Histological Changes of the Rat Liver After Administration of Imatinib Mesylate:An Experimental Study

Iraq J Pharm Vol. 14, No.1, 2014

Histological changes of the rat liver after administration of imatinib mesylate:an experimental study

Luma Ibrahim Khalel Al-Allaf , Hafidh Al-Ashoo Department of Anatomy, College of Medicine, University of Mosul, Mosul, Iraq. Correspondence: [email protected] Received Accepted 14.1.2014 4.3.2014 ABSTRACT Objectives: This study aims to determine the histological changes of the liver of rats after administration of a low dose or a clinically relevant high dose of Imatinib mesylate for one month in comparison to control ones. Study setting and design: This experimental study was performed over a period of four months starting from the 10th march 2013 to the 10th July 2013 and was conducted on male Albino rats purchased from Animal Houses of both Mosul Medical College, and Veterinary College, University of Mosul, Mosul, Northern Iraq. Methods: The first experiment includes40- 45 days aged rats who administered orally daily dose of 75mg/Kg of imatinib mesylate (Glivec®; Novartis) purchased from IBN-SENA Teaching Hospital , Mosul and bought from some private pharmacies for 30 days with age matched control who administered distilled water. The second experiment includes 40- 45 days aged rats who administered daily dose of 200mg/Kg orally)with age matched control who administered distilled water . Liver of rats from each experimental group were obtained. The tissues were embedded in paraffin and stained with hematoxylin-eosin and periodic acid schiff stain. Results: The histological examination of the liver tissues of groups receiving imatinib at doses of 75 mg/kg or 200mg/kg on daily for 30 days duration showed different degrees of various histological changes of damage when compared with the control group . Male rats administered with 75 mg/kg of imatinib resulted moderate degree of several histological changes. The most striking feature is disruption in radial arrangement around central vein, sinusoidal dilatation, and hepatocytes with eosinophilic cytoplasm. Perivenular inflammatory cells, accumulation of inflammatory cells. Loss of cellular outline , and loss of euchromatin of the hepatocytes .Light microscopic examination of sections obtained from liver tissues of groups receiving imatinib at dose of 200mg/kg revealed similar changes, however , these changes were more pronounced in comparison to those in low dose group. Conclusion: Imatinib causes hepatotoxicity even in low dose group (75mg/kg, however, it has a dose dependant effect but to some extent. Appropriate protective measures must be applied with anticancer treatment for improving liver function. Keywords: Imatinib mesylate, chronic myelogenous leukemia, hepatotoxicity. اﻟﺨﻼﺻﺔ أھﺪاف اﻟﺪراﺳﺔ: ﺗﮭﺪف اﻟﺪراﺳﺔ اﻟﻰ ﺗﺤﺪﯾﺪ اﻟﺘﻐﯿﺮات اﻟﻨﺴﯿﺠﯿﺔ اﻟﺤﺎﺻﻠﺔ ﻓﻰ ﻛﺒﺪ اﻟﺠﺮذان ﺟﺮاء اﻟﺘﺠﺮﯾﻊ ﺑﺎﻻﯾﻤﺎﺗﻨﺐ وﺑﺠﺮﻋﺔ ﻗﻠﯿﻠﺔ او ﺟﺮﻋﺔ ﻛﺒﯿﺮة ﺳﺮﯾﺮﯾﺎ وﺑﺎﻟﻤﻘﺎرﻧﺔ ﻣﻊ ﻣﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة. ﻣﻜﺎن اﻟﺪراﺳﺔ: ھﺬه اﻟﺪراﺳﺔ اﻟﺘﺠﺮﯾﺒﯿﺔ اﺟﺮﯾﺖ ﻓﻰ ﻏﻀﻮن ﻓﺘﺮة ارﺑﻌﺔ اﺷﮭﺮ اﺑﺘﺪاءا ﻣﻦ اﻟﻌﺎﺷﺮ ﻣﻦ اذار 2013 وﻟﻐﺎﯾﺔ اﻟﻌﺎﺷﺮ ﻣﻦ ﺗﻤﻮز ﻟﻨﻔﺲ اﻟﻌﺎم وﺷﻤﻠﺖ ﺗﺠﺮﯾﻊ اﻟﺠﺮذان اﻟﻤﮭﻘﺎء واﻟﻤﮭﺪاة ﻣﻦ ﺑﯿﺘﻰ اﻟﺤﯿﻮاﻧﺎت اﻟﺘﺎﺑﻌﯿﻦ ﻟﻜﻠﯿﺘﻰ طﺐ اﻟﻤﻮﺻﻞ واﻟﻄﺐ اﻟﺒﯿﻄﺮى، ﺟﺎﻣﻌﺔ اﻟﻤﻮﺻﻞ ﻓﻰ ﻣﺪﯾﻨﺔ اﻟﻤﻮﺻﻞ ﺷﻤﺎﻟﻰ اﻟﻌﺮاق. طﺮق اﻟﻌﻤﻞ: اول ﺗﺠﺮﺑﺔ ﺗﻀﻤﻨﺖ ﺗﺠﺮﯾﻊ اﻟﺠﺮذان ﺑﻌﻤﺮ 40-45 ﯾﻮﻣﺎ ﻋﻦ طﺮﯾﻖ اﻟﻔﻢ ﯾﻮﻣﯿﺎ ﺑﻤﻘﺪار 75 ﻣﻠﻐﺮام ﻟﻜﻞ ﻛﯿﻠﻮﻏﺮام ﻣﻦ وزن اﻟﺠﺴﻢ ﻣﻦ ﻋﻘﺎر اﻻﯾﻤﺎﺗﻨﺐ ﻣﺴﯿﻠﯿﺖ (ﻛﻠﯿﻔﻚ ،ﻧﻮﻓﺎرﺗﺲ) ﻣﮭﺪاة ﻣﻦ ﻣﺴﺘﺸﻔﻰ اﺑﻦ ﺳﯿﻨﺎ اﻟﺘﻌﻠﯿﻤﻰ ، ﻣﺪﯾﻨﺔ اﻟﻤﻮﺻﻞ اوﻣﺸﺘﺮى ﻣﻦ ﺑﻌﺾ اﻟﺼﯿﺪﻟﯿﺎت اﻟﺨﺎﺻﺔ وﻟﻤﺪة ﺷﮭﺮ ﻣﻊ ﻣﺠﻤﻮﻋﺔ ﺳﯿﻄﺮة وﺑﻨﻔﺲ اﻟﻌﻤﺮ ﺗﻢ ﺗﺠﺮﯾﻌﮭﻢ ﺑﺎﻟﻤﺎء اﻟﻤﻘﻄﺮ.اﻟﺘﺠﺮﺑﺔ اﻟﺜﺎﻧﯿﺔ ﺗﻀﻤﻨﺖ ﺗﺠﺮﯾﻊ اﻟﺠﺮذان ﺑﻌﻤﺮ 40-45 ﯾﻮﻣﺎ ﻋﻦ طﺮﯾﻖ اﻟﻔﻢ ﯾﻮﻣﯿﺎ ﺑﻤﻘﺪار 200ﻣﻠﻐﺮام ﻟﻜﻞ ﻛﯿﻠﻮﻏﺮام ﻣﻦ وزن اﻟﺠﺴﻢ ﻣﻦ ﻋﻘﺎر اﻻﯾﻤﺎﺗﻨﺐ ﻣﺴﯿﻠﯿﺖ وﻟﻤﺪة ﺷﮭﺮ ﻣﻊ ﻣﺠﻤﻮﻋﺔ ﺳﯿﻄﺮة وﺑﻨﻔﺲ

