Perfluorinated alkyl substances impede growth, reproduction, lipid metabolism and lifespan in Daphnia magna 全氟烷基物质阻碍大型蚤的生长、繁殖、脂质代谢和寿命
Asmerom Seyoum, Ajay Pradhan, Jana Jass, Per-Erik Olsson * Asmerom Seyoum、Ajay Pradhan、Jana Jass、Per-Erik Olsson *The Life Science Center-Biology, School of Science and Technology, Örebro University, SE-701 82 Örebro, Sweden 瑞典厄勒布鲁大学科技学院生命科学中心-生物学，SE-701 82 Örebro
H I G H L I G H T S
Per- and polyfluorinated alkyl substances altered lipid metabolism in Daphnia magna. 全氟和多氟烷基物质改变了大型蚤的脂质代谢。
Daphnia magna reproduction is impaired by both PFOS and PFOA. 全氟辛烷磺酸和全氟辛酸都会影响大型蚤的繁殖。
The longevity of Daphnia magna is reduced by PFOS but not PFOA 全氟辛烷磺酸会降低大型蚤的寿命，而全氟辛酸不会
Daphnia magna is a good animal model to investigate pollutants mechanism of action. 大型蚤是研究污染物作用机制的良好动物模型。
A R T I C L E I N F O
Article history: 文章历史：
Received 17 December 2019 2019 年 12 月 17 日收到
Received in revised form 18 May 2020 2020 年 5 月 18 日收到修订稿
Accepted 22 May 2020 接受 2020 年 5 月 22 日
Available online 25 May 2020 可于 2020 年 5 月 25 日在线查阅
Editor: Daniel Wunderlin 编辑丹尼尔-温德林
Keywords: 关键词：
Perfluorooctane sulfonate (PFOS) 全氟辛烷磺酸（PFOS）
Perfluorooctanoic acid (PFOA) 全氟辛酸（PFOA）
Fatty acid 脂肪酸
PFAS toxicity 全氟辛烷磺酸的毒性
Gene expression 基因表达
Fecundity 繁殖力
A B S T R A C T
Per- and polyfluorinated alkyl substances (PFASs) are synthetic organofluorine compounds with unique stability accompanied with hydrophobic and lipophobic properties. Perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) are of high concern due to their wide application in consumer and industrial products, extreme persistence, abundant occurrence in the environment and their toxic effect to humans and animals. However, knowledge on the molecular mechanisms of toxicity and the effects on reproduction output remain scarce. In this study, we analyzed the effects of PFOS and PFOA on Daphnia magna. Acute toxicity, development, reproduction, lipid metabolism (lipid-accumulation) and lifespan was investigated, as well as the expression of genes related to these endpoints. Exposure of PFOS and PFOA at 1, 10 and did not cause acute lethality. Hatching was reduced following exposure to both compounds, and lifespan was decreased following exposure to PFOS. Body length of Daphnia magna was reduced significantly by PFOS following 7 days exposure. Lipid staining revealed that all PFAS exposures increased lipid accumulation. qRT-PCR analysis of genes involved in lipid metabolism suggests that the increase in lipid content could be due to inhibition of genes involved on absorption and catabolism of fatty acids. Exposure to both PFOA and PFOS reduced the fecundity significantly. Downregulation of genes involved in development and reproductive process, including vtg2, vasa, EcRA, EcRB, usp, jhe, HR3, ftz-F1, E74 and E75 were observed. The alterations in developmental and reproductive genes as well as the disturbed lipid metabolism provides mechanistic insight into the possible causes for decreased fecundity and lifespan observed following exposure to both PFOS and PFOA. 全氟和多氟烷基物质（PFASs）是一种合成的有机氟化合物，具有独特的稳定性以及疏水和疏脂特性。全氟辛烷磺酸（PFOS）和全氟辛酸（PFOA）广泛应用于消费品和工业产品，具有极强的持久性，在环境中大量存在，对人类和动物具有毒性作用，因此备受关注。然而，有关其毒性的分子机制以及对生殖产出的影响的知识仍然匮乏。本研究分析了 PFOS 和 PFOA 对大型蚤的影响。研究了急性毒性、发育、繁殖、脂质代谢（脂质积累）和寿命，以及与这些终点相关的基因表达。接触 1、10 和 的全氟辛烷磺酸和全氟辛酸不会导致急性致死。暴露于这两种化合物后，孵化率降低，而暴露于 全氟辛烷磺酸后，寿命缩短。接触 全氟辛烷磺酸 7 天后，大型蚤的体长明显缩短。对参与脂质代谢的基因进行的 qRT-PCR 分析表明，脂质含量的增加可能是由于参与脂肪酸吸收和分解代谢的基因受到了抑制。暴露于全氟辛烷磺酸和全氟辛烷磺酸会显著降低繁殖力。观察到参与发育和生殖过程的基因下调，包括 vtg2、vasa、EcRA、EcRB、usp、jhe、HR3、ftz-F1、E74 和 E75。发育和生殖基因的改变以及脂质代谢的紊乱从机理上揭示了暴露于全氟辛烷磺酸和全氟辛酸后生育能力和寿命下降的可能原因。
Per- and polyfluorinated alkyl substances (PFASs) are synthetic organofluorine compounds where the hydrogen atoms on the carbon 全氟和多氟烷基物质（PFASs）是一种合成的有机氟化合物，其中碳原子上的氢原子和烷基上的氢原子都是烷基。
backbone are replaced by fluorine atoms with a charged functional group of either a carboxylate, alcohol, or sulfonate terminal group (Buck et al., 2011; Conder et al., 2008). Due to the strong electron withdrawing effect of fluorine, the bond is the strongest existing covalent bond ( ), which attributes to the chemical and thermal stability of PFASs (O'Hagan, 2008; Parsons et al., 2008). This unique stability together with their hydrophobic and lipophobic nature, contributes to their use in a wide variety of industrial and commercial products, such as lubricants, fire retardants, polymer additives, pesticides, protective coatings for clothing and other surfactants (Key et al., 1997; Kissa, 2001). The high-energy chemical bond in PFAS makes them resistant to hydrolysis, photolysis, biodegradation, and metabolism. These properties lead to PFAS persistence in the environment and allows bioaccumulation and biomagnification in the food web (Hekster et al., 2003; Schultz et al., 2003). Due to the reported toxicity, both the production and use of Perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) is now restricted in Europe (EC_1907/2006, 2006) and North America (USEPA, 2017). 骨架被带有羧酸盐、醇或磺酸盐末端带电官能团的氟原子取代（Buck 等人，2011 年；Conder 等人，2008 年）。由于氟具有很强的退电子效应， 键是现有最强的共价键（ ），这也是全氟辛烷磺酸具有化学和热稳定性的原因（O'Hagan，2008 年；Parsons 等人，2008 年）。这种独特的稳定性加上其疏水性和疏脂性，使其被广泛应用于各种工业和商业产品中，如润滑剂、阻燃剂、聚合物添加剂、杀虫剂、服装防护涂层和其他表面活性剂（Key 等人，1997 年；Kissa，2001 年）。全氟辛烷磺酸中的高能化学键使其不易水解、光解、生物降解和新陈代谢。这些特性导致 PFAS 在环境中持久存在，并在食物网中产生生物累积和生物放大作用（Hekster 等人，2003 年；Schultz 等人，2003 年）。由于所报告的毒性，全氟辛烷磺酸（PFOS）和全氟辛酸（PFOA）的生产和使用现已在欧洲（EC_1907/2006，2006年）和北美（美国环保局，2017年）受到限制。
Among PFASs, PFOS and PFOA are the two most ubiquitously distributed both in aquatic and terrestrial environment and are therefore of great concern for human and animal health (Ahrens and Bundschuh, 2014; Quinones and Snyder, 2009). PFOA and PFOS have been detected in both humans and animals, causing adverse effects on reproduction, development and immune response (DeWitt et al., 2012). In the environment, PFAS has been detected most commonly in the range of to level, however in highly contaminated areas such as at a fire-training area at Wurtsmith Air Force Base in USA it ranged from 3 to (Eschauzier et al., 2012; Moody et al., 2003) and at commercial airports in Sweden, levels between to were detected (Wennberg and Fridlund, 2015). A compilation of data from different studies estimated the release of PFAS into the aquatic environment in a range from to (Ahrens and Bundschuh, 2014). Studies on humans have shown that these chemicals cross the placental barrier, are transferred to the fetal circulation and also found in maternal milk (Kuklenyik et al., 2004; Midasch et al., 2007). The half-life of PFOS and PFOA in human serum is estimated to be 3.4 years and 2.7 years, respectively (Li et al., 2018). Estimated half-life in the aquatic environment due to hydrolysis has been reported to exceed 41 years for PFOS and 92 years for PFOA (Hekster et al., 2002). 在全氟辛烷磺酸和全氟辛酸中，全氟辛烷磺酸和全氟辛烷磺酰氨是在水生和陆生环境中分布最广的两种物质，因此对人类和动物的健康产生了极大的影响（Ahrens 和 Bundschuh，2014 年；Quinones 和 Snyder，2009 年）。已在人类和动物体内检测到全氟辛烷磺酸和全氟辛烷磺酸，它们会对生殖、发育和免疫反应造成不良影响（DeWitt 等人，2012 年）。在环境中，最常检测到的全氟辛烷磺酸含量在 至 之间，但在高污染地区，如美国沃兹密斯空军基地的消防训练区，全氟辛烷磺酸含量在 3 至 之间（Eschauzier 等人，2012 年；Moody 等人，2003 年），在瑞典的商业机场，检测到的全氟辛烷磺酸含量在 至 之间（Wennberg 和 Fridlund，2015 年）。不同研究的数据汇编估计，全氟辛烷磺酸在水生环境中的释放量在 至 之间（Ahrens 和 Bundschuh，2014 年）。对人类的研究表明，这些化学物质会穿过胎盘屏障，进入胎儿血液循环，并在母体乳汁中发现（Kuklenyik 等人，2004 年；Midasch 等人，2007 年）。据估计，全氟辛烷磺酸和全氟辛酸在人体血清中的半衰期分别为 3.4 年和 2.7 年（Li 等人，2018 年）。据报告，由于水解作用，全氟辛烷磺酸在水生环境中的半衰期估计超过41年，全氟辛酸则超过92年（Hekster等人，2002年）。
The toxicity of PFOS and PFOA has been studied in different model animals. The observed effects include mortality and immobilization, reproductive toxicity and immunotoxicity. However, little is known about their mode of action (Rainieri et al., 2017). Correlating gene expression profiles to physiological endpoints can provide better insights into the biological pathways and mode of action of the pollutants (Liu et al., 2014; Mao et al., 2013). 全氟辛烷磺酸和全氟辛酸的毒性已在不同的模式动物中进行过研究。观察到的影响包括死亡率和固定性、生殖毒性和免疫毒性。然而，人们对它们的作用模式知之甚少（Rainieri 等人，2017 年）。将基因表达谱与生理终点相关联，可以更好地了解污染物的生物途径和作用模式（Liu等人，2014年；Mao等人，2013年）。
Previous studies have shown that the is for PFOS and for PFOA for Daphnia magna (Ji et al., 2008), while the is for PFOS and for PFOA (Li, 2009). In a multigenerational study on Daphnia magna, it was observed that reduced fecundity was not observed below and that the time to first brood increased with exposures above (Jeong et al., 2016). In another study on Daphnia magna it was observed that PFOS at concentrations above and PFOA at concentrations above resulted in inhibition of reproduction (Yang et al., 2019). 先前的研究表明，对于大型蚤而言，全氟辛烷磺酸的 是 ，全氟辛酸的 （Ji 等人，2008年），而全氟辛烷磺酸的 是 ，全氟辛酸的 （Li，2009年）。在对大型蚤进行的一项多代研究中，观察到在 以下未观察到繁殖力降低的情况，而随着暴露量超过 ，第一次育雏的时间会增加（Jeong 等人，2016 年）。在另一项关于大型蚤的研究中观察到，全氟辛烷磺酸浓度高于 和全氟辛酸浓度高于 会导致繁殖受到抑制（Yang 等人，2019 年）。
In the present study, the aim was to determine both physiological effects (acute toxicity, fecundity, hatching, growth, longevity and lipid metabolism) on Daphnia magna and compare these effects with the toxicogenomic responses. In order to make these comparisons we choose to use concentrations of PFOS and PFOA that had earlier been observed to have effects on Daphnia magna, Japanese medaka and C. elegans (Jeong et al., 2016; Kang et al., 2019; Stylianou et al., 2019). 本研究旨在确定对大型水蚤的生理影响（急性毒性、繁殖力、孵化、生长、寿命和脂质代谢），并将这些影响与毒原反应进行比较。为了进行这些比较，我们选择使用早先已观察到对大型水蚤、日本青鳉和秀丽隐杆线虫有影响的全氟辛烷磺酸和全氟辛酸浓度（Jeong 等人，2016 年；Kang 等人，2019 年；Stylianou 等人，2019 年）。
Daphnia magna is widely distributed throughout the planet in all types of freshwater systems and is a part of the food chain of freshwater ecosystems and is therefore an excellent model system to study aquatic toxicology (Koivisto, 1995). Daphnia magna reproduces both by asexual parthenogenesis under favorable conditions and sexual production of dormant encapsulated eggs (ephippia) under unfavorable environments (Hebert, 1978). It is considered a representative species among the aquatic invertebrates and is used in standardized toxicity tests as pollution indicator species (Brown and Thompson, 1982; Le et al., 2016). In the present study, we analyzed the effects of PFOS and PFOA at different concentrations on Daphnia magna. Physiological endpoints were analyzed and compared to the gene expression from several signaling pathways including stress response, immune response, respiration, lipid metabolism, and reproduction. The results show a strong correlation between the physiological responses and the regulation of genes related to these physiological pathways. Analysis of toxicogenomic endpoints of PFOS and PFOA, on Daphnia magna, is important for improving our understanding of the effects elicited by these compounds and to develop adverse outcome pathway maps. 大型蚤广泛分布于地球上的各种淡水系统中，是淡水生态系统食物链的一部分，因此是研究水生毒物学的极佳模式系统（Koivisto，1995 年）。大型水蚤在有利条件下通过无性孤雌生殖繁殖，在不利环境下通过有性繁殖产生休眠包裹卵（ephippia）（Hebert，1978 年）。它被认为是水生无脊椎动物中的代表性物种，在标准化毒性试验中被用作污染指示物种（Brown 和 Thompson，1982 年；Le 等人，2016 年）。在本研究中，我们分析了不同浓度的全氟辛烷磺酸和全氟辛酸对大型蚤的影响。我们对生理终点进行了分析，并与包括应激反应、免疫反应、呼吸、脂质代谢和繁殖在内的几种信号通路的基因表达进行了比较。结果表明，生理反应与这些生理途径相关基因的调控之间存在很强的相关性。分析全氟辛烷磺酸和全氟辛酸对大型蚤的毒性基因组学终点，对于提高我们对这些化合物引发的影响的认识和开发不良后果途径图非常重要。
2. Materials and methods 2.材料和方法
2.1. Chemicals 2.1.化学品
PFOS (CAS No. 1763-23-1) and PFOA (CAS No. 335-67-1) (Fig. 1) were purchased from Sigma (USA) with stated purities in excess of . To obtain experimental exposure concentration i.e. and PFAS were dissolved in dimethyl sulfoxide (DMSO; Sigma). The final assay concentration of DMSO was . 全氟辛烷磺酸（化学文摘社编号：1763-23-1）和全氟辛酸（化学文摘社编号：335-67-1）（图 1）购自 Sigma（美国），纯度超过 。为了获得实验暴露浓度，即 和 ，将 PFAS 溶解在二甲基亚砜（DMSO；Sigma）中。DMSO 的最终检测浓度为 。
A
PFOS 全氟辛烷磺酸
CAS No. 化学文摘社编号
Mol wt. 500.13 摩尔重量 500.13
B
PFOA
CAS No. 335-67-1 化学文摘社编号 335-67-1
Mol wt. 414.07 摩尔重量 414.07
A
B
D
Day 14 第 14 天
PFOS 全氟辛烷磺酸
PFOS 全氟辛烷磺酸
PFOS 全氟辛烷磺酸
PFOA PFOA
PFOA PFOA
ㅂ PFOA ㅂ PFOA
Fig. 2. PFOS and PFOA impede Daphnia magna ephippia hatching, growth and lifespan. Twenty ephippia were exposed in triplicate to 1,10 and PFOS and PFOA and hatching success were recorded at (A). Similarly, 20 neonates ("24-h old) were exposed to PFOS and PFOA for 7 and 14 days and body and tail length were measured (B, C, D and E). Daphnia magna neonates ("24-h old) were exposed to PFOS and PFOA and the lifespan was recorded (F). One-way ANOVA followed by Dunnett post-test ("p ; * ; ***p p 0.001 ). . 图 2.全氟辛烷磺酸和全氟辛酸阻碍大型蚤蜕膜孵化、生长和寿命。将 20 只蜉蝣暴露于 1、10 和 的全氟辛烷磺酸和全氟辛酸中，一式三份，在 （A）记录孵化成功率。同样，20 只新生幼体（"24 小时龄"）分别暴露于全氟辛烷磺酸和全氟辛酸 7 天和 14 天，并测量了体长和尾长（B、C、D 和 E）。将大型蚤新生儿（"24 小时龄"）暴露于全氟辛烷磺酸和全氟辛酸并记录其寿命（F）。单因素方差分析，然后进行邓尼特后检验（"p ； * ； ***p p 0.001 ）。 .
