Data on the uptake and metabolism of the vertebrate steroid estradiol-17β from water by the common mussel, Mytilus spp.

The data presented in this article primarily provide support for the research article entitled “Mussels (Mytilus spp.) display an ability for rapid and high capacity uptake of the vertebrate steroid, estradiol-17β from water” (T.I. Schwarz, I. Katsiadaki, B.H. Maskrey, A.P. Scott, 2016) [1]. Data are presented on the ability of mussels to absorb tritiated estradiol (E2) from water. The data indicate that most of the radioactivity remaining in the water is 1,3,5(10)-estratriene-3,17β-diol 3-sulfate (E2 3-S) and the radioactivity in the mussel tissue is mainly in the form of fatty acid esters. The latter, following saponification, were identified by ultra-high performance liquid chromatography in conjunction with tandem mass spectrometry (UHPLC-MS/MS) as intact E2. Data are included that indicate that the remaining radioactivity in the tissue is composed of E2 3-S and unidentified free metabolites. Experimental data included also relate to a) the efficiency of extraction of radioactivity from tissue, b) the efficiency of separation of free and esterified E2 using solvents and c) possible factors affecting the recovery of radioactivity. Finally, preliminary data are provided on concentrations of immunoreactive E2 in the free and ester fractions of tissue extracts from mussels caged in the field.


Data format Raw and analyzed Experimental factors
Studying the rate of uptake of tritiated E 2 by live mussels, developing a new method for separating free and esterified steroids in tissue extracts, identifying the metabolites Experimental features Measuring the rate of disappearance of tritiated E 2 from water in vessels containing mussels, extracting and then separating the metabolites in both water and tissue by liquid/liquid partition and by chromatography, definitively identifying metabolites by tandem mass spectrometry using a Waters Xevo TQ mass spectrometer Data source location The Retreat, Brancaster Staithe, Norfolk; Menai Strait, Wales; Portland Harbour, Dorset Data accessibility Data presented in this article

Value of the data
The data provide supporting evidence to challenge the assumption that E 2 found in the flesh of mollusks is of endogenous origin.
Identification of E 2 3-S in water and tissue is essential for understanding E 2 uptake and metabolism in Mytilus spp.
The development of a new and simple way to separate free and esterified E 2 from tissue extracts will help those working on the origin and role (if any) of vertebrate steroids in mollusks.

Data
The data presented in this article show the uptake of radiolabeled E 2 ([ 3 H]-E 2 ) from water by mussels under different conditions (Figs. 1-3); the production of E 2 metabolites in water (E 2 3-S; Fig. 4) and tissue extracts (E 2 3-S, Figs. 5-6; E 2 esters, Fig. 7), the identification of intact E 2 in the ester fraction of saponified tissue extracts (Fig. 8) and the concentrations of free and esterified E 2 in tissue extracts of mussels caged in the field (Fig. 9). Original data from experiments that were carried out to determine the best methods for extracting (Table 1) and then separating free and esterified E 2 ( Table 2) are also presented.

Laboratory exposures of mussels to [ 3 H]-E 2
A summary of the exposure conditions (including controls) for the experiments described below are in Table 1 of the research article [1]. All mussels (Mytilus spp.) were acclimatized prior to exposure for at least 1 day, where they were fed Shellfish Diet s 1800 (unless stated otherwise) following manufacturer's instructions and the water was changed daily. The time at which water samples (1 mL) were taken during exposure for scintillation counting are indicated in the relevant Figures.  (Fig. 1). The water was changed after 48 h and the radioactivity refreshed. After exposure, mussel soft tissue was extracted with Method 1 (Table 1) for putative identification of [ 3 H]-E 2 lipid soluble metabolites using normal phase HPLC (Fig. 7).