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اﻟﻌﻤﺮ ﺗﻢ ﺗﺠﺮﯾﻌﮭﻢ ﺑﺎﻟﻤﺎء اﻟﻤﻘﻄﺮ.ﺗﻢ اﺧﺬ اﻟﻜﺒﺪ وطﻤﺮه ﺑﺎﻟﺒﺎراﻓﯿﻦ وﺻﺒﻐﮫ ﺑﻌﺪ ذﻟﻚ ﺑﺎھﯿﻤﺎﺗﻮﻛﺴﻠﯿﻦ اﯾﻮﺳﯿﻦ وﺻﺒﻐﺔ ﺣﺎﻣﺾ اﻟﺒﺮﯾﻮدك –ﺷﯿﻒ . اﻟﻨﺘﺎﺋﺞ: اﺳﻔﺮ اﻟﻔﺤﺺ ﺑﺎﻟﻤﺠﮭﺮ اﻟﻀﻮﺋﻰ ﻟﻠﺸﺮاﺋﺢ اﻟﻨﺴﯿﺠﯿﺔ اﻟﻜﺒﺪﯾﺔ اﻟﺘﻰ اﺧﺬت ﻣﻦ ﻣﺠﻤﻮﻋﺘﻰ اﻟﺠﺮذان اﻟﻠﺘﯿﻦ اﺳﺘﻠﻤﺘﺎ اﻻﯾﻤﺎﺗﻨﺐ ﻋﻨﺪ ﺟﺮﻋﺔ 75 ﻣﻠﻐﺮام ﻟﻜﻞ ﻛﯿﻠﻮﻏﺮام او 200 ﻣﻠﻐﺮام ﻟﻜﻞ ﻛﯿﻠﻮﻏﺮام ﻟﻤﺪة ﺛﻼﺛﯿﻦ ﯾﻮﻣﺎ درﺟﺎت ﻣﺨﺘﻠﻔﺔ ﻟﺘﻐﺒﺮات ﻧﺴﯿﺠﯿﺔ ﻋﺪة ﻟﻠﺘﻀﺮر ﺑﺎﻟﻤﻘﺎرﻧﺔ ﻣﻊ ﺗﻠﻚ اﻟﺘﺎﺑﻌﺔ ﻟﻤﺠﻤﻮﻋﺔ اﻟﺴﯿﻄﺮة.ان ﻣﺠﻤﻮﻋﺔ ذﻛﻮراﻟﺠﺮذان اﻟﺘﻰ اﺳﺘﻠﻤﺖ اﻻﯾﻤﺎﺗﻨﺐ ﻋﻨﺪ ﺟﺮﻋﺔ 75 ﻣﻠﻐﺮام ﻟﻜﻞ ﻛﯿﻠﻮﻏﺮام اظﮭﺮت درﺟﺔ ﻣﺘﻮﺳﻄﺔ ﻟﻠﺘﻐﺒﺮات ﻧﺴﯿﺠﯿﺔ واھﻤﮭﺎ ﻛﺎن اﻟﺘﺸﻮه ﻓﻰ اﻟﺘﻨﻈﯿﻢ اﻟﻘﻄﺮى ﺣﻮل اﻟﻮرﯾﺪ اﻟﻤﺮﻛﺰى،ﺗﻮﺳﻊ اﻟﺠﯿﻮب،وﺟﻮد ﺧﻼﯾﺎ ﻛﺒﺪﯾﺔ ذات ﺳﺎﯾﺘﻮﺑﻼزم ﻣﺤﺐ ﻟﻼﯾﻮﺳﯿﻦ اﻟﺤﻤﻀﻰ ﺑﺸﺪة، وﺟﻮد ﺧﻼﯾﺎ اﻟﺘﮭﺒﯿﺔ ﺣﻮل اﻟﻮرﯾﺪ وﺗﺮاﻛﻤﮭﺎ، ﻓﻘﺪان ﺣﺪود اﻟﺨﻠﯿﺔ اﻟﺨﺎرﺟﯿﺔ وﻓﻘﺪان اﻻﻧﺴﺠﺎم اﻟﻜﺮوﻣﺎﺗﯿﻨﻰ اﻟﻄﺒﯿﻌﻰ. ان اﻟﻔﺤﺺ ﺑﺎﻟﻤﺠﮭﺮ اﻟﻀﻮﺋﻰ ﻟﻠﺸﺮاﺋﺢ اﻟﻨﺴﯿﺠﯿﺔ اﻟﺘﻰ اﺧﺬت ﻣﻦ اﻟﻤﺠﻤﻮﻋﺔ اﻟﺘﻰ اﺳﺘﻠﻤﺖ اﻻﯾﻤﺎﺗﻨﺐ 200 ﻣﻠﻐﺮام ﻟﻜﻞ ﻛﯿﻠﻮﻏﺮام اﺳﻔﺮ ﻋﻦ وﺟﻮد ﻧﻔﺲ اﻟﺘﻐﯿﺮات وﻟﻜﻦ ﻛﺎﻧﺖ اﻛﺜﺮ ﺣﺪة ﻣﻦ اﻟﺘﻰ اﺳﺘﻠﻤﺖ اﻟﺠﺮﻋﺔ اﻟﻘﻠﯿﻠﺔ. اﻻﺳﺘﻨﺘﺎﺟﺎت: ان ﻋﻘﺎر اﻻﯾﻤﺎﺗﻨﺐ اﺳﮭﻢ ﻓﻌﻼ وﺑﺼﻮرة ﻣﻠﺤﻮظﺔ ﻓﻰ ﺣﺪوث ﺳﻤﯿﺔ اﻟﻨﺴﯿﺞ ﻓﻰ اﻟﻜﺒﺪ ﺣﺘﻰ ﻋﻨﺪ اﻟﺠﺮﻋﺔ اﻟﻘﻠﯿﻠﺔ ﻋﻠﻤﺎ اﻧﮫ ﻛﺎن ﺗﺎﺛﯿﺮا ﻏﯿﺮ ﻣﺴﺘﻘﻞ ﻋﻦ ﻣﺴﺘﻮى اﻟﺠﺮﻋﺔ وﻟﻜﻦ اﻟﻰ ﺣﺪ ﻣﻌﯿﻦ. ﯾﺠﺐ ان ﯾﺘﻢ ﺗﻄﺒﯿﻖ ﻗﯿﺎﺳﺎت وﻗﺎﺋﯿﺔ ﻣﻼﺋﻤﺔ ﻣﻊ اﺳﺘﺨﺪام اﻟﻌﻼﺟﺎت اﻟﻤﻀﺎدة ﻟﻠﺴﺮطﺎن ﻟﺘﺤﺴﯿﻦ وظﺎﺋﻒ اﻟﻜﺒﺪ. اﻟﻜﻠﻤﺎت اﻟﻤﻔﺘﺎﺣﯿﺔ: اﻻﯾﻤﺎﺗﻨﺐ ﻣﺴﯿﻠﯿﺖ ، ﺳﻤﯿﺔ اﻟﻜﺒﺪ، اﺑﯿﻀﺎض اﻟﺪم اﻟﻠﻮﻛﯿﻤﻰ اﻟﻤﺰﻣﻦ hronic myelogenous leukemia cells in the body, including, hair and C (CML) is a myelo- proliferative blood cells4. disorder characterized by the presence Imatinib (Gleevec® or Glivec® of translocation t(9;22) (q34;q11) Novartis, NJ), is a selective, rationally which generates the Philadelphia (Ph) designed, c-KIT and Bcr-Abl tyrosine chromosome and the associated fusion kinase inhibitor, approved for the gene (Abelson murine leukemia) treatment of chronic myelogenous (BCR-ABL)1. leukemia (CML)5, gastrointestinal