Intramolecular [2 + 2] Photocycloaddition of Altrenogest: Confirmation of Product Structure, Theoretical Mechanistic Insight, and Bioactivity Assessment.

While studying the environmental fate of potent endocrine-active steroid hormones, we observed the formation of an intramolecular [2 + 2] photocycloaddition product (2) with a novel hexacyclic ring system following the photolysis of altrenogest (1). The structure and absolute configuration were established by X-ray diffraction analysis. Theoretical computations identified a barrierless two-step cyclization mechanism for the formation of 2 upon photoexcitation. 2 exhibited progesterone, estrogen, androgen, and pregnane X receptor activity, albeit generally with reduced potency relative to 1.

Cross talk between androgen and Wnt signaling potentially contributes to age-related skeletal muscle atrophy in rats.

We sought to determine whether age-related gastrocnemius muscle mass loss was associated with parallel decrements in androgen receptor (AR) or select Wnt signaling markers. To test this hypothesis, serum-free and total testosterone (TEST) and gastrocnemius AR and Wnt signaling markers were analyzed in male Fischer 344 rats that were 3, 6, 12, 18, and 24 mo (mo) old ( n = 9 per group). Free and total TEST was greatest in 6 mo rats, and AR protein and Wnt5 protein levels linearly declined with aging. There were associations between Wnt5 protein levels and relative gastrocnemius mass ( r = 0.395, P = 0.007) as well as AR and Wnt5 protein levels (r = 0.670, P < 0.001). We next tested the hypothesis that Wnt5 affects muscle fiber size by treating C2C12-derived myotubes with lower (75 ng/ml) and higher (150 ng/ml) concentrations of recombinant Wnt5a protein. Both treatments increased myotube size ( P < 0.05) suggesting this ligand may affect muscle fiber size in vivo. We next tested if Wnt5a protein levels were androgen-modulated by examining 10-mo-old male Fischer 344 rats ( n = 10-11 per group) that were orchiectomized and treated with testosterone-enanthate (TEST-E); trenbolone enanthate (TREN), a nonaromatizable synthetic testosterone analogue; or a vehicle (ORX only) for 4 wk. Interestingly, TEST-E and TREN treatments increased Wnt5a protein in the androgen-sensitive levator ani/bulbocavernosus muscle compared with ORX only ( P < 0.05). To summarize, aromatizable and nonaromatizable androgens increase Wnt5a protein expression in skeletal muscle, age-related decrements in muscle AR may contribute Wnt5a protein decrements, and our in vitro data imply this mechanism may contribute to age-related muscle loss. NEW & NOTEWORTHY Results from this study demonstrate androgen and Wnt5 protein expression decrease with aging, and this may be a mechanism involved with age-related muscle loss.

Veterinary drug, 17ß-trenbolone promotes the proliferation of human prostate cancer cell line through the Akt/AR signaling pathway.

Trenbolone acetate (TBA) is a synthetic anabolic steroidal growth factor that is used for rapid muscle development in cattle. The absorbed TBA is hydrolyzed to the active form, 17ß-trenbolone (17 TB; 17ß-hydroxy-estra-4,9,11-trien-3-one) in meat and milk products, which can cause adverse health effects in humans. Similar to 5α-dihydrotestosterone (DHT), 17 TB was reported to exhibit endocrine disrupting effects on animals and humans due to its androgenic effect via binding to the androgen receptor. The purpose of this study is to investigate the molecular mechanism of cell proliferation in prostate cancer (PCa) cells treated with 17 TB. We found that 17 TB induces AR-dependent cell proliferation in the human prostate cancer cell line, 22Rv1 in a concentration dependent manner. Treatment with 17 TB increased the expression of cell cycle regulatory proteins, cyclin D2/CDK-4 and cyclin E/CDK-2, whereas the expression of p27 was down-regulated. Furthermore, phosphorylation of Rb and activation of E2F were also induced, which suggests the activation of cyclin D2/CDK-4 and cyclin E/CDK-2 in the cells. When 22Rv1 cells were exposed to 30 pM of 17 TB, which is the effective concentration (EC50) value required to observe proliferative effects on 22Rv1 cells, the expression levels of the phosphorylated forms of Akt and GSK3ß were increased. This study demonstrates that 17 TB induces AR-dependent proliferation through the modulation of cell cycle-related proteins in the Akt signaling pathway. The present study provides an effective methodology for identifying cell proliferation signaling of veterinary drugs that exert AR agonistic effects.

