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J A Heddle

Publications and source records attributed to J A Heddle.

At least 19 recordsLinked to original sources
PubMed2000

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.

Animals↗
PubMed1999

Somatic mutation in the mammary gland: influence of time and estrus.

A critical factor in the quantitation of mutation induction in vivo is the time interval between treatment and sampling. In order to study mutagenesis in the mammary epithelium, the cell type in which breast cancer arises, we have measured the manifestation time, the minimum time required for the maximum mutant frequency to be achieved, in this tissue. The F1 LacZ transgenic mice (Muta MousexSWR) were treated with N-ethyl-N-nitrosourea (ENU) at 50 mg/kg for five consecutive days and then sampled at 1, 2, 4, 6, 9, or 12 weeks after the last treatment. The LacZ- mutant frequency reached a maximum at 4 weeks post-treatment and did not vary significantly thereafter. Dlb-1- mutations in the small intestine reached a maximum at 2 weeks after treatment and did not vary significantly thereafter. Since the stage of estrus cycle during carcinogen exposure influences the mammary tumor incidence and latency, it was expected that it would also affect mutation induction. To test this, F1 LacZ mice in the estrus or di-estrus stage were treated with an acute dose of 250 mg/kg ENU and sampled 10-13 weeks post-treatment. No statistical difference between the two groups was found, indicating that the effect of estrus on carcinogenesis is not due to variation in the sensitivity of the stage of the mammary gland to mutation.

Animals↗
PubMed1999

A comparison of the effects of diverse mutagens at the lacZ transgene and Dlb-1 locus in vivo.

Transgenic assays permit the detection of mutations in any tissue, whereas endogenous mutations can be measured in very few. For this reason comparisons between these loci when both can be measured in the same cells are of considerable interest. Previous comparisons have been inconsistent: usually these loci have responded alike, however, in some cases the endogenous locus has been more sensitive and at other times the transgenic locus has been more sensitive. Here we report a comparison of the lacZ transgene of the MutaMouse and the endogenous Dlb-1 gene in the epithelium of the small intestine after acute exposure to seven mutagens. Benzo[a]pyrene, 5-bromo-2'-deoxyuridine, methyl methane sulphonate, ethyl methane sulphonate, N-ethyl-N-nitrosourea, mitomycin C and N-methyl-N-nitrosourea were all given by gavage to F1 (MutaMouse x SWR) mice. Mutations were quantified 2 weeks after the end of treatment. The data shows that all of the agents induced similar mutant frequencies at the Dlb-1 locus and at the lacZ transgene. The acute treatments generally produced only modest increases in mutant frequency at both loci. The higher background frequency observed at the lacZ transgene reduces the ability of the transgenic assay to detect the same absolute increase in mutant frequency.

Animals↗
PubMed1999

The cII locus in the MutaMouse system.

Here, we report the first application and characterization of the cII locus as a mutational target for use with the Muta(trade mark)Mouse system for quantifying somatic mutations in vivo. This locus can be analyzed for mutations using positive selection and is identical in sequence to the cII in the Big Blue((R)) Mouse. The cII displays similar spontaneous (5.5 x 10(-5)) and induced mutation frequencies when compared to the lacZ gene in the small intestine of MutaMice treated with ENU (N-ethyl-N-nitrosourea). After acute treatment with 250 mg/kg ENU (ip) the mutant frequencies were 127 x 10(-5) at the cII and 147 x 10(-5) at the lacZ loci, reaching a maximal mutant frequency 10 days posttreatment and remaining constant thereafter. These data prove that this transgene is genetically neutral, conferring neither selective advantage nor disadvantage on the host cells. The cII dose response curve was linear (R(2) = 0.93) comparable to the lacZ after treatments with 0, 50, 150, or 250 mg/kg ENU. Use of the cII locus (0.3 kb) addresses the single most significant drawback associated with the MutaMouse system, namely the inability to obtain sequence spectra efficiently, due to the large size of the lacZ gene (3.0 kb). Moreover, a less obvious application, but nevertheless of considerable importance, is the easy identification of jackpot mutations, without sequencing. The cII, identical in both sequence and origin on the transgenic constructs used in producing the Big Blue and MutaMouse systems, provides the first transgenic locus common to the two widely used in vivo mutagenesis assays.

