Chlorothalonil suppresses anterior kidney macrophage activity in mummichog, Fundulus heteroclitus, following in vivo exposure

Chlorothalonil (2,4,5,6-tetrachloro-isophthalonitrile, TCIN) is the most commonly applied fungicide with near-ubiquitous distribution. Previous research has shown that TCIN is a potential immunotoxicant that merits further investigation. In this study, we exposed mummichogs to 0, 0.1, 1.0 and 10 μg/l TCIN for 48 h. At the termination of the exposure, fish were sacrificed and the anterior kidneys and livers were collected. Macrophage-enriched cell pools were derived from the anterior kidneys; these cells were challenged with zymosan and reactive oxygen species (ROS) production was recorded using luminol-augmented chemiluminescence. Livers were homogenized and cytosols were separated from microsomal fractions and stored at −80°C. Cytosolic proteins (10 μg/lane) were separated by polyacrylamide gel electrophoresis, blotted to PVDF membrane, probed with polyclonal antibodies for inducible nitric oxide synthase (iNOS) induction, and visualized with chemiluminescence. The results indicate that TCIN exposure is associated with a dose-dependent decrease in zymosan-stimulated macrophage ROS production. There was no apparent induction of iNOS in the liver. Interestingly, we do not see decreased ROS production in macrophages following in vitro exposure, until significantly higher TCIN concentrations are employed (e.g. 250 μg/l). This suggests that the observed suppression following in vivo exposure may result from TCIN metabolites, and not the parent compound.