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SUMMARY – The expression of HIF-1a and HSF1 was investigated in plasma samples (T0: at birth; T12: 12 hours after birth) of 9 neonates (4 healthy and 5 sick foals); plasma MDA concentration was also evaluated in 16 neonates (10 healthy and 6 sick foals). MDA concentration, HIF-1a and HSF1 expression decreased significantly at T12 if compared with T0 (p< 0.05). HIF-1a was significantly higher in sick foals at all sampling times (p< 0.05) and resulted correlated to pCO2 at T0 and to MDA at T12 (p< 0,05; R=0,83 e R=-0,72, respectively). HSF was significantly correlated to HCT and RBC at T0 (p< 0,05; R=0,68 e R=0,77, respectively). The parameters measured could be considered potential indexes of perinatal hypoxia.

Bidimensional electrophoresis (2-DE) protocols were adapted on Chamelea gallina digestive glands studies by the analysis of Heat Shock Proteins (HSP) compared with monodimensional electrophoresis (1-DE) results. Because polycyclic aromatic hydrocarbons (PAH) act on HSPs, C. gallina specimens were exposed to 0.5 mg/L of benzo[a]pyrene (B[a]P) for 24 h, 7 and 12 days. Immunoblotting after 1-DE showed a single band of 70 kDa significantly induced after 7 days of B[a]P exposure. After 2-DE, eight major high-resolved spots between 17 and 98 kDa were revealed. Three spots fell within the range of 62–98 kDa and of 5–6 pI, parameters which could include HSP70. Two spots of 77 and 72 kDa, obtained after 2-DE immunoblotting, could correspond to constitutive HSC70 and to inducible HSP70 forms respectively. Changes observed in inducible and in constitutive forms might be related to an adaptation to stress and to a normal protein synthesis capability, respectively. Employment of 2-DE and relationship between HSP70 and HSC70 may be useful to clarify their role in molluscs subjected to stress events.

Gli organismi viventi che tollerano ampie variazioni di ossigeno durante episodi di ipossia/anossia (euriossici), presentano adattamenti biochimici e fisiologici che ne consentono la sopravvivenza fino al ripristino delle normali condizioni di ossigenazione. Il ritorno alla normossia è caratterizzato da un aumento repentino del consumo di ossigeno che può indurre la formazione di specie radicaliche dell’ossigeno (ROS) con un conseguente danno ossidativo. Dal momento che i ROS sono continuamente prodotti, gli organismi hanno evoluto un sistema di difesa antiossidante costituito da componenti enzimatiche e non enzimatiche. I sistemi enzimatici maggiormente coinvolti sono rappresentati da superossido dismutasi (SOD), da catalasi (CAT) e dalla glutatione perossidasi Se-dipendente (GPx). Nel presente studio è stato utilizzato come animale sperimentale Scapharca inaequivalvis, una specie di mollusco bivalve presente in Adriatico, dal momento che la risposta alla riossigenazione degli animali euriossici è proponibile come un modello utile per lo studio del danno da ischemia/riperfusione. Gli animali sono stati esposti ad anossia per 3,6,24 ore e riossigenazione per 1,3,5,24 ore. Sulla ghiandola digestiva dei molluschi è stata investigata l’attività di SOD, CAT, GPx e G6PDH (tecniche spettrofotometriche), nonchè l’espressione di due isoforme della SOD, MnSOD e CuZnSOD (immunoblotting). I risultati hanno mostrato alti livelli di SOD sia in anossia che in riosssigenazione; CAT e GPx aumentano significativamente durante il recupero mostrando una correlazione inversa dal momento che competono per lo stesso substrato a dimostrazione di una attività di difesa contro lo stress ossidativo integrata ed efficiente. L’aumento del GPx, corrispondente ad un picco di attività della G6PDH alla dodicesima ora di riossigenazione, potrebbe essere riconducibile a fenomeni di perossidazione lipidica. Dall’immunoblotting si nota che solo la CuZnSOD viene maggiormente espressa dopo 6 e 24h di anossia e 3h di riossigenazione. Nei mammiferi in ischemia si assiste ad una caduta della concentrazione degli enzimi antiossidanti, il ritorno alla normossia (riperfusione o riossigenazione) è caratterizzato da un rapido aumento del consumo di ossigeno che induce la formazione di ROS. Gli episodi degenerativi a livello cellulare iniziano a comparire solo in seguito alla reintroduzione dell’ossigeno molecolare nel tessuto ischemico. La presenza di alti livelli di enzimi anche in anossia indica una risposta di S. inaequivalvis tipica degli animali eurossici e utile per lo studio dei processi ischemici in mammiferi.

