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The main aim of the study was to compare growth, viability and stress in rat HepG2 myocardial cells when cultures in the presence or absence of extra iron. In aerobic cells, glucose is oxidized completely to CO2 and H2O in the presence of O2.Many highly proliferative cells generate almost all ATP via glycolysis despite abundant O2 and a normal complement of fully functional mitochondria, a circumstance known as the Crabtree effect. Such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, such as the inhibitors rotenone, antimycin, oligomycin, and compounds like carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone (FCCP), that uncouple the respiratory electron transfer system from phosphorylation. These cells are also resistant to the toxicity of many drugs whose deleterious side effect profiles are either caused, or exacerbated, by impairment of mitochondrial function. Drug-induced mitochondrial toxicity is shown by members of important drug classes, including the thiazolidinediones, statins, fibrates, antivirals, antibiotics, and anticancer agents. To increase detection of drug induced mitochondrial effects in a preclinical cell-based assay, HepG2 cells were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media. Oxygen consumption doubles in galactose-grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells. Some drugs were equally toxic to both glucose and galactose-grown cells, suggesting that mitochondrial impairment is likely secondary to other cytotoxic mechanisms.
Many drugs are variously injurious to the liver, cardiovascular system, skeletal muscles, nervous system, and kidneys, among others (Amacher, 2005; Chan et al., 2005; Cote et al., 2006; Malhi et al., 2006; Scatena et al., 2004). This toxicity is often idiosyncratic and frequently not discovered until a large population of patients has been exposed. Evidence is rapidly accumulating that such negative side effect profiles are attributable to varying extents to deleterious effects on mitochondrial function (Amacher, 2005; Brunmair et al., 2004; Chan et al., 2005; McKee et al., 2006; Wallace and Starkov, 2000).
In most mammalian cells, mitochondria generate almost all the energy, in the form of ATP, required for survival. Agents that undermine mitochondrial function will correspondingly impair cell viability and, depending on severity, lead to tissue or organ injury. Indeed, several drugs withdrawn from the market because of cytotoxicity to liver (Masubuchi et al., 2006; Ong et al., 2006; Scatena, 2004) and skeletal muscle (Schaefer et al., 2004) have now been found to impair mitochondrial function. Due to their physiologically relevant metabolic poise (at least under short-term culture conditions), primary cells in culture would be the ideal platform for evaluating potential mitochondrial drug toxicities. However, expense, the technical difficulties associated with isolation, and their brief survival in culture, combine to limit the utility of primary cells in the drug development arena.
For these reasons, tumor-derived, immortalized cell lines have become the mainstay of the cell-based assays used in drug discovery and development efforts. Such cell lines are metabolically adapted for rapid growth under hypoxic and acidic conditions, and they derive almost all of their energy from glycolysis rather than via mitochondrial oxidative phosphorylation (OXPHOS), the Crabtree effect (Rodrý´guez-Enrý´quez et al., 2001). This is the case despite the presence of functionally competent mitochondria, and is due to several factors, including allosteric modulation of glycolytic enzymes (Rodrý´guez-Enrý´quez et al., 2001) and binding of hexosekinase to mitochondrial porin (Golshani-Hebroni and Bessman, 1997). In such cells, mitochondrial toxicants have little effect on cell growth or viability, which correspondingly diminishes their utility as predictors of mitochondrial drug liabilities in vivo. Such resistance to perturbed mitochondrial function is exacerbated by contemporary tissue culture practice where cells are typically grown in 25mM glucose, more than fivefold the physiological levels. Despite such limitations, glycolytically poised cell lines have typically been the primary tool for assessment of mitochondrial toxicity.
HepG2 cells are resistant to mitochondrial toxicants due to high glycolytic capacity. Second, HepG2 cells grown in galactose media will increase respiration rates to maintain ATP levels (Warburg et al., 1967).
Oxidation of galactose to pyruvate via glycolysis yields no net ATP, forcing cells to rely on mitochondrial OXPHOS to generate sufficient ATP for survival (Rossignol et al., 2004). Third, aerobically poised cells oxidizing galactose will be correspondingly more susceptible to mitochondrial toxicants and drugs with known mitochondrial liabilities.
