PM2.5中不同组分对人脐静脉内皮细胞的联合毒性作用研究

doi:10.3969/j.issn.1004-616x.2021.02.006

摘要/Abstract

摘要： 目的：探讨PM_2.5中各种组分单独以及联合暴露对于人脐静脉内皮细胞（HUVECs）的毒性作用，并探讨不同组分之间是否存在交互作用。方法：以永生化的HUVECs细胞为研究对象，分别给予0、5、10、20、40、80、160 μg/mL纳米炭黑颗粒（下称炭黑），0、2.5、5、10、20、40、80、160 μg/mL尘土颗粒（下称尘土），0、6.25、12.5、25、50、100、200、400 μmol/L醋酸铅，0、5、10、20、40、80、160 μmol/L亚砷酸钠和氯化镉，染毒24 h，采用CCK-8法测定不同受试物对细胞存活率的影响，并计算不同受试物对HUVECs细胞的半数抑制浓度（IC₅₀）。然后进行联合染毒实验，选用细胞存活率为80%时的剂量，炭黑（或尘土）分别与铅、砷、镉对HUVECs进行联合染毒24 h，用CCK-8测定细胞存活率；另外根据PM_2.5中各组分实际占比，按m_铅：m_{炭黑/尘土}=1：50，m_砷：m_{炭黑/尘土}=1：100，m_镉：m_{炭黑/尘土}=1：500的比例进行两两联合染毒，以及按m_铅：m_砷：m_镉：m_{炭黑/尘土}=10：5：1：500的比例进行四者联合染毒。染毒24 h后，用CCK-8法测定细胞存活率。结果：HUVEC细胞存活率随着炭黑、尘土、醋酸铅、亚砷酸钠、氯化镉染毒浓度的增加而下降（P<0.05），以上各受试物对HUVECs细胞的IC₅₀分别为16 μg/mL、110 μg/mL、184 μmol/L、15 μmol/L、14 μmol/L。炭黑与醋酸铅、亚砷酸钠、氯化镉联合染毒时，细胞存活率比炭黑单独染毒时分别下降了17.8%、43.8%、41.2%；尘土与醋酸铅、亚砷酸钠、氯化镉联合染毒时，细胞存活率比尘土单独染毒时分别下降了11.6%、27.8%、28.3%。联合染毒时细胞存活率比单独染毒时明显下降（P<0.05）。炭黑与亚砷酸钠、炭黑与氯化镉之间存在交互作用（均为P<0.05）。结论：PM_2.5中不同组分均可以对血管内皮细胞产生毒性作用，单独暴露时炭黑的细胞毒性作用比尘土大；但与3种金属联合暴露时，尘土比炭黑的联合暴露细胞毒性更大。铅、砷、镉对细胞的毒性作用大小排序为氯化镉>亚砷酸钠>醋酸铅，且炭黑颗粒与亚砷酸钠、炭黑颗粒与氯化镉之间存在交互作用，表现为协同作用。

关键词: 大气细颗粒物, 超细颗粒, 重金属, 人脐静脉内皮细胞, 联合毒性作用

Abstract: OBJECTIVE: To investigate toxic effects of various components in PM_2.5 and of their combined exposures on human umbilical vein endothelial cells (HUVECs),and to determine their interactive effects. METHODS: Immortalized HUVECs were exposed in culture to 0,5,10,20,40,80,160 μg/mL carbon black,0,2.5,5,10,20,40,80,160 μg/mL dust,0,6.25,12.5,25,50,100,200,400 μmol/L lead acetate,0,5,10,20,40,80,160 μmol/L sodium arsenite and cadmium chloride for 24 h. The CCK-8 assay was used to determine cell viability and IC₅₀ values were calculated. For the combined toxicity study,the doses at which cell viability was 80%,carbon black (or dust) was combined with lead,arsenic,and cadmium for 24 h,and the CCK-8 assay was used to determine cell viability. Additionally,according to the actual proportion of each fraction in PM_2.5,carbon black (or dust) was proportionally (m_lead:m_arsenic:m_cadmium:m_{carbon black/dust}=10:5:1:500) combined with lead,arsenic,and cadmium,respectively,for 24 h,and the CCK-8 assay was used to determine cell viability. RESULTS: The cell viability test shows a decreasing trend with increasing doses of carbon black,dust,lead acetate,sodium arsenite,and cadmium chloride (P<0.05),with IC₅₀ of 16 μg/mL,110 μg/mL,184 μmol/L,15 μmol/L,and 14 μmol/L,respectively. Cell viability was decreased by 17.8%,43.8%,41.2% when carbon black was combined with lead acetate,sodium arsenite,and cadmium chloride,respectively,and by 11.6%,27.8%,28.3% when dust was combined with lead acetate,sodium arsenite,and cadmium chloride,respectively. When carbon black and dust were combined with lead acetate,sodium arsenite,and cadmium chloride,the cell viability was significantly lower than when they were exposed individually (P<0.05). There were interactions between carbon black and sodium arsenite or cadmium chloride (all P<0.05). CONCLUSION: Different components in PM_2.5 can cause toxic effects to the vascular endothelial cells in vitro,the cytotoxic effect of carbon black was greater than dust particles. Upon combined exposure,dust was more cytotoxic upon simultaneous exposure to the other three metals. The order of toxic effect of lead,arsenic and cadmium was cadmium chloride > sodium arsenite > lead acetate,and the interactions between carbon black particles and sodium arsenite and cadmium chloride wa synergistic.

