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Heat-induced liver injury in old rats is associated with exaggerated oxidative stress and altered transcription factor activation ¹

HANNAH J. ZHANG^*, LINJING XU^*, VICTORIA J. DRAKE^*, LITAO XIE^*, LARRY W. OBERLEY and KEVIN C. KREGEL^*,,2

Integrative Physiology Laboratory,
^* Department of Exercise Science and Free Radical and Radiation Biology Program,
Department of Radiation Oncology, The University of Iowa, Iowa City, Iowa, USA

²Correspondence: Integrative Physiology Laboratory, 532 FH, The University of Iowa, Iowa City, IA 52242, USA. E-mail: kevin-kregel{at}uiowa.edu

SPECIFIC AIMS

Little is known about the potential effect of aging on stress-induced oxidative damage in vivo and the extent of any subsequent physiological injury. Thus, we examined the effects of heat stress on steady-state levels of ROS, oxidative injury, changes in redox status, and DNA binding activation of critical stress response transcription factors (AP-1 and NF-B) in old vs. young rats. Experiments focused on the liver because it shows age-dependent evidence of increased ROS levels and is a prime target of tissue injury from environmental challenge. We hypothesized that older animals would have exaggerated oxidative stress and aberrant activation of stress response transcription factors in response to heat stress, leading to cellular dysfunction and age-related reductions in stress tolerance.

PRINCIPAL FINDINGS

1. Heat stress results in substantial hepatic injury in old rats
To determine the extent of liver injury from a physiologically relevant level of heat stress, young and old Fischer 344 rats were exposed to an environmental heating protocol on 2 consecutive days. Liver samples were obtained at several points over the subsequent 24 h. Heat stress produced only a small degree of histopathologic damage in young animals, which peaked at 12 h and recovered at 24 h points after heating. The damage was characterized by cellular vacuolization and sinusoidal congestion. Old animals had widespread liver injury that became more severe over the 24 h postheating period. At 24 h there was severe hepatic damage including sinusoidal congestion, monocyte infiltration, hepatocellular vacuolization, and widespread necrosis.

2. Liver injury in old animals is associated with increased steady-state levels of ROS and prolonged hepatic oxidative damage to lipids and DNA
To evaluate the effect of aging and hyperthermic challenge on steady-state levels of ROS, liver samples were obtained from young and old rats at several time points after heat stress for detection of in situ ROS. Hyperthermic challenge produced increases in the oxidative fluorescent markers dihydroethidium and dihydrofluorescein diacetate in both age groups. In young animals, steady-state levels of ROS increased only in the early stages after heating (i.e., 0, 2, and 6 h), returning to control levels by 12 h. However, fluorescence was markedly higher in the old animals; these elevated levels of staining were maintained throughout the 24 h poststress period.

In line with our histopathology and ROS results, there was exaggerated hepatocellular oxidative damage to lipids and DNA in old rats. The lipid peroxidation products malondialdehyde (MDA) was twofold higher in old vs. young controls. After heat stress, lipid peroxidation damage was significantly increased above control levels in old animals at several time points (Fig. 1 ). In contrast, there was no evidence of heat-induced lipid peroxidation in the young group. Consistent with these results, immunohistochemical analysis of the liver showed a strong accumulation of the lipid peroxidation marker 4-hydroxy-2-noneal (4-HNE) in old rats after heating, localized primarily around hepatic vessels. A similar pattern was noted in the liver for oxidative damage to DNA, with exaggerated heat-induced immunohistochemical staining for the DNA oxidation product 8-hydroxy-2'-deoxyguanosine in hepatic nuclei of the old cohort.

View larger version (27K): [in this window] [in a new window]   Figure 1. Heat stress increases lipid peroxidation damage in the liver of old rats. Tissue samples were obtained in young and old rats in control conditions and 0, 2, 6, 12, and 24 h (n=3 rats/age group at each time point) after a heating protocol. Lipid peroxidation product MDA was measured using a colorimetric assay. Relative changes from young control values for MDA are presented. Liver MDA levels were unchanged in young rats after heat stress, whereas old rats responded to heating with increased levels of MDA. *P < 0.05 vs. young group for a specific time point. P < 0.05 vs. old control group.

3. Alterations in intracellular redox profiles and activation patterns for the stress response transcription factor AP-1
The ratio of glutathione (GSH) to glutathione disulfide (GSSG) was measured in liver samples from control and heated animals to evaluate hepatic redox status. This ratio was significantly lower in old vs. young controls, indicative of an oxidized environment in the liver with aging. After heat stress, the GSH/GSSG ratio transiently decreased in young livers, then returned to control levels by 12 h poststress. Conversely, GSH/GSSG values remained unchanged from their depressed control levels after heating in the old cohort.

DNA binding activities of AP-1 and NF-B were increased in hepatic nuclear extracts from old vs. young control rats. Relative binding activity for AP-1 was sharply elevated in young animals at early time points after heating, reaching a peak at 2 h with an eightfold increase, then returning to control levels by 12 h (Fig. 2 A). In contrast, binding activity in the old group was elevated above control levels only at 1 h; this increase was <threefold (Fig. 2B ). However, there was a trend for higher binding activity levels compared with the control condition at all subsequent times for old animals. In contrast to the AP-1 responses, NF-B binding activity did not change in either young or old animals in response to heat stress.

