Re-evaluation of thyroid hormone signaling antagonism of tetrabromobisphenol A for validating the T3-induced Xenopus metamorphosis assay

We developed the T3-induced Xenopus metamorphosis assay, which is supposed to be able to sensitively detect thyroid hormone(TH) signaling disruption of chemicals. The present study aimed to validate the T3-induced Xenopus metamorphosis assay by re-evaluating the TH signaling antagonism of tetrabromobisphenol A(TBBPA), a known TH signaling disruptor. According to the assay we developed, Xenopus tadpoles at stage 52 were exposed to 10–500 nmol/L TBBPA in the presence of 1 nmol/L T3. After 96 hr of exposure, TBBPA in the range of 10–500 nmol/L was found to significantly inhibit T3-induced morphological changes of Xenopus tadpoles in a concentration-dependent manner in term of body weight and four morphological endpoints including head area(HA), mouth width(MW), unilateral brain width/brain length(ULBW/BL), and hind-limb length/snout-vent length(HLL/SVL).The results show that these endpoints we developed are sensitive for characterizing the antagonistic effects of TBBPA on T3-induced metamorphosis. Following a 24-hr exposure,we found that TBBPA antagonized expression of T3-induced TH-response genes in the tail,which is consistent with previous findings in the intestine. We propose that the tail can be used as an alternative tissue to the intestine for examining molecular endpoints for evaluating TH signaling disruption. In conclusion, our results demonstrate that the T3-induced Xenopus metamorphosis assay we developed is an ideal in vivo assay for detecting TH signaling disruption.     

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone (TH) signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3, whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.     

Optimization of the T3-induced Xenopus metamorphosis assay for detecting thyroid hormone signaling disruption of chemicals

T3-induced Xenopus metamorphosis is an ideal model for detecting thyroid hormone(TH)signaling disruption of chemicals. To optimize the T3-induced Xenopus assay and improve its sensitivity and reproducibility, we intend to develop quantitatively morphological endpoints and choose appropriate concentrations and exposure durations for T3 induction.Xenopus laevis at stage 52 were exposed to series of concentrations of T3(0.31–2.5 nmol/L)for 6 days. By comparing morphological changes induced by T3, we propose head area,mouth width, unilateral brain width/brain length, and hindlimb length/snout-vent length as quantitative parameters for characterizing T3-induced morphological changes, with body weight as a parameter for indicating integrated changes. By analyzing time-response curves, we found that following 4-day exposure, T3-induced grossly morphological changes displayed linear concentration–response curves, with moderate morphological changes resulting from 1.25 nmol/L T3 exposure. When using grossly morphological endpoints to detect TH signaling disruption, we propose 4 days as exposure duration of T3, with concentrations close to 1.25 nmol/L as induction concentrations. However, it is appropriate to examine morphological and molecular changes of the intestine on day 2 due to their early response to T3. The quantitative endpoints and T3 induction concentrations and durations we determined would improve the sensitivity and the reproducibility of the T3-induced Xenopus metamorphosis assay.     

Thyroid disruption by technical decabromodiphenyl ether （DE-83R） at low concentrations in Xenopus laevis

Decabromodiphenyl ether (decaBDE), as a flame retardant, is widely produced and used. To study the thyroid disruption by technical decaBDE at low concentrations, Xenopus laevis tadpoles were exposed to technical decaBDE mixture DE-83R (1–1000 ng/L) in water from stage 46/47 (free swimming larvae, system of Nieuwkoop and Faber) to stage 62. DE-83R at concentration of 1000 ng/L significantly delayed the time to metamorphosis (presented by forelimb emergence, FLE). Histological examination showed that DE- 83R at all tested concentrations caused histological alterations – multilayer follicular epithelial cell and markedly increased follicle size accompanied by partial colloid depletion and increase in the peripheral colloid vacuolation, in thyroid glands. All tested concentrations of DE-83R also induced a down-regulation of thyroid receptor mRNA expression. These results demonstrated that technical decaBDE disrupted the thyroid system in X. laevis tadpoles. Analysis of polybrominated diphenyl ethers (PBDEs) (sum of 39 congeners) in X. laevis indicated that mean concentrations of total PBDEs in X. laevis exposed to 1, 10, 100, 1000 ng/L were 11.0, 128.1, 412.1, 1400.2 ng/g wet weight, respectively. Considering that PBDEs burden of X. laevis tadpoles was close to PBDEs levels in amphibians as reported in previous studies, our study has raised new concerns for thyroid disruption in amphibians of technical decaBDE at environmentally relevant concentrations.     

