Time-course evaluation of ROS-mediated toxicity in mussels, Mytilus galloprovincialis, during a field translocation experiment

Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study several biomarkers were investigated in marine mussels caged for 4 weeks (June–July 2001) into an industrialized harbour of NW Italy. Organisms were collected at different time-intervals to better characterize the sensitivity, temporal variations and interactions of analysed responses. Besides single antioxidants the total oxyradical scavenging capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralize specific forms of radicals: these data were further integrated by measurement of DNA integrity, oxidized bases and the impairment of lysosomal membrane stability in haemocytes. Results showed a biphasic trend for single antioxidants and TOSC, with an increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment.     

Total oxyradical scavenging capacity and cell membrane stability of haemocytes of the Arctic scallop,Chlamys islandicus,following benzo(a)pyrene exposure

Industrial activities, notably oil and gas industries, are expanding in the Arctic. Most of the biomarkers were developed using temperate organisms living at temperatures above 10 degrees C. Little is known about the biomarker responses of organisms living between -1.88 and 5 degrees C. Therefore, assessment of the toxicity of chemicals to cold-water adapted species is required. In this study, the Arctic scallop, Chlamys islandicus, was selected as a key species for bio-monitoring because of wide distribution in Arctic waters and its commercial value. Test animals, stored in seawater at 2 degrees C, were injected with benzo(a)pyrene (diluted in cod liver oil 5 mg ml(-1)) in the adductor muscle every 24 h for four days giving a final dose of 0, 74 and 90.6 mg kg(-1) wet weight for control, low and high dose, respectively. The biomarkers used were total oxyradical scavenging capacity (TOSC) in the digestive gland and cell membrane stability of haemocytes. TOSC values were significantly reduced (ca. 30%) in exposed groups (P < 0.05), indicating a depletion in oxyradical molecular scavengers. The antioxidant defences appeared to be overwhelmed by the reactive oxygen species as the plasma membranes of haemocytes were destabilised (P < 0.05) probably due to lipid peroxidation. These data indicate that reactive oxygen species (ROS) were produced by Arctic scallops via the metabolisation of benzo(a)pyrene at 2 degrees C.     

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic Sea

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, was developed to evaluate the potential impact of an offshore gas platform in the central Adriatic Sea. Reference organisms were collected on a seasonal basis from an unpolluted site and transplanted for four weeks in both the sampling area and to the investigated platform. Chemical analyses of trace metals in mussel tissues were integrated with a multi-biomarker approach for the early detection of biological responses at several cellular targets. Induction of metallothioneins, peroxisomal proliferation and activity of acetylcholinesterase were measured as markers for specific classes of chemicals. Special attention was given to oxyradical metabolism and appearance of oxidative-mediated toxicity to reveal a more general onset of cellular disturbance. In addition to individual antioxidants (superoxide dismutase, catalase, glutathione S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, and levels of total glutathione), the total oxyradical scavenging capacity (TOSC) allowed a quantification of the overall capability to neutralize specific forms of intracellular reactive oxygen species (ROS; i.e. peroxyl and hydroxyl radicals). Cellular damages were evaluated as lysosomal destabilization (membrane stability, accumulation of lipofuscin and neutral lipids), lipid peroxidation products (malondialdehyde) and DNA integrity (strand breaks and micronuclei); the air survival test was finally applied to evaluate the overall physiological condition of mussels. Concentration of trace metals (As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn) revealed only limited variations in transplanted mussels during various experimental periods and such changes appeared partly related to natural fluctuations. Among biological responses, variations of antioxidants and lysosomal stability were confirmed as sensitive early warning signals for biological disturbance of both natural and anthropogenic origin. The presented protocol with caged mussels allowed marked biological effects caused by the investigated platform to be excluded, and represented a useful approach that is easy to extend for monitoring the impact of offshore activities in the Adriatic sea.     

