Effects of lycopene on the model of oleic acid-induced acute lung injury

(1)

Effects of lycopene on the model of oleic acid-induced acute lung injury

Suat TÜRKOĞLU¹, Mehmet Hamdi MUZ², Reşat ÖZERCAN³, Ferit GÜRSU⁴, Gamze KIRKIL²

1SB Batman Bölge Devlet Hastanesi, Göğüs Hastalıkları Kliniği, Batman,

2Fırat Üniversitesi Tıp Fakültesi, Göğüs Hastalıkları Anabilim Dalı, Elazığ,

3Fırat Üniversitesi Tıp Fakültesi, Patoloji Anabilim Dalı, Elazığ,

4Fırat Üniversitesi Tıp Fakültesi, Biyokimya Anabilim Dalı, Elazığ.

ÖZET

Oleik asit ile oluşturulan akut akciğer hasarı modelinde likopenin etkileri

Giriş:Bu çalışmada, akciğer hasarı modelinde likopenin koruyucu etkisinin araştırılması amaçlandı.

Materyal ve Metod:Çalışmaya 28 adet Wistar rat alındı. Kontrol grubuna (n= 7) serum fizyolojik + etanol (9/1) infüzyonu uygulandı. Oleik asit (OA) grubuna (n= 7), OA (100 mg/kg) tek doz intravenöz olarak uygulandı. Mısır yağı + OA grubu- na (n= 7), beş hafta mısır yağı (1 mL/gün) gavajla verildi. Likopen + OA grubuna (n= 7), beş hafta likopen gavajla verildi ve beşinci haftanın sonunda OA (100 mg/kg) uygulandı. OA verildikten dört saat sonra kan ve akciğer doku örnekleri alın- dı. Malondialdehid, süperoksit dismutaz, glutatyon peroksidaz ve doku katalaz enzim aktivite düzeyleri ölçüldü.

Bulgular:Kontrole göre OA ile mısır yağı + OA gruplarında artmış olan serum ve akciğer doku malondialdehid düzeyi, li- kopen + OA grubunda kontrol değeri düzeyinde idi (p< 0.05). Serum ve doku süperoksit dismutaz ve glutatyon peroksi- daz enzim aktivitesi kontrole yakın değerler veya hafif artışlar görülürken, likopen + OA grubunda diğer gruplara göre be- lirgin artış mevcuttu (p< 0.05). Kontrol grubunun histopatolojik değerlendirmesi normalken, OA ve mısır yağı + OA grup- larında perivasküler, alveoler ödem, hemoraji, belirgin nötrofil infiltrasyonu, alveoler yapılarda destrüksiyon saptandı. Li- kopen + OA grubunda daha az nötrofilik infiltrasyon, perivasküler ve alveoler ödem izlendi.

Sonuç:Likopenden zengin diyet akciğer hasarının önlenmesinde önemli role sahip olabilir.

Anahtar Kelimeler: Akut akciğer hasarı, rat model, likopen.

SUMMARY

Effects of lycopene on the model of oleic acid-induced acute lung injury

Suat TÜRKOĞLU¹, Mehmet Hamdi MUZ², Reşat ÖZERCAN³, Ferit GÜRSU⁴, Gamze KIRKIL²

1Clinic of Chest Diseases, Batman Region State Hospital, Batman, Turkey,

2Department of Chest Diseases, Faculty of Medicine, Firat University, Elazig, Turkey,

3Department of Pathology, Faculty of Medicine, Firat University, Elazig, Turkey,

4Department of Biochemistry, Faculty of Medicine, Firat University, Elazig, Turkey.

