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Transcript of The Antioxidant Effect of Aminophylline in Rat Brain and ... · PDF fileThe Antioxidant Effect...
The Antioxidant Effect of Aminophylline in Rat Brainand Spinal Cord Homogenates
Rat Beyin ve Omurilik Homojenatlannda AminofilininAntioksidan Etkisi
ERKAN KAPTANOGLU, iHSAN SOLAROGLU, FiLiz AKBIYIK,
Eoiz DEMiRPEN<;E, M. FiKRET ERGUNGOR
Ankara Numune Education and Research Hospital, Department of Neurosurgery
(EK, is, MFE) Hacettepe University, Faculty of Medicine, Department of
Biochemistry (FA, ED), Ankara, Turkey
Received: 03.07.20020 Accepted: 10.12.2002
Abstract: Object: Free oxygen radicals playa major rolein the pathophysiology of c~ntral nervous system (CNS)trauma, neurodegeneration and aging. Aminophylline,an antiasthmatic drug, was shown to exert some freeradical scavenging effect on lung tissue. The purpose ofthis study was to investigate the free radical scavengingeffect of aminophylline on CNS tissues. The antioxidantaction of different doses of aminophylline was studiedunder oxidant conditions in vitro.
Methods: The whole rat brains and spinal cords obtainedfrom male Wistar rats were homogenized in 1:10 coldpotassium phosphate buffer by using a douncehomogenizer. Peroxidation was induced with ferrousiron (Fe++), ascorbate (Asc) and hydrogen peroxyde(HzOz). Aminophylline was added into the reactionmixtures just before the addition of lipid substrates.Thiobarbituric acid-reacting substances (TBARS) weremeasured as an index of lipid peroxidation.Results: The highest lipid peroxidation was obtainedwith Fe++, Asc and Hz0z-induced group. Addition ofaminophylline showed no free radical scavenging effecton rat brain and spinal cords.Conclusion: Analysis of the results demonstrated that
Ozet: AmaF Serbest oksijen radikalleri merkez srmrsistemi travmasl, norodejenerasyon ve ya~lanmamnpatofizyolojisinde onemli rol oynamaktadlr. Birantiastmatik olan aminofilin akciger dokusunda serbestradikal tutucu etki gostermektedir. Bu <;ah~manm amaClaminofilinin merkez sinir sisteminde serbest radikaltutucu etkisini incelemektir. Aminofilinin farkh dozlan
in vitro antioksidan ko~ullarda <;ah~lldl.Metod: Di~i Wistar ratlardan elde edilen tam beyin veomurilik dokulan 1:10 soguk fosfat tamponundahomojenizatOr ile homojenize edildi. Peroksidasyonferroz demir (Fe++), askorbat (Asc) ve hidrojen peroksit(HzOz) i1e indiiklendi. Arninofilin reaksiyon kan~lmmalipid substratlann eklenmesinden once ilave edildi.Tiyobarbitiirik asit reaktif maddeler (TBARS) lipidperoksidasyonunun bir indeksi olarak ol<;ii1dii.Bulgular: En yiiksek lipid peroksidasyonu Fe++, Asc veHzOz indiikleme grubunda elde edildi. Kan~lmaaminofilin ilave edilmesi rat beyin ve omurilikdokulannda peroksidasyona kar~l koruyucu etkigostermedi.Sonuf: Sonu<;larm analizi aminofilinin rat beyin veomurilik homojenatlannda antioksidan etkisinin
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Turkish Neurosurgery 13: 9-13, 2003
aminophylline has no antioxidant effect on rat brain andspinal cord homogenates in vitro. Further in vitro and invivo studies are needed to evaluate the free radical
scavenging effect of aminophylline.
Key words: Aminophylline, antioxidant, brain, lipidperoxidation, spinal cord
INTRODUCTION
Oxidative stress has been considered as one of
the basic events involved in cell and tissue damage.Production of free oxygen radicals beingconsidered an important component of secondarydamage during ischemia, trauma, andneurodegenerative diseases in the CNS (1,7,11).Re-oxygenation of the ischemic tissues results ingeneration of free oxygen radicals. The generationof free oxygen radicals results in lipid peroxidationof cell membrane, damage of DNA and othersubcellular organels and subsequently cell death(8,12,22). The antioxidant activity of drugs havegreat clinical interest because of the importance ofpreventing or reducing the free radical-mediatedtoxicity in CNS.
There are many clinical and experimentalinvestigations about the free radical scavengingproperties of various antiasthmatic drugs in theliterature such as salbutamol, terbutaline, fenoteroland theophylline (10,19,24). Aminophylline, amixture of theophylline af\d ethylenediamine(85:15%), is a bronchodilatating and antiasthmaticcompound widely used in patients with acuterespiratory insufficiency (3,17,23). Aminophyllinehas been reported to possess free radicalscavenging effect in lung tissue (18). Currentlythere is no information regarding aminophylline'sneuroprotective effect against lipid peroxidation inthe rodent brain and spinal cord. In the presentstudy, we aimed at investigating the antioxidantaction of aminophylline in rat brain and spinal cordhomogenates against in vitro induced lipidperoxidation. For this purpose, we used a hydroxylradical-generating system and we evaluatedwhether aminophylline was protective against thefree radical-induced lipid peroxidation.
