Effect of Amifostine on Lung Oxidative Stress After Cyclophosphamide Therapy

Bull. Vet. Inst. Pulawy 46, 87-94, 2002

EFFECT OF AMIFOSTINE ON LUNG OXIDATIVE STRESS AFTER CYCLOPHOSPHAMIDE THERAPY

ANNA STANKIEWICZ1, ELŻBIETA SKRZYDLEWSKA1, MARIOLA SULKOWSKA2 AND STANISŁAW SULKOWSKI2

Departments of 1Analytical Chemistry and 2Pathological Anatomy, Medical Academy of Bialystok, 15-230 Bialystok, Poland

The ability of amifostine to protect healthy rat lung cells from cyclophosphamide- induced injury was evaluated. It was shown that cyclophosphamide decreased the activity of superoxide dismutase, gluthatione reductase and catalase as well as the level of reduced gluthatione, vitamin C and total antioxidant status and increased lipid peroxidation measured as malondialdehyde. Amifostine alone did not change or cause the increase in the antioxidative parameters and decrease in lipid peroxidation. Amifostine with cyclophosphamide only partially prevented changes in lung cells caused by cyclophosphamide.

Key words: rats, lungs, cyclophosphamide, amifostine, free radicals, antioxidants, lipid peroxidation.

Reactive oxygen forms play an important role in the pathogenesis of acute and chronic lung injuries. Since oxygen is indispensable for life, the cells lining the airways as well as the alveolar surface are continuously in contact with oxygen and do not seem to suffer. The partial reduction of oxygen to reactive oxygen forms during cellular metabolism, and also cellular injury of any type occurs. Among pulmonary defense cells, neutrophils, monocytes and macrophages seem to be particularly predisposed to converting molecular oxygen to reactive oxygen forms which might be related to both their phagocytosis and antimicrobial activities (1). Under physiological conditions generated reactive oxygen forms are in balance with lung antioxidants, but a number of agents, including drugs, have been recognized to potentiate reactive oxygen forms generation. Under such conditions endogenous antioxidants are not able to prevent reactive oxygen forms action and its clinical manifestations. Alkylating compounds, including antineoplastic drugs make a significant group of drugs, which disturb oxidant-antioxidant balance. One of such anticancer drug is cyclophosphamide which is used to treat many types of cancer including microcellular lung cancer (3). Cyclophosphamide is hydroxylated by the cytochrom P- 450 mixed function oxidase (MFO) system mainly in the liver or cooxidated via arachidone acid metabolism through prostaglandin H synthetase (21,22,24). The 88 activity of the enzymes involved in metabolic transformations of cyclophosphamide shows significant tissue differentiation affecting cyclophosphamide selective toxicity (5). Lack of detoxifying enzymes, aldehyde oxidase and aldehyde dehydrogenase in the lungs is a cause of selective cyclophosphamide toxicity to lung tissue. In such a situation using cytoprotective compound may be beneficial, so the aim of this study was to determine the effect of amifostine on the antioxidative system in the lung after cyclophosphamide therapy.

