Download The Intestinal Microflora and tbe Colon Cancer

REVIEWS OF INFECTIOUS DISEASES • VOL. 12, SUPPLEMENT 2 • JANUARY-FEBRUARY 1990
© 1990 by The University of Chicago. All rights reserved. 0162-0886/90/1201-0069$02.00
The Intestinal Microflora and tbe Colon Cancer Connection
Sherwood L. Gorbach and Barry R. Goldin
From the Department of Community Health, Tufts
University School of Medicine, Boston, Massachusetts
The intestinal microflora of humans represents a rich
ecosystem composed of metabolically active microorganisms in close proximity to an absorptive
mucosal surface. Substrates for bacterial transformation can reach the colonic flora through direct
oral ingestion, by biliary secretion into the upper
bowel, or by secretion across the mucosa. There. is
considerable interest in the possibility that bactenal
metabolism of these substrates in the large bowel may
play a role in the etiology of cancers that arise in
the adjacent epithelium.
Metabolic Activities
Microorganisms within the flora possess an impressive array of enzymes that can convert exogenous
and endogenous compounds into a spectrum of metabolites. In addition to producing constitutive enzymes, these microorganisms can undergo enzyme
induction when exposed to high levels of substrate.
Table 1 contains a partial list of recognized reactions
involving intestinal bacteria. (For further details, see
[4, 5].)
The Colon Cancer Connection
Distribution of the Microflora
It is estimated that the intestinal flora of any given
person contains more than 400 different species of
bacteria [1-3]. Strictly anaerobic bacteria predominate over the facultative forms by a factor of 1,000:1.
Among the major anaerobes are Bacteroides, Bifidobacterium, Fusobacterium, Eubacterium, Clostridium, and Peptostreptococcus species.
The major bacterial populations are located in the
large intestine, where the bacterial concentration is
101 1-1012 cfu/mL of fecal material. The major landmark with respect to bacterial populations is the ileocecal valve; proximal to the valve the upper intestine has small numbers of bacteria and the milieu
is rich in oxygen, whereas distal to the valve the bacterial population is dense and a highly anaerobic environment with a low oxidation-reduction potential
is found. Thus, the bacterial enzymatic reactions in
the colon are characteristically reductive.
Please address requests for reprints to Dr. Sherwood L. Gorbach, Department of Community Health, Tufts University School
of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111.
S252
Large-bowel cancer is a major disease in Western
countries but is rather uncommon in Asia, Africa,
and South America [6, 7]. The critical factor that
accounts for the fivefold differences in incidence of
disease among geographic locations may be the characteristic Western-style diet, which is high in fat and
calories and low in dietary fiber [8-10]. By use of
food consumption data in various countries, a positive correlation has been found between the consumption of beef, total fat, animal fat, total calories,
and animal protein and the incidence of colon cancer. Several studies have shown a protective effect
of dietary fiber [11-13], although the relative importance of dietary fat and fiber in the etiology of colon cancer remains controversial.
Severalinvestigators have proposed that the effect
of diet on cancer development is indirect, consisting primarily of an impact on the ability of the host
to metabolize procarcinogens to proximate carcinogens [14-18]. In the case of colon cancer, activation
of carcinogens may be mediated by the bacterial flora
in the large bowel. Several bacterial enzymes have
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
Epidemiologic studies and laboratory research have indicated an association between the
metabolic activity of the intestinal micro flora and cancer of the large bo.wel. It h~s bee~
suggested that activation of procarcinogens could be mediated enzy~atlcally by l~teStl­
nal bacteria. The levelsof incriminated colonic bacterial enzymes are Increased by dleta~y
fat and inhibited by certain dietary fibers. Organic extracts of feces contain a mu~agem.c
substance, presumably derived from bacterial metabolism in the l~rge bo~el, t~at IS pOSItive in the Ames test. Whether this substance or some other organic chemical IS the putative proximate carcinogen remains speculative, but the evidence continues to point to intestinal bacteria as the metabolic intermediary in colon cancer.
Intestinal Microflora and Colon Cancer
5253
Bacterial Enzymes
Reaction
Glycosidases. Hydrolysis ofglycosidic bonds is
one of the best-known examples of bacterial metabolism. Glycosides arc compounds consisting of a
nonsugar moiety (aglycone) bound to a sugar byeither an a- or a J3-glycosidin linkage. Glycosides enter the gut from two major sources: the diet and the
liver (via the bile). The diet contains a large number
of plant glycosides, predominantly flavonoids. Glycosides from the liver include compounds that are
glucuronidated and subsequently secreted into the
bowel via the bile. The intestinal flora can then hydrolyze the J3-glucuronide bond; hydrolysis leads to the
release of biologically active aglycones, some of
which are potentially toxic or carcinogenic.
The principal glycosidases produced by the intestinal flora are J3-glucosidase, J3-galactosidase, and
J3-glucuronidase [21]. Hawksworth et al. [22] concluded on the basis of a study of 50 strains of each
of four bacterial species commonly found in the
bowel that, on a per-cell basis, Escherichia coli and
Clostridium had the highest J3-glucuronidase activity
and Lactobacillus and Bifidobacterium the lowest.
Bacterial J3-glucuronidase seems to play an important role in the metabolism of colon carcinogens.
This substance has a wide substrate specificity and
consequently can hydrolyze a large number of different glucuronides. Several studies have shown that
intestinal bacterial J3-glucuronidasecan alter or amplify the biologic activity of exogenous and endogenous compounds. For example, toxic aglycones
such as methylazoxymethanol from cycasin can be
regenerated in situ in the bowel by bacterial J3-glucuronidase.