Nei teleostei il rame svolge funzioni vitali come cofattore in processi biologici-chiave, ma può anche manifestare effetti tossici, in dipendenza della biodisponibilità e del bioaccumulo. La forma Cu2+ del rame è quella a maggiore biodisponibilità. La tossicità del rame varia a seconda della specie ed è noto che gli stadi larvali dei pesci sono molto più sensibili al metallo rispetto agli adulti della stessa specie. Nella stessa specie tessuti e processi diversi possono inoltre manifestare effetti estremamente diversificati. Le principali vie di assunzione del metallo nei pesci sono rappresentate dalle branchie e dall’apparato digerente. I dati presentati fanno parte di una ricerca multidisciplinare che abbraccia un vasto spettro di indagini su diversi organi e tessuti e sono stati ottenuti in orate (Sparus auratus) esposte a CuSO4 (0,5 ppm nominali di Cu, salinità 33‰, t 10°C) e mantenute per 50 gg in un sistema statico, senza alimentazione. Parellelamente nelle stesse condizioni sono state mantenute due vasche con animali non esposti (controllo). Durante tutta la sperimentazione non è stata riscontrata mortalità, né visibili sofferenze o alterazioni comportamentali degli animali. Lipidi e acidi grassi. Le indagini condotte sul grasso periviscerale, più esposto rispetto ad altri distretti ai cambiamenti che si possono produrre in un organismo, evidenzia uno pettro degli acidi grassi tipico di un teleosteo allevato in acqua salata, alimentato quindi con diete contenenti grassi di origine vegetale come suggerito dalla cospicua presenza di 18:2n-6. Il digiuno protratto per tutti i 50 giorni di sperimentazione non ha modificato i rapporti reciproci tra i vari acidi grassi le cui percentuali al tempo 0 sono sovrapponibili con quelle di fine esperimento. La temperatura complessivamente bassa per tutta la durata dell’esperimento può aver ridotto al minimo le esigenze metaboliche e consentito il mantenimento dei valori iniziali anche dei saturi a corta catena. La stessa ridotta attività metabolica può aver interessato anche i processi attraverso cui si esplica l’azione del rame il cui effetto non è risultato quindi rilevabile su questo parametro. Espressione citocromo P450 (CYP1A): fegato - branchie. Indagini preliminari sul citocromo P450 (CYP 1A), eseguite mediante Western Blotting con anticorpi policlonali anti-fish in campioni di controllo e in campioni prelevati dopo 14 giorni di esposizione, hanno evidenziato in fegato e branchie due bande distinte di 49 e 61 kDa. Nel fegato la banda di 49 kDa non mostra differenze significative fra le condizioni esaminate mentre la banda di 61 kDa risulta più espressa nei trattati. Nelle branchie non si evidenziano differenze significative per quanto concerne la banda di più alto peso molecolare, mentre la banda inferiore sembra significativamente meno espressa nei trattati rispetto ai controlli.

