The Society for Integrative and Comparative Biology
Larval Development and Vitellin-like Protein Expression in Palaemon elegans Larvae Following Xeno-oestrogen Exposure1
1 Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK
2 Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK
3 The Environment Agency, Head Office, Rio House, Waterside Drive, Almondsbury, Bristol BS32 4UD, UK
4 School of Marine Science and Technology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
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Certain anthropogenic chemicals, most notably xeno-oestrogens, are known to have the potential to disrupt vertebrate endocrine systems. For example, induction of the female-specific protein, vitellogenin, in male fish is a well-known effect of exposure to xeno-oestrogens and serves as a biomarker of such exposure. There have been few comparable studies of putative biomarkers of endocrine disruption in invertebrates. An exception is the upregulation of vitellin-like larval storage protein (LSP) expression in the barnacle cypris larva following exposure to oestrogenic chemicals. The current study aimed to establish whether larvae of the glass prawn, Palaemon elegans, are likewise susceptible to xeno-oestrogen exposure. Using a polyclonal antiserum to P. elegans apolipovitellin, an 86 kDa polypeptide was detected by western blotting in the larval and early postlarval stages of this species. An indirect ELISA applied to the soluble protein fraction of larval homogenates determined that the titre of this putative LSP ranged, depending on larval stage, from 0.48–0.67 ng µg–1. Exposure of P. elegans larvae to the xeno-oestrogen 4-n-nonylphenol (4-NP), at 0.2–20 µg L–1, resulted in a significant, concentration-independent increase in putative LSP levels of 5–18%. Conversely, exposure to the natural oestrogen, 17ß-oestradiol (E2), at 0.2 and 20 µg L–1, led to a significant concentration-independent decline (up to 11%) in LSP levels. Whether the effect of 4-NP results from endocrine disruption is not known, however, an oestrogen receptor-mediated effect is unlikely. Other than a slight increase in larval mortality when exposed to 4-NP at 2 µg L–1, neither 4-NP nor E2 significantly affected development, growth or survival of P. elegans larvae.
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INTRODUCTION |
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There is a considerable body of evidence to suggest that many anthropogenic chemicals in the environment are able to disrupt the endocrine system of vertebrates, resulting in adverse effects on hormonally controlled functions like reproduction and development (Jobling and Sumpter, 1993
; Guillette et al., 1999; Bosveld and van den Berg, 2001). Particular concern has been raised by the ability of so called xeno-oestrogens to exhibit oestrogenic activity via the oestrogen receptor in vitro (Soto et al., 1991; Segner et al., 2003).
Several anthropogenic chemicals, particularly alkyphenol polyethoxylates (APnEOs), have been shown to produce significant oestrogenic effects in fish (Jobling and Sumpter, 1993; Harries et al., 1995; Jobling et al., 1996). APnEOs are synthetic surface-active agents (surfactants), commonly used in industrial detergents and plastics manufacturing (Blackburn et al., 1999). Around 80% of all manufactured APnEOs are nonylphenol ethoxylates (NPnEOs) (Naylor, 1998), which degrade to nonylphenol (NP) in sewage treatment plants (Ahel et al., 1994). Concentrations of NP in U.K. rivers are <0.2–12 µg L–1, although concentrations as high as 180 µg L–1 have been reported in water receiving effluent directly from sewage treatment works (Blackburn and Waldock, 1995; Allen et al., 2002). The concentration of NP in marine environments is normally lower with reported values in the range 0.2–5.2 µg L–1 (Blackburn and Waldock, 1995; Blackburn et al., 1999; Lye et al., 1999; Allen et al., 2002).
Monitoring of environmental contaminant levels by chemical analyses has a strong tradition. The associated methodologies are typically costly, however, requiring expensive equipment which may not be available to all laboratories. Moreover, contaminant levels, which exert effects on organisms, may be below the limits of analytical detection. Consequently, there has been a move to identify and develop biomarkers as monitoring tools for environmental pollution (Depledge and Billinghurst, 1999; Handy et al., 2003).