Total oxyradical scavenging capacity and cell membrane stability of haemocytes of the Arctic scallop, Chlamys islandicus, following benzo(a)pyrene exposure

Industrial activities, notably oil and gas industries, are expanding in the Arctic. Most of the biomarkers were developed using temperate organisms living at temperatures above 10 °C. Little is known about the biomarker responses of organisms living between −1.88 and 5 °C. Therefore, assessment of the toxicity of chemicals to cold-water adapted species is required. In this study, the Arctic scallop, Chlamys islandicus, was selected as a key species for bio-monitoring because of wide distribution in Arctic waters and its commercial value. Test animals, stored in seawater at 2 °C, were injected with benzo(a)pyrene (diluted in cod liver oil 5 mg ml⁻¹) in the adductor muscle every 24 h for four days giving a final dose of 0, 74 and 90.6 mg kg⁻¹ wet weight for control, low and high dose, respectively. The biomarkers used were total oxyradical scavenging capacity (TOSC) in the digestive gland and cell membrane stability of haemocytes. TOSC values were significantly reduced (ca. 30%) in exposed groups (P<0.05), indicating a depletion in oxyradical molecular scavengers. The antioxidant defences appeared to be overwhelmed by the reactive oxygen species as the plasma membranes of haemocytes were destabilised (P<0.05) probably due to lipid peroxidation. These data indicate that reactive oxygen species (ROS) were produced by Arctic scallops via the metabolisation of benzo(a)pyrene at 2 °C.     

Total oxyradical scavenging capacity of the deep-sea amphipod Eurythenes gryllus

Environmental concern for the deep-sea ecosystem is increasing as contaminants, originating from anthropogenic activities, have been detected in deep-sea biota. However, little is known on the xenobiotics metabolising capability of deep-sea fauna. In this study, the deep-sea amphipod Eurythenes gryllus was selected as sentinel species to measure the total oxyradical scavenging capacity (TOSC). Individuals of E. gryllus were sampled at 2000 m depth in the Arctic Ocean. The TOSC assay was measured on the cytosolic fraction and the soluble fraction (3 kDa) of the digestive gland and on the cell-free haemolymph toward peroxyl, hydroxyl and peroxynitrite radicals according to the method of Winston et al. [Free Radical Biology and Medicine 24 (3) (1998) 480] and Regoli and Winston [Toxicology and Applied Pharmacology 156 (1999) 96]. These results provide the first baseline data set for total antioxidant capacity in a deep-sea amphipod.     

Total oxyradical scavenging capacity of the deep-sea amphipod Eurythenes gryllus

Environmental concern for the deep-sea ecosystem is increasing as contaminants, originating from anthropogenic activities, have been detected in deep-sea biota. However, little is known on the xenobiotics metabolising capability of deep-sea fauna. In this study, the deep-sea amphipod Eurythenes gryllus was selected as sentinel species to measure the total oxyradical scavenging capacity (TOSC). Individuals of E. gryllus were sampled at 2000 m depth in the Arctic Ocean. The TOSC assay was measured on the cytosolic fraction and the soluble fraction (3 kDa) of the digestive gland and on the cell-free haemolymph toward peroxyl, hydroxyl and peroxynitrite radicals according to the method of Winston et al. [Free Radical Biology and Medicine 24 (3) (1998) 480] and Regoli and Winston [Toxicology and Applied Pharmacology 156 (1999) 96]. These results provide the first baseline data set for total antioxidant capacity in a deep-sea amphipod.     

Long-term effects of tributyltin compounds on the baltic amphipod, Gammarus oceanicus

Amphipod larvae, Gammarus oceanicus, were exposed for 8 weeks to bis(tributyltin) oxide (TBTO) or tributyltin fluoride (TBTF), or for 5 weeks to leachates from tributyltin-containing antibiofouling paints. No larvae survived 8 weeks' exposure to 3 μg/litre TBTO or TBTF. Differential larval survival occurred during exposure to 0·3 gmg/litre of these same compounds. Paint leachates were shown to cause similar toxicity responses at comparable aqueous tributyltin concentrations. An examination of sublethal responses showed slight decreases in growth for larvae exposed to TBTO and to paint leachates, although responses at tributyltin levels below 1 μg/litre were not marked. Whole-animal oxygen consumption rates were not affected by low tributyltin exposure levels. While these studies demonstrate low level toxicity of tributyltins, effects on oxygen consumption and growth do not indicate a mode of action for these compounds.     

