Kenneth R. Tindall

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells.

AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage‐based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of ∼3 × 10−5 mutants/locus and 125,000–300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two‐thirds the MTD, and one‐third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose‐response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control. Environ. Mol. Mutagen. 35:253–259, 2000

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

The long-term effect of exposure to DNA alkylating agents is entwined with the cells genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this “gene–environment interaction” paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA β-polymerase (β-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene–environment interaction. The β-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in β-pol null cells, but not in isogenic wild-type cells; UV light exposure or N-methyl-N′-nitro-N-nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in β-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in β-pol null cells eliminates the effect. In contrast, β-pol/AAG double null cells are slightly more mutable than the β-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure.

Spectrum of spontaneous mutations in liver tissue of lacI transgenic mice

The advent of transgenic technology has greatly facilitated the study of mutation in animals in vivo. The Big Blue® mouse system, transgenic for the lacI gene, permits not only the quantification of mutations in different tissues but also provides for the generation of in vivo‐derived mutational spectra. This report details the sequence alterations of 348 spontaneous mutations recovered from the liver of 6–8‐week‐old male Big Blue® mice. The spectra recovered from two strains of mice, C57Bl/6 and B6C3F1, were compared and found to be very similar. The predominant mutations are G:C → A:T transitions, with 75% of these occurring at 5′‐CpG‐3′ sequences. This mutational bias is consistent with deamination‐directed mutation at methylated cytosine bases. The second most common class of mutations is G:C → T:A transversions. A significant clonal expansion of mutants was found in several animals, and this was used to make an approximate correction of the mutant frequency such that the most conservative estimate of mutation frequency is presented. The establishment of this substantial database of spontaneous mutations in the liver of Big Blue® mice is intended to serve as a reference against which mutations recovered after treatment can be compared. Environ. Mol. Mutagen. 30:273–286, 1997

Specificity of mutations induced by methyl methanesulfonate in mismatch repair-deficient human cancer cell lines.

Recently, we showed that the cytotoxic and mutagenic response in human cells to the model SN2 alkylating agent methyl methanesulfonate (MMS) can be modulated by the mismatch repair (MMR) pathway. That is, human cancer cell lines defective in MMR are more resistant to the cytotoxic effects of MMS exposure and suffer more induced mutations at the HPRT locus than MMR-proficient cell lines. Since MMS produces little O6-methylguanine (O6-meG), the observed hypermutability and resistance to cytotoxicity in MMR-defective cells likely results from lesions other than O6-meG. MMS produces a high yield of N7-methylguanine (N7-meG) and N3-methyladenine (N3-meA), which can lead to the formation of promutagenic abasic sites, and these lesions may be responsible for the observed cytotoxic and/or mutagenic effects of MMS. To further investigate the mechanism of MMS mutagenesis, two MMR-defective human cancer cell lines were treated with MMS and the frequency and the types of mutations produced at the HPRT locus were determined. MMS treatment (1.5 mM) produced a 1.6- and a 2.2-fold increase in mutations above spontaneous levels in HCT116 and DLD-1 cell lines, respectively. An average 3.7-fold increase in transversion mutations was observed, which accounted for greater than one-third of all induced mutations in both cell lines. In contrast, an average 1.6-fold increase was seen among transition mutations (the class expected from O-alkylation products). Since transversion mutations are not produced by O6-meG, these findings suggest that abasic sites may be the lesion responsible for a large proportion of MMS mutagenicity in MMR-defective cells. Furthermore, these data suggest the MMS-induced damage, either abasic site-inducing base alterations (i.e., N7-meG and N3-meA) or the resulting abasic sites themselves, may be substrates for recognition and/or repair by MMR proteins.

Mutagenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the new gptΔ transgenic mouse

Gender differences and organ specificity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptdelta transgenic mouse (T. Nohmi, M. Katoh, H. Suzuki, M. Matsui, M. Yamada, M. Watanabe, M. Suzuki, N. Horiya, O. Ueda, T. Shibuya, H. Ikeda, T. Sofuni, A new transgenic mouse mutagenesis test system using Spi-and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465-470). In this mouse model, two distinct selections are employed to efficiently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi-selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No significant differences in 6-TG and Spi- mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed.

Mutational specificity: Spectrum of mutations in kidney, stomach, and liver from lacl transgenic mice recovered after treatment with tris(2,3‐dibromopropyl)phosphate

The flame retardant tris(2,3‐dibromopropyl)phosphate (TDBP), once used in cotton sleepware for children, is presently banned from commerce. It produces tumors in rodents in both a sex‐ and tissue‐specific manner. The kidney is the main target for tumor formation in male and female rats, as well as in male mice. In contrast, tumors are formed in the liver of female animals. We have used lacl transgenic male B6C3F1 mice (Big Blue®) to examine the induction of mutation in kidney, liver, and stomach after exposure to 150 mg/kg (2 days), 300 mg/kg (4 days), and 600 mg/kg (4 days of TDBP. At the highest dose, the mutant frequency was approximately 50% above control values in the kidney (P < 0.01). A smaller increase was observed in the liver (P = 0.07), while no increase was seen in the stomach (P = 0.28). Sequence analysis of the recovered mutants showed a TDBP‐specific change in mutation spectrum in kidney, which was not observed in liver and stomach. In kidney, a dose‐dependent decrease in G:C → A:T transitions, including at 5′‐CpG‐3′ sites, was observed. This was accompanied by an increase in the loss of single G:C base pairs from approximately 3% to 15%. These results illustrate both the sensitivity and specificity of the lacl transgenic system in the analysis of tissue‐specific mutation. This study also reinforces the importance of examining mutational spectra when mutant induction levels are low.

