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Nutrition and Cancer

A Synbiotic Combination of Resistant Starch and Bifidobacterium lactis Facilitates Apoptotic Deletion of Carcinogen-Damaged Cells in Rat Colon¹

Richard K. Le Leu², Ian L. Brown^*, Ying Hu, Anthony R. Bird, Michelle Jackson, Adrian Esterman^** and Graeme P. Young

Department of Medicine, Flinders University of South Australia, Bedford Park, South Australia 5042; ^* National Starch & Chemical Company, Bridgewater, NJ; CSIRO Health Sciences and Nutrition, Adelaide, South Australia; and ^** Flinders Centre for Epidemiology & Biostatistics, Flinders University, Bedford Park, South Australia 5042

²To whom correspondence should be addressed. E-mail: richard.leleu{at}flinders.edu.au.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Recent reports suggest that combinations of prebiotics and probiotics may be protective against colorectal cancer. We examined in rats the effects of probiotic bacteria, resistant starch (RS), and their interaction on luminal and epithelial events of relevance to the development of colorectal cancer. Lyophilized cultures (1 x 10¹⁰ cfu/g) of Lactobacillus acidophilus and/or Bifidobacterium lactis were added at a concentration of 1% by weight to a semipurified diet containing either low-RS (no supplemented RS) or moderate-RS (10% Hi-maize®) and fed to male Sprague-Dawley rats for 4 wk. Experimental end-points included cecal bacterial enumeration, fecal and cecal pH, SCFA levels, cell proliferation, and the acute apoptotic response to a genotoxic carcinogen (AARGC; measured 6 h after a single azoxymethane injection). A significant interaction between dietary RS and supplemental bacteria was observed for the AARGC in the colon and fecal pH (P < 0.01). Rats fed the moderate-RS diet in combination with B. lactis had a significantly greater AARGC in the colon than those fed that diet without B. lactis. Fecal pH was elevated in the moderate-RS fed rats supplemented with bacteria. The moderate-RS diet increased cell proliferation and crypt column height (P < 0.001) compared with the low-RS diet. SCFA levels and numbers of bifidobacteria and lactobacilli species were also increased (P < 0.001) by the moderate-RS diet, whereas pH levels and total coliforms were lowered (P < 0.001). The synbiotic combination of RS and B. lactis significantly facilitated the apoptotic response to a genotoxic carcinogen in the distal colon of rats. It appears likely that ingested RS acts as a metabolic substrate, thus creating the right conditions for B. lactis to exert its proapoptotic action. Because the synbiotic combination of these agents facilitates the apoptotic response to DNA damage by a cancer initiator in the colon of rats, it warrants further study for its capacity to protect against colorectal cancer.

KEY WORDS: • apoptosis • prebiotics • probiotics • butyrate • colon cancer

Colonic microflora are increasingly being shown to be capable of influencing gastrointestinal diseases and disorders including colorectal cancer (1,2). There exists a potential role for foods that contain probiotics and/or prebiotics, which can change the colonic microflora in a way that might prevent diseases such as colorectal cancer. Probiotics are live microorganisms that are used as dietary supplements with the aim of benefiting health by influencing the intestinal microbial balance (3). Among the major genera of colonic bacteria, bifidobacteria and lactobacilli are thought to have beneficial effects on the human host (4). These probiotic bacteria were shown previously to exert some cancer protective effects in vitro and in vivo (5–7). The precise mechanisms by which certain probiotic bacteria exert their antitumorigenic influence are uncertain, but might involve modifying gut pH and increasing the net production rate of SCFA (mainly acetate, propionate, and butyrate) (8), antagonizing pathogens through production of antimicrobial and antibacterial compounds (such as bacteriocins, cytokines and butyrate), stimulating immunomodulatory cells (9), or competing with pathogens for available nutrients, receptors, and growth factors (10).

