Methodology

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6.1. Human intestinal microflora (Collins & Gibson, 1998)

The identification of factors controlling or influencing the composition of the human intestinal microflora, including prebiotics and probiotics, may be compromised by the precision of current methodologies for determining bacterial composition which are based almost entirely on phenotypic approaches. Whilst these have met with some success, when done properly, they are time-consuming, laborious and lack the resolving power necessary to analyse the complex microbiota at the species or subspecies level.

Traditional gut microbiological methodologies are usually based on morphological and biochemical properties of the organisms (Table 2). Whilst such an approach is cost-effective and allows the processing of replicate samples, the procedures used may be unreliable and may lack resolution. For example, the metabolic plasticity of organisms is problematic and the test used may not be reproducible. Phenotypic characterization does not allow a high degree of fidelity and is most useful for genus level identification. In some cases this situation is eased if the test organism exhibits a specific metabolic trait. For example, bifidobacteria may be detected, on a qualitative basis, by the production of fructose-6-phosphate phosphoketolase activity. An additional problem is that traditional cultivation-based methods may result in underestimation of microbial diversity, due to the presence of organisms that cannot be

Table 2. Methods for study of the human gut microbiota

Method

Advantages

Disadvantages

Morphological and biochemical characteristics

Specific biomarkers, e.g. certain cell-wall antigens, cellular fatty acids, plasmid profiles, serotyping, resistance to antibiotics Ribotyping (RNA polymorphisms)

16 S ribosomal RNA typing

Straightforward to carry out. Can run in parallel a large number of replicates. Relatively inexpensive

Cultural procedures may not be required

Reliable. Very high discriminatory power

High fidelity. Reliable. Cumulative database allows placement of unknown species. Applicable to culturable and non-culturable forms. Allows probe development

Involves operator subjectively to recognize different colonial and cellular morphologies. Lack of discriminatory power and subject to metabolic plasticity of the organisms. Applicable only to culturable bacteria Cannot assign the position of hitherto unknown species. Relies on all test organisms having unique biomarker. Stability of the biomarker may be questionable

Applicable only to culturable forms. Cannot assign the taxonomic position of any unknown species Costly for both reagents and large-scale equipment, e.g. automated sequencers. Recommended for partial use only cultivated, which therefore elude isolation. This has led to the development of alternative strategies for assessing microflora changes.

The detection of biomarkers that may be attributed to certain components of the flora offers some potential. However, to be wholly effective there would be a need for all the major bacterial components of interest to be separable by individual biomarkers, e.g. changes in cellular fatty acids. This may be feasible but would be difficult to prove in a reliable manner.

An attractive solution to the problem of determining microflora changes accurately, lies in the application of modern high-resolution molecular genetic techniques. Recent advances in the field of molecular biology are revolutionizing the characterization and identification of micro-organisms (Pace, 1996). For example, molecular sequence analysis, particularly of ribosomal RNA (rRNA), provides a powerful tool for determining the genetic interrelationships of micro-organisms, and allows systematic monitoring of the gut flora response to dietary intervention. By utilizing diagnostic sequences within the rRNA, it is possible to design gene probes that facilitate precise identification. The use of polymerase chain reaction technologies may also allow access to non-culturable micro-organisms. Over the next few years, 16 S rRNA sequence analysis is expected to rapidly advance our knowledge of the true genetic diversity of the gut micro-biota, including organisms that evade traditional identification, due either to a lack of taxonomic resolution and/or non-culturability.

Molecular approaches have already been used to determine changes in the composition of the microbial gut flora (Langendijk et al. 1995; Kok et al. 1996; McCartney et al. 1996; Wilson & Blitchington, 1996). Clearly, however, before such techniques are routinely used in gut microbiological applications, the fidelity and efficacy of such methods need to be rigorously evaluated. A comparative phylogenetic framework of gut micro-organisms, based on genetic material such as 16 S rRNA, would allow highly discriminatory and dependable diagnostic probes to be developed. Preferably, the probes should be validated using different procedures, such as in situ and dot blot hybridizations.

