الفهرس 
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Abstract
Cadmium is a well-known human carcinogen and a potent nephrotoxic, the presented study was carried to evaluate the protective influences of alpha- lipoic acid and melatonin treatment alone and in combination against red blood cells cadmium toxicity induced oxidative stress in rats.
This study was carried out on One hundred white male albino rats, 12-16 weeks old and average body weight 160-200 gm were used in the experimental investigation of this study. Animals were housed in separate metal cages and kept at constant environmental and nutritional conditions throughout the period of experiment.
Design of the experimental work:
After acclimatization to the laboratory conditions the rats were randomly divided into five equal groups, each one consisting of (20) animals placed in individual cages, and classified as follows:
GroupI (control normal non-treated group): Rats received no drugs, served as control non-treated for all experimental groups.
Group II (Cadmium chloride exposed group): Rats, received cadmium chloride 1/20 of L.D.50 (4.4 mg/kg. body weight) orally and once per day over a period of 10 weeks.
Group III (Cadmium Chloride + Alpha-lipoic acid treated group):Rats, received cadmium chloride (4.4 mg/kg. body weight) and treated daily with Alpha-lipoic acid at a dose level of (54 mg/kg body weight intraperitoneal).
Group IV (Cadmium Chloride + Melatonin treated group):Rats, received cadmium chloride (4.4 mg/kg. body weight) and treated daily with melatonin at oral dose level of (10 mg/kg body weight orally).
Group V (Cadmium Chloride +Alpha-lipoic acid + melatonin treated group):Rats, received cadmium chloride (4.4 mg/kg. body weight) and treated daily with alpha-lipoic acid (54 mg/kg body weight i.p) in combined with melatonin (10 mg/kg body weight orally) for 10 weeks.
Sampling:
Random blood samples were collected from all animal groups (control and experimental groups) three times along the duration of experiment at 2, 4 and 10 weeks from the onset of the experiment. Also tissue specimens (liver and kidney) were taken from each rats groups at 4 and 10 weeks of the experiment from the onset of rats exposed to cadmium and administered with antioxidant compounds.
Blood samples
Blood samples were collected by ocular vein punctureat the end of each experimental period in dry, clean, and screw green caps heparinized tubes. All tubes were mixed gently, and then approximately (100 µl) of blood was isolated into separated epindorf tube, mixed gently and used directly for determination of hemoglobin concentration and G-6-p dehydrogenase activity. While the rest of samples were used for plasma separation by centrifugation at 3500 r.p.m. for 15 minutes and then collected indry sterile caped tubesusing automatic micropipette. All plasma samples were analysed for determination of:
1- Aspartate aminotransferase (AST)
2- Alanine aminotransferase (ALT)
3- Gamma-glutamyl transferase (γ - GT)
4- Urea
5- Creatinine
6- Total cholesterol
7- Phospholipids
• Moreover, after plasma separation, erythrocytes were washedfor preparation of erythrocyte lysate.
Preparation of erythrocyte lysate:
Erythrocytes were separated from blood plasma by centrifugation at 3500 rpm for 15 minutes, and then washed three times with a cold isotonic saline solution (0.9%NaCl).The supernatant and the buffy coat were carefully removed after each wash. One ml of RBCs was lysed with 4 ml distilled water in dry sterile caped tubes, and kept in a deep freeze at -20°C until used for determination of the following biochemical parameters:
1. L-Malondialdehyde (L-MDA)
2. Catalase
3. Superoxide dismutase
4. Glutathione s-transferase
5. Reduced Glutathione (GSH).
Tissue specimen (Liver and Kidney):
a) For Cadmium residue determination:
Liver and kidney specimen were taken from each group of rats two times after had been sacrificed at 4 and 10 weeks from the onset of the experiment. The specimens were quickly removed and washed several times with saline and blotted between two damp filter papers, weighed and then processed for determination of cadmium residues concentrations.
b) For Histopathological examination:
After ten weeks of treatment, Liver and kidney specimen of rats were carefully examined by naked eyes for detection of any abnormalities. Small specimen of each organ were taken and immediately fixed in 10% neutral buffered formalin solution for histopathological examination according to Dray and Walling, (1976).