Prebiotics are nondigestible dietary components that pass through the digestive tract to the colon. Thus, prebiotics are a potential substrate for fermentation by the microbiota and are capable of stimulating proliferation and/or activity of endogenous desirable bacteria (11). Prebiotics such as resistant starch (RS),³ in the form of Hi-maize® (20–30% wt:wt) and oligosaccharides (5–10% wt:wt) (12,13) as well as wheat bran (equivalent to 5 or 10% total dietary fiber) (14,15) were shown to stimulate the acute apoptotic response to a genotoxic carcinogen (AARGC) in the rat colon. The AARGC might regulate mutational load in the colon and eliminate DNA-damaged cells that might otherwise progress to malignancy, thereby exerting a protective effect at the early stages in the onset of cancer. Oligosaccharides also suppress azoxymethane (AOM)-induced preneoplastic aberrant crypt foci (ACF) and colon carcinogenesis in the rat colon (2,16,17) as does wheat bran (18–20). Prebiotics may exert their cancer protective effects via modulation of fermentative events, possibly by increasing SCFA production or by altering gut microbiota toward a more beneficial composition.

It was suggested that a combination of a probiotic and a prebiotic, termed synbiotics, might be more active than either a probiotic or prebiotic alone (21). This idea was supported by Rowland et al. (2) who showed that administration of the prebiotic inulin with the probiotic Bifidobacterium longum to rats resulted in additive effects, with a more potent inhibition of AOM-induced ACF than administration of either inulin or B. longum separately.

These findings raise the possibility that probiotics, prebiotics, or synbiotic combinations thereof might be protective against colorectal cancer by regulating the consequences of carcinogen-induced damage to colonic epithelial cells. Thus, we aimed to determine whether supplementation with either the probiotic bacteria Lactobacillus acidophilus and B. lactis alone or in combination with a moderate amount of RS could influence the acute apoptotic response to a genotoxic carcinogen (AARGC) and fermentative events in the colon rats.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and diets. Male Sprague-Dawley rats (n = 96, 5 wk of age) [body weight (mean ± SEM) 155 ± 2 g] were obtained from the Animal Resource Centre, Perth, Western Australia. After arrival, rats were quarantined for 2 wk during which they were fed a modified AIN-76 diet (22) (see low-RS, Table 1). Rats were then divided randomly into 8 experimental groups and housed 3/plastic cage with wire bottom grills and wire tops in an animal holding room under controlled conditions of 22 ± 2°C, 80 ± 10% humidity, and a 12-h light:dark cycle and fed experimental diets for 4 wk. Rats had free access to water and were weighed weekly throughout the study. The experimental diets were based on the AIN-76 standard for purified diets for rats and mice (22) and contained either low-RS or moderate-RS (10% Hi-maize 958) (Table 1). The moderate-RS diet was equivalent to the low-RS diet with the exception that 100 g/kg diet of Hi-maize 958 was added at the expense of an equal amount of cornstarch. Lyophilized cultures (1 x 10¹⁰ cfu/g) of L. acidophilus (LAFTI® L10), B. lactis (LAFTI B94) (DSM Food Specialties Australia) and a combination of the 2 cultures were added at a concentration of 1% each by weight to semipurified diets containing low-RS or moderate-RS at the expense of sucrose. Hi-maize 958 is a natural, unmodified, food-grade, high-amylose maize starch that is an important source of total dietary fiber (22% analyzed by AOAC) and RS (61.8%) (23). The Hi-maize 958 was supplied by the National Starch and Chemical Company.

