Mutation Research 589 (2005) 47–65

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Review

Bile acids as carcinogens in human gastrointestinal cancers

H. Bernsteina,b, C. Bernsteina,*, C.M. Paynea,b, K. Dvorakovaa,b, H. Garewalb,c,d
a
Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson AZ 85724, USA
b
Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
c
Department of Internal Medicine, Arizona Cancer Center, College of Medicine, University of Arizona, Tucson AZ 85724, USA
d
Tucson Veterans Affairs Medical Center, Section of Hematology/Oncology, Tucson, AZ 85723, USA
Received 26 January 2004; received in revised form 27 July 2004; accepted 6 August 2004
Available online 22 September 2004

Abstract

Bile acids were first proposed to be carcinogens in 1939 and 1940. On the basis of later work with rodent models, bile acids
came to be regarded as cancer promoters rather than carcinogens. However, considerable indirect evidence, obtained more
recently, supports the view that bile acids are carcinogens in humans. At least 15 reports, from 1980 through 2003, indicate that
bile acids cause DNA damage. The mechanism is probably indirect, involving induction of oxidative stress and production of
reactive oxygen species that then damage DNA. Repeated DNA damage likely increases the mutation rate, including the
mutation rate of tumor suppressor genes and oncogenes. Additional reports, from 1994 through 2002, indicate that bile acids, at
the increased concentrations accompanying a high fat diet, induce frequent apoptosis. Those cells within the exposed population
with reduced apoptosis capability tend to survive and selectively proliferate. That bile acids cause DNA damage and may select
for apoptosis-resistant cells (both leading to increased mutation), indicates that bile acids are likely carcinogens. In humans, an
increased incidence of cancer of the laryngopharyngeal tract, esophagus, stomach, pancreas, the small intestine (near the
Ampulla of Vater) and the colon are associated with high levels of bile acids. The much larger number of cell generations in the
colonic (and, likely, other gastrointestinal) epithelia of humans compared to rodents may allow time for induction and selection
of mutations leading to cancer in humans, although not in rodents.
# 2004 Elsevier B.V. All rights reserved.

Keywords: Bile acids; Carcinogen; Apoptosis; Gastrointestinal cancer; DNA damage; Oxidative stress

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

2. Bile acids in the body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

3. Potentially more severe consequences of bile acid exposure in humans than in experimental rodent models . . . 50

* Corresponding author. Tel.: +1 520 626 6069; fax: +1 520 626 2100.
E-mail address: bernstein3@earthlink.net (C. Bernstein).

1383-5742/$ – see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrrev.2004.08.001
48 H. Bernstein et al. / Mutation Research 589 (2005) 47–65

4. Bile acids are implicated in the etiology of human gastrointestinal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 52

4.1. Upper aerodigestive tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.2. Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.3. Stomach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.4. Gallbladder and bile duct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.5. Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.6. Small intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.7. Colon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

5. Effects of vitamin D, the vitamin D receptor, and calcium on colon cancer and bile acid regulation . . . . . . . . 53

6. Mechanisms of actions of bile acids: bile acids introduce oxidative/nitrosative stress . . . . . . . . . . . . . . . . . . . 54

7. Mechanisms of actions of bile acids: bile acids induce DNA damage, a likely direct cause of cancer risk . . . . 55

8. Bile acids as likely mutagens in a pathway to gastrointestinal cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

9. Bile acids cause apoptosis, which may select for apoptosis resistance and increased cancer risk . . . . . . . . . . . 58

10. Is it reasonable to hypothesize that bile acids, natural detergents with an important function in digestion,
can also be carcinogens? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

1. Introduction gens. However, the bile acids, by themselves, were not

The bile acid, deoxycholic acid, was first proposed to
be a carcinogen in 1940 by Cook et al. [1], based on
induction of tumors in mice when injected, in their
flanks, with this bile acid. These authors also cite a 1939
meeting report by Vittorio Ghiron that ‘‘desoxycholic
acid elicited transplantable subcutaneous fibro-sarco-
mas in a high proportion of the mice and rats injected.
This is believed to be the first experimental production
of malignant growths with a compound that exists under
some conditions in the human body.’’ In a 1999 mouse
experiment, using mice with a germ line mutation in Apc
(Min/+) as a model of familial adenomatous polyposis,
administration of chenodeoxycholate increased duode-
nal tumors [2]. This increase occurred without addition
of a standard carcinogen such as MNNG, MNU or
azoxymethane, so that in Min/+ mice, the bile acid
chenodeoxycholate was a carcinogen.
In a number of rat experiments reported between
1974 and 1993, several bile acids were shown to be
promoters, increasing tumorigenesis by known carcino-
carcinogens, failing to induce colon tumors. In
particular, in rats, the bile acids lithocholic, taurodeoxy-
cholic and deoxycholic acids had a promoting effect
on colon carcinogenesis after intrarectal instillation of
N-methyl-N0 -nitro-N-nitrosoguanidine (MNNG) [3–5].
The bile acid, cholic acid, also had a promoting effect on
colon tumor formation in rats after intrarectal instilla-
tion of N-methyl-N-nitrosourea (MNU) [6] or sub-
cutaneous injection of azoxymethane [7]. The promot-
ing effect of cholic acid may have been due to the
formation of deoxycholic acid from cholic acid by
bacterial action in the colon [8]. Further, taurocholate, in
rats, enhanced the induction by MNNG of hyperplastic
and neoplastic lesions in stomach mucosa [9]. Based
largely on these experiments in non-mutated rat model
systems, it has been generally assumed that bile acids act
as promoters, but not as carcinogens, in humans.
Recently, however, considerable indirect evidence
and logical argument supports the view that bile acids
are carcinogens in humans. The evidence and
arguments are detailed below, and include evidence
 H. Bernstein et al. / Mutation Research 589 (2005) 47–65 49

