Stephen G. George

Adaptive differences in gene expression in European flounder (Platichthys flesus)

Population structure was previously believed to be very limited or absent in classical marine fishes, but recently, evidence of weakly differentiated local populations has been accumulating using noncoding microsatellite markers. However, the evolutionary significance of such minute genetic differences remains unknown. Therefore, in order to elucidate the relationship between genetic markers and adaptive divergence among populations of marine fishes, we combined cDNA microarray and microsatellite analysis in European flounders (Platichthys flesus). We demonstrate that despite extremely low levels of neutral genetic divergence, a high number of genes were significantly differentially expressed between North Sea and Baltic Sea flounders maintained in a long‐term reciprocal transplantation experiment mimicking natural salinities. Several of the differentially regulated genes could be directly linked to fitness traits. These findings demonstrate that flounders, despite little neutral genetic divergence between populations, are differently adapted to local environmental conditions and imply that adaptation in gene expression could be common in other marine organisms with similar low levels of population subdivision.

A piscine glutathione S-transferase which efficiently conjugates the end-products of lipid peroxidation

Abstract A cDNA clone for glutathione S -transferaseA (GSTA) from plaice ( Pleuronectes platessa ) was expressed in Eschericia coli (E. coli) and purified to homogeneity by S -hexylglutathione affinity chromatography. When compared to literature values for a variety of purified mammalian GSTs, the heterologously expressed purified plaice enzyme had moderate activity towards the model substrate 1,2-chloro-2,4-dinitrobenzene (CDNB) and exhibited a Km of 2.5 ± 2 mM and Vmax of 30.9 ± 2.3 μmol min −1 mg −1 . It had little or no activity towards several other model GST substrates including 1,2-dinitrochloro-4-benzene (DCNB), ethacrynic acid (EA), and p-nitrobenzylchloride (NBC). However plaice GSTA was a relatively efficient catalyst for the conjugation of a series of alk-2-enals and alk-2,4-dienals and also 4-hydroxynonenal. The highest activity observed with this series of substrates was with trans-non-2-enal with a Km of 17.9 ± 2.2 μM and a Vmax of 3.01 ± 0.57 μmol min −1 mg −1 . These unsaturated alkenals have been identified in cells and cell extracts as highly toxic products arising from peroxidation of unsaturated fatty acids particularly during periods of oxidative stress. Fish are relatively rich in polyunsaturated fatty acids and thus GSTA mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products.

Metabolism of zinc in the mussel, Mytilus edulis (L.): a combined ultrastructural and biochemical study

The uptake, transport, storage and excretion of zinc has been studied in Mytilus edulis . Zinc accumulates in the soft tissues in proportion to its concentration in sea water whilst the concentration in the haemolymph is little above that in the environment. Uptake is via the gut, mantle and gills. The zinc is transported from the gills and gut (t½ ≈ 8 days) via the haemolymph, either as a high molecular weight complex or in the granular amoebocytes, to the kidney. Most of the body zinc is present in the granular amoebocytes (which are found in all the body tissues) or in the gut and kidney. The kidney forms the major storage organ for many trace metals, containing 30% of the body zinc and a concentration of about 1000 μg/g. Zinc is localized as insoluble granules in membranelimited vesicles occupying some 20% of the cell volume. Excretion of zinc is by defaecation, exocytosis of the kidney granules into the urine and diapedesis of the amoebocytes. A multicompartmental model for zinc metabolism which correlates the ultrastructural and kinetic data is proposed.

Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.

Background In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics—Moving into Regulation and Monitoring, held 21–23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). Objectives The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. Discussion Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. Conclusions The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

Cadmium effects on plaice liver xenobiotic and metal detoxication systems: dose-response

Abstract The effects of intraperitoneal administration of varying doses of cadmium on hepatic metal and xenobiotic detoxication systems in the plaice, Pleuronectes platessa , were studied. The results showed that above a threshold of ca. 2 μg Cd/g liver, metallothionein (MT) levels were increased but at high doses the sequestration capacity of induced MT was exceeded and at the highest dose tested (1 mg Cd/kg) MT induction/synthesis was reduced and hepatic Zn levels decreased. Cadmium injection strongly reduced cytochrome P -450 dependent ethoxyresorufin o -deethylase (EROD) activity and preliminary immunological studies indicated that this was due to a decrease in enzyme protein rather than direct inhibition of activity by Cd. At the sampling time of this study (6 days) there was no significant alteration in activity of the phase II enzyme, glutathione- s -transferase.

