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EFFECT OF TEMPOL ON REDOX HOMEOSTASIS AND STRESS TOLERANCE IN MIMETICALLY AGED DROSOPHILA Ugur Aksu Department of Biology, Science Faculty, Zoology Division, Istanbul University, Istanbul, Turkey
Karolin Yanar and Duygu Terzioglu Department of Medical Biochemistry, Cerrahpas¸a Medical Faculty, Istanbul University, Istanbul, Turkey
Tugc¸e Erkol and Evrim Ece Department of Biology, Science Faculty, General Biology Division, Istanbul University, Istanbul, Turkey
Seval Aydin, Ezel Uslu, and Ufuk C¸akatay Department of Medical Biochemistry, Cerrahpas¸a Medical Faculty, Istanbul University, Istanbul, Turkey
We aimed to test our hypothesis that scavenging reactive oxygen species (ROS) with tempol, a membrane permeable antioxidant, affects the type and magnitude of oxidative damage and stress tolerance through mimetic aging process in Drosophila. Drosophila colonies were randomly divided into three groups: (1) no D-galactose, no tempol; (2) D-galactose without tempol; (3) D-galactose, but with tempol. Mimetic aging was induced by D-galactose administration. The tempol-administered flies received tempol at the concentration of 0.2% in addition to D-galactose. Thiobarbituric acid reacting substance (TBARS) concentrations, advanced oxidation protein products (AOPPs), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), sialic acid (SA) were determined. Additionally, stress tolerances were tested. Mimetically aged group without tempol led to a significant decrease in tolerance to heat, cold, and starvation (P < 0.05), but tempol was used for Grant sponsor: Istanbul University; Grant number: 21261. Correspondence to: Dr. Ugur Aksu, Department of Biology, Faculty of Science, The University of Istanbul, Vezneciler-Istanbul 34459, Turkey. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 87, No. 1, 13–25 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21176
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these parameters. The Cu,Zn-SOD activity and SA concentrations were lower in both mimetically aged and tempol-administered Drosophila groups compared to control (P < 0.05), whereas there were no significantly difference between mimetically aged and tempol-administered groups. Mimetically aged group without tempol led to a significant increase in tissue TBARS and AOPPs concentrations (P < 0.05). Coadministration of tempol could prevent these alterations. Scavenging ROS using tempol also restores redox homeostasis in mimetically aged group. Tempol partly C 2014 restores age-related oxidative injury and increases stress tolerance. Wiley Periodicals, Inc. Keywords: tempol; mimetic aging; Drosophila; oxidative stress; sialic acid
INTRODUCTION Free radical theory of aging is one of the widely accepted theories set forth in relation to cellular effects of both natural and mimetic aging (Yanar et al., 2011; Aydin et al., 2012). According to this theory, reactive oxygen species (ROS) may initiate oxidative injury that a living organism is affected through its lifetime. Increased oxidative stress may cause functional decline and various age-related disorders in humans and experimental animals (C¸akatay, 2011). Aerobic organisms continuously produce ROS through their lifespan. It has been seen that those free radicals are the molecules that pose unpaired electron in the outermost molecular orbitals and have caused oxidative damage to cellular macromolecules such as DNA, proteins, and lipids (C¸akatay, 2011). To protect against harmful toxic effects of ROS and modulate physiological effects of ROS, the cell has developed its antioxidant systems. Under normal circumstances, ROS are metabolically formed but they are removed efficiently by antioxidant systems virtually, therefore no macromolecular damage occurs in cell. However, this homeostatic process is declined in favor of ROS with aging (C¸akatay, 2011). Impaired redox homeostasis is originated both by inefficiency of antioxidant systems and increased ROS formation due to aging process. The ability of amphipathic antioxidants to penetrate into cellular lipid bilayers is undoubtedly crucial to the protection against macromolecular oxidation (C¸akatay, 2006; Zhou et al., 2010). In agreement with this idea, several routes of superoxide dismutase administration have been described, and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) cannot easily penetrate biological membranes to attenuate the effects of intracellular production of superoxide radical anion (Fridovich, 1995). Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidineN-oxyl), a low molecular weight piperidine nitroxide, can effectively penetrate biological membranes and scavenge superoxide radicals. Mitochondrial ROS are known to be the main sources of all oxygen-related free radicals, and antioxidant derivatives of tempol are accumulated in mitochondria. Nonetheless, the possible proposed biochemical mechanism overwhelming mitochondrial oxidative stress of tempol is attributed to hydroxylamine reduction of tempol as well as nitroxide formation (Wang et al., 2003; Wilcox, 2010). Moreover, tempol has been reported to improve chronic high salt intake induced kidney injury (Carlstr¨om et al., 2013), and to be effective in preventing several of the adverse consequences of oxidative stress (Wilcox, 2010), and type 1 diabetes induced organ injury (Zheng et al., 2013) in animal models. Here, we demonstrated beneficial effects on
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alleviation of oxidative protein damage by tempol and stress tolerance in mimetic aging model of Drosophila. Biomarkers of oxidative protein damage are often applied when a battery of markers of oxidative stress status is being studied. Several oxidative protein modifications such as advanced oxidation protein products (AOPPs) formation may result from ROS oxidative stress and lead to the formation of the high molecular weight insoluble aggregates that are common in aging and age-related disorders (C¸akatay, 2011). AOPPs contain a variety of protein oxidation products such as protein carbonyl groups, dityrosine, and advanced glycation end products (AGEs; Selmeci, 2011). Besides protein oxidation marker, other oxidative damage markers of lipid peroxidation include malondialdehyde, lipid hydroperoxides, isoprostanes, and thiobarbituric acid reacting substances (TBARS; Buege and Aust, 1978; Hanasand et al., 2012). TBARS is a group of reactive aldehydes resulting from ROS-induced degradation of polyunsaturated membrane lipids (Buege and Aust, 1978; Hanasand et al., 2012). Increase in oxidative stress may be one of the reasons for the decrease in the stress tolerance, which develops through natural and mimetic aging (Yanar et al., 2011; Aydin et al., 2012). The research on D-galactose show that optimum doses that are used for establishing a mimetic aging model of D-galactose affect the redox homeostasis by increasing the formation of hydrogen peroxide, galactitol, and AGEs (Yanar et al., 2011; Aydin et al., 2012). Although majority of the mimetic aging studies related to D-galactose administration were performed by using rodents, D-galactose-induced aging model was also commonly preferred in Drosophila (Cui et al., 2004). In that study, Cui and co-workers pointed out that D-galactose administration shortens the lifespan of Drosophila. Although use of synthetic antioxidants has recently become widespread, their effects on protecting and restoring cellular redox homeostasis are not entirely known (Augustyniak et al., 2010). The organisms as Drosophila are mostly composed of postmitotic cells; the results from this invertebrate are much more supportive of the free radical theory of aging than the results from vertebrates. Additionally, postmitotic cells in vertebrate as neurons and muscles are more sensitive than other type of cells in regards to oxidative stress mediators (C¸akatay, 2011). The aim of this study was to test the hypothesis whether tempol restores impaired redox homeostasis and increases stress tolerance in mimetic aging model of Drosophila. For this reason, we investigated extent of general oxidative stress and, specifically, oxidative protein damage in mimetically aged flies following tempol administration. Therefore, TBARS, AOPPs, Cu,Zn-SOD, and sialic acid (SA) were determined.