Fig. 1. Formation of bile acids. Chenodeoxycholic acid and cholic acid are formed in the liver from cholesterol. The bacterial enzyme 7-a
dehydroxylase converts chenodeoxycholic acid into lithocholic acid and converts cholic acid into deoxycholic acid.

that bile acids cause DNA damage, induce frequent
apoptosis, and, after repeated exposure, select for cells
with reduced apoptosis capability. In addition, high or
abnormal bile acid exposure is associated with
increased incidence of cancer in the laryngophar-
yngeal tract, esophagus, stomach, pancreas, the small
intestine (near the Ampulla of Vater) and the colon.
Bile acids are avidly re-adsorbed by an active bile
salt re-absorptive mechanism in the terminal ileum,
with uptake into ileal columnar epithelium cells,
leaving less than 5% of the bile salt pool to enter the
colon [11,14]. After uptake into enterocytes of the
ileum, bile salts are shuttled to the basolateral domains
of the cells for efflux into the portal circulation,

2. Bile acids in the body

The primary bile acids, cholic acid and cheno-

deoxycholic acid, are derived from cholesterol,
primarily in the liver, through three main pathways
involving 10–14 different enzymes [10]. The struc-
tures of cholesterol, cholic acid and chenodeoxycholic
acid are shown in Fig. 1. After their synthesis in
hepatocytes, bile acids are excreted as C24 amides
conjugated with glycine or taurine [11]. The cyclical
path of bile acids in the body, after their synthesis in
the liver, is shown schematically in Fig. 2. In
particular, the conjugated bile salts are excreted from
the liver into the gall bladder, at a concentration of
approximately 100 mM [12], and then released into
the intestinal tract when fat enters the proximal portion
of the intestine [13]. Within the small intestine, the
bile acids are critically important for lipid absorption
in the ileum [14]. Fig. 2. The enterohepatic circulation of bile acids.
50 H. Bernstein et al. / Mutation Research 589 (2005) 47–65
Table 1
Bile acid concentrations (mM)a in fecal water of normal individuals on a normal diet [18] and a high fat diet [19]
Bile acid
Cholic acid
Chenodeoxycholic acid
Deoxycholic acid
Lithocholic acid
Ursodeoxycholic acid
Total bile acids
a
Median values of bile acids from 25 normal individuals on a normal diet [18] or seven individuals on a high fat diet [19], with the range of
values in parentheses, N.D.: not determined.
transported by portal vein blood to the liver, and
extracted from portal blood plasma by hepatocytes
[11]. Fasting and postprandial levels of bile acids in
the portal venous blood are approximately 0.014 and
0.043 mM, respectively [15]. The liver is efficient in
removing bile acids from the circulation, so that
peripheral arterial blood serum of healthy individuals
has an approximate bile acid concentration of
0.003 mM [15].
There are approximately 6–12 enterohepatic
circulations per day, so that there is an average entry
of 20% of the bile acid pool per day into the colon
[16]. Bile acids entering the colon are metabolized by
the anaerobic bacterial flora. Bacteria of the large
intestine carry out two major and a number of minor
reactions on the bile salts [17]. The first is
deconjugation to release free bile acids, which are
only poorly ionized and are lipophilic. The second
major reaction is 7-a dehydroxylation to yield
deoxycholic and lithocholic acids, respectively, from
cholic and chenodeoxycholic acids (Fig. 1) [17].
Deoxycholic acid is partly absorbed in the colon and
enters the enterohepatic circulation, where it is
conjugated in the liver and secreted in the bile [16].
Lithocholic acid is fairly insoluble and little of it is
reabsorbed [17]. Thus, the circulating bile acid pool
(conjugated when it leaves the gallbladder, and then
de-conjugated by action of bacterial enzymes after it
enters the colon) is composed of about 30–40% each
of cholic acid and chenodeoxycholic acid, about 20–
30% of deoxycholic acid, and less than 5% of
lithocholic acid [16]. Further bacterial degradation in
the colon and alterations in the liver produce tertiary
bile acids, such as ursodeoxycholic acid, which also
enter the circulating bile acid pool. Bile acid
compositions, reported for the fecal water (which
bathes the colon wall), both with a normal diet [18]
Fecal water (normal diet) Fecal water (high fat diet)
0.011 (0–0.058) N.D.
0.004 (0–0.017) N.D.
0.104 (0.046–0.210) 0.17 (0.07–0.73)
0.013 (0.004–0.043) 0.09 (0.04–0.30)
0.013 (0–0.039) N.D.
0.184 (0.088–0.393) 0.36 (0.16–1.96)
and after a high fat diet [19] are shown in Table 1. Bile
acids in the gallbladder [20] (later ejected into the
small intestine, and subsequently diluted with food),
or in esophageal aspirates [21] (from duodenal reflux)
are conjugated with taurine or glycine, and may be
sulfated in addition, as shown in Table 2. For persons
on a high fat diet, both deoxycholic acid and
lithocholic acid may be substantially raised in the
fecal water in the colon (Table 1). Conjugated bile
acids are at much higher levels in the gallbladder, and
upon delivery, near the Ampulla of Vater, than bile
acids in any other region of the body (Tables 1 and 2).
Reflux of duodenal contents into the esophagus of
patients can yield peak (short term) concentrations of
conjugated bile acids at levels as high as the maximum
bile acid concentration reported in the fecal water of
the colon after a high fat meal (Tables 1 and 2). Based
in part on DNA damaging effects, evidence of muta-
genicity, and higher frequency in serum of adenoma
patients, the secondary bile acids, deoxycholic acid and
lithocholic acid are considered the ones most likely to
be implicated in colon carcinogenesis [16,17].
3. Potentially more severe consequences of bile
acid exposure in humans than in experimental
rodent models
On the basis of the observation that bile acids by
themselves do not induce colonic tumors in most
rodent model systems, but do enhance the induction of
tumors by carcinogens such as MNNG, MNU and
azoxymethane, it is ordinarily assumed that in
humans, like rodents, bile acids are promoters, not
carcinogens. However, there is a fundamental differ-
ence between the rodent models and human gastro-
intestinal cancer. The rodent models and humans have
 H. Bernstein et al. / Mutation Research 589 (2005) 47–65 51

