lacI Mutations Research Articles

Comparison of mutant frequencies and types of mutations induced by thiotepa in the endogenous Hprt gene and transgenic lacI gene of Big Blue® rats

The utility of the lacI transgene of Big BluesR rats as a reporter of in vivo mutation was evaluated by comparing the frequency and types of mutations induced by thiotepa in the transgene and the endogenous Hprt gene. Transgenic rats were given i.p. injections of 1.4 mg/kg of thiotepa three times per week over a period of 4 weeks (a total dose of 16.8 mg/kg); 1 week after the last injection, mutation assays were performed on spleen lymphocytes isolated from the animals. Thiotepa treatment increased the lacI mutant frequency from 34.8±4.1×10 −6 in control animals to 140.9 ± 64.8 × 10 −6 ( p = 0.0020) and the Hprt mutant frequency from 3.5 ± 1.5 × 10 −6 to 41.1 ± 23.2 × 10 −6 ( p = 0.0028). Sequence analysis of lacI mutant DNA and Hprt mutant cDNA produced similar overall mutation patterns: G:C → T:A transversion was the most common base pair substitution (32% of independent mutations in the lacI gene and 28% of Hprt mutations), and deletions and insertions accounted for 34% of mutations in the lacI gene and 28% in the Hprt gene. The majority of thiotepa-induced base pair substitutions in the Hprt gene occurred with the mutated purine on the non-transcribed DNA strand, while no strand-related bias was found for mutations in the lacI gene. Substitutions at G:C base pairs in the lacI gene, but not in the Hprt gene, were found disproportionately in CpG sites. In addition, multiplex polymerase chain reaction analysis of genomic DNA from the Hprt mutants indicated that 34% had relatively large deletions; none of these deletions was detected by the cDNA analysis. The results indicate that the frequency of thiotepa-induced mutants in Big Blue® rats was 2.8-fold greater in the lacI gene than in the Hprt gene. Although the Hprt gene recovered a fraction of large deletions not found among the lacI mutants, the effects of transcription-coupled DNA repair in the Hprt gene and the targeting of base pair substitutions to G:C base pairs in CpG sites may have contributed to the higher mutant frequencies induced by thiotepa in the lacI transgene compared with the Hprt gene.

EMS-induced mutant frequency and spectrum in bone marrow of D6-2 transgenic mice

EMS-induced mutant frequency and mutation spectrum as well as background mutant frequency have been characterized fur bone marrow of the D6-2 transgenic mice. ThelacI genes carried on pSPORT1 vectors were recovered from the treated or untreated mouse genomic DNA by excision and circularization, and analyzedin vitro for mutations that occurred in the mouse bone marrow, lacI(-) mutants were positively selected with the M9/L media. The 6 lacI(-) mutants were identified out of 11 935 vectors recovered from genomic DNA of the treated mice (mutant frequency was 50 x 10(5)), while no mutant was found in 11 649 vectors Imm untreated mice (the background mutant frequency wan lower than 8.6 x 10(-5)). Two regions oflacI for each mutant, in which the majority of sensitive sites for inactivation of thelacI gene product have been located, were sequenced and 16 mutation events were identified. The predominant mutations (14/16 or 87.5%) were base substitutions, whereas the remaining 2 mutations were single base deletions (12.5%). Of these base substitutions, transversions made up 9/14 or 64%, and transitions cornprised 5/14 or 36%, These findings were markedly different from the spontaneous spectra characterized by using Big-Blue system, as well as from the EMS-induced mutation spectra obtained within vitro assay systems, where the EMS-induced predominant mutations are CG --> AT transitions. In addition, 45% of mutations analyzed occurred at CpG dinucleotides, which was in accordance with previous studies with other systems. These data show that: (i) the D6-2 transgenic mouse lineage is a suitable mdel for studying mutagenesisin vivo; (ii) a fundamental difference in mutagenesis for EMS betweenin nitro andin vivo assay systems may exist, but more extensive sequence analyses are required to determine the possible differences in mutation spectra.

