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Analysis of testosterone fatty acid esters in the digestive gland of mussels by liquid chromatography-high resolution mass spectrometry
Steroids 123 (2017) 67–72
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Analysis of testosterone fatty acid esters in the digestive gland of mussels by liquid chromatography-high resolution mass spectrometry Cesare Guercia, Piergiorgio Cianciullo, Cinta Porte
MARK
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Department of Environmental Chemistry, IDAEA-CSIC, Barcelona, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Acyl-CoA:testosterone acyltransferase Testosterone esters Mussels UPLC-HRMS Pantetheine thioesters
Several studies have indicated that up to 70% of the total steroids detected in molluscs are in the esterified form and that pollutants, by modifying the esterification of steroids with fatty acids, might act as endocrine disrupters. However, despite the strong physiological significance of this process, there is almost no information on which fatty acids form the steroid esters and how this process is modulated. This study (a) investigates the formation of fatty acid esters of testosterone in digestive gland microsomal fractions of the mussel Mytilus galloprovincialis incubated with either palmitoly-CoA or CoA and ATP, and (b) assesses whether the endocrine disruptor tributyltin (TBT) interferes with the esterification of testosterone. Analysis of testosterone esters was performed by liquid chromatography–high resolution mass spectrometry (UPLC-HRMS). When microsomal fractions were incubated with testosterone and palmitoly-CoA, the formation of testosterone palmitate was detected. However, when microsomes were incubated with CoA and ATP, and no exogenous activated fatty acid was added, the synthesis of 16:0, 16:1, 20:5 and 22:6 testosterone esters was observed. The presence of 100 µM TBT in the incubation mixture did not significantly alter the esterification of testosterone. These results evidence the conjugation of testosterone with the most abundant fatty acids in the digestive gland microsomal fraction of mussels.
1. Introduction Fatty acid conjugation of steroids appears to be a well-conserved conjugation pathway during evolution; it is known to occur in both vertebrate and invertebrates. Fatty acid conjugation (or esterification) renders steroids to an apolar form, which is retained in the lipoidal matrices of the body, while reducing their activity, bioavailability, and susceptibility to elimination [1,2]. Steroid esters do not bind to receptors, but they can be hydrolyzed by esterases to liberate the active steroid [3]. Steroid esters have been reported in molluscs and some studies have suggested that esterification might play a key role in regulating levels of free steroids in these organisms. Thus, Gooding and LeBlanc [2] showed that the mud snail Ilyanassa obsoleta, primarily metabolized free testosterone to non-polar fatty acid ester conjugates via acyl-CoA:testosterone acyltransferase (ATAT), which is localized in the endoplasmic reticulum. The authors showed that esterification was the major biotransformation pathway for testosterone in snails, as exogenously provided testosterone was converted to fatty acid esters and retained in the organism. In addition, irrespective of the amount of testosterone administered to the snails, the amount of free testosterone measured in
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Corresponding author. E-mail address: [email protected] (C. Porte).
http://dx.doi.org/10.1016/j.steroids.2017.05.008 Received 23 February 2017; Received in revised form 2 May 2017; Accepted 8 May 2017 Available online 11 May 2017 0039-128X/ © 2017 Elsevier Inc. All rights reserved.
the tissues remained relatively constant and all excess of testosterone was converted to the fatty acid ester [4]. Similarly, exogenously administered testosterone and estradiol were extensively esterified by the mussel Mytilus galloprovincialis, whereas levels of unconjugated steroids remained almost unaltered [5,6]. On the other hand, few studies have investigated which are the specific fatty acid steroid conjugates formed by molluscs. Janer et al. [7] reported the esterification of estradiol in digestive gland microsomal fractions of Crassostrea virginica with a variety of saturated or unsaturated fatty acids (C16:0; C18:0; C16:1; C18:1; C18:2 and C20:4); however, identification was tentative. Labadie et al. [8] reported for the first time the identity of three estradiol fatty acid conjugates (C16:0; C16:1 and C16:2) in Mytilus edulis by using tandem mass spectrometry with direct probe insertion. The organotin compound tributyltin (TBT) is a well-known endocrine disrupter in molluscs that it has been reported to interfere with the esterification of testosterone. Thus, Gooding et al. [9] showed that females of Ilyanassa obsoleta experimentally exposed to 10 ng/L TBT for 3 months had a lower ability to conjugate testosterone with fatty acid moieties; and females collected in an organotin-polluted site had lower levels of esterified testosterone than those collected in a clean site.
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Table 1 Testosterone esters and pantetheine thioesters detected in the digestive gland microsomal fraction of M. galloprovincialis. Testosterone undecanoate (T-11:0) and laurate (T-12:0) were used as internal standards. Fatty Acid
Molecular ion [M + H]+
Theoretical mass (Da)
Experimental mass (Da)
Mass difference (ppm)
Retention time (min)
Testosterone esters 11:0 12:0 16:0 16:1 20:5 22:6
C30H49O3 C31H51O3 C35H59O3 C35H57O3 C39H57O3 C41H59O3
457.3682 471.3838 527.4459 525.4303 573.4305 599.4464
457.3674 471.3825 527.4467 525.4308 573.4311 599.4440
2.1 2.1 1.1 2.5 2.7 2.8
4.3 4.6 6.9 5.9 5.3 5.7
517.3675 545.3988
517.3668 545.3991
4.7 6.0
3.4 3.8
Acyl-S-pantetheine thioesters 16:0 C27H53N2O5S 18:0 C29H57N2O5S
Table 2 Amounts of testosterone esters formed in digestive gland microsomal fractions co-incubated with 100 µM palmitoyl-CoA (n = 12) or 1 mM CoA and 2 mM ATP (n = 10). Values are pmol/h/mg protein. Results are expressed as mean ± SEM. Minimum and maximum values are shown in parenthesis.
Palmitoyl-CoA CoA + ATP
T-16:0
T-16:1
T-20:5
T-22:6
24.8 ± 5.3 (7–72) 42.6 ± 12.0 (n.d.–76)
– 32.8 ± 8.8 (n.d.–58)
– 15.5 ± 4.8 (0.3–47)
– 23.2 ± 3.0 (n.d.–33)
homogenized in ice-cold 100 mM phosphate buffer pH 7.4, containing 150 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 0.1 mM phenylmetylsulfonyl fluoride (PMSF). Homogenates were centrifuged at 500-g for 15 min at 4 °C. The supernatant was collected and centrifuged at 12,000-g for 45 min and further ultracentrifuged at 100,000-g for 90 min. The resulting pellet was resuspended in homogenization buffer and further centrifuged at 100,000-g for 30 min. Microsomal pellets were then resuspended in a small volume of 100 mM phosphate buffer pH 7.4, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and 20% w/v glycerol. Protein concentrations were determined by the method of Bradford [14], using bovine serum albumin as a standard.
Similarly, Janer et al. [10] reported a 60–85% decrease in esterified testosterone in females of Marisa cornuarietis exposed to TBT; however, the decrease could not be directly linked with a decrease in microsomal acyl-CoA:testosterone acyltransferase (ATAT) activity, the enzyme that catalyzes the conjugation of steroids with different fatty acids. Recent advances in analytical techniques have allowed the sensitive analysis of a wide range of lipids, including lipoidal derivatives of steroids, by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) [11,12]. Different mass spectrometers, including accurate mass Time-of-Flight (TOF) and Orbitrap have been evaluated for the analysis of short chain steroid esters (2 to 11 carbons) [13]. The use of these analyzers would be the method of choice for the detection of target analytes. For a non-target analysis, a mass resolving power high enough to separate analyte ions from isobaric co-eluting ions is necessary to reduce misidentification. This study investigates the use of ultra-high performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS) to assess the formation of fatty acid esters of testosterone in digestive gland microsomal fractions of the mussel Mytilus galloprovincialis incubated with either palmitoly-CoA or CoA and ATP. The study also assess whether the endocrine disruptor tributyltin (TBT) interferes with the esterification of testosterone.
