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Am J Physiol Regul Integr Comp Physiol 294: R1165-R1174, 2008. First published February 13, 2008; doi:10.1152/ajpregu.00719.2007
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APPETITE, OBESITY, DIGESTION, AND METABOLISM

Dysregulation of hepatic iron with aging: implications for heat stress-induced oxidative liver injury

Steven A. Bloomer,1 Kyle E. Brown,2,3,4 Garry R. Buettner,4 and Kevin C. Kregel1,4

1Department of Integrative Physiology, 2Iowa City Veterans Administration Medical Center, Iowa City, Iowa; 3Division of Gastroenterology-Hepatology, Roy J. and Lucille A. Carver College of Medicine, and 4Program in Free Radical and Radiation Biology, Department of Radiation Oncology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa

Submitted 4 October 2007 ; accepted in final form 8 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Environmental heat stress is associated with an age-related increase in hepatic oxidative damage and an exaggerated state of oxidative stress. The purpose of this investigation was to evaluate the regulation of hepatic iron after heat stress. A secondary aim was to determine a potential role for iron in heat stress-induced liver injury. Hyperthermia-induced alterations in hepatic iron were evaluated in young (6 mo) and old (24 mo) Fischer 344 rats by exposing them to a two-heat stress protocol. Livers were harvested at several time points after the second heating and assayed for labile and nonheme iron. In the control condition, there was no difference in labile iron between age groups. Both labile iron and storage iron were not altered by hyperthermia in young rats, but both were increased immediately after heating in old rats. To evaluate a role for iron in liver injury, hepatic iron content was manipulated in young and old rats, and then both groups were exposed to heat stress. Iron administration to young rats significantly increased hepatic iron content and ferritin but did not affect markers of lipid peroxidation under control conditions or after heat stress. In old rats, iron chelation with deferoxamine prevented the increase in nonheme iron, labile iron, ferritin, and lipid peroxidation after heat stress. These results suggest that iron may play a role in hepatic injury after hyperthermia. Thus, dysregulation of iron may contribute to the gradual decline in cellular and physiological function that occurs with aging.

reactive oxygen species; deferoxamine; hyperthermia; electron paramagnetic resonance spectroscopy; inflammation

IRON REPRESENTS A BIOLOGICAL double-edged sword. Although it is essential for life, it may also catalyze deleterious reactions within the cell. Several lines of evidence demonstrate that iron facilitates oxidative injury after various cellular and physiological stressors (1, 19, 27, 33, 38, 46). Iron may also play a similar role with aging, which is associated with chronic oxidative stress. Interestingly, measures of both storage iron (9) and labile iron (the catalytically active form) have been shown to increase with aging in the liver (45). The liver plays a major role in whole body iron metabolism (52), and we have previously observed increased levels of hepatic reactive oxygen species (ROS) and oxidative injury after heat stress in old animals (55). Because labile iron can be mobilized from intracellular sources by ROS (34, 39) and can contribute to oxidative injury, it may play a major role in hyperthermia-induced oxidative stress with aging.

The liver continually uses iron during normal metabolism and recycles a large proportion of iron from the circulation to the bone marrow for erythrocyte production (52). Continuous turnover of intracellular metabolic enzymes, as well as senescent and damaged red blood cells necessitates that the liver maintain tight control over intracellular iron. Conditions that perturb normal regulation of intracellular iron could elevate concentrations of labile iron, which could subsequently facilitate oxidative damage. Specifically, in vivo phorone treatment increases catalytically active iron in the liver (6) and renal ischemia/reperfusion elevates labile iron in the kidney (2). Furthermore, in cell culture, exposure to ultraviolet radiation (37) and H2O2 treatment (36, 53) both result in transient increases in labile iron. Oxidative stress is associated with all of these conditions and is thought to play a role in elevating labile iron.

The catalytically active pool of iron can increase with cellular and physiological stresses, and this elevation in labile iron can facilitate the associated tissue injury. The critical evidence that assigns a causal role to iron are studies that have reported diminished tissue injury after stressors with pretreatment of the iron chelator, deferoxamine (DFO) (1, 10, 30, 35, 42, 49). DFO is protective in animal models of tissue injury (19, 35) and in human cases of iron overload (49) and cardiovascular disease (1). Specifically, in rodents, DFO protects against intestinal (19) and muscular (44) ischemia/reperfusion injury. In humans, chronic treatment with DFO reduced the frequency of cardiac disease in patients with Thalassemia major—an inherited disorder characterized by iron overload (49).

