Gastrointestinal cancer

Best Practice & Research Clinical Gastroenterology Vol. 18, No. 2, pp. 323 –336, 2004 doi:10.1053/ybega.2004.440, available online at http://www.sciencedirect.com

8 Gastrointestinal cancer Patricia M. Heavey* BSc , Dphil Ian R. Rowland BSc, PhD Northern Ireland Centre for Diet and Health, Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Ireland

A combination of both environmental and genetic factors contributes to the vast majority of human cancers and in particular cancers of the gastrointestinal tract, including the stomach, colon and rectum. The mechanisms associated with cancer causation or prevention are largely unknown and the subject of much research. Many of these mechanisms implicate the metabolic activities of the bacterial flora normally resident in the gastrointestinal tract. This paper examines both the detrimental and beneficial consequences of bacterial activity of the gastrointestinal tract, focusing in particular on the stomach and large intestine. Key words: microflora; gastrointestinal cancer; Helicobacter pylori; colorectal cancer.

The human gastrointestinal tract and, in particular, the large intestine, harbours a large and diverse microflora, with over 1012 bacteria per gram of contents1 and so it is not surprising that the activities of this population have a significant impact on the health of the host. The microflora interacts with its host at both the local level (intestinal mucosa) and systemically, resulting in a broad range of immunological, physiological and metabolic effects. From the standpoint of the host, these effects have both beneficial and detrimental outcomes, for nutrition, infections, xenobiotic metabolism, toxicity of ingested chemicals and cancer. The microflora is completely dependent on the host for nutrients, which are made available in the form of undigested dietary residues (especially indigestible carbohydrates such as dietary fibre, resistant starch and non-digestible oligosaccharides), host secretions (intestinal mucins, enzymes, gut hormones) and sloughed mucosal cells. The host animal acquires some nutrients from the microflora in the form of vitamins and short-chain fatty acids (acetic, propionic and butyric acid) which are absorbed and used as energy sources by the liver and other tissues, including the colonic epithelium.2 Mechanisms by which diet is linked with colon cancer aetiology or prevention are at present largely unknown and the subject of much research. Carcinogenic agents may be present in the diet or formed in vivo during digestion. Many * Corresponding author. Tel.: þ44-28-7083-4313; Fax: þ44-28-7083-4965. E-mail address: [email protected] (P.M. Heavey). 1521-6918/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

324 P. M. Heavey and I. R. Rowland

of these mechanisms involve the metabolic activities of the microflora normally resident in the human colon. The participation of bacteria in carcinogenesis continues to be controversial partly due to the lack of agreement on the molecular mechanism(s) involved in the development of this disease. In normal adult tissues, proliferation, apoptosis and DNA repair are in equilibrium and this ensures a steady state of healthy cells. In the adenoma-carcinoma sequence, for a normal epithelial cell to proceed to carcinoma. Extensive studies on colorectal cancer (CRC) have identified specific genetic changes in various proto-oncogenes, tumour suppressor genes and DNA mismatch repair genes, as well as alterations in DNA methylation status and inherited genetic defects. Subsequently, several molecular pathways have been identified which can contribute to the development of CRC. In 1990, Fearon and Vogelstein3 proposed a genetic pathway of colorectal tumorigenesis, which is now generally accepted as the classic model for the development of CRC (ref. 3 and Figure 1). The model postulates that at least five to seven major molecular alterations need to occur for a normal epithelial cell to proceed to carcinoma. This process is now accepted as central to the majority of cancers and has been studied extensively in colorectal cancer. Bacteria have been linked to cancer by two mechanisms: induction of chronic inflammation following bacterial infection and production of toxic bacterial metabolites. Here we discuss both the detrimental and beneficial consequences of bacterial activity of the gastrointestinal tract, focusing on the stomach and large intestine.

