Maria Pia Santacroce

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.

Aquaporin-4-containing astrocytes sustain a temperature- and mercury-insensitive swelling in vitro.

In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32‐kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature‐independent volume changes in response to osmotic gradients, in agreement with a channel‐mediated water transport. Water movement was found to be HgCl2 insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation. GLIA 31:29–38, 2000.

Increased activity of matrix metalloproteinases in the cerebrospinal fluid of patients with HIV-associated neurological diseases

Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in HIV-associated neurological diseases. The activity of the 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) was detected by zymography in the cerebrospinal fluid (CSF) of 138 HIV-infected patients (40 with AIDS dementia, 83 with brain opportunistic infections and 15 neurologically asymptomatic), 26 HIV-seronegative individuals with inflammatory neurological diseases (IND) and 12 HIV-seronegative subjects with noninflammatory neurological diseases (NIND). MMP-2 was present in all CSF samples from HIV-seropositive and HIV-seronegative individuals, including those of subjects with NIND. On the contrary, MMP-9 was absent in the CSF of NIND controls, whereas the activity of this MMP was found in the 77 - 100% of CSF samples from HIV-infected patients, including those with HIV dementia, central nervous system (CNS) opportunistic infections or neurologically asymptomatic subjects. The highest levels of MMP-9 were found in the CSF of patients with cryptococcosis, cytomegalovirus encephalitis and tuberculous meningitis and were comparable with those found in the CSF of HIV-negative patients with multiple sclerosis or meningitis. A significant correlation between CSF MMP-9 activity and CSF cell count was found only in patients with HIV dementia. The increased CSF activity of MMPs capable to degrade components of the extracellular matrix of blood-brain barrier may contribute to the transendothelial migration of virus-infected cells into the CNS and development of HIV-associated neurologic damage.

Regulation of gelatinases in microglia and astrocyte cell cultures by plant lectins

The effects of 25 recently discovered plant lectins on cell proliferation and enzyme release were compared to those of previously known lectins on rat microglia and astrocyte cell cultures. A dose‐dependent proliferation of microglial cells, but not of astrocytes, was induced by seven lectins, whereas five lectins showed dose‐dependent cytotoxicity on both microglia and astrocyte cell cultures. The activity of gelatinase B (MMP‐9) was strongly increased in microglial cells by the aforementioned seven lectins, by concanavalin A, and by phytohemagglutinin (PHA‐E4), whereas gelatinase A (MMP‐2) remained at constitutive levels. The five cytotoxic lectins decreased the activity of gelatinase B in microglia and of gelatinase A in astrocytes, in a dose‐dependent manner. The lectin wheat germ agglutinin induced a decrease in gelatinase B activity in microglia, but stimulated gelatinase A and B activity in astrocytes. These results indicate that lectins possess neuromodulatory effects that may motivate the study of their effects on central nervous system (CNS) function in vivo. This, in turn, may lead to better insight into whether lectin or lectin‐like molecules can interact with glial cells, and whether they have a role in acute toxicity and in multifactorial diseases in which environmental factors may play a role. GLIA 27:53–61, 1999.

Implications for chronic toxicity of benzo[a]pyrene in sea bream cultured hepatocytes: Cytotoxicity, inflammation, and cancerogenesis.

