Micaela Fabbri

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

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.

Lipid unsaturation per se does not explain the physical state of mitochondrial membranes in Mytilus galloprovincialis

Through a multiple approach, the present study on the mitochondrial membranes from mussel gills and swine heart combines some biochemical information on fatty acid composition, sterol pattern, and temperature dependence of the F1FO-ATPase activity (EC 3.6.3.14.) with fluorescence data on mitochondrial membranes and on liposomes obtained from lipid extracts of mitochondria. The physical state of mussel gills and swine heart was investigated by Laurdan steady state fluorescence. Quite surprisingly, the similar temperature dependence of the F1FO complex, illustrated as Arrhenius plot which in both mitochondria exhibits the same discontinuity at approximately 21°C and overlapping activation energies above and below the discontinuity, is apparently compatible with a different composition and physical state of mitochondrial membranes. Accordingly, mussel membranes contain highly unsaturated fatty acids, abundant sterols, including phytosterols, while mammalian membranes only contain cholesterol and in prevalence shorter and less unsaturated fatty acids, leading to a lower membrane unsaturation with respect to mussel mitochondria. As suggested by fluorescence data, the likely formation of peculiar microdomains interacting with the membrane-bound enzyme complex in mussel mitochondria could produce an environment which somehow approaches the physical state of mammalian mitochondrial membranes. Thus, as an adaptive strategy, the interaction between sterols, highly unsaturated phospholipids and proteins in mussel gill mitochondria could allow the F1FO-ATPase activity to maintain the same activation energy as the mammalian enzyme.

Lipid-protein interactions in mitochondrial membranes from bivalve mollusks: molecular strategies in different species

The mitochondrial F1FO-ATPase, the key enzyme in cell bioenergetics, apparently works in the same way in mollusks and in mammals. We previously pointed out a raft-like arrangement in mussel gill mitochondrial membranes, which apparently distinguishes bivalve mollusks from mammals. To explore the relationship between the microenvironmental features and the enzyme activity, the physico-chemical features of mitochondrial membranes and the F1FO-ATPase activity temperature-dependence are here explored in the Manila clam (Ruditapes philippinarum). Similarly to the mussel, clam gill mitochondrial membrane lipids exhibit a high sterol content (42 mg/g protein), mainly due to phytosterols (cholesterol only attains 42% of total sterols), and abundant polyunsaturated fatty acids (PUFA) (70% of total fatty acids), especially of the n-3 family. However, the F1FO-ATPase activation energies above and below the break in the Arrhenius plot (22.1 °C) are lower than in mussel and mammalian mitochondria. Laurdan fluorescence spectroscopy analyses carried out at 10 °C, 20 °C and 30 °C on mitochondrial membranes and on lipid vesicles obtained from total lipid extracts of mitochondria, indicate a physical state without coexisting domains. This mitochondrial membrane constitution, allowed by lipid-lipid and lipidprotein interactions and involving PUFA-rich phospholipids, phytosterols (much more diversified in clams than in mussels) and proteins, enables the maintenance of a homogeneous physical state in the range 10-30 °C. Consistently, this molecular interaction network would somehow extend the temperature range of the F1FO-ATPase activity and may contribute to clam resilience to temperature changes.