Expression in Male Estuarine Toadfish
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AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Vol. 12, pp. 3-8, 2006 Rapid assessment of fish endocrine disruption Booth and Skene PAPERS RAPID ASSESSMENT OF ENDOCRINE DISRUPTION: VITELLOGENIN (VTG) EXPRESSION IN MALE ESTUARINE TOADFISH David J. Booth1* and Caroline D. Skene1,2 1 Department. of Environmental Sciences, University of Technology, Sydney, PO Box 123, NSW, 2007, Australia. 2 Department. of Veterinary Science, University of Melbourne, Melbourne, Corner Park Drive and Flemington Road, Parkville, VIC, 3052, Australia Manuscript received, 22/11/2005; accepted, 22/6/2006. ABSTRACT Increased contamination of waterways has lead to many impacts on organisms, including effects on reproduction. A suite of endocrine-disruptive chemicals (EDC’s) has been shown to mimic sex hormones in vertebrates and their presence is an important bioindicator of environmental degradation. We examined the expression of vitellogenin (Vtg, a female yolk protein) in male toadfish (Tetractenos glaber), as an indicator of EDC presence in estuaries around Sydney, Australia. First, we demonstrated the induction of Vtg in males from unpolluted estuarine sites through injection of 17-β-oestradiol. Secondly, the presence of Vtg in the serum of fish from polluted and unpolluted estuaries was investigated by reducing-polyacryamide gel electrophoresis (SDS-PAGE). While females from polluted (downstream from sewage treatment plants, and subject to urban runoff) and less polluted sites all expressed Vtg in blood serum, males from less polluted sites showed little or no evidence of Vtg expression. However, most males from heavily polluted sites showed moderate to high levels of Vtg expression, indicating that EDC’s were present and affecting normal endocrine function in males. We suggest that simple biochemical examinations of EDC effects, such as Vtg induction in males, are useful rapid assessment methods which can provide evidence upon which, further, more detailed studies may be undertaken. Key words: endocrine-disruptive compounds; estuarine fishes; sewage effluent; toadfish (Tetractenos glaber); vitellogenin. INTRODUCTION been employed as a bioindicator of endocrine disruption Estuaries are sites of high biodiversity and are interfaces and indicator of water quality (e.g., Tyler and Sumpter 1990; between terrestrial and coastal ecosystems. The harsh Purdom et al. 1994; Peters et al. 2001; Damstra 2003). This conditions within estuarine waters (fluctuating salinity, is of particular interest given that a suite of chemicals, many extremes of temperature, variable sediment and nutrient of which are present in sewage effluent, has been shown to influx) have lead to colonisation by unique assemblages of mimic natural hormones including the sex steroid, oestrogen organisms, many of which have great commercial importance (Batty and Lim, 1999; Robinson et al. 2003), which may lead to humans. However, estuaries are also prime sites for to endocrine effects such as Vtg induction. shipping, adjacent agriculture and urban developments. Vtg is a 300-600 kDa lipophosphoglycoprotein dimer Consequently, disturbances such as dredging, sewage and that is normally produced in female oviparous vertebrates nutrient influx, sedimentation and heavy metal accumulation (Denslow et al. 1999). This large, unstable molecule is have caused marked degradation in the water quality of normally synthesised in the liver of sexually mature females estuaries worldwide (Hutchings and Saenger 1987). in response to endogenous 17-β-oestradiol secreted by the Reduced water quality may affect community structure, ovary (Denslow et al. 1999). After transportation in the blood population sizes and physiology of aquatic organisms. At the from the liver to the ovary, Vtg is cleaved to form egg-yolk community level, species diversity of fish assemblages is often proteins (Kime et al. 1999). Although 17-β-oestradiol is not reduced in polluted waters and population densities of some normally produced in males, the cellular oestrogen receptors species may also be depleted (e.g., Lee et al. 1992). While responsible for inducing Vtg in females are present and can lethal effects of point-sources of pollution on fishes have be stimulated by exogenous oestrogens to produce Vtg. been demonstrated, chronic, sublethal effects of degraded The presence of functional oestrogen receptors in males waterways may be of more widespread importance and more and their ability to be stimulated by exogenous oestrogen accurately reflect the health of estuarine communities. makes Vtg induction a useful biomarker of xenoestrogen Biochemical attributes can serve as useful sublethal exposure in male oviparous vertebrates. Numerous studies bioindicators of organism, or ecosystem, health. Mixed- in the