EFFECT OF MACROLIDE ANTIBIOTICS ON NITRIC
OXIDE SYNTHASE AND XANTHINE OXIDASE ACTIVITIES, AND MALONDIALDEHYDE LEVEL IN ERYTHROCYTE OF THE GUINEA PIGS WITH EXPERIMENTAL OTITIS MEDIA WITH EFFUSION
Bülent Aktan
1,#, Seyithan Taysi
2, Kenan Gümüºtekin
3, Harun Üçüncü
1, Ramazan Memiºo ðullari
2, Kürºad Save
4, Nuri Bakan
21Department of Otorhinolaryngology-Head and Neck Surgery,2Department of Biochemistry,
3Department of Physiology, School of Medicine,4Department of Medical Education, Atatürk University, Medical School, Erzurum, Turkey
Effect of macrolide antibiotics on nitric oxide synthase and xanthine oxi- dase activities, and malondialdehyde level in erythrocyte of the guinea pigs with experimental otitis media with effusion. B. AKTAN, S. TAYSI, K. GÜ- MܪTEKIN, H. ÜÇÜNCÜ, R. MEMIªOÐULLARI, K. SAVE, N. BAKAN.
Pol. J. Pharmacol. 2003, 55, 1105–1110.
Although the long-term administrations of macrolide antibiotics are ef- fective for diffuse panbronchiolitis, otitis media with effusion (OME), and some other diseases, their mechanism of action has not been fully under- stood. In order to elucidate the mechanisms of possible effects of macrolide antibiotics on activities of erythrocyte nitric oxide synthase (NOS), xanthine oxidase (XO), and malondialdehyde (MDA) levels in experimental OME, we aimed to evaluate the effect of macrolide antibiotics (erythromycin, azithromycin, roxithromycin, and clarithromycin) using an experimental guinea pig otitis media model.
Erythrocyte NOS, XO activities, and MDA level were measured in all groups. Erythrocyte NOS activities were significantly higher in erythromy- cin-, azithromycin-, roxithromycin-, and clarithromycin-treated groups than in the experimental group. Erythrocyte XO activities were significantly lower in erythromycin-, azithromycin-, roxithromycin-, and clarithromycin- treated groups than in the control group. However, erythrocyte XO activities in experimental group were significantly higher than those of control group.
Erythrocyte MDA levels were significantly lower in erythromycin-, azithro- mycin-, roxithromycin-, and clarithromycin-treated groups than those of the experimental group. The MDA levels in erythromycin- and roxithromycin- treated groups were significantly higher than those of azithromycin-treated group. The MDA levels in azithromycin-treated group were significantly lower than those of roxithromycin-treated group.
In conclusion, the present study shows that the macrolide antibiotics (erythromycin, azithromycin, roxithromycin, and clarithromycin) increase NOS activity, decrease XO activity and MDA level, which is an important indicator of oxidative stress.
Key words: nitric oxide synthase, malondialdehyde, xanthine oxidase, otitis media with effusion, oxidative stress
ISSN 1230-6002
INTRODUCTION
Otitis media with effusion (OME) is a very common disease, especially in childhood and in- fancy and is characterized by non-purulent fluid in the middle ear and fluctuating conductive hearing loss. OME is an inflammatory response of the mid- dle ear caused by multiple factors, such as viral or bacterial infection, eustachian tube dysfunction, or allergy. Inflammatory mediators seem to play a ma- jor role in the pathogenesis of OME. The patho- genesis of OME is still not fully understood [17].
Macrolides are a long-used class of antibiotics which still play an important role in the chemother- apy of infectious diseases. They have been shown to affect several pathways of the inflammatory pro- cess, such as the migration of neutrophils, the oxi- dative burst in phagocytes, and the production of proinflammatory cytokines. Although the precise mechanisms of these effects are not clear, it has been suggested that the interaction between mac- rolides and leukocytes may be important [14].
Some studies have suggested that the antioxidant properties, shared by several macrolides, may play a role in the anti-inflammatory activity of these agents [14, 20].
In response to inflammatory stimuli released during OME, the middle ear mucosa undergoes in- tense cell proliferation and differentiation, the mid- dle ear is rapidly populated by leukocytes, and ef- fusion appears in the middle ear cavity. These re- sponses are mediated by the action of a number of bioactive molecules that interact with the cells of the middle ear mucosa and its vasculature [26].
