环境激素对鱼类生殖毒性的研究进展

文章综述了水环境激素的种类及其对鱼类生殖毒性的常用的研究方法,并以三丁基锡(TBT)和三苯基锡(TPT)等有机锡污染物为例,详述了环境激素对鱼类生殖毒性的研究进展。研究表明,有机锡化合物会导致鱼类性腺结构和功能受损,生殖行为异常,引起雌鱼雄性化,并产生隔代毒性效应。其毒性机理主要是抑制雌激素合成途径关键酶,干扰雌激素核受体基因表达,此外还可能涉及脂毒性、氧化应激反应等其他复杂机制。建议在今后的研究中,加强学科融合,发展新的研究技术手段,开展复杂条件下的生殖毒理研究,结合地区特点和养殖模式开展养殖鱼类生殖毒理研究,不断开展各种新型化合物环境行为和毒理效应研究。     

双酚A和酞酸酯对鱼类的内分泌干扰效应和繁殖毒性的研究进展

环境雌激素作为一类内分泌干扰物进入人和动物体内后能干扰机体内分泌系统的正常功能,通过多种机制表现出拟天然雌激素或者抗天然雄激素的作用。双酚A（bisphenol A, BPA）和酞酸酯（也称作邻苯二甲酸酯,Phthalic Acid Esters, PAEs）是具有雌激素类活性的环境内分泌干扰物,广泛存在于水环境中,并对水生动物,特别是鱼类的安全造成潜在威胁。本文总结了目前BPA和PAEs对鱼类毒性效应的研究成果,特别是内分泌干扰作用和生殖毒性,为评估这两类环境污染物对鱼类的影响提供参考。     

Plasma vitellogenin levels in male common carp Cyprinus carpio and crucian carp Carassius cuvieri of Lake Kasumigaura

In order to investigate the possible influence of estrogenic environmental endocrine disruptors on the reproductive activity of fish in Lake Kasumigaura, plasma levels of vitellogenin (VTG), a biomarker of estrogen exposure, were measured in wild and cultured male common carp Cyprinus carpio and wild crucian carp Carassius cuvieri . Testicular histology and plasma steroid hormone levels were also examined. Fish were collected from June 1998 to August 1999. Plasma VTG levels in most fish examined were below a detection limit (40 ng/mL) throughout the sampling period, and a small amount of VTG (43.5 ng/mL–1680 ng/mL) compared to that in females was detected in some fish. Active spermatogenesis in the testis and increased levels of plasma sex steroids were observed in most of the fish examined. Thus, no marked influence of estrogenic chemicals was detected in the reproductive activity of male common carp and crucian carp in Lake Kasumigaura collected from the sampling sites in the present investigation.     

Identification and expression analysis of two growth hormone receptors in zanzibar tilapia (Oreochromis hornorum)

Growth hormone plays important roles in various physiological processes such as growth, metabolism, and reproduction. In this study, two cDNAs encoding growth hormone receptor (GHR) were isolated from the liver of zanzibar tilapia (Oreochromis hornornum). The two cDNAs were 2,831 and 2,044 bp in length and named GHR1 and GHR2, respectively. GHR1 and GHR2 shared 57.4% similarity in nucleotide sequences and 33.5% similarity in deduced amino acid sequences. Consequently, it was presumed that they were two different genes. Conserved regions of GHR1 and GHR2 in zanzibar tilapia were different from those of other vertebrates. For example, conserved box2 regions of GHR1 and GHR2 in zanzibar tilapia were, respectively, WVELM and WVEFT, while it was WVEFI for GHRs in other vertebrates. Similar to other fish species, GHR1 and GHR2 were expressed in brain, gill, liver, muscle, spleen, gonad, stomach, kidney, and pituitary in zanzibar tilapia. The expression levels were the highest in liver. Unlike fathead minnow (Pimephales promelas) and mossambique tilapia (O. mossambicus), the expression levels of GHR1 in most female fish tissues were higher than those in male fish. No significant difference in GHR2 expression was found in all the tissues in male and female of zanzibar tilapia. Under fasting condition, the expressions of GHRs and IGF-II were significantly up-regulated (P < 0.05) in liver, while the expression of IGF-I remained stable. This observation would contribute to understanding the evolution of the GHR family in further investigation of growth regulation of zanzibar tilapia.     

