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Impact of morin-5′-sulfonic acid sodium salt on cyclophosphamide-induced gastrointestinal toxicity in rats
Pharmacological Reports 67 (2015) 1259–1263
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Impact of morin-50 -sulfonic acid sodium salt on cyclophosphamide-induced gastrointestinal toxicity in rats Anna Merwid-La˛d a,*, Dorota Ksia˛dzyna a, Agnieszka Hałon´ b, Ewa Chlebda-Sieragowska a, Małgorzata Trocha a, Marta Szandruk a, Tomasz Sozan´ski a, Jan Magdalan a, Maria Kopacz c, Anna Kuz´niar c, Dorota Nowak c, Małgorzata Pies´niewska a, Adam Szela˛g a a
Department of Pharmacology, Wroclaw Medical University, Wrocław, Poland Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wrocław, Poland c Department of Inorganic and Analytical Chemistry, Chemical Faculty, University of Technology, Rzeszo´w, Poland b
A R T I C L E I N F O
Article history: Received 29 April 2014 Received in revised form 6 May 2015 Accepted 21 May 2015 Available online 4 June 2015 Keywords: Cyclophosphamide NaMSA Liver Intestines Toxicity
A B S T R A C T
Background: The aim of this study was to evaluate the effect of morin-50 -sulfonic acid sodium salt (NaMSA) on cyclophosphamide-induced gastrointestinal changes in rats. Methods: Rats received intragastrically 0.9% saline (group C), cyclophosphamide (15 mg/kg) (group CX), NaMSA (100 mg/kg) (group M) or cyclophosphamide (15 mg/kg) with NaMSA (100 mg/kg) (group MCX), respectively, for 10 days. Results: No histological lesions were observed in the liver and the large intestine in the control group and group receiving NaMSA. In the cyclophosphamide-treated group, a generalized blurred trabecular structure, hepatocyte apoptosis, focal and diffuse necrosis were noticed in the liver and atypia of epithelial cells or adenoma were noticed in the large intestine. In the group receiving both cyclophosphamide and NaMSA, hepatocyte apoptosis in the liver was observed less frequently. Histological examination of the small intestine revealed: low-grade dysplasia adenoma in the C, M, CX and M-CX group (in 44%, 0%, 100%, and 55.6% of specimens, respectively) with adenocarcinoma in 55.6% of specimens in the cyclophosphamide-receiving group only. Adenoma with high-grade dysplasia was observed in the control and NaMSA-receiving group with a similar frequency (22%). In addition to the histological evaluation, blood cell count parameters, as well as total protein concentration, blood glucose level, amylase, ALT, AST and GGTP activities were evaluated. Cyclophosphamide impaired weight gain, decreased blood cell count parameters and total protein concentration, and increased the GGTP activity. Those changes were not reversed by NaMSA. Conclusions: Summing up, NaMSA may protect against some cyclophosphamide-induced histological abnormalities in the gastrointestinal tract, including intestinal neoplasia in rats. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction
Abbreviations: ALT, alanine aminotransferase; AST, asparagine aminotransferase; CPX, cyclophosphamide; GGTP, gamma-glutamyl transferase; HGB, hemoglobin; HT, hematocrit; i.g., intragastrically; LABQUALITY, External Quality Assessment Scheme; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; NaMSA, morin-50 -sulfonic acid sodium salt; PLT, platelets; RBC, red blood cells; RIQAS, Randox International Quality Assessment Scheme; SD, standard deviation; SNCS IQAS e-CHECK, Sysmex International Quality Assurance System; SOS, sinusoidal obstruction syndrome; WBC, white blood cells. * Corresponding author. E-mail address: [email protected] (A. Merwid-La˛d).
Oral cyclophosphamide (CPX) plays a very important role in the treatment of some autoimmune disorders, but its use is limited by hematological and gastrointestinal adverse effects. Rapidly dividing cells of the bone marrow and the gastrointestinal mucosa are particularly susceptible to toxic effects of alkylating agents such as CPX. Bone marrow toxicity results in decreased leukocyte and platelet counts and, to a lesser extent, red blood cells count. Additionally, gastrointestinal adverse effects, including anorexia, nausea, vomiting, diarrhea, stomatitis, hemorrhagic colitis, acute pancreatitis as well as hepatic injury with jaundice and elevation of liver transaminases and/or GGTP activities are described in
http://dx.doi.org/10.1016/j.pharep.2015.05.020 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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humans. Clinical symptoms and abnormal laboratory tests are accompanied by ultrastructural changes [1]. CPX-induced hepatotoxicity in humans is dose-dependent and characterized by cholestasis and cytolysis with three histological patterns described: massive hepatic necrosis, necrosis of perivenous hepatocytes and diffuse hepatocellular damage with mild steatosis [2]. These effects are also observed in animal models. CPX increased chromosomal aberrations of bone marrow cells, caused myelosuppression, leucopenia, thrombocytopenia and macrocytic normochromic anemia in rats [3]. In rats, mucositis is a wellestablished injury following administration of CPX alone [4] or with other anticancer drugs [5]. Many attempts were made to find substances preventing gastrointestinal toxicity of anticancer drugs [4]. Our previous study demonstrated that morin, a naturally occurring flavonoid, partly reversed CPX-induced decrease in RBC count and completely reversed CPX-induced hypoproteinemia [6]. Morin is practically insoluble in water and that is one of its most important disadvantages. On the other hand, morin-5’sulfonic acid sodium salt (NaMSA) is not only water soluble but also has antioxidative properties and low toxicity similar to the original compound [7,8]. However, to our best knowledge, little is known about its possible protective action against CPX-induced hematological and gastrointestinal toxicity in rats. The aim of the present study was to evaluate the effects of NaMSA on CPX-induced liver, pancreatic and intestinal toxicity in rats, using laboratory tests and histological evaluation.
