Alteration of gene expression profiles in the brain of Japanese medaka (Oryzias latipes) exposed to KC-400 or PCB126

Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals and possibly alter animal behavior. We previously reported that PCB-exposure induced abnormal schooling behavior in Japanese medaka (Oryzias latipes). This abnormal behavior might be caused by the functional alteration of central or terminal nervous system. To understand the mechanism(s) of behavioral change by PCB-exposure, we analyzed the gene expression profiles in the brain of medaka exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) or a PCB mixture (Kanechlor-400: KC-400) using a cDNA microarray that we constructed. Twelve FLF-II strain medaka (six individuals per treatment) were dietary exposed to PCB126 (0.01 μg/g b.w./day) or KC-400 (1 μg/g b.w./day) for three weeks. For the control, six fish were fed a control diet. After the exposure period, fish were dissected, and the brain samples were collected. The samples from control fish were pooled and used as a common reference in the microarray experiment. Microarray data were normalized by the LOWESS method, and we screened the genes whose expression levels were altered more than 1.5-fold. Gene expression profiling showed 97 down-regulated and 379 up-regulated genes in the brain of medaka exposed to PCB126. KC-400 exposure suppressed 15 genes and induced 266 genes in medaka brain. Among these genes, the expression levels of 7 and 188 genes were commonly down- or up-regulated, respectively in both treatment groups. On the other hand, 31 gene expressions were significantly different between PCB126 and KC-400 treatment groups, and three out of 31 genes were received opposite effects. In addition, the microarray data showed that thyroid hormone-responsive genes were up-regulated by PCB-exposure, which may imply that PCBs or their metabolites mimic thyroid hormone effects in the brain of PCB-exposed medaka.     

Alteration of gene expression profiles in the brain of Japanese medaka (Oryzias latipes) exposed to KC-400 or PCB126

Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals and possibly alter animal behavior. We previously reported that PCB-exposure induced abnormal schooling behavior in Japanese medaka (Oryzias latipes). This abnormal behavior might be caused by the functional alteration of central or terminal nervous system. To understand the mechanism(s) of behavioral change by PCB-exposure, we analyzed the gene expression profiles in the brain of medaka exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126) or a PCB mixture (Kanechlor-400: KC-400) using a cDNA microarray that we constructed. Twelve FLF-II strain medaka (six individuals per treatment) were dietary exposed to PCB126 (0.01 microg/g b.w./day) or KC-400 (1 microg/g b.w./day) for three weeks. For the control, six fish were fed a control diet. After the exposure period, fish were dissected, and the brain samples were collected. The samples from control fish were pooled and used as a common reference in the microarray experiment. Microarray data were normalized by the LOWESS method, and we screened the genes whose expression levels were altered more than 1.5-fold. Gene expression profiling showed 97 down-regulated and 379 up-regulated genes in the brain of medaka exposed to PCB126. KC-400 exposure suppressed 15 genes and induced 266 genes in medaka brain. Among these genes, the expression levels of 7 and 188 genes were commonly down- or up-regulated, respectively in both treatment groups. On the other hand, 31 gene expressions were significantly different between PCB126 and KC-400 treatment groups, and three out of 31 genes were received opposite effects. In addition, the microarray data showed that thyroid hormone-responsive genes were up-regulated by PCB-exposure, which may imply that PCBs or their metabolites mimic thyroid hormone effects in the brain of PCB-exposed medaka.     

