Long-term aerobic swimming training by rats reduces the number of aberrant crypt foci in 1 , 2-dimethylhydrazine-induced colon cancer

We determined the effect of long-term aerobic swimming training regimens of different intensities on colonic carcinogenesis in rats. Male Wistar rats (11 weeks old) were given 4 subcutaneous injections (40 mg/kg body weight each) of 1,2-dimethylhydrazine (DMH, dissolved in 0.9% NaCl containing 1.5% EDTA, pH 6.5), at 3-day intervals and divided into three exercise groups that swam with 0% body weight (EG1, N = 11), 2% body weight (EG2, N = 11), and 4% body weight of load (EG3, N = 10), 20 min/day, 5 days/week for 35 weeks, and one sedentary control group (CG, N = 10). At sacrifice, the colon was removed and counted for tumors and aberrant crypt foci. Tumor size was measured and intra-abdominal fat was weighed. The mean number of aberrant crypt foci was reduced only for EG2 compared to CG (26.21 ± 2.99 vs 36.40 ± 1.53 crypts; P < 0.05). Tumor incidence was not significantly different among groups (CG: 90%; EG1: 72.7%; EG2: 90%; EG3: 80%). Swimming training did not affect either tumor multiplicity (CG: 2.30 ± 0.58; EG1: 2.09 ± 0.44; EG2: 1.27 ± 0.19; EG3: 1.50 ± 0.48 tumors) or size (CG: 1.78 ± 0.24; EG1: 1.81 ± 0.14; EG2: 1.55 ± 0.21; EG3: 2.17 ± 0.22 cm3). Intra-abdominal fat was not significantly different among groups (CG: 10.54 ± 2.73; EG1: 6.12 ± 1.15; EG2: 7.85 ± 1.24; EG3: 5.11 ± 0.74 g). Aerobic swimming training with 2% body weight of load protected against the DMH-induced preneoplastic colon lesions, but not against tumor development in the rat.


Introduction
The process of carcinogenesis can be divided into three major stages: initiation, promotion and progression.Aberrant crypt foci (ACF) are considered to be putative preneoplastic colon lesions that may be early indicators of colon carcinogenesis (1).
Descriptive studies have suggested that an inverse association exists between regular physical activity and the risk of developing colon cancer (2).However, such studies suffer from some methodological limitations such as lack or absence of standardization to assess the frequency, duration and intensity of the activity.Experimental studies on the effects of exercise training on colon cancer, however, have reported controversial results.For example, animal studies have shown that both long-term voluntary (3) and low-intensity treadmill (4) running trainings reduced tumor incidence in rats injected with 1,2-dimethylhydrazine (DMH), although, other interventional studies have failed to show the protective effects of short-term moderate-intensity treadmill running training against tumor development in mice with genetic predisposition (APC min mouse) (5,6).Recently, Fuku et al. (7) found that short-term low-intensity treadmill running training reduced the number of DMH-induced ACF in rats.
The positive association of the intensity of physical activity and protection against carcinogenesis has also been suggested (8).However, depending on its intensity exercise may have either harmful or beneficial effects concerning colon carcinogenesis (9).For example, exercise may cause immune function depression or improvement (10), increase in free radical production (11) or improvement in the antioxidant enzymatic defense system (12).Thus, exercise may protect against or promote colorectal carcinogenesis depending on its intensity.For instance, Demarzo and Garcia (13) showed that a single session of exhaustive swimming increased the number of DMH-induced ACF in rat colons.
To date, no experimental study has assessed the effects of long-term aerobic exercise training of different intensities on colon cancer and which step of the carcinogenic process physical exercise training exerts its preventive effects in rodents injected with carcinogens remains unknown.Our aim was therefore to verify the effects of long-term aerobic swimming training regimens of different intensities on the colonic carcinogenic process in rats treated with DMH.

