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.     

Benzo[a]pyrene-dione-stimulated oxyradical production by microsomes of digestive gland of the common mussel, Mytilus edulis L

The potential of benzo[ a]pyrene (BaP) quinones (diones) to stimulate NAD(P)H-dependent hydroxyl radical (·OH) production (2-keto-4-methiolbutyric acid (KMBA) oxidation) by digestive gland microsomes of M. edulis was studied using iron/EDTA as a promotor of the Haber-Weiss reaction (O₂⁻ + H₂O₂ = ·OH + OH⁻ + O₂). Stimulation of basal rates of KMBA oxidation was observed for all three diones. Stimulation was similar for the 1,6- and 3,6-diones, but much less for the 6,12-dione, and greater for the NADH than the NADPH-dependent reaction, viz. % increases of 78 to 333 (NADH) compared to 0 to 78 (NADPH). Maximal KMBA oxidation was obtained at dione concentrations of 12.5 to 50 μm. Inhibition of KMBA oxidation by Superoxide dismutase and catalase indicated the involvement of respectively Superoxide anion radical (O₂⁻) and hydrogen peroxide (H₂O₂) in 1,6-dione stimulated NADPH-dependent · OH formation. The apparent K_m values of xenobiotic-stimulated KMBA oxidation were lower for BaP-diones than for the model redox cycling quinone menadione (2-methyl-1,4-naphthoquinone), viz. in μm, 0.6 to 14.6 (NADH) and 4.4 to 28.5 (NADPH) compared to 315 to 457 (NADH) and 72 to 103 (NADPH). The results are consistent with metabolism of BaP to diones and resultant enhanced generation of oxyradicals being a potential mechanism of pollutant-mediated toxicity in molluscs.     

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.     

Detection of mRNA sequences homologous to the human glutathione peroxidase and rat cytochrome P-450IVA1 genes in Mytilus edulis

In this study we have used cloned gene probes for human glutathione peroxidase (GPX), rat cytochrome P-450IVA1 and rat cytochrome P-450IIE1 to detect homologous sequences in RNA from the hepato-pancreas of Mytilus edulis. The presence of sequences hybridising to the GPX and P-450IVA1 probes, but not to the P-450IIE1 probe, confirms the ancient origin of the former genes and indicates that conserved-sequence DNA probes from higher organisms can be used to examine the structure and function of genes of environmental interest in marine organisms.     

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.     

Cellular responses to polycyclic aromatic hydrocarbons and phenobarbital in Mytilus edulis

Certain polycyclic aromatic hydrocarbons and phenobarbital induced an increase in the activity of microsomal NADPH neotetrazolium reductase (linked to mixed function oxygenase systems) in the blood cells of Mytilus edulis. Phenanthrene and methylated naphthalenes caused lysosomal destabilisation which is believed to be directly related to the mechanism of cytotoxicity in the digestive cells. The use of these cytochemical techniques as indices of aromatic hydrocarbon contamination is discussed.     

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.     

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.     

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.     

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.     

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.     

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.     

三种典型POPs对紫贻贝不同组织DNA损伤的比较研究

采用彗星电泳技术,比较研究了Aroclor 1254、BaP、DDT三种典型POPs对紫贻贝(Mytilus edulis)鳃、性腺、消化腺细胞的DNA损伤效应的差异,以期为进一步评价这三种典型POPs遗传毒理机制及其预警监测提供科学依据.结果表明,Tail DNA%是评价DNA损伤的理想指标,另外,在急性毒性试验中,...     

Microsomal cytochrome P-450 function in fish evaluated with polyclonal and monoclonal antibodies to cytochrome P-450E from scup (Stenotomus chrysops)

Monooxygenase reactions catalyzed by cytochromes P-450 are paramount in the oxidative metabolism of many xenobiotics, determining both the persistence and effects of numerous types of compounds. Immunological probes are proving useful in evaluating the functions of P-450 isozymes in microsomal preparations from many species. The regulation of specific isozymes by endogenous and exogenous factors can also be evaluated with such probes. Here we describe studies on the activities apparently catalyzed by induced P-450 in fish, evaluated with both polyclonal and monoclonal antibodies to cytochrome P-450E, the apparent major β-naphthoflavone(BNF) or methylcholanthrene(MC)-inducible isozyme purified from scup (S. chrysops) liver.     

Characteristics of the hepatic microsomal cytochrome P-450 system of the minke whale (Balaenoptera acutorostrata)

The whales are an interesting group in several aspects of science, including evolutionary biology, marine ecology and toxicology. Some of the whale species, including the minke whale, graze at the top of the food chains, where they become susceptible to the accumulation of marine pollutants ingested by organisms at lower levels. The northern stock of the minke whale grazes in Arctic waters in the summer, an area of increasing interest in the development of new oil fields. Studies on the metabolism, disposition and effects of xenobiotics in marine mammals are few. The only reports in the open literature so far have dealt with these questions in seals.^1–3 Although the marine mammals comprise a particularly interesting group in marine ecology and toxicology, no data have to our knowledge been presented on the xenobiotic metabolising enzyme systems of the whales. In this study, components of the microsomal cytochrome P-450 system were measured in liver samples from several females, one male, and two foetal minke whales, caught in the Norwegian whaling season of 1983. Because of the limited number of samples studied we have treated the samples individually and not performed any statistical analysis of the data. The trends discussed below must therefore be considered with this background.     

Cellular responses in the mussel Mytilus edulis following exposure to diesel oil emulsions: Reproductive and nutrient storage cells

The objectives of this study were to examine, and quantify with the aid of stereological techniques,^1,2 the effects of exposure to hydrocarbons on the reproductive and nutrient storage cell systems in the marine mussel, Mytilus edulis. In addition, the capacity for recovery of these cell systems was quantified following a period of hydrocarbon depuration. The second phase of these investigations examined the significance of nutritional reserves at the time of exposure to hydrocarbons and how this may influence the animal's ability to survive the insult. The results (Table 1) indicated that, first, hydrocarbons had a deleterious effect on the nutritional storage cells leading to reduced fecundity and oocyte atresia (degeneration). Given a period of depuration, however, there were indications of a recovery in both the reproductive and storage cell systems. Second, the ability of mussels to survive hydrocarbon exposure was dependent upon the nutrient reserves at the onset of exposure.