Archives Des Sciences
Vol 66, No. 1;Jan 2013
Antioxidant system in Uromastyx Philbyi at hibernation and activity periods Mohamed Afifi Department of Biological Sciences, Faculty of Science, King Abdul Aziz University, North
Campus,
PO Box 11508, Jeddah, 21463, Saudi Arabia. Permanent address: Department of Biochemistry, Faculty of Veterinary Science, Zagazig University, Egypt. Tel: +966509562637, mail;
[email protected];
[email protected]
Ali ALKALADI Department of Biological Sciences, Faculty of Science, King Abdul Aziz University, North Campus, PO Box 11508, Jeddah, 21463, Saudi Arabia Tel: ++966540424093;
[email protected]
Abstract Hibernation is an extreme physiological stat characterized by profound decreases in oxidative metabolism and body temperature during bouts of prolonged torpor, interrupted by brief periods of arousal with sudden increase of oxidative metabolism. During activity periods the hibernating animals have highly active oxidative metabolism with
increase of oxygen consumption, this extreme condition may be
accompanied with alteration of antioxidant defense, to determine that we monitored the activities of antioxidant enzymes and oxidative stress either during hibernation or during activity in uromastyx philbyi. 20 animals were used and collected from Bisha province at the south of Saudi Arabia, 10 of them collected in hibernation season, group I and the other collected during the active period, group II. After collection the animals directly transported to the lap and blood, liver, brown adipose tissue (BAT) and brain samples were taken for determination of free radicals and antioxidants. The results indicate a significant decrease of free radicals and increase of vitamin C especially in serum in hibernation, in contrast revealed an increase of free radicals and antioxidant in all studied tissues at the active period. It can be concluded that uromastyx philbyi has a strong antioxidant defense system that protect it from the injurious effect of free radicals either at the periods of arousal or the activity periods. Keywords: Antioxidant, Free radicals, Uromastyx philbyi.
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1. Introduction It has been known since the 1990's that free radical metabolism is an integral part of the metabolic depression machinery associated with hibernation, reactive oxygen species (ROS), generated during hibernation especially when the environment is anoxic will mediate lipid peroxidation and DNA damage, a well developed antioxidant defense system can minimizes oxidative damage during hibernation (Qizhang and Xu 2012) . In the case of metabolic depression under anoxia/hypoxia or freezing, the transition towards aerobic metabolism may involve an overproduction of ROS devastates cellular integrity (Adrienne et al. 2009). Direct observation of ROS formation in anoxic-tolerant turtles indicated very low levels of hydroxyl radical generation in brain during anoxia followed by a restoration in ROS formation upon reoxygenation (Milton et al. 2007). However, reperfusion in animals adapted to these challenges anoxia/hypoxia or freezing is a physiological process that is an integral part of their natural life cycle. In the case of transitions from aerobic hypometabolism in hibernation to normal metabolic rate, there is an intermediate phase of very high oxygen consumption where increased mitochondrial ROS production may occur (RamosVasconcelos and Hermes-Lima 2003). Accompanied in several cases by increased markers of oxidative stress: lipid peroxidation, protein carbonyl or oxidized glutathione (GSSG). Furthermore, an increase in antioxidant defenses during anoxia/hypoxia or estivation (Bickler and Buck 2007) indicating that animals emerging from a hypometabolic state may experience oxidative stress. No studies to date have uncovered evidence of oxidative stress in Uromastyx philbyi (Arabian blue uromastyx) during hibernation or active periods so this work was designed to study the antioxidant defense system in Uromastyx philbyi during the hibernation and activity periods. 2. Material and Methods Adult male lizards were collected from different locations near Bisha, Saudi Arabia, Lizards were captured twice in the year, in the active season (july 2010) (Group I) and during hibernation (January 2011) (Group II). Ten animals were picked during summer and ten ones during winter, animals are collected during the day light, after taking the approval of Saudi Arabian wiled life authorities number 120/2010. The animals were transported to the laboratory in plastic boxes on the day of capture. 2.1. Sampling protocol and sample preparation: In the clinical biochemistry lab of medical laboratories department of King Khalid University ( Bisha branch) Saudi Arabia ,animals were anesthetized with ether then, the animals were fixed on dissecting plat, the abdomen and thorax were opened and blood samples were withdrawn by needle directly from the hart
