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Annals of Clinical & Laboratory Science 39:64-70 (2009)
© 2009 Association of Clinical Scientists

Effects of Aminoguanidine on Tissue Oxidative Stress Induced by Hindlimb Unloading in Rats

Parimal Chowdhury, Michael E. Soulsby and Joseph L. Scott
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Address correspondence to Parimal Chowdhury, Ph.D., Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205, USA; tel 501 686 5443; fax 501 686 8167; e-mail pchowdhury{at}uams.edu.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We investigated the effects of hindlimb unloading (HLU) on malondialdehyde (MDA), a biomarker for oxidative stress, and glutathione (GSH) levels in tissues of rats. Aminoguanidine (AG), a nucleophilic hydralazine compound and an in vivo antioxidant against reactive oxygen species (ROS) and lipid peroxidation, was used to confirm the HLU-induced oxidative response. Three groups of rats were used: Group 1 was a loaded control group that was maintained on drinking water only; Groups 2 and 3 were hindlimb unloaded (HLU) groups that were maintained on drinking water and on AG in drinking water, respectively. The hindlimb unloaded rats maintained on tap water had significantly elevated MDA levels in 7 tissues (brain, lung, pancreas, kidney, intestine, heart, liver) when compared to the paired hindlimb loaded controls (p <0.05). In contrast, the hindlimb unloaded rats maintained on AG in drinking water had no increase in tissue MDA levels when compared to the loaded controls; moreover, their tissue MDA levels were significantly reduced from the HLU group on tap water (p <0.05). In HLU rats maintained on AG, there were no changes in tissue GSH levels with the exception of brain, where GSH levels were significantly reduced when compared to the other groups (p <0.05). In summary, HLU induced an oxidative response in rats and this response was reduced significantly by ingestion of AG. These results suggest the potential application of AG in the diet of astronauts living in a stressful environment.

Keywords: hindlimb suspension, malondialdehyde, GSH, oxidative stress, simulated microgravity


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Studies conducted on astronauts and cosmonauts to examine lipid peroxidation (LPO) damage have produced conflicting results [1–4]. These results are attributed to the loss of antioxidant protein defenses secondary to in-flight reductive modeling of skeletal muscle, to decreased work load on the antigravity muscles, and to inadequate dietary intake [1–6].

Studies examining muscle lipid peroxidation (LPO) damage in rats returning from space flight [7] and in hindlimb unloaded (HLU) rats [8] have also been conducted. These studies show that undernutrition is not a likely factor in the post-flight response in rodents, for unlike humans, rats are able to maintain food intake and energy balance after the initial few days of space flight [9] or under hindlimb suspension (HLU) [10]. This suggests that use of rodents instead of humans as model is less complex to assess the effects of microgravity on tissue metabolism.

To study muscle lipid peroxidation damage in following exposure to microgravity, Nikawa et al [11] investigated key genes for elucidating the mechanisms of skeletal muscle wasting in space. They identified two spaceflight-specific gene expression patterns associated with spaceflight: (a) imbalanced expression of genes in the mitochondrial electron transport system, and (b) up-regulated expression of ubiquitin ligase oxidative stress-induced genes. More recently, Onishi et al [12] reported that oxidative stress associated with hindlimb unloading and spaceflight resulted in accumulation of mono-ubiquinated LDH in gastrocnemius skeletal muscle. They concluded that oxidative stress induces mono- and poly-ubiquination of LDH-A, which may be involved in lysosomal degradation during hindlimb unloading. Hence, oxidative stress may play an important role in triggering protein-ubiquitination in skeletal muscles atrophied by weightlessness. Spaceflight-associated skeletal muscle oxidative changes have been well documented in the literature. Matsushima et al [13], Fomina et al [14], and Selsby et al [15] have investigated the effects of hindlimb unloading, reloading, and immobilization on oxidative damage in skeletal muscle.

