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DDAH pathway in rats
Pharmacological Reports 2013, 65, 201207 ISSN 1734-1140
Copyright © 2013 by Institute of Pharmacology Polish Academy of Sciences
Short communication
Effect of cyclophosphamide and morin-5’-sulfonic acid sodium salt, alone or in combination, on ADMA/DDAH pathway in rats Anna Merwid-L¹d1, Ma³gorzata Trocha1, Ewa Chlebda-Sieragowska1, Tomasz Sozañski1, Jan Magdalan1, Dorota Ksi¹dzyna1, Andrzej Szuba2, Maria Kopacz3, Anna KuŸniar3, Dorota Nowak3, Ma³gorzata Pieœniewska1, Lidia Fereniec-Go³êbiewska1, Adam Szel¹g1
Department of Pharmacology, Wroclaw Medical University, Mikulicza-Radeckiego 2, PL 50-345 Wroc³aw, Poland
Department and Clinic of Internal Medicine and Occupational Diseases, and Hypertension, Wroclaw Medical University, Pasteura 4, PL 50-367 Wroc³aw, Poland !
Department of Inorganic and Analytical Chemistry, Chemical Faculty, Rzeszow University of Technology, Powstañców Warszawy 6, PL 35-959 Rzeszów, Poland Correspondence: Anna Merwid-L¹d, e-mail: [email protected]
Abstract: Background: The aim of the study was to evaluate the effect of cyclophosphamide (CPX) and morin-5’-sulfonic acid sodium salt (NaMSA) on plasma asymmetric dimethylarginine (ADMA) level and dimethylarginine dimethylaminohydrolase (DDAH) activity in rat liver. Methods: The study was performed on Wistar rats receiving normal saline, CPX (15 mg/kg/day), NaMSA (100 mg/kg/day) or both CPX and NaMSA for 10 consecutive days. Results: Significant decrease in ADMA level was found in all groups when compared to the control. DDAH activity in the liver was significantly higher in CX group compared to the control group. Conclusion: Obtained results of ADMA/DDAH pathway parameters require further research. Key words: cyclophosphamide, morin-5’-sulfonic acid sodium salt (NaMSA), ADMA, DDAH, rats
Abbreviations: ADMA – asymmetric dimethylarginine, CAT – catalase, CPX – cyclophosphamide, DDAH – dimethylarginine dimethylaminohydrolase, eNOS – endothelial nitric oxide synthase, GPx – glutathione peroxidase, GSH – glutathione, iNOS – inducible nitric oxide synthase, HPLC – high-performance liquid chromatography, MDA – malondialdehyde, NaMSA – morin-5’-sulfonic acid sodium salt, NO – nitric oxide, NOS – nitric oxide synthase, ROS – reactive oxygen species, SOD – superoxide dismutase
Introduction Asymmetric dimethylarginine (ADMA) is a very well known risk factor of cardiovascular complications. ADMA is an endogenous inhibitor of all isoforms of nitric oxide synthase (NOS), but especially of the endothelial isoform of NOS (eNOS) [18]. DimethylPharmacological Reports, 2013, 65, 201207
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arginine dimethylaminohydrolase (DDAH) is an enzyme responsible for the metabolism of ADMA. It is mainly located in the liver but it can also be found in other organs such as kidneys, brain, adrenal glands, pancreas or testes [17, 22]. In case of oxidative stress DDAH activity may be reduced, causing subsequent increase in ADMA level. It was demonstrated that some antioxidants can preserve DDAH activity, which indicates that DDAH activity could be modulated by reactive oxygen species (ROS) [17]. Cyclophosphamide (CPX) is a very well-known anticancer and immunosuppressive drug. Although cardiotoxicity is not the main adverse effect of CPX, sinus tachycardia, cardiomyopathy and heart failure are also noticed following administration of CPX. More rarely, ventricular or supraventricular tachyarrhythmias (including atrial or ventricular fibrillation), angina, myocardial infarction, myocarditis, pericarditis or even cardiac arrest were reported [4]. An increased level of nitric oxide (NO), produced by inducible form of NO synthase (iNOS) in tissues may be an important reason of some severe adverse reactions. Involvement of NO in pathogenesis of those adverse effects of CPX is quite well confirmed [1, 16], but still little is known about the impact of CPX on eNOS activity and/or expression or the influence of CPX on ADMA level as well as on DDAH activity. Various studies have shown that substances belonging to flavonoids exhibit strong antioxidant properties. They may also influence the NO system. Quercetin inhibits iNOS mRNA and protein expression. The compound is also a potent vasodilator in eNOS-dependent manner [15, 25]. Less data are available for water-soluble derivatives of flavonoids, such as morin-5’-sulfonic acid sodium salt (NaMSA). It was demonstrated that those compounds exerted low toxicity, possessed antioxidant properties and might be effective as antidotes in some heavy metals intoxications [3, 12]. However, their influence on ADMA concentration and/or DDAH activity as well as impact on NOS activity is not known. The aim of the study was to evaluate the effect of CPX and NaMSA, given alone or in combination, on plasma ADMA levels and DDAH activity in rat liver.
