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[Cancer Research 64, 6919-6923, October 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Spontaneous Mutations in Digestive Tract of Old Mice Show Tissue-Specific Patterns of Genomic Instability

Tetsuya Ono1, Hironobu Ikehata1, Vishnu Priya Pithani1, Yoshihiko Uehara1, Yali Chen1, Yoshitaka Kinouchi2, Toru Shimosegawa2 and Yoshio Hosoi3

Divisions of 1 Genome and Radiation Biology and 2 Gastroenterology, Graduate School of Medicine, Tohoku University, Sendai, Japan; and 3 Department of Radiation Research, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In an attempt to evaluate the possible role of mutations in the age-dependent increase of tumor incidence, we studied the mutational burden that accumulates in the aging process in different parts of the digestive tract in mice. The mutations were monitored in lacZ genes integrated in the mouse genome. The digestive tract was divided into the esophagus, stomach, proximal, medial, and distal part of the small intestine, and the colon. Epithelial tissues were separated from these tissues with the exception of the esophagus, in which case the whole tissue was examined. At a young age, the mutant frequencies as well as the molecular nature of the mutations were similar among the tissues examined. In old age, on the other hand, mutant frequencies were elevated to different degrees among the tissues; they were high in the small intestine and colon, intermediate in the stomach, and low in the esophagus. The molecular characteristics of the mutations also revealed distinct tissue-specificity; there were elevated rates of a small deletion mutation in the esophagus, G:C to T:A transversion in the proximal small intestine, and multiple mutations in the distal small intestine and colon. The results indicate that different parts of the digestive tract suffer from different kinds of mutational stress in the aging process. The nature of the multiple mutations suggests the presence of a mutator phenotype based on an imbalance in deoxyribonucleotide pools.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although aging is one of the major causes of tumors, what kind of mechanism underlies the relationship has not been clarified. Many lines of evidence have shown that DNA mutations play an important role in tumor development (1, 2, 3) . Detailed analyses of the genomic changes in tumors as well as in precancerous tissues additionally suggest that many alterations of DNA, described as genomic instability, appear in the early stages of tumor development (1 , 2) . Thus, it is possible that genomic instability emerges in normal cells as individuals age, and they become transformed as tumor cells. To prove this hypothesis, however, it would be important to determine what kinds of mutational stress accumulate in the aging process.

In human tumors, extensive studies on DNA have revealed that different types of mutations are observed at high frequency in oncogenes and suppressor oncogenes (4 , 5) . A survey of these mutations reveals specific causes of mutations such as UV light in skin cancers and polycyclic aromatic hydrocarbons in lung cancers of tobacco smokers (4, 5, 6) . However, because such mutations constitute only a small fraction of those observed, many remain to be explained. Studies of mutations at very early stages of tumor development like dysplasia and chronic inflammation in the colon also showed a high frequency of mutations in APC and p53 genes, respectively (7 , 8) . Because the mutations in inflammatory lesions are dominated by G:C to T:A transversion, a typical type of mutation induced by reactive oxygen species, a inflammation-associated reactive oxygen species have been considered to be a possible cause (8 , 9) . The dominant type of mutation observed in the APC gene, on the other hand, is a frameshift, which is a rather rare type of spontaneous mutation (10) . Hence, the type of mutation observed in the APC gene could reflect the result of cellular selection whereby cells containing mutations that give an advantage in terms of cellular proliferation can remain and the mutation observed does not reflect the original ones (11) . Thus, it is difficult to speculate what kinds of mutations are accumulating in normal tissues in the aging process based on the list of mutations observed in precancerous cells as well as in cancers. The other characteristic of the mutations observed in tumor cells is their multiple incidents. A single tumor cell contains many DNA alterations at different positions of the genome (1, 2, 3) . Because the mutation frequency in normal cells is very low and could not explain these multiple mutation events in a single cell, it is referred to as genomic instability. Loeb et al. (12 , 13) proposed the existence of a mutator phenotype, which is observed in the SOS response in Escherichia coli, as a cause of genomic instability. They postulated that an abnormally high rate of mutations (mutator phenotype) could be derived from the suppression of genome maintenance systems. Because a high rate of microsatellite changes is observed in the early stage of tumor development (14) , they speculated that this mutator phenotype could be an early event (13) . Thus, the mutator phenotype could appear in normal tissues of older individuals.