Benzo[a]pyrene (B[a]P) is the most studied dangerous polycyclic aromatic hydrocarbon for its hepatotoxic, carcinogenic, mutagenic, teratogenic, and immunosuppressant effects, which can affect both wild and farmed marine fish through the trophic chain. This study investigated, for the first time, the chronic effects induced in vitro by B[a]P prolonged exposure on gilthead sea bream (Sparus aurata L.) hepatocytes, evaluating the cellular and nuclear latent damage. The purpose was to characterize the kind of B[a]P cyto‐ and genotoxic damage by morphological and immunocytochemical parameters applied in combination with the use of multiple assay endpoints. In light of our results, the short‐term effects at higher B[a]P doses were linked to higher cytotoxicities and necrotic lysis, whereas a sustained inflammatory response at medium–low doses was perceived as a mitochondria‐mediated apoptosis, both by surface and nuclear morphological changes. The strong immunoreactivity for the cleaved caspase‐3 showed that the labeled cells committed suicide by apoptosis. B[a]P involvement on carcinogenesis comes from prolonged exposure at lower doses, establishing the connection between the escape from apoptosis and the selection of a tumoral phenotype. Cells colabeled with proliferating cell nuclear antigen/caspase‐3 within the proliferative foci, were proliferating transformed oval stem cells, which escaped the suicide by apoptosis allowing cancer development. Finally, it was established that sea bream cultured hepatocytes are highly sensitive to chronic B[a]P exposure, as serious genotoxic effects were found even at the lowest doses.

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.

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.

New Development in Aflatoxin Research: From Aquafeed to Marine Cells

Available data on the real impact of aflatoxins on farm aquatic species are very limited. Since long time, aflatoxin B1 (AFB1) has been considered the most potent food-born hepatotoxicant, frequently found in animal feedstuff. At present, it has been reported as responsible agent in unforeseen outbreaks of fish mortality due to acute or chronic aflatoxicosis, mainly well documented in freshwater species. The lack of information on the incidence of aflatoxicosis in marine reared teleosts may be partially due to the difficulty in accurately diagnosing the disease in fish, as well as to the lack of specie-specific in vitro models for toxicity studies. In this work: 1) we have verified that pelletted fish feed might be considered as sources of AFB1 contamination in aquaculture due to the isolation and identification of blue eye fungi (Aspergillus spp., Penicillium spp.) in feed samples, as well as other several genera (Fusarium, Cladosporium, Alternaria, Geotrichum, Mucor, Rizophus, Acremonium); 2) we have performed an in vitro evaluation of AFB1 potential cytotoxic on Sparus aurata hepatocyte primary cultures (SaHePs), using a multiple endpoint screening. Our results demonstrate that seabream hepatocytes are highly sensitive to AFB1 exposure and especially indicate three distinct pathways of cytotoxic response: necrotic cell death, apoptotic cell death and uncontrolled cell proliferation; 3) we have compared the dose response curves obtained by measuring the bioluminescence of Vibrio fischeri upon AFB1 exposure to those obtained from in vitro cell culture system. Results show equivalent and overlapping toxic responses with those from seabream hepatocytes.

The impact of hangingcleaning husbandry practices on Mediterranean mussels, Mytilus galloprovincialis Lmk, cultivated in the Mar Piccolo (Taranto, Ionian Sea, Italy)

Abstract The impact of the stressful hanging-cleaning husbandry practices on the growth conditions of the cultured Mediterranean mussels, Mytilus galloprovincialis Lmk, cultivated in the Mar Piccolo (Ionian Sea) was investigated from November 2005 to June 2006. The experimental strings were randomly organized in four groups, with five replications, exposed to different time periods of hanging: Group A, none; Group B, once every 15 days; Group C, once every 30 days; Group D, once every 60 days. The study, carried out on a total of 2000 mussels, showed that the cleaning of the mussel strings using the hanging-cleaning practice exerted an adverse effect on mussel growth (SGR%=46.62 in Group A; 44.85 in Group D; 43.72 in Group C; 42.12 in Group B). In particular, the highest percentage of meat yield occurred in the mussels more frequently exposed to air (Group B, 4.2±0.43 g) in spite of their lower morphometric variable values. In order to evaluate mussel condition, three different indexes were calculated. The hanging practice in mussel cultivation, commonly used in the basin of Taranto and in many mussel farms all over the world, would provide an economic benefit to the mussel farmers allowing the greatest production and the highest specific growth rate (SGR%) of mussels, together with a more appreciable aesthetic for the consumer. Our findings suggest that the hanging practice every 60 days would provide an economic benefit allowing an increase of about 30% of total string weight compared to the 30-day hanging practice commonly in use.