Yazışma Adresi (Address for Correspondence):

Dr. Gamze KIRKIL, Fırat Üniversitesi Tıp Fakültesi, Göğüs Hastalıkları Anabilim Dalı, ELAZIĞ - TURKEY

e-mail: gamkirkil@yahoo.com

(2)

INTRODUCTION

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), is a syndrome characterized by the acute onset, hypoxemia refractory to oxygen therapy, increased lung microvascular permeability and diffuse alveolar damage (1). ALI/ARDS in which lung injury and cellular elements (neutrophils, macropha- ges/monocytes, lymphocytes, platelets) and humoral components (complement system, cytokines, coagu- lation/fibrinolysis system, kinin system, lipid medi- ators, oxidants, proteases, nitric oxide, growth factors, neuropeptides) that arise as a result of activati- on of the system plays an important role in response mediator. ALI/ARDS in patients with early stage taken in the bronchoalveolar lavage (BAL) fluid increased neutrophils and neutrophil products have been identi- fied. Neutrophiles cause endotelial and epitelial cell damage by excreting free radicals, inflammatory me- diators, proteases (elastase, collagenase, reactive oxygen species), cytokines like tumor necrosis factor alfa (TNF-α) (2). Oxidant-mediated tissue injury of ALI/ARDS has an important role in pathogenesis.

DNA, protein and lipid oxidation causes the formation of free radicals in lung injury are involved. The rele- ase of proteolytic enzymes and production of toxic oxygen radicals are the main factors in tissue damage. Plasma antioxidant levels decreased in patients with ARDS. Free oxygen radicals and neutrophile streams are thought to consume the total antioxidant capacity of lungs (3,4).

Oleic acid (OA)-induced model of lung injury is frequ- ently used in investigation of novel ALI/ARDS treatment methods (5). ALI after the implementation of OA, decreased serum total antioxidant capacity (6).

Caratenoids found in fruits and vegetables, have protective effects against oxidative damages as well as ac- tivate enzymes which prevent oxidants harmful effects, stimulate the immune system, and cell proliferation (7). Lycopene the most important member of the caro- tene group, consisting of only carbon and hydrogen atoms contain carotenoids. Lycopene is a carotenoid which can not be synthesized but can be stored in hu- man body (8). In a study, lycopene have been shown to reduce the risk of lung cancer when daily and regularly consumed (9). In experimentally induced gastric can- cers, lycopene has been showed to decrease the lipid peroxidation by increasing the antioxidant capacity (10).

Many pharmacological agents in the treatment of ARDS have been tried but it has still a high mortality rate. If ALI, which is accepted as a beginning form of ARDS, may be prevented or may be treated, mortality rate will be decreased. In this OA induced lung injury model, we aimed to investigate the protective effects of lycopene in the oxidant-antioxidant systems.

MATERIALS and METHODS Study Design

The local ethics committee of Firat University has app- roved the experimental protocol. Twenty eight female Wistar rats (140-160 g) were received from Experi- mental Research Unit of Firat University, Faculty of Me- dicine. Wistar rats were seperated into four groups as follows:

Control group (n= 7): Animals of control group were fed in standard rat chow for five weeks, and applied PBS + ethanol (9/1) infusion on the last day of examination.

Introduction:This study, we aimed to investigate the protective effect of lycopene in lung injury rat model.

Materials and Methods:Twenty eight Wistar rats were enrolled into the study. Control group (n= 7) were applied PBS + et- hanol (9/1). A single dose of 100 mg/kg oleic acid (OA) intravenously was administrated to OA group (n= 7). One mL of corn oil was given daily to corn oil + OA group (n= 7) by gavage for five weeks. Lycopene was given by gavage to lycopene + OA group (n= 7) for five weeks. At the end of the 5^thweeks, OA were given. Four hour after OA administration, lung tissue, blo- od samples were taken. Malondialdehyde, superoxide dismutase, glutathione-peroxidase, catalase levels were determined.

Results:Malondialdehyde levels of serum, lung tissues were increased in OA, corn oil + OA groups than control, where as decreased to controls levels in lycopene + OA group (p< 0.05). Superoxide dismutase, glutathione-peroxidase activities of serum, tissue increased moderetaly or they were closed with control values. There was significant increase in lycopene + OA group values. Histopathological examination of control group was normal. OA, cornoil + OA groups had perivascular, alveolar edema, hemorrage, prominent neutrophil infiltration, destruction in alveolar structure. Lycopene + OA group had less neutrophilic infiltration, perivascular, alveolar edema.

Conclusion:Lycopene rich diet may have an important role preventing damages in lungs.

Key Words: Acute lung injury, rat model, lycopene, oxidant, antioxidant.

