Effects of morin-5′-sulfonic acid sodium salt (NaMSA) on cyclophosphamide-induced changes in oxido-redox state in rat liver and kidney

Abstract

Cyclophosphamide (CPX) is an anticancer drug with immunosuppressive properties. Its adverse effects are partly connected to the induction of oxidative stress. Some studies indicate that water-soluble derivative of morin—morin-5′-sulfonic acid sodium salt (NaMSA) exhibits strong antioxidant activity. The aim of present study was to evaluate the effect of NaMSA on CPX-induced changes in oxido-redox state in rat. Experiment was carried out on Wistar rats divided in three experimental groups (N = 12) receiving: 0.9% saline, CPX (15 mg/kg) or CPX (15 mg/kg) + NaMSA (100 mg/kg), respectively, and were given intragastrically for 10 days. Malondialdehyde (MDA) and glutathione (GSH) concentrations and superoxide dismutase (SOD) activity were determined in liver and kidneys. Catalase (CAT) activity was assessed only in liver. Treatment with CPX resulted in significant decrease in MDA level in both tissues, which was completely reversed by NaMSA treatment only in liver. In comparison to the control group significant decrease in SOD activity were observed in both tissues of CPX receiving group. In kidneys this parameter was fully restored by NaMSA administration. CPX evoked significant decrease in GSH concentration in kidneys, which was completely reversed by NaMSA treatment. No significant changes were seen in GSH levels and CAT activity between all groups in liver. Results of our study suggest that CPX may exert significant impact on oxido-redox state in both organs. NaMSA fully reversed the CPX-induced changes, especially MDA level in liver, SOD activity and GSH concentration in kidneys and it may be done by enhancement of activity/concentration of endogenous antioxidants.

Introduction

Cyclophosphamide (CPX) is an anticancer agent with strong immunosuppressive properties. It belongs to alkylating agents acting by cross-linking the strands of DNA. CPX is useful in the treatment of some cancers, e.g. breast cancer, leukemias or lymphomas. It also possesses immunosuppressive properties; therefore, it is important in the therapy of some autoimmune diseases such as rheumatoid arthritis or lupus erythematosus. ¹ The long-term use of CPX is however limited by its adverse effects which, at least partly, are connected to the induction of oxidative stress and neutrophil infiltration in tissues. ² It was shown in many studies that CPX induced marked oxidative stress in tissues as shown by increased lipid peroxides (LPOs) ³ or malondialdehyde (MDA) ^{4 –6} levels together with decreased antioxidants activities or concentrations. In many studies, CPX decreased superoxide dismutase (SOD) ^{7 –9} as well as glutathione (GSH) concentration. ^3,6

There is a great necessity to find new, nontoxic substances decreasing CPX-induced adverse effects, especially hemorrhagic cystitis or hematological toxicity. Such substances may allow administering CPX for longer time with minimized adverse effects.

A wide variety of natural compounds with postulated antioxidant activity have been tested for their beneficial effect in CPX or ifosfamide-induced toxicity. Most experiments concerned treatment or prophylaxis of cystitis. ^{10 –12} Flavonoids, available from e.g. fruits and vegetables, inhibit enzymes involved in superoxide anion production. ^13,14 Some of them may also act as peroxynitrite scavengers. ¹⁵ Different studies have shown that substances belonging to polyhydroxyflavones, e.g. quercetin or morin, exhibit strong antioxidant properties. Their water-soluble derivatives, such as morin-5′-sulfonic acid sodium salt (NaMSA), exert low toxicity ¹⁶ and may be potent in reducing CPX-induced toxicity. In our earlier works we have demonstrated protective effects of NaMSA in Cd-intoxicated mice. NaMSA protected from Cd-induced body weight loss and cadmium accumulation in mice liver and kidney. NaMSA also reversed increased LPOs level, prevented from decrease in the GSH level and restored decreased SOD activity in mice liver. ^17,18 NaMSA was also effective as an antidote in acute chromium intoxication in rats ¹⁶ and in acute inorganic mercury poisoning in rats. ¹⁹

Because of the demonstrated antioxidant properties of NaMSA, low toxicity of the substance and effectiveness in acute or subacute intoxications with heavy metals, the aim of the present study was to evaluate the effect of NaMSA on CPX-induced changes in oxido-redox state in rat liver and kidney. It may be useful as a protective factor during long-term treatment with CPX, e.g. in autoimmune disorders, because at least some of the CPX-induced adverse effects may be reactive oxygen species (ROS) dependent.

