original papers

Adv Clin Exp Med 2011, 20, 6, 683–690 © Copyright by Wroclaw Medical University ISSN 1230-025X

Anna Merwid-Ląd, Małgorzata Trocha, Ewa Chlebda, Tomasz Sozański, Jan Magdalan, Dorota Ksiądzyna, Małgorzata Pieśniewska, Adam Szeląg The Effects of Morin, a Naturally Occurring Flavonoid, on -Induced Toxicity in Rats* Wpływ naturalnie występującego flawonoidu – moryny – na wywołaną cyklofosfamidem toksyczność narządową u szczurów

Department of Pharmacology, Wroclaw Medical University, Poland

Abstract Background. The use of cyclophosphamide (CPX) as an anticancer or immunosuppressive drug is strongly limited by its toxicity. Pronounced changes are observed, e.g. in the hematological system, especially decreases in leukocyte and platelet levels. It has been shown that different flavonoids exhibit antioxidant properties and protect against some of the adverse effects caused by anticancer drugs such as cyclophosphamide. Morin is one of the naturally occurring flavonoids with antioxidant properties, but little is known about its possible action against CPX-induced toxicity. Objectives. The aim of the present study was to evaluate the effects of morin (3,5,7,2’,4’-pentahydroxyflavone), a naturally occurring flavonoid, on cyclophosphamide-induced toxicity. Material and Methods. The experiment was carried out on Wistar rats of both sexes (202.6 g ± 18.2 g) divided into three groups of 12: group C, receiving 0.9% saline; group CX, receiving CPX (15 mg/kg); and group M-CX, receiv- ing CPX (15 mg/kg) and morin (100 mg/kg). All the substances were given intragastrically for 10 days. On the 10th day of the study, the animals were placed in metabolic cages for 24 hours for urine collection. On the 11th day blood samples were collected for assessment of biochemical parameters (total protein, glucose, urea, creatinine, AST, ALT, GGTP and amylase) and blood morphology parameters (WBC, RBC, hemoglobin, hematocrit, MCV, MCH, MCHC and PLT). Creatinine and total protein concentrations were also measured in the daily urine collection. Results. CPX significantly inhibited body weight gain, and this was further aggravated by morin. CPX also signifi- cantly decreased leukocyte and platelet levels as well as red blood cell counts and hematocrit values. Only eryth- rocyte levels were partly restored by co-administrating morin. CPX induced significant hypoproteinemia in many biochemical parameters; this was completely brought back to normal by morin administration. Conclusions. Before morin is widely recommended as a dietary supplement it is necessary to clarify its utility as a chemoprotective agent. On the basis of this study morin’s action as a substance decreasing CPX-induced toxicity is rather doubtful (Adv Clin Exp Med 2011, 20, 6, 683–690). Key words: morin, cyclophosphamide, rat, toxicity. Streszczenie Wprowadzenie. Cyklofosfamid (CPX) jest szeroko stosowanym lekiem przeciwnowotworowym mającym dodat- kowo silne właściwości immunosupresyjne. Jego zastosowanie w lecznictwie jest jednak ograniczone znaczną toksycznością, zwłaszcza w układzie moczowym (krwotoczne zapalenie pęcherza moczowego). Obserwuje się również zmniejszenie liczby leukocytów, erytrocytów i płytek krwi. Wykazano, że różne flawonoidy mają działanie antyoksydacyjne i chronią przed niektórymi działaniami niepożądanymi wywołanymi lekami cytostatycznymi, np. cyklofosfamidem. Moryna należy do flawonoidów i ma działanie antyoksydacyjne. Niewiele wiadomo o możliwym działaniu moryny jako związku zmniejszającego toksyczność indukowaną cyklofosfamidem. Cel pracy. Zbadanie wpływu naturalnie występującego flawonoidu – moryny na wywołaną cyklofosfamidem toksyczność narządową.

*The study was financially supported by Wroclaw Medical University research grant No ST-346 and by a research fellowship program within the Wroclaw Medical University Development Program, funded by the European Social Fund’s Human Capital Operating Programme and Cohesion Policy (contract No UDA-POKL.04.01.01-00-010/08-01). 684 A. Merwid-Ląd et al.

