Original Article 281

Protective Effects of Melatonin against Cyclophosphamide-induced Oxidative Lung Toxicity in Mice

Affiliations

Key words

▶ melatonin ● ▶ cyclophosphamide ● ▶ lung toxicity ● ▶ lipid peroxidation ● ▶ catalase ● ▶ superoxide dismutase ● ▶ glutathione ●

received 09.01.2014 accepted 20.02.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1371801 Published online: March 25, 2014 Drug Res 2015; 65: 281–286 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence A. Ahmadi Pharmaceutical Sciences Research Center Faculty of Pharmacy Mazandaran University of Medical Sciences 18 kilometer of Farah Abad Road Sari P. Box: 48175-86 Iran Tel.: + 98/151/3543 084 Fax: + 98/151/3543 084 Amirhossein_pharma@yahoo. com

M. Shokrzadeh1, 2, A. Chabra3, F. Naghshvar4, A. Ahmadi1, M. Jafarinejhad4, Y. Hasani-Nourian2 1

Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran 3 Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran 4 Department of Pathology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran 2

Abstract

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This study was undertaken to evaluate the protective effects of melatonin against cyclophosphamide (CP)-induced oxidative lung toxicity in mice. Mice were pre-treated with various doses of melatonin for 7 consecutive days and were then injected with CP (200 mg/kg b. w.) 1 h after last melatonin injection. After 24 h, the mice were euthanized and their lungs were immediately harvested. Several biomarkers associated with oxidative stress in lung homogenates, such as thiobarbituric acid reactive substances (TBARs) and reduced glutathione (GSH) levels and the activity of superoxide dismutase (SOD) and catalase (CAT) were measured spectrophotometrically. A single dose of CP markedly altered the levels of these oxidative stress biomarkers in lung homogenates. However, increased

Introduction

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Cyclophosphamide (CP) is a valuable chemotherapeutic agent used in the treatment of many neoplastic tumors. Additionally, it has been used as an immunosuppressant prior to organ transplantation and to treat certain autoimmune diseases [1, 2]. However, CP has been reported to cause acute and chronic pulmonary injury in both humans and animals [3]. Furthermore, CP is a common component of multi-drug regimens that have increased pulmonotoxic potential. Although the mechanisms driving pulmonary damage caused by CP or other cytotoxic agents are poorly understood, it is possible that these agents have direct toxic effects or are indirectly toxic through the activation of pulmonary inflammatory cells [3]. CP chemotherapy is often limited by lung and/or bladder injuries in both animals and humans [3–5]. Early histopathological findings in rats demonstrated capillary

lipid peroxidation, measured as TBARs, was significantly inhibited in the lung tissues of the melatonin-pretreated mice compared to the CP alone-injected group. In addition, pretreatment with melatonin also significantly restored GSH levels and SOD and CAT activities. Melatonin also effectively protected animals from CP-induced histological abnormalities in lung tissue. In conclusion, the increase in oxidative stress markers and concomitant adaptations by the antioxidant defense system indicates that oxidative stress plays an important role in CP-induced damage to the lung. Moreover, melatonin is a potent natural antioxidant that helps prevent CP-induced oxidative toxicity in mouse lung tissues. Thus, because melatonin is regarded to be a safe pineal secretory product, it may be used concomitantly as a supplement to reduce lung damage in patients undergoing chemotherapy.

endothelial damage, type II pneumocyte (EP II) injury and necrosis, pulmonary edema, and infiltration of inflammatory cells. Generation of reactive oxygen species (ROS) and lipid peroxide formation in lung microsomes are thought to mediate lung damage in this model [5]. It has been reported that disruption of the redox balance by oxidative stress following CP exposure causes biochemical and physiological changes [6, 7]. Lack of detoxifying enzymes, aldehyde oxidase, and aldehyde dehydrogenase in the lungs is a cause of selective CP-induced lung toxicity. Several classes of antioxidant dietary compounds have been suggested to have health benefits. There is evidence that the consumption of antioxidant compounds decreases various proinflammatory and/or oxidative stress biomarkers [8]. We previously reported that natural products and antioxidant compounds ameliorated CPinduced oxidative stress and genotoxicity in mouse bone marrow cells [9, 10]. Thus, antioxi-