Recently, the age-dependent alteration in spontaneous mutations has been examined directly using transgenic mice that were created for in vivo mutation assay (15, 16, 17, 18, 19, 20, 21, 22, 23) . The results showed that the mutation frequency as well as the molecular nature of the mutations are similar among the tissues when the animals are young. As they age, the mutation frequencies increase in a tissue-specific way. The highest rate, a 3-fold increase, was observed in bladders of 1-year-old lacI-transgenic mice (17) . In liver, spleen, small intestine, kidney, and heart, 2- to 3-fold increases were seen in 2-year-old mice (19, 20, 21, 22, 23) , whereas little or no increase was observed in brain and skin (17 , 19 , 22) . The molecular nature of the mutations or mutation spectra observed in middle-aged or old mice were similar to those found in young mice in all of the tissues studied except for liver, kidney, and small intestine, where some changes in the frequencies of certain types of mutation were observed in old mice (21, 22, 23) . The similarity in the spectra found in most of the mutations suggests that similar mutational stress is working in the aging process but at a different rate according to the tissue. On the other hand, differences in the mutation spectra suggest that the quality of the stress vary according to the tissue. Therefore, studying the age-dependent mutational stress in each tissue should be important. Here, we examined different parts of the digestive tract and found new types of mutational stress, one of which suggests the existence of a mutator phenotype based on an imbalance in the DNA precursor pool.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Young lacZ-transgenic Muta mice (24) were purchased from Covance Research Products (Denver, PA) and kept in our animal room until they reached 2 and 23 months of age. The mice were sacrificed by cervical dislocation, and the digestive tracts were removed. The esophagus, stomach, small intestine, and colon were separated and kept frozen at –70°C. We used mice that did not show any abnormality under macroscopic examination. The experiments were done according to the Guidelines for Animal Welfare and Experimentation of Tohoku University.

Isolation of Genomic DNA.
Frozen tissues were thawed on ice. A whole esophagus was used for DNA extraction because we could not isolate enough epithelial cells for the mutation assay. The stomach was opened by a pair of scissors, rinsed by PBS, and the inner surface was scraped off using a slide glass to remove the epithelial tissue. The tissues were then subjected to DNA extraction. The small intestine was cut into three sections of equal length, the proximal, medial, and distal parts. From each part, epithelial tissue was removed using a slight modification of the method of Bjerknes and Cheng (25) . Briefly, the tube was rinsed both inside and outside with CMF Hanks’ solution followed by EDTA/CMF Hanks’ solution. The tube was soaked in 5 ml of EDTA/CMF Hanks’ solution, kept at 37°C for 15 minutes, and the epithelial tissue was squeezed out from the tube with a pair of forceps. Epithelial tissue of the colon was separated in the same way as that used for the small intestine. DNA was extracted from the tissues by the use of phenol, as described previously (26) .

Mutation Assay.
Extracted mouse genomic DNA was mixed with packaging extract solution prepaired by ourselves or purchased from Stratagene (Transpack Packaging Extract, La Jolla, CA). The two packaging extracts showed indistinguishable results. The retrieved {lambda} phages were counted as plaques on E. coli (lacZ– and galE–). Phages containing mutant lacZ gene were identified as plaques formed in the presence of phenyl-galactoside. The mutants were additionally confirmed by the absence of digestibility of X-gal. The mutant frequency was determined as the number of mutants divided by the total number of phages. The details were reported previously (20) .

DNA Sequencing.
A single plaque was isolated from mutant phages, and DNA was extracted using phenol. The whole lacZ gene was amplified by PCR reaction as 6 overlapping fragments of ~600 bp, and all of the fragments were sequenced with an ABI Prism 377 or 3100 after labeling with fluorescent dye (Big-Dye Terminating Cycle Sequencing System, Applied Biosystems, Foster City, CA). The elucidated DNA sequences were compared with those of the wild-type lacZ gene. For multiple mutations, each mutation was confirmed by sequencing the opposite strand of DNA.

Statistical Analysis.
Mutant frequencies were examined by t test. The incidence of different kinds of mutation was analyzed by chi-square test or Fisher’s exact test. The frequency of multiple mutations was examined by proportion test. In all cases, P < 0.05 was regarded as statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mutant Frequencies in Young and Old Mice.
The mutant frequencies in each tissue of the digestive tract were determined for 3 or 4 mice at 2 and 23 months of age. The averages and the SDs are shown in Fig. 1Citation . The levels at 2 months of age were similar among the tissues examined. At 23 months, the levels increased by 2-fold in the esophagus, 3.5-fold in the stomach, 7-fold in the proximal and medial parts of the small intestine, 5.4-fold in the distal small intestine, and 6.9-fold in the colon. In all cases, the differences between the young and old mice were statistically significant.



