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摘要:背景
磷酸三丁酯(TBP)作为一种有机磷酸酯阻燃剂被广泛应用,然而TBP在低浓度暴露下对水生生物毒性研究有限。
目的以斑马鱼作为模式动物,探讨阻燃剂TBP对斑马鱼早期发育的影响。
方法将受精后2 h(2 hpf)的斑马鱼胚胎随机分为4组,分别为0.01%二甲基亚砜(DMSO)对照组和TBP染毒组(0.02、0.2、2 μg·L−1)。染毒时间为2 hpf至120 hpf,分别观察斑马鱼胚胎72 hpf孵化率、畸形率、心率和体长,24~29 hpf卷尾频率、96 hpf运动能力和120 hpf存活率。染毒结束后利用酶联免疫法检测幼鱼全身三碘甲状腺原氨酸(T3)及甲状腺素(T4)含量,采用实时荧光定量聚合酶链式反应(q-PCR)法检测下丘脑-垂体-甲状腺轴(HPT)和神经发育相关基因的表达水平。
结果TBP染毒组斑马鱼胚胎均出现心率下降(
P <0.001),0.02、2 μg·L−1染毒组的存活率下降(P< 0.05),2 μg·L−1染毒组畸形率上升(P< 0.05),主要表现为心包水肿。各组斑马鱼胚胎卷尾频率在25 hpf达到最高,各染毒组的卷尾频率低于对照组(P< 0.001)。运动行为实验中,暗周期0.02、0.2 μg·L−1染毒组斑马鱼游泳速度下降(P< 0.05),在光周期0.2、2 μg·L−1染毒组斑马鱼游泳速度明显下降(P< 0.05)。与对照组相比,0.2 μg·L−1染毒组幼鱼T3水平上升(P< 0.05)。q-PCR结果表明,染毒组HPT轴相关基因甲状腺激素受体(trα、 $tr\beta $)、甲状腺球蛋白(tg )、钠碘共转运体(nis )表达水平下调,甲状腺转运蛋白(ttr )在0.02 μg·L−1染毒组上调,碘甲腺原氨酸脱碘酶2(dio2 )在0.02 μg·L−1染毒组下调(P< 0.05);染毒组神经发育相关基因乙酰胆碱酯酶(ache )下调,髓鞘碱性蛋白(mbp )和ELAV样神经元特异性RNA结合蛋白3(elavl3 )在0.02 μg·L−1染毒组也下调(P< 0.05)。结论TBP暴露可导致斑马鱼早期发育异常,表现为孵化期和早幼期的发育毒性、甲状腺内分泌破坏和神经毒性。
Abstract:BackgroundTributyl phosphate (TBP) is widely used as an organophosphate flame retardant. However, there are limited studies on the toxicity of TBP to aquatic organisms at low levels of exposure.
ObjectiveTo investigate the effects of TBP on early development of zebrafish (
Danio rerio ).MethodsZebrafish embryos were randomly divided into four groups at 2 h post-fertilisation (2 hpf), namely, the 0.01% dimethyl sulfoxide (DMSO) control group and TBP exposure groups (0.02, 0.2 and 2 μg·L−1). The exposure time was from 2 hpf to 120 hpf and the hatching rate, malformation rate, heart rate and body length of zebrafish embryos at 72 hpf, the frequency of tail curling at 24-29 hpf, the locomotor ability at 96 hpf and the survival rate at 120 hpf were evaluated, respectively. The whole-body triiodothyronine (T3) and tetraiodothyronine (T4) levels of juvenile fish were measured by enzyme immunoassay at the end of the infection, and the expression levels of hypothalamic-pituitary-thyroid axis (HPT) and neurodevelopmental-related genes were detected by quantitative real-time PCR (q-PCR).
