Biomarkers for endocrine disruptors in three species of Mediterranean large pelagic fish

The hypothesis that Mediterranean top predator species, such as large pelagic fish, are potentially at risk due to endocrine disrupting chemicals (EDCs), is investigated. The potential estrogenic effects of PHAHs in three fish species of commercial interest, the top predators bluefin tuna (Thunnus thynnus thynnus), swordfish (Xiphias gladius), and Mediterranean spearfish (Tetrapturus belone), were investigated using vitellogenin (Vtg), zona radiata proteins (Zrp) and mixed function oxidases (EROD, BPMO) as diagnostic tools. High induction of Vtg and Zrp was detected by western blot and ELISA techniques in adult males of X. gladius and T. thynnus thynnus, suggesting that these species are at high toxicological risk in the Mediterranean sea. Comparison of BPMO and EROD activities in the three species indicated, both in male and female, much higher MFO activity in bluefin tuna. This data suggests high exposure of this species to lipophilic xenobiotic contaminants in the Mediterranean environment.     

The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 μ(micro)M) with or without 0.2 μ(micro)g/l of the CYP1A inducer 3,3^′,4,4^′,5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

嗜水气单胞菌(Aeromonas hydrophila)引起斑马鱼肠道生理损伤和肠道菌群失调

研究分析了嗜水气单胞菌(Aeromonas hydrophila)感染斑马鱼后肠道生理健康和肠道微生物的变化。以斑马鱼为研究对象,刮伤皮下真皮,使用105 CFU/mL浓度的嗜水气单胞菌暴露6 h后转入清水,分别在暴露前、暴露后6 h、12 h、24 h取样。使用鱼特异性酶联免疫吸附试验(ELISA)试剂盒,检测肠道中紧密连接蛋白ZO-2 (TJP2)含量和乙氧基异戊二烯-O-脱乙基酶(EROD)酶活性,结果表明,嗜水气单胞菌暴露引起了斑马鱼肠道生理损伤,表现为肠道TJP2含量、EROD酶活性在暴露后显著下调。采用16S rRNA高通量测序检测肠道微生物及其菌群结构变化,嗜水气单胞菌暴露斑马鱼后致病菌不动杆菌属(Acinetobacter)明显增加,OTUs数量明显下降,Alpha多样性降低,结果说明嗜水气单胞菌引起了斑马鱼肠道菌群失调。肠道中微生物的多样性与改善肠道上皮和黏膜屏障功能紧密相关,该研究对从肠道健康的角度评价嗜水气单胞菌毒性,探索嗜水气单胞菌的致病机制具有重要意义。     

饲料蛋白水平对奥尼罗非鱼(Oreochromis niloticus × O. aureus)生长、免疫功能以及抗病力的影响

采用单因素实验设计方法,进行了饲料蛋白水平对奥尼罗非鱼(Oreochromis niloticus×O.aureus)生长、免疫功能和抗无乳链球菌能力影响的研究。结果表明,饲料蛋白水平对奥尼罗非鱼生长和饲料利用率以及成活率不产生显著影响,蛋白质效率和蛋白质沉积率伴随着蛋白水平的升高显著下降。35%(D35)饲料组奥尼罗非鱼肝脏过氧化氢酶含量显著高于其它饲料组,然而奥尼罗非鱼肝脏溶菌酶、超氧化物歧化酶活性以及丙二醛含量并不受饲料蛋白水平的影响。无乳链球菌攻毒实验结果表明,蛋白水平对奥尼罗非鱼在攻毒后24h和48h后的累积死亡率是不存在显著影响。本实验的结果表明,24.8%(D25)蛋白水平饲料能够满足奥尼罗非鱼正常生长、以及维持机体正常的非特异性免疫功能和抗病力的营养需求。     

海洋卡盾藻的致毒机制研究进展

海洋卡盾藻 ( Chattonella marina ) 引起的赤潮近年来在中国频繁爆发,对水产养殖业及海洋生态系统造成了严重危害.海洋卡盾藻产生的神经毒素、溶血毒素、游离脂肪酸和活性氧等次生代谢物可能是造成鱼类大范围死亡的重要原因.对此进行综述,以期为阐明海洋卡盾藻赤潮的致毒机制与危害方式提供参考,为正确认识和评价卡盾藻赤潮的危害提供依据.     