(3)

OA group (n= 7): ALI was performed by a single intra- venous injection of 100 mg/kg OA (cis-9-octadeceno- ic acid; Sigma-Aldrich Germany) on the last day of examination after feeding in standard rat chow for five weeks. The suspension comprised 25 mg/mL pure OA suspended in ethanol, then 0.9% NaCL added to the suspension (ethanol/NaCl= 1/9) (6).

OA + corn oil group (n= 7): One mL of corn oil was gi- ven daily to this group by gavage for five weeks. At the end of the 5^thweek, a single dose of 100 mg/kg OA was administered.

Lycopene + OA group (n= 7): Twenty mg/kg/day Lycopene (Lycopene 10% FS; Roche redivivo) in the corn oil was given by gavage to this group for five weeks. At the end of the 5^thweek, a single dose of 100 mg/kg OA was administered.

Biochemical Analysis

Four hours after OA infusion, rats were decapitated un- der intramuscular 80 mg/kg ketamine anesthesia, according to the ethics guidelines, and blood samples were collected for biochemical analysis. Blood samples were centrifuged at 3500 rpm for 10 minutes, separa- ted serum samples transferred to eppendorf tubes and stored at -80°C until analysis. Right lungs were remo- ved, wrapped separately in aluminum foil, frozen in dry ice and stored at -80°C until the preparation of tissue homogenate samples for the measurement of malondialdehyde (MDA) levels and catalase (CAT), glutathione-peroxidase (GSH-Px), superoxide dismutase (SOD) activities.

Frozen lung tissues dissolved, washed with isotonic NaCl solution and dried in room temperature with ad- sorbent paper. Also the wet weights of tissues were determined. Tissues were kept cold and sliced into small pieces with a bistoury and transferred to the glass tubes. 2 mL cold Tris-HCl buffer solution (pH 7.4; 0.2 M Tris-HCl buffer) was added to the tissues and this buffer used for all studies. Tissues were homogenized in 16.000 rpm for two minutes using Ultra Turrax T25 Ba- sic (Germany) Homogenizer. The homogenization completed to three minutes by adding 4 mL buffer el- se. A portion of the homogenates were vortexed and transferred to eppendorf tubes. The homogenates centrifuged at 3500 g for 45 minutes at +4°C for preparing the supernatants.

Measurement of MDA levels: Serum MDA levels were measured by the thiobarbituric acid (TBA) method, which was modified from methods of Satoh and Yagi (11,12). Peroxidation was measured as the production of MDA, which in combination with TBA forms a pink

chromogen compound whose absorbance was measured spectrophotmetrically at 532 nm. Serum MDA results were expressed as nmol/mL. Lung tissue MDA levels were analyzed by the method of Ohkawa and expressed as nmol/mg protein (13).

Measurement of SOD activity levels: SOD activity in lung tissue and serum samples was measured according to the method of Sun et al. and modification of Durak et al. by determining the reduction of nitro blue tetrazolium (NBT) by superoxide anion produced with xanthine/xanthine oxidase system (14,15). One unit for SOD activity was expressed as the amount of protein that causes 50% inhibition in NBT reduction rate.

Results were defined as units per milligram protein (U/mg protein).

Measurement of GSH-Px activity levels: GSH-Px acti- vity in lung tissue and serum samples were measured according to the method of Paglia and Valentine, by monitoring the oxidation of reduced nicotinamide ade- nine dinucleotid phosphate (NADPH) at 340 nm (16).

Enzyme units were defined as the number of micromo- les of NADPH oxidised per minute. Results were defined as units per milligram protein (U/mg protein).

Measurement of CAT activity levels: CAT activity in lung tissue samples were determined according to the method of Aebi by measuring the decomposition of hydrogen peroxide at 240 nm (17). And results were expressed as rate constant per second per milligram protein (k/mg protein).

Histologic Analysis of the Lung

Left lungs were fixed in 10% formaldehyde. After em- bedding in paraffin, the tissues were cut into 3 µm sec- tions and stained with hematoxylin-eosin methods and assessed by light microscope (Olympus BX-50, Ja- pan).