210.8 ± 16.7 g) obtained from the Animal Laboratory of the Department of Pathological Anatomy, Wroclaw Medical University, Poland. Animals were housed individually in chambers with a 12:12 h light-dark cycle and the ambient temperature was maintained between 21 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 I Local Ethics Commission for Experiments on Animals in Wroc³aw. Chemicals
The following chemicals were used in the study: cyclophosphamide (Sigma, Germany), 0.9% NaCl solution (amp. 10 ml, Polpharma S.A., Poland) and thiopental (amp. 0.5 g, Biochemie, Austria). NaMSA was synthesized in the Department of Inorganic and Analytical Chemistry, University of Technology in Rzeszów, Poland, according to the methods described previously. NaMSA is easily soluble in water and keeps properties of the parent compound [9, 10].
Experiment
Experimental animals were randomly divided into four experimental groups (n = 12 each, 6 females and 6 males): group C – receiving 0.9% saline solution at 9 a.m. and 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.; group M – receiving 0.9% saline solution at 9 a.m. and NaMSA at a dose of 100 mg/kg 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 (4 ml/kg) and were given intragastrically by a tube for 10 consecutive days. Saline solution (4 ml/kg) was also given by the same route. On the 11 day of the experiment, blood samples from the tail vein were collected and thereafter animals were sacrificed in a deep anesthesia with intraperitoneal thiopental injection (70 mg/kg). Half of the liver and one kidney were homogenized on ice, using a lysis buffer (140 mM NaCl, 10 mM EDTA, 10% glycerol, 1% NP40, 20 mM Tris base, pH 7.5) and centrifuged at 14,000 rpm for 25 min at 4°C. DDAH activity was measured in the obtained supernatants. JD
Materials and Methods Animals
Experiment was carried out on Wistar rats of both sexes (203.5 ± 17.6 g; females: 196.3 ± 15.6 g; males:
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Impact of cyclophosphamide and NaMSA on ADMA/DDAH pathway Anna Merwid-L¹d et al.
Measurements of ADMA concentration
ADMA concentration was measured by high-performance liquid chromatography (HPLC) with fluorescence detection according to the method described in our previous paper [21]. Measurements of DDAH activity
DDAH activity was measured spectrophotometrically according to the method of Tain and Baylis [19], adopted to the macromethod for spectrophotometer MARCEL S350 PRO (Marcel Sp. z o.o., Poland). The method is based on the rate of L-citrulline production. Briefly, liver tissue homogenates were mixed with phosphate buffer, pH = 6.5. One mM ADMA was added to the samples and samples were incubated at 37°C for 45 min. After the reaction was stopped with 4% sulfosalicylic acid, samples were centrifuged and oxime reagent (diacetyl monoxime (0.08% w/v) in 5% acetic acid) mixed with antipyrine/H SO" (antipyrine (0.5% w/v) in 50% sulfuric acid) reagent was added. Samples were thereafter incubated at 60°C for 110 min and cooled in ice bath for 10 min. L-citrulline formation was measured at 466 nm and values were subtracted of respective blanks (without ADMA). Standard was prepared as serial dilutions of L-citrulline. DDAH activity was represented as μM L-citrulline formation/g protein/min at 37°C.
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 the mean values ± standard deviation (SD). Statistical analysis of the effect of CPX and NaMSA on ADMA concentration and DDAH activity in the liver and kidney were performed using analysis of variance (ANOVA). Specific comparisons were made with Tukey’s test for the whole population and in the female and male subgroups. Hypotheses were considered positively verified if p < 0.05.
Results ADMA level was significantly lower in the group receiving CPX comparing to the control group and was the lowest in all experimental groups. NaMSA also
Fig. 1. Plasma asymmetric dimethylarginine (ADMA) concentration (μM) and liver dimethylarginine dimethylaminohydrolase (DDAH) activity (μM L-citrulline/mg of protein/min). C – control group, CX – group receiving CPX (15 mg/kg), M – group receiving NaMSA (100 mg/kg), group M-CX – group receiving CPX (15 mg/kg) and NaMSA (100 mg/kg). Data are expressed as the mean ± SD. * p < 0.001 vs. C, ** p < 0.01 vs. C, # p < 0.001 vs. CX, ## p < 0.005 vs. M-CX, $ p < 0.05 vs. C
Plasma ADMA concentration [μM] and liver DDAH activity [μM L-citrulline/g protein/min]
1.6
**
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## #
1.4
CX
M
M-CX
1.2
$
1
*
*
0.8
0.6
0.4
0.2
0
C
CX
M
M-CX
plasma ADMA level
C
CX
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liver DDAH activity
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significantly decreased ADMA concentration compared to the control group. Significant decrease in ADMA level was observed in the group receiving both studied substances and was significantly different from the control group and from the group receiving NaMSA only, but not significantly different from the CPX-receiving group (Fig. 1). In the subgroup of female rats ADMA level was also the lowest in the group receiving CPX and significantly different from the level reported for the control group. Administration of NaMSA alone or of both examined substances (CPX and NaMSA) similarly significantly reduced the level of ADMA in comparison to the control group. In the male subgroup of rats AMDA concentrations were significantly decreased when compared to the control group in CPX-receiving group and in the group receiving both studied substances, but not in the NaMSA-receiving group. Therefore, significant difference was also noticed between M-CXM and MM group. Mean values of ADMA concentrations and detailed comparisons between studied groups are presented in Table 1. Liver DDAH activity was the highest in the group receiving CPX and was significantly higher than in the control group. In the group receiving NaMSA, DDAH activity was also increased but the difference in comparison to the control group was insignificant. Administration of both substances did not significantly changed DDAH activity comparing to the control group (Fig. 1).
