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Published online before print November 7, 2006, 10.1148/radiol.2421060083
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(Radiology 2007;242:189-197.)
© RSNA, 2006

Molecular Imaging

x-Irradiation in Rat Liver: Consequent Upregulation of Hepcidin and Downregulation of Hemojuvelin and Ferroportin-1 Gene Expression1

Hans Christiansen, MD, Nadeem Sheikh, PhD, Bernhard Saile, MD, Felix Reuter, BS, Margret Rave-Fränk, MSc, Robert M. Hermann, MD, Jozsef Dudas, PhD, Andrea Hille, MD, Clemens Friedrich Hess, MD, PhD and Giuliano Ramadori, MD

1 From the Department of Radiation Oncology (H.C., F.R., M.R., R.M.H., A.H., C.F.H.) and Department of Internal Medicine, Section of Gastroenterology and Endocrinology (N.S., B.S., J.D., G.R.), University Hospital Goettingen, Robert-Koch-Strasse 40, 37075 Goettingen, Germany. Received January 16, 2006; revision requested March 21; revision received April 7; final version accepted June 7. Supported by grants SFB 402 TP C6, C7, D3, and GRK 335 from the Deutsche Forschungsgemeinschaft. Address correspondence to H.C. (e-mail: hchrist{at}gwdg.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To prospectively analyze hepcidin, hemojuvelin, and ferroportin-1 expression after x-irradiation of rat liver and isolated rat hepatocytes.

Materials and Methods: The treatment of the rats and this study were approved by the local committee and the public authority on animal welfare. Rat livers in vivo and isolated rat hepatocytes in vitro were irradiated. The total number of rats in this study was 43. RNA extracted from livers (1, 3, 6, 12, 24, and 48 hours after irradiation) and from hepatocytes (1, 3, 6, 12, and 24 hours after irradiation) was analyzed with real-time polymerase chain reaction and Northern blot. Cytokines and prohepcidin in serum of irradiated rats were quantitatively detected with enzyme-linked immunosorbent assay. Sham-irradiated animals served as controls in all experiments. Differences between sham-irradiated and irradiated data groups were tested with analysis of variance and Dunnett post hoc test.

Results: In vivo, a significant radiation-induced increase of hepcidin (P = .034), interleukin (IL) 1ß (P = .008), IL-6 (P < .011), and tumor necrosis factor {alpha} (TNF-{alpha}) (P = .047) expression could be detected within the first 48 hours after irradiation. Expression of hemojuvelin (P = .008) and ferroportin-1 (P = .002) was significantly decreased. Serum iron levels were decreased because of irradiation (P < .058); prohepcidin serum levels were increased (P = .05). In rat hepatocytes in vitro, hepcidin RNA levels were significantly downregulated after irradiation (P < .001). Incubation of irradiated hepatocytes with IL-1ß, IL-6, or TNF-{alpha} led to upregulation of hepcidin expression in vitro up to 6 hours after irradiation, with subsequent significant downregulation for incubation with IL-1ß (P < .001). Hemojuvelin expression behaved in a way opposite to that of hepcidin.

Conclusion: x-Irradiation of the liver induced changes of hepcidin gene expression that are probably induced by acute phase mediators produced within the liver itself.

© RSNA, 2006


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Hepcidin—previously reported as a liver-expressed antimicrobial peptide (1)—is a circulating antimicrobial peptide mainly synthesized in the liver by hepatocytes (2), and it regulates intestinal iron absorption, as well as maternal fetal iron transport across the placenta (3). It affects the release of iron from hepatic stores and from macrophages involved in the recycling of iron from hemoglobin (4). Hepcidin inhibits iron efflux by binding to ferroportin-1 and inducing ferroportin-1 internalization, and this activity leads to decreased iron release (5). Furthermore, hepcidin is an acute phase peptide, and production of hepcidin is increased in inflammation and iron overload (6). As a key protein in iron metabolism, hepcidin is supposed to be a major contributor to the hypoferremia associated with inflammation (7) and is one mediator of anemia in acute and chronic infections, inflammatory disorders, and neoplastic diseases.