MATERIALS AND METHOD Chemicals and Apparatuses Chemicals and solvents used in the experiments were of the highest purity and analytical grade. All chemicals and reagents were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (St Louis, MO). Deionized water was used in the analytical procedures. Reagents were stored at +4°C. The reagents were maintained in equilibrium at room temperature for 0.5 h before use. All centrifugation procedures were performed with a Sigma 3–18 KS centrifuge (SIGMA Laborzentrifugen GmbH, Osterode am Harz, Archives of Insect Biochemistry and Physiology
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Time course of experimental period.
Germany). Oxidative stress parameters were run in duplicate by using the Biotek SynergyTM H1 Hybrid Multi-Mode Microplate Reader (BioTek US, Winooski, VT). Animals In this study Drosophila melanogaster (Diptera: Drosophilidae; fruit fly), the Oregon-R strain, was used. All the individuals making up the experimental groups were exposed to 60% relative humidity and ambient temperature of 25°C during experimental period. Animals were kept into 25 × 100 mm glass bottles containing 2 ml standard nutrient. Experimental Groups
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Control group: In standard feeding environment. Standard feedlot: contained 8.5 g corn flour, 0.75 g agar, 0.75 g dry yeast, 6.5 g sucrose, 0.5 ml 100% propionic acid, and 90 ml distilled water. Mimetically aged Drosophilas: By adding same amount of D-galactose instead of sucrose in the standard feeding environment. Mimetically aged Drosophilas + Tempol administration: By adding tempol in the concentration of 0.2% in addition to D-galactose (Izmalylov and Obukhova, 1996).
Two male and two female individuals were allowed to live for 4 weeks in organized environment described above, and the experimental process was terminated. In this period, the flies were at middle age. At the end of the fourth week, male flies were isolated, both stress tolerance tests and biochemical analyzes were carried out in flies. Female flies were discarded due to possible antioxidant effect of estrogens (Altun et al., 2011; Fig. 1) Stress Tolerance Tests (Cold, Heat, and Starvation) The stress response test needs to be run precisely and carefully to not let other physical factors be the reason for which the flies are dying. Flies exposed to the different diets were assayed simultaneously and percentage of survival ratio was calculated as Ns /Nt × 100, where Ns is the number of survived flies and Nt is the number of total flies. Archives of Insect Biochemistry and Physiology
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Cold Stress. Flies in an empty bottle, in groups of 10–15 individuals, were exposed to a temperature of 0 °C for 2 h. At the end of the period, they were taken into standard diet medium. After 24 h, the flies that survived were counted and the percentage of ratio was calculated by taking the average of three repeated processes into account. Heat Stress. The flies in groups of 10–15 individuals were exposed to 38.3°C and 60% relative humidity for 2 h, and the survivors were counted after 24 h and the percentage of ratio was calculated by taking the average of three repeated processes into account. Starvation Stress. The flies in groups of 10–15 individuals were kept in bottles in which there was no food. Dehydration was prevented by putting water absorbed Whatman filter papers in the bottles. After 24 h, the dead and surviving flies were counted and the percentage of ratio was calculated by taking the average of three repeated processes into account. Biochemical Methods Flies (25 in each group) were sacrificed by exposing to −80 °C for 15 min for three times. For the frozen 25 flies, homogenization was performed in ice-cold phosphate buffer solution by glass homogenizer (Potter-Elvehjem). Afterwards, the resulting homogenates were centrifuged at the rate of 7,000 rcf for 10 min and the resulting supernatants were stored at −80 °C until they were analyzed. The by-product of the centrifugation, supernatants of homogenates was used for the biochemical assays. Measurement of Advanced Oxidative Protein Products (AOPPs). Modified method of Hanasand et al. (2012) was performed for spectrophotometric determination of AOPPs concentrations. According to the procedure, homogenates were diluted with citric acid, 10 μl of 1.16 M KI was added to diluted solution, 2 min later followed by 20 μl acetic acid. The absorbance of the reaction mixture was immediately read at 340 nm against the blank solution. AOPPs concentrations were expressed as micromoles per liter of chloramine-T equivalents. Measurement of Protein-Bound Sialic Acid. SA concentrations were determined by the thiobarbituric acid (TBA) method of Tram et al. (1997), who have made some modifications to previous methods (Aminoff, 1961) that gave improved sensitivity and high reproducibility to SA assay. Homogenate proteins (30 μl) were precipitated with 500 μl trichloroacetic acid with the volume of 20% (w/v). The upper layer was removed and discarded. The precipitated proteins were dissolved in 280 μl H2 SO4 and then incubated in 80°C for 1 h for hydrolysis. N-acetylneuraminic acid was used as the standard. The samples, standards, and blank were treated with 70 μl of periodate reagent (25 mM periodic acid in 0.125N sulfuric acid) and incubated at 37°C for 30 min. The reaction was terminated by adding 70 μl of sodium arsenite (2% sodium arsenite in 0.22 M hydrochloric acid). Once the yellow color of liberated iodine had disappeared, 140 μl of TBA (0.1 M, pH 9.0) was added and the solution was heated in temperature-controlled water bath for 7.5 min, and then cooled in icy water. Dimethyl sulfoxide (560 μl) was added and corresponding absorbances were measured at 549 nm. Assay of Thiobarbituric Acid Reacting Substances. The rate of lipid peroxidation was determined by the procedure of Buege and Aust (1978). One of the major secondary products Archives of Insect Biochemistry and Physiology
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Figure 2. Survival percentage of groups after exposure to heat. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control; # P < 0.05 vs. mimetically aged group.)
