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Mutagenesis Advance Access originally published online on November 28, 2005
Mutagenesis 2006 21(1):29-34; doi:10.1093/mutage/gei066
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Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2006. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Levels of H-ras codon 61 CAA to AAA mutation: response to 4-ABP-treatment and Pms2-deficiency

Barbara L. Parsons1,*, Robert R. Delongchamp2, Frederick A. Beland3 and Robert H. Heflich1

1Division of Genetic and Reproductive Toxicology, 2Division of Biometry and Risk Assessment and 3Division of Biochemical Toxicology, National Center for Toxicological Research, USFDA, Jefferson, AR 72079, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA mismatch repair (MMR) deficiencies result in increased frequencies of spontaneous mutation and tumor formation. In the present study, we tested the hypothesis that a chemically-induced mutational response would be greater in a mouse with an MMR-deficiency than in the MMR-proficient mouse models commonly used to assay for chemical carcinogenicity. To accomplish this, the induction of H-ras codon 61 CAA->AAA mutation was examined in Pms2 knockout mice (Pms2–/–, C57BL/6 background) and sibling wild-type mice (Pms2+/+). Groups of five or six neonatal male mice were treated with 0.3 µmol 4-aminobiphenyl (4-ABP) or the vehicle control, dimethylsulfoxide. Eight months after treatment, liver DNAs were isolated and analysed for levels of H-ras codon 61 CAA->AAA mutation using allele-specific competitive blocker-PCR. In Pms2-proficient and Pms2-deficient mice, 4-ABP treatment caused an increase in mutant fraction (MF) from 1.65 x 10–5 to 2.91 x 10–5 and from 3.40 x 10–5 to 4.70 x 10–5, respectively. Pooling data from 4-ABP-treated and control mice, the ~2-fold increase in MF observed in Pms2-deficient as compared with Pms2-proficient mice was statistically significant (P = 0.0207) and consistent with what has been reported previously in terms of induction of G:C->T:A mutation in a Pms2-deficient background. Pooling data from both genotypes, the increase in H-ras MF in 4-ABP-treated mice, as compared with control mice, did not reach the 95% confidence level of statistical significance (P = 0.0606). The 4-ABP treatment caused a 1.76-fold and 1.38-fold increase in average H-ras MF in Pms2-proficient and Pms2-deficient mice, respectively. Furthermore, the levels of induced mutation in Pms2-proficient and Pms2-deficient mice were nearly identical (1.26 x 10–5 and 1.30 x 10–5, respectively). We conclude that Pms2-deficiency does not result in an amplification of the H-ras codon 61 CAA->AAA mutational response induced by 4-ABP.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
It has been hypothesized that the mutagenic and carcinogenic effects of chemicals could be magnified in mouse strains that are deficient in DNA repair, carry a constitutively-expressed human oncogene or are hemizygous or homozygous for a defect in an apoptotic or chemical detoxification pathway. Consequently, a number of transgenic animal models that possess these traits are being investigated for use in carcinogenicity testing (1Go,2Go). In addition, it has become feasible to quantify levels of specific oncogene and tumor suppressor gene mutations in normal-appearing tissues, using a sensitive allele-specific amplification method called allele-specific competitive blocker-PCR (ACB-PCR) (3GoGo–5Go). The ability to quantify specific oncogene and tumor suppressor gene mutations has potential application in investigating the early events in carcinogenesis and in evaluating the mutagenic potency of chemicals and drugs (6Go). In theory, quantitation of oncogene or tumor suppressor gene mutation can be coupled with transgenic technology to provide greater sensitivity in the detection of a chemical's mutagenic effect in an animal model. Previously, levels of H-ras codon 61 CAA->AAA mutation induced in the liver DNAs of B6C3F1 and C57BL/6N mice treated with 4-aminobiphenyl (4-ABP) were measured by ACB-PCR. In order to investigate whether a greater mutagenic response is elicited by 4-ABP in a DNA mismatch repair (MMR)-deficient background, the ACB-PCR measurement of H-ras codon 61 CAA->AAA mutation was replicated in mice deficient in the MMR protein, Pms2.

