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山东大学学报 (工学版) ›› 2019, Vol. 49 ›› Issue (3): 120-128.doi: 10.6040/j.issn.1672-3961.0.2018.443

• 化学与环境 • 上一篇    

改性超细炭黑诱发氧化应激的毒性效应与机理

贾晨号(),刘汝涛*()   

  1. 山东大学环境科学与工程学院, 山东 青岛 266200
  • 收稿日期:2018-10-18 出版日期:2019-06-20 发布日期:2019-06-27
  • 通讯作者: 刘汝涛 E-mail:1272466157@qq.com;rutaoliu@sdu.edu.cn
  • 作者简介:贾晨号(1993—)，女，河北衡水人，硕士研究生，主要研究方向为环境监测与健康. E-mail:1272466157@qq.com
  • 基金资助:
    国家自然科学基金资助项目(21477067);国家自然科学基金资助项目(21777088);高等学校博士学科点专项科研基金资助项目(708058)

Toxic effects and mechanisms of oxidative stress induced by modified ultrafine carbon black

Chenhao JIA(),Rutao LIU*()   

  1. School of Environment Science and Engineering, Shandong University, Qingdao 266200, Shandong, China
  • Received:2018-10-18 Online:2019-06-20 Published:2019-06-27
  • Contact: Rutao LIU E-mail:1272466157@qq.com;rutaoliu@sdu.edu.cn
  • Supported by:
    国家自然科学基金资助项目(21477067);国家自然科学基金资助项目(21777088);高等学校博士学科点专项科研基金资助项目(708058)

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摘要：

为研究改性超细炭黑(modified ultrafine carbon black, MCB)诱发氧化应激的毒性效应与机理,将小鼠肝细胞和过氧化氢酶(catalase, CAT)暴露于不同质量浓度的MCB溶液中。采用CCK-8、丙二醛(malondialdehyde, MDA)含量和CAT活力检测等方法评价MCB的细胞毒性;利用荧光光谱,紫外-可见吸收光谱,圆二色谱等方法探究MCB对CAT构象的影响。结果表明:细胞活力随MCB质量浓度的升高而降低,低质量浓度的MCB(< 30 mg/L)刺激细胞提高CAT的活力来保护自身免受氧化损伤,高质量浓度的MCB(>30 mg/L)使MDA在细胞内累积并造成氧化还原失衡,造成肝脏的氧化损伤;光谱学研究发现, MCB会改变CAT的二、三级结构和氨基酸微环境,使肽链紧缩极性增强,骨架结构的变化降低了CAT的活性。阐明MCB造成氧化应激效应的毒性机理,同时为纳米材料的毒理研究提供了参考。

关键词: 改性超细炭黑, 氧化应激, 肝细胞, 过氧化氢酶, 荧光光谱, 紫外-可见吸收光谱

Abstract:

To study the toxic effects and mechanisms of oxidative stress induced by modified ultrafine carbon black (MCB), mouse hepatocytes and catalase (CAT) were exposed to MCB solutions. The cytotoxicity of MCB was assessed by CCK-8 kit, malondialdehyde (MDA) kit and CAT activity assay. The effects on CAT structure and function of MCB were investigated by utilizing fluorescence, UV—vis absorption and circular dichroism spectroscopy. The experiment results demonstrated that the hepatocyte viability decreased with the increase of MCB concentration. Low MCB doses (< 30 mg/L) increased CAT activity to protectcells from oxidative damage while high doses of MCB (> 30 mg/L) caused accumulation of MDA and redox imbalance in the cells, which induced oxidative damage in the liver. Spectroscopy studies found that MCB destroyed the secondary and tertiary structure of CAT and changed the microenvironment of amino acids, which made denaturation of the peptide chain. As a result, changes of skeleton structure reduced the activity of CAT. This study clarified the oxidative mechanism of MCB causing oxidative stress effects and provided a reference for the toxicity mechanism of nanomaterials.

Key words: modified ultrafine carbon black, oxidative stress, liver cells, catalase, fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy

中图分类号: 

  • X503.1

图1

MCB的SEM图和TEM图"

图2

改性前后超细炭黑在PB缓冲液中的分散情况"

图3

染毒24 h和48 h后小鼠肝细胞活力的变化"

图4

细胞活力随MCB质量浓度变化的剂量效应拟合曲线"

图5

不同MCB质量浓度下24 h肝细胞MDA生成量"

图6

肝细胞内24 h CAT活性变化"

图7

MCB对CAT荧光光谱的影响 MCB的质量浓度/(mg/L) a-g:0、2.5、5、7.5、10、15、20; pH=7.4, T=310 K "

图8

不同质量浓度的MCB对CAT紫外-可见吸光光谱的影响 MCB的质量浓度/(mg/L) a-g:0, 2.5, 5, 7.5, 10, 15, 20; pH=7.4, T=310 K "

图9

MCB与CAT结合作用示意图"

图10

MCB与CAT作用的三维荧光图谱"

表1

MCB与CAT结合的三维荧光信息"

MCB暴露的质量浓度/(mg·L-1) 峰1 峰2
激发波长/发射波长(nm/nm) 荧光强度 激发波长/发射波长(nm/nm) 荧光强度
0 280/280 4 309.0 280/326 5 600.3
2.5 280/284 9 035.4 280/328 5 006.1
5 280/284 9 078.6 280/328 3 862.7
10 280/284 9 595.5 280/330 2 633.3
15 280/284 9 615.8 280/330 1 493.1
20 280/284 9 680.5 280/332 1 040.7

表2

MCB对CAT二级结构的影响"

MCB暴露的质量浓度/ (mg·L-1) 二级结构含量/%
α-螺旋 β-折叠 β-转角 无序结构
0 43.4 18.8 14.4 23.1
5 45.8 18.0 14.2 22.9
10 47.9 17.4 13.9 21.6
20 49.5 16.7 13.5 21.3

图11

不同质量浓度的MCB对CAT圆二色谱的影响 MCB的质量浓度/(mg/L) a-d:0, 5, 10, 20; pH=7.4, T=310 K "

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