of lipid peroxidation is reactive aldehydes. TBARS, along with other by-products, react with TBA to generate a colored product that absorbs maximally at 535 nm wavelength, representing the color produced by all the TBARS. The coefficients of intra- and interassay variations for TBARS assay were 3.4 and 5.4%, respectively. Assay of Superoxide Dismutase Activity (Cu,Zn-SOD). Determination of Cu,Zn-SOD (EC 1.15.1.1) activity was assayed in supernatant fractions based on the method developed by Sun et al. (1988). This assay involves the inhibition of nitroblue tetrazolium reduction, with xanthine oxidase used as a superoxide radical generator. One unit of Cu,Zn-SOD is defined as the amount of enzyme needed to exhibit a 50% dismutation of superoxide radical. The coefficients of intra- and interassay variations were 2.4 and 2.7%, respectively. Total Protein Assay. Supernatants were stored at −70°C for protein measurement. Total protein was determined by the Folin phenol procedure (Lowry et al., 1951). Statistical Analyses Data sets are shown as mean ± SE. While the results were statistically evaluated, one-way ANOVA and post hoc Bonferroni tests were performed. The significance level of P < 0.05 was considered as significant for the statistical evaluations.
RESULTS Stress Tolerance Test Results Test results are shown in Figures 2–4. After the exposure to heat and cold, more flies in mimetic aging group died compared to respective control group (P < 0.05). Although the ratio of those died of starvation is not too much, it is yet statistically at significant levels (P < 0.05 vs. control). It has been observed in the tempol administration that the percentage of survival in all three tests increased to a level close to control group (P > 0.05 vs. control). Archives of Insect Biochemistry and Physiology
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Figure 3. Survival percentage of groups after exposure to cold. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control; # P < 0.05 vs. mimetically aged group.)
Figure 4. Survival percentage after the starvation stress. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control group.)
Biochemical Results Cu,Zn-SOD activities and TBARS concentrations for the experimental groups are presented, respectively, in Figures 5 and 6. In comparison to the control group, it has been shown that the Cu,Zn-SOD activity has decreased (P < 0.05 vs. control) and TBARS concentration has significantly increased (P < 0.05 vs. control) by D-galactose administration. On the other hand, no significant variation has been observed in Cu,Zn-SOD activities by tempol administration compared to D-galactose administration (P > 0.05), whereas TBARS concentration has dropped to control group concentration (P < 0.05 in comparison to control group, P < 0.05 in comparison to mimetically aged group). SA and AOPPs concentrations for the experiment groups are presented, respectively, in Figures 7 and 8. When the SA concentrations were considered, we observed no significant difference between the mimetically aged and tempol-administered groups (P > 0.05), whereas SA concentrations were found to be lower in both mimetically aged and tempol-administered groups compared to control group (P < 0.05). AOPPs concentrations in the mimetically aged group male flies were significantly higher than those in the male control group (P < 0.05), and additionally tempol administration decreased AOPPs concentrations (P < 0.05 vs. mimetically aged group). Archives of Insect Biochemistry and Physiology
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Figure 5. Superoxide dismutase (Cu,Zn-SOD) activity values of the groups. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control group.)
Figure 6. Thiobarbituric acid reacting substances (TBARS) concentrations of the groups. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control; # P < 0.05 vs. mimetically aged group.)
Figure 7. Protein-bound sialic acid (SA) values of the groups. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control group.)
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Figure 8. Advanced oxidative protein products (AOPPs) concentrations of the groups. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control; # P < 0.05 vs. mimetically aged group.)
DISCUSSION A role for oxidative damage in mimetic aging is mainly supported by studies in rodents (Yanar et al., 2011; Aydin et al., 2012; C¸akatay et al., 2013). Increased oxidative protein damage and free radical mediated desialylation of cellular proteins is another important mechanism for cellular aging in rodents (C¸akatay et al., 2013). On the other hand, Drosophila is used widely to examine the relationship between oxidative stress and aging (Cui et al., 2004; Lushchak et al., 2013; Yamamato et al., 2013) because Drosophila genetic systems are well known and postmitotic tissues (Clancy and Birdsall, 2013). When D-galactose is present at high levels, it can also be converted to aldose hydroperoxides with the catalysis of galactose oxidase, resulting in the generation of a superoxide radical anion and other ROS (Zhong et al., 2009). Previous studies showed that mitochondrial dysfunction may be a key issue in the mechanism of accelerated aging caused by D-galactose (Long et al., 2007; Kumar et al., 2010). Additionally, it was demonstrated that D-galactose causes damage on the integrity of the mitochondria and disturbs the efficiency of ATP production, which in turn contributes to more ROS generation in mitochondria (Long et al., 2007). Prooxidant–antioxidant homeostasis was determined to influence prooxidation due to D-galactose-induced mimetic aging, although there are some differences in results obtained from various mitotic and postmitotic tissues (Yanar et al., 2011; Aydın et al., 2012; C¸akatay et al., 2013). In this study, the effect of tempol on an aging model in terms of stress tolerance, general oxidative stress, and specifically oxidative protein damage has been focused for the first time. According to our current findings, it is possible to state that tempol has a positive impact on the increase of stress tolerance as well as the detrimental effects of oxidative damage in mimetically aged flies. We have found out in our study that stress tolerance levels of older individuals increase with tempol application. Increase in resistance of the individual will ensure their survival and therefore extend lifespan. In the current study, lifespan of the flies colony has not been determined. However, increase in the amount of flies that resist to cold, heat, and hunger more could be explained by the increase of lifespan. Tempol administration improved stress tolerance response against heat and cold. Although, the effect of tempol on starvation response was not statistically significant, there was an increasing trend on mean values. In fact, starvation conditions are accepted as favor for the organisms due to Archives of Insect Biochemistry and Physiology
