药学学报, 2019, 54(6): 1062-1068
引用本文:
黄灿, 何法静, 杨潇, 官丽欢, 张思敏, 周艳莹, 范仕成, 姚欣鹏, 黄民, 毕惠嫦. 环磷酰胺所致小鼠肝损伤的动态变化[J]. 药学学报, 2019, 54(6): 1062-1068.
HUANG Can, HE Fa-jing, YANG Xiao, GUAN Li-huan, ZHANG Si-min, ZHOU Yan-ying, FAN Shi-cheng, YAO Xin-peng, HUANG Min, BI Hui-chang. Dynamic changes of cyclophosphamide-induced liver injury in mice[J]. Acta Pharmaceutica Sinica, 2019, 54(6): 1062-1068.

环磷酰胺所致小鼠肝损伤的动态变化
黄灿, 何法静, 杨潇, 官丽欢, 张思敏, 周艳莹, 范仕成, 姚欣鹏, 黄民, 毕惠嫦
中山大学药学院药物代谢与药动学实验室, 广东 广州 510006
摘要:
环磷酰胺(cyclophosphamide,CPA)是烷化剂类抗肿瘤药物,在体内由细胞色素P450酶代谢为4-羟基环磷酰胺发挥抗肿瘤作用。CPA除引起骨髓抑制、膀胱炎等毒性反应外,还会引起肝损伤。本研究旨在评估CPA在小鼠体内产生肝损伤的动态变化过程。雄性BALB/c小鼠单次腹腔注射CPA(200 mg·kg-1),分别于0、2、6、12和24 h后采集血清和肝脏样本进行生化和病理检测。动物实验方案经中山大学动物伦理委员会批准。结果表明,小鼠给予CPA 2 h后开始出现肝损伤,在12 h肝损伤最严重,血清天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)和丙二醛(MDA)显著升高,还原型谷胱甘肽(GSH)显著下降,广泛可见肝细胞水肿并伴有空泡变性,而24 h之后肝损伤显著改善。由于CPA产生氧化应激损伤,机体应激性激活核因子红细胞系相关因子-2(nuclear factor-erythroid 2-related factor 2,NRF2)信号通路,上调NRF2下游醌氧化还原酶1(quinine oxidoreductase 1,NQO1)、血红素加氧酶-1(heme oxygenase-1,HO-1)、谷氨酰半胱氨酸合成酶催化亚基(glutamate-cysteine ligase catalytic subunit,GCLC)和谷氨酸半胱氨酸连接酶修饰亚基(glutamate cysteine modifier subunit,GCLM)的表达,从而抵抗氧化应激损伤。本研究阐明了CPA所致肝损伤随时间的动态变化过程,并探讨了NRF2介导的机体保护机制的动态变化,为抵抗CPA所致肝损伤提供了科学数据。
关键词:    环磷酰胺      肝损伤      氧化应激      核因子红细胞系相关因子-2     
Dynamic changes of cyclophosphamide-induced liver injury in mice
HUANG Can, HE Fa-jing, YANG Xiao, GUAN Li-huan, ZHANG Si-min, ZHOU Yan-ying, FAN Shi-cheng, YAO Xin-peng, HUANG Min, BI Hui-chang
Lab of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Guangzhou 510006, China
Abstract:
Cyclophosphamide (CPA) is one of the most commonly used alkylating agents in the treatment of malignant cancer. CPA is metabolized by cytochrome P450 enzymes into 4-hydroxycyclophosphamide in vivo which can exhibit anti-tumor activity. Metabolic activation of CPA can cause adverse reactions such as myelosuppression, cystitis, and liver injury. The aim of this study was to evaluate the dynamic changes of hepatic injury induced by CPA in mice. Male BALB/c mice were injected CPA (200 mg·kg-1) intraperitoneally. Both serum and liver samples were collected at 0, 2, 6, 12 and 24 hours after dosing. The animal experiment protocol was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University. The results showed that hepatotoxicity was observed at 2 hours after CPA dosing, and the most serious liver injury was measured at 12 hours. The level of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and malondialdehyde (MDA) was significantly increased, glutathione (GSH) level was significantly decreased, hepatocyte edema and vacuolar degeneration were widely observed in liver tissue, and began to recover 24 hours after dosing. In addition, due to oxidative stress damage caused by CPA, nuclear factor-erythroid 2-related factor 2 (NRF2) signaling pathway was activated and the mRNA and protein expression of its downstream targets such as quinine oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate cysteine modifier subunit (GCLM) were up-regulated, which alleviated oxidative stress injury. In a summary, this study demonstrate the dynamic change of CPA-induced liver injury and the NRF2-mediated protective mechanisms, providing new insights into the CPA-induced liver injury.
