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La continua immissione in ambiente dei farmaci ad uso umano, e la loro incompleta rimozione nei depuratori rende questi composti pseudo-persistenti e potenzialmente pericolosi per gli organismi acquatici. La ricerca ha inteso valutare gli effetti sub-letali di farmaci di rilevanza ambientale su due organismi d’interesse commerciale: il mitilo mediterraneo e l’anguilla europea, tramite lo sviluppo e l’applicazione di indicatori biologici, in laboratorio e in campo. Gli studi sull’anguilla hanno permesso di identificare nella glicogenolisi, valutata in termini di glucosio rilasciato dagli epatociti, il parametro ideale per valutare alterazioni sul metabolismo glucidico. Con l’utilizzo di un metodo in vitro dinamico e sensibile, la perifusione di epatociti isolati in colonna, sono stati valutati gli effetti dei β-bloccanti propranololo ed atenololo sul metabolismo glucidico. I risultati hanno mostrato la maggior potenza del propranololo nell’alterare la glicogenolisi e la maggior sensibilità dei pesci quando esposti al farmaco. Questa metodologia potrebbe permettere di rilevare la presenza di composti adrenergici, noti e non, nelle matrici ambientali. La consolidata batteria di biomarker applicata sui mitili in condizioni controllate, ha permesso di valutare gli effetti specifici della caffeina dopo esposizione di 7 giorni a concentrazioni ambientali. I risultati hanno evidenziato una moderata induzione della sindrome da stress, confermando la bassa pericolosità della caffeina per gli organismi acquatici, rispetto ad altri farmaci. L’applicazione degli stessi biomarker in un sistema naturale soggetto ad effetti antropici (la laguna Piallassa Piombone), ha permesso di valutare la qualità biologica della laguna, ma non ha potuto imputare gli effetti osservati ai residui farmaceutici, risultati minoritari rispetto ad altri inquinanti. In conseguenza di effetti osservati a basse dosi, concludiamo che è necessario aumentare le conoscenze sugli effetti che questi contaminanti hanno sulle specie non target, e di sviluppare nuove e sensibili metodologie applicabili nella valutazione di rischio ambientale derivante da farmaci., The continuous release and incomplete removal of human pharmaceuticals in the environment poses a risk for aquatic wildlife. The research aimed at evaluating sub-lethal effects of pharmaceuticals of environmental concern on two commercial aquatic species: the Mediterranean mussel and the European eel. The effects were assessed through the development and application of biological indicators in experiments under controlled conditions and in the field. Studies on eel allowed to identify that glycogenolysis, assessed in term of glucose released from the hepatocytes, is the best parameter to evaluate the alterations on hepatic glucose metabolism. The development of a sensitive in vitro dynamic method (perifusion of isolated hepatocytes on columns), permitted to assess the effects of two β-blockers (propranolol and atenolol) on fish glucose metabolism. The results showed that propranolol is more potent than atenolol in modifying the glycogenolysis. Moreover, fish resulted more sensitive when exposed to therapeutic concentrations of the pharmaceutical. The use of this technique could allow the search for unknown adrenergic compounds in environmental matrices. The well-established set of biomarkers used in controlled conditions, allowed the evaluation of the specific effects of caffeine on mussels health status, after 7-days exposure to environmentally relevant concentrations. Results showed a moderate induction of the stress syndrome, confirming the low risk posed by caffeine for aquatic species, in comparison with other pharmaceuticals. The application of the same set of biomarkers in a natural impacted ecosystem (the Piallassa Piombone lagoon), allowed to assess the biological quality of the lagoon, but could not attribute the observed effects to pharmaceuticals amongst other pollutants. Considering the effects observed at low pharmaceutical doses, we highlight the need for a wider knowledge regarding the effects that these contaminants on non-target species, and for implementing new and sensitive methodologies to be used in the environmental risk assessment posed by pharmaceuticals.

