Aromatisation of steroids in marine bivalves: characteristic of enzymatic complex and process efficiency under the presence of endocrine disruptors.

In the case of sedentary organisms such as mussels, many of the pathologies observed may be associatedwith toxic effects occurring after direct exposure to pollutants directly in the natural habitats of these animals. Due to increasing anthropogenic pressure, a growing problem concerns a plethora of toxic or potentially toxic compounds, many known as contaminants of emerging concern, entering the Baltic Sea environment. Some of them are characterized by the ability to influence the endocrine functions of living organisms found in the marine environment thus they are called Endocrine Disruptors (EDs). Exposure to EDs results in changes in hormone metabolism and furthermore in the physiology and behavior of individual specimens and the whole populations. Their mechanism of action is the agonistic and/or antagonistic interaction with steroid receptors resulting in disturbances in the regulation of key metabolic pathways. However, the cause-and-effect relationship between the dysfunction of the endocrine system of marine invertebrates and the presence of EDs is unclear and scientifically controversial. What is, however, known is that the exposure of marine molluscs to EDs, both naturally occurring androgens, estrogens and synthetic hormones, results in destabilisation of natural hormones levels, impairing biosynthesis, secretion, transport, metabolism and receptor binding/signal transduction of hormones. That may not only lead to reproduction malfunction, but also affect embryos and ontogenesis, behavior, immune function and genes expression.Despite the fact, that the knowledge on bivalves endocrinology is still poor (e.g. the role and the origin ofsteroids are debatable) mussels are widely used in testing the effects of various emerging pollutants, including those with endocrine disrupting properties. However, the recent detection of active aromatization of androgens by M. trossulus indicates this species as a model organism among marine molluscs for the study of selected EDs. Apart from the aromatase activity, measured indirectly by the amount of tritium water produced in the reaction with 1beta-3H androstenedione, neither the enzymatic protein catalysing the aromatization process nor the gene encoding this protein is known. To this purpose, the next step is to provide full biochemical characteristic on an enzyme behind aromatisation and to develop a map of the M. trossulus transcriptomes needed to search for the aromatase-like gene in the cytochromes P450 superfamily or other genes encoding proteins involved in hormonal or metabolic regulation. Such assembled and annotated protein-coding transcriptome may also serve as a reference for quantitative gene expression studies. Since an involvement of microorganisms forming the mussel microbiome or the algae feed in aromatisation cannot be completely ruled out at this stage, apart from performing expression profiling experiments combined with targeted and untargeted approaches for searching the gene(s) responsible for the aromatization, we also propose to perform a metagenome study of the mussel and water (feed) from the natural environment using metabarcoding based on high-throughput sequencing. Also, the role of steroids and aromatization will be investigated by testing their effects during windows of susceptibility recognized in marine bivalves.