MECHANISMS OF IMMOBILIZATION AND DETOXICATION OF METALS IN MARINE ORGANISMS

Current concepts of metal metabolism have been reviewed with a view to understanding the factors that influence the uptake, storage and elimination of essential and pollutant trace metals in aquatic organisms. Examples from our own studies on estuarine molluscs exposed to heavy metals have been used to illustrate these factors. When metal uptake was combined with electron microscope studies, a novel storage and detoxication mechanism was revealed, where formation of vesicles within the cell enclose the metal within a membrane. This prevents contact of excess metal with vital constituents and effectively detoxifies it until eliminated or passed on to other tissues, as required. The generality of this mechanism has been established.

The occurrence of cadmium in sub-cellular particles in the kidney of the marine mussel, Mytilus edulis, exposed to cadmium: The use of electron microprobe analysis

In mussels (Mytilus edulis) chronically exposed to cadmium, 85% of the Cd2+ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca2+, iron and Zn2+. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd2+ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd2+-binding component of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.

Characterisation of cadmium-binding proteins from mussels, Mytilus edulis (L), exposed to cadmium

Three Cd2+-binding proteins have been purified and partially characterised from the digestive gland of the bivalve mollusc, Mytilus edulis, after exposure to Cd2+. The major protein, which was judged to be pure on polyacrylamide gel electrophoresis, showed many of the characteristics of mammalian metallothionein; having a high -SH content, few aromatic amino acids and a high A250/A280 nm ratio which disappears on acidification. It also contains Zn and Cu, but differs in its higher apparent molecular weight of about 25 000 and high glycine content (12-19%). The two additional Cd2+-binding proteins had lower cysteine contents and different molar proportions of Cd2+, Zn2+ and Cu2+.

The time course of effects of cadmium and 3-methylcholanthrene on activities of enzymes of xenobiotic metabolism and metallothionein levels in the plaice, Pleuronectes platessa

Plaice were treated with an acute dose of a polyaromatic hydrocarbon (3-methylcholanthrene, 3-MC) or cadmium, or 3-MC and cadmium by i.p. injection. The effects on hepatic detoxication systems, cytochrome P-450 (ethoxyresorufin O-deethylase, EROD), UDP-glucuronyl transferase, glutathione S-transferase, glutathione peroxidase activities, total glutathione (GSH), metallothionein and Cd and Zn in the cytosol were studied over a 14 day period. 3-MC increased EROD (7-18-fold), glucuronyl transferase (40%) and GSH transferase (200%) activities, whereas GSH peroxidase activity decreased by 60%. Cd treatment inhibited EROD (90%), GSH transferase (90%) and GSH peroxidase (30%) activities and displaced Zn. Total GSH levels increased (200%) prior to onset of metallothionein synthesis (6 days). Cotreatment with 3-MC and Cd led to a marked increase in GSH levels (300%) but the onset of metallothionein synthesis was delayed by a week. Induction of enzyme activities was abolished, EROD activity was strongly inhibited and there was a transient 50-90% decrease in glucuronyl transferase, GSH transferase and GSH peroxidase activities on days 2 and 3 after treatment. The results indicate that a polyaromatic hydrocarbon could result in increased peroxidative damage, the heavy metal Cd can severely inhibit organic xeno- and endobiotic metabolism and that the effects of both agents may be synergistic.

Regulation of metallothionein in teleosts: Induction of MTmRNA and protein by cadmium in hepatic and extrahepatic tissues of a marine flatfish, the turbot (Scophthalmus maximus)☆

Synthesis of the heavy metal-binding protein metallothionein (MT) was determined in the major organs of accumulation of Cd (liver, kidney and gills) of a marine flatfish, the turbot, after intraperitoneal (i.p.) administration of varying doses of Cd. Synthesis of MTmRNA and MT were linearly related to dose only at low Cd dosages (up to ca. 100 micrograms Cd/kg). Induction of MTmRNA was rapid, peaking 1-2 days after Cd administration in gills and kidneys, at 4 days in liver. In all three tissues, at low doses of Cd, MTmRNA levels declined with an apparent half life of 5-7 days and for a given dose of Cd, similar MTmRNA concentrations were attained. Induction of MT levels temporally followed that of MTmRNA. Steady state levels of MT were attained more quickly at a low dose of Cd. At acute Cd doses of > 200 micrograms Cd/kg, MT gene transcription and protein translation appeared to be progressively reduced, inferring that the rate of MT synthesis was limiting due to cytotoxicity of the high acute Cd dosage. In contrast to MTmRNA levels which were induced to similar levels in the three tissues, MT levels decreased in the order liver > kidneys > gills implying differences in translational processes.