As part of the MutL{alpha} heterodimer, Pms2 is one of several proteins responsible for MMR and eliciting the cellular response to DNA-damaging agents in mammalian cells (7Go,8Go). The MutS heterodimeric subunit of the MMR complex binds a wide range of damaged DNA substrates and is thought to act as a sensor for damaged DNA. DNA containing 5-fluoruracil and bases adducted by aromatic amines, methylating agents and cisplatin are recognized by the MMR system (8GoGoGoGo–12Go). The MutL{alpha} heterodimer is required to induce cell-cycle arrest and apoptosis in response to unrepaired DNA damage, and the Pms2 subunit is critical for this function (8Go,12GoGo–14Go). MMR-deficient transgenic mice are more tolerant of the cytotoxic effects of chemicals and accumulate greater levels of mutation than MMR-proficient cells, presumably because cell division proceeds despite the presence of unrepaired DNA damage in MMR-deficient cells (12GoGo–14Go).

Transgenic mice deficient in Pms2 are more sensitive than MMR-proficient mice in terms of both spontaneous mutagenesis and carcinogenesis (15GoGoGoGo–19Go). Pms2-deficiency results in the spontaneous development of lymphomas and sarcomas (20Go). Using a variety of reporter systems, Pms2-deficient mice exhibit higher levels of microsatellite instability and spontaneous mutation than their MMR-proficient counterparts (16Go,17Go,21Go). The increase in frequency of spontaneous mutation associated with Pms2-deficiency ranges from an ~3-fold increase observed using the Aprt reporter gene to an ~100-fold increase observed using the supFG1 reporter gene (16GoGoGo–19Go). Pms2 mutational spectra are dominated by increases in the frequencies of +1 bp and –1 bp frameshift mutations and increases in A:T->G:C transitions (16GoGo–18Go). Increases in the frequencies of micronuclei and loss of heterozygosity-type mutation have also been observed (19Go). Reported frequencies of G:C->T:A transversion, the type of mutation detected using the H-ras ACB-PCR assay, range from 0 to 7% of the spontaneous mutations in a Pms2-deficient background, with those targets having a high frequency of +1 bp and –1 bp frameshift mutations having a smaller overall percentage of the G:C->T:A transversion (16Go,22GoGo–24Go). In studies where the absolute levels of G:C->T:A mutation can be estimated (based on the levels of spontaneous mutation and percent of G:C->T:A mutation observed in the mutational spectra), Pms2-deficiency resulted in ~2-fold increases in this mutation. In supFG1, G:C->T:A MF increased from 2.9 x 10–5 in Pms2-proficient mice to 7.6 x 10–5 in Pms2-deficient mice (16Go). In lacI, G:C->T:A MF increased from 0.6 x 10–5 in Pms2-proficient mice to 1.2 x 10–5 in Pms2-deficient mice (22Go).

To determine whether or not Pms2-deficiency would amplify the mutational response induced by 4-ABP treatment, neonatal male Pms2+/+ and Pms2–/– mice were treated with 0.3 µmol 4-ABP or the vehicle control, using the same treatment regimen that was previously used in C57BL/6N and B6C3F1 mice (4Go). The levels of H-ras codon 61 CAA->AAA mutation in the livers of Pms2-proficient and Pms2-deficient mice were then measured by ACB-PCR.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Treatment of neonatal mice
These experiments were reviewed and conducted as approved by NCTR's Institutional Animal Care and Use Committee. C57BL/6-Pms2+/– mice were provided by Dr Sean Baker (University of California, Berkeley) and used to establish a Pms2 colony. In order to maintain the Pms2 colony, male Pms2+/– mice were mated with female Pms2+/– and Pms2–/– mice. The Pms2 genotypes of the offspring were determined as previously described (19Go). The mice were housed four per cage and maintained on NIH-31 diet and water ad libitum. Groups of five or six newborn male Pms2-proficient (Pms2+/+) and Pms2-deficient (Pms2–/–) pups were given a total dose of 0.3 µmol of 4-ABP dissolved in DMSO, with one-third of the total dose given in a 10 µl intraperitoneal (i.p.) injection on post-natal day eight and two-thirds of the total dose given in a 20 µl i.p. injection on post-natal day 15. 4-ABP was purchased from Sigma-Aldrich (St Louis, MO) and purified by silica column chromatography, using methylene chloride elution. High-performance liquid chromatography was used to establish the 4-ABP purity as high as 99%. Groups of five or six Pms2-proficient and Pms2-deficient male pups were treated in parallel with the DMSO vehicle. Eight months after the last treatment, the mice were euthanized and their livers harvested.