BCR-ABL1 encodes the chimeric stromal tumors (GIST)6,7, and protein BCR-ABL1 which has unresectable GIST8. deregulated tyrosine kinase activity Currently imatinib is the treatment of and leads to increased cellular choice for the management of the proliferation, resistance to apoptosis chronic and accelerated phases of and genetic instability and it is at the CML with an overall survival rate of center of CML pathogenesis, as 89% after 5 years 6,7. Besides the BCR- attested by mouse models which ABL kinase, imatinib also inhibits the replicate the disease2. CML, once expression of c-Kit tyrosine kinase considered a fatal disease, is now receptor in the gastrointestinal tract essentially a chronic disorder, and most which is involved in the pathogenesis patients can enjoy long-term survival. of gastrointestinal stromal tumors This history of success has been the (GIST) 8. In addition, imatinib inhibits result of development of tyrosine the platelet derived growth factor kinase inhibitors (TKIs), compounds (PDGF) receptor 9. which may allow which suppress the abnormal tyrosine further therapeutic applications kinase (TK) activity of the BCR-ABL1 including dermatofibrosarcoma protein 3. protuberans10, glioblastoma11, and non- Chemotherapy involves the use of cancer related pathologies like chemical agents to stop the growth and rheumatoid arthritis 12and eliminate cancer cells even at distant atherosclerosis13. sites from the origin of primary tumor. Imatinib undergoes P450 mediated However, it does not distinguish metabolism mainly via CYP3A4 and between a cancer and normal cells, and CYP3A5, and CYP1A2, CYP2D6, eliminates not only the fast-growing CYP2C9 and CYP2C19 which play a cancer cells but also other fast-growing minor role14. Imatinib and metabolites are excreted in the bile and only

