GSTA is a major glutathione S-transferase gene responsible for 4-hydroxynonenal conjugation in largemouth bass liver

We have previously shown that largemouth bass (Micropterus salmoides) has a remarkable ability to conjugate 4-hydroxy-2-nonenal (4HNE), a mutagenic and cytotoxic alpha,beta-unsaturated aldehyde produced during the peroxidation of lipids. In addition, we have isolated a glutathione S-transferase cDNA (bass GSTA) that encodes a recombinant protein which is highly active in 4HNE conjugation and structurally similar to plaice (Pleuronectes platessa) GSTA. In the present study, HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with one peak constituting 80% of the total bass liver GST protein. Liquid chromatography mass spectrometry (LC-MS) and electrospray ionization analysis of the major bass GST subunit yielded a molecular weight of 26,396 kDa. Endo-proteinase Lys-C digestion and Edman degradation protein sequencing of this GST peak demonstrated that this protein was encoded by bass GSTA. Analysis of genomic DNA fragments isolated by nested PCR indicated the presence of a GST gene cluster in bass liver that contained GSTA, and was similar to a GST gene cluster characterized by Leaver et al., in plaice. Collectively, our data indicates the presence of a major GST in bass liver involved in the protection against oxidative stress. This GST is part of a gene cluster that may be conserved in certain freshwater and marine fish.     

GSTA is a major glutathione S-transferase gene responsible for 4-hydroxynonenal conjugation in largemouth bass liver

We have previously shown that largemouth bass (Micropterus salmoides) has a remarkable ability to conjugate 4-hydroxy-2-nonenal (4HNE), a mutagenic and cytotoxic α,β-unsaturated aldehyde produced during the peroxidation of lipids. In addition, we have isolated a glutathione S-transferase cDNA (bass GSTA) that encodes a recombinant protein which is highly active in 4HNE conjugation and structurally similar to plaice (Pleuronectes platessa) GSTA. In the present study, HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with one peak constituting 80% of the total bass liver GST protein. Liquid chromatography mass spectrometry (LC–MS) and electrospray ionization analysis of the major bass GST subunit yielded a molecular weight of 26,396 kDa. Endo-proteinase Lys-C digestion and Edman degradation protein sequencing of this GST peak demonstrated that this protein was encoded by bass GSTA. Analysis of genomic DNA fragments isolated by nested PCR indicated the presence of a GST gene cluster in bass liver that contained GSTA, and was similar to a GST gene cluster characterized by Leaver et al., in plaice. Collectively, our data indicates the presence of a major GST in bass liver involved in the protection against oxidative stress. This GST is part of a gene cluster that may be conserved in certain freshwater and marine fish.     

Conjugation of 4-hydroxynonenal by largemouth bass (Micropterus salmoides) glutathione S-transferases

The glutathione S-transferases (GST) are a major group of conjugative enzymes involved in the detoxification of electrophilic compounds and products of oxidative stress. We have previously described the kinetics of hepatic GST conjugation in largemouth bass using a variety of synthetic GST reference substrates. In the present study, we investigated the ability of largemouth bass hepatic GSTs to conjugate 4-hydroxynon-2-enal (4HNE), a mutagenic and cytotoxic alpha-beta-unsaturated aldehyde produced during oxidative injury. Hepatic cytosolic fractions from largemouth bass rapidly catalyzed GSH-dependent 4HNE conjugation, with the rate of GST-4HNE conjugation in bass liver exceeding those of several other mammalian and aquatic species. No apparent sex-related differences in GST-4HNE activity were observed among adult bass. SDS-PAGE and Western blotting analysis of GSH affinity-purified bass liver cytosolic GST revealed the presence of two major GST subunits of approximately 30 and 27 KDa that exhibited slight cross-reactivity when probed with a rat alpha class GST antibody, but not to rat mu, pi or theta class GST. The rapid conjugation of 4HNE by hepatic GST suggests an important role for GSTs in protecting against peroxidation of polyunsaturated fatty acids in bass liver.     