Animals↗
PubMed1999

Effects of extended chronic exposures on endogenous and transgenic loci: implications for low-dose extrapolations.

Although transgenic and endogenous loci generally respond alike to acute mutagenic exposures, those loci that have been tested respond differently to daily or continuous exposures. During chronic exposures, the transgenes accumulate mutations linearly, whereas the endogenous loci are less mutable initially but later accumulate mutations at an accelerating rate. The result is a reverse dose rate effect in which the same total dose is more mutagenic for the endogenous locus when spread over a longer time. This makes extrapolations to still lower chronic exposures uncertain. Here we report extension of a chronic exposure to N-ethyl-N-nitrosourea (ENU) in drinking water to longer times and to lower doses. The F(1) of MutaMouse males x SWR that were used permit detection of mutations at both lacZ and Dlb-1. Both of these mutations were found to be genetically neutral over this period. Extension of the exposure from 30 to 90 days at 94 microg/ml/d showed a continuation of the curves found previously for 10 to 30 days, namely, linear for mutations of the lacZ transgene and concave upward for the Dlb-1 endogenous gene. A simple model for these data is presented. Of the extended exposures, only the highest dose produced a significant increase in Dlb-1 mutant frequency, an increase consistent with the model. The time (at 2.8 microg/ml/d), concentration (after 480 days exposure), and dose (concentration x duration of exposure) response curves were not significantly different from linearity. The data for the transgene are not as convincing, due to the high spontaneous mutant frequency, obscuring the induced response.

Animals↗
PubMed1998

Effect of maternal folate levels on somatic mutation frequency in the developing colon.

Folic acid deficiency is associated with an increase in chromosomal aberrations in adult rodents and humans. Somatic mutations have a critical role in carcinogenesis. Since most mutations arise during early development, the effect of maternal folic acid levels on the spontaneous mutant frequency in the developing colon was examined using lacZ transgenic mice. No significant difference in mutant frequencies at both 3 and 8 weeks of age were found between offspring whose mothers were fed low folate and those on high folate diets during pregnancy. Our results suggest that the correlation between folic acid intake and cancer risk may only be effective at extreme folate deficiencies or in combination with other dietary deficiencies or an underlying predisposition.

Age Factors↗
PubMed1998

The accumulation of chromosome aberrations and Dlb-1 mutations in mice with highly fractionated exposure to gamma radiation.

The dichotomy between the doses at which experimental measurements of genetic effects can be made and the doses to which people are exposed is often different by two or more orders of magnitude. This presents a significant problem when determining the effects of low doses of radiation or chemicals. The solution has usually involved extrapolating the data by curve-fitting or by applying theoretical considerations. Both approaches are unsatisfactory due to uncertainties of the assumptions used in each process. The alternative solution has been to increase the sample size enormously at the lower doses. This is impractical beyond a certain point due to the variation in the spontaneous frequency and the need to quadruple the sample size for a doubling of precision. The development of new methods for measuring stable genetic effects, however, permits a simple and effective approach to this problem: if the genetic events being detected have no effect on survival, i.e., are selectively neutral, then the effects of multiple independent treatments will be additive. If the independent treatments are identical, then the effect of each is easily calculated by dividing the total effect by the number of treatments. Here we report a limited test of this approach using mice. Chromosome aberrations induced in lymphocytes and Dlb-1 mutations induced in the small intestine were measured after daily doses of 0.64, 1.85 or 5.5 cGy 137Cs gamma rays administered for 21, 42 or 63 days. The dose response curve for chromosome translocations obtained in this way, combined with the data from single larger acute doses, shows no evidence for a threshold over a 500-fold dose range. Dlb-1 mutations were increased at each dose and time but the results do not permit reliable extrapolations. The results suggest that translocations might be useful for quantifying the effect of doses below 0.05 cGy and that the effect of dose rate and dose fractionation at much lower doses than reported here could be investigated.

