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Address correspondence to Seyithan Taysi, M.D., Department of Biochemistry, Ataturk University Medical School, 25240 Erzurum, Turkey; tel 90 442 236 1212 2220; fax 90 442 236 0867; e-mail seytaysi{at}hotmail.com.
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Abstract |
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(received 17 August 2002; accepted 30 August 2002)
Keywords: lipid peroxidation, malondialdehyde, oxygen free radicals, staphylococcus aureus, oxidative stress, antioxidant enzymes
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Introduction |
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Oxygen free radicals (OFRs) and lipid peroxides have been implicated in the pathogenesis of many diseases, including diabetes mellitus, cancer, rheumatoid arthritis, systemic lupus erythematosus, Behcet’s disease, infectious diseases, atherosclerosis, and aging [2–7]. Free radicals are chemical species with an unpaired electron and they are generally highly reactive. They are produced continuously in cells, either accidentally as metabolic byproducts or deliberately during processes such as phagocytosis. In aerobic cells, the most important free radicals reactants are oxygen derivates (hydroxyl radical,OH•, superoxide anion, O2•-), hydrogen peroxide (H2O2), and certain transition metals. Cells possess an array of antioxidant defenses that help to prevent the formation of free radicals and to limit their damaging effects [8]. This defense system includes antioxidant molecules, such as glutathione (GSH), and various antioxidant enzymes. Superoxide dismutase (SOD), the first line of defense against oxygen-derived free radicals, catalyses dismutationof O2•- to H2O2. Glutathione peroxidase (GPx), a selenoprotein, reduces both lipid and nonlipid hydroperoxides, as well as H2O2, and oxidizes GSH. Oxidized glutathione (GSSG) is reduced back to GSH by glutathione reductase [9–12].
Reactive free radicals that are formed within cells can oxidize biomolecules, leading to cell death and tissue injury. Free radicals can attack almost any component of the cell, but lipids, proteins, and nucleic acids are particularly important targets. Lipids of cells membranes and organelles are frequently damaged, resulting in lipid peroxidation [8]. The process of lipid peroxidation involves oxidative degradation of polyunsaturated fatty acids to malondialdehyde (MDA), which is commonly measured by the chromogenic thiobarbituric acid reaction and expressed as total thiobarbituric acid reactive substances (TBARS) [13,14].
To the authors’ knowledge, there is no previous study of tissue lipid peroxidation and antioxidant enzyme activities in acute maxillary sinusitis induced in rabbits by treatment with killed S. aureus. In the present study, we used this experimental model for measurements of MDA levels and SOD, GPx, and CAT activities in the maxillary sinus mucosa.
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Material and Methods |
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Induction of rhinosinusitis. Eight New Zealand white rabbits of either sex (body weight, 1.5 to 2.5 kg) that were free of nasal infection were used for this study. The animals were housed under standard conditions at the animal research laboratory of Ataturk University with free access to food and water. The right maxillary sinus of the 8 rabbits was used for the experimental induction of sinusitis and the contralateral maxillary sinus of each rabbit served as a control. The animals were anesthetized by im injection of ketamine (50 mg/kg) and diazem (2 mg/kg). After infiltration of a local anesthetic, jetokain, incisions of the skin and periostium were made on the nasal dorsum. Rhinosinusitis was induced by introducing 0.2 ml of a heat-killed suspension of S. aureus CMF-1 at the concentration of 1 x 109 CFU/ml into the right maxillary sinus cavity; 0.2 ml of physiologic saline solution (0.9 % NaCl) was injected in the left maxillary sinus as a control [15].
All rabbits were kept 7 days after the inoculation for the development of symptomatic rhinosinusitis. At the end of the seventh day, the rabbits were sedated with a respiratory failure dose of pentobarbital sodium (120 mg/kg, iv). The external nasal dorsum was sterilized by swabbing with poviod. After skin elevation, the upper wall of the maxillary sinus was excised and mucosa samples were taken for biochemical studies. Inflammation and various degrees of exudation were grossly evident in the treated maxillary sinuses, but not in the contralateral control sinuses [16].
Biochemical measurements. Each sample of sinus mucosa was homogenized (Omni Tissue Homogenizer, Omni Corp., USA) and the homogenate was centrifuged at 10,000 g for 60 min. The clear supernatant was removed and assayed for SOD, GPx, and CAT activities and for MDA and protein concentrations.
