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Effect of Hibernation on Oxidative and Antioxidant Events under Laboratory Conditions in Anatolian Ground Squirrel, Spermophilus xanthoprymnus (Bennett, 1835) (Mammalia: Sciuridae) from Central Anatolia.

Byline: Emre Avci, Safak Bulut, Filiz Sezen Bircan, Aydin Ozluk and Sule Coskun Cevher

Abstract The oxidative stress is an integral part of the metabolic depression machinery associated with hibernation, estimations and tolerance to freezing, dehydration, hypoxia and anoxia In the present study, we aim to investigate the role of hibernation on lipid peroxidation, nitric oxide production and glutathione levels in various tissues of Anatolian Ground Squirrel Spermophilus xanthoprymnus.

A total of 9 female ground squirrels were collected from different localities in Nigde, Bolkar Mountains, Turkey. All squirrels were in three different conditions: at hibernation, aroused and non-hibernation stage. They were sacrificed under anesthesia.

Glutathione (GSH), reactive nitrogen oxide species (NOx) and malondialdehyde (MDA) levels were measured spectrophotometrically. GSH levels of almost all tissues were lower in the hibernating group compared with the aroused group. NOx levels were found to decrease in all tissues except the brain tissue examined on hibernating group compared to non-hibernating group.

MDA levels were found to increase in brain, lungs and heart tissues examined on hibernating group compared to non- hibernating group. In conclusion, our data show that an impaired balance exists between oxidative stress and antioxidant systems in most organs and tissues during hibernation.

Key words: Hibernation, Spermophilus xanthoprymnus, oxidative stress, antioxidant.

INTRODUCTION

The ground squirrel, Spermophilus xanthoprymnus, is a terrestrial diurnal rodent; an excellent hibernator of the family Cricetidae. Bennet first described S. xanthoprymnus from Erzurum Turkey in 1835 (Yigit et al., 2000), followed by many studies done on different aspects including hibernation (Dogramaci et al., 1995; Yigit et al.,

2000).

Hibernation is a physiological adaptation

characterized by entry into torpor, which involves profound decrease in metabolism, heart rate, respiration, and body temperature (a few degrees above the ambient temperature) (Geiser, 2004; Okamoto et al., 2006).

During hibernation, mammals undergo repeated cycles of torpor and arousal and several physiological and biochemical parameters are reversed to normal euthermic levels

without obvious damages in a short period when they return to aroused state (Okamoto et al., 2006).

Mammalian hibernators are exposed to hypoxia due to rapid increase in oxygen consumption while increasing to 37degC in a few

minutes the body temperature during waking from

hibernation outside receive apneic oxygenation in a deep hibernation (Storey and Storey, 2004).

Besides that, dramatic changes due to wake and deep hibernation cycles result in increasing risk of oxidative stress in sensitive tissues of mammalian hibernators (Carey et al., 2000). Oxidative stress occurs when intracellular concentrations of free radicals increase over the physiologic values.

Mammalian cells have developed elaborate antioxidant defense systems to prevent oxidative damage caused by free radicals (O'Duffy and Goldstein, 1976; Raborn and Grace, 2003). However, hibernators clearly tolerates the cycles of torpor and arousal (Carey et al., 2003a; Okamoto et al., 2006).

The mechanisms of adaptation to the oxidative stress during re-warming of hibernators is not yet clearly understood. Markers of oxidative stress such as glutathione (GSH), nitrous oxide (NO) and malondialdehyde (MDA) are likely to provide such explanation.

GSH is an antioxidant whose role is to prevent damage caused by reactive oxygen species (ROS) (Pompella et al., 2003). In this process, glutathione is converted to its oxidized form glutathione disulfide. The ratio of reduced glutathione to oxidized glutathione within cells is often used as a measure of cellular toxicity.

An increase in glutathione disulfide is considered as an indicator of oxidative stress (Pastore et al., 2003). NO is both a gas and a free radical that reacts with many biological molecules (Bosca et al., 2005). MDA is widely used as an indicator of increased lipid peroxidation. It is a stable end-product of peroxidation of membrane lipids by ROS (Saral et al., 2005).

In the present study, we aim to investigate the effects of hibernation on GSH levels, NO production and lipid peroxidation in various tissues of aroused and non-hibernating of Anatolian ground squirrel.