2.2. Daphnia magna maintenance 2.2.大型蚤的维护
Daphnia magna ephippia were purchased from MicroBioTests Inc. (Belgium), and were activated by rinsing in tap water and hatched by incubating for 72-90 in standard freshwater (MicroBioTests Inc) at a temperature of under continuous illumination of . The artificial media, which was used for Daphnia magna culture included and , with a hardness of . The media was aerated for by bubbling air through a tube connected to an air pump. Newly hatched neonates ( old) were pre-feed with a suspension of Spirulina microalgae before exposure. For long term exposures, Daphnia magna cultures were maintained in controlled laboratory conditions, at a temperature of and a photoperiod cycle of light/10 h dark. Daphnia magna were fed a mixture of Spirulina microalgae and yeast (Saccharomyces cerevisiae) at a concentration of cells day and cells day, respectively. Fifty percent of treatment water was changed every other day. and dissolved oxygen (DO) were measured before starting and during exposure and remained in the range of and DO . Similarly, salinity, nitrite and nitrate were measured throughout the experiment and was maintained at , below and below respectively. 大型蚤(Daphnia magna ephippia)购自 MicroBioTests Inc.（比利时），用自来水冲洗活化，在标准淡水（MicroBioTests Inc）中孵化 72-90 ，温度为 ，持续光照 。用于大型蚤培养的人工培养基包括 和 ，硬度为 。培养基通过与气泵相连的管子鼓气，以便 。新孵化的新生蚤（ ）在暴露前预先投喂螺旋藻微藻类悬浮液 。对于长期暴露，大型蚤培养物保持在受控实验室条件下，温度为 ，光周期为 光/10 小时暗。给大型水蚤喂食螺旋藻微藻和酵母（酿酒酵母）的混合物，浓度分别为 cells day 和 cells day。每隔一天更换 50%的处理水。在开始接触前和接触期间测量 和溶解氧 (DO)，并保持在 和 DO 的范围内。同样，在整个实验过程中对盐度、亚硝酸盐 和硝酸盐 进行了测量，并分别保持在 、 和 以下。
2.3. Hatching, acute toxicity and gene expression analysis 2.3.孵化、急性毒性和基因表达分析
To determine the effects of PFOS and PFOA on hatching success, ephippia were rinsed with tap water and exposed in triplicates ( ) in standard fresh water containing DMSO (control) or PFOS and PFOA ( 1,10 and ). The ephippia were exposed in 6 well plates (BD Falcon, USA). In order to reduce binding of PFAS to the plates, the plates were pretreated with the exposure solutions for . 为确定全氟辛烷磺酸和全氟辛酸对 孵化成功率的影响，用自来水冲洗鳗鲡，并以三重复 ( ) 的方式将鳗鲡置于含有 DMSO（对照）或全氟辛烷磺酸和全氟辛酸 ( 1,10 和 ) 的 标准淡水中。表皮藻暴露在 6 孔板（BD Falcon，美国）中。为了减少 PFAS 与平板的结合，用暴露溶液对平板进行预处理， 。
For survival assay, Daphnia magna neonates ( old) were exposed as outlined above with 5 animals in each well with 5 replicates. Mortality was recorded at 6,24 and 48 h. Daphnia magna were considered dead when there was no mobility and no organ movement under the microscope. For qRT-PCR analysis, 20 Daphnia magna neonates ( old) were exposed to 1,10 and PFOS and PFOA in 6 well plates for . Following exposure Daphnia magna were collected, snap frozen using liquid nitrogen and the samples were stored at until analyzed. 在存活率检测中，按上述方法将大型蚤新生儿（ ）暴露在水中，每孔 5 只，5 次重复。在 6、24 和 48 小时时记录死亡率。当大型水蚤在显微镜下没有活动和器官移动时，则认为其已经死亡。为了进行 qRT-PCR 分析，20 只大型水蚤新体（ ）在 6 孔板中暴露于 1、10 和 全氟辛烷磺酸和全氟辛酸， 。暴露后收集大型蚤，用液氮速冻，并将样本保存在 ，直至分析。
Fig. 3. PFOS and PFOA alter the expression of stress response genes. Daphnia magna neonates ( old) were exposed to 1,10 and PFOS and PFOA for and transcription level of (A), (B), (C), (D), (E), sod1 (F), sod2 (G), cat (H) and gst (I) were analyzed using qRT-PCR. One-way ANOVA followed by Dunnett post-test ( ; **p ; . 图 3.全氟辛烷磺酸和全氟辛酸会改变应激反应基因的表达。将大型水蚤新生儿（ 龄）暴露于 1,10 和 PFOS 和 PFOA， ，并使用 qRT-PCR 分析 (A)、 (B)、 (C)、 (D)、 (E)、sod1 (F)、sod2 (G)、cat (H) 和 gst (I) 的转录水平。单因素方差分析后进行邓尼特后检验 ( ; **p ; 。
2.4. Determination of growth, reproduction and lifespan 2.4.生长、繁殖和寿命的测定
For growth and reproduction analysis, Daphnia magna neonates ( old) were exposed in triplicates in crystallization dish containing of 1,10 and PFOS or PFOA prepared in standard water. Exposure for growth analysis was continued for 14 days with 10 animals per dish and the body and tail lengths were measured using a bright field microscope (Olympus BX51) at day 7 and day 14. To determine the effects on reproduction, Daphnia magna were exposed for 21 days with 10 animals per dish and the number of offspring was recorded daily and removed from the container. For lifespan analysis, exposure continued until all animals were dead and the number of live animals were recorded every day. 在进行生长和繁殖分析时，将大型蚤幼体（ ）置于 结晶皿中，每皿三只，皿中含有 、1,10 和 在标准水中制备的全氟辛烷磺酸或全氟辛酸。在第 7 天和第 14 天，使用明视野显微镜（奥林巴斯 BX51）测量体长和尾长。为了确定对繁殖的影响，对大型水蚤进行了 21 天的暴露，每个皿中有 10 只水蚤，每天记录后代的数量并从容器中取出。在寿命分析中，暴露持续到所有动物死亡，每天记录活体动物的数量。
2.5. isolation and quantitative (qRT-PCR) 2.5. 分离和定量 (qRT-PCR)
Twenty Daphnia magna neonates ( ), were pooled together and lysed in of TRIzol reagent (Sigma) followed by RNA isolation using Direct-zol Kit (Zymo Research, USA) according to the manufacturer's instructions. The RNA concentration was measured using a DeNovix DS-11 spectrophotometer (Wilmington DE, USA) and of RNA was used to prepare cDNA using qScript cDNA synthesis kit (Quanta Biosciences, USA) according to the manufacturer's instructions. qRT-PCR was performed to quantify the expression of the genes using SSoAdvanced universal SYBR Green supermix (BioRad, USA) using a CFX384 Real time PCR detection system (Bio-Rad, USA) with thermocycling profiles of initial denaturation step at for followed by 35 cycles of for and for . Relative gene expression was determined using the Ct method (Schmittgen and Livak, 2008) by normalizing Ct values obtained against the Ct value of the housekeeping gene actin. Primers for selected genes are listed (Table ). 将 20 只大型蚤新生个体( )集中在一起，用 TRIzol 试剂（Sigma 公司）裂解，然后按照生产商的说明使用 Direct-zol Kit（Zymo Research 公司，美国）分离 RNA。使用 DeNovix DS-11 分光光度计（Wilmington DE, USA）测量 RNA 浓度， ，然后按照生产商的说明使用 qScript cDNA 合成试剂盒（Quanta Biosciences, USA）制备 cDNA。使用 CFX384 Real time PCR 检测系统（Bio-Rad，美国），使用 SSoAdvanced universal SYBR Green supermix（BioRad，美国）进行 qRT-PCR 对基因的表达进行定量，热循环曲线为 初始变性步骤（ ），然后进行 35 个循环： （ ）和 （ ）。使用 Ct 法（Schmittgen 和 Livak，2008 年）将获得的 Ct 值与看家基因肌动蛋白的 Ct 值进行归一化，从而确定基因的相对表达量。表 列出了选定基因的引物。
2.6. Lipid staining 2.6.脂质染色
To analyze effect of PFOS and PFOA on lipid metabolism, Daphnia magna were exposed for 4 days in solution as previously described (Seyoum and Pradhan, 2019) and thereafter stained using Oil Red O (ORO; Sigma). In brief, exposed Daphnia magna were collected and washed twice with PBS and fixed with isopropanol. Daphnia magna were then permeabilized using triton X 100 in PBS (PBST). The fixed samples were washed again with PBS and Daphnids were then stained for using an ORO working solution. Images were taken using a microscope with a objective (Olympus BX51). 为了分析全氟辛烷磺酸和全氟辛酸对脂质代谢的影响，按照之前的描述（Seyoum 和 Pradhan，2019 年），将大型蚤暴露于 溶液中 4 天，然后用油红 O（ORO；Sigma）染色。简言之，收集暴露的大型蚤，用 PBS 冲洗两次，然后用 异丙醇固定。然后用 triton X 100 in PBS (PBST) 对水蚤进行渗透。再次用 PBS 冲洗固定样本，然后使用 ORO 工作溶液对水蚤进行染色，以检测 。使用 物镜（奥林巴斯 BX51）拍摄图像。
NOS 2
Fig. 4. PFOS and PFOA alter the expression of immune related genes. Daphnia magna neonates ( old) were exposed to 1,10 and PFOS and PFOA for 24 -h and transcription level of C1qdc (A), a2m (B), GNBP (C), Cst6 (D), dap1 (E), NOS2 (F) and proPO (G) were analyzed using qRT-PCR. One-way ANOVA followed by Dunnett post-test ( ; * ; ). . 图 4.全氟辛烷磺酸和全氟辛酸会改变免疫相关基因的表达。将大型水蚤新生儿（ ）暴露于 1,10 和 PFOS 和 PFOA 24 小时，使用 qRT-PCR 分析 C1qdc (A)、a2m (B)、GNBP (C)、Cst6 (D)、dap1 (E)、NOS2 (F) 和 proPO (G) 的转录水平。单因素方差分析，然后进行 Dunnett 后检验 ( ; * ; )。 .
2.7. Statistical analysis 2.7.统计分析
Statistical analysis was performed using the GraphPad Prism 7 software (GraphPad Software, USA). One-way ANOVA followed by Dunnett's post-test was used to assess statistical significance (* . 使用 GraphPad Prism 7 软件（GraphPad Software，美国）进行统计分析。采用单因素方差分析和 Dunnett 后检验来评估统计显著性（* .
