Chronic iron overload in the development of liver fibrosis

Abstract

The studies described in this thesis investigate chronic iron overload and its relation to the development of hepatic fibrosis. Chapter 1 contains a survey of the literature relevant to studies of iron overload and hepatic fibrosis, and therefore the metabolism of iron in the normal liver is first described, followed by a description of hepatic iron overload in the human iron storage disease, genetic haemochromatosis, and in various animal models. One of the postulated mechanisms for specific hepatocellular injury resulting in fibrosis and cirrhosis in chronic iron overload is lipid peroxidation of organelle membranes, and therefore the process of lipid peroxidation, the role of iron in its initiation, and the methods used to detect it are described prior to the evidence for lipid peroxidation in chronic iron overload. Another postulated mechanism is that excess iron increases hepatic collagen production without the requirement for iron-induced cellular injury and so in the final part of Chapter 1, the structure and hepatic distribution of collagen are described, followed by a discussion of the regulation and detection of hepatic collagen deposition. The effect of chronic iron overload on hepatic fibrogenesis is also discussed. Chapter 2 provides a list of the materials used in the studies described in this thesis and describes some general methodology. Further details of the methods employed in each chapter are found in the Methods section of the same chapter. Chapter 3 describes the results of a preliminary study which examined the effect of cytokines produced by stimulated mononuclear cells on collagen synthesis. Cytokines were isolated from the medium of phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) by selective salt precipitation and gel filtration chromatography and their effect on collagen, total protein and DNA synthesis was tested using human fibroblast monolayers. The results from this preliminary study indicated that supernatants from PHA-stimulated PBMCs (PHAMCF) decreased collagen synthesis by dermal fibroblasts. The net synthesis of total protein and DNA was also decreased, however these decreases were less than those of collagen, indicating a more specific function of fibroblasts had been affected. The inhibition of collagen synthesis was first detected after 12 hours of incubation of PHA-MCF with dermal fibroblasts and this inhibitory effect was not greatly affected by different storage conditions (+4°C and -70°C). Preliminary characterization of the factors in PHA-MCF preparations was investigated using two types of gel filtration chromatography media (Sephacryl S-200 and Superose 6B), however the results were difficult to confirm as detectable amounts of the inhibitory factors after chromatography required large volumes of blood to be processed, which became difficult and unethical to obtain as a routine measure.