When female and male subgroups of rats were analyzed no significant differences in DDAH activity were observed. However, the tendency observed was exactly the same as for the whole population and the highest activity of DDAH was measured in CPXreceiving groups, both in female and male rats. Mean values of DDAH activities and detailed comparisons between studied groups are presented in Table 1.
Discussion It was demonstrated in the experimental studies that CPX administration led to oxidative stress measured as malondialdehyde (MDA) concentration as the indicator of lipid peroxidation [11]. The effect was the most pronounced following administration of large single doses of CPX [1, 11]. Overproduction of ROS and reactive nitrogen leads to the induction of oxidative stress in cells and tissues, DNA injury, cellular damage and necrosis. Moreover, it may contribute to the pathogenesis of some adverse effects of cyclophosphamide, e.g., hemorrhagic cystitis [24]. Cyclophosphamide may also induce changes in endogenous antioxidant system including superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPx) activities and glutathione (GSH) level with SOD as one of the most powerful antioxidant in the
Tab. 1. Plasma asymmetric dimethylarginine (ADMA) concentrations (μM) and liver dimethylarginine dimethylaminohydrolase (DDAH) activities (μM L-citrulline/mg of protein/min) in female and male rats. CF and CM – control group; female and male, respectively, CXF and CXM – group receiving CPX (15 mg/kg); female and male, respectively, MF and MM – group receiving NaMSA (100 mg/kg); female and male, respectively, group M-CXF and M-CXM – group receiving CPX (15 mg/kg) and NaMSA (100 mg/kg); female and male, respectively. Data are expressed as the mean ± SD
Plasma ADMA concentration (μM)
p
Liver DDAH activity (μM L-citrulline/mg of protein/min)
p
Female (CF)
1.291 ± 0.235
–
0.377 ± 0.100
–
Male (CM)
1.168 ± 0.045
–
0.592 ± 0.201
–
Group
Sex
Control group (C) CPX group (CX) NaMSA group (M)
CPX with NaMSA group (M-CX)
204
Female (CXF)
0.688 ± 0.109
p < 0.001 vs. C
0.578 ± 0.222
–
Male (CXM)
0.770 ± 0.117
p < 0.001, vs. CM
0.819 ± 0.291
–
Female (MF)
0.920 ± 0.153
p < 0.005, vs. CF
0.550 ± 0.068
–
Male (MM)
1.151 ± 0.089
p < 0.001 vs. CXM
0.638 ± 0.133
–
Female (M-CXF)
0.805 ± 0.085
p < 0.001 vs. CF
0.448 ± 0.119
–
Male (M-CXM)
0.811 ± 0.081
p < 0.001, vs. CM p < 0.001 vs. MM
0.559 ± 0.183
–
Pharmacological Reports, 2013, 65, 201207
Impact of cyclophosphamide and NaMSA on ADMA/DDAH pathway Anna Merwid-L¹d et al.
cells [11]. In our previous work, we have documented that treatment with CPX in rats resulted in significant decrease in SOD activity and GSH concentration but with no impact on CAT activity [13]. Oxidative stress may subsequently cause decrease in DDAH activity and elevation in ADMA concentration [17, 22]. Considering those facts, increased ADMA concentration along with decreased DDAH activity in the CPXreceiving group was expected. Results obtained in our experimental model were quite opposite in this group. Some authors observed that administration of small doses of CPX (15 mg/kg, 3 times during the study) led rather to decrease than elevation in MDA level [2]. Also in our experimental model of 10 day administration of CPX in a daily dose of 15 mg/kg we did not observe any increase in MDA concentration in CPX-receiving group. We even noticed a significant decrease in MDA level comparing to the control group [13]. It may, at least partly, explain significant decrease in ADMA level and increase in DDAH activity in the liver observed in this study. Because published data concerning the impact of CPX or other cytotoxic and immunosuppressive agents are very scarce, it is difficult to compare results of our study with the results obtained by other authors. Single data show that immunosuppressive therapy may lead to the decrease of elevated ADMA concentration in e.g., patients after kidney transplantation [5]. Influence of flavonoids on ADMA level and/or DDAH activity has been also the subject of many studies. It was demonstrated that various antioxidants caused decrease in ADMA level by preserving DDAH activity. Such effects were demonstrated for both plant-derived and synthetic compounds [22]. In our study, NaMSA, a water soluble derivative of morin, significantly decreased plasma ADMA concentration comparing to the control group, but an increase in DDAH activity in the liver was insignificant. Data concerning effects of flavonoids on ADMA/DDAH are also limited, but it was shown that quercetin decreased ADMA level in healthy volunteers when administered twice a day in a 500 mg dose for 4 weeks [14]. Kaempferol treatment decreased ADMA plasma concentrations and increased DDAH activity in apolipoprotein E-deficient mice [26]. Another flavonoid – epigallocatechin gallate significantly decreased the level of ADMA and increased the activity of DDAH caused by oxidized LDL in endothelial cells [20]. Results of our study are consistent with the results obtained with different flavonoids by other researchers.
