Aldehyde dehydrogenase and glucose-6-phosphate dehydrogenase as markers for enzyme-altered foci in the liver of dab (Limanda limanda L.)

The activities of the most important aldehyde oxidizing enzymes, the aldehyde dehydrogenases (ALDH), were detected histochemically in liver cryosections of dab (Limanda limanda L.) caught at differently polluted stations along the North Sea coast. In parallel, glucose-6-phospate dehydrogenase (G6PDH) as an NADPH-generating enzyme was measured. Increased activities of both enzymes localized putatively preneoplastic hepatocytes (foci), but ALDH in combination with propionaldehyde and NAD marked 50% more foci than G6PDH alone. Nevertheless, at the more contaminated stations with highest prevalences of foci, ALDH activities showed a shift in substrate and coenzyme dependence from propionaldehyde and NAD to benzaldehyde and NADP in altered hepatocytes, a possible result of physiological adaption to the toxic environment. Increased G6PDH activities in focal hepatocytes indicated elevated NADPH production in contrast to the injured extrafocal liver parenchyma showing reduced G6PDH activities compared to normal livers. Using the ALDH inhibitor disulfiram, the existence of a specific tumor-associated isozyme previously found in rats could not be clearly demonstrated in early enzyme-altered hepatocellular foci of dab. However, based on the total inhibition of Bz-NADP-dependent ALDH, the presence of a xenobiotic-induced ALDH isozyme can be anticipated.     

Clonal adaptation of cancer cells in flatfish liver to environmental contamination by changes in expression of P-gp related MXR, CYP450, GST-A and G6PDH activity

Progression from eosinophilic foci to persistent basophilic foci and carcinomas was observed in pollution-induced hepatocellular carcinogenesis in European flounder (Platichthys flesus L.) in a similar sequence as in chemically induced liver cancer in mammals. Image analysis was used to quantify enzyme activity and protein expression as visualised by enzyme and immunohistochemistry in various stages of toxipathic lesions and carcinogenesis. In eosinophilic foci, growth advantage was achieved by increased production of NADPH and pentoses for biosynthesis and cell proliferation due to increased G6PDH capacity during the first steps of clonal adaptation. Simultaneously, oxyradical production by CYP450, phase I was reduced. Overexpression of P-gp mediating multi-xenobiotic resistance was noted in basophilic cell types which persisted during progression towards carcinomas. This was accompanied by increased protein levels of oxyradical scavenging GST-A. These changes are consistent with adaptation and phenotypic expression of the multidrug or xenobiotic resistance (MDR/MXR) type in hepatocarcinomas of European flounder.     

Nuclear changes in blood, early liver anomalies and hepatocellular cancers in flounder (Platichthys flesus L.) as prognostic indicator for a higher cancer risk?

Nuclear changes (micronuclei) in blood are a well-established biomarker of toxic injury and related genotoxicity in mussel, fish and humans, as well. Yet, the relationship between the frequencies of micronuclei in blood and nuclear changes in liver cells itself and the higher potential risk to develop liver cancer is not yet fully implemented. We analysed and diagnosed macroscopic liver changes and H&E and Feulgen stained histological liver sections (2 microm of thickness) together with blood smears (stained with Sytox Green) from same individuals in flounder caught along a well-known contaminant gradient in the North Sea. Results showed an increase in micronuclei frequencies in blood at the more contaminated sites and higher frequencies of macroscopically visible neoplastic liver changes. In livers of the same individuals, nuclear changes were diagnosed in early to degenerative liver lesion, extra-focal tissue surrounding neoplastic lesion and various stages during cancer progression from early neoplastic foci, benign adenomas and malignant carcinomas. The aim of this approach is to expand diagnostic criteria, well-established in the frame of BEQUALM to nuclear changes which might help diagnosis of malignancies in doubtful cases.     

