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. Treatment groups were hydrochloride for 30 min to block endogenous peroxidases, constructed and assigned as before. There were two males and incubated 20 min in 0.5% albumin in PBS, and then stained two females in each group. Mice were sacrificed by cervical with 5 ,ug of D. biflorus agglutinin-peroxidase conjugate dislocation immediately after treatment in the first study and (Sigma) per ml in the PBS/albumin. The peroxidase was 1 week after treatment in all others. The animals were treated developed by using 3,3'-diaminobenzidine (Sigma) at 0.5 daily between 9:00 and 11:00 a.m. Controls were not treated. mg/ml for 20 min. The slides were rinsed twice with PBS and Mutagens. Ethylnitrosourea (ENU) was obtained from stored in PBS until analyzed. Sigma. Test solutions were made by dissolving ENU in di- The slides were coded and then scored with a dissecting methyl sulfoxide at 20 times the highest final concentration and microscope at x50. The Dlb-lb/Dlb-la epithelial cells stained then frozen at -70°C. Each day an individual vial of frozen brown, whereas mutant cells (Dlb-1 -/Dlb-la) were not stained concentrate was diluted to the proper concentration of 1 or 3 and appeared as vertical white ribbons on the villi. Fifty fields, mg/kg with phosphate-buffered saline (PBS) at pH 7.2 and defined by a rectangle in an eyepiece graticule, were scored. injected intraperitoneally within 20 min. The first and last fields were each counted twice and averaged lacI- Mutations. After sacrifice, the entire small intestine to estimate the total number of villi, which was always about was removed and two-thirds of it was used for DNA extraction. 104 per slide. There is no difference in the response at either locus in Statistics. The statistical analyses were conducted with the different regions of the small intestine to acute doses of ENU MINITAB software. All means and standard errors reported are (10). These sections were flushed with PBS (pH 8.0) and based on the mutant frequency observed in each animal, inverted. The inverted small intestine was placed in 3 ml of regardless of the number of plaques or villi analyzed. Douncing buffer containing RNase A at a concentration of 100 jig/ml and pushed in and out of a 5-ml syringe in order to loosen the cells. The cell suspension was then digested with a RESULTS proteinase K solution (2 mg/ml), and the DNA was extracted The mutant frequency as a function of time is shown in Fig. 1 with phenol/chloroform and precipitated with ethanol (9). for both lacI and Dlb-1b. The time shown is the actual The A phage shuttle vector, which contains the entire lacI treatment time less the expression time of 4 days, since in this mutational target gene and the odacZ reporter gene, was case there was only 1 day between the last treatment and the 200 to

x0 >1 150

L)

cs 100 IL.

C4- a 50

30 60 90 0 30 60 90 0 1 2 3 Days of Treatment (ENU) Days of Treatment Dose Rate (mg/kg/day)

FIG. 1. Mutant frequency (± SEM) observed after daily treatments with ENU. In several cases the error bars are smaller than the symbols. (A) ENU at 3 mg/kg per day. v, Individual animals; 0, means. (B) o, 3 mg/kg per day; v, 1 mg/kg per day; *, control. (C) Open symbols represent individual animals; closed symbols represent the means. Downloaded by guest on September 28, 2021 11472 Genetics: Shaver-Walker et al. Proc. Natl. Acad. Sci. USA 92 (1995)