Vaziri et al [16] and Sangha et al [17] have provided evidence that cardiovascular adaptation to microgravity may involve up-regulation of the nitric oxide (NO) vasodilator system. Since it also inhibits renal tubular sodium reabsorption, the NO vasodilator system has a major role in the regulation of systemic vascular resistance and blood pressure. Hence, the vasodilator response could be blocked by the inhibitor of the inducible form of NO synthase, aminoguanidine. In earlier studies from this laboratory we have shown that brain tissues from hindlimb unloaded rats contained significantly higher MDA levels than brains from control hindlimb loaded rats on a soy diet [18,19] suggesting a role of soyproteins as dietary anti-oxidants. Since lipid peroxidative stress-related changes in muscle have been well documented in the literature, and also since we have demonstrated oxidative stress-related changes in brain after hindlimb unloading, in this study we elected to investigate the extent to which oxidative changes occur in other organs in response to simulated weightlessness. We investigated a specific oxidative pathway by employing a specific inhibitor of the nitric oxide synthase pathway.

Oxidative stress induces increased oxidation of membrane fatty acid moieties leading to production of unstable lipid peroxides. These peroxides tend to degrade rapidly into products such as malondialdehyde (MDA) [20]. The formation of these aldehydes contributes significantly to the mechanisms of oxidant-induced injury.

Aminoguanidine (AG) is a compound that reportedly exerts both pro- and antioxidant effects, depending upon concentration [21]. AG is a relatively selective inhibitor of the inducible form of nitric oxide synthase and has been shown to be efficient in animal models [22,23] and in humans [24,25] as an antioxidant against lipid peroxidation by decreasing NO production. Thus the objective of this study was to determine if aminoguanidine (AG) in drinking water reduces the susceptibility of tissue membrane fatty acid particles to oxidation, thereby affecting lipid peroxidation induced by hindlimb suspension of rats.

Improvement of the imbalance in the pro-oxidant/antioxidant defense system may lessen the severity of the oxidative stress documented during space flight. To determine the effect of HLU on the cellular GSH redox system, we measured the activities of total GSH (GSH and GS-SH) in homogenates from harvested tissues following 2-wk intervals of HLU. The GSH assay provides an accurate and reliable measure of GSH production and reflects tissue usage of one of the major antioxidant systems.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The protocol and procedures used in this study were in accordance with the Guiding Principles in the Care and Use of Animals of the American Physiological Society, and were approved by the University of Arkansas for Medical Sciences (UAMS) Institutional Animal Care and Use Committee.

Experiments were performed on male Sprague-Dawley rats (N = 18, initial body weight, 285–338 g). Animals were randomly assigned to 3 groups: non-suspended (LC, N = 6) with Purina chow and water only; tail-suspended (HLU, N = 6) with Purina chow and water only, or HLU with Purina chow and water containing 50 mg/L aminoguanidine. The animals were acclimated for 1 wk prior to the study. Body weights of all animals were monitored on the first day of arrival and again on day 7 (the first day of the experiment). Subsequently, the body weights, food intakes, and fluid intakes were monitored daily until termination of the experiment. The animals were housed in circadian chambers maintained on 12:12 hr light-dark cycles. They were allowed free access to tap water and were fed the diets for 21 days. No surgical interventions were involved in this study.

HLU was achieved using a modification of the Morey [26] tail-suspension model. Rats assigned to suspended groups were housed individually in Plexiglas chambers (10 x 19.5 x 21 inches). HLU was accomplished with a tail harness constructed by looping a 0.5 x 10-inch Skin-Trac (Zimmer, Inc., Charlotte, NC) orthopedic foam strip around a pulley that can travel along a bar that traverses the length of the cage. The adhesive surfaces along the remainder of the Skin-Trac strip were applied to the long axis of opposite sides of the tail, creating a tail-sandwich. This sandwich was encircled by a bias-cut orthopedic stockinette and secured with one-inch glass zip-reinforced strapping tape at the base and tip of the tail. This construction did not interfere with the animal’s ability to use its tail to maintain its core body temperature. Control values were recorded for 7 days before suspension of the animals at a 30° angle with the cage floor.

Twenty-four hours before sacrifice, the animals were deprived of food but not water to lessen intestinal content. At sacrifice, rats were anesthetized with ketamine hydrochloride and euthanized by decapitation. Liver, pancreas, kidney, small intestine, heart, lung, brain, and plasma were harvested. Each organ was washed in ice-cold normal saline solution. After each organ was weighed, each sample was homogenized in 20 mM phosphate buffer (pH = 7.4; tissue/buffer ratio, 1/10 w/v). Then 10 µ l of 0.5 M BHT was added per 1.0 ml of homogenate to prevent sample oxidation.