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around 5% is excreted unchanged in Throughout the investigations the rats urine15,16. The main adverse effects were housed under controlled normal include severe neutropenia and environmental laboratory conditions thrombocytopenia, oedema, fluid and animal facility and were kept in an retention, nausea, mild diarrhoea, skin air-conditioned room with 12-hours rashes, arthralgia, myalgia, bone pain, light and dark cycles, where the acute renal failure and temperature (22 ± 2◦C) and relative hepatotoxicity17,18. humidity(65–70%) were kept constant. Hepatotoxicity has been They were local breaded and put observed in 5% of CML patients which individually in Animal House plastic show cytolytic hepatitis (including cages22.Animals were let to acclimatize spotty and piecemeal necrosis), hepatic for a week before any experiment was necrosis, and acute hepatitis. In spite of performed23, and provided with free the fact that hepatotoxicity resolved access of water ad libitum and pelleted after imatinib discontinuation and stanrdized food24. The experiments steroids administration, in some were performed during the light patients, the hepatic condition further portion 25. deteriorated leading to fatal liver Experimental design and procedures failure19. Histopathological findings Mean bodyweight of all rats revealed severe necrosis, cellular was 70-110 gm. The first experiment canalicular cholestasis, submassive includes 40-45 days aged rats who acute hepatic necrosis and multiacinar administered daily dose of 75mg/Kg of hepatocellular necrosis20,21. The imatinib mesylate (Glivec® Novartis) majority of TKIs approved to date are purchased from IBN-SENA Teaching reported to induce hepatic injury. Hospital or bought from some private Several studies reporting imatinib- pharmacies and were dissolved in induced hepatic lesions describe distilled water and administered orally various types of changes in hepatocyte by gavage with 24 gage needle for 30 morphology. However, It is apparent days (n=8) with age matched control that many important issues regarding who administered distilled water the potential adverse effects of following the same protocol applied to Imatinib on the liver need further imatinib group (n=4). clarification. This study aims to The second experiment evaluate the histopathological changes includes 40- 45 days aged rats who that occur in liver of rats administered administered daily dose of 200mg/Kg different regimens of imatinib mesylate orally by gavage with 24 gage needle (either low dose or high dose imatinib for 30 days (n=8)with age matched mesylate for one month duration) in control who administered distilled comparison to the control ones. water following the same protocol applied to imatinib group (n=4). Materials and Methods Imatinib doses selected were This experimental study was intended to be in the range of those performed over a period of four used in clinical treatment regimens26 months starting from the 10th march (400-800 mg/d or 340-590 mg/m2 2013 to the 10th July 2013 and was based on a weight of 70 kg) dose conducted on male Albino rats surface area adjusted to body-weight, purchased from Animal Houses of both f × mg/kg = mg/m2, where f is a Mosul Medical College, and constant equal to 6.0 in rats27.Each Veterinary College, university of animal was observed for overt signs of Mosul, Mosul, Northern Iraq. toxicity. The animals were firmly

