Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5' nuclease assay (TaqMan).

The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5' nuclease PCR (TaqMan) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7kb beta-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using 32P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas 32P-postlabeling with nuclease P(1) enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B(1). Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing 32P-postlabeling assay with nuclease P(1) enrichment.

Suppression of spontaneous and hydrogen peroxide-induced mutagenesis by the antioxidant ascorbate in mismatch repair-deficient human colon cancer cells.

Genomic instability associated with deficiencies in mismatch repair (MMR) plays a critical role in tumorigenesis. Here we have investigated the contribution of oxidative damage to this instability in MMR-defective cells. Treatment with H(2)O(2) produced less cytotoxicity in MMR-deficient cells than in those proficient in MMR, supporting a role for MMR in the recognition and/or processing of oxidative damage. Additionally, growth of MMR-defective cells in the presence of the antioxidant ascorbate (500 microM) reduced the spontaneous mutation rate at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus by up to 50% and reduced microsatellite instability by 30%. Induction of HPRT mutants by exogenously added H(2)O(2) was also significantly suppressed by ascorbate. Collectively, these results suggest that (i) oxidative damage contributes significantly to the spontaneous mutator phenotype in MMR-defective cells, (ii) this damage may select for MMR-deficient cells due to their increased resistance to cell killing and (iii) dietary antioxidants may help to suppress the mutator phenotype and resulting carcinogenesis in individuals with compromised MMR.

Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4A.

Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan(R) probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3' end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families.

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.

Specificity of mutations induced by the food-associated heterocyclic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine in colon cancer cell lines defective in mismatch repair.

Recently, we have shown a hypermutable response to the food-associated heterocyclic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP) in human cells defective in mismatch repair (MMR). These findings suggest that exogenous compounds such as PhIP may play an important role in the generation of tumors in MMR-defective individuals. The specificity of mutations induced by PhIP exposure at the endogenous HPRT locus was determined in cell lines defective in MMR to better understand the mutagenic effects of PhIP in MMR-defective individuals and to gain insight into the molecular mechanism of carcinogenesis induced by PhIP. Eighty-six induced HPRT mutants from two different cell lines were isolated and sequenced after exposure to 10 microM PhIP. Nineteen (22%) of these mutants contained G:C to T:A transversion mutations, consistent with the promutagenic adduct of PhIP at the C8 position of guanine miscoding with adenine. This level of PhIP-induced G:C to T:A transversions was approximately 4.5-fold higher than spontaneous G:C to T:A frequencies. Additionally, a hotspot for mutation was observed in a run of six guanines in HPRT exon 3, where a total of 23 (27%) of all PhIP-induced mutations occurred. These mutations consisted of transversions, transitions, and frameshift mutations. The increase in mutant frequency at this run of guanines corresponded to a 24-fold elevation above the spontaneous frequency in one cell line and a 3.3-fold increase in the other. These data suggest that PhIP may increase the risk of human carcinogenesis mediated by MMR by increasing mutations at runs of guanine residues. PhIP may thereby promote tumorigenesis by mutating growth-regulating genes that contain runs of guanines in their coding sequences, such as BAX, the insulin-like growth factor II receptor IGFIIR, and even the mismatch repair gene hMSH6.

Biomarkers of exposure and effect as indicators of potential carcinogenic risk arising from in vivo metabolism of ethylene to ethylene oxide.

The purposes of the present study were: (i) to investigate the potential use of several biomarkers as quantitative indicators of the in vivo conversion of ethylene (ET) to ethylene oxide (EO); (ii) to produce molecular dosimetry data that might improve assessment of human risk from exogenous ET exposures. Groups (n = 7/group) of male F344 rats and B6C3F1 mice were exposed by inhalation to 0 and 3000 p. p.m. ET for 1, 2 or 4 weeks (6 h/day, 5 days/week) or to 0, 40, 1000 and 3000 p.p.m. ET for 4 weeks. N:-(2-hydroxyethyl)valine (HEV), N:7-(2-hydroxyethyl) guanine (N7-HEG) and HPRT: mutant frequencies were assessed as potential biomarkers for determining the molecular dose of EO resulting from exogenous ET exposures of rats and mice, compared with background biomarker values. N7-HEG was quantified by gas chromatography coupled with high resolution mass spectrometry (GC-HRMS), HEV was determined by Edman degradation and GC-HRMS and HPRT: mutant frequencies were measured by the T cell cloning assay. N7-HEG accumulated in DNA with repeated exposure of rodents to 3000 p.p.m. ET, reaching steady-state concentrations around 1 week of exposure in most tissues evaluated (brain, liver, lung and spleen). The dose-response curves for N7-HEG and HEV were supralinear in exposed rats and mice, indicating that metabolic activation of ET was saturated at exposures >/=1000 p.p.m. ET. Exposures of mice and rats to 200 p.p.m. EO for 4 weeks (as positive treatment controls) led to significant increases in HPRT: mutant frequencies over background in splenic T cells from exposed rats and mice, however, no significant mutagenic response was observed in the HPRT: gene of ET-exposed animals. Comparisons between the biomarker data for both unexposed and ET-exposed animals, the dose-response curves for the same biomarkers in EO-exposed rats and mice and the results of the rodent carcinogenicity studies of ET and EO suggest that too little EO arises from exogenous ET exposure to produce a significant mutagenic response or a carcinogenic response under standard bioassay conditions.

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 approximately 3 x 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.

A novel assay for allelic discrimination that combines the fluorogenic 5' nuclease polymerase chain reaction (TaqMan) and mismatch amplification mutation assay.