Cloning and developmental expression of kinesin superfamily7 (kif7) in the brackish medaka (Oryzias melastigma), a close relative of the Japanese medaka (Oryzias latipes)

kif7 is a member of the kinesin superfamily members which are molecular motor proteins that move along microtubules in a highly regulated manner through ATP hydrolysis. In this paper, we report on the cloning of the Oryzias melastigma kif7 (omkif7) using primers designed according to the Japanese medaka (Oryzias latipes) database. The cloned omkif7 has an open reading frame of 3762bp and is deduced to encode a polypeptide of 1254 amino acids that possesses the putative ATP-binding and microtubule-binding motifs in its motor domain at the N-terminal region. We characterized the cloned omkif7 by comparison with the zebrafish kif7. Both omkif7 and zebrafish kif7 are shown to be expressed in all embryonic stages and adult tissues examined with higher expression level in the testis and ovary. Whole-mount in situ hybridization revealed that the expression of omkif7 is ubiquitous during the early stages of embryonic development, but became more restrictive and localized to the brain, fin bud and eye at later development. This study suggested that the brackish O.melastigma can serve as a good seawater model organism for developmental studies by utilizing the resources developed from its close relative of the Japanese medaka.     

Differential developmental toxicity of naphthoic acid isomers in medaka (Oryzias latipes) embryos

Polycyclic aromatic hydrocarbons (PAHs) are widespread persistent pollutants that readily undergo biotic and abiotic conversion to numerous transformation products in rivers, lakes and estuarine sediments. Here we characterize the developmental toxicity of four PAH transformation products each structural isomers of hydroxynaphthoic acid: 1H2NA, 2H1NA, 2H3NA, and 6H2NA. Medaka fish (Oryzias latipes) embryos and eleutheroembryos were used to determine toxicity. A 96-well micro-plate format was used to establish a robust, statistically significant platform for assessment of early life stages. Individual naphthoic acid isomers demonstrated a rank order of toxicity with 1H2NA > 2H1NA > 2H3NA > 6H2NA being more toxic. Abnormalities of circulatory system were most pronounced including pericardial edema and tube heart. To determine if HNA isomers were AhR ligands, spatial-temporal expression and activity of CYP1A was measured via in vivo EROD assessments. qPCR measurement of CYP1A induction proved different between isomers dosed at respective concentrations affecting 50% of exposed individuals (EC50s). In vitro, all ANH isomers transactivated mouse AhR using a medaka CYP1A promoter specific reporter assay. Circulatory abnormalities followed P450 induction and response was consistent with PAH toxicity. A 96-well micro-plates proved suitable as exposure chambers and provided statistically sound evaluations as well as efficient toxicity screens. Our results demonstrate the use of medaka embryos for toxicity analysis thereby achieving REACH objectives for the reduction of adult animal testing in toxicity evaluations.     

Toxicogenomic analysis of immune system-related genes in Japanese flounder (Paralichthys olivaceus) exposed to heavy oil

Heavy oil contamination is one of the most important environmental issues. Toxicities of polycyclic aromatic hydrocarbons (PAHs), including immune toxicities, are well characterized, however, the immune toxic effects of heavy oil, as a complex mixture of PAHs, have not been investigated. In the present study, we selected Japanese flounder (Paralichthys olivaceus) as a model organism, and observed alteration of immune function by the exposure to heavy oil. To analyze the expression profiles of immune system-related genes, we selected 309 cDNAs from our flounder EST library, and spotted them on a glass slide. Using this cDNA array, alteration of gene expression profiles was analyzed in the kidneys of flounders exposed to heavy oil. Six Japanese flounders (mean body weight: 197 g) were acclimated to laboratory conditions at 19–20 °C. Three fish were exposed to heavy oil C (bunker C) at a concentration of 3.8 g/L for 3 days, and the others were kept in seawater without heavy oil and used as the control. After the exposure period, the fish were transferred into control seawater and maintained for 4 days, and then they were dissected and their kidneys were removed. Total RNA was extracted from the kidney samples to use in gene expression analyses. The microarray detected alteration of immune system-related genes in the kidneys of heavy oil-exposed flounders, including down-regulation of immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and lysozyme. These results suggest that pathogen resistance may be weakened in heavy oil-exposed fish, causing a subsequent bacterial infection, and then proinflammatory genes may be induced as a defensive response against the infection. Additionally, we found candidate genes for use as biomarkers of heavy oil exposure, such as N-myc downstream regulated gene 1 and heat shock cognate 71 kDa proteins.     