sacrifice. The accumulation of mutations at lacI appears to be 360

linear (F = 0.054; df = 1,14; P = 0.82), as expected, but the I I/IeIaIy accumulation of mutations at Dlb-] is not linear at either 1 320 LI) mg/kg per day (F = 14.6; df = 1,17; P = 0.001) or 3 mg/kg per 280 0~ day (F = 17.1; df = 1,17; P = 0.0007) and appears to be exponential. In acute experiments, the frequencies of muta- a) 240 tions at the two loci are about equal (10), but this was not U- 200 observed at the early samples where there are fewer mutations 160 at Dlb-1 than at lac. The two curves do not have the same 0 r ...... ~~~~~~~~~~~~I = = = shape (F 4.79; df 2,33; P 0.02). In spite of the nonlinear 120 accumulation ofDlb-l mutations with time, the dose-response 1 mg/kg per day/ curve at any one time is linear (Fig. 1C). The nonlinear 80 accumulation of mutants at the Dlb-] locus with continued treatment is reproducible and continues to accelerate past 90 .0 40 days, as shown in Fig. 2. In this experiment, a 1-week expres- o sion time was allowed after the last treatment before mea- 0 60 120 180 240 300 360 surements were made. ENU Dose (mg/kg) A reverse dose rate effect is evident when the mutant FIG. 3. Mutant frequency (+ SEM) observed after daily treat- frequency is plotted as a function of cumulative dose (Fig. 3). ments with ENU. In several cases the error bars are smaller than the The mutant frequencies induced by the same total dose at a dose symbols. rate of 1 mg/kg per day are greater than those accumulated at 3 mg/kg per day. This can also be seen in Fig. 2, where the this hypothesis, animals that had been treated for 90 days with mutant frequency observed after 1 mg/kg for 90 days is higher daily low doses were challenged with a series of high doses, than that after 3 mg/kg for 30 days, although the total doses together with untreated controls. The challenge doses induced are the same. The same effect was observed in the initial approximately the same number of mutations in animals that experiment. had previously been treated for 90 days with either 1 or 3 One explanation for the nonlinear accumulation of Dlb-] mg/kg per day as in controls (Fig. 5). No evidence of increased mutants with time is that the mutations are not neutral, so that sensitivity to high doses could be found. Thus the higher the increasing mutant frequency does not reflect an increasing mutation rate observed is not the result of some overall mutation rate but rather a selective advantage, in spite of sensitization of the animals and is a low-dose effect. previous results. To test this, some animals were exposed for The mutations being detected arise in the stem cells of the 90 days afild then left untreated for 30 or 60 days in addition small intestine, which may divide infrequently. It is thought to the 1-week expression time. As shown in Fig. 4, the mutant that many mutations arise during DNA synthesis and that frequency remained essentially constant in animals whose dividing cells may be more sensitive to mutation as a result treatment had ceased at 90 days, although it continued to (18). We wondered if increased proliferation of the stem cells, increase at an accelerating rate in those animals whose treat- induced by prior treatment, might increase the sensitivity of ment continued. This is consistent with the conclusions of Tao the small intestine to mutation. As a test of this hypothesis, we and Heddle (12) and shows that the increased frequency is not have given animals two doses of 50 mg of ENU per kg the result of selection. It should be noted that even if a mutant separated by different intervals from 0 to 7 days. The results stem cell had a selective advantage, this would be expected to (Fig. 6) show that the treatments were additive at 7 days, as produce a larger mutant ribbon rather than more ribbons. reported earlier for ENU and other mutagens (12). With Thus the higher mutant frequency at the longer times results fractionation intervals of 1-3 days, however, the mutant fre- from a higher mutation rate rather than selection of mutant quency was greater than that produced by the unfractionated cells. dose. This shows that the tissue can be transiently sensitized by The higher mutation rate observed at later times might a previous exposure. Compared to the effect of the initial dose result from some sort of sensitization of the animals, although of 50 mg/kg, the second dose of 50 mg/kg 2 days later the lacI data are not consistent with this. As a further test of produced about twice as many mutations.

LO 360 400 0 U.) 320 o 360 X 280 X 320 c) T 240 o 280 a)LI U) Continued v 240 - treatment 0) 200 200 Stopped treatment 160 T CZ C 160 - 120 D 120 LI 80 Continued 80 treatment 40 10 Q 40 Stopped treatment

0 30 60 90 120 150 0 30 60 90 120 150 180 Days of Treatment (ENU) Day of Experiment FIG. 2. Mutant frequency (+ SEM) observed after daily treat- FIG. 4. Mutant frequency (± SEM) observed after daily treat- ments with ENU. In several cases, the error bars are smaller than the ments with ENU. In several cases, the error bars are smaller than the

symbols. oi, 3 mg/kg per day; , 1 mg/kg per day; 0, control. symbols. m, 3 mg/kg per day; o, 1 mg/kg per day. Downloaded by guest on September 28, 2021 Genetics: Shaver-Walker et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11473

