Yacon (Smallanthus sonchifolius) and Lactobacillus acidophilus CRL 1014 reduce the early phases of colon carcinogenesis in male Wistar rats

Food Research International 74 (2015) 48–54

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Yacon (Smallanthus sonchifolius) and Lactobacillus acidophilus CRL 1014 reduce the early phases of colon carcinogenesis in male Wistar rats Ana Paula da Silva Almeida a, Camilla Martins Avi a, Luís Fernando Barbisan b, Nelci Antunes de Moura b, Brunno Felipe Ramos Caetano b, Guilherme Ribeiro Romualdo b, Kátia Sivieri a,⁎ a b

Department of Food and Nutrition, Faculty of Pharmaceutical Science, UNESP - UnivEstadual Paulista Araraquara, SP, Brazil Department of Morphology, Institute of Biosciences, UNESP—São Paulo State University, Botucatu, SP, Brazil

a r t i c l e

i n f o

Article history: Received 20 February 2015 Received in revised form 16 April 2015 Accepted 18 April 2015 Available online 24 April 2015 Keywords: Chemical carcinogenesis Colon Prebiotic Probiotic Synbiotic

a b s t r a c t The modifying effects of aqueous yacon extract (AYE) and Lactobacillus acidophilus CRL 1014 against colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male Wistar rats were investigated. Animals were allocated into five groups: G1: untreated group; G2: DMH-treated group; G3: DMH + L. acidophilus-treated group; G4: DMH + AYE-treated group; G5: DMH + L. acidophilus and AYE-treated group. A significant reduction (p b 0.05) in leukocyte DNA damage and in colonic cell proliferation was observed after the first DMH administration in G3 (probiotic), G4 (prebiotic) and G5 (synbiotic) groups. In this moment, a significant increase (p b 0.05) in colonic apoptosis was also observed in G3 (probiotic) and G5 (synbiotic) groups. In special, at five months after DMH administrations, a significant reduction (p b 0.05) in ACF development was observed in G3 (probiotic), G4 (prebiotic) and G5 (synbiotic) groups. Incidence of colon tumors was lower at five months in G4 (prebiotic) and G5 (synbiotic) groups but not in eight months after DMH administrations. In conclusion, the findings suggest that the oral treatments have potential effects as a chemopreventive agent against colon carcinogenesis on an early stage of tumor development. © 2015 Published by Elsevier Ltd.

1. Introduction Colon carcinogenesis (CC) is the third most commonly diagnosed cancer and the second leading cause of cancer death in the western world (IARC, 2012; WHO, 2014). While family history of CC is an important risk factor, only 20% of new cases are hereditary. In fact, majority of CC (80%) occur sporadically by acquired risk factors such as lifestyle and diet. Dietary factors that potentially increase the risk of CC include low fruit, vegetable and fiber intake and are associated with high red meat or saturated fat consumption (Fotiadis, Stoidis, Spyropoulus, & Zografos, 2008; Liong, 2008). Although a wide array of compounds derived from the diet has been found to stimulate the development and growth of tumors, there is accumulative evidence to support an inverse relationship through chemoprevention. Chemoprevention is defined as the employment of either natural or synthetic compounds to prevent, reverse or delay the development of cancer (Hou, Huo, & Dignam, 2013). Conversely, probiotics, prebiotics and synbiotics supplementation have been shown to exert a protective effect against colon carcinogenesis (Fotiadis et al., 2008; Liong, 2008). ⁎ Corresponding author at: Department of Food and Nutrition, Faculty of Pharmaceutical Science, UNESP—São Paulo State University, Araraquara, Brazil, km 1 Araraquara-Jau Highway, São Paulo State, Brazil. Tel./fax: +55 16 33016922. E-mail address: [email protected] (K. Sivieri).

http://dx.doi.org/10.1016/j.foodres.2015.04.034 0963-9969/© 2015 Published by Elsevier Ltd.