Mutant frequency of lacI in transgenic mice following benzo[a]pyrene treatment and partial hepatectomy.

The Big Blue, transgenic mouse provides an in vivo mutation system that permits the study of pharmacodynamic parameters on mutant frequency (MF) following xenobiotic exposure. We have studied the effects of cellular proliferation on the frequency of mutations in the lacl transgene by evaluating the MF in the liver of male C57B1/6 Big Blue mice following treatment with benzo[a]pyrene (B[a]P) and a partial hepatectomy. Mice received either 40 mg/kg of B[a]P in corn oil or corn oil alone by i.p. injection on three consecutive days, followed by a partial hepatectomy on the fourth day. Three days later (i.e., 7 days following the initial B[a]P injection), the animals were sacrificed and the MF in the liver was compared to the MF observed in the liver of the same mouse at the time of hepatectomy. Induction of cytochrome P-450 1A (CYP1A) following B[a]P treatment was evident by Western blot analysis. The MF in untreated control animals was not significantly different at hepatectomy (4.7 +/- 0.8 x 10(-5)) and 3 days later, at sacrifice (3.0 +/- 0.4 x 10(-5)). Neither was the MF observed in the B[a]P-treated mice at the time of sacrifice (12.0 +/- 2.1 x 10(-5)) significantly different from the MF observed at the time of hepatectomy (10.6 +/- 5.3 x 10(-5)). However, B[a]P-treatment resulted in a 4.0-fold increase in MF at sacrifice which was significantly different (p < 0.05), when compared to the untreated controls. The B[a]P-treated mice at hepatectomy showed a modest 2.2-fold increase in MF which was not statistically significantly different from the untreated controls. In addition, both control and B[a]P-treated tissues gave sectored mutant plaques. The sectored plaque frequency (SPF) was significantly elevated (p < 0.05) in the B[a]P-treated mice at hepatectomy (4.2 +/- 1.0 x 10(-5)) and sacrifice (7.3 +/- 2.4 x 10(-5)) as compared to the respective frequency in the control mice at hepatectomy (1.9 +/- 0.7 x 10(-5)) and sacrifice (1.4 +/- 0.2 x 10(-5)). One explanation for this data is the persistence of the B[a]P adducts in the mouse genomic DNA that was packaged into the lambda phage, and ultimately fixed as mutations in Escherichia coli.

Genetic instability favoring transversions associated with ErbB2-induced mammary tumorigenesis

It has been argued that genetic instability is required to generate the myriad mutations that fuel tumor initiation and progression and, in fact, patients with heritable cancer susceptibility syndromes harbor defects in specific genes that normally maintain DNA integrity. However, the vast majority of human cancers arise sporadically, in the absence of deficiencies in known “mutator” genes. We used a cII-based mutation detection assay to show that the mean frequency of forward mutations in primary mammary adenocarcinomas arising in mouse mammary tumor virus-c-erbB2 transgenic mice harboring multiple copies of the λ bacteriophage genome was significantly higher than in aged-matched, wild-type mammary tissue. Analysis of the cII mutational spectrum within the mammary tumor genomic DNA demonstrated a >6-fold elevation in transversion mutation frequency, resulting in a highly unusual inversion of the transition/transversion ratio characteristic of normal epithelium; frameshift mutation frequencies were unaltered. Arising oncogenic point mutations within the c-erbB2 transgene of such tumors were predominantly transversions as well. Data from this model system support the notion that elaboration of a mutator phenotype is a consequential event in breast cancer and suggest that a novel DNA replication/repair gene is a relatively early mutational target in c-erbB2-induced mammary tumorigenesis.

Complementation of mismatch repair gene defects by chromosome transfer.

The study of the multiple functions of mismatch repair genes in humans is being facilitated by the use of human tumor cell lines carrying defined MMR gene mutations. Such cell lines have elevated spontaneous mutation rates and may accumulate mutations in other genes, some of which could be causally related to the phenotypes of these cells. One approach to establish a cause-effect relationship between a MMR gene defect and a phenotype is to determine if that phenotype is reversed when a normal chromosome carrying a wild-type MMR gene is introduced by microcell fusion. This approach has the advantage of presenting the gene in its natural chromosomal environment with normal regulatory controls and at a reasonable dosage. The approach also limits candidate genes to only those encoded by the introduced chromosome and not elsewhere in the genome. Here we review studies demonstrating that hMSH2, hMSH3, hMSH6 and hMLH1 gene defects can each be complemented by transferring human chromosome 2, 5, 2 or 3, respectively. These transfers restore MMR activity, sensitivity to killing by MNNG, stability to microsatellite sequences and low spontaneous HPRT gene mutation rates.