الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Chemicals
1) Tramadol
The certified reference of Tramadol was of >99 % purity.
2) Abamectin
A pure reference standard of abamectin (>98.7%, purity.
Experimental Designs
Nighty six (96) male albino rats (Rattus norvegicus) of the same stock were obtained from the animal house of Cairo University, Giza-Egypt.
Exposure protocol
At the start of the study, the male rats were approximately 10 weeks old (weight 180-200 g).
The rats were randomly assigned to 3 groups of 32 animals each and were treated as follows:
group 1: Tramadol-treated rats.
The animal group (Tramadol group, N1 = 32), was divided into 8 sub-groups received tramadol .Each sub-group consisted of 4 rats (n1=4). Were received Tramadol at doses of 40 mg kg-1, administered orally by gavage.
group 2: Abamectin-treated rats.
The animal group (Abamectin group, N2 = 32), was divided into 8 sub-groups received abamectin .Each sub-group consisted of 4 rats (n2=4). Were received Abamectin at doses of 10 mg kg-1, administered orally by gavage.
group 3: Tramadol-Abamectin - treated rats.
The animal group (Tramadol +Abamectin treated group, N3 = 32), was divided into 8 sub-groups ,each sub-group consisted of 4 rats (n3=4) and received Tramadol at a dose of 40 mg kg-1 plus Abamectin at doses of 10 mg kg-1, administered orally by gavage.
Tramadol and Abamectin were administrated orally by gavages using corn oil.
Samples and parameters measured
1) Blood samples
Blood and plasma samples were collected at constant intervals; half hr, hr1 hr, 1.5 hr, 2 hr ,2.5 hr,3 hr 3.5hr and 24 hr, respectively.
samples were used for the determination of:
Urea and creatinine
(AST) and alanine aminotransferase (ALT) enzyme activities.
Brain specimens were taken at constant intervals; half hr, hr1 hr, 1.5 hr, 2 hr ,2.5 hr,3 hr 3.5hr and 24 hr respectively, for the determination the following (Biochemical analysis):
0) ATPase activity
1) Reduced GSH content
2) SOD
3) Acetylcholine Esterase (AChE) Enzyme Activity
Determination of the concentrations of the following neurotransmitters in brain Homogenate:
1. Measurement of serotonin
2. Measurement of nor- epinephrine
3. Measurement of epinephrine
4. Measurement of DOPA
Fecal sampling
Half gm of each animals was taken at constant intervals; half hr, hr1 hr, 1.5 hr, 2 hr ,2.5 hr,3 hr 3.5hr and 24 hr respectively, to determine the concentration of tramadol and abamectin.
Determination of Tramadol
The HPLC analysis was performed with an Agilent 1260 HPLC system (USA), with quaternary pump, autosampler injector, thermostat compartment for the column and fluorescence detector (FLD).
Determination of Abamectin
Abamectin concentration was determined by an Agilent 1260 HPLC system (USA), with quaternary pump, autosampler injector, thermostat compartment for the column and diodearray detector (DAD).
Histopathological Examination
Laparotomy and Histopathological examination:
1. Specimens
Midline laparotomy was performed and brain, liver and kidney specimens were obtained.
2. Histopathological procedures
Histological analysis. Tissues from animals were collected after the animals were killed and then fixed in buffered 10% formaldehyde PH 7.0 (phosphate puffer). After 48 hr, the fixed tissue was paraffin embedded. Paraffin blocks were cut serially in10–15 6-lm slices and stained with hematoxylin and eosin.
The Results revealed the following:
TRAMADOL
Tramadol in blood plasma
The concentration of tramadol begins to decrease (3.64 µg\ml) at two hr post treatment.
It was observed that, the excretion of tramadol in the faecal matter was highest (12.417 µg\g) after half an hr post-treatment. Then the concentration of tramadol begins to decrease to a level 2.50 µg\ml at 24 hr post treatment.
Effects of tramadol on Brain
1. ATPase activity
Tramadol effects on the activity levels of ATPase in Mitochondrial Fractions of rat brain areas.
2. Superoxide dismutase (SOD)
The levels of SOD were found to be significantly higher in treated group as compared to control group (P>0.05) this indicates that Tramadol alone produces a significant increase of SOD in brain
3. Reduced GSH content
The levels of GSH content were found to be significantly higher in treated group as compared to control group (P<0.05) and it was recovered at 24 hr as compared to control group.
Our study favours the hypothesis as reduced Glutathione (GSH) decline in tramadol treated rats can lead to enhanced H2O2 production, stimulation of lipid peroxidation, nitric oxide and protein oxidation.
4. Acetylcholinesterase
Acetylcholine esterase (AChE) activity in the brain increased after the administration of tramadol.
Neurotransmitters
1. Serotonin
Tramadol administration significantly increased the 5-HT levels in the brain examined in the present study.
