Partial purification and properties of cytochrome P450 from digestive gland microsomes of the common mussel, Mytilus edulis L.

Cytochrome P450 from the digestive gland of M. edulis was partially purified by sodium cholate solubilization, 4–15% polyethylene glycol fractionation, and octyl-Sepharose affinity, DEAE-Sephacel ion-exchange and hydroxylapatite chromatography (yields of up to 7–10%). Three peaks were resolved by DEAE-Sephacel chromatography (termed peaks 1–3). P450 specific content was increased from 26 to 800 pmol per mg protein, and the ratio of P450 content to NADPH-cytochrome c (P450) reductase activity reduced by a factor of 250. Oxidised spectrum λmax of P450 was 410.5 ± 1.5 nm. Type II difference spectra were seen with both type II (clotrimazole, metyrapone) and type I (α-naphthoflavone, 7-ethoxycoumarin) compounds. Western blotting with polyclonal anti-P4501A from perch (Perca fluviatilis) gave a single band of approximately 54 kDa molecular weight. A reconstituted system containing peak 2 or 3, rat liver P450 reductase, lipid and NADPH metabolised benzo[a]pyrene to diones, diols, phenols and putative protein adducts. Peak 2 plus cumene hydroperoxide was indicated to produce diones and protein adducts only. Peak 2 alone was indicated to produce diones and phenols. The major free metabolites in all cases were diones (53–100%). The results indicate the existence of a P4501A-like enzyme in M. edulis, possibly with unusual properties as indicated by the difference spectra, metabolism in absence of NADPH and added P450 reductase, and predominance of diones.     

Metabolism and mutagenicity of 4-nitroquinoline N-oxide by microsomes and cytosol of digestive gland of the mussel Mytilus edulis L.

The modulation of the mutagenicity of 4-nitroquinoline N-oxide (4-NQO) by M. edulis digestive gland cytosolic and microsomal fractions was studied by monitoring the induction of the umu operon by 4-NQO in Salmonella typhimurium 1535/pSK1002. The mutagenicity of 4-NQO was increased by M. edulis cytosol and microsomes in dose-dependent manner with respect to protein and toxicant concentration. The mutagenic enhancement reflected increased NAD(P)H-dependent oxygen consumption by microsomes in the presence of 4-NQO and enhanced nitroreductase activity in cytosol. The former was stimulated by azide, indicating 4-NQO-enhanced oxyradical production by microsomes, and the latter was inhibitable by dicoumarol, indicating involvement of DT-diaphorase in nitroreduction by M. edulis cytosol. Neither dicoumarol nor allopurinol (inhibitors of DT-diaphorase and xanthine oxidase, respectively) affected the enhanced mutagenic expression of 4-NQO by cytosol. The results support the possibility of oxidative stress as a pollution-mediated mechanism of toxicity.     

In vivo metabolism of aromatic amines by the bay mussel, Mytilus edulis

The hazard posed to aquatic organisms by exposure to aromatic amines will depend on their ability to activate or detoxify these potentially genotoxic compounds. We studied the metabolism of four aromatic amines (p-toluidine, aniline, 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF)) in Bay mussels (Mytilus edulis) to gain an understanding of their metabolic capabilities in vivo. Mussels rapidly accumulated these compounds from the water column and depurated the majority of their steady-state body burdens as metabolites and unmetabolized parent compounds. The biotransformation pathways of [¹⁴C]-labeled parent compounds were determined by analyses of depurated metabolites and tissue residues. Biotransformation of p-toluidine and aniline resulted in the formation of their corresponding N-acetylated detoxification products. Mussels converted small amounts of 2-AAF into a deacetylated product (2-AF) and a mutagenically active metabolite (N-hydroxy-2-AF). 2-AF was extensively detoxified via N-acetylation, which limited the amount of primary amine available for activation.     

Partitioning of metals among metal-binding proteins in the bay mussel, Mytilus edulis

Metabolism of metals was assessed in populations of Mytilus edulis exposed to metals in the laboratory and in the field. Chronic exposure to metals in both environments resulted in increased concentrations of metals in both the low molecular weight metal-binding protein fraction, which contains metallothioneins, and in the high molecular weight metal-binding protein fraction, which contains metalloenzymes. Our results from analysis of laboratory-exposed populations and from monitoring indigenous and transplanted populations indicate that the capacity for production of metallothioneins is limited and that the quantities present differ greatly with seasonal changes in the environment.     