Regulation of hepatic glutathione S-transferase expression in flounder

The major glutathione S-transferase isoform of flounder liver, an antigenically related structural homologue of plaice GST-A, also displays mRNA homology. A 901bp cRNA probe for plaice GST-A cross-hybridised to a 1100bp flounder mRNA on northern blot analysis. The plaice antibody and cRNA probes were used to study effects of inducer treatment on GST-A expression in flounder liver. Six days after PAH treatment (3-methylcholanthrene) total hepatic GST activity was halved, levels of GST-A were 80% and GST-A mRNA levels were 25% of controls. A commercial PCB mixture (Aroclor 1254^TM) had little effect on total GST or GST-A levels despite halving GST-A mRNA levels. An epoxide, trans-stilbene oxide induced total GST activity 1·4 fold and GST-A protein levels 1·8-fold and its mRNA levels 3-fold. This reduced expression of the major flounder hepatic GST by agents which induce cytochrome P4501A1 may modulate cytoxicity of environmental pollutants in this species.     

Reduction of flatfish habitat as a consequence of the proliferation of an invasive mollusc

Coastal bays provide habitats for juveniles and adults of many marine species. Mont Saint-Michel Bay (MSMB, France) hosts a highly diversified fish community and constitutes one of the most important nursery grounds for many commercially exploited marine species, such as sea bass, flatfish, clupeids and rays in the English Channel. Besides, MSMB also suffers from the massive invasion of an exotic mollusc, the American slipper-limpet (Crepidula fornicata, L.). This species arrived four decades ago and now represents the main filter-feeder biomass in the bay (150 Mt), an order of magnitude larger than local farmed and natural shellfishes. Recent analyses underlined the impact of this small gastropod on the trophic structure of this bay and its negative influence on juvenile sole densities in the nursery grounds. The present study uses a geostatistical approach to explore the effect of the extension of the slipper-limpet on flatfish (common sole Solea solea, L.; plaice Pleuronectes platessa, L.; brill Scophthalmus rhombus, L. and flounder Platichthys flesus, L.) spatial distribution. Data collected during survey of the MSMB at the end of the 1970s and three decades later have been used to build interpolated maps of (1) slipper-limpet and (2) flatfish spatial distributions. Slipper-limpets were concentrated in a small area, in the western part of the MSMB, in the 1970s while today they occupy half of the bay. This rapid proliferation led to the decrease of available surface for flatfishes, which previously occupied the whole bay and are now restricted to its eastern part. The present study highlighted that the negative influence on fish habitat in MSMB is apparently more related to changes in the substratum than to trophic interactions. This invasion has possible consequences on flatfish population renewal at a large scale and may also damage other benthic or demersal species, such as rays.     

Evidence from heterologous expression of glutathione S-transferases A and A1 of the plaice (Pleuronectes platessa) that their endogenous role is in detoxification of lipid peroxidation products

cDNA clones for glutathione S-transferases A (GST-A) and A1 (GST-A1) from plaice (Pleuronectes platessa) were expressed as N-terminally 6XHis tagged proteins in Escherichia coli and purified to homogeneity from Ni-NTA silica. GST-A was an efficient catalyst for conjugation of unsaturated alkenals derived from peroxidation of polyunsaturated fatty acids with the highest activity observed with trans-non-2-enal (8 micromol min(-1) mg(-1)). GST-A1 was a very efficient Se-independent glutathione peroxidase with an activity towards cumene hydroperoxide of 25 micromol min(-1) mg(-1). Although the enzymes exhibited moderately high activities towards the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) they exhibited little or no activity towards other common prototypical xenobiotic substrates. Together with data for ontogeny, tissue distribution and inducibility of these enzymes, we contend that a primary function of these enzymes is protection from the harmful effects of lipid peroxidation products generated naturally or exacerbated by xenobiotic exposure.     

Effects of beta-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to beta-naphthoflavone (beta-NF, 66 mg/kg, i.p.) elicited a 7-9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to beta-NF, and generally tracked the minimal changes observed in GST-CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by beta-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to beta-NF.     

Identification of cytochrome P450 1B-like sequences in two teleost fish species (scup, Stenotomus chrysops and plaice, Pleuronectes platessa) and in a cetacean (striped dolphin, Stenella coeruleoalba).