MDA was assayed by the method of Esterbauer et al [20]. Harvested tissues were washed in ice-cold NaCl solution. After 0.4 to 0.5 g of each tissue was excised, each sample was homogenized in 20 mM phosphate buffer, pH = 7.4 (tissue to buffer ratio, 1:10 w/v). The homogenates were centrifuged at 4,000 g at 4°C for 10 min. Then 100 µ l of supernatant from each homogenate was used to analyze the MDA level. Measurement of MDA was based on reaction of a chromogenic reagent, N-methyl-2-phenylindole (R1), with MDA at 24°C. One molecule of MDA interacts with 2 molecules of R1 to yield a chromophore with maximal absorbance at 586 nm, which is stable for 1 hr at room temperature. The net absorbance measured at 586 nm yields a linear function of MDA concentrations from 0 to 20 µ M. The detection limit of MDA in this assay is 0.1 µ M. The tissue MDA content was expressed as µ mol/mg protein.

GSH concentrations were determined in homogenates of harvested tissues using a glutathione assay kit (Cayman Chemical Co., Ann Arbor, MI). In this procedure the sulfhydryl group of GSH reacts with DTNB (5,5'-dithiobis-2-nitrobenzoic acid) to produce GSTNB (the mixed disulfide), which is reduced by glutathione reductase to recycle the GSH and produce more TNB (5-thio-2-nitrobenzoic acid). The rate of TNB production is directly proportional to this recycling reaction, which in turn is directly proportional to the concentration of GSH in the sample. The assay has a dynamic range of 0–16 µ moles GSH or 0–8 µ moles GSSG. The intra-assay coefficient of variation (CV) is 1.6% and the inter-assay CV is 3.6%.

Tissue protein concentrations were measured by the Bradford method [27]. Results were espressed as mean ± SE. Statistical significance was determined by one-way ANOVA. A value of p <0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Table 1Go shows the effect of drinking water alone, and of aminoguanidine in drinking water, on tissue MDA levels of loaded and unloaded rats. MDA levels in all tissues harvested from loaded control rats on drinking water only were not significantly different from MDA levels in tissues from loaded control rats on water containing aminoguanidine (p >0.05). The MDA levels in all tissues harvested from unloaded (HLU) rats on drinking water only were increased significantly from MDA levels in tissues from loaded control rats on drinking water only (p <0.05). Tissues from unloaded (HLU) rats maintained on water containing aminoguanidine showed significantly decreased MDA levels, when compared to both loaded and unloaded groups without aminoguanidine (p <0.05).


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  		Table 1. Effect of water alone, and of aminoguanidine (AG) in the drinking water, on MDA levels in tissues of hindlimb loaded and unloaded rats.
 
Results showing the effect of drinking water alone, and of AG in the drinking water, on tissue GSH levels of loaded and unloaded rats are shown in Table 2Go. GSH levels in all tissues harvested from loaded control rats on drinking water only were not significantly different from GSH levels in tissues from loaded control rats on water containing aminoguanidine (p >0.05). The GSH levels in tissues harvested from unloaded (HLU) rats on drinking water only were not significantly different from levels in the same tissues harvested from loaded control rats on drinking water only. Tissues harvested from unloaded (HLU) rats maintained on water containing aminoguanidine were not significantly different from GSH levels in similar tissues harvested from loaded and unloaded groups without aminoguanidine, with the exception for brain tissue, where GSH levels were significantly less (p <0.05) than GSH levels in loaded and unloaded rats on drinking water only.


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  		Table 2. Effect of water alone, and of aminoguanidine (AG) in the drinking water, on GSH levels in tissues of hindlimb loaded and unloaded rats.
 
Plasma MDA levels in loaded rats and in unloaded rats maintained on only water, and on AG in the drinking water are shown in Fig. 1Go. MDA levels in plasma from loaded rats on water alone were not significantly changed from the MDA levels in plasma from unloaded rats on water only. Plasma MDA levels of unloaded rats with AG in the drinking water were significantly less than those of the other groups (p <0.05).