Histological apperance of groups were graded as follows (18);

Grade 1: Normal histopathology.

Grade 2: Mild neutrophil infiltration.

Grade 3: Moderate neutrophil infiltration, perivascular edema, alveolar edema, partially destruction in alveolar structure.

Grade 4: Severe neutrophil infiltration, abscess forma- tion, destruction in alveolar structure.

Statistical Analysis

Data are expressed as mean ± SE. For statistical analysis, the non-parametric Kruskal-Wallis test was used.

(4)

Comparisons between groups were performed using the Mann-Whitney Rank Sum test. p value < 0.05 de- notes the presence of a significant statistical difference.

RESULTS

Serum MDA levels of OA group was statistically higher than control group (p< 0.05), when we compared lycopene + OA group with OA group, we saw that serum MDA levels was significantly lower in lycopene + OA group (p< 0.01). Tissue MDA levels in control and lycopene + OA groups was similar, levels of control and lycopene + OA group was significantly lower than cornoil + OA group (p< 0.05).

Serum SOD levels was significantly higher in lycopene + OA group than control group and OA group (for both p<

0.01). Tissue SOD levels of OA group was significantly higher than control group (p< 0.01). Moreover, lycopene + OA group levels was significantly higher than other three groups (p< 0.01, p< 0.05, p< 0.01, respectively).

When evaluated serum GSH-Px levels of groups, although lycopene + OA group and cornoil + OA group levels was higher than control group, no significant difference was seen between groups. Tissue GSH-Px levels of lycopene + OA group was significantly higher than other three groups (p< 0.01, p< 0.01, p< 0.05, respectively).

Tissue CAT levels of OA, cornoil + OA, and lycopene + OA groups was significantly higher than control group (p< 0.01, p< 0.05, p< 0.01, respectively). Lycopene + OA group levels was also significantly higher than OA, and cornoil + OA groups (p< 0.01, p< 0.05, respectively).

Serum and tissue levels of MDA, SOD, GSH-Px, and tissue levels of CAT of all groups is shown on Table 1.

Histopathological examination of lung tissue were as follows; control group had a normal appearance (Figu- re 1), OA, cornoil + OA groups had perivascular edema, alveolar edema, hemorrage, prominent neutrophil infiltration, and destruction in alveolar structure (Figu- re 2,3). Lycopene + OA group had less neutrophilic infiltration, perivascular and alveolar edema, and alveolar structure was prevented (Figure 4).

DISCUSSION

Histopathological changes in acute lung injury begins with increase in neutrophile leucocytes, and the formation of free oxygen radicals in lung cells. Lipid peroxidation is the main cause of the damage caused by free oxygen radicals in cells and tissues. Lipid peroxidation completed with converting of lipid peroxides to active aldehide and other carbonyl compounds. MDA, alco- hol, etane, pentanes were some of the compounds pro-

Table 1. Serum and lung tissue levels of MDA, SOD, GSH-Px, and tissue levels of CAT of all groups.

Group I Group II Group III Group IV

(control) (OA) (cornoil + OA) (lycopene + OA)

Serum MDA levels 5.59 ± 2.11* 9.01 ± 1.62^† 7.12 ± 1.47 5.13 ± 1.96 (nmol/mL)

Tissue MDA levels 41.62 ± 6.34^# 51.48 ± 13.07 66.62 ± 15.28^‡ 41.11 ± 3.75 (nmol/mL)

Serum SOD levels 10.60 ± 1.46 10.83 ± 2.54^† 15.52 ± 5.88 18.51 ± 4.26^§ (U/mg)

Tissue SOD levels 7.09 ± 0.50* 8.71 ± 1.17^† 8.25 ± 1.63^‡ 11.46 ± 2.71^§ (U/mg)

Serum GSH-Px levels 1.90 ± 0.25 1.95 ± 0.33 3.07 ± 1.07 3.02 ± 1.35 (U/mg)

Tissue GSH-Px levels 2.04 ± 0.30 1.98 ± 0.32^† 2.10 ± 0.76^‡ 3.29 ± 0.74^§ (U/mg)

Tissue CAT levels 0.08 ± 0.02*^# 0.12 ± 0.01^† 0.12 ± 0.04^‡ 0.20 ± 0.05^§ (U/mg)

* p< 0.05 when compared group I vs. group II.