It was also reported that other agents belonging to isoflavones e.g., puerarin extracted from Radix puerariae may inhibit LPS-induced iNOS expression by suppression of NF-kB activation in macrophages [7]. Combination of both substances (CPX and NaMSA) caused significant decrease in ADMA level comparing to the control group and the group receiving NaMSA. Mean ADMA concentration in M-CX group was not significantly different from mean values obtained in the CX group. What is more, in the group receiving both compounds, the liver DDAH activity was not significantly increased when compared to the control group, which did not explain the significant decrease in ADMA level. Hypothetical explanation of this effect might be the fact that a decrease in ADMA level in M-CX group depends more on the action of CPX and is not directly related to the influence of the studied substances on the hepatic DDAH activity, but depends also on other factors e.g., protein arginine methyltransferase activity – the enzyme responsible for ADMA synthesis. The explanation of concomitant action of CPX and flavonoids is especially important, because the first substance is widely used as immunosuppressive drug in autoimmune diseases and flavonoids, as dietary supplements, are used in uncontrolled way by many patients who believe in their beneficial action. Besides, many plants and vegetables are a rich source of various flavonoids [12]. When sex subgroups of rats were analyzed, similar changes in ADMA concentrations were observed as in general population especially in CPX-receiving group and the group receiving both substances. Only in males NaMSA alone did not alter the ADMA level. No significant changes were observed in DDAH activity between groups. The phenomenon requires additional studies, because it was observed that in females estrogens decrease ADMA level together with activation of DDAH activity [6, 8]. In our study, no significant differences in DDAH activity as well as ADMA concentrations were observed between female and male rats. The age of rats and lack of significant estrogen stimulation might have been a possible reason, but the level of hormones was not checked. It is quite well documented that elevated plasma level of ADMA plays role in the pathogenesis of many clinical disorders and are observed in such states as hyperglycemia/diabetes, hypercholesterolemia, hypertension or heart failure [22]. Nowadays, many studies focus on intensive investigations of well-known drugs with additional (pleiotropic) action
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restoring increased ADMA level [23]. Much less data are available regarding possible impact of e.g., anticancer/immunosuppressant drugs on AMDA level, which is not increased by the main disease. Results of our study, evaluating influence of CPX on ADMA/ DDAH pathway, however preliminary, are the first step to the further experimental and/or clinical research and may have clinical relevance in the future.
6.
7.
8.
Conclusions 9.
Significant decrease in plasma ADMA concentration in the group receiving multiple small doses of CPX was unexpected and requires further studies especially in the context of other NO pathway parameters and in other models of CPX administration, in order to evaluate the importance and possible mechanism of such changes.
Acknowledgments: The study was financially supported by the statutory means of Wroclaw Medical University (No. ST-346) and by research fellowship within “Development Program of Wroclaw Medical University” funded from European Social Fund, Human Capital, National Cohesion Strategy” (contract no. UDA-POKL.04.01.01-00-010/08-01)”.
10. 11.
12.
13.
14.
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metric dimethylarginine serum levels are associated with acute rejection in kidney transplant recipients. Transplant Proc, 2009, 41, 1570–1573. Holden DP, Cartwrigth JE, Nussey SS, Whitley GS: Estrogen stimulates dimethylarginine dimethylaminohydrolase activity and the metabolism of asymmetric dimethylarginine. Circulation, 2003, 108, 1575–1580. Hu W, Yang X, Zhe C, Zhang Q, Sun L, Cao K: Puerarin inhibits iNOS, COX-2 and CRP expression via suppression of NF-kB activation in LPS-induced RAW264.7 macrophage cells. Pharmacol Rep, 2011, 63, 781–789. Karkanaki A, Vavilis D, Traianos A, Kalogiannidis I, Panidis D: Hormone therapy and asymmetrical dimethylarginine in postmenopausal women. Hormones (Athens), 2010, 9, 127–135. Kopacz M: Quercetin- and morinosulfonates as analytical reagents. J Anal Chem, 2003, 58, 225–229. Kopacz M: Sulfonic Derivatives of morin. Pol J Chem, 1981, 55, 227–229. Korkmaz A, Topal T, Oter S: Pathophysiological aspects of cyclophosphamide and ifosfamide induced hemorrhagic cystitis; implication of reactive oxygen and nitrogen species as well as PARP activation. Cell Biol Toxicol, 2007, 23, 303–312. Magdalan J, Szel¹g A, Chlebda E, Merwid-L¹d A, Trocha M, Kopacz M, KuŸniar A, Nowak D: Quercetin5’-sulfonic acid sodium salt and morin-5’-sulfonic acid sodium salt as antidotes in the subacute cadmium intoxication in mice. Pharmacol Rep, 2007, 59, Suppl 1, 210–216. Merwid-L¹d A, Trocha M, Chlebda E, Sozañski T, Magdalan J, Ksi¹dzyna D, Kopacz M et al.: Effects of morin-5’-sulfonic acid sodium salt (NaMSA) on cyclophosphamide-induced changes in oxido-redox state in rat liver and kidney. Hum Exp Toxicol, 2012, 31, 812–819. Nickel T, Hanssen H, Sisic Z, Pfeiler S, Summo C, Schmauss D, Hoster E, Weis M: Immunoregulatory effects of the flavonol quercetin in vitro and in vivo. Eur J Nutr, 2011, 50, 163–172. Olszanecki R, Gebska A, Kozlovski VI, Gryglewski RJ: Flavonoids and nitric oxide synthase. J Physiol Pharmacol, 2002, 53, 571–584. Oter S, Korkmaz A, Oztas E, Yildrim I, Topal T, Bilgic H: Inducible nitric oxide synthase inhibition in cyclophosphamide induced hemorrhagic cystitis in rats. Urol Res, 2004, 32, 185–189. Palm F, Onozato ML, Luo Z, Wilcox CS: Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems. Am J Physiol Heart Circ Physiol, 2007, 293, H3227–3245. Schnabel R, Blankenberg S, Lubos E, Lackner KJ, Rupprecht HJ, Espinola-Klein C, Jachmann N et al.: Asymmetric dimethylarginine and the risk of cardiovascular events and death in patients with coronary artery disease: results from the AtheroGene Study. Circ Res, 2005, 97, e53–59. Tain YL, Baylis C: Determination of dimethylarginine dimethylaminohydrolase activity in the kidney. Kidney Int, 2007, 72, 886–889.
