雌激素胁迫对萝卜种子萌芽和幼苗生长及其累积效应的影响

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魏瑞成, 李金寒, 何龙翔, 王冉, 邵明诚, 郑勤. 雌激素胁迫对萝卜种子萌芽和幼苗生长及其累积效应的影响. 22(5): 190-197
WEI Rui-cheng, LI Jin-han, HE Long-xiang, WANG Ran, SHAO Ming-cheng, ZHENG Qin. Effects of estrogen stress on seed germination, seedling growth and accumulation in radish. Acta Prataculturae Sinica, 22(5): 190-197 复制到剪切板

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雌激素胁迫对萝卜种子萌芽和幼苗生长及其累积效应的影响

魏瑞成¹, 李金寒², 何龙翔², 王冉^1*,^*, 邵明诚¹, 郑勤¹

1. 江苏省畜禽产品安全性研究重点实验室江苏省食品质量安全重点实验室—省部共建国家重点实验室培育基地江苏省农业科学院,江苏南京 210014

2. 中国药科大学,江苏南京 211198

*通讯作者。E-mail:wangran@jaas.ac.cn

作者简介:魏瑞成(1979-),男,河南商丘人,博士。E-mail:rcwei79@jaas.ac.cn

基金:江苏省自然科学基金(BK2011681)资助

摘要

通过水培试验,研究了不同浓度17β-雌二醇(17β-E2)胁迫对萝卜种子萌芽、幼苗生长和抗氧化酶系统活性的影响,以及17β-E2在幼苗体内的积累。结果发现,萝卜种子的发芽势、发芽率和根长与17β-E2处理浓度呈负相关,在10 μg/L浓度时17β-E2刺激萝卜种子的发芽势和根长明显增加,诱导幼苗体内抗氧化酶SOD、POD和CAT活性显著上升;但在50 μg/L浓度的17β-E2胁迫下,SOD、CAT和POD活性受到明显抑制,MDA含量显著升高,幼苗抗氧化能力显著降低,膜质过氧化程度加剧;受17β-E2污染,萝卜幼苗体内17β-E2积累量随着处理浓度的升高而增加,其在根部累积量要大于地上部分,在转运过程中发生17β-E2代谢,产生具有低雌激素活性的雌酮。萝卜对17β-E2胁迫具有一定的抗性,在17β-E2污染土壤中种植萝卜存在潜在的食用安全风险。

关键词: 17β-雌二醇; 萝卜; 抗氧化酶系统; 丙二醛; 积累

中图分类号:S631.103.4 文献标志码:A 文章编号:1004-5759(2013)05-0190-08

Effects of estrogen stress on seed germination, seedling growth and accumulation in radish

WEI Rui-cheng¹, LI Jin-han², HE Long-xiang², WANG Ran¹, SHAO Ming-cheng¹, ZHENG Qin¹

1.Jiangsu Province Key Laboratory of Animal-Derived Food Safety, Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Jiangsu Academy of Agricultural Science, Nanjing 210014, China

2. China Pharmaceutical University, Nanjing 211198, China

Abstract

The effects of different concentrations of 17β-estradiol (17β-E2) on seed germination, seedling growth, antioxidant enzyme system and 17β-E2 accumulation in radish ( Raphanus sativus) were investigated using water culture method. The results indicated that the germinating potentiality, germination rate and root elongation of radish presented the inverse correlation with 17β-E2 treatment. The germinating potentiality and root elongation of radish were obviously increased and activities of SOD, POD and CAT of radish were significantly enhanced by 17β-E2 at levels of 10 μg/L. The 50 μg/L 17β-E2 treatment inhibited the activities of SOD, POD and CAT and significantly stimulated an increase in the content of MDA. It made seedling oxidation resistance significantly decrease and finally resulted in aggravate of the lipid peroxidation. With rising treatment concentration, the content of 17β-E2 accumulated in seedlings was increased. The content of 17β-E2 in the root was larger than that in aerial part. In the transportation process, 17β-E2 was partly metabolized into the low active estrone in radish. The experiments revealed that radish showed a certain tolerance to 17β-E2 stress, so there was a potential food risk posed to mankind health for radish growing in estrogen-contaminated soils.

Keyword: 17β-estradiol; radish; antioxidant enzyme system; malondiadehyde; accumulation

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环境雌激素, 对野生动物和人体健康具有巨大的潜在危害, 已经愈来愈受到全世界的关注^{[1, 2]}。在诸多天然的雌性荷尔蒙物质中, 雌二醇被认为是最具潜在影响的一种, 即使是很低的浓度也会产生严重影响^{[3, 4]}。污水处理厂出水和畜禽养殖场排泄物是天然雌激素的两大污染源, 前者最先引起了人们的关注和研究, 但是近期研究发现, 集约化养殖场对环境雌激素的贡献很大, 主要源于畜禽动物正常的生理分泌排泄以及饲料中促生长素的添加, 在欧洲养殖废弃物中发现雌二醇含量高达14904 μ g/kg干重, 新西兰奶牛场污水中雌二醇水平为191 360 ng/L, 代谢物雌酮的水平为413 123 ng/L, 养猪场污水中的雌激素含量为46 ng/L, 使用肉鸡粪便的土壤中雌激素浓度有55675 ng/kg^[5], 而施用猪粪3个月后雌激素从植物根区的渗滤使农田排水系统中E1浓度可以达到68.1 ng/L^[6], 雌激素通过畜禽粪便农用的方式进入到土壤、水体中, 对环境构成潜在威胁^{[7, 8]}, 也造成农产品的污染。雌激素在110 ng/L浓度即具有很强的生理功能, 河水中含量仅为1 ng/L时就会造成雄鱼的雌性化, 人类长期食用含有激素的肉制食品或其他农产品, 即使含量甚微, 但由于其作用极强, 亦会影响机体的激素平衡, 从而形成长期潜在的危害。有研究表明, 雌激素对人类的直接危害是干扰内分泌生殖系统的正常功能, 促使婴儿性早熟和造成男性生育能力的下降甚至丧失^{[7, 9, 10]}。

