BIHAREAN BIOLOGIST 12 (2): 97-101 ©Biharean Biologist, Oradea, Romania, 2018 Article No.: e181302 http://biozoojournals.ro/bihbiol/index.html

Morphological characters, antioxidant defenses and oxidative stress in the elegans at different altitudes

Amir Mohammad SOURI and Nasrullah RASTEGAR-POUYANI*

Department of Biology, College of Sciences, Razi University, Kermanshah, Iran *Corresponding author, N. Rastegar-Pouyani, Email: [email protected]

Received: 08. August 2017 / Accepted: 28. February 2018 / Available online: 28. February 2018 / Printed: December 2018

Abstract. In the wild, living at different altitude environment face different stressors, accordingly generating clines for several traits and biochemical changes with altitude. The present study was conducted to examine morphological traits and biochemical biomarkers in the lizard Ophisops elegans at three different altitudes. Biomarkers included antioxidant enzymatic activity of glutathione peroxidase, superoxide dismutase, total antioxidant capacity (TAC) and lipid peroxidation (malondialdehyde) in brain, liver and tail tissue samples. The results indicated that Lipid peroxidation in lowland was significantly increased in brain, liver and tail tissues. Total antioxidant capacity in the liver and glutathione peroxidase activity in tail were much higher in lowland than those in highlands. However, the activity of superoxide dismutase was similar among three elevations. The lizards from highlands were longer than those from lowland. We found significant negative correlation between snout-vent length and lipid peroxidation in the brain and tail. Total antioxidant capacity in liver and glutathione peroxidase in tail also recorded a negative correlation with snout-vent length. By contrast, total antioxidant capacity in liver was positively correlated with lipid peroxidation. These results clearly suggest that oxidative stress and antioxidant defences in O. elegans varies in response to elevation.

Key words: antioxidant capacity, brain, lizard, malondialdehyde, oxidant environment.