Another approach to analyse the genetic diversity of complex microbial populations is denaturing gradient gel electrophoresis or temperature gradient gel electrophoresis. The technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16 S rRNA, all of the same length (Muyzer et al. 1993). This results in unique separation patterns for different microbial populations, and will contribute to the description of changes or differences in microflora composition of uncharacterized microbial populations.

The potential benefits of such technologies in gut microbiology, especially dietary modulation for improved health, are large, particularly when used in conjunction with traditional phenotypic procedures. The use of molecular genetic approaches for qualitative and quantitative monitoring of the human intestinal microbiota will constitute an essential step forward for determining the validity of the functional food concept, when directed towards the role of the gut flora.

6.2. Functional analysis of the gut microflora (Rowland, 1995)

The complexity of the gut microflora, coupled with time-consuming procedures necessary to identify and enumerate the anaerobic components, makes their characterization by conventional methodology difficult and expensive. In particular, such methods are not suited to studies involving large numbers of subjects or treatment regimens. Less comprehensive studies (e.g. identification of major groups) are open to the criticism that any induced changes occurring in genera or species other than those being enumerated will be missed. Furthermore, although bacteriological investigations are useful in describing the basic ecology of the gut, they are of less value in studies of metabolism, nutrition and cancer.

An alternative approach is to use biochemical assays that measure the functional activity of the flora as a whole and thus permit deductions to be made regarding the role of the flora in the metabolism of dietary components. In addition, by selecting microbial enzyme activities or metabolic endpoints resulting in compounds with potentially toxic or beneficial effects, probable health consequences for the host can be assessed.

  1. 2.1. Bacterial enzymes. The bacterial enzymes commonly assayed include /^-glucuronidase (EC 3.2.1.31), /3-glucosidase (EC 3.2.1.21), azoreductase, nitroreductase, nitrate reductase (EC 1.7.99.4), the conversion of pre-carcinogen 2-amino-3-methyl-7H-imidazo[4,5-/]quinoline (IQ) to 7-hydroxy-2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-/]quinoline-7-one (7-OHIQ). The substrates of these enzymes and the functional and health implications of their products have been extensively reviewed (Rowland, 1995). For example, bacterial /3-glucuronidase in the colon is able to release carcinogens from hepatically derived glucuronic acid conjugates and is a critical factor in the enterohepatic circulation of drugs and other foreign compounds. /3-Glucosidase hydrolyses plant glycosides to release the aglycones, many of which are mutagenic, although some exert anti-carcinogenic activity (Rowland, 1995). Azo- and nitroreductases reduce their substrates to amines, which are usually more toxic than the parent compound, and nitrate reductase generates the highly reactive and toxic anion nitrite. Bacterial conversion to 7-OHIQ is one pathway of activation of the pre-carcinogen IQ (formed during cooking of meat and fish) to a genotoxic derivative.
  2. 2.2. Bacterial metabolites in faeces. Faecal metabolites that are indicators of bacterial activity relevant to colonic health include NH3, a toxic product of bacterial breakdown of protein and urea (Clinton, 1992), and phenols and cresols, which are derived from amino acid catabolism by gut bacteria (Macfarlane & Macfarlane, 1995). The production of NH3 is closely related to bacterial activity and is associated with certain toxic events in the gastrointestinal tract. NH3 is considered to be a potential tumour promoter in the colon, and has been postulated to enhance neoplastic transformation in the gut.

Other gut bacterial products with possible adverse effects on the colonic mucosa include /V-nitroso compounds, which are potentially carcinogenic substances formed by bacterial catalysis of the reaction of nitrite and nitrogenous compounds in the colon (Rowland, 1995), diacylglycerol, a putative tumour promoter derived from lipid breakdown, and secondary bile acids, deoxycholic and lithocholic acids, also putative tumour promoters.