THE STOMACH The presence of bacteria in the stomach is determined largely by the pH of the contents. At pH values below 3, bacteria capable of living as commensals in the gut are quickly killed. Because the pH of the gastric contents of the fasting normal human is usually less then 3, such samples are invariably sterile.4 However, during a meal the gastric acid is buffered, which allows bacteria ingested with food to survive at least until the pH falls. Thus, under such conditions, the gastric flora can be considered only transient. However, where gastric acid secretion is impaired, allowing the pH to remain above 3, bacteria can survive longer and even proliferate. Reduced gastric acid secretion (hypochlorhydria) occurs naturally with ageing5, is common after gastric surgery and is associated with certain diseases such as pernicious anaemia and hypogammaglobulinaemia. In the latter two cases, subjects are often achlorhydric, which results in the gastric pH rising to 7 and above.5 This allows a diverse flora with up to 109 organisms per gram to establish; this flora usually consists of species of salivary bacteria of the genera Streptococcus, Neisseria, Staphylococcus and Veillonella, although species of Bacteroides, Lactobacillus and Escherichia are also found.5 Hypochlorhydria is also common in patients with atrophic gastritis associated with chronic Helicobacter pylori (H. pylori) infection. The gastric flora of hypochlorhydric individuals has toxicological significance because it increases the probability of xenobiotic metabolism by the bacteria, particularly as the gastric emptying time of such patients may be up to 5 hours.5 It has been suggested that the increased gastric cancer risk of achlorhydric patients is linked to increased formation of N-nitroso compounds by their gastric bacteria flora.6

Genes located on 18q

APC

Normal epithelium

Hyperplasia

Carcinoma 1?

Changes in DNA methylation patterns

P53

K-ras

2?

Metastasis

3?

Adenoma

Figure 1. Adenoma–carcinoma sequence.3

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Protective processes: Mismatch repair genes, Apoptosis

326 P. M. Heavey and I. R. Rowland

Helicobacter pylori H. pylori is a Gram-negative bacterium found in the human stomach; it plays an important role in the pathogenesis of chronic gastritis and peptic ulcer diseases.7 Both epidemiological and clinical evidence has indicated that H. pylori is associated with an increased risk of gastric carcinoma.8,9 It is the first bacterium to be termed a definitive cause of cancer by the International Agency for Research into Cancer (IARC). Furthermore, in developed countries, strains of H. pylori that carry the cag pathogenicity island are associated with an increased risk of peptic ulcer and adenocarcinoma compared with strains that are negative for the cag island.10 It is generally accepted that H. pylori infection plays a significant role in the aetiology of gastric cancer but the precise mechanisms involved in its pathogenesis have yet to be fully elucidated. Several mechanisms proposed for H. pylori-associated carcinogenesis in humans are listed below: † † † † †

dysregulation of the gastric epithelial cell cycle; the formation of DNA adducts; the generation of free radicals; alterations in growth factor secretion and cytokines; the effect of decreased gastric secretions.

The inflammatory effects of H. pylori infection have been related to cancer due to increased cell proliferation and production of mutagenic free radicals and N-nitroso compounds.11 In the Mongolian gerbil model of H. pylori infection it has been shown that H. pylori inoculation can induce abnormality in gastric mucosal cell proliferation.12 Infection with H. pylori is associated with significant epithelial cell damage as well as an increased level of apoptosis. However, the mechanism for H. pylori-induced apoptosis in gastric epithelial cells remains uncertain. Apoptosis is a genetically programmed mode of cell death that is regulated by many genes, including oncogenes and oncosuppressor genes, which may be mutated, delayed or abnormally expressed in neoplasms, thus altering tumour cell susceptibility to apoptosis.13 The initiation and regulation of apoptosis involves several pathways and has yet to be fully elucidated. There are two different mechanisms by which apoptosis may be induced in a cell. The first is generated by signals arising within the mitochondria, referred to as the ‘intrinsic pathway’ and the other is triggered by death activators binding to receptors at the cell surface (extrinsic pathway). In both pathways, a family of proteases called caspases (cysteine aspartyl-specific proteases) are triggered and cleave cellular substrates, resulting in biochemical and morphological changes characteristic of apoptosis. The role of the p53 tumour suppressor gene in apoptosis is currently of particular interest. Genetic abnormalities in this gene have been observed in a wide range of human cancers and are also closely associated with the transition from adenoma to carcinoma.14 The p53 protein plays an important part in many cell processes such as DNA repair, apoptosis and transcriptional activation. The mutational inactivation of p53 function allows cells to continue with their cell cycle, meaning that damaged or mutated DNA is propagated in the next generation of cells. Zhang et al15 examined the effect of H. pylori on gastric epithelial cells, and the role of p53, by incubating H.pylori strains with gastric epithelial cells. H. pylori induced

