Toxicology 219 (2006) 41–52 Hepatocellular carcinoma caused by iron overload: A possible mechanism of direct hepatocarcinogenicity George A. Asare a , Kensese S. Mossanda b , Michael C. Kew a,∗ , Alan C. Paterson c , Christina P. Kahler-Venter d , Kwanele Siziba e a MRC/University Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa b Department of Chemical Pathology, Medical University of South Africa, Pretoria, South Africa c School of Pathology, Department of Anatomical Pathology, University of the Witwatersrand and National Health Services Laboratories, Johannesburg, South Africa d Department of Pharmacology, Medical University of South Africa, Pretoria, South Africa e National Centre for Occupational Health, Johannesburg, South Africa Received 24 August 2005; received in revised form 1 November 2005; accepted 1 November 2005 Available online 6 December 2005 Abstract Background/aims: Excess hepatic iron may be both directly and indirectly carcinogenic. The aim of this study was to determine if generation of reactive oxygen species and the resulting oxidative damage induced by free hepatic iron is directly hepatocarcinogenic. Methods: Sixty male Wistar albino rats were iron-loaded by ferrocene supplementation of their diet. Biochemical parameters of oxidative damage and lipid peroxidation, DNA unwinding and strand breaks, and the Ames Mutagenesis Test were measured at 4 monthly intervals and correlated with the degree of hepatic iron overload, the presence of iron-free preneoplastic foci in the liver, and the development of hepatocellular carcinoma in comparison with 60 control rats. Results: Levels of lipid hydroperoxides, malonaldehyde, 8-isoprostane and 8-hydroxy-2 ′ -deoxyguanosine increased, reaching peak concentrations at 20–24 months, and correlating with an increase in the rate of DNA unwinding, strand breaks, and positive Ames Tests. Iron-free neoplastic foci became evident at 16 months and thereafter increased in number. Preneoplastic foci were present in five of eight rats remaining at 32 months and HCC had developed in one of the five. Conclusions: Our findings are compatible with the hypothesis that the direct hepatocarcinogenic effect of free iron is mediated by the generation of oxygen reactive species and oxidative damage that are mutagenic and carcinogenic. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Ferrocene; Wistar albino rats; Reactive oxygen species; Oxidative damage; Lipid peroxidation; Hereditary haemochromatosis; African dietary iron overload; Iron-free neoplastic foci 1. Introduction Although essential for the growth of cells, in exces- sive amounts iron is toxic. The liver is especially sub- ∗ Corresponding author. Tel.: +11 488 3628; fax: +11 643 4318. E-mail address: kewmc@medicine.wits.ac.za (M.C. Kew). ject to this toxic effect because it is the major site of iron storage. Portal fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) are late sequelae of hepatic iron over- load in hereditary haemochromatosis (Niederau et al., 1985; Deugnier et al., 1993) and dietary iron overload in the African (previously called Bantu visceral sidero- sis) (Bothwell and Bradlow, 1960; Gordeuk et al., 1996; Moyo et al., 1998; Mandishona et al., 1998). Although 0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.11.006