MATERIALS AND METHODS

Chemicals

All chemicals were of highest analytical

grade, and purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO, USA).

Animals

Nine female ground squirrels were collected from different localities in Nigde, Bolkar Mountains, Turkey. The squirrels were divided into three groups: hibernating (n=3), aroused (n=3) and non- hibernating (n=3) housed individually in a room with an ambient temperature of 21oC with a 12 h light/12 h dark cycle, and were fed standard rodent diet and tap water ad libitum.

They were sacrificed under anesthesia, their brain, heart, kidney, liver and lung tissues were excised, immediately frozen in liquid nitrogen and stored at -80oC until used.

Determination of GSH level

The GSH levels were determined by the modified Elman method (Aykac et al., 1985). Tissues were homogenized in 10% ice-cold

trichloroacetic acid in tissue homogenizer Heidolph Diax 900 (Heidolph-Instruments GMBH and COKG, Scwabach, Germany). After centrifugation at

3000xg for 10 min by Hettich D-78532 (Andreas Hettich GmbH and Co. KG, Tutlingen, Gemany), 0.5 ml of supernatant was added to 2 ml of 0.3 M Na2HPO4 2H2O solution.

Then 0.2 ml of dithiobis nitrobenzoic acid solution (0.4 mg/ml 1% sodium citrate) was added and the absorbance at 412 nm was measured by Spectro UV-VIS RS (Labomed, Inc., CA, USA) immediately after mixing. The GSH levels were calculated unit using an extinction coefficient of 13.600 mol/cm.

Determination of NO levels

NOx (nitrite+nitrate) levels in tissues, stable end products of nitric oxide, were measured by the Griess reaction (Green et al., 1982). The tissue samples were homogenized in five volumes of phosphate buffer (pH 7.5) and centrifuged at

2,000xg for 5 min and 0.25 ml of 0.3 M NaOH were added to the 0.5 ml supernatant.

After incubation for

5 min at room temperature, 0.25 ml of 10% ZnSO4 was added for deproteinization. This mixture was then centrifuged at 14 000 x g for 5 min, and then the supernatants were used for the Griess assay.

The nitrate levels in tissue homogenates were determined spectrophoto-metrically, based on the reduction of nitrate to nitrite by VCI3 (Miranda et al.,

2001).

The nitrite levels were measured by a

standard curve prepared by different concentrations of (1, 10, 50, 100 uM) sodium nitrite and nitrate solutions.

Measurement of lipid peroxidation

Lipid peroxidation was estimated as MDA levels reacting with thiobarbituric acid, according to methods described by Casini et al. (1986) and expressed as nanomole of MDA per gram wet weight. In brief, tissue samples were homogenized in 10% ice cold trichloroacetic acid in a tissue homogenizer.

After centrifugation of the homogenate at 3 000 rpm for 10 min, 750 ul of supernatant was added to an equal volume of 0.67% thiobarbituric acid and heated at 100degC for 15 min. The absorbance of the samples was measured at 535 nm with a spectrophotometer.

Statistical analysis

The statistical differences between the mean values were evaluated by one-way analysis of variance by SPSS 15.0 (SPSS Inc., Chicago). Values of pLess than0.05 were considered to be significant. All data were expressed as the mean +- standard deviation.

RESULTS

Table I shows GSH, NOx and MDA levels in hibernating, aroused and non-hibernating Anatolian ground squirrel.

GSH levels

GSH levels of almost all tissues were lower on hibernating group compared with aroused group. Moreover, GSH levels decreased in all tissues except the brain in hibernating group compared to non-hibernating group.

The GSH level of brain tissue was the lowest in non-hibernating group (1.705+-0.78 umol/g tissue) whereas those of kidney, lung, liver and heart were the lowest in hibernating group (2.260+-1.01, 1.988+-0.78, 11.442+-2.75 and

1.988+-0.94 umol/g, respectively).

GSH levels increased however, in all tissues in aroused state compared with hibernating. When all tissues were evaluated separately, the GSH level of kidney was the lowest in hibernating group (2.26+-1.01 umol/g tissue) compared to both aroused (3.638+-1.13

umol/g tissue) and non-hibernating group (6.345+-1.98 umol/g tissue). GSH level of lung was the lowest in hibernating group (1.988+-0.78 umol/g tissue) compared to both aroused (5.584+-1.96

umol/g tissue) and non-hibernating group (4.081+-1.79 umol/g tissue).