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a time-and dose-dependent inhibition of cell growth and apoptosis over 72 hours. In agreement with other findings16, at low inoculations of H. pylori, cell DNA synthesis was stimulated compared to the controls. These authors also demonstrated no difference in the induction of gastric cell epithelial cell apoptosis and cell proliferation between cells exposed to cagA þ and cagA 2 strains. In addition, H. pylori infection was associated with changes in oncogene and tumour suppressor gene expression, as shown by increased ras p21 expression and p53 mutation in H. pylori-positive cases of gastric cancer.17 Cell cycle regulatory proteins have also been identified as critical targets during carcinogenesis. It has been shown that chronic H. pylori infection is associated with decreased expression of the cyclin-dependent kinase inhibitor (CDI) p27kip1. Another CDI, p16Ink4a (p16) is overexpressed in gastric epithelial cells of H. pylori patients and this is associated with an increase in apoptosis.18 Dietary vitamin C has been associated with a reduced risk of gastric cancer19 and this may be related to its ability to scavenge reactive oxygen species and inhibit NOC formation. High-dose vitamin C has been shown to inhibit H. pylori growth and colonization20 and at physiological concentrations it induced H. pylori associated apoptosis and cell cycle arrest in vitro.21 A case – control study also supports the association of vitamin C with decreased risk of gastric cancer. Others have also reported that high intake of vitamin C, beta-carotene and vitamins B1, B3 or B6 was associated with a decreased risk, whereas a high risk of gastric cancer was associated with a high intake of vitamin A or vitamin B2.22 The subject of antioxidant micronutrients and gastric cancer has been reviewed by Correa et al19 who also reported a consistent association between the risk of gastric cancer and low intakes of fruit and vegetables. Others have implicated cigarette smoking and low levels of dietary vitamin C as a contributing factor in those high-risk individuals with H. pylori infection.23 Overexpression of cyclo-oxygenase-2 (COX-2) has also been observed in tissues of human gastric cancer. There are two isoforms of COX: COX-1 and COX-2. These are key enzymes that convert arachidonic acid to prostaglandins. COX-1 is expressed in most human tissues, whereas COX-2 is usually undetectable. Overexpression of COX-2 has been implicated in a number of cancers, including gastric and colon cancer. It has been shown that COX-2 was overexpressed in 84% of gastric cancer specimens, and those specimens with cagA-positive strain expression had a significantly higher expression of COX-2 than did the specimens with cagAnegative strain expression.24 It has therefore been suggested that the application of COX-2-selective inhibitors may be an effective preventive strategy for gastric cancer and, in particular, those that would not cause gastrointestinal complications. Both the use of non-steroidal anti-inflammatory drugs (NSAIDs) and infection with H. pylori independently and significantly increase the risk of peptic ulcer and ulcer bleeding. In a meta-analysis of the data it was interpreted that there was synergism for the development of peptic ulcer and ulcer bleeding between H. pylori infection and NSAID use.25 The prevalence of H. pylori infection is falling in developing countries and this has been linked to changes in the epidemiology of gastrointestinal diseases, in particular a reduced incidence of gastric cancers in western countries.26,27 Improved nutrition, water supplies and reduced family sizes have been associated with reduced H. pylori colonization.26 Novel treatment of this infection with probiotics is in the initial stages, and results indicate only a slight improvement.28 This is an area for potential research in the future.

328 P. M. Heavey and I. R. Rowland

Practice points † H. pylori is first bacterium to be termed a definitive cause of cancer † H. pylori is involved in the initiation and promotion of gastric neoplasia † cancer risk is believed to be related to H. pylori strain differences. H. pylori populations are extremely diverse

Research agenda † definition of the molecular mechanisms responsible for the carcinogenesis promoted by bacteria such as H. pylori † more research is required into the application of COX-2-selective inhibitors † the development of other potential treatments for this infection, including the use of probiotics

