Sparus aurata L. liver EROD and GST activities, plasma cortisol, lactate, glucose and erythrocytic nuclear anomalies following short-term exposure either to 17β-estradiol (E2) or E2 combined with 4-nonylphenol

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Abstract

Immature Sparus aurata L. (gilthead seabream) were exposed to 17β-estradiol (E2) 4000 ng/l and to the same E2 concentration mixed with 50,000 ng/l 4-nonylphenol (E2+NP) during 4, 8, 12 and 16 h. E2 availability and E2 plasma level variations were assessed. Liver biotransformation capacity was measured as ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST) activities. Plasma cortisol, lactate and glucose were also determined. Genotoxicity was assessed through erythrocytic nuclear anomalies (ENA) frequency. Liver EROD activity significantly decreased during the whole experiment for both treatments, with the exception of 16 h exposure to E2. Liver GST activity was significantly increased after 8 and 12 h of exposure either to E2 or E2+NP. An endocrine disruption expressed as plasma cortisol decrease was observed after 16 h exposure under both tested conditions, concomitantly with a plasma lactate increase. No genotoxic responses, measured as ENA frequency, were detected. Analyzing the E2 water concentration in aquaria without fish it was demonstrated an intense and fast E2 loss, considerably reducing its availability to fish. In the presence of fish, E2 water levels were drastically reduced after 4 h exposure, being this reduction more pronounced in E2 aquarium when compared to E2+NP aquarium. In addition, it was demonstrated a rapid E2 uptake from the water since the highest E2 plasma concentrations were observed after 4 h exposure, followed by a continuous decrease, which became more pronounced between 8 and 12 h of exposure. Furthermore, during the first 8 h exposure to E2 and E2+NP, seabream plasma E2 concentrations were higher than the initial water exposure concentration. Comparing the E2 plasma levels in both seabream-exposed groups, it was clear that its concentration is always higher in E2+NP-treated fish. Despite the previous results, no significant differences were found in the measured responses between E2 and E2+NP.

Introduction

The aquatic environment contamination by xenoestrogens—substances that modulate or mimic the action of sex steroid hormones, mainly 17β-estradiol (E2)—has received considerable scientific, government and public attention White et al., 1994, Brighty, 1996. However, natural estrogens such as E2 and estrone can also be regarded as aquatic contaminants. The discovery of significant E2 concentrations in municipal sewage treatment plants effluents (Brighty, 1996) became a matter of concern, being the assessment of its effects on fish at different levels, strongly recommended.

The degradation products of nonylphenol polyethoxylates, such as 4-nonylphenol (NP), are able to activate fish estrogen-dependent gene expression (Ackermann et al., 2002), revealing estrogenic properties either in vitro (White et al., 1994) or in vivo (Jobling et al., 1996). The polymer industry is the most important source of NP environment contamination, being the major part (95%) released to water. The majority of fish studies carried out on this subject concerned the effects on reproductive aspects. Either E2 or NP elevate plasma vitellogenin and zona radiata proteins in both males and females (Ackermann et al., 2002), induce intersexuality (Jobling et al., 1996), inhibit spermatogenesis and alter gonadosomatic index (Sepúlveda et al., 2003). Besides the known interference on reproduction, it is also important to study the interaction of synthetic and natural steroids with other biological responses. In this perspective, a substantial lack of information concerning the effects of this type of compounds on nonreproductive endocrine responses is evident. Considering the important role of cortisol on fish physiological responses to stressors through intermediary metabolism regulation, immune function and hydromineral homeostasis (Hontela, 1997), its alterations after exposure to xeno/estrogens must be investigated.