Methods

Animals

Experiment was carried out on Wistar rats of both sexes (203.9 ± 18.6 g) obtained from the Animal Laboratory of the Department of Pathological Anatomy, Wroclaw Medical University. The animals were housed individually in chambers with a 12:12 h light–dark cycle and the temperature was maintained between 21°C and 23°C. Before and during the experiment, animals had free access to standard food and water. The experiment was performed with the consent of the Local Ethics Commission for Experiments on Animals in Wroclaw.

Chemicals

Cyclophosphamide (Sigma, Germany), 0.9% NaCl solution (Polpharma S.A., Poland) and thiopental (amp. 0.5 g, Biochemie, Austria) were used in the study. NaMSA was synthesized in the Department of Inorganic and Analytical Chemistry, University of Technology in Rzeszow, Poland, according to the methods described previously. ²⁰ The purity of the obtained compound was checked with thin-layer chromatography. It was demonstrated that NaMSA was homogenous substance and did not contain any untransformed substrates. Spectrophotometric characteristic of NaMSA was found to be consistent with the literature data. ²⁰ NaMSA is easily soluble in water and retains the properties of the parent compounds. Sulfonic morin derivative can be considered to be multiprotonic acids, which dissociate in aqueous solutions yielding respective anions. ^20,21

Experiment

Experimental animals were randomly divided into three experimental groups (N = 12): group C—receiving 0.9% saline solution at 9 a.m. and at 2 p.m.; group CX—receiving CPX at a dose of 15 mg/kg at 9 a.m. and 0.9% saline solution at 2 p.m. and group M-CX—receiving CPX at a dose of 15 mg/kg at 9 a.m. and NaMSA at a dose of 100 mg/kg at 2 p.m. All studied substances were dissolved in 0.9% saline solution in a 4 ml/kg volume and were given intragastrically for 10 consecutive days. Saline solution was also given in a 4 ml/kg volume. On 11th day of the experiment, the animals were killed in deep anesthesia (thiopental intraperitoneal (i.p.) injection, 70 mg/kg). Liver and kidney were homogenized on ice, using lysis buffer (140 mM NaCl, 10 mM ethylenediaminetetraacetic acid, 10% glycerol, 1% NP40, 20 mM Tris base, pH 7.5) and centrifuged at 14,000 r/min for 25 min at 4°C. In the obtained supernatants, MDA and GSH concentrations as well as SOD activity were determined in both liver and kidney. Catalase (CAT) activity was determined only in liver because of the scant amount of tissue homogenates obtained from kidney.

Measurements of the oxido-redox state

All oxido-redox parameters were assessed spectrophotometrically (MARCEL S350 PRO spectrophotometer).

MDA was assayed using BIOXYTECH-MDA-586 kit (OxisResearch, USA) according to manufacturer’s instruction and its level was expressed as μmol/ml.

SOD activity was assayed using Ransod kit (Randox Laboratories, UK), according to manufacturer’s instruction and its activity was expressed as U/mg of protein.

GSH concentration was assayed using BIOXYTECH GSH-400 (OxisResearch, USA), according to manufacturer’s instruction and its level was expressed as μM.

CAT activity was determined following decreases in initial H₂O₂ concentration (30 mM used as initial substrate) at 240 nm and 25°C over the time frame of 60 s, according to the procedure published by Johanson and Borg. ²² Briefly, 100 μl of supernatant isolated from rat liver homogenates was placed in a cuvette and diluted to a final volume of 2 ml with phosphate-buffered saline (50 mM). The decrease in the absorption at 240 nm 60 s after adding 1 ml H₂O₂ was recorded. One unit of CAT was defined as the amount of enzyme that degraded 1 μl H₂O₂ per minute. Values were expressed as U/mg of protein.

Total protein concentrations in supernatants of homogenates were assayed with commercial methods in a certified laboratory on the Dimension RxL-Max apparatus on Flex kit. Briefly, cuprum cation interacts with peptide bond in protein in alkaline solution; amount of Cu(II) complex with blue color, proportional to protein concentration, is measured using bichromatic technique of final point assessment.

Statistical analysis

Data were expressed as mean values ± standard deviation (SD). Statistical analysis of the effect of the drug on antioxidant enzymes activities (SOD and CAT) and MDA and GSH concentration were performed using two-way analysis of variance. Specific comparisons were made with Tukey’s test. Hypotheses were considered positively verified if p < 0.05.

Results

MDA concentration in liver was significantly lower in group receiving CPX than in control group (p < 0.05), and this action was completely reversed by the addition of NaMSA (group M-CX vs. CX, p < 0.005). No significant changes in MDA concentration between M-CX and control groups were found (M-CX vs. C, p = NS (nonsignificant)). Similar changes were observed in kidney. CPX induced significant decrease in MDA level (CX vs. C, p < 0.001), but this was only partly reversed by NaMSA treatment (M-CX vs. CX and M-CX vs. C, p = NS in both cases; Figure 1).