THE LARGE INTESTINE It is becoming increasingly evident that the large intestine plays an extremely important role in both human health and disease and this is primarily due to its resident microflora. Table 15,29,30 summarizes the major bacterial groups that have been identified in the human large intestine. Research has shown that if the microflora is suppressed or compromised, the susceptibility of the host to pathogens is increased.31,32 There is also evidence that bacterial metabolism within the gut may have an important role in the toxicity of ingested chemicals and in cancer.33,34 Bacteria may affect carcinogenesis through several mechanisms. They may have a direct effect through the binding of potential mutagens and thus reduce exposure to the host.35 The normal microflora present in the gut is known to produce and release toxins which can bind specific cell-surface receptors and affect intracellular signal transduction.36 Bacterial involvement in colorectal cancer has been widely studied, most of the information being derived from animal work and some human studies. Evidence from a wide range of sources supports the view that the colonic microflora is involved in the aetiology of cancer; the evidence is summarized in Table 2. Studies of the faecal flora of healthy subjects and colon cancer patients have not revealed any consistent patterns, possibly due to the difficulties in culturing and identifying gut organisms. Increased numbers of Bacteroides have been associated with increased risk of colon cancer in humans.42,43 In another study, lecithinase-negative Clostridium and Lactobacillus were more abundant in colon cancer patients.44 Some Lactobacillus species and Eubacteriumaerofaciens have been associated with a reduced risk.43 In animals, colonic tumour formation is dependent on the presence of intestinal flora.40,45 In a study conducted by Reddy et al45 the rate of tumour formation was much more rapid in conventional rats than in germ-free rats treated with the tumour initiator dimethylhydrazine (DMH). At 20 weeks, 17% of conventional rats had colon carcinomas compared with none of the germ-free animals with either an adenoma or a carcinoma. At 40 weeks a small proportion of germ-free rats had benign adenomas (although still none had carcinomas), but the proportion was much lower than that in conventional rats at 20 weeks; by 40 weeks the proportion of conventional rats with

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Table 1. The bacterial flora of human faeces.5,29,30 Bacterial group

log (bacteria) g21 faeces

Strict anaerobes Bacteroides Eubacteria Bifidobacteria Propionibacteria Peptococci Clostridia Fusobacteria

10– 11 9–10 10– 11 9–10 9–10 6– 9 5–10

Microaerophiles Lactobacilli

4–10

Faculative organisms Enterococci Enterobacteria

7– 8 7– 9

adenomas and carcinomas was 50% higher than at 20 weeks. Thus the gut flora had a tumour-promoting effect when DHM was the tumour initiator. A high incidence of spontaneous colorectal cancer has been demonstrated in the T-cell receptor (TCR) b chain and p53 double knock-out mice. Further work on this model showed that adenocarcinoma of the colon did not occur in germ-free TCR b 2 /2 p53 2 /2 mice but adenocarcinomas were detected in 70% of the conventional animals, showing a major role for intestinal flora.41 Streptococcus bovis has been implicated in colonic neoplasia. Supplements of this strain of bacteria, and antigens extracted from the bacterial cell wall, were shown to induce formation of hyperproliferative aberrant colonic crypts and increase the expression of proliferation markers in carcinogen-treated rats.46 The effect of individual bacteria on cancer risk varies. Mice mono-associated with Mitsuokella multiacida, Clostridium butyricum or Bifidobacterium longum had a higher incidence of colonic adenoma (68% in each case) as compared to those associated with Lactobacillus acidphilus (30%).47 Table 2. Evidence that the microflora is involved in the aetiology of colon cancer. Human faeces have been shown to be mutagenic, and genotoxic substances of bacterial origin have been isolated37 Intestinal bacteria can produce, from dietary components, substances with genotoxic, carcinogenic and tumour-promoting activity38 Gut bacteria can activate procarcinogens to DNA reactive agents Germ-free rats fed human diets exhibit lower levels of DNA adducts in tissues compared to conventional rats39 Germ-free rats fed human with the carcinogen 1,2-dimethylhydrazine have a lower incidence of colon tumours than similarly treated rats having a normal microflora40 Germ-free T-cell receptor chain and p53 double-nockout (TCRb2/2 p532/2 ) mice did not develop adenocarcinoma of the colon at 4 months of age. Adenocarcinomas of the ileocecum and caecum were detected in 70% of the conventional TCRb2/2 p532/2 mice41