Several studies have implicated EDCs, including oestrogens, xeno-oestrogens, insecticides, PCBs and PAHs as having a detrimental effect on moulting, development and the reproduction of invertebrates (Baldwin et al., 1995, 1997; Zou and Fingerman, 1997a, b; Brown et al., 1999; Oberdörster et al., 1999; Nice et al., 2000). However, with the notable exception of imposex in neogastropods due to TBT exposure, (Bryan et al., 1986; Bettin et al., 1996; Evans et al., 2000), there remains a lack of practical biomarkers of EDC exposure in invertebrates.
Billinghurst et al. (2000) reported elevated expression of cyprid major protein (CMP), a vitellin-like protein, in the larvae of the barnacle, Balanus amphitrite, on exposure to the xeno-oestrogen 4-n-nonylphenol (4-NP) at 1 µg L–1. At similar concentrations, 4-NP significantly reduced the settlement rate of B. amphitrite (Billinghurst et al., 1998). Therefore, if vitellin and CMP synthesis are under similar hormonal control (Billinghurst et al., 2000), CMP expression may serve as a biomarker of endocrine disruption.
Though well characterised in insects (Burmester et al., 1998a, b, 1999; Zhu et al., 2002), CMP was the first example of a crustacean larval storage protein. The present study had the dual aim of determining whether a similar vitellin-like protein exists in the larvae of the glass prawn, Palaemon elegans, and if so, whether expression of this protein is modulated by oestrogenic exposure, with concomitant effects on larval development.
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Seawater
Seawater (31–33 ppt.) was obtained via an offshore pipeline and a seabed sand filter at Cullercoats, Tynemouth (U.K.). At the University of Newcastle-upon-Tyne, it was filtered through in-line, 10 µm and 1 µm filters, and UV-irradiated.
Chemicals
4-n-nonylphenol (4-NP, >99.9%) was supplied by Lancaster Chemicals, (U.K.). Unless otherwise stated, all other chemicals were of molecular or analytical grade and were obtained from Sigma-Aldrich Chemical Company Ltd. (Dorset, U.K.). Solvents were of glass distilled or HPLC grade.
Test solutions
Stock solutions of 17ß-oestradiol (E2) and 4-n-nonylphenol (4-NP) were prepared in acetone at 1 mg ml–1 with working solutions prepared daily by dilution with acetone. Test solutions in acute and chronic exposures were prepared by the addition of 25 µl aliquots of the working solution to filtered seawater. A solvent control was used for both acute and chronic exposure trials and contained 25 µl L–1 (0.0025%) acetone.
Analytical methods
To determine the definitive experimental concentrations of 4-NP, experimental solutions underwent solid phase extraction (SPE) using C18 Isolute trifunctional (non-endcapped) cartridges and GC-MS using a Hewlett Packard 5973 MSD fitted with a 30 m DB5MS column (0.25 µm coating with 0.25 mm internal diameter; J&W Scientific) adapted from the methods of Lye et al. (1999) and Billinghurst et al. (1998). Briefly, test solutions of 4-NP were prepared in seawater to give nominal concentrations of 0.02, 0.2, 2 and 20 µg L–1. An additional treatment, containing 4-NP at 20 µg L–1 with Artemia sp. nauplii at 15 ± 1 individuals ml–1, was also prepared. Nauplii were removed prior to SPE by passing the test solution through an 80 µm sieve. A control treatment contained acetone at 1 ppt. At t = 0, 12 and 24 hr, 3 x 1 liter of each test solution was subjected to SPE to isolate 4-NP. 4-tert-octylphenol (4-OP: Fluka, U.K.; >90%) and 2,4,6-tribromophenol (TBP) were used as surrogate (Ss) and internal standards (Is), respectively.
Seven ions, m/z 107, 135, 141, 206, 220, 330 and 332, were monitored, with the selected ions m/z 107, 135 and 330 corresponding to 4-NP, 4-OP and TBP respectively. Concentrations were determined by integrating the peak areas in the m/z 107 (4-NP), 135 (4-OP) and 332 (TBP) mass chromatograms and ratioing the analyte peak areas against the Is, using a response factor from the Ss. Procedural blanks were run for each sample period and a mean extraction recovery (% standard added/% sample recovered) of 97.9 ± 4.6% was determined for this method.
