Gerardo Centoducati

AFLATOXINS IN AQUATIC SPECIES: METABOLISM, TOXICITY AND PERSPECTIVES

Among all known mycotoxins, aflatoxins represent the most investigated, widespread and worrisome source of contamination of foods and feed worldwide. In the early 1960s, soon after the finding of aflatoxin B1 (AFB1) in the feedstuffs of aquacultured rainbow trout that had died in an epizootic of hepatomas, great scientific discoveries were made in several areas by a number of researchers under the direction of scientists like J. Halver, R. 0. Sinnhuber, G. S. Bailey, J. D. Hendricks and colleagues. Since that time, several studies have focused on the identification of new isoenzymes involved in AFB1 metabolism and on the discovery of new modulators in AFB1-induced cancer initiation and progression. However, metabolic and toxicological studies on aflatoxins in marine aquacultured species are fragmented and restricted to a limited number of fish species. Aflatoxins exert a substantial impact on the fish farming production, causing disease with high mortality and a gradual decline of reared fish stock quality, thus representing a significant problem in aquaculture systems. Based on these considerations, the goals of this review article are: (1) to gather the currently available scientific information, summarising existing data on aflatoxin contamination on feeds and fishmeals, and toxicological effects induced in reared aquatic species; (2) to make a comparative analysis of AFB1 metabolism in the most representative species studied; (3) to gain new insights on the risk of DNA damage caused by aflatoxins on fish genomes and their role in cancer development.

Effect of different levels of dietary sweet orange (Citrus sinensis) peel extract on humoral immune system responses in broiler chickens.

This experiment was conducted to evaluate the effects of different levels of sweet orange (Citrus sinensis) peel extract (SOPE) on humoral immune system responses in broiler chickens. Three hundred 1-day broilers (Ross-308) were randomly allocated to treatments varying in supplemental SOPE added in the drinking water. The experimental groups consisted of three treatments fed for 42 days as follows: a control treatment without feed extract, a treatment containing 1000 ppm of SOPE and a treatment containing 1250 ppm of SOPE. All treatments were isocaloric and isonitrogenous. Broilers were vaccinated with Newcastle disease virus (NDV), avian influenza (AI), infectious bursal disease (IBD) and infectious bronchitis virus (IBV) vaccines. Antibody titer response to sheep red blood cells (SRBC) was higher in the group fed 1250 ppm of SOPE (P < 0.05) as well as for immunoglobulin G (IgG) and IgM. Similarly, antibody titer responses to all vaccines were constantly elevated (P < 0.05) by SOPE enrichment in a dose-dependent manner. Relative weights of spleen and bursa of Fabricius were unaffected by treatments. Dietary SOPE supplementation may improve the immune response and diseases resistance, indicating that it can constitute a useful additive in broiler feeding. Thus, supplying SOPE in rations may help to improve relative immune response in broiler chickens.

Effects of Mannan Oligosaccharide and Inulin on Sharpsnout Seabream (Diplodus Puntazzo) in the Context of Partial Fish Meal Substitution by Soybean Meal

One hundred forty-four sharpsnout seabream of about 100 g initial body weight were randomly stocked in 12 experiment tanks (180 L). Testing conditions included 12 fish per tank, with triplicate tanks for treatment. The experimental period lasted 150 days. Average water temperature was 21.9±1.6°C, salinity was 30.0‰ and pH ranged from 7 to 8, throughout the experiment. A control diet (FM) was made from fish meal. One similar diet (SBM) was made with approximately 40% of the protein supplied by soybean meal. The remaining two diets (SBM-MOS and SBM-INU) were formulated adding 8 g of mannanoligosaccharide (MOS) and inulin (INU) per kg of the SBM diet, respectively. The results showed that mean final weight (average values 234.4 g), specific growth rate (average values 0.585), feed conversion rate (average values 2.05) and protein efficiency ratio (average values 1.01) were unaffected by MOS or INU supplementation to SBM diet. Body proximate composition was affected by MOS and INU supplementation. Fish fed SBMMOS and SBM-INU diets showed the highest moisture level and the lowest lipid content. Also the total polyunsaturated fatty of the lipids was reduced by MOS and INU in comparison to SBM diet alone.