Table 2
Mean bile acid concentrations in mM in the gallbladder [20] (126 normal individuals) and in esophageal aspirates [21] (24 patients)
Gallbladder (S.D.)ab Esophagus (S.E.M.)
Glycocholic acid 29.96 (22.56) 0.098 (0.034)
Taurocholic acid 24.62 (27.61) 0.013 (0.006)
Glycochenodeoxychlic acid 29.86 (38.25) 0.069 (0.025)
Taurochenodeoxycholic acid 18.46 (18.49) 0.013 (0.008)
Glycodeoxycholic acid 17.10 (16.25) 0.030 (0.011)
Taurodeoxycholic acid 30.44 (37.91) 0.005 (0.003)
Glycolithocholic acid 0.48 (0.88) 0.002 (0.002)
Taurolithocholic acid 0.40 (0.81) 0.0
Sulfoglycolithocholic acid 11.26 (19.67) N.D.
Sulfotaurolithocholic acid 0.56 (0.55) N.D.
Glycoursodeoxycholic acid 2.48 (1.28) 0.0
Tauroursodeoxycholic acid 0.52 (0.42) 0.0
Total bile acids 166.14 (70.94) N.D.
Maximum peak total bile acids (during reflux of patient) N.D. 2.015
a
Millimolar concentrations of individual bile acids were calculated from values given in ref. [20].
b
Abbreviations: S.D., standard deviation; S.E.M., standard error of the mean; N.D., not determined.

