Archives of Physiology and Biochemistry 2003, Vol. 111, No. 5, pp. 437–442
Influence of Antigen Stimulation on the Oxidative Stress Parameters in Outbred and Inbred Rabbits S. Tanchev1, V. Gadjeva2 and S. Stanilova3 Department of Genetics, Breeding and Reproduction, Faculty of Agriculture; 2Department of Chemistry and Biochemistry; and 3Department of Molecular Biology, Immunology and Genetics, Faculty of Medicine, Trakia University, Stara Zagora, Bulgaria
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Abstract
Introduction
The influence of antigen stimulation on the oxidative stress parameters in two groups of rabbits–inbred and outbred were explored by evaluation of the level of lipid peroxidation products (MDA) in the plasma membrane, and the activity of erythrocyte antioxidant defense enzymes superoxide dismutase (SOD) and catalase (CAT). There was not a significant difference between levels of MDA in inbred and outbred rabbits before immunization. However, SOD activity in inbred rabbits was significantly increased in comparison with that of outbred (p = 0.006). Significantly higher plasma levels of lipid peroxidation products were detected in both inbred and outbred rabbits during immune response in comparison to the corresponding groups before immunization (p = 0.008 and p = 0.002). SOD and CAT activities in erythrocytes of rabbits during immune response were also significantly increased compared to that before immunization. In addition, during immune response SOD and CAT activities were found to be positively correlated to each other in both inbred and outbred rabbits (r = 0.727 and r = 0.916). In conclusion, our results suggest the presence of an increased oxidative stress during the antigen stimulation accompanied by an adaptive increase of SOD and CAT activities. 30 days after immunization, the plasma levels of MDA and the activities of SOD and CAT in erythrocytes decreased and reached values close to the controls.
Reactive oxygen species (ROS), such as O2•-, H2O2 and OH• are highly reactive species generated by the biochemical redox reactions as a part of the normal cell metabolism. Exposure to environmental factors, such as UV light, cigarette smoke, environmental pollutants and gamma radiation, accelerates the generation of ROS (Marks et al., 1996). Some exogenic compounds including drugs can result into increased production of free radicals (Hug et al., 1997). In addition, excessive generation of ROS is observed in monocytes during phagocytosis of foreign particles such as bacteria, old cells and foreign particles (Marks et al., 1996; Mytar et al., 1999). Low levels of ROS are indispensable as mediators in many cell processes including differentiation, cell cycle progression or growth arrest, apoptosis and immunity (Mates & Sanchez-Jimenez, 2000; Shackelford et al., 2000). In contrast, high doses and/or inadequate removal of ROS result in oxidative stress, which may cause severe metabolic malfunctions and damage of biological macromolecules (Hemnani & Parihar, 1998; Toyokuni, 1999). Prime targets of ROS are the polyunsaturated fatty acids (PUFA) in the membrane lipids. This attack causes lipid peroxidation. Furthermore, the decomposition of peroxidized lipids yields a wide variety of end products, including malondialdehyde (MDA) that is widely used as an indicator of free radical damages (Marks et al., 1996). To prevent the damages caused by the ROS, multiple defense systems, collectively called antioxidants, are present
Keywords: Oxidative stress, lipid peroxidation, catalase, superoxide dismutase, inbred, outbred, rabbits.
Accepted: 9 July, 2004 Address correspondence to: Assoc. Prof. Vesselina Gadjeva, Ph.D. Department of Chemistry and Biochemistry, Medical Faculty, 11 Armeiska Str., Stara Zagora 6000, Bulgaria. Tel.: + 359 42 79012; Fax: + 359 42 74112; E-mail:
[email protected] DOI: 10.1080/13813450312331342292
© 2003 Taylor & Francis Ltd.