2. Norepinephrine
The levels of NE were found to be significantly higher in treated group as compared to control group (P>0.05) .
3. Epinephrine
Highest level of EP was recorded in the brain, with maximal elevation at 3.5 hours hr after tramadol administration.
4. Dopamine
Dopamine (DA) activity in the brain increased after the administration of tramadol, revealed a significant increase in the mean concentration of DA activities.
Effects of tramadol on Kidney Activity
1. Urea
Revealed a significant increase in the mean of Urea concentration in serum.
2. Serum Creatinine
Revealed a significant increase in the mean of Serum creatinine concentration.
Effects of tramadol on Liver Enzymes (ALT and AST activity)
There was a significantly increase in ALT and AST. This implies that the drug affects liver of the animals.
Brain
The lesions in the brain of the treated group were gliosis, perineuronal vacuolation, perivascular vacuolation, and neuronal degeneration, discontinuity and shrinkage of the Purkinje cells in the brain and congestion in the meninges apart from the above-mentioned lesions.
Liver
Light microscopy revealed severe centrolobular congestion and focal necrosis in the liver of tramadol groups. The main histopathologic finding was vacuolization in tubular cells in tramadol groups. Our findings pointed out the risk of increased lipid peroxidation, hepatic and renal damage due to use of opioids.
Kidney
Histopathological examination of the kidney showed a normal histological structure in control rats. Glomerular swelling and vacuolization in the lining endothelium In tramadol-treated groups, the main histopathological findings in the kidney samples were vacuolization, swelling of endothelial cells and association with degeneration in cells lining the tubule cortex. Congestion in the tuft of the glomeruli at the cortex), focal degeneration with cystic dilatation and renal cast formation in some tubules of the corticomedullary portion were observed.
ABAMECTIN
Abamectin concentration in blood plasma
The levels of tramadol in blood plasma of treated rats were: 0.04 ± 0.0015, 0.062 ± 0.0016, 0.0715 ± 0.0015, 0.1572 ± 0.001, 0.166 ± 0.0092, 1.104 ± 0.024 and 0.04±0.002µg\ml at 0.5, 1, 1.5, 2, 2.5, 3.3.5 and 24 hrs intervals, respectively. We noticed that, highest concentration of abamectin (4.814 µg\ml) after half an hr of the treatment. Then the concentration of tramadol begins to decrease (1.104 ± 0.024 µg\ml) at 3.5 hrs post treatment.
Abamectin in faecal matter
The levels of abamectin in excreted faecal matter of treated rats were 0.0, 0.177 ± 0.003, 0.645±0. 004, 0.373 ± 0.002, 0.382 ± 0.003, 0.0, 0.0 and 0.0µg\g at 0.5, 1, 1.5, 2, 2.5, 3.3.5 and 24 hrs intervals, respectively.
It is observed that, the excretion of abamectin in the faecal matter was started after an hr post-treatment. Then the concentration of abamectin stopped completely (0.0 µg\ml) after 3 hrs post treatment.
Effects of abamectin on Brain
1. ATPase activity
A significant reduction in the ATPase activity of rat brain mitochondria was observed after 1 - 24h of ABA treatment
2. Reduced GSH content
A significant reduction in the GSH content of rat brain mitochondria was observed after 1 - 24h of ABA treatment
3. Superoxide dismutase (SOD)
The levels of SOD were found to be significantly higher in treated group as compared to control group (P>0.05)
5. Acetylcholinesterase
A significant decrease in the mean of acetylcholine esterase activities.
A highest reduction in the AChE activity of rat brain was observed after 3hr of ABA treatment.
Effects of abamectin on Brain Neurotransmitters
1. Serotonin
Abamectin administration significantly decreased 5-HT levels in the brain examined in the present study. It was not recovered as compared to control group (2.29 μg of 5-HT/g wet wt of tissue).
2. Norepinephrine
The levels of NE were found to be significantly lower in treated group as compared to control group (P<0.05)
3. Epinephrine
The levels of epinephrine were found to be significantly lower in treated group as compared to control group (P<0.05. In the brain the highest reduction of E was recorded at 3.5 hrs. Following this, E level is not reverted back towards the respective control levels at 24 hours.
4. Dopamine
The levels of DA were found to be significantly lower in treated group as compared to control group (P<0.05).
Effects of abamectin on Kidney
1. Urea
The levels of urea concentration in serum were found to be significantly higher in treated group as compared to control group (P<0.05) .
2. Serum Creatinine
Highest levels of Serum creatinine were recorded with maximal elevation at 3.5 hr (at 1.5-24 hrs).
Effects of abamectin on Liver Enzymes (ALT and AST activity)
ALT and AST values showed significantly (p<0.05) increase. This implies that the drug affects liver of the animals.
