Metabolic Activities of the Gut Microora in

Relation to Cancer

Roisin Hughes and Ian R. Rowland

From the School of Biomedical Sciences, University of Ulster, Coleraine, N. Ireland BT52 1SA

Correspondence to: Roisin Hughes, School of Biomedical Sciences, University of Ulster, Coleraine, N. Ireland

BT52 1SA. E-mail: [e-mail]

Microbial Ecology in Health and Disease 2000; Suppl 2: 179–185

The human large intestine is host to a diverse range of bacteria with numbers reaching 10

11

:

ml of faecal material. This population plays

a signiŽcant role in colonic metabolism and health. Undigested dietary substrates and endogenous residues are metabolised by the gut

ora. Some of the products of this metabolism have been associated with carcinogenic processes such as tumour promotion (ammonia,

secondary bile acids), mutagenesis (fecapenaenes) and carcinogenesis (N-nitroso compounds). Bacterial enzymes involved with carcinogen

formation include b-glucuronidase, b-glycosidase, azoreductase, nitroreductase, nitrate reductase, the conversion of pre-carcinogen 2

amino-3-methyl-7H-imidazo[4,5-f]quinoline(IQ) to 7-hydroxy-2-amino-3,6-dihydro-3-methyl-7H-imidazo[4,5-f]quinoline-7-one(7OHIQ).

Protective effects of gut bacterial metabolism include carcinogen binding, detoxiŽcation of methylmercury and formation of lignans and

isoavones. Diet is known to play a role in gut microora metabolism and cancer development. Studies have shown that gut bacterial

enzymes are affected by diet and bacterial metabolism of dietary protein releases toxic products including ammonia, phenols and cresols.

The interrelationships between diet, gut microora metabolism and effects on the host are complicated however they may be important

in the understanding of epidemiological associations between exogenous factors and colorectal cancer risk. Key words: microora

metabolism, colon cancer, toxic end-products, detoxiŽcation.

ORIGINAL ARTICLE

INTRODUCTION

The human gastrointestinal tract possesses a complex
ecosystem, the components of which are generically com-
plex and metabolically diverse with bacterial numbers
reaching 10

11

:

gram intestinal contents in the large intestine

(1). The principal role of the gut ora is to salvage energy
from non digested dietary substrates and endogenous mu-
cus during fermentation. The nature and extent of this
metabolism depends upon the characteristics of the bacte-
rial ora, colonic transit time and the availability of nutri-
ents especially carbohydrates and proteins. Products of
carbohydrate fermentation are thought to beneŽt the host
in contrast to the potentially toxic products of protein
fermentation. The metabolic activities of the gut mi-
croora have also been associated with cancer develop-
ment following the formation of toxic products by
bacterial enzyme activities. The following review discusses
the potentially detrimental and beneŽcial consequences of
gut bacterial activity to the host. Some of these effects are
outlined in Table I.

TOXIC CONSEQUENCES OF GUT BACTERIAL
METABOLISM
Bacterial enzymes associated with cancer
The enzymic activities of the gut microora, towards in-
gested foreign compounds such as nitro-aromatics, azo

compounds, and nitrate can have wide-ranging implica-
tions for health, since bacterial metabolism of such com-
pounds can lead to the generation of genotoxic and
carcinogenic products. Bacterial enzymes commonly as-
sayed include b-glucuronidase, b-glycosidase, azoreduc-
tase, nitroreductase, nitrate reductase, the conversion of
pre-carcinogen 2 amino-3-methyl-7H-imidazo[4,5-f]quino-
line (IQ) to 7-hydroxy-2-amino-3,6-dihydro-3-methyl-7H-
imidazo[4,5-f]quinoline-7-one (7OHIQ).

b

-glucuronidase and b-glycosidase. Many toxic and car-

cinogenic compounds are oxidised in the liver by cy-
tochrome P450 dependent enzymes and then conjugated
by ‘Phase II enzymes’ to glucuronic acid. Because of the
high molecular weight and polar nature of these conju-
gates, they are extensively excreted via the bile into the
small intestine. In the colon, bacterial b-glucuronidase,
can hydrolyse the conjugates releasing the parent com-
pound, or its hepatic metabolite. This process increases
endogenous exposure to carcinogens as the parent com-
pound is absorbed from the intestine entering the entero-
hepatic circulation. Xenophobic compounds removed in
bile as glucuronide conjugates include benzo(a)pyrene glu-
curonide (2), methylazoxymethanol glucuronide (3) and
the N-glucuronides of heterocyclic aromatic amines after
they have undergone N-oxidation in the liver (4). In rats,
caecal and rectal b-glucuronidase activity was dependent