有机磷阻燃剂甲状腺干扰效应及其作用机制研究——以磷酸三(2-氯丙基)酯(TCPP)为例

以典型有机磷阻燃剂磷酸三（2-氯丙基）酯（TCPP）为研究对象,应用GH3细胞增殖实验检测TCPP对甲状腺激素的干扰效应;应用重组甲状腺激素受体（TR）基因酵母实验和GH3（TRβ-）细胞增殖实验结合实时定量PCR技术初步探究TCPP甲状腺激素干扰作用机理.结果表明,TCPP在1×10^-4mol/L和2×10^-4mol/L浓度下对甲状腺激素T3诱导的GH3细胞增殖产生抑制效应;重组TR基因酵母实验和GH3（TRβ-）细胞增殖实验测试结果表明TCPP可能通过TR介导的基因组途径和非基因组途径诱导甲状腺激素干扰效应;实时定量PCR测试结果表明TCPP下调相关基因如：c-fos、TRβ、integrin-av和k-ras的mRNA表达,初步认为TCPP可能通过影响TRβ基因表达和激活α_vβ₃-ERK-1/2信号通路产生甲状腺激素干扰效应.     

改进型重组基因酵母TR-GRIP1检测化合物甲状腺激素干扰活性

应用酵母双杂交技术构建重组TR(甲状腺激素受体)基因酵母,用以检测类/抗甲状腺激素化合物及环境样品的甲状腺激素干扰活性. 提取并纯化含有TR基因的酵母表达质粒pGBT9-TR以及含有TR共激活因子GRIP1基因的酵母表达质粒pGAD424-GRIP1,将pGBT9-TR和pGAD424-GRIP1同时转化至酵母细胞Y187,以营养缺陷型培养基(SD/-Trp/-Leu)筛选阳性菌落,构建TR-GRIP1双杂交酵母. 考察该酵母与天然甲状腺激素——T₃(三碘甲状腺原氨酸)的结合情况,确定最佳暴露时间,建立剂量－效应关系曲线. 结果表明:TR-GRIP1双杂交酵母能够与T₃结合诱导β-半乳糖苷酶活性,选择暴露时间为2 h,T₃诱导酶活性的EC₅₀值为1.1×10-7 mol/L;构建的重组基因酵母TR-GRIP1提供了检测化合物甲状腺激素干扰活性的新方法.      

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.     

Dechloranes exhibit binding potency and activity to thyroid hormone receptors

Dechloranes are a group of halogenated flame retardants with a basic bicyclo[2.2.1]heptene, including Dechlorane Plus(DP), Dechlorane 602(Dec 602), Dechlorane 603(Dec 603) and Dechlorane 604(Dec 604). A few epidemiological investigations and animal experiments have shown that DP exhibited thyroid-interfering effects. In the present study, we investigated whether DP and three other dechloranes could interfere the thyroid function through thyroid hormone receptors(TRs, TR α and TR β) signaling pat...     

镉对黑斑蛙精巢细胞色素P450酶及谷胱甘肽转移酶活性的影响

细胞色素P450酶(CYP450)和谷胱甘肽转移酶(GST)是动物体内主要的解毒酶,在外源毒物的转化和代谢过程中具有重要作用.本文在实验条件下, 将健康性成熟黑斑蛙(Rana nigromaculata)暴露于浓度分别为0.005、0.01、0.05、0.1、0.5和1.0 mg·L^-1的Cd溶液中30 d, 采用荧光分光光度法和紫外分光光度法测定精巢组织乙氧基异酚恶唑脱乙基酶(EROD)、红霉素N-脱甲基酶 (ERND)、氨基比林-N-脱甲基醇酶(APND)和谷胱甘肽转移酶(GST)活性,探讨Cd在精巢的可能代谢过程和毒性机理.结果表明, 与对照组比较, EROD和GST酶活性在0.5和1.0 mg·L^-1 Cd处理组被显著抑制; ERND酶活性在0.01~1.0 mg·L^-1 Cd处理组均被显著抑制;而APND酶活性在各处理组响应变化不明显.结果显示, 精巢EROD、ERND和GST对Cd胁迫的敏感性高于APND, 黑斑蛙精巢中EROD、ERND和GST酶活性的响应可用来评价环境中低水平Cd的污染效应.     

