Analysis of apoptosis of lymphoid cells in fish exposed to immunotoxic compounds

Background: Chemical induction of apoptosis in cells is believed to contrib ute to toxicity. Techniques for measuring apoptosis have increased in both sensitivity and number and in many cases can be readily extended to nontrad itional research species. A comparison of established assays for measuring apoptosis of lymphoid cells has thus far not been performed in the fish and thus would be efficacious in assessing immunotoxicity. Methods: The present study evaluated chemical-induced immune cell apoptosis in fish (tilapia, Oreochromis niloticus) exposed to two known immunotoxic chemicals, azathioprine and T-2 toxin. Cytocentrifugation and light microsc opy of leukocyte-enriched cell samples from the pronephros (i.e., the fish primary hematopoietic compartment) demonstrated chemical-related increases in apoptotic bodies. This observation was examined further with the ApoAler t(TM) Annexin V Apoptosis kit and two DNA-binding dyes employed for detecti ng apoptosis, 7-amino-actinomycin D (7-AAD) and propidium iodide (PI). Results: The apoptotic probes confirmed the microscopic observations of inc reased apoptosis in the chemical-exposed fish. The ApoAlert(TM) annexin V a nd 7-AAD assays, which discriminate early and late apoptosis/necrosis, comp ared well in identifying apoptotic populations. PI staining in Vindelov's s olution was unable to detect early apoptosis. Conclusions:The present data suggest that apoptotic immune cells may be a u seful marker for certain immunotoxicant exposures in fish. These findings a gree with those of previous reports that fish may respond immunologically i n a manner similar to mammals after immunotoxicant challenge. Cytometry 39: 310-318, 2000. (C) 2000 Wiley-Liss, Inc.