Materiał i metody. Badanie zostało przeprowadzone na szczurach szczepu Wistar obojga płci (202.6 g ± 18.2 g) podzielonych na 3 grupy eksperymentalne (N = 12). W grupie C zwierzęta otrzymywały 0.9% roztwór soli fizjolog- icznej, w grupie CX – cyklofosfamid (15 mg/kg), a w grupie M-CX otrzymywały CPX (15 mg/kg) łącznie z moryną (100 mg/kg). Wszystkie substancje były podawane dożołądkowo przez 10 kolejnych dni. W 10. dniu doświadczenia zwierzęta były umieszczane w klatkach metabolicznych w celu całodobowej zbiórki moczu. Jedenastego dnia doświadczenia była pobierana krew do oznaczeń parametrów biochemicznych (białko całkowite, glukoza, moc- znik, kreatynina, AST, ALT, GGTP i amylaza) oraz morfologii krwi obwodowej (WBC, RBC, hemoglobina, hematokryt, MCV, MCH, MCHC i PLT). Stężenie kreatyniny oraz białka było oznaczane również w dobowej zbiórce moczu. Wyniki. CPX istotnie hamował przyrost masy ciała zwierząt, co było wzmacniane przez dodanie moryny. CPX również istotnie zmniejszał liczbę leukocytów i płytek krwi, a także erytrocytów i wartość hematokrytu. Podawanie moryny przywracało jedynie częściowo zmniejszoną liczbę czerwonych krwinek. Wśród wielu badanych param- etrów biochemicznych CPX powodował znaczące zmniejszenie stężenia białka całkowitego, a moryna całkowicie zapobiegała temu zmniejszeniu. Wnioski. Zanim moryna zostanie zarekomendowana jako suplement diety konieczne są dokładniejsze badania jej potencjalnego działania protekcyjnego w przypadku stosowania leków cytostatycznych. Na podstawie obec- nego badania działanie moryny jako związku zmniejszającego indukowaną cyklofosfamidem toksyczność jest raczej wątpliwe (Adv Clin Exp Med 2011, 20, 6, 683–690).

Słowa kluczowe: moryna, cyklofosfamid, szczur, toksyczność.