On the final day of the experimental period, each rat was administered a single i.p. injection of AOM (10 mg/kg body weight, Sigma Chemical) to induce apoptosis (14); 6 h later, rats were killed by CO₂-induced narcosis. The entire colon was rapidly removed and divided into proximal and distal portions; the limit of the proximal portion was defined by the "herring bone" pattern. These were flushed clean with ice-cold saline, and segments (2 cm) were taken from the cecal end of the proximal portion and the rectal end of the distal portion. These segments were placed in 10% buffered formalin for 24 h, then washed and stored in 70% ethanol. The cecum was excised, weighed, and a known weight of digesta placed in 3 volumes of saline for pH measurement; a known weight of digesta was also diluted in 3 volumes of internal standard solution (heptanoic acid, 1.68 mmol/L) and stored at –20°C for later analysis of SCFA. Cecal digesta was also collected using aseptic techniques and diluted in 9 volumes of storage media and stored at –20°C for microbial population determination. The Flinders University of South Australia Animal Welfare Committee approved all experimental procedures.

    Measurement of apoptosis. Colon sections (0.5 cm x 0.5 cm) fixed in 70% ethanol were cut from proximal and distal segments of the colon and embedded in paraffin. Paraffin-embedded sections (5 µm) were stained with hematoxylin and evaluated under a light microscope for apoptotic cells. Apoptotic cells were identified in 20 randomly chosen intact crypts by characteristic morphologic changes of cell shrinkage, the presence of condensed chromatin, and sharply delineated cell borders surrounded with a clear halo (14) as described by Potten et al. (24). In all cases, an independent observer who was unaware of the experimental dietary treatment determined the quantification of apoptotic cells. The percentage of apoptotic nuclei (apoptotic index) was calculated as the mean number of apoptotic cells per crypt column divided by the total number of cells in the column and multiplied by 100. The length of each crypt was determined along with the position of apoptotic cells.

    Measurement of cell proliferation. To assess the proliferative activity and the distribution of proliferating cells in the colonic crypts, the proliferating cell nuclear antigen (PCNA) was performed using standard immunohistochemical procedures. Briefly, deparaffinized sections were rehydrated in a graded series of ethanol from 100 to 50% and then to distilled water. The primary mouse monoclonal antibody (PC-10, Santa Cruz Biotechnology) was placed on the slides (1/500 dilution) and incubated overnight at room temperature. A Level 2 Ultra Streptavidin detection system (Signet Laboratories) was used with biotinylated goat anti-mouse IgG as the secondary antibody. The slides were counterstained for 3 min with hematoxylin. In all cases, an independent observer who was unaware of the experimental dietary treatment determined the quantification of proliferative cells. The scoring for cell proliferation was the same as the method used to score apoptosis.

    SCFA analysis. Fecal and cecal samples were homogenized in 3 volumes of internal standard solution (heptanoic acid, 1.68 mmol/L) and centrifuged at 3000 x g for 10 min. The supernatant was then distilled and 0.3 µL injected into a gas chromatograph (HewlettPackard 5890 Series II A) equipped with a flame ionization detector and a capillary column (Zebron ZB-FFAP, 30 m x 0.53 mm i.d, 1-µm film, SGE). Helium was used as the carrier gas; the initial oven temperature was 120°C and was increased at 30°C/min to 190°C; the injector temperature was 210°C and the detector temperature was 210°C. A standard SCFA mixture containing acetate, propionate, and butyrate was used for calculation and the results are expressed as µmol/g of sample.

    Microbiology. Samples of digesta (1 g) from the cecum were diluted into 9 mL of storage medium containing 20 g/L buffered peptone (Oxoid CM509), 20 g/L lab-lemco powder (Oxoid L29) and 20% v:v glycerol before storage at –20°C. Suspensions were thawed at 4°C, homogenized, and further diluted in 10-fold dilutions using prereduced buffered peptone (20 g/L buffered peptone Oxoid CM509, 0.5% cysteine HCl and 0.1% Tween 80). Four appropriate dilutions (100 µL each) were inoculated in duplicate directly onto Bifidus blood, (25) Rogosa (Oxoid CM627), Columbia blood (Oxoid CM 331), and Chromogenic Escherichia coli/Coliform (Oxoid CM956) medium for the selective enumeration of Bifidobacteria, Lactobacilli, total anaerobes, E. coli, total coliforms, and total aerobes, respectively. Bifidus blood, Columbia Blood, and Rogosa plates were incubated at 37°C under anaerobic conditions using Anaerogen Compact (Oxoid AN010C) for a period of 5 d for Bifidus blood and Columbia Blood plates and 3 d for Rogosa plates. Chromogenic plates were incubated at 37°C under aerobic conditions for 24 h. Colonies characteristic of each bacterial group were counted visually and the concentration calculated as cfu/g wet weight. All colonies were counted on both Rogosa and Columbia agar.

