Project
title / Adaptive markers for environmental pollution.
/ DEFRA
project code / AE1130

Department for Environment, Food and Rural Affairs CSG 15

Research and Development

Final Project Report

(Not to be used for LINK projects)

Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Adaptive markers for environmental pollution.
DEFRA project code / AE1130
Contractor organisation and location / Glasgow Caledonian University
Cowcaddens Road
Glasgow G4 0BA
Total DEFRA project costs / £ 156629
Project start date / 01/12/98 / Project end date / 31/03/02
Executive summary (maximum 2 sides A4)
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CSG 15 (9/01) 4

Project
title / Adaptive markers for environmental pollution.
/ DEFRA
project code / AE1130

1.  The statutory responsibility of UK Government departments to protect the marine environment against adverse effects of environmental contamination and specific concerns about endocrine disrupting chemicals led to the Endocrine Disruption in the Marine Environment (EDMAR) project. EDMAR was started in 1998 as a multi-centre project funded by UK Government Departments, Agencies and the European Chemical Industry Association. The purpose of the project was to assess the extent to which endocrine disruption was affecting wildlife in UK estuaries and to identify endocrine disrupting chemicals and their sources. The project described here was funded separately by MAFF, within its Biological Effects and Impact Assessment programme, to provide analytical support for EDMAR work being conducted at FRS, Aberdeen and at CEFAS, Burnham.

2.  The endocrine systems of animals co-ordinate and regulate growth, development, reproduction and other physiological processes. Recent observations in the UK, USA and elsewhere have shown that exogenous chemicals can interfere with the normal functioning of the endocrine system and thus have the potential to adversely affect marine populations and communities. Although each of the diverse endocrine systems could be affected by environmental chemicals much attention has been focused on systems regulated by oestrogens and the apparent feminisation of fish in rivers and estuaries.

3.  Feminisation of male fish is a consequence of exposure to environmental xeno-oestrogens such as ethynyloestradiol, alkylphenol polyethoxylates and phthalates. Endocrine disrupting chemicals can bind to the oestrogen receptor present in the liver of both male and female fish and mimic the effects of natural oestrogens. In females circulating oestrogens stimulate the synthesis of egg proteins in the liver and these are transported via the vascular system to the ovary. The binding of xeno- or natural oestrogens to the oestrogen receptor of male fish also results in the synthesis of egg proteins in liver and their export but these proteins accumulate in blood in the absence of an ovary. The presence of vitellogenin (VTG), a major component of egg yolk, in the serum of male fish is a diagnostic marker of vitellogenesis as a result of exposure to xeno-oestrogens and acts as a proxy for higher level effects of endocrine disruption. Another marker of the effects of xeno-oestrogens in male fish is the appearance in serum of the an egg envelope component, Zona radiata protein (ZRP).

4.  The classical method of measuring VTG or ZRP are by immunochemical methods such as ELISA. However, there are considerable differences in the structures of VTGs between species and in general antibody preparations are not cross-reactive demanding that for each species VTG is purified. The EDMAR programme identified sand goby (Pomatoschistus minutus) and blenny (Zoarces viviparus) as ideal sentinel species for laboratory experiments and environmental monitoring but anti-VTG antibodies were not available for either. Furthermore the small size of these fish makes it difficult to obtain sufficient serum for VTG purification or for analysis during the experimental and monitoring phases of the programme. We identified an alternative strategy for the measurement of VTG production by looking directly at gene expression in liver and measuring mRNA rather than protein. The aims of this project were to isolate cDNA probes to measure egg protein mRNAs in the two species and to develop biomarker assays using the probes. These would then be applied in experiments assessing the effects of endocrine disrupting chemicals on reproductive success in a laboratory experiment and in fish collected from UK estuarine sites.

