Acta Physiologica Hungarica, Volume 100 (1), pp. 84–88 (2013) DOI: 10.1556/APhysiol.99.2012.001 First published online December 11, 2012 Increased total scavenger capacity in rats fed corticosterone and cortisol on lipid-rich diet J Stark1, Zs Tulassay1, G Lengyel1, D Szombath2, B Székács1, I Ádler1, I Marczell1, P Nagy-Répas1, E Dinya3, K Rácz1, G Békési1 1 nd 2 Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary Institute of Pathophysiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary 3 Department of Health Informatics, Development and Postgraduate Education, Faculty of Medicine, Semmelweis University, Budapest, Hungary 2 Received: August 16, 2011 Accepted after revision: July 17, 2012 Background: In our earlier studies both corticosterone and cortisol had antioxidant effect in vitro. Objectives: Our aim was to clarify whether corticosterone and cortisol oral administration results in beneficial antioxidant changes in Sprague-Dawley adult male rats in vivo. Methods: Experimental animals were fed a lipid rich diet and treated with corticosterone or cortisol in the drinking fluid. Control group was fed only lipid rich diet with untreated drinking water. The untreated group was fed a normal diet with untreated water. Total scavenger capacity (TSC) was measured before and after 4 weeks of treatment in blood samples using a chemiluminometric assay. Results: Both corticosterone and cortisol treatment caused increased TSC. The control group and the untreated group showed no significant changes in TSC. Conclusion: Our results support the hypothesis that corticosterone and cortisol administration can improve the antioxidant status not only in vitro but also in vivo. Keywords: corticosterone, cortisol, antioxidant, free radical, total scavenger capacity, lipid-rich diet, rats Steroid compounds are widely used in medical therapies, often as immune suppressants – an effect that steroids exert in a considerably complex way. One element of this effect is the inhibition of superoxide production in the azurophilic granules of neutrophil granulocytes. The azurophilic granules – also called as primary granules – contain free radicals and a wide variety of anti-microbial defensins that are able to eliminate phagocytosed pathogens after the granule’s fusion with phagocytic vacuoles. As it has been proved certain steroids like dexamethasone, prednisolone, hydrocortisone and estrogen decrease superoxide anion production (4, 14, 16, 17). Although this mechanism is very likely to be present, the relevance of it is presumably secondary in the immune suppressant processes, while it might play pivotal role in the potential treatment of certain free radical mediated disorders such as atherosclerosis. While the highly reactive compounds in the primary granules are essential in the cytotoxic response of phagocytes, due to their high reactivity they can cause direct and indirect damage to the surrounding host tissues as well. Not only immune suppressant glucocorticoids can exert such antioxidant effect. As an example, the protective function of estrogen against atherosclerotic plaque formation is known, and is at least partly mediated through the reduction of oxidative stress (12). Arises Corresponding author: Iván Horváth Heart Institute, University of Pécs, Ifjúság u. 13, H-7624 Pécs, Hungary Phone: +36-72-536-001; Fax: +36-72-536-490; E-mail: ivan.g.horvath@aok.pte.hu 0231–424X/$ 20.00 © 2012 Akadémiai Kiadó, Budapest Some steroids increase total scavenger capacity in vivo 85 the question whether the antioxidant potential can be separated from other steroid effects. To answer this question we systematically tested different steroid hormones and intermediers (2) in order to identify any connection between the molecular structure and the antioxidant capacity. The aim of our present study was to evaluate the effect of corticosterone and cortisol on the oxidative status in an in vivo animal model after having examined their antioxidant effect in vitro (1). Cortisol is considered to be the main glucocorticoid hormone in human, and as such, it increases blood sugar through gluconeogenesis, suppresses the immune system, and aids in fat, protein and carbohydrate metabolism. It also decreases bone formation. During pregnancy, increased production of cortisol between weeks 30–32 initiates production of foetal lung surfactant to promote maturation of the lungs. It is released in response to stress and a low level of blood glucocorticoids. Corticosterone is an intermediate of aldosterone synthesis and it is the main glucocorticoid hormone in rodents and birds, while in humans no specific role has been attributed to it (5, 15). Corticosterone:cortisol ratio was found to be higher in the brain and cerebrospinal fluid than in the blood, and corticosterone penetrated to the brain better, than cortisol. This finding raises the question of the role of corticosterone in the control of human brain function (8, 15). Materials and Methods Study design, experimental animals The study protocol was approved by the local ethical committee for animal experiments at Semmelweis University. Four separated, randomly selected groups of adult male Sprague– Dawley rats (n = 4 for each group), weighing 500–600 g, were studied. Three groups (Corticosterone, Cortisol, Control) were fed lipid-rich food (cholesterol: 1%, olive oil: 10%) and one group (untreated) was fed normal, conventional food for 28 days. Drinking fluid was provided in automated watering devices. In the treated groups of experimental animals corticosterone (150 µg/ml, Sigma-Aldrich, Saint Louis, MO) or cortisol (0.1 mg/ml, SigmaAldrich, Saint Louis, MO) was added to the drinking water. Corticosterone and cortisol doses were calculated according to data from the literature adjusted to the average daily fluid intake and body weight of the experimental animals (3, 18). The untreated and the control group received unaffected water. All groups had free access to food and water. The rats were housed on a controlled 12-h/12-h light/dark schedule (the lights were turned on at 18:00 h) and an ambient temperature of 22 ± 1 °C. Blood sample collection Blood samples were collected at day zero (Day 0) and day 28 (Day 28). Dorsal tail vein puncture was carried out using EDTA vacutainer tubes. Measurement of total scavenger capacity (TSC) TSC was measured using a chemiluminometric assay (DIACHEM Ltd., Budapest, Hungary). This