Biotransformation, stress and genotoxic effects of 17β-estradiol in juvenile sea bass (Dicentrarchus labrax L.)

Abstract

The effects of 17β-estradiol (E₂) on fish became a matter of concern, since significant levels of this hormone were detected in the aquatic environment released mainly by domestic sewage treatment plants. In this perspective, the current study was focused on E₂ effects upon biotransformation, stress and genotoxic responses of juvenile Dicentrarchus labrax L. (sea bass). Fish were exposed to E₂ during 10 days in two different ways: water diluted (200 ng/L or 2000 ng/L) and i.p. injected (0.5 mg/kg or 5 mg/kg). A battery of biological responses was evaluated: liver ethoxyresorufin-O-deethylase (EROD) and alanine transaminase (ALT) activities, liver somatic index (LSI), plasma cortisol, glucose and lactate concentrations, as well as erythrocytic nuclear abnormalities (ENA).

All the exposure conditions induced endocrine disruption, measured as plasma cortisol decrease, and genotoxicity, measured as ENA increase. Thus, no differences were detected either between different exposure routes or tested concentrations. Concerning liver EROD and ALT activities, as well as plasma glucose and lactate concentrations no differences were found between treated and control groups. LSI was the only parameter to respond differently in the two exposure routes, as only E₂ water diluted induced a significant increase in this hepatic indicator.

Introduction

Natural female estrogens are excreted via bile and urine mainly as water-soluble glucuronides, sulfates or, to a minor extent, in its free form (Fürhacker et al., 1999). Before conjugation, estrogens undergo hydroxylation, reduction, methylation or oxidation predominantly in the liver. 17β-Estradiol (E₂), the most effective estrogen secreted by the ovaries, can be oxidized to estrone and further converted into estriol. Polar metabolites like 16-hydroxyestrone, 4-hydroxyestradiol, 16-ketoestrone and/or 16-epiestriol are formed and can also be found in urine and faeces (Ying et al., 2002).

Estrogens and E₂ in particular reach aquatic environments mainly through domestic effluents. Previous measurements of human estrogen excretion estimated that females were excreting 2.3–259 μg/person/day and males 1.6 μg/person/day of E₂ through urine (Johnson et al., 2000). Two other important sources of E₂ are livestock waste (Ying et al., 2002) and agriculture runoff (Céspedes et al., 2004). For example, in poultry waste a concentration ranging from 14 to 533 ng/g dry waste for E₂ was reported by Shore et al. (1995). The E₂ concentration in urine of cattle was found to be 13 ng/L on average (Erb et al., 1977). Recent studies have shown that the utilization of animal manure to agricultural land can lead to movement of E₂ into surface and ground water (Peterson et al., 2001). E₂ has been found mobile and detected in runoff from a pastural land in an average concentration of 3500 ng/L (Nichols et al., 1998). Therefore, in environmental waters E₂ concentration range is between the detection limit up to ng/L (Spengler et al., 2001).

Although E₂ conjugates released into aquatic environments do not possess a direct biological activity, they can act as precursor hormone reservoirs able to be reconverted by bacteria to free E₂. Moreover, this cleavage is particularly relevant in both raw and treated sewages (Baronti et al., 2000).

Taking into consideration the scenario previously described, E₂ has been increasingly reported as an environmental contaminant (Ying et al., 2002, Imai et al., 2005), being also regarded by Dorabawila and Gupta (2005) as “the most potent of all xenoestrogens”, able to affect aquatic organisms, namely fish. Furthermore, E₂ is among the most potent endocrine disrupting chemicals having the potential to exert effects at extremely low concentrations (Bowman et al., 2002).

Studies regarding E₂ effects on fish have been focused on endocrine aspects, mainly reproduction. It is known that it may alter gonadosomatic index in males, reduce egg production in females, induce vitellogenesis in males and juveniles as well as decrease fertility (Mills et al., 2001, Kang et al., 2002). However, there is now ample evidence that non-reproductive endocrine events can be disrupted by E₂, namely the response to stress. Glucocorticoids, such as cortisol, are important for stress responses in fish, particularly in metabolic adjustments by the regulation of energy production, hydro-mineral balance, oxygen uptake and immune competence (Hontela, 1997). Nevertheless, only a few studies have been carried out on E₂ effects on plasma cortisol, leading to non-coincident results (Pottinger et al., 1996, Teles et al., 2005). Similarly, the effects of E₂ on fish secondary stress responses, such as alterations on plasma glucose and lactate concentrations, are still poorly understood (Petersen et al., 1983, Teles et al., 2005).

In the field of non-endocrine E₂ effects on fish, special attention has been paid on regulation of CYP1A expression; hence, several authors observed the suppression of CYP1A-associated 7-ethoxyresorufin-O-deethylase (EROD) activity, in both maturing females Scophthalmus maximus L. and juvenile Sparus aurata L. treated experimentally with E₂ (Arukwe and Goksøyr, 1997, Teles et al., 2005). Moreover, several mammal studies indicated that E₂ is genotoxic (Liehr, 2000, Joosten et al., 2004). Thus, the ecotoxicological relevance of E₂ genotoxicity on fish is worthwhile to study since little is known about this subject.

The present study was undertaken to assess E₂ effects on Dicentrarchus labrax L. (sea bass) at different levels: CYP1A expression, measured as liver EROD activity, genotoxicity, measured as erythrocytic nuclear abnormalities (ENA), as well as stress responses, namely plasma cortisol, glucose and lactate levels. Hepatic indicators, such as liver somatic index (LSI) and alanine transaminase (ALT) activity were also measured. Furthermore, the previous responses were evaluated under two different E₂ exposure routes, i.e., either water diluted or intraperitoneally (i.p.) injected, in order to clarify the influence of the exposure route.