In the last decades, the increase of industrial activities and of the request for the world food requirement, the intensification of natural resources exploitation, directly connected to pollution, have aroused an increasing interest of the public opinion towards initiatives linked to the regulation of food production, as well to the institution of a modern legislation for the consumer guardianship. This work was planned taking into account some important thematics related to marine environment, collecting and showing the data obtained from the studies made on different marine species of commercial interest (Chamelea gallina, Mytilus edulis, Ostrea edulis, Crassostrea gigas, Salmo salar, Gadus morhua). These studies have evaluated the effects of important physic and chemical parameters variations (temperature, xenobiotics like drugs, hydrocarbons and pesticides) on cells involved in the immune defence (haemocytes) and on some important enzymatic systems involved in xenobiotic biotransformation processes (cytochrome P450 complex) and in the related antioxidant defence processes (Superoxide dismutase, Catalase, Heat Shock Protein), from a biochemical and bimolecular point of view. Oxygen is essential in the biological answer of a living organism. Its consume in the normal cellular breathing physiological processes and foreign substances biotransformation, leads to reactive oxygen species (ROS) formation, potentially toxic and responsible of biological macromolecules damages with consequent pathologies worsening. Such processes can bring to a qualitative alteration of the derived products, but also to a general state of suffering that in the most serious cases can provoke the death of the organism, with important repercussions in economic field, in the output of the breedings, of fishing and of aquaculture. In this study it seemed interesting to apply also alternative methodologies currently in use in the medical field (cytofluorimetry) and in proteomic studies (bidimensional electrophoresis, mass spectrometry) with the aim of identify new biomarkers to place beside the traditional methods for the control of the animal origin food quality. From the results it’s possible to point out some relevant aspects from each experiment: 1. The cytofluorimetric techniques applied to O. edulis and C. gigas could bring to important developments in the search of alternative methods that quickly allows to identify with precision the origin of a specific sample, contributing to oppose possible alimentary frauds, in this case for example related to presence of a different species, also under a qualitative profile, but morpholgically similar. A concrete perspective for the application in the inspective field of this method has to be confirmed by further laboratory tests that take also in account in vivo experiments to evaluate the effect in the whole organism of the factors evaluated only on haemocytes in vitro. These elements suggest therefore the possibility to suit the cytofluorimetric methods for the study of animal organisms of food interest, still before these enter the phase of industrial working processes, giving useful information about the possible presence of contaminants sources that can induce an increase of the immune defence and an alteration of normal cellular parameter values. 2. C. gallina immune system has shown an interesting answer to benzo[a]pyrene (B[a]P) exposure, dose and time dependent, with a significant decrease of the expression and of the activity of one of the most important enzymes involved in the antioxidant defence in haemocytes and haemolymph. The data obtained are confirmed by several measurements of physiological parameters, that together with the decrease of the activity of 7-etossi-resourifine-O-deetilase (EROD linked to xenobiotic biotransformation processes) during exposure, underline the major effects of B[a]P action. The identification of basal levels of EROD supports the possible presence of CYP1A subfamily in the invertebrates, still today controversial, never identified previously in C. gallina and never isolated in the immune cells, as confirmed instead in this study with the identification of CYP1A-immunopositive protein (CYP1A-IPP). This protein could reveal a good biomarker at the base of a simple and quick method that could give clear information about specific pollutants presence, even at low concentrations in the environment where usually these organisms are fished before being commercialized. 3. In this experiment it has been evaluated the effect of the antibiotic chloramphenicol (CA) in an important species of commercial interest, Chamelea gallina. Chloramphenicol is a drug still used in some developing countries, also in veterinary field. Controls to evaluate its presence in the alimentary products of animal origin, can reveal ineffective whereas the concentration results to be below the limit of sensitivity of the instruments usually used in this type of analysis. Negative effects of CA towards the CYP1A- IPP proteins, underlined in this work, seem to be due to the attack of free radicals resultant from the action of the antibiotic. This brings to a meaningful alteration of the biotransformation mechanisms through the free radicals. It seems particularly interesting to pay attention to the narrow relationships in C. gallina, between SOD/CAT and CYP450 system, actively involved in detoxification mechanism, especially if compared with the few similar works today present about mollusc, a group that is composed by numerous species that enter in the food field and on which constant controls are necessary to evaluate in a rapid and effective way the presence of possible contaminations. 4. The investigations on fishes (Gadus morhua, and Salmo salar) and on a bivalve mollusc (Mytilus edulis) have allowed to evaluate different aspects related to the possibility to identify a biomarker for the evaluation of the health of organisms of food interest and consequently for the quality of the final product through 2DE methodologies. In the seafood field these techniques are currently used with a discreet success only for vertebrates (fishes), while in the study of the invertebrates (molluscs) there are a lot of difficulties. The results obtained in this work have underline several problems in the correct identification of the isolated proteins in animal organisms of which doesn’t currently exist a complete genomic sequence. This brings to attribute some identities on the base of the comparison with similar proteins in other animal groups, incurring in the possibility to obtain inaccurate data and above all discordant with those obtained on the same animals by other authors. Nevertheless the data obtained in this work after MALDI-ToF analysis, result however objective and the spectra collected could be again analyzed in the future after the update of genomic database related to the species studied. 4-A. The investigation about the presence of HSP70 isoforms directly induced by different phenomena of stress like B[a]P presence, has used bidimensional electrophoresis methods in C. gallina, that have allowed to isolate numerous protein on 2DE gels, allowing the collection of several spots currently in phase of analysis with MALDI-ToF-MS. The present preliminary work has allowed therefore to acquire and to improve important methodologies in the study of cellular parameters and in the proteomic field, that is not only revealed of great potentiality in the application in medical and veterinary field, but also in the field of the inspection of the foods with connections to the toxicology and the environmental pollution. Such study contributes therefore to the search of rapid and new methodologies, that can increase the inspective strategies, integrating themselves with those existing, but improving at the same time the general background of information related to the state of health of the considered animal organism, with the possibility, still hypothetical, to replace in particular cases the employment of the traditional techniques in the alimentary field.

