The Uptake of Ethinyl-Estradiol and Cortisol From Water by Mussels (Mytilus spp.).

Previous toxicokinetic studies have shown that mussels (Mytilus spp.) can readily absorb the three main mammalian sex steroids, estradiol (E2), testosterone (T) and progesterone (P) from water. They also have a strong ability to store E2 and the 5α-reduced metabolites of T and P in the form of fatty acid esters. These esters were shown to have half-lives that were measured in weeks (i.e. they were not subject to fast depuration). The present study looked at the toxicokinetic profile of two other common steroids that are found in water, the potent synthetic oestrogen, (ethinyl-estradiol) (EE2; one of the two components of 'the pill'), and cortisol, a natural stress steroid in vertebrates. In the first three hours of uptake, tritiated EE2 was found to be taken up at a similar rate to tritiated E2. However, the levels in the water plateaued sooner than E2. The ability of the animals to both esterify and sulphate EE2 was found to be much lower than E2, but nevertheless did still take place. After 24 h of exposure, the majority of radiolabelled EE2 in the animals was present in the form of free steroid, contrary to E2, which was esterified. This metabolism was reflected in a much lower half-life (of only 15 h for EE2 in the mussels as opposed to 8 days for E2 and >10 days for T and P). Intriguingly, hardly any cortisol (in fact none at all in one of the experiments) was absorbed by the mussels. The implications of this finding in both toxicokinetic profiling and evolutionary significance (why cortisol might have evolved as a stress steroid in bony fishes) are discussed.

Uptake and metabolism of water-borne progesterone by the mussel, Mytilus spp. (Mollusca).

Previous studies have shown that mussels can pick up 17ß-estradiol [E2] and testosterone [T] from water, metabolize them and conjugate them to fatty acids (esterification), leading to their accumulation in tissue. A key requirement for the esterification process is that a steroid must have a 'reactive' hydroxyl group to conjugate to a fatty acid (which in T, and probably E2, is the ß-hydroxyl group on carbon 17). Progesterone (P) lacks any hydroxyl groups and theoretically cannot be esterified and hence should not accumulate in mussels in the same way as E2 or T. However, it is already known that mussels have an enzyme that can achieve 5α-reduction of the A ring of T and P and that there is also another reductase that can transform the 3-oxo group of the 5α-reduced A ring of T into a hydroxyl group. We hypothesized that, although intact P cannot be directly esterified, it might nevertheless be transformed into metabolites that can. To test this hypothesis, we investigated the rate and capacity of uptake, metabolism and potential depuration of tritiated P by the common mussel, Mytilus spp. We found that tritiated P was taken up from water at a similar rate to E2 and T (mean clearance rate 49mL-1 animal-1h-1) and that, as found with the other steroids, the rate of uptake could not be saturated by the addition of non-radioactive steroid (even at 7.6µgL-1). We found that up to 66% of the radioactivity that was taken up was present in the ester fraction, suggesting that hydroxylation of the P must indeed have occurred. We then definitively identified two metabolites in the ester fraction: 5α-pregnane-3ß,20ß-diol and 3ß-hydroxy-5α-pregnan-20-one. These same two steroids were also present in the free steroid fraction. Intact P was not detected in either of the fractions. When undergoing depuration (under semi-static conditions), the radioactivity in the ester fractions remained at the same concentration in the animals for at least 10 days. Our findings suggest that the lack of reactive hydroxyl groups on P does not preclude it from being taken up, metabolized and subsequently stored. Many questions remain, not least of which is why, when P seems to be so rapidly metabolized, two previous studies on mussels have reported concentrations of up to 30ngg-1 wet weight of P in their flesh.

Data on the uptake and metabolism of testosterone by the common mussel, Mytilus spp.

This article provides data in support of the research article entitled "Rapid uptake, biotransformation, esterification and lack of depuration of testosterone and its metabolites by the common mussel, Mytilus spp." (T.I. Schwarz, I. Katsiadaki, B.H. Maskrey, A.P. Scott, 2017) [1]. The uptake of tritiated testosterone (T) from water by mussels is presented. The two main radioactive peaks formed from T and present in the fatty acid ester fraction of mussel tissues were shown to have the same elution positions on a thin layer chromatography plate as 17ß-hydroxy-5α-androstan-3-one (DHT) and 5α-androstan-3ß,17ß-diol (3ß,17ß-A5α). Reverse phase high performance liquid chromatography of the non-esterified (80% ethanol) fraction of the mussel tissue extracts also presented radioactive peaks at the elution positions of DHT and 3ß,17ß-A5α. There was no evidence for sulfated T in this fraction. It was shown that aeration led to significant losses of radiolabeled testosterone from the water column.