Cyclophosphamide (CPX) is widely used as Material and Methods an anticancer drug with additional strong im- munosuppressive properties. The use of CPX is, The Chemicals however, limited by its toxicity. The most charac- teristic adverse effect induced by CPX is hemor- Cyclophosphamide (CPX) (Sigma, Germany), rhagic cystitis, but pronounced changes are also morin hydrate (Sigma, Germany), 0.9% NaCl so- observed in the hematological system, especially lution (Polpharma S.A., Poland), and thiopental in leukocyte and platelet levels [1, 2]. Many stud- (0.5 g vials, Biochemie, Austria) were used in the ies have focused on the possibility of using differ- study. ent natural and synthetic compounds to decrease CPX-induced toxicity. It has been shown, for example, that citrus extract – particularly its fla- The Animals vonoid constituents with antioxidative activity – The experiment was carried out on Wistar rats of reduces CPX-induced genotoxicity in mouse bone both sexes (202.6 g ± 18.2 g) obtained from the Ani- marrow cells [3]. Administering other flavonoid mal Laboratory of the Department of Pathological antioxidants, such as quercetin or catechin, along Anatomy at Wroclaw Medical University (Wrocław, with resulted in full protection against CPX Poland). During the experiment the animals were toxicity in the urinary bladder [1]. Also, a combi- housed individually in chambers with a 12:12 hour nation of flavonoids (diosminium and quercetin) light-dark cycle and a temperature between 21°C and with CPX clearly reduced the effects of drug toxic- 23°C was maintained. Before and during the experi- ity: CPX-induced leukopenia and thrombocytope- ment, the animals had free access to standard food nia were corrected entirely [4]. Similarly, ternatin and water. The experiment was performed with the (a flavonoid isolated from Egletes viscosa Less.) approval of the First Local Ethics Committee for Ex- prevented cyclophosphamide- and -in- periments on Animals in Wroclaw. duced hemorrhagic cystitis in rats [5]. Some stud- ies indicate that another naturally occurring fla- vonoid – morin (3,5,7,2’,4’-pentahydroxyflavone) The Experiment – has antioxidant properties and may be a possible After a two-week adaptation period the ani- chemopreventive agent against some cancers [6- mals were randomly divided into three groups of 8]. However, little is known about the influence of 12: group C (the control group), receiving 0.9% morin on CPX-induced tissue toxicity. saline solution at 9 AM and at 2 PM; group CX, The aim of the present study was to evaluate receiving CPX at a dose of 15 mg/kg at 9 AM and the effects of morin on cyclophosphamide-induced 0.9% saline solution at 2 PM; and group M-CX, toxicity. receiving CPX at a dose of 15 mg/kg at 9 AM and morin at a dose of 100 mg/kg at 2 PM. All the sub- stances used in the study were dissolved in 0.9% saline solution in a 4 mL/kg volume and were Morin and Cyclophosphamide Toxicity 685 given administered by gastric tube for 10 con- vs. C, p < 0.001). The addition of morin to the CPX secutive days. The saline solution was also given did not reverse this action of CPX; in fact it caused in a 4 mL/kg volume. The animals were weighed a significant decrease in mean body weight (M-CX and observed every day. On the 10th day of the ex- vs. CX, p < 0.01 and M-CX vs. C, p < 0.001). periment, after the morning administration of the In the group receiving CPX alone as well as in substances, the animals were placed in metabolic the group receiving the combination of CPX with cages for 24 hours for urine sample collection. On morin, significant decreases in the weight of criti- the 11th day of the experiment the animals were cal organs such as the liver and kidneys were no- sacrificed by terminal anesthesia with thiopental ticed when compared to the control group (in the (70 mg/kg, administered intraperitoneally). Blood liver the p value for both CX vs. C and M-CX vs. samples were collected from the tail vein for as- C was < 0.001; in the kidneys CX vs. C, p < 0.005 sessing biochemical parameters such as total pro- and M-CX vs. C, p < 0.001). Significant differences tein, glucose, urea, creatinine concentrations and were also observed in the liver index (liver weight alanine and asparagine aminotransferases (ALT per body weight: CX vs. C, p < 0.05, M-CX vs. C, and AST), as well as gammaglutamyltranspepti- p < 0.001). Significant differences were noted be- dase (GGTP) and amylase activity. ALT, AST and tween group M-CX and group CX in liver weight GGTP activities were expressed per gram of liver. and liver index (M-CX vs. CX, p < 0.005 for both A second blood sample was collected for the evalu- parameters). A significant difference in kidney in- ation of blood morphology parameters such as to- dex (kidney weight per body weight) was observed tal white blood cells (WBC), red blood cells (RBC) only between group M-CX and the control group and platelet (PLT) count and the assessment of he- (M-CX vs. C, p < 0.05). matocrit (HCT) and hemoglobin (HGB) levels. In the urine samples, the daily excretion of total pro- tein and creatinine were measured. On the basis of Biochemical Parameters the creatinine concentration in urine and serum, in Blood and Urine and Kidney daily urine excretion and body weight creatinine Function clearance was estimated using the formula (U × V)/(P × 1440 × b.w.), where U is urine creatinine The mean values and respective SDs of the bio- concentration, V is daily urine volume, P is serum chemical parameters are presented in Table 1. CPX creatinine concentration, and b.w. is body weight. caused a significant decrease in total protein level The analyses of the biochemical parameters in as compared to the control group (CX vs. C, p < blood, the protein and creatinine level in urine and 0.001). The addition of morin to the CPX treat- the blood morphology were performed in certified ment completely reversed this action of CPX, and commercial laboratory. a significant difference was noticed between the M-CX group and the CX group, but not between the M-CX and C groups (M-CX vs. CX, p < 0.001, Statistical Analysis M-CX vs. C, p = ns. [non-significant]). Data were expressed as mean values ± standard No statistically important changes were noticed deviation (SD). Statistical analysis of the effects of in glucose levels between groups. However, CPX the drug on the studied parameters was performed caused decrease in glucose level when compared to using analysis of variance (ANOVA). Specific com- the control group and the difference was closed to parisons were tested with Tukey’s test. Hypotheses the significance border (CX vs. C, p = 0.07). were considered positively verified if p < 0.05. AST and ALT activities in group receiving CPX were not significantly higher than in the control group. However, in both cases increase in activities Results of mentioned enzymes were noticed as compared to the control group. What is more interesting in Body Weight the group receiving both CPX and morin activities of both enzymes were the highest and significantly In the control group (C), the mean increase in higher than in the control group (AST and ALT: body weight during the 11 days of the experiment M-CX vs. C, p < 0.05 in both cases). Similar ten- was 57.5 g ± 31.37 g; in the group receiving CPX, dency was observed in case of GGTP activity but the mean increase was 20.8 g ± 17.43 g; while in the difference between groups M-CX and C was in the group receiving CPX with morin a decrease of this case near to significance border (M-CX vs. C, 8.3 g ± 11.15 g in mean body weight was noted. p = 0.078). No significant changes in amylase activ- Compared to the control group, CPX caused a sig- ity were noticed. Concentrations of both urea and nificantly lower increase in mean body weight (CX creatinine in blood were similar in all examined 686 A. Merwid-Ląd et al.