The in vivo mutagenicity and mutational spectrum at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice following a 4-week inhalation exposure to 1,3-butadiene

1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice. We determined the lacI mutant frequency and mutational spectrum in spleen following inhalation exposure to BD at levels that are known to induce tumors. B6C3F1 lacI transgenic mice were exposed to air or to 62.5, 625, or 1250 ppm BD for 4 weeks (6 h/day, 5 days/week) and euthanized 14 days after the last exposure. BD increased the lacI mutant frequency in spleen at all levels of BD examined. In BD-exposed mice, an increased frequency of G:C→A:T transitions occurred at non-5′-CpG-3′ sites. Exposure to BD in B6C3F1 lacI transgenic mice also increased the frequency of base substitution mutations that occurred at A:T base pairs when compared to air controls. The increased frequency of specific mutations at G:C base pairs in spleen was not observed in our previous studies in bone marrow and indicates tissue-specific differences in the BD-induced mutational spectrum. These data demonstrate that in vivo transgenic mouse mutagenicity assays can identify tissue-specific mutagenicity and mutational spectrum responses of genotoxic carcinogens at exposure levels that are known to induce tumors.

No direct correlation between mutant frequencies and cancer incidence induced by MeIQ in various organs of Big Blue® mice

The carcinogenicity of 2-amino-3,4-dimethylimidazo[4,5- f]quinoline (MeIQ) was examined in Big Blue® female mice with the genetic background of C57BL/6N. With the administration of 300 ppm of MeIQ in their diet for 92 weeks, the Big Blue® female mice developed intestinal tumors and hepatocellular carcinomas. The incidences of adenocarcinomas were 42% ( 8 19 ) in the colon and 68% ( 13 19 ) in the cecum. The incidence of hepatocellular carcinomas was 84% ( 16 19 ). No carcinomas of the intestine or the liver were induced in the control group. As we previously reported, administration of 300 ppm of MeIQ in a diet for 12 weeks induced lacI mutants at the highest frequency in colonocytes, and at only less than one-tenth of the colon in cells of the liver, forestomach and bone marrow, indicating no direct correlation between the lacI mutant frequency (MF) and cancer incidence (CI). The fate of cells with lacI mutation in each organ should be taken into consideration to validate MF as an indicator of carcinogenic potency of a chemical in different organs.

Enhanced liver cell mutations in trematode-infected Big Blue ® transgenic mice

Parasite infections in humans have long been associated with specific types of cancers. Schistosoma hematobium is a known inducer of urinary bladder cancer, Helicobacter pylori is a gastric carcinogen, and hepatitis B virus and Opisthorchis viverrini are causative agents of liver cell cancers. Another liver fluke, Fasciola hepatica, has also been identified as a neoplastic risk agent, primarily in animals. We used F. hepatica as a model agent to determine if the presence of an aggressive liver fluke could induce mutagenic events in mammalian tissue. Using the Big Blue ® transgenic mouse assay, we found a two-fold increase in lacI mutations in cells harvested from mice harboring F. hepatica worms when compared to uninfected control animals. These data indicate that biological infections can cause increased genetic damage in surrounding host tissue.

4-Chloro- o-phenylenediamine: a 26-week oral (in feed) mutagenicity study in Big Blue™ mice

4-Chloro- o-phenylenediamine (4-C- o-PDA) is a liver carcinogen in mice and was found to be weakly mutagenic in the liver of female Big Blue™ mice after short term treatment. In the present study the test compound was given subchronically in the diet for 26 weeks at doses of 0, 5000 and 10,000 ppm. The corresponding average test substance intake was 2166 mg kg −1 day −1 (males: 1794 mg kg −1 day −1; females: 2539 mg kg −1 day −1) and 4610 mg kg −1 day −1 (males: 3926 mg kg −1 day −1; females 5925 mg kg −1 day −1) at the low and high dose, respectively. After sacrifice, tissues were flash frozen in liquid nitrogen. The lacI mutant frequency in the liver was determined from three male and three female mice per dose group. The genomically integrated transgene was recovered by packaging into λ phage using Transpack™ packaging extract (Stratagene, La Jolla, USA) followed by infection of Escherichia coli strain SCS-8. Blue mutant plaques were scored against a background of clear non-mutant plaques. Food consumption decreased initially at 10,000 ppm, while no treatment related effect on food intake was observed at 5000 ppm. Body weight gain was found to be decreased in all treated animals. Absolute and relative liver weight increased in a dose-related manner, but only the latter effect was statistically significant. A clear dose dependent increase in lacI mutant frequencies was observed in the liver of both sexes. The following mutant frequencies (×10 −5) were observed: 2.73±1.01 (males, untreated), 7.24±1.50 (females, untreated), 18.91±5.30 (5000 ppm, males), 24.91±7.58 (5000 ppm, females), 20.47±6.68 (10,000 ppm, males) and 36.17±14.98 (10,000 ppm, females). It is therefore concluded that 4-C- o-PDA is a strong mutagen in the liver of mice treated subchronically for 26 weeks.

