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Dietary Emulsifier-Induced Low-Grade Inflammation Promotes Colon Carcinogenesis

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The increased risks conferred by inflammatory bowel disease (IBD) to the development of colorectal cancer (CRC) gave rise to the term "colitis-associated cancer" and the concept that inflammation promotes colon tumorigenesis. A condition more common than IBD is low-grade inflammation, which correlates with altered gut microbiota composition and metabolic syndrome, both present in many cases of CRC. Recent findings suggest that low-grade inflammation in the intestine is promoted by consumption of dietary emulsifiers, a ubiquitous component of processed foods which alter the composition of gut microbiota. Here, we demonstrate in a pre-clinical model of colitis-induced CRC that regular consumption of dietary emulsifiers carboxymethylcellulose or polysorbate-80 exacerbated tumor development. Enhanced tumor development was associated with an altered microbiota metagenome characterized by elevated levels of lipopolysaccharide and flagellin. We found that emulsifier-induced alterations in the microbiome were necessary and sufficient to drive alterations in major proliferation and apoptosis signaling pathways thought to govern tumor development. Overall, our findings support the concept that perturbations in host-microbiota interactions that cause low-grade gut inflammation can promote colon carcinogenesis.
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Microenvironment and Immunology
Dietary EmulsierInduced Low-Grade
Inammation Promotes Colon Carcinogenesis
Emilie Viennois
1
, Didier Merlin
1,2
, Andrew T. Gewirtz
1
, and Benoit Chassaing
1
Abstract
The increased risks conferred by inammatory bowel dis-
ease (IBD) to the development of colorectal cancer gave rise to
the term "colitis-associated cancer" and the concept that
inammation promotes colon tumorigenesis. A condition
more common than IBD is low-grade inammation, which
correlates with altered gut microbiota composition and met-
abolic syndrome, both present in many cases of colorectal
cancer. Recent ndingssuggestthatlow-gradeinammation
in the intestine is promoted by consumption of dietary
emulsiers, a ubiquitous component of processed foods,
which alter the composition of gut microbiota. Here, we
demonstrate in a preclinical model of colitis-induced colorec-
tal cancer that regular consumption of dietary emulsiers,
carboxymethylcellulose or polysorbate-80, exacerbated tumor
development. Enhanced tumor development was associated
with an altered microbiota metagenome characterized by
elevated levels of lipopolysaccharide and agellin. We found
that emulsier-induced alterations in the microbiome were
necessary and sufcient to drive alterations in major prolif-
eration and apoptosis signaling pathways thought to govern
tumor development. Overall, our ndings support the concept
that perturbations in hostmicrobiota interactions that cause
low-grade gut inammation can promote colon carcinogenesis.
Cancer Res; 77(1); 2740. 2016 AACR.
Introduction
Colorectal cancer is among the most common human malig-
nancies (1) and has been rmly linked to chronic intestinal
inammation, giving rise to the term "colitis-associated cancer"
(2, 3). The development of colitis-associated cancer in patients
suffering from inammatory bowel disease (IBD) is one of the
best characterized examples of an association between intestinal
inammation and carcinogenesis (47). Among patients with
ulcerative colitis, the risk of colon cancer has been found to be as
high as 2% at 10 years, 8% at 20 years, and 18% at 30 years after
initial diagnosis (4). In contrast, the lifetime risk of sporadic
colorectal cancer in the United States is only 5% (8).
Gut microbiota is the collective term for the large diverse
community of microorganisms that inhabits the intestine. Gut
microbiota play an important role in health, particularly in
promoting immune system development and aiding metabo-
lism. Alterations in microbiota composition, often referred to
as dysbiosis, are thought to play a central role in the patho-
genesis of numerous intestinal disorders including inamma-
tory bowel disease (IBD; ref. 9), and are associated with
colorectal cancer (10). However, whether microbial dysbiosis
observed in patients with colorectal cancer is a consequence of
the pathology or is causal remains unclear. An altered micro-
biotacanplayaroleinpromotingcolitis-associated cancer,
not only through induction of inammation, but also through
the production of toxins that create a favorable niche for tumor
cells (11). Indeed, commensal organisms can have an enor-
mous impact on tumorigenesis through the production of
tumor-promoting genotoxins that can induce chromosomal
instability (12). For example, certain strains of Escherichia coli
harboring the Pks island, involved in the synthesis of the
colibactin toxin, are frequently associated with human colo-
rectal tumors. These strains inhibit DNA mismatch repair
proteins, and were also reported to have carcinogenic effects
in mice (11, 1316). Besides, treatment of mice with antibiotics
confers some degree of protection against colitis-associated can-
cer, supporting the pivotal role of the gut microbiota during
tumorigenesis (17). Moreover, azoxymethane (AOM)-treated
germ-free IL10
/
mice failed to develop colitis and colorectal
tumors, indicating that the presence of colitogenic bacteria is
essential for the development of colitis-associated cancer (18).
While gut inammation is classically dened histopathologi-
cally, specically by the presence of immune cell inltrates, it is
now appreciated that a much more common form of inamma-
tion is "low-grade intestinal inammation," which is dened by
elevated systemic expression of proinammatory cytokines in the
absence of the classical aggregates of immune cell inltrates.
Alterations in hostmicrobiota relationship have been associated
with and can promote low-grade intestinal inammation (19).
Moreover, it is increasingly appreciated that low-grade chronic
inammation in the gut can promote metabolic disorders such as
type II diabetes, atherosclerosis, and obesity, which is itself
associated with increased incidence of colon cancer (20, 21).
Emulsiers are detergent-like molecules that are incorporat-
ed into most processed foods to improve texture and stability,
and we recently demonstrated that emulsiers disrupted
1
Center for Inammation, Immunity and Infection, Institute for Biomedical
Sciences, Georgia State University, Atlanta, Georgia.
2
Veterans Affairs Medical
Center, Decatur, Georgia.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Benoit Chassaing, Center for Inammation, Immunity,
and Infection, Institute for Biomedical Sciences, Georgia State University,
Atlanta GA 30303. Phone: 404-413-3589; Fax: 404-413-3580; E-mail:
bchassaing@gsu.edu
doi: 10.1158/0008-5472.CAN-16-1359
2016 American Association for Cancer Research.
Cancer
Research
www.aacrjournals.org 27
mucusbacterial interactions, inducing intestinal inammation
(22). In this recent study, we investigated the effect of two
commonly used emulsiers, namely carboxymethylcellulose
(CMC) and polysorbate-80 (P80), on the host's intestine. CMC
was previously described to promote overgrowth and inam-
mation of small intestine in genetically susceptible mice (23),
and P80 is able to increase bacterial translocation across epi-
thelia in vitro (24, 25).
These two emulsiers are indigestible and mainly excreted in
the feces (2629), and we recently found that both CMC and P80
promoted microbiota encroachment and increased levels of
proinammatory agellin and lipopolysaccharide (LPS), which
correlated with a change in microbiota composition and intesti-
nal inammation. Such alterations promoted colitis in mice
genetically predisposed to this disorder, and induced low-grade
inammation and metabolic syndrome in WT mice. Importantly,
such effects were dependent upon the presence of the microbiota
(no phenotype observed in germ-free mice), and fecal microbiota
transplant from emulsier-treated mice to germ-free mice, trans-
ferred some features of intestinal inammation and metabolic
syndrome (22).
In the current study, we hypothesized that emulsiers could be
involved in colorectal cancer development through the promo-
tion of low-grade intestinal inammation and alterations of the
intestinal microbiota. To test this hypothesis, we used the well-
established murine model of colitis-associated cancer using the
carcinogen AOM, followed by two cycles of dextran sulfate
sodium (DSS) in mice subjected to chronic exposures of two
commonly used emulsiers, CMC and P80. We herein report that
dietary emulsifying agents created and maintained a proinam-
matory environment in the colon, associated with alterations of
the proliferation/apoptosis balance that resulted in exacerbated
carcinogenesis. These changes were associated with, and depen-
dent upon, alterations in microbiota composition and diversity
that created a favorable niche for tumorigeneisis. These ndings
support the concept that a perturbed hostmicrobiota interaction
resulting in alterations of the intestinal homeostasis can promote
colonic carcinogenesis.
Materials and Methods
Materials
Sodium carboxymethylcellulose (CMC, average M
W
250,000,
degree of substitution ¼0.7) and polysorbate-80 (P80) were
purchased from Sigma.