very different time periods of exposure to bile acids.
The rodent experiments were carried out for one year
or less (e.g. [1–5]). In humans, cancers of the digestive
tract generally develop after many years of exposure.
In humans, the mean age of occurrence of colon
cancer is 60 years for men, and 59 years for women
[22]. For esophageal adenocarcinoma, the mean age is
64 years [23].
The cell turnover times (defined as the time
necessary to replace the number of cells present in the
entire cell population) of rapidly renewing cell
populations are about the same regardless of life span
of mice (2.5 years), rats (3.5 years) and man (70 years)
[24]. Thus, rapidly renewing cell populations are
replaced many more times during the life span of man
than mice or rats. Colonic epithelium renews itself
roughly 365 times during the life span of mice and rats
and 5110 times in man [24]. At the mean age that
humans develop colon cancer (59–60 years) there
would have been about a 30-fold greater cell turnover
in the human colon than in rodent models where the
animals were exposed to bile acids for a year or less.
Since carcinogenic mutations arise as the result of
errors of DNA replication, the opportunity for such
mutations is at least 30-fold greater in humans than in
the rodent models. Thus, repeated exposure of the
colon to high concentrations of bile acids over a 59–60
year period in humans may be expected to have more
severe consequences than a 1 year exposure in rodents.
The lack of carcinogenic effect in 1 year (or less) in
rodent models does not rule out the possibility of a
carcinogenic effect of bile acids in humans.
That MNNG, MNU and azoxymethane are
effective carcinogens over the time span of these
rodent experiments implies that they are potent
mutagens in rat colon. In contrast, bile acids may
be much weaker mutagens, whose cumulative effect
would only result in carcinogenesis after many years
of exposure, as appears to be the case in humans.
Another factor making these rodent models of colon
carcinogenesis less than ideal for drawing the conclu-
sion that bile acids are non-carcinogens in humans is the
different spectrum of bile acids to which the GI tract of
rodents is ordinarily exposed compared to humans. For
instance, in rat feces, the predominant bile acids are
hyodeoxycholic acid (23%), muricholic acid (b and v
combined) (21%) and deoxycholate (17%) (see con-
ventional rats in [25]), whereas in human fecal water the
predominant bile acids are deoxycholate (58%),
lithocholate (12%) and cholic acid (4%) (calculated
from individuals on a low fiber diet [26]) [see also,
Table 1 (normal diet)]. In the rodent models where bile
acids are administered in the diet [6,7] cholic acid is the
bile acid usually used. When bile acids are administered
by repeated intrarectal instillation, various bile acids
have been used; lithocholate and taurodeoxycholate [3],
deoxycholate [4], and cholate and chenodeoxycholate
[5]. Since the rat GI tract presumably is adapted to cope
with a substantially different spectrum of bile acids than
that of humans, the rat response to these exposures
52 H. Bernstein et al. / Mutation Research 589 (2005) 47–65
probably does not closely mimic the response of the
human GI tract to these bile acids.
4. Bile acids are implicated in the etiology of
human gastrointestinal cancer
Although bile acids have been predominantly
studied in relation to promotion of colon cancer, it
is now evident that bile acids are implicated as playing
a role in carcinogenesis throughout the human gastro-
intestinal (GI) tract. We consider it likely that bile
acids may act by a common underlying mechanism at
various sites within the GI tract. However, conditions
vary widely from site to site within the GI tract. It is
certainly possible that at any given site some factor other
than bile acid exposure, or in combination with bile acid
exposure, is more important in carcinogenesis, or that
the underlying mechanisms of carcinogenesis may be
different from site to site.
4.1. Upper aerodigestive tract
In the upper aerodigestive tract (pharynx and
larynx) bile acid exposure occurs during reflux arising
in the duodenum and passing through the stomach and
esophagus. This reflux contains not only bile acid from
the duodenum, but also acid from the stomach.
Laryngopharyngeal reflux (LPR) is a common event in
patients with head and neck cancer, suggesting that
LPR has a role in the carcinogenic process of
squamous cell carcinoma of the head and neck [27].
The relative importance of acidity and bile acid
exposure has not yet been determined. Bile salts or
acidic conditions, or both, induce cyclooxygenase-2
(COX-2) expression in normal pharyngeal mucosa
[28]. Since it is known that bile acid is associated with
tumor formation in the esophagus and involves over-
expression of COX-2, it was proposed that bile acids
have a similar role in the tumorigenesis of the upper
aerodigestive tract [28].
4.2. Esophagus
Barrett’s metaplasia of the esophagus is a major
risk factor for the development of esophageal
adenocarcinoma. Individuals with Barrett’s metapla-
sia have a higher exposure to bile acids in their
refluxate than normal individuals or those with
minimal esophagitis [29,30]. Furthermore, individuals
with early (T1 tumor stage) adenocarcinoma have an
even higher exposure to bile than individuals with
uncomplicated Barrett’s metaplasia [31]. The concept
that bile acids have a significant role in esophageal
adenocarcinoma has been suggested by numerous
investigators (e.g. [32–35]). Animal models and
human studies suggest that esophageal mucosal injury
can arise from a synergistic damaging effect of
conjugated bile acids (e.g. taurocholate and tauro-
deoxycholate) and HCl, as well as a synergistic
interaction of unconjugated bile acids and trypsin at