Degradation and transformation of 17α-trenbolone in aerobic water-sediment systems.

Synovex® ONE is an extended-release implant containing the active ingredients estradiol benzoate and trenbolone acetate for use in beef steers and heifers. Trenbolone acetate is rapidly hydrolyzed in cattle to form 17ß-trenbolone and its isomer, 17α-trenbolone, which are further transformed to a secondary metabolite, trendione. As part of the environmental assessment for the use of Synovex ONE, data were generated to characterize the fate of 17α-trenbolone, which is the principal metabolite found in cattle excreta, in the environment. A study was conducted to determine the degradation and transformation of [14 C]-17α-trenbolone in 2 representative water-sediment systems under aerobic conditions. The same transformation products, 17ß-trenbolone and trendione, were formed, principally in the sediment phase, in both systems. From the production of these transformation products, the 50% disappearance time (DT50) values of 17ß-trenbolone and trendione were determined, along with the DT50 values of the parent compound and the total drug (17α-trenbolone + 17ß-trenbolone + trendione). The DT50 values for the total system (aqueous and sediment phase) and for the total residues (17α-trenbolone + 17ß-trenbolone + trendione) in the 2 systems were 34.7 d and 53.3 d, respectively. Environ Toxicol Chem 2017;36:630-635. © 2016 SETAC.

Degradation and transformation of 17α-estradiol in water-sediment systems under controlled aerobic and anaerobic conditions.

One of the principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains estradiol benzoate and trenbolone acetate, is 17α-estradiol. As part of the environmental assessment of the use of Synovex ONE, data were generated to characterize the fate of 17α-estradiol in the environment. Studies were conducted to determine the degradation and transformation of 17α-[14 C]-estradiol in 2 representative water-sediment systems each under aerobic and anaerobic conditions. The same transformation products-estriol, 17ß-estradiol, and estrone-were formed, principally in the sediment phase, under both conditions in both systems. From the production of these transformation products, the 50% disappearance time (DT50) values of estrone and 17ß-estradiol were determined, along with the DT50 values of 17α-estradiol and the total drug (17α-estradiol + 17ß-estradiol + estrone). The results indicate that 17 α-[14 C]-estradiol was more persistent under anaerobic conditions than under aerobic conditions and that 17 α-[14 C]-estradiol was less persistent than its transformation products. The DT50 values for the total system (aqueous and sediment phases) and for the total residues (17α-estradiol, 17ß-estradiol, and estrone) were selected for use in modeling the environmental fate of estradiol benzoate. For aerobic degradation in the water-sediment system, the DT50 was 31.1 d, and it was 107.8 d for the anaerobic system. Environ Toxicol Chem 2017;36:621-629. © 2016 SETAC.

Reproductive effects on freshwater fish exposed to 17α-trenbolone and 17α-estradiol.

17α-Trenbolone and 17α-estradiol are principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains trenbolone acetate and estradiol benzoate. As part of the environmental assessment of the use of Synovex® ONE, data were generated to characterize the effects of 17α-trenbolone and 17α-estradiol on the reproduction of freshwater fish. These substances are known endocrine disruptors, so the purpose of testing was not to clarify these properties but to identify concentrations representing population-relevant effects for use in risk characterization. The short-term reproduction assay was conducted with 17α-trenbolone using the fathead minnow (Pimephales promelas) and the medaka (Oryzias latipes) and with 17α-estradiol using the fathead minnow. Adverse effects on the population-relevant endpoints of survival and fecundity were used to establish the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC) for each study. For 17α-trenbolone, adverse effects on fecundity of the fathead minnow occurred at 120 ng/L; this was the LOEC, and the NOEC was 35 ng/L. 17ß-Trenbolone did not adversely affect survival and fecundity of medaka at the concentrations tested, resulting in a NOEC of 110 ng/L and a LOEC of >110 ng/L. 17α-Estradiol did not adversely impact survival and fecundity of the fathead minnow at the concentrations tested, resulting in a NOEC and LOEC of 250 ng/L and >250 ng/L, respectively. Environ Toxicol Chem 2017;36:636-644. © 2016 SETAC.

Sorption and desorption of 17α-trenbolone and trendione on five soils.