Response of the blue mussel Mytilus edulis L. following exposure to PAHs or contaminated sediment

The polycyclic aromatic hydrocarbons fluoranthene and benzo(a)pyrene significantly reduced the feeding rate of mussels. For both compounds the tissue concentration resulting in a 50% reduction of the clearance rate (TEC₅₀) was calculated. At high tissue concentrations both aromatic compounds reduced the tolerance of mussels to aerial exposure, whereas at low tissue concentrations an improved response was noticed. The activities of the antioxidant enzymes Superoxide dismutase (SOD) and catalase were elevated only at low tissue concentrations of fluoranthene and benzo(a)pyrene. At the highest measured tissue concentrations the activity of both enzymes was reduced, possibly due to a narcotic effect. The reproductive success rate of mussels appeared to be affected negatively by the investigated hydrocarbons. The results of a pilot experiment indicate that mussels can be used also for the testing of contaminated sediments.     

Biochemical and ultrastructural observations of long-term silver accumulation in the mussel, Mytilus edulis

The cellular distribution and chemical forms of Ag were determined in mussels after accumulation of the metal from sea water. The major accumulations were either in the vacuoles of connective tissue macrophages, where it was associated with S, or in deposits in the fibrillar layer of the basement membranes of the digestive diverticulum and kidney epithelia, where it was bound to the sulphydryl and sulphate groups of glycoproteins and proteoglycans. In acutely exposed animals, about 10 % of the accumulated Ag was found in a (Ag, Cu)-binding protein with characteristics indicating that it may be a metallothionein. There was a concomitant increase in the body Cu levels after Ag exposure and 40 % of this was bound to this low molecular weight metal-binding protein.     

Responses of the mussel Mytilus edulis to copper and phenanthrene: Interactive effects

Most investigations of the responses of marine organisms to xenobiotics have concentrated on single contaminants and little is known of possible interactive effects of different classes of xenobiotics. As these latter seldom occur in environmental isolation, it is important to understand any interactions (synergistic or antagonistic) which may occur. This problem has been approached in the mussel Mytilus edulis by exposing estuarine mussels to copper (20 μg litre⁻¹) and phenanthrene (100 μg litre⁻¹) both individually and in combination, and measuring cytochemical subcellular and physiological responses after 3 days exposure and 3 days and 12 days recovery period. Results showed that mussels accumulated both xenobiotics during 3 days exposure. Depuration of copper was complete in 3 days recovery period, while loss of phenanthrene ranged from 30% to 70% of the concentration reached after 3 days exposure. There were no interactive effects on depuration.Both copper and phenanthrene reduced lysosomal hydrolase latency in digestive cells, and copper appeared to have a synergistic effect in preventing recovery of latency of lysosomal N-acetyl-β-hexosaminidase during the recovery period. There was evidence, in the digestive cells, of an antagonistic effect of copper on stimulation of activity of the microsomal respiratory chain (measured as NADPH-neotetrazolium reductase) by phenanthrene. Stimulation of this system by phenanthrene persisted after 12 days recovery period. There was a synergistic interaction of copper and phenanthrene on elevation of oxygen consumption and ammonium excretion. Clearance rates and scope for growth (physiological condition) were depressed by copper but not by phenanthrene after 3 days exposure.These findings are discussed in terms of known effects of copper and phenanthrene and the interactions are considered in terms of environmental effects measurements.     

Effects of organic toxicants on the anoxic energy metabolism of the mussel Mytilus edulis

The anoxic rates of heat dissipation by mussels (Mytilus edulis) were enhanced after exposure to pentachlorophenol (PCP) and tributyltin (TBT), both known uncouplers of oxidative phosphorylation. The degree of metabolic activation under anoxia was dependent upon the amount of toxicant accumulated in the tissues, but the concentration-response relationship was different to that under aerobic conditions. Biochemical measurements indicated that the anaerobic metabolic pathways were significantly disturbed by PCP and TBT. There was a decline in succinate, an increase in the fumarate: succinate ratio, and an increase in the accumulation of lactate, indicating a shift from succinate to lactate anaerobic pathways. A consequence of organic toxicant exposure was a reduction in anoxic survival time of mussels.     