河川沙塘鳢GHR和IGF-2基因克隆及mRNA的时序表达

生长激素/胰岛素样生长因子-轴(growth hormone/insulin-like growth-axis,GH/IGF-轴)是调控鱼类生长的主要内分泌轴线。为探讨生长相关基因GHR、IGF-2对河川沙塘鳢(Odontobutis potamophila)生长发育的调控机制,采用cDNA末端快速扩增(RACE)和实时荧光定量PCR等技术,对河川沙塘鳢的GHR、IGF-2基因进行了克隆和表达模式分析。研究结果显示:河川沙塘鳢GHR基因的cDNA全长序列为2 179 bp,开放阅读框为1 830 bp,共编码609个氨基酸,IGF-2基因的cDNA全长序列为1 586 bp,开放阅读框642 bp,共编码213个氨基酸。采用qRT-PCR方法检测了GHR、IGF-2基因在9个不同组织(脑、肝、心、肌肉、脾、肠、性腺、鳃、肾)和8个胚胎不同发育时期(受精卵期、桑椹胚期、原肠胚期、神经胚期、体节期、口裂期、出膜后1 d、出膜后3 d)的表达情况。结果表明:IGF-2和GHR基因在所检测的9种组织中均有表达,其中以肝脏、肌肉、性腺、脑中的表达量较高,在胚胎发育阶段GHR和IGF-2基因表达呈先上升后下降的趋势,在原肠胚期表达量较高,表明其在胚胎发育过程中发挥重要作用。qRT-PCR进一步比较了雌、雄河川沙塘鳢GHR和IGF-2基因在不同生长发育阶段(90、150、210、270、330 d)脑、肝、肌肉mRNA的表达量,结果表明:各时期雄鱼GHR和IGF-2基因在肝组织中表达量均显著高于雌鱼,肌肉和脑GHR基因mRNA的表达量则无显著差异。而IGF-2基因则仅在雌、雄鱼210 d的脑和150、210 d的肌肉存在显著性差异,推测肝GHR和IGF-2基因mRNA表达的雌雄差异是河川沙塘鳢雌雄生长差异的主要原因之一。     

Cichlasoma dimerus responds to refeeding with a partial compensatory growth associated with an increment of the feed conversion efficiency and a rapid recovery of GH/IGFs axis

Many fish species display compensatory growth (CG), a phenomenon by which fasted fish grow faster during refeeding. However, most studies use a group‐housed fish approach that could be problematic in social fish when interaction between individuals is not considered or eliminated. Additionally, the growth hormone (GH)/insulin‐like growth factors’ (IGF‐1 and IGF‐2) axis is implicated in postnatal growth in vertebrates, but its relevance in CG is not fully understood. Thus, the aim of this work was to determine whether CG occurs in a social fish, Cichlasoma dimerus, using an individually held fish approach and secondly, to evaluate the GH/IGFs expression profile during refeeding by 3 days and 3 weeks. C. dimerus showed partial CG. The feed conversion efficiency (FCE) was higher in three‐day‐refed fish, which presented higher GH plasma and mRNA levels than controls but shown no differences in liver and muscle GH receptors (GHR1 and GHR2) and IGFs mRNA levels. Surprisingly, three‐week‐refed fish exhibited GHR1 and IGF‐2 increments, but a reduction in GHR2 expression in muscle. These results show a strong association between GH levels, growth rate and FCE during refeeding, and a long‐lasting effect of refeeding on muscular expression of GHRs and IGF‐2.     