Material and Methods
Eleven-week-old male Wistar rats acquired from the Central Animal House at the Federal University of Viçosa, MG, were maintained in individual cages in a temperaturecontrolled room at 22 ± 2°C with lights on from 7:00 am to 7:00 pm.Animals received standard chow and water ad libitum.All experimentation was conducted in accordance with internationally accepted ethical principles concerning the care and use of laboratory animals and was approved by the animal care and use committee of the Federal University of Viçosa, MG, Brazil.
The animals were randomly divided into 3 exercise groups of 12 animals each (EG1, EG2, EG3) and a control group (CG, N = 10).During the first 2 weeks of the experiment, all rats were given 4 subcutaneous injections of 40 mg/kg body weight DMH (Sigma, USA), two injections in the 1st week, and two in the 2nd week, on nonconsecutive days.DMH was prepared immediately before use, dis-solved in 0.9% NaCl containing 1.5% EDTA as vehicle at a final concentration of 10 mg/mL, and final pH was adjusted to 6.5.
The exercise program was carried out in a tank that was 100 cm high, filled to a depth of 45 cm with water, maintained at 28-30°C.All animals were prevented from floating and made to swim continuously.The rats in the CG were kept in a tank with water (10 cm deep) for the same amount of time as that of the exercised groups.One animal from EG1, 1 from EG2, and 2 from EG3 drowned while exercising.
Twenty-four hours after the first injection of DMH, animals of the 3 exercise groups started exercising.During the first 2 weeks they exercised without additional load, 5 to 15 min/day, 5 days/week.In order to maintain the animals swimming aerobically at different intensities below their anaerobic threshold (4.95% body weight, 7.17 mmol/L) (14), different loads attached to the animal body were used as follows: EG1, 0% body weight (intensity 1), EG2, 2% body weight (intensity 2), and EG3, 4% body weight (intensity 3).From weeks 3 to 5, the animals swam with their respective loads, 5 to 20 min/day, 5 days/week.From week 6 to 35, the animals swam with their respective loads, 20 min/day, 5 days/week.
To avoid any interference of the acute effects of exercise, 72 h after the last exercise session rats were sacrificed and the large intestine was removed, opened longitudinally, washed gently, and fixed flat on a Styrofoam board in 10% buffered formaldehyde for 48 h.Intra-abdominal fat was excised and weighed.
The intestine was then divided into three fragments of equal length: proximal, mid-and distal regions, which were stained with 0.1% methylene blue for approximately 2 min and washed in phosphate buffer.The ACF were identified and counted under a light microscope (100X) by two pathologists unaware of the treatment (15).The ACF were grouped as a function of the size of the foci: small ACF (aberrant crypts per focus ≤3, ACF ≤3), and large ACF (aberrant crypts per focus ≥4, ACF ≥4) (15).The tumors were classified visually and counted.The heights, as well as the smallest and largest diameters of the tumors, were measured to estimate the tumor size.
Data for body weight gain, mean number of ACF, ACF ≤3, tumor multiplicity and size were compared by one-way ANOVA followed by the Tukey test when main effects or interaction were observed.Data for intra-abdominal fat and ACF ≥4 were compared by the Kruskal-Wallis test because data were not normally distributed.Data for tumor incidence were analyzed by the chi-square test.All data were analyzed with Sigma Stat for Windows (SPSS Inc., USA).Differences were considered to be significant at P ≤ 0.05.

Discussion
These are the first observations regarding the effects of different aerobic swimming training intensities on distinct steps of colon carcinogenesis.Our data showed lower values of total ACF number, mean ACF number and number of ACF ≤3 only in animals of EG2 that exercised at intensity 2 for 35 weeks when compared with non-exercised animals (CG).These effects were not observed in rats swimming with either 0% body weight (EG1) or 4% body weight of load (EG3).The EG2 rats also presented better indices for potency, relative risk, ACF reduction percentage, and reduction efficiency in ACF size, compared with EG1 and EG3 animals.These findings suggest that long-term aerobic swimming training protected against the initiation of ACF development in the rat colon and emphasize that there is an optimal exercise intensity to reach this protection, even if exercising below the anaero-Table 1. Table 1.Table 1.Table 1 Data are reported as means ± SEM.Rats received four subcutaneous injections of 1,2-dimethylhydrazine (40 mg/kg body weight each) twice a week, on nonconsecutive days, for 2 weeks and were submitted to exercise intensity 1 (EG1), intensity 2 (EG2), and intensity 3 (EG3) for 35 weeks or were sedentary controls (CG).N = number of animals.*P < 0.05 compared to CG; + P < 0.05 compared to proximal region (ANOVA followed by the Tukey test).Table 2. Table 2. Table 2. Table 2 7) demonstrated a reduced number of DMH-induced ACF in rats submitted to a daily twohour low-intensity treadmill running for 4 weeks (shortterm).The present study differs from theirs in terms of exercise model and duration (35 weeks), although similar effects of exercise on ACF reduction were observed in both studies.Thus, it seems that exercise intensity is important regarding ACF development.In fact, it was demonstrated that long-term moderate-to-vigorous intensity daily exercise of 60 min by men resulted in an increased expression of Bax at the bottom of colon crypts (16).This

Table 1 .
. Effect of swimming training on aberrant crypt foci (ACF) in regions of the large intestine of rats treated with 1,2-dimethylhydrazine.

Table 2 .
. Effect of swimming training on the potency, relative risk, aberrant crypt foci reduction percentage, and reduction efficiency in the aberrant crypt foci size in the large intestine of rats treated with 1,2-dimethylhydrazine.sedentary controls (CG).P = potency (mean ACF of the CG/mean ACF of the respective exercise group); RR = relative risk (1/P); %R = % aberrant crypt foci reduction (100 -100 / P); PRT = reduction efficiency in the aberrant crypt foci size (mean ACF >3 of the CG/mean ACF >3 of the respective exercise group).