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J A Heddle

Publications and source records attributed to J A Heddle.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1996

A test for neutrality of mutations of the lacZ transgene.

Neutral mutations are stable alterations in DNA that are neither beneficial nor damaging to the cell. Once a mutation is fixed in the DNA, the mutant frequency should increase to its maximum and plateau thereafter, provided that enough expression time is given. Evidence has been obtained indicating neutrality for the endogenous Dlb-1 locus and for the lacl in the BigBlue transgenic system, and is reported here for lacZ in the Muta Mouse system. We have found that, after an acute dose of ethylnitrosourea (ENU, 250 mg/kg) the mutant frequency for both lacZ transgene and Dlb-1 remained constant between 10 and 70 days after mutagenesis in the small intestine, where cell turnover is rapid (approximately 7 days). The same concept should apply to other tissues.

Animals↗
PubMed1996

Why do stem cells exist?

Self-renewing tissues have a differentiation hierarchy such that the stem cells are the only permanent residents of the tissue, and it is in these cells that most cancerous mutations arise. The progeny of the stem cells either remain stem cells or enter a transient proliferating cell population that differentiates to produce the functional cells of the tissue. The reason that this differentiation hierarchy exists has not been established. We show here that alternative hierarchies, in which there would be no stem cells, are feasible and biologically plausible. We show that current evidence from somatic mutation frequencies at both transgenic and endogenous loci implicates cell division in the origin of most somatic mutations. We suggest, therefore, that the existence of stem cells is an evolutionary consequence of a selective pressure to avoid cancer by reducing the number of somatic mutations. The stem cell hierarchy reduces the number of cell divisions of those cells that reside permanently in the tissue, which reduces the number of somatic mutations and thus minimizes the cancer rate. In the small intestine, the existence of stem cells reduces the mutant frequency in the stem cells by about one order of magnitude. Since two or more mutations are required to transform a cell, the protective effect may be 100-fold or more. Similar factors may be expected in other tissues.

Age Factors↗
PubMed1996

Risk estimation from somatic mutation assays.

The ability to quantify somatic mutations in vivo provides a new source of toxicological information that is relevant to the assessment of cancer risk. The major experimental factors that influence the mutant frequency are age, time after treatment, treatment protocol, and tissue analyzed. In untreated mice, the mutant frequency increases very rapidly with age from conception to birth, more slowly from birth to adulthood, and very slowly thereafter. All somatic tissues studied so far in adults have similar mutant frequencies. The time after treatment (expression time) is the most important experimental variable. The minimum time for expression varies from one tissue to another. To be valid, comparisons between tissues and treatments must be made after complete expression of the mutations. Unfortunately, the minimum expression time has not been characterized in most tissues. Since carcinogens are tissue specific, and many chemicals are distributed in the body in complex patterns, it is to be expected that there will be differences in the frequency of mutation induced in different tissues. As yet this has not been extensively studied. Since the mutations detected by the transgenic assays are neutral, the mutants should accumulate as the integral of the mutation rate. Hence chronic treatment protocols should be more effective than acute and subacute protocols whenever they permit substantially larger doses to be delivered. Such protocols are more relevant to human exposure and are preferable for dose extrapolations. The importance of transcription in determining mutation rates is not yet known, but it is noteworthy that the transgenes are not transcribed whereas the Ioci involved in carcinogenesis are. The mutation spectrum is important for quantitative risk estimation. Risk estimation must also take into account the spectrum of mutations that are involved in the carcinogenic process in the tissue and the spectrum of mutations that are detectable by the assay. New assays are being used to quantify mutations in vivo in order to understand the carcinogenic process, to search for the environmental factors involved in human cancer, and to evaluate the carcinogenic hazard qualitatively.

Animals↗
PubMed1996

Intestinal mutagenicity of two carcinogenic food mutagens in transgenic mice: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and amino(alpha)carboline.

The heterocyclic amines produced during the cooking of meat, including amino(alpha)carboline (AalphaC) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are potent bacterial mutagens and are carcinogenic in rodents. PhIP is mutagenic in the small intestine, but its mutagenicity in the colon, where most human intestinal cancers arise, has not been reported, nor has the mutagenicity of AalphaC. In this study, AalphaC (800 p.p.m.) was fed for 30 and 45 days and PhIP (100 and 400 p.p.m.) was fed for 30, 60 and 90 days to groups of F1 (C57BL/6 x SWR) mice hemizygous for multiple tandem copies of a lacI transgene (the Big Blue mouse) and heterozygous at the endogenous Dlb-1 locus. The mutant frequencies were assayed at Dlb-1 and at lacI in the small intestine and at lacI in the colon. PhIP induced mutations at both loci in the small intestine and induced slightly fewer mutations in the colon. The accumulation of mutations at both loci appears to be linear with both PhIP concentration and duration of exposure and, thus, with dose (concentration x duration). The linear increase with time is in agreement with predictions about the effectiveness of chronic treatment protocols for tests of in vivo mutagenicity. Unlike PhIP, AalphaC induced mutations specifically in the colon and not in the small intestine, thereby showing a dramatic tissue specificity. The rate (mutations/p.p.m. day) was similar to PhIP.