A disorder that can result in organ damage due to significant and excessive absorption and storage of iron is known and named as hemochromatosis. There are two forms. Primary hemochromatosis is genetic; an autosomal recessive disorder caused by a single site mutation in the HFE gene. Hereditary hemochromatosis is an inherited (genetic) disorder in which there is excessive accumulation of iron in the body (iron overload). It is a common genetic disorder among Caucasians, affecting approximately one in 240 to 300 Caucasians. Individuals affected with hereditary hemochromatosis may have no symptoms or signs (and have normal longevity), or they can have severe symptoms and signs of iron overload that include sexual dysfunction, heart failure, joint pains, liver cirrhosis , diabetes mellitus, fatigue, and darkening of skin.
The normal iron content of the body is three to four grams. The total amount of iron in the body is carefully controlled. The body loses one mg of iron daily from sweat and cells that are shed from the skin and the inner lining of the intestines. Women also lose one mg of iron daily on average from. In normal adults the intestines absorb one mg of iron daily from food to replace the lost iron, and therefore, there is no excess accumulation of iron in the body. When iron losses are greater, more iron is absorbed from food.
In individuals with hereditary hemochromatosis, the daily absorption of iron from the intestines is greater than the amount needed to replace losses. Since the normal body cannot increase iron excretion, the absorbed iron accumulates in the body. At this rate of iron accumulation, a man with hemochromatosis can accumulate 20 gram of total body iron by age 40 to 50. This excess iron deposits in the joints, liver, testicles, and heart, which causes damage to these organs, and causes signs and symptoms of hemochromatosis, this is exactly what will be investigated during the research that will take place in the laboratory, but perhaps the investigation will focus on how the excess iron deposits mainly in the liver. Its also relevant to mention that women with hemochromatosis accumulate iron at a slower rate than men because they lose more iron than men due to iron loss from menstruation and breastfeeding. Therefore, they typically develop signs and symptoms of organ damage due to excess iron 10 years later then men.
The other form of haemochromatosis is simply known as secondary haemochromatosis and it is caused by frequent transfusions of plasma or by an excess of iron in the diet. Iron from the newly-infused rbc's is deposited in the reticuloendothelial system in the liver, spleen, and bone marrow. This can eventually lead to organ failure (like cirrhosis of the liver), heart attack, cancer and pancreatic damage, which can lead to death. In patients who have received more than 40 units of blood, the reticuloendothelial system is typically saturated with iron (10 g), and additional iron deposits are seen in the parenchymal cells of the liver, pancreas, and heart. Iron chelation therapy is used in patients who receive large numbers of transfusions to remove excess iron and prevent organ damage. Patients with thalassemia have increased demand for iron in the bone marrow because of ineffective erythropoiesis. This results in increased absorption of iron. In patients without transfusions, the excess iron is deposited in hepatocytes, not in Kupffer cells. If patients are transfusion-dependent, they also may have abnormal iron deposition in the reticuloendothelial system. Men and women who have haemochromatosis and exhibit a thalassemia trait may be at further risk of fatal myocardial infarction.
There is growing evidence that normal or only mildly increased amounts of iron in the liver can be damaging, particularly when they are combined with other hepatotoxic factors such as alcohol, porphyrogenic drugs, or chronic viral hepatitis. Iron enhances the pathogenicity of microorganisms, adversely affects the function of macrophages and lymphocytes, and enhances fibrogenic pathways, all of which may increase hepatic injury due to iron itself or to iron and other factors. Iron may also be a co-carcinogen or promoter of hepatocellular carcinoma, even in patients without HC or cirrhosis.
The hypothesis that higher levels of iron in the cell increases the level of oxygen radicals generated within mammalian cells will be tested using several methods described in the following page. The aim of the work was to determine protein oxidation in liver cells.
Materials and Methods
Results obtained after measuring fluorescence in the FLUOstar Omega plate-reader (BMG LABTECH Ltd., Aylesbury, UK) with 485nm excitation and 530 nm emission.
Ros Assay -Galactose using 24well plate.(1st replicate)
Ros Assay - Glucose using 24 well plate. (1st replicate)
Ros Assay - Galactose using 24well plate (2nd replicate)
Ros assay - Glucose using 24well plate (2nd replicate)
Ros assay - Glucose using 24well plate (3rd replicate)
Ros assay - Galactose using 24well plate (3rd replicate)