萝卜(Raphanus sativus)作为我国主要蔬菜之一, 在蔬菜的周年均衡供应上占有重要地位。雌激素随粪污进入土壤, 与植物根细胞壁中交换位点相互作用, 能吸附雌激素或将其进一步向地上部分转移, 因此粪污中的雌激素将在根部积累, 通过萝卜的食用进入人体, 对人体健康造成潜在的危害。目前, 有关雌激素污染对萝卜生长影响少有报道。本研究以萝卜为试验材料, 采用水培法研究了不同浓度17β -雌二醇(17β -E2)胁迫对萝卜种子萌芽、幼苗生长和SOD、POD、CAT活性、MDA含量的影响, 以及17β -E2在幼苗体内的积累和代谢物雌酮的生成(图1), 旨在探讨雌激素对非靶标植物生态毒性和残留特征, 以期为揭示植物适应和抵御雌激素污染物的损伤机制以及蔬菜的安全生产和健康风险评价提供依据。

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	图1 17β -雌二醇和代谢物雌酮化学结构Fig.1 General chemical structure of the 17β -E2 and E1

1 材料与方法

1.1 材料与仪器

材料:17β -雌二醇(17β -E2)标准品购自Dr. Ehrenstorfer, 其标准液用甲醇溶解; 甲醇、乙腈和乙酸铵为色谱纯, ROE SCIENTIFIC INC(Newark, USA); 超氧化物歧化酶(superoxide dismutase, SOD)、过氧化物酶(peroxidase, POD)、过氧化氢酶(catalase, CAT)、丙二醛(malondiadehyde, MDA)和蛋白质含量测定试剂盒购自南京建成生物技术公司; 聚乙烯吡咯烷酮(PVPK₃₀)购自德国 BASF公司; 水为超纯水。

仪器:人工气候箱(GXZ, 宁波江南仪器厂); H-1850高速冷冻离心机(湖南湘仪实验室仪器开发有限公司); 紫外-可见分光光度计(Perkin Eimer Instruments); 数显恒温水浴锅HH-6(国华电器有限公司); 漩涡混合器(上海琪特分析仪器有限公司); 固相萃取柱(Agilent C₁₈柱, 500 mg); 超声振荡仪; 氮吹仪(NEVAPTM112); 配备DAD 检测器的Agilent 1200S 高效液相色谱仪和ESI 源的Agilent 6410 三重四极管质谱仪等。

1.2 试验设计

供试蔬菜为萝卜。2012年3月将萝卜种子用0.3%双氧水消毒后蒸馏水洗净, 挑选均一、饱满的种子置于铺3层滤纸的培养皿(直径9 cm)中, 每皿150粒, 每个浓度设3个重复, 放置种子时, 保持种子胚根末端和生长方向呈直线。17β -雌二醇(17β -E2)处理浓度为10, 50和100 μ g/L, 将配制的溶液均匀的加入到培养皿中, 每皿5 mL, 以0.1%甲醇作为对照(CK), 将上述处理置于人工气候箱[温度(25± 1)℃, 相对湿度(75± 5)%, 光期12 h/d, 光强3000 Lux]中。每日更换相应的溶液以保持滤纸湿润。试验第8天时收获幼苗, 用于各项指标的测定。

1.3 测定项目与方法

1.3.1 发芽和生长指标的测定分别在试验第3天和第7天统计种子发芽数, 计算发芽势和发芽率:发芽势(GE, %)=前3 d发芽种子粒数/供试种子总数× 100; 发芽率(GP, %)=前7 d发芽种子粒数/供试种子数× 100。根长的测定:每个重复随机取5株, 用精确度为0.1 mm的游标卡尺测量幼苗的主根长度, 计算平均值。

1.3.2 抗氧化酶活性和MDA含量的测定试验第8天时取萝卜幼苗地上部分0.6 g, 加入6 mL预冷的0.05 mol/L pH 7.8磷酸缓冲液(含1% PVPK₃₀), 在冰浴上研磨成浆, 匀浆液倒入离心管中, 在4℃下5 000 r/min离心10 min, 上清液即为酶提取液, 置于4℃ 保存待用^{[11, 12]}。

蛋白质含量的测定采用考马斯亮蓝G-250法, 测定595 nm下OD 值。超氧化物歧化酶(SOD)采用黄嘌呤氧化酶法测定, 活性的测定以37℃条件下每mL反应液中SOD抑制率达50%时所对应的SOD量为1个SOD活力单位(U), 测定550 nm 下OD值。过氧化物酶(POD)活性的测定以37℃条件下每mg组织蛋白每min内A₄₂₀催化产生1 μ g底物的酶量为1个酶活力单位。过氧化氢酶(CAT)的测定采用钼酸铵法, 以每mg组织蛋白每s内A₄₀₅分解1 μ mol的H₂O₂的量为1个活性单位(U)。丙二醛(MDA)采用硫代巴比妥酸法测定。

1.3.3 幼苗体内17β -雌二醇(17β -E2)残留量的分析取待测幼苗用蒸馏水冲洗除去附在表面的17β -雌二醇(17β -E2), 然后用滤纸吸干表面水分, 研磨后备用。样品处理参照Fiers等^[13]方法并加以改进^{[14, 15]}。准确称取0.20.5 g 研磨样品至10 mL聚丙烯具塞离心管, 加1 mL乙腈, 超声10 min后在4℃下10 000 r/min离心10 min, 取上清液, 45℃水浴中氮气吹近干, 加入1 mL 10%甲醇超声溶解后过C₁₈ SPE柱。将Agilent C₁₈柱分别用6 mL乙腈和6 mL水活化, 将上述样品全部加入小柱中, 用6 mL 10%甲醇淋洗, 然后用6 mL乙腈洗脱, 样品装载和洗脱过程控制流速0.5 mL/min。洗脱后的样品在45℃水浴氮气吹近干, 加入200 μ L乙腈溶解, 溶液过0.22 μ m滤膜后供LC-MS测定。