The metabolites 17α-trenbolone and 17α-estradiol are principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains trenbolone acetate and estradiol benzoate. As part of the environmental assessment of the use of Synovex ONE, data were generated to characterize the fate of 17α-trenbolone, and its metabolite trendione in the environment. Predictions of the fate and environmental concentrations of these hormones after land application require accurate estimates of the sorption of these compounds in soils. The sorption and desorption of 17α-trenbolone and trendione were measured at 5 nominal concentrations in 5 soils from different geologic settings using a batch equilibrium technique following guideline 106 of the Organisation for Economic Co-operation and Development. Both the sorption and desorption of 17α-trenbolone and trendione to soils were adequately described by the Freundlich sorption model and by linear partition coefficients. The mean sorption coefficients were 9.04 mL/g and 32.2 mL/g for 17α-trenbolone and trendione, respectively. The corresponding mean Freundlich sorption exponents were 0.88 and 0.98, respectively. Sorption of 17α-trenbolone and trendione was correlated principally with soil organic carbon. Average sorption coefficients normalized to soil organic carbon content (KOC ) were 460 mL/g and 1804 mL/g for 17α-trenbolone and trendione, respectively. The mean desorption coefficients were 22.1 mL/g and 43.8 mL/g for 17α-trenbolone and trendione, respectively. Calculated hysteresis coefficients based on the difference in the area between sorption and desorption isotherms indicated that sorption equilibrium was not fully reversible and hysteresis of desorption isotherms occurred for both 17α-trenbolone and trendione. Environ Toxicol Chem 2017;36:613-620. © 2016 SETAC.

Impact of sediment particle size on biotransformation of 17ß-estradiol and 17ß-trenbolone.

Soil/sediment particle size has been reported to influence the sorption and bioavailability of steroid hormones in the environment. However, the impact of particle size on biotransformation has not been well elucidated. The present study investigated the dissipation of 17ß-estradiol and 17ß-trenbolone and the formation and degradation of the subsequent transformation products in different size fractions of a sandy and a silt loam sediment. The results showed that the decay of 17ß-estradiol and 17ß-trenbolone associated with fine particles followed a biphasic pattern with more rapid decay in the initial phase followed by a second phase with slower decay of the residues compared to their decay rates in the sand fraction. Estrone and trendione were detected as a primary biotransformation product for 17ß-estradiol and 17ß-trenbolone, respectively. The parent-to-product conversion ratios and the degradation rates of estrone and trendione varied among different size fractions, but no consistent correlation was observed between decay rates and sediment particle size. Estrone and trendione decayed in the whole sediments at rates not statistically different from those associated with the fine fractions. These results indicate that fine particles may play an important role in influencing the persistence of and the potential risk posed by steroid hormones in the aquatic systems.

Sorption, uptake, and biotransformation of 17ß-estradiol, 17α-ethinylestradiol, zeranol, and trenbolone acetate by hybrid poplar.

Hormonally active compounds may move with agricultural runoff from fields with applied manure and biosolids into surface waters where they pose a threat to human and environmental health. Riparian zone plants could remove hormonally active compounds from agricultural runoff. Therefore, sorption to roots, uptake, translocation, and transformation of 3 estrogens (17ß-estradiol, 17α-ethinylestradiol, and zeranol) and 1 androgen (trenbolone acetate) commonly found in animal manure or biosolids were assessed by hydroponically grown hybrid poplar, Populus deltoides x nigra, DN-34, widely used in riparian buffer strips. Results clearly showed that these hormones were rapidly removed from 2 mg L(-1) hydroponic solutions by more than 97% after 10 d of exposure to full poplar plants or live excised poplars (cut-stem, no leaves). Removals by sorption to dead poplar roots that had been autoclaved were significantly less, 71% to 84%. Major transformation products (estrone and estriol for estradiol; zearalanone for zeranol; and 17ß-trenbolone from trenbolone acetate) were detected in the root tissues of all 3 poplar treatments. Root concentrations of metabolites peaked after 1 d to 5 d and then decreased in full and live excised poplars by further transformation. Metabolite concentrations were less in dead poplar treatments and only slowly increased without further transformation. Taken together, these findings show that poplars may be effective in controlling the movement of hormonally active compounds from agricultural fields and avoiding runoff to streams.

