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Redox modulation of the liver with chronic antioxidant enzyme mimetic treatment prevents age-related oxidative damage associated with environmental stress

HANNAH J. ZHANG^*, SUSAN R. DOCTROW, LINJING XU^*, LARRY W. OBERLEY, BENJAMIN BEECHER^*, JOANNA MORRISON^*, TERRY D. OBERLEY and KEVIN C. KREGEL^*,1

^* Integrative Physiology Laboratory, Department of Exercise Science and
Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, Iowa, USA;
Eukarion, Inc., Bedford, Massachusetts, USA; and
Department of Pathology, University of Wisconsin, Madison, Wisconsin, USA

¹ Correspondence: Integrative Physiology Laboratory, 532 FH, University of Iowa, Iowa City, IA 52242, USA. E-mail: kevin-kregel{at}uiowa.edu

SPECIFIC AIMS

A reduction in stress tolerance is a hallmark of the aging process, and the lowered functional capacity observed in aged organisms is associated with an increased rate of oxidative stress and a greater susceptibility of aged tissues to oxidative injury. We investigated whether continuous, low-dose infusion of a synthetic catalytic reactive oxygen species (ROS) scavenger would be efficacious in preventing age-associated hepatic injury generated in response to an environmental challenge. Rats were chronically administered the superoxide dismutase (SOD)/catalase mimetic EUK-189, then exposed to a heat stress protocol. Hepatic function, oxidative injury, redox status, and DNA binding activity of stress response transcription factor AP-1 were then assessed. We hypothesized that EUK-189 treatment would enhance stress tolerance in aged animals by reducing cellular oxidative stress and subsequent accrual of hepatic injury.

PRINCIPAL FINDINGS

1. EUK-189 supplementation improves the redox profile in old rats
Young and old Fischer 344 rats were implanted with mini-osmotic pumps for chronic (30 days) delivery of either EUK-189 or vehicle. Animals were exposed to a heating protocol on each of the last two days of supplementation and liver samples were obtained 2 h after the second heating trial. To evaluate the effect of EUK-189 supplementation on hepatic redox status, the ratio of glutathione (GSH) to glutathione disulfide (GSSG) was measured in the following groups of young and old rats: 1) vehicle-treated and sham-heated (control); 2) vehicle-treated and heat-stressed (V/H); 3) EUK-189-treated and heat-stressed (E/H); and 4) EUK-189-treated and sham-heated (E/NH).

Old rats had a lower GSH/GSSG ratio than their young counterparts in the control condition, indicative of a more oxidized hepatic environment. EUK-189 supplementation increased this ratio in old sham-heated groups to a level similar to that of young controls. EUK-189 also preserved the redox potential of young and old heat-stressed rats by maintaining GSH/GSSG ratios at young control levels.

2. EUK-189 supplementation protects old rats from heat-induced liver injury
Hepatic morphology was evaluated to determine whether EUK-189 supplementation could attenuate extensive histopathologic damage in the liver that accompanies heat stress in old rats (Fig. 1 , top panels). Old V/H rats exhibited widespread and severe liver damage. In sharp contrast, this heat-induced liver damage in the old cohort was prevented by EUK-189 treatment, as demonstrated by normal hepatic morphology that was similar to the control condition.

View larger version (99K): [in this window] [in a new window]   Figure 1. EUK-189 supplementation protects old rats from heat-induced liver injury. Tissue morphology in hematoxylin and eosin stained liver sections obtained under euthermic control conditions and 2 h after a heating protocol in old rats (top panels) was normal in old control rats (A), while old vehicle-treated heat-stressed rats (V/H group; panels C, D) exhibited extensive liver damage, characterized by monocyte infiltration (C), along with sinusoidal congestion and hepatocyte vacuolization (D). EUK-189-treated heat-stressed rats (E/H group) had normal hepatic morphology (B) that was similar to the control condition. x100. EUK-189 supplementation also prevents hepatic ALT release (bottom panel). Plasma samples were obtained from young and old rats in control conditions and 2 h after a heating protocol. Old rats in the V/H group had elevated ALT levels, while old rats in the E/H group had reductions in plasma ALT back to control values (n=5–9 rats/age group for each treatment). *P < 0.05 vs. old control; P < 0.05 vs. young V/H.

Plasma alanine aminotransferase (ALT) levels were also measured as a systemic marker of liver injury (Fig. 1 , bottom panel). In young animals, there were no changes in plasma ALT with heat stress or EUK-189 treatment compared with control levels. However, V/H old animals exhibited a 4-fold increase in plasma ALT levels, which was consistent with severe hepatocellular damage noted in these same animals. Conversely, heat-stressed old rats treated with EUK-189 (E/H) had ALT levels that were similar to those in old sham-heated control rats.

3. EUK-189 supplementation prevents heat-induced hepatic lipid peroxidation
Lipid peroxidation marker malondialdehyde (MDA) was assessed to evaluate the effects of EUK-189 supplementation on heat-induced hepatic oxidative damage. Liver MDA levels in old V/H rats were increased above old control and young heated values (Fig. 2 ). Conversely, heat-stressed old rats that received EUK-189 had MDA levels that were significantly lower than the V/H animals and were similar to those obtained in sham-heated animals with EUK-189 supplementation. The presence of lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) was also examined in the liver using immunohistochemical techniques. 4-HNE immunostaining levels in hepatocytes were consistent with liver MDA results.