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (FAO, 2001). Prebiotics are non-digestible food components that affect the colonic microbiota, stimulating the growth of beneficial bacteria (Roberfroid, 2003). Fructooligosaccharides (FOS) which are prebiotics are found in several plants, including yacon root (Roberfroid, 2003; Santana & Cardoso, 2008). Yacon (Smallanthus sonchifolius) is an Andean tuberous root with the highest known content of FOS in nature (Santana & Cardoso, 2008) and it provides several benefits, such as hypoglycemic (Aybar, Riera, Grau, & Sa'nchez, 2001), hypocholesterolemic (Park, Yang, Hwang, Yoo, & Han, 2009), antioxidant (Yan et al., 1999) and antitumor actions (de Moura et al., 2012). Sivieri et al. (2014) investigated the prebiotic action of an aqueous yacon extract (AYE) in the Simulator of Human Intestinal Microbial Ecosystem (SHIME) and reported a significant increase in the Bifidobacterium spp. and Lactobacillus spp. populations and, consequently, an increase in short chain fatty acids production in SHIME system. In addition, de Preter, Hamer, Windey, and Verbeke (2011) showed that yacon promotes a selective growth of probiotic bacteria, producing less procarcinogenic β-glucuronidase enzyme. Lactobacillus acidophilus has showed anti-carcinogenic effect against colon cancer development (Baldwin et al., 2010; Lee & Lee, 2000; Urbanska, Bhathena, Martoni, & Prakash, 2009). Different strains of L. acidophilus have showed beneficial effects against aberrant crypt foci (ACF) development in chemically-induced colon carcinogenesis models (Lee & Lee, 2000; Rao, Sanders, Indranie, Simi, & Reddy, 1999). Some

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studies showed that microencapsulated L. acidophilus reduces the incidence, multiplicity and size of colonic tumors and cell proliferation in HT-29 colon cancer cell by cell-bound exopolysaccharide from L. acidophilus 606 (Kim, Oh, Yun, Oh, & Kim, 2010; Urbanska et al., 2009). Rodent colon cancer can be induced by 1,2-dimethylhydrazine (DMH), or its metabolite azoxymethane (AOM), showing morphological and genetic similarities with human colon cancer. Therefore, chemically-induced models are powerful tools to evaluate molecular events, risk factors, and prevention strategies for this malignant disease (Perse & Cerar, 2010; Ravnik-Glavac, Cerar, & Glavac, 2000). Colon cancer development includes three stages: initiation, promotion and progression (Terzic, Grivennikov, Karin, & Karin, 2010). DNA damage can lead to the initiation and promotion stages that can be monitored by counting the preneoplastic lesions, while progression stage can be assessed by colonic adenoma and adenocarcinoma development (Bird, 1995; Weinberg, 2007). Several biomarkers have been used in rodent colon carcinogenesis bioassays, such as DNA damage (Klaude, Eriksson, Nygren, & Ahnstrijm, 1996), stereological ACF counting (Bird, 1995) and cell proliferation and apoptosis analysis (Costa et al., 2011; Wu, Yang, & Chen, 2014). Several reports have indicated different biological actions of dietary yacon (Genta et al., 2009; Geyer, Manrique, Degen, & Beglinger, 2008; Habib, Honoré, Genta, & Sánchez, 2011; Lobo, Colli, Alvares, & Filisetti, 2007; Valentová, Moncion, Waziers, & Ulrichová, 2004), but there are few studies showing anti-carcinogenic action on colon carcinogenesis (de Moura et al., 2012). Therefore, the aim of this research was to assess the beneficial effect of aqueous yacon extract alone, or in association with L. acidophilus CRL 1014 on previous and during early and late stages of colon carcinogenesis in male Wistar rats initiated by DMH.

