Seasonal variation of plasmatic and hepatic vitellogenin and EROD activity in carp, Cyprinus carpio, in relation to sewage treatment plants

Abstract

Concern about the health of aquatic fauna living in waters containing biologically active levels of estrogenic compounds is particularly focused on the effects on their reproductive success. To that end, carp, Cyprinus carpio, a feral fish living in warm waters of Southern Europe (NE Spain), were selected for signs of estrogenicity. The study area covered two tributaries (the Anoia and the Cardener) of the Llobregat River both known to be polluted by estrogenic compounds. The estrogenicity in the carp was measured as vitellogenin (VTG) presence in males and alterations in VTG levels in females, over a 6-month period, embracing both the pre- and post-spawning seasons. VTG content was measured in both the plasma and liver, the latter being the organ that synthesizes it. Also, hepatic 7-ethoxyresorufin O-deethylase (EROD) activity was recorded, as interactions of xenoestrogens and oestradiol have been reported to affect this enzymatic activity. The estrogenicity of these rivers was more evident in the Anoia at the location downstream from the sewage treatment plant (STP), by elevated levels of VTG in males and by the presence of some intersex individuals. In the Cardener, no intersex fish were found and male plasmatic VTG was not so highly elevated. However presence of hepatic VTG, in up to 54% of the male fish analyzed, proved exposure to xenoestrogens. In females, VTG fluctuated according to the biological cycle with a plasmatic peak in May and an earlier maximal in the liver. However, this pattern was altered in the locations with higher xenoestrogens presence. EROD activity showed differences between sexes, with higher activity in males than females, as well as site-related differences (up to one order of magnitude) in the same river. These differences were even greater than those detected between rivers. A seasonal trend was also seen in EROD activity with higher induction towards the summer in both males and females.

Introduction

Fish inhabiting polluted waters are exposed to complex mixtures of compounds that might have negative consequences for their fitness and reproductive success. Some of these compounds are known to exert estrogenic activity on humans and wildlife (Colborn et al., 1993, Tyler et al., 1998), whereas others display antiestrogenic properties (Nicolas, 1999). However, their combined effects are controversial, as some studies suggest synergistic responses while others antagonistic interactions (Sumpter and Jobling, 1995, Tyler et al., 1998).

In cyprinid fish, laboratory evidence of estrogenicity due to either exposure to alkylphenol ethoxylates (APs) or to natural and synthetic hormones (Gimeno et al., 1997, Folmar et al., 2000), has been further confirmed in the field mostly in relation to proximity to sewage treatment plants (STPs) (Purdom et al., 1994, Folmar et al., 1996, Routledge et al., 1998, Rodgers-Gray et al., 2000). Estrogenicity is due to interactions of 17β-oestradiol (E₂), or compounds which mimic this natural hormone (xenoestrogens), with its cellular receptor. On the contrary, widespread xenobiotics such as polycyclic aromatic hydrocarbons (PAHs) and some polychlorinated biphenyls (PCBs) are known to act as antiestrogenic compounds. They bind to the aryl hydrocarbon (Ah) receptor and this complex acts preventing the union of E₂ with its target gene. However, consensus has not yet been reached in relation to the proposed mechanism of action (for reviews see Gillesby and Zacharewski, 1998, Nicolas, 1999).

Vitellogenin (VTG) presence and/or content and 7-ethoxyresorufin O-deethylase (EROD) activity are broadly accepted biomarkers of xenoestrogens and xenobiotics exposure, respectively, and both have been applied in monitoring studies (Matthiessen et al., 1998, McArdle et al., 2000). VTG is a phosphoglycoprotein that constitutes an egg yolk precursor, its synthesis is hormonally regulated in oviparous females by E₂, and in male carp it is considered a reliable biomarker of estrogenic exposure (Heppell et al., 1996). Estrogenic exposure and VTG synthesis in male fish has clear negative consequences such as kidney dysfunction and death (Herman and Kincaid, 1988), loss of calcium from scales (Carragher and Sumpter, 1991), inhibition of testicular growth (Jobling et al., 1996, Panter et al., 1998), alterations in testis morphology (Christiansen et al., 1998, Kinnberg et al., 2000), reproductive abnormalities (Lye et al., 1997, Lye et al., 1998), and liver, kidney and spermatogenesis pathologies (Folmar et al., 2001). In females, alterations on the normal VTG pattern have also been proposed as a biomarker of estrogenic exposure (Kime et al., 1999). EROD activity is cytochrome P450 dependent; it is located in the liver endoplasmic reticulum and plays a key role in the oxidative metabolism of endogenous compounds (e.g. steroids), as well as a wide range of xenobiotics. In fish, EROD activity is the most broadly accepted biomarker in pollution monitoring as hazardous widespread industrial pollutants such as PAHs and PCBs clearly induce this activity. Its adequacy in monitoring programs has been repeatedly demonstrated (Bucheli and Fent, 1995, Goksøyr et al., 1996, Whyte et al., 2000). Both EROD activity and the isozyme CYP1A have particularly shown response to estrogens (Stegeman and Hahn, 1994, Arukwe and Goksøyr, 1997, Solé et al., 2000a), although other factors, such as temperature, physiological health and nutritional status of the organism can also modulate EROD expression (Whyte et al., 2000).

Fish are regularly used as valuable surrogates in monitoring programs (Goksøyr and Förlin, 1992, Goksøyr et al., 1996, Whyte et al., 2000). Carp, Cyprinus carpio, is a widespread, robust fish species that resists highly polluted waters. Along with other cyprinids it has been successfully selected for monitoring estrogenicity of freshwater systems (Purdom et al., 1994, Folmar et al., 1996, Jobling et al., 1998, Minier et al., 2000, Solé et al., 2000b). The selection of a representative native species has more interest as it shows the effects on the feral fauna in the target environment and integrates the effects occurred throughout its life history. Moreover, recent in vitro studies on the ability of xenobiotics to bind to the hepatic estrogen receptor have supported the usefulness of carp as a sentinel organism (Kloas et al., 2000).

The aim of this study was to investigate the combined effects of estrogens–xenobiotics exposure in two biomarkers (VTG and EROD) of male and female carp over the reproductive period. This study should allow us to detect the most sensitive period to pollutants exposure for each sex. To this end, carp were sampled every other month for a 6-month period (including the pre- and post-spawning seasons) in two rivers: the Anoia with a high presence of estrogenic compounds and the Cardener with a low to moderate presence of estrogenics. Both rivers also display a chronic and moderate exposure to PAHs and PCBs.