Detection of cyclophosphamide-induced mutations at the Hprt but not the lacl locus in splenic lymphocytes of exposed mice

The relative sensitivities and specificities of the endogenous Hprt gene an d the loci transgene as mutational targets were evaluated in splenic lympho cytes from male standard B6C3F1 mice (only Hprt assayed) and from lacl tran sgenic B6C3F1 mice treated at 6-7 weeks-of-age with the indirect-acting age nt, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were give n single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then nec ropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mb were Found at 6 we eks postexposure, with mean Mf values ranging from 2.27 to 3.27 x 10(-5) us ing two different lots of CP in standard packaging (compared with mean cont rol Mf values of 0.14 to 0.26 x 10(-5) in various experiments). To determin e the dose response For Hprt Mk, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. T hese treatments produced a supralinear dose response curve for CP-induced H prt Mb. Based on these experiments, CP mutagenicities at Hprt and lad were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using anot her lot of CP in ISOPAC(R) bottles; Sigma) and necropsied 6 weeks later. Th ere was a significant increase in Hprt Mb in treated transgenic mice (100 m g CP/kg: 0.75 +/- 0.09 x 10(-5); 25 mg CP/kg: 0.39 +/- 0.05 x 10(-5)) versu s controls (0. 10 +/- 0.01 x 10(-5)); however, the Mb in lacl of lymphocyte s from the same CP-treated animals were not significantly different from co ntrols (100 mg CP/kg: 9.4 +/- 1.1 x 10(-5); 25 mg CP/kg: 6.7 +/- 0.8 x 10(- 5). control: 7.7 +/- 0.7 x 10(-5)). Hprt mutational spectra data in CP-trea ted transgenic and nontransgenic mice were different From those of control mice, whereas the spectra of mutations in lad of lymphocytes from Big Blue( R) transgenic mice were not significantly changed after CP treatment. These data indicate that, under these treatment conditions, CP-induced mutations in splenic lymphocytes were detectable in the Hprt gene but not the lacl t ransgene of this nontarget tissue for CP-induced cancer. (C) 1999 Wiley-Lis s, Inc.