牡蛎体内及其养殖水体中细菌耐药性研究

为进一步了解牡蛎体内外细菌的耐药性，对分离自阳江的香港牡蛎（Crassostrea hongkongensis）养殖水体及其内脏团的637个异养细菌进行了10种抗生素的药敏试验。采用纸片扩散法（Kirby-Bauer，简称K-B），参照NCCLS抗生素敏感试验操作标准，研究细菌的耐药概况。结果显示：健康牡蛎体内细菌对恩诺沙星和庆大霉素耐药率较低，发病牡蛎体内细菌对庆大霉素耐药率最低，次之为复合磺胺和恩诺沙星；水体细菌对氯霉素、恩诺沙星、环丙沙星耐药率较低，水体及体内细菌对呋喃唑酮耐药率最高，而对卡那霉素及青霉素耐药率则波动较大。结果还显示，不同来源的受试菌株多重耐药状况严重，来源于健康和病牡蛎的菌株在7月份的多重耐药率达到峰值，分别为66.7%和64.3%，养殖水体细菌多重耐药率峰值出现在4月份，最高达76.7%。总体来看，源于牡蛎养殖环境水体及其牡蛎体内的异养菌多重耐药比例较高，值得引起注意。而受试菌株对不同药物的抗性存在较大差异，且异养细菌对呋喃唑酮的耐药率明显高于对其它受测抗生素的耐药率。从时间动态上看，水体菌耐药率峰值出现于4月份，而牡蛎体内菌群耐药率峰值出现于7月份。     

紫锥菊提取物对大菱鲆(Scophthalmus maximus)的非特异性免疫功能的影响

本文旨在探讨肌肉注射不同浓度的紫锥菊提取物对大菱鲆(Scophthalmus maximus)的非特异性免疫功能的影响,试验选取体重(250±20)g的大菱鲆作为试验动物,分别注射10、20、40mg/m L浓度的紫锥菊提取物,试验共进行28d,每隔7d采集血液测试非特异性免疫指标,第28天用迟缓爱德华氏菌(Edwardsiella tarda)攻毒,连续观察7d,测定其免疫保护率。结果显示肌肉注射紫锥菊提取物对大菱鲆超氧化物歧化酶(SOD)活力和溶菌酶活力(LSZ)的影响极显著(P0.01);对吞噬细胞吞噬率(PP)的影响显著(0.01P0.05),对吞噬细胞呼吸爆发活力的影响极显著(P0.01)。攻毒试验表明,紫锥菊提取物能够降低迟缓爱德华氏菌攻毒后鱼的死亡率,提高其免疫保护率,尤其是40mg/m L组。本试验研究表明,紫锥菊提取物能显著的提高大菱鲆的非特异性免疫功能,且以注射浓度40mg/m L的效果最好。     

Evidence for resistance to benzo[a]pyrene and 3,4,3'4'-tetrachlorobiphenyl in a chronically polluted Fundulus heteroclitus population

Halogenated aromatic hydrocarbons (HAHs) and polynuclear aromatic hydrocarbons (PAHs) are major environmental contaminants. Fish species that are chronically exposed to these compounds can develop resistance to their toxic effects. In all fish species studied to date, toxicant resistance has been accompanied by decreased inducibility of the xenobiotic metabolizing enzyme, cytochrome P450 1A (CYPIA). CYP1A induction is mediated through the Aryl Hydrocarbon Receptor (AHR). Although these compounds mediate their effects through this pathway, there have been resistant populations in which one chemical class cannot induce CYPIA expression (HAHs) while the other (PAHs) can. Resistance to PAHs was examined in a HAH-resistant population of Fundulus heteroclitus collected from a site contaminated with both compound classes (Newark Bay, NJ). Fish were injected intraperitoneally with the HAH 3,4,3',4'-tetrachlorobiphenyl (PCB77), benzo[a]pyrene (B[a]P, a PAH) or vehicle and sacrificed after 2 (B[a]P) or 5 days (PCB77, vehicle). We found no significant increase in CYP1A mRNA levels in resistant Newark Bay F. heteroclitus treated with either B[a]P or PCB77, while there was a 3.9 fold (PCB77) and 4.2 fold (B[a]P) increase in CYP1A mRNA in Flax fish relative to controls. AHR labeling studies revealed significantly (P < 0.05) lower levels of hepatic AHR in Newark fish (1,770 +/- 1,693.2 DPM) relative to Flax fish (6,082.5 +/- 1,709.9 DPM). Overall, these data suggest Newark F. heteroclitus are resistant to both PAHs and HAHs at the level of CYP1A mRNA, which might be mediated, in part, though lower expression of AHR. We are currently studying the promoter sequence to determine its role in chemical resistance.     

The expression of CYP1A, vitellogenin and zona radiata proteins in Atlantic salmon (Salmo salar) after oral dosing with two commercial PBDE flame retardant mixtures: absence of short-term responses

The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and Octa-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days 1 and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n=5 for each group) and 14 (n=6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYP1A were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17β for Vtg and Zrp expression, and β-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.     

