Use of the Three-Spined Stickleback ( Gasterosteus aculeatus ) As a Sensitive in Vivo Test for Detection of Environmental Antiandrogens

Abstract

We have previously shown that exposure to exogenous androgens causes female sticklebacks (Gasterosteus aculeatus) to produce the glue protein, spiggin, in their kidneys. This protein can be quantified by an enzyme-linked immunosorbent assay developed and validated at the Centre for Environment, Fisheries and Aquaculture Science. Here we report the development of an in vivo test for the detection of environmental antiandrogens. The system involves the simultaneous exposure of female sticklebacks to 17α-methyltestosterone (a model androgen) at 500 ng/L and suspected environmental antiandrogens over a period of 21 days. The spiggin content of the kidneys is then measured, and any antiandrogenic activity is evaluated by comparing the spiggin levels of female fish exposed to antiandrogens to those of female fish exposed solely to the model androgen. The assay detects the antiandrogenic activity of flutamide, vinclozolin (both used at 250 μg/L), linuron (at 150 μg/L), and fenitrothion (at 15 and 150 μg/L). These results provide the first evidence of in vivo antiandrogenic activity of both linuron and fenitrothion in teleosts. Although there are other suggested fish species that could be used for this purpose, the stickleback is the only widely available species in which it is now possible to study both estrogenic and antiandrogenic end points in the same individual. Furthermore, the species is endemic and ubiquitous in Europe, and it possesses many ecological traits that make it better suited than other potential species for field research into endocrine disruption.

Use of chemical mixtures to differentiate mechanisms of endocrine action in a small fish model

“…Environmental Protection Agency, 2007;Organisation for Economic Cooperation and Development, 2009b). While some endocrine mechanisms are reliably detected in these assays, anti-androgens are more difficult to identify in an unambiguous fashion (Seki et al, 2006;Katsiadaki et al, 2006;Filby et al, 2007;U.S. Environmental Protection Agency, 2007;Organisation for Economic Cooperation and Development, 2009b).…”

“…Chemicals that inhibit steroidogenesis consistently decrease VTG levels in female fish. However, the suite of responses produced by anti-androgenic chemicals in sexually mature fish can be somewhat ambiguous, making it difficult to clearly diagnose this important pathway Katsiadaki et al, 2006;Seki et al, 2006;Filby et al, 2007;Organisation for Economic Cooperation and Development, 2009b). A wide variety of known or potential environmental contaminants, including some dicarboximide fungicides (e.g., vinclozolin), conazole fungicides (e.g., prochloraz), organochlorine insecticides (e.g., p,p -DDE), urea-based herbicides (e.g., linuron), polybrominated diphenyl ethers, and pharmaceuticals (e.g., flutamide) have been shown to bind to the AR, and cause anti-androgenic effects in vivo (Gray et al, 2006).…”

“…Martinović et al (2008) used a similar approach to help confirm that vinclozolin affects the reproductive endocrine system of fathead minnows through antagonism of the AR. Katsiadaki et al (2006) have done analogous work with the stickleback (Gasterosteus aculeatus), a fish species in which the male produces an androgen-dependent protein, spiggin (nest-building "glue"), as part of its normal reproductive cycle. Exposure of female sticklebacks to androgens causes the abnormal production of spiggin, while co-exposures with flutamide and anti-androgenic pesticides block production of spiggin in females exposed to exogenous androgens.…”

A potential biomarker of androgen exposure in European bullhead (Cottus sp.) kidney

“…This hypertrophy can be quantified using the Kidney Epithelium Height (KEH) measurement previously developed by (Borg 1993) or the quantification of spiggin production using an ELISA (Katsiadaki et al 2002;Sanchez et al 2008a). Due to this physiological property, the stickleback is considered as a relevant model fish species to assess the (anti)androgenic effects of endocrine-disrupting chemicals (EDCs) in laboratory and field studies (Sanchez et al 2008b;Katsiadaki et al 2006;Pettersson et al 2007;Wartman et al 2009). Chemical analysis has led to the identification of chemicals involved in this biological activity and showed that natural androgenic compounds such as androstenedione, equol, dehydrotestosterone or androsterone represent a large part of measured activity (Hill et al 2010;Thomas et al 2002;Kinani et al 2009).…”

“…Chemical analysis has led to the identification of chemicals involved in this biological activity and showed that natural androgenic compounds such as androstenedione, equol, dehydrotestosterone or androsterone represent a large part of measured activity (Hill et al 2010;Thomas et al 2002;Kinani et al 2009). Field studies have described physiological changes associated with androgen exposure such as spiggin induction in stickleback from contaminated sites (Sanchez et al 2008b;Katsiadaki et al 2006;Pettersson et al 2007;Wartman et al 2009) or elongated anal fin rays, resembling the male gonopodium, in female mosquitofish (Gambusia sp.) collected downstream of a pulp mill effluent discharge (Bortone et al 1989;Howell and Denton 1989).…”