Intercalibration exercise using a stickleback endocrine disrupter screening assay

Abstract

The Organisation for Economic Cooperation and Development (OECD) is currently validating a short-term fish screening protocol for endocrine disrupters (estrogens, androgens, and their antagonists and aromatase inhibitors), using three core species: fathead minnow, Japanese medaka, and zebrafish. The main endpoints proposed for the first phase of validation of the screen are vitellogenin (VTG) concentration, gross morphology (secondary sexual characteristics and gonado-somatic index), and gonadal histopathology. A similar protocol is concurrently being developed in the United Kingdom using the three-spined stickleback, with identical endpoints to those for the core species and, in addition, a unique androgen-specific endpoint in the form of spiggin (glue protein) induction. To assess the suitability of this species for inclusion in the OECD protocol alongside the core species, an intercalibration was conducted using 17beta-estradiol (a natural estrogen) and trenbolone (a synthetic androgen), thus mimicking a previous intercalibration with the core species. All three participating laboratories detected statistically significant increases in VTG in males after 14 d exposure to nominal concentrations of 100 ng/L 17beta-estradiol and statistically significant increases in spiggin in females after 14 d exposure to nominal concentrations of 5,000 ng/L trenbolone. The stickleback screen is reliable, possessing both relevant and reproducible endpoints for the detection of potent estrogens and androgens. Further work is underway to assess the relevance and suitability of the screen for weakly acting estrogens, anti-androgens, and aromatase inhibitors.

Field surveys reveal the presence of anti-androgens in an effluent-receiving river using stickleback-specific biomarkers

“…Therefore it is possible that the between-year variation in VTG concentrations in the male sticklebacks could be a result of interferences from chemicals or from endogenous factors that promote VTG synthesis in males, rather than the presence of oestrogenic substances alone. The lack of male VTG response in the long-term laboratory trial, where sticklebacks were exposed to E2 at concentrations corresponding to the upper and lower levels of oestrogenic activity measured at the study site (Grover et al, 2011a;, agrees with the data obtained from 21-day laboratory exposures in the stickleback (Allen et al, 2008) and other fish (OECD, 2006).…”

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

“…Trenbolone, the major metabolite of TbA, is also recognised as a powerful androgen known to induce spiggin after 14 days of exposure to 5 µg/L (Allen et al 2008). In the present work, TbA induced ultrastructural changes in the kidney of bullheads with an enlargement of proximal tubules.…”

Custom microarray construction and analysis for determining potential biomarkers of subchronic androgen exposure in the Eastern Mosquitofish (Gambusia holbrooki)

“…In female G . aculeatus , this protein was found to be upregulated in the kidneys after exposure to 1 μg/L of methyltestosterone (MT), 5 μg/L of dihydrotestosterone (DHT), and 5 μg/L of TB, with co-treatment of the anti-androgen flutamide (flu) resulting in decreased spiggin protein levels [23,24]. These results indicate that this gene may be a direct target of androgen action and that spiggin has a strong potential to be developed as a robust biomarker for androgen exposure in G .…”