Oral single pulse exposure of flounder Platichthys flesus to 4-tert-octylphenol: Relations between tissue levels and estrogenic effects

The accumulation of 4-tert-octylphenol and the associated estrogenic effects were studied after a single pulse exposure to flounder Platichthys flesus. 4-tert-octylphenol was administered orally in a single dose of 50 mg kg(-1) and tissue (liver, muscle and testis) and plasma concentrations of 4-tert-octylphenol as well as plasma vitellogenin were measured 3, 6, 12, 18, 24, 48, 72, 144 and 216 h after administration of the dose. Concentrations of 4-tert-octylphenol in plasma and tissues were determined by Liquid Chromatography Mass Spectrometry (LC-MS). 4-tert-octylphenol was detectable in liver, testis, muscle and plasma 3h post administration and an accumulation was observed in liver, muscle and plasma up to 12 h and in testis 18 h post administration, respectively. The maximum concentrations of 4-tert-octylphenol in liver, muscle and testis were 67, 3.2 and 6.8 microg g(-1), respectively. An increase in plasma vitellogenin levels was seen 48 h post administration and the vitellogenin level continued to increase (from <100 ng ml(-1) to 1.4 mg ml(-1)) until the end of the experiment 9 days after the administration of 4-tert-octylphenol.     

Short-time induction of oxidative stress in hepatocytes of the European flounder (Platichthys flesus).

Oxidative stress induced by xenobiotic compounds has been studied using primary hepatocytes of juvenile European flounder (Platichthys flesus L.) caught in a low polluted area of the German Bight, Tiefe Rinne (Landwüst et al., 1996.). Cells were exposed to known oxidative stressors such as hydrogen peroxide and benzo[a]pyrene (B[a]p) in various concentrations (50 and 100 microM) up to 6 days. Cell mortality was determined using fluorescent ethidium homodimer-1 and calcein AM. Oxidative stress response was measured by image analysis using dihydrorhodamine 123, which is converted to fluorescent rhodamine 123 in the presence of intracellular ROS. Oxyradical formation was initiated already after 2 h of exposure to low concentrations of B[a]p and hydrogen peroxide. Probably due to a membrane stabilising effect of the serum factors the addition of fetal bovine serum to the culture medium had a protecting influence on the hepatocytes and resulted in (1) an increased cell viability and (2) reduced formation of intracellular ROS during exposure. In conclusion, the assay is a sensitive tool for testing the potential of various xenobiotics to induce oxidative stress in living hepatocytes.     

Comparisons of liver proteomes in the European flounder Platichthys flesus from three contrasted estuaries

Estuaries are important areas highly susceptible to anthropogenic degradations like pollution. Estuarine species have thus to cope with many types of constraints depending on the estuaries' characteristics. The European flounder Platichthys flesus is considered as a sentinel species for the monitoring of estuarine water quality. In this study, juvenile flounders (0+ group) were sampled from three contrasted Channel estuaries, i.e. the Seine, the Canche and the Tamar, and we characterized their liver proteomes by using a two-dimensional electrophoresis based proteomic approach.We showed that 27 protein spots differentially accumulated between the 3 populations. Six of these proteins were identified by MALDI TOF–TOF mass spectrometry. Flounders from the Seine and from the Tamar, two highly polluted estuaries, displayed common differences, i.e. an increase of the energetic- and the glutathione-metabolism. The most accumulated protein in the Seine's samples (6.7-fold) was a Vitelline Membrane Outer layer protein 1 homolog, suggesting oogenesis deregulation in these juvenile (sexually immature) flounders. Future works applying this kind of proteomic approach on flounders experimentally exposed to conditions that mimic environmental constraints will help to better understand the significance of these environmental proteomic signatures.     

Application of real time PCR determination to assess interanimal variabilities in CYP1A induction in the European flounder (Platichthys flesus)

In both laboratory experiments and field investigations with fish a large interanimal variability in CYP1A expression has been observed which may be attributed to variations in environmental inducer exposure and/or inducer response. We are carrying out laboratory investigations to assess the contribution of a potential genetic component in inducer response of flounder (Platichthy sflesus) CYP1A to PCB exposure and in this paper we report development of a sensitive quantitative RT-PCR procedure (real time PCR) where accumulation of the intercalated dye SYBR Green I is followed during cycling. Preliminary experiments using this procedure with artificially reared Arochlor 1254-treated flounders showed large interanimal differences in response for a single family group indicating that variability does have a genetic basis.     

