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The p53 codon 270 CGT to TGT mutation was investigated as a biomarker of sunlight-induced mutagenesis and carcinogenesis. The relationship between tumor development and abundance of this hotspot mutation was analyzed in mouse skin tumors induced by chronic exposure to simulated solar light (SSL). The 24 tumors analyzed had similar growth kinetics, with an average doubling time of approximately 16.4 d. Levels of the p53 codon 270 mutation were quantified in the 24 mouse skin tumors using allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). All tumors contained measurable amounts of the mutation. The p53 codon 270 CGT to TGT mutant fraction (MF) ranged from 2.29 x 10(-3) to 9.42 x 10(-2), with 3.26 x 10(-2) as the median. These p53 MF measurements are lower than expected for an initiating mutation involved in the development of tumors of monoclonal origin. There was no evidence of a correlation between p53 codon 270 MF and either tumor area or an estimate of tumor cell number. Thus, the data do not support the idea that p53 mutation accumulates linearly during tumor development. To investigate how p53 mutation was distributed within tumors, 19 needle biopsies from seven different tumors were analyzed by ACB-PCR. This analysis demonstrated that p53 codon 270 mutation is heterogeneously distributed within tumors. The long-term goal of this research is to combine morphological and p53 MF measurements from tissues corresponding to the various stages of tumor development, in order to derive mathematical models relating the p53 codon 270 mutation to the development of SSL-induced skin tumors.

Sunlight and ultraviolet-induced mutation of the p53 gene is a frequent, possibly obligate step in skin cancer development, making quantitative measurement of p53 mutation an ideal biomarker for sunlight-induced skin carcinogenesis. To understand how the appearance of p53 mutation relates to skin tumor development, SKH-1 hairless mice were exposed 5 d per week to one of four different doses of simulated solar light (SSL; 0, 6.85, 13.70, 20.55 mJ x CIE/cm(2)) previously characterized for their tumorigenic potential. Allele-specific competitive blocker-PCR (ACB-PCR) was used to measure levels of p53 codon 270 CGT to TGT mutation within DNA isolated from dorsal skin of exposed mice. For each dose, p53 mutant fraction (MF) was measured after 4, 16, and 28 wk of exposure. Significant dose- and time-dependent increases in p53 MF were identified. All p53 MF measurements were integrated by relating the observed p53 MF to the cumulative dose of SSL. The increase in the logarithm of p53 MF was described by the linear function: log(10) MF = alpha + 0.0016 x d, where alpha is the spontaneous log(10) MF after a particular time point and d is the dose of SSL in mJ x CIE/cm(2). The p53 MF induced in nontumor bearing skin by 28 wk of exposure at the high dose of SSL was significantly lower than that found in skin tumors induced by approximately 32 wk of exposure to the same dose of SSL. p53 MF showed a strong negative correlation with tumor latency, suggesting this quantitative biomarker has the potential to predict tumorigenicity., ((c) 2008 Wiley-Liss, Inc.)

The utility of liver H-ras codon 61 CAA to AAA mutant fraction as a biomarker of liver tumor development was investigated using neonatal male mice treated with 4-aminobiphenyl (4-ABP). Treatment with 0.1, 0.3, or 1.0 mumol 4-ABP produced dose-dependent increases in liver DNA adducts in B6C3F(1) and C57BL/6N mice. Eight months after treatment with 0.3 mumol 4-ABP or the DMSO vehicle, H-ras codon 61 CAA to AAA mutant fraction was measured in liver DNA samples (n = 12) by allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). A significant increase in average mutant fraction was found in DNA of 4-ABP-treated mice, with an increase from 1.3 x 10(-5) (control) to 44.9 x 10(-5) (treated) in B6C3F(1) mice and from 1.4 x 10(-5) to 7.0 x 10(-5) in C57BL/6N mice. Compared with C57BL/6N mutant fractions, B6C3F(1) mutant fractions were more variable and included some particularly high mutant fractions, consistent with the more rapid development of liver foci expected in B6C3F(1) mouse liver. Twelve months after treatment, liver tumors developed in 79.2% of 4-ABP-treated and 22.2% of control B6C3F(1) mice; thus measurement of H-ras mutant fraction correlated with subsequent tumor development. This study demonstrates that ACB-PCR can directly measure background levels of somatic oncogene mutation and detect a carcinogen-induced increase in such mutation., (Published 2005 Wiley-Liss, Inc.)

