Silvia Franzellitti

The β-blocker propranolol affects cAMP-dependent signaling and induces the stress response in Mediterranean mussels, Mytilus galloprovincialis.

Widespread occurrence of pharmaceuticals is reported in aquatic systems, posing concerns for the health of aquatic wildlife and a theoretical risk to humans. A recent concept was developed for the identification of highly active compounds amongst the environmental pharmaceuticals, based on their mode of action, the homology between human targets and possible targets in the environment, and the importance of the affected pathway for the target species. In line with this approach, this study investigated whether propranolol (PROP) affects the cAMP-dependent pathway in Mediterranean mussels, Mytilus galloprovincialis. PROP is a prototypical β-adrenoceptor antagonist, and these receptors exist in bivalves and show gross pharmacological properties similar to their mammalian counterparts. PROP also acts as a 5-HT1 receptor antagonist, which is the sole 5-HT receptor reported in bivalves to date. Importantly, β-adrenoceptor and 5HT-1 receptor subtypes are positively and negatively coupled to cAMP-mediated signaling, respectively. PROP was administered as either l-PROP or dl-PROP. A wide range of concentrations was tested including low (0.3, 3 and 30ng/L) and high (300ng/L) environmental ranges, and a concentration 5-fold above the maximum reported environmental level (30,000ng/L). After a 7-day exposure, mussel cAMP levels and PKA activities were significantly reduced in digestive gland, increased in mantle/gonads and unaffected in gills. Similar patterns were observed for the mRNA expression of the ABCB1 gene encoding the membrane transporter P-glycoprotein, hypothesised to be under PKA modulation. The effects on the digestive gland are consistent with PROP blocking β-adrenoceptors. The observed increased cAMP levels in the mantle/gonad tissue support PROP blocking 5-HT1 receptors. Catalase and glutathione-S tranferase were differently affected by PROP in the two tissues. Mussel haemocyte lysosome membrane stability, a sensitive biomarker of animal health status, was concentration-dependently reduced following PROP exposure. Our observations provide evidence for PROP affecting cell signaling in M. galloprovincialis. Moreover, the chemical interacts with specific and evolutionally conserved biochemical pathways for which it was designed. The mode of action of PROP in mussels is related with its therapeutic properties in humans, based upon these conserved human targets. It also induced a stress response, and all these effects were displayed at the lowest concentrations tested.

An exploratory investigation of various modes of action and potential adverse outcomes of fluoxetine in marine mussels.

The present study investigated possible adverse outcome pathways (AOPs) of the antidepressant fluoxetine (FX) in the marine mussel Mytilus galloprovincialis. An evaluation of molecular endpoints involved in modes of action (MOAs) of FX and biomarkers for sub-lethal toxicity were explored in mussels after a 7-day administration of nominal FX concentrations encompassing a range of environmentally relevant values (0.03-300ng/L). FX bioaccumulated in mussel tissues after treatment with 30 and 300ng/L FX, resulting in bioconcentration factor (BCF) values ranging from 200 to 800, which were higher than expected based solely on hydrophobic partitioning models. Because FX acts as a selective serotonin (5-HT) re-uptake inhibitor increasing serotonergic neurotransmission at mammalian synapses, cell signaling alterations triggered by 5-HT receptor occupations were assessed. cAMP levels and PKA activities were decreased in digestive gland and mantle/gonads of FX-treated mussels, consistent with an increased occupation of 5-HT1 receptors negatively coupled to the cAMP/PKA pathway. mRNA levels of a ABCB gene encoding the P-glycoprotein were also significantly down-regulated. This membrane transporter acts in detoxification towards xenobiotics and in altering pharmacokinetics of antidepressants; moreover, it is under a cAMP/PKA transcriptional regulation in mussels. Potential stress effects of FX were investigated using a battery of biomarkers for mussel health status that included lysosomal parameters, antioxidant enzyme activities, lipid peroxidation, and acetylcholinesterase activity. FX reduced the health status of mussels and induced lysosomal alterations, as suggested by reduction of lysosomal membrane stability in haemocytes and by lysosomal accumulation of neutral lipids in digestive gland. No clear antioxidant responses to FX were detected in digestive gland, while gills displayed significant increases of catalase and glutathione-s-transferase activities and a significant decrease of acetylcholinesterase activity. Though AOPs associated with mammalian therapeutic MOAs remain important during assessments of pharmaceutical hazards in the environment, this study highlights the importance of considering additional MOAs and AOPs for FX, particularly in marine mussels.