U-) 400 Dlb-1. At this time, we can only speculate as to the mechanism 0 360 involved and the generality and importance of the effect. x The two loci differ in several ways that may affect mutagen- 320 esis. Obviously their sequences and their locations in the c 260 genome differ. In addition, the lacI transgenes are embedded a) in 2 megabases of prokaryotic DNA. Since it was constructed 03 240 U1) without mammalian promoter sequences and is heavily meth- 200 ylated (19), the transgene is probably unexpressed in any tissue. The Dlb-1 locus is a single endogenous locus, which is 160 expressed in the small intestine and many other tissues. Given 120 the coupling of some forms of DNA repair and transcription (20, 21), some differences between the two loci would not be 60 -D surprising. Nevertheless, the two loci respond very similarly to C 40 acute, single-dose mutagenesis by ENU, even quantitatively (10), as do lacI and hprt in splenic lymphocytes (22). X-rays, 0 aD 30 60 90 120 150 180 however, are more mutagenic at Dlb-1 than at lacI, showing that the mutation spectrum differs (10). Obviously deletions ENU Challenge (mg/kg) having one end in the vector will not be recovered as viable FIG. 5. Mutant frequency (± SEM) observed in mice that had been phage, so the large deletions characteristic of x-rays will be chronically exposed to the daily doses shown for 90 days and then underrepresented. treated with a high (challenge) dose of ENU. In several cases the error Unfortunately, no information about the molecular nature bars are smaller than the symbols. of the mutations detectable at Dlb-1 is available, except by inference from the relative responsiveness to different agents. DISCUSSION If the nonlinear component of the Dlb-1 mutant accumulation The were all deletion mutants, then a difference between lacI and results clearly demonstrate that the mutation rate at Dlb-1 Dlb-1 would be expected, but it would not be the observed is not constant exposure during chronic to ENU but increases difference. What would be expected would be a consistent exponentially with time over a 4-month period. It is apparent excess of Dlb-1 mutations at all times, whereas a deficiency that neither a selective advantage of preexisting mutations nor exists at early times. Furthermore, ENU produces primarily a generally increased sensitivity of the animals to the mutagen base substitutions. It is also unclear what mechanism could is responsible for the increasing rate of accumulation of lead to an increasing frequency of deletion mutations but not mutants as a function of exposure time. The selective advan- to sensitization of the cells to a large acute dose. It cannot be tage is ruled out by the constant mutant frequency observed the accumulation of DNA adducts, as the tissue turns over after the cessation of chronic exposure, as had been observed weekly. after both single and weekly exposures. These control exper- The other difference between the loci, transcription ofDlb-1 iments do not rule out the possibility that Dlb-1 - mutants are and nontranscription of lad, is more suggestive. Preferential at a selective advantage during exposure, but this seems repair of transcribed genes does occur in mammalian cells (19). unlikely when a higher rate of cell division would lead to a It has also been shown that quiescent normal cells are more larger ribbon (mutant clone) rather than more ribbons. resistant to mutation by some agents, such as UV, whereas Equally, a general sensitization of the tissue by any mechanism repair-deficient cells are not (18). Since alkylating agents are that changes the effective dose to the DNA is inconsistent with S-phase-dependent mutagens, enhanced mutagenesis can be both the linear accumulation of mutants at the lacI locus and explained by the biology of the stem cell in the small intestine. the normal response to a large acute dose after 90 days of In the small intestine, cells proliferate in the crypts and then exposure. The phenomenon is a low-dose effect and is appar- migrate from the crypts up the villus to be sloughed off at the ent only after an extended exposure. Any proposed mechanism tip (7). Stem cells typically have a much longer cell cycle time must explain these facts and the difference between lacI and than the proliferative population, although this is not certain for the small intestine, where the stem cells have not been 180 to) identified definitively (7). There is evidence for a slowly 0 160 dividing population of cells in the small intestine from the dilution of [14C]thymidine with time (G. Dawod, I. Kogan, J. 140 Moody, P. B. Moens, R. R. Swiger, J. D. Tucker, K. W. Tur- teltaub, and