Hepatocarcinogenesis (Z#2)/mutagenesis during initiation stage

Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human α-1-antitrypsin ( α1AT) Z gene transgenic mouse (Z#2). We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies. We crossed the Z#2 line and mice transgenic for lambda/ lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis. Five month old double transgenic mice (Z#2 +/ lacI +) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10 −5–10 −4) useful to mutagenesis studies. In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (<2×) when compared to age matched negative controls. In the future, it may be important to reconcile possibly limited lacI mutagenesis at the time of initiation and demonstrated high incidence of hepatocarcinogenesis.

The butadiene metabolite, 1,2:3,4-diepoxybutane, induces micronuclei but is only weakly mutagenic atlacI in the Big Blue® Rat2lacI transgenic cell line

1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites, 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB). We investigated the mutagenicity and induction of micronuclei by DEB in vitro in Rat2 lambda/lacI transgenic fibroblasts (Big Blue Rat2 cells, Stratagene, LaJolla, CA). Assays for mutagenicity and micronuclei induction were carried out at concentrations of 0, 2, 5, or 10 microM DEB for 24 hours. Exposure of cells to these concentrations of DEB resulted in approximately 100, 50, and 10% survival, respectively, compared with media controls. In independent replicate experiments, no statistically significant increase in lacI mutant frequency was observed in Rat2 cells at any of the DEB exposure concentrations when compared to media or solvent controls. However, regression analyses indicated a trend toward increasing mutant frequency with increasing DEB exposure concentration. Experiments to examine the induction of micronuclei by DEB revealed a concentration-dependent increase in micronuclei in Rat2 cells following exposure to DEB. These results indicate that DEB induces micronuclei in the absence of detectable gene mutation at lacI in Big Blue Rat2 cells. The induction of micronuclei but only weak mutagenicity at the lacI transgene is likely due to the poor recovery of deletions using this lambda shuttle vector system, demonstrating the need to investigate multiple endpoints of genotoxicity when considering the mutational activity of a compound.

Effects of gender and species on spectra of mutation induced by 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine in the lacI transgene

Feeding of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) to F344 rats induces colon tumors specifically in male rats. Mutant frequencies and mutational spectra of the lacI transgene were studied in male and female Big Blue® transgenic rats after feeding 400 ppm of PhIP in the diet for 60 days. Mutant frequencies in the colon mucosa were increased 20–25 times compared with those of the control rats, being 661.4±33.3×10 −6 and 718.2±16.9×10 −6 in males and females, respectively. No significant differences in types and distribution of the mutations were detected between males and females. One-base deletions were the most frequent mutation, including the characteristic guanine deletion at 5′-GGGA-3′ which is also seen in the Apc gene of rat colon cancers induced by PhIP. Comparison of the lacI mutations in the rat colon with those previously identified in the mouse colon showed that the rate of G to T transversions was significantly higher in the mouse. This is the first report stating that there exist differences in the mutation specificity on the same gene, among mammalian species. However, the characteristic guanine deletion was recovered in both the mouse and the rat. These findings do not offer a mechanistic explanation of the gender specificity of PhIP-induced colon cancer in rats, though the universality of the guanine deletion suggests that this alteration may prove a useful indicator of human exposure.

P XV.5 - P XV.5 Increase in the frequency of LacI mutants accompanies development of hepatitis in the livers of LEC rats

P XV.5 - P XV.5 Increase in the frequency of LacI mutants accompanies development of hepatitis in the livers of LEC rats

Comparative study on organ-specificity of tumorigenicity, mutagenicity and cell proliferative activity induced by dimethylnitrosamine in Big Blue mice.

Recently, we have shown that dimethylnitrosamine (DMN) treatments increase lacI mutant frequency in the liver, kidney and lung but not in other organs, and also enhance cell proliferation only in the bronchial epithelia. In the present study, organ specificity of tumorigenicity induced by DMN was compared to those of lacI mutation and cell proliferation in Big Blue mice. Male 8-week-old Big Blue mice were treated with daily i.p. injections of 1 or 10 mg/kg DMN for 5 days, or a single i.p. injection of 5 or 10 mg/kg DMN. Except for the 10 mg/kg x 5 DMN group, all animals survived until 78 weeks after the first treatment of DMN. In the present study, the induction of cell proliferation in the bronchial epithelia was confirmed in a dose-dependent manner. At the termination of 78 weeks, it was histopathologically shown that the DMN-treated mice developed liver cell tumor in three out of seven (43%) of the 5 mg/kg group, renal tubule dysplasia in three out of seven (43%) of the 1 mg/kg x 5 group, and duodenal adenocarcinoma in one of seven (14%) of the 1 mg/kg x 5 group, although no neoplastic or preneoplastic lesions were found in the control mice. Because non-transgenic C57BL/6 mice are resistant to developing spontaneous liver cell and duodenal tumors, it was speculated that even these low doses of DMN could be sufficient to initiate target cells. Our results thus suggest that organ specificity of tumorigenicity by DMN is in favorable agreement with that of lacI mutation but not with possibly temporal cell proliferation induced by DMN.

