Mepanipyrim induces cardiotoxicity of zebrafish (Danio rerio) larvae via promoting AhR-regulated COX expression pathway

The wide use of pesticides has seriously threatened human health and the survival of beneficial organisms. The fungicide mepanipyrim is widely used in viticulture practices. Studies of mepanipyrim-induced toxicity in organisms are still scarce, especially studies on cardiotoxicity. In this study, we aimed to investigate mepanipyrim-induced cardiotoxicity in zebrafish (Danio rerio) larvae. We found that mepanipyrim could induce cardiotoxicity by altering the heart rate and cardiomyocyte diameter of larvae. Meanwhile, RNA sequencing and RT-qPCR data indicated that mepanipyrim exposure could dramatically alter the mRNA expression of calcium signaling pathway-, cardiac muscle contraction-, and oxidative respiratory chain-related genes. Interestingly, by the CALUX cell bioassay, we found that most cytochrome c oxidase (COX) family genes exhibited potential AhR-regulated activity, suggesting that mepanipyrim induced cardiotoxicity via a novel AhR-regulated manner in larvae. Additionally, the AhR antagonist CH223191 could effectively prevent mepanipyrim-induced cardiotoxicity in zebrafish larvae. In conclusion, the AhR agonist mepanipyrim could induce cardiotoxicity in a novel unreported AhR-regulated manner, which could specifically affect the expression of COX family genes involved in the mitochondrial oxidative respiratory chain. Our data will help explain the toxic effects of mepanipyrim on organisms and provide new insight into the AhR agonistic activity pesticide-induced cardiotoxicity.     

溴氰菊酯对稀有鮈鲫早期生命阶段发育和内分泌干扰毒性

采用半静态水体暴露方式研究了水中溴氰菊酯对稀有鮈鲫早期生命阶段的发育毒性与内分泌干扰效应.结果表明,影响稀有鮈鲫胚胎孵化的LOEC (最低可观察效应浓度)>3.0μg/L;影响稀有鮈鲫仔鱼发育畸形和死亡指标的LOEC和NOEC (无可观察效应浓度)分别为1.0和0.33μg/L.低至0.04μg/L的溴氰菊酯暴露便可显著下调稀有鮈鲫幼鱼体内雄激素受体基因(AR)表达量并上调甲状腺激素受体基因(TRβ)表达量;0.11μg/L的溴氰菊酯暴露可以下调稀有鮈鲫幼鱼体内雄激素受体基因(AR)、雌激素受体基因(ER1、ER2b)和芳香烃受体基因(AhR1a)的表达量,并上调甲状腺激素受体基因(TRβ)表达量;0.33μg/L的溴氰菊酯可以下调稀有鮈鲫幼鱼体内雄激素受体基因(AR)、雌激素受体基因(ER1、ER2b)、芳香烃受体基因(AhR1a、AhR1b、AhR2)的表达量,并诱导甲状腺激素受体基因(TRβ)表达量的上调.上述作用浓度水平已经处于多个天然水体中溴氰菊酯的检出浓度范围,因此,关于水体残留溴氰菊酯对鱼类的内分泌干扰效应必须予以重视.     

Kin recognition and cannibalistic behaviours by adult male fathead minnows (<Emphasis Type="Italic">Pimephales promelas</Emphasis>)

Parental care is an energetically demanding activity that ensures genes are efficiently passed from one generation to the next. According to evolutionary theory, the greatest energetic investment should be directed towards offspring that are most closely related to the parent. Male fathead minnows, Pimephales promelas, provide this parental investment to developing embryos but not newly hatched larvae. Therefore, selection should favour recognition of embryonic kin to ensure energetic expenditure is optimally invested. In this study, adult male fathead minnows were tested using behavioural assays, with egg cannibalism as an endpoint, to determine whether adult males could discriminate between related and unrelated embryos. Egg cannibalism was highest when adult male fathead minnows were presented with unrelated eggs and lowest when presented with eggs fertilized by the test subject (related eggs). The degree of cannibalism was also a function of breeding status. Unrelated males in breeding condition showed an intermediate response between the low cannibalism demonstrated by related males and the high cannibalism demonstrated by unrelated males in a nonbreeding condition. These results suggest that although male fathead minnows can discriminate between unrelated and related embryos, at least some component of parental investment is a simple function of breeding status.     