The mode of action (MOA) approach reveals interactive effects of environmental pharmaceuticals on Mytilus galloprovincialis

Aquatic organisms are unintentionally exposed to a large number of pharmaceutical residues in their natural habitats. Ecotoxicological studies have agreed that these compounds are not harmful to aquatic organisms, as their environmental concentrations are typically too low. However, recent reports have shown biological effects at such low concentrations when biological endpoints related to the therapeutic effects are assessed. Therefore, conservation of molecular targets is now addressed as a key aspect for the development of more efficient test strategies for pharmaceutical environmental risk assessment, providing the rationale for the mode of action (MOA) approach. In the present study the MOA approach was used to investigate the interactive effects of fluoxetine (FX) and propranolol (PROP) on the Mediterranean mussels (Mytilus galloprovincialis). Indeed, organisms in the environment are exposed to pharmaceutical mixtures throughout their lifetime, and particular combinations may be of concern. The antidepressant FX increases serotonin (5-HT) levels in the synaptic cleft by inhibiting 5-HT reuptake. PROP, a prototypical β-adrenoceptor antagonist, also blocks 5-HT1 receptors, which are negatively coupled to cAMP-mediated signaling. Cell signaling alterations potentially triggered by 5-HT1 receptor occupation were therefore assessed after a 7-day mussel exposure to FX or PROP, alone or in combination, each at 0.3 ng/L concentration. FX decreased cAMP levels and PKA activities in digestive gland and mantle/gonads, in agreement with an increased occupation of 5-HT1 receptors. PROP caused a decrease in cAMP levels and PKA activities in digestive gland and an increase in cAMP levels in mantle/gonads, consistent with a differential expression of adrenergic and 5-HT receptors in the two tissues. Co-exposure to FX and PROP provides significant indications for antagonistic effects of the pharmaceuticals, consistent with a direct (PROP) and indirect (FX) action on the same molecular target. Interestingly, FX induced over-expression of a 5-HT1 gene product, and PROP counteracted such increase when the mixture was administered, while having per se no effect. Finally, mRNA expression of the ABCB gene encoding the MXR-related transporter P-glycoprotein was reduced by both pharmaceuticals in the digestive gland, while decreased by FX, increased by PROP, and not affected by the mixture in mantle/gonads. Since transcription of this gene product is under cAMP/PKA modulation, the impairment of regulatory pathways triggered by low concentrations of pharmaceuticals have the potential to affect the ability of animals to elaborate strategies of defense or adaptation toward further stress factors. In this specific case, the pharmaceutical mixture limits the detrimental effects of the single compounds.

Exposure of mussels to a polluted environment: Insights into the stress syndrome development

Coastal environments are often subjected to contamination, whose biological impact is profitably evaluated through sentinel organisms and biomarkers. mRNA profiling was also proposed as a potential biomarker, whose relevance is still under discussion. Indeed, correlation between molecular and cell-organism responses need further investigations, especially under field conditions. In this study, we followed the development of physiological alterations in Mytilus galloprovincialis transplanted into a polluted coastal lagoon for 2, 4, 7, 14 and 30 days. Three consolidated biomarkers were measured, i.e. lysosomal membrane stability, lipofuscin and metallothionein contents. In parallel, the expressions of stress-related genes encoding metallothioneins (mt10 and mt20), 70-kDa heat shock proteins (MgHSC70 and MgHSP70), and Multi Xenobiotic Resistance-related transporters (MgPgp, MgMrp2, and MgMvp) were analyzed, to have a greater insight into the time-related evolution of the response. Significant (p<0.05) biomarker responses were induced after 7 days of exposure and further increased with time, whereas gene expression profiles were dramatically altered 2 days after transplanting. Biomarkers and gene expression profiles indicated a stress syndrome development in mussels, although with different temporal patterns. Their combined application provided insights into the molecular and cellular basis of mussel responses to challenging environments, and may have far-reaching implications for monitoring environmental health.