Weisburger et al. have studied the metabolism of
the carcinogen N-hydroxyfluorenylacetamide when
administered parenterally to conventional and germfree rats [23]. Germfree rats excreted appreciably
larger amounts of the glucuronide of N-hydroxyfluorenylacetamide in their feces than did conventional animals. The cecal and fecal metabolites in
conventional rats were mostly free unconjugated
compounds, whereas the major fraction in germfree
animals was conjugated with glucuronic acid or
sulfate.
Morotomi et al. [24J reported that cell-freeextracts
of some strains of intestinal bacteria, including Bacteroidesfragilis, Bacteroides vulgatus, Bacteroides
Hydrolysis
Glucuronides
Glycosides
Sulfamates
Amides
Esters
Nitrates
Dehydroxylation
C-Hydroxy groups
N-Hydroxy groups
Decarboxylation
d-Demethylation
Deamination
Dehydrogenase
Dehalogenation
Reduction
Nitro groups
Double bonds
Aw groups
Aldehydes
Alcohols
N-Oxides
Nitrosamine formation
Aromatization
Acetylation
Esterification
Representative substrate
Estradiol 3-glucuronide
Cycasin
Cyclamate, amygdalin
Methotrexate
Acetyldigoxin
Pentaerythritol trinitrate
Bile acids
N- Hydroxyfluorenylacetamide
Amino acids
Biochanin A
Amino acids
Cholesterol, bile acids
DDT (chlorophenothane)
p-Nitrobenzoic acid
Unsaturated fatty acids
Food dyes
Benzaldehydes
Benzyl alcohols
4-Nitroquinoline l-oxide
Dimethylnitrosamine
Quinic acid
Histamine
Gallic acid
been implicated in the generation of mutagens, carcinogens, and various tumor promoters: Jl-glucuronidase, J3-glucosidase, J3-galactosidase, nitro reductase,
azoreductase, 7·a-steroid dehydrogenase, and 7-ahydroxy-steroid dehydroxylase.
The carcinogenic potential of bacterial enzymes
in the intestinal micro flora has been illustrated in
a series of studies involving experimental colon cancer induced by cycasin. This substance is a naturally
occurring J3-glucoside of the methylazoxymethanol
extractable from the seeds and roots of cycad plants
such as tropical ferns. Laqueur and Spatz discovered that feeding cycasin to infant rats caused hepatomas, renal sarcomas, squamous cell carcinomas
of the ear duct, and - in greatest frequency - intestinal adenocarcinomas that were located almost exclusively in the large bowel [19]. The genetic strain
of rat had little influence on the carcinogenic effect
of cycasin; similar tumors were induced in OsborneMendel, Sprague-Dawley, Fischer, and Wistar rats.
The intestinal flora was required for the carcinogenic
activity of cycasin: the compound was completely
inactive when given orally to germfree rats [20].
thetaiotaomicron, Eubacterium lentum, Peptostrep-
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
Table 1. Biochemical reactions of intestinal bacteria.
Gorbach and Go/din
8254
is mutagenic in the Ames assay, is also carcinogenic
[31]. Ponceau 3R, another biologic stain, is reduced
in vitro by Fusobacterium to a 2, 4, 5-trimethylaniline
that has been determined to be mutagenic [32]. Incubation of methyl orange or methyl yellow with intestinal anaerobes and testing with Salmonella TA1538
in the presenceof a microsomal activating system have
produced a positive result for mutagenicity. Both dyes
are reductively cleaved to the mutagen N,N-dimethylp-phenylene diamine [33].
Other azo dyes that have been shown to undergo
bacterial reduction to mutagenic or carcinogenic
products are direct black 38, direct red 2, and direct
blue 15. These dyes are converted to benzidine, 3,3dimethylbenzidine, and 3,3-dimethoxybenzidine,
respectively. Congo red is also reduced by rat cecal
bacteria to benzidine [34]. In the absence of a bacterial reductase system, congo red is not mutagenic
for Salmonella TA1538in the presence of a liver activating system; however, preincubation of congo red
with cecal bacteria results in a positive mutagenic
response.
Nitroreductase. The reduction of nitro groups by
intestinal bacteria is another source of aromatic
amines. Nitroreductase causes the formation of reactive nitroso and N-hydroxy intermediates in the
course of converting aromatic nitro compounds to
the aromatic amines. The precursor aromatic nitro
compounds are commonly found in factory effluents
as industrial chemical pollutants. Wheeler et al. [35]
studied a similar reaction, the reduction of p-nitrobenzoic acid, in conventional and germfree rats.
Conventional animals rapidly converted p-nitrobenzoic acid, whereas germfree rats reduced little of
the nitrocompound.
A nitrated polynuclear aromatic hydrocarbon, 1nitropyrene, is readily formed by reaction of nitrogen oxides with the combustion product pyrene. Its
presence in diesel engine exhaust represents a potential health hazard because of its high mutagenicity
in bacterial test systems and its carcinogenicity in rats.
When l-nitropyrene was administered orally to conventional rats, 5070-6070 of the dose was detected in
the feces as I-aminopyrene [36]. When a similar experiment was performed in germfree rats, l-aminopyrene was not found in the feces. Since reduction
of l-nitropyrene to l-aminopyrene is an activation process, the results indicate that the intestinal microflora
is important in the metabolic activation of 1nitropyrene,
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
tococcus species, and E. coli, enhanced the mutagenicity of bile from rats given l-nitropyrene via stomach tube. These bacterial cell-freeextracts hydrolyzed
the synthetic l3-n-glucuronides of phenolphthalein
and/or p-nitrophenol. Cell-free extracts of bacteria
not capable of increasing mutagenicity did not
hydrolyze the glucuronides. These data indicate that
the glucuronides of I-nitropyrene metabolites secreted into the bile can be hydrolyzed in the intestine by bacterial B-glucuronidases to potent mutagenic aglycones.