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restrained (the animal was grasped by with molten paraffin wax at 65ºC. The the loose skin of the neck and back) to mould was transferred to a cold plate at immobilize the head and maintained in -5ºC, the tissue adjusted to the desired an upright (vertical) position. The orientation and the cassette base placed gavage needle was passed through the on top of the mould, filled with molten side of the mouth, followed the roof of wax and let to solidify for 1h, removed the mouth, and advanced into the and then stored at -20ºC until esophagus toward the stomach. After sectioning. The frozen embedded wax the needle was passed to the correct blocks were sectioned at 3-5 micron length, imatinib was injected28. thickness using Reichert-Jung Study termination procedures microtom (Austria), placed on frosted Animals in each experiment were glass slides and dried overnight at euthanized with ether22,29 24 h after the 37°C. Prior to modified Harris final dose was given at laboratory of Haematoxylin and Eosin staining and postgraduate studies of Department of periodic acid schiff stain (PAS) for Anatomy, Mosul College of Medicine, general liver structure and periodic University of Mosul. acid schiff (PAS) to demonstrate the Tissue and organs collection: Liver glycogen deposition in hepatocytes portions of rats from each experimental respectively, the samples were washed group were obtained using longitudinal in xylene twice (3 min each), hydrated thoracoabdominal incision and they in five sequential changes of alcohol were immersed in Nacl solution 0.9% 100%, 100%, 95%, 80% and 70% for 3 for few seconds in order to get rid of min each, rinsed with water for 3 min superficial blood. The liver was and stained. Finally, the stained slides excised and examined were dehydrated in three sequential 1- macroscopically. min changes of alcohol 70%,80% and Preparation of histological sections 95% and two changes of alcohol 100% Liver were fixed in 10% Neutral for three minutes each. The sections buffered formalin30, the tissues were were then dried and mounted onto the 31,32 embedded in paraffin (Merck, clean glass slides and labeled . Germany) and stained with Harris Histopathological analysis hematoxylin-eosin (Scarlau, Spain) and Histopathology changes were observed periodic acid schiff stain (PAS). The for changes in toxicokinetic assessment evaluation was blinded to treatment including sinusoidal congestion, loss of and any data. The tissue of hepatic cellular outline and lobular samples were grossed and transferred architecture, nuclear changes, and fatty 31 into cassettes and processed as follows: changes . On the other hand, (1)– Two consecutive changes of disruption in radial arrangement solution of 10% Neutral buffered around central vein, sinusoidal formalin 1 h and 1.5 h; (2)85% Ethyl dilatation, hepatocytes with alcohol (Thomas baker,(chemicals), eosinophilic cytoplasm, hydropic limited, UK)– 1.5 h; (3) 95% alcohol – degeneration (cytoplasmic 1.5 h; (4) 100% Alcohol – three vacuolization and swelling of consecutive changes of 1.5 h each; (5) hepatocyte), and loss of the glycogen xylene (Thomas baker, (chemicals), deposition in hepatocytes. These limited, UK)– three consecutive changes were grouped based on two changes of 1.5 h each; and (6) paraffin main criteria: vascular changes bath at 55ºC – two changes of 1.5 h including vessel congestion, sinusoidal each. Upon completion, the tissues dilatation, extravasation of red blood were placed in 1.5x2cm moulds lined cells and hematoma formation; and