6.2.3. Assessment of cytotoxicity, genotoxicity and mutagenicity of faeces. An alternative approach to assaying enzymes or metabolites in faeces is to assess toxicological activity of fractions using short-term tests for toxicity, genotoxicity and mutagenicity. This provides a direct estimate of the potential of the faecal sample to damage the colonic mucosa and has been used to provide insights into possible processes involved in colon cancer. Usually, the aqueous phase of human faeces (faecal water) is used (Rafter et al. 1987). Cellular toxicity can be assessed using rapid colorimetric assays in multiwell plates. For example the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983) involves the pH-dependent conversion of water-soluble MTT to water-insoluble formazan and provides a reliable estimate of viable cell number.

Recently, using the Comet assay, it has been shown that the genotoxicity of faecal water varies markedly between individuals, with at least some of the DNA

damage occurring via an oxidative mechanism (Venturi etal. 1997).

6.2.4. Susceptibility of functional markers to dietary change. In animal models, major changes in activities of bacterial enzymes and in levels of bacterial metabolic products have been seen after a wide range of dietary changes. These include the type and level of dietary fat and protein, supplementation with dietary fibre and resistant starch, and addition of oligosaccharides (Rowland, 1991). In many cases changes were seen in the absence of alteration in composition of the gut flora.

In contrast, modification by diet of bacterial metabolism in human subjects has proved more difficult. However, changes in enzyme activities and concentrations of NH3, phenol and cresol have been detected in volunteers consuming lactulose (Terada et al. 1992) and lactobacilli (Goldin & Gorbach, 1984).

A low level of NH3 production in the gut is associated with low-protein, high-fibre diets, which appear to be protective against cancer of the colon. NH3 levels have been shown to be elevated in rats consuming a diet containing high-risk factors for colon cancer (Hambly et al. 1997).

The cytotoxicity of faecal water strongly correlates with bile acid concentration in faeces and is increased in individuals on high-fat diets (Rafter et al. 1987) and decreased in subjects on high-resistant-starch diets (van Munster et al. 1994).

6.3. Digestibility and bioavailability of foods

The methodology for studying non-digestibility of foods is an important area for understanding the effects of food and food components on intestinal microflora and physiology. A compilation of current methodology is given in Table 3. It is important to identify the nature of molecules, their chemical bonds and molecular size to understand digestibility. In vitro digestion studies and markers for absorption and excretion are of value. Animal models offer a means of simulating different digestion extremes and human volunteer studies enhance the understanding of in vivo digestibility of foods and bioavailability of nutrients.

6.4. Large-bowel function

Study of large-bowel function is extremely difficult, mainly because of its inaccessibility. However, the large bowel has unique aspects of metabolism; the principal events in the lumen are anaerobic and end-products such as H2 and SCFA are not produced by other biochemical reactions in the body. These products are absorbed and appear in blood and breath, and can be used to study intraluminal events. Many investigators have used faeces and their composition as a guide to intracolonic events. Such studies have often been criticized because of the lack of representativeness of faeces, but for some aspects of colonic metabolism, such as the gut microflora of the lumen, they are probably acceptable. The study of the large intestine has spawned a large number of in vitro models, particularly of fermentation. A short summary of methods is given in Table 4.

Table 3. Methods to study the digestibility and bioavailability of foods

Method

Advantages

Disadvantages

  1. Chemistry
  2. In vitro model
  3. Blood appearance

4. Breath

  1. Ileostomy model
  2. Intestinal intubation: jejunum, ileum, colon, perfusion/aspiration, single or multilumen tubes
  3. Faecal analysis
  4. In vitro fermentation
  5. Animal models: germ-free/conventional, fistulated

Identifies nature of molecules, chemical bonds, molecular size, etc. (e.g. most /3-glucans are not digested)

Studies with pancreatic and other enzymes

Glucose tolerance, chylomicrons. Applicable to a large number of samples

H2 or CH4,13C02. Simple non-invasive for human studies

Probably the gold standard for study of digestion in stomach and small bowel

Valuable for dynamic studies of flow rate, site of digestion in small bowel, nutrient concentrations and physical form, and identification of intermediate products of digestion

Relatively straightforward and gold standard for overall digestion and fermentation Various batch and continuous culture methods available.