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the lesser mitochondrial electron leakage during decreased glucose uptake and utilization (Gredilla and Barja, 2005). At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, however, nitric oxide (NO), superoxide anion, and related ROS play an important role as regulatory mediators in nuclear signaling processes. Many of the ROS-mediated responses actually protect the cells against oxidative stress and reestablish “redox homeostasis.” Higher organisms, however, have evolved the use of NO and ROS also as signaling molecules for other physiological functions (Dr¨oge, 2002). There are many irreversible degenerative molecular processes in proteome of aging cells (C¸akatay, 2011). The most important process is the formation of ROS and the fact that they cause damage to lipid, protein, and DNA in cells (Huangfu et al., 2013; Na et al., 2013). In addition, more reactive secondary derived metabolites from such macromolecules form in time due to disruption in redox homeostasis in aging cells. In our study, this case has been observed by means of the increase in TBARS concentration, which is the indicator of cellular membrane damage. Moreover, it has been originally shown in our study that mimetic aging significantly accelerates free radical mediated deterioration of redox homeostasis in various stress conditions. Cu,Zn-SOD is an essential antioxidant enzyme in front line defense system that dismutates superoxide radical anion to hydrogen peroxide and molecular oxygen within the mitochondrial matrix (Maiese and Chong, 2004). It is well known that during aging process, there is a reduced Cu,Zn-SOD activity (Lawler et al., 2009) and antioxidant enzyme expression levels (Fleenor et al., 2012; Ramesh et al., 2012). In our study, tempol administration in the mimetically aged flies had no significant effect on the activity of Cu,Zn-SOD, which was already at lower levels compared with the untreated galactoseadministered rodents. Since it has been shown that free radicals have an impact on the aging process, the notion to increase the lifespan by the inhibition of those molecules has arisen. Therefore, the studies with synthetic antioxidants are still kept up-to-date. Although the use of antioxidants seems to be beneficial, yet we need to be alert in terms of causing endogenic defense system to become of secondary importance. This has been observed in our study as well and no significant change has been determined in Cu,ZnSOD activity in the tempol application. This may certainly be in relation to decreased Cu,Zn-SOD activity and/or expression level through aging process (Uzun et al., 2013). The spectral characteristics of AOPPs correspond to several chromophores, which include dityrosine, carbonyls, and pentosidine, however nitrotyrosine is not in this group (Breusing et al., 2010). Oxidative modifications of cellular proteins, as in AOPPs formation, usually result in loss of protein function. When mimetically aged flies are compared to tempol-administered group, the impaired redox homeostasis was reversed by tempol by means of decreased AOPPs concentrations. Impaired protein redox homeostasis, which appears to occur in mimetically aged group, may be an enhancing factor in the propagation of protein oxidation, as indicated by the AOPPs concentrations. Protein-bound SA residues play significant roles in various biological functions (Li and Chen, 2012). Desialylation shows its effect not only by altering the structure of glycoforms and also function of glycoproteins, but also by increasing the amount of SA concentrations, which leads to the emergence of pathologies in tissues (Goswami and Koner, 2002). SA residues occupying terminal positions in N-linked oligosaccharides of glycoproteins have been shown to play important roles in a variety of biological functions (Aminoff, 1961). In our study, there was no significant change in protein-bound SA concentrations in mimetically aged flies compared to tempol-administered groups. To maintain the Archives of Insect Biochemistry and Physiology
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critical functions of SA groups mentioned above, this finding might be explained by the importance of strict maintenance of the redox homeostasis of glycoproteins in proteomes of flies. In other words, the current results of our study suggest that in D-galactoseinduced mimetic aging, there is an association between desialylation of protein-bound SA and increased protein oxidation that leads to clustering of age-related disorder results obtained in these aged flies.
CONCLUSION In conclusion, our study demonstrated that scavenging ROS using tempol not only partly reduced organism oxidative damage during aging, but also directly scavenge the mediators related oxidative stress rather than improve reduced endogenous defense system, thereby improved endurance against environmental stress. Taken together, these effects led to a modest improvement of aging-related frailty.
ACKNOWLEDGMENT We declare that there is no conflict of interest.
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Tram TH, Brand Miller JC, McNeil Y, McVeagh P. 1997. Sialic acid content of infant saliva: comparison of breast fed with formula fed infants. Arch Dis Child 77:315–318. Uzun D, Korkmaz GG, Sitar ME, Cebe T, Yanar K, C¸akatay U, Aydın S. 2013. Oxidative damage parameters in renal tissues of aged and young rats based on gender. Clin Interv Aging 8:809– 815. Wang W, Jittikanont S, Falk SA, Li P, Feng L, Gengaro PE, Poole BD, Bowler RP, Day BJ, Crapo JD, Schrie RW. 2003. Interaction among nitric oxide, reactive oxygen species, and antioxidants during endotoxemia-related acute renal failure. Am J Physiol Renal Physiol 284:F532–F537. Wilcox CS. 2010. Effects of tempol and redox-cycling nitroxides in models of oxidative stress. Pharmacol Ther 126:119–145. Yamamoto R, Bai H, Dolezal AG, Amdam G, Tatar M. 2013. Juvenile hormone regulation of Drosophila aging. BMC Biol 11:85. doi:10.1186/1741-7007-11-85 Available at. http://www.ncbi.nlm.nih.gov/pubmed?term=Yamamoto%20R%255BAuthor%255D&cauthor =true&cauthor uid=23866071 Yanar K, Aydin S, C¸akatay U, Mengi M, Buyukpinarbasili N, Atukeren P, Sitar ME, Sonmez A, Uslu E. 2011. Protein and DNA oxidation in different anatomic regions of rat brain in a mimetic ageing model. Basic Clin Pharmacol Toxicol 109:423–433. Zheng H, Liu X, Patel KP. 2013. Centrally mediated erectile dysfunction in rats with type 1 diabetes: role of angiotensin II and superoxide. J Sex Med 10(9):2165–2176. Zhong SZ, Ge QH, Qu R, Li Q, Ma SP. 2009. Paeonol attenuates neurotoxicity and ameliorates cognitive impairment induced by D-galactose in ICR mice. J Neurol Sci 277:58–64. Zhou Z, Lenk RP, Dellinger A, Wilson SR, Sadler R, Kepley CL. Liposomal formulation of amphiphilic fullerene antioxidants. Bioconjug Chem 21:1656–1661.
