应用基因重组细胞模型研究苯并[a]芘的细胞毒性和遗传损伤效应
응용기인중조세포모형연구분병[a]비적세포독성화유전손상효응
Cytotoxicity and genomic damage of benzo[a] pyrene in gene transformed cell model
  • 万方数据
    中华预防医学杂志 중화예방의학잡지
    CHINESE JOURNAL OF
    2010年 4期 314-318 ,共5页

    段化伟%宾萍%刘清君%王亚东%牛勇%刘庆%戴宇飞%陈雯%郑玉新

    단화위%빈평%류청군%왕아동%우용%류경%대우비%진문%정옥신

    苯并芘%毒性试验%细胞核分裂%微核,染色体不完整 苯併芘%毒性試驗%細胞覈分裂%微覈,染色體不完整
    분병비%독성시험%세포핵분렬%미핵,염색체불완정
    Benzo (a) pyrene%Toxicity tests%Cell nucleus division%Micronuclei,chromosome-defective
    目的 利用经基因重组构建的具有细胞色素P450 1A1(CYP1A1)代谢活性的人支气管上皮细胞(16HBE)为体外模型,研究苯并[a]芘[B(a)P]的细胞毒性和遗传效应改变.方法 应用实时定量PCR方法检测细胞的CYP1A1和微粒体环氧化物水解酶(mEH)的mRNA水平.细胞分0、1、5、10、20 μmol/L B(a)P处理组,应用胞质分裂阻滞微核细胞组学技术综合评价B(a)P的有害生物效应,其中核分裂指数、细胞凋亡率和细胞坏死率等指标评价B(a)P的细胞毒性,微核、核质桥和核芽的发生率检测B(a)P的遗传损伤效应.结果 CYP1A1和mEH在16HBE-CYP1A1细胞中有较高表达,mRNA相对含量分别是7.8×10~(-4)和0.030.B(a)P作用后,16HBE-CYP1A1细胞1、5、10、20μmol/L处理组核分裂指数分别为1.92±0.04,1.71±0.01,1.61±0.04,1.41±0.01,低于对照组(2.08±0.03);双核细胞率分别为(76.33±3.51)%、(66.33±0.58)%、(51.67±1.53)%、(39.00±1.00)%,低于对照组的(82.67±6.66)%;细胞坏死率分别为(1.93±0.42)%、(2.20±0.53)%、(8.07±0.90)%、(15.27±2.80)%,高于对照组的(0.47±0.11)%,差异均有统计学意义(F值分别为899.94、303.33、240.87,P值均<0.01).而凋亡细胞随着B(a)P作用剂量的增加出现先增高后降低的趋势,分别为(1.20±0.53)%、(2.00±0.20)%、(1.47±0.12)%、(1.20±0.00)%、(1.20±0.00)%.遗传损伤效应分析中发现,随B(a)P作用浓度的增加,核质桥发生率随之增加,分别为(4.67±2.89)‰、(7.33±1.53)‰、(10.67±2.08)‰、(11.00±1.00)‰;核芽发生率也随之增加,分别为(2.33±0.58)‰、(4.00±1.00)‰、(5.00±1.00)‰、(7.67±1.16)‰,均有剂量-效应关系(F值分别为50.23、121.09,P值均<0.01).在B(a)P低于10 μmol/L组中,微核率也随B(a)P作用浓度的增加而升高,分别为(8.33±3.21)‰、(14.67±1.15)‰,但在20 μmol/L组,微核率为(16.67±2.88)‰,低于10 μmol/L处理组[(17.67±2.08)‰].在空载质粒细胞16HBEV中,细胞毒性出现较晚,在5、10、20 μmol/L组中,微核、核质桥和核芽的发生率[分别为(6.37±2.08)‰、(9.33±1.52)‰、(9.33±3.21)‰;(4.33±1.53)‰、(6.00±2.65)‰、(5.33±1.53)‰;(2.33±0.58)‰、(3.33±1.16)‰、(3.67±1.16)‰]没有明显的组间改变.结论 B(a)P代谢活化后可导致遗传损伤效应增加,这可能与细胞的核分裂指数降低、细胞坏死率增加或细胞凋亡受抑制有关.
    목적 이용경기인중조구건적구유세포색소P450 1A1(CYP1A1)대사활성적인지기관상피세포(16HBE)위체외모형,연구분병[a]비[B(a)P]적세포독성화유전효응개변.방법 응용실시정량PCR방법검측세포적CYP1A1화미립체배양화물수해매(mEH)적mRNA수평.세포분0、1、5、10、20 μmol/L B(a)P처리조,응용포질분렬조체미핵세포조학기술종합평개B(a)P적유해생물효응,기중핵분렬지수、세포조망솔화세포배사솔등지표평개B(a)P적세포독성,미핵、핵질교화핵아적발생솔검측B(a)P적유전손상효응.결과 CYP1A1화mEH재16HBE-CYP1A1세포중유교고표체,mRNA상대함량분별시7.8×10~(-4)화0.030.B(a)P작용후,16HBE-CYP1A1세포1、5、10、20μmol/L처리조핵분렬지수분별위1.92±0.04,1.71±0.01,1.61±0.04,1.41±0.01,저우대조조(2.08±0.03);쌍핵세포솔분별위(76.33±3.51)%、(66.33±0.58)%、(51.67±1.53)%、(39.00±1.00)%,저우대조조적(82.67±6.66)%;세포배사솔분별위(1.93±0.42)%、(2.20±0.53)%、(8.07±0.90)%、(15.27±2.80)%,고우대조조적(0.47±0.11)%,차이균유통계학의의(F치분별위899.94、303.33、240.87,P치균<0.01).이조망세포수착B(a)P작용제량적증가출현선증고후강저적추세,분별위(1.20±0.53)%、(2.00±0.20)%、(1.47±0.12)%、(1.20±0.00)%、(1.20±0.00)%.유전손상효응분석중발현,수B(a)P작용농도적증가,핵질교발생솔수지증가,분별위(4.67±2.89)‰、(7.33±1.53)‰、(10.67±2.08)‰、(11.00±1.00)‰;핵아발생솔야수지증가,분별위(2.33±0.58)‰、(4.00±1.00)‰、(5.00±1.00)‰、(7.67±1.16)‰,균유제량-효응관계(F치분별위50.23、121.09,P치균<0.01).재B(a)P저우10 μmol/L조중,미핵솔야수B(a)P작용농도적증가이승고,분별위(8.33±3.21)‰、(14.67±1.15)‰,단재20 μmol/L조,미핵솔위(16.67±2.88)‰,저우10 μmol/L처리조[(17.67±2.08)‰].재공재질립세포16HBEV중,세포독성출현교만,재5、10、20 μmol/L조중,미핵、핵질교화핵아적발생솔[분별위(6.37±2.08)‰、(9.33±1.52)‰、(9.33±3.21)‰;(4.33±1.53)‰、(6.00±2.65)‰、(5.33±1.53)‰;(2.33±0.58)‰、(3.33±1.16)‰、(3.67±1.16)‰]몰유명현적조간개변.결론 B(a)P대사활화후가도치유전손상효응증가,저가능여세포적핵분렬지수강저、세포배사솔증가혹세포조망수억제유관.