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in human serum and erythrocytes (Esterbauer et al., 1992; Patterson & Leake, 1998). Erythrocytes are excellently equipped to handle intracellular oxidative stress through the combined activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione (GSH). SOD is believed to play a major role in the first line of antioxidant defense (Siems et al., 2000). Although catalase is not essential for some cell types under normal conditions, it plays an important role in the acquisition of tolerance to oxidative stress in the adaptive response of cells (Mates et al., 1999). Offspring born to closely related parents commonly show reduced fitness (Crnokrak & Roff, 1999; Husband & Schemske, 1996; Keller, 1998; Saccheri et al., 1996), particularly under stressful conditions (Dahlgaard & Hoffman, 2000; Hauser & Loeschke, 1996). This phenomenon has long been recognized and is referred to as inbreeding depression (Darwin 1868; Charlesworth & Charlesworth, 1987) and is thought to be one of the primary selective forces opposing the build-up of deleterious recessive mutations (Lynch, 1993). So far, only limited number of reports have described the influence of pathogens on the levels of the oxidative stress parameters in blood which are considered to be a criterion for the oxidative status of entire organism. The aim of the present study was to determine the effect of antigen stimulation on the parameters of oxidative stress through the levels of lipid peroxidation products (MDA) in plasma membrane, and the activities of antioxidant defense enzymes superoxide dismutase (SOD) and catalase (CAT) in erythrocytes of both inbred and outbred rabbits.
Materials and methods Experimental animals Two groups of rabbits, bred in the animal facilities of Trakia University, were used for this study. The first group of 13 outbred rabbits was heterogeneous and was named the control group. The second group contained 12 inbred rabbits, which were the first inbred generation from sibs – 1 male and 3 female rabbits. All animals received food and water ad libitum and were in good health. They were 10-months old with 3.5–4 kg body weight at the start of the experiment. Immunization protocol Experimental rabbits were immunized with 50 mg keyhole limpet hemocyanin (KLH) in 0.5 ml saline, injected subcutaneously. Weekly boosters of antigen in saline were administered 3 times for primary humoral responses. Blood was collected before the first injection of antigen, after the 3rd antigen injection, and 30 days later.
and the plasma was carefully separated. Erythrocyte lysate was prepared by the method of Ivanov (1999). In brief: after the erythrocyte pellet was washed three times with saline, and 0.5 ml of the cell suspension was diluted with 2 ml cold water to lyse the erythrocytes. To 0.2 ml lysate 1.8 ml water and ethanol/chloroform (3 : 5 v : v) were then added to precipitate hemoglobin. The tubes were shaken vigorously for 5 min and centrifuged at 2500 rpm for 20 min. The supernatant was used for determination of enzyme activity. Estimation of products of lipid peroxidation The total amount of lipid peroxidation products in the plasma of inbred and outbred rabbits was estimated using the thiobarbituric acid (TBA) method, which measures the malondialdehyde (MDA) reactive products (Plaser et al., 1966). In brief, 1.0 ml of plasma, 1.0 ml of normal saline and 1.0 ml of 25% trichloro-acetic acid (TCA) were mixed and centrifuged at 2000 for 20 min. 1 ml of protein free supernatant was taken, mixed with 0.25 ml of 1% TBA and boiled for 1 h at 95°C. After cooling, the intensity of the pink color of the obtained fraction product was read at 532 nm. Measurement of antioxidant enzymes in erythrocytes SOD activity was estimated as described by Sun et al., (1988) with minor modifications. The xanthine/xanthine oxidase system was used to generate the superoxide anion. This anion reduces nitroblue tetrazolium (NBT) to formazan, which is monitored at 560 nm. SOD in the sample removes the superoxide anion and inhibits the reduction. The level of this reduction is used as a measure of SOD activity. The final concentrations of xanthine, xanthine oxidase and nitroblue tetrazolium in the assay were 50 mM, 10 U/ml and 0.125 mM. One unit of SOD activity is defined as the amount of enzyme causing 50% inhibition of the reduction of NBT to formazan observed. CAT activity was assessed in the erythrocyte lysate by the method described by Beers and Sizer (1952). Briefly, hydrogen peroxide (30 mM) was used as a substrate and the decrease in H2O2 concentration at 22°C in phosphate buffer (50 mM, pH 7.0) was followed spectroscopically at 240 nm. One unit of CAT activity is defined as the amount of enzyme that degrades 1 mM H2O2 per min. Results are presented as units per g hemoglobin. The hemoglobin concentration of lysate was determined by the cyanmethemoglobin method (Mahoney et al., 1993). The results are reported as means ± SE. Statistical analysis was performed with Student’s t-test and multiple regression analysis. p < 0.05 was considered statistically significant.