Histological results
Brain
The lesions in the brain of the treated group were gliosis, perineuronal vacuolation, perivascular vacuolation, and neuronal degeneration.
The severity revealed discontinuity and shrinkage of the Purkinje cells in the cerebellum and congestion in the meninges.
Liver
Histopathological changes were more intense in rats treated with abamectin than those in control. In conclusion, the results of this study demonstrate that acute- oral administration of abamectin altered some biochemical parameters which correlated with histopathological changes. The liver of control group revealed normal hepatocytes arranged in cords which are separated from each other by sinusoids. Kupffer cells are also present along the sinusoidal spaces.
Kidney
Varying degrees of degeneration extending to necrosis was observed in the tubular epithelial cells of animals of all treatment groups. The findings were congestion and diffuse atrophy of the glomeruli, multiple haemorrhages in both the cortical and medullary tubules, besides the interstitium, infiltration of mononuclear cells between proximal tubules and around blood vessels and vacuolation of cytoplasm and rarefaction in the parenchyma of tubular epithelial cells of the kidneys, along with intense congestion.
The interaction of Tramadol + Abamectin in combination in male Rats
Blood and Fecal matter
• The interaction between Tramadol and Abamectin in blood plasma
We noticed that, the levels of tramadol and ABM were decreased in blood and the tramadol concentration was maintained to constant levels (2.9 µg\ml) during all the intervals of measurement.
• The interaction between Tramadol and ABM in fecal matter
We noticed that, the levels of tramadol excretion in fecal matter were significantly (p>0.05) decreased while there was excretion for ABM in fecal matter during all the intervals of measurement.
Brain Activities
1. ATPase activity
This indicates that Tramadol +ABM produce a significant reduction of ATPase in brain than that alone.
2. SOD content
This indicates that Tramadol alone produces a significant increase of SOD in brain relatively higher than ABM alone and in combination with Tramadol in brain
3. GSH content
This indicates that Tramadol alone produces a significant increase of GSH in brain higher than ABM alone and in combination with Tramadol in brain.
4. AchE of brain
This indicates that Tramadol alone produces a significant increase of AChE in brain higher than that of ABM alone and in combination with Tramadol in brain.
Brain Neurotransmitters
1. Serotonin in brain
This indicates that ABM and Tramadol-+Abamectin in combination produce the same significant reduction of Serotonin in brain .
Nor-Epinephrine
2. Norepinephrine in brain
This indicates that Tramadol and Tramadol +ABM produce the same significant increasing of NE of brain tissue.
3. Epinephrine
This indicates that Tramadol not affected in induction of Epinephrine in brain when used in combination with ABM but it antagonizes the reduction effect of ABM.
4. Dopamine
This indicates that Tramadol and Tramadol +ABM produce the same significant increasing of DA of brain tissue.
Effects on Kidney
1. Serum Urea
The levels of Urea were found to be significantly higher in Tramadol + Abamectin treated group, but it was much higher in ABM-treated group (P>0.05).
2. Serum Creatinine.
The levels of Urea were found to be significantly higher in Tramadol+Abamectin treated group. This indicates that Abamectin and Tramadol+Abamectin produce a significant increasing of Creatinine in serum plasma of treated rats.
Effects on Serum ALT and AST
This indicates that Tramadol, Abamectin and Tramadol+Abamectin produce a significant increasing of ALT and AST in serum plasma of treated rats. Histopathlogical effect of Tramadol, Abamectin and their combination on liver, kidney and brain in male rats.
Effects on Histological Examination (Brain-Liver –Kidney)
Brain
Light microscope examination of hematoxylin and eosin (H&E) stained sections of the brain cerebellum of the Normal Control group rats revealed that the brain tissues were normal with no shrunken cells. On the other hand, stained with H&E stain in the tramadol + abamectin rats group showed that, there was a shrunken purkinje cell, some cells had pykinotic nuclei and majority of the cells of granular layer were non-nucleated, few of them had pykinotic nuclei.
Liver
Liver hepatocyte architecture was affected by in rats. The most striking histological findings in the liver were congestion, dilated central and portal veins, dilated hepatic sinusoids with collagen proliferation, Kupffer cell proliferation between hepatocytes and apoptosis and degeneration of hepatocytes.
Kidney
Histopathological examination of the kidney showed a normal histological structure in control rats of the normal set. In tramadol +abamectin treated groups, the main histopathological findings in the kidney samples were vacuolization, swelling of endothelial cells and association with degeneration in cells lining the tubule cortex. Congestion in the tuft of the glomeruli at the cortex, focal degeneration with cystic dilatation and renal cast formation in some tubules of the corticomedullary portion were observed. Tramadol + Abamectin exert together exert toxic effect via two distinct and synergistic mechanisms of actions. This may reflect the greater frequency of decreasing hepatic and renal functions.