    Objective To investigate cytotoxicity and genotoxicity of benzo(a) pyrene (B(a) P) by 16HBE-CYP1A1 cells which are human bronchial epithelial cell with CYP1A1 transformed. Methods Expression of CYP1A1 and mEH of cell models were tested by real-time quantitative polymerase chain reaction. Cells were treated with 0,1,5,10 and 20 μmol/L B(a) P for 24 h. Adverse effects of B(a) P were tested by cytokinesis-block micronucleus (CBMN) cytome assays. Cytotoxicity was assessed by the nuclear division index (NDI), frequency of necrotic and apoptotic cells. Genetic damages were assessed by frequencies of CBMN,nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs). Results High levels of CYP1A1 and mEH were found in 16HBE-CYP1A1 cells (relative mRNA content was 7.8×10~(-4) and 0.030 respectively). In 16HBE-CYP1A1 cells,NDl were decreased in 1,5,10 and 20 μmol/L B(a)P treated groups,1.92±0.04,1.71±0.01,1.61±0. 04,and 1.41±0. 01 ,respectively; and lower than control group (2.08±0.03 ) . Compared with control group ( (82. 67±6. 66 ) %), the binucleated cells ratios were decreased,(76.33±3.51)%, (66.33±0.58)%, (51.67±1.53)% and (39.0±1.0)%respectively. Necrotic cells ratios were (1.93±0.42) %, (2.20±0. 53) %, (8.07±0.90) % and (15.27±2. 80 )%, respectively, higher than control group ((0.47±0. 11 )% ). The differences were significant( F values were 899. 94,303.33,240. 87, P < 0. 01). Apoptotic cells were increased at lower groups and decreased to normal at higher groups treated by B (a) P. They were (1.20±0.53) %, (2. 00± 0. 20) %, (1.47±0.12) %, (1.20±0.00) % and (1.20±0.00) %, respectively. Analysis on biomarkers of genetic damage,the significant dose-effect relationship were observed in NPBs and NBUDs (F values were 50. 23,121.09,P<0. 01, respectively). Frequencies of NPBs were (4.67±2. 89)‰, (7.33±1.53)‰, (10.67±2.08)‰ and (11.00±1.00)‰ respectively. Frequencies of NBUDs were (2.33±0.58)‰, (4.00±1.00)‰, ( 5.00±1.00 )‰, and (7.67±1.16 )‰ respectively. However, the dose-relationship of CBMN last only to 10 μmol/L B (a) P treated groups in 16HBE-CYP1A1 cells, and frequencies of CBMN were(8.33±3. 21)‰, ( 14. 67±1.15 )‰, respectively. Frequency of CBMN was ( 16. 67±2. 88 )‰ in 20 μmol/L B(a) P treated group, lower than 10 μmol/L B (a) P treated group ( (17. 67±2. 08)‰). In 16HBEV control cells,the cytotoxicity was found only in higher B(a)P treated groups and frequencies of CBMN, NPBs and NBUDs were increased also. While no significant differences were observed between 5,10, 20 μmol/L B(a) P treated groups (they were (6.37±2.08)‰, (9. 33±1.52)‰, (9. 33±3. 21)‰; (4. 33±1.53)‰, (6. 00±2. 65)‰, (5.33±1.53)‰ and (2.33±0. 58)‰, (3.33±1.16)‰, (3.67±1.16)‰,respectively). Conclusions The genetic damages were more severe after treated with activated B(a) P,which may be induced by decreased NDI,increased necrotic cells and inhibition of apoptosis.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문