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Authors

282 Original Article

Materials and Methods

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control, mice were administered a single toxic dose of CP (200 mg/kg b.w. i. p.) in normal saline (10 ml/kg b.w.); groups 3–6, mice were treated with melatonin at different concentrations (2.5, 5, 10, and 20 mg/kg b. w. by i. p. injection) in normal saline (10 ml/kg b.w.) each day for 7 consecutive days followed by a single i. p. dose of CP 1 h after the last dose of melatonin. All doses of CP and melatonin used in the experiment were obtained from our previous study [20]. 24 h after CP injection, all animals were sacrificed by an overdose of ether. The lungs were then removed and washed 3 times with normal saline for complete blood removal. The left lungs were used for biochemical analysis, and the right lungs were used for histopathological examination.

Homogenate preparation The left lung tissues were weighed and homogenized in chilled potassium chloride (1.15 %). The homogenates were centrifuged at 6 000 g for 15 min at 4 °C, and the supernatant was used for biochemical analysis.

Lung biochemical analysis Lipid peroxidation assay The presence of thiobarbituric acid reactive substances (TBARs) in the lung was evaluated by reaction with thiobarbituric acid [23]. Briefly, a reaction mixture (1.0 mL) contained 0.1 M phosphate buffer (0.58 mL; pH 7.4), tissue homogenate (0.2 mL), 100 mM of ascorbic acid (0.2 mL) and 100 mM of ferric chloride (0.02 mL). After incubation at 37 °C for 60 min, the reaction was stopped by the addition of 10 % trichloroacetic acid (1.0 mL). Next, 1.0 mL of thiobarbituric acid (0.67 %) was added to the reaction mixture and placed in a boiling water bath for 20 min. TBARS formation was examined by recording the absorbance of the reaction mixture at 535 nm using a spectrophotometer blanked to the reagent.

Chemicals CP (Endoxan®) was obtained from Baxter Oncology GMBH (Westfalen, Germany). Melatonin, 5,5'-dithiobis (2-nitrobenzoic acid), glacial acetic acid, heparin, nitro blue tetrazolium chloride, potassium dihydrogen phosphate, reduced glutathione, sodium dihydrogen phosphate, sodium fluoride, trichloroacetic acid, thiobarbituric acid, hydrogen peroxide, ferric chloride, and hydroxylamine hydrochloride were purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). All other chemicals were either equal to or more pure than analytical grade.

Glutathione (GSH) content

Animals

Superoxide dismutase (SOD) activity

NMRI mice weighing 28 ± 4 g were obtained from the Pasteur Institute of Iran (Amol). The mice were housed in good condition at the university animal facility and were maintained under a controlled 12 h light/dark cycle and temperature (23 ± 1 °C). The animals were acclimatized for 1 week before the study and were given standard food pellets and water ad libitum. All procedures were performed according to the ‘Care and Use of Laboratory Animals’ prepared by the Mazandaran University of Medical Sciences, Sari, Iran. The study protocol was approved by the Research Committee of the University.

SOD activity was evaluated by the method described in our previous study [20]. Reaction mixtures contained sodium carbonate (1 ml, 50 mM), nitroblue tetrazolium (0.4 ml, 25 mm), and freshly prepared hydroxylamine hydrochloride (0.2 ml, 0.1 mM). The reaction mixtures were mixed by inversion followed by the addition of the clear tissue homogenate supernatant (0.1 ml, 1:10, w/v). The change in absorbance of the samples was recorded at 560 nm.