3. Results and discussion 3.结果和讨论
3.1. PFOS and PFOA impede hatching, growth and lifespan 3.1.全氟辛烷磺酸和全氟辛酸阻碍孵化、生长和寿命
Exposure of Daphnia magna neonates ( old) to PFOS and PFOA at 1,10 and for after hatching did not result in any mortality (data not shown). As these concentrations did not cause acute lethality, they were considered to be relevant for the subsequent physiological and toxicogenomic experiments. Earlier studies have detected up to PFOS and PFOA in humans (Olsen et al., 2007), up to PFOS (wet wt) in the liver of Ornate Jobfish (Tropidinius amoenus) (Taniyasu et al., 2003) and PFOS in plasma of Bottlenose dolphin (Tursiops truncatus) (Houde et al., 2005). As previous studies have shown that these levels have been detected in the environment and in blood and serum of animals, including humans they can be considered environmentally relevant (DeWitt et al., 2012; Houde et al., 2005). Thus, the exposure conditions used in the present study are within the range of observed concentrations in humans and animals. 孵化后的大型蚤新生儿（ ）接触浓度为 1、10 和 的全氟辛烷磺酸和全氟辛酸， ，并未导致任何死亡（数据未显示）。由于这些浓度不会导致急性死亡，因此被认为与随后的生理和毒物基因组学实验相关。早先的研究在人类体内检测到了高达 的全氟辛烷磺酸和 的全氟辛基醚（Olsen 等人，2007 年），在鸟鲣（Tropidinius amoenus）肝脏中检测到了高达 的全氟辛烷磺酸 （湿重）（Taniyasu 等人，2003 年），在瓶鼻海豚（Tursiops truncatus）血浆中检测到了 的全氟辛烷磺酸（Houde 等人，2005 年）。先前的研究表明，在环境以及包括人类在内的动物血液和血清中都检测到了这些水平，因此可以认为它们与环境相关（DeWitt 等人，2012 年；Houde 等人，2005 年）。因此，本研究中使用的接触条件属于在人类和动物体内观测到的浓度范围。
Exposure of Daphnia magna ephippia to PFOS and PFOA resulted in significantly reduced hatching following exposure to and PFOS and PFOA (Fig. 2A) indicating that PFOS is more toxic than PFOA to this life stage. While light and changes in photoperiod and temperature play an important role in stimulating the hatching of Daphnid dormant eggs (Davison, 1969; Paes et al., 2016; Pancella and Stross, 1963), earlier studies have shown that environmental toxicants can also influence hatching (Radzikowski et al., 2016). The pesticides fenoxycarb inhibit hatching at concentrations between 2 and (Navis et al., 2013). Daphnia curvirostris ephippia hatching success was reduced by the fire-retardant, Fire-Trol 934 (Angeler et al., 2005). PFOS has also been shown to reduce hatching of tree swallow (Tachycineta bicolor) and Caenorhabditis elegans eggs (Custer et al., 2014; Guo et al., 2016). The present results are in line with the previous studies as both PFAS compounds affected Daphnia magna hatching success. 将大型水蚤蜕膜暴露于全氟辛烷磺酸和全氟辛酸后，其孵化率在暴露于 和 全氟辛烷磺酸以及 全氟辛酸后显著降低（图 2A），这表明全氟辛烷磺酸对这一生命阶段的毒性大于全氟辛酸。虽然光照以及光周期和温度的变化在刺激水蚤休眠卵的孵化过程中起着重要作用（Davison，1969 年；Paes 等人，2016 年；Pancella 和 Stross，1963 年），但早期研究表明，环境毒物也会影响孵化（Radzikowski 等人，2016 年）。浓度在 2 到 之间的杀虫剂芬氧威会抑制孵化（Navis 等人，2013 年）。阻燃剂 Fire-Trol 934 会降低水蚤的孵化成功率（Angeler 等人，2005 年）。研究还表明，全氟辛烷磺酸会降低树燕（Tachycineta bicolor）和秀丽隐杆线虫卵的孵化率（Custer等人，2014年；Guo等人，2016年）。本研究结果与之前的研究结果一致，因为这两种全氟辛烷磺酸化合物都会影响大型水蚤的孵化成功率。
Daphnia magna growth was analyzed by measuring body and tail length after 7- and 14-day exposures (Fig. 2B-E). Daphnia magna exposed to PFOS showed high mortality after day 7 . As this resulted in a drastic reduction of adult Daphnia magna the PFOS exposure was excluded from growth and reproduction analysis. While there was a reduction of body length following exposure to PFOS after 7 days, this effect was no longer visible after 14 days. This suggests that PFOS could lead to delayed development. A increase in tail length was observed after exposure to PFOS for 14 days, demonstrating that PFOS disrupted developmental processes in Daphnia magna. Studies in mice have shown that PFOS leads to delayed development in utero and reduced postnatal growth and survival (Abbott et al., 2009). PFOS has also been found to cause developmental toxicity in both zebrafish and Caenorhabditis elegans (Guo et al., 2016; Shi et al., 2008). 通过测量暴露 7 天和 14 天后的体长和尾长，分析了大型蚤的生长情况（图 2B-E）。接触 全氟辛烷磺酸的大型蚤在第 7 天后死亡率很高。由于这导致大型水蚤成虫数量急剧下降，因此 全氟辛烷磺酸暴露被排除在生长和繁殖分析之外。虽然 在接触 全氟辛烷磺酸 7 天后体长会缩短，但这种影响在 14 天后就不再明显。这表明全氟辛烷磺酸可能会导致发育延迟。接触 全氟辛烷磺酸 14 天后，观察到 尾长增加，这表明全氟辛烷磺酸干扰了大型蚤的发育过程。对小鼠的研究表明，全氟辛烷磺酸会导致小鼠在子宫内发育迟缓，产后生长和存活率降低（Abbott 等人，2009 年）。研究还发现，全氟辛烷磺酸会对斑马鱼和秀丽隐杆线虫造成发育毒性（Guo 等人，2016 年；Shi 等人，2008 年）。
PFOS 全氟辛烷磺酸
Fig. 5. PFOS and PFOA increase lipid accumulation. Daphnia magna neonates ( old) were exposed to 1,10 and PFOS and PFOA for 4 days after which the lipid accumulation was analyzed using Oil Red O staining. Scale bar represents . 图 5.全氟辛烷磺酸和全氟辛酸会增加脂质积累。将大型水蚤新生儿（ ）暴露于 1,10 和 PFOS 和 PFOA 4 天后，使用油红 O 染色法分析脂质积累情况。刻度条代表 。
Thus, our results are in line with the earlier studies and demonstrate that PFOS disturbs developmental processes in Daphnia magna. 因此，我们的研究结果与之前的研究结果一致，证明了全氟辛烷磺酸会干扰大型蚤的发育过程。
To determine PFAS effect on longevity, Daphnia magna were exposed through a complete lifecycle. The control animals showed a lifespan of 45 days while animals exposed to 1 and PFOS and all concentrations of PFOA showed lifespans between 39 and 41 days. Daphnia magna exposed to PFOS showed severely reduced lifespan of 23 days, with an early onset of mortality from day 5 (Fig. 2F). Chronic exposure of Caenorhabditis elegans to 0.2-200 PFOS have also shown concentration dependent decrease in lifespan (Xu et al., 2016). Furthermore, in the same study, it was observed that exposure to PFOS quickened the aging process by affecting the insulin signaling pathway, including the daf-16 and age-1 genes, resulting in a shortened lifespan (Xu et al., 2016). As the insulin signaling pathway is evolutionarily conserved and plays an important role in regulating animal development and lifespan (Tatar et al., 2003), it is possible that the PFOS mediated shortened lifespan observed for Daphnia magna could also be due to disruption of this pathway. 为了确定全氟辛烷磺酸对寿命的影响，大型水蚤在整个生命周期中都接触了全氟辛烷磺酸。对照组动物的寿命为 45 天，而暴露于 1 和 全氟辛烷磺酸以及所有浓度全氟辛酸的动物的寿命为 39 至 41 天。大型蚤暴露于 全氟辛烷磺酸后，寿命严重缩短为 23 天，并从第 5 天开始死亡（图 2F）。草履虫长期暴露于 0.2-200 全氟辛烷磺酸也会导致寿命缩短（Xu 等人，2016 年）。此外，在同一研究中还观察到，暴露于全氟辛烷磺酸会影响胰岛素信号通路，包括 daf-16 和 age-1 基因，从而加速衰老过程，导致寿命缩短（Xu 等人，2016 年）。由于胰岛素信号通路在进化过程中是保守的，并且在调节动物发育和寿命方面发挥着重要作用（Tatar 等人，2003 年），因此在大型水蚤中观察到的由全氟辛烷磺酸介导的寿命缩短也可能是由于该通路被破坏所致。
To determine toxicogenomic responses of Daphnia magna following exposure to PFAS, stress and immune response genes were analyzed. The stress response genes included 2 heat shock response genes (hsp70 and hsp90), 3 metallothionein genes ( and ) and 4 oxidative response genes copper/zinc-superoxide dismutase (sod1), manganese-superoxide dismutase (sod2), (glutathione S transferase (gst) and catalase (cat)). No dose dependent effects were observed for hsp70 or hsp90 (Fig. 3). All 3 mt genes showed a dose dependent response to PFOS, while, only showed dose dependent regulation following exposure to PFOA. MT's are small, cysteine-rich heavy metalbinding proteins present in most organisms, from invertebrates to mammals that participate in metal homeostasis, protection against heavy metals and oxidative stress and metabolic regulation (Amiard et al., 2006; Andrews, 2000; Dong et al., 2015; Olsson et al., 1998). Apart from responding to metals stress, MTs are also known to respond to other xenobiotic compounds (Pradhan et al., 2020; Sato et al., 2013; Seyoum and Pradhan, 2019). It has been suggested that the glucocorticoid receptor and aryl hydrocarbon receptor can activate MTs (Pradhan et al., 2020; Sato et al., 2013). Studies have shown that downregulation of genes leads to susceptibility of organisms to metal toxicity, oxidative stress as well as metabolic dysregulations (Kling and Olsson, 2000; Park et al., 2001; Rodriguez-Menendez et al., 2018; Sato et al., 2010). Thus, the downregulation of the genes by PFOS may contribute to the physiological effects observed for Daphnia magna exposed to PFOS 为了确定大型蚤接触全氟辛烷磺酸后的毒性基因组反应，分析了应激和免疫反应基因。应激反应基因包括 2 个热休克反应基因（hsp70 和 hsp90）、3 个金属硫蛋白基因（ 和 ）和 4 个氧化反应基因铜/锌-超氧化物歧化酶（sod1）、锰-超氧化物歧化酶（sod2）、谷胱甘肽 S 转移酶（gst）和过氧化氢酶（cat）。对 hsp70 和 hsp90 没有观察到剂量依赖效应（图 3）。所有 3 个 MT 基因对全氟辛烷磺酸都表现出剂量依赖性反应，而只有 在暴露于全氟辛酸后表现出剂量依赖性调节。MT是一种富含半胱氨酸的小型重金属结合蛋白，存在于从无脊椎动物到哺乳动物的大多数生物体中，参与金属平衡、防止重金属和氧化应激以及代谢调节（Amiard等人，2006年；Andrews，2000年；Dong等人，2015年；Olsson等人，1998年）。除了对金属应激做出反应外，已知 MTs 还能对其他异生物化合物做出反应（Pradhan 等人，2020 年；Sato 等人，2013 年；Seyoum 和 Pradhan，2019 年）。有研究表明，糖皮质激素受体和芳基烃受体可激活 MTs（Pradhan 等人，2020 年；Sato 等人，2013 年）。研究表明， 基因的下调会导致生物体易受金属毒性、氧化应激以及代谢失调的影响（Kling 和 Olsson，2000 年；Park 等人，2001 年；Rodriguez-Menendez 等人，2018 年；Sato 等人，2010 年）。因此，全氟辛烷磺酸对 基因的下调可能是导致大型蚤暴露于 全氟辛烷磺酸的生理效应的原因。
Cellular oxidative stress is a state of imbalance between the level of cellular reactive oxygen species (ROS) production and the antioxidant defense mechanisms (Flora, 2009). Environmental pollutants and irradiations can trigger excess generation of ROS and free radicals that can lead to oxidative damage including lipid peroxidation, protein and DNA oxidation, and enzyme inactivation (Livingstone, 2001; MacFadyen et al., 2004; Sarkar et al., 2014; Slaninova et al., 2009). Antioxidant enzymes with a central role to overcome the imposed oxidative stress includes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). In the SOD-CAT system SOD catalytically converts the superoxide radical ( ) or singlet oxygen radical generated in the cells, to molecular oxygen and toxic hydrogen peroxide (Ighodaro and Akinloye, 2018). However, the antioxidant enzyme, CAT and GST, can synergistically convert the intracellular free radical into water and molecular oxygen, consequently counteracting free radical-induced damage (Ighodaro and Akinloye, 2018; Wang et al., 2011). In the present study, sod1 was upregulated following exposure to 1 and PFOS and downregulated by PFOA, whereas the expression of sod2 was only upregulated by 10 and PFOS. However, the transcript level of gst and cat were not altered by PFOS and PFOA. In an earlier study on Daphnia magna it was observed that F0 daphnia exposed to PFOS did not show any significant change in GST activity (Jeong et al., 细胞氧化应激是细胞活性氧（ROS）生成水平与抗氧化防御机制之间的一种失衡状态（Flora，2009 年）。环境污染物和辐照会引发过量生成 ROS 和自由基，从而导致氧化损伤，包括脂质过氧化、蛋白质和 DNA 氧化以及酶失活（Livingstone，2001 年；MacFadyen 等人，2004 年；Sarkar 等人，2014 年；Slaninova 等人，2009 年）。在克服强加的氧化应激方面发挥核心作用的抗氧化酶包括超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GPx）和谷胱甘肽 S-转移酶（GST）。在 SOD-CAT 系统中，SOD 催化将细胞中产生的超氧自由基 ( ) 或单线态氧自由基 转化为分子氧 和有毒的过氧化氢 （Ighodaro 和 Akinloye，2018 年）。然而，抗氧化酶 CAT 和 GST 可协同将细胞内自由基 转化为水和分子氧，从而抵消自由基引起的损伤（Ighodaro 和 Akinloye，2018；Wang 等人，2011）。在本研究中，sod1 在暴露于 1 和 全氟辛烷磺酸后上调，在暴露于 全氟辛酸后下调，而 sod2 仅在暴露于 10 和 全氟辛烷磺酸后上调。然而，全氟辛烷磺酸和全氟辛酸不会改变 gst 和 cat 的转录水平。早先对大型水蚤进行的一项研究观察到，暴露于全氟辛烷磺酸的 F0 型水蚤的 GST 活性没有发生任何显著变化（Jeong 等人："水蚤的 GST 活性"）、