Considering the abundance of studies in both animals and humans assigning a causative role to iron in tissue injury, it was of interest to determine the role of iron in our model of aging and physiological stress. We have previously observed that old rats display hepatocellular injury after a repeated, physiologically relevant heat stress (16, 18, 54, 55); hence, this model provides a valuable in vivo model for the high morbidity and mortality rates observed in older humans with stress (28, 43). Changes in liver iron stores with aging have been well characterized (9, 45), but the effects of a physiologically relevant stress on hepatic iron are less clear. Hence, the first goal of the present study was to evaluate the effects of heat stress on hepatic iron in young and old rats. It is unknown whether iron manipulation would affect liver injury after heat stress in young or old rats; therefore, a secondary aim was to evaluate a potential role for iron in hyperthermia-induced hepatic damage. On the basis of current evidence demonstrating that cellular stresses elevate labile iron (34, 36, 39), combined with our previous observations of increased steady-state levels of ROS after heat stress (55), we hypothesized that heat stress would cause an elevation in hepatic iron. Furthermore, because there is an exaggerated increase in hepatic ROS in old rats, it was postulated that they would experience a greater increase in labile iron after heat stress compared with young rats.

To address whether iron plays a role in mediating hepatic injury after heat stress, we increased hepatic iron stores in young rats to levels observed in old rats and then exposed them to heat stress. Conversely, we lowered hepatic iron with deferoxamine in old rats before heat stress. We hypothesized that iron administration to young rats would exacerbate liver injury after heat stress and that iron chelation in old rats would ameliorate liver injury.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal protocols. Young (6 mo old; 300–400 g) and old (24 mo old; 350–450 g) male Fischer 344 rats (National Institute on Aging) were used in these experiments. Rats were housed in The University of Iowa Animal Care Facility, and all experimental procedures were approved by the institutional animal care and use committee. Animals were maintained at 22–24°C on a 12:12-h light-dark cycle and provided food (standard rat chow) and water ad libitum.

Separate sets of young and old rats were used in four different experimental protocols. The first set of animals was used to compare the parenchymal and nonparenchymal distribution of storage iron between young and old rats under nonstressed conditions. Perl's Prussian Blue staining for iron deposits in fixed liver sections and a nonheme iron assay were used for this purpose. The nonheme iron assay measures iron contained predominantly in ferritin and hemosiderin (47) and is therefore a measure of storage iron in an organ. Hepatocytes were isolated from young and old rats according to Seglen's method, as previously described (17) and then assayed for nonheme iron. Whole livers were harvested from a separate group of young and old animals and also assayed for nonheme iron.

The second set of animals was used to evaluate the effects of a two-heat stress protocol on hepatic labile iron and hepatic storage iron. Young and old rats were randomly divided into heated and sham-heated groups. Livers were harvested from sham-heated control animals and from animals euthanized immediately after (0 h), and at 2 h and 24 h after the second heat stress.

The third set of animals was used to determine the effects of iron administration on heat stress-induced oxidative damage in young rats. A two-heat stress protocol was used for this purpose, and animals that underwent heat stress were euthanized immediately (0 h) after the second heat stress. Only young rats were used in this protocol, and the iron treatment procedure is described below. Animals were divided into four groups: 1) vehicle-treated, sham-heated control (V/C); 2) iron-treated, sham-heated control (Fe/C); 3) vehicle-treated, heated (V/H); and 4) iron-treated, heated (Fe/H).

The final set of animals was an old cohort used to determine the effects of iron chelation on heat stress-induced oxidative damage in old rats. A two-heat stress protocol was employed for this purpose, and animals that underwent heat stress were euthanized immediately after (0 h) the second heat stress. Only old rats were included in this protocol, and these animals were treated with deferoxamine (DFO), as described below. Animals were divided into four groups: 1) vehicle-treated, sham-heated control (V/C); 2) DFO-treated, sham-heated control (DFO/C); 3) vehicle-treated, heated (V/H); and 4) DFO-treated, heated (DFO/H).

Heat stress protocols. All rats were handled daily and familiarized with a colonic temperature probe during the week preceding the heat stress protocol. The two-heat stress protocol was performed as previously described (55). On the day of the first heat exposure, each rat was fitted with a thermistor temperature probe inserted 6–7 cm into the distal colon and then placed in a plastic cage, conscious and unrestrained. Core temperature (Tco) was continuously monitored on a digital display. A baseline Tco (37.0–38.0°C for both age groups) was established over a 5-min control period for each rat. Following the baseline period, Tco was increased to 41°C over 60 min with an infrared lamp placed ~40 cm above each rat. The lamp was raised or lowered to obtain a stable ambient temperature of 38–40°C and movement of the lamp permitted a constant heating rate (~0.06°C/min). Tco was maintained at 41°C for an additional 30 min, at which point, the thermistor probe was removed and rats were allowed to cool passively in a cage at room temperature. Twenty-four hours after the first heating, a second identical heating protocol was administered. Control animals underwent sham heatings at times that were similar to those of the heat stress groups; they were also fitted with a thermistor, placed in a plastic cage, and remained under euthermic conditions for the duration of the heat stresses. Animals were euthanized at the indicated times after the second sham-heating trial or after the second heat stress (Fig. 1A).