Quantitation of CYP1A expression in two flatfish species showing different prevalences of contaminant-induced hepatic disease

English sole (Pleuronectes vetulus) and starry flounder (Platichthys stellatus) are two sympatric flatfish species which show markedly different responses to chemical contaminant exposure. Whereas English sole develop hepatic neoplasms, the prevalences of which are strongly linked to exposure to polycyclic aromatic hydrocarbons (PAHs), evidence of neoplasia is virtually nonexistent in starry flounder, even those residing in areas with very high levels of PAH in the sediments. Because PAHs are activated to genotoxic forms by the action of cytochrome P450 1A (CYP1A) in teleosts, we are examining the hypothesis that variation in the hepatic expression of this major inducible cytochrome P450 isozyme may contribute to the differential contaminant response. These two species were captured in the Duwamish Waterway, a contaminated area of Puget Sound, Washington. Catalytic activity of CYP1A [determined as aryl hydrocarbon hydroxylase (AHH) activity] was measured in the liver to quantitatively assess the relative induction of CYP1A in these two species, and AHH activity was significantly higher in English sole than in starry flounder (p = 0.015). Cellular expression of CYP1A was determined by immunohistochemical localization of tissues, using an avidin-biotin peroxidase complex method, with polyclonal rabbit anti-cod CYP1A. The results showed a markedly reduced expression of CYP1A in hepatocytes of starry flounder, which is consistent with the apparent resistance of this species to the development of hepatocellular neoplasia. This reduced expression of CYP1A in hepatocytes of contaminant-exposed fish was previously seen in oyster toadfish from the Elizabeth River, Virginia, and this species is also apparently resistant to hepatocellular neoplasia.     

牙鲆幼鱼对n-3高度不饱和脂肪酸的吸收及与生长关系的初步研究

于1996年9-10月在中国水产科学研究院黄海水产研究所小麦岛实验基地,采用人工投喂的方法,给牙鲆幼鱼投喂含不同含量n-3高度不饱和脂肪酸（n-3HUFA）的人工饵料,研究牙鲆幼鱼对饵料中n-3HUFA的消化、吸收以及n-3HUFA的含量与日增重率、饵料效率的关系。结果表明,在饵料中添加0.60％—2.40％的n-3HUFA可有效提高牙鲆幼鱼的日增重率和饵料效率;肝脏中n-3HUFA的含量与饵料中n-3HUFA（尤其是DHA）的含量成正比;牙鲆幼鱼对饵料中n-3HUFA的表观消化吸收率随其含量的增加呈下降趋势。     

应用单克隆抗体检测牙鲆血清中抗淋巴囊肿病毒的特异性抗体

应用杂交瘤单克隆抗体技术制备10株分泌抗牙鲆免疫球蛋白单克隆抗体的杂交瘤细胞。利用单抗2H4,通过间接酶联免疫法(ELISA)对牙鲆血清中抗淋巴囊肿病毒(LCDV)特异性抗体进行了检测。结果表明:无外观症状的牙鲆血清中特异性抗体水平很低(平均OD值为0.092),个别鱼体偏高,证明已感染LCDV,处于潜伏期;患淋巴囊肿病且症状显现的牙鲆血清中特异性抗体水平升高(平均OD值为0.165),患淋巴囊肿病后处于恢复期的牙鲆抗体水平最高(平均OD值为0.231);健康牙鲆接种LCDV灭活疫苗后,特异性抗体水平显著升高。     

Exposure to 2,4-decadienal negatively impacts upon marine invertebrate larval fitness

Diatoms liberate volatile, biologically active unsaturated aldehydes following cell damage, which negatively impact upon invertebrate reproductive processes such as fertilization, embryogenesis and larval survival. 2,4-Decadienal is frequently identified among the aldehydes produced and is one of the more biologically active. The majority of studies which have examined the toxic effects of diatom aldehydes to invertebrate reproduction have scored egg production and/or hatching success as indicators of biological impacts. There are very few studies which have dealt specifically with the impacts of diatom-derived aldehydes on larval fitness. Larval stages of the polychaetes Arenicola marina and Nereis virens and the echinoderms Asterias rubens and Psammechinus miliaris exposed to 2,4-decadienal at sub 1 microg ml(-1) concentrations suffered reduced survival over the incubation period (day 1-8 post fertilization) with detectable differences for the polychates at a concentration of 0.005 and 0.01-0.1 microg ml(-1) for the echinoderms. Susceptibility of larval N. virens was investigated using stage specific 24 h exposures at 2,4-decadienal concentrations up to 1.5 microg ml(-1). A clear stage specific effect was found, with earlier larval stages most vulnerable. Nectochaete larvae (9-10 d) showed no reduction in survival at the concentrations assayed. Fluctuating asymmetry (FA), defined as random deviations from perfect bilateral symmetry, was used to analyse fitness of larval P. miliaris exposed to 2,4-decadienal at concentrations of 0.1, 0.5 and 1 microg ml(-1). The degree and frequency of asymmetrical development increased with increasing 2,4-decadienal concentration. Equally, as FA increased larval survival decreased. These results provide further support for the teratogenic nature of 2,4-decadienal and its negative impact on invertebrate larval fitness.     

