Effect of probiotics on the development of dimethylhydrazine-induced preneoplastic lesions in the mice colon 1

PURPOSE: To determine the effect of probiotics on the development of chemically induced (1, 2-dimethylhydrazine) colonic preneoplastic lesions, in mice. METHODS: The animals were divided into five groups. The control group was injected with carcinogen alone and the other groups also received probiotics (1Lactobacillus delbrueckii UFV-H2b20; 2Bifidobacterium animalis var. lactis Bb12; 3L. delbrueckii UFVH2b20 plus B. animalis var. lactis Bb12; and 4Saccharomyces boulardii) administered orally in drinking water throughout fourteen weeks. RESULTS: Consumption of lactobacilli and bifidobacteria alone resulted in a significant reduction of the total number of aberrant crypt foci (55.7% and 45.1%, respectively). Significant reduction in the number of these small foci (≤3 aberrant crypts) was only observed in the group treated with lactobacilli (52.2%) in comparison to control group. The number of larger foci (>3 aberrant crypts) crypts had no significant reduction. CONCLUSION: L. delbrueckii UFV-H2b20 and B. animalis var. lactis Bb12 administered alone protect colonic preneoplastic lesions in mice, while the combined treatment of these bacteria and the administration of S.boulardii were not effective in reducing such colonic lesions.


Introduction
The incidence of cancer is increasing worldwide, and this trend is expected to continue.New cases of cancer are estimated to rise from 11.3 million in 2007 to 15.5 million in 2030.The number of global cancer deaths is projected to increase 45% from 2007 to 2030 (from 7.9 million to 11.5 million deaths), influenced, in part, by an increase in the global elderly population.One of the most frequent types of cancer is colorectal cancer, which has contributed significantly to increased mortality.Thus, the existence of evidence-based strategies for early detection, treatment, and especially for prevention of the disease is extremely important as approximately 40% of all cancer deaths can be prevented 1 .Hence, it is extremely important to implement strategies to prevent new cases of cancer.
It has been observed that the general population of intestinal bacteria is associated with initiation of colon cancer 2 .This has led to interest in factors that can modulate the gut microbiota and, consequently, prevent the disease.Of special interest is the beneficial effect of certain probiotics, defined as live microorganisms that, when administered in adequate amounts confer health benefit to the host 3 .Some studies indicate that the ingestion of certain microorganisms reduces both the risk of developing certain types of cancer and tumor growth [4][5][6][7][8] .Attention One week after the beginning of probiotic treatment, the mice were injected sub-cutaneously with the carcinogen 1,2-dimethylhydrazine (Sigma, Saint Louis, USA) (25 mg/kg, 37 mg/100 mL EDTA, s.c.) once a week, throughout six weeks.
Probiotic supplementation was administered continually throughout the entire experiment, except on the first day that DMH was administered.This is because in our pilot study, mice treated with lactobacilli, bifidobacteria, and the mixture of lactobacilli and bifidobacteria presented with 40%, 30%, and 60% mortality, respectively, between 48h and 72h after the first injection of DMH.And this was related to increased bacterial translocation to mesenteric lymph nodes, spleen, and liver observed in mice injected with the carcinogen 9 .
On the fourteenth week of the experimental period, the mice were euthanized by cervical dislocation, under sedation, and the entire colon was collected for aberrant crypt analysis.The experimental design for this study is show in Figure 1A.The probiotics were administered orally in the water given every day at 3x10 8 c.f.u mL -1 , except for Lac/Bif group that received 3x10 8 c.f.u mL -1 of each bacteria.All groups where allowed to consume water (with probiotics for the Lac, Bif, Lac/ Bif and Sb groups and without probiotics for the Cont group), as explained below and commercial chow (Labina, Brazil) ad libitum.The mice were housed in plastic cages with wood chips for bedding in a room with a 12h light/dark cycle.The study was approved by the Ethics Committee for Animal Experimentation

Effect of probiotics on the development of dimethylhydrazine-induced preneoplastic lesions in the mice colon
of the Universidade Federal de Minas Gerais (protocol number 189/07) and the institutional guidelines for the care and use of laboratory animals was followed.