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

Existing regulatory frameworks for aquatic pollutants in the United States are idealized, often lacking mechanisms to account for contaminants characterized by (1) bioactivity of both the parent and transformation products and (2) reversible transformations (that is, metastable products) driven by chemical or physical heterogeneities. Here, we modelled a newly discovered product-to-parent reversion pathway for trenbolone acetate (TBA) metabolites. We show increased exposure to the primary metabolite, 17α-trenbolone (17α-TBOH), and elevated concentrations of the still-bioactive primary photoproduct hydroxylated 17α-TBOH, produced via phototransformation and then converted back to 17α-trenbolone in perpetually dark hyporheic zones that exchange continuously with surface water photic zones. The increased persistence equates to a greater potential hazard from parent-product joint bioactivity at locations and times when reversion is a dominant trenbolone fate pathway. Our study highlights uncertainties and vulnerabilities with current paradigms in risk characterization.

Rates and product identification for trenbolone acetate metabolite biotransformation under aerobic conditions.

Trenbolone acetate metabolites are endocrine-active contaminants discharged into the aquatic environment in runoff from agricultural fields, rangelands, and concentrated animal feeding operations. To investigate the environmental fate of these compounds and their biotransformation mechanisms, the authors used inocula from a variety of different water sources and dosed biologically active microcosms with approximately 1400 ng/L of trenbolone acetate metabolites, including 17ß-trenbolone, trendione, and 17α-trenbolone. To investigate aerobic biotransformation rates and interconversions between known trenbolone acetate metabolites, gas chromatography-tandem mass spectrometry was used to measure concentrations and assess product distributions as a function of time. High-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to characterize novel transformation products and potential transformation pathways. Kinetic analysis yields observed half-lives of approximately 0.9 d, 1.3 d, and 2.2 d for 17ß-trenbolone, trendione, and 17α-trenbolone, respectively, at 20 °C, although colder conditions increased half-lives to 8.5 d and biphasic transformation was observed. Relative to reported faster attenuation rates in soils, trenbolone acetate metabolites are likely more persistent in aqueous systems. Product distributions indicate an enzymatic preference for biotransformation between trendione and 17ß-trenbolone. The LC-MS/MS characterization indicates dehydrogenation products as the major detectable products and demonstrates that major structural elements responsible for bioactivity in steroids are likely retained during biotransformation.

Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers.

Biotransformation of trenbolone acetate metabolites and estrogens derived from animal feeding operations in soils, waste storage systems, and in land applied manure has been well characterized. Yet recent data demonstrate potential for steroid transport into the environment directly from feedyard pens via runoff or airborne particulate matter. Therefore, the objective of this study was to determine steroid transformation rates in beef cattle excreta. Feces and urine were collected from steers recently treated with steroidal implants. Excreta were stored and periodically extracted over 112 d then analyzed for trenbolone acetate metabolites and estrogens by liquid chromatography mass spectrometry. Conjugated steroids were present primarily in urine, and conjugates quickly degraded to free steroid with a half-life of 0.6-1.0 d. The primary trenbolone acetate metabolite, 17α-trenbolone, had a half-life of 5.1-9.5 d. Likewise, 17α-estradiol was the predominant estrogen, with a half-life of 8.6-53 d. Secondary trenbolone metabolites formed from 17α-trenbolone biotransformation were observed at low concentrations less than 10% initial 17α-trenbolone concentrations. Estrone was the primary metabolite of 17α-estradiol and concentrations of estrone exceeded initial 17α-estradiol concentration in all sample types. These results suggest manure-borne steroids are more stable in excreta than in soil microcosms.

17ß-trenbolone, an anabolic-androgenic steroid as well as an environmental hormone, contributes to neurodegeneration.