Analysis of mutations in the p53 tumor suppressor gene and Ki- and Ha-ras proto-oncogenes in hepatic tumors of European flounder (Platichthys flesus).

DNA from five tumors, three other-injured livers and two normal liver tissue samples from the European flounder were analyzed for mutations in exons 5-8 of the tumor suppressor gene p53 and at codons 12, 13 and 61 of Ha- and Ki-ras proto-oncogenes. No tumor-specific mutations were identified by direct sequencing and single-strand conformation polymorphism of these genes. A number of silent polymorphisms were noted in p53. In addition to a need for more extensive analyses of flounder liver tumor samples for ras or p53 mutations, other cancer-related genes should be investigated.     

半滑舌鳎仔稚鱼消化酶活性的变化

对半滑舌鳎不同发育阶段主要消化酶（酸性蛋白酶、碱性蛋白酶、淀粉酶、脂肪酶）以及与消化吸收相关的碱性磷酸酶的活性变化进行了测定。结果表明，半滑舌鳎在仔鱼发育早期即可检出酸性蛋白酶和碱性蛋白酶的活力，碱性蛋白酶的2个极低值分别出现在仔鱼开口期和仔稚鱼的转交期，从孵化后23d左右胃腺出现时，酸性蛋白酶的活性开始明显升高。早期仔鱼体内表现出较强的淀粉酶活性，孵化后12d其活性急剧下降，并一直保持较低的比活力状态。脂肪酶的活性在半滑舌鳎早期仔鱼体内一直较低，进入稚鱼期后开始缓慢上升。碱性磷酸酶活性在变态结束后出现大幅度的升高，这标志着肠组织结构和肠道消化功能的完善。     

Isozyme changes during larval development of Penaeus chinensis

Isozyme changes during early development of Penaeus chinensis are described for six enzymes (EST, AMY MDH, GDH, Gd and LDH) by means of polyacrylamide gel vertical plate electrophoresis. The results show that the number of isozymes varies considerably and the enzyme activity also rises gradually with development. The band number of EST increases from 1 in nauplius stage to 6 in postlarval stage. There are three types of AMY, α-AMY, R-AMY and Q-AMY. The phenotype of LDH changes slightly, showing the same pattern except nauplius in all the stages. The remaining three enzymes (MDH, Gd and GDH) have more bands and their isozyme number also increases with development.     

Egg and yolk sac stages of the sand flounder (Teleostei: Pleuronectidae)

The developmental stages of the eggs and yolk‐sac larvae of the common sand flounder, Rhombosolea plebeia (Richardson), are described and illustrated.     

方斑东风螺早期发育过程中几种消化酶的活性

将方斑东风螺Babylonia areolata(Link)的早期发育从早期面盘幼体到变态后15d的稚贝分成6个阶段,分别测定各阶段蛋白酶(P)、脂肪酶(L)、淀粉酶(A)和纤维素酶(C)活性。研究结果表明,随着幼体发育的推进,蛋白酶比活力逐渐减小,脂肪酶比活力逐渐增加,淀粉酶和纤维素酶比活力则在变态前逐渐增加,而变态后逐渐减小。比较A/P和(A C)/(P L)两种指标指示方斑东风螺幼体发育过程中的食性变化,提出(A C)/(P L)用于指示方斑东风螺食性变化更合理、准确。综合比较幼体发育各阶段4种消化酶活性,发现在面盘幼体向稚贝变态期间,幼体消化能力最弱,原因与幼体的变态过程有关。实验结果也验证了贝类具有分泌纤维素酶的能力,该酶在东风螺对纤维素分解中占主导地位。     

Comparative histopathological effects of chemically contaminated sediment on marine organisms

