Effects of environmental cocaine concentrations on COX and caspase-3 activity, GRP-78, ALT, CRP and blood glucose levels in the liver and kidney of the European eel (Anguilla anguilla)

Abstract Cocaine is one of the most widely used illicit drugs in the world, and as a result of incomplete removal by sewage treatment plants it is found in surface waters, where it represents a new potential risk for aquatic organisms. In this study we evaluated the influence of environmental concentrations of cocaine on the liver and the kidney of the European eel (Anguilla anguilla). The eels were exposed to 20 ng L−1 of cocaine for fifty days, after which, three and ten days after the interruption of cocaine exposure their livers and kidneys were compared to controls. The general morphology of the two organs was evaluated, as well as the following parameters: cytochrome oxidase (COX) and caspase-3 activities, as markers of oxidative metabolism and apoptosis activation, respectively; glucose-regulated protein (GRP)78 levels, as a marker of endoplasmic reticulum (ER)-stress; blood glucose level, as stress marker; serum levels of alanine aminotransferase (ALT), as a marker of liver injury and serum levels of C-reactive protein (CRP), as a marker of the inflammatory process. The liver showed morphologic alterations such as necrotic areas, karyolysis and pyknotic nuclei, while the kidneys had dilated glomeruli and the renal tubules showed pyknotic nuclei and karyolysis. In the kidney, the alterations persisted after the interruption of cocaine exposure. In the liver, COX and caspase-3 activities increased (COX: P = 0.01; caspase-3: P = 0.032); ten days after the interruption of cocaine exposure, COX activity returned to control levels (P = 0.06) whereas caspase-3 activity decreased further (P = 0.012); GRP78 expression increased only in post-exposure recovery specimens (three days: P = 0.007 and ten days: P = 0.008 after the interruption of cocaine exposure, respectively). In the kidney, COX and caspase-3 activities increased (COX: P = 0.02; caspase-3: P = 0.019); after the interruption of cocaine exposure, COX activity remained high (three days: P = 0.02 and ten days: P = 0.029 after the interruption of cocaine exposure, respectively) whereas caspase-3 activity returned to control values (three days: P = 0.69 and ten days: P = 0.67 after the interruption of cocaine exposure, respectively). Blood glucose and serum ALT and CRP levels increased (blood glucose: P = 0.01; ALT: P = 0.001; CRP: 0.015) and remained high also ten days after the interruption of cocaine exposure (blood glucose: P = 0.009; ALT: P = 0.0031; CRP: 0.036). These results suggest that environmental cocaine concentrations adversely affected liver and kidney of this species

L’enzima feniletanolamina-N-metiltransferasi (PNMT) nelle cellule cromaffini della ghiandola interrenale di Triturus carnifex (Amphibia, Urodela). Fattori in grado di modulare l’attività dell’enzima e sequenziamento del suo mRNA

Lo studio è stato affrontato 1) localizzando l’enzima per via immunocitochimica, sia in M.O. che in M.E., e localizzando il suo mRNA mediante ibridazione in situ in M.O. 2) verificando l’eventuale sensibilità dell’enzima PNMT ai fattori in grado di regolare il ciclo riproduttivo del tritone, attraverso un trattamento di esemplari di T. carnifex con FSH (ormone follicolo-stimolante); infatti l’enzima PNMT di T. carnifex, come riportato in letteratura per gli Anfibi Anuri, appare insensibile all’azione stimolante dei glucocorticoidi, che si osserva invece in altre classi di Vertebrati 3) sequenziando l’mRNA dell’enzima PNMT per ottenere le sequenze nucleotidica e aminoacidica dedotte ( sulle quali sono assenti dati bibliografici negli Anfibi). La presenza dell’enzima PNMT nelle cellule cromaffini del tritone è stata dimostrata sia per via immunocitochimica che mediante ibridazione in situ. I risultati del trattamento con FSH hanno evidenziato un’influenza inibitoria di quest’ultimo sull’enzima PNMT, e quindi una sensibilità dell’enzima ai fattori che regolano il ciclo riproduttivo di questa specie. Questo risultato confermerebbe la relazione tra ciclo funzionale delle cellule cromaffini e ciclo riproduttivo, già supposta a seguito di precedenti ricerche. Infine, mediante il sequenziamento dell’mRNA, la candidata ha potuto confrontare le sequenze nucleotidiche e aminoacidiche dedotte con quelle adrenaliche note (uomo, bovino, ratto e topo). Il confronto ha evidenziato un 17% di omologia, localizzato prevalentemente tra i nucleotidi 241-421, e probabilmente relativo alle funzioni più essenziali dell’enzima, presumibilmente più conservate nel corso dell’evoluzione. Il lavoro sperimentale ha richiesto l’utilizzo delle tecniche di base di microscopia ottica e elettronica, nonché di immunocitochimica. Inoltre, tecniche di biologia molecolare, quali elettroforesi su gel, ibridazione in situ, estrazione di acidi nucleici, screening di library genomiche e di cDNA, PCR e RT-PCR, sequenziamento di frammenti di cDNA, clonaggi in vettori diversi