Oxidative stress in ecotoxicology: from the analysis of individual antioxidants to a more integrated approach

An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxicity. While the analysis of individual antioxidants is useful for their sensitivity and to understand the mode of action of a stressor, the integration with the analysis of the total antioxidant capacity provides a more holistic assessment of the overall biological significance of such variations. TOSC has a greater predictive value on the health condition of the organisms and allows to discriminate the different role of specific ROS in oxidative stress syndrome.     

Cellular responses of oysters, Crassostrea virginica, to metal-contaminated sediments

Elevation of metal concentrations in coastal environments associated with anthropogenic enrichment pose a significant threat to estuarine organisms. The purpose of this study was to evaluate the relationships between cellular responses that may be potentially valuable as indicators of chronic stress and metal-contaminated sediments. For these studies, hatchery-reared juvenile oysters were deployed in situ at 15 sites for approximately 1 month around Charleston Harbor, SC. The effects on lysosomal destabilization and glutathione concentrations were determined; and the relationships between the cellular responses and sediment metal concentrations were described. Both single metal and multiple metal parameters (based on total metal concentrations, aluminum normalizations, and summed sediment quality guidelines) were considered. Generally, significant correlations were observed for individual metal analytes and multiple metal parameters. Since many of the individual metal analytes covary, the responses may reflect overall contaminant loading rather than responses to individual metals. Methods for estimating overall contaminant loading based on multiple analytes provide a more realistic estimate of potential adverse effects.     

Antioxidant efficiency and detoxification enzymes in spotted dogfish Scyliorhinus canicula

Although elasmobranchs are widely distributed species, commonly found on sandy, gravely or muddy bottoms, several biological aspects of their metabolism still remain poorly investigated. In this work the efficiency of antioxidant system and detoxification enzymes were investigated in the coastal spotted dogfish Scyliorhinus canicula and in the red mullet Mullus barbatus for comparison with a teleost species. Organisms were sampled during a bottom trawl survey and analyzed for the biotransformation activity (EROD), levels of metallothioneins, catalase, glutathione S-transferases and total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals. EROD activity in the elasmobranchs was more than one order of magnitude lower than in the red mullets, while similar levels of metallothioneins were measured in these species. S. canicula showed significantly lower antioxidant enzymes and a more reduced efficiency in neutralizing *OH; on the other hand the scavenging capability toward ROO* was comparable in S. canicula and M. barbatus.     

Field application of biochemical markers and a physiological index in the mussel,Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy)

A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter.     

Assessment of environmental quality and inland water pollution using biomarker responses in caged carp (Cyprinus carpio): Use of a bioactivation:detoxication ratio as a Biotransformation Index (BTI)

In the present study the bioaccumulation of poly chlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) as well as the responses of a suite of biochemical parameters were investigated in a standardized carp (Cyprinus carpio) fish line. Carps were caged for 2 to 8 weeks at two Amsterdam freshwater sites: the relatively unpolluted Outer IJ and the heavily polluted Volgermeerpolder. The primary objective of the experiments was to develop a reliable and reproducible method to monitor the inland water pollution and to classify the environmental quality of freshwater sites. The highest tissue levels of organic trace pollutants were generally found in carp that was caged for at least four weeks at the polluted site. Hepatic phase I enzymes showed the highest pollution-induced responses, while phase II enzymes and serum transaminases were less sensitive. The antioxidant enzymes showed virtually no response to pollutants. In order to assess effects on the balance between bioactivation and detoxication the ratio between phase I (EROD) and phase II (GST or UDPGT) activities was introduced as a biotransformation index (BTI). Highest BTI values were observed in carp that was caged for 4 to 6 weeks at the polluted site. The BTI values decreased after eight weeks of exposure due to elevated phase II activities.     

鳗弧菌灭活疫苗对牙鲆外周血细胞吞噬活性的影响

牙鲆(Paralichthys olivaceus)是我国名贵的海产鱼类，也是我国北方沿海地区大规模增养殖的经济鱼种之一。目前随着牙鲆养殖业的蓬勃发展,养殖规模也不断扩大,养殖中各种病害的爆发也相继增多、由此给养殖业户造成了巨大的经济损失。为此科学家们研究出了各种防病、治病方法。其中,通过接种疫苗对鱼类病害进行免疫防治被认为是最重要、有效和绿色环保的方法之一,许多针对鱼类病原的灭活、减毒和基因工程疫苗相继研制成功,这些疫苗的应用在不同程度上都可以提高鱼类的抗病性,减少发病率。作者利用制备鳗弧菌灭活疫苗对牙鲆进行注射免疫的方法,从而观察牙鲆外周血细胞吞噬活性的变化，探讨了鱼类非特异性细胞免疫的保护机制,为今后进一步深入研究提供了可靠依据。     