1.4 数据分析

试验数据用SPASS 16.0和Excel 2003进行统计分析和作图。

2 结果与分析

2.1 高效液相色谱-串联质谱分析

17β -雌二醇(17β -E2)和其代谢物雌酮(E1)的分离对分析是相当重要的。本试验比较了0.1%甲酸水-甲醇和10 mmol/L乙酸铵-乙腈作为流动相、C₈与C₁₈为分离柱对目标组分分离的影响, 经过优化, 采用C₁₈色谱柱和10 mmol/L乙酸铵-乙腈在0.30 mL/min的流速下实现了雌激素以及杂质峰的分离(图2)。质谱参数获得采用1 mg/L的17β -E2和E1标准液以流动注射的方式在ESI(-)模式下进行母离子全扫描, 确定17β -E2和E1分子离子, 然后分别以分子离子为母离子, 对其子离子进行全扫描, 选取丰度较强, 干扰较小的离子为定性离子, 最后以多反应检测(MRM)确定碰撞能量和碎裂电压, 优化的质谱条件见表1。萝卜植株根和地上部分的17β -E2和E1回收率分别在70%85%和78%91%, 相对标准偏差均小于10%, 最低检出限分别为10和1 μ g/kg, 能够满足本试验的检测需求。

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	图2 17β -雌二醇和雌酮质谱图Fig.2 Mass chromatograms of 17β -E2 and E1

表1 17β -雌二醇和雌酮的MRM监测质谱条件 Table 1 MS/MS multiple reaction monitoring (MRM) analysis conditions of 17β -E2 and E1

2.2 不同浓度17β -雌二醇对萝卜生长的影响

2.2.1 不同浓度17β -雌二醇对萝卜的发芽势和发芽率的影响萝卜种子的发芽势受到加入17β -E2的影响(图3), 低浓度刺激有促进种子发芽的趋势, 以10 μ g/L 处理组效果最为显著, 10和50 μ g/L 17β -E2处理组的发芽势比对照组分别上升了9.76%和4.16%, 而100 μ g/L 17β -E2处理组对萝卜的发芽势有抑制作用, 使其比对照组下降了5.28%。虽然雌激素的3个浓度处理组中种子的发芽率与对照组之间不存在显著差异, 但从数据看低浓度组刺激有促使发芽率升高的能力, 比对照组分别升高了3.25%和4.73%; 高浓度组刺激可以降低种子的发芽趋势, 比对照组下降了0.89%。上述结果可能与雌激素透过种皮影响种子萌发相关的酶有关, 这种影响存在一个较低浓度下刺激效应和高浓度下的抑制效应^{[11, 16, 17]}。

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	图3 不同浓度17β -雌二醇对萝卜发芽的影响Fig.3 Effects of different concentrations of 17β -E2 on the germination of radish

2.2.2 不同浓度17β -雌二醇对萝卜根长的影响 17β -E2对萝卜根长的影响趋势与发芽势相同(图4), 10和50 μ g/L 17β -E2处理组促进了根长的生长, 比对照组长了17.31%和8.86%, 10 μ g/L 处理组与对照组相比具显著性差异; 而17β -E2浓度为100 μ g/L处理组的根长比对照组减少了1.53%, 其下降程度同10 μ g/L 处理组比较有显著性差异。说明在低浓度(010 μ g/L)范围内17β -E2对萝卜幼苗根长的生长具有促进作用, 而在高浓度(50100 μ g/L)范围内17β -E2抑制了萝卜幼苗根长的生长, 根长的发育与加入的污染物17β -E2浓度呈负相关。

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	图4 不同浓度17β -雌二醇对萝卜根长的影响Fig.4 Effects of different concentrations of 17β -E2 on the root length of radish

2.3 不同浓度17β -雌二醇对萝卜抗氧化酶活性及MDA含量的影响

2.3.1 不同浓度17β -雌二醇对萝卜SOD影响超氧化物歧化酶(superoxide dismutase, SOD)是1969年由Mccord 和Fridovich发现, 催化超氧化物自由基 $\begin{matrix} {O_{2}}^{- \cdot} \end{matrix}$ 和氢离子反应形成过氧化氢(H₂O₂)和分子氧(O₂), 消除生物体在新陈代谢过程中产生的有害物质— — 自由基。在10 μ g/L的17β -E2胁迫下, 萝卜中SOD活性被激活, 比对照组升高了20.54%, 与对照组差异显著。而50和100 μ g/L处理组则抑制其活性, 使其分别下降了10.52%和6.09%, 其中50 μ g/L处理组与对照组差异显著(图5)。可能是由于17β -E2对萝卜造成了胁迫, 促进了活性氧自由基产生, 低浓度(10 μ g/L)诱导了SOD活性升高, 而高浓度对于SOD活性的升高具有一定的抑制作用, 在自由基清除能力上受到阻碍, 如果长时间处于高浓度胁迫状态, 将造成萝卜幼苗的生理功能损伤, 影响产量。

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	图5 不同浓度17β -雌二醇对萝卜幼苗SOD活性的影响Fig.5 Effects of different concentrations of 17β -E2 on the SOD activity in radish

2.3.2 不同浓度17β -雌二醇对萝卜CAT影响过氧化氢酶(catalase, CAT)是以铁卟啉为辅基、催化过氧化氢(H₂O₂)分解为分子氧和水的重要酶, 与SOD、POD等协同作用, 使细胞免于遭受H₂O₂的毒害。10和50 μ g/L 浓度的17β -E2使萝卜CAT的活性分别比对照下降了5.84%和13.78%, 其中50 μ g/L 浓度处理组与对照差异显著; 100 μ g/L 浓度的17β -E2使萝卜CAT活性比对照上升1.85%, 但与对照差异不显著(图6)。一方面可能是由于萝卜在50 μ g/L 浓度的17β -E2胁迫下, 抑制了SOD的产生, 致使H₂O₂生成减少, 而CAT专一作用于H₂O₂, 但反应的米氏常数(Km)较大, 只能在H₂O₂浓度或酶活性较高时发挥作用^[18], 而本试验中H₂O₂生成减少, 使得CAT活性降低; 另一方面可能是因为植物体吸收的17β -E2直接对CAT的生成产生抑制, 具体机制需要进一步探讨。

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	图6 不同浓度17β -雌二醇对萝卜幼苗CAT活性的影响Fig.6 Effects of different concentrations of 17β -E2 on the CAT activity in radish