低剂量全氟辛酸对雄性黑斑蛙生殖毒效应及机理研究

针对新型环境有机污染物全氟辛酸(PFOA)对两栖动物的毒效应问题,以黑斑蛙(Rana nigromaculata)为实验对象,研究了低剂量PFOA暴露对雄性黑斑蛙生殖毒效应及机理.结果表明,在0.001～1mg.L-1低剂量体外暴露20d条件下,PFOA能够显著诱导黑斑蛙精子发生畸形,相对于对照组,精子畸形率呈现明显的剂量-效应关系.酶联免疫试剂盒检测发现,除0.001mg.L-1诱导组外,0.01、0.1和1mg.L-1暴露组的血清雄性激素睾酮和雌性激素雌二醇含量分别显著降低(p<0.01,F=288.7)和上升(p<0.01,F=289.0),说明PFOA作用下黑斑蛙体内性激素水平受到改变.采用荧光定量PCR技术进行进一步的分子生物学检测,发现与性激素水平密切相关的两个基因P450芳香化酶和类固醇激素合成因子(SF-1)均增强表达(0.001mg.L-1诱导组,p<0.05;0.01、0.1和1mg.L-1暴露组,p<0.01,F=140.8).全氟辛酸对两栖类动物的雄性生殖毒效应机理主要是通过介导与性激素分泌相关的两个重要基因P450芳香化酶和类固醇激素合成因子,使其表达上调,从而一方面抑制睾酮的产生,另一方面促进性激素雌二醇的分泌.在0.001mg.L-1的体外暴露剂量下,PFOA就能够明显诱导黑斑蛙精子产生畸形毒性及增强P450芳香化酶和SF-1基因的表达(p<0.05),因此,为保护两栖动物种群,在制订PFOA地表水环境质量标准时应当考虑其下限不能低于0.001mg.L-1.     

Species-dependent effects of the phenolic herbicide ioxynil with potential thyroid hormone disrupting activity: modulation of its cellular uptake and activity by interaction with serum thyroid hormone-binding proteins

Ioxynil, a phenolic herbicide, is known to exert thyroid hormone (TH) disrupting activity by interfering with TH-binding to plasma proteins and a step of the cellular TH-signaling pathway in restricted animal species. However, comparative studies are still lacking on the TH disruption.We investigated the interaction of [¹²⁵I]ioxynil with serum proteins from rainbow trout, bullfrog, chicken, pig, rat, and mouse, using native polyacrylamide gel electrophoresis. Candidate ioxynil-binding proteins, which included lipoproteins, albumin and transthyretin (TTR), differed among the vertebrates tested. Rainbow trout and bullfrog tadpole serum had the lowest binding activity for ioxynil, whereas the eutherian serum had the highest binding activity. The cellular uptake of, and response to, ioxynil were suppressed by rat serum greater than by tadpole serum. The cellular uptake of [¹²⁵I]ioxynil competed strongly with phenols with a single ring, but not with THs. Our results suggested that ioxynil interferes with TH homeostasis in plasma and with a step of cellular TH-signaling pathway other than TH-uptake system, in a species-specific manner.     

Direct calorimetry of free-moving eels with manipulated thyroid status

In birds and mammals, the thyroid gland secretes the iodothyronine hormones of which tetraiodothyronine (T4) is less active than triiodothyronine (T3). The action of T3 and T4 is calorigenic and is involved in the control of metabolic rate. Across all vertebrates, thyroid hormones also play a major role in differentiation, development and growth. Although the fish thyroidal system has been researched extensively, its role in thermogenesis is unclear. In this study, we measured overall heat production to an accuracy of 0.1 mW by direct calorimetry in a free-moving European eel (Anguilla anguilla L.) with different thyroid status. Hyperthyroidism was induced by injection of T3 and T4, and hypothyroidism was induced with phenylthiourea. The results show for the first time at the organismal level, using direct calorimetry, that neither overall heat production nor overall oxygen consumption in eels is affected by hyperthyroidism. Therefore, we conclude that the thermogenic metabolism-stimulating effect of thyroid hormones (TH) is not present with a cold-blooded fish species like the European eel. This supports the concept that TH does not stimulate thermogenesis in poikilothermic species.     