© Taylor & Francis 2000. ISSN 1403-4174

Microbial Ecology in Health and Disease

R

. Hughes and I. R. Rowland

180

upon bile ow suggesting that the enzymic activity was
induced in response to the glucuronides secreted in bile (5).
Methylazoxymethanol (MAM) is formed following bacte-
rial deconjugation of the carcinogen 1,2-dimethylhydrazine
(DMH) which is used to induce colon cancer in animal
models. Germ-free rats have fewer colon tumours after
treatment with DMH or with MAM-glucuronic acid con-
jugate as compared to conventional ora animals (6).

Plant glycosides comprise a wide variety of low-molecu-

lar weight substances linked to sugar moieties (7). Human
intakes range from 50 to 1000 mg:day (7) and dietary
sources include fruits and vegetables and beverages, such
as tea and wine, derived from plants. In their glycosidic
form most of these substances are relatively harmless.
Most plant glycosides are poorly digested and pass to the
colon where they are hydrolysed by bacterial b-glycosi-
dases which cleave the sugar moiety for energy require-
ments and release aglycones. Aglycones have a variety of
biological activities including toxicity, mutagenicity and
carcinogenicity (8, 9). Assessment of the toxicological sig-
niŽcance of glycoside hydrolysis by intestinal microora is
complicated by reports of potential anti-carcinogenic and

anti-mutagenic effects of avonoid aglycones. It is clear
therefore that hydrolysis of plant glycosides in the gut can
lead, potentially, to both adverse and beneŽcial conse-
quences for man.

Nitroreductase and nitrate reductase

. There is extensive

human exposure to nitrocompounds. Nitroaromatics are
found in diesel exhaust, cigarette smoke and airborne
particulates and heterocyclic and aromatic nitro com-
pounds are extensively used in industry during the manu-
facture of consumer products. These compounds often
possess toxic, mutagenic and carcinogenic activity. Aro-
matic and heterocyclic nitrocompounds are reduced by
bacterial nitroreductases to potentially toxic N-nitroso and
N

-hydroxy compounds before conversion to aromatic

amines. The toxicity of the important chemical intermedi-
ates, dinitrotoluenes, has been shown to be dependent
upon the reductive activity of the intestinal microora as
genotoxicity and the ability to bind covalently to macro-
molecules (a critical early step in tumorigenesis) was lower
in germ-free than in conventional rats (10).

Nitrate, ingested via diet and drinking water, is readily

converted to nitrite in the human colon via nitrate reduc-
tase activity of the intestinal microora. The anaerobic
reduction of nitrate to nitrite is the most important route
of nitrate dissimilation by human faecal bacteria (11). This
reaction has important consequences for the host as oxides
of nitrogen which are derived from nitrite, can react with
nitrogenous compounds such as amines, amides and
methylureas to produce N-nitroso compounds, many of
which are highly carcinogenic. In vitro work has shown
that denitrifying bacteria are more potent nitrosators than
non-denitrifying bacteria following induction under anaer-
obic conditions in the presence of nitrate or nitrite (12).
Bacterial strains belonging to Escherichia, Pseudomonas,
Proteus

, Klebsiella and Neissera families have been shown

to N-nitrosate nitrogenous precursors in vitro and N-nitro-
sation activity is dependent upon the presence of nitrate
and nitrite reductase genes (13– 15).

IQ

. Heterocyclic amines (HAA) are formed from amino

acids when food is cooked at a high temperature (16).
Some HAA have been shown to be carcinogenic. Colon
cancer was enhanced in animals fed well cooked meat
containing high levels of HAA (17, 18). 2-Amino-3-
methyl-3H-imidazo[4,5-f ]quinoline (IQ) is one of several
HAA formed in small quantities when meat and Žsh are
grilled or fried. It induces tumours at various sites in
rodents including the large intestine, suggesting that it may
play a role in the aetiology of colon cancer in man (19).
Incubation of IQ with a human faecal suspension yields
the 7-keto derivative, 2-amino-3,6-dihydro-3-methyl-7H-
imidazo[4,5-f]quinoline-7-one (7-OHIQ).