The specific function of recently identified hemojuvelin is currently unknown (8,9). Findings in in vitro studies revealed that it may inhibit hepcidin synthesis (10). Mutations in the hemojuvelin gene seem to be involved in the pathogenesis of juvenile hemochromatosis (11). Although hepcidin has been described as an acute phase protein (12), hemojuvelin is considered as one of the few anti–acute phase proteins known so far (13).

The liver is a highly radiosensitive organ because of the danger of development of radiation-induced liver disease. Because isolated primary hepatocytes in vitro are known to be radioresistant (14–20), cell-cell interactions between different cell systems occurring in the liver probably play a decisive role in the development of radiation-induced liver disease. The threshold dose for whole-liver irradiation without chemotherapy is supposed to be 20–30 Gy (21,22). In patients who undergo combined radiation therapy and chemotherapy or combined radiation therapy and immunotherapy, the risk for development of radiation-induced liver disease is higher (23,24). Findings in studies in animals have suggested that liver irradiation at a dose higher than the threshold dose is followed by progressive liver fibrosis and cirrhosis (25).

The molecular pathogenesis of hepatocellular damage after irradiation is obscure. Cytokines are important for hepatocellular damage, repair, and fibrosis development in other toxic liver injuries (26–28). Similarly, cell types of different organs interact by way of cytokines in the development of normal tissue reactions after radiation therapy (29). Investigators have shown that hepatocytes are not substantially damaged by radiation. Still, radiation can weaken the defense mechanisms of hepatocytes, and this weakening can lead to the susceptibility to apoptosis mediated by tumor necrosis factor {alpha} (TNF-{alpha}) in vitro in some hepatocytes (30). Furthermore, results of a recent study indicate that whole-liver irradiation in the rat leads to development of steatosis within the first 48 hours after irradiation (31).

We hypothesized that cytokines influence radiation-induced hepcidin and hemojuvelin expression. Thus, the purpose of our study was to prospectively analyze hepcidin, hemojuvelin, and ferroportin-1 expression after x-irradiation of rat liver and isolated rat hepatocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Animals
Male Wistar rats that were 8–10 weeks of age and weighed 180–200 g were purchased from a supplier (Harlan-Winkelmann, Borchen, Germany). Rats were maintained with pathogen-free conditions in our animal facility, with access to food and tap water ad libitum. We adhered to the institutional policies and the relevant guidelines for care and use of laboratory animals. The treatment of the rats and our study were approved by a local committee and the public authority on animal welfare. According to the approval, five animals were examined at each time. The total number of rats in this study was 43 (35 were used for the in vivo experiments; hepatocytes were obtained from eight rats for the in vitro studies).

Whole-Liver Irradiation in Vivo
In vivo experiments were performed by several authors (H.C., F.R., M.R., R.M.H., A.H.) in consensus, as described previously by Christiansen et al (31): Planning computed tomography (CT) was performed with a scanner (Somatom Balance; Siemens Medical Solutions, Erlangen, Germany) in each rat to delineate the livers of the animals. The rats were intraperitoneally anesthetized with 90 mg per kilogram of body weight of ketamine (Intervet, Unterschleissheim, Germany) and 7.5 mg/kg of 2% xylazine (Serumwerk Bernburg, Bernburg/Saale, Germany). The margins of the liver were marked on the skin of each animal, and a dose distribution was calculated.

The livers were irradiated selectively with 6–MeV photons (dose rate, 2.4 Gy/min) by using an accelerator (Clinac 600 C; Varian, Palo Alto, Calif). A dose of 25 Gy in a single treatment was delivered by using an anteroposterior and posteroanterior treatment technique. Normal sham-irradiated animals, which were transported and anesthetized simultaneously with the irradiated animals, served as specific controls. Animals were observed at specified times up to 48 hours after irradiation and seemed to behave normally without signs of discomfort. Livers and serum samples were taken from five animals each at 1, 3, 6, 12, 24, and 48 hours after irradiation and frozen.

Cell Isolation and in Vitro Treatment
Hepatocytes were isolated from normal animals by several individuals (H.C., N.S., B.S., R.M.H., J.D.) in consensus, according to Seglen (32) as described previously by Ramadori et al (33). Purity of cell isolations was determined by using phase-contrast microscopy and immunocytochemistry with antibodies against laminin or extraneural glial fibrillary acidic protein, both of which were from the same supplier (Sigma, Deisenhofen, Germany), to identify stellate cells or ectodysplasin-1 and ectodysplasin-2 (gift from Dijkstra et al [34]) for macrophages (35). Four independent hepatocyte isolations were performed. Thereby, hepatocytes were obtained from a total of eight animals.

