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J A Heddle

Publications and source records attributed to J A Heddle.

At least 55 records · Page 3Linked to original sources
PubMed1995

Factors affecting somatic mutation frequencies in vivo.

The factors that influence the spontaneous mutant frequencies in mammalian tissues have been ranked on the basis of data from our laboratory together with published data. Some of the data come from the endogenous hprt and Dlb-1 loci, but most come from transgenic mice carrying the bacterial lacI and lacZ genes in recoverable lambda phage vectors. Since there is evidence that these bacterial loci are selectively neutral, the mutant frequency observed is the integral of the mutation rates from the formation of the zygote. The factors that affect the inferred mutation rate, in decreasing order of importance are: site of integration of the transgene, age, tissue, and strain. Insufficient data exist to determine the influence of gender (probably small) and inter-laboratory variables (probably at least as important as age). The two most surprising results are (1) that about half of all mutations arise during development (and half of these in utero) and (2) that most somatic tissues, whether queiscent or actively proliferating, have similar mutant frequencies and similar increases during adult life.

Aging↗
PubMed1995

An assay for loss of heterozygosity in vivo at the Dlb-1 locus.

Loss of heterozygosity (LOH) is a frequent event in many tumours, resulting in the loss of tumour suppressor genes and ultimately magnifying the number of cells with a mutant phenotype. We have used the Dlb-1 locus as a simple quantitative assay for LOH in vivo. Mutations of the dominant Dlb-1b allele are readily detected in heterozygous (Dlb-1a/Dlb-1b) mice by the loss of histochemical staining, which results in unstained, white (Dlb-1a/Dlb-1-) ribbons on a stained background. Such ribbons are extremely rare in untreated C57BL mice which are homozygous for the dominant allele, as would be expected when two independent mutations are required. To test for LOH, we first treated the animals with a high dose of ethylnitrosourea (ENU) which induces many mutations and thus many heterozygous cells, and allowed 2 weeks for gene expression. Then the animals were treated with the test agent to determine if it could cause LOH and thus convert heterozygous mutant cells, which would not produce detectable ribbons, into homozygotes that would produce white, non-staining ribbons. Treatment with ENU alone produced a low, but detectable, frequency of mutant ribbons. Treatment with X-rays alone produced no detectable increase in the frequency of mutant ribbons. Combinations of these treatments produced additive effects, thus showing that no significant LOH was induced. The additivity of the two equal ENU treatments was unexpected, since double mutants should increase as the square of the mutant frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1994

Revelling in cytogenetics.

A fascination with chromosomes, especially chromosomal aberrations, linked many at the Oak Ridge National Laboratory and the Medical Research Council Radiobiological Research Unit at Harwell, but they disagreed about the correctness of Revell's exchange hypothesis. This is a personal story of how a critical test of this hypothesis was conceived, with glimpses of life in the two centres.

Chromosome Aberrations↗
PubMed1994

The accumulation and persistence of somatic mutations in vivo.

Mutant frequencies at the native Dlb-1 and the lacI transgene reach a plateau within 1 week of mutagenesis in the small intestine of mice. This indicates that these mutations are selectively neutral and should accumulate additively. In a test with three mutagens, one potent (ethylnitrosourea) and two weak (1,2-dimethylhydrazine and methyl methanesulphonate), ten weekly treatments induced ten times the frequency of Dlb-1 mutations induced by a single treatment in each case, a result that is consistent with perfect additivity. These observations indicate that the mutation frequency at neutral loci will be an integrated measure of the effects of aging, endogenous mutagens, environmental mutagens and antimutagens. They suggest a simple protocol for transgenic mutation assays which would increase sensitivity, require fewer animals, and conform to standard toxicological practice.

1,2-Dimethylhydrazine↗
PubMed1993

Comparison of somatic mutation in a transgenic versus host locus.

Somatic mutations can now be quantified in almost any cell type in mice carrying bacterial genes in a lambda phage shuttle vector. Mutations induced in vivo are detectable ex vivo, after packaging host-cell DNA into phage that are grown on suitable bacteria. However, the transgenic DNA differs from many host loci in several ways: it (i) is prokaryotic DNA, (ii) is present in multiple tandem copies, and (iii) is heavily methylated and probably not expressed. Thus, mutation of a transgene may not be a suitable model of the host loci, which are eukaryotic, unique, and expressed. To test the relevance of the transgene mutation model, the frequencies of the bacterial lacI+ to lacI- mutations induced in half of the small intestine were compared with the frequencies of the host Dlb-1b to Dlb-1a mutations induced in the other half. The loci responded similarly to ethyl nitrosourea (ENU) with respect to the animal's age and sex, sex of the parent transmitting the transgene, and expression time. ENU dose-response curves were similar. Furthermore, no difference was found at the Dlb-1 locus between transgenic and nontransgenic siblings. In contrast, x-rays induced few lacI mutations but many Dlb-1 mutations. Probably few large deletions are detectable at lacI, but many are detectable at Dlb-1. If so, an important class of mutation is not readily detected in these transgenic mice. With this exception, the transgene and host gene responded similarly in this somewhat limited trial, as is necessary if the transgenic mice are to be a useful model.