Preparation of the samples:
Samples were fixed in 10 % neutral formalin for twenty four hours; and then washed by running water over night. The washed samples were dehydrated by using graded ascending concentrations of ethyl alcohol starting with 50% and ending with absolute alcohol. The dehydrated samples were cleared in xylol for 6 hours.
The samples were placed in a crucible containing soft paraffin and kept in an oven at 56°C for 12 hours. The samples were then blocked in hard paraffin and cut into sections of 5 microns in thickness.
Procedure of staining:
Paraffin was removed from the sections by two changes of absolute alcohol (five minutes in each) which was removed by washing with tap water. Sections were stained with Harris haematoxylin and eosin for 10 minutes, and then washed with running water for 15 minutes for general histological examination. The sections were dehydrated with two changes of absolute alcohol (five minutes in each), then they were cleared with xylol. They were mounted with Canada balsam and covered with cover slides to be ready for microscopical examination.
Histological changes in the stained sections were viewed under the light microscope by an expert pathologist without prior knowledge of the groups; Digital photos were taken to demonstrate the histological changes between groups after different treatments.
The obtained results summarized as the followings:
1- Plasma alanine aminotransferase (ALT):
Cadmium exposed rats showed significant increase in plasma (ALT) activity allover the periods of the experiment when compared with normal control group.
The intraperitoneal administration of Alpha-lipoic acid in cadmium intoxicated male rats caused a significant decrease in plasma (ALT) activity allover the periods of the experiment. Also, oral Melatonin treatment caused a significant decrease in plasma (ALT) activity allover the periods of the experiment when compared with cadmium exposed group.
Treatment with alpha-lipoic acid and Melatonin combination in cadmium intoxicated rats caused a significant decrease in plasma (ALT) activity allover the periods of the experiment when compared with cadmium exposed group.
2- Plasma aspartate aminotransferase (AST):
Cadmium exposed rats showed a significant increase in plasma (AST) activity allover the periods of the experiment when compared with normal control group.
Administration of alpha-lipoic acid in cadmium intoxicated male rats resulted in a significant decrease in plasma (AST) activity after two and ten weeks, and non-significantly decreased after four weeks. However, oral melatonin administration showed a significant decrease in plasma (AST) activity allover the periods of the experiment when compared with cadmium exposed group.
Co-adminstration of Alpha-lipoic acid and Melatonin treatment caused a significant decrease in plasma (AST) activity allover the periods of the experiment when compared with cadmium intoxicated group.
3- Plasma Gamma-glutamyl transferase (γ - GT):
A significant increase in plasma Gamma-glutamyl transferase (γ - GT) activity was observed in cadmium exposed rats allover the periods of the experiment when compared with normal control group.
Administration of alpha-lipoic acid in cadmium intoxicated male rats showed a non-significant decrease in plasma (γ - GT) activity after two and four weeks, followed by a significant decrease after ten weeks. However, oral melatonin administration showed a significant decrease in plasma (γ - GT) activity allover the periods of the experiment, when compared with cadmium exposed group.
Treatment with alpha-lipoic acid and melatonin combination in cadmium intoxicated rats showed a significant decrease in plasma (γ - GT) activity allover the periods of the experiment when compared with cadmium exposed group.
4- Plasma urea:
Cadmium intoxicated rats showed a significant increase in plasma urea concentration allover the periods of the experiment when compared with normal control group.
Alpha-lipoic acid administration in cadmium intoxicated male rats exhibited a non-significant decrease in plasma urea concentration after two weeks, and this decrease became significant after four weeks, followed by a non-significant increase after ten weeks. However, melatonin treatment in cadmium exposed rats caused a significant decrease in plasma urea concentration allover the periods of the experiment when compared with cadmium intoxicated group.