The cells were incubated at 37°C in 5% CO2 atmosphere. Dulbecco's modified Eagle's medium (Biochrom, Berlin, Germany) was supplemented with 10% fetal calf serum (PAA Laboratories, Cölbe, Germany), 0.05% insulin (Roche, Mannheim, Germany), and 7–10 mol/L dexamethasone (Sigma).

Irradiation was performed 24 hours after the hepatocytes were plated, and the culture medium was replaced immediately before irradiation: On the first day after isolation, hepatocytes were irradiated with 8 Gy (6–megaelectron volt photons) at a dose rate of 2.4 Gy/min by using the accelerator mentioned before. Hepatocytes were either irradiated or irradiated and additionally exposed to 500 ng of recombinant interleukin (IL) 1ß, IL-6, or TNF-{alpha} (PeproTech, Rocky Hill, NJ) in 3-mL cell culture supernatants immediately before irradiation. RNA from all in vitro experiments was extracted at 1, 3, 6, 12, and 24 hours after irradiation. Sham-irradiated cells served as controls in all experiments.

Cytokine and Prohepcidin Serum Measurement
Detection of cytokines and prohepcidin in serum was performed by a few authors (H.C., N.S., F.R.) in consensus with enzyme-linked immunosorbent assay kits (Quantikine; R&D Systems, Minneapolis, Minn) for IL-6, IL-1ß, and TNF-{alpha} and with enzyme-linked immunosorbent assay kits manufactured by another company (DRG International, Marburg, Germany) for prohepcidin. Samples were processed according to suppliers' instructions. Samples contained serum from irradiated rats removed at 1, 3, 6, 12, 24, and 48 hours after irradiation and serum of sham-irradiated control animals.

Detection of Serum Iron Levels
In serum of irradiated rats and sham-irradiated controls, iron levels were detected by one individual (N.S.) with a kit (Rolf Greiner BioChemica, Flacht, Germany).

RNA Isolation, Real-time Polymerase Chain Reaction, and Northern Blot Analysis
RNA isolation, real-time polymerase chain reaction (PCR), and Northern blot analysis were performed by several authors (H.C., N.S., F.R., J.D.) in consensus. Total RNA from isolated irradiated hepatocytes and from rat liver from irradiated and sham-irradiated controls was isolated after homogenization in guanidinium isothiocyanate (Sigma) by using the CsCl ultracentrifugation method (36), as previously described by Ramadori et al (37).

With real-time PCR analysis, we analyzed the expression of hepcidin, hemojuvelin, ferroportin-1, ferritin, transferrin, transferrin receptor 1, and transferrin receptor 2 as main proteins in iron metabolism. Because we wanted to test the hypothesis that cytokines are involved in radiation-induced changes of proteins in iron metabolism, the expression of the cytokines Il-1ß, IL-6, and TNF-{alpha} was also analyzed. For analysis of real-time PCR, reverse transcription of RNA samples was performed by using a kit (Superscript; Invitrogen, Groningen, the Netherlands). The instructions of the manufacturer were followed. Real-time PCR analysis of complementary DNA was performed with a sequence detection system (ABI Prism 7600; Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instructions by using a PCR mix (SYBR Green Master Mix; Applied Biosystems) and primers (Table). All our primers were synthesized by one manufacturer (MWG Biotech; Ebersberg, Germany).


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  		Primers for Quantitative Real-time PCR Analysis

 
For Northern blot analysis, total RNA (5 µg per probe) was size fractionated with electrophoresis in 1% agarose-formaldehyde gels, transferred to nylon membranes (Amersham Pharmacia Biotech, Freiburg, Germany) by using the capillary transfer systems, and crosslinked by using ultraviolet light. Rat hepcidin complementary DNA was generated with a PCR from rat hepatic RNA with the following primers: forward primer of 5'-AGGACAGAAGGCAAGATGGCA-3' and reverse primer of 5'-TGTTGAGAGGTCAGGACAAGGC-3'. Rat hemojuvelin complementary DNA was generated with a PCR from rat hepatic RNA with the following primers: forward primer of 5'-ATGCCGTGTCCAAGGAGCTT-3' and reverse primer of 5'-TCCACCTCAGCCTGGTAGAC-3'. Hybridization was performed at 68°C for 2 hours with random-primed phosphorus 32–labeled complementary DNA probes for hepcidin or hemojuvelin (Amersham Pharmacia Biotech) and overnight at 42°C for 28S ribosomal RNA.