Mice
Four-week-old male C57BL/6 WT mice were used in this study.
All mice were bred and housed at Georgia State University,
(Atlanta, GA) under institutionally approved protocols (IACUC
# A14033). Mice were housed in specic pathogen-free conditions
and fed ad libitum with regular chow diet. Animals used in Figs.
16 and Supplementary Fig. S1S10 were housed in Helicobacter
positive room, and animals used in Fig. 7 and Supplementary
Fig. S11 were housed in Helicobacter negative room.
Emulsier agent treatment
Mice were exposed to CMC or P80 diluted in the drinking water
(1.0%). The same water (reverse-osmosis treated Atlanta city
water) was used for the water-treated (control) group. These
solutions were changed every week. Body weights were measured
every week and expressed as percentages of the initial body weight
(day 0 dened as 100%) to study emulsier effect on body weight
gain. Fresh feces were collected every week for downstream
analysis.
Colitis-associated cancer model
Colitis-associated cancer was induced as previously described
with some modications (30). As schematized in Supplementary
Fig. S1, after 13 weeks (91 days) of emulsier administration,
mice were intraperitoneally injected with AOM (10 mg/kg body
weight; Sigma-Aldrich) diluted in PBS and maintained on regular
chow diet and water or emulsier-supplemented water for 5 days.
Mice were then subjected to two cycles of DSS treatment (MP
Biomedicals), in which each cycle consisted of 2.5% DSS for 7
days followed by a 14-day recovery period with regular water or
emulsier-supplemented water. Body weights were measured
every week and expressed as percentages of the initial body weight
(day 91 ¼post emulsier dened as 100%) to study AOM-DSS
protocol effect on body weight gain. After treatment, mice were
fasted for 5 hours, at which time blood was collected by retro-
bulbar intraorbital capillary plexus. Hemolysis-free serum was
generated by centrifugation of blood using serum separator tubes
(Becton Dickinson). After colitis-associated cancer protocol, mice
were euthanized, and colon length, colon weight, spleen weight,
and adipose weight were measured. Organs and blood were
collected for downstream analysis. Colonic tumors were counted
and surface measured using a dissecting microscope. The total
area of tumors for each colon was determined.
As schematized in Supplementary Fig. S11, emulsier-treated
animals were also weekly treated with AOM (10 mg/kg body
weight) diluted in PBS. At the end of the experiment, mice were
fasted for 15 hours, colonoscopy procedure was performed (Karl
Storz Endoskope) and mice were euthanized and organs collected
as previously described.
Germ-free experiments
Germ-free Swiss Webster mice were kept under germfree con-
ditions in a Park Bioservices isolator in our germ-free facility. CMC
and P80 were diluted to 1% in water and then autoclaved for
germ-free purpose. The same water was used for the water-treated
(control) group. Conventional age-matched and sex-matched
Swiss Webster mice were used in parallel. After 3 months of
emulsier agent treatment, terminal analyses were performed.
Microbiota transplantation
Cecal contents from Swiss Webster detergent-treated mice were
suspended in 30% glycerol diluted in PBS (1.0 mL) and stocked at
80C until analysis. Germ-free Swiss Webster mice (4 weeks old)
were removed from the isolator and were orally administered
200 mL of fecalsuspension made usingglycerol stocks.Transplanted
mice were monitored for 3 months before terminal analysis.
Colonic myeloperoxidase assay, quantication of fecal Lcn-2
and serum CXCL-1 and IL6 by ELISA
For details, see Supplementary Methods section.
Fecal agellin and LPS load quantication
We quantied agellin and LPS as previously described (31)
using human embryonic kidney (HEK)-Blue-mTLR5 and HEK-
BluemTLR4 cells, respectively (Invivogen). We resuspended
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research28
fecalmaterialinPBStoanal concentration of 100 mg/mL
and homogenized using a Mini-Beadbeater-24 without the
addition of beads to avoid bacteria disruption. Supernatants
were serially diluted and applied to mammalian cells. Puried
E. coli agellin and LPS (Sigma) were used for standard curve
determination. After 24 hours of stimulation, we applied cell
culture supernatant to QUANTI-Blue medium (Invivogen) and
measured alkaline phosphatase activity at 620 nm after 30
minutes.
RNA extraction, real-time RT-PCR, and bacterial quantication
by qPCR
For details, see Supplementary Methods section and Supple-
mentary Table S1.
Fecal microbiota analysis by 16S rRNA gene sequencing using
Illumina technology and metagenome prediction
16S rRNA gene sequencing was performed as previously
described (22), with data deposited in the European Nucleotide
Archive under accession number PRJEB8035. For details, please
see Supplementary Methods.
Ki67 immunohistochemistry
Mouse proximal colon devoid of tumor were xed in 10%
buffered formalin for 24 hours at room temperature and subse-
quently embedded in parafn. Tissues were sectioned at 5-mm
thickness and deparafnized. Sections were incubated in sodium
citrate buffer and cooked in a pressure cooker for 10 minutes for
antigen retrieval. Sections were then blocked with 5% goat serum
in TBS followed by one hour incubation with anti-Ki67 (1:100,
Vector Laboratories) at 37C. After washing with TBS, sections
were treated with biotinylated secondary antibodies for 30 min-
utes at 37C, and color development was performed using the
Vectastain ABC kit (Vector Laboratories). Sections were then
counterstained with hematoxylin, dehydrated, and coverslipped.
Ki67-positive cells were counted per crypt.
Terminal deoxynucleotidyl transferase deoxyuridine
triphosphate nick-end labeling assay
To quantitate the number of apoptotic cells in colonic
epithelial cells, mouse proximal colon devoid of tumor were
xed in 10% buffered formalin for 24 hours at room temper-
ature, embedded in parafn, sectioned at 5-mm thickness,
deparafnized, and stained for apoptotic nuclei according to
the manufacturer's instructions using the In Situ Cell Death
Detection Kit (Roche Diagnostics). Terminal deoxynucleotidyl
transferase deoxyuridine triphosphate nick-end labeling
(TUNEL)-positive cells overlapping with DAPI nuclear staining
were counted per crypt.
Statistical analysis
Data are presented as means SEM. Signicance was deter-
mined using ttests, with each treatment group compared with
the control group. Two-way group ANOVA corrected for mul-
tiple comparisons using Bonferroni posttest was used for body
weight over time and alpha diversity analysis (GraphPad Prism
software, version 6.01). and # indicate statistically signicant
differences.
Results
Dietary emulsifying agents induce low-grade intestinal
inammation associated with metabolic syndrome
Multiple mouse litters were equally split at weaning into three
groups that received either water, CMC, or P80 in drinking water
(1.0% w/v or v/v, respectively) for 13 weeks, as reported previ-
ously (Supplementary Fig. S1; ref. 22). In accord with our previous
work, emulsier consumption resulted in features of chronic low-
grade intestinal inammation, including shortened colons and
splenomegaly (Supplementary Fig. S2). Fecal lipocalin-2 (Lcn2),
which is a sensitive and broadly dynamic marker of intestinal
inammation in mice (32), was used to quantify the intestinal
inammation, and showed that emulsier-treated mice exhibited
elevated fecal Lcn2 levels after 9 weeks of dietary emulsier
consumption (day 63; Supplementary Fig. S2AS2C), conrming
the induction of low-grade inammation. As expected, both CMC
and P80 induced a modest but statistically signicant increase in
body mass (Supplementary Fig. S2D). Emulsier treatment also
impaired glycemic control as assessed by fasting blood glucose
concentration (Supplementary Fig. S2E), and was associated with
increased food consumption (Supplementary Fig. S2F), conrm-
ing our previous observation that emulsiers induce low-grade
intestinal inammation and impair glucose metabolism (22).
Emulsier consumption exacerbates carcinogenesis in a colitis-
associated cancer model
In an effort to investigate whether low-grade intestinal inam-
mation induced by emulsier consumption might predispose to
the development of colitis-associated carcinogenesis, mice that
had consumed emulsiers for 90 days were subsequently admin-
istered AOM and DSS while maintaining emulsier consumption
except during DSS administration (Supplementary Fig. S1). All
groups of AOM/DSS-treated mice displayed acute weight loss
during DSS treatment and, when euthanized, exhibited gross
features of inammation, including increased colon and spleen
weights (Fig. 1AE). While emulsier treatment, by itself, induced
some indicators of inammation such as colon shortening and
mild splenomegaly, the extent of inammatory changes induced
by AOM/DSS treatment (i.e., fold change induced by AOM/DSS
treatment) was not greater in mice that had consumed emulsiers.