more neutral pH values [36].
4.3. Stomach
In the remnant stomach of rats after gastrectomy,
bile acids, the main component of the duodenal juice,
have been implicated in gastric cancer due to
duodenogastric reflux [37]. In humans, duodenogas-
tric reflux also appears to be implicated in gastric
stump carcinoma [38].
4.4. Gallbladder and bile duct
The gallbladder and bile duct are exposed to high
concentrations of bile acids, as discussed above in
Section 2. The role of bile acids in gallbladder and bile
duct carcinogenesis is unclear. In a study carried out in
India, raised biliary deoxycholic acid and lithocholic
acid concentrations were present in patients with
carcinoma of the gallbladder, and it was suggested that
this may be a factor in gallbladder carcinogenesis [39].
On the other hand, in a study carried out in Mexico and
Bolivia, patients with carcinoma of the gallbladder did
not have significantly raised conjugated deoxycholic
or lithocholic acids in the gallbladder [20]. Dietary
differences between India on the one hand, and Mexico
and Bolivia on the other, may have contributed to these
different observations.
Choledochal cysts are dilated lesions in the biliary
tree, and biliary tract carcinoma occurs 5–35 times
more frequently in patients with these cysts than for
those without them [40]. The bile acid composition of
the cyst contents, in one case, showed 2% lithocholate,
88% deoxycholate, 5% chenodeoxycholate, and 5%
cholate, with virtually all the bile acids in unconju-
 H. Bernstein et al. / Mutation Research 589 (2005) 47–65
gated form. In contrast, the bile acids found in the
hepatic bile of this individual were fully conjugated,
and were composed of 0% lithocholate, 34%
deoxycholate, 43% chenodeoxycholate and 23%
cholate. The patient was found to have metaplasia
of the epithelial lining of the cyst. These data
suggested that stasis of bile within choledochal cysts
contributes to bacterial overgrowth and generation of
unconjugated secondary bile acids [40]. However,
another study of secondary bile acids in choledochal
cysts in eleven patients did not find alterations in
relative compsition or absolute concentrations of
secondary bile acids [41].
4.5. Pancreas
Epidemiological studies have demonstrated a
positive correlation between ingestion of a western-
style high fat diet and the incidence of pancreatic
cancer [42–44]. Most adenocarcinomas of the
pancreas occur in the head of the gland, which is in
close proximity to bile. On the basis of the finding that
bile acids induce cyclooxygenase-2 expression in
human pancreatic cancer cell lines, a possible role for
bile acids in the pathogenesis of pancreatic cancer has
been suggested [45].
4.6. Small intestine
About 57% of all adenocarcinomas of the small
intestine of humans occur in a 7 cm length which
comprises less than 1% of the entire length of the
small intestine [46]. The majority of these adeno-
carcinomas were pinpointed to a small region around
the Ampulla of Vater, where bile and pancreatic
secretions enter the small intestine. This finding led to
the suggestion that bile acids may be carcinogenic
[46]. Individuals with familial adenomatous polyposis
(FAP) have an increased risk for adenomas and
cancers of the small and large intestines. In the small
intestine these lesions also occur mainly around the
Ampulla of Vater, where their distribution parallels
mucosal exposure to bile [47,48].
4.7. Colon
The development of colon cancer in humans
involves genetic and environmental factors. A major
53
environmental factor appears to be diet. Epidemiolo-
gical studies indicate a positive association between
dietary fat consumption and colon cancer incidence
(e.g. [49–54]). A high fat diet is associated with
increased bile acid secretion [55]. This association
presumably reflects the role of bile acids in
emulsifying dietary fat for absorption by the small
intestine. Epidemiological studies have found that
fecal bile acid concentrations are elevated in popula-
tions with a high incidence of colon cancer (e.g.
[17,54,56–63]). The most important bile acids in the
etiology of colon cancer in humans appear to be the
secondary bile acids, deoxycholic acid and lithocholic
acid [17]. Deoxycholate is also higher in the serum of
patients with colorectal adenomas than in individuals
without adenomas [64,65].
5. Effects of vitamin D, the vitamin D receptor,
and calcium on colon cancer and bile acid
regulation
Epidemiologic evidence indicates that dietary
intake of vitamin D and exposure to sunlight
contribute to the prevention of colon cancer [66,67].
The mechanisms by which vitamin D3 prevents colon
cancer may involve increased apoptosis [68],
decreased proliferation [69,70], enhanced cellular
differentiation [71] and/or increased transcription that
results from activation of the vitamin D3 receptor
[72,73]. In addition, vitamin D3 may protect against
oxidative DNA damage [69]. Some of the beneficial
activities of vitamin D3 may ameliorate damaging
effects of bile acids. Of particular interest is the role of
the vitamin D3 receptor as a bile acid sensor [74].
Activation of the vitamin D3 receptor by the secondary
bile acid, lithocholic acid, or by vitamin D3 induces
expression of CYP3A, a cytochrome P450 enzyme
that detoxifies lithocholic acid [74] through catabolic
reactions (see review by Wolf [75]).
Epidemiologic and animal studies indicate that
increased dietary intake of calcium lowers the
incidence of colon cancer [72,76–88]. The mechan-
isms by which calcium reduces colon cancer risk are
probably multifactorial. Calcium may interact directly
with bile acids in the intestinal lumen [82] to form
insoluble soaps [76], thereby reducing the deleterious
effects of bile acid interactions with epithelial cells.
54 H. Bernstein et al. / Mutation Research 589 (2005) 47–65