Cyclic-AMP Mediated Regulation of ABCB mRNA Expression in Mussel Haemocytes

Background The multixenobiotic resistance system (MXR) allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp). In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified. Methodology/Principal Findings cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis) exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX) alone or in combination with 0.3 ng/L propranolol (PROP). FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin) and pharmacological modulators (PROP, forskolin, dbcAMP, and H89) of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT) decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89. Conclusions This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels.

Molecular and Cellular Effects Induced in Mytilus galloprovincialis Treated with Oxytetracycline at Different Temperatures.

The present study evaluatedthe interactive effects of temperature (16°C and 24°C) and a 4-day treatment with the antibiotic oxytetracycline (OTC) at 1 and 100μg/L on cellular and molecular parameters in the mussel Mytilus galloprovincialis. Lysosomal membrane stability (LMS), a sensitive biomarker of impaired health status in this organism, was assessed in the digestive glands. In addition, oxidative stress markers and the expression of mRNAs encoding proteins involved in antioxidant defense (catalase (cat) and glutathione-S-transferase (gst)) and the heat shock response (hsp90, hsp70, and hsp27) were evaluated in the gills, the target tissue of soluble chemicals. Finally, cAMP levels, which represent an important cell signaling pathway related to oxidative stress and the response to temperature challenges, were also determined in the gills. Exposure to heat stress as well as to OTC rendered a decrease in LMS and an increase in malonedialdehyde accumulation (MDA). CAT activity was not significantly modified, whereas GST activity decreased at 24°C. Cat and gst expression levels were reduced in animals kept at 24°C compared to 16°C in the presence or absence of OTC. At 16°C, treatment with OTC caused a significant increase in cat and gst transcript levels. Hsp27 mRNA was significantly up-regulated at all conditions compared to controls at 16°C. cAMP levels were increased at 24°C independent of the presence of OTC. PCA analysis showed that 37.21% and 25.89% of the total variance was explained by temperature and OTC treatment, respectively. Interestingly, a clear interaction was observed in animals exposed to both stressors increasing LMS and MDA accumulation and reducing hsp27 gene expression regulation. These interactions may suggest a risk for the organisms due to temperature increases in contaminated seawaters.

Bioaccumulation of algal toxins and changes in physiological parameters in Mediterranean mussels from the North Adriatic Sea (Italy)

The Northwestern Adriatic Sea is a commercially important area in aquaculture, accounting for about 90% of the Italian mussel production, and it was subjected to recurring cases of mussel farm closures due to toxic algae poisoning. A spatial and temporal survey of four sites along the North Adriatic Sea coasts of Emilia Romagna (Italy) was undertaken to study the possible impairments of physiological parameters in Mytilus galloprovincialis naturally exposed to algal toxins. The sites were selected as part of the monitoring network for the assessment of algal toxins bioaccumulation by the competent Authority. Samples positive to paralytic shellfish toxins and to lipophilic toxins were detected through the mouse bioassay. Lipophilic toxins were assessed by HPLC. Decreasing yessotoxins (YTX) levels were observed in mussels from June to December, while homo‐YTX contents increased concomitantly. Lysosome membrane stability (LMS), glutathione S‐transferase and catalase activities, and multixenobiotic resistance (MXR)‐related gene expressions were assessed as parameters related to the mussel health status and widely utilized in environmental biomonitoring. Levels of cAMP were also measured, as possibly involved in the algal toxin mechanisms of action. Low LMS values were observed in hemocytes from mussels positive to the mouse bioassay. MXR‐related gene expressions were greatly inhibited in mussels positive to the mouse bioassay. Clear correlations were established between increasing homo‐YTX contents (and decreasing YTX) and increasing cAMP levels in the tissues. Similarly, significant correlations were established between the increase of homo‐YTX and cAMP levels, and the expressions of three MXR‐related genes at submaximal toxin concentrations. In conclusion, YTXs may affect mussel physiological parameters, including hemocyte functionality, gene expression and cell signaling.

Molecular and cellular effects induced by hexavalent chromium in Mediterranean mussels.