However, emulsier consumption increased tumor development
in response to AOM/DSS compared with AOM/DSS control
animals, as assessed by number and size of tumors (Fig.
1FH). Histologic examination revealed the presence of larger
adenomas and increased areas of inammatory cell inltration in
emulsier/AOM/DSS-treated animals compared with the water/
AOM/DSS group (Supplementary Fig. S3). Histologically, no
gross difference between water-treated and emulsier-treated
mice was observed in the absence of AOM/DSS (Supplementary
Fig. S3). We next examined the expression of proinammatory
cytokines by qRT-PCR using mRNA extracted from whole distal
colon devoid of tumor. mRNAs were thus isolated from a mixture
of epithelial cells, immune cells, and other colonic cells, allowing
the analysis of global molecular alterations of the colon. Emul-
sier consumption by itself resulted in increased expression of the
proinammatory cytokine CXCL1, conrming that emulsiers
induce the development of low-grade intestinal inammation
(Fig. 2AE). AOM/DSS signicantly increased the expression
levels of all the tested cytokines/chemokines (IL6, CXCL1, CXCL2,
IL22, and TNFa) compared with the corresponding control
Emulsifying Agents Promote Colonic Carcinogenesis
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Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research30
groups (Fig. 2AE), as were the levels of circulating CXCL1 (Fig.
2F). After AOM/DSS-induced carcinogenesis, both CXCL1 and
CXCL2 were signicantly increased in emulsier-treated group
compared with water-treated group (Fig. 2B and C), supporting
the previous observation that AOM/DSS-induced tumors were
inltrated by inammatory cells. Importantly, we noted that mice
receiving P80 exhibited the highest tumor and a greater increase of
the CXCL1 and CXCL2 compared with CMC (Fig. 2B and C). As a
further readout of the inammatory state of the intestine, we next
examined colonic myeloperoxidase (MPO) activity and fecal Lcn2
level following AOM/DSS treatment (Fig. 3A and B). These
analyses conrmed the observation that emulsier's consump-
tion per se was sufcient to drive intestinal inammation, as
revealed by a moderate but nonetheless signicant increase in
MPO in emulsier-only treated groups compared with water-only
treated group (Fig. 3A). Furthermore, emulsiers led to an exacer-
bation of the intestinal inammation observed after AOM/DSS, as
revealed by both MPO and Lcn2 measurements at day 141 (Fig.
3A and B).
Dietary emulsifying agents alter the intestinal microbiota
composition, leading to a proinammatory intestinal
environment
We next considered the possibility that emulsier-induced
alterations of the gut microbiota might underlie its tumor-pro-
moting effects. Microbiota composition analysis were previously
performed on CMC- and P80-treated animals and revealed a
strong clustering following treatment irrespective of cage group-
ing, thus conrming our observation that emulsiers alter micro-
biota composition (22). On the basis of previous reports that
some pathovars of Escherichia coli can produce the procarcino-
genic toxin colibactin (11, 14, 33), we aimed to elucidate whether
such pathovars were playing a role in the observed emulsier-
induced exacerbation of tumorigenesis. The quantication of
g-Proteobacteria, Enterobacteriaceae, Escherichia coli, or ClbB
encoding gene (colibactin polyketide synthesis system) revealed
that none of these phylotypes were signicantly altered following
emulsier consumption (day 0, day 21, and day 63) or after
tumorigenesis induction (day 141), suggesting that colibactin-
producing bacteria were not involved in the aforementioned
emulsier-induced exacerbation of tumorigenesis (Supplemen-
tary Fig. S4). We next performed a more in-depth analysis of the
intestinal microbiota after emulsier treatment, and found that
both CMC and P80 led to a signicant reduction of microbiota
diversity after 9 weeks (day 63) of treatment (Supplementary Fig.
S5A and S5B), as well as profound alteration of the bacterial
community at the phylum, class, and order levels (Supplementary
Fig. S5CS5E). Such alterations were characterized by an increase
in Bacteroidales and a decrease in Clostridiales orders upon CMC
or P80 consumption (Supplementary Fig. S5CS5E). LEfSe (LDA
effect size), used to identify the most differentially abundant
taxons and OTUs between water and emulsier-treated groups,
revealed a decrease of numerous Firmicutes members, such as
Lactobacillus, upon emulsier consumption, together with an
increase of Bacteroidetes members (Supplementary Fig. S6).
We next wanted to investigate whether such alterations of the
microbiota were associated with any modication of its inherent
ability to induce proinammatory gene expression in the host.
Hence, we measured the capacity of feces from control and
emulsier-treated mice to activate proinammatory gene expres-
sion via the LPS and agellin receptors Toll-like receptor 4 (TLR4)
and TLR5, respectively. As previously reported, at day 63, exposure
to emulsiers increased levels of bioactive fecal LPS and agellin
(Fig. 3CH). Elevated fecal agellin and LPS were not associated
with an elevation in the total fecal bacterial load (Supplementary
Fig. S7), indicating that this increase in microbiota proinamma-
tory potential was independent of bacterial load and, rather likely
a consequence of altered species composition. The induction of
tumorigenesis by AOM/DSS was found to further increase the
proinammatory potential of the microbiota, in both water and
emulsier-treated groups (Fig. 3E and H). The analysis of the
predicted metagenome indicated the presence of an altered meta-
genome in emulsier-treated animals compared with water-only
treated animals (Fig. 4; Supplementary Figs. S8 and S9; ref. 34).
Using principal coordinate analysis, a strong and distinct cluster-
ing (P¼0.01) was observed between metagenomes of water and
CMC or P80-treated groups at day 63 (Fig. 4C and D), while
predicted metagenomes of all groups were similar at day 0 (Fig. 4A
and B). This was further conrmed using Volcano plots of Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathways abun-
dance, demonstrating a drastic alteration in microbiota metagen-
omes following emulsier consumption at day 63 (Fig. 4E). Such
alterations were characterized, in emulsier-treated animals, by a
decrease in the richness of signaling pathways, in accord with the
observed decrease in bacterial community richness (Supple-
mentary Fig. S5). Moreover, the analysis of signicantly altered
metabolic pathways revealed that emulsier consumption led
to an increased proportion of bacterial genes involved in LPS
biosynthesis, bacterial motility, and secretion systems (Fig. 4F
and G; Supplementary Figs. S8 and S9), correlating with the
observationofanincreasedproinammatory microbiota under
emulsier consumption. Altogether, these data demonstrate
that emulsier consumption drastically altered the intestinal
microbiota composition, resulting in a basal low-grade proin-
ammatory environment in the intestine that predisposed to
subsequent tumorigenesis.
Dietary emulsifying agents disturb the proliferation/apoptosis
balance of epithelial cells
The increased tumor burden observed in emulsier-treated
animals suggested the possibility of increased cell proliferation
in those animals. Hence, proliferation of colonic epithelial cells
was analyzed by Ki67 staining, which revealed that the consump-
tion of emulsiers by itself (i.e., no AOM/DSS) resulted in
increased cell proliferation relative to the water-treated control
group (Fig. 5). AOM/DSS treatment increased the number of
Figure 1.
Dietary emulsiers promote colitis-associated cancer. WT mice were exposed to drinking water containing CMC or P80 (1.0%) for 13 weeks. Mice were then injected
intraperitoneally with AOM (10 mg/kg body weight), maintained for 7 days, and then subjected to a two-cycle DSS treatment (each cycle consisted of 7 days
of 2.5% DSS and 14 days of H2O). Colon weights (A), colon lengths (B), spleen weights (C), fat-pad mass (D), body weight over time (E), representative colon samples
from each experimental group at the end of the AOM/DSS protocol (F), number of tumor per mouse (G), total tumor surface determined using a dissecting
microscope tted with an ocular micrometer (H). Data are the means SEM (n¼58). Signicance was determined using ttest (,P<0.05) or two-way group
ANOVA corrected for multiple comparisons with a Bonferroni test (#, statistical signicance).
Emulsifying Agents Promote Colonic Carcinogenesis
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Figure 2.
