SD大鼠灌胃盐酸丙卡巴肼与乌拉坦的多脏器碱性彗星实验研究
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摘要
目的:通过碱性彗星实验评价盐酸丙卡巴肼(PCZ)和乌拉坦(EC)对大鼠不同脏器的DNA损伤效应,验证多脏器碱性彗星实验的可行性。方法:取30只SD大鼠按体质量随机分成6组,每组5只,分别为阴性对照组(超纯水)、PCZ 75 mg/kg组、PCZ150 mg/kg组、EC 400 mg/kg组、EC 800 mg/kg组以及阳性对照组(N-乙基-N-亚硝基脲,40 mg/kg)。大鼠连续灌胃给药4 d,实验期间观察其临床症状并记录体质量,末次给药后3 h内处死大鼠,称肝、肾、肺质量,收集肝、肾、肺及外周血淋巴细胞,制备成单细胞悬液进行碱性彗星实验。样本分别经裂解、解旋、电泳、染色等步骤后,使用Komet 6.0软件进行图像分析尾DNA含量百分率(尾DNA%)、尾距。结果:与阴性对照组比较,PCZ 75、150 mg/kg组和阳性对照组大鼠给药4 d后体质量和肝、肾质量均明显降低(P<0.05或P<0.01),EC 800 mg/kg组大鼠给药4 d后仅体质量明显降低(P<0.05或P<0.01),其余差异均无统计学意义(P>0.05)。与阴性对照组比较,PCZ 75、150 mg/kg组和阳性对照组大鼠肝、肾、肺和外周血淋巴细胞的尾DNA%和尾距均明显增加(P<0.05或P<0.01),其中PCZ对肝、肺的影响程度更明显;EC 800 mg/kg组大鼠肝、肾和外周血淋巴细胞的尾DNA%和尾距均明显增加(P<0.05或P<0.01),EC 400 mg/kg组大鼠仅肾组织的尾DNA%和尾距明显增加(P<0.05)。结论:PCZ诱导的细胞DNA损伤较强,肝和肺是其遗传毒性作用的易感器官;EC诱导的细胞DNA损伤相对较弱,肾是其遗传毒性作用最敏感的器官。
OBJECTIVE:To evaluate the DNA damage response of procarbazine hydrochloride(PCZ) and ethyl carbamate(EC)to different tissues in rats by performing alkaline comet assay,to validate the feasibility of alkaline comet assay of various tissues. METHODS:Totally 30 SD rats were randomly divided into 6 groups according to body weight,with 5 rats in each group,such as negative control group(hyperpure water),PCZ 75 mg/kg group,PCZ 150 mg/kg group,EC 400 mg/kg group,EC 800 mg/kg group,positive control group(N-ethyl-N-nitrosourea 40 mg/kg). Those rats were given relevant medicine intragasttrically for4 d;clinical symptoms of rats were observed and body weight was recorded during experiment. Within 3 h after last medication,the rats were sacrificed;liver,renal and lung weight were weighed;liver,kidney,lung and peripheral blood lymphocytes were collected. The single cell suspension was prepared to perform alkaline comet assay. After lysis,unwind,electrophoresis and dying,tail DNA% and tail distance of samples were analyzed by Komet 6.0 software. RESULTS:Compared with negative control group,body weight,liver and renal weight of rats were decreased significantly in PCZ 75 mg/kg group,PCZ 150 mg/kg group and positive control group 4 d after medication(P<0.05 or P<0.01). Body weight of rats were decreased significantly in EC 800 mg/kg 4 d after medication(P<0.05 or P<0.01);there was no statistical significance(P>0.05). Compared with negative control group,tail DNA% and tail distance in liver,kidney and peripheral blood lymphocytes were increased significantly in PCZ 75 mg/kg group,PCZ 150 mg/kg group and positive control group(P<0.05 or P<0.01);PCZ showed more significant effects on liver and lung. Tail DNA% and tail distance of liver,kidney and peripheral blood lymphocytes were increased significantly in EC800 mg/kg group(P<0.05 or P<0.01),and tail DNA% and tail distance of renal tissue was increased significantly in EC 400mg/kg group(P<0.05). CONCLUSIONS:PCZ induced stronger DNA damage;liver and lung are the major genotoxicity target of PCZ. EC-induced DNA damage is relatively weak,and kidney is the most sensitive organ for EC-induced genotoxicity.