Introduction is considered as one of the most abundant lacertid lizards, mainly distributed along the Mid-Zagros area at different al- Environmental stressors that affect wildlife come in many titudes from 1000 to 2000 meters above sea level, m a.s.l. forms including hypoxia (He et al. 2013), extreme low or (Anderson 1999). Although some information is available on high temperature (Li et al. 2011, Wang et al. 2016), environ- reproductive cycle (Torki 2007), sexual dimorphism (Oraie et mental contaminants (McFarland et al. 2011), food limitation al. 2013), digestive characteristics (Çakici & Akat 2013) and (Stamps & Tanaka 1981) and intense ultraviolet radiation distribution pattern (Oraie et al. 2012) but, there are no re- (Reguera et al. 2015). It is now well known that the cumula- port of characterization of oxidative stress and antioxidant tive number of stress events varied with the altitude and defences in O. elegans from Iranian Plateau. The main goal of therefore, animals living at different altitudes face different this study was thus, to investigate the oxidative stress and oxidant environment. For animals that live at severe oxidant antioxidant capacity of this lizard at different altitudes in environment, survival requires possessing certain character- Iranian Plateau. istics and behaviors that allow them to adapt to the envi- ronmental challenges. In living organisms, cells can generate harmful substances called free radicals, including reactive Material and methods oxygen (ROS) and reactive nitrogen species (RNS) from endogenous and exogenous sources (Durackova 2010). Study site Samplings were performed in western regions of the Iranian Plateau, Excess of free radicals can lead to cumulative damage in pro- western slopes of the Mid-Zagros Mountain during a two weeks pe- teins, lipids, and DNA (Halliwell 2007). In order to maintain riod of October 2016. Three sample sites were situated at different al- the delicate balance of free radicals generation and minimize titudes; Sarv-Abad (35.31 N and 46.36 E and 1134 m a.s.l.), Has- oxidant damages, biological systems display a network of san-Abad (35.24 N and 46.92 E and 1995 m a.s.l) and Mahidasht enzymatic and non-enzymatic antioxidants defences (Higu- (34 14 N and 46.83 E and 1907 m a.s.l.). chi et al. 2012, Szczepańska et al. 2003). Oxidative stress oc- curs when the productions of oxidants overwhelm antioxi- sampling and procedures dants (Niki 2016). In fact, oxidative stress emerges from an Eighteen adult male lizards (Ophisops elegans, six from each location) were caught by hand and transported to the nearest laboratory in enhanced reactive oxygen and nitrogen species generation or each location, 20-30 Km far from the place of capture. The lizard sex from a decay of the antioxidant protective ability, being was characterized based on femoral pores which were larger in characterized by the reduced capacity of endogenous sys- males than in females. We considered as adult those males with SVL tems to fight against the oxidative attack directed towards longer or equal to 40 mm. This experiment and all the experimental target biomolecules (Pisoschi & Pop 2015). During recent procedures were approved by the animal ethical committee of Razi years, researchers have taken to studying oxidative stress in University. The animals were euthanized with an overdose of ether field population of lizards because it has been proposed as a and measurement was carried out on metric traits; snout-vent length, (SVL), tail length (TL), length of between forelimb and major challenge for the survival of lizards living at different hindlimb (LHF), head length (HL), Head width (HW), length of fore- altitudes (Reguera et al. 2014). limb (LFL), length of hindlimb (LHL), length of eye (LE); width of The snake-eyed lizard, Ophisops elegans Menetries, 1832 or cloaca (LV) and meristic traits; number of supralabial (NSL), number field lizard, is widely distributed throughout the Eastern of infralabial (NIL), subdigital lamellae under of fourth toe (SDLT), Mediterranean region and Southwestern Asia and North Af- number of ventral scales (NVS), number of dorsal scales (NDS) rica (Kyriazi et al. 2008). In Iran, this ground-dwelling lizard number of gular scales (NGS) femoral pores (FP) and subdigital la- 98 A.M. Souri & N. Rastegar-Pouyani mellae under of fourth finger (SDLF). Metric traits were measured by ues were expressed as means ± standard error (s.e.). Differences in using a digital caliper (accuracy 0.01 mm) and meristic traits were morphometric characters, oxidative stress biomarker (MDA), total examined through a dissecting microscope. Then, the lizards were antioxidant capacity (TAC) and antioxidant enzymatic activity (GPX quickly dissected out and tissue samples; tail, brain and liver were and SOD) between animals from three different sampling sites ana- taken and frozen in liquid nitrogen until carried to the laboratory at lysed by Tukey’s test. Pearson’s correlation analyses were used to Razi University, where they were kept at -80 ºC for biochemical determine the correlation among oxidative stress and antioxidant analysis. biomarkers in brain, liver and tail tissues, themselves and with SVL as an indication of body size of the lizards separately. Test values Biochemical analyses with P < 0.05 were considered significantly different. All data analy- To measure cytosolic enzyme activity, total antioxidant capacity, ses were performed using Minitab Statistical software (version 17.1, lipid peroxidation (MDA) and soluble protein concentrations, the 2013). frozen tail, brain and liver samples were thawed and homogenized in 1.15% KCl solution. Homogenates were centrifuged at -40 °C for 15 minutes and supernatants were stored at -80°C until used for Results measuring of the biochemical parameters. Glutathione peroxidase (GPX) activity was measured according to Paglia and Valentine The values for morphometric characters of the lizards are (Paglia & Valentine 1967), using a GPX commercial assay kit (Cat. presented in Table 1. Except for the SVL, no significant dif- No RS 504, Randox Laboratories Ltd., UK). GPX was catalyzed by the oxidation of reduced glutathione in the presence of cumene hy- ferences were observed in other characters. The snout-vent droxyperoxide. length (SVL) of male O. elegans in different sites varied from Tissue superoxide dismutase (SOD) activity was determined as 46.0 to 52.8 mm. Lizards had longer SVL at 1907 and 1995 m described by Sun et al. 1988. This method depends on the inhibition a.s.l. (MA and HA) than those at 1134 m a.s.l. (SA, P<0.05). of nitroblue tetrazolium (NBT) reduction by xanthine-xanthine oxi- However, SVL was similar between lizards at MA and HA. dase used as a superoxide generator. SOD activity was expressed as Values for MDA, TAC, GPX and SOD in brain, liver and the amount of enzyme that causes 50% inhibition of the rate of NBT tail tissues of lizards at different altitudes are presented in reduction. GPX and SOD activity was designated as unit for mg pro- tein of tissue. Table 2. We found significant differences in lipid peroxida- Malondialdehyde (MDA) levels as product of lipid peroxide tion (MDA concentration) in all three tissues (brain, liver and degradation were measured using the thiobarbituric acid reactive tail) with altitude. The MDA contents of brain, liver and tail substances (TBARS) method (Miller et al. 1993). tissues in lizards at low altitude (SA) were significantly The soluble protein concentration was determined by Bradford higher (P<0.01, P<0.05 and P<0.001 respectively) compared protein assay reagent (Bradford 1976), using albumin as the standard with those at both high altitudes (MA and HA), but MDA to estimate the enzymatic specific activity. contents of all three tissues were similar between both high Quantitative determination of TAC was measured in tissue su- elevations (MA and HA). In the case of TAC, only value for pernatant using a TAC commercial assay kit (Cat. No 2331, Randox Laboratories Ltd., UK), according to the method of Miller et al. 1993. liver tissue was significantly different among the lizards at different altitudes (P<0.005). In liver the level of TAC for liz- Statistical analyses ards at low altitude (SA) was more than two fold higher than The statistical analysis was performed by one-way ANOVA. All val- those for lizards at both high altitudes (MA and HA). GPX