The use of transgenic targets for measuring mutant frequencies in mammalian tissue requires an estimate of the mutant frequency that results from recovery of the transgene in bacterial recovery systems. In this study, we have determined the spontaneous mutant frequency, estimated the mutation rate, and ascertained the mutation spectrum for gene A of phiX174 grown in E. coli strain CQ2 from 156 small independent cultures. The mutant frequency of 12 of the 156 cultures was 17 +/- 1.0 x 10(-6) and the estimated mutation rate per gene replication was 7.4 +/- 2.3 x 10(-6). The mutant frequency and spectrum from E. coli were not significantly different from that of solvent-treated embryonic mouse cells in culture, 19 +/- 0.5 x 10(-6) (Valentine CR et al. [2002]: Environ Mol Mutagen 39:55-68), indicating that those spontaneous mutants were primarily derived from E. coli. The E. coli spectrum was heavily weighted toward two major target sites (hot spots), 4225A-->G (56%) and 4218G-->A or C (20%). Four new target sites and one new mutational event were recovered by the gene A forward assay. A mutant spectrum from an expanded phage stock was also determined to assess the effects of propagating the virus. This mutant frequency was higher (6 x 10(-4)), contained more double mutants (15% compared to 0.6%), and had a significantly different spectrum from the spectrum for independent cultures (fewer A:T-->G:C and G:C-->C:G changes and more G:C-->A:T; P < 0.002). The E. coli mutation spectrum will be useful for determining the origin of gene A mutation in tissues of phiX174 transgenic mice., (Copyright 2004 Wiley-Liss, Inc.)

Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants., (Copyright 2004 Wiley-Liss, Inc.)

Transgenic systems for measuring mammalian mutagenesis often use recoverable viral vectors. We hypothesize that mutations in these transgenic systems can arise from three different origins of DNA damage and replication errors and that these three origins of mutations (in vivo, ex vivo, and in vitro) can be differentiated in the PhiX174 am3, cs70 single burst assay (SBA) on the basis of burst size (BS). In vivo mutations are fixed in the animal, ex vivo mutations are fixed in bacterial cells during recovery of the phage, and in vitro revertants arise during the first replications of nonmutant phages under selective conditions. PX-2 cells, derived from a homozygous embryo of a PhiX174 transgenic mouse, were treated with vehicle or N-ethyl-N-nitrosourea (ENU). An algorithm was developed to estimate the BS that resulted in the highest induced revertant frequency; the estimate was 56. In vivo revertants were defined as having BS >55, ex vivo revertants as having a BS of 13-56, and in vitro revertants as having a BS of <14. The frequencies of in vivo revertants at 0, 100, and 200 mg/kg ENU were 0.06, 0.36, and 4.10 x 10(-6) (dose response, P = 0.004); ex vivo revertants were 0.36, 0.46, and 0.41 x 10(-6) (P = 0.37), and in vitro revertants were 0.39, 0.46, and 0.41 x 10(-6) (P = 0.55), respectively. These results show that only in vivo revertants reflect mutagen treatment. They also provide a basis for identifying PhiX174 am3 revertants induced in vivo and may increase the sensitivity of the assay for in vivo mutation.

