Evaluation of cadmium induced cytotoxicity in osteosarcoma cell line

Cadmium exerts harmful effect on public health. Environmental and occupational exposure, are the two main ways for human exposure. It was also shown that even low levels of cadmium exposure can leave to renal and other organs failure. Exposure to cadmium is associated with various bone problems and diseases like bone loss, fragilization and osteoporosis. Moreover, it was reported that cadmium exposure may perturb calcium homeostasis, Vitamin D formation and interact directly on bone metabolism. Reactive oxygen species have an important function in bone resorption, so disruption of the oxidative balance in bone tissue may result in disorders in bone metabolism. Although the deleterious effects of cadmium in bone are known, the mechanisms of action in bone cell are still not completely understood. The aims of this research study were to evaluate cadmium role in promoting cellular alterations, namely inducing oxidative stress imbalance and ultimately apoptosis. To investigate cadmium effects the human osteoblast-like cell line MG-63 was used. Inhibition of cell viability by cadmium was assessed by MTT assay. Based on these results, the sublethal doses of cadmium, 20 and 50 μM were chosen, for further studies. Cells were exposed to those concentrations of cadmium chloride for two periods of time (24 and 48 hours). After that, cells were harvested and several parameters were evaluated. The cytotoxic parameters followed a comprehensive set of oxidative stress assays with enzymatic parameters, namely determination of the activity of superoxide dismutase, catalase glutathione reductase and total glutathione content. Moreover, cadmium-induced apoptosis was assessed with annexin V assay. We observed differences in all anti-oxidant enzymes, namely an increase, catalase activities with the increased of cadmium dose and a glutathione reductase activity inhibition and also depletion on glutathione content, which may result in imbalance in oxidative stress. Finally, for the doses and periods tested, cadmium induced cell apoptosis. In conclusion, cadmium chloride at 20 μM and 50 μM induced changes on human MG-63 cell line physiology, as it cause oxidative stress imbalance which lead to cell death by apoptosis. INTRODUCTION. 1 1. CADMIUM: GENERAL CONSIDERATIONS . 1 1.1. Cadmium as a Health Hazard . 2 1.1.1. Cadmium intake . 2 1.1.2. Cadmium and bone . 3 1.2. Cadmium and Cell Biology . 5 1.2.1. Cadmium as a carcinogen . 5 1.2.2. Cadmium and oxidative stress . 6 2. OXIDATIVE STRESS . 7 2.1. Free Radicals . 7 2.2. Anti-Oxidant Defenses . 9 2.2.1. Enzymatic defenses . 9 2.2.2. Non-enzymatic defenses . 10 2.2.3. Others defenses . 10 2.3. Oxidative Damage to Biomolecules . 11 2.3.1. DNA damage . 11 2.3. 2. Lipid peroxidation . 11 2.3. 3. Protein oxidation . 12 2.4. ROS and Cancer . 13 2.4. 1. Cadmium, ROS and apoptosis . 14 3. CADMIUM AND OSTEOBLAST METABOLISM . 14 3.1. Osteoblast in vitro Culture. 15 OBJECTIVES . 17 MATERIAL AND METHODS . 18 4. MATERIAL . 18 5. METHODS . 19 5.1. Cell Culture . 19 5.1.1. Harvesting cells . 19 5.1.2. Cell exposure . 19 5.2. Protein Quantification . 19 5.3. Cell Viability . 20 5.4. Enzymatic Parameters . 20 5.4. 1. Superoxide Dismutase . 21 5.4. 2. Catalase . 22 5.4. 3. Glutathione Reductase . 22 5.4. 4. Total Glutathione quantification . 23 5.5. Apoptose . 24 5.5.1. Annexin V assay . 24 5.6. Data and Statistical Analysis . 25 RESULTS . 26 6. GLOBAL SCREENING OF CELL CULTURES EXPOSED TO CADMIUM . 26 7. VIABILITY ASSAY . 28 8. ENZYMATIC ASSAYS . 29 8.1. Superoxide dismutase activity . 29 8.2. Catalase activity . 30 8.3. Glutathione Reductase . 30 8.4. Total glutathione content . 31 8.5. Apoptosis assay . 32 DISCUSSION . 33 CONCLUSIONS AND FUTURE PERSPECTIVES . 37 REFERENCES . 39