Results Blood samples Blood was collected in tubes containing ethylendiaminetetraacetic acid (EDTA), centrifuged at 3000 rpm for 15 min,
The results of MDA-reactive products estimated in the inbred rabbits and control group are given in Figure 1. The level of MDA in inbred rabbits before immunization was not signifi-
Oxidative Stress in Outbred and Inbred Rabbits
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Fig. 1. Comparison of MDA levels in plasma of inbred rabbits and controls. The results are expressed as mean ± S.D. * p = 0.008, compared to controls before antigen stimulation. ** p = 0.002, compared to inbred rabbits before antigen stimulation.
cantly different than that of the control group (mean 1.465 ± 0.235 mmol/l vs 1.619 ± 0.264 mmol/l, p > 0.05). Significantly higher plasma levels of lipid peroxidation products were detected in both inbred and outbred rabbits after last injection of antigen compared to the corresponding groups before immunization (mean 2.020 ± 0.508 mmol/l, p = 0.002 and 1.919 ± 0.214 mmol/l, p = 0.008, respectively, t-test). There was no significant difference between levels of MDA in inbred and outbred rabbits during immune response (p > 0.05). However, 30 days after immunization, the plasma levels of MDA for both inbred and outbred rabbits decreased and reached values close to that before immunization (mean 1.409 ± 0.170359 mmol/l and 1.527 ± 0.249198 mmol/l, p > 0.05). When erythrocyte SOD activity was studied, it was significantly higher in inbred rabbits compared to the control group (mean 3772.23 ± 1467.18 U/mgHb vs 1801.29 ± 655.52 U/mgHb, p = 0.006), (Fig. 2). Significantly higher level of SOD in both inbred and control rabbits during immune response was detected compared to the level before immunization (mean 6988.28 ± 1763.75 U/gHb, p = 0.002 and 8091.97 ± 2520.53 U/gHb, p = 0.0001, respectively, ttest). There was a significant difference between SOD activities in inbred and outbred rabbits during immune response (p < 0.05). However, 30 days after immunization SOD activities were decreased. For inbred rabbits SOD activity reached a level close to that before immunization (mean 3375.88 ± 560.61 U/gHb, p > 0.05). This is in contrast to controls, where twice higher levels were observed compared to before
immunization (mean 3773.62 ± 551.78 U/gHb, p = 0.33, t-test). The results of catalase activity estimated in the inbred and outbred rabbits are given in Figure 3. There was no significant difference between CAT in inbred rabbits and controls before immunization (mean 44,543.14 ± 18,670.60 U/gHb vs 43,219.8 ± 12,402.05 U/gHb, p > 0.05). CAT activity in both groups during antigen stimulation was significantly increased compared before immunization (mean 50,789.17 ± 15,722.87 U/gHb, p = 0.001 and 60,025.67 ± 11,975.18 U/gHb, p = 0.04). There was not significant difference between CAT activities in inbred rabbits and controls during immune response (p > 0.05). However, 30 days after immunization CAT activities in inbred rabbits and controls decreased and reached levels significantly lower than these before immunization (mean 29,137.14 ± 6098.98 U/gHb, p = 0.002 and 29,460.48 ± 6484.97 U/gHb, p = 0.06) and significantly lower than the corresponding group during antigen stimulation (p < 0.0000). Investigating relationships among the levels of MDA, SOD and CAT showed the following correlations. A significant negative correlation was observed in the inbred rabbits (SOD vs MDA, r = -0.58) whilst no such significant correlations between these parameters in the outbred rabbits (SOD vs MDA r = -0.24; CAT vs MDA r = -0.12) were observed before antigen stimulation. Negative correlations for both inbred and outbred rabbits were also found between MDA, SOD and CAT 30 days after antigen stimulation (SOD vs MDA for outbred rabbits r = -0.793 and CAT vs MDA for
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Fig. 2. Comparison of SOD activity in erythrocytes of inbred rabbits and controls. The results are expressed as mean ± S.D. * p = 0.006, compared to controls before antigen stimulation. ** p = 0.002, compared to controls before antigen stimulation. *** p < 0.0001, compared to inbred rabbis before antigen stimulation and controls with antigen stimulation.
Fig. 3. Comparison of CAT activity in erythrocytes in erythrocytes of inbred rabbits and controls. The results are expressed as mean ± S.D. * p = 0.001, compared to controls before antigen stimulation. ** p = 0.04, compared to inbred rabbits before antigen stimulation. *** p = 0.0000, compared to the corresponding group during antigen stimulation.
