Transgenic B6C3F1 Mice Research Articles

28-Day somatic gene mutation study of 1-bromopropane in female Big Blue® B6C3F1 mice via whole-body inhalation: Support for a carcinogenic threshold

A 2-year inhalation rat and mouse cancer study by the National Toxicology Program (NTP) on 1-bromopropane, a brominated solvent most commonly used as a vapor degreaser, showed significant increase in tumors in the lung of female mice and in the large intestine of male and female rats. The most sensitive endpoint was lung tumors in female mice. Mice of both sexes had hyperplasia and inflammation of the nose and showed regeneration of lung tissue. The NTP assumed that these tumors were due to genotoxic effects and that a linear dose-response relationship was appropriate. It is plausible that, similar to chloroform, hyperplasia and inflammation are required as initial events for tumor development. If true, then a threshold-based model may be more appropriate for 1-bromopropane. To test this hypothesis, a 28-day repeat dose inhalation Big Blue® Assay was conducted using female transgenic B6C3F1 mice. The target exposure concentrations and the exposure regimen were identical to those used by the NTP. Results demonstrated no elevation in mutant frequency of the cII transgene in lung, colon, or liver. Positive controls produced statistically significant increases in mutant frequencies across all tested tissues. These results demonstrate that 1-bromopropane does not induce cII mutants in lungs, colon, or liver under the testing conditions. These data have important ramifications in the quantitative evaluation of tumor results for this chemical and support a mechanism of action where a threshold for carcinogenicity is plausible.

Evaluation of mutant frequencies of chemically induced tumors and normal tissues in ?/cII transgenic mice

Genomic instability has been implicated as an important component in tumor progression. Evaluation of mutant frequencies (MFs) in tumors of transgenic mice containing nontranscribed marker genes should be useful for quantitating mutation rates in tumors as the physiologically inactive transgene provides neither a positive nor a negative selective pressure on the tumor. We have conducted long-term carcinogenicity studies in lambda/cII transgenic B6C3F1 mice using a variety of genotoxic and nongenotoxic test agents and have evaluated the mutant frequencies in both tumors and normal tissues from these animals. Mice were administered diethylnitrosamine (DEN) as three intraperitoneal injections of 15 mg/kg; phenobarbital (PB) or oxazepam (OXP) provided ad libitum at 0.1% or 0.25% in the diet, respectively; DEN initiation plus PB in the diet; or urethane (UTH) provided ad libitum at 0.2% in the drinking water. Normal tissues and tumors were isolated at various times over a 2-year period and half of each tissue/tumor was evaluated histopathologically and the other half was evaluated for MF in the cII transgene. Approximately 20 mutants from each of 166 individual tissues (tumor and nontumor) were sequenced to determine whether increases in MF represented unique mutations or were due to clonal expansion. UTH produced significant increases in MF in normal liver and lung. DEN either with or without PB promotion produced significant increases in MF in liver and correction of MF for clonality produced little change in the overall MF in these groups. PB produced a twofold increase in liver MF over controls after 27 weeks of treatment, but a similar increase was not observed with longer dosing times; at later time points, the MF in the PB groups was lower than that of the control group, suggesting that PB is not producing direct DNA damage in the liver. OXP failed to produce an increase in MF over controls, even after 78 weeks of treatment. Selected cases of genomic instability were observed in tumors from all treatments except OXP, with individual liver tumors showing very high MF values even after clonal correction. One rare and interesting finding was noted in a single mouse treated with UTH, where a mammary metastasis had an MF approximately 10-fold greater than the parent tumor, with 75% of the mutations independent, providing strong evidence of genomic instability. There was no clear correlation between tumor phenotype and MF except that pulmonary adenomas generally had higher MFs than normal lung in both genotoxic and nongenotoxic treatment groups. Likewise, there was no correlation between tumor size and MF after correction for clonality. The results presented here demonstrate that individual tumors can show significant genomic instability, with very significant increases in MF that are not attributed to clonal expansion of a single mutant cell.