Figure 1
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  		Fig. 1. Time line of experimental protocols. A: two-heat stress protocol used for both young and old rats. Time points at which tissue was harvested are indicated below the time line. B: iron treatment and heat stress protocol used for young rats. C: deferoxamine (DFO) treatment and heat stress protocol used for old rats. Arrows with dashed lines indicate administration of DFO.

 
This heat stress protocol was designed to provide novel insights into the physiology of thermoregulation and the impact of aging on acute stress responses at cellular and systemic levels. For instance, the heating rate used simulates physiological conditions and generates a variety of systemic adjustments typically observed in humans (24, 25, 40). The Tco response of 41°C was selected because it is below the Tco threshold for heat-induced mortality (20) or heat stroke (7) in young rats. The rationale for heating rats to a Tco of 41°C on two consecutive days is based on epidemiological data (28, 43), studies in our own laboratory (18, 55), and clinical accounts indicating that humans who survive an initial heat challenge are subsequently less heat tolerant and more susceptible to thermal injury than unexposed subjects (22).

At the designated time points after heat stress, animals were administered an overdose of pentobarbital sodium (80 mg/kg ip). Livers were collected and rinsed twice in PBS, then immediately frozen in liquid nitrogen or fixed overnight in 10% neutral buffered formalin for subsequent analysis.

Hepatic iron administration to young rats. A pilot iron administration experiment was performed to determine the concentration of iron-dextran needed to approximately double hepatic iron concentration in young rats. On the basis of previously published results (3, 4), young rats were treated with intraperitoneal injections of 10, 15, 20, and 50 mg/kg iron-dextran to increase hepatic storage iron to levels similar to those observed in old rats. A dose of 15 mg/kg iron-dextran was used for subsequent experiments since it most closely approximated the hepatic iron concentration in old rats. Furthermore, iron-dextran administration to young rats mimicked the pattern of iron deposition (predominantly nonparenchymal) observed in old rats. The preliminary dose-response experiments also established that hepatic storage iron was increased in young rats at 24 h after iron-dextran injection and stayed elevated for at least 7 days after a single injection (data not shown). Dextran in saline was used as a control for iron treatment, and these animals were randomly assigned to heat stress and sham groups. Rats were injected with iron-dextran on day 1 and subjected to the first of two heat stresses on day 5. This time point after iron injection was chosen to obviate a potential acute phase response (APR) caused by iron injection, which might confound results obtained from heating experiments. Twenty-four hours after the first heat stress, a second heating commenced, and rats were euthanized immediately (0 h) after the second heating. Nonheated iron-treated and dextran-treated rats were euthanized on day 6, which corresponds to the time point when the 0 h postheat stress group was euthanized. Figure 1B illustrates this protocol.

Iron chelation in old rats. Deferoxamine mesylate (DFO; Sigma Aldrich, St. Louis, MO) was used to decrease hepatic iron content in old rats. Pilot studies established that a repeated dosing regimen of 200 mg/kg DFO per dose was necessary to lower hepatic storage iron in old rats. Hence, for the heat stress study, treatment began in the morning on day 1, and rats were injected every 12 h until the end of the study. DFO was weighed and dissolved in sterile saline immediately before each intraperitoneal injection. On day 4, animals were injected in the morning with DFO, and 2 h after the injection, they were exposed to the first of two heat stresses. DFO treatment continued during the two-heat stress protocol. On day 5, animals were injected at a similar time in the morning, then euthanized immediately after the second heat stress. Old animals injected with saline served as treatment controls and were randomly assigned to sham-heated and heated groups. Figure 1C depicts the timeline of events for this experiment.

Histology. To visualize iron deposits in liver samples, Perl's Prussian Blue staining was used as previously described (4). Slides were counterstained with nuclear fast red and coverslipped for microscopic analysis. Micrographs were taken in two liver regions: the area adjacent to the portal triad (periportal region) and the area adjacent to the terminal hepatic venule (perivenous region).

Histological damage was assessed by hematoxylin-and-eosin staining as previously described (55). Several fields from six animals in each group were scored by a reviewer blinded to the experimental condition.

