Abstract
The aim is to investigate the sub-acute neurotoxic effects of copper sulfate in chicks on motor and neurobehavioral activity and its relation to oxidative stress and histopathological changes in chick brain tissue. Thirty chicks were employed in this experiment, randomly separated into 5 groups of 6 chicks. They were given the following concentration 2.5, 5, 10, and 15% of LD50. Each of the chicks is put through the following behavioral tests response to tonic immobility test, righting reflex, testing the motor activity of the chicks inside the open field box. Orally LD50 was 772 mg/kg, Recording an inhibition in the animal's movement in the open field and an increase in the chicks' dormancy duration. The effects are directly proportional to the increase in the chicks' dose. Copper sulfate in 2.5, 5, 10, and 15% of the LD50 showed a significant increase in malondialdehyde concentration, while 15% of LD50 recorded a significant decrease in glutathione and cholinesterase activity. All doses substantially decreased total antioxidant capacity in brain and liver tissue. Chick brain of copper sulfate 15% of LD50 shows in the cortex of cerebrum severe gliosis, satellitosis, perivascular and periaxonal edema, necrosis (karyorrhexis) of neuron, and apoptosis. The rest of the concentrations had histopathological alterations proportionate to the rise in the given dose. We concluded from this work that high concentrations of copper sulfate in the brain generated oxidative stress and histopathological alterations, which influenced chicks' neurobehavior and motor activity in the open environment.
Main Subjects
Full Text
Introduction
Heavy metals refer to any metallic chemical element with a relatively high density and are toxic at a low concentration (1). Continuous exposure to many heavy metals that may contain copper, cadmium, zinc, nickel, lead, and arsenic has a wide range of harmful effects on living organisms (2). Although some heavy metals have a role in biochemistry and affect low concentrations, they negatively affect physiological functions at high concentrations due to bioaccumulation in vital organs (3-5). Except in rare hereditary diseases and deficiencies that result in the inability to eliminate copper. Copper is not considered a dangerous substance for living creatures, but it can cause liver, kidney, and brain damage if consumed excessively unless special conditions exist (6-8). Copper-containing enzymes are required for several important activities. Copper is received from food, and the liver ensures that it is transported safely. On the other hand, inorganic copper essentially bypasses the liver and enters the bloodstream directly, which is potentially harmful because copper can cross the blood-brain barrier (9-11). Even though copper attaches to the protein, it can uncouple and release it and accelerate the generation of highly reactive hydroxyl and other reactive oxygen species (12-14). The current study aimed to investigate the sub-acute neurotoxic effects of copper sulfate in chick's neurobehavioral and motor activity in the open field-moreover, the effect of previous treatments on oxidative stress levels by measuring glutathione and malondialdehyde. The total antioxidant capacity and cholinesterase activity support the result histopathological examination of changes in changes in chick brain tissue will be done.
Materials and methods
Ethical approval
The College of Veterinary Medicine at the University of Mosul provided all ethical clearances for the humane treatment of laboratory animals, and the form number was UM.VET.2021.35.
The animals
Local chickens of both sexes were used in this study, with an estimated number of 40 chicks (10 chicks for LD50) raised in the poultry hall in the animal house of the College of Veterinary Medicine, University of Mosul, for 7 days before the experiment began. Specially adapted for this purpose and given plenty of water and forage and the proper circumstances.
The Chemicals
The following chemical substances were used, hydrated copper sulfate from Karlsruhe, Germany, Zeppelinstrassa Thaiobarbturic Acid (TBA) Merk Company, HCl at a concentration of 0.25 N, Trisodium-citrate buffer at a concentration of 10%, Na2HPO4 buffer (0.3 M, 5.5 Th) DTNB (L-Glutathione, formalin, neutralized 10% utilized diagnostic kit) 2- Nitrobenzoic acid. Elabscience produced a Total Antioxidant Capacity (T-AOC) colorimetric measurement kit.
Preparing the potion
After estimating the specific dose for each item, copper sulfate doses were produced and dissolved in distilled water, then administered to the animals using a gavage oral syringe, with a 5ml volume of administration.