全氟辛烷磺酸(PFOS)对斑马鱼卵黄蛋白原mRNA水平的影响

为了研究环境低剂量全氟辛烷磺酸（perfluorooctane sulfonate,PFOS）对水生生物的内分泌干扰效应和可能的作用机制,测定了PFOS对斑马鱼（Brachydanio rerio）肝脏中卵黄蛋白原（vitellogenin,VTG）mRNA水平的影响.将斑马鱼暴露于4个PFOS的环境低剂量浓度组（0.1、1、10、100μg.L-1）中进行21d毒性试验,收集肝脏样品,提取RNA,采用荧光定量PCR（qRT-PCR）分别检测VTG1和VTG3的mRNA水平.结果表明：①PFOS暴露引起雄性斑马鱼肝脏VTG1和VTG3 mRNA水平升高,VTG1 mRNA水平升高与剂量呈正相关,在100μg.L-1暴露浓度处与对照组呈现显著性差异;VTG3的mRNA水平变化与剂量呈倒U型曲线,呈现典型的毒物刺激荷尔蒙效应,在10和100μg.L-1暴露浓度处与对照组呈现显著性差异;②PFOS暴露引起雌性斑马鱼肝脏中VTG1 mRNA水平升高,在10μg.L-1暴露浓度处与对照组呈现显著性差异,但在高浓度（10和100μg.L-1）处试验结果误差较大;VTG3 mRNA水平只在10μg.L-1暴露浓度处升高,但相比于对照组均没有显著性差异.试验结果表明PFOS暴露对斑马鱼的内分泌干扰作用明显,其毒性作用机制可能是类雌激素效应,而肝脏中VTG1和VTG3mRNA水平可能作为PFOS内分泌干扰效应评价的敏感生物标志物,但VTG1和VTG3 mRNA水平的响应曲线呈现基因亚型和性别差异.     

氯苯对斑马鱼胚胎发育和仔鱼的毒性效应研究

选取健康的雌雄斑马鱼按1:1或1:2的比例进行交配产卵。采用静态法,以丙酮为助溶剂,分别用不同浓度的CB进行斑马鱼受精卵和刚平衡游动仔鱼的暴露试验,记录胚胎孵化数和胚胎、仔鱼畸形数以及仔鱼死亡数。不同浓度的CB可导致斑马鱼胚胎孵化率降低及孵出的仔鱼畸形率增加,且毒性呈现剂量-效应关系。当CB浓度高于0.88μg/L时,斑马鱼胚胎及其孵出的仔鱼畸形率开始增加;当CB为108.9μg/L时,暴露24 h的胚胎出现全致畸效应。能平衡游动的斑马仔鱼对CB的毒性也很敏感,24 h、72 h和96 h的LC50值分别为95.35、80.51和62.28μg/L;110.0μg/L CB使处理24 h的仔鱼全部死亡。较低浓度的CB就使斑马鱼胚胎发育畸形;其对仔鱼的毒性也很显著,达到一定浓度可使其死亡。     

氟吡菌胺对斑马鱼的毒性效应

采用斑马鱼胚胎发育技术和传统毒理学方法, 研究了氟吡菌胺对斑马鱼胚胎、成鱼及仔鱼的毒性效应.结果发现,氟吡菌胺对3个阶段斑马鱼均具有致死能力,对仔鱼LC50(48h)值为0.204mg/L,成鱼LC50(96h)为0.286mg/L,幼鱼LC50(96h)为1.489mg/L.研究表明,0.0596mg/L以上浓度的氟吡菌胺对斑马鱼胚胎均有一定程度的致死效应,高浓度处理组胚胎出现心包囊肿、卵黄囊不吸收、黑色素沉积少及鱼体弯曲等症状;氟吡菌胺对斑马鱼成鱼进行14d暴露后,处理组成鱼体重下降,产卵量减少,有效卵量降低; 0.0298mg/L处理组144h子代仔鱼存活率虽然比较高,但存活鱼大部分出现心包囊肿、体弯曲等畸形.上述结果说明,水体中残留的氟吡菌胺对于斑马鱼各生命阶段的生长发育均具有潜在的危害.     

不同生长阶段斑马鱼对Cu2+的毒性响应差异

为探究鱼类不同生长阶段对污染物的毒性响应差异,以斑马鱼为试验生物,初步建立了一套实验室分阶段培养方法,通过培养时间确定斑马鱼的不同生长阶段,研究了标准稀释水和模拟自来水条件下不同生长阶段斑马鱼对Cu2+的毒性响应差异,并利用物种敏感度分布法表达不同生长阶段斑马鱼对Cu2+的敏感性差异.结果表明:斑马鱼体长、体质量的变化均符合logistic增长方程（R2>0.97）,斑马鱼全生命周期按日龄可细分为卵黄囊仔鱼期（1 d）、晚期仔鱼期（10 d）、稚鱼期（17 d）、早幼期（24 d）、幼鱼期（31 d）、发育期（60 d）、成熟期（90 d）、成鱼期（120 d）等8个不同生长阶段.标准稀释水条件下,Cu2+对不同生长阶段斑马鱼的96 h-LC₅₀（96 h半致死浓度）分别为0.425 mg/L（1 d）、0.768 mg/L（10 d）、0.550 mg/L（17 d）、0.309 mg/L（24 d）、0.334 mg/L（31 d）、0.327 mg/L（60 d）、0.230 mg/L（90 d）和0.180 mg/L（120 d）,随着鱼龄的增加,Cu2+对斑马鱼的毒性逐渐增大;模拟自来水条件下,Cu2+对不同生长阶段斑马鱼的96 h-LC₅₀分别为0.377 mg/L（1 d）、0.438 mg/L（10 d）、0.366 mg/L（17 d）、0.201 mg/L（24 d）、0.206 mg/L（31 d）、0.189 mg/L（60 d）、0.167 mg/L（90 d）和0.144 mg/L（120 d）,随着鱼龄的增加,Cu2+对斑马鱼的毒性逐渐增大.根据96 h-LC₅₀得出不同生长阶段斑马鱼对Cu2+的敏感性顺序为120 d > 90 d > 24 d > 60 d > 31 d > 1 d > 17 d > 10 d,可见,斑马鱼成鱼期阶段对Cu2+较敏感,而成熟期和早幼期的敏感性次之,晚期仔鱼期最不敏感.研究显示,鉴于斑马鱼对Cu的物种敏感度的阶段性差异,采用24 d早幼期斑马鱼研究金属水质基准时既敏感又节约受试生物的培养成本,此外水环境参数也是水质基准研究需考虑的重要影响因素之一.      

