邻苯二甲酸二(2-乙基己基)酯对秀丽线虫生殖能力的影响
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摘要
目的:探讨邻苯二甲酸二(2-乙基己基)酯(DEHP)对秀丽线虫生殖能力的影响。方法:设置对照组(K溶液)和DEHP低(0.1mg/L)、中(1.0 mg/L)、高(10.0 mg/L)3个剂量的染毒组,20℃下恒温培养染毒48 h,观察DEHP对秀丽线虫后代数目、世代时间以及瞬时产卵量的影响;利用二脒基苯基吲哚(DAPI)染色,观察并计数线虫有丝分裂区、减数分裂区的生殖细胞数;利用MD701突变株,在荧光显微镜下观察粗线期细胞的凋亡情况。结果:与对照组相比,L4期线虫暴露DEHP 48 h后,秀丽线虫的后代数目在1.0和10.0 mg/L剂量组显著下降(P<0.05),但对世代时间无明显影响(P>0.05)。随着DEHP暴露剂量的增加,线虫的瞬时产卵量下降(P<0.05)。与对照组相比,在DEHP 1.0和10.0 mg/L的暴露剂量下,秀丽线虫总生殖细胞数和有丝分裂区生殖细胞数均显著下降(P<0.01),在10.0 mg/L暴露剂量下减数分裂区生殖细胞显著减少(P<0.01)。随着染毒剂量的升高,粗线期生殖细胞凋亡数明显增加(P<0.01)。结论:DEHP可以导致秀丽线虫有丝分裂区增殖阻滞以及减数分裂区生殖细胞数减少,这种作用可能和DEHP导致秀丽线虫粗线期凋亡数目增多有关。
OBJECTIVE: To investigate the effect of di(2-ethylhexyl) phthalate(DEHP) on the reproductive capacity of nematode Caenorhabditis elegans(C. elegans). METHODS : C. elegans cultures were organized into several groups : control(K solution) and three DEHP exposure groups : low(0.1 mg/L),medium(1.0 mg/L) and high(10.0 mg/L). Reproductive capacities were evaluated based on brood size, generation time and egg-laying rate/h. Dianamidophenylindole(DAPI) staining was used to observe the number of germline cells in the mitotic zone and the meiotic region of nematodes. Using transgenic MD701 nematodes, apoptotic cells in the pachytene zone were observed with the aid of fluorescence microscopy. RESULTS: Compared with control group, brood sizes of C. elegans were significantly decreased in the 1.0 mg/L and 10.0 mg/L exposed groups(P<0.05), but had no significant effect on generation time(P>0.05). With the increase of exposure dose, the egg-laying rates were significantly decreased(P<0.01) and showed a dose-effect relationship. In addition, the total number of germline cells and the number of germline cells in the mitotic region were significantly decreased in the 1.0 mg/L(P<0.01) and 10.0 mg/L(P<0.01) exposed group. The number of germline cells in the meiotic region also showed a downward trend. Compared with the control group, the germline cell numbers in the meiotic region were decreased significantly at 10.0 mg/L exposure concentration group(P<0.01).With the increase of the dose, the germ cells showing apoptosis in the pachytene zone were also significantly increased(P<0.01). CONCLUSION: DEHP caused arrest in mitotic proliferation and reduction in the number of germ cells in the meiotic zone. The observed toxicity was related to the increased number of apoptosis caused by DEHP in the pachytene of C. elegans.
OBJECTIVE: To investigate the effect of di(2-ethylhexyl) phthalate(DEHP) on the reproductive capacity of nematode Caenorhabditis elegans(C. elegans). METHODS : C. elegans cultures were organized into several groups : control(K solution) and three DEHP exposure groups : low(0.1 mg/L),medium(1.0 mg/L) and high(10.0 mg/L). Reproductive capacities were evaluated based on brood size, generation time and egg-laying rate/h. Dianamidophenylindole(DAPI) staining was used to observe the number of germline cells in the mitotic zone and the meiotic region of nematodes. Using transgenic MD701 nematodes, apoptotic cells in the pachytene zone were observed with the aid of fluorescence microscopy. RESULTS: Compared with control group, brood sizes of C. elegans were significantly decreased in the 1.0 mg/L and 10.0 mg/L exposed groups(P<0.05), but had no significant effect on generation time(P>0.05). With the increase of exposure dose, the egg-laying rates were significantly decreased(P<0.01) and showed a dose-effect relationship. In addition, the total number of germline cells and the number of germline cells in the mitotic region were significantly decreased in the 1.0 mg/L(P<0.01) and 10.0 mg/L(P<0.01) exposed group. The number of germline cells in the meiotic region also showed a downward trend. Compared with the control group, the germline cell numbers in the meiotic region were decreased significantly at 10.0 mg/L exposure concentration group(P<0.01).With the increase of the dose, the germ cells showing apoptosis in the pachytene zone were also significantly increased(P<0.01). CONCLUSION: DEHP caused arrest in mitotic proliferation and reduction in the number of germ cells in the meiotic zone. The observed toxicity was related to the increased number of apoptosis caused by DEHP in the pachytene of C. elegans.
引文
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[15]SEDHA S,KUMAR S,SHUKLA S.Role of oxidative stress in male reproductive dysfunctions with reference to phthalate compounds[J].Urol J,2015,12(5):2304-2316.