Blood sample collection
The chicks' jugular veins were cut to collect blood in special tubes, and the blood was separated into two groups, one with anticoagulants to extract blood plasma and the other without anticoagulants to extract blood serum. The serum and plasma samples were collected for 15 minutes and centrifuged at 3000 rpm for 15 minutes. Before being stored in special plastic tubes and frozen at -20°C until laboratory biochemical testing was completed.
Organ collection
After the chickens were slaughtered, the brains and livers were taken. Each sample was divided into 2 sections and placed in numbered bags in the freezer at -20ºC (for use in Tissue Homogenisis). The remaining piece of each sample was placed in special containers containing diluted formalin at a concentration of 10% for preservation until it was completed, taking into account the numbering of each group separately. A brain histopathological investigation will be performed.
Experiment design
The first experiment was conducted to determine the median lethal dose (LD50) of copper sulfate in chicks. The up and down approach was used to determine the LD50 (15). Second, encounter neurobehavioral tests used to determine the response to different dosages of copper sulfate. Thirty chicks were employed in this experiment, randomly separated into five groups of six chicks each. Several dosages of copper sulfate were administered to the chicks in this experiment, based on the median lethal dose determined in the preceding experiment. They were given the following concentrations: 2.5, 5, 10, and 15 % of LD50 equal to 19.3 mg/kg, 38.6 mg/kg, 77.5 mg/kg and, 115.8 mg/kg. The therapy lasted three days before being recorded on the fourth day, which marked the end of the experiment's treatments. Each of the chicks is put through the following behavioral tests:
Response to tonic immobility test
The test is performed by placing each chick alone on its right side and attempting to calm it for 15 seconds. Then quietly withdraw the hand and measure the time of the chick to remain silent until resistance and movement began, with a maximum period of 300 seconds, and repeat the chick that failed to calm for five attempts, with a 30-second gap between each try (16).
Righting reflex
The chicks in the preceding groups are placed on one of their sides and calculated the time it takes to adjust their body position or their failure to do so (17).
Testing the motor activity of the chicks inside the open field box
In an isolated room, a rectangular wooden box with dimensions of 60*60*30 cm and a floor partitioned into 24 equal squares with a side length of around 15 cm was employed. This assessment examines the chicks' general movement and activity within the open field box for each chick (18).
Measuring some biochemical variables
Some biochemical parameters of treated chicks' plasma and blood serum were measured by studying oxidative stress by measuring malondialdehyde (19), serum glutathione (20), total antioxidant status (TAC), and measurement of cholinesterase activity in plasma.
Statistical analysis
For non-parametric data, use the Mann-Whitney test. Parametric data were statistically evaluated using the SPSS one-way analysis of the variance test program, after which the LSD test was used to determine the level of significant difference. The probability was less than 0.05 (21).
Results
The first experiment determined the median lethal dose of copper sulfate (LD50) in chicks. When given orally, LD50 was 772 mg/kg, with a 200 mg/kg value for the rise and decrease in dose. The presence of puffy feathers, tiredness in animals, lack of movement and eating, difficulty breathing, bloody diarrhea, and eventually death were all indicators of poisoning in chicks (Table 1).
Table 1: The oral median lethal dose of copper sulfate in chicks
|
Variable |
Results |
|
LD50 (orally) |
772 mg/kg |
|
Dosage range |
1200-600 mg/kg |
|
First dose |
1200 mg/kg |
|
Last dose |
600 mg/kg |
|
No. chicks used |
(xxoxxo) 6 |
|
Dose change |
200 mg/kg |
|
Time of toxicity |
minutes 3-10 |
|
Signs of poisoning |
Fluffy feathers, breath difficulty, bloody diarrhea, slimy secretions from the mouth, and finally, death. |
X means the chick is dead and 0 means survives the chick.