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UNESP, Brazil (Protocol 16/2010). Hundred 4-week old male Wistar rats were obtained from the UNESP animal facility and maintained in opaque propylene boxes, in a room with a controlled photoperiod (12 h of darkness) under air filtration and controlled temperature (22 °C ± 2 °C) with free access to water and commercial rat chow (23% protein, 49% carbohydrate, 4% fat, 5% fiber, 7% ashes and 6% vitamin C). After a 2-week acclimation period to the housing environment, the animals were randomly allocated into five groups (n = 20 each): G1: untreated group; G2: DMH-treated group; G3 (probiotic): DMH + L. acidophilus CRL 1014treated group; G4 (prebiotic): DMH + aqueous yacon extract (AYE)treated group; and G5 (synbiotic): DMH + L. acidophilus CRL 1014 and AYE treated group (Fig. 1). Two weeks after the beginning of the treatments with probiotic, prebiotic and synbiotic, G2 to G5 groups received four subcutaneous injections of 40 mg kg−1 of DMH (Sigma-Aldrich, CO, USA, pH 6.5 in NaCl solution), twice a week for two weeks (Dias et al., 2010; Sivieri et al., 2008), G1 group received similar injections of 0.9% NaCl solution (DMH vehicle). All treatments were administered by gavage for 8 months, L. acidophilus CRL 1014 was administered at 109 CFU/mL in 3 mL kg−1 body weight (Sivieri et al., 2008). Amount of AYE administered was calculated in relation of its FOS content. All animals ingested daily 2.2 mL of AYE with 1% of FOS content (Pool-Zobel, 2005). Five animals of each group were sacrificed in a CO2 chamber, 24 h after the first DMH administration (cell proliferation and apoptosis in the colonic crypt) and at the third, fifth and eighth months of the experimental period (ACF and tumor analysis) (Fig. 1). All macroscopic tumors were measured and classified into small (b 0.22 cm3), medium (0.22– 0.84 cm3) and large (N 0.85 cm3). The body weights of animals in all groups were recorded once a week throughout the experiment. 2.2. Probiotic and prebiotic preparation

2. Material and methods 2.1. Animals and treatment The animals used in this study were handled in accordance with Brazilian Animal Experimentation College, and approved by the Ethics Committee of the Faculty of Pharmaceutical Science of Araraquara-SP

L. acidophilus CRL 1014 was administered to probiotic (G3) and synbiotic groups (G5) at a concentration of 109 CFU mL−1 of sterile peptone water by adding 3 mL kg−1 bw. A pure culture of L. acidophilus CRL 1014 (CERELA, San Miguel de Tucumán, Argentina) was inoculated into De Man, Rogosa and Sharpe (MRS) broth (Acumedia, Baltimore, USA). Cultures were harvested during the exponential growth and

Fig. 1. Experimental design (for details see Materials and methods section).

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subsequently, they were centrifuged (5000 ×g, 10 min, 4 °C) and washed with sterile peptone water. The L. acidophilus CRL1014 cells were kept at the concentration of 109 CFU/mL in sterile peptone water until use (Sivieri et al., 2007; Sivieri et al., 2013). AYE was administered to prebiotic (G4) and synbiotic (G5) groups. Yacon root (purchased in a local marketing) was pasteurized at 95 °C/15 min immediately after yacon cutting, and yacon was ground to obtain an aqueous extract (Pauly-Silveira et al., 2010; Roselino et al., 2012). FOS content in AYE was determined with fructan assay kit (Megazyme, Ireland) (Korakli, Hinrichs, Ehrmann, & Vogel, 2003).

2.3. Comet assay This technique was performed as described by Singh, Mccoy, Tice, and Schneider (1988), with modifications. Briefly, four hours after the first injection of DMH or vehicles, slides were prepared by depositing 5 μL of blood of retro orbital plexus mixed with 100 μL of lowmelting-point agarose (1.5%) on a glass slide coated with normal agar, at 37 °C. Slides (covered with glass coverslip) were chilled at 10 °C for 5 min. Slides (after removal of the coverslips) were immersed in freshly prepared lysis solution, composed of 100 mL lysis stock solution (2.5 M NaCl, 100.0 mM EDTA, 10.0 mM Tris, pH adjusted to 10.0 with solid NaOH, 1000 mL of distilled water and 1% N-laurosylsarcosine sodium), 1.0 mL Triton X-100 and 10.0 mL DMSO (dimethyl sulfoxide). Protected from light, lysis proceeds for at least 24 h at 4 °C. Slides (washed with phosphate buffered saline (PBS)) were placed in an electrophoresis chamber (25.0 V and 300.0 mA (1.25 V cm−1), and using alkaline electrophoresis buffer (0.3 M NaOH, 1 mM Na2EDTA, pH N 13) at 4 °C for 20 min. Slides were neutralized with 0.4 M Tris–HCl buffer, pH 7.5, fixed in absolute ethanol for 10 min and saved for later analysis. For staining, the slides were covered with 50 μL of ethidium bromide (20 μg/mL) and a coverslip. The material was examined with an epiillumination fluorescence microscope at 400× magnification connected to an image analysis system (Comet Assay II, Perspective Instruments, Suffolk, UK). A total of 100 nucleoids (two slides per animal) per treatment were analyzed and software used to calculate comet tail length.