Population ecology and habitat preferences of juvenile flounder Platichthys flesus (Actinopterygii: Pleuronectidae) in a temperate estuary

The European flounder Platichthys flesus is a widely distributed epibenthic species and an important component of demersal fish assemblages in the European Atlantic coastal waters. In Portuguese estuaries, this species reaches high densities, especially in Minho estuary (NW Iberian Peninsula, Europe), potentially playing an important role in the system's ecology. In this context, the population structure, production and the habitat use of juvenile P. flesus were investigated. Sampling took place monthly, from February 2009 until July 2010 along the entire estuarine gradient (5 sampling stations distributed in the first 29 km from the river mouth, with S1 located near the river mouth, S2 inside a salt marsh, S3 in a salinity transition zone, while S4 and S5 were located in the upper estuary). Flounder's density varied significantly among sampling stations and seasons (two-way PERMANOVA: p < 0.001), with the majority of the individuals being found during the spring (30.1%) and in S3 and S4 (72.6%). Males and females presented an even distribution, with a higher proportion of males observed during summer. Fish length also differed among sampling stations and seasons (two-way PERMANOVA: p < 0.001), with larger fishes being found in S1 during the autumn (168.50 ± 59.50 mm) and the smallest in S4 during the spring (33.80 ± 3.12 mm). Size classes associated differently with environmental variables, with larger juveniles being more abundant in the downstream areas of the estuary, whereas smaller juveniles were related to higher water temperatures, suggesting a habitat segregation of P. flesus of different sizes. The fish condition of P. flesus in Minho estuary was higher than in other systems, probably due to the dominance of juveniles on the population. Also, the densities found in this estuary were up to 32 times higher than in other locations, suggesting that Minho estuary is an important nursery area for the species. The estimated secondary production of P. flesus was lower than previous studies acknowledged in the system (0.037 g.WWm^− 2.year^− 1), indicating that the production estimates of this species in estuaries can vary considerably depending on of several factors such as the sampling year and strategy, population and fish size.     

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17 beta-estradiol to 19 +/- 11%, 27 +/- 7%, 63 +/- 20% and 61 +/- 12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17 beta-estradiol and 17 alpha-hydroxyprogesterone levels (r = -0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.     

Availability of cadmium and zinc from sewage sludge to the flounder, Platichthys flesus, via a marine food chain

This paper examines the potential availability to a demersal fish of cadmium and zinc associated with digested sewage sludge, via a food chain as well as directly from the sludge, and the tissue distribution and possible excretion of any accumulated cadmium and zinc. Radioactive tracer techniques were used in order to follow the food chain transfer of the metals. Flounder (Platichthys flesus) accumulated cadmium both directly from sludge (delivered in newly ingested unassimilated gutfuls in the amphipods) and in assimilated form from the tissues of the amphipod crustacean Corophium volutator that had been feeding on a sludge-sediment mixture. Cadmium from both sources could be excreted by the fish. The concentration of cadmium within the whole fish increased as the input of cadmium in the diet increased. Zinc, similarly, is available to the flounder both directly from sludge and from zinc accumulated in C. volutator. Only a limited increase in net accumulation of zinc by the flounder was observed upon increased inputs of zinc through the diet, perhaps indicating some regulation of body zinc concentration by the flounder.     

Disorder and recovery of environmental health monitored by means of lysosomal stability in liver of European flounder (Platichthys flesus L.)

From 1995 to 2000 biological effects were studied in liver of flounder (Platichtysflesus L.) from the German Bight. During the study period deleterious consequences of acute discharges of DDT and PCBs in early spring 1996 and after 1998 due to remobilization of contaminants from riverbed deepening of the River Elbe became evident. As core biomarker which reflects toxically induced liver pathologies and integrates effects of various classes of pollutants we measured the integrity of lysosomal membranes in individual flounder liver. During the study period, twice statistically significant disturbances of lysosomal function was detected in fish from the River Elbe: in summer 1996 and in spring 1999. Yet, the detrimental contaminant effects were not only restricted to individuals from the Elbe but expanded to those flounder inhabiting formerly less polluted reference areas. In contrast to flounder of the Elbe, their ability to recover from the lysosomal disorders were limited. While in autumn 2000 Elbe individuals showed clear signs of recovery, those fish caught in areas more distant to the source of toxicant input still maintained significantly decreased lysosomal membrane integrity. It can be speculated that fish populations which are not continuously exposed to chronic anthropogenic stress may have a lower potential or need a longer period to recover from the effects of pollution.     