5.  Analysis of VTG DNA sequences of fish species available from the sequence databases (GenBank/EMBL) allowed the identification of conserved structures and the design of primers. Fragments of VTG cDNA were generated by Reverse Trancriptase-PCR using as starting material RNA from the livers of oestrogen-treated fish or from gravid females. In initial experiments RNA from flounder (Platichthys flesus) and plaice (Pleuronectes platessa) were used since these species were relevant to previous and continuing EDMAR-related work. The fragments were cloned and sequenced confirming that VTGs had indeed been obtained. The sequence data allowed the design of further PCR primers which led to the generation and cloning of VTG fragments from blenny and eventually from sand goby. A number of modifications of the methodology had to be introduced before success with the sand goby was achieved. Comparison of the sequences of the four VTGs revealed why. Plaice and flounder share a sequence identity of 97 % and blenny is 70 % identical to flounder. However, sand goby shares only 48 % identity with flounder. In contrast to VTG, obtaining cDNA for ZRP from sand goby was straight forward and the resulting sequence showed greater than 60 % identity to sequences of this protein in carp, trout and salmon.

6.  Northern blots were used to established the specificity of reaction of the VTG and ZRP cDNA reagents with mRNAs in fish liver. The cDNAs reacted with mRNA but only from the same species other than flounder and plaice for which VTGs cross-hybridised. cDNAs reacted with a single mRNA species, the size of which was similar to VTG and ZRP mRNAs in fish species previously characterized. The cDNAs reacted with mRNA from the livers of females but only when samples were taken in those parts of the year when egg production was occurring. No reaction occurred with mRNA from livers of untreated males at any time of the year. The cDNAs did react with mRNA from the livers of male animals when those animals had been exposed to oestrogens. Reaction also occurred with mRNA from oestrogen-exposed females regardless of the time of year.

7.  A method was developed for the measurement of VTG and ZRP mRNAs in preparations of RNA from fish liver using slot blots which thus allowed parallel processing of multiple samples. The method had a sensitivity of four orders of magnitude and thus could detect small increases of VTG expression at 0.1 % of a fully expressing female.

8.  The methodology has been deployed for analysis of sand goby undergoing experimental exposures to ethynyloestradiol (EE2), sewage effluent, octylphenol (OP) or oestradiol (E2). The effects of exposure on egg-protein mRNA expression in male and female animals has been investigated in parallel with studies of reproductive success. EE2, OP and E2 caused parallel increases in VTG and ZRP mRNA in males which was time and concentration dependent and with threshold concentrations of < 6 ng l-1 (EE2), 30 mg l-1 (OP) and 17-70 ng l-1 (E2). Associated with the changes caused by EE2 was a 95 % decrease in breeding success. The effects of sewage effluent were complex and a low concentration (0.03 % v/v) enhanced fecundity without alteration of VTG and ZRP expression in either gender. However, higher concentrations of effluent (0.3 % v/v) increased female VTG and ZRP while decreasing egg production relative to the lower concentration.

9.  In the environmental work sand goby and blenny were sentinel species and sampled from 12 UK estuaries. Inappropriate egg-protein production in male fish showed clear signs of endocrine disruption at locations suspected of being contaminated but only in blenny. Surprisingly sand goby collected from these same sites and at the same times showed no enhanced production of VTG or ZRP mRNAs.

10.  Differences in VTG expression between blenny and sand goby from the same environmental sites could be explained by different kinetics of induction following exposure and/or kinetics of depuration following removal of the oestrogen. An on-going “temporal response” study is underway in which males and female fish of four species (plaice, flounder, sand goby and blenny) are being followed during exposure to EE2 for 21 days and then for a further 31 days in the absence of oestrogen. Fish are sampled at intervals and hepatic VTG mRNA and serum VTG protein measured. Data is complete for plaice and showed similar responses for male and female animals. The induction of mRNA was faster than protein but both parameters reached a maximum by 16-20 days. There-after mRNA declined rapidly with a half life of approximately 3 days while the decay in protein levels was slower with a half life of 35 days.

11.  An assessment of the use of Differential Display, Reverse Transcriptase PCR (DDRT PCR) to detect altered gene expression in hermit crab (Pagurus bernhardus) exposed to polyaromatic hydrocarbons formed a minor part of this project. Differentially expressed genes were detected in crabs exposed to benzo[a]pyrene and induction and suppression of expression was found.