Hepatic labile iron. Labile iron in liver samples was detected with electron paramagnetic resonance spectroscopy (EPR). Approximately 0.2 g of frozen liver tissue was homogenized in two volumes (0.4 ml) of 20 mM Tris buffer with a glass-on-glass homogenizer. To produce a unique EPR signal for this labile iron, the homogenate (400 µl) was incubated with 1 mM DFO for 45 min before being frozen in a quartz EPR tube (3 mm OD) with liquid nitrogen. A separate aliquot of the liver homogenate was set aside for protein determination via the Bradford assay. Samples were stored at –80 C until EPR analysis. EPR spectra were obtained with a Bruker EMX spectrometer operated at 9.43 GHz with 100-kHz modulation frequency. All measurements were made at 100 K using the ER4111VT variable temperature apparatus. Five scans of each sample were used for signal-averaging to improve the signal-to-noise ratio. The EPR spectrometer settings were microwave power, 20.4 mW; modulation amplitude, 16 G; time constant, 1.6 s; scan rate, 500 G/84 s; receiver gain, 2.5 x 105. A standard curve was generated by adding FeSO4 to aliquots of one liver homogenate in 5-µM increments. DFO drives the oxidation of the Fe2+ in FeSO4 to Fe3+ and binds Fe3+ with high affinity, hence the signal measured by EPR is referred to as a ferrioxamine signal. The g = 4.3 (magnetic field of ~1,550 Gauss) signal of the resulting ferrioxamine complex of each standard was readily detectable, increased proportionately with increasing iron concentration, and therefore yielded a linear standard curve. The labile iron concentration in each sample was determined by measuring the signal height of the ferrioxamine signal and comparing it to the standard curve, yielding an iron concentration in micromoles. This concentration was then converted to nanomoles of iron and normalized by the protein concentration of the sample. Results are expressed as nanomoles of iron per milligram protein.

Hepatic storage iron. Storage iron was assessed in liver samples using a nonheme iron assay as described by Torrance and Bothwell (47) with some modifications as previously described (4). Results are expressed as micrograms of iron per gram of dry weight liver. Primary hepatocytes that had been in frozen storage were subjected to the same protocol as whole liver tissue for determination of nonheme iron.

Hepatic malondialdehyde. Hepatic malondialdehyde (MDA) was assessed in liver homogenates using a commercially available kit (Oxis International, Foster City, CA) according to the manufacturer's instructions, as described previously (55). The MDA standard from the kit was diluted appropriately to yield a five-point standard curve and was subjected to the same steps as the liver homogenates. An aliquot of the same sample homogenate used for MDA determination was also used for determination of protein concentration via the Bradford assay. Results are expressed as nanomoles of MDA per milligram of protein.

Immunoblots. Samples of rat liver were homogenized in 10 volumes of RIPA buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 0.25% sodium deoxycholate, 1% SDS, 1 mM EDTA, 1 mM Na3VO4] and 10 µl/ml of a protease inhibitor cocktail (Sigma, St. Louis, MO). Immunoblotting was performed as previously described (5) with a primary antibody against human ferritin (F5012, used at 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) or 4-hydroxynonenal (4-HNE; ALX210-767, used at 1:1,000; Alexis Biochemicals, San Diego, CA). Membranes were stored at 4°C for 1 wk and then reprobed with a primary antibody against GAPDH (1:20,000; Abcam, Cambridge, MA) as a loading control. Protein bands were quantified with Gel Pro software (Media Cybernetics, Bethesda, MD), and results are expressed as the ratio of ferritin or 4-HNE optical density to GAPDH optical density in arbitrary units.

Statistical analyses. Results from the first set of studies characterizing the effects of age and heat stress on hepatic iron were analyzed for statistical significance using a two-factor ANOVA with the SPSS statistical package (SPSS, Chicago, IL). Results from the iron administration study (young rats only) and the iron chelation study (old rats only) were compared separately with a two-factor ANOVA to evaluate the effects of iron manipulation and heat stress within each age group. Bonferroni-adjusted P values were calculated for each variable to control for multiple comparisons. Results were considered significant when adjusted P values were less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of age on hepatic storage iron. To examine the effects of aging on the distribution of hepatic storage iron, liver sections were stained using Perl's Prussian Blue method. Figure 2A shows representative micrographs of periportal and perivenous regions from young and old rats. Stainable iron was frequently observed in nonparenchymal cells in both regions, as well as in hepatocytes in the perivenous region in old rats. (Fig. 2A, bottom right). Young rats had very little stainable iron in hepatocytes and displayed rare nonparenchymal iron deposits.