苯并[a]芘(BaP)对褐菖鲉(Sebasticus marmoratus)肝CYP1A1酶活性、基因表达及蛋白表达的影响

为了了解苯并[a]芘(BaP)对鱼类细胞色素P4501A1(CYP1A1)表达的影响,以褐菖鲉(Sebasticus marmoratus)为实验材料,采用体内实验,研究其在经过不同浓度(0.1、1、10、20、50mg/kg鱼体重量)的BaP诱导后,鱼体肝脏研究CYP1A1基因表达的情况,筛选出后续时间-效应实验中BaP注射的最佳浓度,研究BaP诱导6h、12h、1d、3d、7d后(质量浓度为20mg/kg鱼体重量)鱼体肝脏CYP1A1酶活性、基因表达和蛋白表达的情况。结果表明:剂量-效应实验中,20mg/kg鱼体重量为最佳浓度,此浓度下,基因表达在各组中变化最显著。时间-效应实验中,较空白对照组而言,染毒6h、12h和1d后,EROD酶活性显著增加。3d后开始下降,与对照组相比变化不大,7d后酶活性又发生上调。半定量RT-PCR结果表明,各染毒组与对照组相比,CYP1A1基因表达量都发生了上调,呈现先上升后下降的趋势。其中,6h和12h组相对表达量极显著增加,1d后开始下降且与3d和7d组相比变化不明显。Western blot结果表明,蛋白表达量在染毒12h后表现出显著的诱导效应,随着时间的延长略有回落,但与对照组相比仍有显著性差异。研究表明:BaP对褐菖鲉CYP1A1具有较强的诱导作用。一定质量浓度的BaP注射于褐菖鲉不同的时间后,能诱导褐菖鲉活体EROD酶活性、CYP1A1基因m RNA表达及蛋白表达,并随着时间的延长呈现先诱导后抑制的趋势。这说明BaP作为诱导剂对CYP1A1酶活性和蛋白表达的作用机制可能与调控CYP1A1的转录水平有关。     

Apparent lack of CYP1A response to high PCB body burdens in fish from a chronically contaminated PCB site

Chronic exposure to organic contaminants such as polychlorinated biphenyls (PCBs) can lead to the development of resistance to these chemicals, a condition associated with reduced response of CYP1A1, a pollutant-inducible biomarker. We measured CYP1A activity (ethoxyresorufin o-deethylase, EROD) and PCB concentrations in feral fish from the Town Branch/Mud River system (Logan County, KY), a stream historically contaminated with PCBs and partially remediated. As a first step in evaluating the possible development of resistant populations in this system, we measured CYP1A expression and PCB body burdens in resident fish from sites we previously characterized as containing biologically significant levels of CYP1A inducing compounds. Mean PCB concentrations in edible flesh ranged from 75.2 to 16.7 microg/g in fish collected from Town Branch remediated sites and were relatively low (1.23 microg/g) in Town Branch reference site fish. However, hepatic CYP1A activity was similar among individuals of most species collected from reference and contaminated/remediated sites. The absence of elevated CYP1A levels in resident fish species despite the presence of significant PCB body burdens may indicate these fish have developed reduced sensitivity to CYP1A induction, a condition associated with acquired resistance to toxicants.     

Elucidating cellular and behavioural effects of contaminant impact (polycyclic aromatic hydrocarbons,PAHs) in both laboratory-exposed and field-collected shore crabs,Carcinus maenas (Crustacea: Decapoda)

Monitoring effects of contaminants at lower levels of biological organisation (e.g. biochemical and cellular) allows for mechanistic evaluation of effects of contaminant exposure through laboratory exposures. However, higher level organism effects (e.g. physiological and behavioural) are deemed more ecologically-relevant. In the present study, cellular (cell viability and immune function), physiological (cardiac activity) and behavioural (foraging behaviour) responses were evaluated in field-collected shore crabs Carcinus maenas from three estuaries [a ‘relatively’ (polycyclic aromatic hydrocarbon) PAH-contaminated site (Plym Estuary) and two ‘comparatively clean’ field sites (Avon and Yealm Estuaries)] and compared with responses of crabs exposed in the laboratory to a model organic contaminant (PAH) pyrene (200 μg l⁻¹ for 28 days). The hypothesis that behavioural indicators may be more sensitive than other more traditional methods was tested. No significant impacts were observed at the cellular or physiological level in Plym-collected crabs (the ‘relatively’ contaminated site), but foraging behaviour was significantly altered (increased prey handling time) compared to individuals collected from the ‘relatively’ uncontaminated sites (Avon and Yealm). When given a cockle as a prey item, both Plym-collected and laboratory-exposed crabs took longer to handle and break into cockle shells. Therefore, ecologically-relevant behavioural observations may serve as valuableindicators of environmental quality.     