The

7-keto

metabolite has been found in faeces of individuals consum-
ing a diet containing a high level of fried meat indicating

Table I

Potentially toxic effects of gut bacterial metabolism

Activity

Example of gut bacterial
metabolism

Activation to toxicants,

Reduction of Azo and

mutagens and carcinogens

Nitro compounds.

Formation of Aglycones

from Plant glycosides.

Conversion of IQ to

7-OHIQ.

Synthesis of carcinogens and

Formation of N-nitroso

mutagens

compounds from nitrogenous
residues and nitrate:nitrite.

Formation of fecapentaenes

from ether phospholipids.

Synthesis of promoters

Metabolism of cholic and

chenodeoxycholic acid to
deoxycholic and lithocholic
acid.

Protein degradation to

ammonia, phenols and cresols.

Formation of fecapentaenes

from ether phospholipids.

Enterohepatic circulation and

Hydrolysis of conjugated

deconjugation

steroid hormones, drugs and
carcinogens.

DetoxiŽcation:protection

Metabolism and thus

excretion of methylmercury.

ModiŽcation of plant

phyto-oestrogens to
mammalian derivatives.

Metabolism of plant

glycosides.

Gut microora metabolism and cancer

181

that the formation of 7-OHIQ can occur in vivo in man
(20). Unlike IQ itself, the bacterial metabolite is a direct-
acting and potent mutagen in Salmonella typhimurium and
induces DNA damage in colon cells in vitro (20, 21). Thus
there is strong evidence for the bacterial formation in the
human gut of a directly genotoxic derivative of a dietary
carcinogen. However present estimates of risk from these
compounds are rather low when extrapolated from animal
carcinogenicity data (17).

Effect of prebiotics on gut bacterial activities

. A number

of studies have shown that diet and antibiotics can change
bacterial enzyme activities including the enzymes involved
in carcinogen formation i.e. b-glucuronidase, b-glycosi-
dases and nitroreductases (8). More recently prebiotic non
digestible oligosaccharides (NDOs) have been shown to
suppress carcinogen metabolising enzyme activities in rats.
This is probably due to their stimulatory effects on probi-
otic lactic acid bacterial (LAB) growth. Species of
BiŽdobacterium

and Lactobacillus have low activities of the

enzymes involved in carcinogen formation and metabolism
by comparison to other major anaerobes in the gut such as
Bacteroides

, Eubacteria and Clostridia (22). Therefore in-

creasing the proportion of lactic acid bacteria in the gut
could modify, beneŽcially, the levels of xenobiotic
metabolising enzymes. Supplements of galacto-oligosac-
charide (TOS) and the synthetic NDO lactulose have been
shown to decrease b-glucuronidase activities and increase
faecal LAB counts in rats (23–25). The study by Rowland
and Tanaka, (23) also showed reduced bacterial nitrate
reductase activities, pH and conversion of the cooked food
carcinogen 2-amino-3-methyl-3H-imidazo(4,5-f)quinoline
(IQ) to its directly genotoxic 7-hydroxy derivative in the
caecal contents of TOS-fed rats. In contrast, b-glycosidase
activity increased, presumably as a consequence of the
elevated numbers of biŽdobacteria which have high levels
of this enzyme (22). In humans, Neosugar containing 4
g:day fructooligosaccharide (FOS), increased intestinal
biŽdobacteria and reduced faecal activities of enzymes
involved in genotoxin formation i.e. b-glucuronidase and
glycholic acid hydroxylase (26). Two recent studies have
shown reduced caecal b-glucuronidase activity in animals
fed a commercial derivative of the NDO Inulin. Changes
in enzyme activity occurred alongside a suppression of
preneoplastic colonic aberrant crypt foci (ACF) (27, 28).
In the later study, dietary effects were more potent when
the NDO was administered with BiŽdobacterium Longum.
The evidence so far shows therefore that one mechanism
to explain the cancer protective effects of NDOs may be
the suppression of enzymes involved in producing
genotoxic metabolites.