2.4. Colon processing and histopathological analysis The colon was removed, opened longitudinally, residual bowel contents was removed with 0.9% NaCl and colon fixed flat in 10% buffered formalin (pH 6.9–7.1) for 48 h, then maintained in 70% ethanol solution (Dias et al., 2010). The localization and volume of each gross macroscopic tumor were registered using a caliper of 0.01 mm precision. Measured width (W) and length (L) were used to calculate tumor volume (cm3) (V) according mathematical equation V = (L × W2) / 2 (Cheng et al., 2014). Tumors were classed as small, medium or large (de Moura et al., 2012). The number of ACF was identified according to Bird's criteria (Bird, 1995). The colons were stained with Leishman (Merck®, USA) for 2 min to evaluate the ACF count, using an equipped microscope with image capture, at 10 and 40 × magnifications. Then, colon and tumor samples were embedded in paraffin, cut in 5 μm sections and stained with hematoxylin–eosin (HE) for histological analysis (Dias et al., 2010). Colon tumors were classified as adenomas or adenocarcinomas using well-established criteria (Hamilton & Aaltonen, 2000). 2.5. Apoptosis analysis Apoptosis index was performed on slides from animals sacrificed 24 h after first DMH administration. Slides stained with HE were used to determined apoptosis index and twenty normal appearing colonic crypts were analyzed in longitudinal section of each slide. Apoptosis index (%): (number of epithelial cells in apoptosis in half of crypt / number of epithelial cells in half of crypt) × 100 (Leu et al., 2005).

2.6. Immunohistochemical analysis Ki-67 and p53 labeling indexes were determined on slides from animals sacrificed 24 h after first DMH administration. Using a labeled polymer system (Universal Dako LSAB® + kit Peroxidase, Denmark A/S), immunoreactivity of Ki-67 (Rabbit monoclonal antibody [SP6], Abcam, USA) and p53 (anti-p53, rabbit polyclonal antibody Abcam, USA) in the colon sections was detected. Briefly, colon sections were dewaxed in xylol and hydrated in absolute alcohol. Slides were treated with 0.01 M citrate buffer (pH 6.0) at 120 °C for 5 min in a Pascal pressure chamber (Dako Cytomation A/S, Denmark); 3% H2O2 in PBS for 10 min; 1% nonfat milk in PBS for 60 min; anti-Ki-67 (1:100 dilution in BSA-bovine serum albumin) or p53 (1:100 in BSA) antibodies overnight at 4 °C; slides were incubated with Dako Biotinylated Universal link and Streptavidin separately for 15 min, each one at room temperature. Chromogen color was developed and accomplished with 3.3-diaminobenzidine tetrahydrochoride (Sigma-Aldrich, Co, USA). The slides were counter-stained with Harris's hematoxylin. Twenty crypts were analyzed in longitudinal section of each slide. Ki-67 index (%): (number of labeled epithelial cells in half of crypt / number of epithelial cells in half of crypt) × 100 (de Moura et al., 2012). p53 staining was regarded as positive when more than 5% of the nuclei showed intense staining (Dias et al., 2010). 2.7. Statistical analysis Quantitative data were compared among experimental groups using ANOVA or Kruskal–Wallis tests followed by Turkey's contrast test. Incidence of tumor volume and adenocarcinomas (malignant tumors) was subjected to a chi-square test. The statistical difference among the parameters was considered when p b 0.05. 3. Results and discussion 3.1. General findings The chronic oral treatments with prebiotic (AYE), probiotic (L. acidophilus CRL 1014) and synbiotic (L. acidophilus CRL 1014 + AYE) did not significantly modify the body weight gain or food consumption during the experimental period (data not shown). In fact, various experimental evidences have demonstrated that prebiotics and probiotics are safe for human use (Aybar et al., 2001; Campos et al., 2012; Delgado, Thomé, Gabriel, Tamashiro, & Pastore, 2012; Leu et al., 2005). 3.2. Comet assay Since carcinogenesis is a complex multistep process initiated by DNA mutation (Humphries & Wright, 2008), the oral treatments with prebiotic (AYE), probiotic (L. acidophilus CRL 1014) and synbiotic (L. acidophilus CRL 1014 + AYE) were used trying to inhibit or reduce the early induction of DNA damage caused by the genotoxic carcinogen DMH. The comet assay has been shown to be sensitive in detecting DNA damage at various levels in different tissue samples (Olive & Banáth, 2006). The amount of DNA damage induced by DMH can be verified by the length of tails of the nucleoids produced on peripheral leukocytes after electrophoresis (Fig. 2A). A significant increase (p b 0.05) in the length of the comet tail was observed in all groups exposure to DMH (G2–G5 group) when compared to the G1 group (untreated animals). On the other hand, a significant reduction (p b 0.05) in the DNA damage in peripheral leukocytes from the G3 (probiotic), G4 (prebiotic) and G5 (synbiotic) groups was observed in comparison to the G2 group (DMH alone) (Fig. 2A). This finding indicates that these oral treatments, before and during DMH exposure, reduced the extent DNA damage in leukocytes and, probably, in colonic epithelial cells target susceptibly to colon carcinogenesis. These results are in accordance