Figure 2
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  		Fig. 2. Age-related and region-specific alterations in hepatic storage iron. A: Perl's Prussian Blue staining for ferric iron deposition demonstrated frequent nonparenchymal iron deposits (arrows) in liver sections from old (bottom panels), but not young rats (top). Hepatocellular staining (open arrow) was evident only in the perivenous region of old rats (bottom right panel). B: quantitation of nonheme iron in primary hepatocytes and whole livers of young and old rats. The scale used to express nonheme iron is consistent with subsequent figures to allow for comparison of values between figures. Results are presented as means ± SE; n = 4–9 animals per group. *Significant difference (P < 0.05) between young and old rats within a preparation (primary hepatocytes or whole liver); {dagger}Significant difference between preparations within an age group.

 
Because of the pattern of iron deposits, samples of whole-liver tissue and primary hepatocytes were assayed for nonheme iron to quantitatively determine differences in parenchymal and nonparenchymal iron storage that occur with aging (Fig. 2B). Both primary hepatocytes and whole liver tissue samples from old rats contained more nonheme iron than similar samples from young rats. In old rats, whole-tissue samples contained significantly more nonheme iron than primary hepatocytes, which is consistent with the staining pattern shown in Fig. 2A. In young rats, there was no significant difference in iron content between hepatocytes and whole liver samples.

Labile iron and storage iron after a two-heat stress protocol. Because we observed striking differences between young and old rats in hepatic iron storage under nonstressed conditions, we asked whether hepatic iron was altered with a two-heat stress protocol. Accordingly, labile iron was measured via EPR, and storage iron was measured with the nonheme iron assay. Liver samples from young and old animals harvested under nonstressed conditions and at 0, 2, and 24 h were used for analysis. Figure 3A shows original EPR spectra of representative liver samples from young and old rats. In the nonstressed condition, there was no difference in labile iron concentrations between young and old rats. However, immediately after heat stress (0 h), labile iron was significantly elevated in old rats (Fig. 3, A and B). Labile iron was not altered at any time point assessed after heat stress in young rats.


Figure 3
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  		Fig. 3. Labile iron is elevated transiently in the liver of old rats after a two-heat stress protocol. A: representative ferrioxamine electron paramagnetic resonance (EPR) spectra from young and old rats under control conditions and at the indicated time points after a two-heat stress protocol. S.H., signal height in arbitrary units; [G], magnetic field in Gauss. B: quantitation of hepatic labile iron in young and old rats after heat stress. Results are presented as means ± SE; n = 4–7 animals per group. *Significant difference (P < 0.05) between young and old rats. {dagger}Significant effect of heat stress within an age group.

 
The increase in hepatic labile iron after a two-heat stress protocol in old rats prompted further investigation into the source of the catalytically active iron. Because iron can be released from intracellular storage proteins, we measured hepatic storage iron with the nonheme iron assay. This assay measures tissue iron that is stored in ferritin and hemosiderin (47); therefore, measurement of storage iron combined with labile iron might provide information regarding iron flux in the liver after heat stress. Old rats had higher concentrations of storage iron than young rats at all time points examined (Fig. 4). Hepatic storage iron was elevated in old rats that were exposed to a two-heat stress protocol immediately after the second heating (Fig. 4) but was not statistically different at any other time point. In contrast to old rats, young rats did not experience a change in storage iron with heat stress.


Figure 4
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  		Fig. 4. Hepatic storage iron increases with aging and is transiently elevated after heat stress in old rats. Liver samples from young and old rats were assayed for nonheme iron under control conditions and at the indicated time points after heat stress. Results are presented as means ± SE; n = 7–9 animals per group. *Significant (P < 0.05) difference between young and old rats within a time point. {dagger}Significant effect of heat stress within an age group.

 
Effects of iron treatment on nonheme iron and liver damage after heat stress in young rats. Iron administration to young rats increased hepatic storage iron to levels observed in old rats (Fig. 5A). In contrast to their old counterparts, young animals given iron did not experience a further increase in storage iron after heat stress (Fig. 5A). To examine the effects of increased hepatic iron content in the young rats on oxidative damage, liver samples were assayed for the lipid peroxidation markers, malondialdehyde (MDA) and 4-HNE-modified proteins. Iron administration did not result in a significant change in MDA levels in young animals under nonheated conditions or after heat stress (Fig. 5B). Hepatic 4-HNE-modified proteins and liver histology were similarly unaltered by heat stress and iron manipulation in young rats (not shown).


Figure 5
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  		Fig. 5. Iron administration in young rats increases hepatic storage iron but does not affect hepatic malondialdehyde (MDA). Quantitation of hepatic nonheme iron (A) and hepatic MDA (B) in vehicle-treated and iron-treated young rats under control conditions and immediately after a two-heat stress protocol. The scale used for MDA concentrations is consistent with Fig. 7 to allow for comparison of values between figures. Results are expressed as means ± SE; n = 5–9 animals per group. *Significant (P < 0.05) difference between vehicle-treated and iron-treated rats.