ResultsThe heart rates of zebrafish embryos were significantly decreased in all TBP-treated groups (
P< 0.001), the survival rates of the 0.02 and 2 μg·L−1 TBP groups were significantly decreased (P< 0.05), and the malformation rate of the 2 μg·L−1 treated group was significantly increased (P< 0.05), which was mainly manifested by pericardial oedema. The frequency of tail curling of zebrafish embryos in all groups reached the highest at 25 hpf, which was significantly lower (P< 0.001) in all exposure groups than in the control group (P< 0.001). In the locomotor behaviour experiments, the swimming speed of zebrafish larvae in the dark cycle was significantly decreased in the 0.02 and 0.2 μg·L−1 TBP groups (P <0.05), and similar results were found for the light cycle in the 0.2 and 2 μg·L−1 TBP groups (P< 0.05). Compared with the control group, the T3 level of zebrafish juveniles in the 0.2 μg·L−1 TBP group increased significantly (P< 0.05). The q-PCR results showed that the expression levels of HTP axis-related genes [thyroid hormone receptors (trα, $tr\beta $) , thyroglobulin (tg ), and sodium/iodide co-transporter (nis )] were significantly down-regulated in the exposure groups, the expression level of transthyretin (ttr ) was significantly up-regulated in the 0.02 μg·L−1 TBP group, and the iodothyronine deiodinase 2 (dio2 ) expression level was significantly down-regulated in the 0.02 μg·L−1 TBP group (P< 0.05); the neurodevelopment-related gene acetylcholinesterase (ache ) was significantly down-regulated in the exposure groups, and the expression levels of myelin basic protein (mbp ) and Elav like neuron-specific RNA binding protein 3 (elavl3 ) were significantly down-regulated in the 0.02 μg·L−1 TBP group (P< 0.05).ConclusionTBP exposure can lead to early developmental abnormalities in zebrafish, manifested as developmental toxicity, thyroid endocrine disruption and neurotoxicity during hatching and early juvenile stages.
