Marta Monari

Anoxic survival potential of bivalves: (arte)facts

The anoxic survival time of the bivalves Chamelea gallina, Cerastoderma edule and Scapharca inaequivalvis from two different ecosystems and differing anoxia tolerances was studied in static (closed) and flow-through systems. The antibiotics chloramphenicol, penicillin and polymyxin were added, and molybdate (specific inhibitor of the process of sulfate reduction). Survival in (near) anoxic seawater of Chamelea was studied in a static system by comparing untreated seawater with autoclaved seawater and untreated clams with clams incubated in well-aerated seawater, containing the broad-spectrum antibiotic chloramphenicol, prior to the anoxic survival test. With untreated clams and natural seawater (median mortality time 2.4 days) a decrease in pH and exponential accumulation of sulfide and ammonium was observed in the anoxic medium, indicating excessive growth of (sulfate reducing) bacteria. In sterilized seawater LT50 (2.1 days) was not significantly different and again considerable amounts of ammonium and sulfide accumulated. However, pre-treatment of clams with chloramphenicol resulted in an increase of LT50 (11.0 days) by approximately fivefold. Accumulation of ammonium and sulfide was retarded, but was finally even stronger than in the medium containing untreated clams. Median mortality times were 2.5 and 2.4 days for Chamelea and 2.7 and 2.9 days for Cerastoderma for static and flow-through incubations, respectively. Addition of chloramphenicol increased strongly survival time in both systems with corresponding values of 11.0 and 16.3 days for Chamelea, and 6.4 and 6.5 days for Cerastoderma. LT50 of Scapharca in anoxic seawater was 14.4 days. Chloramphenicol and penicillin increased median survival time to 28.5 and 28.7 days, respectively, whereas polymyxin displayed no effect (LT50=13.6 days). Molybdate added to artificial sulfate free seawater blocked biotic sulfide formation, but did not improve survival time (LT50=13.7 days). Overall the results indicate that proliferation of anaerobic pathogenic bacteria, firmly associated with the bivalves, is a main cause of death besides lack of oxygen. Bacterial damage is probably caused by injury of the tissues of the clams and not by the release of noxious compounds to the medium.

Effect of Copper Exposure on the Antioxidant Enzymes in Bivalve Mollusc Scapharca inaequivalvis

G. Isani, M. Monari, G. Andreani, M. Fabbri and E. Carpenè* Department of Biochemistry, Veterinary Section, Alma Mater Studiorum, University of Bologna, V ia T olara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy *Correspondence: Dipartimento di Biochimica ‘G. Moruzzi ’, Sezione di Biochimica Veterinaria, Facoltà di Medicina Veterinaria, Alma Mater Studiorum, Università di Bologna, V ia T olara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy E-mail: carpene@vet.unibo.it

Superoxide dismutase in gastric adenocarcinoma: is it a clinical biomarker in the development of cancer?

Abstract Gastric cancer is the second most common cancer worldwide. The involvement of reactive oxygen species (ROS) in the pathogenesis of gastric malignancies is well known. Many human tumours have shown significant changes in the activity and expression of superoxide dismutase (SOD), which might be correlated with clinical–pathological parameters for the prognosis of human carcinoma. The aim of this study is the detection of MnSOD and CuZnSOD activity and their expression in gastric adenocarcinoma and healthy tissues. Gastric samples (adenocarcinoma and healthy tissues) harvested during endoscopy or resected during surgery were used to determine MnSOD and CuZnSOD activity and expression by spectrophotometric and Western blotting assays. The total SOD activity was significantly higher (p<0.05) in healthy mucosa with respect to gastric adenocarcinomas. No differences were found in MnSOD activity and, on the contrary, CuZnSOD activity was significantly lower (p<0.001) in cancer samples with respect to normal mucosa. The rate of MnSOD/CuZnSOD activity in adenocarcinoma was over ninefold higher than that registered in healthy tissues (p<0.05). Moreover, in adenocarcinoma MnSOD activity represented the 83% of total SOD with respect to healthy tissues where the ratio was 52% (p<0.001). On the contrary, in cancer tissues, CuZnSOD activity accounted for only 17% of the total SOD (p<0.001 if compared with the values recorded in normal mucosa). After immunoblotting, MnSOD was more expressed in adenocarcinoma with respect to normal mucosa (p<0.001), while CuZnSOD was similarly expressed in adenocarcinoma and healthy tissues. The SOD activity assay might provide a specific and sensitive method of analysis that allows the differentiation of healthy tissue from tumour tissue. The MnSOD to CuZnSOD activity ratio, and the ratio between these two isoforms and total SOD, presented in this preliminary study might be considered in the identification of cancerous from healthy control tissue.

