D.M. Zimmer

Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue® transgenic mice

Big Blue® mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor‐intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073–9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C → A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3′ flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G → A and G → T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G → A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations. Environ. Mol. Mutagen. 33:132–143, 1999

Culture and propagation of Hprt mutant T-lymphocytes isolated from mouse spleen.

The optimization of the mouse lymphocyte Hprt mutation assay has been impeded by the relatively poor growth potential of mouse T‐cells in vitro, which leads to low cloning efficiencies (CEs) and limited expansion of Hprt mutant clones for molecular analysis of mutations occurring in control and treated mice. In this study, the addition and manipulation of concanavalin A (Con A), mouse interleukin‐2 (IL‐2), and a commercially available culture supplement, rat T‐STIMTM with Con A, were used to identify growth conditions producing relatively high CEs for mouse T‐cells. Supplementation of medium with 10% rat T‐STIMTM, along with appropriate amounts of Con A for priming and exogenous IL‐2 for cloning, resulted in average CEs of 15–16% in lymphocytes isolated from spleens of control mice (n = 32) or mice exposed to 1,3‐butadiene (n = 27). In addition, several reagents were assessed for their potential to stimulate long‐term growth of Hprt mutant clones; these T‐cell stimulatory agents included Con A, phytohemagglutinin, and a calcium ionophore ionomycin combined with a tumor promoter phorbol 12‐myristate 13‐acetate. In a pilot study, stimulation with Con A proved to be the most effective means for propagating mouse T‐cell clones under the various conditions tested. In follow‐up experiments, transfer of mutant clones to 24‐well plates and repeated stimulation with Con A in IL‐2 and rat T‐STIMTM supplemented medium was found to expand 76% of 536 mutant clones to about 400,000 to several million cells per clone. These data indicate that rat T‐STIMTM‐supplemented medium enhances the initial outgrowth of mouse T‐cells, and that repeated mitogenic stimulation with Con A in the presence of IL‐2 and rat T‐STIMTM provides a means for propagating mouse T‐cell clones for mutation analyses by a variety of methods. Environ. Mol. Mutagen. 32:236–243, 1998

Comparison of mutant frequencies at the transgenic lambda LacI and cII/cI loci in control and ENU-treated Big Blue mice.

We compared the lambda cII/cI transgenic mutation assay described by Jakubczak et al. [(1996): Proc Natl Acad Sci USA 93:9073–9078] to the previously established Big Blue® assay. Genomic DNA isolated from liver, spleen, and lung tissue of control or ethylnitrosourea (ENU)‐treated Big Blue® mice (100 mg/kg i.p., single dose) was packaged into phage (five animals, two packagings per DNA sample) which were simultaneously plated for lacI and cII/cI mutant frequency (MF) and titer. Mean MF of control animals was higher for cII/cI than lacI for all three tissues examined (spontaneous cII/cI MF divided by spontaneous lacI MF = 2.9, 3.1, and 1.7 for liver, spleen, and lung, respectively). The differences were statistically significant for liver and spleen, but not lung. The ENU‐induced MF measured by subtracting control MFs from ENU‐treated MFs was higher in the cII/cI assay than lacI (liver = 23.0 × 10−5 for cII/cI vs. 15.1 × 10−5 for lacI; spleen = 64.8 × 10−5 for cII/cI vs. 36.1 × 10−5 for lacI; lung = 17.1 × 10−5 for cII/cI vs. 15.8 × 10−5 for lacI). Fold increase over control values measured by dividing MF of ENU‐treated animals by appropriate control values was higher for lacI than cII/cI (liver = 4.4‐fold for lacI vs. 2.7 for cII/cI; spleen = 13.1‐fold for lacI vs. 8.4 for cII/cI; and lung = 5.6‐fold for lacI vs. 4.0 for cII/cI). Despite these differences, overall results were similar for the two mutational endpoints. These results suggest that the cII/cI assay may be an acceptable alternative to lacI where transgenic mutation studies are indicated. Environ. Mol. Mutagen. 33:249–256, 1999

System issue: Spontaneous and ethylnitrosourea-induced mutation fixation and molecular spectra at the lacl transgene in the Big Blue® Rat-2 embryo cell line

Big Blue™ Rat‐2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea [ENU]‐induced). Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined. Exposures were 100, 200, 400, 600, and 1,000 μg ENU/ml. The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 × 10−5. Dose‐related increases in mutant frequency were observed following ENU treatment. Kinetics (time course, of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 μg ENU/ml. Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C → A:T transitions (five at CpG sites), six (23%) G:C → T:A transversions, three (11.5%) G:C → C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion + insertion) mutation. Among 46 ENU‐induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C → A:T transitions (four at CpG sites), five (13.5%) A:T → G:C transitions, four (10.8%) G:C → T:A transversions, 11 (30%) A:T → T:A transversions, and two (5.4%) A:T → C:G transversions. Nearly 50% of the base substitutions in the ENU‐treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found. Both the spontaneous and the ENU‐induced mutational spectra were similar to that reported in vivo and for other cells. An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 μg/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity.

Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay.

We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue® transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU‐treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4°C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4°C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4°C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37°C or 24°C showed highest titers on G1217 at 37°C. The fraction of the G1217, 37°C titer seen in the other strains and conditions varied widely with the cII mutation. Environ. Mol. Mutagen. 32: 325–330, 1998

In vivo mutagenesis in the cynomolgus monkey: time course of HPRT mutant frequency at long time points following ethylnitrosourea exposure.

We have monitored mutant frequency at the HPRT locus in peripheral blood lymphocytes of cynomolgus monkeys using a clonal assay in which mutants are selected by resistance to 6‐thioguanine. Among untreated animals, the mean spontaneous mutant frequency was 2.9 ± 2.9 × 10‐6 (standard deviation, based on 131 determinations in 33 animals), in good agreement with HPRT mutant frequencies in other species. In four animals treated with a single intraperitoneal dose of 77 mg/kg ethylnitrosourea, mutant frequency increased with time, peaking 70 to 100 days after treatment. Mutant frequency in two of the four animals was monitored at intervals for 6 years, and a second identical treatment was given about 830 days after the first. Mutant frequency again peaked in these two animals 70 days after the second dose and decreased following peak values, declining to a plateau that was higher than the predose mutant frequency in both animals. This pattern was repeated following the second ethylnitrosourea treatment. Fractionating the dose of ethylnitrosourea into five equal daily injections had no effect on mutant frequency in two animals when compared to a single dose. Environ. Mol. Mutagen. 29:117‐123, 1997.

DNA sequence analysis of hprt mutants persisting in peripheral blood of cynomolgus monkeys more than two years after ENU treatment.

We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T‐lymphocytes. This primate model allows us to study mutations and their kinetics under well‐controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg). At 832 days after that first treatment, the monkey received a second dose of 77 mg/kg ENU. Up to 1,331 days after the second treatment, the T‐cell mutant frequency (44.2 × 10−6) was still 26‐fold higher than background (1.7 × 10−6), suggesting that mutants persisted in the peripheral blood. Mutant clones from Days 974, 1,164, and 1,311 after the second treatment were selected in thioguanine. Hprt cDNA was prepared from a cell lysate, PCR‐amplified, and sequenced. Of 45 mutants, 30 yielded PCR product and 26 were sequenced. Base substitutions were found in 21 (81%) of the 26 mutants and consisted of one G:C → A:T and five A:T → G:C transitions, one G:C → C:G, eight A:T → T:A, and six A:T → C:G transversions. Therefore, most base substitutions occurred at A:T basepairs, characteristic of ENU‐induced mutations in vivo, and were detected up to 3.6 years after the second treatment. Deletions of exons 2 and 3 occurred in two mutants and exon 7 was deleted in one mutant. There were two insertion mutants: one was a single base insertion and the other contained an insertion of 277 basepairs which was nearly identical to a simian retroviral sequence. Environ. Mol. Mutagen. 33:42–48, 1999

Lack of response to multiple genotoxic agents at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys

We tested the ability of a series of known genotoxic agents to cause mutations at the hprt locus in peripheral blood T‐lymphocytes of cynomolgus monkeys as measured by the ability to form clones in the presence of 6‐thioguanine. Ethylmethane sulfonate (EMS, 300 mg/kg IP), chloroethylmethane sulfonate (Cl‐EMS, 35 or 50 mg/kg IP), and the Pharmacia & Upjohn antitumor agents adozelesin (1.6, 4, 6, or 8 μg/kg IV) and CC‐1065 (6 μg/kg IV) were all negative in the hprt mutation test. Results with cyclophosphamide (CP, 75 mg/kg IV) were equivocal. Adozelesin, CC‐1065, and Cl‐EMS treatments increased the percentage of T‐lymphocytes with chromosome aberrations, as well as inducing types of aberrations not seen in control cells. EMS and CP were not tested for chromosome aberrations. We have previously shown that treatment of monkeys with 77 mg/kg ENU substantially increased the hprt mutant frequency, with a lag time of approximately 77 days between treatment and peak MF values. The results of the present study suggest a low sensitivity of the hprt mutation assay to certain classes of genotoxic agents in cynomolgus monkeys. Environ. Mol. Mutagen. 33:123–131, 1999