Animals↗
PubMed1993

Mutagenicity of peroxyacetyl nitrate (PAN) in vivo: tests for somatic mutations and chromosomal aberrations.

A series of experiments was conducted in which Chinese hamsters inhaled PAN, an ubiquitous pollutant that is present in the atmosphere at concentrations that are as high as, or higher than, other known genotoxic agents. The animals were exposed to PAN in air at concentrations of approximately 3 ppm for up to 1 month and then examined for somatic mutations and chromosomal aberrations. Mutations were assayed by measuring the frequency of thioguanine-resistant lung fibroblasts (isolated de novo and cultured). Chromosomal aberrations were assayed by measuring the frequency of micronuclei in either the bone marrow (polychromatic erythrocytes) or the lungs (binucleate lung fibroblasts cultured in the presence of cytochalasin B). The results for the test animals were compared to those from animals exposed similarly, but without PAN. Although in each experiment the mutation frequencies for the test animals were higher than the corresponding controls, the mutation frequencies were not significantly different from the concurrent negative controls (P > .05) or the historical controls, except for experiment C. In experiment C, there was a significant regression of mutation frequency versus dose (P < 0.001) if all of the historical controls for pooled animals are included at zero dose. No reproducible evidence of chromosomal breakage was found in either lung or bone marrow. Thus, although PAN has been found to be a bacterial mutagen, we did not find statistically significant evidence of mutagenicity in vivo. The toxicity of PAN limited the exposure concentration that could be used. When all of the PAN data were used, the best estimate of the mutagenic potency proved to be comparable to that of ethylene dibromide, a carcinogenic atmospheric pollutant.

Air Pollutants↗
PubMed1993

Mutagenicity of methyl methanesulfonate (MMS) in vivo at the Dlb-1 native locus and a lacI transgene.

Methyl methanesulfonate (MMS) is an extraordinarily poor mutagen compared to ethylnitrosourea (ENU) or even X-rays. In lung fibroblasts in vivo, MMS has been shown to induce many micronuclei but few, if any, mutations at the hpt locus. We wondered if the lack of mutations might be due to the lack of division and DNA synthesis in fibroblasts in vivo, which would permit substantial time for differential repair of DNA lesions. This idea was tested in the small intestine, a tissue in which the cells are actively dividing. Two loci were examined: a native locus (Dlb-1) which determines the presence or absence of a lectin binding site on the surface of the epithelial cells, and a lacl transgene which controls beta-galactosidase synthesis. Locl mutations were detected after in vitro packaging of DNA isolated from the intestinal epithelium into lambda phage and expression in suitable bacteria. Although the epithelial cells are proliferating, acute treatments produced no significant increase in mutations at either locus. Subacute treatments produced low but significant increases in mutation frequency at both loci. The results confirm that MMS is a far more potent clastogen than it is a mutagen and should be regarded as a super-clastogen in the same manner as ENU is a super-mutagen. The carcinogenicity of MMS is probably the result of its potent clastogenicity rather than its weak activity as a point mutagen.

Animals↗
PubMed1992

Optimization of the concurrent assay for gene mutations and chromosomal aberrations in vivo: expression time in rats.

Ethylnitrosourea was used as a model mutagen to determine the time at which the most mutants and chromosomal aberrations could be detected. Primary fibroblasts derived from Fischer (F344) rats that had been mutagenized by ethylnitrosourea in vivo, were grown in culture and sampled at increasing intervals to analyze, quantitatively, gene mutations and chromosomal aberrations. Since ethylnitrosourea reacts very quickly and the cells were isolated five hours after treatment, expression time represents the intracellular processes involved, not the pharmacokinetics of the mutagen. A thioguanine selection procedure was used to quantify gene mutations. A cytochalasin B technique for micronuclei was used to quantify chromosomal aberrations. A significant increase in the number of mutations was observed on the ninth day in an experiment wherein sampling was done at increasing intervals of three days. One of two experiments wherein sampling was done at increasing intervals of five days showed maximum number of mutations in dishes plated with cells, exposed to maximum dose (200 mg/kg), on the tenth day for selection. Cells that received dose 100 mg/kg on the contrary produced maximum number of mutant colonies in dishes that were plated on the 20th day for selection. A repeat of the 5-day interval experiment showed maximum number of mutant colonies in dishes selected on the tenth day, irrespective of the dose used. The number of micronuclei reached a maximum on the third day.

Animals↗
PubMed1992

Detection of somatic mutations in vivo in lung fibroblasts, I. Spontaneous frequencies in Chinese hamsters and F344 rats.

