不同焦油释放量卷烟诱导TK基因突变能力的比较研究
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摘要
目的:研究卷烟烟气冷凝物对TK基因的致突变作用,并比较不同焦油释放量卷烟样品的TK基因致突变能力。方法:以盒标焦油为5、8和11 mg的市售卷烟(分别为1、2和3号样品)烟气冷凝物为受试物,分别以20、40、60、80、100、150μg/mL的卷烟烟气冷凝物对小鼠淋巴瘤细胞L5178Y tk+/-3.7.2C进行染毒处理,以15μL/mL的二甲基亚砜为溶剂对照,3μg/mL的环磷酰胺为阳性对照,处理3 h,细胞洗涤重新接种培养2 d,加入三氟胸苷继续培养12 d后,计数有突变集落生长的孔数,计算各样品剂量组的三氟胸苷抗性突变频率,并比较不同样品的致突变强度。结果:1号样品在剂量为150μg/mL时、2号样品和3号样品在剂量为100μg/mL时,小鼠淋巴瘤细胞三氟胸苷抗性突变频率是溶剂对照组3倍以上,并且随着卷烟烟气冷凝物剂量的增加,三氟胸苷抗性突变频率呈上升趋势,呈现明显的剂量-反应关系,3种卷烟样品的TK基因致突变强度分别为0.37、0.36和0.38。结论:3种卷烟样品的烟气冷凝物均对小鼠淋巴瘤细胞表现出TK基因致突变作用,比较3种样品的致突变强度发现,卷烟烟气的致突变强度与焦油释放量无明显相关关系。
OBJECTIVE:To study the mutagenicity of cigarette smoke condensates(CSC) in the TK gene mutation assay and to compare the mutagenicity of TK gene in cigarette samples with different tar release.METHODS:3 commercial cigarettes of different tar releases,5 mg(1#),8 mg(2#) and 11 mg(3#),weretested.The mouse lymphoma cell line L5178 Y tk+/-3.7.2C was treated with 20 μg/mL,40 μg/mL,60 μg/mL,80 μg/mL,100 μg/mL and 150 μg/mL CSC from the 3 cigarette samples.15 μL/mL DMSO treatment wasused as the solvent control,and 3 μg/mL cyclophosphamide treatment was used as the positive control.Thetest protocol includeds the 4-h treatment,2-day expression and 12-day cloning phases.Then the mutationfrequency of trifluorothymidine resistance(TMF) of each dose group was calculated.Finally,the mutagenicpotency of different cigarette samples was compared.RESULTS:At the dose of 150 μg/mL of 1#,100 μg/mLof 2#and 3#,the TMF were more than 3 times higher than that in the solvent control group.In addition,theTMF increased with increase of the CSC doses.The mutagenic potency of the CSCs for 3 cigarette sampleswere 0.37,0.36 and 0.38,respectively.CONCLUSION:All CSCs induced dose-dependent increases ofmutagenicity in the mouse lymphoma assay.Comparing the mutagenicity of the 3 cigarette samples,there wasno significant correlation between TK gene mutagenic potency and tar release in cigarette smoke.
OBJECTIVE:To study the mutagenicity of cigarette smoke condensates(CSC) in the TK gene mutation assay and to compare the mutagenicity of TK gene in cigarette samples with different tar release.METHODS:3 commercial cigarettes of different tar releases,5 mg(1#),8 mg(2#) and 11 mg(3#),weretested.The mouse lymphoma cell line L5178 Y tk+/-3.7.2C was treated with 20 μg/mL,40 μg/mL,60 μg/mL,80 μg/mL,100 μg/mL and 150 μg/mL CSC from the 3 cigarette samples.15 μL/mL DMSO treatment wasused as the solvent control,and 3 μg/mL cyclophosphamide treatment was used as the positive control.Thetest protocol includeds the 4-h treatment,2-day expression and 12-day cloning phases.Then the mutationfrequency of trifluorothymidine resistance(TMF) of each dose group was calculated.Finally,the mutagenicpotency of different cigarette samples was compared.RESULTS:At the dose of 150 μg/mL of 1#,100 μg/mLof 2#and 3#,the TMF were more than 3 times higher than that in the solvent control group.In addition,theTMF increased with increase of the CSC doses.The mutagenic potency of the CSCs for 3 cigarette sampleswere 0.37,0.36 and 0.38,respectively.CONCLUSION:All CSCs induced dose-dependent increases ofmutagenicity in the mouse lymphoma assay.Comparing the mutagenicity of the 3 cigarette samples,there wasno significant correlation between TK gene mutagenic potency and tar release in cigarette smoke.