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Keywords:
- organophosphate /
- tributyl phosphate /
- zebrafish /
- endocrine disruption /
- neurotoxicity /
- developmental toxicity
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随着溴代阻燃剂的禁用,有机磷酸酯阻燃剂(organophosphate flame retardants, OPFRs)因具备优良的阻燃效能及化学稳定性,被广泛应用。磷酸三丁酯(tributyl phosphate, TBP)作为一种常见的有机磷化合物,因其优良的萃取性能和润滑性能,在石油加工、金属提取、塑料制造及农药生产等多个领域得到广泛应用。由于TBP等OPFRs以物理方式与基质材料结合,它们可通过挥发、浸出和磨损等多种途径释放到环境中[1],最终对生物体和生态环境造成破坏。最新研究报告显示,各种环境样本中均能检测到高浓度TBP。例如,室内空气检测到的TBP浓度达1.7~29 ng·m−3,其中居民区室内空气TBP的浓度最高(1.0~30 ng·m−3)[2]。中国太湖沉积物OPFRs污染调查发现,TBP、磷酸三苯酯(triphenyl phosphate, TPP)等5种为主要污染类型,其总浓度区间为3.38~14.25 g·kg−1[3]。研究表明,我国多地人群尿液中TBP代谢产物的检出率为66%,浓度中位数为0.84 ng·mL−1[4]。然而,目前毒理学研究中所用到的TBP暴露浓度主要在25~
3125 μg·L−1,远高于其环境浓度。研究发现,TBP在高浓度暴露下可以影响斑马鱼神经发育,降低其游泳速度,改变体内乙酰胺胆碱酯酶活性和相关基因的转录水平[5]。然而关于TBP在低浓度暴露下对水生生物的神经毒性及内分泌毒性研究有限,需要开展基于环境暴露水平下TBP的相关毒性实验。斑马鱼逐渐成为毒理学研究的理想动物模型[6],具有易于维持、生命周期短、繁殖力高、发育快速、对环境毒素敏感等优点。本研究以斑马鱼胚胎为模型,研究TBP对其早期胚胎发育、甲状腺激素破坏和神经发育的影响,为了解TBP对水生生物的潜在健康风险提供数据支持。
1. 对象与方法
1.1 实验材料与仪器
实验用AB系野生型斑马鱼(中国木芮生物科技有限公司),TBP(美国Sigma),三碘甲状腺原氨酸(triiodothyronine, T3)和甲状腺素(tetraiodothyronine, T4)酶联免疫试剂盒(中国华美生物公司),蛋白浓度测定试剂盒(美国Biosharp),反转录试剂盒(中国诺唯赞),实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, q-PCR)试剂盒(中国诺唯赞)。
体式显微镜(SMZ18,日本尼康),斑马鱼(幼鱼)行为追踪分析仪(Noldus,中国诺达思),超微量分光光度计(NANODROP 2000,美国Thermo Scientifid),多功能酶标仪(Infinite M200PRO,瑞士TECAN),实时荧光定量PCR仪(LightCycler480Ⅱ,瑞士Roche),双量程电子天平(ME155DU,美国梅特勒),台式离心机(AllegraX-30R,德国贝克曼)。
1.2 实验方法
1.2.1 工作液配制
研究团队前期对无锡市太湖水源生活饮用水监测发现TBP含量为1.71~50.82 ng·L−1,平均浓度为20.44 ng·L−1,本研究以0.02 μg·L−1作为最低染毒剂量。准确称取TBP标准品0.125 g,溶入1 mL的二甲基亚砜(dimethyl sulfoxide, DMSO),配制成浓度为1.25×105 mg·L−1的标准储存溶液,4 ℃保存备用。使用胚胎培养液将TBP标准储存溶液梯度稀释为0.02、0.2、2 μg·L−1,置于−20 ℃避光保存。
1.2.2 斑马鱼胚胎染毒实验
