How important are glutathione and thiol reductases to oyster hemocyte function?

• Oyster hemocytes were exposed in vitro to the thiol-depleting compound CDNB.
• CDNB affected neutral red retention, but not MTT reduction in oyster hemocytes.
• CDNB effectively impaired thiol metabolism, depleting GSH and inhibiting GR and TrxR.
• Adhesion, phagocytosis and ROS production was unaltered in GSH and GR deficient cells.
• Parallel impairment of lysosome, GSH, GR and TrxR correlated with a lower immunocompetence.

Bivalves are animals with worldwide distribution. Although they play key roles in economic activities, human feeding and environmental studies, there is a considerable lack of knowledge about the relationship between their immune system and antioxidant defenses. Here, we performed an in vitro experiment where Crassostrea gigas hemocytes were exposed to the electrophilic compound 1-chloro-2,4-dinitrobenzene (CDNB, 0.1–50 μM) for one hour. CDNB treatment clearly disturbed thiol homeostasis, causing a concentration-dependent decrease in the glutathione (GSH) content and a decrease in the activity of two thiol reductases, glutathione reductase (GR – 2.5 and 50 μM CDNB) and thioredoxin reductase (TrxR – only 50 μM CDNB). The MTT reduction assay showed that none of the CDNB concentrations tested significantly altered cell viability. However, there was a decrease in the hemocyte's ability to uptake the neutral red dye, which indicates lysosomal impairment (≥12.5 μM CDNB). Cellular immunocompetence was further investigated and, despite the lower GSH content, GR activity and impairment in lysosome integrity, hemocyte functions (adhesion capacity, phagocytosis of latex beads and laminarin-induced ROS production) were preserved in the 2.5 and 12.5 μM CDNB treatments. These results suggest a minor importance of thiol pools and GR activity in C. gigas hemocyte's immunocompetence, in an in vitro acute exposure model. The 50 μM CDNB treatment, however, significantly compromised all the measured hemocyte functions. This response was associated with TrxR inhibition, increased lysosome impairment, decreased GSH content, and lower GR activity. Therefore, it seems that TrxR may be particularly important for the hemocyte function, or, alternatively, it is only affected when a deeply aggravated scenario in thiol homeostasis is set up. Such findings point out the need for further studies towards a better understanding of antioxidant and immune defenses interactions in bivalve cellular systems.

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