Broodstock and larval culture
Gravid Palaemon elegans, collected from Wembury Beach (Devon, U.K.), were individually maintained in 1-liter glass beakers containing 1 liter of filtered seawater and fed fresh Mytilus edulis tissue until the eggs hatched. Within 8 hr of hatching, zoea 1 larvae (Z1) from 3–10 adults were pooled and the healthiest larvae were collected by phototaxis to a single point light source (first to light selected) prior to use. Larvae were reared in batch cultures, at 50 individuals L–1 and fed newly hatched Artemia sp. nauplii, (Sanders Co. Utah) at 15 ± 1 individuals ml–1 until moulting to the required stage. Batch cultures were maintained at 15°C, with daily seawater changes and overhead lighting set to an ambient square wave pattern during development (approx. 16L:8D summer –7L:17D winter).
Acute toxicity
Acute toxicity trials were only conducted on newly moulted larvae (<6 hr) with similar moult histories. Larval stages were exposed in 1 liter glass beakers to 4-NP or E2, in a geometric range of 30–1730 µg L–1, at 14 ± 1°C. The seawater and solvent (acetone <1 ppt) controls, and each of the test concentrations were replicated (n = 3) and 15 larvae were added to each. A complete water exchange was performed daily, at which point dead larvae were removed and the surviving larvae counted after 48 hr and 96 hr. No food was provided during this period.
Chronic toxicity
Z1 were exposed to sublethal concentrations of 4-NP (0.2, 2 or 20 µg L–1) or E2 (0.2 or 20 µg L–1), with seawater and solvent controls in 1-liter glass beakers at 50 individuals L–1. Each treatment and control was run in triplicate, illuminated by overhead lights, fed daily with newly hatched Artemia sp., and gently aerated via glass tubes.
A complete water exchange was performed daily, at which point, the surviving larvae were counted. Zoeal larval stages 1–7 and the post larval stage were identified according to Fincham (1977) and Carli (1978). The length, from the tip of the rostrum to the tip of the tail, of 3 individuals from each replicate was recorded under a binocular microscope fitted with a calibrated eyepiece graticule. Once more than 50% of the population, in each replicate, had developed to the next stage, larvae of the most advanced stage were measured.
Total soluble protein assay of larval homogenates
All the larvae from each replicate were pooled before homogenisation and the soluble protein extracted in a homogenisation buffer (0.5 mN Tris, pH 6.8), using a PTFE pencil mixer (DX, IUCHI, Japan). Extracts were centrifuged at 10, 000 g for 10 min and the resultant supernatant filtered through a 0.2 µm Vectaspin micro-centrifuge filter (6833-0201, Whatman International, U.K.) prior to being assayed for total soluble protein and stored at –80°C. Total soluble protein was determined by the Bradford method (Bradford, 1976), according to Billinghurst et al. (2000), with bovine serum albumin (BSA) as the standard. According to this method only 3.7 ± 0.2% of the total soluble protein extracted from larvae was lost during sample preparation.
Electrophoresis
Samples were mixed with an equal volume of cathode buffer (1% SDS, 1% mercaptoethanol, 50 mM Tris, pH 6.8, 20% glycerol, 0.001% bromophenol blue), heated to 100°C for 3 min and then subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970). Reference molecular weight markers of 205, 116, 97.4, 66, 45 and 29 kDa (SDS-6H, Sigma) were applied as required. Gels were stained with Coomassie Brilliant Blue R (CBB-R). Holoproteins were resolved on polyacrylamide gels under non-denaturing conditions in the absence of SDS (Native-PAGE). Samples were diluted to 1 mg ml–1 and mixed in equal volume with a non-denaturing cathode buffer (50 mM Tris, pH 6.8, 20% glycerol, 0.001% bromophenol blue) and subjected to electrophoresis on 3–12% native gradient gels (15 x 15 cm) at 40 mA for 4 hr. The molecular mass of holoproteins was estimated by comparison of the mobility of the subject proteins with that of jack bean urease (hexamer, 545 kDa; trimer, 272 kDa) and bovine serum albumin (dimer, 132 kDa; monomer, 66 kDa) (Sigma).
