*Efecto modulador del berro sobre el estrés oxidativo inducido por ciclofosfamida en ratones

Watercress (Nasturtium officinale, Cruciferae; W. Aiton) is a widely consumed cruciferous vegetable with nutritional and potentially chemopreventive properties. More specifically, this vegetable reduces oxidative DNA damage and modulates antioxidant enzymes in human cells, according to the results obtained in diverse in vitro and in vivo models1-4. This ability has been attributed to isothiocyanates, the main phytochemical compounds present in cruciferae. However, paradoxical effects of isothiocyanates have been reported in relation with oxidative stress and the metabolites involved5.

Cyclophosphamide (CP), a bifunctional alkylating agent, is widely used in the treatment of cancer and other conditions such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, among others. It is classified as a known human carcinogen based on the extensive evidence found not only in experimental animals but also in humans6.

CP is classified and used as a model for the study of indirect mutagens due to the fact that the molecule needs to be metabolized to generate its toxic effects. The drug is converted into products with cytotoxic and alkylating activity via the P-450-dependent mixed-function oxidase system. CP is transformed into 4-hydroxicyclophosphamide and aldophosphamide, which in turn generate phosphoramide mustard and acrolein, among other metabolites7. These metabolites interact with DNA and form adducts and cross-links that interfere with DNA replication and repair mechanisms8, 9. Likewise, CP induces oxidative stress and cell death10. Thus, it is important to highlight that the CP alkylating activity is responsible for its therapeutic activity. However, metabolites like acrolein induce oxidative stress which leads to DNA damage of normal cells and toxicity to various target organs11.

Chemoprevention is a substantial strategy to lessen or avoid damage of normal cells that inevitably occur by exposure to genotoxic and oxidative agents present in air pollutants, chemotherapy or diet. In previous studies, our group demonstrated that watercress juice seems to have a protective effect on peripheral blood lymphocytes against hydrogen peroxide-induced DNA damage in vitro12. Furthermore, we found that watercress-supplemented diet exhibited protective activity against the DNA damaging effect induced by the indirectly acting alkylating agent CP, as measured in vivo by comet and micronucleus assays13. Encouraged by these results, we designed the present study to investigate the effects of watercress on the oxidative damage induced in vivo by CP in Swiss albino mice. The experimental end points included catalase activity, superoxide dismutase activity, levels of thiobarbituric acid reactive substances as expression of lipid peroxidation, and ratio of reduced glutathione to oxidized glutathione.

Materials and methods

All chemicals were purchased from Sigma (St. Louis, MO, USA) unless otherwise indicated.

Watercress was purchased from an organic market garden located in Luján, Buenos Aires, Argentina, and voucher specimens were kept at the Museum of Pharmacobotany Juan A. Domínguez, School of Pharmacy and Biochemistry, University of Buenos Aires. The juice of watercress leaves was prepared on ice and protected from light using a commercial processor (Ultracomb). The homogenate was centrifuged at 10 000 g for 20 minutes at 4 °C. The supernatants were clarified by filtration using 0.45 μm pore membranes (Millipore) and sterilized by microfiltration using 0.22 μm pore membranes (Millipore).

Seven to eight weeks old Swiss mice (25-30 g) supplied by the Animal House of the School of Pharmacy and Biochemistry, University of Buenos Aires, were housed in plastic cages at 20-25 °C under 12 h light-dark cycles. The animals were acclimated for a week before the study onset and received food and water ad libitum through the entire experimental period. In all experiments, animal use and care were in accordance with ethical laws on animal manipulation.

A previous study from our lab showed that watercress protected against the genotoxic effects of CP, considering Nasturtium officinale human daily consumption (75-125 g)12. The dose of CP (100 mg/kg b.w.) was selected on the basis of a pilot study and reports from other groups16, 17. The doses of watercress used in this experimental design were 0.5 and 1g/kg b.w.18. The mice (n = 4 per group) were given either saline solution (groups I and II) or watercress juice (groups III and IV) by gavage for 15 consecutive days prior to the intraperitoneal administration of saline solution (I) or CP (II, III and IV). All animals were killed by cervical dislocation 24 h after CP administration.

Blood samples were collected by cardiac puncture with a heparinized syringe immediately after death. Red blood cells were separated by centrifugation at 1000 g for 15 min at 4 °C followed by removal of plasma and buffy coat. The erythrocytes were washed three times with saline solution, and stored at -20 °C until use. Hemoglobin concentration was determined with a Sysmex XS 1000i automatic hematology analyzer at 555 nm.