Statistical Analysis
Data are given as the mean ± standard deviation (SD); corresponding figures were created by using software (Excel for Windows 2003; Microsoft, Redmond, Wash). A Student t test was performed to find out significant differences among sham-irradiated and irradiated data groups obtained in at least three (in vivo) or four (in vitro) independent experiments. Differences found were also tested with analysis of variance. Because all values at specified times after irradiation were compared with values of sham-irradiated controls, the Dunnett post hoc test was performed subsequent to analysis of variance. Differences with a value of P ≤ .05 after adjustment for multiple comparisons were considered to be significant. Statistical comparisons were performed by using software (KaleidaGraphSoftware, version 4.01 for Macintosh; Synergy Software, Reading, Pa).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Real-time PCR Analysis of Rat Liver in Vivo
We could detect statistically significant radiation-induced upregulation of hepcidin 24 hours after irradiation (mean, 29.2-fold ± 17.9 [SD]; P = .034) (Figs 1, 2). In contrast, hemojuvelin gene expression was significantly downregulated 3 hours after irradiation (mean, 0.51-fold ± 0.22; P = .008). For IL-1ß (mean, 5.83-fold ± 2.78; P = .008), IL-6 (mean, 26.23-fold ± 20.98; P < .011), and TNF-{alpha} (mean, 6.61-fold ± 5.81; P = .047), statistically significant upregulation could be detected 6 hours after irradiation. Furthermore, we measured statistically significant upregulation of ferritin 6 hours (mean, 2.73-fold ± 0.7; P = .016) and 12 hours (mean, 3.5-fold ± 1.35; P < .001) after irradiation and of transferrin 6 hours after irradiation (mean, 2.03-fold ± 0.07; P < .001), whereas downregulation of ferroportin-1 was significant 3 hours after irradiation (mean, 0.67-fold ± 0.07; P = .002). For transferrin receptors 1 and 2, no statistically significant changes could be detected.


Figure 1
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  		Figure 1: Graph of gene expression in irradiated liver tissue measured with quantitative real-time PCR shows radiation-induced upregulation of IL-1ß, IL-6, TNF-{alpha}, and hepcidin and radiation-induced downregulation of hemojuvelin. Data are shown relative to expression of ubiquitin (levels of sham-irradiated controls were normalized to one and are not directly represented). Values are means ± SD for analyzed RNA extracted from at least four animals. P values were calculated compared with levels in sham-irradiated controls. * = P ≤ .05, ** = P ≤ .01.

 

Figure 2
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  		Figure 2: Graph of gene expression in irradiated liver tissue measured with quantitative real-time PCR shows radiation-induced upregulation of ferritin and transferrin and radiation-induced downregulation of ferroportin-1. Data are shown relative to expression of ubiquitin (levels of sham-irradiated controls were normalized to one and are not directly represented). Values are means ± SD for analyzed RNA extracted from at least three animals. P values were calculated compared with levels in sham-irradiated controls. TRF 1 = transferrin receptor 1, TRF 2 = transferrin receptor 2, * = P ≤ .05, ** = P ≤ .01.

 
Northern Blot Analysis of Rat Liver in Vivo
With Northern blot analysis (Fig 3), the hepcidin-specific transcript level increased up to 24 hours after radiation therapy, and the hemojuvelin-specific transcript level initially decreased at 1, 3, and 6 hours after irradiation and returned to control levels thereafter, and this activity supports our real-time data.


Figure 3
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  		Figure 3: Northern blot analysis results of two experiments for hepcidin-specific (top row) and hemojuvelin-specific (middle row) transcripts (black = amount of specific transcript) in rat liver in sham-irradiated controls (c) and at 1, 3, 6, 12, and 24 hours after 25 Gy of liver irradiation show radiation-induced upregulation for hepcidin and radiation-induced downregulation of hemojuvelin. Total RNA was electrophoresed, blotted onto nylon membranes, and hybridized with complement DNA specific for rat hepcidin, hemojuvelin, or an oligonucleotide recognizing the 28S ribosomal RNA (bottom row).