HSP70, HSP27 and TNF-a were measured by immunoblotting in samples of pathological and healthy cutaneous and gland tissues (20 dogs of different ages and sex). HSP70 and HSP27 expressions are respectively characterized by a significant increase (p

The expression of inducible and constitutive forms of HSP70s, respectively HSP70 and HSC70, were investigated in skin samples of pathological and healthy tissues (28 dogs of different ages and sex). HSP70 increased in 54.3% (p

To prove CYP1A-immunopositive protein presence and to evaluate MnSOD behaviour in haemocytes and haemolymph, specimens of C. gallina were exposed to 0.5 mg/L of Benzo[a]pyrene for 12 days. CYP1A-immunopositive protein showed two bands of 53.5 kDa and 63.8 kDa not differently expressed, whereas MnSOD activity decreased after 7 and 12 days of exposure.

Environmental stresses are responsible of HSPs induction, that guarantee tolerance of otherwise lethal conditions. This study was performed to evaluate HSP70 expression in Chamelea gallina (Bivalvia) digestive gland in response to oxygen, temperature, salinity changes and B[a]P exposure. The results obtained confirm general induction of HSP70 in all the stress conditions tested.

SOD activity and expression were evaluated in haemocytes of Chamelea gallina exposed for seven days to three different temperatures. The values recorded for the acitivity of Total SOD, Mn-SOD and CuZn-SOD were significantly lower in specimens kept at 30° C. Even the results of immumological analysis (Western Blotting) revealed a minor expression at the end of the exposure at the same temperature for Mn-SOD, CuZn-SOD and EC-SOD. The high mortality recorded at 30° C is probably related to a lower phagocytosis activity and to a lower production of ROS, a useful defence against foreign elements.

The major animal responses to acute shock are known to involve changes in gene transcription and translation of heat shock protein (HSPs) . HSPs show different roles as chaperones in normal and damaged protein folding and in maintenance of receptor conformation. Marine littoral invertebrates tolerate a remarkable array of natural stresses, including anoxia, thermal and osmotic fluctuations. Several of these environmental stresses are responsible of HSP induction, that contribute to the tolerance of otherwise lethal conditions. This study was aimed at detecting HSP70 expression in Chamelea gallina tissues in response to oxygen, temperature and salinity changes.