Dietary emulsiers promote intestinal inammation and carcinogenesis. Analysis of IL6 (A), CXCL1 (B), CXCL2 (C), IL22 (D), and TN FamRNA expression (E) by qRT-
PCR in the colon following emulsier treatment and following the induction of colonic neoplasia. F, Analysis of serum CXCL1 level by ELISA following emulsier
treatment and following the induction of colonic neoplasia. Data are the means SEM (n¼58). Signicance was determined using ttest (,P<0.05).
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research32
Figure 3.
Dietary emulsiers favor a
proinammatory microbiota. Colonic
MPO levels (A), fecal Lcn2 concentration at
day 141 (B), and bioactive levels of fecal
agellin (FliC; CE) and LPS (FH) assayed
with TLR5 and TLR4 reporter cells,
respectively, at day 0 (C, F), day 63 (D, G),
and day 126 (E, H). Data are the means
SEM (n¼58). Signicance was
determined using ttest (,P<0.05).
Emulsifying Agents Promote Colonic Carcinogenesis
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Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research34
Ki67-positive cells in all groups of mice, but the proliferation level
remained signicantly higher in AOM/DSS-treated mice that had
consumed emulsiers, in accord with the notion that emulsier
promotion of tumorigeneis involves increased cell proliferation
(Fig. 5). To further address the role of cell turnover in this process,
we performed TUNEL-based quantication of apoptosis in colon-
ic sections from emulsier-treated mice. Analogous to our results
for cell proliferation, we observed that consumption of emulsi-
ers, alone, increased the basal level of TUNEL
þ
cells. Moreover,
this difference between the water and emulsier-consuming
groups was further increased in response to AOM/DSS treatment.
For both proliferative and apoptotic cells counts, similar results
were obtained when subdividing the crypts (top/middle/bottom,
data not shown). Together, these results indicate that emulsier
consumption upregulates both apoptosis/proliferation in the
intestinal epithelium, resulting in an increased cell turnover, and
creating an opportune milieu for the tumorigenesis.
Dietary emulsiers alter epithelial cell proliferation and
apoptosis in a microbiota-dependent manner
To further investigate how emulsier consumption impacted
proliferation/apoptosis, we next analyzed by qRT-PCR the expres-
sion levels of genes that control proliferation (cyclin D1, D2,
Ki67), apoptosis (BCL2 and BAD), and angiogenesis (VEGFA). As
shown in Fig. 7, we observed that, following dietary emulsier
consumption, the expression of cyclin D1, cyclin D2, and Ki67-
encoding genes were signicantly deregulated (Fig. 7AC), while
the b-catenin pathway were found unaltered (Supplementary Fig.
S10). In the AOM/DSS-treated groups, the expression levels of
these genes were further deregulated, without any difference
observed between water- and emulsier-treated groups (data not
shown). The anti- and proapoptotic encoding genes Bcl2 and Bad
(Fig. 7D and E) and the marker of angiogenesis VEGFA (Fig. 7F)
remained unaltered under emulsier's consumption.
We next sought to investigate whether the unbalanced prolif-
eration/apoptosis and the molecular alterations observed in
emulsier-treated mice were driven by alterations in gut micro-
biota. We therefore analyzed the expression of the same genes in
germ-free animals treated with dietary emulsiers, and impor-
tantly found that none of those genes have an altered expression
under germ-free conditions (Fig. 7AF), thus indicating that the
presence of an altered microbiota is a prerequisite for subsequent
perturbations in proliferation and apoptosis processes. Finally,
we investigated whether the altered microbiota driven by emul-
sier consumption was sufcient to alter intestinal expression of
genes involved in the proliferative/apoptosis balance. As pre-
sented in Fig. 7GL, we found that transfer of microbiota from
emulsier-consuming mice to germ-free mice, that were not fed
emulsiers, recapitulated perturbations of cyclin D1 and D2
expression, thus suggesting that emulsier-induced alterations of
intestinal microbiota composition plays a central and direct role
in the promotion of carcinogenesis.
Dietary emulsiers induce carcinogenesis in AOM-treated
animals
We next sought to investigate whether emulsier consumption
may be sufcient to drive colonic carcinogenesis in animals
treated with AOM but without exogenously induced severe intes-
tinal inammation. For this purpose, WT mice were exposed to
drinking water containing CMC or P80 (1.0%) for 12 weeks, and
were injected intraperitoneally with AOM (10 mg/kg body
weight) weekly, for a total of seven injections. The combination
of AOM injections and emulsier consumption leads to substan-
tial intestinal inammation, as characterized by increase in colon
and spleen weights and by increased proinammatory cytokine
expressions and fecal Lcn2 levels (Fig. 7AL). Importantly, CMC-
induced intestinal inammation was sufcient to induce carci-
nogenesis in a subset of animals, that was associated with altera-
tions of proliferative pathways (Fig. 7MR), suggesting that, at
least in presence of some mutagens, emulsier-induced low grade
inammation and/or alteration of proliferation pathways is suf-
cient to drive colon carcinogenesis.
Discussion
Mounting evidence implicates alteration of the gut microbiota,
that is, dysbiosis, as an important determinant of colon cancer. A
major tenet in this indictment is that the microbial dysbiosis is a
major driver of gut inammation, which, when occurring chron-
ically, is strongly associated with an increased incidence of colon
cancer. More recently, it has been shown that the microbiota
composition can inuence tumor development beyond merely
driving inammation (11, 14, 33, 35). Herein, we add to this body
of knowledge in several ways thus providing new insights into
factors that may drive microbial dysbiosis in the rst place and,
moreover, elucidate how an altered microbiota can result in
increased tumor development. Specically, we report that, in
mice, consumption of dietary emulsiers resulted in an altered
gut microbiota composition associated with increased levels of
agellin and LPS, hence creating a low-grade proinammatory
environment. The latter was associated with altered rates of
proliferation/apoptosis that predisposed animals to exacerbated
tumor development when subjected to a chemical-induced model
of colitis-associated cancer. In addition, emulsier consumption
Figure 4.
Profound metagenome alteration following CMC and P80 consumption. WT mice were exposed to drinking water containing CMC or P80 (1.0%) for 13 weeks.
PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to predict the metagenomes, subsequently
analyzed by principal coordinates analysis of the beta diversity using binary Jaccard method at day 0 (Aand B) and day 63 (Cand D). E, KEGG pathways were
visualized on a volcano plot. From left to right, water-treated versus CMC-treated at day 0; water-treated versus CMC-treated at day 63; water-treated
versus P80-treated at day 0; water-treated versus P80-treated at day 63. For each KEGG identier, the difference in abundance between the two groups is
indicated in log
2
fold change on x-axis (with positive values corresponding to an increase in emulsier-treated group compared with water-treated group, and
negative values corresponding to a decrease in emulsier-treated group compared with water-treated group), and signicance between the two groups is
indicated by log
10
Pvalue on the y-axis. Red dots correspond to KEGG identiers with a Pvalue <0.05 between emulsier-treated and water-treated groups. Orange
dots correspond to KEGG identiers with at least a 2-fold decreased or increased abundance in emulsier-treated group compared with water-treated group.
Green dots correspond to KEGG identiers with at least a 2-fold decreased or increased abundance in emulsier-treated group compared with water-treated
group and with a Pvalue <0.05. Fand G, Predicted metagenomes were categorized at level 3 of the KEGG pathways, and pathways involved in LPS biosynthesis,
secretion system synthesis, and motility were graphed. Data are the means SEM (n¼58). Signicance was determined using ttest (,P<0.05).
Emulsifying Agents Promote Colonic Carcinogenesis
www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 35
Figure 5.
Dietary emulsiers alter epithelial cell proliferation and apoptosis during colitis-associated cancer development. Aand B, Epithelial cell proliferation was analyzed by
IHC using the proliferation marker Ki67 in colonic tissue sections. A, Representative images of Ki67 staining. Scale bar, 200 mm. B, Ki67
þ
cells were counted
and averaged per crypt. Cand D, Epithelial cell apoptosis was analyzed by TUNEL assay. C, Representative confocal images of TUNEL assay. TUNEL, green;
DNA, blue. Scale bar, 25 mm. D, TUNEL
þ
DAPI
þ
cells were counted and averaged per crypt. Data are the means SEM (n¼58). Signicance was determined using
ttest (,P<0.05).