Animals↗
PubMed1998

Spontaneous mutation during fetal development and post-natal growth.

Somatic mutations seem to accumulate slowly with age during adult life in both mice and men. There is, however, a substantial mutant frequency at birth, suggesting that the rate of accumulation is much higher before birth. This suggests that DNA replication plays an important role in the generation of spontaneous mutations. Since most cell division and accompanying DNA replication occurs early in development, more mutations would arise during growth and development. Indeed, if the mutations are genetically neutral, the mutant frequency would rise very rapidly during early fetal growth, more slowly during later fetal growth and development and still more slowly after birth. To test this hypothesis, we have assayed the mutant frequencies from before birth to 28 days after birth, by which time most growth has occurred. We have used the F1 mice generated by crossing SWR females and MutaMouse males. The MutaMouse has a rescuable lacZ/lambda shuttle vector that can be assayed for an in vivo mutation in an in vitro system. Up to and including birth we assayed the entire animal for mutants; at 14 and 28 days after birth we assayed the small intestine. The data show that, as expected, many mutations arise early in development, by 12.5 days after conception, and confirms the non-linearity of mutation with age. In these mice, about one third of mutations arise before birth, about one third during growth to adulthood and the remaining during the rest of the animal's life, although this depends somewhat on the tissue.

Animals↗
PubMed1997

On the origin of spontaneous somatic mutations and sectored plaques detected in transgenic mice.

The use of transgenic mice with bacterial genes that can be readily recovered and analysed for mutation has made it possible to measure mutant frequencies in many tissues. The mutations are detected by packaging the murine DNA into lambda phage and then growing these phage on a bacterial lawn under conditions such that the mutants are distinguishable from nonmutants. In the lacI mouse assay, the mutant plaques are blue whereas the nonmutant plaques are clear. The mutations detected in the lacI Big Blue Mouse are, in principle, a mixture of mutations that arose in the mouse (in vivo mutations), mutations that arose in the bacterium from lesions pre-existing in the murine DNA (ex vivo mutations), and mutations that arose during growth of the phage on the bacteria (in vitro mutations). It has been suggested that plaque morphology can be used to visually distinguish in vivo mutations (which would be seen as wholly blue plaques) from ex vivo mutations (which would produce blue and white sectored plaques) and in vitro mutations (which would produce sectored or pin-point plaques). We show here that this is not the case: ex vivo mutations produce plaques that are a homogeneous blue. By superinfection of bacteria with mutant and nonmutant phage and by in vitro mutagenesis, we found that blue plaques may contain large proportions of nonmutant phage. None of the mutant plaques seen after in vitro mutagenesis were sectored but most contained nonmutant phage. In addition, we show that most spontaneous mutations from the small intestine, which has a higher than normal mutant frequency, arose in vivo, since 17 of 17 mutants were homogeneous mutants. Sectored plaques must arise in some way other than by ex vivo mutation, like perhaps by the confluence of a mutant and nonmutant plaque.

Animals↗
PubMed1997

Agreement of mutational characteristics of heterocyclic amines in lacI of the Big Blue mouse with those in tumor related genes in rodents.

The mutational spectra of carcinogenic heterocyclic amines (HCAs), 2-amino-3,4-dimethylimidazo[4,5-b]quinoline (MeIQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-9H-pyrido[2,3-b]indole (A alphaC) were studied in the colon of Big Blue mice. In 90, 115 and 105 lacI mutants from mice fed 300 p.p.m. MeIQ, 400 p.p.m. PhIP and 800 p.p.m. A alphaC, respectively, 92, 115 and 105 mutations were identified. G:C-->T:A transversions predominated with these HCAs. Mutational hot spots for base-substitution mutations caused by MeIQ, PhIP and A alphaC were in distinct sequence contexts; at 5'-GC-3', in runs of guanine and in 5'-CGT-3', respectively. Further, 30 of 115 (26%) PhIP-induced mutations were G:C base pair deletions, and eight of these deletions were in 5'-GGGA-3'. The mutational characteristics of MeIQ in the lacI gene coincided well with those in the Ha-ras gene of MeIQ-induced mouse forestomach tumors and rat Zymbal gland tumors. The characteristic single-base deletion induced by PhIP in the lacI gene also coincided well with those in the Apc gene of PhIP-induced rat colon tumors. These results suggest that the mutational characteristics of each chemical are conserved across different genes in different species.

