3A,E) that colocalized predominately, but not exclusively, with the iron storage protein, ferritin, in periportal regions of the liver (Supporting Fig. 2). The number of CD45+ inflammatory cells was significantly increased in the livers from Hfe−/−×Tfr2mut mice, compared with the other groups of mice (P < 0.05), whereas the number of CD45+ cells in Hfe−/−, Tfr2mut, and iron-loaded WT mice was not significantly different from those in non-iron-loaded WT mice (Fig. 3F). Another unique feature of Hfe−/− ×Tfr2mut mice was the evidence Ixazomib order of inflammatory sideronecrosis of hepatocytes, which was not observed in any other group of mice (Fig. 3E). Liver injury

was assessed by examining plasma ALT as well as hepatic SOD and F2-isoprostane levels. Plasma ALT activity was increased in Hfe−/−×Tfr2mut mice by at least 1.8-fold, compared with all other types of mice (P < 0.001; Fig. 4A). Both hepatic copper/zinc (cytosolic) and manganese (mitochondrial) SOD activities were significantly decreased in all HH mice. In Hfe−/−×Tfr2mut mice copper/zinc SOD levels were similar, whereas manganese SOD levels were significantly lower than Hfe−/−

and Tfr2mut mice (P < 0.01; Fig. 4B). Liver F2-isoprostanes were elevated in all groups of HH mice, compared with non-iron-loaded WT mice (P < 0.01), with Hfe−/− ×Tfr2mut mice having similar liver F2-isoprostane levels to iron-loaded WT mice and significantly higher AZD9668 research buy levels than either Hfe−/− or Tfr2mut mice (P < 0.01; Fig. 4C). Hepatic collagen deposition, a marker of fibrosis, was examined by histology using Sirius red and Masson's trichrome staining and by biochemical measurement of hydroxyproline levels. Hydroxyproline levels were increased

in all iron-loaded mice. In Hfe−/−×Tfr2mut mice, hydroxyproline levels were significantly increased, compared with Tfr2mut mice, and both were elevated, compared with Hfe−/− and iron-loaded WT mice (Fig. 4D; P < 0.05). Likewise, Hfe−/−×Tfr2mut mice had significantly increased Sirius red staining, compared with Hfe−/−, Tfr2mut, and iron-loaded WT mice (P < Y-27632 ic50 0.05), which, in turn, exhibited greater collagen deposition than non-iron-loaded WT mice (P < 0.01; Fig. 5A-F). Sirius red staining revealed portal tract thickening and periportal fibrosis in Hfe−/−×Tfr2mut mice. In addition, there was evidence of portal tract bridging in Hfe−/− ×Tfr2mut mice, which was not evident in other groups. Quantification of Sirius red staining correlated with HIC (r2 = 0.98; P = 0.001), plasma NTBI (r2 = 0.82; P = 0.033), as well as hydroxyproline (r2 = 0.89; P = 0.015) and F2-isoprotane levels (r2 = 0.77; P = 0.048) in HH mice. This suggests that the collagen levels measured by biochemical assay were consistent with histological observations using Sirius red staining and were dependent on both plasma NTBI and HIC in HH mice. Furthermore, the intensity of trichrome staining, a commonly used, but less sensitive, marker of fibrosis, was also significantly enhanced in Hfe−/−×Tfr2mut and Tfr2mut mice (Fig.