MATERIALS AND METHODS
Chemicals
Thiobarbituric acid (TBA), ascorbic acid (Asc)
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KaplmlOg/lI: Ti,e Alllioxidmll Effecl of Amillophyllille 011 eNS
olmadlgml gosterdi. Aminofilinin antioksidan ozelliginidegerlendirmek i~in in vitro ve in vivo ileri ~ah~malaragereksinim vardJr.
Anahtar Kelimeler: Aminofilin, antioksidan, beyin, lipidperoksidasyon, omurilik
and hydrogen peroxide (H202) were from SigmaChemical Co. (St Louis, MO, USA). Trichloroaceticacid (TCA) and ferrous sulfate were from Merck
(Germany). Aminophylline was from Turfarma(Turkey).
Preperation of lipid substrates forperoxidation
Brain and spinal cord homogenates wereused to investigate the antioxidant actions ofaminophylline. Two male, 2 month old Wistar rats,approximately 200 g were used for tissuepreperation. The rats were anesthetized withcombination of 60 mg/kg ketamine hydrochloride(Parke Davis, Istanbul,) and 10 mg/kg xylazine(Bayer Birle~ik Alman ila<; Fabrikalan A.$.,Istanbul), and placed prone to the operation table.Brains were obtained following wide craniectomyand spinal cords were obtained following C3-T12laminectomy. Dura was removed from the neuraltissues with scalpel, and the samples were rinsedwith physiologic saline. The whole rat brains andspinal cords were homogenized in 1:10 (w:v) coldpotassium phosphate buffer (25 mM, pH 7.4) byusing a dounce homogenizer. The resulting 10%homogenates were used in lipid peroxidationstudies (6).
Determination of lipid peroxidation
Thiobarbituric acid-reacting substances(TBARS) were measured as an index of lipidperoxidation (2). Peroxidation was induced withFe++(0.02 mM), Asc (1 mM) and H202 (0.5 mM) ina final volume of 0.5 ml. Aminophylline was addedinto the reaction mixtures at indicated
concentrations just before the addition of lipidsubstrates. The reaction mixture was incubated for
30 min at room temperature. At the end ofincubation, 0.5 ml of TCA (10%) was added to stopthe reaction. Samples were centrifuged at 1500 x gfor 10 min and 0.5 ml of supernatant was mixed
Turkish Neurosurgery 13: 9-13, 2003
with 0.5 ml of TBA (0.67%). Tubes were placed intoboiling water for 15 min. After cooling the tubes,the absorbance of the samples was measured at 532run.
RESULTS
Fe++,Asc and HzOz were used to induce lipidperoxidation. The ferrous iron was the mosteffective agent in induction, although eachcomponent, when used alone, was able to inducelipid peroxidation. Combination of two agentsshowed that the highest peroxidation was obtainedwith Fe++and Asc combination. The overall highestperoxidation levels were measured in the presenceof Fe++,Asc and HzOz (Table 1). The iron-Asc-HzOzsystem was very effective for generating oxidantconditions.
The results of spectrophotometry showed thatthere was a gradual increase in lipid peroxidationlevels with increasing amounts of aminophylline.The absorbance of the samples is shown in Table 2.Results are expressed as the mean ± SO.
DISCUSSION
In intense ischemic state followed by reoxygenation could result in irreversible organdamage (21). Mitochondrial damage due tocalcium overload of the cells and excessive
generation of free oxygen radicals are theunderlying mechanisms of reperfusion injury (4).Increased cytoplasmic calcium results in theproduction of reactive oxygen species (15). Freeoxygen radicals are reactive species which candestroy the tissues via lipid peroxidation of the cellmembranes (13,14). Excessive generation of freeoxygen radicals causes DNA damage, lipidperoxidation and inactivation of proteins andfinally leads to severe tissue injury (8,9,12,22).Pharmacological agents which can prevent orreduce free oxygen radical mediated toxicity can beclinically useful.
Although ~z-agonists seem to have a directoxidant scavenger function, which antiasthmaticdrugs have antioxidant effect remains speculative.Glissen et aL (10) demonstrated that ~z-agonistshave a potent antioxidative function in HzOzmediated cytotoxicity in vitro. They also suggested
Kaptanoglu: The Antioxidant Effect of Aminophylline all eNS
that corti costeroids and theophylline had noantioxidant effect. Aminophylline is a saltcomposed of two molecules of theophylline andone molecule of ethylenediamine and is a wellknown antiasthmatic drug (3,17,23). Kang et al.reported that aminophylline exert someantioxidant activity in vitro (16). Similarly,Lapenna et al. reported the free radical scavengingeffect of aminophylline, but not theophylline inlung epithelial tissue (18). They showed that bothaminophylline and theophylline are scavengers ofhydroxyl radical (OH), but they are ineffectiveagainst superoxide anion and hydrogen peroxide.Aminophylline was found to be effective in lowerconcentrations, because of ethylenediamine'smarked OH scavenging activity. Moreover, theydemonstrated that therapeutic concentrations ofaminophylline, but not of theophylline, are capableof antagonizing hypochlorous acid (HOCl); thiseffect was entirely due to presence ofethylenediamine. They hypothesized thatantioxidant properties of aminophylline, partly ortotally, is attributable to ethylenediamine which ispresent in the aminophylline molecule.