Different authors Pajor et al., 1990, Arukwe et al., 1997 proposed a cross-talk mechanism between P450 activities and steroid synthetic pathways. Thus, cytochrome P450-based mixed-function oxygenase (MFO) system is involved in xenobiotics and endogenous steroids metabolism. Despite the lack of information concerning CYP1A regulation by endogenous compounds, it was demonstrated that both EROD activity and CYP1A respond to estrogens (Solé et al., 2000). A significant reduction in EROD activity and CYP1A protein was reported in several fish species, namely Pseudopleuronectes americanus, Stenotomus chrysops (Gray et al., 1991) and Salmo salar (Arukwe and Goksøyr, 1997) after E2 exposure. The same pattern of response was also observed in S. salar (Arukwe et al., 1997) after NP exposure. Metabolites resulting from biotransformation processes (phase I) may be conjugated with endogenous molecules (phase II) making them more readily excretable (GoksØyr and Förlin, 1992). Gallagher et al. (2001) found that conjugase activities are also modulated by endogenous steroids. Additionally, effects of synthetic or natural steroids include genotoxicity (Liehr, 2000) as it was proposed that they could induce genetic damage and gene mutations in mammals Roy and Liehr, 1999, Yared et al., 2002. However, to our knowledge only a few studies are available concerning genotoxicity of these compounds in nonmammal species. NP was reported as genomic DNA damaging agent to crustacean larvae (Atienzar et al., 2002), and genotoxic to sea bass, measured as erythrocytic nuclear abnormalities (ENA) (Teles et al., 2004a).

Gilthead seabream, Sparus aurata L., seawater teleost widespread in Atlantic and Mediterranean coastal waters, is one of the most commercially important species, and as protandrous hermaphrodite is particularly appropriate as a biological model to endocrine studies.

Environmental waters are frequently contaminated simultaneously by different natural and synthetic steroids, being therefore relevant to study xeno/estrogen mixtures effects, as well as their eventual interference on single compounds effects, in fish.

The aims of the present research work, using juvenile seabream, were to assess the waterborne E2 or E2 mixed with NP (E2+NP) effects on biotransformation, measured as total P450 content, ethoxyresorufin-O-deethylase (EROD) activity (phase I) and glutathione-S-transferase (GST) activity (phase II). The variation on E2 concentrations in aquaria water and in seabream plasma was determined during the whole experiment. Liver somatic index (LSI) and alanine transaminase (ALT) activity were measured as general hepatic condition indicators. Additionally, the stress responses were evaluated as plasma cortisol, glucose and lactate, and genotoxicity was investigated using the ENA assay.

Section snippets

Chemicals

NP and E2 were purchased from Sigma-Aldrich (Germany). Marine salt was supplied by Sera Premium (Germany). All the other chemicals were of analytical grade obtained from Sigma-Aldrich, Roche (Germany) and E. Merck-Darmstadt (Germany).

Test animals

The experiment was carried out with juvenile S. aurata specimens obtained from a local fish farm, Materaqua- Ílhavo, Portugal. Seabream weighing 34±0.3 g were transported in aerated saltwater and acclimated to laboratory conditions for 1 week prior to

Water and plasma 17β-estradiol quantification (Table 1)

A time-related reduction in 17β-estradiol concentrations was observed either in aquaria with fish (E2 and E2+NP) or without fish (E2nf and E2+NPnf), during the entire experiment. However, this E2 reduction was more pronounced in aquaria containing fish. On the other hand, the E2+NP aquaria always presented clearly higher E2 concentrations than the E2 aquaria, either in the fish presence or absence. After 4 h exposure E2 levels in the aquaria with fish were drastically reduced, i.e., a 99%

17β-Estradiol measurements

E2 has been detected in effluent sewage treatment plants in different countries such as USA, Japan, Germany, Italy and the Netherlands, at concentrations up to 64 ng/l (Spengler et al., 2001). Considering the previous environmental data, a spiking concentration was adopted in the current study, to ensure a substantial uptake in a short period. Thus, this fact should be kept in mind in eventual extrapolations of the current findings to environmental situations.

The NP concentrations detected in

Conclusions

The current data concerning seabream exposure either to E2 or to its mixture with NP revealed:

  • –

    a liver EROD activity depression after both treatments, while GST activity was elevated;

  • –

    an endocrine disruption, expressed as plasma cortisol decrease after exposure either to E2 or E2+NP. Furthermore, a plasma lactate increase was measured for both conditions;

  • –

    that despite the higher E2 levels measured in plasma of E2+NP comparing to E2-treated fish, no significant differences were detected on the

Acknowledgements

The authors express their appreciation for the financial support provided by the Aveiro University Research Institute (CESAM) and by the “Fundação para a Ciência e Tecnologia” (FCT-Grant no. SFRH/BD/6607/2001).

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