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necrotic changes including necrosis, degrees of various histological changes fibrosis, nuclear changes, abscesses of damage when compared with the and cell regeneration33. baseline-control group. Male rats The morphological changes administered with 75 mg/kg of were assessed semi-quantitatively to imatinib resulted moderate degree of assess the extent of the several histological changes (Table 1). histopathological changes, blind by an The most striking feature is disruption independent assessor and graded as in radial arrangement around central follows: No change - 0 (no vein, sinusoidal dilatation, and distinguishable change, 0%); mild hepatocytes with eosinophilic change - 1 (initiation of changes, up to cytoplasm (Figure 3). 30%); moderate change - 2 (patent Perivenular inflammatory cells, changes, 31-60%); severe change - 3 accumulation of inflammatory cells (wide spread changes, 61-100%) for were shown in Figures 4,5,6. each criterion. Maximum score is Loss of cellular outline, and noted as 934. loss of euchromatin of the hepatocytes Photography were noticed in sections obtained from All sections were visualized in Bright the liver tissues of group receiving field Olympus microscope(Japan). imatinib at doses of 75 mg/kg (Figures Photomicrographs of representative 5,6,7). changes were taken using digital Light microscopic examination camera (Optika, Italy, HD 1080, of sections obtained from liver tissues resolution 8.0 Mega pixels) attached of groups receiving imatinib at dose of using planapochromatic objectives. 200mg/kg revealed similar changes; The magnifications of however, these changes were more photomicrograph will be indicated with pronounced in comparison to those in the legends for the photograph. low dose group (Figures 8,9,10,11,12,13). Results Moreover, high dose group The histopathology assessment in liver showed various features of nuclear was performed blindly for all groups. alterations of hepatocytes including At necropsy, no obvious tissue anisocytosis (Figure 9), presence of abnormalities were noted in the liver of dense apoptotic nuclei (Figure 12).In any animal. This study revealed that addition, swelling of hepatocytes were the light microscopic examination of also noticed (Figure 9,13). the liver sections obtained from of the Feathery degeneration was control group showed a normal shown obviously especially in sections appearance of the liver cells which obtained from liver tissues of groups appeared as normal large polygonal receiving imatinib at dose of 200mg/kg cells with prominent round nuclei and (Figure 11). eosinophilic cytoplasm, and few Microscopic damage score for spaced hepatic sinusoids arranged in- each group was determined and results between the hepatic cords with fine were given in Table 1. arrangement of Kupffer cells (Figure Figures 14,15 shows the 1,2). decreased amount of glycogen in the The histological examination of sections obtained from the liver tissues the liver tissues of groups receiving of groups receiving imatinib at doses imatinib at doses of 75 or 200mg/kg of 75 or 200mg/kg respectively. daily for 30 days showed different