Lipovitellin extraction and purification
Ovaries, containing developed oocytes, were removed from 15 females under Pantin's crustacean solution (Pantin, 1933). Approximately 3 g of the excised tissue was homogenised in 10 ml of extraction buffer (50 mM Tris-HCl, pH 7.5), containing 100 µl of protease inhibitor cocktail (Sigma, P8340) and centrifuged at 10,000 g for 15 min. Protein was precipitated from the supernatant by the addition of an equal volume of saturated ammonium sulphate, producing a crude vitellin extract (Oberdörster et al., 2000). The final pellet was re-suspended in 3 ml of extraction buffer, prior to ion-exchange chromatography following a procedure modified from Pateraki and Stratakis (1997). In brief, 3 ml of crude vitellin extract was dialysed overnight against 1 liter of elution buffer containing 0.5 mM PMSF and aprotinin at 10 KIU ml–1 (Denslow et al., 1999). Exactly 1 ml of the dialysed sample was loaded onto a 10 x 1 cm column of CM Sepharose CL-6B (Amersham Biopharmacia) and the unbound proteins were eluted with approximately 5 bed heights of 0.1 M sodium acetate containing 10 mM EDTA. Vitellin was eluted with 0.5 M NaCl at 1 ml min–1 according to Allen et al. (2002) and fractions, monitored at 280 nm, were collected in 2 ml increments.
Fractions containing vitellin of the highest concentration and purity, based on molecular weight and sub-unit composition ascertained by SDS-PAGE, were pooled and reduced to 2.5 ml (Centricon, model 100; Amicon Ltd., U.K). Desalting was performed using PD10 columns (Amersham Biopharmacia), eluting with 0.1 M phosphate buffered saline (PBS). The overall volume was reduced to 1 ml (Centricon, model 30) and the purity of the final vitellin extract confirmed by SDS-PAGE. Gels were stained with Silver Stain Plus (Bio-Rad Laboratories Ltd.).
Lipid removal
Lipid was removed from lipovitellin according to Spaziani et al. (1995). In brief, lipovitellin was added to ethanol:diethylether (1:1), mixed, left to stand on ice for 10 min and centrifuged at 10,000 g for 10 min at 4°C. The pellet was resuspended in 30 ml of ethanol: diethylether and the process repeated. The final pellet was suspended in 5 ml of 0.153 M NaCl, containing 1% SDS (w/v), and reduced to 1 ml (Centricon, model 30) over 1 hr at 20°C. Remaining SDS was removed using SDS-Out (20310, Pierce Il. U.S.A.). Lipid removal was confirmed by staining gels for lipid, following Native-PAGE, with Sudan Black B (Rainwater, 1998) with marker proteins stained with CBB-R.
Polyclonal antisera production
Polyclonal antibodies were generated, in rabbits, against lipovitellin and apolipovitellin by Immune Systems Ltd. (Paignton, U.K.).
Immunoblotting
Protein transfer (Western blotting) to Flurotrans polyvinylidene-difluride (PVDF) membrane was conducted using a semi-dry procedure with CAPS buffer (10 mM CAPS, pH 11). Membranes were blocked in casein solution (SP5020, Vector Laboratories Inc, U.S.A.) overnight at 4°C. Membranes were first incubated for 30 min with the polyclonal antiserum to apolipovitellin, which was diluted (1:3,000) with PBS and then in a secondary anti-rabbit (IgG) alkaline phosphatase conjugated antibody (Sigma, A3687) diluted 1:3,000 with casein solution. Bands were visualised with 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) using the Sigma-FAST substrate system (B5655).
Sample preparation for ELISA
Z1 were exposed to 4-NP at 0.2, 2.0, 20.0 µg L–1 or E2 at 0.2 and 20.0 µg L–1 with seawater and solvent controls. Solutions were changed daily and newly hatched Artemia sp. nauplii were added as food. On moulting to the required stage, 100 larvae from each treatment and control were sampled for soluble protein extraction for ELISA.