Table 1. Biochemical blood parameters of rats receiving saline (group C), those receiving cyclophosphamide in doses of 15 mg/kg (group CX) and those receiving cyclophosphamide in doses of 15 mg/kg with morin in doses of 100 mg/kg (group M-CX) Tabela 1. Parametry biochemiczne we krwi szczurów otrzymujących sól fizjologiczną (grupa C), cyklofosfamid w dawce 15 mg/kg (grupa CX) oraz cyklofosfamid w dawce 15 mg/kg łącznie z moryną w dawce 100 mg/kg (grupa M-CX)

Group C CX M-CX (Grupa) Parameter Mean ± SD Mean ± SD Mean ± SD (Parametr) (Średnia ± SD) (Średnia ± SD) (Średnia ± SD) Total protein [g%] 5.57 ± 0.20 5.17 ± 0.32* 5.79 ± 0.17** (Białko całkowite) [g%] Glucose [mg%] 181.75 ± 20.40 165.42 ± 17.06 180.67 ± 14.75 (Glukoza) [mg%] Urea [mg%] 48.00 ± 8.93 46.25 ± 8.64 47.58 ± 10.66 (Mocznik) [mg%] Creatinine [mg%] 0.23 ± 0.05 0.23 ± 0.08 0.22 ± 0.03 (Kreatynina) [mg%] AST [U/L/g liver] 10.35 ± 4.22 24.53 ± 20.03 29.00 ± 21.20# (GOT) [U/L/g wątroby] ALT [U/L/g liver] 4.09 ± 1.19 5.24 ± 1.91 7.43 ± 4.88# (GPT) [U/L/g wątroby] GGTP [U/L/g liver] 0.26 ± 0.07 0.39 ± 0.2 0.75 ± 0.91 (GGTP) [U/L/g wątroby] Amylase [U/L] 853.25 ± 149.1 708.00 ± 127.83 746.92 ± 196.38 (Amylaza) [U/L] * – CX vs. C, p < 0.001. ** – M-CX vs. CX, p < 0.001. # – M-CX vs. C, p < 0.05. groups and no significant changes were observed and the difference was borderline significant (CX vs. between different groups (p = ns. in all compari- C, p = 0.057). In group M-CX, CrCl was estimated sons in both parameters). as 14.10 ± 3.54 mL/min/kg, and the difference from In the group receiving CPX, the urine creatinine the control group was significant (M-CX vs. C, p < concentration was not significantly different from 0.001). No significant differences between groups the control group (group C: 31.86 ± 10.66 mg%; were noticed in daily urine protein excretion, which group CX: 32.04 ± 12.51 mg%; p = ns.), but in the was 2.12 mg ± 0.69 mg in the control group, 2.42 group receiving both substances (CPX and morin) mg ± 0.66 mg in group CX, and 1.67 mg ± 2.88 mg the urine creatinine concentration was 89.18 ± 32.97 in group M-CX. mg% – significantly higher than in the control group and in the CPX-only group (p < 0.001 for both M- CX vs. C and M-CX vs. CX). In the group receiving Blood Morphology Parameters CPX with morin, lower daily urine excretion was The mean values and respective SDs of blood observed than in the two other groups (group C: morphology parameters are presented in Table 2. 21.2 ± 6.9 mL; group CX: 24.2 ± 6.6 mL; group M- CPX significantly decreased white blood cell count CX: 10.3 ± 5.2 mL). The differences between group (WBC) compared to the control group (CX vs. C, p M-CX and groups C and CX were significant (p < < 0.001). The addition of morin to the experimen- 0.001 for both M-CX vs. C and M-CX vs. CX). No tal protocol did not reverse this action of CPX: The significant differences in daily urine excretion were difference between the group receiving CPX with noticed between the control group and the CPX- morin and the control group was also significant, only group (C vs. CX, p = ns.). Creatinine clearance but no difference was observed between group M- (CrCl; mL/min/kg) increased in both experimental CX and group CX (M-CX vs. C, p < 0.001, M-CX groups as compared to the control group: The con- vs. CX, p = ns.). trol group’s CrCl was calculated as 7.58 ± 2.38 mL/ Very similar results were obtained in case of min/kg; in group CX it was 11.04 ± 4.39 mL/min/kg; platelet level (PLT). CPX significantly decreased Morin and Cyclophosphamide Toxicity 687

