Fatal attraction: synthetic musk fragrances compromise multixenobiotic defense systems in mussels

We studied interactions of nitromusk compounds musk ketone and musk xylene and polycyclic musks Galaxolide trade mark (HHCB), Celestolide trade mark (ADBI), Tetralide trade mark (AHTN), and Traseolide trade mark (AITI) with multixenobiotic resistance (mxr) transporters in gill tissue of the marine mussel Mytilus californianus (Conrad, 1837). A competitive substrate transport test with rhodamine B was used to assay modulation of transport activity by musks. All tested musks inhibited the transport activity in the low microm range as indicated by increased accumulation of rhodamine B in the tissue. Compared to known substrates of mxr transporters, the effective concentration range was similar to quinidine and about 100 times higher than verapamil. Musk ketone and musk xylene also inhibited efflux of rhodamine B from gill tissue which was loaded with the dye and subsequently incubated with these compounds. Synthetic musk compounds are persistent environmental pollutants in aquatic environments with a high potential to bioaccumulate. As potent inhibitors of mxr transporters they may also play a role as chemosensitizers that enable toxic mxr substrates to accumulate in cells of aquatic organisms.     

Characterisation of choline esterases and their tissue and subcellular distribution in mussel (Mytilus edulis)

Acetylcholinesterase in mussel is potentially a useful biomarker of exposure to organophosphates (OP) in the marine environment. This study looked at cholinesterase activity in subcellular fractions of various tissues from the common mussel, Mytilus edulis. Measurement of enzyme rates demonstrated that although highest specific activity was found in foot 'mitochondrial' fraction, recovery of activity was very low. Gill 'microsomal' fraction had the second highest specific activity with a useful level of recovery and therefore was the most suitable tissue fraction for biomarker applications. Comparative studies of alternative alkylthiocholine substrates and competitive inhibitors suggest there is a single cholinesterase enzyme type present in this fraction. Inhibition of alkylcholine hydrolysis by BW284C51, specific to acetylcholinesterase in vertebrates, showed that cholinesterase activity in gill 'microsomal' fraction is inhibited by this compound but to a lesser extent than in vertebrate AChE. Inhibition of cholinesterase activity by azamethiphos in gill 'microsomal' fraction gave an IC50 of approximately 100 microM and showed both time and concentration dependence. However this indicates a lower potency compared to other animals and it is debatable whether mussel cholinesterase activity is useful as a biomarker of exposure in the field.     

Identification of the multidrug resistance-associated protein (mrp) related gene in red mullet (Mullus barbatus)

Multixenobiotic resistance mechanism (MXR) in aquatic organisms is mediated by the activity of the P-glycoprotein (Pgp) transporter that binds and actively effluxes different chemicals out of cell. In addition to the Pgp, several other, non-Pgp transport proteins have been recently identified in different human and animal tissues. Given their characteristics and tissue distribution we hypothesized that members of the so-called multidrug resistance-associated protein (MRP) family may be expressed in aquatic organisms. This study attempted to identify MRP related genes in different tissues of several marine and freshwater bivalves (Mytilus galloprovincialis, Dreissena polymorpha, Anodonta cygnea) and fish species (Mullus barbatus, Cyprinus carpio, Salmo trutta). Following an alignment of known MRP1 and MRP2 human sequences, as well as the GenBank available mrp2 sequences from different animals, we determined highly conserved regions and used them to design three pairs of consensus primers. Total RNA was isolated, reverse transcribed to cDNA and the obtained cDNAs were PCR amplified with the corresponding primers. The amplified PCR products were sequenced and their homology compared with Pgp and MRP protein sequences from different species. The expression of MRP related mRNA was clearly identified only in liver tissue isolated from red mullet, with homologies at the protein level ranging from 75% to 76%. Described results clearly pointed at the possibility that at least in the red mullet MXR as a general defense mechanism may be mediated by the activities of at least two different types of transport proteins.     