Calcium may also activate signal-transduction path-
ways that reduce cellular proliferation, enhance
cellular differentiation and induce apoptosis [81–87].
6. Mechanisms of actions of bile acids:
bile acids introduce oxidative/nitrosative
stress
Deoxycholate and other hydrophobic bile acids
increase oxidative/nitrosative stress through the
generation of reactive oxygen/nitrogen species
(ROS/RNS). The source of the ROS/RNS is likely
multifactorial (see Fig. 3). As indicated in Fig. 3,
some of the generation of ROS results from a direct
detergent effect of bile acids on membrane enzymes,
such as phospholipase A2 (PLA2), which upon
activation release arachidonic acid. Arachidonic
acid is then acted on by enzymes such as cycloox-
ygenase (COX) and lipoxygenase (LOX) to release
ROS through the partial reduction of molecular
oxygen (O2), which accompanies formation of
prostaglandins and leukotrienes. Bile acid produc-
tion of ROS has been shown to depend, in part, on
COX and LOX activity [89–92]. The ROS produced
are likely, in turn, to cause oxidative DNA damage
[93], in part, through the iron-catalyzed Fenton
reaction [92].

Fig. 3. Formation of ROS and RNS by hydrophobic bile acids and the pathways by which they induce DNA damage.
 H. Bernstein et al. / Mutation Research 589 (2005) 47–65
ROS are also produced by mitochondria, as
indicated in Fig. 3. Mitochondria are known to be
damaged by bile acids [94–98], although the
mechanism of this damage is not known. Mitochon-
drial damage due to bile acids can arise from several
causes, as illustrated in Fig. 3; (1) the endogenous
generation of arachidonic acid [99], (2) truncated bid
(tbid) [100] from the ligand-independent activation of
the Fas receptor (FasR) [101], (3) Bak released from
the stressed endoplasmic reticulum (ER), (4) ROS
themselves, or (5) the decline of mitochondrial NAD+
levels as a result of DNA damage-induced poly(ADP-
ribose) polymerase (PARP) activity.
Nitrosative stress, as illustrated in Fig. 3, can be
generated through the activities of both NOS2
(inducible nitric oxide synthase) and NOS3 (endothe-
lial NOS). Deoxycholate activates the redox-sensitive
transcription factor, NF-kB [102], resulting in
increased levels of NOS2 [103]. Bile acids also
induce the activation of phospholipase C, which
releases inositol 3-phosphate (IP3) [104,105] resulting
in the release of Ca2+ from the ER [106]. The increase
in cytosolic Ca2+ can, in turn, activate the Ca2+/
calmodulin-dependent NOS3 [107], known to be
expressed in colon epithelial cells [108] in addition to
endothelial cells. NOS3, which produces RNS, can
also be activated by hydrogen peroxide (H2O2) [109]
resulting from the dismutation of O2. Deoxycholate
activates the peroxynitrite (ONOO) pathway, as
evidenced by the formation of nitrotyrosine residues
[110]. DNA damage may be caused by the direct
action of NO and ONOO or the generation of the
hydroxyl radical (OH) through Fenton chemistry. NO
can also enhance oxidative DNA damage through the
S-nitrosylation and inhibition of DNA repair enzymes
[111] that have a critical thiol group in their catalytic
site or interact with DNA through zinc-finger domains.
In addition, an important source of ROS generated
by high levels of hydrophobic bile acids, is from the
action of microflora within the intestinal lumen. The
bile tolerant Bacteroides strains, for example, synthe-
size and utilize menaquinones, and vitamin K1 is a co-
factor in the dehydrogenation of bile acid substrates to
form reduced K vitamins [112]. It has been suggested
that these reduced K vitamins can be transported into
mature colonocytes as mixed micelles and initiate
superoxide (O2) generating redox cycling reactions
[112].
55
7. Mechanisms of actions of bile acids: bile acids
induce DNA damage, a likely direct cause of
cancer risk
As described above, bile acids cause oxidative/
nitrosative stress and liberate ROS and RNS
[91,93,95,96,98,102,113–115]. ROS have a plethora
of biological effects, but perhaps of greatest sig-
nificance, in terms of carcinogenesis, is their ability to
induce DNA damage. Oxidative DNA damage can
cause mutation [116]. Bile acids also increase NOS2
expression [117] leading to increased production of
reactive nitrogen species and increased DNA damage
[118].
Table 3 lists 15 studies showing that the bile acids
deoxycholate and lithocholate cause DNA damage in
mammalian cells. These studies, plus additional
studies with bacteria and with isolated DNA, are
described next.
The bile acid lithocholic acid produces single-
strand breaks in the DNA of mouse lymphoblastoma
cells or of colon crypt cells [119–121]. Foreskin
fibroblasts treated with chenodeoxycholate and deox-
ycholate were found to experience significantly more
DNA damage than untreated fibroblasts (as indicated
by increased DNA repair, measured by unscheduled
DNA synthesis) [122]. DNA repair-deficient Chinese
hamster ovary (CHO) cells were found to be more
sensitive than wild-type cells to killing by cheno-
deoxycholate and deoxycholate, which again indicates
induction of DNA damage by these agents [122]. Bile
acids have also been shown to induce a DNA repair
response in bacteria [122,123]. Bile acids, or their
activated metabolites, have been reported to interact
directly with naked DNA [124–126]. However, these
interactions may not be physiologically relevant to
bile acid exposure of mammalian cells, since bile
acids may not gain direct access to nuclear DNA. In
HeLa cells, deoxycholate and lithocholate activate the
gadd153 promoter, a promoter activated by DNA
damaging agents [127]. Deoxycholate also activates
the gadd153 promoter in HepG2 cells [128]. Canine
bile containing a high level of deoxycholic acid,
obtained in an experimental model for the anomalous
arrangement of the pancreaticobiliary ducts, induced
DNA strand breaks in HeLa cells [129].
Extensive DNA damage was introduced into
colonic cells by treatment, at physiologic concentra-
 56
Table 3
Bile acids cause DNA damage in mammalian cells
Bile acid(s)/salt(s) Evidence for DNA damage Source of DNA Year [ref.]
Lithocholate Single strand breaks (nucleoid Mouse lymphoblastoma cells 1980 [119]
sedimentation and alkaline elution)
Lithocholate Single strand breaks (nucleoid sedimentation) Mouse lymphoblastoma cells 1982 [120]
Lithocholate Single strand breaks (nucleoid sedimentation) Colon crypt cells 1985 [121]
Chenodeoxycholate Unscheduled DNA synthesis Human foreskin fibroblasts 1991 [122]