Microorganisms and preparation of water
Lactobacillus delbrueckii UFV-H2b20 was isolated from the feces of a newborn at the Viçosa Federal University (Viçosa-MG, Brazil); Bifidobacterium animalis var.lactis Bb12 was provided by the Christian Hansen Lab (Horsholm, Denmark); and Saccharomyces boulardii was provided by Merck S.A. (Rio de Janeiro, Brazil).
For the preparation of water, L. delbrueckii UFV-H2b20 and B. animalis var.lactis Bb12 were cultured for 24 hours at 37ºC in MRS broth (Difco, Sparks, USA); S. boulardii were cultured in YPG broth (1% yeast extract, 2% peptone, 3% glycerol) for 48 hours at 37ºC.Bifidobacteria were incubated in an anaerobic chamber (Forma Scientific Company, Marietta, USA; containing an atmosphere of 85% N 2 , 10% H 2 and 5% CO 2 ).After cultivation, the culture was centrifuged at 5,000 rpm for 15 minutes, and the supernatants were discarded.The cells were washed with 5mL of water and centrifuged under the same conditions; then, the supernatants were discarded.The cells were resuspended in 100mL of drinking water placed in each box with 10 animals.
Fresh cultures were prepared daily and the water changed every 24h when found to be about 10 8 c.f.u mL -1 .

Aberrant Crypt Foci analysis
At the end of the 14 week experiment, after euthanasia, the colons were immediately removed from the cecum down to the anus, rinsed in saline to remove the faeces, slit longitudinally and opened on paraffin plates with the mucosa facing up and fixed for 24 hours in 10% formaldehyde.Next, the sample was measured and divided in three equal fragments referred to as proximal, medial and distal in relation to the cecum, stained with 1% methylene blue for 2 minutes and rinsed with a phosphate buffer (PBS), in order to identify the foci of aberrant crypts.The foci throughout the entire mucosal surface of the large bowel were blindly counted by two independent and trained observers using a light microscope at 40x magnification according to the technique used by Bird 10 (Figure 1B).Because there was no significant difference between the evaluations of the two observers (p >0.05) (data not shown), the average of two counts was used for the analysis.The total number of aberrant crypt foci (ACF), its distribution along the colon and the multiplicity of the crypts were analyzed.The distribution among the proximal, middle and distal colon was evaluated, and multiplicity was determined by counting the foci containing three or fewer aberrant crypts and foci with more than three aberrant.

Statistical analysis
Data were analyzed using the SigmaPlot (Version 11.0).
Student's t-test was utilized to compare the counts of two observers and for further analysis of aberrant crypts between groups treated with probiotics and the Cont group.Analysis of ACF in the proximal, middle and distal regions of mouse colon was carried out using ANOVA followed by Holm-Sidak test.The differences were considered significant at p<0.05.

Results
There were no significant differences in the initial and after 14 weeks body weight and food intake among the experimental groups (data not shown).
All mice developed ACF in the colon (Figure 1C).Data on the total number of ACF, multiplicity of aberrant crypts and the distribution of ACF in the colon after 14 weeks of treatment in the different groups are summarized in Table 1.The most potent protective effect against large ACF was observed in the Bif group, in which the number of foci with more than three aberrant crypts was decreased by 74% (0.6 ± 0.

