ferric-ammonium-citrate and thiazolyl-blue

Iron overload can cause liver toxicity and increase the risk of liver failure or hepatocellular carcinoma in humans. Curcumin (diferuloylmethane), a component of the food spice turmeric, has antioxidant, iron binding and hepatoprotective properties. The aim of this study was to quantify its effects on iron overload and the resulting downstream toxic effects in cultured T51B rat liver epithelial cells.. T51B cells were loaded with ferric ammonium citrate (FAC) with or without the iron delivery agent 8-hydroxyquinoline. Cytotoxicity was measured by methylthiazolyldiphenyl-tetrazolium bromide assay. Iron uptake and iron bioavailability were documented by chemical assay, quench of calcein fluorescence and ferritin induction. Reactive oxygen species (ROS) were measured by a fluorescence assay using 2',7'-dichlorodihydrofluorescein diacetate. Oxidative stress signalling to jnk, c-jun and p38 was measured by a Western blot with phospho-specific antibodies.. Curcumin bound iron, but did not block iron uptake or bioavailability in T51B cells given FAC. However, it reduced cytotoxicity, blocked the generation of ROS and eliminated signalling to cellular stress pathways caused by iron. Inhibition was observed over a wide range of FAC concentrations (50-500 microM), with an apparent IC(50) in all cases between 5 and 10 microM curcumin. In contrast, desferoxamine blocked both iron uptake and toxic effects of iron at concentrations that depended on the FAC concentration. The effects of curcumin also differed from those of alpha-tocopherol, which did not bind iron and was less effective at blocking iron-stimulated ROS generation.. Curcumin reduced iron-dependent oxidative stress and iron toxicity in T51B cells without blocking iron uptake.

Topics: Animals; Cells, Cultured; Curcumin; Deferoxamine; Epithelial Cells; Ferric Compounds; Inhibitory Concentration 50; Liver; Oxidative Stress; Quaternary Ammonium Compounds; Rats; Reactive Oxygen Species; Signal Transduction; Tetrazolium Salts; Thiazoles

The reasons underlying the high iron content found in the substantia nigra (SN) of Parkinson's disease (PD) are largely unknown. We suppose, based on our previous studies, that the newly discovered iron transporter divalent metal transporter 1 (DMT1) might be involved in this SN iron accumulation process. To investigate this, we first observed the cellular expression of DMT1 in rat SN, both with the iron response element (+IRE) and without the IRE (-IRE) forms. The results showed that both forms of DMT1 were expressed on neurons, astrocytes, and microglia but not on oligodendrocytes. We further observed the relationship between the increased iron influx and DMT1 expression in 6-hydroxydopamine (6-OHDA)-treated C6 cells. 6-OHDA (10 micromol/liter) caused a significant increase in ferrous iron influx, with the increased expression of DMT1+IRE, both in protein and in mRNA levels, whereas no change was observed for DMT1-IRE. To clarify further that the increased expression of DMT1 was not due to the increased intracellular iron content, C6 cells were overloaded with ferric ammonium citrate (100 microg/ml). Decreased expression of both forms of DMT1 was observed. Our data suggest that DMT1 is highly expressed in rat SN in a cell-specific manner. Increased DMT1+IRE expression is the mechanism behind ferrous iron influx induced by 6-OHDA treatment in C6 cells. This may give some evidence for the involvement of DMT1 in the iron accumulation in PD.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Analysis of Variance; Animals; Cation Transport Proteins; CD11b Antigen; Cell Count; Cells, Cultured; Ferric Compounds; Fluoresceins; Glial Fibrillary Acidic Protein; Iron; Iron-Regulatory Proteins; Mice; Neuroglia; Neurons; Oxidopamine; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Substantia Nigra; Sympatholytics; Tetrazolium Salts; Thiazoles; Up-Regulation