Comparative pathological effects in three species of fish and six species of invertebrates were investigated using a chemically contaminated sediment from Black Rock Harbor (BRH), Connecticut, USA. Substances contained in BRH sediment are known to be genotoxic, carcinogenic, co-carcinogenic and tumor-promoting. Proliferative lesions were observed in winter flounder (Pseudopleuronectes americanus) external, oral and esophageal epithelial surfaces, renal vascular and nephroblastic elements, and the pancreatic islets. Islet lesions included cystic adenomas and a diffuse islet proliferative condition, nesidioblastosis. In addition, pathology was enhanced when winter flounder were fed mussels (Mytilus edulis) previously exposed to BRH sediment. Neoplastic lesions developed in kidney tubule epithelia, gills, some regions of the gastrointestinal tract, neural elements and heart of oysters (Crassostrea virginica) exposed to BRH sediment for 30 days in the laboratory. In addition, renal carcinomas in three oysters had metastasized to the viseral ganglion. Lesions were also found in oysters exposed in situ in BRH and Long Island Sound for 30 or 36 days. Digestive diverticula, gills and kidneys appeared to be the prime organs affected in mussels, soft-shelled clams (Mya arenaria) and oysters exposed to BRH sediment. Mucous cell activity increased greatly in corals (Astrangia densa) while no microscopic lesions were detectable in lobster (Homarus americanus) exposed in the laboratory. Myxomas were also found in the hearts of < 5% of the mussels exposed in the laboratory.     

条斑星鲽幼鱼变态期间核酸及蛋白的变化

本试验测定了条斑星鲽(Verasper moseri)幼鱼变态过程中DNA、RNA、蛋白及其比值的变化,作为评价幼鱼变态阶段生长潜能的指标。结果表明,试验期间培育水温为16.5～18.0°C,DNA、RNA及蛋白的变化都带有发育阶段特异性。DNA含量在34～42日龄增加缓慢,然后快速增加直至44日龄,在45日龄和46日龄分别保持下降和上升趋势。蛋白含量和RNA含量的变化趋势相似,在试验期间保持快速增长的趋势。RNA/DNA比值从试验开始至35日龄呈上升状态,36日龄时下降,38日龄时达到最高值5.01,然后显著下降(P0.05),在试验结束时达到最低值1.76。蛋白/DNA在40日龄时达到最高值58.64,在46日龄时达到最低值21.28。RNA、DNA及蛋白含量跟全长和体质量有明显的线性关系(P0.05)。RNA/DNA与全长和体质量的关系比蛋白/DNA与全长和体质量的关系密切。蛋白/DNA比值的变化趋势跟RNA/DNA比值类似,但前者的变化滞后于后者,表明RNA/DNA比值是评价条斑星鲽生理状况的更有效的生理指标。     

Natural modulation of hepatic metallothionein and cytochrome P4501A in flounder, Platichthys flesus L.

The use of biomarkers such as cytochrome P4501A (CYP1A) or metallothionein (MT) for pollution monitoring is based on the assumption that an increased activity or concentration is primarily caused by exposure to contaminants. Previous studies have shown that the response of biomarkers may be affected by factors such as season, temperature, gender, nutritional status or size. The objective of the present study was to identify natural factors that affect the hepatic activity of CYP1A and hepatic concentration of MT in flounder (Platichthys flesus L.). Thirty flounder were sampled at each of two sites in the Hvaler archipelago, southern Norway, monthly through one year. A set of variables were recorded for each sampling and individual including water temperature, salinity, external and internal lesions, intestinal content, sex, size and organ weights. Hepatic CYP1A activity (EROD), MT and metal concentrations were determined for each individual flounder. The influence of environmental and endogenous factors on the response of these two biomarkers was assessed in multiple regression models. For both biomarkers, 50–60% of the total variability could be explained from factors directly related to season, gender and maturation. Season contributed significantly in the model for EROD, as did external lesions. Size, condition and diet did not contribute greatly when the above factors were included. The results confirm previous findings that season, gender and maturation must be taken into account in biomarker monitoring, but also indicate that other factors such as external lesions should be considered.     

