Effects of erythropoietin on methotrexate induced lung injury in rats

1 Chest Disease, Faculty of Medicine, Kahramanmaras Sutcuimam University, Kahramanmaras, Turkey. 2 Pediatric Surgery, Faculty of Medicine, Kahramanmaras Sutcuimam University, Kahramanmaras, Turkey. 3 Biochemistry, Faculty of Medicine, Kahramanmaras Sutcuimam University, Kahramanmaras, Turkey. 4 Chest Surgery, Faculty of Medicine, Kahramanmaras Sutcuimam University, Kahramanmaras, Turkey. 5 Pathology, Faculty of Medicine, Kahramanmaras Sutcuimam University, Kahramanmaras, Turkey.

been used for the treatment of different diseases and malignancies like leukemia, breast, head, neck, and lung carcinomas, and rheumatoid arthritis (Cooper et al., 1986).Sixty to ninety-three percentage of patients treated with MTX eventually develop adverse reactions and most of these reactions are not life-threatening, but up to 30% of patients treated with MTX for more than five years, eventually discontinue the MTX therapy, because of unacceptable toxicity (Goodman and Polisson, 1994).Pulmonary, hepatic, and hematologic side-effects are the major and potentially life-threatening toxicities (Salach and Cash, 1996).Studies generally suggest that pulmonary toxicities arise in 2 to 8% of patients receiving MTX, but others define as high as 33% (Salach and Cash, 1996).The precise mechanisms of MTX-induced pulmonary injury are not known.Most researchers think that MTX generates a type IV delayed hypersensitivity pneumonitis presenting lymphocytic proliferation and alveolitis (Chikura et al., 2008;Cooper et al., 1986).Some side effects are thought to emerge from idiosyncratic mechanisms unrelated to folate antagonism, because pulmonary toxicities occur with both low and high doses and occur by different routes of administration (Ohbayashi et al., 2010).
Reactive oxygen species (ROS) are produced as a result of normal cellular metabolism (Ergur et al., 2008;Thannickal and Fanburg, 2000).ROS aroused from the partial reduction of oxygen, represent their cytotoxic effects by directly modifying cellular and extracellular components.It is well known that chemical-induced cellular injury can lead to oxidative stress, because of an imbalance between the generation of ROS and their detoxification in tissues.ROS-induced injury is well characterized and includes DNA base oxidation, lipid peroxidation, and protein oxidation (Laskin et al., 2010).Malondialdehyde (MDA) is a marker of oxidative stress and is formed during lipid peroxidation of tissue (Naziroglu, 2007).Erythropoietin (EPO), a hypoxia-inducible hematopoietic growth factor, has antiapoptotic, antioxidant, anti-inflammatory, and angiogenic effects.In addition to its traditional hematopoietic role, recently, it is highlighted that EPO has cytoprotective effects (Jelkmann, 2007;Mori et al., 2008).Wu et al. (2006) demonstrated that pretreatment with EPO appears to attenuate ischemia-reperfusion-induced lung injury.In another study, EPO was shown to reduce oxidative stress-associated lipid peroxidation in lung tissue (Tascilar et al., 2007).
The role of ROS and the protective effect of EPO on MTX-induced lung injury have not been investigated yet.On the basis of the aforementioned findings, we conducted this study to examine biochemically and histopathologically the protective effects of EPO on MTX induced lung damage in a rat model.