动物化学防御系统的组成及作用机制

化学防御系统是生物抵御环境中化学胁迫和维持内环境稳态的重要机制,由长期进化出的一系列基因家族、蛋白和相关反应通路组成。该系统的核心组分包括:(1)受体和配体激活转录因子,其负责感知有毒化合物;(2)生物转化酶,其通过氧化、还原和结合反应使有毒物质的毒性降低,增加亲水性,使化合物更易排出;(3)外排转运蛋白,其负责将外源物质或生物转化产生的代谢物排出细胞;(4)抗氧化酶,其作用是保护细胞免受外部和内部产生的活性氧或自由基的伤害。本综述对动物化学防御系统的组成及作用机制进行概述,为研究海洋无脊椎动物的化学防御系统提供参考。     

The activation of aromatic amines in some marine invertebrates

Recently we found that the postmitochondrial fraction of marine mussel (Mytilus edulis) digestive gland metabolizes aromatic amines readily but not polycyclic aromatic hydrocarbons.¹ Here we present some properties of similar biotransforming capacity of the mussel Mytilus galloprovincialis and preliminary evidence for the presence of similar exclusive metabolism of aromatic amines in all marine invertebrates tested so far.     

Measurement of DNA single-strand breaks in gill and hemolymph cells of mussels, Mytilus sp., collected on the French Atlantic Coast

DNA single-strand breaks were measured by the comet assay in both gill and hemolymph cells of mussels collected in 3 sampling areas of the French coast (Pointe du Castelli, Pen Bron and Saint-Nazaire Harbour). Whole mussel tissue samples were also collected for the chemical determination of PAH, PCB and heavy metal concentrations. In mussel, a higher level of DNA strand breaks was measured in gill than in hemolymph cells (p < 0.01). Despite a factor of contamination from 2 to 3 between sites, no difference in the extent of mussel DNA strand breaks was shown between sampling locations (p > 0.05), questioning the sensitivity of the assays used in biomonitoring studies.     

Tolerance of five species of tropical marine mussels to continuous chlorination

The paper examines the relative lethal and sublethal response of five important tropical marine mussels (Perna viridis, Perna perna, Brachidontes striatulus, Brachidontes variabilis and Modiolus philippinarum) to different chlorine concentrations varying from 0.25 to 15 mg l(-1). The mussels were observed to co-exist in the cooling water circuits of a coastal power station that adopted intermittent chlorination as a fouling control technique. The five mussel species showed, in response to chlorination, 100% mortality at significantly different exposure times, indicating significant species-specific variability in chlorine tolerance. For example, at 1 mg l(-1) residual chlorine, B. variabilis and P. viridis took 288 and 816 h, respectively, to achieve 100% mortality. The time taken for 100% mortality decreased with increasing chlorine residual concentration. The effect of mussel size (= mussel age) of P. viridis, P. perna, B. striatulus and M. philippinarum on mortality was significant between 1 and 5 mg l(-1) residual chlorine, with larger mussels showing greater resistance than smaller ones. All mussel species showed progressive reduction in physiological activities when chlorine residuals were increased from 0 to 1 mg l(-1). However, species-specific differences in the relative rate of physiological activities were observed. Accordingly, relative reduction in physiological activities in response to chlorination was the lowest in P. viridis and the highest in B. variabilis. The data clearly indicate significant differences in the lethal and sublethal responses of the five mussel species to chlorination. The results, therefore, suggest that for effective fouling control, chlorine treatment against mussels has to be employed judiciously, depending on the mussel species involved.     

