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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

On the differential responsiveness of males and females in the micronucleus assay.

The primary question asked was whether the increased sensitivity of female mice to the induction of micronuclei by ethyl methanesulphonate, compared to males, would vanish if the doses were adjusted for any difference in the LD50 between the sexes. The second question was whether the frequency of micronuclei after 4 daily treatments would differ from 2 daily treatments. We measured the LD50 in the two sexes separately and found the female value to be lower. We also repeated the micronucleus measurements in each sex separately. At any given dose expressed in mg/kg the response in females was greater than in males. This was still true when the dose was expressed as a fraction of the LD50, but the difference was reduced. The uncertainty in the LD50 measurements was such that there may be no meaningful difference between males and females when the dose is expressed in this way. The 2-day and 4-day results differed very little for ethyl methanesulphonate, but were quite different for the positive control, dimethylbenz[a]anthracene, being higher at 4 days than at 2 days. We argue that the results of our experiments, which confirm the results of others, demonstrate the advisability of using both sexes in the assay.