Thomas R. Skopek

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

DNA synthesis inhibition as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA-reactive clastogens

Positive results in the in vitro assay for chromosome aberrations sometimes occur with test chemicals that apparently do not react with DNA, being negative in tests for mutation in bacteria, for DNA strand breaks, and for covalent binding to DNA. These chromosome aberrations typically occur over a narrow concentration range at toxic doses, and with mitotic inhibition. Indirect mechanisms, including oxidative damage, cytotoxicity and inhibition of DNA synthesis induced by chemical exposure, may be involved. Understanding when such mechanisms are operating is important in evaluating potential mutagenic hazards, since the effects may occur only above a certain threshold dose. Here, we used two-parameter flow cytometry to assess DNA synthesis inhibition (uptake of bromodeoxyuridine [BrdUrd]) associated with the induction of aberrations in CHO cells by DNA-reactive and non-reactive chemicals, and to follow cell cycle progression. Aphidicolin (APC), a DNA polymerase inhibitor, induces aberrations without reacting with DNA; 50 microM APC suppressed BrdUrd uptake during a 3-h treatment to <10% of control levels. Several new drug candidates induced aberrations concomitant with marked reductions in cell counts at 20 h (to 50-60% of controls) and suppression of BrdUrd uptake (<15% of control). Several non-mutagenic chemicals and a metabolic poison, which induce DNA double strand breaks and chromosome aberrations at toxic dose levels, also suppressed DNA synthesis. In contrast, the alkylating agents 4-nitroquinoline-1-oxide, mitomycin C, methylnitrosourea, ethylnitrosourea, methylmethane sulfonate and ethylmethane sulfonate, and a topoisomerase II inhibitor, etoposide, produced many aberrations at concentrations that were less toxic (cell counts >/=73% of controls) and gave little inhibition of DNA synthesis during treatment (BrdUrd uptake >/=85% of controls), although cell cycle delay was seen following the 3-h treatment. Thus, inhibition of DNA synthesis at the time of treatment is supporting evidence for an indirect mechanism of aberrations, when there is no direct DNA reactivity.

System Issues: Mutagenic response of the endogenous hprt gene and lacl transgene in benzo[a]pyrene-treated Big Blue™ B6C3F1 mice

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 lacl 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 × 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 × 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 offer B[a]P exposure. Western blot analysis of liver samples from B[a]P‐treated mice suggests that the reduced adducts 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 × 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 × 10−6). To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified λ‐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 lacl transgene than at the endogenous hprt locus.

A comparative study of in vivo mutation assays: analysis of hprt, lacI, and cII/cI as mutational targets for N-nitroso-N-methylurea and benzo[a]pyrene in Big Blue™ mice

We have compared the response of the native hprt gene and the lacI, cII, and cI transgenes in Big Blue B6C3F1 mice following treatment with either N-nitroso-N-methylurea (MNU) or benzo[a]pyrene (BaP). Three weeks after mutagen treatment splenic T cells were isolated from the animals, and samples were either cultured to measure mutation at the native hprt locus or used to extract genomic DNA for transgene mutation analysis. Phage rescued from extracted DNA were plated in the presence of 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) to score lacI mutations, or plated on a hflAB lawn to score cII and cI mutants. With MNU hprt mutant frequency increased in a dose-related, sublinear manner up to 78-fold above background at the highest dose tested (20 mg/kg). In comparison, the lacI transgene yielded only a 3.1-fold increase at this dose, and the cII and cI transgenes did not show any increase. With 150 mg/kg BaP a 5.8- and 8.7-fold increase in mutant frequency was observed at hprt and lacI, respectively, while only a 1.3-fold increase was observed at cII. DNA sequencing revealed an increase in GC-->TA transversions among the cII mutants, suggesting that the increase was related to BaP exposure. No significant increase in cI mutant frequency was observed. Therefore, the order of mutation assay sensitivity was hprt>lacI>cII/cI with MNU, and hprt approximately lacI> cII/cI with BaP. While the hflAB selection system offers significant advantages with respect to cost and effort when compared to the lacI assay, additional evaluation of its sensitivity is warranted.

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

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.

Comparison of motility and membrane integrity to assess rat sperm viability.