Key words:    cyclophosphamide    liver injury    oxidative stress    nuclear factor-erythroid 2-related factor 2   
收稿日期: 2019-04-03
DOI: 10.16438/j.0513-4870.2019-0237
基金项目: 国家自然科学基金资助项目(81573489);广东省自然科学基金资助项目(2017A030311018).
通讯作者: 毕惠嫦
Email: bihchang@mail.sysu.edu.cn
相关功能
PDF(515KB) Free
打印本文
0
作者相关文章
黄灿  在本刊中的所有文章
何法静  在本刊中的所有文章
杨潇  在本刊中的所有文章
官丽欢  在本刊中的所有文章
张思敏  在本刊中的所有文章
周艳莹  在本刊中的所有文章
范仕成  在本刊中的所有文章
姚欣鹏  在本刊中的所有文章
黄民  在本刊中的所有文章
毕惠嫦  在本刊中的所有文章

参考文献:
[1] Jones SE, Collea R, Paul D, et al. Adjuvant docetaxel and cyclophosphamide plus trastuzumab in patients with HER2-amplified early stage breast cancer:a single-group, open-label, phase 2 study[J]. Lancet Oncol, 2013, 14:1121-1128.
[2] Geisler CH. Cyclophosphamide for CLL:to be or not CYP2B activated?[J]. Blood, 2013, 122:4156-4157.
[3] Chen LY, Wang XD, Huang M. Advances in the research of pharmacogenomics of cyclophosphamide[J]. Acta Pharm Sin (药学学报), 2014, 49:971-976.
[4] Zhang J, Tian Q, Zhou SF. Clinical pharmacology of cyclophosphamide and ifosfamide[J]. Curr Drug Ther, 2006, 1:55-84.
[5] Xie HJ, Yasar U, Lundgren S, et al. Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation[J]. Pharmacogenomics J, 2003, 3:53-61.
[6] Ekhart C, Gebretensae A, Rosing H, et al. Simultaneous quantification of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS)[J]. J Chromatogr B, 2007, 854:345-349.
[7] Lehman TJ, Singh C, Ramanathan A, et al. Prolonged improvement of childhood onset systemic lupus erythematosus following systematic administration of rituximab and cyclophosphamide[J]. Pediatr Rheumatol Online J, 2014, 12:3.
[8] de Jonge ME, Huitema AD, Rodenhuis S, et al. Clinical pharmacokinetics of cyclophosphamide[J]. Clin Pharmacokinet, 2005, 44:1135-1164.
[9] Subramaniam SR, Cader RA, Mohd R, et al. Low-dose cyclophosphamide-induced acute hepatotoxicity[J]. Am J Case Rep, 2013, 14:345-349.
[10] de Jonge ME, Huitema ADR, Beijnen JH, et al. High exposures to bioactivated cyclophosphamide are related to the occurrence of veno-occlusive disease of the liver following high-dose chemotherapy[J]. Br J Cancer, 2006, 94:1226-1230.
[11] McDonald GB, Slattery JT, Bouvier ME, et al. Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation[J]. Blood, 2003, 101:2043-2048.
[12] Hauck AK, Bernlohr DA. Oxidative stress and lipotoxicity[J]. J Lipid Res, 2016, 57:1976-1986.
[13] Ishii T, Itoh K, Takahashi S, et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages[J]. J Biol Chem, 2000, 275:16023-16029.
[14] Kwak MK, Wakabayashi N, Kensler TW. Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme inducers[J]. Mutat Res, 2004, 555:133-148.
[15] Min KJ, Kim JH, Jou I, et al. Adenosine induces hemeoxygenase-1 expression in microglia through the activation of phosphatidylinositol 3-kinase and nuclear factor E2-related factor 2[J]. Glia, 2008, 56:1028-1037.
[16] Fan X, Chen P, Tan H, et al. Dynamic and coordinated regulation of KEAP1-NRF2-ARE and p53/p21 signaling pathways is associated with acetaminophen injury responsive liver regeneration[J]. Drug Metab Dispos, 2014, 42:1532-1539.