2.3. Enzyme assays Digestive gland microsomal proteins (400–500 µg) were incubated in 0.1 M sodium acetate buffer pH 6.0 with 2 μM testosterone, 100 μM palmitoyl-CoA and 5 mM MgCl2 in a final volume of 500 μL. Endogenous esterification was assayed in the presence of 1 mM CoA and 2 mM ATP instead of 100 μM palmitoyl-CoA. Reactions were initiated by the addition of palmitoyl-CoA or CoA, and stopped after 90 min incubation at 30 °C by the addition of 1 mL of ethyl acetate. Blank reactions, which consisted of microsomal fractions incubated in the absence of co-factors and/or testosterone, were included in every run. The interaction of TBT with the esterification reaction was investigated by incubating the microsomal fraction with testosterone, the corresponding co-factors and 100 µM TBT. TBT was delivered in absolute methanol and the solvent removed from the assay by evaporation under a gentle nitrogen stream, prior to the addition of microsomes.
2. Experimental 2.1. Reagents Palmitoyl-CoA lithium salt, coenzyme A trilithium salt, adenosine 5′-triphosphate disodium salt hydrate, tributyltin chloride and testosterone were purchased from Sigma Aldrich (Steinheim, Germany). All solvents were of analytical grade from Merck (Darmstadt, Germany). Testosterone laurate (T-12:0) and undecanoate (T-11:0) were from Steraloids (Wilton, NH, USA).
2.4. Analysis of testosterone esters
2.2. Tissue preparation
Testosterone esters were extracted with 1 mL of ethyl acetate (x3), the extracts collected and evaporated to dryness under a gentle nitrogen stream and reconstituted in methanol. Separation and identification of the esterified metabolites was achieved in an Acquity UPLC system (Waters, USA) connected to a Time-of-Flight Detector (LCT Premier XE) with an Acquity UPLC BEH C8 column (1.7 μm particle size, 100 mm × 2.1 mm, Waters, Ireland) at a flow rate of 0.3 mL/min and column temperature of 30 °C. The mobile phases were (A) methanol
Mussels, Mytilus galloprovincialis (3 to 5 cm) were collected from the bivalve farms located in the Ebro Delta (NE Spain) at different times of the year. Mussels were carried to the laboratory, the digestive glands immediately dissected, frozen in liquid nitrogen and stored at −80 °C. Subcellular fractions were prepared as described in Fernandes et al. [6]. Digestive glands (each sample a pool of 4 digestive glands) were 68
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A
T-16:0 16:0 pantetheine thioester
B
16:0 pantetheine thioester
C
Fig. 1. Reconstructed ion chromatograms corresponding to the theoretical m/z of the protonated molecule of testosterone palmitate (T-16:0) (m/z 527.4459) and 16:0 pantetheine thioester (m/z 517.3679) synthetized by digestive gland microsomal fractions incubated with (A) testostosterone and palmitoyl-CoA, (B) palmitoyl-CoA, (C) testosterone. B & C correspond to blank reactions.
10 ppm and its relative retention time. They were annotated as total fatty acyl chain length:total number of double bonds (e.g. testosterone palmitate was annotated as T-16:0). Addition of internal standards (T11:0; T-12:0) to individual samples at concentrations similar to the analytes was used for the assessment of mass tolerance windows and to perform the semi-quantitative analysis of the detected testosterone esters. These compounds were used since longer chain testosterone esters were not commercially available.
with 1 mM ammonium formate and 0.2% formic acid, and (B) water with 2 mM ammonium formate and 0.2% formic acid. Gradient elution started at 80% of A, increased to 90% A in 3 min, held for 3 min, increased to 99% A in 9 min and held for 3 min. Initial conditions were attained in 2 min and the system was stabilized for 3 min. A positive ESI interface was used to detect the compounds in a full scan acquisition mode. The mass resolving power of the TOF-MS (determined from the [M + H]+ ion of leucine at m/z 556.2771) was 10,000 FWHM (full width at half maximum). The theoretical exact mass of the putative testosterone esters (2 – 24 carbon chain, 0 to 6 double bonds) were determined using a spectrum simulation tool of MassLynx 4.1 software, and the obtained list was used as a reference database. Individual chromatographic peaks were isolated from full-scan MS spectra when selecting their theoretical exact mass. Then, a list of possible candidates fitting the specific exact mass was generated using formula determination tools (elemental composition search) of MassLynx software. The elemental number was restricted to include C, H and O. Testosterone esters were identified by accurate mass measurement with an error <
3. Results Testosterone esters were detected as [M + H]+ adducts and separated from other lipids by normal phase chromatography using ammonium acetate as an ion source to improve ionization efficiency. LC separation provided good selectivity for testosterone esters that eluted between 5 and 7 min, in an area of the chromatogram with low presence of other lipoidal compounds. This enhanced MS capabilities for an adequate confirmation of steroid esters in the microsomal matrix. 69
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TOF of several testosterone–fatty acid conjugates formed in microsomal fractions isolated from the digestive gland of the mussel Mytilus galloprovincialis. Usually, the identification of the steroid esters is hampered by the fact that they are present at low concentrations, they co-extract with highly abundant neutral lipids and have low volatility and poor ionization, which precludes their analysis by GC- or LCcoupled to MS. By using HPLC coupled to radiometric detector, Janer et al. [7] reported the formation of several estradiol esters by digestive gland microsomal fractions of the Eastern oyster Crassostrea virginica incubated with CoA and ATP. The esters were tentatively identified as C16:0, C16:1, C18:0, C18:1 and C18:2 estradiol esters by their retention time in the radio-HPLC system and by comparison with synthetized standards. The authors also reported the formation of other esters that were suggested to be polyunsaturated fatty acid esters, but couldn’t be identified. In the present work, the analysis of digestive gland microsomal fraction of mussels by UPLC-TOF has allowed the detection of minor amounts of T-22:6 and T-20:5 esters in some microsomal preparations, and the formation of T-16:0; T-16:1; T-20:5 and T-22:6 esters after activation of endogenous fatty acids with ATP and CoA. The similar fatty acid composition of the steroid esters (DHEA, estradiol, testosterone) reported in oysters and mussels supports the hypothesis of a single isoenzyme catalysing the conjugation reaction of different steroids with endogenous activated fatty acids, with no preference for specific fatty acid acyl-CoA substrates. Labadie et al. [8] reported the detection of 16:0, 16:1 and 16:2 estradiol conjugates by GC-MS/MS after exposure of Mytilus edulis to 10 ng/L estradiol for 13 days. This study did not detect the formation of longer chain fatty acid esters of estradiol, and the authors suggested a selective esterification of estradiol that was not dependent on the most abundant fatty acids. However, this contrast with our results, as the fatty acid esters of testosterone found in the present study (T-16:0; T16:1; T-20:5 and T-22:6) have been reported to be major fatty acids in mussels. Namely, palmitic acid (16:0), palmitoleic acid (16:1), eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) represented up to 19, 7, 25, and 15%, respectively, of the fatty acid composition of total lipids detected in microsomal membranes of the bivalve M. galloprovincialis [15]. Similarly, the fatty acids 16:0, 18:0, 22:6 and 20:5 were the most abundant in the digestive gland microsomal fractions from the present study; they represented 28, 12, 16 and 9% of the detected fatty acids (unpublished results). Interestingly, acyl-CoA acyltransferases catalyse the esterification of steroids with fatty acids, but other compounds, such as okadaic acid and related toxins in molluscs, and ecdysteroids in arthropods are also esterified as a part of their excretion mechanism [16,17]. Compounds that alter esterification or ester cleavage will subsequently alter their inactivation and change the availability of free (non esterified) steroids or toxins. In fact, esterification of testosterone has already been considered as a possible site of action for the endocrine disrupter TBT [18]. However, our results indicate no direct effect of TBT on ATAT activity in mussels, as previously reported for the gastropod Marisa cornuarietis [19]. These results contrast with the obtained for other species, where a strong inhibitory effect of TBT on ATAT activity was reported. Thus, the rate of conversion of [14C]testosterone to T-16:0 in digestive gland S-9 fractions of the zebra mussel Dreissena polymorpha was significantly inhibited by 10 µM TBT (27 ± 10%) and the percentage of inhibition increased up to 93 ± 7% in the presence of 100 μM TBT (Loi et al., unpublished results). Similarly, TBT acted as a competitive inhibitor of the activity ATAT (Ki = ∼9 µM) in the gastropod Ilyanassa obsoleta, supporting the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting the regulatory process for maintaining free testosterone homeostasis [20]. Interestingly, the UPLC/TOFMS system allowed the detection of two other molecules, which were tentatively identified as C16:0 and C18:0 acyl-S-pantetheine. Trams et al. [21] reported for the first time that a substantial portion of palmityl-14C coenzyme A was converted to Spalmityl-pantetheine possibly by the action of a nucleotide pyropho-
Fig. 2. Effect of 100 µM TBT on the synthesis of fatty acid testosterone conjugates in digestive gland microsomal fractions of the Mytilus gallopronviancialis incubated in the presence of palmitoyl-CoA (black bar) or CoA + ATP (blue bars). Values are mean ± SEM (n = 3).