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EFFECT OF TEMPOL ON REDOX HOMEOSTASIS AND STRESS TOLERANCE IN MIMETICALLY AGED DROSOPHILA Ugur Aksu Department of Biology, Science Faculty, Zoology Division, Istanbul University, Istanbul, Turkey
Karolin Yanar and Duygu Terzioglu Department of Medical Biochemistry, Cerrahpas¸a Medical Faculty, Istanbul University, Istanbul, Turkey
Tugc¸e Erkol and Evrim Ece Department of Biology, Science Faculty, General Biology Division, Istanbul University, Istanbul, Turkey
Seval Aydin, Ezel Uslu, and Ufuk C¸akatay Department of Medical Biochemistry, Cerrahpas¸a Medical Faculty, Istanbul University, Istanbul, Turkey
We aimed to test our hypothesis that scavenging reactive oxygen species (ROS) with tempol, a membrane permeable antioxidant, affects the type and magnitude of oxidative damage and stress tolerance through mimetic aging process in Drosophila. Drosophila colonies were randomly divided into three groups: (1) no D-galactose, no tempol; (2) D-galactose without tempol; (3) D-galactose, but with tempol. Mimetic aging was induced by D-galactose administration. The tempol-administered flies received tempol at the concentration of 0.2% in addition to D-galactose. Thiobarbituric acid reacting substance (TBARS) concentrations, advanced oxidation protein products (AOPPs), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), sialic acid (SA) were determined. Additionally, stress tolerances were tested. Mimetically aged group without tempol led to a significant decrease in tolerance to heat, cold, and starvation (P < 0.05), but tempol was used for Grant sponsor: Istanbul University; Grant number: 21261. Correspondence to: Dr. Ugur Aksu, Department of Biology, Faculty of Science, The University of Istanbul, Vezneciler-Istanbul 34459, Turkey. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 87, No. 1, 13–25 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21176
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these parameters. The Cu,Zn-SOD activity and SA concentrations were lower in both mimetically aged and tempol-administered Drosophila groups compared to control (P < 0.05), whereas there were no significantly difference between mimetically aged and tempol-administered groups. Mimetically aged group without tempol led to a significant increase in tissue TBARS and AOPPs concentrations (P < 0.05). Coadministration of tempol could prevent these alterations. Scavenging ROS using tempol also restores redox homeostasis in mimetically aged group. Tempol partly C 2014 restores age-related oxidative injury and increases stress tolerance. Wiley Periodicals, Inc. Keywords: tempol; mimetic aging; Drosophila; oxidative stress; sialic acid
INTRODUCTION Free radical theory of aging is one of the widely accepted theories set forth in relation to cellular effects of both natural and mimetic aging (Yanar et al., 2011; Aydin et al., 2012). According to this theory, reactive oxygen species (ROS) may initiate oxidative injury that a living organism is affected through its lifetime. Increased oxidative stress may cause functional decline and various age-related disorders in humans and experimental animals (C¸akatay, 2011). Aerobic organisms continuously produce ROS through their lifespan. It has been seen that those free radicals are the molecules that pose unpaired electron in the outermost molecular orbitals and have caused oxidative damage to cellular macromolecules such as DNA, proteins, and lipids (C¸akatay, 2011). To protect against harmful toxic effects of ROS and modulate physiological effects of ROS, the cell has developed its antioxidant systems. Under normal circumstances, ROS are metabolically formed but they are removed efficiently by antioxidant systems virtually, therefore no macromolecular damage occurs in cell. However, this homeostatic process is declined in favor of ROS with aging (C¸akatay, 2011). Impaired redox homeostasis is originated both by inefficiency of antioxidant systems and increased ROS formation due to aging process. The ability of amphipathic antioxidants to penetrate into cellular lipid bilayers is undoubtedly crucial to the protection against macromolecular oxidation (C¸akatay, 2006; Zhou et al., 2010). In agreement with this idea, several routes of superoxide dismutase administration have been described, and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) cannot easily penetrate biological membranes to attenuate the effects of intracellular production of superoxide radical anion (Fridovich, 1995). Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidineN-oxyl), a low molecular weight piperidine nitroxide, can effectively penetrate biological membranes and scavenge superoxide radicals. Mitochondrial ROS are known to be the main sources of all oxygen-related free radicals, and antioxidant derivatives of tempol are accumulated in mitochondria. Nonetheless, the possible proposed biochemical mechanism overwhelming mitochondrial oxidative stress of tempol is attributed to hydroxylamine reduction of tempol as well as nitroxide formation (Wang et al., 2003; Wilcox, 2010). Moreover, tempol has been reported to improve chronic high salt intake induced kidney injury (Carlstr¨om et al., 2013), and to be effective in preventing several of the adverse consequences of oxidative stress (Wilcox, 2010), and type 1 diabetes induced organ injury (Zheng et al., 2013) in animal models. Here, we demonstrated beneficial effects on
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alleviation of oxidative protein damage by tempol and stress tolerance in mimetic aging model of Drosophila. Biomarkers of oxidative protein damage are often applied when a battery of markers of oxidative stress status is being studied. Several oxidative protein modifications such as advanced oxidation protein products (AOPPs) formation may result from ROS oxidative stress and lead to the formation of the high molecular weight insoluble aggregates that are common in aging and age-related disorders (C¸akatay, 2011). AOPPs contain a variety of protein oxidation products such as protein carbonyl groups, dityrosine, and advanced glycation end products (AGEs; Selmeci, 2011). Besides protein oxidation marker, other oxidative damage markers of lipid peroxidation include malondialdehyde, lipid hydroperoxides, isoprostanes, and thiobarbituric acid reacting substances (TBARS; Buege and Aust, 1978; Hanasand et al., 2012). TBARS is a group of reactive aldehydes resulting from ROS-induced degradation of polyunsaturated membrane lipids (Buege and Aust, 1978; Hanasand et al., 2012). Increase in oxidative stress may be one of the reasons for the decrease in the stress tolerance, which develops through natural and mimetic aging (Yanar et al., 2011; Aydin et al., 2012). The research on D-galactose show that optimum doses that are used for establishing a mimetic aging model of D-galactose affect the redox homeostasis by increasing the formation of hydrogen peroxide, galactitol, and AGEs (Yanar et al., 2011; Aydin et al., 2012). Although majority of the mimetic aging studies related to D-galactose administration were performed by using rodents, D-galactose-induced aging model was also commonly preferred in Drosophila (Cui et al., 2004). In that study, Cui and co-workers pointed out that D-galactose administration shortens the lifespan of Drosophila. Although use of synthetic antioxidants has recently become widespread, their effects on protecting and restoring cellular redox homeostasis are not entirely known (Augustyniak et al., 2010). The organisms as Drosophila are mostly composed of postmitotic cells; the results from this invertebrate are much more supportive of the free radical theory of aging than the results from vertebrates. Additionally, postmitotic cells in vertebrate as neurons and muscles are more sensitive than other type of cells in regards to oxidative stress mediators (C¸akatay, 2011). The aim of this study was to test the hypothesis whether tempol restores impaired redox homeostasis and increases stress tolerance in mimetic aging model of Drosophila. For this reason, we investigated extent of general oxidative stress and, specifically, oxidative protein damage in mimetically aged flies following tempol administration. Therefore, TBARS, AOPPs, Cu,Zn-SOD, and sialic acid (SA) were determined.