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research36
was sufcient to drive development of colonic tumors in some
animals treated with AOM (without DSS) and was associated with
altered proliferation. The level of exposure used herein in mice is
intended to approximately model the case of humans who eat
large amounts of processed foods, many of which contain CMC,
P80, and/or other emulsiers exceeding 2% of the product by
weight (23).
While human studies of microbial dysbiosis, inammation,
and cancer are primarily limited to associations, that germ-free
mice are protected against colonic carcinogenesis supports the
notion that dysbiosis is not purely a consequence of disease but,
rather, also plays a role in driving the development of colorectal
cancer (9, 3638). Moreover, in this study, we demonstrated that
microbiota dysbiosis that precedes the initiation of colonic
tumorigenesis was sufcient to promote cancer development.
Importantly, the induction of colonic tumorigenesis similarly
induced intestinal inammation in emulsier- and water-treated
groups, showing that the observed phenotypes were not only due
to an exacerbation of inammation during DSS, but that the low-
grade intestinal inammation that precedes the initiation of
carcinogenesis was playing a central role. While, in mice, emul-
sier consumption promotes increased food consumption and
adiposity and, in humans, obesity itself is associated with colon
cancer, we speculate that the link between emulsiers and cancer
is not obesity per se but rather low-grade inammation. Accord-
ingly, emulsiers increased food consumption in AOM/DSS-
treated mice without a corresponding increase in weight, likely
reecting increased energy demand, suggest that inammation
and associated increases in cell turnover are more germane.
However, it is possible that emulsier-induced alterations in
microbiota can cause increased energy intake that might promote
carcinogenesis irrespective of adiposity.
One mechanism by which microbiota was previously reported
to promote colorectal cancer is via a transient contact between
colibactin-producing E. coli and epithelial cells that subsequently
became malignant (11, 14, 33, 35). We thus hypothesized that
colibactin-producing E. coli could be involved in the emulsier-
induced exacerbation of tumorigenesis. However, the quantica-
tion of Proteobacteria, Enterobacteriaceae, Escherichia coli, and
ClbB-encoding gene negated this possibility. Although emulsi-
ers did not drive substantial changes in abundance of such
pathovars, inammation, alteration of microbiota and its capacity
to generate proinammatory signals were critical for tumor devel-
opment. Indeed, upon emulsier's exposure, the diversity of
microbiota was decreased and the abundance of some phyla,
class, and orders was altered (decrease of Firmicutes, notably
Clostridiales class and Lactobacillus member, and increase of
Bacteroidetes). Lactobacilli have been associated with protection
against colonic carcinogenesis via antioxidant, antiproliferation
properties and immunomodulatory and antitumor effects
(3941). We observed a greater abundance of bacteroidetes in
both CMC- and P80-treated mice, correlating with the observa-
tion that intestinal mucosal surface of patients with adenoma
displayed increased abundance of Bacteroidetes (42). However,
this same study reported higher abundance of Firmicutes in
adenoma patients while we observed a decrease of this phylum
upon emulsier's consumption (42). While little is known about
how emulsiers impact microbiota, the ndings of our current
study, in conjunction with previous reports, suggest that the
consumption of emulsiers induce a shift of the gut microbial
population creating a favorable environment for colonic
Figure 6.
Dietary emulsiers alter epithelial cell proliferation and apoptosis in a
microbiota-dependent manner. Conventional and germfree Swiss-Webster WT
mice were exposed to drinking water containing CMC or P80 (1.0%) for 13 weeks.
Intestinal microbiota from conventional Swiss-Webster WT mice exposed to
drinking water containing CMC or P80 (1.0%) for 13 weeks were transplanted to
germfree Swiss-Webster WT mice. Analysis of cyclin D1 (Aand G), cyclin D2
(Band H), Ki67 (Cand I), BCL2 (Dand J), BAD (Eand K), and VEGFA
mRNA expression (Fand L) by qRT-PCR in the colon following emulsier
treatment under germ-free conditions (AF) and following microbiota
transplantation (GL).
Emulsifying Agents Promote Colonic Carcinogenesis
www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 37
Figure 7.
Dietary emulsiers promote intestinal inammation and carcinogenesis in the absence of DSS. WT mice were exposed to drinking water containing CMC or P80 (1.0%)
for 12 weeks. Mice were injected intraperitoneally with AOM (10 mg/kg body weight) weekly for a total of 7 injections. Colon weights (A), colon lengths (B),
spleen weights (C), and fat-pad mass (D). EI, Analysis of IL6 (E), CXCL1 (F), CXCL2 (G), IL22 (H), and TNFamRNA expression (I) by qRT-PCR in colons of emulsier-
AOMtreated mice. JL, Fecal Lcn2 concentration at day 0 (J), 56 (K), and 84 (L). M, Representative colonoscopy from each experimental group at the end
of the protocol. Nand O, Number of tumor per mouse (N) and total tumor surface (O) determined using a dissecting microscope tted with an ocular micrometer.
PR, Analysis of cyclin D1 (P), cyclin D2 (Q), and Ki67 mRNA expression (R) by qRT-PCR in colons of emulsier-AOMtreated mice. Data are the means SEM
(n¼10). Signicance was determined using ttest (,P<0.05).
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research38
carcinogenesis (22, 23). Gut microbes could indeed act through
various pathways including proliferation, immune system, or
inammation. We quantied the proinammatory potential of
the microbiota associated with the consumption of CMC or P80
and showed increased level of bioactive agellin and LPS. It was
recently reported that in humans, serum anti-agellin and anti-
LPS antibody concentrations positively correlate with colorectal
cancer risk (43). Importantly, TLR4-decient mice, unable to
recognize bacterial LPS, are protected from colon carcinogenesis
(44), further highlighting the central role played by increased fecal
agellin and/or LPS loads in the carcinogenesis initiation and
promotion.
Comparative metagenomes analyses of CMC- or P80-treated
mice and water control mice showed that the shift of bacteria was
accompanied by alterations of several metabolic pathways, with a
decrease in overall metabolic pathway richness and an increased
proportion of bacterial genes involved in LPS biosynthesis, as well
as bacterial motility and secretion systems. These observations
were correlating with the increased proinammatory ability of
microbiota measured under emulsier consumption. Important-
ly, in addition of promoting and maintaining low-grade intestinal
inammation, we observed in the current study that the con-
sumption of dietary emulsiers also induced alterations of some
major proliferation and apoptosis actors in a microbiota-depen-
dent manner. The observation that such disruptions were ef-
ciently transferred to mice receiving fecal microbiota of emulsier-
treated donors revealed that emulsier-induced alteration of
microbiota composition plays a central role in the promotion
of carcinogenesis. The previous observation that genetically engi-
neered animals that develop intestinal inammation, such as
TLR5KO and NLRP3 mice, are not necessarily predisposed to
colonic carcinogenesis (unpublished data and ref. 45, respective-
ly) highlights the concept that alterations of proliferation and
apoptosis pathways in a microbiota-dependent manner may be
the central mechanism that mediates the emulsier-dependent
increase of carcinogenesis.
An important distinction between this study and others exam-
ining microbiota, inammation, and cancer, is that emulsier
consumption does not induce histopathologically evident (i.e.,
classic) inammation, but rather induces only low-grade inam-
mation. Such low-grade inammation is associated with and may
cause obesity and its interrelated metabolic diseases, that is,
metabolic syndrome. Hence, the mechanisms described herein
may be relevant not only to the potential promotion of colon
cancer by one class of food additive, emulsiers, but may be a
broad mechanism whereby any inducer of low-grade inamma-
tion, including obesity itself, may increase potential for cancer
development. While the increased risk of cancer development in
obese are modestly less than that of IBD patients (46), given the
very high and increasing prevalence of obesity in all developed
countries, low-grade inammation may prove to be a major factor
that underlies the increasing incidence of colon cancer. Hence, we
propose that numerous factors that induce low-grade inamma-
tion, including consumption of dietary emulsiers, may promote
a hostile environment in the colon by modifying the microbiota
composition, leading to low-grade intestinal inammation and
alterations in the colonic proliferation/apoptosis balance, there-
fore creating a favorable niche for colonic tumorigenesis.
Disclosure of Potential Conicts of Interest
No potential conicts of interest were disclosed.