 
Real-time PCR Analysis of Isolated Rat Hepatocytes in Vitro
Irradiation alone led to statistically significant downregulation of hepcidin gene expression in hepatocytes, with a maximum of 12 hours after irradiation (mean, 0.4-fold ± 0.11; P < .001). In contrast, hemojuvelin gene expression was significantly upregulated 24 hours after irradiation (mean, 42.16-fold ± 14.38; P = .003) (Fig 4).


Figure 4
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  		Figure 4: Graph of gene expression in irradiated hepatocytes measured with quantitative real-time PCR shows radiation-induced downregulation of hepcidin and radiation-induced upregulation of hemojuvelin. Data are shown relative to expression of ubiquitin (levels of sham-irradiated controls were normalized to one and are not directly represented). Values are means ± SD for analyzed RNA extracted from four independent hepatocyte isolations. P values were calculated compared with levels in sham-irradiated controls. ** = P ≤ .01.

 
An increase of hepcidin expression, however, could be detected in irradiated hepatocytes that were incubated, in addition, with recombinant IL-1ß, IL-6, or TNF-{alpha} up to 6 hours after irradiation, with subsequent downregulation (Fig 5). This was statistically significant for incubation with IL-1ß 6 hours after irradiation (mean, 8.09-fold ± 1.24; P < .001). Afterward, hepcidin expression was downregulated at 12 hours (mean, 0.26-fold ± 0.05) and 24 hours (mean, 0.37-fold ± 0.13) after irradiation (P < .001).


Figure 5
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  		Figure 5: Graphs of gene expression in irradiated hepatocytes incubated additionally with IL-1ß, IL-6, or TNF-{alpha} measured with quantitative real-time PCR. Top: Hepcidin expression revealed initial upregulation 1–6 hours after irradiation, followed by downregulation up to 24 hours after irradiation. Bottom: Hemojuvelin expression behaved in a way opposite to that of hepcidin. Data are relative to expression of ß-actin (levels of sham-irradiated controls were normalized to one and are not directly represented). Values are means ± SD for analyzed RNA extracted from four independent hepatocyte isolations. P values were calculated compared with levels in sham-irradiated controls. ** = P ≤ .01.

 
For hemojuvelin expression in irradiated hepatocytes after incubation with recombinant IL-1ß, IL-6, or TNF-{alpha}, effects contrary to those for hepcidin expression could be observed (Fig 5): After initial downregulation up to 6 hours after irradiation, hemojuvelin was upregulated. Changes in hemojuvelin expression after incubation with recombinant IL-1ß, IL-6, or TNF-{alpha}, however, were not statistically significant.

Ferritin showed upregulation 24 hours after irradiation (mean, 4.43-fold ± 0.39; P < .001), and transferrin protein was upregulated 12 hours after irradiation (mean, 3.11-fold ± 1.08; P = .015) (Fig 6). Ferroportin-1, which was initially downregulated at 1 hour after irradiation (mean, 0.63-fold ± 0.13; P = .003), was found to be upregulated at 24 hours (mean, 7.63-fold ± 2.67; P = .005). For both transferrin receptor 1 (mean, 0.51-fold ± 0.19; P = .004) and transferrin receptor 2 (mean, 0.42-fold ± 0.16; P < .001), downregulation 6 hours after irradiation was followed by upregulation at 24 hours for transferrin receptor 1 (mean, 4.11-fold ± 1.89; P = .029) and transferrin receptor 2 (mean, 24.84-fold ± 18.49; P = .033).


Figure 6
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  		Figure 6: Graph of gene expression in irradiated hepatocytes measured with quantitative real-time PCR shows initial radiation-induced downregulation of ferroportin-1, transferrin receptor 1 (TRF 1), and transferrin receptor 2 (TRF 2), followed by upregulation of ferritin, transferrin, ferroportin-1, and transferrin receptors 1 and 2. Data are relative to expression of ubiquitin (levels of sham-irradiated controls were normalized to one and are not directly represented). Values are means ± SD for analyzed RNA extracted from four independent hepatocyte isolations. P values were calculated compared with levels in sham-irradiated controls. * = P ≤ .05, ** = P ≤ .01.