The molecular formula, accurate mass, error and retention time of the internal standards and testosterone esters detected are shown in Table 1. The analysis of digestive gland microsomes allowed the detection of two lipoidal derivatives of testosterone with 20 and 22 carbons and 5 and 6 double bonds that were tentatively identified as docosahexaenoic DHA-testosterone ester (T-22:6) and eicosapentaenoic EPA-testosterone ester (T-20:5). Relative quantification was done by comparison of peak areas in extracted ion chromatograms with those of the internal standard (T-12:0). T-22:6 was detected at concentrations ranging from n.d. to 152 pmol/mg protein, while T-20:5 ranged from n.d. to 92 pmol/mg protein. When digestive gland microsomal fractions were incubated with palmitoyl-CoA, the synthesis of testosterone palmitate (T-16:0) at concentrations ranging from 7 to 72 pmol/h/mg protein (n = 12) was detected (Table 2). Reconstructed ion chromatograms are shown in Fig. 1. T-16:0 was only detected when both, testosterone and palmitoylCoA, were added to the incubation mixture (Fig 1A). No T-16:0 was synthetized in the absence of testosterone (Fig. 1B) or palmitoyl-CoA (Fig. 1C). Moreover, the formation of another metabolite tentatively identified as C16:0 pantetheine thioester was observed (Fig. 1, Table 1). This metabolite was only detected in those incubations containing palmitoyl-CoA and the synthesis was not dependent on the presence of testosterone (Fig. 1A & B). The analysis of palmitoyl-CoA by UPLC/TOF excluded the possibility that C16:0 pantetheine thioester was an impurity of palmitoyl-CoA or an artefact of the analytical process. When digestive gland microsomal fractions were incubated with CoA and ATP, the synthesis of four testosterone esters with saturated, monounsaturated and polyunsaturated endogenous fatty acids was observed, namely: T-16:0 (n.d. to 76 pmol/h/mg protein); T-16:1 (n.d. to 58 pmol/h/mg protein); T-20:5 (0.3 to 47 pmol/h/mg protein) and T-22:6 (n.d. to 33 pmol/h/mg protein) (Table 2). Those metabolites were not synthetized in the absence of testosterone. Moreover, the synthesis of C16:0 pantetheine thioester and a second metabolite, tentatively identified as stearyl (C18:0) pantetheine thioester was also observed. The presence of 100 µM TBT in the palmitoyl-CoA/CoA + ATP incubation mixture did not significantly alter the esterification of testosterone (Fig. 2). 4. Discussion In this study, we report for the first time the identification by UPLC70
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Fig. 3. Reconstructed ion chromatograms corresponding to the theoretical m/z of the NH4+ adduct of DHT-palmitate (DHT-16:0) (m/z 546.4886) and 16:0 5α-androstane-3β,17β-diol ester (m/z 548.5043) detected in digestive gland microsomal fractions. Mass difference with theoretical mass given in ppm.
diol esters (i.e. C16:1, C18:0, C18:1, C20:5, C22:6) was detected. The finding of these esters support the observations by Schwarz et al. [26] that testosterone undergoes extensive metabolism in mussels and testosterone esters are minor components in comparison to DHT and 5α-androstane-3β,17β-diol esters. Nonetheless, due to the absence of standards for these compounds and the insufficient mass resolution of the instrument to resolve isobaric ions (e.g. DHT-16:0 from 16:1 5αandrostane-3β,17β-diol ester), the identification is tentative [27]. Overall, this work describes the synthesis of testosterone esters (T16:0; T-16:1; T-20:5 and T-22:6) in the digestive gland microsomal fraction of mussels as well as the formation of two acyl-S-pantetheine derivatives (C16:0, C18:0) by using UPLC combined with full scan accurate mass TOF analysis. The formation of testosterone esters in the digestive gland of mussels depends largely on the availability of activated fatty acids for the esterification reaction and it was not modulated by TBT. Although the technique has proved to useful for the non-target analysis of fatty acid testosterone conjugates, the very low concentration of these compounds in a complex lipidic matrix makes necessary the use of a more selective sample extraction procedure, but also a higher resolving and the optimization of existing approaches, like the ability to model MS/MS spectra, to improve detection limits and accuracy. Future advances in this area will hopefully contribute to discover the subcellular and tissue distribution (digestive gland, gonads) of specific steroid esters in molluscs as well as to understand the role of esterification as an excretion or inactivation/storage mechanism of different types of toxins and xenobiotics.