The European eel (Anguilla anguilla) has a complex life cycle that includes migrations to and from seawater at different life stages. Yellow eels grow in freshwater, then metamorphose in silver eels and start a long migration to the Sargasso Sea for reproduction. The regulation of energetic metabolism in the life stages is an interesting issue, since silver specimens migrate for 6,000 km without eating. We investigated the adrenergic control of glucose metabolism in liver of eels collected from the Comacchio lagoon (North Adriatic Sea, Italy) at different silvering stages. Eels were captured in Oct-Nov and Dec-Jan. Morphometric parameters were measured and animals were classified as silver eels at the 3rd and 4th stage, respectively. For comparison, yellow eels were collected during autumn and spring. Glycogen content measured in liver was significantly higher in yellow than in silver eels. Hepatocytes were isolated through collagenase perfusion and glycogen levels and glucose release were evaluated. As expected, glycogen levels were significantly higher in yellow eel hepatocytes. Both basal and epinephrine stimulated glucose release were significantly higher in yellow than silver eels. To evaluate whether epinephrine sensitivity is related to a differential expression of adrenergic receptors in hepatocytes, expression of α1- and β2- AR mRNAs in yellow eels and silver at the 4th stage was evaluated, revealing a higher AR mRNA expression in silver than in yellow eels. The analysis of AR mRNA expressions in silver eels at the 3rd stage will provide a complete assessment of AR mRNA expression changes across life stages.

European eels spend up to twenty years in fresh or brackish water as “yellow” eels. Then, part of the population change into migrant “silver” eels, moving towards the spawning grounds at the Sargasso sea. Migrating eels do not feed, and show higher levels of cortisol, causing lipolysis in muscle and liver and higher free fatty acids into the blood. Further hormones change their levels and modulate metabolism in migrating eels, including insulin, IGFs, leptin, and ghrelin. Our investigation compared for the first time catecholamine regulation of liver metabolism in yellow and silver eels. Expression of α1- and β2-adrenergic receptor (AR) mRNAs in eel hepatocytes was assessed by qRT-PCR. Levels of both α1- and β2-AR mRNAs were significantly higher in hepatocytes from silver than from yellow eels, with a 5-fold and a 2-fold increase, respectively. As a result, the mean ratio α1-AR/β2-AR transcripts was significantly higher in silver than in yellow eel. In a first approach, the β2-AR coupled signal transduction was investigated. Epinephrine dose-dependently increased cAMP levels in eel hepatocytes, an effect counteracted by propranolol. Under the same stimuli, the extent of cAMP increase was similar at the two life-stages; differently, the induced glucose release was significantly higher in silver than in yellow eels. Since α1-AR mediated pathway concurs to modulate liver glycogenolysis, further studies will evaluate the contribution of calcium signaling in silver eel response.

Biomass is one of the most interesting sources of organic carbon that can be used for obtaining renewable chemicals. This chapter focuses on the potential offered by a combination of two approaches, which are often considered as an alternative or sometimes even competing: thermochemical processing and biological processing. Understanding of hybrid thermochemical–biological processes requires a steep change in the view of pyrolysis processes. Because most of pyrolysis studies aim to directly obtain a fuel, Pyrolysis Products (PyP) are usually characterized in terms of average fuel proprieties, neglecting details on molecular structures. Microbial Mixed Consortia (MMC) conversions of PyP is something relatively new; thus, some data were back-calculated from experimental results to provide a general indication of the maximum yields and rates obtainable from biological conversions of pyrolysis products with MMC. The metabolic potential of a MMC system can address the conversion of the complex mixture produced by pyrolysis.

Coenzyme Q10 (CoQ, ubiquinone) is a redox-active lipid endogenously synthesized by the cells. The final stage of CoQ biosynthesis is performed at the mitochondrial level by the 'complex Q', where coq2 is responsible for the prenylation of the benzoquinone ring of the molecule. We report that the competitive coq2 inhibitor 4-nitrobenzoate (4-NB) decreased the cellular CoQ content and caused severe impairment of mitochondrial function in the T67 human glioma cell line. In parallel with the reduction in CoQ biosynthesis, the cholesterol level increased, leading to significant perturbation of the plasma membrane physicochemical properties. We show that 4-NB treatment did not significantly affect the cell viability, because of an adaptive metabolic rewiring toward glycolysis. Hypoxia-inducible factor 1α (HIF-1α) stabilization was detected in 4-NB-treated cells, possibly due to the contribution of both reduction in intracellular oxygen tension and ROS overproduction. Exogenous CoQ supplementation partially recovered cholesterol content, HIF-1α degradation, and ROS production, whereas only weakly improved the bioenergetic impairment induced by the CoQ depletion. Our data provide new insights on the effect of CoQ depletion and contribute to shed light on the pathogenic mechanisms of ubiquinone deficiency syndrome.