View larger version (60K): [in this window] [in a new window]   Figure 2. The increase in AP-1 binding activity in response to heat stress is more pronounced in young rats. Liver samples were obtained from young and old rats in euthermic control conditions and 1, 2, 6, 12, and 24 h after a heat stress protocol (n=3 rats/age group at each time point). Nuclear extracts were analyzed by electrophoretic mobility shift assay (EMSA) using AP-1-specific 32P-labeled oligonucleotides with representative images presented for young (A) and old (B) rats. Densitometry values for AP-1 DNA binding were obtained from EMSA gels using NIH image software. Values presented in the bar graphs represent the mean and SE of 3 animals/time point for both young and old rats. Con, control; Mut, mutant probe. *P < 0.05 vs. control condition.

CONCLUSIONS

In the current studies we demonstrate that the reduction in stress tolerance that accompanies aging is associated with a complex set of integrated alterations in hepatic steady-state levels of ROS, macromolecular damage, redox buffering, and transcription factor regulation. We used an environmental heating protocol that produced several distinct differences in hepatic responses between young and old animals. First, heat stress produced clear evidence of a small, transient rise in oxidative stress in the liver of young rats. In contrast, old rats had an impressive increase in steady-state levels of ROS after heating that was maintained up to 24 h.

Second, this increase in ROS was associated with a distinct temporal pattern of liver injury in the old animals, including hepatic vacuolization, necrosis, and inflammation, which became progressively worse over the 24 h postheating period. The exaggerated ROS accumulation in the old group was also associated with marked oxidative damage to lipids and DNA during the 24 h following hyperthermic challenge. The most pronounced response among oxidative damage products measured was for lipid peroxidation, with significantly higher levels present in the livers of aged rats after heat stress. This is an important observation because the peroxidation of polyunsaturated fatty acids can lead to changes in cellular membrane permeability and even membrane leakage. The time course for the lipid peroxidation increase after heat stress in old animals preceded the liver injury, suggesting that exaggerated steady-state levels of ROS and oxidative damage in old animals contributes to the cellular injury and concomitant decrease in stress tolerance associated with aging. In contrast, there were only a small, transient ROS accumulation and lipid peroxidation responses after heat stress in the young rats. These data suggest a strong functional linkage between cellular oxidative stress and the age-related pathophysiological responses produced from hyperthermic challenge.

Third, old animals had aberrant intracellular redox profiles compared with their young counterparts. It was not surprising that the old group had a lower liver GSH/GSSG ratio than their young counterparts in control conditions. However, young animals had a reduced GSH/GSSG ratio in the early stages of recovery, then a progressive recovery back to control levels at 24 h postheating, whereas old animals failed to produce any changes in the GSH/GSSG ratio in response to heating. The observation that heat stress transiently reduced the in vivo GSH/GSSG ratio in young animals is supportive of the involvement of oxidative stress in hyperthermia. We postulate that GSH functions as an effective redox buffer in environmentally challenged young animals to prevent oxidative damage to intracellular macromolecules, whereas in old animals the GSH/GSSG ratio has been lowered to a threshold level where any further decline in this ratio may endanger the survival of an organism. In this scenario, an expected outcome of a decreased GSH/GSSG ratio in older animals would be a blunted ability to buffer ROS accumulation, resulting in increased oxidative stress and an exaggerated accumulation of macromolecular damage products—outcomes consistent with results in the current study.

Finally, old rats had altered DNA binding activation patterns for AP-1, a critical stress response transcription factor. This activation preceded hepatic injury. These results suggest that intracellular signal transduction responses modulated by AP-1 may play an active role in determining the extent of age-associated liver damage.

In conclusion, the present data show that hyperthermic challenge in young rats produces a small, transient increase in oxidative stress in the liver that is associated with a decline in the ratio of GSH to GSSG, suggesting that these animals have an effective redox buffering system that provides protection from oxidative damage to intracellular macromolecules in a stressful situation. In contrast, widespread and prolonged liver injury is present in aged rats after heat stress, which is associated with exaggerate in vivo levels of ROS and oxidative damage to hepatic lipids and DNA (Fig. 3 ). Heat-stressed older animals also have an aberrant intracellular redox profile and an altered activation pattern for the critical stress response transcription factor AP-1. Taken together, these data suggest that environmental stress in older animals produces exaggerated steady-state levels of ROS along with extensive hepatocellular oxidative damage, an aberrant redox buffering system, and alterations in intracellular signal transduction that can contribute to cellular dysfunction and age-related reductions in stress tolerance.

View larger version (41K): [in this window] [in a new window]   Figure 3. Schematic summary of proposed mechanisms for heat-induced liver injury in old rats. An environmental stress such as heat exposure results in an increase in reactive oxygen species (ROS) production in hepatocytes of young and old animals. In young animals, intracellular redox buffering mechanisms (e.g., antioxidants, glutathione) are able to prevent ROS accumulation. Activation of immediate early response transcription factors (TF) such as AP-1 modulates the expression of various stress response genes. The result is preservation and recovery of physiological function. In contrast, old rats demonstrate a decline in redox protection, leading to extensive oxidative damage to intracellular macromolecules and an attenuated stress gene response. These alterations can result in cellular dysfunction and reduced stress tolerance in older organisms.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0139fje; doi: 10.1096/fj.03-0139fje

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This Article Full Text (PDF) All Versions of this Article: 17/15/2293 03-0139fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZHANG, H. J. Articles by KREGEL, K. C. Search for Related Content PubMed PubMed Citation Articles by ZHANG, H. J. Articles by KREGEL, K. C.