Good for modelling physiology Gives greater access to tissues. Germ-free, human associated and conventional flora

Requires dedicated carbohydrate chemistry laboratory

Care needed to mimic conditions in gut and hence rate and extent of digestion Requires human subjects and can be affected by factors other than those determining digestion Difficult to quantify. Prolonged studies (16-24h)

needed for fermentation. Large subject variability Requires access to patient population. May underestimate losses to caecum because of microbial colonization of ileum and fermentation in bag

Very invasive human studies. Need X-ray control. Presence of tube may alter normal physiology. Difficult to make quantitative

Requires accurate faecal collections with balance markers and good methods validated for faeces Needs well-founded microbiology laboratory

Animal facilities needed. Extrapolation to man

Table 4. Methods for studying large-bowel function

Focus of study

Method

Bowel habit

Diary record

Motor function

Motility recording

Recto-anal manometry and pelvic floor

neurophysiology

Transit time:

whole gut - non-absorbable markers

Partial transit time:

small bowel - isotope-labelled meal and

gamma scanning

- lactose breath H2

large bowel - X-ray following marker ingestion

Faecal analysis

Microflora, and enzymic activities

Fat, N, biomass, carbohydrate, DM, bile acids,

sterols, pH

Faecal water: osmolarity, pH

electrolytes

bile acids

genotoxicity/cytotoxicity

Occult blood

In vitro models

Transport physiology

Batch cultures, single chemostats,

multi-chamber chemostats

Blood (including

Short-chain fatty acids, branched-chain fatty

portal blood)

acids, bile acids

Breath

H2, ch4, C02

Mucosal biopsies

General histology and immuno-stalning

Exfoliated cells

DNA adducts

Mutational analysis (e.g. Kras)

Proliferation markers

Genotyping

Apoptosis

Mucus

Composition and structure

Thickness

Breakdown

X-ray

Barium studies

Plain film of abdomen

Endoscopy

Proctoscopy

Sigmoidoscopy (including flexible)

Colonoscopy

Angiography

Vasculature

Radiotelemetry

PH

Motility

6.5. Gut-associated lymphoid tissue

The presence of the digestive flora has a considerable influence on the immune system of the host (see section 3.5) and the principal methodology to study this is the use of germ-free animals, also termed axenic animals. By comparing germ-free and conventional animals, it is possible to highlight the role of the digestive flora in immune function. Moreover, the role played by bacteria isolated from the digestive flora, or used as probiotics, can be analysed by inoculating the gut of germ-free animals with these bacteria. These are called gnotobiotic animals. Recently, germ-free mice associated with human flora have been developed allowing in vivo studies of functional properties of probiotics and prebiotics used in human nutrition.

The advantages of gnotobiotic animal studies are to determine which kind of immune response a given bacteria established in the gut is able to exert, e.g. non-specific and/or specific immune response; inductive or suppressive immune response. Many methodologies are available. They are summarized in Table 5 and marked with (H) when they are applicable to human studies. The disadvantages of gnotobiotic models are the expensive animal facilities they need and the limits of the animal species studied. Moreover, it is not certain that a modulating bacterial effect observed in gnotobiotic conditions will be expressed in conventional conditions. Thus, other studies using conventional animals are needed. Current methods utilize in vitro cultures of systemic or intestinal lymphoid cells (De Simone et al. 1993), cellular assays with colonic cell lines (Schiffrin et al. 1995) and in vivo assays with conventional animal models (Perdigon et al. 1996). Two types of specific immune response can be assessed at the intestinal level. These include the suppression of humoral and cellular immune responses to chronically administered antigens at the systemic level (immune regulation) and the induction of a protective IgA antibody response at the mucosal level