Table 2. Blood morphology parameters of rats receiving saline (group C), those receiving cyclophosphamide in doses of 15 mg/ kg (group CX) and those receiving cyclophosphamide in doses of 15 mg/kg with morin in doses of 100 mg/kg (group M-CX) Tabela 2. Parametry morfologii krwi szczurów otrzymujących sól fizjologiczną (grupa C), cyklofosfamid w dawce 15 mg/kg (grupa CX) oraz cyklofosfamid w dawce 15 mg/kg łącznie z moryną w dawce 100 mg/kg (grupa M-CX)

Group C CX M-CX (Grupa) Parameter Mean ± SD Mean ± SD Mean ± SD (Parametr) (Średnia ± SD) (Średnia ± SD) (Średnia ± SD) WBC [cells/L] 3.6 × 109 ± 1.64 × 109 0.83 × 109 ± 0.47 × 109* 0.47 × 109 ± 0.36 × 109** (WBC [kom./L]) RBC [cells/L] 6.92 × 1012 ± 0.23 × 1012 6.45 × 1012 ± 0.43 × 1012# 6.67 × 1012 ± 0.56 × 1012 (RBC [kom./L]) Hemoglobin [g%] 13.77 ± 0.41 12.93 ± 0.90 12.72 ± 1.58 (Hemoglobina [g%]) Hematocrit [%] 40.14 ± 1.53 37.48 ± 2.70# 36.66 ± 3.40## (Hematokryt [%]) MCV [fL] 58.03 ± 1.57 58.16 ± 3.30 55.01 ± 2.79 MCH [pg] 19.93 ± 0.44 20.04 ± 1.03 19.10 ± 1.92 MCHC [pg/dL] 34.34 ± 0.36 34.48 ± 0.46 34.84 ± 4.69 PLT [cells/L] 931.92 × 109 ± 246.06 × 109 396.36 × 109 ± 232.85 × 109* 512.20 × 109 ± 216.16 × 109** PLT [kom./L] * – CX vs. C, p < 0.001. ** – M-CX vs. C, p < 0.001. # – CX vs. C, p < 0.05. ## – M-CX vs. C, p < 0.01.