[16]李娜,刘特,叶琳,等.邻苯二甲酸二(2-乙基己基)酯对雌性小鼠子宫内膜雌激素受体α表达影响的研究[J].环境与健康杂志,2011,28(8):692-694.
[17]XU C,CHEN J A,QIU Z,et al.Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl)phthalate[J].Toxicol Lett,2010,199(3):323-332.
[18]OZPINAR H,OZPINAR N,KARAKUS S.The effects of erzincan grape(Vitis viniferaspp.cimin)and benzothiazol on a Caenorhabditis elegans organism model[J].Pharmacogn Mag,2017,13(Suppl 2):380-384.
[19]ANGELES-ALBORES D,LEIGHTON D H W,TSOU T,et al.The Caenorhabditis elegans female-like state:Decoupling the transcriptomic effects of aging and sperm status[J].G3,2017,7(9):2969-2977.
[20]YIN J,LIU R,JIAN Z,et al.Di(2-ethylhexyl)phthalateinduced reproductive toxicity involved in DNA damagedependent oocyte apoptosis and oxidative stress in Caenorhabditis elegans[J].Ecotox Environ Safe,2018,163(16):298-306.
[21]ANDUX S,ELLIS R E.Apoptosis maintains oocyte quality in aging Caenorhabditis elegans females[J].PLoSGenet,2008,4(12):e1000295.
[2]MAGDOULI S,DAGHRIR R,BRAR S K,et al.Di 2-ethylhexyl phtalate in the aquatic and terrestrial environment:a critical review[J].J Environ Manage,2013,127(15):36-49.
[3]HEUDORF U,MERSCH-SUNDERMANN V,ANGERERJ.Phthalates:toxicology and exposure[J].Int J Hyg Envir Heal,2007,210(5):623-634.
[4]CALDWELL J C.DEHP:genotoxicity and potential carcinogenic mechanisms-a review[J].Mut Res,2012,751(2):82-157.
[5]谭琴,秦逍云,徐新云,等.邻苯二甲酸二(2-乙基己)酯对大鼠卵巢病理和超微结构的影响[J].癌变·畸变·突变,2014,26(5):369-273.
[6]张燕芬,王大勇.利用模式动物秀丽线虫建立环境毒物毒性的评估研究体系[J].生态毒理学报,2008,3(4):313-322.
[7]LUI D Y,COLAIACOVO M P.Meiotic development in Caenorhabditis elegans[J].Adv Exp Med Biol,2013,757(6):133-170.
[8]LESCH B J,PAGE D C.Genetics of germ cell development[J].Nat Rev Genet,2012,13(11):781-794.
[9]刘恩岐,仓林让.模型动物--秀丽隐杆线虫研究进展[J].猪业科学,2003,20(10):23-25.
[10]ZHAO Y L,GONG Q,WANG D Y,et al.An epigenetic signal encoded protection mechanism is activated by graphene oxide to inhibit its induced reproductive toxicity in Caenorhabditis elegans[J].Biomaterials,2015,79:15-24.
[11]杨栋.邻苯二甲酸酯对秀丽隐杆线虫的雌性生殖毒性[D].南京:东南大学,2015.
[12]张敏辉.三唑类杀菌剂对雄性秀丽线虫生殖功能的影响及作用机制[D].南京:东南大学,2016.
[13]SWAIN S C,KEUSEKOTTEN K,BAUMEISTER R,et al.C.elegans metallothioneins:new insights into the phenotypic effects of cadmium toxicosis[J].J Mol Biol,2004,341(4):951-959.
[14]SCHUMACHER B,SCHERTEL C,WITTENBURG N,et al.C.elegans ced-13 can promote apoptosis and is induced in response to DNA damage[J].Cell Death Differ,2005,12(2):153-161.
[15]SEDHA S,KUMAR S,SHUKLA S.Role of oxidative stress in male reproductive dysfunctions with reference to phthalate compounds[J].Urol J,2015,12(5):2304-2316.
[16]李娜,刘特,叶琳,等.邻苯二甲酸二(2-乙基己基)酯对雌性小鼠子宫内膜雌激素受体α表达影响的研究[J].环境与健康杂志,2011,28(8):692-694.
[17]XU C,CHEN J A,QIU Z,et al.Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl)phthalate[J].Toxicol Lett,2010,199(3):323-332.
[18]OZPINAR H,OZPINAR N,KARAKUS S.The effects of erzincan grape(Vitis viniferaspp.cimin)and benzothiazol on a Caenorhabditis elegans organism model[J].Pharmacogn Mag,2017,13(Suppl 2):380-384.
[19]ANGELES-ALBORES D,LEIGHTON D H W,TSOU T,et al.The Caenorhabditis elegans female-like state:Decoupling the transcriptomic effects of aging and sperm status[J].G3,2017,7(9):2969-2977.
[20]YIN J,LIU R,JIAN Z,et al.Di(2-ethylhexyl)phthalateinduced reproductive toxicity involved in DNA damagedependent oocyte apoptosis and oxidative stress in Caenorhabditis elegans[J].Ecotox Environ Safe,2018,163(16):298-306.
[21]ANDUX S,ELLIS R E.Apoptosis maintains oocyte quality in aging Caenorhabditis elegans females[J].PLoSGenet,2008,4(12):e1000295.