六溴环十二烷转甲状腺素蛋白结合活性及其发育期暴露的甲状腺激素干扰效应研究

为了探讨新型溴代阻燃剂六溴环十二烷(HBCDs)的潜在健康危害,采用体外研究与动物暴露实验相结合的方法,分析了HBCDs与转甲状腺素蛋白(TTR)的结合活性,以及不同剂量HBCDs暴露对发育期大鼠体内甲状腺激素水平的干扰作用.TTR竞争结合实验显示,HBCDs与125 I-T4竞争结合TTR的能力随溶液浓度的增加而升高,但即使在80μmol.L-1的高浓度下,125I-T4与TTR的结合率仍高达75.08%,表明HBCDs抑制甲状腺激素T4与转运蛋白结合的能力较弱.动物实验结果表明,新生3 d大鼠暴露于0.2 mg/kg及1 mg/kg剂量的HBCDs 21 d后,与对照组相比,暴露组大鼠血清中总三碘甲状腺原氨酸TT3、游离三碘甲状腺原氨酸FT3的含量显著升高(p0.05、p0.05);总甲状腺激素TT4、游离甲状腺激素FT4含量下降约20%,促甲状腺激素水平上升30%～230%,但3个指标变化均不具统计学意义.结合体内实验及动物实验结果,HBCDs可能通过对甲状腺激素T3的协同或替代作用产生直接和间接的甲状腺干扰效应.低剂量的HBCDs暴露即可导致发育期大鼠甲状腺激素内稳态失衡,对HBCDs发育期暴露的毒性作用值得关注.     

Physiological cellular responses and adaptations of Rhodococcus erythropolis IBBPo1 to toxic organic solvents

A new Gram-positive bacterium, Rhodococcus erythropolis IBBPo1（KF059972.1） was isolated from a crude oil-contaminated soil sample by enrichment culture method. R. erythropolis IBBPo1 was able to tolerate a wide range of toxic compounds, such as antibiotics（800–1000 μg/mL）,synthetic surfactants（50–200 μg/mL）, and organic solvents（40%–100%）. R. erythropolis IBBPo1 showed good tolerance to both alkanes（cyclohexane, n-hexane, n-decane） and aromatics（toluene, styrene, ethylbenzene） with logPOW（logarithm of the partition coefficient of the solvent in octanol–water mixture） values between 2.64 and 5.98. However, alkanes were less toxic for R. erythropolis IBBPo1 cells, compared with aromatics. The high organic solvent tolerance of R. erythropolis IBBPo1 could be due to the presence in their large genome of some catabolic（alkB, alkB1, todC1, todM, xylM）, transporter（HAE1） and trehalose-6-phosphate synthase（otsA1, KF059973.1） genes. Numerous and complex physiological cellular responses and adaptations involved in organic solvent tolerance were revealed in R. erythropolis IBBPo1 cells exposed 1 and 24 hr to 1% organic solvents. R. erythropolis IBBPo1 cells adapt to 1% organic solvents by changing surface hydrophobicity, morphology and their metabolic fingerprinting.Considerable modifications in otsA1 gene sequence were also observed in cells exposed to organic solvents（except ethylbenzene）.     