UK, Europe and America report the successful use function oxygenase (MFO) activity (Holdway et al. 1991, of Vtg as a biomarker of xenoestrogens in a wide range of Smith and Gagnon 2000), bile metabolite analysis (Lin et al. situations including monitoring oil refinery effluents, sewage 1996) and serum hormones (Nash et al. 2000) have all been effluent and the quality of enclosed water bodies (Folmar et used as biomarkers in a range of applications. In addition, al. 1996; Johnson et al., 1995; Knudsen at al. 1997). Folmar the appearance of Vtg in blood plasma of male fishes has et al. (2001) reported measurable levels of Vtg in the blood *Author for correspondence, email: David.Booth@uts,edu,au 3 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Vol. 12, pp. 3-8, 2006 Rapid assessment of fish endocrine disruption Booth and Skene Figure 1. Sydney Harbour (Port Jackson) and Hawkesbury River, Sydney, showing sampling sites (Berowra Creek, Cowan Creek, Homebush Bay). Black squares indicate location of Sewage Treatment Plants. of both male and female freshwater walleye (Cyprinus disruption in the species under investigation, before large carpio) collected from the effluent channel of a major sewage outlays of time and funds have been made. The aims of this treatment plant (STP), but males contained no serum Vtg study were, therefore, first to provide preliminary evidence away from the point source of sewage. of the capability of Vtg induction in an Australian estuarine fish (Tetractenos glaber F. Tetraodontdae) by exposure to 17- Few studies have definitively demonstrated EDC effects, β-oestradiol in the laboratory, and secondly, if successful, to especially for estuarine organisms. One shortcoming of use the evidence gathered to make a primary assessment of serum Vtg as an indicator of estuarine pollution is that Vtg induction in fish captured from estuarine sites suspected the development of an assay such as ELISA or RIA for to be contaminated by EDC’s. quantitative detection is a complex and expensive procedure. Assay development involves Vtg induction experiments over MATERIALS AND METHODS a number of weeks, purification and characterisation of Vtg from fish serum, induction of Vtg-specific antibodies (poly- Tetractenos glaber (Fréminville 1813) is a small fish or monoclonal) and potentially the use of radioactivity (e.g., inhabiting estuaries and coastal bays on the south-east coast Sumpter 1995; Tyler and Sumpter 1990). of Australia (Kuiter 1994). They are found in aggregations in mud-flat shallows in estuaries and may enter freshwater, but Apart from the expense of quantitative ELISAs and RIAs, it are not fished commercially due to the toxin carried on their is also potentially hazardous to experimenters if radioactive skin and in their internal organs. Few studies have focussed isotopes are used and neither protocol is compatible with on tetraodontid fish ecology, despite their major contribution rapid assessment. We propose that a quantitative assay may to fish biomass in estuaries and other habitats (e.g., Thresher not be necessary to establish the presence or absence of Vtg. 1984). Toadfish are common in the estuaries of central New A simpler, qualitative initial test would help determine the South Wales, and consume benthic crabs and bivalves (Booth relevance of further study of Vtg induction and endocrine and Shultz 1999). Gonadal development occurs in late autumn 4 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Vol. 12, pp. 3-8, 2006 Rapid assessment of fish endocrine disruption Booth and Skene (April-May, Booth and Schultz 1999). T. glaber are site- cavity using 1-mL syringes and 25 gauge needles (Terumo). specific, common throughout the year and have previously A second injection was administered seven days after the first. been used as bioindicators (Booth and Schultz 1999; Alquezar Serum was collected by cardiac puncture ten days post second et al. 2006a,b). injection after lethal anaesthesia using clove oil. Blood was allowed to clot on ice before centrifugation at 5000 g, 4°C Toadfish were sampled from two major estuarine arms of the for 15 minutes. Serum samples were then sub-aliquoted into Hawkesbury River (33°30’S, 151°10’E) near Sydney and in multiple separate tubes and stored at -20°C. Fish total weight, Sydney Harbour. Cowan Creek (Reference Site) and Berowra total length, liver weight, gonad weight and carcass weight Creek (Impacted Site), are both in the Hawkesbury River were also recorded. Sex was determined after dissection or estuary, and Homebush Bay (Impacted Site), is an estuarine histological examination of gonads. arm of Sydney Harbour (see Figure 1). The Hawkesbury- Nepean River system extends over 200 km and flanks the Whole serum samples were run on reducing SDS-PAGE Sydney Basin to the north and west. It supports significant (sodium dodecyl sulfate polyacrylamide