Introduction

Pharmaceuticals based on steroid hormones and their derivatives are increasingly produced, consumed, and finally released into the aquatic environment via wastewater (Herzog and Oliveto 1992, Hogg 1992). The influence of various anthropogenic steroids on river water wildlife is of concern and is extensively studied (Vos et al. 2000). We have published a study on metabolism of obeticholic acid, an engineered bile acid used to treat a number of human liver diseases, in brown bullhead recently (Mach et al. 2019). In this paper, we focus on a derivative of dehydroepiandrosterone (DHEA), abiraterone acetate (ABR-acetate), prescribed to castration-resistant prostate cancer patients. ABR-acetate prodrug is converted in vivo to abiraterone (ABR), an androgen biosynthesis inhibitor (Janssen Inc. 2012). It affects not only cancerous tissue but also natural synthesis of androgens. Toxicological studies in rats and monkeys report reduction in circulating testosterone and decrease in organ weights in the reproductive system after 13 weeks of administration of a half of the human clinical exposure (Janssen Inc. 2012). Toxicological studies in fish are not known (Di Giulio and Hinton 2008). Nevertheless, its action is probably similar compared with mammals. Hinfray et al. (2013) studied antiandrogenic action of exogenous17-β-estradiol in zebrafish. They found out that exposure to 17-β-estradiol specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis.

As for the metabolism of steroids in fish, 2 phases are discerned (James 2011): the first phase is hydroxylation. Various positions of hydroxylation are possible (Scornaienchi et al. 2010). The second phase is the formation of sulfate and glucuronide conjugates (James 2011).

The two main circulating metabolites of ABR in human plasma are ABR-sulfate and ABR-sulfate-N-oxide, which account for about 43% of exposure each (FDA 2011; Janssen Inc. 2012). In rats and humans, ABR-acetate was excreted mainly (≥ 90%) via the feces as metabolites, unchanged prodrug, and ABR, with minor amounts of metabolites (mainly the N-oxide) excreted in urine. Other metabolites were not described. Nevertheless, glucuronides could be also likely formed similarly as from other steroids (Itaaho et al. 2008; Dutton 1966).

The aim of the paper was to study the metabolism of ABR-acetate in the brown bullhead as a model fish in comparison with metabolism of other steroid compounds in fish and ABR-acetate metabolism in humans.

Materials and methods

Chemicals

Dichloromethane (p.a. grade) was purchased from Blue Cube Germany, 1-butanol (p.a. grade) and NH4H2PO4 (p.a. grade) were purchased from Lach-Ner, n-hexane (puriss. p.a. grade) was purchased from Riedel-de Haën, acetonitrile (LC/MS grade) and water (LC/MS grade) were purchased from Fisher Scientific, formic acid (LC/MS grade) was purchased from Fluka, and NaOH (puriss. p.a. grade) was purchased from Sigma-Aldrich. ABR-acetate and standards of 16,17-alpha-epoxy-ABR-acetate and 16,17-beta-epoxy-ABR-acetate were from Teva research and development department internal sources, were fully characterized (MS, NMR, IR) and their HPLC purity was in case of ABR-acetate better than 99% (API, i.e., active pharmaceutical ingredient, quality) and in case of standards 16,17-alpha-epoxy-ABR-acetate and 16,17-beta-epoxy-ABR-acetate better than 95%.

Animals

Juvenile brown bullhead (average weight 77 g) were obtained from Přelouč fish hatchery and were maintained in 100-L tanks at outdoor temperature, i.e., app. 12–13 °C. Two days starvation period preceded all experimental use. Twenty individuals were used for the experiment.

ABR-acetate administration

Basic experimental design was the same as with obeticholic acid (Mach et al. 2019). Exposure experiment was carried out in aerated 40-L aquarium placed in the laboratory. First, the aquarium was filled with 14 L of water from the tank placed outdoor and the twenty individuals were transferred into it. In this way, the temperature of water grew gradually from outdoor temperature to laboratory temperature and thermal shock of animals was prevented. ABR-acetate was applied, dissolved in olive oil via gastric probe. A dose was calculated so as to represent 3 mg/10 g fish weight. One by one, an individual was taken from the aquarium, and the dose was applied and, after feeding fish, it was maintained for 3 min under a wet towel so as to prevent vomiting. Then it was let out into the laboratory aquarium and kept there for 3 days.

