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.     

Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)

Several genes normally induced by estradiol (E₂) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E₂ and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E₂ and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.     

Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)

Several genes normally induced by estradiol (E(2)) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E(2) and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E(2) and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.     

Kinetics of vitellogenin protein and mRNA induction and depuration in fish following laboratory and environmental exposure to oestrogens

Plaice, flounder and sand goby were exposed to ethynylestradiol (EE2) for 21 days and then followed for up to 31 days after removal of the oestrogen. Plasma vitellogenin (VTG) and hepatic VTG mRNA were determined in groups of fish sampled during the induction and post-exposure phases. VTG mRNA increased slightly earlier than plasma protein, but both reached maxima by 21 days. In contrast, VTG mRNA decayed much more rapidly than protein after EE2 exposure was terminated (typical values t(1/2) mRNA 3 days, protein 15-30 days). Vitellogenin and VTG mRNA thus measure different temporal events and this is illustrated by field data of male flounder in which both parameters have been determined. Few fish show co-ordinate increased VTG mRNA and vitellogenin but rather more fish have increased vitellogenin. Low level increases of VTG mRNA (5 X) is observed in some fish without increased vitellogenin and this may represent polymorphic differences between individual fish.     

Structural Studies of a UDP-glucuronosyltransferase gene from the plaice (Pleuronectes platessa)

A partial length cDNA coding for the putative PAH-inducible phenol-conjugating UDP-glucururonosyltransferases (UGT) isoform of plaice was used to isolate overlapping clones from a plaice genomic library. Sequence analysis revealed the presence of a complete gene spanning 4.1 kb for a plaice UGT which showed a strong conservation in exon structure, amino acid character and amino acid sequence with mammalian UGT1 family genes, although additional alternative upstream exon 1s were not identified in the present study. Southern blot analysis revealed a low copy number for the gene and some degree of structural polymorphism in gene structure between individuals. This was also reflected in the finding that there were significant variations between the nucleotide sequences of the plaice gene and the cDNA previously isolated from a different individual fish. Future studies will investigate the possibility that there may be phenotypic variations which could lead to alterations in susceptibility to pollutant toxicity.     

Differential sensitivity of flounder (Platichthys flesus) in response to oestrogenic chemical exposure: an issue for design and interpretation of monitoring and research programmes

This study was conducted as an initial investigation of 'differential response' in one of the main sentinel organisms used for monitoring programmes in United Kingdom estuaries, the flounder Platichthys flesus. It has been hypothesised that monitoring using species with a wide geographical spread and limited migration, such as flounder, might result in the comparison of different genetic stocks and certainly of populations with differing early life stage contaminant exposure histories. Furthermore, it is probable that these pre-exposure and genetic differences could manifest themselves in an ability to respond differently to contaminant exposure, so-called 'differential response'. It is important that the extent and nature of this response is understood, if we want to be able to fully interpret the monitoring data from such programmes. During this study, flounder were collected from four separate sources; wild caught fish from the estuaries of the Rivers Alde, Mersey and Tyne, and farmed flounder from Port Erin Farm, Isle of Man. Under controlled laboratory conditions, groups of fish from each source were exposed to water-borne concentrations of the synthetic oestrogen ethynylestradiol (EE2) at a nominal concentration of 50 ng/l. Plasma was taken from each male fish after 6 and 10 days exposure and analysed for the presence of vitellogenin (VTG) using an ELISA technique. Significant levels of VTG induction were evident in fish from all sources after both 6 and 10 days exposure. Flounder from the Mersey were the only fish with significantly elevated initial background levels of VTG (day 0) and this appeared to be reflected in that these specimens showed the highest induction response after day 6. However, after day 10, fish from all other sites had a slightly higher mean VTG than those from the Mersey which showed significantly (p < 0.05) lower mean plasma VTG. It is suggested that other differential responses may have been masked by the use of a high dose of EE2 which produced maximum induction in nearly all fish. The findings of the study are discussed in terms of implications for further research into the differential response issue and how the initial plasma VTG figures contribute to a time-series from the Mersey, Tyne and Alde estuaries.     

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.     

Comparative vitellogenic responses in three teleost species: extrapolation to in situ field studies.

Induction of vitellogenin (VTG) was compared among three teleostean species to determine their relative sensitivity of exposure to 17 beta-estradiol (E2). Japanese medaka (Oryzias latipes), sunshine bass (Morone saxatalis x Morone chrysops) and channel catfish (Ictalurus punctatus) were exposed to aqueous concentrations of E2 ranging from 10 to 100,000 ng/l for 21 days. Respective EC50 values for plasma VTG detected by western blot in medaka, catfish and bass were 200, 170 and 1560 ng E2/l. Since these EC50 values are based on VTG induction curves calculated relative to control values, they indicate differences in species' sensitivity to E2 exposure. Catfish and bass VTG responses obtained in laboratory exposures were compared to VTG responses previously observed with 21-day wastewater treatment plant effluent exposures. Plasma VTG induction in effluent-exposed fish ranged from 14 to 82% above reference values depending on species. Extrapolation of field responses with laboratory-exposed fish indicate catfish and bass were exposed to the equivalent of 27-240 ng E2/l in sewage effluent.     

