Analysis of apoptosis of lymphoid cells in fish exposed to immunotoxic compounds.
BACKGROUND
Chemical induction of apoptosis in cells is believed to contribute to toxicity. Techniques for measuring apoptosis have increased in both sensitivity and number and in many cases can be readily extended to nontraditional 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 microscopy 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 ApoAlert Annexin V Apoptosis kit and two DNA-binding dyes employed for detecting apoptosis, 7-aminoactinomycin D (7-AAD) and propidium iodide (PI).


RESULTS
The apoptotic probes confirmed the microscopic observations of increased apoptosis in the chemical-exposed fish. The ApoAlerttrade mark annexin V and 7-AAD assays, which discriminate early and late apoptosis/necrosis, compared well in identifying apoptotic populations. PI staining in Vindelov's solution was unable to detect early apoptosis.


CONCLUSIONS
The present data suggest that apoptotic immune cells may be a useful marker for certain immunotoxicant exposures in fish. These findings agree with those of previous reports that fish may respond immunologically in a manner similar to mammals after immunotoxicant challenge.
Gogal
R M
RM
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24061-0342, USA. rgogal@vt.edu
Smith
B J
BJ
Kalnitsky
J
J
Holladay
S D
SD
D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D001379:Azathioprine / Q000494:pharmacology; D015415:Biomarkers / Q000032:analysis; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000922:Immunotoxins / Q000494:pharmacology*; D008214:Lymphocytes / Q000187:drug effects*; D011419:Propidium / Q000494:pharmacology; D013605:T-2 Toxin / Q000494:pharmacology; D017210:Tilapia / Q000276:immunology*
D017304:Annexin A5; D015415:Biomarkers; D000922:Immunotoxins; D003609:Dactinomycin; D011419:Propidium; C025942:7-aminoactinomycin D; D013605:T-2 Toxin; D001379:Azathioprine