Experimental treatment Animals were divided into the following 6 groups of 5 mice (Groups 1–6, n = 5) each for the experiments: negative control, mice were administered normal saline (10 ml/ kg b. w.) via intraperitoneal (i. p.) injection for 7 days; positive

GSH content was evaluated according to the method described by Ellman (1959). The homogenate sample (720 μL) was diluted and trichloroacetic acid (5 %) was added to the reaction mixture to precipitate tissue homogenate proteins. The reaction was centrifuged at 10 000 g for 5 min, and the supernatant was taken. Ellman’s reagent [5,5'-dithiobis (2-nitrobenzoic acid) solution] was added to the sample. Finally, the absorbance of the sample was recorded at 417 nm.

Catalase (CAT) activity CAT activity was evaluated according to the method of Nabavi et al. Briefly, a reaction mixture (3 mL) containing 50 mM phosphate buffer (2.5 mL; pH 5.0), 5.9 mM H2O2 (0.4 mL) and lung homogenate (0.1 mL) was incubated for 1 min, and the absorbance of the sample was recorded at 240 nm [24].

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dant biological compounds may help protect cells and tissues from the deleterious effects of CP-induced ROS and other free radicals [11, 12]. Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone, which is secreted from the pineal gland in all mammals [13]. Numerous experimental studies have documented that melatonin has antioxidant, anti-apoptotic, anti-carcinogenic, and anti-aging properties. In addition, melatonin affects the immune system [13–15]. Thus, melatonin is a versatile antioxidant. In addition to directly neutralizing the number of free radicals and reactive oxygen/nitrogen species, melatonin also stimulates several antioxidant enzymes, which increases its efficiency as an antioxidant [16–18]. Furthermore, melatonin diminishes free radical production. The ability to cross all biological membranes and reach the cell nucleus is what allows melatonin to decrease oxidative stress [19]. In our recent study, melatonin reduced CPinduced testicular toxicity by its antioxidant and free radicalscavenging activities [20]. In addition, we showed that melatonin helped prevent diazinon-induced genotoxicity in human blood lymphocyte cells because of its free radical-scavenging properties [21]. Zhong et al. observed that pretreatment with melatonin reduced lung capillary permeability and pulmonary edema. These results demonstrated a marked decrease in inflammatory cell infiltration and capillary permeability in reexpansion pulmonary edema (RPE), a rare but life-threatening complication of many lung diseases, in animals treated with melatonin, which reflects its broad anti-inflammatory and antioxidant effects [22]. Therefore, this study was undertaken to assess the protective effects of melatonin against CP-induced oxidative lung toxicity.

Original Article 283

Table 1 Effect of CP (200 mg/kg) and/or melatonin on lung lipid peroxide formation measured by TBARS and GSH levels and the activity of GSH and SOD. Treatment Groups

TBARSa (nmol MDA eq/g tissue)

Control CP Melatonin 2.5 mg/kg + CP Melatonin 5 mg/kg + CP Melatonin 10 mg/kg + CP Melatonin 20 mg/kg + CP

38.63 ± 2.21 78.07 ± 7.39 b 57.42 ± 6.68 c 46.13 ± 6.23 d 45.83 ± 4.67 d 35.47 ± 3.83 e,f

GSHa (μg/mg protein) 7.59 ± 0.25 4.23 ± 0.03 b 4.29 ± 0.09 4.63 ± 0.38 6.68 ± 0.31 d 7.89 ± 0.21 e,f

SODa (U/mg protein) 93.28 ± 4.19 48.43 ± 1.07 b 48.26 ± 1.84 68.19 ± 2.75 d 71.28 ± 3.92 d 95.26 ± 3.58 e,f

CATa (μmol/min/mg protein) 46.38 ± 3.24 28.83 ± 2.69 b 30.41 ± 4.81 35.09 ± 2.15 c 44.92 ± 1.96 e,f 45.88 ± 4.26 e.f

CP = cyclophosphamide; TBARS = thiobarbituric acid-reactive substances; GSH = reduced glutathione; SOD = superoxide dismutase a