Molecular evidence for multiple UDP-glucuronosyltransferase gene familes in fish

The presence of multiple distinct UGT genes in fish was derived by analysis of DNA sequence data derived the zebrafish EST project, confirming indications from previous protein purification studies in another fish species, the plaice, for a diversity of isoforms in lower vertebrates. At least 10 different UGTs can be identified from nucleotide sequence data in zebrafish. Phylogenetic analysis of exon 1 sequences of the zebrafish, plaice and human UGTs indicates that six of these genes are related to the 1A, 1B and 2 families and that a further four genes were of more ancient lineage. Importantly data for the 3' sequences of the zebrafish clones, both from the database and our own sequences of the publicly available clones did not provide any evidence for elaboration of family 1A genes by alternative splicing in this lower vertebrate.     

Searching for novel CYP members using cDNA library from a minke whale liver

The contaminant-induced cytochrome P450 (CYP) members in minke whale (Balaenoptera acutorostrata) can be potential biomarkers of the contaminant exposure and toxic effects. In this study, we constructed a cDNA library from the liver of minke whale from the North Pacific, and further screened a total of 6930 clones randomly selected in the library for the isolation of cDNA clones encoding novel members of CYP superfamily. The screening revealed the isolation of six novel CYP cDNA clones that are classified into CYP1A, CYP2C, CYP2E, CYP3A, CYP4, and CYP4A subfamilies. The BLAST homology search using the partial cDNA fragments of four CYP subfamilies (CYP1A, CYP2C, CYP2E and CYP4A) demonstrated that the minke whale CYPs were most closely related to pig CYPs (81-91%). Identification of multiple CYP genes in marine mammal species such as minke whale will provide new insights into the metabolic or toxicological functions of individual CYP members.     

The effect of estrogen on cadmium distribution in rainbow trout (Oncorhynchus mykiss)

The effects of injections of 17β-estradiol (E2) and Cd, on the distribution of Cd and the induction of metallothionein (MT) mRNA and vitellogenin mRNA was investigated. Bone and liver were the main organs accumulating Cd. However, E2 redirected the metal accumulation from the bone and liver to the gill, gut and muscle upon exposure to E2. Cd did not induce the hepatic MT mRNA levels in animals treated with E2. The VTG mRNA levels were also reduced following co-injection of E2 and Cd. However, the kidney responded to Cd exposure by upregulating MT mRNA even in the presence of E2 treatment. In the liver the reduced MT mRNA induction led to a redistribution of CD from MT to non-MT proteins. The toxicological significance of these alterations in Cd handling remains to be determined.     

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

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.     

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.     

Characterization and molecular analysis of hepatic microsomal UDP-glucuronosyltransferases in the marine fish, Pleuronectes platessa

Here we report the results of the first molecular enzymological study of fish UDP-glucuronosyltransferase (UDPGT). UDPGT activities of plaice liver microsomes were greatest for planar phenols and there was low but measurable conjugation of bilirubin, testosterone and androsterone. A highly purified preparation was isolated which possessed activities towards 1-naphthol, bilirubin and testosterone, containing several molecular weight species of M_r 52–56 kDa. On immunoblot analysis these proteins cross-reacted with a polyspecific anti-rat UDPGT antibody, suggesting that a number of plaice UDPGT isoforms with epitopes in common with the corresponding rat enzymes had been purified.     

Exposure to environmentally relevant concentrations of different nonylphenol formulations in Japanese medaka.

The time course of exposure to p-nonylphenol (NP) from two different sources was compared to equivalent exposures of 17-beta-estradiol (E2) and a solvent control (ethanol; EtOH). Japanese medaka were exposed for 4 days to a nominal concentration of 20 micrograms/l of either NP-I (Schenectady International, Inc.), NP-II (Aldrich), or E2, and were then placed in untreated water for 5 days. Tissue samples were taken at two time points during the 4-day exposure and two time points during the 5 days following exposure. Liver homogenates were analyzed using a western blot to detect vitellogenin (VTG) and quantified by measuring the optical density for each lane. Preliminary results indicate that E2 significantly increased VTG staining above the level observed in EtOH-treated controls for both males and females. A two-way analysis of variance (ANOVA) indicates that NP from both sources, as well as E2, significantly increased VTG staining in males (ANOVA, n = 48, P < 0.001; Tukey pairwise tests, all P < 0.008). A significant increase in VTG was observed in E2-treated males and females the first day following transfer into toxicant-free water (two-way ANOVAs, both n = 48, P < 0.003; Tukey pairwise tests, all P < 0.019). If confirmed, this extended response observed for low-level exposures may represent a significant factor for sampling scenarios following pulsitile exposure.