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

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 × 10–5 using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 × 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® 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 × 10–5; 25 mg CP/kg: 0.39 ± 0.05 × 10–5) versus controls (0.10 ± 0.01 × 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 × 10–5; 25 mg CP/kg: 6.7 ± 0.8 × 10–5; control: 7.7 ± 0.7 × 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® 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. Environ. Mol. Mutagen. 34:167–181, 1999 © 1999 Wiley-Liss, Inc.

Detection of cyclophosphamide-induced mutations at theHprt but not thelacI locus in splenic lymphocytes of exposed mice

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.

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver.

Dichloroacetic acid (DCA) is a chlorination byproduct found in finished drinking water. When administered in drinking water this chemical has been shown to produce hepatocellular adenomas and carcinomas in B6C3F1 mice over the animal's lifetime. In this study, we investigated whether mutant frequencies were increased in mouse liver using treatment protocols that yielded significant tumor induction. DCA was administered continuously at either 1.0 or 3.5 g/l in drinking water to male transgenic B6C3F1 mice harboring the bacterial lacI gene. Groups of five or six animals were killed at 4, 10 or 60 weeks and livers removed. At both 4 and 10 weeks of treatment, there was no significant difference in mutant frequency between the treated and control animals at either dose level. At 60 weeks, mice treated with 1.0 g/l DCA showed a 1.3-fold increase in mutant frequency over concurrent controls (P = 0.05). Mice treated with 3.5 g/l DCA for 60 weeks had a 2.3-fold increase in mutant frequency over the concurrent controls (P = 0.002). The mutation spectrum recovered from mice treated with 3.5 g/l DCA for 60 weeks contained G:C-->A:T transitions (32.79%) and G:C-->T:A transversions (21.31%). In contrast, G:C-->A:T transitions comprised 53.19% of the recovered mutants among control animals. Although only 19.15% of mutations among the controls were at T:A sites, 32.79% of the mutations from DCA-treated animals were at T:A sites. This is consistent with the previous observation that the proportion of mutations at T:A sites in codon 61 of the H-ras gene was increased in DCA-induced liver tumors in B6C3F1 mice. The present study demonstrates DCA-associated mutagenicity in the mouse liver under conditions in which DCA produces hepatic tumors.

P XV.9 - P XV.9 Dimethylnitrosamine and chloroform induced mutagenic profiles in lacI transgenic B6C3F1 mice over 180 days

P XV.9 - P XV.9 Dimethylnitrosamine and chloroform induced mutagenic profiles in lacI transgenic B6C3F1 mice over 180 days

A selectable system for mutation detection in the Big Blue ®lacI transgenic mouse system: what happens to the mutational spectra over time

Transgenic animals offer a powerful tool to study the mechanisms of spontaneous and induced mutagenesis in vivo. Herein we used a test version of a growth selectable assay to obtain spontaneous mutants in a lacI target transgene recovered from lacI transgenic B6C3F1 mice (Big Blue ®). This selection system may have certain advantages relative to the more established plaque screening system for mutation detection because: (1) the plating density of the phage is up to 60 times higher in the selectable assay, reducing the number of plates needed to be screened for a comparable amount of mutants; and (2) the mutant frequency obtained from the selectable assay is higher compared to the plaque assay, possibly due to a higher sensitivity for weaker mutants. However, the longer incubation time of the growth selectable assay might allow E. coli host derived mutants to appear. To address this issue, we investigated the sequence changes in the amino-terminal domain of the lacI gene of 405 mutants derived from the liver, spleen, brain, germ cells and skin of five untreated 6-week-old mice. The mutant colonies were isolated after 60, 84, 108 and 150 h of incubation under growth selectable conditions. Tissue-specific differences in the mutational pattern obtained after 60 and 84 h disappear after a longer time of incubation, possibly due to an increasing contribution of E. coli derived mutants. The evolving selectable systems offer the potential to increase screening efficiency, but the results suggest caution in interpreting data from this system because repair by E. coli of DNA lesions or mismatched heteroduplexes either originating in mouse in vivo or produced by ex vivo manipulation as well as de novo mutations in E. coli might contribute significantly to the observed mutational spectra at each timepoint.

Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F1 mice.