sphatase or a phosphatase upon the acyl-CoA. As coenzyme A plays a key role in the metabolism of carboxylic acids, including short- and long-chain fatty acids, the concentration of fatty acid CoA thioesters should be tightly controlled. Moreover, some of these fatty acid CoA thioesters show high biological activity and actively participate in the regulation of cell function through regulation of gene expression, the formation of signalling molecules and modulation of ion channel activities [22–24]. It has been described that an excess of 16:0-CoA or other saturated fatty acids could harm cell viability and induce apoptosis [24]. Thus, the formation of C16:0 pantetheine thioester (palmitoyl-CoA incubations) and C:16 and C:18 pantetheine thioester (CoA + ATP incubations) appears as a specific inactivation mechanism of these two activated fatty acids, as it has been shown that acyl-Spantetheines do not act as an acyl donor in the acylation of sn-glycerol3-phosphate, 1,2-diacylglycerol, or lysolecithin [25]. Despite these findings, the analytical approach has some major limitations. Due to the lack of analytical standards, it is not possible to prove whether a compound present in a sample will be identified in the chromatogram, as it could get lost during any step of the analytical procedure or might not be ionized as anticipated. For the analysis of testosterone esters, the use of T-11:0 and T-12:0 standards has been useful to know the ionization pattern, to assess mass tolerance windows and to perform a semi-quantitative analysis of the longer chain testosterone esters. Moreover, the low matrix background in the area of the chromatogram where testosterone esters occur (retention times between 5 and 7 min) has increased the chance to resolve the suspected ions from the matrix. Nonetheless, the presence and synthesis of other testosterone esters than the ones reported in this study cannot be discarded as these esters are minor components within the extracted microsomal lipids and for low concentration esters, it might be difficult to distinguish their molecular ions from background ions. Method improvement, including selective extraction and derivatization prior to tandem mass spectrometry analysis, will hopefully increase the sensitivity of the analysis [12]. Regarding advantages, this method enables retrospective re-analysis of the acquired datafiles in case a further analyte needs to be screened. Recently, Schwarz et al. [26] reported that when mussels were exposed to radioactive testosterone in vivo, most of the testosterone (ca. 90%) in the ester fraction was present in the form of 5α-dihydrotestosterone (DHT) and 5α-androstane-3β,17β-diol (3β-diol), while T was a minor component. Fig. 3 shows the reconstructed ion chromatogram corresponding to the theoretical m/z of the NH4+ adduct of DHT palmitate (m/z 546.4886) and 5α-androstane-3β,17β-diol-palmitate (m/z 548.5043) that eluted at 6.02 and 6.61 min, respectively, in a typical digestive gland microsomal sample. These esters were much more abundant (up to 20-fold) than testosterones esters (i.e. T-20:5, T-22:6). No evidence of the presence of other DHT and 5α-androstane-3β,17β-
5. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the Spanish National Plan for Research (Project Ref. CGL2014-52144-P). CG acknowledges a grant from La Sapienza, University of Rome. Biology Scholarship. Specialization Activities Abroad. References [1] W. Borg, C.H. Shackleton, S.L. Pahuja, R.B. Hochberg, Long-lived testosterone esters in the rat, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 1545–1549. [2] M.P. Gooding, G.A. LeBlanc, Biotransformation and disposition of testosterone in the eastern mud snail Ilyanassa obsoleta, Gen. Comp. Endocrinol. 122 (2001) 172–180. [3] R.B. Hochberg, Biological esterification of steroids, Endocr. Rev. 19 (1998) 331–348. [4] M.P. Gooding, G.A. LeBlanc, Seasonal variation in the regulation of testosterone
71
Steroids 123 (2017) 67–72
C. Guercia et al.
[5]
[6]
[7] [8]
[9]
[10]
[11]
[12] [13]
[14]
[15]
levels in the eastern mud snail (Ilyanassa obsoleta), Invertebr. Biol. 123 (2004) 237–243. G. Janer, R. Lavado, R. Thibaut, C. Porte, Effects of 17beta-estradiol exposure in the mussel Mytilus galloprovincialis: a possible regulatory role for steroid acyltransferases, Aquat. Toxicol. 75 (2005) 32–42. D. Fernandes, J.C. Navarro, C. Riva, S. Bordonali, C. Porte, Does exposure to testosterone significantly alter endogenous metabolism in the marine mussel Mytilus galloprovinciallis? Aquat. Toxicol. 100 (2010) 313–320. G. Janer, S. Mesia-Vela, C. Porte, F.C. Kaufman, Esterification of vertebrate-type steroids in the Eastern oyster (Crassostrea virginica), Steroids 69 (2004) 129–136. P. Labadie, M.R. Peck, C. Minier, E.M. Hill, Identification of the steroid fatty acid ester conjugates formed in vivo in Mytilus edulis as a result of exposure to estrogens, Steroids 72 (2007) 41–49. M.P. Gooding, V.S. Wilson, L.C. Folmar, D.T. Marcovich, G.A. LeBlanc, The biocide tributyltin reduces the accumulation of testosterone as fatty acid esters in the mud snail (Ilyanassa obsoleta), Environ. Health Perspect. 111 (2003) 426–430. G. Janer, A. Lyssimachou, J. Bachmann, J. Oehlmann, U. Schulte-Oehlmann, C. Porte, Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: the effect of xenoandrogenic compounds, Steroids 71 (2006) 435–444. M.W.F. Nielen, J.P.C. Vissers, R.E.M. Fuchs, J.W. van Velde, A. Lommen, Screening for anabolic steroids and related compounds in illegal cocktails by liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/ quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement, Rapid Commun. Mass Spectrom. 15 (2001) 1577–1585. G. Forsdahl, H.K. Vatne, T. Geisendorfer, G. Gmeiner, Screening of testosterone esters in human plasma, Drug Test. Anal. 5 (2013) 826–833. E. van der Heeft, Y.J.C. Bolck, B. Beumer, A.W.J.M. Nijrolder, A.A.M. Stolker, M.W.F. Nielen, Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis, J. Am. Soc. Mass Spectrom. 20 (2009) 451–463. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. V. Ventrella, M. Pirini, A. Pagliarani, F. Trombetti, M.P. Manuzzi, A.R. Borgatti,
[16] [17] [18]
[19]
[20]
[21]
[22]
[23] [24] [25] [26]
[27]
72
Effect of temporal and geographical factors on fatty acid composition of M. galloprovincialis from the Adriatic sea, Comp. Biochem. Physiol. 149B (2008) 241–250. A.J. Slinger, R.E. Isaac, Synthesis of apolar ecdysone esters by ovaries of the cockroach Periplaneta americana, Gen. Comp. Endocrinol. 70 (1988) 74–82. A.E. Rossignoli, D. Fernández, J. Regueiro, C. Mariño, J. Blanco, Esterification of okadaic acid in the mussel Mytilus galloprovincialis, Toxicon 57 (2011) 712–720. G.A. Leblanc, M.P. Gooding, R.M. Sternberg, Testosterone-Fatty Acid esterification: a unique target for the endocrine toxicity of tributyltin to gastropods, Integr. Comp. Biol. 45 (2005) 81–87. G. Janer, R.M. Sternberg, G.A. LeBlanc, C. Porte, Testosterone conjugating activities in invertebrates: are they targets for endocrine disruptors? Aquat. Toxicol. 71 (2005) 273–282. R.M. Sternberg, G.A. LeBlanc, Kinetic characterization of the inhibition of acyl coenzyme A: steroid acyltransferases by tributyltin in the eastern mud snail (Ilyanassa obsoleta), Aquat. Toxicol. 78 (2006) 233–242. E.G. Trams, H.A. Fales, A.E. Gal, S-palmityl pantetheine as an intermediate in the metabolism of palmityl Coenzyme A by rat liver plasma membrane preparations, Biochem. Biophys. Res. Comm. 3 (1968) 973–976. P.N. Black, N.J. Faergeman, C.C. DiRusso, Long-chain acyl-CoA-dependent regulation of gene expression in bacteria, yeast and mammals, J. Nutr. 130 (2000) 305S–309S. R. Leonardi, Y.-M. Zhang, C.O. Rock, S. Jackowski, Coenzyme A: back in action, Prog. Lip. Res. 44 (2005) 125–153. L.O. Li, E.L. Klett, R.A. Coleman, Acyl-CoA synthesis, lipid metabolism and lipotoxicity, Biochim. Biophys. Acta 2010 (1801) 246–251. M. Sribney, J.L. Dove, Synthesis of acyi-S-pantetheine by rat liver microsomes, Lipids 13 (1978) 422–426. T.I. Schwarz, I. Katsiadaki, B.H. Maskrey, A.P. Scott, Rapid uptake, biotransformation, esterification and lack of depuration of testosterone and its metabolites by the common mussel, Mytilus spp. J. Steroid Biochem. Mol. Biol. (2017), http://dx. doi.org/10.1016/j.jsbmb.2017.02.016. M. Krauss, H. Singer, J. Hollender, LC-high resolution MS in environmental analysis: from target screening to the identification of unknowns, Anal. Bional. Chem. 397 (2010) 943–951.
