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Frequency and Spectrum of Ethylnitrosourea-induced Mutation at the hprt and lacI Loci in Splenic Lymphocytes of Exposed lacI Transgenic Mice¹

Departments of Pathology [V. E. W., T. R. S.] and Environmental Sciences and Engineering [T. R. S., T. R. C.], University of North Carolina, Chapel Hill, North Carolina, 27599; The Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45253-8707 [N. J. G., J. L. A.]; Biology Department, Center for Environmental Health, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada [J. G. d., B. W. G.]; and Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509 [V. E. W.]

The development of mouse models with the endogenous hypoxanthineguanine phosphoribosyl transferase (hprt) gene and lacI transgene as mutational targets provides an excellent opportunity to compare the mutant frequency (Mf) and types of mutations induced in vivo in different sequence contexts. To this end, a study was conducted to determine the Mfs and spectrum of mutations induced at these loci in splenic T cells from male B6C3F1 Big Blue mice (6 weeks old) exposed to N-ethyl-N-nitrosourea (ENU). Six weeks after i.p. injection of 40 mg ENU/kg, T cells were isolated from control (n = 7) and treated (n = 8) mice for the culture of hprt mutants and for the extraction of DNA and recovery of lacI mutants. Mutations in hprt exon 3 and in lacI were quantified and analyzed using published procedures (S. W. Kohler et al., Proc. Natl. Acad. Sci. USA, 88: 7958–7962, 1991; T. R. Skopek et al., Proc. Natl. Acad. Sci. USA, 89: 7866–7870, 1992). In treated mice, the Mfs (average ± SE) in hprt (6.0 ± 0.2 x 10^-5) and lacI (11.4 ± 1.8 x 10^-5) were approximately 16.2-fold (P = 0.096) and 3.4-fold (P = 0.009), respectively, above controls. However, the average induced Mfs (i.e., induced Mf = treatment Mf — background Mf) in hprt and lacI were similar, with the respective increases in Mf being 5.6 ± 0.2 x 10^-5 and 8.0 ± 2.3 x 10^-5 over background. Eleven of the 107 hprt mutants from treated Big Blue mice had mutations in exon 3, with 73% being substitutions at A·T bp. These data are similar to those observed in ENU-exposed nontransgenic B6C3F1 mice, in which 62 of 69 exon 3 mutations were substitutions at A·T bp (T. R. Skopek et al., Proc. Natl. Acad. Sci. USA, 89: 7866–7870, 1992). For comparison, the sequences of the lacI genes in two to five mutants from each mouse were determined, and a total of 75 mutations (70 different mutations) was detected. In exposed mice, 55% (24 of 44) of the mutations in lacI were substitutions at A·T bp. In controls, substitutions at AT bp comprised only 20% of the recovered mutations in either hprt exon 3 (1 of 5) or lacI (5 of 26). These data indicate that the lacI mutation assay is less sensitive than the hprt assay for detecting increases in Mf induced by ENU exposure of mice as indicated by the lower relative increase in Mf in the lacI gene, but, given a 6-week expression time, the types of mutations induced by ENU in the transgene reflect those observed in the native transcribed gene.

¹ Funded in part by grants from the Procter and Gamble Co. and National Institute of Environmental Health Sciences Grant P42-ES0594801.

² To whom requests for reprints should be addressed, at the Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 122010509.

Received 4/22/96. Accepted 8/14/96.

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