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and used for serum separation, then as early as possible before the animal death liver, brown adipose tissue ( BAT ) and brain tissues immediately were isolated, cleaned from adhering matters, washed with saline solution, where 0.5 g of each sample was homogenized in 5 ml of D.W. (Sidhu 2004) using electrical homogenizer, centrifuged at 3000 r.p.m. for 15 minutes, the resulting supernatant were collected and kept in deep freeze (-20C0) until be used for estimation of the biochemical parameters . 2.2. Biochemical assays In the liver, BAT, brain homogenate and serum, Malondialdehyde (MDA) levels were determined spectrophotometrically by the method of Packer and Glazer (1990). GSH, GSSG and total GSH levels were determined according to the method of Ellman (1959). GPx activities were determined by the method of Paglia and Valentine (1967). The rate of reduction of NADPH was observed at 340 nm. One unit of GPx activity was defined as the oxidation of 1 μmol NADPH and expressed as µM /min/gm tissue. Glutathione reductase (GR) activity was determined by the amount of NADPH consumed in the conversion of oxidized glutathione (GSSG) to GSH following the method of Racker (1955). SOD activities were determined according to the method of Winterbourn et al. (1975). Catalase activity was determined according the method of Sinha (1972). Vitamin C according to Kyaw (1978). And Vitamin E was determined according to Baker and Frank (1978). 2.3. Statistical analysis The data were statistically analyzed by SPSS version 20. statistical packages (IBM 1 New Orchard Road Armonk, New York 10504-1722 United States) . Data were presented as a mean ± SD, n = 10. Statistical differences between groups were performed using student's t-test. Differences considered significant when P < 0.05 (Steel
and Torrie 1960).
3. Result The concentration of MDA, GSH, total GSH, vitamin E and the activities of GPX, GR, SOD and CAT were significantly increased in liver, BAT and serum at active period when compared with hibernation period , in contrast the animals showed a significant increase in GSSG and vitamin C at the hibernation period. Brian tissue showed the same result except that there is no a significant change in GSH, total GSH and vitamin C concentration. 4. Discussion No studies have monitored oxidative stress during hibernation or following arousal with focusing on different tissues in Arabian blue Uromastix. Here we assessed oxidative stress in liver, as it the main organ of detoxification and metabolism., Brain as the most consuming organ of oxygen, the first one affected by
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oxidative stresses and it manly depends on oxidative metabolism " man source of oxidative stresses" as the source of energy. BAT the main thermogenic tissue in the body and finally , serum as it carry the most of catabolites and oxidative stress west products, during hibernation in winter and arousal in summer. There is no evidence of increased oxidative stresses was observed in any studded organs during hibernation ( Table 1,2,3 and 4). These data suggested that , liver , brain ,BAT and serum avoid oxidative stress during hibernation through decreasing it main sources, oxidative metabolism. Adrienne et al.(2009) Mentioned that, the oxidative stress in Arctic ground squirrel brain decreases during hibernation along with a fall in oxidative metabolism. During arousal, their was an evidence of increased oxidative stress in all studded tissues manifested by increase in MDA and GSSG ( Tables 1,2,3 and 4) in concomitant with increase of all studded parameters of antioxidant defense system
( GPX, GR, SOD, CAT and vitamin E) except vitamin C ( Tables 1,2,3 and 4).