Authors' Contributions
Conception and design: E. Viennois, D. Merlin, A.T. Gewirtz, B. Chassaing
Development of methodology: E. Viennois, B. Chassaing
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): E. Viennois, B. Chassaing
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): E. Viennois, B. Chassaing
Writing, review, and/or revision of the manuscript: E. Viennois, D. Merlin,
A.T. Gewirtz, B. Chassaing
Study supervision: B. Chassaing
Grant Support
This work was supported by NIH grants DK099071 and DK083890 (A.T.
Gewirtz). B. Chassaing is a recipient of the Career Development Award from the
Crohn's and Colitis Foundation of America (CCFA). E. Viennois is a recipient of
the Research Fellowship Award from the CCFA. D. Merlin is a recipient of a
Research Career Scientist Award from the Department of Veterans Affairs.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received May 13, 2016; revised October 25, 2016; accepted October 26, 2016;
published OnlineFirst November 7, 2016.
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Cancer Res; 77(1) January 1, 2017 Cancer Research40
Viennois et al.
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... A number of studies have reported that some of the approved food emulsifiers behave like detergents and can increase intestinal permeability, leading to a plethora of diseases including inflammatory bowel diseases, colitis, metabolic syndrome, obesity, and cancer. [42][43][44][45][46] Tang et al 47 reported that dietary emulsifier polysorbate-80 can cause cerebral cavernous malformation in mice through changes in the gut barrier. Similar to RNA-seq results, targeted proteomics suggested activation of cell proliferation, inflammatory and immune responses, cell migration, and cell adhesion processes in response to rinse aid in 1:40,000 dilution. ...
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Background The increased prevalence of many chronic inflammatory diseases linked to gut epithelial barrier leakiness has prompted us to investigate the role of extensive use of dishwasher detergents, among other factors. Objective We sought to investigate the effects of professional and household dishwashers, and rinse agents, on cytotoxicity, barrier function, transcriptome, and protein expression in gastrointestinal epithelial cells. Methods Enterocytic liquid-liquid interfaces were established on permeable supports, and direct cellular cytotoxicity, transepithelial electrical resistance, paracellular flux, immunofluorescence staining, RNA-sequencing transcriptome, and targeted proteomics were performed. Results The observed detergent toxicity was attributed to exposure to rinse aid in a dose-dependent manner up to 1:20,000 v/v dilution. A disrupted epithelial barrier, particularly by rinse aid, was observed in liquid-liquid interface cultures, organoids, and gut-on-a-chip, demonstrating decreased transepithelial electrical resistance, increased paracellular flux, and irregular and heterogeneous tight junction immunostaining. When individual components of the rinse aid were investigated separately, alcohol ethoxylates elicited a strong toxic and barrier-damaging effect. RNA-sequencing transcriptome and proteomics data revealed upregulation in cell death, signaling and communication, development, metabolism, proliferation, and immune and inflammatory responses of epithelial cells. Interestingly, detergent residue from professional dishwashers demonstrated the remnant of a significant amount of cytotoxic and epithelial barrier–damaging rinse aid remaining on washed and ready-to-use dishware. Conclusions The expression of genes involved in cell survival, epithelial barrier, cytokine signaling, and metabolism was altered by rinse aid in concentrations used in professional dishwashers. The alcohol ethoxylates present in the rinse aid were identified as the culprit component causing the epithelial inflammation and barrier damage.
... Cependant, l'exposition aux additifs alimentaires pourrait induire une dysbiose et une dérégulation de l'homéostasie intestinale avec une altération de l'intégrité de la barrière intestinale et de la couche de mucus, ainsi qu'une activation de la réponse immunitaire (Chassaing et al., 2015;Gültekin, 2019). Les émulsifiants tels que la carboxyméthylcellulose et le polysorbate-80 ont démontré des effets avérés sur la composition du microbiote intestinal des souris, avec notamment une augmentation des bactéries sulfato-réductrices (Chassaing et al., 2015;Viennois et al., 2017). Or, ces dernières pourraient jouer un rôle important dans la modulation de la douleur viscérale (Crouzet et al., 2013). ...
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Les perturbations de l'axe cerveau-intestin-microbiote font l’objet de nombreuses études afin de mettre au point de nouveaux traitements pour les pathologies intestinales chroniques telles que le syndrome de l’intestin irritable (SII). Dans certains cas, à la suite d’une infection gastro-intestinale par des Entérobactéries et malgré l'élimination de l'agent pathogène, des troubles du transit et des douleurs abdominales chroniques persistent et peuvent favoriser le développement de symptômes anxio-dépressifs. Ceci est alors appelé SII post-infectieux (SII-PI). En développant un modèle préclinique d'infection à Citrobacter rodentium, mes travaux se sont focalisés sur (1) l’étude de l'impact de la cytokine IL-22 sur les symptômes associés au SII-PI, (2) l’étude du métabolisme du tryptophane (Trp), en particulier l'expression d’AhR induite par ses ligands dérivés du microbiote et, (3) l’étude du microbiote et du métabolome fécal sur les troubles de l'homéostasie suite à la résolution d’une infection gastro-intestinale. Ainsi, nous avons montré que le modèle murin d'infection à C. rodentium développe en période post-infectieuse, une hypersensibilité viscérale d’origine colique (HSVC) persistante, un comportement de type anxieux ainsi que des altérations cognitivo-émotionnelles, associés à une dysbiose, une inflammation à bas bruit et à une perméabilité intestinale augmentée montrant qu’il s’agit d’un modèle pertinent pour étudier les mécanismes physiopathologiques du SII-PI. Dans un premier temps, des analyses de métabolomique dirigée ont dévoilé des perturbations fonctionnelles au niveau fécal en phase post-infectieuse. Le métabolisme du Trp est altéré avec une diminution de la voie indole et de l'activité d’AhR ainsi qu’une diminution de la production du tryptophol. La production de la cytokine IL-22 peut être activée par la voie AhR et représente un acteur essentiel de l’homéostasie intestinale. Ainsi l’efficacité de la vectorisation d’IL-22 à l’aide d’une souche de Lactococcus lactis portant un plasmide d'expression eucaryote pour l’IL-22 murine (L. lactisIL-22) a été testée dans ce modèle animal de SII-PI. Le traitement avec L. lactisIL-22 permet d’améliorer les perturbations associées à l’infection par C. rodentium durant la phase post-infectieuse. Nos résultats suggèrent que le ciblage de la voie de signalisation AhR/IL-22 atténue les symptômes du SII-PI en agissant sur l'intégrité de la barrière épithéliale intestinale et les métabolites du Trp dérivés du microbiote. Les études de métabolomique ont été élargies afin d’identifier les perturbations fonctionnelles du microbiote fécal induite par l’infection. Ces travaux ont permis de préciser la physiopathologie du SII-PI et d’identifier de nouvelles cibles thérapeutiques potentielles telle que la voie AhR/IL-22. De plus, le rôle d’autres infections sur les comportements cognitivo-émotionnels de l’hôte a été étudié dans deux modèles animaux. Une infection chronique parasitaire à Blastocystis chez le rat a été utilisé afin de mimer un SII, ainsi qu’une infection chronique aux Escherichia coli producteurs de colibactine (CoPEC), Entérobactéries associées au cancer colorectal (CCR).
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Background Accumulating evidence indicates that some non-absorbed food additives, including emulsifiers carboxymethylcellulose (CMC) and polysorbate 80 (P80), can negatively impact intestinal microbiota, leading to microbiota encroachment, chronic low-grade intestinal inflammation and, subsequently, promotion of metabolic dysregulations. Detrimental impacts of emulsifier consumption on gut microbiota include depletion of the health-associated mucus-fortifying bacteria, Akkermansia muciniphila . Objective Investigate, in mice, the potential of administration of exogenous A. muciniphila as a means to protect against detrimental impacts of emulsifiers. Results Daily oral administration of A. muciniphila prevented phenotypic consequences of consumption of both CMC and P80, including hyperphagia, weight gain and dysglycaemia. A. muciniphila administration also counteracted the low-grade intestinal inflammation-induced CMC and P80. Furthermore, A. muciniphila supplementation prevented the proximal impacts of CMC and P80 on gut microbiota that are thought to drive low-grade chronic inflammation and metabolic dysregulations. Specifically, A. muciniphila prevented alterations in species composition and encroachment of gut microbiota that were otherwise induced by CMC and P80. Remarkably, we finally report that CMC and P80 altered the colonic transcriptome, while A. muciniphila largely protected against these alterations. Conclusion Daily administration of A. muciniphila protects against the detrimental impact of emulsifiers on both the microbiota and host. These results support the notion that use of A. muciniphila as a probiotic can help maintain intestinal and metabolic health amidst the broad array of modern stresses that can promote chronic inflammatory diseases.
