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Asia Pacific J Clin Nutr (1996) 5: 48-52

Probiotics and colon cancer prevention

Graeme H McIntosh, BUSc, PhD (ANU)

CSIRO Division of Human Nutrition

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This review examines some of the evidence regarding probiotic bacteria as agents to reduce the risk of colon cancer in humans. While some of the evidence using rodent models of colon cancer is convincing for a reduction in cancer incidence and burden with the introduction orally of such bacteria as Bifidobacterium longum, Lactobacillus acidophilus and gg, convincing evidence in humans is more difficult to find. It consists of epidemiological studies or marker intervention studies using faecal enzymes, faecal bile acids or urinary/ faecal mutagens from microbial activity as measures of cancer risk, following probiotic introduction. Taken together these sources of data provide limited support for the hypothesis that probiotic bacteria are effective in cancer prevention.

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Introduction

Colon cancer is a major health problem in Westernised cultures like Australia, and diet is considered to be a major factor influencing its prevalence. Diets containing high animal proteins and fat and low dietary fibre have been identified as being associated with greatest risk. Recent research has also focused on the influence that gastrointestinal microflorae have on outcome. A large and complex microbiological population inhabiting the colon was seen by some as being established early in life and relatively unchangeable by external factors. However, other research has suggested that it is manipulatable by dietary and microbiological means as well as by antibiotic therapy. The side effects of modern antibiotic therapy may include significant disturbances of the gut microflora and have been part of the motivation to find ways of achieving treatment of disease using ‘desirable bacteria’ as an alternative therapy. Insofar as the microflora can influence the immune system, nutrient metabolism, detoxification and carcinogen activation mechanisms and thereby the expression of a number of disease processes affecting the bowel, a better knowledge of their contribution to health and disease is warranted.

Probiotics are defined as live microbial food supplements which benefit the host by improving its intestinal microbial balance¹. Yoghurt is a traditional and common vehicle for such probiotics (Lactobacilli and Bifidobacteria species being most often used in this role). They have a significant target, gastrointestinal disturbances and diseases. Wider claims include their value as a life extender, an elixir of life. Elie Metchnikoff², the Russian Nobel Prize biologist, popularised the view that some lactic acid bacteria were capable of increasing length of life, supporting his theory with observations of Bulgarians who ate yoghurt regularly and showed remarkable longevity. At the time, 1000 it created a world-wide interest in yoghurt but in the ensuing 70 or so years little attention was given to this claim. However, in the last 20 years, with the upswing in colon cancer and inflammatory bowel diseases in Westernised countries, it has been given increasing attention by researchers.

Table 1. Chemical colon cancer studies in rats and mice         % rats with colon carcinoma Goldin and DMH Beef   77% Gorbach (S/C) rats Beef +   40% (17) n =22 L. acidophilus             % dead at 36 wks (colon carcinoma) Shakelford DMH Skim milk   28% et al (26) (S/C) n = 25 SM + L. bulgaricus   7%   F344 rats SM + S. thermophilus   10%       Tumours/tba % rats with colon tumours Takano et DMH (S/C) Control 2.6 100% al (27) rats n = 9 + L. helveticus and C. utilis 1.0* 66%       Aberrant crypts/colon       Wk 20 Wk 30 Koo and DMH (S/C) • C 14 20 Rao (28) CF1 mice +B. longum 7* 10*   n = 21 + 5% neosugar             Aberrant crypts/colon Kulkarni and AOM (S/C) • C   249 Reddy (29) rats +B. longum 1.5%   142*   n = 11 +B. longum 3.0%   130* S/C = subcutaneous; DMH = dimethylhydrazine; C= control; tba = tumour bearing animals * Significance at p< .05 Table 2. The effects of oral consumption of lactic cultures on faecal enzyme activity < 1000 /tr> Reference Bacteria used Reduction of faecal enzyme activity Goldin et al L. acidophilus + nitroreductase (18) (7 subjects) - azoreductase     + b -glucuronidase     - steroid 7- a -dehydroxylase Goldin and L. acidophilus + nitroreductase Gorbach (20) (7 subjects) - azoreductase     + b -glucuronidase Goldin and L. acidophilus NCFM + nitroreductase Gorbach (19) L. acidophilus N-2 + azoreductase   (22 subjects) + b -glucuronidase Marteau et al Milk fermented with + nitroreductase (30) L. acidophilus B.bifidum, + azoreductase   and mesophilic - b -glucuronidase   cultures(9 subjects) b -glucosidase Goldin et al Lactobacillus GG frozen + b -glucuronidase (31) concentrate (8 subjects)   Lidbeck et al L. acidophilus milk ? b -glucuronidase (12) (12 subjects)   Ling et al L.gg +b -glucuronidase (32) (64 subjects) + nitroreductase     + glycocholic acid hydrolase Kulkarni and B. longum + b -glucuronidase Reddy (29) F344 Rats     (33 rats)   + = statistically significant positive, results; - = negative results; ? = results not definitive. Table 3. Some oligosaccharides used to promote bifidobacteria in vivo40 Lactulose, Lactitol, Lactobionic Acid Neosugar P® Transgalactosylated oligosaccharides Galactooligosaccharides, Oligomate® Gluconic Acid Xylooligosaccharides Fructooligosaccharides Maltooligosaccharides Stachyose, Raffinose

Useful reviews by Mitsuoka³, Adachi⁴, Marteau et al⁵, and Ballongue⁶, have discussed much of the groundwork research studies, in an area where Japanese and French researchers have made significant contributions.