Experiment 2
Mussels were obtained from 'Deepdock Mussels' in the Menai Strait, Walesa catchment which also holds a long term class B shellfish harvesting classification. The animals were harvested in April 2014, transported in a cool-box overnight and immediately placed in a flow-through system of Removal of radiolabel from water by Mytilus spp. (Experiment 2) in the presence of food (▼) and low and high concentration of cold E 2 (7.1 ng L À 1 E 2 , ☐ and 35.7 ng L À 1 E 2 : △) compared with a [ 3 H]-E 2 -only treatment (O) during a 48 h exposure under the following conditionsglass tanks, 7 L seawater tank À 1 , 5 animals tank À 1 , 0.7 mCi L À 1 (1.36 ng L À 1 ) [ 3 H]-E 2 . Data are presented as mean percentage7 S.E.M. of radioactivity remaining in the water (n¼ 3 tanks). The lines represent the same data. ([ 3 H]-E 2 -only, solid; feed, dot-dash; low cold, dotted; high cold, dash) fitted to a three parameter hyperbolic decay equation. A single sorption control with radiolabel but no animals and two feed sorption controls with radiolabel and algae but no animals were included, but as there was no evidence for any losses, no adjustments were made to the data. Removal of radiolabel from water by Mytilus spp. (Experiment 1) exposed for two consecutive 48 h periods (1st¼ ○; 2nd¼ □) under the following conditionsglass tanks, 13 L seawater tank À 1 , 5 animals tank À 1 , 0.7 mCi L À 1 (1.36 ng L À 1 ) [ 3 H]-E 2 , water change. Data are presented as mean percentage7 S.E.M. of radiolabel remaining in the water (n ¼10 tanks per time point). The lines show the best fit of the data for each period (1st, solid; 2nd, dash) to a three parameter hyperbolic decay equation. A single sorption control with radiolabel but no animals was included for each exposure period, but as there was no evidence for any losses in the 1st period, no adjustments were made to the data. Two of the exposures (Experiments 4 & 5; ○, □) were carried out in duplicate with aeration and 5 animals in 2 L water (they were set up as positive controls to examine the uptake of other steroids). The final exposure (Experiment 6; △) was carried out in a single vessel with 18 animals in 3.6 L water (and was set up for a subsequent depuration experiment). The lines represent the same data fitted to a three parameter hyperbolic decay equation. Fig. 4. Thin layer chromatographic separation of a putative sulfate peak (fraction 38) obtained via reverse phase HPLC separation of water that had been collected from mussels exposed to [ 3 H]-E 2 for 24 h (Fig. 2 in [1], original water sample from Experiment 4 and 5). Top graph: pattern of separation of radioactivity without any treatment; standards (horizontal black bars under the x axis) were run concurrently, from left to right: cortisol glucuronide, E 2 17β-S, E 2 3-S and E 2 . Bottom graph: pattern of separation of radioactivity after removal of the sulfate group with sulfatase; E 2 (black bar on the left) and estrone (black bar on the right) standards were run concurrently. NB. The two TLC separations were run at separate times with different mobile phases.
seawater. Animals were suspended (in nets) in aerated glass tanks at 16 71°C with a 16:8 h light: dark photoperiod for exposure to [ 3 H]-E 2 in the presence of food or cold (unlabeled) E 2 (Fig. 2). The water was changed daily and the animals in the feeding treatment were fed a combination of live algae (Isochrysis spp. and Tetraselmis spp.) three times a day at a concentration of 95 cells mL À 1 . The amount of feed required was calculated as the equivalent to 2.5% of mean expected mussel dry weight and the concentration was based on the range of 50-100 cells mL À 1 . The ratio Isochrysis:Tetraselmis was 1:3 in order to achieve the appropriate mass and concentration.

Experiment 3
Mussels were collected from Portland Harbour, Dorset in May 2014. The nearby northeast Portland Harbour breakwater is a catchment holding a long term class B shellfish harvesting classification. The data for this exposure (for which temperature was not controlled and ranged between 17.7-21.2°C) are presented in the research article [1].

Experiments 4 and 5
Mussels were collected from Portland Harbour in October 2014. Animals were placed in a bucket lined with a polyethylene bag with aerated seawater at 16 71°C with a 16:8 h light:dark photoperiod for exposure to [ 3 H]-E 2 (Fig. 3). Water (50 mL) samples were taken at 24 h and extracted [1] for examination of [ 3 H]-E 2 metabolites (see Section 2.3 and Fig. 4).

Experiment 6
Mussels were collected from Portland Harbour in November 2015 and placed in a bucket lined with a polythene bag with aerated seawater for exposure to [ 3 H]-E 2 (Fig. 3). After exposure, mussel soft tissue was extracted using Method 2 (Table 1) and separated (see research article [1] and Table 2 for method optimization) for identification of putative water soluble [ 3 H]-E 2 metabolites by HPLC (Fig. 5).

Experiment 7
Mussels were collected from Portland Harbour in May 2015 and placed in beakers lined with polythene bags with aerated seawater for exposure to cold E 2 . Animals were then extracted (Method 1) and separated for identification of both lipid soluble metabolites in the saponified ester fraction (heptane phase) and water soluble metabolites (in the 80% ethanol phase) by UHPLC-MS/MS (Fig. 8 and Fig. 6 respectively). ]-E 2 radioactivity derived from the 80% ethanol (free and sulfate) fraction of a pooled mussel extract (from Experiment 6). Data are presented as radioactivity (solid line) and UV absorption at 280 nm of E 2 17β-S and E 2 standards that were run concurrently (▲; from left to right).