the PLT count in comparison to the control group phosphamide or other anticancer agents has been (CX vs. C, p < 0.001) and, as in case of WBC, this evaluated in many studies [9-12]. However, little adverse effect was not reversed by morin (M-CX has been published about the impact of morin, vs. C, p < 0.001, M-CX vs. CX, p = ns.). a natural flavonoid, on CPX-induced toxicity – the The impact of CPX on red blood cell param- subject of the current study. eters (RBC) was not as pronounced as in the case In the current study it was noted that admin- of WBC or platelets. CPX significantly decreased istering CPX for 10 days at 15 mg/kg b.w. caused RBC count (CX vs. C, p < 0.05), and the addition of significant inhibition of body weight gain, and that morin only partially reversed this (p = ns. for both the addition of morin to the therapeutic schedule M-CX vs. C and M-CX vs. CX). The mean value did not prevent this. On the contrary, with the of the RBC count in the group receiving CPX with addition of morin a significant decrease in body morin was between the mean values obtained in the weight was observed. Cyclophosphamide is a cyto- control group and the CPX-only group. However, toxic drug that causes severe damage to the gastro- in the case of CPX-induced lowering of hematocrit intestinal systems, including mucositis [13], which values (HCT, CX vs. C, p < 0.05), the addition of may lead to impaired food absorption in the gas- morin did not cause even a partial reverse (M-CX trointestinal tract and impaired body weight gain vs. C, p < 0.01, M-CX vs. CX, p = ns.). No influence in CPX-treated rats. Other mechanisms may also of CPX on other parameters such as hemoglobin be involved in the inhibition of body weight gain concentration (HGB), mean corpuscular volume after CPX administration. A reduction in body (MCV), mean corpuscular hemoglobin (MCH) weight after single-dose cyclophosphamide treat- or mean corpuscular hemoglobin concentration ment was noted by Latha and Panikkar [14]. Al- (MCHC) were noticed. though in the current study morin failed to reverse CPX-induced body weight loss, in various other studies different natural substances, including fla- Discussion vonoids, decreased or even prevented mucositis induced by anticancer drugs, and this effect was The influence of different natural and syn- probably connected to their antioxidant activity thetic substances on the toxicity induced by cyclo- [15–17]. Generation of oxidative stress in gastro- 688 A. Merwid-Ląd et al. intestinal tissues may not be the only reason for excluded as a reason for hypoproteinemia, because mucositis; interleukins may also be involved. Xi- creatinine and urea concentrations did not differ ang et al. found that interleukin-1 receptor antago- between the control group and the animals receiv- nist reduced cyclophosphamide-induced mucositis ing CPX, and the daily protein excretion was simi- in a murine model [13]. On the other hand it has lar in all three groups. also been shown that some substances, e.g. green Quite interesting changes in creatinine clear- tea extract, aggravate cyclophosphamide-induced ance were observed in this study. Creatinine clear- reductions in fetal weight in a rat model by modu- ance was the highest in the group receiving both lating the expression of cytochrome P-450 mRNA CPX and morin, and it was a significant difference [18]. Morin also exerts antioxidant activity, but did from the control group. This was consistent with not prevent the body weight loss induced by CPX. the highest daily creatinine excretion in urine in Unfortunately, little is known about the action of the same experimental group. However, the vol- morin in the gastrointestinal tract. ume of daily excreted urine in the M-CX group The CPX-induced changes in the level of blood was the smallest, and this was also significantly cells in this study were similar to those observed different from the control group. The amount of in humans during treatment with this drug. The creatinine excreted during the 24-hour collection most common abnormalities found in humans are period was similar in all three groups. It cannot be leukopenia and anemia; thrombocytopenia is ob- excluded that in longer-term experiments signifi- served after longer courses of treatment [2]. Simi- cant kidney damage may be revealed. lar results were obtained in rats after a single in- Very little is known about morin’s potential traperitoneal dose of CPX (50 mg/kg): Significant for protecting against the adverse effects of cyclo- decreases in WBC count and HGB concentration phosphamide. There is some evidence indicating were noted [14]. In the current study the most pro- that morin not only exerts antioxidant properties, nounced changes were in WBC and platelet levels. but may also be a possible chemopreventive agent It has been shown that some flavonoids, such as against some cancers [6–8]. Morin hydrate co-ad- diosminum and quercetin, correct cyclophosph- ministrated with either or mitomycin