Material and methods Chemicals Cyclophosphamide (CPX) (Sigma, Seelze, Germany), 0.9% NaCl solution (Polpharma S.A., Starogard Gdan´ski, Poland), and thiopental (0.5 g vials, Biochemie, Kundl, Austria) were used in the study. Morin-50 -sulfonic acid sodium salt (NaMSA) was synthesized in the Department of Inorganic and Analytical Chemistry, University of Technology in Rzeszow, Poland, according to the methodology described previously. Thin-layer chromatography on alumina plates from MERCK (covered with silica gel 60 WF254s) was used to check the purity of the NaMSA. The compounds of mobile phase were as follows: n-butanol:acetic acid:water (4:1:5). Synthetized NaMSA was a homogenous compound not containing untransformed morin. Elemental analysis for C, H, and S contents was used to determine the molecular formula of NaMSA. Derivatographic and gravimetric methods were used to check the number of crystalline water molecules. Sodium amount was measured using atomic absorption spectrometry. Aqueous NaMSA solution had the electronic absorption spectrum between 200 and 800 nm (two bands from pp* electronic transitions). Parameters: lI = 370 nm (e = 12,700) for band I and lII = 262 nm (e = 20,500) for band II were observed. Obtained findings confirmed the identity of morin-50 -sulfonic acid sodium salt (molecular formula: C15H9O10SNa2H2O; molar mass: 440.319) [7– 9].
The experiment was performed with the approval of the First Local Ethics Committee for Experiments on Animals in Wroclaw. The experiment After an adaptation period the animals were randomly divided into four groups of 12 animals each (6 males, 6 females): group C (the control group), receiving 0.9% saline solution at 9 a.m. and 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.; 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 the substances were dissolved in 0.9% saline solution, and the 4 mL/kg volume was administered intragastrically (i.g.) via a gastric tube for 10 consecutive days. The volume of administered saline solution was also 4 mL/kg. A 100 mg/kg dose of NaMSA was chosen on the base of the previous works, indicating that NaMSA has protective effects against acute chromium poisoning [9]. Animals were weighed and monitored every day. On the Day 11 of the experiment blood samples were collected from the tail vein for assessment of blood cell count parameters such as total white blood cell (WBC), red blood cell (RBC) and platelet (PLT) count as well as the hematocrit (HT) and hemoglobin (HGB) level, and biochemical parameters, including total protein and glucose concentrations, alanine and asparagine aminotransferases (ALT and AST), gamma-glutamyl transferase (GGTP) and amylase activities. ALT, AST and GGTP activities were expressed per gram of the liver; amylase activity was expressed per gram of protein. Then the animals were sacrificed under deep thiopental anesthesia (70 mg/kg, administered intraperitoneally). Livers were isolated and weighted. Liver index (expressed as the percentage of the body weight) was also calculated. Analyses of blood cell count parameters and biochemical parameters in serum were performed by a certified laboratory according to the commercially available methods (International certificates: RIQAS, LABQUALITY, SNCS IQAS e-CHECK). Histological assessment The liver, the pancreas, the small and large intestine were collected post mortem for histological examination. Hepatic, pancreatic and gut sections were fixed in a 4% buffered formalin solution (pH 7.4), embedded in paraffin and cut into 5 mm thick slices with a microtome. All specimens were stained with hematoxylin and eosin and blindly examined under a light microscope (40, 100 and 200 magnifications) by an experienced pathomorphologist. Hepatic and pancreatic specimens were assessed for the presence of apoptosis, necrosis, inflammatory infiltrate and hyperemia. Each intestinal segment was evaluated principally for lymphoid cell infiltrations and the presence of cellular atypia, dysplasia, adenomas or adenocarcinomas. Some microscopic abnormalities, for example, shedding of epithelium, hyperemia, undetermined intestinal inflammation, qualified as unspecific and difficult to distinguish from processing-related artifacts, were not considered for further evaluation.
Animals Statistical analysis The experiment was performed on male and female Wistar rats (aged 9–10 weeks, 203.5 g 17.6 g) obtained from the Animal Laboratory of the Department of Pathomorphology Wroclaw Medical University (Wrocław, Poland). During the experiment the animals were housed individually in chambers with a 12:12 h light-dark cycle and a temperature between 21 8C and 23 8C. Before and during the experiment, the animals had free access to standard feed and water.
Data were expressed as mean values standard deviation (SD). The statistical analysis of the effect of the drug on studied parameters was performed using the analysis of variance (ANOVA). Specific comparisons were tested with the Tukey’s test. Hypotheses were considered positively verified if p < 0.05. Statistical analysis was performed using STATISTICA 9 PL (StatSoft, Krako´w, Poland).