# p< 0.05 when compared group I vs. group III.

§ p< 0.05 when compared group I vs. group IV.

† p< 0.05 when compared group II vs. group IV.

‡ p< 0.05 when compared group III vs. group IV.

OA: Oleic acid, MDA: Malondialdehyde, SOD: Superoxide dismutase, GSH-Px: Glutathione-peroxidase, CAT: Catalase.

(5)

duced at the end of the reaction. So, MDA is used as an indirect indicator of lipid peroxidation (19,20).

Plasma and BAL levels of MDA have been shown to increase significantly in experimental studies of acute lung injury (21,22). Septic lung injury models in rats

showed the increase in lung tissue MDA levels which decreased after application of N-acetylsistein or methylene blue (18,23). Karahan et al. reported that lycopene decreased plasma and sera MDA levels that increased in experimental oxidative stress induced by cisplatin and gentamycine (24). Another study in which gastric carcinogenesis was induced by N- methyl-N-nitro–N-nitrosoguanidin showed the increase in lipid peroxidation, and the investigators reported that lycopene decreased the lipid peroxidation products in blood (25). In an acute lung injury model in rats, Kök- sel et al. reported the increase in MDA levels in lung tissue, plasma and BAL, and decrease in these levels after applying an antioxidant, caffeic acid phenethyl ester (CAPE) (26).

In our study, higher sera and tissue MDA levels of group II and III proved the lung injury formation, and serum and tissue MDA levels of lycopene given group (group IV) even below the levels found in the control group to suggest that lycopene prevents lipid peroxidation.

It is known that in order to prevent the lipid peroxidation in ARDS patients, increase in total antioxidant capacity and decrease in glutation specific antioxidants oc- curs and this causes a spesific decrease in antioxidant defence of lung (27). Liu et al. determined a decrease in SOD enzyme activity at early stages of ARDS induced by oleic acide (28). In an other study conducted in ARDS patients, no changes were seen in SOD, GSH-Px enzyme activities but a little increase in CAT enzyme activities (3). In other studies carried out in ARDS patients and patients with sepsis found the increased serum levels of CAT, and SOD, decreased levels of glutation (29-31). It is also reported that decreases in GSH- Figure 1. Normal lung tissue of control group (HE, x200).

Figure 2. Perivascular edema, neutrophilic infiltration, and destruction in alveolar structure in OA group (HE, x200).

Figure 3. Prominent neutrophilic infiltration, hemorrage, al- veolar destruction in cornoil + OA group (HE, x200).

Figure 4. Less perivascular edema, neutrophilic infiltration, prevented alveolar structure in lycopene + OA group (HE, x100).

(6)

Px, SOD, and CAT enzyme activities in oxidative stress can be regulated by lycopene (25,32).

In our study, a little increase determined in tissue, and serum SOD, GSH-Px enzyme activities and tissue CAT activities in OA and corn oil + OA groups when compared with control group. However, significant incre- ases were determined in lycopene + OA group when compared with other groups.

Dose of lycopene intake in the studies shows heteroge- neity. While 6.5 mg/day lycopene decreases lung cancer risk in nonsmoker women, the dose in nonsmoker men is 12 mg/day (9). 30 mg lycopene per day is in- dicated for prevention of exercise-induced asthma (33). Although high levels of lycopene and other carotenoids in lungs provide an additional protection against to oxidative damage, no dose-dependent relations- hip between increased tomato consumption and reduced risk of lung cancer was found (34). Lower values of serum carotenoids were found in dead lung cancer patients and it is reported that carotenoid support slowed down the disease progression in lung cancer patients (35). The dose of lycopene as an antioxidant used in our study (20 mg/kg) may be accepted as an effective dose and there were no side effects reported for this dose.