amide-induced leukopenia and thrombocytopenia C could minimize the toxicity of these anti-tumor in rats [4]. The current authors did not observe drugs against cardiovascular cells; however, it did significant improvement of these two parameters not attenuate the cytotoxicity of those drugs on when CPX was administered with morin for 10 human hepatocellular carcinoma cells (HepG2) days. In this study morin partially reversed the [19]. It has been shown that flavonoids reduced decreased RBC count, but not the decreased HCT CPX-induced genotoxicity in mice bone marrow level. cells [3] and protect from CPX- or ifosfamide-in- Hypoproteinemia is not very frequently men- duced hemorrhagic cystitis in rats [5]. In the cur- tioned as a complication of CPX treatment in rent study, however, CPX did not cause significant humans [2]. In the current study, however, CPX changes in the urinary tract. No marked erythro- caused a significant decrease in the total blood cyturia or significant kidney disorders manifested protein level. In contrast to the body weight gain by increased blood creatinine or urea concentra- inhibition, hypoproteinemia was completely re- tions were observed. These differences in the oc- versed by concomitant administration of morin. currence of one of the major adverse effects of This suggests that diverse mechanisms are prob- CPX treatment may be connected to differences ably involved in these two complications. Mucosi- in the regimen of CPX administration. In the cur- tis caused by CPX is one of the possible reasons rent study cyclophosphamide was administered for protein loss. It cannot be excluded that morin’s intragastrically in a 15 mg/kg daily dose for 10 action in the gastrointestinal tract is strong enough days, which resembles the chronic administration to restore protein reabsorption, but insufficient to of the drug as an immunosuppressant rather than prevent body weight loss. In the current study, as an anticancer agent. In most other studies CPX liver function was not significantly impaired dur- was administered intraperitoneally in higher single ing CPX administration, and the activities of liver doses (75–200 mg/kg) [9, 10, 20–22]. The cumu- enzymes such as ALT, AST and GGTP were not lative dose in the current experiment was similar significantly elevated, which indicates that hypo- (150 mg/kg) to those previously reported [9–11]. proteinemia was not likely to be a result of liver It may be that smaller daily doses cause different damage. Albumin levels are definitely a more sen- acute adverse effects, but not chronic effects such sitive indicator of liver injury and dysfunction than as leukopenia and thrombocytopenia. total serum protein level, but the albumin concen- Some studies have focused on the impact of tration was not assessed in this study. Additionally, morin on the pharmacokinetics of anticancer urinary protein loss due to kidney damage may be drugs or its intracellular penetration. Hong et al. Morin and Cyclophosphamide Toxicity 689 found that morin retards renal and total clearance which may enhance the sensitivity of the tumor of methotrexate (MTX) and significantly reduces cells to CMB [27]. cellular MTX uptake, which may be connected to In conclusion the authors suggest that morin the inhibition of OAT1-mediated renal excretion may partly reverse some CPX-induced toxic [23]. Fang et al. noted that in mice, morin markedly changes such as hypoproteinemia, but fails to re- decreased cyclosporin A (CsA) concentration, but verse or protect from important CPX-induced tox- had no significant effect on the immune response icity on the hematopoietic system or prevent body suppressed by CsA administration [24]. Ikegawa et weight loss. The latter may even be even worsened al. found that an esther derivative of morin – pen- by morin co-administration. Flavonoids are gener- taethylmorin – significantly increased the uptake ally considered promising dietary supplements for of [3H]vincristine by K562/ADM (adriamycin- patients undergoing anticancer or immunosup- resistant human myelogenous leukemia) cells by pressant therapy. It is necessary to verify the use of inhibiting the expression of P glycoprotein (PgP), morin as a chemoprotective agent before it can be adding that flavonoid derivatives may be useful in recommended as a dietary supplement. It is pos- as multidrug resistance-reversing sible that morin or its derivatives influencing PgP agents [25]. It has also been shown that morin may be useful for potentiating the intracellular ac- acts as a PgP inhibitor and inhibited the efflux of tion of anticancer drugs. chlorambucil (CMB) from colon cancer cells [26],

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Address for correspondence: Anna Merwid-Ląd Department of Pharmacology Wroclaw Medical University Mikulicza-Radeckiego 2 50-345 Wrocław Poland Tel.: +48 71 784 14 42 E-mail: [email protected]

Conflict of interest: None declared

Received: 14.03.2011 Revised: 7.04.2011 Accepted: 3.10.2011