The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing. A forward mutational assay exists for Phi X174 that requires an altered, functional Phi X174 protein and therefore should have fewer targets (sense, base-pair substitutions) than forward assays that inactivate a protein. We investigated this assay to determine the number of targets and their suitability for detecting a known mutagen, N-ethyl-N-nitrosourea (ENU). We identified 25 target sites and 33 different mutations in Phi X174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for Phi X174 am3, cs70 (line 54). All six types of base-pair substitution were represented among both the spontaneous and ENU-treated mutant spectra. The mutant spectra from cells treated with 200 and 400 microg/ml ENU were both highly different from the spontaneous spectrum (P < 0.000001) but not from each other. The dose trend was significant (P < 0.0001) for a linear regression of mutant frequencies (R(2) = 0.79), with a ninefold increase in mutant frequency at the 400 microg/ml dose. The spontaneous mutant frequency was 1.9 x 10(-5) and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations. Thirteen mutations at 12 targets were identified only from ENU-treated cells. Seven mutations had highly significant increases with ENU treatment (P < 0.0001) and 15 showed significant increases. The results suggest that the Phi X174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.

Target genes in most transgenic systems have higher spontaneous mutation frequencies than do endogenous mammalian genes. Spontaneous mutations in transgenes predominantly arise from three sources: (1) mutations fixed in the animals, (2) mutations arising from replication errors caused by damage to the DNA that may have occurred in vivo or in vitro and then was fixed during amplification of the vector in vitro, and (3) mutations arising during replication of non-revertant phages in non-permissive bacteria. An assay based on single bursts was developed to directly distinguish between the in vivo and in vitro origins of revertants. The size of the aliquot is determined by mutant frequency and is adjusted so that ideally no more than 10 to 20% of the aliquots contain a bacterial cell transformed with a mutant phage. Mutations are detected as revertants of an amber mutation (am3) in phiX174 am3, cs70. The minimum burst size of non-revertant phiX am3, cs70 from splenic DNA on a permissive bacterial strain was larger than 30 plaque-forming units (pfu). Based on this observation, a burst size of 31 plaque-forming revertants was chosen as the minimum burst size of a fixed mutation. The single burst assay was tested on DNA from spleens of animals that were treated with 150 mg/kg 1-ethyl-1 nitrosurea. Only the fraction of aliquots with single bursts of revertants (> 30) increased in the treated animals compared to the controls. In contrast, there was no difference between treated and control animals for revertant frequencies calculated for burst sizes < or =30 pfu. Among the spontaneous mutations, only 30% were caused by mutations fixed in animals (i.e. burst size >30 pfu). Total average revertant frequency measured in DNA from treated animals was less than twofold more than the average spontaneous frequency (P = 0.048). When frequencies were based on burst sizes >30, there was a 4.6-fold increase among treated animals compared with controls (P = 0.026). The single burst-assay resulted in a more sensitive test for mutagenicity because it eliminated noise from in-vitro mutations.

In mutation assays using transgenic mice, with recoverable vectors such as PhiX174 am3, cs70, mutations originate from two sources: (1) in vivo mutations, that is, mutations that were fixed in the mouse, or (2) ex vivo mutations, that is, mutations that were fixed during recovery or plating. When a bacteriophage infects a bacterium, it multiplies and bursts the cell, releasing a number of phages referred to as the burst size. Our method for distinguishing between in vivo mutations and ex vivo mutations estimates the average number of bursts, the denominator of in vivo mutant frequencies, by dividing the total plaque-forming units (PFU) by the average number of phages in a burst. Herein, we outline a probability model relating observed plaque counts to the burst size and present the statistical method used to estimate the burst size. The average size of a single burst from nonrevertant phages was estimated in eight studies under the conditions of our mutation assay. The average burst size was stable across studies at 182.5 plaques per burst (standard error, 14.25). The probability that a burst is a specific size was approximated by a negative binomial distribution, which implies a Poisson-Pascal distribution for the observed plaque counts. The observed plaque counts were adequately fit by this approximation. Environ. Mol. Mutagen. 37:356-360, 2001 Published 2001 Wiley-Liss, Inc.