A
D
B B
Fig. 6. PFOS and PFOA alter the expression of genes involved in lipid metabolism. Daphnia magna neonates ( ) were exposed to 1,10 and PFOS and PFOA for and qRT-PCR analysis was performed for hr96 (A), NPC1b (B), magro (C), SM3 (D), cer2 (E) and man (F). One-way ANOVA followed by Dunnett post-test was performed to determine statistical significance . 图 6.全氟辛烷磺酸和全氟辛酸会改变脂质代谢相关基因的表达。将大型蚤新生儿 ( ) 暴露于 1,10 和 PFOS 和 PFOA， ，并对 hr96 (A)、NPC1b (B)、magro (C)、SM3 (D)、cer2 (E) 和 man (F) 进行 qRT-PCR 分析。进行单因素方差分析和邓尼特后检验以确定统计显著性 。
A
2016). An increased GST activity from F2 to F4 at different exposure concentration was also observed, but mainly in response to a high PFOS concentrations ( exposure (Jeong et al., 2016). Overall, our qRT-PCR results are in agreement with the observations by Jeong and coworkers. 2016).在不同的暴露浓度下，也观察到从 F2 到 F4 的 GST 活性增加，但主要是对高浓度全氟辛烷磺酸的反应（ ）（Jeong 等人，2016 年）。总体而言，我们的 qRT-PCR 结果与 Jeong 及其同事的观察结果一致。
3.2. PFOS and PFOA alterations of immune response gene expression 3.2.全氟辛烷磺酸和全氟辛酸对免疫反应基因表达的改变
The immune system is essential for survival of organisms and is an important response to environmental exposures (MacGillivray and Kollmann, 2014). PFOS and PFOA have been shown to increase mortality through disruption of the immune system in different organisms including Caenorhabditiselegans, zebrafish, mice and humans (Li et al., 2017; Stylianou et al., 2019). To obtain insights into immune toxicity of PFOS and PFOA in Daphnia magna, the transcript levels of six immune response genes were analyzed. 免疫系统对生物的生存至关重要，也是对环境暴露的重要反应（MacGillivray 和 Kollmann，2014 年）。研究表明，全氟辛烷磺酸和全氟辛酸会通过破坏不同生物体（包括鲤科动物、斑马鱼、小鼠和人类）的免疫系统而增加死亡率（Li 等人，2017 年；Stylianou 等人，2019 年）。为了深入了解全氟辛烷磺酸和全氟辛酸对大型蚤的免疫毒性，我们分析了六个免疫反应基因的转录水平。
The genes C1q-domain-containing gene (C1qdc), Alpha-2Macroglobulin (a2m) and, Gram-negative bacteria binding-protein (GNBP) were all downregulated by PFOS and at all PFOA exposures (Fig. 4A-C). C1qdc proteins bind to various pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycan and glucan, and function as signaling molecules to regulate initialization of inflammation response, adaptive immunity, apoptotic cells clearance, bacteria and retrovirus recognition and energy homeostasis (Kishore et al., 2004; Wang et al., 2012). a2m bind pathogen serine proteases and GNBPs recognize polysaccharides on pathogen surfaces and play important roles as pathogen protease inhibitors and in non-selfrecognition to stimulate the subsequent immune signal transduction pathways, such as the prophenoloxidase (proPO) cascade, Toll and/or immune deficiency pathways (Jin et al., 2012; Ponprateep et al., 2017). It is interesting to note that PFOA despite having lower toxicity than PFOS, showed strong effects on C1qdc, , and GNBP genes. This indicates that PFOA exposure could make the animal highly immunocompromised. The different toxicity profiles of PFOS and PFOA may be due to differences in hydrophilic functional groups which could result in different affinity to biomolecules (Gao et al., 2013; and Hungerbuehler, 2015). Xia and coworkers showed that PFOS can bioaccumulate around 2 times higher than PFOA (Xia et al., 2013). A study on Escherichia coli indicated that PFOA is more toxic than PFOS. The authors suggested that the toxicity mechanism of PFOS is more direct by disrupting the bacterial cell membrane and inducing oxidative stress, while PFOA induce toxicity through cell inactivation and/or death mainly through a genetic interruption pathway (Liu et al., 2016). PFOS 和所有 PFOA 暴露条件下，C1q-domain-containing gene (C1qdc)、Alpha-2Macroglobulin (a2m) 和革兰氏阴性菌结合蛋白 (GNBP) 等基因均出现下调（图 4A-C）。C1qdc 蛋白与各种病原体相关分子模式（如脂多糖、肽聚糖和 葡聚糖）结合，并作为信号分子调节炎症反应的初始化、适应性免疫、凋亡细胞清除、细菌和逆转录病毒识别以及能量平衡（Kishore 等人，2004 年；Wang 等人，2012 年）、A2m 结合病原体丝氨酸蛋白酶，GNBPs 识别病原体表面的多糖，作为病原体蛋白酶抑制剂和在非自我识别中发挥重要作用，以刺激后续的免疫信号转导途径，如丙酚氧化酶（proPO）级联、Toll 和/或免疫缺陷途径（Jin 等人，2012 年；Ponprateep 等人，2017 年）。值得注意的是，尽管全氟辛烷磺酸的毒性低于全氟辛烷磺酸，但它对 C1qdc、 和 GNBP 基因却有很强的影响。这表明，接触全氟辛烷磺酸会使动物的免疫力高度下降。全氟辛烷磺酸和全氟辛酸不同的毒性特征可能是由于亲水官能团的不同，这可能导致与生物大分子的亲和力不同（Gao 等，2013 年； 和 Hungerbuehler，2015 年）。Xia和同事的研究表明，全氟辛烷磺酸的生物累积能力约为全氟辛酸的2倍（Xia等人，2013年）。对大肠杆菌的研究表明，全氟辛烷磺酸比全氟辛烷磺酸毒性更强。作者认为，全氟辛烷磺酸的毒性机制更直接，是通过破坏细菌细胞膜和诱发氧化应激，而全氟辛酸则主要通过基因干扰途径，通过细胞失活和/或死亡诱发毒性（Liu等人，2016年）。
Multicystatin 6 (Cst6) was downregulated by 10 and PFOS but not by PFOA (Fig. 4D). Multicystatins belongs to a family of cysteine protease inhibitors that provide protective functions through inhibition of host proteases (Hartmann and Lucius, 2003). Death associated protein (dap1) which is a small proline-rich cytoplasmic protein involved in apoptotic pathway, was upregulated by PFOS only (Fig. 4E). Nitric oxide synthase 2 (NOS2) and prophenoloxidase (proPO) were not altered by the exposures (Fig. 4F-G). The observed downregulation of immune response genes in the present study indicates that PFAS exposure could make Daphnia magna more susceptible to various infections, which in turn may compromise longevity, growth and reproduction. 全氟辛烷磺酸 10 和 会导致多囊蛋白酶 6（Cst6）下调，但全氟辛酸不会（图 4D）。多囊蛋白属于半胱氨酸蛋白酶抑制剂家族，可通过抑制宿主蛋白酶提供保护功能（Hartmann 和 Lucius，2003 年）。死亡相关蛋白（dap1）是一种富含脯氨酸的小型细胞质蛋白，参与细胞凋亡途径，仅在 全氟辛烷磺酸的作用下上调（图 4E）。一氧化氮合酶 2（NOS2）和丙酚氧化酶（proPO）没有因暴露而改变（图 4F-G）。本研究中观察到的免疫反应基因下调表明，接触全氟辛烷磺酸会使大型水蚤更容易受到各种感染，进而可能影响其寿命、生长和繁殖。
3.3. PFOS and PFOA alter lipid metabolism 3.3.全氟辛烷磺酸和全氟辛酸会改变脂质代谢
The ORO staining analysis revealed that PFOS and PFOA exposure resulted in lipid accumulation in Daphnia magna (Fig.5). To characterize the mechanisms behind the lipid accumulation, genes involved in lipid metabolism pathways including nuclear hormone receptor 96 ( ), ORO 染色分析表明，接触全氟辛烷磺酸和全氟辛酸会导致大型蚤体内脂质积累（图 5）。为了确定脂质积累背后的机制，参与脂质代谢途径的基因包括核激素受体 96 ( )、
Niemann Pick type C1b (NPC1b), magro, sphingomyelinase3 (SM3), mannosidase (man), and ceramidase (cer2) were analyzed. 分析了 Niemann Pick C1b 型（NPC1b）、magro、鞘磷脂酶 3（SM3）、甘露糖苷酶（man）和神经酰胺酶（cer2）。
The gene was upregulated by PFOS and downregulated by 1 and PFOA (Fig. 6A). Both NCP1b and magro were downregulated by PFOS and PFOA (Fig. 6B & C). HR96 regulates several genes involved in absorption of lipids and mediates energy metabolism through homeostasis and transport of triacylglycerols and cholesterol (Sengupta et al., 2017; Sieber and Thummel, 2009). It plays a key role in regulating the NPC gene family that is involved in cholesterol and fatty acid homeostasis. The mammalian gastric lipase homolog, magro, which is essential for hydrolysis of cholesterol esters and stimulation of cholesterol clearance from the intestine of Drosophila melanogaster is also regulated by HR96 (Sieber and Thummel, 2012). 受 PFOS 的影响而上调，受 1 和 PFOA 的影响而下调（图 6A）。NCP1b 和 magro 受全氟辛烷磺酸和全氟辛酸的影响而下调（图 6B 和 C）。HR96 通过三酰甘油和胆固醇的平衡和转运，调节多个参与脂质吸收的基因并介导能量代谢（Sengupta 等人，2017 年；Sieber 和 Thummel，2009 年）。它在调节参与胆固醇和脂肪酸平衡的 NPC 基因家族中发挥着关键作用。哺乳动物胃脂肪酶同源物 magro 对水解胆固醇酯和刺激黑腹果蝇肠道清除胆固醇至关重要，它也受 HR96 的调控（Sieber 和 Thummel，2012 年）。
SM3, which is responsible for the sphingomyelinase enzyme that hydrolyze sphingomyelin into ceramide, was not affected by PFOS but downregulated by all PFOA exposures (Fig. 6D). However, cer2, that cleaves fatty acids from ceramide to generate sphingosines, was downregulated by both PFOS and PFOA (Fig. 6E). The gene man, which is involved in the breakdown of the complex sugars found on glycoproteins (Sieber and Thummel, 2009), was upregulated by all PFOS exposure (Fig. 6F), however, expression of man was not altered by PFOA exposure. SM3 是负责将鞘磷脂水解为神经酰胺的鞘磷脂酶，不受全氟辛烷磺酸的影响，但受所有全氟辛烷磺酸暴露的影响而下调（图 6D）。然而，从神经酰胺中裂解脂肪酸生成鞘磷脂的 cer2 受全氟辛烷磺酸和全氟辛酸的影响而下调（图 6E）。参与分解糖蛋白上的复合糖的基因 man（Sieber 和 Thummel，2009 年）在所有 PFOS 暴露中都出现上调（图 6F），然而，PFOA 暴露并没有改变 man 的表达。
Lipid metabolism is regulated throughout the life of invertebrates, and proper regulation is needed for survival and fecundity in juvenile Daphnia magna (Lee et al., 2018). Studies have shown that disruption of lipid metabolism or hormonal control by obesogenic chemicals lead to increase in energy storage as lipid droplets (Janesick and Blumberg, 2011; Lee et al., 2018). 脂质代谢在无脊椎动物的整个生命过程中都受到调控，幼年大型水蚤的生存和繁殖需要适当的调控（Lee 等人，2018 年）。研究表明，致肥化学物质对脂质代谢或激素控制的破坏会导致以脂滴形式储存的能量增加（Janesick 和 Blumberg，2011 年；Lee 等人，2018 年）。
PFAS has been shown to disrupt metabolic functions in zebrafish, mice and humans (Fletcher et al., 2013). The mechanisms of lipid droplet accumulation and storage in Daphnia magna in response to exogenous substances has been suggested to be similar to vertebrates (Fuertes et al., 2018; Grun and Blumberg, 2009; Jordao et al., 2016). In a recent study it was shown that triclosan reduce the metabolism of SM into ceramides or sphingosine, thereby promoting SM accumulation in Daphnia magna which results in delayed development and maturation of the neonates and reduced fecundity of the adults (Sengupta et al., 2017). Similarly, we have previously reported that the three phthalates (DEHP, DBP and DEP) lead to increased lipid accumulation in Daphnia magna, and gene expression analysis suggested that the accumulation was due to inhibition of enzymes involved in fatty acid uptake and catabolism (Seyoum and Pradhan, 2019). Thus, the altered expression of these genes, in the present study, which are involved in the hydrolysis and uptake of lipids, suggests that PFOS and PFOA may disrupt lipid catabolism and absorption which ultimately contribute for the excessive accumulation of lipid droplets in the body of Daphnia magna. 研究表明，PFAS 会破坏斑马鱼、小鼠和人类的代谢功能（Fletcher 等人，2013 年）。有研究表明，大型蚤体内脂滴积累和储存对外源物质的反应机制与脊椎动物类似（Fuertes 等人，2018 年；Grun 和 Blumberg，2009 年；Jordao 等人，2016 年）。最近的一项研究表明，三氯生会减少 SM 向神经酰胺或鞘磷脂的代谢，从而促进 SM 在大型蚤体内的积累，导致新生儿发育和成熟延迟以及成虫繁殖力降低（Sengupta 等人，2017 年）。同样，我们之前也报道过，三种邻苯二甲酸盐（DEHP、DBP 和 DEP）会导致大型蚤体内脂质积累增加，基因表达分析表明，这种积累是由于参与脂肪酸摄取和分解代谢的酶受到抑制所致（Seyoum 和 Pradhan，2019 年）。因此，在本研究中，这些参与脂质水解和吸收的基因的表达发生了改变，这表明全氟辛烷磺酸和全氟辛酸可能会破坏脂质的分解和吸收，最终导致大型蚤体内脂滴的过度积累。