Animals
This study was approved by the Ethics Committee on Animal Research at the Faculty of Medicine of Kahramanmaraş Sütçü Imam University.For the study, 21 female Sprague-Dawley rats, weighing 210 to 230 g each were selected and acclimatized for 10 days in the animal laboratory of our university research center, receiving a standard diet and water ad libitum.The rats were divided into 3 groups: sham group (sham operation, n = 7) animals were administered single-dose subcutaneous (SC) injections of 0.3 ml serum physiologic; control group animals (MTX group, n = 7) were administered single-dose SC injections of MTX (20 mg/kg); and study animals (EPO group, n = 7) were administered singledose SC injections of MTX (20 mg/kg) and EPO (5000 IU/kg, Recormon, Roche Diagnostics GmbH, Mannheim, Germany) separately.On the 5th day, animals in all groups were sacrificed by decapitation and the right lungs were removed.One halves of Kahraman et al. 3281 removed lungs were stored at -80°C until biochemical analysis and the other halves were fixed in 10% neutral-buffered formaldehyde solution for histological evaluation (Ohbayashi et al., 2010;Rubio et al., 1998;Pesce et al., 1985).

evaluation
Oxidative stress was evaluated by measuring the levels of MDA.Tissues were weighed and placed in 1.15% potassium chloride (KCl) solution, then homogenized for 30 min at 14,000 rpm.The aliquots homogenate were centrifugated at 10,000 rpm for 30 min and the supernatants were analyzed for MDA.The concentration of plasma lipid peroxidation (total MDA, expressed in nanomoles per mg protein) was measured by the Ohkawa method (Ohkawa et al., 1979).

Histopathologic evaluation
The samples were fixed in 10% neutral buffered formalin solution and embedded in paraffin.Serial sections were cut in 4 µm thick slices, stained with hematoxylin-eosin, and examined by light microscopy for the presence of tissue damage.A single pathologist examined and scored the lung sections in a blinded fashion.Five microscopy fields were evaluated for the presence of tissue congestion and inflammation.For congestion scale was scored as 0 representing no pathologic findings and 1, 2, and 3 representing pathologic findings of less than 25%, 25 to 75%, and more than 75% of the fields, respectively.For inflammation or leukocytic infiltration scale was scored as follows: 0, no extravascular leukocytes; 1, <10 leukocytes; 2, 10 to 45 leukocytes; 3, >45 leukocytes.An average of the numbers was used for comparison (Calikoglu et al., 2003).

Statistical analysis
Statistical analyses were carried out using the statistical package of SPSS 15.0 for Windows (SPSS, Inc., Chicago, IL, USA).Analysis of variance (ANOVA) was carried out on the biochemical data to examine differences among groups.When a significant group effect was found, Kruskal-Wallis followed by Mann-Whitney U tests were performed.Statistical significance was defined as P < 0.05.Tissue damage scores were compared by nonparametric analysis, and statistical significance was determined by Kruskal-Wallis followed by Mann-Whitney U test.The data were expressed as mean ± standard deviation (SD).

Biochemical
MDA levels of the sham, MTX and EPO groups were measured.MDA levels of sham group was significantly lower than those of MTX and EPO groups (both P = 0.002).MDA level in the EPO group was lower than in the MTX group, but the difference was not significant statistically (P > 0.05) (Figure 1).

Histopathological
Inflammation score (IS) and congestion score (CS) of sham, MTX and EPO groups were measured.The IS of Protein (nmol/g) Figure 1.Malondialdehyde (MDA) levels in rat lung tissue.The groups were sham, MTX (one dose methotrexate), and EPO (methotrexate plus erythropoietin given) groups.*MDA level of sham was significantly lower than those of MTX and EPO groups (P<0.05).
the sham group was significantly lower than the MTX group (P = 0.02) (Figure 2).The IS of the EPO group was lower than the MTX group, but the decrease was not statistically significant (P > 0.05).Congestions and inflammations of rat lung are shown in Figures 3 and 4.