Values are the mean ± standard deviation for each group of 5 mice. b P < 0.001 compared to the control; c P < 0.05 compared with the CP treated group; d P < 0.01 compared

with the CP treated group; e P < 0.001 compared with the CP treated group; f No significant difference compared to the control group The data were analyzed with one-way ANOVA and the Tukey’s HSD test

The right lung lobes were fixed in 10 % neutral buffered formalin, sliced transversely, embedded in paraffin, and prepared as 5-μm-thick sections that were then stained with hematoxylin and eosin (H&E) for light microscopic evaluation. 3 factors (thickening of the alveolar septa, pneumocyte hyperplasia, and immune cell infiltration) were measured using a semi-quantitative method. The level of damage was based on a graded scale of 0–4, in which grade 0 = no damage, 1 = very low levels of damage, 2 = mild damage, 3 = moderate damage and 4 = severe damage. Slides were viewed and photographed using a camera attached to a microscope (Labomed, LX400) at 400 × magnification in at least 3 random microscopic fields from each animal by 2 expert pathologists without knowledge of the treatment groups.

Statistical analysis The data are presented as the means ± the standard deviation (SD). One-way analysis of variance and Tukey’s honest significant difference (HSD) tests were used for multiple comparisons of the data. A p-value less than 0.05 was considered to be statistically significant. All measurements were replicated 3 times.

Fig. 1 Normal group; a section in the mouse lung tissue showing a normal pneumocyte in the alveolar septa A, normal alveolar space B and a capillary filled by erythrocytes in the alveolar septa C. (hematoxylin and eosin-stained paraffin sections; H&E × 400).

SOD activity ▶ Table 1 shows the activity of SOD in mouse lung homogenates. ●

Results

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Lung biochemical analysis Lipid peroxidation level The analysis of the lung lipid peroxidation studies are shown ▶ Table 1. Accordingly, a single injection of CP significantly in ● increased (p < 0.001) the lipid peroxidation levels of lung homogenates compared to the negative control group. A 7-day pretreatment with melatonin reduced the elevated levels of lipid peroxidation in the lung following administration of CP at all of the administered doses of melatonin. The maximal inhibition of lipid peroxidation (p < 0.001) was observed with a 20 mg/kg dose of melatonin before challenging animals with CP.

GSH content The GSH levels in the lungs of animals treated with CP were lower than mice in the negative control group (P < 0.001) ▶ Table 1). Pre-administration of melatonin at different doses (● before CP injection significantly attenuated the loss of GSH in the lung following CP administration. The maximum effect was observed after a 7 day pretreatment with 20 mg/kg of melatonin, which restored GSH levels in lung tissues compared to the CP alone-treated group (p < 0.001).

Accordingly, the SOD activity in the lung homogenates of CPtreated mice was significantly (p < 0.001) lower than that of the negative control group. In contrast, SOD activity was significantly higher in the lung homogenates of melatonin-treated mice compared to the CP alone-treated group with a maximal effect observed after a pretreatment with 20 mg/kg of melatonin (p < 0.001).

CAT activity The CAT activity in the lung homogenates of CP-administered mice was significantly (p < 0.001) lower than the negative con▶ Table 1), and melatonin pretreatment significantly trol group (● restored this activity. The maximum effect was observed after a 20 mg/kg melatonin pretreatment, which significantly prevented losses in CAT activity compared to the CP alone-treated group (p < 0.001).

Histopathological examination of the lung Light microscopy photomicrographs of representative lung histological sections showing the optimal melatonin pretreatment dose 24 h after CP administration (200 mg/kg) compared with the administration of melatonin (20 mg/kg for 7 consecutive ▶ Fig. 1–3). The lung tissue from control mice has normal days) (●

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Histopathological examination

284 Original Article

the lung tissues 24 h after CP administration. Pretreatment of mice with melatonin for 7 consecutive days prior to CP treatment reduced lung tissue damage in a dose-dependent manner. These results are in agreement with the measured biochemical parameters.