Discussion
In the present study, we demonstrate that consumption of Lactobacillus delbrueckii UFV-H2B20 and Bifidobacterium animalis var.lactis Bb12 resulted in significant inhibition of DMH-

Effect of probiotics on the development of dimethylhydrazine-induced preneoplastic lesions in the mice colon
Acta Cirúrgica Brasileira -Vol.28 (5) 2013 -371 induced ACF formation in mice, indicating a chemopreventive efficacy of these probiotics against colonic tumorigenesis, a shortterm effect and without any noticeable adverse effects.Although the mechanism of inhibition of colon cancer by probiotics is not clear, we believe that this effect may proceed through diverse mechanisms, including alteration of intestinal microbiota; estimulation of immune response and inactivation of carcinogenic compounds.
Other studies in animal models of colon cancer have also shown that the consumption of some probiotics could inhibit preneoplastic lesions and tumor development.An important reduction was observed in azoxymethane-induced ACF and colon crypt multiplicity after animals were fed B. longum 11 .Bolognani et al. 7 observed a significant decrease in azoxymethaneinduced colonic ACF in rats given L. acidophilus.A symbiotic combination was also demonstrated to have a beneficial effect, observed by the reduction in ACF with the use of inulin and B. longum 4 or of oligofructose and bifidobacteria 5 .In another study, the combined administration of B. lactis and L. rhamnosus with inulin and oligofructose in rats was able to reduce the occurrence of malignant tumors induced by the carcinogen azoxymethane 8 .
We also used a mixture containing 10 8 CFU/mL of each bacteria (L.delbrueckii UFV-H2B20 plus B. animalis var.lactis Bb12) to verify whether feeding mice a large amount of a combination of bacterial species would exert better effects than the use of a single species or the use of a small amount.However, we were not able to demonstrate the positive effects of such a combination.It was not possible to determine the reason why the combination of probiotics showed no positive result, but it's our impression that this may be due to the used dose that may have caused a derangement in the intestinal microbiota or in the immune system.
Aberrant crypt foci, that are enlarged and elevated relative to normal crypts, have also been detected in both rodents and humans and can be recognized as early neoplastic lesions.
Furthermore, crypt multiplicity (number of crypts per focus) has been considered to be one of the consistent predictors of tumor incidence.It has been suggested that large ACF are more predictive of tumor formation than small ACF 15 .In the present study, we observed that small ACF were predominant in all groups.The formation of small ACF was mostly inhibited in the Lac group, while the most potent inhibition of large ACF was observed in the Bif group, when both were compared to the Cont group.However, the reduction seen in the Bif group was not significant, probably due to the high standard deviation.
In addition to crypt multiplicity, the location of ACF is also an interesting feature.In this study, the majority of ACF was present in the medial and distal colon in the Cont/DMH group.
According to the literature, in both human and animal colons, ACF are located in distal segments more often than in proximal regions, a characteristic also observed in colorectal cancers 16 .Interestingly, the reduction of ACF occurred in middle and distal regions in all groups treated with probiotics, except in Lac/Bif group that showed no significant positive effect.
To our knowledge, only one study has evaluated the effect of S. boulardii in cancer and analyzed its effect on tumor development.In this work, Chen et al. 17  protected against colon carcinogenesis.
has been focused on lactobacilli and bifidobacteria.However, it is still necessary to demonstrate the effectiveness of these and other species to suppress the development of preneoplastic lesions and to determine the ideal formulation of probiotics.The aim of the present study was to determine the effect of Lactobacillus delbrueckii UFV-H2b20, Bifidobacterium animalis var.lactis Bb12 and Saccharomyces boulardii on the prevention of pre-neoplasic lesions in mouse colon.In addition, we aimed to determine whether the combined administration of L. delbrueckii UFV-H2b20 and B. animalis var.lactis Bb12 would display a potential additive effect.Methods Eight week old male Swiss mice were distributed into five groups (n=10/group) as follows: Control group received injections of the carcinogen dimethylhydrazine; Lac group received injections of carcinogen and Lactobacillus delbrueckii UFV-H2b20; Bif group received injections of carcinogen and Bifidobacterium animalis var.lactis Bb12; Lac/Bif group received the carcionogen and Lactobacillus delbrueckii UFV-H2b20 plus Bifidobacterium animalis var.lactis Bb12; and Sb group received the carcinogen and Saccharomyces boulardii.