In the second experiment, a neurobehavioral test was used to measure the reaction to different dosages of copper sulfate. This experiment was carried out with concentrations of 2.5 % (19.3 mg/kg), 5% (38.6 mg/kg), 10% (77.5 mg/kg) and 15% (115.8 mg/kg) of the median lethal dose. The number of squares crossed by chicks was directly related to the increase in the provided dose compared to the control. The behavioral changes reflected by the delay in the time of starting movement were recorded with a significant decrease in the number of squares crossed (Table 2). In the body posture correction (Righting Test), the administered dose resulted in a substantial increase in posture correction time of 5, 10, and 15% compared to the control and the other groups. The groups treated with concentrations of 5, 10, and 15% of the mean lethal dose recorded a significant and apparent increase in the dormancy period of the chicks compared to the control group in the Tonic immobility test. At the same time, the 2.5 percent group also recorded an increase in the chicks' dormancy period, but less than the other concentrations (Table 2).
Table 2: Motor and neurobehavioral response to different doses of copper sulfate
|
Neurobehavior |
Control |
19.5 mg/kg |
38.6 mg/kg |
77.5 mg/kg |
115.8 mg/kg |
|
Start movement/sec |
1±0.1 |
0.1 ±3* |
1±5*a |
2±17*ab |
33±30 *abc |
|
Number of squares |
2±20 |
2±14* |
1.1±4*a |
1 ±4 *ab |
1±2 *ab |
|
Body correction/sec |
1±0.1 |
0.2±2 |
0.1 ±3* |
0.3±3* |
1.4 ±3 * |
|
Tonic immobility/sec |
56±5 |
5.6±66* |
23±232*a |
35±300*ab |
66±300*ab |
Each group consists of 6 animals. Data as mean ± SE. *This means different in the control group. a- This means a significant difference from 19.5 mg/kg. b- This means a significant difference from 38.6 mg/kg. c- This means a significant difference from 77.5 mg/kg.
Copper sulfate in 2.5, 5, 10, and 15% of the LD50 showed a significant increase in malondialdehyde concentration compared with the control group. While 15% of LD50 recorded a significant decrease in glutathione compared to the other groups, all doses substantially decreased total antioxidant capacity (TAC) in brain and liver tissue compared with the control group (Table 3).
Table 3: Oxidative stress status by measuring malondialdehyde, glutathione, in serum and TAC in the tissue of chicks
|
Groups |
In Serum |
TAC (U/ml) |
||
|
MDA (nmol/ml) |
GSH (nmol/ml) |
Brain Tissue |
Liver Tissue |
|
|
Control |
2.29±1 |
0.08±0.01 |
3.1±0.4 |
4.1±1 |
|
19.5 mg/kg |
3.89±1 *d |
0.02±0.01 *d |
1.1±0.03* |
1.0±0.02* |
|
38.6 mg/kg |
3±0.5 *d |
0.021±0.001 *d |
0.7±0.05* |
0.9±0.01* |
|
77.5 mg/kg |
94.3±1 *d |
0.01±0.005 *d |
0.5±0.04* |
0.7±0.02* |
|
115.8 mg/kg |
5.69±1 * |
0.005±0.002 * |
0.3±0.02* |
0.4±0.02* |
Each group consists of 6 animals. Data as mean ± SE. *This means different in the control group. d- This means a significant difference from 115.8 mg/kg
The level of inhibition of cholinesterase activity in concentrations of 2.5, 5, 10, and 15% of the lethal median dose resulted in a significant decrease in cholinesterase activity compared to the control group. While the concentrations of 10 and 15% of the lethal median dose resulted in a significant decrease in cholinesterase activity compared to the control group and two groups of 2.5 and 5% (Table 4).
Table 4: Effect on cholinesterase activity in plasma of chicks treated with different concentrations of LD50
|
Groups |
Cholinesterase activity in plasma |
|
Control |
2.95 ± 0.01 |
|
19.5 mg/kg |
0.96 ± 0.007* |
|
38.6 mg/kg |
0.90 ± 0.004* |
|
77.5 mg/kg |
0.80 ± 0.002*ab |
|
115.8 mg/kg |
0.69 ± 0.004*ab |
Each group consists of 6 animals. Data represented as mean ± SE. *This means different in the control group. a- This means a significant difference from 19.5 mg/kg. b- This means a significant difference from 38.6 mg/kg.