Material and Methods

Male Wistar rats (approximately 230 g b.w.) fed a standard diet (containing 0.55% of cysteine and methionine) were used for the experiment. The experimental protocol was approved by the Ethical Committee on Human and Animal Experimentation of our Medical Academy. The rats were divided into 4 groups: A. Control group (n=18). B. Cyclophosphamide group. The rats were treated intraperitoneally with cyclophosphamide in a dose of 150 mg/kg b.w. (n=18). C. Amifostine group. The rats received intraperitoneally amifostine in a dose of 200 mg/kg b.w. (n=18). D. Cyclophosphamide and amifostine group. The rats were treated intraperitoneally with amifostine in a dose of 200 mg/kg b.w. and subsequently with cyclophosphamide in a dose of 150 mg/kg b.w. (n=18) One, 5, and 14 days after the treatment rats were sacrified following ether anaesthesia (six animals in each group). The lungs were removed, perfused with the 0.15 M NaCl solution to remove blood cells, weighed and homogenized in 0.25 M sucrose with the addition of 6 l 250 mM BHT (butylated hydroxytoluene) in ethanol, to prevent formation of new peroxides during the assay. Homogenization procedure was performed under standardized conditions. Ten per cent homogenates were centrifugated at 10.000 x g for 15 min at 4 0C and the supernatant was kept on ice until assayed. Superoxide dismutase (Cu,Zn-SOD; EC.1.15.1.1) activity was determined by the method of Misra and Fridovich (16) modified by Sykes (25). Catalase (CAT; EC.1.11.1.9) activity was measured spectrophotometrically by the determination of H2O2 decomposition (2). Glutathione peroxidase (GSH-Px; EC.1.11.1.6) activity was measured spectrophotometrically using a technique applied by Paglia and Valentine (20). Glutathione reductase (GSSG-R; EC.1.6.4.2) activity was measured by monitoring the oxidation of NADPH at 340 nm (17). Reduced glutathione (GSH) concentration was measured using Bioxytech GSH-400 test (Bioxytech S.A., Bonneuil/Marne Cedex, France). Total antioxidant status (TAS) was measured with 2,2’-azino-di-[3- ethylbenzthiazoline] sulphonate (ABTS) using Randox test (15). Lipid peroxidation in lung was assayed as MDA by direct HPLC method (6). Statistic data were expressed as mean ± SD. One way ANOVA with Scheffe’s F test for multiple comparisons was used to determine significance between different groups. The differences were considered statistically at P< 0.05.

Results

Administration of cyclophosphamide caused a decrease in lung Cu,Zn-SOD activity, while amifostine caused an increase in its activity throughout the experiment (Table 1). After cyclophosphamide and amifostine injection, the activity of this enzyme was similar to the values of control group but was significantly increased in comparison to cyclophosphamide group. The activity of GSH-Px was significantly increased during 5 days after cyclophosphamide administration and during two weeks after amifostine injection. After both drugs injections, the GSH-Px activity was increased during 5 days. The activity of glutathione reductase was significantly decreased only after 5 days from beginning of the experiment. Amifostine caused an increase in GSSG-R activity at the beginning and a progressive decrease during the next days. The injection of cyclophosphamide and amifostine induced an increase in reductase activity during the first day. Cyclophosphamide injection caused a decrease in CAT activity observed two weeks after the onset of the experiment, while amifostine caused the increase in its activity. During 5 days after cyclophosphamide and amifostine administration, the CAT activity was higher than that in the control group, while after 5 and 14 days its activity was significantly increased in comparison to cyclophosphamide group. Administration of cyclophosphamide caused a significant decrease in GSH level 1 and 5 days after administration (Table 2). Amifostine alone did not significantly influence these parameters, while given with cyclophosphamide prevented such significant changes as cyclophosphamide alone did. The administration of cyclophosphamide caused significant decrease in vitamin C level and total antioxidant status throughout the experiment and amifostine only partially prevented this drug action. Changes in antioxidant parameters after cyclophosphamide administration were accompanied by enhanced lipid peroxidation and significant increase in the content of lipid peroxidation product - malondialdehyde. Amifostine alone decreased these parameters, while administrated with the examined antineoplastic drug caused partial prevention of its action.

Discussion

It has been observed that the administration of cyclophosphamide, an antineoplastic and immunosuppressive drug, induced lung injury as evidenced by histological findings which included endothelial cell destruction, type I and type II alveolar epithelial cell damage, alveolitis, alveolar edema, haemorrhage and extensive infiltration by inflammatory cells (9). The mechanism of cyclophosphamide mediated lung injury is not completely understood. It is suggested that the early effect of cyclophosphamide on the rat lungs is connected with acrolein generation during drug metabolism and severe inflammatory reaction connected with accumulation of neutrophils and in consequence with reactive oxygen forms generation and lipid peroxidation enhancement (22, 26).