Isolation and quantitation of ACB-PCR MF standards and H-ras-specific PCR products
The methods used were identical to those previously published (4Go). Liver DNA was isolated, digested with the restriction enzymes Pvu II and Pst I, and 1 µg of DNA (~3 x 105 copies of H-ras sequence) was used as template to synthesize a 354 bp H-ras PCR product. Restriction fragments containing mutant (AAA) or wild-type (CAA) codon 61 DNA sequence were prepared from plasmid DNAs. Restriction fragments and PCR products generated from liver DNA were agarose gel-purified, aliquoted and the DNA concentration of the purified fragments was determined. The H-ras codon 61 CAA->AAA MF of each first-round PCR product was measured by ACB-PCR, based on comparison with MF standards analysed concurrently. The MF standards were constructed by mixing restriction fragments containing mutant (AAA) or wild-type (CAA) codon 61 DNA sequence (4Go). The 354 bp Pvu II to Rsa I restriction fragments comprised the same DNA sequence as the PCR products amplified from genomic DNA. The MF mixtures (corresponding to MFs of 10–2, 10–3, 10–4 and 10–5) were prepared at a concentration of 7.65 pg/µl and 10 µl of each standard were incorporated as template for ACB-PCR. Each set of MF standards also included a no-mutant control comprising only the wild-type restriction fragment.

ACB-PCR analyses of H-ras codon 61 CAA->AAA MF
The ACB-PCR was performed as previously described (4Go). Each 50 µl reaction contained 2 x 108 copies of template DNA, 10 mM KCl, 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 1 mg/ml Triton X-100, 0.1 mg/ml gelatin, 20 µM dNTPs, 400 nM primer TR10 (5'-fluorescein-ATGGCACTATACTCTTGTCT-3'), 330 nM primer TR31 (5'-TGGGGAGACATGTCTACTG-3'), 200 nM TR27 (5'-ATGGCACTATACTCTTCTAddG-3'), 0.06 units of Perfect Match PCR Enhancer (Stratagene, La Jolla, CA) and 3 units of Taq DNA polymerase Stoffel Fragment (Applied Biosystems, Foster City, CA). ACB-PCR reactions were initiated by diluting the Taq DNA polymerase Stoffel fragment and Perfect Match PCR Enhancer into 5 µl of reaction buffer, warming at 94°C for 20 s, then quickly adding the enzyme mixture to 45 µl of DNA-containing reaction mix that had been incubated for 2 min at 94°C [for details see Ref. (5Go)]. The cycling conditions were 37 cycles of 30 s at 94°C, 45 s at 46°C and 1 min at 72°C.

For each ACB-PCR experiment, the first-round PCR products generated from the 22 liver DNA samples were analysed in parallel with replicate samples of each MF standard (10–2–10–5), a no-mutant control and a no-DNA control. Using this format, each first-round PCR product was analysed in three independent ACB-PCR experiments. The fluorescent ACB-PCR products were visualized and quantified after electrophoresis through 8% polyacrylamide/TAE gels using a Molecular Dynamics FluorImager and ImageQuaNT software (GE Healthcare, Piscataway, NJ). For each gel, a standard curve relating MF to fluorescence in terms of pixel intensity of the diagnostic 60 bp band was constructed and used to calculate the H-ras codon 61 CAA->AAA MF in each unknown sample. The 10–2 MF standards were not used to construct the standard curves because none of the unknown samples had MFs >10–3.

Statistical analyses
Log-transformed MF measurements were averaged for each mouse and reported as the geometric mean (Table I). A two-way analysis of variance (ANOVA) was performed using log-transformed MF measurements. The significance of strain, treatment and their interaction was evaluated by this analysis. However, because 4-ABP treatment and loss of Pms2 should increase MF, significance was based on one-sided P-values. Effects of treatment and strain were estimated by the least squares means. For reporting and discussion, these least squares means were transformed from log MF to MF (geometric means, Table II).