Histopathological changes
Figure 1 appeared the chick brain of the control group shows the cortex of the cerebrum with standard architecture represented by neurons, glial cells, and blood vessels. In figure 2 chick brain of copper sulfate, 2.5% of LD50 dose treated group shows the cerebrum cortex with intact neurons with mild cytogenic edema and mild congestion of blood vessels with mild perivascular edema. Figure 3 for chick brain of copper sulfate 5% of LD50 dose treated group shows the cortex of cerebrum with intact neurons and gliosis, congestion of blood vessels, and perivascular edema. In figure 4, the chick brain of copper sulfate 10% of LD50 dose treated group shows the cortex of cerebrum with congestion of blood vessels, diffuse perivascular edema, periaxonal edema, satellitosis by glial cells around neurons, and neuronophagia. Also Figure 5 showed chick brain of copper sulfate 15% of LD50 dose treated group the cortex of cerebrum appeared with liquefactive necrosis of neurons and neuropil, gliosis, neuronophagia congestion of blood vessels, perivascular edema and periaxonal edema. Figure 6 for chick brain of copper sulfate 15% of LD50 dose treated group shows the cortex of cerebrum with severe gliosis, satellitosis by glial cells around neurons, congestion of blood vessels, perivascular edema, periaxonal edema, necrosis (karyorrhexis) of neuron and apoptosis.
Figure 1: photomicrograph of chick brain of control group shows the cortex of cerebrum with standard architecture represented by neurons (A), glial cells (B) vessels(C). H&E stain, Scale bar = 100μm.
Figure 2: photomicrograph of chick brain of copper sulfate 2.5% of LD50 dose treated group shows the cortex of cerebrum with intact neurons (A) with mild cytogenic edema (B) with mild congestion of blood vessels (C) and mild perivascular edema (D). H&E stain, Scale bar =100 μm.
Figure 3: photomicrograph of chick brain of copper sulfate 5% of LD50 dose treated group shows the cortex of cerebrum with intact neurons (A) with gliosis (B), congestion of blood vessels (C), and perivascular edema (D). H&E stain, Scale bar = 100μm.
Figure 4: photomicrograph of chick brain of copper sulfate 10% of LD50 dose treated group shows the cortex of cerebrum with a cluster of sizeable multiple pyramidal cells with necrosis (A), satellitosis by glial cells around neurons (B), hemorrhage (C), apoptosis (D) and periaxonal edema (E). H&E stain, Scale bar = 100μm.
Figure 5: photomicrograph of chick brain of copper sulfate 15% of LD50 dose treated group shows the cortex of cerebrum with severe gliosis (A), diffuse perivascular edema (B), congestion of blood vessels (C) and periaxonal edema (D), satellitosis by glial cells around neurons (D) and neuronophagia (E). H&E stain, Scale bar = 100μm.
Figure 6: photomicrograph of chick brain of copper sulfate 15% of LD50 dose treated group shows the cortex of cerebrum with severe gliosis (A), satellitosis by glial cells around neurons (B), congestion of blood vessels (C), perivascular edema (D) periaxonal edema (E), necrosis (karyorrhexis) of neuron (F) and apoptosis (G). H&E stain, Scale bar = 100μm.
Discussion
Behavior is an appropriate end aim for environmental toxicology research because it integrates information from various levels of biological information and has direct and intuitive relevance to environmentally significant outcomes such as growth, reproduction, and survival. It is also an excellent early warning indication, with a sensitivity of 10-1000 times that of more severe endpoints like mortality (22,23). In our current investigation, the median lethal dose of copper sulfate in chicks was 772 mg/kg, with acute clinical signs. The goal of utilizing several percentages of the median lethal dosage of copper sulfate in chicks (2.5 percent, 5 percent, 10 percent, and 15 percent) was to discover the lowest hazardous dose that could indicate the latent effects on the nervous system without causing visible toxic effects on the chicks. In the tensile immobility test, there was a decrease in the chicks' activity and movement inside the open field and an extension of the chicks' dormancy phase.