Assessing neuroprotective P-glycoprotein activity at the blood-brain barrier in killifish (Fundulus heteroclitus) using behavioural profiles

The neuroprotective role of P-glycoprotein, a multixenobiotic resistance transporter (ABCB1/MDR1), in the blood-brain barrier in fish was examined using behavioural toxicological assays. P-glycoprotein acts as cellular efflux pump to prevent substrates from accumulating in the brain, including environmental contaminants such as ivermectin, a common aquaculture pesticide and mammalian anti-parasitic drug. The behavioural toxicological assays were developed to determine the neuropathological effect of ivermectin in killifish (Fundulus heteroclitus). P-glycoprotein function and thus blood-brain barrier integrity can be compromised by chemosensitizers that inhibit transport activity. Fish treated with ivermectin and the P-glycoprotein inhibitor cyclosporin A were significantly more sensitive and succumbed more rapidly to tilting, lethargy, slowing of pectoral-fin movement and loss of haptic-reactivity compared to fish treated with ivermectin-only. P-glycoprotein inhibition is associated with significantly earlier onset and increased mortality in ivermectin-exposed fish. Our results suggest that P-glycoprotein confers resistance against ivermectin-induced behavioural neuropathology and mortality in fish. This assay provides us with a non-invasive tool to study P-glycoprotein function in the blood-brain barrier and evaluate the behavioural effects of potential environmental neurotoxins.     

Influence of parasitism in controlling the health, reproduction and PAH body burden of petroleum seep mussels

Petroleum seep mussels are often exposed to high hydrocarbon concentrations in their natural habitat and, thus, offer the opportunity to examine the relationship between parasitism, disease and contaminant exposure under natural conditions. This is the first report on the histopathology of cold-seep mussels. Seep mussels were collected by submersible from four primary sites in the Gulf of Mexico, lease blocks Green Canyon (GC) 184, GC-234, GC-233, and Garden Banks 425 in 550–650 m water depth. Five types of parasites were identified in section: (1) gill “rosettes” of unknown affinity associated with the gill bacteriocytes, (2) gill “inclusions” similar to chlamydia/rickettsia inclusions, (3) extracellular gill ciliates, (4) body “inclusions” that also resemble chlamydial/rickettsial inclusions, and (5) Bucephalus-like trematodes. Comparison to shallow-water mytilids demonstrates that: (1) both have similar parasite faunas; (2) seep mytilids are relatively heavily parasitized; and (3) infection intensities are extremely high in comparison to shallow-water mytilids for Bucephalus and chlamydia/rickettsia. In this study, the lowest prevalence for chlamydia/rickettsia was 67%. Prevalences of 100% were recorded from three populations. Bucephalus prevalence was 70% in three of 10 populations. The parasite fauna was highly variable between populations. Some important parasites were not observed in some primary sites. Even within primary sites, some important parasites were not observed in some populations. Bucephalus may exert a significant influence on seep mussel population dynamics. Forty percent of the populations in this study are severely reproductively compromised by Bucephalus infection. Only a fraction of petroleum seep mussel populations are maintaining the entire beta-level population structure of this species. Variation in two parasites, gill ciliates and Bucephalus, explained most of the variation in PAH body burden between mussel populations. PAHs are known to be sequestered preferentially in gametic tissue. Bucephalus would be expected to reduce overall body burden, at high infection intensities, by replacing gametic tissue. PAH concentrations exceeded 1 ppm in 4 of 9 populations, a ratio significantly higher than the 8 of 30 mussel locales in the NOAA Mussel Watch Program. Only five Mussel Watch locales exceeded the highest value for a petroleum seep population. Digestive gland and gill tissue atrophy were not significantly correlated with PAH body burden, even though some populations were characterized by body burdens exceeding 1 ppm, suggesting that seep mussels may not be as sensitive to PAH exposure as are some shallow-water mytilid populations.     

Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay

The single-cell microgel electrophoresis assay or the comet assay was used to evaluate DNA damage of dispersed crude oil on sea urchins (Strongylocentrotus droebachiensis) and mussels (Mytilus edulis L.). Sea urchins were exposed to 0.06 and 0.25 mg/L dispersed crude oil in a continuous flow system, while the mussels were exposed to 0.015, 0.06 and 0.25 mg/L dispersed crude oil. Sea urchin coelomocytes and mussel haemocytes were sampled after 4 and 5 weeks exposure, respectively. In the sea urchin coelomocytes, there was a significant concentration-related increase in the percentage of DNA in comet tail. In mussel haemocytes, there was a significantly higher percentage of DNA in comet tail for all treatments compared to the control. The responses were concentration-related up to 0.06 mg/L oil. The two highest exposure concentrations of mussels were not significantly different from each other. These results indicate that the comet assay can be used for biomonitoring of DNA damage in marine invertebrates following oil contamination.     