Modulation of monoamine neurotransmitters in fighting fish <Emphasis Type="Italic">Betta splendens</Emphasis> exposed to waterborne phytoestrogens

Endogenous estrogens are known to affect the activity of monoamine neurotransmitters in vertebrate animals, but the effects of exogenous estrogens on neurotransmitters are relatively poorly understood. We exposed sexually mature male fighting fish Betta splendens to environmentally relevant and pharmacological doses of three phytoestrogens that are potential endocrine disruptors in wild fish populations: genistein, equol, and β-sitosterol. We also exposed fish to two doses of the endogenous estrogen 17β-estradiol, which we selected as a positive control because phytoestrogens are putative estrogen mimics. Our results were variable, but the effects were generally modest. Genistein increased dopamine levels in the forebrains of B. splendens at both environmentally relevant and pharmacological doses. The environmentally relevant dose of equol increased dopamine levels in B. splendens forebrains, and the pharmacological dose decreased norepinephrine (forebrain), dopamine (hindbrain), and serotonin (forebrain) levels. The environmentally relevant dose of β-sitosterol decreased norepinephrine and dopamine in the forebrain and hindbrain, respectively. Our results suggest that sources of environmental phytoestrogens, such as runoff or effluent from agricultural fields, wood pulp mills, and sewage treatment plants, have the potential to modulate neurotransmitter activity in free-living fishes in a way that could interfere with normal behavioral processes.     

Towards strategic use of fungicides against Saprolegnia parasitica in salmonid fish hatcheries

Abstract. The life-cycle of Saprolegnia parasitica was considered in relation to the use of protective fungicides against it in salmonid fish hatcheries. Malachite green oxalate was the example fungicide, and all experiments were at 10°C. If the fungicide is to be used as a continuous addition to the water, as a protectant, then target stages of the fungus are identified as zoospores and secondary zoospore cysts: the latter, although physically sedimentary, have a remarkably high degree of buoyancy in the water and hence pose a constant threat to the fish. Continuous addition to the water, to give 0·25 mgl⁻¹ malachite green oxalate, would be expected to kill zoospores and zoospore cysts, but 0·05 mgl⁻¹, while allowing zoospore cyst germination, was a strong inhibitor to fungal growth and, aided by natural defences of the fish, might well succeed in disease prevention. If the fungicide is to be used as an intermittent addition to the water (every 24 h), then target stages of the fungus are small mycelia on the fish in addition to zoospores and zoospore cysts in the water. An experimental model was made to consider the vulnerability of these small mycelia, growing on the fish over the preceding 24 h, to malachite green oxalate. If mycelia are of narrow diameter, as expected, then 0·25 mgl⁻¹ malachite green oxalate, applied for 15 min, should control their growth and protect the fish.     

银鲳ghr和igfbp1基因表达特征、组织分布及操作胁迫对其的影响（英文）

为探讨急性操作胁迫对银鲳生长的影响,首次克隆了3种生长调控相关基因-生长激素受体1(ghr1)、生长激素受体2(ghr2)和胰岛素样受体1(igfbp1),并对3种基因在银鲳肝脏、心脏、性腺、肌肉和肾脏等不同组织中的表达进行了比较。定量表达结果表明3种基因均广泛分布于所研究的各个组织中,而其中肾脏、性腺和心脏中的表达量较其它组织低。在银鲳受到外界操作胁迫刺激后,3种基因呈现不同的表达模式:其中ghr2和igfbp1的表达下调,而ghr2的表达量与对照组无显著性差异。结果表明,ghr2和igfbp1在银鲳生长调节中起到重要的作用;在胁迫环境中,外界刺激可通过下调生长相关基因表达以影响鱼体的生长。     

Toxicity of four commonly used chemotherapeutic compounds to fry of the African catfish, Clarias gariepinus (Burchell)

Acute toxicity tests were carried out for four compounds commonly used against various fish pathogens to determine their 24-h LC₅₀ values to fry of the African catfish, Clarias gariepinus (Burchell). Compounds tested and their calculated 24-h LC₅₀ values (mg l^?1) were as follows: praziquantel (13.4); malachite green oxalate (0.14); acriflavin neutral (10.0); and mebendazole (315). The results show that dosage levels of malachite green oxalate, praziquantel and acriflavin neutral recommended in the literature for the control of most fish ectoparasitoses caused substantial mortalities and cannot be applied to Clarias gariepinus fry.     