I ROS rivestono un importante ruolo microbicida e allo stesso tempo sono responsabili di molteplici danni a diverse macromolecole biologiche (stress ossidativi). Per contrastare lo stress ossidativo gli organismi aerobi dispongono di sistemi di difesa. In particolare la superossido dismutasi (SOD) è il principale enzima coinvolto nella dismutazione dello ione superossido (O2-•). Dal momento che le variazioni della tensione di ossigeno e della temperatura sono responsabili di potenziali rischi immunologici, con la presente ricerca ci proponiamo di valutare gli effetti della anossia/riossigenazione e della temperatura sull'attività ed espressione della SOD negli emociti del bivalve Chamelea gallina.

Aim: reactive oxigen species (ROS) have a well-estabilished role in the athogenesis of many forms of malignancy and superoxide dismutase(SODs) are the only enzymes dismuting one of the main radicals: the superoxide anion. Since many human tumors have shown significant changes in the activity and expression of SODs, this enzyme might be correlated with survival and clinical-pathological parameters for the prognosis of human carcinoma. This preliminary study were performed to assess the SODs(CuZnSOD and MnSOD) activity and expression in gastric adenocarcinoma with respect to healthy tissue using western blotting (W-B) technique as a specific and sensible clinical test.

Nel presente lavoro è stata monitorata l’attività della SOD, come importante biomarker di alterazioni dovute alla variazione del tenore di ossigeno, nel Mollusco Bivalve Tapes philippinarum (euriossico) in seguito ad anossia e riossigenazione, utilizzando tecniche enzimatiche e immunologiche.

Most of the enzymes involved in such biotransformation, including cytochrome P450 monooxygenase or mixed function oxygenase (MFO) systems have been identified in marine vertebrates and invertebrates. Several studies suggest that during phase I reactions cytochrome P450 and the flavoprotein reductase components of the MFO system are involved in the formation of oxygen free radicals or metabolite radicals. Since the oxyradical generation causes biological damage, the integrity of the cell is protected by antioxidant defences. Antioxidant enzymes as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), play a crucial role in maintaining cell homeostasis. The aim of this work is to study the induction of cytochrome P450 after B[a]P exposure in Scapharca inaequivalvis and the relationship between the level of the antioxidant defences and P450 content

Hypoxia and anoxia are stress conditions that may affect physiological and biochemical responses in infaunal marine organisms, such as bivalves. Depending on the species, bivalves generally show a variable tolerance (measured as anoxic survival response) to hypoxia/anoxia periods, according to ability of organisms to modulate their metabolism. Low oxygen conditions have also been shown to affect bivalve immune responses (Hawkins et al., 1993; Pampanin et al., 2002). In this work, the effects of anoxia on functional responses of haemocytes were investigated in the commercially important species, Chamelea gallina. The capability of clams to recover after anoxic stress was also evaluated. Total haemocytes count (THC), phagocytosis and lysozyme activity were chosen as cellular biomarkers of exposure to anoxic conditions

The immune system of the animals consists of cellular and humoral elements involved in defence mechanisms from pathogens, parasites and foreign elements. Concerning to invertebrates, the immune responses are mediated by haemocytes through several internal defence mechanisms. As mammalian monocytes and macrophages, bivalve haemocytes show high phagocytosys activity versus non-self molecules and cells. Haemocytes activation determines an increasing oxygen consumption (respiratory burst) resulting in a reactive oxygen species (ROS) generation, that play a very important role in microbiocidal activity. However an excess of ROS production during an infection, can be dangerous for the cells, causing lipid peroxidation and DNA damages. The cell defence versus oxidative stress rely on enzymatic and non-enzymatic antioxidant systems. The superoxide dismutase (SOD) is one of the main enzyme involved in antioxidant activity. In this research will be evaluated the production of ROS (indirectly through SOD activity detection) by Chamelea gallina haemocytes during anoxia and following recovery and its role in immune responses.