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Fig. 2. Effects of AYE alone or associated with L. acidophilus CRL 1014 on comet tail length (A), apoptosis index (B) and Ki-67 labeling index (C). Values represent mean ± S.D. (D) Apoptotic bodies and (E) Ki-67 immunostaining in colonic epithelium (arrows) (40× objective). (E) (G1) untreated group; (G2) DMH; (G3) (probiotic) DMH + L. acidophilus CRL 1014; (G4) (prebiotic) DMH + AYE; (G5) (synbiotic): DMH + L. acidophilus CRL 1014 + AYE. Different letters correspond to statistical difference among groups by ANOVA and post hoc Tukey Test (p ≤ 0.05).

to Pool-Zobel et al. (1996) which demonstrated that animals treated with lactic-acid bacteria (L. acidophilus, Lactobacillus confuses, Lactobacillus gasseri, Bifidobacterium longum and Bifidobacterium breve), before DMH administration, presented lower response to the genotoxic effect this colon carcinogen. This is the first finding showing that yacon has a protective effect against DNA damage in leukocytes. Interestingly, beneficial effects on genotoxicity have been reported for certain prebiotics (lactulose and polidextrose) in animal and human studies (Costabile, Fava, Röytiö, Forssten, & Olli, 2011; Rowland, Bearne, Fischer, & Pool-Zobel, 1996) and for probiotic/prebiotic mixtures (oligofructose-enriched inulin + Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12) in humans (Rafter et al., 2008). However, no additional or synergic effects were observed using our synbiotic formulation.

of ACF/colon in both treatments after DMH administrations. In addition, L. acidophilus has demonstrated protective effects in reducing the number of ACF in the colon of animals initiated for colon carcinogenesis (Bolognani, Rumn, Pool-Zobel, & Rowland, 2001; Lee & Lee, 2000). However, it should be noted that these studies differed in the extent of reduction of ACF, indicating that the probiotic action was strain dependent. Furthermore, our study and these studies used distinct experimental protocols, with different treatment periods, doses of DMH and type and amounts of prebiotic and probiotic administered. In the present study, the oral treatments (G2 to G4) had no longer reducing effects on the number of ACF per colon when compared to the DMH-treated group (G2) at eighth month of experimental protocol (Table 1). In fact, Takayama et al. (1998) demonstrated that either the number and multiplicity of ACF and their evolution for malignancy increase with aging.

3.3. Classical ACF analysis In rodent models of colon carcinogenesis, putative preneoplastic lesions (ACF) are the earliest detectable colonic abnormality that can precede adenoma/adenocarcinoma development (Khare, Chaudhary, Bissonnette, & Carroll, 2009). Classical ACF were evaluated in methylene blue-stained whole mount colon at the end of third, fifth and eight months of experimental period (Table 1). There was a significant reduction (p b 0.05) in mean number of ACF/colon in G5 group (synbiotic) when compared to the G2 group (DMH) in the third month. In the fifth month, a significant reduction (p b 0.05) in mean number of ACF/ colon was detected in G3 (probiotic), G4 (prebiotic) and G5 (synbiotic) groups in relation to the G2 group (DMH). Moreover, no significant difference was observed among DMH-treated groups at late stage of colon carcinogenesis (Table 1). According these results, de Moura et al. (2012), using 1% yacon root, alone or in a synbiotic combination with Lactobacillus casei in chow, showed a significant decrease in the number