Mutagenic consequences of the incorporation of 6-thioguanine into DNA

6-Thioguanine (S 6G) has been used in the treatment of acute leukemias because of its cytotoxic effect on proliferating leukemic cells. The cytotoxicity of S 6G is thought to derive from its incorporation into DNA in place of guanine. The deoxyribonucleoside triphosphate of S 6G, SdGTP, is a good substrate for bacterial and human DNA polymerases (Ling et al., Mol Pharmacol 40: 508–514, 1991). Since SdGTP was observed to misincorporate in place of adenine at a greater frequency than did dGTP, it appeared plausible that this analog could produce more subtle effects (mutations) due to mispairing with thymine. To assess whether such mutations occur, SdGTP was incorporated into the lacI gene of phage M131acISaXb in reactions that omitted dGTP ( −G) or dATP (−A). LacI mutation frequency was determined by β-galactosidase colorimetric staining (inactivation of the lac repressor results in blue plaques in the absence of inducet). When a high concentration of SdGTP (24 μM) was used in the DNA polymerase reaction, phage infectivity was inhibited. When a relatively low concentration (2.4 nM) was added to the −G and −A reactions, mutagenic effects were observed. DNA sequencing of mutant progeny arising from the −G + S 6G reaction revealed C-to-T base transitions and some C-to-A transversions. Similarly, the presence of SdGTP in the −A reactions led to mutants with T-to-C transitions. No insertions or deletions were observed. These data indicate that mispairing of S 6G with thymine leads to mutagenic effects in this assay.

In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure

Ethylene oxide (EO) is a direct-acting alkylating agent with the potential to induce cytogenetic alterations, mutations, and cancer. In the present study, the in vivo mutagenicity of EO at the hypoxanthine guanine phosphoribosyltransferase ( hprt) locus of T-lymphocytes was evaluated following inhalation exposure of male B6C3F1 lacI transgenic mice. For this purpose, groups of male Big Blue ® mice at 6–8 ( n=4/group) and 8–10 ( n=5/group) weeks of age were exposed to 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week). At necropsy, T-cells were isolated from thymus and/or spleen and cultured in the presence of concanavalin A, IL-2, and 6-thioguanine [Skopek, T.R., V.E. Walker, J.E. Cochrane et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 7866–7870]. The time course for expression of hprt-negative lymphocytes in thymus was determined in mice necropsied 2 h, 2 weeks, and 8 weeks after exposure to 200 ppm EO. The dose-response for hprt mutant T-cells in thymus and spleen was defined in mice necropsied 2 and 8 weeks post-exposure, respectively. The hprt mutant frequency (M f) in thymus of exposed mice was increased 2 h after exposure and reached a maximum of 7.5±0.9×10 −6 (average M f±SE) at 2 weeks post-exposure, compared with 2.3±0.8×10 −6 in thymus of control mice. Dose-related increases in hprt M fs were found in thymus from mice exposed to 100 and 200 ppm EO. In addition, a nonlinear dose-dependent increase in hprt M fs was observed in splenic T-cells, with greater mutagenic efficiency (mutations per unit dose) found at higher concentrations than at lower concentrations of EO. Average induced M fs (i.e. induced M f=treatment M f−background M f) in splenic T-cells were 1.6, 4.6, and 11.9×10 −6 following exposures to 50, 100, or 200 ppm EO, respectively, while the average control M f value was 2.2±0.3×10 −6. In aliquots of lymphocytes (both B- and T-cells) isolated from spleen for analysis of lacI mutations in the same animals, only two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI M f and these elevations were apparently due to the in vivo replication of preexisting mutants and not due to the induction of new mutations associated with EO exposure [Sisk, S., L.J. Pluta, K.G. Meyer and L. Recio (1996) Mutation Res., submitted]. These data demonstrate that repeated inhalation exposures to high concentrations of EO produce dose-related increases in mutations at the hprt locus of T-lymphocytes in male lacI transgenic mice of B6C3F1 origin.