3.4. Reproductive effects of PFOS and PFOA 3.4.全氟辛烷磺酸和全氟辛酸对生殖的影响
In order to understand the mechanisms behind the decreased fecundity, we analyzed the expression of genes related to reproduction and molting. These included vitellogenin (vtg1 and vtg2), vasa, juvenile hormone esterase (jhe), ecdysone receptor (EcRA), ecdysone receptor (EcRB), ultraspiracle (usp), hormone receptor 3 (HR3), ecdysoneinducible gene 74 (E74), ecdysone-inducible gene 75 (E75), Fushi-tarazu transcription factor 1 (Ftz-F1) and cytochrome P450-18a1 (Cyp18a1). 为了了解繁殖力下降背后的机制，我们分析了与繁殖和蜕皮有关的基因表达。这些基因包括卵黄素（vtg1 和 vtg2）、卵黄腺、幼年激素酯酶（jhe）、蜕皮激素受体 (EcRA)、蜕皮激素受体 (EcRB)、ultraspiracle (usp)、激素受体 3 (HR3)、蜕皮激素诱导基因 74 (E74)、蜕皮激素诱导基因 75 (E75)、Fushi-tarazu 转录因子 1 (Ftz-F1) 和细胞色素 P450-18a1 (Cyp18a1)。
In the present study we observed that tg1 expression was only downregulated by exposure to PFOA (Fig. 7B). On the other hand, vtg 2 was downregulated both by 10 and PFOS and 1 and PFOA (Fig. 7C). Vtg is the major egg yolk precursor protein and 在本研究中，我们观察到 tg1 的表达仅在暴露于 PFOA 后才会下调（图 7B）。另一方面，10 和 PFOS 以及 1 和 PFOA 都会下调 vtg 2 的表达（图 7C）。Vtg 是蛋黄的主要前体蛋白，也是蛋黄的重要组成部分。
Fig. 7. PFOS and PFOA decrease fecundity and alter expression of genes related to reproduction. Daphnia magna neonates ( old) were exposed to PFOS and PFOA for 21 days in triplicates and number of offspring were recorded (A). Following exposure, qRT-PCR analysis was performed for vtg1 (B), vtg2 (C), vasa (D), EcRA (E), EcRB (F), usp (G), jhe (H), HR3 (I), ftz-F1 (J), 774 (K), E75 (L) and Cyp18A1 (M). One-way ANOVA followed by Dunnett post-test ( 图 7.全氟辛烷磺酸和全氟辛酸会降低繁殖力并改变生殖相关基因的表达。将大型水蚤新生儿（ ）暴露于 PFOS 和 PFOA 21 天，记录子代数量（A）。 暴露后，对 vtg1 (B)、vtg2 (C)、vasa (D)、EcRA (E)、EcRB (F)、usp (G)、jhe (H)、HR3 (I)、ftz-F1 (J)、774 (K)、E75 (L) 和 Cyp18A1 (M) 进行 qRT-PCR 分析。单因素方差分析，然后进行邓尼特后验（P<0.05）。
is required for egg maturation and provides protein- and lipid-rich nutrients for the developing embryos and larvae (Levi et al., 2012; Sun and Zhang, 2015). 卵成熟所需的营养物质，并为发育中的胚胎和幼虫提供富含蛋白质和脂质的营养物质（Levi 等人，2012；Sun 和 Zhang，2015）。
The vasa gene was only downregulated by PFOA exposure (Fig. 7D). Vasa is a highly conserved ATP-dependent RNA helicase that belongs to the DEAD-box family and is required for germ-cell development (Hay et al., 1988; Lasko and Ashburner, 1988; Pradhan and Olsson, 2018). Since vasa expression is limited to the germ-cells, it is an important marker for germ line development (Noce et al., 2001; Raz, 2000). Recently a study on Drosophila melanogaster showed that absence of vasa during the germarial stages caused checkpoint kinase 2 (Chk2)dependent oogenesis arrest (Durdevic and Ephrussi, 2019). Similarly, PFOS decreased germ-cell proliferation in Caenorhabditis elegans in a dose-dependent manner and resulted in reduced reproductive output through impaired gonadal development (Guo et al., 2016). This indicates that downregulation of vasa by PFOA exposure (Fig. 7D) results in oogenesis arrest and as a consequence contribute to the reduced fecundity (Fig. 7A). Vasa 基因只在暴露于 PFOA 后才下调（图 7D）。Vasa 是一种高度保守的 ATP 依赖性 RNA 螺旋酶，属于 DEAD-box 家族，是生殖细胞发育所必需的（Hay 等人，1988 年；Lasko 和 Ashburner，1988 年；Pradhan 和 Olsson，2018 年）。由于 vasa 的表达仅限于生殖细胞，因此它是生殖系发育的一个重要标记（Noce 等人，2001 年；Raz，2000 年）。最近一项关于黑腹果蝇的研究表明，在生殖阶段，vasa的缺失会导致检查点激酶2（Chk2）依赖性的卵子发生停滞（Durdevic和Ephrussi，2019年）。同样，全氟辛烷磺酸以剂量依赖性的方式减少了秀丽隐杆线虫的生殖细胞增殖，并通过性腺发育受损导致生殖能力下降（Guo 等人，2016 年）。这表明，暴露于全氟辛烷磺酸（图 7D）会导致输精管下调，导致卵子生成停止，从而导致繁殖力降低（图 7A）。
The and genes were downregulated by all PFOA exposures and 1 and of PFOS (Fig. 7E-F). On the other hand, expression of usp, that form a heterodimer with EcR in the nucleus, was downregulated by PFOS and all PFOA exposures (Fig. 7G). jhe is the enzyme that catalyzes the hydrolysis of JH (LeBoeuf et al., 2018) and regulate the concentration of JH. It was downregulated by all PFOS and PFOA exposures ( . ). 和 基因受所有 PFOA 暴露以及 1 和 的 PFOS 影响而下调（图 7E-F）。另一方面，在细胞核中与 EcR 形成异源二聚体的 usp 的表达受 PFOS 和所有 PFOA 暴露的影响而下调（图 7G）。Jhe 是催化 JH 水解并调节 JH 浓度的酶（LeBoeuf 等人，2018 年）。所有 PFOS 和 PFOA 暴露都会下调该酶的活性 ( . )。
Reproduction and development in crustaceans is associated with molting and are regulated by two major hormones, ecdysteroid (20-hydroxyecdysone, 20E) and juvenile hormone (JH) as well as by transcription of the nuclear EcR receptors and USP (Dai et al., 2016; Riddiford et al., 2001). In the presence of and USP form a nuclear heterodimer complex (EcR/USP ) and upregulate the transcription of genes such as , and that coordinates development and metamorphic transitions (Kato et al., 2007). In the presence of JH, EcR/USP-20E induce transcription of genes that regulate molting, whereas in the absence of JH, the EcR/USP-20E complex direct the onset of metamorphosis (Kim et al., 2005; Lenaerts et al., 2016; Yao et al., 1993). JH also plays a key role in regulating molting and reproduction through its involvement in vitellogenesis (Gilbert et al., 2000; Lenaerts et al., 2016). JH regulates daphnid vitellogenesis by suppressing expression of vtg gene through binding to the upstream JH-responsive elements (Tokishita et al., 2006). Clearance of JH is associated with an increase in the level of JHE that hydrolyzes into the biologically inactive -acid (Touhara et al., 1995). 甲壳类的繁殖和发育与蜕皮有关，受两种主要激素--蜕皮激素（20-羟基蜕皮激素，20E）和幼体激素（JH）以及核 EcR 受体和 USP 的转录调控（Dai 等人，2016 年；Riddiford 等人，2001 年）。在 和 USP 存在的情况下，它们会形成核异源二聚体复合物（EcR/USP ），并上调 、 和 等基因的转录，从而协调发育和蜕变（Kato 等人，2007 年）。在有 JH 的情况下，EcR/USP-20E 会诱导调控蜕皮的基因转录，而在没有 JH 的情况下，EcR/USP-20E 复合物则会引导变态的开始（Kim 等人，2005 年；Lenaerts 等人，2016 年；Yao 等人，1993 年）。JH 还通过参与卵黄发生，在调控蜕皮和繁殖中发挥关键作用（Gilbert 等人，2000 年；Lenaerts 等人，2016 年）。JH 通过与上游的 JH 响应元件结合，抑制 vtg 基因的表达，从而调节水蚤的卵黄发生（Tokishita 等人，2006 年）。JH 的清除与 JHE 水平的增加有关，JHE 可将 水解为无生物活性的 -酸（Touhara 等人，1995 年）。
The observed downregulation of he following exposure to PFAS indicates that the levels of should increase. In line with this we observed that the Vtg expression was downregulated. This downregulation of tg following PFAS exposure could be a contributing factor for the reduced fecundity. 暴露于全氟辛烷磺酸后，观察到 he 的下调，这表明 的水平应该上升。与此相一致，我们观察到 Vtg 表达下调。暴露于 PFAS 后， tg 表达下调，这可能是导致繁殖力降低的一个因素。
Nuclear receptor , a gene responsible for controlling molting through its involvement in chitin synthesis and degradation, was significantly downregulated by both PFOS and PFOA (Fig. 7I). The E74 and genes serve as repressors of HR3-mediated gene regulation (Swevers et al., 2002) and were downregulated by high doses of PFOS as well as by all PFOA exposures (Fig. 7K-L). Cyp18a1 controls molting by inactivation of through a degradation pathway (Guittard et al., 2011). Fluctuation of ecdysteroid concentrations as a result of biosynthesis and degradation at specific timing regulates metamorphosis and molting (Gilbert, 2012; LeBoeuf et al., 2018). In the present study we did not observe any effect on the expression of Cyp18a1 following exposure to PFAS (Fig. 7M). Disruption of the ecdysteroid signaling pathway that regulate reproduction and molting may be a contributing factor to the decreased fecundity in response to PFOA and PFOS exposure. Overall, both PFOS and PFOA disrupted several genes in the reproductive signaling pathways of Daphnia magna. 核受体 是一个通过参与几丁质的合成和降解来控制蜕皮的基因，该基因在 PFOS 和 PFOA 的作用下均显著下调（图 7I）。E74 和 基因是 HR3 介导的基因调控的抑制因子（Swevers 等人，2002 年），在高剂量 PFOS 和所有 PFOA 暴露下均出现下调（图 7K-L）。Cyp18a1 通过降解途径使 失活，从而控制蜕皮（Guittard 等，2011 年）。蜕皮激素浓度的波动是生物合成和降解在特定时间进行的结果，可调节变态和蜕皮（Gilbert，2012；LeBoeuf 等人，2018）。在本研究中，我们没有观察到暴露于 PFAS 后对 Cyp18a1 表达的任何影响（图 7M）。调节繁殖和蜕皮的蜕皮激素信号通路的中断可能是导致暴露于 PFOA 和 PFOS 后繁殖力下降的一个因素。总体而言，全氟辛烷磺酸和全氟辛酸都干扰了大型蚤生殖信号通路中的多个基因。
4. Conclusions 4.结论
In the present study we evaluated the effects of two widely detected perfluoroalkyl substances, PFOS and PFOA on the freshwater crustacean Daphnia magna using different physiological and toxicogenomic endpoints to understand their mechanisms of action. PFOS and PFOA differed in their effects on Daphnia magna, suggesting that they have different but overlapping modes of action. The results indicate that PFOS is more toxic than PFOA, and that the toxicity of PFOS and PFOA is strongly correlated to oxidative stress and the disruption of immune response genes. The results also indicate that the fatty acid accumulation following PFAS exposure in Daphnia magna may be due to inhibition of enzymes that are involved in fatty acid uptake and catabolism. The decreased fecundity following exposure to PFOA and PFOS exposure is correlated to the disruption of the ecdysteroid signaling pathway that regulates reproduction and molting. This study provides valuable toxicogenomic data on PFOS and PFOA toxicity in Daphnia magna. Additional toxicogenomic studies using other animal model is needed to determine species specific characteristics of PFOS and PFOA toxicity. 在本研究中，我们采用不同的生理和毒物基因组学终点，评估了两种广泛检测到的全氟烷基物质（PFOS 和 PFOA）对淡水甲壳动物大型蚤的影响，以了解它们的作用机制。全氟辛烷磺酸和全氟辛酸对大型蚤的影响各不相同，这表明它们具有不同但重叠的作用模式。结果表明，全氟辛烷磺酸比全氟辛酸毒性更大，全氟辛烷磺酸和全氟辛酸的毒性与氧化应激和免疫反应基因的干扰密切相关。研究结果还表明，大型水蚤接触全氟辛烷磺酸后出现脂肪酸积累，可能是由于参与脂肪酸摄取和分解代谢的酶受到了抑制。暴露于全氟辛烷磺酸和全氟辛烷磺酸后繁殖力下降，这与调节繁殖和蜕皮的蜕皮激素信号通路被破坏有关。这项研究为全氟辛烷磺酸和全氟辛酸在大型水蚤中的毒性提供了宝贵的毒原组学数据。要确定全氟辛烷磺酸和全氟辛酸毒性的物种特异性，还需要使用其他动物模型进行更多的毒原组学研究。
Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2020.139682. 本文的补充数据可在线查阅：https://doi. org/10.1016/j.scitotenv.2020.139682。
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 作者声明，他们没有任何可能会影响本文所报告工作的已知经济利益或个人关系。
Acknowledgements 致谢
This study was financed by the Knowledge Foundation Sweden, grants 20170118 (to PEO) and 20180027 (to JJ) and Örebro University. 本研究由瑞典知识基金会（Knowledge Foundation Sweden）资助，资助金额为20170118（PEO）和20180027（JJ），以及厄勒布鲁大学（Örebro University）。
References 参考资料
Abbott, B.D., Wolf, C.J., Das, K.P., Zehr, R.D., Schmid, J.E., Lindstrom, A.B., et al., 2009. Developmental toxicity of perfluorooctane sulfonate (PFOS) is not dependent on expression of peroxisome proliferator activated receptor-alpha (PPAR alpha) in the mouse. Reprod. Toxicol. 27, 258-265. Abbott, B.D., Wolf, C.J., Das, K.P., Zehr, R.D., Schmid, J.E., Lindstrom, A.B., et al., 2009.全氟辛烷磺酸（PFOS）的发育毒性不依赖于小鼠过氧化物酶体增殖激活受体-α（PPAR α）的表达。Reprod.Toxicol.27, 258-265.