2.3.3 不同浓度17β -雌二醇对萝卜POD影响过氧化物酶(peroxidase, POD)是一族以过氧化氢(H₂O₂)为电子受体催化底物氧化的酶, 铁卟啉为辅基, 具有消除过氧化氢毒性的作用, 与植物的生长、发育、病性、衰老等有密切关系。萝卜POD活性受到加入17β -E2影响, 10 μ g/L处理组明显促进萝卜POD活性上升, 比对照组升高了32.87%, 而50和100 μ g/L浓度处理组的POD活性受到抑制, 活性分别下降了15.50%和5.64%, 其中以50 μ g/L浓度处理最为明显, 与对照差异显著(图7)。17β -E2对萝卜中POD的影响趋势与SOD基本相同, 主要协助SOD清除由SOD催化自由基 $\begin{matrix} {O_{2}}^{- \cdot} \end{matrix}$ 形成的过氧化氢(H₂O₂), 从而对细胞起保护作用。

	Figure Option View Download New Window
	图7 不同浓度17β -雌二醇对萝卜幼苗POD活性的影响Fig.7 Effects of different concentrations of 17β -E2 on the POD activity in radish

2.3.4 不同浓度17β -雌二醇对萝卜MDA影响丙二醛(malondialdehyde, MDA)是自由基作用于膜脂发生过氧化反应的产物, MDA的积累会引起蛋白质、核酸等生命大分子的交联聚合, 且具有细胞毒性。随着17β -E2处理浓度升高, MDA含量逐渐增加, 10, 50和100 μ g/L浓度处理的萝卜中MDA含量比对照升高了42.41%、52.65%和35.22%, 尤其以50 μ g/L浓度处理组的萝卜膜质过氧化程度严重, 3个处理组试验结果与对照组差异显著(图8)。

	Figure Option View Download New Window
	图8 不同浓度17β -雌二醇对萝卜幼苗MDA生成的影响Fig.8 Effects of different concentrations of 17β -E2 on the levels of MDA in radish

2.4 不同浓度17β -雌二醇在萝卜幼苗中的累积

17β -E2处理浓度在10100 μ g/L时, 随着浓度的升高, 单位重量的地上部分和根中累积的雌激素的量相应增加, 各组间差异显著, 同组内根部吸收的17β -E2比地上部分累积的量高1.11.7倍(图9)。检测发现, 17β -E2处理的幼苗除了有17β -E2残留, 还在地上部分和根部检测到代谢物雌酮(E1)的生成, 说明17β -E2可以随着萝卜的生长被机体吸收, 而吸收的17β -E2在机体某种机制下可发生代谢转化为E1。

	Figure Option View Download New Window
	图9 17β -雌二醇和代谢物雌酮在萝卜幼苗体内的累积Fig.9 The contents of 17β -E2 and E1 in radish

3 讨论

不同浓度的雌激素处理对萝卜种子萌发的影响程度不同。低浓度处理时, 对萝卜种子萌发有促进作用, 但随着17β -E2处理浓度的升高, 种子发芽势和发芽率均表现出逐渐降低的趋势, 而对发芽势的影响明显大于发芽率。萝卜根的生长也受到加入17β -E2的影响, 其生长与浓度呈负相关。17β -E2对种子发芽势、发芽率与根长影响的差异可能与种子发育过程有关。通常种子发芽过程与胚内营养成分和各种酶活动有关, 生长环境污染导致的影响只有在环境严重污染时与种子发芽有关的酶才能完全被抑制, 否则存在一个低浓度下刺激效应和高浓度下的抑制效应^{[11, 16, 17]}, 本试验结果与此相似。植物在生长和发育过程中, 根是始终暴露于17β -E2等污染的环境, 其对污染物的反应较为敏感^{[18, 19, 20, 21]}, 主要因为根是植物同周围环境交换和吸收营养物质的场所, 根细胞壁中交换位点能将17β -E2等污染物固定在这些位点上, 阻止或减少污染物进一步向地上部分转移^{[22, 23, 24]}, 试验中发现17β -E2在萝卜根部吸收的量要高于地上部分累积的量, 积累的雌激素使得其对根的抑制作用较为明显, 本研究也发现雌激素对萝卜幼苗根的抑制要大于对种子萌发的影响。17β -E2被萝卜吸收后在体内因素参与下发生了代谢, 随着萝卜幼苗生长期延长, 17β -E2是否会继续积累以及物质代谢发生的程度, 还需要进一步研究和探讨。

植物在正常代谢中会产生 $\begin{matrix} {O_{2}}^{- \cdot} \end{matrix}$ 、H₂O₂和· OH等活性氧, 而活性氧具有毒害作用。通常在逆境胁迫下, 植物活性氧产生和清除系统间的平衡被打破, 从而引起细胞膜、细胞器膜及DNA的伤害。SOD、POD和CAT是植物体构成过氧化物酶体的3种重要抗氧化酶, SOD的功能是将超氧阴离子自由基 $\begin{matrix} {O_{2}}^{- \cdot} \end{matrix}$ 歧化为H₂O₂, POD和CAT能够进一步把H₂O₂降解成无毒的H₂O和O₂, 三者间相互协调, 有效地清除代谢过程产生的活性氧, 从而防止了活性氧引起的膜脂过氧化及其他伤害。因此, SOD、POD和CAT活性的升降, 反映了植物在逆境因子作用下, 通过自身防御机制对环境胁迫做出保护性应激反应^{[18, 25]}; 而MDA则是脂质过氧化的产物, 直接反映了活性氧对植物的损伤情况^[26]。本试验中, 在010 μ g/L浓度的17β -E2胁迫下, 萝卜幼苗SOD活性增加, 清除 $\begin{matrix} {O_{2}}^{- \cdot} \end{matrix}$ 能力增强, 进而诱导CAT和POD协同清除氧自由基, 使其对机体的损伤减少到最小, 但在50 μ g/L浓度的17β -E2胁迫下, SOD、CAT和POD活性明显受到抑制, MDA含量显著升高, 膜质过氧化程度加剧, 究其原因, 一方面17β -E2诱导机体产生大量的活性氧自由基导致膜质发生氧化损伤; 另一方面可能是17β -E2抑制了抗氧化酶活力, 降低了线粒体清除活性氧自由基的能力, 如果长时间处于此种胁迫状态, 可能造成萝卜幼苗的生理功能损伤。

综上所述, 从萝卜种子的发芽势、发芽率、根长、抗氧化酶活性及MDA含量来看, 在17β -E2胁迫下, 萝卜的种子萌发和幼苗生长会受到影响, 植物从周围环境吸收养分的能力也导致17β -E2在根部和地上部分的累积并伴随代谢活动的发生, 从而可能造成农产品潜在的食用安全风险, 对于17β -E2在非靶标植物的运转机制和作用机理还需要进一步的探讨和研究。

The authors have declared that no competing interests exist.