Figure 1
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  		Fig. 1. Effect of water alone, and of aminoguanidine in drinking water, on plasma MDA levels in the experimental groups. Controls = non-suspended (loaded) rats; HLU = hindlimb suspended rats on water alone; HLU + AG = suspended rats with aminoguanidine (AG) in drinking water. * p <0.05 vs HLU group. The data are means ± SE, N = 6 rats/group.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Membrane lipid peroxides tend to degrade rapidly into products such as malondialdehyde (MDA) [20]. Therefore, tissue MDA levels are often used to monitor cellular stress as indicators of the degree of oxidation of membrane fatty acids [20]. Since lipid peroxidative stress-related changes in muscle have been well documented in the literature, we elected in this study to investigate the extent to which oxidative changes are present in other organs following simulated weightlessness. We measured MDA levels in selected organs harvested from time-matched non-suspended control rats, and in time-matched tail-suspended unloaded (HLU) rats. The MDA levels were increased in all tissues harvested from HLU rats, providing a measure for the effect on tissue oxidation and subsequent MDA levels of the stress of suspension (unloading).

Aminoguanidine (AG) decreases nitric oxide production, leading to decreased production of lipid peroxides. AG has been shown to be effective in animal models as an antioxidant against lipid peroxidation [22,23]. AG is thought to protect LDLs against lipid peroxidation by its scavenging effect toward lipid peroxyl radicals [21]. AG also acts as an anti-oxidant in vivo preventing ROS formation and lipid peroxidation in cells and tissues, and preventing oxidant-induced apoptosis [31]. In the current study, administration of AG in drinking water significantly decreased tissue MDA levels, when compared to both loaded and unloaded groups without AG (p <0.05), thus confirming its role as antioxidant to lipid peroxidation induced by HLU. These results further suggest that lipid peroxidation induced by hindlimb suspension is activated at least in part by iNOS pathways, since the MDA levels were significantly decreased in the presence of AG, a relatively specific inhibitor of inducible nitric oxide synthase. The antioxidative effect of AG is perhaps exerted by scavenging peroxyl radicals, hydroxyl radicals, and superoxide radicals, which are dismutated to hydrogen peroxide by superoxide dismutases.

Numerous investigators have shown that cell lines adapted to a highly peroxidative environment are resistant to the cytotoxicity of aldehydes like MDA formed during lipid peroxidation [28]. This resistance appears to be related to increased cellular metabolism of the aldehydes, possibly through the glutathione peroxidases and catalases, which reduce the hydrogen peroxide product of the superoxide dismutases. Glutathione reacts with the hydrogen peroxide, reducing it to water and oxygen and lessening the H2O2-mediated cytotoxicity [29]. To determine the effect of HLU on the cellular GSH redox system, we measured the activities of both GSH and GS-SG (total glutathione activities) in homogenates from tissues harvested following 2-wk intervals of HLU.

The GSH levels in tissues harvested from HLU rats on drinking water alone were not significantly different from levels in the same tissues harvested from control loaded rats on drinking water alone. Tissues harvested from HLU rats maintained on water containing AG were also not significantly different from GSH levels in similar tissues harvested from control and unloaded groups without AG, except for brain tissue, where GSH levels were significantly less (p <0.05) than GSH levels in control and unloaded rats on drinking water only. Possibly the brain, with its psychological stress component, is more susceptible to membrane lipid peroxidation than other tissues studied, resulting in an elevation in radical production upon unloading, and a need for the GSH redox system to eliminate these additional radicals.

A recent study in our laboratory [32], similar to the one described here, monitored superoxide dismutase (SOD) levels in addition to GSH and MDA in hindlimb unloaded, animals, followed immediately by hindlimb reloading. The SOD levels in all tissues harvested rose with unloading and continued to rise further upon reloading. This result was interpreted as continued production of LPO during the short reloading period. Increased superoxide generation, by several mechanisms, is thought to be an initial component of the cellular membrane peroxidation process. Following this, nitric oxide acts as a potent inhibitor of the propagation process.

In summary, AG reduces the susceptibility of fatty acid particles to oxidation, as evidenced by the universal reduction in tissue MDA levels after unloading in the presence of AG. The mechanism of AG action is probably via reduction of tissue peroxyl and hydroxyl radicals using its free-radical scavenging activity, as well as its inhibition of inducible nitric oxide synthase. Evidence for this mechanism is strengthened by the lack of need for the GSH redox system after unloading in the presence of AG. If the concentration of peroxyl radicals were elevated after unloading in the presence of AG, the cell’s natural GSH redox system would have been called upon to eliminate these radicals. This action would reduce tissue GSH levels after unloading in the presence of AG. The absence of GSH reduction after unloading suggests that other mechanisms for radical reduction must have been activated. Cells utilize multiple layers of antioxidant defenses in order to cope with the stress that oxygen engenders [30].


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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