受精后2 h(2 hours of fertilization, 2hpf)内收集斑马鱼胚胎,随机分配到6孔板中,每孔30只,加入3 mL工作液以进行斑马鱼的生长发育观察、运动行为检测及q-PCR分析。另外在10 cm培养皿中随机放置100只鱼卵,每个培养皿的液体总体积为10 mL[7],用于检测幼鱼全身T3、T4的含量。分组情况为0.01% DMSO对照组,0.02、0.2、2 μg·L−1 TBP染毒组,每组3个重复。将胚胎置于培养箱中,14 h光照与10 h黑暗循环,温度保持28 ℃,至120 hpf暗周期暴露结束。暴露期间,每天更换暴露液体,保持TBP暴露浓度恒定,每天及时挑除并记录死亡胚胎及幼鱼。
按照经济合作与发展组织(Organisation for Economic Co-operation and Development, OECD)制订的有关使用斑马鱼进行急性发育毒性测试的指南TestGuideline 236(TG236),记录斑马鱼72 hpf孵化数、畸形数、心率、体长,记录120 hpf存活率[8]。
1.2.3 幼鱼运动行为检测
从24 hpf开始记录尾巴的交替卷绕或弯曲直到29 hpf,每隔1 h进行一次记录,每次持续1 min。在84 hpf时,每组随机挑选24条幼鱼经干净的胚胎培养液清洗后按次序放入96孔培养板中,每孔1条。于96 h置于行为追踪分析仪中[9],设置仪器20 min亮-10 min暗-10 min亮-10 min暗,开始记录前适应10 min。记录50 min内幼鱼的游动距离及速度。
1.2.4 甲状腺激素含量测定
暴露结束后,每组取100条斑马鱼幼鱼放于离心管中,匀浆并收集上清液用于检测。在进行激素含量测量之前,取适量上清液测量蛋白浓度,以备后续激素含量的标化。然后按说明书进行激素含量的测定操作,用酶标仪在450 nm波长下依序测得各孔的光密度(optical density, OD)值。根据浓度和OD值用软件拟合,算出标准曲线的回归方程。该试剂盒T3、T4的检出限分别为0.5~8 ng·mL−1、20~320 ng·mL−1。
1.2.5 引物设计及合成
本研究所用引物均查阅以往文献[5,10],包括下丘脑-垂体-甲状腺(hypothalamic-pituitary-thyroid, HPT)轴与神经发育相关基因,引物序列见表1。
表 1 斑马鱼荧光定量PCR引物序列Table 1. The sequences of zebrafish qPCR primers目的基因
(Target gene)引物序列
(Primer sequence)β肌动蛋白($\beta $-actin) F: 5′-CAGTGCCCATCTACGAGGGTTAT-3′
R: 5′-CGGCTGTGGTGGTGAAGGAGT-3′甲状腺激素受体α(Thyroid hormone receptor α, trα) F: 5′-CGAGAAGTGTCAGGAGAT-3′
R: 5′-GTTCGTCACCTTCATCAG-3′甲状腺激素受体β(Thyroid hormone receptor β, tr$\beta $) F: 5′-ACTTGGACGATTCAGAGG-3′
R: 5′-CCTTGTGCTTACGGTAGT-3′甲状腺球蛋白(Thyroglobulin, tg) F: 5′-GTGAAGAGGATGGTGAGT-3′
R: 5′-GATGGCTGGTTGAATGAC-3′钠碘共转运体(Sodium-iodide transporter, nis) F: 5′-GGTGGCATGAAGGCTGTAAT-3′
R: 5′-GATACGGCATCCATTGTTGG-3′甲状腺转运蛋白(Transthyretin, ttr) F: 5′-CTCCTGGTGTGTATCGGGTG-3′
R: 5′-AGGATGTCAGTCATGTGCCTT-3′尿苷二磷酸葡萄糖醛酸转移酶(Udp-glucuronosyltransferase family 1 member A1, ugt1ab) F: 5′-CCACCAAGTCTTTCCGTGTT-3′
R: 5′-GCAGTCCTTCACAGGCTTTC-3′碘甲状腺原氨酸脱碘酶1(Iodothyronine deiodinase 1, dio1) F: 5′-CTGGACCGACAGAAGACGAG-3′
R: 5′-TGCGACATTGCTGAAGTCCT-3′碘甲状腺原氨酸脱碘酶2(Iodothyronine deiodinase 2, dio2) F: 5′-CTCGGACACTTGGCTTGACT-3′