Mahomed et al. suggested that theophyllinehas antioxidant effects due to its
phosphodiesterase inhibitory properties in humanneutrophils (19). Aminophylline, also a nonselective phosphodiesterase inhibitor, inhibitsphosphodiesterase enzyme that metabolize andinactivate cyclic adenosine monophosphate(cAMP) and cyclic guanosine monophosphate(cGMP) (20). Increase in the intracellularconcentration of either cAMP or cGMP results in
relaxation of airway smooth muscle. It was alsoexpected that inhibition of these enzymes results inreduction of free radicals in the tissues. In the
present study, aminophylline itself does not haveantioxidant effect in vitro. But its effect on
phosphodiesterase enzyme may results in decreasein tissue free radical levels after traumatic orischeamic insult in vivo. However, translocation ofintracellular calcium and the antagonistic blockadeof adenosine receptors are the otherpharmacological actions of aminophylline (5).
The aim of our present study was toinvestigate whether aminophylline had anantioxidant action. We analyzed the effect ofaminophylline toward lipid peroxidation in vitro,
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Turkish Neurosurgery 13: 9-13, 2003
using the Fe++-Asc-H202 system to generateoxidant conditions. This system generates the mostreactive radical species, the hydroxyl radical,through the reaction of ferrous iron with H202
(Fenton reaction). The iron is oxidized to its ferric
form and in the presence of ascorbate as a reducingagent, ferrous iron is recovered from its oxidizedform. As a consequence, hydroxyl radicalgeneration and peroxidation persists. In this study,
when Fe++-Asc-H202 was added, levels of lipidperoxidation products in the homogenates weresignificantly increased (Table I). The Fe++-Asc-H202
system was found to be very effective forgenerating oxidant conditions (12-14). Since ahydroxyl radical scavenging effect was previouslydescribed for aminophylline (18), different
Table I: Table shows the basal TBARS levels of the
rat brain and spinal cord homogenates. Increase intissue TBARS levels are seen with induction of one,two or all of Fe++,Asc and H202. The best inductionwas observed with addition of all chemicals. The
values are the absorbances of the samples wasmeasured at 532 nm., and are exspressed as themean ± SD.
BrainSpinal cord
Basal
40,48±0,9617,01±0,00
Fe++288,44±7,43158,79±0,28
Asc58,28±6,9831,48±0,28
H?O?
174,24±1,92107,36±4,57
Fe+Asc434,92±1,54307,61±8,05
Fe+ H202
343±13,17142,95±0,73
Asc+H202
283,36±0,48303,70±14,13
Fe+Asc+ H202
715,93±36,17629,30±6,47
Kaptalloglu: Tire Alltioxidallt Effect of AlIlillophyllille 011 eNS
concentrations of the drug were added to thehomogenates in order to evaluate a possibleprotective action. The results showed thataminophylline did not inhibit lipid peroxidation inrat brain and spinal cord homogenates (Table II).We concluded that aminophylline was not anefficient scavenger against Fe++-Asc-H202 -inducedradical generation, at least in our experimentalconditions.
In summary, the role of free oxygen radicals insevere tissue injury, especially in ischemiareperfusion induced organ damage and trauma toCNS, is well known. Importance of preventing andreducing the free oxygen radical inducedsecondary neuronal damage must be considered invarious clinical conditions. Pharmacological agentswhich have radical scavenging properties areneeded to be studied. Further in vitro and in vivostudies are needed to evaluate the free radical
scavenging effect of aminophylline.
Correspondence: Erkan KaptanogluAnkara Numune Egitim veAra~hrma HastanesiN6ro~irurji KlinigiSamanpazan 06100 Ankara / TURKEY
E-mail: [email protected]: 312-310 3640Phone : 532-435 1057
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Table II: Table shows the effect of aminophylline on free radicals in brain and spinal cord homogenates. Thevalues are the absorbances of the samples was measured at 532 nm, and are exspressed as the mean ± SD.
BasalInductionAminophyllineAminophyllineAminophylline
25 f.lM
50f.lM100 f.lM
Brain
40,48±0,96715,93±36,17762,67±42,58898,77±85,781089,05±169,25
Spinal cord
17,01±0,00629,30±6,47781,64±33,78888,00±40,67957,05±30,51
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Turkislr Neurosurgery 13: 9-13, 2003
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Kaptalloglu: The Antioxidant Effect of Aminophylline on eNS
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