Table 1

The activity of antioxidant enzymes in the lung of control rat (C) and rats treated with cyclophosphamide (CP), amifostine (A) and cyclophosphamide with amifostine (CP+A)

Analyzed Time C CP A CP+A enzyme

SOD 1d 4.61 ±0.27 4.02 ±0.34a 4.98 ±0.32b 4.76±0.36b (U/mg protein) 5d 4.38 ±0.25 3.91 ±0.35a 4.64 ±0.34b 4.38±0.36b a b b 14d 4.25 ±0.25 3.58 ±0.34 4.35 ±0.34 4.35±0.35 a GSH–PX 1d 65.2 4.4 70.3 5.6 68.4 5.1 75.2 5.9 9 ± ± ± ± 0

a a a (U/mg protein) 5d 63.7 ±4.1 81.2 ±6.5 73.4 ±5.7 77.1±6.1 14d 65.9 ±4.1 62.3 ±5.8 81.5 ±6.2ab 65.5±5.5c GSSG–R 1d 24.5 ±1.7 26.4 ±1.9 29.1 ±2.1ab 28.7±2.5a a b (U/mg protein) 5d 26.1 ±1.9 22.7 ±1.8 26.6 ±2.1 24.6±2.0 14d 25.0 ±1.9 24.1 ±2.0 22.2 ±1.7a 26.1±2.1c CAT 1d 47.5 ±3.1 49.5 ±3.1 59.9±4.7ab 54.3±4.4a (U/mg protein) 5d 45.3 ±3.4 44.3 ±3.8 53.3±4.5ab 55.3±4.4ab 14d 48.9 ±3.4 38.2 ±3.3a 52.4±4.2b 48.5±3.4b

Data points represent mean ± SD; n = 6; (a P< 0.05 in comparison with control group; b P< 0.05 in comparison with cyclophosphamide group; c P< 0.05 in comparison with amifostine group.

Table 2

The level of nonenzymatic antioxidants and lipid peroxidation product - malondialdehyde in the lung of control rats (C) and rats treated with cyclophosphamide (CP), amifostine (A) and cyclophosphamide with amifostine (CP+A)

Analyzed Time C CP A CP+A parameter GSH 1d 13.5±0.9 8.4±0.8a 13.2±1.0b 9.7±0.9abc (nmol/g tissue) 5d 13.1±0.9 4.2±0.4a 12.5±1.0b 8.7±0.8abc 14d 14.0±0.9 13.9±0.9 14.5±1.0 13.9±1.1 Vitamin C 1d 71±5.0 38±3.1a 68±5.3 54±4.5 (mg/g tissue) 5d 70±5.0 30±2.5a 70±6.0b 60±4.9

a b 9 14d 73±5.0 21±1.9 69±6.0 51±4.5 1

TAS 1d 2.73±0.19 1.18±0.1a 2.97±0.21b 2.07±0.19abc (nmol/g tissue) 5d 3.1±0.2 1.38±0.11a 2.75±0.2ab 2.31±0.20abc 14d 2.94±0.2 1.99±0.14a 2.90±0.22b 2.50±0.22abc MDA 1d 18.5±1.2 22.7±1.8a 18.9±1.5b 20.1±1.7 (nmol/g tissue) 5d 20.6±1.4 29.1±2.1a 18.1±1.6ab 24.9±1.9abc 14d 19.2±1.4 21.8±1.9a 17.5±1.3b 20.9±1.5ac

Data points represent mean ± SD; n = 6; (a P< 0.05 in comparison with control group; b P< 0.05 in comparison with cyclophosphamide group; c P< 0.05 in comparison with amifostine group).