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Background: Growing evidence indicates the adverse effect of ultra-processed food (UPF) consumption. However, it remains unknown whether UPF consumption influences the risk of colorectal cancer precursors, namely conventional adenomas and serrated lesions. Methods: We drew data from the Nurses' Health Study (NHS), NHS II, and Health Professionals Follow-up Study (HPFS), comprising 142,052 participants who had undergone at least one lower gastrointestinal endoscopy during follow-up. To handle multiple records per participants, we used multivariable logistic regression for clustered data to calculate odds ratios (OR) and 95% confidence intervals (CIs) of colorectal polyps in relation to cumulative average consumption of UPFs. All statistical tests were two-sided. Results: We documented 11,644 cases with conventional adenomas and 10,478 with serrated lesions during 18-20 years of follow up. Compared to participants in the lowest quintile of UPF consumption, those in the highest quintile had an increased risk of conventional adenomas (OR = 1.18, 95% CI = 1.11 to 1.26) and serrated lesions (OR = 1.20, 95% CI = 1.13 to 1.28). Similar results were found for high-risk polyps (ie, advanced adenomas and ≥10 mm serrated lesions; OR = 1.17, 95% CI = 1.07 to 1.28). These associations were slightly attenuated but remained statistically significant after further adjusting for body mass index, Western dietary pattern score, or individual dietary factors (fiber, folate, calcium, and vitamin D). The results remained essentially unchanged after excluding processed meat from total UPF intake. Conclusions: Higher consumption of UPFs is associated with an increased risk of colorectal cancer precursors. UPFs might be a modifiable target for early prevention of colorectal cancer.
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Traditional Med diets consumed around the Mediterranean basin reflect the local environment and so may vary quite widely in food content. This chapter discusses the importance of minimising consumption of foods typical of a Western diet. The Western diet includes substantial amounts of meat and other animal products, refined grains and HFSS foods. Most of the packaged foods that typify the Western diet are highly calorific and of low nutritional value. The higher fibre content and lower level of free sugars in the Med diet compared to the Western diet result in different effects on the microbiome. The food industry is responding to the demand for pre prepared vegan/vegetarian foods with a massive expansion of new lines. Current evidence suggests that to ensure nutritional adequacy, it is easier for consumers to transition to flexitarian diets such as the Med diet than to vegetarian/vegan diets.
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This chapter discusses barriers and opportunities associated with consumption of the main foods and food groups of the Med diet. Price is a major reason why extra virgin olive oil (EVOO) is often replaced by other cooking oils. Raw and cooked EVOO are not normally distinguished in epidemiological studies and so their relative health merits are not well established. Frying creates desirable flavours, improves digestibility and can have health benefits. Preparing a Mediterranean stew usually begins by frying onions and other ingredients. Rapeseed oil is associated with health benefits. Fruit and vegetable selection in Mediterranean cuisine is rooted in local and seasonal produce. Pulses have amongst the highest nutritional value in relation to cost of any food group. Even though seafood is generally perceived as a healthy food, many consumers fail to meet dietary targets.
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Background and aims: Controversy surrounds the risk of colorectal cancer (CRC) in ulcerative colitis (UC). Many studies have investigated this risk and reported widely varying rates. Methods: A literature search using Medline with the explosion of references identified 194 studies. Of these, 116 met our inclusion criteria from which the number of patients and cancers detected could be extracted. Overall pooled estimates, with 95% confidence intervals (CI), of cancer prevalence and incidence were obtained using a random effects model on either the log odds or log incidence scale, as appropriate. Results: The overall prevalence of CRC in any UC patient, based on 116 studies, was estimated to be 3.7% (95% CI 3.2-4.2%). Of the 116 studies, 41 reported colitis duration. From these the overall incidence rate was 3/1000 person years duration (pyd), (95% CI 2/1000 to 4/1000). The overall incidence rate for any child was 6/1000 pyd (95% CI 3/1000 to 13/1000). Of the 41 studies, 19 reported results stratified into 10 year intervals of disease duration. For the first 10 years the incidence rate was 2/1000 pyd (95% CI 1/1000 to 2/1000), for the second decade the incidence rate was estimated to be 7/1000 pyd (95% CI 4/1000 to 12/1000), and in the third decade the incidence rate was 12/1000 pyd (95% CI 7/1000 to 19/1000). These incidence rates corresponded to cumulative probabilities of 2% by 10 years, 8% by 20 years, and 18% by 30 years. The worldwide cancer incidence rates varied geographically, being 5/1000 pyd in the USA, 4/1000 pyd in the UK, and 2/1000 pyd in Scandinavia and other countries. Over time the cancer risk has increased since 1955 but this finding was not significant (p=0.8). Conclusions: Using new meta-analysis techniques we determined the risk of CRC in UC by decade of disease and defined the risk in pancolitics and children. We found a non-significant increase in risk over time and estimated how risk varies with geography.
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The intestinal tract is inhabited by a large and diverse community of microbes collectively referred to as the gut microbiota. While the gut microbiota provides important benefits to its host, especially in metabolism and immune development, disturbance of the microbiota-host relationship is associated with numerous chronic inflammatory diseases, including inflammatory bowel disease and the group of obesity-associated diseases collectively referred to as metabolic syndrome. A primary means by which the intestine is protected from its microbiota is via multi-layered mucus structures that cover the intestinal surface, thereby allowing the vast majority of gut bacteria to be kept at a safe distance from epithelial cells that line the intestine. Thus, agents that disrupt mucus-bacterial interactions might have the potential to promote diseases associated with gut inflammation. Consequently, it has been hypothesized that emulsifiers, detergent-like molecules that are a ubiquitous component of processed foods and that can increase bacterial translocation across epithelia in vitro, might be promoting the increase in inflammatory bowel disease observed since the mid-twentieth century. Here we report that, in mice, relatively low concentrations of two commonly used emulsifiers, namely carboxymethylcellulose and polysorbate-80, induced low-grade inflammation and obesity/metabolic syndrome in wild-type hosts and promoted robust colitis in mice predisposed to this disorder. Emulsifier-induced metabolic syndrome was associated with microbiota encroachment, altered species composition and increased pro-inflammatory potential. Use of germ-free mice and faecal transplants indicated that such changes in microbiota were necessary and sufficient for both low-grade inflammation and metabolic syndrome. These results support the emerging concept that perturbed host-microbiota interactions resulting in low-grade inflammation can promote adiposity and its associated metabolic effects. Moreover, they suggest that the broad use of emulsifying agents might be contributing to an increased societal incidence of obesity/metabolic syndrome and other chronic inflammatory diseases.
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Colorectal cancer (CRC) is the third leading cause of cancer deaths worldwide and the fourth most common cancer diagnosed among men and women in the United States. Considering the risk factors of CRC, dietary therapy has become one of the most effective approaches in reducing CRC morbidity and mortality. The use of probiotics is increasing in popularity for both the prevention and treatment of a variety of diseases. As the most common types of microbes used as probiotics, lactic acid bacteria (LAB) are comprised of an ecologically diverse group of microorganisms united by formation of lactic acid as the primary metabolite of sugar metabolism. LAB have been successfully used in managing diarrhea, food allergies, and inflammatory bowel disease. LAB also demonstrated a host of properties in preventing colorectal cancer development by inhibiting initiation or progression through multiple pathways. In this review, we discuss recent insights into cellular and molecular mechanisms of LAB in CRC prevention including apoptosis, antioxidant DNA damages, immune responses, and epigenetics. The emerging experimental findings from clinical trials as well as the proposed mechanisms of gut microbiota in carcinogenesis will also be briefly discussed.
