Oxytetracycline-induced inflammatory process without oxidative stress in blue mussels Mytilus trossulus.

Potentially harmful compounds including pharmaceuticals are commonly found in marine waters and sediments. Amongst those, antibiotics and their metabolites are detected worldwide in various abiotic (at concentrations as high as µg/L) and biotic matrices at ng/gram of tissue, posing a risk to non-target species exposed to them such as blue mussels. Amongst those, oxytetracycline (OTC) belongs to the most detected antibiotics in the marine environment. In this work, we concentrated on studying the potential induction of oxidative stress, activation of cellular detoxification processes (including Phase I and Phase II xenobiotic biotransformation enzymes) and multixenobiotic resistance pumps (Phase III) as well as changes in the aromatisation efficiency in Mytilus trossulus exposed to 100 µg/L OTC. Our results show that 100 µg/L OTC concentration did not provoke cellular oxidative stress and did not affect the expression of genes involved in detoxification processes in our model. Moreover, no effect of OTC on aromatisation efficiency was found. Instead, phenoloxidase activity measured in haemolymph was significantly higher in OTC exposed mussels than in those from the control (30.95 ± 3.33 U/L and 17.95 ± 2.75 U/L, respectively). OTC exposed mussels were also characterised by a tissue-dependant activation of major vault protein (MVP) gene expression (1.5 times higher in gills and 2.4 times higher in the digestive system) and a decreased expression of the nuclear factor kappa B-a (NF-κB) gene (3.4 times lower in the digestive system) when compared to those from the control. Additionally, an elevated number of regressive changes and inflammatory responses in tissues such as gills, digestive system and mantle (gonads) was observed underlining the worsening of bivalves' general health. Therefore, instead of a free-radical effect of OTC, we for the first time describe the occurrence of typical changes resulting from antibiotic therapy in non-target organisms like M. trossulus exposed to antibiotics such as OTC.

Bivalves Transmissible Neoplasia: Biochemical Aspects of Contagious Cancer in a Clam Macoma Balthica.

BACKGROUND/AIMS: Occurring in marine invertebrates infectious haemic neoplasia (bivalves transmissible neoplasia, BTN) arises from genome instabilities leading to multilevel malfunctions and unregulated cell division of presumably haemocyte precursors. As its biochemical characterisation remains unknown, we here present the first data describing selected aspects of the physiology and biochemistry of the disease a in model clam Macoma balthica. We chose free amino acids (FAA) composition, mitochondrial respiration and enzymatic activity, oxidative stress enzymes activities and corticosteroids profile as markers of this contagious cancer. METHODS: Selected markers were measured in neoplastic and healthy clams and two tissue types, haemolymph and solid tissue. FAA composition was assessed in the haemolymph samples using high performance liquid chromatography-mass spectrometry (LC/MS). Mitochondrial respiration analysis was performed on haemocytes using oxygen electrodes integrated system Seahorse XFp. Mitochondrial enzymes activities were measured using spectrophotometry (cytochrome oxidase, COX) and commercial kit (succinate dehydrogenase, SDH). Total Antioxidant Capacity (TAC), Acetylocholinesterase (AChE), Protein Carbonyl Content (CBO) and Malondialdehyde (MDA) levels were measured in the solid tissue using analytical kits, and glutathione (GSH) was measured spectrophotometrically. Corticosteroids profile, measured in the solid tissue, was obtained with Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS/MS) technique. RESULTS: In both clam groups nine FAAs were detected with Asp, Glu, Pro, Ser constituting over 90% of total FAA content. Significantly higher Gln level was detected in BTN positive clams. In neoplastic clams, an impairment of mitochondrial metabolism was observed as a decrease in mitochondrial oxygen consumption and lower cytochrome c oxidase activity. In the neoplastic clams significantly higher concentration of low molecular weight antioxidants was found. Finally, we report high level of corticosterone and lower levels of dehydrocorticosterone, cortisol and cortisone in healthy clams and elevated cortisol level in BTN individuals. CONCLUSION: Neoplastic clams are characterized by altered mitochondrial metabolism, with a potential key role of glutamine (Gln) in cancer cells energy production. Despite low aerobic respiration, BTN cells have efficient antioxidative response to elevated concentration of ROS. Elevated cortisol level in BTN-positive clams may indicate an important role of this corticosteroid in cancer biochemistry. Thus, we here provide the first results of selected physiological and biochemical aspects of BTN, making an important step in studying cancer epidemiology in wildlife.