H. Bernstein et al. / Mutation Research 589 (2005) 47–65

deoxycholate
Chenodeoxycholate Increased cytotoxity when applied to two mutant Chinese Hamster Ovary cells 1991 [122]
deoxycholate cell lines with different DNA repair defects
Deoxycholate lithocholate Activation of gadd153 promoter, a promoter Human HeLa cells 1996 [127]
activated by DNA damage
Lithocholate deoxycholate DNA breakage seen by single cell electrophoresis Human colonic epithelial HT-29 cells 1997 [92]
‘‘Comet’’ assay; likely
oxidative DNA damage
Lithocholate deoxycholate Oxidative DNA damage seen by endonuclease III + Human colonic epithelial CACO-2 cells 1997 [93]
‘‘Comet’’ assay
Deoxycholate DNA strand breaks seen by nick translation method Canine bile used to test strand breaks in HeLa cells 1997 [129]
Lithocholate DNA breakage seen by single cell electrophoresis Human colon mucosal cells (from biopsies) 1997 [132]
‘‘Comet’’ assay
Lithocholate deoxycholate DNA breakage seen by single cell electrophoresis Human colonic epithelial HT-29 cells 1998 [133]
‘‘Comet’’ assay; likely
oxidative DNA damage
Deoxycholate Induction of DNA repair enzyme PARP (responds Human Jurkat cells, rat colon epithelial cells 1998 [102]
to breaks in DNA)
Deoxycholate Activation of gadd153 promoter, a promoter HepG2 cells 1999 [128]
activated by DNA damage
Deoxycholate DNA breakage seen by single cell electrophoresis Human colonic epithelial HCT-116 cells (p53+/+), HCT-15 cells (p53/) 2001 [130]
‘‘Comet’’ assay
Deoxycholate Single strand breaks seen by single cell electrophoresis Human colonic epithelial HT-29 cells, HCT-116 cells 2002 [131]
‘‘Comet’’ assay
Deoxycholate Induces DNA repair gene BRCA-1 Human colonic epithelial HCT-116 cells 2003 [138]
 H. Bernstein et al. / Mutation Research 589 (2005) 47–65
tions, with deoxycholate [92,130,131] or lithocholate
[92,132] as measured by the comet assay to detect and
visualize DNA damage (strand breaks). Deoxycho-
late-induced DNA damage could be reduced by
inhibiting release of a membrane-bound phospholipid,
arachidonic acid, and by inhibiting metabolism of
arachidonic acid through the lipoxygenase pathway
[133]. The lipoxygenase pathway, through a one
electron reduction of molecular O2 produces the
superoxide free radical. Similarly, incubation of cells
with CuDIPSH [copper(II) 3,5-diisopropyl salicylate
hydrate], a superoxide dismutase mimetic compound
(which removes superoxide), also protects cells
against deoxycholate-induced DNA damage [133].
In addition, the antioxidant, vitamin E, a potent
inhibitor of lipid peroxidation, reduced deoxycholate-
and lithocholate-induced DNA damage close to
negative control values [92]. These results indicate
that ROS produced during lipid peroxidation may be a
major source of DNA damage produced by deox-
ycholate and lithocholate. Deoxycholate and litho-
cholate, at levels often found in patients with colon
cancer associated pathology, produced DNA damage
as measured by the comet assay [93]. Endonuclease
III, an enzyme which nicks DNA specifically at sites of
oxidized pyrimidines, is used in the comet assay to
determine whether the DNA damage is oxidative in
nature. Endonuclease III treatment significantly
increased the genotoxicity of the bile acids [93],
again suggesting that the DNA damage is caused by
ROS.
Activation of poly(ADP-ribose) polymerase
(PARP) is an immediate cellular reaction to DNA
strand breakage as induced by oxidizing agents as well
as other agents [134]. The resulting formation of
protein-coupled poly(ADP-ribose) facilitates survival
of proliferating cells under conditions of DNA
damage, probably via its contribution to DNA base-
excision repair [134]. Deoxycholate activates PARP,
and specific inhibition of PARP sensitizes cells to the
induction of apoptosis by deoxycholate [102]. These
findings suggest that deoxycholate produces DNA
strand breaks whose repair is mediated by PARP.
Deoxycholate and chenodeoxycholate, at physiologic
concentrations, stimulate hydroxylation of DNA bases
mediated by the hydroxyl radical (a highly reactive
oxygen species) to form 8-hydroxydeoxyadenine and
8-hydroxydeoxyguanine [135,136].
57
Lithocholate inhibits polymerase b, an enzyme
required for DNA base excision repair [137]. This
inhibition should have the effect of increasing the level
of unrepaired DNA damages. Deoxycholate, at low
doses, increases expression of the DNA repair gene
BRCA-1 at the mRNA and protein level, suggesting
that DNA damage caused by deoxycholate may up-
regulate this repair protein [138].
Deoxycholate has also been shown to cause the
degradation of p53 [139]. The functions of p53 in
response to DNA damage include, first, inducing cell
cycle arrest to allow time for DNA repair; second,
acting directly in DNA repair processes, and third,
promoting apoptosis when DNA damage is excessive
[140]. Thus, a reduction in p53 by deoxycholate
should increase genomic instability, because of
reduced DNA repair and reduced apoptotic removal
of cells with DNA damage.
8. Bile acids as likely mutagens in a pathway to
gastrointestinal cancer
Spontaneous mutagenesis in mammalian cells
appears to be caused mainly by oxidative events
[141]. We reviewed, above, the evidence that bile
acids cause DNA damage, particularly oxidative
DNA damage. A certain fraction of the oxidative
DNA damages induced by repeated exposure to high
levels of bile acids can be expected to remain
unrepaired until the next cycle of DNA replication.
Such damages will cause replication errors as the
replication apparatus inaccurately copies the
damaged template [142]. Such replication errors lead
to mutation, and some of the mutations will cause
aberrant expression of oncogenes and tumor sup-
pressor genes, leading to cancer (Fig. 4). In addition,
oxidative stress inactivates the human mismatch
repair system [143] and this would also be expected
to enhance mutagenesis. In an azoxymethane-treated
rat model of colon tumorigenesis, deoxycholate
increased the incidence of colon tumors as well as
the incidence of K-ras point mutations in the tumors
[144]. This suggests that the promotion effect of bile
acids on colon carcinogenesis in rodents may involve
induction of K-ras mutations. The bile acid litho-
cholic acid has also been reported to induce cell
transformation of CHO cells [145], which could
58 H. Bernstein et al. / Mutation Research 589 (2005) 47–65
Fig. 4. Pathway to GI cancer through bile acid induced DNA
damage.
indicate induction of mutations or stable epigenetic
alterations. Bile acids were also reported to increase
the mutation frequency in transgenic rat fibroblasts at
pH 5, but not at pH 7 [146].
Attempts have been made to measure the muta-
genicity of bile acids using the Ames Salmonella
mutagen test system. In the standard experiment,
which measures induced reversion to amino acid
independence in an auxotrophic strain of bacteria, the
cells are treated with mutagen and plated on selective
agar containing a growth limiting supplement of the
particular amino acid. Of 30 bile acids tested in the
standard Ames Salmonella mutagen test system, none
was mutagenic by itself, but lithocholic acid was co-
mutagenic with 2-aminoanthracene activated by
phenobarbital-stimulated rat liver homogenate
[147]. Deoxycholic acid and lithocholic acid were
also co-mutagenic towards 1,2-dimethylhydrazine