 
Effects of deferoxamine on labile iron, nonheme iron, and liver damage after heat stress in old rats. In vivo treatment with DFO greatly amplified the hepatic ferrioxamine signal in the control condition. Although the EPR signal is a measure of labile iron, the spectrum itself is the DFO-iron spectrum (i.e., the ferrioxamine signal). This apparent amplification is due to the presence of DFO in the liver, which will continuously bind transit iron as it becomes available during normal metabolism. Thus, the EPR signal of the ferrioxamine complex from the in vivo treatment with DFO is a result of the basal level of chelatable iron plus the integrated flux of the transient, labile iron that becomes available during the time of treatment with DFO. Hence, the signal amplification observed is not necessarily an increase in the labile iron, but an increase in the DFO-iron signal due to the in vivo DFO treatment. The effects of DFO on labile iron after heat stress were, therefore, normalized to the nonheated group in each treatment group (saline vs DFO). Figure 6 shows representative EPR spectra from saline-treated (Fig. 6A) and DFO-treated (Fig. 6B) animals. DFO treatment prevented the increase in hepatic labile iron observed in the saline-treated groups (Fig. 6C).


Figure 6
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  		Fig. 6. DFO treatment prevents the increase in hepatic labile iron in old rats after heat stress. Representative EPR spectra from saline-treated (A) and DFO-treated (B) rats; spectra from heated and control conditions are superimposed. S.H. = signal height in arbitrary units; [G] = magnetic field in Gauss. C: effects of DFO on hepatic labile iron in old rats after heat stress. Results were normalized as described in METHODS and are presented as means ± SE; n = 7 rats per group. *Significant (P < 0.05) effect of DFO treatment.

 
DFO treatment also lowered storage iron under control conditions and prevented the increase in nonheme iron after heat stress (Fig. 7A). In vehicle-treated old rats, hepatic MDA was elevated after heat stress. In contrast, animals that were treated with DFO did not experience a hyperthermia-induced increase in MDA (Fig. 7B). Hepatic 4-HNE-modified proteins were two-fold higher in old rats compared with young rats at all time points; however, they were not significantly altered by heat stress or iron manipulation in old rats (data not shown).


Figure 7
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  		Fig. 7. DFO treatment blunts the increase in storage iron and MDA in the liver after heat stress in old rats. Quantitation of hepatic nonheme iron (A) and hepatic MDA (B) in vehicle-treated and DFO-treated old rats under control conditions and immediately after a two-heat stress protocol. Results are expressed as means ± SE; n = 7–10 animals per group. *Significant (P < 0.05) difference between vehicle-treated and DFO-treated rats within a given time point. {dagger}Significant effect of heat stress within a treatment group.

 
As previously reported (55), the histological damage score was higher in old rats compared with young. However, neither heat stress nor iron manipulation significantly affected liver histology in old rats (not shown).

Hepatic ferritin protein levels after iron manipulation and heat stress. As expected, iron administration to young rats resulted in a significant increase in hepatic ferritin expression. However, no effect of heat stress on ferritin protein was observed in either the vehicle-treated or iron-treated young rats (Fig. 8A). Heat stress significantly increased ferritin protein levels in vehicle-treated old rats. Treatment of old rats with DFO significantly reduced ferritin protein in the control condition and prevented the increase after heat stress (Fig. 8B).


Figure 8
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  		Fig. 8. Ferritin protein expression is altered by heat stress and iron manipulation. A: immunoblot analysis of ferritin protein in young and young iron-treated rats after heat stress. B: immunoblot analysis of ferritin protein in old and old DFO-treated rats after heat stress. Top: representative immunoblots of ferritin and GAPDH protein. Bottom: differences in ferritin expression were quantified by normalizing the optical density of ferritin to the optical density of GAPDH in arbitrary units. V/C, vehicle control; Fe/C, iron-treated control; V/H, vehicle heated; Fe/H, iron-treated heated; DFO/C, deferoxamine-treated control; DFO/H, deferoxamine-treated heated. Results are presented as means ± SE; n = 5–6 rats per group. Ordinate scales used in A and B are consistent to allow for qualitative comparison of young and old groups. *Significant (P < 0.05) difference between vehicle-treated and iron/DFO-treated groups. {dagger}Significant effect of heat stress within a treatment group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results from the present investigation suggest that an impairment in the regulation of iron homeostasis may play a causal role in the gradual physiological deterioration that occurs with aging. Studies from other laboratories have shown that aging is associated with increased labile iron in the liver (45) and increased hepatic iron storage (9). Our results are consistent with the observations of increased iron stores and also demonstrate that there are both regional and cell-specific differences in iron deposition. Specifically, more hepatocellular iron deposition was observed in perivenous regions compared with periportal regions in old rats. The stainable iron present in hepatocytes in this area is likely due to increased ferritin iron stores (47). Interestingly, the perivenous zones also show elevated levels of lipid peroxidation damage after heat stress in old rats (55). While the current study does not definitively show that the higher iron storage in this region causes oxidative damage, a mechanistic link can be postulated. The perivenous liver region becomes hypoxic after heat stress (14), and hypoxia causes ROS generation from the mitochondrial respiratory chain (8). Iron can be released from ferritin by ROS (39), creating a scenario in which labile iron is available to react with the ROS and cause lipid peroxidation. Thus, higher iron in this region may contribute to liver injury when old rats are subjected to a physiological challenge.