The increase in oxidative stress my be due to the over production of reactive oxygen species (ROS) that accompanied the transition toward the aerobic metabolism during arousal (Hermes-Lima 2004). The increase of antioxidant defense system refers to the ability of the animal for adaptation to oxidative stress. Voituron (2006) Suggested that, the activation of the antioxidant enzymes was an effective ecological strategy in response to excessive oxygen free radicals after arousal. Current estimates indicate that 0.1% of oxygen consumed by mammalian mitochondria leads to ROS formation (Fridovich 2004). A four-fold increase in oxygen consumption during rewarming in AGS (from ∼0.5 mL O2/g/h in hibernation to ∼2.0 mL O2/g/h (Ma et al. 2005) would therefore supply a reasonable rate of ROS formation to prompt oxidative stress as observed in the case of free radical protein damage and lipid peroxidation in BAT. While Vitamin C, GSH and total GSH remain stable through hibernation and arousal in brain, they decreased in liver, BAT and serum in hibernation, that not associated with oxidative stress ( no increase in MDA) ( Tables 1,3 and 4). This may be attributed to decrease carbon flux through glycolysis and the pentose phosphate pathway. Decreased carbon flux through glucose utilization in torpid hibernators (Frerichs et al. 1995) would be expected to lead to a decrease in NADPH contents and subsequent decrease in glutathione reductase (GR) activity ( Tables 1,3 and 4) (such a decrease in activity would be due to a reduction in carbon flux through the pathway and not necessarily in GR concentration). Decreased GR activity would lead to an augment in GSSG levels
( Table 1,3 and 4). Interestingly, Carey et al. (2003a) observed decreased GR activity in the
gut of hibernating 13-lined ground squirrels. Decreased biosynthesis and/or increased export of liver GSH (for amino-acid transport) could also account for the decrease in hepatic GSH in torpid hibernators.
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Biosynthesis of GSH is an energy consuming process (Hermes-Lima and Zenteno-Savin 2002) and may be diminished during torpor in several organs. In contrast to all antioxidant Vitamin C was increased in all studded tissue (especially plasma) except brain in hibernation period. Vit. C increased three to four folds during tropor in AGS and 13-lined ground Squirrels, this might provide an antioxidant source that could be taken by tissues to prevent oxidative stress during the increase in O2 consumption that accompanies arousal (Tqien et al. 2001). It can be concluded that, the hibernation in Uromastyx philbyi not accompanied by oxidative stress and this may be due to increase of vit. C and decrease of oxidative metabolism, in contrast the arousal accompanied by increase of oxidative stress and the animal have high ability for adaption through induction of all antioxidative defense system. References Adrienne, L. et al. (2009). Physiological oxidative stress after arousal from hibernation in Arctic ground squirrel. Comp Biochem Physiol A, 153 (2), 213–221. doi: 10.1016/j.cbpa.2009.02.016 Baker, H., & Frank, F. (1978). Determination in serum tocopherol. In Practical Clinical. Bickler, P., & Buck, L. (2007). Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability. Annu Rev Physiol, 69,145–170. Carey, H., Rhoads, C., & Aw, T. (2003a). Hibernation induces glutathione redox imbalance in ground squirrel intestine. J Comp Physiol B,173,269–276. Ellman, L. (1959). Tissue sulfhydryl group. Arch Biochem Biophys, 82,70–77. Frerichs, K. et al. (1995). Rates of glucose utilization in brain of active and hibernating ground squirrels. Am J Physiol, 268, R445–453. Fridovich, I. (2004). Mitochondria: are they the seat of senescence? Aging Cell, 3,13–16. Hermes-Lima, M. (2004). Oxygen in biology and biochemistry: role of free radicals. In: Storey KB, editor. Functional Metabolism: Regulation and Adaptation. John Wiley & Sons; New Jersey. pp. 319–368. Hermes-Lima, M., & Zenteno-Savin, T. (2002). Animal response to drastic changes in oxygen availability and physiological oxidative stress. Comp Biochem Physiol C, 133,537–556. Kyaw, A. (1978). A simple colorimetric method for ascorbic acid determination in blood plasma. Clin. Chim. Acta, 86,153-157.