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Steroids journal homepage: www.elsevier.com/locate/steroids
Analysis of testosterone fatty acid esters in the digestive gland of mussels by liquid chromatography-high resolution mass spectrometry Cesare Guercia, Piergiorgio Cianciullo, Cinta Porte
MARK
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Department of Environmental Chemistry, IDAEA-CSIC, Barcelona, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Acyl-CoA:testosterone acyltransferase Testosterone esters Mussels UPLC-HRMS Pantetheine thioesters
Several studies have indicated that up to 70% of the total steroids detected in molluscs are in the esterified form and that pollutants, by modifying the esterification of steroids with fatty acids, might act as endocrine disrupters. However, despite the strong physiological significance of this process, there is almost no information on which fatty acids form the steroid esters and how this process is modulated. This study (a) investigates the formation of fatty acid esters of testosterone in digestive gland microsomal fractions of the mussel Mytilus galloprovincialis incubated with either palmitoly-CoA or CoA and ATP, and (b) assesses whether the endocrine disruptor tributyltin (TBT) interferes with the esterification of testosterone. Analysis of testosterone esters was performed by liquid chromatography–high resolution mass spectrometry (UPLC-HRMS). When microsomal fractions were incubated with testosterone and palmitoly-CoA, the formation of testosterone palmitate was detected. However, when microsomes were incubated with CoA and ATP, and no exogenous activated fatty acid was added, the synthesis of 16:0, 16:1, 20:5 and 22:6 testosterone esters was observed. The presence of 100 µM TBT in the incubation mixture did not significantly alter the esterification of testosterone. These results evidence the conjugation of testosterone with the most abundant fatty acids in the digestive gland microsomal fraction of mussels.
1. Introduction Fatty acid conjugation of steroids appears to be a well-conserved conjugation pathway during evolution; it is known to occur in both vertebrate and invertebrates. Fatty acid conjugation (or esterification) renders steroids to an apolar form, which is retained in the lipoidal matrices of the body, while reducing their activity, bioavailability, and susceptibility to elimination [1,2]. Steroid esters do not bind to receptors, but they can be hydrolyzed by esterases to liberate the active steroid [3]. Steroid esters have been reported in molluscs and some studies have suggested that esterification might play a key role in regulating levels of free steroids in these organisms. Thus, Gooding and LeBlanc [2] showed that the mud snail Ilyanassa obsoleta, primarily metabolized free testosterone to non-polar fatty acid ester conjugates via acyl-CoA:testosterone acyltransferase (ATAT), which is localized in the endoplasmic reticulum. The authors showed that esterification was the major biotransformation pathway for testosterone in snails, as exogenously provided testosterone was converted to fatty acid esters and retained in the organism. In addition, irrespective of the amount of testosterone administered to the snails, the amount of free testosterone measured in
⁎
Corresponding author. E-mail address: [email protected] (C. Porte).
http://dx.doi.org/10.1016/j.steroids.2017.05.008 Received 23 February 2017; Received in revised form 2 May 2017; Accepted 8 May 2017 Available online 11 May 2017 0039-128X/ © 2017 Elsevier Inc. All rights reserved.
the tissues remained relatively constant and all excess of testosterone was converted to the fatty acid ester [4]. Similarly, exogenously administered testosterone and estradiol were extensively esterified by the mussel Mytilus galloprovincialis, whereas levels of unconjugated steroids remained almost unaltered [5,6]. On the other hand, few studies have investigated which are the specific fatty acid steroid conjugates formed by molluscs. Janer et al. [7] reported the esterification of estradiol in digestive gland microsomal fractions of Crassostrea virginica with a variety of saturated or unsaturated fatty acids (C16:0; C18:0; C16:1; C18:1; C18:2 and C20:4); however, identification was tentative. Labadie et al. [8] reported for the first time the identity of three estradiol fatty acid conjugates (C16:0; C16:1 and C16:2) in Mytilus edulis by using tandem mass spectrometry with direct probe insertion. The organotin compound tributyltin (TBT) is a well-known endocrine disrupter in molluscs that it has been reported to interfere with the esterification of testosterone. Thus, Gooding et al. [9] showed that females of Ilyanassa obsoleta experimentally exposed to 10 ng/L TBT for 3 months had a lower ability to conjugate testosterone with fatty acid moieties; and females collected in an organotin-polluted site had lower levels of esterified testosterone than those collected in a clean site.
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Table 1 Testosterone esters and pantetheine thioesters detected in the digestive gland microsomal fraction of M. galloprovincialis. Testosterone undecanoate (T-11:0) and laurate (T-12:0) were used as internal standards. Fatty Acid
Molecular ion [M + H]+
Theoretical mass (Da)
Experimental mass (Da)
Mass difference (ppm)
Retention time (min)
Testosterone esters 11:0 12:0 16:0 16:1 20:5 22:6
C30H49O3 C31H51O3 C35H59O3 C35H57O3 C39H57O3 C41H59O3
457.3682 471.3838 527.4459 525.4303 573.4305 599.4464
457.3674 471.3825 527.4467 525.4308 573.4311 599.4440
2.1 2.1 1.1 2.5 2.7 2.8
4.3 4.6 6.9 5.9 5.3 5.7
517.3675 545.3988
517.3668 545.3991
4.7 6.0
3.4 3.8
Acyl-S-pantetheine thioesters 16:0 C27H53N2O5S 18:0 C29H57N2O5S
Table 2 Amounts of testosterone esters formed in digestive gland microsomal fractions co-incubated with 100 µM palmitoyl-CoA (n = 12) or 1 mM CoA and 2 mM ATP (n = 10). Values are pmol/h/mg protein. Results are expressed as mean ± SEM. Minimum and maximum values are shown in parenthesis.
Palmitoyl-CoA CoA + ATP
T-16:0
T-16:1
T-20:5
T-22:6
24.8 ± 5.3 (7–72) 42.6 ± 12.0 (n.d.–76)
– 32.8 ± 8.8 (n.d.–58)
– 15.5 ± 4.8 (0.3–47)
– 23.2 ± 3.0 (n.d.–33)
homogenized in ice-cold 100 mM phosphate buffer pH 7.4, containing 150 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 0.1 mM phenylmetylsulfonyl fluoride (PMSF). Homogenates were centrifuged at 500-g for 15 min at 4 °C. The supernatant was collected and centrifuged at 12,000-g for 45 min and further ultracentrifuged at 100,000-g for 90 min. The resulting pellet was resuspended in homogenization buffer and further centrifuged at 100,000-g for 30 min. Microsomal pellets were then resuspended in a small volume of 100 mM phosphate buffer pH 7.4, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and 20% w/v glycerol. Protein concentrations were determined by the method of Bradford [14], using bovine serum albumin as a standard.
Similarly, Janer et al. [10] reported a 60–85% decrease in esterified testosterone in females of Marisa cornuarietis exposed to TBT; however, the decrease could not be directly linked with a decrease in microsomal acyl-CoA:testosterone acyltransferase (ATAT) activity, the enzyme that catalyzes the conjugation of steroids with different fatty acids. Recent advances in analytical techniques have allowed the sensitive analysis of a wide range of lipids, including lipoidal derivatives of steroids, by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) [11,12]. Different mass spectrometers, including accurate mass Time-of-Flight (TOF) and Orbitrap have been evaluated for the analysis of short chain steroid esters (2 to 11 carbons) [13]. The use of these analyzers would be the method of choice for the detection of target analytes. For a non-target analysis, a mass resolving power high enough to separate analyte ions from isobaric co-eluting ions is necessary to reduce misidentification. This study investigates the use of ultra-high performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS) to assess the formation of fatty acid esters of testosterone in digestive gland microsomal fractions of the mussel Mytilus galloprovincialis incubated with either palmitoly-CoA or CoA and ATP. The study also assess whether the endocrine disruptor tributyltin (TBT) interferes with the esterification of testosterone.