Impact of cyclophosphamide and NaMSA on ADMA/DDAH pathway Anna Merwid-L¹d et al.
20. Tang WJ, Hu CP, Chen MF, Deng PY, Li YJ: Epigallocatechin gallate preserves endothelial function by reducing the endogenous nitric oxide synthase inhibitor level. Can J Physiol Pharmacol, 2006, 84, 163–171. 21. Trocha M, Merwid-L¹d A, Szuba A, Chlebda E, Pieœniewska M, Sozañski T, Szel¹g A: Effect of simvastatin on nitric oxide synthases (eNOS, iNOS) and arginine and its derivatives (ADMA, SDMA) in ischemia/reperfusion injury in rat liver. Pharmacol Rep, 2010, 62, 343–351. 22. Trocha M, Merwid-L¹d A, Szuba A, Sozañski T, Magdalan J, Szel¹g A: Asymmetric dimethylarginine. Synthesis and degradation under physiological and pathological conditions. Adv Clin Exp Med, 2010, 19, 233–243. 23. Trocha M, Szuba A, Merwid-L¹d A, Sozañski T: Effect of selected drugs on plasma asymmetric dimethylarginine (ADMA) levels. Pharmazie, 2010, 65, 562–571.
24. Virág L, Szabó E, Gergely P, Szabó C: Peroxynitriteinduced cytotoxicity: mechanism and opportunities for intervention. Toxicol Lett, 2003, 140-141, 113–124. 25. Wan LL, Xia J, Ye D, Liu J, Chen J, Wang G: Effects of quercetin on gene and protein expression of NOX and NOS after myocardial ischemia and reperfusion in rabbit. Cardiovasc Ther, 2009, 27, 28–33. 26. Xiao HB, Jun-Fang, Lu XY, Chen XJ, Chao-Tan, Sun ZL: Protective effects of kaempferol against endothelial damage by an improvement in nitric oxide production and a decrease in asymmetric dimethylarginine level. Eur J Pharmacol, 2009, 15, 213–222.
Received: July 5, 2011; in the revised form: September 17, 2012; accepted: October 8, 2012.
Pharmacological Reports, 2013, 65, 201207
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Copyright © 2013 by Institute of Pharmacology Polish Academy of Sciences
Short communication
Effect of cyclophosphamide and morin-5’-sulfonic acid sodium salt, alone or in combination, on ADMA/DDAH pathway in rats Anna Merwid-L¹d1, Ma³gorzata Trocha1, Ewa Chlebda-Sieragowska1, Tomasz Sozañski1, Jan Magdalan1, Dorota Ksi¹dzyna1, Andrzej Szuba2, Maria Kopacz3, Anna KuŸniar3, Dorota Nowak3, Ma³gorzata Pieœniewska1, Lidia Fereniec-Go³êbiewska1, Adam Szel¹g1
Department of Pharmacology, Wroclaw Medical University, Mikulicza-Radeckiego 2, PL 50-345 Wroc³aw, Poland
Department and Clinic of Internal Medicine and Occupational Diseases, and Hypertension, Wroclaw Medical University, Pasteura 4, PL 50-367 Wroc³aw, Poland !