Geochronology and geochemistry of lavas from the 1996 North Gorda Ridge eruption

Radiometric dating of three North Gorda Ridge lavas by the ²¹⁰Po–²¹⁰Pb method confirms that an eruption occurred during a period of increased seismic activity along the ridge during late February/early March 1996. These lavas were collected following detection of enhanced T-phase seismicity and subsequent ocean bottom photographs documented the existence of a large pillow mound of fresh-appearing lavas. ²¹⁰Po–²¹⁰Pb dating of these lavas indicates that an eruption coinciding with this seismicity did occur (within analytical error) and that followup efforts to sample the recent lava flows were successful. Compositions of the three confirmed young lavas and eleven other samples of this contiguous “new flow” sequence are distinct from older lavas from this area but are variable at a level outside analytical uncertainty. These intraflow variations can not easily be related to a single, common parent magma. Compositional variability within the new flow is compared to that of other recently documented individual flow sequences, and this comparison reveals a strong positive correlation of compositional variance with flow volumes spanning a range of >2 orders of magnitude. The geochemical heterogeneity in the North Gorda new flow probably reflects incomplete mixing of magmas generated from a heterogeneous mantle source or from slightly different melting conditions of a single source. The compositional variability, range in sample ages (up to 6 weeks) and range in active seismicity (4 weeks) imply that this relatively large flow was erupted over an interval of several weeks.     

Biochemical and histochemical responses to environmental contaminants in clam, Tapes philippinarum, transplanted to different polluted areas of Venice Lagoon, Italy.

In this study the clam, Tapes philippinarum, a filter-feeding bivalve living in soft bottoms, was used to investigate the water/sediment pollution in Venice Lagoon (Venice, Italy), a heavily urbanised and industrialised area. To this end, clams collected from indigenous populations in a clean farming area (Val Dogà) were transplanted into a relatively low contaminated site (Palude della Rosa) and in a heavily polluted area (Porto Marghera), for 5 weeks. A range of possible induced stress indices were measured, on the basis of potential toxicity mechanisms, including specific enzymatic activities [Bap hydroxylase and NADPH cytochrome c reductase, superoxide dismutase, catalase, glutathione peroxidase and propionaldehyde dehydrogenase (PDH)] and general cellular responses (lysosomal latency). Moreover, chemical analyses (polychlorinated biphenyls, polycyclic aromatic hydrocarbon, DDTs) were carried out on sediments and animal tissues at the beginning and end of the transplantation experiments. The chemical results show an active bio-accumulation activity from sediment in clams translocated to the most polluted area. Correspondingly, biological data indicate marked effects on PDH activity and latency in T. philippinarum from the same site. In contrast, no similar pattern has been observed in the other considered indices, possibly due to antagonistic effects of the complex contaminant mixture present in the environment.     

Semipermeable membrane devices link site-specific contaminants to effects: Part 1 - Induction of CYP1A in rainbow trout from contaminants in Prince William Sound, Alaska

Extracts from semi-permeable membrane devices (SPMDs) deployed on beaches in Prince William Sound (PWS), Alaska, were used to evaluate if complex contaminant mixtures from different sources can be distinguished by the resulting cytochrome P450 1A (CYP1A) activity in exposed test animals. Deployment sites included canneries, salmon hatcheries, and beaches where lingering oil remains from discharges during the 1964 earthquake or the 1989 Exxon Valdez oil spill. Other sites were selected at random to evaluate region-wide contaminant inputs or were located in salmon streams to evaluate contaminants carried and released by migrating salmon carcasses following reproduction. Following standard deployments of approximately 28 d, an aliquot of the accumulated contaminants was intraperitoneally injected without cleanup into juvenile rainbow trout (Oncorhynchus mykiss). After 2 d and 7 d, the activity of CYP1A was measured by the ethoxyresorufin-o-deethylase (EROD) assay. Exposure to extracts from the oiled sites and one hatchery site with numerous creosote pilings elicited strong EROD responses, whereas fish exposed to salmon stream extracts elicited weak but significant responses during late summer compared to late spring. Responses from the other sites were not significant, indicating contaminants from these sources are unlikely to cause CYP1A induction in resident biota. Rather than simply assessing extant contaminants, this method evaluates the potency of the different sites for bringing about aryl hydrocarbon receptor responses in resident biota.     

