Cytogenetic mechanisms in the selective toxicity of cyclophosphamide analogs and metabolites towards avian embryonic B lymphocytes in vivo.
Cyclophosphamide (CP) is selectively toxic to avian and mammalian B lymphocytes, but the mechanisms of action are incompletely understood. We used a structure-activity approach to determine the cytogenetic mechanisms underlying the selective lymphoid toxicity in chicken embryos at 18-19 days of incubation. Two doses of 5-bromo-2'-deoxyuridine (BrdU; 3 mg/200 microliters x 2) were pipetted onto the inner shell membrane to label lymphocyte DNA over 20 h. A single dose of the CP analogs or metabolites was given 1 h after the initial BrdU application. After a terminal 3-h exposure to demecolcine to block cells in metaphase, the embryos were sacrificed at hour 20, and their bursae and thymi were removed for cytogenetic processing. Microscope slide preparations of metaphases were stained by the fluorescence-plus-Giemsa technique to differentiate the sister chromatids for an assessment of sister-chromatid exchange (SCE) induction and cell cycle progression based on replication cycle-specific staining patterns. Isophosphamide (1.25-40 mg/kg), phosphoramide mustard (0.7-45.7 mg/kg), and 4-methylcyclophosphamide (1.3-42.1 mg/kg) selectively damaged B cells as shown by dose-related reductions in the mitotic activity, inhibition of cell cycle kinetics, and approximately 9-15-fold increases in the SCE frequency above control. B cells were up to 392 times more susceptible to the toxicity of these three bifunctional alkylating agents compared to T cells based on reductions in the mitotic activity. At most of the drug doses tested, the T-cell mitotic index was not depressed significantly and was usually higher than the control value by as much as 50-60%. Importantly, monochloroethylcyclophosphamide (70-245 mg/kg; monofunctional alkylation) did not induce differential lymphoid toxicity, although a 9-fold increase in the SCE frequency of B cells was observed at the highest dose. Didechlorocyclophosphamide (181-422 mg/kg; acrolein generation only) was a weak SCE inducer (approximately 1.8-fold increase) and was not selectively toxic to B cells. Our data show that selective toxicity to B lymphocytes is strongly associated with bifunctional alkylation via the chloroethyl groups rather than with monofunctional alkylation and acrolein-mediated damage. In addition, the results with phosphoramide mustard and 4-methylcyclophosphamide emphasize that aldehyde dehydrogenase activity is not the primary determinant in the relative sparing of T lymphocytes in vivo.
Wilmer
J L
JL
Department of Avian and Aquatic Animal Medicine, Cornell University, Ithaca, NY 14853.
Colvin
O M
OM
Bloom
S E
SE
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D002642:Chick Embryo; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D007069:Ifosfamide / Q000633:toxicity*; D008938:Mitosis / Q000187:drug effects / Q000235:genetics; D009152:Mutagenicity Tests; D010752:Phosphoramide Mustards / Q000633:toxicity*; D012854:Sister Chromatid Exchange / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects; D013723:Teratogens / Q000633:toxicity
D010752:Phosphoramide Mustards; D013723:Teratogens; C030090:phosphoramide mustard; C014668:4-methylcyclophosphamide; C040102:N,N-diethylcyclophosphamide; D003520:Cyclophosphamide; D007069:Ifosfamide