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2003

0.0

... 环境雌激素,对野生动物和人体健康具有巨大的潜在危害,已经愈来愈受到全世界的关注^[1,2] ...

2006

3.137

0.0

. 2006, 65(8):1265-1280 DOI:10.1016/j.chemosphere.2006.08.003

Biologically directed environmental monitoring, fate, and transport of estrogenic endocrine disrupting compounds in water: A review

Abstract Endocrine disrupting compounds (EDCs) are contaminants that may be hormonally active at low concentrations and are emerging as a major concern for water quality. Estrogenic EDCs (e-EDCs) are a subclass of EDCs that, when organisms are exposed to them, function as estrogens. Given that there are numerous e-EDCs that can negatively affect humans and wildlife, general screening techniques like biologically based assays (BBAs) may provide major advantages by estimating the total estrogenic effects of many e-EDCs in the environment. These techniques may potentially be adapted for field portable biologically directed sampling and analyses. This article summarizes available BBAs used to measure estrogenic e-EDCs in the environmental samples and also presents results relating to fate and transport of e-EDCs. Estrogenic EDCs appear to be almost ubiquitous in the environment, despite low solubility and high affinity of organic matter. Potential transport mechanisms may include: (1) transport of more soluble precursors, (2) colloid facilitated transport, (3) enhanced solubility through elevated pH, and (4) the formation of micelles by longer-chain ethoxylates. Due to their persistent and ubiquitous nature, source control strategies for e-EDCs may reduce influent concentration to wastewater treatment plants so that the post treatment effluent will decrease concentrations to estrogenically inactive levels. Alternatively if source reduction is not possible, then more testing is needed on tertiary treatment technologies and treatment efficiencies for e-EDCs. There is still a need for research on remediation and restoration approaches for habitats disturbed by elevated e-EDC concentrations.

... 环境雌激素,对野生动物和人体健康具有巨大的潜在危害,已经愈来愈受到全世界的关注^[1,2] ...

2000

0.0

... 在诸多天然的雌性荷尔蒙物质中,雌二醇被认为是最具潜在影响的一种,即使是很低的浓度也会产生严重影响^[3,4] ...

2003

3.258

0.0

. 2003, 302(1-3):199-209

Fate of natural estrogen conjugates in municipal sewage transport and treatment facilities

Dipartimento di Chimica, Università La Sapienza di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.

The aim of this study was to investigate the fate of the conjugated forms of the three most common natural estrogens in the municipal aqueous environment. Levels of conjugated and free estrogens in (1) female urine; (2) a septic tank collecting domestic wastewater; (3) influents and effluents of six activated sludge sewage treatment plants (STPs) were measured. The analytical method was based on solid-phase extraction by using a Carbograph 4 cartridge and Liquid Chromatography-tandem Mass Spectrometry. On average, a group of 73 women selected to represent a typical cross section of the female inhabitants of a Roman condominium, excreted 106, 14 and 32 microg/day of conjugated estriol (E(3)), estradiol (E(2)) and estrone (E(1)), respectively. Apart from some E(3) in pregnancy urine, free estrogens were never detected in urine samples. Estrogen sulfates represented 21% of the total conjugated estrogens. This situation changed markedly in the condominium collecting tank. Here, significant amounts of free estrogens were observed and the estrogen sulfate to estrogen glucuronated ratio rose to 55/45. A laboratory biodegradation test confirmed that glucuronated estrogens are readily deconjugated in unmodified domestic wastewater, presumably due to the large amounts of the beta-glucuronidase enzyme produced by fecal bacteria (Escherichia coli). Deconjugation continued in sewer transit. At the STP entrance, free estrogens and sulfated estrogens were the dominant species. The sewage treatment completely removed residues of estrogen glucuronates and with good efficiency (84-97%) the other analytes, but not E(1) (61%) and estrone-3-sulfate (E(1)-3S) (64%). Considering that (1) E(1) has half the estrogenic potency of E(2), (2) the amount of the former species discharged from STPs into the receiving water was more than ten times larger than the latter one and (3) a certain fraction of E(1)-3S could be converted to E(1) in the aquatic environment, E(1) appears to be the most important natural endocrine disrupter.

PMID: 12526909

... 在诸多天然的雌性荷尔蒙物质中,雌二醇被认为是最具潜在影响的一种,即使是很低的浓度也会产生严重影响^[3,4] ...

2010

0.0

... g/kg干重,新西兰奶牛场污水中雌二醇水平为191 360 ng/L,代谢物雌酮的水平为413 123 ng/L,养猪场污水中的雌激素含量为46 ng/L,使用肉鸡粪便的土壤中雌激素浓度有55675 ng/kg^[5],而施用猪粪3个月后雌激素从植物根区的渗滤使农田排水系统中E1浓度可以达到68 ...

2007

0.0

. 2007, 41(11):3911-3917

Leaching of estrogenic hormones from manure-treated structured soils

Geological Survey of Denmark and Greenland, Oster Voldgade 10, DK-1350 Copenhagen, Denmark. jkj@geus.dk

The threat to the aquatic environment posed by root zone leaching of estrogens from manure-treated fields has hitherto been overlooked. The steroid hormones 17beta-estradiol (E2) and its degradation product estrone (E1) are of particular environmental concern as both are abundant in slurryfrom pregnant and cycling pigs and both are potential endocrine disruptors (lowest observable effect level (LOEL) 14 and 3.3 ng/L, respectively). The present one-year study examines the transport of E1 and E2 from manure to tile drainage systems at two field sites on structured, loamy soil. The estrogens leached from the root zone to tile drainage water in concentrations exceeding the LOEL for as long as 3 months after application, with the maximum recorded concentration of E1 and E2 being 68.1 and 2.5 ng/ L, respectively. Transport of estrogens from the soil to the aquatic environment was governed by pronounced macropore flow and consequent rapid movement of the estrogens to the tile drains. These findings suggest that the application of manure to structured soils poses a potential contamination risk to the aquatic environment with estrogen, particularly when manure is applied to areas where the majority of streamwater derives from drainage water.