iTRAQ-based proteomic profiling of the marine medaka (Oryzias melastigma) gonad exposed to BDE-47

A recent study demonstrated that 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) may have an adverse effect on the reproduction in marine medaka (Oryzias melastigma), but the molecular mechanisms remain largely unknown. In this study, we investigated the protein expression profiles of male and female gonads of O. melastigma exposed to dietary BDE-47 at two dosages (0.65 and 1.30 μg/g/day, respectively) for 21 days. Extracted proteins were labeled with iTRAQ and analyzed on a MALDI TOF/TOF analyzer, as results, 133 and 144 unique proteins were identified in testis and ovary, respective, and they exerted dose- and sex-dependent expression patterns. In testis, among the 42 differentially expressed proteins; down-regulation of histone variants and parvalbumins implicated BDE-47 may disrupt the spermatogenesis and induce sterility in fishes. In ovary, 38 proteins were differentially expressed; the elevation of vitellogenins and apolipoprotein A–I expression indicated BDE-47 acts as an estrogen-mimicking compound and led to reproductive impairment in O. melastigma.     

Changes in the scope for growth of blood cockles (Anadara granosa) exposed to industrial discharge

Blood cockles (Anadara granosa) were placed in plastic cages and deployed at eight locations along a pollution gradient. A control site was chosen a few kilometres away. At 2-week intervals for 8 weeks, the scope for growth of the cockles was determined. A total of 11 environmental parameters were measured during each sampling exercise for correlation with the growth index. Values of scope for growth for all the eight stations, except for stations 7 and 8, were significantly lower than the value for the control site; station 1 (nearest the discharge point) had the lowest value. Strong correlations were found between the scope for growth of the cockles and seven of the environmental parameters. The pattern of the impact of the discharge on the scope for growth of the cockles suggests that longshore currents may play a dominant role in the distribution of the industrial wastes.     

Screening of Mercury and Persistent Organochlorine Pollutants in Long-Finned Pilot Whale (Globicephala melas) in the Faroe Islands

Results of mercury and organochlorine analyses in pooled muscle and blubber samples representing in all 417 long-finned pilot whales (Globicephala melas) sampled in 1997 in the Faroe Islands are presented. The 28 pooled muscle samples from (in all) 9 pods were analysed for total mercury and the same number of pooled blubber samples were analysed for PCB (IUPAC CBs no. 28, 52, 99, 101, 105, 118, 128, 138, 153, 170, 180, 183 and 187), p,p′-DDT and metabolites and o,p′-DDT and metabolites, cis () and trans (γ)-chlordane, cis- and trans-nonachlor, oxychlordane, mirex, hexachlorobenzene, β-HCH and toxaphene (Parlars no. 26 (T2), 32, 50 (T12), 62 (T20) and 69.     

PCB, PCDD/F and PBDE levels and profiles in crustaceans from the coastal waters of Brittany and Normandy (France)

Polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) were analysed in the muscle of various edible marine crustaceans (spider crab, edible crab, velvet swimming crab and Norway lobster) from the Brittany and Normandy coasts (France). The highest concentrations were measured in species collected from Antifer (Seine Bay). PCB and PBDE patterns in crustacean muscles were similar and independent of the geographical area with the predominance of the high chlorinated PCBs (CB153, 138, 118 and 180), and of a few PBDE congeners (BDE47, BDE99, BDE100 and BDE28). Oppositely, dioxin contamination differed with site. The major component in crustaceans from the Seine Bay was 2378-TCDF, whereas specimens from cleaner areas had higher relative concentrations of OCDD. Finally, the comparison of the spider crab contaminant profiles to those measured in mussel and sea bass highlighted two different trends: decapod crustaceans possess relatively strong capacity to metabolise PCBs and PBDEs; however these species might be used as bioindicators for dioxin pollution monitoring in the marine coastal environment.     