Discussion

Fig. 2 A single-dose injection of the CP (200 mg/kg) group; a section of the lung tissues from a mouse showing thickened alveolar septa lined with hyperplastic pneumocytes A and small alveolar space filled by lymphoplasma cells, alveolar macrophages and necrotic debris B. (hematoxylin and eosin-stained paraffin sections; H&E × 400).

Fig. 3 Pretreatment with 20 mg/kg of melatonin for 7 days before CP administration; a section of mouse lung tissue showing alveolar space with scattered RBC extravasation A and alveolar septa with focally hyperplastic pneumocytes and congestion B. (hematoxylin and eosin-stained paraffin sections; H&E × 400).

alveolar spaces, normal pneumocytes in the alveolar septa, and ▶ Fig. 1). ● ▶ Fig. 2 septal capillaries filled with erythrocytes (● shows a lung section with small alveolar spaces filled by lymphoplasma cells, alveolar macrophages, necrotic debris, and thickened alveolar septa lined with hyperplastic pneumocytes, which is representative of mouse lungs pretreated with a single ▶ Fig. 3 shows the lung section of a mouse administration of CP. ● pretreated with 20 mg/kg of melatonin for 7 days prior to CP injection, demonstrating the alveolar space with scattered red blood cell (RBC) extravasation and alveolar septa with focally hyperplastic pneumocytes and congestion. The results of the semi-quantitative histopathological examina▶ Table 2. Briefly, the grades of tion of the lung are shown in ● damage for all 3 factors were 0 and 4 for the untreated control animals and animals receiving a single dose of CP at 200 mg/kg, respectively. This result indicates severe damage was incurred in

Cancer patients usually suffer from lung toxicity after CP therapy, which is characterized by hypoxemia, non-cardiogenic pulmonary edema, low lung compliance, and widespread capillary leakage [4]. Several studies have indicated that CP has pro-oxidant characteristics, and CP-induced oxidative stress decreases antioxidant enzyme activities and increases lipid peroxidation in the lungs of animals [25, 26]. Induction of lipid peroxidation has been reported in different tissues of experimental animals after CP administration [27, 28]. CP and its metabolite acrolein inactivate microsomal enzymes and increase ROS and lipid peroxidation [29, 30]. Previously, we reported that CP increases TBARS levels and therefore the extent of lipid peroxidation in the testis tissues from experimental animals [20]. In this current study, the production of malondialdehyde (MDA), which is an index of lipid peroxidation, was significantly increased in mouse lung homogenate compared to the control group after CP administration. This observation is in accordance with many reports that demonstrated an apparent elevation in lung TBARS following CP administration [26, 31–33]. Melatonin is principally synthesized by the pineal gland of mammals and has been suggested to have antioxidant and prophylactic properties [34]. Melatonin exerts direct antioxidant effects by directly scavenging hydroxyl and peroxyl radicals, singlet oxygen, peroxynitrite anions, and superoxide anions [35]. Melatonin also increases the levels of potential antioxidants, such as SOD, CAT, and glutathione peroxidase (GSH-Px) by an indirect mechanism [36]. Furthermore, melatonin prevents lipid peroxidation and the oxidative destruction of lipids [37]. In our previous study, melatonin had dose-dependent inhibitor effects on CP-induced lipid peroxidation in mouse testicular tissue [20]. We further showed that melatonin had a potent genoprotective effect in preventing DNA damage induced by diazinon in human blood lymphocyte cells, and this protective effect may result from its free radical-scavenging properties [21]. Therefore, we evaluated the pulmonoprotective effects of melatonin against CP-induced oxidative lung toxicity in mice. Administration of melatonin for 7 consecutive days before CP injection significantly inhibited the levels of lung lipid peroxidation compared to CP-only-treated mice. This activity could be due to the ability of melatonin to scavenge the free radicals generated by CPinduced oxidative stress where melatonin efficiently inhibited lipid peroxidation. GSH is the principal intracellular non-enzymatic antioxidant. It is able to detoxify endogenous and exogenous substances, including free radicals and xenobiotics [38]. CP metabolism produces highly reactive electrophiles, which decreases the levels of GSH in CP-treated mice because of increased cellular electrophilic burden and acrolein formation [39]. In our study, administration of CP decreased GSH levels in the lung by approximately 92 %. This reduction could be due to decreased expression of this antioxidant during bronchial cell damage [40]. This supposition is in accordance with other reports that demonstrated GSH depletion following CP treatment in animals [11, 41].

