Cancer of the large intestine is classified into colon and rectal cancers. The incidence of colon cancer is increasing at a faster rate than that of rectal cancer in recent years in advanced countries, including Japan. Colon cancers develop after the multistep accumulation of genetic and epigenetic induction of oncogenes in both humans and experimental animals (9,14,19).
The proposed multisteps of colon carcinogenesis may start when aberrant crypt foci (ACF) appear in the colon (37). ACF were defined as lesions composed of enlarged crypts, slightly elevated above the surrounding mucosa and more densely stained with methylene blue than normal crypts (3). ACF are considered to be putative preneoplastic colon lesions that may be early indicators of colon carcinogenesis (4,10,18).
Epidemiological evidence has suggested that physical activity has a protective effect on colon cancer incidence (11,13,30). However, few experimental studies have been conducted to elucidate the mechanisms of exercise-related effects on colon cancer. For example, a few earlier animal studies found that both voluntary (1,26) and treadmill (36) running training reduced tumor incidence after the administration of 1,2-dimethylhydrazine (DMH) or azoxymethane. Furthermore, no studies have examined the effects of physical exercise training on colon cancer as they might be related to the multistep nature of colon carcinogenesis, although Demarzo et al. (7) recently reported that a single session of exhaustive exercise increased the number of ACF DMH-induced rat colons. Up to now, there is no study demonstrating that exercise training affects the number of ACF, which is a putative initial step of colon carcinogenesis of rats. Therefore, we investigated the effects of running exercise training on the number of DMH-induced ACF, because previous studies suggested that physical training of this type has a protective effect on colon tumor incidence in rats.
MATERIALS AND METHODS
All chemicals, including 1,2-dimethylhydrazine (DMH), a carcinogenic chemical of the colon, was purchased from SIGMA (St. Louis, MO).
All experimentation was conducted in accordance with policy statement of the American College of Sports Medicine on research with experimental animals and was approved by the animal care and use committee of National Institute of Health and Nutrition. Four-week-old Fischer 344 male rats were purchased from CLEA Japan, Tokyo. The animals were housed in rooms lighted from 7 a.m. to 7 p.m. and were maintained on an ad libitum diet of standard chow and water. The room temperature was maintained at 20-22°C.
After 1 wk of acclimatization to the housing environment (5 wk of age), the rats were randomly assigned to one of two groups: the treadmill-running training group (N = 19) or the control group (N = 19). All rats were given a subcutaneous injection of DMH at a dose level of 20 mg·kg−1 body weight, once a week for 2 wk. The DMH was dissolved in 0.1 mM EDTA (pH 6.5) immediately before the administration.
One week after the last injection of DMH (i.e., at age 7 wk), running training was started. The training rats ran for 120 min·d−1 (two 60-min bouts separated by 10 min of rest) on a flat motorized treadmill (Natsume, Tokyo, Japan). On the first day, the rats were accustomed to running at a speed of 10 m·min−1 by gradually increasing the treadmill speed to the fixed speed. The running speed was maintained for the following 4 wk of training. The intensity of this training was considered to be low because this exercise could be continued for more than 6 h without exhaustion, as reported elsewhere (34).
Two or three days after the last bout of exercise, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg·kg−1 body weight), and the heart and soleus muscles were excised, weighed, quickly clamp-frozen in liquid nitrogen, and stored at −80°C until analysis. Then, the colons were dissected and gently flushed with 10% neutralized formalin to remove residual bowel contents, cut open longitudinally, fixed flat between filter papers, and submerged in 10% neutralized formalin overnight at 4°C (23). Peritoneal fat, epididymides fat, and brown adipose tissue (BAT) were excised and weighed.
Detection of ACF.
Fixed colons were stained with 0.2% methylene blue, as described by Bird (3). The number of ACF and total number of aberrant crypts (AC) comprising ACF were counted for each colon. The ratio of total AC/ACF was calculated to assess ACF multiplicity. As shown in Figure 1, ACF were identified as lesions composed of enlarged crypts, with an increased pericryptal area, a slightly elevated appearance above the surrounding mucosa with an oval or slitlike orifice, and a higher staining intensity with 0.2% methylene blue than normal crypts (3).