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Table 1 Body weight, liver weight, liver index and biochemical parameters of rats. Group C – rats receiving saline solution, group M – rats receiving with NaMSA at the dose of 100 mg/ kg, group CX – rats receiving cyclophosphamide at the dose of 15 mg/kg and group M-CX – rats receiving cyclophosphamide at the dose of 15 mg/kg with NaMSA at the dose of 100 mg/kg. Parameter
Body weight increase (g) Liver weight (g) Liver index (% of body weight) Total protein (g%) Glucose (mg%) AST (U/L/g liver) ALT (U/L/g liver) GGTP (U/L/g liver) Amylase (U/L/g protein) a b c d e f
Group C Mean SD
M Mean SD
CX Mean SD
M-CX Mean SD
57.5 31.4 14.5 3.0 5.4 0.4 5.57 0.20 181.8 20.4 10.35 4.22 4.09 1.19 0.26 0.07 15.36 2.84
51.7 42.4 12.4 2.6 4.8 0.5 5.55 0.3 192.0 16.2 11.0 3.38 4.11 0.72 0.33 0.24 15.02 3.30
18.3 13.4a 10.8 2.0c 4.9 0.4 5.07 0.18a 170.5 10.1 12.84 10.70 5.23 4.49 0.46 0.20e 13.72 3.86
23.3 13.0b 11.0 1.6b 4.9 0.5 5.13 0.22d 185.6 22.7 15.04 8.45 3.85 1.51 0.45 0.20f 12.91 3.44
CX vs. C, p < 0.001. M-CX vs. C, p < 0.005. CX vs. C, p < 0.005. M-CX vs. C, p < 0.001. CX vs. C, p < 0.05. M-CX vs. C, p < 0.05.
Results Blood cell count parameters CPX significantly decreased WBC, RBC and PLT count as well as HGB and HT levels compared to the control group. WBC count did not exceed 20% of that in the control group, PLT level was decreased to 48% of the control value and the least advanced, but still significant changes were observed in the RBC count. The addition of NaMSA partly reversed the decreased RBC level only. NaMSA alone did not exert any impact on blood cell count parameters. Body weight and blood biochemical parameters Mean values and respective SDs of the body weight, liver weight, liver index and biochemical parameters are presented in the Table 1. CPX caused a significant retardation of the mean body weight gain in comparison to the control group. Co-administration of NaMSA did not reverse that effect of CPX and a difference between the M-CX and the control group was significant, contrary to the difference between M-CX and CX groups. NaMSA alone did not alter the body weight gain. Similar changes were observed in the mean liver weight. However, when liver weight was expressed as liver index no statistically significant differences between studied groups were noticed. Moreover, loose stools were observed in the CPX-receiving group. In the M-CX group stools were formed and more regular. In the control and NaMSA-receiving groups no abnormalities in stools were noticed throughout the experiment. CPX caused a significant decrease in the mean total protein level as compared to the control group. The addition of NaMSA to the CPX did not reverse that effect of CPX. The difference between the M-CX and C group was statistically significant, but insignificant between M-CX and CX groups. NaMSA did not have any effect on the total protein level. CPX caused an increase in activities of AST, ALT and GGTP (by 24.3%, 27.9%, and 76.9%, respectively), but only in the case of GGTP the difference between CPX-receiving group and the control group was statistically significant. That effect of CPX was not reversed by NaMSA. Therefore, the difference between M-CX and C group was significant whereas the difference between the M-CX and CX group was insignificant.
No statistically important changes were noticed in the mean glucose levels and the mean amylase activity (expressed per gram of total protein) between all groups. Histological evaluation Detailed histological changes observed in the liver, the small and large intestines, presented as a percentage of evaluated samples are presented in the Table 2. Histology evaluation detected no hepatic lesions in the control group. In CPX-receiving group, blurred trabecular structure, hepatocyte apoptosis, focal and diffuse necrosis (Fig. 1) were observed. In the group receiving CPX and NaMSA hepatocyte apoptosis was also observed but in a lower number of specimens. In group M none of the mentioned abnormalities were found. Histological examination of the small intestinal specimens in the control group (group C) revealed small superficial adenomas with low-grade dysplasia and larger adenomas with high-grade dysplasia. In the CX group adenomas were found in all animals. Those precancerous changes were vast in nature and comprised the whole depth of the mucous membrane. In addition, in over 50% of specimens adenocarcinoma was revealed. In the group receiving CPX combined with NaMSA (the M-CX group) small superficial Table 2 Results of the histological examination of livers, small and large intestines. Group C – rats receiving saline solution, group M – rats receiving NaMSA at the dose of 100 mg/kg, group CX – rats receiving cyclophosphamide at the dose of 15 mg/kg and M-CX – rats receiving cyclophosphamide at the dose of 15 mg/kg with NaMSA at the dose of 100 mg/kg. Histological changes expressed as % of the studied samples
Group C
Group M
0%
0%
16.7%
36.4%
0% 0% 0%
0% 0% 0%
41.7% 25% 33.3%
18.2% 0% 0%
Small intestine Adenoma with low-grade dysplasia Adenoma with high-grade dysplasia Adenocarcinoma
44.4% 22.2% 0%
0% 0% 0%
100% 0% 55.6%
55.6% 22.2% 0%
Large intestine Atypia of the epithelial cells Adenoma
0% 0%
0% 0%
71.4% 14.3%
Liver Generalized blurred trabecular structure Hepatocyte apoptosis Focal hepatocyte necrosis Diffuse hepatocyte necrosis
Group CX
Group M-CX
0% 0%
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Fig. 1. Microscopic morphology of the liver. Focal necrosis. Group CX, receiving cyclophosphamide i.g. at the daily dose of 15 mg/kg for 10 days (hematoxylin-eosin staining, 200 magnification).
adenomas were also found, as well as extensive adenomas with high-grade dysplasia. That kind of changes were not present in the M group. Microscopic assessment of the large intestine showed no abnormalities in the control group, in the group receiving M and in the M-CX group. In the CX group the presence of atypical epithelial cells and adenomas was found (Fig. 2). Microscopic evaluation of the pancreas revealed no abnormalities in any of the studied groups. Discussion The objective of this study was to assess the impact of coadministration of NaMSA on CPX-related adverse reactions in the hematological system and the gastrointestinal tract. To our best knowledge no paper has been published on this subject. Changes in blood cells count observed in the current study were very similar to those disclosed in our earlier work [6] and also to those noticed in humans during the treatment with CPX. Pronounced leukopenia and thrombocytopenia are commonly observed during the first 7–14 days of treatment, with anemia observed later, after several cycles of treatment [1]. The effect on
Fig. 2. Microscopic morphology of the large intestine. Adenoma. Group CX, receiving cyclophosphamide i.g. at the daily dose of 15 mg/kg for 10 days (hematoxylin–eosin staining, 40 magnification).