View larger version (13K): [in this window] [in a new window]   Figure 2. EUK-189 supplementation prevents heat-induced hepatic lipid peroxidation. Tissue samples were obtained from young and old rats in control conditions and 2 h after a heating protocol. Liver MDA levels were unchanged from control values for all three treatments in the young rats. MDA level increased over 2-fold after heat stress in the V/H rats compared to old controls, while heat-stressed old rats that were chronically treated with EUK-189 (E/H group) had MDA levels that were similar to those obtained in sham-heated control conditions. *P < 0.05 vs. young rats within a treatment group; P < 0.05 vs. old control, E/H, and E/NH groups.

4. EUK-189 supplementation normalizes DNA binding activity of AP-1
DNA binding activity of redox-sensitive transcription factor AP-1 was determined in hepatic nuclear extracts from young and old rats to evaluate the effects of heat stress and EUK-189 treatment on intracellular signal regulation. Old rats in the V/H group had a 2-fold increase in relative AP-1 DNA binding activity compared with control levels. However, the corresponding old heat-stressed group that had received chronic EUK-189 treatment instead of vehicle showed significantly lower AP-1 DNA binding activity, with values approximating those in old control animals.

CONCLUSIONS AND SIGNIFICANCE

In the current study we demonstrate for the first time that substantial oxidative stress-mediated injury associated with an acute physiological stress in old animals can be prevented with a chronic treatment protocol that utilizes low-concentration delivery of a synthetic catalytic scavenger of ROS. Old rats that received chronic SOD/catalase mimetic supplementation had integrated cellular changes that were associated with dramatically improved tolerance to heat stress. For instance, cellular redox potential, which is an important factor governing the fate of cells, was significantly improved in the liver of old rats that received EUK-189. Previous experiments from our laboratory suggest that decreased redox potential with aging is associated with loss of stress tolerance. Findings from the present study reinforce these observations and provide a potential therapeutic approach for aged organisms to preserve intracellular redox potential and increase antioxidant capacity.

Our results indicate that pretreatment of old rats with EUK-189 for 1 month prevents oxidative stress and produces protection against pathophysiological manifestations of heat stress in the liver. The novel findings from this in vivo stress protocol suggest a strong causal relationship between exaggerated oxidative stress in old animals and the marked cellular injury and reduced tolerance to stress with aging. These results provide strong support for the postulate that oxidative stress plays a critical role in triggering heat-induced hepatic damage, and that older organisms are especially susceptible to this type of injury.

Oxidative damage has long been thought to play a key role in many pathophysiological manifestations of aging, and numerous studies have reported increases in oxidation of intracellular macromolecules throughout the body. These findings have led to the postulate that an older organism lacks sufficient antioxidant defense mechanisms to adequately cope with age-related increases in oxidative stress. To address this possibility, recent investigations have used synthetic antioxidant enzyme mimetics in a variety of models and demonstrated that these molecules can alleviate oxidative damage associated with several disease states.

We have previously demonstrated that an acute heat stress manifests dramatic liver injury, elevates steady state levels of ROS, and increases lipid peroxidation in old rodents. In the current study, instead of addressing potential therapeutic properties of an antioxidant mimetic in dealing with an existing pathological condition, we used EUK-189 as a systemic treatment to prevent oxidative-mediated cell injury and systemic dysfunction that accompanies an environmental challenge in highly susceptible older animals. These results suggest that ROS play a key role in age-related hepatic damage manifested with heat stress and that EUK-189 is protective via an antioxidant mechanism (Fig. 3 ).

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic summary of proposed mechanisms for heat-induced liver injury in old rats and prevention of oxidative-mediated damage with chronic SOD/catalase treatment. An environmental stress such as heat exposure results in an increase in production of reactive oxygen species (ROS) such as O2·– and peroxides in hepatocytes of old animals (right). A decline in redox protection with old age leads to extensive oxidative damage to intracellular macromolecules as well as exaggerated and prolonged activation of selected "stress response" transcription factors such as AP-1. These alterations can result in cellular dysfunction in older organisms. In old animals supplemented with ROS scavenger EUK-189 (left side), intracellular redox buffering is enhanced, providing protection from oxidative damage. Immediate early response transcription factors are not activated. The result is preservation and recovery of physiological function.

AP-1 is a stress response transcription factor that is sensitive to intracellular redox balance and oxidative stress. We have recently shown that there is an age-related alteration in DNA binding activity of AP-1 during a time course of recovery from heat stress that is associated with hepatic injury. In the current study, EUK-189 supplementation abolished the activation of AP-1 in response to acute heat stress in both young and old animals. This suppression of AP-1 activation was mirrored by protection from hepatic injury and elimination of oxidative damage in old animals. Since AP-1 is an early-response transcriptional factor that can up-regulate many downstream stress response genes, including those of potentially injurious agents such as cytokines, our observations strongly suggest that a connection exists between redox regulation of signal transduction pathways and oxidative stress-mediated tissue injury. Moreover, these results imply that AP-1 activation may be important in determining the onset and extent of heat stress-induced hepatic injury.

This study demonstrates that long-term synthetic SOD/catalase mimetic administration protects against acute stress-induced hepatic injury in aged animals through mechanisms involving the elimination of oxidative damage to macromolecules and the modulation of signal transduction pathways. These results also implicate a role for intracellular redox modulation in the reduced tolerance to physiological stress that accompanies aging. Overall, these results demonstrate that oxidative stress is involved in age-related hyperthermic injury and suggest that oxidative-mediated injury associated with environmental stress can be prevented with a chronic treatment protocol that utilizes SOD/catalase mimetics.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1629fje;

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