Oxidative Stress in Outbred and Inbred Rabbits
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B Fig. 4. (A) Correlation between SOD and CAT in outbred rabbits during immune response (r = 0.727). (B) Correlation between SOD and CAT in inbred rabbits during immune response (r = 0.916).
outbred rabbits r = -0.6057). As shown in Figure 4A and B, SOD and CAT activities were found to be positively correlated during immune response in both inbred and outbred rabbits rabbits (r = 0.916 and r = 0.727, respectively). This positive correlation between SOD and CAT was not significant either before, or 30 days after, antigen stimulation (r = 0.21 and r = 0.38, respectively).
Discussion Inbreeding in animals can increase their susceptibility to pathogens, but direct evidence from wild population is scarce (Acevevo, 2003) and it is unclear whether all pathogens are affected equally. In the current study we explored to our knowledge for the first time the influence of antigen stimulation on antioxidant enzyme activities and MDA levels in inbred rabbits and outbred, heterogeneous rabbits. Our results showed that both
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outbred and inbred rabbits have low level of MDA prior antigen stimulation. However, the erythrocyte CAT was higher and SOD was significantly higher in inbred rabbits compared to outbred, which might be due to primary increased capacity of antioxidant enzyme system in inbred rabbits. Significantly higher plasma levels of lipid peroxidation products were detected in both inbred rabbits and in the controls during antigen stimulation. This suggests the presence of increased oxidative stress during immune response. We report, a 20% increase of MDA in inbred rabbits compared to controls, thus presenting inbred rabbits as about 20% more sensitive to oxidative stress than outbred rabbits. This increase could possibly be in response to the production of ROS during antigen stimulation and need SOD and CAT for detoxication. Important sources of increased ROS production might be the monocytes (Marks et al., 1996; Mytar et al., 1999) as well as lymphocytes during the mounting of immune response. The results from evaluation of anti-KLH demonstrate that all rabbits responded upon immunization with KLH and reached its maximum four weeks after primary injection. Moreover, the first inbred generation can lead to differential adaptive humoral immune responses to primary immunization (to be published). We suggest that the differences in the parameters of oxidative stress, particularly SOD activity might be related with outcome of the immune response. The observed decrease in MDA levels 30 days after immunization suppose a reduced ROS production that might be attributed to attenuation of the immune response. According to our results, this increase of MDA levels was accompanied by significant increase in the activity of the antioxidant defense enzymes SOD and CAT during immune response. The observed increase of SOD was 2.4 times higher in the outbred compared to inbred rabbits. This suggests that inbred rabbits are less adaptive to the oxidative stress than outbred rabbits. In addition we observed a direct positive correlation between SOD and CAT activities immediately after immunization in both inbred rabbits and controls (r = 0.727 and r = 0.90 respectively). Recently, it was suggested that H2O2 have a stimulatory effect on SOD activity (Kosenko et al., 1997). In addition, activation of CAT by superoxide anion was also shown (Tauler et al., 1999). In this sense, the observed increase of SOD activity in inbred rabbits immediately after antigen stimulation in comparison with controls in our study could indicate the compensated decomposition of H2O2 through increased activity of CAT, which in turn could be activated by the increased production of O2•during antigen stimulation. Thus, CAT, that is known to be not essential for some cell types under normal conditions, may play an important role in the adaptive response to oxidative stress, as was suggested earlier. However, the increased SOD and CAT activities appeared to be sufficient to inactivate the increased ROS; we measured reduced levels of MDA 30 days after immunization. Thus, we suggest that increased oxidative stress exists during antigen stimulation, and was accompanied by a relatively suf-
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ficient increase in the activity of antioxidant enzyme system. When immune response fell down, the observed SOD and CAT activities tended to return to the values before immunization that might be connected with the decreased oxidative burden. A confirmation of this suggestion is provided by our results, showing a negative correlation between MDA level SOD and CAT 30 days after last injection of antigen for inbred and outbred rabbits. However, CAT was about 1.5 times lower than that before immunization. This result suggests that both inbred and outbred rabbits actually are in a risk of oxidative injury after immunization. Our further aim of the research is to follow up the oxidative status of the second inbred generation from sibs.