FIGURE 1 -
FIGURE 1 -(A) Experimental design.(B) Colon preparation for analysis of aberrant crypts: 1-removing of colon; 2-washing with saline; 3opening on paraffin plates with the mucosa facing up and fixing for 24 hours in 10% formaldehyde (C) A dimethylhydrazine-induced aberrant crypt foci presenting two aberrant crypts methylene blue-stained (40x magnification).

FIGURE 2 -
FIGURE 2 -Percentage of aberrant crypt foci induced by dimethylhydrazine in the mice colon and percentual of reduction of aberrant crypt foci in groups treated with Lactobacillus delbrueckii UFV-H2B20 (Lacto), Bifidobacterium animalis var.lactis Bb12 (Bifido), L. delbrueckii UFV-H2B20 plus B. animalis var.lactis Bb12 (Lacto/Bifido) and Saccharomyces boulardii (Sb) in relation to control group.FIGURE 2 -Percentage of aberrant crypt foci induced by dimethylhydrazine in the mice colon and percentual of reduction of aberrant crypt foci in groups treated with Lactobacillus delbrueckii UFV-H2B20 (Lacto), Bifidobacterium animalis var.lactis Bb12 (Bifido), L. delbrueckii UFV-H2B20 plus B. animalis var.lactis Bb12 (Lacto/Bifido) and Saccharomyces boulardii (Sb) in relation to control group.Values were obtained in relation to average of aberrant crypt foci found in the Cont group, considered as 100%.*, p < 0.05, the group was significantly different from Cont group.(A) Total of aberrant crypt foci; (B) Foci containing three or fewer aberrant crypts; (C) Foci with more than three aberrant crypts.
observed the prevention of cancer cell formation through the reduction of epidermal growth factor-mediated cell proliferation and increased apoptosis, as well as by a 50% decrease in tumor number and volume in the distal small intestine of C57BL/6J Min/+ (Apc Min ) mice treated with S.boulardii.These results pointed to the potential role of the yeast in the prevention or treatment of intestinal adenomatous polyps and/ or adenocarcinoma.S. boulardii is a yeast that has been widely used as a probiotic agent in human medicine, and its efficacy in the prevention and treatment of various intestinal disorders has been demonstrated18,19 .However, in the present study, S. boulardii did not exert a marked effect on the formation of preneoplastic lesions.The best result was observed in the reduced number of ACF with more than three aberrant crypts (21.07%) in the group receiving S. boulardii as compared to the Cont/DMH group, but the difference was not significant.In summary, our study suggests that only certain probiotic bacteria (L.delbrueckii UFV-H2b20 and B. animalis var.lactis Bb12) are capable of diminishing preneoplastic lesions development carcinogen-induced, but this beneficial effect seems to be related, under the experimental conditions, to the type of microorganism used.Our results confirm what has been pointed out by Azcárate-Peril et al.20  in their review on the beneficial effects of probiotics in prevention of colorectal cancer, that the role of probiotic is species and strain dependent.More probiotic studies are needed to examine possible mechanisms for their potential benefit.ConclusionAmong the three tested probiotics, only L. delbrueckii UFV-H2b20 and B. animalis var.lactis Bb12 administered aloneLiboredo JC et al.372 -Acta Cirúrgica Brasileira -Vol.28 (5) 2013

TABLE 1 -
Effects of on the formation of aberrant crypt foci (ACF) induced by DMH in the mice colon.
The mean number of ACF per colon in the DMH alone group (Cont) was 62.0 ± 7.9 whereas mice treated with Lactobacillus and Bifidobacteria showed a significantly lower number of ACF.A 55.7% and 45.1% reduction of ACF was found in the Lac and Bif groups, respectively.However, we did not find a synergistic effect of Lactobacillus plus Bifidobacteria