Transgenic B6C3F1 mice carrying a lacI target gene were exposed to acute and multiple doses of ethylnitrosourea (ENU), and germ cells from the seminiferous tubules were assayed for mutation 3 and 90 days after treatment. Relative to untreated controls, the mutation frequency increased 3.2- and 19.9-fold at 3 and 90 days after treatment, respectively. Mutant lacI genes recovered from untreated and treated groups were sequenced, and the spectra of mutations were determined. Eighty-five percent (11/13) of the spontaneous mutations resulted in G.C-->A.T transitions, all of which occurred at CpG dinucleotides. Fifteen of 22 sites (68%) found mutated 3 days after ENU treatment occurred at G.C base pairs, although some of these are expected to be spontaneous mutations. Ninety days after treatment, 13 of 19 sites (68%) found mutated occurred at A.T base pairs. The mutation spectra seen are consistent with proposed mechanisms of ENU mutagenesis and correlate with the in vivo spectra seen in ENU studies by using transmissibility assays and the hprt gene. These findings represent significant progress toward defining the in vivo spectra of ENU mutagenesis in mammalian germ cells.

Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice

Ionizing radiation induces gene mutations (point mutations, deletions and insertions) as well as chromosome damage in mammalian cells. Although these effects have been studied extensively in cells in culture, until recently it has not been possible to analyze the mutagenic potential of ionizing radiation in vivo, especially at the molecular level. The development of transgenic mutagenesis has now made it possible to study the effects of ionizing radiation at both the molecular and chromosomal levels in the same animal. In this report we present preliminary data on the response of Big Blue TM lacI transgenic mice to ionizing radiation as measured by lacI mutations and micronuclei: C57B1/6 transgenic mice were irradiated with 137Cs γ-rays at doses ranging from 0.1 to 14 Gy, and expression times ranging from 2 to 14 days. Dose-related increases in the mutant frequency were observed after irradiations with longer expression times. Mutant plaques were analyzed by restriction enzyme digestion to detect large structural changes in the target sequence. Of 34 γ-ray-induced mutations analyzed, 4 were large-scale rearrangements. 3 of these rearrangements were deletions within the lacI gene characterized by the presence of short regions of homology at the breakpoint junctions. The fourth rearrangement was a deletion that extended from within the αlacZ gene into downstream sequences and that had 43 bp of homology at the junction. These data indicate that the Big Blue TM lacI transgenic mouse system is sensitive to the types of mutations induced by ionizing radiation. To determine whether the presence of the transgene effects micronucleus induction we compared the response of nontransgenic to hemizygous transgenic B6C3F1 mice and the response of nontransgenic to hemizygous and homozygous transgenic C57B1/6 mice. The presence or absence of the lacI transgene had no effect on spontaneous micronucleus frequencies for either strain. However, radiation-induced micronucleus frequencies were significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; the converse was true in C57B1/6 mice. These data suggest that the lacI transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.

Mutagenicity of o-anisidine to the bladder of lacI- transgenic B6C3F1 mice: absence of 14C or 32P bladder DNA adduction.

Earlier studies have established that the rodent bladder carcinogen o-anisidine (OA) gives negative results in all of the standard rodent genetic toxicity assays. In the present study, a single oral administration of the maximum tolerated dose level (750 mg/kg) of OA to B6C3F1 mice yielded negative results in 32P-post-labelling assays of bladder and liver DNA (24 h after dosing). Likewise, 14C-ring-labelled OA administered orally to B6C3F1 mice gave no evidence of DNA binding 6, 12 or 24 h later. Administration of OA (750 mg/kg) to transgenic lacI- mice (Big Blue) led to a small increase in mutation frequency (MF) in the bladder, but not in the liver. Increased MFs were observed in the bladder following 1, 3 or 10 daily doses with sampling times of 1 or 2 weeks after the final dose. However, statistical significance (P < 0.01) was only reached 2 weeks after either 3 or 10 daily administrations of OA. The positive control chemical (dimethylnitrosamine) gave a positive result (P < 0.01) in the liver, but not the bladder, 7 days after a single administration of 10 mg/kg. The possibility that OA is mutagenic and carcinogenic to the rodent bladder via formation of radical species is suggested.