Bone marrow cells from both femurs of each animal were flushed in the form of fine suspension into a centrifuge tube containing RPMI 1640 medium (Gibco BRL). Cell suspensions were centrifuged at 1000 g for 10 min and pellets were lysed by two freezing-thawing cycles followed by homogenization by repeated passage through a 21G needle and centrifugation at 1000 g. Protein concentration was assayed by a colorimetric method19.

Livers were properly cleaned, freed from connective tissue, and dried on filter paper. With livers preserved at -80 °C, homogenates were prepared according to Sabatini et al.20. Briefly, livers were homogenized with 0.134 M KCl (1:5 w/v) containing 0.5 mM phenylmethylsulfonyl fluoride and 0.2 mM benzamidine (protease inhibitors) to study oxidative stress parameters. Homogenates were centrifuged at 11 000 g for 20 min. Biochemical determinations were carried out in supernatants of total homogenates and total soluble protein content was determined in each homogenate19.

For enzyme assays, erythrocytes were hemolyzed by adding ice-cold water at a 1/100 dilution; bone marrow suspensions and liver homogenates were diluted 1/10. Catalase activity was evaluated according to Aebi21. Briefly, a mixture composed of 50 mM phosphate buffer pH 7.0, 53 mM hydrogen peroxide and samples were sequentially measured at 240 nm with a Hitachi 3000 spectrophotometer for 1 min at 25 °C to assess the reduction rate of hydrogen peroxide. One unit of catalase was defined as the amount of enzyme that decomposed 1 mole of hydrogen peroxide per min. The specific activity was expressed as kU/g Hb in erythrocytes and as U/mg protein in tissues. Superoxide dismutase activity was measured using a commercial kit (SOD determination kit, Sigma Aldrich) according to the manufacturer’s instructions and expressed as percentage inhibition.

Lipid peroxidation was determined as malondialdehyde according to Ohkawa et al.22. Each sample was added to a 8% SDS solution, followed by addition of 20% acetic acid and 0.8% thiobarbituric acid. Distilled water was added to a final volume of 1 ml. Then, samples were incubated in a 100 °C water bath for 1h. The mixture was cooled and centrifuged at 10 000 g for 15 min. The supernatant was used to determine the absorbance at 532 nm with a Hitachi 3000 spectrophotometer.

Lipid peroxidation was measured as concentration of thiobarbituric acid reactive substances, calculated using the extinction coefficient 1.56 x 105/M x cm and expressed as nmol/g Hb or nmol/g protein.

The ratio of reduced glutathione to oxidized glutathione was assayed using the method described earlier23. Briefly, reduced glutathione was measured by a modification of Ellman´s procedure24 with 1.5 mM dithiobisnitrobenzoate in 0.25 M phosphate buffer (pH 8.0) and 0.1 ml of supernatant (total volume: 1.1ml). The mixture was incubated for 2 minutes at 25 °C and the absorbance was measured at 412 nm using a Hitachi 3000 spectrophotometer.

In order to estimate the total content of glutathione (GSSG + GSH) the oxidized form was reduced with glutathione reductase (EC 1.6.4.2) and 0.21 mM NADPH in 143 mM phosphate buffer plus EDTA (pH 7.5). After 30 minutes of incubation at 25 °C, the reaction was stopped with trichloroacetic acid and the supernatant was used to determine the absorbance as described above.

Results were shown as mean ± SD for all the endpoints. Differences between controls and treatments were analyzed by one-way analysis of variance and the post-hoc Student-Newman-Keuls test. A value of p < 0.05 was considered as statistically significant (Sigma Stat 9.0 software).

Results

Erythrocyte catalase did not show any significant change in its activity for any of the groups, whereas the activity of superoxide dismutase was altered in the groups treated with CP (p < 0.001) and combined treatment (p < 0.001) when compared with the control group (Tables 1 and 2). In bone marrow, CP administration increased the activity of both enzymes as compared to the control group (p < 0.001), while cress juice attenuated these effects (p < 0.001) (Tables 1 and 2). A similar pattern was observed for the activity of both antioxidant enzymes in liver tissue (catalase: p < 0.05; superoxide dismutase: p < 0.01; Tables 1 and 2). CP led to a considerable increase in the levels of thiobarbituric acid reactive substances (p < 0.001); significant restoration was observed in animals that received watercress supplementation as compared to animals that received CP alone (1 g/kg: p < 0.001; 0.5 g/kg in erythrocytes (p < 0.005), bone marrow (p < 0.001), and liver tissue (p < 0.05) (Table 3). Moreover, CP altered the ratio of reduced glutathione to oxidized glutathione and watercress supplementation restored, at least in part, the balance in blood cells and bone marrow (p < 0.05; p < 0.001 respectively) (Table 4).