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Enhanced Somatic Mutation Rates Induced in Stem Cells of Mice by Low Chronic Exposure to Ethylnitrosourea (Dose Rate/Intestine/D1b-1/Laci) P

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Enhanced Somatic Mutation Rates Induced in Stem Cells of Mice by Low Chronic Exposure to Ethylnitrosourea (Dose Rate/Intestine/D1b-1/Laci) P Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11470-11474, December 1995 Genetics Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea (dose rate/intestine/D1b-1/lacI) P. M. SHAVER-WALKER, C. URLANDO, K. S. TAO*, X. B. ZHANG, AND JOHN A. HEDDLEt Department of Biology, York University, Toronto, ON, Canada M3J 1P3 Communicated by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY, August 21, 1995 (received for review April 28, 1995) ABSTRACT We have found that the somatic mutation these sites can be detected by means of peroxidase staining of rate at the Dlb-1 locus increases exponentially during low daily the small intestine after reaction with peroxidase-bound lectin. exposure to ethylnitrosourea over 4 months. This effect, In the small intestine, the Dlb-1b allele (presence of the binding enhanced mutagenesis, was not observed at a lacI transgene in site) is dominant over the Dlb-la allele (absence of the binding the same tissue, although the two loci respond very similarly site). In heterozygous mice (Dlb-lb/Dlb-la), mutation of the to acute doses. Since both mutations are neutral, the mutant dominant Dlb-1b allele in an intestinal stem cell results in a frequency was expected to increase linearly with time in mutant clone that lacks the lectin binding site and is unstained response to a constant mutagenic exposure, as it did for lacI. by the peroxidase. Since the stem cells of the small intestine are Enhanced mutagenesis does not result from an overall sen- located in the crypts (invaginations of the cell sheet) and feed sitization ofthe animals, since mice that had first been treated cells inward to the villi (projections of the cell sheet into the with a low daily dose for 90 days and then challenged with a lumen of the intestine), a mutant stem cell produces a ribbon large acute dose were not sensitized to the acute dose. Nor was of nonstaining cells extending from the crypt to the tip of the the increased mutant frequency due to selection, since animals villus. There are about 10 crypts, each with a single stem cell, that were treated for 90 days and then left untreated for up to maintaining each villus. The time required for the appearance 60 days showed little change from the 90-day frequency. The of these mutant ribbons corresponds roughly to the 5-7 days effect is substantial: about 8 times as many Dlb-1 mutants required for the progeny of a stem cell to reach the tip of the were induced between 90 and 120 days as in the first 30 days. villus (7). This resulted in a reverse dose rate effect such that 90 mg/kg Similarly, mutations arising at the transgenic lacI locus in the induced more mutants when delivered at 1 mg/kg per day stem cell can be measured by allowing at least 1 week after the than at 3 mg/kg per day. We postulate that enhanced mu- end of treatment for the epithelium to turn over. Hemizygous tagenesis arises from increased stem cell proliferation and the transgenic mice carry a A vector that contains the lacI target preferential repair of transcribed genes. Enhanced mutagen- gene and an a-complementing lacZ reporter gene (8, 9). This esis may be important for risk evaluation, as the results show vector can be recovered from the DNA of the mice in the form that chronic exposures can be more mutagenic than acute ones of a viable A phage. When these phage are plated on Esche- and raise the possibility of synergism between chemicals at richia coli on plates containing 5-bromo-4-chloro-3-indolyl low doses. ,3-D-galactopyranoside (X-Gal), those that carry a functional lacI will form clear plaques, whereas those in which the Somatic mutations are important in carcinogenesis, yet little is repressor has been mutated will produce 03-galactosidase, known about their origins. The large number of mutations which cleaves the X-Gal to produce blue plaques (8, 9). found in tumors seems to be at variance with the estimated Comparisons of the behavior of the lacI transgene and the spontaneous mutation rates (1). There are several possible Dlb-l host locus in the small intestine showed that they explanations for this. Loeb (1) suggests that one of the early behaved very similarly in most respects (10). Indeed, both events may be a mutation that inactivates a DNA repair gene behaved as if mutations at these loci are neutral: the frequency and creates a mutator phenotype. This is supported by the of mutations was constant from 1 to 8 weeks after mutagenesis finding of high mutant frequencies in murine tumors at a locus (10), and the