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The induction and nature of mutations in the lacI transgene were evaluated in multiple tissues after exposure of adult male B6C3F1 lacI transgenic mice to cyclophosphamide (CP). Mice were given a single i.p. injection of 25 mg CP/kg, 100 mg CP/kg, or vehicle (PBS) and then necropsied 6 weeks after treatment to allow DNA extraction and lacI mutant recovery. Tissues evaluated included target tissues for tumorigenesis (lung, urinary bladder) and sites not susceptible to tumor formation in B6C3F1 mice (kidney, bone marrow, splenic T-lymphocytes). After exposure to the high dose of CP, a significant increase in the mutant frequency (Mf) was detected in the lungs and urinary bladders, compared to the respective tissues from vehicle-treated controls. In contrast, the Mfs in kidney, bone marrow, and splenic T cells from CP-treated mice were not significantly different from controls. The spectra of mutations in lacI from lung and urinary bladder were significantly changed after high-dose CP treatment, with a significant increase in the frequency of A. T --> T. A transversions found in both tissues and a significantly elevated frequency of deletions in the lungs. Conversely, in vehicle-treated mice, the two predominant classes of lacI mutations recovered in lung and urinary bladder were G. C --> A. T transitions at CpG sites and G. C --> T. A transversions. These CP exposures were also genotoxic as measured by the significant induction of micronuclei in peripheral blood 48 hr after exposure. These data indicate that under these study conditions, CP-induced mutations are detectable in the lacI transgene in the target tissues, but not in nontarget tissues for CP-induced cancer. With the lacI assay it is possible to study mutagenicity in a variety of critical tissues to provide mechanistic information related to genotoxicity and carcinogenicity in vivo., (Copyright 1999 Wiley-Liss, Inc.)

The relative sensitivities and specificities of the endogenous Hprt gene and the lacI transgene as mutational targets were evaluated in splenic lymphocytes from male standard B6C3F1 mice (only Hprt assayed) and from lacI transgenic B6C3F1 mice treated at 6-7 weeks- of-age with the indirect-acting agent, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were given single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then necropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mfs were found at 6 weeks postexposure, with mean Mf values ranging from 2.27 to 3.27 x 10(-5) using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 x 10(-5) in various experiments). To determine the dose response for Hprt Mfs, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. These treatments produced a supralinear dose response curve for CP-induced Hprt Mfs. Based on these experiments, CP mutagenicities at Hprt and lacI were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using another lot of CP in ISOPAC((R)) bottles; Sigma) and necropsied 6 weeks later. There was a significant increase in Hprt Mfs in treated transgenic mice (100 mg CP/kg: 0.75 +/- 0.09 x 10(-5); 25 mg CP/kg: 0.39 +/- 0.05 x 10(-5)) versus controls (0.10 +/- 0.01 x 10(-5)); however, the Mfs in lacI of lymphocytes from the same CP-treated animals were not significantly different from controls (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-treated transgenic and nontransgenic mice were different from those of control mice, whereas the spectra of mutations in lacI 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 lacI transgene of this nontarget tissue for CP-induced cancer., (Copyright 1999 Wiley-Liss, Inc.)

Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study. Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle. Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations. The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213). Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries. As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice. The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%). In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995). These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment. Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells.

The Big Blue lacI transgenic rodent assay, which uses the lambda LIZ/lacI gene as the target for mutation, provides a convenient short-term assay for the study of mutation in vivo [Kohler et al. (1991): Proc Natl Acad Sci USA 88:7958-7962; Provost et al. (1993): Mutat Res 288:133-149). However, the interpretation of data from transgenic animal assays is sometimes complicated by mutants that appear as sectored mutant lambda plaques. These mutants can form a significant fraction of the mutant plaques [Hayward et al. (1995): Carcinogenesis 16:2429-2433]. Thus, in order to accurately determine in vivo mutant frequencies and mutational specificities, it is necessary to score sectored plaques and partition them from the rest of the data. In this study, the specificity of mutation in sectored plaques recovered from untreated and UVB-treated Big Blue mouse skin was analyzed and compared to mutations recovered from lambda LIZ/lacI grown on the Escherichia coli host. The mutational spectra of sectored plaques from untreated and UVB-treated mice were remarkably similar to each other and resembled those recovered from the lambda LIZ/lacI phage plated directly on E. coli. Both the sectored mutants and those recovered in lambda LIZ/lacI phage differed from the spectra of spontaneous mutants in E. coli and in Big Blue mouse skin. While sectored mutants from UVB-treated mouse skin and lambda LIZ/lacI mutants were also different from spontaneous mutants recovered from Big Blue liver, these was little difference between sectored mutants from untreated mouse skin and spontaneous liver mutants (P = 0.07). The mutational spectra of sectored plaques is thus largely consistent with their origin as spontaneous mutations arising in vitro during growth of the lambda LIZ/lacI shuttle vector DNA on the E. coli host, although the potential contribution from lesions in mouse DNA being expressed ex vivo in the E. coli host cannot be excluded.

Transgenic mice carrying the bacterial lacl gene in a lambda shuttle vector were used to isolate and characterize background and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutations in skin. Adult male mice were treated once topically with either DMBA or acetone or were left untreated. Seven days later, DMBA treatment had significantly increased the mutant frequency in the skin (mean +/- SEM, 36 +/- 3 x 10(-5)) versus in vehicle-treated (6.4 +/- 1.2 x 10(-5)) and untreated mice (7.1 x 1.0 x 10(-5)). At least 10 mutants from each of three DMBA-treated and three untreated mice were selected for DNA sequence analysis. In each case, the entire 1080-bp target gene was sequenced. Base-pair substitutions predominated (86 of 96 mutations), although frameshift and deletion mutations were also detected. Twelve percent of the mutants carried more than one mutation. In controls, the mutations were predominantly GC-->AT transitions (26 of 42), and no AT-->TA transversions were recovered. In contrast, in the DMBA-treated mice, AT-->TA transversions represented 42% of the mutations (23 of 54) and GC-->AT transitions accounted for only 11%. The AT-->TA transversions occurred mostly at 5'-CA sites. This class of mutation has been recovered frequently in ras genes from DMBA-treated mice and probably represents an early event in carcinogenesis (Nelson MA et al., Proc Natl Acad Sci USA 89:6398-6402, 1992). Our present results are consistent with the types of DNA damage induced by DMBA. The observation of different mutant frequencies and spectra in treated and control mice demonstrates the utility of this approach in the study of mutagenesis in vivo.