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Ma Y. et al. (2005). Absence of cellular stress in brain after hypoxia induced by arousal from hibernation in Arctic ground squirrels. Am J Physiol Regul, 289, R1297–1306. doi: 10.1152/ajpregu.00260.2005 Milton, S. et al. (2007). Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta. J Neurochem, 101, 993–1001. doi: 10.1111/j.1471-4159.2007.04466.x Packer, L., & Glazer, A. (1990). Method in enzymology,186 B, Academic press Inc. new York .PP. 251. Paglia, D., & Valentine, W. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med, 70, 158-169. Qizhang, C., & Xu, W. (2012). Effect of dietary vitamin C on the antioxidant defense system of hibernating juvenile Three keeled pond Turtles. Asian Herpetological Research, 3(2), 151-156. Racker, E. (1955). Glutathione reductase (liver and yeast). In: Sidney PC, Nathan OK, editors. Methods in enzymology. Vol. 3. New York: Academic Press; pp.72225. Ramos-Vasconcelos, G., & Hermes-Lima, M. (2003). Hypometabolism, antioxidant defenses and free radical metabolism in the pulmonate land snail Helix aspersa. J Exp Biol, 206, 675–685. Sidhu, P., Garg, L., & Dhawan, D. (2004). Protective effects of zinc on oxidative stress enzymes in liver of protein deficient rats. Nutr. Hosp, 6, 341-347. Sinha, K. (1972). Colorimetric assay of catalase .Analytical biochemistry. 47:389. Steel, R., & Torrie, J. (1990). Principles and procedures of statistics. Mc Graw- Hill Book Comp. Inc., New York. Tqien Q. et al. (2001). Ascorbate dynamics and oxygen consumption during arousal from hibernation in Arctic Ground Squirrels. Am J Physiol Reg Comp Physiol, 281, R572-R583. Voituron Y. et al. (2006). Oxidative DNA damage and antioxidant defenses in the European common lizard in super cooled and frozen states. Cryobiology, 52 (1), 74-82. http://dx.doi.org/10.1016/j.cryobiol.2005.09.006. Winterbourn C. et al. (1975). The estimation of red cell superoxide dismutase activity. J Lab Clin Med, 85, 337–342.
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Table 1. The antioxidants and free radicals in hepatic tissue of uromastyx during hibernation and activity periods. G
MDA
GSH
II
GPX µM
GR
SOD
CAT (µM
Vit C mg/ml
Vit.E mg/L
homogenate
homogenate
6.7 ± 0.6*
934
nmol
umol/g
total
umol/g
/min/g
unit/g
ug/g
/g
tissue
umol/g
tissue
tissue
tissue
tissue
decomposed
tissue
1.16±
3.02±
0.45
0.7
2.3±
5.9±
0.4 P
GSSG
H O 2 2)
tissue
I
GSH
***
1.2
0.000
5.1± 0.6
**
0.001
2.1 ± 0.1
7.1± 1.4
/g tissue
*
0.01
**
1.2±0. 2 0.002
34 ±2 42 ± 4* 0.014
47.7
2.8 ±
± 2.7
0.9
57 ±
4.9 ±
4.3 ±
***
**
5
**
0.5
0.006
3.6 ± 0.2
0.8
0.000
1676 ±
5.5 ± 0.4
0.005
±27
172***
0.049
0.000
G, group. MDA, Malondialdehyd. GSH, reduced glutathione. GSH total, total glutathione. GSSG, oxidized glutathione. GPX, glutathione oxidase. GR, glutathione reductase. SOD, super oxide dismutase. CAT, catalase. Vit C, vitamin C. Vit. E, Vitamin E. Results are expressed as mean ± SD of ten animals. Statistical analyses were performed using two tail Student's t-test * p < 0.05
**
P
