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, , PO Box 123, NSW, 2007, . 2 Department. of Veterinary Science, University of , 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 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 ( 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 , 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

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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 gel electrophoresis) recreational and commercial fisheries, particularly prawns gels (6%) and silver stained using Amersham Pharmacia and is utilised for mariculture of oysters along the foreshores Biotech AB (Sweden) kits (Ref. # 17-1150-01) with a (Mercer 1984). Berowra Creek is a sheltered waterway with a detection limit of 1-5 ng. Results of the SDS-PAGE were limited tidal flow in which urban development has generated scored as either presence or absence of presumptive Vtg band considerable sediment movement. Its tributaries are sites and statistically analysed using the G-test (Zar 1996). for two sewage treatment plants (Figure 1). Urban and farm Fish collected from each field site were bled as described for run-off cause significant pollution and may be major factors the Vtg induction experiment described above upon return affecting the quality of the water and sediment. Cowan Creek, to the laboratory and whole serum was analysed by reducing part of the Ku-ring-gai Chase National Park, is located on SDS-PAGE. For statistical analysis, due to low numbers of the southern side of Broken Bay, approximately 5 km east of fish available during the study, it was necessary to pool data Berowra Creek (Figure 1). The major habitats found within among sites for the “impacted” treatment. this Creek include and sandflats with sparse areas. While similar in morphology and habitats, RESULTS AND DISCUSSION Cowan Creek is generally cleaner than Berowra Creek, due to reduced urban and agricultural runoff, and lack of sewage There was clear induction of a protein of approximately 115 treatment plant effluent (Hornsby Council, unpub. report). kDa in male fish treated with 17-β-oestradiol relative to the However, it receives considerable recreational boat traffic. reference fish (Figures 2 and 3). The protein was found in Natural oyster beds are prolific along the rocky shores of untreated and treated females, but not in untreated males. The Cowan Creek, compared to Berowra Creek, where rocky presumption that this protein was Vtg was based on numerous foreshores that once supported oysters are now covered with studies in the literature that cite the approximate size of the green algae. Both creeks have sediment “hotspots” for organic dimeric molecule to be between 300-600 kDa (160-200 kDa contaminants such as DDD and other derivatives of DDT subunit), and that Vtg is inducible by 17-β-oestradiol (eg. along with high concentrations of nitrogen and phosphate Núñez-Rodriguez et al. 1989; Mañanós et al. 1994; Tyler et nutrients (Birch et al. 1998). Sydney Harbour drains the al., 1996; Heppell and Sullivan, 1999). central Sydney Basin, an area of high population density By demonstrating the capacity of male toadfish to produce and industrial effluent, including heavy metal accumulation presumptive plasma Vtg in response to oestrogen exposure, in sediments (Macfarlane et al. 2000). the induction experiment fulfilled the primary aim of the Fish were collected in April 2001 using a 15.5 x 1.75 m beach- present study. Although the 115 kDa molecule was not seine net with a mesh of 16 mm stretch. The method involved characterised beyond SDS-PAGE analysis, the evidence in manually hauling the net across the estuary floor, covering a the literature strongly suggests that a serum protein is likely depth of up to 1.5m and a width of 8-10 m, encompassing an to be Vtg if within the expected size range of other Vtg area of approximately 10-15 m2. From each haul, a random monomers and is induced in males by a strong oestrogen sample of up to ten T. glaber were kept alive in aerated water (17-β-oestradiol). Furthermore, given reducing SDS-PAGE and returned to the laboratory where they were either bled analysis was used, it is very likely that the 115 kDa protein immediately or kept in aerated 40-L aquaria for induction we detected is a monomer, as dimeric molecules would have experiments (see below). been separated during processing of the sample. Fish were fed ad libitum in aquaria every day except at Examination of sera from male toadfish collected from weekends, with freeze-dried brine . Seawater was reference and polluted sites clearly indicated that significantly partially changed at least twice per week. On day one, fish more males from polluted sites expressed the 115 kDa protein were injected with 10 μg of 17-β-oestradiol (Sigma, Australia) in their serum, relative to males from reference sites (Figure per gram of their body weight (sensu Tyler et al. 1996). 3; 2-tailed G-test, P<0.02), despite relatively low sample Oestradiol was prepared in a corn oil carrier using minimal sizes. Given the absence of the protein in all males from the dimethyl sulphoxide (DMSO) (Sigma, Australia) to dissolve reference site, and compared with existing literature (Wiley the oestradiol (Teasdale, unpub. thesis). Prior to weighing et al. 1979; Núñez-Rodriguez et al. 1989; Mañanós et al. and injection, fish were anaesthetised with clove oil to reduce 1994; Heppell and Sullivan 1999) the presence of the 115 kDa stress. Injections were administered to the intra-peritoneal protein in all but one male from the polluted sites suggests the action of EDCs in estuarine waters around Sydney.