MATERIALS AND METHOD Chemicals and Apparatuses Chemicals and solvents used in the experiments were of the highest purity and analytical grade. All chemicals and reagents were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (St Louis, MO). Deionized water was used in the analytical procedures. Reagents were stored at +4°C. The reagents were maintained in equilibrium at room temperature for 0.5 h before use. All centrifugation procedures were performed with a Sigma 3–18 KS centrifuge (SIGMA Laborzentrifugen GmbH, Osterode am Harz, Archives of Insect Biochemistry and Physiology
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Time course of experimental period.
Germany). Oxidative stress parameters were run in duplicate by using the Biotek SynergyTM H1 Hybrid Multi-Mode Microplate Reader (BioTek US, Winooski, VT). Animals In this study Drosophila melanogaster (Diptera: Drosophilidae; fruit fly), the Oregon-R strain, was used. All the individuals making up the experimental groups were exposed to 60% relative humidity and ambient temperature of 25°C during experimental period. Animals were kept into 25 × 100 mm glass bottles containing 2 ml standard nutrient. Experimental Groups
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Control group: In standard feeding environment. Standard feedlot: contained 8.5 g corn flour, 0.75 g agar, 0.75 g dry yeast, 6.5 g sucrose, 0.5 ml 100% propionic acid, and 90 ml distilled water. Mimetically aged Drosophilas: By adding same amount of D-galactose instead of sucrose in the standard feeding environment. Mimetically aged Drosophilas + Tempol administration: By adding tempol in the concentration of 0.2% in addition to D-galactose (Izmalylov and Obukhova, 1996).
Two male and two female individuals were allowed to live for 4 weeks in organized environment described above, and the experimental process was terminated. In this period, the flies were at middle age. At the end of the fourth week, male flies were isolated, both stress tolerance tests and biochemical analyzes were carried out in flies. Female flies were discarded due to possible antioxidant effect of estrogens (Altun et al., 2011; Fig. 1) Stress Tolerance Tests (Cold, Heat, and Starvation) The stress response test needs to be run precisely and carefully to not let other physical factors be the reason for which the flies are dying. Flies exposed to the different diets were assayed simultaneously and percentage of survival ratio was calculated as Ns /Nt × 100, where Ns is the number of survived flies and Nt is the number of total flies. Archives of Insect Biochemistry and Physiology
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Cold Stress. Flies in an empty bottle, in groups of 10–15 individuals, were exposed to a temperature of 0 °C for 2 h. At the end of the period, they were taken into standard diet medium. After 24 h, the flies that survived were counted and the percentage of ratio was calculated by taking the average of three repeated processes into account. Heat Stress. The flies in groups of 10–15 individuals were exposed to 38.3°C and 60% relative humidity for 2 h, and the survivors were counted after 24 h and the percentage of ratio was calculated by taking the average of three repeated processes into account. Starvation Stress. The flies in groups of 10–15 individuals were kept in bottles in which there was no food. Dehydration was prevented by putting water absorbed Whatman filter papers in the bottles. After 24 h, the dead and surviving flies were counted and the percentage of ratio was calculated by taking the average of three repeated processes into account. Biochemical Methods Flies (25 in each group) were sacrificed by exposing to −80 °C for 15 min for three times. For the frozen 25 flies, homogenization was performed in ice-cold phosphate buffer solution by glass homogenizer (Potter-Elvehjem). Afterwards, the resulting homogenates were centrifuged at the rate of 7,000 rcf for 10 min and the resulting supernatants were stored at −80 °C until they were analyzed. The by-product of the centrifugation, supernatants of homogenates was used for the biochemical assays. Measurement of Advanced Oxidative Protein Products (AOPPs). Modified method of Hanasand et al. (2012) was performed for spectrophotometric determination of AOPPs concentrations. According to the procedure, homogenates were diluted with citric acid, 10 μl of 1.16 M KI was added to diluted solution, 2 min later followed by 20 μl acetic acid. The absorbance of the reaction mixture was immediately read at 340 nm against the blank solution. AOPPs concentrations were expressed as micromoles per liter of chloramine-T equivalents. Measurement of Protein-Bound Sialic Acid. SA concentrations were determined by the thiobarbituric acid (TBA) method of Tram et al. (1997), who have made some modifications to previous methods (Aminoff, 1961) that gave improved sensitivity and high reproducibility to SA assay. Homogenate proteins (30 μl) were precipitated with 500 μl trichloroacetic acid with the volume of 20% (w/v). The upper layer was removed and discarded. The precipitated proteins were dissolved in 280 μl H2 SO4 and then incubated in 80°C for 1 h for hydrolysis. N-acetylneuraminic acid was used as the standard. The samples, standards, and blank were treated with 70 μl of periodate reagent (25 mM periodic acid in 0.125N sulfuric acid) and incubated at 37°C for 30 min. The reaction was terminated by adding 70 μl of sodium arsenite (2% sodium arsenite in 0.22 M hydrochloric acid). Once the yellow color of liberated iodine had disappeared, 140 μl of TBA (0.1 M, pH 9.0) was added and the solution was heated in temperature-controlled water bath for 7.5 min, and then cooled in icy water. Dimethyl sulfoxide (560 μl) was added and corresponding absorbances were measured at 549 nm. Assay of Thiobarbituric Acid Reacting Substances. The rate of lipid peroxidation was determined by the procedure of Buege and Aust (1978). One of the major secondary products Archives of Insect Biochemistry and Physiology
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Figure 2. Survival percentage of groups after exposure to heat. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control; # P < 0.05 vs. mimetically aged group.)