Department of Inorganic and Analytical Chemistry, Chemical Faculty, Rzeszow University of Technology, Powstañców Warszawy 6, PL 35-959 Rzeszów, Poland Correspondence: Anna Merwid-L¹d, e-mail: [email protected]
Abstract: Background: The aim of the study was to evaluate the effect of cyclophosphamide (CPX) and morin-5’-sulfonic acid sodium salt (NaMSA) on plasma asymmetric dimethylarginine (ADMA) level and dimethylarginine dimethylaminohydrolase (DDAH) activity in rat liver. Methods: The study was performed on Wistar rats receiving normal saline, CPX (15 mg/kg/day), NaMSA (100 mg/kg/day) or both CPX and NaMSA for 10 consecutive days. Results: Significant decrease in ADMA level was found in all groups when compared to the control. DDAH activity in the liver was significantly higher in CX group compared to the control group. Conclusion: Obtained results of ADMA/DDAH pathway parameters require further research. Key words: cyclophosphamide, morin-5’-sulfonic acid sodium salt (NaMSA), ADMA, DDAH, rats
Abbreviations: ADMA – asymmetric dimethylarginine, CAT – catalase, CPX – cyclophosphamide, DDAH – dimethylarginine dimethylaminohydrolase, eNOS – endothelial nitric oxide synthase, GPx – glutathione peroxidase, GSH – glutathione, iNOS – inducible nitric oxide synthase, HPLC – high-performance liquid chromatography, MDA – malondialdehyde, NaMSA – morin-5’-sulfonic acid sodium salt, NO – nitric oxide, NOS – nitric oxide synthase, ROS – reactive oxygen species, SOD – superoxide dismutase
Introduction Asymmetric dimethylarginine (ADMA) is a very well known risk factor of cardiovascular complications. ADMA is an endogenous inhibitor of all isoforms of nitric oxide synthase (NOS), but especially of the endothelial isoform of NOS (eNOS) [18]. DimethylPharmacological Reports, 2013, 65, 201207
201
arginine dimethylaminohydrolase (DDAH) is an enzyme responsible for the metabolism of ADMA. It is mainly located in the liver but it can also be found in other organs such as kidneys, brain, adrenal glands, pancreas or testes [17, 22]. In case of oxidative stress DDAH activity may be reduced, causing subsequent increase in ADMA level. It was demonstrated that some antioxidants can preserve DDAH activity, which indicates that DDAH activity could be modulated by reactive oxygen species (ROS) [17]. Cyclophosphamide (CPX) is a very well-known anticancer and immunosuppressive drug. Although cardiotoxicity is not the main adverse effect of CPX, sinus tachycardia, cardiomyopathy and heart failure are also noticed following administration of CPX. More rarely, ventricular or supraventricular tachyarrhythmias (including atrial or ventricular fibrillation), angina, myocardial infarction, myocarditis, pericarditis or even cardiac arrest were reported [4]. An increased level of nitric oxide (NO), produced by inducible form of NO synthase (iNOS) in tissues may be an important reason of some severe adverse reactions. Involvement of NO in pathogenesis of those adverse effects of CPX is quite well confirmed [1, 16], but still little is known about the impact of CPX on eNOS activity and/or expression or the influence of CPX on ADMA level as well as on DDAH activity. Various studies have shown that substances belonging to flavonoids exhibit strong antioxidant properties. They may also influence the NO system. Quercetin inhibits iNOS mRNA and protein expression. The compound is also a potent vasodilator in eNOS-dependent manner [15, 25]. Less data are available for water-soluble derivatives of flavonoids, such as morin-5’-sulfonic acid sodium salt (NaMSA). It was demonstrated that those compounds exerted low toxicity, possessed antioxidant properties and might be effective as antidotes in some heavy metals intoxications [3, 12]. However, their influence on ADMA concentration and/or DDAH activity as well as impact on NOS activity is not known. The aim of the study was to evaluate the effect of CPX and NaMSA, given alone or in combination, on plasma ADMA levels and DDAH activity in rat liver.
210.8 ± 16.7 g) obtained from the Animal Laboratory of the Department of Pathological Anatomy, Wroclaw Medical University, Poland. Animals were housed individually in chambers with a 12:12 h light-dark cycle and the ambient temperature was maintained between 21 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 I Local Ethics Commission for Experiments on Animals in Wroc³aw. Chemicals
The following chemicals were used in the study: cyclophosphamide (Sigma, Germany), 0.9% NaCl solution (amp. 10 ml, Polpharma S.A., Poland) and thiopental (amp. 0.5 g, Biochemie, Austria). NaMSA was synthesized in the Department of Inorganic and Analytical Chemistry, University of Technology in Rzeszów, Poland, according to the methods described previously. NaMSA is easily soluble in water and keeps properties of the parent compound [9, 10].
Experiment
Experimental animals were randomly divided into four experimental groups (n = 12 each, 6 females and 6 males): group C – receiving 0.9% saline solution at 9 a.m. and 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.; group M – receiving 0.9% saline solution at 9 a.m. and NaMSA at a dose of 100 mg/kg 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 (4 ml/kg) and were given intragastrically by a tube for 10 consecutive days. Saline solution (4 ml/kg) was also given by the same route. On the 11 day of the experiment, blood samples from the tail vein were collected and thereafter animals were sacrificed in a deep anesthesia with intraperitoneal thiopental injection (70 mg/kg). Half of the liver and one kidney were homogenized on ice, using a lysis buffer (140 mM NaCl, 10 mM EDTA, 10% glycerol, 1% NP40, 20 mM Tris base, pH 7.5) and centrifuged at 14,000 rpm for 25 min at 4°C. DDAH activity was measured in the obtained supernatants. JD
Materials and Methods Animals
Experiment was carried out on Wistar rats of both sexes (203.5 ± 17.6 g; females: 196.3 ± 15.6 g; males:
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Impact of cyclophosphamide and NaMSA on ADMA/DDAH pathway Anna Merwid-L¹d et al.
Measurements of ADMA concentration
ADMA concentration was measured by high-performance liquid chromatography (HPLC) with fluorescence detection according to the method described in our previous paper [21]. Measurements of DDAH activity
DDAH activity was measured spectrophotometrically according to the method of Tain and Baylis [19], adopted to the macromethod for spectrophotometer MARCEL S350 PRO (Marcel Sp. z o.o., Poland). The method is based on the rate of L-citrulline production. Briefly, liver tissue homogenates were mixed with phosphate buffer, pH = 6.5. One mM ADMA was added to the samples and samples were incubated at 37°C for 45 min. After the reaction was stopped with 4% sulfosalicylic acid, samples were centrifuged and oxime reagent (diacetyl monoxime (0.08% w/v) in 5% acetic acid) mixed with antipyrine/H SO" (antipyrine (0.5% w/v) in 50% sulfuric acid) reagent was added. Samples were thereafter incubated at 60°C for 110 min and cooled in ice bath for 10 min. L-citrulline formation was measured at 466 nm and values were subtracted of respective blanks (without ADMA). Standard was prepared as serial dilutions of L-citrulline. DDAH activity was represented as μM L-citrulline formation/g protein/min at 37°C.