Azoreductase. Most of the artificial coloring additives used in the food, printing, and textile industries are dyes that contain a single azo bond (monoazo dyes). The water-soluble dyes are not absorbed
well from the intestine and are subject to bacterial
action in the large bowel [25]. A number of studies
have shown that the bacterial flora can reductively
hydrolyze the azo bond by the action of azoreductase (an enzyme confined largely to intestinal bacteria); this reaction results in the formation of substituted aromatic amines [26], a class of bacterially
generated compounds including a number of wellestablished carcinogens [27].
Large-bowel cancer occurs more commonly in
Western countries than elsewhere, and the extent of
the use of azo dyes is related to the degree of industrialization of the country. A connection may exist
between the number of cases of cancer and the use
of azo dyes [28].
The reduction of azocompounds by azoreductase
is believed to be mediated through a free-radical
mechanism, which produces intermediates that react with proteins and amino acids. Azoreductase also
can reduce food dyes, releasing phenyl- and naphthylsubstituted amines. These compounds have been implicated as chemical carcinogens [29]. The amines
generated in the bowel via the azoreductase reaction
are probably further oxidized to proximal carcinogens
by microsomal enzymes in the intestinal mucosa.
The role of bacteria in the generation of mutagens
from a number of azo dyes is noteworthy. There is
a 90070 correlation between carcinogenicityand mutagenicity for aromatic amines and azo dyes tested with
the salmonella/microsomal mutagenicity test. The
transformation of azo dyes by intestinal bacteria may
be a necessary prerequisite of carcinogenicity.Trypan
blue is widely used as a biologic stain and is not mutagenic, but reduction by a cell-freeextract of Fusobacterium produces a mutagen. O-Toluidine [30], which
Intestinal Microflora and Colon Cancer
Miller and Miller [37] and Weisburger and Weisburger [29], after reviewing the evidence, have suggested that the products of these reactions are extremely important in chemical carcinogenesis.
Diet and Bacterial Enzymes
Bile Acids
Bile acids have been studied extensively as candidate
carcinogens because of their structural similarity to
the carcinogenic polycyclic aromatic hydrocarbons
[42]. The concentration of fecal bile acids is increased
in people eating a meat (high-fat) diet. High concentrations of fecal bile acids cause colonic bacteria
to produce larger amounts of 7-a-dehydroxylase, the
enzyme involved in conversion of primary to secondary bile acids [43]. This finding was confirmed by
the addition of chenodeoxycholic acid to a growing
culture of Eubacterium species, resulting in a striking increase in 7-a-dehydroxylase activity [44]. Salvioli and co-workers found increased fecal concentrations of cholic and chenodeoxycholic acid and
decreased fecal concentrations of secondary bile
acids after daily administration 'of lyophilized Streptococcus jaecalis to healthy volunteers [45].
Demographic studies have demonstrated a correlation between high fecal concentrations of secondary bile acids and the Western high-beef diet [46].
Hill et al. noted that the fecal microflora of North
Americans and Western Europeans contained more
bacterial strains capable of 7-a-dehydroxylation than
did the fecal microflora of Ugandans or Indians [47].
In studies by Mowar et al. involving Japanese living
in Akita, Japan, and Japanese living in Hawaii and
consuming a Western-style diet, the latter group had
higher levels of fecal deoxycholic acid; however, little difference was noted in the other fecal bile acids
[48]. These authors also found that fecal concentrations of coprostanol and coprostanone, degradation
products of cholesterol, were higher in people eating a Western-style diet [48]. Mastromarino and coworkers studied patients with colon cancer and
found elevated levels of both 7-a-dehydroxylase and
cholesterol dehydrogenase compared with those in
healthycontrols [49J. Elevations of both enzymes
also were noted in patients with nonhereditary colonic polyps [17J.
MacDonald et al. [50] measured the activity of fecal NAD- and NADP-dependent 7-a-hydroxysteroid
dehydrogenase, which converts hydroxy-bile acids to
keto-bile acids, in vegetarian Seventh Day Adventists and subjects consuming a mixed Western diet.
The activity was lower in the vegetarian group. The
authors suggested that increased fecal NAD- and
NADP-dependent 7-a-hydroxysteroid dehydrogenase
is associated with a risk of large-bowel cancer.
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
The effect of diet on fecal bacterial B-glucuronidase,
nitroreductase, and azoreductase activities has been
studied in rats [14, 38]. Rats initially maintained on
a grain diet and then shifted after several weeks to
a meat diet showed a twofold rise in fecal l3-glucuronidase and nitroreductase activitieson the meat diet.
This increase started within 6 days, although the total effect required 12-17days. Fecal azoreductase also
increased by approximately twofold when rats were
shifted to the meat diet. An increase in specific activity of this enzyme was noted 4-10 days after the dietary change. Similar increases in fecal bacterial enzymes were noted when the grain diet was
supplemented with 30070 beef fat without other beef
products [39].
Variations in diet influence the activity of fecal bacterial enzymes in humans in a manner similar to that
reported in rats. Fecal bacterial enzymes were studied in omnivorous individuals who ate a mixed Western diet, lactovegetarians who consumed a diet that
excluded all animal foods except milk products, and
strict vegetarians [40]. Omnivores had considerably
higher levelsof fecal B-glucuronidase, nitroreductase,
and 7-a-dehydroxylase than did lactovegetarians or
strict vegetarians. Azoreductase levels were significantly lower among the strict vegetarians than among
omnivores. Elimination of red meat from the diet of
omnivores and fiber supplementation of the mixed
Western diet produced no significant change in any
enzyme activity except that of 7-a-dehydroxylase. In
both cases levels of this enzyme fell significantly during the period of dietary adjustment.
