Despite the widespread use of the anabolic androgen trenbolone acetate (TBA) in animal agriculture, evidence demonstrating the occurrence of TBA metabolites such as 17β-trenbolone (17β-TBOH), 17α-trenbolone (17α-TBOH), and trendione (TBO) is relatively scarce, potentially due to rapid transformation processes such as direct photolysis. Therefore, we investigated the phototransformation of TBA metabolites and associated ecological implications by characterizing the photoproducts arising from the direct photolysis of 17β-TBOH, 17α-TBOH, and TBO and their associated ecotoxicity. LC-HRMS/MS analysis identified a range of hydroxylated products that were no longer photoactive, with primary photoproducts consisting of monohydroxy species and presumptive diastereomers. Also observed were higher-order hydroxylated products probably formed via subsequent reaction of primary photoproducts. NMR analysis confirmed the formation of 12,17-dihydroxy-estra-5(10),9(11),dien-3-one (12-hydroxy-TBOH; 2.2 mg), 10,12,17-trihydroxy-estra-4,9(11),dien-3-one (10,12-dihydroxy-TBOH; 0.7 mg), and a ring-opened 11,12-dialdehyde oxidation product (TBOH-11,12-dialdehyde; 1.0 mg) after irradiation of ∼14 mg of 17β-trenbolone. Though unconfirmed by NMR, our data suggest that the formation of additional isomeric products may occur, likely due to the reactivity of the unique 4,9,11 conjugated triene structure of trenbolone. In vivo exposure studies employing Japanese medaka (Oryzias latipes) indicate that low concentrations of 17α-TBOH photoproduct mixtures can alter ovarian follicular development, and photoproducts alter whole-body 17β-estradiol levels. Therefore, direct photolysis yields photoproducts with strong structural similarity to parent steroids, and these photoproducts still retain enough biological activity to elicit observable changes to endocrine function at trace concentrations. These data indicate that environmental transformation processes do not necessarily reduce steroid hormone ecotoxicity.

The cholesterol-lowering pharmaceutical gemfibrozil is a relevant environmental contaminant because of its frequency of detection in U.S. wastewaters at concentrations which have been shown to disrupt endocrine function in aquatic species. The treatment of gemfibrozil solutions with sodium hypochlorite yielded a 4'-chlorinated gemfibrozil analog (chlorogemfibrozil). In the presence of bromide ion, as is often encountered in municipal wastewater, hypobromous acid generated through a halogen exchange reaction produced an additional 4'-brominated gemfibrozil product (bromogemfibrozil). Standards of chloro- and bromogemfibrozil were synthesized, isolated and characterized using mass spectrometry and NMR spectroscopy. Mass spectrometry was used to follow the in situ halogenation reaction of gemfibrozil in deionized water and wastewater matrices, and to measure levels of gemfibrozil (254 ± 20 ng/L), chlorogemfibrozil (166 ± 121 ng/L), and bromogemfibrozil (50 ± 11 ng/L) in advanced primary wastewater treatment effluent treated by chlorination. Chlorogemfibrozil demonstrated a significant (p < 0.05) reduction in the levels of 11-ketotestosterone at 55.1 μg/L and bromogemfibrozil demonstrated a significant (p < 0.05) reduction in the levels of testosterone at 58.8 μg/L in vivo in Japanese medaka in a 21 day exposure. These results indicated that aqueous exposure to halogenated degradates of gemfibrozil enhanced the antiandrogenicity of the parent compound in a model fish species, demonstrating that chlorination may increase the toxicity of pharmaceutically active compounds in surface water.

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Treated wastewater effluent containing endocrine-disrupting chemicals is discharged into the coastal waters of the Southern California Bight (SCB) daily. The present study investigated changes in indicators of reproductive health and environmental estrogen exposure in hornyhead turbot (Pleuronichthys verticalis) near wastewater outfalls. Fish were collected from discharge areas, farfield stations, and a reference location in the SCB to examine spatial and temporal patterns. Fish from the Orange County outfall farfield site were younger and less sexually mature than fish from other sites. The sex ratio was skewed in some fish from outfall sites as well as from the Dana Point reference site. However, no consistent pattern in sex ratio was present over time. Low-level induction of vitellogenin was frequently observed in male fish from all sites, suggesting widespread exposure to estrogenic compounds, but did not appear to impact reproductive function as there was no incidence of gonad abnormalities (ova-testis). Analysis of historical hornyhead turbot trawl data indicated that populations are either increasing or stable in the SCB; thus, environmental estrogen exposure was not adversely impacting fish abundance. Additional research is needed to determine the cause of the estrogenic response in hornyhead turbot and whether the source of the estrogenic compounds is a consequence of historical contamination or of ongoing sources or representative of baseline characteristic of this species.

An in vitro system was used to analyze the effects of sex steroids on the development of primary (late perinucleolar stage) and early secondary, previtellogenic (early cortical alveolus stage) ovarian follicles of coho salmon cultured for up to 21 days. Late perinucleolar-stage follicles increased significantly in size after 7 days of treatment with low concentrations of 11-ketotestosterone (11-KT), a nonaromatizable androgen. An androgen receptor antagonist (flutamide) inhibited this growth-promoting effect, and the highest concentration resulted in atresia of follicles, implicating androgens as survival factors at this stage. Testosterone (T) was less effective than 11-KT in promoting growth, but blocking aromatization with exemestane resulted in a growth response similar to that of 11-KT. Estradiol-17beta (E2) had no effect on growth at this stage. After 21 days of culture, E2 was the most potent steroid in increasing the number of follicles containing cortical alveoli and the number of cortical alveoli within those follicles. At the early cortical alveolus stage, low doses of E2 promoted growth and strongly stimulated synthesis of cortical alveoli, actions that were inhibited by an estrogen receptor antagonist (tamoxifen). 11-KT displayed moderate growth-promoting effects, and 11-KT and T stimulated moderate to substantial increases in abundance of cortical alveoli. This study shows that the predominant role of androgens is the promotion of growth of late perinucleolar-stage follicles, while E2 stimulates both the growth and accumulation of cortical alveoli in early cortical alveolus-stage follicles.