations, may be attributed to the differences in the experimental protocols, dosages of the xenobiotics administered, etc.
Conclusion
The findings of this research revealed that the treatment of male Wistar rats with imidacloprid (71 mg/kg) for 14 days evoked significant adverse effects on the levels of total protein, globulin, aspartate aminotransferase and creatinine. Imidacloprid did not evoke marked oxidative stress at the dosage used in this research. However, L-arginine exerted partial ameliorative effects on imidacloprid toxicity. Therefore, further studies are recommended for the elucidation of the mechanisms of toxicity of imidacloprid and bioprotection by L-arginine.
Conflict of interest statement
The authors declare that there is no conflict of interest.
Acknowledgements
The authors acknowledge the support of the staff of the Faculty of Veterinary Medicine, University of Abuja, Nigeria.
Funding
The research was funded by the authors.
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