In another study [41], rats on either grain or beef
diets were fed Lactobacillus acidophi/us. The activities of fecaljl-glucuronidase, azoreductase, and nitroreductase were significantly reduced in the beef-fed
rats, but no changes were seen in the grain-fed animals, whose fecal enzyme activities were already low.
The results cited above indicate that diet and other
factors can alter levels of bacterial enzymes in the
gastrointestinal tract, perhaps affecting the formation of carcinogens and the concentration of tumorpromoting substances.
8255
8256
High-Fat Diets
Dietary Fiber
A protective effect against colon cancer has been
ascribed by some authors to consumption of dietary
fiber [11]. The authors of a recent study found the
risk of colon cancer to be relatively low in people
who ate larger amounts of crude dietary fiber, fruits,
and vegetables, although intake of grains did not appear to be protective [53].
The effect of fiber on fecal bacterial enzymes was
studied in animals fed either a grain or a beef diet;
the grain diet was associated with lower activity of
~glucuronidase, nitroreductase, and azoreductase
[14]. The specific components of fiber have been
studied more intensively in an effort to discern their
effects on fecal enzymes. Fecal bacterial glycosidase
activity was lower in animals fed a high-eellulose diet
than in those fed a standard laboratory diet [54]. In
another study three single sources of dietary fibercellulose, hemicellulose, and Pectin- were fed to lab-
oratory animals, and the effects on fecal bacterial
enzymes, fecal bacterial counts, and dimethylhydrazine-induced colon cancer were observed [55].
Cellulose and hemicellulose decreased the activity
of fecal enzymes, whereas pectin increased that activity. Fecal bacterial counts were not altered by any
of the three fiber sources. Protection against chemically induced colon cancer was related to bacterial
enzyme levels in that celluloseand hemicellulose protected the animals, whereas pectin had no effect.
Protection Against Experimental Colon Cancer by
Manipulation of the Intestinal Microflora
The evidence presented above implicating the microflora in colon cancer is circumstantial- either from
in vitro laboratory studies or from metabolic epidemiology. More direct evidence implicating bacteria
in colon cancer is derived from studies in animal
models involving chemical induction of large-bowel
cancer.
Dimethylhydrazine, an experimental carcinogen
that is metabolized by the liverto methylazoxymethanol, is similar to cycasin in its structure and carcinogenic. effects [56]. Rats fed a high-beef diet were
more susceptible to the carcinogenic effects of
dimethylhydrazine than were rats fed a grain diet.
When dimethylhydrazine was fed to rats on high-beef
and high-grain diets, the rates of tumor production
were 83070 and 31070, respectively [16].
In studies in which germfree and conventional rats
werechallenged with 3,2-dimethyl-4-aminobiphenyl
(DMAB), there were significantly fewer colonic
tumors among germfree animals [57]. When the role
of dietary fat in promoting colonic carcinogenesis
was studied during challenge with DMAB, more
animals fed a high-fat diet than animals fed a lowfat diet had colonic tumors. Dietary fat had no effect on the incidence of tumors when germfree
animals were studied; this observation further emphasizes the role of the microflora in this process.
The administration of oral antibiotics influences
the metabolic activity of the intestinal flora and the
induction of cancer. Three groups of beef-fed rats
were studied: a control group, a group receiving
tetracycline, and a group receivingerythromycin [58].
There was a striking reduction in the incidence of
intestinal tumors in the antibiotic-treated groups after challenge with dimethylhydrazine: colonic tumors
developed in 74070 of control animals but in only 20070
of the tetracycline group and 22070 of the erythro-
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
Several studies have implicated dietary fat as the nutrient most closely associated with the high incidence
of colon cancer in Western populations [7-10]. Various theories have been advanced to explain the role
of dietary fat in colonic carcinogenesis [8]. A highfat diet causes increased bile flow and higher fecal
levels of bile acids; as noted above, bile acids have
been implicated in colon cancer, possibly as a cocarcinogen. It has been suggested that dietary fat can
increase mucosal enzyme systems, an effect that may
promote activation of carcinogens [51]. A high-fat
diet may also increase bacterial enzyme activities in
the large bowel.
Temple and EI-Khatib [52] studied the effects of
a high-fat diet in mice, measuring both intestinal
mucosal and fecal enzymes. An increase was noted
in mucosal levels of 5'-nucleotidase but not in
mucosal levels of P-glucuronidase. There was an increase in fecal bacterial reductase in Swiss mice fed
the high-fat diet but not in C57BL/l mice. These
bacterial enzyme changes correlated with susceptibility of the strain of animal to dimethylhydrazine-induced colon cancer: Swiss mice, which had
increased levelsof bacterial enzymes,werehighly susceptible to tumor formation, whereas C57BL/l mice
were resistant to cancer induction. As noted above,
beef fat, when added to a grain diet, caused increases
in the activities of several bacterial enzymes [39].
Gorbach and Goldin
Intestinal Microflora and Colon Cancer
Fecal Mutagens
Distributions in different populations. Bruce et
al. [60] first reported that organic extracts of human
feces contained material positive in the bacterial
mutagenic test developed by Ames et al. [61]. Using
a number of separation techniques, Bruce and associates found that mutagenic activity was associated
with a single compound. Several groups investigated
the occurrence and levels of fecal mutagens in populations at different risk for colon cancer. In a study
conducted in South Africa, fecal specimens were obtained from low-risk rural blacks and from high-risk
whites in Johannesburg [62]. The frequency of mutagenic substrates found in the feces differed significantly between the two populations. Only 20/0 of the
fecal specimens obtained from the rural black population contained mutagens; in contrast, 15% of those
from the white population tested positive.
Reddy et al. [63] measured fecal mutagenic activity in three populations: residents of New York City
who consumed a typical Western diet (considered
at high risk for colon cancer), Seventh Day Adventists, and residents of the rural Finnish town of Kuopio (both considered at low risk for colon cancer).