The lung antioxidant system disturbances observed in response to cyclophosphamide administration are characterized by changes in enzymatic and nonenzymatic antioxidant parameters. After cyclophosphamide injection the lung superoxide dismutase, catalase and glutathione reductase activities have decreased, while glutathione peroxidase activity has increased. Similar changes have also been observed by other authors (26). The reduction activity is probably connected with damage to the structure of these proteins molecules by reactive oxygen forms or aldehydes generated during cyclophosphamide metabolism. Cellular proteins are susceptible to free radicals oxidative modification which can have a significant impact on cellular function (8). Cyclophosphamide metabolite - acrolein and lipid peroxidation product - MDA belongs to carbonyl compounds that are very reactive and may also modify amino acids of proteins causing changes in the structure and functions of enzymes (10). The reduction of Cu,Zn-SOD activity may also be due to an inhibited biosynthesis of enzyme molecules by cyclophosphamide or its metabolites and/or to the effect of hydrogen peroxide, which may directly alter its activity. On the other hand, Russel et al. (23) revealed a decrease in Mn-SOD expression in type II cells isolated from nonventilated and hypoperfused lungs. SOD selectively scavenges superoxide anion radicals and generated hydrogen peroxide is removed by the GSH-Px system (21) what suggests an important role of the NADPH-GSH-Px system in scavenging toxic forms of oxygen. Glutathione peroxidase is increased after cyclophosphamide administration but it acts in cooperation with co-substrate such as GSH whose level is decreased by reaction with acrolein (7). The removal of hydrogen peroxide is mainly performed by CAT, whose activity was reduced in this experiment. Another important redox enzyme, which catalyses the conversion of GSSG to GSH, is glutathione reductase. This enzyme contains one or more sulphydryl group residues which are essential for catalytic activity (17). In such a situation, the inactivation of glutathione reductase may be caused by free radicals as well as aldehydes. This is a reason of GSH lung deficit during cyclophosphamide administration. It is very harmful, because GSH play an important role in protecting the lung surface from oxidative attack (12). GSH reduction can explain additionally a decreased concentration of the second of the nonenzymatic antioxidant – vitamin C, which enters the cell mainly in the oxidized form where it is reduced by GSH (12). The diminution of this vitamin is very dangerous because additionally to antioxidant function vitamin C plays a role in sparing of other antioxidants (13). The above changes result in the reduction of total antioxidant status, what testifies the possibility of reactive oxygen forms reactions with cell components. Regarding the above changes in the lungs of animals treated with cyclophosphamide, it is significant to find a compound capable of protecting the healthy lung tissue against the activity of cyclophosphamide metabolites - acrolein and free radicals. Amifostine has been shown to protect normal tissues from the cytotoxic damage induced by chemo- and radiation therapy (4). It was proved that amifostine shows protective action in cases of administration of another antineoplastic drug - bleomycin which induced lung injury (19). The present study has demonstrated that amifostine also partially prevents lung injury observed after cyclophosphamide administration. Being an organic thiophosphate amifostine is an inactive predrug (4). The active sulphydryl compound (WR-1065) is generated by dephosforylation cell

93 membrane-bound by alkaline phosphatase and incorporated into normal tissues where it exerts its cytoprotective role. Cytoprotective level of amifostine or its metabolites was found in many organs including the lungs (4). Amifostine active metabolite (WR-1065) has free sulphydryl group so it can react with electrophillic compounds preventing cyclophosphamide metabolite reaction with such important cell components as GSH, proteins, lipids and DNA (4). Moreover, an in vitro study demonstrated that the active metabolite of amifostine, W-1065, was able to scavenge superoxide anions and peroxyl radicals and especially strong hydroxyl radicals (14,18). It is very important because hydroxyl radical is an aggressive form, reacting with extremely high rate constants with nearly every type of cell molecule and probably with disulfide compounds such as amifostine. The known preferential activity of amifostine as a selective scavenger of hydroxyl radicals appears to be the key element in the drug’s efficacy, since hydroxyl radicals are surely the most dangerous free radicals from a biological view point. Indeed, they are extremely aggressive against cell structures and may both initiate and next self-propagate cell damage while superoxide dismutase seems to play a secondary role (11). Free radicals demonstrate, however, destructive activity in cases of antioxidative system insufficiency. It is evident from the study that amifostine administration reduces changes caused probably by cyclophosphamide metabolites - electrophilic compounds such as acrolein and free radicals - mainly due to easy access to free sulphydryl group. Reacting with amifostine these compounds attack cell components to a lesser degree. In conclusion, the results indicate that amifostine partially protects healthy lung cells against cyclophosphamide injury. It can be expected that administration of cyclophosphamide together with amifostine will not change antineoplastic activity of the drug simultaneously alleviating the side effects. These results seem to be particularly important for their clinical purposes and for the possibility to use them in chemotherapy.

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Corresponding author: Elżbieta Skrzydlewska Department of Analytical Chemistry Medical Academy of Białystok 15–230 Białystok 8, P. O. Box 14 Poland tel/fax.: (48–085)7447376 e–mail: [email protected]łystok.pl.