Effects of chlorothalonil on phagocyte function in fish (Morone saxatilis and Fundulus heteroclitus) and oysters (Crassostrea virginica): Mechanisms of immunotoxicity

Abstract

The pesticide chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile, TCIN is the most commonly applied fungicide in the United States. A thiol-reactive agent, TCIN inactivates enzymes by binding to functional sulfhydryl groups. It is detoxified by glutathione (GSH) conjugation. The purpose of this research was to evaluate the effects of TCIN on immune system function in fish (striped bass, Morone saxatilus and mummichogs, Fundulus heteroclitus ) and oysters (Crassostrea virginica). Four separate exposure studies were performed. In the first study, phagocytes from the anterior kidneys of striped bass were exposed to TCIN (10-500 mug/L = 0.04-2.0 muM) in vitro for 20h. Cell viability was evaluated by the trypan blue exclusion assay. Phagocytosis was measured using fluorescent-labeled yeast. Reactive oxygen species (ROS) production was evaluated using chemiluminescent assays, and NADPH production was measured using a colorimetric assay based on the reduction of a tetrazolium. For the second study, this experiment was repeated using oyster hemocytes. The third study examined the effect of sulfhydryl-modifying agents (buthionine sulfoximine, BSO, and dithiothreitol, DTT) on TCIN-induced immunomodulation. Cell viability, superoxide generation, NADPH production, and lipid peroxidation were measured. The final experiments evaluated the effects of 48h in vivo exposure to TCIN (1.0-20 mug/L) using mummichogs. The endpoints tested included anterior kidney macrophage ROS production and hepatic inducible nitric oxide synthase (NOS) expression. Results of these studies indicate that TCIN had no effect on phagocytosis, but did suppress ROS generation. NADPH production was enhanced at the 50-100 mug/L TCIN concentrations, but was decreased at higher concentrations. Oyster hemocytes were found to respond in a manner very similar to that of striped bass macrophages. In fish phagocytes, BSO was found to enhance the toxicity of TCIN, as measured by ROS production, whereas DTT reduced toxicity. TCIN did not induce lipid peroxidation, even when BSO-pre-incubation was employed. In vivo exposure of mummichogs to TCIN resulted in decreased ROS production in male fish, but not in females. Hepatic NOS expression exhibited a dose-dependent decrease in baseline expression. In conclusion, environmental exposures to TCIN have the potential to modulate phagocyte function; since multiple species are affected the effect may be ubiquitous.