From North America reviews are provided by Fernandes et al⁷, Fernandes and Shahani⁸, Gorbach and Goldin⁹, Sanders¹⁰; while from Scandinavia Lidbeck et al¹¹, Salminen¹², Rafter¹³ useful reviews have also been provided.

Research with regard to use of probiotics in prevention of colon cancer is reviewed in this paper. It must be appreciated that lack of knowledge of the carcinogenic process, the complexity of colonic function, and lack of techniques for adequately identifying specific strains of bacteria, has held back progress significantly. Nevertheless there have been some impressive advances, which I believe are bringing us nearer to predicting a protective diet and/ or probiotic strategy for reducing high rates of colon cancer. The anti-tumour action of probiotics have been proposed as:

• direct suppression of the carcinogens and/ or procarcinogens by binding, blocking, removing;
• inhibition of bacteria which directly or indirectly convert procarcinogens to carcinogens by enzyme activity etc.
• activation of the host’s immune system to antitumourigenesis
• reduction of the intestinal pH, thereby altering microbial activity, solubility of bile acids, mucus secretion etc.
• alteration of colonic motility and transit time.

Malhotra¹⁴, a medical officer with the Indian railways, reported on the gastrointestinal cancers in India, and proposed that the much lower incidence of colon cancers in northern people was associated with the significant consumption of dairy (including fermented) foods, cereals and vegetable dietary fibres in the regular diet. Southern diets by contrast were low residue highly digestible diets and tended to create a more alkaline colonic milieu. Similar differences have been noted for rural northern versus southern urban populations of Sweden¹⁶, and in Finland versus Denmark¹⁵. The difference in each case was a reduction to one half or one third the colon cancer incidence and mortality.

Research up to the mid 1980s was mainly concerned with the direct or indirect anti-tumour action of streptococci, lactobacilli, and bifidobacteria studied in animals and to a lesser extent in man^17-20. To induce the effect, bacteria were often injected systemically and/ or cancers were transplanted into mice. Bifidobacterium longum had a direct inhibitory effect on liver tumours in the mouse²¹. In the BALB/C m 1000 ouse, B. infantis and B. adolescentis injected subcutaneously or intraperitonelly had an antitumour effect²². The number of tumours developed by mice with an intestinal flora including Eschericia coli, Enterococcus faecalis, and Clostridium paraputrificum was considerably reduced if B. longum was present²¹. Feeding fermented milks or cultures containing Lactobacillus acidophilus, L. bulgaricus and/ or L. casei inhibited Ehrlich ascites tumour cell growth or growth of Sarcoma 180 in mice^23,24.

Goldin and Gorbach^17,19,25 used the dimethyl-hydrazine (DMH) rat model to help assess the impact of lactobacilli on intestinal tumours and their studies and others are presented in Table 1. It was shown¹⁷ that the high incidence of DMH induced colon carcinomas in rats fed beef could be lowered from 77 to 40% when L. acidophilus was fed simultaneously with the beef diet.

Other studies using the same or similar experimental cancer models have largely confirmed this early observation, although as can be seen there have been differing bacteria tested and endpoints of assessment used. More recently, Lactobacillus GG, a known human gut commensurate has been shown also to reduce incidence and tumour numbers of chemically induced colon cancers⁹.

These animal studies have been paralleled by human and animal faecal enzyme studies, assessing nitro-reductase, b glucuronidase, azoreductase and/ or urease activity to predict risk of colon cancer. The hypothesis relies on the assumption that modulation of deconjugating and/ or dehydroxylating enzymes found in certain colonic bacteria but not in others will alter risk of carcinogens being generated from procarcinogenic agents or released from bound form into the gut contents, as they traverse the large intestine. Displacement by probiotic bacteria (of undesirable bacteria) will effect significant change. Significant results (Table 2) have been achieved with this approach to assessment of risk.

For example, Lidbeck et al¹¹, produced a significant increase in lactobacilli and dietary calcium by feeding L. acidophilus fermented milk to colon cancer patients for 6 weeks. Faecal enzyme activity was reduced 14% and soluble faecal bile acids 38%, but both results were not significant. They attributed this result to small number (n=12) of patients and the large variability in enzyme activity between patients. Ling et al³² have shown a greater reduction in faecal enzymes (40%) with the feeding for 4 weeks of lyophylised lactobacillus GG and dietary fibre as cereal rye, relative to controls. Urinary paracresol, a mutagenic metabolite of protein, was also significantly reduced (18% p<0.05). Bartram et al³³ showed they could increase the faecal excretion of B. longum with oral supplements of the bacteria via yoghurt(>10⁹ cfu/L) and lactulose, and that breath hydrogen increased and mouth-cecum transit time increased, but no other changes (such as bile acids, SCFA, pH) were observed. They 1000 attributed this to significant gut microflora stability.