    Statistical analysis. Results are presented as means ± SEM. Data were tested by 2-way ANOVA using the SPSS version 12 statistical package. The 2 factors were diet (Low-RS and Moderate-RS) and bacteria (Control, L. acidophilus, B lactis, L. acidophilus + B lactis). Differences were considered significant at P 0.05. When the diet x bacteria interaction term was not significant, the interaction term was removed from the model. Where there was a significant interaction, each diet was analyzed separately for differences due to bacteria using 1-way ANOVA and the Sidak post hoc test.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Rats. Body weight gains did not differ among the rats fed the different experimental diets. During the 4-wk experiment, weight gains were as follows: Low-RS, 49.0 ± 1.6 g; Low-RS + L. acidophilus, 50.0 ± 3.5 g; Low-RS + B. lactis, 49.8 ± 2.2 g; Low-RS + L. acidophilus + B. lactis, 46.8 ± 2.0 g; Moderate-RS, 49.9 ± 1.6 g; Moderate-RS + L. acidophilus, 50.1 ± 2.1 g; Moderate-RS + B. lactis, 49.4 ± 2.4; Moderate-RS + L. acidophilus + B. lactis, 48.9 ± 2.7 g.

    Fecal fermentation variables. Fecal pH was used as an indicator of distal lumenal pH (26) (Table 2). For fecal pH, there was a significant interaction between diet and bacteria. Fecal pH did not differ among the bacteria-treated groups fed the low-RS diets (P = 0.094), but in rats fed the moderate-RS diet, it was elevated in those treated with bacteria (P < 0.05).

    Cecal fermentation variables. Cecal pH was lower in rats fed the moderate-RS diets (P < 0.001) compared with rats fed the low-RS diets. Supplementation of probiotics to the RS diet did not affect pH (Table 3).

    Microbiological studies. The level of dietary RS affected total anaerobes (P = 0.021), total aerobes (P < 0.001), lactobacilli (P < 0.001), bifidobacteria (P < 0.001), and total coliforms (P < 0.001) in the cecal digesta (Table 4). Furthermore, the type of bacteria supplemented to the diet had a significant effect on lactobacillus species numbers (P = 0.004). B. lactis supplementation increased Lactobacilli numbers.

View larger version (45K): [in this window] [in a new window]   FIGURE 1 Apoptotic index in the distal colon of rats fed a low-RS or moderate-RS diet supplemented with L. acidophilus, B. lactis, or both. Data are expressed as means ± SEM, n = 12. There was a significant interaction (P = 0.002) between diet and bacteria (2-way ANOVA). Each diet was analyzed separately by 1-way ANOVA and Sidak’s test for differences due to bacteria. Means for the moderate-RS diet without a common letter differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

There are several reports in which a synbiotic combination showed biological effects beyond those of the individual components. Rowland et al. (2) demonstrated a reduction of preneoplastic ACF using the synbiotic combination of inulin and B. longum. Gallaher and Khil (27) also showed a reduction in ACF using oligofructose and bifidobacteria. In another study, Femia et al. (17) found that the combined administration of B. lactis and L. rhamnosus with inulin enriched with oligofructose in rats was able to further reduce the occurrence of malignant tumors induced by the carcinogen AOM compared with the individual components alone.