R: 5′-TTGGATCAGGACGGAGAGGT-3′乙酰胆碱酯酶(Acetylcholinesterase, ache) F: 5′-CCCTCCAGTGGGTACAAGAA-3′
R: 5′-GGGCCTCATCAAAGGTAACA-3′髓鞘碱性蛋白(Myelin basic protein, mbp) F: 5′-AATCAGCAGGTTCTTCGGAGGAGA-3′
R: 5′-AAGAAATGCACGACAGGGTTGACG-3′突触素Ⅱa(Synapsin Ⅱa, syn2a) F: 5′-GTGACCATGCCAGCATTTC-3′
R: 5′-TGGTTCTCCACTTTCACCTT-3′生长相关蛋白43(Growth associated protein 43, gap43) F: 5′-TGCTGCATCAGAAGAACTAA-3′
R: 5′-CCTCCGGTTTGATTCCATC-3′ELAV样神经元特异性RNA结合蛋白3
(ELAV like neuron-specific RNA binding protein 3, elavl3)F: 5′-AGACAAGATCACAGGCCAGAGCTT-3′
R: 5′-TGGTCTGCAGTTTGAGACCGTTGA-3′胶质纤维酸性蛋白(Glial fibrillary acidic protein, gfap) F: 5′-GGATGCAGCCAATCGTAAT-3′
R: 5′-TTCCAGGTCACAGGTCAG-3′音猬因子a(Sonic hedgehog signaling molecule a, shha) F: 5′-GCAAGATAACGCGCAATTCGGAGA-3′
R: 5′-TGCATCTCTGTGTCATGAGCCTGT-3′1.2.6 斑马鱼胚胎总RNA提取及基因检测
暴露结束后,提取每组25条幼鱼的RNA,采用反转录试剂盒合成互补脱氧核糖核酸(complementary deoxyribonucleic acid, cDNA),按照说明配置反应体系,使用q-PCR仪进行扩增。结果采用对照基因β肌动蛋白对目的基因转录表达进行归一化处理,分析目的基因表达的相对变化。
1.3 统计学分析
实验数据通过SPSS 20.0软件进行处理分析,对照组和染毒组的差异采用单因素方差分析进行检验。根据方差齐性结果,如果方差齐,两两比较采用LSD-t检验;如果方差不齐,两两比较则采用Dunnett-t检验。P值小于0.05,认为差异有统计学意义。利用专业作图软件OriginPro 8.0进行作图。
2. 结果
2.1 TBP暴露对斑马鱼胚胎发育的影响
各染毒组的斑马鱼孵化率与对照组相比差异无统计学意义(P>0.05)(图1A);与对照组相比,各染毒组斑马鱼的心率分别下降4.8%、9.5%和7.5%(P<0.001)(图1B);2 μg·L−1染毒组斑马鱼畸形率较对照组升高49.4%(P<0.05),主要表现为心包水肿,0.02、0.2 μg·L−1染毒组斑马鱼的畸形率较对照组分别升高30.1%和33.3%,但差异无统计学意义(P>0.05)(图1C),另外,各染毒组斑马鱼的心包腔均有不同程度的增大,心包面积增加(图2);斑马鱼胚胎的体长与对照组相比,差异无统计学意义(P>0.05)(图1D)。120 hpf时,0.02、2 μg·L−1染毒组斑马鱼的存活率较对照组分别下降6.2%和8.2%(P<0.05)(图1E)。
2.2 TBP暴露对斑马鱼发育早期运动行为的影响
24~29 hpf卷尾行为检测发现,各组斑马鱼胚胎每分钟的卷尾次数在25 hpf时达到最高。25 hpf时,与对照组相比,各染毒组的斑马鱼胚胎卷尾频率分别下降19.6%、32.6%和43.5%(P<0.001)(图3A)。
暴露后各组幼鱼自由游泳行为变化结果如图3B所示。染毒组的幼鱼游泳速度的变化差异无统计学意义(P>0.05)。幼鱼光暗周期刺激下的游泳行为变化如图3B所示,与对照组相比,暗周期时0.02、0.2 μg·L−1的染毒组斑马鱼幼鱼游泳速度下降(P<0.05),光周期时0.2、2 μg·L−1染毒组幼鱼游泳速度有明显的下降(P<0.05)。
2.3 TBP暴露对斑马鱼幼鱼甲状腺激素含量的影响
暴露结束后,对幼鱼体内的甲状腺激素含量进行了测定,结果如图4。0.2 μg·L−1染毒组幼鱼体内T3含量与对照组相比有上升(P<0.05),各染毒组幼鱼体内T4含量差异无统计学意义(P>0.05)。