The cytochromes P450 (CYP) constitute a multigene family of enzymes playing a critical role in the oxidation of many endogenous and xenobiotic substrates. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and aryl amines. Three members of the CYP1 family, CYP1A1, CYP1A2, and CYP1B1, have been identified in mammals. We report here on the identification and cloning of cytochrome P4501B-like sequences from two teleost fish species and a marine mammal. Sequences clustering with CYP1B1 in phylogenetic analysis were obtained from liver cDNA of scup (Stenotomus chrysops), genomic DNA of plaice (Pleuronectes platessa), and liver cDNA of striped dolphin (Stenella coeruleoalba).     

A comparative analysis of recruitment variability in North Atlantic flatfishes — testing the species range hypothesis

The hypothesis that recruitment variation in flatfishes should be most variable at the northern edge of the species range, least near the centre of the range, and intermediate near the southern limit was tested using stock and recruitment data generated from sequential population analysis for several different flatfish stocks involving four species (plaice Pleuronectes platessa, sole Solea vulgaris from the eastern Atlantic, American plaice Hippoglossoides platessoides, and yellowtail flounder Limanda ferruginae from the western Atlantic). Several groundfish species have been found to conform to this so-called species range hypothesis with the suggestion that density-independent processes predominate at the edges of the distributional range and density-dependent processes dominate in the centre of the range. Our results were generally inconsistent with the hypothesis: the coefficient of variation (CV) of recruitment for plaice in the eastern Atlantic was independent of latitude, the CV of recruitment for sole exhibited a dome-shaped relationship with latitude with the highest CVs occurring at the mid-point of the range, and the CV of recruitment for the western Atlantic stocks exhibited a monotonic decrease with latitude. We extended our latitudinal analyses by assessing both the degree of dependency of recruitment on spawning stock biomass and the spatial and temporal scales of variability in recruitment and pre-recruit survival for the eastern Atlantic stocks. In general our analysis revealed no evidence of a strong stock and recruitment relationship for any of the stocks examined, and previously published analyses revealed no such patterns with latitude. Analysis of both de-trended recruitment and pre-recruit survival time series over the species ranges of sole and plaice revealed strong positive correlations among adjacent stocks and inverse correlations among stocks at the extremes of the range. Recruitment variation in the flatfish stocks examined appears to be dominated by density-independent factors, operating at a local scale, on the egg and larval stages.     

Effects of β-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to β-naphthoflavone (β-NF, 66 mg/kg, i.p.) elicited a 7–9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to β-NF, and generally tracked the minimal changes observed in GST–CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by β-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to β-NF.     

Cloning of the major 3-MC inducible phase I and phase II detoxification enzymes of plaice liver

cDNA's coding for cytochrome P4501A1 (CYP1A1), phenol UDP-glucuronosyltransferase (UDPGT) and glutathione S-transferase (GST-A) were cloned and sequenced from an expression library prepared from the liver of a 3-methylcholanthrene (3-MC) induced plaice. Plaice CYP1A1 and Phenol UDPGT display a high degree of structural conservation with homologous mammalian isoforms and their mRNAs were shown to be highly induced in liver after 3-MC treatment of fish. Expression of plaice GST-A, which displays closer homology to GSTs from plants and invertebrates than those of mammals, is repressed after 3-MC treatment.     

Environmental factors and interactions affecting the temporal abundance of juvenile flatfish in the Thames Estuary