Rat sperm motility and membrane integrity were compared as endpoints for viability. Sperm motility was measured by computer-assisted semen analysis (CASA), whereas membrane integrity was assessed by flow cytometric analysis of sperm stained with two nucleic acid stains, SYBR-14 and propidium iodide. The two techniques were compared in experiments that examined sperm viability over time and by analysis of known mixtures of control and freeze/thaw-killed sperm. Results from the two approaches were quantitatively very similar. Sperm from rats treated with dibromoacetic acid (600 or 1200 mg/kg) or alpha-chlorhyrin (100 mg/kg) were also analyzed. Neither technique detected a treatment-related effect with dibromoacetic acid. CASA identified a significant decrease in sperm motility in alpha-chlorhyrin-treated rats, whereas flow cytometric analysis did not find a measureable change in sperm membrane integrity. Because decreases in sperm motility would be expected to directly affect fertility, CASA may be a more robust endpoint for risk assessment in reproductive toxicology studies than flow cytometric analysis of membrane integrity.

Molecular analyses of a Lesch-Nyhan syndrome mutation (hprtMontreal) by use of T-lymphocyte cultures

SummaryThe frequency of hprt mutants in peripheral blood T-lymphocytes of two putative Lesch-Nyhan individuals and their parents was determined by a cell cloning assay to quantify the frequency of thioguanine-resistant mutants. The results confirmed the Lesch-Nyhan diagnosis and demonstrated that the mother has an elevated mutant frequency consistent with being heterozygous for an hprt mutation. Mass cultures of T-lymphocytes from both the children and their mother, as well as cultures of hprt mutant clones from the mother, were employed as sources of mRNA for cDNA sequence analysis. These hprt mutants show a single base substitution (T→C transition) at position 170 (exon 3). The predicted amino acid change is the substitution of threonine for methionine56. We have designated this new Lesch-Nyhan mutation hprtMontreal. The use of T-lymphocyte cultures allows rapid sequence analyses of hprt mutations, as well as family studies to define the origin of a particular mutation.

Extent of CpG methylation is not proportional to the in vivo spontaneous mutation frequency at transgenic loci in Big Blue™ rodents

The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5x10(-5) range. Recently, the bone marrow and bladder of the Big Blue rat were reported to have, by an order of magnitude, the lowest spontaneous mutation frequencies ever observed for lacI in a transgenic animal, approaching the value for endogenous targets such as hprt ( approximately 10(-6)). Since spontaneous mutations in transgenes have been attributed in part to deamination of 5-methylcytosine in CpG sequences, we have investigated the methylation status of the lacI transgene in bone marrow of BB rats and compared it to that present in other tissues including liver, spleen, and breast. The first 400 bases of the lacI gene were investigated using bisulfite genomic sequencing since this region contains the majority of both spontaneous and induced mutations. Surprisingly, all the CpG cytosines in the lacI sequence were fully methylated in all the tissues examined from both 2- and 14-week-old rats. Thus, there is no correlation between 5-methylcytosine content at CpG sites in lacI and the frequency of spontaneous mutation at this marker. We also investigated the methylation status of another widely used transgenic mutation target, the cII gene. The CpG sites in cII in BB rats were fully methylated while those in BB mice were partially methylated (each site approximately 50% methylated). Since spontaneous mutation frequency at cII is comparable in rat and mouse, the methylation status of CpG sequences in this gene also does not correlate with spontaneous frequency. We conclude that other mechanisms besides spontaneous deamination of 5-methylcytosine at CpG sites are driving spontaneous mutation at BB transgenic loci.

Analysis of sequence alterations in a defined DNA region: comparison of temperature-modulated heteroduplex analysis and denaturing gradient gel electrophoresis.

The ability to detect DNA sequence heterogeneity quickly and reliably is becoming increasingly important as more genes involved in disease processes are discovered. We have assessed the ability of a high pressure liquid chromatography technique (HPLC) termed temperature-modulated heteroduplex analysis (TMHA) to detect a collection of 20 point mutations distributed throughout a 279 base pair fragment spanning the exon 8 region of the human HPRT gene. All mutant/wild type heteroduplexes formed from mutations in the lowest temperature melting domain of the fragment were easily resolved from the corresponding mutant and wt homoduplexes, while those generated from mutants in the next higher melting domain barely resolved from their parental homoduplexes. For comparison, identical heteroduplex samples were subjected to denaturing gradient gel electrophoresis (DGGE). Heteroduplexes in the lowest temperature melting domain were easily resolved, while no resolution was achieved with those in the next higher melting domain. These results suggest that TMHA and DGGE are measuring similar melting characteristics in heteroduplex molecules. TMHA appears to be a robust approach for detecting and/or purifying a wide variety of mutations in a defined region of DNA, provided that the melting characteristics of the fragment under study are carefully considered.