Likewise, GSH level of liver was the lowest in hibernating group (11.442+-2.75 umol/g tissue) compared to both aroused (12.076+-2.78 umol/g tissue) and non- hibernating group (13.726+-2.98 umol/g tissue). GSH level of heart was the lowest in hibernating group (1.988+-0.94 umol/g tissue) compared to both aroused group (2.136+-0.98 umol/g tissue) and non- hibernating group (3.955+-1.12 umol/g tissue).

NOx levels

Table I shows that NOx levels increased in brain and kidney tissues (0.183+-0.02 and 0.582+-0.05 umol/g tissue, respectively), but decreased in lung, liver and heart (0.189+-0.01, 1.238+-0.12 and 0.115+-0.01 umol/g tissue, respectively) in hibernating group compared with aroused group.

NOx levels were found to decrease in all tissues except the brain in hibernating group compared to non-hibernating group. NOx levels on the other hand, increased in the brain tissue of hibernating group (0.183+-0.02 umol/g tissue) compared with both aroused (0.148+-0.05 umol/g tissue) and non-hibernating group (0.156+-0.04 umol/g tissue).

MDA levels

MDA levels were found to increase in brain and liver tissues, and decrease in kidney, lung and heart of aroused group compared with hibernating group. Table I shows that MDA levels increase in brain, lung and heart tissues in hibernating group compared to non-hibernating group.

In contrast, MDA levels decreased in kidney and liver in hibernating group (19.531+-3.21 and 21.745+-3.71 umol/g tissue, respectively) compared to non- hibernating group (28.402+-3.01 and 30.745+-7.81 umol/g tissue, respectively). The MDA level ofbrain tissue was the lowest in non-hibernating group (10.875+-1.19 umol/g tissue). MDA level of heart tissue was observed the highest in hibernating group (45.942+-9.16 umol/g tissue).

DISCUSSION

Hibernation is widely regarded as an adaptation to seasonal energy shortage, but the actual influence of energy availability on hibernation patterns is rarely considered. The presumed benefits of metabolic depression for hibernators have led some authors to interpret factors that lead to increased torpor expression as eliminating constraints that prevent torpor from being expressed at its ideal, maximal level (Frank et al., 2000; Harlow and Frank, 2001).

Hibernating mammals exhibit oxidative stress resistance in brain, liver and other tissues. In many animals, cellular oxidative stress resistance is associated with enhanced expression of intracellular antioxidant enzymes (Page et al., 2009).

Table I.- MDA, GSH and NOx levels of the brain, kidney, lungs, liver and heart tissues

Tissues GSH NOx MDA

(umol/g tissue) (umol/g tissue) (nmol/g tissue)

Brain

Hibernationa 1.776 +- 0.29 0.183 +- 0.02 11.663 +- 1.89

Arousedb 3.169 +- 1.11 0.148 +- 0.05 29.997 +- 3.27

Non-hibernationc 1.705 +- 0.78 0.156 +- 0.04 10.875 +- 1.19

Kidney

Hibernationa 2.260 +- 1.01 0.582 +- 0.05 19.531 +- 3.21

Arousedb 3.638 +- 1.13 0.339 +- 0.07 19.134 +- 2.27

Non-hibernationc 6.345 +- 1.98 0.644 +- 0.12 28.402 +- 3.01

Lung

Hibernationa 1.988 +- 0.78 0.189 +- 0.01 17.846 +- 2.89

Arousedb 5.584 +- 1.96 0.289 +- 0.06 12.058 +- 1.97

Non-hibernationc 4.081 +- 1.79 0.617 +- 0. 21 13.753 +- 1.73

Liver

Hibernationa 11.442 +- 2.75 1.238 +- 0.12 21.745 +- 3.71

Arousedb 12.076 +- 2.78 1.385 +- 0.24 25.042 +- 4.61

Non-hibernationc 13.726 +- 2.98 1.515 +- 0.65 30.846 +- 7.81

Heart

Hibernationa 1.988 +- 0.94 0.115 +- 0.01 45.942 +- 9.16

Arousedb 2.136 +- 0.98 0.154 +- 0.03 29.979 +- 3.14

Non-hibernationc 3.955 +- 1.12 0.162 +- 0.07 16.344 +- 2.21

A hibernator needs to protect its cells against oxidative damage occurring both over long periods while in torpor and during arousal as a result of the huge increase in oxygen consumption. Several studies have indicated seasonal and state-dependent changes in antioxidant defenses in ground squirrels (Buzadzic et al., 1997; Blagojevic et al., 1998).