环境光色对白条双锯鱼(Amphiprion frenatus)幼鱼生长和体色的影响

本试验研究了循环水养殖条件下白条双锯鱼(Amphiprion frenatus)幼鱼在不同光照环境中的生长状况和体色变化特点。设置白色、红色、黄色、蓝色、绿色五个光照试验组,每组三个重复,试验周期120d。结果表明,各试验组幼鱼存活率和特定生长率均无显著性差异(P0.05),增重率在红光组和绿光组间差异显著(P0.05)。试验鱼红光组红色皮肤胡萝卜素含量显著高于蓝光、黄光和绿光组(P0.05);白光组红皮中胡萝卜素含量显著高于蓝光组(P0.05);在眼、肝、肌肉和白色皮肤组织中胡萝卜素含量在各试验组间无显著性差异(P0.05)。白光组肝组织中酪氨酸酶活性活性显著高于红光组(P0.05);在眼、皮肤、肌肉组织中各试验组酪氨酸酶活性无显著性差异(P0.05)。研究表明,在循环水养殖条件下,白条双锯鱼在红光环境下体表类胡萝卜素含量增加,而酪氨酸酶活性减弱,有提高鱼体色泽鲜艳度的作用,此研究结果可为人工养殖海水观赏鱼的体色改善提供理论依据。     

Cell responses to xenobiotics: comparison of MCF7 multi-drug- and mussel blood cell multi-xenobiotic-defense mechanisms

Multi-drug resistance (MDR) in MCF7 breast cancer cells and multi-xenobiotic resistance (MXR) in mussel (Mytilus edulis) blood cells (MBC) are well known mechanisms that contribute to the decrease in intracellular concentrations of many unrelated but cytotoxic compounds. In the present work, we have carried out comparative investigations of the MDR/MXR protective mechanisms using a rapid colorimetric assay for cell viability and calcein accumulation for MDR/MXR activities. These studies were performed using cultured MCF7 and MBC before and after in vitro exposure to xenobiotics. Our results indicate that a 5-day exposure to doxorubicin or vincristine decreased calcein accumulation in MBC which is consistent with an induction of multi-xenobiotic resistance. The increase in calcein accumulation provoked by 1-h treatment with 50 microM verapamil was much lower in MBC when compared to the P-glycoprotein overexpressing MCF7 cell line. We conclude that such microplate assays could be used in primary cultures of MBC to estimate the effects of various chemicals on MXR activity.     

水产养殖环境常见四环素类抗生素抗性基因tet（M）的演化及传播的初步分析

利用分子系统学方法分析了20 条水产养殖环境中常见的四环素抗性tet（M）基因的序列特点、多态性及系统发育, 并对其细菌寄主分类、环境介质来源作相关性分析。结果表明, 20 条基因序列有4种单倍型, 其中tet（M）-b 和d 是两种远缘基因型, 而a 和c 则是二者之间的序列变异。系统发育树分成三大分支, tet（M...     

Embryonic exposure to environmentally relevant concentrations of PCB126 affect prey capture ability of Fundulus heteroclitus larvae

Early life stages from a marine fish species, Fundulus heteroclitus, were exposed to sublethal doses of 3,3',4,4',5 pentachlorobiphenyl (PCB126) to evaluate its effects on ecologically relevant responses: growth and behavior. A few hours after fertilisation, eggs were treated topically with PCB126 (2.5-50 pg egg?1). Four days post-hatching (dph), morphological changes (body length and malformations), spontaneous locomotor activity (active swimming speed, rate of travel, % inactivity), prey capture ability (Artemia franciscana nauplii) and whole body EROD activity were evaluated in larvae. Untreated larvae collected at 0.5, 1, 2, 3, 4 dph were also examined. PCB126 did not increase the mortality or malformation rates. Body length and spontaneous locomotor activity were altered only in larvae treated with the highest dose. Treatment with PCB126 caused a dose-responsive reduction in prey capture ability (rate of decline in the number of Artemia) and induction of EROD activity. The lowest observed effective dose for both of these responses was 5.0 pg PCB126 egg?1 or 5.0 TCDD-toxic equivalents pg g?1 egg, using a TCDD-toxic equivalent factor of 0.005 and an egg mass of 5 mg. Prey capture efficiency (number of Artemia captured per feeding strike) was reduced at ≥ 10.0 pg egg?1. In untreated developing larvae, prey capture ability and efficiency increased as post-hatching development progressed and EROD activity remained low. The pattern of behavioral responses observed in PCB126-exposed Fundulus larvae differed from that observed in less-developed larvae indicating that other mechanisms than retarded development were involved. Behavioral dysfunction was a more sensitive response to PCB126 than morphological alterations and it occurred at environmentally relevant concentrations.     