DISCUSSION
In this study, the 5 th day effects of one dose subcutaneous administration of MTX on the rat lung were evaluated early.The effects of 5000 IU/kg dose of EPO administration on MTX-induced lung injury were also investigated.Our biochemical results suggested that subcutaneous MTX administration increases the oxidative stress marker significantly and addition of exogenous EPO decreases the oxidative stress marker, but the observed effect did not reach statistically significant level.In MTX given group, pathological investigation of inter-alveolar septa showed higher lymphocytic inflammation and congestion scores than those in MTX plus EPO given group, but the difference was not significant.These results have similarities with previous experimental studies on other tissues (Dasgupta et al., 2011;Garipardic et al., 2010).
The most common pulmonary toxicity associated with the use of MTX is hypersensitivity pneumonitis.The other pulmonary conditions are bronchiolitis obliterans with organizing pneumonia (BOOP), acute lung injury with noncardiogenic pulmonary edema, pulmonary fibrosis, and bronchitis with airway hyperreactivity (Cooper et al., 1986;Cronstein, 1996).Most researchers think that MTX generates a type IV delayed hypersensitivity pneumonitis presenting lymphocytic proliferation and alveolitis  (Chikura et al., 2008;Cooper et al., 1986).In our study, we determined that MTX increases significantly lymphocytic inflammation and congestion in histopathological examination of rat lung.Our results support the thought that MTX generates a type IV delayed hypersensitivity pneumonitis.
In the last decade, researches have shown that EPO and its receptors are expressed in tissues other than those concerned in erythropoiesis.Non-erythropoietic functions of EPO are widespread and play a role in organogenesis during early embryonic development and have effects on recovery of tissues injured by ischemia, mechanical trauma, excitotoxins, and other stressors (Sasaki et al., 2001;Sepodes et al., 2006;Vogel and Gassmann, 2011).It has been shown that EPO prevents inflammation by preserving the cellular membrane asymmetry (Maiese et al., 2005).Also in humans EPO has potential cytoprotective effects on lung tissues (Tascilar et al., 2007;Wu et al., 2006;Yildirim et al., 2005).EPO has been demonstrated to decrease lipid peroxidation levels in hypoxic-ischemic brain injury (Kumral et al., 2005) and in MTX-induced esophageal damage (Garipardic et al., 2010).In one study, acute lung injury was induced by experimental acute pancreatitis and EPO was given to measure its effect.It is found that EPO reduced the MDA levels significantly and is determined to have cytoprotective effect (Tascilar et al., 2007).In the present study, MDA level in MTX group increased significantly than in sham group.In EPO group, MDA level was lower than those in MTX group, but the difference was not significant.So we cannot conclude that EPO has anti-inflammatory effects on lung tissue.Reasons of this result may be due to be animals' low number or may be due to giving EPO in inappropriate dose or duration.
In order to protect a tissue, the required serum concentration of EPO is higher than that for erythropoiesis.Preclinical data suggest that, the minimum therapeutic level needed for tissue protection against injury appears to be 300 to 500 IU/kg body weight for the organs to be adequately investigated.In one study at a dose of 1000 IU/kg, EPO was administered and it had protective effects against acute lung injury in a rat model of acute necrotizing pancreatitis (Tascilar et al., 2007).Higher doses of EPO like 5000 IU/kg are necessary for cardioprotection and neuroprotection (Bogoyevitch, 2004).We administered EPO at a dose of 5000 IU/kg to achieve the cytoprotective effect on the lung tissue.The presence of a therapeutic window dictates specific time limitations for effectiveness of exogenous EPO as a cytoprotectant (Coleman and Brines, 2004).Therefore, we administered EPO immediately after the MTX administration.EPO treatment in dose of 5000 IU/kg or being given only once may be the reason for failure to determine the anti-inflammatory effect.
In conclusion, our results showed that EPO administration decreases the tissue inflammation and congestion scores, and the tissue MDA level, but these decreases were not statistically significant.Being statistically insignificant may be related to the dose and duration of administered EPO.Our study is the first and preliminary study in this area.Protective effects of EPO on MTXinduced lung injury should be investigated with different doses and durations and the net results should be presented.

Figure 2 .
Figure 2. Inflammation and congestion scores of all three groups were shown; *Inflammation score of sham was significantly lower than those of MTX group (P = 0.02); **Congestion score of sham was significantly lower than those of MTX group (P = 0.01).