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  		Table I.. H-ras codon 61 CAA->AAA MFs in Pms2-proficient and Pms2-deficient mice treated with DMSO or 4-ABP

 

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  		Table II.. Summary of H-ras codon 61 CAA->AAA MF measurements

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Using groups of five or six mice for each treatment, male Pms2+/+ and Pms2–/– neonatal mice were treated with 0.3 µmol 4-ABP or the DMSO vehicle, as described in Materials and methods. The animals were euthanized eight months after the dosing was completed and their livers removed. Liver DNAs were isolated and used to synthesize H-ras-specific, first-round PCR products. Each first-round PCR product was analysed in three independent ACB-PCR experiments along with MF standards, a no-mutant control and a no-DNA control. Two replicate ACB-PCR analyses of the 22 samples are shown in Figure 1. The MF standards were used to construct a standard curve relating fluorescence (measured in pixels) to MF for each independent experiment. The correlation coefficients of the standard curves ranged from 0.93 to 0.99. Using the power function defined by each standard curve and the fluorescence intensity (in pixels) of the diagnostic 60 bp band generated in the unknown samples, the H-ras codon 61 CAA->AAA MF of each unknown sample was calculated.


Figure 1
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  		Fig. 1.. Fluorescent detection of ACB-PCR products generated from MF standards, controls and first-round PCR products synthesized from liver DNA samples. Two of the three independent ACB-PCR analyses of the 22 DNA samples are shown (Pms2-proficient and Pms2-deficient mice treated with DMSO in the top panel, Pms2-proficient and Pms2-deficient mice treated with 4-ABP in the bottom panel). Although the 10–2 MF standards were included in the ACB-PCR, they were not used for MF quantitation because none of the unknown samples had MFs >10–3.

 
The levels of H-ras codon 61 CAA->AAA MF measured in 4-ABP-treated and control Pms2-proficient and Pms2-deficient mice are given in Table I and presented graphically in Figure 2. Through repeated ACB-PCR measurement, it was possible to determine an average H-ras MF for each sample and calculate the standard deviation for each measurement. The combined variation among the triplicate MF measurements for each mouse (variance = 0.044, measurement error) was significantly lower (P < 0.0001, ANOVA) than the combined variation among mice within treatment groups (variance = 0.231). Based upon the mean MF measured in each sample and the amount of genomic DNA that was analysed, the number of H-ras mutant copies present in each original genomic liver DNA sample (1 µg or 3 x 105 copies) was estimated to be between 0 and 30 mutant molecules (see Table I). It should be noted that, using this ACB-PCR approach, the measurement of one or two mutant copies is not clearly distinguishable from zero mutant copies (6Go). This is because the lowest MF standard used was 10–5 and because measurements of very small numbers of mutant molecules may be significantly influenced by sampling error (25Go). Table I shows that only two Pms2-proficient mice had H-ras liver DNA MFs below the level of reliable detection (10Go–5Go). Consequently, the prevalence of the H-ras mutation (reported in Table II as the percentage of animals with a detectable level of mutation) was 83% for vehicle-treated Pms2-proficient mice and 80% for 4-ABP-treated Pms2-proficient mice, but 100% for both Pms2-deficient treatment groups.


Figure 2
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  		Fig. 2.. Geometric mean H-ras MFs from the three repeated ACB-PCR measurements plotted by treatment group. ACB-PCR can reliably quantify MFs ≥ 10–5. Because the lowest MF standard used was 10–5 and because measurement of one or two mutant molecules may cause significant sampling errors, measurements <10–5 cannot be distinguished from zero with certainty.

 
The statistical significance of the observed differences between the strains and the 4-ABP treatments were evaluated by a two-way ANOVA. This analysis was performed on the logarithms of the MFs because the variance within treatments was relatively stable on this scale. Table II gives the geometric means with 90% confidence bounds, which were computed from the pooled within-treatment variance estimated by the ANOVA.

A statistically significant, 1.82-fold (Table II: 4.00/2.19) increase in H-ras codon 61 CAA->AAA mutation was observed when Pms2-deficient mice were compared with Pms2-proficient mice (one-sided P = 0.0207, 90% CI: 1.13, 2.93). A 1.56-fold (Table II: 3.70/2.37) increase in H-ras codon 61 CAA->AAA mutation was observed when 4-ABP-treated mice were compared with DMSO-treated controls (one-sided P = 0.0606, 90% CI: 0.97, 2.51). Considering Pms2-deficient mice and Pms2-proficient mice separately, 4-ABP treatment increased the MF by 1.38-fold and 1.76-fold, respectively. The difference between strains with regard to the 4-ABP treatment effect was not significant (one-sided P = 0.6667 for strain by treatment interaction). This relatively small difference in fold-increase primarily reflects the strain difference in the frequency of spontaneous mutations because the level of induced mutations (treated MF minus untreated MF) was almost identical, 1.30 x 10–5 for Pms2-deficient mice versus 1.26 x 10–5 for Pms2-proficient mice.