2.4 TBP暴露对斑马鱼mRNA转录水平的影响
2.4.1 TBP暴露对斑马鱼HPT轴相关基因表达的影响
与对照组相比,trα、trβ、tg、nis基因的表达在各染毒组均下调(P<0.05),ttr基因的表达在0.02 μg·L−1染毒组上调(P<0.05),dio2基因的表达在0.02 μg·L−1染毒组下调(P<0.05)。而与对照组相比,ugt1ab和dio1基因表达在各染毒组差异均无统计学意义(P>0.05)(图5A)。
图 5 TBP暴露对斑马鱼HPT轴及神经发育相关基因表达的影响($\bar x \pm s$,n=3)A~B:HPT轴相关基因、神经发育相关基因。*:与对照组比较,P<0.05。Figure 5. The effects of TBP exposure on the expression of genes related to zebrafish HPT axis and neurodevelopment ($\bar x \pm s$, n=3)A-B: HPT axis-related genes and neurodevelopment-related genes. *: Compared with the control group, P<0.05.2.4.2 TBP暴露对斑马鱼神经发育相关基因表达的影响
与对照组相比,ache基因的表达在各染毒组下调(P<0.05),0.02 μg·L−1染毒组mbp和elavl3基因的表达下调(P<0.05),其他神经发育相关基因的表达差异均无统计学意义(P>0.05),见图5B。
3. 讨论
生命早期的毒性试验经常被用来评估环境污染物对鱼类的不良影响,因为它们在这一阶段对化学物质暴露极为敏感。一些致命的和非致命的终点,如存活率、心率、孵化率、畸形率和体长等,通常被用于评估发育毒性。在本实验中,TBP对斑马鱼早期发育造成了负面的效应,具体表现为各染毒组斑马鱼心率均出现降低,同时,在2 μg·L−1染毒组心包水肿的畸形发生增加,可能是TBP对斑马鱼心脏的发育造成抑制,导致心囊水肿,加大了心脏对血液的传导压力所导致的[11]。研究发现,斑马鱼暴露于90或100 μg·L−1的TPP也会增加斑马鱼卵黄囊水肿,心包水肿和脊柱变形等畸形的发生[12–13]。另外,本实验中暴露于0.02、2 μg·L−1 TBP会降低斑马鱼生存率,这也可能与染毒组幼鱼的心率降低以及心包水肿等畸形发生有关。孵化是胚胎发育的一个重要步骤,但在本研究未发现斑马鱼胚胎的孵化率和体长存在明显变化。以往研究发现,在高剂量TBP(25~
3125 μg·L−1)的暴露下斑马鱼的孵化率和体长均没有影响[5],与本研究一致。因此,暴露于环境相关浓度的TBP可能会通过降低幼鱼心率,增加心包水肿畸形进而对斑马鱼的胚胎发育产生不利影响。运动行为被认为是斑马鱼的重要神经发育指标[14]。在斑马鱼中,肌源性自发运动(即卷尾行为)作为发育早期的第一个运动行为,只涉及脊髓神经元的神经支配,独立于大脑神经支配[15–16]。本实验中,各染毒组斑马鱼卷尾频率均有降低。对其他阻燃剂(如得克隆、纳米二氧化钛与TPP联合暴露)的研究也报告了类似的结果[8,17]。游泳行为是在发育后期发生的一种更为复杂的运动行为,其依赖于后脑的输入和化学信号的传递来实现[14]。光暗周期刺激实验是最常用来测试焦虑行为的实验模型[18]。毒性研究发现,斑马鱼幼鱼暴露于三(1, 3-二氯-2-丙基)磷酸酯[tris(1, 3-dichloro-2-propyl)phosphate, TDCIPP]会影响黑暗和光亮环境下的游泳速度,暴露于磷酸三(2-氯乙基)酯[tris(2-chloroethyl)phosphate, TCEP]会导致幼鱼在光亮环境下的游泳速度下降[19]。本实验与以往研究一致,染毒组的斑马鱼在光暗周期游泳速度均有的下降,提示低剂量的TBP也可以对神经系统产生毒性作用。
斑马鱼的早期发育异常可能与甲状腺激素分泌有关[16]。由于甲状腺激素的作用,早期阶段的鱼类幼鱼比成鱼对毒性化学物质更敏感[20]。在本研究中,染毒组的T4浓度未发现明显变化,暴露于0.2 μg·L−1 TBP升高了幼鱼体内T3的水平。但该染毒组的心率、存活率及运动能力下降,可能的原因是暴露于TBP导致前期斑马鱼全身T3含量低,促甲状腺激素开始负反馈上升,导致T3含量在120 h升高[21]。之前的研究显示,暴露于TDCIPP和TPP降低雄性斑马鱼血浆中T3和T4的浓度,并改变下丘脑、脑垂体和甲状腺的受体转录[22–27],可能是暴露浓度和斑马鱼暴露时期与本研究不同,导致对甲状腺激素的影响也不同。