不同冷冻贮藏时间对三种鱼类肌肉中孔雀石绿残留量的影响

研究鲤鱼、鳗鲡、鳜鱼在加入不同浓度的孔雀石绿标准样品且在不同冷冻贮藏时间下对孔雀石绿残留量的影响。三种基质中,鳗鲡组织中添加有无色孔雀石绿降解速率最慢、鳜鱼次之、鲤鱼较快。三种基质中添加10μg/kg、50μg/kg、100μg/kg有色孔雀石绿平均降解率分别为57.9%、48.0%、23.0%;无色孔雀石绿平均降解率分别为46.6%、43.4%、19.8%,有色孔雀石绿较无色孔雀石绿降解快,低浓度较高浓度降解快。在三种基质鲤鱼、鳗鲡、鳜鱼组织中有色和无色孔雀石绿降解反应均符合准一级反应动力学。分析可能由于鳗鲡组织脂肪较多,其结合方式影响了有色无色孔雀石绿的降解。     

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.     

Effects of cysteamine on mRNA levels of growth hormone and its receptors and growth in orange-spotted grouper (Epinephelus coioides)

Effects of cysteamine (CS) on growth hormone (GH) mRNA, two types of growth hormone receptor (GHR) mRNAs and growth rate in orange-spotted grouper (Epinephelus coioides) were investigated. CS could cause a modification in the structure of somatostatin, which is the most important neuroendocrine inhibitor of basal and stimulated growth hormone synthesis and release, and renders it nonimmunoreactive probably through interaction with the disulfide bonds. In the present study, cysteamine hydrochloride (CSH) enhanced the level of pituitary GH mRNA in a dose-dependent manner through attenuating or deleting the inhibiting action of somatostatin on GH mRNA expression. CSH at relatively low doses (from 1 to 3 mg/g diet) enhanced the levels of two types of GHR mRNAs in dose-dependent manner, whereas the stimulation induced by CSH declined from the peak at higher dose of CSH (4 mg/g diet). It might be attributed to the variation in GH-induced up-regulation of GHRs at different doses of GH. Feeding of CSH could induce remarkable enhancement of growth rate in orange-spotted grouper. In addition, the stimulatory effect of CSH could be potentiated by the additive effect of luteinizing hormone-releasing hormone analog (LHRH-A). Compared with individual treatments, combined feeding of CSH and LHRH-A caused more efficient elevation of growth rate after 8 weeks of feeding. CSH and LHRH-A individually and in combination remarkably increased the levels of GH and GHR mRNAs compared with the control. The combined administration of CSH and LHRH-A in diet was most effective to enhance the level of GH and GHR1 mRNA. The morphological characteristics of the experimental fish were evaluated. Compared with control, the ratios of muscle RNA/DNA, condition factors (CF) and feed conversion efficiency (FCE) were significantly enhanced in the treated groups, while the highest values were observed in the combined treatment. All the results suggested that CSH (1–3 mg/g diet) is an effective, economical and feasible feed additive in orange-spotted grouper culture.     

Considerations on the Use of Malachite Green in Aquaculture and Analytical Aspects of Determining the Residues in Fish: A Review

Aquaculture in Brazil has shown expressive development since the 1990s with growth rates superior to those of cattle and poultry. In order to achieve greater productivity, intensive fish cultivation systems are employed, which can cause greater susceptibility to diseases caused by viruses, bacteria, fungi, and parasites. The reduced availability of veterinary medications approved for use in aquaculture in Brazil has lead fish farmers to the indiscriminate use of several chemical substances with antimicrobial activity, such as the dye malachite green (MG). As a result of this use, residues of MG and its main biotransformation product, leucomalachite green (LMG), may be present in fish available for consumption. The presence of residues of these compounds represents a risk to human health due to their toxicity, as well as a potential impact on the environment, and could also raise barriers for commercialization in the country and for exportation. The objective of this review is to provide the context and evidence of the use of MG in aquaculture and of its toxicological and legislative aspects. A review of the analytical methods used to determine MG residues in fish, with emphasis on mass spectrometry, is also presented.     