Assessment of the in vivo mutagenicity of ethylene oxide in the tissues of B6C3F1 lacI transgenic mice following inhalation exposure

In the present study, the lacI mutant frequency was determined in the tissues of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of 15 male transgenic lacI B6C3F1 mice were exposed to either 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week) and were sacrificed at 0, 2, or 8 weeks after the last EO exposure. The lacI transgene was recovered from lung, bone marrow, spleen, and germ cells for determination of the lacI mutant frequency. The tissues selected for analysis were tumor target site tissues in chronic bioassays (lung tumors and lymphomas) and germ cells. The lacI mutant frequency in lung was significantly increased at 8 weeks post exposure to 200 ppm EO (6.2±2.2 vs. 9.1±1.5, p=0.046). In contrast, the lacI mutant frequency in spleen and bone marrow at 2 and 8 weeks was not significantly increased in mice exposed to 200 ppm EO. The lacI mutant frequencies in male germ cells for 200 ppm EO-exposed mice were not increased compared to air controls at 2 and 8 weeks post-exposure. In a spleen cell fraction two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI mutant frequency. The increased lacI mutant frequency in these animals was likely due to mutant siblings that contained background G:C→A:T transitions at CpG sites. These results demonstrate that a 4-week inhalation exposure to EO is mutagenic in lung. However, EO did not increase the frequency of mutations recovered at the lacI transgene in other tissues examined under the conditions used in the present studies. Since the mutational spectrum for EO in other systems consists of an increased proportion of large deletions, the lack of a mutagenic response in the tissues examined is likely due to the lack of recovery of large deletions in lambda-based shuttle vector systems. These data indicate that a primary mechanism of EO-induced mutagenicity in vivo is likely through the induction of deletions, not specific point mutations.

Insertion mutagenesis of the lac repressor and its implications for structure-function analysis.

We recently developed a simple technique for the generation of relatively large (31-codon) insertion mutations in cloned genes. To test whether the analysis of such mutations could provide insight into structure-function relationships in proteins, we examined a set of insertion mutants of the Escherichia coli lac repressor (LacI). Representatives of several LacI mutant classes were recovered, including mutants which exhibit fully active, inducer-insensitive, or weak dominant-negative phenotypes. The various properties of the recovered mutants agree with previous biophysical, biochemical, and genetic data for the protein. In particular, the results support the prior designation of mutationally tolerant spacer regions of LacI as well as proposed differences in dimerization interactions among regions of the protein core domain. These findings suggest that the analysis of 31-codon insertion mutations may provide a simple approach for characterizing structure-function relationships in proteins for which high-resolution structures are not available.

Agreement of mutational characteristics of heterocyclic amines in lacI of the Big Blue mouse with those in tumor related genes in rodents

The mutational spectra of carcinogenic heterocyclic amines (HCAs), 2-amino-3,4-dimethylimidazo[4,5-b]quinoline (MeIQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-9H-pyrido[2,3-b]indole (A alphaC) were studied in the colon of Big Blue mice. In 90, 115 and 105 lacI mutants from mice fed 300 p.p.m. MeIQ, 400 p.p.m. PhIP and 800 p.p.m. A alphaC, respectively, 92, 115 and 105 mutations were identified. G:C-->T:A transversions predominated with these HCAs. Mutational hot spots for base-substitution mutations caused by MeIQ, PhIP and A alphaC were in distinct sequence contexts; at 5'-GC-3', in runs of guanine and in 5'-CGT-3', respectively. Further, 30 of 115 (26%) PhIP-induced mutations were G:C base pair deletions, and eight of these deletions were in 5'-GGGA-3'. The mutational characteristics of MeIQ in the lacI gene coincided well with those in the Ha-ras gene of MeIQ-induced mouse forestomach tumors and rat Zymbal gland tumors. The characteristic single-base deletion induced by PhIP in the lacI gene also coincided well with those in the Apc gene of PhIP-induced rat colon tumors. These results suggest that the mutational characteristics of each chemical are conserved across different genes in different species.

Evaluation of lacI mutation in germ cells and micronuclei in peripheral blood after treatment of male lacI transgenic mice with ethylnitrosourea, isopropylmethane sulfonate or methylmethane sulfonate

Male C57Bl/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±0.7×10 −5) following treatment with ENU (11.7±0.8×10 −5, p=0.003) or with IPMS (9.6±0.5×10 −5, p=0.018) but not following treatment with MMS (8.1±0.8×10 −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.