330 P. M. Heavey and I. R. Rowland

The enormous numbers and diversity of the human gut microflora is reflected in a large and varied metabolic capacity, particularly in relation to xenobiotic biotransformation, carcinogen synthesis and activation. The metabolic activities of the gut microflora can have wide-ranging implications for the health of the host, resulting in both beneficial and detrimental effects.48 To date the vast majority of mechanisms whereby bacteria are involved in carcinogenesis involve toxic or protective products of bacterial metabolism. Such metabolic activities include numerous enzymic reactions and degradation of undigested residue for energy. Diet can substantially modulate these activities by providing a vast array of substrates. A wide range of enzyme activities capable of generating potentially carcinogenic metabolites in the colon are associated with the gut microflora, including b-glucuronidase, b-glucosidase, nitrate reductase and nitro-reductase. These are usually assayed in faecal suspensions and appear to be present in many types of bacteria.49,50 Table 3 summarizes the major faecal bacterial enzymes and their relationship with cancer.

Table 3. Faecal bacterial enzymes. Enzyme

Relationship with cancer

Modulation by diet

References

b-Glucuronidase (GN)

De-conjugates carcinogens in the colon. Populations at high risk of CRC have high GN activity. Colon cancer patients have significantly higher GN activity than in healthy controls

High-risk diets for CRC increase GN in human and animal studies

38,51–54

b-Glucosidase

Hydrolyses glycoside conjugates of plant mutagens and carcinogens. Remains controversial due to reports of potential anti-carcinogenic and anti-mutagenic effects of flavonoid aglycones

Poor relationship to high-risk diets

38,55

Nitrate reductase

Nitrate, is reduced by this enzyme to nitrite which reacts with nitrogenous compounds in the body to produce N-nitroso compounds (NOC), many of which are highly carcinogenic, DNA alkylating agents.

This bacteiral N-nitrosation process can be monitored by determining apparent total NOC (ATNC) in faeces. Faecal ATNC excretion is increased by consumption of red meat

56–58

Nitroreductase

Reduces nitro compounds to amines (some of which are carcinogenic)

No consistent response to dietary change

56

IQ oxidoreductase

Converts IQ to 7-OHIQ (direct-acting carcinogen)

Increased by high-risk CRC diet in rats

38

Gastrointestinal cancer 331

A major role for the intestinal microflora has been identified in the metabolism of the bile acids—which are thought to possess tumour-promoting activity. The primary bile acids, chenodeoxycholic acid and cholic acid are subject to extensive metabolism by the intestinal microflora59, predominantly 7-a-dehydroxylation, which converts cholic to deoxycholic acid (DCA) and chenodeoxycholic acid to lithocholic acid (LCA). These secondary bile acids, exert a range of biological and metabolic effects in vitro and in vivo, including cell necrosis, hyperplasia and tumour-promoting activity in the colon, induction of DNA damage and apoptosis.60 It has also been suggested that secondary bile acids influence colorectal cancer by selecting for apoptosis-resistant cells or by interacting with various secondary messenger signalling systems. A number of human observational studies in patients with adenomas or CRC have reported a correlation between faecal bile acid concentrations and the risk of CRC.61,62 Some studies have also suggested that a high concentration of DCA, and a high DCAto-LCA ratio, are associated with increased risk of CRC.63 However, not all studies have confirmed this relationship between bile acids and the risk of CRC.64 Formation of protective agents during fermentation Both exogenous (dietary) and endogenous substrates are hydrolysed by bacterial enzymes in the gut produce the short-chain fatty acids acetate, propionate and butyrate. These short-chain fatty acids (SCFAs) provide an energy source for the intestinal cells and are also thought to confer beneficial effects on the host. SCFAs decrease colonic and faecal pH and this acidic environment is thought to be beneficial to the host.65 Specific oligosaccharides and resistant starch that result in SCFAs, and in particular butyrate, may have the potential to decrease the risk of colorectal cancer. Butyrate is of specific interest because it has been shown to induce apoptosis in colon adenoma and cancer cell lines. In vitro studies have shown that an increased butyrate supply to colon cells induces growth of the gut epithelium whereas reduced butyrate supply causes gut atrophy and functional impairments.66 However, the majority of these results have come from experiments conducted in vitro and, again, there have been conflicting views.67 Sodium butyrate (NaB) has been observed to induce apoptosis and to alter the resistance of colonic tumour cells to apoptosis.68 It has been hypothesized that the ingestion of indigestible carbohydrates resulting in the production of butyrate in the large intestine may be beneficial in terms of reducing risk factors for colorectal cancer. Recently, it has been recognized that resistant starch, in particular, induces a high production of butyrate and propionate.69 Butyrate is known to have beneficial effects on the reduction of risk factors involved in the aetiology of colon cancer and adenoma development.70 It follows from the above that modification of the gut microflora may exert a beneficial effect on the process of carcinogenesis and this opens up the possibility for dietary modification of the risk of clon cancer. Probiotics and prebiotics, which modify the microflora by increasing the numbers of lactobacilli and/or bifidobacteria in the colon, have been a particular focus of attention in this regard. In general, species of Bifidobacterium and Lactobacillus have low activities of those enzymes involved in carcinogen formation and metabolism by comparison with other major anaerobes in the gut such, as bacteroides, eubacteria and clostridia.50 This suggests that increasing the proportion of lactic acid bacteria (LAB) in the gut could modify, beneficially, the levels of xenobiotic-metabolizing enzymes. This manipulation of the gut is discussed in greater detail in other chapters within this book. However, Tables 4– 6 outline evidence for a protective effect of pro- and prebiotics on the risk of colon cancer risk—which is