red blood cells may be related to the CPX-induced lower level of mRNA for erythropoietin, described in other papers [10]. Although some flavonoids were able to reverse the CPX-induced leukopenia and thrombocytopenia in rats [11], we did not observe any significant improvement of those two parameters when CPX was administered with NaMSA for 10 days. CPX inhibited body weight gain and NaMSA did not reverse that trend. In contrast to natural morin, tested in our previous study [6], NaMSA did not aggravate body weight loss. NaMSA given alone did not affect body weight gain. It is well known that the use of many cytotoxic drugs causes nausea and/or vomiting resulting in anorectic action and therefore, slower body weight increase [1]. In our study loose stools were also observed in the CPX group. Lower body weight gain in the CX group may be, at least partly, caused by dehydration due to both lower water intake and/or mucositis in the oral cavity of animals. Further studies require a model involving metabolic cases and detailed measurements of water and food intake. Similarly to our earlier work with natural morin, we did not observe any differences in glucose levels between studied groups. In our work, neither endocrine nor exocrine function of the pancreas was influenced by CPX. The activity of amylase was not significantly changed in any of the groups, similarly to the previous work [6]. No histological changes of the organ were observed as well. No abnormalities were found in the group receiving NaMSA alone. That may indicate that the substance demonstrates no tissue toxicity. A significant intestinal protein loss following a single, intraperitoneal dose (80 mg/kg) of CPX was described nearly 30 years ago by Bero and Javor [12]. Similar changes are probably present with repeated administration of lower doses, therefore CPX in the current work caused a significant decrease in total serum protein level that was not reversed by addition of NaMSA. CPX caused also significant increase of the GGTP activity not prevented by NaMSA, but in comparison with our previous work [6] NaMSA did not aggravate changes of liver enzyme activities. It is a quite well-known fact that drug toxicity is rarely limited only to one particular organ [1]. Alkylating agents are very often toxic to many tissues and organs, e.g. liver [13], and any noticed changes in the liver structure are of the great clinical importance [14]. As it is pointed out [13], crucial changes in histopathological evaluation are not always associated with alterations in biochemical parameters such as bilirubin and albumin levels or ALT activity. As it was also demonstrated in our work, some significant changes in histological structure may be accompanied by only minor biochemical findings. Sinusoidal obstruction syndrome (SOS) also known as venoocclusive disease and an increase in the total serum bilirubin level are probably the most characteristic clinical manifestations of CPXrelated hepatotoxicity. In vitro, hepatic sinusoidal endothelial cells become injured by metabolites of CPX generated within hepatocytes, and damage of the hepatic microcirculation leads to the development of liver dysfunction following anticancer chemotherapy [15]. Toxic metabolites formed in consequence of administration of CPX are phosphoramide mustard and acrolein. Depletion of reduced glutathione in hepatocytes and sinusoidal endothelial cells by CPX makes them more vulnerable [16]. In our study significant histological changes were observed in almost all specimens in the CX group. We believe that the absence of typical signs of SOS in our study results from administration of CPX alone – insufficient for development of extensive sinusoidal injury. Moreover, in our experimental model we did not observe any significant glutathione depletion in the liver, but in kidneys [17]. Data concerning intestinal toxicity of CPX are scant. Moreover, damage to intestinal mucosa is not easy to detect. Mucositis is a very serious and common complication of chemotherapy [5,18]. In animal models of mucositis following administration of various
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anticancer drugs, alone or in combination, massive crypt disruption and lymphoid cell infiltration in the mucosa were observed. Those changes were accompanied by severe villus atrophy and thickening of muscularis externa and were more pronounced in the jejunum than in the ileum [5]. Decreased villus height and crypt depth were noticed following the administration of a high single dose of CPX (300 mg/kg intraperitoneally) [4]. In a very early work of Bero and Javor [12] necrotic cells and mitosis arrest in metaphase as well as severe villous atrophy and mucosal erosions were noticed in the small intestine, 24 h and 48 h after a single peritoneal administration of CPX, respectively. In addition, exposure to CPX involves genotoxic hazards and may lead to malignancies. CPX belongs to the group 1 of human carcinogens [19]. In our study we also observed some histological changes in the small intestine such as adenocarcinoma in the CPXreceiving group, that were not detected in any specimen in the control or M-CX group. In addition, in the large intestine atypia of epithelial cells, adenomas and lymphoma-like infiltrations were noticed in the CPX-receiving group only. Data regarding secondary neoplasm development following a CPX administration are quite well known but concern mainly the urinary bladder or urinary tract cancer, as well as acute leukemias [1]. To our best knowledge there are no data concerning NaMSA protection against genotoxic potential of CPX. Conclusions To sum up, CPX induces some significant changes in peripheral blood cell count parameters and in the structure and function of the liver and intestines. Presented results demonstrate that NaMSA may diminish diarrhea and offer protection against some CPXinduced histological abnormalities, including intestinal neoplasia in rats, but it seems that NaMSA is not a promising protective agent against CPX-induced hepatotoxicity or hematological changes in rats. Funding The study was financially supported by Wroclaw Medical University research grant number ST-346. Conflict of interest We confirm that there is no conflict of interest.