Table 1. Metric (mm) and meristic (number) characteristics for the lizard Ophisops ele- gans at different altitude (SA, Sarvabad, 1134 m a.s.l.; HA, Hassan abad, 1995m a.s.l.; MA, Mahidasht, 1907 m a.s.l.).

SA HA MA P-value SVL 48.15 ± 1.01 51.13 ± 0.41 51.19 ± 0.63 0.021 TL 90.16 ± 0.55 95.35 ± 2.14 94.71 ± 2.61 0.276 LHF 23.61 ± 0.34 22.18 ± 0.86 22.68 ± 0.76 0.462 HL 13.67 ± 1.34 14.07 ± 0.83 13.76 ± 0 .46 0.944 HW 7.07 ± 0.40 7.23 ± 0.28 7.81 ± 0.50 0.448 LFL 15.11 ± 0.82 16.75 ± 0 .86 15.98 ± 0 .54 0.371 LHL 29.13 ± 1.93 28.28 ± 1.37 29.20 ± 1.35 0.887 LE 2.13 ± 0 .12 2.03 ± 0 .14 2.08 ± 0 .09 0.845 LV 5.34 ± 0.28 4.77 ± .0.42 4.84 ± 0.23 0.493 NSL 7.67 ± 0.33 8.00 ± 0.00 8.00 ± 0.00 0.289 NIL 7.67 ± 0.33 8.00 ± 0.00 8.00 ± 0.00 0.289 SDLT 23.33 ± 1.45 22.75 ± 0.85 23.75 ± 1.18 0.814 NVS 40.67 ± 0.88 40.25 ± 0.85 40.50 ± 0.64 0.935 NDS 23.67 ± 0.67 23.75 ± 0.48 23.75 ± 0.48 0.993 NGS 18.67 ± 0.33 18.50 ± 0.29 18.5 ± 0.29 0.914 FP 10.33 ± 0.33 10.25 ± 0.25 10.5 ± 0.29 0.812 SDLF 17.33 ± 0.88 17.25 ± 0.0.48 17.25 ± 0.48 0.994 SVL, snout-vent length; TL, tail length; LHF, length of between forelimb and hindlimb; HL, head length; HW, Head width; LFL, length of forelimb ; LHL, length of hindlimb; LE, length of eye; LV, width of cloaca; NSL, number of supralabial; NIL, number of infralabial; SDLT, subdigital lamellae under of fourth toe; NVS, number of ventral scales, NDS, num- ber of dorsal scales; NGS, number of gular scales; FP, femoral pores; SDLF, subdigital la- mellae under of fourth finger. Table shows mean values and standard error (±SE) and sig- nification value (P-value). Morphological characters, antioxidant defenses and oxidative stress in the Ophisops elegans 99