Evaluation of spontaneous and chemical-induced lacI mutations in germ cells from lambda/ lacI transgenic mice

The spontaneous mutant frequency in germ cells isolated from seminiferous tubules of two lambda/ lacI transgenic mouse strains, C57BL/6 and B6C3F 1 was evaluated. At least 500 000 phage were screened for mutation at lacI for each animal using standardized assay procedures. The germ cell spontaneous lacI mutant frequency was 17.8±8.1×10 −6 in C57BL/6 mice and 17.0±10.0×10 −6 in B6C3F 1 mice. The induction of germ cell mutations by three well characterized alkylating agents were also evaluated in C57BL/6 mice on day 3 after a single dose administration. The lacI mutant frequencies were significantly elevated in transgenic mice dosed with ENU at 150 mg/kg (2-fold increase above control) and iPMS at 200 mg/kg (3-fold increase above control) but not in those receiving MMS at 40 mg/kg. These findings suggest that single dose studies using the lambda/ lacI transgenic system may be capable of detecting germ mutations induced by chemicals characterized either by point mutations or small, intragenic deletions but not those characterized by a predominance of multi-locus deletions.

Molecular analysis of lacI mutations in Rat2 ™ cells exposed to 7,12-dimethylbenz[ a]anthracene: evidence for DNA sequence and DNA strand biases for mutation

The Rat2 ™ cell line carries 50–70 stably integrated copies per cell of a λ/lacI shuttle vector as a target for mutagenicity testing. Rat2 ™ cells were exposed to 1 and 10 μg/ml of 7,12-dimethylbenz[ a]anthracene (DMBA) for 24 h at 37 °C in the presence of primary rat hepatocytes, and grown to confluence. The shuttle vector was rescued from untreated and mutagen-treated cells and mutant frequencies were determined. The low and high doses of DMBA induced mutant frequencies that were 7-fold (25 ±4.9 × 10 −5) and 33-fold (127 ±19.9 × 10 −5) higher, respectively, than the spontaneous mutant frequency (3.8 + 0.7 × 10 −5). DNA sequence analysis of the DMBA-induced lacI −1 mutants indicated that they contained mainly basepair substitution mutations at A : T and G : C, and that A : T → T : A and G : C → T : A transversions were the predominant types. In addition, 23 of 28 (82%) A : T basepair substitution mutations occurred with the mutated dA, the putatively adducted base, on the coding strand. Furthermore, 20 of the 28 (71%) A:T mutations had the mutated dA flanked 5′ by a dC, and 17 of these were A : T → T:A transversions, suggesting a sequence preference for this mutation. Except for a higher proportion of G : C → A : T transitions in the low dose data, the mutational profiles from low and high doses of DMBA were similar. These results indicate that DMBA mutagenesis in the lacI gene of Rat2 ™ cells displays distinct DNA sequence and DNA strand preferences.

A novel lacI transgenic mutation-detection system and its application to establish baseline mutation frequencies in the scid mouse

To assess DNA mutations in vivo, we have established a new transgenic mouse line, BC-1, carrying a lacI target gene for mutation detection within a bacteriophage shuttle-vector. The lacI gene was positioned within sequences derived from a rearranged murine immunoglobulin gene locus, a feature that distinguishes the BC-1 transgene from other shuttle vector systems. As mutations in lacI transgenes likely reflect mutations occurring throughout the genome, these systems have been successfully used to investigate spontaneous and induced mutations in a variety of tissues. An important additional application of the transgenic systems is the characterization of lacI mutations occurring in murine strains having specific DNA repair defects. For this study, scid (severe combined immunodeficiency) mice were selected as animals with this mutation have a defect in double-strand DNA break repair. To determine what impact the scid mutation might have on spontaneous mutation frequencies within DNA recovered from various tissues, these mice were crossed with the BC-1 line. Interestingly, mutation frequencies within BC-1/ scid mouse DNA were not significantly different from those of BC-1 control mice. Furthermore, spontaneous lacI mutations obtained from BC-1 and from BC-1/ scid liver DNA were similar in spectrum. As spontaneous BC-1 liver mutations were similar to those reported previously for other lacI systems, such as the Big Blue ® transgenic line, this suggested that the nature of the DNA sequences flanking the reporter gene did not modify lacI mutation rate or character.