Table 5. Selected methods to study intestinal immune function

Immune response

Methodology

Advantages

Disadvantages

Proliferative assays

Cytokine production Phagocytic activity

Modulation of molecular expression in intestinal cell lines

IgA antibody response

Oral tolerance to dietary antigens

In vitro: measurement of the proliferation of systemic or intestinal lymphoid cells after stimulation with mitogens or cell components

In vitro: after proliferative assays

In vitro: peritoneal cells, circulating cells

In vitro: HT-29, CaCo-2 cell line cultures

Facs analysis, histochemical methods

Measured by ELISA or ELISPOT at several levels:

  • 1) In serum: soluble IgA antibodies
  • 2) In blood: circulating IgA-producing cells
  • 3) In faeces
  • 4) In saliva
  • 5) Whole gut lavage fluid

Ex vivo: proliferative assays of lymphoid blood cells with specific antigen

Inflammatory cytokine production by lymphoid blood cells

In vivo: intestinal permeability

In vitro: Ussing chamber

Easy for systemic assays: from blood samples (H)

Development of new methodologies (H) Biomarkers (H)

Easy for systemic assays: from blood samples (H)

Cell lines originated from human intestine

Specialized line cells Biomarkers

Easy in human subjects (H) Reflects intestinal response (H) Easy, allows kinetic studies (H)

Invasive, but avoids intestinal biopsy (H)

Direct measure of the unresponsiveness state (H)

Biomarkers: TNF-a (H)

Increase in food hypersensitivities (H)

Direct correlation with antigen transfer

Not easy for intestinal assays in human subjects: need biopsy Technical difficulties Need correlation with in vivo assays to give a biological significance Need correlation with in vivo assays to give a biological significance Correlation with specific immune response poorly understood Technical difficulties for intestinal phagocytes Adenocarcinoma lines Absence of correlation with intestinal cellular environment

Does not reflect the intestinal response Does not reflect transepithelial transport into gut lumen Individual and daily variations, proteolytic activity, reflects only colonic response Individual and daily variations Needs hospitalization

Cause or consequence of oral tolerance breakdown? Not specific to immunological changes Invasive and difficult technology

H, applicable to human studies; immunospot assay.

IgA, immunoglobulin A; TNF-a, tumour necrosis factor-a; Facs, fluorescence-activated cell-sorting; ELISPOT, enzyme-linked

(immune exclusion). The definition of biomarkers is still incomplete. Several methodologies have been developed for IgA response measurements. Sampling needs blood collection or intestinal biopsies or stool and saliva collection. Problems in faeces and saliva handling and daily variations in expression of IgA activities have not been solved. In the case of oral tolerance, the biomarkers available in human subjects are defined as the absence of proliferation of cultured blood cells with antigen or down-regulation of inflammatory cytokines involved with food allergies (Benlounes et al. 1996; Siitas et al. 1996a,b). Other methodologies must be developed according to further knowledge about oral tolerance mechanisms (Weiner et al. 1994).

  1. 6. Epithelial cell proliferation and colon carcinogenesis
  2. 6.1. Biological markers for colorectal carcinogenesis. A relationship between colorectal carcinogenesis and abnormal cell proliferation has been demonstrated (Lipkin, 1988) in studies of the mucosa in patient groups at high risk of cancer, e.g. ulcerative colitis and familial adenomatous polyposis, and in animals exposed to carcinogens that target the colon. Two changes in colonic cell proliferation have been described (Risio, 1992): an increase in the total number of proliferating cells, or hyperproliferation, which is not a specific marker of cancer risk, and a progressive shift of proliferating cells to the crypt surface (stage II abnormality) which is more specific for tumour risk.
  3. 6.2. Cell proliferation. Several techniques have been developed to measure cell proliferation in colonic mucosa and assess the influence of diet. Measurement of crypt-cell production rate in microdissected crypts is considered to provide the best assessment of proliferation with the fewest artifacts. However, since it requires in vivo treatment with vincristine, it is not suitable for human studies.