Table 1 Effects of aqueous yacon extract (AYE) and in association with L. acidophilus CRL 1014 or L. acidophilus CRL 1014 on development of colonic aberrant crypt foci (ACF) (number ACF per colon) during the 8 months of experimental protocol in groups G2, G3, G4, G5 (n = 5 rats per group). Month

Groups G2

Third Fifth Eighth

G3 a

12.00 ± 2.68 36.00 ± 4.00a 15.25 ± 6.17a

G4 ab

5.00 ± 1.96 10.00 ± 1.53b 23.00 ± 5.53a

G5 ab

4.50 ± 2.84 7.67 ± 1.45b 12.00 ± 4.16a

0.75 ± 0.25b 18.75 ± 2.53b 15.00 ± 5.84a

G1: untreated group; G2: DMH-treated group; G3 (probiotic): DMH + L. acidophilus CRL 1014-treated group; G4 (prebiotic): DMH + AYE-treated group; G5 (synbiotic): DMH + L. acidophilus CRL 1014 + AYE-treated group. Different letters in the same line indicate significantly different results (p b 0.05). Values are mean ± Standard error.

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3.4. Histopathological analysis At sacrifices, there were more adenocarcinomas on medial and distal colons, representing 44% and 45% of total tumors detected, respectively. No difference among groups was observed in relationship to the incidence of macroscopic tumors as classified by tumor sizes (Fig. 3A and B). In the initial phase of tumor progression, groups that ingested AYE (G4) and its association with L. acidophilus CRL 1014 (G5) showed better suppressor response when compared to DMH group (G2) and probiotic (G3), once only 50 and 66.67% of macroscopic tumors in G4 (prebiotic) and G5 (synbiotic) were adenocarcinomas (Fig. 3A). Likewise, de Moura et al. (2012) reported lower proportion of adenocarcinomas induced by DMH in groups receiving a synbiotic formulation (1% yacon root and L. casei at 2.5 × 10−10 CFU per gram). However, no additional or synergic effects were observed using our synbiotic formulation. At eighth month of experimental protocol, all oral treatments (G3, G4, G5) had no reducing effect on either incidence of macroscopic tumors or tumor size (Fig. 3B). This result corroborates with Capurso, Marignani, and Fave (2006), who described animal models to assess colorectal cancer risk and probiotic treatments, suggesting that these treatments have beneficial effects during an early stage of the colon cancer, which become less effective in the promotion and progression stages of colon carcinogenesis. Pool-Zobel (2005) reported that prebiotic (with inulin-type fructan) and synbiotic treatments are more effective and the beneficial action more pronounced when they are chronically administered throughout the carcinogenic process.

3.5. Apoptosis index Apoptosis is an important regulatory process in the protection against the development of cancer. It provides an innate cellular defense against carcinogenesis by removing cells with genomic instability and by deleting cells with DNA damage induced by genotoxic agents such as DMH (Hu, Martin, Leu, & Young, 2002). In fact, an increase in apoptosis during initiation events of chemical carcinogenesis helps in eliminating mutated cells that may otherwise progress to malignancy after a long period of latency (Hu et al., 2002). Apoptosis index was analyzed in the colonic crypts 24 h after first DMH administration. The results showed that groups treated with L. acidophilus CRL 1014 (G3) and AYE associated with L. acidophilus CRL 1014 (G5) showed higher apoptotic indexes (22.79% and 20.41%, respectively) when compared to the DMH-treated group (G2) (p b 0.05) (Fig. 2B). Leu et al., (2005) showed that a synbiotic combination of resistant starch and probiotic can facilitate apoptotic deletion of acutely carcinogen-damaged cells in the rat colon. However, the findings of