Combination of alpha-lipoic acid and melatonin treatment in cadmium intoxicated rats showed a significant decrease in plasma urea concentration allover the periods of the experiment when compared with cadmium exposed group.
5- Plasma creatinine:
In cadmium exposed rats a significant increase in plasma creatinine concentration was observed allover the experimental periods when compared with normal control group.
Intraperitoneal administration of alpha-lipoic acid to cadmium intoxicated male rats caused a non-significant decrease in plasma creatinine concentration after two weeks, this decrease became a significant after four and ten weeks. Also, melatonin treatment in cadmium exposed rats caused a non-significant decrease in plasma creatinine concentration after two weeks, followed by a significant decrease after four and ten weeks when compared with cadmium exposed group.
Alpha-lipoic acid and Melatonin combination treatment to cadmium exposed rats showed a non-significant decrease in plasma creatinine concentration after two weeks, followed by a significant decrease after four and ten weeks, when compared with cadmium intoxicated non treated group.
6- Plasma total cholesterol:
A non-significant increase in plasma total cholesterol concentration was observed in cadmium exposed rats after two weeks, and this decrease became significant increase after four and ten weeks when compared with normal control group.
Administration of alpha-lipoic acid in cadmium intoxicated rats exhibited a non-significant decrease in plasma total cholesterol concentration after two weeks, followed by a significant decrease after four and ten weeks. Also, Melatonin treatment to cadmium exposed rats caused a non-significant decrease in plasma total cholesterol concentration after two weeks, followed by a significant decrease after four and ten weeks when compared with cadmium exposed group.
Co-treatment with alpha-lipoic acid and Melatonin administration to cadmium exposed rats resulted in a significant decrease in plasma total cholesterol concentration allover the periods of the experiment when compared with cadmium treated group.
7- Plasma phospholipids:
Cadmium exposed rats showed a significant increase in plasma phospholipids concentration allover the periods of the experiment when compared with normal control group.
Intraperitoneal administration of alpha-lipoic acid in cadmium intoxicated male rats showed a significant decrease in plasma phospholipids concentration allover the periods of the experiment. Also, oral Melatonin treatment in cadmium intoxicated rats caused a significant decrease in plasma phospholipids concentration allover the periods of the experiment when compared with cadmium exposed group.
Treatment with alpha-lipoic acid and melatonin combination to cadmium intoxicated rats resulted in a significant decrease in plasma phospholipids concentration allover the periods of the experiment when compared with cadmium treated group.
8- Blood Haemoglobin (Hb):
Cadmium exposed rats showed a significant decrease in blood haemoglobin concentration allover the experimental periods when compared with normal control group.
Treatment with alpha-lipoic acid in cadmium intoxicated rats exhibited a significant increase in blood haemoglobin concentration. Also, melatonin treatment in cadmium intoxicated rats showed a significant increase in blood haemoglobin concentration allover the periods of the experiment, when compared with cadmium treated group.
The treatment with alpha-lipoic acid and melatonin combination in cadmium intoxicated rats exhibited a significant increase in blood haemoglobin concentration allover the periods of the experiment, when compared with cadmium exposed group.
9- Erythrocyte L-Malondialdehyde (L-MDA):
In cadmium exposed rats a significant increase in erythrocyte (L-MDA) concentration was observed allover the periods of the experiment when compared with normal control group.
Intraperitoneal administration of alpha-lipoic acid in cadmium intoxicated male rats showed a significant decrease in Erythrocyte (L-MDA) concentration. Also, Melatonin treatment to cadmium exposed rats resulted in a significant decrease in Erythrocyte (L-MDA) concentration allover the periods of the experiment when compared with cadmium exposed group.
Moreover, Alpha-lipoic acid and Melatonin combination treatment to cadmium exposed rats caused a significant decrease in Erythrocyte (L-MDA) level allover the periods of the experiment when compared with cadmium exposed group.