2.3. Enzyme assays Digestive gland microsomal proteins (400–500 µg) were incubated in 0.1 M sodium acetate buffer pH 6.0 with 2 μM testosterone, 100 μM palmitoyl-CoA and 5 mM MgCl2 in a final volume of 500 μL. Endogenous esterification was assayed in the presence of 1 mM CoA and 2 mM ATP instead of 100 μM palmitoyl-CoA. Reactions were initiated by the addition of palmitoyl-CoA or CoA, and stopped after 90 min incubation at 30 °C by the addition of 1 mL of ethyl acetate. Blank reactions, which consisted of microsomal fractions incubated in the absence of co-factors and/or testosterone, were included in every run. The interaction of TBT with the esterification reaction was investigated by incubating the microsomal fraction with testosterone, the corresponding co-factors and 100 µM TBT. TBT was delivered in absolute methanol and the solvent removed from the assay by evaporation under a gentle nitrogen stream, prior to the addition of microsomes.
2. Experimental 2.1. Reagents Palmitoyl-CoA lithium salt, coenzyme A trilithium salt, adenosine 5′-triphosphate disodium salt hydrate, tributyltin chloride and testosterone were purchased from Sigma Aldrich (Steinheim, Germany). All solvents were of analytical grade from Merck (Darmstadt, Germany). Testosterone laurate (T-12:0) and undecanoate (T-11:0) were from Steraloids (Wilton, NH, USA).
2.4. Analysis of testosterone esters
2.2. Tissue preparation
Testosterone esters were extracted with 1 mL of ethyl acetate (x3), the extracts collected and evaporated to dryness under a gentle nitrogen stream and reconstituted in methanol. Separation and identification of the esterified metabolites was achieved in an Acquity UPLC system (Waters, USA) connected to a Time-of-Flight Detector (LCT Premier XE) with an Acquity UPLC BEH C8 column (1.7 μm particle size, 100 mm × 2.1 mm, Waters, Ireland) at a flow rate of 0.3 mL/min and column temperature of 30 °C. The mobile phases were (A) methanol
Mussels, Mytilus galloprovincialis (3 to 5 cm) were collected from the bivalve farms located in the Ebro Delta (NE Spain) at different times of the year. Mussels were carried to the laboratory, the digestive glands immediately dissected, frozen in liquid nitrogen and stored at −80 °C. Subcellular fractions were prepared as described in Fernandes et al. [6]. Digestive glands (each sample a pool of 4 digestive glands) were 68
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A
T-16:0 16:0 pantetheine thioester
B
16:0 pantetheine thioester
C
Fig. 1. Reconstructed ion chromatograms corresponding to the theoretical m/z of the protonated molecule of testosterone palmitate (T-16:0) (m/z 527.4459) and 16:0 pantetheine thioester (m/z 517.3679) synthetized by digestive gland microsomal fractions incubated with (A) testostosterone and palmitoyl-CoA, (B) palmitoyl-CoA, (C) testosterone. B & C correspond to blank reactions.
10 ppm and its relative retention time. They were annotated as total fatty acyl chain length:total number of double bonds (e.g. testosterone palmitate was annotated as T-16:0). Addition of internal standards (T11:0; T-12:0) to individual samples at concentrations similar to the analytes was used for the assessment of mass tolerance windows and to perform the semi-quantitative analysis of the detected testosterone esters. These compounds were used since longer chain testosterone esters were not commercially available.
with 1 mM ammonium formate and 0.2% formic acid, and (B) water with 2 mM ammonium formate and 0.2% formic acid. Gradient elution started at 80% of A, increased to 90% A in 3 min, held for 3 min, increased to 99% A in 9 min and held for 3 min. Initial conditions were attained in 2 min and the system was stabilized for 3 min. A positive ESI interface was used to detect the compounds in a full scan acquisition mode. The mass resolving power of the TOF-MS (determined from the [M + H]+ ion of leucine at m/z 556.2771) was 10,000 FWHM (full width at half maximum). The theoretical exact mass of the putative testosterone esters (2 – 24 carbon chain, 0 to 6 double bonds) were determined using a spectrum simulation tool of MassLynx 4.1 software, and the obtained list was used as a reference database. Individual chromatographic peaks were isolated from full-scan MS spectra when selecting their theoretical exact mass. Then, a list of possible candidates fitting the specific exact mass was generated using formula determination tools (elemental composition search) of MassLynx software. The elemental number was restricted to include C, H and O. Testosterone esters were identified by accurate mass measurement with an error <
3. Results Testosterone esters were detected as [M + H]+ adducts and separated from other lipids by normal phase chromatography using ammonium acetate as an ion source to improve ionization efficiency. LC separation provided good selectivity for testosterone esters that eluted between 5 and 7 min, in an area of the chromatogram with low presence of other lipoidal compounds. This enhanced MS capabilities for an adequate confirmation of steroid esters in the microsomal matrix. 69
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TOF of several testosterone–fatty acid conjugates formed in microsomal fractions isolated from the digestive gland of the mussel Mytilus galloprovincialis. Usually, the identification of the steroid esters is hampered by the fact that they are present at low concentrations, they co-extract with highly abundant neutral lipids and have low volatility and poor ionization, which precludes their analysis by GC- or LCcoupled to MS. By using HPLC coupled to radiometric detector, Janer et al. [7] reported the formation of several estradiol esters by digestive gland microsomal fractions of the Eastern oyster Crassostrea virginica incubated with CoA and ATP. The esters were tentatively identified as C16:0, C16:1, C18:0, C18:1 and C18:2 estradiol esters by their retention time in the radio-HPLC system and by comparison with synthetized standards. The authors also reported the formation of other esters that were suggested to be polyunsaturated fatty acid esters, but couldn’t be identified. In the present work, the analysis of digestive gland microsomal fraction of mussels by UPLC-TOF has allowed the detection of minor amounts of T-22:6 and T-20:5 esters in some microsomal preparations, and the formation of T-16:0; T-16:1; T-20:5 and T-22:6 esters after activation of endogenous fatty acids with ATP and CoA. The similar fatty acid composition of the steroid esters (DHEA, estradiol, testosterone) reported in oysters and mussels supports the hypothesis of a single isoenzyme catalysing the conjugation reaction of different steroids with endogenous activated fatty acids, with no preference for specific fatty acid acyl-CoA substrates. Labadie et al. [8] reported the detection of 16:0, 16:1 and 16:2 estradiol conjugates by GC-MS/MS after exposure of Mytilus edulis to 10 ng/L estradiol for 13 days. This study did not detect the formation of longer chain fatty acid esters of estradiol, and the authors suggested a selective esterification of estradiol that was not dependent on the most abundant fatty acids. However, this contrast with our results, as the fatty acid esters of testosterone found in the present study (T-16:0; T16:1; T-20:5 and T-22:6) have been reported to be major fatty acids in mussels. Namely, palmitic acid (16:0), palmitoleic acid (16:1), eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) represented up to 19, 7, 25, and 15%, respectively, of the fatty acid composition of total lipids detected in microsomal membranes of the bivalve M. galloprovincialis [15]. Similarly, the fatty acids 16:0, 18:0, 22:6 and 20:5 were the most abundant in the digestive gland microsomal fractions from the present study; they represented 28, 12, 16 and 9% of the detected fatty acids (unpublished results). Interestingly, acyl-CoA acyltransferases catalyse the esterification of steroids with fatty acids, but other compounds, such as okadaic acid and related toxins in molluscs, and ecdysteroids in arthropods are also esterified as a part of their excretion mechanism [16,17]. Compounds that alter esterification or ester cleavage will subsequently alter their inactivation and change the availability of free (non esterified) steroids or toxins. In fact, esterification of testosterone has already been considered as a possible site of action for the endocrine disrupter TBT [18]. However, our results indicate no direct effect of TBT on ATAT activity in mussels, as previously reported for the gastropod Marisa cornuarietis [19]. These results contrast with the obtained for other species, where a strong inhibitory effect of TBT on ATAT activity was reported. Thus, the rate of conversion of [14C]testosterone to T-16:0 in digestive gland S-9 fractions of the zebra mussel Dreissena polymorpha was significantly inhibited by 10 µM TBT (27 ± 10%) and the percentage of inhibition increased up to 93 ± 7% in the presence of 100 μM TBT (Loi et al., unpublished results). Similarly, TBT acted as a competitive inhibitor of the activity ATAT (Ki = ∼9 µM) in the gastropod Ilyanassa obsoleta, supporting the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting the regulatory process for maintaining free testosterone homeostasis [20]. Interestingly, the UPLC/TOFMS system allowed the detection of two other molecules, which were tentatively identified as C16:0 and C18:0 acyl-S-pantetheine. Trams et al. [21] reported for the first time that a substantial portion of palmityl-14C coenzyme A was converted to Spalmityl-pantetheine possibly by the action of a nucleotide pyropho-
Fig. 2. Effect of 100 µM TBT on the synthesis of fatty acid testosterone conjugates in digestive gland microsomal fractions of the Mytilus gallopronviancialis incubated in the presence of palmitoyl-CoA (black bar) or CoA + ATP (blue bars). Values are mean ± SEM (n = 3).