ELISA
An indirect ELISA, modified from Allen et al. (2002), was used to quantify vitellin-like proteins in P. elegans zoeae. For each sample, 15 µg of total soluble protein in 200 µl of coating buffer (50 mM carbonate, pH 9.6) was added to 3 wells of a 96-well polystyrene microplate (Elkay Laboratory products, U.K.) which was incubated overnight at 4°C. The wells were washed four times with Tween-20 phosphate buffered saline (TPBS) (10 mM sodium phosphate, pH 7.3, 0.05% Tween-20) and incubated for 1 hr at 21°C with 200 µl blocking buffer (10 mM sodium phosphate, pH 7.3, 3.5% powered milk, Tesco). After washing with TPBS antiserum to P. elegans apolipovitellin, diluted 1:3,000 in blocking buffer, was added to each well and incubated overnight at 4°C. Wells were subsequently rinsed thoroughly with TPBS and incubated with 200 µl of PBS (10 mM sodium phosphate, pH 7.3), containing horseradish peroxidase conjugated anti-rabbit IgG (Sigma, A6154), for 2 hr at 37°C. Following a further three washes in TPBS, 200 µl of citrate buffer (0.1 M citrate buffer, 0.03% H2O2, pH. 4.2), containing 0.5 mg ml–1 of 2,2'azinobis[3-ethylbenzothiazo line-6-sulphonic acid] diammonium salt (ABTS), was added to each well. After incubating for 90 min at 21°C the absorbance was recorded at 405 nm using a microplate reader (Molecular Devices).
A range of lipovitellin standards (5–70 ng) was added to each plate, in triplicate, to obtain a standard curve. From this, the amount of vitellin-like protein present in larval homogenates could be expressed as vitellin equivalents. Assay sensitivity was defined as the point two standard deviations from the mean of 15 replicates of the blank. Assay robustness, associated with its reproducibility, was defined by intra-plate coefficients of variation (CV) below 10%.
Data analysis
The 48-hr and 96-hr LC50 values and associated confidence intervals for 4-NP and E2 respectively were calculated, according to the Trimmed Spearman-Karber method (Hamilton et al., 1977), using a computer programme supplied by the Environmental Monitoring Systems Laboratory (E.P.A., U.S.A.). For the analysis, data for seawater and solvent controls were pooled. Statistical analyses were performed using Minitab (v. 10.2). One-way analysis of variance (ANOVA) was performed after checking for homogeneity of variance with Bartlett's test. Significant differences (P < 0.05) were analysed by post-hoc Scheffe's or Tukeys methods. Where required, to meet normality requirements, data were arcsine transformed and ANOVA performed on the transformed data.
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Chemical analysis
Definitive concentrations of freshly prepared test solutions were either in close agreement with the nominal values or slightly lower (Table 1). Excluding the treatment of 0.02 µg L–1, which was not used in subsequent acute or chronic exposure experiments, definitive concentrations after 24 hr were 17.8–55.6% lower than initial levels. A greater loss of 86.4%, between the initial and the final measurement of 4-n-nonylphenol (4-NP), was recorded in the highest test solution (20 µg L–1) when Artemia sp. nauplii were present.
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Acute toxicity
The 48-hr LC50s for 4-NP are presented, for each larval stage, in Table 2. Based on the 95% confidence intervals, the initial larval stages (Z1–Z3; 59.3–96.1 µg L–1) were less sensitive to 4-NP than the intermediate stages (Z4–Z7; 34.3–52.8 µg L–1). The 1st postlarval stage was as sensitive as Z2 and Z3. All 96-hr LC50 values for larvae exposed to E2 were >2 mg L–1.
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Chronic exposure
Within the range 0.2–20 µg L–1, neither 4-NP nor E2 had a significant effect on the size of larvae at each stage (Fig. 1). There was also no effect on the timing of development, with successive larval stages appearing in the different treatments within 8 hr of each other. Survival to the first postlarval stage in 2 µg L–1 was significantly reduced (F6,20 = 2.88, P < 0.05) compared to both controls (Fig. 2). There were no significant differences, however, between any of the other treatments.
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Lipovitellin characterisation
P. elegans lipoprotein was approximately 540 kDa and, as judged by staining with Sudan Black B, the associated lipid fraction was successfully removed by solvent extraction to produce apolipovitellin (Fig. 3). After screening the polyclonal antisera against their respective antigens, the antiserum to apolipovitellin produced the strongest response and was used to examine proteins in larval homogenates.
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Identification of vitellin-like proteins in P. elegans larvae
SDS-PAGE of lipovitellin and homogenates of each of the larval stages and the 1st postlarval stage is presented in Figure 4. Lipovitellin was comprised of 5 sub-units of approximately 182.9, 103.7, 97.4, 86.4 and 79.6 kDa. All larval homogenates contained primary polypeptides of approximately 86 and 76 kDa with minor polypeptides between 81–41 kDa (Fig. 4). Smaller peptides ran off the gel. In each of the larval stages a single protein band of approximately 86 kDa cross reacted strongly with antiserum to apolipovitellin (Fig. 5).