The differences in the effects of heat stress on hepatic iron in young and old rats were striking. Young rats did not show excursions in iron after heat stress, while old rats demonstrated marked fluctuations in hepatic iron. The simultaneous increase in both iron pools could have several explanations involving both extracellular and intracellular mechanisms. For example, previous studies suggest that heat stress is a severe circulatory challenge (15, 25) that is associated with an elevation in heme-NO complexes in the portal blood (15). Therefore, intravascular hemolysis is a possible source of the elevation in both labile and storage iron observed in the present study. Increased catalysis of heme by hepatic cells (via heme oxygenase) would lead to subsequent liberation of iron (46). Some of the labile iron may then be stored and contribute to the increase in nonheme iron observed. The simultaneous increase in labile iron immediately after a physiological challenge suggests that the mechanisms for sequestration of iron may be impaired or delayed in aged livers.

The elevation in storage iron with aging and the increase in labile iron after heat stress in old rats led us to investigate whether age-related differences in iron regulation contributed to liver injury after heat stress. Treatment of old rats with DFO prevented the hyperthermia-induced increase in both labile iron and MDA, which suggests that iron plays a role in heat stress-induced oxidative liver injury. Iron administration to young rats and iron chelation in old rats had differential effects on oxidative liver injury. We postulate that in young rats, the additional iron was directed to storage molecules, tightly sequestered and not available to catalyze oxidative injury. Support for this concept is seen in the induction of ferritin with iron treatment in young rats. The differential effects of iron manipulation on young and old rats suggest that the combination of aging plus a physiological challenge results in iron dysregulation and liver injury, rather than iron being the sole contributor. It is postulated that changes in the physiological environment that accompany aging, such as an increase in the prooxidative state, predisposes organisms to iron dysregulation and allows iron to contribute to oxidative injury.

While changes in the redox environment occur with aging and could affect hepatic iron regulation, changes in the inflammatory milieu might also contribute to the alterations in hepatic iron homeostasis with aging and heat stress. Aging is considered a proinflammatory state marked by an increase in circulating concentrations of proinflammatory cytokines (11, 12). The reticuloendothelial system continuously recycles iron, and under inflammatory conditions, iron export from tissue macrophages is blocked (23). The liver contains a large population of macrophages (Kupffer cells) that play a major role in whole body iron metabolism. Thus, the increase in nonparenchymal iron deposition observed with aging might be due to an inflammation-mediated blockade of Kupffer cell iron recycling.

The elevation in hepatic storage iron after heat stress in old rats suggests that an APR is induced by hyperthermia. Because an APR is associated with lowered serum iron and a reduction in macrophage iron recycling (13, 41, 48), we propose that during heat stress Kupffer cell iron recycling is decreased, thus increasing hepatic iron stores. In support of this concept, elevated levels of ceruloplasmin (an acute phase protein) have been observed in portal blood of rats after heat stress (15). Furthermore, plasma concentrations of the inflammatory cytokines IL-6 and TNF-{alpha} are increased in old rats under control conditions and after heat stress, compared with their younger counterparts (unpublished observations). IL-6 induces hepcidin (31, 50), a peptide that blocks iron export in macrophages (31). Both IL-6 and hepcidin are acute phase reactants (32); hence, this axis is a plausible mechanism for increased storage iron under control conditions and with heat stress. Thus, humoral factors might also contribute to the dysregulation of hepatic iron observed with both aging and heat stress by blocking the continuous export of iron by liver macrophages.

