In vitro effects of tributyltin on functional responses of haemocytes in the clam Tapes philippinarum

Abstract

The effects of tributyltin (TBT, as the chloride) on circulating cells from the clam Tapes philippinarum were investigated, in order to set up quick and reproducible in vitro bioassays to evaluate TBT toxicity in bivalve molluscs. Haemocytes, collected from the adductor muscle of clams, were exposed for 60 min at 25°C to sublethal concentrations of TBT (0.01, 0.05, 0.1 mM) and the effects on uptake of the vital dye Neutral Red, and both superoxide dismutase (antioxidant enzyme) and lysozyme (bacteriolytic enzyme) activities were spectrophotometrically evaluated. Exposure of haemocytes to 0.05 mM TBT caused a significant increase (P < 0.05) in Neutral Red dye uptake compared with controls, whereas no differences resulted after exposure to 0.01 and 0.1 mM TBT. Enlarged lysosomes were observed in haemocytes exposed to 0.05 mM TBT. Moreover, in haemocytes treated with 0.05 mM and 0.1 mM TBT, superoxide dismutase activity significantly decreased (P < 0.05 and P < 0.01 respectively) with respect to that of controls. A significant decrease in lysozyme activity was also observed in haemocytes exposed to 0.05 (P < 0.01) and 0.1 mM TBT (P < 0.001). These results suggest a relationship between TBT exposure and alterations in functional responses of haemocytes in T. philippinarum. The proposed assays are sensitive, rapid and reproducible. They may be proposed as biomarkers, although their responsiveness needs to be more fully evaluated in haemocytes collected after clam exposure in both laboratory and field conditions.

4-Nonylphenol induces immunomodulation and apoptotic events in the clam Tapes philippinarum

“…NP exposure also decreased SOD activity in the digestive gland and gills of Tapes philippinarum (Matozzo et al 2004). Decreased SOD activity was previously observed in haemocytes from T. philippinarum after 7 d exposure to copper (Matozzo et al 2001), and in in vitro TBT-exposed haemocytes (Matozzo et al 2002). We hypothesise that the inhibition of SOD observed in the present study is due to ROS-mediated oxidation of the enzyme SH groups.…”

“…This is in agreement with our previous findings. Indeed, increased NR uptake and enlarged lysosomes were observed in haemocytes from Tapes philippinarum exposed in vitro to TBT (Matozzo et al 2002).…”

“…The cationic probe, NR (Merck), was used to evaluate haemocyte membrane stability after NP exposure. Alterations in its uptake are commonly recognised as an indicator of cell damage in in vitro studies (Gómez-Mendikute et al 2002, Matozzo et al 2002. Haemolymph (final volume = 1 ml) from both NP-treated and untreated clams (controls) was centrifuged at 780 × g for 10 min, haemocytes were resuspended in an equal volume of 8 mg l -1 NR dye solution in filtered seawater (FSW) and then incubated at 25°C for 30 min.…”

“…Alterations in immunocompetence have been reported for bivalves after exposure, both in vitro and in vivo, to organic and inorganic contaminants, such as heavy metals (Cheng & Sullivan 1984, Cheng 1988, Viarengo et al 1994, Pipe et al 1999, Matozzo et al 2001, Sauvé et al 2002, organotins (Fisher et al 1990, Auffret & Oubella 1997, Cima et al 1998, Matozzo et al 2002, fungicides (Alvarez & Friedl 1992), polycyclic aromatic hydrocarbons (PAHs) (Sami et al 1992, Coles et al 1994, Grundy et al 1996, Gómez-Mendikute et al 2002 and chlorinated phenols (Florence et al 1997). Among the functional responses of bivalve circulating cells, total haemocyte count (THC), viability, motility, aggregation and adhesion capability, phagocytosis, production of reactive oxygen species (ROS), hydrolytic and oxidative enzyme activities, and lysosomal and cell membrane stability are all generally recognised as useful biomarkers in immunotoxicity studies.…”

Effects of pyrene exposure on immune response and oxidative stress in the pearl oyster, Pinctada martensii

“…LPO can interfere with membrane structure and function by changing membrane fluidity, breaking the membrane integrity, passivating membrane-bound enzymes and disrupting surface receptor molecules [81]. Many other contaminants, including tributyltinoxide and cadmium, can induce alterations in cell membranes and cause destabilization and reduce cell viability [5,26,52,67]. Additionally, lipid hydroperoxides form at the early stages of LPO, these can take part in redox reactions and exacerbate peroxidative cell injury [25].…”