Although each of these studies suggested several mechanisms of action by which synbiotics might be protective, there is little direct evidence to implicate specific effects on biological events of relevance to oncogenesis. Apoptosis is an important regulatory process in the protection against the development of cancer. Apoptosis provides an innate cellular defense against oncogenesis by processes that include removal of cells with genomic instability that have developed during oncogenesis (28) and by deletion of cells suffering DNA insult from genotoxic agents such as carcinogens (29). Upregulation or facilitation of apoptosis during initiation events might increase the elimination of mutated cells that might otherwise progress to malignancy (14,15). Such an effect might be the mechanism by which prebiotics and/or probiotics act to protect against colorectal cancer.

We showed previously that the moderate amount of RS used in the present study did not affect the AARGC, whereas higher amounts (i.e., 20 or 30 g Hi-maize 958/100 g diet) do have an effect (12). Thus we used this dose of RS to test for a synbiotic effect. Because it was suggested that a mixture of probiotics might be more effective than individual strains, (21) we combined the RS with 2 probiotic strains. The synbiotic combination of RS with B. lactis clearly enhanced the apoptotic response, i.e., by 33%. This is likely to be biologically relevant because it was estimated that only a small change (<1%) in the proportion of apoptotic cells in the crypt is required to influence development of colorectal tumors (24,30,31). This might have important implications for humans because the proportion of starch consumed as RS (10 g Hi-maize 958 per 100 g diet) is feasible in the context of the human diet and not likely to create a serious problem of side effects such as flatulence and bloating (32).

A possible mechanism by which the B. lactis in combination with RS facilitated AARGC may be through the immunomodulating properties of probiotic bacteria (33). Cytokines such as tumor necrosis factor (TNF-) are capable of inducing apoptosis (34). Although cytokine levels were not measured in the present study, other studies have shown that the levels of cytokines such as TNF-, interferon- and interleukin-10 may be increased with probiotic supplementation (35).

RS influences fermentation variables such as pH and SCFA concentrations, including butyrate (14,36,37). The RS concentration used in the present study consistently altered all fermentation variables measured, including significant increases in butyrate concentrations in the cecum and feces. Butyrate is associated with induction of differentiation, suppression of proliferation, and enhanced apoptosis in vitro (38–41) and may be associated with enhanced apoptosis in vivo (12,14). Increased luminal concentrations have been directly linked to protection in the rodent model (19). Although the probiotic strains used did not influence SCFA production, and with only minor alterations in pH, it seems likely that RS-induced changes in the luminal microenvironment (such as acidification of digesta, SCFA production, or changed balance of microfloral species) might create a situation that enables B. lactis to exert a proapoptotic affect. RS in the form of high-amylose maize starch was reported to increase survival of bifidobacteria at low pH in the intestinal tract of mice through adhesion and utilization of the starch granules as an energy source to sustain their growth (42). This might increase amounts of metabolically active B. lactis in the colon.

Lactobacilli are not apparently sustained by RS in the rat. Wang et al. (42) reported that none of the lactobacillus stains that they tested showed any starch-degrading activity. This may well explain why L. acidophilus in combination with RS had no effect on the AARGC.

In conclusion, the synbiotic combination of RS and B. lactis significantly facilitated the apoptotic response to a genotoxic carcinogen in the distal colon of rats. It appears likely that ingested RS acts as a metabolic substrate, thus creating the right conditions for B. lactis to exert its proapoptotic action. Because the synbiotic combination of these agents facilitates the apoptotic response to DNA damage by a cancer initiator in the colon of rats, it warrants further study for its capacity to protect against colorectal cancer.

	FOOTNOTES

 
¹ Support by the National Starch and Chemical Company, Bridgewater, NJ.

³ Abbreviations used: AARGC, acute apoptotic response to a genotoxic carcinogen; ACF, aberrant crypt foci; AOM, azoxymethane; low-RS, no supplemented resistant starch; moderate-RS, supplemented with 10% Hi-maize®; NSP, nonstarch polysaccharide; RS, resistant starch; TNF-, tumor necrosis factor .

Manuscript received 16 November 2004. Initial review completed 13 December 2004. Revision accepted 15 February 2005.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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