Cocaine effects on the gills of the European eel, Anguilla anguilla”

Many illicit drugs and their breakdown products are detected in the aquatic environment due to the worldwide use of these substances and the variable efficiency with which they are removed from sewage effluent in sewage treatment plants.1 These substances have strong pharmacological activity; indeed, despite their low concentrations in surface waters, the first studies showed toxic effects for aquatic organisms.2,3,4,5 The aim of this study was to evaluate the influence of environmental concentrations of cocaine, an illicit drug widespread in surface waters, on the gills of the European eel (Anguilla anguilla). A stock solution of 0.006 mg mL-1 cocaine free-base in ethanol was prepared. Male silver eels were exposed for fifty days to 20 ng L-1 of cocaine, corresponding to the mean cocaine concentration detected in surface waters.1 The gills of cocaine exposed eels were compared to untreated control and vehicle control groups. The morphology of the gills and plasma levels of prolactin and cortisol, involved in gill physiology regulation, were evaluated. The gills of cocaine-exposed eels showed an hyperplasic epithelium, in which many mucous cells were observed, and lamellar fusion. Moreover, cocaine increased plasma prolactin and cortisol levels. The changes observed in the gills epithelium agree with those observed in the intestine4 of cocaine-exposed eels. Since in fish prolactin and cortisol stimulate epithelial cell proliferation and the differentiation and proliferation of mucous cells, these hormones might be involved in the changes induced by cocaine. Fish gills are involved in gas exchange, in the exchange of salt and water and excretion of nitrogenous waste products; therefore, even slight structural changes can render a fish vulnerable to osmoregulatory and respiratory difficulties. This study shows that even low cocaine concentrations affect the gills, suggesting potential impact on the survival of this species. 1. Rosi Marshall EJ et al. J Hazard Mater 2015,282;18-25. 2. Capaldo A et al. Water Air Soil Pollut 2012, 223:2137-2143. 3. Gay F et al. Water Air Soil Pollut 2013, 224:1579. 4. Gay F et al. J Fish Dis 2016, 39:295-308. 5. Monaco A et al. Eur J Histochem 2016,9

Human follicle- stimulating hormone modulation of adrenal gland activity in the Italian crested newt, Triturus carnifex (Amphibia, Urodela)

ABSTRACT The aim of the present study was to verify if human FSH influences the adrenal gland of the newt, Triturus carnifex. Newts were given intraperitoneal injections of human FSH throughout the periods of February–March, and December–January; periods in which newt FSH levels are normally very low. The effects of human FSH on adrenal gland activity were observed in the morphological features of the steroidogenic and chromaffin adrenal cells, and in the serum levels of aldosterone, corticosterone, norepinephrine and epinephrine. The effect of human FSH on the steroidogenic cells was significant during the February–March period; the quantity of cytoplasmic lipids decreased, and the corticosteroid serum levels increased. During the December–January period, the human FSH effects were negligible. The effect of human FSH on the chromaffin cells was significant during both the February–March, and the December–January periods. During February–March, the human FSH increased the numeric ratio of norepinephrine granules to epinephrine granules, and increased the epinephrine serum levels. During December–January, the human FSH decreased the numeric ratio of norepinephrine granules to epinephrine granules, and increased the norepinephrine serum levels. The results of the present study show that human follicle-stimulating hormone influences the activity of the newt adrenal gland, thus indicating a relationship between the annual sexual cycle and the annual adrenal cycle of the newt

Environmental effects of cocaine addiction: the muscle of the European eel (Anguilla anguilla) exposed to environmental cocaine concentrations.

