Three-week-old Big Blue (BB) B6C3F1 mice were given a single i.p. injection of ENU. Three weeks later, splenic T cells were isolated from each animal by ficoll gradient centrifugation and divided into two samples. One sample was cultured to measure hprt- mutation and the other was used to extract DNA for lacI- analysis. T cells from BB mice exposed to 0, 4.5, 13.5, and 40 mg ENU/kg (9 or 10 animals per group) displayed dose-related increases in the frequency of both hprt- and lacI- mutations. Within each treatment group, the ENU-induced mutation frequency (average observed mutation frequency minus average control frequency) was remarkably similar at the two loci. This suggests that treatments that increase mutation frequency at the endogenous hprt gene also produce similar incremental increases at the BB lacI transgene. However, because of the ten-fold higher spontaneous mutation rate at lacI, the fold-increase over background produced by ENU at this locus was significantly less than the fold-increase produced at hprt. For example, the 4.5 mg ENU/kg treatment produced a 5.2-fold increase above background at hprt (P = 0.001), whereas only a 1.5-fold increase was produced at lacI (P = 0.140). Consequently, mutagenic insults that produce up to a fivefold increase in mutation frequency at an endogenous locus may be difficult to detect at the lacI transgene. Finally, the ENU-induced response at hprt in BB mice was identical to that in generic B6C3F1 mice, suggesting that there are no inherent differences between transgenic and normal mice in their response to this mutagenic agent.

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Big Blue (BB) and generic B6C3F1 mice were given one to three i.p. injections of 50 mg/kg benzo[a]pyrene (B[a]P) in DMSO every other day to achieve cumulative doses of 50 to 150 mg/kg. Three weeks after treatment, the mutation frequency at the endogenous hprt gene and lacI transgene was measured in splenic T cells. Generic mice given 50, 100, and 150 mg/kg B[a]P displayed induced hprt frequencies (observed hprt frequency minus control frequency) of 5.5 +/- 1.0, 11 +/- 2.0, and 19 +/- 2.6 x 10(-6), respectively (average +/- SEM). In contrast, BB mice given 50 and 150 mg/kg B[a]P displayed induced hprt frequencies of 0.9 +/- 0.6 and 9.1 +/- 1.5 x 10(-6). 32P postlabelling revealed that the lower hprt response in BB mice correlated with lower amounts of BP-DNA adducts in spleen, liver, and lung 24 hours after B[a]P exposure. Western blot analysis of liver samples from B[a]P-treated mice suggests that the reduced adduct load in turn may be due to lower P450 1A1 levels in BB mice. The frequency of induced, nonsectored blue plaques (observed blue plaque frequency minus control frequency) in BB mice receiving 50 and 150 mg/kg B[a]P was 41 +/- 9 and 134 +/- 10 x 10(-6) (15- to 40-fold higher than the induced hprt frequency in the same treated animals). Sectored plaques were observed in both control and B[a]P groups but their frequency showed no relationship to dose (sectored frequency in all groups was approximately 20 x 10(-6)). To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified lambda-LIZ DNA was treated in vitro with B[a]P diol epoxide (BPDE), packaged, and plated on E. coli lawn cells. Treatment with BPDE did not produce significant increases in homogeneous blue plaques, suggesting that the majority of mutants obtained from B[a]P-treated BB mice occurred in vivo. These results indicate that B[a]P exposure produces many more mutations at the lacI transgene than at the endogenous hprt locus.

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