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 the mean values ± standard deviation (SD). Statistical analysis of the effect of CPX and NaMSA on ADMA concentration and DDAH activity in the liver and kidney were performed using analysis of variance (ANOVA). Specific comparisons were made with Tukey’s test for the whole population and in the female and male subgroups. Hypotheses were considered positively verified if p < 0.05.
Results ADMA level was significantly lower in the group receiving CPX comparing to the control group and was the lowest in all experimental groups. NaMSA also
Fig. 1. Plasma asymmetric dimethylarginine (ADMA) concentration (μM) and liver dimethylarginine dimethylaminohydrolase (DDAH) activity (μM L-citrulline/mg of protein/min). C – control group, CX – group receiving CPX (15 mg/kg), M – group receiving NaMSA (100 mg/kg), group M-CX – group receiving CPX (15 mg/kg) and NaMSA (100 mg/kg). Data are expressed as the mean ± SD. * p < 0.001 vs. C, ** p < 0.01 vs. C, # p < 0.001 vs. CX, ## p < 0.005 vs. M-CX, $ p < 0.05 vs. C
Plasma ADMA concentration [μM] and liver DDAH activity [μM L-citrulline/g protein/min]
1.6
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## #
1.4
CX
M
M-CX
1.2
$
1
*
*
0.8
0.6
0.4
0.2
0
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CX
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M-CX
plasma ADMA level
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liver DDAH activity
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significantly decreased ADMA concentration compared to the control group. Significant decrease in ADMA level was observed in the group receiving both studied substances and was significantly different from the control group and from the group receiving NaMSA only, but not significantly different from the CPX-receiving group (Fig. 1). In the subgroup of female rats ADMA level was also the lowest in the group receiving CPX and significantly different from the level reported for the control group. Administration of NaMSA alone or of both examined substances (CPX and NaMSA) similarly significantly reduced the level of ADMA in comparison to the control group. In the male subgroup of rats AMDA concentrations were significantly decreased when compared to the control group in CPX-receiving group and in the group receiving both studied substances, but not in the NaMSA-receiving group. Therefore, significant difference was also noticed between M-CXM and MM group. Mean values of ADMA concentrations and detailed comparisons between studied groups are presented in Table 1. Liver DDAH activity was the highest in the group receiving CPX and was significantly higher than in the control group. In the group receiving NaMSA, DDAH activity was also increased but the difference in comparison to the control group was insignificant. Administration of both substances did not significantly changed DDAH activity comparing to the control group (Fig. 1).
When female and male subgroups of rats were analyzed no significant differences in DDAH activity were observed. However, the tendency observed was exactly the same as for the whole population and the highest activity of DDAH was measured in CPXreceiving groups, both in female and male rats. Mean values of DDAH activities and detailed comparisons between studied groups are presented in Table 1.
Discussion It was demonstrated in the experimental studies that CPX administration led to oxidative stress measured as malondialdehyde (MDA) concentration as the indicator of lipid peroxidation [11]. The effect was the most pronounced following administration of large single doses of CPX [1, 11]. Overproduction of ROS and reactive nitrogen leads to the induction of oxidative stress in cells and tissues, DNA injury, cellular damage and necrosis. Moreover, it may contribute to the pathogenesis of some adverse effects of cyclophosphamide, e.g., hemorrhagic cystitis [24]. Cyclophosphamide may also induce changes in endogenous antioxidant system including superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPx) activities and glutathione (GSH) level with SOD as one of the most powerful antioxidant in the
Tab. 1. Plasma asymmetric dimethylarginine (ADMA) concentrations (μM) and liver dimethylarginine dimethylaminohydrolase (DDAH) activities (μM L-citrulline/mg of protein/min) in female and male rats. CF and CM – control group; female and male, respectively, CXF and CXM – group receiving CPX (15 mg/kg); female and male, respectively, MF and MM – group receiving NaMSA (100 mg/kg); female and male, respectively, group M-CXF and M-CXM – group receiving CPX (15 mg/kg) and NaMSA (100 mg/kg); female and male, respectively. Data are expressed as the mean ± SD
Plasma ADMA concentration (μM)
p
Liver DDAH activity (μM L-citrulline/mg of protein/min)
p
Female (CF)
1.291 ± 0.235
–
0.377 ± 0.100
–
Male (CM)
1.168 ± 0.045
–
0.592 ± 0.201
–
Group
Sex
Control group (C) CPX group (CX) NaMSA group (M)
CPX with NaMSA group (M-CX)
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Female (CXF)
0.688 ± 0.109
p < 0.001 vs. C
0.578 ± 0.222
–
Male (CXM)
0.770 ± 0.117
p < 0.001, vs. CM
0.819 ± 0.291
–
Female (MF)
0.920 ± 0.153
p < 0.005, vs. CF
0.550 ± 0.068
–
Male (MM)
1.151 ± 0.089
p < 0.001 vs. CXM
0.638 ± 0.133
–
Female (M-CXF)
0.805 ± 0.085
p < 0.001 vs. CF
0.448 ± 0.119
–
Male (M-CXM)
0.811 ± 0.081
p < 0.001, vs. CM p < 0.001 vs. MM
0.559 ± 0.183
–
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Impact of cyclophosphamide and NaMSA on ADMA/DDAH pathway Anna Merwid-L¹d et al.
