Abstract
Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2). Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining) remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01) and CYP356A1 (p < 0.05) transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx) remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP) and stress (Hsp70), but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD) related genes.
Highlights
Brevetoxins represent a group of polyether compounds known to cause neurotoxic shellfish poisoning (NSP) through the consumption of brevetoxin-containing shellfish
In order to investigate whether PbTx-2 could cause hemocyte toxicity, cell viability assays were performed using the MTT and neutral red (NR) methods
No significant differences were observed in the viability of C. gigas hemocytes after incubation with PbTx-2 concentrations ranging from 3 to 1000 μg/L for 1, 4 and 12 h, according to either viability assay (Figure 1)
Summary
Brevetoxins represent a group of polyether compounds known to cause neurotoxic shellfish poisoning (NSP) through the consumption of brevetoxin-containing shellfish. They are produced mainly by the marine dinoflagellate Karena brevis, but recently brevetoxin-like compounds were found in other microalgae species such as Chatonella marina, Chatonella antiqua, Fibrocapsa japonica and Heterosigma akashiwo [1]. Shellfish appear to be only toxin vectors unaffected by harmful algal blooms (HABs), some behavioral, physiological and cellular responses of bivalves to harmful algae have already been described, along with mortality events [2,6]. The HAB species H. akashiwo, described to produce brevetoxin-like compounds [12], affected significantly bivalve immune cells viability and phagocytosis in vitro [13]
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