Figure 1.

Effects of cyclophosphamide (CPX) and morin-5′-sulfonic acid sodium salt (NaMSA) on malondialdehyde (MDA) concentrations in rat liver and kidney. Data are presented as mean values ± SD. C: control group; CX: cyclophosphamide 15 mg/kg, M-CX: cyclophosphamide 15 mg/kg and NaMSA 100 mg/kg. *Significant difference between group C and CX. **Significant difference between group CX and M-CX. Detailed p values are presented in Results section.

In comparison to the control group, CPX caused significant decrease in SOD activity in rat liver (C vs. CX, p < 0.01) what was only partly restored by NaMSA coadministration (M-CX vs. CX and M-CX vs. C, p = NS in both cases). In rat kidney, changes in SOD activity were even more pronounced. CPX significantly decreased SOD activity comparing to the control group (CX vs. C, p < 0.001) whereas NaMSA fully prevented CPX-induced changes in rat kidney (M-CX vs. CX, p < 0.001, M-CX vs. C, p = NS; Figure 2).

Figure 2.

Effects of cyclophosphamide (CPX) and morin-5′-sulfonic acid sodium salt (NaMSA) on superoxide dismutase (SOD) activity in rat liver and kidney. Data are presented as mean values ± SD. C: control group; CX: cyclophosphamide 15 mg/kg, M-CX: cyclophosphamide 15 mg/kg and NaMSA 100 mg/kg. *Significant difference between group C and CX. **Significant difference between group CX and M-CX. Detailed p values are presented in Results section.

In kidneys, CPX evoked significant decrease in GSH concentration (C vs. CX, p < 0.01). This action of CPX was fully reversed by NaMSA treatment (M-CX vs. CX, p < 0.05 and M-CX vs. C, p = NS). No significant changes were seen in GSH levels between all groups in liver. However, CPX alone caused slight decrease in GSH concentration comparing to control group but GSH level in the group receiving both CPX and NaMSA was insignificantly higher than in CX and C group (Figure 3).

Figure 3.

Effects of cyclophosphamide (CPX) and morin-5′-sulfonic acid sodium salt (NaMSA) on glutathione (GSH) concentrations in rat liver and kidney. Data are presented as mean values ± SD. C: control group; CX: cyclophosphamide 15 mg/kg, M-CX: cyclophosphamide 15 mg/kg and NaMSA 100 mg/kg. *Significant difference between group C and CX. **Significant difference between group CX and M-CX. Detailed p values are presented in Results section.

The highest CAT activity in liver was observed in M-CX group (0.101 ± 0.042 U/mg of protein) and the lowest in CX group (0.085 ± 0.023 U/mg of protein). In the control group, CAT activity was 0.091 ± 0.021 U/mg of protein. No significant differences were found between all examined groups (p = NS in all comparisons).

Discussion

In most of experimental studies CPX caused significant increase in MDA concentration, which confirmed its pro-oxidative action. ^4,5,23,24 Overproduction of reactive nitrogen and ROS leads to the induction of oxidative stress in tissues, cellular injury and necrosis and may contribute to the pathogenesis of some adverse effects of CPX, especially cystitis. ²⁵

In our study, we did not notice increased MDA concentrations in examined tissues. Contrary, significant decrease in MDA level in the group receiving CPX when compared to the control group was observed in both liver and kidneys. Concomitant administration of NaMSA reversed these CPX-induced changes completely in liver and only partly in kidneys.

Such important differences may result from different experimental schedules used in our study, when compared to other authors’ works. In most of experimental models on rats, CPX is used as i.p. injection of high single dose (100–200 mg/kg), which causes quickly marked adverse effects, mainly acute hemorrhagic cystitis. ^2,6,26,27 It probably causes acute oxidative stress resulting in increased MDA level as a consequence of augmented lipid peroxidation. However, in our work the total cumulative dose of CPX was comparable (150 mg/kg) to other studies but was administered in different schedules (15 mg/kg/day, during 10 consecutive days). CPX administered in lower daily doses are rather used in the treatment of autoimmune disorders than in anticancer therapy. In experimental studies, there are only single evidences that administration of CPX led to the decrease in MDA concentration, instead of its increase. Askar et al ²⁸ demonstrated that CPX may exert protective effect in ischemia–reperfusion (I/R) injury in epigastric island skin flaps. In this experiment, CPX administered 3 times in a daily dose of 15 mg/kg decreased MDA concentration in CPX-receiving group compared to the control group submitted only to I/R injury. It could not be excluded that in conditions of increased oxidative stress such as I/R model, CPX may exert protective action, resulting from e.g. decreased neutrophils accumulation. However, it was also noticed that single injection of CPX in a dose of 150 mg/kg did not alter MDA concentration in rat liver 6, 16 or 24 h after CPX administration, ²⁹ but increased MDA level in kidneys 16 hours after exposure to CPX in a similar model of experiment. ²³