Fish populations of the Avon‐Heathcote Estuary

Breeding seasons and sizes at first maturity for nine species of fish in the Avon‐Heathcote Estuary, Christchurch, New Zealand, were: sand flounder, Rhombosolea plebeia, mid‐winter to spring at 200 mm+; yellow‐bellied flounder, Rhombosolea leporina, winter and spring at 260 mm+; common sole, Peltorhamphus novaeieelandiae, did not breed in the estuary but reached first maturity at 220 mm +; yellow‐eyed mullet, Aldrichetta forsteri, spawned twice a year, winter and summer, at 220 mm +; kahawai, Arripis trutta, bred outside the estuary at 520–540 mm +; spotty, Pseudolabrus celidotus, spring to autumn at 200 mm for males and 160 mm for females; cockabully, Tripterygion nigripenne, spring and early summer at 52–57 mm +; common bully, Gobiomorphus basalis, spring and early summer at 45–50 mm+; and globefish, Speroides richei, spring to autumn at 147 mm + for males and 120 mm + for females. The successive stages in gonad development are described for these species.

In pelagic species, e.g., flatfish and kahawai, x² analyses showed that for most months females are more numerous than males, and that migration is of major importance. In (littoral species, e.g., common bully and cockabully, there is a 1 : 1 sex ratio in the breeding season, but this ratio subsequently breaks down.

The Avon‐Heathcote estuary was used mainly as a nursery area by juvenile fish, although some species, such as sand flounder, yellow‐bellied flounder, yellow‐eyed mullet, and cockabully spawned in the estuary.     

Spatial and temporal shifts in suitable habitat of juvenile southern flounder (Paralichthys lethostigma)

Factors influencing suitable habitats of juvenile southern flounder (Paralichthys lethostigma) within the Galveston Bay Complex (GBC), Texas, were assessed using generalized additive models (GAM). Fishery independent data collected with bag seines throughout the GBC from 1999 to 2009 were used to predict the probability of southern flounder occurrence. Binomial GAMs were used to assess presence/absence of southern flounder and models included temporal variables, benthic variables such as distance to habitats generated within a geographic information system, and physicochemical conditions of the water column. Separate models were generated for newly settled southern flounder, young-of-the-year (YOY) southern flounder observed in the summer, and YOY southern flounder observed in fall based on size and collection month. Factors affecting southern flounder occurrence changed seasonally, as did the corresponding shifts in the spatial distribution of suitable habitat. Temporal effects (year and month) were retained in all models. Physicochemical conditions (temperature, turbidity, and measures of environmental variability), and the presence of seagrass beds were influential for newly settled southern flounder. Distance to marine and/or freshwater sources were found to be important for YOY southern flounder in the summer and fall seasons. The abundance of brown shrimp was found to only influence the distribution of YOY southern flounder in the fall, when intermediate abundances of the potential prey item increased the occurrence of southern flounder. After model completion, the availability and spatial distribution of suitable habitat within the GBC was predicted using available environmental and spatial data for 2005. Spatial distributions of predicted suitable habitat stress the relative importance of West Bay during the newly settled stage and in the fall season, and Upper Bay during the summer and fall of the first year of life. These models demonstrate the potential dynamics of suitable habitats for juvenile southern flounder and provide insight into ontogenetic shifts in habitat preference during the first year of life.     

雌核发育牙鲆同工酶基因的重组及父方基因的表达

于1998年4、5月在山东省荣成市寻出水产公司养鱼场人工采集牙Ping成熟精卵，紫外线灭活精子遗传物质，冷休克诱导，获得雌核发育牙Ping。1999年5月收集57尾雌核发育牙Ping生化样品，采用淀粉胶电泳分析了LDH、IDHP、PGM、ACP、ADH、SDH、GDH、CAT等8种在野生种群中为多态的同工酶。结果表明，牙Ping雌核发育群体的Ldh-C、Cat两个基因座位发生了重组，基因座位与着丝点之间的重组率分别为52．6％和29．8％；Idhp－1有父方基因表达现象，说明雌核发育过程中父方基因可能在分子水平整合。牙Ping雌核发育群体的多态座位比例和平均杂合度分别为7．0％和0．035，远低于野生种群；研究表明，通过抑制第二极体排出获得的牙Ping雌核发子代尚有一定基因杂合，不能用于培育纯系。     

Flounder health status in the Seine Bay. A multibiomarker study.

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.