Fecal specimens collected from the three groups were
freeze-dried, extracted with ether, partially purified
by silica gel chromatography, and assayed with
Salmonella test strains TA9S and TAloo, with and
without a liver microsomal activating system. Of the
fecal specimens from the high-risk population, 22070
were directly mutagenic on strain TA9S, 11 070 were
directly mutagenic on strain TAloo, and 6070 were'
mutagenic with S-9 activation on strain TAloo. Of
the Kuopio samples, 13070 were mutagenic on strain
TA9S, with microsomal activation required in all
cases; no mutagenicity was observed on strain TA100.
None of the samples from Seventh Day Adventists
showed mutagenicity with any test strain. These data
indicate that the incidence of colon cancer may be
correlated with the presence of mutagens in the fecal stream.
In a subsequent study by Reddy et al. [64], fecal
comutagens were measured in two of the three populations discussed above: the New York City residents
and the Seventh Day Adventists. Fecal specimens
were selected on the basis of having shown no mutagenic activity in the previous study. Fecal extracts
were prepared and tested for their ability to enhance
the indirect-acting mutagen and carcinogen 2-acetylaminofluorene (2-AAF) and the direct-acting mutagen N-methyl-N -nitro-N-nitroguanide (MNNG).
Dose-related enhancement of 2-AAF on strains TA9S
and TA100 was observed with fecal extracts from
both populations, but extracts from the vegetarian
group had significantly lower comutagenic activity
on strains TA9S. Comutagenic activity with MNNG
was the same for both groups.
Kuhnlein et al. [65] observed significant differences in the mutagenicity of water extracts from feces of lactoovovegetarians. The mutagenic activity
was assayed with the fluctuation test described by
Green and Muriel [66]. Activity was detected in concentrations as low as 1 mg of fecal supernatant/mL,
and the level of activity was significantly lower for
vegetarians than for nonvegetarians. There was, however, considerable variation among individuals. For
example, the subjects with the highest and lowest activities were both from the vegetarian group. Thus
the results are difficult to interpret. Varied mutagenic
activity of the water extracts toward the test strains
indicated the presence of several different mutagens.
Ferguson et al. [67] used repair-proficient and
repair-deficient strains of E. coli to investigate the
DNA-damaging activity of ethanol-soluble fecal extracts. Feces from European patients with colorectal cancer, age-matched controls, Maoris, Samoans,
and lactoovovegetarian Seventh Day Adventists were
analyzed for DNA-modifying activity. The Europeans had the highest activity regardless of whether
or not they had cancer. The rate of positive samples
was lower in the Polynesian groups, and there were
no unequivocally positive samples in the Seventh Day
Adventist group.
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
mycin group. Fecal J3-glucuronidase activity was also
significantly reduced in antibiotic-treated animals.
Similarly, administration of a J3-glucuronidase inhibitor decreased the formation of colon cancer in
conventional animals treated with the experimental
carcinogen azoxymethane, a compound similar to
dimethylhydrazine [59].
In another study, beef-fed rats were given dimethylhydrazine to induce colonic tumors; viable
Lactobacillus was included in the diet [16]. At the
2Q-week observation time, the rate of colon cancers
was significantly lower among beef-fed animals given
Lactobacillus than among beef-fed controls; however, no difference was noted at 36 weeks.These findings suggest that Lactobacillus increases the latency
or induction time for colon cancers, probably by suppressing the metabolic activity of the colonic flora.
8257
Gorbach and Goldin
8258
icallyat 37°C, mutagen was produced. Studies on
pure cultures revealed that five species of Bacteroides
(B. fragilis, B. ovatus, B. uniformis, B. thetaiotaomicron, and strain 3452A) produced the mutagen.
These strains are commonly found in human feces.
The in vitro production of mutagen was inhibited
by fermentable carbohydrates such as glucose, starch,
and dextran. Some 40 other species of intestinal
anaerobes tested for production of the mutagen all
proved negative. The facts that Bacteroides species
are common in the human colon and that only a relatively small percentage of individuals produce the
mutagen indicate that the precursor is the determining factor in its production. This possibility was confirmed by the demonstration that bacteria from feces
not containing mutagen were capable of producing
it in the presence of the precursor compound. The
precursor - which is a product of other bacteria in
the colon, a result of the diet, or a metabolite derived from the host - is necessary, in combination
with Bacteroides, for mutagen production. It has
subsequently been demonstrated that cell-free extracts of Bacteroides in the presence of bile can produce the mutagen. The bacterial enzyme system is
not oxygen sensitive; however, the mutagen is produced under only anaerobic conditions [69].
Structure offecal mutagen. The structure of the
fecal mutagen was first announced in 1982 [72] and
was confirmed independently in the following year
(73]. The characteristic ultraviolet spectra and shift
on oxidation implied that the compound was a pentaene. Chemical ionization, mass spectrometry, and
nuclear magnetic resonance analysis elucidated the
structure of the compound, called (8)-3-(1,3,5,7,9dodecapentaenyloxy)-1,2-propanediol (figure 1).
The compound is a vinyl ether and is a conjugated
laurylglyceroI. There are no known mutagens with
similar structures. The mutagen structurally resembles a group of ether-linked lipids that are found in
anaerobic bacteria and in low concentrations in some
mammalian tissues [74].
Conclusion
There is no direct evidence that the human intestinal microflora influences the risk of colon cancer.
What has emerged from the various studies cited in
this review is that the intestinal microflora is capable of engaging in reactions that can generate carcinogens, mutagens, or tumor promoters in the large
bowel. Furthermore, various treatments that alter the
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
Isolation ofether-soluble mutagen from human
feces. Bruce et al. [6OJ described the presence of
mutagenic activity in an ether extract of freeze-dried
feces. The ether extract demonstrated maximal activity when washed with aqueous sodium hydroxide
and then neutralized prior to testing. The washed
ether extract was subsequently dried, suspended in
benzene, placed on a silica gel column, and eluted
with benzene and ether. A single fraction contained
most of the activity. This was the first evidence that
the mutagenic activity in the ether fraction was associated with a single compound or a class of similar compounds.