It is apparent from the above studies that there are differences of opinion as to which bacteria offer most potential for human health and cancer prevention, as well as considerable variation in background diets which could significantly influence outcome of such studies. This could account for some of the large differences in results, and present a possible obstacle to progress. To help sort out the bacteria most likely to be effective against colon cancer cells Baricault and co-workers³⁴ introduced the use of an in vitro cultured colon cancer cell (HT-29) assay. The test relied on inhibition of cells to grow into a confluent layer, or to differentiate under the influence of inhibitory bacteria. In an examination of a number of the probiotic bacteria being used currently they identified Lactobacillus helveticus and Bifidobacterium (species not named) as being effective, whereas Lactobacillus acidophilus was not.

Study of the growth requirements of Lactobacilli and of optimal healthy diets have led to the recognition of some desirable substrates for fermentative bacteria in vivo, which when fed alter significantly the proportion of beneficial bacteria present in the colon. without the need to orally supplement bacteria. It could be argued that this aspect of diet may have a bigger impact on health objectives than the provision of probiotic bacteria orally. Ecological studies of faecal microflora support this well^35,36. These 2 reports point very clearly to the impact of diet on colonic microflora and colon cancer risk but come to opposite conclusions regarding the relevance of Bifidobacteria species to colon cancer risk. In a workshop summary report Roberfroid et al³⁷ referred to the circumstantial evidence of colonic microflora on cancer risk, and proposed the absorption and metabolism of mutagens and carcinogens as the primary role in prevention, while SCFA production from carbohydrate fermentation as seen as secondary in its influence.

Studies in my laboratory have identified the potential of whey proteins to significantly reduce cancer incidence (to one half) relative to red meat and soybean protein in the DMH rat cancer model³⁸. This raises the possibility that dairy foods may offer, apart from any probiotic influence, high quality proteins which protect the rat gut from chemical carcinogenesis by an as yet undefined mechanism. It also highlights undesirable characteristics shared by two disparate sources of protein, soybean and red meat. In several of the studies reviewed, grilled or dried beef is used as a background diet to enable a significant improvement to be achieved with probiotics. A recent study by Reddy and Rivenson³⁹ is of interest in this regard. They have used the now well characterised meat mutagen IQ (2-Amino-3 Methylimidazo) (4,5 -f) quinoline to induce cancers in male and female rats. It is capable of producing breast, liver and colon tumours in rats and mice. When B. longum was fed at 0.5% as a lyophilised culture to rats there was 100% suppression of colon tumours, 80% suppression of liver tumours and in females 50% suppression of mammary tumours. Whatever the mechanism for this inhibitory influence, it is an impressive demonstration of a probiotic effect.

1000 A number of studies have reported the use of specific agents to improve the growth of desirable gut microflora such as Bifidus growth factors⁴⁶. They fall into the category of dietary fibre or fibre like components (such as resistant starch, oligosaccharides) which have the attribute of passing undigested through the small intestine to supply a substrate for the colonic bifidobacteria^41-44. A list of some of the agents reported to be beneficial is shown in Table 3. In general their presence in the diet significantly influences the total counts of bifidobacteria in faeces. For example, with 9g/day gluconic acid, 10 healthy volunteers showed a significant increase (p<0.001), while less desirable bacteria like C. perfringens fell in number and Enterobacteriaceae stayed constant⁴⁴.

Provided that increasing bifidobacteria can be identified with reducing risk of cancer, these types of studies support a view that such perturbation of gut flora is in a desirable direction.

Finally there has been considerable research investigating the bacterial and plant cell wall components (peptidoglycans, b -glucans and other polysaccharides) for their influence as an anticancer-strategy in stimulating the immune system via the gut associated lymphoid tissue⁴⁵. This represents a relatively new and challenging area for future research.

Conclusion

There is a promising future for research into probiotic bacteria, to open up a better understanding of the contribution to health of a well constituted balanced microflora in the large intestine. Its potential for prevention of colon cancer is currently under active investigation, with both animal and human studies contributing. Both approaches appear to be valid and necessary, albeit caution should be exercised in extrapolating animal results directly to humans.

While some of the data supports the view that probiotics as freeze dried powder/ capsules or as yoghurts provide protection from colon cancer, the nature of the diet and/ or components provided by the yoghurt vehicle must also be taken into account.

This means carefully controlled experiments are needed to provide reliable interpretation.

Acknowledgements

I wish to acknowledge the assistance of Leanne Griffiths, Librarian, CSIRO Division of Human Nutrition, Dr Martin Playne of the CSIRO Division of Food Science and Technology, Highett Victoria, for help with this task, and the Dairy Research and Development Corporation for its research grant support.

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Chinese abstract

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