Influence of circulating GH levels on GH-binding capacity measurements in the hepatic membrane of rainbow trout (Oncorhynchus mykiss): importance of normalization of results

In fish, regulation of the growth hormone (GH) receptor has been mainly analyzed by binding studies, with some discrepancies in the results. The present work aims at determining whether circulating GH levels influence the measurement of hepatic GH-binding capacities. To do this, the effectiveness of the dissociation of the GH/GH receptor (GHR) complex by an MgCl₂ treatment was assessed in rainbow trout, and data on GH-binding capacities under various physiological conditions were studied using different means of expression. Our results reveal that MgCl₂ treatment dissociated the liver GH/GHR complex formed under in vitro conditions (85 ± 23 vs. 361 ± 16 fmol/g of liver; p<0.001) but not in vivo, showing such treatment in trout is not applicable. A comparison of fasted (3 weeks) and fed fish revealed that GH-binding capacities, expressed as femtomoles per milligram protein or femtomoles per gram of liver, were similar in both fed and fasted fish. However, when changes in liver and body size were taken into account, the total GH-binding capacities were lower in the fasted fish (0.026 ± 0.006 vs. 0.062 ± 0.009 fmol/cm³ liver; p<0.05). One day after hypophysectomy or GH injection, changes in the plasma GH levels increased or decreased GH-binding capacities, respectively. Five days later, GH-binding capacities increased in GH-injected fish (527 ± 38 vs. 399 ± 38 fmol/g liver; p<0.01). Our interpretation is that acute treatment modified GH-binding capacities through receptor occupancy and that GH stimulated the synthesis of its own receptor. On the other hand, long-term treatment through successive injections of GH lowered the total binding capacities (approx. 40%), which could result from receptor occupancy. We conclude that circulating GH levels strongly influence the measurement of GH-binding capacities in the liver, thereby limiting interpretation of the binding data and preventing accurate conclusions to be drawn on GHR regulation.     

中国大鲵生长激素受体的克隆与表达分析

利用中国大鲵(Andrias davidianus)性腺转录组测序获得的生长激素受体(GHR)基因部分序列,克隆获得基因全长2992 bp,开放阅读框1812 bp,编码604个氨基酸,该蛋白具有高度保守的FGEFS基序与BOX框。系统进化分析结果显示,中国大鲵GHR氨基酸序列与两栖类非洲爪蟾(Xenopus laevis)同源性最高,蜥形纲锦龟(Chrysemys picta bellii)次之,哺乳类水牛(Bubalus bubalis)和鱼类半滑舌鳎(Cynoglossus semilaevis))最低。实时定量PCR结果表明,GHR基因在肝中表达最高,肌肉、垂体、肾、性腺中的表达量次之,其他各组织表达量较低。在精巢发育早期GHR表达较高,随后表达量逐渐降低,在卵巢中表达量随时间增长而增加;17α-甲基睾丸酮(MT)与芳香化酶抑制剂来曲唑(LE)短暂处理后GHR基因在脑与卵巢中表达量发生变化,MT处理后,脑与卵巢中GHR表达量增加,LE处理后脑与卵巢中表达量降低。研究表明,GHR基因在大鲵性腺发育中可能发挥作用,且MT与LE可能通过不同的途径调节GHR基因的表达。     

Gene expression levels of growth hormone receptor and insulin-like growth factor-I in gonads of maturing coho salmon (Oncorhynchus kisutch)

The expression levels of growth hormone receptor (GHR) and insulin-like growth factor (IGF-I) in gonads of maturing coho salmon were measured by real-time PCR. The GHR and IGF-I mRNAs showed different gene expression pattern between male and female. These results suggest that a sexual dimorphism may exist in the GHR and IGF-I mRNA expressions of coho salmon during gonadal maturation