cells [11]. In our previous work, we have documented that treatment with CPX in rats resulted in significant decrease in SOD activity and GSH concentration but with no impact on CAT activity [13]. Oxidative stress may subsequently cause decrease in DDAH activity and elevation in ADMA concentration [17, 22]. Considering those facts, increased ADMA concentration along with decreased DDAH activity in the CPXreceiving group was expected. Results obtained in our experimental model were quite opposite in this group. Some authors observed that administration of small doses of CPX (15 mg/kg, 3 times during the study) led rather to decrease than elevation in MDA level [2]. Also in our experimental model of 10 day administration of CPX in a daily dose of 15 mg/kg we did not observe any increase in MDA concentration in CPX-receiving group. We even noticed a significant decrease in MDA level comparing to the control group [13]. It may, at least partly, explain significant decrease in ADMA level and increase in DDAH activity in the liver observed in this study. Because published data concerning the impact of CPX or other cytotoxic and immunosuppressive agents are very scarce, it is difficult to compare results of our study with the results obtained by other authors. Single data show that immunosuppressive therapy may lead to the decrease of elevated ADMA concentration in e.g., patients after kidney transplantation [5]. Influence of flavonoids on ADMA level and/or DDAH activity has been also the subject of many studies. It was demonstrated that various antioxidants caused decrease in ADMA level by preserving DDAH activity. Such effects were demonstrated for both plant-derived and synthetic compounds [22]. In our study, NaMSA, a water soluble derivative of morin, significantly decreased plasma ADMA concentration comparing to the control group, but an increase in DDAH activity in the liver was insignificant. Data concerning effects of flavonoids on ADMA/DDAH are also limited, but it was shown that quercetin decreased ADMA level in healthy volunteers when administered twice a day in a 500 mg dose for 4 weeks [14]. Kaempferol treatment decreased ADMA plasma concentrations and increased DDAH activity in apolipoprotein E-deficient mice [26]. Another flavonoid – epigallocatechin gallate significantly decreased the level of ADMA and increased the activity of DDAH caused by oxidized LDL in endothelial cells [20]. Results of our study are consistent with the results obtained with different flavonoids by other researchers.
It was also reported that other agents belonging to isoflavones e.g., puerarin extracted from Radix puerariae may inhibit LPS-induced iNOS expression by suppression of NF-kB activation in macrophages [7]. Combination of both substances (CPX and NaMSA) caused significant decrease in ADMA level comparing to the control group and the group receiving NaMSA. Mean ADMA concentration in M-CX group was not significantly different from mean values obtained in the CX group. What is more, in the group receiving both compounds, the liver DDAH activity was not significantly increased when compared to the control group, which did not explain the significant decrease in ADMA level. Hypothetical explanation of this effect might be the fact that a decrease in ADMA level in M-CX group depends more on the action of CPX and is not directly related to the influence of the studied substances on the hepatic DDAH activity, but depends also on other factors e.g., protein arginine methyltransferase activity – the enzyme responsible for ADMA synthesis. The explanation of concomitant action of CPX and flavonoids is especially important, because the first substance is widely used as immunosuppressive drug in autoimmune diseases and flavonoids, as dietary supplements, are used in uncontrolled way by many patients who believe in their beneficial action. Besides, many plants and vegetables are a rich source of various flavonoids [12]. When sex subgroups of rats were analyzed, similar changes in ADMA concentrations were observed as in general population especially in CPX-receiving group and the group receiving both substances. Only in males NaMSA alone did not alter the ADMA level. No significant changes were observed in DDAH activity between groups. The phenomenon requires additional studies, because it was observed that in females estrogens decrease ADMA level together with activation of DDAH activity [6, 8]. In our study, no significant differences in DDAH activity as well as ADMA concentrations were observed between female and male rats. The age of rats and lack of significant estrogen stimulation might have been a possible reason, but the level of hormones was not checked. It is quite well documented that elevated plasma level of ADMA plays role in the pathogenesis of many clinical disorders and are observed in such states as hyperglycemia/diabetes, hypercholesterolemia, hypertension or heart failure [22]. Nowadays, many studies focus on intensive investigations of well-known drugs with additional (pleiotropic) action
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restoring increased ADMA level [23]. Much less data are available regarding possible impact of e.g., anticancer/immunosuppressant drugs on AMDA level, which is not increased by the main disease. Results of our study, evaluating influence of CPX on ADMA/ DDAH pathway, however preliminary, are the first step to the further experimental and/or clinical research and may have clinical relevance in the future.
6.
7.
8.
Conclusions 9.
Significant decrease in plasma ADMA concentration in the group receiving multiple small doses of CPX was unexpected and requires further studies especially in the context of other NO pathway parameters and in other models of CPX administration, in order to evaluate the importance and possible mechanism of such changes.
Acknowledgments: The study was financially supported by the statutory means of Wroclaw Medical University (No. ST-346) and by research fellowship within “Development Program of Wroclaw Medical University” funded from European Social Fund, Human Capital, National Cohesion Strategy” (contract no. UDA-POKL.04.01.01-00-010/08-01)”.
10. 11.
12.
13.
14.
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Received: July 5, 2011; in the revised form: September 17, 2012; accepted: October 8, 2012.
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