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ELISA optimisation
Compared to the standard curve acquired using antiserum to lipovitellin, which only had a linear response above 20 ng, the standard curve obtained with antiserum to apolipovitellin was linear within the range 5–70 ng (Fig. 6). Lipovitellin standards (5–70 ng) were incubated with serial dilutions of primary and secondary antisera in the range 1:100–1:15,000. Maximum OD values, from 70 ng of lipovitellin, were measured using the antiserum to apolipovitellin and the secondary antiserum at dilutions of 1:3,000. Using these dilutions, the curve obtained for serial dilutions of larval homogenates was also linear up to a loading of 20 µg of total soluble protein per well. Inter- and intra-assay variations were 2.9–6.8% and 3.6–12%, respectively. The limit of detection (LOD) was calculated as 6.3 ng.
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Quantification of vitellin-like larval proteins
Vitellin-like protein concentrations in P. elegans larvae following exposure to 4-NP and E2 are presented in Figure 7. At each larval stage there were no significant differences (p < 0.05) between the concentrations of the vitellin-like protein in larvae from the seawater or acetone control and these data were pooled. The highest concentration of 0.67 ng µg–1, (based on 15 µg per sample) was recorded in Z1. The levels declined in the control larvae to 0.48 ng µg–1 between Z2 and Z4 and remained relatively constant thereafter. The concentration in the first postlarval stage was comparable to Z1 (
0.67 ng µg–1). With the exception of Z3, vitellin-like protein levels were elevated in all the larval stages exposed to 0.2 µg L–1 4-NP (Fig. 7A). In Z2, the corresponding titre was 108% of the control and increased to 118% by Z6, after which it fell to 107% in the postlarval stage. In contrast, only Z4 larvae exposed to 2 µg L–1 4-NP had significantly (F5,15 = 65.63, P < 0.001) elevated levels of the vitellin-like protein (107%). Exposure to 20 µg L–1 4-NP significantly reduced the protein titre of Z3 to 91% of the control larvae (F5,15 = 12.79, P < 0.001), while levels in Z4, Z5 and Z6 were 105, 108 and 113% of the control respectively. Significantly lower levels (F5,15 = 12.79, P < 0.001) in Z3 (91%), and significantly higher levels in Z5 (104%; F5,15 = 88.38, P < 0.001) and Z6 (105%; F5,15 = 88.78, P < 0.001), were measured in larvae exposed to 0.2 µg L–1 E2. At each stage concentrations recorded in the larvae exposed to 20 µg L–1 E2 were significantly lower than the control larvae (Fig. 7B).
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This study has provided evidence that proteins, immunologically similar to lipovitellin, are present in the larval stages of P. elegans; the titres of which are significantly modified by exposure to both the xeno-oestrogen 4-n-nonylphenol (4-NP) and the natural oestrogen 17ß-oestradiol (E2). Furthermore, while 4-NP is acutely toxic to the larval stages of P. elegans at lower, environmentally realistic, concentrations 4-NP has little effect on their survival or development.
Exposures to test chemicals in this study were conducted by static renewal. Under these conditions the concentrations of 4-NP declined by 17.8–55.6% over 24 hr. Similar time dependent losses have previously been attributed to sorption of 4-NP to test containers (McLeese et al., 1980; Bechmann, 1999). Comber et al. (1993) proposed that a 60% fall in the concentration of NP (86% 4-NP) in test cultures of the freshwater cladoceran, Daphnia magna, was due to sorption of nonylphenol onto either algae (present as food) or the test subjects themselves. In the current study the loss of 86.4% of 4-NP in test media containing live Artemia sp., compared with only 17.8% in the absence of Artemia sp., supports this proposition. Therefore, nominal concentrations, as referred to in this study, are an overestimation of actual exposure levels.