HPT轴在调节甲状腺激素的合成、运输和代谢中起着重要作用[28–29],本研究检测了HPT轴的基因表达情况。trα和trβ可以通过与HPT轴上的特定DNA序列结合,调节与斑马鱼幼鱼早期发育相关的靶基因的转录[30–31]。实验结果表明,斑马鱼胚胎暴露于不同浓度的TBP后,trα和trβ基因的表达水平降低可能影响甲状腺激素与之结合,使下游级联反应激活失败,较少的甲状腺激素能发挥作用进而抑制斑马鱼的心率和存活率[32]。tg和nis用于合成甲状腺素[33];ttr在甲状腺素转运及代谢中起重要作用[16]。研究表明,环境污染物可以通过竞争性结合ttr来影响血清中THs的水平[34]。在本研究中,TBP染毒组的tg和nis的转录水平下调,但是T3含量上升。这可能与ttr转录上调有关,ttr转录上调可以导致游离的THs含量减少,使其不容易被肝脏代谢清除,从而导致T3总含量上升。碘甲状腺原氨酸脱碘酶(iodothyronine deiodinase 3, dios)负责甲状腺激素的激活和失活。在鱼类中,dio1影响碘的恢复和甲状腺激素的降解;而鱼类dio2的功能与人类相似,将外周游离T4转化为活性T3;而dio3将T4和T3分别转化为无生物活性的反三碘甲状腺原氨酸(reverse triiodothyronine, rT3)和二碘甲状腺原氨酸(diiodothyronine, T2)[35]。在本研究中,斑马鱼胚胎经TBP暴露后,低剂量组的dio2表达增加,但是该染毒组T3水平并未升高,可能原因是dio3表达上调,加快T3转化为rT3和T2[36]。
基于与有机磷农药的结构相似性,TBP对鱼类的潜在神经毒性越来越备受关注[23]。本研究已发现斑马鱼的卷尾频率和游泳速度下降,所以我们进而在分子水平上量化与斑马鱼120 hpf神经发育相关基因的表达。与对照组相比,只有ache转录水平在各染毒组均有性的下调。先前的研究已揭示,TBP能够提升鸡胚胎血液中ache浓度,然而具体分子机制尚待进一步阐明[37]。在鱼的中枢神经系统发育时,mbp作为髓鞘形成的标志性生物分子,在轴突髓鞘化的关键环节中发挥着不可或缺的作用[38]。神经系统中elavl3在神经板区域的神经前体细胞内呈现出高度表达的特性,这一发现进一步强调了其在神经系统早期发育中的潜在重要性[39]。在本实验中,0.02 μg·L−1的染毒组mbp和elavl3表达下调,提示TBP可能对轴突髓鞘造成损伤;0.2、2 μg·L−1染毒组与对照组相比差异无统计学意义,这可能是机体出现代偿行为,导致mbp和elavl3表达出现上升。本研究中仍有一些不足:首先,虽然斑马鱼基因与人类基因高度相似,但也存在差异,可采用斑马鱼模型结合其他动物模型深入地探讨差异基因的具体作用及其潜在影响。其次,本研究只观察了斑马鱼早期的急性发育毒性,今后还需研究长期在环境暴露剂量下对于成鱼的毒性。
综上所述,TBP暴露可导致斑马鱼早期发育异常,可能是通过影响斑马鱼的HPT轴和神经发育相关基因的表达,下调trα、trβ、tg、nis、dio2、ache、mbp和elavl3的表达,上调ttr的表达;干扰了机体内T3的含量,导致斑马鱼心率、生存率的降低和畸形率的上升;卷尾频率下降,在光暗周期游泳速度均下降。本研究基于环境暴露剂量水平研究TBP毒性,结果为进一步深入研究TBP对鱼类生长发育的影响提供支持,也为TBP的生态毒性效应及风险性评价积累资料。
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图 5 TBP暴露对斑马鱼HPT轴及神经发育相关基因表达的影响($\bar x \pm s$,n=3)
A~B:HPT轴相关基因、神经发育相关基因。*:与对照组比较,P<0.05。
Figure 5. The effects of TBP exposure on the expression of genes related to zebrafish HPT axis and neurodevelopment ($\bar x \pm s$, n=3)
A-B: HPT axis-related genes and neurodevelopment-related genes. *: Compared with the control group, P<0.05.