Investigation of EROD, CYP1A immunopositive proteins and SOD in haemocytes of Chamelea gallina and their role in response to B[a]P

CYP1A sub-family represents the main form of cytochrome P450 involved in benzo[a]pyrene (B[a]P) detoxification, but there are no clear evidences about its presence in invertebrates. 7-Ethoxy resorufin O-deethylase (EROD) activity is strictly related to CYP1A presence, at the same time P450-dependent oxidative metabolism leads to reactive oxygen species (ROS) production, thought to be an important mechanism of pollutant-mediated toxicity in aquatic organisms. Superoxide dismutases (SODs), EROD and CYP1A activities and/or expressions were detected in haemocytes of pooled clams (Chamelea gallina) and cell-free haemolymph after 24 h, 7 and 12 days of exposure to 0.5 mg/L of B[a]P. After 24 h, B[a]P content was maximum in whole tissues. A 61 kDa band was recognized in haemocytes and cell-free haemolymph by polyclonal anti-fish CYP1A, while 53.5 and 63.8 kDa CYP1A immunopositive proteins were discriminate without differences of expression. Differently, EROD, MnSOD activity/expression and ECSOD expression decreased in haemocytes and haemolymph. C. gallina immune system presents an interesting response dose/time exposure of B[a]P and the 7 days condition highlights the major effects of xenobiotic action. The identification of basal EROD levels supports the possible presence of the CYP1A, never identified in C. gallina and more specifically never isolated in immune cells, as confirmed by CYP1A-immunopositive proteins identification.

Chloramphenicol influence on antioxidant enzymes with preliminary approach on microsomal CYP1A immunopositive-protein in Chamelea gallina

Chloramphenicol (CA) is a largely used antibiotic and it is an inhibitor of protein synthesis that also induces ROS production. In this work there were investigated activities and expressions in the Adriatic bivalve Chamelea gallina of some antioxidant and detoxification proteins like superoxide dismutase (Mn-SOD, Cu/Zn-SOD), catalase (CAT) and Cytochrome P450 (CYP1A). Clams exposed to 5mgl(-1) of chloramphenicol were sampled 2, 4 and 8 days after treatment (CA2, CA4 and CA8). SODs, CAT, and CYP1A activity and/or expression were detected in pooled digestive glands by Western blotting and by spectrophotometrical analysis. Enzymes activities increase during the entire antibiotic exposure. With respect to the control Cu/Zn-SOD expression increases, while Mn-SOD expression decreases significantly after 4 days. Two CYP1A immunopositive-proteins (57.7 and 59.8kDa) were detected. The lower band significantly decreases in CA8, the upper one also in CA4 condition. High levels of Mn-SOD, CAT activity and Cu/Zn-SOD expression, indicate intense ROS production while Mn-SOD expression inhibition might be ascribable to mitochondrial alterations due to CA and indirectly to ROS. CYP1A1 action determines H2O2 production that would contribute to a CYP1A1 gene promoter down regulation, a response to oxidative stress with the antioxidant enzymes activation as a final result. This study highlights the close association, in C. gallina, in presence of chloramphenicol, between SOD/CAT and CYP system, and it appear particularly interesting to the lack of similar researches on mollusc species.

Biochemical changes during post-larval growth in white muscle of gilthead sea bream (Sparus aurata) fed zinc-fortified diets.

E. Carpenè*, G. Andreani, M. Monari, M. Kindt and G. Isani Department of Biochemistry, Veterinary Section, Alma Mater Studiorum, University of Bologna, Ozzano Emilia, Bologna,Italy *Correspondence: Dipartimento di Biochimica ‘G. Moruzzi ’, Sezione di Biochimica Veterinaria, Facoltà di Medicina Veterinaria, Alma Mater Studiorum, Università di Bologna, V ia T olara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy E-mail: carpene@vet.unibo.it

Early advances of α1-antitrypsin precursor using the proteomic approach in gastric juice

Evaluation of: Hsu PI, Chen CH, Hsieh CS et al.: alpha1-antitrypsin precursor in gastric juice is a novel biomarker for gastric cancer and ulcer. Clin. Cancer Res. 13(3), 876-883 (2007) [1] . The authors have identified the alpha1-antitrypsin precursor as a new protein marker of gastric cancer in gastric juice by 2D electrophoresis gel and mass spectrometric analyses. Three different pathologic classes of patients were considered with duodenal ulcer, gastric ulcer and gastric cancer and compared with healthy cases. alpha1-antitrypsin precursor was present, like the main peptide, in the specific band pattern, and seemed to be more expressed not only in advanced gastric cancer, but also in early forms of tumor. The study provides a fine contribution to this field of medicine, significantly correlating the alpha1-antrypsin precursor with gastric hypoacidity, gastric ulcer and gastric cancer. In addition, the utilization of gastric juice for biomarker recognition avoids the invasive resection of tissues. In conclusion, the research underlines, with an innovative and thorough approach, the role of the alpha1-antitrypsin precursor as a valid biomarker in gastric cancer and ulcers. However, it does not contribute to the advancement of research aimed at discovering the possible origin of the protein, and this could be the aim of future investigations.

Evaluation of HSP70, 27 and TNF-α espression in different canine cutaneous and gland diseases

Evaluation of HSP70, 27 and TNF-α espression in different canine cutaneous and gland diseases M. Monari & J. Foschi & E. Bichi Ruspoli Forteguerri & S. Valgimigli & M. Zanatta & O. Capitani & G. P. Serrazanetti Published online: 4 July 2009 # Springer Science + Business Media B.V. 2009