of lipid peroxidation is reactive aldehydes. TBARS, along with other by-products, react with TBA to generate a colored product that absorbs maximally at 535 nm wavelength, representing the color produced by all the TBARS. The coefficients of intra- and interassay variations for TBARS assay were 3.4 and 5.4%, respectively. Assay of Superoxide Dismutase Activity (Cu,Zn-SOD). Determination of Cu,Zn-SOD (EC 1.15.1.1) activity was assayed in supernatant fractions based on the method developed by Sun et al. (1988). This assay involves the inhibition of nitroblue tetrazolium reduction, with xanthine oxidase used as a superoxide radical generator. One unit of Cu,Zn-SOD is defined as the amount of enzyme needed to exhibit a 50% dismutation of superoxide radical. The coefficients of intra- and interassay variations were 2.4 and 2.7%, respectively. Total Protein Assay. Supernatants were stored at −70°C for protein measurement. Total protein was determined by the Folin phenol procedure (Lowry et al., 1951). Statistical Analyses Data sets are shown as mean ± SE. While the results were statistically evaluated, one-way ANOVA and post hoc Bonferroni tests were performed. The significance level of P < 0.05 was considered as significant for the statistical evaluations.
RESULTS Stress Tolerance Test Results Test results are shown in Figures 2–4. After the exposure to heat and cold, more flies in mimetic aging group died compared to respective control group (P < 0.05). Although the ratio of those died of starvation is not too much, it is yet statistically at significant levels (P < 0.05 vs. control). It has been observed in the tempol administration that the percentage of survival in all three tests increased to a level close to control group (P > 0.05 vs. control). Archives of Insect Biochemistry and Physiology
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Figure 3. Survival percentage of groups after exposure to cold. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control; # P < 0.05 vs. mimetically aged group.)
Figure 4. Survival percentage after the starvation stress. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control group.)
Biochemical Results Cu,Zn-SOD activities and TBARS concentrations for the experimental groups are presented, respectively, in Figures 5 and 6. In comparison to the control group, it has been shown that the Cu,Zn-SOD activity has decreased (P < 0.05 vs. control) and TBARS concentration has significantly increased (P < 0.05 vs. control) by D-galactose administration. On the other hand, no significant variation has been observed in Cu,Zn-SOD activities by tempol administration compared to D-galactose administration (P > 0.05), whereas TBARS concentration has dropped to control group concentration (P < 0.05 in comparison to control group, P < 0.05 in comparison to mimetically aged group). SA and AOPPs concentrations for the experiment groups are presented, respectively, in Figures 7 and 8. When the SA concentrations were considered, we observed no significant difference between the mimetically aged and tempol-administered groups (P > 0.05), whereas SA concentrations were found to be lower in both mimetically aged and tempol-administered groups compared to control group (P < 0.05). AOPPs concentrations in the mimetically aged group male flies were significantly higher than those in the male control group (P < 0.05), and additionally tempol administration decreased AOPPs concentrations (P < 0.05 vs. mimetically aged group). Archives of Insect Biochemistry and Physiology
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Figure 5. Superoxide dismutase (Cu,Zn-SOD) activity values of the groups. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control group.)
Figure 6. Thiobarbituric acid reacting substances (TBARS) concentrations of the groups. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control; # P < 0.05 vs. mimetically aged group.)
Figure 7. Protein-bound sialic acid (SA) values of the groups. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control group.)
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Figure 8. Advanced oxidative protein products (AOPPs) concentrations of the groups. (The bars represent mean of 25 animals ± SE. *P < 0.05 vs. control; # P < 0.05 vs. mimetically aged group.)
DISCUSSION A role for oxidative damage in mimetic aging is mainly supported by studies in rodents (Yanar et al., 2011; Aydin et al., 2012; C¸akatay et al., 2013). Increased oxidative protein damage and free radical mediated desialylation of cellular proteins is another important mechanism for cellular aging in rodents (C¸akatay et al., 2013). On the other hand, Drosophila is used widely to examine the relationship between oxidative stress and aging (Cui et al., 2004; Lushchak et al., 2013; Yamamato et al., 2013) because Drosophila genetic systems are well known and postmitotic tissues (Clancy and Birdsall, 2013). When D-galactose is present at high levels, it can also be converted to aldose hydroperoxides with the catalysis of galactose oxidase, resulting in the generation of a superoxide radical anion and other ROS (Zhong et al., 2009). Previous studies showed that mitochondrial dysfunction may be a key issue in the mechanism of accelerated aging caused by D-galactose (Long et al., 2007; Kumar et al., 2010). Additionally, it was demonstrated that D-galactose causes damage on the integrity of the mitochondria and disturbs the efficiency of ATP production, which in turn contributes to more ROS generation in mitochondria (Long et al., 2007). Prooxidant–antioxidant homeostasis was determined to influence prooxidation due to D-galactose-induced mimetic aging, although there are some differences in results obtained from various mitotic and postmitotic tissues (Yanar et al., 2011; Aydın et al., 2012; C¸akatay et al., 2013). In this study, the effect of tempol on an aging model in terms of stress tolerance, general oxidative stress, and specifically oxidative protein damage has been focused for the first time. According to our current findings, it is possible to state that tempol has a positive impact on the increase of stress tolerance as well as the detrimental effects of oxidative damage in mimetically aged flies. We have found out in our study that stress tolerance levels of older individuals increase with tempol application. Increase in resistance of the individual will ensure their survival and therefore extend lifespan. In the current study, lifespan of the flies colony has not been determined. However, increase in the amount of flies that resist to cold, heat, and hunger more could be explained by the increase of lifespan. Tempol administration improved stress tolerance response against heat and cold. Although, the effect of tempol on starvation response was not statistically significant, there was an increasing trend on mean values. In fact, starvation conditions are accepted as favor for the organisms due to Archives of Insect Biochemistry and Physiology