10- Erythrocyte reduced glutathione (GSH):
A significant decrease in erythrocyte reduced glutathione (GSH) concentration was observed in cadmium exposed rats allover the period of the experiment when compared with normal control group.
The treatment with alpha-lipoic acid in cadmium intoxicated male rats resulted in a significant increase in erythrocyte (GSH) concentration. Also, Melatonin treatment in cadmium exposed rats exhibited a significant increase in erythrocyte (GSH) concentration allover the period of the experiment when compared with cadmium exposed group.
Co-administration of alpha-lipoic acid and melatonin treatment to cadmium exposed rats exhibited a significant increase in erythrocyte (GSH) concentration allover the period of the experiment when compared with cadmium exposed non treated group.
11- Erythrocyte catalase (CAT):
Cadmium exposed rats showed a significant decrease in erythrocyte (CAT) activity allover the periods of the experiment when compared with normal control group.
The administration of alpha-lipoic acid in cadmium intoxicated male rats showed a significant increase in erythrocyte (CAT) activity after two and four weeks, followed by a non significant decrease after ten weeks. Also, melatonin treatment resulted in a significant increase in erythrocyte (CAT) activity after two and four weeks, followed by a non significant decrease after ten weeks when compared with cadmium exposed group.
Treatment with alpha-lipoic acid and melatonin combination in cadmium exposed rats showed a significant increase in erythrocyte (CAT) activity after two and four weeks, followed by a non significant increase after ten weeks when compared with cadmium exposed group.
12- Erythrocyte Superoxide dismutase (SOD):
In cadmium exposed rats a significant increase in erythrocyte (SOD) activity was observed after two and ten weeks and non-significantly decreased after four weeks, when compared with normal control group.
Alpha-lipoic acid administration in cadmium intoxicated male rats exhibited a significant decrease in erythrocyte (SOD) activity allover the period of the experiment. However, melatonin treatment in cadmium intoxicated rats showed a non-significant increase in (SOD) activity after two weeks. However a non-significant decrease in SOD activity was observed after four weeks, and this decrease became significant after ten weeks when compared with cadmium exposed group.
Alpha-lipoic acid and Melatonin combination treatment resulted in a non-significant increase in erythrocyte (SOD) activity after two weeks and significant decrease after four weeks. This decrease became non-significant after ten weeks when compared with cadmium exposed group.
13- Erythrocyte Glutathione s-transferase (GST):
A significant decrease in erythrocyte GST activity was observed in cadmium exposed rats allover the period of the experiment when compared with normal control group.
Administration of alpha-lipoic acid in cadmium intoxicated rats exhibited significant increase in erythrocyte GST activity after two and four weeks, and this decrease became non-significant after ten weeks. However, melatonin administration caused a significant increase in erythrocyte GST activity allover the period of the experiment when compared with cadmium exposed group.
Co-adminstration of alpha-lipoic acid and melatonin treatment in cadmium exposed rats caused a significant increase in erythrocyte GST activity allover the period of the experiment when compared with cadmium intoxicated group.
14- Blood Glucose-6-Phosphate Dehydrogenase (G-6-PDH):
A significant decrease in blood Glucose-6-Phosphate Dehydrogenase activity (G-6-PDH) was observed in cadmium exposed rats after ten weeks of the experiment when compared with normal control group.
Alpha-lipoic acid administration in cadmium intoxicated rats caused a non-significant increase in blood (G-6-PDH) activity after ten weeks. However, oral administration of melatonin treatment resulted in a non-significant increase in (G-6-PDH) activity after ten weeks when compared with cadmium exposed group.
Treatment with alpha-lipoic acid and melatonin in combination in cadmium exposed rats exhibited a significant increase in blood (G-6-PDH) activity after ten weeks when compared with cadmium intoxicated group.