饲料中过量添加晶体蛋氨酸和蛋氨酸羟基类似物对大菱鲆生长、饲料利用和抗氧化反应的影响

以初始体质量为(5.49±0.01)g的大菱鲆幼鱼为实验对象,探讨饲料中过量添加晶体蛋氨酸或蛋氨酸羟基类似物对大菱鲆生长、饲料利用和抗氧化反应的影响.以白鱼粉和豆粕为主要蛋白源,饲料中同时添加0.25％ DL-蛋氨酸和0.3％蛋氨酸羟基类似物钙盐(MHA)作为对照,在此基础上分别添加0.5％ DL-蛋氨酸、1.25％ DL-蛋氨酸、0.59％MHA、1.49％ MHA、1.25％ DL-蛋氨酸+0.85％柠檬酸钙,配制出6种实验饲料,使饲料中蛋氨酸和蛋氨酸羟基类似物(HMTBA)含量分别是1.35％、1.83％、2.50％、1.45％、1.37％、2.51％和0.22％、0.22％、0.22％、0.63％、1.38％、0.29％.养殖试验在室内流水系统中进行,持续10周,每个处理设5个重复,每个重复放养大菱鲆幼鱼30尾.结果显示,过量添加DL-蛋氨酸和HMTBA对大菱鲆幼鱼生长无显著影响(P＞0.05).过量添加DL-蛋氨酸和HMTBA对大菱鲆体组分、肥满度(CF)、脏体比(VSI)和肝体比(HSI)均无显著影响(P＞0.05),但柠檬酸钙添加组(处理6)体蛋白含量与基础饲料组相比显著降低(P＜0.05).对大菱鲆肝脏过氧化氢酶( CAT)、超氧化物歧化酶(SOD)和肝脏硫代巴比妥酸反应产物量(TBARS)进行测定,各处理组间均无显著性差异(P＞0.05).结果显示,在该实验条件下,过量添加晶体蛋氨酸(1.31～1.73倍)和HMTBA(1.32～1.75倍)对大菱鲆幼鱼的生长、饲料利用和抗氧化反应没有显著影响.     

蓝藻钙信号的研究进展

近年来对钙离子在蓝藻信号传导中的潜在作用进行了研究。大量证据表明,蓝藻能够“感知”与“区分”不同的环境刺激,并以钙瞬变的形式产生反应。这是不同的环境刺激所引起的钙离子的内流或外流的结果。由氮缺乏所引起的钙信号对丝状蓝藻鱼腥藻异型胞的分化非常关键。鱼腥藻PCC7120中钙结合蛋白（CcbP）的发现为钙信号在蓝藻中的作用提供了进一步的证据,CcbP的降解或表达下调是氮缺乏时钙信号产生的主要原因。但是与真核生物相比,蓝藻钙信号的编码与解码机制还不清楚。因此,为了解钙信号如何在蓝藻中发挥作用,还要进行系统的、深入的研究,特别是从细胞水平了解钙信号的动力学特征。     

Antioxidant efficiency and detoxification enzymes in spotted dogfish Scyliorhinus canicula

Although elasmobranchs are widely distributed species, commonly found on sandy, gravely or muddy bottoms, several biological aspects of their metabolism still remain poorly investigated. In this work the efficiency of antioxidant system and detoxification enzymes were investigated in the coastal spotted dogfish Scyliorhinus canicula and in the red mullet Mullus barbatus for comparison with a teleost species.Organisms were sampled during a bottom trawl survey and analyzed for the biotransformation activity (EROD), levels of metallothioneins, catalase, glutathione S-transferases and total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals.EROD activity in the elasmobranchs was more than one order of magnitude lower than in the red mullets, while similar levels of metallothioneins were measured in these species. S. canicula showed significantly lower antioxidant enzymes and a more reduced efficiency in neutralizing ^√OH; on the other hand the scavenging capability toward ROO^√ was comparable in S. canicula and M. barbatus.