Koksel et al. showed alveolar edema, congestion, neutrophil infiltration and damage in pulmonary structures in oleic acid lung injury model. These authors reported that pulmonary injury was decreased by giving antioxidant CAPE and NAC (6,26). In an experimental model of sepsis, Ozdulger et al. determined interstitiel edema, inflammatory cell infiltration and degeneration in pulmonary structure and also they reported edema, infiltration and pulmonary degeneration decreased with NAC therapy Liu and his colleagues (18,28) have cre- ated an ARDS model in rats with OA, and they reported that pulmonary interstitial edema and pulmonary hemorrhage were reduced when SOD was given befo- re OA application. Gultekin and colleagues have cre- ated an experimental acute pancreatitis in rats and showed neutrophil infiltration, alveolar edema, enlarge- ment and wall thickening in lungs when decreased by giving leptin (36).

In our study, we determined that corn oil + OA, and OA groups had intense neutrophil infiltration, prominent perivascular and alveolar edema, hemorrhage, and im- paired alveolar structure consistent with the literature.

Lycopene + OA group showed mild neutrophil infiltration, perivascular and alveolar edema, and preserved alveolar structure, suggests that lycopene may prevent the progression of lung damage.

As a result, lycopene rich diet has an important role in preventing damages in lungs that is open to oxidative stress, and we can say that extensive clinical studies will better explain the subject.

CONFLICT of INTEREST None declared.

REFERENCES

1. Repine JE. Scientific perspectives on adult respiratory distress syndrome. Lancet 1992; 339: 466-9.

2. Ware LB, Matthay MA. The acute respiratory distress syndro- me. N Engl J Med 2000; 342: 1334-9.

3. Metnitz PG, Bartens C, Fischer M, Fridrich P, Steltzer H, Druml W. Antioxidant status in patients with acute respiratory dist- ress syndrome. Intensive Care Med 1999; 25: 180-5.

4. Pittet JF, Mackersie RC, Martin TR, Matthay MA. Biological markers of acute lung injury: prognostic and pathogenetic significance. Am J Respir Crit Care Med 1997; 155: 1187-205.

5. Schuster DP. ARDS: clinical lessons from oleic acid model of acute lung injury. Am J Respir Crit Care Med 1994; 149: 245-60.

6. Koksel O, Cinel I, Tamer L, Cinel L, Ozdulger A, Kanik A, et al.

N-acetylcysteine inhibits peroxynitrite-mediated damage in oleic acid-induced lung injury. Pulm Pharmacol Ther 2004;

17: 263-70.

7. Stahl W, Sies H. Bioactivity and protective effects of natural carotenoids. Biochim Biophys Acta 2005; 1740: 101-7.

8. Bramley PM. Regulation of carotenoid formation during toma- to fruit ripening and development. J Exp Bot 2002; 53: 2107-13.

9. Michaud DS, Feskanich D, Rimm EB, Colditz GA, Speizer FE, Willett WC, et al. Intake of specific carotenoids and risk of lung cancer in 2 prospective US cohorts. Am J Clin Nutr 2000; 72:

990-7.

10. Velmurugan B, Bhuvaneswari V, Nagini S. Antiperoxidative ef- fects of lycopene during N-methyl-N'-nitro-N-nitrosoguanidine- induced gastric carcinogenesis. Fitoterapia 2002; 73: 604-11.

11. Satoh K. Serum lipid peroxide in cerebrovascular disorders de- terminde by a new colorimetric method. Clin Chim Acta 1978;

90: 37-43.

12. Yagi K. Assay for blood plasma or serum. Methods in Enzymol 1984; 105: 328-31.

13. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in ani- mal tissues by thiobarbituric acid reaction. Analytical Bioche- mistry 1979; 95; 351-8.

14. Sun Y, Oberley LW, Ying L. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988; 34: 497-500.

15. Durak I, Yurtarslanı Z, Canpolat O, Akyol O. A methodologi- cal approach to superoxide dismutase activity assay based on inhibition of nitroblue tetrazolium reduction. Clin Chim Acta 1993; 214: 103-4.

16. Paglia DE, Valentine WN. Studies on the quantitative and qu- alitative characterization of erythrocyte glutathione peroxida- se. J Lab Clin Med 1967; 70: 158-69.