effect of weekly treatments was additive (11, 12). not involved in carcinogenesis (a lacI transgene) and by the The concept of neutrality led to the suggestion that chronic frequent occurrence of mutations at a gene involved in mis- protocols would enhance the sensitivity of transgenic mutation match repair in human colonic tumors (2, 3). Others have assays, provided time is allowed for tissue turnover (12-14). It suggested that sequential expansion of selected clones pro- also inspired these experiments, which began as an attempt to vides a cell that the mutation rate is quantify the mutant frequency induced at much lower doses by large enough population treating the animals many times with such a dose and assuming not limiting (4). Environmental mutagens may also influence additivity. This approach has been used for thioguanine- the mutation rate, as epidemiological studies show that envi- resistant mutants in cultured TK6 cells (15). The doses used ronmental factors are important for human cancer rates (5). correspond to about 1% and 0.3% of the acute LD50, whereas Clearly it is the mutation rate in the stem cells that is important most previous studies have used doses about 20 times higher for carcinogenesis, as differentiated cells are often unable to or more. divide and are lost from the epithelial cell populations where many cancers arise. Until recently, it has been difficult to study somatic muta- MATERIALS AND METHODS tions in stem cells, but the Dlb-1 mutation assay makes this easy Animals. An independent animal care committee approved for the small intestine (6). Dlb-1 determines the presence or all experimental protocols in advance. All mice were housed in absence of Dolichos biflorus lectin binding sites on the surface of cells of the small intestine and elsewhere. The presence of Abbreviations: ENU, ethylnitrosourea; X-Gal, 5-bromo-4-chloro-3- indolyl ,B-D-galactopyranoside. The publication costs of this article were defrayed in part by page charge *Present address: Hospital for Sick Children, Toronto, ON, Canada payment. This article must therefore be hereby marked "advertisement" in M5G 1X8. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 11470 Downloaded by guest on September 28, 2021 Genetics: Shaver-Walker et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11471 plastic cages with wood chip bedding at 70% humidity and 22 recovered by in vitro packaging with Transpack packaging ± 2°C and a 12-h light/12-h dark cycle. Water and food were extract (Stratagene). The extracts were incubated with SCS-8 supplied ad libitum. The hemizygous lacI C57BL/6 (Dlb-1b/ E. coli (Stratagene), which produce the complementing car- Dlb-1b) transgenic mice were obtained from Stratagene and boxyl-terminal end of the lacZ gene. The infected bacteria bred with SWR (Dlb-la/Dlb-la) mice, obtained from The were grown on NZY agar containing 70 mg of X-Gal per 25-cm2 Jackson Laboratory. Those F1 that carry the transgene are agar plate. When a mutation inactivates the lacI repressor, the suitable for mutation detection at both loci. Their nontrans- alacZ reporter gene is produced and complementation results genic siblings were also used for some of these experiments. in a functional ,3-galactosidase, which cleaves the X-Gal, and For the initial measurements, only the high-dose group con- blue plaques result. The number of plaques recovered from tained transgenic animals. Since the animals differed some- each animal differed greatly: the mean was 16,400 plaques; the what in age, they were stratified into four age groups, 41, range was 6100-50,000 plaques. 43-44, 47-52, and 53-56 weeks old. There is very little Dlb-) Assay. Whole mounts were prepared as described in difference in the spontaneous mutation frequency in adult Winton et al. (6) with a few modifications (10). The middle mice in this age range (16, 17). The test groups were structured one-third of the small intestine was flushed with PBS, inflated, to contain similar contributions from each age group, and the and fixed with 10% formal saline [0.85% (wt/vol) NaCl in 10% treatments were assigned to each group randomly after their buffered formalin]. It was then cut open and clipped, villi up, composition had been determined. Initially, each group con- to a microscope slide with plastic-coated paper clips. The slides tained five animals, two males and three females or three males were fixed for 1 h in 10% formal saline, rinsed with PBS, and and two females. Subsequently, somewhat younger animals then incubated in 20 mM dithiothreitol for 45 min to remove were used, but, again, it was difficult to obtain enough animals the mucus. They were stored in 10% formal saline until of the same age, and they were stratified into three groups of staining. The slides were incubated in 0.1% phenylhydrazine 17-19, 23, and 28-29 weeks of age.
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