Extraction of metabolites

After finishing the experiment, all 14 L of water from the aquarium was extracted two times with dichloromethane (dichloromethane:water = 1:10) and consequently, after the addition of 100 g of NH4H2PO4 per 1 L of water, the same water sample was extracted two times with 1-butanol (1-butanol:water = 1:10). This procedure is recommended for the extraction of polar metabolites of steroids (Makin et al. 2010). The dichloromethane (DCM) and 1-butanol extracts were further treated separately. Both the DCM and 1-butanol extracts were evaporated to dryness, redissolved in 80% aqueous solution of methanol, and partitioned three times with n-hexane (80% aqueous methanol:n-hexane = 3:1) to remove lipidic substances. Hexane layer with lipids was removed each time. Finally, the aqueous phase was evaporated to dryness again.

The purified DCM extract evaporation residue was then redissolved in 20 mL methanol. One milliliter was taken into the analytical vial and was analyzed by the HPLC/MS method described further.

The purified 1-butanol extract evaporation residue was redissolved in 1 L of 20% aqueous methanol. It was deprived of the residual NH4H2PO4 by preparative chromatography performed on Labio Biospher PSI 100 C18 column, 250 × 50 mm, particle size 15 μm, using two VWR LaPrep P110 preparative pumps, Knauer UV detector 2500 and Knauer valve drive directed by Clarity software. Solid phase extraction principle was applied. Column was conditioned by 95% aqueous methanol at flow rate 30 mL/min for 15 min, followed by 20% aqueous methanol for 15 min, then the sample was applied. More polar impurities including residual NH4H2PO4 were washed out by another 450 mL of 20% aqueous methanol. Elution was performed by 95% aqueous methanol and flow rate was reduced to 10 mL/min. Fractions were taken every 5 min, altogether 10 fractions. All fractions were monitored by the HPLC/MS method described further for the presence of possible polar metabolites (data not shown). Fractions containing glucuronides and sulfates of ABR were put together and these formed the sample denoted as 1-BuOH extract in the part results and discussion. One milliliter of this sample was taken into the analytical vial and was analyzed by the HPLC/MS method described further.

HPLC/MS analysis

Samples were analyzed by Accela HPLC pump with Thermo LTQ XL linear ion trap mass spectrometric detector directed by X-calibur software. Chromatographic conditions were as follows: column Supelco Ascentis Express C8, 100 × 2.1 mm, particle size 2.7 μm, column temperature ambient, gradient elution with mobile phase A 10 mM formic acid in water and mobile phase B acetonitrile. The detailed gradient process was as follows: 0–0.5 min 20% B, 0.5–15 min 20–100% B, 15.1–20 min re-equilibration at 20% B. Flow rate of mobile phase was 200 μL/min. MS detector was interfaced with HPLC via electrospray ionization in positive ion mode. Mass spectrometric conditions were spray voltage 2.5 kV, sheath gas flow rate 35, auxiliary gas flow rate 10, capillary voltage 43 V, capillary temperature 210 °C, tube lens offset 115 V, and full-scan mass range 200–1200; MS2- and MS3-dependent scan data were also recorded.

The aim of this study was mostly to perform qualitative analysis of metabolites formed from ABR-acetate in brown bullhead. It was not designed to prepare a validated method and to quantitate the metabolites. Estimation of relative quantities of metabolites is based on intensities of MS signal only.

NMR spectroscopy

The most abundant metabolite from DCM extract with [M+H]+ m/z 366, eluting in RT 9.2 min was isolated by preparative chromatography and its structure was further studied by NMR on a Bruker Avance III 600 MHz (600.23 MHz for 1H, 150.93 MHz for 13C) in CDCl3 at 20 °C and Bruker Avance III 700 MHz (700.13 MHz for 1H, 176.05 MHz for 13C) in CDCl3 at 20 °C. The following NMR experiments were performed: 1H NMR, 13C NMR, gCOSY, 1H-13C gHSQC, 1H-13C gHMBC, and 1H-13C gHMQC using the standard manufacturer software TopSpin 3.5 (Bruker, Rheinstetten, Germany).

Results and discussion

ABR-acetate administration

Brown bullhead was used as easily feasible model for the metabolism of drugs (Jegorov et al. 2000). Administration of ABR-acetate to the fish at the dose 3 mg/10 g fish weight did not reveal any significant toxicity and no one fish was lost within 3-day exposure, even though the amount applied was considerably higher than the therapeutic dose used in humans. Extraction of water in which the fish were kept provided very simple and efficient method for obtaining ABR-acetate metabolites. Similar principle was already applied for monitoring of various fish metabolites of other steroid compounds (Scott and Ellis 2007; Mach et al. 2019).