J.A.H., unpublished and for the existence of 120 data) 0~ a brief sensitive period in the life of these stem cells in the small intestine (23). When an animal is a c 100 initially treated with very low dose of ENU, few of the stem cells are in S phase, and thus 0- 80 few are susceptible to mutation and killing. Most of the stem 60 cells are nonproliferating and resistant. If a stem cell is killed, L-n one of the cells from the proliferating pool must replace it in S4 4 40 order to maintain the flow of epithelial cells toward the villus. This possibility is well established from radiation experiments 20 that show there are many more potential than actual stem cells in the small intestine (7). The death of cells in the proliferating 0 0 1 2 3 4 5 6 7 8 pool may also stimulate the stem cells to proliferate more Days Between Treatments frequently. When these concepts are applied to the chronic of 50 mg/kg low-dose exposure, the stem cells will be stimulated to prolif- erate FIG. 6. Mutant frequency (± SEM) observed when animals were slightly more often after the initial exposure to compen- treated with a single dose of 50 mg of ENU per kg (0) or two doses sate for a small amount of cell death, so slightly more cells will (0) separated by the intervals shown. The horizontal line is that be in the sensitive phase at the time of the next dose. This expected if the two doses were additive (= twice that observed for 50 second dose will thus induce a few more mutations than the mg/kg minus the control frequency). first dose, slightly more cell death, and still more proliferation. Downloaded by guest on September 28, 2021 11474 Genetics: Shaver-Walker et al. Proc. Natl. Acad. Sci. USA 92 (1995) With continuing exposures, this process will continue so that ation would increase the sensitivity to mutation by other agents the mutant frequency will rise exponentially. and to further cell proliferation, thus initiating the enhanced The split-dose experiments are consistent with a transient mutagenesis. Further experiments are required to test this sensitization induced by an acute exposure to a high dose of possibility. It may also be that more or less continuous ENU, as would be expected under the mechanism proposed exposures to low doses are more hazardous than intermittent above. However, the results of the challenge experiment, in ones, even if the total dose is the same. Possibly enhanced which animals treated daily for 90 days responded normally to mutagenesis is one of the factors involved in the origin of large high doses of ENU, do not seem consistent with this hypoth- numbers of mutations, both point mutations and chromosomal esis. They can be made compatible by assuming (i) that the deletions and rearrangements, found in human cancer. In any preferential repair of transcribed genes is readily saturable and case, enhanced mutagenesis indicates that the existing trans- (ii) that high doses induce more proliferation. It is reasonable, genic loci in widespread use for assessing somatic mutation although untested, that the preferential repair of transcribed may not reflect all of the biological phenomena of interest in genes is readily saturated; why else would unexpressed genes vivo. not be repaired as well? Such a saturable repair would lead to a large difference in sensitivity to low doses between S-phase We are grateful to Stratagene for determining which of the F1 cells, where the time available for repair before a replication carried the transgene. We thank Gemma Vomiero-Highton and Jason fork reaches the lesion is short, and non-S cells, where the time Halberstadt for their help with these experiments. This work was available for repair is much longer. At high doses, the repair supported by grants from the National Cancer Institute of Canada and system is saturated in all cells, so that many lesions survive until the National Science and Engineering Research Council. a replication fork arrives, even in cells exposed while not in S and non-S 1. Loeb, L. A. (1991) Cancer Res. 51, 3075-3079. phase, and the difference in sensitivity between S 2. Fishel, R., Lescoe, M. K., Rao, M. R. S., Copeland, N. G., Jen- cells is small. At high doses the sensitivity is observable in a kins, N. A., Garber, J., Kane, M. & Kolodner, R. (1993) Cell 75, split-dose experiment, even though small, as many cells are 1027-1038. stimulated to enter S phase. In the chronic experiments, 3. Leach, F. S., Nicolaides, N. C., Papadopoulos, N., Liu, B., Jen, J., relatively few cells are involved, and the overall increased Parsons, R., Peltomaki, P., Sistonen, P., Altonen, L. A., Nystrom- sensitivity to the high challenge doses is negligible. But in the Lahti, M., Guan, X.