Recovery of Mytilus edulis L. from chronic oil exposure

Previous laboratory studies¹ have shown that physiological and cellular processes of Mytilus edulis are affected by exposure to low and environmentally realistic concentrations of oil. However, there is little information concerning the rate of recovery from oil exposure and the extent to which physiological recovery may be related to the depuration of hydrocarbons from the tissues. The present study has shown a marked reduction in the feeding rate and scope for growth of mussels exposed to two concentrations of diesel oil (30 and 130 μg/litre) for 8 months. During recovery from oil exposure the depuration of hydrocarbons from the tissues was concomitant with the recovery of physiological performance. Mussels exposed to high oil concentrations (‘high-oil’ mussels) were found to recover more rapidly than those exposed to low oil concentrations (‘low-oil’ mussels), both in terms of depuration and scope for growth, and there was evidence of ‘catch-up’ growth. Recovery of both low- and high-oil mussels was complete after approximately 55 days.     

Interactions of cadmium with the reproductive cycle of Mytilus edulis L.

Sublethal effects of cadmium on reproduction can have detrimental consequences for the long-term survival, even of species with high LC₅₀ values. The sea mussel, Mytilus edulis, is well-known for its use in biomonitoring work due to its relatively low sensitivity towards many toxicants, including cadmium.¹ Because little is known on sublethal effects of this metal in invertebrates,^2–6 we have studied these effects, with emphasis on reproduction, by exposing mussels to 100 ppb cadmium in sea water. This concentration significantly inhibits follicle development in both male and female gonads, but has subsequently a stimulating effect on the spawning frequency. Due to these opposite effects the consequences of cadmium stress for gamete production are expected to be small, as the average number of gametes released by spawning animals was not significantly changed.     

CYP1A- and CYP3A-immunopositive protein levels in digestive gland of the mussel Mytilus galloprovincialis from the Mediterranean Sea

CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6β-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r²=0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.     

Quantitative assessment of intertidal blue mussel (Mytilus edulis L.) stocks: combined methods of remote sensing, field investigation and sampling

The aim of this investigation was to develop appropriate methods for quantitative assessment of blue mussel beds. Combined methods of remote sensing, ground truth investigation and sampling in the field were applied to mature and young intertidal mussel beds. Three variables were measured to obtain reliable quantitative estimates of biomass and abundance: the total area covered by a bed; the cover, i.e. the area of mussel-covered patches in relation to the total mussel bed area; and the proportion, i.e. the area covered by mussels within the patches in relation to the total area of mussel patches. Cover and proportion of intertidal blue mussel beds were measured in the field by the transection method. Aerial photographs enabled the total area of the beds to be determined and large-scale surveys of mussel beds to be carried out synchronously. By examining large-scale aerial photographs it was possible also to determine the cover by remote sensing. The proportion could not be derived from aerial photographs, even from pictures at the largest scale tested during this investigation (scale 1:2500). By statistical methods it was estimated that 12 sub-samples (each covering 177 cm²) from mature beds, and 12 sub-samples (each covering 38 cm²) from young beds are needed to obtain reliable data on biomass and abundance.     

Some effects of copper on the veliger larvae of the mussel Mytilus edulis and the Scallop Pecten maximus (Mollusca, Bivalvia)

During 1983 and 1985 several batches of laboratory reared veliger larvae of Mytilus edulis and Pecten maximum here subjected to a rank of concentrations of added copper (CuCl₂) over a 15-day period. M. edulis larvae were less sensitive, measured both as mortality (15-day LC₅₀) of 400 μglitre⁻¹) and reduced growth, than P. maximus larvae (15-day LC₅₀ of 85μg litre⁻¹). Both species appeared less sensitive to Cu than other bivallve larvae previously studied. Veliger larvae of M. edulis are from 7 to 10 times more tolerant of Cu than juveniles or adults and this unexpected finding is discussed in relation to the recent literatures on Cu toxicity and accumulation in mussels.     