Ahrens, L., Bundschuh, M., 2014. Fate and effects of poly- and perfluoroalkyl substances in the aquatic environment: a review. Environ. Toxicol. Chem. 33, 1921-1929. Ahrens, L., Bundschuh, M., 2014年。多氟和全氟烷基物质在水生环境中的归宿和影响：综述。Environ.Toxicol.Chem.33, 1921-1929.
Amiard, J.C., Amiard-Triquet, C., Barka, S., Pellerin, J., Rainbow, P.S., 2006. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat. Toxicol. 76, 160-202. Amiard, J.C., Amiard-Triquet, C., Barka, S., Pellerin, J., Rainbow, P.S., 2006.水生无脊椎动物中的金属硫蛋白：它们在金属解毒中的作用及其作为生物标志物的用途。Aquat.Toxicol.76, 160-202.
Andrews, G.K., 2000. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 59, 95-104. Andrews, G.K., 2000.氧化应激和金属离子对金属硫蛋白基因表达的调控。Biochem.Pharmacol.59, 95-104.
Angeler, D.G., Martin, S., Moreno, J.M., 2005. Daphnia emergence: a sensitive indicator of fire-retardant stress in temporary wetlands. Environ. Int. 31, 615-620. Angeler, D.G., Martin, S., Moreno, J.M., 2005.水蚤的出现：临时湿地阻燃剂压力的敏感指标。环境。31, 615-620.
Brown, D., Thompson, R.S., 1982. Phthalates and the aquatic environment. 1. The effect of di-2-ethylhexyl phthalate (Dehp) and di-isodecyl phthalate (Didp) on the reproduction of Daphnia-magna and observations on their bioconcentration. Chemosphere 11 . Brown, D., Thompson, R.S., 1982.邻苯二甲酸盐与水生环境。1.邻苯二甲酸二-2-乙基己酯 (Dehp) 和邻苯二甲酸二-异癸酯 (Didp) 对水蚤繁殖的影响及其生物浓缩观察。Chemosphere 11 。
Buck, R.C., Franklin, J., Berger, U., Conder, J.M., Cousins, I.T., de Voogt, P., et al., 2011. Perfluoroalkyl and polyfluoroalkyl substances in the environment: terminology, classification, and origins. Integr. Environ. Assess. Manag. 7, 513-541. Buck, R.C., Franklin, J., Berger, U., Conder, J.M., Cousins, I.T., de Voogt, P., et al., 2011.环境中的全氟烷基和多氟烷基物质：术语、分类和起源。Integr.Environ.Assess.Manag.7, 513-541.
Conder, J.M., Hoke, R.A., De Wolf, W., Russell, M.H., Buck, R.C., 2008. Are PFCAs bioaccumulative? A critical review and comparison with regulatory criteria and persistent lipophilic compounds. Environ Sci Technol 42, 995-1003. Conder, J.M., Hoke, R.A., De Wolf, W., Russell, M.H., Buck, R.C., 2008年。全氟辛烷磺酸具有生物累积性吗？评论以及与监管标准和持久性亲脂化合物的比较。Environ Sci Technol 42, 995-1003.
Custer, C.M., Custer, T.W., Dummer, P.M., Etterson, M.A., Thogmartin, W.E., Wu, Q., et al., 2014. Exposure and effects of perfluoroalkyl substances in tree swallows nesting in Minnesota and Wisconsin, USA. Arch. Environ. Contam. Toxicol. 66, 120-138. Custer, C.M., Custer, T.W., Dummer, P.M., Etterson, M.A., Thogmartin, W.E., Wu, Q., et al., 2014.在美国明尼苏达州和威斯康星州筑巢的树燕接触全氟烷基物质及其影响。Arch.Environ。Contam.Toxicol.66, 120-138.
Dai, T.H., Sserwadda, A., Song, K., Zang, Y.N., Shen, H.S., 2016. Cloning and expression of ecdysone receptor and retinoid receptor from Procambarus clarkii: induction by eyestalk ablation. Int. J. Mol. Sci. 17. Dai, T.H., Sserwadda, A., Song, K., Zang, Y.N., Shen, H.S., 2016.Procambarus clarkii的蜕皮激素受体和视黄醇 受体的克隆与表达：眼茎消融诱导。Int.J. Mol.Sci.
Davison, J., 1969. Activation of the ephippial egg of Daphnia pulex. J Gen Physiol 53, 562-575. Davison, J., 1969.水蚤卵的活化。J Gen Physiol 53, 562-575.
Dong, G., Chen, H., Qi, M.Y., Dou, Y., Wang, Q.L., 2015. Balance between metallothionein and metal response element binding transcription factor 1 is mediated by zinc ions. Mol. Med. Rep. 11, 1582-1586. Dong, G., Chen, H., Qi, M.Y., Dou, Y., Wang, Q.L., 2015.金属硫蛋白与金属反应元件结合转录因子1之间的平衡由锌离子介导。Mol. Med.Rep. 11, 1582-1586.11, 1582-1586.
Durdevic, Z., Ephrussi, A., 2019. Germ cell lineage homeostasis in Drosophila requires the vasa RNA helicase. Genetics . Durdevic, Z., Ephrussi, A., 2019.果蝇生殖细胞系平衡需要 vasa RNA 螺旋酶。遗传学 。
EC_1907/2006, 2006. REGULATION (EC) No 1907/2006 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL. Off. J. Eur. Union L 136, 3-280. EC_1907/2006，2006 年。欧洲议会和欧盟委员会第 1907/2006 号条例。Off.J. Eur.Union L 136, 3-280.
Eschauzier, C., De Voogt, P., Brauch, H.-J., Lange, F.T., 2012. Polyfluorinated Chemicals in European Surface Waters, Ground-and Drinking Waters. The Handbook of Environmental Chemistry. 17, pp. 73-102. Eschauzier, C., De Voogt, P., Brauch, H.-J., Lange, F.T., 2012.欧洲地表水、地下水和饮用水中的多氟化学品。环境化学手册》。17, pp.
Fletcher, T., Galloway, T.S., Melzer, D., Holcroft, P., Cipelli, R., Pilling, L.C., et al., 2013. Associations between PFOA, PFOS and changes in the expression of genes involved in cholesterol metabolism in humans. Environ. Int. 57-58, 2-10. Fletcher, T.、Galloway, T.S.、Melzer, D.、Holcroft, P.、Cipelli, R.、Pilling, L.C.等人，2013年。全氟辛烷磺酸、全氟辛烷磺酸与人类胆固醇代谢相关基因表达变化之间的关联。环境。57-58, 2-10.
Flora, S.J., 2009. Structural, chemical and biological aspects of antioxidants for strategies against metal and metalloid exposure. Oxidative Med. Cell. Longev. 2, 191-206. Flora, S.J., 2009.抗氧化剂的结构、化学和生物方面，以应对金属和类金属暴露。氧化医学。细胞。Longev.2, 191-206.
Fuertes, I., Jordao, R., Casas, F., Barata, C., 2018. Allocation of glycerolipids and glycerophospholipids from adults to eggs in Daphnia magna: perturbations by compounds that enhance lipid droplet accumulation. Environ. Pollut. 242, 1702-1710, Fuertes, I., Jordao, R., Casas, F., Barata, C., 2018.大型蚤从成虫到卵的甘油脂和甘油磷脂分配：增强脂滴积累的化合物的扰动。Environ.Pollut.242, 1702-1710,
Gao, Y., Li, X., Guo, L.H., 2013. Assessment of estrogenic activity of perfluoroalkyl acids based on ligand-induced conformation state of human estrogen receptor. Environ Sci Technol 47, 634-641. Gao, Y., Li, X., Guo, L.H., 2013.基于配体诱导人类雌激素受体构象状态的全氟烷基酸雌激素活性评估。Environ Sci Technol 47, 634-641.
Guittard, E., Blais, C., Maria, A., Parvy, J.P.,, Pasricha, S., Lumb, C., et al., 2011. CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis. Dev. Biol. 349, 35-45. Guittard, E., Blais, C., Maria, A., Parvy, J.P.,, Pasricha, S., Lumb, C., et al., 2011.果蝇类固醇激素灭活的关键酶 CYP18A1 对变态至关重要。Dev.349, 35-45.
Guo, X.Y., Li, Q.Q., Shi, J., Shi, L.L., Li, B.Q., Xu, A., et al., 2016. Perfluorooctane sulfonate exposure causes gonadal developmental toxicity in Caenorhabditis elegans through ROS-induced DNA damage. Chemosphere 155, 115-126. Guo, X.Y., Li, Q.Q., Shi, J., Shi, L.L., Li, B.Q., Xu, A., et al., 2016.全氟辛烷磺酸暴露通过ROS诱导的DNA损伤导致秀丽隐杆线虫性腺发育毒性。Chemosphere 155, 115-126.
Hartmann, S., Lucius, R., 2003. Modulation of host immune responses by nematode cystatins. Int. J. Parasitol. 33, 1291-1302. Hartmann, S., Lucius, R., 2003.线虫嚢虫素对宿主免疫反应的调节。Int.Parasitol.33, 1291-1302.
Hay, B., Jan, L.Y., Jan, Y.N., 1988. A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATP-dependent helicases. Cell 55, 577-587. Hay, B., Jan, L.Y., Jan, Y.N., 1988.果蝇极性颗粒的一种蛋白质成分由 vasa 编码，与 ATP 依赖性螺旋酶具有广泛的序列相似性。细胞 55，577-587。
Hekster, F.M., Pijnenburg, A.M.C.M., Laane, R.W.P.M., de Voogt, P., 2002. Perfluoroalkylated Substances: Aquatic Environmental Assessment. RIKZ Report. Institute for Coastal and Marine Management, p. 99. Hekster, F.M., Pijnenburg, A.M.C.M., Laane, R.W.P.M., de Voogt, P., 2002.全氟烷基化物质：水生环境评估。RIKZ Report.沿海和海洋管理研究所，第 99 页。
Hekster, F.M., Laane, R.W.P.M., de Voogt, P., 2003. Environmental and toxicity effects of perfluoroalkylated substances. Rev. Environ. Contam. Toxicol. 179 (179), 99-121. Hekster, F.M., Laane, R.W.P.M., de Voogt, P., 2003.全氟烷基化物质对环境和毒性的影响。Rev. Environ.Contam.Toxicol.179 (179), 99-121.
Houde, M., Wells, R.S., Fair, P.A., Bossart, G.D., Hohn, A.A., Rowles, T.K., et al., 2005 Polyfluoroalkyl compounds in free-ranging bottlenose dolphins (Tursiops truncatus) from the Gulf of Mexico and the Atlantic Ocean. Environ Sci Technol 39, 6591-6598. Houde，M.、Wells，R.S.、Fair，P.A.、Bossart，G.D.、Hohn，A.A.、Rowles，T.K.等人，2005 年墨西哥湾和大西洋散养瓶鼻海豚（Tursiops truncatus）体内的多氟烷基化合物。Environ Sci Technol 39, 6591-6598.
Ighodaro, O.M., Akinloye, O.A., 2018. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): their fundamental role in the entire antioxidant defence grid. Alexandria J Med 54, 287-293. Ighodaro, O.M., Akinloye, O.A., 2018.一线防御抗氧化剂--超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPX）：它们在整个抗氧化防御网中的基本作用。亚历山大医学杂志》54，287-293。
Janesick, A., Blumberg, B., 2011. Endocrine disrupting chemicals and the developmental programming of adipogenesis and obesity. Birth Defects Res C-Embryo TodayReviews 93, 34-50. Janesick, A., Blumberg, B., 2011.内分泌干扰化学品与脂肪生成和肥胖的发育过程。Birth Defects Res C-Embryo TodayReviews 93, 34-50.
Jeong, T.Y., Yuk, M.S., Jeon, J., Kim, S.D., 2016. Multigenerational effect of perfluorooctane sulfonate (PFOS) on the individual fitness and population growth of Daphnia magna. Sci. Total Environ. . Jeong, T.Y., Yuk, M.S., Jeon, J., Kim, S.D., 2016.全氟辛烷磺酸（PFOS）对大型蚤个体适应性和种群增长的多代效应。 .
Ji, K., Kim, Y., Oh, S., Ahn, B., Jo, H., Choi, K., 2008. Toxicity of perfluorooctane sulfonic acid and perfluorooctanoic acid on freshwater macroinvertebrates (Daphnia magna and Moina macrocopa) and fish (Oryzias latipes). Environ. Toxicol. Chem. 27, 2159-2168 Ji, K., Kim, Y., Oh, S., Ahn, B., Jo, H., Choi, K., 2008.全氟辛烷磺酸和全氟辛酸对淡水大型无脊椎动物（大型蚤和大型鳅）和鱼类（鹗）的毒性。Environ.Toxicol.Chem.27, 2159-2168
Jin, P., Zhou, L., Song, X., Qian, J., Chen, L., Ma, F., 2012. Particularity and universality of a putative Gram-negative bacteria-binding protein (GNBP) gene from amphioxus (Branchiostoma belcheri): insights into the function and evolution of GNBP. Fish Shellfish Immunol 33, 835-845. Jin, P., Zhou, L., Song, X., Qian, J., Chen, L., Ma, F., 2012.文昌鱼推测革兰氏阴性菌结合蛋白（GNBP）基因的特殊性和普遍性：对GNBP功能和进化的启示。Fish Shellfish Immunol 33, 835-845.
Jordao, R., Garreta, E., Campos, B., Lemos, M.F.L., Soares, A.M.V.M., Tauler, R., et al., 2016. Compounds altering fat storage in Daphnia magna. Sci. Total Environ. 545, 127-136. Jordao, R.、Garreta, E.、Campos, B.、Lemos, M.F.L.、Soares, A.M.V.M.、Tauler, R.等人，2016年。改变大型蚤脂肪储存的化合物。总环境科学》，545, 127-136.545, 127-136.
Kang, J.S., Ahn, T.G., Park, J.W., 2019. Perfluorooctanoic acid (PFOA) and perfluooctane sulfonate (PFOS) induce different modes of action in reproduction to Japanese medaka (Oryzias latipes). J. Hazard Mater. 368, 97-103. Kang, J.S., Ahn, T.G., Park, J.W., 2019.全氟辛酸（PFOA）和全氟辛烷磺酸（PFOS）对日本青鳉（Oryzias latipes）繁殖的不同诱导作用模式。J. Hazard Mater.368, 97-103.