PMID: 17612168

... 1 ng/L^[6],雌激素通过畜禽粪便农用的方式进入到土壤、水体中,对环境构成潜在威胁^[7,8],也造成农产品的污染 ...

2010

0.0

. 2010, 30(4):1058-1065

Ninety Breathalyzer instruments (Model 1000) and twenty instruments (Models 900, 900A) were studied using a protocol described by the Department of Transportation's "Standard for Devices to Measure Breath Alcohol." Although the mean of each of three concentrations tested (0.05, 0.10, and 0.15 g/210 L) compared favorably in both series, the standard deviation was consistently higher for the Model 1000 instruments. The Model 1000 instruments also produced a significant number of test results which exceeded the normally expected scientific deviation.

PMID: 4067535

... 1 ng/L^[6],雌激素通过畜禽粪便农用的方式进入到土壤、水体中,对环境构成潜在威胁^[7,8],也造成农产品的污染 ...

... 有研究表明,雌激素对人类的直接危害是干扰内分泌生殖系统的正常功能,促使婴儿性早熟和造成男性生育能力的下降甚至丧失^[7,9,10] ...

2009

3.137

0.0

. 2009, 74(4):551-555 DOI:10.1016/j.chemosphere.2008.09.055

Biochemical and biological assays of endocrine disrupting compounds in various manure matrices

Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.

Wastes generated by animal agriculture have complex matrices and present a significant challenge for achieving accurate measurements of estrogens. The objective of this study was to compare two different extraction methods and two different biochemical and biological estrogen assays on two dairy manure matrices. The two extraction methods studied in this research were a two-step base-toluene extraction and a one-step cyclohexane extraction. The two assays assessed were an enzyme-linked immunosorbent assay (ELISA) and a yeast estrogen screen (YES) bioassay. Estrogenic activity was measured directly as 17beta-estradiol (E2) through the ELISA method or as E2 equivalents (E2-eq) through the YES bioassay. Both extraction methods yielded reasonable recoveries from distilled water in the absence of matrix interferences. In manure samples, ELISA resulted in recoveries slightly higher than 100% in two types of dairy manures, but YES recoveries varied. The YES bioassay detected much higher estrogenic activities in dairy manure compared to the E2 concentrations measured by ELISA. The base-solvent extraction yielded higher E2 concentrations in dairy manure compared to the one-step cyclohexane extraction. These results suggest that manure matrices vary sufficiently that extraction methods must be optimized for specific assays utilized to quantify estrogens in manures.

PMID: 18992909

... 1 ng/L^[6],雌激素通过畜禽粪便农用的方式进入到土壤、水体中,对环境构成潜在威胁^[7,8],也造成农产品的污染 ...

2008

0.0

0.99

... 有研究表明,雌激素对人类的直接危害是干扰内分泌生殖系统的正常功能,促使婴儿性早熟和造成男性生育能力的下降甚至丧失^[7,9,10] ...

2001

4.328

0.0

. 2001, 60(2):296-304

Defining the impact of weakly estrogenic chemicals on the action of steroidal estrogens

Centre for Toxicology, School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom.

We tested whether bisphenol A (BPA) or o,p'-DDT, when combined with 17beta-estradiol (E2), would contribute to the overall mixture effect using a yeast reporter gene assay, the yeast estrogen screen. Following comprehensive concentration-response analyses of the single agents, the pharmacologically well-founded models of concentration addition and independent action were used to predict entire concentration-response relationships for mixtures of the agents with a variety of fixed mixture ratios, assuming additivity. For molar mixture ratios proportional to the levels normally found in human tissues (i.e., below 1:5000, E2:BPA or o,p'-DDT), these predictions suggest that the effects of individual xenoestrogens are too weak to create an impact on the actions of steroidal hormones. However, at mixture ratios more in favor of the xenoestrogens, a significant contribution to the overall mixture effect was predicted. The predictions were tested experimentally. The observed combined effects of mixtures of E2 with either BPA or o,p'-DDT did not deviate from the additivity expectation. On combining E2 with either BPA or o,p'-DDT at approximately equieffective concentrations corresponding to molar mixture ratios between 1:20,000 and 1:100,000 (E2:BPA or o,p'-DDT), substantial modulations of the effects of E2 became discernible. The assumption that weak xenoestrogens are generally unable to create an impact upon the already strong effects of endogenous steroidal estrogens is not supported by our observations. Our studies indicate that the potential health implication of additive combination effects between xenoestrogens and steroidal estrogens deserve serious consideration.

PMID: 11248142

... 有研究表明,雌激素对人类的直接危害是干扰内分泌生殖系统的正常功能,促使婴儿性早熟和造成男性生育能力的下降甚至丧失^[7,9,10] ...

2011

0.0

... 8磷酸缓冲液(含1% PVPK₃₀),在冰浴上研磨成浆,匀浆液倒入离心管中,在4℃下5 000 r/min离心10 min,上清液即为酶提取液,置于4℃ 保存待用^[11,12] ...

... 上述结果可能与雌激素透过种皮影响种子萌发相关的酶有关,这种影响存在一个较低浓度下刺激效应和高浓度下的抑制效应^[11,16,17] ...

... 通常种子发芽过程与胚内营养成分和各种酶活动有关,生长环境污染导致的影响只有在环境严重污染时与种子发芽有关的酶才能完全被抑制,否则存在一个低浓度下刺激效应和高浓度下的抑制效应^[11,16,17],本试验结果与此相似 ...