OBJECTIVE:To evaluate the DNA damage response of procarbazine hydrochloride(PCZ) and ethyl carbamate(EC)to different tissues in rats by performing alkaline comet assay,to validate the feasibility of alkaline comet assay of various tissues. METHODS:Totally 30 SD rats were randomly divided into 6 groups according to body weight,with 5 rats in each group,such as negative control group(hyperpure water),PCZ 75 mg/kg group,PCZ 150 mg/kg group,EC 400 mg/kg group,EC 800 mg/kg group,positive control group(N-ethyl-N-nitrosourea 40 mg/kg). Those rats were given relevant medicine intragasttrically for4 d;clinical symptoms of rats were observed and body weight was recorded during experiment. Within 3 h after last medication,the rats were sacrificed;liver,renal and lung weight were weighed;liver,kidney,lung and peripheral blood lymphocytes were collected. The single cell suspension was prepared to perform alkaline comet assay. After lysis,unwind,electrophoresis and dying,tail DNA% and tail distance of samples were analyzed by Komet 6.0 software. RESULTS:Compared with negative control group,body weight,liver and renal weight of rats were decreased significantly in PCZ 75 mg/kg group,PCZ 150 mg/kg group and positive control group 4 d after medication(P<0.05 or P<0.01). Body weight of rats were decreased significantly in EC 800 mg/kg 4 d after medication(P<0.05 or P<0.01);there was no statistical significance(P>0.05). Compared with negative control group,tail DNA% and tail distance in liver,kidney and peripheral blood lymphocytes were increased significantly in PCZ 75 mg/kg group,PCZ 150 mg/kg group and positive control group(P<0.05 or P<0.01);PCZ showed more significant effects on liver and lung. Tail DNA% and tail distance of liver,kidney and peripheral blood lymphocytes were increased significantly in EC800 mg/kg group(P<0.05 or P<0.01),and tail DNA% and tail distance of renal tissue was increased significantly in EC 400mg/kg group(P<0.05). CONCLUSIONS:PCZ induced stronger DNA damage;liver and lung are the major genotoxicity target of PCZ. EC-induced DNA damage is relatively weak,and kidney is the most sensitive organ for EC-induced genotoxicity.
引文
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[2]NIKOLOVA T,MARINI F,KAINA B.Genotoxicity testing:comparison of theγH2AX focus assay with the alkaline and neutral comet assays[J].Mutat Res,2017.DOI:10.1016/j.mrgentox.2017.07.004.
[3]王欣,王晨,宋捷,等.体内彗星实验联合微核实验检测化合物的遗传毒性[J].中国药房,2014,25(33):3107-3109.
[4]KAINA B.甲基化抗肿瘤药物治疗中的DNA修复:治疗效果与健康结局[J].癌变畸变突变,2016,28(5):333-341.
[5]霍桂桃,杨艳伟,刘甦苏,等.P53～(+/-)基因敲除小鼠对尿烷致癌性验证试验的敏感性研究[J].药物分析杂志,2017,32(7):1188-1195.
[6]PRADO-OCHOA MG,ABREGOREYES VH,RAMIREZ-NOGUERA P,et al.Subchronic toxicity study in rats of two new ethyl-carbamates with ixodicidal activity[J].Biomed Res Int,2014.DOI:10.1155/2014/467105.
[7]GOWD V,SU H,KARLOVSKY P,et al.Ethyl carbamate:an emerging food and environmental toxicant[J].Food Chem,2018.DOI:10.1016/j.foodchem.2017.12.072.
[8]OECD.Guidelines for the testing of chemicals No.407:repeated dose 28-day oral toxicity study in rodents[EB/OL].(2008-10-03)[2018-06-27].https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecdtg407-2008.pdf.
[9]文海若,毛志慧,陈高峰,等.SD大鼠连续灌胃给药对碱性彗星电泳和骨髓微核的影响[J].药物分析杂志,2017,37(6):1063-1070.
