Studies on genetic hemochromatosis and the hepatotoxicity of iron

The aim of this study was to investigate the mutations and clinical expression of genetic hemochromatosis in patients and mechanisms involved in the hepatotoxicity of iron in animal and cell- culture models.

In Swedish patients with a clinical diagnosis of genetic hemochromatosis, the C282Y mutation of the HFE gene was present in 94.2% of chromosomes, and 92.0% of the patients were homozygous. It is a well-established fact that the clinical expression of the disease varies. In patients, we concluded that the amount of body iron stores correlates negatively with CD8+, a subpopulation of T-lymphocytes, both in peripheral blood and liver lobuli, and that the lowest counts are seen in patients with cirrhosis. Thus, apart from genotype, other factors influence the expression of the disease.

For studies on iron toxicity, an experimental model with rats fed a carbonyl-iron-enriched diet resulting in hepatic iron overload was used. In rats fed diets with carbonyl iron followed by ethanol, a synergistic effect on liver toxicity was seen. When treating rats with desferrioxamine, serum alanine transferases decreased, showing that the toxicity was dependant on chelatable iron. The synergistic effect was not caused by depleted liver glutathione levels, nor by differences in induction of cytochrome P450 2E1.

As the extracellular matrix undergoes changes in the development of liver fibrosis with deposition of collagen fibrils, we studied the role of the extracellular matrix in iron loading, using primary cultures of hepatocytes from rats fed a diet with carbonyl iron. We compared hepatocytes plated on a laminin- rich extracellular matrix with hepatocytes plated on collagen type 1. The rate of lipid peroxidation in hepatocytes from iron-loaded rats was higher in cells plated on collagen and when exposed to oxidative stress. When comparing hepatocytes from iron-loaded rats with hepatocytes from normal rats, cells from iron-loaded rats were more susceptible to oxidative stress. As lipid peroxidation diminished when iron was chelated, we concluded that the combination of iron overload and collagen matrix leads to an increased susceptibility to oxidative stress.

Since signs of microinflammation are seen in iron-overloaded livers, the effects of tumour necrosis factor-ct on hepatocytes in culture were studied. Tumour necrosis factor-alpha increased the synthesis of ferritin H, decreased the pool of chelatable iron and diminished the susceptibility to a hydroperoxide. This was true in cells from rats with a normal iron load, but not in cells from iron- loaded rats. The cytokine also induced manganese-superoxide dismutase transcription in both cell types.

In livers from patients with genetic hemochromatosis, expression of the collagen type I gene and staining for ct-smooth muscle actin was seen as evidence of hepatic stellate cell activation. When examining a second liver biopsy after treatment with venesection, a reversal of fibrosis was observed. The strongest collagen gene expression was observed in livers from patients investigated shortly after treatment completion.

List of scientific papers

I. Cardoso EM, Stal P, Hagen K, Cabeda JM, Esin S, de Sousa M, Hultcrantz R (1998). "HFE mutations in patients with hereditary haemochromatosis in Sweden. " J Intern Med 243(3): 203-8
https://pubmed.ncbi.nlm.nih.gov/9627157

II. Cardoso EM, Hagen K, de Sousa M, Hultcrantz R (2001). "Hepatic damage in C282Y homozygotes relates to low numbers of CD8+ cells in the liver lobuli. " Eur J Clin Invest 31(1): 45-53
https://pubmed.ncbi.nlm.nih.gov/11168438

III. Stal P, Johansson I, Ingelman-Sundberg M, Hagen K, Hultcrantz R (1996). "Hepatotoxicity induced by iron overload and alcohol. Studies on the role of chelatable iron, cytochrome P450 2E1 and lipid peroxidation. " J Hepatol 25(4): 538-46
https://pubmed.ncbi.nlm.nih.gov/8912154

IV. Hagen K, Zhu C, Melefors O, Hultcrantz R (1999). "Susceptibility of cultured rat hepatocytes to oxidative stress by peroxides and iron. The extracellular matrix affects the toxicity of tert-butyl hydroperoxide. " Int J Biochem Cell Biol 31(3-4): 499-508
https://pubmed.ncbi.nlm.nih.gov/10224673

V. Hagen K, Eckes K, Melefors O, Hultcrantz R (2002). "Iron overload decreases the protective effect of tumor necrosis factor-alpha on rat hepatocytes exposed to oxidative stress." Scand J Gastroenterol (In Print)

VI. Hagen K, Garuti C, Montosi G, Stal P, Lindholm J, Hultcrantz R, Pietrangelo A (2002). "Effects of iron removal on fibrosis in genetic hemochromatosis." (Manuscript)