The molecular formula, accurate mass, error and retention time of the internal standards and testosterone esters detected are shown in Table 1. The analysis of digestive gland microsomes allowed the detection of two lipoidal derivatives of testosterone with 20 and 22 carbons and 5 and 6 double bonds that were tentatively identified as docosahexaenoic DHA-testosterone ester (T-22:6) and eicosapentaenoic EPA-testosterone ester (T-20:5). Relative quantification was done by comparison of peak areas in extracted ion chromatograms with those of the internal standard (T-12:0). T-22:6 was detected at concentrations ranging from n.d. to 152 pmol/mg protein, while T-20:5 ranged from n.d. to 92 pmol/mg protein. When digestive gland microsomal fractions were incubated with palmitoyl-CoA, the synthesis of testosterone palmitate (T-16:0) at concentrations ranging from 7 to 72 pmol/h/mg protein (n = 12) was detected (Table 2). Reconstructed ion chromatograms are shown in Fig. 1. T-16:0 was only detected when both, testosterone and palmitoylCoA, were added to the incubation mixture (Fig 1A). No T-16:0 was synthetized in the absence of testosterone (Fig. 1B) or palmitoyl-CoA (Fig. 1C). Moreover, the formation of another metabolite tentatively identified as C16:0 pantetheine thioester was observed (Fig. 1, Table 1). This metabolite was only detected in those incubations containing palmitoyl-CoA and the synthesis was not dependent on the presence of testosterone (Fig. 1A & B). The analysis of palmitoyl-CoA by UPLC/TOF excluded the possibility that C16:0 pantetheine thioester was an impurity of palmitoyl-CoA or an artefact of the analytical process. When digestive gland microsomal fractions were incubated with CoA and ATP, the synthesis of four testosterone esters with saturated, monounsaturated and polyunsaturated endogenous fatty acids was observed, namely: T-16:0 (n.d. to 76 pmol/h/mg protein); T-16:1 (n.d. to 58 pmol/h/mg protein); T-20:5 (0.3 to 47 pmol/h/mg protein) and T-22:6 (n.d. to 33 pmol/h/mg protein) (Table 2). Those metabolites were not synthetized in the absence of testosterone. Moreover, the synthesis of C16:0 pantetheine thioester and a second metabolite, tentatively identified as stearyl (C18:0) pantetheine thioester was also observed. The presence of 100 µM TBT in the palmitoyl-CoA/CoA + ATP incubation mixture did not significantly alter the esterification of testosterone (Fig. 2). 4. Discussion In this study, we report for the first time the identification by UPLC70
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Fig. 3. Reconstructed ion chromatograms corresponding to the theoretical m/z of the NH4+ adduct of DHT-palmitate (DHT-16:0) (m/z 546.4886) and 16:0 5α-androstane-3β,17β-diol ester (m/z 548.5043) detected in digestive gland microsomal fractions. Mass difference with theoretical mass given in ppm.
diol esters (i.e. C16:1, C18:0, C18:1, C20:5, C22:6) was detected. The finding of these esters support the observations by Schwarz et al. [26] that testosterone undergoes extensive metabolism in mussels and testosterone esters are minor components in comparison to DHT and 5α-androstane-3β,17β-diol esters. Nonetheless, due to the absence of standards for these compounds and the insufficient mass resolution of the instrument to resolve isobaric ions (e.g. DHT-16:0 from 16:1 5αandrostane-3β,17β-diol ester), the identification is tentative [27]. Overall, this work describes the synthesis of testosterone esters (T16:0; T-16:1; T-20:5 and T-22:6) in the digestive gland microsomal fraction of mussels as well as the formation of two acyl-S-pantetheine derivatives (C16:0, C18:0) by using UPLC combined with full scan accurate mass TOF analysis. The formation of testosterone esters in the digestive gland of mussels depends largely on the availability of activated fatty acids for the esterification reaction and it was not modulated by TBT. Although the technique has proved to useful for the non-target analysis of fatty acid testosterone conjugates, the very low concentration of these compounds in a complex lipidic matrix makes necessary the use of a more selective sample extraction procedure, but also a higher resolving and the optimization of existing approaches, like the ability to model MS/MS spectra, to improve detection limits and accuracy. Future advances in this area will hopefully contribute to discover the subcellular and tissue distribution (digestive gland, gonads) of specific steroid esters in molluscs as well as to understand the role of esterification as an excretion or inactivation/storage mechanism of different types of toxins and xenobiotics.
