![]() | Only 14 pages are availabe for public view |
Abstract Biochemical influence of alpha-lipoic acid on antioxidant enzymes in blood and renal tissue of lead toxicity induced oxidative stress in rats were investigated. Moreover, the possible protective effects of alpha lipoic acid on serum nitric oxide, proinflammatory cytokines (TNF-α, IL-6, IL1β), liver marker enzyme (ALT, AST), renal function tests (urea ,creatinine), erythrocytes and renal tissue antioxidant enzymes (CAT, SOD and GPX), L-MDA and GSH, in addition renal tissue DNA fragmentation , caspase 3 , NF-KBP65, 8-ohdg and Cox-2 concentrations of lead toxicity induced oxidative stress in rats were also evaluated. Eighty white male albino rats of 12-16 weeks old and weighting 220-250 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 laboratory conditions the rats were randomly divided into four equal groups, 20 rats each placed in individual cages and classified as follows: Group I (control normal group): Comprised 20 male rats, received no drugs, served as control non-treated for all experimental groups. Group II (Lead acetate exposed group): Included 20 male rats, received lead acetate 1/20 of L.D.50 orally and once per day over a period of 10 weeks (30 mg/kg. body weight). Group III (Lead acetate + Alpha-lipoic acid (treatment group)): Comprised 20 male rats, received lead acetate orally and daily at a dose level of (30 mg/kg. body weight) and treated daily with alpha-lipoic acid intraperitoneal at a dose level of (54 mg/kg. body weight). Group IV (alpha-lipoic acid treated normal group): Included 20 male rats, administrated daily with alpha-lipoic acid at a dose level of (54 mg/kg body weight/IP). Sampling: Random blood samples and tissue specimens (liver, kidney) were collected from all animals groups (control and experimental groups) two times along the duration of experiment at 8 and 10 weeks from the onset of treatment with lead and antioxidants. Blood samples were collected by ocular vein puncture from all animal groups and after overnight fasting. In screw capped heparnized tubes and plasma were separated by centrifugation at 2500 r.p.m for 15 minutes. After plasma separation, erythrocytes were washed 3 times with an equal volume of cold saline. 1ml RBC lyses with 4 ml distilled water in dry sterile caped tubes for subsequent biochemical analysis. All hemolysate samples were analyzed for the following parameters: L-Malondialdhyde (L-MDA), catalase, superoxide dismutase, Glutathione peroxidase and reduced Glutathione (GSH). The rest of blood samples were collected in dry, clean test tubes and allowed to clot for 30 minutes and serum was separated by centrifugation at 3000 r.p.m for 15 minute. The serum was separated by automatic pippte and received in dry srile tubes, processed directly for ALT and AST determination, then kept in a deep freeze at -20ْC until use for subsequent biochemical analysis. All sera were analyzed for the following parameters: NO, Creatinine, Urea, TNF-α, IL-6 and IL-1b. Tissue sampling: For biochemical analysis: After eight and ten weeks of treatment with alpha-lipoic acid the rats were sacrificed. Livers and kidneys were removed, rinsed in ice-cold 0.9% sodium chloride solution, quick frozen in a deep freeze at -20°C for subsequent biochemical analyses. Renal tissue preparation: Briefly, renal tissues were cut, weighed and minced into small pieces, homogenized with a glass homogenizer in 9 volume of ice-cold 0.05 mm potassium phosphate buffer (pH7.4) to make 10% homogenates. The homogenates were centrifuged at 6000 r.p.m for 15 minutes at 4°C then the resultant supernatant were used for the determination of the following parameters: L-malondialdehyde (L-MDA), antioxidant enzymes (Catalase, superoxide dismutase, Glutathione peroxidase), DNA fragmentation, Caspase 3, 8-ohdg, NF-KBP 65 and Cox-2 . For lead residue : Liver and kidney specimen were taken two times from each group of rats after had been sacrificed at 8 and 10 weeks from the onset of the experiment. The specimens were immediately removed and washed several times with saline and blotted between two damp filter papers, weighed and then processed for determination of lead residues concentrations. Histopathological finding: After ten weeks of treatment, Liver and kidney specimen of rats were carefully examined by naked eyes for detection of any abnormalities. The specimens were preserved in 10% buffered neutral formalin and subjected for microscopically examination. |