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Table 1. Comparison of the effect of imatinib on microscopic structure in liver in all groups Parameter Control Group Low dose Group High dose Group Microscopic damage 0.6 4.9 5.1

Figure 1. A photomicrograph of a section obtained from the liver of rat of control group with normal histological appearance (H&E ×400).

Figure 2. A photomicrograph of section obtained from the liver of rat of control group showed The PAS-positive reaction shows a magenta staining where glycogen is present within hepatocytes (PAS ×250).

Figure 3. A photomicrograph of section obtained from the liver of rat treated with low dose of imatinib showed disruption of radial arrangement of hepatic cords, dilation of sinusoid , presence of esinophilic stained hepatocytes (H&E ×250).

Figure 4. A photomicrograph of section obtained from the liver of rat treated with low dose of imatinib showed disruption of radial arrangement of hepatic cords , presence of few perivenular inflammatory cells (H&E ×250).

Figure 5. A photomicrograph of section obtained from the liver of rat treated with low dose of imatinib showed disruption of radial arrangement of hepatic cords , dilated sinusoids, accumulation of inflammatory cells and esinophilic hepatocytes with loss of euchromatin and cellular outline(H&E ×400).

Figure 6. A photomicrograph of section obtained from the liver of rat treated with low dose of imatinib showed disruption of radial arrangement of hepatic cords , dilated sinusoids, accumulation of inflammatory cells in perivenular area and esinophilic hepatocytes with loss of euchromatin (H&E ×250).

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Figure 7. A photomicrograph of section obtained from the liver of rat treated with low dose of imatinib showed disruption of radial arrangement of hepatic cords , obliterated sinusoids, and esinophilic hepatocytes with loss of cellular outline (H&E ×400).

Figure 8. A photomicrograph of section obtained from the liver of rat treated with high dose of imatinib showed congested portal vein and sinusoids, presence of accumulated inflammatory cells with esinophilic stained hepatocytes (H&E ×400).

Figure 9. A photomicrograph of section obtained from the liver of rat treated with high dose of imatinib showed swelling of hepatocytes with degeneration and loss of euchromatin, area of dissolution of hepatic cords was noticed (H&E ×600).

Figure 10. A photomicrograph of section obtained from the liver of rat treated with high dose of imatinib showed congested portal vein, dilation of sinusoid , presence of esinophilic stained hepatocytes (H&E ×250).

Figure 11. A photomicrograph of section obtained from the liver of rat treated with high dose of imatinib showed disruption of radial arrangement of hepatic cords , esinophilic hepatocytes with loss of euchromatin and cellular outline, and feathery degeneration (H&E ×250).

Figure 12. A photomicrograph of section obtained from the liver of rat treated with high dose of imatinib showed esinophilic hepatocytes with loss of euchromatin and cellular outline, hepatocytes with apoptotic nuclei are also seen(H&E ×400).

Figure 13. A photomicrograph of section obtained from the liver of rat treated with high dose of imatinib showed esinophilic hepatocytes with loss of euchromatin and cellular outline, swelling of hepatocytes with anisocytosis are also noticed(H&E ×400).

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Figure 14. A photomicrograph of section obtained from the liver of rat treated with low dose of imatinib showed decreasing in the glycogen amount(PAS×400).

Figure 15. A photomicrograph of section obtained from the liver of rat treated with high dose of imatinib showed decreasing in the glycogen amount(PAS×250).

