| BackgroundAflatoxicosis is considered as the most important health crisis in developing world which is produced by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus and affects growing poultry (especially ducklings and turkey poults), young pigs, pregnant sows, calves, and dogs. Adult cattle, sheep, and goats are comparatively resistant to the acute form of the disease but are prone if toxic diets fed over a long time periods.Selenium is a mineral found in a small magnitude that is essential to the diet and possesses many exceptional and novel nutritional, biochemical, and molecular biology properties that continue to make it an exciting target for nutritional research. Selenium contains antioxidative selenoprotein GPX1 which works against oxidative stress and make it as a significant micronutrient with antioxidative properties.Beta-carotene is the most powerful provitamin A carotenoid and has an ability to exert antioxidative properties. It could wield its antioxidant functions in lipid phases by reducing free radicals. It is an effective antioxidant agent which exerts antioxidant effect in lipid systems by quenching singlet oxygen, peroxyl radical and inhibits lipid peroxidationAim of studyThis thesis addresses two studies,First study was conducted to examine the potential of AFB1 to induce cell cytotoxicity and oxidative stress in MDCK cells and potential of selenium (SeMet and SS) to protect from AFB1 induced oxidative stress, and also compare the antioxidative potential of organic (SeMet) and inorganic (SS) selenium.Second study was conducted to investigate the potential of AFB1 to induce cell cytopathogenicity, oxidative stress and apoptosis in MDCK cells and potential of beta-carotene to protect from AFB1 induced oxidative stress and apoptosis.MethodsMDCK cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% serum and 1% antibiotics (10,000 IU/ml penicillin and 10,000 μg/ml streptomycin, Gibco) in 37℃ and humidified atmosphere. After confluent cells were detached by trypsinization and were re-suspended according to the requirements.In the first study the cells were cultured in 94 (For MTT),24 (LDH, GSH) and 6 (MDA, and GPX1, activity and mRNA) well plates.24 hours after plating the media were replaced with new media containing selenium (Either SeMet or SS) with different doses and cultured for 24 hours, then the media were changed again with new media containing 0.25μg/ml AFB1 (Dissolved in DMSO at the concentration of lmg/ml) and cultured for 48 hours. In the experiments with only selenium, after 24 hours media were changed with selenium and were grew for only 24 hours. The groups were divided as A (Control, cells without any stimulant) B (Toxin control,0.25μg/ml toxin) C (0.2μM selenium+0.25μg/ml toxin), D (0.8μM selenium+0.25μ g/ml toxin), E (1μM selenium+ 0.25μg/ml toxin), F (2μM selenium+0.25μg/ml toxin), G (4μM selenium+0.25μg/ml toxin), and H (8μM selenium+0.25μg/ml toxin). In the experiments only with selenium the concentrations of the selenium were the same but without toxin.In second study the cells were cultured in 94 (For MTT),24 (GSH) and 6 (MDA, caspase-3 activity and Bcl-2 mRNA) well plates. For cytopathological examination the cells were grew on the glass cover slips (pre-coated with poly-1-lysine). The groups were divided as A (Control, Cells without any stimulant) B (Toxin control,0.25μg/ml toxin), C (0.05μM Beta-carotene+0.25μg/ml toxin), D (0.1 μM Beta-carotene+0.25μg/ml toxin), E (0.2μM Beta-carotene+0.25μg/ml toxin), F (0.4μM Beta-carotene+0.25μg/ml toxin), and G (0.8μM Beta-carotene+0.25μg/ml toxin). In the experiments only with beta-carotene the concentrations of beta-carotene were the same but without toxin.After completion of culture time samples were prepared according to requirement of each test. Like for GSH, MDA, and GPX1 activity cell lysates were prepared by sonication, MTT were done spectrophotometrically on cells directly, and for extracellular ldh culture medium were collected. For gpxl and Bcl-2 mRNA cDNA was prepared according to protocol of kits.ResultsToxic effect of AFB1 on MDCK cells were both dose and time dependent, half maximal toxic concentration (IC50) was noted at 0.25 μg/ml of AFB1. Hence for future experiment 025μg/ml was used.The results from first study showed that 0.25μg/ml of AFB1 causes significant increase in oxidative stress, which was demonstrated by significant increase of malondialdehyde (MDA) level, reduction of intracellular GSH level, as well as GPX1 activity and mRNA level in MDCK cells when compared with control. SeMet protected the cells from AFB1-induced oxidative damage in a dose-dependant manner. Good protection could be achieved between 1 and 4 μM of concentration. Amid this range, MDA level significantly decreased while intracellular GSH level and GPX1 activity in addition to mRNA level significantly increased. Moreover, cell viability was significantly improved, but when SS was used it can be observed that only small concentration of SS could protect the cells against AFB1 induced damage, in higher concentrations SS can act as a pro-oxidant and the damage in the groups with higher concentration of SS were even higher then only toxin group.The results of second study showed that 0.25 μg/ml of AFB1 caused significant increase in oxidative stress, which was demonstrated by significant increase of malondialdehyde (MDA) level, reduction of intracellular GSH. As a result of oxidative stress cells undergoes apoptosis which was demonstrated by reduction in Bcl-2 mRNA and increase in caspase-3 activity (markers widely used for detection of apoptosis). Apoptotic changes in MDCK cells after AFB1 treatment were confirmed through cytopathological examination of the slides. Administration of AFB1 to the cells causes decrease in cell number and there is denaturation of protein and rupture of cell membraneFive concentration of beta-carotene were used against IC50 dose of AFB1. It was observed that beta-carotene administration causes significant protection against AFB1 induced oxidative stress and apoptosis. When cells were pre-treated with beta-carotene it was observed that MDA level and caspase-3 activity significantly decreased while intracellular GSH level in addition to mRNA level of Bcl-2 increased. Moreover, cell viability was significantly improved. Protective effect of beta-carotene against AFB1 can also be confirmed through cytopathological examination of slides.Conclusion1. Aflatoxin treatment causes alteration in oxidative stress parameters in MDCK cells.2. Selenium treatment can effectively protect from oxidative stress by decreasing MDA and increasing GSH level and GPX 1 activity in the cells.3. Organic selenium as SeMet is more powerful antioxidant as compared to inorganic selenium (SS) in averting oxidative stress.4. Care should be taken when use inorganic selenium because in higher concentration then physiologic limit it can be toxic.5. Oxidative stress as a result of AFB1 could promote apoptosis in MDCK cells6. Increase in caspase-3-activity and decrease in Bcl-2 mRNA is responsible of apoptosis caused by AFB17. Beta-carotene is a powerful antioxidant which can protect from AFB1 induced oxidative stress and apoptosis... |
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