Screening for ABR-acetate metabolites by HPLC/MS

Based on the metabolism of ABR-acetate in humans and rats, expected both primary and secondary metabolites (Fig. 1) were monitored at their respective mass traces of protonated molecules.

Fig. 1
figure 1

Chemical structures of ABR-acetate and its expected metabolites. Not all possible positional isomers are shown, as well as possible combinations (e.g., dihydroxides)

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In DCM extract (Fig. 2), compounds corresponding to monitored m/z included the following: ABR-acetate (m/z 392), ABR (m/z 350), hydroxy-ABR or ABR-N-oxide (m/z 366; 2 peaks), hydroxy-ABR-acetate or ABR-acetate-N-oxide (m/z 408; 3 peaks), dihydroxy-ABR or hydroxy-ABR-N-oxide (m/z 382; several peaks), and dihydroxy-ABR-acetate or hydroxy-ABR-acetate-N-oxide (m/z 424; several peaks).

Fig. 2
figure 2

HPLC/MS analysis of the purified DCM extract. TIC m/z 200–1200 at the top, below selected ion chromatograms of [M+H]+ of ABR-acetate and expected metabolites

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In 1-BuOH extract (Fig. 3), compounds corresponding to monitored m/z included the following: ABR-sulfate (m/z 430), hydroxy-ABR-sulfate or ABR-sulfate-N-oxide (m/z 446; several peaks), ABR-glucuronide (m/z 526), and hydroxy-ABR-glucuronide or ABR-glucuronide-N-oxide (m/z 542; several peaks).

Fig. 3
figure 3

HPLC/MS analysis of the purified 1-butanol extract. Base peak chromatogram m/z 200–1200 at the top, below selected ion chromatograms of [M+H]+ of expected metabolites

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Hence, in contrast to the expectation, ABR-acetate metabolites excreted by fish seem to be different than those found in rats or humans. Besides abiraterone and its sulfate, also glucuronide, hydroxy-sulfate, and hydroxy-glucuronide were found. The presence of hydroxy-ABR-acetates shows that deacetylation does not necessarily precede other metabolic pathways. Further attention was paid to presumed hydroxy-ABR (m/z 366, RT 9.2 min.), which was isolated by preparative chromatography and its structure was further determined by NMR.

NMR structure analysis of metabolite from DCM extract with [M+H]+ m/z 366 eluting in RT 9.2 min

The combination of the 13C NMR and 1H-13C multiplicity-edited HSQC allowed us to identify two methyls, seven methylenes, ten methines (five sp2- and five sp3-hybridized), and four quaternary carbons (three sp3- and one sp2-hybridized). The remaining carbon was detected only indirectly by 1H-13C HMBC spectrum at 131.5 ppm. The 1H NMR spectrum contains two singlets of isolated tertiary methyls together with two spin systems, which were evaluated by COSY in combination with 1H-13C HSQC and 1H-13C HMBC as CH(O)CH2CHCH(CHCH2CH2)CH2CH=CCH2CH(O)CH2CH2- and an ABCD spin system.

The heteronuclear couplings from 18-methyl proton to C-12, C-14, and quaternary carbons at 42.90 ppm (C-13) and 70.02 ppm (C-17) together with coupling coming from H-16 to C-13 allow the closing of the C and D rings and approve the bond between C-13 and C-14. The correlations from 19-Me protons to C-1, C-9, and quaternary carbons at 140.99 ppm (C-5) and 36.70 ppm (C-10) allow the closure of the rings A and B and confirm the bond between C-5 and C-10. The methylene C-4 is coupled to methine H-6, which confirms the connection between the A and B rings.

The ABCD spin system due to its proton-coupling pattern can be identified as 3-substituted pyridine. The quaternary carbon at 131.5 ppm was identified as C-3’ by its HMBC correlation with H-5’. The connection between the steroid moiety and the 3-pyridyl group was demonstrated by the 1H-13C HMBC correlation between H-2’ and H-4’ of the 3-pyridyl group and C-17 of the steroid moiety. The chemical shift of C-16 and C-17 can be explained by attachment to an oxygen atom. The value of 1J(C-16, H-16) = 183 Hz well agrees with the epoxide formation.