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Rapid assessment of fish endocrine disruption Booth and Skene

Figure 2. Examples of SDS-PAGE gels, illustrating absence or presence of presumptive Vtg bands at 115 kDa. Arrows indicate presence of presumptive Vtg band. The lanes are: A. 1 = female, from reference site; 2 = marker lane (numbers indicate kDa size); 3 = male, from reference site; 4 = male, from reference site; 5 = female, induced; 6 = male, induced; 7 = female, from reference site; 8 = male, from reference site. B. 1 = female, from reference site; 2 = marker lane; 3 = male, from reference site; 4 = female, from reference site; 5 = female, induced; 6 = male, from polluted site; 7 = female, from polluted; 8 = female, from polluted; 9 = male, induced; 10 = male, from polluted site.

Whilst most (9/10) of the male fish collected from polluted experimental concentrations of sewage effluent (containing sites exhibited the presence of Vtg, the protein was absent EDC’s) in a laboratory study. While some liver atrophy was from one fish. This may have occurred if the fish had recently measured in males and females subjected to the highest moved into the area from a less impacted site, and not been effluent concentrations, no obvious effect on gonadosomatic sufficiently exposed to ECD’s for Vtg induction to occur. index was found. This suggests that subtle indications of This particular fish was about 6 g in weight, the smallest fish endocrine disruption, such as Vtg expression in males, may tested, and may have been an immature male. In addition, occur in the absence of more obvious effects and be more differences in individual physiology probably lead to variation sensitive indicators of pollution. It should be noted that in responses to EDC’s. Such variation may be particularly presence of plasma Vtg is only suggestive, not indicative, of useful in making an initial assessment of the health of an endocrine disruption (Allen et al. 2002) estuary. For example, if all (100%) fish are found positive The importance of investigating the relevance of a rapid for presumptive Vtg, it may indicate a highly contaminated assessment for serum Vtg is to minimise time and resources site; if 50%, a moderately polluted site; and if none (0%), a invested in developing highly specific, quantitative measures clean site. While numbers of males were low in this study, in a species that may not be sensitive to endocrine disruptors, results were highly significant (P=0.02), indicating strong and to determine whether a specific species or site warrants effects of EDC’s on these fish. further study. This study has clearly shown that there is a Actual changes in reproductive physiology of toadfish due basis for further investigation of T. glaber as a bioindicator to EDCs have not been found to date. Booth and Longman of endocrine disruption, using a rapid assessment for serum (unpub.data) compared gonad and liver morphometry in Vtg, and suggests that there may be a role for this kind of rapid males and females that had been subjected to different assessment method for other potential bioindicator species.

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Rapid assessment of fish endocrine disruption Booth and Skene

Figure 3. Tetractenos glaber exhibiting Vtg presence in blood serum in each of three treatments for both males and females: Induced = induction with 17-β-oestradiol; Reference = fish derived from Cowan Creek field site; Polluted = fish derived from Berowra Creek and Homebush Bay field sites (combined). Proportion of males exhibiting Vtg presence in Reference vs. Polluted treatments differed (G-test, G=5.9, P=0.02)

CONCLUSIONS AND FUTURE DIRECTIONS ACKNOWLEDGEMENTS We successfully identified a presumptive Vtg protein of This study was financially supported through a grant from approximately 115 kDa in T. glaber that was subsequently the University of Technology, Sydney, to DJB. We are used to rapidly assess the health of estuaries in and around grateful to the Molecular Parasitology Unit at The University Sydney. Estuarine toadfish males at sites downstream from of Technology, Sydney for use of laborataory space and sewage treatment plants exhibited Vtg in their blood, whereas equipment, and to two anonymous reviewers of an earlier those from a similar site without a nearby sewage treatment draft of this manuscript. We thank Ciaran Longman and plant did not. The results of this study suggest that further Ralph Alquezar for assistance with toadfish collection. This characterisation of the 115 kDa protein induced in T. glaber is contribution No.1 from The Sydney Harbour Institute of males would be worthwhile to further validate the methods Marine Science. used in addition to studies in other species of oviparous fish to substantiate this new, fast approach to the assessment of REFERENCES estuarine health. The protocol developed to measure the Allen K, Blaam J, plus 34 authors. 2002. Endocrine presence/absence of Vtg allows more rapid assessment of Vtg Disruptors in the Marine Environment UK Environment presence than the more elaborate and expensive quantitative Agency 70 pp. methods employed to date and will prove useful in identifying endocrine disruption in fish. Alquezar R, Markich SJ and Booth DJ. 2006a. Metal accumulation in the smooth toadfish, Tetractenos glaber, in estuaries around Sydney, Australia. Environmental Pollution 142, 116-122. Alquezar R, Markich SJ and Booth DJ. 2006b. Effects of metals on condition and reproductive output of the smooth toadfish in Sydney estuaries, south-eastern Australia. Environmental Pollution 142, 123-131. 7 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Vol. 12, pp. 3-8, 2006

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