However, only a few studies have monitored oxidative stress during hibernation or following arousal in different tissues (Carey et al., 2003b; Ma et al., 2005) or different animals (Osborne and Hashimoto, 2007). The effect of oxidative stress on hibernating animals of this species is limited.

ROS are highly damaging to biomacromolecules, hence, effective antioxidant defense mechanisms are needed. Indeed, early reports showed an increase in the activities of superoxide dismutase and glutathione peroxidase, two major antioxidant enzymes, in arousing hibernators (Chance et al., 1979).

GSH is the major thiol-disulfide redox buffer within cells that plays a key role in the detoxification of endogenous and exogenous ROS (Aw et al., 1997). Cellular oxidative stress is often manifested as a shift in the ratio of glutathione from its reduced (GSH) to oxidized (GSSG) forms.

Several events that occur during the hibernation season place the intestine at particular risk for oxidative stress including drastic alterations in blood flow, thermal stress and nutritional deprivation (Granger and Korthuis, 1995; Jonas et al., 1999).

GSH levels of almost all tissues were lower on hibernating group compared with aroused group in our study. The increase in GSH levels may be protective against any oxidative stress resulting from arousal.

Organisms are exposed to oxidative stress with continuous generated ROS. Organisms use enzymatic and non-enzymatic defense mechanisms to protect themselves from these products. These ROS listed as superoxide radicals, hydrogen peroxide, hydroxyl radicals, molecular oxygen, ozone, nitric oxide and peroxynitrite (Hermes-Lima and Zenteno, 2002).

NO is a bioactive molecule known to be involved in inflammation as well as having a demonstrated stimulatory effect on vascular cells (Coskun et al., 2007). It is also a potential candidate for such regulation because it is capable of modulating contractile function under a variety of circumstances (Canty, 2000).

It is released by the endothelium in response to shear stress and plays an important role in flow-mediated vasodilatation (Avci et al., 2008). In this study, NOx levels determined to increase in the brain tissue at hibernating group when compared with both aroused and non-hibernating groups.

The increase of NOx levels is an indicator of increased oxidative stress on brain tissues during hibernating.

If the balance between the productions of free oxygen radicals and antioxidant defense mechanisms becomes corrupted, free oxygen radicals increase and cause tissue damage (Yariktas et al., 2004). Free radicals harm such basic molecular as nucleic acids, proteins, free amino acids, lipids and carbohydrates.

Furthermore, the free radicals affect the permeability of the cell membrane via lipid peroxidation and oxidation of protein sulfhydryl groups by starting (Kurata et al., 1993; Portoles et al., 1996).

Lipid peroxidation by free radicals results in the formation of several end products, including MDA. This presence is commonly used as an indicator of oxidative damage (Baker et al., 2007).

In our study, MDA levels increased in brain, lung and heart tissues in hibernating group. We show that there is more oxidative stress in these tissues during hibernation. Orr et al. (2009) evaluated lipid peroxidation in brown adipose tissue during hibernation. Lipid peroxidation was higher in eurotherm period but declined during hibernation.

However, lipid peroxidation increased during the period of waking up late. At the same time, lipid peroxidation damage was found higher in liver tissue during hibernation than eurotherm period. L'vova and Gasangadzhieva (2003) reported high MDA levels during hibernating period compared to before hibernation. Carey et al. (2003b) showed that hibernation is associated with increased lipid peroxidation as well as the expression of stress-activated signaling pathways in ground squirrel intestine.

In conclusion, hibernation is being increasingly recognized as a valuable model for natural resistance to stress induced by extreme changes. Studies show that the effect of hibernation on oxidative stress in this species is limited, and there is an impaired balance between oxidative stress and antioxidant systems in most organs and tissues during hibernation.

Conflict of interest declaration

The authors have no conflict of interests to declare.

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