The degree of iron overload, however, varies between strains, which is consistent with previous observations that iron metabolism is modified by genetic background.29 Our HH mice were generated on an AKR background and have relatively high plasma and liver iron levels, compared with other strains of mice. Colocalization of a more marked fibrotic process in areas of greatest iron deposition in the hepatic periportal regions in our Hfe−/−×Tfr2mut mice provides further evidence of the importance of genetic background and phenotypic expression of iron overload in the pathogenesis of liver injury in HH. Rodents

are generally relatively resistant to iron-induced Ensartinib liver injury. Dietary carbonyl iron loading of rats for 3 months produced iron loading in hepatocytes, similar to the levels observed in the Hfe−/− ×Tfr2mut mice in the present study, but demonstrated only early signs of liver injury, including NVP-BGJ398 datasheet increased LPO and collagen gene expression. Long-term iron loading was required for up to 12 months before morphological evidence of fibrosis

was observed.30, 31 Dietary iron supplementation in combination with hepatotoxins, such as ethanol and carbon tetrachloride, was required to accelerate liver injury.32, 33 In the present study, the degree of liver fibrosis observed in Hfe−/− ×Tfr2mut mice at 3 months of age was similar to that observed after dietary loading of rodents for 12 months.30, 31 In our Hfe−/−×Tfr2mut mice, hepatic inflammation, fibrosis, and LPO occurred in the presence of marked elevation of both plasma NTBI and hepatic iron levels, similar to those observed in human HFE-related HH.34, 35 Furthermore,

the degree of fibrosis observed in the HH mice was dependent on both HIC and NTBI levels. The observation that Hfe−/−×Tfr2mut mice have increased plasma ALT levels is consistent with previous observations in HH patients, where the majority of patients had mildly elevated ALT levels.36 Levels of the antioxidant enzymes, cytosolic copper/zinc and mitochondrial manganese PIK-5 SOD, were both decreased in Hfe−/−×Tfr2mut mice consistent with increased oxidative stress. Earlier studies have also reported decreased copper/zinc SOD in dietary iron-overloaded animals, whereas manganese SOD was decreased in Hfe knockout and increased in iron-loaded rodents.11, 20, 37 Furthermore, LPO was increased in HH mice. Unexpectedly, the level of F2-isoprostanes in dietary iron-loaded mice was greater than in HH mice with similar HIC. This may be the result of differences between dietary iron (i.e., high HAMP) and genetic HH (i.e., low HAMP) models of liver iron overload where variation in cellular iron distribution between parenchymal and Kupffer cells occurs, despite similar total HIC.