Effects of the Antidepressant Amitriptyline on Juvenile Brown Trout and Their Modulation by Microplastics.

Pharmaceuticals such as antidepressants are designed to be bioactive at low concentrations. According to their mode of action, they can also influence non-target organisms due to the phylogenetic conservation of molecular targets. In addition to the pollution by environmental chemicals, the topic of microplastics (MP) in the aquatic environment came into the focus of scientific and public interest. The aim of the present study was to investigate the influence of the antidepressant amitriptyline in the presence and absence of irregularly shaped polystyrene MP as well as the effects of MP alone on juvenile brown trout (Salmo trutta f. fario). Fish were exposed to different concentrations of amitriptyline (nominal concentrations between 1 and 1000 µg/L) and two concentrations of MP (104 and 105 particles/L; <50 µm) for three weeks. Tissue cortisol concentration, oxidative stress, and the activity of two carboxylesterases and of acetylcholinesterase were assessed. Furthermore, the swimming behavior was analyzed in situations with different stress levels. Exposure to amitriptyline altered the behavior and increased the activity of acetylcholinesterase. Moreover, nominal amitriptyline concentrations above 300 µg/L caused severe acute adverse effects in fish. MP alone did not affect any of the investigated endpoints. Co-exposure caused largely similar effects such as the exposure to solely amitriptyline. However, the effect of amitriptyline on the swimming behavior during the experiment was alleviated by the higher MP concentration.

Impact of the NSAID diclofenac on survival, development, behaviour and health of embryonic and juvenile stages of brown trout, Salmo trutta f. fario.

The NSAID diclofenac is controversially discussed with respect to its environmental relevance. Since further information is need to assess whether diclofenac should be included as substance of priority in the EU water framework directive, we investigated the impact of this analgesic on the embryonic development of brown trout (Salmo trutta f. fario) from fertilized egg until the end of sac-fry stage and studied effects in juvenile fish six months post hatch. Embryos were exposed to five test concentrations (0.1, 0.5, 1, 10, 100µg/L) over 127days at 7°C. None of the treatments affected mortality, hatching, development or heart rate. Six months old juveniles exposed to five concentrations (0.1, 1, 10, 100, 200µg/L) over 25days at 7°C, however, showed increased mortality, reaching significance at 100µg/L. Furthermore, a significantly higher proportion of juvenile animals bore injuries at concentrations higher 10µg/L. Neither the levels of the stress protein Hsp70, nor the amount of lipid peroxides was affected by any of the treatments. Histological analyses of gill, liver and kidney revealed visible tissue reactions in fish from all experimental groups. Histological responses in livers of diclofenac-exposed fish outstripped the status of laboratory control fish, particularly when exposed to the two highest concentrations. Chemical analyses of fish muscle tissue revealed concentration-dependent uptake of DCF into the animal, but no relevant bioconcentration. Our study supports earlier findings indicating a lower sensitivity of trout early life stages compared to older individuals, suggesting that studies for risk assessment of diclofenac should predominantly focus on later life stages. Furthermore, fish mortality was found to increase with rising diclofenac concentrations, and the lowest observed effect concentration of 10µg/L on the organismic level emphasises the classification of diclofenac as a micropollutant that requires close attention.

EGFR overexpression induces activation of telomerase via PI3K/AKT-mediated phosphorylation and transcriptional regulation through Hif1-alpha in a cellular model of oral-esophageal carcinogenesis.

Telomerase plays an important role during immortalization and malignant transformation as crucial steps in the development of human cancer. In a cellular model of oral-esophageal carcinogenesis, recapitulating the human disease, immortalization occurred independent of the activation of telomerase but through the recombination-based alternative lengthening of telomeres (ALT). In this stepwise model, additional overexpression of EGFR led to in vitro transformation and activation of telomerase with homogeneous telomere elongation in already immortalized oral squamous epithelial cells (OKF6-D1_dnp53). More interestingly, EGFR overexpression activated the PI3K/AKT pathway. This strongly suggested a role for telomerase in tumor progression in addition to just elongating telomeres and inferring an immortalized state. Therefore, we sought to identify the regulatory mechanisms involved in this activation of telomerase and in vitro transformation induced by EGFR. In the present study we demonstrate that telomerase expression and activity are induced through both direct phosphorylation of hTERT by phospho-AKT as well as PI3K-dependent transcriptional regulation involving Hif1-alpha as a key transcription factor. Furthermore, EGFR overexpression enhanced cell cycle progression and proliferation via phosphorylation and translocation of p21. Whereas immortalization was induced by ALT, in vitro transformation was associated with telomerase activation, supporting an additional role for telomerase in tumor progression besides elongating telomeres.