-Y., Zhang;J., Meltzer, P. S., Yu, J.-W., Kao, chronic experiments, the relative sensitivity difference is large F.-T., Chen, D. J., Cerosaletti, K. M., Fournier, R. E. K., Todd, and so detectable when summed over many daily repetitions. S., Lewis, T., Leach, R. J., Naylor, S. L., Weissenbach, J., Meck- The magnitude of the sensitization would be in proportion to lin, J.-P., Jarvinen, H., Petesen, G. M., Hamilton, S. R., Green, J., the increase in the rate of proliferation of the stem cells; this Jass, J., Watson, P., Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler, K. W. & Vogelstein, B. (1993) Cell 75, 1215-1225. prediction is testable. 4. Nowell, P. C. (1993) Adv. Cancer Res. 62, 1-17. The generality of enhanced mutagenesis is not known. If the 5. Doll, R. & Peto, R. (1981) J. Natl. Cancer Inst. 66, 1191-1308. effect is the result of differential sensitivity of cycling and 6. Winton, D. J., Blount, M. A. & Ponder, B. A. J. (1988) Nature noncycling stem cells, then only agents with S-phase depen- (London) 333, 443-446. dence would show enhanced mutagenesis. A single experiment 7. Potten, C. S. & Loeffler, M. (1990) Development (Cambridge, with x-rays, which mutate cells at all stages of the cell cycle, U.K) 110, 1001-1020. showed no indication of enhanced mutagenesis (data not 8. Kohler, S. W., Provost, G. S., Fieck, A., Kretz, P. L., Bullock, shown). Similarly chronic exposures to 2-amino-1-methyl-6- W. O., Putman, D. L., Sorge, J. A. & Short, J. M. (1991) Environ. phenylimidazo[4,5-b]pyridine (PhIP; X.B.Z., J. D. Felton, Mol. Mutagen. 18, 316-321. C. U. Tucker, and J.A.H., unpublished data) show a linear 9. Kohler, S. W., Provost, G. S., Fieck, A., Kretz, P. L., Bullock, W. O., Putman, D. L., Sorge, J. A. & Short, J. M. (1991) Proc. accumulation of mutants with time at both lacI and at Dlb-1 Natl. Acad. Sci. USA 88, 7958-7962. over a 90-day period, but the mechanism by which PhIP is 10. Tao, K. S., Urlando, C. & Heddle, J. A. (1993) Proc. Natl. Acad. mutagenic is not known. Sci. USA 90, 10681-10685. Ames and Gold (24) have suggested that the usual method 11. Tao, K. S., Urlando, C. & Heddle, J. A. (1993) Environ. Mol. of running the cancer bioassay produces artifactual positive Mutagen. 22, 293-296. results because very high doses are used. If these doses induce 12. Tao, K. S. & Heddle, J. A. (1994) Mutagenesis 9, 187-191. cellular toxicity and increased cellular proliferation as a re- 13. Shephard, S. E., Lutz, W. K. & Schlatter, C. (1994) Mutat. Res. sponse, then the cells might be more sensitive to mutation and, 306, 119-128. as a consequence, more cancers may result. Implicit in this 14. Heddle, J. A., Shaver-Walker, P., Tao, K. S. & Zhang, X. B. suggestion is the assumption that the mutable cells are not (1995) Mutagenesis, in press. 15. Grosovsky, A. J. & Little, J. B. (1985) Proc. Natl. Acad. Sci. USA proliferating at their maximum rate and that the cells are more 82, 2092-2095. sensitive when proliferating. Our assumptions are identical, 16. Lee, A. T., DeSimone, C., Cerami, A. & Bucala, R. (1994) but we suggest that a similar effect can occur at low doses if the FASEB J. 8, 545-550. exposure is chronic. This would mean that extrapolations from 17. Zhang, X. B., Urlando, C., Tao, K. S. & Heddle, J. A. (1995) high to low dose would require consideration of the pattern of Mutat. Res., in press. exposure. Possibly intermittent exposure is less hazardous than 18. McGregor, W. G., Chen, R. H., Lukash, L., Maher, V. M. & chronic exposure at low doses. It is noteworthy that the chronic McCormick, J. J. (1991) Mol. Cell. Biol. 11, 1927-1934. dose-response curves observed at any one time were linear in 19. Kohler, S. W., Provost, G. S., Kretz, P. L., Dycaico, M. J., Sorge, all of our experiments. A sequential series of measurements is J. A. & Short, J. M. (1990) Nucleic Acids Res. 18, 3007-3013. 20. Bohr, V. A., Smith, C. A., Okumoto, D. S. & Hanawalt, P. C. required to detect the enhanced mutagenesis. (1985) Cell 40, 359-369. It has often been thought that combinations of mutagens 21. Hanawalt, P. C. (1994) Science 266, 1957-1958. would be additive at low environmental doses. These data 22. Skopek, T. R., Kort, K. L. & Marino, D. R. (1995) Environ. Mol. suggest that combinations of mutagens could be synergistic, Mutagen. 26, 9-15. with each one sensitizing the cells to the other by inducing 23. Vomiero-Highton, G. & Heddle, J. A. (1995) Mutagenesis, in enhanced mutagenesis. If increased proliferation is involved, press. any agent, mutagenic or not, that increased cellular prolifer- 24. Ames, B. N. & Gold, L. S. (1990) Science 249, 970-971. Downloaded by guest on September 28, 2021