Mussels caged in the field
Mussels (Mytilus spp.) were kindly provided by Dr Tim Bean of the Cefas Laboratory (with thanks to the Port London Authority, The Historic Dockyard and Chatham and Southend Council for allowing the placement of cages). The mussels were originally collected from Morston shellfishery in Norfolk (reference site) and depurated for two weeks before being deployed for eight weeks in cages in three locations in the Thames (Gravesend, Southend-on-Sea and Chatham) and one at an offshore site (Wharp). The animals were delivered to the laboratory in a cool-box and stored at À20°C; four animals from each site (20 in total) were processed. Mussel soft tissue was extracted (Method 1) and separated into free, ester and sulfate fractions with the same method employed for radioactive residues [1]. The ester fraction was then subjected to saponification and the sulfate fraction to acid solvolysis as described in [1]. All three fractions were then reconstituted in radioimmunoassay (RIA) buffer for subsequent quantification of immunoactive E 2 concentrations using RIA [2] (Fig. 9).  6. Identification of E 2 3-S in 80% ethanol fraction of tissue extract from five mussels exposed to cold E 2 for 24 h (Experiment 7). Representative UHPLC-MS/MS chromatograms of a negative ion MRM transition of 351 4271 of authentic E 2 3-S standard (panel A), 80% ethanol fraction from E 2 -treated mussel extract (panel B) and 80% ethanol fraction from solvent control treated mussel extract (panel C). Fig. 7. Normal phase HPLC chromatogram of tissue extract from mussels that had been exposed to [ 3 H]-E 2 for two consecutive 48 h periods (animals from Experiment 1). Data are presented as radioactivity (solid line) and UV absorption at 280 nm of E 2 standard that was run concurrently (▲). Fig. 8. Identification of E 2 in the saponified heptane fraction (hydrolysate) of tissue extract from five mussels exposed to cold E 2 for 24 h (Experiment 7). Representative UHPLC-MS/MS chromatograms of a positive ion MRM transition of 255 4159 of authentic E 2 standard (panel A), hydrolysate from E 2 -treated mussel extract (panel B) and hydrolysate from solvent control treated mussel extract (panel C).

Thin layer chromatography
A portion of the major HPLC peak found in water at 24 h (presumptive sulfated [ 3 H]-E 2 ; see Fig. 2 in [1]) was mixed with 10 mg each of E 2 3-S, estradiol 17β-sulfate (E 2 17β-S), T glucuronide and E 2 and loaded as described previously [3] onto one lane of a TLC plate (catalog no. LK6DF; Whatman Labsales; www.whatman.com; but no longer manufactured). Standards were also separately loaded onto adjacent lanes. The plate was developed for 45 min with a mixture of ethyl acetate:ethanol:ammonia solution (45:45:15, v-v:v), which enables not just free, but also sulfated and glucuronidated steroids to migrate on the silica gel [3] (Fig. 4, top graph). The plate was sprayed with 10% phosphomolybdic acid in ethanol and heated at 150°C for 5 min to display the positions of the standards. Lanes were then divided into 5 mm bands (ensuring that the two sulfates, that ran close together, were in different bands) and the silica gel from each band was scraped off the plate. The scraped gel bands were mixed with 500 μL ethanol, 500 μL water and 7 mL scintillation fluid and placed in the scintillation counter. Free [ 3 H]-E 2 metabolites were also separated on a TLC plate but using chloroform:ethanol (98:2, v-v) as a mobile phase (Fig. 4, bottom graph).
2.4. Experiments to investigate factors affecting recovery of radioactivity from tissue extracts a) Color quenching? All tissue extracts had an orange color (of varying intensity). The efficiency of color-quench correction (an option provided by the manufacturers of the scintillation counter) Fig. 9. Immunoactive E 2 concentrations (ng g À 1 wet weight) in the ester and free fractions of twenty mussels caged at three sites in the Thames estuary, an offshore site and a reference site. The horizontal dotted line indicates the detection limit of the radioimmunoassay. Table 1 Steroid extraction (from tissue) procedure development.
Step Action Radioactivity recovery (mean % 7 SD) was tested by selecting a heavily colored extract from a mussel that had not been exposed to radiolabel. A range of volumes of extract (0, 0.1, 0.25, 0.5, 1, 2 and 3 mL) were placed in duplicate into scintillation vials and left to evaporate overnight. The dry extracts were then mixed with an identical amount of radioactivity (c. 50,000 dpm), 0.5 mL of water and 7 mL scintillation fluid and counted using color quench correction. The extracts had a mean þS.E.M. of 52,769þ 2208 dpm (coefficient of variation¼ 11%; n ¼7 in duplicate). b) Adsorption to mussel shells? Empty shells from Experiment 6 were washed twice in diethyl ether.
The solvent was decanted directly into a scintillation vial, dried, reconstituted in 1 mL of 80% ethanol and 7 mL scintillation fluid and counted. The amounts of radioactivity adsorbed to shells were insignificant (mean 7S.E.M. 1 70.15% of the total radioactivity in the soft tissue). c) Tritiated water in the extracts? To test this, nine tissue extracts were counted with and without drying them before adding scintillation fluid. No differences were found between extracts (mean 10,596 vs 10,133 dpm; n ¼9) that had been counted with and withoutdrying (i.e. there was no evidence for any tritiated water in tissue extracts).