(7)

17. Aebi H. Catalase in vitro. Methods Enzymol 1984; 105: 121-6.

18. Ozdulger A, Cinel I, Koksel O, Cinel L, Avlan D, Unlu A, et al.

The protective effect of N-acetylcysteine on apoptotic lung in- jury in cecal ligation and puncture-induced sepsis model.

Shock 2003; 19: 366-72.

19. MacNee W, Rahman I. Oxidants and antioxidants as therape- utic targets in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160: 558-65.

20. Ozaras R, Tahan V, Turkmen S, Talay F, Besirli K, Aydin S, et al. Changes in malondialdehyde levels in bronchoalveolar flu- id and serum by the treatment of asthma with inhaled stero- id and beta2-agonist. Respirology 2000; 5: 289-92.

21. Oliveira-Junior IS, Brunialti MK, Koh IH, Junqueira VB, Salo- mão R. Effect of pentoxifylline on lung inflammation and gas exchange in a sepsis-induced acute lung injury model. Braz J Med Biol Res 2006; 39: 1455-63.

22. Meyancı G, Arıcıoglu F, Oz H, Aydemir A. The effects of int- ratrcheal dexamethasone on lipid peroxidation in acute lung injury. Cerrahpasa J Med 2001; 32: 20-4.

23. Demirbilek S, Sizanli EE, Karaman A, et al. Effects of Methyle- ne Blue on Lung Injury in Septic Rats. Journal of Inonu Uni- versity Medical Faculty 2004; 11: 207-11.

24. Karahan I, Yılmaz S, Atessahin A. Effects of Lycopene on cisp- latin and gentamicin induced oxidative stres in blood and li- ver in rats. Firat University Medical Journal of Health Sciences 2006; 20: 39-43.

25. Velmurugan B, Nagini S. Combination chemoprevention of ex- perimental gastric carcinogenesis by s-allylcysteine and lyco- pene: modulatory effects on glutathione redox cycle antioxi- dants. J Med Food 2005; 8: 494-501.

26. Koksel O, Kaplan MB, Ozdulger A, Tamer L, Degirmenci U, Ci- nel L, et al. Oleic acid-induced lung injury in rats and effects of caffeic acid phenethyl ester. Exp Lung Res 2005; 31: 483-96.

27. Gadek JE, Pacht ER. The interdependence of lung antioxi- dants and antiprotease defense in ARDS. Chest 1996; 110 (Suppl 6): S273-S7.

28. Liu H, Zhang D, Zhao B, Zhao J. Superoxide anion, the main species of ROS in the development of ARDS induced by oleic acid. Free Radic Res 2004; 38: 1281-7.

29. Leff JA, Parsons PE, Day CE, Moore EE, Moore FA, Oppegard MA, et al. Increased serum catalase activity in septic patients with the adult respiratory distress syndrome. Am Rev Respir Dis 1992; 146: 985-9.

30. Pacht ER, Timerman AP, Lykens MG, Merola AJ. Deficiency of alveolar fluid glutathione in patients with sepsis and the adult respiratory distress syndrome. Chest 1991; 100: 1397-403.

31. Brigham KL. Role of free radicals in lung injury. Chest 1986;

9: 59-63.

32. Breinholt V, Lauridsen ST, Daneshvar B, Jakobsen J. Dose res- ponse effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat. Cancer Letters 2000; 154: 201- 10.

33. Neuman I, Nahum H, Ben-Amotz A. Reduction of exercise-in- duced asthma oxidative stress by lycopene a natural antioxi- dant. Allergy 2000; 55: 1184-9.

34. Arab L, Steck-Scott S, Fleishauer AT. Lycopene and the lung.

Exp Biol Med (Maywood) 2002; 227: 894-9.

35. Heber D, Lu QY. Overview of mechanisms of action of lycope- ne. Exp Biol Med (Maywood) 2002; 227: 920-3.

36. Gultekin FA, Kerem M, Tatlicioglu E, Aricioglu A, Unsal C, Bu- kan N. Leptin treatment ameliorates acute lung injury in rats with cerulein-induced acute pancreatitis. World J Gastroente- rol 2007; 13: 2932-8.