Potentially toxic end products of gut bacterial metabolism

Products of protein degradation in the large intestine

. On

average, 12 g of proteinaceous material or 0.5–4g total
nitrogen, enters the large intestine each day mainly in the

form of protein (48–51%) and peptides (20–30%) (29).
Dietary sources make up at least 50% of this protein
material, however the amount may vary due to protein
intake and the physical form of the food (30). The remain-
ing 50% includes pancreatic enzymes, mucus and exfoli-
ated epithelial cells. The large intestine contains numerous
proteolytic bacteria with species of Bacteroides and Propi-
onibacterium

being the most predominant (31). Products of

protein fermentation include short chain fatty acids
(SCFA), hydrogen, CO

2

and biomass in addition to

branched chain fatty acids such as isobutyrate, isovalerate
and 2-methylbutyrate together with other organic acids.
Ammonia, amines, phenols and indoles are also formed
following deamination, decarboxylation, fermentation and
h

or b elimination reactions. Concentrations of protein

degradation products are higher in distal regions of the
colon as compared to proximal regions. This subsite distri-
bution may be related to concentrations of fermentation
substrates. Carbohydrate is the favoured energy supplying
nutrient for the gut microora and proteinaceous material
is typically degraded for energy once carbohydrate sources
are exhausted (32).

The concentration of ammonia in human faecal samples

ranges from 12 to 30 mM and is positively related to
dietary protein intake (33– 35). Ammonia concentrations
as low as 5– 10 mM have been shown to alter the morphol-
ogy and intermediary metabolism of intestinal cells, affect
DNA synthesis and reduce the lifespan of cells (36). Such
effects suggest a role in tumour promotion via increased
mucosal cell turnover and hence the likelihood of multipli-
cation of damaged cells. Phenols and indoles are formed
following bacterial degradation of aromatic amino acids.
Intestinal bacteria involved in these processes include
Clostridia

(37) Bacteroides (38), Enterobacteria (39)

BiŽdobacteria

(40) and Lactobacilli (41). Phenolic com-

pounds are absorbed in the colon, detoxiŽed by the liver
and excreted in urine (phenol, p-cresol and 4-ethylphenol)
(31). Physiological levels of phenolic compounds in colonic
contents are normally low making the relation of these
compounds to colorectal mucosal damage unclear. In vitro
work, however has shown that phenol may enhance N-ni-
trosation of dimethylamine by nitrite and the reaction
between phenol and nitrite produces the mutagen diazo-
quinone (42). Amines are produced by large intestinal
bacteria following hydrolysis and decarboxylation of par-
ent nitrogenous residues. Amines formed in the large
intestine include methylamine, pyrrolidine, butylamine, pu-
trescine, histidine and taurine (29). These compounds may
exert toxic effects following reaction with nitrosating
agents to form N-nitrososcompounds.

N

-nitrosocompounds.

Many

N

-nitroso

compounds

(NOC) are known to exert carcinogenic:mutagenic effects
following the formation of potent DNA alkylating agents
during metabolism. Preformed NOC are found in cosmet-
ics, pharmaceutical products and occupational sources.

R

. Hughes and I. R. Rowland

182

However endogenous formation provides the most potent
source of exposure for humans (43). N-nitrosation may be
acid catalysed, bacterially catalysed at a neutral pH or cell
mediated. Therefore NOC formation may occur at a num-
ber of sites in the body. The large intestine provides a site
for N-nitrosation reactions due to the presence of nitrosat-
ing agents from dissimilatory nitrate metabolism and ni-
trosatable substrates from dietary residues. Endogenous
large intestinal NOC formation has been demonstrated in
rats (44). In this study a signiŽcantly higher level of NOC
were detected in gut contents of conventional ora rats as
compared to germ free rats, implicating a role for micro-
bial metabolism. More recently NOC have been detected
in human faecal samples at levels ranging from 82 vg:kg
to 1010 vg:kg and excretion is positively related to dietary
nitrate (45) and red meat (34, 46). Each of these studies
reported a high interindividual variation in faecal NOC
excretion and this was related to individual variations in
gut microora compositions especially nitrate and nitrite
reducing populations. NOC detected in these studies were
determined using a group selective approach so no infor-
mation on the individual NOC present is available. At-
tempts to characterise the compounds have shown that
they are water-soluble, and 50% have a molecular weight
less than 3000. Such compounds can cross cell walls and
exert effects at the cellular level. Compounds known to be
present include acidic and basic nitrosamines which may
be genotoxic upon activation by cytochrome P450 enzymes
(34). Further work is needed in this area to determine the
risk associated with increased faecal NOC excretion.