Both genetic and environmental factors contribute to neurodegenerative disorders. In a large number of neurodegenerative diseases (for example, Alzheimer's disease (AD)), patients do not carry the mutant genes. Other risk factors, for example the environmental factors, should be evaluated. 17ß-trenbolone is a kind of environmental hormone as well as an anabolic-androgenic steroid. 17ß-trenbolone is used as a growth promoter for livestock in the USA. Also, a large portion of recreational exercisers inject 17ß-trenbolone in large doses and for very long time to increase muscle and strength. 17ß-trenbolone is stable in the environment after being excreted. In the present study, 17ß-trenbolone was administered to adult and pregnant rats and the primary hippocampal neurons. 17ß-trenbolone's distribution and its effects on serum hormone levels and Aß42 accumulation in vivo and its effects on AD related parameters in vitro were assessed. 17ß-trenbolone accumulated in adult rat brain, especially in the hippocampus, and in the fetus brain. It altered Aß42 accumulation. 17ß-trenbolone induced apoptosis of primary hippocampal neurons in vitro and resisted neuroprotective function of testosterone. Presenilin-1 protein expression was down-regulated while ß-amyloid peptide 42 (Aß42) production and caspase-3 activities were increased. Both androgen and estrogen receptors mediated the processes. 17ß-trenbolone played critical roles in neurodegeneration. Exercisers who inject large doses of trenbolone and common people who are exposed to 17ß-trenbolone by various ways are all influenced chronically and continually. Identification of such environmental risk factors will help us take early prevention measure to slow down the onset of neurodegenerative disorders.

Characterization of trenbolone acetate and estradiol metabolite excretion profiles in implanted steers.

Exogenous growth promoters have been used in US beef cattle production for over 50 yr. The environmental fate and transport of steroid growth promoters suggest potential for endocrine-disrupting effects among ecological receptors; however, the initial excretion of steroid metabolites from cattle administered growth promoters has not been well characterized. To better characterize excretion of trenbolone acetate and estrogen metabolites, steers were assigned to 1 of the following treatment groups: control, given no implant, or treatment, administered a combination implant (200 mg trenbolone acetate, 40 mg estradiol). Blood, urine, and fecal samples were collected over the course of 112 d following implantation. Samples were extracted and analyzed by liquid chromatography tandem mass spectrometry for trenbolone acetate and estrogen metabolites. In both urine and feces, 17α-trenbolone and 17α-estradiol were the predominant metabolites following implantation. Mean concentrations of 17α-trenbolone and 17α-estradiol in feces of implanted steers were 5.9 ± 0.37 ng/g and 2.7 ± 0.22 ng/g, respectively. A best-fit model is presented to predict 17α-trenbolone and 17α-estradiol excretion from steers receiving implants. The present study provides the first characterization of both trenbolone and estrogen metabolites in excreta from implanted cattle and will help provide estimates of steroid production from feedyards in the United States.

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

Endocrine disrupting effects in aquatic organisms have been observed in systems influenced by steroid hormones. Associating endocrine disruption with aqueous concentrations of steroids alone may overlook the influence of source-sink dynamics in sediments on steroid hormone bioavailability. The objective of this study was to determine the fate of 17ß-estradiol and 17ß-trenbolone in two field sediments and to evaluate the corresponding bioavailability of the compounds to the fathead minnow (Pimephales promelas). Steroid fate was evaluated using analytical chemistry and verified by assessing the biological activity using yeast based in vitro assays. Effective bioavailability of the steroids was inferred from changes in hepatic vitellogenin expression (increased expression in males exposed to 17ß-estradiol, and reduced expression in females exposed to 17ß-trenbolone). In experiments conducted with 17ß-estradiol, no induction of hepatic vitellogenin mRNA expression was observed in male fish exposed to sediment-associated 17ß-estradiol. In contrast, female minnows exposed to sediment-associated 17ß-trenbolone experienced significant reductions in hepatic vitellogenin compared to negative controls. In both systems, the parent compounds were shown to degrade rapidly to the more persistent metabolites, estrone and trendione, both of which were found predominantly associated with the sediments. Results from the yeast estrogen screen indicate a reduction in biological activity as biotransformation of 17ß-estradiol occurs; results from the yeast anti-estrogen screen were inconclusive and unable to substantiate 17ß-trenbolone fate in aquatic systems. Collectively, these data support the contention that steroid hormones associated with the sediment can become bioavailable to fish, and that sediment characteristics influence the observed bioavailability of these compounds.

Trenbolone acetate metabolites promote ovarian growth and development in adult Japanese medaka (Oryzias latipes).

Trenbolone acetate, a synthetic androgen, has been used as a growth promoter in beef cattle in the US since 1987. While several teleost studies have investigated the masculinization effects of the metabolite 17ß-trenbolone, few have focused on the reproductive impacts of all three trenbolone acetate (TBA) metabolites including trendione. Adult female medaka (Oryzias latipes) were exposed to TBA metabolites (10, 100, and 1000ng/L) for 14days (n=3). Histological examination revealed that TBA metabolites (1000ng/L) significantly reduced the percentage of primary ovarian follicles and increased the percentage of vitellogenic follicles compared to control fish. 17α-Trenbolone significantly increased whereas trendione reduced whole body levels of estradiol-17ß. Testosterone was significantly reduced by trendione treatment and only the highest dose of 17ß-trenbolone and lowest dose of trendione altered 11-ketotestosterone. Additionally, TBA metabolites may be further broken down and/or metabolized or converted by the animal influencing both sex steroid levels and ovarian development.

