PubMed Health⌕ Search

Biomedical subjects

J A Heddle

Publications and source records attributed to J A Heddle.

At least 19 recordsLinked to original sources
PubMed2001

A test of the mutagenicity of cooked meats in vivo.

There is a correlation between intestinal cancer and diets high in meat, so fried beef, chicken, lamb, pork and fish were tested for their ability to induce mutations in the small intestine of mice. The mice were bred to be heterozygous at the Dlb-1 locus so that loss of the dominant Dlb-1 b allele by mutation could be detected. Mice were fed the AIN-76A diet (which contains 50% of the calories in the form of sucrose) or an isocaloric diet in which the sucrose was replaced by meat or fish, for 5 or 9 weeks. Manifestation of mutants requires approximately 1 week in this system, so this corresponds to an effective exposure of 4 and 8 weeks, respectively. There was no significant difference in the weights of animals on the different diets, and no difference in mutant frequency. Several food mutagens were present, but at low levels. These results, when considered in the light of tests of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and amino(alpha)carboline at much higher doses (Zhang,X.-B., Tao,K.S., Urlando,C., Shaver-Walker,P. and Heddle,J.A. (1996) MUTAGENESIS:, 11, 43-48), indicate that there is no highly mutagenic compound missed by previous testing with bacterial assays and that mixtures of heterocyclic amines at low levels do not show great synergy.

Administration, Oral↗
PubMed2000

Differential mutation of transgenic and endogenous loci in vivo.

Although chemicals usually induce very similar frequencies of mutations in transgenes and endogenous genes in vivo when given acutely, chronic exposure to N-ethyl-N-nitrosourea (ENU) produced a more complex pattern in which the endogenous locus was spared many mutations. Here, we demonstrate that the effect is neither ENU-specific nor locus-specific, and thus, may be important in the extrapolations of risk assessment and in understanding mutational mechanisms. During chronic mutagen exposure, mutations at the transgene accumulate linearly with time, i.e. in direct proportion to the dose received. In contrast, mutations at the endogenous gene are much less frequent than those of the transgene early in the exposure period and the accumulation is not linear with time, but rather accelerates as the exposure continues. Previous comparisons involved the endogenous Dlb-1 locus and the lacI transgene from the Big BlueMouse in the small intestine. These experiments involved the Dlb-1 locus and the lacZ transgene from the MutaMouse in the small intestine and the hprt locus and the lacZ transgene in splenocytes. Comparisons were made in both tissues after acute and chronic exposures to ENU, the original mutagen, and in the small intestine after exposures to benzo(a)pyrene. All comparisons showed that during chronic exposures mutations at the transgene accumulate linearly with the increasing duration of exposure, whereas induced mutations of the endogenous gene initially accumulate at a slower rate. Thus, the difference in mutational response observed during low chronic treatment is not unique to a particular transgene, endogenous gene, tissue, or mutagen used, but may be a general phenomenon of such genes.

Animals↗
PubMed2000

Dietary restriction during murine development provides protection against MNU-induced mutations.

The developmental stage is the most rapid period for the accumulation of somatic mutations. Epidemiological studies have also suggested a significant role of early life for cancer susceptibility, showing a protective effect of modest dietary restriction early in life. To determine if mutation rate, diet, and cancer risk are related, we have investigated the effect of dietary restriction on somatic mutations early in life. The diet of mouse dams was restricted during pregnancy and lactation by 10% from ad libitum control. F(1) pups (SWRxMutaMouse) were weaned at 3 weeks of age. Pups from dams that were on a restricted diet were kept under dietary restriction (40% until 5 weeks of age and then 20% until sacrifice). Only females from litters of seven or eight were used in this study. A portion of pups from both groups were treated with N-methyl-N-nitrosourea (MNU, 50mg/kg, i.p.) at 5 weeks of age and all mice were sacrificed at 10 weeks of age. The frequency of induced mutations was reduced by about 30% at the three loci studied, lacZ (P=0.028) and cII (P=0.042) and Dlb-1 (P=0.032) in the small intestine in the restricted group. A similar decrease in the lacZ mutant frequency was observed in the bone marrow, but the results did not reach statistical significance (P=0.074). Few differences in the lacZ mutant frequency were observed in the colon and the mammary epithelium, but variability of the mutant frequencies was such that an effect of similar magnitude could not be excluded statistically. Analysis of 47 cII mutants revealed that the majority of MNU-induced mutations were G:C to A:T transition at non-CpG sites, with no difference in the mutation spectrum between the two dietary groups.

Animals↗
PubMed2000

Proliferation is necessary for both repair and mutation in transgenic mouse cells.

Proliferating cells are often presumed to be more mutable than quiescent cells because they have less time to repair DNA damage before DNA replication. Direct tests of this hypothesis have been confounded by the need for cell division before a mutation can be detected. We have avoided this problem by showing that the Big Blue mouse cell line permits the dynamic quantification of both lesions and mutations in the complete absence of cell division. These cells carry the bacterial lacI gene in a lambda shuttle vector. Mutant plaques recovered by in vitro packaging of the mouse DNA can arise from mutations sustained either in mouse cells or in the bacteria. The proportion of mutant phage contained within a mutant plaque can distinguish these two types of mutation. Mutations formed in mouse cells yield >90% mutant phage because both DNA strands are mutant. On the other hand, mutations formed in the bacteria from adducted DNA yield </=50% mutant phage, because one of the DNA strands is wild type. Immediately after exposure to a test mutagen, ethylnitrosourea, all induced mutations were formed in the bacteria, but after approximately one cell division, the reverse was true and all mutations arose in the mouse cells. Only one-fifth as many mutations were recovered from quiescent cells and all arose in the bacteria, showing that the mouse cells made no mutations in the absence of proliferation. Surprisingly, the mouse cells did not repair any of the premutagenic damage during 4 days of quiescence. When these quiescent cells were induced to proliferate, however, both repair and mutation fixation ensued.

Animals↗
PubMed2000

ENU induces mutations in the heart of lacZ transgenic mice.

The use of transgenic mouse models as somatic mutation assays allows determination of mutation in all tissues of the mouse, including non-dividing tissues. In this regard, these models can be used to study the possibility that mutations can be induced in mitotically quiescent organs such as the heart. Mutations are generally thought to be associated with mitotic processes of DNA replication. Mutations, however, are also postulated to occur in the absence of mitosis as the result of DNA repair. In order to determine whether or not mutations could be induced in the heart, we analyzed the mutant frequency in the hearts of F(1) (Muta Mouse X SWR) mice that had been treated acutely with 250 mg/kg ENU and sampled at days 10, 35, and 70 post-treatment. A significant increase in mutant frequency at day 70 shows that mutations can be induced in the heart. Since the heart contains small numbers of non-muscle cells, additional mechanisms that could explain these results were also considered. The effect of ENU-induced cell proliferation or a sub-population of rapidly dividing cells is ruled out by C(14)-thymidine uptake studies which showed minimal proliferation. By the same token, the influence of ex vivo mutations (i.e., DNA adducts fixed as mutations during replication in the bacteria) is ruled out by the observed time course of mutations, as well as experimental evidence showing that such mutations are not detected in the lacZ assay.

Animals↗
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↗