Rapid uptake, biotransformation, esterification and lack of depuration of testosterone and its metabolites by the common mussel, Mytilus spp.

The presence of the vertebrate steroids, testosterone (T) and 17ß-estradiol in mollusks is often cited as evidence that they are involved in the control of their reproduction. In this paper, we show that a likely source of T in at least one species, the common mussel (Mytilus spp.), is from uptake from water. When mussels were exposed to waterborne tritiated T ([3H]-T) in a closed container, the radioactivity decreased rapidly and exponentially until, by 24h, approximately 35% remained in the water. The rate of uptake of radiolabel could not be saturated by concentrations as high as 16.5µgL-1 (mean measured) of non-radiolabeled T, showing that the animals have a very high capacity for uptake of T. At least 30% of the applied radioactivity could be extracted from the tissues of the animals with organic solvents and most of this (26% of the total applied radioactivity) was in the fatty acid ester fraction. Following alkaline hydrolysis, reverse phase HPLC and TLC, this fraction was shown to consist predominantly of 5α-dihydrotestosterone and 5α-androstane-3ß,17ß-diol, while T was a minor component. These steroids were definitively identified in the fatty acid ester fraction by mass spectrometry. Overall, less than 5% of the [3H]-T applied to the system remained untransformed at the end of exposure. After ten days of depuration there was no reduction in the total amount of radioactivity in the tissues, nor any changes in the ratio of the metabolites in the ester fraction. These findings show that any association between T presence and reproductive status or sex is confounded by their significant capacity for uptake, and that T undergoes extensive metabolism in mussels in vivo and therefore may not be representative of the androgenic burden of the animals. Consequently, measurements of T in mussel tissue offer little utility as an indicator of reproductive status or sex.

Mussels (Mytilus spp.) display an ability for rapid and high capacity uptake of the vertebrate steroid, estradiol-17ß from water.

Six experiments were carried out to define the optimum conditions for investigating the dynamics of uptake and metabolism of tritiated E2 from water by adult blue mussels, Mytilus spp. Optimum uptake was achieved using 400mL aerated sea water animal-1 and an incubation period of no more than 24h. The pattern of disappearance conformed closest to an inverse hyperbolic curve with the percentage of radiolabel that could be measured in the water reaching an asymptote that was on average 50% of the original. This apparent inability of the animals to absorb all the radiolabel was investigated further. Solvent partition and chromatography revealed that, after 24h, c. 60% of the radiolabel still present in the water was composed of water soluble conjugates, c. 25% was composed of tritiated water and only 15% ran on and around the chromatographic position of E2. The major water soluble constituent was identified by chromatography and mass-spectrometry as 1,3,5(10)-estratriene-3,17ß-diol 3-sulfate (estradiol 3-S). The clearance rate of radiolabel was 46.9±1.8mLanimal-1h-1. This was not significantly affected by the addition of as much as 25µgL-1 cold E2 to the water, demonstrating that mussels have a large capacity for E2 uptake. A new procedure involving solvent partition was developed for separating the free, esterified and sulfated forms of E2 present in the flesh of mussels. This involved extracting the soft tissue with organic solvents and then treating a portion of dried extract with a combination of heptane (dissolved fatty acid esters of E2) and 80% ethanol (dissolved free and sulfated E2). The latter fraction was further partitioned between water (sulfate) and diethyl ether (free steroid). This procedure was much cheaper and less time-consuming than chromatography. Approximately 80% of the radioactivity that was taken up by the animals was present in the form of ester. Moreover, E2 was the only steroid identified after saponification of these esters. Of the remaining radioactivity, c. 10% was in the form of unidentified free steroids and c. 10% was estradiol 3-S. In order to determine how rapidly mussels were able to depurate tritiated E2 and its metabolites, two experiments were carried out. Animals from the first experiment purged up to 63% of radioactivity in 20days under flow-through conditions; whereas animals from the second experiment released only 16% of radioactivity in 10days under semi-static conditions. The ratios of the different forms of E2 did not change substantially during the course of depuration.

Data on the uptake and metabolism of the vertebrate steroid estradiol-17ß 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-17ß 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-3,17ß-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.