[17] Uskudar Cansu D, Oztas E, Yilmaz E, et al. Cyclophosphamide-induced severe acute hepatitis in a rheumatic disease:case-based review[J]. Rheumatol Int, 2019, 39:377-385.
[18] Snyder LS, Heigh RI, Anderson ML. Cyclophosphamide-induced hepatotoxicity in a patient with Wegener's granulomatosis[J]. Mayo Clin Proc, 1993, 68:1203-1204.
[19] Pinto N, Ludeman SM, Dolan ME. Drug focus:pharmacogenetic studies related to cyclophosphamide-based therapy[J]. Pharmacogenomics, 2009, 10:1897-1903.
[20] Naranjo CA, Busto U, Sellers EM, et al. A method for estimating the probability of adverse drug reactions[J]. Clin Pharmacol Ther, 1981, 30:239-245.
[21] Mok CC, Wong WM, Shek TW, et al. Cumulative hepatotoxicity induced by continuous low-dose cyclophosphamide therapy[J]. Am J Gastroenterol, 2000, 95:845-846.
[22] Martinez-Gabarron M, Enriquez R, Sirvent AE, et al. Hepatotoxicity following cyclophosphamide treatment in a patient with MPO-ANCA vasculitis[J]. Nefrologia, 2011, 31:496-498.
[23] Gustafsson LL, Eriksson LS, Dahl ML, et al. Cyclophosphamide-induced acute liver failure requiring transplantation in a patient with genetically deficient debrisoquine metabolism:a causal relationship?[J]. J Intern Med, 1996, 240:311-314.
[24] Cleland BD, Pokorny CS. Cyclophosphamide related hepatotoxicity[J]. Aust N Z J Med, 1993, 23:408.
[25] Bacon AM, Rosenberg SA. Cyclophosphamide hepatotoxicity in a patient with systemic lupus erythematosus[J]. Ann Intern Med, 1982, 97:62-63.
[26] Meister A, Anderson ME. Glutathione[J]. Annu Rev Biochem, 1983, 52:711-760.
[27] Bhat N, Kalthur SG, Padmashali S, et al. Toxic effects of different doses of cyclophosphamide on liver and kidney tissue in swiss albino mice:a histopathological study[J]. Ethiop J Health Sci, 2018, 28:711-716.
[28] Weijl NI,Cleton FJ, Osanto S. Free radicals and antioxidants in chemotherapy-induced toxicity[J]. Cancer Treat Rev, 1997, 23:209-240.
[29] Bhatia K, Kaur M, Atif F, et al. Aqueous extract of Trigonella foenum-graecum L. ameliorates additive urotoxicity of buthionine sulfoximine and cyclophosphamide in mice[J]. Food Chem Toxicol, 2006, 44:1744-1750.
[30] Srivastava A, Shivanandappa T. Hepatoprotective effect of the root extract of Decalepis hamiltonii against carbon tetrachloride-induced oxidative stress in rats[J]. Food Chem, 2010, 118:411-417.
[31] Anderstam B, Vaca C, Harms-Ringdahl M. Lipid peroxide levels in a murine adenocarcinoma exposed to hyperthermia:the role of glutathione depletion[J]. Radiat Res, 1992, 132:296-300.
[32] Fraiser LH, Kanekal S, Kehrer JP. Cyclophosphamide toxicity. Characterising and avoiding the problem[J]. Drugs, 1991, 42:781-795.
[33] Gutteridge JMC. Lipid peroxidation and antioxidants as biomarkers of tissue damage[J]. Clin Chem, 1995, 41:1819-1828.
[34] Farombi EO, Shrotriya S, Na HK, et al. Curcumin attenuates dimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induction of heme oxygenase-1[J]. Food Chem Toxicol, 2008, 46:1279-1287.
[35] Copple IM, Goldring CE, Kitteringham NR, et al. The keap1-nrf2 cellular defense pathway:mechanisms of regulation and role in protection against drug-induced toxicity[J]. Handb Exp Pharmacol, 2010, 196:233-266.
相关文献:
1.罗飘, 楚世峰, 高岩, 罗林明, 彭兰, 陈乃宏.人参皂苷Rg1在肝脏疾病中的药理作用研究进展[J]. 药学学报, 2018,53(1): 21-27