In the present investigation, 29 ng L–1 of 4-NP was detected in untreated seawater sampled off the coast of Cullercoats (NW. England). Billinghurst et al. (1998), using a similar extraction protocol, recorded concentrations of 5 ng L–1 of 4-NP in water sampled 12 miles off the coast of southwest England. Differences between the two values may be attributable to the increased sensitivity of the GM-MS instrumentation used in this study. It is possible, however, that the values represent actual differences in the distribution of nonylphenol ethoxylates in the coastal waters of the U.K. and supports the understanding that estuarine and coastal environments are particularly susceptible.
For a variety of saltwater animals the 96-hr LC50 acute toxicity values for para-nonylphenol are between 17–195 µg L–1 (Lussier et al., 2000). In the current study the acute toxicity (48-hr LC50) of 4-NP to the larval and the postlarval stages of P. elegans ranged from 41 to 87.5 µg L–1. These values are similar to those reported for nauplii of the marine copepod, Tisbe battagliai (Bechmann, 1999), but 2–3 times lower than the 48-hr LC50 of 190 µg L–1 reported for the adult stage of D. magna (Comber et al., 1993) and 20–40 times lower than 96-hr LC50 of 1,670 µg L–1 reported for the adult stage of the marine amphipod, Corophium volutator (Brown et al., 1999). Consequently, P. elegans larvae appear to be among those species most susceptible to the toxic effects of 4-NP.
In contrast, the acute toxicity (96-hr LC50) of E2 to P. elegans larvae was >2 mg L–1 which is similar to the 96-hr LC50 of 1.6 mg L–1 (1.3–2.0) reported for adults of the harpacticoid copepod, Nitocra spinipes (Breitholtz and Bengtsson, 2001). Since, in estuarine and coastal waters, concentrations of E2 are several orders of magnitude below these levels (Allen et al., 2002), E2 likely represents a low toxic risk in the marine environment.
The moulting and growth of arthropods is regulated by a multi-hormonal system, primarily under the control of ecdysteroids (Quackenbush, 1986; Chang et al., 1993; Lachaise et al., 1993; Huberman, 2000). In some Crustacea, ecdysteroids may also play a role in the control of vitellogenesis and embryogenesis (Subramoniam, 2000). While a functional role for vertebrate-like steroids remains debatable in Crustacea, there are similarities between oestrogens and ecdysteroids in the way they bind to nuclear receptors (Chung et al., 1998). Historically, this supported the suggestion that oestrogens and xeno-oestrogens may affect moulting in Crustacea by interacting with the nuclear ecdysteroid receptor (EcR) (Zou and Fingerman, 1997a, b). More recent evidence from an ecdysone receptor assay has indicated that neither natural nor synthetic oestrogens/androgens bind to the EcR. However, certain xeno-oestrogens, including Bisphenol A, diethylphthalate, lindane and 4-NP, are weak EcR antagonists possibly via interaction with the EcR ligand-binding site (Dinan, 2001). Moreover, in vitro binding assays, based on the Sf-9 cell line, have indicated that E2 and the synthetic oestrogen, diethylstilboestrol (DES), are both capable of acting as weak antagonists by inhibiting the binding of ponasterone A (Nakagawa et al., 2000). While such activity has been reported in vitro at relatively high concentrations it remains to be seen whether such antagonistic interaction occurs in vivo.
The rapid cellular differentiation and growth of larval stages makes them particularly susceptible to the effects of chemicals interfering with hormonally regulated processes (Kennedy, 1996). Exposure to 4-NP, at concentrations as low as 0.1–10 µg L–1, inhibits the larval development of the oyster, Crassostrea gigas (Nice et al., 2000). Although endocrine disruption was not demonstrated in this case, it was hypothesised that 4-NP may have been responsible for morphological deformities via abnormal calcium mobilisation (Nice et al., 2000). Comber et al. (1993) reported reduced growth in D. magna exposed to 4-NP at concentrations of 30 and 71 µg L–1, while growth rates of the amphipod, Corophium volutator, were retarded in populations exposed to 10 µg L–1 (Brown et al., 1999).