Since the stereochemistry of isolated metabolite was not revealed by NMR, standards of 16,17-epoxy derivatives of ABR-acetate were used for comparison and their absolute configuration was verified by X-ray structure determination (see supplementary data). Their NMR data are also deposited as supplementary tables S2 and S3. Table S4 shows differences among chemical shifts of isolated metabolite and 16,17-alpha-epoxy-ABR-acetate and 16,17-beta-epoxy-ABR-acetate. The differences in the case of 16,17-alpha-epoxy-ABR-acetate do not exceed 0.5 ppm for the rings B, C, and D. The higher differences of the A ring carbons are attributed to the fact that the epoxide derivative was acetylated at C-3. Therefore, we can assume that the orientation of the epoxy group is alpha and the orientation of other chiral centers remains retained (Figs. 4 and 5).

Fig. 4
figure 4

Structure of metabolite from DCM extract eluting in RT 9.2 min as determined by NMR

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Fig. 5
figure 5

The spin systems and the crucial HMBC correlations

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Discussion of structure determined by NMR

The structure determined by NMR is rather surprising as epoxidation of steroids in fish has not been apparently described until now. Hydroxylation is reported as usual first-step metabolism (James 2011). On the other hand, epoxidation of other substances is reported (Ludke et al. 1972, Yang et al. 2000) and epoxidation of some sterols was observed, e.g., by rat microsomes (Watabe et al. 1986).

Discussion of MS fragments

An interesting link can be found among first-step and second-step metabolites found in extracts. While metabolite from DCM extract with [M+H]+ m/z 366 eluting in RT 9.2 min (its structure was determined by NMR as ABR-16,17-epoxide) provides MS2 fragments m/z 120 and 134, the other isobaric metabolite eluting in RT 8 min does not provide these fragments (Figs. 6 and 7). Possible structure of fragments m/z 120 and 134 is given in Fig. 8.

Fig. 6
figure 6

Full-scan MS and fragments in RT 9.2 min, purified DCM extract

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Fig. 7
figure 7

Full-scan MS and fragments in RT 8 min, purified DCM extract

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Fig. 8
figure 8

Possible structure of fragments m/z 120 and 134

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Similarly, presumed hydroxy-ABR-acetate with [M+H]+ m/z 408 eluting in RT 12.67 min provides MS3 fragments m/z 120 and 134 (Fig. 9; in MS2 only acetate is eliminated to m/z 348) and no other remaining isobaric metabolite provides these fragments (data not shown). Metabolite with [M+H]+ m/z 408 eluting in RT 12.67 min probably contains 16,17-epoxide.

Fig. 9
figure 9

Full-scan MS and fragments in RT 12.67 min, purified DCM extract

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Analogously, metabolite from DCM extract with [M+H]+ m/z 382 eluting in RT 6.25 min (corresponds to dihydroxy-ABR) provides MS2 fragments m/z 120 and 134 (Fig. 10) and no other remaining isobaric metabolite provides these fragments. Metabolite with [M+H]+ m/z 382 eluting in RT 6.25 min probably contains 16,17-epoxide. Metabolite from 1-BuOH extract with [M+H]+ m/z 446 (corresponds to hydroxy-ABR-sulfate) eluting in RT 6.9 min provides MS3 fragments m/z 120 and 134 (Fig. 11; in MS2 only sulfate is eliminated to m/z 348) and no other remaining isobaric metabolite provides these fragments; data shown only for RT 4.2 min (Fig. 12). Metabolite with [M+H]+ m/z 446 eluting in RT 6.9 min probably contains 16,17-epoxide.

Fig. 10
figure 10

Full-scan MS and fragments in RT 6.25 min, purified DCM extract

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Fig. 11
figure 11

Full-scan MS and fragments in RT 6.9 min, purified 1-BuOH extract

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Fig. 12
figure 12

Full-scan MS and fragments in RT 4.2 min, purified 1-BuOH extract

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Conclusion

Feeding of brown bullhead (Ameiurus nebulosus) with ABR-acetate and extraction of water in which the fish were kept provided an easy way of obtaining various ABR-acetate metabolites. Various hydroxy-ABRs were among expected first-stage metabolites. NMR measurement of one of the prevailing metabolites presumed to be one of possible hydroxy-ABRs (based on MS data) discovered that it is not hydroxy-ABR but ABR-16,17-epoxide. Although epoxidation of a double bond is a well-recognized pathway and in many cases the epoxide is then further metabolized to diols by epoxide hydrolase, epoxidation of steroids was likely not described in fish until now. Closer analysis of MS2 and MS3 spectra revealed that one of presumed hydroxy-ABR-acetates (based on MS data) and also some secondary metabolites are probably 16,17-epoxides. The fact that epoxidation is the preferred pathway of first-stage metabolism of ABR-acetate in brown bullhead is thus quite unexpected. Further experiments are needed to investigate if this finding can be generalized for more fish species and more steroid compounds.