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Escherichia coli strains harbouring the pks island (pks+ E. coli) are often seen in human colorectal tumours and have a carcinogenic effect independent of inflammation in an AOM/IL-10(-/-) (azoxymethane/interleukin) mouse model. To investigate the mechanism sustaining pks+ E. coli-induced carcinogenesis. Underlying cell processes were investigated in vitro and in vivo (xenograft model) using intestinal epithelial cells infected by pks+ E. coli or by an isogenic mutant defective for pks (pks- E. coli). The results were supported by data obtained from an AOM/DSS (azoxymethane/dextran sodium sulphate) colon cancer mouse model and from human colon cancer biopsy specimens colonised by pks+ E. coli or pks- E. coli. Colibactin-producing E. coli enhanced tumour growth in both xenograft and AOM/DSS models. Growth was sustained by cellular senescence (a direct consequence of small ubiquitin-like modifier (SUMO)-conjugated p53 accumulation), which was accompanied by the production of hepatocyte growth factor (HGF). The underlying mechanisms involve microRNA-20a-5p, which targets SENP1, a key protein regulating p53 deSUMOylation. These results are consistent with the expression of SENP1, microRNA-20a-5p, HGF and phosphorylation of HGF receptor found in human and mouse colon cancers colonised by pks+ E. coli. These data reveal a new paradigm for carcinogenesis, in which colibactin-induced senescence has an important role.
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BACKGROUND: Chronic inflammation and oxidative stress are thought to be involved in colorectal cancer development. These processes may contribute to leakage of bacterial products, such as lipopolysaccharide (LPS) and flagellin, across the gut barrier. The objective of this study, nested within a prospective cohort, was to examine associations between circulating LPS and flagellin serum antibody levels and colorectal cancer risk. METHODS: A total of 1,065 incident colorectal cancer cases (colon, n = 667; rectal, n = 398) were matched (1:1) to control subjects. Serum flagellin- and LPS-specific IgA and IgG levels were quantitated by ELISA. Multivariable conditional logistic regression models were used to calculate ORs and 95% confidence intervals (CI), adjusting for multiple relevant confouding factors. RESULTS: Overall, elevated anti-LPS and anti-flagellin biomarker levels were not associated with colorectal cancer risk. After testing potential interactions by various factors relevant for colorectal cancer risk and anti-LPS and anti-flagellin, sex was identified as a statistically significant interaction factor (Pinteraction < 0.05 for all the biomarkers). Analyses stratified by sex showed a statistically significant positive colorectal cancer risk association for men (fully-adjusted OR for highest vs. lowest quartile for total anti-LPS + flagellin, 1.66; 95% CI, 1.10-2.51; Ptrend, 0.049), whereas a borderline statistically significant inverse association was observed for women (fully-adjusted OR, 0.70; 95% CI, 0.47-1.02; Ptrend, 0.18). CONCLUSION: In this prospective study on European populations, we found bacterial exposure levels to be positively associated to colorectal cancer risk among men, whereas in women, a possible inverse association may exist. IMPACT: Further studies are warranted to better clarify these preliminary observations.
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Background: Chronic inflammation and oxidative stress are thought to be involved in colorectal cancer development. These processes may contribute to leakage of bacterial products, such as lipopolysaccharide (LPS) and flagellin, across the gut barrier. The objective of this study, nested within a prospective cohort, was to examine associations between circulating LPS and flagellin serum antibody levels and colorectal cancer risk. Methods: A total of 1,065 incident colorectal cancer cases (colon, n = 667; rectal, n = 398) were matched (1:1) to control subjects. Serum flagellin- and LPS-specific IgA and IgG levels were quantitated by ELISA. Multivariable conditional logistic regression models were used to calculate ORs and 95% confidence intervals (CI), adjusting for multiple relevant confouding factors. Results: Overall, elevated anti-LPS and anti-flagellin biomarker levels were not associated with colorectal cancer risk. After testing potential interactions by various factors relevant for colorectal cancer risk and anti-LPS and anti-flagellin, sex was identified as a statistically significant interaction factor (Pinteraction < 0.05 for all the biomarkers). Analyses stratified by sex showed a statistically significant positive colorectal cancer risk association for men (fully-adjusted OR for highest vs. lowest quartile for total anti-LPS + flagellin, 1.66; 95% CI, 1.10-2.51; Ptrend, 0.049), whereas a borderline statistically significant inverse association was observed for women (fully-adjusted OR, 0.70; 95% CI, 0.47-1.02; Ptrend, 0.18). Conclusion: In this prospective study on European populations, we found bacterial exposure levels to be positively associated to colorectal cancer risk among men, whereas in women, a possible inverse association may exist. Impact: Further studies are warranted to better clarify these preliminary observations.
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Background: The American Cancer Society, the Centers for Disease Control and Prevention (CDC), the National Cancer Institute (NCI), and the North American Association of Central Cancer Registries (NAACCR) collaborate annually to update cancer rates and trends in the United States. This report updates statistics on lung, female breast, prostate, and colorectal cancers and highlights the uses of selected surveillance data to assist development of state-based cancer control plans. Methods: Age-adjusted incidence rates from 1996 through 2000 are from state and metropolitan area cancer registries that met NAACCR criteria for highest quality. Death rates are based on underlying cause-of-death data. Long-term trends and rates for major racial and ethnic populations are based on NCI and CDC data. Incidence trends from 1975 through 2000 were adjusted for reporting delays. State-specific screening and risk factor survey data are from the CDC and other federal and private organizations. Results: Cancer incidence rates for all cancer sites combined increased from the mid-1970s through 1992 and then decreased from 1992 through 1995. Observed incidence rates for all cancers combined were essentially stable from 1995 through 2000, whereas the delay-adjusted trend showed an increase that had borderline statistical significance (P = .05). Increases in the incidence rates of breast cancer in women and prostate cancer in men offset a long-term decrease in lung cancer in men. Death rates for all cancer sites combined decreased beginning in 1994 and stabilized from 1998 through 2000, resulting in part from recent revisions in cause-of-death codes. Death rates among men continued to decline throughout the 1990s, whereas trends in death rates among women were essentially unchanged from 1998 through 2000. Analysis of state data for the leading cancers revealed mixed progress in achieving national objectives for improving cancer screening, risk factor reduction, and decreases in mortality. Conclusions: Overall cancer incidence and death rates began to stabilize in the mid- to late 1990s. The recent increase in the delay-adjusted trend will require monitoring with additional years of data. Further reduction in the burden of cancer is possible but will require the continuation of strong federal, state, local, and private partnerships to increase dissemination of evidence-based cancer control programs to all segments of the population.
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Cougnoux and colleagues1 report a new mechanism by which select bacteria can drive colon cancer. Specifically, they discovered that colobactin-producing E. coli strains can activate growth-arrested (ie, senescent) cells to produce growth factors that drive tumor growth. These findings may, in part, underlie the association of such bacteria with carcinomas and could result in novel strategies to slow tumor growth. Approximately 20% of cancers are considered to be a consequence of infection by bacteria and/or viruses typically classified as pathogens. Moreover, many cancers occur in tissues with high exposure to microbiota, such as colorectal cancers (CRC), suggesting microbes not typically thought of as pathogens in promoting carcinogenesis. For example, mucosa-associated Escherichia coli are present more frequently in colon tissue from patients with adenocarcinomas than in control subjects. While E. coli are typically present in the intestine of healthy persons, various strains of E. coli have ‘pathogen-like features’ and associate with disease resulting in them being referred …
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Overweight and obesity have reached pandemic levels on a worldwide basis and are associated with increased risk and worse prognosis for many but not all malignancies. Pathophysiologic processes that affect this association are reviewed, with a focus on the relationship between type 2 diabetes mellitus and cancer, lessons learned from the use of murine models to study the association, the impact of obesity on pancreatic cancer, the effects of dietary fats and cholesterol on cancer promotion, and the mechanisms by which the intestinal microbiome affects obesity and cancer.
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Colorectal cancer (CRC) presents a considerable disease burden worldwide. The human colon is also an anatomical location with the largest number of microbes. It is natural, therefore, to anticipate a role for microbes, particularly bacteria, in colorectal carcinogenesis. The increasing accessibility of microbial meta'omics is fueling a surge in our understanding of the role that microbes and the microbiota play in CRC. In this review, we will discuss recent insights into contributions of the microbiota to CRC and explore conceptual frameworks for evaluating the role of microbes in cancer causation. We also highlight new findings on candidate CRC-potentiating species and current knowledge gaps. Finally, we explore the roles of microbial metabolism as it relates to bile acids, xenobiotics, and diet in the etiology and therapeutics of CRC.