Temporal study of estrogenic responses of mussel (Mytilus galloprovinciallis) extracts applied to reporter cell lines

In vitro bioassays would facilitate monitoring of estrogen-like compounds in mussels (Mytilus galloprovincialis) since they tend to accumulate lipophilic compounds in their fat and muscle tissue. However, estradiol (E2) steroid (already identified in mussels) could induce false positive responses. This study focuses on temporal variability in estrogenic responses of mussel extracts and the possible relation of this response with E2 levels. The reproductive cycle and growth were thus monitored for six months in sexed mussels. E2 levels were similar between genders and these levels varied with reproductive stages. human estrogen receptor (hER) activities were similar at all stages except in February. E2 present in male and female extracts accounted for part of the hER activation observed, but no correlation was found between E2 levels and hER activities.     

Bacterial killing by Mytilus hemocyte monolayers as a model for investigating the signaling pathways involved in mussel immune defence

The signaling pathways involved in mussel immune defence were investigated utilizing a model of killing of Escherichia coli by Mytilus galloprovincialis hemocytes in a co-culture setting. In particular, the role played by different mitogen activated protein kinases (MAPKs) and by the production of eicosanoids were investigated utilising specific cell permeant, pharmacological enzyme inhibitors. Hemocyte pretreatment with the p38 MAPK inhibitor SB203580 significantly reduced bacterial killing, whereas PD98059 (an inhibitor of ERK--extracellularly regulated kinase--MAPK activation) had no significant effect. Wortmannin also inhibited bacterial killing, indicating a crucial role for PI3-kinase activation in the immune response. Killing of E. coli was also reduced by inhibitors of both PLA2 and cyclooxygenase activities, indicating that eicosanoid production is involved in mediating the response to bacterial challenge. The results demonstrate that bacterial killing by mussel hemocytes is particularly sensitive to inhibitors of the key steps involved in the transduction of bacterial signals into the host cell. Moreover, these data indicate that the hemocyte bactericidal activity can be suitably utilized not only for identifying the signaling pathways involved in the response to bacterial infection, but also as a potential investigative-toxicology model to test drugs and contaminants for their effect on the overall mussel immune defence.     

褐菖鲉鳃丝EROD生物标志物监测技术的研究与应用

以褐菖鲉为实验鱼类,以其鳃丝EROD为研究对象,进行了室内鳃丝EROD活性测定方法条件的摸索和现场海域石油类污染监测的初步应用研究.室内EROD活性测定条件摸索实验结果表明：褐菖鲉鳃丝EROD活性测定的最适pH值为7.8、最适温度为35℃、最适鳃丝用量为18.8±1.9 mm;在鳃丝总长相近（约20 mm）的条件下,剪切成2 mm长的鳃丝片段与否对于EROD活性无显著差异;反应50 min内EROD具有较高活性,随后呈现下降趋势.2次厦门西海域污染现场监测结果表明：褐菖鲉鳃丝EROD活性与海水石油类含量具有良好的正相关趋势,与对照组比较总体上具有显著的差异.因此,褐菖鲉鳃丝的EROD活性测定方法与褐菖鲉肝的一样,能够作为生物标志物应用于现场海区的石油类污染及其生化效应的监测.     

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.     