The purification and ultraviolet spectra of the
compound were described by two collaborating
groups [68, 69]. The compound has a peak in the
ultraviolet at 365 nm and a high extinction coefficient. It has a lime-green fluorescence when exposed
to long-wavelength ultraviolet light. The high absorbance allowed investigators [69] to devise a rapid
high-performance liquid chromatography method
based on the area of optimal density in column fractions; this method is 10 times more sensitive than
the Ames test. The compound is highly sensitive to
oxidation; it is inactivated by liver microsomal preparations normally added to bacterial mutagen assays
to activate compounds by oxidation. Oxidation can
be inhibited by inclusion of antioxidants such as
butylated hydroxytoluene in the organic solvents and
can be prevented completely if purification is performed under an atmosphere of argon in solvents
containing butylated hydroxytoluene. The mutagen
is very stable under strictly anaerobic conditions.
When oxidation is allowed to proceed under controlled conditions, the three ultraviolet peaks shift
to lower wavelength and the fluorescence disappears.
Bacterial production of fecal mutagen. The
amount of mutagenic activity increases dramatically
if a fecal specimen already containing mutagen is
incubated anaerobically at 37°C for several days.
Mutagen production is inhibited by exposure of the
feces to oxygen, low temperature, autoclaving, or
radiation [70].
It was not possible to generate a mutagen in vitro
when fecal specimens were suspended in bacteriologic media. However, the addition of bile to the media resulted in the production of mutagen [71]. It
was then demonstrated that a precursor was present
in feces containing mutagen. When this precursor
was added to bacteriologic media containing bile,
inoculated with fresh feces, and incubated anaerob-
Intestinal Micro/lora and Colon Cancer
H2CO(CH-CH)5CH2CH 3
I
H-C-OH
I
CH 20H
Figure 1. Structure of fecal mutagen (8)-3-(1,3,5,7,9dodecapentaenyloxy)-1,2-propanediol.
References
1. Simon GL, Gorbach SL. The human intestinal microflora.
Dig Dis Sci 1986;31:147S-62S
2. Finegold SM, Attebery HR, Sutter VL. Effect of diet on human fecal flora: comparison of Japanese and American
diets. Am J Clin Nutr 1974;27:1456-69
3. Moore WEC, Holdeman LV. Human fecal flora: the normal
flora of 20 Japanese-Hawaiians. Appl Microbiol 1974;
27:961-79
4. Scheline RR. Metabolism of foreign compounds by gastrointestinal microorganisms. Pharmacol Rev 1973;25:451-523
5. Scheline RR. Drug metabolism by the gastrointestinal
microflora. In: Gram TE, ed. Monographs in pharmacology and physiology. Vol 5. Extrahepatic metabolism of
drugs and other foreign compounds. Jamaica, NY: Spectrum Publications, Inc., 1980:551-80
6. Doll R. The geographical distribution of cancer. Br J Cancer 1969;23:1-8
7. Burkitt DP. Epidemiology of cancer of the colon and rectum. Cancer 1971;28:3-13
8. Reddy BS, Cohen LA, McCoy GO, Hill P, Weisburger JH,
Wynder EL. Nutrition and its relationship to cancer. Adv
Cancer Res 1980;32:237-345
9. Miller AB. Risk factors from geographic epidemiology for
gastrointestinal cancer. Cancer 1982;50:2533-40
10. Correa P. Epidemiological correlations between diet and cancer frequency. Cancer Res 1981;41:3685-90
11. Burkitt DP. Colonic-rectal cancer: fiber and other dietary factors. Am J Clin Nutr 1978;31(10Suppl):58-64
12. Modan B, Barell V,Lubin F, Modan M, Greenberg RA, Graham S. Low-fiber intake as an etiologic factor in cancer
of the colon. Journal of the National Cancer Institute
1975;55:15-8
13. Dales W, Friedman GO, Ury HK, Grossman S, Williams
SR. A case-control study of relationships of diet and other
traits to colorectal cancer in American blacks. Am J
Epidemiol 1979;109:132-44
14. Goldin BR, Gorbach SL. The relationship between diet and
rat fecalbacterial enzymesimplicated in colon cancer. Journal of the National Cancer Institute 1976;57:371-5
15. Goldin BR, Sullivan CE, Gorbach SL. Etiologic factors in
the development of colonic cancer: bacteria, beef and animal fat. In: Kurtz RC, ed. Nutrition in gastrointestinal
Disease. New York: Churchill Livingstone, 1981:29-44
16. Goldin BR, Gorbach SL..Effect of Lactobacillus acidophilus
dietary supplements on 1,2-dimethylhydrazine dihydrochloride-induced intestinal cancer in rats. Journal of the
National Cancer Institute 1980;64:263-5
17. Mastromarino AJ, Reddy BS, Wynder EL. Fecal profiles of
anaerobic micro flora of large bowel cancer patients and
patients with nonhereditary large bowel polyps. Cancer
Res 1978;38:4458-62
18. Hill MJ, Crowther JS, Drasar BS, Hawksworth G, Aries V,
Williams REO. Bacteria and aetiology of cancer of large
bowel. Lancet 1971;1:95-100
19. Laqueur GL, Spatz M. Oncogenicity of cycasin and
methylazoxymethanol. Gann Monographs on Cancer Research 1975;17:189-204
20. Laqueur GL, McDaniel EG, Matsumoto H. Thmor induction in germfree rats with methylazoxymethanol (MAM)
and synthetic MAM acetate. Journal of the National Cancer Institute 1975;17:189-204