[10]TAO ZF,WANG X,SOUERS AJ,et al.Apoptosis-inducing agents for the treatment of cancer and immune and autoimmune diseases[J].J Physiol,2017,586(18):4541-4557.
[11]JUTRAS G,BELANGER K,LETARTE N,et al.Procarbazine,lomustine and vincristine toxicity in low-grade gliomas[J].Curr Oncol,2018,25(1):e33-e39.
[12]陈晨,盛云华,王宇,等.乌拉坦致C57BL/6J小鼠肺腺瘤动物模型方法研究[J].中国药学杂志,2018,53(9):701-706.
[13]IGAMI T,NAKAMURAY,HIROSE T,et al.Application of a three-dimensional print of a liver in hepatectomy for small tumors invisible by intraoperative ultrasonography:preliminary experience[J].World J Surg,2014,38(12):3163-3166.
[14]PARODI S,TANINGHER M,RUSSO P,et al.DNA-damaging activity in vivo and bacterial mutagenicity of sixteen aromatic amines and azo-derivatives,as related quantitatively to their carcinogenicity[J].Carcinogenesis,1981,2(12):1317-1326.
[15]HUBNER P,GROUX PM,WEIBEL B,et al.Genotoxicity of ethyl carbamate(urethane)in Salmonella,yeast and human lymphoblastoid cells[J].Mutat Res,1997,390(1/2):11-19.
[16]PU J,DENG Y,TAN X,et al.The in vivo Pig-a gene mutation assay is applied to study the genotoxicity of procarbazine hydrochloride in Sprague-Dawley rats[J].Fundam Toxicol Sci,2016,3(4):167-175.
[17]KOUL A,ABRAHAM SK.Efficacy of crocin and safranal as protective agents against genotoxic stress induced by gamma radiation,urethane and procarbazine in mice[J].Hum Exp Toxicol,2017,37(1):13-20.
[18]LABASH C,AVLASEVICH SL,CARLSON K,et al.Mouse Pig-a and micronucleus assays respond to N-ethyl-N-nitrosourea,benzo[a]pyrene,and ethyl carbamate,but not pyrene or methyl carbamate[J].Environ Mol Mutagen,2016,57(1):28-40.
[19]SUZUKI T,ITOH S,NAKAJIMA M,et al.Target organ and time-course in the mutagenicity of five carcinogens in MutaMouse:a summary report of the second collaborative study of the transgenic mouse mutation assay by JEMS/MMS[J].Mutat Res,1999,444(2):259-268.
[20]STANKOWSKI LF JR,ARADEMA MJ,LAWLOR TE,et al.Integration of Pig-a,micronucleus,chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane[J].Mutagenesis,2015,30(3):335-342.
[21]WILLIAMS CV,FLETCHER K,TIINWELL H,et al.Mutagenicity of ethyl carbamate to lacZ-transgenic mice[J].Mutagenesis,1998,13(2):133-137.
[22]INTERNATIONAL AGENCY FOR RESEARCH ON CA-NCER(IARC).IARC Monographs on the evaluation of the carcinogenic risk of chemicals to man:some anti-thyroid and related substances,nitrofurans and industrial chemicals:volume 7[EB/OL].[2018-11-09].http://publications.iarc.fr/book-and-report-series/iarc-monographs-onthe-evaluation-of-carcinogenic-risks-to-humans/some-antithyroid-and-related-substances-nitrofurans-and-industrialchemicals-1974.
[23]INTERNATIONAL CONFERENCE ON HARMONIZA-TION(ICH).Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1):step 4 version[EB/OL].(2011-11-09)[2018-06-27].http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S2_R1/Step4/S2R1_Step4.pdf.
[24]OECD.Guidelines for the Testing of Chemicals No.489:In vivo mammalian alkaline comet assay[EB/OL].(2014-09-26)[2018-06-27].https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecdtg489-2014.pdf.
[25]国家食品药品监督管理总局.药物遗传毒性研究技术指导原则[S].2018.
[26]UNO Y,KOJIMA H,HAYASHI M.The JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay[J].Mutat Res,2015.DOI:10.1016/j.mrgentox.2015.04.010.