present study indicate that AYE treatment did not alter apoptotic index in carcinogen-damaged cells in rat colon. de Moura et al. (2012) observed no effect on the apoptotic index in tumor colon in groups DMH-initiated and fed 1.0% yacon root and L. casei. On the other hand, it has to be taken into account that, between 6 and 24 h after carcinogen administration, there is a temporary rise in the apoptotic index, which later (at 72 h) falls to a lower value (Ohyama, Okada, Fujiishi, Narumi, & Yasutake, 2013). The damage suffered by the DNA and, therefore, changes in the process of apoptosis, have their origin in the phase of initiation of carcinogenesis. Now, the transformation of an adenoma into an adenocarcinoma was related to genetic changes and, while there is no exclusive and predetermined sequence of such changes, among those linked to tumor development is a reduction in the rate of apoptosis (Liu, 2006; Turner, 2012). 3.6. Ki-67 and p53 immunohistochemical analysis Ki-67 antigen, which is expressed in nuclei during all phases of the cell cycle except G0, is a suitable biomarker of cell proliferation in various tissues (Tsamandas et al., 2009) while p53 is a transcription factor that accumulates in the nucleus in response to DNA damage induced by chemical carcinogens (Hanahan & Weinberg, 2011) . Ki-67 and p53 were analyzed in the colonic crypts 24 h after first DMH administration. Ki-67 labeling index (LI%) was higher in DMHtreated group (G2) when compared to the untreated group (G1). Besides, a significant reduction (p b 0.05) in Ki-67 LI% in colonic crypts in L. acidophilus CRL 1014 (G3) and AYE + L. acidophilus CRL 1014 (G5) groups was observed in relation to the DMH-treated group (G2) (Fig. 2C). Wu et al. (2014) observed a reduction in cell proliferation index after AOM administration in mice given with inulin as a dietary supplement for 3 weeks. However, our findings did not show a protective effect from AYE treatment. Cell proliferation may lead to an increased risk of developing cancer whereas apoptosis is a protective innate mechanism taking to elimination of the epithelial cells with DNA damage or genomic instability (Hanahan & Weinberg, 2011). In agreement with ours results, Linsalata et al. (2005) also observed the protective action of a probiotic treatment on the cell proliferation and apoptotic indexes in colonic mucosa from male Fisher 344. This work showed that a probiotic cocktail (2.8 × 108 CFU/mL, consisting of 9 × 1010 CFU/g bifidobacteria, 8 × 1010 CFU/g lactobacilli and 20 × 1010 CFU/g Streptococcus thermophilus) induced a lower Ki-67 index and higher apoptotic index, relative to the control untreated group in healthy animals. Differently from DMH-treated groups (G2 to G4), there was no wildtype p53 staining in colonic epithelial cells in the untreated group (G1) (data not shown). Similarly, Sakuma, Fujimori, Hirabayashi, and Terano

Fig. 3. Effects of AYE alone or associated with L. acidophilus CRL 1014 on Tumor Macroscopic (volume) and Histopathological Evaluation at (A) early and (B) late stages of carcinogenesis progression. Values represent the proportion of tumors (percentage). Macroscopic gross tumors were classified as malignant (Adenocarcinomas) after Histopathological Evaluation. (G1) untreated group; (G2) DMH; (G3) (probiotic) DMH + L. acidophilus CRL 1014; (G4) (prebiotic) DMH + AYE; (G5) (synbiotic): DMH + L. acidophilus CRL 1014 + AYE.