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  		Fig. 1. Spontaneous mutant frequencies in different parts of the digestive tract at 2 months ({square}) and 23 months ({blacksquare}) of age. Except for the esophagus, epithelial tissues were examined. Means of three or four individuals are shown. Bars, ±SD.

 
Molecular Nature of Mutations.
About 20 mutant clones were separated randomly from each tissue sample, and the lacZ gene was sequenced. The tissues from three individual mice were analyzed. The total number of mutants analyzed and the number of mutations found in each tissue are shown in Table 1Citation . Among the mutations found, the redundant mutations, which showed identical alterations as one of the other mutations in the same part of the tissue from the same mouse in terms of the molecular nature, and the position of the mutation in the lacZ gene were eliminated from additional analysis, based on the assumption that these redundant mutations could be produced by copying pre-existing mutations through DNA replication and thus were not appropriate for the assessment of mutations created originally. The numbers of mutations after the elimination of the redundant mutations are listed as independent mutations in Table 1Citation .


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  		Table 1 The numbers of mutants examined and mutations found

 
Multiple Mutations.
Among the mutants, we found the clones showing two mutations at separate positions of a single lacZ gene. We classified these as multiple mutations and the others as single mutations. The frequencies of the multiple mutations were low in most parts of the digestive tract at young and old ages. However, it was elevated in the distal part of the small intestine and in the colon of old mice when compared with those of young mice (Table 1)Citation . The occurrence was 4 among 49 independent mutant clones in the old distal small intestine and 1 of 44 in the young tissue, whereas it was 4 of 43 in old colon and 0 of 39 in young colon. The frequencies in young and old mice were not different statistically; P = 0.216 in distal small intestine and P = 0.0706 in colon (Fisher’s exact test). However, when the events in the two tissues were combined, the difference between young and old became significant (1 case in 93 independent mutant clones in young tissues versus 8 cases in 92 clones in old tissues, P = 0.0165). This indicates that the frequency of multiple mutations becomes significantly higher in old age in the distal part of the digestive tract.

Although the frequencies were low, multiple mutations were found in other parts of the digestive tract. The molecular natures of these multiple mutations are summarized in Table 2Citation . All of the mutations were one base substitutions appearing twice at separate positions in the lacZ gene. The distance between the two mutations varied from 10 bp (DO1–2) to 2173 bp (CO1–19). When the changes of the bases were examined closely, 9 mutant clones among the 18 clones showed identical base substitutions at different positions in the lacZ (indicated in the last column of Table 2Citation ). The incidence rate (9 of 18) was higher than the expected rate of 1 of 16 (1/4 x 1/4), which is the probability for identical base substitutions at two positions (P < 10–5, proportion test). The locations in the lacZ gene where the multiple mutations were found were scattered along the gene (Table 2)Citation . Among them, a T to C base substitution at 2181 was found 6 times and a T to G base substitution was found 3 times at both 1904 and 2803. These might represent mutational "hot spots" for multiple mutations. They were not observed in single mutations in the digestive tract or in other tissues examined previously (20) . Analysis of the frequencies of the different kinds of mutations found among the multiple mutations showed a high frequency of base substitutions of A:T to G:C (P < 10–5) and A:T to C:G (P < 10–4) when compared with the mutations found among the single mutations (Fig. 2Citation ; see also Table 3Citation ; {chi}2 test). The frequencies of G:C to T:A and deletion were reduced in multiple mutations (P = 0.0122 and P = 0.0198, respectively, Fisher’s exact test). Thus, the multiple mutations seemed to have different characteristics from those found in single mutations.


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  		Table 2 List of multiple mutations

 


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  		Fig. 2. Comparison of mutation spectra of single ({square}) and multiple ({blacksquare}) mutations found in the digestive tract of young and old mice. Frequencies of different types of mutations are taken from Tables 2Citation and 3Citation . One case of a complex mutation observed in a single mutation was a change of C to GG at 843 found in old esophagus. *, statistically significant differences (see text).