Change in the rate of cell proliferation in the normal mucosa may be less reliable as a biomarker for diet-related cancer risk (Wasan & Goodlad, 1996) and is just one of the processes contributing to colonic mucosal crypt architecture. Other events are differentiation, exfoliation and apoptosis. It is likely that the best predictor of cancer risk is an overall assessment of these events.

Markers of early epithelial events have been used in animal models. These include DNA damage, microadenomas and aberrant crypt foci in the mucosa. The microgel electrophoresis (Comet) assay has been used for assessing

DNA damage in the colonic mucosa. Induction of aberrant crypt foci has been particularly widely used, as it is easy to observe macroscopically. However, its reliability as a marker of colorectal tumour risk is a matter of debate. In man, aberrant crypts and microadenomas, similar to those described in animals, have been described (Roncucci, 1992) but their correlation with other well-known markers of risk has not been established. Other markers of cell proliferation include PCNA and Mib 1; both are proteins appearing at specific stages in the cell cycle.

  1. 6.3. Differentiation. Methods exist to measure the state of differentiation of the mucosal epithelial cells by histochemical staining of mucins using binding to specific lectins. The methods can be applied to tissue sections from human and animal biopsies after fixation. Such approaches have been used to study colonic epithelium in rats and human subjects at various stages of neoplasia. They have not achieved widespread use for investigating dietary modification of neoplastic processes.
  2. 6.4. Apoptosis. Identification of oligonucleotide fragments by in situ end labelling using immunoperoxidase techniques forms the basis for various methods that can be applied to sections of colonic tissue from human subjects and animals and to cell suspensions (Ansari et al. 1993). Apoptosis in cell cultures from transformed colon tissues has been determined by measuring cell loss from monolayers (Hague et al. 1993).
  3. 6.5. Products used in experimental carcinogenesis (Martin et al. 1981). Spontaneous colorectal cancer is exceptional in animal models, but tumours can be created easily in rats, mice or hamsters using chemicals such as yV-methylnitrosourea and iV-methyl-W-nitro-Af-nitroso-guanidine (Table 6). These products are direct carcinogens, which explains their local efficacy and their specificity when administered intra-rectally. Others such as 1,2-dimethylhydrazine or azoxymethane must be first metabolized by the liver, then at the level of the target cell, in order to be carcinogenic.

1,2-Dimethylhydrazine and derived azo and azoxy alkanes represent the carcinogens most commonly used to induce intestinal carcinomas in rats, mice and hamsters, by the oral or subcutaneous route. In rats, precancerous lesions such as adenomas are exceptional, cancers arising most commonly without any precursor adenoma, which is the opposite to what is observed in man. In mice, it is easier to induce adenomas than carcinomas (Maskens, 1976).

Secondary bile acids are cocarcinogens. They have been shown to promote colorectal carcinogenesis in animal models by increasing tumour formation rate induced by carcinogens, or increasing colonic cell hyperproliferation through the production of diacylglycerol and stimulation of protein kinase.

6.6.6. Types of lesion (Weisburger, 1973). The most studied lesion is adenocarcinoma induced either directly or, more often, indirectly. When azoxymethane is used by the subcutaneous or intramuscular route, intestinal carcinomas are formed in 100% of cases. Small doses induce tumours in the proximal colon and the caecum, whereas larger doses produce tumours mainly in the distal colon. Such tumours arise on flat mucosa and form plaques, thus mimicking human infiltrating tumours, which arise without any detectable adenomatous tissue and are named de novo cancers. However, this type of tumour is rare in human carcinogenesis, particularly in Western countries where tumours are mainly of the fungating type and arise in a pre-existing adenoma. Aberrant foci are interesting to study as they can be easily observed macroscopically. After a single injection of azoxymethane, aberrant foci have been described after early slaughter of the animals. These lesions are considered by some authors to be an early marker of tumour risk. They are more common in the rectum (90% of animals) than the caecum (10% of animals), but tend to migrate with time, with a decrease in rectal lesions and an increase in caecal lesions after 4 weeks.