Because the Pms2 knockout was transferred to the C57BL/6 genetic background, the spontaneous and 4-ABP-induced levels of H-ras codon 61 CAA->AAA mutation determined in the Pms2+/+ mice should be comparable with those determined previously for C57BL/6N mice. An ANOVA, analogous to that described earlier, was used to compare these strains and the effect of 4-ABP treatment within these strains. The MFs for these strains were not significantly different (C57BL/6N MF/Pms2+/+ MF = 0.91, two-sided P = 0.7250, 90% CI: 0.56, 1.47). The MF was significantly increased by 4-ABP treatment (4-ABP MF/DMSO MF = 2.47, one-sided P = 0.0024, 90% CI: 1.52, 4.03). This statistical significance is driven in large part by the highly significant 4-ABP-treatment effect established previously for the C57BL/6N mice [one-sided P = 0.0008 (2Go)]. However, the observed difference between these strains in their respective 4-ABP-induced increase in mutations is not significant (strain by 4-ABP-treatment interaction two-sided P = 0.2344).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
This study was designed to evaluate whether a MMR-deficient mouse model provides greater sensitivity in detecting a mutagenic response than a MMR-proficient model. Because ACB-PCR had already been used to examine the 4-ABP-induced H-ras codon 61 CAA->AAA mutational response in male B6C3F1 and C57BL/6N mice treated neonatally (4Go), this approach was replicated using mice deficient in Pms2. The aromatic amine, 4-ABP primarily induces G:C->T:A mutations that arise through misinsertion of adenine opposite the major DNA adduct, N-(deoxyguanosin-8-yl)-4-ABP (4Go,26Go). Conformational changes caused by the presence of these adducts in DNA may be recognized by the MutS{alpha} heterodimer, which binds DNA adducts created by aromatic amines (10Go). Recognition of this DNA damage probably occurs in the context of a replication fork and may lead to futile rounds of abortive repair that could in turn trigger the damage response (8Go). In the presence of a proficient MMR system, this process leads to cell-cycle arrest and apoptosis, thereby preventing the conversion of damaged DNA into mutation (8Go,12GoGo–14Go). In the absence of proficient MMR, however, the misinsertion of adenine opposite the adducted guanine base, if not corrected by another DNA repair pathway, would result in G:C->T:A mutation. It was expected, therefore, that Pms2-deficiency might impact the mutagenic response to 4-ABP.

Groups of only five or six animals were sufficient to ascribe a significant ~2-fold increase in MF to the Pms2–/– genotype. This result fits quite well with the 2-fold increases in the absolute levels of G:C->T:A transversions observed using other mutational reporters (16Go,22Go). In the previous study (4Go), using 12 animals per group, a statistically significant induction of mutation by 4-ABP was demonstrated in the C57BL/6N mice (P = 0.0008). In the B6C3F1 treatment groups, the results were more variable and, consequently, the induction of mutation by 4-ABP was only marginally significant (P = 0.0515). When the induction of mutation was estimated as fold-change in the geometric mean (a relatively stable metric to use when analysing samples with different levels of variability), almost identical levels of 4-ABP-induced mutation were seen in the two strains (3.46-fold for B6C3F1 and 3.48-fold for C57BL/6N).

In the current study, the induction of mutation in 4-ABP-treated mice was only marginally significant (P = 0.0606). Furthermore, the magnitude of the 4-ABP-associated increase in average MF was smaller in the current experiment than that observed previously for B6C3F1 or C57BL/6N mice. The 4-ABP-treated Pms2+/+ mice showed only 1.76-fold increase and the Pms2–/– mice showed 1.38-fold increase, as compared with the ~3.4-fold increases observed previously (4Go). However, when the results obtained using the Pms2+/+ mice were compared with the comparable C57BL/6N data, the results in the current and previous studies were not statistically distinguishable. There are a number of possible explanations for the smaller, although not significantly smaller, effect of 4-ABP-treatment in this study. One explanation is that an insufficient number of animals were used and if more animals were analysed a larger 4-ABP-associated increase in MF would have been observed. A second possibility is that the genetic background of the 129 embryonic stem cells used to generate the Pms2 knockout mice affected the response to 4-ABP, even though the mice were backcrossed for seven or eight generations onto a C57BL/6N background.