15- Cadmium residues in liver and kidney:
Kidney cadmium:
A significant increase in cadmium concentration was observed in kidney tissue of cadmium exposed rats along the periods of the experiment when compared with control group.
A significant decrease in kidney cadmium concentration was observed in cadmium exposed rats administrated with alpha-lipoic acid allover the experiment periods. Also, melatonin treatment in cadmium exposed rats caused a significant decrease in kidney tissue cadmium concentration after four and ten weeks in comparison with cadmium intoxicated group.
Treatment of cadmium exposed rats with alpha-lipoic acid and melatonin in combination caused a significant decrease in kidney cadmium concentration along the periods of the experiment when compared with cadmium exposed group.
Liver cadmium:
A significant increase in liver cadmium concentration was observed in cadmium exposed rats allover the periods of the experiment when compared with normal control group.
However, alpha-lipoic acid administration in cadmium intoxicated male rats caused a significant decrease in cadmium concentration in liver tissue. Also, Melatonin treatment in cadmium exposed rats caused a significant decrease in liver cadmium concentration after four and ten weeks of the experiment when compared with cadmium exposed group.
A significant decrease in liver cadmium concentration was observed in cadmium exposed rats treated with alpha-lipoic acid and melatonin in combination along the periods of experiment in comparison with cadmium intoxicated group.
16- Histopathological findings
1)Group I: (Control normal group).
Normal liver and kidney was seen.
2) Group II: (Cadmium chloride exposed group )
Kidney:
The microscopical examination of the kidney revealed severe degenerative changes in the renal tubules which manifested by degeneration of the lining epithelium with cystic dilation of the renal tubules which become showing a wide lumen and lined by flattened epithelium. Some section showing diffuse glomerulonephritis also seen. The renal glomeruli showing severe thickening of its Bowman capsule with peri-golumerular mononuclear infiltration. Focal necrotic areas were seen scattered in the renal medulla. The renal tubules were completely destructed and showing loss of the lining epithelium with the presence of hyalinized eosinophilic cast in their lumina.
Liver:
The microscopical examination of the liver revealed severe congestion and dilatation of hepatic sinusoids and portal blood vessels. Moreover, some sections revealed the presence of focal areas of coagulative necrosis which taken granular eosinophilic and basophilic coloration and infiltrated with mononuclear leukocyte.
3) Group III: Rats exposed to (cadmium chloride + Alpha-lipoic acid group).
Kidney:
The kidney of rats in this group showing severe destructive changes and the renal tubules showing complete desquamation of their lining epithelium with the presence of focal areas of dystrophic calcification which taken dark bluish coloration by H&E particularly in the renal medulla. Moreover, focal mononuclear aggregation and infiltration were also seen.
The renal medulla showing the presence of eosinophilic depris in the degenerated tubules.
Liver:
The liver of rats in this group was showing severe congestion and dilatation of the central vein and sinusoids. Moreover the hepatocytes were suffered from degenerative changes in the form of the vacuolar degeneration of the cytoplasm of the hepatocyts.
4) Group IV: Rats exposed to (cadmium chloride + melatonin treatment group).
Kidney:
The kidney of rats in this group showing degenerative changes in the glomerular tuft with shrinkage or complete destruction of some of them. Cystic dilation and desquamation of the renal epithelium in cortex were also seen. Some sections showing coagulative necrosis of the renal tubules in which the renal tubules showing homogenous eosinophilic cytoplasm and pyknoyic nuclei.
Liver:
The liver of rats in this group showing congestion and dilatation of the central vein with mild degenerative changes of the hepatocytes which suffering from hydropic degeneration.
5) Group V: Rats exposed to (cadmium chloride + alpha-lipoic acid + melatonin treatment group).
Kidney:
Microscopically the kidney of rats in this group is showing mild degeneration changes in the form of mild desquamation of the epithelial cells lining the tubules.
Liver:
The liver of rats in this group is showing mild degenerative changes in the form of hydropic degeneration of hepatocyts.