Hemochromatosis is the termed used to describe a state of iron overload in an individual. Our goal is to address the isolated issue of how iron- overload alters the function of well-differentiated hepatocytes in the absence of the other liver cell types. We have carried out preliminary studies to determine whether primary hepatocytes in long-term DMSO culture can be loaded with iron. We conclude from these studies: (1) Primary rat hepatocytes in long-term DMSO culture can be iron-induced by exposure to iron in the form of ferrous sulfate or (3,5,5-trimethylhexanoyl) ferrocene (TMH-ferrocene) but not with holotransferrin at the concentrations tested. (2) Because iron loading can be carried out over long time periods (months) in hepatocytes in DMSO it is possible to obtain iron loading using concentrations as low as 2.5 muM TMH-ferrocene. When exposed to 25muM TMH-ferrocene, hepatocytes continued to load increasing amounts for iron for two months before the cells died; when exposed to lower concentrations such as 2.5 or 5.0 muM TMH-ferrocene, hepatocytes were able to continuously load iron and remain viable for more than two months. (3) The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared to those exposed to ferrous sulfate; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosome. (4) Iron loading distorted nuclear shape in hepatocytes; the amount of nuclear distortion was greater in hepatocytes exposed to ferrous sulfate than in those exposed to TMH-ferrocene. (5) TMH-ferrocene produced a normal physiologic induction of ferritin. In summary, we have demonstrated that hepatocytes in long-term DMSO culture can be iron loaded and represent a flexible system for studying the effects of chronic iron loading on the cells. The hypothesis being tested in this proposal is: Iron loading of hepatocytes in long-term DMSO culture induces specific types of cellular changes may be potentiated is the cells are treated with cytokines. We will use iron over-loaded hepatocytes in long term DMSO culture: 1. To characterize the time- and concentration-dependent characteristics of TMH-ferrocene treatment with respect to cellular iron content, ferritin expression, IRE function, and markers of oxidative damage. 2. To characterize the mechanisms whereby alpha-tocopherol produces an increase in ferritin expression. 3. To assess the ability of our changes induced by iron overload are reversed by iron chelation. 4. To characterize the effects of chronic iron loading on the TNF-alpha signaling pathway with regard to NK-kappaB expression and induction of apoptosis.
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