Key words: fine particulate matter, ultrafine particles, heavy metals, HUVECs, combined toxicity

中图分类号:

R122.7

刘洁仪, 张睿, 夏冬, 李镕, 李晓雯, 陈姿旭, 魏青, 赵志强, 何云. PM_2.5中不同组分对人脐静脉内皮细胞的联合毒性作用研究[J]. 癌变·畸变·突变, 2021, 33(2): 116-123,128.

LIU Jieyi, ZHANG Rui, XIA Dong, LI Rong, LI Xiaowen, CHEN Zixu, WEI Qing, ZHAO Zhiqiang, HE Yun. Combined toxicity of different PM_2.5 components in human umbilical vein endothelial cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(2): 116-123,128.

参考文献

[1] KIM K H, KABIR E, KABIR S. A review on the human health impact of airborne particulate matter[J]. Environ Int, 2015, 74:136-143.
[2] ARIAS-PÉREZ R D, TABORDA N A, GÓMEZ D M, et al. Inflammatory effects of particulate matter air pollution[J]. Environ Sci Pollut Res Int, 2020, 27(34):42390-42404.
[3] CALDERÓN-GARCIDUEÑAS L, REYNOSO-ROBLES R, VARGAS-MARTÍNEZ J, et al. Prefrontal white matter pathology in air pollution exposed Mexico City young urbanites and their potential impact on neurovascular unit dysfunction and the development of Alzheimer's disease[J]. Environ Res, 2016, 146:404-417.
[4] LIU F, HUANG Y L, ZHANG F, et al. Macrophages treated with particulate matter PM_2.5 induce selective neurotoxicity through glutaminase-mediated glutamate generation[J]. J Neurochem, 2015, 134(2):315-326.
[5] HYMAN B T, PHELPS C H, BEACH T G, et al. National Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease[J]. Alzheimers Dement, 2012, 8(1):1-13.
[6] MARTINELLI N, OLIVIERI O, GIRELLI D. Air particulate matter and cardiovascular disease:a narrative review[J]. Eur J Intern Med, 2013, 24(4):295-302.
[7] FATTORE E, PAIANO V, BORGINI A, et al. Human health risk in relation to air quality in two municipalities in an industrialized area of Northern Italy[J]. Environ Res, 2011, 111(8):1321-1327.
[8] LEVY J I, DIEZ D, DOU Y, et al. A meta-analysis and multisite time-series analysis of the differential toxicity of major fine particulate matter constituents[J]. Am J Epidemiol, 2012, 175(11):1091-1099.
[9] FONKEN L K, XU X, WEIL Z M, et al. Air pollution impairs cognition, provokes depressive-like behaviors and alters hippocampal cytokine expression and morphology[J]. Mol Psychiatry, 2011, 16(10):987-995, 973.
[10] GATTO N M, HENDERSON V W, HODIS H N, et al. Components of air pollution and cognitive function in middle-aged and older adults in Los Angeles[J]. Neurotoxicology, 2014, 40:1-7.
[11] BHATT D P, PUIG K L, GORR M W, et al. A pilot study to assess effects of long-term inhalation of airborne particulate matter on early Alzheimer-like changes in the mouse brain[J]. PLoS One, 2015, 10(5):e0127102.
[12] JIANG N, LI Q, SU F C, et al. Chemical characteristics and source apportionment of PM_2.5 between heavily polluted days and other days in Zhengzhou, China[J]. J Environ Sci, 2018, 66:188-198.
[13] ZANOBETTI A, COULL B A, GRYPARIS A, et al. Associations between arrhythmia episodes and temporally and spatially resolved black carbon and particulate matter in elderly patients[J]. Occup Environ Med, 2014, 71(3):201-207.