Objective: Cocaine is a common organic contaminant of the aquatic environment (Pal et al. 2013). Due to its pharmacological activity, its presence in surface waters suggests new hazards, still unknown, for fish, living in contaminated waters. A chronic exposure to environmental cocaine concentrations (20 ng/l) alters the endocrine system of European eels (Gay et al. 2013) that accumulate cocaine into their tissues, especially muscle (Capaldo et al. 2012), the edible part of the animal, suggesting risks for this species and potentially for humans, consuming contaminated fish. Therefore, we aimed to verify whether environmental cocaine concentrations could damage the muscle of the European eel. Method: Adult eels were divided into four groups (control, carrier, treated, post-exposure recovery), each containing ten specimens. A stock solution of 3 mg/500 ml cocaine free-base in ethanol was prepared. Control, treated and carrier groups were exposed daily, during 50 days, to: tap water; 20 ng/l cocaine; ethanol, respectively; a post-exposure recovery group was exposed to cocaine, as treated group, and then deprived of cocaine and exposed to tap water, during ten days. Plasma levels of the following enzymes and hormones were determined: lactate dehydrogenase (LDH) and M1 muscle-specific creatine kinase (M1-CK), well-known indicators of tissue injury; growth hormone- releasing hormone (GHRH) and corticotrophin- releasing hormone (CRH), regulating in the eel growth hormone (GH) release; GH and insulin-like growth factor 1 (IGF-1), involved in eel muscle metabolism. Samples of muscles were fixed in Bouin solution, embedded in Paraplast and stained with Mallory trichromic stain for histological examination. Results: Cocaine significantly (P < 0.001) increased the levels of LDH (from 45.30 U/ml in controls to 62.60 U/ml in treated); M1-CK (from 38.52 U/ml to 151.28 U/ml); GHRH (from 2.15 ng/ml to 7.33 ng/ml); CRH (from 50.13 ng/ml to 82.13 ng/ml); GH (from 0.90 ng/ml to 3.27 ng/ml); IGF-1 (from 0.28 ng/ml to 0.85 ng/ml). All the values in post-exposure recovery eels were still significantly (P < 0.001) higher than control values (LDH: 102.50 U/ml; M1-CK: 188.78 U/ml; GHRH: 4.31 ng/ml; CRH: 93.81 ng/ml; GH: 5.37 ng/ml; IGF-1: 0.71 ng/ml). Histological examination revealed striated muscle damages in both treated and post-exposure recovery eels. Conclusion: The present results show that cocaine, at environmental concentrations, damages the eel muscle physiology and morphology, as shown by the strong increase in plasma enzyme and hormone levels and the alterations of the muscle, also evident in the recovery eels. These results agree with those of previous studies, showing the presence of cocaine (20,17 ± 0.47 pg/g) in the muscle of eels chronically exposed to environmental cocaine concentrations (Capaldo et al. 2012) and showing human muscle damages induced by cocaine (Tóth and Varga, 2009). Moreover, these results indicate that cocaine addiction is not only a human, but also an environmental problem, since cocaine (and probably the other illicit drugs) directly affects fish health, disrupting their muscle and endocrine system. Finally, taken together, the presence of cocaine in the eel muscle, and the alterations, cocaine-induced, in this tissue, suggest probable risks to the human health, through the food chain

Endocrine Disruption in the European Eel, Anguilla anguilla, Exposed to an Environmental Cocaine Concentration

The aim of the present study was to verify if cocaine, at environmental concentrations, influences the endocrine system of the European eel. Silver eels (a stage of the eel life cycle preparing the fish for the oceanic reproductive migration) were exposed to a nominal cocaine concentration of 20 ng/l during thirty days; at the same time, control, carrier and post-exposure recovery groups were made. The effects of cocaine were observed in 1) brain dopamine content 2) plasma catecholamine levels: dopamine, norepinephrine and epinephrine 3) pituitary-adrenal axis activity: plasma adrenocorticotropic hormone (ACTH), corticosterone, cortisol and aldosterone levels 4) pituitary-thyroid axis activity: plasma thyroid-stimulating hormone (TSH), triiodothyronine and thyroxine levels. In the treated group, brain dopamine, plasma catecholamines, cortisol and TSH levels were higher, whereas ACTH, corticosterone and triiodothyronine levels were lower than controls. In the post-exposure recovery group, brain dopamine, plasma dopamine and epinephrine, and thyroxine levels further increased, whereas plasma norepinephrine, cortisol and corticosterone levels were similar to treated values. Finally, ACTH and TSH were similar, whereas triiodothyronine levels were lower than controls. Aldosterone levels were unaffected by cocaine exposure. The results of the present study show that cocaine, at environmental concentrations, behaves like an endocrine disruptor changing brain dopamine and plasma catecholamine levels, and the activity of pituitary-adrenal/thyroid axes. Since the endocrine system plays a key role in the metabolic and reproductive processes of the eel, our results suggest that environmental cocaine could be considered another cause for the decline in the European eel

The pituitary-adrenal gland activity of Triturus carnifex (Amphibia, Urodela) exposed to environmental cadmium concentrations

The aim of the present study was to verify whether cadmium (Cd), at environmental concentrations, influences the pituitary-adrenal activity of the newt, Triturus carnifex. Newts were exposed to 5 µg and 20 µg doses of cadmium (limits fixed by Italian laws in drinking and surface waters, respectively) during 3 months. The effects of cadmium were observed in the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine. At both doses, ACTH and corticosterone serum levels decreased, whereas aldosterone serum levels increased. Epinephrine serum levels increased whereas norepinephrine serum levels remained unchanged. The results of the present study show that cadmium, at environmental concentrations, behaves as an endocrine disruptor influencing the pituitary-adrenal activity of the newt. This suggests that cadmium could contribute to amphibian decline