Samples taken regularly from the intake screens of West Thurrock power station between January 1977 and November 1992 were used to investigate the factors controlling flatfish abundance (flounder: Platichthys flesus; dab: Limanda limanda; plaice: Pleuronectes platessa; sole: Solea solea) in the middle Thames estuary. Most sampled fish were age-0. All species followed regular patterns of seasonal occurrence, had distinctive seasonal abundance peaks and demonstrated only minimal temporal overlap. Multiple-regression models were used to study the relationship between physico-chemical variables, physico-chemical variable interactions, trends and seasonal factors on fluctuations in flatfish sample abundance. Flounder abundance was significantly influenced by temperature, suspended solids, oxygen-flow and oxygen-temperature interactions, trend and seasonal variables. Dab abundance was significantly related to suspended solids, oxygen-flow, oxygen-temperature and flow-chlorinity interactions, Crangon crangon abundance and seasonal variables. Sole abundance was controlled only by seasonal variables, whilst plaice abundance could not be adequately explained by the available physico-chemical data. The combination of variables affecting flatfish abundance indicates species using the estuary were affected by a complex set of events within the estuary. The interactions between routinely measured environmental variables further suggest that estuarine monitoring studies must attempt to understand the nature of possible interactions between variables if an improved understanding of the changes in estuarine fish communities resulting from human activity is to be gained.     

Histopathological observations on liver,kidney and gonad of plaice (Pleuronectes platessa) taken from the Mersey estuary

The Mersey estuary is the most contaminated estuary in British waters. Detailed studies are underway on the pathology of flounder (Platichthys flesus) from the Mersey and on flounder from the nearby, but less contaminated, Dee estuary. Flounder breed offshore but spend a lot of time in the estuaries, penetrating into freshwater. These flatfish are in close contact with sediments and will be exposed to exenobiotic stored in the sediments. Plaice (Pleuronectes platessa) are flatfish which enter the Mersey with the tidal flow but spend less time in the estuary than flounder and are less likely to show evidence of pollutant exposure. Over 20 plaice were collected from the Mersey. Samples of the liver, kidney and gonad were fixed in 10% formal saline, processed into paraffin wax and 5 microm sections cut and stained with haematoxylin and eosin. Liver changes consisted of variable amounts of glycogen/lipid storage product, minor perivenular and perivascular fibrosis, and helminth parasitisation. No tumours or foci of cellular alteration, necrosis and inflammation were seen. In the gonad only changes consistent with normal gonadal cycling was seen. These studies provide evidence that plaice show little or no evidence of toxic pathological damage resulting from their stay in the estuary.     

Identification of transcriptional effects of ethynyl oestradiol in male plaice (Pleuronectes platessa) by suppression subtractive hybridisation and a nylon macroarray

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.     

Polychaete assemblage as surrogate for prey availability in assessing southeastern Bering Sea flatfish habitat

The flatfish yellowfin sole (Limanda aspera), northern rock sole (Lepidopsetta polyxystra), and Alaska plaice (Pleuronectes quadrituberculatus) in the southeastern Bering Sea prey mainly on infauna. Spatial correspondence between their stomach contents and infauna assemblages across habitat types was examined to identify indices of prey availability for flatfish habitat characterization and quality assessment. Benthic samples and flatfish stomachs were collected in 2009 near the Alaska Peninsula in the southeastern Bering Sea. Polychaetes and bivalves were the most dominant infauna groups, each comprising 35–60% by weight in each infauna sample. These two were also the only prey groups that frequently averaged > 50% of stomach content by weight. Bivalves dominated the infauna biomass on the relatively sandy inner shelf (0–50 m depth). The muddier middle shelf (50–100 m) had the highest infauna biomass, which was dominated by polychaetes. Diet compositions of the flatfish varied spatially in correspondence with the infauna assemblage. Polychaetes were prevalent in all flatfish diets on the middle shelf, even yellowfin sole whose typical primary prey are amphipods and bivalves. Polychaete-rich habitats are potentially prime for flatfish as polychaetes are readily utilized where available and generally have high nutritional value. Flatfish did not select for specific polychaete taxa, so an index of habitat quality could be based on the biomass of aggregate polychaetes or on dominant polychaete families of the region. Under normal environmental conditions, the three flatfish have slightly-offset spatial distributions, enabling each to utilize different infauna assemblages across the shelf. However, during cold phases in the Bering Sea ecosystem, as when this study was conducted, a cold pool of < 2 °C bottom water from the spring ice melt extends over the middle shelf in summer. This physiological barrier displaces all three flatfish to the inner shelf, intensifying competition for prey resources.     

Identification of transcriptional effects of ethynyl oestradiol in male plaice (Pleuronectes platessa) by suppression subtractive hybridisation and a nylon macroarray

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.     