Bioaccumulation of polybrominated diphenyl ethers (PBDEs) in flounder (Platichthys flesus) in the southern Baltic Sea

Concentrations of seven polybrominated diphenyl ether (PBDE) congeners were examined in flounder (Platichthys flesus) and sediment in three southern Baltic Sea sites, representing a range of exposure conditions, in order to evaluate spatial differences in PBDE contamination. Additionally, PBDEs were measured in muscle, liver, and gonads of flounder from one of the sites in order to examine inter-tissue distribution. Mean muscle Σ₇PBDE levels, in the range of 10–21 ng g⁻¹ lipid, showed inter-site differences attributed to the distance from the Gulf of Gdańsk, and were overall lower than reported earlier in herring, sprat, and salmon. Biota sediment accumulation factors (BSAFs) for Σ₇PBDE and individual BDE congeners, in the range of 0.5–24.5, were generally consistent with predicted models for persistent hydrophobic halogenated contaminants. Wet weight (wet wt) PBDE levels in muscle and liver, but not in gonads, were related to tissue lipid content and did not correlate with the fish length and weight. These tissues differed in PBDE levels and profiles as a result of varying lipid content and presumably lipid composition and congener-specific physico-chemical properties.     

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.     

Pathology of the liver, kidney and gonad of flounder (Platichthys flesus) from a UK estuary impacted by endocrine disrupting chemicals.

The Mersey estuary is highly contaminated with xenobiotics compared to the nearby Dee estuary. Male flounder, a migratory flatfish caught in the Mersey frequently contains high blood concentrations of the oestrogen controlled, female protein vitellogenin, suggesting that Mersey flounder have been exposed to endocrine disrupting contaminants. Males caught from the Dee contain lower blood vitellogenin levels. Preliminary histopathological examination of 410 flounder from both estuaries, focuses on the liver, kidney and gonads. Hepatic tubular vacuolation, foci of cellular alteration, and hepatocellular tumours were seen. Renal and gonadal pathology included the presence of two Mersey fish with enlarged and abnormal glomeruli, three phenotypic male Mersey flounder with unilateral intersex gonads and one male with bilateral, abnormal ovarian follicular components in the testis. These initial results provide pathological evidence of xenobiotic exposure in flounder sampled from both estuaries. Preliminary findings from flounder sampled from Millport, an offshore site in Southwest Scotland, showed no histopathological evidence of xenobiotic exposure.     

Analysis of mutations in the p53 tumor suppressor gene and Ki- and Ha-ras proto-oncogenes in hepatic tumors of European flounder (Platichthys flesus).

DNA from five tumors, three other-injured livers and two normal liver tissue samples from the European flounder were analyzed for mutations in exons 5-8 of the tumor suppressor gene p53 and at codons 12, 13 and 61 of Ha- and Ki-ras proto-oncogenes. No tumor-specific mutations were identified by direct sequencing and single-strand conformation polymorphism of these genes. A number of silent polymorphisms were noted in p53. In addition to a need for more extensive analyses of flounder liver tumor samples for ras or p53 mutations, other cancer-related genes should be investigated.     

Bioconcentration and distribution of 4-tert-octylphenol residues in tissues of the rainbow trout (Oncorhynchus mykiss)

Branched chain alkylphenols are weak oestrogen mimics which are present in the aquatic environment and have been implicated in the feminisation of fish. This study reports the biotransformation, bioconcentration and tissue distribution of the xenoestrogen 4-tert-octylphenol (t-OP) in juvenile rainbow trout. Fish were exposed for 10 days to a concentration of 4 micrograms/l of [14C] t-OP in a flow-through system and were sampled after 1, 4, 7 and 10 days of exposure. t-OP residues were extracted from all tissues and analysed by radio-high-performance liquid chromatography. After 1 day of exposure radioactive residues were detected in all tissues and reached steady state conditions in the whole fish after 4 days of exposure. The concentration of t-OP residues were highest in bile, followed by faeces, pyloric caeca, liver and intestine. In these tissues the majority of alkylphenol was in the form of two metabolites which were identified by gas chromatography-mass spectroscopy as the glucuronide conjugates of t-OP and t-octylcatechol. t-OP accumulated as the parent compound in fat with a bioconcentration factor (BCF) of 1190, and in brain, muscle, skin, bone, gills, and eye with BCFs of between 100 and 260. This study suggests that exposure to water-borne alkylphenols results in rapid conjugation and elimination of the chemical via the liver/bile route, but that high amounts of the parent xenoestrogen can accumulate in a variety of other fish tissues.