Superoxide anion generation by Crassostrea virginica hemocytes as measured by nitroblue tetrazolium reduction

Abstract

Hemocytes of the oyster ( Crassostrea virginica) were shown to produce superoxide anion (O 2 −) by the superoxide dismutase-sensitive reduction of nitroblue tetrazolium (NBT). This reaction was observed in adherent hemocytes by cytochemistry and was quantified in suspension cultures by spectrophotometry. Weak NBT reduction, as indicated by pale blue staining of the cytoplasm, was present in about 40% of unchallenged hemocytes. After ingesting heat-killed yeast particles, dark blue formazan deposits formed frequently in yeast-containing phagosomes. NBT formazan was extracted from the cells with pyridine, permitting accurate quantitation of O 2 −. Superoxide production was measured in both resting and stimulated hemocytes and was dependent on environmental temperature. Statistically significant phagocytic induction of O 2 − over resting levels occurred only in hemocytes collected from oysters living at temperatures >21°C. Generation of oxygen intermediates is probably a component of the internal defense mechanisms of oysters involved in cytotoxic responses to microorganisms and parasites.