Iron chelation with DFO attenuated the fluctuations in hepatic iron after heat stress in old rats, which suggests that it has a beneficial effect on iron homeostasis, and may serve a protective role with other physiological challenges in aged organisms. It is notable that treatment of old rats with DFO resulted in only a small lowering of hepatic nonheme iron content in the control condition. This observation may reflect the difficulty of chelating iron from storage molecules once it is sequestered. DFO also prevented the increase in MDA after heat stress, providing evidence for a causal role of iron in mediating oxidative damage in this model. Since DFO tightly binds iron, it likely decreased the amount of labile iron available to catalyze oxidative damage. By binding iron, it seems that DFO prevented the enhanced iron fluxes from reaching storage, thus explaining the decrease in storage iron under control conditions and after heat stress.

There are potential secondary effects of DFO treatment on liver storage iron in old rats as well. Specifically, DFO may modulate the proinflammatory environment that occurs with aging. Data to support this theory come from studies showing that DFO inhibits NF-{kappa}B activation (51). NF-{kappa}B is a redox-sensitive transcription factor that facilitates transcription of inflammatory mediators (29), and its activity is increased in old compared with young rats (26, 55). An inflammatory environment blocks iron efflux in macrophages (23); therefore, NF-{kappa}B-dependent increases in inflammatory mediators might explain the iron retention observed in old rats under control conditions. Further activation of NF-{kappa}B as might occur in old rats in response to heat stress would increase inflammatory cytokine transcription, which could block iron export and increase liver iron storage. Because iron is needed for NF-{kappa}B activation in Kupffer cells and DFO inhibits its activation (51), it is possible that DFO attenuates inflammatory cytokine expression in old rats under control conditions and during heat stress. The modulation of the proinflammatory environment by DFO would also contribute to its effects on hepatic iron regulation in old rats.

Hepatic ferritin protein levels were profoundly affected by heat stress and by DFO in old rats. The effects of DFO on ferritin paralleled its effects on labile and nonheme iron; DFO prevented the increase in ferritin in old rats after heat stress. The induction of ferritin protein with heat stress in old rats was a unique and unexpected finding, particularly since an elegant study by Cairo et al. (6) has shown degradation of hepatic ferritin with in vivo oxidative stress. In their study, an increase in labile iron occurred at the same time as ferritin protein degradation after phorone treatment. It is likely that the differences in stress models could account for the differences in ferritin regulation observed between studies.

While 4-HNE-modified proteins were not altered with iron manipulation or heat stress, the elevation in labile iron and the effects of DFO on MDA suggest a causal role of iron in this model. Furthermore, there is evidence for differential responses of various oxidative damage markers to a particular oxidative stressor (21). For instance, in response to carbon tetrachloride (an agent commonly used to induce oxidative stress), plasma MDA increased, while thiobarbituric acid reactive species and protein carbonyls remained unchanged (21). Hence, the accumulation of specific oxidative biomarkers is dependent on the stress used. Although we observed differential accumulation of lipid peroxidation markers with stress, it is noteworthy that the physiologically relevant stress employed caused a marked alteration in iron homeostasis in old rats.

Perspectives and Significance

This study demonstrates clear effects of age and hyperthermia on hepatic iron homeostasis and suggests that fluctuations in iron after heat stress contribute to oxidative liver injury in old rats. Importantly, the alteration in iron regulation in old organisms occurs with a sublethal stressor. The moderate heat stress employed does not cause mortality but clearly elicits dysfunction at the cellular and physiological levels in old rats. This highlights the possibility that other moderate stressors may have similar deleterious effects in aged organisms. Impaired regulation of hepatic iron might also contribute to the gradual decline in physiological function that occurs with aging. For instance, repeated increases in labile iron over time could result in DNA damage, which would alter transcription of proteins that are crucial for homeostasis. Thus, interventions such as iron chelation, antioxidants, and anti-inflammatory agents, which beneficially affect the physiological environment and prevent fluctuations in iron, may improve physiological function with aging. Delineating the cellular and systemic mechanisms that contribute to elevations in labile iron with a physiological stressor will be important areas of investigation for future studies. These approaches will identify potential therapeutic modalities to ameliorate physiological dysfunction with aging.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
The research performed in the authors’ laboratory was supported by National Institutes of Health Grant AG-12350. K. E. Brown was supported by a Merit Review Grant from the Veterans Administration. The University of Iowa College of Medicine ESR facility also supported this research.


   ACKNOWLEDGMENTS
 
The authors wish to thank Kim Broadhurst and Brett Wagner for excellent technical support and Joan Seye for assistance with manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. C. Kregel, Dept. of Integrative Physiology, 532 Field House, The Univ. of Iowa, Iowa City, IA (e-mail: kevin-kregel{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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S. A. Bloomer, H. J. Zhang, K. E. Brown, and K. C. Kregel
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