Discussion observed in the reported cases may be Drugs targeting tumor-specific contributed by imatinib accumulation pathways are believed to be more due to the multiple dosage effective than conventional administration31,36. On the other hand chemotherapeutic drugs, which tend to ,in humans, signs of liver dysfunction affect rapidly dividing cells in both were found in CML patients with grade normal and cancerous tissues. Targeted 3. small molecule drugs have In addition, fatal liver failure occurred revolutionized treatment of CML over in several cases21,37. The pathogenic the last decade35.The introduction of mechanisms of imatinib-induced small-molecule TKIs in clinical hepatotoxicity are unknown31. oncology has transformed the Research on the pathogenic treatment of certain forms of cancers. mechanisms of imatinib-induced However, their use has been found to hepatotoxicity suggests that toxicity be associated with serious toxic effects may be related to the P450 mediated on a number of vital organs including metabolic pathway or idiosyncratic the liver 35,36. reactions in susceptible individuals This study revealed many 38.A study which was done by Sherif histopathological abnormalities in the et al29 showed comparable results were liver. These findings are in accordance obtained with the rat’s liver following with that of Cohen et al36, who treatment with imatinib ,he suggested reported presence of hepatotoxicity that the histopathological investigation after 2 weeks of imatinib showed hydropic changes and administration in dogs. The attributed that to the increase in NO histological assessment revealed mild production (reflected in the total NOx focal hepatocellular necrosis, single content level). Augmentation of cell bile duct necrosis and bile duct cardiotoxicity following treatment with hyperplasia (associated with peribiliary imatinib with arsenic might be fibrosis)36. However, Seggara et al31 attributed to imatinib- induced PDGF revealed the histological findings in receptor and c-Ab1 blockade29. mice with lesser degree of toxicity On the other hand, chemotherapeutic which may be anticipated since they agents such as cisplatin, doxorubicin, used a single oral dose of imatinib (100 and 5-FU cause direct hepatic toxicity mg/kg); while the greater toxicity including inflammatory cells forming

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granulomatous lesions and periportal precede necrosis, as in the fibrosis and apoptosis4. hepatotoxicity induced by It has been suggested that Doxorubicin thioacetamide4 , or it may occur has been shown to induce concurrently with necrosis as in accumulation of inflammatory cells39, hepatotoxicity associated with associated with increased activities of acetaminophen31.This study showed tissue aminotransferases, lactate that the hepatotoxic effect of imatinib dehydrogenase and alkaline was more pronounced in high dose phosphatase, indicating hepatic group in comparison with that in low damage via induction of apoptosis and dose, these finding is in accordance generation of reactive oxygen with that of Kerkela et al in 200631 radicals40,41. and Sook Han et al in 2009 who A recent article suggested that reported that the response to imatinib activation of endoplasmic reticulum may be dose and tissue dependent 42 . stress after imatinib is a cause of In conclusion Appropriate protective imatinib-induced cardiomyopathy42,43. measures must be applied with Any pathological state that leads to anticancer treatment for improving increased production and/or ineffective liver function. This study provide an in scavenging of reactive oxygen species -vivo evidence, at light microscopic, of may play a crucial role in determining direct chemotherapeutic hepatotoxicity tissue injury44,45. The administration of caused by imatinib. Furthermore, this cyclophosphamide damages the study identified pathological features at liver46,47. Tissue damage due to structural level, which could be used as cyclophosphamide might be alleviated the basis for determining the due to the antioxidant property and appropriate dose of these drugs to membrane stabilizing property of reduce their hepatotoxic effects. Post- melatonin48,49,50. It is known that marketing experience with drugs such eosinophilic-stained cells show the as imatinib, lapatinib and sorafenib starting irreversible damage in the suggests that the hepatotoxic safety of tissue32. On the other hand, there were all the TKIs requires diligent degenerative changes as swelling of surveillance. hepatocytes (reversible damage) in the rat treated with methotraxate ,and that References is attributed to oxidative stress32,51. This study showed many 1. Santos FPS, Kantarjian H, Alfonso histopathological abnormalities in the Quintás-Cardama A, et al. Evolution of liver including inflammatory Therapies for Chronic Myelogenous infiltration, hyperplasia, periportal Leukemia Cancer J. 2011;17(6): 465– fibrosis, marked disruption of hepatic 476. cords and dilated blood sinusoids. 2. Gishizky ML, Johnson-White J, Many hepatocytes showed Witte ON. Efficient transplantation of karyomegaly and pyknotic nuclei BCR-ABL-induced chronic- indicating apoptosis. Cell death can myelogenous leukemia-like syndrome result from naturally occurring in mice. Proc Natl Acad Sci U S A. apoptosis(physiological apoptosis) or 1993;90(8):3755–3759. from irreparable cell injury 3. Druker BJ, Guilhot F, O'Brien SG, (pathological apoptosis) as described et al. Five-year follow-up of patients by Farber in 199452. Apoptosis is a receiving imatinib for chronic myeloid common feature of hepatotoxicity leukemia. N Engl J Med 2006; induced by many chemicals; it may 355(23):2408–2417.

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