2008

1.232

0.8103

. 2008, 18:766-774 DOI:10.1016/S1002-0160(08)60072-7

Protective role of Ca against NaCl toxicity in Jerusalem artichoke by up-regulation of antioxidant enzymes

Abstract The ameliorative effect of external Ca 2+ on Jerusalem artichoke (Helianthus tuberosus L.) under salt stress was studied through biochemical and physiological analyses of Jerusalem artichoke seedlings treated with or without 10 mol L −1 CaCl 2 , 150 mmol L −1 NaCl, and/or 5 mmol L −1 ethylene-bis(oxyethylenenitrilo)-tetraacetic acid (EGTA) for five days. Exposure to NaCl (150 mmol L −1 ) decreased growth, leaf chlorophyll content, and photosynthetic rate of Jerusalem artichoke seedlings. NaCl treatment showed 59% and 37% higher lipid peroxidation and electrolyte leakage, respectively, than the control. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were decreased by NaCl, indicating an impeded antioxidant defense mechanism of Jerusalem artichoke grown under salt stress. Addition of 10 mmol L −1 CaCl 2 to the salt solutions significantly decreased the damaging effect of NaCl on growth and chlorophyll content and simultaneously restored the rate of photosynthesis almost to the level of the control. Ca 2+ addition decreased the leaf malondialdehyde (MDA) content and electrolyte leakage from NaCl-treated seedlings by 47% and 24%, respectively, and significantly improved the activities of SOD, POD, and CAT in NaCl-treated plants. Addition of EGTA, a specific chelator of Ca 2+ , decreased the growth, chlorophyll content, and photosynthesis, and increased level of MDA and electrolyte leakage from NaCl-treated plants and from the control plants. EGTA addition to the growth medium also repressed the activities of SOD, POD, and CAT in NaCl-treated and control seedlings. External Ca 2+ might protect Jerusalem artichoke against NaCl stress by up-regulating the activities of antioxidant enzymes and thereby decreasing the oxidative stress.

... 8磷酸缓冲液(含1% PVPK₃₀),在冰浴上研磨成浆,匀浆液倒入离心管中,在4℃下5 000 r/min离心10 min,上清液即为酶提取液,置于4℃ 保存待用^[11,12] ...

2012

0.0

. 2012, 893-894:57-62 DOI:10.1016/j.jchromb.2012.02.034

Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization

Ghent University Hospital, Department of Hormonology, De Pintelaan 185, 9000 Gent, Belgium. tom.fiers@uzgent.be

Measurement of estrone (E1) and estradiol (E2) values <1 pg/mL (3.7 pmol/L) is necessary for postmenopausal, pediatric and male serum samples. Until now this was rarely reached and only through derivatization which can present problems for estradiol. A very sensitive LC-MS/MS method was developed avoiding derivatization, convenient for large-scale studies. The desired sensitivity and specificity were achieved using ESI negative mode, LLE and a 2D chromatography consisting of a trapping column and a second dimension reverse-phase C8 analytical column. A mixture of an aqueous solution of ammonium fluoride at 0.2mM and methanol was used on the analytical column to further increase the sensitivity. Serum LOQ was <0.5 pg/mL (1.9 pmol/L) for E2 and E1 and recoveries ranged from 95 to 105%. No carry-over was detectable. Inter assay CV's were 4.0% at 21 pg/mL (77 pmol/L) for E2, 7.6% at 25 pg/mL (93 pmol/L) for E1. Comparison with commercial direct estrogen assays (Roche Diagnostics E170 for E2, Bioline RIA for E1) exposed analytical unsuitability (due to a combined lack of sensitivity and specificity) for the assay of male, postmenopausal or pediatric samples.

PMID: 22429877

... 样品处理参照Fiers等^[13]方法并加以改进^[14,15] ...

2010

4.612

0.0

. 2010, 1217(29):4784-4795 DOI:10.1016/j.chroma.2010.05.024

A systematic investigation to optimize simultaneous extraction and liquid chromatography tandem mass spectrometry analysis of estrogens and their conjugated metabolites in milk

Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260, USA.

In this study, the simultaneous extraction of estrone (E1), 17beta-estradiol (E2), estriol (E3), ethinylestradiol (EE2), and their glucuronated and sulfated metabolites in milk was optimized using solid-phase extraction (SPE). The aim of this research was to analyze estrogens and their conjugated metabolites by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in a single run, without the need to perform enzymatic cleavage and derivatization. Two SPE cartridges in tandem were used, consisting of sorbents based on the hydrophilic-lipophilic balance and amine-functionalized packing materials. To monitor analyte loss at every step of the SPE procedure (14)C-labeled E2 was spiked into the milk sample and the radioactivity was monitored at all stages of the SPE. In addition, non-radiolabeled standards of estrogens and metabolites were used to optimize solvent systems for the SPE and LC-MS/MS. The optimized method described in this paper can achieve recoveries ranging from 72% to 117% for the free estrogens (E1, E2, E3, and EE2), and 62% to 112% for seven conjugated metabolites. The three doubly conjugated, highly polar metabolites included in this study gave lower recoveries (< or = 43%) due to poor retention in SPE. Finally, commercial milk samples were analyzed for the presence of estrogens and their conjugated metabolites. Estrone (concentration range: 23-67 ng/L) was found to be the major free estrogen present in all milk samples. Estradiol was consistently observed in milk, but the concentrations were below the limit of detection (LOD of 10 ng/L), and no estriol and ethinylestradiol were detected. Several conjugated estrogen metabolites were identified, 17beta-estradiol-3-glucuronide (71-289 ng/L), estrone-3-sulfate (60-240 ng/L), 17beta-estradiol-3,17beta-sulfate (< LOD to 30 ng/L), and estrone-3-glucuronide (< LOQ of 25 ng/L). This method proved efficient in the simultaneous analysis of estrogens and their metabolites in milk.

PMID: 20541211

... 样品处理参照Fiers等^[13]方法并加以改进^[14,15] ...

2009

3.137

0.0

. 2009, 74(4):508-514 DOI:10.1016/j.chemosphere.2008.09.089

Determination of estrogenic compounds in wastewater using liquid chromatography-tandem mass spectrometry with electrospray and atmospheric pressure photoionization following desalting extraction

Department of Chemistry, National Central University, Chung-Li 32054, Taiwan.

Two complementary LC-MS ionization methods, electrospray (ESI) and atmospheric pressure photoionization (APPI), have been optimized to determine three natural estrogenic compounds (estrone, 17beta-estradiol and estriol) and two synthetic estrogenic compounds (17alpha-ethynylestradiol and diethylstilbestrol) in the influent and effluent of wastewater treatment plants (WWTPs). The wastewater samples were first subjected to solid-phase extraction coupled with desalting extraction to remove matrix interference. The analytes were then detected using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with ESI and dopant-assisted (DA) APPI to evaluate the ion suppression effect and to complement the detection and quantification of estrogenic compounds in complex wastewater samples. The average ion suppression factors for the extracts of the WWTP influent analyzed using ESI and APPI were 52+/-5% and 27+/-7%, respectively. The sensitivity and ionization efficiency of the LC-ESI-MS-MS system decreased dramatically when a complex matrix was present in the WWTP influent sample. Estrogenic compounds could be detected in the WWTP influent and effluent samples at concentrations below the parts-per-billion level. The lower detection limits obtained when using ESI and the higher matrix tolerance of the APPI method allowed the complete quantification of estrogenic compounds in very complex samples in a complementary manner.