In view of the potential use of pyrolysis-based technologies, it is crucial to understand the environmental hazards of pyrolysis-derived products, in particular bio-oils. Here, three bio-oils were produced from fast pyrolysis of pine wood and intermediate pyrolysis of corn stalk and poultry litter. They were fully characterized by chemical analysis and tested for their biodegradability and their ecotoxicity on the crustacean Daphnia magna and the green alga Raphidocelis subcapitata. These tests were chosen as required by the European REACH regulation. These three bio-oils were biodegradable, with 40-60% of biodegradation after 28 days, and had EC50 values above 100mgL(-1) for the crustacean and above 10mgL(-1) for the alga, showing low toxicity to the aquatic life. The toxic unit approach was applied to verify whether the observed toxicity could be predicted from the data available for the substances detected in the bio-oils. The predicted values largely underestimated the experimental values.

This study aimed at evaluating the environmental quality of a coastal lagoon (Pialassa Piomboni, NW-Adriatic, Italy) by combining analyses of biomarkers of environmental stress and bioaccumulation of contaminants in marine mussels (Mytilus galloprovincialis) transplanted for 28 days to six selected sites. Assessed biomarkers encompassed lysosomal endpoints, oxidative stress and detoxification parameters, specific responses to metals, neuro- and genotoxic substances; chemical analyses focused on PAHs, metals, pesticide and pharmaceuticals. Results showed up to a 67-fold bioaccumulation of 4- to 6-ring PAHs, including pyrene, fluoranthene, chrysene and benzo(ghi)perylene in transplanted mussels compared to reference conditions (T0). A 10-fold increase of Fe, Cr and Mn was observed, while pesticides and pharmaceuticals were not or slightly detected. The onset of a significant (p < 0.05) general stress syndrome occurred in exposed mussels, as outlined by a 50–57.7% decrease in haemocytes lysosomal membrane stability and an increased lysosomal volume (22.6–26.9%) and neutral lipid storage (18.9–48.8%) observed in digestive gland. Data also revealed a diffuse lipofuscin accumulation (86.5–139.3%; p < 0.05) in digestive gland, occasionally associated to a catalase activity inhibition in gill, indicating an increased vulnerability toward pro-oxidant factors. Higher levels of primary DNA damage (258%; p < 0.05) and PAH accumulation were found in mussels exposed along the eastern shoreline, hosting a petrochemical settlement. Bioaccumulated metals showed a positive correlation with increased metallothionein content (85–208%; p < 0.05) observed in mussels from most sites. Overall, the use of physiological and chemical analyses detected chronic alterations of the mussel health status induced by specific toxicological pathways, proving a suitable approach in the framework of biomonitoring programs of coastal lagoons.

The occurrence of caffeine (CF), a biologically active drug, has widely been documented in coastal waters, and whether its environmental concentrations do represent a threat for marine organisms is unclear. The present study aimed at assessing sub-lethal effects induced by a 7-day exposure to environmentally relevant concentrations of CF (5, 50 and 500 ng/L) in the Mediterranean mussel, Mytilus galloprovincialis. CF in water and mussel tissues, and a battery of biomarkers, including lysosomal parameters of general stress, oxidative stress responses and endpoints of neurological and genetic damages, were evaluated and tested for significance vs controls (p

The focus of the present study was to set a methodological approach for evaluating molecular mechanisms underlying silvering transformation in the European eel, Anguilla anguilla. Silvering is a tightly controlled process during which eels undergo significant morphological, physiological and behavioral changes, pre-adapting for the oceanic spawning migration. Female eels showing different silver indexes were caught in different seasons in the Comacchio Lagoon (North Adriatic Sea, Italy). Isolated hepatocytes from these eels were selected as the experimental model given the relevant role of these cells in metabolic functions potentially altered during silvering. Expression profiles of 7 candidate reference transcripts were analyzed seeking the most viable and robust strategies for accurate qPCR data normalization during silvering. Stability analysis and further statistical validation identified transcripts encoding the ribosomal proteins L13 and ARP as the appropriate reference genes in studies on A. anguilla through silvering. The identified reference transcripts were further used to evaluate expression profiles of target transcripts encoding the thyroid hormone receptor β (THRβ) and vitellogenin (vtg), known to be involved in silvering processes. To the best of our knowledge, this is the first study comparing THRβ expression in European eels across silvering.