重金属镉对拟柱孢藻(Cylindrospermopsis raciborskii)PSII及能量分配特征的影响效应

为了研究重金属镉对藻类光合系统毒性效应及能量分配特征,以拟柱孢藻(Cylindrospermopsis raciborskii)为研究对象,分析了不同镉浓度下拟柱孢藻的生长、单位叶绿素a含量、光合系统Ⅱ(PSⅡ)叶绿素荧光及其能量分配模型.结果表明:不同浓度镉培养拟柱孢藻96 h后,0.2 mg/L Cd~(2+)对拟柱孢藻单位细胞叶绿素a含量及PSⅡ活性无显著影响,而0.5mg/L Cd~(2+)培养时拟柱孢藻的单位细胞叶绿素a含量为对照组的62.97%.当Cd~(2+)浓度高于5 mg/L时,拟柱孢藻的PSⅡ活性受到抑制并导致表征量子产额分配的φEo和ψo分别下降了15.35%和13.52%.此外,当Cd~(2+)浓度高于5 mg/L时,藻体PSⅡ能量分配表现为TRO/CSm和ETO/CSm显著减小,DIO/CSm差异不显著,类囊体能量分配表现为ABS/RC、TRO/RC和DIO/RC显著增大,明显区别于0.2、0.5和1 mg/L Cd~(2+)处理组.这些结果表明PSⅡ反应中心是镉对拟柱孢藻光合作用影响的重要作用位点,且Cd~(2+)高于5 mg/L时显著影响了拟柱孢藻PSⅡ反应中心并且抑制了PSⅡ电子传递链供体侧的电子传递,而拟柱孢藻则可能通过改变PSⅡ的能量分配来保护和缓解镉对其光合结构的损伤.     

Cloning and expression of alpha class glutathione S-transferase gene from the small hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

In order to assess its potential as a biomarker of aquatic pollution, an alpha class glutathione S-transferase gene (GSTα gene) was cloned from the small hermaphroditic fish Rivulus marmoratus. The R. marmoratus GSTα gene spanned 1.3 kb, consisting of 6 exons encoding 221 amino acid residues. It showed high similarity to zebrafish GST. We named this R. marmoratus GSTα gene as rm-GSTα. The cDNA of the rm-GSTα gene was also investigated for its phylogeny, tissue-specific and chemical-induced expression. Rm-GSTα was subcloned into a 6 × His-tagged pCR^®T7 TOPO TA expression vector to produce the recombinant 6 × His-tagged rm-GST protein. This will be used in future to raise an rm-GSTα antibody for use in the study of phase II metabolism involved in detoxification. We also exposed R. marmoratus to 300 μg/l of 4-nonylphenol in water, and found approximately 4-fold induction of R. marmoratus GSTα mRNA in the treated animals. In this paper, we discuss the characteristics of the R. marmoratus GSTα gene as well as its potential use in relation to environmental pollution.     

Does sediment resuspension by storms affect the fate of polychlorobiphenyls (PCBs) in the benthic food chain? Interactions between changes in POM characteristics, adsorption and absorption by the mussel Mytilus galloprovincialis

Environmental parameters and gross sedimentation rates (GSR) were monitored at a fixed site located in the Bay of Banyuls-sur-Mer (NW Mediterranean), between March 1997 and April 1998, together with the main biochemical characteristics of both sedimenting and sedimented particulate organic matter (POM). Three storms which occurred during this time period resulted in natural sediment resuspension. This is indicated by the corresponding increase in GSR and a decrease in the enzymatically hydrolysable amino acids/totally hydrolysable amino acids ratio (EHAA/THAA), within the sedimenting POM. Only the strongest storm resulted in (1) a transitory increase in fine-grained particles, (2) concomitant increases in organic carbon, carbohydrates, lipids and THAA, and (3) a decrease in the EHAA/THAA ratio in surficial sediments. For most of the assayed parameters, the values recorded after the December 1997 storm corresponded to extremes for the whole period under study. This emphasises the role of storms in controlling the characteristics of sedimented and sedimenting POM.Ten sediment types, with contrasting biochemical characteristics, were selected for experiments; these were based on the results of the monitoring survey and were used during adsorption and absorption experiments involving ¹⁴C tetrachlorobiphenyl (TCB). Adsorption rates differed significantly between the sediment types, but did not correlate with any of the assayed biochemical parameters. Absorption efficiency by the mussel Mytilus galloprovincialis also differed between the sediment types; it correlated positively with all the assayed biochemical parameters, except lipids. Comparison between the magnitudes of the increase in GSR, together with the decrease in absorption efficiency during resuspension events, suggests that resuspension tends to enhance the transfer of organic pollutants in the benthic food chain.     

Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata)

This study presents full-length cDNA sequences of CYP1A1 and 1A2, in common minke whale (Balaenoptera acutorostrata) from the North Pacific. Both CYP1A1 and CYP1A2 cDNAs had an open reading frame of 516 amino acid residues, and predicted molecular masses were 58.3 kDa and 58.1 kDa, respectively. The deduced full-length amino acid sequence of CYP1A1 revealed higher identities with those of sheep (86%) and pig (87%), and that of CYP1A2 was most closely related to human (82%) and monkey CYP1A2 (82%) among species from which CYP1A2 has been isolated so far. Differences in certain conserved and functional amino acid residues of CYP1A1 and 1A2 between common minke whale and other mammalian species indicate the possibility of their specific metabolic function. Concentrations of organochlorine compounds (OCs) including PCBs and DDTs analyzed in common minke whale liver showed no significant correlation with hepatic mRNA expression levels of CYP1A1 and CYP1A2, indicating no induction of these enzymes by such OCs.     

葛洲坝至胭脂坝长江干流段江豚活动规律

长江江豚是我国一级保护野生动物,也是长江生态系统的指示性物种,其种群数量恢复情况及活动规律一直备受关注。本文选择长江宜昌段江豚活动频繁的葛洲坝下游至胭脂坝江段为研究区域,于2021年6月-2022年5月采用定点目测、水上流动监测与无人机监测相结合的方法分区监测长江江豚的游泳行为,分析长江江豚的活动规律,构建了长江江豚出水头次的零膨胀泊松回归模型,识别影响长江江豚出水头次的关键因素,建立长江江豚出水头次与各因素间的相关关系。结果表明:(1)葛洲坝至胭脂坝长江干流段监测到最大江豚出水头次为19头次,长江江豚集群规模以2~3头最为常见,占比达58.1%;长江江豚主要表现出4种行为特征,各行为占比从大到小依次为:玩耍＞休息＞摄食＞抚幼。(2)长江江豚在葛洲坝下游近坝区(A区,葛洲坝至至喜长江大桥)出水头次最多,且在秋冬季节累计出水头次多于春夏季节,冬季累计出水头次最高,达252头次。(3)长江江豚出水头次与电站下泄流量呈显著负相关;浊度增大,长江江豚出水的概率减小。本文研究结果对长江江豚生态保护策略及长江十年禁渔效果评估具有重要参考价值。     

太湖湖鲚渔获量变化与生物环境间相互关系

2003-2004年在汉江中游江段共监测654船次,统计渔获物6914．16 kg,生物学测定1682尾鱼,共采集鱼类78 种,隶属18科58属．目前汉江中游渔获物组成和结构与20世纪70年代相比发生了较大变化,翘嘴红鲌、瓦氏黄颡鱼、鲶、 鲢、黄尾鲴、大鳍蠖等在渔获物中比例已经很少;草鱼资源也明显下降;铜鱼、青鱼、蒙古红鲐、鳡、细鳞斜颌鲴、吻鮈 、长吻 鮠、拟尖头红鲌、鳙、鳜等在渔获物中已基本消失;而鲤、鲫、黄颡鱼、长春鳊、赤服鳟等中小型鱼类在渔获物中的比例却相 对上升;渔获物中个体大的鱼减少,低龄鱼及幼鱼个体比重增加．与70年代资料相比,汉江中游鱼类资源已呈衰退趋势．