铅在五指山猪组织器官中的分布与积累研究

研究了铅在五指山猪（WZSP）组织器官中的分布与累积,及饲料铅含量与组织器官中铅含量的关系.将20头五指山猪随机分为5组,分别饲喂含铅量为0.046 mg·kg-1（Ⅰ组）、6.790 mg·kg-1（Ⅱ组）、17.370 mg·kg-1（Ⅲ组）、53.600 mg·kg-1（Ⅳ组）、154.000 mg·kg-1（Ⅴ组）的饲料,持续喂养100d.屠宰后取心脏、肝脏、肾脏、肺、胃、小肠、大肠、猪耳、猪舌、猪尾、里脊肉、肋排肉、股骨骨质、股骨骨髓、肋骨和猪毛,测定其中的铅含量.结果表明, 肝脏、肾脏、股骨骨质、股骨骨髓、肋骨、猪毛等组织器官的铅含量随攻毒剂量的提高而明显上升,且与饲料中的铅含量呈正相关;当饲喂铅含量低于国家标准（5mg·kg-1）时,这些组织器官内的铅含量仍有可能超标.心脏、肺、胃、大肠、里脊肉、猪舌、猪尾中的铅含量与饲料中的铅含量没有相关性,即使饲喂铅含量超过国家标准30倍以上的饲料（铅含量154.000mg·kg-1）,这些组织器官的铅含量仍低于国家限量标准.骨组织、肝脏、肾脏对铅有着较强的富集能力,而猪肉、猪耳、猪舌、猪尾、猪心、猪肺等其它组织器官中的铅累积量低于限量标准.本研究旨在为制定猪肉制品,特别是可食用组织器官的铅限量标准提供理论依据,同时,为禽畜可食用组织器官的重金属风险评估提供参考.     

环境中的PAES对蟾蜍蝌蚪红细胞微核的影响研究

通过对邻苯二甲酸酯对蟾蜍蝌蚪红细胞微核的影响进行了研究。目的探讨邻苯二甲酸酯对蟾蜍蝌蚪的致突变作用。方法以邻苯二甲酸二甲酯为污染物对蟾蜍蝌蚪染毒24小时,并观察其细胞核变化。所用蟾蜍蝌蚪处于变态期,对污染物较为敏感。结果邻苯二甲酸酯使蟾蜍蝌蚪红细胞微核率以及核异常细胞率上升,微核率随着浓度的增大而上升,在浓度为78 ug/ml,对照组相比有极显著差异P〈0.01,浓度在18.5 ug/ml时试验组突变的细胞核的形态发生显著改变,核异常细胞率与对照组核异常细胞率有显著差异P〈0.05。此时显微镜下观察可见微核、双核、核内凹、核碎裂、无丝分裂时核分裂不均等核的形态变化。结论邻苯二甲酸酯对蟾蜍蝌蚪具有明显的致突变作用。     

Integument and defence in larva and prepupa of a sawfly living on a semi-aquatic plant

The larvae of the sawfly Rhadinoceraea micans live and feed on a semi-aquatic plant, Iris pseudacorus, and their integument is strongly hydrophobic. The hydrophobicity is part of a chemical defence strategy, easy bleeding, also known from congeners. The prepupae burrow into the soil where they form a cocoon in which they pupate, thus implying different micro-environmental conditions. The cuticle structure and wetting defensive effectiveness of R. micans were compared between larvae and prepupae. The two stages were similarly well defended against attacking ants by the bleeding of a deterrent hemolymph, whereas they were dissimilar in the cuticle surface that presented sculptures and wax crystals at the larval stage only. The integument of prepupae was less structured, and hydrophilic. Larvae of R. micans exhibit, among sawflies, an exceptional cuticle structuring and we assume that they occupy this particular niche of a semi-aquatic environment to avoid encounters with ground-dwelling predators whereas prepupae may benefit from the chemical defence acquired at larval stage.     

Intraspecific competition and high food availability are associated with insular gigantism in a lizard

Resource availability, competition, and predation commonly drive body size evolution. We assess the impact of high food availability and the consequent increased intraspecific competition, as expressed by tail injuries and cannibalism, on body size in Skyros wall lizards (Podarcis gaigeae). Lizard populations on islets surrounding Skyros (Aegean Sea) all have fewer predators and competitors than on Skyros but differ in the numbers of nesting seabirds. We predicted the following: (1) the presence of breeding seabirds (providing nutrients) will increase lizard population densities; (2) dense lizard populations will experience stronger intraspecific competition; and (3) such aggression, will be associated with larger average body size. We found a positive correlation between seabird and lizard densities. Cannibalism and tail injuries were considerably higher in dense populations. Increases in cannibalism and tail loss were associated with large body sizes. Adult cannibalism on juveniles may select for rapid growth, fuelled by high food abundance, setting thus the stage for the evolution of gigantism.