The frequency of mutant cells observed in a series of experiments in which primary cells were isolated from untreated or solvent control animals are reported. The mutants detected are thioguanine-resistant lung fibroblasts isolated de novo from Chinese hamsters or Fischer 344 rats. The results in the two species were very similar. The distribution of mutant colonies in cells isolated from untreated animals is not random (Poissonian) but rather shows an excess of mutant clusters. No significant difference was detected between males and females. The results provide the information necessary to define the appropriate conditions for the negative controls for a routine assay for mutations induced in vivo.

Animals↗
PubMed1991

Micronuclei as an index of cytogenetic damage: past, present, and future.

The workshop was designed to present what is known about the production of micronuclei, what protocols are now accepted or proposed internationally, what new results have been obtained, and what new methods and protocols are likely to be forthcoming. This report is designed to convey the flavour of the workshop and to provide the essence of the new information. After the workshop an effort was made to determine what single protocol would satisfy the requirements set for the micronucleus test by as many regulatory agencies as possible. The result, reported here, includes the requirements of six regulatory authorities in Canada, the European Economic Community, the Organization for Economic Co-operation and Development, Japan, and the United States.

Animals↗
PubMed1991

Implications for genetic toxicology of the chromosomal breakage syndromes.

The activation of oncogenes and our knowledge of the chromosome breakage syndromes show that both intragenic mutations and chromosomal aberrations are important in carcinogenesis. Each suggests that an agent could produce genetic changes in a tissue without producing cancer there, if the types of genetic change do not match: chromosomal aberrations may be irrelevant in the mammary epithelium but be very significant in the bone marrow, and vice versa. This has vital implications for genetic toxicology: (1) both gene mutations and chromosomal aberrations should be measured, and (2) carcinogens may be mutagenic in tissues in which they are not carcinogenic. One might therefore expect in vivo assays for mutagenicity to correlate rather well with cancer bioassays; unfortunately, the bioassays themselves seem faulty. If cancer bioassays are valid, they would be reproducible. If bioassays are reproducible, they would be internally consistent. The information supplied by Tennant et al. (1987) for their validation of in vitro assays gives data from both sexes in rats and mice for 70 chemicals. When the data are analyzed site-by-site, positive results were not replicated in the other sex or in the other species much of the time: in half the cases the other sex does not give the same result; in two-thirds of the cases the other species does not give the same result. There are 3 potential explanations for these differing results: (1) genuine sex-specific carcinogens are common, (2) genuine species-specific carcinogens are common, or (3) the bioassay does not replicate well, i.e., is erratic. The third possibility best explains the data. The apparent inability of short-term in vitro tests to discriminate well between carcinogens and non-carcinogens may be more a reflection of the cancer bioassays that were used to determine which chemicals were carcinogenic than any defect in the assays. In this situation in vivo assays can scarcely be expected to do better even if they are better.

Carcinogenicity Tests↗
PubMed1991

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures.

A recommended protocol has been developed for chromosomal aberration and sister-chromatid exchange assays in CHO, V79 and human lymphocyte cultures. The protocol was based on the responses to a detailed questionnaire completed by North-American and European governmental, university, and contract laboratories using these tests. This report identifies those modifications to previously described methods that are used on a regular basis and clarifies confusing or inconsistent practices. These protocols can be modified for use in other types of cells.

Animals↗
PubMed1991

Chemical induction of somatic gene mutations and chromosomal aberrations in lung fibroblasts of rats.

Rats can be used to detect both somatic gene mutations and chromosomal aberrations induced in vivo. The technique adapted from Chinese hamsters for the isolation and analysis of lung fibroblasts confirm the surprising results obtained in hamsters; namely, methyl methanesulphonate induces many micronuclei but no significant increase in gene mutations. In contrast ethylnitrosourea induces both micronuclei and gene mutations.

Animals↗
PubMed1991

Measuring gene mutation in vivo.

Both chromosomal aberrations and intragenic mutations are important in the activation of oncogenes and inactivation of antioncogenes. It is now possible to measure both types of mutational events in vivo in a single cell type. The results obtained in both Chinese hamsters and rats are surprising: mutations can be induced in the absence of detectable chromosomal aberrations by some agents and vice versa by others. Preliminary results from a validation study are presented.

Animals↗
PubMed1990

The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The protocol recommended for the micronucleus assay in mammalian bone marrow has been revised and simplified. The number of sample times has been reduced to one or two, depending upon the dosing protocol. The minimum number of cells to be scored per treatment group has been increased to 20,000 to increase the ability of the assay to detect a doubling of the control micronucleus frequency. Use of both male and female animals is recommended. Scoring of micronuclei in polychromatic erythrocytes of peripheral blood is included as a variation of the bone marrow assay. Published data on chemicals tested by the micronucleus assay have been reviewed and are summarized.

Animals↗