基于全基因组重测序技术的浙江近岸海域耐盐金黄色葡萄球菌(Staphylococcus aureus)耐药机制解析<

金黄色葡萄球菌(Staphylococcus aureus)是近岸海域海水中的主要病原菌,严重威胁接触者的安全。抗生素处理是治疗金黄色葡萄球菌感染的重要手段,其耐药性的发生受到了高度重视。采用全基因组重测序与KEGG富集分析结合的方法,对红霉素(erythromycin)、氯霉素(chloramphenicol)和万古霉素(vancomycin)处理后的耐盐金黄色葡萄球菌(S.aureus ZS01)和不耐盐金黄色葡萄球菌(S.aureus 502A)进行耐药机制研究。结果表明,S.aureus 502A经抗生素处理后发生突变的程度大于S.aureus ZS01,二者在经过氯霉素处理发生了更大程度的突变。红霉素、氯霉素和万古霉素处理主要影响了金黄色葡萄球菌的致病能力;红霉素和氯霉素可能通过影响金黄色葡萄球菌脂类的代谢引起其耐药性的变化。除此之外,三种抗生素处理均出现了较多TIGR01741家族蛋白和假设蛋白基因的突变,推测与菌株的耐药性和致病性相关。耐盐金黄色葡萄球菌可通过外排系统作用产生红霉素耐药性,不耐盐菌株因细胞壁成分相关基因的突变提高了对万古霉素的耐受性。研究结果可为耐盐和不耐盐金黄色葡萄球菌的耐药机制及抗生素对金黄色葡萄球菌致病性影响的研究提供基础数据。     

光唇鱼(Acrossocheilus fasciatus)Leptin基因克隆及禁食-再投喂对其表达的影响

瘦素(Leptin)是在动物摄食和能量代谢调节中具有重要作用的一种蛋白。为研究光唇鱼(Acrossocheilus fasciatus)中Leptin基因(AfLep)的结构和功能,作者克隆了其c DNA序列全长。结果显示AfLep由1315个核苷酸组成,含1个长度为516bp的开放阅读框,预测编码蛋白由171个氨基酸残基组成并具有长度为20aa的信号肽。系统进化树中AfLep与其他鲤科鱼类的Leptin蛋白聚为一簇,与齐口裂腹鱼(Schizothorax prenanti)进化相关性最高。多重序列比对显示AfLep与齐口裂腹鱼Leptin蛋白相似性最高(86.7%),与鲤科其他鱼类间的相似性均在80%以上。AfLep具有脊椎动物Leptin蛋白的4个保守?螺旋结构,其三级结构与人Leptin相似。AfLep在光唇鱼肝脏中的表达量最高,其次是脑和肾,其他组织中仅有微弱表达。实时荧光定量PCR检测显示禁食后光唇鱼肝脏AfLep表达量降低(P0.05),禁食1d、3d、7d时的表达量分别比与禁食前降低了58.25%、73.82%和92.05%;禁食1d和3d的鱼经再投喂后肝脏AfLep表达量均显著升高(P0.05),但禁食7d的鱼再投喂后AfLep表达量仅略有升高(P0.05)。上述结果表明AfLep参与了光唇鱼的摄食管理和能量代谢调控。     

喂食当归多糖对点带石斑鱼非特异性免疫力和抗病力的影响

在饵料中添加0、500、3 000mg/kg的当归多糖(ASP)喂食点带石斑鱼(Epinephelus malabaricus)(初始体重(158.64±2.14)g),分别在喂食4和8周后取样,研究ASP对点带石斑鱼非特异性免疫力和抗病力的影响。结果表明,ASP能显著提高血液白细胞吞噬率和血清LZM活力(P<0.05),对血清一氧化氮(NO)、体表黏液溶菌酶(LZM)活力和抗菌活力无显著影响。ASP能促进头肾白细胞增殖、呼吸爆发活力和吞噬活力,降低用迟缓爱德华菌(Edwardsiella tar-da)攻毒后试验鱼的累积死亡率,3 000mg/kg组累积死亡率低于500mg/kg组,连续喂食ASP 8周的保护效果优于4周。研究表明,用含ASP 3 000mg/kg的饵料连续喂食点带石斑鱼8周能有效提高其非特异性免疫力和抗病力。