Fecapentaenes

. Fecapentaenes (FP) have been detected

in faecal samples from humans consuming Western meat
containing diets and are thought to contribute to the
mutagenicity of these samples (47). Fecapentaenes 12 and
14 (FP12 and FP14) have been identiŽed and consist of a
glyceryl ether compound containing a pentaene moiety
with a chain length of 12 or 14 (48). The gut microora
particularly species of Bacteroides, have been implicated in
FP formation as FP were produced in vitro by faecal
suspensions under anaerobic conditions. Synthesis was
inhibited by antibiotics and heat sterilization (Hirai et al.,
1982). Average FP concentration in human faecal samples
is approximately 500 ng:g dry weight although concentra-
tions up to 10000 ng:g have been detected (49). In vitro
work has shown that FPs are potent direct-acting muta-
gens. At low concentrations (0.6–10 vg:ml), FP12 induces
single strand DNA breaks, gene mutations, chromosome
aberrations, sister chromatid exchanges and unscheduled
DNA synthesis in human Žbroblasts in vitro (50). Prolifer-
ative effects of FP12 were demonstrated in rat colon
epithelia (51). Evidence from animal work however does
not support these Žndings. Rodent bioassays have indi-
cated that FP12 does not have carcinogenic or tumour-ini-
tiating activity (52–54). However, Zarkovic et al., (55)
showed that fecapentaenes may possess tumour-promoting

activity in a rat colon carcinogenesis model using N-
methyl-N-nitrosurea (MNU) as an initiating agent. Epi-
demiological work has also revealed some anomalies. For
example, lower FP levels were found in faeces from col-
orectal cancer patients than in controls and faecal excre-
tion of FP is higher in vegetarians, a population at low
risk from colon cancer (49, 56). The role of fecapentaenes
as colon carcinogens is thus unclear.

Secondary bile acids

. The bile acids cholic and

chenodeoxycholic acids are secreted by the liver, deconju-
gated and dehydroxylated by the anaerobic ora in the
large bowel forming the secondary bile acids-deoxycholic
acid and lithocholic acid. These two secondary bile acids,
which comprise over 80% of faecal bile acids, are postu-
lated to play an important role in the aetiology of colon
cancer by acting as promoters of the tumorigenic process.
In experimental animal models they have been shown to
disrupt colonic mucosal cell membrane integrity leading to
a compensatory increase in mucosal proliferation (57).
Recent in vitro work has shown that secondary bile acids
inuence cell growth by interacting with two important cell
signalling systems, i.e. Prostaglandin E2 and Protein Ki-
nase C. Both systems are known to be regulators of cell
growth, differentiation and apoptosis (58). There is also
evidence to show that secondary bile acids, albeit at rela-
tively high concentrations, can induce DNA damage in
colon cells in culture (59). In human studies however the
role of bile acids as colon carcinogens is less clear as
several case-control studies have reported no difference in
faecal bile acid excretion between colorectal adenoma or
cancer patients as compared to controls (60).

DETOXIFICATION AND PROTECTIVE EFFECTS
OF GUT BACTERIAL METABOLISM
Phytoestrogens

Dietary phytoestrogens including isoavonoids and lig-
nans, undergo extensive metabolism in the human body,
with the intestinal ora being the major site of biotransfor-
mation. The glycosides of isoavonoids are rapidly hy-
drolysed by gut bacteria to release the aglycones genistein
and daidzein which are further metabolised by colonic
bacteria to equol, desmethylangolensin and p-ethylphenol
(61). Plant lignans are converted to enterolactone and
enterodiol by hydrolysis, dehydroxylation, demethylation,
and oxidation reactions catalysed by the facultative anaer-
obes of the intestinal tract (62). Microbial involvement in
phytoestrogen metabolism has been demonstrated in rats
fed phytoestrogens (63). In this study, the isoavone
metabolites, equol and desmethylangolensin and the lignan
metabolites, enterolactone and enterodiol, were detected in
urine from HFA rats and not from germ free rats. Phy-
toestrogens have numerous biological effects including an-
tiviral, antiproliferative and growth inhibiting activities.
Lignans and isoavones are structurally similar to potent