Moreover, in our work we showed that coadministration of NaMSA with CPX caused reversion of CPX action. Mean values of MDA concentration in a group receiving CPX with NaMSA was not significantly different from mean values obtained in control group both in liver and in kidneys. Such action of NaMSA is difficult to explain, because natural flavonoids as well as their synthetic derivatives are considered as antioxidative compounds. ^12,17,18 In our earlier work, NaMSA given alone did not influence oxido-redox state parameters in mice but reversed unfavorable changes in LPOs level caused by subacute cadmium intoxication. ¹⁷ Single reports also indicated antiperoxidative potential of morin on CPX/flutamide-induced lipid peroxidation in rabbits confirmed by MDA level. ³⁰

Influence of CPX on SOD activity was consistent with the results obtained in other studies. ^{24,31 –33} In both examined organs significant decrease in SOD activity in CPX-receiving group was noticed which was fully (kidneys) or partly (liver) reversed by concomitant NaMSA administration. It may confirm the protective antioxidant effect of NaMSA in our model of CPX intoxication. It is also consistent with our earlier observations from subacute cadmium intoxication model in mice. ¹⁸ Little is known about the influence of other flavonoids on SOD activity during CPX treatment. In one study in breast cancer-bearing rats, treatment with luteolin and CPX caused increase in liver SOD activity compared to the cancer-bearing animals (breast cancer control) and it was very similar to the normal control (without tumor). However, in that experiment high activity of SOD was also observed in the group of rats with breast tumor treated only with CPX. Similar changes in SOD activity was also observed in kidneys. ³⁴

In case of GSH concentration, significant influence of CPX administration was observed only in kidneys (in liver such action was insignificant), which may suggest the depletion of endogenous GSH supplies caused by CPX. This is concordant with other data. ^24,31,35 Concomitant administration of NaMSA with CPX fully reversed CPX-induced GSH depletion in kidneys. It may also provide evidences for antioxidative action of NaMSA. Similar effect of NaMSA administration on decreased GSH level in mice liver was observed in subacute cadmium poisoning described in our previous work. ¹⁸ Moreover, in earlier experiment of Ray et al. ³⁰ antioxidant action of naturally occurring morin was revealed in CPX/flutamide-induced lipid peroxidation. Flavonoid constituents of citrus extract (Citrus aurantium var. amara) may restore the GSH level to normal in CPX-induced stress in mice liver. ³⁶ Also other flavonoids (diosmine and quercetin under shape of propolis) may decrease CPX-induced increased MDA concentration as well as CPX-induced GSH depletion in rat liver. ⁵ Some limitation of our study was evaluation of pro- and antioxidant parameters only in one end point of the study which did not reveal the dynamic changes during the longer experiment period.

In different experimental models, decrease in CAT activity was the most often observed change after CPX administration in both liver and kidney. ^31,32,35,37 However, impact of CPX on CAT activity may depend on the studied tissue. ³ Samy et al. demonstrated that in breast cancer rat model the CAT activity in liver and kidney in groups receiving CPX was higher than in tumor-bearing animals without any treatment and was similar to the values observed in the group of rats without tumor. ³⁴ In our experimental model, administration of CPX at a daily dose of 15 mg/kg for 10 days insignificantly decreased CAT activity and NaMSA did not modify significantly its activity. Because CAT activity was not determined in kidneys, it is difficult to predict the effect of CPX and NaMSA on CAT activity in this organ in our model. The differences observed in the impact of CPX on CAT activity between our study and previously cited studies may result from different experimental protocol.

Results of our study suggest that in both organs (liver and kidney) significant impact of CPX of selected parameters of oxido-redox state was observed. NaMSA fully reversed CPX-induced changes, the most pronounced in case of MDA level in liver, SOD activity in kidney and GSH level in kidney. NaMSA may decrease harmful effect of CX on oxido-redox state by enhancement of activity/concentration of endogenous antioxidants. Further studies are required to assess the influence of CPX on lipid peroxidation, because of unexpected results obtained with MDA liver and kidney concentration. In our experiment, the observation that changes in oxido-redox state may influence the tissue protection of NaMSA in CPX-induced toxicity also requires further evaluation. Our experiment may reflect this stage/moment of the CPX action when lipid peroxidation (expressed as MDA concentration) is not yet observed, but depletion of some endogenous enzymatic as well as nonenzymatic antioxidants are already noticed.