氟对斑马鱼甲状腺内分泌功能的干扰效应

为探究氟对斑马鱼甲状腺内分泌功能的干扰效应,本研究以雌性斑马鱼成鱼为研究对象,分别以不同浓度氟(0、20、 40、80 mg·L~(-1))暴露45 d和90 d,对斑马鱼的生长发育指数进行测定,用组织学方法对斑马鱼甲状腺组织结构进行显微观察,用酶联免疫吸附法检测血浆中T3和T4激素水平,并用实时荧光定量PCR方法检测HPT轴上内分泌相关基因的表达.结果显示,与对照组相比,氟暴露组雌性斑马鱼的生长发育指数随着暴露浓度和时间的增加而呈下降趋势(p0.05);甲状腺组织病变程度随着氟暴露浓度的增大和时间的延长而加重;氟暴露45 d时,T3和T4水平呈升高趋势,90 d时,T4水平呈下降趋势且在80 mg·L~(-1)组下降显著(p0.05);氟暴露45 d时,40 mg·L~(-1)组除crh处,其他mRNA表达均显著下降(p0.05),80 mg·L~(-1)组除tg、nis、ttr外其他mRNA表达均显著下降(p0.05);暴露90 d时tg、dio1、dio2的mRNA表达水平在40 mg·L~(-1)和80 mg·L~(-1)组显著上升(p0.05),ugt1ab mRNA表达在80 mg·L~(-1)组显著下降(p0.05).综上,氟可通过影响雌性斑马鱼的生长发育、甲状腺组织结构、激素水平及HPT轴上内分泌相关基因的表达,从而对其甲状腺内分泌功能产生一定程度的干扰,进而影响机体的生长发育.本研究结果对水环境中氟的生态毒性效应及风险评估提供了理论资料.     

Effects of ZnO nanoparticles on perfluorooctane sulfonate induced thyroid-disrupting on zebrafish larvae

Perfluorooctane sulfonate(PFOS) and ZnO nanoparticles(nano-ZnO) are widely distributed in the environment.However,the potential toxicity of co-exposure to PFOS and nano-ZnO remains to be fully elucidated.The test investigated the effects of co-exposure to PFOS and nano-ZnO on the hypothalamic–pituitary–thyroid(HPT) axis in zebrafish.Zebrafish embryos were exposed to a combination of PFOS(0.2,0.4,0.8 mg/L) and nano-ZnO(50 mg/L)from their early stages of life(0–14 days).The whole-body content of TH and the expression of genes and proteins related to the HPT axis were analyzed.The co-exposure decreased the body length and increased the malformation rates compared with exposure to PFOS alone.Co-exposure also increased the triiodothyronine(T3) levels,whereas the thyroxine(T4)content remained unchanged.Compared with the exposure to PFOS alone,exposure to both PFOS(0.8 mg/L) and nano-ZnO(50 mg/L) significantly up-regulated the expression of corticotropin-releasing factor,sodium/iodidesymporter,iodothyronine deiodinases and thyroid receptors and significantly down-regulated the expression of thyroid-stimulating hormone,thyroglobulin(TG),transthyretin(TTR) and thyroid receptors.The protein expression levels of TG and TTR were also significantly down-regulated in the co-exposure groups.In addition,the expression of the thyroid peroxidase gene was unchanged in all groups.The results demonstrated that PFOS and nano-ZnO co-exposure could cause more serious thyroid-disrupting effects in zebrafish than exposure to PFOS alone.Our results also provide insight into the mechanism of disruption of the thyroid status by PFOS and nano-ZnO.     

环境雌激素的男性生殖健康效应

概述了近年来国内外学者对内分泌干扰物健康效应的解释，及其对环境雌激素作用的新认识和男性生殖健康的影响。重点讨论了环境雌激素对男性生殖系统结构和功能的影响，以及造成男性生殖健康不良效应的可能作用机理及其最新研究进展，并简要介绍了对环境雌激素健康机制的进一步研究方向。根据报道，环境雌激素可以通过模拟天然雌激素的受体结合机制，干扰生物体内正常的内分泌生物机制，并使生殖系统的肿瘤发病率上升。