[148]. The N-nitroso bile acid conjugates N-nitroso-
taurocholic acid and N-nitrosoglycocholic acid are
formed naturally by a reaction of bile acids with N-
nitrosamides catalyzed by bacterial enzymes. These
conjugates were mutagenic in the standard Ames
Salmonella test system and in a human lymphoblast
cell line [149].
The standard mutation experiment, performed on
petri dishes, is effective for detecting an induced
mutation rate only if it is considerably more than 10
times the spontaneous mutation rate [150]. Thus, this
approach is relatively insensitive to moderate or weak
mutagens. To avoid this limitation, a test was
developed based on the classical Luria and Delbruck
fluctuation test, which is approximately 100-fold more
sensitive than the conventional tests [150]. Using this
more sensitive bacterial fluctuation test, deoxycholate,
chenodeoxycholate and cholic acid were found to be
significantly mutagenic in one study [151], and
deoxycholate and cholic acid were non-mutagenic
in another study [152].
9. Bile acids cause apoptosis, which may select for
apoptosis resistance and increased cancer risk
Deoxycholate is the bile acid present at highest
concentration in the human colon (Table 1 and [153]).
Deoxycholate causes production of ROS (see Section
6, above), and ROS are a common mediator of
apoptosis [154]. Apoptosis is induced by deoxycholate
in mammalian cells at concentrations comparable to
the bile acid concentration found in the colon after a
high fat meal [127,130,131,155–166]. DNA damage
appears to be an initiating event in deoxycholate-
induced apoptosis [131].
It might be expected that frequent exposure of the
colonic epithelium to high concentrations of cytotoxic
bile acids would allow selective growth of cells
resistant to bile acid-induced apoptosis, leading to an
apoptosis resistant cell population. In a rat model,
repeated feeding of a diet containing 0.2% cholic acid
resulted in the development of increased resistance to
apoptosis of the colon crypt cells both in aberrant crypt
foci and normal crypts [167]. In addition, repeated
long-term exposure of a human colonic epithelial cell
line to sublethal concentrations of deoxycholate
selected for cells that are resistant to deoxycholate
induced apoptosis [168]. Most importantly, when
epithelial cells of the flat non-neoplastic colonic
mucosa of individuals with colon cancer are evaluated,
they are found to have reduced capacity to undergo
 H. Bernstein et al. / Mutation Research 589 (2005) 47–65
deoxycholate-induced apoptosis [154,160,164,165].
The reduction in apoptosis capability in cancer patients
would be expected if repeated long term exposure to
high levels of bile acids selected for apoptosis resis-
tance. Loss of apoptosis capability is associated with an
increased rate of mutation [169–171]. This may
predispose to development of malignancy. However,
it is not known if the development of apoptosis
resistance precedes development of malignancy as
expected by this view, or that the apoptosis resistance
results from selection by bile acids.
10. Is it reasonable to hypothesize that bile acids,
natural detergents with an important function in
digestion, can also be carcinogens?
A possible objection to the hypothesis that bile
acids are carcinogenic could be made on evolutionary
grounds. It may seem contrary to our understanding of
how natural selection operates that a natural substance
produced by the body for a beneficial purpose (the
emulsification of dietary fats to aid in their digestion)
could be carcinogenic. Would not natural selection
have operated to allow an alternative benign solution
to the problem of fat digestion, if bile acids reduce
fitness by causing cancer? In answer to this objection
we make the following points.
Colon cancer is mainly a disease of individuals over
age 50 [172]. However, the hunter-gatherer ancestors of
modern humans probably had an average life span of
about 40 years ([173], p. 15), and those individuals who
survived past 50 years were well past their reproductive
prime. Thus, in our ancestors, the influence of colon
cancer on natural selection would have been weak.
Life span in Western societies began to increase in
the 18th century, apparently due to such factors as
improved sanitation and agricultural development
[173]. The substantial increase in life span in the 19th
and 20th centuries resulted largely from a decline in
death rates due to infectious disease [173]. Important
factors influencing the incidence of gastrointestinal
cancers in current, longer lived populations may be a
decline in beneficial calorie restriction and increased
meat consumption [174]. The exposure of the
gastrointesinal tract to bile acids in the ancestors of
Western man may have been similar to that of
contemporary populations in underdeveloped coun-
59
tries or of vegetarians, where the incidence of colon
cancer is low [175,176]. Thus, the high colon cancer
incidence in Western countries appears to be largely a
reflection of recent improvements associated with
increased life span. Cancer of the digestive tract was
probably a negligible problem for our hunter-gatherer
ancestors ([173], p. 10) and thus would have exerted
insignificant selective pressure for an alternative
manner of fat digestion.
11. Conclusions
The evidence reviewed here indicates that bile
acids play a key etiologic role in gastrointestinal
cancers. Experiments in rodent model systems
indicate that bile acids act as promoters, but are
not, by themselves, carcinogenic. However, the
rodent experiments do not preclude the possibility
that bile acids act as carcinogens in humans,
considering the great difference in time of tumor
development in rodent models compared to humans.
Many different studies indicate that bile acids cause
DNA damage, strongly suggesting mutagenic and
carcinogenic potential. The DNA damaging mechan-
ism seems to be indirect, involving production of
ROS/RNS. Several mutagenesis experiments indi-
cate that bile acids are mutagenic. On the basis of
several lines of evidence we consider it likely that
bile acids are carcinogens in human gastrointestinal
cancers.
Some common types of non-gastrointestinal
cancers (e.g. lung and bronchus, prostate, urinary
bladder) are strongly age-related, where a 70-year-old
human is roughly 50–100 times as likely to be
diagnosed with a malignancy as a 27-year-old [172].
Major gastrointestinal cancers also are strongly age-
related [e.g. the relative incidence for a 70-year-old
compared to a 27-year-old is approximately 27-fold
(colon and rectum), 62-fold (esophagus), 71-fold
(pancreas) and 79-fold (larynx)] [172]. Thus, age-
related gastrointestinal cancers are members of
a larger group of strongly age-related cancers.
Perhaps age-related cancers generally reflect chronic
(decades-long) exposure to specific agents that cause
cellular stress, DNA damage and accumulation of
mutations, as appears to be the case for gastrointestinal
cancers.
60 H. Bernstein et al. / Mutation Research 589 (2005) 47–65
Acknowledgments
This work was supported in part by NIH
Institutional Core Grant #CA23074, NIHPPG
#CA72008, Arizona Disease Control Research Com-
mission Grants #10016 and #6002, VAH Merit Review
Grant 2HG, NCI SPORE Grant 1 P50CA95060-01
and Biomedical Diagnostics & Research Inc., Tucson,
AZ.
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