Effects of the partial substitution of fish meal by soy bean meal with or without mannanoligosaccharide and fructooligosaccharide on the growth and feed utilization of sharpsnout seabream, Diplodus puntazzo (Cetti, 1777): preliminary results

The present study was carried out in order to investigate the effects of the mannanoligosaccharide (MOS) and fructooligosaccharide (FOS) on sharpsnout seabream, Diplodus puntazzo in the context of partial fish meal substitution by soybean meal. One-hundred-forty-four sharpsnout seabream of about 100 g initial body weight were randomly divided in 12 experimental tanks (180 litre each). Testing conditions included 12 fish per tank, with triplicate tanks for treatment. The experimental period lasted 114 days. Average water temperature was 21.9±1.6°C, salinity was 30.0%0 and pH ranged from 7 to 8. Four isonitrogenousisolipidic diets were tested: a control diet (FM) with fish meal as the sole protein source; a second diet (SBM) with approximately 40% of the protein supplied by soybean meal. The remaining two diets were formulated adding 8g of MOS and FOS per kg of the SBM diet respectively. Average final weight, specific growth rate, feed conversion ratio and protein efficiency ratio remained unaffected by partial fish meal substitution and by MOS or FOS supplementation. Also apparent digestibility coefficients values for organic matter, protein, lipid and energy were not significantly affected by dietary treatment.

Diablo/SMAC: A novel biomarker of pollutant exposure in European flounder (Platichthys flesus)

Diablo (or SMAC) is a protein released from mitochondria following apoptotic stimuli and inhibits the actions of Inhibitors of Apoptosis (IAP) proteins. IAPs regulate the activity of caspases and NFkB, the primary executioners of apoptosis and of inflammation, respectively. Thus, Diablo is important for the regulation of cellular responses to damage. In Northern Europe, statutory governmental marine monitoring programs measure various biomarkers in flounder to indicate biological effects of pollutant exposure. More recently transcriptomic techniques have been applied in flounder to gain a more comprehensive understanding of pollutant effects, and to discover novel biomarkers. In most of these studies utilising flounder, Diablo was amongst the most highly increased transcripts identified. The aim of this study was to further examine piscine Diablo, at the gene level and mRNA level, after exposure to prototypical pollutants, and in flounder caught from polluted environments. The results show that two genes encoding Diablo exist in fish species, and in flounder one of these genes is increased in liver after exposure to polyaromatic hydrocarbons and polychlorinated biphenyls, and also in livers from fish living on contaminated estuarine sediments. Therefore, Diablo measurement has potential as a biomarker of pollutant exposure, and could indicate damaging effects of chemical contaminants.

Molluscs and echinoderms aquaculture: Biological aspects, current status, technical progress and future perspectives for the most promising species in Italy

Shellfish aquaculture is a widespread activity in the Italian peninsula. However, only two bivalve species are mainly cultured along the coastline of that country: the Mediterranean mussel Mytilus galloprovincialis and the Manila clam Venerupis philippinarum (Ruditapes philippinarum). By contrast, just a few other mollusc species of commercial interest are scarcely reared at a small-scale level. After analysing the current status of Italian shellfish production, this paper reports and discusses the potential for culturing several different invertebrate species [i.e., the European flat oyster Ostrea edulis, the grooved carpet shell Venerupis decussata (Ruditapes decussatus), the razor clams Ensis minor and Solen marginatus, the cephalopod Octopus vulgaris, and the purple sea urchin Paracentrotus lividus] in this country. In addition, a detailed overview of the progress made in aquacultural techniques for these species in the Mediterranean basin is presented, highlighting the most relevant bottlenecks and the way forward to shift from the experimental to the aquaculture phase. Finally, an outlook of the main economic and environmental benefits arising from these shellfish culture practices is also given.

Effects of lycopene on in vitro quality and lipid peroxidation in refrigerated and cryopreserved turkey spermatozoa

1. The effects of lycopene-enriched extenders on the in vitro quality of turkey semen including lipid peroxidation were examined after chilled and frozen storage. 2. Five pools of semen diluted in extenders containing 0, 0·05 or 0·1 mg/ml of lycopene were stored at 5°C for 48 h or cryopreserved as pellets and the following variables determined in fresh samples and samples stored chilled or frozen: sperm motility, viability, osmotic resistance, DNA integrity and lipid peroxidation (as malonaldehyde production). 3. Semen quality was generally compromised after storage, especially post-freezing. However, in the presence of the highest dose of lycopene, both the viability and osmotic-resistance of chilled spermatozoa and the DNA integrity of frozen spermatozoa were similar to those of fresh spermatozoa. 4. Greater lipid peroxidation was detected in refrigerated compared to fresh or cryopreserved spermatozoa. However, spermatozoa chilled in lycopene-enriched extenders showed significantly lower malonaldehyde levels than those chilled without lycopene, while the addition of lycopene to the freezing medium served to maintain the lipid peroxidation levels observed in fresh semen. 5. In conclusion, the presence of lycopene in the extender improved the survival of turkey spermatozoa after liquid-storage and protected DNA integrity against cryodamage. The beneficial effects of lycopene observed could be related to its capacity to diminish sperm lipid peroxidation during refrigeration or cryopreservation.