332 P. M. Heavey and I. R. Rowland

Table 4. Effects of probiotics in animal models of colon cancer. Effect

Probiotic

Reduced incidence of colon tumour Reduced ACF formation Suppression of bacterial enzyme activities Reduced DNA damage

L. acidophilus B. longum L. acidophilus B. longum L. casei Str. thermophilus

Reference 71 72 73 74 75 76

Table 5. Effects of probiotics in human intervention trials. Study group

Organism

Healthy Heathy Bladder cancer patients Colon adenoma patients

L. L. L. L.

casei fermented milk rhamnosus GG casei acidophilus

Effect Reduced faecal enzyme activity Reduced faecal enzyme activity Reduced tumour recurrence Reduced mucosal cell proliferation and decreased faecal pH

Reference 77 78 79 80

B. bifidum

Table 6. Effects of prebiotics in animal models of colon cancer. Effect

Prebiotic

Less genotoxic damage to colon cells No effect on anerrant crypt foci (ACF) Decrease in Azoxymethane (AOM)-induced ACF Significantly higher apoptotic cells per crypt compared to controls

Lactulose Inulin Inulin and Fructo-oligsaccharides (FOS) Oligofructose and inulin

Reference 72 81 82 83

particularly evident in animal studies. The evidence in humans is not as strong, but some studies have had positive results (Table 5). Overall, experimental and animal research shows encouraging effects from several probiotic strains in decreasing colon cancer, leading the way to the development of well designed human intervention trials.

Practice points † the resident microflora of the large intestine plays a major role in the health and disease state of the host † evidence from several sources confirms that the colonic microflora is involved in the aetiology of colorectal cancer † the metabolic activities of the gut microflora may confer either toxic or protective effects on the host. These effects may be modified by diet

Gastrointestinal cancer 333

Research agenda † further research is required on the precise mechanisms by which the gut microflora is involved in the initiation and progression of colorectal cancer † development of techniques to enable more rapid and accurate quantification and identification of the gut bacteria † the anticancer effects of pro- and prebiotics should be confirmed in carefully controlled human intervention studies using validated biomarkers of colorectal cancer risk

SUMMARY It is becoming increasingly evident that the microflora of the gastrointestinal tract and, in particular, that of the large intestine, interacts with its host and may exert either harmful or protective effects, thus participating in the aetiology of cancer. Gastric adenocarcinoma is the second leading cause of cancer-related deaths in the world and has been associated with the presence of H. pylori in the stomach. Several mechanisms of how this bacterium may affect tumorigenesis have been identified—as well as dietary and environmental agents which may confer either protective or detrimental effects. Colon cancer is the fourth most common cancer worldwide and, again, environmental factors—and in particular, diet—play an important role in this disease. It has been shown that the microflora of the gut interacts with the host both locally and systemically, resulting in a broad range of effects which may have both beneficial and detrimental outcomes for nutrition, infections, xenobiotic metabolism, toxicity of ingested chemicals and cancer. It is important to gain more insight into the pathogenesis of these cancers in order to develop more effective preventive and treatment strategies for these common cancers. The use of pro- and prebiotics may serve to induce beneficial effects on the host. Further research from well-planned intervention trials is required to further our understanding of the role of these agents in human carcinogenesis.

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