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the lesser mitochondrial electron leakage during decreased glucose uptake and utilization (Gredilla and Barja, 2005). At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, however, nitric oxide (NO), superoxide anion, and related ROS play an important role as regulatory mediators in nuclear signaling processes. Many of the ROS-mediated responses actually protect the cells against oxidative stress and reestablish “redox homeostasis.” Higher organisms, however, have evolved the use of NO and ROS also as signaling molecules for other physiological functions (Dr¨oge, 2002). There are many irreversible degenerative molecular processes in proteome of aging cells (C¸akatay, 2011). The most important process is the formation of ROS and the fact that they cause damage to lipid, protein, and DNA in cells (Huangfu et al., 2013; Na et al., 2013). In addition, more reactive secondary derived metabolites from such macromolecules form in time due to disruption in redox homeostasis in aging cells. In our study, this case has been observed by means of the increase in TBARS concentration, which is the indicator of cellular membrane damage. Moreover, it has been originally shown in our study that mimetic aging significantly accelerates free radical mediated deterioration of redox homeostasis in various stress conditions. Cu,Zn-SOD is an essential antioxidant enzyme in front line defense system that dismutates superoxide radical anion to hydrogen peroxide and molecular oxygen within the mitochondrial matrix (Maiese and Chong, 2004). It is well known that during aging process, there is a reduced Cu,Zn-SOD activity (Lawler et al., 2009) and antioxidant enzyme expression levels (Fleenor et al., 2012; Ramesh et al., 2012). In our study, tempol administration in the mimetically aged flies had no significant effect on the activity of Cu,Zn-SOD, which was already at lower levels compared with the untreated galactoseadministered rodents. Since it has been shown that free radicals have an impact on the aging process, the notion to increase the lifespan by the inhibition of those molecules has arisen. Therefore, the studies with synthetic antioxidants are still kept up-to-date. Although the use of antioxidants seems to be beneficial, yet we need to be alert in terms of causing endogenic defense system to become of secondary importance. This has been observed in our study as well and no significant change has been determined in Cu,ZnSOD activity in the tempol application. This may certainly be in relation to decreased Cu,Zn-SOD activity and/or expression level through aging process (Uzun et al., 2013). The spectral characteristics of AOPPs correspond to several chromophores, which include dityrosine, carbonyls, and pentosidine, however nitrotyrosine is not in this group (Breusing et al., 2010). Oxidative modifications of cellular proteins, as in AOPPs formation, usually result in loss of protein function. When mimetically aged flies are compared to tempol-administered group, the impaired redox homeostasis was reversed by tempol by means of decreased AOPPs concentrations. Impaired protein redox homeostasis, which appears to occur in mimetically aged group, may be an enhancing factor in the propagation of protein oxidation, as indicated by the AOPPs concentrations. Protein-bound SA residues play significant roles in various biological functions (Li and Chen, 2012). Desialylation shows its effect not only by altering the structure of glycoforms and also function of glycoproteins, but also by increasing the amount of SA concentrations, which leads to the emergence of pathologies in tissues (Goswami and Koner, 2002). SA residues occupying terminal positions in N-linked oligosaccharides of glycoproteins have been shown to play important roles in a variety of biological functions (Aminoff, 1961). In our study, there was no significant change in protein-bound SA concentrations in mimetically aged flies compared to tempol-administered groups. To maintain the Archives of Insect Biochemistry and Physiology
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critical functions of SA groups mentioned above, this finding might be explained by the importance of strict maintenance of the redox homeostasis of glycoproteins in proteomes of flies. In other words, the current results of our study suggest that in D-galactoseinduced mimetic aging, there is an association between desialylation of protein-bound SA and increased protein oxidation that leads to clustering of age-related disorder results obtained in these aged flies.
CONCLUSION In conclusion, our study demonstrated that scavenging ROS using tempol not only partly reduced organism oxidative damage during aging, but also directly scavenge the mediators related oxidative stress rather than improve reduced endogenous defense system, thereby improved endurance against environmental stress. Taken together, these effects led to a modest improvement of aging-related frailty.
ACKNOWLEDGMENT We declare that there is no conflict of interest.
LITERATURE CITED Altun D, Uysal H, Askin H, Ayar A. 2011. Determination of the effects of genistein on the longevity of Drosophila melanogaster meigen (Diptera; Drosophilidae). Bull Environ Contam Toxicol 86:120– 123. Aminoff D. 1961. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J 81:384–392. Augustyniak A, Bartosz G, Cipak A, Duburs G, Horakova L, Luczaj W, Majekova M, Odysseos AD, Rackova L, Skrzydlewska E, Stefek M, Strosov´a M, Tirzitis G, Venskutonis PR, Viskupicova J, Vraka PS, Zarkovi´c N. 2010. Natural and synthetic antioxidants: an updated overview. Free Radic Res 44:1216–1262. Aydin S, Yanar K, Atukeren P, Dalo E, Sitar ME, Uslu E, Caf N, C¸akatay U. 2012. Comparison of oxidative stress biomarkers in renal tissues of D-galactose induced, naturally aged and young rats. Biogerontology 13:251–260. Breusing N, Grune T. 2010. Biomarkers of protein oxidation from a chemical, biological and medical point of view. Exp Gerontol 45:733–737. Buege JA, Aust SD. 1978. Microsomal lipid peroxidation. Methods Enzymol 52:302–310. C¸akatay U. 2006. Pro-oxidant actions of alpha-lipoic acid and dihydrolipoic acid. Med Hypotheses 66:110–117. C¸akatay U. 2011. Protein redox-regulation mechanisms in aging. In: Bondy S, Maiese K, editors. Aging and age related disorders. New York: Springer, p 3–25. C¸akatay U, Aydin S, Atukeren P, Yanar K, Sitar ME, Dalo E, Uslu E. 2013. Increased protein oxidation and loss of protein-bound sialic acid in hepatic tissues of D-galactose induced aged rats. Curr Aging Sci 6:135–141. Carlstrom M, Brown RD, Yang T, Hezel M, Larsson E, Scheffer PG, Teerlink T, Lundberg JO, Persson AE. 2013. L-arginine or tempol supplementation improves renal and cardiovascular function in rats with reduced renal mass and chronic high salt intake. Acta Physiol (Oxf) 207:732–741. Archives of Insect Biochemistry and Physiology
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