A cytogenetic approach for detecting the selective toxicity of drugs in avian embryonic B and T lymphocytes

Abstract

The developing immune system of late stage embryos and neonates may be particularly susceptible to the toxicity of drugs and environmental contaminants due to high rates of cell proliferation and ongoing processes of differentiation. We have developed a cytogenetic assay to study the mechanisms of the selective targeting of cyclophosphamide (CP) to B lymphocytes compared to T lymphocytes in chicken embryos at days 18–19 of incubation. 5-Bromo-2′-deoxyuridine (BrdU; 3 mg/200 μl PBS; two doses; 3-h interval) was pipetted onto the inner shell membrane in order to label DNA of replicating lymphoid cells. CP (1.25–40 mg/kg) was injected 1 h after the initial BrdU dose, and the embryos were exposed to colcemid (10 μg/100 μl H 2O) at hour 17. Three hours later, the bursa and thymus were removed, and the lymphocytes were swollen in hypotonic solution, fixed, and processed through a fluorescence-plus-Giemsa technique to differentiate sister chromatids. Based on reductions in mitotic indices, B cells were ∼ 213 times more susceptible than T cells to the cytotoxicity of CP. Because the mitotic indices of B and T cells were comparable (21.3 ± 3.7%, vs. 25.5 ± 6.9%), the differential toxicity cannot be ascribed to greater numbers of B cells being in mitosis. CP induced a dose-related increase in the sister-chromatid exchange frequency in B cells of up to 10.4-fold above controls, representing one of the most sensitive vertebrate systems for detecting the genotoxicity of CP. The average generation time was slowed from 9.8 ± 0.3 h in control B cells to 19.4 ± 0.9 h in embryos exposed to 10 mg CP/kg. Furthermore, an analysis of control SCE data from 56 embryos indicated that there was a significant overdispersion of B cells exhibiting relatively high SCE frequencies compared to a Poisson distribution. Our data indicate that the chicken embryo in the late developmental stage is a good model for detecting the presence and selective toxicity of drugs and environmental toxins in differentiating B and T lymphocytes in vivo.