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Original Article 285

Table 2 The results of semi-quantitative histopathological examination of the lung showing reduced CP-induced tissue damage after melatonin (MLT) administration at various doses. Groups Factors/Grades Thickening of the alveolar septa

Hyperplasia of pneumocytes

Infiltration by inflammatory cells

Control Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Grade 0 Grade 1 Grade 2 Grade 3 Grade 4

CP 200 mg/

MLT 2.5 mg/

MLT 5 mg/

MLT 10 mg/

MLT 20 mg/

kg

kg + CP

kg + CP

kg + CP

kg + CP

+

+

+

+

+ + + + + + + + + + + +

+

+

However, administration of melatonin for 7 days prior to CP treatment significantly inhibited GSH depletion compared to the CP-only-treated group. In addition, we show that the changes in GSH are accompanied by a concomitant decrease in the activity of the SOD and CAT antioxidant enzymes following CP administration. SOD and CAT are considered to be first line cellular antioxidant defense enzymes that protect cells from oxidative damage. These cellular antioxidants play an important role in eliminating free radicals, and equilibrium exists between these enzymes under normal physiological conditions. When excess free radicals are manufactured because of toxin exposure, this balance is lost and subsequent oxidative insults occur [42]. In the current study, a single administration of 200 mg/kg of CP significantly decreased the activity of SOD and CAT in lung tissues. However, pretreatment of melatonin reversed this decrease, suggesting that melatonin was able to restore the activities of these enzymes. These results are in accordance with our recent study in which administration of melatonin to mice prior to the injection of CP restored the antioxidant enzyme levels of SOD and CAT and reduced GSH depletion following CP injection in mouse testis tissue [20]. Therefore, melatonin treatment reduced oxidative damage, likely by its ability to quickly and efficiently scavenge lipid peroxyl radicals and free radicals before they attack membrane lipids [43]. CP induced lung injury has been observed histologically by endothelial cell destruction, type I and type II alveolar epithelial cell damage, alveolitis, alveolar edema, hemorrhage and extensive inflammatory cell infiltration [44]. Sulkowska et al. [41] demonstrated that congestion and edema is because of CP-induced changes in epithelial cell structure and alveolocapillary permeability. A recent study demonstrated that melatonin treatment remarkably reduced alveolar epithelial injury, inflammation and interalveolar septal thickening in a rat mechlorethamine-induced lung toxicity model. This result was because melatonin has both antioxidant and anti-inflammatory effects [45]. In our study, melatonin treatment resulted in minimal lung damage with no areas of intralobular necrosis or significant inflammatory cell infiltration. This histopathological result is consistent with the results obtained from the measured biochemical parameters.

Conclusion

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Melatonin may reduce CP-induced lung toxicity in mice through its ability to increase the activity of the antioxidant defense system, scavenge ROS, which induce lipid peroxidation and peroxidative damage, and quench free radicals. Because melatonin has been used extensively as a supplement in several diseases and is regarded to be safe, it could be a potent supplement for preventing lung toxicity side effects in patients undergoing chemotherapy.

Acknowledgments

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This study was supported by a grant from the Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.

Conflict of Interest

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The authors declare no conflict of interest.

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CP = cyclophosphamide; MLT = melatonin

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286 Original Article