表 1 斑马鱼荧光定量PCR引物序列
Table 1 The sequences of zebrafish qPCR primers
目的基因
(Target gene)引物序列
(Primer sequence)β肌动蛋白($\beta $-actin) F: 5′-CAGTGCCCATCTACGAGGGTTAT-3′
R: 5′-CGGCTGTGGTGGTGAAGGAGT-3′甲状腺激素受体α(Thyroid hormone receptor α, trα) F: 5′-CGAGAAGTGTCAGGAGAT-3′
R: 5′-GTTCGTCACCTTCATCAG-3′甲状腺激素受体β(Thyroid hormone receptor β, tr$\beta $) F: 5′-ACTTGGACGATTCAGAGG-3′
R: 5′-CCTTGTGCTTACGGTAGT-3′甲状腺球蛋白(Thyroglobulin, tg) F: 5′-GTGAAGAGGATGGTGAGT-3′
R: 5′-GATGGCTGGTTGAATGAC-3′钠碘共转运体(Sodium-iodide transporter, nis) F: 5′-GGTGGCATGAAGGCTGTAAT-3′
R: 5′-GATACGGCATCCATTGTTGG-3′甲状腺转运蛋白(Transthyretin, ttr) F: 5′-CTCCTGGTGTGTATCGGGTG-3′
R: 5′-AGGATGTCAGTCATGTGCCTT-3′尿苷二磷酸葡萄糖醛酸转移酶(Udp-glucuronosyltransferase family 1 member A1, ugt1ab) F: 5′-CCACCAAGTCTTTCCGTGTT-3′
R: 5′-GCAGTCCTTCACAGGCTTTC-3′碘甲状腺原氨酸脱碘酶1(Iodothyronine deiodinase 1, dio1) F: 5′-CTGGACCGACAGAAGACGAG-3′
R: 5′-TGCGACATTGCTGAAGTCCT-3′碘甲状腺原氨酸脱碘酶2(Iodothyronine deiodinase 2, dio2) F: 5′-CTCGGACACTTGGCTTGACT-3′
R: 5′-TTGGATCAGGACGGAGAGGT-3′乙酰胆碱酯酶(Acetylcholinesterase, ache) F: 5′-CCCTCCAGTGGGTACAAGAA-3′
R: 5′-GGGCCTCATCAAAGGTAACA-3′髓鞘碱性蛋白(Myelin basic protein, mbp) F: 5′-AATCAGCAGGTTCTTCGGAGGAGA-3′
R: 5′-AAGAAATGCACGACAGGGTTGACG-3′突触素Ⅱa(Synapsin Ⅱa, syn2a) F: 5′-GTGACCATGCCAGCATTTC-3′
R: 5′-TGGTTCTCCACTTTCACCTT-3′生长相关蛋白43(Growth associated protein 43, gap43) F: 5′-TGCTGCATCAGAAGAACTAA-3′
R: 5′-CCTCCGGTTTGATTCCATC-3′ELAV样神经元特异性RNA结合蛋白3
(ELAV like neuron-specific RNA binding protein 3, elavl3)F: 5′-AGACAAGATCACAGGCCAGAGCTT-3′
R: 5′-TGGTCTGCAGTTTGAGACCGTTGA-3′胶质纤维酸性蛋白(Glial fibrillary acidic protein, gfap) F: 5′-GGATGCAGCCAATCGTAAT-3′
R: 5′-TTCCAGGTCACAGGTCAG-3′音猬因子a(Sonic hedgehog signaling molecule a, shha) F: 5′-GCAAGATAACGCGCAATTCGGAGA-3′
R: 5′-TGCATCTCTGTGTCATGAGCCTGT-3′ -
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