Isolation and Identification of Ligands for the Goldfish Testis Androgen Receptor in Chemical Recovery Condensates from a Canadian Bleached Kraft Pulp and Paper Mill

Abstract

This study builds on a series of investigations characterizing substances in kraft mill chemical recovery condensates that depress sex steroids in fish. Here, incubations of goldfish testis androgen receptors (AR) with condensate extracts were used to investigate the potential role of androgens in hormone depressions. Condensates contained variable levels of AR ligands, with the highest amounts in nonpolar extracts of filtered solids prior to solid phase extraction (SPE). High pressure liquid chromatography (HPLC) fractionation recovered the majority of activity in one fraction, with ligands detected in three additional fractions. Gas chromatography mass spectrometry analysis of the most active fraction confirmed the two most abundant components as the diterpenes manool and geranyl linalool. Manool exhibited a relative affinity for the AR that was 300 fold less than testosterone and accounted for 26% of total filtered solids activity. Geranyl linalool exhibited no affinity for the AR. Three additional diterpenoid families were tentatively identified as principal components of the three other androgenic HPLC fractions. Compared to condensates, final effluent had 3000 fold less androgenic activity, with <1% attributable to manool. Putative androgens previously associated with mill effluents (androstenedione and androstadienedione) and progesterone were not detected; however, additional condensate diterpenes suspected as androgens were identified in final effluent. This study is the first to confirm nonsteroidal cyclic diterpenes as androgenic at pulp mills. A major in-mill source of these substances was identified, and the role of androgens in mill effluents affecting fish reproduction was reinforced.

Evaluating the potential of effluent extracts from pulp and paper mills in Canada, Brazil, and New Zealand to affect fish reproduction: Estrogenic effects in fish

“…The apparent role of metabolic activation in estrogenic responses could also be necessary to activate androgenic effects. A new class of diterpene ligands for the goldfish testicular AR has been identified ; however, it remains unclear if these compounds are not simply potent ligands for the AR but outright androgens or precursors to androgens involved in well‐documented masculinization studies at kraft mills in the United States , New Zealand , and Canada .…”

Effects of model aromatizable (17α-methyltestosterone) and non-aromatizable (5α-dihydrotestosterone) androgens on the adult mummichog (Fundulus heteroclitus) in a short-term reproductive endocrine bioassay

“…Introduction of androgen agonists and antagonists into the environment is via sources such as pulp and paper mills (Scott et al, 2011), sewage treatment facilities (Batty and Lim, 1999) and agricultural runoff (Kolok and Sellin, 2008). Recently, androgenic activities of 11 pulp and paper mills in Canada, Brazil and New Zealand were compared, with two Canadian mills having the highest androgen activities (Milestone et al, 2012).…”

“…The ongoing interest in the androgenic effects of PME and feedlot runoff (Scott et al, 2011;Bartelt-Hunt et al, 2012;Milestone et al, 2012) warrants improved understanding of the effects and modes of action of androgens in mummichog, a species identified as an excellent model for determining the interactions of environmental variables and reproductive contaminants on fish (Burnett et al, 2007;Lister et al, 2011;Gillio Meina et al, 2013). To build on previous work in mummichog, a short-term adult reproductive endocrine bioassay was used to test the effects of MT, an aromatizable androgen and 5α-dihydrotestosterone (DHT), a non-aromatizable androgen, on plasma sex steroid levels and gonadal gene expression.…”

The Relationship between Organic Loading and Effects on Fish Reproduction for Pulp Mill Effluents across Canada

“…At Kraft mills, such reductions can be realized through spill control efforts, chemical recovery condensate handling, and brownstock washing efficiencies. 18 A series of in-mill studies have linked chemical recovery condensates with high BOD 5 loadings to effects on circulating levels of sex steroids in fish 19 including the identification of diterpene androgens, 20 indicating condensate-specific treatment as an important option.…”