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Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Impact of morin-50 -sulfonic acid sodium salt on cyclophosphamide-induced gastrointestinal toxicity in rats Anna Merwid-La˛d a,*, Dorota Ksia˛dzyna a, Agnieszka Hałon´ b, Ewa Chlebda-Sieragowska a, Małgorzata Trocha a, Marta Szandruk a, Tomasz Sozan´ski a, Jan Magdalan a, Maria Kopacz c, Anna Kuz´niar c, Dorota Nowak c, Małgorzata Pies´niewska a, Adam Szela˛g a a
Department of Pharmacology, Wroclaw Medical University, Wrocław, Poland Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wrocław, Poland c Department of Inorganic and Analytical Chemistry, Chemical Faculty, University of Technology, Rzeszo´w, Poland b
A R T I C L E I N F O
Article history: Received 29 April 2014 Received in revised form 6 May 2015 Accepted 21 May 2015 Available online 4 June 2015 Keywords: Cyclophosphamide NaMSA Liver Intestines Toxicity
A B S T R A C T
Background: The aim of this study was to evaluate the effect of morin-50 -sulfonic acid sodium salt (NaMSA) on cyclophosphamide-induced gastrointestinal changes in rats. Methods: Rats received intragastrically 0.9% saline (group C), cyclophosphamide (15 mg/kg) (group CX), NaMSA (100 mg/kg) (group M) or cyclophosphamide (15 mg/kg) with NaMSA (100 mg/kg) (group MCX), respectively, for 10 days. Results: No histological lesions were observed in the liver and the large intestine in the control group and group receiving NaMSA. In the cyclophosphamide-treated group, a generalized blurred trabecular structure, hepatocyte apoptosis, focal and diffuse necrosis were noticed in the liver and atypia of epithelial cells or adenoma were noticed in the large intestine. In the group receiving both cyclophosphamide and NaMSA, hepatocyte apoptosis in the liver was observed less frequently. Histological examination of the small intestine revealed: low-grade dysplasia adenoma in the C, M, CX and M-CX group (in 44%, 0%, 100%, and 55.6% of specimens, respectively) with adenocarcinoma in 55.6% of specimens in the cyclophosphamide-receiving group only. Adenoma with high-grade dysplasia was observed in the control and NaMSA-receiving group with a similar frequency (22%). In addition to the histological evaluation, blood cell count parameters, as well as total protein concentration, blood glucose level, amylase, ALT, AST and GGTP activities were evaluated. Cyclophosphamide impaired weight gain, decreased blood cell count parameters and total protein concentration, and increased the GGTP activity. Those changes were not reversed by NaMSA. Conclusions: Summing up, NaMSA may protect against some cyclophosphamide-induced histological abnormalities in the gastrointestinal tract, including intestinal neoplasia in rats. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction
Abbreviations: ALT, alanine aminotransferase; AST, asparagine aminotransferase; CPX, cyclophosphamide; GGTP, gamma-glutamyl transferase; HGB, hemoglobin; HT, hematocrit; i.g., intragastrically; LABQUALITY, External Quality Assessment Scheme; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; NaMSA, morin-50 -sulfonic acid sodium salt; PLT, platelets; RBC, red blood cells; RIQAS, Randox International Quality Assessment Scheme; SD, standard deviation; SNCS IQAS e-CHECK, Sysmex International Quality Assurance System; SOS, sinusoidal obstruction syndrome; WBC, white blood cells. * Corresponding author. E-mail address: [email protected] (A. Merwid-La˛d).
Oral cyclophosphamide (CPX) plays a very important role in the treatment of some autoimmune disorders, but its use is limited by hematological and gastrointestinal adverse effects. Rapidly dividing cells of the bone marrow and the gastrointestinal mucosa are particularly susceptible to toxic effects of alkylating agents such as CPX. Bone marrow toxicity results in decreased leukocyte and platelet counts and, to a lesser extent, red blood cells count. Additionally, gastrointestinal adverse effects, including anorexia, nausea, vomiting, diarrhea, stomatitis, hemorrhagic colitis, acute pancreatitis as well as hepatic injury with jaundice and elevation of liver transaminases and/or GGTP activities are described in
http://dx.doi.org/10.1016/j.pharep.2015.05.020 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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humans. Clinical symptoms and abnormal laboratory tests are accompanied by ultrastructural changes [1]. CPX-induced hepatotoxicity in humans is dose-dependent and characterized by cholestasis and cytolysis with three histological patterns described: massive hepatic necrosis, necrosis of perivenous hepatocytes and diffuse hepatocellular damage with mild steatosis [2]. These effects are also observed in animal models. CPX increased chromosomal aberrations of bone marrow cells, caused myelosuppression, leucopenia, thrombocytopenia and macrocytic normochromic anemia in rats [3]. In rats, mucositis is a wellestablished injury following administration of CPX alone [4] or with other anticancer drugs [5]. Many attempts were made to find substances preventing gastrointestinal toxicity of anticancer drugs [4]. Our previous study demonstrated that morin, a naturally occurring flavonoid, partly reversed CPX-induced decrease in RBC count and completely reversed CPX-induced hypoproteinemia [6]. Morin is practically insoluble in water and that is one of its most important disadvantages. On the other hand, morin-5’sulfonic acid sodium salt (NaMSA) is not only water soluble but also has antioxidative properties and low toxicity similar to the original compound [7,8]. However, to our best knowledge, little is known about its possible protective action against CPX-induced hematological and gastrointestinal toxicity in rats. The aim of the present study was to evaluate the effects of NaMSA on CPX-induced liver, pancreatic and intestinal toxicity in rats, using laboratory tests and histological evaluation.