Kato, Y., Kobayashi, K., Oda, S., Tatarazako, N., Watanabe, H., Iguchi, T., 2007. Cloning and characterization of the ecdysone receptor and ultraspiracle protein from the water flea Daphnia magna. J. Endocrinol. 193, 183-194 Kato, Y., Kobayashi, K., Oda, S., Tatarazako, N., Watanabe, H., Iguchi, T., 2007.来自水蚤大型蚤的蜕皮激素受体和超螺旋蛋白的克隆与表征。J. Endocrinol.193, 183-194
Key, B.D., Howell, R.D., Criddle, C.S., 1997. Fluorinated organics in the biosphere. Environ Sci Technol 31, 2445-2454. Key, B.D., Howell, R.D., Criddle, C.S., 1997.生物圈中的含氟有机物。Environ Sci Technol 31, 2445-2454.
Kim, H.W., Lee, S.G., Mykles, D.L., 2005. Ecdysteroid-responsive genes, RXR and E75, in the tropical land crab, Gecarcinus lateralis: differential tissue expression of multiple RXR isoforms generated at three alternative splicing sites in the hinge and ligand-binding domains. Mol. Cell. Endocrinol. 242, 80-95. Kim, H.W., Lee, S.G., Mykles, D.L., 2005.热带陆蟹 Gecarcinus lateralis 的蜕皮激素反应基因 RXR 和 E75：铰链和配体结合域的三个替代剪接位点产生的多种 RXR 异构体的不同组织表达。Mol.Cell.内分泌。242, 80-95.
Kishore, U., Gaboriaud, C., Waters, P., Shrive, A.K., Greenhough, T.J., Reid, K.B.M., et al., 2004. C1q and tumor necrosis factor superfamily: modularity and versatility. Trends Immunol. 25, 551-561. Kishore, U., Gaboriaud, C., Waters, P., Shrive, A.K., Greenhough, T.J., Reid, K.B.M., et al., 2004.C1q 和肿瘤坏死因子超家族：模块化和多功能性。Trends Immunol.25, 551-561.
Kissa, E., 2001. Fluorinated Surfactants and Repellents. Marcel Dekker, New York. Kissa, E., 2001.氟化表面活性剂和驱虫剂》。Marcel Dekker，纽约。
Kling, P.G., Olsson, P., 2000. Involvement of differential metallothionein expression in free radical sensitivity of RTG-2 and CHSE-214 cells. Free Radic. Biol. Med. 28, 1628-1637. Kling, P.G., Olsson, P., 2000.不同金属硫蛋白表达参与 RTG-2 和 CHSE-214 细胞对自由基的敏感性。Free Radic.Biol.Biol.28, 1628-1637.
Koivisto, S., 1995. Is Daphnia-magna an ecologically representative zooplankton species in toxicity tests. Environ. Pollut. 90, 263-267. Koivisto, S., 1995.Daphnia-magna 是毒性试验中具有生态代表性的浮游动物物种吗？Environ.Pollut.90, 263-267.
Kuklenyik, Z., Reich, J.A., Tully, J.S., Needham, L.L., Calafat, A.M., 2004. Automated solidphase extraction and measurement of perfluorinated organic acids and amides in human serum and milk. Environ Sci Technol 38, 3698-3704. Kuklenyik, Z., Reich, J.A., Tully, J.S., Needham, L.L., Calafat, A.M., 2004.人血清和牛奶中全氟有机酸和酰胺的自动固相萃取与测量。Environ Sci Technol 38, 3698-3704.
Lasko, P.F., Ashburner, M., 1988. The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4A. Nature 335, 611-617. Lasko, P.F., Ashburner, M., 1988.果蝇基因 vasa 的产物与真核生物启动因子-4A 非常相似。Nature 335, 611-617.
Le, Q.A.V., Sekhon, S.S., Lee, L., Ko, J.H.,, Min, J., 2016. Daphnia in Water Quality Biomonitoring - "Omic" Approaches. vol 8. Le, Q.A.V., Sekhon, S.S., Lee, L., Ko, J.H.,, Min, J., 2016.水质生物监测中的水蚤--"Omic "方法》，第 8 卷。
LeBoeuf, A.C., Cohanim, A.B., Stoffel, C., Brent, C.S., Waridel, P., Privman, E., et al., 2018. Molecular evolution of juvenile hormone esterase-like proteins in a socially exchanged fluid. Sci. Rep. 8. LeBoeuf, A.C., Cohanim, A.B., Stoffel, C., Brent, C.S., Waridel, P., Privman, E., et al., 2018.社会交换流体中幼年激素酯酶样蛋白的分子进化。科学报告 8.
Lee, M.C., Park, J.C., Lee, J.S., 2018. Effects of environmental stressors on lipid metabolism in aquatic invertebrates. Aquat. Toxicol. 200, 83-92. Lee, M.C., Park, J.C., Lee, J.S., 2018.环境胁迫对水生无脊椎动物脂质代谢的影响。Aquat.Toxicol.200, 83-92.
Lenaerts, C., van Wielendaele, P., Peeters, P., Vanden Broeck, J., Marchal, E., 2016. Ecdysteroid signalling components in metamorphosis and development of the desert locust, Schistocerca gregaria. Insect Biochem. Mol. Biol. 75, 10-23. Lenaerts, C., van Wielendaele, P., Peeters, P., Vanden Broeck, J., Marchal, E., 2016.沙漠蝗虫（Schistocerca gregaria）变态和发育过程中的类蜕皮激素信号成分。Insect Biochem.Mol.Biol. 75, 10-23.
Levi, L., Ziv, T., Admon, A., Levavi-Sivan, B., Lubzens, E., 2012. Insight into molecular pathways of retinal metabolism, associated with vitellogenesis in zebrafish. Am. J. Physiol. Endocrinol. Metab. 302, E626-E644. Levi, L., Ziv, T., Admon, A., Levavi-Sivan, B., Lubzens, E., 2012.洞察斑马鱼视网膜新陈代谢与卵黄发生相关的分子途径。Am.J. Physiol.Metab.302，e626-e644。
Li, M.H., 2009. Toxicity of perfluorooctane sulfonate and perfluorooctanoic acid to plants and aquatic invertebrates. Environ. Toxicol. 24, 95-101. Li, M.H., 2009.全氟辛烷磺酸和全氟辛酸对植物和水生无脊椎动物的毒性。Environ.Toxicol.24, 95-101.
Li, K., Gao, P., Xiang, P., Zhang, X.X., Cui, X.Y., Ma, L.N.Q., 2017. Molecular mechanisms of PFOA-induced toxicity in animals and humans: implications for health risks. Environ. Int. 99, 43-54. Li, K., Gao, P., Xiang, P., Zhang, X.X., Cui, X.Y., Ma, L.N.Q., 2017.全氟辛酸诱导动物和人类毒性的分子机制：对健康风险的影响。Environ.99, 43-54.
Li, Y., Fletcher, T., Mucs, D., Scott, K., Lindh, C.H., Tallving, P., et al., 2018. Half-lives of PFOS, PFHxS and PFOA after end of exposure to contaminated drinking water. Occup. Environ. . 7 . . Li, Y., Fletcher, T., Mucs, D., Scott, K., Lindh, C.H., Tallving, P., et al., 2018.受污染饮用水暴露结束后全氟辛烷磺酸、全氟己烷磺酸和全氟辛酸的半衰期。职业。 .7 . .
Liu, C.H., Chang, V.W.C., Gin, K.Y.H., Nguyen, V.T., 2014. Genotoxicity of perfluorinated chemicals (PFCS) to the green mussel (Perna viridis). Sci. Total Environ. 487, Liu, C.H., Chang, V.W.C., Gin, K.Y.H., Nguyen, V.T., 2014.全氟化学品（PFCS）对绿贻贝（Perna viridis）的遗传毒性。Sci.487,
Liu, G., Zhang, S., Yang, K., Zhu, L., Lin, D., 2016. Toxicity of perfluorooctane sulfonate and perfluorooctanoic acid to Escherichia coli: membrane disruption, oxidative stress, and DNA damage induced cell inactivation and/or death. Environ. Pollut. 214, . Liu, G., Zhang, S., Yang, K., Zhu, L., Lin, D., 2016.全氟辛烷磺酸和全氟辛酸对大肠杆菌的毒性：膜破坏、氧化应激和 DNA 损伤诱导细胞失活和/或死亡。Environ.Pollut.214, .
Livingstone, D.R., 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Pollut. Bull. 42, 656-666. Livingstone, D.R., 2001.Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms.Mar.Pollut.Bull.42, 656-666.
MacFadyen, E.J., Williamson, C.E., Grad, G., Lowery, M., Jeffrey, W.H., Mitchell, D.L., 2004. Molecular response to climate change: temperature dependence of UV-induced DNA damage and repair in the freshwater crustacean Daphnia pulicaria. Glob. Chang. Biol. 10, 408-416. MacFadyen, E.J., Williamson, C.E., Grad, G., Lowery, M., Jeffrey, W.H., Mitchell, D.L., 2004.Molecular response to climate change: temperature dependence of UV-induced DNA damage and repair in the freshwater crustacean Daphnia pulicaria.Glob.Biol.10, 408-416.
MacGillivray, D.M., Kollmann, T.R., 2014. The role of environmental factors in modulating immune responses in early life. Front. Immunol. 5 MacGillivray, D.M., Kollmann, T.R., 2014.环境因素在调节生命早期免疫反应中的作用。Front.Immunol.5
Mao, Z.X., Xia, W., Wang, J., Chen, T., Zeng, Q.Q., Xu, B., et al., 2013. Perfluorooctane sulfonate induces apoptosis in lung cancer A549 cells through reactive oxygen speciesmediated mitochondrion-dependent pathway. J. Appl. Toxicol. 33, 1268-1276. Mao, Z.X., Xia, W., Wang, J., Chen, T., Zeng, Q.Q., Xu, B., et al., 2013.全氟辛烷磺酸通过活性氧介导的线粒体依赖途径诱导肺癌 A549 细胞凋亡。J. Appl.33, 1268-1276.
Midasch, O., Drexler, H., Hart, N., Beckmann, M.W., Angerer, J., 2007. Transplacental exposure of neonates to perfluorooctanesulfonate and perfluorooctanoate: a pilot study. Int. Arch. Occup. Environ. Health 80, 643-648. Midasch, O., Drexler, H., Hart, N., Beckmann, M.W., Angerer, J., 2007年。新生儿经胎盘暴露于全氟辛烷磺酸和全氟辛酸：一项试点研究。Int.Arch.Occup.环境。健康 80，643-648。
Moody, C.A., Hebert, G.N., Strauss, S.H., Field, J.A., 2003. Occurrence and persistence of perfluorooctanesulfonate and other perfluorinated surfactants in groundwater at a fire-training area at Wurtsmith Air Force Base, Michigan, USA. J. Environ. Monit. 5, Moody, C.A., Hebert, G.N., Strauss, S.H., Field, J.A., 2003年。美国密歇根州 Wurtsmith 空军基地消防训练区地下水中全氟辛烷磺酸及其他全氟表面活性剂的存在和持久性。J. Environ.Monit.5,
Navis, S., Waterkeyn, A., Voet, T., De Meester, L., Brendonck, L., 2013. Pesticide exposure impacts not only hatching of dormant eggs, but also hatchling survival and performance in the water flea Daphnia magna. Ecotoxicology 22, 803-814. Navis, S., Waterkeyn, A., Voet, T., De Meester, L., Brendonck, L., 2013.农药暴露不仅会影响休眠卵的孵化，还会影响大型水蚤幼体的存活率和表现。生态毒理学》22，803-814。
Ng, C.A., Hungerbuehler, K., 2015. Exploring the use of molecular docking to identify bioaccumulative perfluorinated alkyl acids (PFAAs). Environ Sci Technol 49, Ng, C.A., Hungerbuehler, K., 2015.探索使用分子对接法识别生物累积性全氟烷基酸（PFAAs）。Environ Sci Technol 49、
O'Hagan, D., 2008. Understanding organofluorine chemistry. An introduction to the C-F bond. Chem. Soc. Rev. 37, 308-319. O'Hagan, D., 2008.了解有机氟化学。C-F键简介。Chem.Soc. Rev. 37, 308-319.
Olsen, G.W., Burris, J.M., Ehresman, D.J., Froehlich, J.W., Seacat, A.M., Butenhoff, J.L., et al., 2007. Half-life of serum elimination of perfluorooctanesulfonate, perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ. Health Perspect. 115, 1298-1305. Olsen, G.W., Burris, J.M., Ehresman, D.J., Froehlich, J.W., Seacat, A.M., Butenhoff, J.L., 等人，2007 年。退休氟化工生产工人血清中全氟辛烷磺酸、全氟己烷磺酸和全氟辛酸的半衰期。Environ.Health Perspect.115, 1298-1305.
Olsson, P.-E., Kling, A., Hogstand, C., 1998. Mechanisms of heavy metal accumulation and toxicity in fish. In: Langston, W.J., Bebianno, M.J. (Eds.), Metal Metabolism in Aquatic Environments. Chapman and Hall, London, pp. 321-350. Olsson, P.-E., Kling, A., Hogstand, C., 1998.鱼类体内重金属积累和毒性的机理。In. Langston, W.J., Bebianno：Langston, W.J., Bebianno, M.J. (Eds.), Metal Metabolism in Aquatic Environments.Chapman and Hall, London, pp.
Paes, T.A.S.V., Rietzler, A.C., Pujoni, D.G.F., Maia-Barbosa, P.M., 2016. High temperatures and absence of light affect the hatching of resting eggs of Daphnia in the tropics. Anais Da Academia Brasileira De Ciencias 88, 179-186 Paes, T.A.S.V., Rietzler, A.C., Pujoni, D.G.F., Maia-Barbosa, P.M., 2016.高温和无光影响热带水蚤休眠卵的孵化。Anais Da Academia Brasileira De Ciencias 88, 179-186
Park, J.D., Liu, Y., Klaassen, C.D., 2001. Protective effect of metallothionein against the toxicity of cadmium and other metals(1). Toxicology 163, 93-100. Park, J.D., Liu, Y., Klaassen, C.D., 2001.金属硫蛋白对镉和其他金属毒性的保护作用(1)。毒理学》，163，93-100。
Parsons, J.R., Saez, M., Dolfing, J., de Voogt, P., 2008. Biodegradation of perfluorinated compounds. Rev. Environ. Contam. Toxicol. 196, 53-71. Parsons, J.R., Saez, M., Dolfing, J., de Voogt, P., 2008.全氟化合物的生物降解。Rev. Environ.Contam.Toxicol.196, 53-71.