Animals↗
PubMed1996

Mutagenicity of high fat diets in the colon and small intestine of transgenic mice.

Dietary fat has been implicated as a cause of colon cancer by epidemiological studies. Unfortunately, these studies are compatible with high fat as either initiator or promoter. Since initiators are normally mutagenic, we have tested the mutagenicity of high fat diets in the intestinal epithelium at two loci. The Dlb-1 assay, used in the small intestine, detects a wide spectrum of mutations. The lacI assay (Big Blue Mouse assay) is not as sensitive to some types of mutation as is Dlb-1 but was used in the colonic epithelium. Mice suitable for both assays were fed isocaloric high fat diets and subsequently assayed for somatic mutation. The diets consisted of: (i) a mixture of beef tallow, butter and lard totalling to 31% w/w of the diet (AIN-76A) up to 17 weeks; and (ii) corn oil, beef tallow, lard or butter individually, at 31% w/w of the diet for 5 and 9 weeks. These diets provided 50% of the calories from fat. The weights of the experimental and control mice were similar throughout the experiment. No significant increases in mutant frequencies were observed on any high fat diet compared to controls, so we conclude that uncooked fats are not mutagenic and are not initiators of carcinogenesis in the intestinal epithelium.

Animals↗
PubMed1995

Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea.

We have found that the somatic mutation rate at the Dlb-1 locus increases exponentially during low daily exposure to ethylnitrosourea over 4 months. This effect, enhanced mutagenesis, was not observed at a lacI transgene in the same tissue, although the two loci respond very similarly to acute doses. Since both mutations are neutral, the mutant frequency was expected to increase linearly with time in response to a constant mutagenic exposure, as it did for lacI. Enhanced mutagenesis does not result from an overall sensitization of the animals, since mice that had first been treated with a low daily dose for 90 days and then challenged with a large acute dose were not sensitized to the acute dose. Nor was the increased mutant frequency due to selection, since animals that were treated for 90 days and then left untreated for up to 60 days showed little change from the 90-day frequency. The effect is substantial: about 8 times as many Dlb-1 mutants were induced between 90 and 120 days as in the first 30 days. This resulted in a reverse dose rate effect such that 90 mg/kg induced more mutants when delivered at 1 mg/kg per day than at 3 mg/kg per day. We postulate that enhanced mutagenesis arises from increased stem cell proliferation and the preferential repair of transcribed genes. Enhanced mutagenesis may be important for risk evaluation, as the results show that chronic exposures can be more mutagenic than acute ones and raise the possibility of synergism between chemicals at low doses.

Animals↗
PubMed1995

New molecular endpoints and methods for routine toxicity testing.

New molecular and instrumental techniques have made available many markers of cellular damage that can be evaluated in multiple tissues in vivo at low cost without compromising the normal conduct of in vivo toxicity evaluations, and without the need for substitution of new species or strains of animals. These techniques include (1) the activation of stress genes that respond to general classes of toxic agents and cellular damage at doses below those that cause frank toxicity; (2) electrophoretic methods for the detection of DNA strand breakage due to DNA degradation resulting from cell death or genotoxic damage; (3) the use of fluorescent chromosome-specific DNA probes that allow evaluation of stable chromosomal rearrangements, chromosomal breaks, and aneuploidy in laboratory animals; and (4) endogenous and exogenous (transgenic) reporter genes for the evaluation of in vivo gene mutation. Additionally, powerful new analytical techniques such as accelerator mass spectrometry make possible ultrasensitive measurements of metabolite binding to specific macromolecular targets and permit pharmacokinetics studies at very low doses. Often, identical or analogous endpoints can be measured in cellular models, in laboratory animals, and in humans, an approach that allows in vitro screening for product development, in vivo hazard identification, and early risk assessments in animal models and direct risk assessment in humans. These new in vivo techniques will greatly enhance our ability to extrapolate laboratory data to human health risk.

Animals↗
PubMed1995

The transmission rate of the lacI transgene from the Big Blue mouse.

Since transgenic mice are being used to analyze somatic and germinal mutation rates in vivo, it is of interest to know to what extent these mice are normal or abnormal in any way. During experiments designed to compare the mutational response of the transgene and an endogenous gene, Big Blue mice hemizygous for the transgene were bred to create a hybrid mouse in which the comparisons could be made. The fraction of these mice that inherited the transgene was 37% rather than the Mendelian expectation of 50%.

Animals↗
PubMed1995

The induction of dominant somatic mutations at the Dlb-1 locus.

In the small intestine of heterozygous mice (Dlb-1b/Dlb-1a), the Dlb-1b allele results in a stainable epithelium. The mutation or loss of the dominant Dlb-1b allele in a stem cell results in a non-staining ribbon of cells on a villus of the small intestine. To determine if dominant mutations resulting in the gain of staining--the induction of a Dlb-1b-like allele--could also be detected, we examined Dlb-1a homozygous mice (SWR) 2 weeks after a single treatment with 250 mg/kg ethylnitrosourea. Mutations to the dominant allele should appear as brown ribbons on unstained villi. Such ribbons were observed in the treated group but not in controls. The mutant frequency was low compared to the frequency of Dlb-1a-like mutations reported at the Dlb-1b allele in heterozygous mice.

Animals↗