[14] LOUWIES T, NAWROT T, COX B, et al. Blood pressure changes in association with black carbon exposure in a panel of healthy adults are independent of retinal microcirculation[J]. Environ Int, 2015, 75:81-86.
[15] CHEN W H, LIU Y W, WANG H J, et al. Long-term exposure to silica dust and risk of total and cause-specific mortality in Chinese workers:a cohort study[J]. PLoS Med, 2012, 9(4):e1001206.
[16] SHANG Y, LIU M Y, WANG T T, et al. Modifications of autophagy influenced the Alzheimer-like changes in SH-SY5Y cells promoted by ultrafine black carbon[J]. Environ Pollut, 2019, 246:763-771.
[17] ZHANG W J, SUN Y L, ZHUANG G S, et al. Characteristics and seasonal variations of PM_2.5, PM₁₀, and TSP aerosol in Beijing[J]. Biomed Environ Sci, 2006, 19(6):461-468.
[18] KU T T, ZHANG Y Y, JI X T, et al. PM_2.5-bound metal metabolic distribution and coupled lipid abnormality at different developmental windows[J]. Environ Pollut, 2017, 228:354-362.
[19] PAITHANKAR J G, SAINI S, DWIVEDI S, et al. Heavy metal associated health hazards:an interplay of oxidative stress and signal transduction[J]. Chemosphere, 2021, 262:128350.
[20] 朱振坤, 李金泉, 曾燕. 重金属暴露与阿尔茨海默病发生发展关系的研究进展[J]. 华中科技大学学报:医学版, 2020, 49(6):770-776, 783.
[21] KALARIA R N. Small vessel disease and Alzheimer's dementia:pathological considerations[J]. Cerebrovasc Dis, 2002, 13(Suppl 2):48-52.
[22] SWEENEY M D, SAGARE A P, ZLOKOVIC B V. Blood-brain barrier breakdown in Alzheimer disease and other neurodegenerative disorders[J]. Nat Rev Neurol, 2018, 14(3):133-150.
[23] ZHANG J, ZHOU X, WANG Z, et al. Trace elements in PM_2.5 in Shandong Province:Source identification and health risk assessment[J]. Sci Total Environ, 2018, 621:558-577.
[24] YIN Z X, HUANG X F, HE L Y, et al. Trends in ambient air pollution levels and PM_2.5 chemical compositions in four Chinese cities from 1995 to 2017[J]. J Thorac Dis, 2020, 12(10):6396-6410.
[25] CHO C C, HSIEH W Y, TSAI C H, et al. In vitro and in vivo experimental studies of PM_2.5 on disease progression[J]. Int J Environ Res Public Heal, 2018, 15(7):1380.
[26] OUDIN A, SEGERSSON D, ADOLFSSON R, et al. Association between air pollution from residential wood burning and dementia incidence in a longitudinal study in Northern Sweden[J]. PLoS One, 2018, 13(6):e0198283.
[27] ZHANG Y Y, JI X T, KU T T, et al. Heavy metals bound to fine particulate matter from Northern China induce season-dependent health risks:a study based on myocardial toxicity[J]. Environ Pollut, 2016, 216:380-390.
[28] ALVES C A, EVTYUGINA M, VICENTE A M P, et al. Chemical profiling of PM₁₀ from urban road dust[J]. Sci Total Environ, 2018, 634:41-51.
[29] 汪浪, 杨海龙, 李晓燕. 中国部分省会城市PM_2.5中重金属水平及影响因素分析[J]. 环境化学, 2017, 36(1):72-83.
[30] 陈冲, 焦宁, 靖景艳, 等. 台盼蓝拒染法、MTT法、CCK-8法在研究As₂O₃细胞毒性作用中的意义[J]. 中国医药导报, 2013, 10(12):24-26.
[31] 蔡文涛. MTT法和CCK-8法检测中药抗病毒活性成分细胞毒性的比较[J]. 湖北大学学报:自然科学版, 2017, 39(3):305-310.
[32] 赵婧, 杨磊. 不同粒径二氧化硅对人脐静脉内皮细胞损伤的比较[J]. 微量元素与健康研究, 2017, 34(4):4-5, 9.
[33] CASSEE F R, GROTEN J P, VAN BLADEREN P J, et al. Toxicological evaluation and risk assessment of chemical mixtures[J]. Crit Rev Toxicol, 1998, 28(1):73-101.
[34] 杨蓉, 李娜, 饶凯锋, 等. 环境混合物的联合毒性研究方法[J]. 生态毒理学报, 2016, 11(1):1-13.