Relationship between metal levels in the vent mussel Bathymodiolus azoricus and local microhabitat chemical characteristics of Eiffel Tower (Lucky Strike)

The turbulent mixing of hydrothermal hot fluid with cold seawater creates large chemical gradients at a small spatial scale that may induce variable physiological and biochemical adaptations within the vent fauna. The adaptation to such a variable environment by the vent mussel Bathymodiolus azoricus relies on a dual symbiosis hosted in the gills, and digestion of particulate organic matter. The surrounding environment not only provides the necessary energy sources and suspended organic particles for the vent mussel nutrition, but also potentially toxic compounds such as metals. Our main goal was to see if there is a relation between metal accumulation in mussel organs and the chemical characteristics of their close environment. Mussels were collected at six locations in a cold part of the Eiffel Tower fluid-seawater mixing zone, characterized by distinct chemical compositions. Metals (Cd, Cu, Fe and Zn) and metallothioneins were quantified in the gills and digestive gland. The physiological condition of the sampled mussels was also evaluated using tissues and gill indices. Our study indicates that the accumulation of metals in B. azoricus is related to their spatial distribution and linked to fine scale environmental conditions that influence the physiological status of the organism.     

大竹蛏同工酶组织特异性与多态性初步研究

用不连续垂直聚丙烯酰胺凝胶电泳对大竹蛏(Solen grandis Dunker)外套膜、鳃、性腺、消化腺、斧足5种组织6种同工酶(LDH、ADH、EST、MDH、ACP、SOD)进行组织特异性分析,并对吕四渔场海域大竹蛏自然种群生化遗传多样性进行了初步研究.结果表明:大竹蛏同工酶的活性及酶谱特征具有明显的组织特异性,6种酶在消化腺中的活性最强,LDH、EST、MDH和SOD酶谱具有明显的组织特异性;6种酶在消化腺和斧足中共检测到12个基因位点,其中Ldh-1、Ldh-2、Adh-1、Est-1、Mdh-1、Mdh-2、Mdh-3、Sod-2等8个基因位点为多态,多态位点比例为66.67%,平均每个位点的等位基因数N<,a>为2.5,有效等位基因数(N<,e>)为1.9,预期杂合度(H<,c>)为O.373 7,实际杂合度(H<,o>)为0.333 3,香农信息指数(I)为0.624 2.     

Synchronous fluorescence spectrometry of 1-hydroxypyrene: a rapid screening method for identification of PAH exposure in tissue from marine polychaetes

The uptake of polycyclic aromatic hydrocarbons (PAHs) by marine deposit-feeding invertebrates can be determined by screening for PAH-derived metabolites. We identified 1-hydroxypyrene as the only intermediate metabolite in tissue of four species of deposit-feeding polychaetes, Nereis diversicolor, Nereis virens, Arenicola marina, and Capitella sp. I exposed to pyrene spiked sediment. Synchronous fluorescence spectroscopy (SFS) provides a fast and simple method for both qualitative and quantitative analysis of 1-hydroxypyrene in all four species. The SFS assay was validated using HPLC with ultraviolet detection. A good correlation between 1-hydroxypyrene concentrations determined by the two methods was observed. We used HPLC with fluorescence detection combined with enzymatic hydrolysis of conjugated metabolites to investigate species specific metabolite patterns. A tentative aqueous metabolite identification scheme indicates that Nereid polychaetes predominately make use of glucuronide conjugation whereas Capitella sp. I. and Arenicola marina appear to utilize predominantly sulfate and/or glucoside conjugation. The usefulness of 1-hydroxypyrene as a biomarker for PAH exposure in deposit-feeding invertebrates is discussed.     

海洋无脊椎动物细胞培养与细胞干性的捕获

细胞系的构建在生物学领域具有重要意义,与其他动物(如昆虫)不同,经过数十年的发展,海洋无脊椎动物细胞研究仍停留在原代培养水平,没有建立起克隆化的永生细胞系.本文主要介绍了近年来海洋无脊椎动物细胞培养的研究进展,还介绍了一个基本原理,即海洋无脊椎动物永生细胞系的建立,可以通过捕获细胞的干性而实现.捕获细胞干性的第一条途径...