 
Cytokine and Prohepcidin Serum Measurement and Iron Levels of Rat Serum in Vivo
With enzyme-linked immunosorbent assay, elevated serum levels after irradiation could be detected for the cytokines (Fig 7) and prohepcidin (Fig 8), and the levels were statistically significant for TNF-{alpha} 1 hour after irradiation (mean, 5.26 pg/mL ± 3.1 vs 0.34 pg/mL ± 0.37 [sham-irradiated controls]; P = .034) and for prohepcidin 3 hours after irradiation (mean, 565.48 ng/mL ± 279.93 vs 278.07 ng/mL ± 17.31 [sham-irradiated controls]; P = .05). The increase of IL-1ß and IL-6 serum level was not statistically significant at any time after irradiation. Because of irradiation, mean serum iron levels decreased within 48 hours after irradiation compared with the levels in sham-irradiated controls from a maximum of 65.3 µg/dL ± 13.51 (control) to a minimum of 46.5 µg/dL ± 6.15 at 1 hour after irradiation. The changes, however, showed no statistically significant difference but a strong trend toward decrease (P < .058) (Fig 9).


Figure 7
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  		Figure 7: Graph of IL-1ß, IL-6, and TNF-{alpha} levels in serum of irradiated rats and sham-irradiated controls. Values on y-axis are serum values of each cytokine measured with enzyme-linked immunosorbent assay. The increase in serum levels was statistically significant for TNF-{alpha} 1 hour after irradiation. Values are means ± SD from at least three animals. P values were calculated compared with levels in sham-irradiated controls. * = P ≤ .05.

 

Figure 8
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  		Figure 8: Graph of prohepcidin levels in serum of irradiated rats and sham-irradiated controls. Values on y-axis are serum values of prohepcidin measured with enzyme-linked immunosorbent assay. The increase in serum levels 3 hours after irradiation was statistically significant. Values are means ± SD from at least four animals. P values were calculated compared with levels in sham-irradiated controls. * = P ≤ .05.

 

Figure 9
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  		Figure 9: Graph of iron levels in serum of irradiated rats and sham-irradiated controls. Values on y-axis are serum values of iron and show a trend for decreased iron levels after irradiation. Values are means ± SD from at least three animals.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
To our knowledge, our report is the first about the effect of x-irradiation applied to the liver on gene expression of several proteins of iron metabolism and the first report in which x-irradiation–induced upregulation of hepcidin and downregulation of hemojuvelin and ferroportin-1 are described.

In our model, the expression of the proinflammatory cytokines IL-1ß, IL-6, and TNF-{alpha} was also upregulated after liver irradiation in vivo. Because the effect of irradiation of isolated hepatocytes was opposite to that observed in vivo, we suggest that inflammatory cytokines may be responsible for the changes observed in vivo. In fact, hepcidin is upregulated up to 6 hours after irradiation in hepatocytes incubated with recombinant IL-1ß, IL-6, or TNF-{alpha}. Thereby, IL-1ß was more potent than IL-6 or TNF-{alpha}. This effect has similarities to that observed in the so-called acute phase condition observed after endotoxin administration or after tissue damage induction at a site distant from the liver. In these cases, however, IL-6 has been shown to be the main mediator (38). One reason for the minor role of IL-6 may be the downregulation of IL-6-receptor caused by liver irradiation in vivo (31).

Proinflammatory cytokines are stimulators of early hepcidin expression in inflammatory processes (39). The first cytokine described as a modulator of hepcidin expression was IL-6, which induces hepcidin expression in vitro and in vivo (12,38,40). The involvement of IL-1 has been discussed controversially. Although researchers in some studies could not demonstrate any IL-1 effect on hepcidin gene expression (12), others showed IL-1 involvement in regulation of hepcidin transcription (41). Our in vitro data confirmed that IL-1ß had an effect on hepcidin expression in vitro, and this finding suggests an important role for IL-1ß in hepatic radiation-induced hepcidin expression. Furthermore, we report an influence of TNF-{alpha} on radiation-induced hepcidin expression, which has not been reported in the literature to date, to our knowledge.