12.  This project has demonstrated the ease with which cDNA reagents can be generated for the measurement of biomarkers in fish species. These provided sensitive tools to assess the induction of egg proteins following (xeno-)oestrogen exposure. Tank experiments with oestrogen-treated fish revealed a complex association between altered VTG expression in both male and female and decreased reproductive success. In combination with other observations of morphological changes in sand goby exposed in the laboratory or collected from environmental sites it would appear that exposure at critical developmental stages may be significant in determining the severity of endocrine disruption. Clarification of the relationship between VTG expression and the critical exposure window merit further investigation. A temporal study undertaken to explore the relationship between VTG mRNA and protein during induction and depuration point to the value of both parameters. The relatively short half-life of mRNA commend this as the measure of choice to identify endocrine disrupting chemicals.

CSG 15 (9/01) 4

Project
title / Adaptive markers for environmental pollution.
/ DEFRA
project code / AE1130
Scientific report (maximum 20 sides A4)
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CSG 15 (9/01) 4

Project
title / Adaptive markers for environmental pollution.
/ MAFF
project code / AE1130

Introduction

The UK has international obligations under the London Convention and the OSPAR Convention to protect the marine environment and this responsibility is devolved to UK-Government Departments and to Environmental Agencies. A key element of fulfilling these responsibilities is in monitoring the condition of the sea and the well-being of the many plants and animals that live there. These are complex issues and the former MAFF commissioned a variety of programmes to research and thus to better understand the impact of discharges on the marine environment. The AE11 programme considered “Biological effects and impact assessment” and the project described here was funded as a part of that programme. The main aim of the project (AE1130) concerned the development of monitoring tools to assess the effects of endocrine disruption in fish (see below) and developed and molecular analytical techniques based on the newer approaches of recombinant DNA technology.

The project was carried out in the context of the UK-wide programme to evaluate Endocrine Disruption in the Marine Environment (EDMAR). EDMAR was started in 1998 as a multi-centre investigation funded by UK Government Departments, Agencies and the European Chemical Industry Association. The purpose of the project was to assess the extent to which endocrine disruption was affecting UK estuaries and to identify endocrine disrupting chemicals (EDCs) and their sources. The project described here was funded outwith EDMAR but had as its main focus support for EDMAR work being conducted at FRS, Aberdeen and at CEFAS, Burnham. A minor objective, not related to EDMAR, was to evaluate a novel technique for the impact of hydrocarbons on invertebrates.

The endocrine systems of animals co-ordinate and regulate growth, development, reproduction and other physiological processes. Recent observations in the UK, USA and elsewhere have shown that exogenous chemicals can interfere with the normal functioning of these systems and thus have the potential to adversely affect populations and communities. Although each of the diverse endocrine systems could be affected by environmental chemicals attention has focused in the UK on systems regulated by oestrogens because of the apparent feminisation of fish in rivers and estuaries as evidenced by the presence of a female yolk protein in male serum and occurrence of ovotestis.

The presence of vitellogenin (VTG) in the serum of male fish is the classic biomarker to diagnose exposure of animals to environmental oestrogens and thus is a proxy for endocrine disruption. The level of the protein in male animals from uncontaminated sites is very low (Purdom et al., 1994) but exposure to oestrogens or oestrogen mimics elicits a very large increase (Purdom et al., 1994, Harries et al., 1996, 1997, Folmar et al., 1996) by as much as a factor of 105. Traditionally the measurement of serum VTG is made by an immuno- chemical methodology (ELISA, RIA) which offers an economic and sensitive assay but lack of antibody cross-reactivity between species demands the purification of VTG from each new species with attendant method development. While the purification process is technically undemanding it requires a reasonable quantity of starting serum and thus is limited to relatively large fish and like all protein purifications from serum cannot entirely eliminate albumin. In turn this results in antibodies with anti-albumin immunoglobulin contamination which compromises ELISA measurements particularly at low VTG levels (Palmer et al., 1998).

An alternative strategy for the development of new biomarkers for different species is to develop nucleic acid probes and measure increased VTG expression at the level of mRNA. This approach exploits the resources of sequence databases and associated software to identify sequences conserved across species and based on this to design primers allowing the generation of species-specific cDNA fragments by PCR. Previous work from this laboratory has demonstrated the viability of this approach by the cloning of CYP1A from five teleost species (Craft et al., 2001) and the use of cDNA probes to measure CYP1A mRNA expression (Stagg et al., 2000).