Identification and Environmental Implications of Photo-Transformation Products of Trenbolone Acetate Metabolites

Abstract

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.

Bioavailability and fate of sediment-associated trenbolone and estradiol in aquatic systems

“…Recent research has shown that cycling of trenbolone metabolites may occur in surface waters either due to diurnal light progression and/or due to differences in light penetration as water circulates between depths. These studies have documented a regeneration of 17β-Tb, 17α-Tb, and Td from structurally similar photoproducts (12-hydroxy-trenbolone, 10,12-dihydroxy-trenbolone, and trenbolone-11,12-dialdehyde) during periods of darkness with significant implications for trenbolone bioavailability in natural systems (Oliver et al, 2010;Kolodziej et al, 2013;Qu et al, 2012). However, it is important to note that these studies evaluated surface water systems with low suspended solids.…”

Product-to-Parent Reversion of Trenbolone: Unrecognized Risks for Endocrine Disruption

“…Accordingly, we previously identified the structure and bioactivity of TBA metabolite photoproducts, showing that 17β-TBOH undergoes rapid photohydration (13) to yield 12,17-dihydroxy-estra-5(10),9(11)-diene-3-one (or simply 12-hydroxy-17β-TBOH) (14, 15). An analogous C12-hydroxylated photoproduct was identified for TBO (15), whereas 17α-TBOH yields both major C12 [12-hydroxy-17α-TBOH (15)] and minor C5 (5-hydroxy-17α-TBOH) hydroxylated photoproducts (supplementary text and figs.…”

Coupled reversion and stream-hyporheic exchange processes increase environmental persistence of trenbolone metabolites

“…On the basis of observations in Qu et al 1 , photolysis of 17a-TBOH was assumed to yield 90% revertible products (that is, hydroxy-17a-TBOH) and 10% other products not subject to reversion and assumed non-bioactive (for example, species with a substantially modified structure, such as the 11,12-dialdehyde product we previously reported 15 ). All other parameters were assumed constant in space and time to isolate the effect of the reversion process on results.…”

“…One benefit of our modelling approach is that we can explore fate and risk scenarios that might otherwise be too difficult to assess via traditional approaches wholly reliant on demanding and expensive analytical measurements. For example, a scenario that complicates risk management for TBA metabolites is that their photoproducts seem to retain some aspects of bioactivity distinct from their parent metabolites 15 . Thus, confining ecological risk considerations only to the known bioactivity of 17a-TBOH would underestimate the ecological hazard posed by the mixture of parent and product bioactivity.…”

“…We hypothesize that the net effect of these processes operating in stream-hyporheic systems is increased persistence of TBA metabolites in the stream system, resulting in enhanced hydraulic transport and higher concentrations. This increased persistence is particularly troublesome for compounds like TBA metabolites where both the parent species and transformation products retain bioactivity 15 . Our second objective is to characterize expected joint bioactivity (JBA) arising from the suite of TBA metabolites and their photoproducts, including their spatial and temporal occurrence, partitioning and interactions with hydrological dynamics in stream systems.…”