St well-characterized heme importer and exporter respectively. As shown in Figure 5A and 5B, PCFT/HCP1 and FLVCR1 mRNA and PCFT/HCP1 protein resulted expressed at similar levels in the 10781694 duodenum of Hx-null and wild-type mice. In addition, when mRNA levels of other heme exporters were evaluated, it was found that the ATPbinding cassette, subfamily G, member 2 (ABCG2), as well as the intracellular heme transporter Heme Regulated Gene (HRG)-1 were unchanged in Hx-null duodenum (Figure 5A).Hx deficiency does not affect the expression of duodenal iron transportersTo get more insight into the molecular mechanisms underlying iron overload, the expression of DcytB, DMT1, Fpn1, TfR1 and Heph in the duodenum has been assessed. qRT-PCR analysis showed that DcytB, DMT1, Fpn1 and Heph mRNA levels were not altered in Hx-null mice compared to wild-type animals, while TfR1 mRNA level was higher in Hx-nullLack of Hemopexin Results in Duodenal Iron LoadFigure 1. Hx deficiency results in Title Loaded From File increased iron deposits in the duodenum. (A) Total iron content in the duodenum of 2 month-old wild-type and Hx-null mice measured by ICP-MS. Values are expressed as g iron/g wet tissue. Data Ind both molybdate and the adenylated form of cyclic pyranopterin monophosphate represent mean ?SEM; n=10; * = P<0.05. (B) Duodenal sections of a wild-type mouse (i, ii) and an Hx-null mouse (iii, iv) at 2 months of age stained with DAB enhanced Perls' reaction. Note the more intense staining in the duodenum of the Hx-null mouse. Bar i, iii = 500 ; bar ii, iv = 100 .doi: 10.1371/journal.pone.0068146.gLack of Hemopexin Results in Duodenal Iron LoadFigure 2. Increased H-Ft expression in Hx-null mice duodenum. Representative Western blots of H-Ft (A) and L-Ft (C) expression in the duodenum of wild-type and Hx-null mice. Band intensities were measured by densitometry and normalized to actin expression. Densitometry data represent mean ?SEM; n=3 for each genotype. (B) Duodenal sections of a wild-type mouse (i) and an Hx-null mouse (ii) processed by immunohistochemistry with an anti-H-Ft antibody. The H-Ft-positive signal is increased in the duodenum of the Hx-null animal. Bar= 100 .doi: 10.1371/journal.pone.0068146.gLack of Hemopexin Results in Duodenal Iron LoadThus, the increased heme catabolism in duodenum cells of Hx-null mice cannot be accounted for by an impaired expression of heme transporters.Hx deficiency results in an enhanced iron uptake in the duodenumTo assess the rate of iron absorption, an oral dose of FeSO4 or of 57Fe-labelled heme was administered to Hx-null and wild-type mice. 57Fe content in duodenum, liver, bone marrow and kidney was determined by inductively coupled plasma mass spectrometry (ICP-MS) analysis [18] at different times after the administration. Thirty minutes after 57FeSO4 administration, a higher amount of 57Fe was detected in the duodenal mucosa of treated mice as compared with controls, and the quantity of 57Fe retained by duodenum further increased at ninety minutes after the oral administration. The amount of 57Fe retained in the mucosa was significantly higher in the duodenum of Hx-null mice than in that of wild-type animals (Figure 6A). Similar results were obtained when 57Fe labelled heme (57Feheme) was orally administered (Figure 6B). Analyses of liver and bone marrow showed comparable quantities of 57Fe in both Hx-null and wild-type mice after 57 FeSO4 administration (Figure 7A, B). The transfer of 57Fe to the kidney, an organ only marginally involved in iron handling, was negligible in both genotypes (Figure 7C). Collective.St well-characterized heme importer and exporter respectively. As shown in Figure 5A and 5B, PCFT/HCP1 and FLVCR1 mRNA and PCFT/HCP1 protein resulted expressed at similar levels in the 10781694 duodenum of Hx-null and wild-type mice. In addition, when mRNA levels of other heme exporters were evaluated, it was found that the ATPbinding cassette, subfamily G, member 2 (ABCG2), as well as the intracellular heme transporter Heme Regulated Gene (HRG)-1 were unchanged in Hx-null duodenum (Figure 5A).Hx deficiency does not affect the expression of duodenal iron transportersTo get more insight into the molecular mechanisms underlying iron overload, the expression of DcytB, DMT1, Fpn1, TfR1 and Heph in the duodenum has been assessed. qRT-PCR analysis showed that DcytB, DMT1, Fpn1 and Heph mRNA levels were not altered in Hx-null mice compared to wild-type animals, while TfR1 mRNA level was higher in Hx-nullLack of Hemopexin Results in Duodenal Iron LoadFigure 1. Hx deficiency results in increased iron deposits in the duodenum. (A) Total iron content in the duodenum of 2 month-old wild-type and Hx-null mice measured by ICP-MS. Values are expressed as g iron/g wet tissue. Data represent mean ?SEM; n=10; * = P<0.05. (B) Duodenal sections of a wild-type mouse (i, ii) and an Hx-null mouse (iii, iv) at 2 months of age stained with DAB enhanced Perls' reaction. Note the more intense staining in the duodenum of the Hx-null mouse. Bar i, iii = 500 ; bar ii, iv = 100 .doi: 10.1371/journal.pone.0068146.gLack of Hemopexin Results in Duodenal Iron LoadFigure 2. Increased H-Ft expression in Hx-null mice duodenum. Representative Western blots of H-Ft (A) and L-Ft (C) expression in the duodenum of wild-type and Hx-null mice. Band intensities were measured by densitometry and normalized to actin expression. Densitometry data represent mean ?SEM; n=3 for each genotype. (B) Duodenal sections of a wild-type mouse (i) and an Hx-null mouse (ii) processed by immunohistochemistry with an anti-H-Ft antibody. The H-Ft-positive signal is increased in the duodenum of the Hx-null animal. Bar= 100 .doi: 10.1371/journal.pone.0068146.gLack of Hemopexin Results in Duodenal Iron LoadThus, the increased heme catabolism in duodenum cells of Hx-null mice cannot be accounted for by an impaired expression of heme transporters.Hx deficiency results in an enhanced iron uptake in the duodenumTo assess the rate of iron absorption, an oral dose of FeSO4 or of 57Fe-labelled heme was administered to Hx-null and wild-type mice. 57Fe content in duodenum, liver, bone marrow and kidney was determined by inductively coupled plasma mass spectrometry (ICP-MS) analysis [18] at different times after the administration. Thirty minutes after 57FeSO4 administration, a higher amount of 57Fe was detected in the duodenal mucosa of treated mice as compared with controls, and the quantity of 57Fe retained by duodenum further increased at ninety minutes after the oral administration. The amount of 57Fe retained in the mucosa was significantly higher in the duodenum of Hx-null mice than in that of wild-type animals (Figure 6A). Similar results were obtained when 57Fe labelled heme (57Feheme) was orally administered (Figure 6B). Analyses of liver and bone marrow showed comparable quantities of 57Fe in both Hx-null and wild-type mice after 57 FeSO4 administration (Figure 7A, B). The transfer of 57Fe to the kidney, an organ only marginally involved in iron handling, was negligible in both genotypes (Figure 7C). Collective.