Some studies have documented the roles of several cytokines, inflammatory mediators, and growth factors in contributing to inflammatory responses in the middle ear [5, 26, 27]. The interaction of cy- tokines and growth factors with their receptors has multiple intra- and extracellular effects. They can include the production and release of the small free radical nitric oxide (NO•), which is produced by ni- tric oxide synthases (NOSs) [26]. Recently, atten- tion has focused on the importance of NO• in the respiratory system, and, in particular, the impor- tance of epithelial derived NO•in the regulation of airway function [26]. NO• is known, together with reactive oxygen species (ROS), to induce cytotoxi- city and cytostasis. Several studies with NO•- and H2O2-induced oxidative damage have shown simi- larities in their cytotoxicity [1]. NO•is an inorganic
free radical gas produced from L-arginine by a fa- mily of isoenzymes called NOSs. Two of them are constitutively expressed and the third is inducible by immunological stimuli. NO•, produced by the constitutive enzymes, that acts as an important sig- naling molecule in the cardiovascular and nervous systems, and NO• induced by the inducible NOS (iNOS) and generated for prolonged periods by cells of the immune system among others, are cyto- static/cytotoxic for tumor cells and a variety of mi- croorganisms [4, 16, 29, 32].
Xanthine oxidase (XO) is the last enzyme of purine catabolism. It catalyzes conversion of xan- thine and hypoxanthine to uric acid and the produc- tion of superoxide radical anion (O•–2), which is potentially toxic to cellular structures [6]. Lipid peroxidation is one of oxidative conversions of po- lyunsaturated fatty acids to such products as ma- londialdehyde (MDA), which is usually measured as thiobarbituric acid-reactive substances (TBARS), or lipid peroxides, and it is the most studied, bio- logically relevant, free radical reaction [4, 32]. Free radicals (FR) and lipid peroxides have been impli- cated in the pathogenesis of a wide variety of dis- eases ranging from infectious, inflammatory and autoimmune diseases to atherosclerosis and cancer [23, 25, 28, 30, 31, 34].
In this study, we aimed to investigate the effect of four macrolide antibiotics (erythromycin, azi- thromycin, roxithromycin, and clarithromycin) on erythrocyte NOS and XO activities, and MDA levels in experimental OME.
MATERIALS and METHODS
Animals
Forty-two albino guinea pigs (650–700 g) ob- tained from the Research Institute of Animal Dis- eases, Erzurum, Turkey, were used in this study.
Guinea pigs were acclimated to the facility for a period of at least two weeks before being in- cluded in the study. Each animal was separately caged and had free access to standard pelleted food and water throughout the experiment. It was deter- mined that there was no middle ear pathology in guinea pigs by otoscopy and tympanometry. The animals were divided into six groups, two of which were the control and experimental groups, the oth- ers were the treatment groups.
Medicines and treatments
The animals were treated with erythromycin, or azithromycin, or clarithromycin, or roxythromycin for three consecutive days. Erythromycin (30 mg/
kg/day), azithromycin (10 mg/kg/day), clarithro- mycin (10 mg/kg/day) and roxythromycin (5 mg/
kg/day) were administered by the use of gastric tubes. An equal volume of isotonic saline was ad- ministered via a gastric tube to the control group.
After treatment, histamine dihydrochloride (Sigma, Germany) was added to normal saline solution (1 mg/ml), and the pH of this solution was adjusted to 7.4 with sodium hydroxide. The guinea pigs were anesthetized (100 mg/kg ketamine hydrochlo- ride and 3 mg/kg diazepam ip). Histamine (0.1 ml) was injected via a 27-gauge needle through the right tympanic membrane to the middle ear cavity [7]. The same procedure was applied in the control subjects but they were given only normal saline so- lution (0.1 ml). Three hours later [7], the animals were anesthetized again (100 mg/kg ketamine hy- drochloride and 3 mg/kg diazepam ip), and blood was collected by cardiac puncture after thora- cotomy. The blood samples were collected in vacu- tainer tubes with K3EDTA as anticoagulant.
Biochemical measurements
Erythrocyte sediments were prepared for the analyses. Erythrocytes were then hemolyzed by di- luting with deionized water (50-fold), and the ana- lyses were carried out on this hemolyzed super- natant fraction. Hemoglobin (Hb) values of the samples were measured by a GEN-S counter hema- tology analyzer. Hemolyzed samples were kept at –80°C until biochemical determinations.
NOS activity assay is based on the diazotization of sulfanilic acid by NO•at acid pH and subsequent coupling to N-(1-naphthyl)ethylenediamine. To 0.1 ml of sample, 0.2 ml of 0.2 M arginine was added and incubated at 37°C for 1h. Then, the following combinations were added: 0.2 ml of 10 mM HCl, 100 mM sulfanilic acid, and 60 mM N-(1-naphthyl)- ethylenediamine. After 30 min, the absorbance of the sample tube was measured against a blank tube at 540 nm [11]. Results are expressed as interna- tional unit (IU)/g Hb.