Horizontal transmission of disseminated neoplasia in the widespread clam Macoma balthica from the Southern Baltic Sea.

Disseminated neoplasia (DN) is one of the most challenging and unrecognised diseases occurring in aquatic fauna. It has been diagnosed in four bivalve species from the Gulf of Gdansk (Southern Baltic Sea) with the highest frequency in Macoma balthica (formerly Limecola balthica), reaching up to 94% in some populations. The aetiology of DN in the Baltic Sea has not yet been identified, with earlier studies trying to link its occurrence with environmental pollution. Taking into account recent research providing evidence that DN is horizontally transmitted as clonal cells between individuals in some bivalve species, we aimed to test whether DN is a bivalve transmissible neoplasia (BTN) in the population of M. balthica from the Gulf of Gdansk highly affected with cancer. We examined mitochondrial cytochrome c oxidase I (mtCOI) and elongation factor 1α (EF1α) sequences of genomes obtained from haemolymph and tissues of neoplastic and healthy individuals. Sequence analysis resulted in detection of an independent transmissible cancer lineage occurring in four neoplastic clams that is not present in healthy animals. This study describes the first case of BTN in the clam M. balthica (MbaBTN), providing further insights for studies on this disease.

Exposure of Mytilus trossulus to diclofenac and 4'-hydroxydiclofenac: Uptake, bioconcentration and mass balance for the evaluation of their environmental fate.

Diclofenac (DIC) is one of the most widely consumed drugs in the world, and its presence in the environment as well as potential effects on organisms are the subject of numerous recent scientific works. However, it is becoming clear that the risk posed by pharmaceuticals in the environment needs to be viewed more broadly and their numerous derivatives should also be considered. In fact, already published results confirm that the transformation products of NSAIDs including DIC may cause a variety of potentially negative effects on marine organisms, sometimes showing increased biological activity. To date, however, little is known about bioconcentration of DIC and DIC metabolites and the role of sex in this process. Therefore, the present study for the first time evaluates sex-related differences in DIC bioconcentration and estimates bioconcentration potential of DIC metabolite, 4-OH DIC, in the Mytilus trossulus tissues. In the experiment lasting 7 days, mussels were exposed to DIC and 4-OH DIC at concentrations 68.22 and 20.85 µg/L, respectively. Our study confirms that DIC can be taken up by organisms not only in its native form, but also as a metabolite, and metabolised further. Furthermore, in the present work, mass balance was performed and the stability of both studied compounds under experimental conditions was analysed. Obtained results suggest that DIC is more stable than its derivative under the tested conditions, but further analyses of the environmental fate of these compounds are necessary.

Toxic effects of NSAIDs in non-target species: A review from the perspective of the aquatic environment.

The presence of pharmaceuticals in the aquatic environment, both in marine and freshwater reservoirs, is a major concern of global environmental protection. Among the drugs that are most commonly used, NSAIDs tend to dominate. Currently, being aware of the problem caused by drug contamination, it is extremely important to evaluate the scale and the full spectrum of its consequences, from short-term to long-term effects. The influence on non-target aquatic animals can take place at many levels, and the effects can be seen both in behaviour and physiology, but also in genetic alterations or reproduction disorders, affecting the development of entire populations. This review summarises all the advances made to estimate the impact of NSAIDs on aquatic animals. Multicellular animals from all trophic levels, inhabiting both inland waters, seas and oceans, have been considered. Particular attention has been paid to chronic studies, conducted at low, environmentally-relevant concentrations, to estimate the real effects of the present pollution. The number of such studies has indeed increased in recent years, allowing for a better insight into the possible consequences of pharmaceutical pollution. It should be stressed, however, that our knowledge is still limited to a few model species, while there are many groups of organisms completely unexplored regarding the effects of drugs. Therefore, the main aim of this paper was to summarise the current state of knowledge on the toxicity of NSAIDs in aquatic animals, also identifying important gaps and major issues requiring further analysis.