Recently much attention has been focused on single nucleotide polymorphisms (SNPs) within fundamentally important genes, such as those involved in metabolism, cell growth regulation, and other disease-associated genes. Methodologies for discriminating different alleles need to be specific (robust detection of an altered sequence in the presence of wild-type DNA) and preferably, amenable to high throughput screening. We have combined the fluorogenic 5' nuclease polymerase chain reaction (TaqMan) and the mismatch amplification mutation assay (MAMA) to form a novel assay, TaqMAMA, that can quickly and specifically detect single base changes in genomic DNA. TaqMan chemistry utilizes fluorescence detection during PCR to precisely measure the starting template concentration, while the MAMA assay exploits mismatched bases between the PCR primers and the wild-type template to selectively amplify specific mutant or polymorphic sequences. By combining these assays, the amplification of the mutant DNA can be readily detected by fluorescence in a single PCR reaction in 2 hours. Using the human TK6 cell line and specific HPRT-mutant clones as a model system, we have optimized the TaqMAMA technique to discriminate between mutant and wild-type DNA. Here we demonstrate that appropriately designed MAMA primer pairs preferentially amplify mutant genomic DNA even in the presence of a 1,000-fold excess of wild-type DNA. The ability to selectively amplify DNAs with single nucleotide changes, or the specific amplification of a low copy number mutant DNA in a 1,000-fold excess of wild-type DNA, is certain to be a valuable technique for applications such as allelic discrimination, detection of single nucleotide polymorphisms or gene isoforms, and for assessing hotspot mutations in tumor-associated genes from biopsies contaminated with normal tissue.

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.

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 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.

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.

Cytotoxic and mutagenic response of mismatch repair-defective human cancer cells exposed to a food-associated heterocyclic amine.

The cytotoxic and mutagenic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP), a food-associated heterocyclic amine, were measured in three human cancer cell lines possessing different mismatch repair (MMR) defects and in matched cell lines corrected for the MMR deficiencies by specific chromosome transfer. Cells deficient in MMR were more resistant to PhIP-induced cytotoxicity and displayed approximately 3-fold more induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. These results suggest that defects in MMR carried by patients with hereditary nonpolyposis colorectal cancer syndrome may result in enhanced sensitivity to certain dietary and environmental carcinogens such as PhIP.

Relationships between exposure, cell loss and proliferation, and manifestation of Hprt mutant T cells following treatment of preweanling, weanling, and adult male mice with N-ethyl-N-nitrosourea.

Experiments were performed to characterize the age-related patterns of appearance and frequency of hypoxanthine-guanine phosphoribosyl transferase (Hprt) mutant T lymphocytes in thymus and spleen following exposure of preweanling (12-day-old), weanling (22-day-old), and young adult (8-week-old) male B6C3F1 mice to ethylnitrosourea (ENU). Mice were given single i.p. injections of 0 or 40 mg ENU/kg and then groups of animals were necropsied from 2 h to 116 days after treatment to examine the relationships between exposure, cell loss and proliferation, and the frequency of Hprt mutant T cells in thymus and spleen. Hprt mutant frequency (Mf) data for thymus of ENU-exposed (0, 11.7, 35, 58, or 72 mg/kg, or five weekly doses of 1.7 mg/kg i.p.) male C57BL/6 mice (12- or 62-week-old), obtained during an earlier study of spleen cells [I.M. Jones, K. Burkhart-Schultz, C.L. Strout, T.L. Crippen, Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea, Environ. Mutagen, 9 (1987) 317-329.], were compared to results in B6C3F1 mice. Isolated T cells were cultured in the presence of mitogen, growth factor, and 6-thioguanine to detect Hprt mutants. The time required to achieve maximum Mfs in thymus was uniformly found at 2 weeks after ENU treatment, while the times needed to reach peak values in spleen were proportional to animal age at treatment. These data indicate that age-related differences in the appearance of Hprt mutant cells in spleen are largely defined by the physiologically based, age-dependent trafficking of mutant cells from or through the thymus. Three modes of handling the resulting Hprt Mf data were evaluated: (i) comparing the Mfs at a single time point, (ii) comparing the maximum Mfs observed, and (iii) comparing the change in Mfs over time (or the mutant T cell 'manifestation' curves in treated vs. control mice) in each age group post-exposure. Measuring the Mfs in spleen at multiple time points after cessation of exposure and integrating the frequency of mutants as a function of time appeared to be the superior method for comparing mutagenic responses in different age groups. Some of the underlying assumptions of this approach, as well as its strengths and weaknesses, are discussed.

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-STIM with Con A, were used to identify growth conditions producing relatively high CEs for mouse T-cells. Supplementation of medium with 10% rat T-STIM, 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-STIM 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-STIM-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-STIM provides a means for propagating mouse T-cell clones for mutation analyses by a variety of methods.

A comparative study of in vivo mutation assays: analysis of hprt, lacI, cII/cI and 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.

Effect of target gene CpG content on spontaneous mutation in299 transgenic mice.

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct transgenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10-5 range and were predominantly base pair substitutions, similar to results seen in Big Blue. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC-->AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.

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.

Genetic instability in human T-lymphocytes.

Mutations arising in vivo in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of T-lymphocytes provide a measure of mutation induction in human somatic cells. Studies of measured background HPRT mutant frequency (MF) values show wide inter-individual variation. At the extremes are individuals with 'outlier' MF values, i.e., non-exposed individuals with MF>100x10-6 [Robinson et al., Mutation Res. 313 (1994) 227-247.]. The elevated HPRT MF in one well-studied outlier is due to the in vivo expansion of mutant cells possessing an identical T-cell receptor (TCR) gene rearrangement pattern. We report here that this in vivo expanding TCR clone shows multiple different HPRT mutations and thus possesses a mutator phenotype. Other individuals with T-cell mutator phenotypes have been found, suggesting that this phenomenon may contribute to the extremes of variation in HPRT MFs in the human population.

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.