Mass balance approaches to characterizing the leaching potential of trenbolone acetate metabolites in agro-ecosystems.

Several studies have documented the occurrence and fate of trenbolone acetate (TBA) metabolites in soil and water. However, considerable uncertainty still exists with respect to TBA risk in agro-ecosystems because limited data are available to quantify excretion, transformation, and leaching processes. To address these uncertainties, we used experimental mesocosms and a mass balance approach to estimate the TBA metabolite leaching potential from manure excreted by implanted (40 mg TBA, 8 mg 17ß-estradiol) beef cattle. Manure sample analysis indicates that over 113 days, a maximum of 9.3% (3,200 µg/animal unit [AU]) of the implant dose was excreted as 17α-trenbolone (17α-TBOH), and <1% was excreted as 17ß-trenbolone (65 µg/AU) or trendione (3 µg/AU). While most (>97%) of the total excreted mass of 17α-TBOH transforms to uncharacterized products, 0.3-0.6% (100-220 µg/AU) of the implant dose accumulates on land surfaces and is available for subsequent transport. During rainfall or irrigation events, a maximum of 0.005-0.06% (1.6-22 µg/AU 17α-TBOH) or 0.005-0.012% (1.8-4 µg/AU 17α-TBOH) of the dose leached into runoff, respectively. Leaching potentials peak at 5-30 days postimplantation, suggesting that targeted timing of implantation and irrigation could minimize steroid leaching during rainfall and irrigation events.

Product-to-parent reversion of trenbolone: unrecognized risks for endocrine disruption.

Trenbolone acetate (TBA) is a high-value steroidal growth promoter often administered to beef cattle, whose metabolites are potent endocrine-disrupting compounds. We performed laboratory and field phototransformation experiments to assess the fate of TBA metabolites and their photoproducts. Unexpectedly, we observed that the rapid photohydration of TBA metabolites is reversible under conditions representative of those in surface waters (pH 7, 25°C). This product-to-parent reversion mechanism results in diurnal cycling and substantial regeneration of TBA metabolites at rates that are strongly temperature- and pH-dependent. Photoproducts can also react to produce structural analogs of TBA metabolites. These reactions also occur in structurally similar steroids, including human pharmaceuticals, which suggests that predictive fate models and regulatory risk assessment paradigms must account for transformation products of high-risk environmental contaminants such as endocrine-disrupting steroids.

Identification and environmental implications of photo-transformation products of trenbolone acetate metabolites.

Despite the widespread use of the anabolic androgen trenbolone acetate (TBA) in animal agriculture, evidence demonstrating the occurrence of TBA metabolites such as 17ß-trenbolone (17ß-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 17ß-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 17ß-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 17ß-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.

Evidence of small modulation of ethinylestradiol induced effects by concurrent exposure to trenbolone in male eelpout Zoarces viviparus.

The interaction of xenobiotics is common in aquatic ecosystems; therefore, we wanted to evaluate if trenbolone (TB) modulates the effects of 17α-ethinylestradiol (EE2). Male eelpout (Zoarces viviparus) were exposed to 5 ng L(-1) EE2 continuously for 19 d (EE2-C) or discontinuously (11 d, EE2-D) alone or in combination with low (50 ng L(-1), TBL) or high (500 ng L(-1), TBH) concentrations of TB (19 d). Exposure to EE2 caused reduced gonadosomatic index, increased plasma vitellogenin concentrations, up-regulated vtg and era mRNA expression and severe alterations in gonadal histology. TBL and TBH did not affect plasma vitellogenin, era or vtg mRNA expression. TBL and TBH did not counteract the EE2-induced increase in plasma vitellogenin and reduction in 11-ketotestosterone whereas TBH counteracted the EE2 induced increase in vtg and era mRNA expression. Exposure to TBH and EE2-C + TBH lead to severe gonadal histology alterations. TBL and EE2-D + TBH exposed fish showed less histopathological alterations.