In the case of sole incubation of isolated hepatocytes with the proinflammatory cytokines (IL-6, IL-1ß, or TNF-{alpha}) without irradiation, only IL-6 could induce hepcidin expression 3 hours after exposure (42). Therefore, we suggest that, because of irradiation, intracellular processes in hepatocytes must be acting, and the activity leads to increased sensitivity to cytokine-induced effects such as hepcidin expression. In accordance with this idea, researchers in previous in vitro studies have shown that radiation leads to susceptibility of hepatocytes to cytokine-mediated apoptosis in vitro (30). It is well known that cell-cell interactions by way of cytokines play a role in the development of normal tissue reactions after radiation therapy. They can lead to apoptosis and chronic fibrotic reactions (29,30,43). Cytokine-modulated radiation-induced hepcidin expression after liver irradiation is a particular aspect of normal tissue reactions after radiation therapy. Hepcidin expression profiles were converse to hemojuvelin expression after irradiation alone both in vivo and in vitro. In our in vivo model, hemojuvelin expression was significantly downregulated. In contrast, it was significantly upregulated in isolated hepatocytes in vitro.

Incubation of irradiated hepatocytes with recombinant IL-1ß, IL-6, or TNF-{alpha} led to contrary effects concerning hemojuvelin expression in comparison with hepcidin expression, too: After initial downregulation, hemojuvelin was upregulated. In contrast, hepcidin expression showed initial upregulation with subsequent downregulation.

Researchers have supposed that hemojuvelin can downregulate hepcidin gene expression with a mechanism independent of IL-6 (10). We provide data for a further mechanism of regulation, as both genes are modified at the same time in the opposite direction. Further investigations are needed to evaluate the intracellular pathways involved in this process.

Upregulation of hepcidin has been reported in the context of acute phase processes—such as endotoxic reactions induced by administration of lipopolysaccharide (44)—in which upregulation of hepcidin expression has been reported (19,20). Hepcidin has been in fact identified as a type II acute phase protein (12).

Hepcidin is known to be a key iron regulatory protein because it helps adjust iron flow into plasma and aids intestinal iron absorption, an activity that leads to decreased serum iron levels (45). It also inhibits the release of recycled iron from macrophages (46). Therefore, hepcidin is supposed to be one mediator of anemia in acute and chronic infections, inflammatory disorders, and neoplastic diseases (47,48), as the pathogenesis of inflammatory anemia is associated with decreased iron absorption and impaired mobilization of iron stores (49).

Another protein involved in iron metabolism is ferroportin-1. Molecular studies have shown that control of iron homeostasis is centered mainly on hepcidin–ferroportin-1 interaction (50). It is suggested that ferroportin-1 mediates iron export in hepatocytes (51). Thereby, ferroportin-1 is regulated through systemic regulators that likely include hepcidin (52). In our animal model, liver irradiation led to increased hepcidin expression and to decreased ferroportin-1 expression. These data suggest that hepcidin–ferroportin-1 interactions play a role in the decrease of serum iron levels after liver irradiation. Decreased iron levels in hepatocytes may have disastrous consequences for the cell and its resistance to injury, as iron is a crucial element for vital biochemical activities (32).

Our data show that altered hepcidin, hemojuvelin, and ferroportin-1 expression may be involved in the processes following liver irradiation. Changes in the levels of proinflammatory cytokines (IL-1ß, IL-6, TNF-{alpha}) induced by irradiation in the liver influence radiation-induced hepcidin, hemojuvelin, and ferroportin-1 expression in vitro, and this activity suggests that a similar mechanism may act in vivo with fundamental consequences on overall iron metabolism. Moreover, it is likely that the regulation of iron metabolism plays an underestimated role in cellular or tissue reaction to anticancer treatment. More effort to link iron metabolism with human radiation responses is essential.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   FOOTNOTES
 

Abbreviations: IL = interleukin • PCR = polymerase chain reaction • SD = standard deviation • TNF-{alpha} = tumor necrosis factor {alpha}

Authors stated no financial relationship to disclose.

See also Science to Practice in this issue.

Author contributions: Guarantors of integrity of entire study, H.C., N.S., G.R.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, all authors; experimental studies, H.C., N.S., B.S., F.R., R.M.H., J.D., G.R.; statistical analysis, H.C., N.S., F.R., M.R., R.M.H., G.R.; and manuscript editing, all authors


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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