In the current study neither 4-NP nor E2 inhibited the growth of P. elegans larvae, although survival was reduced in larvae exposed to 4-NP at 2 µg L–1. The lack of significant effects on growth and survival at the other concentrations tested is consistent with results reported for barnacle larvae exposed to 4-NP (Billinghurst et al., 1998). Both Nitocra spinipes (Hutchinson et al., 1999; Breitholtz and Bengtsson, 2001) and Tisbe battagliai (Bechmann, 1999) were also found to be insensitive to exposure to a range of natural, synthetic and xeno-oestrogens, as determined by monitoring survival, development, reproductive capability and sex ratios. The ability of many invertebrates to biotransform steroids and xenobiotics, like 4-NP (Baldwin and Leblanc, 1994; Baldwin et al., 1997; Shurin and Dodson, 1997; Baldwin et al., 1998; Oberdörster et al., 1998), possibly via glucose and sulphate conjugation (Parks and LeBlanc, 1996), may account for the lack of developmental effects observed in this and other investigations. An alternative mode of action for xeno-oestrogens has been reported in the fiddler crab, Uca pugilator, where disturbances in the moulting pattern were linked to inhibition of chitobiase activity rather than an interaction with the EcR (Zou and Fingerman, 1999a, b). The lack of a significant effect on the moult frequency of P. elegans larvae exposed to 4-NP and E2, reported in this study, lends further support to the contention that crustaceans are relatively insensitive to xeno-oestrogen exposure.
E2 and 4-NP had contrasting effects on the expression of the vitellin-like protein in P. elegans. Relatively high concentrations of E2 significantly reduced expression of the protein, while 4-NP produced a concentration-independent increase; the lowest concentration tested, 0.2 µg L–1, exerted the most consistent stimulatory effect. A concentration-independent upregulation of CMP was also observed in barnacle larvae exposed to 4-NP at 0.01–1.0 µg L–1 (Billinghurst et al., 2000). In contrast to the present study, however, upregulation of CMP was also observed in larvae exposed to E2 at 1.0 µg L–1. It is therefore unlikely that the oestrogenic properties of 4-NP are solely responsible for the upregulation of the vitellin-like protein in P. elegans larvae.
The biochemical, structural and amino acid sequence similarities between CMP and barnacle vitellin (Shimizu et al., 1996a) suggest that the former may be analogous to insect larval storage proteins (LSPs); the hexamerins. These proteins typically have a molecular mass of approximately 500 kDa with subunits of 70–90 kDa (Telfer and Kunkel, 1991). Could it be that the P. elegans larval vitellin-like protein is also a LSP? Certainly the immunological results and subunit size are consistent with this hypothesis. However, insect LSPs typically appear during the latter larval instars, peaking in titre prior to metamorphosis and fall dramatically thereafter (Burmester et al., 1998a). Expression of CMP follows a similar pattern (Shimizu et al., 1996b; Billinghurst et al., 2000). In contrast, the vitellin-like protein in P. elegans larvae was present at relatively low concentrations in all the larval stages and tended to decline with development. This pattern is inconsistent with a LSP function. Clearly sequence information will be required to resolve this issue and this work is in progress.
While 96% of the total soluble protein of larvae was retained during the extraction process, it is necessary to report that the exact recovery efficiency of specifically vitellin-like proteins was not assessed and may have contributed a small level of unaccounted for variability in the vitellin-like protein titres between treatments. However, variations of up to 18% in the vitellin-like protein titres of exposed larvae cannot be exclusively attributed to losses in the extraction procedure.
Overall, although significantly altered vitellin-like protein titres were recorded in P. elegans larvae exposed to 4-NP and E2, it is possible that the variation, attributed to exposure, is within the natural physiological range of the larvae. The lack of consistent effects on the development, growth and survival of exposed larvae tends to support this and suggests that 4-NP and oestradiol pose little risk to P. elegans at environmentally realistic concentrations. It remains to be investigated whether exposure to other xeno-oestrogens affects expression of the vitellin-like protein and whether this effect is specific to such exposure.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Martin Jones for assistance with the analytical procedures and Drs. Shaw Bamber and Tamara Galloway for assistance in the purification of lipovitellin. This work was supported by a grant (GST/02/2170) from the Natural Environment Research Council (UK) awarded to ASC, MHD and ZB.
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FOOTNOTES |
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1 From the Symposium EcoPhysiology and Conservation: The Contribution of Endocrinology and Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
2 Present address: The Centre for Environment, Fisheries and Aquaculture Science, Weymouth Laboratory, Barrack Road, Weymouth, DT4 8LB, UK; E-mail: M.B.Sanders{at}CEFAS.co.uk
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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