Citrate synthase activity in skeletal muscle.
Ten percent homogenates were made from the muscle in 175 mM KCl, 10 mM glutathione, and 2 mM EDTA, pH 7.4. The homogenate was frozen and thawed four times and mixed thoroughly before enzymatic measurements. As a maker of oxidative enzyme, citrate synthase (CS) activity in the soleus muscle was measured using Srere's method (31).
All data are shown as the mean ± SE. Quantitative clinical data were compared between run-trained rats and controls by use of the unpaired t-test. ACF-related data were also compared by use of the Mann-Whitney rank sum test because the numbers of ACF were not normally distributed. These data were analyzed by use of SigmaStat for Windows (SPSS Inc., Chicago, IL). Differences were considered significant when the P value was less than 0.05.
Physical characteristics and citrate synthase activity.
The body weight of the training rats was significantly less than that of the control rats (P < 0.01) (Table 1). The weight of heart and soleus, expressed relative to body weight, was significantly heavier in the training group compared with the control group (P < 0.05 and P < 0.001, respectively), whereas the relative adipose tissue weight of the peritoneum and epididymides was significantly lighter in the training group compared with the control group (P < 0.001 and P < 0.05, respectively). The relative BAT weight of the training rats tended to be lower than that of the control group (P = 0.07). CS activity in the soleus muscle of the training group was significantly higher than in the control group (P < 0.05).
Induction of ACF and AC.
As shown in Figure 2 upper panel, the number of ACF of training-group rats was significantly less than that observed in the control group (P < 0.05). The number of total AC was also significantly less in the training group than in the control group (P < 0.05; Fig. 2 lower panel). As shown in Figure 3, the occurrences of one, three, and five aberrant crypts per focus were significantly smaller in the training group than in the control group (P < 0.05). Furthermore, running training decreased the number of not only small ACF, which consists of less than or equal to three AC (P < 0.05), but also large ACF (≥ 4 AC) (P < 0.05), as compared with the control group (Fig. 4). However, the ratio of total AC/ACF, which indicates the average size of induced ACF, did not significantly differ between the training and control groups (2.9 ± 0.2 vs 2.9 ± 0.7, P > 0.10).
The main finding of the present study was that short-term, low-intensity running training reduced DMH-induced ACF production in the rat colon.
Colon carcinogenesis is well known to be a multistep process involving multiple genetic alterations (15,37,38). In humans, mutation of adenomatous polyposis coli (APC) gene is regarded as the initial event in ACF, followed by additional mutation of K-ras gene in adenomas; further mutation of the p53 gene is the progressive event in carcinomas (14,33). In rodents, β-catenin mutations are frequently observed in colon tumors and in dysplastic ACF induced by azoxymethane (32). APC and/or K-ras mutations are also occasionally observed in rodents, as they are in humans (32), and ACF has been considered a very initial lesion in a multistep colorectal tumorigenesis model (14). After ACF were first described in animals, similar lesions were found in humans (28). Because previous studies have suggested that low-intensity, long-term treadmill-running training prevented the incidence or development of colon tumors in a rat model injected subcutaneously with azoxymethane (36), it is reasonable to speculate that exercise training may exert its effect on one or more steps in colon carcinogenesis. To date, however, it remains unknown at which step of the carcinogenic process (e.g., ACF, adenomas (early, intermediate, and late), or carcinomas) physical exercise training exerts its preventive effect in rodents injected with carcinogens. The present results suggest that training in rats suppressed the first step, the initiation of ACF development in the rat colon. This is the first observation regarding the effect of physical exercise on ACF, the development of which is considered the first step in colon carcinogenesis. Furthermore, Colbert et al. (5), using the APCMin mouse model, reported a trend toward fewer polyps in the colon in treadmill-exercised males compared with nonexercised mice. From the present investigation, it is obvious that the earlier phase of colon carcinogenesis is inhibited by exercise training. Therefore, it is of great importance when considering efficient chemopreventive effects on cancer development. Several hypotheses have been suggested to explain the preventive effects of exercise/physical activity on colon carcinogenesis-for example, shortened gastrointestinal transit time as a result of exercise (6); energy balance (16); reduced levels of blood insulin, which might be a growth factor for colon cancer cells (12); enhanced immune activity-related NK cells (20); enhanced free-radical scavenger system (8); changed prostaglandin levels (17); and decreased obesity (25). However, mechanisms related to the exercise-induced decrease in AC and/or ACF are not known at all. Therefore, only a few hypotheses can be raised. First, as Lasko and Bird et al. (16) have reported that caloric restriction-induced weight loss suppressed the increase in the number of ACF after the injection of azoxymethane in rats, it may be possible that energy balance (29), including energy expenditure and reduced food intake (24) or reduced nonexercise activity level (35) by exercise training, may exert a suppressive effect similar to that of caloric restriction, inhibiting the initiation or proliferation of ACF on the colonic mucosa. In fact, the results of the present study indicate that the body weight and/or adipose tissue weight of the peritoneum and epididymides were significantly lower in the running group than in the control group. Therefore, it is plausible that body- or fat weight loss yielded by physical exercise may reduce the initiation of ACF. Another plausible mechanism is that exercise might exhibit its preventive effects on mutation induction in the APC, K-ras, and/or p53 genes through the induction of some detoxification enzymes for oxidative stresses. A third possibility is the commitment of moderate levels of physical exercise on the improvement of lipid metabolism. High fat levels in serum and low expression levels of lipid metabolism-related genes such as lipoprotein lipase in the liver and colon are now considered to have some significant impact on the development of intestinal tumors in the APCMin mouse model (21,22). Further studies are expected to investigate the molecular mechanisms underlying exercise-induced effects on AC/ACF formation.
Recently, Demarzo and Garcia (7) reported that a single bout of exhaustive swimming exercise with a 2% weight tied to the tail was related to an elevated number of colonic ACF in untrained rats treated with DMH, when compared with a control group. Because this report included no description of the exercise protocol, such as a period of acclimatization that is usually given before acute bouts of exercise (27), it is not known how much "stress" was imposed on the exercised rats in the study. Therefore, we could not discuss the different effects of ACF production between the two studies in terms of exercise training-induced stress. On the other hand, the intensity of the swimming exercise with a 2% weight tied to the tail might be higher than that of the running training adopted in the present investigation. Thus, the exercise intensity may be a key factor determining the number of ACF after exercise. In fact, unaccustomed exhaustive and/or high-intensity exercise increases systemic free-radical generation in experimental animals (2). On the other hand, in the present study, we showed that low-intensity physical exercise for 2 h may decrease the development of colonic ACF in experimental animals. Furthermore, stress related to exercise at times during which the rats normally sleep may influence the ACF number in the colon. Therefore, voluntary exercise during the night cycle may be a better alternative exercise protocol than the "forced daytime" treadmill running adopted in the present investigation. However, because the number of ACF in the trained rats in the present study was actually lower than in the nonexercise control group, the overall effects of treadmill running on ACF production are favorable. Therefore, the benefits of the exercise training adopted in the present study are thought to outweigh the disadvantages of exercise-related stress. A future study using voluntary exercise should be conducted to clarify this issue.
The number of AC with a specific number of ACF (1,3,5) in the trained rats was lower than in the control group in the present investigation. However, running training did not affect the overall mean AC/ACF ratios of the rat colon induced by DMH. Thus, it is suggested that the physical exercise training adopted in the present investigation may not be effective for preventing the proliferation of ACF in rat colonic mucosa.
In conclusion, the results of the present investigation demonstrated that low-intensity running training inhibits the initiation of ACF in the rat colon induced by DMH. Furthermore, the present investigation suggests that increasing physical activity might be effective for primary prevention of colon cancer incidence, not only for rats but also for humans, by affecting the first step of cancer induction. However, the clinical implications and pathophysiological mechanisms of these findings warrant further investigation.
This work was supported in part by the Grants-in-Aid for Scientific Research on Exploratory Areas (16650160 to I.T.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the Sasakawa Scientific Research Grant (to N.F.) from The Japan Science Society. The present addresses of Drs. N. Fuku and S. Terada are Division of Genomics for Longevity and Health, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; and Washington University School of Medicine, St. Louis, MO, respectively.
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