Material and methods Chemicals Cyclophosphamide (CPX) (Sigma, Seelze, Germany), 0.9% NaCl solution (Polpharma S.A., Starogard Gdan´ski, Poland), and thiopental (0.5 g vials, Biochemie, Kundl, Austria) were used in the study. Morin-50 -sulfonic acid sodium salt (NaMSA) was synthesized in the Department of Inorganic and Analytical Chemistry, University of Technology in Rzeszow, Poland, according to the methodology described previously. Thin-layer chromatography on alumina plates from MERCK (covered with silica gel 60 WF254s) was used to check the purity of the NaMSA. The compounds of mobile phase were as follows: n-butanol:acetic acid:water (4:1:5). Synthetized NaMSA was a homogenous compound not containing untransformed morin. Elemental analysis for C, H, and S contents was used to determine the molecular formula of NaMSA. Derivatographic and gravimetric methods were used to check the number of crystalline water molecules. Sodium amount was measured using atomic absorption spectrometry. Aqueous NaMSA solution had the electronic absorption spectrum between 200 and 800 nm (two bands from pp* electronic transitions). Parameters: lI = 370 nm (e = 12,700) for band I and lII = 262 nm (e = 20,500) for band II were observed. Obtained findings confirmed the identity of morin-50 -sulfonic acid sodium salt (molecular formula: C15H9O10SNa2H2O; molar mass: 440.319) [7– 9].
The experiment was performed with the approval of the First Local Ethics Committee for Experiments on Animals in Wroclaw. The experiment After an adaptation period the animals were randomly divided into four groups of 12 animals each (6 males, 6 females): group C (the control group), receiving 0.9% saline solution at 9 a.m. and 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.; 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 the substances were dissolved in 0.9% saline solution, and the 4 mL/kg volume was administered intragastrically (i.g.) via a gastric tube for 10 consecutive days. The volume of administered saline solution was also 4 mL/kg. A 100 mg/kg dose of NaMSA was chosen on the base of the previous works, indicating that NaMSA has protective effects against acute chromium poisoning [9]. Animals were weighed and monitored every day. On the Day 11 of the experiment blood samples were collected from the tail vein for assessment of blood cell count parameters such as total white blood cell (WBC), red blood cell (RBC) and platelet (PLT) count as well as the hematocrit (HT) and hemoglobin (HGB) level, and biochemical parameters, including total protein and glucose concentrations, alanine and asparagine aminotransferases (ALT and AST), gamma-glutamyl transferase (GGTP) and amylase activities. ALT, AST and GGTP activities were expressed per gram of the liver; amylase activity was expressed per gram of protein. Then the animals were sacrificed under deep thiopental anesthesia (70 mg/kg, administered intraperitoneally). Livers were isolated and weighted. Liver index (expressed as the percentage of the body weight) was also calculated. Analyses of blood cell count parameters and biochemical parameters in serum were performed by a certified laboratory according to the commercially available methods (International certificates: RIQAS, LABQUALITY, SNCS IQAS e-CHECK). Histological assessment The liver, the pancreas, the small and large intestine were collected post mortem for histological examination. Hepatic, pancreatic and gut sections were fixed in a 4% buffered formalin solution (pH 7.4), embedded in paraffin and cut into 5 mm thick slices with a microtome. All specimens were stained with hematoxylin and eosin and blindly examined under a light microscope (40, 100 and 200 magnifications) by an experienced pathomorphologist. Hepatic and pancreatic specimens were assessed for the presence of apoptosis, necrosis, inflammatory infiltrate and hyperemia. Each intestinal segment was evaluated principally for lymphoid cell infiltrations and the presence of cellular atypia, dysplasia, adenomas or adenocarcinomas. Some microscopic abnormalities, for example, shedding of epithelium, hyperemia, undetermined intestinal inflammation, qualified as unspecific and difficult to distinguish from processing-related artifacts, were not considered for further evaluation.
Animals Statistical analysis The experiment was performed on male and female Wistar rats (aged 9–10 weeks, 203.5 g 17.6 g) obtained from the Animal Laboratory of the Department of Pathomorphology Wroclaw Medical University (Wrocław, Poland). During the experiment the animals were housed individually in chambers with a 12:12 h light-dark cycle and a temperature between 21 8C and 23 8C. Before and during the experiment, the animals had free access to standard feed and water.
Data were expressed as mean values standard deviation (SD). The statistical analysis of the effect of the drug on studied parameters was performed using the analysis of variance (ANOVA). Specific comparisons were tested with the Tukey’s test. Hypotheses were considered positively verified if p < 0.05. Statistical analysis was performed using STATISTICA 9 PL (StatSoft, Krako´w, Poland).