MDA was determined by the thiobarbituric acid method [17]. Aliquots (0.2 ml) of tissue supernatant were mixed with 0.8 ml of phosphate-buffered saline (pH 7.4) and 0.025 ml of butylated hydroxytoluene. After addition of 0.5 ml of 30% (w/v) trichloroacetic acid, the samples were placed on ice for 2 hr and then centrifuged (2000 g, 25°C, 15 min). One ml of supernatant was mixed with 0.075 ml of 0.1 M EDTA solution and 0.25 ml of 1% (w/v) thiobarbituric acid in 0.05 N NaOH. The resulting solution was placed in boiling water for 15 min, cooled to room temperature, and its absorbance at 532 nm was determined. Total thiobarbituric acid-reactive substances (TBARS) were expressed as MDA, using a molar extinction coefficient for MDA of 1.56 x 105 cm-1M-1. Results were expressed as nmol MDA/mg protein.
SOD activity was measured according to Sun et al [18]. Xanthine-xanthine oxidase complex produces superoxide radicals that react with nitrobluetetrazolium (NBT) to produce a colored formazone. SOD activity is assayed at 560 nm by the inhibition of this reaction. A blank contains all ingredients except the tissue supernatant. One unit of SOD activity is the enzyme amount that causes 50% inhibition of NBTH2 reduction in the blank. The SOD activity is expressed as U/mg protein.
GPx activity was measured according to the Paglia and Valentina [19]. GPx catalyses the oxidation of glutathione in the presence of tert-butyl hydroperoxide (tBH). Oxidized glutathione is converted to the reduced form in the presence of glutathione reductase and NADPH, while NADPH is oxidized to NADP. Reduction in the absorbance of NADPH at 340 nm is measured. By measuring the absorbance change per min and using the molar extinction coefficient of NADPH, GPx activity is calculated and expressed as mU/mg protein.
CAT activity was measured at 20°C according to Aebi [20]. Briefly, H2O2 was used as the substrate and the decrease of H2O2 concentration at 20°C in phosphate buffer was assayed by spectrophotometry at 240 nm. One unit of CAT activity is the amount of enzyme that degrades 1 µmol of H2O2/min; CAT activity was expressed as U/mg protein.
Absorbance measurements were made at room temperature using a model CE-3041 spectrophotometer (Cecil Instruments, Cambridge, UK). Protein concentrations were assayed by Lowry’s method [21].
Statistics. Results were expressed as mean ± SD. The paired-sample t-test was performed by a computer program (SPSS-for-Windows, version 10.0, SPSS, Inc., Chicago, IL). A p-value <0.05 was considered significant.
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Results |
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, SOD activity was significantly higher and GPx activity was significantly lower in the treated maxillary sinus mucosa, compared to the paired samples of mucosa from the contralateral control sinuses (p <0.01 and p < 0.005, respectively). On the other hand, no significant differences were found in CAT activity or in MDA level in the inflammed sinus mucosa, compared to the controls.
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Discussion |
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OFRs are known to play an important role in the intracellular killing of microorganisms by leukocytes. Challenges to polymorphnuclear cells by many activating agents, including immune complement, evoke a potent response that produces toxic OFRs (eg, O2•- and H2O2). During phagocytosis, OFRs are also produced extracellularly, however they are directly involved in inflammation. Leucocytes that reach the area of inflammation produce OFRs by consuming oxygen and OFRs levels increase locally, causing tissue damage [22–24].
Excessive lipid peroxidation in the experimental sinus mucosa can arise due to factors favoring the formation of ROS. Parks et al [25] demonstrated that mucosal MDA level was increased in experimental otitis media and OFRs in mucosa of infected middle ear might cause damage by lipid peroxidation. Döner et al [23] reported that MDA levels of serum and infected maxillary sinus mucosa were significantly higher than that of controls. However, in the present study, we found that MDA level of inflammed maxillary sinus mucosa was not significantly different from the control maxillary mucosa in the same rabbits.
Grisham and Granger [26] reported that reactive oxygen metabolites generated from xanthine oxidase and inflammatory leukocytes may play an important role in mediating mucosal injury during an active episode of ulcerative colitis. Verspaget et al [27] showed that the neutrophil content of SOD was markedly diminished in Crohn’s disease and ulcerative colitis compared to a control group. When neutrophils accumulated, however, they may produce local toxic levels of OFRs and can cause an inflammatory process.
We found that tissue SOD activity in the treated maxillary sinus mucosa was significantly higher than in the control sinus mucosa of the same rabbit. In agreement with our finding, Döner et al [23] reported increased SOD activity in the serum and erythrocytes of rabbits with experimental sinusitis. A high concentration of superoxide produced by xanthine oxidase (or from other sources) may be responsible for the increased SOD activity in treated maxillary sinus mucosa.
In the control maxillary sinus mucosas of the rabbits, GPx activity was higher than in the treated maxillary sinus mucosas. We cannot explain this interesting observation.
In conclusion, this study demonstrates that experimental induction of acute maxillary sinusitis in rabbits by instillation of killed S. aureus does not increase lipid peroxidation in the sinus mucosa, but does alter the activities of some antioxidant enzymes.
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