 

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  		Table 3 Distribution of different kinds of mutations in different part of digestive tract

 
G:C to T:A Mutation.
The frequencies of the different kinds of mutations found among the independent mutants showing a single mutation in the lacZ gene are summarized in Table 3Citation . By comparing young and old tissues, we found that the frequency of the G:C to T:A transition was elevated in the proximal part of the small intestine. The incidence rates were 19 of 51 in old tissues and 5 of 34 in young tissues. The difference was statistically significant (P = 0.0200, Fisher’s exact test). A similar trend was also observed in the medial small intestine (Table 3)Citation .

Deletion Mutation.
The frequency of deletion mutations was elevated in old esophagus; 9 of 44 in old tissue and 2 of 38 in young tissue (P = 0.0429). Most of the mutations were 1-base deletions at regions of a single base repeat (Table 4)Citation . One was a deletion of 28 bp between the base positions of 573 and 600, which contained GC sequences at one terminal of the deleted fragment and also at the flanking sequence at the other side of the deleted fragment (mutant number EO 2.8 in Table 4Citation ). The other deletion was 8 bp long and did not show any repeated sequences at the terminal regions (EO 1–12).


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  		Table 4 List of deletion mutations in esophagus

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented show that different parts of the digestive tract suffer different degrees of mutational stress in the aging process. The mutant frequencies as well as the molecular nature of mutations were similar at 2 months of age in all parts of the tract examined. They were also similar to the spontaneous mutations found in other tissues such as the liver, spleen, and brain of newborn mice and mice at 2 months of age (19 , 24 , 27) . This suggests that the mutational burden shortly after birth and at a young age is similar among many somatic tissues. During old age, however, the mutant frequencies as well as the mutation spectra show distinct tissue-specificity. The levels of mutant frequency observed at old age were high in the small intestine and colon, intermediate in the stomach, and low in the esophagus. These levels did not correspond directly to those of the spontaneous tumor incidence rate of BALB/c and DBA2 mice, whose genetic backgrounds are shared by the Muta mice used in the present study (24) . BALB/c shows a low incidence of stomach cancer, and DBA2 shows a moderate rate of cancer in the small intestine (28 , 29) . However, no cancer is reported for esophagus and colon in these mice. Thus, it seems likely that the high levels of the mutation itself are not directly related to the spontaneous tumor incidence.

In esophagus, the level of the increase of the mutant frequency in old mice was similar to those observed in spleen and liver in our previous study (20) . The frequency of small deletion type mutations was elevated in old esophagus. An increase of deletion mutations was not found before in any tissue of old mice (19, 20, 21, 22, 23) . Because most of the deletion type mutations were observed at repeated sequences, slippage in DNA synthesis at the repeat and/or an insufficient mismatch repair system to correct the slippage could be related to the mutation formation (30 , 31) . It should be remembered that the tissue we examined was a whole esophagus. Thus, the results do not necessarily represent the events specific in the epithelial tissue.

In stomach epithelium, the mutant frequency increased by about 3.5-fold in the old mice, which was higher than that in the esophagus. The spectrum of mutations did not show any noticeable difference between the young and old mice. This might suggest that the mutational stress working in the aging process after adulthood is similar to that working in the fetal and postnatal growth phases.

The epithelial tissues in the three parts of the small intestine and that in the colon showed the highest rate of increase in the old mice. The rates were higher than those of any other tissues examined thus far (19, 20, 21, 22, 23) . The mutation spectra showed that the frequency of G:C to T:A transversions was higher in the proximal small intestine of old mice than in that of the young mice. A similar trend was observed in the medial part of the small intestine. This type of mutation has been shown to be induced by reactive oxygen (32) and chemical carcinogens (33) . Because the old mice we examined were not exposed to carcinogens, reactive oxygen could account for the mutation. It is noteworthy that bile acids have been reported to create reactive oxygen species (34) .

Mutations in the small intestine of old mice have been reported previously by Dollé et al. (21) . They found that a 2.5-fold increase in the mutant frequency occurred during the period of from 3 months to 25 months of age. The sequencing of the mutants revealed an elevation of all kinds of base substitutions except a G:C to A:T change at the CpG sequence (21) . These results are slightly different from ours. It could be based on the difference in the mouse strains used, differences in the environment in which the mice were kept, and/or the tissues examined. We used epithelial tissue, whereas Dollé et al. (21) used whole small intestine.