Given that the 4-ABP-induced MF was almost identical between the two strains and that the statistical test for a strain: treatment interaction gave a P-value of 0.6667 (ANOVA), the data from this study clearly show that the 4-ABP mutational response was not any greater in the Pms2-deficient background than in a Pms2-proficient background. Although our results indicate that Pms2-deficiency did not amplify the mutational response to 4-ABP, it is still possible that under different circumstances a DNA repair deficient model could provide increased sensitivity in detecting a mutational response. Because the ACB-PCR assay we employed recognizes a mutation that represents only a relatively minor fraction of the Pms2 spontaneous mutational spectra, different results might be obtained if a different mutational specificity was examined. It is also possible that different results might be obtained using different DNA repair deficient transgenic mice, such as mice deficient in nucleotide or base excision repair. Thus, additional studies will be needed to determine with certainty whether the mutagenic response to a chemical treatment can be amplified in a DNA repair deficient background and how particular DNA repair deficiencies impact the mutational specificities that can be detected.

This is the second study in which ACB-PCR has been used to measure the levels of H-ras codon 61 CAA->AAA mutation within mouse liver. The data presented in Table II illustrate that the ability to quantify levels of mutation provides greater power in evaluating a chemical's mutational consequences than does detecting the presence or absence of mutation, the metric that is used more commonly. In terms of the background level of spontaneous H-ras codon 61 mutation, the results obtained in the Pms2+/+ mice were consistent with previous findings in B6C3F1 and C57BL/6N mice and indicate a surprisingly high burden of H-ras codon 61 CAA->AAA mutation. The average H-ras MF in 8.5-month-old untreated, Pms2-proficient mice was ~1.65 x 10–5 (Table II). Based on this MF and assuming an adult mouse liver contains ~1.2 x 108 tetraploid or octaploid hepatocytes (27Go,28Go), it can be calculated that there are between 7920 and 15 800 cells in the mouse liver that carry this mutation. The burden of H-ras mutant cells was higher in the Pms2-deficient mice, with between 16 320 and 32 640 mutant cells, and was the highest in 4-ABP-treated, Pms2-deficient mice, with between 22 560 and 45 120 mutant cells. 4-ABP-treatment increases the frequency of the H-ras codon 61 CAA->AAA mutation and causes an increase in tumors carrying this mutation. DNA sequencing detected the H-ras codon 61 CAA->AAA mutation in 42% of liver tumors of untreated B6C3F1 mice (29Go) and in 73% of liver tumors of B6C3F1 mice treated with 0.3 µmol 4-ABP (30Go). 4-ABP also induces liver tumors in the C57BL/6 mice (31Go). Based upon these results, it is surprising that spontaneous liver tumors have not been reported in Pms2-deficient mice, and it would be interesting to determine if liver tumors can be induced by 4-ABP-treatment in Pms2-deficient mice.

The use of ACB-PCR in this manner, to estimate the burden of oncogene and/or tumor suppressor gene mutations in normal-appearing tissues, provides an illustration of how this approach could be applied in the future in the clinical setting. ACB-PCR could be applied to the quantitation of K-ras mutation in exfoliated epithelial cells in stool samples or sputum samples, for example, as a screening approach for the early identification of individuals at risk for developing colon or lung cancer, respectively. However, considerable validation and technical development will be needed before ACB-PCR can be applied to assess an individual's risk of developing cancer. And, although the approach seems promising, considerable validation will be needed to establish that ACB-PCR can be used to quantify a chemical's mutagenic and carcinogenic potential in an animal model in a manner that will be useful for cancer risk assessment.


   Acknowledgments
 
We thank Dr. Sean Baker of The University of California at Berkeley for providing the Pms2 mice. We also thank Vicky Thompson, Delbert Law and the Building 53 animal care staff of the Bionetics Corporation for their contributions to this study. The views presented in this article do not necessarily reflect those of the US Food and Drug Administration.


   Notes
 
* To whom correspondence should be addressed. Barbara Parsons, Division of Genetic and Reproductive Toxicology, HFT-120, 3900 NCTR Road, Jefferson, AR 72079, USA; Tel: 870 543 7946; Fax: 870 543 7393; Email: bparsons{at}nctr.fda.gov


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received on June 1, 2005; revised on October 26, 2005; accepted on October 31, 2005.


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