Table 2. Lipid peroxidation (MDA), and antioxidant levels in brain, liver and tail tissues for the lizard Ophisops elegans at different altitude (SA, Sarvabad, 1134 m a.s.l.; HA, Hassan abad, 1995 m a.s.l.; MA, Mahidasht, 1907 m a.s.l.). SA HA MA P-value MDA (nmol/mg protein): Brain 2.2 ± 0 .22 1.29 ± 0 .10 1.26 ± 0 .16 0.003 Liver 15.07 ± 0.63 11.77 ± 0.68 11.90 ± 0.45 0.013 Tail 2.22± 0 .12 1.17 ± 0 .11 1.37 ± 0 .08 0.000 TAC (mmol/mg Protein): Brain 2.14 ± 0 .28 2.02 ± 0 .29 1.69 ± 0 .24 0.554 Liver 22.90 ± 3.32 9.30 ± 3.17 6.92 ± 1.71 0.009 Tail 3.97 ± 0 .26 3.69 ± 0 .34 4.08 ± 0 .33 0.683 GPX (U/mg protein): Brain 39.33 ± 2.00 41.04 ± 1.97 40.76 ± 2.35 0.832 Liver 82.59 ± 9.73 79.89 ± 16.40 75.82 ± 8.55 0.855 Tail 22.35 ± 1.99 12.92 ± 0.80 13.65 ± 0.88 0.007 SOD (U/mg protein): Brain 46.94 ± 4.11 48.70 ± 2.99 46.11 ± 3.52 0.846 Liver 107.19 ± 3.23 104.85 ± 1.60 103.24 ± 2.53 0.565 Tail 34.18 ± 0.81 31.21 ± 1.11 31.61 ± 0.85 0.201

MDA, malondialdehyde; TAC, total antioxidant capacity; GPX, glutathione peroxidase; SOD, superoxide dismutase. Table shows mean values and standard error (±SE) and signification value (P-value).

Table 3. The correlation (Spearman’s correlation) coefficients among tively). Likewise, there was a significant negative correlation SVL, MDA (nmol/mg protein), TAC (mmol/mg protein), GPX between SVL and TAC in liver tissue (P<0.05). By contrast (U/mg protein) and SOD (U/mg protein) in brain, liver and tail GPX and SOD activity was not significantly correlated with tissues for the lizard Ophisops elegans. SVL, excepting GPX in tail tissue, which was negatively cor- SVL MDA TAC GPX related with SVL (P<0.05). Furthermore, in tail tissue, GPX In brain tissue: and SOD activity was positively correlated to MDA concen- MDA r = -0.71 tration (P<0.001 and P<0.05). There was no significant corre- 0.02* lation between TAC and any enzyme activity. TAC r = -0.35 r = 0.04 0.32 0.88 GPX r = 0.63 r = -0.07 r = -0.29 0.05 0.81 0.30 Discussion SOD r = 0.00 r = 0.00 r = 0.47 r = 0.03 0.99 0.99 0.09 0.91 To the best of our knowledge, this is the first report on inves- In liver tissue: tigating oxidative stress in the lizard in Iranian Plateau. In MDA r = -0.56 the wild, animals living at different altitude environment 0.09 TAC r = -0.80 r = 0.71 face different stressors that can amplify the generation of re- 0.01* 0.01* active oxygen species (ROS) in the body causing serious GPX r = -0.14 r = -0.17 r = -0.26 damage to macromolecules of organism such as nucleic ac- 0.70 0.55 0.40 ids, proteins and lipids (McCord 2000). The severity of SOD r = -0.02 r = 0.38 r = 0.09 r = 0.07 stressors increases with elevation (Cichoż-Lach & Michalak 0.52 0.18 0.77 0.80 2014). Thus, native species who live at high altitude must In tail tissue: MDA r = -0.74 possess certain adaptive characteristics and behaviors in- 0.01* cluding changes in free radical metabolism that make them TAC r = -0.18 r = 0.26 more tolerant to oxidant environment and provide the 0.62 0.37 means to survive (Jefferson et al. 2004). GPX r = -0.67 r = 0.81 r = -0.04 The current study has highlighted several important 0.03* 0.00** 0.89 findings and indicated that SVL, as an indicator of body size, SOD r = -0.17 r = 0.58 r = 0.27 r = 0.53 the level of oxidative stress and antioxidant statues of Ophi- 0.64 0.03* 0.34 0.05 sops elegans varied with elevation. Lizards at high altitude SVL, snout-vent length; MDA, malondialdehyde; TAC, total antioxidant capac- showed larger SVL. Larger body size at higher altitude has ity; GPX, glutathione peroxidase; SOD, superoxide dismutase. P values are given below the correlation coefficients. * significant at P<0.05, ** significant been reported in other lizards (Reguera et al. 2014). It is well at P<0.01. known that metabolic and free radical production rates are higher in smaller organisms (Speakman 2005). Under these

