In vitro immunotoxicological assays for detection of compounds requiring metabolic activation

A system for metabolic activation of cyclophosphamide (CP), consisting of a crude microsomal fraction of mouse liver and necessary cofactors (S9 mix), was interfaced with three murine cell culture assays for immunotoxicity. These assays were: the Mishell-Dutton assay for in vitro antibody formation, splenic lymphocyte responsiveness to mitogens and bone marrow cell cultures. There was no effect of CP at doses up to 261 microgram/ml (lmM) on any of the parameters measured unless S9 mix was included. Much greater potency was achieved if the S9 mix was prepared from livers of mice pretreated with phenobarbital. Under these conditions and dose-related inhibition of plaque-forming cells (PFC) in the Mishell-Dutton assay was observed, yielding an ED50 of 6.3 microgram/ml. When splenic lymphocytes were exposed to CP in the presence of induced S9 mix, a dose related inhibition of the response to the B-cell mitogen, lipopolysaccharide (LPS), and to the T-cell mitogen, concanavalin A (Con A), was observed. For the optimum LPS concentration, the ED50 for CP was 8.1 microgram/ml; for the optimum concentration of Con A, the ED50 was 6.7 microgram/ml. DNA synthesis was not inhibited by the doses used. When bone marrow cells were exposed to CP in the presence of induced S9 mix, the stem cell population, enumerated by colonization in semisolid medium, was reduced in a dose-dependent manner, with an ED50 of 5.2 microgram/ml. Again, DNA synthesis was not affected unless higher doses of CP were used.