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(1999) observed an absence of p53 staining in normal colonic mucosa. The lack of difference in p53 staining in colonic crypt among DMHtreated groups (G2 to G5) will be due to loss transient peak of p53 expression after DNA damage (Lukin, Carvajal, Liu, Resnick-Silverman, & Manfredi, 2015). 4. Conclusion This study shows that oral administration of either AYE or L. acidophilus CRL 1014 may have a protective effect against colon carcinogenesis on the early phases of tumor development. Nevertheless, no synergistic effect was found using our symbiotic formulation. When administered before, during and after DMH initiation, AYE treatment protected against DMH-induced DNA damage in leukocytes and decreased ACF incidence, while L. acidophilus CRL 1014 treatment also protected against DNA damage but induced apoptosis and decreased colonic proliferation and ACF incidence on early tumor development. In conclusion, our results have demonstrated that both AYE and L. acidophilus CRL 1014 oral supplementations showed a potential protective effect against colon DMH-induced tumorigenesis, albeit the AYE exhibited a less pronounced effect. Acknowledgments This work was supported by grants from FAPESP (São Paulo Research Foundation). FAPESP Process number 2009/53878-8. References [WHO] World Health Organization (2014). Cancer Fact sheet N° 297. Feb 2014. Available from: http://www.who.int/mediacentre/factsheets/fs297/en/ (Accessed 02 Jul 2014) Aybar, M.J., Riera, A.N.S., Grau, A., & Sa'nchez, S.S. (2001). Hypoglycemic effect of the water extract of Smallantus sonchifolius (yacon) leaves in normal and diabetic rats. Journal of Ethnopharmacology, 74, 125–132. Baldwin, C., Millette, M., Oth, D., Ruiz, M.T., Luquet, F.M., & Lacroix, M. (2010). Probiotic Lactobacillus acidophilus and Lactobacillus casei mix sensitive colorectal tumoral cells to 5-fluorouracil-induced apoptosis. Nutrition and Cancer, 62, 371–378. Bird, R.P. (1995). Role of aberrant crypt foci in understand the pathogenesis of colon cancer. Cancer Letters, 93, 55–71. Bolognani, F., Rumn, C.J., Pool-Zobel, B., & Rowland, I.R. (2001). Effect of lactobacilli, bifidobacteria and inulin on the formation of aberrant crypt foci in rats. European Journal of Nutrition, 40, 293–300. Campos, D., Betalleluz-Pallardel, I., Chirinos, R., Aguilar-Galvez, A., Noratto, G., & Pedreschi, R. (2012). Prebiotic effects of yacon (Smallanthus sonchifolius Poepp. & Endl), a source of fructooligosaccharides and phenolic compounds with antioxidant activity. Food Chemistry, 135, 1592–1599. Capurso, G., Marignani, M., & Fave, C.D. (2006). Probiotics and the incidence of colorectal cancer: When evidence is not evident. Digestive and Liver Disease, 38, S277–S282. Cheng, T.C., Lai, C.S., Chung, M.C., Kalyanam, N., Majeed, M., Ho, C.T., et al. (2014). Potent anti-cancer effect of 39 hydroxypterostilbene in human colon xenograft tumors. PLoS One, 9, e111814. Costa, H., Lima, F.O., Barrezueta, L.F.M., Oshima, C.T.F., Silva, J.A., Jr., Gomes, T.S., et al. (2011). Correlation between immunoexpression of BCL-2 protein family with apoptosis index, cellular proliferation and survival in colorectal carcinomas. Applied Cancer Research, 31, 74–80. Costabile, A.F., Fava, H., Röytiö, S.D., Forssten, K., & Olli, J.K. (2011). Impact of polydextrose on the faecal microbiota: A double-blind, crossover, placebo-controlled feeding study in healthy human subjects. British Journal of Nutrition, 108, 471–481. de Moura, N.A., Caetano, B.F.R., Sivieri, K., Urbano, L.H., Cabello, C., Rodrigues, M.A.M., et al. (2012). Protective effects of yacon (Smallanthus sonchifolius) intake on experimental colon carcinogenesis. Food and Chemical Toxicology, 50, 2902–2910. de Preter, V., Hamer, H.M., Windey, K., & Verbeke, K. (2011). The impact of pre- and/or probiotics on human colonic metabolism: Does it affect human health? Molecular Nutrition & Food Research, 55, 46–57. Delgado, G.T.C., Thomé, R., Gabriel, D.L., Tamashiro, W.M.S.C., & Pastore, G.M. (2012). Yacon (Smallanthus sonchifolius)-derived fructooligosaccharides improves the immune parameters in the mouse. Nutrition Research, 32, 884–892. Dias, M.C., Vieiralves, N.F., Gomes, M.I., Salvadori, D.M., Rodrigues, M.A., & Barbisan, L.F. (2010). Effects of lycopene, synbiotic and their association on early biomarkers of rat colon carcinogenesis. Food and Chemical Toxicology, 48, 772–780. Food and Agriculture Organization of the United Nations, & World Health Organization (2001). Evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. (Córdoba 34p. Available from: ftp://ftp. fao.org/docrep/fao/009/a0512e/a0512e00.pdf. Accessed 15 Sep 2011 [Report of a Joint FAO/WHO Expert Consultation]). Fotiadis, C.I., Stoidis, C.N., Spyropoulus, B.G., & Zografos, E.D. (2008). Role of probiotics, prebiotics and synbiotics in chemoprevention for colorectalcancer. World Journal of Gastroenterology, 14, 6453–6645.

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