These effects were directly proportional to the dose value, as the dose of 2.5 percent was substantially better than the other doses, although the toxic effects were quite apparent in the other doses. The remaining doses and results are consistent with several studies that have shown that copper sulfate has harmful effects on the central nervous system as a result of its capacity to cross the blood-brain barrier, where copper showed an effect on human astrocytes exposed to copper (24,25).
According to one study, the effect of copper chloride on membrane function and integrity in astrocytes in vitro after low doses of copper chloride was applied. According to these tests, the copper content of astrocytes exponentially with time and in a concentration-dependent manner (26,27). Chronic copper toxicity generates copper accumulation in the cortex, striatum, and cerebellum areas of male Wistar rats, which is linked to cognitive deficits (28,29). Excessive copper administration via oral administration increased the amount of copper unbound to ceruloplasmin in plasma, causing oxidative damage through excessive production of reactive oxygen species in the cortex and hippocampus (30,31).
The cerebellum is essential in balance, motor control, and different brain functions such as emotional regulation and cognition (32,33). Oxidative stress appears to be the primary source of neurological impairments following copper accumulation. Because the cerebellum is one of the brain regions affected by copper toxicity, it is logical to believe that elevated copper levels in the cerebellum could impact its structure and function (34,35). Also, because our results showed a significant decrease in the cholinesterase enzyme activity in plasma of all treated groups compared to the control group, cholinesterase inhibition may play a role in the decreased activity of chick movement inside the open field to muscle fatigue (weakness). This is consistent with other researchers who found that metal ions in high concentrations (especially copper) can inhibit cholinesterase activity by binding to azole imide. Metal ions also interact with other amino acids in the active protein, such as tryptophan, 9-metho, cysteine, and d phenylalanine (36,37).
Copper is also one of the few heavy metals that inhibits cholinesterase activity in a non-competitive manner (38,39). The prior effects were not isolated from the oxidative stress generated by various concentrations of copper sulfate, where the concentration of malondialdehyde increased significantly and was linked to a drop in serum glutathione levels. The overall antioxidant capacity in liver and brain tissue was much lower than in the control group. These findings are consistent with other research that suggests the cause of this is due to free copper ions acting as potent catalysts for the creation of (ROS), (Cu2+) to copper (Cu+) in the presence of superoxide (O2-), which can catalyze the production of reactive hydroxyl radicals (OH) from the decomposition of hydrogen peroxide (H2O2) (40,41,42). Hydroxyl radicals are the most potent oxidizing radicals found in living systems and can catalyze lipid peroxidation, which causes tissue injury (39,43,44). The histological study confirmed the findings, revealing apparent degenerative alterations on the neurons whose intensity correlated with the dose administered, with the effects being more severe at a concentration of 15% of the typical lethal dose. These findings show that oxidative stress and free radicals produced by high copper dosages play a role in cell damage, neuronal degeneration, and other pathological alterations, including programmed death.
Necrosis is a type of cell death that occurs when tissue is damaged accidentally and includes activating a specific cellular program (40). Copper-generated free radicals appear to have caused the early loss of plasma membrane integrity. Moreover, the expansion of the cell body is followed by the cell's explosion, and as the cell explodes, hazardous compounds are released from the mitochondria, causing damage to the surrounding cells. Dying cells shrink, compress, and eventually break apart during apoptosis, releasing small membrane-bound apoptosis bodies filled by immune cells (macrophages).
Conclusion
We concluded that copper sulfate had toxicity at high doses, as evidenced by neurobehavioral and motor activity, several serum indices of oxidative stress and tissue in the brain, and several enzymes and histological alterations.
Acknowledgment
The researchers are appreciative of the University of Mosul's College of Vets. Medicine for providing all of the resources needed to complete this work
Conflict of interest
The researchers promise that they have no links to any of the parties involved in this study.
)