Animals↗
PubMed1997

Identical megabase transgenes on mouse chromosomes 3 and 4 do not promote ectopic pairing or synapsis at meiosis.

To investigate ectopic interactions at the chromatin level, we examined the meiotic organization of 1-2 mb phage lambda transgenes on mouse chromosomes 3 and 4 by fluorescence in situ hybridization in combination with immunocytology of meiotic chromosomes. At early meiotic prophase, the transgenes are sufficiently dispersed in the nuclear volume to permit potential DNA-DNA interactions, but no synaptonemal complexes form between the sites of transgenes residing on different chromosomes. At later stages, when the chromatin is more condensed, the transgenes on different chromosomes are not preferentially associated as they are when they are on the same chromosome. At diplotene and metaphase I, no formations were observed that could be interpreted as reciprocal crossovers or chiasmata between the transgenes located on chromosomes 3 and 4. It appears that in normal fertile mice, a I- to 2-mb homology is insufficient to initiate synapsis between nonhomologs, and it is concluded that homology is assessed within the broader context of the chromosome to initiate synapsis at meiotic prophase.

Animals↗
PubMed1996

Assessment of the flexed-tail mouse as a possible model for Fanconi anemia: analysis of mitomycin C-induced micronuclei.

Fanconi anemia (FA) is a rare, autosomal recessive disorder characterized by elevated frequencies of chromosome aberrations, hypersensitivity to DNA cross-linking agents and predisposition to cancer. At least 5 complementation groups (FA-A to FA-E) underlie FA and the gene defective in FA-C (FAC) has been cloned. The mouse orthologue, Fac, maps in close proximity to the f locus, on chromosome 13, which codes for the flexed-tail mouse phenotype, raising the possibility that f and Fac are synonymous. If this were the case flexed-tail mice could be used as mouse models for FA-C to help determine the basic defect and to evaluate clinical intervention and gene therapy. To further characterize the flexed-tail mouse, the frequency of micronuclei (a measure of chromosomal aberrations) induced by mitomycin C (MMC), an alkylating and DNA cross-linking agent, was analyzed in peripheral blood and bone marrow erythrocytes. Although a higher spontaneous micronucleus frequency was seen in flexed tail mice in comparison to wild-type mice, the sensitivity to MMC was not elevated. This result suggests that f and Fac are different genes and that the flexed-tail mouse is not a model for FA-C.

Animals↗
PubMed1996

The relationships among stem cells, crypts, and villi in the small intestine of mice as determined by mutation tagging.

The number of stem cells that maintain a crypt of the small intestine is uncertain. Although the number of stem cells per crypt had been thought to be 10-20, current estimates indicate a much smaller number, possibly 1 stem cell per crypt. We report here that in SWR and C57BL/6 X SWR F1 mice, the results of mutation tagging are inconsistent with the existence of more than one stem cell per crypt. Mutations at the Dlb-1 locus mark the progeny of a single epithelial stem cell in the small intestine and reveal the size of the mutant clone. The epithelial cells are produced in the crypts and migrate up the villi to be sloughed off at the tip so that a mutant clone takes the form of a ribbon on the villus. Because the size of the mutant clones must be inversely proportional to the number of stem cells contributing to the villus, this provides a means of counting the stem cells (ethylnitrosourea was used to induce mutations in the intestine). In the duodenum the ribbons average 0.099 +/- 0.004 villus circumferences, indicating that there are 1/0.099 = 10.2 stem cells per villus. Because there are 10.5 +/- 0.67 crypts/villus, the estimate gives 10.2/10.5 = 0.97 +/- 0.1 stem cells/crypt. Because each crypt must have at least one stem cell, the vast majority can have only one stem cell and very few may have two or more.

Animals↗