sphatase or a phosphatase upon the acyl-CoA. As coenzyme A plays a key role in the metabolism of carboxylic acids, including short- and long-chain fatty acids, the concentration of fatty acid CoA thioesters should be tightly controlled. Moreover, some of these fatty acid CoA thioesters show high biological activity and actively participate in the regulation of cell function through regulation of gene expression, the formation of signalling molecules and modulation of ion channel activities [22–24]. It has been described that an excess of 16:0-CoA or other saturated fatty acids could harm cell viability and induce apoptosis [24]. Thus, the formation of C16:0 pantetheine thioester (palmitoyl-CoA incubations) and C:16 and C:18 pantetheine thioester (CoA + ATP incubations) appears as a specific inactivation mechanism of these two activated fatty acids, as it has been shown that acyl-Spantetheines do not act as an acyl donor in the acylation of sn-glycerol3-phosphate, 1,2-diacylglycerol, or lysolecithin [25]. Despite these findings, the analytical approach has some major limitations. Due to the lack of analytical standards, it is not possible to prove whether a compound present in a sample will be identified in the chromatogram, as it could get lost during any step of the analytical procedure or might not be ionized as anticipated. For the analysis of testosterone esters, the use of T-11:0 and T-12:0 standards has been useful to know the ionization pattern, to assess mass tolerance windows and to perform a semi-quantitative analysis of the longer chain testosterone esters. Moreover, the low matrix background in the area of the chromatogram where testosterone esters occur (retention times between 5 and 7 min) has increased the chance to resolve the suspected ions from the matrix. Nonetheless, the presence and synthesis of other testosterone esters than the ones reported in this study cannot be discarded as these esters are minor components within the extracted microsomal lipids and for low concentration esters, it might be difficult to distinguish their molecular ions from background ions. Method improvement, including selective extraction and derivatization prior to tandem mass spectrometry analysis, will hopefully increase the sensitivity of the analysis [12]. Regarding advantages, this method enables retrospective re-analysis of the acquired datafiles in case a further analyte needs to be screened. Recently, Schwarz et al. [26] reported that when mussels were exposed to radioactive testosterone in vivo, most of the testosterone (ca. 90%) in the ester fraction was present in the form of 5α-dihydrotestosterone (DHT) and 5α-androstane-3β,17β-diol (3β-diol), while T was a minor component. Fig. 3 shows the reconstructed ion chromatogram corresponding to the theoretical m/z of the NH4+ adduct of DHT palmitate (m/z 546.4886) and 5α-androstane-3β,17β-diol-palmitate (m/z 548.5043) that eluted at 6.02 and 6.61 min, respectively, in a typical digestive gland microsomal sample. These esters were much more abundant (up to 20-fold) than testosterones esters (i.e. T-20:5, T-22:6). No evidence of the presence of other DHT and 5α-androstane-3β,17β-
5. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the Spanish National Plan for Research (Project Ref. CGL2014-52144-P). CG acknowledges a grant from La Sapienza, University of Rome. Biology Scholarship. Specialization Activities Abroad. References [1] W. Borg, C.H. Shackleton, S.L. Pahuja, R.B. Hochberg, Long-lived testosterone esters in the rat, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 1545–1549. [2] M.P. Gooding, G.A. LeBlanc, Biotransformation and disposition of testosterone in the eastern mud snail Ilyanassa obsoleta, Gen. Comp. Endocrinol. 122 (2001) 172–180. [3] R.B. Hochberg, Biological esterification of steroids, Endocr. Rev. 19 (1998) 331–348. [4] M.P. Gooding, G.A. LeBlanc, Seasonal variation in the regulation of testosterone
71
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[5]
[6]
[7] [8]
[9]
[10]
[11]
[12] [13]
[14]
[15]
levels in the eastern mud snail (Ilyanassa obsoleta), Invertebr. Biol. 123 (2004) 237–243. G. Janer, R. Lavado, R. Thibaut, C. Porte, Effects of 17beta-estradiol exposure in the mussel Mytilus galloprovincialis: a possible regulatory role for steroid acyltransferases, Aquat. Toxicol. 75 (2005) 32–42. D. Fernandes, J.C. Navarro, C. Riva, S. Bordonali, C. Porte, Does exposure to testosterone significantly alter endogenous metabolism in the marine mussel Mytilus galloprovinciallis? Aquat. Toxicol. 100 (2010) 313–320. G. Janer, S. Mesia-Vela, C. Porte, F.C. Kaufman, Esterification of vertebrate-type steroids in the Eastern oyster (Crassostrea virginica), Steroids 69 (2004) 129–136. P. Labadie, M.R. Peck, C. Minier, E.M. Hill, Identification of the steroid fatty acid ester conjugates formed in vivo in Mytilus edulis as a result of exposure to estrogens, Steroids 72 (2007) 41–49. M.P. Gooding, V.S. Wilson, L.C. Folmar, D.T. Marcovich, G.A. LeBlanc, The biocide tributyltin reduces the accumulation of testosterone as fatty acid esters in the mud snail (Ilyanassa obsoleta), Environ. Health Perspect. 111 (2003) 426–430. G. Janer, A. Lyssimachou, J. Bachmann, J. Oehlmann, U. Schulte-Oehlmann, C. Porte, Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: the effect of xenoandrogenic compounds, Steroids 71 (2006) 435–444. M.W.F. Nielen, J.P.C. Vissers, R.E.M. Fuchs, J.W. van Velde, A. Lommen, Screening for anabolic steroids and related compounds in illegal cocktails by liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/ quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement, Rapid Commun. Mass Spectrom. 15 (2001) 1577–1585. G. Forsdahl, H.K. Vatne, T. Geisendorfer, G. Gmeiner, Screening of testosterone esters in human plasma, Drug Test. Anal. 5 (2013) 826–833. E. van der Heeft, Y.J.C. Bolck, B. Beumer, A.W.J.M. Nijrolder, A.A.M. Stolker, M.W.F. Nielen, Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis, J. Am. Soc. Mass Spectrom. 20 (2009) 451–463. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. V. Ventrella, M. Pirini, A. Pagliarani, F. Trombetti, M.P. Manuzzi, A.R. Borgatti,
[16] [17] [18]
[19]
[20]
[21]
[22]
[23] [24] [25] [26]
[27]
72
Effect of temporal and geographical factors on fatty acid composition of M. galloprovincialis from the Adriatic sea, Comp. Biochem. Physiol. 149B (2008) 241–250. A.J. Slinger, R.E. Isaac, Synthesis of apolar ecdysone esters by ovaries of the cockroach Periplaneta americana, Gen. Comp. Endocrinol. 70 (1988) 74–82. A.E. Rossignoli, D. Fernández, J. Regueiro, C. Mariño, J. Blanco, Esterification of okadaic acid in the mussel Mytilus galloprovincialis, Toxicon 57 (2011) 712–720. G.A. Leblanc, M.P. Gooding, R.M. Sternberg, Testosterone-Fatty Acid esterification: a unique target for the endocrine toxicity of tributyltin to gastropods, Integr. Comp. Biol. 45 (2005) 81–87. G. Janer, R.M. Sternberg, G.A. LeBlanc, C. Porte, Testosterone conjugating activities in invertebrates: are they targets for endocrine disruptors? Aquat. Toxicol. 71 (2005) 273–282. R.M. Sternberg, G.A. LeBlanc, Kinetic characterization of the inhibition of acyl coenzyme A: steroid acyltransferases by tributyltin in the eastern mud snail (Ilyanassa obsoleta), Aquat. Toxicol. 78 (2006) 233–242. E.G. Trams, H.A. Fales, A.E. Gal, S-palmityl pantetheine as an intermediate in the metabolism of palmityl Coenzyme A by rat liver plasma membrane preparations, Biochem. Biophys. Res. Comm. 3 (1968) 973–976. P.N. Black, N.J. Faergeman, C.C. DiRusso, Long-chain acyl-CoA-dependent regulation of gene expression in bacteria, yeast and mammals, J. Nutr. 130 (2000) 305S–309S. R. Leonardi, Y.-M. Zhang, C.O. Rock, S. Jackowski, Coenzyme A: back in action, Prog. Lip. Res. 44 (2005) 125–153. L.O. Li, E.L. Klett, R.A. Coleman, Acyl-CoA synthesis, lipid metabolism and lipotoxicity, Biochim. Biophys. Acta 2010 (1801) 246–251. M. Sribney, J.L. Dove, Synthesis of acyi-S-pantetheine by rat liver microsomes, Lipids 13 (1978) 422–426. T.I. Schwarz, I. Katsiadaki, B.H. Maskrey, A.P. Scott, Rapid uptake, biotransformation, esterification and lack of depuration of testosterone and its metabolites by the common mussel, Mytilus spp. J. Steroid Biochem. Mol. Biol. (2017), http://dx. doi.org/10.1016/j.jsbmb.2017.02.016. M. Krauss, H. Singer, J. Hollender, LC-high resolution MS in environmental analysis: from target screening to the identification of unknowns, Anal. Bional. Chem. 397 (2010) 943–951.