Estimated intensity needed for sampling flatfish assemblages in reference areas of tidal mud flats systems may be disproportionately costly and deleterious

Beam trawl surveys were carried out in a tidal stream system of the German Wadden Sea on two to three successive days of comparable weather conditions in three summers. The variability in time and space detected for plaice (Pleuronectes platessa), flounder (Platichthys flesus), dab (Limanda limanda), sole (Solea solea) and lemon sole (Microstomus kitt) was considerable even for species with a very high abundance. Other flatfish species such as turbot (Scophthalmus maximus) and brill (Scophthalmus rhombus) occurred only occasionally in the catches. Sampling effort calculated to detect a 50 or 20% difference between the surveys caused by immigration, emigration or mortality appears to be disproportionate. It is argued that such a major sampling effort of reference areas in tidal mud flat systems is likely to influence the results, to harm the environment, and to be very expensive.     

From sediment bioassay to fish biomarker--connecting the dots using simple trophic relationships.

Two common problems in applying and interpreting invertebrate bioassays and fish biomarkers in sediment toxicology are the wide gap between significant effects concentrations determined by these two approaches, and a general lack of ecological context. We have devised an exposure system that is able to reconcile much of the disparity between invertebrate bioassay and fish biomarker results by incorporating realistic ecological processes based on deposit feeding and predator-prey interactions. This system relates the disturbance of interest (sediment contamination) to biologically meaningful effects in a resource of interest (marine flatfish) via a realistic contaminant vector (a deposit-feeding polychaete worm). In this pilot study, polychaetes (Armandia brevis) were exposed for 28 days to clean sediments supplemented with benzo(a)pyrene (BaP), para-para dichlorodiphenyldichloroethylene (pp'DDE), Aroclor 1254, or field sediments collected from two sites in Puget Sound, Washington, contaminated predominantly with polcyclic aromatic hydrocarbons (PAHs) or chlorinated compounds. Exposed worms were then fed live to juvenile English sole (Pleuronectes vetulus) for 10 or 12 days. At the end of the exposure period, fish were measured for length and weight, sacrificed, and preserved for either routine histopathology and immunohistochemical analysis of cytochrome P450 1A induction, or 32P post-labeling determination of hepatic PAH-DNA adducts. Growth of predatory flatfish was lower than reference in all but one of eight groups fed contaminant-exposed polychaetes; however, statistically significant reductions in growth were only observed in three of these eight groups, at least in part due to low statistical power. Juvenile sole from all contaminant-exposed groups showed increased expression of CYP1A, and fish exposed to BaP-exposed worms showed clear evidence of hepatic PAH-DNA adducts. This method allows the concurrent evaluation of sediment contamination at multiple biological and ecological levels. These results indicate that sediments determined to be nontoxic by common invertebrate bioassays may have the potential to cause adverse effects at higher trophic levels.     

Kinetics of hepatic phase I and II biotransformation reactions in eight finfish species

Hepatic microsomes and cytosols of channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), red tilapia (Oreochromis sp.), largemouth bass (Micropterus salmoides), striped bass (Morone saxatilis), hybrid striped bass (M. saxatilis × M. crysops), and bluegill (Lepomis macrochuris) (n = 8) were used to study the kinetics of phase I (ECOD, EROD, PROD, BROD) and phase II (UDP-glucuronosyltransferase (UDPGT)-, sulfotransferase (ST)- and glutathione-s-transferase (GST)-mediated) reactions. The best catalytic efficiency for ECOD and GST activities was performed by channel catfish, Atlantic salmon, rainbow trout and tilapia. The highest EROD catalytic efficiency was for Atlantic salmon. None of the species had either PROD or BROD activities. Rainbow trout had very similar UDPGT catalytic efficiency to tilapia, channel catfish, Atlantic salmon, largemouth bass and bluegill. Sulfotransferase conjugation had no significant differences among the species. In summary, tilapia, channel catfish, Atlantic salmon and rainbow trout had the best biotransforming capabilities; striped bass, hybrid striped bass and bluegill were low metabolizers and largemouth bass shared some capabilities with both groups.