21. Drasar BS, Hill MJ. Human intestinal flora. New York: Academic Press, 1974;38:54-68
22. Hawksworth G, Drasar BS, Hill MJ. Intestinal bacteria and
the hydrolysis of glycosidic bonds. J Med Microbiol
1971;4:451-9
23. Weisburger JH, Grantham PH, Horton RE, Weisburger EK.
Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide in germfree rats. Biochem Pharmacol 1970;
19:151-62
24. Morotomi M, Nanno M, Watanabe T, Sakurai T, Mutai M.
Mutagenic activation of biliary metabolites of I-nitropyrene
by intestinal microflora. Mutat Res 1985;149:171-8
25. Chung K-T. The significance of azo-reduction in the mutagenesis and carcinogenesis of azo dyes. Mutat Res
1983;114:269-81
26. Parkinson TM, Brown JP. Metabolic fate of food colorants.
Annu Rev Nutr 1981;1:175-205
27. Combes RD, Haveland-Smith RB. A review of the genotoxicity of food, drug and cosmetic colours and other azo,
triphenylmethane and xanthene dyes. Mutat Res
1982;98:101-248
28. Wolff AH. Oehme FW. Carcinogenic chemicals in food as
an environmental health issue. J Am Vet Med Assoc
1974;164:623-9
29. Weisburger JH, Weisburger EK. Biochemical formation and
pharmacological, toxicological and pathological properties of hydroxylamines and hydroxamic acids. Pharmacol
Rev 1973;25:1-66
30. Hartman CP, Fulk GE, Andrews AW. Azo reduction of trypan
blue to a known carcinogen by a cell-free extract of a human intestinal anaerobe. Mutat Res 1978;58:125-32
31. Weisburger JH, Weisburger EK. Chemicals as causes of cancer. Chemical Engineering News 1966;44(6):124-42
32. Hartman CP, Andrews AW, Chung K-T. Production of a mutagen from ponceau 3R by a human intestinal anaerobe. Infect Immun 1979;23:686-9
33. Chung K-T, Fulk GE, Andrews AW. The mutagenicity of
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
microflora can influence the production of carcinogens and the development of colon cancers in experimental animal models. These findings are in accord with observed geographic variations in the
incidence of colon cancer and with the results of
studies in migrant populations. The ultimate proof
of the relation between the intestinal microflora and
colon cancer awaits further studies in human populations.
S259
S260
by colonic microsomes: a process influenced by type of
dietary fat. Nutr Cancer 1982;4:146-53
52. Temple NJ, El-Khatib SM. High-fat diets and fecal level of
reductase and colon musocallevel of ornithine decarboxylase, J}-glucuronidase, 5'-nucleotidase, ATPase, and esterase in mice. J Natl Cancer Inst 1984;72:679-84
53. Slattery ML, Sorenson AW, Mahoney AW,French TK, Kritchevsky D, Street JC. Diet and colon cancer: assessment of
risk by fiber type and food source. J Nat! Cancer Inst
1988;80:1474-80
54. Prizont R. Influence of high dietary cellulose on fecal glycosidases in experimental rat colon carcinogenesis. Cancer Res
1984;44:557-61
55. Freeman HJ. Effects of differing purified cellulose, pectin,
and hemicellulose fiber diets on fecal enzymes in 1,2dimethylhydrazine-induced rat colon carcinogenesis. Cancer Res 1986;46:5529-32
56. Druckrey H, Preussmann R, Matzkies F, Ivankovic S. Selektive Erzeugung Von Darmkrebs bei Ratten durch 1,2dimethylhydrazin. Naturwissenschaften 1967;54:285-6
57. Reddy BS, Watanabe K. Effect, of intestinal microflora on
3,2'-dimenthyl-4-aminobiphenyl-induced carcinogenesis in
F344 rats. J Natl Cancer Inst 1978;61:1269-71
58. Goldin BR, Gorbach SL. Effect of antibiotics on incidence
of rat intestinal tumors induced by 1,2-dimethylhydrazine
dihydrochloride. J Natl Cancer Inst 1981;67:877-80
59. Takada H, Hirooka T, Hiramatsu Y, Yamamoto M. Effect
of J}-glucuronidase inhibitor on azoxymethane-induced colonic carcinogenesis in rats. Cancer Res 1982;42:331-4
60. Bruce WR, Varghese AJ, Furrer R, Land PC. A mutagen
in the feces of normal humans. In: Hiatt HH, Watson JD,
Winsten JA, eds. Origins of human cancer. Vol 4. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratories,
1977:1641-6
61. Ames BN, McCann J, Yamasaki E. Methods for detecting
carcinogens and mutagens with the salmonella/mammalian-microsome mutagenicity test. Mutat Res 1975;
31:347-64
62. Ehrich M, Aswell:JE, Van Tassell RL, Wilkins TD. Mutagens in the feces of 3 South African populations at different levelsof risk for colon cancer. Mutat Res 1979;64:231-40
63. Reddy BS, Sharma C, Darby L, Laakso K, Wynder EL. Metabolic epidemiology of large bowel cancer: fecal mutagens
in high- and low-risk populations for colon cancer. A preliminary report. Mutat Res 1980;72:511-22
64. Reddy BS, Sharma C, Wynder EL. Fecal factors which modify
the formation of fecal co-mutagens in high- and low-risk
populations for colon cancer. Cancer Lett 1980;10:123
65. Kuhnlein V, Bergstrom D, Kuhnlein H. Mutagens in feces
from vegetarians and non-vegetarians. Mutat Res 1981;
85:1-12
66. Green MHL, Muriel WJ. Mutagen testing using Trp" reversion in Escherichia coli. Mutat Res 1976;38:3-32
67. Ferguson LR, Alley PG, Gribben BM. DNA-damaging activity in ethanol-soluble fractions of feces from New
Zealand groups at varying risks of colorectal cancer. Nutr
Cancer 1985;7:93-103
68. Dion P, Bruce WR. Mutagenicity of different fractions of
extracts of human feces. Mutat Res 1983;119:151-60
69. Wilkins TD, Lederman M, Van 'Iassell RL. Isolation of a
mutagen produced in the human colon by bacterial action.