PMID: 18996561

... 样品处理参照Fiers等^[13]方法并加以改进^[14,15] ...

2010

0.0

... 上述结果可能与雌激素透过种皮影响种子萌发相关的酶有关,这种影响存在一个较低浓度下刺激效应和高浓度下的抑制效应^[11,16,17] ...

2010

0.0

... 上述结果可能与雌激素透过种皮影响种子萌发相关的酶有关,这种影响存在一个较低浓度下刺激效应和高浓度下的抑制效应^[11,16,17] ...

2008

0.0

... -E2胁迫下,抑制了SOD的产生,致使H₂O₂生成减少,而CAT专一作用于H₂O₂,但反应的米氏常数(Km)较大,只能在H₂O₂浓度或酶活性较高时发挥作用^[18],而本试验中H₂O₂生成减少,使得CAT活性降低 ...

... -E2等污染的环境,其对污染物的反应较为敏感^{[18,19,20,21]},主要因为根是植物同周围环境交换和吸收营养物质的场所,根细胞壁中交换位点能将17#cod#x003b2 ...

... 因此,SOD、POD和CAT活性的升降,反映了植物在逆境因子作用下,通过自身防御机制对环境胁迫做出保护性应激反应^[18,25] ...

2008

0.0

2009

0.0

2011

0.0

2009

0.0

... -E2等污染物固定在这些位点上,阻止或减少污染物进一步向地上部分转移^[22,23,24],试验中发现17#cod#x003b2 ...

2010

0.0

. 2010, 78:1416-1421 DOI:10.1016/j.chemosphere.2009.12.048

Uptake of human pharmaceuticals by plants grown under hydroponic conditions

Colorado State University-Pueblo, Biology Department, 2200 Bonforte Blvd., Pueblo, CO 81001, USA.

Cabbage (Brassica rapa var. pekinensis) and Wisconsin Fast Plants (Brassica rapa) were chosen for a proof of concept study to determine the potential uptake and accumulation of human pharmaceuticals by plants. These plants were grown hydroponically under high-pressure sodium lamps in one of two groups including a control and test group exposed to pharmaceuticals. The control plants were irrigated with a recirculating Hoagland's nutrient solution while the test plants were irrigated with a Hoagland's nutrient solution fortified with the pharmaceuticals carbamazepine, salbutamol, sulfamethoxazole, and trimethoprim at 232.5 microg L(-1). When plants reached maturity, nine entire plants of each species were separated into components such as roots, leaves, stems, and seedpods where applicable. An analytical method for quantifying pharmaceuticals and personal care products was developed using pressurized liquid extraction and liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) in positive and negative ion modes using single ion monitoring. The method detection limits ranged from 3.13 ng g(-1) to 29.78 ng g(-1) with recoveries ranging from 66.83% to 113.62% from plant matrices. All four of the pharmaceuticals were detected in the roots and leaves of the cabbage. The maximum wet weight concentrations of the pharmaceuticals were detected in the root structure of the cabbage plants at 98.87 ng g(-1) carbamazepine, 114.72 ng g(-1) salbutamol, 138.26 ng g(-1) sulfamethoxazole, and 91.33 ng g(-1) trimethoprim. Carbamazepine and salbutamol were detected in the seedpods of the Wisconsin Fast Plants while all four of the pharmaceuticals were detected in the leaf/stem/root of the Wisconsin Fast Plants. Phloroglucinol staining of root cross-sections was used to verify the existence of an intact endodermis, suggesting that pharmaceuticals found in the leaf and seedpods of the plants were transported symplastically.

PMID: 20096438

... -E2等污染物固定在这些位点上,阻止或减少污染物进一步向地上部分转移^[22,23,24],试验中发现17#cod#x003b2 ...

2010

3.73

0.0

. 2010, 158:129-134 DOI:10.1016/j.envpol.2009.07.039

Hormetic effect(s) of tetracyclines as environmental contaminant on Zea mays

Dipartimento di Biologia, Università Tor Vergata, Rome, Italy.

Animal wastes from intensive pig farming as fertilizers may expose crops to antimicrobials. Zea mays cultivations were carried out on a virgin field, subjected to dressing with pig slurries contaminated at 15 mg L(-1) of Oxy- and 5 mg L(-1) of Chlor-tetracycline, and at 8 mg L(-1) of Oxy and 3 mg L(-1) of Chlor, respectively. Pot cultivation was performed outdoor (Oxy in the range 62.5-1000 ng g(-1) dry soil) and plants harvested after 45 days. Tetracyclines analyses on soils and on field plants (roots, stalks, and leaves) did not determine the appreciable presence of tetracyclines. Residues were found in the 45-day pot corn only, in the range of 1-50 ng g(-1) for Oxy in roots, accounting for a 5% carry-over rate, on average. Although no detectable residues in plants from on land cultivations, both experimental batches showed the same biphasic growth form corresponding to a dose/response hormetic curve.

PMID: 19695752

... -E2等污染物固定在这些位点上,阻止或减少污染物进一步向地上部分转移^[22,23,24],试验中发现17#cod#x003b2 ...

2007

0.0

0.4047

. 2007, 11:59-60

Effects of low temperature stress on physiological process of Galega L

以东方山羊豆及敖汉苜蓿为材料,研究低温胁迫对东方山羊豆和敖汉苜蓿可溶性蛋白含量、保护酶活性、渗透调节物质的影响.结果表明:在低温胁迫下,东方山羊豆及敖汉苜蓿脯氨酸含量、SOD、POD活性、可溶性蛋白含量先上升后下降.经对几项生理指标的综合分析,认为东方山羊豆比敖汉苜蓿具较强的抗低温能力.

... 因此,SOD、POD和CAT活性的升降,反映了植物在逆境因子作用下,通过自身防御机制对环境胁迫做出保护性应激反应^[18,25] ...

2012

0.0

... 而MDA则是脂质过氧化的产物,直接反映了活性氧对植物的损伤情况^[26] ...