method is suitable for measuring the total free radical production irrespectively of its origin. In this assay samples were incubated with a fresh-prepared solution containing microperoxidase, hydrogen peroxide and luminol, in TRIS-HCl buffer. The luminol component can be excitated by hydroxyl radicals which are produced by the microperoxidasehydrogen peroxide system in Fenton reaction. If a sample (blood, tissue suspension) is added Acta Physiologica Hungarica 100, 2013 Stark J et al. 86 to this system the chemiluminescence can be reduced. The blocking effect of the sample correlates with its redox potential and total scavenger capacity. Twenty μl plasma samples were used and the light emission of luminol was measured at 425 nm for 30 seconds on 37 °C on alkaline pH. The relative luminescence of every plasma sample was determined by comparing it to a blank control (sample/blank quotients). Blank controls contained all reagents but no plasma samples were added. Decrease in sample/blank quotient means better antioxidant status (increased TSC value). Statistical method The matched sample/blank quotients of individual samples were analyzed. The TSC data from day zero (Day 0) and day 28 (Day 28) were compared with paired sample two-tailed t-test (SPSS v. 12. Chicago, IL). Significance level was set to < 0.05. Results Our results are shown in Table I. TSC changes (Day 0 vs. Day 28) are expressed as the mean sample/blank ratios in every four-member group. The decrease of the quotient marks the increase of TSC. There was no significant change in the untreated group. The TSC value was reduced in the control group on the lipid-rich diet most likely because of the oxidative stress caused by the lipid load; however, this change was not significant. The groups on the lipidrich diet both with corticosterone (0.172 ± 0.052 vs. 0.084 ± 0.066) and with cortisol (0.110 ± 0.021 vs. 0.076 ± 0.037) showed significantly increased TSC. Table I. TSC results of the treatments in the study groups Food Drink Mean sample/blank quotients on Day 0 (± SD) Mean sample/blank quotients on Day 28 (± SD) normal water 0.140 ± 0.039 0.188 ± 0.140 Control lipid-rich water 0.257 ± 0.148 0.625 ± 0.911 Corticosterone lipid-rich corticosterone+water 0.172 ± 0.052 0.084 ± 0.066* Cortisol lipid-rich cortisol+water 0.110 ± 0.021 0.076 ± 0.037* Group Untreated *Significant change during the treatment (p < 0.05) Discussion In line with our expectations chronic (28 days) corticosterone and cortisol treatment improved the antioxidant state in rats on lipid-rich diet, however, due to the small number of experimental animals the study provides only limited information. Decreased total scavenger capacity of the control group (water + lipid-rich diet) can be considered as a logical consequence of the lipid load, but once again, because of the low number of experimental animals and great inter-animal variability, it did not reach the criteria of significance. In literature contradictory data have been published concerning the in vivo antioxidant effects of corticosterone. In rats 4 weeks of treatment did not increase reactive oxygen species (ROS) production in liver mitochondria and decreased the level of two lipid peroxidation Acta Physiologica Hungarica 100, 2013 Some steroids increase total scavenger capacity in vivo 87 protein markers, but increased oxidative damage to mtDNA (3). On the contrary, according to Dhanabalan et al. 15 days of corticosterone treatment in rats increased the levels of lipid peroxidation and hydrogen peroxide and decreased the activity of the antioxidant superoxide dismutase (SOD) and catalase enzymes (7). When corticosterone was administered to broiler chickens for 3 hours, lipid peroxidation of the liver and heart did not change, while plasma lipid peroxidation decreased. Furthermore, the plasma level of the nonenzymatic antioxidant uric acid, total antioxidant capacity and SOD activity in heart were elevated (13). Chronic (14 days) corticosterone administration to broiler chickens initially increased plasma lipid peroxidation and the formation of ROS. However, after 3 days of treatment nonenzymatic antioxidant capacity and SOD activity were enhanced and thus oxidative damage was alleviated (13). In another experiment after a 14-day corticosterone treatment of kestrels ROS formation and oxidative stress increased, while total serum antioxidant capacity did not change (5). Corticosterone administered to common lizards increased lipid peroxidation, decreased SOD activity, increased glutathione peroxidase activity and did not change the activity of catalase in the first days of treatment. As a result, the oxidant–antioxidant balance was biased toward oxidative stress (6). The effects of cortisol on neutrophil function in terms of superoxide production is surprisingly unclear although there are results that suggest that psychological stress – probably through cortisol synthesis – reduces superoxide production of human neutrophils (10). Our earlier results about the effect of cortisol (1) on superoxide production have been recently confirmed by another study, in which Khanfer examined changes in cortisol:DHEAS ratio and its consequences on neutrophil function (9). In conclusion, our data confirm the antioxidant effect of corticosterone and cortisol reported previously by our study group but now amongst in vivo circumstances. Although previous data in literature are contradictory, and our current study due to financial issues was carried out with a low number of experimental animals, we think the results support our conception about antioxidant steroids and indicate the importance of the topic. The significant antioxidant effect of cortisol in our model emphasize the overlapping spectrum-like receptor specificity of the structurally related steroid compounds, since cortisol is not synthesized normally in rats. The aim of our experiment was to underline this property so in the future such attributes could be detached from the complex effects of steroids. We think that the antioxidant capacity of steroid compounds can have innovative pharmacological relevance in the future, and more generally the precise mapping of the complex effects of steroids can open new scopes for these drugs. Acknowledgements The authors are thankful for the chemiluminometric assay provided by DIACHEM Ltd. 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