Gut microora metabolism and cancer

183

synthetic estrogens such as diethylstilboestrol, and possess
hormonal activity mediated by intestinal microora. Such
activities include modulation

of

steroid hormone

metabolism and reduced proliferation of hormone depen-
dant cancer cells (64). Epidemiological studies provide
good evidence for a protective role of soy phytoestrogens
against hormonal cancers such as breast and prostate
cancer (65). In terms of colon cancer the evidence stems
mainly from case-control studies and is as yet unclear as
approximately equal numbers of studies show, protective,
causative or no effect (64). Animal work has however
shown that lignans may be protective in rats treated with
the carcinogen azoxymethane. In rats fed a basal diet
supplemented with axseed oil, a rich source of lignans,
the incidence of colonic aberrant crypt foci was reduced by
50% as compared to those fed the basal diet alone (66).
Other sources of phytoestrogens, namely, linseed and
genistein, have shown a similar suppressive effect on the
number of colonic aberrant crypt foci in animals (67, 68).
In vitro

work has shown that genistein suppressed prolifer-

ation of the colon cancer cells HT29 and CACO-2 in a
dose dependant manner (69). Genistein may also prevent
endogenous N-nitrosation as it has been shown to inhibit
the expression of the enzyme inducible nitric oxide syn-
thase which is a source of nitric oxide (70). Although large
scale epidemiological evidence is lacking, emerging in vitro
and animal work suggests that bacterial metabolites of
dietary phytoestrogens may exert effects at the cellular
level which is relevant to colon cancer. Further work is
needed in this area to determine the importance of these
metabolites to the host.

Reduced exposure to toxic compounds

. Certain metabolic

activities of the gut microora may reduce human expo-
sure to toxic compounds and certain dietary carcinogens.
For example, endogenous exposure to the neurotoxin
methylmercury (MeHg) is determined largely by its rate of
elimination, the main route of which is the faeces (71). The
gut microora appear to be involved in the demethylation
of MeHg into its mercuric form for ultimate excretion.
This process has been reviewed by Rowland, 1995 (72) and
has been demonstrated in animal models. Studies have
reported the ability of freeze dried preparations of intesti-
nal bacteria, especially lactic acid bacteria, to bind dietary
carcinogens including heterocyclic amines, the fungal toxin
aatoxin B

1

(AFB

1

) and the food contaminant AF2 (73).

In some studies, binding was associated with a concomi-
tant decrease in mutagenic activity in vitro (73, 74). In
theory, carcinogen binding should decrease the bio-
availability of ingested carcinogens in the gut, reducing
their capacity to damage the intestinal mucosa. However a
recent study showed that lactic acid bacteria administered
to rats, had no effect on absorption or genotoxic activity in
vivo

of carcinogens (75). Conditions required for carcino-

gen binding in vivo may differ from those showing success-
ful binding in vitro. In addition, the toxicological

signiŽcance of carcinogen binding remains to be
established.

CONCLUSIONS

The contribution of gut microora activities to cancer
development have been discussed in this review. Such
activities include formation of tumour promotors, (ammo-
nia and secondary bile acids), mutagens (fecapentaenes)
carcinogens (N-nitrosocompounds and deconjugated xeno-
phobic compounds) and cancer protective effects following
the formation of isoavones and lignans, detoxiŽcation of
methylmercury and possibly binding carcinogens. Epi-
demiological evidence shows that diet is associated with
80% of colorectal cancer cases (76). Mechanisms for this
association are unknown however diet is important for gut
microbial activity via the provision of substrates for
metabolism. Interrelationships between diet, bacterial
metabolism and effects on the host remain to be fully
established. Such relationships may provide a link with
epidemiological work showing associations between exoge-
nous factors and colorectal cancer risk.

ACKNOWLEDGEMENTS

This review has been carried out with Žnancial support from the
Commission of the European Communities, Agriculture, and
Fisheries (FAIR) speciŽc RTD programme PL98 423O ‘Intestinal
Flora: Colonistaion Resistance and Other effects’. It does not
reect its views and in no way anticipates the Commission’s future
policy in this area.

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