References Acevevo K (2003): Disease susceptibility in California sea lions. Nature 422(6): 35–64. Beers R, Sizer T (1952): Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195: 133–138. Charlesworth D, Charlesworth B (1987): Inbreeding depression and its evolutionary consequences. A Rev Ecol Syst 18: 237–268. Crnokrak P, Roff DA (1999): Inbreeding depression in the wild. Heredity 83: 260–270. Dahlgaard J, Hoffmann AA (2000): Stress resistance and environmental dependency of inbreeding depression in Drosophilia melanogaster. Consent Biol 14: 1187–1192. Darwin CR (1868): The variation of animals and plants under domestication. London: John Murray. Esterbauer H, Gebicki J, Puhl H, Jurgens G (1992): The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med 13: 341–390. Hauser TP, Loeschke V (1996): Drought stress and inbreeding depression in lychnis flos-cucli (Caryophyllaccae). Evolution 50: 1119–1126. Hemnani T, Parihar MS (1998): Reactive oxygen species and oxidative DNA damage. Indian J Physiol Pharmacol 42: 440–452. Husband BC, Schemske DW (1996): Evolution of the magnitude and timing of inbreeding depression in plants. Evolution 50: 54–70. Hug H, Strand S, Grambihler A, Galle J, Hack V, Stremmel W, Krammer PH, Galle PR (1997): Reactive oxygen intermediates are involved in the induction of CD95 ligand mRNA expression by cytostatic drugs in hepatoma cells. J Biol Chem 272: 28191–28193. Ivanov IT (1999): Low pH-induced hemolysis of erythrocytes is related to the entry of the acid into cytosole and oxidative stress on cellular membranes. Biochem Biophys Acta 1415: 349–360.
Kellcr L (1998): Inbreeding and its fitness effects in an insular population of song sparrows (Melosfiiw melodia). Evolution 52: 240–250. Kosenko EA, Kaminsky Yu G, Stavrovskaya IG, Sirota TV, Kondrashova MN (1997): The stimulatory effect of negative air ions and hydrogen peroxide on the activity of superoxide dismutase. FEBS Lett 410: 309–312. Lynch M (1993): The mutational meltdown in asexual populations. J Hmd 84: 339–344. Mahoney JJ, Vreman HJ, Stevenson DK, Van Kessel AL (1993): Measurement of carboxyhemoglobin and total hemoglobin by five specialized spectrophotometers (CO-oximeters) in comparison with reference methods. Clin Chem 39: 1693–1700. Marks DB, Marks AD, Smith CM (1996): Oxygen metabolism and oxygen toxicity. In: Velker J, ed., Basic Medical Biochemistry. A Clinical Approach, Baltimore, MD, Williams & Wilkin. Mates JM, Perez-Gomez C, Nunez de Castro I (1999): Antioxidant enzymes and human diseases. Clin Biochem 32: 595–603. Mates JM, Sanchez-Jimenez FM (2000): Role of reactive oxygen species in apoptosis: implications for cancer therapy. Int J Biochem Cell Biol 32: 157–170. Mytar B, Siedlar M, Woloszyn M, Ruggiero I, Pryjma J, Zembala M (1999): Induction of reactive oxygen intermediates in human monocytes by tumour cells and their role in spontaneous monocyte cytotoxicity. Br J Cancer 79: 737–743. Patterson RA, Leake DS (1998): Human serum, cysteine and histidine inhibit the oxidation of low density lipoprotein less at acidic pH. FEBS Lett 434: 317–321. Plaser ZA, Cushman LL, Janson BC (1966): Estimation of product of lipid peroxidation (Malonyl Dialdehyde) in biochemical systems. Anal Biochem 16: 359–364. Saccheri IJ, Brakefield PM, Nichols RA (1996): Severe inbreeding depression and rapid fitness rebound in the butterfly Bicyclus anynana (Satyridae). Evolution 50: 2000– 2013. Shackelford RE, Kaufmann WK, Paules RS (2000): Oxidative stress and cell cycle checkpoint function. Free Rad Biol Med 28: 1387–1404. Siems WG, Sommerburg O, Grune T (2000): Erythrocyte free radical and energy metabolism. Clin Nephrol 53: S9–17. Sun Y, Oberley LW, Li Y (1988): A simple method for clinical assay of superoxide dismutase. Clin Chem 34: 497–500. Tauler P, Gimeno I, Aguilo A, Guix MP, Pons A (1999): Regulation of erythrocyte antioxidant enzyme activities in athletes during competition and short-term recovery. Pflugers Archiv – Eur J Physiol 438: 782–787. Toyokuni S (1999): Reactive oxygen species-induced molecular damage and its application in pathology. Pathol Int 49: 91–102.