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Table 1 Body weight, liver weight, liver index and biochemical parameters of rats. Group C – rats receiving saline solution, group M – rats receiving with NaMSA at the dose of 100 mg/ kg, group CX – rats receiving cyclophosphamide at the dose of 15 mg/kg and group M-CX – rats receiving cyclophosphamide at the dose of 15 mg/kg with NaMSA at the dose of 100 mg/kg. Parameter
Body weight increase (g) Liver weight (g) Liver index (% of body weight) Total protein (g%) Glucose (mg%) AST (U/L/g liver) ALT (U/L/g liver) GGTP (U/L/g liver) Amylase (U/L/g protein) a b c d e f
Group C Mean SD
M Mean SD
CX Mean SD
M-CX Mean SD
57.5 31.4 14.5 3.0 5.4 0.4 5.57 0.20 181.8 20.4 10.35 4.22 4.09 1.19 0.26 0.07 15.36 2.84
51.7 42.4 12.4 2.6 4.8 0.5 5.55 0.3 192.0 16.2 11.0 3.38 4.11 0.72 0.33 0.24 15.02 3.30
18.3 13.4a 10.8 2.0c 4.9 0.4 5.07 0.18a 170.5 10.1 12.84 10.70 5.23 4.49 0.46 0.20e 13.72 3.86
23.3 13.0b 11.0 1.6b 4.9 0.5 5.13 0.22d 185.6 22.7 15.04 8.45 3.85 1.51 0.45 0.20f 12.91 3.44
CX vs. C, p < 0.001. M-CX vs. C, p < 0.005. CX vs. C, p < 0.005. M-CX vs. C, p < 0.001. CX vs. C, p < 0.05. M-CX vs. C, p < 0.05.
Results Blood cell count parameters CPX significantly decreased WBC, RBC and PLT count as well as HGB and HT levels compared to the control group. WBC count did not exceed 20% of that in the control group, PLT level was decreased to 48% of the control value and the least advanced, but still significant changes were observed in the RBC count. The addition of NaMSA partly reversed the decreased RBC level only. NaMSA alone did not exert any impact on blood cell count parameters. Body weight and blood biochemical parameters Mean values and respective SDs of the body weight, liver weight, liver index and biochemical parameters are presented in the Table 1. CPX caused a significant retardation of the mean body weight gain in comparison to the control group. Co-administration of NaMSA did not reverse that effect of CPX and a difference between the M-CX and the control group was significant, contrary to the difference between M-CX and CX groups. NaMSA alone did not alter the body weight gain. Similar changes were observed in the mean liver weight. However, when liver weight was expressed as liver index no statistically significant differences between studied groups were noticed. Moreover, loose stools were observed in the CPX-receiving group. In the M-CX group stools were formed and more regular. In the control and NaMSA-receiving groups no abnormalities in stools were noticed throughout the experiment. CPX caused a significant decrease in the mean total protein level as compared to the control group. The addition of NaMSA to the CPX did not reverse that effect of CPX. The difference between the M-CX and C group was statistically significant, but insignificant between M-CX and CX groups. NaMSA did not have any effect on the total protein level. CPX caused an increase in activities of AST, ALT and GGTP (by 24.3%, 27.9%, and 76.9%, respectively), but only in the case of GGTP the difference between CPX-receiving group and the control group was statistically significant. That effect of CPX was not reversed by NaMSA. Therefore, the difference between M-CX and C group was significant whereas the difference between the M-CX and CX group was insignificant.
No statistically important changes were noticed in the mean glucose levels and the mean amylase activity (expressed per gram of total protein) between all groups. Histological evaluation Detailed histological changes observed in the liver, the small and large intestines, presented as a percentage of evaluated samples are presented in the Table 2. Histology evaluation detected no hepatic lesions in the control group. In CPX-receiving group, blurred trabecular structure, hepatocyte apoptosis, focal and diffuse necrosis (Fig. 1) were observed. In the group receiving CPX and NaMSA hepatocyte apoptosis was also observed but in a lower number of specimens. In group M none of the mentioned abnormalities were found. Histological examination of the small intestinal specimens in the control group (group C) revealed small superficial adenomas with low-grade dysplasia and larger adenomas with high-grade dysplasia. In the CX group adenomas were found in all animals. Those precancerous changes were vast in nature and comprised the whole depth of the mucous membrane. In addition, in over 50% of specimens adenocarcinoma was revealed. In the group receiving CPX combined with NaMSA (the M-CX group) small superficial Table 2 Results of the histological examination of livers, small and large intestines. Group C – rats receiving saline solution, group M – rats receiving NaMSA at the dose of 100 mg/kg, group CX – rats receiving cyclophosphamide at the dose of 15 mg/kg and M-CX – rats receiving cyclophosphamide at the dose of 15 mg/kg with NaMSA at the dose of 100 mg/kg. Histological changes expressed as % of the studied samples
Group C
Group M
0%
0%
16.7%
36.4%
0% 0% 0%
0% 0% 0%
41.7% 25% 33.3%
18.2% 0% 0%
Small intestine Adenoma with low-grade dysplasia Adenoma with high-grade dysplasia Adenocarcinoma
44.4% 22.2% 0%
0% 0% 0%
100% 0% 55.6%
55.6% 22.2% 0%
Large intestine Atypia of the epithelial cells Adenoma
0% 0%
0% 0%
71.4% 14.3%
Liver Generalized blurred trabecular structure Hepatocyte apoptosis Focal hepatocyte necrosis Diffuse hepatocyte necrosis
Group CX
Group M-CX
0% 0%
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Fig. 1. Microscopic morphology of the liver. Focal necrosis. Group CX, receiving cyclophosphamide i.g. at the daily dose of 15 mg/kg for 10 days (hematoxylin-eosin staining, 200 magnification).
adenomas were also found, as well as extensive adenomas with high-grade dysplasia. That kind of changes were not present in the M group. Microscopic assessment of the large intestine showed no abnormalities in the control group, in the group receiving M and in the M-CX group. In the CX group the presence of atypical epithelial cells and adenomas was found (Fig. 2). Microscopic evaluation of the pancreas revealed no abnormalities in any of the studied groups. Discussion The objective of this study was to assess the impact of coadministration of NaMSA on CPX-related adverse reactions in the hematological system and the gastrointestinal tract. To our best knowledge no paper has been published on this subject. Changes in blood cells count observed in the current study were very similar to those disclosed in our earlier work [6] and also to those noticed in humans during the treatment with CPX. Pronounced leukopenia and thrombocytopenia are commonly observed during the first 7–14 days of treatment, with anemia observed later, after several cycles of treatment [1]. The effect on
Fig. 2. Microscopic morphology of the large intestine. Adenoma. Group CX, receiving cyclophosphamide i.g. at the daily dose of 15 mg/kg for 10 days (hematoxylin–eosin staining, 40 magnification).