Previous studies have suggested that reserpine treatment may result in altered heart development. In order to more fully investigate this possibility, reserpine was administered s.c. at 0, 0.375, or 0.75 mg/kg/day to pregnant rats on gestation days 12-15. Maternal weight gain, as well as pup weight on postnatal day (PND) 1, was significantly reduced in a dose-dependent manner. Litter size was unaffected, but reserpine-treated dams had more dead pups than did control dams. On PND 1, litters were randomly standardized at ten pups each for analysis on PNDs 5, 8, 15, and 22. Pup body weight and heart weight were reduced in a dose-related manner at all ages measured. The decreased heart weights were probably due to decreases in cell number. Beta-adrenergic receptor concentration was significantly reduced only on PND 5, at the low reserpine dose, and was not considered to be a treatment effect. Prenatal reserpine exposure had no effect on levels of basal cardiac ornithine decarboxylase (ODC), an enzyme associated with growth and development. Cardiac ODC stimulation by insulin and isoproterenol also showed no effects of maternal reserpine treatment. The results suggest that maternal reserpine treatment may lead to adverse effects in the developing offspring.

Imipramine (IMI) was administered s.c. at 0, 5, or 10 mg/kg/day to pregnant rats on gestation days 8-20 to assess possible alterations in postnatal heart and brain development. Maternal weight gain was significantly reduced in a dose-response manner, but litter size and pup weight on postnatal day (PND) 1 were unaffected. On PND 1, litters were culled to 10 pups for analysis on PNDs 4/5, 7/8, 14/15, and 21/22. Pup body weight was not affected at any age measured, but heart weight was significantly reduced at 10 mg/kg IMI on PNDs 4/5 and 7/8. Brain weight was increased in a dose-related pattern on PNDs 4/5 and 7/8 and was significantly higher at 5 mg/kg IMI on PND 14/15. No significant effect was observed in heart or brain protein and DNA content or in cardiac beta-adrenergic receptor concentration. Prenatal IMI exposure had no effect on basal cardiac ornithine decarboxylase (ODC), an enzyme associated with growth and development, but basal brain ODC was lower at 5 mg/kg IMI at all ages measured. Cardiac ODC stimulation by insulin was unaffected by prenatal exposure to IMI, but isoproterenol-stimulated ODC was increased on PND 21/22 at 5 mg/kg IMI. In conclusion, the IMI-related changes in several parameters suggest that when maternal IMI treatment is used, alterations in postnatal heart and brain development must be considered as possible outcomes.

Maternal propranolol (PRO) treatment has previously been associated with adverse effects on the fetus and neonate. In the present study, pregnant rats were treated with PRO (25 or 50 mg/kg/day s.c.) on gestation days 8-20 to assess its possible effects on the developing heart. Maternal weight gain and pup weight on postnatal day (PND) 1 were reduced in a dose-dependent manner; litter size was unaffected. Pup body weight and heart weight both showed a dose-related decrease at all ages tested (PNDs 5/6, 8/9, 15/16, and 22/23). Since heart protein, but not DNA, was similarly reduced, the decrease seen in heart weight most likely reflects a decrease in cell size instead of cell number. Basal ornithine decarboxylase (ODC), an enzyme associated with growth and development, was unaffected by maternal PRO treatment. Insulin and isoproterenol stimulation of ODC, suggested markers for testing the function of the sympathetic pathway to the heart and of the heart's ODC response system, respectively, also showed no PRO-related response. In conclusion, prenatal PRO exposure resulted in reduced body weight, heart weight, and heart protein, but had little effect on heart DNA or ODC activity. Since PRO treatment also reduced maternal weight gain, the adverse effects seen in the pups may be due to generalized PRO toxicity. The results suggest that when high PRO doses were used clinically, the careful monitoring of maternal weight gain during pregnancy might be useful in predicting adverse fetal effects.