Effects of PCB 126 on Primary Immune Organs and Thymocyte Apoptosis in Chicken Embryos

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyl (PCB) 126 produce thymic atrophy and immunosuppression. This study explored the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 is associated with an increase in apoptotic thymocytes at the end of incubation in chicken embryos. Eggs were injected via the air cell with PCB 126 (0.05, 0.13, 0.32, 0.64, and 0.80 ng/g egg) on d 0 of incubation, and tissues were collected on d 20. Controls included noninjected and vehicle-injected (sunflower oil) eggs. Thymocytes were cultured for 6 h and analyzed by flow cytometry for decreased DNA content (propidium iodide staining) and cell size (forward scatter), which indicate apoptosis. PCB 126 induced dose-dependent mortality with an LD50 of 1.01 ng/g and lowest-observed-effect concentration (LOEC) of 0.32 ng/g. Teratogenic effects commonly associated with TCDD and planar PCBs, including cranial and foot deformities and subcutaneous edema, tended to increase with dose of PCB 126. PCB 126 reduced thymus mass by approximately 20% at 0.64 and 0.8 ng/g, the number of viable thymocytes by approximately 20–24% at and above 0.13 ng/g, and the number of bursal lymphoid cells by 57% at 0.64 ng/g. The percentage of apoptotic thymocytes increased with dose, reaching levels 2 times greater than controls at 0.8 ng/g. Electrophoresis of low-molecular-weight DNA from thymocytes of all doses demonstrated fragments in multiples of 180 bp. This DNA laddering is a hallmark of apoptosis. At all doses, thymocytes exhibited caspase-3 activation, another indicator of apoptosis. The results of this experiment supported the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 in chicken embryos is associated with an increase in apoptotic thymocytes on embryonic d 20.

This project was supported by the U.S. Environmental Protection Agency (GR825216-01-0) and the Biomedical Sciences PhD Program at Wright State University. E. Lavoie, R. Garrett, A. Handy, and E. Reaves helped with laboratory work. Drs. Nancy Bigley, Thomas Brown, G. Allen Burton, and Robert Grubbs provided technical assistance and reviewed earlier drafts of this article.

Notes

This project was supported by the U.S. Environmental Protection Agency (GR825216-01-0) and the Biomedical Sciences PhD Program at Wright State University. E. Lavoie, R. Garrett, A. Handy, and E. Reaves helped with laboratory work. Drs. Nancy Bigley, Thomas Brown, G. Allen Burton, and Robert Grubbs provided technical assistance and reviewed earlier drafts of this article.