Ponprateep, S., Vatanavicharn, T., Lo, C.F., Tassanakajon, A., Rimphanitchayakit, V., 2017. Alpha-2-macroglobulin is a modulator of prophenoloxidase system in pacific white shrimp Litopenaeus vannamai. Fish Shellfish Immunol 62, 68-74. Ponprateep, S., Vatanavicharn, T., Lo, C.F., Tassanakajon, A., Rimphanitchayakit, V., 2017.α-2-巨球蛋白是太平洋南美白对虾丙酚氧化酶系统的调节剂。Fish Shellfish Immunol 62, 68-74.
Pradhan, A., Olsson, P.E., 2018. Germ cell depletion in zebrafish leads to incomplete masculinization of the brain. Gen. Comp. Endocrinol. 265, 15-21. Pradhan, A., Olsson, P.E., 2018.斑马鱼生殖细胞耗竭导致大脑不完全男性化。Gen.Endocrinol.265, 15-21.
Quinones, O., Snyder, S.A., 2009. Occurrence of perfluoroalkyl carboxylates and sulfonates in drinking water utilities and related waters from the United States. Environ Sci Technol 43, 9089-9095. Quinones, O., Snyder, S.A., 2009年。美国饮用水设施及相关水域中全氟烷基羧酸盐和磺酸盐的存在。Environ Sci Technol 43, 9089-9095。
Radzikowski, J., Sikora, A., Slusarczyk, M., 2016. The effect of lake sediment on the hatching success of Daphnia ephippial eggs. J. Limnol. 75, 597-605. Radzikowski, J., Sikora, A., Slusarczyk, M., 2016.湖泊沉积物对水蚤卵孵化成功率的影响。J. Limnol.75, 597-605.
Rainieri, S., Conlledo, N., Langerholc, T., Madorran, E., Sala, M., Barranco, A., 2017. Toxic effects of perfluorinated compounds at human cellular level and on a model vertebrate. Food Chem. Toxicol. 104, 14-25. Rainieri, S., Conlledo, N., Langerholc, T., Madorran, E., Sala, M., Barranco, A., 2017.全氟化合物在人体细胞水平和对模型脊椎动物的毒性效应。Food Chem.Toxicol.104, 14-25.
Raz, E., 2000. The function and regulation of vasa-like genes in germ-cell development, Genome Biol. 1, 1017. Raz, E., 2000.生殖细胞发育中血管样基因的功能和调控》，Genome Biol. 1, 1017.
Riddiford, L.M., Cherbas, P., Truman, J.W., 2001. Ecdysone receptors and their biological actions. Vitamins and Hormones - Advances in Research and Applications 60 (60), . Riddiford, L.M., Cherbas, P., Truman, J.W., 2001.蜕皮激素受体及其生物作用。维生素与激素--研究与应用进展 60 (60), .
Rodriguez-Menendez, S., Garcia, M., Fernandez, B., Alvarez, L., Fernandez-Vega-Cueto, A., Coca-Prados, M., et al., 2018. The zinc-metallothionein redox system reduces oxidative stress in retinal pigment epithelial cells. Nutrients 10 . Rodriguez-Menendez, S., Garcia, M., Fernandez, B., Alvarez, L., Fernandez-Vega-Cueto, A., Coca-Prados, M., et al., 2018.锌-金属硫蛋白氧化还原系统可降低视网膜色素上皮细胞的氧化应激。营养素 10 .
Sarkar, S., Mukherjee, S., Chattopadhyay, A., Bhattacharya, S., 2014. Low dose of arsenic trioxide triggers oxidative stress in zebrafish brain: expression of antioxidant genes. Ecotoxicol. Environ. Saf. 107, 1-8. Sarkar, S., Mukherjee, S., Chattopadhyay, A., Bhattacharya, S., 2014.低剂量三氧化二砷引发斑马鱼大脑氧化应激：抗氧化基因的表达。Ecotoxicol.Ecotoxicol.Saf.107, 1-8.
Sato, M., Kawakami, T., Kondoh, M., Takiguchi, M., Kadota, Y., Himeno, S., et al., 2010. Development of high-fat-diet-induced obesity in female metallothionein-null mice. FASEB J. 24, 2375-2384. Sato, M., Kawakami, T., Kondoh, M., Takiguchi, M., Kadota, Y., Himeno, S., et al., 2010.雌性金属硫蛋白缺失小鼠高脂饮食诱发肥胖的发展。FASEB J. 24, 2375-2384.
Sato, S., Shirakawa, H., Tomita, S., Tohkin, M., Gonzalez, F.J., Komai, M., 2013. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A. Toxicol. Appl. Pharmacol. 273, 90-99. Sato, S., Shirakawa, H., Tomita, S., Tohkin, M., Gonzalez, F.J., Komai, M., 2013.芳基烃受体和糖皮质激素受体相互作用激活人金属硫蛋白 2A。Toxicol.Appl.273, 90-99.
Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative C-T method. Nature Protoc 3, 1101-1108. Schmittgen, T.D., Livak, K.J., 2008.用 C-T 比较法分析实时 PCR 数据。Nature Protoc 3, 1101-1108.
Sengupta, N., Reardon, D.C., Gerard, P.D., Baldwin, W.S., 2017. Exchange of polar lipids from adults to neonates in Daphnia magna: perturbations in sphingomyelin allocation by dietary lipids and environmental toxicants. PLoS One 12. Sengupta, N., Reardon, D.C., Gerard, P.D., Baldwin, W.S., 2017.大型蚤极性脂质从成体到新生儿的交换：膳食脂质和环境毒物对鞘磷脂分配的扰动。PLoS One 12.
Seyoum, A., Pradhan, A., 2019. Effect of phthalates on development, reproduction, fat metabolism and lifespan in Daphnia magna. Sci. Total Environ. 654, 969-977. Seyoum, A., Pradhan, A., 2019.邻苯二甲酸盐对大型蚤发育、繁殖、脂肪代谢和寿命的影响。Sci.654, 969-977.
Shi, X., Du, Y., Lam, P.K., Wu, R.S., Zhou, B., 2008. Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS. Toxicol. Appl. Pharmacol. . Shi, X., Du, Y., Lam, P.K., Wu, R.S., Zhou, B., 2008.暴露于全氟辛烷磺酸的斑马鱼胚胎的发育毒性和基因表达改变。Toxicol.Appl. .
Sieber, M.H., Thummel, C.S., 2009. The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila. Cell Metab. 10, 481-490. Sieber, M.H., Thummel, C.S., 2009.DHR96核受体控制果蝇的三酰甘油平衡。细胞代谢。10, 481-490.
Sieber, MH, Thummel, CS, 2012. Coordination of triacylglycerol and cholesterol homeostasis by DHR96 and the Drosophila LipA homolog magro. Cell Metab. (15), 122-127. Sieber, MH, Thummel, CS, 2012.DHR96和果蝇LipA同源物magro协调三酰甘油和胆固醇的平衡。细胞代谢》（15），122-127。
Slaninova, A., Smutna, M., Modra, H., Svobodova, Z., 2009. A review: oxidative stress in fish induced by pesticides. Neuro Endocrinol Lett 30 (Suppl. 1), 2-12. Slaninova, A., Smutna, M., Modra, H., Svobodova, Z., 2009.A review: oxidative stress in fish induced by pesticides.Neuro Endocrinol Lett 30 (Suppl. 1), 2-12.
Stylianou, M., Bjornsdotter, M.K., Olsson, P.E., Ericson Jogsten, I., Jass, J., 2019. Distinct transcriptional response of Caenorhabditis elegans to different exposure routes of perfluorooctane sulfonic acid. Environ. Res. 168, 406-413. Stylianou, M., Bjornsdotter, M.K., Olsson, P.E., Ericson Jogsten, I., Jass, J., 2019.秀丽隐杆线虫对全氟辛烷磺酸不同暴露途径的转录反应差异。Environ.168, 406-413.
Sun, C., Zhang, S.C., 2015. Immune-relevant and antioxidant activities of vitellogenin and yolk proteins in fish. Nutrients 7, 8818-8829. Sun, C., Zhang, S.C., 2015.鱼类卵黄素和卵黄蛋白的免疫相关性和抗氧化活性。Nutrients 7, 8818-8829.
Swevers, L., Ito, K., Iatrou, K., 2002. The BmE75 nuclear receptors function as dominant repressors of the nuclear receptor BmHR3A. J. Biol. Chem. 277, 41637-41644. Swevers, L., Ito, K., Iatrou, K., 2002.BmE75 核受体作为核受体 BmHR3A 的显性抑制因子发挥作用。J. Biol.Chem.277, 41637-41644.
Taniyasu, S., Kannan, K., Horii, Y., Hanari, N., Yamashita, N., 2003. A survey of perfluorooctane sulfonate and related perfluorinated organic compounds in water, fish, birds, and humans from Japan. Environ Sci Technol 37, 2634-2639. Taniyasu, S., Kannan, K., Horii, Y., Hanari, N., Yamashita, N., 2003.日本水、鱼类、鸟类和人类体内全氟辛烷磺酸及相关全氟有机化合物的调查。Environ Sci Technol 37, 2634-2639.
Tatar, M., Bartke, A., Antebi, A., 2003. The endocrine regulation of aging by insulin-like signals. Science 299, 1346-1351 Tatar, M., Bartke, A., Antebi, A., 2003.胰岛素样信号对衰老的内分泌调节。科学》299 期，1346-1351 页
Tokishita, S., Kato, Y., Kobayashi, T., Nakamura, S., Ohta, T., Yamagata, H., 2006. Organization and repression by juvenile hormone of a vitellogenin gene cluster in the crustacean, Daphnia magna. Biochem. Biophys. Res. Commun. 345, 362-370. Tokishita, S., Kato, Y., Kobayashi, T., Nakamura, S., Ohta, T., Yamagata, H., 2006.甲壳动物大型蚤卵黄素基因簇的组织和幼年激素的抑制。Biochem.Biophys.Res. Commun.345, 362-370.
Touhara, K., Bonning, B.C., Hammock, B.D., Prestwich, G.D., 1995. Action of juvenilehormone (Jh) esterase on the Jh-Jh binding-protein complex - an in-vitro model of Jh metabolism in a caterpillar. Insect Biochem. Mol. Biol. 25, 727-734. Touhara, K., Bonning, B.C., Hammock, B.D., Prestwich, G.D., 1995.幼虫激素（Jh）酯酶对 Jh-Jh 结合蛋白复合物的作用--毛虫体内 Jh 代谢的体外模型。Insect Biochem.Mol.25, 727-734.
Wang, H.F., Zhong, X.H., Shi, W.Y., Guo, B., 2011. Study of malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in chickens infected with avian infectious bronchitis virus. African J Biotechnol 10 , 9213-9217. Wang, H.F., Zhong, X.H., Shi, W.Y., Guo, B., 2011.鸡感染禽传染性支气管炎病毒后丙二醛（MDA）含量、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）活性的研究。African J Biotechnol 10 , 9213-9217。
Wang, L.L., Wang, L.L., Zhang, H., Zhou, Z., Siva, V.S., Song, L.S., 2012. A C1q domain containing protein from scallop Chlamys farreri serving as pattern recognition receptor with heat-aggregated IgG binding activity. PLoS One 7. Wang, L.L., Wang, L.L., Zhang, H., Zhou, Z., Siva, V.S., Song, L.S., 2012.一种来自扇贝 Chlamys farreri 的含 C1q 结构域的蛋白作为模式识别受体，具有热聚集 IgG 结合活性。PLoS One 7.
Wennberg, L., Fridlund, M., 2015. Swedavias work with the PFAS issue. Dialogmöte om PFAS, Stockholm in Swedish. Wennberg, L., Fridlund, M., 2015.瑞典在全氟辛烷磺酸问题上的工作。Dialogmöte om PFAS，斯德哥尔摩，瑞典语。
Xia, X., Rabearisoa, A.H., Jiang, X., Dai, Z., 2013. Bioaccumulation of perfluoroalkyl substances by Daphnia magna in water with different types and concentrations of protein. Environ Sci Technol 47, 10955-10963. Xia, X., Rabearisoa, A.H., Jiang, X., Dai, Z., 2013.大型蚤在不同类型和浓度蛋白质水中对全氟烷基物质的生物累积。环境科学与技术》47，10955-10963。
Xu, T., Li, P., Wu, S., Li, D., Wu, J., Raley-Susman, K.M., et al., 2016. Chronic exposure to perfluorooctane sulfonate reduces lifespan of Caenorhabditis elegans through insulin/IGF-1 signaling. Bull. Environ. Contam. Toxicol. 97, 119-123. Xu, T., Li, P., Wu, S., Li, D., Wu, J., Raley-Susman, K.M., et al., 2016.慢性暴露于全氟辛烷磺酸会通过胰岛素/IGF-1信号传导降低秀丽隐杆线虫的寿命。Bull.Environ.Contam.Toxicol.97, 119-123.
Yang, H.B., Zhao, Y.Z., Tang, Y., Gong, H.Q., Guo, F., Sun, W.H., et al., 2019. Antioxidant defence system is responsible for the toxicological interactions of mixtures: a case study on PFOS and PFOA in Daphnia magna. Sci. Total Environ. 667, 435-443. Yang, H.B., Zhao, Y.Z., Tang, Y., Gong, H.Q., Guo, F., Sun, W.H., et al., 2019.抗氧化防御系统对混合物毒理学相互作用的影响：大型蚤体内全氟辛烷磺酸和全氟辛酸的案例研究。Sci.667, 435-443.
Yao, T.P., Forman, B.M., Jiang, Z.Y., Cherbas, L., Chen, J.D., Mckeown, M., et al., 1993. Functional Ecdysone receptor is the product of Ecr and ultraspiracle genes. Nature 366, . Yao, T.P., Forman, B.M., Jiang, Z.Y., Cherbas, L., Chen, J.D., Mckeown, M., et al., 1993.功能性蜕皮激素受体是 Ecr 和 ultraspiracle 基因的产物。自然 366, .