In the distal part of the small intestine and in the colon, the frequency of multiple mutations was elevated in old mice. The multiple mutation is interesting because it has been predicted to be a typical type of mutation created under a mutator phenotype (13) . When the mutations found in the distal part of the small intestine and in the colon are summarized, the frequencies of single mutations are shown to be 7 x 10–5{[(43 + 39)/(51 + 60)] x 9.48 x 10–5}; the ratio of the number of single mutations to the total number of mutant clones is multiplied by the average of mutant frequencies} for young mice and 41 x 10–5{[(45 + 39)/(60 + 59)] x 58.04 x 10–5} for old mice. The averages of the mutant frequencies of the two tissues at the same age were taken because they showed similar levels (Fig. 1Citation ; Table 1Citation ). If the multiple mutation is only the result of a coincidence of two single mutation events, the expected frequencies would be calculated by the squares of the single mutation frequencies, 4.9 x 10–9 and 1.7 x 10–7 for young and old mice, respectively. On the other hand, the observed frequencies of multiple mutations were 8.54 x 10–7 [1 of 111 (51 + 60) sequenced mutants among the mutants appeared at a ratio of 9.48 x 10–5] and 3.90 x 10–5 [8 of 119 (60 + 59) sequenced mutants among the mutants appeared at a rate of 58.04 x 10–5] in the young and old mice, respectively. These values are higher than those estimated from the calculated values by two orders of magnitude. The multiple mutations found in the other tissues of the digestive tract showed similarly high values (Table 1Citation ; Fig. 1Citation ). Thus, it is likely that the multiple mutations could not be explained merely by the coincidence of two single mutations. The characteristics of these mutations were different from those of single mutations in terms of the spectra and the positions of probable hot spot mutations. It appeared that some unique mechanism is working to create such mutations. In 1991, Harwood et al. (35) found 4 cases of multiple mutations in the APRT gene of a cultured human colorectal carcinoma cell line. Possible mechanisms for the creation of these multiple mutations were speculated to be an imbalance in the deoxyribonucleotide triphosphate pool or mistakes in long patch repair such as mismatch repair (35 , 36) . The high incidence of identical base substitutions in a single gene (Table 2)Citation might be explained by an imbalance of one kind of deoxyribonucleotide triphosphate pool during DNA replication. Phear and Meuth (37) reported that the cultured Chinese hamster ovary cells suffering excess dCTP pool showed an elevation of A:T to C:G and A:T to G:C base substitutions. This corresponds well to the mutation spectrum of the multiple mutations observed in the digestive tract (Fig. 2)Citation . Hence, it is possible that the digestive tract suffers mutational stress based on excess dCTP, and the stress increases with age in the distal part of digestive tract. If this is the case, controlling the balance of the nucleotide pool could be one way to prevent the age-related accumulation of mutations in the digestive tract. Additionally, if the multiple mutations observed took place in one replication cycle of DNA because of excess dCTP, the frequency of mutations per cell could be tremendous, because lacZ is only 3 kbp long and the genome is 3 x 106 kbp. Two mutations in 3 kbp correspond to 2 x 106 mutations per genome or 4 x 106 per cell. On the other hand, the frequencies of A:T to G:C and A:T to C:G base substitution in the single mutations are not high (Table 3)Citation . Because the excess dCTP pool size will affect the single mutations as well as the multiple mutations, the data do not seem to support the pool size hypothesis. The other possibility would be that the multiple mutations are derived by an instability in DNA polymerization apparatus especially in long patch repair. In any rate, these hypotheses will require additional study.

Overall, the present data would provide a new clue to understand a relationship between aging and cancer from a viewpoint of genomic instability.


   ACKNOWLEDGMENTS
 
We thank Yasuko Syono, Yukiko Ikeda, and Hiroyuki Tamogami for technical assistance, Sakiko Kikuchi for preparation of the manuscript, Brent Bell for his editorial help, and Peter M. Glazer for providing the E. coli NM759 and BHB2688, which were needed for preparation of the packaging extract.


   FOOTNOTES
 
Grant support: Grants-in-Aid from Japan Society for Promotion of Sciences and Yakult Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Tetsuya Ono, Division of Genome and Radiation Biology, Graduate School of Medicine, Tohoku University, Seiryo-machi, Sendai 980-8575, Japan. Phone: 81-22-717-8131; Fax: 81-22-717-8136; E-mail: tono{at}mail.tains.tohoku.ac.jp

Received 4/26/04. Revised 6/23/04. Accepted 7/19/04.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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