XO activity was measured spectrophotometri- cally by the formation of uric acid from xanthine through the increase in absorbance at 293 nm, as described previously [13]. Results are expressed as
mIU/g. MDA was determined on the basis of spec- trophotometric absorbance measurement of the pink colored product of the TBARS complex [15].
Total TBARS were expressed as MDA. Results are expressed as nmol/g Hb. Biochemical measurements were carried out at room temperature using a spec- trophotometer (CECIL CE 3041, Cambridge, UK).
Statistical analysis
The findings were expressed as the means ± SD.
Statistical analyses were undertaken using one-way ANOVA. LSD (least significant difference) multi- ple range test was used to compare the mean values (acceptable significance was recorded when p val- ues were < 0.05). Statistical analysis was perfor- med with Statistical Package for the Social Scien- ces for Windows (SPSS, version 10.0, Chicago, IL, USA).
RESULTS
All parameters are shown in Table 1. As can be seen in the Table, erythrocyte NOS activities were significantly higher in experimental, erythromy- cin-, azithromycin-, roxithromycin-, and clarithro- mycin-treated groups than in the of control group (p < 0.01, for the first one, p < 0.001, for the next four groups). Erythrocyte NOS activities were sig- nificantly higher in erythromycin- (p < 0.01), azi- thromycin- (p < 0.05), roxithromycin- (p < 0.001), and clarithromycin-treated (p < 0.001) groups than those of experimental group. Erythrocyte NOS ac- tivities were significantly higher in clarithromycin- treated group than in azithromycin-treated group (p < 0.05).
Erythrocyte XO activities were significantly lower in erythromycin- (p < 0.001), azithromycin- (p < 0.001), roxithromycin- (p < 0.05), and cla- rithromycin-treated (p < 0.001) groups than in the control group. However, erythrocyte XO activities in experimental group were significantly higher than in the control group. Erythrocyte XO activities were significantly lower in erythromycin-, azithro- mycin-, roxithromycin-, and clarithromycin-treated groups than in the experimental group (p < 0.001, for all groups). The erythrocyte XO activities in erythromycin- and azithromycin-treated groups were significantly lower than in roxithromycin- treated group (p < 0.05, for all groups). The eryth- rocyte XO activities in roxithromycin-treated group were significantly higher than in erythromycin-
treated group (p < 0.05). The erythrocyte XO ac- tivities in clarithromycin-treated group were sig- nificantly higher than in azithromycin-treated group.
Erythrocyte MDA levels were significantly lower in erythromycin- (p < 0.05), azithromycin- (p < 0.001), roxithromycin- (p < 0.05), and cla- rithromycin-treated (p < 0.001) groups than in the experimental group. The MDA levels in erythro- mycin- and roxithromycin-treated groups were sig- nificantly higher than in azithromycin-treated group (p < 0.05). The MDA levels in azithromy- cin-treated group were significantly lower than in roxithromycin-treated group (p < 0.05).
DISCUSSION
Many investigators have shown that middle ear effusion (MEE) in patients with OME can be caused by several pathogenic factors, such as microorgan- isms, endotoxins, immune complexes, and arachi- donic acid metabolites. Conditions sufficient to produce the release of histamine are known to exist in the middle ear and during OME. Mast cells are generously distributed in normal middle ear mu- cosa. The free mast cell granules have been re- ported in the mucosa of OME patients, which im- plies ongoing degranulation in this chronic disease state. Histamine is released when the mast cells de- granulate. This condition triggers inflammation [2, 7]. Although the long-term administrations of mac- rolide antibiotics are effective for diffuse panbron- chiolitis [18], and OME [12], their mechanism of action has not been fully understood. In order to elucidate the mechanisms of effect of macrolide an- tibiotics on erythrocyte NOS, XO activities, and
MDA levels, which is an important indicator of oxidative stress, in experimental OME, we evalu- ated the effect of macrolide antibiotics (erythromy- cin, azithromycin, roxithromycin, and clarithromy- cin) using an experimental guinea pig otitis media model.
FRs are known to play an important role in the intracellular killing of microorganisms by leuko- cytes. The challenge of polymorphnuclear cells with many activating agents, including immune complement, evokes a potent response that produ- ces toxic oxygen species, such as O•–2, and hydro- gen peroxide. During phagocytosis, FRs are also produced extracellularly, however, they are directly involved in inflammation. Thus, leukocytes reach- ing the inflammatory area produce an excessive amount of FRs by consuming oxygen (“respiratory burst”) and FR levels increase in the inflammation.