引文
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[2]华辰凤,谢复炜,尚平平,等.体外遗传毒性测试方法在烟草制品毒理学评价中的应用[J].癌变·畸变·突变,2017,29(3):241-244.
[3]李翔,华辰凤,尚平平,等.遗传毒性测试方法在烟草制品体外毒理学评价中的应用[C].杭州:第二届毒性测试替代方法与转化毒理学(国际)学术研讨会暨有害结局路径(AOP)与风险评估培训会议,2016:164-165.
[4]谢剑平.形势与未来:烟草科技发展展望[J].中国烟草学报,2017,23(3):1-6.
[5]米娜.中草药金圣香降低卷烟危害及机理研究[D].北京:中国人民解放军军事医学科学院,2007.
[6]孙学辉,杨松,孙培健,等.基于卷烟降焦减害的滤嘴添加剂研究与应用进展[J].烟草科技,2017,50(2):86-96.
[7]尚平平,李翔,彭斌,等.部分国内外卷烟全烟气暴露的体外致突变作用[J].烟草科技,2013,49(5):45-49.
[8]尚平平,李翔,聂聪,等.卷烟全烟气的Ames试验[J].癌变·畸变·突变,2013,25(5):227-231.
[9]GUO X,VERKLER T L,CHEN Y,et al.Mutagenicity of 11 cigarette smoke condensates in two versions of the mouse lymphoma assay[J].Mutagenesis,2011,26(2):273.
[10]CROOKS I,DILLON D M,SCOTT J K,et al.The effect of long term storage on tobacco smoke particulate matter in vitro,genotoxicity and cytotoxicity assays[J].Regul Toxicol Pharmacol,2013,65(2):196-200.
[11]BREHENY D,CUNNINGHAM F,KILFORD J,et al.Application of a modified gaseous exposure system to the in vitro,toxicological assessment of tobacco smoke toxicants[J].Environ Mol Mutagen,2014,55(8):662-672.
[12]GARCIA-CANTON C,ERRINGTON G,ANADON A,et al.Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro,γH2AX assay by high content screening[J].BMC Pharmacol Toxicol,2014,15(1):41-41.
[13]GARCIA-CANTON C,ERRINGTON G,MEREDITH C.Assessment of the in vitro gamma-H2AX assay by High Content Screening(HCS)as a novel genotoxicity test:application for the evaluation of single toxicants and mixtures[C].United Kingdom:Environmental Mutagen Society Meeting,2014,768.
[14]GARCIA-CANTON C,ANADON A,MEREDITH C.Genotoxicity evaluation of individual cigarette smoke toxicants using the in vitro,γH2AX assay by high content screening[J].Toxicol Lett,2013,223(1):81-87.
[15]LOU J,ZHOU G,CHU G,et al.Studying the cytogenotoxic effects of 12 cigarette smoke condensates on human lymphoblastoid cell line in vitro[J].Mut Res,2010,696(1):48-54.
[16]陆叶珍.卷烟烟气冷凝物加S9与不加S9细胞-遗传毒性体外试验的比较研究[D].杭州:浙江大学医学院,2010.
[17]CLIVE D,SPECTOR J F.Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells[J].Mut Res,1975,31(1):17-29.
[18]HARRIS J E,THUN M J,MONDUL A M,et al.Cigarette tar yields in relation to mortality from lung cancer in the cancer prevention study II prospective cohort,1982-8[J].Brit Med J,2004,328(7431):72-79.
[19]SS HECHT,SE MURPHY,SG CARMELLA,et al.Similar uptake of lung carcinogens by smokers of regular,light,and ultralight cigarettes[J].Cancer Epidem,Biomark Prevent,2005,14(3):693-698.
[20]BURNS D M.Cigarettes and cigarette smoking[J].Clin Chest Med,1991,12(4):631-642.