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
methyl orange and metabolites produced by intestinal
anaerobes. Mutat Res 1978;58:375-9
34. Reid TM, Morton KC, Wang CY, King CM. Conversion of
Congo red and 2-azoxyfluorene to mutagens following in
vitro reduction by whole-cell rat cecal bacteria. Mutat Res
1983;117:105-12
35. Wheeler LA, Soderberg FB, Goldman P. The relationship
between nitro group reduction and the intestinal microflora.
J Pharmacol Exp Ther 1975;194:135-44
36. EI-Bayoumy K, Sharma C, Louis YM, Reddy B, Hecht SS.
The role of intestinal microflora in the metabolic reduction of 1-nitropyreneto l-aminopyrene in conventional and
germfree rats and in humans. Cancer Lett 1983;19:311-6
37. Miller JA, Miller EC. The metabolic activation of carcinogenic aromatic amines and amides. Prog Exp Tumor Res
1969;11:273-301
38. Goldin BR, Dwyer J, Gorbach SL, Gordon W, Swenson L.
Influence of diet and age on fecal bacterial enzymes. Am
J Clin Nutr 1978;31(10 Suppl):S136-40
39. Goldin BR. The role of diet and the intestinal flora in the
etiology of large bowel cancer. In: Winawer SJ, Schottenfeld D, Sherlock P, eds. Progress in cancer research and
therapy. Vol 13. Colorectal cancer prevention, epidemiology and screening. New York: Raven, 1980:43-50
40. Goldin BR, Swenson L, Dwyer J, Sexton M, Gorbach SL.
Effect of diet and Lactobacillus acidophilus supplements
on human fecal bacterial enzymes. J Natl Cancer Inst
1980;64:255-61
41. Goldin B, Gorbach SL. Alterations in fecal micro flora enzymes related to diet, age, lactobacillus supplements, and
dimethylhydrazine. Cancer 1977;40:2421-6
42. Weisburger JH. Colon carcinogens: their metabolism and
mode of action. Cancer 1971;28:60-70
43. Kay RM. Effects of diet on the fecal excretion and bacterial
modification of acidic and neutral steroids, and implications for colon carcinogenesis. Cancer Res 1981;41:3774-7
44. White BA, Lipsky RL, Fricke RJ, Hylemon PB. Bile acid
induction specificity of 7-a-dehydroxylase activity in an
intestinal Eubacterium sp, Steroids 1980;35:103-9
45. Salvioli G, Salati R, Bondi M, Fratalocchi A, Sala BM, Gilbertini A. Bile acid transformation by the intestinal flora
and cholesterol saturation in bile. Effects of Streptococcus
faecium administration. Digestion 1982;23:80-8
46. Reddy B~ Wynder EL. Large-bowelcarcinogenesis: fecal constituents of populations with diverse incidence rates of colon cancer. J Natl Cancer Inst 1973;50:1437-42
47. Hill MJ, Crowther JS, Drasar BS, Hawksworth GM, Aries
VC, Williams REO. Bacteria and etiology of cancer of large
bowel. Lancet 1971;1:95-100
48. Mower HF, Ray RM, Shoff R, Stemmermann GN, Nomura
A, Glober GA, Kamiyama S, Shimada A, Yamakawa H.
Fecal bile acids in two Japanese populations with different colon cancer risks. Cancer Res 1979;39:328-31
49. Mastromarino A, Reddy BS, Wynder EL. Metabolic epidemiology of colon cancer: enzymic activity of fecal flora. Am
J Clin Nutr 1976;29:1455-60
SO. MacDonald lA, Webb GR, Mahoney DC. Fecal hydroxysteroid dehydrogenase activities in vegetarian Seventh-Day
Adventists, control subjects and bowel cancer patients. Am
J Clin Nutr 1978;31(10Suppl):S233-8
51. Wargovich MJ, Felkner tc, Metabolic activation of DMH
Gorbach and Goldin
Intestinal Microflora and Colon Cancer
In: Bruce WR, Correa P, Lipkin M, 'Iannenbaum SR,
Wilkins TD, eds. Banbury report. Vol 7. Gastrointestinal
cancer: endogenous factors. Cold Spring Harbor, NY:Cold
Spring Harbor Laboratories, 1981:205-14
70. Lederman M, Van lassell R, West SEH, Ehrich MF, Wilkins
TD. In-vitro production of human fecal mutagen. Mutat
Res 1980;79:115-24
71. Van Tassell RL, MacDonald DK, Wilkins TD. Stimulation
of mutagen production in human feces by bile and bile
acids. Mutat Res 1982;103:233-9
S261
72. Hirai N, Kingston DGI, Van lassell RL, Wilkins TD. Structure elucidation of a potent mutagen from human feces.
J Am Chern Soc 1982;104:6149-50
73. Gupta I, Baptista J, Bruce WR, Che cr, Furrer R, Gingerich
JS, Grey AA, Marai L, Yates P, Krepinsky JJ. Structures
of fecapentaenes, the mutagens of bacterial origin isolated
from human feces. Biochemistry 1983;22:241-5
74. Wilkins TD, Van Tassell RL. Production of intestinal mutagens. In: Hentges DJ, ed. Human intestinal microflora in
health and disease.New York:AcademicPress, 1983:265-88
Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016