Increased FRs may cause cell and tissue damage [8, 9, 33]. Parks et al. [24] reported that both lipid hy- droperoxide and MDA, two different indicators of oxidative cell and tissue damage, were significantly elevated in the middle ear mucosa of guinea pigs infected with pneumococci in comparison with normal middle ear mucosa. Döner et al. [9] re- ported that MDA levels of erythrocytes and in- fected otitis media mucosa were significantly higher than in the control group. We found that erythrocyte MDA levels were significantly lower in erythromycin-, azithromycin-, roxithromycin-, and clarithromycin-treated groups than in the ex- perimental group.
A major source of radicals in biological systems is molecular oxygen (O2). The radicals originating from molecular oxygen are generally named reac- tive oxygen species (ROS). XO is an important
Table 1. NOS, XO activities, and MDA levels in the guinea pig erythrocytes
NOS (IU/g Hb) XO (mIU/g Hb) MDA (nmol/g Hb)
Control group 408.7 ± 76.8 41.2 ± 7.8 15.7 ± 2.2
Experimental group 534.6 ± 99.5b 65.3 ± 8.8c 20.0 ± 2.1a
Erythromycin-treated group 687.4 ± 103.5c,e 17.8 ± 6.0c,f,l 17.7 ± 1.9d
Azithromycin-treated group 636.0 ± 62.3c,d 21.0 ± 8.2c,f,l 15.0 ± 2.0f,g
Roxithromycin-treated group 732.7 ± 74.2c,f,k 29.2 ± 14.8a,f,g 17.5 ± 1.8d,k Clarithromycin-treated group 772.8 ± 94.9c,f,l 22.3 ± 8.5c,f,k 14.8 ± 1.6f,h,l
ap < 0.05,bp < 0.01,cp < 0.001 vs. control group,dp < 0.05,ep < 0.01fp < 0.001, vs. experimental group,gp < 0.05,hp < 0.01 vs.
erythromycin-treated group,kp < 0.05, vs. azithromycin-treated group,lp < 0.05, vs. roxithromycin-treated group. Data are given as the means ± SD of 7 guinea pigs per group
source of O•–2in cells and tissues. This enzyme ca- talyses the conversion of hypoxanthine and xan- thine to uric acid and the rate-limiting step in pu- rine nucleotide catabolism [3]. We found that XO activities were significantly lower in erythromy- cin-, azithromycin-, roxithromycin-, and clarithro- mycin-treated groups, while XO activities in ex- perimental group were significantly higher than in the control group.
In general, NO•, a free radical produced by iNOS, appears to regulate several steps of the in- flammatory process. As a potent vasodilator, NO• modulates the early vascular responses of the acute inflammatory reaction. In addition, NO• is one of the cytostatic-cytotoxic defence mechanisms against a pathogen, in the non-specific immune response.
FR production by the interaction of NO•with O•–2 has both protective (microbial killing, neutralizing O•–2) and toxic effects by the formation of the per- oxynitrite (ONOO–), which is now generally con- sidered a more toxic species than either NO•or O•–2 alone, and hydroxyl radical (•OH). Furthermore, NO• synthesized by activated inflammatory cells regulates the functions of other cells involved in the inflammatory process and appears to act as a secondary mediator of some actions of pro- inflammatory cytokines, such as interleukin-1 [19].
We found that erythrocyte NOS activities were sig- nificantly higher in erythromycin-, azithromycin-, roxithromycin-, and clarithromycin-treated groups than in the experimental and control groups. The erythrocytes cannot synthesize NOS. The blood flow to inflamed regions increases. Possibly, NOS released by injured cells may be absorbed by eryth- rocytes.
The macrolide antibiotics increase NOS activi- ties that results in increased NO• synthesis, which combines with O•–2to form ONOO–. Recently, Lee et al. [22] showed that ONOO– bound the element molybdenum, the cofactor of XO. They demon- strated that ONOO– down-regulated O•–2 genera- tion from XO. ONOO–was shown to decrease both XO activities and O•–2generation in vitro [22]. So, these conditions reduce MDA formation, which is an important indicator of oxidative stress.
In conclusion, this is the first study that investi- gates oxidant system in the erythrocyte samples from animals with experimental OME to elucidate the mechanisms of effect of macrolide antibiotics on NOS, XO activities, and MDA levels, which is
an important indicator of oxidative stress. We found the increased NOS activity, and decreased XO activity and MDA level in macrolide-treated groups compared to experimental OME group. Fur- ther studies including determination of 4-hydroxy- 2-nonenal (HNE), a more specific peroxidation product of essential fatty acids, particularly arachi- donic acid, in addition to MDA assay are needed to provide definitive information about the relation- ships between lipid peroxidation and membrane fatty acid composition in OME.
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Received: September 4, 2003; in revised form: November 18, 2003.