贻贝附着基海带苗种运输技术探究

中介生物辅助大型海藻海底基质附着技术,是采用海面撒播苗种方法,进行大型海藻海底增殖的核心技术。研究发现,该贝、藻复合体苗种的运输比普通水产苗种运输难度显著增加。本文采取传统的干运、水运以及发明的淋水运输3种不同方式,进行了贻贝附着基海带苗种运输技术研究。结果表明:干运3h内苗种的存活率和生长速度与对照组差异不显著(P0.05);水运1h以上苗种的存活率与对照组差异显著(P0.05);淋水运输24 h内苗种的存活率和生长速度与对照组差异不显著(P0.05)。实验说明:水运不适合该苗种运输;在露空时间3 h以内,干运是最经济、最便捷的苗种运输方法;在露空24 h以内,采取淋水运输能够保证苗种的成活率,可以作为该苗种常规的运输方法。     

Effects of long-term exposure to silver or copper on growth, bioaccumulation and histopathology in the blue mussel Mytilus edulis

The purpose of this study was to determine the long-term accumulation of either silver or copper from low concentrations in seawater by blue mussels, Mytilus edulis. Mussels raised from eggs in the laboratory to the age of 2·5 months (approximately 4·5 mm in length) were continuously exposed to 0, 1, 5 and 10μg/liter of either silver (nitrate) or copper (chloride) and sampled at 12, 18 and 21 months for growth studies, measurements of metal accumulation and histopathological examination.Whole-body soft tissues were analyzed for the presence of both silver and copper, as background levels of copper in the incoming seawater averaged 2–4 μg/liter. Mussels exposed to silver had accumulated significant amounts of silver only at the highest test concentration (10 μg/liter Ag) after 12 months, but at 18 and 21 months significant levels were accumulated at all three test concentrations. Mussels exposed to copper accumulated significant amounts of copper at 5 and 10 μg/liter Cu after all three sampling periods, but not at 1μg/liter. Silver-exposed animals also accumulated significantly greater amounts of copper than control animals.In a comparative study, field-collected juvenile mussels (approximately 16·1 mm in shell length) and adult mussels (approximately 53·4 mm in shell length) were exposed for 12 months to 0, 5, 25 and 50 μg/liter silver only and subsequently sampled for metal-accumulation analyses and growth measurements. Juvenile mussels accumulated significant amounts of silver at all test concentrations, with the exception of mussels exposed to 5 μg/liter Ag for 6 months. Copper accumulation in the silver-exposed juveniles was significant only at 50 μg/liter Ag after 6 months, but at all test concentrations after 12 months. Adult mussels exposed to silver accumulated significant levels of both silver and copper, but at somewhat lower levels than juveniles.In the growth study, silver had no effect on laboratory reared mussels at the highest concentration of 10 μg/liter tested, whereas copper at 10 μg/liter did appear to affect growth as early as 4 months after the start of experimental exposure. Field-collected juvenile mussels did show inhibition in growth after 6 months' exposure to 25 and 50 μg/liter Ag, with some growth occurring after 12 months. Adults also showed inhibition in growth after 6 months but not at 12 months.Histopathological examination of mussels exposed to either 5 or 10 μg/liter of copper for 18 months showed changes in the digestive diverticula, gastrointestinal tract, reproductive tract and muscle tissues. These changes were more noticeable in mussels exposed to 5 μg/liter Cu than in those exposed to 10 μg/liter. Mussels exposed to silver for 21 months showed yellowish to black particulate deposition in the basement membrane and connective tissue of the various organs and tissues. Silver deposition increased with increasing test concentration.     

In vivo metabolism of benz[a]anthracene by the polychaete Nereis virens

Polycyclic aromatic hydrocarbons (PAH) can be metabolically activated to reactive intermediates with mutagenic potential. Although marine sediments serve as the major repository for PAH released into the environment, little is known of the capabilities of benthic organisms, particularly infaunal invertebrates to metabolize PAH.¹ In this study, in vivo metabolism of of the PAH benz[a]anthracene (BA) by the polychaete Nereis virens was investigated. Worms maintained in flow-through benthic microcosms were exposed to [¹⁴C-12]-BA sorbed to sediment, introduced directly to the water column, and in labeled food for periods of 4 to 25 days. Regardless of mode of exposure, most radiolabel recovered from Nereis was present as metabolic products with only 2 to 23% remaining as parent compound. In addition, a significant proportion (from 33 to 51%) of accumulated radioactivity was neither solvent extractable nor water soluble, suggesting that incorporation into macromolecular pools is a major fate of accumulated BA in Nereis virens.