red blood cells may be related to the CPX-induced lower level of mRNA for erythropoietin, described in other papers [10]. Although some flavonoids were able to reverse the CPX-induced leukopenia and thrombocytopenia in rats [11], we did not observe any significant improvement of those two parameters when CPX was administered with NaMSA for 10 days. CPX inhibited body weight gain and NaMSA did not reverse that trend. In contrast to natural morin, tested in our previous study [6], NaMSA did not aggravate body weight loss. NaMSA given alone did not affect body weight gain. It is well known that the use of many cytotoxic drugs causes nausea and/or vomiting resulting in anorectic action and therefore, slower body weight increase [1]. In our study loose stools were also observed in the CPX group. Lower body weight gain in the CX group may be, at least partly, caused by dehydration due to both lower water intake and/or mucositis in the oral cavity of animals. Further studies require a model involving metabolic cases and detailed measurements of water and food intake. Similarly to our earlier work with natural morin, we did not observe any differences in glucose levels between studied groups. In our work, neither endocrine nor exocrine function of the pancreas was influenced by CPX. The activity of amylase was not significantly changed in any of the groups, similarly to the previous work [6]. No histological changes of the organ were observed as well. No abnormalities were found in the group receiving NaMSA alone. That may indicate that the substance demonstrates no tissue toxicity. A significant intestinal protein loss following a single, intraperitoneal dose (80 mg/kg) of CPX was described nearly 30 years ago by Bero and Javor [12]. Similar changes are probably present with repeated administration of lower doses, therefore CPX in the current work caused a significant decrease in total serum protein level that was not reversed by addition of NaMSA. CPX caused also significant increase of the GGTP activity not prevented by NaMSA, but in comparison with our previous work [6] NaMSA did not aggravate changes of liver enzyme activities. It is a quite well-known fact that drug toxicity is rarely limited only to one particular organ [1]. Alkylating agents are very often toxic to many tissues and organs, e.g. liver [13], and any noticed changes in the liver structure are of the great clinical importance [14]. As it is pointed out [13], crucial changes in histopathological evaluation are not always associated with alterations in biochemical parameters such as bilirubin and albumin levels or ALT activity. As it was also demonstrated in our work, some significant changes in histological structure may be accompanied by only minor biochemical findings. Sinusoidal obstruction syndrome (SOS) also known as venoocclusive disease and an increase in the total serum bilirubin level are probably the most characteristic clinical manifestations of CPXrelated hepatotoxicity. In vitro, hepatic sinusoidal endothelial cells become injured by metabolites of CPX generated within hepatocytes, and damage of the hepatic microcirculation leads to the development of liver dysfunction following anticancer chemotherapy [15]. Toxic metabolites formed in consequence of administration of CPX are phosphoramide mustard and acrolein. Depletion of reduced glutathione in hepatocytes and sinusoidal endothelial cells by CPX makes them more vulnerable [16]. In our study significant histological changes were observed in almost all specimens in the CX group. We believe that the absence of typical signs of SOS in our study results from administration of CPX alone – insufficient for development of extensive sinusoidal injury. Moreover, in our experimental model we did not observe any significant glutathione depletion in the liver, but in kidneys [17]. Data concerning intestinal toxicity of CPX are scant. Moreover, damage to intestinal mucosa is not easy to detect. Mucositis is a very serious and common complication of chemotherapy [5,18]. In animal models of mucositis following administration of various
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anticancer drugs, alone or in combination, massive crypt disruption and lymphoid cell infiltration in the mucosa were observed. Those changes were accompanied by severe villus atrophy and thickening of muscularis externa and were more pronounced in the jejunum than in the ileum [5]. Decreased villus height and crypt depth were noticed following the administration of a high single dose of CPX (300 mg/kg intraperitoneally) [4]. In a very early work of Bero and Javor [12] necrotic cells and mitosis arrest in metaphase as well as severe villous atrophy and mucosal erosions were noticed in the small intestine, 24 h and 48 h after a single peritoneal administration of CPX, respectively. In addition, exposure to CPX involves genotoxic hazards and may lead to malignancies. CPX belongs to the group 1 of human carcinogens [19]. In our study we also observed some histological changes in the small intestine such as adenocarcinoma in the CPXreceiving group, that were not detected in any specimen in the control or M-CX group. In addition, in the large intestine atypia of epithelial cells, adenomas and lymphoma-like infiltrations were noticed in the CPX-receiving group only. Data regarding secondary neoplasm development following a CPX administration are quite well known but concern mainly the urinary bladder or urinary tract cancer, as well as acute leukemias [1]. To our best knowledge there are no data concerning NaMSA protection against genotoxic potential of CPX. Conclusions To sum up, CPX induces some significant changes in peripheral blood cell count parameters and in the structure and function of the liver and intestines. Presented results demonstrate that NaMSA may diminish diarrhea and offer protection against some CPXinduced histological abnormalities, including intestinal neoplasia in rats, but it seems that NaMSA is not a promising protective agent against CPX-induced hepatotoxicity or hematological changes in rats. Funding The study was financially supported by Wroclaw Medical University research grant number ST-346. Conflict of interest We confirm that there is no conflict of interest.
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