circumstances lipids are susceptible targets of peroxidation and SOD enzymes but GPX in tail tissue had similar activity (Porter et al. 1995). Two of the most well studied markers of in all three tissues of lizards from different altitudes. lipid peroxidation are isoprostanes (IsoPs) and malondial- As shown in Table 3, MDA concentration was negatively dehyde (Ho et al. 2013). In this study malondialdehyde lev- correlated to the SVL, although only correlations in brain els were determined as product of lipid peroxide degrada- and tail tissues were significant (P<0.05 and P<0.05 respec- tion. We found that levels of lipid peroxidation increased in 100 A.M. Souri & N. Rastegar-Pouyani all three brain, liver and tail tissues of lizards at low altitude, Bradford, M.M. (1976): A rapid and sensitive method for the quantitation of suggesting an increase of oxidative damage. Although, over- microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72(1-2): 248-254. all increasing MDA negatively correlated with SVL, however Çakici, Ö., Akat, E. (2013): Some histomorphological and histochemical considering each elevation separately, no significant correla- characteristics of the digestive tract of the snake-eyed lizard, Ophisops elegans tion observed between SVL and MDA levels. This may indi- Menetries, 1832 (: ). North-Western Journal of Zoology 9(2): 257-263. cate that higher level of oxidative stress in lizards from low- Cichoż-Lach, H., Michalak, A. (2014): Oxidative stress as a crucial factor in liver land is not due to smaller body size. This result can be ex- diseases. The World Journal of Gastroenterology 20(25): 8082-8091. plained by the fact that the activities of ectotherms are Durackova, Z. (2010): Some current insights into oxidative stress. Physiological Research 59(4): 459-469. greatly depend on the environmental temperature for ther- Halliwell, B. (2007): Biochemistry of oxidative stress. Biochemical Society moregulation (Hawkins 1995). At low elevation, higher envi- Transactions 35(5): 1147-1150. ronmental temperature increases metabolic rate and thus, Hawkins, A.J. (1995): Effects of temperature change on ectotherm metabolism and evolution: metabolic and physiological interrelations underlying the oxidative stress increases in ectotherms (Heise et al. 2007). superiority of multi-locus heterozygotes in heterogeneous environments. Moreover, the increase of oxidative damage may be the re- Journal of Thermal Biology 20 (1-2): 23-33. sult of increased free radical production or decreased anti- He, J., Xiu, M., Tang, X., Wang, N., Xin, Y., Li, W., Chen, Q. (2013): oxidant defenses induced by more contaminants at low alti- Thermoregulatory and metabolic responses to hypoxia in the oviparous lizard, Phrynocephalus przewalskii. Comparative Biochemistry and Physiology tude (Loumbourdis 1997). Consistent with our results, pre- Part A: Molecular & Integrative Physiology 165(2): 207-213. vious study reported that lizards from high elevation have Heise, K., Estevez, M.S., Puntarulo, S., Galleano, M., Nikinmaa, M., Pörtner, H. less oxidative stress levels than those from low elevation O., Abele, D. (2007): Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in (Reguera et al. 2014). They suggested that highland envi- zoarcid fish. Journal of Comparative Physiology 177(7): 765-777. ronment is less stressful than lowlands. In the current study, Higuchi, M., Celino, F.T., Shimizu-Yamaguchi, S., Miura, C., Miura, T. (2012): TAC in liver was higher in the lowland lizards and posi- Taurine plays an important role in the protection of spermatogonia from oxidative stress. Amino Acids 43(6): 2359-2369. tively correlated with MDA. 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