Effects of benzo[a]pyrene on gilthead seabream (Sparus aurata L.) hepatocytes exposed in vitro to short and long term trials

In the present study, cytotoxic effects of the polycyclic aromatic hydrocarbons benzo[a] pyrene (B[a]P) were investigated in Sparus aurata hepatocytes primary cultures after acute and chronic exposure. Cells were treated with a wide range of B[a]P doses (1 pg/mL to 100 μg/mL) for 24, 48 and 72 h. B[a]P toxicity was quantified in sea bream hepatocytes by MTT assay and immunofluorescence analysis of apoptosis after the various exposure periods, in order to evaluate the hepatic damage and toxicity range. Results showed three cytotoxic responses: B[a]P cell death for primary necrosis after exposure to high concentrations for short times, apoptosis induction with the use of sublethal doses and cell proliferation allied with neoplastic foci formation after exposure to low concentrations for long times. This responses provided an interesting correlation between the damage caused on hepatocytes and the metabolism of this toxic compound, to date mainly studied in vivo. Additionally, the statistical analysis revealed that the effects of time and dose were significant for both parameters and especially the time was extremely significant (P<0.0001), in fact B[a]P induced damage that increased over time. Our findings demonstrated and confirmed that S. aurata is a very sensitive species to B[a]P exposure since adverse effects were found at all tested doses. Furthermore, the new in vitro animal model can be considered a useful tool for studying the cellular effects induced by any contaminant harmful for farmed fish.

A multidisciplinary study of the extracutaneous pigment system of European sea bass (Dicentrarchus labrax L.). A possible relationship between kidney disease and dopa oxidase activity level

Infectious diseases and breeding conditions can influence fish health status. Furthermore it is well known that human and animal health are strongly correlated. In lower vertebrates melano-macrophage centres, clusters of pigment-containing cells forming the extracutaneous pigment system, are widespread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. Hence, they are employed as biomarker of fish health status. We have investigated this cell system in the European sea bass (Dicentrarchus labrax L.) following the enzyme activities involved in melanin biosynthesis. We have found a possible relationship between kidney disease of farmed fishes and dopa oxidase activity level, suggesting it as an indicator of kidney disease. Moreover variations of dopa oxidase activity in extracutaneous pigment system have been observed with respect to environmental temperature. At last, for the first time, using femtosecond transient absorption spectroscopy (Femto-TA), we pointed out that pigment-containing cells of fish kidney tissue present melanin pigments.

Characterization of the cellular damage induced by Aflatoxin B1 in sea bream (Sparus aurata Linnaeus, 1758) hepatocytes

Abstract Gilthead sea bream (Sparus aurata L.) is one of the most intensively farmed fish species in the Mediterranean, greatly studied for the relevant economic value, although its sensitivity to Aflatoxin B1 (AFB1) has to be investigated, yet. The aim of this study was to perform an in vitro evaluation of cytotoxic potential of AFB1 on S. aurata hepatocytes in order to grade the range of AFB1 toxicity, and the boundary between acute and long-term toxicity. Primary monolayer cultures of hepatocytes from S. aurata juveniles were treated with a wide range of concentrations from 5x103 ng/ml to 2x10-5 ng/ml of AFB1 for a different period of exposure (24, 48, 72 hours). The cytotoxic activity was characterized by MTT reduction assay. After each exposition hepatocytes were examined for morphologic alterations and apoptosis induction. AFB1 exposure significantly reduced cell viability in a dose- and time-dependent manner. Dose-response curves obtained after 24, 48 and 72 hrs revealed that prolonged exposure times lead to a significant increase of the toxic potency of AFB1. Our results demonstrate that S. aurata hepatocytes are highly sensitive to AFB1 exposure. Such scientific findings could provide new insights to investigate the real impact of aflatoxin on marine farmed fish.