Changes in colonic cell proliferation, which will be described in man elsewhere, are largely used to test the protective effect of products, e.g. Ca, against secondary bile acid-induced proliferation.

6.6.7. Transgenic mouse models for colon cancer studies. A number of inbred mouse models carrying germ-line mutations at the Ape gene (the murine homologue of APC, which is mutated in patients with familial adenomatous polyposis) have been developed. These animals exhibit spontaneous tumours throughout the intestinal tract, usually in the first few months of life. They are of use in studies of the interaction of diet and colon cancer and provide a model that dispenses with the need for chemical induction of carcinogenesis. The susceptibility of tumour incidence in these mice to dietary modulation is under investigation in a number of laboratories. Table 7 lists four such mouse models in current use.

Table 6. Current methods for studying colon carcinogenesis in animals

Products Route Lesions

MNNG (A/-methyl-A/'-nitro-A/- Intra-rectal instillations Colon adenocarcinomas, squamous nitrosoguanidine) and MNU cell anal carcinomas, spleen and

(N-methylnitrosourea) liver haemangiomas

DMH (1,2-dimethylhydrazine) Oral or subcutaneous Multiple colorectal adenocarcinomas and a few adenomas in rats

Azo and azoxy alkane derivatives Subcutaneous or intramuscular In mice adenomas, and a few carcinomas; adenocarcinomas in plaques {de novo)

Secondary bile acids Intra-rectal or oral Alone: colonic cell hyperproliferation

After DMH: increased number of carcinomas

Table 7. Transgenic mouse models currently in use in colon cancer studies

Model

Mutation

Tumour yield per animal

Min (multiple

Germline nonsense

About 100

intestinal

mutation at

adenomas with

neoplasia), usually

codon 850 of Ape

the majority in the

used in the hetero

small intestine

zygous cross with

C57B16/J mice

Apc1638N

Ape disrupted at

About five intestinal

codon 1638,

tumours

probably leading

to a null allele for

Ape

Ape1638T

Ape disrupted at

No intestinal

codon 1638

tumours at 5

leading to a

months

truncated

polypeptide

Ape A716

Ape disrupted at

200-500 intestinal

codon 716,

adenomas

leading to a

truncated protein

6.6.8. Limits of experimental models (Maskens & Dujardin Loits, 1981). Experimental carcinogenesis in animals creates mainly infiltrating de novo carcinomas, and is an indirect process in most cases, i.e. multiple metabolic transformations are needed, the last occurring in the colon itself and involving bacterial enzymes such as 0-glucuronidase, azoreductase or nitroreductase. In man, in particular in high-risk countries such as Western Europe, North America or Australia, most colorectal tumours are polypoid, fungating, and arise on a pre-existing adenoma. It has been estimated that, in the distal colon and rectum, where most tumours arise, over 80% of cancers arise through the adenoma-carcinoma pathway. Indirect evidence, such as the type of mutation of the p53 protein, points to a major role played by secondary bile acids, i.e. endogenous carcinogenesis, whereas carcinogenesis in animals is called exogenous carcinogenesis. The role of the latter may be more important in low-risk countries such as Japan, but is likely to be of little importance in high-risk countries. Therefore, it is difficult to extrapolate from animal models to man, in particular regarding the importance of bacterial enzymes, apart from the major role played by the 7 a-dehydroxylase, which converts primary into secondary bile acids.

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