The present study evaluated the effects of Cr(VI) in digestive gland of the Mediterranean mussel (Mytilus galloprovincialis) exposed for 1 week to the metal at 1, 10, and 50 ng/L. Tissue accumulation of Cr and lysosomal biomarkers were measured. Moreover, a low-density DNA microarray was used to identify early molecular markers of metal exposure. A concentration-dependent increase in tissue Cr concentrations was observed in both digestive gland and remaining soft tissues. A reduction of lysosomal membrane stability was detected in digestive gland at 10 and 50 ng/L of Cr(VI), indicating a loss of cell functional integrity. The expression of mRNAs encoding 13 genes involved in metal resistance (mt10, mt20), molecular chaperoning (hsp70), immune response (mytlB, mytcA and lys), transcriptional (histones h1, h2-a and h4), and antioxidant/detoxification (cat, mrp2, mvp) processes were significantly altered already at the lowest Cr(VI) concentration, where the effects at the histological level were nonsignificant. Altogether, data point out that depending on the exposure concentration Cr(VI) may cause or not oxidative stress altering the efficiency of the antioxidant system in counteracting the effects of Cr as a redox-active metal. Moreover, changes of mRNA expression profiles induced by Cr(VI) concentrations as low as 1-50 ng/L were related to altered immunomodulation, DNA stability, and stress response pathways previously proven to be affected by the metal. The molecular targets presently identified may drive the development of new biomarkers for Cr exposure or help their interpretation.

Effects of the exposure to intermittent 1.8 GHz radio frequency electromagnetic fields on HSP70 expression and MAPK signaling pathways in PC12 cells.

Abstract Purpose: We previously reported effects on heat shock protein 70 (HSP70) mRNA expression, a cytoprotective protein induced under stressful condition, in human trophoblast cells exposed to amplitude-modulated Global System for Mobile Communication (GSM) signals. In the present work the same experimental conditions were applied to the rat PC12 cells, in order to assess the stress responses mediated by HSP70 and by the Mitogen Activated Protein Kinases (MAPK) in neuronal-like cells, an interesting model to study possible effects of mobile phone frequencies exposure. Materials and methods: HSP70 gene expression level was evaluated by reverse transcriptase polymerase chain reaction, HSP70 protein expression and MAPK phosphorylation were assessed by Western blotting. PC12 cells were exposed for 4, 16 or 24 h to 1.8 GHz continuous wave signal (CW, carrier frequency without modulation) or to two different GSM modulation schemes, GSM-217Hz and GSM-Talk (which generates temporal changes between two different GSM signals, active during talking or listening phases, respectively, thus simulating a typical conversation). Specific adsorption rate (SAR) was 2 W/kg. Results: After PC12 cells exposure to the GSM-217Hz signal for 16 or 24 h, HSP70 transcription significantly increased, whereas no effect was observed in cells exposed to the CW or GSM-Talk signals. HSP70 protein expression and three different MAPK signaling pathways were not affected by the exposure to any of the three different 1.8 GHz signals. Conclusion: The positive effect on HSP70 mRNA expression, observed only in cells exposed to the GSM-217Hz signal, is a repeatable response previously reported in human trophoblast cells and now confirmed in PC12 cells. Further investigations towards a possible role of 1.8 GHz signal modulation are therefore advisable.

Effects of cadmium and 17β-estradiol on Mytilus galloprovincialis redox status. Prooxidant–antioxidant balance (PAB) as a novel approach in biomonitoring of marine environments

Cadmium and 17β-estradiol are rapidly accumulated in mussel tissues, making mussels excellent pollution sentinel organisms. The aim of the present study was to compare the oxidative responses of the mussels after 1, 3 and 7 days of exposure to cadmium with those to 17β-estradiol and subsequently, to suggest a multi-parametric approach for biomonitoring studies. Our results showed that environmentally relevant concentrations of either cadmium or 17β-estradiol for 1, 3 and 7 days induced oxidative stress in hemocytes of exposed mussels. The latter was determined by significantly increased ROS levels and apoptosis, by suppression of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) expression levels and subsequent increased prooxidant levels, as measured by prooxidant-antioxidant balance (PAB) assay. To our knowledge this is the first time that prooxidant-antioxidant balance is evaluated in invertebrates as an index of oxidative stress. The simultaneous use of the parameters of prooxidant-antioxidant balance and antioxidant enzymes expression patterns, in combination with ROS production levels and apoptosis, in mussel hemocytes is suggested as an approach that may help to better evaluate the impact of environmental pollution on marine organisms and thereupon ecosystems.